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We have examined the levels of expression of mRNA species encoding cholesterol side-chain cleavage cytochrome P-450 (P-450_scc), 17-hydroxylase cytochrome P-450 (P-450₁₇ ), aromatase cytochrome P-450 (P-450_AROM) and 3β-hydroxy-steroid dehydrogenase (3β-HSD) in rat ovaries throughout the oestrous cycle, during pregnancy and in immature animals treated with pregnant mare serum gonadotrophin (PMSG). Total or poly(A)⁺-enriched RNA was prepared from adult rat ovaries throughout the oestrous cycle, from immature rat ovaries 24 and 48 h after treatment and from adult rat ovaries on days 10, 14, 17 and 21 of gestation. Expression of the mRNA species was examined by Northern analysis using specific [³²P]cDNA probes. During the oestrous cycle P-450_scc mRNA of 1·9 kb was detected at low levels, while 3β-HSD mRNA of 1·7 kb was in relatively high abundance throughout the oestrous cycle. While P-450₁₇ mRNA of 1·9 kb and P-450_AROM of 2·7, 2·2 and 1·7 kb were highly abundant during dioestrus, pro-oestrus and oestrus, the levels of these mRNA species decreased markedly to be nearly undetectable during metoestrus. During pregnancy there was considerably more variation in the expression of the mRNA species examined. Expression of P-450_scc mRNA was at low, but detectable, levels until day 14, thereafter expression increased to high levels (day 14–21 of gestation). Levels of P-450₁₇ mRNA on day 10 of gestation were lower than at pro-oestrus during the oestrous cycle and decreased further on days 14 and 17. Expression of 3β-HSD was decreased on day 10, but on days 14, 17 and 21 of gestation high mRNA levels were detectable. Ovarian expression of the three P-450_AROM species was dramatically increased between days 14 and 17 of pregnancy, but declined by day 21. In immature rats, P-450_scc mRNA was detected at low levels in unstimulated animals and increased markedly after treatment with PMSG, while subsequent treatment with human chorionic gonadotrophin (hCG) had a minimal effect on expression. Expression of P-450₁₇ mRNA was high in unstimulated immature and PMSG-treated rats, but diminished after treatment with hCG. All three P-450_AROM mRNA species were undetectable in ovaries from unstimulated immature animals; however, induction of all three was observed in PMSG-treated rats, but this expression decreased to undetectable levels upon subsequent administration of hCG. Further RNA blot analysis utilizing a 3' portion of the P-450_AROM cDNA as probe, which contains an intronic segment instead of the steroid-binding region, revealed that the two mRNA species of lower molecular weight hybridized to this probe. In contrast, only the largest aromatase mRNA band hybridized to a probe specific for the steroid-binding region. These findings indicate that a majority of the P-450_AROM mRNA species in rat ovarian tissue during the oestrous cycle and throughout pregnancy represents alternatively spliced products, which presumably lack the ability to encode for aromatase activity. These results indicate that the pattern of steroid secretion in the rat can be explained, in part, by the differential expression of mRNA species encoding the various key steroidogenic enzymes throughout the ovarian cycle, during pregnancy and following administration of gonadotrophins.