Evolution of 2 reproductive proteins, ZP3 and PKDREJ, in cetaceans

The rapid evolution of proteins involved in reproduction has been documented in several animal taxa. This is thought to be the result of forces involved in sexual selection and is expected to be particularly strong in promiscuous mating systems. In this study, a range of cetacean species were used to analyze the patterns of evolution in 2 reproductive proteins involved in fertilization: the zona pellucida 3 (ZP3), present in the egg coat, and PKDREJ, localized in the sperm head. We targeted exons 6 and 7 of ZP3 and a part of the REJ domain in PKDREJ for a total of 958 bp in 18 species. We found very low levels of amino acid sequence divergence in both proteins, a very weak signal of positive selection in ZP3 and no signal in PKDREJ. These results were consistent with previous reports of a slow rate of molecular evolution in cetaceans but unexpected due to the existence of promiscuous mating systems in these species. The results raise questions about the evolution of reproductive isolation and species recognition in whales and dolphins.     

Evolution of modern humans: evidence from nuclear DNA polymorphisms.

Previously we have described studies of the evolution of modern humans based upon data for classical genetic markers and for nuclear DNA polymorphisms. Such polymorphisms provide a different point of view regarding human evolution than do mitochondrial DNA sequences. Here we compare revised dates for major migrations of anatomically modern humans, estimated from archaeological data, with separations suggested by a genetic tree constructed from classical marker allele frequencies. Analyses of DNA polymorphisms have now been extended and compared with those of classical markers; genetic trees continue to support the hypothesis of an initial African and non-African divergence for modern humans. We have also begun testing non-human primates for a set of human DNA polymorphisms. For most polymorphisms tested so far, humans share a single allele with other primates; such shared alleles are likely to be ancestral. Populations living in humid tropical environments have significantly higher frequencies of ancestral alleles than do other populations, supporting the hypothesis that natural selection acts to maintain high frequencies of particular alleles in some environments.     

Evaluation of Sequence Variation and Selection in the Bindin Locus of the Red Sea Urchin, Strongylocentrotus franciscanus

Recent evidence suggests that gamete recognition proteins may be subjected to directed evolutionary pressure that enhances sequence variability. We evaluated whether diversity enhancing selection is operating on a marine invertebrate fertilization protein by examining the intraspecific DNA sequence variation of a 273-base pair region located at the 5′ end of the sperm bindin locus in 134 adult red sea urchins (Strongylocentrotus franciscanus). Bindin is a sperm recognition protein that mediates species-specific gamete interactions in sea urchins. The region of the bindin locus examined was found to be polymorphic with 14 alleles. Mean pairwise comparison of the 14 alleles indicates moderate sequence diversity (p-distance = 1.06). No evidence of diversity enhancing selection was found. It was not possible to reject the null hypothesis that the sequence variation observed in S. franciscanus bindin is a result of neutral evolution. Statistical evaluation of expected proportions of replacement and silent nucleotide substitutions, observed versus expected proportions of radical replacement substitutions, and conformance to the McDonald and Kreitman test of neutral evolution all indicate that random mutation followed by genetic drift created the polymorphisms observed in bindin. Observed frequencies were also highly similar to results expected for a neutrally evolving locus, suggesting that the polymorphism observed in the 5′ region of S. franciscanus bindin is a result of neutral evolution. Received: 19 June 1998 / Accepted: 2 August 2000     

Sexual Selection and the Molecular Evolution of ADAM Proteins

Rapid evolution has been identified for many reproductive genes and recent studies have combined phylogenetic tests and information on species mating systems to test sexual selection. Here we examined the molecular evolution of the ADAM gene family, a diverse group of 35 proteins capable of adhesion to and cleavage of other proteins, using sequence data from 25 mammalian genes. Out of the 25 genes analyzed, all those expressed in male reproductive tissue showed evidence of positive selection. Positively selected amino acids within the protein adhesion domain were only found in sperm surface ADAM proteins (ADAMs 1, 2, 3, 4, and 32) suggesting selection driven by male × female interactions. We tested heterogeneity in rates of evolution of the adhesion domain of ADAM proteins by using sequence data from Hominidae and macaques. The use of the branch and branch-site models (PAML) showed evidence of higher d _N/d _S and/or positive selection linked to branches experiencing high postmating selective pressures (chimpanzee and macaque) for Adams 2, 18, and 23. Moreover, we found consistent higher proportion of nonsynonymous relative to synonymous and noncoding sequence substitutions in chimpanzee and/or macaque only for Adams 2, 18, and 23. Our results suggest that lineage-specific sexual selection bouts might have driven the evolution of the adhesion sperm protein surface domains of ADAMs 2 and 18 in primates. Adams 2 and 18 are localized in chromosome 8 of primates and adjacent to each other, so their evolution might have also been influenced by their common genome localization.     

