Regulation of spermatogenesis by sumoylation

描述（由申请人提供）：精子发生的成功进展对于正常配子形成和将遗传信息传递给下一代至关重要。不幸的是，在人类中，不孕症影响着全球约15%的夫妇，男性伴侣在所有病例中至少有一半是不孕症的原因。类小泛素化已成为细胞功能中的关键调节事件，并与包括癌症在内的各种疾病有关。SUMO蛋白最近被定位于生殖和睾丸体细胞的特定亚结构域，并且所获得的证据表明SUMO化在正常和受损的精子发生的不同方面。然而，与体细胞不同，在体细胞中已经鉴定和研究了许多SUMO化的蛋白质，SUMO在睾丸中的靶点和调控大多是未知的。本研究的目的是确定并初步表征有丝分裂精原细胞，减数分裂精母细胞，分化精子细胞和睾丸体细胞中sumoylation的特定目标。将从青春期小鼠（经历精子发生的初始波）和成年小鼠睾丸中获得富集特定细胞类型的群体。使用抗SUMO抗体的高度验证的免疫沉淀程序将随后进行液相色谱-质谱法蛋白质鉴定和生物信息学分析。精原细胞相关的实验，使用新鲜分离的细胞将补充和扩展使用细胞系和稳定的同位素标记与细胞培养物中的氨基酸（SILAC），以研究在SUMO化的精原细胞蛋白质组的变化，因为他们经历分化。从不同的实验环境中获得的结果将进一步确认使用免疫共沉淀和定位研究。总之，拟议的研究将通过阐明精子发生所必需的新蛋白质网络和调控途径来推进生殖细胞生物学领域的知识。 公共卫生相关性：在至少一半的人类不育病例中（每六对试图怀孕的夫妇中就有一对），男性生精障碍是一个主要或促成因素。这项工作重点研究新型蛋白质（SUMO）在个体靶点和相应途径水平上的生物学功能，从而更好地了解男性不育的可能原因并开发新型安全避孕药，从而改善人类医疗保健。

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.