Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M501430200 on July 21, 2005

J. Biol. Chem., Vol. 280, Issue 37, 32238-32244, September 16, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/37/32238    most recent
M501430200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Carlson, A. E.
Right arrow Articles by Babcock, D. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carlson, A. E.
Right arrow Articles by Babcock, D. F.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Identical Phenotypes of CatSper1 and CatSper2 Null Sperm*{boxs}

Anne E. Carlson{ddagger}1, Timothy A. Quill¶, Ruth E. Westenbroek||, Sonya M. Schuh{ddagger}, Bertil Hille{ddagger}, and Donner F. Babcock{ddagger}2

From the {ddagger}Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195, the Department of Pharmacology, Cecil and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, and the ||Department of Pharmacology, University of Washington, Seattle, Washington 98195

Received for publication, February 7, 2005 , and in revised form, June 17, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Among several candidate Ca2+ entry channels in sperm, only CatSper1 and CatSper2 are known to have required roles in male fertility. Past work with CatSper1 null sperm indicates that a critical lesion in hyperactivated motility underlies the infertility phenotype and is associated with an absence of depolarization-evoked Ca2+entry. Here we show that failure of hyperactivation of CatSper2 null sperm similarly correlates with an absence of depolarization evoked Ca2+ entry. Additional shared aspects of the phenotypes of CatSper1 and -2 null sperm include unperturbed regional distributions of conventional voltage-gated Ca2+ channel proteins and robust acceleration of the flagellar beat by bicarbonate. Further study reveals that treatment of both wild-type and CatSper2 null sperm with procaine increases beat asymmetry, a characteristic of the flagellar waveform of hyperactivation. This partial rescue of the loss-of-hyperactivation phenotype suggests that an absence of CatSper2 precludes hyperactivation by preventing delivery of needed Ca2+ messenger rather than by preventing flagellar responses to Ca2+. CatSper2 null sperm also have an increased basal cAMP content and beat frequency. Protein kinase A inhibitor H89 lowers beat frequency to that of wild-type sperm, suggesting that CatSper2 is required for protein kinase A-mediated, tonic control of resting cAMP content. Relative to wild-type testis, CatSper1 and -2 null testes contain normal amounts of CatSper2 and -1 transcripts, respectively. However, CatSper1 null sperm lack CatSper2 protein and CatSper2 null sperm lack CatSper1 protein. Hence, stable expression of CatSper1 protein requires CatSper2 and vice versa. This co-dependent expression dictates identical loss-of-function sperm phenotypes for CatSper1 and -2 null mutants.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The four members of the CatSper family are cation-channel-like proteins found exclusively in sperm and spermatogenic cells (1–3). The pore-lining residues and overall sequences of CatSper1 and CatSper2 most resemble those of a single repeat from a conventional four-repeat voltage-gated Ca2+ channel (1, 2). Thus, CatSper1 and CatSper2 are proposed to form all or part of a novel, hetero- or homotetrameric, Ca2+-selective channel.

Several of the conventional four-repeat voltage-gated Ca2+ channel proteins that also are detected immunologically in sperm (4–6) are not required for male fertility as revealed by targeted gene disruption. These include CaV1.3 (7), CaV2.2 (8), CaV2.3 (9), CaV3.1 (10), and CaV3.2 (11). Mice carrying null mutations for CNGA3 (12), TRPC2 (13), and TRPC3 (14), which also are candidates for sperm Ca2+ entry channels, likewise have no reported fertility deficits. Null mutants for CaV1.2 (15) and CaV2.1 (16) are embryonic lethal and thus unsuitable for fertility studies. Only CatSper1 (2) and CatSper2 (17) are required for male fertility as determined by targeted gene disruption.

Ca2+ is an important messenger in capacitation, the processes that prepare sperm for fertilization while they reside in the female reproductive tract after mating (18). Past work indicates that capacitation includes obligatory changes in sperm swimming behavior that are mediated by elevation of [Ca2+]i (19–23). The requirement of CatSper1 and CatSper2 for male fertility suggests that these putative channels open to allow Ca2+ entry to generate such instructive Ca2+ signals. Localization of CatSper1 and -2 to the membrane of the principal piece of the flagellum (2, 17) indicates a role in the control of flagellar function rather than in acrosomal exocytosis, another essential Ca2+-dependent component of capacitation. Indeed, a defect in sperm hyperactivation is prominent in the phenotypes of CatSper1 and -2 null sperm (17, 24, 25). Although engagement of the protein tyrosine phosphorylation cascade, the zona pellucida-induced acrosome reaction, and several other landmarks in capacitation do not require CatSper1 or CatSper2 (1, 2, 17, 25), the extent of identity in the phenotypes of CatSper1 and -2 null sperm has remained unclear. Here, we further examine the phenotype of CatSper2 null sperm and document additional similarities to those of sperm of the CatSper1 null mutant. We propose that these nearly or completely identical phenotypes result from a co-dependent expression of CatSper1 and CatSper2 proteins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Fura-2 acetoxymethyl (AM)3 ester and Pluronic F127 were from Molecular Probes, Inc. (Eugene, OR), and H89 from Calbiochem. Antibodies to CatSper1 and {alpha}-tubulin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and InnoGenex (San Ramon, CA), respectively. The CatSper2 polyclonal antibody used here was directed against the carboxyl-terminal epitope, as described previously (1). Unless noted, other chemicals were from Sigma.

