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.     

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.     

Protamine 2 deficiency leads to sperm DNA damage and embryo death in mice

Cytokinesis is incomplete in spermatogenic cells, and the descendants of each stem cell form a clonal syncytium. As a result, a heterozygous mutation in a gene expressed postmeiotically affects all of the haploid spermatids within a syncytium. Previously, we have found that disruption of one copy of the gene for either protamine 1 (PRM1) or protamine 2 (PRM2) in the mouse results in a reduction in the amount of the respective protein, abnormal processing of PRM2, and inability of male chimeras to transmit either the mutant or wild-type allele derived from the 129-genotype embryonic stem cells to the next generation. Although it is believed that protamines are essential for compaction of the sperm nucleus and to protect the DNA from damage, this has not been proven experimentally. To test the hypothesis that failure of chimeras to transmit the 129 genotype to offspring was due to alterations in the organization and integrity of sperm DNA, we used the single-cell DNA electrophoresis (comet) assay, ultrastructural analysis, and the intracytoplasmic sperm injection (ICSI) procedure. Comet assay demonstrated a direct correlation between the fraction of sperm with haploinsufficiency of PRM2 and the frequency of sperm with damaged DNA. Ultrastructural analysis revealed reduced compaction of the chromatin. ICSI with PRM2-deficient sperm resulted in activation of most metaphase II-arrested mouse eggs, but few were able to develop to the blastocyst stage. These findings suggest that development fails because of damage to paternal DNA and that PRM2 is crucial for maintaining the integrity of sperm chromatin.     

The protamine family of sperm nuclear proteins

The protamines are a diverse family of small arginine-rich proteins that are synthesized in the late-stage spermatids of many animals and plants and bind to DNA, condensing the spermatid genome into a genetically inactive state. Vertebrates have from one to 15 protamine genes per haploid genome, which are clustered together on the same chromosome. Comparison of protamine gene and amino-acid sequences suggests that the family evolved from specialized histones through protamine-like proteins to the true protamines. Structural elements present in all true protamines are a series of arginine-rich DNA-anchoring domains (often containing a mixture of arginine and lysine residues in non-mammalian protamines) and multiple phosphorylation sites. The two protamines found in mammals, P1 and P2, are the most widely studied. P1 packages sperm DNA in all mammals, whereas protamine P2 is present only in the sperm of primates, many rodents and a subset of other placental mammals. P2, but not P1, is synthesized as a precursor that undergoes proteolytic processing after binding to DNA and also binds a zinc atom, the function of which is not known. P1 and P2 are phosphorylated soon after their synthesis, but after binding to DNA most of the phosphate groups are removed and cysteine residues are oxidized, forming disulfide bridges that link the protamines together. Both P1 and P2 have been shown to be required for normal sperm function in primates and many rodents.     

Analysis of hamster protamines: primary sequence and species distribution.

Basic nuclear proteins were isolated from the sperm of the Syrian hamster Mesocricetus auratus and characterized by gel electrophoresis, amino acid analysis, and sequencing. Analyses of the proteins by gel electrophoresis show that sperm of this species contain both protamines 1 and 2. The two proteins were purified by HPLC and the complete primary sequence of hamster protamine 1 was determined by automated amino acid sequence analysis. The protein sequence was subsequently confirmed by sequencing the PCR-amplified protamine 1 gene. The first forty-two residues of the hamster protamine 2 sequence were obtained by amino acid sequence analysis of the isolated protein, and this sequence was also confirmed and extended by sequencing the gene. Total basic nuclear protein was also isolated from sperm of six other species of hamsters, the protamines were identified by HPLC and amino acid analysis, and the proportion of protamines 1 and 2 in each species was determined. Marked differences in the protamine 2 content of sperm were observed among the different species of hamster. This variation and the high level of sequence similarity between mouse and hamster protamines provide insight into how the two protamines may be organized in sperm chromatin. Mol. Reprod. Dev. 54:273-282, 1999. Published 1999 Wiley-Liss, Inc.     

