Rapid evolution of a primate sperm protein: relaxation of functional constraint or positive Darwinian selection?

Protamines are arginine-rich proteins that replace histones and bind sperm DNA during spermatogenesis in vertebrates. Previous studies have shown that protamine exons evolve faster than does the protamine intron. It has been suggested that this is a result of a relaxation of functional constraint. However, a more likely explanation is that the evolutionary rate of exons has been accelerated by positive Darwinian selection, because introns are generally believed to evolve in a neutral fashion. Therefore, we examined the possibility that positive selection has been acting on the protamine genes of three groups of placental mammals: primates (hominoids and Old World monkeys), rodents (mice, rats, and guinea pigs), and pecoran ruminants (deer and bovids). We found that the nucleotide substitution rate at nonsynonymous sites is significantly higher than the rate at synonymous and intron sites for protamine P1 of hominoids and Old World monkeys. This result suggests that positive selection has been operating on protamine P1 of these species. In contrast, no clear-cut evidence of positive selection was found for protamine P1 of ruminants and rodents or protamine P2 of primates. The agent of positive selection on primate protamine P1 remains unknown, though sperm competition is a possibility. Further investigations on the function and intraspecific polymorphism of this protein are needed in order to identify the selection agent.     

Sexual selection on protamine and transition nuclear protein expression in mouse species

Post-copulatory sexual selection in the form of sperm competition is known to influence the evolution of male reproductive proteins in mammals. The relationship between sperm competition and regulatory evolution, however, remains to be explored. Protamines and transition nuclear proteins are involved in the condensation of sperm chromatin and are expected to affect the shape of the sperm head. A hydrodynamically efficient head allows for fast swimming velocity and, therefore, more competitive sperm. Previous comparative studies in rodents have documented a significant association between the level of sperm competition (as measured by relative testes mass) and DNA sequence evolution in both the coding and promoter sequences of protamine 2. Here, we investigate the influence of sexual selection on protamine and transition nuclear protein mRNA expression in the testes of eight mouse species that differ widely in levels of sperm competition. We also examined the relationship between relative gene expression levels and sperm head shape, assessed using geometric morphometrics. We found that species with higher levels of sperm competition express less protamine 2 in relation to protamine 1 and transition nuclear proteins. Moreover, there was a significant association between relative protamine 2 expression and sperm head shape. Reduction in the relative abundance of protamine 2 may increase the competitive ability of sperm in mice, possibly by affecting sperm head shape. Changes in gene regulatory sequences thus seem to be the basis of the evolutionary response to sexual selection in these proteins.     

Sexual selection drives weak positive selection in protamine genes and high promoter divergence,enhancing sperm competitiveness

Phenotypic adaptations may be the result of changes in gene structure or gene regulation, but little is known about the evolution of gene expression. In addition, it is unclear whether the same selective forces may operate at both levels simultaneously. Reproductive proteins evolve rapidly, but the underlying selective forces promoting such rapid changes are still a matter of debate. In particular, the role of sexual selection in driving positive selection among reproductive proteins remains controversial, whereas its potential influence on changes in promoter regions has not been explored. Protamines are responsible for maintaining DNA in a compacted form in chromosomes in sperm and the available evidence suggests that they evolve rapidly. Because protamines condense DNA within the sperm nucleus, they influence sperm head shape. Here, we examine the influence of sperm competition upon protamine 1 and protamine 2 genes and their promoters, by comparing closely related species of Mus that differ in relative testes size, a reliable indicator of levels of sperm competition. We find evidence of positive selection in the protamine 2 gene in the species with the highest inferred levels of sperm competition. In addition, sperm competition levels across all species are strongly associated with high divergence in protamine 2 promoters that, in turn, are associated with sperm swimming speed. We suggest that changes in protamine 2 promoters are likely to enhance sperm swimming speed by making sperm heads more hydrodynamic. Such phenotypic changes are adaptive because sperm swimming speed may be a major determinant of fertilization success under sperm competition. Thus, when species have diverged recently, few changes in gene-coding sequences are found, while high divergence in promoters seems to be associated with the intensity of sexual selection.     

Is the hook of muroid rodent's sperm related to sperm train formation?

