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The effect of anthropogenic noise on relative levels of luteinizing hormone receptor and testosterone in the testes of Litoria caerulea

A thesis submitted in partial fulfillment of the requirements for a degree of Bachelor of Arts at Pomona College

Department of Biology

By

Neha Savant May 2014

 

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ABSTRACT

Studies investigating causes of global amphibian declines often focus on the ecology of

declines, but a complete understanding of declines requires an integrative perspective,

incorporating an animal's physiological response as well as ecological and behavioral. Indeed,

many of the leading causes that declines have been attributed to are likely to be perceived by

animals as stressors, leading to secretion of corticosterone (CORT), a stress-responsive hormone.

Chronic elevations of plasma CORT levels are associated with pathology. Previously, we have

shown that exposure to anthropogenic noise, a generalized environmental stressor, increased

levels of corticosterone, and decreased sperm count and sperm viability in male frogs exposed to

chronic noise. The mechanism by which this occurs likely involves interactions between the

hormone axes that govern stress responses and reproduction a successful spermatogenesis

requires secretion of testosterone (T) in the testes. In order to elucidate the factors that mediate

reproductive suppression in response to chronic stress in amphibians, we subjected male White's

treefrogs (Litoria caerulea) to anthropogenic noise and chorus noise for eight nights and

compared the relative abundance of testicular T to that of frogs presented with only chorus noise.

We fixed, sectioned, and stained testes using immunohistochemical techniques and quantified

fluorescence using Fiji. We observed a significant increase in testicular T of frogs exposed to

anthropogenic noise, suggesting that testicular T may contribute to the decrease in sperm health

and production. The portion of the endocrine pathway responsible for this pathology, however,

remains unknown.

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INTRODUCTION Global Amphibian Populations

Amphibians are excellent indicators of environmental changes and ecological problems

(Blaustein et al., 1994; Blaustein & Wake, 1995). Their naked, permeable skin makes them

vulnerable to environmental contaminants, pollution and ultraviolet radiation (Ankley et al.,

1998; Hayes et al., 2006; Licht & Grant, 1997; Sparling 2000). Due to the terrestrial and aquatic

nature of most amphibian life cycles, they can also be exposed to a variety of problems with

habitat, disease, temperature, and predation (Wassersug, 1997). Additionally, since many

amphibians live in the same geographical area almost their entire lives, they can be monitored

and studied consistently over time (Rowe et al., 2003). Ecologically, amphibians are vital.

Amphibians are often described as keystone species for moving energy, nutrients and minerals

around ecosystems and to new ecosystems (Murphy et al., 2000). Also, a number of different

terrestrial and aquatic food webs include amphibians: adults are important carnivores, while

tadpoles are vital aquatic herbivores and carnivores (Rowe et al., 2003).

Despite the ecological importance of amphibians, global populations have been in decline

since the 1970s (Sherman & Morton, 1993; Stuart et al., 2004). Amphibians comprise about 25%

of the world’s vertebrates and in Neotropical areas, where amphibian diversity is the highest,

63% of amphibian species are categorized as rapidly declining (Hoffman et al., 2010; Stuart et

al., 2004). Here in the United States, at least a third of known amphibian species are thought to

be in danger (Bury et al., 1995).

There are several causes for these amphibian declines including habitat destruction,

introduced species, pesticides, and disease (Alford & Richards, 2007; Berger et al., 1998;

Bradford, 1991; Johnson et al., 2002; Kiesecker & Blaustein, 1997; Kindermann et al., 2012;

Petranka et al., 1993; Semlitsch, 1998). The species richness of salamanders in North Carolina

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was five times higher in mature forests than in recently clearcut forests: a direct result of habitat

destruction (Petranka et al., 1993). Introduced species, like the predatory fish encroaching on

Rana mucosa habitats in the lakes of Sierra Nevada, can cause decreases in local frog

populations (Bradford, 1991). The bullfrog, a commonly introduced frog species, also has

negative impacts on native frog populations (Kiesecker & Blaustein, 1996). Pesticides and

contaminants can alter an amphibian’s endocrine system, reproductive ability, growth, behavior,

or kill amphibians directly (Alford & Richards, 2007; Hayes et al., 2002). These individual

causes, most originating from habitat degradation, are essential to understanding why amphibian

declines are occurring; however this loss is likely a result of a complex interaction of these and

other unknown causes.

