umi - university of hawaii...i dedicate this dissertation to my wife, sue, in appreciation of her...
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REGULATION OF PROLACTIN AND CHANGES IN PROLACTIN AND GROWTH
HORMONE IN OSMOREGULATION, METABOLISM, AND REPRODUCTION IN
THE TILAPIA, OREOCHROMIS MOSSAMBICUS.
A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THEUNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
ZOOLOGY
MAY 1995
By
Gregory Martin Weber
Dissertation Committee:
E. Gordon Grau, ChairmanShannon Atkinson
Christopher L. BrownGillian D. Bryant-Greenwood
Fred I. Kamemoto
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UMI Number: 9532638
OMI Microform 9532638Copyright 1995, by OMI Company. All rights reserved.
This microform edition is protected against unauthorizedcopying under Title 17, United States Code.
UMI300 North Zeeb RoadAnn Arbor, MI 48103
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I dedicate this dissertation to my wife, Sue, in
appreciation of her constant support, and to my daughter,
Katelyn, who likes to see her name in books.
(MMS)
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ACKNOWLEDGEMENTS
I wish to extend my thanks to my thesis committee,
and especially to my advisor, Prof. E. Gordon Grau for his
support that allowed me to pursue a diversity of projects,
including some which are not encompassed in this disserta
tion. I would like to acknowledge the contributions of
Buel Rodgers with whom I ran the first experiments of this
work. This dissertation also includes studies conducted
with Drs. Hal Richman and Russell Borski. I would like to
thank Drs. Tatsuya Sakamoto and Takashi Yada for teaching
me the RIA procedures and Prof. Tetsuya Hirano for provid
ing the antibodies. Among the many other people who have
helped me along the way and may have also contributed to
this work are: Benny Ron, Craig Morrey, Brian Shepherd,
Steve Shimoda, Prof. Milton Stetson, Prof. Howard Bern,
Prof. Yoshitaka Nagahama, Dr. Richard Nishioka, Prof.
Nancy Sherwood, Dr. Matthew Grober, and Philippa Melamed.
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ABSTRACT
These studies addressed the regulation of the prolac
tins (PRL) and growth hormone (GH) in the tilapia, Oreo
chromis mossambicus. Regulation of the PRL cell was
investigated in the context of its potential roles in
osmoregulation, metabolism, and reproduction. The in
vitro release of the tPRLs (tPRL1 77 and tPRL1 SS) was
measured as well as changes in serum concentrations and
pituitary content of the PRLs and GH.
These studies provide evidence that it is the osmotic
gradient across the cell membrane that leads to an in
crease in osmotic pressure, and not osmolality per se,
which accounts for the osmoreceptivity of the tilapia PRL
cell. Prolactin release from pituitary tissues (rostral
pars distalis; RPD) was inhibited when RPD were incubated
in medium made hyperosmotic by the addition of NaCI or the
membrane-impermeant molecule, mannitol; but not by the
addition of the permeant molecules, urea or ethanol.
Injections of a gonadotropin-releasing hormone (GnRH)
analog elevated serum concentrations of the tPRLs. Fur
thermore, native forms of GnRH stimulated PRL release in
vitro with the following order of potency: chicken-GnRH-II
> salmon-GnRH > seabream-GnRH. Prolactin release was
accompanied by an increase in intracellular free ca2 + ,
suggesting Ca2 + operates as a second messenger in mediat-
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ing the effects of GnRH on PRL release. Estradiol-178 and
testosterone potentiated the effects of GnRH on PRL re
lease in vi tro.
Serum concentrations of the tPRLs were elevated in
response to fasting, followed by increases in serum con
centrations and pituitary content of GH. Serum concentra
tions and pituitary content of GH and serum concentrations
of PRL were highest late in the brooding phase of the
reproductive cycle. Reduced food intake during brooding
may contribute to changes in PRL and GH serum concentra
tions and pituitary content. Nevertheless, patterns of
changes in serum and pituitary levels of the tPRLs and GH
observed during the reproductive cycle had characteristics
that were both similar to and distinct from patterns
observed during fasting, suggesting the hormones may have
actions in both metabolism and reproduction. In addition,
salinity altered the patterns of changes in serum and
pituitary levels of the tPRLs, but not GH, observed during
the reproductive cycle.
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TABLE OF CONTENTS
DEDICATION iiiACKNOWLEDGMENTS i vABSTRACT vLIST OF TABLES viiiLIST OF FIGURES ixLIST OF ABBREVIATIONS xiiiCHAPTER I: Introduction , 1CHAPTER II: Alteration in osmotic pressure and not
osmolality are coupled to the sustained release ofthe osmoregulatory hormone, prolactin, in theeuryhaline teleost, tilapia(Oreochromis mossambicus) 34
INTRODUCTION 34MATERIALS AND METHODS 40RESULTS 45DISCUSSION 57
CHAPTER III: Gonadotropin-releasing hormone functionsas a prolactin-releasing factor in the tilapia,Oreochromis mossambicus 62
INTRODUCTION 62MATERIALS AND METHODS 65RESULTS 72DISCUSSION 97
CHAPTER IV: Changes in serum concentrations andpituitary content of the prolactins and growthhormone with fasting in the tilapia,Oreochromis mossambicus '" 108
INTRODUCTION 108MATERIALS AND METHODS '" 112RESULTS 117DISCUSSION 136
CHAPTER V: Changes in serum concentrations andpituitary content of the prolactins and growthhormone during the reproductive cycle of femaletilapia, Oreochromis mossambicus, adapted tofresh water and seawater 145
INTRODUCTION 145MATERIALS AND METHODS 150RESULTS 156DISCUSSION 175
CHAPTER IV: Conclusions 196REFERENCES 201
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LIST OF TABLES
Table Page1. The effects of fasting up to 21 days on condition
factor, body weight and standard length in maleand female tilapia with a high condition factor ... 123
2. The effects of fasting up to 31 days on conditionfactor, body weight and standard length in maletilapia with a low condition factor 132
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LIST OF FIGURES
Figure Page1. The effects of increasing medium osmolality by
the addition of membrane permeant (urea) andimpermeant (mannitol) molecules on PRL releasefrom the tilapia RPD during 18-20 hr staticincubation 48
2. The effects of increasing medium osmolality bythe addition of the membrane permeant moleculeurea, on PRL release from the tilapia RPD during18-20 hr static incubation '" 50
3. The effects of increasing medium osmolality bythe addition of the membrane permeant moleculeethanol, on PRL release from the tilapia RPDduring 18-20 hr static incubation 53
4. The effects of increasing medium osmolality bythe addition of membrane permeant (urea) andimpermeant (mannitol) molecules on PRL releasefrom the tilapia RPD during perifusion incubation .. 56
5. The effects of intraperitoneal injections ofmGnRHa on serum PRL concentrations in male andfemale tilapia..................................... 74
6. The effects of mGnRHa on PRL release from thetilapia RPD during 18-20 hr static incubation inisosmotic medium 77
7. The effects of mGnRHa on PRL release from thetilapia RPD during 18-20 hr static incubation inhyposmotic medium, isosmotic medium andhyperosmotic medium................................ 79
8. The effects of cGnRH-I, cGnRH-II and sGnRH on PRLrelease from the tilapia RPD during 18-20 hrstatic incubation '" 82
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9. The effects of cGnRH-II, sGnRH and sbGnRH on PRLrelease from the tilapia RPD during 18-20 hrstatic incubation 85
10. The effects of sbGnRH on PRL release from thetilapia RPD during perifusion incubation 87
11. The effects of cGnRH-II and changes in mediumosmolality on [Ca++]i in dispersed PRL cellsduring perifusion incubation 89
12. The effects of decreasing concentrations ofcGnRH-II and changes in medium osmolality on[Ca++]i in dispersed PRL cells and PRL releasefrom tilapia RPD during perifusion incubation 91
13. The effects of increasing concentrations ofcGnRH-II and changes in medium osmolality on[Ca++]i in dispersed PRL cells and PRL releasefrom tilapia RPD during perifusion incubation 93
14. The effects of E2, T, sGnRH and combinationsof the steroid hormones and sGnRH on PRL releasefrom the tilapia RPD during 3 hr static incubation. 96
15. The effects of 2-phenoxyethanol on serumconcentrations of the tPRLs and GH in tpe tilapia .. 115
16. Changes in serum concentrations of the tPRLs andGH during fasting in male and female tilapia witha high condition factor 119
17. Changes in pituitary content of the tPRLs and GHduring fasting in male and female tilapia witha high condition factor , 122
18. Changes in gonadosomatic index during fasting inmale and female tilapia with a high conditionfactor , 126
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19. Changes in serum concentrations of the tPRLs andGH during fasting in male tilapia with a lowcondition factor 128
20. Changes in pituitary content of the tPRLs and GHduring fasting in maletilapia with a lowcondition factor 131
21. Changes in hepatosomatic index during fasting inmale tilapia with a low condition factor 135
22. Changes in follicle mass during the brooding phaseof the reproductive cycle in the female tilapia ... 158
23. Changes in condition factor and hepatosomaticindex during the brooding phase of thereproductive cycle in FW-adapted female tilapiamaintained in an outdoor tank 161
24. Comparison of condition factors and hepatosomaticindices for FW- and 8W-adapted female tilapiawhich were either brooding post-yolksac larvae ornot brooding and at the end of vitellogenesis 163
25. Changes in serum concentrations of the tPRLs andGH during the brooding phase of the reproductivecycle in FW-adapted female tilapia maintained in anoutdoor tank 166
26. Changes in pituitary content of the tPRLs and GHduring the brooding phase of the reproductive cyclein FW-adapted female tilapia maintained in anoutdoor tank 168
27. Changes in serum concentrations of the tPRLs and GHduring the reproductive cycle in FW-adapted femaletilapia maintained in indoor tanks 171
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28. Changes in serum concentrations of the tPRLs andGH during the brooding phase of the reproductivecycle in 8W-adapted female tilapia maintained inindoor tanks...................................... 174
29. Changes in pituitary content of the tPRLs and GHduring the brooding phase of the reproductive cyclein 8W-adapted female tilapia maintained in indoortanks ' 177
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LIST OF ABBREVIATIONS
BW " Body weightcAMP '" 3'S'-cyclic adenosine
monophosphatecGnRH-I '" Chicken-gonadotropin-releasing
hormonecGnRH-II Chicken-gonadotropin-releasing
hormoneCF Condition factorcfGnRH Catfish-gonadotropin-releasing
hormoneE2 Estradiol-178FFA " Free fatty acidsFW Fresh waterGH Growth hormoneGHRH Growth hormone-releasing hormoneGnRH '" Gonadotropin-releasing hormoneGSI Gonadosomatic indexGtH GonadotropinHSI Hepatosomatic indexmGnRHa Mammal-gonadotropin-releasing
hormone analogmOsmolal MilliosmolalmOsm MilliosmolalPRL ProlactinRPD Rostral pars distalissbGnRH Seabream-gonadotropin-releasing
hormonesGnRH Salmon-gonadotropin-releasing
hormoneSL 'Standard lengthSW SeawaterT Testosterone
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Chapter I
INTRODUCTION
Prolactin (PRL) and growth hormone (GH) are members
of the same polypeptide family and are thought to be
derived from a common ancestral gene (Niall et al., 1971)
Not surprisingly, PRL and GH have many overlapping actions
and are regulated by many of the same factors, although
often in opposite directions. The spectrum of actions of
each of these hormones throughout the vertebrates includes
effects on reproduction, growth and development, metabo
lism, and osmoregulation (Clarke and Bern, 1980; Loretz
and Bern, 1982; Sakamoto et al., 1993; Nicoll, 1982).
Often, the hormones regulate these different functions
simultaneously. The cells secreting the hormones must,
therefore, receive regulatory information from a variety
of sources to be used in determining the secretion rate of
the hormones. In order to understand how PRL and GH
accomplish the regulation of diverse functions, changes in
hormones that are related to these activities must be
characterized and the factors regulating PRL and GH must
be identified. This has been the thrust of my research
and is the subject of this thesis.
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Two tiiapia proiactins
The rostral pars distalis (RPD) of the adenohypophy
sis of the tilapia releases two distinct PRL molecules
which are encoded by different genes and are not derived
through the differential processing of the same transla
tion product (Specker et ai., 1985b; Yamaguchi et ai.,
1988). The larger PRL (tPRL1 88) contains 188 amino acid
residues and has a molecular weight of 20.8 kDa, whereas
the smaller PRL (tPRL1 77) contains 177 amino acid residues
and has a molecular weight of 19.6 kDa (Yamaguchi et ai.,
1988) .
There is limited evidence that the two tPRLs may have
distinct as well as overlapping functions. The two PRL
molecules show similar effects in the tilapia sodium
retaining assay, a well characterized bioassay that
measures the osmoregulatory activity of PRL (Specker et
ai., 1985b). Nevertheless, Borski and colleagues (1992)
have shown that the ratio of the total amount of tPRL1 8 8
to tPRL1 77 is greater in the pituitary of freshwater
(FW)-adapted tilapia, Oreochromis mossambicus, compared
with seawater (SW)-adapted tilapia. Specker and col
leagues (1985a) found that tPRL1 88 and not tPRL177 has
growth-promoting activity in the tilapia. On the other
hand, Shepherd and colleagues (1994) found that injections
of tPRL1 77 and not tPRL1 88 are effective at restoring
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sulfate and thymidine incorporation in vi'tro by branchial
cartilage of hypophysectomized tilapia. The assay is a
well characterized assay for skeletal growth. Shepherd
and colleagues also found that tPRL1 77, but not tPRL1 88,
ovine-PRL or salmon-PRL, can displace GH from high
affinity, low-capacity binding sites in the tilapia liver.
A functional distinction between the two PRLs in regards
to reproduction or metabolism within the tilapia has not
been elucidated. Rubin and Specker (1992) found similar
activities of the two PRLs in affecting steroid release
from testicular tissues of courting and non-courting male
tilapia.
Tilapia pituitary pland morphology
The anatomical arrangement of the tilapia pituitary
gland imparts certain advantages as a model to study PRL
and GH cell physiology. The distinctive features of the
tilapia pituitary are described below in the next section,
followed with a description of how these features have
been exploited to study PRL and GH'cell function. The
morphology of the tilapia pituitary gland has been de
scribed in detail (Dharmamba and Nishioka, 1968; Bern et
al., 1975; Nishioka et al., 1988). Two features of the
pituitary gland common to most teleosts including the
tilapia, are strikingly different from those of mammals.
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They are: 1) hormone-producing cell types are segregated
into discrete areas of the pituitary gland, and 2) hypo
thalamic fibers innervate the adenohypophysis (Dharmamba
and Nishioka, 1968; Bern et al., 1975; Ball, 1981). In
the tilapia, the proximal pars distalis contains GH cells
in addition to gonadotropin (GtH) and thyrotropin cells.
The RPD contains PRL and adrenocorticotropin cells. The
PRL cells are located in the anterior region of the RPD
and adrenocorticotropin cells are located close to the
neurohypophysis. Hypothalamic fibers innervate the proxi
mal pars distalis and terminate in close proximity to the
hormone-secreting cells. In contrast, hypothalamic fibers
do not leave the neurohypophysis in the RPD, but terminate
on an adjacent basement membrane. Separating the PRL
cells and the hypothalamic fibers are the basement
membrane, the adrenocorticotropin cells, and a layer of
stellate cell processes (see review, Nishioka et al.,
1988). Nishioka and colleagues (1988) pose the possibili
ty that the stellate cells are involved in the movement of
neurohormonal factors from the neurohypophysial nerve
endings to the PRL cells.
Tilapia PRL and GH cells as models for studying PRL and GH
cell function
Interactions among hormones in heterogeneous cell
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populations complicate the study of the regulation of
individual cell types. Segregation of cell types within
the teleost pituitary facilitates separation of certain
cell types for study. A tissue containing a nearly homo
geneous population of PRL cells can be obtained for in
vitro study by a simple dissection of the anterior RPD.
The RPD tissue obtained in this way is 95-99% PRL cells.
An additional attribute of the PRL cell which lends itself
to study is its sensitivity to small physiological changes
in osmolality. This sensitivity allows for the control of
baseline release to facilitate the investigation of poten
tially important regulators of PRL secretion (cf. Grau et
al., 1982). Cells of the proximal pars distalis can also
be separated from cells of the pars intermedia and RPD by
simple dissection. Separation of GH, GtH and thyrotropin
cells from each other is more difficult. Currently, most
investigators utilize mammalian clonal cell lines derived
from PRL tumors to study the regulation of PRL cells
(Tashjian et al., 1970; Lamberts and MacLeod, 1990; Sato
et al., 1990). A concern with the use of tumor cell lines
is that one can not be sure that they are studying normal
cell function as opposed to tumor cell function.
Direct innervation of the tilapia adenohypophysis by
hypothalamic neurosecretory fibers enables the tracing of
fibers from the hypothalamus to the region of the
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pituitary where the hormone is released. Since the hor
mone-secreting cells of the pituitary are segregated,
neurosecretory fibers can be followed to determine at
which cells the neurosecretory factors are released, and
therefore, may regulate. Furthermore, neurosecretory
factors reaching the pituitary can be easily identified
due to the high quantity of the factors in the'nerve
terminals in the pituitary. I have taken advantage of
this feature and determined with colleagues, that sea
bream-gonadotropin-releasing hormone is the most abundant
form of gonadotropin-releasing hormone (GnRH) in the
tilapia pituitary (Weber et al., 1994).
Hormonal regulation of teleost PRL and GH cell activity
The teleost PRL cell responds to many molecules in a
way that is similar to the manner in which the molecules
have been shown to be effective in mammals. Basal PRL
secretion in teleosts and mammals is under inhibitory
control from the hypothalamus. When connections with the
hypothalamus are severed at the pituitary stalk, as in
auto-transplants, the pituitary secretes high levels of
PRL. In mammals, this inhibition appears to be controlled
predominantly by dopamine (cf. Lamberts and MacLeod, 1990)
while in the tilapia, somatostatin appears to be the
predominant suppressor molecule (Grau et al., 1982, 1985).
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Thyrotropin-releasing hormone stimulates PRL release in
mammals and tilapia, while vasoactive intestinal peptide
is a stimulator in mammals and an inhibitor in tilapia
(Barry and Grau, 1986; Kelley et al., 1988; cf. Lamberts
and MacLeod, 1990). A non-hypothalamic hormone, cortisol,
has been shown to be a potent inhibitor of PRL release in
the tilapia (Wigham et al., 1977; Borski et al., 1991).
More related to reproduction, it has been shown that
the tilapia PRL cells are stimulated to release PRL in
response to estradiol-17B (E2) and testosterone (T),
(Wigham et al., 1977; Barry and Grau, 1986; Borski et al.,
1991). Other steroids including progestins have been
found to have no effect on PRL release (Borski et al.,
1991). Furthermore, stimulation of PRL cells by thyrotro
pin-releasing hormone was only observable following pre
treatment with E2 (Barry and Grau, 1986). Stimulation of
PRL release from PRL cells by E2 and T suggests an
increase in PRL activity during phases of the reproductive
cycle when circulating E2 and T levels are elevated.
Control of GH cell activity in teleosts is not as
well characterized as control of PRL cell activity (see
review, Nishioka et ai, 1988). In mammals, GH secretion
is primarily under the control of hypothalamic regulators.
Growth hormone-releasing hormone (GHRH) stimulates GH
secretion and somatostatin inhibits GH secretion. It is
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clear that the inhibitory role of somatostatin on GH cell
activity has been conserved in teleosts including the
tilapia (Fryer et ai., 1979). Studies examining the
effects of GHRH on GH release have been mixed. Neverthe
less, GHRH was found to be a potent stimulator of GH
release in the only study to use native GHRH (Vaughan et
ai., 1992). Vaughan and colleagues (1992) isolated and
synthesized GHRH from the common carp and found it able to
elevate circulating GH levels and stimulate release of GH
from pituitary cells. In this same study a native GnRH,
salmon GnRH (sGnRH), was found to be equipotent with carp
GHRH at the one concentration compared (100 nm). Marchant
et ai. (1989) have shown that GnRH is a potent stimulator
of GH release in the goldfish. Injections of a human GHRH
fragment, fragment 1-29, elevated plasma GH levels in a
tilapia hybrid. Furthermore, GnRH fragment 1-29 and carp
GHRH stimulated GH release from pituitary fragments with
similar potency (Melamed et ai., 1995). In the same
study, injections of a sGnRH superactive analog was more
effective than the human GHRH fragment in elevating GH
levels. In addition, sGnRH was more potent than either
GHRH form in stimulating GH release in vitro.
Cortisol is a potent stimulator of GH release, yet
direct effects of sex steroids on GH secretion in the
tilapia have not been demonstrated (Nishioka et ai., 1985;
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Helms et al., 1987). There is evidence, however, that
androgens have modulatory effects on GH-releasing factors,
similar to the effects of E2 on the induction of PRL
release by thyrotropin-releasing hormone described earlier
(Barry and Grau, 1986). Melamed (1993) has shown that
GHRH and GnRH are only effective in vivo and in vitro in a
tilapia hybrid when the fish are reproductively mature.
Furthermore, the releasing factors can be effective in
vivo with reproductively immature animals if the fish are
first injected with T or the synthetic androgen, 17a
methyltestosterone. Growth hormone-releasing hormone and
GnRH are also effective in vitro with tissues from
reproductively immature tilapia, if the tissues are
co-incubated with these steroids (Melamed, 1993). In con
trast to sex steroids, the glucocorticoid cortisol has a
direct and potent stimulatory effect on GH secretion
(Nishioka et al., 1985; Helms et al., 1987). Finally,
insulin-like growth factor-I inhibits GH release in tele
osts as in mammals (Perez-Sanchez et al., 1992).
Possible role of GnRH in PRL regulation
One area of regulation of reproduction in which the
teleost system may differ from the mammalian system is
with regard to GnRH. First, while most eutherian mammals
have only one form of GnRH, mammalian-GnRH, teleosts, like
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most vertebrates examined to date, have multiple forms
(see review, Sherwood et ai., 1994). By convention, the
GnRH molecules are named for the animals from which they
were first identified. There is evidence for specificity
of function for the different forms of the peptide.
Chicken-GnRH II (cGnRH-II) was found to be more potent in
releasing GtH from peri fused fragments and dispersed cells
of the goldfish pituitary than sGnRH, while sGnRH was
found to be the more potent of the two in releasing growth
hormone (GH) from the pituitary fragments, although equip
otent with dispersed cells (Chang et ai., 1989; Peter et
ai., 1990).
The possibility that one or more of the multiple
forms of GnRH present in the tilapia may stimulate PRL
release was investigated as part of the studies described
in this dissertation. Marchant and colleagues (1989) have
shown that GnRH stimulates GH release in the goldfish, and
Melamed and colleagues (1995) have shown the same for a
tilapia hybrid. However, Cook and colleagues (1991) were
not able to detect GnRH binding to PRL cells of the gold-
fish despite binding to GtH and GH cells.
The involvement of GnRH in PRL regulation is not well
understood in mammals or in fishes. In mammals, GnRH has
been shown to stimulate PRL release in vivo when basal PRL
release is within normal ranges, but inhibit PRL release
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when basal PRI release is unusually high as in hyperpro
lactinemia (Debeljuk et al., 1985j Kugu et al., 1988j
Sridaran et al., 1988). Injections of GnRH have been
shown to increase PRL cell activity in the Atlantic salm
on, based on an immunocytological and electron microscopi
cal study (Ekengren et al., 1978). The authors suggest
that this effect was mediated via GnRH stimulation of GtH
release which in turn, stimulated E2 release, a potent
stimulator of PRL cell activity.
One example of PRL stimulation by GnRH in a mammal
may not fit the teleost. Denef and Andries (1983) have
shown that in the fourteen-day-old rat, GnRH stimulates
gonadotroph cells to release a paracrine factor that
stimulates PRL release. This factor is not one of the
GtHs. They demonstrated that PRL release is stimulated
when a population of dispersed pituitary cells, including
both PRL and GtH cells, are treated with GnRH. However,
if GtH cells are removed from the culture, GnRH has no
effect. Furthermore, when PRL cells are incubated in
media from GnRH stimulated GtH cells, PRL release is
increased. Recent studies suggest that angiotensin II may
be this paracrine factor (Becu-Villalobes et al., 1994).
The anatomical arrangement of the tilapia pituitary makes
this same paracrine interaction less likely. The PRL
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cells of the tilapia are segregated into the RPD while the
GtH cells are in the proximal pars distalis.
