Analysis of the protein composition of the mouse sperm perinuclear theca and characterization of its major protein constituent
Analysis of the protein composition of the mouse sperm perinuclear theca and characterization of its major protein constituent
复制标题
DOI：
10.1095/biolreprod57.6.1426
复制
发表时间：
1997-12-01
影响因子：
3.6
通讯作者：
Oko, R
中科院分区：
文献类型：
--
作者：
Korley, R;Pouresmaeili, F;Oko, R
核周膜(PT)是一种覆盖在哺乳动物精子核上的细胞骨架结构，被认为具有膜结合作用。本研究的目的是分析小鼠PT的蛋白质组成，鉴定和测序其主要蛋白质组分，并表征该蛋白质在精子发生过程中的转录和翻译来源。PT是从去膜和顶体耗尽的小鼠精子头中通过碱性处理提取的。PT提取物的蛋白质谱由几个多肽组成，其中15 kDa的顶体下蛋白与先前克隆的大鼠15 kDa PT蛋白(PERF 15)具有免疫交叉反应，PERF 15属于脂结合蛋白家族。根据大鼠PERF-15基因设计一对引物，通过聚合酶链式反应(PCR)筛选小鼠睾丸cDNA文库。扩增产物的氨基酸序列与大鼠睾丸特异表达的PERF-15的氨基酸序列基本一致。用编码小鼠PERF-15的核糖核酸探针进行的发育Northern杂交和原位杂交研究表明，在圆形和早期伸长的小鼠精子细胞中，mRNA的表达水平最高。免疫组织化学结果显示，PERF-15在粗线期中期精母细胞胞浆中开始表达，在晚期伸长的小鼠精母细胞远端胞浆中表达最强，在转录停止后很长一段时间内表达。在圆形和早期细长精子细胞的发育过程中，免疫标记逐渐集中在精子细胞核的前半部分，这表明在小鼠精子发生的这一阶段，PERF 15在顶体下沉积。
The perinuclear theca (PT) is a cytoskeletal structure that covers the nucleus of mammalian spermatozoa and is believed to have a membrane-binding role. The objectives of this study were to analyze the protein composition of the mouse PT, to identify and sequence its major protein component, and to characterize this protein's transcriptional and translational origins during spermatogenesis. The PT was extracted from demembranated and acrosome-depleted mouse sperm heads by alkaline treatment. The protein profile of the PT extract was composed of several polypeptides, of which a 15-kDa subacrosomal protein predominated and was found to be immunocross-reactive with a previously cloned 15-kDa PT protein of the rat (PERF 15) that belongs to a family of lipid-binding proteins. A primer pair designed from rat PERF 15 cDNA was then used to screen a mouse testicular cDNA library by polymerase chain reaction (PCR). The deduced amino acid sequence obtained from the PCR product was almost identical to the testicular-specific rat PERF 15. Developmental Northern blots and in situ hybridization studies performed with riboprobes encoding the mouse PERF 15 cDNA revealed that mRNA levels were highest in round and early elongating mouse spermatids. Immunohistochemistry indicated that PERF 15 began to be expressed in the cytoplasm of mid-pachytene spermatocytes and appeared to reach maximum expression in the distal cytoplasm of late elongating mouse spermatids, long after transcriptional arrest. During the development of round and early elongated spermatids, the immunolabel became progressively concentrated over the anterior half of the spermatid nucleus, suggesting a subacrosomal deposition of PERF 15 during this phase of mouse spermiogenesis.