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: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=630464
• 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力的调节。我检测到几个蛋白质差异表达异常精子，包括睾丸特异性亚型的Na+/K+-ATP酶（ATP 1A 4），诱导睾丸温度升高（NSERC DG）。Sertoli细胞含有Na+/K+-ATP酶同工酶，哇巴因（它们的特异性配体）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+-ATP酶异构体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ATP 1A 4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+-ATP酶亚型在支持细胞紧密连接形成和功能中的作用）：Na+/K+-ATP酶调节相邻上皮细胞间紧密连接的形成和功能。将使用来自青春期大鼠睾丸（当TJ形成时）的Sertoli细胞来检验Na+/K+-ATP酶同工型调节Sertoli细胞中TJ的组装和功能的假设。哇巴因处理将暗示Na+/K+-ATP酶同工型在TJ形成和功能中的作用。目的2（Na+/K+-ATP酶在牛精子获能过程中的作用）：脂质筏调节多种动物精子的功能; Na+/K+-ATP酶参与体细胞中筏介导的信号转导。在牛精子中发现了Na+/K+-ATP酶的筏池和非筏池。我推测，筏池的Na+/K+-ATP酶亚型主要相互作用，并激活多种信号分子，导致哇巴因诱导获能在公牛精子。我们将鉴定获能精子筏组分中Na+/K+-ATP酶的结合伴侣（免疫沉淀和质谱）。这些分子的相对丰度和活化（磷酸化）将决定Na+/K+-ATP酶亚型的筏池和非筏池的贡献及其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ATP 1A 4基因转录的CpG岛甲基化模式的影响）：射精精子不能合成蛋白质;因此，睾丸温度升高诱导的异常精子中ATP 1A 4含量降低归因于精子发育过程中基因组的改变。可能ATP 1A 4基因表达受特定基因组区域（CpG岛）的化学修饰（DNA甲基化）调节，以响应温度升高。这一假说将通过比较CpG岛的甲基化模式来研究。目的4（异常精子胚胎的发育能力）：正常精子与异常精子中几种蛋白质的含量不同，暗示胚胎中基因组的改变。我假设，精子暴露于睾丸温度升高产生的胚胎：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受精。从原核到胚泡阶段的发育，一组父系表达基因的表达（8细胞期，即，基因表达的母体-合子转变）和潜在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控中的作用，并促进对男性生育力调控的认识。ATP 1A 4的表观遗传调节可能是生育力标志物，精子基因组的表观遗传变化和对发育中胚胎的影响将对提高生育力产生影响。将为HQP提供极好的合作培训机会。