The role of Ca2 + in tilapia PRL release
Indirect evidence suggests that PRL release in
response to an osmotic signal is Ca2+ dependent in the
tilapia (see reviews, Grau and Helms, 1989; Grau et al.,
1994). Recently, Borski (1993) has used the Ca 2+
sensitive fluorescent dye, fura-2, to show that intracel
lular free Ca 2+ concentrations ([Ca2+]i) increase within
20 sec after exposure to reduced osmolality medium,
further supporting a role for Ca2+ in PRL release. Now
that homologous radioimmunoassays are available for tila
pia PRLs, the time-course for PRL release in conjunction
with changes in [Ca2+]i, in response to osmotic stimuli,
should be investigated. Jobin and Chang (1992) have also
shown that native GnRH stimulates increases in [Ca 2+]i in
GtH and GH cells of goldfish. As part of my studies, I
examined simultaneous changes in [Ca2+]i and the release
of the two tPRLs in response to reductions in medium
osmolality and GnRH.
Roles and regulation of PRL and GH in osmoregulation
The tilapia, o. mossambicus, is a euryhaline teleost
fish that reproduces and thrives in habitats ranging in
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salinity from fresh water (FW) to hypersaline seawater
(SW). Prolactin and GH are regulators of physiological
processes in the tilapia, which confer the ability to
adapt to various salinities.
Prolactin plays a central role in FW adaptation and
GH is increasingly believed to play a role in SW adapta
tion in tilapia and other euryhaline teleost fishes.
Prolactin maintains extracellular salt and water balance
when the fish are in a low salinity environment by acting
on virtually all osmoregulatory tissues including the
gills, integument, urinary bladder, intestine and kidney
to reduce water permeability and increase sodium retention
(Clarke and Bern, 1980; Hirano, 1986). Growth hormone has
been shown to act in SW adaptation although its mode of
action is not as well known as for PRL. Studies suggest
that GH affects ion transport at the gills and may be
working in conjunction with cortisol and insulin-like
growth factors, or through a stimulation of cortisol and
insulin-like-growth factors (see review, Sakamoto et al.,
1993, Sakamoto et al., 1994).
Consistent with the roles of PRL and GH in osmoregu
lation, PRL cell activity is enhanced when tilapia are
maintained in FW and is reduced in SW fish, while the
reverse is true for GH cell activity (Dharmamba and
Nishioka, 1968; Clarke et al., 1973; Nagahama et al.,
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1975; Nishioka et al., 1993; Borski et al., 1994). Small
changes in medium osmolality, well within the physiologi
cal range of the tilapia, alter PRL and GH release from
pituitary tissues (Nagahama et al., 1975; Grau et al.,
1982; Helms et ali 1987). Grau and colleagues (1982) have
shown that PRL release from PRL cells is increased in
response to reductions in medium osmolality within 10-20
mini time-course studies have not been conducted for GH
cells. Nagahama and colleagues (1975) have shown that the
PRL cells respond to changes in medium osmolality and not
sodium or chloride ions per se, by demonstrating that the
response of PRL cells to changes in medium osmolality are
the same whether the changes in medium osmolality are due
to differences in NaCl concentration or are made by
changes in the membrane-impermeant molecule, mannitol. It
has not been determined whether changes in medium osmolal
ity or the osmotic gradient across the cell membrane
leading to a change in osmotic pressure, evoke the osmotic
response. This question was addressed as part of this
dissertation.
Circulating levels of GH and PRL change during adap
tation of o. mossambicus to different salinities.
Circulating and pituitary levels of PRL were observed to
decrease when tilapia were transferred or acclimated from
FW to SW and to increase when transferred or acclimated
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from SW to FW (Nicoll et ai., 1981; Borski et ai., 1992;
Ayson et ai., 1993; Yada et ai., 1994).
Borski and colleagues (1994) found that the pituitary
content of GH was almost twice as high in male tilapia
raised in SW for 7 months compared with male tilapia
raised in FW fQr 7 months. In addition, pituitary GH
levels were reduced in male fish transferred from SW to FW
and increased in fish transferred from FW to SW after 49
days. Ayson and colleagues (1993) did not see an increase
in pituitary GH in O. mossambicus (sex not reported) 3-4
weeks after transfer from FW to SW. Yada et ai. (1994)
observed an increase in plasma GH concentrations shortly
after transfer of male o. mossambicus from FW to 70~-SW
but no change in levels in females. Growth hormone con
centrations in the plasma were reduced shortly after
transfer from SW to FW in both sexes, compared to animals
transferred from SW to SW, at the same time points.
Circulating levels of GH were no longer significantly
different from controls, in either direction of transfer
and either sex, by 7 days after transfer. Sakamoto and
colleagues (1994) also observed an increase in plasma GH
concentrations following transfer of O. mossambicus from
FW to SW, whereas plasma GH was not altered following
transfer from SW to FW. Furthermore, the increase in
plasma GH was observed in both males and females and at
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the end of the study, day 14 after transfer, plasma GH was
elevated in males but not in females (Sakamoto, personal
communication) .
The patterns of circulating GH concentrations
observed in o. mossambicus following transfer from FW to
SW or SW to FW are similar to those observed in salmonids
undergoing comparable salinity changes (see review,
Sakamoto et al., 1993; Yada et al., 1994; Sakamoto et al.,
1994). Circulating levels of GH increase transiently or
do not change following transfer from FW to SW or SW to FW
in salmonids (see review, Sakamoto et al., 1993). Trans
fer of rainbow trout and coho salmon to SW was accompanied
by an increase in metabolic clearance rate and the
calculated secretion rate of GH whereas clearance kinetics
did not change in coho salmon transferred from SW to FW
(Sakamoto et al., 1990, 1991). Whether there are changes
in metabolic clearance rate or secretion rate of GH in
tilapia following adaptation to FW or SW has not been
determined.
Roles and regulation of PRL and GH in metabolism
Prolactin and GH have effects on protein, carbohy
drate, and lipid metabolism in mammals (see reviews,
Nicoll, 1974; Fain, 1980; Davidson, 1987). These metabol
ic actions appear to be conserved for GH in teleosts,
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while the actions of PRL in metabolism have received
little attention.
Prolactin can be lipolytic or lipogenic in teleosts,
depending on the influences of temperature and photoperi
od, circadian rhythms, and development (Lee and Meier,
1967; de Vlaming and Pardo, 1974; de Vlaming et ai., 1975;
Horseman and Meier, 1979; Sheridan, 1986). There has been
only one study examining circulating PRL concentrations
with changes in metabolic state. In this study, circulat
ing PRL levels were not altered in Kokanee salmon
(Oncorhynchus nerka), fasted for 30 days (McKeown et ai.,
1975). Nevertheless, Rodgers and colleagues (1992) found
both pituitary content of PRL and basal release from RPD
in vitro are decreased after 2 weeks of fasting in O.
mossambicus. In this same study, PRL release in vitro was
negatively correlated with the concentration of essential
amino acids added to the incubation medium and was
unaffected by D-glucose concentration. Although PRL
appears to have metabolic actions in teleosts, the roles
of PRL in regulating metabolism are still unclear. I
examined changes in serum and pituitary levels of PRL in
response to fasting as part" of this thesis.
The role of GH in metabolism has received more atten
tion in teleosts than has the role of PRL, but is not
without controversy. Growth hormone treatment has been
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shown to increase free fatty acids (FFA) in the circula
tion of rainbow trout and goldfish and to mobilize lipid
reserves from the liver of rainbow trout and coho salmon
(Minick and Chavin, 1970; Leatherland and Nuti, 1981;
Sheridan, 1986). In addition, GH injections increased
muscle FFAs but not plasma FFAs or glucose, and increased
liver glycogen in Kokanee salmon (McKeown et al., 1975).
Different responses to GH treatment were observed with the
eel, Anguilla japonica, and tilapia, o. mossambicus.
Injections of bovine-GH in hypophysectomized eels
increased serum amino acid concentration and did not alter
plasma lipid concentration (Inui et al., 1985). Injec
tions of bovine-GH stimulate the release of glycogen and
not lipid from liver reserves of the tilapia, and increase
serum concentrations of amino acids and glucose but not
protein, lipid or cholesterol (Leung et al., 1991).
Consistent with this elevation in circulating amino acids
and glucose with fasting, Rodgers and colleagues (1992)
found an increase in GH release from pituitary tissues in
response to reductions in essential amino acids and
glucose in the incubation medium, in the same species.
Together the results of these studies suggest that GH
cells act in the regulation of, and are directly respon
sive to changes in the blood concentrations of amino acids
and glucose in the tilapia.
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Increases in circulating and pituitary levels of GH
have been observed in fasted teleosts. Increases in
plasma GH levels have been observed within 3 weeks of
fasting in rainbow trout by Wagner and McKeown (1986), and
within 1 week by Sumpter et al. (1991). No change in
plasma GH was observed after 30 days of fasting in Kokanee
salmon (McKeown et al., 1975). Farbridge and Leatherland
(1992b) observed a bi-modal response to fasting in rainbow
trout. Plasma GH concentrations were lower in fasted fish
than in fed fish at 2 weeks of fasting, followed by an
elevation in circulating GH concentrations in fasted fish
at 4 weeks. Plasma GH concentrations were elevated at 2
weeks of fasting in a repeat of the study. Melamed (1993)
observed a rise in plasma GH within 16 days of fasting in
a tilapia hybrid. Also in tilapia, o. mossambicus, Rogers
and colleagues (1992) found that pituitary content of GH
was elevated with 2 weeks of fasting.
Farbridge et al. (1992) found that feeding rainbow
trout only once every five days resulted in reduced plasma
GH and suggested that GH concentrations are depressed by
low feeding rates. This is not consistent with the study
on the tilapia hybrid, by Melamed (1993). The tilapia at
the start of the experiment by Melamed, were described as
"sub-optimally fed". When the fish were switched to an
increased feeding rate, plasma GH levels fell within 8
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days. The nutritional state of the animal prior to fast
ing and changes in nutritional state during fasting is
likely to affect the hormonal response to subsequent
fasting. For this reason, I examined changes in serum and
pituitary levels of GH and PRL, in response to fasting, in
tilapia which had been well-fed and tilapia which had been
on a restricted diet. I also measured changes in body
parameters including body weight, length, gonad weight,
and liver weight in response to fasting as a means of
assessing changes in the nutritional state of the animals.
The reproductive cycle of the female tilapia
Prolactin and GH have actions in reproduction in
teleosts. Before reviewing these actions, I will first
discuss what is known about the reproductive cycle of the
female tilapia. Tilapias are important food fishes world
wide and therefore their reproductive habits and physiolo
gy have received much attention. The tilapia o. mossambi
cus, can reproduce in FW and in SW. After the female lays
her eggs and they are fertilized by the male, she picks up
the eggs and broods them in her buccal cavity. Brooding
continues for about 3 weeks. The female tilapia o. mos
sambicus, can reproduce every 20-25 days if they do not
brood. If the female does brood, the inter-spawn period
is extended to approximately 40 days. Smith and Haley
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(1987, 1988) have described sex steroid and ovarian
morphology profiles of the reproductive cycle for brooding
and non-brooding female O. mossambicus. Plasma steroid
hormone levels and ovarian tissue responsiveness to GtH at
different phases of the breeding cycle have also been
examined for another tilapia species, O. aureus, by
Bogomolnaya and colleagues (1984).
Smith and Haley (1987) found that postovulatory
follicles remain viable over the length of the brooding
period in fish that brood, but regress quickly in those
females which do not. They have provided evidence that
these structures produce steroid hormones, most strongly
during the first 7 days after spawning. In addition,
blood levels of E2 and T are elevated during the latter
phase of the brooding cycle, even though oocytes consti
tuting the next clutch to be spawned are arrested in early
vitellogenesis during this time. Both E2 and T are also
elevated during brooding in O. aureus (Bogomolnaya and
colleagues, 1984). Smith and Haley (1987) found that
ovarian growth is not arrested in non-brooding female
tilapia that are fasted, however, the oocytes show signs
of atresia at latter stages. Smith and Haley (1987, 1988)
suggest that E2 and testosterone, and postovulatory folli
cles, may be involved in parental care behavior, as well
as the arresting of oocyte growth during brooding and the
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protection of oocytes from atresia in the tilapia. Based
on what is known about the actions of PRL and GH in
reproduction and metabolism, PRL and GH should also be
considered in these roles. Furthermore, PRL and GH may be
involved in the elevation of the steroid hormone levels
and in prolonging the life of the postovulatory follicles.
In addition to the reproductive roles PRL and GH may
have during the reproductive cycle of the tilapia, the
hormones may be called upon to regulate osmotic homeosta
sis and metabolism. This would depend on both the salini
ty in which the animals reproduce and on metabolic changes
that may occur in response to reduced feeding during the
brooding phase of the reproductive cycle. For this reason
I characterized changes in serum and pituitary levels of
the tPRLs and GH during the reproductive cycle of FW- and
SW-adapted female tilapia as well as fed and fasted
tilapia as part of my studies.
Prolactin and teleost perentzeI care behaviors
The roles of PRL in vertebrate reproduction are
diverse; often they are associated with the nurturing of
young. These include nestbuilding and protective behav
iors as in birds, rabbits and rats, and mitogenic effects
associated with maternal tissue derived feeding, such as
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mammary development and lactation and pigeon cropsac
development (cf. Nicoll and Bern, 1971).
Similar actions have been attributed to PRL in
teleosts; these include parental care behaviors such as
fanning of eggs and nests by sticklebacks, bluegills and
cichlids. Also in cichlids, "calling movements" to fry, a
behavior exhibited by o. mossambicus, and reduced feeding
behavior suggested to prevent the cannibalism of young
during brooding, have been attributed to PRL. Prolactin
has been shown to induce mucus secretion in the discus
fish, a source of nutrition for developing fry (Noble et
al., 1938; Blum and Fielder, 1965; Slijkhuis et al., 1984;
DeRuiter et al., 1986; Kindler et al., 1991). The tilapia
is a cichlid and displays parental care behaviors similar
to those attributed to PRL in other cichlids. Thus, PRL
may be involved in the regulation of parental care behav
ior in the tilapia.
Prolactin cell activity was shown to be increased in
male sticklebacks displaying fanning behavior, based on
ultrastructure morphometry and r3H] - l y s i ne incorporation
rate of PRL cells (Slijkhuis et al., 1984). However, the
same group (Wendelaar Bonga et al., 1984) found no differ
ences in PRL cell activity between brooding and non-brood
ing female tilapia using these same techniques. While
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this conclusion may be correct, further investigation is
warranted.
Roles of PRL in teleost reproduction
Evidence suggests that PRL has roles in reproduction
in teleosts in addition to regulating behavior. As I have
discussed earlier, the sex steroids E2 and T stimulate PRL
cell activity directly and sensitize PRL cells to thyro
tropin-releasing hormone stimulation. Studies examining
changes in circulating or pituitary PRL levels with
reproductive cycles are limited but do provide support for
PRL having roles in reproduction. Prolactin has been
implicated in the regulation of vitellogenesis (covered in
detail in the next section) and steroidogenesis. Finally,
specific binding of ovine-PRL has been observed in mem
brane preparations of ovary and testis of tilapia (Edery
et al., 1984).
Hirano et al. (1986) and Prunet et al. (1990) both
cite preliminary data suggesting plasma PRL concentrations
change during the reproductive cycle in female salmonids
and suggest an inverse correlation with plasma progestins.
The rise in PRL in rainbow trout described by Prunet and
colleagues (1990) did not occur until 4 weeks after
ovulation. Changes in prolactin bioactivity in sera and
pituitary of the FW catfish, Clarias batrachus, assessed
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using a pigeon crop sac assay, paralleled changes in GtH
concentrations (Singh and Singh, 1981). In contrast, no
changes in ultrastructure of PRL cells were observed
during the reproductive cycle of the sailfin molly,
Poecilia latipinna (Young and Ball, 1983).
To date, there have been only three studies on the
effects of teleost PRL on steroidogenesis. Singh and
colleagues (1988) used purified salmon PRL to examine
gonadal steroidogenesis in hypophysectomized Fundulus
heteroclitus, and found salmon PRL significantly increased
plasma concentrations of T in males but had no effect on
steroid levels in females and no effect on steroid release
in vitro by either testes or ovaries despite preventing
the decline in gonadal weight usually associated with
hypophysectomy. Both tPRLs, tPRL1 88 and tPRL1 7 7, were
tested for their effects on E2 production by vitellogenic
oocytes of the guppy, Poecilia reticulata, (Tan et al.,
1988). Prolactin1 88 stimulated E2 production from all
stages of vitellogenic oocytes of the guppy with the
strongest response elicited from oocytes at the beginning
of vitellogenesis. Prolactin177 was found to have no
consistent effect. Neither of these studies used homolo
gous PRL, and therefore, the specificity of the responses
to the hormones is open to question.
Rubin and Specker (1992) conducted the only study to
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examine the effects of homologous PRL on steroidogenesis.
They examined the effects of the two tPRLs on steroid
production by testicular tissues of courting and non
courting male tilapia. Rubin and Specker (1992) found
similar effects with both tPRLs. The tPRls stimulated T
production in testicular tissue from courting males, but
not in testicular tissue from non-courting males. The
tPRLs also increased ovine-luteinizing hormone-stimulated
T production in courting males but were inhibitory in
non-courting males.
Blum and Weber (1968), showed that injections of
ovine-PRL increased steroid-3g-ol-dehydrogenase activity
in the cichlid, Aquideus pulcher. Young and colleagues
(1983), reported that ovine-PRL increases E2 and 17a,20B
dihydroxy-4-pregnen-3-one production by ovarian follicles.
Collectively, changes in circulating PRL levels during the
reproductive cycle, effects of exogenous PRL on steroido
genesis and vitellogenesis, and detection of ovarian and
hepatic PRL receptors strongly indicate a role for PRL in
control of fish reproduction.
Roles of GH in teleost reproduction
Several lines of evidence suggest GH may have roles
in fish reproduction. Circulating GH levels change during
the reproductive cycle of fishes; GH preparations affect
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steroidogenesis and gonadal development; and GH receptors
are present in the ovary and in the liver, the site of
vitellogenin production.
The recent availability of homologous radioimmunoas
says led to the discovery that GH is present at moderate
levels in the circulation during vitellogenesis (oocyte
growth) or spermatogenesis in several fish species, and
then increases abruptly during or just prior to the spawn
ing period (Stacey et ai., 1984; Marchant and Peter 1986;
Bjornsson et ai., 1991; Swanson 1991). In pituitaries of
vitellogenic striped bass (genus Morone) , immunoreactive
GH cells were strongly labeled with a heterologous
antiserum to fish GH (Huang and Specker, 1994). The
density of GH cells and their intensity of staining then
decreased in spawning fish, suggesting changes in GH
secretion are linked to final maturation. Changes in
serum GH and GtH levels are closely correlated during the
ovulatory GtH surge in goldfish (Yu et ai., 1991). Sumpt
er and colleagues, (1991b) on the other hand, found no
significant elevations in GH with reproductive cycle in
female rainbow trout until after ovulation. They suggest
the rise in GH after ovulation and the rise in GH observed
in maturing males, were due to starvation effects and were
not associated with reproduction per se.
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In salmonids, mammalian GH preparations have long
been known to enhance ovarian growth, increase circulating
levels of sex steroids and promote in vitro ovarian ste
roidogenesis (Higgs et al., 1976; Fostier et al.,
1983; Young et al., 1983). Similar effects of bovine-GH
were recently observed in spotted seatrout (Singh and
Thomas, 1993). In combined treatments, stimulation of
steroidogenesis by bovine-GH and human chorionic GtH
were additive, confirming that bovine-GH did not merely
potentiate GtH action. Furthermore, bovine-GH increased
follicular aromatase activity, an action dependent upon
the synthesis of new RNA and regulatory protein(s). The
gonadotropic and steroidogenic actions of mammalian GH
have been confirmed using purified and recombinant fish
hormones. Injections of recombinant GH stimulate gonadal
growth in hypophysectomized killifish and elevate circu
lating sex steroids in immature rainbow trout (Singh et
al., 1988; Danzmann et al., 1990). The recombinant GH
stimulated steroidogenesis by follicles isolated from
hypophysectomized killifish or trout, even in the absence
of GtH preparations (Singh et al., 1988). Purified carp
or chum salmon GH potentiated carp GtH-II stimulation of
in vitro steroidogenesis by both vitellogenic and preovu
latory goldfish follicles (Van Der Kraak et al., 1990).
However, GH was ineffective when used alone.
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Most oocyte growth in fishes can be accounted for by
the uptake of a yolk precursor protein (vitellogenin) that
is synthesized and secreted by the liver of maturing
females (vitellogenesis) under the influence of circulat
ing estrogens, primarily E2 (Specker and Sullivan, 1994).
In frogs and turtles, GH stimulates hepatic synthesis of
vitellogenin in vitro (Carnevali et al., 1992; Ho et al.,
1985). The effect is also seen in vivo in hypophysecto
mized turtles (Ho et ai., 1982), implying direct action of
GH on the liver independent of circulating GtH. Recently,
Kwon and Mugiya (1994) demonstrated that GH or PRL as well
as E2 are essential for vitellogenin synthesis in the eel.
Although E2 injections could induce vitellogenin synthesis
in intact and sham operated immature eels, they were
ineffective in hypophysectomized animals, confirming that
pituitary hormones play a role in initiating vitellogenin
synthesis. Growth hormone or PRL added to the incubation
medium of cultured eel hepatocytes greatly potentiated
weak responsiveness to E2 alone. Growth hormone and
insulin are known to regulate uptake of vitellogenin
(growth) by cultured oocytes of various vertebrates,
including fishes (reviewed by Specker and Sullivan, 1994)
Finally, specific membrane receptors for GH have
recently been detected in salmon ovary and testes (Le Gac
et ai., 1991; Mourot et al., 1992), confirming that the
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teleost gonad is a GH target. Hepatic receptors for GH
were described earlier for a variety of teleosts (Gray and
Kelley, 1991; Hirano, 1991; Sakamoto and Hirano, 1991; Yao
et al., 1991). Collectively, the maturational changes in
circulating GH levels, effects of exogenous GH on steroi
dogenesis and vitellogenesis or oocyte growth, and detec
tion of ovarian and hepatic GH receptors strongly indicate
a role for GH in control of fish reproduction.
Research ojectives
The spectrum of actions of PRL and GH in teleosts
include effects on osmoregulation, metabolism and repro
duction. Often, the hormones are called upon to regulate
these different functions simultaneously. The cells
secreting the hormones must therefore receive regulatory
information from a variety of sources to be used in
determining the secretion rate of the hormones. In order
to understand how PRL and GH may be involved in regulating
diverse functions, hormone changes with these functions
must be characterized and the factors regulating PRL and
GH must be identified.
Changes in serum and pituitary levels of the tPRLs
and GH with changes in environmental salinity have been
well characterized in the tilapia O. mossambicus, but
changes with metabolic or reproductive state have received
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little attention. There is still much to'learn about the
regulation of the tPRLs and GH in osmoregulation,
metabolism, and reproduction. The overall objectives of
my thesis research were to characterize changes in serum
concentrations and pituitary content of the tPRLs and GH
with changes in metabolic and reproductive states and to
characterize further the regulation of these hormones.
Prolactin and GH cells of the tilapia can detect
changes in the osmotic concentration of extracellular
fluids and respond by altering the release of their
hormones (Helms et ai., 1987; cf. Grau et al., 1994). It
is not known whether osmoreceptive cells, such as the PRL
and GH cells, detect changes in the osmolality of fluids
or changes in the osmotic gradient across the cell mem
brane which lead to an increase in osmotic pressure on the
cell membrane. The first objective of my thesis research
was to make this determination.
Gonadotropin-releasing hormone has been shown to
stimulate not only GtH release in teleosts, including the
tilapia, but also GH release (Marchant et al., 1989;
Melamed et al., 1995). Since PRL and GH are closely
related molecules, derived from a common ancestral gene
and regulated by many of the same factors, my second
objective was to determine whether GnRH has effects on PRL
release. As part of this study, it was also my objective
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to determine whether calcium operates as a second messen
ger in mediating the effects of GnRH on PRL release.
Little is known about the roles of PRL and GH in
the regulation of metabolism in teleosts. Treatment of
teleosts including the tilapia, with heterologous PRLs and
GHs indicates that these hormones have effects on the
mobilization of metabolites. There have been few studies
inquiring into the existence of correlations between
changes in circulating and pituitary levels of GH with
alterations in metabolic state in teleosts. Furthermore,
there has been only 1 study describing changes in circu
lating PRL concentrations and 1 describing changes in
pituitary PRL content with changes in metabolic state
(McKweon et al., 1975; Rodgers et al., 1992). The third
objective of my research was to characterize changes in
serum concentrations and pituitary content of the tPRLs
and GH in relation to alterations in metabolic state
induced by fasting.
