Li H, Papadopoulos V, Vidic B, Dym M, Culty M
Department of Cell Biology, Georgetown University Medical Center, Washington, D.C. 20007, USA.
Endocrinology. 1997 Mar;138(3):1289-98. doi: 10.1210/endo.138.3.5021.
To determine what factors regulate gonocyte proliferation in newborn rats, we first examined the expression of several signal transduction molecules by immunocytochemistry in 3-day-old rat testis sections. We found that gonocytes specifically expressed the iota and zeta isoforms of protein kinase (PK) C (PKC) and the phosphatidylinositol 3-kinase (PI3-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 proliferation, we examined the effects of PDGF on gonocytes. For this, we developed a method to obtain highly purified and viable gonocytes in culture. After enzymatic digestion, differential adhesion, and two successive gradient fractionations, the gonocyte suspension obtained was over 90% pure, as assessed by light microscopy. The viability of cultured gonocytes exceeded 90% after 48 h in the presence of 2.5% FBS used as a survival factor. Immunodetection studies showed that isolated gonocytes expressed zeta PKC, PI 3-K, and the PDGF receptor. Treatment with 10 ng/ml PDGF induced a 4-fold increase of bromodeoxyuridine incorporation into gonocytes (from 5% proliferative gonocytes under basal conditions to 20% in the presence of PDGF). Because neonatal Sertoli cells secrete high levels of the growth promoting steroid, 17 beta-estradiol, we also tested its effect and found that it induced gonocyte proliferation at a level comparable with that of PDGF and that this effect was blocked by the estrogen receptor antagonist, ICI 164384. The combination of PDGF and estradiol, however, was not additive, suggesting that their effects were mediated by common molecular target(s). These results demonstrate that PDGF and estradiol activate gonocyte proliferation in vitro, suggesting that they may act as the physiological regulators of gonocyte development in vivo.
为了确定哪些因素调节新生大鼠生殖母细胞的增殖，我们首先通过免疫细胞化学方法检测了3日龄大鼠睾丸切片中几种信号转导分子的表达。我们发现生殖母细胞特异性表达蛋白激酶（PK）C的iota和zeta亚型（PKC）以及磷脂酰肌醇3激酶（PI3-K）。由于zeta PKC和PI 3-K已被证明在血小板衍生生长因子（PDGF）诱导的细胞增殖中起作用，我们检测了PDGF对生殖母细胞的影响。为此，我们开发了一种在培养中获得高度纯化且有活力的生殖母细胞的方法。经过酶消化、差异黏附以及两次连续的梯度分级分离后，通过光学显微镜评估，获得的生殖母细胞悬液纯度超过90%。在用作存活因子的2.5%胎牛血清存在下培养48小时后，培养的生殖母细胞活力超过90%。免疫检测研究表明，分离的生殖母细胞表达zeta PKC、PI 3-K和PDGF受体。用10 ng/ml PDGF处理可使生殖母细胞中溴脱氧尿苷掺入量增加4倍（从基础条件下5%的增殖生殖母细胞增加到存在PDGF时的20%）。由于新生支持细胞分泌高水平的促生长类固醇17β-雌二醇，我们也检测了其作用，发现它诱导生殖母细胞增殖的水平与PDGF相当，并且这种作用被雌激素受体拮抗剂ICI 164384阻断。然而，PDGF和雌二醇的联合作用并非相加的，这表明它们的作用是由共同的分子靶点介导的。这些结果表明，PDGF和雌二醇在体外激活生殖母细胞增殖，提示它们可能作为体内生殖母细胞发育的生理调节因子。
