The evolution of reproductive proteins and the spermatozoa diversification

1. Introduction

The evolution of sex has been a subject of interest and scientific debate since the Theory of Evolution. At the cellular level, the last aides in charge of culminating sexual reproduction are the gametes -the spermatozoon and the ovum. Given their vital importance for all species survival and prevalence, the understanding of these two cells has been a main concern throughout our history. For the most part, many hypotheses and sex studies have focused on the spermatozoon, due to its vital role as a carrier of male hereditary material and its involvement in fertility.

Spermatozoa are extremely specialized cells which undergo genetic, molecular and physiological changes not detected in other cells (Roldan and Gomendio, 2006). From the evolutionary point of view, spermatozoa constitute a cellular model of exceptional interest, since they are among the most divergent cells of living beings, presenting very different sizes and shapes (Pitnick et al., 2006). For the last 50 years, numerous studies have been carried out to try to understand the basic principles and the evolutionary forces that govern the great spermatozoa diversification. The heap of these investigations has resulted in the identification of various selective forces that direct the adaptation of morphology related traits, the storage and functioning of spermatozoa (Birkhead et al., 2008). A determining factor in sperm diversification are the postcopulatory sexual selection (PCSS) forces. PCSS involves all those barriers and challenges that spermatozoa face when entering the female tract towards fertilization. PCSS will therefore impose a selective control on the probability of spermatozoa fertilization based on its genetic endowments and its phenotypic qualities (Birkhead and Pizzari, 2002). Despite extensive knowledge on the traits that have allowed spermatozoa and ejaculates to adapt to both their reproductive environments and the forces of PCSS, the molecular basis of these adaptive changes have remained unexplored for a long time.

The reproductive proteins, here widely defined as all those proteins that take action after the copulation and intervene in the transportation, storage and functioning of the gametes, as well as in the fertilization, are crucial in the biological sense because they contribute to the generation of new individuals. In evolutionary terms, reproductive proteins are transcendental elements because of three main reasons: 1) they have an effect on the outcome of postcopulatory sexual selection forces, 2) they mediate reproductive conflicts between the sexes, and 3) they contribute to the formation of postcopulatory and prezygotic (PCPZ) reproductive isolation barriers, which leads to the formation of new species. An interesting fact about reproductive proteins is that the genes that code them (reproductive genes) evolve very quickly (Swanson and Vacquier, 2002). However, there is few evidence to demonstrate a direct impact of PCSS on the adaptive evolution of the reproductive genes.

The latest advances in genomic tools have allowed the identification of gene collections expressed in reproductive tissues. However, a mature spermatozoon is a transcriptionally inactive cell, and therefore transcriptomic methods have not been effective on this system. This obstacle has been overcome through the application of proteomics to the study of spermatozoa. The development of high performance tools based on mass spectrometry (MS) has allowed its application to characterize the spermatozoa molecular composition in many species (McDonough et al., 2016). In this chapter, we review the great progress made in the mass identification of spermatozoa proteins through the use of MS- based techniques, and how these methods have contributed to a better understanding of the male gamete molecular composition. We will subsequently discuss how transcriptomic and proteomic techniques can be integrated with comparative genomics analysis to identify groups of reproductive genes subject to positive selection, and how these findings give us an idea of the selective forces that have driven the evolution of reproductive systems. Finally, we will talk about how emergent methods of quantitative proteomics can be applied to know the primary responses of spermatozoa to PCSS episodes.

2.  Characterization of Spermatozoa Proteome

A mature spermatozoon is an ideal cell for proteomics studies development because of three reasons: 1) it is accessible since it can be easily extracted from the ejaculate or from storage tissues, 2) it can be easily purified, and 3) it supposedly has no transcriptional or translational activity. Traditional methods to analyze the spermatozoon composition normally were based on the use of antibodies or 2D gels, identifying a very limited number of proteins. However, the development of proteomics techniques based on mass spectrometry (MS) has made it possible to identify much wider collections of spermatozoa proteins with greater precision. That is why along the last decade prodigious advances have been made in the understanding of the spermatozoa molecular composition (Oliva et al., 2009; Karr and Dorus, 2012; McDonough et al., 2016).

