Abstract 17β-Hydroxysteroid dehydrogenases (17HSDs)/17-ketosteroid reductases (17KSRs) modulate the biological activity of certain estrogens and androgens by catalyzing dehydrogenase and reductase reactions between 17β-hydroxy and 17-ketosteroids. In the present study, cDNAs encoding mouse and rat 17HSD/KSR1 were cloned in order to study the role of rodent type 1 enzyme in ovarian estradiol (E2) biosynthesis and its enzymatic characteristics. Both rat and mouse 17HSD/KSR1 were expressed in granulosa cells of developing follicles, where diethylstilbestrol and follicle-stimulating hormone stimulated follicular maturation and up-regulated the expression of 17HSD/KSR1, whereas human chorionic gonadotropin caused luteinization of follicles and down-regulation of the enzyme. In line with this, the rodent type 1 enzymes are not expressed in the corpus luteum (CL). Mouse 17HSD/KSR1 showed substrate specificity different from that of the human counterpart. The mouse type 1 enzyme catalyzed the reaction from androstenedione to testosterone at least as efficiently as estrone (E1) to E2, while human 17HSD/KSR1 clearly preferred the E1 to E2 reaction. A mouse mammary epithelial cell line was found to possess strong estrogenic 17KSR activity. A novel type of 17HSD/KSR responsible for this activity was expression-cloned on the basis of its ability to convert E1 to E2 and it was chronologically named 17HSD/KSR7. Interestingly, it showed 89 % identity with a rat protein called prolactin receptor-associated protein (PRAP), which is expressed in the CL. Enzymatic characterization showed that both mouse 17HSD/KSR7 and PRAP efficiently catalyzed the reaction from E1 to E2. The mouse type 7 enzyme was most abundantly expressed in the ovary and placenta. Similar primary structure, enzymatic characteristics, and tissue distribution of mouse 17HSD/KSR7 and PRAP suggest that PRAP is rat 17HSD/KSR7. Further studies showed that in rat ovaries 17HSD/KSR7 is primarily expressed in the middle and second half of pregnancy, in parallel with E2 secretion from the CL. Using in situ hybridization, cell-specific expression of 17HSD/KSR7 was studied in the mouse ovary, uterus and placenta. In the mouse ovary, the enzyme was expressed exclusively in the CL. In the uterus on day 5 post coitum (p.c.), the type 7 enzyme was expressed in the decidua, mostly in the inner zone of antimesometrial decidua. Between day 8 and 9 p.c. the enzyme was abundant in decidua capsularis of the developing placenta, after which expression moved to the basal zone. On days 12 and 14 p.c., mouse type 7 enzyme was abundantly expressed in the spongiotrophoblasts, where expression decreased towards parturition. Altogether, rodent 17HSD/KSR7 is a new 17HSD/KSR which is involved in the biosynthesis of E2 in the ovaries. In addition, E2 produced locally in the decidua and placenta by the type 7 enzyme may have a role in decidualization and/or implantation and placentation.

No description available.

No description available.

