Abstract

X-linked genes can evolve slower or faster depending on whether most recessive, or at least partially recessive alleles are deleterious or beneficial due to their hemizygous expression in males. Molecular studies of X chromosome divergence have provided conflicting evidence for both a higher and lower rate of nucleotide substitution at both synonymous and nonsynonymous sites, depending on the nucleotide sites sampled. Using human and mouse orthologous genes, we tested the hypothesis that genes encoding male-specific sperm proteins are evolving faster on the X chromosome compared with autosomes. X-linked sperm proteins have an average nonsynonymous mutation rate almost twice as high as sperm genes found on autosomes, unlike other tissue-specific genes, where no significant difference in the nonsynonymous mutation rate between the X chromosome and autosomes was found. However, no difference was found in the average synonymous mutation rate of X-linked versus autosomal sperm proteins, which along with corresponding higher values of Ka/Ks in X-linked sperm proteins suggest that differences in selective forces and not mutation rates are the underlying cause of higher X-linked mammalian sperm protein divergence.

Introduction

Sex chromosomes and sex-linked genes have become a central focus of research in many areas of evolutionary biology, including the molecular evolution of sex determining pathways (Pask and Graves 1999; Nagai 2001), segregation distortion (de La Casa et al. 2002), antagonistic sexual evolution (Gibson, Chippindale, and Rice 2002), and Haldane's rule and speciation (Zeng and Singh 1993; Turelli and Begun 1997). However, there is conflicting evidence as to whether the evolutionary rate of the X chromosome is accelerated or reduced compared with that of autosomes.

In the first scenario, there are greater selective constraints on X-linked genes compared with autosomal genes, as any recessive deleterious mutations will be expressed in males and may be subject to stronger purifying selection. Two studies in mammals support this theory and have found X-linked genes to have a significantly lower nonsynonymous substitution rate (Wolfe and Sharp 1993; McVean and Hurst 1997). However, the latter study suggests nonsynonymous substitution rates may in fact be higher than expected on the X chromosome after controlling for a reduced mutation rate (McVean and Hurst 1997).

Alternatively, there are certain conditions where genes on the X chromosome can evolve faster than those on autosomes. If the majority of new mutations are beneficial and are at least partially recessive, haploid expression in the heterogametic sex will result in higher rates of sequence divergence (Charlesworth, Coyne, and Barton 1987). Two studies support the second scenario of faster X evolution due to an increased likelihood of positive selection or a selective sweep of beneficial recessive mutations on the X chromosome. First, reduced polymorphism on the X chromosome has been reported in Drosophila simulans, suggesting that a form of positive selection may be acting on sex chromosomes (Begun and Whitley 2000). Second, gene duplications on the X chromosome in D. melanogaster are highly diverged due to relaxed selective constraint and likely positive selection acting on duplicate copies (Thornton and Long 2002). However, Betancourt, Presgraves, and Swanson (2002) found no evidence for faster X evolution in male-specific genes in Drosophila, but this has not been tested in mammals.

There is little evidence in mammals to suggest a higher divergence of X-linked genes in general; however, there have been no attempts to identify candidate X-linked genes that may be under rapid evolution due to selection on beneficial alleles. The faster evolution of the X chromosome is expected to be enhanced for genes with male-specific expression, as they are always haploid expressed, allowing advantageous recessive mutations to be selected for. The majority of deleterious mutations are thought to be recessive (Crow and Temin 1964; Mukai et al. 1972); however, for male-specific haploid expressed genes, it is not known whether most mutations are deleterious. Previous reports show that many mammalian sperm proteins are evolving rapidly under positive selection (Torgerson, Kulathinal, and Singh 2002; Swanson, Nielsen, and Yang 2003), suggesting that mutations in these genes may often be nondeleterious or even beneficial to rapidly adapting sperm proteins. Moreover, there is a disproportionately high number of sperm proteins found on the mammalian X chromosome (Wang et al. 2001), which may suggest adaptive advantages for sperm proteins to be located on the X chromosome. With the combined evidence of positive selection acting on many sperm proteins and a higher abundance of mammalian sperm proteins on the X chromosome, we hypothesized that sperm proteins on the X chromosome are evolving faster than sperm proteins on the autosomes due to selection acting on beneficial alleles. We provide evidence here that sperm proteins are significantly more diverged on the mammalian X chromosome than sperm proteins located on the autosomes and discuss why this might be so.

