Gill-Sharma Manjeet Kaur, Choudhuri Jyoti, D'Souza Serena
Department of Neuroendocrinology, National Institute for Research in Reproductive Health, Parel, Mumbai, India.
Protein Pept Lett. 2011 Aug;18(8):786-801. doi: 10.2174/092986611795714005.
During spermiogenesis, the elongating rat spermatid chromatin undergoes a gradual process of condensation which is initiated in the round spermatids at "step 7" of cytodifferentation in stage VII and extending to elongated spermatids at "step 19" of cytodifferentiation in stage VIII. The mechanism of chromatin condensation in the elongating spermatids is an elaborate process that encompasses several biochemical and biological aspects culminating in the deposition of protamine in DNA grooves. The protamination of sperm chromatin involves expression and storage of proteins involved in condensation, removal and degradation of nuclear histones and their replacement by transition proteins and protamine 1, transcriptional silencing and DNA repair, reduction of nuclear volume, repackaging of protaminated chromatin in torroids and development of a characteristic head shape and perforatorium. A study was undertaken in my laboratory to delineate the role of follicle stimulating hormone (FSH) and testosterone in the condensation of nuclear chromatin in the elongating spermatids of sexually competent species of rat. Towards this end, sexually competent male Holtzmann rats were treated with 20 mg/Kg/d per os (oral supplementum) of cyproterone acetate and 3 mg/Kg/d i.p (intra peritoneal) of fluphenazine decanoate to induce a functional deficiency in either testosterone or FSH. In both rat models, membrane-impermeable CMA3 fluorescent dye uptake assay for GC-rich prospective sites of DNA protamination, was indicative of insufficiency of protamine 1 in spermatozoa taken from caput epididymides of treated rats whereas a fluorescent TUNEL assay indicated the presence of nicked chromatin strands only in protamine-deficient spermatozoa derived from caput epididymides of fluphenazine-treated rats with functional deficiency of FSH. Western blotting of acid-soluble sperm basic proteins had confirmed the near absence of protamine 1 in treated rat spermatozoa in both models. Electron Microscopic evaluation too revealed fine ultrastructural changes in the nuclear membrane of cyproterone acetate as well as fluphenazine decanoate treated spermatozoa derived from caput epididymides. Electrophoretic analysis of caput sperm nuclear basic proteins substantiated the observations at cellular level and revealed a pattern of abnormal persistence of acid-soluble nuclear basic proteins in both rat models, the levels being more prominent in fluphenazine treated rats. Our studies suggest that adequate levels of both FSH and testosterone could be essential during the stages of spermatidal condensation and led us to hypothesize the existence of an endocrine-regulated molecular mechanism for histone to protamine transition and maintenance of chromatin integrity during chromatin condensation in the testis during spermiogenesis.
在精子发生过程中，正在伸长的大鼠精子细胞染色质经历一个逐渐浓缩的过程，该过程始于细胞分化VII期“第7步”的圆形精子细胞，并延伸至细胞分化VIII期“第19步”的伸长精子细胞。正在伸长的精子细胞中染色质浓缩的机制是一个复杂的过程，涉及几个生化和生物学方面，最终导致鱼精蛋白沉积在DNA凹槽中。精子染色质的鱼精蛋白化涉及参与浓缩的蛋白质的表达和储存、核组蛋白的去除和降解以及它们被过渡蛋白和鱼精蛋白1取代、转录沉默和DNA修复、核体积减小、鱼精蛋白化染色质在环面中的重新包装以及特征性头部形状和穿孔器的发育。我的实验室进行了一项研究，以阐明促卵泡激素（FSH）和睾酮在性成熟大鼠正在伸长的精子细胞核染色质浓缩中的作用。为此，对性成熟的雄性霍尔茨曼大鼠口服醋酸环丙孕酮20mg/Kg/d（口服补充剂）和腹腔注射癸酸氟奋乃静3mg/Kg/d，以诱导睾酮或FSH功能缺陷。在这两种大鼠模型中，用于富含GC的DNA鱼精蛋白化潜在位点的膜不可渗透CMA3荧光染料摄取试验表明，从处理过的大鼠附睾头采集的精子中鱼精蛋白1不足，而荧光TUNEL试验表明，仅在来自癸酸氟奋乃静处理的FSH功能缺陷大鼠附睾头的鱼精蛋白缺乏精子中存在切口染色质链。对酸溶性精子碱性蛋白的蛋白质印迹分析证实，在两种模型中，处理过的大鼠精子中几乎不存在鱼精蛋白1。电子显微镜评估也揭示了醋酸环丙孕酮以及癸酸氟奋乃静处理的来自附睾头的精子细胞核膜的细微超微结构变化。附睾头精子核碱性蛋白的电泳分析证实了细胞水平的观察结果，并揭示了两种大鼠模型中酸溶性核碱性蛋白异常持续存在的模式，在癸酸氟奋乃静处理的大鼠中水平更为突出。我们的研究表明，在精子细胞浓缩阶段，足够水平的FSH和睾酮可能是必不可少的，并使我们推测存在一种内分泌调节的分子机制，用于在精子发生过程中睾丸染色质浓缩期间组蛋白向鱼精蛋白的转变以及染色质完整性的维持。
