The Sertoli Cell Product, Doppel, and Male Fertility

描述(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进行空间组织，而且还分泌大量的旁分泌因子、载体蛋白、蛋白水解酶和蛋白水解酶抑制物。虽然这些分泌产物被认为影响生殖细胞的发育，但值得注意的是，到目前为止，还没有证明支持细胞分泌产物是产生可育雄配子所必需的。这一应用验证了一种假说，即支持细胞分泌的蛋白质多谱勒是发育可育精子所必需的。这一假设的理论基础来自四个观察结果。首先，两个实验室公布的数据表明，编码多谱勒(PrND)基因敲除的雄性小鼠是不育的，因为它们的精子不能经历顶体反应。其次，睾丸表达比其他任何器官都要高得多的Prnd基因。第三，睾丸内的多谱勒主要定位于支持细胞。第四，我们实验室的最新数据表明，Sertoli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该转录本。然而，应该注意的是，Prnd mRNA也在小鼠的附睾头表达，尽管表达水平比睾丸低得多。因此，尽管所有数据都认为支持细胞是男性生育所需的多谱勒的最有可能的来源，但人们不能先验地排除附睾产生的多谱勒的作用。这项R03应用的目的是专门评估支持细胞分泌的多谱勒对生育的重要性。我们建议建立携带FLOXPRND等位基因的转基因小鼠，并将这些小鼠培育成在Sertoli细胞中特异性表达Cre重组酶的AMH-Cre小鼠。他们后代的Sertoli细胞将有条件地敲除PrND基因。我们还建议将表达Cre重组酶的EIIA-Cre小鼠的PrND等位基因培育成EIIA-Cre小鼠作为植入前胚胎。 与公共卫生相关：它们后代的所有细胞都将有条件地敲除PrND基因。然后，我们将比较支持细胞特异性多谱勒分泌丧失对生育力的影响和所有细胞多谱勒分泌丧失对生育的影响。通过进行这种比较，我们将直接检验支持细胞的分泌产物多谱勒是发育可育精子所必需的假设。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，在生精上皮内进行空间组织，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有着明显的密切关系，但支持细胞支持精子发生的关键机制大多尚不清楚。这项应用将确定支持细胞分泌蛋白多谱勒是否对男性生育至关重要。我们建议在支持细胞中产生编码多谱勒的基因的条件性敲除小鼠，并确定支持细胞分泌多谱勒的缺失是否会导致雄性小鼠不育。这些拟议实验的完成有望提供第一个直接证据，证明支持细胞产品是男性生育所必需的。这些证据可能会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道路。

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.