Sperm mobility: mechanisms of fertilizing efficiency, genetic variation and phenotypic relationship with male status in the domestic fowl, Gallus gallus domesticus

When females are sexually promiscuous, sexual selection continues after insemination through sperm competition and cryptic female choice, and male traits conveying an advantage in competitive fertilization are selected for. Although individual male and ejaculate traits are known to influence paternity in a competitive scenario, multiple mechanisms co-occur and interact to determine paternity. The way in which different traits interact with each other and the mechanisms through which their heritability is maintained despite selection remain unresolved. In the promiscuous fowl, paternity is determined by the number of sperm inseminated into a female, which is mediated by male social dominance, and by the quality of the sperm inseminated, measured as sperm mobility. Here we show that: (i) the number of sperm inseminated determines how many sperm reach the female sperm-storage sites, and that sperm mobility mediates the fertilizing efficiency of inseminated sperm, mainly by determining the rate at which sperm are released from the female storage sites, (ii) like social status, sperm mobility is heritable, and (iii) subdominant males are significantly more likely to have higher sperm mobility than dominant males. This study indicates that although the functions of social status and sperm mobility are highly interdependent, the lack of phenotypic integration of these traits may maintain the variability of male fitness and heritability of fertilizing efficiency.     

Sperm mobility determines the outcome of sperm competition in the domestic fowl

The aim of this study was to establish whether the mobility of sperm of the domestic fowl, as measured by an in vitro assay, predicted the outcome of sperm competition. Thirteen pairs of New Hampshire roosters, comprising one male categorized as having high-mobility sperm and the other as having average-mobility sperm, were used. Each male provided 25 x 10(6) sperm, which were mixed and artificially inseminated into between four and seven New Hampshire hens, each of which produced 2-11 offspring. The experiment was conducted twice, such that the same pair of males inseminated the same females. Paternity was assigned by using microsatellite markers. There was a clear effect of sperm-mobility phenotype on the outcome of sperm competition: in all 13 pairs the high-mobility male fathered the majority of offspring (75.3% overall; p < 0.0001). The proportion of offspring fathered by the high-mobility male within pairs varied significantly between male pairs (p < 0.0005). This effect was associated with the difference in sperm-mobility scores between males within pairs; there was a significant positive relationship between the proportion of offspring fathered by the high-mobility male and the ratio of mobility scores between males (p < 0.05). In addition, compared with their success predicted from the non-competitive situation, in the competitive situation high-mobility males were disproportionately successful in fertilizing eggs compared with average-mobility males. This may occur because female sperm storage is limited in some way and a greater proportion of high-mobility sperm gain access to the female's sperm storage tubules. There was no evidence that female effects accounted for any of the variation in paternity.     

Random sperm use and genetic effects on worker caste fate in Atta colombica leaf-cutting ants

Sperm competition can produce fascinating adaptations with far-reaching evolutionary consequences. Social taxa make particularly interesting models, because the outcome of sexual selection determines the genetic composition of groups, with attendant sociobiological consequences. Here, we use molecular tools to uncover some of the mechanisms and consequences of sperm competition in the leaf-cutting ant Atta colombica, a species with extreme worker size polymorphism. Competitive PCR allowed quantification of the relative numbers of sperm stored by queens from different males, and offspring genotyping revealed how sperm number translated into paternity of eggs and adult workers. We demonstrate that fertilization success is directly related to sperm numbers, that stored sperm are well-mixed and that egg paternity is constant over time. Moreover, worker size was found to have a considerable genetic component, despite expectations that genetic effects on caste fate should be minor in species with a low degree of polyandry. Our data suggest that sexual conflict over paternity is largely resolved by the lifetime commitment between mates generated by long-term sperm storage, and show that genetic variation for caste can persist in societies with comparatively high relatedness.     

