The Sertoli Cell Product, Doppel, and Male Fertility

描述（由申请人提供）：精子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精质上皮内进行空间组织，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会影响生殖细胞的发育，但值得注意的是，迄今为止还没有证据表明支持细胞分泌产物是产生可育雄性配子所必需的。这一应用验证了一个假设，即支持细胞分泌的Doppel蛋白对可育精子的发育是绝对必要的。这一假设的基本原理来自四个观察结果。首先，来自两个实验室的公开数据表明，敲除编码Doppel Prnd的基因的雄性小鼠是不育的，因为它们的精子无法进行顶体反应。其次，睾丸表达的Prnd mRNA比其他任何器官都要高得多。第三，睾丸的大部分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、精母细胞和精原细胞不表达可检测水平的这种转录物。然而，应该注意的是，Prnd mRNA也在小鼠附睾头中表达，尽管其表达水平远低于睾丸。因此，虽然所有的数据都表明支持细胞是男性生育所需的Doppel最可能的来源，但我们不能先验地排除附睾产生的Doppel的作用。本R03应用的目的是专门评估Sertoli细胞分泌Doppel对生育的重要性。我们打算产生携带固定Prnd等位基因的转基因小鼠，并将这些小鼠培育成Amh-Cre小鼠，后者在Sertoli细胞中特异性表达Cre重组酶。它们后代的Sertoli细胞会有条件地敲除Prnd基因。我们还建议将带带flxed Prnd等位基因的小鼠与表达Cre重组酶的EIIa-Cre小鼠作为着床前胚胎进行繁殖。

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.