Bioactive lipid profiling of mammalian spermatozoa

 描述(申请人提供)：不育症是一个全球性的医学问题，其中男性不育症占所有病例的一半。此外，超过70%的男性不育病例被认为是特发性的。我们对男性不育病因认识上的巨大差距部分归因于我们对精子，特别是人类精子的生理学知识不足。虽然精子生理的微妙调节发生在许多水平上，但其受生物活性脂类的调节值得特别关注。我们的初步数据表明，几种生物活性脂类影响精子离子通道的活性，进而调节精子。 运动性和顶体反应。例如，类固醇激素孕酮刺激人类精子的初级钙通道CatSper的开放，并控制精子细胞内的钙离子。黄体酮也抑制钾精子通道KSper。我们的初步数据表明，CatSper通道可以被某些脂肪酸抑制，但除了 孕酮可以被一种未知的精子内源性脂肪上调，这种内源性脂肪很容易被无脂肪酸牛血清白蛋白(Faf BSA)和环糊精提取出来。将精子与Faf BSA或β-环糊精短暂孵育会导致CatSper活性的抑制。这些实验 是在去除精子细胞质，但精子膜保持完整的条件下进行的。此外，这种作用是可逆的，表明负责激活的分子 在没有次级信使的情况下，可以由完整的精子质膜合成CatSper。此外，我们最近发表的数据表明，许多精子离子通道和调节其活性的分子是人类精子所特有的，或者没有被观察到，或者它们的功能在小鼠精子中发生了改变。这突显了这项建议集中精力研究人类精子中的生物活性脂质信号的必要性。为了确定负责敏化CatSper通道的分子(S)，我们将用？-环糊精从人精子质膜中提取脂质，以便从CatSper增敏剂中将其去除，等待后者恢复，然后再用？-环糊精提取脂质。新合成的脂类将被鉴定并测试它们调节CatSper的能力。我们还将获得人类射精和获能精子的生物活性图谱。从拟议的研究中获得的知识将填补我们对生物活性脂质调节人类精子的基本机制的了解的空白，可能导致开发新的男性不育诊断测试，并开发男性避孕药。

 DESCRIPTION (provided by applicant): Infertility constitutes a global medical problem, with male infertility contributing to half of all cases. Moreover, more than 70% of male infertility cass are considered idiopathic. This huge gap in our understanding of etiology of male infertility is partially attributed to our insufficient knowledge of physiology of spermatozoa, and human sperm cells in particular. While delicate regulation of sperm physiology occurs on many levels, its regulation by bioactive lipids deserves special attention. Our preliminary data indicate that several bioactive lipids influence the activity of sperm ion channels, which in turn regulate sperm motility and the acrosome reaction. For example, the steroid hormone progesterone stimulate the opening of the primary calcium channel of human sperm, CatSper, and controls sperm intracellular Ca2+. Progesterone also inhibits potassium sperm channel KSper. Our preliminary data indicate that CatSper channel can be inhibited by certain fatty acids, but also in addition to progesterone, can be up-regulated by an unknown sperm endogenous lipid easily extracted by fatty acid free bovine serum albumin (faf BSA) and ß-cyclodextrin. A brief incubation of sperm with either faf BSA or ß-cyclodextrin results in inhibition of CatSper activity. These experiments were done under conditions when sperm cytoplasm is removed, but sperm membrane stays intact. Moreover, such action is reversible indicating that the molecule responsible for activation of CatSper can be synthesized by intact sperm plasma membrane in the absence of secondary messengers. In addition, our recently published data indicate that many sperm ion channels and the molecules that modulate their activity are specific to human spermatozoa, and either are not observed, or their functions are altered in murine spermatozoa. This highlights the necessity to concentrate efforts of this proposal on studying bioactive lipid signaling in human spermatozoa specifically. In order to identify the molecule(s) responsible for sensitizing CatSper channel we will perform lipid extraction from human sperm plasma membrane by ß-cyclodextrin in order to deplete them from CatSper sensitizer, wait for the later to recover, and again extract lipids with ß-cyclodextrin. The newly synthesized lipids will be identified and tested for their ability to modulate CatSper. We will also obtain bioactive profiling of human ejaculated and capacitated spermatozoa. The knowledge gained from the proposed research will fill in gaps in our understanding of the basic mechanisms underlying the regulation of human sperm by bioactive lipids, may lead to the development of novel diagnostic tests for male infertility, and to the development of male contraceptives.