Demonstration of a suppressive effect of inhibin alpha-subunit on the developmental competence of in vitro matured bovine oocytes.

Abstract

Changes in intrafollicular concentrations of different forms of inhibin (free alpha-subunits and alpha beta dimers) occur during follicle development and may influence the oocyte maturation process. The aim of this study was to investigate the effects of inhibin A and free alpha-subunit (pro-alpha C) isolated from bovine follicular fluid on maturation of bovine cumulus-oocyte complexes, as reflected by their competence for embryo development after in vitro fertilization. Bovine cumulus-oocyte complexes were isolated from ovaries obtained from an abattoir and were cultured for 22-24 h at 38.5 degrees C in TCM-199 medium supplemented with 10% oestrous cow serum, pregnant mares' serum gonadotrophin (2.5 iu ml-1) and either inhibin A (0, 0.2 and 1.0 microgram ml-1) or pro-alpha C (0, 2 and 10 micrograms ml-1). Neither inhibin A nor free alpha-subunit affected the cleavage rate of cumulus-oocyte complexes after fertilization (approximately 60%). Inhibin A reduced the proportion of cleaved oocytes reaching the eight-cell stage by 19% (P < 0.05), but did not affect the yield of blastocysts. However, pro-alpha C decreased the proportion of cleaved oocytes that reached the eight-cell (25%; P < 0.05) and blastocyst (28%; P < 0.05) stages. In addition, a negative correlation (r = -0.55, P < 0.001) was found between concentrations of total immunoreactive (ir) alpha-inhibin (measured by radioimmunoassay) produced by untreated control cumulus-oocyte complexes and their post-cleavage development to the blastocyst stage. In a second experiment, mouse monoclonal antibodies (20 micrograms ml-1) against two different regions of the inhibin alpha-subunit precursor (pro-region and alpha C fragment) were tested for their ability to neutralize endogenous inhibin alpha-subunit-related molecules produced by cumulus cells; control cumulus-oocyte complexes were treated with normal mouse IgG (20 micrograms ml-1). Although the cleavage rate was not affected, the yield of blastocysts was significantly higher in the presence of mouse monoclonal antibodies to both pro-alpha (77% increase; P < 0.05) and alpha C (48% increase; P < 0.05). None of the treatments tested affected endogenous production of activin-A or follistatin by cumulus-oocyte complexes. Overall, these results indicate that the inhibin alpha-subunit (pro-alpha C) has an inhibitory role in oocyte maturation which is independent of the modulatory effects of activin and follistatin.