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425
Journal of Andrology, Vol. 10, No. 6, November/December, 1989
Copyright © American Society of Andrology
Ultrastructural Studies on the Development of theBlood-Epididymis Barrier in Immature Rats
ASHOK AGARWAL AND ANITA P. HOFFER
The development of the blood-epididymis barrier inimmature rats (8, 11, 14, 18, and 21 days old) wasexamined with an electron microscope using lanthanumnitrate as an electron dense tracer. A gradual increasein the development of the blood-epididymis barrier wasnoted with age. On Day 8, lanthanum was frequentlydetected in both the intercellular spaces and the lumen.On day 14, no lanthanum penetration into the lumenwas observed in 75% of the junctions in the caput, 40.3%
in corpus, and 30% in cauda epididymidis. On Day 18,only 7.5%, 9%, and 15%, of the junctions in the caput,corpus, and cauda epididymidis, respectively, remainedpermeable to lanthanum. No lanthanum was observedin the lumen of any tubules in the 21-day-old ratepididymis. These findings indicate that the postnataldevelopment of the blood-epididymis barrier is gradual,and that its formation is virtually completed by Day21. As with adult rats, the zonula occludens is theultimate structural component of the blood-epididymisbarrier in immature rats (Agarwal and Hoffer, 1985).
Key words: Blood-epididymis barrier, ultrastructure,immature.
J Androl 1989;10:425-431
Supported by a grant from the Rockefeller Foundation, New
York, NY.
Reprint requests: Dr. Ashok Agarwal, Brigham AndrologyLaboratory, Medical Research Building, Rm. 413, Brigham &
Women’s Hospital, 75 Francis Street, Boston, MA 02115.
Received for publication March 18, 1988; revised April 4, 1989;
accepted for publication May 26, 1989.
From the Harvard Program in Urology andBrigham Andrology Laboratory, Harvard
Medical School and Brigham & Women’sHospital, Boston, Massachusetts
The presence of a blood-tissue barrier restricting
movement of certain substances (e.g. protein, amino
acids, ions) in some body organs has been known
for many years (Waites and Setchell, 1969). The most
important examples are the blood-brain (Reese and
Karnovsky, 1967), blood-thymus (Raviola and
Karnovsky, 1972) and blood-testis (Fawcett et al,
1970; Dym and Fawcett, 1970) barriers. Few
ultrastructural studies on the blood-epididymis
barrier have been published to date (Howards et
al, 1976; Hoffer and Hinton, 1984). In the rat
epididymis, peritubular myoid cell layers, elaboratetight junctions (occluding junction of zonula
occludens), and desmosomes (zonula adhaerens)
serve as a barrier between luminal fluid and blood
capillaries. The epididymal-tight junctions are highly
developed among other epithelial cell contacts
studied. They form a continuous zonule, or gasket,
around the cell, sealing the spaces between the
epithelial cells so that the luminal space and the
intercellular spaces become separate physiological
I
Fig. 1. Electron micrograph of the epididymal epithelium in
the caput of an 8-day-old rat intravascularly perfused with
collidine-buffered glutaraldehyde containing 2% lanthanum
nitrate. Electron-dense tracer is freely distributed along the basal
lamina (not shown) and in the intercellular compartment near
the base of the principal cells.
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- .
426 Journal of Andrology . November/December 1989 Vol. i
compartments (Friend and Gilula, 1972). Zonula
occludens at the apicolateral surface of the
epididymal epithelium serve as the ultimate
structural component of the rat blood-epididymis
barrier. The flow of intravascularly perfused
lanthanum is not significantly impeded by the
vascular endothelium, peritubular myoid layer, or
other lateral cell surface specializations (Hoffer and
Hinton, 1984).
The present investigation studied for the first
time, the development of the blood-epididymis
barrier in immature rats to determine the exact
morphological location of the structural component
of the blood-epididymis barrier in the head, body,
and tail of the developing epididymis by using an
electron-dense tracer, lanthanum nitrate.
Materials and Methods
Immature Sprague-Dawley rats (Charles RiverLaboratories, Wilmington, MA) 8, 11, 14, 18, and 21 daysold were used. The animals were maintained in accordancewith the guidelines of the Committee on Animals of theHarvard Medical School and those prepared by theCommittee on Care and Use of Animal Resources,National Research Council (DHEW Publication No. (NIH)78-23, revised 1978). Immature rat testes were fixed byantegrade perfusion through the thoracic aorta using 5%
glutaraldehyde with 0.16 M collidine (pH 7.4) containing
2% lanthanum nitrate according to previous methods(Hoffer and Hinton, 1984); except that 2.5 g/100 ml ofpolyvinylpyrollidone (PVP) were dissolved in the collidinebuffer containing lanthanum nitrate by stirring. The pH
of the final fixative (5% glutaraldehyde buffered with 0.16
M collidine containing a final concentration of 2%
lanthanum nitrate and 2.5% PVP) was 6.9. This solution
Fig. 2. Electron micrograph showing
the cauda epididymidis of an 8-day-oldrat. Lanthanum particles can be seen in
the intercellular space near the apical
surface of the principal cells. The absence
of tight junctions at this time intervalallows the lanthanum to penetrate into
the tubular lumen.
