GENE EXPRESSION IN SPERMATOGENIC CELLS

生精细胞中的基因表达

• 批准号: 6106765
• 负责人: EDWARD MITCHELL EDDY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6106765
• 关键词: adenosine triphosphate; cytoskeleton; developmental genetics; endopeptidases; enzyme activity; enzyme induction /repression; flagellin; gene mutation; genetic promoter element; genetically modified animals; glyceraldehyde 3 phosphate dehydrogenase; laboratory mouse; male; protamines; site directed mutagenesis; spermatogenesis

Summary of Work: The aim of these studies is to determine the mechanisms that regulate developmental expression of genes in male germ cells and the roles of the proteins they encode in gamete development and function. The focus is on genes encoding proteins that are either unique to spermatogenic cells, are germ cell-specific members of a protein family, or produce spermatogenic cell-specific alternative transcripts and protein isoforms. These studies use the gene knockout approach and yeast two-hybrid assays to examine the roles of some proteins believed to be important in male gamete development and function. One example is fertilin beta, a sperm surface glycoprotein which is a member of the ADAM (a disintegrin and metalloprotease domain) family. It has been found that fertilin beta has a key role in sperm-egg interaction and binds to an alpha 6-beta 1 integrin on the egg surface. Mice heterozygous for a targeted mutation in the fertilin beta gene are deficient in sperm-egg membrane adhesion, sperm-egg fusion, migration from the uterus into the oviduct, and binding to the egg zona pellucida. Other examples are protamines 1 and 2, highly basic nuclear proteins that replace the histones and are thought to be essential for DNA compaction in the absence of nucleosomes during sperm development. They have been shown to have developmentally and transcriptionally regulated expression in spermatids. Chimeric mice have been produced that carry a targeted mutation in the protamine 1 or 2 gene and are being mated to produce heterozygous offspring. It is expected that disruption of one or both genes will lead to abnormal nuclear compaction and infertility in homozygous males. A final example is glyceraldehyde 3-phosphate dehydrogenase, a key glycolytic enzyme. A germ cell homolog (Gapd-s) is expressed only in spermatids and has a key role in regulating the generation of ATP required for fertilization. The enzyme is also the likely target for (S)-3-chlorolactaldehyde, a male reproductive toxicant that is a metabolite of the industrial solvent epichlorohydrin. Studies with transgenic mice determined that sequences required for correct expression of Gapd-s are within 230 bp of the transcription initiation site. A knock-out construct for Gapd-s is being prepared and the predicted phenotype of the knock-out is normal appearing sperm that are unable to activate glycolysis, achieve hyper-activated motility, or to fertilize eggs.

工作总结：这些研究的目的是 确定调节发育表达的机制 男性生殖细胞中的基因和蛋白质的作用， 在配子发育和功能中编码。重点是基因 编码蛋白质是生精细胞所特有的， 蛋白质家族的生殖细胞特异性成员，或产生 生精细胞特异性替代转录物和蛋白 同种型。这些研究使用基因敲除方法和酵母 双杂交试验，以检查一些蛋白质的作用， 对雄配子的发育和功能很重要。一 例子是受精素β，一种精子表面糖蛋白， ADAM（解整合素和金属蛋白酶结构域）的成员 家人已经发现，受精素β在以下方面具有关键作用： 精子-卵子相互作用，并结合到α 6-β 1整合素上， 鸡蛋表面小鼠中靶向突变的杂合子 受精素β基因缺乏精卵膜粘附， 精卵融合，从子宫迁移到输卵管， 与卵透明质酸结合其他例子是鱼精蛋白1 和2，高度碱性核蛋白，取代组蛋白， 被认为是必要的DNA压缩在缺乏 精子发育过程中的核小体。刻意为 有发育和转录调控的表达， 精子细胞已经产生了嵌合小鼠，其携带靶向的 在鱼精蛋白1或2基因突变，并正在交配， 产生杂合子后代。据估计， 一个或两个基因将导致异常的核致密化， 纯合子男性不育。最后一个例子是甘油醛 3-磷酸脱氢酶，一种关键的糖酵解酶。生殖细胞 同源物（Gapd-s）仅在精子细胞中表达， 在调节受精所需的ATP的产生中的作用。 这种酶也可能是（S）-3-氯乙醛的目标， 一种男性生殖毒物，是工业 溶剂环氧氯丙烷。转基因小鼠研究确定 Gapd-s正确表达所需的序列在 230 bp的转录起始位点。一种敲除构建体， GAPD-S正在制备中， 敲除是正常出现的精子，无法激活 糖酵解，实现超激活运动，或受精卵。