The Sertoli Cell Product, Doppel, and Male Fertility

支持细胞产品、Doppel 和男性生育能力

• 批准号: 7669398
• 负责人: WILLIAM Wallace WRIGHT
• 金额: $ 8.2万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-15 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7669398
• 关键词: Acrosome Reaction; Affect; Age; Alleles; Amino Acids; Binding; Breeding; Carrier Proteins; Cells; Code; Data; Development; Eels; Environment; Epididymis; Exons; Fertility; Genes; Germ Cells; Goals; Infertility; Knock-out; Laboratories; Life; Messenger RNA; Mus; Organ; Peptide Hydrolases; Population; Production; Protease Inhibitor; Proteins; Publishing; Role; Running; Site; Somatic Cell; Source; Spermatids; Spermatocytes; Spermatogenesis; Spermatogenic Cell; Spermatogonia; Testing; Testis; Transcript; Transgenic Mice; United States; blastocyst; doppel protein; knockout gene; male; men; paracrine; public health relevance; recombinase; research study; sertoli cell; sertoli cell secretory proteins; sperm cell; spermatogenic epithelium structure

DESCRIPTION (provided by applicant): Spermatogenesis occurs in a unique environment that is created by the somatic Sertoli cells. Not only do these cells bind to the developing male germ cells and spatially organize them within the seminiferous epithelium, they also secrete a large number of paracrine factors, carrier proteins, proteases and protease inhibitors. While it is assumed that these secretory products affect germ cell development, it is remarkable that to date no Sertoli cell secretory product has been proven to be required for production of fertile male gametes. This application tests the hypothesis that the secretion by Sertoli cells of the protein, Doppel, is absolutely required for development of fertile sperm. The rationale for this hypothesis comes from four observations. First, published data from two laboratories demonstrate that male mice with a knockout of the gene encoding Doppel, Prnd, are infertile because their sperm are incapable of undergoing the acrosome reaction. Second, the testis expresses substantially higher concentrations of Prnd mRNA than any other organ. Third, most of the Doppel in the testis is localized to Sertoli cells. Fourth, recent data from our laboratory indicate that Sertoli cells express very high levels of Prnd mRNA while spermatids, spermatocytes and spermatogonia do not express detectable levels of this transcript. It should be noted, however, that Prnd mRNA is also expressed in the caput epididymis of the mouse, albeit at much lower levels than in the testis. Thus, while all data identify Sertoli cells as the most likely source of the Doppel that is required for male fertility, one cannot a priori exclude a role for Doppel produced by the epididymis. The goal of this R03 application is to specifically evaluate the importance to fertility of the secretion of Doppel by Sertoli cells. We propose to generate transgenic mice that carry a floxed Prnd allele and to breed these mice to Amh-Cre mice, which express Cre recombinase specifically in Sertoli cells. The Sertoli cells of their progeny will have a conditional knockout of the Prnd gene. We also propose to breed mice with the floxed Prnd allele to EIIa-Cre mice, which express Cre recombinase as preimplantation embryos. Public Health Relevance: All cells of their progeny will have a conditional knockout of the Prnd gene. We will then compare the effects on fertility of the specific loss of Doppel secretion by Sertoli cells to the effects of the loss of Doppel secretion by all cells. By making this comparison, we will directly test the hypothesis that the secretory product of Sertoli cells, Doppel, is required for development of fertile sperm. Sertoli cells are the somatic cells that support and nourish developing male gametes. These cells bind to spermatogenic cells, spatially organize them within the seminiferous epithelium and secrete a large repertoire of products that are assumed to be required for male fertility. However, despite the obvious intimate relationship between Sertoli cells and spermatogenic cells, the crucial mechanisms by which Sertoli cells support spermatogenesis are mostly unknown. This application will determine if the Sertoli cell secretory protein, Doppel, is essential for male fertility. We propose to generate mice with a conditional knockout in Sertoli cells of the gene that encodes Doppel and to determine if loss of Doppel secretion by Sertoli cells renders male mice infertile. Completion of these proposed experiments hold promise for providing the first direct evidence that a Sertoli cell product is required for male fertility. Such evidence may open new avenues to the study of male fertility and, thus, to the treatment of the large population of infertile men that live in the United States.

描述（由申请人提供）：精子发生发生在由体细胞支持细胞创造的独特环境中。这些细胞不仅与发育中的雄性生殖细胞结合并在生精上皮内空间组织它们，它们还分泌大量的旁分泌因子、载体蛋白、蛋白酶和蛋白酶抑制剂。虽然假设这些分泌产物影响生殖细胞发育，但值得注意的是，迄今为止，没有支持细胞分泌产物被证明是产生可育雄配子所必需的。该应用测试了支持细胞分泌的蛋白质Doppel是受精精子发育所必需的假设。这一假设的基本原理来自四个观察结果。首先，来自两个实验室的已发表数据表明，敲除编码Doppel，Prnd的基因的雄性小鼠是不育的，因为它们的精子不能进行顶体反应。第二，睾丸表达的Prnd mRNA浓度比任何其他器官都高。第三，睾丸中的大多数Doppel定位于支持细胞。第四，我们实验室最近的数据表明，支持细胞表达非常高水平的Prnd mRNA，而精子细胞，精母细胞和精原细胞不表达可检测水平的这种转录。然而，应该注意的是，Prnd mRNA也在小鼠附睾头中表达，尽管水平比睾丸中低得多。因此，虽然所有的数据确定支持细胞作为最有可能的来源的Doppel是所需的男性生育能力，不能先验排除的作用，为Doppel产生的附睾。本R 03申请的目的是专门评价支持细胞分泌Doppel对生育力的重要性。我们建议产生转基因小鼠，携带一个floxed Prnd等位基因，并将这些小鼠繁殖为Amh-Cre小鼠，表达Cre重组酶，特别是在支持细胞。其后代的支持细胞将具有Prnd基因的条件性敲除。我们还建议将具有floxed Prnd等位基因的小鼠培育为EIIa-Cre小鼠，其表达Cre重组酶作为植入前胚胎。 公共卫生相关性：其后代的所有细胞将具有Prnd基因的条件性敲除。然后，我们将比较支持细胞Doppel分泌特定损失对生育能力的影响与所有细胞Doppel分泌损失的影响。通过这种比较，我们将直接检验支持细胞的分泌产物Doppel是受精精子发育所必需的这一假设。支持细胞是支持和滋养发育中的雄配子的体细胞。这些细胞与生精细胞结合，在生精上皮内空间组织它们，并分泌大量被认为是男性生育所需的产物。然而，尽管支持细胞和生精细胞之间存在明显的密切关系，支持细胞支持精子发生的关键机制大多是未知的。该应用将确定支持细胞分泌蛋白Doppel是否对男性生育力至关重要。我们建议产生小鼠的支持细胞中的编码Doppel的基因的条件性敲除，并确定如果支持细胞的Doppel分泌的损失，使雄性小鼠不育。这些实验的完成有望提供第一个直接证据，证明支持细胞产物是男性生育所必需的。这些证据可能为研究男性生育能力开辟新的途径，从而为治疗生活在美国的大量不育男性开辟新的途径。