1
.
“Jo. 6 BLOOD-EPIDIDYMIS BARRIER IN IMMATURE RATS . Agarwal and Hoffer 427
was routinely filtered through a Buchner funnel(Whatman No. 1) before use. Following a brief rinse incollidine buffer, tissue blocks from the caput, corpus, andcauda were postosmicated and stained en bloc with uranylacetate (Terzakis, 1968) to enhance the contrast ofmembranes and to reveal their trilaminar structure. Afteren bloc staining, the blocks were dehydrated in increasingconcentrations of acetone and embedded in araldite byroutine methods. In some cases, the same lanthanum
concentration was maintained in all subsequent sectionsup to and including the osmium tetroxide; in others,lanthanum was omitted from the immersion solutions to
confirm that the observed path of tracer was causedexclusively by extravasated lanthanum and not by random
diffusion of the tracer from the immersion solutions intothe interstitium.
Sections showing silver to pale-gold interference colors
were cut with a diamond knife on a Sorval MT 6000
microtome. The sections were stained twice with uranylacetate (Watson, 1958) and lead citrate (Venable andCoggeshall, 1965). A Philips 200 or a Jeol lOOs electron
microscope was used to examine the sections. Thepercentage of patent junctions was calculated as follows:
o/p S .o aen -tight junction with lanthanum leaks X 100
Junctions - total number of junctions
A total of 1500 junctions from sections of immature
rat epididymis were counted under a transmission electronmicroscope. In each group, 300 junctions were observed.Chi-square tests were used to compare the percentage
of patency between different age groups (8, 11, 14, 18,and 21 days) and regions of the epididymis (caput, corpus,
and cauda). The null hypothesis was rejected if the chi-square value was � 6.6349 (DF = 1). P values less than
0.05 were considered to be significantly different.
Results
All intercellular contacts between epididymal
epithelial cells, capillary endothelial cells, and
peritubular myoid cells were considered as possible
sites of the blood-epididymis barrier. Each site was
analyzed in the caput, corpus, and cauda epididymidis
to determine whether any variations in the
permeability of the barrier could be detected along
the epididymal duct.
Days 8 and 11
On Day 8, lanthanum was detected in the
intercellular spaces leading to the lumen. The
presence of lanthanum in the basal lamina and in
the intercellular spaces near the apical and the basal
surfaces of the principal cells was observed (Figs.
1 and 2). A paucity of tight junctions, at this time,
allowed the lanthanum to penetrate into the tubular
lumen. Sixty-three percent of the junctions were
patent in the caput, 46% in the corpus, and 32%
Fig. 3. Electron micrograph of the apical cytoplasm of principal
cells in the cauda epididymidis of an 11-day-old rat showing
lanthanum particles in the lumen (arrows).
in the cauda. The most significant decrease in the
percentage of patent junctions was in the caput
region, where it fell from 65% on Day 8 to 3% on
Day 21.
There were no histological differences between
the epididymis of 8 and li-day-old rats. On day
11, 60% of the caput junctions, 36% of the corpus
junctions, and 43% of the cauda junctions were
patent and allowed the passage of lanthanum
particles in the lumen (Fig. 3). Except in the corpus,
there was no signficant difference in the percentage
of patency between 8 and li-day-old rats.
Day 14
In the epididymis of 14-day-old rats, light
microscopic observations of sections from the three
regions revealed an increase in tubular diameter, as
well as an increase in epithelial cell height. Mitotic
division was observed in some of the epithelial cell
428 Journal of Andrology . November/December 1989 Vol. l(
nuclei. The density of cells and fibers in the
intertubular connective tissue had increased, and
tubules were more compactly arranged than in 8-
and li-day-old rats.
In 14-day-old rats, a significant decrease in the
number of patent junctions was observed in the
caput; the corpus and cauda did not reveal any
change. The lanthanum particles were prevented
from reaching the lumen by multiple points of fusion
between the outer leaflets of opposing cell
membranes (Figs. 4 and 5).
