Abstract
Building on our recent discovery of the zinc signature phenomenon present in boar, bull, and human spermatozoa, we have further characterized the role of zinc ions in the spermatozoa’s pathway to fertilization. In boar, the zinc signature differed between the three major boar ejaculate fractions, the initial pre-rich, the sperm-rich, and the post-sperm-rich fraction. These differences set in the sperm ejaculatory sequence establish two major sperm cohorts with marked differences in their sperm capacitation progress. On the subcellular level, we show that the capacitation-induced Zn-ion efflux allows for sperm release from oviductal glycans as analyzed with the oviductal epithelium mimicking glycan binding assay. Sperm zinc efflux also activates zinc-containing enzymes and proteases involved in sperm penetration of the zona pellucida, such as the inner acrosomal membrane matrix metalloproteinase 2 (MMP2). Both MMP2 and the 26S proteasome showed severely reduced activity in the presence of zinc ions, through studies using by gel zymography and the fluorogenic substrates, respectively. In the context of the fertilization-induced oocyte zinc spark and the ensuing oocyte-issued polyspermy-blocking zinc shield, the inhibitory effect of zinc on sperm-borne enzymes may contribute to the fast block of polyspermy. Altogether, our findings establish a new paradigm on the role of zinc ions in sperm function and pave the way for the optimization of animal semen analysis, artificial insemination (AI), and human male-factor infertility diagnostics.
Highlights
It is widely understood that zinc ions (Zn2+) play an important role in male fertility, in species ranging from with C. elegans [1] through higher order mammals [2,3,4], but its role in creating subpopulations of fertilization competent spermatozoa was not known until the discovery of the zinc signature in boar, bull, and human spermatozoa [8]
Utilizing high-throughput image-based flow cytometry (IBFC), we examined the sperm zinc signature as reported by fluorescent Zn-tracer FluoZin TM-3 AM (FZ3) across the three main fractions of the boar ejaculate, separated into pre-rich, rich, and post-rich fractions
We found that the zinc signature is significantly different between ejaculate fractions, with most pre-sperm rich/vanguard group spermatozoa expressing the non-capacitated signature 1 (93.5 ± 1.4%)
Summary
It is widely understood that zinc ions (Zn2+) play an important role in male fertility, in species ranging from with C. elegans [1] through higher order mammals [2,3,4] (for review, see [5,6,7]), but its role in creating subpopulations of fertilization competent spermatozoa was not known until the discovery of the zinc signature in boar, bull, and human spermatozoa [8]. Spermatozoa from the initial pre-rich fraction are more fit for cryopreservation survival [15] and are overrepresented in the sperm reservoir [16], earning its nickname, the “vanguard cohort” [16]. This cohort is much more sensitive to the 26S proteasome inhibition of capacitation than the rich/post-rich fractions [8], indicating a previously unknown, distinct early capacitation event occurring at ejaculation and regulated by sperm-borne proteasomes. Vanguard cohort-friendly semen supplements or extenders have not been developed, which could increase sperm capability to bind to the oviductal sperm reservoir, especially given that sperm processing techniques, such as sex sorting, produce capacitation-like changes that are detrimental to sperm oviductal reservoir binding [17]
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