Materials and Methods

We defined sperm-specific genes as genes that code for proteins that are only known to be present or expressed in the spermatogonia or mature sperm, including those known to have specialized functions in the sperm. Genes were selected through a primary literature search for sperm proteins that fit the above criteria; however, many sperm-specific genes were identified in Wang et al. (2001) using RT-PCR. Human and mouse sperm-specific orthologs were retrieved and aligned as described in Torgerson, Kulathinal, and Singh (2002). The sperm-specific human and mouse orthologous genes included in this study are as follows, with GenBank accession numbers for human/mouse: ACRV1 (NM_001612/NM_007391), ADAM18 (AJ133004/AF167405), ADAM2 (NM_001464/U16242), ACTRT1 (AF440739/AK006855), CYLI (Z22780/XM_285518), FTHL17 (AF285592/AF285569), GAPDS (NM_014364/NM_008085), LDH (J02938/L10389), MOV10L1 (AF285604/AF285587), MTL5 (U86074/NM_010841), NASP (NM_002482/NM_016777), NR6A1 (U64876/U14666), NXF (AF285596/AF285575), ODF1 (Q14990/Q61999), ODF2 (NM_002540/NM_013615), PRKA1 (NM_003488/NM_009648), PRKA4 (NM_003886/NM_009651), PRM2 and PRM3 (Z46940/Z47352), SAM1 (X84347/U33958), SPAG1 (AF311312/AF181252), SPAG17 (NM_017425/NM_011449), SPAG6 (AF079363/AF173866), STK31 (AF285599/AF285580), TAF2Q (AF285595/AF285574), TDRD1 (AF285606/AF285591), TEX11 (AF285594/AF285572), TEX12 (AF285600/AF285582), TEX14 (AF285601/AF285584), TEX15 (AF285605/AF285589), TP1 (M59924/X12521), USP26 (AF285593/AF285570), and ZPBP (NM_007009/D17569).

The expected numbers of substitutions per site at synonymous sites (Ks) and at nonsynonymous sites (Ka) were calculated using Li's method (1993). Estimates of Ka and Ks for other tissue-specific human/mouse orthologs were kindly provided by Laurent Duret at the Université Claude Bernard, France (http://pbil.univ-Lyon1.fr/datasets/Duret_Mouchiroud_1999/data.html), which were also calculated using the method of Li (1993). Only genes expressed exclusively in a single adult tissue were selected for analysis, and any genes expressed in the sperm were excluded. Using a random sample, recalculated divergence estimates were equal to estimates of Ka and Ks from the original data set. Chromosomal location was retrieved from UniGene at NCBI (http://www.ncbi.nlm.nih.gov/UniGene/). An analysis of variance was used to compare the estimates of Ka, Ks, and Ka/Ks between tissue-specific genes on the X chromosome versus autosomes, as well as to compare values of Ks among autosomes.

Results and Discussion

A total of 8 out of 33 human/mouse orthologous sperm-expressed genes were found to be located on the X chromosome, similar to the findings of Wang et al. (2001) that there is a high abundance of sperm-expressed genes found on the mammalian X chromosome. We do not find this trend among any other tissue-specific genes, including several testis-specific genes (fig. 1), supporting the idea that the mammalian X chromosome has become specialized in sperm function.

X-linked sperm proteins have generally higher rates of amino acid replacement substitutions (Ka) and values of Ka/Ks than sperm proteins on autosomes (fig. 2). In fact, sperm-specific proteins on the X chromosome have rates of amino acid replacement substitutions more than twice as high as the rate on autosomes (Ka = 0.147 versus 0.309, P < 0.001), showing a significantly higher divergence of X-linked sperm proteins (table 1). However, this is not a general trend among tissue-specific genes and appears to only be true for sperm proteins, as no differences were found in nonsynonymous substitution rates between the X chromosome and autosomes for tissue-specific data after sperm proteins were removed (table 1). Controversy also surrounds the question of whether the synonymous substitution rate (Ks) is different between the X chromosome and autosomes (Bauer and Aquadro 1997; McVean and Hurst 1997; Lercher, Williams, and Hurst 2001). We therefore corrected values of Ka by the synonymous mutation rate and consistently found a significantly higher nonsynonymous divergence of X-linked sperm genes compared with those on the autosomes. Therefore, possible differences in the mutation rate between the X chromosome and autosomes do not appear to be a factor in nonsynonymous divergence.

