title: Role of meiosis-activating sterols in rat oocyte maturation: effects of specific inhibitors and changes in the expression of lanosterol 14alpha-demethylase during the preovulatory period.
abstract: 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.
authors.affiliation: The Bernhard Zondek Hormone Research Laboratory, Department of Biological Regulation, the Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: K M
authors.initials: KM
authors.lastname: Vaknin
authors.forename: S
authors.initials: S
authors.lastname: Lazar
authors.forename: M
authors.initials: M
authors.lastname: Popliker
authors.forename: A
authors.initials: A
authors.lastname: Tsafriri
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D046508:Culture Techniques; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D007986:Luteinizing Hormone / Q000494:pharmacology; D008540:Meiosis / Q000187:drug effects*; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D010053:Ovary / Q000201:enzymology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D058886:Sterol 14-Demethylase; D013261:Sterols / Q000494:pharmacology*; D013440:Sulfides / Q000494:pharmacology; D001371:trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride / Q000494:pharmacology
chemical_list: D000814:Aniline Compounds; D006063:Chorionic Gonadotropin; C548417:Cyp51 protein, rat; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D013261:Sterols; D013440:Sulfides; C082410:azalanstat; D001371:trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride; D007986:Luteinizing Hormone; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C548415:Cyp51 protein, mouse; D058886:Sterol 14-Demethylase