Mammalian Sperm Proteins Are Rapidly Evolving: Evidence of Positive Selection in Functionally Diverse Genes

Dara G. Torgerson, Rob J. Kulathinal1 and Rama S. Singh

Department of Biology, McMaster University, Hamilton, Ontario, Canada


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
A growing number of genes involved in sex and reproduction have been demonstrated to be rapidly evolving. Here, we show that genes expressed solely in spermatozoa represent a highly diverged subset among mouse and human tissue-specific orthologs. The average rate of nonsynonymous substitutions per site (Ka) is significantly higher in sperm proteins (mean Ka = 0.18; N = 35) than in proteins expressed specifically in all other tissues (mean Ka = 0.074; N = 473). No differences, however, are found in the synonymous substitution rate (Ks) between tissues, suggesting that selective forces, and not mutation rate, explain the high rate of replacement substitutions in sperm proteins. Four out of 19 sperm-specific genes with characterized function demonstrated evidence of strong positive Darwinian selection, including a protein involved in gene regulation, Protamine-1 (PRM1), a protein involved in glycolysis, GAPDS, and two egg-binding proteins, Adam-2 precursor (ADAM2) and sperm-adhesion molecule-1 (SAM1). These results demonstrate the rapid evolution of sperm-specific genes and highlight the molecular action of sexual selection on a variety of characters involved in mammalian sperm function.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
A fundamental question in evolutionary biology asks which processes generate the enormous diversity of species that are observed in nature. One approach to examine the molecular foundations of species diversity has been to identify the genes that show high divergence between species because these genes may have played an important role in the early stages of species formation. But the rate of nucleotide substitution that a particular gene may experience is contingent on a variety of factors such as its physical location in the genome (Wolfe, Sharp, and Li 1989Citation ; Casane et al. 1997Citation ; Matassi, Sharp, and Gautier 1999Citation ; Williams and Hurst 2000Citation ), base composition (Wolfe and Sharp 1993Citation ), and dispensability (Hirsh and Fraser 2001Citation ). A gene's spatial pattern of expression is also a major determinant of its evolutionary rate (Civetta and Singh 1995Citation ; Duret and Mouchiroud 2000Citation ) because genes of different functional classes may evolve under different selective pressures. For example, genes expressed in the brain and nervous system may possess significant functional constraints and may tend to evolve slowly (Kuma, Iwabe, and Miyata 1995Citation ; Duret and Mouchiroud 2000Citation ), whereas genes expressed in the immune system play a role in antigen recognition and coevolution and tend to evolve more rapidly (Hughes and Nei 1988Citation ; Kuma, Iwabe, and Miyata 1995Citation ; Hughes 1997Citation ; Duret and Mouchiroud 2000Citation ). Other evidence suggests that genes involved in sex and reproduction also are evolving rapidly (Singh and Kulathinal 2000Citation ; Swanson and Vacquier 2002Citation ), particularly proteins found in the male reproductive tract (Coulthart and Singh 1988Citation ; Wyckoff, Wang, and Wu 2000Citation ; Swanson et al. 2001aCitation ).

