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Molecular Mechanisms of the Remodeling of Sperm Nuclear Basic Proteins

精子核碱性蛋白重塑的分子机制

基本信息

批准号：
01480022
负责人：
KATAGIRI Chiaki
金额：
$ 4.42万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

项目摘要

The sperm nuclear bsic proteins (SBPs) in two anuran amphibians, Xenopus laevis and Bufo japonicus, were characterized, and their synthesis during spermatogenesis and removal during fertilization were studied. The SBPs in Bufo consist exclusively of two protamines (Pl, P2), and those in Xenopus consist of 6 proteins (SPl-6) in addition to four nucleosomal core histones. Amino acid sequence analyses based on cloned cDNAs indicated that the Bufo P1 and P2 contain 39 amino acid residues, with a high sequence homology with fish protamines, and differ with each other only in the 28th amino acid residue (Pl, Asp ; P2, Glu). The Xenopus SP4 comprises 78 amino acid residues whose sequence has no indications of homology with protamines found in many other classes of vertebrates. During spermatogenesis, the histonesare synthesized before the end of primary spermatocyte stage, while the SBPs are first detectable in nuclei at the beginning of chromatin granulation and increase sharply at the last … More step of spermiogenesis. Both Northern analyses and in situ hybridization studies employing cDNA clones encoding Bufo P2 and Xenopus SP4 as probes revealed that in Bufo the transcripts of PI genes are present in the round spermatids (haploid stage), while in Xenopus the mRNAs for SP4 are present in the pachytene stage spermatocytes (tetraploid stage), and round spermatids. Thus the results suggested the occurrence of different regulatory mechanisms for transcription of SBP genes between Xenopus and Buf, as well as translational regulation of SBP gene products during spermiogenesis. When lysolecithinpermeabilized sperm were induced to form pronucleus by incubation with the egg extract, protamines were lost from nuclei within 1 min, accompanied by nuclear decondensation. The activities that induce both selective removal of SBPs and decondensation in sperm nuclei were found in extracts from growing and mature oocytes and pregastrula embryos, but not in postneurula embryos and adult tissues. The protamine-removing activity (PRA) was purified from egg extracts as the unique, heat-stable proteins with mobilities of l4OkD and 36kD on native- and SDS-PAGE, respectively, with the isoelectric points in the range 4.2 - 4.5, and possessing amino acid composition quite similar to that reported for Xenopus nucleoplasmin. We propose that in fertilized eggs the protamines are removed from sperm nuclei by nucleoplasmin by binding to but not by enzymatic degradation of the protamine. Less

本文报道了非洲爪蟾和中华蟾蜍精子核基蛋白（SBPs）的特征，并对其在精子发生过程中的合成和受精过程中的去除进行了研究。蟾蜍中的SBP仅由两种鱼精蛋白（P1，P2）组成，而非洲爪蟾中的SBP除了四种核小体核心组蛋白外，还由6种蛋白质（SP1 -6）组成。氨基酸序列分析表明，中华蟾蜍P1和P2均含有39个氨基酸残基，与鱼类鱼精蛋白有很高的同源性，仅第28位氨基酸残基（P1，Asp ; P2，Glu）不同。非洲爪蟾SP 4包含78个氨基酸残基，其序列与在许多其他种类的脊椎动物中发现的鱼精蛋白没有同源性的迹象。在精子发生过程中，组蛋白在初级精母细胞阶段结束前合成，而SBPs在染色质颗粒化开始时首先在细胞核中检测到，最后急剧增加 ...更多信息精子形成步骤。北方分析和原位杂交研究采用cDNA克隆编码蟾蜍P2和非洲爪蟾SP 4作为探针显示，在蟾蜍的PI基因的转录本存在于圆形精子细胞（单倍体阶段），而在非洲爪蟾的mRNA的SP 4存在于粗线期阶段精母细胞（四倍体阶段），和圆形精子细胞。因此，研究结果表明，非洲爪蟾和Buf之间的SBP基因的转录调控机制不同，以及SBP基因产物在精子发生过程中的翻译调控。当溶血卵磷脂透化的精子通过与卵提取物孵育诱导形成原核时，在1分钟内鱼精蛋白从核中丢失，伴随着核去凝聚。在生长和成熟的卵母细胞和前原肠胚胚胎的提取物中发现了诱导选择性去除SBPs和精子核去凝聚的活动，但在后神经胚胚胎和成人组织中没有发现。从卵提取物中分离纯化出具有鱼精蛋白去除活性的蛋白质（PRA），其电泳迁移率为140 kD和36 kD，等电点为4.2 - 4.5，氨基酸组成与已报道的爪蟾核质蛋白相似。我们认为，在受精卵中，精蛋白是通过核质蛋白与精蛋白结合而不是通过酶降解从精核中去除的。少