Physiology Informing the Ecology

A difficult issue in addressing amphibian declines is finding the most influential

anthropogenic factors affecting amphibians (Rowe et al., 2003). As human populations continue

to increase, deforestation and habitat degradation are also increasing (Laurance et al., 2002;

Rudel et al., 2005). As a result, the external environment of amphibians is changing. Most

studies investigating the causes of amphibian decline, like the ones described above, focus on

ecological effects. However, not much is known about how these changes affect the physiology

of amphibians. By investigating how an amphibians’ physiology responds to environmental

changes, we can gain a more complete understanding about which factors may underlie declines

most and how they influence populations.

The Stress Response

Amphibians can perceive changes in the environment as stressors, and as a result,

hormonal pathways can be up regulated, down-regulated or hormonal interactions can be altered.

One of the main hormonal pathways activated by a stressor is the hypothalamic-pituitary-adrenal

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(HPA) axis (Reeder & Kramer, 2005). The HPA axis is well conserved in vertebrates, so

comparisons across vertebrate species are feasible (Denver, 2009). In the HPA axis, a stressor

stimulates the hypothalamus resulting in the release of corticotrophin-releasing factor (CRF),

which travels to the anterior pituitary gland and up-regulates the expression of pro-

opiomelanocortin (POMC), a polypeptide precursor of several hormones (Norris & Carr, 2013).

Prohormone convertase enzymes cleave POMCs to produce adrenocorticotropic hormone

(ACTH) and ß-lipotropin (Norris & Carr, 2013). ß-lipotropin is cleaved again to form ß-

endorphin (Norris & Carr, 2013). While ß-endorphin, an endogenous neurotransmitter, travels to

the neurons of the peripheral and central nervous system, ACTH moves through the blood to the

adrenal gland. The amphibian adrenal gland differs from mammalian adrenal glands because

amphibians do not have a discrete gland. Instead, adrenal tissue is closely associated with kidney

tissue and is composed mainly of a series of adrenocortical cells arranged in cords or interrenal

glands (Heatwole, 2005). In amphibians, adrenal tissue produces glucocortioids (GC), such as

corticosterone (CORT) and aldosterone, in response to ACTH. These glucocorticoids can down-

regulate several points on the HPA-axis, causing a negative feedback loop (Reeder & Kramer,

2005; Figure 1).

Catecholamines, like epinephrine and norepinephrine, are neurotransmitters that are also

important in the stress response (Olson et al., 2013). These factors are released from the brain

and adrenal tissue, and initiate several responses in the body including shutting down digestion,

increasing brain blood flow and increasing muscle vasodilation. All of these changes result in

increasing an organism’s alertness, energy, and ability to respond to the stressor (Romero &

Butler, 2007). Catecholamines are much faster at exerting their effect on the body compared to

GCs. Catecholamines and GCs are released immediately after the stimulus is perceived;

however, while catecholamines induce a response in the body immediately, getting GCs into

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circulation can take three to thirty minutes due to the steps of the axis (Romero & Butler, 2007).

The lag time observed with GCs is likely a result of the time it takes to go through the

transcription and translation needed to produce the GCs (Romero & Butler, 2007).

  .

Amphibians perceive environmental stressors in several different ways, but most studies

focus on two responses: acute and chronic. Although most studies treat these responses as either

an acute or chronic response, the response to stressors ranges on a continuum. The type of

stressor can cause different physiological and behavioral responses in vertebrates. Acute stress

Figure 1: Summary illustration of the vertebrate HPA and HPG axes and their generalized interactions. Blue arrows indicate inhibition; green arrows indicate activation. Elevated GCs have been shown to inhibit hormones highlighted in blue.  

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results in a quick up-regulation of the HPA-axis and a release of catecholamines, which increases

an organism’s ability respond to a stressor (Reeder & Kramer, 2005). In contrast, chronic stress

or a continuous state of stress causes an increase in the activation of the HPA-axis resulting in

consistently elevated circulating GCs (Fowler et al. 1991; Moore et al. 2000). However, chronic

stress is difficult to define explicitly: there is no established length of exposure that qualifies as

chronic stress because the finer responses to stress is so species specific and dependent on the

natural history of different species (Dickens & Romero, 2013).

The theory of allostatic load is another way to categorize the stress response. Allostatic

load is a general term used to describe the cost to an organism’s body to maintain stability in a

changing environment (McEwen & Wingfield, 2003). Any stressor (disease, predation, weather,

habitat modification, etc…) can inflict change and cause an organism to enter an allostatic state.