Identification of embryo paternity using polymorphic DNA markers to assess fertilizing capacity of spermatozoa after heterospermic insemination in boars

Differences in sperm fertilizing capacity of males often remain undetected by routine semen parameters. Heterospermic insemination with equal numbers of spermatozoa from 2 males is an accurate method for assessing differences in fertility. Use of heterospermic insemination depends on a reliable, efficient assay to identify paternity of conceptuses or offspring. In this study, polymorphic DNA markers amplified by PCR were tested to determine paternity of Day 5 to 6 embryos. The fertilizing capacity of 2 boars (A and B) with similar semen parameters was compared after homospermic (n=14 gilts) and heterospermic (n=11 gilts) insemination. Single AI's were performed under suboptimal conditions using 1 x 10(9) spermatozoa at 12 to 24 h before ovulation to prompt differences in fertilization and to stimulate sperm competition. The fertilization rate and the number of accessory spermatozoa were determined in Day 5 to 6 embryos. Using 5 different polymorphic DNA markers, paternity could be determined in 95.8% of the embryos. Boar B sired significantly (P<0.05) more offspring than Boar A after insemination with pooled semen, and this was reflected by a significantly (P<0.05) higher number of accessory spermatozoa following homospermic insemination with semen from Boar B, although fertilization rates did not differ between the 2 boars after homospermic insemination. The results suggest that the viability of spermatozoa in the female reproductive tract contributes to differences in fertility rates of males with similar in vitro sperm quality parameters. The number of accessory spermatozoa is a more sensitive measure of boar fertility than the fertilization rate. Polymorphic DNA markers are suitable for verification of parentage even at a very early stage of embryonic development.     

FORMATION OF THE CORTICAL CONCAVITY AT FERTILIZATION IN THE SEA URCHIN EGG

During the initial stages of fertilization envelope elevation in eggs of Strongylocentrotus pur puratus and S. droebachiensis a large concavity of the egg cortex was observed in the light microscope. This concavity corresponded in shape and size with the elevating fertilization envelope. However, after the vitelline layers of eggs were disrupted and the eggs inseminated, the concavity failed to develop although the eggs were fertilized and developed normally. We propose that the concavity is formed owing to increased hydrostatic pressure within the perivitelline space. To further support this hypothesis we measured total egg protein secreted during fertilization, and found that 98% was retained within the perivitelline space. Furthermore, 80% of the total protein was contributed by the hyaline layer. Presumably, colloidal osmotic pressure and/or hydration of fertilization product, trapped beneath the fertilization envelope, is responsible for increased hydrostatic pressure within the perivitelline space, and therefore promotes not only fertilization envelope elevation, but the cortical concavity as well.     

Quantitative aspects of sperm:egg interaction in chickens and turkeys

Spermatozoa embedded in the outer perivitelline layer and points of hydrolysis (holes) produced by spermatozoa in the inner perivitelline layer of chicken and turkey eggs were found to be evenly distributed and linearly correlated (r = 0.80 for both species) throughout the layers from most regions of the egg, except from those directly over the germinal disc, in which there were more holes. In turkey eggs there appeared to be relatively fewer perivitelline spermatozoa, since many had degenerated beyond recognition. In eggs from both species, there were approximately 25 times more holes mm⁻² in the inner perivitelline layer from over the germinal disc region than that from other regions of the egg. The relationship between these two frequencies could also be described as linear (r = 0.81 for chicken and 0.78 for turkey eggs), although there was some evidence for a saturation effect for holes over the germinal disc. The fertile status of eggs was shown to be a function of all of the above parameters. Eggs from both species had a 50% probability of being fertile when around 3 spermatozoa penetrated the inner perivitelline layer over the germinal disc and showed maximum fertility when more than 6 spermatozoa penetrated this region. Spermatozoa in the outer perivitelline layer and holes in the inner perivitelline layer from regions other than over the germinal disc could also be used to predict fertility, although with less certainty. Since the number of spermatozoa interacting with the egg reflects the numbers of those stored in the uterovaginal sperm storage tubules, the relationships derived in this work should be useful for understanding how fertility in chickens and turkeys is a function of oviducal sperm storage and transport.     

