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This study was undertaken to develop a system that simulated, to some extent, the environment of the immature follicle and which reversibly inhibited the spontaneous maturation of porcine oocytes. The hypothesis was tested that a physiological concentration of testosterone (0.5 microM) in the presence of the cyclic nucleotide derivative, dibutyryl cAMP (1 mM), would prevent germinal vesicle breakdown and subsequent chromatin condensation of intact oocytes. Liberated cumulus-enclosed (intact) and cumulus-free (denuded) oocytes were cultured in a complex serum-based medium supplemented with the steroid and/or the nucleotide analogue. After 24 h, chromatin spreads were prepared and meiotic maturation was scored. In the presence of testosterone or dibutyryl cAMP, meiotic maturation of intact oocytes was similarly and significantly inhibited (57 and 58% matured, respectively) compared with controls (74% matured). This inhibition, however, was not nearly as marked as when both compounds were present together (19% matured), indicating a synergistic inhibitory relationship between the steroid and nucleotide. The inhibition exerted by the two compounds on the intact oocyte was reversible, a finding consistent with the observation that fewer germinal vesicles were degenerate in 'inhibited' oocytes than in oocytes cultured in control, unsupplemented medium. Although maturation of denuded oocytes was likewise inhibited by testosterone + dibutyryl cAMP (15% matured compared with 68% in control medium), this inhibition was due to a direct action of the cAMP derivative on the oocyte, with testosterone playing a passive role. These results suggest, therefore, that the cumulus cell mediated the inhibitory action of testosterone and dibutyryl cAMP on the maturation of the intact oocyte. In the light of reports that FSH or cAMP stimulate aromatase activity in cultured granulosa cells, our results suggest that oestrogen may be implicated in the observed inhibition of oocyte maturation.

Effect of testosterone and dibutyryl cAMP on the spontaneous maturation of pig oocytes