The standard protocol of an MS-based proteomics analysis includes: 1) the proteins extraction from the cell or tissue, 2) the proteins digestion by proteases, 3) the separation of the peptides, 4) the peptides analysis in a mass spectrometer, and 5) the identification of peptides and the corresponding proteins based on the spectra information (Fig. 1). The application of MS-based high performance techniques allows the characterization of the spermatozoa proteome in various model organisms such as the human (Homo sapiens) (Amaral et al., 2014), the mouse (Mus musculus) (Baker, Hetherington, GM Reeves, et al., 2008; Dorus et al., 2010; Chauvin et al., 2012), the rat (Rattus norvegicus) (Baker, Hetherington, G. Reeves, et al., 2008), the bull (Bos taurus) (Peddinti et al., 2008), the fruit fly (Drosophila melanogaster) (Dorus et al., 2006; Wasbrough et al., 2010) or the Caenorhabditis elegans nematode (Ma et al., 2014). On account of the advance of genomics and the great growth of species whose genome has been sequenced and annotated, in recent years the proteome has also been obtained from species that are not experimental models, such as the rhesus macaque (Macaca mulatta) (Skerget et al., 2013), the rainbow trout (Nynca et al., 2014) or the Manduca sexta butterfly (Whittington et al., 2015). Recent studies have shown that a large proportion of the spermatozoa proteome is conserved among phylogenetically distant species (Dorus et al., 2006; Whittington et al., 2015; Bayram et al., 2016). This will allow carrying out a comparative proteomics analysis that includes non- model organisms often used in ecological and evolutionary studies (see paragraph 6).

Figure 1. Protocol to extract and identify proteins in the spermatozoon. The first step is to isolate the spermatozoa from its environment (it can be from the ejaculate or from the storage tissue, depending on the organism); proteins are extracted from total cells or from subcellular compartments, for which a prior subcellular fractionation must be performed. The extracted proteins are normally separated in polyacrylamide gels (PAGE), by using 1D (1D-PAGE) or 2D (2D-PAGE) gels. The proteins are cleaved from the gels and digested (regularly with trypsin) to generate the peptides, which are separated (commonly by liquid chromatography) and analyzed by mass spectrometry. The spectrometry data are finally identified with proteins by means of database search algorithms. Figure based on Amaral et al., 2014. Images taken from Van der Velden et al. 2011 (gel 1D), Wikimedia Commons (gel 2D), Wikipedia (Spectrometer), Separation Science (spectra) and Findlay & Swanson, 2010 (protein identification).

The characterization of the complete spermatozoa proteome, in some cases, has been complemented with studies where subcellular regions have been analyzed, such as the head surface (Brewis and Gadella, 2010), the acrosomal matrix (Guyonnet et al., 2012), or the flagellum accessory structures (Cao et al., 2006). This kind of analysis has allowed the identification of proteins that, being less abundant, were previously unfound in the complete proteome studies, providing collections of new proteins. Furthermore, the subcellular structures fractionation allows identifying proteins that have specific reproductive functions. One example is the characterization of the spermatozoon surface proteome to identify proteins involved in the interaction of gametes (Stein et al., 2006; Zigo et al., 2013; Kongmanas et al., 2015). In contrast, some studies have focused on analyzing the post-translational modifications that spermatozoa experience during capacitation (Ficarro et al., 2003; Arcelay et al., 2008), as well as proteins that undergo changes during the transit of spermatozoa through the epididymis (Labas et al., 2015; Skerget et al., 2015).

An advantage about identifying proteins with MS, with respect to transcriptomic methods, is that the protein presence in a specific tissue is ensured. This advantage is even more remarkable in the case of spermatozoa, since the testicle is the tissue with the highest transcriptional activity, and since gene expression during spermatogenesis is regulated by extremely complex mechanisms (Chocu et al., 2012; Soumillon et al., 2013). The complexity of the regulation mechanisms for genes and transcripts during spermatogenesis is the reason why the testicle is the tissue with the lowest correlation between the levels of mRNA abundance and protein (Cagney et al., 2005; Chocu et al. 2012; Vicens et al., 2017). For this reason, an adequate study of spermatogenesis should integrate both massive gene expression analysis and proteomics methods.

3. Evolution of Reproductive Proteins

Ever since Darwin formulated his theory on sexual selection (Darwin, 1871), many researchers have taken an interest in studying the evolution of sex in order to understand the factors that have generated the wide diversity of reproductive systems observed in nature. This field produced a greater interest when researchers began to characterize the first reproductive proteins, and it was observed that they present very divergent sequences among close species (Swanson and Vacquier, 2002). This was a surprise finding, because, due to the essential role played by sexual reproduction for the survival of the species, it was expected that the reproductive proteins would be highly conserved. However, this phenomenon could be observed from an opposite perspective: reproductive proteins can evolve to be more effective and competitive, and therefore contribute to the reproductive success of organisms. The fast evolution of reproductive proteins is a recurrent pattern that has been observed in groups of both animals and plants, as well as in different stages of reproduction (Swanson and Vacquier, 2002; Clark et al., 2006; Turner and Hoekstra, 2008a; Karr et al., 2009; Wilburn and Swanson, 2015).