As for direct comparisons of the synonymous mutation rate, we find significantly lower values of Ks on the X chromosome for tissue-specific genes, consistent with the hypothesis of male-driven evolution or of a reduced mutation rate on the X chromosome (table 1). However, when tissue-specific genes are compared between human chromosomes, we also find chromosomes 14 and 17 to have significantly lower values of Ks compared with other autosomes (data not shown), as previously reported by Lercher, Williams, and Hurst (2001), so we cannot conclude that the X chromosome is evolving any slower than autosomes with the data we have. However, the question of reduced substitution rate on the X chromosome does not affect our findings of highly diverged sperm genes being located on the X chromosome. If there were a reduction in the overall nucleotide substitution rate on the X chromosome, it would suggest that X-linked sperm proteins could be even more highly diverged without a conflicting reduction in mutation rate. We therefore suggest that X-linked sperm proteins are rapidly evolving due to selection and not due to elevated mutation rates compared with autosomes. Values of Ka/Ks can be interpreted as an indication of whether selective constraints are acting on a gene, with higher ratios indicating lower selective constraints. When ratios of Ka/Ks are compared across sperm proteins (fig. 2), values of Ka/Ks are significantly higher for X-linked sperm proteins (table 1), consistent with the idea that sperm proteins on the X chromosome are more rapidly evolving due to relaxed selective constraint or stronger positive selection acting on beneficial alleles.

Rapid evolution, including positive selection, has been reported to act on genes involved in the immune response (Hughes, Ota, and Nei 1990; Zhang and Nei 2000) and is particularly widespread in genes involved in sex and reproduction (Singh and Kulathinal 2000; Swanson and Vacquier 2002). To determine why sperm proteins on the X chromosome are evolving faster than sperm proteins on autosomes, a look at the functions of X-linked sperm proteins may give more insight. First, meiotic drive or sexually antagonistic genes may be preferentially located on the X chromosome and may explain why there is a high abundance of sperm proteins on the X chromosome (discussed in Wang et al. 2001). If either meiotic drive or sexually antagonistic genes are evolving rapidly, it may explain a higher rate of amino acid substitution on the X chromosome for these sperm-expressed proteins. Alternatively, rapidly evolving sperm proteins not restricted to meiotic drive or sexually antagonistic genes may have adaptive advantages to being located on the X chromosome to allow for more rapid changes in amino acid composition. Our previous findings show sperm proteins of diverse functional classes are rapidly evolving (Torgerson, Kulathinal, and Singh 2002); therefore, highly diverged sperm proteins on the X chromosome may not be limited to sexually antagonistic or meiotic drive genes.

The functions of X-linked sperm proteins are variable (table 2); however, their past or current involvement in sexual antagonism or in meiotic drive is for the most part unknown. Two of these genes, cylicin I and actin-related protein T1, may be important in sperm head structure, which may affect a sperm's ability to fertilize the egg. The contribution of sperm-egg coevolution in driving the rapid evolution of male traits has been shown previously (see Swanson and Vacquier 2002); however, we find the majority of genes that are involved in direct sperm-egg interactions to be autosomal. It is therefore becoming more apparent that diverse and complex forces of natural and sexual selection are influencing the rapid evolution of male-expressed genes, including chromosomal location.

In conclusion, we have shown that sperm proteins on the X-chromosome are highly diverged compared with those on autosomes for human and mouse comparisons, and we hypothesize that these differences are likely due to adaptive evolution driven by diverse aspects of selection acting on X-linked sperm proteins. Sexual selection can influence sex gene-pool evolution in terms of broad-sense sexual selection and may not be limited to secondary sexual traits and mating behavior (Civetta and Singh 1999). The hemizygosity of the X chromosome, X-autosomal interaction, higher proportion of sex genes on the X chromosome, and stronger antagonistic and/or adaptive sexual selection in the male, can all collectively mount their effects to drive X-linked sperm protein evolution at a faster rate than the rest of the genome.

Mark Springer, Associate Editor

Fig. 1.

The proportions of human X-linked, tissue-specific genes for various tissues. The number of genes sampled for each tissue is in brackets. A high abundance of sperm-specific genes are found on the X chromosome (eight out of 33), consistent with previous findings by Wang et al. 2001. The proportion of X-linked sperm genes is higher than that found in any other tissue-specific genes, including the testes, supporting the idea that the mammalian X chromosome has specialized in sperm function

Fig. 1.

The proportions of human X-linked, tissue-specific genes for various tissues. The number of genes sampled for each tissue is in brackets. A high abundance of sperm-specific genes are found on the X chromosome (eight out of 33), consistent with previous findings by Wang et al. 2001. The proportion of X-linked sperm genes is higher than that found in any other tissue-specific genes, including the testes, supporting the idea that the mammalian X chromosome has specialized in sperm function

Fig. 2.