Artificial insemination using cryopreserved sperm in the silkworm,Bombyx mori

We report in this paper that female moths artificially inseminated with cryopreserved sperm (-196 degrees C) could oviposit eggs when the sperm was preserved for 356days, and that the fertilization rate and the number of eggs laid were almost equivalent to those obtained in normally mated moths. The optimal cooling rate for sperm freezing was 5-65 degrees C/min for maintaining a high fertility of sperm. The simple and reliable method of cryopreservation was to put the semen first in a deep freezer at -80 degrees C and thereafter put them in liquid nitrogen. When female moths of 'white 2' egg-color mutant strain were inseminated with a mixture of frozen-thawed sperm from males of normal-colored egg strain and non-frozen sperm from males of the 'white 2', female moths deposited a majority of 'white 2' eggs and a very small number of eggs of normal color. The result shows that there was a competitive fertilization of sperm between the two strains of the silkworm, and that sperm fertility was reduced to a considerable extent by freezing at -196 degrees C. These results may contribute not only to basic studies on fertilization in Lepidoptera but also to the development of long-term preservation procedure of genetic resources by using cryopreserved sperm of Bombyx mori.     

Delineating the roles of males and females in sperm competition

Disentangling the relative roles of males, females and their interactive effects on competitive fertilization success remains a challenge in sperm competition. In this study, we apply a novel experimental framework to an ideally suited externally fertilizing model system in order to delineate these roles. We focus on the chinook salmon, Oncorhynchus tshawytscha, a species in which ovarian fluid (OF) has been implicated as a potential arbiter of cryptic female choice for genetically compatible mates. We evaluated this predicted sexually selected function of OF using a series of factorial competitive fertilization trials. Our design involved a series of 10 factorial crosses, each involving two ‘focal’ rival males whose sperm competed against those from a single ‘standardized’ (non-focal) rival for a genetically uniform set of eggs in the presence of OF from two focal females. This design enabled us to attribute variation in competitive fertilization success among focal males, females (OF) and their interacting effects, while controlling for variation attributable to differences in the sperm competitive ability of rival males, and male-by-female genotypic interactions. Using this experimental framework, we found that variation in sperm competitiveness could be attributed exclusively to differences in the sperm competitive ability of focal males, a conclusion supported by subsequent analyses revealing that variation in sperm swimming velocity predicts paternity success. Together, these findings provide evidence that variation in paternity success can be attributed to intrinsic differences in the sperm competitive ability of rival males, and reveal that sperm swimming velocity is a key target of sexual selection.     

Quantification of spermatozoa in the sperm-storage tubules of turkey hens and the relation to sperm numbers in the perivitelline layer of eggs

This study was conducted to determine the number of spermatozoa residing in the oviduct sperm-storage tubules (SST) and the relationship between these numbers and the number of spermatozoa embedded in the perivitelline layer of oviductal eggs after a single insemination of 200 x 10(6) spermatozoa. The SST of hens inseminated within one week before the expected onset of egg production were filled faster (4 h vs. 2 days) and possessed more spermatozoa (4.1 vs. 2.0 x 10(6)) than the SST of hens inseminated after the onset of egg production. Furthermore, for hens in egg production, significantly fewer spermatozoa were recovered from the SST if the hen was inseminated within 2 h before or after oviposition than if inseminated more than 2 h before or after the oviposition. There was a strong positive correlation between the number of spermatozoa in the SST and the number of spermatozoa embedded in the perivitelline layer of the oviductal eggs (r = 0.85, p less than 0.01). These data show that the population of spermatozoa actually accepted by the SST is quite small relative to the number of spermatozoa inseminated and that maximum sperm-storage is achieved when the hen is inseminated just prior to the onset of egg production. It is suggested that the sperm-storage capacity of the oviduct and the quality of the semen sample can be estimated on the basis of numbers of spermatozoa embedded in the egg perivitelline layer.     

Determinants of paternity in a butterfly

Success in sperm competition is of fundamental importance to males, yet little is known about what factors determine paternity. Theory predicts that males producing high sperm numbers have an advantage in sperm competition. Large spermatophore size (the sperm containing package) also correlates with paternity in some species, but the relative importance of spermatophore size and sperm numbers has remained unexplored. Males of the small white butterfly, Pieris rapae (Lepidoptera: Pieridae), produce large nutritious spermatophores on their first mating. On their second mating, spermatophores are only about half the size of the first, but with almost twice the sperm number. We manipulated male mating history to examine the effect of spermatophore size and sperm numbers on male fertilization success. Overall, paternity shows either first male or, more frequently, second male sperm precedence. Previously mated males have significantly higher fertilization success in competition with males mating for the first time, strongly suggesting that high sperm number is advantageous in sperm competition. Male size also affects paternity with relatively larger males having higher fertilization success. This may indicate that spermatophore size influences paternity, because in virgin males spermatophore size correlates with male size. The paternity of an individual male is also inversely correlated with the mass of his spermatophore remains dissected out of the female. This suggests that females may influence paternity by affecting the rate of spermatophore drainage. Although the possibility of female postcopulatory choice remains to be explored, these results clearly show that males maximize their fertilization success by increasing the number of sperm in their second mating.     

