Sertoli cell injury and mechanisms of testicular germ cell apoptosis

支持细胞损伤及睾丸生殖细胞凋亡机制

• 批准号: 7730349
• 负责人: JOHN H RICHBURG
• 金额: $ 28.93万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7730349
• 关键词: Accounting; Age; Apoptosis; Apoptotic; CD95 Antigens; Cell Death Process; Cell physiology; Cells; Cessation of life; Chemical Exposure; Chemicals; Chromatin; Data; Development; Diethylhexyl Phthalate; Environment; Enzymes; Evaluation; Event; Experimental Models; Exposure to; Family member; Genes; Germ Cells; Goals; Grant; Histopathology; Human; In Vitro; Incidence; Infertility; Inflammatory Response; Laboratories; Lead; Ligands; Male Infertility; Mediating; Metalloproteases; Modeling; Molecular; Mono-S; Mus; Phase; Phenotype; Physical condensation; Physiological; Principal Investigator; Process; Production; Proteins; Regulation; Reproductive Health; Research; Research Project Grants; Research Proposals; Risk; Rodent; Role; Secondary to; Signal Pathway; Signal Transduction; Source; Spermatids; Spermatocytes; Spermatogenesis; Staging; TNFRSF1A gene; TP53 gene; Testicular Diseases; Testing; Testis; Tissue Inhibitor of Metalloproteinases; Toxic Environmental Substances; Tumor Necrosis Factor Receptor; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Ubiquitination; Work; base; cell injury; cellular targeting; critical period; design; environmental agent; in vivo; insight; member; mono-(2-ethylhexyl)phthalate; novel; phthalates; postnatal; prevent; programs; protein activation; public health relevance; research study; response; sertoli cell; toxicant; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the cellular and molecular signals instigated in the testis that lead to germ cell apoptosis as a result of environmental toxicant-induced Sertoli cell injury. Since analogous decreases in Sertoli cell support and germ cell apoptosis occur during early postnatal testis development, revealing the mechanisms by which germ cell apoptosis is regulated will enhance our understanding of both the physiological importance of this process during distinctive periods of testicular development as well as to provide insights into cellular targets of environmental toxicants and possible mechanisms of infertility. We have previously revealed the participation of FasL and Fas, two members of the tumor necrosis factor (TNF) superfamily of proteins, in triggering apoptosis of specific germ cell sub- types after exposure to the Sertoli cell toxicant mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the widely dispersed environmental agent DEHP. Here we propose to use this established Sertoli cell injury model to decipher the mechanism(s) responsible for conferring the unique sensitivity of spermatocytes/early round spermatids to undergo apoptosis after MEHP exposure. Our intriguing preliminary findings show that a soluble form of TNF-? (sTNF-?) is elicited by germ cells, via the action of distinct metalloproteinase (MP) enzymes, after MEHP-treatment and that sTNF-? may act upon TNFR1 on Sertoli cells to stimulate an increase in their expression of FasL. Our preliminary findings further indicate that Sertoli cells regulate the activity of MPs via their secretion of TIMP (tissue inhibitor of metalloproteinase) proteins into the adluminal space. Finally, the sensitivity of germ cells to FasL-mediated apoptosis is largely dependent on the ubiquitination, and subsequent degradation, of the anti-apoptotic protein c-FLIP. Our preliminary data suggest that the E3 ligase, Itch, ubiquitinates c-FLIP in germ cells secondary to MEHP- induced Sertoli cell injury. Taken together, these findings led to the development of the central hypothesis of this research proposal that decreases in Sertoli cell-derived TIMP proteins into the adluminal space, and the consequent activation of MPs, is a critical key event that instigates ensuing changes in specific germ cell sub-types that accounts for their sensitivity to undergo apoptosis during periods of reduced Sertoli cell supportive capacity. The first specific aim is designed to delineate the MP and TIMP family members in the testes and assess their role in the regulation of germ cell apoptosis. In the second aim, the functional participation of sTNF-? in FasL-mediated germ cell apoptosis will be challenged. In the last aim, experiments are focused on revealing the mechanisms regulating the levels of the c-FLIP protein and its role in altering the sensitivity of germ cell sub-types to undergo FasL-triggered apoptosis. PUBLIC HEALTH RELEVANCE: The focus of this grant project is to decipher the molecular and cellular mechanisms that regulate the death of testicular germ cells by the process of apoptosis. Apoptosis is an active process of cell death characterized by sequential phases of chromatin condensation, fragmentation and cell disintegration that leads to the orderly destruction and disposal of a cell without a consequent inflammatory response. Although many studies in humans have associated alterations in the incidence of germ cell apoptosis with conditions of abnormal spermatogenesis and male infertility, the cellular processes that regulate germ cell apoptosis in the testis remain poorly characterized. It is anticipated that the mechanistic insights provided from this work will be useful for predicting and preventing human reproductive health risks to chemicals, such as the phthalates, found ubiquitously in the environment.

