Figures
Abstract
The mammalian CatSper ion channel family consists of four sperm-specific voltage-gated Ca2+ channels that are crucial for sperm hyperactivation and male fertility. All four CatSper subunits are believed to assemble into a heteromultimeric channel complex, together with an auxiliary subunit, CatSperβ. Here, we report a comprehensive comparative genomics study and evolutionary analysis of CatSpers and CatSperβ, with important correlation to physiological significance of molecular evolution of the CatSper channel complex. The development of the CatSper channel complex with four CatSpers and CatSperβ originated as early as primitive metazoans such as the Cnidarian Nematostella vectensis. Comparative genomics revealed extensive lineage-specific gene loss of all four CatSpers and CatSperβ through metazoan evolution, especially in vertebrates. The CatSper channel complex underwent rapid evolution and functional divergence, while distinct evolutionary constraints appear to have acted on different domains and specific sites of the four CatSper genes. These results reveal unique evolutionary characteristics of sperm-specific Ca2+ channels and their adaptation to sperm biology through metazoan evolution.
Citation: Cai X, Clapham DE (2008) Evolutionary Genomics Reveals Lineage-Specific Gene Loss and Rapid Evolution of a Sperm-Specific Ion Channel Complex: CatSpers and CatSperβ. PLoS ONE 3(10): e3569. https://doi.org/10.1371/journal.pone.0003569
Editor: Arnold Schwartz, University of Cincinnati, United States of America
Received: September 3, 2008; Accepted: October 9, 2008; Published: October 30, 2008
Copyright: © 2008 Cai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported, in part, by National Institutes of Health Grants HD045339 and NIH/U01 45857 (D.E.C.), and a Postdoctoral Fellowship Award (0625403U) from the American Heart Association (X.C.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
In spermatozoa, Ca2+ influxes through plasma membrane Ca2+ channels play a key role in mediating sperm maturation, motility, and the acrosome reaction [1]–[3]. Sperm motility is driven by flagellar ATP-dependent dynein motor proteins. Basic flagellar architecture is conserved across species; flagellar movement does not require calcium, or even a plasma membrane. In general, sperm acquire motility after they exit the male reproductive system.
Both initiation of motility and modulation of motility vary in a species-specific fashion. Initiation of sperm motility display many features distinct from modulation of motility. In many mammals, motility is characterized by symmetrical movement of the tail and progressive movement (normal swimming), but the initiating signal or signals are poorly understood. Hyperactivated motility in mammals occurs later, as the sperms encounter progressively more alkaline environments as they ascend the reproductive tract. The primary characteristic of sperm hyperactivated motility in well-characterized mammalian species is a large bend angle between head and tail [4], [5]. This larger sweep of the tail results in substantially more force than the force of sperm cells swimming before hyperactivation [6]. This force has been proposed to free sperm from surfaces or trapping spaces in the uterus and oviduct, and/or penetrate the cumulus and the thick protective wall of the zona pellucida [7]. In sea urchin, the chemoattractant peptide, Resact, initiates an increase in intracellular [Ca2+] and change in the bend angle, resulting in larger swimming arcs [8]. In contrast, marine fish spermatozoa motility [9] is initiated by the large change in osmolality as they exit the fish and encounter seawater, but this initiation of motility is not Ca2+-dependent [10]. These marine teleosts have up to 10 times the beat frequency of mammalian sperm cells, but swim only for short distances to eggs deposited in seawater [9]. A change in flagellar bend angle, equivalent to hyperactivation in mammals, has not been reported, and indeed may not be required for sperm docking to the marine fish egg pyle. In birds and reptiles, sperm are often stored in specialized tubules connected to the oviduct. In chickens, initiation, or re-initiation of motility may be related to temperature sensitivity of a plasma membrane Ca2+ ATPase [11]. Thus, although all spermatozoa have the basic ATP-dynein motor required for normal swimming, they appear to differ in modulation of motility in a species-specific manner.
Several types of Ca2+-permeant channel proteins, such as voltage-gated Ca2+ (CaV), CatSper, cyclic nucleotide-gated (CNG), and Transient Receptor Potential (TRP) channels, have been detected in spermatocytes or spermatozoa [1]–[3], [12], but until recently their function could only be assayed in spermatocytes. Among the Ca2+-permeant channels, CatSper channels mediate the dominant Ca2+-carrying current in mouse epididymal spermatozoa and induce sperm hyperactivation during sperm capacitation [13]. Intracellular alkalinization triggers CatSper activation and subsequent hyperactivation of motility in mice. In addition, in the absence of CatSper current, sperm cell motility endurance declines [12]–[15].
The mammalian CatSper family of ion channels is composed of four members, CatSper1-4 [14], [16]–[19]. All four CatSper proteins are expressed in sperm cells and functionally localized to the principal piece of the sperm tail [12]. Like most of the voltage-gated ion channels [20], the six-transmembrane-spanning (6-TMS) CatSpers are believed to form a tetrameric structure. All four CatSpers seem to be required to mediate functional alkalinization-activated Ca2+-selective sperm currents [12]. Targeted deletion of any of the four CatSper genes results in mouse male infertility and an identical sperm cell phenotype including loss of sperm hyperactivation [12], [14], [21]–[23], while mice lacking other ion channel genes, if viable, are fertile [1]. Interestingly, a sperm-specific two-TMS protein, CatSperβ, is associated with the CatSper1 channel subunit in mouse testis [24]. CatSperβ displays a similar localization pattern with CatSpers, copurifies with CatSper proteins, and is absent in CatSper1−/− sperm. Thus, CatSperβ is an auxiliary subunit of the CatSper channel complex [24], but its role in channel activity is unknown.