Between 10-15% of women suffer from polycystic ovary syndrome (PCOS), making it the most common cause of female infertility. Clinical features of PCOS include high circulating levels of ovarian androgens (T and A4), anovulation and obesity. The aetiology of this reproductive endocrinopathy is likely to be multifactorial, through the interplay of genetics, epigenetics and environmental factors. Primate research into sexual behaviour development noted that fetally androgenised monkeys developed symptoms like those of PCOS. There are now multiple animal models of PCOS using primates, sheep, rats and transgenic mice. The investigations described in this thesis use rodent models to examine the role of androgens in the pathogenesis of female infertility. An attempt to generate a granulosa cell specific androgen receptor knockout mouse model will first be described, followed by several studies into the developmental programming of female Wistar rat infertility and metabolism by steroid hormones. Initial investigations showed that testosterone proprionate (TP) administered to female rats during different windows of fetal and neonatal life alters the reproductive and metabolic axes of the adult animals. Fetal plus neonatal TP exposure led to complete ovarian dysgenesis, while postnatal exposure produced a PCOS-like phenotype. Animals which received TP postnatally were heavier and had an increased proportion of primordial follicles in their ovaries by postnatal day (pnd) 90 of life. Evaluation of this PCOS model showed that neonatally androgenised rats had ovarian follicles with larger antra and a greater ovarian stromal compartment. In addition, these animals were heavier when compared to controls. However, unlike human studies, neonatally androgenised rats showed no differences in circulating gonadotrophin or ovarian androgen levels. Nor did they show any programming effect of neonatal TP upon the theca interna by pnd 90. Further investigations to narrow the windows and dose of TP required to produce a PCOS phenotype showed that TP administered in an early window of neonatal life, between postnatal days (pnd) 1-6 not only led to anovulation, but potentially reprogrammed the hypothalamic-pituitary axis, as there was minimal gonadotrophin response to reduced ovarian negative feedback (inhibin B and estradiol) in these rats. Neonatal TP also affected the rat metabolic axis with adult animals becoming heavier after weaning without any change in food intake. Animals developed mesenteric and retroperitoneal obesity along with insulin resistance (IR). Increased hepatic glucocorticoid turnover and altered adipokine expression were also noted in neonatally androgenised females, possibly contributing to the pathogenesis of obesity. No effect of TP dose upon the severity of infertility or metabolic abnormalities in adult animals was observed. To delineate which features of the rat PCOS model resulted from androgenic, estrogenic or corticosteroid action, a final study used administration of different steroids during the early window of postnatal life: TP, estradiol valerate (EV), dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA) and dexamethasone (DEX). The anovulatory PCO-like phenotype observed with TP was also seen in animals which received EV, but not those which received DHT, DHEA or DEX. TP and EV treatment also resulted in a reduction of ovarian follicle numbers and activated follicle proportions, with an increase in primordial follicle proportions. Although glucose tolerant, animals treated with TP and EV were highly IR. Unlike dexamethasone, DHT and DHEA also produced IR in adult animals, to a lesser extent than TP and EV. Taken collectively, the results described in this thesis demonstrate that the PCOS-like phenotype observed in the neonatally androgenised female rat is likely to be due to the estrogenic actions of testosterone, potentially through as yet unknown epigenetic mechanisms. The programming of the metabolic components described may additionally be due to the actions of androgens. Furthermore, these studies demonstrate a novel estrogenic effect of neonatal steroids upon primordial follicle populations and show that the neonatally androgenised rat may be a rational PCOS model in a poly-ovulatory species.

Background Polycystic ovary syndrome (PCOS) is a common endocrine condition in women associated with obesity, reproductive and metabolic abnormalities. It improves with weight loss, however currently no specific dietary recommendations exist and there may be abnormalities in appetite regulation in PCOS that contribute to difficulty in weight management. Aims To assess the effect of 1) short and long-term weight loss and weight maintenance strategies on weight loss, reproductive and metabolic parameters in overweight women with PCOS and to 2) assess the relative effect of weight loss on cardiovascular risk factors and 3) postprandial appetite, appetite hormones (ghrelin, CCK, PYY) and food intake in overweight women with and without PCOS. Results Overweight women with PCOS followed an 8-week weight loss (2 meal replacements/day, 4904.4±127 kJ, n=32) followed by a 6 month carbohydrate (<120 g/day) or fat restricted (<50 g/day) weight maintenance regime (n=23). Reductions in weight (5.6±2.4 kg) and improvements in body composition, insulin, reproductive hormones and menstrual cyclicity occurred and were sustained equivalently for both diet groups. We then assessed the effect of weight loss (4.2±0.7 kg over 8 weeks as described above) in overweight women with (n=15) and without (n=17) PCOS on cardiovascular risk factors. All subjects had similar improvements in body composition, triglycerides, reproductive hormones and fasting and post-prandial insulin. C-reactive protein decreased with weight loss for non-PCOS women (-1.2±0.5 mg/L, P=0.025) but not for PCOS women. We finally assessed appetite regulation in PCOS. Women with (n=20) and without (n=12) PCOS followed a standard protein (55% carbohydrate, 15% protein) or high protein diet (40% carbohydrate, 30% protein) for 16 weeks (~6000 kJ/day). Non-PCOS subjects were more satiated (P=0.001) and less hungry (P=0.007) after the test meals and had a 70% higher fasting baseline ghrelin (P=0.011), a greater increase in fasting ghrelin (57.5 versus 34.0%, P=0.033), a greater post-prandial ghrelin decrease at week 16 (113.5±46.3 versus 49.3±12.2 pg/mL, P=0.05) and a greater maximal decrease in post-prandial ghrelin (-144.1±58.4 versus -28.9±14.2 pg/mL, P=0.02) following weight loss than subjects with PCOS. Lastly, women with (n=14) and without (n=14) PCOS undertook an 8-week weight loss regime (4.2±0.7 kg as described above). At week 0 and 8, women with PCOS again displayed lower ghrelin levels (P=0.01 and P=0.097 respectively) and a lesser post-prandial ghrelin decrease (P=0.048 and P=0.069 respectively) but similar post-prandial appetite, buffet consumption and fasting or post-prandial peptide YY and cholecystokinin compared to women without PCOS. Conclusion Meal replacements and moderate macronutrient restriction are effective strategies for the dietary management of PCOS. Equivalent weight losses improved cardiovascular risk factors similarly for overweight women with and without PCOS with the exception of CRP which did not decrease with weight loss for overweight women with PCOS. PCOS status is associated with altered fasting and post-prandial ghrelin levels but is not consistently associated with other impairments in post-prandial gut peptides or food intake. Further investigation is required to assess if appetite regulation is impaired in PCOS and the optimal strategies and amount of weight loss for improvement of reproductive and metabolic parameters in PCOS. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1282329 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2007