Competition between spermatozoa of rival males to gain fertilizations has led to a wide array of modifications in sperm structure and function. Sperm cells of most muroid rodents have hook‐shaped extensions in the apical–ventral tip of the head, but the function of this structure is largely unknown. These ‘hooks’ may facilitate aggregation of spermatozoa in so‐called ‘trains’, as an adaptation to sperm competition, because sperm in trains may swim faster than free‐swimming cells. However, there is controversy regarding the role of the hook in train formation, and in relation to whether it is selected by sperm competition. We examined spermatozoa from muroid rodents with varying levels of sperm competition to assess whether (i) sperm aggregates are common in these taxa, (ii) presence of a hook relates to the formation of sperm aggregations, and (iii) formation of sperm aggregations is explained by sperm competition. Our analyses in 25 muroid species revealed that > 92% of spermatozoa swim individually in all species, with the exception of the wood mouse, Apodemus sylvaticus, which has ~50% spermatozoa swimming freely. Species with hooked spermatozoa had higher sperm competition levels and longer sperm than species whose sperm lack a hook. Neither the presence of hook nor sperm competition levels were related to the percentage of sperm in aggregations. Thus, (i) sperm aggregates in muroid rodents are an exceptional trait found only in a few species, (ii) evolution of the sperm hook is associated to sperm competition levels, but (iii) the hook is unlikely to be related to the formation of sperm aggregates. The evolutionary significance of the sperm head hook thus remains elusive, and future studies should examine potential roles of this pervasive structure in sperm's hydrodynamic efficiency and sperm–female tract interactions.     

The molecular evolution of sperm zonadhesin

Based on pioneering work of Hardy and Garbers, zonadhesin has become one of the best studied sperm ligands in boreoeutherian mammals, both from a biochemical and evolutionary perspective. Zonadhesin is a mosaic-type protein that localizes to the apical head of spermatozoa. In pig, cattle, rabbit and primates, zonadhesin precursor essentially consists of two or three MAM (meprin/A5 antigen/mu receptor tyrosine phosphatase) domains, one mucin-like domain, one incomplete and four complete D domains (homologous to vWFD). Mouse zonadhesin is distinguished from this general pattern by 20 extra partial D3 domains. While concerted evolution drives the divergence of the mucin-like domain in the ortholog comparison, MAM and D domains mainly diverge under the influence of drift and positive selection, both in the paralog and ortholog comparison. As can be seen particularly well within a putative binding region in the most C-terminal MAM domain, positive selection not only causes amino acid exchanges, but also promotes changes in the pattern of predicted posttranslational modification. Moving window and correlation analyses of sequence evolution and sexual body dimorphism further suggest that sexual selection, especially sperm competition, drives zonadhesin divergence. However, considering its zona pellucida avidity, female cryptic choice might as well contribute to zonadhesin evolution. Despite the general tendency for divergence of zonadhesin, conservation by negative selection dominates the evolution of most codon sites. In accordance, the distribution of EGF (epidermal growth factor)-like motifs, DP-doublets, single cysteines and CGLC motifs suggests a wide conservation of processing, folding and oligomerization of zonadhesin in pig, rabbit and primates.     

Protamine 3 shows evidence of weak, positive selection in mouse species (genus Mus)--but it is not a protamine

Protamines are short and highly basic sperm-specific nuclear proteins that replace somatic histones during spermiogenesis in a process that is crucial for sperm formation and function. Many mammals have two protamine genes (PRM1 and PRM2) located in a gene cluster, which appears to evolve fast. Another gene in this cluster (designated protamine 3 [PRM3]) encodes a protein that is conserved among mammals but that does not seem to be involved in chromatin condensation. We have compared protein sequences and amino acid compositions of protamines in this gene cluster, searched for evidence of positive selection of PRM3, and examined whether sexual selection (sperm competition) may drive the evolution of the PRM3 gene. Nucleotide and amino acid analyses of mouse sequences revealed that PRM3 was very different from PRM1 and from both the precursor and the mature sequences of PRM2. Among 10 mouse species, PRM3 showed weak evidence of positive selection in two species, but there was no clear association with levels of sperm competition. In analyses from among mammalian species, no evidence of positive selection was found in PRM3. We conclude that PRM3 exhibits several clear differences from other protamines and, furthermore, that it cannot be regarded as a true protamine.     