In the absence of suitable heterologous expression systems, our current understanding of the physiological role of CatSpers is primarily based on studies using mouse models [12]–[14], [21]–[23]. Reproductive proteins usually undergo rapid evolution and functional divergence because selective evolutionary forces such as sperm competition, sexual selection and sexual conflict may act on reproductive proteins to generate phenotypic diversity through speciation [25]–[27]. In addition, comparative genomics studies and evolutionary analyses can provide insights into molecular, biophysical, and biochemical analyses of functional and regulatory mechanisms of ion channels and transporters [28]–[33]. Therefore, to better understand the functional role of the CatSper channel complex, we set out to determine the evolutionary origins and patterns of CatSpers and CatSperβ using extensive database mining and rigorous phylogenetic analyses, and to analyze the evolutionary rates and functional divergence of CatSpers and CatSperβ.
Materials and Methods
Database mining, sequence alignment and phylogenetic analysis
TBlastN and BlastP searches [34] using protein sequences of Homo sapiens CatSper and CatSperβ protein sequences were performed on the genomic and protein databases of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/blast/), Ensembl (http://www.ensembl.org/Multi/blastview), and the Joint Genome Institute (JGI) (http://www.jgi.doe.gov/). H. sapiens and Nematostella vectensis CatSper and CatSperβ sequences were also used for BlastN and TBlastN searches of three sponge databases - Amphimedon queenslandica genomic traces at NCBI, SpongeBase (http://spongebase.uni-mainz.de/), and Oscarella carmela EST database [35]. TBlastN searches were also conducted on the survey genomic database of the elephant shark, Callorhinchus milii (http://esharkgenome.imcb.a-star.edu.sg/). Protein sequences of the bacterial NaV channel superfamily were obtained from the bacterial protein cluster CLS1187052 at the NCBI protein cluster database and sequences characterized previously [36].
Sequence alignments, manual editing, and phylogenetic analysis for the collected dataset were carried out essentially as previously described [29], [32], [37].
Chromosome synteny
The Evolutionarily Conserved Regions (ECR) browser (http://ecrbrowser.dcode.org/) was used for initial screening of syntenic chromosomal regions among H. sapiens, Mus musculus, Gallus gallus, Fugu rubripes, and Xenopus tropicalis genomes. However, only M. musculus and G. gallus genomes showed substantial synteny in H. sapiens chromosomal regions flanking the CatSpers and CatSperβ genes, and were used for further analysis. The flanking orthologous genes were identified on the ECR browser and/or the NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/).
Estimation of non-synonymous (dN) to synonymous (dS) nucleotide substitution ratio
H. sapiens and M. musculus mRNA sequences of CatSper and CatSperβ genes were retrieved from the NCBI database, and then converted to codon alignments by the PAL2NAL server [38], using corresponding protein sequence alignments. dN and dS values were calculated with the codeml program implemented in the PAML package [39].
Results and Discussion
Development of four distinct CatSper genes and CatSperβ in early Eumetazoa
Mouse gene knockout studies indicate that all four CatSper subunits are required to mediate functional Ca2+-selective sperm currents necessary for sperm hyperactivation [12]–[14], [21]. However, previous reports have suggested the presence of fewer copies of CatSper homologues in early deuterostomes: three in Ciona intestinalis [40] and two in sea urchin testis [24]. Here, extensive genomic analysis of two sea squirts, C. intestinalis and Ciona savignyi, the sea urchin, Strongylocentrotus purpuratus, and the most basal extant chordate lineage, the amphioxus Branchiostoma floridae [41], [42], demonstrated the presence of four CatSper subunits and single copies of CatSperβ in these four species (Fig. 1A and Supplementary Table S1). Therefore, the CatSper channel complex containing four CatSper subunits and one CatSperβ had developed in early deuterostomes.
The phylogenetic trees of the CatSper protein family (A) and the CatSperβ protein family (B) were constructed using the maximum likelihood approach [37]. Two putative primitive Ca2+ channels (MbrCaVS5 and NveCaVS17) were used as the outgroup for the CatSper family. Bootstrap values of more than 60 are shown at corresponding branches. The CatSper1-4 groups are indicated by rectangular bars, with invertebrate CatSpers filled with white and vertebrate CatSpers with gray. Note that protein sequences that failed in the chi-square test in Tree-Puzzle [69] or contained more than 15% gaps in the refined alignments were not subjected to phylogenetic analysis (Table S1). Abbreviations used: Aca, A. carolinensis; Bfl, B. floridae; Bta, B. taurus; Cfa, C. familiaris; Cin, C. intestinalis; Cja, C. jacchus; Cpo, C. porcellus; Csa, C. savignyi; Eca, E. caballus; Hsa, H. sapiens; Mdo, M. domestica; Mmu, M. mulatta; Mus, M. musculus; Nve, N. vectensis; Oan, O. anatinus; Pan, P. anubis; Ptr, P. troglodytes; Rno, R. norvegicus; Spu, S. purpuratus.