Halo cells were first observed at this time. A few
round mitochondria, vesicles, and ribosomes were
present in the cytoplasm. Cytoplasmic processes
extended between the adjacent epithelial cells, but
no junctional complexes were seen between halo
cells and columnar cells.
Day 18 and 21
In the principal cells, the chromatin appeared
diffused as euchromatin in the nucleus. Endoplasmic
reticulum was smooth in the apical cytoplasm, but
granular in the basal region of the cell. Lysosomes
appeared as dense membrane bound sacs. The most
significant change during the third postnatal week
was the appearance of narrow cells in all the three
segments of the epididymis. Basal cells were not
observed. The narrow cells had various features that
set them apart from the rest of the epididymal
epithelium. These cells were clearly identifiable in
light microscopic preparations by their shape and
intense toluidine blue staining. Other components
of the narrow cells included endoplasmic reticulum
consisting of short elements, multi-vesicular bodies,
dense lysosomes, and a small golgi apparatus in the
Figs. 4 and 5. In the caput epididymidis, a signficant decrease in the number of patent junctions is first observed at 14 days
after birth. These electron micrographs of the epididymal epithelium at Day 14 show a tight junction (Fig. 4) and a patent junction
(Fig. 5) in the caput and corpus respectively. Fig. 4: In the junction between the caput epithelial cells, the lanthanum is prevented
from reaching the lumen by multiple points of fusion (arrows) between the outer leaflets of opposing cell membranes. Fig. 5: The
junction between corpus principal cells is still patent and lanthanum can be observed as it extends all the way to the lumen.
a’
No.6 BLOOD-EPIDIDYMIS BARRIER IN IMMATURE RATS . Agarwal and Hoffer 429
Fig. 6. Electron micrograph of the peritubular myoid layer
and basal lamina of the caput epididymidis of the 21-day-oldrat. Extravasated particles of tracer are observed adjacent to
the basal lamina of the epididymal epithelium and appear to
encounter little resistance along the basal and mid portions of
the lateral cell surfaces.
supranuclear cytoplasm. The nucleus was long, and
the chromatin appeared dense compared to that of
surrounding cells.
There was a signficant decrease in the percentage
of patent junctions between 8- and i8-day-old rats,
as well as between 8- and 2i-day-old rats. In the
caput and corpus of 18-day-old rats, about 92% of
the junctions became impermeable to lanthanum
passage (Figs. 6 and 7). In 2i-day-old rats, 97% of
the junctions in the caput and corpus, and 93% in
the cauda became impermeable to lanthanum
passage. In the caput, corpus, and cauda of 2i-day-
old rats, the percentage of patency in tight junctions
did not differ. There was, however, a significant
difference in the percentage of patency between the
caput, corpus, and cauda of i8 and 2i-day-old rats.
The development of the blood-epididymis barrier
was virtually completed by Day 21 (Fig. 8).
Fig. 7. Electron micrograph of the corpus epididymidis of 21-
day-old rat intravascularly perfused with lanthanum. The
passage of tracer particles in the intercellular spaces is obstructedby the zonula occludentes; 97% of the occluding junctions areimpermeable to lanthanum at this time.
Extravasated particles of tracers were observed
adjacent to the basal lamina of the epididymal
epithelium. The particles appeared to encounter little
resistance along the basal and mid portions of the
lateral cell surfaces.
Discussion
The junctional complex is the most elaborate
contact zone between mammalian epithelial cells. It
includes the zonula occludens (tight junction), zonula
adhaerens (intermediate junction), and macula
adhaerens (desmosomes). The junctional complex,
in particular, zonula occludens, is found in epithelia,
which restrict interchange of large molecules
between extracellular and intercellular space (Friend
and Gilula, 1972; Howards et al, 1976; Turner et
al, i980). The zonula occludens of the epididymis
is one of the best developed tight junctions anywhere
8 II 4 IS 21 8 II 14 8 21 8 II 4 lB 21
[ CAPUT I I CORPUS 1 [ CAUDA J
AGE IN DAYS
430 Journal of Andrology . November/December 1989 Vol. 10
FREQUENCY OF EPITHELIAL TIGHT JUNCTIONSIN IMMATURE RAT EPIDIDYMIS
Fig. 8. Frequency of epithelial tight junctions in immaturerat epididymis, ‘significantly different from 8-day interval at
P < 0.01, 1’not significantly different from 8-day interval at P
<0.01, ‘Patent junctions =
Occluding junctions with lanthanum leaks X 100
total number of junctions
in mammalian tissues (Friend and Gilula, 1972). The
ultimate site of the blood-epididymis barrier differs
from the blood-tissue barriers of other organs, such
as the thymus (Raviola and Karnovsky, 1972), the
brain (Reese and Karnovsky, 1967), and the testis
(Fawcett et al, 1970; Dym and Fawcett, i970). In
the epididymis, the zonula occludens near the
luminal surface of the epididymal epithelium are
exclusively responsible for the maintenance of the
blood-epididymis barrier, since neither the capillary
endothelium nor the peritubular myoid layer
significantly impedes the flow of tracers towards
the epididymal lumen (Hoffer and Hinton, i984).
