REGULATION AND FUNCTION OF SPARC IN MALE REPRODUCTION

哺乳动物睾丸中的精子发生部分受一种复合体的调节 非终末Sertoli、Leydig和 肾小管周围细胞为成熟期创造独特的环境 精子。钙结合蛋白SPARC(分泌型蛋白，酸性 和富含半胱氨酸的)已在一种 孕育晚期精子细胞的小鼠支持细胞群。两者都有 SPARC基因和蛋白定位于小鼠间质 细胞。此外，几种与小鼠反应的单抗 精子表面抗原和抑制体外受精被证明是 与SPARC发生交叉反应。SPARC，最初被描述为一种差异化- 小鼠胚胎外内胚层与畸胎癌的相关产物 细胞，已经被分子克隆。完整的cdna序列揭示了一个 富含半胱氨酸的“Finger”序列与蛋白酶部分同源 抑制卵粘蛋白，以及与钙离子结合的“EF手”类似于 存在于钙调蛋白和小白蛋白中。 我们的观察表明，SPARC在男性生殖中发挥了作用，我们 建议研究在小鼠睾丸中的功能和调节。 这个特性很好的分子。相应地，SPARC的分布 胚胎、青春期前、成体和成熟睾丸中的mRNA和蛋白质 精子将通过免疫细胞化学和原位核酸来确定 光镜和电子显微镜水平的杂交。结合，吸收 并将监测睾丸组织对放射性标记SPARC的处理 在体内，可能的SPARC结合受体将从 睾丸组织和体液。我们将确定SPARC是否能与培养的 间质和支持细胞诱导细胞形态和蛋白质的变化 生物合成。我们将研究SPARC基因的激素调节 人绒毛膜促性腺激素对间质细胞培养及转录相互作用的影响 影响SPARC启动子结构域的因素。在培养的间质细胞中， 外源SPARC改变类固醇合成的能力 将对HCG进行检查。最后，我们将研究与精子相关的命运 体外获能过程中的SPARC及添加SPARC和抗SPARC的效果 SPARC抗体对小鼠体外受精的影响。 这些实验的重点是一种特性良好的分子，它的功能 基因调控将在睾丸发育和 精子发生。

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.