However, when the damaging effects of stress occur over longer time intervals, allostatic

overload is achieved and hormone secretion and other bodily functions can be dysregulated

(McEwen, 1998). In this way, chronic stress, as described above, and allostatic overload are

comparable terms.

Allostatic overload leads to prolonged exposure to high levels of GCs can decrease

immune function, reproductive function and even cause neuronal cell death (Brann & Mahesh,

1991; Carragher et al., 1989; Reeder & Kramer 2005; Salvante and Williams, 2003). Yet

physiological responses to allostatic overload are not always consistent. One study by Rich &

Romero (2005) showed an attenuation of CORT in birds when exposed to chronic stress, most

likely to prevent CORT from disrupting normal functions. Additionally, depending on a species’

range, their response to environmental disturbances can vary. For example, the sensitivity of the

HPA axis to stressors is usually lower in Arctic species compared to temperate species because

Arctic species are more accustomed to severe environmental conditions (Wingfield & Sapolsky,

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2003). Because the GC response is still an enigma, their role in the stress response is still being

studied in many species.

Hypothalamic-Pituitary-Gondal Axis and Spermatogenesis

The hypothalamic-pituitary-gonadal (HPG) axis is a hormonal pathway vital to the

reproduction of an organism. Like the HPA axis, the HPG axis is well conserved among

vertebrates (Maruska & Fernald, 2011). After an environmental stimulus, through the HPG axis,

the hypothalamus in response to hypothalamic neurohormones like kisspeptin, releases

gonadotrophic-releasing hormone (GnRH) to the anterior pituitary (Clarke 2011; Norris & Carr,

2013). GnRH regulates the release of the gonadotropins: follicle-stimulating hormone (FSH) and

luteinizing hormone (LH) from the pituitary (Reeder & Kramer, 2005). These two hormones

travel to the gonads and regulate the development and release of androgens (Wingfield &

Sapolsky, 2003). In females, LH and FSH stimulate the ovary to produce steroids like estradiol

from testosterone (T). In males, LH stimulates the interstitial Leydig cells to produce T (in

females, T is produced in Theca cells), while FSH stimulates spermatogenesis (Griswold 1998).

Spermatogenesis occurs in the testes and is the process by which spermatagonia

differentiate into spermatocytes, then haploid spermatids and eventually sperm (Figure 2). This

process is closely associated with Sertoli cells, a type of nurse cell that nourishes developing

sperm cells (Weinbauer et al., 2004). The Sertoli cells are located just inside the seminiferous

tubules, while Leydig cells are situated just outside the tubules (Figure 2). The Leydig cells

contain LH receptors (LHR), which bind LH and govern the secretion of T. FSH acts within the

seminiferous tubules to stimulate Leydig cell production and maturation (Haywood et al., 2003).

Thus, FSH, LH and T, are all vital for successful spermatogenesis (Weinbaer et al., 2004).

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Interactions Between the HPA and HPG axes

CORT and other hormones produced in the up-regulated HPA axis are hypothesized to

have a disruptive effect on the HPG axis and vertebrate reproductive physiology during chronic

stress (Bambino & Hsueh 1981; Brann and Mahesh, 1991; Carragher et al. , 1989; Hardy et al.,

2005; Salvante and Williams, 2003; 1981; Viau 2002). In their review of the interplay between

the HPA and HPG axes, Mastorakos et al. (2006) conclude that GCs, when chronically up-

regulated, can inhibit many points on both hormone axes (Figure 1). Chronic GC treatment,

induced by a cortisol implants, decreased plasma LH in female rats, illustrating that GC levels

alone can affect the HPG axis (Baldwin, 1979). Additionally, increased plasma GCs caused by

chronic stress can lead to decreased testicular T secretion in rats (Hardy et al., 2005; Monder et

al., 1994). These alterations in reproductive hormones can modify the reproductive abilities of

stressed organisms (Salvante and Williams, 2003; Kaiser et al., unpublished results. However,

Figure 2: Physiology of testes. Leydig cells surround seminiferous tubules, where spermatogenesis occurs. The spermatogonia mature into primary spermatocytes. Then meiosis I occurs and they divide into secondary spermatocytes and during meiosis II, the secondary spermatocytes turn into spermatids, which mature into sperm. Sertoli cells line the inner border of the tubules providing nutrients for developing sperm. Image courtesy of Sinauer Associates.  

