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Hormonal Regulation of Prostaglandin F2α Receptor Expression: Identifying Mechanisms of Female Reproductive Behavior in a

Anusha Kumar

May 2014

HORMONAL REGULATION OF PROSTAGLANDIN F2α RECEPTOR EXPRESSION: IDENTIFYING MECHANISMS OF FEMALE REPRODUCTIVE BEHAVIOR IN A CICHLID FISH

An Honors Thesis Submitted to the Department of Biology in partial fulfillment of the Honors Program STANFORD UNIVERSITY

by Anusha Kumar May 2014

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Acknowledgements

I would like to thank my postdoctoral mentor Scott Juntti for guiding me through the experimental design process, for overseeing my use of molecular biology techniques, and for providing invaluable suggestions as I analyzed my data. I also thank postdoctoral fellow Mariana Jimenez for helping me develop the ovariectomy procedure I make extensive use of in this study. Special thanks to my PI Russell Fernald, for providing me continuing support and a friendly lab environment to conduct my research. I will treasure the memories from these four, stellar years, Fernald Lab. Also, many thanks to all my readers, Scott Juntti and Professors Russ Fernald and Craig H. Heller, who helped me edit and revise this document. Finally, this project would not have been possible with out the generous funding provided by the UAR Major Grant, for the 2013-2014 year.

! 4 Table of Contents

ABSTRACT...... 7! INTRODUCTION...... 8! METHODS ...... 11! RESULTS...... 16! DISCUSSION ...... 18! REFERENCES...... 23! FIGURES ...... 26!

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! 5 List of Figures

Figure 1. Ovarian cycle in the A. burtoni female

Figure 2. Timeline for hormonal and vehicle injections of OVX A. burtoni

Figure 3. Identification and confirmation of DHP receptor (PR) sequence in A. burtoni genome

Figure 4. Ptgfr expression in the POA of OVX+DHP treated female A. burtoni is significantly higher than in OVX females

Figure 5. Ptgfr expression in the POA of OVX A. burtoni females

Figure 6. PR expression in the female A. burtoni brain is localized in two areas, the POA and NLT

Figure 7. Overlapping Ptgfr and PR expression in the POA of female A. burtoni

! 6 Abstract Several work in concert to mediate female reproduction, a behavior that drives evolution. Previous studies in mammals have identified the ovarian steroids estradiol and progesterone as hormones that act together to induce female mating. In other organisms, the identity of and relationship between reproductive hormones can vary, and this divergence is informative from an evolutionary and molecular standpoint. For example, though ovarian steroids are involved in the reproduction of teleost fish, the post-ovulatory hormone prostaglandin F2α (PGF2α) is sufficient to induce spawning (mating) behavior in females of many teleost species. PGF2α thus activates a distinct, important pathway for reproduction. However, the regulation of this pathway has yet to be defined. Here, we sought to address whether and how ovarian steroids regulate PGF2α -mediated female reproductive behavior. To do so, we used the African cichlid fish burtoni, a for the study of social behaviors and neural plasticity. In female A. burtoni, the PGF2α receptor (Ptgfr) is expressed in a behaviorally relevant brain region, the preoptic hypothalamus (POA), and increases expression during spawning. This spatio-temporal expression pattern implicates the POA as a locus of PGF2α signaling during reproductive behaviors. We investigated the mechanism of PGF2α-mediated spawning by searching for the regulators of POA Ptgfr expression in female A. burtoni. We hypothesized that ovarian steroids account for the observed variation in Ptgfr mRNA expression. Using in situ hybridization, we demonstrated upregulated POA Ptgfr expression in ovariectomized females treated with teleost progesterone 17α,20β- dihydroxy-4-pregnen-3-one (DHP). We further demonstrated that DHP receptors are expressed in the POA, suggesting DHP directly regulates Ptgfr expression in that region.

These results are consistent with a model where DHP-regulated PGF2α signaling mediates reproductive behavior. This model can help identify conserved PGF2α pathways in other vertebrates or track the diversification of PGF2α’s reproductive role over evolutionary time.

Key Words: Biology, prostaglandin F2α (PGF2α), receptor, social behavior, reproductive regulation, teleost, 17α,20β-dihydroxy-4-pregnen-3-one (DHP)

! 7 Introduction Reproductive behaviors comprise an important subset of social behaviors (1). These behaviors are key substrates upon which selection pressures act to maximize reproductive success (28); they are a vehicle for adaptation and drive evolution. Reproductive behaviors arise from signaling through multiple pathways, which integrate environmental and internal cues to activate hardwired neural substrates (1). Many of the molecules that constitute these reproductive pathways are conserved across species. Even when reproductive molecules and signaling pathways differ between organisms, this divergence is informative from an evolutionary standpoint, helping us understand the mechanisms by which molecules and neural circuits accommodate adaptive behaviors. Some progress has been made in elucidating the control of reproductive behaviors, since some of the neural circuitry involved in reproduction feeds into a conserved hypothalamic-pituitary-gonad (HPG) axis. The HPG axis produces several steroid hormones (e.g. estrogen, , etc.) that have been established as mediators of gonadal and gametic maturation (2-4). Through considerable research in mammals, many of these same hormones have since been identified as molecular effectors of reproductive behavior. In particular, estrogen and progesterone elicit lordosis (female sexual behavior) in mice, thereby implicating these hormones as the key translators of reproductive state to sexual and receptive behaviors (2-4). Nonetheless, the precise mechanisms by which these steroid hormones signal in the brain and influence reproductive behaviors have yet to be determined. These hormones could act through several pathways, in conjunction with many other hormones, to mediate the different stages of sexual behavior. Extending reproductive research to other vertebrates, such as teleost fish, will broaden the understanding of these various hormonal mechanisms and better define evolutionarily conserved or divergent pathways controlling reproductive behaviors. To this end, we have chosen to use a social behavioral model system, the African cichlid Astatotilapia burtoni, to investigate the mechanisms of reproductive behavior. This teleost species lends itself to such investigations for a variety of reasons. A. burtoni requires only simple maintenance and has a relatively short generation time. Moreover, unlike other traditional model organisms that suffer from a stunted repertoire of

! 8 reproductive behaviors in laboratory housing, A. burtoni continues to display a rich suite of well-defined social behaviors (6). In addition, molecular investigations in this species have been aided in recent years by the sequencing of the A. burtoni genome. Thus, behavioral, neural, and molecular changes that proceed or follow changes in reproductive state can be readily assayed using A. burtoni.