Resolving mechanisms of short-term competitive fertilization success in the red flour beetle

Postcopulatory sexual selection occurs when sperm from multiple males occupy a female’s reproductive tract at the same time and is expected to generate strong selection pressures on traits related to competitive fertilization success. However, knowledge of competitive fertilization success mechanisms and characters targeted by resulting selection is limited, partially due to the difficulty of discriminating among sperm from different males within the female reproductive tract. Here, we resolved mechanisms of competitive fertilization success in the promiscuous flour beetle Tribolium castaneum. Through creation of transgenic lines with fluorescent-tagged sperm heads, we followed the fate of focal male sperm in female reproductive tracts while tracking paternity across numerous rematings. Our results indicate that a given male’s sperm persist and fertilize eggs through at least seven rematings. Additionally, the proportion of a male’s sperm in the bursa (the site of spermatophore deposition), which is influenced by both timing of female’s ejecting excess sperm and male size, significantly predicted paternity share in the 24 h following a mating. Contrary to expectation, proportional representation of sperm within the female’s specialized sperm-storage organ did not significantly predict paternity, though spermathecal sperm may play a role in fertilization when females do not have access to mates for longer time periods. We address the adaptive significance of the identified reproductive mechanisms in the context of T. castaneum’s unique mating system and ecology.     

Effects of ovarian fluid and genetic differences on sperm performance and fertilization success of alternative reproductive tactics in Chinook salmon

In many species, sperm velocity affects variation in the outcome of male competitive fertilization success. In fishes, ovarian fluid (OF) released with the eggs can increase male sperm velocity and potentially facilitate cryptic female choice for males of specific phenotypes and/or genotypes. Therefore, to investigate the effect of OF on fertilization success, we measured sperm velocity and conducted in vitro competitive fertilizations with paired Chinook salmon (Oncorhynchus tshawytscha) males representing two alternative reproductive tactics, jacks (small sneaker males) and hooknoses (large guarding males), in the presence of river water alone and OF mixed with river water. To determine the effect of genetic differences on fertilization success, we genotyped fish at neutral (microsatellites) and functional [major histocompatibility complex (MHC) II ß1] markers. We found that when sperm were competed in river water, jacks sired significantly more offspring than hooknoses; however, in OF, there was no difference in paternity between the tactics. Sperm velocity was significantly correlated with paternity success in river water, but not in ovarian fluid. Paternity success in OF, but not in river water alone, was correlated with genetic relatedness between male and female, where males that were less related to the female attained greater paternity. We found no relationship between MHC II ß1 divergence between mates and paternity success in water or OF. Our results indicate that OF can influence the outcome of sperm competition in Chinook salmon, where OF provides both male tactics with fertilization opportunities, which may in part explain what maintains both tactics in nature.     

The evolution of polyandry: an examination of the genetic incompatibility and good‐sperm hypotheses

I have examined the adaptive significance of polyandry using the Australian field cricket Teleogryllus oceanicus. Previous studies of polyandry have examined differences in offspring production by females mated multiply to a single male or females mated multiply to different males. Here I combine this approach with a study of parentage of offspring produced in the later group. Females mated to two different males had a higher proportion of their eggs hatching than did females mating twice with a single male. Offspring fitness parameters were not effected. There was little evidence to suggest that females elevate their hatching success via fertilizing their eggs with sperm from genetically compatible males. Although the average paternity points towards random sperm mixing, there was considerable individual variation in sperm competition success. Patterns of parentage were consistent across females mating twice or four times. Sperm competition success was not related to offspring viability or performance. Thus, the notion that competitively superior sperm produce competitively superior offspring is not supported either. The mechanism underlying increased hatching success with polyandry requires further study.     