As one of the unicellular ancestors of Metazoa [43], choanoflagellates possess homologues of many components of animal Ca2+ signaling and amplification pathways (the Ca2+ signaling ‘toolkit’ [44]) but not CatSper channels [33]. To further determine the evolutionary origins of CatSper channels, we next searched the currently available databases of metazoans that branched off the metazoan stem before the radiation of bilaterians – the genome sequences of Cnidarian Nematostella vectensis [45] and Placozoa Trichoplax adhaerens at JGI, the EST database of Ctenophora Pleurobrachia pileus, and EST and genomic trace databases of ancestral metazoan Porifera sponges.
Indeed, N. vectensis has four CatSper gene homologues, which can be classified into CatSper1-4 based on phylogenetic analysis, and a single homolog of CatSperβ (Fig. 1A and Table S1). Three EST sequences were also identified in the P. pileus EST database, which were further categorized as CatSper1 and CatSper2 (Table S1). No significant hits were found in the sponge EST and genomic trace databases and in the Placozoa genome. It should be noted that the negative results from currently available sponge databases should be viewed as provisional until the complete genome or sperm EST sequences from sponges have been examined.
The transmembrane segments (TMS) and the putative pore region of human and mouse CatSpers are highly conserved [17]. Sequence alignment of CatSper sequences identified in this study showed similar observations across invertebrate and vertebrate species (Fig. 2). The key aspartate residue of CatSpers, presumably corresponding to the highly conserved glutamate residues (or occasionally aspartate residues) in the pore loop regions that underlie Ca2+ selectivity in many CaV channels [46], is absolutely conserved (Fig. 2).
The putative pore regions of CatSpers from selected species, corresponding to amino acids 210–239 of human CatSper3 (GenBank Accession No., NP_821138.1), were aligned and manually edited to improve alignments. Invertebrate CatSpers are indicated with white rectangular bars and vertebrate CatSpers with gray rectangular bars. The key aspartate residue of CatSpers is overlined with an asterisk symbol. For abbreviations, please refer to the Fig. 1 legend.
The identification of four CatSper homologues and CatSperβ in Cnidarians, but not in choanoflagellates, suggests that the origin of the CatSper complex could be dated back to early Eumetazoa, much earlier than previously thought [24]. Many other ion channels and transporters usually underwent further expansion in basal vertebrates after divergence from Urochordata [29], [31], [32], [47], [48]. However, the CatSper channel complex appears to be preserved from early metazoans to vertebrates without any further duplication events. The stringent requirement for four different CatSpers to form a putative heterotetrameric channel complex might render any further gene duplication of CatSpers excessive and the gene then be degenerated. For instance, a recent primate-specific duplication of CatSper2 resulted in a CatSper2 pseudogene (Table S4).
Lineage-specific gene loss of CatSper and CatSperβ across the metazoan evolution
Sperm cells express a variety of Ca2+ channels and transporters [2], the majority of which are also expressed in other tissues and are often highly conserved from early metazoans to mammals, such as TRP channels [49], [50], Na+/Ca2+ exchangers (NCX) [29], [48], and sarco/endoplasmic reticulum Ca2+ ATPases [51], [52]. In contrast, little is known about the detailed evolutionary pattern of sperm-specific ion channels.
Fig. 3 shows the comprehensive evolutionary genomics of the CatSper ion channel complex. We have identified novel CatSper sequences from non-mammal vertebrates, such as the Anole lizard Anolis carolinensis, and cartilaginous fishes Callorhinchus milii and Leucoraja erinacea (Table S1). The CatSper channel complex is completely absent from a diverse sampling of protostome genomes. Interestingly, even though they are present in basal Chordata (amphioxus and sea squirts), all four CatSpers and CatSperβ appear to be lacking in several vertebrate lineages such as Agnatha (jawless fishes), Teleostei (bony fishes), Amphibia (frogs), and Aves (birds). This distinctive lineage distribution pattern of the CatSper channel complex (Fig. 3) is in drastic contrast to that of most ion channels and transporters characterized previously, which are usually highly conserved in metazoans [29], [31], [32], [40], [47], [48].
The phylogenetic branching patterns of metazoans (not proportional to the evolutionary rates) were extracted from the Tree of Life project (http://www.tolweb.org/tree/) as of July 30, 2008. The metazoan lineages known to contain the CatSper channel complex are indicated by boxes with a solid line and gray background, while those believed to lack the channel complex are marked by boxes with a broken line and white background. Two lineages (Porifera and cartilaginous fishes) are underlined since the results obtained from these lineages are based solely on survey genome or genomic traces rather than complete genome sequences. The availability of more metazoan genome sequences in the future will probably expand and/or refine the lineage branches shown here.
Two main evolutionary mechanisms might account for the intermittent pattern of presence or absence of genes in the genomes: lineage-specific gene loss and horizontal gene transfer [53], [54]. A bacterial voltage-gated Na+ channel, NaVBP, is believed to have the closest relationship with CatSpers, before the mammalian CaV and NaV channel classes [19]. Similar to CatSpers, NaVBP is activated by alkalinization and is important for motility [55]. Horizontal gene transfer between prokaryotes and metazoans is not common, but possible [56]. Perhaps during the process of metazoan evolution, a bacterial channel protein like NaVBP became incorporated into sperm cells of some, but not all metazoan genomes. Such a bacterial channel would have undergone further functional divergence to enhance sperm motility by inducing Ca2+-dependent hyperactivation and subsequent species-dependent adaptation to reflect phylogeny. Alternatively, the absence of the CatSper complex in the specific metazoan lineages described above could be explained as a result of differential gene loss. Thus, in sperm cells of those metazoan lineages, Ca2+ influx across plasma membrane might not be important for sperm motility endurance or hyperactivated motility. Of course, other types of Ca2+ channels/sources might have substituted for the CatSper channel complex.
