The acquisition of FSH receptor during preantral folliculogenesis is believed to be a key step in the subsequent development of follicles. We examined the interaction between activin and cAMP in FSH receptor induction in rat granulosa cells by measuring 125I-FSH binding and FSH receptor mRNA. In the 125I-FSH binding study, 0.2 mM 8-Br-cAMP and 1 microM forskolin were maximally effective in FSH receptor induction (169 and 220% respectively of control), while higher concentrations gave attenuated responses. It appears that cAMP has ambivalent effects on FSH receptor induction depending on the concentration and length of exposure. Activin alone dramatically increased the number of FSH receptors (314% of control). Moreover, synergistic effects of activin and 8-Br-cAMP or forskolin were observed on FSH receptor induction: a combination of activin (80 ng/ml) and low doses of 8-Br-cAMP (0.2 mM) or forskolin (1 microM) was most effective (160 or 140% of that induced by activin alone) and receptor levels reached a maximum at 24 h. These levels than markedly decreased after 72 h of incubation. Northern blot analysis revealed that the combination of activin (80 ng/ml) and 8-Br-cAMP (0.2 mM) or forskolin (1 microM) increased FSH receptor mRNA to about 140% of that induced by activin alone. These results indicate that activin and cAMP induced FSH receptor synergistically. However, activin did not enhance the production of cAMP induced by forskolin. In addition, a protein kinase A inhibitor (H89) (2 microM), which inhibited the effects of forskolin, had no effect on the action of activin.(ABSTRACT TRUNCATED AT 250 WORDS)
The gonadotrophins follicle stimulating hormone (FSH) and luteinizing hormone (LH) are key hormones in the regulation of ovarian function. In the present study, the expression of LH/human chorionic gonadotrophin (HCG) receptor mRNAs in the human ovary was examined. Northern blot analysis was used to measure relative amounts of LH/HCG receptor mRNA, and in-situ hybridization was used to localize LH/HCG receptor transcripts. Northern blot analysis of human ovaries detected three transcripts (5.4, 3.6 and 2.4 kb) for the LH/HCG receptor. LH/HCG receptor mRNA concentrations increased from preovulatory follicles to the corpus luteum of the midluteal phase, and decreased at the late luteal phase. Using in-situ hybridization, LH/HCG receptor mRNA was located predominantly in granulosa cells in the same follicle. Cloning of the human LH/HCG receptor cDNA previously revealed the existence of two alternative forms of the receptor differing by the presence (HLH-Ra) and absence (HLH-Rb) of 62 amino acids by exon 9. We have studied the functional significance of these receptor isoforms and have confirmed that they are generated by alternative splicing. A reverse transcription-polymerase chain reaction amplification was used to detect different isoforms of LH receptor mRNAs in ovary and placenta. The expression of the two mRNA forms of LH/HCG receptor were detected in ovary, and at very low concentrations in placenta. Treatment with HCG caused a dose-dependent increase in cAMP production with an initial response evident at approximately 1 ng/ml HCG in COS-7 cells expressing HLH-Ra. However, a complete loss of signal transduction was found in cells transfected with the truncated HLH-Rb.
The present study was undertaken to identify the mechanisms underlying the effect of retinoic acid (RA) on follicle-stimulating hormone receptor (FSH-R) in rat granulosa cells. Treatment with FSH produced a substantial increase in FSH-R mRNA level, as was expected, while concurrent treatment with increasing concentrations of RA brought about dose-dependent decreases in FSH-induced FSH-R mRNA, with a maximal inhibition one-third lower than that induced by FSH alone. RA, either alone or in combination with FSH, did not affect intracellular cAMP levels, while it inhibited the effect of 8-Br-cAMP on FSH-R mRNA production. These results suggested that RA diminished the action of FSH on FSH-R expression at sites distal to cAMP generation in the granulosa cells. Whether the effect of RA and FSH on FSH-R mRNA levels was the result of decreased transcription and/or altered mRNA stability was also investigated. The rate of FSH receptor mRNA gene transcription, assessed by nuclear run-on transcription assay, was found to decrease by the addition of RA. On the other hand, the decay curves for the 2.4 kb FSH-R mRNA transcript in primary granulosa cells did not alter the slope of the FSH-R mRNA decay curve in the presence of RA. Our data suggests for the first time that the effect of RA on FSH-R expression is possibly mediated by the reduction of the FSH-R mRNA level due to a negative regulation of the FSH-R gene in the presence of FSH. These findings assist in understanding the molecular mechanism underlying the effect of RA on reproductive function in rat granulosa cells.
The gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are key hormones in the regulation of ovarian function. The acquisition of FSH receptors during folliculogenesis is believed to be a key event in the subsequent development of the follicle. However, the binding and biochemical properties of the human FSH receptor are not well-characterized owing to the low abundance of these receptors and the limited availability of human tissue. The binding experiments show that, while the affinity of the FSH receptor does not change through the menstrual cycle, the total number of FSH receptors in the leading follicles increases by about two-fold at mid-follicular phase compared with those at other periods. Northern blot analysis was used to measure relative levels of FSH receptor mRNA, and in situ hybridization was used to localize FSH receptor transcripts. Northern blot analysis of human ovaries detected two transcripts (4.1 and 2.4 kb) for the FSH receptor. The FSH receptor mRNA was abundant in the preovulatory follicle, with expression decreased by about 50% in the corpus luteum. Using in situ hybridization, FSH receptor mRNA was found to be confined to the granulosa cells of developing follicles. A reverse transcription-polymerase chain reaction amplification was used to detect the expression of different isoforms of the FSH receptor mRNA in human corpus luteum and placenta.
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