Prolactin and GH have reproductive actions in tele
osts. There have been few studies which examine changes
in these hormones with the reproductive cycle of teleosts.
The fourth objective of my thesis work was to characterize
changes in serum concentrations and pituitary content of
the tPRLs and GH during the reproductive cycle of the
female tilapia. As part of this study, my objective was
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to determine whether environmental salinity alters the
patterns of serum and pituitary levels of the tPRLs and GH
observed during the reproductive cycle.
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Chapter II
ALTERATIONS IN OSMOTIC PRESSURE AND NOT OSMOLALITY ARE
COUPLED TO THE SUSTAINED RELEASE OF THE OSMOREGULATORY
HORMONE, PROLACTIN, IN THE EURYHALINE TELEOST, TILAPIA
(OREOCHROMIS MOSSAMBICUS)
INTRODUCTION
Prolactin is the FW-adapting hormone of many euryha
line teleosts including the tilapia, O. mossambicus.
Prolactin acts on all osmoregulatory tissues to stimulate
ion transport and to reduce ion and water permeability
(see reviews by Clarke and Bern, 1980; Hirano, 1986; Brown
and Brown, 1987; Grau and Helms, 1990). Consistent with
this osmoregulatory action, prolactin cell activity, and
blood and pituitary PRL levels are higher in FW-adapted
tilapia than in SW-adapted tilapia, (Dharmamba and Nishio
ka, 1968; Nicoll et al., 1981; Borski et al., 1992; Ayson
et al., 1993). Clearly defined osmoregulatory actions
have yet to be defined in mammals.
In addition to the osmoregulatory actions of PRL in
the tilapia, the tilapia PRL cell appears to be an osmore
ceptor. Support for this notion comes from 3 lines of
evidence. First, studies have demonstrated changes in
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sustained PRL release, PRL synthesis and PRL mRNA levels
in direct response to small changes in medium osmolality
that are well within the range of plasma osmolalities
observed in vivo (Nagahama et al., 1975; Wigham et al.,
1977; Grau et al., 1981; Kelly et al., 1988; Grau et al.,
1994; Yoshikawa et al., 1995). Furthermore, the effect of
changes in osmolality on PRL release appears to be mediat
ed by second messenger systems. Prolactin release in
response to changes in medium osmolality is Ca2 + and cAMP
dependent and is accompanied by an increase in intracellu
lar Ca 2 + (Grau et al., 1981, 1982; Richman et al., 1990,
1991; Borski, 1992; Grau et al., 1994). The increased
release of PRL in response to decreases in osmolality is
specific to PRL cells. The secretory response of PRL
cells to changes in osmolality is not shared by the close
ly related GH cells of the tilapia. The release of GH
from GH cells of the tilapia is either unchanged or inhib
ited by decreases in medium osmolality (Helms et al.,
1987; c.f. Grau and Helms, 1989).
The studies demonstrating that the PRL cells of the
tilapia are osmoreceptive cells were conducted using
pituitary tissues of the tilapia which are a nearly homo
geneous population of PRL cells (95-95%; Nishioka et al.,
1988). These tissues are from the anterior most region of
the rostral pars distalis (RPD). Thus, the PRL cells were
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responding directly to changes in medium osmolality. The
ability to isolate a nearly homogeneous population of PRL
cells obviates the impediments to investigating most
osmoreceptive cells.
The vasopressin cells of the mammalian hypothalamus
are an example of the complexity of most osmoreceptive
cells. The cell bodies of these neurosecretory cells are
located mainly in the supraoptic nucleus with a smaller
number in the paraventricular nucleus. In both sites, the
vasopressin cells are found among a variety of cell types
and synapse with many others. The axons of vasopressin
neurons project a considerable distance from these loca
tions to capillaries in the posterior pituitary, the site
of vasopressin secretion. This kind of complexity in
morphology and arrangement has virtually blocked attempts
to clarify the mechanisms by which the osmotic signals
alter vasopressin secretion. In fact, it is still unknown
whether vasopressin neurons are directly osmosensitive, or
are governed by adjoining osmoreceptive cells, possibly
around the anteroventral border of the third ventricle
(see Guyton, 1991).
The combination of PRL's actions in regulating osmot
ic homeostasis together with the PRL cell1s ability to
respond directly to changes in osmolality demonstrates
that the PRL cell both monitors and regulates the osmolal-
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ity of extracellular fluids. Little is known about the
cellular mechanisms which monitor the osmolality of extra
cellular fluids and lead to changes in the release of
osmoregulatory hormones. Basic to this understanding is
the identification of the osmotic signal recognized by the
osmoreceptive cell. Nagahama and colleagues (1975) using
the tilapia PRL cell as a model, showed that osmoreceptive
cells respond to changes in osmolality and not sodium.
They demonstrated that changes in medium osmolality by the
addition of NaCI or the membrane-impermeant molecule
mannitol, result in similar reductions in PRL releases
from PRL cells. Since this study, there has been little
progress towards characterizing the osmotic signal recog
nized by osmoreceptive cells. Studies on cells which are
not involved in the osmoregulation of extracellular fluids
point to the possibility that osmotic pressure and not
osmolality may be the osmotic signal recognized by osmore
ceptive cells.
Studies have shown that many cells, including mamma
lian PRL cells, release hormones in response to changes in
medium osmotic pressure and not medium osmolality per se
(Blackard et al., 1975; Sato et al., 1991; Wang et al.,
1991). These cells include adenohypophysial cells and B
cells of the pancreatic islets of the rat. Thyrotropin is
released by thyrotropin cells and insulin is released from
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pancreatic cells in response to reductions in medium
osmolality. These studies have shown that when the NaCI
concentration of isosmotic media is reduced and replaced
with membrane-impermeant molecules, release is not al
tered, however, when the same amount of NaCI is replaced
with membrane-permeant molecules release is increased.
Unlike membrane-impermeant molecules, membrane-permeant
molecules reach an equilibrium across the cell membrane
and do not contribute appreciably to the osmotic pressure
on the cell membrane. The osmolality inside and outside
the cell are increased equally by membrane-permeant
molecules.
Unlike the tilapia PRL cells, the release of hormones
by the rat PRL, thyrotropin and B-pancreatic cells is not
sustained with continued reduced osmolality (Blackard et
al., 1975; Sato et al., 1991; Wang et al., 1991). There
is a burst of release followed by a return to baseline
levels of release within 10 min of exposure to reduced
osmolality. This temporally limited response to changes
in osmolality is inconsistent with osmolality or osmotic
pressure being a signal which regulates the release of
hormones to serve osmoregulatory functions. Unlike PRL in
the tilapia, the hormones released by these cells have not
been shown to posses clearly defined osmoregulatory ac
tions in mammals. Thus, it is not likely that a sustained
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response to osmotic stimuli would be required. Blackard
and colleagues (1975) noted that a reduction in serum
osmolality of the magnitude required to increase insulin
release (20 mOsmolal) does not occur physiologically in
the rat and therefore the significance of this response is
questionable. Furthermore, Sato and colleagues (1991),
also working with the rat, made isotonic medium containing
the membrane-permeant molecule ethanol by removing NaCI to
reduce the osmolality of the medium 80 mOsmolal and then
replacing it with ethanol. The reduction in medium osmo
lality before the addition of ethanol in the studies by
Sato and colleagues (1991) was an even greater change in
osmolality than in the studies by Blackard and colleagues
(1975) .
The studies just described raise the question of
whether the sustained release of PRL from PRL cells of the
tilapia, in response to reductions in medium osmolality,
results from differences in osmotic pressure and not
osmolality per se. Do cells respond to the overall osmo
lality of the medium or do they respond to the osmotic
pressure of the medium which results from the osmotic
gradient between the cell cytoplasm and the medium? To
this end, I examined the response of PRL cells to various
medium osmolalities achieved by varying the concentrations
of membrane-permeant or membrane-impermeant molecules.
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Tilapia RPD were incubated individually in wells for 18-20
hr to examine whether the effect of reduced medium osmotic
pressure on PRL release is sustained. Prolactin tissues
were also incubated in peri fusion to characterize the
short-term response of PRL cells to reduced medium osmotic
pressure and osmolality. Prolactin release in response to
a reduction in osmotic pressure must be sustained for
osmotic pressure to be the physiologically important
signal recognized by osmosensitive cells in monitoring
extracellular osmotic conditions. Sustained increases in
PRL cell activity and hormone effects are necessary for
PRL to maintain osmotic homeostasis in the tilapia.
MATERIALS AND METHODS
Animals
Tilapia, o. mossambicus were collected from brackish
water streams and maintained in FW in outdoor tanks at a
temperature of 22-25°C for at least 2 months before use.
The fish were fed to satiation twice daily with Purina
trout chow (Purina Mills, Inc., St. Louis, MO).
Static incubations
Mature male tilapia 30-60 g were decapitated and
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their pituitaries removed. Each pituitary was dissected
and the RPD was placed into a well of a Falcon 96-well
culture plate with 100 ~l of Kreb's bicarbonate-Ringer
solution containing glucose (500 mg/l) , L-glutamine (290
mg/l) , and Eagles's minimal essential medium (50X MEM, 20
ml/l: GIBCO; Grand Island, NY) (Wigham et ai., 1977). The
osmolality of the incubation medium was adjusted by
varying -the concentration of NaCl (or treatment molecules)
and measured using a Wescor vapor osmometer (Wescor;
Logan, UT). Treatment media were hyposmotic medium, (300
mOsmolal), or hyposmotic medium made hyperosmotic (355
mOsmolal) by the addition of either NaCl, the permeant
molecules; urea or ethanol, or the impermeant molecule;
D-mannitol. The medium was gassed for 10 min with 95%
O2/5% CO2 (pH = 7.3). The tissues were incubated at 28 ±
1°C under a humidified atmosphere of 95% O2/5% CO2 and
placed on a gyratory platform (80 rpm) for 18-20 hr. All
chemicals were purchased from Sigma, St. Louis, MO unless
otherwise indicated.
Perifusion incubations
Mature male tilapia 200-300 g were used for the
perifusion studies. The RPD were dissected and placed 8
per chamber in parallel chambers with 3 chambers per
treatment. The perifusion apparatus consisted of a peris-
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taltic pump (Technicon proportional pump model III,
Dublin, Ireland) connected to a fraction collector (ISCO
model 328, Lincoln, NE). The chambers containing the
tissues consisted of a 3.5 cm length of glass tubing (cut
from a 100-200 ~l microdispenser tube; Drummond, Broomall,
PAl sealed at both ends with 120 ~l mesh Nitex screen.
The chambers were placed after the pump. The dead volume
between the introduction of the test media and the
fraction collector was 1.1 mI. Tissues were preincubated
in control medium (355 mOsmolal) overnight and continued
until the introduction of treatment media. Unlike the
static culture medium, the medium used in the perifusion
studies contained 50 I.U./ml penicillin and 0.05 mg/ml
streptomycin. Fractions (100 ~l) were collected every 2
minutes into 1.5-ml polypropylene microcentrifuge tubes
containing 50 ~l of 2% bovine serum albumin in phosphate
buffered saline.
Electrophoresis
Prolactin release during 18-20 hr incubations was
quantified using a combination of gel electrophoresis and
densitometry (Specker et al., 1985a; modified by Kelley et
al, 1988). At the termination of the incubations, media
and tissue were placed in sodium dodecyl sulfate (SDS)-2
mercaptoethanol buffer, ultrasonically disrupted (Heat
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Systems W-385 sonicator, Heat Systems-Ultrasonics, Inc.;
Farmingdale, NY) and boiled for 3 min. Samples were then
stored at -80 aC or measured immediately.
The tPRLs were separated using SDS-polyacrylamide gel
electrophoresis (PAGE). A vertical slab gel electrophore
sis apparatus (Bio-Rad; Richmond, CA) was used. The
samples were stacked in a 4% 37.5:1 acrylamide:bis-acryla
mide gel and separated in a 15% 37.5:1 acrylamide:bis
acrylamide gel (12 cm long, 0.15 cm thick). Samples were
subject to electrophoresis at 30 rnA constant current/gel
for 4-5 hr using a voltage- and current-regulated power
supply (ISCOi Lincoln, NE). The gels were stained with
Coomassie blue R-250 dissolved in a 10% methanol, 5%
acetic acid solution. The gels were destained in a 10%
methanol, 7% acetic acid solution until clearly
discernible bands were observed and then stored in a 7%
acetic acid solution. Bands of both tPRLs were quantified
by using a densitometer and proprietary software (Hoefer
Scientific, San Francisco, CA). For all static
incubations, the release of PRL into the incubation medium
was normalized as a percentage of the total PRL in the
tissue and the medium.
Radioimmunoassays
Prolactins in medium collected during peri fusion
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incubations were measured using the homologous radioimmu
noassays developed by Ayson et al., (1993) as modified by
Yada et al. (1994). These radioimmunoassays were
also used in studies described in following chapters for
measuring the tPRLs and GH in serum and pituitaries. r
will describe all three assays at this time for the sake
of clarity. Hormones used as labeled-ligand were labeled
with 125r using chloromine-T. The assays were performed
using a double antibody method under disequilibrium condi
tions. Samples were run in duplicate. The tPRL1 7 7
antiserum (P177-9-4) does not cross-react with either
tPRL1 88 or GH. The tPRL1 8 8 antiserum (P188-1-3) does not
cross-react with tPRL1 7 7 and only slightly (3.1%) with GH.
The GH antiserum (G-4-4) shows slight cross-reactivity
with tPRL1 7 7 (0.4%) and tPRL1 88 (1.6%). Sample values
were corrected for cross-reactivity using the formula:
Corrected value = measured value - %cross-reaction X value
of cross-reacting hormone/laO.
Repeated measurements within the same assay and in
subsequent assays of a pool of FW o. mossambicus gave
intra- and interassay coefficients of variation of 5.9 and
8.8% for tPRL1 77, 6.3 and 9.5% for tPRL1 88, and 7.8 and
10.4% for GH. The sensitivity of the assays, defined as
the amount of measured hormone that can be distinguished
from zero dose and calculated as twice the standard devia-
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tion at zero dose, was 0.18-0.40 ng/ml for tPRL1 77,
0.19-0.45 ng/ml for tPRL1 88, and 0.14-0.30 ng/ml for GH
when 50 ~l of serum was used. Non-specific binding did
not exceed 5% of total counts.
Statistical analysis
Differences among groups were determined using analy
sis of variance and the least significant difference test
for a priori pairwise comparisons (Steel and Torrie,
1980). Experiments with only two groups were analyzed
using the unpaired Student's t-test. Data are expressed as
means ± SE.
RESULTS
Static Incubations
Effects of increasing medium osmolality by the addition of
membrane-permeant (urea) and impermeant (mannitol) mole
cules on PRL release from RPD of the tilapia
The purpose of this study was to determine whether
changes in medium osmolality or changes in the osmotic
gradient across the cell membrane evoke sustained changes
in PRL release. The release of both tPRLs was greater for
tissues incubated for 18-20 hr in medium with reduced
osmolality (300 mOsmolal) compared with release from
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tissues incubated in medium with increased osmolality (355
mOsmolal), when the increase in osmolality is due to the
addition of NaCl or mannitol (Fig. 1; P < 0.001). In
creased medium osmolality did not affect PRL release when
the difference in osmolality was derived from the addition
of the membrane-permeant molecule, urea. Mannitol and
NaCl were equally effective at reducing PRL release when
added to medium to increase the osmolality of the medium.
Effects of increasing medium osmolality by the addition of
the membrane-permeant molecule urea, on PRL release from
RPD of the tilapia
The effect of urea on PRL release was investigated.
The membrane-permeant molecule urea had no effect on PRL
release when added to hyposmotic medium (300 mOsmolal),
raising the osmolality 20 mOsmolals and making the medium
isosmotic (320 mOsmolal), or when added to isosmotic
medium, raising the osmolality 35 mOsmolals and making the
medium hyperosmotic (355 mOsmolal) to tilapia plasma (Fig.
2) .
Effects of increasing medium osmolality by the addition of
the membrane-permeant molecule ethanol, on PRL release
from RPD of the tilapia
The purpose of this study was to examine the effects
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Figure 1. Effects of increasing medium osmolality by theaddition of membrane-permeant (urea) and impermeant(mannitol) molecules on PRL release from RPD of tilapia.Treatment media were made by adding NaCl, mannitol or ureato hyposmotic medium (300 mOsmolal) to increase theosmolality of the media to 355 mOsmolal, numbers indicatebasal osmolality of media plus additional mOsmolalscontributed by mannitol or urea (mean ± BE; N = 12 pertreatment; *** P < 0.001, ns = not significant atP < 0.05).
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o tPRl188
[] tPRL177 ns
80
***70 I
***
0
NaCI NaCI NaCI NaCI300 355 300 300
+ +Mannitol Urea
55 55
10
60
(I)tn 50ca(I)-(I)'- 40..J~D-
30'#.
20
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Figure 2. Effects of increasing medium osmolality by theaddition of the membrane permeant molecule urea, on PRLrelease from RPD of tilapia. Treatment media were made byadding NaCI or urea to hyposmotic medium (300 mOsmolal) toincrease the osmolality of the media to the designatedosmolalities, numbers indicate basal osmolality of mediaplus additional mOsmolals contributed by urea (mean ± BE;N = 12 per treatment; ns = not significant at P < 0.05).
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70 D tPRL188
EJ tPRL177ns ,,'
60 I
50
CDtncaCD 40-e~
0::: 30C.
"eft.20
10
NaCI300
NaCI300
+Urea20
50
NaCI320
nsI
"....
NaCI320
+Urea35
NaCI355
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of a second membrane-permeant molecule on PRL release from
RPD. The addition of the membrane-permeant molecule,
ethanol, to medium of varying osmolalities had no effect
on PRL release from RPD (Fig. 3). Prolactin release from
tissues incubated in 300 mOsmolal medium without ethanol
was not significantly different from PRL release from
tissues incubated in the same medium increased to 330
mOsmolal by the addition of ethanol. Prolactin release
from tissues incubated in 330 mOsmolal medium without
ethanol was not significantly different from PRL release
from tissues incubated in the same medium increased to 355
mOsmolal by the addition of ethanol. Prolactin release
from tissues incubated in 355 mOsmolal medium without
ethanol was not significantly different from PRL release
from tissues incubated in the same medium increased to 405
mOsmolal by the addition of ethanol.
Peri fusion Incubations
Time-course for PRL release in response to a change in
medium osmolality from hyperosmotic (355 mOsmolal) to
hyposmotic (300 mOsmolal) , or to hyposmotic medium made
hyperosmotic by the addition of the permeant molecule
urea, or the impermeant molecule mannitol
This study was aimed at investigating the time-course
of possible changes in the release of the tPRLs from PRL
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Figure 3. Effects of increasing medium osmolality by theaddition of the membrane permeant molecule ethanol on PRLrelease from RPD of tilapia. Media was made by adding NaCIor ethanol to hyposmotic medium (300 mOsmolal) to increasethe osmolality of the media to the designated osmolalities, numbers indicate basal osmolality of media plusadditional mOsmolals contributed by ethanol (mean ± BE;
N = 12 per treatment; ns = not significant at P < 0.05).
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70 D tPRL188nsI
[] tPRL17760
...
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cells when medium was altered from hyperosmotic medium to
either hyposmotic medium (300 mOsmolal), hyposmotic medium
made hyperosmotic with the addition of the membrane
permeant molecule, urea (355 mOsmolal), or hyposmotic
medium made hyperosmotic by the addition of the membrane
impermeant molecule, mannitol. The insert in Figure 4
shows the changes in osmolality of the medium in response
to the introduction of hyposmotic medium (300 mOsmolal)
into the perifusion system, following hyperosmotic medium
(355 mOsmolal). The hyposmotic medium was introduced
at the beginning of fraction 1 and the drop in medium
osmolality in the collection tubes occurs primarily in
fraction 4, or 6-8 minutes later.
Prolactin release was not altered when the medium was
switched from hyperosmotic medium to hyperosmotic medium
containing mannitol (Fig. 4). Prolactin release increased
when the medium was switched from hyperosmotic medium to
hyposmotic medium, or to hyperosmotic medium containing
urea. Prolactin release increased more rapidly when RPD
were exposed to hyposmotic medium than when RPD were
exposed to hyperosmotic medium containing urea. Prolac
tin177 release was greater in RPD exposed to hyposmotic
medium than in RPD exposed to hyperosmotic medium contain
ing urea, within 2 min after the onset of exposure to the
new medium (fraction 4), but was no longer different at 4
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Figure 4. Effects of increased medium osmolality by theaddition of membrane permeant (urea) and impermeant(mannitol) molecules on tPRL1 77 release (top graph) andtPRL1 88 (bottom graph) from RPD of tilapia. Media wasmade by adding NaCl, mannitol or urea to hyposmotic medium(300 mOsmolal) to increase the osmolality of the media to355 mOsmolal (hyperosmotic media). Tissues were incubatedin hyperosmotic medium raised to 355 mOsmolal with NaClbefore the media was switch to the treatment media whichwere 300 mOsmolal or hyposmotic medium made hyperosmoticwith mannitol or urea. Fractions are 2 min fractions withnumbering starting with the first sample collected at thetime the treatment medium was added to the peri fusionsystem. The insert shows the rate of osmolality change inthe collected medium when hyposmotic medium is introducedinto the perifusion system (mean ± SE; N = 12 per treat) .
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~360iii'0 340E
16 Mannitol~ 320-- ---- § 3000
II) 14 :a)( -e- Urea ~ 280- 0 1 2 3 4 5 6 7 8- 12E -*- 300-0') 10c--.. 8cec
60e.......... 4~
-I2c::
a....0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
16--0II) 14X::-12E-0') 10c-.....c 8ec
60eco 4co~
-I0::: 2a....
00 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Fraction (2 min)
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min (fraction 5) after the onset of exposure to the new
medium. Prolactin1 88 release was greater in RPD exposed
to hyposmotic medium than in RPD exposed to hyperosmotic
medium containing urea, within 2 and 4 min after the RPD
were first exposed to the new medium (fractions 4 and 5) ,
but was no longer different at 6 min (fraction 6) after
the onset of exposure to the new medium. Peak PRL release
was observed earlier in RPD exposed to hyposmotic medium
than in RPD exposed to hyperosmotic medium containing
urea. Prolactin1 77 release peaked by 4 min after the
onset of exposure to hyposmotic medium and about 10-12 min
after the onset of exposure to hyperosmotic medium with
urea. Prolactin1 88 release also peaked by 4 min after the
onset of exposure to hyposmotic medium and about 8-10 min
after the onset of exposure to hyperosmotic medium con
taining urea. At 22 min after the initiation of exposure
(fraction 19) to treatment media, the release of both
tPRLs was similar for RPD exposed to hyposmotic medium and
RPD exposed to hyperosmotic medium containing urea.
DISCUSSION
Prolactin release was high in RPD incubated for 18-20
hr in hyposmotic medium (300 mOsmolal) or medium made
hyperosmotic (355 mOsmolal) with the membrane-permeant
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molecules; urea or ethanol, but was low in RPD incubated
in medium made hyperosmotic with NaCI or the impermeant
molecule, mannitol. These data suggest that it is the
osmotic gradient that leads to an increase in osmotic
pressure, and not the medium osmolality per se, which
accounts for the osmoreceptivity of the tilapia PRL cell.
Furthermore, the greater release from tissues incubated in
the medium with urea compared with mannitol and NaCI was
not due to a stimulatory effect of the urea. This was
demonstrated by showing that the addition of urea to media
of various osmolalities, including osmolalities with
reduced baseline release of the tPRLs, did not increase
PRL release (Fig. 2).
Results similar to these obtained with urea were
obtained with an additional permeant molecule, ethanol.
Ethanol had no effect on PRL release, further supporting
the notion that PRL cells of the tilapia respond to
changes in osmotic pressure (Fig. 3). Contrary to our
findings, Sato et al., (1991) reported isotonic ethanol
solutions stimulate PRL and thyrotropin release from rat
adenohypophysial cells along with inducing cell swelling.
In the study by Sato and colleagues, medium at 300 mOsmo
lal was diluted 80 mOsmolal with distilled water and the
osmolality restored to 300 mOsmolal with ethanol. The
induced release as well as the observed swelling could
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have been induced by the reduced osmolality of the diluted
medium with the permeant ethanol molecules having no
effect.
Prolactin177 and tPRL18 8 release from RPD incubated
in hyposmotic medium and in hyperosmotic medium containing
urea were the same at 22 min after the onset of exposure
to these media. Furthermore, release of both tPRLs in RPD
exposed to both media, were greater than the release of
both PRLs in RPD exposed to hyperosmotic medium containing
mannitol. These data support the results of the my static
incubation experiments and the conclusion that PRL release
in response to changes in osmotic pressure is sustained
beyond the 10 min response observed in cells which secrete
hormones that are not known to regulate the osmolarity of
extracellular fluids, specifically: mammalian PRL, thyro
tropin and pancreatic B-cells (Blackard et al., 1975; Sato
et al., 1991; Wang et al., 1991).