If lineage-specific gene loss had occurred, it might first render the gene degenerate and non-functional. Subsequent neutral evolution could then mask a substantial portion of the gene sequence, but short fragments in the coding sequence of the degenerated gene might still be present, with flanking functional genes on the syntenic chromosomes conserved across closely related species. Therefore, we compared syntenic chromosomal regions containing CatSper and CatSperβ genes in mouse and human genomes and those in bird, amphibian, and bony fish genomes.
By examining genomic regions with the ECR browser and the NCBI genomic database, we found highly conserved synteny between selected regions of the mouse and human genomes and those of the chicken genome for CatSper2-4 and CatSperβ (Fig. 4 and Table S2, S3, S4, S5, S6). As shown in Fig. 4A and Table S2, 10 genes flanking the 5′- and 3′-end of CatSperβ on human chromosome 14 and corresponding orthologous genes on chicken chromosome 5 are syntenic. By using three different gene-finding programs [37], subsequent examination of chicken genomic sequence located between SMEK1 and TC2N genes did not yield any obvious gene coding region, but a portion of the genomic sequence could be translated into a 66-aa fragment with high similarity to human CatSperβ (Fig. 4A) and other CatSperβ sequences (data not shown). Similar observations have also been observed for CatSper2 and CatSper3 (Tables S4 and S5). Thus, CatSper sequences might have been degenerated in chick genomes, with short fragments of coding sequences still present.
A, chromosomal regions harboring degenerate DNA fragment of putative CatSperβ on chicken chromosome 5 with synteny to human chromosome 14. Shown here are ten genes flanking H. sapiens CatSperβ on human chromosome 14 and syntenic genes on chicken chromosome 5 (not to the scale of base-pair length). CatSperβ is indicated with a black rectangle and other genes with gray rectangles. The location of the degenerate genomic fragment of putative chicken CatSperβ is specified with an open rectangle, and the translated sequence is aligned with HsaCatSperβ. B, H. sapiens CatSper3 is aligned with the short stretch of sequences translated from putative degenerate chicken and zebra finch CatSper3 fragments. Abbreviations for flanking genes can be found in Table S2.
We next examined the preliminary genome sequence of the zebra finch Taeniopygia guttata. We also found a stretch of genomic sequence that could be translated into a short CatSper3 fragment sharing sequence similarity with a region in human CatSper3 and the putative chicken CatSper3 fragment (Fig. 4B). Such a short CatSper4 fragment was also made in the genome of the snail Lottia gigantean (data not shown). In addition, the EST libraries of a non-insect arthropod, the Arctic springtail Onychiurus arcticus, contain an EST clone (GenBank Accession No. EW749693.1) encoding a partial protein sequence in which the C-terminal (aa 68–218) displayed high similarity to TMS domains of CatSper4, with N. vectensis CatSper4 as the most significant hit (E-value, 3×e−27). The N-terminal 67 aa share no sequence homology to other CatSpers or known proteins.
Taken together, we conclude that the intermittent presence/absence pattern of the CatSper channel complex in metazoan genomes was likely derived from lineage-specific gene loss. Importantly, CatSperβ is present in genomes in which CatSpers are identified, but is missing in genomes that lack CatSpers. Thus, functional association between CatSperβ and CatSpers is not only critical for CatSperβ stability at the cellular level, i.e., mouse sperm cells [24], but might also relate to the all-or-none presence of CatSpers and CatSperβ at the genomic level.
Mouse spermatozoa have only two primary ion channel currents in normal saline solution, ICatSper and IKsper [15], [57]. The Ca2+ current is mediated by CatSpers while the K+ current is mediated presumably by mSlo3. Both currents are effectively gated by alkalinization and voltage changes. Activated K+ current hyperpolarizes the flagellar plasma membrane and thus maximizes the entry of Ca2+ via Catsper by increasing driving force on Ca2+. In sea urchin, direct whole-sperm voltage clamp recordings have not been possible, but indirect evidence suggests that the chemoattractant peptide Resact activates a cyclic-nucleotide-gated (CNG) K+-selective conductance [8] (presumably, Sp-tetraKCNG channel [58]) . This K+ current may act in place of IKSper to enhance Ca2+ entry. Mammalian spermatocytes may contain CNGB1 and perhaps CNGA3 channel proteins [59], but to date, currents consistent with CNG channels have not been observed in mouse sperm [57]. The simplest interpretation is that CatSpers are critical for the alteration of motility patterns for chemotaxis (sea urchin) and hyperactivation (mammals), but that the type of K channel activated (CNG or mSlo3) modulates CatSper's roles specific to the species. Thus, sea urchin eggs, with their thin walls, are easy for sperm to penetrate but sperm must be guided to the egg in the less restricted ocean environment by chemotactic peptide alteration of sea urchin sperm motility. In contrast, mouse spermatozoa require more force, and thus hyperactivated motility, to penetrate the thicker oocyte wall. Whether there is an additional chemotactic factor guiding spermatozoa in mammals is not established. Since birds and fish have thin oocyte cell walls, CatSper may have provided no evolutionary advantage and was degenerated in these species.
