330 THE PROTEIN EXPRESSION PROFILE (SECRETOME) OF INDIVIDUAL CUMULUS–OOCYTE COMPLEXES DURING IVM IN THE MOUSE IS AFFECTED BY FSH

Abstract

Studies of the protein expression profile into the surrounding medium (secretome) of in vitro-matured cumulus-oocyte complexes (COCs) have the potential to elucidate biochemical pathways involved in oogenesis, including the complex dialogue between the oocyte and its supporting cells. The understanding of these processes should assist in improving IVM success and fertility outcome, as early embryo development reflects the quality of the oocyte and its cumulus cells. Through the analysis of the individual COC secretome, we have investigated the effects of adding follicle stimulating hormone (FSH) to a defined maturation medium during IVM. COCs were collected from 3-week-old female mice (C57BL/6 × CBA) 48 h post-pregnant mare serum gonadotropin (PMSG) (5/iu) injection. Individual COCs were cultured in 5-¼L drops of a defined maturation medium (0.25 mg/mL recombinant albumin) with the addition of 0, 2, 20, or 200 ng/mL FSH, under oil for 17 h. Oocytes were denuded and maturity recorded. Each microdrop of media (n = 8 oocytes per group) was collected, processed through an optimized series of buffers and washes prior to analysis by time-of-flight mass spectrometry (TOF-MS). Differential protein expression profiles were obtained from the secretome of individual COCs producing MII oocytes after maturation in differing doses of FSH. Statistical analysis revealed significant differences observed across 10 proteins/biomarkers with mass-to-charge (m/z) ratios ranging from 2.7 to 6 kDa (Mann-Whitney non-parametric test; P < 0.05). In addition, hierarchical and horizontal clustering analysis identified unique clusters of both up-regulated and down-regulated proteins/biomarkers within the m/z range of 2 to 18 kDa in the 2 ng/mL FSH group. Several of the individual COCs from the 20 ng/mL FSH group were also clustered alongside the 2 ng/mL group with similar protein expression profiles. In contrast, COCs cultured in the presence of 0 ng/mL and 200 ng/mL FSH were observed to cluster as a separate branch with distinctly different protein expression profiles. This study has determined for the first time the secretome profiles of individual COCs after IVM. These data have shown that the FSH dose in a defined maturation medium affects the secretome of an individual COC. Further investigation is currently underway to characterize these protein differences. The development of this proteomics approach will assist in revealing the intricate cellular function of an individual COC and elucidate critical pathways involved in mammalian oocyte maturation.