The purpose of the studies reported in this thesis was to increase our understanding of the reproductive physiology of South American camelids. Studies were conducted in llamas and alpacas to investigate methods to electively control ovarian follicular dynamics, to determine the effects of hormone preparations or biological factors derived from seminal plasma on ovulation induction, and to evaluate the establishment of superstimulatory protocols to induce a consistent ovarian follicular response for oocyte collection. The first study was designed to compare the efficacy of treatments intended to induce follicular wave synchronization among llamas, and to determine the effect of these treatments on pregnancy rates after fixed-time natural mating. In the first experiment, lutenizing hormone (LH) and follicular ablation treatments were most effective for inducing follicular wave synchronization, while estradiol plus progesterone (E/P) treatment was intermediate. In the second experiment, llamas were assigned randomly to Control, (E/P), and LH groups. A single, fixed-time natural mating was permitted 10 to 12 days after treatment. The pregnancy rate was higher (P<0.05) for synchronized llamas (LH and E/P groups combined) than for non-synchronized llamas (Control group). The second study was done to compare the effects of hormonal treatments and natural mating on ovulation induction, interval to ovulation, and luteal development in llamas. No differences were detected among groups (mated, LH, and GnRH) in ovulation rate (80%, 91%, 80%, respectively; P = 0.6), or interval from treatment to ovulation (30.0 ¡À 0.5, 29.3 ¡À 0.6, 29.3 ¡À 0.7 h, respectively; P = 0.9). Similarly, no differences were detected among groups (mated, LH, and GnRH) in maximum corpus luteum (CL) diameter. The third study documents the existence of an ovulation-inducing factor (OIF) in the seminal plasma of alpacas and llamas. In Experiment 1, female alpacas were given alpaca seminal plasma or saline intramuscularly (im) or by intrauterine infusion. Only alpacas that were given seminal plasma im ovulated. In Experiment 2, ovulation was detected in 9/10 (90%) llamas at a mean of 29.3 ¡À 0.7 hours after seminal plasma treatment. In Experiment 3, female llamas were given llama seminal plasma, GnRH, or saline im, and ovulation was detected in 6/6, 5/6, and 0/6 llamas, respectively (P < 0.001). Treatment was followed by a surge (P < 0.01) in plasma LH concentration beginning 15 minutes and 75 minutes after treatment with GnRH and seminal plasma, respectively. Plasma LH remained elevated longer in the seminal plasma group (P < 0.05), and plasma progesterone concentration was twice as high in the seminal plasma group (P < 0.01). The fourth study describes the presence of an OIF in the seminal plasma of Bos taurus ¨C a species conventionally considered to ovulate spontaneously - contains OIF. Bull seminal plasma induced ovulations in 26% (5/19) of llamas compared to 0% (0/19) in PBS group (P < 0.001). The ovulation rate was lower (P < 0.01) in bull seminal plasma group compared to that in the groups treated with alpaca or llama seminal plasma (100%). The fifth study was conducted to determine a local versus systemic effect of ovulation-inducing factor in seminal plasma. Ovulation rate in the seminal plasma intramuscular group (93%) was higher (P < 0.01) than seminal plasma intrauterine group (41%), while the seminal plasma intrauterine curettage group was intermediate (67%). The sixth study was done to determine the time required for llama oocyte to reach the maturation stage, and to establish a superstimulatory treatment for oocyte collection. Llama oocytes reached second metaphase as early as 28 h after in vitro culture. The FSH- and eCG-treated groups did not differ (P = 0.85) with respect to the number of follicles ¡Ý6 mm at the time of cumulus-oocyte complex (COC) collection (17.9 ¡À 2.2 vs 17.7 ¡À 2.2), the number of COC collected (10.7 ¡À 2.1 vs 11.2 ¡À 2.3 per llama), or the collection rate per follicle aspirated (71 vs 74%). Finally, in the last study, the effect of two superstimulatory treatments was evaluated on ovarian response and COC collection efficiency and oocyte maturation in alpacas. No difference (P = 0.54) was observed between FSH and eCG- treated alpacas in the number of expanded (11.5 ¡À 2.9 vs 8.8 ¡À 2.8) or compact COC collected with ¡Ý3 layers of cumulus cells (12.5 ¡À 4.3 vs 14.3 ¡À 2.6; P = 0.72). No difference (P = 0.1) was detected between FSH and eCG groups in the number of expanded COC at first metaphase (1.2 ¡À 1.2 vs 1.7 ¡À 0.6) or second metaphase stage (8.5 ¡À 1.9 vs 6.0 ¡À 2.1) respectively. In conclusion, these studies demonstrated that the control of ovarian follicular wave emergence and ovulation induction in llamas will contribute consistently to the establishment of fixed-time natural or artificial insemination as well as recipient synchronization in embryo transfer programs. The discovery of an ovulatory molecule in the semen of this species generates a new area of research regarding the ovulation mechanism in induced ovulators. Characterization of this factor may have important implications in the diagnosis and treatment of male and female infertility. Finally, the superstimulatory treatments and oocyte development studies will establish the baseline for the development of an in vitro embryo production system in llamas and alpacas.

