Morphologically abnormal bovine sperm: A model for the study of spermatogenesis, sperm function and sperm contributions to preimplantation embryo development

形态异常的牛精子：研究精子发生、精子功能和精子对植入前胚胎发育的贡献的模型

• 批准号: RGPIN-2014-04850
• 负责人: Thundathil, Jacob
• 金额: $ 2.19万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=588057
• 关键词: Morphologically; abnormal; bovine; sperm; model

My long-term goal is elucidating regulation of male fertility. I detected several proteins differentially expressed in abnormal sperm, including a testis-specific isoform of Na+/K+-ATPase (ATP1A4), induced by increased testicular temperature (NSERC DG). Sertoli cells contain Na+/K+-ATPase isoforms and ouabain (their specific ligand) induces capacitation of bull sperm. The current proposal (NSERC DG renewal) is to elucidate roles of Na+/K+-ATPase isoforms in regulation of Sertoli cell function and capacitation, effects of elevated testicular temperature on mechanisms regulating expression of ATP1A4 during sperm development, and effects of abnormal sperm on embryo development. Objective 1 (roles of Na+/K+-ATPase isoforms in formation and function of Sertoli cell tight junctions; TJ): Na+/K+-ATPase regulates formation and function of TJs between adjacent epithelial cells. Sertoli cells from peri-pubertal rat testis (when TJ formation occurs) will be used to test the hypothesis that Na+/K+-ATPase isoforms regulate assembly and function of TJs in Sertoli cells. Ouabain treatment will implicate roles of Na+/K+-ATPase isoforms in TJ formation and function. Objective 2 (roles of raft and non-raft pools of Na+/K+-ATPase isoforms during bull sperm capacitation): Lipid rafts regulate sperm function in several species; Na+/K+-ATPase is involved in raft-mediated signaling in somatic cells. We discovered raft and non-raft pools of Na+/K+-ATPase isoforms in bull sperm. I hypothesize that the raft pool of Na+/K+-ATPase isoforms predominantly interact with and activate multiple signaling molecules, leading to ouabain-induced capacitation in bull sperm. We will identify binding partners for Na+/K+-ATPase in raft fractions of capacitated sperm (mmunoprecipitation and mass spectrometry). Relative abundance and activation (phosphorylation) of these molecules will determine contributions of raft and non-raft pools of Na+/K+-ATPase isoforms and their potential interaction during capacitation. Objective 3 (Effects of elevated testicular temperature on methylation patterns of CpG islands potentially regulating transcription of ATP1A4 gene): Ejaculated sperm cannot synthesize proteins; therefore, reduced ATP1A4 content in abnormal sperm induced by elevated testicular temperature was attributed to an altered genome during sperm development. Perhaps ATP1A4 gene expression is regulated by chemical modifications (DNA methylation) of specific genomic regions (CpG islands) in response to elevated temperature. This hypothesis will be investigated by comparing methylation patterns of CpG islands. Objective 4 (Developmental competence of embryos resulting from abnormal sperm): Content of several proteins differed in normal vs abnormal sperm, implicating genome alterations in resulting embryos. I hypothesize that embryos resulting from sperm exposed to elevated testicular temperature have: 1) reduced ability to develop in vitro; and 2) differential expression of paternally expressed genes. Oocytes will be fertilized by microinjecting abnormal sperm. Development from pronuclei to blastocyst stages, expression of a cohort of paternally expressed genes (8-cell stage, i.e., maternal-zygotic transition of gene expression) and methylation patterns of CpG islands potentially regulating expression of these genes, will be determined. These studies will elucidate roles of Na+/K+-ATPase in molecular regulation of Sertoli cell function and sperm capacitation and advance knowledge regarding regulation of male fertility. Epigenetic regulation of ATP1A4 may be a fertility marker and epigenetic changes in the sperm genome and effects on developing embryos will have implications for improving fertility. There will be excellent collaborative training opportunities for HQP.

我的长期目标是阐明男性生育能力的调节。我检测到在异常精子中差异表达的几种蛋白质，包括睾丸温度升高（NSERC DG）诱导的Na+/K+-ATPase（ATP1A4）的睾丸特异性亚型（ATP1A4）。 Sertoli细胞包含Na+/K+-ATPase同工型和Ouabain（其特定配体）诱导牛精子的电容。当前的建议（NSERC DG更新）是阐明Na+/K+-ATPase同工型在Sertoli细胞功能调节中的作用和电容，睾丸温度升高对调节精子发育过程中ATP1A4表达的机制的影响以及异常精子对胚胎发育的影响。 目标1（Na+/K+-ATPase同工型在Sertoli细胞紧密连接的形成和功能中的作用； TJ）：Na+/K+-ATPase调节相邻上皮细胞之间TJ的形成和功能。来自骨周围大鼠睾丸的Sertoli细胞（发生TJ形成时）将用于检验以下假设：Na+/K+-ATPase同工型调节Sertoli细胞中TJ的组装和功能。 Ouabain处理将暗示Na+/K+-ATPase同工型在TJ形成和功能中的作用。 目标2（牛+/k+-ATPase同工型的筏和非系列库的作用）：脂质筏调节了几种物种的精子功能； Na+/K+-ATPase参与了体细胞中筏介导的信号传导。我们在牛精子中发现了Na+/k+-ATPase同工型的筏和非收割池。我假设Na+/K+-ATPase同工型的筏池主要与多个信号分子相互作用并激活多个信号分子，从而导致ouabain诱导的牛精子中的电容。我们将在电容精子（Mmunoprecipitation和质谱法）的筏分数中鉴定Na+/K+-ATPase的结合伴侣。这些分子的相对丰度和激活（磷酸化）将确定Na+/K+-ATPase同工型的筏和非收割机的贡献及其在电容过程中的潜在相互作用。 目标3（睾丸温度升高对CpG岛的甲基化模式的影响可能调节ATP1A4基因的转录）：射精的精子无法合成蛋白质；因此，睾丸温度升高的异常精子中的ATP1A4含量降低归因于精子发育过程中基因组的改变。也许ATP1A4基因表达受到特定基因组区域（CPG岛）的化学修饰（DNA甲基化）的调节，以响应升高温度。该假设将通过比较CpG岛的甲基化模式来研究。 目标4（由异常精子引起的胚胎的发育能力）：正常人与异常精子中的几种蛋白质含量不同，这暗示了产生的胚胎的基因组改变。我假设由暴露于睾丸温度升高的精子引起的胚胎具有：1）体外发展的能力降低； 2）亲子表达基因的差异表达。卵母细胞将通过微注射异常精子受精。将确定从原始囊肿到胚泡阶段的发育，含位表达基因的同类群体（即基因表达的母体 - 杂型旋转过渡）的表达以及CPG岛的甲基化模式潜在地调节了这些基因的表达。 这些研究将阐明Na+/K+-ATPase在Sertoli细胞功能和精子电容的分子调节中的作用，并提高有关男性生育能力的调节知识。 ATP1A4的表观遗传调节可能是生育标记，精子基因组的表观遗传学变化，对发展胚胎的影响将对改善生育能力产生影响。 HQP将有出色的协作培训机会。