Androgen receptor localization inthe Haplochromis burtoni male brain

Rosa Navarro

May 2002

Androgen receptor localization in theHaplochromis burtoni male brain

An Honors Thesis submitted tothe Department of Biological Sciences

in partial fulfillment of the Honors ProgramSTANFORD UNIVERSITY

byRosa Navarro

May 2002

Androgen receptor localization in theHaplochromis burtoni male brain

byRosa Navarro

Approved for submittal to the Department of Biological Sciencesfor consideration of granting graduation with honors:

Research Sponsor:Russell Fernald, PhD _____________________ Date _________

Second Reader:Robert Sapolsky, PhD _____________________ Date _________

i

ACKNOWLEDGEMENTS

I would like to give a big thanks to Mike Vagell for being the best mentor I could

have asked for. I really appreciate all the countless hours he spent training and helping

me; since day one he has been there to help guide me through the frustrations and

accomplishments of the project. His endless patience, support and understanding of my

busy schedule outside of the lab meant a lot to me as well, and was vital in the

completion of this project. The support and mentorship Mike provided me exceeded that

of which was required for the project, and I feel very fortunate to have found in him not

only a great mentor but also a great friend. I would also like to give thanks to Professor

Russ Fernald for giving me the opportunity to join his lab, allowing me to work with such

a wonderful group of people, and providing me with one of the most challenging and

meaningful experiences I’ve had while at Stanford. Russ has also been a great mentor

(not to mention one of the coolest professors I’ve ever known) and I really appreciate the

encouragement and guidance he has given me since joining his lab. Thanks also to the

rest of the Fernald lab members, for creating such a warm and friendly environment- and,

of course, for supplying the degree of wackiness that makes the Fernald lab so unique.

Also, thanks to Professor Robert Sapolsky for taking the time out of his busy schedule to

be my second reader. I would also like to give a special thanks to my parents, who may

not have always understood all the details of my research, but who have always

supported and encouraged me. And finally, thanks to the Howard Hughes Medical

Institute and the Undergraduate Research Opportunity program for their help in funding

this research project.

ii

TABLE OF CONTENTS

Acknowledgement............................................................................................................... i

Table of Contents............................................................................................................... ii

List of Tables and Figures................................................................................................. iii

Abstract.............................................................................................................................. iv

Introduction......................................................................................................................... 1

Materials and Methods........................................................................................................ 3

Results................................................................................................................................. 7

Discussion........................................................................................................................…9

Figures................................................................................................…………………... 15

References.........................................................................…............................……........ 34

Appendix: Immunocytochemistry protocol.............................................………….....… 35

iii

LIST OF TABLES AND FIGURES

Table 1: AR types and antibodies used..........................................................................5

Figure 1: Territorial vs. Nonterritorial H. burtoni males.................................................15

Figure 2: Steroid-receptor activation...............................................................................15

Figure 3: Model for GnRH-1 regulation..........................................................................16

Figure 4: Drawing of H. burtoni brain.............................................................................16

Figure 5: Schematic illustration of androgen receptor domains.................................….17

Figure 6: Diagram of immunolocalization method………..............................................17

Figure 7: Low power micrograph of H. burtoni brain, showing relative location in brainof AR localization shown in Figures 8-9B.................................................….18

Figure 8: Hb AR-α staining (with W57 Ab) and negative control of forebrain andpreoptic area (10x)...........................................................................................21

Figure 8B: Hb AR-α staining (with W57 Ab) and negative control of forebrain andpreoptic area (40x)...........................................................................................22

Figure 9: Hb AR-β staining (with tAR Ab) and negative control of brain (10x)............23

Figure 9B: Hb AR-β staining (with tAR Ab) and negative control of brain (40x) ..........23

Figure 10: Hb AR-α staining (with W275 Ab) of testes at various concentrations andTSA times........................................................................................................24

Figure 11: Hb AR-α receptor localization in the testes (with W275 Ab).........................25

Table 2-4: Measurements of mean pixel intensities and standard deviations for primaryAb stained regions and negative controls……………………………………26

Figures 12-14: Graphs of differences in mean pixel intensities and standard deviationsobserved for primary Ab stained regions and negative controls…………….29