Recently, a number of studies have focused on the rapid evolution of seminal proteins in Drosophila and have reported the presence of extensive directional selection (Aguadé, Miyashita, and Langley 1992Citation ; Clark et al. 1995Citation ; Tsaur, Ting, and Wu 1998Citation ; Swanson et al. 2001aCitation ). One explanation is that sperm competition between different males results in the rapid divergence of sperm-associated proteins, increasing the probability of successful fertilization. In marine invertebrates, male- and female-expressed genes involved in sperm-egg recognition and binding also have been shown to evolve rapidly (Swanson and Vacquier 1995Citation ; Metz and Palumbi 1996Citation ; Swanson and Vacquier 1998Citation ; Swanson et al. 2001bCitation ; Swanson and Vacquier 2002Citation ), demonstrating that molecular coevolutionary processes may drive the evolution of sex- and reproduction-related traits. Similarly, positive Darwinian selection may often be the prevailing force acting on male reproductive genes in humans and primates, including several sperm genes (Rooney and Zhang 1999Citation ; Wyckoff, Wang, and Wu 2000Citation ). It is therefore expected that sperm-expressed genes, as a group, may show higher divergence because of intersexual selective pressures acting on these genes. Morphological evidence supports this hypothesis because sperm morphology reveals substantial diversity, even between closely related species (Eberhard 1985Citation ; Pitnick 1996Citation ). Yet, even with these examples, it has not been systematically tested whether sperm genes, as a general class, are rapidly evolving. In fact, an alternative explanation is that because sperm proteins perform a critical reproductive function, the vast majority of sperm-associated genes may possess significant selective constraints and thus may follow a distribution of divergence similar to other tissue-specific genes. Here, we report that proteins specifically found in mammalian sperm are rapidly evolving when compared with a large sample of tissue-specific orthologous genes from human and mouse lineages and further demonstrate that positive Darwinian selection has acted on a variety of sperm components.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
Human and mouse sperm-specific orthologs were retrieved from HomoloGene at the NCBI database, which uses a reciprocal best hits criterion against two or more taxa to infer putative sequence orthology (http://www.ncbi.nlm.nih.gov/HomoloGene/). Sequence orthology was further confirmed by using BLASTp (Altschul et al. 1990Citation ) to retrieve human and mouse protein sequences, which were aligned using ClustalX, Version 1.81 (Thompson et al. 1997Citation ). Gene trees were drawn using the neighbor-joining algorithm (Saitou and Nei 1987Citation ) and were examined manually to confirm orthologs and exclude paralogs. Sperm specificity was determined through a critical review of the primary literature for each gene. DNA sequences coding for each sperm protein were then successfully aligned and each manually inspected using the amino acid alignment as a template. The expected numbers of substitutions per site at synonymous sites (Ks) and nonsynonymous sites (Ka) were calculated using Li's method (1993)Citation . For calculations of Ks, doublet substitutions were removed to avoid neighboring effects (substitutions between adjacent codon positions). Previous studies in mammals have found the correlation between Ks and Ka to be influenced by doublet substitutions (Duret and Mouchiroud 2000Citation ); thus, removing such substitutions reduces bias in the synonymous substitution rate because of mutations at nonsynonymous sites. A list of sperm-specific human-mouse orthologous genes with accession numbers and values of Ka and Ks are found in table 1 .


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Table 1 Sperm-Specific Human and Mouse Orthologous Genes

 
Estimates of Ka and Ks for other tissue-specific human-mouse orthologs were kindly provided by Laurent Duret at the Université Claude Bernard, Villeurbanne, France (also calculated using the method of Li [1993]Citation with doublet substitutions removed). Only genes expressed exclusively in a single adult tissue were selected for analysis, and in the testis-specific data set, genes expressed in the sperm were excluded. Using a random sample of genes, calculated divergence estimates were equal to estimates of Ka and Ks from the original data set. A Student's t-test with a Bonferroni correction for multiple tests compared the estimates of Ka and Ks between sperm-specific and other tissue-specific proteins.