The rise in release of the tPRLs upon exposure of RPD
to hyposmotic medium made hyperosmotic with urea was
slower than that observed after the introduction of hypos
motic medium, ~lthough PRL release in both treatments
converged by 22 min after the initiation of exposure (Fiq.
4). Thus, urea affected only the early phase of the
response and not the later phase of the response.
The difference in response of tissues incubated in
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hyposmotic medium compared with tissues incubated in
hyposmoticmedium made hyperosmotic with urea is consist
ent with urea having a diffusion rate across the cell
membrane of the PRL cells that is slower than that of
water. Until the urea molecules diffuse across the
membrane, there is an osmotic gradient across the cell
membrane. The osmotic pressure on the membrane due to the
medium made hyperosmotic with NaCI or mannitol, and the
medium made hyperosmotic with urea would be similar imme
diately after exposure and thus, PRL release would be
expected to be the same. As the urea molecules diffuse
across the membrane, the osmotic gradient across the cell
membrane decreases as does the osmotic pressure. As this
decrease in osmotic pressure occurs, PRL release in
creases. The time required for the urea to diffuse across
the membrane is the cause of the delay in PRL release
observed between the RPD exposed to hyposmotic medium and
RPD exposed to hyposmotic medium with added urea. Once
the urea comes into equilibrium across the membrane, only
the impermeant molecules in the initial hyposmotic medium
affect the osmotic pressure across the cell membrane.
This is the same in both the hyposmotic medium and the
hyperosmotic medium containing urea. At this point,
release of the PRLs would be expected to be equal in RPD
incubated in the two media, which is what is observed at
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22 min after the initiation of exposure to the two media.
In summary, alterations in osmotic pressure and not
osmolality are coupled to the sustained release of the
osmoregulatory hormone, prolactin, in the euryhaline
teleost, O. mossambicus.
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Chapter III
GONADOTROPIN-RELEASING HORMONE FUNCTIONS AS A
PROLACTIN-RELEASING FACTOR IN THE TILAPIA,
OREOCHROMIS MOSSAMBICUS
INTRODUCTION
Teleosts, like most vertebrates other than placental
mammals, have multiple forms of GnRH in the brain which
differ in their spatial distribution throughout the brain.
Three endogenous forms of GnRH have been identified in the
brain of tilapia: salmon-GnRH (sGnRH), chicken-GnRH-II
(cGnRH-II) and seabream-GnRH (sbGnRH) (Weber et al.,
1994). The GnRH molecules are named for the first animal
from which they were identified. The presence of multiple
forms of GnRH together with multifarious distributions of
these forms has led to speculation that separate functions
may be subserved by distinct GnRH peptides. These func
tions may extend beyond the regulation of GtHs since GnRH
has also been shown to function as a regulator of GH in
several teleosts, including tilapia (Marchant et al.,
1989; Melamed et al., 1995).
Growth hormone and PRL are part of the same polypep
tide family and are thought to be derived from a common
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ancestral gene (Niall et al., 1971). Not surprisingly,
PRL and GH are regulated by many of the same factors:
somatostatin, cortisol, and changes in osmotic pressure
(cf. Nishioka et al., 1988). Despite these commonalities
between GH and PRL, and the knowledge that GnRH regulates
GH release, the effects of GnRH on PRL release in teleosts
have received little attention.
There is little direct evidence to support or oppose
a role for GnRH as a regulator of PRL in teleosts. In the
Atlantic salmon, injections of GnRH have been shown to
increase PRL cell activity, based on light and electron
microscopical evidence (Ekengren et al., 1978). There was
an increase in the synthetic and exocytotic activity of
the cells. It was thought, however, that this effect was
mediated via GtH and E2, with GnRH inducing rises in
circulating concentrations of GtH and then E2 which in
turn stimulated the increase in PRL cell activity. Gona
dotropin-releasing hormone did not bind to PRL cells of
the goldfish, suggesting that GnRH may not be a direct
regulator of PRL cell function in this species (Cook et
al., 1991).
In mammals, GnRH has been found to stimulate PRL
release indirectly via paracrine secretions of GtH cells
(Denef and Andries, 1983; Lamberts et al., 1989). There
is strong evidence that angiotensin II from gonadotrophs
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is a paracrine mediator of this action (cf. Saavedra,
1992; Becu-Villalobos et al., 1994). In mammals,
prolactin cells are found in very close association with
GtH cells (Sato, 1980). In contrast, the PRL cells in the
tilapia are segregated into the anterior most region of
the RPD as an almost homogeneous mass, while the GtH cells
are located in the proximal pars distalis (Bern et al.,
1975). This anatomical arrangement of the tilapia pitui
tary gland makes a paracrine-mediated effect through GtH
cells less likely.
Jobin and Chang (1992) have recently shown that
native GnRHs stimulate increases in [Ca2+]i in GtH and GH
cells of the goldfish. Borski (1993) has also recently
shown that [Ca2+]i is increased in PRL cells of the
tilapia with exposure to reduced medium osmolality. The
effects of GnRH on changes in [Ca2+]i in PRL cells of a
teleost have not been investigated.
The purpose of the present study was to: 1) determine
whether GnRH injections can affect circulating levels of
PRL; 2) determine whether GnRH can alter the release of
PRL from tilapia RPD and to compare potencies of the
native GnRH forms in releasing PRL; 3) characterize the
effect of medium osmolality on the ability of GnRH to
stimulate PRL release; 4) determine whether GnRH alters
[Ca2+]i and compare temporal changes in [Ca2+]i with
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temporal changes in PRL release; and 5) determine whether
the steroid hormones, E2 or T, alter the affect of GnRH on
PRL cells; both steroids stimulate PRL release.
MATERIALS AND METHODS
Animals
Tilapia, o. mossambicus, were collected from brackish
water streams and maintained in FW in outdoor tanks at a
temperature of 22-25°C for at least 2 months before use.
The tanks were supplied with a continuous flow of FW and
constant aeration. The fish were fed to satiation twice
daily with Purina trout chow. All fish were mature adults
and ranged in size from 70-120 g body weight.
Injection study
Mature male and female tilapia were used in the
study. Fish were moved to indoor oval fiberglass tanks
(60 1), 8 individuals per tank, and allowed to acclimate
under a fixed photoperiod of 14L:10D to the tanks for at
least 2 weeks before injections. The tanks were supplied
with a continuous flow of FW and constant aeration. The
fish were not fed on the day of the experiment. Fish were
anesthetized with 2-phenoxyethanol at a concentration of 1
mlll for 1 min before being weighed and given intraperito-
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neal injections of an analog of mammalian~GnRH (mGnRHai
des-Glyl0, [d-Ala6JmGnRHi Sigma, St. Louis, MO) dissolved
in 0.9% saline (0.1 ~g mGnRHa/g body weight) or saline
for injected controls. Non-injected controls were also
anesthetized. Injected animals received an injection
volume of 1 ~l/g body weight. Fish to be sampled at 11 hr
post-injection were injected 9 hr before the other treat
ments so that all treatments were sampled between 3-6 PM.
Fish were also anesthetized for blood collection.
Static incubations
Procedures for static incubations are described in
detail in chapter II. Mature male tilapia were used in
the study. The sGnRH, cGnRH-II and chicken GnRH-I (cGnRH
I) used in the study comparing these 3 forms, was pur
chased from Peninsula Laboratories, San Carlos, CA. The
sGnRH, cGnRH-II and sbGnRH used in the study comparing
these 3 forms were a gift from the Salk Institute. The
GnRHs were dissolved in hyperosmotic medium to 1 roM and
the steroid hormones (Sigma) were dissolved in absolute
ethanol to 1 roM before dilution in medium. In studies
which included steroid hormones which were dissolved in
100% ethanol, all treatments received equal volumes of
ethanol.
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Perifusion incubations
Procedures for perifusion incubations are described
in detail in chapter II. Eight RPD from mature males were
placed in each of 6 parallel chambers. The tissues were
preincubated in control medium (355 mOsmolal) overnight.
Seabream-GnRH (Salk Institute) was introduced as 5 min
pulses in graded doses of 0, 0.1, 1, 10, 100, and
1000 nm with 2 hr between pulses. Ten min fractions were
collected. Half of the chambers received increasing
concentrations of GnRH and the other half decreasing
concentrations.
Cell dispersion for fCa 2+Ji measurement
Cells from RPD were dispersed following the method
described by Borski (1993). Prolactin cells of the RPD
were dispersed by first placing a single tissue in 0.5 ml
of a 0.25% solution of porcine trypsin 1-300 (US Biochem,
Cleveland, OH) in phosphate buffered saline (PBS; pH =
7.5; 355 mOsmolal) for 45 min at 28 ± ICC and then gently
passing the tissues through a 1 ml pipette 5-10 times.
The cells were centrifuged (250 X g, 5 min, 25 ± ICC), the
supernatant was decanted, and the cells were resuspended
and triturated in 1 ml of trypsin free phosphate buffered
saline. The cells were rinsed in this way 3 additional
times and then resuspended in culture medium adjusted to
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355 mOsmolal. The cells were plated onto a glass cover
slip coated with 0.1 mg/ml of poly-L-lysine. Cells were
preincubated in 355 mOsmolal medium for at least 12 hr
prior to the determination of [Ca2+]i.
Monitoring fCa 2+Ji by dual microspectrofluorometry with
fura-2
Validations and procedures used in this study were
described in detail by Borski (1993). The procedures are
described again, briefly. Prolactin cells, plated
on poly-L-Iysine coated cover slips and incubated in
hyperosmotic medium, were loaded with 10 ~M of fura-2/AM
(Molecular Probes, Eugene, OR), for 90 min at 28 ± 1°C.
The fura-2/AM was solubilized in anhydrous dimethyl
sulfoxide (DMSOj Aldrich Chemical, Milwaukee, WI) to a
concentration of 10 mM prior to its final dilution to 10
~M « 0.10% DMSO v/v). The fura-2/AM is cleaved to its
impermeable form, fura-2 by endogenous esterases within
the PRL cells. The cover slips were then mounted in a
chamber that allows the cell to be peri fused and the
chamber was in turn mounted on a microscope stage. The
PRL cells were perfused for at least 30 min in hyperosmot
ic medium to allow the fura-2/AM to further deesterify
(Grynkiewcz et al., 1985j Lewis et al., 1988).
Single cell measurements of the fura-2 ratio were
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made with a dual excitation spectrofluorometer (ARCM-MIC
N, Spex Industries, Edison, NJ) interfaced with a
Diaphot-TMD inverted microscope (CF 40 X oil immersion
fluorite objective, Nikon). The microscope was equipped
with fluorescence optics, a 50 watt halogen illuminator,
epifluorescence illumination, and a quartz nose-piece (for
UV). Excitation light alternated between 340 and 380 nM
(narrow bandpass filters, SPEX) by a computer-controlled
chopper mirror. Fluorescent emission intensity was
transduced every 2 sec by a photomultiplier tube focused
on a single PRL cell after it had passed through a 500 nM
emission filter. A pinhole (1 mm) placed in the epiillu
mination path restricted the UV illumination to only the
cell of interest.
All data are expressed as the relative intensity of
the ratio of fura-2 fluorescence excited at 340 nm (fura-2
bound to Ca 2+) to that excited by 380 nm (free fura-2)
from which background (autofluorescence) was subtracted.
Shifts in this ratio (340/380) result directly from
changes in [ca2+]i which are independent of
dye concentration, cell thickness, and absolute optical
efficiency of the instrument (Tsien et ai., 1985;
Grynkiewicz et ai., 1985; cf. Poenie et al., 1986).
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Incubations for measuring changes in [Ca2+]i in
conjunction with PRL release
Experimental media (hyperosmotic medium with or
without cGnRH-II, and hyposmotic medium) were maintained
at 28 ± 1°C in hanging 60 ml plastic syringes connected to
an eight-point manifold perifusate selector (Hamilton Co.,
Reno, NV) via one-way stop cocks and polyethylene tubing.
The manifold output is connected to the input port of the
perifusion chamber by another piece of polyethylene tub
ing. The rate of perifusion through the chamber was
maintained at 300 ~l/min by keeping the height of the
syringes and volume of all solutions in the syringes
constant throughout the experiment. Chicken-GnRH-II was
used in the study because it showed the widest range of
stimulation in our static incubation studies. The
cGnRH-II was dissolved in hyperosmotic medium (0.1 pM
cGnRH-II, 355 mOsmolal). Hyposmotic medium (300 mOsmolal)
was introduced to the system at the end of each run.
The amount of the tPRLs released from the dispersed
cells was insufficient to measure tPRL release into the
media. For this reason, in 2 runs of the experiment, 4
RPD were placed in a chamber taken from the perifusion
system described earlier and placed inline after the
chamber with the dispersed cell preparation used for
[ca2+]i measurements. The experimental medium flowed
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through the chamber with the cells for [ca2+]i
measurements and then through polyethylene tubing to the
chamber with the 4 RPD, and finally to a fraction
collector. One min fractions were collected and tPRL in
the media were measured as described for the perifusion
system earlier.
Quantification of PRLs
Prolactin release was quantified using a combination
of gel electrophoresis and densitometry for 18-20 hr
cultures and radioimmunoassay for 3 hr cultures and serum.
The electrophoresis and radioimmunoassay procedures are
described in detail in chapter II. For perifusion incuba
tions, the data are expressed as percent of baseline
release which is the sum of the concentrations of hormone
in each of the three fractions following and including the
introduction of the GnRH (b1,b2,b3), divided by the sum of
the hormone in the three fractions preceding the introduc
tion of the GnRH (a1,a2,a3), expressed as percentage
((b1+b2+b3)/(a1+a2+a3) X 100).
Statistical analysis
Differences among groups were determined using analy
sis of variance and the least significant difference test
for a priori pairwise comparisons (LSD) analysis. Due to
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the much greater response to GnRH than to the steroid
hormones in the experiment comparing these responses, the
mean sum of squares used in the LSD analysis was derived
using only values from groups being compared (Snedecor and
Cochran, 1980). Experiments with only two groups were
analyzed using the unpaired Student's t-test. Data are
expressed as means ± SE.
RESULTS
The effects of mGnRHa injections on serum concentrations
of PRL
Intraperitoneal injections of 0.1 ~g mGnRHa/g body
weight elevated serum tPRL1 88 and tPRL1 77 concentrations
of mature male and female tilapia at 1 hr after injection,
over concentrations observed in vehicle-injected and non
injected controls (Fig. 5; P < 0.01). Serum hormone
concentrations were not significantly different from
controls at 11 hr after injection. Injections did not
alter GH serum concentrations.
The effects of mGnRHa on the in vitro release of PRL from
tilapia RPD incubated in isosmotic medium
To determine the response of PRL cells to GnRH, RPD
were incubated for 18-20 hr in isosmotic medium
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Figure 5. Effects of intraperitoneal injections of0.1 ~g/ g body weight of mGnRHa on serum PRL levels inmale (M) and female (F) O. mossambicus at 1 and 11 hrspost-injection (non-injected, black bars; vehicleinjected, open bars; mGnRHa injected, diagonal bars)(mean ± SE, N = 10-12 per treatment from 2 replicateexperiments, ** P < 0.01 and *** P < 0.001, comparisonsare to injected controls) .
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1 hr
40tPRL188
- *- *E 35 *- *C')
..:. 30 *c0 25+:if!
20 11 hr-c Non-(1)o 15 injectedc0o 10E:::s 5~
CDen0
F M F M F M
tPRL177
M
11 hr
FM
1 hr
***
FMF
Noninjected
_18-.€ 16C')
":'14
5 12+:ie 10-; 8o5 6(,) 4E2 2(1)
en 0
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(320 mOsmolal) containing graded concentrations of mGnRHa
ranging from 0.01 nM to 1 ~M. Tissues were incubated in
isosmotic medium (320 mOsmolal) which elicits moderate
baseline PRL release so that either a stimulatory or
inhibitory effect of GnRH on PRL release could be ob
served. Incubation with mGnRHa stimulated the release of
tPRL177 and tPRL188 with the greatest response elicited at
a concentration of 10 nM mGnRHa (Fig. 6; P < 0.01).
Release is variable with isosmotic medium. For this
reason, data from each of two :replicate experiments were
normalized as a percent of controls before being combined.
The effects of medium osmolality on mGnRHa stimulated
release of PRL from tilapia RPD
To determine whether GnRH could overcome the effects
of medium osmolality on PRL release, RPD were incubated
for 18-20 hr in hyposmotic medium (300 mOsmolal), isosmot
ic medium (320 mOsmolal), or hyperosmotic medium (355
mOsmolal), with graded concentrations of mGnRHa. The
release of both PRLs showed a typically inverse correla
tion with medium osmolality in the absence of GnRH (Fig.
7). Release of both PRLs was stimulated by mGnRHa at a
concentration of 100 nM when incubated in isosmotic and
hyperosmotic medium (P < 0.01 and 0.001 respectively)
There was no significant difference among treatments
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Figure 6. Prolactin release from RPD of o. mossambicusduring 18-20 hr incubations in isosmotic medium(320 mOsmolal), over a range of mGnRHa concentrations.
Due to variability of baseline release in isosmoticmedium, the data are expressed as a percent change fromthe control values for each replicate of the experiment(mean ± SE, N = 10-12 per treatment from 2 replicateexperiments, ** P < 0.01).
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200
160(I)tilCU(I)-e..I 1200::C.e.-(I)
en 80cns
..c:o?ft.
40
D tPRL188
D tPRL177***
*
o 0.01 0.1 1 10 100 1000
log mGnRHa (nM)
77
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Figure 7. Prolactin release from RPD of O.mossambicusduring 18-20 hr incubations with mGnRHa, in hyposmoticmedium (300 mOsmolal), isosmotic medium (320 mOsmolal) andhyperosmotic medium (355 mOsmolal; mean ± SE, N : 10-12per treatment from 2 replicate experiments, ** P < 0.01and *** P < 0.001) .
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***355320300
-
- + **rt- f+-
~-
r--
+- +-
II I I I I I I I I I I
60
o
~ 50ns(1)- 40e~30"t"'"
....Ia:: 20D....,';f!. 10
o 10 100 o 10 100 o 10 100
60300 320 355
***~ 50eu(1)
Q) 40a..
~30~
....IIX: 20a..+of
~ 10o
**
o 10 100 o 10 100 o 10 100
mGnRHa (nM)
79
~~ ... _- -~-_._--------
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exhibiting the greatest stimulation for each osmolality,
possibly because stimulation was near maximal under each
condition. Better separation of treatment effects was
observable with hyperosmotic medium due to a lower base
line release. For this reason, subsequent studies were
conducted in hyperosmotic medium.
The relative potency of various GnRH molecules on the
release of PRL from tilapia RPD
The potency of the GnRH forms were compared using
18-20 hr static incubations of RPD in hyperosmotic medium.
These incubations differ from the previous incubations in
that the tissues were preincubated in hyperosmotic control
medium overnight and the medium was changed and tissues
rinsed 3 times before the treatment medium was added.
This long preincubation reduced baseline release and
provided a better separation of responses. The response
of tPRLs were similar to each other in both studies. In
the first set of experiments (Fig. 8), cGnRH-I, a form of
GnRH which was not found in the tilapia, was the least
potent form of GnRH tested. Chicken-GnRH-I did not elic
iting a significant increase in the release of either PRL
until a concentration of 1000 nM. Both native forms of
GnRH, sGnRH and cGnRH-II were more potent than cGnRH-I. A
significant response with sGnRH was observed with 1 nM
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Figure 8. Prolactin release from RPD of O. mossambicusduring 18-20 hr incubations in hyperosmotic medium(355 mOsmolal), over a range of cGnRH-I, cGnRH-II andsGnRH concentrations (mean ± SE, N = 10-12 per treatmentfrom 2 replicate experiments, * P < 0.05 and*** P < 0.001).
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sGnRH. Chicken GnRH-II was found to be the most potent of
the three forms tested. A significant increase in the
release of both tPRLs was observed with the lowest concen
tration tested, 0.01 nm cGnRH-II. All three forms evoked
the maximum response observed. When three forms of GnRH
that are native to the tilapia were cOTI~ared (Fig. 9),
cGnRH-II was again found to be the most potent form of the
native GnRHs, followed by sGnRH. Seabream GnRH was the
least potent for stimulating the release of both tPRLs.
Effects of sbGnRH on PRL release from tilapia RPD in
perifusion incubation
Single short exposures (5 min pulse) of RPD to sbGnRH
stimulated the release of both PRLs in a dose-related
manner at concentrations similar to that observed with the
18-20 hr static incubations (Fig. 10). A stimulation was
first observed at a concentration of 10 nM sbGnRH
(P < 0.05).
The effects of cGnRH-II on [ca2+J i and PRL release
Chicken GnRH-II and hyposmotic medium evoked in
creases in [ca2+]i and PRL release (Figs. 11, 12, 13).
Chicken GnRH-II was effective at a concentration as low as
0.1 pM. An increase in [ca2+]i was observed within 1 min
of exposure to cGnRH-II or hyposmotic medium. A reduction
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Figure 9. Prolactin release from RPD of o. mossambicusduring 18-20 hr incubations in hyperosmotic medium(355 mOsmolal), over a range of cGnRH-II, sGnRH, andsbGnRH concentrations (mean ± SE, N = 6 per treatment,* P < 0.05, ** P < 0.01 and *** P < 0.001).
84
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85
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Figure 10. Prolactin release from RPD of o. mossambicusduring perifusion incubation in hyperosmotic medium(355 mOsmolal), in response to a range of sbGnRHconcentrations delivered in 5 min pulses. Fractions werecollected at 10 min intervals and the data are presentedas the percent of total hormone released into the mediumin the 3 fractions following the introduction of thesbGnRH, compared to the total hormone in the 3 fractionspreceding the introduction of the sbGnRH, at eachconcentration (mean ± SE, N = 10 RPD per column and 6columns per GnRH form, * P < 0.05 and ** P < 0.001).
86
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220-e- tPRL188
200 --II- tPRL177
-- **eu 180 ***tneu..c **'eft. 160-(I)tneuCI) 140-G)~
..Ia:: 120a..
100
80 -l.--,---.,.----r----r---,-------r-
o 0.1 1 10 100 1000
Log sbGnRH (nM)
87
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Figure 11. Changes in [Ca++]i from dispersed PRL cells ofO. mossambicus during perifusion incubation inhyperosmotic medium (355 mOsmolal) I in response to a rangeof cGnRH-II concentrations and a switch to hyposmoticmedium (300 mOsmolal). The [ca++]i is expressed as therelative intensity of the ratio of fura-2 fluorescenceexcited at 340 nm (fura-2 bound to Ca++) to that excitedby 380 nm (free fura-2) .
88
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89
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\Do
Figure 12. Prolactin release from 4 RPD and changes in [Ca++]ifrom dispersed PRL cells of O.mossambicus during peri fusionincubation in hyperosmotic medium (355 mOsmolal), in response toa range of decreasing cGnRH-II concentrations and a switch tohyposmotic medium (300 mOsmolal). Fractions were collected at 1min and the PRL data are presented as ng/ml of medium. The[Ca++]i is expressed as the relative intensity of the ratio offura-2 fluorescence excited at 340 nm (fura-2 bound to Ca++) to~hat excited by 380 nm (free fura-2).
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:::- 180 ---tPRL188.E 160Cl -tPRL177..s 140 Control5 120 GnRH GnRH 300
~ 100 GnRH 0.01 nM 0.0001 nM Control mQsm... 355 mQsm.... 1.0 nM 355 rnOsm 355c 80CD 355 mOsmuc 600e 40E 20::s...(I) 0en
0 10 20 30 40 50 60 70\D Fractions (1 min)I-'
5.5~GnRH
_ 5 0.01 nM
g 4.5 ~ 355 mQsm
~ 40~M 3.5
~ 3~~GnRH i GnRH "~r" I Control~ 2.5 i 1.0nm 0.0001 nM
~ 2 355mQsm 355 mQsm300mQsm
1.5 ..... - ... - ..... - - - .. - ............
0 10 20 30 40 50 60 70Minutes
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\DN
Figure 13. Prolactin release from 4 RPD and changes in [Ca++]ifrom dispersed PRL cells of o. mossambicus during peri fusionincubation in hyperosmotic medium (355 mOsmolal) I in response toa range of increasing cGnRH-II concentrations alternating withhyperosmotic medium without cGnRH-II and a switch to hyposmoticmedium (300 mOsmolal). Fractions were collected at 1 min and thePRL data are presented as ng/ml of medium. The [Ca++]iis expressed as the relative intensity of the ratio of fura-2fluorescence excited at 340 nm (fura-2 bound to .Ca++) to thatexcited by 380 nm (free fura-2) .
