Captive populations of Asian elephants (Elephas maximus) in North America are not self-sustaining, and increasing reproductive success within captive populations is a high priority. The ability to accurately predict parturition can have a direct impact on elephant welfare. Elephants in captivity often require significant preparation and management throughout the birthing process, and complications during labor and delivery can necessitate immediate intervention, including stillbirth, protracted labor, maternal aggression towards a newborn calf, and dystocia. Being able to predict when parturition will commence can ensure appropriate staff is available and adequate monitoring is performed. Routine endocrine sampling can be used to predict parturition in Asian elephants, with a drop in progesterone (P4) to baseline levels signaling parturition in 2-5 days. However, we determined this method is not without limitations, and it is not used in all institutions that house elephants. As changes in hormones regulate and alter behaviors, we investigated behavioral indicators as an additional management tool for predicting parturition, a time of drastic hormone changes. We conducted a study of five pregnancies in Asian elephants at the Oregon Zoo, U.S.A, and Taronga Zoo, Australia, between 2008 and 2012. In Chapter 2, I evaluated progesterone (P4) and cortisol levels across three time periods: Baseline; Pre, (the week preceding the drop in P4); and Post, (the period after the P4 drop). Levels of P4 were significantly lower, and levels of cortisol were significantly higher in the days just prior to parturition. I found considerable intra- and inter-individual variation in both endocrine profiles, which can make endocrine assessments difficult to interpret in real time. In Chapter 3, I investigated whether behaviors in the preparturition period could be predictive of impending parturition in the Asian elephant. ANOVA results indicated a significant difference in the amount of time that elephants spent walking backwards across three time periods (F(2) = 3.723, p = 0.033), with the behavior increasing as parturition approached. These results were supported by a non-parametric Kruskal- Wallis. Using a generalized linear mixed model (GLMM), I found that as P4 levels decrease, walking backwards behavior significantly increases. In Chapter 4, I evaluated investigative trunk behaviors, or "trunk checks", directed towards the temporal gland near the ear, mammary glands, vulva and anus of the pregnant dam. Investigative behaviors included both self-directed behaviors and those sent from herd mates towards the pregnant dam. Self-directed behaviors are most likely associated with physical changes in the pregnant dam, such as using the trunk to pull on swollen teats. Other-directed behaviors may stem from chemo-sensory signaling or other types of communication between herd mates, such as detecting changes in progesterone or cortisol. I ran GLMM and found that four trunk-check behaviors varied significantly with P4 and/or cortisol profiles. These were: self-checks of mammary glands increased with decreasing P4 levels; herd-mate-checks of mammary glands increased with decreasing P4 levels; self-checks of vulva increased with decreasing P4 levels and increasing cortisol levels; herd-mate-checks of anus increased with increasing cortisol levels. In Chapter 5, I evaluated activity budget behaviors in the pregnant elephants. Generalized comparisons were made to published activity budgets of typical captive Asian elephants. I report that activity budgets are within the range of normal activity, though I note a high level of inter-individual variation. In addition, I compared two sampling techniques, including one-zero and instantaneous sampling, that were used for activity budget data collection. I discuss the different results obtained by each sampling technique. These results are a very promising indication that behaviors, including walking backwards and multiple trunk-check behaviors, are changing over time or with parturition-related hormone profiles. We recommend that keepers, veterinary staff, and other observers that are familiar with the regular behavioral repertoire of a pregnant female should pay close attention to these highlighted behaviors. Keeping track of these behaviors, especially in conjunction with P4 and cortisol tracking, can help staff refine existing windows of expected parturition.

Several studies suggest that the administration of GnRH near the time of insemination improves pregnancy rates in cattle. It has also been reported that there is greater improvement in repeat-breeder animals than at first service. The mechanism for this observation has not been established. Twenty-eight lactating Holstein cows that returned to estrus after one or more inseminations from the usu caine Dairy were used in the study. Anilrals were rarxiomly divide:i into tW'O treatment groups, intrarmJscular administration of 100 ug GnRH or saline oontrol at the tirre of insemination. Blood samples were collected at o, 1, 1.5, 2, 2.5, 3 and 4 hours post-insemination for LH determination and on days 0 through 7, 10, 16 and 22 for progesterone determination. Pregnancy status was detennined by rectal palpation 40 to 47 days post insemination. serum IR concentrations reached peak concentrations (9.33 ± 5.5 ng/ml) by one hour following GnRH administration. This was significantly different from saline controls (p Serum progesterone levels increased as expected. from day o to day 16 in all animals. Animals treated with GnRH that became pregnant tended to have the highest progesterone levels beginning from day 4. Animals treated with GnRH that were non-pregnant at 40 to 47 days tended to have the lowest progesterone levels from days 4 through 10 but were high on day 16. Pregnant animals had higher progesterone levels than non-pregnant animals from days 4 to 16. These differences approached significance (0.25 > p < 0.10). These results support the contention that GnRH administration affects progesterone levels rut do not conclusively establish increased early progesterone levels as the mechanism for improved pregnancy rates. Other hormonal andl functional factors may be involved.