The post-ovulatory hormone prostaglandin F2α (PGF2α) plays a critical role in regulating reproductive behaviors in teleosts including A. burtoni. In female fish, ovulation triggers synthesis of PGF2α, which in turn activates spawning behavior (7,8). Spawning in A. burtoni comprises several stereotyped mating behaviors, including male courtship, female following into the male’s territory, egg laying, and female mouth- brooding after fertilization (9). Thus, in teleosts, PGF2α appears to be a key translator of internal state (the occurrence of ovulation) into outward reproductive behavior, a role associated with steroid hormones in mammals.

Functional tests confirm that this postovulatory PGF2α acts as a -inducing hormone in several teleosts (5, 10). In both goldfish and A. burtoni, treating non- reproductive females with PGF2α is sufficient to induce spawning behaviors (11, 12, 5).

The necessity of PGF2α for such behaviors was further confirmed in goldfish by treating mature, gravid (post-ovulation) females with a prostaglandin synthesis inhibitor, which prevented the females from displaying spawning behaviors (12). However, the neural and molecular mechanisms by which PGF2α stimulates reproductive behaviors has yet to be demonstrated.

As a step towards bridging this gap, recent work identified the PGF2α receptor (Ptgfr) in A. burtoni (5). Ptgfr expression is localized to four brain areas: the ventral telencephalon (Vd-c), the preoptic area (POA), the lateral tuberal nucleus (NLT), and the vagal lobe (VL) (5). Of these areas, the POA has been identified as a crucial node in the mating neural circuit. Evidence for this conclusion includes immediate early gene expression, markers of cell activity, found in POA neurons following social behaviors (13). In addition, lesion studies across vertebrates reveal impaired reproductive behavior in POA disrupted (14). Thus, Ptgfr in the POA likely plays an important role in the control of reproductive behaviors. This suggestion is further supported by in situ hybridization results, which show

! 9 that the expression of Ptgfr in the POA varies across the female reproductive cycle (5). In female A. burtoni, Ptgfr expression in the POA spikes in spawning females. The increase in Ptgfr expression at the time of spawning reflects a potential increase in sensitivity to

PGF2α. This finding is consistent with a model that PGF2α is a key coordinator between internal ovarian state and reproductive behaviors in female A. burtoni. Since Ptgfr is expressed in a behaviorally relevant brain region and varies in expression across the reproductive cycle, PGF2α likely activates spawning behavior through pathways downstream of Ptgfr in the POA. The differential Ptgfr expression across the reproductive cycle also suggests that

PGF2α does not act alone to stimulate spawning. Additional molecules work upstream or in conjunction with PGF2α to coordinate female sexual behaviors. These molecules include the factor(s) that regulate Ptgfr expression and thereby regulate PGF2α signaling. Since these factor(s) must also be able to translate internal reproductive state into behavior, ovarian steroid hormones (i.e. estrogen, progestin) are good candidates. This hypothesis is supported by studies on hypophysectomized (pituitary-removed) female goldfish. When treated with PGF2α, hypophysectomized goldfish do not spawn without addition of gonadotropin (15,12). This indicates necessary factor(s) in PGF2α signaling are downstream gonadotropin activity, implicating gonadal steroid hormones.

Furthermore, adding a steroid synthesis inhibitor to hypophysectomized, PGF2α and gonadotropin treated goldfish blocks female spawning, more evidence that steroid hormones are involved in PGF2α signaling (12). Overall, these results indicate that some steroid downstream of gonadotropin signaling is necessary for PGF2α-mediated female sexual behavior. This necessary factor(s) may be responsible for the regulation of Ptgfr expression in the POA.

Thus in this study, we sought to understand the regulation of PGF2α signaling by searching for and investigating the factor(s) responsible for the regulation of POA Ptgfr expression. Since Ptgfr is expressed in a behaviorally relevant neural population and is upregulated during spawning, the signaling pathways downstream of Ptgfr in the POA likely play a role in the control of reproductive behaviors. In turn, the factors regulating Ptgfr expression, the factors upstream of this pathway, are also relevant to reproductive behavior control. We hypothesize that these regulators are other ovarian steroid

! 10 hormones, the estrogen estradiol (E2) and teleost progesterone 17α-hydroxy,20β- dihydroprogesterone (DHP). Previous studies have shown that both of these hormones peak in the ovaries prior to the release of PGF2α at the time of spawning (Figure 1). This hormonal profile suggests that one or both of these hormones may regulate subsequent sensitivity to PGF2α at the level of PGF2α receptor expression. Here we evaluate this hypothesis, by creating a background devoid of ovarian steroids, ovariectomized female A. burtoni. We inject these fish with different ovarian steroids to test the sufficiency of these hormones to upregulate Ptgfr, and find that DHP is sufficient to upregulate POA Ptgfr expression. We then identify the sequence for the DHP receptor, PR, the only ortholog for the mammalian nuclear progesterone receptor in the A. burtoni genome. Analysis of PR expression in the brain revealed that PR is also expressed in the POA, consistent with a mechanism where DHP binds to PR and upregulates Ptgfr. Overall, our findings define the following model: in A. burtoni, DHP regulates PGF2α signaling and thus female reproductive behavior by acting at the level of Ptgfr expression in the POA. This mechanism will inform our understanding of the evolution of social behaviors, in particular the conservation or divergence of PGF2α - dependent behaviors.