Sperm proteins with more than two orthologs and with characterized function were further tested for evidence of positive selection. A maximum likelihood approach was implemented using the program codeml (Yang 1997Citation ), which uses a codon substitution model of evolutionary change. This method detects positive selection at the level of the codon (Yang 1994Citation ) and uses a likelihood ratio test to test various models of selection against a neutral model. Orthologous sperm-specific sequences from different mammalian species were extracted from GenBank. DNA sequences producing significant matches (E < 10-6) were then aligned with ClustalX, Version 1.81 (Thompson et al. 1997Citation ) and neighbor-joining trees (Saitou and Nei 1987Citation ) drawn. Sequences were bootstrapped 1,000 times, and a consensus tree was drawn and examined manually to select orthologous genes and exclude paralogs. A bootstrap level of 90% was used as a cut-off value for a significant node. Bayes theorem was also used to calculate the posterior probability that each codon belongs to a certain class of {omega} (where {omega} = dN/dS). Codons that identify with classes of {omega} > 1 are purported to be under positive selection. Because of the high divergence of many genes, we confirmed tests of positive selection by using a more conservative DNA sequence alignment by removing the immediate regions flanking indels until all sequences revealed identical nucleotides at one codon.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
Sperm-specific genes show a significantly higher nonsynonymous substitution rate (Ka) than do other tissue-specific genes combined [mean Ka (sperm) = 0.18 (N = 35) versus mean Ka (nonsperm) = 0.073 (N = 473); P < 0.001]. Similarly, if we examine each tissue type separately, we find a significantly higher Ka in sperm-specific genes than in genes expressed in most of the other tissue types (excluding the liver and lymphocyte, where lower values of Ka are found but are not significantly lower; fig. 1 ). Using a second axis of divergence, we find that sperm-specific proteins have evolved the largest change in protein size, indicating the occurrence of a larger number of indel events compared with genes from other tissues (fig. 1 ). But the correlation of higher rates of amino acid substitutions to greater differences in coding region size is weak and may not represent a common pattern among mammalian genes (R2 = 0.23, P = 0.082). Ka has been correlated previously to Ks (Wolfe and Sharp 1993Citation ; Mouchiroud, Gautier, and Bernardi 1995Citation ; Makalowski and Boguski 1998Citation ; Duret and Mouchiroud 2000Citation ), but a higher synonymous substitution rate in sperm-expressed genes is not correlated to the observed higher rate of amino acid substitution because there is no difference in Ks between sperm- and other tissue-specific genes [mean Ks (sperm) = 0.45 (N = 35) versus mean Ks (nonsperm) = 0.41 (N = 473); P = 0.123]. We also find that values of Ks do not vary between tissue types (fig. 2 ), suggesting that different selective forces and not differential mutation rates explain the differences in Ka between tissues.



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Fig. 1.—Comparison of mean protein divergence among various tissue-specific orthologs from mouse and human. Mean Ka, nonsynonymous substitution rate, and the mean length difference between orthologous coding regions were calculated. Error bars represent twice the standard error of mean. Sperm-specific genes have a significantly higher Ka than do genes expressed in most other tissue types, excluding the liver and lymphocyte, where lower values of Ka are found but are not significantly lower. Sperm-specific proteins also have the highest protein-coding length differences, indicating the occurrence of a larger number of insertion and deletion events during divergence than with other tissue types

 


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Fig. 2.—Mean synonymous substitution rate (Ks) and mean ratio of nonsynonymous to synonymous substitution rates (Ka/Ks) for various tissue-specific orthologs in mouse and human. Error bars represent twice the standard error of mean. Mean values of Ks do not significantly vary between tissue types, but sperm-specific genes have a significantly higher mean ratio of Ka/Ks than do proteins from five other tissues

 
In the immune system, high rates of nonsynonymous nucleotide substitution have been found in the major histocompatibility complex (Hughes, Ota, and Nei 1990Citation ) and immunoglobulins (Tanaka and Nei 1989Citation ; Hughes 1997Citation ), both of which are directly involved in the identification of foreign antigens. The selective forces promoting amino acid diversity in the immune system may be coevolving with foreign or toxic substances. To maintain their ability to recognize antigens, lymphocyte proteins may also experience a high rate of amino acid substitution. Similar mechanisms have also been proposed to explain the rapid evolution of sperm proteins (Swanson and Vacquier 1998Citation ; Swanson, Aquadro, and Vacquier 2001Citation ; Swanson et al. 2001bCitation ; Swanson and Vacquier 2002Citation ). Sperms must recognize and interact with proteins on the oocyte surface for successful fertilization, and such proteins in the female may also be subjected to rapid evolutionary change. For example, in the marine invertebrate, abalone, sperm lysin is believed to evolve rapidly by selective pressure caused by the rapid concerted evolution of the egg receptor (Swanson and Vacquier 1998Citation ). In mating systems such as those found in many aquatic organisms, where sperms are released freely to seek out female oocytes, a selective drive toward specificity may be expected because of the sperm's increased chance of encountering foreign material or unsuitable oocytes. But in mammalian systems, complex behavioral and ecological premating barriers exist, so we might not expect sperm-egg coevolution to drive nonsynonymous substitutions to the extent seen in free-spawning fertilization systems. But positive selection is often a primary force acting on male reproductive genes in humans and primates (Wyckoff, Wang, and Wu 2000Citation ) and appears to be driving the evolution of several mammalian egg surface proteins (Swanson et al. 2001bCitation ). Because sperm genes, in general, have a higher proportion of replacement substitutions than do proteins from other tissues, positive selection may be acting on a wider array of mammalian sperm genes other than those involved in primary sperm-egg interactions. We therefore tested for signatures of positive selection against sperm genes with characterized function to examine precise mechanisms of rapid evolution and to determine the extent of positive selection on sperm genes.