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chronically elevated CORT does not always lead to a down-regulation in the HPG axis. Some

animals, to adapt to permanent changes in the environment or consistent inclement weather and

avoid reproductive or immune suppression, may be able to de-couple the HPA and HPG axes

(Wingfield & Sapolsky, 2003).

The HPG axis can also manipulate the HPA axis. T inhibits the HPA axis in rats,

specifically decreasing ACTH and AVP (Viau & Meany, 1996). Even with the possibility of

decoupling, interactions between the two hormone axes may lead to clues as to why stress elicits

many different responses in the body. A change in one body system draws on the body’s limited

energy supply, taking away from other systems; therefore a change in reproductive function

could influence cardiovascular, immune or neurological function. Habitat change, disease and

introduced species are few examples of stressors that can cause chronic stress and a subsequent

interaction between the HPA and HPG axes (Viau & Meany, 1996; Peterson et al., 2013).

Importance of Advertisement Calls in Male Amphibians

Amphibian reproduction, like most vertebrates, is controlled by the timed release of sex

hormones. As a result, both males and females undergo energetically taxing behavioral and

physiological changes. In frogs, females must use a large amount of their stored energy to

generate eggs during vitellogenesis (Jorgensen, 1981). In many species, males exert large

amounts of energy to call and attract mates (in some species, females call as well though with

less energy investment). During the reproductive season, males, such as Physalaemus pustulosus

and Ranidella can sustain daily calling for two to three months, which results in a six-fold

increase in energy expenditure and an increase in O2 consumption (Bucher et al., 1982;

MacNally, 1981, 1984; Wells & Taigen, 1986). Hyla versicolor has even been shown to have the

most energetically expensive call among all ectothermic vertebrates (Taigen & Wells, 1985;

Emerson & Hess, 2001). Clearly, calling is a vital component of frog behavior and physiology.

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Exogenous Noise as a Stressor

The expensive behaviors described above can be increased or suppressed by sonic

interference by exogenous sound. As human populations continue to increase, deforestation and

habitat degradation also increases (Laurance et al., 2002; Rudel et al., 2005). Therefore, more

roads and traffic encroach on the area, which then can introduce anthropogenic noise. The

addition of non-natural sound to complex habitats can cause significant changes in behavior in

frogs, as well as other vertebrates (Bayne et al., 2008; Blickley et al., 2012; Kaiser & Hammers,

2009; Kaiser et al., 2011; Wells & Taigen, 1986; Zelick & Narins, 1983).

Zelick & Narins (1983) conducted field acoustic playback experiments with

Eleutherodactylus coqui and E. portoricensis to find if periodic bursts of added natural noise (to

create sonic interference) altered the frogs’ calling behavior. They observed males suppress

vocalizations during sound bursts, and call more in the silence between bursts. Another study

added chorus calls to the environment: H. versicolor males exposed to dense chorus playbacks

tended to give calls about twice as long, but half the rate as isolated males (Wells & Taigen,

1986). Similar behavior with both natural and unnatural sounds was observed in Dendropsophus

triangulum: male vocalizations increased with both an increase in music playbacks and

motorcycle noise playbacks (Kaiser & Hammers, 2009). Another study showed that D.

microcephalus exposed to anthropogenic noise called for a shorter duration and returned to the

breeding aggregation for fewer days in comparison to control frogs (Kaiser et al., 2011). Clearly,

the acoustic environment of amphibians is important in determining amphibian behavior.

The physiological effect of anthropogenic noise on amphibian physiology, however, has

not been extensively explored. Since 2011, the Kaiser lab has been investigating the effect of

anthropogenic noise on the physiology of Litoria caerulea. At present, we have discovered that

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circulating CORT levels increases in response to chronic anthropogenic noise, suggesting that

frogs perceive such anthropogenic noise as a stressor (Kaiser, unpublished data). We have also

found that sperm viability and sperm count decrease during this same exposure to anthropogenic

noise (Kaiser, unpublished data). To further explore the role of CORT in decreasing reproductive

and immune function, we did another study: exogenous CORT was chronically applied to L.

caerulea. Though there was evidence of potential immunosupression, there was no difference in

sperm count or sperm viability (Kaiser et al., in review). Therefore, CORT does not appear to be

responsible for the observed reduction in male fertility. All these results lead us closer to the

elucidating the mechanism by which anthropogenic noise causes reproductive suppression but

more evidence needs to be collected.

