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It has been recently shown in mice that sperm undergo acrosome reaction (AR) by passing through cumulus cells; furthermore, the acrosome-reacted sperm can bind to zona pellucida and consequently fertilise the egg. During AR, the relocation of the primary fusion protein IZUMO1 into the equatorial segment is crucial for sperm–egg fusion. There is a high rate of spontaneous AR in rodents, with up to 60% in promiscuous species. The aim of this study was to clarify whether the IZUMO1 relocation in sperm after spontaneous and induced AR is the same, and whether there is a correlation between the speed of IZUMO1 relocation and species-specific mating behaviour in field mice. Immunofluorescent detection of IZUMO1 dynamics during the in vitro capacitation, spontaneous, calcium ionophore and progesterone-induced AR was monitored. Our results show that during spontaneous AR, there is a clear IZUMO1 relocation from the acrosomal cap to the equatorial segment, and further over the whole sperm head. In addition, there is positive tail tyrosine phosphorylation (TyrP) associated with hyperactive motility. Moreover, the beginning and the progress of IZUMO1 relocation and tail TyrP positively correlate with the level of promiscuity and the acrosome instability in promiscuous species. The findings that crucial molecular changes essential for sperm–egg fusion represented by dynamic movements of IZUMO1 also happen during spontaneous AR are vital for understanding fertilisation in mice.

In order for mammalian sperm to obtain a fertilizing ability, they must undergo a complex of molecular changes, called capacitation. During capacitation, steroidal compounds can exert a fast nongenomic response in sperm through their interaction with plasma membrane receptors, and activate crucial signaling pathways leading to time-dependent protein tyrosine phosphorylation (TyrP). Estrogen receptor beta was detected in epididymal mouse sperm; therefore, the effect of 17B-estradiol, estrone, estriol, and 17A-ethynylestradiol on mouse sperm capacitation in vitro was investigated. The effect was evaluated by positive TyrP in sperm heads and in the whole sperm lysates. Simultaneously, the state of the acrosome after the calcium ionophore-induced acrosome reaction was assessed. Generally, estrogens displayed a time and concentration-dependent stimulatory effect on sperm TyrP during capacitation. In contrast, the number of sperm that underwent the acrosome reaction was lower in the experimental groups. It has been demonstrated that both natural and synthetic estrogens can modify the physiological progress of mouse sperm capacitation. The potential risk in the procapacitation effect of estrogens can also be seen in the decreased ability of sperm to undergo the acrosome reaction. In conclusion, the capacitating ability of sperm can be significantly lowered by increasing the level of estrogens in the environment.

Estrogens play a crucial role in spermatogenesis and estrogen receptor α knock-out male mice are infertile. It has been demonstrated that estrogens significantly increase the speed of capacitation in vitro; however this may lead to the reduction of reproductive potential due to the decreased ability of these sperm to undergo the acrosome reaction. To date the in vivo effect of estrogens on the ability of sperm to capacitate has not been investigated. Therefore, in this study, we exposed mice (n=24) to 17β-estradiol (E₂) at the concentration of 20 ng/ml either during puberty from the fourth to seventh week of age (n=8), or continuously from birth for a period of 12 weeks (n=8) at which age the animals from both groups were killed. The capacitation status of epididymal and testicular sperm was analysed by tyrosine phosphorylation (TyrP) antibody (immunofluorescence and western blot) and chlortetracycline (CTC) assay. According to our results, in vivo exposure to increased E₂ concentrations caused premature sperm capacitation in the epididymis. The effect of E₂, however, seems reversible because after the termination of the exposure premature epididymal sperm capacitation is decreased in animals treated during puberty. Furthermore the changes in epididymal sperm capacitation status detected by TyrP and CTC positively correlate with plasma levels of E₂ and the expression of the estrogen-dependent trefoil factor 1 (Tff1) gene in testicular tissue. Therefore, our data implicate that in vivo exposure to E₂ under specific conditions leads to the premature capacitation of mouse sperm in epididymis with a potential negative impact on the sperm reproductive fitness in the female reproductive tract.

There is pronounced promiscuity and sperm competition in long-tailed field mice (Apodemus sylvaticus). These mice have evolved unusual sperm behaviour favouring rapid fertilisation, including dynamic formation of sperm trains and their subsequent dissociation. The cell surface complement regulatory (CReg) protein CD46 is broadly expressed in eutherian mammals other than rodents, in which it is expressed solely on the spermatozoal acrosomal membrane. Ablation of the CD46 gene has been associated with a faster acrosome reaction (AR) rate in inbred laboratory mice. Here, we demonstrate that wild-caught field mice of three species, A. sylvaticus, A. flavicollis and A. microps, exhibit a more rapid AR than wild-caught house mice Mus musculus or inbred laboratory BALB/c mice. We also demonstrate that wild-caught field mice of these three species, unlike house mice, produce alternatively spliced transcripts of testicular CD46 mRNA lacking exons 5–7 or 6–7, together with an extended 3′ - and often truncated 5′-utr, leading to failure to express any sperm CD46 protein in both the testis and epididymis. Male field mice may therefore have traded expression of this CReg protein for acrosomal instability, providing a novel genus-specific strategy to favour rapid fertilisation and competitive advantage in the promiscuous reproductive behaviour of wild field mice.

Sperm–egg interaction and fusion represent a key moment of fertilization. In mammals, it is not possible without the interaction of the tetraspanin superfamily proteins including CD81. A detailed immunohistochemical localization of CD81 was monitored in bovine oocytes during different maturation stages, as well as during early embryogenesis. In addition, characterization of CD81 was carried out in bovine and mouse sperm. In bovine eggs, CD81 was detected on the plasma membrane of the germinal vesicle, metaphase I and metaphase II oocytes. During fertilization, accumulation of CD81 molecules in the perivitelline space of fertilized oocytes, which appeared as vesicles associated with plasma membrane, was observed. In majority of bull-ejaculated sperm and caput, corpus and cauda epididymal sperm, as well as mouse cauda epididymal sperm, CD81 was found on the plasma membrane covering the apical acrosome. Although the process of capacitation did not influence the localization of CD81, it was lost from the surface of the acrosome-reacted spermatozoa in bull, in contrast to mouse sperm where there was a relocalization of the CD81 protein during acrosome reaction across the equatorial segment and later over the whole sperm head. The presented results highlight conservative unifying aspects of CD81 expression between cattle and mouse, together with mouse-specific traits in sperm CD81 behaviour, which emphasizes certain species-specific mechanisms of fertilization to be considered.