| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Research Article |
Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557
ABSTRACT
CATSPER1 and CATSPER2 are two cation channel-like proteins exclusively expressed in the testis and essential for normal sperm motility and male fertility. Using in silico subtraction and database mining, we identified expressed sequence tags encoding two previously uncharacterized cation channel-like proteins structurally homologous to CATSPER1 and CATSPER2. Similar to CATSPER1 and CATSPER2, these two proteins contain a single-ion transport domain comprised of six transmembrane spanning regions, in which the fourth transmembrane region resembles a voltage sensor and a pore-forming region lies between transmembrane regions 5 and 6. The pore contains the consensus sequence T x D x W, which is indicative of a potential calcium-selective channel. The mRNAs for Catsper3 and Catsper4 were detected exclusively in the testis using multitissue Northern blot and RT-PCR analyses. The onsets of both genes coincide with the first appearance of spermatids during testicular development. In situ hybridization analyses revealed that Catsper3 and Catsper4 mRNAs displayed identical localization patterns and were confined to spermatids of steps 1–8. Immunofluorescence and immunohistochemistry analyses demonstrated that these two proteins were expressed within the acrosome of late spermatids and spermatozoa. Our data suggest that CATSPER3 and CATSPER4 are two cation-channel proteins and have roles in acrosome reaction and male fertility.
acrosome reaction, calcium, sperm, sperm motility and transport, spermatogenesis
INTRODUCTION
Many ion channels, especially calcium channels, are present in sperm. These channels include high voltage-gated calcium channels (Cav 1.2, 2.1/2/3) [1]; cyclic nucleotide-gated channels [2]; the transient receptor potential channel [3, 4]; the voltage-operated Ca2+-channels 
1A, 1C, and 1E [5, 6]; the T-type voltage-operated Ca2+-channels 1G and 1H [7–10]; the N-type, R-type [11], and L-type voltage operated Ca2+ channels [12]; and pkD2 cation channel [13]. However, the molecular identity and/or physiological roles of these channels remain largely undefined.
CATSPER1 and CATSPER2 are putative six-transmembrane, voltage-gated ion channels located in the sperm flagellum [14–18]. Gene targeting studies show that both are required for cAMP-induced Ca2+ current essential for normal sperm motility and male fertility [14]. Similar structure and expression patterns of these two proteins, as well as identical phenotypes in the Catsper1 and Catsper2 knockout mice, strongly suggest that they may work together as a heterodimer. However, heterologous expression of either or both fails to induce detectable ionic current and to modify intracellular Ca2+ concentration [14–16]. It has been suggested that CATSPER1 and CATSPER2 require additional subunits and/or interacting partners to function, because the pore-forming units of voltage-gated cation channels form a tetramer [14–16, 19]. Recently, Lobley et al. reported the in silico identification of Catsper3 and Catsper4 cDNAs [19]. However, little is known about their potential function because of the lack of experimental data.
In a search for male germ cell-specific genes using in silico subtraction and database mining [20–24], we identified ESTs encoding CATSPER3 and CATSPER4 from several testis libraries. Here, we report the expressions and localizations of Catsper3 and Catsper4 mRNAs and proteins. Our data suggest that CATSPER3 and CATSPER4 may not form a tetramer with CATSPER1 and CATSPER2 because of their different expression and localization patterns, and CATSPER3 and CATSPER4 may be involved in acrosome reaction.
MATERIALS AND METHODS
Animals
C57BL/6J F1 mice were maintained under constant temperature (22°C) in a standard animal facility with free access to pelleted food and water at the University of Nevada School of Medicine. All experimental procedures were performed according to the guidelines of the Institutional Animal Care and Use Committee of the University of Nevada, Reno.
In Silico Subtraction and Database Mining
Expressed sequence tags (ESTs; n = 49,064) from three libraries (Lib.6786 [round spermatids], Lib.2547 [adult testis], and Lib.2511 [adult testis]) were downloaded from the mouse Unigene database (http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Mm) at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov). In silico subtraction was performed as described previously [22]. Using the BLAST program at the NCBI, mouse Catsper3 and Catsper4 cDNA and protein sequences were used as baits to search against the nucleotide and protein databases and to identify orthologs of these two proteins in other species, including the rat, dog, chimpanzee, and human.
Protein Alignment Analyses
Alignments of CATSPER3 and CATSPER4 proteins of different species were performed using the MegAlign program of the DNASTAR software package (DNASTAR, Inc.). The sequence identity analyses were performed using the same program.