Tables 5-7: T-test statistical analysis for significance of primary antibody stainingobserved in tissue …………………………………….…….……….……...32

iv

ABSTRACT

The African cichlid fish Haplochromis burtoni has proven useful for studyinghow social interactions are turned into functional changes in the brain. Males of thisspecies exist in either of two behavioral states: territorial or nonterritorial. An individualH. burtoni male is able to change from one social status state to the other and back againdepending on social interactions with other fish. Preoptic gonadotropin releasinghormone (GnRH-I) neurons in the male H. burtoni have been shown to integrate socialcues and neuroendocrine information. Androgens are an integral part of the homeostaticregulation of preoptic GnRH-I neurons, controlling how much GnRH-I is released to thepituitary. There are two types of androgen receptors in H. burtoni that may be involved inthis regulation, the alpha form and a beta form (Hb AR-α and Hb AR-β). To discoverhow androgens interact with behavior to regulate preoptic GnRH-I neurons, it isimportant to discover where the androgen receptors are located relative to preopticGnRH-I neurons. The expression of androgen receptor protein was assessed usingimmunocytochemistry. Two chicken polyclonal antibodies (W57 and W275) made fromdistinct H. burtoni androgen receptor alpha peptides were used to localize Hb AR-α. Athird primary antibody (tAR) raised against the first 200 amino acids of the rainbow troutandrogen receptor was used to localize Hb AR-β. Distinct staining observed with theW57 primary antibody (peptide made against amino acid 57-74 starting from the aminoterminus) suggests that Hb AR-α is present in the ventral forebrain and the preopticregion of the H. burtoni territorial male brain. Hb AR-α staining was also observed intestis tissue when using the W275 primary antibody (peptide made against amino acid275-294 starting from the amino terminus). The tAR antibody was observed to stain forHb AR-β in the octavolateral nucleus region, which is below the facial lobe and abovethe medulla in mid-sagittal sections. This is an area which is important in lateral linemechanoreception. Androgen receptors in this region may be significant in regulating thesensitivity of H. burtoni territorial males to attacks from other fish, thereby playing animportant role in the transitioning and maintenance of their social status. Further studieswill include double label staining for androgen receptors and GnRH-I. This will be usefulin determining whether the androgen receptors are co-expressed in the same cells asGnRH-I. Discovering where androgen receptors are located in the brain will addsignificantly to our understanding of how hormonal information is integrated with socialinformation at preoptic GnRH-I neurons.

1

INTRODUCTION

The African cichlid fish Haplochromis burtoni has proven useful for studying how

social interactions cause functional changes in the brain. Males of this species, which are

found along the edges of Lake Tanganyika in Africa, exist in either of two behavioral

states: territorial or nonterritorial (Figure 1). Territorial males are brightly colored with

distinct markings, are reproductively mature, and defend their territories with aggressive

behavior. However, the majority of males in a population are nonterritorial, lacking

distinct sexually dimorphic markings, are not reproductively mature, and flee when

challenged by a dominant male. Nonterritorial males look very much like the females, and

tend to spend most of their time schooling with females.

Interestingly, an individual H. burtoni male is able to change from one social status

state to the other and back again. This phenomenon is controlled by social interactions

with other fish. In the absence of other males, or in the presence of much smaller males,

nonterritorial males acquire the phenotype of territorial males. Large nonterritorial males

can also displace smaller territorial males, who then take on the attributes of nonterritorial

males over the course of several weeks. Therefore, the process of acquiring the behavioral

and physical characteristics of either territorial or nonterritorial status is a reversible

process. In contrast, females of this species do not display socially-mediated changes in

behavior or appearance (Fernald 1995).

Preoptic gonadotropin releasing hormone (GnRH-I) neurons in the male teleost

Haplochromis burtoni have been shown to integrate social cues and neuroendocrine

information. Androgens, steroid hormones that regulate cell function through the control

of gene expression (Figure 2), play an important role in the regulation of preoptic GnRH-I

2

neurons. Androgens help control how much GnRH-I is released into the pituitary, which

stimulates the gonadotroph cells to release gonadotropins into the bloodstream. These

gonadotropins then signal the gonads to release more testosterone (an androgen) which

travels back to the brain turning off the GnRH-I neurons through interaction with

androgen receptors (Figures 3 and 4). However, the exact mechanism through which

androgens regulate GnRH-I expression is not known.

In contrast to terrestrial vertebrates, fish have two androgen receptor genes. These

two types of androgen receptors, an alpha form (Hb AR-α) and a beta form (Hb AR-β), in

H. burtoni may be involved in this negative feedback control of androgen secretion. To

discover how androgens interact with behavior to regulate preoptic GnRH-I neurons in H.

burtoni males, it is important to first determine where the androgen receptors are located

relative to preoptic GnRH-I neurons.

