bioRxiv2024
In mammals, fertilization takes place followed by fetus development in the maternal body, so the number of fetuses that exceeds the mothers capacity increases the frequency of stunted growth and stillbirths, while an excessive number of pups increases postnatal stunting and maternal child-rearing stress, which is detrimental to offspring procreation and species maintenance. Therefore, various control mechanisms are thought to be at work to ensure that fertilization occurs in appropriate numbers. Sperm are not capable of fertilization immediately after they are produced in the testis, and must undergo a stepwise activation process including capacitation, hyper motility activation or acrosome reaction before they meet the egg in the oviduct, where fertilization takes place, contributing positively on fertilization, which in turn ensures the number of fetuses for the maintenance of species. In contrast, in this study, we found that a subpopulation of sperm is derived in a Ca2+-dependent manner during the process for sperm to acquire fertility, which may regulate the number of fetuses. When sperm were harvested from the epididymis of mice and activated in vitro, a subpopulation of sperm emerged from the sperm population in which Lypd4 was expressed on the sperm surface at a ratio up to 30%. The sperm in this subpopulation were rather small in size, more permeable to dyes, and had already ceased motility. We further characterized this sperm population by surface antigen screening using various monoclonal antibodies and found the expression of several proteins, such as CD55, ICOS or Ccr3, specific to this population. The emergence of this sperm population was induced at a concentration of about 4% of Ca2+ in body fluids and was independent of capacitation or acrosome reaction, and also apoptotic process. This subpopulation also appeared over time in sperm ejaculated into the female body, accounting for about 50% of the sperm that reached the oviduct in an hour. When this sperm subpopulation was then removed from the entire population using anti-Lypd4 antibody followed by in utero insemination, the fertilization rate of the oocytes collected from the oviducts doubled. Such a sperm subpopulation was also observed in macaque monkeys, and removal of this subpopulation increased the egg penetration rate of sperm, suggesting that this sperm subpopulation exists commonly in mammals and that the mothers acceptable fetal number is adjusted by systematically sterilizing a certain number of sperm.