Sperm Preparation and Incubation—Sperm were prepared as in prior work (6, 26, 27). Briefly, caudae epididymides and vasa deferentia were excised from male mice that were euthanized by CO2 asphyxiation. After rinsing with medium Na7.4 (135 mM NaCl, 5 mM KCl, 2 mM CaCl2,1mM MgSO4,20mM HEPES, 5 mM glucose, 10 mM lactic acid, 1 mM pyruvic acid, adjusted to pH 7.4 with NaOH), the tissue was transferred to 1 ml of a "swimout/capacitation" medium (medium Na7.4 with 5 mg of bovine serum albumin/ml and 15 mM NaHCO3). Semen was allowed to exude (15 min at 37 °C, 5% CO2) from several small incisions. All subsequent operations were at room temperature (22–25 °C) in medium Na7.4, unless noted otherwise. Sperm were washed twice and then dispersed and stored at 1–2 x 107 cells ml–1. Potassium-evoked responses were produced with medium K8.6 (135 mM KCl, 5 mM NaCl, 2 mM CaCl2, 1 mM MgSO4, 30 mM TAPS, 10 glucose, 10 lactic acid, 1 pyruvic acid), adjusted to pH 8.6 with NaOH. For in vitro capacitation, sperm were transferred to the "swimout/capacitation" medium and incubated for 90 min at 37 °C in a 95% air, 5% CO2 atmosphere. Sperm then were washed twice in medium Na7.4 and examined at room temperature.

Dye Loading and Photometry—Fura-2 AM was dispensed from 2 mMstocks in Me2SO, dispersed in 10–15% Pluronic F127, diluted to 20 µM in 0.25 ml medium Na7.4, and then immediately mixed with an equal volume of the sperm suspension. After 15–20 min, medium Na7.4 (0.5 ml) was added, and the cells were sedimented. After resuspension in 0.25 ml of fresh medium, incubation continued for 45 min before use. Ten microliters of cell suspension was added to the surface of an uncoated glass coverslip resting in a glass-bottomed incubation chamber containing ~3 ml of medium. After ~5 min, test solutions were applied with a multibarreled local perfusion device (estimated exchange time of <0.8 s). Excitation light of 340 and 380 nm was provided from a computer-controlled monochromator (T.I.L.L., Gräfelfing Germany), and >450-nm emitted light was collected by a photodiode detector from an adjustable viewfinder that selected a rectangular region containing a small cluster (3–5 cells) of loosely tethered sperm, each pivoting about a single point of attachment at the base of the head. The raw photometric signals were corrected for cell-free background, collected prior to each series of measurements. The ratio of the corrected signals was calibrated (5) with the constants Rmin (0.380), Rmax (1.795), and K* (1228 nM) obtained from cells equilibrated in solutions fortified with ionomycin (10 µM) and containing 20 mM EGTA, 15 mM CaCl2, or 20 mM EGTA with 15 mM CaCl2 (calculated free Ca2+ concentration of 226 nM). The calibrated signal reports spatially averaged internal [Ca2+] from the head and proximal flagellum of several sperm. Further analyses were performed in Igor (Wavemetrics, Lake Oswego, OR). Statistical analyses were performed in Excel (Microsoft, Redmond, WA). All results are presented as mean ± S.E., except where noted.

Ester Loading of cAMP—The cAMP-AM was dispensed from a 20 mM stock in Me2SO, dispersed in 10–15% Pluronic F127, diluted in 0.35 ml of medium Na7.4, and then immediately mixed with 0.15 ml of sperm suspension for a final concentration of 60 µM. After 30 min, an aliquot was added to the sample chamber containing medium Na7.4 for imaging. Data were collected within 5 min to preclude loss of signal due to declining cAMP content following dilution of external cAMP-AM.

Waveform Analysis—The flagellar waveform was analyzed as described (27). Briefly, stop-motion digital images were collected at 30 Hz from a 128 x 128-pixel region of the camera chip (larger regions were used for asymmetry measurements), under the direction of Metamorph (Universal Imaging, West Chester PA). Images were stored in TIFF format for subsequent semiautomated tracing of the flagellum. Additional software routines analyzed flagellar images to (i) determine the flagellar beat frequency; (ii) tabulate the distance along the flagellum (arc length), the angular deviation (tangent angle) along the flagellum, and the time-averaged tangent angle; (iii) present the time-averaged tangent angle versus arc length data (shear curves) as a measure of flagellar beat asymmetry; (iv) determine the flagellar beat envelope and the beat amplitude at regular intervals along the beat axis; and (v) calculate the maximal curvature of the 20-µm midpiece in both the pro-hook and anti-hook directions (28).

Immunocytochemistry and Immunoblotting—As in prior work (5, 6, 25), sperm were probed with the affinity-purified antibodies CNB1, CNC1, or CNE2, directed respectively against the pore-forming {alpha} subunits of the CaV2.2, CaV1.2, and CaV2.3 channels that specify N-, L-, and R-type currents in somatic cells, using methods for confocal immunomicroscopy as described (5, 6). Briefly, fixed and permeabilized sperm were washed, blocked, and rinsed before incubation with diluted (1:15) antibody. Samples were rinsed again, treated with biotinylated anti-rabbit IgG (1:300), rinsed, treated with avidin D fluorescein (1:300), rinsed, and mounted for examination by confocal microscopy. For immunoblotting, spermatozoa were boiled in Laemmli sample buffer (1 x 106 cells/20 µl), separated by SDS-PAGE, transferred to nitrocellulose, probed with the indicated antibodies diluted in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.4) containing 2.5% nonfat milk, and treated with enhanced chemiluminescence reagents (Pierce).

Quantitative Real-time PCR—Real-time PCR analysis was performed as described using previously validated primers for CatSper1 and CatSper2 (29). Testicular total RNA was treated with TURBO DNA-free reagent (Ambion) and reverse transcribed (2 µg) with random primers and Superscript III (Invitrogen) according to the manufacturer's protocol. The resultant cDNA samples were diluted 10-fold, and 3 µl was used as a template for amplification with the SYBR Green PCR Master Mix (Applied Biosystems). All samples were normalized to the 18 S ribosomal RNA signal for determination of relative expression levels, which were calculated according to the Applied Biosystems Comparative CT method, assuming that a 2-fold difference in concentration changes CT by ±1.