Both P1 and P2 protamine genes are expressed in mouse, hamster, and rat

To date, in mammals except for the mouse and human, only one protamine variant has been isolated from sperm. These mammalian protamines share amino acid sequence homology with mouse protamine 1 (mP1), the tyrosine-containing variant. Southern blot analysis of restriction enzyme digests of hamster and rat liver DNA reveals the presence of sequences homologous to mP1, and also to mouse protamine 2 (mP2) cDNAs. Northern blots of hamster and rat total testis RNA probed with mP2 cDNA confirm that the protamine 2 gene in these species is transcribed into two size classes of mRNA of approximately 830 and 700 nucleotides. However, the relative abundance of the rat and hamster protamine 2 mRNAs (rP2 and hP2) in total testis is approximately 50-fold lower and 2- to 5-fold lower, respectively, than the mouse protamine 2 mRNA. Northern blot analysis of hamster and rat testis polysome gradients demonstrates that although the amount of rP2 mRNA and hP2 mRNA is reduced, both are present on polysomes. The decreased expression of rat and hamster protamine 2 mRNA relative to their protamine 1 counterparts contrasts protamine expression in the mouse testis, where approximately equal amounts of mP1 and mP2 protamine mRNAs are present. These results suggest differential expression of the P1 and P2 protamine genes in three closely related mammals.     

Sexual selection halts the relaxation of protamine 2 among rodents

Sexual selection has been proposed as the driving force promoting the rapid evolutionary changes observed in some reproductive genes including protamines. We test this hypothesis in a group of rodents which show marked differences in the intensity of sexual selection. Levels of sperm competition were not associated with the evolutionary rates of protamine 1 but, contrary to expectations, were negatively related to the evolutionary rate of cleaved- and mature-protamine 2. Since both domains were found to be under relaxation, our findings reveal an unforeseen role of sexual selection: to halt the degree of degeneration that proteins within families may experience due to functional redundancy. The degree of relaxation of protamine 2 in this group of rodents is such that in some species it has become dysfunctional and it is not expressed in mature spermatozoa. In contrast, protamine 1 is functionally conserved but shows directed positive selection on specific sites which are functionally relevant such as DNA-anchoring domains and phosphorylation sites. We conclude that in rodents protamine 2 is under relaxation and that sexual selection removes deleterious mutations among species with high levels of sperm competition to maintain the protein functional and the spermatozoa competitive.     

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.     

Convergent evolution of cysteine residues in sperm protamines of one genus of marsupials, the Planigales

A characteristic feature of the sperm P1 protamines of eutherian mammals is the constant presence of six to nine cysteine residues per molecule. During spermiogenesis these residues become oxidized to form a three-dimensional network of disulfide bridges between, and within, protamine molecules in the sperm chromatin. This covalent cross linking strongly stabilizes eutherian sperm nuclei. In contrast, protamines sequenced from teleost fish, birds, monotremes, and marsupials all lack cysteine residues and their sperm nuclei, without the stabilizing cross links, are easily decondensed in vitro. We have now found that one genus of tiny, shrewlike dasyurid marsupials, the Planigales, possess P1 protamines containing five to six cysteine residues. These residues appear to have evolved since the divergence of Planigales from other members of the family Dasyuridae, such as the marsupial mouse, Sminthopsis crassicaudata. We believe this constitutes a case of convergent evolution in a subfamily of dasyurid marsupials toward the cysteine-rich eutherian form of sperm protamine P1.      

Zinc is sufficiently abundant within mammalian sperm nuclei to bind stoichiometrically with protamine 2

Although studies have demonstrated that zinc can bind to sperm nuclear proteins, specifically protamine 2, it has not been shown that the metal is sufficiently abundant inside the sperm nucleus to interact stoichiometrically with these proteins. In this study proton-induced X-ray emission (PIXE) has been used to measure the amount of sulfur and zinc within the nuclei of individual sperm cells to infer the stoichiometry of zinc binding to protamine 2 in six species of mammal: bull, chinchilla, stallion, hamster, human, and mouse (protamine 2 comprises from 0% (bull) to 67% (mouse) of the protamine present in the sperm of these animals). Using the sulfur mass and electrophoretic data on the relative proportion of protamine 1 and protamine 2 in the sperm chromatin of these species, the protamine 1, protamine 2, and total protamine contents within each species sperm nuclei have been determined. The PIXE measurements reveal that the zinc content of the sperm nucleus varies proportionately with the protamine 2 content of sperm chromatin. PIXE analyses of hamster protamines extracted under conditions that appear to at least partially preserve zinc binding also confirm that the majority of the metal is bound to protamine. In five of the species examined, sufficient zinc is present for each protamine 2 molecule to bind one zinc. The results obtained for chinchilla sperm, conversely, indicate the chinchilla protamine 2 molecule may interact differently with zinc. Chinchilla sperm only contain enough zinc for one atom to be bound to two protamine 2 molecules.     