This research is important because it will help provide a foundation for the

molecular understanding of how social status and neuroendocrine feedback are integrated

at a specific site to control a particular gene. Since androgens can regulate the abundance

of GnRH-I mRNA, which is in the preoptic area, at least one of the following hypotheses

must be correct: either androgens activate receptors within preoptic neurons coexpressing

GnRH-I, or the androgen receptors are in neurons that do not coexpress GnRH-I but are in

the same area or they are located in neurons in a different area which then project to

GnRH-I containing neurons. It is important to note that these hypotheses are not mutually

exclusive.

3

MATERIALS AND METHODS

Fish Care

H. burtoni used in this experiment were progeny of a laboratory-bred stock

derived from fish caught in Lake Tanganyika, Africa. The fish were kept in tanks

resembling their natural environment. Water temperature was kept at 29°C and pH at 8.0.

Full spectrum illumination operated on a 12-hr light/ 12-hr dark cycle. For identification

purposes some males were individually tagged by securing plastic tags with colored

beads through the dorsal muscle. All experiments were done in accordance with IACUC

protocols.

Tissue Preparation

Territorial social status of male fish was determined by observation of

characteristic territorial behavior and of defining physical characteristics on the day of

sacrifice. Males were sacrificed by rapid cervical transection of the spinal cord. Brains

were removed and fixed in 4% paraformaldehyde at 4°C overnight, saturated in 30%

sucrose solution, mounted in OCT and then sectioned at –20°C with a cryostat. Frozen

sections (20 µm) were thaw-mounted on precoated (Fisher Superfrost) slides. The tissue

was stored at –80°C.

Antibody Staining

The expression of androgen receptor protein was assessed using

immunocytochemistry. This highly sensitive procedure allows observation of where the

receptors are located in the tissue by amplification of the signal. Two polyclonal

4

antibodies (made in chicken against two different H. burtoni androgen receptor alpha

peptides, Fig. 5 ) to the androgen receptor alpha were used. Richard White, a former

postdoc in the Fernald Lab, was responsible for all the AR-α antibody preparation work

(Table 1). Territorial male fish tissue was used for these experiments, because they have

larger testes and have greater levels of testosterone (Fraley and Fernald, 1982; Soma et al.,

1986), so one can reasonably expect to find measurable amounts of androgen receptors in

them due to the larger amount of testosterone available. A TSA-Direct Kit (Tyramide

Signal Amplification, NEN Life Sciences) was used for this immunocytochemistry

procedure, which is highly sensitive and routinely used in the Fernald laboratory

(complete protocol in Appendix). The chicken polyclonal antibodies generated against the

androgen receptor alpha peptides were used as primary antibodies. A second biotinylated

polyclonal goat anti-chicken antibody was then used as a secondary antibody.

Streptavidin- horseradish peroxidase (Vector Laboratories) is then added at the tertiary

step, which binds to the secondary antibody further amplifying the signal. Finally a

tyramide conjugated fluorophore is added for 5-10 minutes, which again leads to even

more amplification of the signal (Figure 6). Testis tissue was mounted on the same slides

alongside the brain sections, serving as the positive control. It is known that testes contain

a high abundance of androgen receptors (Bond, Biology of Fishes) so staining in these

tissue sections served to verify that the staining was working.

In the most recent experiments, a primary antibody raised against the first 200

amino acids of the rainbow trout androgen receptor-alpha was used to localize Hb AR-β.

This antibody was synthesized by Jiro Takeo in the Central Research Laboratory in

Tokyo, Japan (Takeo and Yamashita, 1999). The first 200 amino acids (amino terminus)

5

of the rainbow trout AR-α is 43% identical (and 55% similar) to a 115 amino acid region

of Hb AR-β. However the trout AR antibody (tAR) does not have significant similarity to

Hb AR-α, as shown with direct sequence comparison using Basic Local Alignment Search

Tool (BLAST). BLAST is a similarity search program which uses a heuristic algorithm to

seek local alignments, and is able to detect relationships among sequences which may

share only isolated regions of similarity (Altschul et al., 1990). Therefore any staining

observed with tAR in our tissue is possibly due to the presence of Hb androgen receptor

beta.

Hb AR TypeRecognized

Ab Name Source

White 57 (W57)Peptide raised against amino acid 57-74 startingfrom the amino terminus of H. burtoni AR-α.Chicken polyclonal antibody.α

White 275 (W275)Peptide raised against amino acid 57-74 startingfrom the amino terminus of H. burtoni AR-α.Chicken polyclonal antibody.

β tARRaised against the first 200 amino acids (from theamino terminus) of rainbow trout AR-α. Thispeptide is 43% identical (and 55% similar) to a 115amino acid region of Hb AR-β, which is why it isused to localize Hb AR-β. Rabbit polyclonalantibody.