A method to estimate a protein evolutionary rate is to compare the coding DNA sequence between different species. This lets us estimate the dN/dS parameter, also known as omega (ω), which is the quotient between non-synonymous mutations -those that modify the amino acid for which it codes the codon- in non-synonymous sites (dN) and the synonymous mutations -those that conserve the amino acid due to the degeneration of the genetic code- in synonymous sites (dS). From the ω value one can infer the protein evolutionary profile. A value of ω < 1 indicates that the protein is conserved, since the selection has been eliminating the mutations that alter the amino acids, which is known as negative or purifying selection. A ω ~ 1 value is obtained in genes that have accumulated mutations by selection relaxation, and are considered to have evolved neutrally. An example is the pseudogenes that lose functionality by fixing deleterious mutations. There are genes that show a ω > 1 value, indicating that mutations in the protein have been favored by selection, which is known as positive selection or adaptive evolution. The estimates of ω for a complete sequence are usually < 1 since most proteins are subject to strong selective pressures. It is also frequent that adaptive evolution only acts in some episodes of the protein evolutionary history. For these reasons, to detect positive selection in proteins and organisms, methods capable of estimating ω in specific codons or lineages have been developed (Yang, 2002).

The constant identification of reproductive proteins that undergo positive selection led us to assume that most proteins involved in reproduction evolve rapidly, in response to the sexual selection forces. However, recent advances in genomics and proteomics methods have allowed the identification of the genes and proteins repertoire expressed in reproductive tissues, such as the testicle (Turner et al., 2008), epididymis (Dean et al., 2008) or accessory glands (Dean et al., 2009). The studies revealed that most proteins expressed in reproductive tissues are highly conserved, while only a small proportion of proteins presents signs of positive selection. These studies revealed that proteins with a positive selection signal are almost exclusively those mixed with spermatozoa in the ejaculate, such as seminal fluid proteins in mammals (Clark and Swanson, 2005; Dean et al., 2009), seminal proteins accessory in Drosophila (Swanson et al., 2001; Wolfner 2002; Findlay et al., 2009), or proteins secreted by the epididymis that bind spermatozoa during maturation (Dean et al., 2008).

In the case of spermatozoa, a first study analyzed the evolution of a group of sperm-specific mammalian proteins (Torgerson et al., 2002). The analysis revealed that spermatozoa proteins show a greater divergence (measured as dN/dS) than the genes expressed in other tissues. However, only positive selection evidence was detected for 4 out of the 19 analyzed genes. The first study in which proteomics analysis and molecular evolution were combined was carried out in Drosophila melanogaster spermatozoa (Dorus et al., 2006). In this work it was detected that spermatozoa proteins evolve, in average, similarly to non-reproductive tissues. This implies that most spermatozoa proteins are conserved and subject to purifying selection (Fig. 2A). Moreover, no spermatozoa proteins were identified under positive selection. In a later study, the D. melanogaster spermatozoa proteome was re-analyzed using more sophisticated proteomic tools (Wasbrough et al., 2010). These analyses let us identify a much broader proteins catalog, and thanks to the availability of a greater number of sequenced genomes in Drosophila species, much more robust evolutionary analyses were performed, showing that the average evolutionary rate of spermatozoa proteins is significantly lower than that of accessory gland proteins. On the other hand, statistical selection analyses detected that only 77 spermatozoa genes (8% of the total) present evidence of positive selection. These results corroborate that the spermatozoon is subject to a strong purifying selection.

Subsequently, Dorus et al. focused on knowing the selective forces that have guided the evolution of mammalian spermatozoa. To do this, they combined the protein catalogs that had been identified in mice spermatozoa by previous proteomic studies with a self-collection obtained by MS/MS analysis (Dorus et al., 2010). In this study, the evolutionary rate of about 1000 genes was determined by interspecific analysis, using mouse sequences and other mammalian species. It was observed that genes coding for the spermatozoon surface proteins (including membrane proteins and acrosome) evolve significantly faster than the rest of the proteome (Fig. 2B). In addition, a higher proportion of proteins from the spermatozoon surface shows evidence of positive selection. These results suggest that diverse selective forces, such as sexual selection or immune response, could be directing the adaptive evolution of membrane proteins and acrosomes, since these are the proteins eventually exposed to the extracellular environment and the interaction with other cells.