An ultrastructural study of epididymal mouse spermatozoa binding to zonae pellucidae in vitro: sequential relationship to the acrosome reaction

Mouse sperm bind to the zona pellucida of the egg prior to penetration of the zona and entry into the perivitelline space. The question then arises: when does the acrosome reaction occur relative to these processes? An ultrastructural study of mouse epididymal sperm bound to the surface of the zona and in the privitelline space was undertaken to clarify this point. Cumulus-free mouse eggs were inseminated in either a complete defined culture medium capable of supporting in vitro fertilization or in Tris/NaCl buffer containing Ca+2. Both media support sperm binding to the zona to the same extent; binding is complete in 15 minutes. Unbound sperm were removed by a step gradient density centrifugation to yield a preparation of eggs with sperm firmly bound. All sperm in the perivitelline space had undergone the acrosome reaction. Sperm bound at the surface of the zonae pellucidae of eggs recovered at ten minutes after insemination all had intact acrosomes. At 40 minutes after insemination, half of the sperm were intact; the other half were in the initial stages of the acrosome reaction. At 90 minutes after insemination, 12% of the sperm had undergone the full acrosome reaction and were starting to penetrate the zona; of the balance, half were in various stages of the acrosome reaction, while half were still intact. These findings support the hypothesis that the sequence of the early reactions leading to fertilization in the mouse is: intact sperm binding to zona; acrosome reaction at the zona surface; penetration of the zona.     

Sperm competition in a fish with external fertilization: the contribution of sperm number, speed and length

The role of sperm number and quality in male competitiveness was investigated using in vitro fertilization experiments with bluegill (Lepomis macrochirus). Bluegill males use one of three mating tactics: 'sneakers', which streak spawn; 'satellites', which mimic females; and 'parentals', which are territorial. The in vitro experiments mimicked natural spawning by incorporating these males' mean proximity to eggs and timing of sperm release. Using a maximum-likelihood algorithm, raffle equations were fit to paternity data, which revealed a strong effect of sperm number on male competitiveness. There was no difference in sperm flagellum length, curvilinear swim speed or path linearity among the three male mating types, and these traits did not explain any additional variation in male competitiveness. It was estimated that, given closer proximity to eggs, satellites need release only 0.34 times as many sperm as parentals to obtain equal paternity. Despite being farther from the eggs and releasing sperm about half a second after parentals, sneakers need only release 0.58 times as many sperm as parentals to obtain equal paternity. Thus, the increased competitiveness of sneakers' sperm must come from a component of sperm quality other than speed or length.     

The sperm entry site during fertilization of the zebrafish egg: localization of actin.

The sperm entry site (SES) of zebrafish (Brachydanio rerio) eggs was studied before and during fertilization by fluorescence, scanning, and transmission electron microscopy. Rhodamine phalloidin (RhPh), used to detect polymerized filamentous actin, was localized to microvilli of the SES and to cytoplasm subjacent to the plasma membrane in the unfertilized egg. The distribution of RhPh staining at the SES correlated with the ultrastructural localization of a submembranous electrondense layer of cortical cytoplasm approximately 500 nm thick and containing 5- to 6-nm filaments. Actin, therefore, was organized at the SES as a tightly knit meshwork of filaments prior to fertilization. Contact between the fertilizing sperm and the filamentous actin network was observed by 15-20 sec postinsemination or just before the onset of fertilization cone formation. Growing fertilization cones of either artificially activated or inseminated eggs exhibited intense RhPh staining and substantial increase in thickness of the actin meshwork. Collectively, TEM and RhPh fluorescence images of inseminated eggs demonstrated that the submembranous actin became rearranged in fertilization cones to form a thickened meshwork around the sperm nucleus during incorporation. The results reported here suggest that activation of the egg triggers a dramatic polymerization of actin beneath the plasma membrane of the fertilization cone. Furthermore, the actin involved in sperm incorporation is sensitive to the action of cytochalasins.     

