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首页> 外文学位 >Molecular genetic analysis of the flagellar axoneme and fibrous sheath in sperm motility and male infertility.
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Molecular genetic analysis of the flagellar axoneme and fibrous sheath in sperm motility and male infertility.

机译：鞭毛轴突和纤维鞘在精子运动和男性不育中的分子遗传学分析。

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摘要

Sperm motility is a critical component of male fertility; however the mechanisms which underlie the normal assembly and function of the mammalian flagellum are not well-characterized. To better understand the genes and proteins involved in sperm motility, I utilized the AE24 mouse model in which a transgene insertion has mutated an unidentified endogenous gene. Sperm from these mice possess an unstable axoneme, resulting in flagellar paralysis. I attempted to clone the endogenous gene from wild-type and AE24 genomic libraries. Although regions of wild-type and mutant DNA surrounding the site of the transgene insertion were cloned, I have not yet identified a relevant open reading frame or a candidate gene. In addition, mapping the site of the transgene insertion using Fluorescence In Situ Hybridization (FISH) on metaphase chromosomes from AE24 cells was unsuccessful, probably due to the probe's small size. Finally, I performed differential display analysis to compare mRNA expression between wild-type and AE24 testes. While several differentially expressed sequences were identified, northern analysis needs to confirm these results before studies are continued.; As another approach to identifying proteins involved in flagellar function, I cloned the gene and cDNA for a major human sperm fibrous sheath (FS) protein, hAKAP82 and the gene for its mouse homologue. The human AKAP82 protein is tyrosine-phosphorylated in a capacitation-dependent manner. I showed that the human protein was an A-Kinase Anchor Protein (AKAP) and mapped its PK-A binding site. I demonstrated that both the mouse and human genes were alternatively spliced and that the human gene mapped to the X-chromosome by FISH. I showed that the protein was made as a precursor that is processed into the mature protein. Immunoblotting and immunofluorescence analyses showed that this processing was less complete in the human than the mouse. However, I found no evidence for an association between either processing or tyrosine phosphorylation of this protein and motility in sperm from normal men. In addition, I found no evidence to support a role for hAKAP82 in Dysplasia of the Fibrous Sheath, a naturally-occurring, heritable human disorder that results in abnormal development of the FS and infertility.
机译：精子运动是男性生育的重要组成部分。然而，哺乳动物鞭毛正常组装和功能的基础机制尚不十分清楚。为了更好地了解与精子运动有关的基因和蛋白质，我利用了AE24小鼠模型，其中转基因插入已使未鉴定的内源基因突变。这些小鼠的精子具有不稳定的轴突，导致鞭毛麻痹。我试图从野生型和AE24基因组文库中克隆内源基因。尽管克隆了转基因插入位点周围的野生型和突变型DNA区域，但我尚未发现相关的开放阅读框或候选基因。此外，在AE24细胞的中期染色体上使用荧光原位杂交（FISH）绘制转基因插入位点的图谱是不成功的，这可能是由于探针的尺寸很小。最后，我进行了差异显示分析以比较野生型和AE24睾丸之间的mRNA表达。虽然鉴定出了几种差异表达的序列，但在继续研究之前，Northern分析需要确认这些结果。作为鉴定与鞭毛功能有关的蛋白质的另一种方法，我克隆了人类主要精子纤维鞘（FS）蛋白hAKAP82的基因和cDNA及其小鼠同源基因。人AKAP82蛋白以酪氨酸磷酸化（capacitation-dependent）的方式被酪氨酸磷酸化。我证明了人类蛋白质是A激酶锚蛋白（AKAP），并绘制了其PK-A结合位点。我证明了小鼠和人类基因都被选择性剪接，并且人类基因通过FISH定位到X染色体。我证明该蛋白质是作为前体制成的，可加工成成熟蛋白质。免疫印迹和免疫荧光分析表明，该过程在人中不如在小鼠中完成。但是，我没有发现该蛋白质的加工或酪氨酸磷酸化与正常男性精子运动之间存在关联的证据。此外，我发现没有证据支持hAKAP82在纤维鞘发育异常中的作用，纤维鞘是一种自然发生的遗传性人类疾病，可导致FS异常发育和不育。

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  • 作者

    Turner, Regina Marie Oristaglio.;

  • 作者单位

    University of Pennsylvania.;

  • 授予单位 University of Pennsylvania.;
  • 学科 Biology Molecular.; Biology Cell.
  • 学位 Ph.D.
  • 年度 1999
  • 页码 346 p.
  • 总页数 346
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;细胞生物学;
  • 关键词

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