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.     

Polymorphism in abalone fertilization proteins is consistent with the neutral evolution of the egg's receptor for lysin (VERL) and positive darwinian selection of sperm lysin

The evolution of species-specific fertilization in free-spawning marine invertebrates is important for reproductive isolation and may contribute to speciation. The biochemistry and evolution of proteins mediating species-specific fertilization have been extensively studied in the abalone (genus Haliotis). The nonenzymatic sperm protein lysin creates a hole in the egg vitelline envelope by species-specifically binding to its egg receptor, VERL. The divergence of lysin is promoted by positive Darwinian selection. In contrast, the evolution of VERL does not depart from neutrality. Here, we cloned a novel nonrepetitive region of VERL and performed an intraspecific polymorphism survey for red (Haliotis rufescens) and pink (Haliotis corrugata) abalones to explore the evolutionary forces affecting VERL. Six statistical tests showed that the evolution of VERL did not depart from neutrality. Interestingly, there was a subdivision in the VERL sequences in the pink abalone and a lack of heterozygous individuals between groups, suggesting that the evolution of assortative mating may be in progress. These results are consistent with a model which posits that egg VERL is neutrally evolving, perhaps due to its repetitive structure, while sperm lysin is subjected to positive Darwinian selection to maintain efficient interaction of the two proteins during sperm competition.     

Positive selection is a general phenomenon in the evolution of abalone sperm lysin

Lysin is a 16kDa acrosomal protein used by abalone sperm to create a hole in the egg vitelline envelope (VE). The interaction of lysin with the VE is species-selective and is one step in the multistep fertilization process that restricts heterospecific (cross-species) fertilization. For this reason, the evolution of lysin could play a role in establishing prezygotic reproductive isolation between species. Previously, we sequenced sperm lysin cDNAs from seven California abalone species and showed that positive Darwinian selection promotes their divergence. In this paper an additional 13 lysin sequences are presented representing species from Japan, Taiwan, Australia, New Zealand, South Africa, and Europe. The total of 20 sequences represents the most extensive analysis of a fertilization protein to date. The phylogenetic analysis divides the sequences into two major clades, one composed of species from the northern Pacific (California and Japan) and the other composed of species from other parts of the world. Analysis of nucleotide substitution demonstrates that positive selection is a general process in the evolution of this fertilization protein. Analysis of nucleotide and codon usage bias shows that neither parameter can account for the robust data supporting positive selection. The selection pressure responsible for the positive selection on lysin remains unknown.      

Signatures of selection in the human olfactory receptor OR5I1 gene

The human olfactory receptor (OR) repertoire is reduced in comparison to other mammals and to other nonhuman primates. Nonetheless, this olfactory decline opens an opportunity for evolutionary innovation and improvement. In the present study, we focus on an OR gene, OR5I1, which had previously been shown to present an excess of amino acid replacement substitutions between humans and chimpanzees. We analyze the genetic variation in OR5I1 in a large worldwide human panel and find an excess of derived alleles segregating at relatively high frequencies in all populations. Additional evidence for selection includes departures from neutrality in allele frequency spectra tests but no unusually extended haplotype structure. Moreover, molecular structural inference suggests that one of the nonsynonymous polymorphisms defining the presumably adaptive protein form of OR5I1 may alter the functional binding properties of the OR. These results are compatible with positive selection having modeled the pattern of variation found in the OR5I1 gene and with a relatively ancient, mild selective sweep predating the "Out of Africa" expansion of modern humans.     

