Polycystic ovary syndrome (PCOS) is a common and complex endocrine condition with reproductive and metabolic complications, affecting up to 10% of reproductive-age women. Hyperandrogenemia, ovulatory dysfunction, and luteinising hormone hypersecretion are characteristic traits of PCOS however, it seems that the most concerning long-term key issues are metabolic problems associated with the syndrome, such as hyperinsulinemia, insulin resistance, obesity, dyslipidaemia and non-alcoholic liver disease. Despite the numerous studies on PCOS, its origin and pathophysiology are still not fully understood. However, there is increasing evidence that the adult PCOS phenotype is programmed in fetal life by androgen excess. Exposure to increased levels of testosterone in utero in rodents, sheep and monkeys result in adult reproductive and metabolic pathologies that parallel those seen in PCOS women. Since hyperandrogenemia is a hallmark of PCOS and daughters of PCOS mothers have elevated levels of androgens at birth, it is likely that prenatal androgenisation during early life predispose to the future development of PCOS. Animal models of PCOS provide an opportunity to examine the developmental aetiology and molecular mechanisms underlying the pathogenesis of this condition. Over last 10 years our lab has successfully utilised a well-established ovine model of PCOS, where pregnant ewes were treated with testosterone propionate (TP) through mid-gestation. From this model, we had a large sample bank of fixed and frozen tissues from the fetal, lamb and adolescent prenatally androgenised animals that allowed to carry a broad range of experiments. In addition, a new cohort of prenatally androgenised adult sheep enabled additional in vivo analysis. Past research documented that prenatal androgenisation result in hyperinsulinemia with altered pancreas structure and function, and early fatty liver without difference in body weight in adolescent sheep. This thesis examines the effects and consequences of increased in utero androgen exposure on metabolic dysregulation in adolescent and adult female sheep. During puberty, but not fetal or early life, there was decreased adipogenesis in subcutaneous adipose tissue (SAT), but not visceral adipose tissue (VAT), accompanied by decreased circulating concentrations of fibroblast growth factor 21 (FGF21), leptin and adiponectin, and increased concentrations of fasting free fatty acids (FFA) in prenatally androgenised sheep. This was countered by upregulated expression of FFA transporters in liver. As adults, TP-exposed animals had increased body weight, elevated fasting insulin and FFA concentrations but normal FGF21, leptin and adiponectin levels. Histological analysis revealed that adult TP-exposed animals had SAT hypertrophy, which was associated with increased expression of inflammatory markers and correlated with increased fasting FFA. Therefore, it is likely that impaired preadipocyte differentiation in SAT during adolescence resulted in hypertrophy and inflammation of adult SAT. This consequently lowered capacity of SAT to safely store fat and potentially explains metabolic perturbations observed in PCOS-like female sheep. To further investigate potential causes of obesity in adult PCOS-like sheep postprandial thermogenesis (PPT), an important constituent of energy expenditure, was measured through implantation of datalogger thermometers into interscapular adipose tissue. Adult prenatally androgenised sheep had decreased amplitude of PPT, without difference in basal body temperature, despite receiving the same caloric intake, and independent of obesity. These findings indicate that adult PCOS-like sheep have reduced capacity for energy expenditure, which is mirrored in women with PCOS. This reduced capacity for postprandial thermogenesis was correlated with hyperinsulinemia decreased noradrenaline levels and reduced thermogenic potential of brown and/or beige adipose tissue. This suggests that women with PCOS might be prenatally programmed to become obese. In summary, findings documented in this thesis provide better understanding into the pathophysiology of PCOS from puberty to adulthood and give opportunities for early clinical intervention to ameliorate the metabolic phenotype of PCOS.

