Regulation of sperm motility by the Rho signaling pathway

描述(由申请者提供)：在所有人类不育症中，精子活力不佳的比例接近30%。因此，更好地了解调节精子运动的生化机制可能会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，处于不育或不育状态。弱精子症患者ROPN1的表达水平显著低于正常精子症患者。ROPN1最初被鉴定为一种与Rho的结合伙伴--嗜鼻素结合的蛋白质。利用酵母双杂交分析，我们发现ROPN1也与精子A-激酶锚定蛋白(AKAP)结合。ROPN1含有一个二聚化/对接结构域，类似于蛋白激酶A(PKA)的调节亚单位。然而，在对接结构域之外，ROPN1与PKA几乎没有相似性，这表明它们具有不同的功能。两项观察表明，ROPN1是鞭毛运动所必需的：1)添加干扰AKAP与ROPN1之间相互作用的肽(Ht31)会导致蛋白磷酸酶1(PP1)活性改变、GSK3磷酸化和精子活力受阻；2)缺乏ROPN1(Ropn1-/-)突变小鼠的精子活力降低。因此，ROPN1似乎是位于cAMP和Rho通路之间的一个关键分子。虽然关于cAMP/PKA在精子中的作用已经发表了很多文章，但关于Rho在精子中的作用的文献相对较少。Rho-GTP酶失活可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Rho途径的下游效应器PP1会显著改变精子的运动能力。然而，据我们所知，还没有人测量过精子中Rho-GTP酶的活性。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是否作为这一途径的一部分发挥作用。这项建议的成功完成将增进我们对这一重要调控机制的了解，从而可能促进男性不育治疗方法的发展。 与公共健康相关：这项提议的目标是加强对调节鞭毛功能的信号通路的了解，这对定义正常的男性生育能力至关重要。这种对正常过程的理解应该能够更准确地诊断不孕不育的根本原因(S)，也将为治疗和合理的药物设计提供更多的靶点。鞭毛和纤毛在功能、蛋白表达和信号通路上的共性表明，这些发现也可能对睫状结构疾病的诊断和治疗具有更广泛的意义。

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. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to enhance knowledge of signaling pathways that regulate flagellar function, which is essential to defining normal male fertility. This increased understanding of normal processes should allow for more precise diagnoses of the root cause(s) of infertility, and will also provide more targets for treatment and rational drug design. Commonalities between flagella and cilia in function, protein expression and signaling pathways suggest that these findings may also have wider implications in diagnosis and treatment of ciliary diseases.