Effect of sex-sorting on the ability of fresh and cryopreserved bull sperm to undergo an acrosome reaction

Previous studies indicate that sex-sorted sperm exhibit different physiology, including fertilizing capacity, from non-sorted sperm. However, differences between X- and Y-bearing sperm in their ability to undergo an acrosome reaction have never been investigated. This study determined the ability of non-sorted and sex-sorted sperm to undergo the acrosome reaction prior to and after cryopreservation. Sperm were treated with dilauroylphosphatidylcholine (PC12) to induce the acrosome reaction and the percentages of live-acrosome-reacted sperm and dead sperm were evaluated. The X- and Y-bearing sperm reacted similarly to the PC12 treatment, regardless of whether sperm were assessed prior to or after cryopreservation. Fresh control sperm exhibited lower percentages of live sperm (60%) than either X- or Y- sorted sperm (69-74%, P<0.05). Percentages of live control sperm were also lower after thawing (29-35%) than sex-sorted sperm (55-58%, P<0.05). Control and sex-sorted fresh sperm responded similarly to PC12 treatment. However, sex-sorted cryopreserved sperm exhibited higher percentages of live-acrosome-reacted sperm (23%) than control sperm (9%, P<0.05) after 40 min without PC12 treatment. In addition, cryopreserved control sperm treated with 79 microM PC12 exhibited higher percentages of live-acrosome-reacted sperm than sex-sorted sperm. In conclusion, X- and Y-bearing sperm responded similarly to PC12 treatment. In addition, fresh sexed and non-sorted sperm responded similarly to PC12 treatment. However, cryopreserved sex-sorted sperm underwent an acrosome reaction more rapidly in the absence of PC12 (over a 40 min period) than the non-sorted sperm. Therefore, sex-sorting induced changes in sperm membranes that accelerated the acrosome reaction process in sperm after cryopreservation.     

Peristaltic squeezing of sperm bundles at the late stage of spermatogenesis in the silkworm, Bombyx mori

Silkworm (Lepidoptera) males produce dimorphic sperm, nucleate eupyrene sperm, and anucleate apyrene sperm. The eupyrene sperm is the ordinary sperm fertilizing eggs, while the function of the apyrene sperm, which are about four times as numerous as the eupyrene sperm, is still uncertain. We found the peristaltic phenomenon at the very late stage of spermatogenesis. Peristalsis occurs in both eupyrene and apyrene sperm bundles. Through peristaltic action, cytoplasm of the eupyrene sperm and both cytoplasm and nuclei of the apyrene sperm are discarded from the posterior end of the sperm bundles. Peristaltic squeezing seems to be a process to eliminate the irregular nuclei of apyrene sperm while preserving the nuclei of eupyrene sperm.     

Significance of peristaltic squeezing of sperm bundles in the silkworm, Bombyx mori: elimination of irregular eupyrene sperm nuclei of the triploid

Silkworm (Lepidoptera) males produce dimorphic sperm: nucleate eupyrene sperm and anucleate apyrene sperm. The eupyrene sperm are ordinary sperm to fertilise the eggs, while the function of apyrene sperm remains uncertain. After meiosis, 256 sperm cells are enclosed by a layer of cyst cells, forming a sperm bundle. We have previously documented that the nucleus of eupyrene sperm anchors to the head cyst cell, which locates at the anterior apex of the bundle, by an acrosome tubule-basal body assembly. Neither the basal body attachment to the nucleus nor the acrosome is seen in apyrene sperm, and the nuclei remain in the middle region of the bundle. Peristaltic squeezing starts from the anterior of the bundles in both types of sperm, and cytoplasmic debris of the eupyrene sperm, and both the nuclei and debris of apyrene sperm, are eliminated at the final stage of spermatogenesis. Since the irregularity of meiotic division in apyrene sperm is known, we used triploid silkworm males that show irregular meiotic division even in eupyrene spermatocytes and are highly sterile. The irregular nuclei of the triploid are discarded by the peristaltic squeezing just as those of the apyrene sperm. Transmission electron microscopic observations disclose the abnormality in the acrosome tubule and in the connection to the basal body. The peristaltic squeezing of sperm bundles in the silkworm appears to be the final control mechanism to eliminate irregular nuclei before they enter female reproductive organs.     