The shock prod defensive burying test has proven incredibly reliable and instrumental in determining the underpinnings of normal anxiety in rodents. Largely ignored in tests of defensive burying, however, is the capacity for individual animals to display marked variations in active and passive coping behaviors. To unmask the neurobiological correlates of this behavioral differentiation, rats were exposed to a mousetrap that was remotely triggered upon approach to remove the quality of pain. This design invited striking variations in defensive burying behavior levels, in which some rats either buried robustly or showed little to no levels of defensive burying. Furthermore, differences in burying behavior were associated with marked differences in the central expression of arginine vasopressin (AVP) and oxytocin (OT). Thus, relative to animals showing no significant levels of defensive burying activity, rats showing sustained elevations in defensive burying expressed higher levels of AVP mRNA and increased numbers of androgen receptor positive cells in the medial amygdala and posterior bed nuclei of the stria terminalis, brain regions that integrate emotional appraisal and sensory information. In contrast, animals showing little to no defensive burying responses expressed relatively higher levels of AVP and OT mRNA within the supraoptic nucleus and subregions of the paraventricular nucleus of the hypothalamus responsible for neuroendocrine and autonomic function. CRH mRNA levels did not vary as a function of burying activity in the central nucleus of the amygdala, the anterior division of the bed nuclei of the stria terminalis, nor in the paraventricular nucleus. These findings suggest a role for central AVP and OT in mediating differential defensive behaviors, and demonstrate the utility of using a pain free test of conditioned defensive burying as a framework for exploring individual differences in behavioral coping and neuroendocrine capacity. / Medicine, Faculty of / Graduate

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BACKGROUND: Polycystic ovary syndrome is the most common endocrine disorder affecting reproductive-aged women. It is diagnosed using a combination of menstrual irregularity, clinical and/or biochemical hyperandrogenism and polycystic ovary morphology upon ultrasound. Hyperandrogenism in females may clinically manifest as hirsutism, acne, alopecia, or other masculinization of features. Assessing total/free testosterone, dehydroepiandrosterone sulfate, and 17-hydroxyprogesterone provides biochemical evidence of hyperandrogenism. OBJECTIVE: To determine self-reported clinical signs of androgen excess using data from the Ovulation and Menstruation Health (OM) Study, a diverse, multi-ethnic cohort study being conducted at Boston University School of Medicine. METHODS: Data was collected from participants enrolled in the Ovulation and Menstruation Health Study pilot cohort. This epidemiologic survey captured demographics, menstrual cycle patterns, PCOS histories, reproductive histories and manifestations of androgen excess in a diverse patient population. Participants were women ages 18-45 who had the capacity to ovulate/menstruate at the time of the study, had no history of chemotherapy, radiation, or surgical menopause, and were not pregnant at the time of the study. To assess androgen excess, participants were asked to self-report hair growth in nine body areas, acne on the face and back and hair loss on the scalp. The nine body areas were scored using the modified Ferriman-Gallwey (mFG) scoring system. Reference images created by a medical illustrator were used for hirsutism and alopecia grading while clear descriptions were provided for grading acne severity. Clinical hirsutism was defined as total mFG score of ≥ 8, or ethnic specific cutoff for East Asian (≥ 2) and Southeast Asian (≥ 3) women. Alopecia was defined as scalp hair loss ≥ 2. For participants that consented to medical record validation total, free and bioavailable testosterone lab levels were assessed for biochemical hyperandrogenism evaluation. RESULTS: Beginning August 9, the day the study opened to the public, 249 participants completed the pilot survey questionnaire. These participants were 66.8% white (n=165), 6.5% Hispanic or Spanish origin (n=16), 10.5% Black or African-American (n=26), 1.6% East Asian (n=4), 2.0% Southeast Asian (n=5), 2.4% South Asian (n=6), and 10.9% were of mixed ethnic backgrounds (n=27). 22.5% (55/245) of these women had clinical hirsutism by total mFG score. Mean total mFG scores were highest in women who were South Asian at 13.8±9.1 (n=6) and Hispanic at 8.6±8.7 (n=16). Moderate-severe acne was reported in 23.6% (58/246) of respondents, 24.8% (41/165) of white women, 26.7% (4/15) of Hispanic women, 15.4% (4/26) of Black women, 0.0% (0/4) of East Asian women, 20.0% (1/5) of Southeast Asian women, 50% of South Asian women (3/6) and 20% (5/25) of women of mixed ethnicities. 9.4% (23/246) of all pilot women reported alopecia, highest in Black (26.9%, 7/26) and East Asian women (25%, 1/4). Among women that had a PCOS diagnosis there was a higher presence of clinical hirsutism, higher acne severity, and higher prevalence of alopecia when compared to non-PCOS women. In addition, 33%(4/12) of the 44 women that consented to medical record validation had total testosterone levels above the normal range. CONCLUSIONS: This pilot population demonstrated an ethnic dependent pattern of development for hirsutism, acne and alopecia. Additionally, women who had a PCOS diagnosis were more likely to report having the clinical signs of androgen excess than those without a diagnosis. / 2020-06-14T00:00:00Z

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No description available.