Such an extensive lineage-specific gene loss of an entire ion channel complex through metazoan evolution, especially in vertebrates, has not been documented in other channels and transporters. In some cases, such as for Na+/Ca2+ exchangers, one member, NCX4, which arose in basal vertebrates, persevered in teleost, amphibian and reptilian species but was lost in mammals and birds. Most of NCX members, NCX1-3, however, were still retained in all vertebrates examined [48]. Interestingly, a sperm-specific and unusual putative Na+/H+ exchanger required for sperm motility and fertility [60] also shows similar extensive lineage-specific gene loss in metazoans (Cai, X, unpublished observation). Thus, further evolutionary genomics studies of sperm-specific channels and transporters will no doubt shed novel insights into physiological roles of ion transport in sperm biology.
Our database search did not identify the putative primordial CatSper sequence. It remains possible that the introduction of the primordial, probably distantly related, CatSper channel could be made through horizontal gene transfer between prokaryotes and primitive metazoan species, such as single 6-TMS domain NaV channels NaVBP [19], NaChBac [61], and 11 bacterial NaChBac homologues [36]. All these bacterial NaV channel homologues share a glutamate residue as the key acidic residue in the putative channel pore region [36], in contrast to the key aspartate residue conserved in CatSpers (Fig. 2). We analyzed 40 more bacterial protein sequences (protein cluster CLS1187052), which display high sequence homology and structural similarity to NaChBac. None of these 40 bacterial homologues grouped with CatSper sequences (Fig. 5). However, two (GI No. 56963529 and 134099759) have the key aspartate residue in the putative pore region, and it would be interesting compare their Ca2+-selectivity, voltage-dependence and kinetics with the CatSper channel.
A bootstrapped maximum parsimony tree was constructed with a NaV channel homologue in choanoflagellates (MbrNaV-S3L) as an outgroup. Two CatSper sequences, NveCatSper2 and HsaCatSper2, and two putative CaV channel homologues, MbrCaVS5-1 and NveCaV-S17, are also included. The position of the key aspartate residue in the pore region of two bacterial proteins is marked by gray circles. Bootstrap values of >50 are shown at corresponding branches. Each branch of the tree is labeled with the GI numbers in the NCBI protein database for most organisms. NaChBac, NaVSP and NaVPZ channels were functionally characterized previously [36], [61]. Abbreviations: Hsa, H. sapiens; Mbr, M. brevicollis; Nve, N. vectensis.
Rapid evolution of the CatSper channel complex
Reproductive system proteins are subjected to selective evolutionary forces and often undergo accelerated evolution and functional divergence [25]–[27]. Indeed, mammalian sperm-specific proteins, mostly cell surface binding proteins and enzymes, are rapidly evolving [26]. Sperm-specific channels and transporters have not been extensively studied, but positive selection on indel substitutions does occur in the first exon of the CatSper1 gene in mammals [27], [62].
Non-synonymous (dN) and synonymous (dS) nucleotide substitution values as well as dN/dS ratios are often used for detecting evolutionary rates [63]. We calculated dN and dS values of human-mouse orthologous gene pairs of CatSper and CatSerβ genes, using the codeml program implemented in the PAML package [39]. The dN value of CatSper and CatSerβ genes (average = 0.268, Table 1) is 5.8- and 3.2-fold larger, respectively, than housekeeping genes and non-sperm tissue-specific genes [64], and 1.5 fold larger than other sperm-specific genes [26]. Thus, both CatSpers and CatSperβ appear to be subjected to high selective forces that promote amino acid diversity.
The dS values (Table 1) indicate that CatSper and CatSerβ genes contain high mutation rates, with an ∼2-fold increase over those of housekeeping genes, other tissue-specific genes and sperm-specific genes. Thus, CatSper and CatSerβ genes have an average dN/dS ratio that is smaller than other sperm-specific genes. Nevertheless, the average dN/dS ratio of CatSper and CatSerβ genes is ∼3- and 1.6-fold larger than housekeeping genes and other tissue-specific genes, respectively (Table 1). In addition, even though mammalian synonymous mutation rates (dS values) are generally considered selectively neutral, recent studies suggest that synonymous mutations might also be subjected to selection, possibly through their effects on splicing and/or mRNA stability [65]. Thus, CatSper and CatSerβ mRNA might also be processed, as shown in recent studies on human and mouse homologues of the cystic fibrosis transmembrane conductance regulator Cl− channel [66]. Interestingly, CatSperβ has the highest dN/dS ratio of the genes examined (0.409; Table 1). The exact number of CatSperβ subunits in the channel complex is not yet known. Presumably, based on the observations of other tetrameric 6-TMS voltage-gated ion channels such as K+ channels [67], multiple CatSperβ proteins may be in the CatSper1-4 channel complex.
Rapid evolution of all four CatSper proteins appeared to have occurred at different domains in CatSpers. For instance, CatSper1 underwent strong positive selection in the N-terminal cytoplasmic domain [27], [62], but displayed evolutionary constraint in the 6-TMS domains (dN/dS, 0.062) (Table 1). CatSper 4 displays similar constraints on its TMS domains with a dN/dS value of 0.077. In contrast, the TMS domains of CatSper2 and CatSper3 were driven by higher selective forces (dN/dS, 0.336 and 0.143, respectively). The distinct evolutionary patterns in CatSper channel domains may promote channel diversity in modulating potential protein or second-messenger interactions [12]. CatSperβ, as perhaps the sole accessory protein, may have evolved even faster to accommodate concerted evolution at differential evolutionary sites of CatSper proteins, resulting in a high dN/dS ratio.