L’implantation est caractérisée par les premiers contacts cellulaires permanents entre l’endomètre, tapissant l’utérus, et le conceptus (disque embryonnaire et tissus extra-embryonnaires). Cette étape se trouve être l’un des plus importants points de contrôle de la gestation nécessitant un dialogue finement régulé entre ces deux entités. Concernant les ruminants, un déclin de la fertilité a été observé notamment chez les vaches laitières hautes productrices. La moitié des gestations s’arrête pendant la période pré-implantatoire due à des mortalités embryonnaires précoces ainsi qu’à des défauts utérins. Depuis 10 ans, des analyses exploratoires ont été mises en place dans le but d’étudier les profils d’expression de gènes endométriaux sous l’influence du cycle oestral, de la gestation précoce ou encore des stéroïdes ovariens comme la progestérone et les oestrogènes. Ces études sont essentielles pour l’identification des gènes endométriaux clés pour la survie et la croissance du conceptus avant l’implantation. Notre laboratoire a réalisé une analyse transcriptomique à partir d’échantillons endométriaux collectés sur des vaches cycliques et gestantes au 20 ème jour post-oestrus correspondant respectivement à la phase folliculaire et au premier jour d’implantation. Plusieurs familles de facteurs de transcription apparaissent différentiellement exprimées dans cette étude, notamment FOXL2, un membre de la famille des Forkhead Box transcription factor considéré comme le gène clé de la différenciation ovarienne. Ce travail de thèse s’est intéressé à l’implication de FOXL2 dans la physiologie endométriale. FOXL2 est exprimé et régulé pendant le cycle oestral et la gestation précoce dans l’endomètre de ruminants. De plus, la progestérone a été identifiée comme le régulateur majeur de l’expression endométriale de FOXL2 chez la vache et la brebis alors que l’effet des estrogènes n’a pas été démontré. A partir d’une approche gènes candidats, la surexpression de FOXL2 induit la régulation différentielle de onze gènes potentiellement cibles de FOXL2 dans des cultures primaires endométriales de cellules stromales et épithéliales glandulaires. En particulier, PTGS2 qui est un gène impliqué dans la réceptivité utérine apparait inhibé par FOXL2 alors que SCARA5 et RSAD2, tout deux impliqués dans la réponse immunitaire sont stimulés. Enfin, DLX5 apparait différentiellement régulé entre les cellules stromales et épithéliales glandulaires sous l’impact d’une surexpression de FOXL2. Pour conclure, l’expression endométriale de FOXL2 est fortement liée au processus de réceptivité utérine qui se déroule avant l’implantation et peut moduler l’expression de gènes endométriaux essentiels. De nouvelles analyses sont nécessaires pour déterminer si FOXL2 est le gardien de la physiologie reproductive femelle. / Implantation is characterized by the first permanent cellular interactions between the endometrium, lining the uterus, and the conceptus (embryonic disk and extra-embryonic tissues) and appears to be one of the most important checkpoints of successful pregnancy. Regarding ruminant species, and more specifically dairy cows, half of pregnancies abort during the pre-implantation period due to early embryonic death and uterine defects. In the last decade, exploratory approaches have been developed to study endometrial genes expression under the influence of oestrous cycle, early pregnancy, and ovarian steroid hormones in order to identify systematically crucial endometrial genes for conceptus growth and survival leading to a successful implantation in ruminant specifically. A microarray analyse made at the laboratory based on endometrial samples collected from cyclic and pregnant cows at 20 days post-oestrous, corresponding respectively to the follicular phase and the implantation initiation. Several members of the transcription factor families appeared to be differentially expressed in this study including FOXL2, a member of the Forkhead box L sub-class originally considered as a key gene for ovarian differentiation. My PhD thesis focused on the implication of FOXL2 gene in endometrial physiology. FOXL2 gene had been demonstrated to be expressed and regulated during the oestrous cycle and early pregnancy in ruminant endometrium. Moreover, progesterone was identified as a master regulator of FOXL2 endometrial expression in both cattle and sheep whereas estrogens have no impact. Based on candidate genes approach, over-expression of FOXL2 gene induces a regulation of eleven putative FOXL2 target genes in primary endometrial stromal and glandular epithelial cells. In particular, PTGS2 which is a positive regulator gene for uterine receptivity was shown to be inhibited whereas SCARA5 and RSAD2 expressions that were involved in immune response were shown to be stimulated as well as DLX5 expression was differentially regulated between stromal and glandular epithelial cells. Collectively, FOXL2 endometrial expression is strongly linked to the uterine receptivity process prior to the implantation and modulates the expression of essential endometrial genes. Further investigations will be required to investigate whether FOXL2 is the gatekeeper of female reproduction in the vertebrate species.