Materials and Methods Animals A. burtoni females were bred and grown to sexual maturity (at least 4 months of age). These fish were descendants of a wild stock native to Tanganyika, Africa and were housed in tanks mimicking their natural environment (water 29°C, pH 8.0, light dark cycle 12D/12L) (9). The tanks contained gravel-covered bottoms, halved terracotta pots, and aquarium water mixed with cichlid salt and Tanganyika buffer (Seachem). The fish were fed every morning with cichlid pellets and flakes (AquaDine). All protocols concerning these fish were approved by the Stanford Administrative Panel for Laboratory Care. Prior to surgery, females were housed in the presence of males of similar age in 60 liter tanks, 10-20 total fish per tank.

! 11 Preparation of ovariectomized (OVX) females Adult female A. burtoni were prepared for the OVX surgery by an initial 20-30 second anesthetization in tricaine mesylate, (MS-222; 0.1 g/ 200 mL). After the fish were fully sedated, as determined by unresponsiveness to a tail pinch, a long incision was made along their ventral surface between the pectoral fins and the cloaca. Ovaries were then detached at their anterior and posterior ends in OVX females. OVX fish were then sutured, revived in oxygenated water, and allowed to recover in isolation for approximately 1 week in methylene blue anti-fungal (1 mg/mL stock diluted 1:1000; Sigma Aldrich) and Furan-2 antibiotic (1 packet of 60 mg Nitrofurazone and 25 mg Furazolidone per 10 gallons; API) treated 8-gallon recovery tanks. During the recovery period, the operated females were group housed with other operated females in a 16- gallon tank until hormone injections at 1-2 weeks post-surgery.

Hormone treatment of OVX females OVX females were treated with different combinations of reproductive steroids estradiol

(E2; 5 µg in corn oil) and 17α-hydroxy,20β-dihydroprogesterone (DHP; 1.25 µg in DMSO/saline). The combination of treatments used produced 5 experimental groups:

OVX, OVX + E2, OVX + DHP, OVX + E2(48), and OVX+E2(48)+DHP. The experimenter was blinded to the identity of the groups. All fish were subject to a series of three intraperitoneal injections, consisting of either treatment (steroid hormones) or vehicle (corn oil or DMSO diluted in saline, 0.6% NaCl). The OVX+ extended E2 fish were subject to two injections of E2, each separated from the next injection by 24 hours, followed by a DMSO vehicle injection 3 hours prior to dissection; the

OVX+E2(48)+DHP fish were treated with the same two-day regimen of E2 as the

OVX+E2(48) females, followed instead by a single dose of DHP 3 hours prior to dissection; and the OVX+DHP and OVX+E2 fish were treated with a two day regimen of corn oil vehicle instead of E2, followed by an injection of DHP or E2 respectively 3 hours prior to dissection. OVX fish were subject to vehicle injections at the same time points the other fish received their treatments. A summary of the treatment time courses can be found in Figure 2.

! 12 Tissue collection and preparation After the hormonal treatment regimen and prior to sacrifice, the injected, operated fish were weighed and measured for standard length. Blood was collected by caudal severance within seconds of capture. Serum was spun for 10 min at 8000 rpm at RT to isolate serum, which was stored at -80ºC. Fish were euthanized through decapitation and OVX effectiveness was determined by ensuring all experimental fish had their ovaries completely removed. Fish with residual ovaries were eliminated from the subsequent data analysis. Brains from operated A. burtoni were also dissected out and fixed in 4% paraformaldehyde (PFA) overnight at 4ºC, cryoprotected for the next 1-2 days in 30% sucrose (in PBS) at 4ºC, and then embedded in Neg 50 sectioning medium (Richard- Allan Scientific). Embedded brains were frozen at -80ºC until they were sectioned. Sections were cut on a cryostat at 30 µm and mounted onto slides (Tissue Path Superfrost Plus Gold, Fisherbrand). Slides were then kept at -80ºC in desiccant until in situ hybridization (ISH).

Generation of Ptgfr probe for in situ hybridization (ISH) Ptgfr antisense RNA probe was synthesized through PCR-mediated amplification and transcription (5). Briefly, RT-PCR amplification of the Ptgfr coding sequence generated complementary DNA, which was then cloned into a pCR-TOPO4 vector (Life Technologies). The vector was then transcribed using a T3 RNA polymerase to generate the antisense RNA probe specific for the Ptgfr RNA. The probe was labeled with digoxygenin to allow for antibody labeling and visualization of the probes’ localization after hybridization.

ISH for POA Ptgfr expression Slides ready for ISH were then subjected to a three day protocol. On day 1, slides were air dried at room temperature, before being washed 3 times for 5 min each in 1X filtered PBS. The slides were then fixed in 4% PFA for 20 minutes. Slides were again washed twice in 1X PBS before treatment for 15 minutes with Proteinase K (10 µg/ml in PK Buffer, 1:2000). Another wash in PBS followed, before slides were fixed again in 4% PFA for 15 minutes. After two more washes in PBS and a single rinse in DI water, slides

