Korley R, Pouresmaeili F, Oko R
McGill University, Montreal, Quebec, Canada.
Biol Reprod. 1997 Dec;57(6):1426-32. doi: 10.1095/biolreprod57.6.1426.
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.
核周膜（PT）是一种细胞骨架结构，覆盖在哺乳动物精子的细胞核上，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，鉴定并测序其主要蛋白质成分，并表征该蛋白质在精子发生过程中的转录和翻译起源。通过碱性处理从去膜和去顶体的小鼠精子头部提取PT。PT提取物的蛋白质谱由几种多肽组成，其中一种15 kDa的顶体下蛋白质占主导地位，并且发现它与先前克隆的大鼠15 kDa PT蛋白质（PERF 15）具有免疫交叉反应性，该蛋白质属于脂质结合蛋白家族。然后使用从大鼠PERF 15 cDNA设计的引物对通过聚合酶链反应（PCR）筛选小鼠睾丸cDNA文库。从PCR产物获得的推导氨基酸序列与睾丸特异性大鼠PERF 15几乎相同。用编码小鼠PERF 15 cDNA的核糖探针进行的发育Northern印迹和原位杂交研究表明，mRNA水平在圆形和早期伸长的小鼠精子细胞中最高。免疫组织化学表明，PERF 15在粗线期中期精子细胞的细胞质中开始表达，并且在转录停滞很久之后，在晚期伸长的小鼠精子细胞的远端细胞质中似乎达到最大表达。在圆形和早期伸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个阶段，PERF 15在顶体下沉积。
