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重要作用。然而，ESH在颗粒细胞（GC）中的作用机制仍不清楚。因此，为了研究FSH在GC中的细胞内信号转导途径，我们在体内和体外测定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。将PMSG注射到未成熟雌性大鼠后，从卵巢中提取总RNA，并使用cDNA进行北方印迹。从猪小卵泡（直径1 -2 mm）获得的GCs也与FSH、P1 AA或毛喉素在体外孵育。还将FSII加入到具有星形孢菌素或Rp-cAMP的细胞培养物中，利用特异性测定试剂盒测定细胞裂解物中的MAPK活性。从培养的GC中提取总RNA并进行北方印迹。注射PMSG后30 mm，c-fos和junB mRNA在大鼠卵巢中显著表达，60 min后达到平台期。junD的表达最低，整个实验期间未检测到c-jun。加入ESH后30 min，FSH再次刺激培养的GC中的c-fos和jun 13 mRNA。结果表明，PMA刺激GCs中c-fos和junB基因的表达，而forskolin对其无影响，Staurosporine可明显抑制ESH引起的这些基因mRNA的表达，而Rp-cAMP则无抑制作用，加入FSH后5 min，MAPK活性被激活，15 min后达到平台，PMA刺激MAPK活性，而forskolin则无此作用。Staurosporine可显著抑制FSH引起的NIAPK活性的升高，而Rp-cAMP则无此作用，提示MAPK、c-fos和junB在FSH作用于颗粒细胞中起重要作用，蛋白激酶C途径参与颗粒细胞的信号转导途径。

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,