Northern Blot Analyses
Total RNA (15 µg) isolated from multiple mouse tissues was fractionated on a 1.2% formaldehyde-agarose gel and transferred to Hybond-N nylon membrane (Amersham Pharmacia Biotech). A 369-bp PCR fragment corresponding to nucleotides 98–466 of the Catsper3 cDNA (GenBank accession no. BC089518) and a 532-bp PCR fragment corresponding to nucleotides 364–895 of the Catsper4 cDNA (GenBank accession no. NM_177866) were labeled with [-32P]dCTP using a RediPrime TM II labeling system (Amersham Pharmacia Biotech). Membrane hybridization, washing, and autoradiography, as well as stripping and reprobing, were performed according to the manufacturer's instructions. Blots were stripped and hybridized with an 18S rRNA cDNA labeled with [-32P]dCTP to control for equal RNA loading.
Semiquantitative RT-PCR
Reverse transcription reaction was performed in a 50-µl volume containing 2 µg of total RNA, 1x RT Buffer, 100 pmol of random primer, 1 mM deoxynucleoside triphosphate, 10 U of RNase inhibitor (RNasin), and 5 U of avian myeloblastosis virus reverse transcriptase at room temperature overnight. All RT reagents were purchased from Promega Corporation. An aliquot (2 µl) of the cDNAs synthesized as described above was used as template for a semiquantitative PCR analysis. Primers (upstream, 5'-TTCACCACAACCCTGTACGA-3'; downstream, 5'-ATGCAGAATGATTCCCCTTG-3') were used to amplify a 369-bp fragment of Catsper3 cDNA, and another pair of primers (upstream, 5'-CCTTATCTGCGAGGTTCTGC-3'; downstream, 5'-TTGTTCCAGGTTTGTGGTCA-3') were employed to yield a 532-bp fragment of Catsper4 cDNA. As a loading control, a housekeeping gene, Hprt, was amplified using a primer set described previously [22].
In Situ Hybridization
In situ hybridization was performed as described previously [25]. Briefly, paraffin-embedded testes were cut into sections (thickness, 5 µm), and cDNA fragments of Catsper3 and Catsper4 were subcloned into a pGEM-T vector (Promega). Next, [-35S]UTP-labeled sense and antisense riboprobes were generated using a Riboprobe In Vitro Transcription System (Promega). Hybridization signals were detected by autoradiography using NTB-2 emulsion (Eastman Kodak Co.). After development and fixation, the slides were counterstained with hematoxylin and mounted for photography.
Productions of CATSPER3 and CATSPER4 Recombinant Proteins and Polyclonal Antibodies
A pET protein production system (EMD Biosciences, Inc.) was employed to produce mouse CATSPER3 and CATSPER4 proteins. A cDNA fragment (1108 bp) from the coding region of Catsper3 was amplified by PCR using the primers 5'-GGATCCCACCACAACCCTGTACGA-3' (with BamHI adaptor, GGATCC) and 5'-CTCGAGGCTCTCCTTGGGCATGTCTTC-3' (with XhoI adapter, CTCGAG). For Catsper4, a cDNA fragment (1261 bp) from the coding region was amplified by PCR using the primers 5'-GGATCCGCAGGTGGAGAACATCGAC-3' (with BamHI adaptor, GGATCC) and 5'-AAGCTTATCAGACACCTTTTCCCTGCT-3' (with HindIII adapter, AAGCTT).
The PCR products were subcloned into pGEM-T vector (Promega Corp.) and sequenced to confirm the sequence accuracy. The BamHI/XhoI fragment of Catsper3 and the BamHI/HindIII fragment of Catsper4 were then subcloned into pET-23b vector (EMD Biosciences), respectively. Protein induction and purification were performed according to the manufacturer's instructions. Fusion proteins of CATSPER3 and CATSPER4 containing the full-length protein, N-terminal T7 flag, and C-terminal histidine tag were used to immunize rabbits to produce polyclonal antibodies (Cocalico Biologicals, Inc.).
Western Blot Analyses
Proteins were isolated from adult mice tissues, including heart, liver, spleen, lung, kidney, brain, testis, and ovary. Western blot analyses were performed as described previously [26]. Rabbit anti-CATSPER3 and anti-CATSPER4 polyclonal antibodies were used at a dilution of 1:1000.
Immunofluorescence and Confocal Microscopy
Testis cryosections were prepared as described previously [27]. Sections were incubated with anti-CATSPER3 and anti-CATSPER4 antibodies (diluted at 1:1000) for 1 h, followed by three washes (15 min each) in TBST (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, and 0.1% Tween 20). The slides were then incubated with 3 µg/ml of fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) for 45 min. The DNA was counterstained with propidium iodide (1 µg/ml) for 10 min. Preimmune serum was used for controls. The staining patterns were observed and photographed using a confocal laser-scanning microscope (model LSM500; Carl Zeiss Microimaging) and its imaging system.