! 13 were acetylated for 10 minutes in a solution of acetic anhydride and 0.1M triethanolamine-HCl, pH 8.0 at 0.25% volume. Slides were removed from this solution and washed in PBS before immersing slides in pre-hybridization buffer for 1-4 hours at 62ºC. Slides were transferred to hybridization buffer containing RNA digoxygenin-labled probe (0.2 µg/ml). Hybridization occurred overnight at 62ºC. On day 2, slides were washed twice in 50% formamide and 2x SSC solution for 30 minutes at 62ºC. Slides were then washed 3 times for 10 minutes in 2x SSC at 37ºC before slides were treated with RNase A (0.2 µg/ml in 2X SSC) solution for 30 minutes at 37ºC. Slides were again washed twice in 2xSSC at 37ºC. Slides were transferred to 62ºC and washed twice in a 1:1 solution of 2x SSC and maleic acid buffer (MABT; 100 mM Maleic Acid, 150 mM NaCl, 0.1% Tween-20). Slides were washed another two times in MABT for 10 minutes each at 62ºC. Then, slides were blocked in a solution of MABT and 2% bovine serum albumin (BSA) for 1-3 hours. After this incubation, slides were incubated with a MABT-BSA solution containing anti-digoxygenin antibody conjugated to alkaline phosphatase (Roche; 1:5000) overnight at 4ºC. On day 3, unbound antibody was removed by washing three times for 30 minutes each in MABT followed by two 5 minute washes in alkaline phosphatase buffer (100 mM

Tris pH 9.5, 100 mM NaCl, 50 mM MgCl2, 0.1% Tween-20, 5 mM levamisole). Finally, the slides were stained during incubation in a NBT/BCIP solution (3.75 µL NBT + 18.75 µL BCIP per 10 mL of alkaline phosphatase buffer) for 5 hours in the dark at 37ºC, after which the slides were washed in PBS to stop staining reaction. The slides were then fixed in 4% PFA for 10 minutes at room temperature and then rinsed in PBS before Aqua Poly Mount media (PolySciences) was applied. The slides were then mounted with coverslips and observed for the presence of anti-digoxygenin antibody, which marked the presence of Ptgfr mRNA by staining purple.

Identification of DHP receptor (PR) in A. burtoni genome Using a tBLASTn algorithm (16), the A. burtoni genome (Broad Institute, Cambridge, USA) was queried for sequences homologous to the nuclear progesterone receptor (PR) protein in mouse (Mus musculus; NP_032855), zebrafish (Danio rerio; NP_001159807), and tilapia (Oreochromis niloticus; BAC77019) (Figure 3).

! 14 The following primers were constructed within the putative fourth (fwd) and eighth (rev) exons of the A. burtoni PR sequence, to yield a predicted PCR product of 624 bp: forward primer (5’-TGCTCACCAGTCTCAACAGG-3’) and reverse primer (5’- GAAATTAATACGACTCACTATAGGGGAGGCAATGACCTCTGACATC-3’). The bolded sequence in the reverse primer corresponds to a T7 promoter sequence, incorporated into the PCR product for subsequent transcription of the RNA probe. After separating the amplified PCR product from the reaction mix by gel electrophoresis,

Sanger sequencing (ElimBio) was used to confirm the product was the PR sequence.

DHP receptor (PR) mRNA probe synthesis and PR ISH The PR PCR product, containing a T7 promoter, was transcribed into antisense mRNA to be used as the PR probe. 400 ng of PR cDNA template was incubated with 10x digoxygenin, dithiothreitol (DTT), RNase inhibitor, and T7 polymerase and buffer, in a final volume of 30µl. This mix was then incubated at 37ºC for an hour before adding more T7 polymerase. Incubation continued for another hour before adding DNase to degrade the template DNA. The generated probe was then quantified and examined by gel electrophoresis to confirm correct transcription. Following PR probe synthesis, the ISH protocol described above was again used to visualize progesterone (PR) receptor expression in the female A. burtoni. The incubation in NBT/BCIP solution was 3 hrs. Slides containing brain sections from both intact (non-gravid, gravid, spawning, and brooding females at the time of sacrifice) and OVX females were stained for PR (total n=7). After performing ISH with PR probes, PR receptor expression in sections within each and across females was visualized. Then, PR stained sections were compared to their Ptgfr stained, analogous sections.

ISH visualization and quantification Sections were viewed under a Zeiss Axioskop microscope using brightfield optics. Images of each section with staining were captured using SPOT software. For Ptgfr stained slides, mRNA expression level in each POA section was quantified using the ImageJ program (NIH). Quantification was performed blind to the hormonal regimen of the samples. A polygon was drawn around the stained region in each hemisphere of every

! 15 section of the POA, on slides for each OVX female. The area within the polygon and mean pixel intensity were calculated for each hemisphere in each section independently. Background intensity was subtracted by first drawing a control polygon in an unstained region neighboring each polygon containing staining. The average pixel count of the control polygon was subtracted from the corresponding polygon with staining in order to determine a mean staining intensity above background. This value was then multiplied by the area of the stained region to calculate total Ptgfr staining per hemisphere. Ptgfr staining was averaged between hemispheres and totaled across POA sections for a given female. This final total was compared across females, grouped according to hormone treatment.

Statistical analysis POA Ptgfr mRNA levels in different hormonal treatment and control groups were then subject to a preliminary non-parametric multi-group comparison Kruskal-Wallis test to determine whether there were any significant differences in receptor expression among the hormone treatment groups. Statistical analyses were performed using GraphPad PRISM software. The OVX treatment and control groups included in the analysis were:

(OVX, OVX+DHP, OVX+E2, OVX+E2(48), OVX+E2(48)+DHP. A post-hoc statistical test, a Dunn’s test, was then used to determine which treatments significantly differ from each other in Ptgfr mRNA expression. After increasing the sample size of treatment groups, a Mann-Whitney U-test was then used to compare receptor expression between hormone treatment groups identified as significantly different from the preliminary statistical analysis.

