REGULATION AND FUNCTION OF SPARC IN MALE REPRODUCTION

SPARC 在男性生殖中的调节和功能

• 批准号: 3325990
• 负责人: ROBERT B VERNON
• 金额: $ 12.67万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3325990
• 关键词: Leydig cells; Sertoli cells; affinity chromatography; calcium binding protein; chorionic gonadotropin; cysteine; electron microscopy; embryo /fetus cell /tissue; genetic transcription; immunocytochemistry; in situ hybridization; in vitro fertilization; laboratory mouse; laboratory rabbit; messenger RNA; monoclonal antibody; protein biosynthesis; sperm; sperm capacitation; spermatogenesis; testis

Spermatogenesis in the mammalian testis is regulated in part by a complex set of interactions among the nongerminal Sertoli, Leydig, and peritubular cells that create a unique environment for the maturation of spermatozoa. A calcium-binding protein, SPARC (Secreted Protein, Acidic and Rich in Cysteine) has been identified in the cytoplasm of a population of murine Sertoli cells bearing Late-stage spermatids. Both SPARC mRNA and protein have been localized to murine interstitial Leydig cells. In addition, several monoclonal antibodies that reacted with mouse sperm surface antigens and inhibited in vitro fertilization were shown to cross-react with SPARC. SPARC, initially described as a differentiation- associated product of murine extraembryonic endoderm and teratocarcinoma cells, has been molecularly cloned. The complete cDNA sequence revealed a cysteine-rich "finger" sequence partially homologous to the protease inhibitor ovomucoid, and a Ca+2 binding "EF Hand" similar to those present in calmodulin and parvalbumin. Our observations suggest SPARC plays a role in male reproduction, and we propose to study, in the murine testis, the function and regulation of this well-characterized molecule. Accordingly, the distribution of SPARC mRNA and protein in embryonic, prepubertal and adult testes, and mature sperm will be determined by immunocytochemistry and in situ nucleic acid hybridization at light and electron microscopic levels. Binding, uptake and processing of radiolabeled SPARC by testis tissue will be monitored in vivo, and putative SPARC-binding receptors will be isolated from testis tissue and fluids. We will determine if SPARC can bind to cultured Leydig and Sertoli cells to induce changes in cell shape and protein biosynthesis. We will study hormonal regulation of the Sparc gene in Leydig cell cultures exposed to hCG, and interaction of transcription factors with domains of the SPARC promoter. In cultured Leydig cells, the ability of exogeneous SPARC to alter steroid production in response to hCG will be examined. Finally, we will study the fate of sperm-associated SPARC during capacitation in vitro and effects of added SPARC and anti- SPARC antibodies on mouse in vitro fertilization. These experiments focus on a well-characterized molecule whose function and genetic regulation will be examined during testicular development and spermatogenesis.

哺乳动物睾丸中的精子发生部分由复合物调节 Nongerminal Sertoli，Leydig和 周围细胞为成熟而创造独特的环境 精子。钙结合蛋白SPARC（分泌蛋白，酸性 在A的细胞质中已经鉴定出富含半胱氨酸） 鼠类塞托利细胞的种群具有晚期精子。两个都 SPARC mRNA和蛋白质已定位为鼠间隙leydig 细胞。另外，几种与小鼠反应的单克隆抗体 精子表面抗原和抑制体外受精被证明 与SPARC进行交叉反应。 Sparc，最初被描述为分化 鼠外胚膜内胚层和畸胎瘤的相关产物 细胞已被分子克隆。完整的cDNA序列显示 半胱氨酸富的“手指”序列与蛋白酶部分同源 抑制剂卵球形和Ca+2结合的“ EF手”类似于那些 存在于钙调蛋白和白蛋白酶中。 我们的观察结果表明，SPARC在男性繁殖中起作用，我们 建议在鼠睾丸中研究 这个良好的分子。因此，Sparc的分布 胚胎，青春期和成人睾丸中的mRNA和蛋白质以及成熟 精子将通过免疫细胞化学和原位核酸确定 光和电子显微镜水平的杂交。绑定，吸收 将监测睾丸组织对放射性标记的SPARC的处理 体内和假定的SPARC结合受体将从 睾丸组织和液体。我们将确定SPARC是否可以与培养 Leydig和Sertoli细胞诱导细胞形状和蛋白质变化 生物合成。我们将研究SPARC基因的激素调节 暴露于HCG的Leydig细胞培养物和转录的相互作用 SPARC启动子域的因素。在培养的leydig细胞中 外生SPARC改变类固醇产生的能力 将检查HCG。最后，我们将研究精子相关的命运 SPARC在体外电容期间以及添加的SPARC和抗 小鼠体外受精的SPARC抗体。 这些实验集中于一个特征良好的分子，其功能 在睾丸发育和 精子发生。