Biochemical and molecular biological studies on intracellular signal transduction mechanisms of follicle-stimulating hormone in ovarian granulosa cells

卵泡刺激素(FSH)在卵泡成熟过程中起着重要作用。然而，ESH在颗粒细胞(GC)中的作用机制尚不清楚。因此，为了研究GCs中FSH的细胞内信号转导途径，我们在体内和体外检测了丝裂原活化蛋白激酶(MAPK)活性以及c-fos和Jun家族基因的表达。未成熟雌性大鼠注射PMSG后，提取卵巢总RNA，进行Northern杂交。从猪小卵泡(直径L-2 mm)获得的GCs与卵泡刺激素(FSH)、P1AA或Forsklin体外孵育。在含有星状孢子素或RP-cAMP的细胞培养物中加入FSII，用特定的试剂盒检测细胞裂解物中MAPK的活性。从培养的GC中提取总RNA，进行Northern杂交。注射PMSG后30 mm，大鼠卵巢中c-fos和JunB基因表达明显增强，60min达到高峰。在整个实验过程中，Jund的表达很低，c-Jun未被检测到。在加入ESH 30 mM后，FSH再次激活培养的GCs中c-fos和Jun13mRNAs。虽然PMA促进c-fos和JunB的表达，但Forsklin对其无影响。星形孢子素可显著抑制ESH引起的上述基因表达的增加，但RP-cAMP对此无明显抑制作用，加入FSH后5 mM可刺激MAPK活性，15 mM后达平台期。PMA可刺激MAPK活性，而Forsklin则不能。上述结果表明，MAPK、c-fos和JunB在颗粒细胞的促卵泡激素作用中起重要作用，蛋白激酶C通路参与颗粒细胞的信号转导。

Follicle-stimulating hormone (FSH) plays an important role in the ovarian follicle maturation. However, the mechanisms of ESH action in the granulosa cells (GC) still remain unclear. Therefore, in order to investigate intracellular signal transduction pathways of FSH in GCs, we determined in vivo and in vitro the expression of mitogen-activated protein kinase (MAPK) activity, and c-fos and Jun family genes. After PMSG was injected to immature female rats, total RNA was extracted from ovaries and subjected to Northern blottings using cDNAs. GCs obtained from porcine small follicles (l-2 mm in diameter) were also incubated in vitro with FSH, P1AA or forskolin. FSII was also added to cell cultures with staurosporine or Rp-cAMP.MAPK activities in cell lysates were determined utilizing specific assay kits. Total RNA was extracted from cultured GCs and subjected to Northern blottings. c-fos and junB mRNAs were significantly expressed in rat ovaries 30 mm after PMSG injection, and reached plateau after 60 min. The expression of junD was minimal, and c-jun was not detected throughout the experimental period. FSH again stimualted c-fos and jun13 mRNAs in cultured GCs 30 mm after ESH addition. Although PMA stimulated the expression of c-fos and junB, forskolin did not influence it. Staurosporine significantly inhibited the increase in mRNAs of these genes by ESH.but Rp-cAMP did not inhibit it, MAPK activiteies were stimulated 5 mm after FSH addition to cultured GCs, and reached plateau after 15 mm. PMA stimulated MAPK activities, while forskolin did not. Staurosporinesignificantly inhibited the increase in NIAPK activities by FSH, whereas Rp-cAMP had no influence.These data suggest that MAPK, c-fos and junB play an important role in FSH action in granulosa cells, and that protein kinase C pathway is invovled in the signal transduction pathways in granulosa cells,