Ability of hamster spermatozoa to digest their own DNA

Mammalian sperm chromatin is bound by protamines into highly condensed toroids with approximately 50 kilobases (kb) of DNA. It is also organized into loop domains of about the same size that are attached at their bases to the proteinaceous nuclear matrix. In this work, we test our model that each sperm DNA-loop domain is condensed into a single protamine toroid. Our model predicts that the protamine toroids are linked by chromatin that is more sensitive to nucleases than the DNA within the toroids. To test this model, we treated hamster sperm nuclei with DNase I and found that the sperm chromatin was digested into fragments with an average size of about 50 kb, by pulse-field gel electrophoresis (PFGE). Surprisingly, we also found that spermatozoa treated with 0.25% Triton X-100 (TX) and 20 mM MgCl2 overnight resulted in the same type of degradation, suggesting that sperm nuclei have a mechanism for digesting their own DNA at the bases of the loop domains. We extracted the nuclei with 2 M NaCl and 10 mM dithiothreitol (DTT) to make nuclear halos. Nuclear matrices prepared from DNase I-treated spermatozoa had no DNA attached, suggesting that DNase I digested the DNA at the bases of the loop domains. TX-treated spermatozoa still had their entire DNA associated with the nuclear matrix, even though the DNA was digested into 50-kb fragments as revealed by PFGE. The data support our donut-loop model for sperm chromatin structure and suggest a functional role for this type of organization in that sperm can digest its own DNA at the sites of attachment to the nuclear matrix.     

EXPERIMENTAL EVOLUTION OF SPERM QUALITY VIA POSTCOPULATORY SEXUAL SELECTION IN HOUSE MICE

Individuals of many species copulate with multiple mates (polygamy). Multiple mating by females (polyandry) promotes sperm competition, which has broad implications for the evolution of the ejaculate. Multigenerational studies of polygamous insects have shown that the removal of sexual selection has profound fitness consequences for females, and can lead to an evolutionary divergence in ejaculate traits. However, the evolutionary implications of polygamous mating across successive generations have not before been demonstrated in a vertebrate. By manipulating the mating system we were able to reinstate postcopulatory sexual selection in a house mouse population that had a long history of enforced monogamy. Following eight generations of selection, we performed sperm quality assays on males from both the polygamous and monogamous selection lines. We applied a principal component analysis to summarize the variation among 12 correlated sperm traits, and found that males evolving under sperm competition had significantly larger scores on the first axis of variation, reflecting greater numbers of epididymal sperm and increased sperm motility, compared to males from lines under relaxed selection. Moreover, we found a correlated response in the size of litters born to females in lines subject to sperm competition. Thus, we present significant evidence that sperm competition has profound fitness consequences for both male and female house mice.     

Postcopulatory sexual selection is associated with accelerated evolution of sperm morphology

Rapid diversification of sexual traits is frequently attributed to sexual selection, though explicit tests of this hypothesis remain limited. Spermatozoa exhibit remarkable variability in size and shape, and studies report a correlation between sperm morphology (sperm length and shape) and sperm competition risk or female reproductive tract morphology. However, whether postcopulatory processes (e.g., sperm competition and cryptic female choice) influence the speed of evolutionary diversification in sperm form is unknown. Using passerine birds, we quantified evolutionary rates of sperm length divergence among lineages (i.e., species pairs) and determined whether these rates varied with the level of sperm competition (estimated as relative testes mass). We found that relative testes mass was significantly and positively associated with more rapid phenotypic divergence in sperm midpiece and flagellum lengths, as well as total sperm length. In contrast, there was no association between relative testes mass and rates of evolutionary divergence in sperm head size, and models suggested that head length is evolutionarily constrained. Our results are the first to show an association between the strength of sperm competition and the speed of sperm evolution, and suggest that postcopulatory sexual selection promotes rapid evolutionary diversification of sperm morphology.     

Comparative analysis of testis protein evolution in rodents

Genes expressed in testes are critical to male reproductive success, affecting spermatogenesis, sperm competition, and sperm-egg interaction. Comparing the evolution of testis proteins at different taxonomic levels can reveal which genes and functional classes are targets of natural and sexual selection and whether the same genes are targets among taxa. Here we examine the evolution of testis-expressed proteins at different levels of divergence among three rodents, mouse (Mus musculus), rat (Rattus norvegicus), and deer mouse (Peromyscus maniculatus), to identify rapidly evolving genes. Comparison of expressed sequence tags (ESTs) from testes suggests that proteins with testis-specific expression evolve more rapidly on average than proteins with maximal expression in other tissues. Genes with the highest rates of evolution have a variety of functional roles including signal transduction, DNA binding, and egg-sperm interaction. Most of these rapidly evolving genes have not been identified previously as targets of selection in comparisons among more divergent mammals. To determine if these genes are evolving rapidly among closely related species, we sequenced 11 of these genes in six Peromyscus species and found evidence for positive selection in five of them. Together, these results demonstrate rapid evolution of functionally diverse testis-expressed proteins in rodents, including the identification of amino acids under lineage-specific selection in Peromyscus. Evidence for positive selection among closely related species suggests that changes in these proteins may have consequences for reproductive isolation.     