Multiple sperm storage organs facilitate female control of paternity

It has been proposed that multiple sperm storage organs (spermathecae) could allow polyandrous females to control paternity. There is little conclusive evidence for this since insemination of individual spermathecae is generally not experimentally manipulable. Here, we examined sperm use patterns in the Australian redback spider (Latrodectus hasselti), which has paired, independent spermathecae. We assessed paternity when two rivals were forced to inseminate a single storage organ or opposite storage organs. When males inseminated a single spermatheca, mean paternity of the female's first mate was 79.8% (median 89.4%), and 38% of first mates achieved 100% paternity. In contrast, when males inseminated opposite organs, the mean paternity of the first mate was 49.3% (median 49.9%), only 10% of males achieved complete precedence, and paternity was normally distributed, suggesting sperm mixing. Males responded to this difference by avoiding previously inseminated female reproductive tracts. Complete sperm precedence can only be achieved if females permit males to copulate with both reproductive tracts. Females often cannibalize smaller males during their first copulation, thus limiting their paternity to 50%. These data show that multiple sperm storage organs can increase female control of paternity.     

Paternity assurance before and after fertilization by male burying beetles (Nicrophorus quadripunctatus)

Parental care requires a large investment of time and energy. This can reduce future parental survival and opportunities for mating. Because males are usually more uncertain of their parentage with respect to the caring of offspring than are females, the reduction in reproductive success is thought to be greater in males. Therefore, males are under selection to ensure paternity of the offspring for which they care. Males can increase paternity before and after fertilization. Before fertilization, males can increase paternity by increasing their competitive ability for fertilization. After fertilization, males can increase paternity by cannibalizing unrelated offspring. Here, we investigated the stage at which male burying beetles, Nicrophorus quadripunctatus, increase their paternity by evaluating the number of offspring sired by a nursing male in asynchronously hatched broods in relation to hatching time. We found that nursing males assure a very high level of the paternity of hatching offspring. We also found that the paternity of non-nursing and nursing males remained constant across hatching time within a brood, indicating that it is unlikely that filial cannibalism plays a role in increasing the paternity of offspring. We concluded that ensuring paternity before fertilization is more important in increasing the paternity of offspring.     

The outermost layer of egg-jelly is crucial to successful fertilization in the newt, Cynops pyrrhogaster

The significance of egg-jelly layers in internal fertilization was evaluated in the newt, Cynops pyrrhogaster. In this species, six egg-jelly layers, J1, J2, J3, J4, J5 and the outermost J6 layers, are accumulated on the surface of the fertilizable eggs in pars convoluta of the oviduct. When a large number of sperm (about 6 x 10(5)) were placed on eggs having different numbers of jelly layers, all the eggs were fully fertilized, although many of the eggs developed abnormally. Upon insemination using about 600 sperm, only eggs with the full set of jelly layers were fertilized at a high rate with normal development. Since around 300 (the range of 48-1,192) sperm were observed on and in the egg-jelly in naturally spawned eggs, we conclude that the J6 layer must be present on the outermost surface of the egg-jelly for successful internal fertilization of the newt. Previous studies have suggested that the J6 layer is a prerequisite for the initiation of sperm motility and the acrosome reaction. In the present study, the fertilization rate decreased in eggs with a full set of jelly layers when inseminated using acrosome-reacted and motile sperm. However, the fertilization rate was high when motile sperm with intact acrosome was used. These results suggest that induction of the sperm acrosome reaction in the J6 layer is an important step in the internal fertilization of the newt.     

Repeated inseminations required for natural fertility in a wild bird population

In most bird species, pairs copulate many times before egg laying. The exact function of repeated inseminations (i.e. successful copulations) is unknown, but several suggestions have been made. We tested the hypothesis that repeated inseminations are required to ensure fertilization of eggs, by using an experimental method where free-ranging male collared flycatchers (Ficedula albicollis) were prevented from inseminating their mates. We show that egg fertility was lower when females had not copulated during the studied part of their fertile period. By counting sperm on the inner perivitelline layer of eggs, we estimated that a minimum of 86 sperm must reach the site of fertilization to ensure average fertility. Using the timing of inseminations and the numbers of sperm on successive eggs, we show that repeated copulations are necessary to achieve an average rate of fertilization of a single clutch. Our results thus provide evidence that repeated inseminations function to ensure fertilization success. We discuss possible constraints on sperm production and utilization that may have contributed to this pattern.