Table 1. Describes the three different antibodies used in these experiments. W57 andW275 was used for Hb AR-α staining, while tAR was used to show Hb AR-β staining.

Data Analysis

Stained sections were observed using a fluorescent microscope (Zeiss Axioskop),

images were captured using a digital camera (Spot II, Diagnostic Instruments) and

downloaded onto a computer, using SPOT imaging software. Localization of androgen

6

receptors was determined by comparing tissue to known neuroanatomical regions in the H.

burtoni brain (Fernald and Shelton, 1985).

Regions displaying a greater signal intensity over background nonspecific staining

were measured (NIH ImageJ 1.27z). Intensity of specific staining was quantified by

measuring pixel intensities. Circles were drawn (average pixel area of all circles = 37822

pixels, Standard Deviation= 1527 pixels), using the circle drawing tool, over areas where

staining of interest was located, and over areas determined as being the background level

of staining. The average mean pixel intensity was measured for the area enclosed by the

circle, and then the background measurement was subtracted from the stained area

measurement to obtain a quantification of specific staining in a tissue region. Similar

tissue areas where measured in the negative control sections, determining the position of

where to draw the circle of analysis based on known anatomical structures. For each tissue

region where consistent staining was observed, mean pixel intensity differences were

compared between the tissue sections that received the primary antibody and the negative

control sections (no primary antibody) to further support visual observations of specific

primary antibody staining in tissue. Statistical analysis using a t-test was performed to

determine significance level of observed specific staining in tissue regions that received

the primary antibody.

7

RESULTS

Distinct, punctuate staining for Hb AR-α was observed in the forebrain and

preoptic regions when W57 antibody was added to the brain tissue (Figures 8 and 8B).

The negative control did not display such distinctive staining in these regions. The

quantitative analysis also showed that generally (except with the 1:100 W57 staining in

the forebrain) the intensity of specific staining was greater in the tissue that was stained

with W57 compared to the negative controls, which showed a small difference between

the background levels and the areas where staining was observed with the primary

stained sections (Table 2 and Figure 12). Also the difference between the standard

deviations, from the mean pixel intensities, for the stained areas and the background level

were consistently greater in the sections that received the W57 primary antibody

compared to the negative control sections (Table 2 and Figure 13).

Hb AR-β staining was observed in the octavolateral nucleus below the facial lobe

and above the medulla area, consistently throughout mid-sagital brain sections stained

with tAR primary antibody. The staining in this area appeared very distinctively and

punctuate compared to background as well as compared to the negative control, in which

the primary antibody was omitted (Figures 9 and 9B). The quantitative analysis also

supported that the intensity of specific staining was greater in the tissue that was stained

with tAR compared to the negative controls, with increasing mean pixel intensity

differences corresponding with increasing tAR concentrations used (Table 3 and Figure

13). Also the difference between the standard deviations, for the stained areas and the

background level increases with increasing tAR concentrations used (Table 3 and Figure

8

13). tAR also showed threadlike staining along the axis of the spinal cord in some tissue

sections, which was not observed in the negative controls.

The W275 Ab primary antibody did not show any consistent staining in the brain,

however it gave excellent staining of Hb AR-α in testis tissue (Figure 10). Greater

staining occurred with increasing W275 concentration as well as with increasing time for

the tyramide signal amplification step (Appendix, immunocytochemistry protocol, step

13). The testis tissue sections that did not receive W275 antibody treatment remained

dark and did not display any significant staining (Figure 10). Again, the quantitative

analysis also showed that generally (except for the tissue that received 1:2500 W275) the

intensity of specific staining was greater in the testis tissue that was stained with W275

compared to the negative control, with increasing mean pixel intensity differences

corresponding with increasing W275 concentrations used as well as with increasing

Tyramide Signal Amplification (TSA) step time (Table 4 and Figure 14). Also the

difference between the measured standard deviations, for the stained areas and the

background level increases with increasing W275 concentrations and with increasing

TSA time (Table 4 and Figure 14). Also, when stained tissue was viewed under Nomarski

optics, spermatic cysts could be observed in the tissue in regions that did not overlap with

the regions stained for androgen receptors (Figure 11).

Negative controls, which were tissue sections that had no primary antibody, show

very low to no distinct punctuate staining in the regions mentioned above. However faint-

fingerlike projections of unspecific staining were observed uniformly throughout

negative control sections in all regions (Figures 8-9B). These fingerlike projections were

9

also observed in tissue that did receive a primary antibody, though not nearly as notable

as in the negative controls.