Sexual selection and the evolution of sperm morphology in sharks

Post‐copulatory sexual selection, and sperm competition in particular, is a powerful selective force shaping the evolution of sperm morphology. Although mounting evidence suggests that post‐copulatory sexual selection influences the evolution of sperm morphology among species, recent evidence also suggests that sperm competition influences variation in sperm morphology at the intraspecific level. However, contradictory empirical results and limited taxonomic scope have led to difficulty in assessing the generality of sperm morphological responses to variation in the strength of sperm competition. Here, we use phylogenetically controlled analyses to explore the effects of sperm competition on sperm morphology and variance in sharks, a basal vertebrate group characterized by wide variation in rates of multiple mating by females, and consequently sperm competition risk. Our analyses reveal that shark species experiencing greater levels of sperm competition produce sperm with longer flagella and that sperm flagellum length is less variable in species under higher sperm competition risk. In contrast, neither the length of the sperm head and midpiece nor variation in sperm head and midpiece length was associated with sperm competition risk. Our findings demonstrate that selection influences both the inter‐ and intraspecific variation in sperm morphology and suggest that the flagellum is an important target of sexual selection in sharks. These findings provide important insight into patterns of selection on the ejaculate in a basal vertebrate lineage.     

Evolution of sperm size in nematodes: sperm competition favours larger sperm

In the free-living rhabditid nematode Caenorhabditis elegans, sperm size is a determinant of sperm competitiveness. Larger sperm crawl faster and physically displace smaller sperm to take fertilization priority, but not without a cost: larger sperm are produced at a slower rate. Here, we investigate the evolution of sperm size in the family Rhabditidae by comparing sperm among 19 species, seven of which are hermaphroditic (self-fertile hermaphrodites and males), the rest being gonochoristic (females and males). We found that sperm size differed significantly with reproductive mode: males of gonochoristic species had significantly larger sperm than did males of the hermaphroditic species. Because males compose 50% of the populations of gonochoristic species but are rare in hermaphroditic species, the risk of male-male sperm competition is greater in gonochoristic species. Larger sperm have thus evolved in species with a greater risk of sperm competition. Our results support recent studies contending that sperm size may increase in response to sperm competition.     

Movement characteristics of bovine epididymal spermatozoa: effects of forward motility protein and epididymal maturation

Movement characteristics of untreated bovine caudal epididymal spermatozoa were compared by high-speed cinemicrography with those of theophylline-activated caput epididymal spermatozoa with and without added forward motility protein (FMP). Comparison of individual movement characteristics clearly established the importance of FMP in converting the nonprogressive motility of theophylline-activated caput sperm into the progressive swimming of mature caudal sperm. Although the total or curvilinear distance traveled in 1 sec by theophylline-activated caput sperm was not changed by the addition of FMP, the linear progression was doubled and the percentage of progressively motile sperm was tripled by this protein. Untreated caudal sperm were 80% motile and theophylline-activated caput sperm were nearly 50% motile; the percentage of motile sperm that were progressive was the same for theophylline-activated caput sperm with FMP and for untreated caudal sperm. Caput sperm without FMP roll infrequently, if at all, but caput sperm with FMP and caudal sperm roll at 4.7 Hz. The beat frequency increases significantly with the addition of FMP and is even higher for caudal sperm. The hydrodynamic power output rises concomitantly with the beat frequency. Perhaps the most striking difference between caput sperm without FMP and those with it is in the swimming paths they follow. Caput sperm without FMP exhibit frequent reversals in direction, or yawing of the sperm heads as they loop back and cross over their tails in an apparently very flexible bending. Their average swimming paths are circles. Caput sperm with FMP and caudal sperm do not show this behavior, but swim in average paths which are linear. The minimum radius of curvature of the tail of caput sperm without FMP is much smaller than that for the other two cell types. These studies clarify the role of FMP in epididymal development of sperm motility.     

