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.

SynopsisEstimation of the isotopic turnover rate in the tissues of organisms allows us to estimate the temporal relationship between the isotope ratio of an organism and its prey, and to analyze seasonal variations of food webs and migratory patterns of animals. We analyzed the isotopic turnover rate in the tissue of lacustrine sockeye salmon, Oncorhynchus nerka, of three age classes in a laboratory food alteration experiment to clarify the isotopic turnover rate. The contribution of growth to isotopic turnover was higher in young and fast-growing lacustrine sockeye salmon than in older and slow-growing fish. Maturation masks the relationship between the contributions of growth and metabolism to isotopic change. Our estimate of the isotopic time lag of lacustrine sockeye salmon can be used to research trophic relationships of anadromous sockeye salmon.

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.

In May 2008, we found five individuals of an acoel flatworm species, which is not found in the literatures or webs, on a branch piece of the coral Acropora humilis off Maja Beach, Aka Island, Okinawa, Japan (26 11 N, 127 17 E). They were approximately 1 mm in length, and had white dots and brown symbiotic algae (Fig. 1A). Identical sequences of the 18SrDNA were obtained from two individuals so far analyzed (accession no. AB539806). DNA phylogenetic analyses assigned its position in the genus Waminoa (data not shown), and the highest BLAST match at present is W. brickneri (accession no. AJ875221, Ogunlana et al. 2005). But, large sequence differences (150 base substitutions and 14 gaps per 1464bp) and morphological differences (Ogunlana et al. 2005) suggest that the flatworm we found is distinct from W. brickneri. Detailed description is a subject remained. There are several reports on Waminoa being epizoic on hard and soft corals in the Red Sea and SouthWest Pacific (e.g. Ogunlana et al. 2005). In particular, the species in this study seems to be camouflagic on Acropora corals (Fig. 1B and Online Supplementary Material). Interests would be taken in their ecological aspects.

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In Salmonidae, subordinate males are exposed to higher risks of sperm competition than dominant males and thus are expected to improve the sperm characteristics (sperm concentrations, sperm velocity and sperm longevity). In this study, we investigated the relationships between body size and secondary sexual characters (breeding colour, hump height and snout length), and sperm characteristics of one-year-old (newly matured) Dolly Varden char. Small males displayed higher sperm concentrations than large males. Moreover, males with dull breeding colours, but not with lesser snout length and hump height, displayed an increased sperm velocity compared to males with bright colours, suggesting a trade-off between sperm quantity and the investment in breeding colour. In addition, sperm longevity decreased as sperm swimming velocity increased. These findings indicate that small males with dull breeding colours improve the quantity and quality of their sperm to a great extent to enhance their chances of reproductive success.

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