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_ 600
15001 --..-- tPRL188n -- tPRL177S 400+:ica GnRH Controlb 300 GnRHr:: GnRH Control 1.0nm 300(1) 0.01 nM Controlg 200 0.0001 nM 355 355 355 3550 355 \ rnOJm mOsm rnOsm rnOsmuE 100 rnOsm ¢~ ---~- ~a.. - ----(1) 0CIJ
0 10 20 30 40 50 60 70~
w Fractions (1 min)4.
GnRHGnRH-g 3.5 0.0001 nM 1.0nmM 355 355-0 3 rnOsm rnOsm ~~M-;2.5 Control+:i 300a:I 2c:= mOsm rnOsm
1.5 ........ - ... -
0 10 20 30 40 50 60 70Minutes
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in [ca2+]i was observed within 1 min after exposure to
control medium (hyperosmotic medium without cGnRH-II, Fig.
13). Prolactin release in response to both GnRH and
hyposmotic medium appeared to be biphasic. An initial,
large response was followed by lower levels that were
still above baseline, even though [ca2+]i remained high
(Figs. 12 and 13). A dose response to cGnRH-II was not
observed for [ca2+]i. Repeated exposures to cGnRH-II with
10 min breaks between exposures evoked increases in
[ca2+]i that were similar in magnitude to the first re
sponse but PRL release did not appear to be restimulated
(Fig 13). Exposure to hyposmotic medium was able to evoke
an increase in PRL release following a failure in repeated
GnRH exposures even though both elicited similar responses
in [Ca2+] i (Fig. 13).
The effects of T and E2 on sGnRH induced PRL release
Treatment with sGnRH (100 nm), T (10 nM), and E2
(10 nM) stimulated the release of both PRLs from RPD
during 3 hr incubations in hyperosmotic medium, following
an overnight preincubation in control medium (Fig. 14; all
P < 0.05). Combined treatments resulted in greater re
lease of both PRLs than either sGnRH or steroid treatments
alone (P < 0.05 for comparisons for each individual treat-
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Figure 14. Prolactin release from RPD of O. mossambicusduring 3 hr incubations in hyperosmotic medium(355 mOsmolal), with 10 nM E2, 10 nM T, 100 nM sGnRH or acombination of the sGnRH and the steroid hormones(mean ± SE, N = 10-12 per treatment from 2 replicateexperiments, * P < 0.05, ** P < 0.01 and *** P < 0.001).
95
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*
sGnRH sGnRH sG nR H&E2 &T
***
***
**
Tc
6
7 D tPRl188
[] tPRL177
1
96
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ment compared with treatments in which they were used in
combination with one of the other hormones) .
DISCUSSION
Effects of GnRH injections on PRL release
Injections of the tilapia with the GnRH analog,
mGnRHa, elevated serum concentrations of the tPRLs. These
data provide evidence that GnRH either stimulates the PRL
cells to release PRL directly, or stimulates less direct
pathways that lead to PRL release from PRL cells. The
injections almost certainly lead to an increase in GtH
release which in turn leads to an increase in the release
of steroids from the gonads. I present data that confirm
earlier findings that T and E2 stimulate PRL release in
the tilapia (Barry and Grau, 1986; Borski et al., 1991).
Taken together, the rise in circulating concentrations of
PRL in the tilapia in response to GnRH injections, could
have been mediated via GtH and then steroid hormones.
Ekengren and colleagues (1978) suggested this pathway to
explain how GnRH injections increased PRL cell activity in
the Atlantic salmon.
The rise in circulating PRL levels within 1 hr of a
single injection of mGnRHa (0.1 ~g/g body weight) occurred
within a time frame consistent with a GtH and steroid
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mediated response. Intraperitoneal injections of
catfish-GnRH (0.125 ~g/g body weight) elevated plasma
concentrations of GtH-II and 11-ketotestosterone in
African catfish, (Clarias gariepinus), within 1 hr post
injection, and cGnRH-II injections (dosage as low as 0.002
~g/g body weight) elevated plasma GtH-II concentrations
within 0.5 hr (Schulz et al., 1993). Each of these time
points represent the earliest time points examined. Both
forms of GnRH are found in the pituitary of the African
catfish with catfish-GnRH being 37-fold more abundant than
cGnRH-II.
The rapid elevation in steroid concentrations in
response to GnRH injections makes in vivo studies of the
effects of GnRH on PRL release difficult to interpret in
intact sexually mature fish. A stimulation of PRL levels
in gonadectomized fish in response to GnRH injections may
be a better indication that GnRH directly stimulates PRL
release, however, the GnRH response may be mediated via
other factors. In the rat, GnRH has been shown to stimu
late PRL release via paracrine action involving GtH cells
(Denef and Andries, 1983). For these reasons, in vitro
examinations of the effects of GnRH on PRL cells was
emphasized in my study.
The rise in serum PRL concentrations induced by GnRH
injections demonstrates that increases in circulating GnRH
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can affect PRL levels, regardless of the mechanism. Thus,
the interpretation of in vivo treatments with GnRHs,
intended to elevate GtH or GH, should consider the effects
of changes in PRL levels on the observed responses to the
treatments.
In vitro evidence that GnRH functions as a PRL-releasing
factor
All three forms of GnRH that are native to the tila
pia stimulated the release of tPRL1 88 and tPRL1 7 7 from RPD
in vitro. Concentrations of GnRH found to stimulate PRL
release were well within the range for receptor-mediated
action. Furthermore, the range of concentrations of GnRH
capable of stimulating PRL release was similar to those
shown to stimulate GtH and GH release from pituitary
fragments of teleosts including tilapia (Levavi-Sivan and
Yaron, 1989; Peter et al, 1990; Melamed et al., 1995).
These data, and the fact that the RPD is an almost homoge
neous mass of PRL cells, suggest that GnRH is a direct
regulator of PRL cell activity in the tilapia, o. mossam
bicus.
Consistent with the notion that GnRH's effects on PRL
release are direct and receptor mediated, treatment with
cGnRH-II evoked an increase in [ca2+]i within 1 min of
exposure and an elevation in PRL release within 2 min.
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These data suggest that calcium is operating as a second
messenger in mediating the effects of GnRH on PRL release.
The pathway of GnRH action on PRL cells of the tila
pia clearly differs from that of the rat. In the rat,
GnRH has been shown to stimulate PRL release via paracrine
action involving GtH cells (Denef and Andries, 1983). The
RPD of the tilapia does not contain either GtH cells or GH
cells. Growth hormone cell have also been to respond to
GnRH in teleosts, including the tilapia (Marchant et ai.,
1989; Melamed et ai., 1995).
The pathway of GnRH action on PRL cells of the
tilapia may also differ from that of the goldfish. Cook
and colleagues found negligible binding of a sGnRH analog
to prolactin cells of the goldfish compared with binding
to GtH and GH cells. Either the PRL cells of the goldfish
do not respond to GnRH or the receptors are only expressed
during certain periods, for example, discrete stages of
development or reproduction. Prolactin is a FW-adapting
hormone in teleosts and may always be expressed in euryha
line teleosts such as the tilapia (Grau et ai., 1994).
Alternatively, the direct regulation of PRL cells by GnRH
may be a late development in the evolution of teleosts.
Direct regulation of PRL cells by GnRH may exist in highly
derived teleosts as the tilapia but not in lower teleosts
as the goldfish or in mammals. An investigation of GnRH
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receptors on PRL cells of the tilapia needs to be conduct
ed as well as studies on the effects of GnRH on PRL
release in other fishes, including the goldfish.
Potencies of the 3 native forms of GnRH
The tilapia and seabream have the same 3 native forms
of GnRH (Powell et al., 1994; Weber et al., 1994).
Interestingly, their order of potency are the same for in
vivo stimulation of GtH-II release in seabream (Zohar et
al., 1995) and in vitro stimulation of PRL release in
tilapia. Seabream GnRH is by far the most abundant form
in the pituitary of the tilapia (N. Sherwood, personal
communication) and seabream (Powell, et al., 1994).
Seabream-GnRH is also the least potent in stimulating PRL
or GtH release, and cGnRH-II is the most potent in both
circumstances. The cause for differences in potency in
stimulating PRL release is at the pituitary level. Dif
ferences in peripheral degradation by specific peptidases
at the liver or kidney, as listed among the possible
factors contributing to the same order of potency in
seabream, can not be a factor in my in vitro studies.
Reasons for the differing potencies of the various
GnRH molecules include differences in receptor affinities,
differences in intracellular degradation rates of the GnRH
molecules within the pituitary, and activation of differ-
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ent signal transduction cascades. A single type of GnRH
receptor has been found in the pituitary of all fish
examined to date with the exception of the goldfish. This
suggests that the different GnRH forms are competing for
the same receptor. Fish found to have a single type of
GnRH receptor in the pituitary include the African catfish
(Leeuw et ai., 1988), winter flounder (Crim et ai., 1988),
stickleback (Andersson et ai., 1989), and seabream (Pagel
son and Zohar, 1992). More than one type of receptor has
been identified in the goldfish (Habibi et ai., 1987).
The native GnRH peptide with the greater GtH-II
releasing activity has been found to have greater receptor
affinity in African catfish (cGnRH-II > catfish-GnRH;
Schulz et ai. 1993) and goldfish (cGnRH-II > sGnRH; Peter
et ai. 1990; Habibi, 1991). Nevertheless, in the same
study in the goldfish, sGnRH was more active in releasing
GH.
Gonadotropin-releasing hormone is degraded within the
pituitary cells of teleosts (Goren et ai., 1990; Zohar et
ai., 1990). The pituitary cells posses cytosolic pepti
dases that degrade GnRH after it has been internalized.
Differences in biological activities of various GnRH
molecules have been shown to be attributable to their
differing susceptibility to degradation by these
peptidases (Goren et ai., 1990; Zohar et ai., 1990).
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Chang and colleagues (1993) have demonstrated that
different native GnRH forms activate different signal
transduction pathways in GtH cells of the goldfish. They
suggest that binding of the two GnRH molecules to the same
receptor leads to distinct receptor-conformation changes
and different G-protein linkages. These different path
ways may result in different potencies for the different
GnRH molecules.
The order of potency appears to parallel the
evolution of the GnRH peptides. The cGnRH-II peptide is
present in cartilaginous fishes, sGnRH first appears in
early teleosts and sbGnRH in late teleosts (cf. Sherwood
et al. 1994). The presence of a single GnRH receptor in
most teleosts suggests the peptides may have evolved
independently of the receptor or receptors and therefore
the older peptides may have a greater affinity to the
receptor or receptors. On the other hand, if the pitui
tary cells which respond to GnRH have evolved specific
peptidases to break down the biologically relevant GnRH
form, then the biologically relevant GnRH peptide would
have the shorter half life and lower biological activity
(Goren et al., 1990; Zohar et al., 1990). Either
possibility is consistent with sbGnRH being the biologi
cally relevant regulator of GtHs, GH and PRLs in the
pituitary. Furthermore, since sbGnRH is delivered by
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neurosecretory fibers directly to the cells of the
pituitary, sbGnRH can be delivered in high concentrations,
and thus, a greater potency may not be necessary for the
hormone to have its effect.
Roles for the different forms of GnRH
The dominance of sbGnRH in the pituitaries suggests
that sbGnRH may be the GnRH form which is the regulator of
GtHs, GH and PRLs. Nevertheless, examinations of pitui
tary and circulating levels of native GnRHs under many
varying physiological and developmental conditions are
needed before regulation of pituitary cells by other GnRH
forms can be dismissed. Schulz and colleagues (1993)
found catfish-GnRH to be in greater abundance than cGnRH
II in the pituitaries of the African catfish, Clarias
gariepinus, with cGnRH-II being the more potent stimulator
of GtH-II release in vivo and in vitro. The researchers
present the possibility that catfish-GnRH may be the
regulator of moderate levels of circulating GtH and the
more potent cGnRH-II may be the regulator of GtH surges,
such as those associated with spawning.
Injections and implants of native forms of GnRH and
analogs have been shown to stimulate GtH and GH release.
Thus, pituitary tissues can respond to GnRH in the blood.
Whether circulating GnRH has biological significance in
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most teleosts has yet to be determined. Gonadotropin
releasing hormone and a GnRH binding protein have been
identified in the circulation of the goldfish. Further
more, the concentrations of GnRH in the circulation were
within a range found to stimulate GtH and GH release in
vitro in the same species (Huang and Peter, 1988; Peter et
al., 1990). In preliminary studies GnRH was not detected
in the serum of tilapia (N. Sherwood, personal communica
tion). Nonetheless, my in vitro studies suggest that
cGnRH-II, which is endogenous in the tilapia (Weber et
al., 1994), may be able to stimulate PRL release at
concentrations which are below the detectable limits of
the radioimmunoassay used to measure the GnRH.
Effects of T and E2 on GnRH stimulated PRL release
The steroid hormones, E2 and T, are able to
enhance the response of PRL cells to GnRH, increasing
percent release over 3-fold. The steroids may regulate
the sensitivity of the PRL cells to GnRH stimulation
during the reproductive cycle. Alternatively, GnRH
molecules may regulate the sensitivity of PRL cells to T
and E2 stimulation. The potentiation of the response of
PRL cells by GnRH and thyrotropin-releasing hormone by sex
steroids supports the suggestion by Barry and Grau (1986)
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that there may be a shift in the control of PRL secretion
with changes in the reproductive state of the tilapia.
Hypothalamic stimulators of PRL cells in the tilapia
Prolactin secretion in teleosts is thought to be
predominantly under inhibitory control, however, evidence
had suggested that a hypothalamic prolactin-releasing
factor was involved in the control of PRL secretion in
fish (see reviews, Clarke and Bern, 1980; Ball, 1981).
Barry and Grau (1986) have shown that thyrotropin
releasing hormone stimulates PRL release in the tilapia
but only following E2 preincubation. Until the present
study, thyrotropin-releasing hormone was the only hypotha
lamic factor shown to have the capability to stimulate PRL
release in the tilapia.
Summary
Gonadotropin-releasing hormone appears to function as
a PRL-releasing factor in the tilapia, o. mossambicus, and
this effect appears to be direct. All endogenous GnRH
molecules stimulate the release of the two tPRLs, tPRL1 77
and tPRL1 88, from RPD with the following order of potency:
cGnRH-II > sGnRH > sbGnRH. Furthermore, the data suggest
that calcium is operating as a second messenger in mediat-
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ing the effects of GnRH on PRL release and the sex ster
oids, °E2 and T, potentiate the response of PRL cells to
GnRH.
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Chapter IV
CHANGES IN SERUM AND PITUITARY LEVELS OF PROLACTIN AND
GROWTH HORMONE WITH FASTING IN THE TILAPIA,
OREOCHROMIS MOSSAMBICUS
INTRODUCTION
Prolactin and GH have effects on protein, carbohy
drate, and lipid metabolism in mammals (see reviews,
Nicoll, 1974; Fain, 1980; Davidson, 1987). The +oles of
PRL and GH in metabolism in teleosts are not well charac
terized. Prolactin has been shown to be either lipolytic
or lipogenic depending on many factors including tempera
ture, photoperiod, circadian rhythms, development and
circulating levels of other hormones (review see, Baker
and Wigham, 1979; Sheridan, 1986). Interestingly, plasma
PRL concentrations are not altered by 30 days of fasting
in Kokanee salmon, (Oncorhynchus nerka; McKeown et al.,
1975). This is the only study to measure circulating PRL
levels during fasting in a teleost. Nevertheless, Rodgers
and colleagues (1992) found both pituitary concentrations
of PRL and basal PRL release from RPD in vitro to be
decreased after 2 weeks of fasting in o. mossambicus. In
this same study, PRL release in vitro was negatively
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correlated with essential amino acids concentration but
was unaffected by D-glucose concentration in the medium.
Although actions of PRL in metabolism appear to be con
served, evidence for a change in secretion rate with
fasting is limited.
The roles of GH in metabolism and fasting in teleosts
have received more attention than those of PRL, but are
not without controversy. Growth hormone treatment has
been shown to increase circulating levels of free fatty
acids (FFAs) in the rainbow trout and goldfish and to
mobilize lipid reserves from the liver of rainbow trout
and coho salmon (Minick and Chavin, 1970; Leatherland and
Nuti, 1981; Sheridan, 1986). In addition, GH injections
increase liver glycogen and muscle FFAs in Kokanee salmon,
but not plasma FFAs or glucose (McKeown et al., 1975).
Growth hormone injections do not appear to stimulate
lipolysis in the eel, Anguilla japonica, and tilapia, o.
mossambicus, but instead appear to stimulate the mobiliza
tion of amino acids and glucose. Injections of bovine-GH
in hypophysectomized eels increase serum amino acid con
centrations and do not alter plasma lipid concentrations
(Inui et al., 1985). Injections of bovine-GH stimulate
the release of glycogen and not lipid from liver reserves
and increases serum concentrations of amino acids and
glucose but not protein, lipid or cholesterol in the
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tilapia (Leung et ai., 1991). Consistent with this
elevation in circulating amino acids and glucose, Rodgers
and colleagues (1992) found an increase in GH release from
the proximal pars distalis of tilapia in response to
reductions in essential amino acids and glucose in the
medium. Together these studies suggest that GH cells in
the tilapia act in the regulation of, and are directly
responsive to, changes in the blood concentrations of
amino acids and glucose. Thus, GH would be expected to
increase with fasting in the tilapia, as in mammals.
Increases in circulating and pituitary levels of GH
have been observed in fasted teleosts. Increases in
plasma GH concentrations in rainbow trout have been ob
served within 3 weeks of fasting in studies by Wagner and
McKeown (1986), and within 1 week in studies by Sumpter et
ai. (1991a). No change in plasma GH concentrations was
observed after 30 days of fasting in Kokanee salmon
(McKeown et ai., 1975). Farbridge and Leatherland (1992)
observed a bi-modal response to fasting in rainbow trout,
first exhibiting a decline in plasma GH concentrations at
2 weeks compared with fed controls, followed by an eleva
tion at 4 weeks. Plasma GH concentrations in the fed
controls may not have been different from initial values
and plasma GH levels were elevated at 2 weeks of fasting
in a repeat of the study. Melamed (1993) observed an
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increase in plasma GH concentrations within 16 days of
fasting in "sub-optimally" fed tilapia hybrids (0. niloti
cus X O. aureus). Also in tilapia, o. mossambicus,
pituitary content of GH was elevated with 2 weeks of
fasting (Rodgers et al., 1992).
Farbridge et al. (1992) found that feeding rainbow
trout only once every five days resulted in reduced plasma
GH and suggested that GH concentrations are depressed by
low feeding rates. This is not consistent with the study
on the tilapia hybrid, by Melamed (1993). The tilapia at
the start of the experiment by Melamed, were described as
"sub-optimally fed". When the fish were switched to an
increased feeding rate, plasma GH concentrations fell
within 8 days. Clearly, the nutritional state of the
animal prior to fasting and changes in nutritional state
during fasting affects the hormonal response to subsequent
fasting.
In order to learn more about the roles of PRL and GH
in tilapia metabolism, I have characterized changes in
serum concentrations and pituitary content of the tPRLs
and GH with 21 and 31 days of fasting in well-fed tilapia
with a high condition factor (CF; fat tilapia) and tilapia
with a low CF (thin tilapia). The fasting should lead to
an alteration in metabolic state requiring the mobiliza
tion of energy stores. If the tPRLs or GH are involved in
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the mobilization of these energy stores, then their serum
concentrations or content in the pituitary may be altered.
Changes in body weight (BW) , standard length (SL) , gonad
weight, and liver weight were recorded to gauge the
effects of fasting on the metabolic state of the animals.
MATERIALS AND METHODS
Animals
Tilapia, o. mossambicus, were collected from brackish
water streams and maintained in FW in outdoor tanks (22
25°C), for at least 3 months before use. The fish were
fed to satiation twice daily with Purina trout chow. Fish
were not fed on the day of sampling.
Fish were transferred to an indoor facility with a
fixed photoperiod of 14L:10D. Fish were placed in oval
fiberglass tanks (60 1). The tanks were outfitted with a
continuous water flow (-23°C) and constant aeration. The
fish ranged in size from about 60-100 g. Fish were al
lowed to acclimate to the tanks for at least 2 weeks
before the start of the experiments.
Fish were anesthetized with 2-phenoxyethanol (1 mIll)
for blood collection. Blood was drawn from the hemal arch
within 5 min of introduction to the anesthesia, using a
28-gauge needle attached to a 1-ml disposable syringe.
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Blood was stored on ice and the serum was separated from
the formed elements by centrifugation and then stored at
-Boec until it was analyzed for PRLs and GH by radioimmu
noassay. The radioimmunoassays are detailed in chapter
II. Anesthetization with 2-phenoxyethanol did not affect
serum PRL and GH levels (Fig. 15).
Additional measurements and collections of tissues
varied with experiments. Body weight, SL, gonad weight
and liver weight were recorded and pituitaries were col
lected and placed in 20 ~l of distilled water in 1.5-ml
microcentrifuge tubes and stored at -Boec until they were
analyzed for PRLs and GH by radioimmunoassay. Pituitaries
were sonicated in phosphate buffered saline and diluted in
phosphate buffer with 1% bovine serum albumin (Sigma).
Protein content was determined in the undiluted samples
using a Bio-Rad (Richman CA) protein assay kit. Data were
expressed as micrograms of hormone per pituitary (~g/pit.)
as opposed to micrograms of hormone per milligram of
protein for 2 reasons. First, the fish in each study were
similar in size and therefore should have similar size
pituitaries. Secondly, pituitary proteins; PRLs, GH, and
GtHs would be expected to change dramatically under the
conditions of the studies, possibly affecting pituitary
protein content. For example, PRL content will appear to
decrease if there is a large increase in pituitary GH
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Figure 15. Effects of 2-phenoxyethanol (1 mIll) onprolactin and growth hormone serum concentrations in thetilapia, Oreochromis mossambicus (mean ± SE,N 10-15 per treatment, ns = not significant atP < 0.05).
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15
14
13
12
:=- 11E......C) 10c:---c: 9o.-...E 8...; 7CJ
§ 6Co)
E 5:s...Q) 4en
3
2
1
ns D control
EJ 2-phenoxyethanol
tPRL188 tPRL177 GH
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content and PRL is normalized to pituitary protein.
Gonadosomatic index (GS1; ((gonad weight (g)/ (g)) X 100)
hepatosomatic index (HS1; ((liver weight (g)/BW (g)) X
100) and condition factor (CF; ((BW (g)/(SL (cm))3) X 100)
were calculated. When sampling required killing the
animals, the fish were quickly decapitated following
anesthetization.
Statistical analysis
Differences among groups were determined using analy
sis of variance and the least significant difference test
for a priori pairwise comparisons analysis (Steel and
Torrie, 1980). Comparisons between two means were per
formed using the unpaired Student's t-test. Data are
expressed as means ± SE.
Study 1
Two fasting studies were conducted. The first study
examined the effects of 10 and 21 days of fasting on
female and 21 days of fasting on male tilapia with a high
condition factor (-3.6). The study was replicated in two
trials. Body weights, SL and gonad weight measurements
were recorded for the second trial of the study. In both
trials of the study fish were placed 5 to a tank and there
were two tanks per treatment (10 treatments). Fasting
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started on a staggered basis so that all fish were sampled
on the same day between 1300 and 1600. Fish were fed to
satiation twice daily with Purina trout chow when not
fasting.
Study 2
The second study examined the effects of 10, 21, and
31 days of fasting on male tilapia with a low condition
factor (-3.2). Study 2 was conducted following the same
protocol as Study 1 except 6 fish were placed in each tank
and were fed once daily when not fasted as part of the
study. The fish used in the second study were fed a
limited ration once daily for 2 months prior to the start
of the study. Body weights, SL, gonad weight and liver
weight measurements were recorded for the study.
RESULTS
The effects of fasting on serum concentrations and
pituitary content of the PRLs and GH in male and female
tilapia with a high condition factor
Serum levels of tPRL1 7 7 increased from 1.8 ± 0.3
ng/ml in fed female tilapia, to 6.2 ± 1.3 ng/ml with 10
days of fasting (P < 0.001) and 5.3 ± 0.5 ng/ml with 21
days of fasting (Fig. 16; P < 0.01). A similar trend
117
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Figure 16. Changes in serum concentrations of prolactinand growth hormone during fasting in male (M) and female(F) tilapia Oreochromis mossambicus with a high conditionfactor (mean ± SE, N = 10-12 per treatment; * P < 0.05,** P < 0.01, *** P < 0.001 ).
