BIOREGULATION OF SPERMATOGENESIS

精子发生的生物调节

基本信息

批准号：
3311710
负责人：
ABRAHAM L KIERSZENBAUM
金额：
$ 14.63万
依托单位：
CITY COLLEGE OF NEW YORK
依托单位国家：
美国
项目类别：
财政年份：
1978
资助国家：
美国
起止时间：
1978-04-01 至 1992-06-30
项目状态：
已结题

项目摘要

We have found that Sertoli cell secretory protein S70 share antigenic homology with polypeptide components of Sertoli cell secretory protein S45-S35 heterodimeric protein. We have made the unexpected observations that polyclonal antisera raised against these two Sertoli cell secretory proteins crossreact with outer dense fiber polypeptides of sperm tail and that a polyclonal antiserum generated against a major polypeptide of outer dense fibers of sperm tail crossreacts with protein S70 and its breakdown products. These findings suggest a possible product-precursor relationship between S70 and S45-S35 heterodimeric protein and raise important questions concerning the cell origin and molecular organization of outer dense fiber polypeptides of the sperm tail. To determine whether S70 is a precursor for S45-S35 heterodimeric protein, we will analyze the structure and expression of S70 and S45-S35 heterodimeric protein genes and search for regions of genomic homology in spermatogenic cells. Polyclonal antibodies raised against these two Sertoli cell secretory proteins will be used to screen rat testis cDNA expression libraries in lambda gtll phage expression vector. Inserts from positive clones will be subcloned into a multifunctional plasmid vector to determine the complete cDNA sequence and derive protein amino acid sequence. Derived amino acid sequence will be compared with N- terminus amino acid sequence analysis of protein S70 and outer dense fiber polypeptides. cDNA will be used for in situ cytohybridization and Northern blot experiments to determine whether spermatogenic cells, mainly spermatids, contain genome sequences and transcripts complementary to Sertoli cell secretory proteins. To determine whether Sertoli cell secretory proteins are related to outer dense fiber polypeptides, we will raise polyclonal antibodies against purified outer dense fiber polypeptides from sperm tail and search for crossreactive epitopes and N-terminus amino acid sequence homology of Sertoli cell secretory protein S70 and S45-S35 heterodimeric protein and outer dense fiber polypeptides. The subcellular localization and fate of both Sertoli cell secretory proteins and outer dense fiber polypeptides will be determined in rat testis by immunogold electron microscopy. Ultimately, the ontogeny, hormonal and spermatogenic cell dependency of Sertoli cell secretory proteins S70 and S45-S35 heterodimeric protein gene activity and expression will be determined in rat testis during development and in cultured Sertoli cells using an automated cell perifusion system combined with radioimmunoassay.

我们发现支持细胞分泌蛋白S70具有相同的抗原性与支持细胞分泌蛋白的多肽成分同源 S45-S35异二聚体蛋白。我们做出了意想不到的观察针对这两种支持细胞分泌的多克隆抗血清蛋白质与精子尾部外部致密纤维多肽发生交叉反应并且针对主要多肽产生的多克隆抗血清精子尾部外层致密纤维与蛋白质S70及其蛋白发生交叉反应分解产品。这些发现表明可能的产物前体 S70与S45-S35异二聚体蛋白的关系及提高有关细胞起源和分子的重要问题精子尾部外部致密纤维多肽的组织。确定 S70 是否是 S45-S35 异二聚体的前体蛋白，我们将分析S70和S45-S35的结构和表达异二聚体蛋白质基因和基因组同源性区域的搜索在生精细胞中。针对这两种病毒产生的多克隆抗体支持细胞分泌蛋白将用于筛选大鼠睾丸 cDNA lambda gtll 噬菌体表达载体中的表达文库。刀片阳性克隆将被亚克隆成多功能质粒载体以确定完整的 cDNA 序列并推导蛋白质氨基酸序列。衍生的氨基酸序列将与N-进行比较蛋白S70及外层致密末端氨基酸序列分析纤维多肽。 cDNA将用于原位细胞杂交和 Northern 印迹实验确定生精细胞是否主要是精子细胞，包含基因组序列和转录本与支持细胞分泌蛋白互补。确定支持细胞分泌蛋白是否与外层密纤维多肽，我们将培育多克隆抗体对抗来自精子尾部的纯化外层致密纤维多肽，以及搜索交叉反应表位和 N 端氨基酸序列支持细胞分泌蛋白 S70 和 S45-S35 异二聚体的同源性蛋白质和外层致密纤维多肽。亚细胞支持细胞分泌蛋白和外部蛋白的定位和命运免疫金法测定大鼠睾丸中的致密纤维多肽电子显微镜。最终，个体发育、荷尔蒙和支持细胞分泌蛋白 S70 和生精细胞依赖性 S45-S35异二聚体蛋白基因活性和表达将在发育过程中的大鼠睾丸和培养的支持氏细胞中测定使用自动细胞灌注系统结合的细胞放射免疫测定法。