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的规律。我检测到了几种在异常精子中差异表达的蛋白质，包括睾丸特异的Na/K-ATPase亚型(ATP1A4)，它是由睾丸温度升高(NSERC DG)诱导的。Sertoli细胞含有Na/K-ATPase亚型，哇巴因(其特异性配体)可诱导公牛精子获能。目前的建议(NSERC DG更新)是为了阐明Na/K-ATPase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。目的1(Na/K-ATPase亚型在支持细胞紧密连接形成和功能中的作用)：Na/K-ATPase调节相邻上皮细胞间紧密连接的形成和功能。将利用青春期大鼠睾丸的支持细胞(TJ形成时)来验证Na/K-ATPase亚型调节TJ在支持细胞中组装和功能的假说。哇巴因处理将涉及Na/K-ATPase亚型在TJ的形成和功能中的作用。目的2(Na/K-ATPase异构体RAFT和非RAFT池在公牛精子获能过程中的作用)：脂筏调节多种物种的精子功能；Na/K-ATPase参与RAFT介导的体细胞信号转导。我们在公牛精子中发现了RAFT和非RAFT的Na/K-ATPase异构体。我推测，Na/K-ATPase异构体的RAFT池主要与多个信号分子相互作用并激活多个信号分子，导致哇巴因诱导公牛精子获能。我们将在获能精子的RAFT部分中确定Na/K-ATPase的结合伙伴(免疫沉淀和质谱仪)。这些分子的相对丰度和激活(磷酸化)将决定RAFT和非RAFT的Na/K-ATPase亚型池的贡献以及它们在获能过程中的潜在相互作用。目的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)：射出的精子不能合成蛋白质，因此，睾丸温度升高导致的异常精子中ATP1A4含量的降低被归因于精子发育过程中基因组的改变。可能ATP1A4基因的表达是通过特定基因组区域(CpG岛)的化学修饰(DNA甲基化)来调节的，以响应温度的升高。这一假说将通过比较CpG岛的甲基化模式来研究。目的4(异常精子所致胚胎的发育能力)：正常精子和异常精子中几种蛋白质的含量不同，可能是导致胚胎基因组改变的原因。我推测，暴露在高睾丸温度下的精子产生的胚胎有：1)体外发育能力降低；2)父系表达基因的差异表达。卵母细胞将通过显微注射异常精子来受精。将确定从原核到囊胚期的发育，一组父系表达的基因的表达(8细胞期，即基因表达的母体-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-ATPase在支持细胞功能和精子获能的分子调控中的作用，并增进对男性生育能力调节的认识。ATP1A4的表观遗传调控可能是一种生育标记，精子基因组的表观遗传变化及其对发育胚胎的影响将对提高生育能力具有重要意义。HQP将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.