Role of T and LHR in the Reproductive Suppression of Litoria caerulea T, an HPG axis end product, is a vital sex hormone necessary for successful

spermatogenesis (Steinberger, 1971; Wingfield & Sapolsky, 2003). Therefore an investigation of

testicular T distribution could give insight into the mechanism of the lowered sperm count and

sperm viability in frogs exposed to chronic anthropogenic noise. Since LH stimulates T

production, a change in the distribution of LHR in the testes could also alter the levels of T

secretion (Bambino & Hsueh, 1981).

Therefore, I measured the relative levels of T and LHR in the testes of L. caerulea

exposed to anthropogenic noise (treatment) and compared them to the levels in frogs exposed to

chorus calls (control). Since increased GCs have been shown to decrease plasma T levels in

vertebrates, I expected to see less T in the treatment frogs compared to control frogs (Hardy et al,

2005; Moore et al., 1991; Monder et al., 1994; Pickering et al., 1987; Wingfield & Sapolsky,

2003). Additionally, GCs result in a direct inhibitory effect on testicular LHR content in rats

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(Bambino & Hsueh, 1981). Therefore, although it is possible that T could decrease without a

decrease in LHR, I anticipated a similar decrease in relative LHR levels in the testes of treatment

frogs.

Immunohistochemistry In order to investigate T and LHR distribution in L. caerulea testes, T and LHR need to

be identified. Immunohistochemistry (IHC) is an increasingly relevant technique used to detect

antigens in thinly sliced tissues (Ramos-Vara, 2005). In IHC, a specific primary antibody is used

to bind antigens in the tissue, followed by a secondary antibody that attaches to the primary

antibodies. The use of both antibodies decreases the chances of non-specific staining. Secondary

antibodies often have attached labels including fluorescent reporters, enzymes and metals

(Ramos-Vara, 2005). In this experiment, I use fluorescent-labeled secondary antibodies to reveal

T and LHR distribution in the testes.

METHODS Animal Husbandry for L. caerulea

L. caerulea, native to Australia, Indonesia, and Papua New Guinea, is listed as Least

Concern by IUCN (IUCN, 2013). This species, commonly known as White’s treefrog, has also

been used in many different physiological studies and as such is an ideal model organism for this

experiment (Buttemer, 1990, Peterson et al., 2013, Pressier et al., 1999; Smith et al., 2003;

Voyles et al., 2007).

The study was completed during the North American breeding season of L. caerulea

from September to October. Twenty captive-bred adult male White’s treefrogs were obtained

from Sandfire Dragon Ranch in Bonsall, California, and individually housed in plastic tanks

(Kritter Keepers size XL, San Marcos, CA). A 10 cm PVC pipe section was included in the tank

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for enrichment along with a bowl of dechlorinated tap water. The frogs were kept at 20 to 23°C

with a 12h:12h, light:dark cycle (lights turning on at 09:00h). The University of California,

Riverside Institutional Animal Care and Use Committee, where this portion of the study was

conducted, and the Pomona College IACUC approved all methods.

Exposure to Anthropogenic Noise

The procedure described in Kaiser et al. (2011) was followed to prepare the stimulus. Ten

3-minute recordings of different automobiles were selected and using Audition (Adobe v. 2.0) to

pitch-shift each recording, 70 noise files were created. To simulate a natural habitat, a playlist of

seventy 3-min conspecific chorus-call tracks and an equivalent number of 3-min silent tracks

was shuffled and played to a total of 19 chorus control frogs. To simulate anthropogenic noise,

the 10 experimental frogs were exposed to two sources of noise: one shuffled playlist consisting

of 70 three-minute car-noise tracks and 70 three-minute silent tracks; and the other from the

previously described chorus call/silence shuffled playlist. Therefore, during the experimental

treatment, frogs were exposed to chorus noise or anthropogenic noise, neither or both. A 3rd

generation (for anthropogenic noise) and 6th generation (for chorus noise) iPod Nano (Apple

Corporation, Cupertino, CA) were used for playbacks. Playbacks were calibrated to 70dB SPL at

one minute with a sound level meter with C-weighing (RadioShack 33-2055) and amplifiers

(Pignose 7-100, Las Vegas, NV). All frogs were exposed to noise from 20:00 to 08:00 from Day

0 to Day 7 (Table 1).

