To fuse with oocytes, spermatozoa of eutherian mammals must pass through extracellular coats, the cumulus cell layer, and the zona pellucida (ZP). It is generally believed that the acrosome reaction (AR) of spermatozoa, essential for zona penetration and fusion with oocytes, is triggered by sperm contact with the zona pellucida. Therefore, in most previous studies of sperm-oocyte interactions in the mouse, the cumulus has been removed before insemination to facilitate the examination of sperm-zona interactions. We used transgenic mouse spermatozoa, which enabled us to detect the onset of the acrosome reaction using fluorescence microscopy. We found that the spermatozoa that began the acrosome reaction before reaching the zona were able to penetrate the zona and fused with the oocyte's plasma membrane. In fact, most fertilizing spermatozoa underwent the acrosome reaction before reaching the zona pellucida of cumulus-enclosed oocytes, at least under the experimental conditions we used. The incidence of in vitro fertilization of cumulus-free oocytes was increased by coincubating oocytes with cumulus cells, suggesting an important role for cumulus cells and their matrix in natural fertilization.sperm-egg interaction | acrosomal exocytosis | real-time imaging | video microscopy F ertilization is a critical moment in animal sexual reproduction and comprises sequential steps in the interaction of sperm with an egg. In eutherian mammals, natural fertilization is accomplished by sperm ascent to the oviduct ampulla, passage through the cumulus oophorus surrounding oocytes, loose binding to the oocyte's outer envelope, the zona pellucida (ZP), followed by tight sperm binding to the ZP and penetration through the ZP, culminating in sperm-oocyte membrane fusion (1). Before penetrating into the ZP, the fertilizing spermatozoon must undergo a morphological change involving disruption of the acrosome called the acrosome reaction (AR). In most studies of mouse sperm-oocyte interactions in vitro, the cumulus oophorus is removed by hyaluronidase treatment before insemination, allowing direct sperm contact with the ZP upon insemination. However, under natural in vivo conditions, spermatozoa first encounter the cumulus before reaching the ZP surface. The question this paper addresses is: Where does the fertilizing mouse spermatozoon begin the AR-in the cumulus or at the ZP?In the mouse, it has been reported that a ZP glycoprotein, ZP3, has both sperm-binding (sperm receptor) and AR-inducing activities and is essential for formation of the zona pellucida and therefore fertility (2, 3). The specificity of sperm binding to homologous zona might be conferred by the supramolecular structure (reviewed in ref. 4) or cleavage status of ZP glycoproteins (5). It is generally assumed that-at least in the mouse-the AR occurs after sperm binding to the ZP (6, 7). Even though spermatozoa of several species other than the mouse are known to be able to undergo the AR on the ZP surface (8-10), the presence of motile spermatozoa within the cumulus at...

BackgroundSperm cells are the target of strong sexual selection that may drive changes in sperm structure and function to maximize fertilisation success. Sperm evolution is regarded to be one of the major consequences of sperm competition in polyandrous species, however it can also be driven by adaptation to the environmental conditions at the site of fertilization. Strong stabilizing selection limits intra-specific variation, and therefore polymorphism, among fertile sperm (eusperm). Here we analyzed reproductive morphology differences among males employing characteristic alternative mating behaviours, and so potentially different conditions of sperm competition and fertilization environment, in the squid Loligo bleekeri.ResultsLarge consort males transfer smaller (average total length = 73 μm) sperm to a female's internal sperm storage location, inside the oviduct; whereas small sneaker males transfer larger (99 μm) sperm to an external location around the seminal receptacle near the mouth. No significant difference in swimming speed was observed between consort and sneaker sperm. Furthermore, sperm precedence in the seminal receptacle was not biased toward longer sperm, suggesting no evidence for large sperm being favoured in competition for space in the sperm storage organ among sneaker males.ConclusionsHere we report the first case, in the squid Loligo bleekeri, where distinctly dimorphic eusperm are produced by different sized males that employ alternative mating behaviours. Our results found no evidence that the distinct sperm dimorphism was driven by between- and within-tactic sperm competition. We propose that presence of alternative fertilization environments with distinct characteristics (i.e. internal or external), whether or not in combination with the effects of sperm competition, can drive the disruptive evolution of sperm size.

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Behavioral traits of sperm are adapted to the reproductive strategy that each species employs. In polyandrous species, spermatozoa often form motile clusters, which might be advantageous for competing with sperm from other males. Despite this presumed advantage for reproductive success, little is known about how sperm form such functional assemblies. Previously, we reported that males of the coastal squid Loligo bleekeri produce two morphologically different euspermatozoa that are linked to distinctly different mating behaviors. Consort and sneaker males use two distinct insemination sites, one inside and one outside the female's body, respectively. Here, we show that sperm release a self-attracting molecule that causes only sneaker sperm to swarm. We identified CO2 as the sperm chemoattractant and membrane-bound flagellar carbonic anhydrase as its sensor. Downstream signaling results from the generation of extracellular H(+), intracellular acidosis, and recovery from acidosis. These signaling events elicit Ca(2+)-dependent turning behavior, resulting in chemotactic swarming. These results illuminate the bifurcating evolution of sperm underlying the distinct fertilization strategies of this species.