Consequently, Vicens et al. carried out a comparative study of the mice spermatozoa proteome. Then, the evolution of different groups of proteins that were classified based on their functional role in the spermatozoon was compared (Vicens, Lüke et al., 2014). Evolutionary analyses revealed that the spermatozoa proteins involved in the interaction with the ovum present a more accelerated evolution (Fig. 2C). Additionally, it was observed that the group of gametes interaction presents a higher proteins percentage under positive selection with respect to the other functional groups (Fig. 2D). These results are based on retrospective studies in which positive selection has been identified in various proteins involved in mammalian fertilization (Swanson et al., 2003; Turner and Hoekstra, 2008b; Vicens, Montoto et al., 2014). Another group that showed a high proportion of proteins under positive selection was the one involved in spermatozoa motility. These results suggest that the speed of spermatozoon swimming can be an adaptive feature in competition scenarios between males (see paragraph 4), and are supported by previous evidence that some proteins that regulate sperm motility exhibit adaptive changes (Torgerson et al., 2002; Podlaha et al., 2005; Dorus et al., 2010).

Figure 2. Evolution of the spermatozoon proteome in different species. A) Estimation of the average evolutionary rate of the sperm proteome of Drosophila Melanogaster and its comparison with non-reproductive proteins, proteome of the accessory glands, and accessory proteins (ACPs). In the lower part, statistical comparisons are shown regarding the proteome of the spermatozoon and the accessory glands. NS: not significant, ++: p <0.01. Modified by Dorus et al., 2006. B) Estimation of the evolutionary rates of the different subcellular components of mice spermatozoa. The average estimates for the complete spermatozoon proteome, the membrane proteins and the flagellum are shown. Evolutionary rates for mice were calculated by comparison with orthologs of other mammals; there are significant differences between membrane proteins and the rest of the proteome (* p <0.01). Modified by Dorus et al., 2010. C) Average evolutionary rate for genes classified in different functional spermatozoon categories. D) Proportion of genes subject to positive selection in the different functional categories. SMG: spermatogenesis; MET: metabolism; MOT: motility; CAP: capacitation; AR: acrosomal reaction; SEI: sperm-egg interaction. Modified from Vicens et al., 2014.

The two evolutionary analyses on mice spermatozoa proteome described here, along with the evidence shown by retrospective studies, suggest that the molecular interactions that occur between the spermatozoon and the ovum during fertilization could be a primary target of sexual selection forces. This is consistent with what has been observed in marine invertebrates, where it has been shown that the proteins involved in the recognition and the fusion of gametes evolve rapidly and present intense signals of positive selection (Vacquier and Swanson, 2011). The fast divergence of spermatozoa proteins involved in fertilization seems to be the result of a coevolution process with proteins from the ovum surface (Clark et al., 2009; Claw et al., 2014; Vicens and Roldan, 2014) and this could have implications for the gametic compatibility and the reproductive isolation (Palumbi, 2009; Vacquier and Swanson, 2011).

In conclusion, it can be said that although the first studies that analyzed the few proteins that had a known spermatic function identified a great impact of positive selection (Wyckoff et al., 2000; Swanson and Vacquier, 2002; Torgerson et al., 2002; Clark et al., 2006; Turner and Hoekstra, 2008a); results obtained with wide-scale analyses suggest that only a small portion of the proteome of reproductive tissues and sperm undergo adaptive evolution. In addition, adaptive foci seem to concentrate on small protein subgroups that have specific locations and functions, such as the spermatozoa membrane proteins involved in gametes recognition. The evolutionary heterogeneity observed within male reproductive tissues and spermatozoa could be the result of a compartmentalization of adaptation in response to PCSS forces, which we will address in the next chapter.

4. Postcopulatory Sexual Selection Forces

Because of their function, spermatozoa and reproductive tissues must be a focus for action, regarding the forces of postcopulatory sexual selection (PCSS). PCSS forces involve competition between male ejaculates, a phenomenon known as spermatic competition (Pizarri and Parker, 2009), as well as a differential selection of the ejaculate by the female, which may be either the result of a sexual conflict, or a cryptic choice by the female (Pitnick et al., 2009).