描述（由申请人提供）：该项目的长期目标是了解睾丸中激发的细胞和分子信号，这些信号会因环境毒物诱导的支持细胞损伤而导致生殖细胞凋亡。由于支持细胞支持和生殖细胞凋亡的类似减少发生在出生后早期睾丸发育过程中，揭示生殖细胞凋亡的调节机制将增强我们对睾丸发育独特时期这一过程的生理重要性的理解，并提供对环境毒物的细胞靶点和不孕不育可能机制的见解。我们之前已经揭示了FasL和Fas（肿瘤坏死因子（TNF）蛋白超家族的两个成员）在接触支持细胞毒性物质邻苯二甲酸单（2-乙基己基）酯（MEHP）（广泛分布的环境剂DEHP的活性代谢物）后触发特定生殖细胞亚型的凋亡。在这里，我们建议使用这种已建立的支持细胞损伤模型来破译负责赋予精母细胞/早期圆形精子细胞在 MEHP 暴露后经历细胞凋亡的独特敏感性的机制。我们有趣的初步发现表明，可溶形式的 TNF-α在 MEHP 处理后，生殖细胞通过不同金属蛋白酶 (MP) 的作用引发 s​​TNF-α (sTNF-α)，并且 sTNF-α可能作用于支持细胞上的 TNFR1，刺激其 FasL 表达增加。我们的初步研究结果进一步表明，支持细胞通过将 TIMP（金属蛋白酶组织抑制剂）蛋白分泌到腔内来调节 MP 的活性。最后，生殖细胞对 FasL 介导的细胞凋亡的敏感性很大程度上取决于抗凋亡蛋白 c-FLIP 的泛素化和随后的降解。我们的初步数据表明，E3 连接酶 Itch 泛素化继发于 MEHP 诱导的支持细胞损伤的生殖细胞中的 c-FLIP。总而言之，这些发现导致了本研究提案的中心假设的发展，即支持细胞衍生的 TIMP 蛋白进入腔内空间的减少，以及随后的 MP 激活，是一个关键的关键事件，它引发了特定生殖细胞亚型的后续变化，这解释了它们在支持细胞支持能力降低期间对细胞凋亡的敏感性。第一个具体目标是描述睾丸中的 MP 和 TIMP 家族成员，并评估它们在调节生殖细胞凋亡中的作用。第二个目标是sTNF-α的功能参与。 FasL介导的生殖细胞凋亡将受到挑战。最后一个目标是，实验的重点是揭示调节 c-FLIP 蛋白水平的机制及其在改变生殖细胞亚型对 FasL 触发的细胞凋亡的敏感性中的作用。公共健康相关性：该资助项目的重点是破译通过细胞凋亡过程调节睾丸生殖细胞死亡的分子和细胞机制。细胞凋亡是一种活跃的细胞死亡过程，其特征是染色质浓缩、断裂和细胞解体的连续阶段，导致细胞有序破坏和处置，而不会产生随后的炎症反应。尽管许多人类研究将生殖细胞凋亡发生率的变化与精子发生异常和男性不育的情况联系起来，但调节睾丸中生殖细胞凋亡的细胞过程仍然知之甚少。预计这项工作提供的机制见解将有助于预测和预防环境中普遍存在的邻苯二甲酸盐等化学品对人类生殖健康的风险。