Global protein profiling of porcine cumulus cells in response to native oocyte secreted factors in vitro.

Abstract

Until recently, the traditional view was that the oocyte played a passive role in folliculogenesis, relying on paracrine signalling from surrounding somatic cells to acquire developmental competence. However, recent evidence suggests that the oocyte plays an active role in the process of folliculogenesis by secreting soluble factors that act on the neighboring somatic cells. Studies using rodent and ruminant animal models have shown the importance of oocytesecreted factors for cell proliferation and differentiation, steroidogenesis, metabolism, and, in certain species, in determining ovulation rate. However, little information is available in the pig species. Therefore, the objective of the current study was to determine the effects of native oocyte-secreted factors on cumulus cell protein expression in the pig follicle. Three groups of four sows were euthanized on day 19 ± 1 after the 1° post-weaning oestrus at a time point when the pre-ovulatory follicle population was established and the oocytes should have acquired full developmental competence. Follicle size and follicular fluid oestradiol concentration were used to confirm that oocytes were derived from highly oestrogenic follicles that had not been exposed to the preovulatory-LH surge. Cumulus-oocyte complexes (n 19 ± 2 per sow) were aspirated from these large pre-ovulatory follicles, washed three times in PVA TL-HEPES, and denuded (DO) from their cumulus cells. Concomitantly, gilt ovaries were obtained from a local slaughterhouse and oocytectomized cumulus complexes (00X) were prepared by microsurgically removing the oocytes from their surrounding cumulus cells. Groups of 1600X were then cultured for 22h with or without the DOs obtained from individual sows (n = 4 groups of 00X per treatment per replicate) in 36 pl droplets of modified M199 medium under mineral oil at 38.5°C in a humidified atmosphere of 5% CO2. For each of the three replicate cultures, 3 groups of 16 00X incubated with or without DOs were pooled and subjected to 2-dimensional gel electrophoresis on 7cm IPGstrips pH 3-10 in the first dimension and 100/0SDS-PAGE slab gels in the second dimension. The SYPRO Ruby (BioRad, CA) stained gels were imaged on a Typhoon Trio (GE Healthcare). Due to incomplete isoelectric focusing for one replicate, only the gel images from the remaining two replicates were finally analysed using Progenesis SameSpots software (Nonlinear Dynamics, UK). Individual spot intensities were normalized with the total spot volume from the gel of origin. A preparative gel containing the protein from the cumulus cells of 500 cumulus-oocyte complexes was also run under the same conditions but using an 18 cm IPGstrip pH 3-10. The preparative gel was matched with the analytical gels and protein spots of interest were manually excised and sent to a mass spectrometry facility for identification by LC MS/MS (Centre Genomique du Quebec, Sainte-Foy, Canada). To confirm proper matching between the analytical and preparative gel, 6 abundant protein spots of interest from the analytical gel were also sent for identification. More than 600 spots were matched across the 4 gels of which 14 proteins were found to be differentially expressed when the 00X were incubated alone or with DOs. Interestingly, 9 of