The purpose of the studies reported in this thesis was to increase our understanding of the reproductive physiology of South American camelids. Studies were conducted in llamas and alpacas to investigate methods to electively control ovarian follicular dynamics, to determine the effects of hormone preparations or biological factors derived from seminal plasma on ovulation induction, and to evaluate the establishment of superstimulatory protocols to induce a consistent ovarian follicular response for oocyte collection. The first study was designed to compare the efficacy of treatments intended to induce follicular wave synchronization among llamas, and to determine the effect of these treatments on pregnancy rates after fixed-time natural mating. In the first experiment, lutenizing hormone (LH) and follicular ablation treatments were most effective for inducing follicular wave synchronization, while estradiol plus progesterone (E/P) treatment was intermediate. In the second experiment, llamas were assigned randomly to Control, (E/P), and LH groups. A single, fixed-time natural mating was permitted 10 to 12 days after treatment. The pregnancy rate was higher (P<0.05) for synchronized llamas (LH and E/P groups combined) than for non-synchronized llamas (Control group). The second study was done to compare the effects of hormonal treatments and natural mating on ovulation induction, interval to ovulation, and luteal development in llamas. No differences were detected among groups (mated, LH, and GnRH) in ovulation rate (80%, 91%, 80%, respectively; P = 0.6), or interval from treatment to ovulation (30.0 ¡À 0.5, 29.3 ¡À 0.6, 29.3 ¡À 0.7 h, respectively; P = 0.9). Similarly, no differences were detected among groups (mated, LH, and GnRH) in maximum corpus luteum (CL) diameter. The third study documents the existence of an ovulation-inducing factor (OIF) in the seminal plasma of alpacas and llamas. In Experiment 1, female alpacas were given alpaca seminal plasma or saline intramuscularly (im) or by intrauterine infusion. Only alpacas that were given seminal plasma im ovulated. In Experiment 2, ovulation was detected in 9/10 (90%) llamas at a mean of 29.3 ¡À 0.7 hours after seminal plasma treatment. In Experiment 3, female llamas were given llama seminal plasma, GnRH, or saline im, and ovulation was detected in 6/6, 5/6, and 0/6 llamas, respectively (P < 0.001). Treatment was followed by a surge (P < 0.01) in plasma LH concentration beginning 15 minutes and 75 minutes after treatment with GnRH and seminal plasma, respectively. Plasma LH remained elevated longer in the seminal plasma group (P < 0.05), and plasma progesterone concentration was twice as high in the seminal plasma group (P < 0.01). The fourth study describes the presence of an OIF in the seminal plasma of Bos taurus ¨C a species conventionally considered to ovulate spontaneously - contains OIF. Bull seminal plasma induced ovulations in 26% (5/19) of llamas compared to 0% (0/19) in PBS group (P < 0.001). The ovulation rate was lower (P < 0.01) in bull seminal plasma group compared to that in the groups treated with alpaca or llama seminal plasma (100%). The fifth study was conducted to determine a local versus systemic effect of ovulation-inducing factor in seminal plasma. Ovulation rate in the seminal plasma intramuscular group (93%) was higher (P < 0.01) than seminal plasma intrauterine group (41%), while the seminal plasma intrauterine curettage group was intermediate (67%). The sixth study was done to determine the time required for llama oocyte to reach the maturation stage, and to establish a superstimulatory treatment for oocyte collection. Llama oocytes reached second metaphase as early as 28 h after in vitro culture. The FSH- and eCG-treated groups did not differ (P = 0.85) with respect to the number of follicles ¡Ý6 mm at the time of cumulus-oocyte complex (COC) collection (17.9 ¡À 2.2 vs 17.7 ¡À 2.2), the number of COC collected (10.7 ¡À 2.1 vs 11.2 ¡À 2.3 per llama), or the collection rate per follicle aspirated (71 vs 74%). Finally, in the last study, the effect of two superstimulatory treatments was evaluated on ovarian response and COC collection efficiency and oocyte maturation in alpacas. No difference (P = 0.54) was observed between FSH and eCG- treated alpacas in the number of expanded (11.5 ¡À 2.9 vs 8.8 ¡À 2.8) or compact COC collected with ¡Ý3 layers of cumulus cells (12.5 ¡À 4.3 vs 14.3 ¡À 2.6; P = 0.72). No difference (P = 0.1) was detected between FSH and eCG groups in the number of expanded COC at first metaphase (1.2 ¡À 1.2 vs 1.7 ¡À 0.6) or second metaphase stage (8.5 ¡À 1.9 vs 6.0 ¡À 2.1) respectively. In conclusion, these studies demonstrated that the control of ovarian follicular wave emergence and ovulation induction in llamas will contribute consistently to the establishment of fixed-time natural or artificial insemination as well as recipient synchronization in embryo transfer programs. The discovery of an ovulatory molecule in the semen of this species generates a new area of research regarding the ovulation mechanism in induced ovulators. Characterization of this factor may have important implications in the diagnosis and treatment of male and female infertility. Finally, the superstimulatory treatments and oocyte development studies will establish the baseline for the development of an in vitro embryo production system in llamas and alpacas.