Factors Affecting Sperm Quality Before and After Mating of Calopterygid Damselflies

Damselflies (Odonata: Zygoptera) have a more complex sperm transfer system than other internally ejaculating insects. Males translocate sperm from the internal reproductive organs to the specific sperm vesicles, a small cavity on the body surface, and then transfer them into the female. To examine how the additional steps of sperm transfer contribute to decreases in sperm quality, we assessed sperm viability (the proportion of live sperm) at each stage of mating and after different storage times in male and female reproductive organs in two damselfly species, Mnais pruinosa and Calopteryx cornelia. Viability of stored sperm in females was lower than that of male stores even just after copulation. Male sperm vesicles were not equipped to maintain sperm quality for longer periods than the internal reproductive organs. However, the sperm vesicles were only used for short-term storage; therefore, this process appeared unlikely to reduce sperm viability when transferred to the female. Males remove rival sperm prior to transfer of their own ejaculate using a peculiar-shaped aedeagus, but sperm removal by males is not always complete. Thus, dilution occurs between newly received sperm and aged sperm already stored in the female, causing lower viability of sperm inside the female than that of sperm transferred by males. If females do not remate, sperm viability gradually decreases with the duration of storage. Frequent mating of females may therefore contribute to the maintenance of high sperm quality.     

Sperm competition in the absence of fertilization in Caenorhabditis elegans

Hermaphrodite self-fertilization is the primary mode of reproduction in the nematode Caenorhabditis elegans. However, when a hermaphrodite is crossed with a male, nearly all of the oocytes are fertilized by male-derived sperm. This sperm precedence during reproduction is due to the competitive superiority of male-derived sperm and results in a functional suppression of hermaphrodite self-fertility. In this study, mutant males that inseminate fertilization-defective sperm were used to reveal that sperm competition within a hermaphrodite does not require successful fertilization. However, sperm competition does require normal sperm motility. Additionally, sperm competition is not an absolute process because oocytes not fertilized by male-derived sperm can sometimes be fertilized by hermaphrodite-derived sperm. These results indicate that outcrossed progeny result from a wild-type cross because male-derived sperm are competitively superior and hermaphrodite-derived sperm become unavailable to oocytes. The sperm competition assays described in this study will be useful in further classifying the large number of currently identified mutations that alter sperm function and development in C. elegans.     

No evidence of trade‐offs in the evolution of sperm numbers and sperm size in mammals

Post‐copulatory sexual selection, in the form sperm competition, has influenced the evolution of several male reproductive traits. However, theory predicts that sperm competition would lead to trade‐offs between numbers and size of spermatozoa because increased costs per cell would result in a reduction of sperm number if both traits share the same energetic budget. Theoretical models have proposed that, in large animals, increased sperm size would have minimal fitness advantage compared with increased sperm numbers. Thus, sperm numbers would evolve more rapidly than sperm size under sperm competition pressure. We tested in mammals whether sperm competition maximizes sperm numbers and size, and whether there is a trade‐off between these traits. Our results showed that sperm competition maximizes sperm numbers in eutherian and metatherian mammals. There was no evidence of a trade‐off between sperm numbers and sperm size in any of the two mammalian clades as we did not observe any significant relationship between sperm numbers and sperm size once the effect of sperm competition was taken into account. Maximization of both numbers and size in mammals may occur because each trait is crucial at different stages in sperm's life; for example size‐determined sperm velocity is a key determinant of fertilization success. In addition, numbers and size may also be influenced by diverse energetic budgets required at different stages of sperm formation.     

Conditions for the evolution of soldier sperm classes

Abstract.— There has been wide disagreement as to whether sperm competition among animals can produce a soldier class of sperm to fight against other males. Utilizing mathematical models, we analyze the appropriate conditions for the evolution and maintenance of a soldier sperm class. We conclude that: (1) soldier sperm evolve even if one soldier sperm can kill or block less than one competing sperm; (2) soldier sperm evolve faster when there is a large variance in the number of competing sperm; (3) soldier ratio increases until reproductive sperm are too scarce to fertilize all ova or a sperm intensely refuses to become a soldier; and (4) soldier sperm are more likely to be smaller than reproductive sperm. Our models suggest that the conditions for the evolution of a soldier sperm class are not stringent.     

