BIOREGULATION OF SPERMATOGENESIS

精子发生的生物调节

• 批准号: 3311707
• 负责人: ABRAHAM L KIERSZENBAUM
• 金额: $ 13.27万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-04-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3311707
• 关键词: DNA; SDS polyacrylamide gel electrophoresis; Sertoli cells; affinity chromatography; chromatin; crosslink; electron microscopy; gene expression; genetic markers; genetic regulation; genetic regulatory element; genetic transcription; laboratory rat; messenger RNA; molecular cloning; northern blottings; nuclear matrix; nucleic acid sequence; polymerase chain reaction; posttranslational modifications; protein biosynthesis; protein sequence; protein structure; secretory protein; southern blotting; sperm; spermatogenesis; testis; tissue /cell culture; western blottings

Past studies from our laboratory have shown that, during rat testicular development, Sertoli cells synthesize and secrete a protein (S45-S35 heterodimeric protein; synonymous names: SGP-2, clusterin, TRPM-2) which then acquires a spermatogenic stage-dependent expression pattern. The purpose of this renewal application is to continue our study of S45-S35 function in spermatogenesis. During the previous funding periods, we have shown that components of the head and tail of developing spermatids and epididymal sperm contain proteins with antigenic homology to S45-S35. We have also examined the expression of S35-S45 mRNA during testicular development and spermatogenesis. During the current funding period we have found that pachytene and spermatids also express S35-S45 mRNA. However, while in Sertoli cells the somatic-type S35-S45 encoded precursor gives rise to the disulfide linked S45-S35 heterodimer, the spermatogenic-type S35-S45 gene generates a similar precursor but its post-translational processing yields monomers. We have also shown that proteins associated with both outer dense fiber (ODF) polymers of sperm tail and sperm protamine-DNA chromatin are antigenically homologous to the S45-S35 precursor. As a result of these studies we have identified structural components in sperm with a role in the morphogenesis of ODF polymers and packaging of sperm chromatin in an orderly fashion. Therefore, we are in a unique situation to analyze molecular aspects of spermiogenesis, in particular the marking genes which may be required during spermiogenesis and, eventually, at fertilization and during early embryogenesis. During the next funding period, we will address specific aspects of the structure of the spermatogenic-type S35-S45 gene and its function in sperm formation through five specific aims: (1) to determine the structure and functional regulation of the spermatogenic-type S35-S45 gene, in particular, the characterization of alternative polyadenylation and splicing sites and identification of nuclear regulatory elements controlling the in vitro transcription of somatic- and spermatogenic-type S35-S45 genes. (2) To characterize molecular aspects of the coiled-coil subunits of ODF polymers and ODF-associated protein(s) and to examine more rigorously the molecular basis for the antigenic homology of ODF-associated proteins with the common somatic and spermatogenic precursor. (3) To elucidate early stages of the assembly of the ODF polymer by in vitro reconstitution and chemical crosslinking of ODF subunits and ODF-associated protein(s). (4) Isolate sperm nuclear matrix proteins bound to a distinct portion of the sperm DNA loop, define DNA molecular characteristics and determine the primary structure of a 38.5 kD nuclear matrix protein antigenically homologous to the somatic/spermatogenic common precursor. (5) Isolate and identify DNA sequences specifically associated with segments of sperm DNA loops bound to or free of nuclear matrix proteins and approach the question of gene marking during spermiogenesis. Results from these studies are significant for understanding molecular mechanisms whereby anomalous transcripts can lead to the formation of abnormal and unfertile sperm as well as testicular malignancies.

我们实验室过去的研究表明，在大鼠睾丸 发育过程中，支持细胞合成并分泌一种蛋白质（S45-S35 异二聚体蛋白；同义名称：SGP-2、clusterin、TRPM-2） 然后获得生精阶段依赖的表达模式。 这 此更新申请的目的是继续我们对 S45-S35 的研究 在精子发生中发挥作用。 在之前的资助期间，我们 研究表明，发育中的精子细胞的头部和尾部的成分 附睾精子中含有与S45-S35具有抗原同源性的蛋白质。 我们还检测了睾丸发育过程中 S35-S45 mRNA 的表达。 发育和精子发生。 在当前的资助期间，我们发现粗线期和 精子细胞也表达S35-S45 mRNA。 然而，在支持细胞中 体细胞型 S35-S45 编码前体产生二硫键 连接S45-S35异二聚体，生精型S35-S45基因产生 类似的前体，但其翻译后加工产生 单体。 我们还表明，与外部和外部相关的蛋白质 精子尾部和精子鱼精蛋白-DNA 的致密纤维 (ODF) 聚合物 染色质与 S45-S35 前体在抗原上同源。 作为一个 这些研究的结果我们已经确定了精子的结构成分 在 ODF 聚合物的形态发生和精子包装中发挥作用 染色质有序排列。 因此，我们处于一个独特的境地 分析精子发生的分子方面，特别是标记 精子发生过程中可能需要的基因，并最终在 受精和早期胚胎发生期间。 在下一个资助期内，我们将解决以下具体问题： 生精型S35-S45基因的结构及其功能 精子的形成通过五个具体目标：（1）确定 生精型S35-S45的结构和功能调控 基因，特别是选择性多腺苷酸化的表征 和剪接位点以及核调控元件的鉴定 控制体细胞和生精型的体外转录 S35-S45 基因。 (2) 表征卷曲螺旋的分子方面 ODF 聚合物和 ODF 相关蛋白的亚基并检查 更严格地确定抗原同源性的分子基础 ODF 相关蛋白与常见的体细胞和生精蛋白 前驱。 (3) 阐明ODF组装的早期阶段 通过 ODF 的体外重构和化学交联制备聚合物 亚基和 ODF 相关蛋白。 (4) 分离精子核基质 与精子 DNA 环的不同部分结合的蛋白质，定义了 DNA 38.5 分子特征并确定一级结构 kD 核基质蛋白与 体细胞/生精共同前体。 (5) DNA的分离和鉴定 与结合的精子 DNA 环片段特异性相关的序列 含有或不含核基质蛋白并解决基因问题 精子发生过程中的标记。 这些研究的结果是 对于理解异常的分子机制具有重要意义 转录物可能导致异常和不育精子的形成 还有睾丸恶性肿瘤。