COMMENTARY

Wrath of the wraiths of CatSper3 and CatSper4

Donner F. Babcock* Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195-7290

he spermatozoon is a marvel- CatSper3 and CatSper4 mRNAs were tive but will require adequate caution ous machine designed by nature found in extracts of testis prepared at because of the possibly incomplete for the single task of finding an day 20 but not in those prepared at day specificity of the available antibodies oocyte, delivering its genetic 10 (10). Using in situ hybridization in revealed here in the residual immunore- Tpayload, and initiating the formation of et al. activity of CatSper4Ϫ/Ϫ sperm probed testis tissue sections, Qi (7) now a new embryo. Like many of the most provide additional evidence that with anti-CatSper4 antibody. An attrac- specialized and sophisticated mechanical CatSper3 and CatSper4 transcription is tive alternate approach may come from creations of humans, the sperm is fitted restricted to the late-stage germ-line recent improvements in proteomic with unique components and stripped of cells that line the seminiferous tubules. methods to study sperm membrane unneeded hardware, including most of This finding eliminates the possibility composition (12). its biosynthetic equipment. As a conse- that CatSper3 and CatSper4 are re- quence, for mice, humans, and other quired in the Sertoli cells that support Restricted Roles for CatSper Channels mammals, the male stores sperm in a the developing sperm. Past work also shows that CatSper1 and quiescent state to conserve irreplaceable Past work established that the CatSper1 CatSper2 are not required for produc- resources. Upon release into the female mRNA contents of testes from adult tion of morphologically normal sperm, reproductive tract, the ejaculated sperm wild-type and CatSper2Ϫ/Ϫ mice are sim- which can mature in vitro to reach sev- ‘‘awaken’’ to begin the obligatory matu- ilar, as are CatSper2 mRNA contents of eral other milepost events in capacita- rational sequence, called ‘‘capacitation,’’ wild-type and CatSper1Ϫ/Ϫ testis (11). tion (11, 13). These include the Ca2ϩ- that is required to complete their vital Nevertheless, the CatSper1Ϫ/Ϫ sperm dependent activation of motility that mission. Several of the components of lack CatSper2 protein, and CatSper2Ϫ/Ϫ occurs when sperm encounter the capacitation require extracellular Ca2ϩ. Ϫ sperm lack CatSper1 protein, indicating HCO3 anion (14) and the delayed These include an early activation of Ca2ϩ-dependent engagement of a pro- sperm motility, thought to be necessary tein kinase cascade (15). Presumably, for entry of sperm into the oviduct, and All four CatSper sperm possess one or more additional a subsequent peculiar ‘‘hyperactivation’’ routes for Ca2ϩ entry that are unper- of motility, thought to be necessary for proteins are required turbed by the absence of CatSper1 and sperm to penetrate the cumulus oopho- CatSper2. Immunological methods sug- rus and zona pellucida that surround the for sperm to form gested several candidates (11, 13), but oocyte (1, 2). Our understanding of the 2ϩ so far none have been verified by cur- Ca requirement for hyperactivation flagellar ion channels. rent records from patch-clamped sperm. has increased greatly since the discovery Ϫ Ϫ Qi et al. (7) found that CatSper3 / of the CatSpers (3–5), a unique family Ϫ Ϫ and CatSper4 / sperm examined in of four sperm-specific ion channel pro- that stable expression of the CatSper1 a medium with NaHCO initially had teins. In landmark work last year (6), 3 protein requires CatSper2 and vice versa swimming speeds similar to those of the Clapham laboratory reported (11). Qi et al. (7) now show that the wild-type sperm, indicating that the acti- successful whole-cell patch-clamp ϩ CatSper1 protein is found together with vation of motility also does not require recording of a Ca2 -selective, alkaline- CatSper3 and CatSper4 proteins in im- the CatSper3 or CatSper4 proteins. promoted current that is present in wild- type sperm but absent in sperm from munoprecipitates prepared from wild- However, both swimming speeds and CatSper1Ϫ/Ϫ mice. In this issue of type testis, indicating that these three the proportion of motile cells declined proteins are physically associated in the during prolonged incubation of sperm PNAS, Qi et al. (7) reveal that all four Ϫ/Ϫ CatSper proteins are required for spermatogenic cells. Likewise, evidence from each of the four CatSper mu- sperm to form the flagellar ion channels of physical association of CatSper pro- tants. The meaning and cause of this that provide the route of entry for the teins was found when CatSper2, decline are unclear. If the ghosts