Onoda M, Pflug B, Djakiew D
Department of Anatomy and Cell Biology, Georgetown University School of Medicine, Washington, D.C. 20007.
J Cell Physiol. 1991 Dec;149(3):536-43. doi: 10.1002/jcp.1041490324.
The mitogenicity of germ cell proteins released from round spermatids (RS) and pachytene spermatocytes (PS) was investigated. Germ cells were isolated by centrifugal elutriation from 90-day-old rat testes and incubated in a supplement enriched culture media that lacked exogenous proteins. The conditioned culture media of RS and PS were dialysed/concentrated and lyophilized to prepare RS protein (RSP) and PS protein (PSP). Mitogenic activity of RSP and PSP was determined by 3H-thymidine incorporation into Swiss 3T3 fibroblasts. RSP and PSP stimulated 3H-thymidine incorporation by fibroblasts in a dose-dependent manner. At a higher concentration of RSP (300 micrograms/ml), fibroblast proliferation was stimulated from 6- to 20-fold of control cultures, whereas PSP (300 micrograms/ml) stimulated fibroblast proliferation 2.5-fold of control cultures. Since RSP exhibited substantially greater mitogenic activity than PSP we further investigated the RSP mitogenic substance(s) by immunoneutralization with antibodies against several growth factors. The mitogenic activity of RSP was significantly reduced by treatment with nerve growth factor (NGF) antibody, while neither the treatment of RSP with acidic fibroblast growth factor (aFGF) antibody, nor basic fibroblast growth factor (bFGF) antibody significantly modified the mitogenic activity of RSP. Interestingly, murine NGF-beta, recombinant human NGF-beta, and bovine serum albumin (BSA) did not exhibit mitogenic activity on 3T3 fibroblasts. Nevertheless, the presence of a NGF-like protein in RS and PS was confirmed by indirect immunofluorescence staining with a murine NGF antibody. Subsequently, a Western blot analysis with the NGF antibody identified two immunoreactive bands of 41 +/- 2 kDa and 51 +/- 1 kDa in both RSP and PSP under reduced conditions. These germ cell NGF-like proteins were apparently different from similarly prepared murine and human NGFs (13 kDa) in their molecular weight. Furthermore, neurite outgrowth from pheochromocytoma cells (PC-12), a functional bioassay for NGF-like activity, was stimulated by addition of RSP and PSP to the culture media of the PC-12 cells. These results demonstrate mitogenic activity in germ cell proteins (RSP and PSP) and identify a NGF-like protein(s) which is associated with most of this activity.
研究了从圆形精子细胞（RS）和粗线期精母细胞（PS）释放的生殖细胞蛋白的促有丝分裂活性。通过离心淘析从90日龄大鼠睾丸中分离出生殖细胞，并在缺乏外源蛋白的补充富集培养基中培养。将RS和PS的条件培养基进行透析/浓缩并冻干，以制备RS蛋白（RSP）和PS蛋白（PSP）。通过将3H-胸腺嘧啶掺入瑞士3T3成纤维细胞中来测定RSP和PSP的促有丝分裂活性。RSP和PSP以剂量依赖的方式刺激成纤维细胞掺入3H-胸腺嘧啶。在较高浓度的RSP（300微克/毫升）下，成纤维细胞增殖被刺激至对照培养物的6至20倍，而PSP（300微克/毫升）刺激成纤维细胞增殖为对照培养物的2.5倍。由于RSP表现出比PSP显著更高的促有丝分裂活性，我们用针对几种生长因子的抗体通过免疫中和进一步研究了RSP促有丝分裂物质。用神经生长因子（NGF）抗体处理显著降低了RSP的促有丝分裂活性，而用酸性成纤维细胞生长因子（aFGF）抗体或碱性成纤维细胞生长因子（bFGF）抗体处理RSP均未显著改变其促有丝分裂活性。有趣的是，小鼠NGF-β、重组人NGF-β和牛血清白蛋白（BSA）对3T3成纤维细胞均未表现出促有丝分裂活性。然而，用小鼠NGF抗体进行间接免疫荧光染色证实了RS和PS中存在一种NGF样蛋白。随后，用NGF抗体进行的蛋白质印迹分析在还原条件下在RSP和PSP中均鉴定出两条免疫反应带，分子量分别为41±2 kDa和51±1 kDa。这些生殖细胞NGF样蛋白在分子量上明显不同于同样制备的小鼠和人NGF（13 kDa）。此外，通过向嗜铬细胞瘤细胞（PC-12）的培养基中添加RSP和PSP，刺激了PC-12细胞的神经突生长，这是一种对NGF样活性的功能性生物测定。这些结果证明了生殖细胞蛋白（RSP和PSP）中的促有丝分裂活性，并鉴定出一种与大部分这种活性相关的NGF样蛋白。
