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

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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.