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tPRL188
*
..-.-E.......C) 10c---co;:; 8e....,s::(1)(J 6co(J
E 4::s'-(I,)
tn
o 1021 0 21
F Mo 10 21 0 21
F M
Days Fasted
o 10 21 0 21
F M
-
-
- tPRl177
**- ***
- t*. GH
-
-~ rf &r.f. rfl
li rJI I I I I I I I I I I I
14
o
12
2
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was not significant for tPRL1 88 (ANOVA P = 0.059). Serum
tPRL1 77 and tPRL1 8 8 concentrations were elevated in male
tilapia fasted for 21 days. Prolactin177 concentrations
increased from 1.7 ± 0.2 to 6.7 ± 1.3 ng/ml with 21 days
of fasting (P < 0.01) and tPRL1 88 serum concentrations
increased from 7.5 ± 0.8 to 12.6 ± 2.1 ng/ml (P < 0.05).
Serum GH concentrations were not affected by 21 days of
fasting in either sex.
Pituitary content of tPRL177 was elevated in female
tilapia fasted 21 days compared with females fasted 10
days or fed (Fig. 17; P < 0.01). Fasting did not affect
pituitary tPRL1 77 content in male tilapia or tPRL1 88
content in male or female tilapia. Pituitary content of
GH increased from 3.9 ± 0.6 ~g/pit. in fed female tilapia
to 6.1 ± 0.6 ~g/pit. in female tilapia fasted for 21 days,
and increased from 3.9 ± 0.2 ~g/pit. in fed male tilapia
to 5.4 ± 0.4 ~g/pit. in male tilapia fasted for 21 days
(P < 0.001 and P < 0.01 respectively) .
The effects of fasting on condition factor, body weight,
standard length and GSI in male and female tilapia with a
high condition factor
At the start of the study, CFs were similar for male
and female tilapia, ranging from 3.58 to 3.77 (Table 1).
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Figure 17. Changes in pituitary content of prolactin andgrowth hormone during fasting in male (M) and female (F)tilapia Oreochromis mossambicus with a high conditionfactor (mean ± SE, N = 10-12 per treatment; ** P < 0.01,*** P < 0.001).
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tPRL188
***
F M
o 1021 0 10o 1021 0 10
F M
Days Fasted
F M
o 1021 0 10
-
-
-
rfGH
-tPRL177
d **-
~a* ~ai~
- r!l
rIlrf-
I I II I I 1 -II I -Io
14
12
.-.......'0. 10--..en~-..... 8c
CI)......C0(J 6~C\1.....-::s 4.....-e,
2
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Table 1. Effects of fasting up to 21 days on condition factor,body weight (g) and standard length (cm) in male and femaletilapia with a high condition factor (CF > 3.6; mean ~ SE).
FemalesDays BW-B BW-E %CH t- SL-B SL-E %CH t- CF-B CF-E %CH t-T
f-'NW
fed
10
21
ANOVA
Malesfed
21
t-T
81. 66.1
79.62.5
74.95.4ns
73.23.5
79.94.4ns
80.75.6
75.12.5
69.04.7ns
78.63.0
74.24.1ns
-1. 2 ns 12.920.35
-5.6 ns 12.820.18
- 7 . 9 ns 12. 600.31
ns
7.3 ns 12.590.22
-7.0 ns 13.030.26
ns
13.170.35
12.880.17
12.600.32
ns
13.070.20
13.060.25
ns
1. 90 ns
0.47 ns
-0.07 ns
3.76 ns
0.20 ns
3.720.053.770.063.690.06
ns
3.650.043.580.08
ns
3.490.053.510.063.410.06
ns
3.520.063.310.07
*
-6.3 **
-7.0 **
-7.4 **
-3.6 ns
-7.7 *
(B, beginning; E, end; BW, body weight; SL, standard length; %CH, % change;t-T, t-Test comparisons; * P < 0.05, ** P < 0.01; ns, not significant)
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Condition factors were reduced by 21 days of fasting in
males compared with values for the same animals at the
start of the study (P < 0.05), and with fed males at the
end of the study (P < 0.05). The CFs for females in all
treatments were lower at the end of the study compared
with initial values (all P < 0.01). Condition factors
were not different among treatments with females, at the
end of fasting. There were no significant changes in BW
or SL with treatment. Nevertheless, there was a signifi
cant drop in GSI for females fasted for 21 days (Fig. 18;
P < 0.01). The follicles of fasted fish were soft, sug
gesting that atresia was beginning. The GSI for males did
not change with fasting.
The effects of fasting up to 31 days on serum
concentrations and pituitary content of the PRLs and GH in
male tilapia with a low condition factor
I did not see an expected increase in serum GH with
21 days of fasting with fish that had a high CF. For this
reason, the study was repeated with animals with a lower
CF, and fasting was extended to 31 days.
Serum concentrations of tPRL1 77 and tPRL1 88, but not
GH, were elevated in tilapia fasted for 10 days (Fig. 19;
both P < 0.05). Serum tPRL177 increased from 0.6 ± 0.1
ng/ml to 9.0 ± 2.8ng/ml and tPRL1 88 increased from
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Figure 18. Changes in gonadosomatic index (8S1) duringfasting in male (M) and female (F) tilapia Oreochromismossambicus with a high condition factor (mean ± SE,N = 10 per treatment; ** P < 0.01).
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2110
Females
o21o
-
-r-- f-
-r-- :-
-
**r- I--
-
-
-
Males
- rr- ~
o
4
2
3
1
1.5
0.5
4.5
3.5
2.5-ene
Days Fasted
126
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Figure 19. Changes serum concentrations of prolactin andgrowth hormone during fasting in male tilapia Oreochromismossambicus with a low condition factor (mean ± SE,N = 9-10 per treatment; * P < 0.05, ** P < 0.01,*** P < 0.001).
127
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**
o 10 21 31o 10 21 31o 10 21 31
- tPRL177 tPRL188
-** **
- *
*r-
- * -I-
*-r-
-
- GH
- +*-
r!--
III r-lrlrlI I I r -, J I I I I I I I I
20
18
-..-E 16........C)
..:.. 14e0.-~ 12COL.~
s::CI> 10CJe0 8CJ
E::s 6L.CI>en
4
2
0
Days Fasted
128
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5.7 ± 0.3 ng/ml to 12.2 ± 1.3 ng/ml, comparing fish that
had been fed to fish that had been fasted. Serum PRL
concentrations were elevated in fish fasted for 21 and 31
days, compared with fed fish. Growth hormone concentra
tions were less than 1 ng/ml in fish fasted up to 21 days
and then increased to 3.5 ± 0.7 ng/ml in fish fasted for
31 days (P < 0.001).
Pituitary tPRL1 8 8 levels were not different among fed
animals and animals fasted for 10 and 21 days. Pituitary
content of tPRL1 8 8 decreased with day 31 of fasting (Fig.
20; P < 0.01). Pituitary content of tPRL1 77 was not
significantly affected by fasting. Pituitary GH content
increased with fasting. Growth hormone rose from 7.9 ±
0.8 ~g/pit. in fed animals, to 13.0 ± 1.9 ~g/pit. in
animals fasted for 21 days (P < 0.05), and 14.3 ± 2.1
~g/pit. in tilapia fasted for 31 days (P < 0.01).
The effects of fasting up to 31 days on condition factor,
body weight, standard length, GSI and RSI in male tilapia
with a low condition factor
Body weight and SL were measured at the start of the
experiment, at the beginning of the fasting period, and at
the end of the fasting period (Table 2). Changes in BW,
SL and CF were compared from the start of the fasting
period to the end of the fasting period, as well as from
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Figure 20. Changes pituitary content of prolactin andgrowth hor~one during fasting in male tilapia Oreochromismossambicus with a low condition factor (mean ± SE,N = 9-10 per treatment; * P < 0.05, ** P < 0.01).
130
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25 -tPRL188
.
. 20 - GH...-c.---.CD
**=:t-...., tPRL177 *s:: 15 - **(I)....,c:0CJ
~
-t tC'O 10...-~ t rr iI-...-'l.
5 -
I I I Io 10 21 31I I I I I I I I I Io 10 21 31 0 10 21 31
Days Fasted
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Table 2. Effects of fasting up tQ 31 days on condition factor, body weight (g)and stnadard length (cm) in male tilapia with a low condition factor
(CF > 3.2; Mean ± BE)
Start of fasting and end of fastingDays BW-B BW-E %CH t-T SL-B BL-E %CH t-T CF-B CF-E %CH t-TFed 8S.2 89.2 14.21 14.21 3.07 3.07
5.7 5.7 0.31 0.31 0.05 0.0510 89.5 86.6 -3.3 ns 14.14 14.11 -0.23 ns 3.10 3.02 -2.7 ns
6.2 6.6 0.35 0.41 0.03 0.0321 80.4 72.0 -10.5 ns 13.70 13.58 -0.87 ns 3.11 2.86 -8.1 **
3.6 3.2 0.23 0.23 0.05 0.0431 68.4 60.6 -11.3 ns 12.78 12.73 -0.39 ns 3.21 2.88 -10.3 ***
I-'w 5.50 4.70 0.37 0.36 0.05 0.04N
ANOVA * *** * ** n ***
Start of study and end of fastingFed 74.6 89.2 19.6 ns 13.31 14.21 6.75 ns 3.10 3.07 -1.1 ns
5.5 5.7 0.35 0.31 0.04 0.0510 72.7 86.6 19.0 ns 13.28 14.11 6.20 ns 3.07 3.02 -1.5 ns
4.4 6.6 0.32 0.41 0.04 0.0321 74.4 72.0 -3.2 ns 13.44 13.58 1. 03 ns 3.05 2.86 -6.1 **
3.5 3.2 0.23 0.23 0.04 0.0431 68.4 60.6 -11.3 ns 12.78 12.73 -0.39 ns 3.21 2.88 -10.3 ***
5.50 4.70 0.37 0.36 0.05 0.04ANOVA ns *** ns ** ns ***
(B, beginning; E, end; BW, body weight; SL, standard length; %CH, % change; t-T,t-Test comparisons; * p < 0.05, ** P < 0.01, *** P < 0.001;ns = not significant)
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the start of the experiment to the end of the fasting
period, and the start of the experiment to the start of
the fasting period. The CFs did not differ among treat
ment groups from the start of the experiment to the start
of fasting. This consistency in CF suggests that the
nutritional status of the animals remained unchanged
throughout the period before fasting was initiated.
Condition factor was reduced from 3.11 ± 0.05 to 2.86 ±
0.04 (P < 0.01) with 21 days of fasting, and from 3.21 ±
0.05 to 2.88 ± 0.04 (P < 0.001) with 31 days of fasting.
The difference (P < 0.05) in BW and SL among treat
ment groups at the start of the fasting period is due to
the slightly smaller size of the fish in the group to be
fasted for 31 days at the start of the study, combined
with the growth of the fish during the interval between
the start of the experiment and the beginning of the
fasting period in the other groups. Body weight, SL and
CF decreased as fasting increased (P < 0.001, P < 0.01, P
< 0.001 respectively). There was a significant increase
in BW in the group fasted for 10 days that occurred be
tween the start of the experiment and the start of the
fasting period (P < 0.05).
Hepatosomatic index was reduced at 10 days of fasting
(Fig. 21; P < 0.001). The HSI of fed fish was 1.72 ± 0.07
compared with 0.97 ± 0.05 for fasted fish. There was no
133
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Figure 21. Changes in hepatosomatic index (HSI) duringfasting in male tilapia Oreochromis mossambicus with a lowcondition factor (mean ± SE, N = 10 per treatment;*** p < 0.001 ).
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*
312110o
-
-- -
-
-
-
***
rf-***-
+ **
rl--
-
-
-
2
o
1.8
1.4
1.6
1.2
0.4
0.8
0.2
0.6
-UJ 1J:
Days Fasted
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further reduction in HSI as fasting continued. There were
no differences in GSI among treatments at the end of the
study.
DISCUSSION
Serum concentrations and pituitary content of GH and
serum concentrations of tPRL1 7 7 and tPRL1 8 8 were elevated
in response to fasting in o. mossambicus. These data
suggest that GH and the tPRLs have roles in the regulation
of intermediary metabolism in the tilapia; o. mossambicus.
Furthermore, serum concentrations of the tPRLs increased
earlier in response to fasting than GH, and thus, the
tPRLs and GH may have different roles in regulating metab
olism.
An increase in circulating GH appeared later in
response to fasting in o. mossambicus than has been ob
served in most other teleosts. Serum GH concentrations
were elevated at 31 days of fasting in male tilapia with a
low CF. Serum concentrations of GH were unchanged for the
first 21 days of fasting in female o. mossambicus, with a
high CF, and in males with a high or a low CF. Increases
in circulating GH concentrations were observed within 1-3
weeks in rainbow trout (Oncorhynchus mykiss; Wagner and
McKeown, 1986; Sumpter et al., 1991) and within 16 days in
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a tilapia hybrid (0. niloticus X o. aureus; Melamed,
1993). On the other hand, McKeown et ale (1975) did not
see an elevation in plasma GH concentrations in Kokanee
salmon fasted for 30 days.
The difference in the timing of the increase in
circulating GH levels among the studies may be due to a
variety of factors. Among these factors are differences
in basal metabolic rates of the different animals. These
differences may be due simply to species or species dif
ferences combined with differences in developmental stage
or environment. For example, in my study there was only a
-31% difference in BW change between male tilapia that had
been fed and male tilapia that had been fasted for 31
days. The rainbow trout in the study by Wagner and
McKeown (1986) that had been fed were -250% greater in BW
than the fish that had been fasted for 21 days. The
greater weight loss observed in the trout compared with
the tilapia is likely due to differences in metabolism
related to the tilapia being a herbivore and the trout
being a carnivore. A second factor Gontributing to the
timing of an increase in circulating GH levels in response
to fasting is the metabolic state of the animals at the
start of the fasting period. The tilapia hybrid used in
the fasting study by Melamed (1993) were described as
IIsub-optimallyll fed. There is no further description of
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the condition of the animals. In that study, the plasma
GH concentrations started at over 200 ng/ml and increased
to over 500 ng/ml following 16 days of fasting, or dropped
to 15-20 ng/ml following 16 days of "optimal feeding". In
my study, fish with low and high CFs had serum GH
concentrations below 2 ng/ml before fasting. The elevated
GH levels prior to fasting in the tilapia hybrids may
suggest that their energy reserves were more depleted than
any of the fish in the present study. Thus, the hybrid
tilapia may have required an increase in GH sooner after a
further reduction in feeding, to mobilize additional
energy stores or protect essential resources from catabo
lism. Alternatively, the difference in response between
the 2 tilapias may be due to species differences in the
hormonal regulation of metabolism. The hybrid is selected
for fast growth and has higher baseline GH levels. Re
gardless of the differences in the timing of the increases
in circulating GH levels, circulating concentrations of GH
were elevated in the tilapia in response to fasting as in
most species of teleosts examined.
The pituitary content of GH was elevated at 21 days
of fasting in all groups in both studies; male and female
tilapia that had a high CF (-3.6) prior to fasting, and
male tilapia that had a low CF (-3.2) prior to fasting.
The pituitary content of GH was not elevated at 10 days of
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fasting in any of these groups. This is consistent with a
previous report by Rodgers and colleagues (1992) that
pituitary GH content was elevated with 2 weeks of fasting
in this same species. Increased pituitary GH content was
observed prior to an increase in serum GH concentration in
male tilapia fasted for 31 days. These data suggest that
pituitary GH may increase before release from the pitui
tary is increased.
An earlier change in pituitary GH content with
fasting, than serum concentration with fasting, suggests
that changes in pituitary GH content may be the better
indicator of changes in GH cell activity with changes in
metabolic state accompanying fasting. Consistent with
this notion, pituitary GH content, but not serum concen
tration, was elevated at 21 days of fasting, and at 21
days of fasting there were signs of changes in metabolic
state in the tilapia which had been fasted. In both
studies CF was reduced in male tilapia fasted for 21 days
compared with fed fish and compared with values from the
same fish prior to fasting. In addition, HSI was reduced
by 10 days of fasting in male tilapia suggesting that
liver energy stores were mobilized. The GSls of female
tilapia fasted for 21 days were reduced compared to those
of fed fish, suggesting ovarian growth was compromised.
Serum tPRL177 concentrations were increased with 10
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days of fasting in male and female tilapia and in tilapia
with a low and a high CF. This is the first report of an
increase in circulating PRL concentrations in response to
fasting in a teleost. Prolactin177 appears more sensitive
to fasting than tPRLI 8 8. Unlike serum tPRL1 77
concentrations, serum tPRL1 8 8 concentrations were not
elevated in female tilapia with a high CF, which had been
fasted for 10 or 21 days. In the same study, serum
tPRL1 7 7 concentrations were increased 3 fold and tPRL1 8 8
levels were increased by about 70% in male tilapia at 21
days of fasting compared with fed tilapia. Serum tPRL1 7 7
and tPRL1 8 8 concentrations were elevated with 10 days of
fasting in male tilapia with a low CF.
Rodgers and colleagues (1992) found that RPD removed
from tilapia (0. mossambicus) fasted for 2 weeks had
reduced spontaneous release of PRL. The pituitary content
of PRL together with the hormone released into the medium
during 20 hr incubations, was also lower for the fasted o.
mossambicus (sex not identified). The increase in serum
PRL concentrations I observed in response to fasting may
at first appear to be inconsistent with the reduction in
spontaneous PRL release from RPD with fasting, reported by
Rodgers and colleagues (1992). It is possible that the
reduction in PRL release from RPD of fasted fish, observed
by Rodgers and colleagues, was the result of a depletion
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of stored PRL in the RPD, prior to removal of the tissues.
This interpretation is consistent with the increase in
circulating tPRLs I observed with fasting and the reduced
PRL content in the pituitaries of the fasted tilapia in
the studies by Rodgers and colleagues.
Pituitary content of tPRL1 77 was elevated at 21 days
of fasting in female tilapia with a high CF whereas pitui
tary content of tPRL177 in males and tPRL1 88 in males and
females were not altered by fasting. On the other hand,
pituitary content of tPRL1 88 was reduced in male tilapia
with a low CF at 31 days of fasting while there was no
change in pituitary tPRL1 77 content. The effect of
fasting on pituitary PRL content appears to be sex specif
ic, however, more data are needed before this notion can
be confirmed.
The differences in serum concentration and pituitary
content of PRL and GH among fed and fasted tilapia ob
served in this study are assumed to be in response to a
need to mobilize or protect energy reserves in response to
a switch from anabolic to catabolic activities, or at
least an increase in catabolic activities with fasting.
Several lines of evidence support that there were changes
in metabolic state with the imposed fasting. In the
second study with tilapia with a low CF, there was a
decline in CF at 21 and 31 days of fasting. Also in the
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second study, there was a dramatic decrease in HSI by day
10 of fasting suggesting a mobilization and possible
depletion of available energy reserves in the liver. In
the first study, there was a decline in CF for males but
no decline in CF related to fasting for females. The
females may not have been in the same nutritional state at
the end of the study even though there were no differences
in CF between fed and fasted female tilapia. Although not
significant, there appeared to be a trend towards a de
crease in BW and SL with increased fasting time. There
may have been reduced linear growth with fasting keeping
pace with any decline in body weight, thus, a decline in
CF with fasting was not observed. There may not have been
sufficient resources for maintaining linear growth in fish
which started with a low CF in Study 2 and therefore
changes in nutritional state were more easily detected in
changes in CF with fasting. A decline in GSI together
with an apparent softening of the follicles suggests that
the females in Study 1 were suffering from a nutritional
deficit, enough to impact ovarian development.
Although there were differences in changes in CF in
response to fasting between the two studies, suggesting
that there were differences between the two studies as to
the changes in metabolic states, there were no discernible
differences in hormone response. In both studies, one
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with low CF (-3.2) and one with high CF (-3.6), pituitary
content but not serum concentrations of GH increased with
21 days of fasting. In addition, serum concentrations of
tPRL1 7 7 were increased by 10 days of fasting in both
studies and serum concentrations of tPRL1 8 8 were elevated
in males with 21 days of fasting in both studies. The
consistency of the responses may be due to the limited
sampling. Serum PRL concentrations may have increased
earlier within the first 10 days of fasting, and pituitary
GH content may have increased earlier between days 10 and
21 of fasting in the tilapia with a lower CF than in the
tilapia with a higher CF. On the other hand, the
metabolic or nutritional state of the tilapia at the start
of fasting may be of less importance than a change in
nutritional state.
The differences in response to fasting by PRL and GH
suggest the hormones may have disparate actions. Serum
tPRL1 7 7 increased much earlier than serum GH concentra
tions. Prolactin may be mobilizing energy stores from the
liver which undergoes most of its weight loss within 10
days. Growth hormone may then rise to mobilize other
energy reserves or to inhibit the mobilizing or cataboliz
ing of needed resources. Further study is needed to
understand the roles of PRL and GH in metabolic processes
associated with fasting in the tilapia. Among these
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studies are kinetic studies to see if there are changes in
the metabolic clearance rates of the tPRLs or GH associat
ed with fasting and studies on changes in body composition
and blood metabolites in response to fasting and homolo
gous hormone treatment. Even then, these studies must be
conducted under different physiological conditions and at
different developmental stages.
In summary, serum concentrations of tPRL1 77
and tPRL1 88 increased soon after the initiation of
fasting while changes in pituitary content of GH and serum
concentrations of GH were not observed until late in
fasting. Prolactin1 77 appeared to be more sensitive to
changes in nutritional state than tPRL1 88. Changes in
pituitary content of GH were observed before changes in
serum concentrations of the hormone while the opposite was
true with PRL. In addition, the PRL response to fasting
may be sex specific. Prolactin1 77 content in the
pituitary increased with fasting in female tilapia with no
change in male tilapia. A decline in pituitary tPRL1 88
content was observed in males with a low CF at 31 days of
fasting but there were not females in the experiment for
comparison. These studies provide evidence that the tPRLs
and GH are active in the regulation of metabolism in the
tilapia, O. mossambicus and that the tPRLs and GH may have
disparate functions.
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Chapter V
CHANGES IN SERUM CONCENTRATIONS AND PITUITARY CONTENT OF
PROLACTIN AND GROWTH HORMONE DURING THE REPRODUCTIVE CYCLE
OF FEMALE TILAPIA, OREOCHROMIS MOSSAMBICUS ADAPTED TO
FRESH WATER AND SEAWATER
INTRODUCTION
Prolactin and GH have actions in osmoregulation,
metabolism, and reproduction (Nicoll, 1974; Clarke and
Bern, 1980; Loretz and Bern, 1982; Hirano, 1986). These
processes are intertwined within the reproductive habits
of the tilapia, O. mossambicus. Oreochromis mossambicus
can reproduce in both FW and SW. Furthermore, feeding is
reduced in the female tilapia when it broods offspring in
its buccal cavity. Brooding lasts for approximately 3
weeks. The subject of this study was the roles of the
tPRLs and GH in the regulation of these disparate though
overlapping processes during reproduction in the female
tilapia.
Prolactin and GH may regulate reproductive functions
in the female tilapia. Prolactin has been implicated in
parental care in teleosts including cichlids. Behaviors
shown to be elicited by PRL are expressed in female tila-
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pia. These behaviors include "calling movements" to fry
and reduced feeding behavior which may prevent the canni
balism of young (Noble et al., 1938; Neil, 1964; Blum and
Fielder, 1965; Blum, 1966). Prolactin has also been shown
to induce mucus secretion as a source of nutrition for
developing fry (see review, Nicoll, 1974).
Fish PRLs and GHs have been shown to stimulate ovar
ian growth and steroidogenesis in teleosts (Singh et al.,
1988; Tan et al., 1988; Danzmann et al., 1990; Van Der
Kraak et al., 1990). In the tilapia, tPRLs have been
shown to alter steroid release from testicular tissues of
courting and non-courting males in vitro (Rubin and
Specker, 1992). The tPRLs stimulate T production from
testis fragments of courting males but have no effect on
testicular tissues from non-courting males. The tPRLs
augment the response of testicular tissues from courting
males to ovine-luteinizing hormone but inhibit this
response in testis from non-courting males. In addition,
tPRL1 8 8 has been shown to stimulate E2 production by
vitellogenic oocytes of the guppy, Poecilia reticulata
(Tan et al., 1988). Further supporting a direct action of
PRL and GH in regulating steroidogenesis in teleosts is
the identification of PRL and GH binding sites in the
ovary. Prolactin binding sites have been identified in
the ovary of the tilapia and GH receptors have been iden-
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tified in the ovaries of salmonids (Edery et al., 1984;
Mourot et al., 1992).