The commonest reproductive disorders of young men (namely low sperm counts, testicular germ cell cancer) may originate in fetal life similar to established disorders (cryptorchidism, hypospadias) that manifest at birth. These disorders are interlinked and may comprise a testicular dysgenesis syndrome (TDS), a concept supported by animal model studies. The latter have identified the likely time-frame within which TDS disorders may be induced, namely within the so-called masculinisation programming window (MPW). During this critical period, sufficient testosterone (androgen) must be produced by the fetal testis to program the male reproductive tract so that it will differentiate and grow normally after the MPW. Impaired androgen production or action within the MPW can result in smaller reproductive organs and their abnormal formation and function (e.g. cryptorchidism, hypospadias). The MPW is thus of fundamental importance in determining normal, or abnormal, male reproductive development and function for later life. There are two big unanswered questions about the MPW. First, what determines its timing? Second, what mechanisms are controlled by androgens specifically within this time-window and not at later time points? Three approaches were undertaken to address the first question experimentally in rats. First, investigation of whether the availability of androgens and or androgen receptors (AR) plays a role in determining the onset or ‘opening’ of the MPW. Second, investigation of whether the expression of AR co-regulators was a factor in determining androgen sensitivity during the MPW. Third, investigation of whether prostaglandins played a role in mediating androgen action in the MPW, as studies in the 1980s had suggested this possibility. To address what mechanisms are controlled by androgens specifically within the MPW, the expression of selected genes in the genital tubercle was investigated before, during and after the MPW in fetuses that had been exposed to treatments that modulated androgen action. Selection of genes was based on microarray studies and data reported in the literature (ie candidate genes). The studies reported in this thesis show that neither availability of androgens nor the AR are important in determining onset of the MPW, and providing exogenous androgens either prior to or during the MPW does not advance or enhance masculinisation. These studies also showed that females may have a slightly different window of susceptibility to androgen action than do males. Key AR co-regulators have been characterized in the male reproductive tract for the first time, two of which (BRG1, CBP) show changes in expression through development of the testis consistent with a role in Sertoli cells. Another AR co-regulator, RWDD1, was found to switch off in the absence of androgen action in the genital tubercle, pointing to a potential role during and/or after the MPW. Studies involving gestational exposure to indomethacin (a compound which inhibits prostaglandin synthesis) during the MPW showed no detectable effect on masculinisation. Finally, evaluation of candidate genes for mediating androgen action in the genital tubercle during the MPW, failed to identify their key involvement, thus they are unlikely to be involved in penis development and disorders such as hypospadias.