Statistical analysis, using the t-test, of mean pixel intensities of the regions

discussed above where specific staining was observed and the corresponding

measurements in their negative controls all gave double-sided p-values less than .01

(Tables 5-7).

DISCUSSION

The distinct staining observed (Figures 8 and 8B) with the W57 primary antibody

suggests that Hb AR-α is present in the lower forebrain and the preoptic region of the H.

burtoni territorial male brain. The fact that staining in these areas is not observed in the

negative control sections further support this conclusion. The results from the mean pixel

intensity analysis (Table 2 and Figure 12), showing that the differences between staining

observed and background levels were greater in the tissue that was stained with W57 than

in the negative control tissue, also supports that the staining with W57 in these areas is

specific and significant. It is also important to note that all sections used in these

experiments were from the mid-sagital plane of the brain, and therefore it’s possible that

AR-α is present in the same area, or even inside, the GnRH-I cells that are in the preoptic

area (Figure 4 and 7). However, we have not yet stained the tissue with GnRH

antibodies, because additional time was needed to optimize the AR staining protocol.

The consistency and distinctiveness of the tAR antibody staining (Figures 9 and

9B) observed in the octavolateral nucleus, below the facial lobe and above the medulla in

10

mid-sagital sections, show that this region is likely to contain Hb AR-β. The mean pixel

intensity analysis showing much greater mean pixel intensity differences with increasing

tAR concentrations (Table 3 and Figure 13) further supports the conclusion that this

staining is significant. One caveat is that the tAR antibody was made to rainbow trout

AR-α (43% identical to our 115 aa region of Hb AR-β) and not directly to a H. burtoni

AR-β peptide. Therefore, the staining observed with tAR does not completely prove the

presence of androgen receptor beta, but does suggest that as a possibility. It is important

to note that the W57 or W275 antibodies, did not stain this area.

The localization of Hb AR-β in the octavolateral nucleus is a particularly exciting

result. Lateral line nerves are cranial nerves through which mechanosensory information

reaches the brain. Lateral line nerves project to a dorsal medullary area between

cerebellum and vagal lobe (Evans, pg. 254), which is the region where the Hb AR-β

staining with tAR was observed. The mechanosensory neuromasts that run along the

length of the fish body can detect the relative movement (acceleration) between water

and the body at low frequencies (1 to 200 Hz) and at relatively short distances (1 to 2

body lengths) in various biological contexts such as prey localization, navigation, and

schooling behavior (Evans, pg. 254). Therefore, androgen receptors in the octavolateral

nucleus area which is important for mechanoreception may be significant in regulating

the sensitivity of H. burtoni territorial males to attacks from other fish, potentially

playing an important role in the transitioning and maintenance of their social status states.

For example, it is possible that androgen regulation in this area may lead to a lower

sensitivity of territorial males to nonterritorial males attacks, therefore making them more

likely to endure a confrontation with another fish. On the other hand, the opposite effect

11

is also a possibility, androgen regulation in the lateral line nerves could also lead to an

increase of sensitivity to interactions with other fish. Following this hypothesis along, a

territorial male would be more easily agitated to nonterritorial intruders and this may be

what results in its more aggressive behavior. Finally, it is also likely that there is a

mechanism that may combine both effects at different points in a male fish that is

transitioning from one social status state to another.

The W275 Ab staining of the testis tissue shows that Hb AR-α is also present in

the testes, which was expected since testosterone is produced in the testes. The mean

pixel intensity analysis of the testis tissue (Table 4 and Figure 14) shows increasing

mean pixel intensity differences (between stained areas and background level) with

increasing W275 concentrations, as well as with increasing TSA times. Therefore this

staining intensity analysis supports the conclusion that this staining of Hb AR-α with the

W275 primary antibody is specific. Figure 11 shows that the staining for Hb AR-α does

not overlap with spermatic cyst regions, further supporting that the staining is specific to

androgen receptors which should only be found in the interstitial tissue and not in the

spermatic cyst areas (Grier, 1981). The W275 primary antibody was very effective in

localizing androgen receptors in testis tissue, however it did not show consistent staining

in the brain, perhaps due to the Hb AR-α region recognized by the W275 antibody being

more accessible in the testis tissue. Another possible explanation for staining by Hb AR-

α in the forebrain and preoptic area with one antibody (W57), while observed only in the

testes with the second (W275) may be due to differences in binding affinity of Hb AR-α

in the different tissue. This is a reasonable conclusion since the same receptor, Hb AR-α,

12

may have different regulatory functions, involved in different processes, and therefore

may have different sensitivities.