Results Ovarian steroid DHP upregulates POA Ptgfr expression in OVX A. burtoni ISH sections of POA from OVX females treated with ovarian steroids were stained for Ptgfr and imaged (Figure 4). Quantification of staining across females within each treatment group was performed (Figure 4C; Figure 5). Ptgfr expression in OVX females treated with DHP was significantly higher than Ptgfr expression levels in control OVX females (P = 0.0286, Mann-Whitney U-test). Treatment of OVX females with E2, either

! 16 the acute 3 hr dose or the two-day extended treatment, did not cause an increase in POA Ptgfr expression above that in control OVX females (Figure 5). Co-treatment of OVX females with the extended E2 and 3 hr DHP increased Ptgfr expression to levels higher than control OVX females, though this expression was not significantly more that that in the OVX+DHP females (Figure 5). Thus, we can conclude that DHP alone is sufficient to upregulate Ptgfr expression in OVX A. burtoni females.

Confirmation of PR sequence in A. burtoni genome tBLASTn searches of the A. burtoni genome identified scaffold 78 as the site of maximal homology to the queried PR sequences, E value 1.4e-13 when run against the tilapia PR sequence (Figure 3A). After identifying this putative A. burtoni PR sequence, we sought confirmation that this sequence was transcribed in vivo. PCR for the putative PR sequence on A. burtoni cDNA, from tissues suspected to express PR, yielded a product of the expected length (Figure 3B). Among the different tissue types, the testes were particularly enriched in PR transcripts, while PCR product was not visibly detected in the whole brain sample. The lack of PCR product in the latter tissue sample was likely due to the dilution of PR transcript in the whole brain, since the hypothalamic sample did yield the PR sequence PCR product. The PCR product isolated from the testes was then sequenced, confirming integrity of the product. The product was transcribed to synthesize antisense PR RNA probe for in situ hybridization.

PR is expressed in two brain regions of the female A. burtoni brain Brain sections from female A. burtoni females of various groups (non-gravid, gravid, spawning, brooding, and OVX; n=7) were stained for PR mRNA and imaged (Figure 6). In all female brains, PR expression was visible in two specific areas, the POA (Figure 6A,A’) and in the nucleus lateralis tuberis (NLT), a more posterior region of the hypothalamus (Figure 6B, B’). PR ISH staining was not detectable in any other brain regions. PR expression also did not visibly vary in intensity across females of different conditions.

! 17 Overlapping Ptgfr and PR expression in the POA of female A. burtoni ISH sections of POA from female A. burtoni were stained for Ptgfr and PR in sequential sections. In Figure 7A-B, serial POA sections from a gravid female show expression of Ptgfr and PR in the same compartment of the POA. PR expression tended to extend across a larger area of the POA in a given section than Ptgfr expression. In addition, PR expression appeared to begin more anterior to the POA than Ptgfr expression (Data not shown).

Discussion

In this study, we sought to identify and characterize the components of the PGF2α pathway involved in female reproduction. Activation of this pathway is sufficient to elicit spawning behaviors in a variety of teleost fish, including our model system, A. burtoni (11, 12, 5). Previously, we demonstrated the spatial and temporal expression pattern of the PGF2α receptor Ptgfr and its overlap with markers of neuronal activity (immediate early genes) at the time of spawning (5). This expression pattern identified the probable neural circuitry through which PGF2α primes spawning, the POA of the hypothalamus.

Using Ptgfr expression in the POA as a readout of sensitivity to PGF2α, we asked what hormones regulate Ptgfr expression and in turn reproductive behaviors in female A. burtoni. Specifically, we asked: 1) do ovarian hormones regulate Ptgfr expression in the POA, and 2) if so, what is the mechanism by which they regulate Ptgfr (i.e. do they act directly upstream)?

Our study provides evidence that the ovarian hormone, DHP, regulates PGF2α mediated reproductive behaviors in female A. burtoni. Ovariectomized females treated with DHP show a significant increase in Ptgfr mRNA expression in the POA. This upregulation of Ptgfr parallels the natural upregulation of Ptgfr expression that coincides with spawning, which is suggestive of increased sensitivity to PGF2α and increased activity of the PGF2α -mediated reproductive pathway (5). Furthermore, our results are consistent with preexisting knowledge about the ovarian hormone cycle in teleost fish. In teleosts such as A. burtoni, DHP is believed to act as the oocyte maturation-inducing hormone (17-19). It is released in the ovary at high levels after yolk formation and accumulation (vitellogenesis) in the oocyte (Figure 1) (20). Elevated ovarian DHP levels

! 18 directly precede the increase in PGF2α that accompanies ovulation. In that regard, the mechanism we propose explains the consequences of this ovarian hormone profile (Figure 1) on the spawning circuitry in the brain: the increasing levels of DHP during oocyte maturation leads to an increase of DHP binding to PR in the POA, which in turn upregulates Ptgfr expression in that region. The higher Ptgfr levels may afford sensitivity to the subsequent PGF2α spike at ovulation, priming the female to respond to male courtship.

Our results indicate that E2, either alone or in combination with DHP, does not upregulate Ptgfr (Figure 5). These results differ from studies in mammals, which have found that both E2 and progesterone are necessary to upregulate uterine prostaglandin receptors in rats (21). In teleosts, there is evidence that estrogen regulates PR in the brain (22). The link between estrogen and PR established by pre-existing literature led us to suspect that a combination of E2 and DHP activity would regulate Ptgfr in the POA. Such a finding would have been further consistent with the ovarian hormone cycle (Figure 1), where an increase in ovarian E2 precedes the increase in DHP. This hypothesis, though, was not supported by our results. E2 treatment did not increase POA Ptgfr expression beyond control levels, nor did the combined DHP and E2 treatment significantly increase Ptgfr expression beyond that of DHP treatment alone (Figure 5). Yet, we cannot conclusively reject the hypothesis that E2 plays a role in POA Ptgfr regulation. To do so, we will first need to increase the sample size of OVX females subject to combined DHP and E2 treatment and once again demonstrate the observed Ptgfr expression is not significantly greater than that from DHP treatment alone.

