REGULATION OF RAT TESTIS GONOCYTE PROLIFERATION BY PLATELET-DERIVED GROWTH-FACTOR AND ESTRADIOL - IDENTIFICATION OF SIGNALING MECHANISMS INVOLVED

To determine what factors regulate gonocyte proliferation in newborn r ats, we first examined the expression of several signal transduction m olecules by immunocytochemistry in 3-day-old rat testis sections. We f ound that gonocytes specifically expressed the iota and zeta isoforms of protein kinase (PK) C (PKC) and the phosphatidylinositol 3-kinase ( PI 3-K). Because both the zeta PKC and PI 3-K have been shown to play a role in platelet-derived growth factor (PDGF)-induced cell prolifera tion, we examined the effects of PDGF on gonocytes. For this, we devel oped a method to obtain highly purified and viable gonocytes in cultur e. After enzymatic digestion, differential adhesion, and two successiv e gradient fractionations, the gonocyte suspension obtained was over 9 0% pure, as assessed by light microscopy. The viability of cultured go nocytes exceeded 90% after 48 h in the presence of 2.5% FBS used as a survival factor. Immunodetection studies showed that isolated gonocyte s expressed zeta PKC, PI 3-K, and the PDGF receptor. Treatment with 10 ng/ml PDGF induced a 4-fold increase of bromodeoxyuridine incorporati on into gonocytes (from 5% proliferative gonocytes under basal conditi ons to 20% in the presence of PDGF). Because neonatal Sertoli cells se crete high levels of the growth promoting steroid, 17 beta-estradiol, we also tested its effect and found that it induced gonocyte prolifera tion at a level comparable with that of PDGF and that this effect was blocked by the estrogen receptor antagonist, ICI 164384. The combinati on of PDGF and estradiol, however, was not additive, suggesting that t heir effects were mediated by common molecular target(s). These result s demonstrate that PDGF and estradiol activate gonocyte proliferation in vitro, suggesting that they may act as the physiological regulators of gonocyte development in vivo.