Cyclic AMP Measurements—Epididymal sperm were harvested at 37 °C in a "swimout/capacitation" medium that lacked NaHCO3 and Ca2+. After washing in the same modified medium, sperm were diluted 12-fold into medium Na7.4 with 5 mg of bovine serum albumin/ml containing or lacking Ca2+, NaHCO3, or both. After the indicated times at 37 °C, aliquots were diluted into an equal volume of cold 1 N perchloric acid, mixed, and frozen in liquid nitrogen. For the t = 0 samples, sperm were diluted into the various media already mixed with cold 1 N perchloric acid. After disruption by five freeze/thaw cycles (dry ice/ethanol bath and then 37 °C), cAMP was isolated and determined by radioimmunoassay as described (30).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
CatSper2 Null Sperm Lack Evoked Ca2+ Entry—Ca2+ entry in sperm is evoked by treatment with a high potassium, high pH medium (31), and the rate of depolarization-evoked Ca2+ entry, reported by Ca2+ photometry, indicates the relative number of open, voltage-gated Ca2+ channels (27). In Fig. 1, fura-2 monitored spatially averaged [Ca2+]i from small clusters of 3–7 motile sperm loosely adhered to a coverslip. The cells were perfused with medium Na7.4 alone or with 15 mM NaHCO3, except during 30-s depolarizing stimuli with medium K8.6. For wild-type sperm (Fig. 1A), [Ca2+]i rose abruptly during each stimulus and then returned slowly toward the initial resting level. As in past work (5, 25, 27, 32), channel opening was facilitated by incubation with the bicarbonate anion. The K8.6-evoked rate of rise was 21 ± 3nM s–1 before and 33 ± 6nM s–1 after conditioning with HCO–3 (Fig. 1C). The modest facilitation observed here presumably results from channel inactivation during the initial 30-s stimulus. For CatSper2 null sperm, depolarization evoked little or no increase in Ca2+ before or after conditioning with HCO–3 (Fig. 1B). Rates of rise were <1nM s–1 under both conditions. As for CatSper1 (25), CatSper2 is required for depolarization-evoked Ca2+ entry in sperm.

Unaffected Regional Distributions of CaV Channels in CatSper2 Null Sperm—A requirement of CatSper2 for evoked Ca2+ entry suggests, but does not demonstrate, that the CatSper2 protein functions as a Ca2+ entry channel. Therefore, we considered the alternate hypothesis that the CatSper2 protein instead is required for membrane targeting and functional expression of conventional voltage-gated Ca2+ channels, which also are candidates for the route of depolarization-evoked Ca2+ entry in sperm (5, 6). However, we find indistinguishable regional localizations of CaV1.2, CaV2.2, and CaV2.3 immunoreactivity in wild-type and CatSper2 null sperm (Fig. 2). The loss of Ca2+ entry channel function in CatSper2 null sperm apparently does not result from disrupted regional distributions of CaV channel proteins.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 1.
No evoked Ca2+ entry in CatSper2 null sperm. Shown is intracellular free Ca2+ concentration reported by fura-2 ratiometric photometry. Averaged responses are shown from five small clusters of cells locally perfused with Na7.4 or with Na7.4 supplemented with 15 mM NaHCO3 (15 BC), except during 30-s depolarizing stimuli with medium K8.6, as indicated. A, wild-type sperm (wt). B, CatSper2 null sperm. Rates of rise (nM s–1) are noted. C, averaged rates of rise evoked by depolarization applied before (0 BC; circles) and after (15 BC; squares) conditioning of wild-type (open symbols) and CatSper2 null (closed symbols) sperm with 15 mM NaHCO3. n = 20 clusters of cells, in four independent experiments. Boxes indicate the mean and S.E.

 
No Hyperactivated Motility for CatSper2 Null Sperm—We also examined whether CatSper2 is required for the highly asymmetrical flagellar waveform that is a hallmark of sperm hyperactivation. In Fig. 3 the time-averaged distribution of bending along the flagellum provides a quantitative measure of asymmetry. For wild-type sperm bathed in Na7.4, asymmetry was low initially but became greater after incubation under capacitating conditions (Fig. 3A). The mean value for asymmetry at 40 µm along the flagellum increased from <0.3 to >1.0 radians. In contrast, the asymmetry of CatSper2 null sperm changed little after capacitating incubations (Fig. 3B). The mean value for asymmetry at 40 µm along the flagellum remained <0.2 radians.

The hyperactivated waveform also has a larger beat amplitude, here measured by the maximal excursion from the flagellar beat axis for each point along the flagellum (Fig. 3, C and D). The amplitude at 30 µm for wild-type sperm in Na7.4 increased from 28 ± 2to35 ± 2 µm after capacitating incubations. The beat amplitude of CatSper2 null sperm decreased slightly from 25 ± 1 to 23 ± 2 µm after capacitating incubations.



View larger version (60K):
[in this window]
[in a new window]
  		FIGURE 2.
CatSper2 is not required for regional distribution of CaV channel proteins. Confocal immunofluorescence images shown in reverse contrast for wild-type (wt) and CatSper2 null sperm treated with antibodies directed to the CaV1.2 (A and B); CaV2.2 (C and D); and CaV2.3 channel proteins (E and F). Each panel contains representative images of central optical sections from the head (above) and the proximal flagellum (below).

 



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 3.
CatSper2 is required for hyperactivated waveform. A and B, flagellar asymmetry reported by time-averaged tangent angles for wild-type (wt) and null sperm, examined before (gray) and after (black) capacitating incubations. C and D, the beat amplitude, averaged for all cells examined, measured by the maximal excursion over two or more beat cycles from the flagellar beat axis for each point along the flagellum. n = 17–23 in four independent experiments.