Immunohistochemistry

The left testis of each frog was flash frozen in isopentene on dry ice and stored at -80°C

until needed for immunohistochemical analysis. Testis cross sections (20nm) were cut on a

cryostat (Leica CM1850 UV) and mounted. Hematoxylin & eosin (H& E) stains were performed

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on select samples immediately after sectioning the testes and freshly sectioned tissue was used

for immunohistochemistry.

The following procedure was validated in our lab for White’s treefrog. Sections were

fixed with a 3:1 acetone:ethanol solution for ten minutes and blocked with 5% donkey serum for

one hour. For localization of LHR and T, tissues were incubated with the primary antibodies,

Lutropin-choriogonadotropic hormone receptor antibody (1:1000; GenWay, San Diego, CA) or

T antibody (1:200; GeneTex, Irvine, CA) for three hours. The tissues were incubated in

AlexaFluor 568 secondary antibody (1:500; Life Technologies, Eugene, OR) and then FITC-

conjugated Concanavalin-A (1:200; Sigma-Aldrich, Milwaukee, WI) to label cell membranes.

Slides were mounted with Prolong Gold (Life Technologies, Eugene, OR).

Image & Statistical Analysis Five to ten representative images of seminiferous tubules were taken from each sample

on a florescent microscope at 100x by using a TRITC filter (emission wavelength: 572 nm) to

observe T staining. Testicular T and LHR were quantified using Fiji, an image-processing

package for ImageJ (NIH, Bethesda, MD). A box of consistent size was used to measure each

image using the “Set Measurements” function. “Integrated Density”, or the sum of intensity

values for all pixels within the boxed area, was taken as the measurement for fluorescence. The

measurements for each section were averaged and compared with sections from the opposite

treatment prepared on the same day. Using SPSS, I used a paired t-test to test for statistical

significance between the relative T in treatment and control frog testes. A statistical test for

testicular LHR levels could not be performed due to an insufficient sample size.

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Table 1: Experimental design. Blood samples collected between 12:30 and 13:30h.

RESULTS A total of 22 frog testes were sliced and stained; however, five testes were unusable due

to freezer burn. In the middle of the study, GenWay discontinued the Lutropin-

choriogonadotropic hormone receptor antibody, so we were only able to detect LHR in half the

testes. Due to scheduling complications, only seven pairs of T-stained testes and one pair of LH-

stained testes were processed on the same day and could be compared.

H & E-stained sections illustrate the anatomy of the L. caerulea testis (Figure 3). The

seminiferous tubules are clearly visible with the interstitial spaces highlighted in a lighter pink

color. The same outlined section from the H & E stain is shown after immunohistochemistry

(Figure 4). T staining (Figure 4B) is localized in the Leydig cells and sparsely scattered in the

seminiferous tubules. Notably, T staining has a distinct distribution when compared to membrane

staining (Figure 4A).

Almost all testis pairs, except for 90:77, displayed a higher T level in frogs exposed to

anthropogenic noise (Figure 5). After running a paired t-test, we found that anthropogenic noise

did have a significant effect on testicular T (t(6) = -2.832, p = 0.03, Figure 5, Figure 6).

The one pair of testes analyzed for LHR showed a decrease in LHR in the noise treatment

(Figure 7).

Group Day -5 Day 0 Day 7 Chorus-control Baseline blood sample Chorus begins Sacrifice + blood

sample Anthro-treatment Baseline blood sample Anthro + chorus

begins Sacrifice + blood sample

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A   B  

Figure 4. Membrane (A) and Testosterone (B) labeled White’s treefrog testis sections using immunohistochemistry. Testosterone is primarily localized in the Leydig cells of the testes throughout the seminiferous tubules. Images were taken at 100x with a fluorescent microscope.

Figure 3. Hematoxylin & Eosin stain of a White’s treefrog testis. The insert shows a larger version of testis anatomy: seminiferous tubules and interstitial spaces (pink outlined areas) are the most distinctive. Dark purple areas inside seminiferous tubules are maturing sperm.

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0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

91:72 84:74 68:76 90:77 87:78 89:71 86:70

Rel

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f T (p

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8 )

Testis Pairs

Chorus Control Noise

Figure 5: Quantified testosterone (T) expression in White’s treefrog testes (n=7). T expression increased in the noise treatment in most paired samples, with the exception of 90:77. Values are expressed as means ± SE. There was no significant difference between noise and chorus control treatments (t(6) =-2.832, p = 0.03).

