Regulation of sperm motility by the Rho signaling pathway

描述（申请人提供）：精子活力不佳占所有人类不孕症的近30%。因此，更好地了解调节精子运动的生化机制可能为不育夫妇提供治疗选择。我们最近发现了一种基因产物（ROPN1），它似乎对精子活力和生育能力都至关重要。ROPN1在睾丸和精子中高度表达，缺乏ROPN1的小鼠精子活力受损，生育能力低下或不育。ROPN1在弱精子男性中的表达水平明显低于正常精子男性。ROPN1最初被鉴定为一种与Rho的结合伙伴rhophilin结合的蛋白质。通过酵母双杂交分析，我们发现ROPN1也与精子a激酶锚定蛋白（AKAPs）结合。ROPN1含有一个二聚化/对接结构域，类似于蛋白激酶a （PKA）的调控亚基。然而，在对接结构域之外，ROPN1与PKA几乎没有相似之处，这表明它们具有不同的功能。两个观察结果表明，鞭毛运动需要ROPN1: 1)添加肽（Ht31）破坏AKAPs和ROPN1之间的相互作用，导致蛋白磷酸酶1 （PP1）活性改变，GSK3磷酸化和精子运动受阻；2)缺乏ROPN1 （ROPN1 -/-）的突变小鼠精子运动能力降低。因此，ROPN1似乎是位于cAMP和Rho通路交汇处的关键分子。虽然关于cAMP/PKA在精子中的作用已经发表了很多文章，但是关于Rho在精子中的作用的文献相对较少。Rho- gtpase的失活已被证明会降低哺乳动物精子的活力，我们最近发现精子含有Rho信号通路的所有蛋白质成分。抑制PP1 （Rho通路的下游效应物）可显著改变精子活力。然而，据我们所知，还没有人测量过精子中Rho-GTPase的活性。该项目的目标是确定Rho是否是调节精子活力的生化机制的关键组成部分，以及ROPN1是否作为该途径的一部分起作用。这一建议的成功完成将增强我们对这一重要调节机制的认识，从而可能促进男性不育症治疗的发展。

DESCRIPTION (provided by applicant): Suboptimal sperm motility accounts for nearly 30% of all human infertility. As such, a better understanding of the biochemical mechanisms regulating sperm motility may provide therapeutic options for infertile couples. We have recently identified a gene product (ROPN1) that appears to be critical for both sperm motility and fertility. ROPN1 is highly expressed in testis and sperm, and mice lacking ROPN1 have impaired sperm motility and are subfertile or infertile. The expression level of ROPN1 is significantly lower in asthenozoospermic men than in normozoospermic controls. ROPN1 was originally identified as a protein that binds to rhophilin, a binding partner of Rho. Using a yeast two-hybrid analysis, we discovered that ROPN1 also binds to sperm A-kinase anchoring proteins (AKAPs). ROPN1 contains a dimerization/docking domain similar to the regulatory subunit of protein kinase A (PKA). However, outside the docking domain, ROPN1 bears little or no similarity to PKA suggesting they have distinct functions. Two observations suggest that ROPN1 is required for flagellar motility: 1) adding peptides (Ht31) that disrupt the interaction between AKAPs and ROPN1 results in altered protein phosphatase 1 (PP1) activity, GSK3 phosphorylation and arrested sperm motility, and 2) sperm from mutant mice lacking ROPN1 (Ropn1-/-) have reduced motility. Thus, ROPN1 appears to be a key molecule located at the intersection between the cAMP and Rho pathways. Although many articles have been published on the role of cAMP/PKA in sperm, the literature on Rho in sperm is relatively sparse. Inactivation of Rho-GTPase has been shown to reduce mammalian sperm motility and we have recently shown that sperm contain all the protein components of the Rho signaling pathway. Inhibition of PP1, a downstream effector in the Rho pathway, dramatically alters sperm motility. However, to our knowledge, no one has measured the activity of Rho-GTPase from sperm. The goal of this project is to determine if Rho is a critical component of the biochemical machinery regulating sperm motility and if ROPN1 functions as part of this pathway. Successful completion of this proposal will enhance our knowledge of this important regulatory mechanism and thus may facilitate development of treatments for male infertility.