The t-test statistical analysis gave double-sided p-values <.01 (Tables 5-7) for all

the previously discussed primary antibody staining when compared to the same regions in

the negative control sections (at the same magnification). In other words, there is less

than one percent probability that the difference between the mean pixel intensity

measurements (proportional to amount of fluorescence in the area) of the primary

antibody stained tissue regions and those of the negative controls is caused by chance.

Therefore, the statistical calculations further support the visual observations of specific

staining in the previously discussed tissue regions, and show that the tissue staining with

all three primary antibodies is significant.

It is important to point out that the negative control sections, which did not

receive any primary antibody, generally did not have significant staining in the brain.

This is as expected, because if the tissue did not receive any primary antibody in the first

place, none of the other added reagents that follow should be able to adhere, since ideally

they are dependent on the initial presence of the primary antibody (Figure 6). The faint

finger-like projections observed in the tissue are unspecific staining, The fact that they

are more noticeable in sections that did not receive the primary antibody is most likely

due to the fact that this was the only fluorescent signal detected. Omission of the

biotinylated secondary antibody or the S-HRP conjugated tertiary antibody indicates that

this staining is probably due to the tyramide reagent reacting with the tissue itself.

Also in slides that underwent the antigen-retrieval, unmasking step (Appendix,

immunocytochemistry protocol, step 3) the presence of these fingerlike projections

13

decreased. Since the antigen-retrieval step involves adding boiling sodium citrate

solution, which breaks some of the formaldehyde cross-linking and thereby exposing

different epitopes, more primary antibody is expected to bind to “unmasked” androgen

receptors and accordingly a decrease in unspecific staining is expected.

It is also important to state that we do not know if the antibodies recognize

androgen receptors before of after binding with androgen. Since we have only used

territorial males in these experiments, who are known to have a higher level of

testosterone than nonterritorial males, it is reasonable to expect that the antibodies are

recognizing occupied receptors as opposed to unoccupied ones. There is also the

possibility that the antibodies are recognizing the occupied and/or dimerized form of the

androgen receptors, which are expected to be found in the nucleus of cells. Staining with

DAPI should let us discover which form of the androgen receptor is being recognized, by

letting us observe if the androgen receptor form is found in the nucleus or not.

To understand where androgen receptors are in relation to GnRH-I neurons,

further studies will include colocalization of GnRH-I and AR staining. Adjacent sections

(on different slides) will be labeled with androgen receptor and GnRH-I antibodies. This

would be useful in determining whether the androgen receptors are co-expressed in the

same cells as GnRH-I, which have already been determined to be localized in the

preoptic area (Davis and Fernald, 1990). If similar regions display staining for androgen

receptors and GnRH-I, then a double-labeling protocol will be used to determine if the

same cells co-express these two proteins. Double-labeling for GnRH-I and androgen

receptors involves using two different fluorophores (secondary antibodies), one green in

color (fluorescein) and the second red in color (rhodamine). Where these two

14

fluorophores are colocalized yellow staining will be observed with fluorescence

microscopy. Therefore localization of androgen receptors will be further investigated by

continuing to compare tissue to known neuroanatomical regions, and then through

comparison within the slides themselves by staining cells with GnRH-I antibodies.

After confirming androgen receptor localization in territorial males, further studies

can also include using males of different social status states: territorial, nonterritorial,

individuals transitioning into territorial status (or into nonterritorial status) to investigate if

changes in social status affect androgen receptor localization or expression level. Since,

territorial and nonterritorial males have different levels of circulating testosterone, it is

likely that the expression of androgen receptors could also differ in animals varying in

social status. Since territorial males have larger testes and higher testosterone levels than

nonterritorial males it is reasonable to expect them to also have greater expression of

androgen receptors in order to be more sensitive to changes of the higher testosterone

concentrations.

The determination of androgen receptor localization in the brain will add

significantly to our understanding of how hormonal information is integrated with social

information at preoptic GnRH-I neurons. Since preoptic GnRH-I neurons have been

highly conserved throughout vertebrate evolution, discoveries in this model system could

have far-reaching applications in behavioral neuroendocrinology.

15

FIGURES

Figure 1. Territorial male fish (on top) has distinct markings andbody coloring compared to a nonterritorial male (on bottom).

Figure 2. Steroid-receptor activation leads to changes in target geneexpression.