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Figure 7: Quantified luteinizing hormone (LH) expression in White’s treefrog testes (n=1). Green represents chorus control; blue represents noise treatment. Only one pair of testis was compared. Values are expressed as means ± SE.

0

0.5

1

1.5

2

2.5

87 (chorus) 73 (noise)

Rel

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H R

ecep

tor

Abu

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Testis Pair

Figure 6: Testosterone (T) expression in White’s treefrog testes. A-C: Chorus control; D-F: Noise Treatment. A and D are shown under a FITC filter illuminating cell membranes. B and E are shown under a TRITC filter illustrating T expression. C and F are merged images of the two previous images. Images taken at 100x with a fluorescent microscope.

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DISCUSSION

The physiological effects of anthropogenic noise on amphibians, and specifically L.

caerulea, have not been categorized. When exposed to anthropogenic noise, sperm count and

sperm viability decreases while CORT increases significantly (Kaiser et al., unpublished data).

Since CORT is one end product of the HPA axis and interacts with the HPG axis, CORT was

hypothesized to be responsible for this decrease in reproductive health, but no change in sperm

was observed with application of exogenous CORT (Kaiser et al., under review). Therefore,

CORT is most likely not responsible for the observed reproductive suppression.

Testosterone was examined next. In this study, testicular T significantly increased in

frogs exposed to anthropogenic noise. This increase in T was contrary to my prediction that I

would see a decrease in testicular T. Furthermore, the observed increase in testicular T is

opposite of the findings of several studies investigating the relationship between stress, CORT,

and T (Hardy et al., 2005; Moore et al., 1991; Monder et al., 1994; Pickering et al., 1987;

Wingfield & Sapolsky, 2003). Stress, stimulated by anthropogenic noise in this study, has been

shown to both inhibit and stimulate T production depending on the situation and species. In

newts, lizards and rats, stress can inhibit T production, but in humans and hamsters stress can

stimulate T production (Moore et al., 1991; Remes et al., 1985; Retana-Marquez et al., 2003;

Sapolsky et al., 1986). However, most studies looking at the relationship between stress and T

examine circulating T, not testicular T (Weinbaur et al., 2004).

Testicular T is necessary for spermatogenesis and a direct relationship exists between

intratesticular T concentration and sperm production in rats and most vertebrates (Steinberger,

1971; Zirkin et al., 1989). Therefore, the change in testicular T seen in this study is likely to be

involved in affecting sperm health in L. caerulea. One scenario is that the over abundance of

intratesticular T inhibits spermatogenesis. Basu (1968) observed this phenomenon when he

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inserted a T pellet in male Rana hexadactyla and observed spermatogenetic suppression.

Handelsman et al. (1992) performed a similar experiment in humans and saw the same effect:

spermatogenesis success decreased. Furthermore, Weinbauer et al. (2004) also found that high

intratesticular T can exert an inhibitory effect on sperm development in rats. However, the effect

of increased T on spermatogenesis varies in amphibians (Rastogi, 1976). Spermatogenic

stimulation was observed in Bufo fowleri and B. arenarum, no effect was observed in R. pipens

and B. americanus, and inhibition was observed in R. hexadactyla and R. pipens (Basu, 1968;

Basu and Nandi, 1965; Blair, 1946; Penhos, 1956; Puckett, 1939). Clearly, the relationship

between T and spermatogenesis is species specific. Therefore, an increase in testicular T in L.

caerulea could mean a decrease in spermatogenesis for this species specifically. However, this

inhibition by T explains the observed decrease in sperm count, not the decrease in sperm

viability: processes beyond spermatogenesis are necessary to finish creating viable sperm.

As mentioned before, most studies looking at the relationship between spermatogenesis

and T do so by examining circulating T (Weinbaur et al., 2004). To gain more insight into the

role of T in the observed decrease in sperm health, the Kaiser lab measured circulating T in the

same frogs analyzed in this study. Opposite to the trend of testicular T, circulating T significantly

decreased in frogs exposed to anthropogenic noise (t=2.319 df = 9,4, p=0.041, unpublished data).

Unfortunately, there is a dearth of studies investigating the relationship between circulating and

testicular T and so it is unknown whether they parallel each other. More studies are needed to

elucidate this connection.