Ca2ϩ that is needed for hyperactivation. CatSper3,orCatSper4 cDNA was coex- (wraiths) of the departed CatSpers were The lack of functional CatSper channels pressed with CatSper1 in a cultured cell angry (wrathful), vengeful, and capable prevents hyperactivation and produces line. Unexpectedly, CatSper3 and of wreaking scientific mischief, they the male infertility phenotype shared CatSper4 proteins also were detected in might create just such a partial-loss of Ϫ/Ϫ by mice carrying mutations that pre- CatSper1 testis, raising the possibility complex cellular function. More rational vent transcription of full-length Catsper1 that stable expression of CatSper3 and explanations probably apply, such as a (4), CatSper2 (3), or CatSper3 or CatSper4 proteins might have less strin- subtle defect in formation of the flagel- CatSper4 (7). gent requirements than those for lum or of the metabolic machinery that CatSper1 and CatSper2. If so, then fuels it, but most are difficult to test. Stable Complexes of CatSper Proteins CatSper3 and CatSper4 proteins might Although the cAMP-mediated path- Ϫ Ϫ Previous studies of testicular gene ex- be retained in mature CatSper1 / way for activation of sperm motility pression by microarray analysis found sperm. However, if CatSper3 and CatSper2 mRNA in the pachytene sper- CatSper4 are present, they do not seem matocytes that appear at Ϸ12 days post- to form functional cation channels de- Author contributions: D.F.B. wrote the paper. partum in the developing mouse testis tectable in patch-clamp recordings from The author declares no conflict of interest. but detected CatSper1 mRNA only after CatSper1Ϫ/Ϫ sperm. Direct examination See companion article on page 1219. round spermatids were produced at ap- of the CatSper3 and CatSper4 content *E-mail: [email protected]. Ϫ Ϫ proximately day 20 (8, 9). Similarly, of CatSper1 / sperm will be informa- © 2007 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610909104 PNAS ͉ January 23, 2007 ͉ vol. 104 ͉ no. 4 ͉ 1107–1108 Downloaded by guest on September 29, 2021 does not require the CatSper proteins, functional activity has not been demon- roles of cAMP, Ca2ϩ, and pH, the rul- Ca2ϩ entry although the CatSper chan- strated. In contrast, the pH sensitivity ing triumvirate of mediators, are coordi- nel of intact wild-type sperm is pro- of the CatSper channel suggested by nated to control the major alterations in moted by a mechanism that requires the high histidine content of the flagellar functions and swimming behav- Ϫ both sAC (16), the atypical HCO3 - CatSper1 predicted protein sequence (4) ior that occur during capacitation. It is stimulated adenylyl cyclase of sperm, was confirmed in the current records likely that the rate-limiting step in meet- and the sperm-specific C␣2 catalytic from wild-type sperm (6). The promot- ing this challenge will be development subunit of cAMP-dependent protein ing effect of alkalinization on opening of a new generation of tools for multi- kinase (17). In the simplest explanation, the CatSper channel further suggested parametric monitoring of sperm func- opening of the CatSper channel is facili- that formation of a functional channel tions and signaling processes. Another unmet challenge will be applying to the tated by phosphorylation of one or might require association of the nascent CatSper channels those existing biophys- more of its subunits. The demonstration channel proteins with sNHE, the puta- ical and molecular biological tools that by Qi et al. (7) that CatSper proteins tive mediator of increases in flagellar Ϫ/Ϫ have been so successful in revealing how can be recovered in immunoprecipitates pH. By showing that sNHE sperm more familiar cation channels operate suggests one path to test of this have apparently normal CatSper cur- and are controlled. A lack of heterolo- hypothesis. rents, Qi et al. (7) effectively discredit gous functional expression of the The cAMP messenger likely also this hypothesis. CatSper channel has blocked progress modulates the function of sNHE, an- Analysis of the phenotype of mice toward this goal. In closing, it should be other sperm-specific flagellar membrane that carry targeted disruptions of key noted that Qi et al. (7) have made protein that is required for male fertility components has provided much insight highly significant progress toward re- (18, 19). Although the predicted se- into the major signaling pathways of moval of that blockade by showing that quence of sNHE suggests that it oper- sperm. A daunting challenge that re- the four CatSper proteins coexpress an ates as a sodium–proton exchanger, mains will be to learn how the shifting interacting protein complex.

