BIOREGULATION OF SPERMATOGENESIS

我们实验室过去的研究表明， 在发育过程中，支持细胞合成并分泌蛋白质（S45-S35 异二聚体蛋白;同义名称：SGP-2、丛生蛋白、TRPM-2）， 然后获得生精阶段依赖性表达模式。 的 本更新申请的目的是继续我们对S45-S35的研究 在精子发生中的作用 在过去的几年中，我们 已经表明精子细胞的头部和尾部的组成部分 附睾精子含有与S45-S35抗原同源的蛋白质。 我们还检测了S35-S45 mRNA在睾丸发育过程中的表达， 发育和精子发生。 在目前的资助期间，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编码的前体产生二硫化物 连接的S45-S35异二聚体，精子发生型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，与两种外膜蛋白相关的蛋白质， 精子尾部和精子精蛋白-DNA的致密纤维（ODF）聚合物 染色质与S45-S35前体抗原同源。 作为 通过这些研究，我们已经确定了精子中的结构成分 在ODF聚合物的形态发生和精子包装中起作用 染色质中的有序排列。 因此，我们处于一种独特的情况 分析精子形成的分子方面，特别是标记 这些基因在精子发生过程中可能是必需的， 受精和早期胚胎发生。 在下一个拨款期内，我们会处理 精子发生型S35-S45基因的结构及其在精子发生中的功能 精子形成通过五个具体目标：（1）确定 精子发生型S35-S45的结构和功能调节 基因，特别是，交替多聚腺苷酸化的表征 和剪接位点以及核调控元件的鉴定 控制体细胞和精子发生型的体外转录 S35-S45基因。(2)为了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 更严格地说， ODF相关蛋白与体细胞和生精细胞共同表达 先驱(3)阐明ODF组装的早期阶段 通过体外重构和ODF的化学交联聚合物 亚基和ODF相关蛋白。(4)分离精子核基质 蛋白质结合到精子DNA环的不同部分，定义DNA 分子特征，并确定了一级结构的38.5 kD核基质蛋白抗原同源的 体细胞/精子发生共同前体。(5)分离并鉴定DNA 与精子DNA环结合片段特异性相关的序列 或不含核基质蛋白，并探讨基因 精子形成过程中的标记。 这些研究的结果 对于理解异常的分子机制 转录本可以导致异常和不育精子的形成， 以及睾丸恶性肿瘤

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.