Includes list of articles published or accepted for publication during the period of PhD candidature. / "July, 2004" / Includes bibliographical references (leaves 255-293) / xxviii, 293 leaves : ill., plates (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pathology, 2004?

WW-domain binding protein-2 (WBP-2) was cloned as an E6-associated protein (E6-AP) interacting protein and its role in steroid hormone receptor (SHR) function was investigated. We show that WBP-2 differs from other SHR coactivators, as it specifically enhanced the transactivation functions of progesterone receptor (PR) and estrogen receptor (ER alpha), whereas it had no significant effect on the androgen receptor, glucocorticoid receptor or the activation functions of p53 or VP-16. We also demonstrated that, like other well characterized coactivators, WBP-2 contains an intrinsic activation domain. Depletion of endogenous WBP-2 with small interfering RNAs indicated that normal physiological protein level of WBP-2 was required for the proper functioning of ER alpha and PR. Moreover, chromatin immunoprecipitation (ChIP) assays demonstrate the hormone-dependent recruitment of WBP-2 onto an estrogen-responsive promoter. As we initially identified WBP-2 as an E6-AP interacting protein, we investigated whether WBP-2 and E6-AP function in concert. Our data shows that WBP-2 and E6-AP each enhance PR function and when co-expressed they additively enhance the transactivation functions of PR. However, WBP-2 was also able to enhance the transactivation functions of ER alpha and PR in mouse embryonic fibroblast cells generated from E6-AP knockout mice lines, suggesting that the coactivation functions of WBP-2 was not dependent on E6-AP. The further elucidate the molecular mechanism of action of WBP-2; we dissected the functional importance of the polyproline (PY) motifs contained within the WBP-2 protein. Mutational analysis suggests that one of three PY motifs, PY3 of WBP-2 was essential for its coactivation and intrinsic activation functions. In this study, we also demonstrate that the WBP-2 binding protein, Yes-kinase associated protein 1 (YAP1) acts as a secondary coactivator of ER alpha and PR. However, the coactivation function of YAP1 is revealed only in the presence of wild-type WBP-2 and not with the PY motif 3 mutant WBP-2. This is consistent with our observations that, unlike the wild-type WBP-2, the PY motif 3 mutant WBP-2 does not interact with YAP1. Our quantitative reChIP assays demonstrates an estrogen-dependent recruitment and association of ER alpha with both WBP-2 and YAP1. The hormone-dependent recruitment of YAP1 to ER alpha responsive promoter is dependent on the physiological expression levels of WBP-2. This is consistent with, our observation that the coactivation functions of YAP1 is dependent on WBP-2, and is also in agreement with other known secondary coactivators that get recruited to SHR responsive promoter via their interaction with primary coactivators. Surprisingly, the association of WBP-2 with ER alpha and its recruitment to the ER alpha target promoter was abrogated by YAP1 knock-down, suggesting that WBP-2 and YAP1 may stabilize each other at the promoter, and consequently, are functionally interdependent. Taken together our data establish the role of WBP-2 and YAP1 as selective coactivators for ER alpha and PR transactivation pathways.