No description available.

Thesis (M.Sc. (Anatomy)) -- Mahidol University, 1969.

AN ABSTRACT OF THE THESIS OF Crystal Marie Welsh, for the Masters of Science degree in Equine Reproductive Physiology, presented on October 6, 2009, at Southern Illinois University Carbondale. TITLE: IMMUNOHISTOCHEMICAL IDENTIFICATION OF DOPAMINERGIC AND ADRENERGIC NERVES IN THE EQUINE OVARY MAJOR PROFESSOR: Dr. Sheryl S. King Dopamine (DA) appears to play a role in seasonal reproduction in the mare. We hypothesize that DA is delivered to the ovary by nerves. The objective of this study was to use immunohistochemical (IHC) localization to identify ovarian dopaminergic and adrenergic nerves. Ovaries were collected during anestrus (n=9) and cyclicity (n=11). Tissue was prepared for IHC using antibodies specific to tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DβH). Microscopic evaluation was performed, comparing the numbers and distribution of TH– versus DβH–specific immunoreactive (IR) neurons. Catecholaminergic neurons were present in all ovaries. The preponderance (P<0.0001) of IR neurons were dopaminergic. Neurons were moderately dispersed throughout the ovarian medulla and lightly in the cortex. Moderate to heavy neuron density was also noted around blood vessels. Neither dopaminergic nor adrenergic neuron type was consistently associated with specific reproductive structures. These results indicate that catecholaminergic innervation does exist in the equine ovary. The presence of ovarian DA nerves supports our hypothesis that DA has a direct role in ovarian function in the equine, although the mechanism of this function requires further study.