1. Yanagimachi R (1994) in The Physiology of Repro- Quill TA, Clapham DE (2007) Proc Natl Acad Sci 14. Wennemuth G, Carlson AE, Harper AJ, Babcock duction, eds Knobil E, Neill JD (Raven, New USA 104:1219–1223. DF (2003) Development (Cambridge, UK) York), pp 189–317. 8. Schultz N, Hamra FK, Garbers DL (2003) Proc 130:1317–1326. 2. Suarez SS, Pacey AA (2006) Hum Reprod Update Natl Acad Sci USA 100:12201–12206. 15. Visconti P, Moore G, Bailey J, Leclerc P, Connors 12:23–37. 9. Shima JE, McLean DJ, McCarrey JR, Griswold S, Pan D, Olds-Clarke P, Kopf GS (1995) Devel- 3. Quill TA, Ren D, Clapham DE, Garbers DL MD (2004) Biol Reprod 71:319–303. opment (Cambridge, UK) 121:1139–1150. (2001) Proc Natl Acad Sci USA 98:12527–12531. 10. Jin JL, O’Doherty AM, Wang S, Zheng H, Sand- 16. Xie F, Garcia MA, Carlson AE, Schuh SM, Bab- 4. Ren D, Navarro B, Perez G, Jackson AC, Hsu S, ers KM, Yan W (2005) Biol Reprod 73:1235–1242. cock DF, Jaiswal BS, Gossen JA, Esposito G, van Shi Q, Tilly JL, Clapham DE (2001) Nature 11. Carlson AE, Quill TA, Westenbroek RE, Schuh Duin M, Conti M (2006) Dev Biol 296:353–362. 413:603–609. SM, Hille B, Babcock DF (2005) J Biol Chem 17. Nolan MA, Babcock DF, Wennemuth G, Brown 5. Lobley A, Pierron V, Reynolds L, Allen L, Micha- 280:32238–32244. W, Burton KA, McKnight GS (2004) Proc Natl lovich D (2003) Reprod Biol Endocrinol 1:53–67. 12. Stein KK, Go JC, Lane WS, Primakoff P, Myles Acad Sci USA 101:13483–13488. 6. Kirichok Y, Navarro B, Clapham DE (2006) Na- DG (2006) Proteomics 6:3533–3543. 18. Quill TA, Wang D, Garbers DL (2006) Mol Cell ture 439:737–740. 13. Carlson AE, Westenbroek RE, Quill T, Ren D, Endocrinol 250:84–92. 7. Qi H, Moran MM, Navarro B, Chong JA, Krapiv- Clapham DE, Hille B, Garbers DL (2003) Proc 19. Wang D, King SM, Quill TA, Doolittle LK, Gar- insky G, Krapivinsky L, Kirichok Y, Ramsey IS, Natl Acad Sci USA 100:4864–14868. bers DL (2003) Nat Cell Biol 5:1117–1122.

1108 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610909104 Babcock Downloaded by guest on September 29, 2021