 
Procaine Partially Rescues Flagellar Asymmetry in CatSper2 Null Sperm—We asked whether CatSper2 null sperm lack an asymmetrical waveform after capacitating incubations due to a defective flagellar motor or due to defects in the mechanisms that control it. As a test, we applied an alternative, pharmacological method to induce flagellar asymmetry. Past work reports that local anesthetics such as procaine evoke hyperactivation in sperm (33–35). Procaine action requires external Ca2+ but may be independent of cAMP and tyrosine phosphorylation (36). We find that with a 5-min exposure to 10 mM procaine, both wild-type and CatSper2 null sperm display a highly asymmetrical flagellar beat (Fig. 4A). Thus, the lesion that prevents capacitating incubations from producing waveform asymmetry is not in the flagellar axoneme of the CatSper2 null sperm but instead in the signals that control it.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 4.
Partial rescue of hyperactivation deficit in CatSper2 null sperm by procaine. A, flagellar asymmetry; C, beat amplitude for wild-type (wt)(white and gray) and null (black) sperm, examined after 5-min treatment with 10 mM procaine. B, beat frequency of wild-type (open) or CatSper2 null (closed) sperm randomly sampled before or after incubation with capacitating conditions or with 10 mM procaine treatment. D, midpiece curvature of wild-type (open) and CatSper2 null (closed) sperm before and after treatment with capacitating conditions or after treatment with 10 mM procaine. n = 12–23 cells in four independent experiments. Different letters indicate p < 0.05 for identity of the indicated groups of data.

 
The waveform of procaine-treated sperm was similar but not identical to that of the hyperactivated waveform of wild-type sperm. Whereas capacitating incubations decreased the beat frequency of wild-type sperm, procaine treatment did not (Fig. 4B). For CatSper2 null sperm, capacitating incubations marginally increased and procaine marginally decreased beat frequency. In addition, procaine treatment decreased beat amplitude of both wild-type and CatSper2 null sperm (Fig. 4C), whereas hyperactivating conditions increased only the beat amplitude of wild-type sperm (Fig. 3C). We further characterized the procaine-induced waveform by measuring the maximal curvature of the flagellar midpiece in both the pro-hook and anti-hook bend directions (i.e. in the same or opposite direction as the hook of the head (28)). After capacitating incubations, wild-type sperm displayed increased pro-hook curvature (arbitrarily assigned a negative value) of the midpiece. Procaine treatment did not increase pro-hook curvature but decreased anti-hook curvature (indicated by a positive value) of both wild-type and CatSper2 null sperm (Fig. 4D). Thus, the similar asymmetry of the waveform of hyperactivation produced by incubation under capacitating conditions and the waveform produced by procaine (cf. Figs. 3A and 4A) are produced by different distributions of bending in pro- and anti-hook directions.

Activation of CatSper2 Null Sperm by Bicarbonate—One of the earliest stages of capacitation is the acceleration of sperm motility that occurs shortly after mating, a process termed activation. Activation is probably signaled by the high HCO–3 concentrations in male and female reproductive fluids. In vitro, the flagellar beat frequency increases severalfold within seconds of exposing sperm to HCO–3 (25, 27). This Ca2+-dependent action of HCO–3 (25) occurs by stimulation of the atypical adenylyl cyclase of sperm (37), and by cAMP-mediated activation of protein kinase A (PKA) (32). Past work found that bicarbonate-evoked activation of motility is not impaired in CatSper1 null sperm (25). We now find that, like wild-type sperm, the CatSper2 null sperm also increase their beat frequency nearly 3-fold upon exposure to 15 mM NaHCO3 or upon incubation with the membrane-permeant cAMP-AM ester (Fig. 5). Thus, CatSper2 is not required for signaling events downstream of cAMP in the bicarbonate-evoked acceleration of the flagellar beat.



View larger version (7K):
[in this window]
[in a new window]
  		FIGURE 5.
CatSper2 is not required for HCO–3 to accelerate flagellar beat. Parallel experiments monitored wild-type (wt)(open) and CatSper2 null (closed) sperm randomly sampled after a 1–10-min incubation in media lacking or containing 15 mM NaHCO3 (to stimulate cAMP production). Ester-generated cAMP was supplied by incubating sperm for 30 min with the membrane-permeant cAMP-AM (60 µM). n = 21–31 in four independent experiments.

 
Increased Basal Beat Frequency of CatSper2 Null Sperm Due to Elevated cAMP—Fig. 5 also shows that, as for CatSper1 null sperm (25), the CatSper2 null cells have an elevated basal (before exposure to HCO–3) beat frequency (3.8 ± 0.1 versus 2.8 ± 0.2 Hz for wild-type sperm). Examining the cause of the accelerated beat, we measured the cAMP content of CatSper2 null and wild-type sperm before and during treatment with HCO–3 (Fig. 6A). The CatSper2 null sperm had an elevated basal cAMP content (inset). In Ca2+-deficient medium, HCO–3 had little or no effect on cAMP content. In the presence of external Ca2+,HCO–3 altered the cAMP content of both wild-type and CatSper2 null sperm with similar but not identical time courses. For sperm of both types, cAMP content rapidly increased >5-fold within the first 1 min of exposure, declined to ~3-fold of the basal value by 5 min, and then again rose to >5-fold the initial value by 30 min. However, the secondary rise was more rapid for CatSper2 null sperm, whose contents at 15 and 30 min were similarly elevated.

To examine whether the elevated basal cAMP increases the basal flagellar beat frequency of CatSper2 null sperm by a PKA-mediated pathway, we applied pharmacological blockade with PKA inhibitor H89, shown previously (27) to block HCO–3-evoked acceleration of the beat of wild-type sperm. Fig. 6B compares the beat frequency before and during exposure to a 30 µM concentration of the PKA inhibitor H89. The H89 had little or no effect on the 2.5 ± 0.2-Hz basal beat of wild-type sperm but decreased the beat of CatSper2 null sperm from 4.1 ± 0.2 to 2.5 ± 0.2 Hz, the same value as the basal beat of wild-type sperm (Fig. 6B). We conclude that the increased basal cAMP content of CatSper2 null sperm raises the resting beat frequency by PKA-mediated protein phosphorylation.

