Intra-ovarian factors regulating follicular development, steroidogenesis and angiogenesis

Reproduction is an indispensable function that is under the control of a sophisticated network of regulatory signals that originates from and is integrated by the hypothalamic-pituitary-gonadal axis, which is primarily driven by gonadotropin releasing hormone (GnRH) from the hypothalamus. Compelling evidence accumulating over the last few decades has revealed that kisspeptin, a hypothalamic neuropeptide encoded by KiSS-1 gene, has a key role in promoting the release of GnRH and luteinizing hormone (LH) in various mammalian species. However, the possibility that kisspeptin exerts additional ‘peripheral’ actions at the level of the gonad has received little attention and no studies have been directed at the bovine ovary. Another neuropeptide, neuromedin B (NMB), belonging to the bombesinrelated peptide family, has been shown to have various physiological roles including the regulation of various exocrine and endocrine secretions but its potential action at the gonadal level has not been explored. This thesis reports a series of experiments designed to investigate: (1) whether KiSS-1 and NMB and their cognate receptors are expressed in bovine endocrine tissues; (2) whether kisspeptin and NMB, alone and in combination with their antagonist, can influence the steroidogenesis in cultured ovarian cells; (3) whether expression of KiSS-1 and NMB by cultured ovarian cells is regulated by gonadotropins and other factors; (4) whether NMB, kisspeptin-10, TGF- β -1, BMP-6 and TSP-1 and their respective antagonist modulate capillary network formation in a follicular angiogenesis model; (5) whether TGF-β-1 and BMP-6 (alone and in combination with their antagonists) affect ovarian steroidogenesis in vitro; (6) whether expression of steroidogenesis transcripts and other angiogeneic factors by cultured ovarian cells is regulated by TGF-β-1 and BMP-6. The laboratory techniques used to address the above included primary ovarian cell culture systems (bovine ovarian theca and granulosa cells under non-luteinized and luteinized conditions, cell migration and follicular angiogenesis models), steroid immunoassays (androstenedione, oestradiol and progesterone), real-time PCR and immunohistochemistry. The results of RT-PCR confirmed that KiSS-1 and its receptor (GPR54) and NMB are expressed in different bovine endocrine tissues including pituitary, adrenal, testis, ovarian corpus luteum, theca cells and granulosa cells. Moreover, changing levels of thecal and granulosal expressions were detected during different stages of follicle development. However, cell culture experiments offered no evidence to support the hypothesis that kisspeptin and NMB have a direct intra-ovarian role to modulate follicular or luteal steroidogenesis or cell proliferation under basal or gonadotrophin stimulated conditions. Neither did they affect ovarian theca cell migration evaluated using a wound-healing ‘scratch’ assay. Results from the follicular angiogenesis model indicate that while TGF-β-1 and BMP-6 reduced VEGF/FGF-induced capillary network formation, kisspeptin, NMB and TSP-1 were without effect. In conclusion, the results provided no evidence to support intrafollicular roles of kisspeptin or NMB peptides in modulating steroidogenesis, cell proliferation, cell migration or angiogenesis. However, both TGF-β-1 and BMP-6 were implicated as negative regulators of follicular angiogenesis, a finding that warrants further research, given their inhibitory action on thecal steroidogenesis.