Zinc ion fluxes on the pathway to mammalian sperm fertilization competency
For decades, the role of the divalent ion calcium was the focus of understanding mechanisms leading to sperm fertilization competency and management of semen storage. Little focus has been placed on other divalent ions, including zinc ion (Zn2+). Further, the ultimate maturation event preparing mammalian spermatozoa for fertilization, sperm capacitation, was first described in 1951, yet its regulatory mechanisms remain poorly understood. Here, we document a novel biological phenomenon of a unique Zn2+ distribution (further zinc signature) associated with mammalian spermatozoa from the round spermatid stage of spermiogenesis, to epididymal maturation, ejaculation, and up to 72 hours of liquid semen storage. Using image-based flow cytometry (IBFC), we identified four distinct sperm zinc signatures present in boar, bull, and human spermatozoa. The zinc signature was altered after sperm capacitation, reduced by proteasomal inhibitors, removed by zinc chelation, and maintained with addition of external ZnCl2. The zinc signature differed between the three major boar ejaculate fractions. These differences set in sperm ejaculatory sequence likely establish two major sperm cohorts; one destined for populating the sperm oviductal reservoir and the other that is capable of fertilizing mature, ovulated oocytes at the time of mating/insemination. Management of the sperm zinc signature prevented spontaneous, pathological capacitation, by day 3 of extended liquid boar semen storage and could allow for use of fewer sperm per artificial insemination (AI) dose to increase the usage of high genetic value sires. A newly formulated semen extender was able to mimic qualities of the pre-sperm rich fraction. On a subcellular level, the capacitation induced Zn2+ efflux allows for release from oviductal glycans studied with the oviductal epithelium mimicking glycan binding assay. Sperm Zn2+ efflux also activates zinc-containing enzymes involved in sperm penetration of the zona pellucida, such as the inner acrosomal membrane metalloproteinase MMP2 that had a severely reduced activity in the presence of Zn2+ by gel zymography. In context of the fertilization-induced oocyte zinc spark and the ensuing, oocyteissued polyspermy-blocking zinc shield, the inhibitory effect of Zn2+ on spermborne enzymes may contribute to the fast block of polyspermy. Altogether, our findings establish a new paradigm on the role of Zn2+ in sperm function, paving the way for improved semen analysis, in vitro fertilization (IVF) and the optimization of AI and semen distribution.
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