Spermatic competition is defined as the contest between spermatozoa from different males to fertilize an oocyte (Parker, 1970; Birkhead and Moller, 1998; Pizarri and Parker, 2009). Therefore, this phenomenon affects polygamous species, in which the oocytes can encounter the ejaculates of several males. Spermatic competition has played a very important role in the evolution of the reproductive systems, and has promoted the acquisition and adaptation of numerous anatomical, physiological and behavioral features aimed at increasing the probability of successful fecundation (Dixson and Anderson, 2004; Pizarri and Parker, 2009). A primary response to spermatic competition is an increase in the testicles size, which leads to increasing the spermatic production (Soulsbury, 2010; Ramm et al., 2014). On the other hand, it has been observed that spermatic competition has promoted the evolution of various spermatozoa qualitative features, which gives the males of promiscuous species greater fertilization efficiency (Pizarri and Parker, 2009; Fitzpatrick and Lupold, 2014). These traits include viability, motility, head morphology, the dimensions of different spermatozoa components, the efficiency for capacitation developing, and the acrosome reaction. All these traits have been correlated with both fertility and levels of promiscuity (Hunter and Birkhead, 2002; Gomendio et al., 2006; Gómez Montoto, Magaña, et al., 2011; Gómez Montoto, Varea Sánchez, et al., 2011; Lupold, 2013; Tourmente et al., 2013). As a result of the spermatic competition, the male ejaculates have a more efficient fecundation. However, in many cases this is not beneficial for females, especially when it is a high amount of spermatozoa converging in the female tract. This situation will cause an increased risk that an ovum will merge with more than one spermatozoon (polyspermia). Polyspermy is detrimental to the female, since it gives rise to non-viable embryos that will not develop. Therefore, there is a scenario in which the male adaptations to increase their probability of fertilizing in response to spermatic competition will favor adaptations in the female to reduce the rate of fertilization. This phenomenon is known as sexual conflict, since the interests and adaptations of each sex to increase their fitness reduce those of the opposite sex (Hosken and Stockley, 2005; Chapman, 2006). On the other hand, females can impose mechanisms so that their oocytes are fertilized only by the spermatozoa of certain males. This is known as the cryptic choice of the female (Eberhard, 1996; Gavrilets et al., 2001).

We only have limited knowledge about the molecular basis of sexual conflict and the cryptic choice, due to the difficulty of studying the interactions between the ejaculate and the female reproductive tract in vivo (although see Pitnick et al., 2009; McDonough et al., 2016). Because of this, most studies trying to see adaptive responses to PCSS have focused on determining the role of spermatic competition in the evolution of the male ejaculate (Birkhead and Moller, 1998; Pizarri and Parker, 2009).

5. The Impact of Sexual Selection on the Evolution of Reproductive Genes

After the previous paragraph we have concluded that PCSS forces, especially spermatic competition, play an important role in the adaptation of reproductive traits. However, since phenotypic adaptations must be the result of genetic variations fixed by positive selection, we can ascertain that PCSS will primarily direct adaptive changes in the reproductive genes. There have been different approaches carried out to try to examine the effects of variation in mating systems on the evolution of reproductive genes -attempts to correlate the evolutionary rate of the coding and regulatory sequences with the variation in the intensity of the PCSS, or to compare the expression levels of extensive gene collections between sexes and species with different mating systems. In the following paragraphs we will briefly describe each approach, their most important results, and their limitations.

5.1 Impact on the Evolution of Reproductive Proteins

The first studies trying to measure the effects of PCSS on the evolution of reproductive proteins were based on the hypothesis that the evolutionary rate -or positive selection intensity- of a reproductive gene must increase with the PCSS levels, which are measured with a behavioral index (e.g., number of matings per reproductive cycle), or an anatomical index (e.g., relative size of testicles, an index of spermatic competition).