Prm3, the fourth gene in the mouse protamine gene cluster, encodes a conserved acidic protein that affects sperm motility

The protamine gene cluster containing the Prm1, Prm2, Prm3, and Tnp2 genes is present in humans, mice, and rats. The Prm1, Prm2, and Tnp2 genes have been extensively studied, but almost nothing is known about the function and regulation of the Prm3 gene. Here we demonstrate that an intronless Prm3 gene encoding a distinctive small acidic protein is present in 13 species from seven orders of mammals. We also demonstrate that the Prm3 gene has not generated retroposons, which supports the contention that genes that are expressed in meiotic and haploid spermatogenic cells do not generate retroposons. The Prm3 mRNA is first detected in early round spermatids, while the PRM3 protein is first detected in late spermatids. Thus, translation of the Prm3 mRNA is developmentally delayed similar to the Prm1, Prm2, and Tnp2 mRNAs. In contrast to PRM1, PRM2, and TNP2, PRM3 is an acidic protein that is localized in the cytoplasm of elongated spermatids and transfected NIH-3T3 cells. To elucidate the function of PRM3, the Prm3 gene was disrupted by homologous recombination. Sperm from Prm3(-/-) males exhibited reductions in motility, but the fertility of Prm3(-/-) and Prm3(+/+) males was similar in matings of one male and one female. We have developed a competition test in which a mutant male has to compete with a rival wild-type male to fertilize a female; the implications of these results are also discussed.     

Mapping and Measuring DNA to Protein Ratios in Mammalian Sperm Head by XANES Imaging

We have used XANES imaging, which combines X-ray absorption near edge spectral features (XANES) with 50-nm-resolution X-ray microscopy, to examine the content and distribution of DNA and protein in mature sperm cells. Sperm nuclei from five different species of mammals were examined; these species were chosen for analysis because their sperm contain marked differences in their protamine 1 and protamine 2 contents. The data we've obtained for bull, stallion, hamster, and mouse sperm suggest that the total nuclear protein to DNA ratio is similar in the sperm of many eutherian mammals. Since protamine constitutes the majority of the sperm nuclear protein, these results indicate that the total protamine content of sperm chromatin must be constant among mammalian species, independent of the extent of expression of the protamine 2 gene.     

Thermostability of sperm nuclei assessed by microinjection into hamster oocytes

Nuclei isolated from spermatozoa of various species (golden hamster, mouse, human, rooster, and the fish tilapia) were heated at 60 degrees-125 degrees C for 20-120 min and then microinjected into hamster oocytes to determine whether they could decondense and develop into pronuclei. Mature, mammalian sperm nuclei, which are stabilized by protamine disulfide bonds, were moderately heat resistant. For example, they remained capable of pronucleus formation even after pretreatment for 30 min at 90 degrees C. Indeed, a temperature of 125 degrees C (steam) was required to inactivate hamster sperm nuclei completely. On the other hand, nuclei of rooster and tilapia spermatozoa and those of immature hamster and mouse spermatozoa, which are not stabilized by protamine disulfide bonds, were sensitive to heating; although some of them decondensed after exposure to 90 degrees C, none formed male pronuclei. Furthermore, nuclei of mature hamster sperm became heat labile when they were pretreated with dithiothreitol to reduce their protamine disulfide bonds. These observations suggest that the thermostability shown by the nuclei of mature spermatozoa of eutherian mammals is related to disulfide cross-linking of sperm protamines.     