Accelerated rate of gene gain and loss in primates

The molecular changes responsible for the evolution of modern humans have primarily been discussed in terms of individual nucleotide substitutions in regulatory or protein coding sequences. However, rates of nucleotide substitution are slowed in primates, and thus humans and chimpanzees are highly similar at the nucleotide level. We find that a third source of molecular evolution, gene gain and loss, is accelerated in primates relative to other mammals. Using a novel method that allows estimation of rate heterogeneity among lineages, we find that the rate of gene turnover in humans is more than 2.5 times faster than in other mammals and may be due to both mutational and selective forces. By reconciling the gene trees for all of the gene families included in the analysis, we are able to independently verify the numbers of inferred duplications. We also use two methods based on the genome assembly of rhesus macaque to further verify our results. Our analyses identify several gene families that have expanded or contracted more rapidly than is expected even after accounting for an overall rate acceleration in primates, including brain-related families that have more than doubled in size in humans. Many of the families showing large expansions also show evidence for positive selection on their nucleotide sequences, suggesting that selection has been important in shaping copy-number differences among mammals. These findings may help explain why humans and chimpanzees show high similarity between orthologous nucleotides yet great morphological and behavioral differences.     

Positive selection in the carbohydrate recognition domains of sea urchin sperm receptor for egg jelly (suREJ) proteins

A wealth of evidence shows that protein-carbohydrate recognition mediates the steps of gamete interaction during fertilization. Carbohydrate-recognition domains (CRDs) comprise a large family of ancient protein modules of approximately 120 amino acids, having the same protein fold, that bind terminal sugar residues on glycoproteins and polysaccharides. Sea urchin sperm express three suREJ (sea urchin receptor for egg jelly) proteins on their plasma membranes. suREJ1 has two CRDs, whereas suREJ2 and suREJ3 both have one CRD. suREJ1 binds the fucose sulfate polymer (FSP) of egg jelly to induce the sperm acrosome reaction. The structure of FSP is species specific. Therefore, the suREJ1 CRDs could encode molecular recognition between sperm and egg underlying the species-specific induction of the acrosome reaction. The functions of suREJ2 and suREJ3 have not been explored, but suREJ3 is exclusively localized on the plasma membrane over the sperm acrosomal vesicle and is physically associated with sea urchin polycystin-2, a known cation channel. An evolutionary analysis of these four CRDs was performed for six sea urchin species. Phylogenetic analysis shows that these CRDs were already differentiated in the common ancestor of these six sea urchins. The CRD phylogeny agrees with previous work on these species based on one nuclear gene and several mitochondrial genes. Maximum likelihood shows that positive selection acts on these four CRDs. Threading the suREJ CRDs onto the prototypic CRD crystal structure shows that many of the sites under positive selection are on extended loops, which are involved in saccharide binding. This is the first demonstration of positive selection in CRDs and is another example of positive selection acting on the evolution of gamete-recognition proteins.     

Pervasive adaptive evolution in mammalian fertilization proteins

Mammalian fertilization exhibits species specificity, and the proteins mediating sperm-egg interactions evolve rapidly between species. In this study, we demonstrate that the evolution of seven genes involved in mammalian fertilization is promoted by positive Darwinian selection by using likelihood ratio tests (LRTs). Several of these proteins are sperm proteins that have been implicated in binding the mammalian egg coat zona pellucida glycoproteins, which were shown previously to be subjected to positive selection. Taken together, these represent the major candidates involved in mammalian fertilization, indicating positive selection is pervasive amongst mammalian reproductive proteins. A new LRT is implemented to determine if the d(N)/d(S) ratio is significantly greater than one. This is a more refined test of positive selection than the previous LRTs which only identified if there was a class of sites with a d(N)/d(S) ratio >1 but did not test if that ratio was significantly greater than one.     

Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization

Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.     

Rates of Evolution of Hominoid Seminal Proteins are Correlated with Function and Expression, Rather than Mating System

In species where females mate with multiple males during a single ovulatory cycle, sperm competition is hypothesized to increase the rate of adaptive evolution of proteins expressed in male reproductive tissues through recurrent selective sweeps (positive selection). The hominoids, comprising apes and humans, are a group of closely related primates with extensive variation in mating behaviors and predicted levels of sperm competition. Since previous studies of individual male reproductive genes have shown evidence of positive selection, we estimated rates of evolution of a comprehensive set of proteins expressed in ejaculated semen. Our results show that these proteins in hominoids do not have elevated rates of nonsynonymous substitutions (Ka) compared with a control dataset of nonreproductive genes. Species with greater sperm competition do not have faster rates of seminal protein evolution. Although at these broad levels our hypotheses were not confirmed, further analyses indicate specific patterns of molecular evolution. Namely, the Ka of seminal genes is more strongly correlated with measures of tissue specificity than nonreproductive genes, suggesting that the former may more readily adapt to tissue-specific functions. Proteins expressed from the seminal vesicles evolve more rapidly than those from other male reproductive tissues. Also, several gene ontology categories show elevated rates of protein evolution, not seen in the control data set. While the generalization that male reproductive genes evolve rapidly in hominoids is an oversimplification, a subset of proteins can be identified that are likely targets for adaptive evolution driven by sexual selection.     