Foreheadstripes

Lachrymal stripe Anal fin spots

Humeral scalesOpercular spot

Steroid diffusioninto the cell

16

Figure 3. Model for GnRH-I regulation in H. burtoni males (White and Fernald 1997).The +/- symbol for Social Setpoint refers to territorial (+) and nonterritorial (-) status.GnRH-I upregulates the release of pituitary gonadotropins, which in turn, upregulates thesecretion of androgens from the testes. Gonadal androgens then complete the loop bynegatively regulating the release of preoptic GnRH-I.

Figure 4. This figure shows the general regions of the Haplochromis burtoni brain, in themid-sagital plane of section. GNRH-I cells are located in the preoptic area .

SocialSocial

SetpointSetpoint +/-+/- GnRHGnRH -I -I ++ Pituitary

GonadsAndrogensAndrogens

GonadotropinsGonadotropins

+ +

----

PreopticArea

(Forebrain)

17

Figure 5. Schematic illustration of androgen receptor domains, in vertebrates, showingthe regions to which antibodies were generated. These regions were chosen because theyare predicted to be particularly antigenic. The peptides from the Hb AR-α were used togenerate two antipeptide polyclonal antibodies in chicken, called White 57 and White275. A purified protein fragment made from the rainbow trout AR-α (43% identical to a115 amino acid region of Hb AR-β) was used to generate a rabbit polyclonal antibody,tAR.

Figure 6. The immunocytochemistry techniques used in these experiments providedamplification of the signal with each step, thereby making the low abundance ofandrogen receptors detectable.

Androgen Receptor

Chicken Polyclonal Primary Ab

Biotinylated polyclonalgoat anti-chicken Secondary Ab

FITC- tyramide

Streptavidin-HRP

TransactivationTransactivationDNADNA

BindingBinding HingeHinge Ligand BindingLigand Binding

tAR200 aa

TransactivationTransactivationDNADNA

BindingBinding HingeHinge Ligand BindingLigand Binding

White 5717 aa

White 27519 aa

H. burtoni AR-α

Rainbow trout AR-α ( H. burtoni AR-β )

18

DOTT

OC

M

C SC

H

9

9B

8Ba

8Bb

8a8b

8c

Figure 7. Photomicrograph of the H. burtoni brain, mid-sagital section, (taken at 2.5xmagnification) with key of major regions below. The larger white boxes outline regionsof pictures taken at 10x magnification, while the smaller black boxes outline regions ofpictures taken at 40x magnification. The numbers in the boxes refer to theircorresponding figures. Distinct staining is not observed in this picture due to the lowresolution of the photomicrograph.

P

C cerebellumF facial lobeH hypothalamusM medula oblongataO octavolateral nucleusOC optic chiasmOT optic tectumP preoptic areaSC spinal cordT telencephalonV vagal lobe

OTT

OC

M

C

SC

H

9

9B

8Ba

8a8b

8c

P 8B

b

FRostral

Ventral

Dorsal

Caudal

100 µm

V

O

19

Captions for figures 8-11

Figure 8. Photomicrographs showing staining observed for W57 Ab (1:100) and the

negative control (which had no primary Ab added), in the left and right column

respectively, of similar regions in two brains at 10x magnification. Row A shows the tip

of the forebrain, row B shows the bottom half of the forebrain, moving back to the middle

of the brain, and row C shows the preoptic region anterior to the optic chiasm. Distinct

staining is observed in the sections that were stained with W57and not in negative

controls.

Figure 8B. These pictures show the staining observed for W57 Ab (1:100) and the

negative control (which had no primary Ab added), in the left and right column

respectively, of similar regions in two brains at 40x magnification. These are greater

magnification pictures of the areas shown in Figure 8. Distinct staining is observed in the

sections that were stained with W57 and not in negative controls.

Figure 9. Pictures of staining observed for negative control and for tAR staining in the

octavolateral nucleus region, located below the facial lobe, at 10x magnification (left) and

40x (right). The top row shows the corresponding area in a negative control section,

while the middle and bottom row show distinct staining with the tAR primary Ab in two

different brain sections (with 1:4000 tAR and 1:8000 tAR concentration respectively).

Distinct staining is observed in the sections that received tAR Ab but not in the negative

control sections.

20

Figure 9B. 40x magnification (left three pictures) of staining area observed in Figure 9.

Distinct staining is observed in the sections with tAR Ab, while only faint nonspecific

staining is observed in the negative control.

Figure 10. Pictures of testis sections stained with different concentrations of W275 Ab,

and either reacted with the Tyramide Signal Amplification step for 5 minutes (the top

three pictures in left column) or 10 minutes (the three pictures in right column). A

negative control section, which received no primary antibody and incubated in the TSA

step for 10 minutes is shown on the bottom left. Note an increase in signal observed with

increasing primary antibody concentration as well as with an increase in TSA time. All

pictures taken at 40x objective magnification.