Characterization and evolutionary relevance of the sperm nuclear basic proteins from stickleback fish

We have characterized the sperm nuclear basic proteins (SNBPs) of the sticklebacks in the suborder Gasterosteoidei. The complete amino acid sequence of the protamines from Aulorhynchus flavidus, Pungitius pungitius, Gasterosteus aculeatus, (anadromous) and G. wheatlandi, as well as the sequences of the protamines of several species pairs of freshwater G. aculeatus, have been determined. Analysis of the primary structure of these proteins has shown that: a) despite the relatively low amino acid complexity and small molecular mass of these basic proteins, they are very good molecular markers at the generic level. The bootstrap parsimony analysis using their sequences provides a phylogenetic relationship for the old anadromous species of Gasterosteoidei which is identical to that obtained from morphological and behavioral analysis; b) the comparison of the sequences also suggests that protamines from the suborder Gasterosteoidei have most likely evolved from a common gene in the early Acanthopterygii by an extension of the carboxy terminal portion of the molecule; c) protamines are not good markers for recent postglacial freshwater isolates of G. aculeatus. However, in the unique case of Enos Lake (British Columbia), we have been able to detect an additional minor protamine component in the benthic forms of G. aculeatus that is not present in the limnetic forms. Thus, this new protamine must have appeared during the past 12,000 years concomitantly with the speciation of benthics and limnetics in this lake.     

Recent origins of sperm genes in Drosophila

Newly created genes often acquire testis-specific or enhanced expression but neither the mechanisms responsible for this specificity nor the functional consequences of these evolutionary processes are well understood. Genomic analyses of the Drosophila melanogaster sperm proteome has identified 2 recently evolved gene families on the melanogaster lineage and 4 genes created by retrotransposition during the evolution of the melanogaster group that encode novel sperm components. The expanded Mst35B (protamine) and tektin gene families are the result of tandem duplication events with all family members displaying testis-specific expression. The Mst35B family encodes rapidly evolving protamines that display a robust signature of positive selection within the DNA-binding high-mobility group box consistent with functional diversification in genome repackaging during sperm nuclear remodeling. The Mst35B paralogs also reside in a significant regional cluster of testis-overexpressed genes. Tektins, known components of the axoneme, are encoded by 3 nearly identical X-linked genes, a finding consistent with very recent gene family expansion. In addition to localized duplication events, the evolution of the sperm proteome has also been driven by recent retrotransposition events resulting in Cdlc2, CG13340, Vha36, and CG4706. Cdlc2, CG13340, and Vha36 all display high levels of overexpression in the testis, and Cdlc2 and CG13340 reside within testis-overexpressed gene clusters. Thus, gene creation is a dynamic force in the evolution of sperm composition and possibly function, which further suggests that acquisition of molecular functionality in sperm may be an influential pathway in the fixation of new genes.     

Chromosomal assignment of four rat genes coding for the spermatid-specific proteins proacrosin (ACR), transition proteins 1 (TNP1) and 2 (TNP2), and protamine 1 (PRM1)

The genes for proacrosin, protamines, and transition proteins are exclusively expressed in haploid spermatogenic cells. From the analysis of mouse x rat cell hybrids which segregate rat chromosomes, the rat gene for proacrosin (ACR) was assigned to chromosome 7, that for transition protein 1 (TNP1) to chromosome 9, and the genes for transition protein 2 (TNP2) and protamine 1 (PRM1) to chromosome 10.     

Phosphorylation state of protamines 1 and 2 in human spermatids and spermatozoa

The basic nuclear proteins of a fraction of elongating spermatids from human tests and of a fraction of motile spermatozoa from the ejaculate, separated by ion-exchange chromatography, were compared. Analysis by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) showed that, in both fractions, four proteins of lower mobility were coeluted with protamine 1 by 23% guanidinium chloride (GuCI) while protamine 2 alone was eluted by 50% GuCI. Treatment with alkaline phosphatase identified those four proteins as phosphorylated protamines, and cyanogen bromide (CNBr) treatment of the dephosphorylated protamines distinguished them as variants of protamine 2 and not of protamine 1. Thus far, phosphorylated forms of protamine 1 have not been detected in either spermatids or spermatozoa. Those observations indicate that protamine 2 functions in the cycle of phosphorylation-dephosphorylation, which is essential to the process of sperm chromatin condensation, while the role of protamine 1 in human spermiogenesis is not yet defined. The presence of phosphorylated protamine in motile, presumably mature spermatozoa appears to be characteristic of human sperm but not of the sperm of other mammals and is probably the basis for the heterogeneity of chromatin condensation frequently observed in human spermatozoa.