Little is known about the genetic basis of evolutionary changes in sperm phenotype. Postcopulatory sexual selection is associated with differences in protamine gene sequences and promoters and is a powerful force acting on sperm form and function, although links between protamine evolution and sperm phenotype are scarce. Protamines are involved in sperm chromatin condensation, and protamine deficiency negatively affects sperm morphology and male fertility, thus suggesting that they are important for sperm design and function. We examined changes in protamine genes and sperm phenotype in rodents to understand the role of sexual selection on protamine evolution and sperm design. We performed a genotype-phenotype association study using root-to-tip dN/dS (nonsynonymous/synonymous substitutions rate ratio) to account for evolutionary rates and phylogenetic generalized least squares analyses to compare genetic and morphometric data. Evolutionary rates of protamine 1 and the protamine 2 domain cleaved off during chromatin condensation correlated with head size and elongation. Protamine 1 exhibited restricted positive selection on some functional sites, which seemed sufficient to preserve its role in head design. The cleaved-protamine 2, whose relaxation is halted by sexual selection, seems to ensure small, elongated heads that would make sperm more competitive. No association existed between mature-protamine 2 and head phenotype, suggesting little involvement during chromatin condensation and a likely role maintaining the condensed state. Our results suggest that evolutionary changes in protamines could be related to complex developmental modifications in the sperm head. This represents an important step toward understanding the role of changes in gene coding sequences in the divergence of germ cell phenotype.

Proteomic studies of spermatozoa have identified a large catalog of integral sperm proteins. Rapid evolution of these proteins may underlie adaptive changes of sperm traits involved in different events leading to fertilization, although the selective forces underlying such rapid evolution are not well understood. A variety of selective forces may differentially affect several steps ending in fertilization, thus resulting in a compartmentalized adaptation of sperm proteins. Here we analyzed the evolution of genes associated to various events in the sperm’s life, from sperm formation to sperm-egg interaction. Evolutionary analyses were performed on gene sequences from 17 mouse strains whose genomes have been sequenced. Four of these are derived from wild Mus musculus, M. domesticus, M. castaneus and M. spretus. We found a higher proportion of genes exhibiting a signature of positive selection among those related to sperm motility and sperm-egg interaction. Furthermore, sperm proteins involved in sperm-egg interaction exhibited accelerated evolution in comparison to those involved in other events. Thus, we identified a large set of candidate proteins for future comparative analyses of genotype-phenotype associations in spermatozoa of species subjected to different sexual selection pressures. Adaptive evolution of proteins involved in motility could be driven by sperm competition, since this selective force is known to increase the proportion of motile sperm and their swimming velocity. On the other hand, sperm proteins involved in gamete interaction could be coevolving with their egg partners through episodes of sexual selection or sexual conflict resulting in species-specific sperm-egg interactions and barriers preventing interspecies fertilization.

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.

Sexual selection is the pervasive force underlying the dramatic divergence of sperm form and function. Although it has been demonstrated that testis gene expression evolves rapidly, exploration of the proteomic basis of sperm diversity is in its infancy. We have employed a whole-cell proteomics approach to characterize sperm divergence among closely related Mus species that experience different sperm competition regimes and exhibit pronounced variation in sperm energetics, motility and fertilization capacity. Interspecific comparisons revealed significant abundance differences amongst proteins involved in fertilization capacity, including those that govern sperm-zona pellucida interactions, axoneme components and metabolic proteins. Ancestral reconstruction of relative testis size suggests that the reduction of zona pellucida binding proteins and heavy-chain dyneins was associated with a relaxation in sperm competition in the M. musculus lineage. Additionally, the decreased reliance on ATP derived from glycolysis in high sperm competition species was reflected in abundance decreases in glycolytic proteins of the principle piece in M. spretus and M. spicilegus. Comparison of protein abundance and stage-specific testis expression revealed a significant correlation during spermatid development when dynamic morphological changes occur. Proteins underlying sperm diversification were also more likely to be subject to translational repression, suggesting that sperm composition is influenced by the evolution of translation control mechanisms. The identification of functionally coherent classes of proteins relating to sperm competition highlights the utility of evolutionary proteomic analyses and reveals that both intensified and relaxed sperm competition can have a pronounced impact on the molecular composition of the male gamete.

Cancer is a disease driven by both somatic mutations that increase survival and proliferation of cell lineages and the evolution of genes associated with cancer risk in populations. Several genes associated with cancer in humans, hereafter cancer genes, show evidence of germline positive selection among species. Taking advantage of a large collection of mammalian genomes, we systematically looked for signatures of germline positive selection in 430 cancer genes available in COSMIC. We identified 40 cancer genes with a robust signal of positive selection in mammals. We found evidence for fewer selective constraints—higher number of non-synonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site (dN/dS)—and higher incidence of positive selection—more positively selected sites—in cancer genes bearing germline and recessive mutations that predispose to cancer. This finding suggests a potential association between relaxed selection, positive selection, and risk of hereditary cancer. On the other hand, we did not find significant differences in terms of tissue or gene type. Human cancer genes under germline positive selection in mammals are significantly enriched in the processes of DNA repair, with high presence of Fanconi anaemia/Breast Cancer A (FA/BRCA) pathway components and T cell proliferation genes. We also show that the inferred positively selected sites in the two genes with the strongest signal of positive selection, i.e., BRCA2 and PTPRC, are in regions of functional relevance, which could be relevant to cancer susceptibility.