Another reason we cannot formally rule out the possibility that E2 plays a role in

POA Ptgfr regulation is because we were unable to conduct a dose response study for E2 due to time and resource constraints. Thus, it is possible that our negative results with respect to E2 regulation of Ptgfr were the consequence of either not treating with a sufficient concentration of E2 and/or not timing our E2 injections appropriately relative to sacrifice. The latter explanation comes into question because treatment of OVX females with E2 3 hours before sacrifice appeared to differ in terms of Ptgfr mRNA expression from OVX females treated with E2 for two days prior to sacrifice (Figure 5). We believe that this difference is rather accounted for by a systemic difference in the size of females

! 19 used in each condition (the females in the E2, 3hr condition were consistently smaller and thus had less POA matter to quantify for Ptgfr expression; data not shown). Overall, further analysis is pending to rule out E2’s role in POA Ptgfr regulation. We wished to tease apart the DHP regulation of POA Ptgfr at the level of neural circuitry. In contrast to the study by O’Connell et. al. 2013, which reported widespread PR expression in the A. burtoni brain (23), we localized PR expression to two specific areas. The two areas of PR expression, the POA and the NLT, also overlap with the regions of Ptgfr expression. In particular, the POA is the site of spawning dependent changes in Ptgfr expression. The overlap between PR and Ptgfr expression in this region suggests that signaling through PR could explain the changes in Ptgfr expression at spawning. The difference in observed ISH PR expression between our and the O’Connell et. al. (2013) study could be accounted for by a difference in ISH development time. If we develop our PR stained sections longer, we may discover additional PR+ populations in the brain. Another explanation for the difference in observed staining relates to the use of different antisense ISH PR probes. Overall, the combination of our ISH results for Ptgfr expression in DHP treated OVX females and our PR expression analysis provides evidence that DHP acts upstream of PGF2α activity to prime spawning in female A. burtoni. Further studies can be performed to extend this claim. First, we have yet to establish whether DHP via its PR receptor acts directly to upregulate PGF2α. Despite localization of the nuclear PR in the POA (Figure 6 & 7), we have not confirmed that the PR expressed in the POA is expressed in the same cells as POA Ptgfr. Additionally, we cannot at present rule out the possibility that DHP acts through another progesterone receptor, such as a membrane progesterone receptor, to regulate Ptgfr levels (29). We have, though, identified a putative PR binding site within the Ptgfr sequence ~600 bp 5’ from the transcriptional start site (S. Juntti, data not shown). The mechanism suggested is consistent with a model of direct regulation of Ptgfr by nuclear PR: DHP bound PR receptors in the POA act as transcription factors to drive expression of Ptgfr in the same cells. To test this hypothesis, we could conduct a luciferase reporter assay in HEK293 cells and observe whether PR can transcriptionally upregulate Ptgfr through this putative PR binding site. We would create two constructs, 1) a reporter construct that

! 20 would fuse the luciferase reporter gene with the Ptgfr promoter and surrounding regulatory sites, and 2) a construct containing the PR sequence under a ubiquitous promoter. These constructs would be co-transfected into cultured HEK293 cells and expressed. Luciferase induction in doubly transfected cells would suggest that the PR- Ptgfr relationship in the POA is cell autonomous and direct. Second, several other questions remain unanswered regarding how ovarian state information is integrated and communicated to the spawning neural circuitry. The model we propose suggests that DHP mediated regulation of Ptgfr controls sensitivity to PGF2α signaling. This model raises the question of how ovarian hormones DHP and PGF2α reach the brain, a site far removed from the gonads. If we suppose the DHP and PGF2α are synthesized in the ovary, we have yet to explain how these hydrophobic hormones molecules are transported through the aqueous bloodstream to the POA. Thus the site of

DHP and PGF2α synthesis and its mechanism of transport warrant further investigation. In particular, future studies will seek blood-borne carriers for prostaglandins. Finally, by identifying PR and Ptgfr expressing POA neurons as mediators of female spawning, we have found an important foothold in the neural circuitry of reproduction. Nonetheless, functional tests must be performed to demonstrate the necessity of the PR and Ptgfr neurons for reproductive behaviors. The stage has recently been set for such functional studies, since reverse genetics has been extended to the A. burtoni system (24). A Tol2 mediated transgenesis protocol has been established and paves the way for future loss of function and gain of function analyses of Ptgfr and PR. In addition, use of CRISPR technology could be harnessed to create Ptgfr and PR knockouts and thus assess their role of their downstream signaling in reproductive behaviors. The phenotypic consequences of such knockouts would shed light on what aspects of reproductive behaviors these receptors control. While our present and future investigations will continue to extend our understanding of the PGF2α pathway in teleosts, our work is relevant to mammalian systems. Prostaglandins direct reproductive behaviors in mammals as well. In mice and rats, the prostaglandin PGE2 appears to prime female reproductive behaviors, analogous to the role of PGF2α in teleosts (25). In mammals PGF2α appears to function primarily in maternal behaviors (26-27). It is unknown whether this difference between teleosts and

! 21 mammals constitutes a switch in PGF2α reproductive role or simply a narrowing of PGF2α activity. Since mating and parenting are temporally coupled in A. burtoni, we cannot be sure that the PGF2α signaling through POA Ptgfr is confined to mating behavior and does not encompass parental behaviors. Furthermore, Ptgfr expression in different brain regions could mediate different aspects of PGF2α reproductive signaling. For instance, PR and Ptgfr expression in the POA, and possibly the NLT, could mediate spawning while Ptgfr expression in other areas could mediate maternal behaviors.

