Polyspermy prevention: facts and artifacts?

In mammals, the sperm : oocyte ratio at origin can be as high as 109:1 [113]. Despite this, behavioural adaptations are required to ensure fertilization, such as the deposition of sperm in the female tract and the synchrony of mating. Regardless of whether spermatozoa are deposited in the vagina (e.g., humans) or directly into the uterus (e.g., mice), the vast majority are rapidly eliminated from the tract [94, 113]. Only a minute fraction successfully migrate to the site of fertilization, the ampulla or ampulla-isthmic junction and there is evidence that the female tract tends to prevent morphologically abnormal sperm from reaching the ampullae [77, 94].

In mice, the utero-tubal junction is the major barrier for sperm ascent. Spermatozoa are then sequestered in the lower part of the oviductal isthmus until ovulation begins when they are progressively released. Sperm ascent and oocyte descent to the ampulla occur synchronously, avoiding premature aging in the oocyte, which would lead to abnormal embryonic development. In humans, the first barrier is the highly folded mucus filled cervix, which retains sperm for later migration to the upper tract. The release of spermatozoa from the cervix may continue for days [47]. Sperm ascend though the uterus primarily by the contractile activity of the uterine wall, and also here sperm appear to be sequestered in the lower isthmus until ovulation (see [52, 53] for domestic animals). In the pig, spermatozoa stored in the isthmus are in close contact with the epithelium [37]. The mechanism by which isthmus bound sperm are selectively released is not clear; however it seems to be associated with changes in the sperm head plasma membrane and capacitation (see also [44]). Migration from the isthmus to the ampullae appears to be due to sperm motility and contractile activity of the oviduct [53]. In mammals, as the sperm passes through the female tract a series of progressive changes to its physiology occur, termed capacitation, which are a pre-requisite for fertilization. This process is essentially the acquisition of fertilizing potential of the spermatozoon and involves cell membrane alterations, changes in protein phosphorylation and modulation to intracellular Ca2+.

In studies in situ, 700 spermatozoa were found in sheep ampullae [8] and five in man [34], while in rodents, sperm : oocyte ratios at the site of fertilization are usually unity or below [54, 92, 113].

In conclusion, the female reproductive tract in mammals modulates and controls the gradual encounter of spermatozoa with oocytes often leading to unity.

After reaching the oocyte in the ampullae, the spermatozoa must traverse and interact with the outer oocyte investment, the cumulus oophorus and then bind to and penetrate the zona pellucida before finally fusing with the plasma membrane. Although the extracellular coats may be removed in some animals without inhibiting fertilization and these initial gamete interactions may be bypassed by micro-injecting the spermatozoon directly into the oocyte, these events cannot and are not bypassed in nature. The zona pellucida composed of several glycoproteins (ZP) that differ in number between species [4, 38, 42, 57, 79, 106] serves to modulate sperm binding and to protect the embryo during early development. Whether or not mammalian spermatozoa respond to chemotactic stimuli is very much open to debate [110]; however there is data to suggest that an odorant receptor gene expressed in the testis may be involved in sperm chemotaxis in humans [90, 105]. Progression through the outer layers of the oocyte depends on successive molecular interactions that change sperm physiology step-by step promoting fertilization competence. Although there is much information in the literature on the genetics and biochemistry of the outer layers, in particular the zona pellucida [4, 38, 42, 57, 79, 106], we know little about their topographical constitution and if indeed sperm entry is piloted to a specific site. Recently chemical modulation of the zona by oviductal-specific glycoproteins before the oocyte encounters the spermatozoon has been described and this may also be involved in the fine tuning of sperm-oocyte interactions in mammals [14, 15]. Finally, although the plasma membrane of the human metaphase ll oocyte appears at the scanning electron microscope to be unpolarized, a study with surface lectins may be instrumental in showing whether or not spermatozoa may fuse at any site around the plasma membrane [87]. In contrast to the situation in the human, the plasma membrane in other mammalian oocytes is polarized with a flat microvillus-free area overlying the metaphase spindle. In mouse rat and hamster, spermatozoa do not fuse with this area of the plasma membrane [60, 78, 89].

The first indication of activation in mammalian oocytes is an electrical change at the plasma membrane followed by the release of Ca2+ from intracellular stores [93, 98–100]. As in other animals, this increase in intracellular calcium triggers the activation of the cell cycle, through the degradation of MPF and the exocytosis of cortical granules. The cortical reaction in mammals is less dramatic and slower than in its invertebrate counterparts, but appears to follow the same principles. For example in the mouse oocyte plasma membrane, the lateral diffusion of proteins and lipids is strongly restricted after fertilization [59]. A protease released from the cortical granules partially hydrolyses the zona pellucida glycoproteins, ZP2 and ZP3, removing sperm binding capacity but also leading to a general hardening of the zona pellucida, even though transient [35, 38, 73]. The cortical reaction is a slow structural change that changes the receptive outer investment of the oocyte into a hardened protective layer for the developing embryo.

Timing of fertilization in mammals is critical to avoid oocyte aging and abnormal activation and development [111]. Timing is also of the essence in situ in mammals, since insemination of post ovulatory pigs may also lead to an increase in polyspermy, either by oocyte aging or the dynamics of sperm transport in the oviduct [51]. Polyspermy is a fact in aged oocytes in vivo and in vitro, whereas the concept of “polyspermy-blocking mechanisms” in mammalian oocytes is mainly based on experiments in vitro using oocytes deprived of their external coats. Within the mammals there are of course species differences with regards to the constitution of the zona pellucida [42] and this reflects in the kinetics and final outcome of the zona reaction. It is the general consensus that polyspermy blocking mechanisms vary between species being either located at the plasma membrane, as in the rabbit [8, 102], at the zona pellucida as in the sheep and pig [3], or a combination of both as in the mouse [3]. Since such conclusions were originally based on experiments using oocytes deprived of their outer investments we would like to offer an alternative explanation. That is, that the species difference is not due to the different location of different sperm repelling mechanisms, but to the different behaviour of gametes under experimental conditions. In particular, how sperm react physiologically to an incorrect sequence of oocyte signals due to investment removal, how oocytes age differently, and how sperm are able to capacitate in vitro.

Since the kinetics of the activation events do not depend on whether one spermatozoon or several spermatozoa approach the oocyte, we suggest they serve solely to change the quiescent oocyte into a dynamic zygote.