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

我的长期目标是阐明男性生育力的调节。我检测到几种在异常精子中差异表达的蛋白质，包括由睾丸温度升高诱导的睾丸特异性 Na /K -ATP 酶亚型 (ATP1A4) (NSERC DG)。支持细胞含有 Na /K -ATP 酶亚型，哇巴因（其特定配体）可诱导公牛精子获能。目前的提案（NSERC DG 更新）旨在阐明 Na /K -ATP 酶亚型在支持细胞功能和获能调节中的作用、睾丸温度升高对精子发育过程中 ATP1A4 表达调节机制的影响，以及异常精子对胚胎发育的影响。目标 1（Na /K -ATP 酶亚型在支持细胞紧密连接的形成和功能中的作用；TJ）：Na /K -ATP 酶调节相邻上皮细胞之间 TJ 的形成和功能。来自青春期周围大鼠睾丸（当发生 TJ 形成时）的支持细胞将用于测试 Na /K -ATP 酶亚型调节支持细胞中 TJ 的组装和功能的假设。哇巴因治疗将涉及 Na /K -ATP 酶亚型在 TJ 形成和功能中的作用。目标 2（公牛精子获能期间 Na /K -ATP 酶亚型的筏和非筏池的作用）：脂筏调节多个物种的精子功能； Na /K -ATP 酶参与体细胞中筏介导的信号传导。我们在公牛精子中发现了 Na /K -ATP 酶亚型的筏池和非筏池。我假设 Na /K -ATP 酶异构体的筏池主要与多种信号分子相互作用并激活多种信号分子，从而导致公牛精子中哇巴因诱导的获能。我们将鉴定获能精子筏部分中 Na /K -ATP 酶的结合伴侣（免疫沉淀和质谱法）。这些分子的相对丰度和激活（磷酸化）将决定 Na /K -ATP 酶亚型的筏池和非筏池的贡献以及它们在获能期间的潜在相互作用。目标3（睾丸温度升高对可能调节ATP1A4基因转录的CpG岛甲基化模式的影响）：射精的精子不能合成蛋白质；因此，睾丸温度升高引起的异常精子中 ATP1A4 含量的减少归因于精子发育过程中基因组的改变。也许 ATP1A4 基因表达是通过响应升高的温度而对特定基因组区域（CpG 岛）进行化学修饰（DNA 甲基化）来调节的。我们将通过比较 CpG 岛的甲基化模式来研究这一假设。目标 4（异常精子产生的胚胎的发育能力）：正常精子和异常精子中几种蛋白质的含量不同，这表明胚胎中的基因组发生了改变。我假设精子暴露于升高的睾丸温度所产生的胚胎具有：1）体外发育能力降低； 2)父系表达基因的差异表达。通过显微注射异常精子使卵母细胞受精。将确定从原核到囊胚阶段的发育、一组父系表达基因的表达（8细胞阶段，即基因表达的母系-合子转变）以及可能调节这些基因表达的CpG岛的甲基化模式。这些研究将阐明 Na /K -ATP 酶在支持细胞功能和精子获能的分子调节中的作用，并增进有关男性生育力调节的知识。 ATP1A4 的表观遗传调控可能是生育力标志物，精子基因组的表观遗传变化以及对发育中胚胎的影响将对提高生育力产生影响。总部将会有极好的协作培训机会。

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.