Zygote 2002 Nov;10(4):333-40

Expression of Fas, p53 and AFP in development of human fetal germ cells in vitro.

Wu J, Chen Y, Li T.

Department of Obstetrics and Gynecology, The Third Medical College, Beijing Medical University, China. jwu00004@temple.edu

Abstract: In the present study we employed a two-step culture system to study the expression of Fas, p53 and alpha-fetoprotein (AFP) in the development in vitro of human fetal germ cells. p53 mRNA was determined by Northern blotting, and Fas content was assessed by western blotting. RT-nested polymerase chain reaction (RT-nPCR) analysis was performed to determine the expression of AFP mRNA in different stages of fetal follicular development. Follicular cell apoptosis was evaluated by DNA fragmentation analyses (DNA ladder). The results showed that by day 7 of culture approximately one-sixth of fetal germ cells grew to class C oocytes (primary oocytes) from class B oocytes (primordial oocytes) or class A oocytes. On day 45 of culture, one-third of these primary follicles doubled in size. In the meantime, there was a high proportion apoptosis of follicular cells on days 35 or 45 of culture, as evident by a clear ladder pattern of DNA fragmentation upon electrophoretic analysis. Expression of Fas antigen and p53 mRNA increased in a time-dependent manner, while AFP mRNA was expressed on days 10 to 35, and disappeared on day 45. These results indicate that human fetal germ cells can develop in a two-step culture system and AFP may play an active role in the proliferation of these germ cells. At the late stage of follicular development in vitro, a number of follicular cells became apoptotic. Moreover, apoptosis may be the mechanism responsible for fetal germ cell regression and the Fas antigen and/or p53-mediated death pathway may be central in the induction of germ cell regression.

受精卵 2002 Nov;10(4): 333-40

离体人胚胎细胞发育过程中Fas，p53 和 AFP的表达

吴际 陈研 李潭

摘要：在此研究中我们应用了一个两步培养系统来观察Fas, p53和α-胎蛋白（AFP）在离体人胚胎细胞发育过程中的表达。p53 mRNA和Fas的含量分别用Northern blotting和western blotting的方法进行评估。AFP mRNA在胎儿卵泡发育过程中不同时期的表达水平则用逆转录巢式聚合酶链反应(RT-nPCR)进行分析。卵泡细胞的凋亡用DNA破碎分析（DNA阶梯）评估。结果显示在培养的第七天大约有六分之一的胎胚细胞从C类卵母细胞（初级卵母细胞）长成B类卵母细胞（原始卵母细胞）或A类卵母细胞。在第45天，三分之一的卵泡大小变为原来的两倍。与此同时，在培养的第35或45天，大部分卵泡细胞出现凋亡。Fas抗原和p53 mRNA的表达则以时间依赖性的方式增加，而AFP mRNA的表达在第10到35天出现，第45天消失。这些结果表明人胚胎细胞可以在两步培养系统中生长并且AFP可能在这些胚胎细胞的增生中发挥了积极的作用。在离体卵泡发育的晚期，一定数量的卵泡细胞开始凋亡。而且，凋亡可能参与了胚胎细胞的衰退，Fas抗原和/或p53介导的死亡通路也可能在诱导胚胎细胞衰退的过程中起到了重要作用。