Figure 11. Top picture shows W275 Ab staining in a testis section. Bottom picture of

same tissue taken with Nomarski optics allows observation of testis morphology. The

middle picture is the top and the bottom picture merged together. Note that regions were

distinct staining for androgen receptors is observed in the interstitial cells does not

overlap with the spermatic cysts.

REFERENCES

Altschul, S.F, et al. (1990). “Basic local alignment search tool.” J Mol Biol 215 (3): 403-410.

Bond, Carl E. Biology of Fishes. (1979) W.B Saunders Company, Philadelphia, pg. 100-101.

Davis, M.R. and R.D. Fernald (1990). “Social Control of Neuronal Soma Size.” JNeurobiology 21 (8): 1180-8.

Evans, David H. The Physiology of Fishes. (1998) CRC Press, New York, pg. 254-255.

Fernald, R.D. (1995). “Social Control of Cell Size: Males and Females Are Different.”Prog Brain Res 105:171-7.

Fernald, R. D. and L. C. Shelton (1985). “The Organization of the Diencephalon and thePretectum in the Cichlid Fish, Haplochromis burtoni.” J Comp Neurol 238 (2):202-17.

Fernald, R. D. and S. A White (1999). Social control of brains: From behavior to genes.In: The Cognitive Neurosciences, 2nd Edition, Ed. M.S Gazzaniga, MIT Press,Cambridge, pp. 1193-1208.

Fraley, N. B and R.D. Fernald (1982). “Social Control of Developmental Rate in theAfrican Cichlid, Haplochromis burtoni.” Ethology 60: 62-82.

Francis, R.C., K. Soma and R. D. Fernald (1993). “Social Regulation of the Brain-Pituitary-Gonadal Axis.” Proc Natl Acad Sci USA 90(16): 7794-8.

Grier, H. J. (1981). “Cellular Organization of the Testes and Spermatogenesis in Fishes.”American Zoology 21: 345-357.

Schulz, Rudiger and V. Blum (1988). “Testosterone Immunoreactivity in Rainbow Trout(Salmo gairdneri) testis.” General and Comparative Endocrinology 72: 80-89.

Soma, K.K, R.C. Francis, J.C Wingfield and R. D. Fernald (1996). “Androgen Regulationof Hypothalamic Neurons Containing Gonadotropin-Releasing Hormone in aCichlid Fish: Integration with Social Cues.” Horm Behav 30(3): 216-26.

Takeo, J. and S. Yamashita (1999). “Two Distinct Isoforms of cDNA Encoding RainbowTrout Androgen Receptors”. J Biol Chem 274: 5674-5680.

White, S.A. and R. D. Fernald (1997) “Changing through doing: Behavioral influenceson the brain.” Recent Progress in Hormone Research, 52: 455-474.

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APPENDIX

Immunocytochemistry protocol (TSA Fluorescence Kit)

Day 1

1. Dry slides at 37 °C for 40 minutes

2. Rehydrate slides for 10 minutes at room temperature (RT) in PBS/Tween solution ( 1xPhosphate Buffer Saline and %.05 Polyoxyethylene Sorbitan Monolaurate (Tween))

3. Add unmasking solution (10mM sodium citrate) to slides, 2 washes, and let them cooldown to room temperature (about 20 minutes)

4. PBS/Tween + 3% hydrogen peroxide for 30 min at RT

5. PBS/Tween was for 10 min at RT

6. Blocking step. Add 250 µl of PBSB, Phosphate Buffer Saline Block Buffer, (with 4drops of Avidin per 1 mL of PBSB) for 1-2hr at RT

7. Incubate slides in 1° Ab overnight at 4 °C (dilute 1° in PBSB with 4 drops of Biotinper 1 mL of PBSB)

Day 2

8. Wash slides in PBS/Tween at RT, 4 times for 10 min washes

9. Incubate slides in 2° Ab (1:200, diluted in PBSB) for 2 hrs at RT

10. Wash slides in PBS/Tween at RT, 4 times for 10 min washes

11. Incubate slides in 1:200 Streptavidin-Horse Radish Peroxidase (S-HRP) at RT for 30minutes

12. Wash slides in PBS/Tween at RT, 4 times for 10 min washes

13. Incubate slides in fluorophore conjugated tyramide (Amplification Reagent, 1:50) atRT for 5-10 minutes only

14. Wash slides in PBS/Tween at RT, 4 times for 10 min washes

15. Mount coverslips on slides using Fluoromount G

35