A cost for high levels of sperm competition in rodents: increased sperm DNA fragmentation

Sperm competition, a prevalent evolutionary process in which the spermatozoa of two or more males compete for the fertilization of the same ovum, leads to morphological and physiological adaptations, including increases in energetic metabolism that may serve to propel sperm faster but that may have negative effects on DNA integrity. Sperm DNA damage is associated with reduced rates of fertilization, embryo and fetal loss, offspring mortality, and mutations leading to genetic disease. We tested whether high levels of sperm competition affect sperm DNA integrity. We evaluated sperm DNA integrity in 18 species of rodents that differ in their levels of sperm competition using the sperm chromatin structure assay. DNA integrity was assessed upon sperm collection, in response to incubation under capacitating or non-capacitating conditions, and after exposure to physical and chemical stressors. Sperm DNA was very resistant to physical and chemical stressors, whereas incubation in non-capacitating and capacitating conditions resulted in only a small increase in sperm DNA damage. Importantly, levels of sperm competition were positively associated with sperm DNA fragmentation across rodent species. This is the first evidence showing that high levels of sperm competition lead to an important cost in the form of increased sperm DNA damage.     

Why mammalian lineages respond differently to sexual selection: metabolic rate constrains the evolution of sperm size

The hypothesis that sperm competition should favour increases in sperm size, because it results in faster swimming speeds, has received support from studies on many taxa, but remains contentious for mammals. We suggest that this may be because mammalian lineages respond differently to sexual selection, owing to major differences in body size, which are associated with differences in mass-specific metabolic rate. Recent evidence suggests that cellular metabolic rate also scales with body size, so that small mammals have cells that process energy and resources from the environment at a faster rate. We develop the 'metabolic rate constraint hypothesis' which proposes that low mass-specific metabolic rate among large mammals may limit their ability to respond to sexual selection by increasing sperm size, while this constraint does not exist among small mammals. Here we show that among rodents, which have high mass-specific metabolic rates, sperm size increases under sperm competition, reaching the longest sperm sizes found in eutherian mammals. By contrast, mammalian lineages with large body sizes have small sperm, and while metabolic rate (corrected for body size) influences sperm size, sperm competition levels do not. When all eutherian mammals are analysed jointly, our results suggest that as mass-specific metabolic rate increases, so does maximum sperm size. In addition, species with low mass-specific metabolic rates produce uniformly small sperm, while species with high mass-specific metabolic rates produce a wide range of sperm sizes. These findings support the hypothesis that mass-specific metabolic rates determine the budget available for sperm production: at high levels, sperm size increases in response to sexual selection, while low levels constrain the ability to respond to sexual selection by increasing sperm size. Thus, adaptive and costly traits, such as sperm size, may only evolve under sexual selection when metabolic rate does not constrain cellular budgets.     

Variation in Apical Hook Length Reflects the Intensity of Sperm Competition in Murine Rodents

Background

Post-copulatory sexual selection has been shown to shape morphology of male gametes. Both directional and stabilizing selection on sperm phenotype have been documented in vertebrates in response to sexual promiscuity.

Methodology

Here we investigated the degree of variance in apical hook length and tail length in six taxa of murine rodents.

Conclusions

Tail sperm length and apical hook length were positively associated with relative testis mass, our proxy for levels of sperm competition, thus indicating directional post-copulatory selection on sperm phenotypes. Moreover, our study shows that increased levels of sperm competition lead to the reduction of variance in the hook length, indicating stabilizing selection. Hence, the higher risk of sperm competition affects increasing hook length together with decreasing variance in the hook length. Species-specific post-copulatory sexual selection likely optimizes sperm morphology.     

Polyandry reduces sperm length variation in social insects

Postcopulatory sexual selection, either in the form of sperm competition or cryptic female choice, is an important selective force that is thought to have generated the enormous variation in sperm morphology observed interspecifically. However, the evolutionary significance of intraspecific variation in sperm morphology, and the role that postcopulatory sexual selection plays in influencing this variation, remains poorly investigated in invertebrates. Here, we tested the hypothesis that postcopulatory sexual selection reduces variation in sperm morphology, both between and within males, in 27 species of eusocial ants and bees. These eusocial species offer an unusual opportunity to assess how selection acts on variance in sperm morphology, as haploid males produce clonal, haploid sperm that does not experience haploid-diploid conflict. We provide solid evidence that males of polyandrous ant and bee species indeed produce less-variable sperm, indicating that sperm competition selected for sperm of superior quality. Our results offer a mechanistic explanation for the evolution of high-quality sperm and provide comprehensive evidence that sperm morphology of social insects is influenced by sexual selection.     

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.     

