Wrath of the wraiths of CatSper3 and CatSper4

The spermatozoon is a marvelous machine designed by nature for the single task of finding an oocyte, delivering its genetic payload, and initiating the formation of a new embryo. Like many of the most specialized and sophisticated mechanical creations of humans, the sperm is fitted with unique components and stripped of unneeded hardware, including most of its biosynthetic equipment. As a consequence, for mice, humans, and other mammals, the male stores sperm in a quiescent state to conserve irreplaceable resources. Upon release into the female reproductive tract, the ejaculated sperm “awaken” to begin the obligatory maturational sequence, called “capacitation,” that is required to complete their vital mission. Several of the components of capacitation require extracellular Ca²⁺. These include an early activation of sperm motility, thought to be necessary for entry of sperm into the oviduct, and a subsequent peculiar “hyperactivation” of motility, thought to be necessary for sperm to penetrate the cumulus oophorus and zona pellucida that surround the oocyte (1, 2). Our understanding of the Ca²⁺ requirement for hyperactivation has increased greatly since the discovery of the CatSpers (3–5), a unique family of four sperm-specific ion channel proteins. In landmark work last year (6), the Clapham laboratory reported successful whole-cell patch-clamp recording of a Ca²⁺-selective, alkaline-promoted current that is present in wild-type sperm but absent in sperm from CatSper1^−/− mice. In this issue of PNAS, Qi et al. (7) reveal that all four CatSper proteins are required for sperm to form the flagellar ion channels that provide the route of entry for the Ca²⁺ that is needed for hyperactivation. The lack of functional CatSper channels prevents hyperactivation and produces the male infertility phenotype shared by mice carrying mutations that prevent transcription of full-length Catsper1 (4), CatSper2 (3), or CatSper3 or CatSper4 (7).

Stable Complexes of CatSper Proteins

Previous studies of testicular gene expression by microarray analysis found CatSper2 mRNA in the pachytene spermatocytes that appear at ≈12 days postpartum in the developing mouse testis but detected CatSper1 mRNA only after round spermatids were produced at approximately day 20 (8, 9). Similarly, CatSper3 and CatSper4 mRNAs were found in extracts of testis prepared at day 20 but not in those prepared at day 10 (10). Using in situ hybridization in testis tissue sections, Qi et al. (7) now provide additional evidence that CatSper3 and CatSper4 transcription is restricted to the late-stage germ-line cells that line the seminiferous tubules. This finding eliminates the possibility that CatSper3 and CatSper4 are required in the Sertoli cells that support the developing sperm.

Past work established that the CatSper1 mRNA contents of testes from adult wild-type and CatSper2^−/− mice are similar, as are CatSper2 mRNA contents of wild-type and CatSper1^−/− testis (11). Nevertheless, the CatSper1^−/− sperm lack CatSper2 protein, and CatSper2^−/− sperm lack CatSper1 protein, indicating that stable expression of the CatSper1 protein requires CatSper2 and vice versa (11). Qi et al. (7) now show that the CatSper1 protein is found together with CatSper3 and CatSper4 proteins in immunoprecipitates prepared from wild-type testis, indicating that these three proteins are physically associated in the spermatogenic cells. Likewise, evidence of physical association of CatSper proteins was found when CatSper2, CatSper3, or CatSper4 cDNA was coexpressed with CatSper1 in a cultured cell line. Unexpectedly, CatSper3 and CatSper4 proteins also were detected in CatSper1^−/− testis, raising the possibility that stable expression of CatSper3 and CatSper4 proteins might have less stringent requirements than those for CatSper1 and CatSper2. If so, then CatSper3 and CatSper4 proteins might be retained in mature CatSper1^−/− sperm. However, if CatSper3 and CatSper4 are present, they do not seem to form functional cation channels detectable in patch-clamp recordings from CatSper1^−/− sperm. Direct examination of the CatSper3 and CatSper4 content of CatSper1^−/− sperm will be informative but will require adequate caution because of the possibly incomplete specificity of the available antibodies revealed here in the residual immunoreactivity of CatSper4^−/− sperm probed with anti-CatSper4 antibody. An attractive alternate approach may come from recent improvements in proteomic methods to study sperm membrane composition (12).

