Novel functions of natriuretic peptides in mammalian cumulus-oocyte complexes

Abstract

Natriuretic peptides are physiological regulators of oocyte meiosis, existing within ovarian follicles and are being exploited to reveal the mechanisms regulating meiosis. To date, assessment of natriuretic peptides have been examined during culture of COCs from many mammals including mice, rats, pigs, cows, goats, horses and cats. Mouse models have dominated this research, elucidating the functions of CNP and NPR2 signalling within the COC and which maternal and oocyte-derived factors regulate their activity. Three in vitro models are utilised to explore novel functions of natriuretic peptides. These include: sheep IVM, pig IVM from large antral follicles, and pig IVM from small developmentally incompetent follicles. This thesis shows that NPR2 is the predominant cGMP-generating natriuretic peptide receptor in cumulus cells and is likely responsible for the majority of cGMP produced in the sheep ovarian follicle. OSFs and IGF-1 may inhibit NPR2 expression in cumulus cells whilst promoting NPR1 expression. Furthermore, CNP has the ability to inhibit the resumption of meiosis in ovine COCs in vitro, however is dose dependant. Using pig COCs, I determine CNP and cGMP have the ability to activate ERK1/2 in COCs from large follicles cultured in vitro. This action was independent of cAMP and CREB activation. However, it required functional MMPs and EGFR without altering the expression of the EGF-like peptides; AREG, EREG or EGFR. CNP and cGMP also induced the breakdown of gap junctions in COCs from large follicles. In pig COCs from small follicles, CNP and cGMP had no effect of ERK1/2 activation or gap junction communication. In COCs collected from both small and large follicles, CNP promoted meiotic resumption of oocytes cultured in vitro. However, when cultured in the presence of a highly stimulated system (FSH+pFF), CNP could inhibit the meiotic resumption of oocytes in vitro. I further show CNP stimulates ERK1/2 activation to levels comparable to FSH and pFF, which therefore could explain why my results may vary from current published research. Our knowledge of the molecular mechanisms which regulate oocyte maturation has grown significantly over the past few years. The findings in this thesis illustrate a major difference between cellular signalling between porcine COCs from small follicles, compared to COCs collected from larger follicle and therefore have importance to research into ovarian follicle development. Furthermore, this thesis supports the notion that there may be more to the CNP/cGMP pathway than simply the inhibition of meiosis; it may promote oocyte development by propagating cumulus cell signals. This dissertation advances our understanding of the process of oocyte maturation, and could be applied to provide better opportunities for the application of IVM in assisted reproductive technology for humans, and in agriculture.