Immunohistochemistry
Cauda epididymal sperm were spread onto Superfrost Plus slides (Fisher Scientific, Inc.) and air-dried. Microwave antigen retrieval was employed as described previously [25]. An aliquot of 100 µl of primary antibody diluted at 1:1000 was applied to each slide and incubated at 4°C overnight. Incubation with secondary antibody and visualization of positive cells were performed using a VectaStain Elite kit (Vector Laboratories, Inc.) according to the manufacturer's instructions. Preimmune serum was used in control slides.
RESULTS
Characteristics of CATSPER3 and CATSPER4
Among the 10 ESTs identified to contain open reading frames (ORFs) encoding a protein structurally homologous to CATSPER1 and CATSPER2, one clone contained an 1149-bp ORF. We designed primers and successfully amplified the entire ORF (data not shown), indicating that this EST contained the full-length cDNA for Catsper3 (GenBank accession no. BC089518). Similarly, we found another EST (of 17 identified) with a 1329-bp ORF encoding another CATSPER homolog that contained the full-length cDNA for Catsper4 (GenBank accession no. NM_177866).
Similar to CATSPER1 and CATSPER2, both CATSPER3 and CATSPER4 contain six transmembrane regions, which are close together and joined by short loop regions. A short, conserved hydrophobic stretch representing the pore-forming region is present in a longer loop region between the fifth and sixth transmembrane domains (Figs. 1A and 2A). The specific arrangement of these transmembrane helices is characteristic of the voltage-gated channel ion-transport domain found in the voltage-gated K+, Ca2+, and Na+ channels and also reported for CATSPER1 and CATSPER2 [14–16].
|
|
The voltage sensor is located within the fourth transmembrane helix and participates in channel activation through positively charged residues positioned every three to four amino acids [28]. Ion specificity is determined by a pore consensus sequence, [T/S] x [D/E] x W, in voltage-gated calcium channels [29]. Sequence analyses of this region revealed the presence of a similar conserved motif, T x D x W, suggesting that CATSPER3 and CATSPER4 may be selective for calcium ions, as discussed previously for CATSPER1 and CATSPER2 [14–16]. Interestingly, a coiled-coil motif recently described and believed to be a protein-protein interaction interface [19] is also present in CATSPER3 and CATSPER4 (Figs. 1A and 2A). Therefore, CATSPER1 through CATSPER4 comprise a CATSPER protein family characteristic of a single-ion transport domain and a C-terminal coiled-coil motif. Despite the structural similarities, the four CATSPER proteins are distinct in sequence relationship; sequence identity ranges between 16% and 27% across the ion-transport domain (data not shown). This low sequence identity contrasts with that observed for the voltage-gated sodium- and calcium-channel families, which display higher sequence identity between their corresponding ion-transport repeat regions [19]. Interestingly, we could not detect CATSPER-like proteins in species lower than the mouse. These data strongly suggest that the CATSPER family proteins neither were derived from an early duplication event nor resulted from convergent evolution of ion-channel genes at different chromosomes toward a common function. Lobley et al. [19] suggest that Catsper genes may result from a more recent evolutionary event or rapid evolution. Nevertheless, when we compare orthologous CATSPER3 and CATSPER4 in several mammalian species, we found that these two proteins were highly conserved during evolution. The sequence identity of CATSPER3 orthologs ranges from 61.1% (mouse vs. rat) to 99.2% (chimpanzee vs. human), and the sequence identity of CATSPER4 orthologs varies from 71.8% (mouse vs. human) to 87.4% (mouse vs. rat) (Figs. 1B and 2B).
Expression and Localization of Mouse Catsper3 and Catsper4 mRNAs
The PCR fragments amplified from mouse Catsper3 and Catsper4 cDNAs were used as probes for multitissue Northern blot analyses. The Catsper3 probe hybridized to an approximately 1.6-kb transcript, and the Catsper4 probe detected an approximately 2.0-kb transcript. Both transcripts were detected exclusively in the testis (Fig. 3A). To reveal potential low-level expression in other tissues, we employed RT-PCR analysis in which Catsper3 and Catsper4 cDNAs were amplified for 40 cycles, but the housekeeping gene Hprt (loading controls) was amplified for 20 cycles (in the exponential range). In this way, we can achieve maximal sensitivity in amplifying target templates with very low expression levels. As shown in Figure 3B, no signals were detected from multiple tissues, except for the testis.