To determine the association between reproductive genes evolution and the mating systems, mainly two methods have been used. The first is based on performing a linear regression analysis between the evolutionary rate (ω) estimated for phylogeny branches with a continuous index of PCSS (Fig. 3A). In this way, obtaining significant correlations implies that the protein in question evolves in response to sexual selection. With such methodology, this study showed that the evolutionary rate of semenogelin 2 (SEMG2), the main component in seminal fluid and in the formation of primate seminiferous clot, correlates positively with levels of spermatic competition (Dorus et al., 2004). It was additionally observed that the evolutionary rate of SEMG2 is associated with the semen coagulation rate in different primate species. This represented the first evidence of a reproductive protein evolving under PCSS influence; it also demonstrated the effect of this evolution at phenotypic level. Herlyn and Zischler analyzed the molecular evolution of the ZAN gene that codes for zonadhesin, a protein involved in gametic recognition and fertilization (Herlyn and Zischler, 2007). They found a negative correlation between ω and the level of sexual dimorphism, which is interpreted as an inverse index of spermatic competition. Recently, Vicens et al. analyzed the molecular evolution of PKDREJ, a protein involved in transport and acrosomal reaction. In our analysis we found a positive correlation between the evolutionary rate and the relative size of the testicle in rodent species (Vicens, Montoto, et al., 2014). It is possible that PCSS not only direct changes in the amino acid composition, but that it could also favor the fixation of insertions and deletions. The adaptive potential of these changes was raised after observing that the size of a protein from the seminal fluid (SVS II) and another from the spermatozoon (Catsper1) correlate significantly with the levels of spermatic competition (Ramm et al., 2009; Vicens, Tourmente, et al., 2014).

Although the first hypothesis implied that the evolutionary rate of a reproductive protein must correlate positively with the levels of spermatic competition, Lüke et al. found an inverse correlation between the evolving rate of protamine 2 (PRM2) and the relative size of the rodents testicles (Lüke et al, 2011). Since PRM2 is codified by a duplicate gene of PRM1, which is the main protamine in charge of compacting chromatin during spermiogenesis, the PRM2 gene could have a redundant function, and therefore be subject to a functional relaxation. A later study revealed that a greater conservation of PRM2 correlates to a greater spermatozoon head elongation, a trait that makes the spermatozoon faster and therefore more competitive (Lüke et al., 2014). Based on these findings, it has been suggested that sexual selection can act by conserving copies of sperm- specific genes to enhance some functions.

The second approach focuses on determining whether there is a greater intensity of positive selection in those lineages with higher PCSS. In this method, phylogeny branches are classified in two categories based on their PCSS index value (Fig. 3B). Different evolutionary models of maximum likelihood are applied to this phylogeny, to evaluate whether there is positive selection in the branches corresponding to high PCSS lineages as opposed to those with low PCSS (for a more detailed description of the method, see Ramm et al., 2008). A study conducted by Ramm and others found evidence of positive selection in 5 out of 7 proteins involved in rodent reproduction (Ramm et al., 2008); out of these 5, only SVS2, a component of the seminal fluid involved in the formation of the mating plug, showed positive selection in the lineages of greater spermatic competition using the aforementioned methodology. Finn and Civetta analyzed the evolution of the family of ADAMs proteins membrane, involved in the interaction with other cells or extracellular components (Finn and Civetta, 2010). In this study, they found evidence of positive selection concentrated in the most polyandrous primate species for three ADAMs expressed on the spermatozoon surface. In the previously cited paper by Vicens et al. (2014), evidence of positive selection in PKDREJ was also detected for rodent species with higher levels of sperm competition.

Figure 3. Comparative methods to identify reproductive genes under the influence of postcopulatory sexual selection (SSPC). A) Estimation of evolutionary rates (ω) in the terminal branches of a phylogeny and correlation of ω with an index of SSPC, e.g. the relative size of the testicle. The length of the branches is proportional to the evolutionary rates. B) Division of the branches between lineages with high SSPC (e.g. polyandrous species) or low SSPC (e.g. monogamous species). Evolutionary models of maximum verisimilitude that assume purifying selection on all branches (ω <_1, null) with models that consider positive selection in branches of high SSPC (ω > 1, alternative) are statistically compared. Figure based in Wong, 2011.

In Table 1, all proteins in which a PCSS effect has been identified are displayed. Although the evolution of numerous reproductive genes has been analyzed, there have been very few studies in which a significant association has been found between their evolutionary rate (or their positive selection signal) and PCSS levels. The difficulty in determining the molecular impact of PCSS by these methodologies has been attributed to four possible (and non-exclusive) factors: 1) reproductive traits are normally regulated by multiple genes with additive effects, 2) sexual selection forces can act on different genes in different species, 3) other selective forces unrelated to sex (e.g. immunological responses in the female tract) can direct the evolution of reproductive genes, and 4) methods to detect covariance between the evolutionary rate of a reproductive protein and mating systems show important limitations (Wong, 2011). The first two can be solved by conducting massive analyses that determine the evolution of extensive gene and protein catalogs between species with different mating systems, as we will see below.