Experimental evidence for the evolution of numerous,tiny sperm via sperm competition

Sperm competition, when sperm from different males compete to fertilize a female's ova, is a widespread and fundamental force in the evolution of animal reproduction. The earliest prediction of sperm competition theory was that sperm competition selected for the evolution of numerous, tiny sperm, and that this force maintained anisogamy. Here, we empirically test this prediction directly by using selective breeding to generate controlled and independent variance in sperm size and number traits in the cricket Gryllus bimaculatus. We find that sperm size and number are male specific and vary independently and significantly. We can therefore noninvasively screen individuals and then run sperm competition experiments between males that differ specifically in sperm size and number traits. Paternity success across 77 two-male sperm competitions (each running over 30-day oviposition periods) shows that males producing both relatively small sperm and relatively numerous sperm win competitions for fertilization. Decreased sperm size and increased sperm number both independently predicted sperm precedence. Our findings provide direct experimental support for the theory that sperm competition selects for maximal numbers of miniaturized sperm. However, our study does not explain why G. bimaculatus sperm length persists naturally at approximately 1 mm; we discuss possibilities for this sperm size maintenance.     

Sperm competition in fishes: the evolution of testis size and ejaculate characteristics

Fishes show one of the widest ranges of sperm competition intensity of any animal group. Here we present a comparative study whose aim is to investigate the effect of relative intensity of sperm competition on investment in spermatogenesis and the number and size of sperm produced. We find that both the gonadosomatic index (GSI = [gonad weight/body weight] x 100) and sperm numbers increase with intensity of sperm competition across species but that sperm length decreases. These new findings are consistent with a raffle-based mode of sperm competition in fishes. Most of these results (positive correlation of the GSI and sperm number with sperm competition intensity) concur with the predictions of current sperm competition theory. However, we also find that sperm longevity decreases with sperm length across species. Current models for continuous fertilization suggest that if length increases a sperm's speed but decreases its longevity, sperm length should increase with sperm competition intensity, whereas models for instant fertilization suggest that sperm length should remain constant. The negative relationship found between sperm competition and sperm length therefore does not fit predictions of either model.     

The evolution of sperm morphometry in pheasants

Post-copulatory sexual selection is thought to be a potent evolutionary force driving the diversification of sperm shape and function across species. In birds, insemination and fertilization are separated in time and sperm storage increases the duration of sperm-female interaction and hence the opportunity for sperm competition and cryptic female choice. We performed a comparative study of 24 pheasant species (Phasianidae, Galliformes) to establish the relative importance of sperm competition and the duration of sperm storage for the evolution of sperm morphometry (i.e. size of different sperm traits). We found that sperm size traits were negatively associated with the duration of sperm storage but were independent of the risk of sperm competition estimated from relative testis mass. Our study emphasizes the importance of female reproductive biology for the evolution of sperm morphometry particularly in sperm-storing taxa.     

Species specificity of hybridoma antibodies to surface antigens of guinea pig sperm

The species specificity of hybridoma antibodies to sperm surface antigens was studied. A collection of over 50 hybridoma antibodies that bind to the guinea pig sperm surface was tested for binding to mouse, rat, hamster, and human sperm by indirect immunofluorescence. None of the antibodies bind to mouse sperm. rat sperm, or human sperm. All but three of the antibodies also fail to bind to hamster sperm. AH-30, AH-31, and AH-1032, the three antibodies that crossreact with hamster sperm, show a different topographical localization on hamster sperm from that seen on guinea pig sperm. The three antibodies do not precipitate a ¹²⁵I surface-labeled antigen from hamster sperm extracts. However, from guinea pig sperm extracts, all three antibodies precipitate ¹²⁵I surface-labeled polypeptides with molecular weights (M_r) of 62,000, 52,000, and 38,000. This result suggests that the crossreacting antibodies may be recognizing different antigens on hamster and guinea pig sperm.     