Co-dependent Expression of CatSper1 and CatSper2 Proteins—The numerous similarities in the phenotypes of CatSper1 null and CatSper2 null sperm indicate that the two proteins are required components in the same pathway(s) that controls Ca2+ entry and flagellar waveform asymmetry. In the simplest interpretation, the sperm of both null mutants lack functional channels for an entry of Ca2+ that is required to produce the flagellar asymmetry of hyperactivation. Such functional co-dependence could have molecular explanation at several levels: formation of a functional membrane channel; trafficking of the putative channel proteins to the membrane; translation and post-translational modification of the channel proteins, or production and processing of the mRNA that encode them.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 6.
Elevated cAMP, PKA activity, and basal beat of CatSper2 null sperm. A, cAMP content of wild-type (wt)(open) and CatSper2 null (closed) sperm at the indicated times of exposure to 15 mM NaHCO3 in the presence (squares) or nominal absence (circles) of Ca2+ (n = 3–4). Inset, basal cAMP content of wild-type (open) and CatSper2 null (closed) sperm before exposure to NaHCO3. B, beat frequency of wild-type (open) or CatSper2 null (closed) sperm randomly sampled after a 5–10-min incubation in media lacking or containing 30 µM H89. n = 13–14 in two independent experiments.

 
Previous studies of testicular gene expression by microarray analyses indicate that the transcription of CatSper2 begins several days before that of CatSper1 in the developing mouse testis (29, 38). To determine whether CatSper2 transcription is required for subsequent production of CatSper1 mRNA, we examined adult CatSper1 and -2 null testes by quantitative real-time PCR. Compared with wild-type testes, neither the relative content of CatSper1 mRNA in the CatSper2 null testes (1.65 ± 1.64-fold; n = 3) nor the CatSper2 mRNA content of CatSper1 null testes (1.01 ± 1.12-fold; n = 3) was significantly altered. Next, we considered whether CatSper1 and -2 proteins stabilize each other through an interaction at the protein level. Fig. 7 shows immunoblots of wild-type sperm and CatSper1 and -2 null sperm probed with antibodies directed against the CatSper1 (Fig. 7A) and CatSper2 proteins (Fig. 7B). The wild-type sperm show prominent immunoreactive ~82 kDa CatSper1 and ~72 kDa CatSper2 bands. Neither of these bands was detected in either the CatSper1 or -2 null sperm examined at similar protein loading as determined with an antibody for {alpha}-tubulin (Fig. 7C). Fig. 7D shows PCR analysis of genomic DNA from the animals used in the immunoblotting experiments, providing confirmation of the assigned genotype.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The database-mining strategy that led to the discovery of CatSper1 (2) and CatSper2 (1) was followed (3) by the identification of two other members of this new family of putative cation channels. Although demonstration of channel function by heterologous expression has remained a difficult and unmet challenge, interest in the CatSpers has remained high due to (i) their unique expression in sperm (1, 2), (ii) their requirement for male fertility (2, 17) and possible role(s) in heritable male infertility (39, 40), and (iii) the unique and informative loss-of-function sperm phenotype produced by their targeted disruption (2, 17, 25). Here, we have further characterized and compared the phenotypes of CatSper1 and -2 null sperm. Our results reveal new aspects of the CatSper2 null phenotype and show that several previously described characteristics of CatSper1 null sperm are shared by the CatSper2 null mutants. These findings lead to the proposal that the phenotypes in fact are identical and to a demonstration that such identity of phenotype is a consequence of co-dependent expression of the two proteins.



View larger version (52K):
[in this window]
[in a new window]
  		FIGURE 7.
CatSper1 protein is absent from CatSper2 null sperm, and CatSper2 protein is absent from CatSper1 sperm. The expression of CatSper1 and CatSper2 in spermatozoa is co-dependent. Duplicate immunoblots of wild-type (wt), CatSper1 null, and CatSper2 null spermatozoa were probed with antibodies to CatSper1 diluted 1:750 (A) and CatSper2 diluted 1:3000 (B). Following the removal of the CatSper2 antibody, this blot was reprobed with antibodies to {alpha}-tubulin diluted 1:10,000 (C). One million sperm cells were loaded in each lane. Relative molecular weights are indicated at the left of A–C. The expected sizes of CatSper1 and CatSper2 based on primary sequence are 79 and 68 kDa, respectively. The dye front is indicated as d.f. D, genotype confirmation of the samples used in the shown immunoblot experiment. Top, CatSper1 gene PCR analysis: wild-type allele, 2100 bp; null allele, 1700 bp. Bottom, CatSper2 gene PCR analysis: wild-type allele, 641 bp; null allele, 831 bp.

 
Past work found immunological evidence that the pore-forming {alpha} subunits of several conventional voltage-gated (CaV) channels are regionally distributed in distinctive patterns in both the sperm head and flagellum (4–6) and that depolarization-evoked entry of Ca2+ has a pharmacological sensitivity profile consistent with involvement of CaV2.2 and CaV2.3 channels (5). Unexpectedly, CatSper1 null sperm lacked the evoked entry of Ca2+ (25), indicating that CatSper1 is required directly or indirectly for functional channels. Regional distributions of CaV1.2, CaV2.2, and CaV2.3 channel proteins in CatSper1 null sperm were not distinguishable from those of wild-type sperm, suggesting that the absence of CatSper1 does not affect expression and trafficking of CaV channel proteins. Here we find that CatSper2 null sperm also lack evoked Ca2+ entry (Fig. 1) and have similarly unperturbed distributions of CaV channel protein (Fig. 2). In the simplest interpretation, the CatSper1 and -2 proteins present in the membrane of the principal piece (1, 2) form voltage-gated Ca2+ channels that are the major route for depolarization-evoked entry of Ca2+ into the flagellum. The photometric methods used here and in past studies of evoked entry of Ca2+ in wild-type sperm (5, 25–27) report changes in spatially averaged [Ca2+] from the heads and proximal flagella of several cells. Evoked Ca2+ entry also occurs when the detection window is limited to the flagella of clusters of wild-type sperm4 and when Ca2+ is monitored by imaging of individual immobilized sperm (41). We also note that the flagellum must possess at least one other route of Ca2+ entry, necessary to explain the preserved Ca2+ dependence of the stimulatory action of bicarbonate on the flagellar beat of CatSper1 null sperm (25) and of the cAMP accumulation by CatSper2 null sperm (Fig. 6). Perhaps these Ca2+-dependent, CatSper-independent actions of bicarbonate are mediated by a direct action of Ca2+ on the sperm adenylyl cyclase (37).