Table 1. List of male reproductive proteins that have evolved as a response to postcopulatory sexual selection.
* Comparative method to evaluate the association of gene evolution with mating systems: correlation of evolutionary rate (Cor), classification of branches by categories in terms of the sexual selection level (Cat).

5.2 Impact on the Evolution of the Expression of the Reproductive Genes

Phenotypic adaptations may result from changes in the genes coding sequence; changes in the regulation of gene expression may have a more important effect on adaptive changes, since regulatory sequences and expression levels are more labile and have fewer functional restrictions than the coding sequences. While the structural evolution of reproductive proteins was examined in most studies during the past decade, very few studies took interest in evaluating the phenotypic effects of changes in regulatory regions and the expression levels of reproductive genes. This has been mainly due to the greater difficulty of identifying the regulatory elements and the limitations of the models that determine the evolution mode of gene expression (Gilad et al., 2006). However, there is evidence that adaptive mutations in regulatory elements can cause changes in the genes expression levels, and that such variation can generate other variations in phenotypic traits (Sucena and Stern, 2000; Shapiro et al., 2004; Rockman et al., 2005).

It has been proven that genes with biased expression between sexes are highly divergent both at the structure and at the expression level (Ellegren and Parsch, 2007). On the other hand, a study by Brawand et al. (2011) analyzed the evolution of gene expression in various mammalian tissues, showing that the evolution of testicular expression has been much faster than in somatic tissues (Brawand et al., 2011). These characteristics have led to the conclusion that the rapid evolution of genes with restricted expression to the male or female sexes is a product of sexual selection, although this has not been evaluated until recently. In a first attempt to evaluate whether PCSS influence the evolution of the expression of male reproductive genes, Martín-Coello et al. (2009) compared the regulatory sequence of the genes that code for protamines between rodent species with different levels of spermatic competition. They found that the divergence of the PRM2 promoter correlates positively with the levels of sperm competition. Subsequently, Lüke et al. detected a negative association between the transcription levels of PRM2 and the elongation of the head. Since a more elongated head increases the spermatozoa swimming speed, it is possible that the PCSS favors a lower expression of PRM2 in the most promiscuous species to generate faster and more competitive spermatozoa. This suggests that PCSS forces could direct regulatory changes in the expression of reproductive genes.

In a recent study by Harrison et al. (2015), transcriptome evolution was compared between bird species with different levels of sexual selection. These analyses revealed that PCSS indices predict the proportion of genes that have a male-restricted expression, a phenomenon not observed in females. In said paper it was also noted that male-restricted genes present a higher divergence from their coding sequences. However, there was no association found between the evolutionary rate of the coding sequences and the levels of sexual selection. This suggests that sexual selection has, at least in a short term, a greater influence on the evolution of the expression of the genes than on the divergence of the coding sequences.

It has been proposed that several forces and mechanisms could contribute to the biased genes expression between the sexes (Parsch and Ellegren, 2013). In the case of male-biased genes, a proposed mechanism, for which there is increasing evidence, is the acquisition of a specific or enriched expression in the testicle of a new copy of a gene originated by a duplication event in tandem or retroduplication (Karr and Dorus, 2012; Parsch and Ellegren, 2013). This may be the main reason that the evolution of testicular expression is much faster than in somatic tissues (Brawand et al., 2011). The acquisition of gene expression in the testicle can lead to the gain of new proteins and functions in the spermatozoon, as previously observed in some studies (Dorus et al., 2008; Dorus et al., 2011).

6. Comparative Proteomics as a Tool to Study the Impact of Sexual Selection on Spermatozoa

As discussed in the previous sections, PCSS forces have had a strong impact on the evolution of gene expression in males (see paragraph 5.2). Although the transcriptome of the testicle seems to be a focus of action of sexual selection, the evolution of gene expression in this tissue may not show a clear correlation with spermatozoa diversification, given the high complexity of the regulatory processes that occur along spermatogenesis (see paragraph 2).