In summary, the results of this study define a model for the regulation of PGF2α- mediated female reproductive behavior. We propose that DHP increases sensitivity to

PGF2α signaling, thereby preparing the female to engage in spawning behaviors. Pending, however, is a comprehensive picture of the circuitry through which these molecular factors act and are integrated. Pursuing such an investigation will lead us to ask whether reproductive circuits in teleosts have been conserved across vertebrates. Demonstrating conservation or divergence would shed light on the mechanisms that guide the evolution of reproductive circuits and behavioral neural circuits in general. While the specific hormones mediating different behaviors may have changed over time, we may find that the same neural circuits mediate the same modules of behavior; though PGF2α mediates different behaviors in mammals, perhaps this corresponds to a shift in which neural circuits are responsive to PGF2α instead of the evolution of new circuits. This study and future related investigations push us to ask such questions and make progress towards answering them.

! 22 References 1. Breedlove, S. M., Watson, N.V., & Rosenzweig, M. R. (2010). “Chapter 12: Sexual Behavior.” Biological psychology. Sinauer Associates.

2. Zemlan, F. P., & Adler, N. T. (1977). Hormonal control of female sexual behavior in the rat. Hormones and behavior, 9(3), 345-357.

3. Beach, F. A. (1976). Sexual attractivity, proceptivity, and receptivity in female mammals. Hormones and behavior, 7(1), 105-138.

4. Kauffman, A.S. (2010) “Mammalian Female Sexual Behavior and Hormones.” Encyclopedia of Animal Behavior. Editors-in-Chief: Michael D. Breed and Janice Moore, Oxford. Pages 355–369.

5. Juntti, S.A., Loveland, J., Fernald, R.D. (In review). Prostaglandin F2α receptors in discrete cell populations regulate female reproductive behavior in a teleost fish.

6. Fernald, R. D., & Maruska, K. P. (2012). Social information changes the brain. Proceedings of the National Academy of Sciences, 109(Supplement 2), 17194-17199.

7. Munakata, A., & Kobayashi, M. (2010). Endocrine control of sexual behavior in teleost fish. General and comparative endocrinology, 165(3), 456-468.

8. Nagahama, Y., & Yamashita, M. (2008). Regulation of oocyte maturation in fish. Development, growth & differentiation, 50, S195-S219.

9. Fernald, R. D. and N. R. Hirata (1977). Field study of Haplochromis burtoni : Quantitative behavioral observations. Animal Behaviour. 25:964-975.

10. Kobayashi, M., P.W. Sorensen, and N.E. Stacey. 2002. Hormonal and pheromonal control of spawning in goldfish. Fish Physiology and Biochemistry 26: 71-84.

11. Kobayashi, M. and Stacey, N. E. 1993. Prostaglandin-induced female spawning behavior in goldfish (Carassius auratus) appears independent of ovarian influence. Horm. Behav. 27: 38–55

12. Stacey, N.E (1977) The regulation of spawning behavior in the female goldfish Carassius auratus. Ph.D. Thesis. University of British Columbia, Vancouver, B.C. Canada

13. Christensen, L. W., Nance, D. M., & Gorski, R. A. (1977). Effects of hypothalamic and preoptic lesions on reproductive behavior in male rats. Brain research bulletin, 2(2), 137-141.

14. Burmeister, S. S., Jarvis, E. D., & Fernald, R. D. (2005). Rapid behavioral and

! 23 genomic responses to social opportunity. PLoS biology, 3(11), e363.

15. Stacey, N.E. (1987) “Chapter 2: Roles of hormones and pheromones in fish reproductive behaviors.” The Psychobiology of Reproductive Behavior: An Evolutionary Perspective. Prentice-Hall, Inc: Englewood Cliffs, N.J.

16. Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI- BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402.

17. Lister, A. L., & Van Der Kraak, G. J. (2009). Regulation of prostaglandin synthesis in ovaries of sexually‐mature zebrafish (Danio rerio). Molecular reproduction and development, 76(11), 1064-1075.

18. Antonopoulou E., Tsikliras A.C., Kocour M., Zlabek V., Flajshans M., Gela D., Piackova V., Scott A.P. (2011). Teleost maturation-inducing hormone, 17,20β- dihydroxypregn-4-en-3-one, peaks after spawning in Tinca tinca. General and Comparative Endocrinology, 172 (2) , pp. 234-242.

19. Miura, C., Higashino, T., & Miura, T. (2007). A progestin and an estrogen regulate early stages of oogenesis in fish. Biology of Reproduction, 77(5), 822-828

20. Sorensen, P. W., & Stacey, N. E. (2004). Brief review of fish pheromones and discussion of their possible uses in the control of non‐indigenous teleost . New Zealand Journal of Marine and Freshwater Research, 38(3), 399-417.

21. Blesson, C. S., Büttner, E., Masironi, B., & Sahlin, L. (2012). Prostaglandin receptors EP and FP are regulated by estradiol and progesterone in the uterus of ovariectomized rats. Reproductive Biology and Endocrinology, 10(1), 3.

22. Diotel, N., Servili, A., Gueguen, M. M., Mironov, S., Pellegrini, E., Vaillant, C., ... & Anglade, I. (2011). Nuclear progesterone receptors are up-regulated by estrogens in neurons and radial glial progenitors in the brain of zebrafish. PloS one, 6(11), e28375.

23. O'Connell LA, Ding JH, Hofmann HA (2013) Sex differences and similarities in the neuroendocrine regulation of social behavior in an African cichlid fish. Hormones and Behavior, 64:468-476.

24. Juntti, S. A., Hu, C. K., & Fernald, R. D. (2013). Tol2-Mediated Generation of a Transgenic Cichlid, Astatotilapia burtoni. PloS one, 8(10), e77647.

25. Rodriguez-Sierra, J. F., & Komisaruk, B. R. (1977). Effects of prostaglandin E2 and indomethacin on sexual behavior in the female rat. Hormones and behavior, 9(3), 281- 289.

