Comparison of oocyte factors and transforming growth factor-beta in the regulation of DNA synthesis in bovine granulosa cells.

Oocytes are powerful local modulators of follicular cell functions and many of the activities of oocytes are attributed to members of the transforming growth factor-beta (TGF-beta) superfamily. Whilst in the mouse it is known that members of this family are able to mimic many of the effects of oocytes on follicular cells, the relative importance of any of these factors is unknown in bovine follicles. The objectives of this study were to determine if bovine oocytes express and secrete TGF-beta and to compare oocyte-secreted factor(s) to TGF-beta in terms of their capacities to stimulate mural granulosa cell (MGC) DNA synthesis. Bovine ovaries were collected from an abattoir and RNA was extracted from isolated MGC, cumulus cells, cumulus-oocyte complexes and denuded oocytes (DO). Using RT-PCR, all cell types were found to express TGF-beta1 and TGF-beta2 mRNA transcripts. However, no TGF-beta bioactivity could be detected from DO using a sensitive (40 pg/ml) and specific mink lung fibroblast cell bioassay. MGC were cultured with various combinations and doses of TGF-beta2 and DO for 18 h, followed by a 6-h pulse of [3H]-thymidine as an indicator of cellular DNA synthesis. MGC DNA synthesis was stimulated by both TGF-beta2 and DO. However in response to increasing doses of TGF-beta2, [3H]-thymidine levels plateaued at <2-fold above control levels, whereas levels continued to increase over the dose range of DO tested (up to 3.4-fold). Addition of a TGF-beta pan-specific neutralising antibody to MGC cultures eliminated the TGF-beta2-stimulatory effects on DNA synthesis and the TGF-beta2-suppressive effects on progesterone production, but the antibody was unable to neutralise the same responses when induced by DO. These results support a role for TGF-beta1, TGF-beta2 and DO in paracrine/autocrine regulation of bovine granulosa cell function, but indicate that neither TGF-beta1 nor TGF-beta2 can account for the actions of bovine oocytes on granulosa cells.