One suggestion for the opposing levels of T is that the effects of anthropogenic noise,

including increased CORT, are somehow influencing the release of T from the testes into the

bloodstream. Under normal conditions, after LH travels to the Leydig cells and T is synthesized,

some T enters the bloodstream while some T stays in the testes and is directed into seminiferous

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tubules for spermatogenesis (Ogielska & Bartmanska, 2009). Several enzymes are necessary for

steroid production (Figure 8). A few important enzymes include 5- α reductase that catalyzes T

to dihydrotestosterone (DHT), aromatase that catalyzes T to estradiol and 17- α hydroxylase, an

early enzyme vital to steroid production, that catalyzes pregnenolone to 17- α

hydroxypregnenalone and progesterone to 17- α hydroxyprogestoerone (Hadley & Levine,

2006). A change in any of these enzymes could lead to differential expression of T. Since

transcription factors (TF) are one regulator of enzymes, a change in TFs could affect enzymes

involved in steroid production (Latchman, 1993). Additionally, stress has been linked to TF

regulation in the inflammatory response (DeZawaan-McCabe, 2013). In the same vein, heat

shock proteins (HSP), often involved in the folding and unfolding of other proteins, could control

enzyme activity (Santoro, 2000). Heat shock and other types of stressors have been shown to

induce HSPs (Santoro, 2000). Thus, we hypothesize that stress can change enzyme activity,

regulated by TFs or HSPs, and subsequently affect the release of T from testes.

Additionally, LH is an important regulator of T production. Although I stained for

testicular LHRs, I could not draw conclusions about the relative abundance of LH with a sample

size of one. Therefore with a further analysis of LHR, we could determine if the increase in T is

associated with a change in LHR.

Further analysis of seminiferous tubule sizes would also be useful to understand how

stress affects the testes. In a brief survey of the sectioned testes, I did not see a difference in

seminiferous tubule size. However, if one treatment had smaller tubules, more tubules would be

captured in one image, thus increasing the estimate of labeled T in the testes. Since tubule size

may influence the IHC quantification method, the next step in this study should explore the

difference in tubule size between control and noise treated frogs.

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The challenge hypothesis, put forward by Wingfield et al. (1990), states that T level is not

always indicative of an organism’s reproductive state. Instead, T secretion and an organism’s

response to environmental cues can depend on a species’ breeding strategy, degree of parental

care and traditional social interactions (Wingfield et al., 1990). Therefore in the analysis of these

results we also have to ask if T is a suitable proxy of reproductive health in this specific species.

Figure 8: Steroid pathways. Vital enzymes could be controlled by TFs and HSPs during chronic stress and influence the release of testosterone from the testes. Important enzymes include 5- α reductase, aromatase, and 17- α hydroxylase.

Savant 23

Overall, further research is necessary to relate chronic environmental stress to amphibian

declines, and more specifically to understand how stressors and T affect sperm health. Frogs are

clearly experiencing allostatic overload from anthropogenic noise and with the findings of this

study, we are one step closer to deriving the mechanism of allostatic overload. More research on

the interaction between spermatogenesis and T, and testicular T and circulating T would shed

light on the results found in the Kaiser lab. My results show a significant increase in testicular T

after exposure to anthropogenic noise, which is correlated to reproductive suppression in male L.

caerulea. Undoubtedly, equilibrium in amphibians’ acoustic environment is vital for their

continued success, but increasing habitat modification disturbs the established equilibrium.

Anthropogenic noise is just one stressor, but the physiological effects of noise are generalizable

to other types of stressors too. If we can understand the mechanism by which anthropogenic

noise affects amphibian reproduction, we can relate these stressors to global amphibian declines.

AWCKNOWLEDGEMENTS

First and foremost, I would like to thank my advisor, Dr. Kristine Kaiser, for her

guidance patience, and humor throughout this entire process. I’d also like to thank Dr. Nina

Karnovsky, my academic advisor, for introducing me to Dr. Kaiser and encouraging me to

follow my passions. I’d also like to thank Cassandra Owen, Jon Feingold, Jessica Hernandez,

and Taylor Beckwith-Ferguson for being awesome fellow lab toadies and my brother for helping

me edit my work. Chris Campbell for his guidance on Fiji, Sara Olson for help with the

fluorescence microscope, Kathryn McGovern for IHC advice, and Mark Sbertole for being the

master of the vivarium. And lastly, this thesis project would not have been possible without

generous funding from the Pomona College Biology Department.

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