The regulation of steroidogenic acute regulatory protein (StAR) in vitro by gonadotropins was investigated in granulosa cells from prehierarchal and preovulatory hen follicles. Basal levels of StAR messenger RNA (mRNA) in undifferentiated granulosa cells from prehierarchal (6- to 8-mm) follicles were consistently low, but detectable, and were significantly increased by treatment with 8-bromo-cAMP and FSH (but not LH) within 3-6 h of culture. After 20 h of culture, 8-bromo-cAMP, FSH, and LH each increased StAR mRNA levels above those in control cultured cells, and the delayed response to LH treatment was associated with increased levels of LH receptor (LH-R) mRNA. On the other hand, inhibition of mitogen-activated protein (MAP) kinase signaling, using the MAP kinase kinase inhibitors U0126 and PD98059, in the presence of FSH further increased StAR mRNA and protein levels, LH-R mRNA levels, and progesterone synthesis compared with those in cells cultured with FSH alone. The highest basal expression of StAR mRNA during follicle development was found in granulosa from the largest (F1) preovulatory follicle, with comparatively lower levels in granulosa from less mature (F2 plus F3) preovulatory follicles. Treatment with LH rapidly increased StAR mRNA and protein (but not LH-R mRNA) expression in cultures of F1 granulosa and in combined F2 plus F3 granulosa within 3 h, although the magnitude of stimulation was greater in F2 plus F3 granulosa. Compared with results from granulosa cells from prehierarchal follicles cultured for 20 h, inhibition of MAP kinase signaling in the presence of LH for 1 h failed to further enhance levels of StAR or LH-R expression or progesterone production in F2 plus F3 follicle granulosa compared with the effect of LH treatment alone. These results demonstrate that StAR expression in the hen ovary is up-regulated by goaadotropins at least in part via cAMP signaling. The ability of MAP kinase kinase inhibitors to potentiate gonadotropin-induced StAR and LH-R expression plus progesterone synthesis in prehierarchal follicle granulosa cells in vitro suggests that inhibition of paracrine or autocrine factor-mediated MAP kinase signaling in vivo may be a prerequisite for the full potentiation of granulosa cell steroidogenesis that occurs after recruitment into the preovulatory hierarchy. Finally, these results fail to support a role for MAP kinase signaling in acutely modulating LH-mediated StAR expression or progesterone production in hierarchal follicles, such as occurs during the preovulatory surge of progesterone.
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