Prior analysis of the flagellar waveform (25) and the swimming behavior (2, 17) of CatSper1 (2, 25) and CatSper2 (17) null sperm indicated that CatSper1 and -2 are each required for sperm hyperactivation as variously assessed by flagellar beat asymmetry, by the profile of path parameters from automated analysis of swimming tracks (2), and by penetration of viscous media (17) and of zona-free but not zona-intact eggs during fertilization in vitro (2, 17). Here we show (Fig. 3) that CatSper2, like CatSper1 (25), is required for the asymmetrical waveform that underlies the swimming behavior of hyperactivated sperm. Several other sperm characteristics were found not to require CatSper1 (25) or CatSper2 (Figs. 2, 4, 5, and 6A). All available evidence is consistent with the hypothesis that the hyperactivation deficit is the critical lesion in the infertility of CatSper1 and -2 sperm.

In permeabilized sperm preparations, Ca2+ mediates transition from a symmetrical to an asymmetric flagellar waveform (35, 42), which is similar to that observed for intact sperm hyperactivated during Ca2+-dependent capacitation in vitro. In the simplest explanation, CatSper1 and -2 form functional channels that open to allow an entry of Ca2+, which is required to initiate or sustain hyperactivation. In a more complicated explanation, CatSper1 and -2 might function to maintain or refill a putative mobilizable internal store proposed by others (43) to provide a Ca2+ signal for hyperactivation. Tests to distinguish between these hypotheses have not been made.

In yet another possible explanation for the hyperactivation deficit of CatSper1 and -2 null sperm, these proteins could be required during spermiogenesis for formation of a hyperactivation-competent flagellar axoneme, capable of responding to the Ca2+ signal with waveform asymmetry. Past studies found that procaine (33, 35, 43) and other membrane active agents (33, 35, 44) produce Ca2+-dependent, hyperactivation-like responses in sperm. We now find that the loss-of-flagel-lar-asymmetry phenotype of CatSper2 null sperm can be rescued pharmacologically by procaine treatment (Fig. 4A), indicating that defects in the responses of the flagellum to Ca2+ do not explain the hyperactivation deficit of the mutant sperm. We note, however, that procaine treatment and capacitating incubations of wild-type sperm produce flagellar waveforms that differ in several ways (Figs. 3 and 4).

In contrast to the requirement of CatSper1 (25) and CatSper2 (Fig. 3) for Ca2+-mediated control of flagellar asymmetry, CatSper1 (25) and CatSper2 (Figs. 5 and 6A) are not required for the cAMP-mediated control of beat frequency. Despite this clear separation of functional roles, the cAMP and Ca2+ signaling systems of sperm seem to be intricately interrelated. For example, past work finds that PKA-mediated phosphorylation is required to facilitate Ca2+ channel activity (27, 32) and to limit the early accumulation of cAMP (32). Conversely, external Ca2+ and presumably Ca2+ entry are required for bicarbonate-evoked increases in cAMP content (Fig. 6A; also see Ref. 45) and acceleration of the flagellar beat (25). In addition, we now see that a presumptive entry of Ca2+ through a CatSper2-dependent pathway may determine the set point for resting cAMP content (Fig. 6).

Previous attempts to demonstrate a functional or physical association of CatSper1 and -2 were unsuccessful (1). Here, we find a reciprocal requirement of CatSper1 and CatSper2 to stabilize both proteins in cauda epididymal sperm. A simple explanation of this codependency of stable expression is that the two proteins are components of a single heteromeric ion channel that regulates sperm hyperactivation. However, functional channels have not yet been produced by co-expression of the CatSper1 and -2 proteins in somatic cells. A possible explanation is that another component(s) is required for the appropriate folding, trafficking, or assembly of a functional CatSper channel.

In summary, knowledge of sperm physiology has increased rapidly from applications of a genetic approach to study of the roles of CatSper1 and -2 and of other sperm components that include sNHE, the novel putative Na+/H+ exchanger of sperm (46), atypical adenylyl cyclase of sperm (37), and unique PKA C{alpha}2 (32). Continued study of these and other yet uncharacterized sperm-specific signaling proteins (29) holds significant promise for ultimate applications to control male fertility.


   FOOTNOTES
 
* This work was supported by U54-HD12629 of the Specialized Cooperative Centers Program in Reproduction Research of NICHD, National Institutes of Health, Grant 5R01-HD36022 and the Cecil and Ida Green Center for Reproductive Biology Sciences at the University of Texas Southwestern Medical Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Table I. Back

1 Supported in part by NIGMS Public Health Service National Research Service Award T32 GM07270. Back

2 To whom correspondence should be addressed: Dept. of Physiology and Biophysics, MS 357290, University of Washington, Seattle, WA 98195-7290. Tel.: 206-543-6661; Fax: 206-685-0619; E-mail: donner{at}u.washington.edu.

