CELLULAR AND MOLECULAR MECHANISMS THAT MEDIATE INSULIN-DEPENDENT RAT GRANULOSA-CELL MITOSIS

Rat ovarian follicles are composed of small and large granulosa cells (GC). The present studies demonstrate that small GCs undergo insulin- or phorbol ester-dependent mitosis in vitro. In order to examine the c ellular and molecular events that account for insulin's mitogenic acti on, small GCs were cultured with either insulin, phorbol ester (TPA), or both insulin and TPA. Insulin and TPA increased GC numbers by 21 +/ - 3% and 20 +/- 2% over control values, respectively (p < 0.05). Simul taneous addition of insulin and TPA increased GC numbers by 20 +/- 3% (P < 0.05). In a second experiment, small GCs were exposed to control medium, insulin, staurosporine (a protein kinase C [PKC] inhibitor), o r both insulin and staurosporine. These studies revealed that insulin induced a 21 +/- 5% increase in GC numbers and that staurosporine bloc ked insulin's mitogenic action. These observations suggest that insuli n mediates its mitogenic action through a PKC-dependent mechanism. Sin ce the proto-oncogenes, c-fos and c-jun, are expressed during GC mitos is, studies were undertaken to determine whether or not the expression of these two proto-oncogenes products was enhanced by insulin. The ex pression of c-fos and c-jun proteins was assessed by immunocytochemist ry. These studies showed that after 5 h, insulin increased the percent age of cells that stained far c-fos and c-jun by 15 +/- 2% and 19 +/- 4, respectively (P < 0.05). The expression of these proto-oncogenes wa s blocked by staurosporine. Both progesterone and 8-br-cAMP, which blo ck insulin-dependent GC mitosis, also inhibited the expression of c-fo s and c-jun. Finally, small GCs were cultured with insulin in the pres ence of sense and antisense oligonucleotides specific for c-fos and c- jun. Under these conditions, insulin induced a 32 +/- 10% and 24 +/- 5 % increase in cell numbers in the presence of c-fos and c-jun sense ol igonucleotides, respectively. Insulin-dependent GC proliferation was c ompletely prevented in the presence of either c-fos or c-jun antisense oligonucleotide. Taken together, these experiments support the concep t that insulin stimulates small GC mitosis by activating PKC, which in duces the expression of both c-fos and c-jun. Both of these genes play essential roles in GC mitosis.