Functional divergence of the CatSper protein family
The identification of many CatSper sequences, especially from invertebrates, allows us to calculate the functional divergence between CatSper1-4 (as shown previously [32], [68]). The coefficients of functional divergence between pairs of CatSper1-4 groups were estimated to be significant, ranging from 0.336±0.092 (CatSper3 vs. CatSper4) to 0.536±0.116 (CatSper2 vs. CatSper4) (Table S7). Thus, each CatSper group appears to have experienced altered evolutionary constraints after divergence from a putative ancestral duplication. Consistently, functional distance analysis also shows similarly long functional branch lengths (Fig. S1), implying that all four CatSpers diverged at comparable overall evolutionary rates from the putative primordial CatSper protein. Nevertheless, despite the similar overall evolutionary rates, the evolutionary processes might act differentially on specific sites that are unique in each CatSper gene (data not shown). Our data suggest that CatSper1-4 have acquired evolutionary novelties through substantial altered functional constraints after possible ancestral replication. Finally, rapid evolution of CatSpers (Table 1) still plays an important role in modulating CatSper functions in mammalian lineages.
In conclusion, we have carried out a comprehensive evolutionary genomics study of CatSper and CatSperβ proteins that constitutes the Ca2+ channel complex critical for sperm Ca2+ hyperactivation in mammals. With continued advances in genome biology, the evolutionary genomics approaches undertaken here will further illuminate the lineage-specific distribution of Ca2+ channels and transporters at the genomic scale and greatly facilitate deciphering the Ca2+ signaling codes in a species- and/or tissue-specific manner.
Supporting Information
Figure S1.
Tree topology of functional distance analysis of the CatSper protein family. Type I functional branch length bF was calculated as described in Materials and methods in Table S7. bF is an estimation of evolutionary distance of each CatSper group (1–4) to the putative primordial CatSper protein before replication (center circle).
https://doi.org/10.1371/journal.pone.0003569.s001
(2.31 MB TIF)
Table S1.
List of CatSper Proteins Used for Analyses
https://doi.org/10.1371/journal.pone.0003569.s002
(0.31 MB PDF)
Acknowledgments
X.C. thanks Yanhong Zhang for excellent technical assistance and for critically reading the manuscript. This project was initiated during a discussion session at the 38th workshop of the International School of Biophysics on Transporters and Channels, held in Erice, Sicily, Italy on 11–19 May 2008. We thank Drs. Louis J. DeFelice and Baruch I. Kanner for organizing this outstanding workshop on transporters and channels.
Author Contributions
Conceived and designed the experiments: XC DEC. Performed the experiments: XC. Analyzed the data: XC. Contributed reagents/materials/analysis tools: XC DEC. Wrote the paper: XC DEC.
References
- 1. Darszon A, Acevedo JJ, Galindo BE, Hernandez-Gonzalez EO, Nishigaki T, et al. (2006) Sperm channel diversity and functional multiplicity. Reproduction 131: 977–988.
- 2. Jimenez-Gonzalez C, Michelangeli F, Harper CV, Barratt CL, Publicover SJ (2006) Calcium signalling in human spermatozoa: a specialized ‘toolkit’ of channels, transporters and stores. Hum Reprod Update 12: 253–267.
- 3. Whitaker M (2006) Calcium at fertilization and in early development. Physiol Rev 86: 25–88.
- 4. Ishijima S, Baba SA, Mohri H, Suarez SS (2002) Quantitative analysis of flagellar movement in hyperactivated and acrosome-reacted golden hamster spermatozoa. Mol Reprod Dev 61: 376–384.
- 5. Carlson AE, Westenbroek RE, Quill T, Ren D, Clapham DE, et al. (2003) CatSper1 required for evoked Ca2+ entry and control of flagellar function in sperm. Proc Natl Acad Sci U S A 100: 14864–14868.
- 6. Fauci LJ, Dillon R (2006) Biofluid mechanics of reproduction. Annual Review of Fluid Mechanics 38: 371–394.
- 7. Suarez SS (2008) Regulation of sperm storage and movement in the mammalian oviduct. Int J Dev Biol 52: 455–462.
- 8. Strunker T, Weyand I, Bonigk W, Van Q, Loogen A, et al. (2006) A K+-selective cGMP-gated ion channel controls chemosensation of sperm. Nat Cell Biol 8: 1149–1154.
- 9. Cosson J, Groison AL, Suquet M, Fauvel C, Dreanno C, et al. (2008) Marine fish spermatozoa: racing ephemeral swimmers. Reproduction 136: 277–294.
- 10. Oda S, Morisawa M (1993) Rises of intracellular Ca2+ and pH mediate the initiation of sperm motility by hyperosmolality in marine teleosts. Cell Motil Cytoskeleton 25: 171–178.
- 11. Thomson MF, Wishart GJ (1991) Temperature-mediated regulation of calcium flux and motility in fowl spermatozoa. J Reprod Fertil 93: 385–391.
- 12. Qi H, Moran MM, Navarro B, Chong JA, Krapivinsky G, et al. (2007) All four CatSper ion channel proteins are required for male fertility and sperm cell hyperactivated motility. Proc Natl Acad Sci U S A 104: 1219–1223.
