Identical phenotypes of CatSper1 and CatSper2 null sperm.

Among several candidate Ca(2+) 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 Ca(2+)entry. Here we show that failure of hyperactivation of CatSper2 null sperm similarly correlates with an absence of depolarization evoked Ca(2+) entry. Additional shared aspects of the phenotypes of CatSper1 and -2 null sperm include unperturbed regional distributions of conventional voltage-gated Ca(2+) 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 Ca(2+) messenger rather than by preventing flagellar responses to Ca(2+). 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.


* 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. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Table I 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 ϫ 10 7 cells ml Ϫ1 . Potassium-evoked responses were produced with medium K8.6 (135 mM KCl, 5 mM NaCl, 2 mM CaCl 2 , 1 mM MgSO 4 , 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% CO 2 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 mM stocks in Me 2 SO, dispersed in 10 -15% Pluronic F127, diluted to 20 M 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 ϳ3 ml of medium. After ϳ5 min, test solutions were applied with a multibarreled local perfusion device (estimated exchange time of Ͻ0.8 s). Excitation light of 340 and 380 nm was provided from a computer-controlled monochromator (T.I.L.L., Gräfelfing Germany), and Ͼ450-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 R min (0.380), R max (1.795), and K* (1228 nM) obtained from cells equilibrated in solutions fortified with ionomycin (10 M) and containing 20 mM EGTA, 15 mM CaCl 2 , or 20 mM EGTA with 15 mM CaCl 2 (calculated free Ca 2ϩ concentration of 226 nM). The calibrated signal reports spatially averaged internal [Ca 2ϩ ] 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 Ϯ S.E., except where noted.
Ester Loading of cAMP-The cAMP-AM was dispensed from a 20 mM stock in Me 2 SO, 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 M. 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 ϫ 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-m midpiece in both the prohook and anti-hook directions (28).
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 DNAfree reagent (Ambion) and reverse transcribed (2 g) with random primers and Superscript III (Invitrogen) according to the manufacturer's protocol. The resultant cDNA samples were diluted 10-fold, and 3 l 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 C T method, assuming that a 2-fold difference in concentration changes C T by Ϯ1.
Cyclic AMP Measurements-Epididymal sperm were harvested at 37°C in a "swimout/capacitation" medium that lacked NaHCO 3 and Ca 2ϩ . 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 Ca 2ϩ , NaHCO 3 , 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 ϭ 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
CatSper2 Null Sperm Lack Evoked Ca 2ϩ Entry-Ca 2ϩ entry in sperm is evoked by treatment with a high potassium, high pH medium (31), and the rate of depolarization-evoked Ca 2ϩ entry, reported by Ca 2ϩ photometry, indicates the relative number of open, voltage-gated Ca 2ϩ channels (27). In Fig. 1, fura-2 monitored spatially averaged [Ca 2ϩ ] 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 NaHCO 3 , except during 30-s depolarizing stimuli with medium K8.6. For wild-type sperm (Fig. 1A), [Ca 2ϩ ] 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 Ϯ 3 nM s Ϫ1 before and 33 Ϯ 6 nM s Ϫ1 after conditioning with HCO 3 Ϫ (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 Ca 2ϩ before or after conditioning with HCO 3 Ϫ (Fig. 1B). Rates of rise were Ͻ1 nM s Ϫ1 under both conditions. As for CatSper1 (25), CatSper2 is required for depolarization-evoked Ca 2ϩ entry in sperm.

Unaffected Regional Distributions of Ca V Channels in CatSper2
Null Sperm-A requirement of CatSper2 for evoked Ca 2ϩ entry suggests, but does not demonstrate, that the CatSper2 protein functions as a Ca 2ϩ 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 Ca 2ϩ channels, which also are candidates for the route of depolarization-evoked Ca 2ϩ entry in sperm (5,6). However, we find indistinguishable regional localizations of Ca V 1.2, Ca V 2.2, and Ca V 2.3 immunoreactivity in wild-type and CatSper2 null sperm (Fig. 2). The loss of Ca 2ϩ entry channel function in CatSper2 null sperm apparently does not result from disrupted regional distributions of Ca V channel proteins.
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 m along the flagellum increased from Ͻ0.3 to Ͼ1.0 radians. In contrast, the asymmetry of CatSper2 null sperm changed little after capacitating incubations (Fig. 3B). The mean value for asymmetry at 40 m along the flagellum remained Ͻ0.2 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 m for wild-type sperm in Na7.4 increased from 28 Ϯ 2 to 35 Ϯ 2 m after capacitating incubations. The beat amplitude of CatSper2 null sperm decreased slightly from 25 Ϯ 1 to 23 Ϯ 2 m after capacitating incubations.
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)(34)(35). Procaine action requires external Ca 2ϩ 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.
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 procaineinduced 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 antihook 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 Ϫ concentrations in male and female reproductive fluids. In vitro, the flagellar beat frequency increases severalfold within seconds of exposing sperm to HCO 3 Ϫ (25, 27). This Ca 2ϩdependent action of HCO 3 Ϫ (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 NaHCO 3 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. Fig. 5 also shows that, as for CatSper1 null sperm (25), the CatSper2 null cells have an elevated basal (before exposure to HCO 3 Ϫ ) beat frequency (3.8 Ϯ 0.1 versus 2.8 Ϯ 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 Ϫ (Fig. 6A). The CatSper2 null sperm had an elevated basal cAMP content (inset). In Ca 2ϩ -deficient medium, HCO 3 Ϫ had little or no effect on cAMP content. In the presence of external Ca 2ϩ , HCO 3