Intraspecific variation in sperm length is negatively related to sperm competition in passerine birds

Spermatozoa are among the most diversified cells in the animal kingdom, but the underlying evolutionary forces affecting intraspecific variation in sperm morphology are poorly understood. It has been hypothesized that sperm competition is a potent selection pressure on sperm variation within species. Here, we examine intraspecific variation in total sperm length of 22 wild passerine bird species (21 genera, 11 families) in relation to the risk of sperm competition, as expressed by the frequency of extrapair paternity and relative testis size. We demonstrate, by using phylogenetic comparative methods, that between-male variation in sperm length within species is closely and negatively linked to the risk of sperm competition. This relationship was even stronger when only considering species in which data on sperm length and extrapair paternity originated from the same populations. Intramale variation in sperm length within species was also negatively, although nonsignificantly, related to sperm competition risk. Our findings suggest that postcopulatory sexual selection is a powerful evolutionary force reducing the intraspecific phenotypic variation in sperm-size traits, potentially driving the diversification of sperm morphology across populations and species.     

Comparative studies of epididymal morphology and sperm distribution in dasyurid marsupials during the breeding season

The structural features of the epididymis and the number and distribution of spermatozoa along the duct, during the breeding season, were examined in two semelparous and three iteroparous dasyurid marsupials. Total numbers of epididymal spermatozoa were extremely low in all of these species when compared with epididymal sperm numbers in most other marsupials and eutherian mammals. Although semelparous dasyurids had significantly more epididymal spermatozoa than itcroparous species, very few spermatozoa were seen in the distal cauda epididymidis of any of the species examined. This coincided with distinct changes in duct shape and the surface area of the lumen in caudal regions which resulted in a reduced sperm storage capacity in the cauda epididymidis of these species. The data suggest that, like Antechinus stuartii (Taggart & Temple-Smith, 1990a), sperm content of the ejaculates in these species will be extremely low, and that sperm motility and/or transport in the female tract is highly efficient. The functional and evolutionary significance of the reproductive strategies of semelparous and iteroparous dasyurid marsupials is still obscure and further study is needed to determine if the length of sperm storage in the female and sperm competition for storage sites is related to sperm distribution in the male and mating activities. This study does, however, clearly indicate that large numbers of spermatozoa are not required to ensure successful fertilization in either semelparous or iteroparous members of the family Dasyuridae.     

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.     

Sperm competition does not influence sperm hook morphology in selection lines of house mice

Sperm show a remarkable degree of variation in size, shape and complexity. Murine rodents exhibit a sperm head morphology that differs greatly from the ovoid shape that is characteristic of most mammals. These rodents have sperm that bear one or more apical hooks, the function of which is currently surrounded by much controversy. It has been suggested that the hook serves to facilitate the formation of sperm groups, which in some species exhibit relatively faster velocities than single cells and thus, may provide an advantage when ejaculates are competing for fertilisations. In support of this hypothesis, a comparative study reported a positive association between the strength of sperm competition (estimated from testes size) and the curvature of the sperm hook amongst 37 murine species. Here, we assessed whether sperm competition influences sperm hookedness at the intra-specific level. Following 16 generations of selection, we used geometric morphometry (GM) to describe sperm head morphology in selection lines of house mice evolving with (polygamous) and without (monogamous) sperm competition. Although the GM analysis returned two relative warps that described variation in the curvature of the sperm hook, we found no evidence of divergence between the selection lines. Thus, we can conclude that sperm competition does not influence the degree of sperm hookedness in house mice.     

Rapid evolution of testis size relative to sperm morphology suggests that post‐copulatory selection targets sperm number in Anolis lizards

Post‐copulatory sexual selection is thought to be responsible for much of the extraordinary diversity in sperm morphology across metazoans. However, the extent to which post‐copulatory selection targets sperm morphology versus sperm production is generally unknown. To address this issue, we simultaneously characterized the evolution of sperm morphology (length of the sperm head, midpiece and flagellum) and testis size (a proxy for sperm production) across 26 species of Anolis lizards, a group in which sperm competition is likely. We found that the length of the sperm midpiece has evolved 2–3 times faster than that of the sperm head or flagellum, suggesting that midpiece size may be the most important aspect of sperm morphology with respect to post‐copulatory sexual selection. However, testis size has evolved faster than any aspect of sperm morphology or body size, supporting the hypothesis that post‐copulatory sexual selection acts more strongly upon sperm production than upon sperm morphology. Likewise, evolutionary increases in testis size, which typically indicate increased sperm competition, are not associated with predictable changes in sperm morphology, suggesting that any effects of post‐copulatory selection on sperm morphology are either weak or variable in direction across anoles. Collectively, our results suggest that sperm production is the primary target of post‐copulatory sexual selection in this lineage.