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

促卵泡激素（FSH）在卵巢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FSH在GCs细胞内的信号转导途径，我们在体内和体外检测了丝裂原活化蛋白激酶（MAPK）活性、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，用cdna进行Northern印迹。从猪小卵泡（直径1 - 2mm）中获得的GCs也与FSH、P1AA或forskolin一起体外孵育。FSII也加入到staurosporine或Rp-cAMP的细胞培养中。利用特异性试剂盒测定细胞裂解物中的MAPK活性。从培养的GCs中提取总RNA，并进行Northern印迹。PMSG注射后30mm大鼠卵巢中c-fos和junB mrna显著表达，60min后达到平稳期。junD的表达极低，整个实验期间均未检测到c-jun。在加入ESH后30mm， FSH再次刺激培养GCs中的c-fos和jun13 mrna。PMA虽然刺激了c-fos和junB的表达，但forskolin对其没有影响。Staurosporine通过ESH显著抑制这些基因mrna的增加。而Rp-cAMP对MAPK没有抑制作用，在FSH加入培养的GCs后5mm， MAPK活性受到刺激，15mm后达到平稳状态。PMA刺激MAPK活性，而forskolin则没有。staurosporin显著抑制FSH对NIAPK活性的增加，而Rp-cAMP对NIAPK活性无影响。这些数据表明，MAPK、C -fos和junB在颗粒细胞FSH的作用中起重要作用，蛋白激酶C通路参与颗粒细胞的信号转导通路。

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,