Coevolution of Interacting Fertilization Proteins

Reproductive proteins are among the fastest evolving in the proteome, often due to the consequences of positive selection, and their rapid evolution is frequently attributed to a coevolutionary process between interacting female and male proteins. Such a process could leave characteristic signatures at coevolving genes. One signature of coevolution, predicted by sexual selection theory, is an association of alleles between the two genes. Another predicted signature is a correlation of evolutionary rates during divergence due to compensatory evolution. We studied female–male coevolution in the abalone by resequencing sperm lysin and its interacting egg coat protein, VERL, in populations of two species. As predicted, we found intergenic linkage disequilibrium between lysin and VERL, despite our demonstration that they are not physically linked. This finding supports a central prediction of sexual selection using actual genotypes, that of an association between a male trait and its female preference locus. We also created a novel likelihood method to show that lysin and VERL have experienced correlated rates of evolution. These two signatures of coevolution can provide statistical rigor to hypotheses of coevolution and could be exploited for identifying coevolving proteins a priori. We also present polymorphism-based evidence for positive selection and implicate recent selective events at the specific structural regions of lysin and VERL responsible for their species-specific interaction. Finally, we observed deep subdivision between VERL alleles in one species, which matches a theoretical prediction of sexual conflict. Thus, abalone fertilization proteins illustrate how coevolution can lead to reproductive barriers and potentially drive speciation.     

Sexual selection and the adaptive evolution of mammalian ejaculate proteins

An elevated rate of substitution characterizes the molecular evolution of reproductive proteins from a wide range of taxa. Although the selective pressures explaining this rapid evolution are yet to be resolved, recent evidence implicates sexual selection as a potentially important explanatory factor. To investigate this hypothesis, we sought evidence of a high rate of adaptive gene evolution linked to postcopulatory sexual selection in muroid rodents, a model vertebrate group displaying a broad range of mating systems. Specifically, we sequenced 7 genes from diverse rodents that are expressed in the testes, prostate, or seminal vesicles, products of which have the potential to act in sperm competition. We inferred positive Darwinian selection in these genes by estimation of the ratio of nonsynonymous (d(N), amino acid changing) to synonymous (d(S), amino acid retaining) substitution rates (omega = d(N)/d(S)). Next, we tested whether variation in this ratio among lineages could be attributed to interspecific variation in mating systems, as inferred from the variation in these rodents' relative testis sizes (RTS). Four of the 7 genes examined (Prm1, Sva, Acrv1, and Svs2, but not Svp2, Msmb, or Spink3) exhibit unambiguous evidence of positive selection. One of these, the seminal vesicle-derived protein Svs2, also shows some evidence for a concentration of positive selection in those lineages in which sperm competition is common. However, this was not a general trend among all the rodent genes we examined. Using the same methods, we then reanalyzed previously published data on 2 primate genes, SEMG1 and SEMG2. Although SEMG2 also shows evidence of positive selection concentrated in lineages subject to high levels of sperm competition, no such trend was found for SEMG1. Overall, despite a high rate of positive selection being a feature of many ejaculate proteins, these results indicate that the action of sexual selection potentially responsible for elevated evolutionary rates may be difficult to detect on a gene-by-gene basis. Although the extreme diversity of reproductive phenotypes exhibited in nature attests to the power of sexual selection, the extent to which this force predominates in driving the rapid molecular evolution of reproductive genes therefore remains to be determined.     

Functional characterization of PKDREJ, a male germ cell-restricted polycystin

Polycystin-1 regulates a number of cellular processes through the formation of complexes with the polycystin-2 ion channel or with other signal transduction proteins. Polycystin-1 is expressed in many tissues but other members of this gene family are distributed in a more restricted fashion. PKDREJ expression has been detected only in the mammalian testis, where it is restricted to the spermatogenic lineage and retained in mature sperm. However, the functional characteristics of this protein and its role in sperm biology are not well understood. In this study it is shown that PKDREJ can modulate G protein signaling and associates with several members of the polycystin-2 family. These interactions, as well as polycystin-2 association with TRPC channels, are consistent with a role of this protein in the regulation of the acrosome reaction and in other aspects of sperm physiology.