175. EFFECTS OF SPECIES DIFFERENCES ON OOCYTE REGULATION OF GRANULOSA CELL PROLIFERATION

J Lin,J Juengel,K Mcnatty

doi:10.1071/srb10abs175

Abstract

The role of oocytes in regulating ovulation quota between species is not fully understood. In sheep, the oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) have profound effects on ovarian follicular development and ovulation-quota. The aim of these studies was to compare the ability of oocytes from sheep (low ovulation-rate) and rat (poly-ovulator) to stimulate radiolabelled thymidine uptake by granulosa cells (GC) both within and between the two species. For these experiments, 32 oocytes denuded of cumulus-cells were co-incubated with 20 000 GC from either species. Rat or sheep oocytes stimulated thymidine uptake by GC from the same species (P < 0.005). Sheep oocytes also stimulated thymidine uptake by rat GC (P < 0.001) but not vice versa. To investigate this further, oocytes and GC were co-incubated with 3.2 µg/mL or 7.6 mg/mL monoclonal antibodies specific to GDF9 or BMP15 respectively and to a hydatids antigen (control). Both sheep and rat oocyte stimulation of thymidine uptake by GC was inhibited with the GDF9 antibody (P < 0.05) but not control, irrespective of species of GC. Sheep oocyte stimulation of rat GC was also inhibited using an antibody to BMP15 (P < 0.05). However, when using the BMP15 antibody to block the effects of rat oocytes on rat GC, the inhibition of thymidine uptake was modest (i.e. ~10%) albeit significant (P < 0.05). The molecular forms of GDF9 and BMP15 in spent media were examined by Western blotting under reducing conditions. For both species, oocyte-secreted GDF9 was present in the mature form. For sheep oocytes, secreted BMP15 was present as promature and monomeric mature forms whereas from rats, trace amounts of mature form was sometimes, but not always, detected. Thus, in sheep both BMP15 and GDF9 are essential for regulating GC proliferation whereas in rats, although responsive to BMP15, GC proliferation is likely regulated primarily by GDF9.

Highlights

1.1 Female Reproduction 1.1.1 The mammalian ovary The mammalian ovary is enclosed within a layer of epithelium known as the surface epithelium (Peters and McNatty 1980)
An alternative explanation is that rat oocytes produce very little bone morphogenetic protein 15 (BMP15) protein and that granulosa cells (GC) proliferation is regulated by growth differentiation factor 9 (GDF9)
GDF9 and BMP15 mRNA from denuded oocytes (DO) while variable showed no significant changes over time during incubation

Summary

Introduction

1.1 Female Reproduction 1.1.1 The mammalian ovary The mammalian ovary is enclosed within a layer of epithelium known as the surface epithelium (Peters and McNatty 1980). Studies to address the second objective included evaluating the optimal number of oocytes and volume of media within each co-incubation well In both high and low ovulation-rate phenotypes, such as mice and sheep, inactivating GDF9 in vivo, either by mutation or immunization, prevented normal follicular growth and ovulation (Dong, Albertini et al 1996; Carabatsos, Elvin et al 1998; Yan, Wang et al 2001; Juengel, Hudson et al 2002; Hanrahan, Gregan et al 2004; McNatty, Hudson et al 2007; Nicol, Bishop et al 2009). Western blotting procedures were used to identify possible pro-mature and/or mature regions of GDF9 and BMP15 in rat and sheep oocyte lysates and spent media after 24h of incubation. Correlations between these genes and GDF9 and BMP15 mRNA expression in the COC, as reported in Chapter 7, were investigated

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