! 24 26. Rodriguez-Sierra, J. F., & Rosenblatt, J. S. (1982). Pregnancy termination by prostaglandin F2α stimulates maternal behavior in the rat. Hormones and behavior, 16(3), 343-351.

27. McCarthy, M. M., Bare, J. E., & Vom Saal, F. S. (1986). Infanticide and parental behavior in wild female house mice: Effects of ovariectomy, adrenalectomy and administration of oxytocin and prostaglandin F2α. Physiology & behavior, 36(1), 17-23.

28. Crews, D., & Moore, M. C. (1986). Evolution of mechanisms controlling mating behavior. Science, 231(4734), 121-125.

29. Zhu, Y., Rice, C. D., Pang, Y., Pace, M., & Thomas, P. (2003). Cloning, expression, and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes. Proceedings of the National Academy of Sciences, 100(5), 2231-2236.

! 25 Figures

9

9 Figure91:99Ovarian!hormone!cycle!in!the!A.#burtoni!female.!E2,!DHP,!and!PGF2α!levels! rise!and!fall!of!over!the!course!of!the!ovarian!cycle,!beginning!with!initial!oocyte! development!(vitellogenesis;!left)!till!ovulation!and!egg!laying!(oviposition;!right).!S.! Juntti,!modified!from![20].!Note!the!additional!gray!curve!representing!our!working! model!for!Ptgfr!expression!in!the!POA,!based!on!the!findings!in!this!study!and!in#situ! hybridization!results!from![5].!!

! 26

Figure92:9Timeline!for!hormonal!and!vehicle!injections!of!OVX!A.#burtoni. The!treatment!timecourse!depicted!applies!to!all!5!operated!fish!groups!(OVX,! OVX+DHP,!OVX+E2,!OVX+E2(48),!OVX+E2(48)+DHP).!Injection!timepoints!are!shown! where!E2/V!or!DHP/V!is!indicated!above!the!timeline.!All!fish!experienced!injections! at!the!three!indicated!timepoints,!though!the!identity!of!these!injections!varied! based!on!the!group.!For!example,!OVX+E2(48)!fish!received!E2!for!their!first!two! injections,!followed!by!V!(vehicle)!for!their!last!injection.!However,!though! OVX+E2(48)+DHP!fish!also!received!E2!for!their!first!injection,!they!received!DHP!for! their!third.!OVX!fish!received!vehicle!for!all!three!injections.!The!total!time!from!OVX! until!dissection!was!kept!constant!despite!the!treatment!being!performed.!The!first! injection!was!performed!at!a!constant!time!(~2!weeks)!after!the!OVX!surgery!and!a! constant!time!from!dissection!(~2!days).! !

! ! ! !

! 27

Figure93:9Identification!and!confirmation!of!DHP!receptor!(PR)!sequence!in!A.!burtoni!genome.!! A)!tBLASTn!output!identifying!the!region!of!highest!homology!to!the!tilapia!PR!sequence!(scaffold! 78).!Tilapia!PR!reference!sequence!shown!below!in!blue.!Regions!in!bold!blue!correspond!to!exons! in!the!tilapia!sequence.!B)!PR!sequence!amplified!through!PCR!on!A.#burtoni!cDNA!from!(1)! hypothalamus;!(2)!ovary;!(3)!testes;!and!(4)!whole!brain!samples.!Primers!were!designed!against! exons!in!the!putative!PR!sequence,!and!predicted!to!yield!a!624bp!product.!All!samples!except!the! whole!brain!show!the!presence!of!desired!PCR!product.!

! 28

Figure94:9Ptgfr!expression!in!the!POA!of!OVX!+!DHP!treated!female!A.#burtoni!is! significantly!higher!than!OVX!treated!females.!A,B)!In#situ#staining!of!Ptgfr!in! representative!POA!sections!of!(A)!OVX!control!females!and!(B)!OVX!females! treated!with!DHP!3!hr.!before!sacrifice.!C)!Quantification!of!average!POA!Ptgfr! expression!in!OVX!and!OVX,!DHP!treated!females.!N=4!in!each!category.!Graph! shows!mean!staining!+/f!SEM.!Staining!between!groups!was!significantly! different!(*!indicates!significant!difference,!P=!0.0286)!

! 29

Figure95:9Ptgfr!expression!in!the!POA!of!OVX#A.#burtoni# females.!Females!were!treated! with!different!hormones,!at!different!time!intervals,!a ccording!to!the!labels!defined!in! Figure!2.!Ptgfr!mRNA!(ISH!quantification)!in!DHP!and!E48+DHP!treated!OVX!females!is! significantly!higher!than!Ptgfr!mRNA!levels!in!vehicle!treated!OVX.!All!other!groups!are! not!significantly!different!from!each!other.!N=4! in!vehicle,!DHP,!and!E48+DHP!treatment! groups.!N=3!in!E3!and!E48!treatment!groups. !

! 30

Figure96:9PR!expression!in!the!female!A.#burtoni!brain!is!localized!in!two! areas,!the!POA!and!NLT.!9 (AfA’)!In#situ!labeling!of!PR!mRNA!in!a!representative!POA!section,!shown!at! two!magnifications! (BfB’)!In#situ!labeling!of!PR!mRNA!in!a!representative!NLT!section,!shown!at! two!magnifications.!

! 31

Figure97:9Overlapping!Ptgfr!and!PR!expression!in!the!POA!of!female!A.#burtoni.!! A)!In#situ#staining!of!Ptgfr!in!the!POA!of!a!gravid,!intact,!and!untreated!female;!! B)!In#situ#staining!of!PR!in!the!POA!of!the!same!female!pictured!in!A).!The!sections! shown!are!serial!duplicates!from!the!same!brain,!and!thus!correspond!to!the!same! region!of!the!POA.!

! 32