title: Developmental programming : prenatal androgen excess disrupts antral follicle function in sheep
Creators: name: Grant, Jonathan Charles
Publisher: name: University of Glasgow
Year: 2011

description: The experiments detailed in this thesis were conducted to investigate the effects of the prenatal environment, specifically excess aromatisable testosterone and the non-aromatisable 5α-dihydrotestosterone, on early adult reproductive function in female sheep, focusing on ovarian antral follicle development. In Chapter 3, blood samples and the largest antral follicles were obtained from sheep prenatally exposed to excess testosterone propionate (TP) (tissue collection over 5 years) and 5α-dihydrotestosterone (DHT) (tissue collection in 1 year) and controls. The aim was to determine the effect of prenatal androgen treatment on ovarian function in the young adult ewe, specifically on circulating steroid concentrations (oestradiol and progesterone), ovarian weight, and on large antral follicle characteristics (follicle diameter, follicular fluid oestradiol, progesterone and testosterone concentrations). Prenatal androgenisation by TP reduced the proportion of ewes undergoing at least one reproductive cycle during the first breeding season. Compared with controls, ovarian weight and peripheral concentrations of oestradiol were increased in ewes prenatally treated with TP. In addition, the largest (generally two) antral follicles recovered from ovaries of ewes exposed prenatally to aromatisable testosterone demonstrated an increase in size, and an increase in follicular fluid oestradiol and progesterone, both markers of follicle differentiation. In Chapter 4, ovaries were harvested from control and TP androgenised ewes at 10- and 22-months of age to investigate the effect of prenatal treatment with TP on small antral follicle health and steroidogenesis in adulthood. In early antral follicles following antrum formation follicle health was improved in 10 month old ewes and more follicles had acquired the ability to produce oestradiol in 22 month old prenatally TP treated ewes. In Chapter 5, the (generally two) largest antral follicles were obtained from ovaries from control and prenatally androgenised (TP) 10 and 12 month old ewes to determine the mRNA expression profile for differentiation, steroidogenic, survival and apoptotic genes in granulosa cells. Granulosa cells of TP ewes demonstrated increased mRNA expression for LHR and HSD3B1 and reduced mRNA expression for FSHR. Gene expression levels of various proliferation and apoptotic makers in granulosa cells were similar between control and androgenised ewes. In Chapter 6, granulosa cells were isolated from the (generally two) largest antral follicles recovered from control and prenatally androgenised (TP and DHT) ewes of 10-months of age. These were cultured in vitro under different gonadotrophin conditions to determine if granulosa cell steroid production is intrinsically different between androgenised and control ewes, or whether the external (hormonal) environment causes the differences detected in antral follicle function. Unfortunately, investigation into the mRNA expression profiles of FSH- and LH-responsive genes in granulosa cells after culture was not possible due to low cell numbers. Oestradiol production was increased independent of gonadotrophins, and progesterone production in response to LH was increased in cells from TP-treated ewes compared with control cells, while prenatal androgenisation by DHT resulted in reduced granulosa cell oestradiol production compared with cells from both control and TP androgenised ewes. Prenatal androgenisation through DHT resulted in reduced granulosa cell progesterone production when compared with cells from TP treated animals. The results presented in this thesis provide evidence that prenatal androgen treatment causes programming of adult ovarian function in sheep. Exposure to excess TP results in altered large antral follicle function such as increased growth and steroidogenesis. Specifically, androgenisation using TP and not DHT leads to increased steroidogenesis (oestradiol and progesterone) in large antral follicles, therefore these abnormalities are programmed through oestrogen action. Increased LHR and reduced FSHR mRNA expression, together with an earlier acquisition of steroidogenic capability provide evidence for the first time that follicles recovered from TP treated ewes are further differentiated. Future studies should be directed towards establishing whether intra-ovarian levels of gene expression equate with protein production. Furthermore, further studies need to address any molecular changes in somatic cells of early antral follicles. Finally, a study abolishing gonadotrophins using a GnRH antagonist using supplementation treatments should be performed, to truly determine if it is indeed LH that is responsible for abnormal antral follicle growth and development in prenatally TP-treated ewes.