Measures of Ka and Ks (above) and Ka/Ks (below) for sperm-specific proteins in mammals. Ka and Ks were calculated using the method of Li (1993). Sperm-specific genes on the X chromosome (in black) show a tendency towards higher values of Ka and on average have a significantly higher Ka than sperm-specific genes found on the autosomes (P < 0.001). However, values of Ks are more scattered and on average show no significant differences between X-linked and autosomal sperm proteins (P = 0.90)

Fig. 2.

Measures of Ka and Ks (above) and Ka/Ks (below) for sperm-specific proteins in mammals. Ka and Ks were calculated using the method of Li (1993). Sperm-specific genes on the X chromosome (in black) show a tendency towards higher values of Ka and on average have a significantly higher Ka than sperm-specific genes found on the autosomes (P < 0.001). However, values of Ks are more scattered and on average show no significant differences between X-linked and autosomal sperm proteins (P = 0.90)

Table 1

A Comparison of the Synonymous (Ks) and Nonsynonymous (Ka) Nucleotide Substitution Rates on Autosomes Versus X-Chromosome for Tissue-Specific Genes Using an ANOVA.

 Autosome X Chromosome P-value 
Sperm     
    N 25  
    Ks 0.64 0.63 0.90 
    Ka 0.15 0.31 <0.001** 
    Ka/Ks 0.25 0.50 0.0019* 
All tissue (no sperm)     
    N 388 20  
    Ks 0.50 0.39 <0.001** 
    Ka 0.072 0.074 0.88 
    Ka/Ks 0.13 0.17 0.15 
 Autosome X Chromosome P-value 
Sperm     
    N 25  
    Ks 0.64 0.63 0.90 
    Ka 0.15 0.31 <0.001** 
    Ka/Ks 0.25 0.50 0.0019* 
All tissue (no sperm)     
    N 388 20  
    Ks 0.50 0.39 <0.001** 
    Ka 0.072 0.074 0.88 
    Ka/Ks 0.13 0.17 0.15 

Note.—*Significant difference (P < 0.05); **highly significant difference (P < 0.001).

Table 2

List of Human Sperm Genes on the X Chromosome with Possible Functions and References.

Gene Possible Function Reference 
A kinase anchor protein 4 (PRKA4) Sperm motility; anchors enzymes near its physiological substrates, regulates signal transduction pathways  Turner et al. 1998 
Actin-related protein T1 (ACTRT1) Component of the cytoskeletal calyx of the mammalian sperm head Heid et al. 2002 
Cylicin I (CYLI) Contributes to the cytoskeletal structure and morphogenesis of the sperm head Hess et al. 1995 
Ferritin heavy polypeptide–like 17 (FTHL17) Iron metabolism Lawson et al. 1991 
Nuclear RNA export factor 2 (NXF)  Homolog encodes nuclear RNA export factors Segref et al. 1997 
  Gruter et al. 1998 
  Kang and Cullen 1999 
  Herold et al. 2000 
TBP-associated factor, RNA polymerase II, Q (TAF2Q) Autosomal homolog interacts with transcriptional activators Chiang and Roeder 1995 
Testis-expressed gene 11 (TEX11) Unknown Wang et al. 2001 
Ubiquitin-specific protease 26 (USP26) Deubiquitinating enzyme Baker, Tobias, and Varshavsky 1992 
Gene Possible Function Reference 
A kinase anchor protein 4 (PRKA4) Sperm motility; anchors enzymes near its physiological substrates, regulates signal transduction pathways  Turner et al. 1998 
Actin-related protein T1 (ACTRT1) Component of the cytoskeletal calyx of the mammalian sperm head Heid et al. 2002 
Cylicin I (CYLI) Contributes to the cytoskeletal structure and morphogenesis of the sperm head Hess et al. 1995 
Ferritin heavy polypeptide–like 17 (FTHL17) Iron metabolism Lawson et al. 1991 
Nuclear RNA export factor 2 (NXF)  Homolog encodes nuclear RNA export factors Segref et al. 1997 
  Gruter et al. 1998 
  Kang and Cullen 1999 
  Herold et al. 2000 
TBP-associated factor, RNA polymerase II, Q (TAF2Q) Autosomal homolog interacts with transcriptional activators Chiang and Roeder 1995 
Testis-expressed gene 11 (TEX11) Unknown Wang et al. 2001 
Ubiquitin-specific protease 26 (USP26) Deubiquitinating enzyme Baker, Tobias, and Varshavsky 1992 

We would like to thank Laurent Duret for providing us with data on tissue-specific genes. Also, many thanks to Richard A. Morton, Associate Editor Mark Springer, and two anonymous reviewers for their helpful comments and suggestions. The Natural Sciences and Engineering Research Council of Canada supported this work through a graduate scholarship to D.G.T and through a research grant to R.S.S.

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