Sex steroid exposure constitutes a risk factor for breast cancer, but little is known about the effects of sex steroids on factors mediating angiogenesis, the development of new blood vessels, in normal and malignant breast tissue. In this thesis we have investigated the effects of estradiol, progesterone, and the nonsteroidal anti-estrogen tamoxifen on vascular endothelial growth factor (VEGF) and its receptors (VEGFR-1 and VEGFR-2) in normal human breast tissue, endothelial cells, and breast cancer. We have applied the technique of microdialysis to provide in situ sampling of estradiol and VEGF in tumors and normal breast tissue of breast cancer patients in vivo. Furthermore, we present a novel method of culturing normal human breast tissue ex vivo. Our results suggest a pro-angiogenic effect of estradiol and an anti-angiogenic effect of tamoxifen in the breast. Estradiol increased extracellular levels of VEGF in normal human breast tissue and breast cancer cells in vitro. In addition, estradiol decreased sVEGFR-1 in breast cancer cells and indirectly increased VEGFR-2 in endothelial cells. Compared to estradiol treatment alone, estradiol + tamoxifen increased sVEGFR-1 and decreased VEGF in breast cancer cells in vitro. Furthermore, estradiol + tamoxifen decreased tumor VEGF levels and tumor vasculature in human breast cancer xenografts in vivo. In breast cancer patients, a significant correlation was found between in vivo levels of estradiol and VEGF sampled by microdialysis in normal human breast tissue, suggesting that estradiol may be a potent regulator of VEGF in the breast in vivo. Tumor levels of VEGF were significantly higher than in normal breast tissue in vivo, supporting the role of VEGF in tumor angiogenesis. For studies of normal human breast, whole breast tissue may be cultured in vitro for up to one week with preserved morphology. Using this method, estradiol, and not progesterone, appears to be the main sex steroid regulator of extracellular VEGF in normal breast tissue. In conclusion, the data suggest that sex steroids and tamoxifen exert pro- and anti-angiogenic effects in normal breast tissue and breast cancer.

The aim of this thesis was to determine any association between plasma oxytocin and vasopressin concentrations and renal water and sodium excretion during normal pregnancy. In addition to investigate changes in concentrations of estradiol, progesterone, oxytocin, cortisol, and glucose in the blood before and in the nearest hours after delivery and if treatment with oxytocin affected these concentrations and the fluid balance during the different stages of labour. Oxytocin, vasopressin, estradiol, progesterone, and cortisol were analysed in blood plasma or serum by radioimmunoassay or ELISA: serum glucose, and osmolality, and sodium in plasma and urine were  analysed by standard laboratory techniques. Fifty-seven women were studied during pregnancy and fifty-one during parturition and post partum. The low plasma vasopressin and increasing plasma oxytocin concentrations with unchanged water and sodium excretion indicate that oxytocin assists vasopressin in concentrating urine during pregnancy. Plasma vasopressin concentration continued to be low during parturition and post partum. Urine flow and concentration was unrelated to changes in plasma sodium concentration, indicating regulation of fluid balance during parturition was different to the non-gravid state. Women with weak myometrial contractions during parturition (slow progress of labour) reacted differently than women with normal parturition and a group of women with fast progress of labour. The group with slow labour had lower serum estradiol concentration in the latency phase and became hyponatremic. Pulsatile and continuous oxytocin infusions were both effective in the treatment of slow progress of labour. A lower amount of oxytocin was needed to affect delivery when given as pulsatile infusion. Serum cortisol and glucose concentrations were high during labour and cortisol level remained elevated after delivery and glucose concentration reached the highest levels (12 mmol/L) at the same time. Insulin resistance together with the long time of elevated cortisol concentration partly explained the high glucose concentration. In conclusion, fluid balance is not regulated according to the usual sensitive osmotic and volumetric influence on vasopressin release from the neurohypophysis during pregnancy and parturition. Parturition involves a change from one demanding condition, pregnancy, to another, lactation. Parturition and the hours directly after delivery are a turbulent period involving considerable stress.