All four CatSper proteins are required for sperm to form flagellar ion channels.

Restricted Roles for CatSper Channels

Past work also shows that CatSper1 and CatSper2 are not required for production of morphologically normal sperm, which can mature in vitro to reach several other milepost events in capacitation (11, 13). These include the Ca²⁺-dependent activation of motility that occurs when sperm encounter the HCO₃⁻ anion (14) and the delayed Ca²⁺-dependent engagement of a protein kinase cascade (15). Presumably, sperm possess one or more additional routes for Ca²⁺ entry that are unperturbed by the absence of CatSper1 and CatSper2. Immunological methods suggested several candidates (11, 13), but so far none have been verified by current records from patch-clamped sperm.

Qi et al. (7) found that CatSper3^−/− and CatSper4^−/− sperm examined in a medium with NaHCO₃ initially had swimming speeds similar to those of wild-type sperm, indicating that the activation of motility also does not require the CatSper3 or CatSper4 proteins. However, both swimming speeds and the proportion of motile cells declined during prolonged incubation of sperm from each of the four CatSper^−/− mutants. The meaning and cause of this decline are unclear. If the ghosts (wraiths) of the departed CatSpers were angry (wrathful), vengeful, and capable of wreaking scientific mischief, they might create just such a partial-loss of complex cellular function. More rational explanations probably apply, such as a subtle defect in formation of the flagellum or of the metabolic machinery that fuels it, but most are difficult to test.

Although the cAMP-mediated pathway for activation of sperm motility does not require the CatSper proteins, Ca²⁺ entry although the CatSper channel of intact wild-type sperm is promoted by a mechanism that requires both sAC (16), the atypical HCO₃⁻-stimulated adenylyl cyclase of sperm, and the sperm-specific Cα2 catalytic subunit of cAMP-dependent protein kinase (17). In the simplest explanation, opening of the CatSper channel is facilitated by phosphorylation of one or more of its subunits. The demonstration by Qi et al. (7) that CatSper proteins can be recovered in immunoprecipitates suggests one path to test of this hypothesis.

The cAMP messenger likely also modulates the function of sNHE, another sperm-specific flagellar membrane protein that is required for male fertility (18, 19). Although the predicted sequence of sNHE suggests that it operates as a sodium–proton exchanger, functional activity has not been demonstrated. In contrast, the pH sensitivity of the CatSper channel suggested by the high histidine content of the CatSper1 predicted protein sequence (4) was confirmed in the current records from wild-type sperm (6). The promoting effect of alkalinization on opening the CatSper channel further suggested that formation of a functional channel might require association of the nascent channel proteins with sNHE, the putative mediator of increases in flagellar pH. By showing that sNHE^−/− sperm have apparently normal CatSper currents, Qi et al. (7) effectively discredit this hypothesis.

Analysis of the phenotype of mice that carry targeted disruptions of key components has provided much insight into the major signaling pathways of sperm. A daunting challenge that remains will be to learn how the shifting roles of cAMP, Ca²⁺, and pH, the ruling triumvirate of mediators, are coordinated to control the major alterations in flagellar functions and swimming behavior that occur during capacitation. It is likely that the rate-limiting step in meeting this challenge will be development of a new generation of tools for multiparametric monitoring of sperm functions and signaling processes. Another unmet challenge will be applying to the CatSper channels those existing biophysical and molecular biological tools that have been so successful in revealing how more familiar cation channels operate and are controlled. A lack of heterologous functional expression of the CatSper channel has blocked progress toward this goal. In closing, it should be noted that Qi et al. (7) have made highly significant progress toward removal of that blockade by showing that the four CatSper proteins coexpress an interacting protein complex.