|
We further examined levels of Catsper3 and Catsper4 mRNAs during testicular development using a semiquantitative RT-PCR analysis [22]. Neither Catsper3 nor Catsper4 mRNA was detected before Postnatal Day (P) 10 (Fig. 3C). They were first detected at P20, and the levels increased at P40 (Fig. 3C). The onset of Catsper3 and Catsper4 mRNA expression coincides with the first appearance of haploid cells in the developing testis (P18), suggesting that spermatids are the germ cell type expressing these two transcripts. To confirm further the expression sites of Catsper3 and Catsper4 mRNAs, we performed in situ hybridization analyses. We found that hybridization signals were confined to spermatids of steps 1–8, and the hybridization patterns of Catsper3 and Catsper4 were identical (Fig. 4). The intensity of hybridization signals displayed a stage-specific pattern, in which both mRNA levels gradually increased from step 1 through step 7, reached a peak in step 8, and diminished thereafter (Fig. 4).
|
Productions of CATSPER3 and CATSPER4 Proteins and Antibodies
Full-length mouse CATSPER3 and CATSPER4 proteins fused with histidine tags at their C termini were produced using the pET protein production system. The proteins were predominantly expressed in inclusion bodies in Escherichia coli (strain BL-21). Using a His affinity purification method [22], we purified the recombinant CATSPER3 and CATSPER4 proteins, which were then used to immunize rabbits to generate polyclonal antibodies. To test further the specificity and confirm the tissue distribution of CATSPER3 and CATSPER4 in the mouse, we performed Western blot analysis using proteins isolated from multiple mouse tissues (Fig. 5). The rabbit anti-CATSPER3 and anti-CATSPER4 antibodies detected as little as 1 ng of recombinant CATSPER3 and CATSPER4 proteins, respectively, and the preimmune sera did not react with the recombinant protein (data not shown). Western blot analyses revealed an approximately 44-kDa band for CATSPER3 and an approximately 51-kDa band for CATSPER4, which are consistent with the predicted sizes of both proteins. These results are consistent with our Northern blot (Fig. 3A) and RT-PCR (Fig. 3B) results, which showed that these two proteins were exclusively expressed in the testis and demonstrated that the polyclonal antibodies raised against CATSPER3 and CATSPER4 were specific.
|
Expressions and Localizations of CATSPER3 and CATSPER4 Proteins in Mouse Testis and Sperm
Indirect immunofluorescent labeling with the anti-CATSPER3 and anti-CATSPER4 antisera and confocal microscopy were used to assess protein expressions in the testis. Both CATSPER3 and CATSPER4 were detected in the acrosomal region of elongated spermatides (steps 13–16) (Fig. 6, A–D). The immunoreactivity for CATSPER3 and CATSPER4 was detected mainly in the acrosome of sperm isolated from cauda epididymis (Fig. 6, E and F). No immunoreactivity was detected in control slides using preimmune sera as the primary antibodies (data not shown).
|
DISCUSSION
Our data demonstrated that Catsper3 and Catsper4 mRNAs were expressed in round spermatids (steps 1–8), whereas CATSPER3 and CATSPER4 proteins were located in the acrosome of the late spermatids and spermatozoa. The delayed protein expression reflects the fact that transcription ceases when spermatid elongation and chromatin condensation start in step 9. Therefore, mRNAs for proteins needed during late spermiogenesis must be produced before step-9 spermatids. This is a phenomenon common to many spermatid-specific genes [30–35].
Both CATSPER3 and CATSPER4, together with previously well-studied CATSPER1 and CATSPER2, constitute a CATSPER protein family. Its members share similar domain structure but display low amino acid sequence identity. Both CATSPER1 and CATSPER2 localize to the flagellum of sperm, and disruption of either Catsper1 or Catsper2 results in infertility because of defects in sperm motility. These data suggest that these two CATSPER proteins may form a single heterodimeric channel. However, attempts to coimmunoprecipitate these two proteins have failed [15], and to our knowledge, neither CATSPER1 nor CATSPER2 has been shown to function as a cation channel when transfected into cells either singly or in conjunction. The presence of CATSPER3 and CATSPER4 prompted suggestions that the four CATSPER proteins form a functional heterotetrameric channel in sperm [16, 19]. However, our data are not supportive to this hypothesis, because CATSPER3 and CATSPER4 are present in the acrosome of sperm but CATSPER1 and CATSPER2 are located in the flagellum of sperm. Distinct localization patterns between CATSPER1/2 and CATSPER3/4 suggest that CATSPER3 and CATSPER4 have different roles in sperm. Based on their acrosomal localization, CATSPER3 and CATSPER4 may be involved in the acrosome reaction, which is a Ca2+-dependent secretory event and an essential early step in the fertilization process [36, 37]. It has been reported that Ca2+ and cyclic nucleotides can induce the acrosome reaction [38–40]. Voltage-gated Ca2+ channels are believed to mediate the entry of extracellular calcium [41–43]. Therefore, CATSPER3 and CATSPER4 may have a role in regulating intracellular Ca2+ levels during the acrosome reaction.