- 13. Kirichok Y, Navarro B, Clapham DE (2006) Whole-cell patch-clamp measurements of spermatozoa reveal an alkaline-activated Ca2+ channel. Nature 439: 737–740.
- 14. Ren D, Navarro B, Perez G, Jackson AC, Hsu S, et al. (2001) A sperm ion channel required for sperm motility and male fertility. Nature 413: 603–609.
- 15. Navarro B, Kirichok Y, Chung JJ, Clapham DE (2008) Ion channels that control fertility in mammalian spermatozoa. Int J Dev Biol 52: 607–613.
- 16. Quill TA, Ren D, Clapham DE, Garbers DL (2001) A voltage-gated ion channel expressed specifically in spermatozoa. Proc Natl Acad Sci U S A 98: 12527–12531.
- 17. Lobley A, Pierron V, Reynolds L, Allen L, Michalovich D (2003) Identification of human and mouse CatSper3 and CatSper4 genes: characterisation of a common interaction domain and evidence for expression in testis. Reprod Biol Endocrinol 1: 53.
- 18. Jin JL, O'Doherty AM, Wang S, Zheng H, Sanders KM, et al. (2005) Catsper3 and catsper4 encode two cation channel-like proteins exclusively expressed in the testis. Biol Reprod 73: 1235–1242.
- 19. Clapham DE, Garbers DL (2005) International Union of Pharmacology. L. Nomenclature and structure-function relationships of CatSper and two-pore channels. Pharmacol Rev 57: 451–454.
- 20. Cai X (2008) Subunit stoichiometry and channel pore structure of ion channels: all for one, or one for one? J Physiol 586: 925–926.
- 21. Quill TA, Sugden SA, Rossi KL, Doolittle LK, Hammer RE, et al. (2003) Hyperactivated sperm motility driven by CatSper2 is required for fertilization. Proc Natl Acad Sci U S A 100: 14869–14874.
- 22. Carlson AE, Quill TA, Westenbroek RE, Schuh SM, Hille B, et al. (2005) Identical phenotypes of CatSper1 and CatSper2 null sperm. J Biol Chem 280: 32238–32244.
- 23. Jin J, Jin N, Zheng H, Ro S, Tafolla D, et al. (2007) Catsper3 and Catsper4 are essential for sperm hyperactivated motility and male fertility in the mouse. Biol Reprod 77: 37–44.
- 24. Liu J, Xia J, Cho KH, Clapham DE, Ren D (2007) CatSperbeta, a novel transmembrane protein in the CatSper channel complex. J Biol Chem 282: 18945–18952.
- 25. Swanson WJ, Vacquier VD (2002) The rapid evolution of reproductive proteins. Nat Rev Genet 3: 137–144.
- 26. Torgerson DG, Kulathinal RJ, Singh RS (2002) Mammalian sperm proteins are rapidly evolving: evidence of positive selection in functionally diverse genes. Mol Biol Evol 19: 1973–1980.
- 27. Podlaha O, Webb DM, Tucker PK, Zhang J (2005) Positive selection for indel substitutions in the rodent sperm protein catsper1. Mol Biol Evol 22: 1845–1852.
- 28.
Hille B (2001) Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer Associates Inc.
- 29. Cai X, Lytton J (2004) The cation/Ca2+ exchanger superfamily: phylogenetic analysis and structural implications. Mol Biol Evol 21: 1692–1703.
- 30. Yu FH, Yarov-Yarovoy V, Gutman GA, Catterall WA (2005) Overview of molecular relationships in the voltage-gated ion channel superfamily. Pharmacol Rev 57: 387–395.
- 31. Cai X (2007) Molecular evolution and structural analysis of the Ca2+ release-activated Ca2+ channel subunit, orai. J Mol Biol 368: 1284–1291.
- 32. Cai X (2007) Molecular evolution and functional divergence of the Ca2+ sensor protein in store-operated Ca2+ entry: stromal interaction molecule. PLoS ONE 2: e609.
- 33. Cai X (2008) Unicellular Ca2+ signaling ‘toolkit’ at the origin of Metazoa. Mol Biol Evol 25: 1357–1361.
- 34. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402.
- 35. Nichols SA, Dirks W, Pearse JS, King N (2006) Early evolution of animal cell signaling and adhesion genes. Proc Natl Acad Sci U S A 103: 12451–12456.
- 36. Koishi R, Xu H, Ren D, Navarro B, Spiller BW, et al. (2004) A superfamily of voltage-gated sodium channels in bacteria. J Biol Chem 279: 9532–9538.
- 37. Cai X, Zhang Y (2006) Molecular evolution of the ankyrin gene family. Mol Biol Evol 23: 550–558.
- 38. Suyama M, Torrents D, Bork P (2006) PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Res 34: W609–612.
- 39. Yang Z (2007) PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol 24: 1586–1591.
- 40. Okamura Y, Nishino A, Murata Y, Nakajo K, Iwasaki H, et al. (2005) Comprehensive analysis of the ascidian genome reveals novel insights into the molecular evolution of ion channel genes. Physiol Genomics 22: 269–282.
- 41. Delsuc F, Brinkmann H, Chourrout D, Philippe H (2006) Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 439: 965–968.
- 42. Putnam NH, Butts T, Ferrier DE, Furlong RF, Hellsten U, et al. (2008) The amphioxus genome and the evolution of the chordate karyotype. Nature 453: 1064–1071.