Although ultrastructural studies of the blood-
epididymis barrier in adult rats have been reported
earlier from this laboratory (Hoffer and Hinton,
1984), the development 0f the blood-epididymis
barrier in the mammalian epididymis is described
here for the first time.
The results of the present study indicate that the
development of the blood-epididymis barrier is
virtually complete by the end of the third post-natal
week. Even though factors controlling the overall
completion of the blood-epididymis barrier by this
time are not completely understood, it is noteworthy
that the blood-epididymis barrier is completed at
approximately the same time as the blood-testis
barrier. Vitale et a! (1973) have demonstrated that
in the rat testes, the blood-testis barrier is established
between post-natal Days 16 and i9 in close temporal
correlation with the appearance of junctional
complexes between Sertoli cells, the onset of fluid
secretion by the seminiferous epithelium, the
stratification of germinal epithelium, and the
development of a lumen in the seminiferous tubule.
Using acridine dyes, Kormano (i967) found that the
permeability barrier in testes develops gradually
during the first 20 post-natal days. During the third
post-natal week there is a steep rise in serum
gonadotrophins and testosterone (Miyachi et al,
1973; Swerdloff et al, 1971), epididymal secretion
of glycerylphosphorylcholine, sialic acid and alkaline
phosphatase (Goyal and Dhingra, 1974; Setty and
Jehan, i977), and ABP in the principal cells of the
caput epididymidis (Hansson et al, i974; White et
al, 1982). These changes reflect the early activation
of pituitary and Leydig cells, which may play a role
in initiating sexual maturation and increased
impermeability of most junctions throughout the
epididymis by Day 2i. Of interest is the observation
that many of the intercellular junctions in the corpus
and cauda are already tight by post-natal Day 8.
In the rat brain, the blood-brain barrier to protein
develops early in fetal life and is well-established
by birth (Olsson et al, 1968). The exact time when
most (-2/3) intercellular junctions in the cauda
epididymidis first became impermeable to
lanthanum is not known, since the development of
the blood-epididymis barrier in animals less than 8-
days-old has not been examined to date.
Another intriguing observation is that differences
in barrier development rate between epididymal
zones can be noted as early as the first 2 post-natal
weeks. In fact, by post-natal Day 8, the proportion
of intercellular junctions that are permeable to
lanthanum in the caput, corpus, and cauda varies
from approximately 2/3 to i/2 to 1/3, respectively.
By week 2, there is a disproportionately large drop
in the number of patent junctions in the caput,
compared to the more distal regions of the
epididymal duct. Declines of comparable magnitude
do not take place until Day 18 in the corpus and
cauda. Early maturation of the blood-epididymis
barrier in the caput is consistent with the
observation of Sun and Flickinger (i979) that
histological maturation occurs earlier in the proximal
regions of the epididymis than in the distal ones.
The reasons for the initial differences in maturity
and the subsequent differential rate of blood-
epididymis barrier development, however, are not
clear. In the adult rat, maintenance of the initial
segment requires more intraluminal testosterone
No.6 BLOOD-EPIDIDYMIS BARRIER IN IMMATURE RATS . Agarwal and Hoffer 431
than the cauda (Fawcett and Hoffer, 1979), but
whether this is relevant to the neonatal epididymis
is not known. The greater immaturity of the caput
blood-epididymis barrier on Day 8, relative to the
distal gential duct, is consistent with this possibility.
It is also noteworthy that during post-natal days
0-14, there is a steady decline of all endogenous
steroids except testosterone and androstanediol,
whereas, during days 10-15, the testicular level of
androstanediol increases significantly until it
emerges as the predominant endogenous androgen
of testes in rats 15 days and older (Tappanien et
al, 1984). It is tempting to speculate that blood-
epididymis barrier development in the prepubertal
epididymis, especially the caput, has a specific need
for androstanediol or some other unknown factor(s),
and that it cannot mature until this factor(s) is
available around the second post-natal week. An
alternate hypothesis is that, since the lumen of the
seminiferous tubules is formed more or less
concurrently with the establishment of the blood-
epididymis barrier, the intraluminal presence of
testicular secretory products in the epididymis may
be required for completion of blood-epididymis
barrier development.
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