Based on their identical patterns of mRNA and protein expression as well as localization, we postulate that CATSPER3 and CATSPER4 may form a heterodimer and, thus, function as a single channel. The interactions between CATSPER3 and CATSPER4 could be mediated directly through their C-terminal coiled-coil domains or indirectly via other intracellular adaptor proteins binding to the coiled-coil domains of CATSPER3 and CATSPER4. The -helical coiled-coil motifs have been shown to mediate subunit multimerization of a large number of proteins [44, 45]. Further biochemical and genetic studies are needed to reveal the physiological roles of CATSPER3 and CATSPER4. A widely used calcium-channel blocker, nifedipine, has been shown to display a reversible contraceptive effect [46], which likely is mediated via voltage-gated calcium channels, primarily the L-type voltage-gated channels. Therefore, CATSPER ion channels appear to be attractive targets for nonhormonal contraceptives.
FOOTNOTES
1 Correspondence: Wei Yan, Department of Physiology and Cell Biology, University of Nevada School of Medicine, 1664 North Virginia Street, MS 352, Reno, NV 89557. FAX: 775 784 6903; weiyan{at}unr.edu 
Received: 12 July 2005.
First decision: 29 July 2005.
Accepted: 15 August 2005.
REFERENCES
1 subunits in sperm. Dev Biol 1999 207:457-469[CrossRef][Medline]1H T-type calcium channels. Mol Hum Reprod 2000 6:893-8971G) calcium channel: CaV3.2 (1H) is the main functional calcium channel in wild-type spermatogenic cells. J Cell Physiol 2004 200:116-124[CrossRef][Medline] motif, and a basic leucine zipper. Mol Endocrinol 2002 16:1168-1184-helices. Biochemistry 1999 38:13263-13269[CrossRef][Medline]This article has been cited by other articles:
|
|
 |
|
  S. Ro, C. Park, R. Song, D. Nguyen, J. Jin, K. M. Sanders, J. R. McCarrey, and W. Yan Cloning and expression profiling of testis-expressed piRNA-like RNAs RNA, October 1, 2007; 13(10): 1693 - 1702. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Xia, D. Reigada, C. H Mitchell, and D. Ren CATSPER Channel-Mediated Ca2+ Entry into Mouse Sperm Triggers a Tail-to-Head Propagation Biol Reprod, September 1, 2007; 77(3): 551 - 559. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Jin, N. Jin, H. Zheng, S. Ro, D. Tafolla, K. M. Sanders, and W. Yan Catsper3 and Catsper4 Are Essential for Sperm Hyperactivated Motility and Male Fertility in the Mouse Biol Reprod, July 1, 2007; 77(1): 37 - 44. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-G. Li, X.-F. Ding, A.-H. Liao, X.-B. Kong, and C.-L. Xiong Expression of CatSper family transcripts in the mouse testis during post-natal development and human ejaculated spermatozoa: relationship to sperm motility Mol. Hum. Reprod., May 1, 2007; 13(5): 299 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zheng, C. J. Stratton, K. Morozumi, J. Jin, R. Yanagimachi, and W. Yan Lack of Spem1 causes aberrant cytoplasm removal, sperm deformation, and male infertility PNAS, April 17, 2007; 104(16): 6852 - 6857. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. F. Babcock Wrath of the wraiths of CatSper3 and CatSper4 PNAS, January 23, 2007; 104(4): 1107 - 1108. [Full Text] [PDF]
|
|
|
|
|
|
H. Qi, M. M. Moran, B. Navarro, J. A. Chong, G. Krapivinsky, L. Krapivinsky, Y. Kirichok, I. S. Ramsey, T. A. Quill, and D. E. Clapham From the Cover: All four CatSper ion channel proteins are required for male fertility and sperm cell hyperactivated motility PNAS, January 23, 2007; 104(4): 1219 - 1223. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Darszon, J. J Acevedo, B. E Galindo, E. O Hernandez-Gonzalez, T. Nishigaki, C. L Trevino, C. Wood, and C. Beltran Sperm channel diversity and functional multiplicity. Reproduction, June 1, 2006; 131(6): 977 - 988. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