Helical nature of sperm swimming affects the fit of fertilization-kinetics models to empirical data

Models of fertilization kinetics rely upon estimates of the swimming velocity of sperm to predict collision rates between egg and sperm. Most investigators measure sperm swimming velocity without accounting for the helical motion of sperm, thereby obtaining an inflated estimate of the velocity with which sperm approach eggs. In turn, models of fertilization predict inflated rates of sperm/egg collision. I observed sea urchin sperm colliding with eggs, quantified the rate of sperm/egg collision, and measured sperm velocity as a component of the helix through which they swim. I also adjusted the "target size" of eggs to reflect the diameter of the helix. My estimate of sperm swimming velocity is an order of magnitude lower than other estimates for the same species. By using helical parameters in fertilization kinetics models and accounting for dead sperm in laboratory trials, I was able to accurately predict lower rates of sperm/egg collision. Moreover, making these adjustments in the model increased the estimated proportion of sperm that initiate fertilization by 6- to 7-fold, suggesting that a better understanding of sperm swimming might lead to a more complete understanding of fertilization biology and natural selection on gamete traits.     

Motility and dynamics of eupyrene and apyrene sperm in the spermatheca of the monandrous swallowtail butterfly Byasa alcinous

Lepidopteran males produce two sperm types: nucleated eupyrene sperm and non‐nucleated apyrene sperm. Although apyrene sperm are infertile, both sperm types migrate from the spermatophore to the spermathecal after copulation. As a dominant adaptive explanation for migration of apyrene sperm in polyandrous species, the cheap filler hypothesis suggests that the presence of a large number of motile apyrene sperm in the spermatheca reduces female receptivity to re‐mating. However, apyrene sperm are also produced in males of the monandrous swallowtail butterfly Byasa alcinous Klug. To identify the role of apyrene sperm in these males, the present study examines the number of spermatozoa produced and transferred and the dynamics and motility of spermatozoa in the spermatheca for each type of sperm. Apyrene sperm represents approximatey 89% of the sperm produced and transferred, which is comparable to polyandrous species. Two‐day‐old males transfer approximately 17 000 eupyrene and 230 000 apyrene spermatozoa to a spermatophore; approximately 5000 eupyrene and 47 000 apyrene spermatozoa arrive at the spermatheca. Eight days after copulation, most eupyrene spermatozoa remain in the spermatheca and a quarter of them are still active. However, the number of apyrene spermatozoa decreases and those remaining lose their motility after the arriving at the spermatheca. Consequently, 8 days after copulation, no motile apyrene sperm are found. The high proportion of apyrene sperm in the spermatophore, as well as in sperm migration, suggests that the production and migration of apyrene sperm is not simply an evolutionary vestigial trait. The possible functions of apyrene sperm in monandrous species are discussed.     

Sperm competition and the evolution of the sperm hook in house mice

Sperm morphology varies considerably both between and within species. The sperm of many muroid rodents bear an apical hook at the proximal end of the head. The curvature of the sperm hook varies greatly across species, however the adaptive significance of the sperm hook is currently not known. In wood mice the apical hooks intertwine to form sperm ‘trains’, which exhibit faster swimming velocities than single cells. Thus, it has been suggested that if sperm ‘trains’ were advantageous in a competitive situation, then the apical sperm hook might be an evolutionary product of selection via sperm competition. A comparative study of rodent species provided support for the hypothesis, and showed that species with higher levels of sperm competition had more reflected sperm hooks. Here, we tested this hypothesis at the intraspecific level. We quantified sperm hook morphology from seven house mouse populations, and found that interpopulation variation in hook curvature was not explained by variation in sperm competition risk. Furthermore, observations of ejaculated sperm revealed that sperm groups are not a common characteristic of mouse ejaculates. We suggest that selection for sperm attachment to the oviduct epithelium, and thus better retainment of sperm fertilizing potential, may provide a more general explanation of the evolutionary relationship between sperm competition risk and the curvature of the sperm hook among rodents, and provide a phylogenetic comparison among rodent species that supports our hypothesis.     

