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) 中的作用机制仍不清楚。因此，为了研究GC中FSH的细胞内信号转导途径，我们测定了体内和体外丝裂原激活蛋白激酶（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体内后，从卵巢中提取总 RNA，并使用 cDNA 进行 Northern 印迹分析。从猪小卵泡（直径1-2毫米）获得的GC也在体外与FSH、P1AA或毛喉素一起孵育。 FSII 也与星形孢菌素或 Rp-cAMP 一起添加到细胞培养物中。利用特定的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 Northern 印迹分析。 PMSG注射后30分钟，c-fos和junB mRNA在大鼠卵巢中显着表达，并在60分钟后达到平台。 junD的表达很少，并且在整个实验期间没有检测到c-jun。添加 ESH 后 30 mm，FSH 再次刺激培养的 GC 中的 c-fos 和 jun13 mRNA。尽管PMA刺激c-fos和junB的表达，但毛喉素对其没有影响。星形孢菌素显着抑制ESH引起的这些基因mRNA的增加，但Rp-cAMP不抑制它，在培养的GC中添加FSH后5mm，MAPK活性受到刺激，并在15mm后达到平台。 PMA 刺激 MAPK 活性，而毛喉素则不然。 Staurosporine显着抑制FSH引起的NIAPK活性的增加，而Rp-cAMP则没有影响。这些数据表明MAPK、c-fos和junB在颗粒细胞的FSH作用中发挥重要作用，并且蛋白激酶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,