Therefore, proteomic analyses seem an adequate tool to associate the testicles changes in gene expression with the spermatozoon phenotypic diversification. Despite the wide collection of studies that have characterized the spermatozoa composition in various species (see paragraph 2), the molecular basis of male gamete diversification is yet to be evaluated. This has been mainly due to the lack of quantitative tools that allow comparing the proteome of species with different mating systems. In a recent study carried out by Vicens et al., quantitative proteomics tools were applied for the first time to compare the spermatozoon proteome between 3 species of rodents with different levels of spermatic competition (Mus musculus, M. spretus and M. spicilegus) (Vicens et al., 2017). In said study more than 1000 proteins were compared quantitatively, and those spermatozoa proteins that showed divergence patterns correlated with changes in the intensity of sperm competition were analyzed by clustering analysis (Fig. 4A). Ontology analysis on the proteins of each cluster revealed those subcellular and functional groups that respond to variation in spermatic competition. An interesting finding was the enrichment of acrosomal proteins and the receptor complex of the zona pellucida within cluster 2 (Fig. 4B), indicating that species that showed a sexual selection relaxation from the common ancestor (M. musculus) reduced the proteins expression involved in the recognition of, and interaction with, the zona pellucida. This could justify the fecundation asymmetries previously observed between these species (Martin-Coello et al., 2009). Regarding the flagellum, we observed an enrichment of the proteins that constitute the dynein framework and the axonena central apparatus, as well as a group of proteins that contribute to the microtubule motive activity, in clusters 1 and 2, positively correlated with the levels of spermatic competition (Fig. 4B). On the other hand, in clusters 3 and 4, which correspond to an abundance decrease in species with greater spermatic competition, a high representation of proteins involved in glycolysis was observed (Fig. 4C). This would reflect a lesser dependence of the species M. spretus and M. spicilegus on this metabolic pathway for ATP production (Tourmente et al., 2015).

Figure 4. Diversity of the spermatozoon proteome associated with the intensity of sperm competition. A) Clustering analysis identified patterns of divergence associated with sperm competition levels, estimated with the difference in the relative size of the testicles. The significance of the proteins belonging to each cluster is indicated by colors (purple: higher probability, green: lower probability), as well as the percentage of deviation in the protein amount associated to each cluster with respect to the expected from a uniform distribution (60 proteins per cluster).

Figure 4B&C. Diversity of the spermatozoon proteome associated with the intensity of sperm competition. B-C) Enrichment of proteins belonging to different functional groups related to the head (B) and the flagellum (C). Significant differences between clusters are marked with asterisks. The enrichment values were estimated based on the expected protein frequency, within the sperm proteome category. Figures taken and modified from Vicens et al., 2017.

Therefore, our comparative proteomic analyses suggest that some protein groups may alter their abundance level in the spermatozoon as a primary response to an increase or reduction in spermatic competition. However, we want to be cautious when interpreting our results, since we only worked with 3 species. In addition, it should be taken into account that spermatic competition is probably not the only force generating the observed proteomic variation, and that other neutral or selective processes should be considered to better understand the molecular diversification of the spermatozoon.

7. Future Prospects

Advances in the genomics and proteomics tools achieved during the last decade have allowed expanding what we know about the molecular composition of different reproductive tissues and spermatozoa. However, studying the forces and mechanisms that drive the evolution of reproductive genes still has a long way to go. The first studies integrating massive methods with analysis of comparative genomics and molecular evolution have allowed knowing the impact of positive selection on the proteome of reproductive tissues and on the spermatozoon. However, the adaptive evolution of the reproductive genes seems to be the result of heterogeneous selective forces, and among these, the degree of contribution of sexual selection has not yet been determined. Consequently, to rigorously evaluate the impact of PCSS forces on the evolution of reproductive systems and gametes, approaches that compare broad gene or protein catalogs among phylogenetically close species spanning a range of mating systems are required. However, it should always be kept in mind that selective forces may be more prominent than sexual selection, and other functions could be the result of the adaptive evolution of reproductive genes, such as the immune response.

On the other hand, the vast majority of studies that have analyzed the role of sexual selection in reproductive traits have focused only on male components, leaving aside the female counterparts. Future evolutionary and functional studies on reproductive systems should take into account the interaction and conflict between the two sexes. For example, to have a more precise understanding about the influence of PCSS forces, the spermatozoon must be studied jointly with elements of the female reproductive tract (FRT). Considering the interactions between the male and female components is also necessary to understand how the reproductive systems co-evolve.

Although inverse genetic approaches have traditionally been used to investigate the molecular basis of adaptive reproductive traits, direct genetics tools can be very valuable for identifying genes associated with differences in reproductive success. A good example is the recent work of Fisher et al., where they identified that the variation of the PRKAR1A locus predicts differences in the length of the middle piece of the spermatozoon and the efficiency of fertilization (Fisher et al., 2016).