Several studies suggest profound effects on mood and cognition by neuroactive steroids. Estrogen alone or in combination with antidepressant drugs affecting the serotonin system has been used to treat mood disorders. On the other hand, progesterone is related to negative effects on mood and memory. A major part of the progesterone effects on the brain can be mediated by its metabolite allopregnanolone, which is also de novo synthesized in the brain, and affects the GABAA receptors. It would be of great importance to find a substance that antagonize allopregnanolone adverse effects. To investigate how long term supplementation of estradiol and progesterone, resembling postmenopausal hormone replacement therapy, affects serotonin receptors in different brain areas important for mood and memory functions, we used ovariectomized female rats. After 2 weeks of supplementation with 17β-estradiol alone or in combination with progesterone, or placebo pellets, estradiol alone decreases but estradiol supplemented together with progesterone increases 5HT1A mRNA expression in the hippocampus. Estradiol decreases the 5HT2C receptor gene expression, while estradiol in combination with progesterone increases the 5HT2A mRNA expression in the ventral hippocampus. Thus, estradiol alone has opposite effects compared to the estradiol/progesterone combination. To detect if acute tolerance develops to allopregnanolone, an EEG method was used where male rats by continuous allopregnanolone infusion were kept on anesthesia level of the silent second (SS). After different time intervals (first SS, 30 min or 90 min of anesthesia) several GABAA receptor subunit mRNAs were measured for detecting if changed expression of any GABAA receptor subunits is involved in development of acute tolerance. There is development of acute tolerance to allopregnanolone and brain regions of importance are hippocampus, thalamus and hypothalamus. The GABAA receptor alpha4 subunit in thalamus and alpha2 subunit in the dorsal hippocampus are related to development of acute tolerance. For assessing allopregnanolone behavioral effects, we studied how this neurosteroid affects spatial learning in the Morris water maze task Allopregnanolone inhibits spatial learning short after the injection and shows a specific behavioral pattern with swimming close to the pool wall. The steroid UC1011 can inhibit the increase in chloride ion uptake induced by allopregnanolone. UC1011 decreases allopregnanoloneinduced impairment of spatial learning in the water maze, as well as the specific behavioral swim pattern. In conclusion, the present work demonstrates that neuroactive steroids affect the 5HT and GABA systems in a brain region specific way. GABAA receptor subunit changes in hippocampus and thalamus are related to acute allopregnanolone tolerance. Allopregnanolone induces cognitive deficits, like spatial learning impairment and UC1011 can inhibit allopregnanolone-induced effects in vitro and in vivo. Key words: Estradiol, progesterone, HRT, allopregnanolone, UC1011, serotonin receptor, GABAA receptor, mRNA, Morris water maze, silent second, tolerance.

Estrogen therapy (ET) is most effective to reduce menopausal symptoms and prevent other disorders associated with estrogen deficiency. However, Women's Health Initiative studies found that hormone therapy (HT) containing estrogen plus progestogen, but not estrogen-alone increases breast cancer (BC) risk. To prevent the increase in BC risk and yet relieve menopausal symptoms, a novel HT with 17&#946;-estradiol (E2) for symptom relief, progesterone (P4) for uterine protection and melatonin (Mel) for both BC and uterine protection was designed. Inclusion of Mel was postulated to offer uterine protection with lower P4 dose and protect against BC. The goal of this study was to assess the efficacy of E2, P4 and Mel Therapy (EPMT) on mammary cancer (MC) and uterine protection in MMTV-Neu mouse model that mimics HER2 BC. Starting at 2 months age, female mice received Mel in drinking water at night to supplement endogenous Mel surge; while E2 and P4 Therapy (EPT) was provided continuously in diet until 14 months with weekly MC onset and growth monitoring. Normal mammary, uterus and mammary tumors harvested by month 14 were analyzed for potential mechanisms. The results from this study revealed that EPMT delayed tumor onset leading to a decrease in MC incidence. In addition, mice in the EPMT group had no increase in relative uterine weight as opposite to an increase of this parameter in EPT group versus control. The percent tumor-bearing mice with gross metastatic lung lesions were reduced in Mel, EPT and EPMT groups. Mel receptor, estrogen receptor (ER) and progesterone receptor (PR) expression revealed that all tissues examined have Mel receptors. However, ER and PR expression varied. In normal mammary tissue, both ER&#945; and PR were detected by immunohistochemistry. However, no ER&#945; and PR were detected in mammary tumors of same mice. In uterus, mice given Mel or EPMT had significant decreases in PR expression but no change in ER&#945; expression compared to control suggesting that Mel-mediated inhibition of ER binding to estrogen response elements may be involved in the down regulation of uterine PRs. Overall, this study reveal that EPMT prevents mammary cancer and may protect against uterotrophy. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Pharmacology / PhD; / Dissertation;

Osmotic swelling of glial cells may contribute to the development of retinal edema. We investigated whether sex steroids inhibit the swelling of glial somata in acutely isolated retinal slices and glial cells of the rat. Superfusion of retinal slices or cells from control animals with a hypoosmolar solution did not induce glial swelling, whereas glial swelling was observed in slices of postis- chemic and diabetic retinas. Progesterone, testosterone, estriol, and 17ß-estradiol prevented glial swelling with half-maximal effects at approximately 0.3, 0.6, 6, and 20 lM, respectively. The effect of progesterone was apparently mediated by transactivation of metabotropic glutamate receptors, P2Y1, and adenosine A1 receptors. The data suggest that sex steroids may inhibit cytotoxic edema in the retina.