- 43. King N, Westbrook MJ, Young SL, Kuo A, Abedin M, et al. (2008) The genome of the choanoflagellate Monosiga brevicollis and the origins of metazoan multicellularity. Nature 451: 783–788.
- 44. Berridge MJ, Bootman MD, Roderick HL (2003) Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4: 517–529.
- 45. Putnam NH, Srivastava M, Hellsten U, Dirks B, Chapman J, et al. (2007) Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Science 317: 86–94.
- 46. Ellinor PT, Yang J, Sather WA, Zhang JF, Tsien RW (1995) Ca2+ channel selectivity at a single locus for high-affinity Ca2+ interactions. Neuron 15: 1121–1132.
- 47. Jackson HA, Marshall CR, Accili EA (2007) Evolution and structural diversification of hyperpolarization-activated cyclic nucleotide-gated channel genes. Physiol Genomics 29: 231–245.
- 48. On C, Marshall CR, Chen N, Moyes CD, Tibbits GF (2008) Gene structure evolution of the Na+-Ca2+ exchanger (NCX) family. BMC Evol Biol 8: 127.
- 49. Trevino CL, Serrano CJ, Beltran C, Felix R, Darszon A (2001) Identification of mouse trp homologs and lipid rafts from spermatogenic cells and sperm. FEBS Lett 509: 119–125.
- 50. Venkatachalam K, Montell C (2007) TRP channels. Annu Rev Biochem 76: 387–417.
- 51. Lawson C, Dorval V, Goupil S, Leclerc P (2007) Identification and localisation of SERCA 2 isoforms in mammalian sperm. Mol Hum Reprod 13: 307–316.
- 52. Vazquez-Martinez O, Canedo-Merino R, Diaz-Munoz M, Riesgo-Escovar JR (2003) Biochemical characterization, distribution and phylogenetic analysis of Drosophila melanogaster ryanodine and IP3 receptors, and thapsigargin-sensitive Ca2+ ATPase. J Cell Sci 116: 2483–2494.
- 53. Krylov DM, Wolf YI, Rogozin IB, Koonin EV (2003) Gene loss, protein sequence divergence, gene dispensability, expression level, and interactivity are correlated in eukaryotic evolution. Genome Res 13: 2229–2235.
- 54. Salzberg SL, White O, Peterson J, Eisen JA (2001) Microbial genes in the human genome: lateral transfer or gene loss? Science 292: 1903–1906.
- 55. Ito M, Xu H, Guffanti AA, Wei Y, Zvi L, et al. (2004) The voltage-gated Na+ channel NaVBP has a role in motility, chemotaxis, and pH homeostasis of an alkaliphilic Bacillus. Proc Natl Acad Sci U S A 101: 10566–10571.
- 56. Andersson JO, Doolittle WF, Nesbo CL (2001) Genomics. Are there bugs in our genome? Science 292: 1848–1850.
- 57. Navarro B, Kirichok Y, Clapham DE (2007) KSper, a pH-sensitive K+ current that controls sperm membrane potential. Proc Natl Acad Sci U S A 104: 7688–7692.
- 58. Galindo BE, de la Vega-Beltran JL, Labarca P, Vacquier VD, Darszon A (2007) Sp-tetraKCNG: A novel cyclic nucleotide gated K+ channel. Biochem Biophys Res Commun 354: 668–675.
- 59. Hofmann F, Biel M, Kaupp UB (2005) International Union of Pharmacology. LI. Nomenclature and structure-function relationships of cyclic nucleotide-regulated channels. Pharmacol Rev 57: 455–462.
- 60. Wang D, King SM, Quill TA, Doolittle LK, Garbers DL (2003) A new sperm-specific Na+/H+ exchanger required for sperm motility and fertility. Nat Cell Biol 5: 1117–1122.
- 61. Ren D, Navarro B, Xu H, Yue L, Shi Q, et al. (2001) A prokaryotic voltage-gated sodium channel. Science 294: 2372–2375.
- 62. Podlaha O, Zhang J (2003) Positive selection on protein-length in the evolution of a primate sperm ion channel. Proc Natl Acad Sci U S A 100: 12241–12246.
- 63.
Li WH (1997) Molecular Evolution. Sunderland, Massachusetts: Sinauer Associates.
- 64. Zhang L, Li WH (2004) Mammalian housekeeping genes evolve more slowly than tissue-specific genes. Mol Biol Evol 21: 236–239.
- 65. Chamary JV, Parmley JL, Hurst LD (2006) Hearing silence: non-neutral evolution at synonymous sites in mammals. Nat Rev Genet 7: 98–108.
- 66. Pagani F, Raponi M, Baralle FE (2005) Synonymous mutations in CFTR exon 12 affect splicing and are not neutral in evolution. Proc Natl Acad Sci U S A 102: 6368–6372.
- 67. Torres YP, Morera FJ, Carvacho I, Latorre R (2007) A marriage of convenience: beta-subunits and voltage-dependent K+ channels. J Biol Chem 282: 24485–24489.
- 68. Wang Y, Gu X (2001) Functional divergence in the caspase gene family and altered functional constraints: statistical analysis and prediction. Genetics 158: 1311–1320.
- 69. Schmidt HA, Strimmer K, Vingron M, von Haeseler A (2002) TREE-PUZZLE: maximum likelihood phylegenetic analysis using quartet and parallel computing. Bioinformatics 18: 502–504.