Effect of hormone implantation on cryopreservation of Atlantic halibut (Hippoglossus hippoglossus L.) sperm

Hormone implantation is widely applied in halibut (Hippoglossus hippoglossus L.) aquaculture to extend the sperm production season of broodstock males. The ability to combine this technique with cryopreservation would increase sperm availability, thereby improving reproduction success and facilitating gene management. In this paper, the cryopreservation ability of sperm from hormone-treated males was examined at three times post-implantation and compared with that of sperm from males that were not hormone-treated. All sperm samples were cryopreserved using the same method. The effectiveness of these techniques was assessed by examining the fertilization rate and motility of thawed sperm. The spermotocrit and concentration of fresh sperm samples were measured to reveal the effect of hormone implantation on sperm characteristics. The reported results indicate that hormone implantation did not affect cryopreservation efficiency. The fertilization rate resulting from thawed sperm of hormone-treated males showed no significant difference from that of untreated males or from fresh sperm. A significant positive relationship was demonstrated between the spermatocrit and concentration of sperm; and a significant decrease of spermatocrit was found in sperm collected from hormone-treated males 14days post-implantation. No significant linear relationship between spermotocrit and fertilization rate of thawed sperm was shown.     

A model of constant random sperm displacement during mating: evidence from Scatophaga.

This paper extends the sperm displacement model of Parker et al. (Behav. Ecol. Sociobiol. 27, 55 (1990)), in which sperm displacement is viewed as a process in which one unit of sperm introduced displaces one unit of sperm from the female's sperm stores. Here this process is envisaged in terms of the change in density of sperm in the sperm stores. In matings with virgin females, only sperm store fluid is displaced at the start of sperm transfer, but if there is swift random mixing of seminal and sperm store fluid, the fluid displaced will contain sperm at the same average density as that in the sperm stores (random displacement). In mating of the same female by two or more males, the sperm density of the last male to mate is assumed to be independent of the presence of previous sperm; P2 (the proportion of eggs fertilized by the last male) thus equals the density of the last male's sperm divided by the current total density of sperm in the sperm stores. Once the sperm stores have reached the asymptotic density (equivalent to the input density, i.e. the density of sperm in the seminal fluid), the present model becomes equivalent to that of Parker et al. (1990). Predictions for this model are tested using all available data from the dung fly, Scatophaga stercoraria. They are based on the assumption that sperm are transferred at a constant rate with copulation time. The data concur with this model, and we conclude that it is better than various other simple alternatives for explaining P2 in Scatophaga.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ability of sulfated glycoconjugates and disulfide-reductants to release bovine epididymal sperm bound to the oviductal epithelium in vitro

In Bos taurus, at ejaculation, epididymal sperm acquire a number of proteins secreted in the seminal plasma that increase their ability to interact with the female reproductive tract. Sperm-oviduct interaction comprises a transient sperm adhesion to the isthmus, the lower portion of the oviduct, followed by sperm release around ovulation. Oviductal fluid molecules, such as sulfated glycoconjugates and disulfide-reductants, are able to release bovine ejaculated sperm bound to the oviductal epithelium in vitro through the reduction of sperm surface protein disulfides to sulfhydryls. To understand whether the sperm molecules sensitive to releasing signals are already exposed on the surface of epididymal sperm, we studied the ability of cauda epididymal sperm to adhere to the oviductal epithelium and to be released by sulfated glycoconjugates and the disulfide-reductant penicillamine. Surface protein sulfhydryls in cauda epididymal sperm were analyzed in the initial suspension, in sperm bound to the in vitro-cultured oviductal epithelium, and in released sperm. Results showed that epididymal sperm are able to bind the oviductal epithelium in vitro, although at a lower extent than frozen-thawed ejaculated sperm; the interaction is mediated by oviductal cell microvilli that closely bind to the plasma membrane of the sperm head rostral region, as previously shown for ejaculated sperm. The sulfated glycoconjugates heparin, fucoidan, and dextran sulfate, as well as the disulfide-reductant penicillamine, are all powerful inducers of sperm release. The level of sulfhydryls in sperm surface proteins was (1) high in the initial sperm suspension; (2) low in bound sperm; (3) markedly increased in sperm released by heparin or by penicillamine. In conclusion, epididymal sperm are already able to bind the oviductal epithelium and to respond to the inducers of release through the reduction of sperm surface protein disulfides to sulfhydryls.