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ABSTRACT

Oestrogen receptor mRNA expression in mouse ovaries was analysed by Northern blotting of total RNA using ³²P-labelled RNA probes complementary to different functional domains of the oestrogen receptor. The ~6·5 kb mouse oestrogen receptor mRNA transcript was present in immature and adult ovaries at extremely low abundance compared with uterus and oviduct. Using a probe complementary to the steroid-binding domain of the oestrogen receptor (probe EF), a novel RNA transcript of ~1·5 kb was also found in the ovaries but was absent from uterus and oviduct. The melting temperature of the hybrid produced by the ~1·5 kb transcript with probe EF was ~10°C lower than that produced by authentic oestrogen receptor mRNA, which demonstrates incomplete sequence homology between the two transcripts and indicates that the ~1·5 kb RNA is not a truncated form of oestrogen receptor mRNA. Furthermore, the ~1·5 kb RNA lacks the DNA-binding domain found in the oestrogen receptor. The ~1·5 kb RNA, but not oestrogen receptor mRNA, was enriched in total RNA from isolated granulosa cells compared with residual ovarian tissue. The encoded product of this novel oestrogen receptor-related RNA could be a steroid-binding protein involved in oestrogen action in the ovaries.

ABSTRACT

Current understanding of the endocrine and paracrine regulation of follicular oestrogen synthesis predicts that aromatase cytochrome P450 (P450arom) mRNA is inducible by FSH in granulosa cells. LH receptor mRNA is constitutively expressed in thecal/interstital cells, and is also thought to be induced in granulosa cells in response to joint stimulation by FSH and oestrogen. This study provides direct evidence that FSH induces the ovarian P450arom gene selectively, perhaps exclusively, in the granulosa cells of Graafian follicles. FSH-induction of LH receptor mRNA occurs simultaneously but is independent of oestrogen synthesis per se.

ABSTRACT

The co-ordinated biosynthesis of progesterone and oestradiol in the human ovary is critical for reproductive cyclicity and eventual pregnancy. The crucial regulatory enzymes for progesterone and oestradiol biosynthesis in granulosa cells are the cholesterol side-chain cleavage (P450scc) and aromatase (P450arom) enzymes respectively. We utilized the cDNA sequences encoding P450arom and P450scc to examine the roles of FSH and LH, and their intracellular second messenger, cyclic AMP (cAMP), in regulating steroidogenic gene expression. Mature granulosa cells (aspirated before the onset of the endogenous LH surge) and granulosa lutein cells (obtained after an ovulatory dose of human chorionic gonadotrophin) were cultured for 4 days with FSH, LH or dibutyryl cAMP (dbcAMP). After the period of culture, total RNA was extracted from granulosa cells and Northern analyses were performed utilizing ³²P-labelled cDNAs encoding P450arom and P450scc. Spent culture media were analysed for steroid and cAMP content.

Both FSH and LH strongly stimulated P450arom mRNA expression and oestradiol production in mature granulosa cells. On the other hand, P450scc mRNA expression and progesterone biosynthesis were weakly induced by FSH; maximal synthesis occurred only in the presence of LH. With both gonadotrophins at equivalent concentrations, LH generated a 30-fold higher level of cAMP than FSH. Furthermore, the differential effects of FSH and LH on P450 mRNA expression were reproduced by the presence of low and high concentrations of dbcAMP respectively. LH (and high levels of dbcAMP) increased P450arom mRNA expression in mature granulosa cells but inhibited its accumulation in granulosa lutein cells. In contrast, it stimulated P450scc mRNA expression and progesterone synthesis in both mature granulosa and granulosa lutein cells. Therefore, FSH/low cAMP levels stimulated P450arom gene expression and oestradiol production, while LH/high cAMP levels maximally induced P450scc gene expression and function, in a development-related manner consistent with steroid production in vivo. These findings support the hypothesis that one set of genes (like P450arom) in human granulosa cells is regulated by FSH/low cAMP levels and another (like P450scc) by LH/high cAMP levels.