Increased Basal Beat Frequency of CatSper2 Null Sperm Due to Elevated cAMP-
Ϫ 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 Ͼ5-fold within the first 1 min of exposure, declined to ϳ3-fold of the basal value by 5 min, and then again rose to Ͼ5-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 Ϫ -evoked acceleration of the beat of wild-type sperm. Fig. 6B compares the beat frequency before and during exposure to a 30 M concentration of the PKA inhibitor H89. The H89 had little or no effect on the 2.5 Ϯ 0.2-Hz basal beat of wildtype sperm but decreased the beat of CatSper2 null sperm from 4.1 Ϯ 0.2 to 2.5 Ϯ 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 Ca 2ϩ entry and flagellar waveform asymmetry. In the simplest interpretation, the sperm of both null mutants lack functional channels for an entry of Ca 2ϩ 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.
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 Ϯ 1.64-fold; n ϭ 3) nor the CatSper2 mRNA content of CatSper1 null testes (1.01 Ϯ 1.12-fold; n ϭ 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 wildtype 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 ϳ82 kDa CatSper1 and ϳ72 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 ␣-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
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-offunction 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.
Past work found immunological evidence that the pore-forming ␣ subunits of several conventional voltage-gated (Ca V ) channels are regionally distributed in distinctive patterns in both the sperm head and flagellum (4 -6) and that depolarization-evoked entry of Ca 2ϩ has a pharmacological sensitivity profile consistent with involvement of Ca V 2.2 and Ca V 2.3 channels (5). Unexpectedly, CatSper1 null sperm lacked the evoked entry of Ca 2ϩ (25), indicating that CatSper1 is required directly or indirectly for functional channels. Regional distributions of Ca V 1.2, Ca V 2.2, and Ca V 2.3 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 Ca V channel proteins. Here we find that CatSper2 null sperm also lack evoked Ca 2ϩ entry (Fig. 1) and have similarly unperturbed distributions of Ca V 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 Ca 2ϩ channels that are the major route for depolarization-evoked entry of Ca 2ϩ into the flagellum. The photometric methods used here and in past studies of evoked entry of Ca 2ϩ in wild-type sperm (5,(25)(26)(27) report changes in spatially averaged [Ca 2ϩ ] from the heads and proximal flagella of several cells. Evoked Ca 2ϩ entry also occurs when the detection window is limited to the flagella of clus-  ters of wild-type sperm 4 and when Ca 2ϩ is monitored by imaging of individual immobilized sperm (41). We also note that the flagellum must possess at least one other route of Ca 2ϩ entry, necessary to explain the preserved Ca 2ϩ 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 Ca 2ϩ -dependent, CatSper-independent actions of bicarbonate are mediated by a direct action of Ca 2ϩ 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, Ca 2ϩ mediates transition from a symmetrical to an asymmetric flagellar waveform (35,42), which is similar to that observed for intact sperm hyperactivated during Ca 2ϩdependent capacitation in vitro. In the simplest explanation, CatSper1 and -2 form functional channels that open to allow an entry of Ca 2ϩ , 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 Ca 2ϩ 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 Ca 2ϩ signal with waveform asymmetry. Past studies found that procaine (33,35,43) and other membrane active agents (33,35,44) produce Ca 2ϩ -dependent, hyperactivation-like responses in sperm. We now find that the loss-of-flagellar-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 Ca 2ϩ 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 Ca 2ϩ -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 Ca 2ϩ signaling systems of sperm seem to be intricately interrelated. For example, past work finds that PKA-mediated phosphorylation is required to facilitate Ca 2ϩ channel activity (27,32) and to limit the early accumulation of cAMP (32). Conversely, external Ca 2ϩ and presumably Ca 2ϩ 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 Ca 2ϩ 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 ϩ /H ϩ exchanger of sperm (46), atypical adenylyl cyclase of sperm (37), and unique PKA C␣ 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.