3 The abbreviations used are: AM, acetoxymethyl; PKA, protein kinase A; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid. Back

4 A. E. Carlson, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Drs. David L. Garbers and G. Stanley McKnight for critically reviewing the manuscript. T. A. Q. also thanks David L. Garbers for providing both space and resources for carrying out some of the work reported here.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Quill, T. A., Ren, D., Clapham, D. E., and Garbers, D. L. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 12527–12531[Abstract/Free Full Text]
  2. Ren, D., Navarro, B., Perez, G., Jackson, A. C., Hsu, S., Shi, Q., Tilly, J. L., and Clapham, D. E. (2001) Nature 413, 603–609[CrossRef][Medline] [Order article via Infotrieve]
  3. Lobley, A., Pierron, V., Reynolds, L., Allen, L., and Michalovich, D. (2003) Reprod. Biol. Endocrinol. 1, 53–68[CrossRef][Medline] [Order article via Infotrieve]
  4. Serrano, C. J., Trevino, C. L., Felix, R., and Darszon, A. (1999) FEBS Lett. 462, 171–176[CrossRef][Medline] [Order article via Infotrieve]
  5. Wennemuth, G., Westenbroek, R. E., Xu, T., Hille, B., and Babcock, D. F. (2000) J. Biol. Chem. 275, 21210–21217[Abstract/Free Full Text]
  6. Westenbroek, R. E., and Babcock, D. F. (1999) Dev. Biol. 207, 457–469[CrossRef][Medline] [Order article via Infotrieve]
  7. Platzer, J., Engel, J., Schrott-Fischer, A., Stephan, K., Bova, S., Chen, H., Zheng, H., and Striessnig, J. (2000) Cell 102, 89–97[CrossRef][Medline] [Order article via Infotrieve]
  8. Ino, M., Yoshinaga, T., Wakamori, M., Miyamoto, N., Takahashi, E., Sonoda, J., Kagaya, T., Oki, T., Nagasu, T., Nishizawa, Y., Tanaka, I., Imoto, K., Aizawa, S., Koch, S., Schwartz, A., Niidome, T., Sawada, K., and Mori, Y. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 5323–5328[Abstract/Free Full Text]
  9. Saegusa, H., Kurihara, T., Zong, S., Minowa, O., Kazuno, A., Han, W., Matsuda, Y., Yamanaka, H., Osanai, M., Noda, T., and Tanabe, T. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6132–6137[Abstract/Free Full Text]
  10. Kim, D., Song, I., Keum, S., Lee, T., Jeong, M. J., Kim, S. S., McEnery, M. W., and Shin, H. S. (2001) Neuron 31, 35–45[CrossRef][Medline] [Order article via Infotrieve]
  11. Chen, C. C., Lamping, K. G., Nuno, D. W., Barresi, R., Prouty, S. J., Lavoie, J. L., Cribbs, L. L., England, S. K., Sigmund, C. D., Weiss, R. M., Williamson, R. A., Hill, J. A., and Campbell, K. P. (2003) Science 302, 1416–1418[Abstract/Free Full Text]
  12. Biel, M., Seeliger, M., Pfeifer, A., Kohler, K., Gerstner, A., Ludwig, A., Jaissle, G., Fauser, S., Zrenner, E., and Hofmann, F. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 7553–7557[Abstract/Free Full Text]
  13. Stowers, L., Holy, T. E., Meister, M., Dulac, C., and Koentges, G. (2002) Science 295, 1493–1500[Abstract/Free Full Text]
  14. Freichel, M., Suh, S. H., Pfeifer, A., Schweig, U., Trost, C., Weissgerber, P., Biel, M., Philipp, S., Freise, D., Droogmans, G., Hofmann, F., Flockerzi, V., and Nilius, B. (2001) Nat. Cell Biol. 3, 121–127[CrossRef][Medline] [Order article via Infotrieve]
  15. Seisenberger, C., Specht, V., Welling, A., Platzer, J., Pfeifer, A., Kuhbandner, S., Striessnig, J., Klugbauer, N., Feil, R., and Hofmann, F. (2000) J. Biol. Chem. 275, 39193–39199[Abstract/Free Full Text]
  16. Jun, K., Piedras-Renteria, E. S., Smith, S. M., Wheeler, D. B., Lee, S. B., Lee, T. G., Chin, H., Adams, M. E., Scheller, R. H., Tsien, R. W., and Shin, H. S. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 15245–15250[Abstract/Free Full Text]
  17. Quill, T. A., Sugden, S. A., Rossi, K. L., Doolittle, L. K., Hammer, R. E., and Garbers, D. L. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 14869–14874[Abstract/Free Full Text]
  18. Yanagimachi, R. (1994) in The Physiology of Reproduction (Knobil, E., Neill, J. D., eds) pp. 189–317, Raven Press, New York
  19. Cooper, T. G. (1984) Gamete Res. 9, 55–74
  20. Fraser, L. R. (1982) J. Androl. 3, 412–419[Abstract]
  21. Suarez, S. S., Katz, D. F., and Overstreet, J. W. (1983) Biol. Reprod. 29, 1277–1287[Abstract]
  22. Yanagimachi, R. (1970) J. Reprod. Fertil. 23, 193–196[Abstract/Free Full Text]
  23. Yanagimachi, R., and Usui, N. (1974) Exp. Cell Res. 89, 161–174[CrossRef][Medline] [Order article via Infotrieve]
  24. Garbers, D. L. (2001) Nature 413, 579, 581–582[CrossRef][Medline] [Order article via Infotrieve]
  25. Carlson, A. E., Westenbroek, R. E., Quill, T., Ren, D., Clapham, D. E., Hille, B., Garbers, D. L., and Babcock, D. F. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 14864–14868[Abstract/Free Full Text]
  26. Wennemuth, G., Babcock, D. F., and Hille, B. (2003) J. Gen. Physiol. 122, 115–128[Abstract/Free Full Text]
  27. Wennemuth, G., Carlson, A. E., Harper, A. J., and Babcock, D. F. (2003) Development 130, 1317–1326[Abstract/Free Full Text]
  28. Ishijima, S., Baba, S. A., Mohri, H., and Suarez, S. S. (2002) Mol. Reprod. Dev. 61, 376–384[CrossRef][Medline] [Order article via Infotrieve]
  29. Schultz, N., Hamra, F. K., and Garbers, D. L. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 12201–12206[Abstract/Free Full Text]
  30. Domino, S. E., Tubb, D. J., and Garbers, D. L. (1991) Methods Enzymol. 195, 345–355[Medline] [Order article via Infotrieve]
  31. Babcock, D. F., and Pfeiffer, D. R. (1987) J. Biol. Chem. 262, 15041–15047[Abstract/Free Full Text]
  32. Nolan, M. A., Babcock, D. F., Wennemuth, G., Brown, W., Burton, K. A., and McKnight, G. S. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 13483–13488[Abstract/Free Full Text]
  33. Singh, J. P., Babcock, D. F., and Lardy, H. A. (1983) Arch. Biochem. Biophys. 221, 291–303[CrossRef][Medline] [Order article via Infotrieve]
  34. Vijayaraghavan, S., Critchlow, L. M., and Hoskins, D. D. (1985) Biol. Reprod. 32, 489–500[Abstract]
  35. Lindemann, C. B., Goltz, J. S., and Kanous, K. S. (1987) Cell Motil. Cytoskeleton 8, 324–332[CrossRef][Medline] [Order article via Infotrieve]
  36. Marquez, B., and Suarez, S. S. (2004) Biol. Reprod. 70, 1626–1633[Abstract/Free Full Text]
  37. Esposito, G., Jaiswal, B. S., Xie, F., Krajnc-Franken, M. A., Robben, T. J., Strik, A. M., Kuil, C., Philipsen, R. L., van Duin, M., Conti, M., and Gossen, J. A. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2993–2998[Abstract/Free Full Text]
  38. Shima, J. E., McLean, D. J., McCarrey, J. R., and Griswold, M. D. (2004) Biol. Reprod. 71, 319–330[Abstract/Free Full Text]
  39. Avidan, N., Tamary, H., Dgany, O., Cattan, D., Pariente, A., Thulliez, M., Borot, N., Moati, L., Barthelme, A., Shalmon, L., Krasnov, T., Ben-Asher, E., Olender, T., Khen, M., Yaniv, I., Zaizov, R., Shalev, H., Delaunay, J., Fellous, M., Lancet, D., and Beckmann, J. S. (2003) Eur. J. Hum. Genet. 11, 497–502[CrossRef][Medline] [Order article via Infotrieve]
  40. Nikpoor, P., Mowla, S. J., Movahedin, M., Ziaee, S. A., and Tiraihi, T. (2004) Hum. Reprod. 19, 124–128[Abstract/Free Full Text]
  41. Fukuda, N., Yomogida, K., Okabe, M., and Touhara, K. (2004) J. Cell Sci. 117, 5835–5845[Abstract/Free Full Text]
  42. Brokaw, C. J. (1979) J. Cell Biol. 82, 401–411[Abstract/Free Full Text]
  43. Ho, H. C., and Suarez, S. S. (2003) Biol. Reprod. 68, 1590–1596[Abstract/Free Full Text]
  44. Gu, Y., Kirkman-Brown, J. C., Korchev, Y., Barratt, C. L., and Publicover, S. J. (2004) Dev. Biol. 274, 308–317[CrossRef][Medline] [Order article via Infotrieve]
  45. Garbers, D. L., Tubb, D. J., and Hyne, R. V. (1982) J. Biol. Chem. 257, 8980–8984[Abstract/Free Full Text]
  46. Wang, D., King, S. M., Quill, T. A., Doolittle, L. K., and Garbers, D. L. (2003) Nat. Cell Biol. 5, 1117–1122[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
L.A. McPartlin, S.S. Suarez, C.A. Czaya, K. Hinrichs, and S.J. Bedford-Guaus
Hyperactivation of Stallion Sperm Is Required for Successful In Vitro Fertilization of Equine Oocytes
Biol Reprod, July 1, 2009; 81(1): 199 - 206.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
S. S. Suarez
Control of hyperactivation in sperm
Hum. Reprod. Update, November 1, 2008; 14(6): 647 - 657.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
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]

