Regulation of spermatogenesis by sumoylation

描述(申请人提供)：通过精子发生的成功进展对于正常的配子形成和将遗传信息传递给下一代是至关重要的。不幸的是，在人类中，全世界大约15%的夫妇受到不孕不育的影响，在所有病例中，至少有一半是男性伴侣造成的。苏莫化已成为细胞功能的关键调节事件，并与包括癌症在内的多种疾病有关。最近，SUMO蛋白被定位于生殖细胞和体细胞睾丸细胞的特定亚区，并且获得的证据表明，SUMO在正常和受损的精子发生的不同方面都有作用。然而，与体细胞不同的是，在体细胞中已经鉴定和研究了大量的相扑蛋白，而睾丸相扑的靶标和调控大多是未知的。本研究的目的是鉴定和初步鉴定有丝分裂精原细胞、减数分裂精母细胞、分化精子细胞和睾丸体细胞中的苏莫化的特异性靶点。特定细胞类型的丰富种群将从经历第一波精子发生的青春期小鼠和成年小鼠睾丸中获得。使用抗相扑抗体的高度有效的免疫沉淀程序将随后进行液相色谱-质谱仪、蛋白质鉴定和生物信息学分析。使用新鲜分离的细胞进行的精原细胞相关实验将使用细胞系和细胞培养中氨基酸的稳定同位素标记(SILAC)来补充和扩展，以研究精原细胞在分化过程中总和化蛋白质组的变化。来自不同实验环境的结果将通过免疫共沉淀和定位研究得到进一步证实。总之，这项拟议的研究将通过阐明精子发生所必需的新的蛋白质网络和调控途径，促进生殖细胞生物学领域的知识进步。 与公共卫生相关：在人类不育的所有病例中，至少有一半(每六对夫妇中就有一对试图怀孕)男性生精失败是主要或促成原因。这项工作主要从个体靶点和相应途径的水平上研究新蛋白(SUMO)的生物学功能，从而更好地了解男性不育的可能原因，开发新的安全避孕药，从而改善人类健康。

DESCRIPTION (provided by applicant): Successful progression through spermatogenesis is crucial for normal gamete formation and for transferring the genetic information to the next generation. Unfortunately, in humans, infertility affects approximately 15% of couples worldwide and the male partner is responsible for the infertility in at least half of all cases. Sumoylation has emerged as a critical regulatory event in cell function and has been implicated in various diseases including cancer. SUMO proteins have recently been localized to specific subdomains of germ and somatic testicular cells and the obtained evidence implicated sumoylation in different aspects of normal and impaired spermatogenesis. However, unlike somatic cells, in which numerous sumoylated proteins have been identified and studied, targets and regulation of SUMO in the testis are mostly uncharacterized. The objective of this study is to identify and initially characterize specific targets of sumoylation in mitotic spermatogonia, meiotic spermatocytes, differentiating spermatids, and testicular somatic cells. Populations enriched for specific cell types will be obtained from both pubertal mice, undergoing their initial wave of spermatogenesis, and adult mouse testis. Highly validated immunoprecipitation procedures using anti-SUMO antibodies will be followed by liquid chromatography-mass spectrometry protein identification and bioinformatic analysis. Spermatogonia-related experiments using freshly isolated cells will be supplemented and extended using cell lines and stable isotope labeling with amino acids in cell culture (SILAC) to study changes in the sumoylated proteomes of spermatogonia as they undergo differentiation. The results obtained from different experimental settings will be further confirmed using co-immunoprecipitation and localization studies. Together, the proposed research will advance knowledge across the field of germ cell biology by elucidating the new protein networks and regulatory pathways that are necessary for progression through spermatogenesis. PUBLIC HEALTH RELEVANCE: In at least half of all cases of human infertility (one in every six couples who are trying to conceive) male spermatogenic failure is a major or contributing cause. This work focuses on studies of the biological functions of novel proteins (SUMO) at the level of individual targets and corresponding pathways, leading to the better understanding of possible causes of male infertility and development of novel safe contraceptives, thus improving human healthcare.