Reproductive hormones have been shown to stimulate or
modulate the release of PRL and GH in tilapia. The ster
oid hormones, E2 and T have been shown to stimulate PRL
cells (Nagahama et al., 1975; Wigham et al., 1977; Barry
and Grau, 1986; Borski et al., 1991) and modulate the
response of PRL and GH cells to hypothalamic factors
including thyrotropin-releasing hormone, GnRH and GHRH
(Barry and Grau, 1986; Melamed et al., 1995).
Prolactin and GH regulate osmoregulatory functions in
the tilapia and may be active in these roles depending on
the salinity in which the tilapia is reproducing (cf.
Nishioka et al., 1988; cf. Grau et al., 1994). Pituitary
content and blood concentrations of the tPRLs are greater
in FW- than in SW-adapted tilapia (Nicoll et al., 1981;
Borski et al., 1992; Ayson et al.; 1993, Yada et al.,
1994). Furthermore, Borski and colleagues (1992) have
shown that the two tPRLs are differentially regulated by
environmental salinity (Borski et al., 1992). Pituitary
content of GH is greater in SW- than in FW-adapted tilapia
(Borski et al., 1994). Yada et al. (1994) observed an
increase in circulating GH shortly after transfer of male
o. mossambicus from FW to 70%-SW but no change in females.
In the same study, GH levels in the plasma were reduced
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shortly after transfer from SW to FW in both sexes,
compared to animals at the same time point which had been
transferred from SW to SW. Interestingly, circulating
levels of GH were no longer different from controls, in
either direction of transfer and either sex, by 7 days
after transfer. Sakamoto and colleagues (1994) also
observed an increase in plasma GH following transfer of o.
mossambicus from FW to SW whereas plasma GH was not
altered following transfer from SW to FW. Furthermore,
the increase in plasma GH was observed in both males and
females and at the end of the study, day 14 after trans
fer, plasma GH was elevated in males but not in females
(Sakamoto, personal communication).
Finally, the tPRLs and GH may regulate metabolic
activities in the brooding female tilapia which experi
ences a reduction in food intake. I have shown in chapter
IV that serum and pituitary levels of the tPRLs and GH
increase in response to fasting. Changes in hormone
levels observed during the reproductive cycle of the
female tilapia may therefore derive from changes in nutri
tional state. Sumpter and colleagues (1991a,b) have
determined that changes in circulating GH concentrations
during the reproductive cycle of male and female rainbow
trout are a consequence of changes in metabolic state and
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not due to reproduction per se. A similar situation may
exist with the tilapia.
The foregoing discussion suggests that reproductive,
metabolic and osmoregulatory requirements of the female
tilapia may contribute to PRL and GH cell activity during
the reproductive cycle. In chapter IV, I have described
changes in serum concentrations and pituitary content of
the tPRLs and GH that occur with changes in metabolic
state induced by fasting. In the studies described in
the present chapter I characterized changes in serum
concentrations and pituitary content of the tPRLs and GH
that occur over the course of the reproductive cycle of
the female tilapia. I also examined the effects of the
salinity on the patterns of changes in the tPRLs and GH in
the serum and pituitary, observed during the reproductive
cycle. Changes in BW, SL, CF, GSI and HSI that occur over
the course of the reproductive cycle were also examined in
an attempt to evaluate the contribution of changes in
metabolic state to hormone changes. The aim was to gain
insight into how the tilapia balances the regulation of
different functions by the same hormones, by comparing
hormone patterns for female tilapia reproducing in FW and
SW, and hormone patterns observed during fasting.
Comparing changes in hormones during the reproductive
cycle of fish maintained under various conditions may help
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to clarify which aspects of the hormone patterns are
intrinsic to the reproductive cycle. Three studies were
conducted. Study 1 was conducted in an outdoor tank with
FW animals. This study was conducted to examine changes
in serum concentrations and pituitary content of the tPRLs
and GH in tilapia that are reproducing in an environment
that is conducive to their natural behavior. Studies 2
and 3 were conducted in indoor tanks. The fish and envi
ronmental conditions for studies 2 and 3 were similar
except that in Study 2, the fish were adapted to FW and in
Study 3 the fish were adapted to SW. Study 2 was also
designed to expand and refine sampling to include earlier
stages of the reproductive cycle. The purpose of Study 3
was to provide for a comparison of serum and pituitary
patterns of PRL and GH between FW- and SW-adapted tilapia.
MATERIALS AND METHODS
Animals
Tilapia, O. mossambicus were collected from brackish
water streams and maintained in FW in outdoor tanks at a
temperature of 22-25°C for at least 2 months before use.
The fish were fed to satiation twice daily with Purina
trout chow. Fish were handled and sampled using proce
dures described in chapter IV. Prolactins and GH in serum
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and pituitaries were measured using the homologous ra
dioimmunoassays developed by Ayson et al., (1993) and
modified by Yada et al. (1994) as described in chapter
III. Pituitaries were collected and prepared for measure
ment by radioimmunoassay as described in chapter IV.
The reproductive stage of the animals was classified
using two criteria. Follicle mass of the largest clutch
of follicles in the ovary was used to classify non
brooding females, and stage of larval development of the
young was used to classify brooding females. Follicle
mass was determined by collecting and weighing a group of
20 follicles from the most advanced clutch of follicles
within the ovary. Follicle growth was found to be linear
over time starting from spawning in o. mossambicus
(Weber et al., 1992). Non-brooding females were grouped
according to follicle mass, to the nearest mg. Follicle
mass and brooding stage were recorded to investigate
changes in ovarian growth during the brooding phase of the
reproductive cycle. The days from spawning for each stage
were estimated from the stage of development of the eggs
and larvae, based on previous studies that examined the
rate of larval development in o. mossambicus (Weber et
al., 1989; Okimoto et al., 1993).
Initially, brooding females were classified into 10
stages based on the developmental stage of the young.
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These stages are: early-egg, eyes not pigmented; mid-egg,
eyes pigmented but no spinal movement; late-egg, spinal
movement; early-yolksac, up until 50% of the larvae can
swim in the water column; mid-yolksac, up until pigmenta
tion covers the sides of the larvae and yolk can only be
seen through the bottom of the fish; late-yolksac, up
until yolk can not be found in the larvae; females
brooding post-yolksac larvae. In most of the present
studies, these stages have been collapsed into just 3
brooding stages: females brooding eggs, females brooding
yolksac larvae, and females brooding post-yolksac larvae.
Females designated as simply non-brooding females were
females with follicles greater than 4 mg. Four mg
follicles are not found in females which are brooding.
Follicle mass does not usually exceed 3 mg in brooding
tilapia (Fig. 22). Follicles weigh about 6 mg at
ovulation.
Statistical analysis
Differences among groups were determined using analy
sis of variance and the least significant difference test
for a priori pairwise comparisons analysis (Steel and
Torrie, 1980). Comparisons between two means were per
formed using the unpaired Student's t-test. Data are
expressed as means ± SEe
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Study 1
Fish were transferred to an outdoor vinyl-lined
broodstock tank (18 ft diameter) with a slow, continuous
flow of FW. The fish were not sampled for at least 2
months after transfer to allow the females time to begin
reproducing. Females averaged about 150-200 g. Water
temperature was dependent on ambient temperatures and
ranged from -22-26°C. An algal bloom was maintained in
the tank as a dietary supplement by controlling water
exchange rate. The tank also accumulated detritus upon
which the tilapia can feed. Detritus-like matter was seen
in the gut of brooding and non-brooding tilapia. The
females in this tank were observed to stay near the center
of the tank with the males establishing nest cites around
the perimeter.
Females were sampled from the outdoor tank on 3
occasions, once each in July, November and April, for a
total of 126 fish. Fish were collected by hand net and
placed immediately in anesthesia. Blood samples were
collected from all fish, and pituitaries were collected in
April and November as described in chapter IV. Also in
November; SL and liver weight was recorded for all females
for the determination of HSI and CF, and follicle mass was
recorded for brooding females for the examination of
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ovarian growth during the brooding phase. Only females
with follicles larger than 4 mg were identified as non
brooding females to insure that they were not brooding
females which had released their clutch before capture.
During the April collection, the time of capture was
recorded. Prolactin and GH levels were not correlated
with time from the start of collection, suggesting that
the collection method did not affect hormone levels (data
not shown).
Study 2
Fish were transferred to an indoor facility with a
fixed photoperiod of 14L:10D. Fish were distributed among
3 fiberglass tanks (500 1) fitted with a plexiglass win
dow. In each tank, 20 females were housed with 2 males.
Females weighed 60-100 g. The tanks were outfitted with a
continuous flow of FW, constant aeration, and submersible
heaters that maintained the water temperature at 26.5 ±
0.5°C. The indoor tanks had limited detritus buildup.
The fish were not sampled for at least 8 weeks following
their introduction to the tanks. At the time of sampling,
females that were brooding were identified through the
windows of the tanks and removed first. This prevented
the females from releasing the clutches of young they were
brooding before I had a chance to identify the females as
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brooding females. Females which had not been brooding but
ware in stages earlier than late vitellogenesis can not be
distinguished with certainty from brooding females with
similar size follicles that had been brooding but released
their clutch prior to capture. Therefore, sampling of
females during the early stages of ovarian growth required
that all females be identified as brooding females or non
brooding females prior to collection.
Only non-brooding females whose follicle mass exceed
ed 1.5 mg were included in the study. This is because
females with a follicle mass of less than 1.5 mg may not
have been reproductively mature or may have terminated
brooding prematurely. A mass of 1.5 mg was chosen as the
lower limit because FW females brooding post-yolksac
larvae were found to have follicles with a mass of just
under 1.5 mg (Fig. 22). Samples were collected on 5
occasions from July through December, from a total of 177
females. Body weight, SL, liver weight and gonad weight
were recorded during the final sample collection for
determination of HSI, CF and CF with gonad weight
subtracted from BW.
Study 3
Study 3 was the same as Study 2 except the fish were
adapted to SW. Fish were introduced into the tanks as in
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Study 2 and the salinity of the water was slowly increased
to full-strength SW over a 1 week period. The tanks were
then supplied with a continuous flow of SW. The fish
were not sampled for at least 8 weeks following this
acclimation to SW. The non-brooding females were all
females which had a follicle mass greater than 4 mg.
Follicle mass was determined for all brooding SW fish for
the examination of ovarian growth during the brooding
phase. Samples were collected on 2 occasions in October
and 1 in November from a total of 53 females. Body
weight, SL and liver weight were recorded during the final
sample collection for the determination of RSI and CF.
RESULTS
Follicle growth during the brooding phase of the
reproductive cycle
Follicle growth during the brooding phase of the
reproductive cycle was examined in fish collected in Study
1 (FW, outdoor tank) and Study 3 (SW, indoor tank) .
Follicles in fish from Study 1 increased in size from 0.22
± 0.07 mg in females brooding early stage eggs to 1.31 ±
0.17 mg in females brooding post-yolksac larvae (Fig. 22).
Follicles in fis~ from Study 3 increased in size from 0.30
± 0.01 mg in females brooding early stage eggs to
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Figure 22. Changes in follicle mass during the broodingperiod of the tilapia Oreochromis mossambicus. For eachstudy, points marked by different letters are significantly different (P < 0.05). Points without letters are N=land were not included in the analysis (mean ± SE; study 1N: EE=3, ME=5, LE=l, EY=6, MY=l, LY=6, PY=6, 28 fishtotal; study 3 N: EE=3, ME=4, LE=2 EY=3, MY=3, LY=5, PY=9,29 fish total). EE, early-egg; ME, mid-egg; LE, late-egg;EY, early-yolksac larvae; MY, mid-yolksac larvae; LY,late-yolksac larvae; PY, post-yolksac larvae broodingfemales.
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3-. Study 1
b
2.5• Study 3 b
.-.tn
2E-tnU'J
beu 1.5:ECI)-U.--- 10
LL
0.5
aO-+--r---1r---T~--"'--'---r--1r---T~--,--.--.....--,.....-,.--r-~-'--r-I
o 2 4 6 8 10 12 14 16 18 20( EE ME LE EY MY LY PY)
Approximate day from spawning(Stage)
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2.34 ± 0.31 mg in females brooding post-yolksac larvae.
Follicles in females brooding late-yolksac larvae were
similar in size to follicles in females brooding post
yolksac larvae in both studies.
Changes in condition factor and hepatosomatic index with
brooding
The CF of FW females maintained in the outdoor tank
(study 1) did not significantly change with brooding
stage. The HSI was reduced in females carrying
early-yolksac larvae, about day 5-6 after spawning,
compared with non-brooding females (Fig. 23; P < 0.05).
Detritus-like matter was seen in the gut of brooding and
non-brooding tilapia in Study 1 but not in any of the
brooding fish in studies 2 or 3. Condition factor and HSI
were reduced in FW (Study 2) and SW (Study 3) females
brooding post-yolksac larvae compared with non-brooding
females (Fig. 24; P < 0.05). The drop in CF in FW fish
(Study 2) was significant even when gonad weights were
subtracted from body weights before CF was calculated
(P < 0.05).
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Figure 23. Changes in condition factor (CF) andhepatosomatic index (HSI) during the brooding period ofthe tilapia Oreochromis mossambicus. For each subject,points marked by different letters are significantlydifferent (P < 0.05). Points without letter are N=l andwere not included in the analysis (mean ± SE; study 1 N:NB=8, EE=3, ME=S, LE=l, EY=6, MY=l, LY=6, PY=6, 28 fishtotal; study 3 N: NB=7, EE=3, ME=4, LE=2 EY=3, MY=3, LY=S,PY=9, 36 fish total). NB, non-brooding; EE, early-egg; ME,mid-egg; LE, late-egg; EY, early-yolksac larvae; MY, midyolksac larvae; LY, late-yolksac larvae; PY, post-yolksaclarvae brooding females.
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4
-e- HSI
1.5 CF
3.5 +
b
a
a
f2
3
-en:J:...... 2.5LLo
o 2 4 6 8 10 12 14 16 18 20
( NB EE ME LE EY MY LY py)
Approximate day from spawning
(Stage)
161
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Figure 24. Changes in condition factor (CF), CFcalculated without gonad weights included in body weightvalues (CF-GW), and hepatosomatic index (HSI) in nonbrooding (NB) female tilapia Oreochromis mossambicus andfemale tilapia brooding post-yolksac-larvae, adapted tofresh water (FW) and seawater (SW) (mean ± SE, FW: N = 7-9per time point; SW: N = 4; * P < 0.05, ** P < 0.01,*** P < 0.001).
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4 CF-GW
3.5
3
2.5-en~ 2LL(J
1.5
1
0.5
FW
CF
FW SW
163
D NB
D py
HSI
FW SW
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Serum concentrations and pituitary content of PRL and GH
during the brooding cycle in FW-adapted female tilapia,
maintained in outdoor tanks (Study 1)
This study was conducted to examine changes in serum
concentrations and pituitary content of the tPRLs and GH
in tilapia that are reproducing in an environment that is
conducive to their natural behavior. Serum tPRL1 7 7
concentrations but not tPRL1 8 8 concentrations were
elevated in female tilapia at the end of the brooding
phase over levels in non-brooding females (Fig. 25).
Serum concentrations of tPRL1 77 were elevated in
females brooding post-yolksac larvae over those observed
in non-brooding females or in females brooding either eggs
or yolksac-larvae (P < 0.05). Serum concentrations of
tPRL1 8 8 remained at about 3-4 ng/ml throughout the brood
ing cycle. Serum GH concentrations were also elevated in
female tilapia at the end of the brooding phase compared
with concentrations in non-brooding females. Serum GH
concentrations were below 1 ng/ml in non-brooding females
and in egg-brooding females before increasing to 2.3 ± 0.3
ng/ml in females brooding yolksac larvae (P < 0.01) and
4.9 ± 0.7 ng/ml in females brooding post-yolksac larvae
(P < 0.001).
Pituitary content of tPRL1 77 changed over the brood
ing cycle whereas tPRL1 88 did not (Fig. 26). Pituitary
164
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Figure 25. Changes in serum concentrations of prolactinand growth hormone during the brooding period of thetilapia Oreochromis mossambicus in fresh water.(mean ± SE, N = 30-47 per treatment for serum levels,
N = 24-35 for pituitary levels; 126 fish total;* p < 0.05, ** P < 0.01, *** P < 0.001 ). N, non-broodingfemales; E, females brooding eggs; Y, females broodingyolksac larvae; PY, females brooding post-yolksac larvae.
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6 -
***tPRL177 tPRL188 GH
5 -.-.-E
*--ene- 4-e0.-..CO.....e 3 -CI)CJ **C0CJ ...~
E 2 -:::sa..Q)
tJ)
1 - rt rt
N E V PY N E Y PY
Stage
166
N E Y PY
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Figure 26. Changes in pituitary content of prolactin andgrowth hormone during the brooding period of the tilapiaOreochromis mossambicus in fresh water. (mean ± SE,N = 30-47 per treatment for serum levels, N = 24-35 forpituitary levels; 126 fish total; * P < 0.05, ** P < 0.01,*** P < 0.001 ). N, non-brooding females; E, femalesbrooding eggs; Y, females brooding yolksac larvae; PY,females brooding post-yolksac larvae.
167
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20-
18-tPRL177 tPRL188 GH ***
16-
.12-
_ 14-....-c.--C):0:-
8-
** *6- ~ If4- t rf
.....s::: 10(1).....s::::ou~ca......-:::s......-n.
2-
N E Y PYN E Y PYo....L..l-....L.-L--I...-.a..-...L-....L.-L_--'--L-I.--I...-.L..-..I-...L.-L_---L..-L-I.--J-...L-.L......J.-L...
N E Y PY
Stage
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content of tPRL1 77 increased from 4.3 ± 0.3 ~g/pit. in
non-brooding females to 6.0 ± 0.5 ~g/pit. in females
brooding eggs (P < 0.01), and 5.8 ± 0.3 ~g/pit. in females
brooding yolksac-larvae (P < 0.05). Pituitary tPRL1 77
content in females brooding post-yolksac larvae was not
different from those at any other stage. Pituitary con
tent of tPRL188 remained at about 8-10 ~g/pit. throughout
the brooding cycle. Pituitary content of GH increased
from 10.5 ± 1.3 ~g/pit. in non-brooding females to 17.6 ±
1.1 ~g/pit. in females brooding post-yolksac larvae
(P < 0.001).
Serum PRL and GH concentrations during the reproductive
cycle of the female tilapia adapted to FW and maintained
in indoor tanks (Study 2).
The purpose of this study was to expand and refine
sampling to include earlier stages of the reproductive
cycle. Serum tPRL1 77 concentrations were lowest at the
end of ovarian growth, when the follicles average 5-6 mg
(4.6 ng/ml). Serum tPRL17 7 then increased to 7.8 ± 0.6
ng/ml in egg-brooding females (P < 0.01) and remained
elevated throughout the remainder of the brooding phase
(Fig. 27). The concentrations were not different among
the non-brooding stages or between the early ovarian
growth stages and the brooding stages. A significant
169
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Figure 27. Changes in serum prolactin and growth hormoneconcentrations during the reproductive cycle of the femaletilapia Oreochromis mossambicus (mean ± SE,N = 12-48 per time point, 177 fish total, for each hormone, points marked by different letters are significantlydifferent at P < 0.05). Stages are classified by folliclemass (mg) and developmental stage of brooded young. E,females brooding eggs; Y, females brooding yolksac larvae;PY, females brooding post-yolksac larvae.
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15 -.- GH
~ tPRL188
.-. 12--II- tPRL177
-E--C)c:.....e0 9.-...,f!.... bc(I)uc 60oE~ c~
(I)
UJ 3a
a a a
02 3 4 5 6 E y py
Stage
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effect of reproductive stage with tPRL1 8 8 concentration
was not detected by analysis of variance (P = 0.14),
however, the pattern for tPRL1 88 appeared similar to that
of tPRL1 77. Growth hormone concentrations were low
throughout the ovarian growth phase in non-brooding fe
males «1.5 ng/ml), and increased with the continuation of
brooding to reach 2.5 ± 0.3 ng/ml in females brooding
yolksac larvae (P < 0.001 compared with egg-brooding
females) and 3.3 ± 0.5 in females brooding post-yolksac
larvae (P < 0.05 compared with yolksac larvae brooding
females) .
Serum concentrations and pituitary content of PRL and GH
during the brooding cycle in SW-adapted female tilapia,
maintained in indoor tanks (Study 3)
The purpose of this study was to provide for a com
parison of serum and pituitary patterns of PRL and GH
between FW- and SW-adapted tilapia. Only those non
brooding females with follicles larger than 4 mg were
included in the study, for the reasons discussed above.
The acclimation of tilapia to SW for a minimum of 8
weeks reduced circulating tPRL1 77 to undetectable levels.
Serum tPRL1 88 concentration was reduced to -4 ng/ml, from
-9 ng/ml measured in FW tilapia under similar conditions
(from Study 2; Fig. 28). Serum tPRL1 8 8 in non-brooding
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Figure 28. Changes in serum concntrations of prolactinand growth hormone during the brooding phase of the reproductive cycle in SW-adapted female tilapia, Oreochromismossambicus, maintained in indoor tanks; (mean ± SE,N = 6-20 per treatment, 53 fish total; * P < 0.05,*** P < 0.001 compared with non-brooding females unlessotherwise indicated). N, non-brooding females; E, femalesbrooding eggs; Y, females brooding yolksac larvae; PY,females brooding post-yolksac larvae.
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tPRL188
*
**
N E Y PYN E Y PY
-GH '*
-r-
-
-*
..-1-
-
t +
5
o
4--E--C)s:::-e0 3.-....CO=-....s:::CI)us:::
20oE::J...(1)
en1
Stage
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females was not significantly different from females
at any stage of the brooding phase. Serum tPRL1 8 8
concentrations were increased in females brooding
post-yolksac larvae over levels in females brooding eggs.
Serum GH concentrations increased as brooding continued.
Serum GH increased from 1 ng/ml in non-brooding and in egg
brooding females to 1.5 ± 0.2 ng/ml in females brooding
yolksac larvae (P < 0.05) and 3.7 ± 0.9 ng/ml in females
brooding post-yolksac larvae (P < 0.001, compared with
non-brooding females). Serum GH concentrations and
changes in concentrations were similar in all studies.
In SW, pituitary content of tPRL1 77 and tPRL1 88 did
not differ among females at various stages of the brooding
cycle (Fig. 29). Growth hormone content was elevated in
females brooding post-yolksac larvae compared with all
other stages (P < 0.05).
DISCUSSION
Patterns of changes in the tPRLs in serum and
pituitaries of the female tilapia, O. mossambicus, during
the reproductive cycle were affected by the salinity to
which the fish were adapted. Patterns of changes in GH,
on the other hand, were not affected by salinity. Fur
thermore, the patterns for the tPRLs and GH observed
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Figure 29. Changes in pituitary content of prolactin andgrowth hormone during the brooding phase of thereproductive cycle in SW-adapted female tilapia, Oreochromis mossambicus, maintained in indoor tanks; (mean ± SE,N = 6-20 per treatment, 53 fish total; * P < 0.05 comparedwith non-brooding females). N, non-brooding females; E,females brooding eggs; Y, females brooding yolksac larvae;PY, females brooding post-yolksac larvae.
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14-
tPRL177 tPRL188 GH *12-
-
4-
6-
::s...-
~:! 10-.-::s...-Q.--.C)== 8-..c:CI)..c:ou~CO...-a.
2-
N E Y PY N E Y PY N E Y PY
Stage
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during the reproductive cycle had characteristics that
were both similar to and distinct from patterns observed
during fasting as described in chapter IV. These findings
suggest that changes in metabolic state may influence tPRL
and GH cell activity during the reproductive cycle al
though they can not account for all the changes in serum
concentration and pituitary content of the hormones.
Overall, serum PRL concentrations were highest in
females brooding post-yolksac larvae in each of the three
studies. Nonetheless, changes in serum and pituitary
levels with the reproductive cycle appeared to be small
and there were differences among studies as to when PRL
levels increased during the brooding phase. The observed
patterns of serum concentration and pituitary content of
the tPRLs do not support the notion that changes in serum
concentrations of the tPRLs are driving parental care
behavior or ovarian function in the female tilapia. On
the other hand, the patterns of serum concentration and
pituitary content of GH during the reproductive cycle were
consistent in all three studies. Serum GH concentrations
increased during the brooding phase of the reproductive
cycle followed by an increase in pituitary content of the
hormone. These changes in serum and pituitary GH levels
appear to be a characteristic component of the reproduc
tive cycle of the female tilapia and suggest that GH has
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functions intrinsic to reproduction. In the following
segments, I will first discuss changes in the physiology
of the female tilapia during the reproductive cycle and
then relate these changes to the patterns of serum and
pituitary tPRLs and GH levels observed during the repro-
ductive cycle.
