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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与支持细胞分泌蛋白S70具有相同的抗原性，与支持细胞分泌蛋白多肽组分的同源性 S45-S35异二聚体蛋白。我们做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与精子尾部外致密纤维多肽交叉反应的蛋白质并且针对主要多肽产生的多克隆抗血清精子尾部外致密纤维与S70蛋白交叉反应，分解产物。这些发现表明一种可能的产物前体 S70和S45-S35异二聚体蛋白与提高关于细胞起源和分子生物学的重要问题精子尾部外部致密纤维多肽的组织。为了确定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寻找基因组同源性区域在生精细胞中针对这两种病毒的多克隆抗体支持细胞分泌蛋白将用于筛选大鼠睾丸cDNA 在λ gtll噬菌体表达载体中的表达文库。插入件将来自阳性克隆的质粒亚克隆到多功能质粒中载体，以确定完整的cDNA序列并衍生蛋白质氨基酸性序列衍生的氨基酸序列将与N- S70蛋白末端氨基酸序列分析及外致密蛋白纤维多肽 cDNA将用于原位细胞杂交，北方印迹实验以确定生精细胞，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。为了确定支持细胞分泌蛋白是否与外密纤维多肽，我们将提出多克隆抗体针对来自精子尾部的纯化的外部致密纤维多肽，寻找交叉反应性表位和N-末端氨基酸序列支持细胞分泌蛋白S70与S45-S35异源二聚体同源性蛋白质和外部致密纤维多肽。亚细胞支持细胞分泌蛋白和外膜蛋白的定位和命运用免疫金法测定大鼠睾丸中致密纤维多肽电镜最终，个体发育，激素和 Sertoli细胞分泌蛋白S70和 S45-S35异源二聚体蛋白基因活性和表达将被在发育期间的大鼠睾丸和培养的Sertoli中测定使用自动化细胞灌注系统结合放射免疫法