The comparison of the rate of nonsynonymous substitutions with the rate of synonymous substitutions has commonly been used to measure the selective pressures on a gene (i.e., Ka/Ks). If the rate of amino acid replacement is higher than the rate of synonymous changes, the gene is typically thought to have evolved under positive selection. But there is no clear cutoff between neutral evolution, Ka/Ks = 1, and positive selection, Ka/Ks > 1. For example, a ratio of Ka/Ks = 1.1 may be interpreted as either positive or neutral selection. In our human and mouse comparison of orthologous genes, most of the values of Ka/Ks are less than 1, providing no indication of positive selection driving sperm protein evolution but rather a relaxed selective constraint. Yet on average, sperm-specific genes have a significantly higher ratio of Ka/Ks in sperm proteins than in genes from five other tissues (fig. 2 ), and there is a twofold increase in the average Ka/Ks of sperm-specific genes when compared with all tissue-specific genes [mean Ka/Ks (sperm) = 0.50 (N = 35) versus mean Ka/Ks (nonsperm) = 0.19 (N = 473); P = 0.001]. Although the data may suggest prematurely that sperm genes have lower selective constraints than do other tissue-specific genes, ratios of Ka/Ks are generally not powerful predictors of positive selection because they represent an average value over all codons and cannot identify positively selected amino acid sites. A maximum likelihood approach has been more successful in detecting positively selected sites within a gene, even when the average Ka/Ks over the entire gene is less than 1 (Yang 1994Citation ; Swanson et al. 2001bCitation ). This method is preferred over average measures of Ka/Ks in detecting positive selection because it can test whether similar selective forces are acting over the entire length of a gene.

Using this approach, sperm-specific genes with known functions were tested for the presence of positively selected codon sites (table 2 ). The first test compares an evolutionary model that estimates a single class of the parameter, {omega} = dN/dS, with the constraint, 0 < {omega} < 1 (M0), to a model that allows for three site classes, including one that estimates {omega} greater than unity (M3). A total of 13 out of 19 sperm-specific genes show a significantly better fit to model M3 (table 2 ), suggesting heterogeneity in {omega} across most of the sperm-specific genes. A second, more conservative test compared models that assume a beta distribution on the parameter {omega}: one model, where 0 < {omega} < 1 (M7), is compared with a model that contains the additional site class, {omega} > 1 (M8). Four out of 19 sperm-specific genes show a significantly better fit to model M8 over M7 with relatively large values of {omega} (table 2 ), indicating that these genes contain positively selected amino acids. Surprisingly, these four sperm genes represent diverse functional classes, including a protein involved in chromatin condensation, protamine-1 (PRM1), a protein involved in glycolysis, sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS), and two proteins involved in sperm-egg binding, Adam 2 precursor (ADAM2) and sperm adhesion molecule 1 (SAM1).


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Table 2 Likelihood Ratio Test of Positive Selection in Sperm Proteins with Different Functions

