Effect of prolactin on the expression of luteinizing hormone receptors during cell differentiation in cultured rat granulosa cells

Chronic and transient hyperprolactinemia has been associated with luteal ph ase dysfunction. Recently, evidence has emerged to suggest that elevated PR L may exert its antigonadal effects through reducing available ovarian LH r eceptors. We have now examined the influences of PRL on LH receptor inducti on in cultured granulosa cells. Basal specific LH binding was negligible an d remained unchanged in response to treatment with PRL by itself. Whereas t reatment with FSH produced, as expected, a substantial increase in specific LH binding, concurrent treatment with PRL resulted in no significant chang e during the first 4 days of culture, followed by a significant decrease in LH binding on days 5 and 6 as well as an approximately 50% inhibition of F SH effect on day 6. Scatchard plot analysis showed that concurrent treatmen t with PRL resulted in inhibition of the granulosa cell LH binding capacity , whereas no difference could be detected in the binding affinity of LH to its receptor. Treatment with 8-bromo-cAMP produced a significant increase i n specific LH binding; concurrent treatment with PRL (30 ng/ml) produced a significant attenuation of 8-bromo-cAMP action. In addition, treatment with FSH increased the intracellular accumulation of cAMP, and concurrent treat ment with PRL did not result in inhibition of the FSH action, as assessed b y the generation of intracellular cAMP. Taken together, these findings sugg est that the ability of PRL to interfere with FSH action with regard to the induction of LH receptors is exerted at sites distal to those involved in cAMP generation. The effect of PRL on LH receptor messenger RNA (mRNA) leve ls was not significant during the increase in receptors, whereas after the maximal level of receptor expression was reached, the effect of PRL was app arent. Cotreatment with FSH (30 ng/ml) and increasing doses of PRL inhibite d the levels of FSH-induced LH receptor mRNA in a dose-dependent manner, wh ereas PRL did not inhibit the effect of FSH on the FSH receptor mRNA. To in vestigate the hormonal regulation of the 5'-flanking region, we analyzed th e effect of FSH on 1379 bp of LH receptor promoter in rat granulosa cells. Treatment with FSH (1-100 ng/ml) significantly enhanced the activity of 137 9 bp of the LH receptor 5'-flanking region in dose-dependent manner. Treatm ent with 30 ng/ml PRL alone did not significantly influence the activity of the LH receptor promoter and did not affect the increased promoter activit y induced by FSH. In addition, the rates of LH receptor mRNA gene transcrip tion assessed by nuclear run-on transcription assay increased by the additi on of FSH and were not affected by the addition of PRL in the presence of F SH. These data showed that PRL might not effect LH receptor gene transcript ion in the regulation of LH receptor mRNA. Next, an attempt was made to det ermine the effect of PRL on LH receptor mRNA stability by measuring the dec ay of LH receptor mRNA under conditions known to inhibit transcription. How ever, inhibitors of transcription were found to have a stabilizing effect o n the LH receptor mRNA, thus potentially masking the effect of PRL. Accordi ng to the expression of LH receptor mRNA, PRL might not affect the maximum level induced by FSH, but thereafter the maximum levels of LH receptor mRNA decreased faster than those of the control. Therefore, it may be possible that PRL acts to stimulate labile LH receptor mRNA-destabilizing factors.