Home page
 Biol. Reprod.Home page
T. Harris, B. Marquez, S. Suarez, and J. Schimenti
Sperm Motility Defects and Infertility in Male Mice with a Mutation in Nsun7, a Member of the Sun Domain-Containing Family of Putative RNA Methyltransferases
Biol Reprod, August 1, 2007; 77(2): 376 - 382.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
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]

Home page
 Mol Hum ReprodHome page
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]

Home page
 Biol. Reprod.Home page
B. Marquez and S. S. Suarez
Bovine Sperm Hyperactivation Is Promoted by Alkaline-Stimulated Ca2+ Influx
Biol Reprod, April 1, 2007; 76(4): 660 - 665.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
K. A. Burton and G. S. McKnight
PKA, Germ Cells, and Fertility
Physiology, February 1, 2007; 22(1): 40 - 46.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
D. F. Babcock
Wrath of the wraiths of CatSper3 and CatSper4
PNAS, January 23, 2007; 104(4): 1107 - 1108.
[Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
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]

Home page
 J. Biol. Chem.Home page
D. Zhang, J. Chen, A. Saraf, S. Cassar, P. Han, J. C. Rogers, J. D. Brioni, J. P. Sullivan, and M. Gopalakrishnan
Association of Catsper1 or -2 with Cav3.3 Leads to Suppression of T-type Calcium Channel Activity
J. Biol. Chem., August 4, 2006; 281(31): 22332 - 22341.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
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 page
 Biol. Reprod.Home page
S. M. Schuh, A. E. Carlson, G. S. McKnight, M. Conti, B. Hille, and D. F. Babcock
Signaling Pathways for Modulation of Mouse Sperm Motility by Adenosine and Catecholamine Agonists
Biol Reprod, March 1, 2006; 74(3): 492 - 500.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/37/32238    most recent
M501430200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Carlson, A. E.
Right arrow Articles by Babcock, D. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carlson, A. E.
Right arrow Articles by Babcock, D. F.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement