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的形成和遗传信息的传递至关重要。不幸的是，在人类中，不育影响了全世界大约15%的夫妇，而男性伴侣至少要对所有病例中的一半负责。Sumoylation已成为细胞功能的关键调控事件，并与包括癌症在内的多种疾病有关。SUMO蛋白最近被定位于生殖和体细胞睾丸细胞的特定亚域，并且获得的证据表明SUMO化在正常和受损精子发生的不同方面。然而，与体细胞不同的是，睾丸中SUMO的靶点和调控大多是未知的，而体细胞中已经鉴定和研究了许多SUMO化蛋白。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、分化精母细胞和睾丸体细胞中sumoylation的特定靶点。从处于初始精子发生阶段的发育期小鼠和成年小鼠的睾丸中都可以获得富集特定细胞类型的种群。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。使用新分离细胞的精原细胞相关实验将补充和扩展使用细胞系和细胞培养氨基酸稳定同位素标记（SILAC）来研究精原细胞在分化过程中磷酸化蛋白质组的变化。不同实验环境下获得的结果将通过共免疫沉淀和定位研究进一步证实。总之，拟议的研究将通过阐明精子发生过程中必需的新蛋白质网络和调节途径，推进生殖细胞生物学领域的知识。

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.