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In-vitro studies of mouse oocytes have provided evidence that two closely related sterols, subsequently named meiosis-activating sterols (MAS), can overcome the inhibitory effect of hypoxanthine on resumption of meiosis. These sterols are synthesized by cytochrome P450 (CYP) lanosterol 14alpha-demethylase (LDM), a key enzyme in cholesterol biosynthesis. Our studies in the rat with specific inhibitors and molecular approaches did not support the hypothesis that MAS is an obligatory step in the stimulation of the resumption of meiosis. (i) Specific inhibitors of MAS synthesizing enzymes did not prevent spontaneous or LH-stimulated meiosis at doses that have previously been shown to effectively suppress LDM activity. At higher doses, they caused degeneration of oocytes. (ii) The timing of LDM expression in the ovary was incompatible with a role for MAS in meiosis. (iii) The preferential localization of LDM protein in the oocytes suggests MAS production in oocytes, rather than its transport from the somatic compartment as expected by the suggested role of MAS in the regulation of meiosis as a putative cumulus-oocyte signal molecule. (iv) AY-9944, which supposedly increases MAS levels by inhibiting its metabolism, induced the maturation of follicle-enclosed oocytes that was much delayed as compared with gonadotropic stimulation. Thus, the resumption of meiosis induced by added MAS [Biol. Reprod. 61 (1999) 1362, Biol. Reprod. 64 (2001) 418] or presumed endogenous MAS accumulation by AY-9944, resulted in oocyte maturation with remarkably slower kinetics than observed with LH stimulation. This delay in meiosis after MAS stimulation, the studies with LDM inhibitors and its spatial and temporal expression, cast serious doubts whether MAS is indeed mediating the meiosis inducing action of the gonadotropins, as suggested.

In vitro studies on mouse oocytes have shown that two closely related sterols, subsequently named meiosis-activating sterols (MAS), can overcome the inhibitory effect of hypoxanthine on the resumption of meiosis. These sterols are synthesized by cytochrome P(450) lanosterol 14alpha-demethylase (LDM), a key enzyme in cholesterol biosynthesis. We have used specific inhibitors of LDM, azalanstat (RS-21607) and RS-21745, to test whether MAS is an obligatory mediator in the resumption of meiosis in the rat. Addition of azalanstat and RS-21745 (1-200 microM) to culture medium of rat isolated cumulus-enclosed oocyte and preovulatory follicle-enclosed oocyte stimulated by LH/hCG did not allow separation between their inhibition of the resumption of meiosis and the degeneration of oocytes. In both models, doses of the drug that inhibited oocyte maturation also increased oocyte degeneration. The inhibitors only partially suppressed follicular progesterone production. We have examined by reverse transcriptase-polymerase chain reaction, Western blotting, and immunocytochemistry the ovarian expression of LDM mRNA and protein during the preovulatory period. We did not find evidence for the stimulation of this enzyme by LH/hCG. The strongest staining by LDM antiserum was obtained in primordial and primary oocytes, and the staining was reduced with oocyte growth. In addition, strong LDM staining could be observed in some of the granulosa cells, especially of the corona radiata localized in close proximity to the oocyte. In conclusion, our results with specific inhibitors and molecular approaches do not reveal evidence to support the hypothesis that MAS is an obligatory step in the stimulation of the resumption of meiosis. Specific inhibitors of MAS synthesis did not prevent spontaneous or LH-stimulated meiosis at doses that have previously been shown to effectively suppress LDM activity. Much higher concentrations of the inhibitors, which affected meiosis, were detrimental to oocytes, leading to their degeneration. The timing of LDM expression in the ovary was incompatible with a role for MAS in meiosis. Finally, the preferential localization of LDM protein to the oocytes suggests MAS production in oocytes rather than its transport from the somatic compartment as implied by the proposed role of MAS as a cumulus-oocyte signal molecule.