 
Further analyses of these four genes reveal many interesting aspects of the selective process in mammalian sperm evolution. Codons were identified in each gene that are likely, under a Bayesian framework, to be under positive selection (fig. 3 ). Many of the positively selected sites in the two egg-binding proteins, ADAM2 and SAM1, lie in the regions of the protein believed to interact with the female egg. In ADAM2, sperm disintegrins are thought to interact with egg integrins allowing for secondary binding, suggesting that coevolution with egg integrins may drive positive selection in this region. In SAM1 most of the protein may interact with the egg, coinciding with a more even dispersal of positively selected sites: the hyaluronidase domain digests the hyaluronic acid of the egg, whereas the inferred zona pellucida–binding domain may be involved in egg recognition. Our findings also demonstrate that positive selection resulting from intersexual forces does not always present itself in the most obvious way, such as in sperm-egg binding. PRM1 replaces transitional protein-2 (TP2) in the sperm head for condensation of the chromatin during spermatogenesis and, at first glance, does not appear to directly interact with egg proteins. Once fertilization takes place, PRM1 interacts with an acidic amino acid motif of the ß subunit of casein kinase II (Ohtsuki et al. 1996Citation ), an important regulatory protein found in the fertilized egg responsible for cellular metabolic alteration. Also, chromatin condensation may be important in determining the shape of the sperm head (Curry and Watson 1995Citation ), which may affect the ability of the spermatid to fertilize the egg. On the other hand, positive selection in sperm proteins may not solely be male-female coevolutionary processes. The enzyme GAPDS is not known to interact directly with female proteins but is likely to have an essential role in regulating energy production for motility (Welch et al. 2000Citation ). A distinct clustering of positively selected sites in GAPDS occurs in the proline-rich 72 amino acid segment that does not have a homologous region in the somatic GAPD, suggesting that this region may have a unique and adaptive function in sperms. Proline-rich regions may mediate protein-protein interactions (Williamson 1994Citation ), and in sperms this region is hypothesized to anchor GAPDS to other glycolytic enzymes or to the fibrous sheath for efficient ATP diffusion to motor proteins of the flagellum (Welch et al. 2000Citation ). Positive selection in GAPDS may therefore be driven by sperm competition for energy production in fertilization or by coevolution with other sperm proteins rather than with female proteins as suggested by ADAM2, SAM1, and PRM1. Overall, the identification of positive selection in such a wide array of spermatozoan functional classes demonstrates the highly cryptic nature of sexual selection and emphasizes the far-reaching effects of sexual selection on a variety of molecular mechanisms.



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Fig. 3.—Location of codons that are likely to be under positive selection in four sperm-specific proteins in mammals. Codon numbering is based on the aligned human and mouse sequences with indels removed, and the alignments are available by request from D.G.T. In SAM1 and ADAM2, positively selected sites coincide with some regions hypothesized to interact with egg proteins. In GAPDS the first 72 amino acids are unique to the sperm-specific isoform where the majority of codons under positive selection are located

 
Selection on sexual traits may be an important and ubiquitous driver of evolutionary change (Carson 1997Citation ; Singh and Kulathinal 2000Citation ). Ever since Darwin (1871)Citation first attempted to explain the often-extreme sexual dimorphism between males and females, sexual selection has been proposed to play a major role in the generation of phenotypic diversity by speciation (Lande 1981Citation ; Carson 1997Citation ). Recently, the focus of sexually selected targets has broadened from male secondary sexual characters to a whole range of traits involved in sex and reproduction. In particular, sperm morphology has become an excellent example of a highly diverged male structure (Eberhard 1985Citation ; Pitnick 1996Citation ), and sexual selection has been suggested to drive its evolution through such processes as sperm competition (Karr and Pitnick 1999Citation ), male-female coevolution (Swanson et al. 2001bCitation ), and sexual conflict (Rice 1996Citation ). Sexual selection has been recently implicated at the molecular level on Drosophila accessory gland proteins used to assist sperm in successful fertilization (Cirera and Aguadé 1997Citation ; Tsaur, Ting, and Wu 1998Citation ) and de novo genes expressed exclusively in the Drosophila testis (Nurminsky et al. 1998Citation ). In mammals, female egg proteins used to bind sperm have been recently demonstrated to evolve through positive selection (Swanson et al. 2001bCitation ), suggesting a molecular coevolutionary arms race. Our study demonstrates positive Darwinian selection on various mammalian sperm components and suggests that sexual selective mechanisms in the form of male-female coevolution, sperm competition, and sexual conflict may have played a critical role in the generation of phenotypic diversity in mammalian lineages.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
We thank G. B. Golding and R. A. Morton for their invaluable suggestions and an anonymous reviewer for helpful comments on the manuscript. This work was supported by the Natural Sciences and Engineering Research Council of Canada through a graduate scholarship to D.G.T and a research grant to R.S.S.


    Footnotes
 
Antony Dean, Reviewing Editor

1 Present address: Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts Back

Keywords: tissue-specific genes sperm rapid evolution positive Darwinian selection Back

Address for correspondence and reprints: Dara G. Torgerson, Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1. torgerdg{at}mcmaster.ca Back


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 Introduction
 Materials and Methods
 Results and Discussion
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Accepted for publication July 17, 2002.