Ovarian growth during the brooding phase of the
reproductive cycle
Ovarian follicles grew in size throughout most of the
brooding phase in the 2 studies in which it was examined,
Studies 1 and 3. Follicles reached their maximum weight
when the females were brooding late-yolksac larvae, about
day 12 days post-spawning, and then stopped growing
throughout the remainder of the brooding phase. Follicles
increased in weight from less than 0.4 mg the day after
spawning up to 1.3 and 2.3 mg by the end of the brooding
phase in Study 1 and Study 3 fish respectively. The
difference in growth between Study 1 and Study 3 may be
due to a combination of factors including salinity, water
temperature, and size of the fish. The females in Study 1
were adapted to FW, maintained in at outdoor tank at
ambient temperatures (-22-26), and were about 150-200 g.
The females in Study 3 were adapted to SW, maintained
indoors in tanks heated to 26.5 ± 0.5°C, and were
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60-100 g. What is of interest is that the follicles
stopped growing at about the same stage of the reproduc
tive cycle in both studies and not at the same size.
Smith and Haley (1987, 1988) gave a similar account
of ovarian growth during the brooding phase of this
species following a histological examination of ovarian
development. They reported that ovarian growth was
arrested between days 15-25 after spawning. In addition,
they found that ovarian growth did not cease in females
which did not brood, even when fasted for 20 days. The
oocytes of some of the fasted females showed softening, an
early sign of atresia. I also observed a reduction in GSI
and softening of follicles in fasted female tilapia
(chapter IV). Smith and Haley (1988) suggested that the
interruption of oocyte growth is not due simply to a
reduction in food intake. This notion is supported by the
consistency in the timing of the interruption in oocyte
growth in both the studies by Smith and Haley (1987, 1988)
and in my studies; and the finding that oocyte growth is
interrupted at a particular stage, regardless of follicle
size. The size of the follicles and the stage of the
brooded young were not recorded in the studies by Smith
and Haley (1987, 1988).
Smith and Haley (1988) showed that E2 and T were
elevated during brooding and suggested that these steroids
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may be involved in either brooding behavior or in
protecting the oocytes from atresia. Furthermore, Smith
and Haley (1987) found that postovulatory follicles
are steroidogenic and persist longer in brooding than non
brooding females. They suggested that these structures
may also be involved in either brooding behavior,
protecting the oocytes from atresia or inhibiting oocyte
growth in the latter part of the brooding phase. I have
found that PRL and GH are elevated during the brooding
phase, and thus, may participate in these actions. More
over, PRL and GH may have roles in regulating circulating
concentrations of levels of E2 and T, and in prolonging
the life of the postovulatory follicles. Alternatively,
PRL and GH may have metabolic roles during the brooding
phase, mobilizing and directing energy reserves.
Changes in condition factor and hepatosomatic index with
brooding in FW-and SW-adapted tilapia
Condition factor and RSI declined in females as
brooding continued. These data suggest that nutritional
status declined during the brooding phase of the reproduc
tive cycle. The decline in CF between non-brooding
females and females brooding post-yolksac larvae was
significant for animals sampled from the indoor tanks
(Studies 2 and 3) but not for females from the outdoor
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tank (Study 1). One possible reason CF was not reduced in
females brooding post-yolksac larvae in the outdoor tank
is that the females had resumed feeding. The outdoor tank
had detritus on the bottom and the fish had detritus-like
matter in their guts. Furthermore, there was more space
in the outdoor tank, possibly allowing the brooding fe
males to release their clutches and feed. I think the
females in Study 1 did undergo changes in condition during
brooding that I was unable to detect. Although CF did not
decline significantly in females in Study 1, the mean CF
in females brooding yolksac larvae from the outdoor tank
(Study 1) was 12% lower than in non-brooding females.
This is the same difference in mean CF for non-brooding
females and females brooding post-yolksac larvae in
studies 2 and 3, where those differences were significant.
The 12% decline in CF observed during the brooding phase
is equal to or greater than reductions in CF that occurred
over the course of 21 days of fasting in female tilapia
(7.5%) and 31 days of fasting in male tilapia (10.5%;
chapter IV) .
In the present study, RBI was reduced from 2.3 in
non-brooding females to below 1.5 in females brooding
post-yolksac larvae, in all studies. A significant
reduction was observed in females brooding early-yolksac
larvae, about 6 days after spawning, (Study 1). The early
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decline in HSI with brooding is comparable to a decline in
HSI observed with 10 days of fasting (chapter IV). The
drop in HSI during brooding was most likely due to nutri
tional demands rather than a decline associated with a
decrease in vitellogenin synthesis activity. In support
of this notion, HSI was reduced from 2.4 in non-brooding
females to 1.7 in females brooding early-yolksac larvae,
even though oocyte growth continues beyond this stage
(Study 1). In addition, studies suggest that tilapia
produce vitellogenin which is secreted into mucus as a
source of nutrition for developing post-yolksac larvae
(Kishida and Specker, 1994).
Effect of salinity on serum and pituitary patterns of tPRL
during the brooding cycle
Salinity affected baseline serum concentrations and
the patterns of the tPRLs in serum and pituitaries during
the brooding cycle of the tilapia. Circulating titers
of the tPRLs were greatly reduced in SW fish (Study 3)
compared with FW fish (Study 2) held under similar condi
tions. Prolactin177 was usually not detectable in the
serum of the SW-adapted tilapia. Reduced serum concentra
tions of the tPRLs in the SW tilapia compared with levels
in FW tilapia is consistent with previous findings in o.
mossambicus (Nicoll et al., 1981; Borski et al., 1992;
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Ayson et al., 1993; Yada et al., 1994). Differences in
levels and patterns of the tPRLs in serum and pituitaries
with salinity suggest that the tPRLs perform osmoregulato
ry actions during the reproductive cycle of female tilapia
adapted to FW. Nevertheless, large differences in serum
tPRL concentrations in FW- compared with SW-adapted
tilapia do not imply that the hormones do not have actions
other than in osmoregulation. Low levels of the tPRLs may
be adequate for the actions of the tPRLs in reproduction
or metabolism.
Changes in serum tPRL1 8 8 were also different in the
FW- and SW-adapted tilapia during the reproductive cycle.
In FW, serum concentrations of tPRL1 88 did not change over
the reproductive cycle. In SW, by contrast, serum tPRL1 88
concentrations were elevated in females that were brooding
post-yolksac larvae over levels in females brooding eggs.
No changes in pituitary content of tPRL1 77 or tPRL1 88 were
observed in the SW-adapted tilapia. No changes in pitui
tary content of tPRL1 88 were observed in the FW fish from
the outdoor tank (Study 1), however, pituitary tPRL1 77
content was elevated in females brooding eggs and females
brooding yolksac larvae, over levels in non-brooding
females.
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Effect of salinity on patterns of GH in serum and
pituitaries during the brooding cycle
Salinity did not influence patterns of changes in
serum or pituitary GH during the brooding cycle of the
tilapia. Increases in circulating GH concentrations,
in response to transfer from FW to SW, have yet to be
observed in the tilapia, o. mossambicus, to last longer
than 14 days. Yada et al. (1994) observed a transient
increase in plasma GH concentrations in male tilapia after
transfer from FW to 70%-SW but no change in females.
Sakamoto and colleagues (1994) also observed an increase
in plasma GH following transfer of o. mossambicus from
FW to SW, whereas plasma GH was not altered following
transfer from SW to FW. Furthermore, the increase in
plasma GH following transfer to SW was observed in both
males and females, and at the end of the study, day 14
after transfer, plasma GH was elevated in males but not in
females (Sakamoto, personal communication). Transient
increases in blood GH occur after SW transfer in coho
salmon and rainbow trout. These rises are accompanied by
increases in metabolic clearance rate and secretion rates
of GH (Sakamoto et al., 1990, 1991). Whether there are
salinity-specific differences in metabolic clearance and
in the secretion of GH in the tilapia remains to be deter-
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mined. Borski and colleagues (1994) found that pituitary
GH content was elevated in o. mossambicus reared in SW for
7 months and in tilapia transferred from FW to SW for 49
days. Nonetheless, in the present studies, changes in
serum and pituitary levels of GH during the brooding cycle
were similar in FW- and SW-adapted tilapia.
A final consideration is that female tilapia may not
respond to changes in salinity as do males. Only males
were used in the studies by Borski and colleague (1994).
Furthermore, Yada et al. (1994) did not observe changes in
plasma GH concentrations in female tilapia following
transfer from FW to SW despite seeing increases with
males.
Changes in serum concentrations and pituitary content of
PRL during the reproductive cycle of the female tilapia
Overall, serum PRL concentrations were highest in
females brooding post-yolksac larvae in each of the three
studies. In the FW-adapted fish, serum concentrations of
tPRL1 77 increased in brooding females over levels in
those in the later stages of vitellogenesis. Nonetheless,
the patterns of change in tPRL serum and pituitary levels
varied with rearing condition. In fish reared in the
outdoor tank, serum tPRL1 7 7 concentrations were not sig
nificantly elevated until late in the brooding phase, that
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is when females were brooding post-yolksac larvae. Serum
concentrations of tPRL1 77 were elevated early in the
brooding phase when tilapia were held in indoor tanks.
Serum concentrations of tPRL188 did not change sig
nificantly in tilapia adapted to FW. In SW, by contrast,
serum concentrations of tPRL1 88 were elevated when females
were brooding post-yolksac larvae above those measured in
females brooding eggs. Prolactin1 77 was not detectable
in most of the serum samples taken from the SW-adapted
tilapia. For this reasn, it could not be determined
whether serum tPRL1 77 concentrations might vary in a
physiologically important manner.
The pattern of serum tPRL concentrations in non
brooding tilapia during the oocyte growth phase may be a
continuation of the pattern observed in females brooding
post-yolksac larvae (Study 2). Female O. mossambicus
can spawn at 22 to 40 day intervals with oocyte growth
immediately following the termination of brooding (Neil,
1966; Smith and Haley, 1987). Serum tPRL concentrations
may increase with brooding and then, following the termi
nation of brooding, slowly decrease as ovarian growth
progresses. Changes in serum tPRL1 88 in the FW fish
reared in the indoor tanks appear to parallel changes in
tPRL1 77, Thus, the same factors which control tPRL1 77
levels may also govern tPRL1 8 8.
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Few conclusions can be drawn from the pituitary
patterns of the tPRLs observed during the reproductive
cycle. Pituitary content of tPRL1 77 was elevated in FW
females brooding eggs or brooding yolksac larvae compared
with levels in non-brooding females, late in vitellogene
sis. On the other hand, there were no changes in tPRL1 7 7
content in the pituitaries from SW tilapia. There were no
changes in pituitary content of tPRL1 88 in FW or SW ani
mals during the brooding cycle.
Although serum PRL concentration was highest in
females brooding post-yolksac larvae in all three studies,
these changes were relatively small compared with changes
observed with fasting (chapter IV) and transfer between FW
and SW (Yada et al., 1994). There was also an inconsist
ency regarding when serum PRL concentrations begin to
increase in the brooding phase. This suggests that
changes in serum PRL concentrations do not drive parental
care or ovarian function in the female tilapia. This view
is supported by the conclusions of Wendelaar Bonga and
colleagues (1984) who found that PRL cell activity did not
vary between non-brooding and brooding female tilapia,
whether adapted to FW or SW. Their conclusions were based
on ultrastructure morphometry and incorporation rates of
[3 H]-lysine into PRL cells.
This conclusion does not preclude PRL from having
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roles in reproduction in the tilapia, including actions in
parental care or ovarian function. It is not necessary
that there be large changes in PRL for PRL to have
affects. Changes may be occurring in other aspects of the
physiology of the animals associated with reproduction
that makes target tissues either less receptive or more
receptive to PRL stimulation. This may include changes in
PRL receptors in target tissues or changes in hormones
that either potentiate or antagonize the actions of PRL.
Changes in serum concentrations and pituitary content of
GH during the reproductive cycle of the female tilapia
The patterns of change in serum and pituitary GH were
consistent in all 3 studies. Serum GH concentrations
increased beginning when females were brooding yolksac
larvae and continued to increase further in females
brooding post-yolksac larvae. Pituitary GH content was
elevated in females brooding post-yolksac larvae both in
SW and in FW. This suggests that an increase (> 3 fold)
in serum GH during the brooding stages of the reproductive
cycle is a characteristic component of the reproductive
cycle of the female tilapia.
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Factors affecting PRL and GH cell activity during the
reproductive cycle of the female tilapia
Prolactin and GH cell activity is likely to be influ
enced by many factors during reproduction. Among these
are salinity, steroid hormone concentrations, and nutri
tion.
Salinity appeared to influence baseline PRL serum and
pituitary levels as well as the patterns in which serum
and pituitary PRL changed over the reproductive cycle.
Salinity did not influence the patterns of changes in
serum and pituitary GH over the reproductive cycle. Both
E2 and T are elevated in brooding tilapia (Smith and
Haley, 1988). The steroid hormones, E2 and T, have been
shown to stimulate PRL release directly and potentiate the
stimulatory effects of GnRH and thyrotropin-releasing
hormone on PRL release in the tilapia (Barry and Grau,
1986; Borski et al., 1991; chapter III). These same
steroid hormones have also been shown to potentiate the
stimulatory actions of GnRH and GHRH on GH release in a
tilapia hybrid (Melamed 1993) .
Changes in serum tPRL concentrations that occur over
the course of the reproductive cycle in female tilapia
vary with rearing condition. This may suggest that PRL
serves functions other than, or in addition to, regulating
aspects of reproduction, that are nonetheless tied to the
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reproductive cycle. This may include metabolic actions.
Changes in CF and HSI observed during the reproductive
cycle were similar or greater than changes observed with
21 days of fasting in studies described in chapter IV. In
those studies, serum concentrations of the tPRLs were
increased with 10 days of fasting in male and female
tilapia. Thus, changes in metabolic state during the
reproductive cycle may be contributing to the changes in
serum tPRL concentrations observed during the course of
the reproductive cycle.
Sumpter et al. (1991a,b) found increases in
circulating GH concentrations were inversely correlated
with changes in CF during fasting and CF during the
reproductive cycle of male and female rainbow trout. They
concluded that changes in GH observed during the reproduc
tive cycle were due to a loss of condition and not a
consequence of reproduction per se. Reductions in CF and
HSI during the brooding phase of the reproductive cycle in
tilapia suggest that a similar situation may exist in this
species.
The 12% decline in CF observed during the brooding
phase is equal to or greater than reductions in CF that
occurred over the course of 21 days of fasting in female
tilapia (7.5%) and 31 days of fasting in male tilapia
(10.5%; chapter IV). Pituitary content of GH were elevat-
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ed in the female tilapia fasted for 21 days compared with
females which were fed, and serum concentrations of GH and
pituitary contents of GH were elevated in the male tilapia
fasted for 31 days. Overall, the decreases in HSI and CF
during the brooding phase of the reproductive cycle are
consistent with the idea that serum and pituitary levels
of GH are elevated in response to changes in metabolic
state occurring during the brooding phase of the reproduc
tive cycle.
Although tPRL and GH serum concentrations and
pituitary content were elevated during brooding, the
patterns were different than with fasting. Serum tPRL17 7
concentrations were elevated prior to increases in tPRL1 77
in pituitaries of female tilapia during fasting. By con
trast, pituitary tPRL1 7 7 levels were elevated prior to an
elevation in serum levels during the brooding cycle.
Serum concentrations of tPRL1 88 appeared to change in a
manner which is similar to tPRL17 7 during fasting and the
reproductive cycle. In both instances, these changes were
not statistically significant (chapter IV) .
Fasting caused elevations in serum and pituitary
levels of GH in o. mossambicus (chapter IV) . Increases in
pituitary content of GH were first observed at 21 days
of fasting in female tilapia and in females brooding
post-yolksac larvae (about 20 days post-spawning). Thus,
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changes in pituitary content of GH occurred over a similar
time-course in both fasting and brooding female tilapia.
Nevertheless, serum levels of GH increased before pitui
tary levels in females during the brooding cycle whereas
the reverse was found during fasting. These differences
in patterns between fasting and brooding animals suggest
that the changes in serum concentration and pituitary
content of the tPRLs and GH observed during the brooding
phase of the reproductive cycle are not due simply to a
decrease in food intake.
Possible actions of PRL and GH during the reproductive
cycle of the female tilapia
The present study suggests that the tPRLs have ac
tions in osmoregulation during the reproductive cycle in
FW adapted-tilapia. The present study also suggests that
the tPRLs and GH may have actions associated with the
brooding phase of the reproductive cycle. It is unclear,
however, whether these actions are more closely aligned to
the regulation of metabolism or reproduction.
Prolactin and GH may have metabolic roles closely
associated with reproduction, ushering resources to or
away from ovarian growth and vitellogenin production. The
tPRLs and GH may release metabolites for vitellogenin
production or protect energy stores and protein from
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utilization during this period when food intake is low.
The tPRLs and GH may have actions in parental behav
ior, steroidogenesis, or maintenance of postovulatory
follicles. Possible actions in parental care include
"calling movements" to fry, reduced feeding behavior to
prevent feeding on young, and mucus production. Wedelaar
Bonga and colleagues (1984) dismissed a role for PRL in
parental care in the tilapia due to the absence of a
change in PRL cell activity. r have shown that PRL levels
are elevated in FW tilapia at the end of the brooding
period. Furthermore, as r have argued previously, large
changes in PRL release may not be necessary for PRL to
have actions. Changes in target tissue receptors and in
interacting hormones which may modulate PRL1s actions, may
change during the reproductive cycle.
r have mentioned previously that, Smith and Haley
(1988) have shown that E2 and T levels are elevated
during the brooding phase and steroidogenic postovulatory
follicles persist longer in brooding females than in
non-brooding females. The tPRLs or GH may be involved in
regulating these events. Fish PRLs including tPRLs, and
GHs have been shown to stimulate ovarian growth and ste
roidogenesis in teleosts (Singh et al., 1988; Tan et al.,
1988; Danzmann et al., 1990; Van Der Kraak et al., 1990;
Rubin and Specker, 1992).
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Clearly, more work is required before the actions of
PRL and GH during the reproductive cycle of the female
tilapia are clear. Studies should include: 1) metabolic
clearance studies for the tPRLs, GH and steroid hormones;
2) studies on the efficacy of native hormones to elicit or
maintain parental behavior and mucus production; 3) in
vitro studies on the effects of the tPRLs and GH on ste
roidogenesis from ovarian tissues, including postovulatory
follicles; 4) studies on the efficacy of the tPRL and GH
to maintain postovulatory follicle function in non-brood
ing tilapia; and 5) studies to determine whether tPRLs, GH
or steroid hormones can protect oocytes from atresia
during fasting.
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Chapter VI
CONCLUSIONS
Consistent with PRL's role in FW osmoregulation, PRL
cells of the tilapia release PRL in response to small
changes in medium osmolality that are well within the
range of plasma osmolalities observed in vivo (Nagahama et
al., 1975; Wigham et al., 1977; Grau et al., 1981, Kelly
et al., 1988). I have shown that alterations in osmotic
pressure and not osmolality are coupled to the sustained
release of the osmoregulatory hormones, tPRL1 77 and
tPRL1 88. This is the first study to describe a direct
response to osmotic pressure by a cell with a clearly
demonstrated osmotic output. Prolactin release was high
in RPD incubated for 18-20 hr in hyposmotic medium (300
mOsmolal) or medium made hyperosmotic (355 mOsmolal) with
the mernbrane-permeant molecules, urea or ethanol, but was
low in RPD incubated in medium made hyperosmotic with NaCl
or the mernbrane-impermeant molecule, mannitol. These data
suggest that it is the osmotic gradient across the cell
membrane that leads to an increase in osmotic pressure,
and not the medium osmolality per se, which accounts for
the osmoreceptivity of the tilapia PRL cell. In perifu
sion studies, tPRL1 77 and PRL1 88 release was the same from
RPD incubated in hyposmotic medium and in hyperosmotic
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medium containing urea, at 22 min after the onset of expo
sure to these media. Furthermore, release was greater in
both media compared with tPRL release from RPD incubated
in hyposmotic media made hyperosmotic with the membrane
impermeant molecule, mannitol. These data provide addi
tional evidence that the increase in tPRL release in
response to reductions in osmotic pressure is sustained.
I have shown that injections of mGnRHa elevate serum
concentrations of the tPRLs and incubation of RPD in
medium containing GnRH molecules that are native to the
tilapia, increases PRL release into the medium. These
data provide evidence that GnRH functions as a PRL
releasing factor in the tilapia, O. mossambicus. The
forms of GnRH endogenous to the tilapia stimulated the
release of the tPRLs from RPD with the following order of
potency: cGnRH-II > sGnRH > sbGnRH. This action appears
to be directly on the PRL cells. Support for this notion
comes from several lines of evidence. First, RPD are a
nearly homogeneous population of PRL cells (95-99% pure) ,
GtH and GH cells which have been shown to respond to GnRH
are not present in the RPD. Second, the range of GnRH
concentrations used in the studies were compatible with
receptor mediated action. Thirdly, cGnRH-II evoked an in
crease in [ca2+]i within 1 min of exposure and an eleva
tion in PRL release within 2 min. These data suggest that
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calcium operates as a second messenger in mediating the
effects of GnRH on PRL release.
Prolactin secretion in teleosts is thought to be
predominantly under inhibitory control, however, evidence
had suggested that a hypothalamic prolactin-releasing
factor was involved in the control of PRL secretion in
fish (reviewed by Clarke and Bern, 1980; Ball, 1981)
Barry and Grau (1986) have shown that thyrotropin
releasing hormone stimulates PRL release in the tilapia
but only following E2 preincubation. Until the present
study, thyrotropin-releasing hormone was the only hypotha
lamic factor shown to have the capability to stimulate PRL
release in the tilapia. Furthermore, the steroid hormones
E2 and T are able to potentiate the response of PRL cells
to GnRH, increasing percent release over 3 fold. The
steroids may regulate the sensitivity of the PRL cells to
GnRH stimulation durIng the reproductive cycle. Alterna
tively, GnRH molecules may regulate the sensitivity of PRL
cells to T and E2 stimulation. The potentiation of the
response of PRL cells to GnRH and thyrotropin-releasing
hormone by sex steroids supports the suggestion by Barry
and Grau (1986) that there may be a shift in the control
of PRL secretion with changes in the reproductive state of
the tilapia.
Serum concentration and pituitary content of GH and
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serum concentrations of tPRL1 77 and tPRL1 88 were elevated
in response to fasting in o. mossambicus. These data
suggest that GH and the tPRLs have roles in the regulation
of intermediary metabolism in the tilapia, o. mossambicus.
This is the first report of changes in circulating PRL
levels in response to fasting in a teleost. Furthermore,
serum concentrations of the tPRLs increased earlier in
response to fasting than GH, and thus, the tPRLs and GH
may have different roles in regulating metabolism.
Patterns of changes in serum and pituitary tPRL
levels observed during the reproductive cycle of the
female tilapia, o. mossambicus, were affected by the
salinity to which the fish were adapted, whereas GH pat
terns were not affected by salinity. The patterns for the
tPRLs and GH observed during the reproductive cycle had
characteristics that were both similar to and distinct
from patterns observed during fasting. In addition,
changes in CF and HSI observed during the brooding phase
of the reproductive cycle were similar in magnitude to
changes observed with fasting which were accompanied by
changes in serum tPRL and GH concentrations and GH pitui
tary content. These findings suggest that changes in
metabolic state may influence tPRL and GH cell activity
during the reproductive cycle although they can not ac-
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count for all the changes in serum concentrations and
pituitary content of the hormones.
Overall, serum PRL concentrations were highest in
females brooding post-yolksac larvae. Nonetheless,
changes in serum and pituitary levels with the reproduc
tive cycle appeared small and there were discrepancies
among studies as to when serum PRL concentrations in
creased during the brooding phase. The observed patterns
of serum concentrations and pituitary content of the tPRLs
do not support the notion that changes in serum concentra
tions of the tPRLs are driving parental care behaviors or
ovarian function in the female tilapia. On the other
hand, the pattern of serum and pituitary GH levels during
the reproductive cycle was consistent among three studies.
Serum GH concentrations increased during the brooding
phase of the reproductive cycle, followed by an increase
in pituitary content of the hormone. These changes in
serum and pituitary GH levels appear to be a characteris
tic component of hormone changes during the reproductive
cycle of the female tilapia and suggest that GH has func
tions intrinsic to reproduction.
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