The teleost ovarian follicle undergoes extensive remodeling and regression during the reproductive cycle—a process involving apoptosis and cell death. However, the hormonal regulation of these processes remains unclear. In the current study the role of testosterone in regulating regression of Atlantic croaker (Micropogonias undulatus) ovarian follicles was investigated in co-cultured granulosa/theca (G/T) cells. Testosterone (T) treatment enhanced serum starvation-induced cell death and apoptosis of G/T cells during the mature stage of oocyte maturation. This effect was mimicked by a cell-impermeable T conjugate, T-bovine serum albumin, indicating that this androgen action is initiated at the cell surface. Mibolerone, a nuclear androgen receptor agonist, was ineffective in promoting apoptosis and cell death, which suggests that T actions are independent from the nuclear receptor. Together, the data suggests that T-induction of apoptosis and cell death are through a novel membrane androgen receptor in the croaker ovary. T treatment also increased expression of a pro-apoptotic member of the Bcl-2 gene family, Bax, and two Bax upstream regulators, JNK and p53. These results suggest that T induces cell death of G/T cells in croaker through the apoptotic pathway involving JNK, p53 and Bax. An opposite response of cell death protection by T was also observed in G/T cells cultured from late-stage ovaries. This response was accompanied by a rapid increased ERK-1/-2 phosphorylation not seen in Mibolerone treatment. By examining the role of T in croaker follicle cell death and elucidating the corresponding basic mechanisms of androgen action, we are learning more about the regulatory components involved in the breakdown and remodeling stages of the teleost reproductive cycle. / text

The growth and survival of normal breast epithelial cells and breast cancer cells is promoted by estrogens. In contrast, androgens inhibit the proliferation of normal and malignant breast epithelial cells. While this effect of androgens on breast cells appears to be androgen receptor (AR) dependent, the precise mechanism of inhibition and its functional significance are unknown. The aims of this thesis were to investigate the effect of androgen signalling on growth of normal and malignant breast epithelial cells, and to assess the interactions between androgen and estrogen signalling in the breast. To investigate the role of androgen signalling in the growth and development of the normal mammary gland, female mice were treated with either the native androgen 5α- dihydrotestosterone (DHT) or the antiandrogen, flutamide. Analysis of the mammary glands at the end of the treatment period demonstrated that DHT reduced ductal branching and mammary epithelial cell proliferation when treatment commenced mid-puberty. Conversely, flutamide treatment that commenced post-puberty significantly increased ductal branching and proliferation of mammary epithelial cells. This data demonstrates that androgen signalling inhibits proliferation in the normal mammary gland, and may therefore oppose to the growth stimulatory effects of estrogen signalling to regulate breast growth and development. The antiproliferative effects of androgens on breast epithelial cells may be due in part to direct AR-mediated activation of androgen regulated genes, or alternatively, androgens could act indirectly through AR to inhibit estrogen receptor alpha (ERα) activity. Expression of fulllength AR or a truncated, constitutively active AR (AR-T707) significantly inhibited the activity of ectopically expressed ERα in MDA-MB-231 breast cancer cells (ERα- and ARnegative), in a dose-dependent manner. The functional consequences of inhibition of estrogen signalling by overexpressing AR were investigated in the T-47D breast cancer cell line (ERα- and AR-positive). Expression of AR-T707 in T-47D cells resulted in inhibition of both basal and estradiol-induced cell proliferation and a marked reduction in the steady-state protein levels of the estrogen regulated gene, PR. The final chapter investigated the mechanism by which AR inhibits ERα activity. A coimmunoprecipitation assay demonstrated an interaction between ectopically expressed AR and ERα in COS-1 cells, but not endogenous AR and ERα in a breast cancer cell line. To delineate the regions of AR required for inhibition of ERα signalling, various functional domains of the AR were mutated or deleted. Reporter gene assays showed that the inhibitory effects of AR were abrogated by deletion or mutation of the DNA binding domain (DBD). Furthermore, overexpression of the AR-DBD alone was sufficient to inhibit ERα activity. Consistent with a requirement for the DBD of AR to inhibit ERα activity, mobility shift assays demonstrated binding of AR to the Xenopus vitellogenin A2 consensus estrogen response element (cERE); however AR/ERα heterodimers were not detected on a cERE. Consistent with these findings, molecular modelling demonstrated that it is feasible for the DBD of AR to bind to a cERE and that it is unlikely that AR/ERα heterodimers could bind. Chromatin immunoprecipitation demonstrated recruitment of AR to the promoters of endogenous estrogen regulated genes. The findings suggest that the inhibitory effect of AR on ERα activity may occur either via formation of non-functional AR/ERα heterodimers that are unable to bind to EREs, or AR homodimers competing effectively for binding to EREs, in ERα target genes. The results in this thesis demonstrate an inhibitory effect of androgen signalling on growth of normal and malignant breast epithelial cells. Additionally, the inhibition of breast epithelial cell proliferation by androgen signalling can be attributed, at least in part, to inhibition of ERα activity. These studies have provided insight into androgen action in the breast, and support a model whereby androgens balance the stimulatory effects of estrogen signalling in normal and malignant breast epithelial cells. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008