Epidermal growth factor-like peptide signalling and oocyte in vitro maturation.

Abstract

A growing body of evidence has recently implicated follicular epidermal growth factor (EGF)-like peptide signalling as essential for the propagation within the ovarian follicle of the LH stimulus that induces oocyte maturation and ovulation. The EGF-like peptides amphiregulin, epiregulin, and betacellulin are produced in mural granulosa and cumulus cells in response to LH, and signal via the EGF receptor (EGFR) in these cells to ultimately induce oocyte maturation, cumulus expansion, and ovulation. Although the function and impact of EGF-like peptide signalling on oocyte maturation in vivo has been characterised, little is currently known about the effect of oocyte in vitro maturation (IVM) on this important signalling network. This thesis aimed to investigate the regulation of EGF-like peptide signalling in mouse cumulus cells and the effect of various IVM models on this network. FSH is a universal IVM additive, and EGF is occasionally used in animal IVM. The effect of FSH-stimulated IVM, EGF-stimulated IVM versus in vivo maturation (IVV) on cumulus cell EGF-like peptide mRNA and/or protein expression, the activity of EGFR, and its classic downstream effector, ERK1/2, were examined. EGF-like peptide mRNA expression, amphiregulin protein expression, and EGFR phosphorylation were significantly lower using FSH-stimulated IVM than during IVV. EGF stimulated significantly lower EGFR phosphorylation, but not EGF-like peptide mRNA expression. These data demonstrate that this signalling network is perturbed in IVM cumulus cells. The effect of FSH, EGF, amphiregulin and epiregulin in IVM on subsequent blastocyst development revealed that epiregulin and amphiregulin significantly increased blastocyst yield and/or the proportion of inner cell mass in blastocysts, than FSH or EGF. Examination of the metabolic profiles of IVM cumulus-oocyte complexes (COCs) matured in the presence of these stimulants revealed that EGF-like peptides and EGF induced significantly higher COC glucose metabolism via the hexosamine biosynthesis pathway than FSH, consequently enabling more hyaluronic acid synthesis and protein β-O-linked glycosylation in the cumulus cells. Epiregulin significantly increased intra-oocyte FAD⁺⁺ and the REDOX ratio compared to FSH, and all three EGF-like peptides induced more oocyte mitochondrial activity than EGF or FSH. Evidence has shown that increasing 3'-5'-cyclic adenosine monophosphate (cAMP) using pharmacological agents significantly increases IVM oocyte developmental competence. This concept, in the form of a pre-IVM culture period with cAMP modulators, was examined in conjunction with IVM in the presence of epiregulin, amphiregulin, EGF or FSH. A pre-IVM phase in conjunction with IVM with EGF-like peptides endowed greater oocyte developmental competence than with FSH or EGF, as evidenced by increased embryo yield and/or quality, which were comparable in embryo development and/or quality rates from IVV oocytes. This thesis provides the physiological basis for, and evidence that, EGF-like peptides are more appropriate IVM additives than FSH or EGF. EGF-like peptides endow greater oocyte developmental competence than FSH, possibly by regulating important aspects of COC metabolism. Combining this concept with cAMP modulation of IVM COCs may represent a more physiological IVM system than existing IVM approaches, as it yields more blastocysts of higher quality. The knowledge provides new opportunities for the treatment of infertility in women and for the in vitro production of embryos and advanced breeding in animals.