Cystic fibrosis transmembrane conductance regulator is vital to sperm fertilizing capacity and male fertility

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1 Cysti firosis transmemrane ondutane regulator is vital to sperm fertilizing apaity and male fertility Wen Ming Xu, Qi Xian Shi, Wen Ying Chen, Chen Xi Zhou, Ya Ni, Dewi Kenneth Rowlands, Guo Yi Liu, Hu Zhu, Ze Gang Ma, Xiao Fei Wang, Zhang Hui Chen, Si Chang Zhou, Hong Shan Dong, Xiao Hu Zhang, Yiu Wa Chung, Yu Ying Yuan, Wan Xi Yang, and Hsiao Chang Chan PNAS pulished online May 22, 27; doi:1.173/pnas This information is urrent as of May 27. Alerts Rights & Permissions Reprints This artile has een ited y other artiles: Reeive free alerts when new artiles ite this artile - sign up in the ox at the top right orner of the artile or lik here. To reprodue this artile in part (figures, tales) or in entirety, see: To order reprints, see: Notes:

2 Cysti firosis transmemrane ondutane regulator is vital to sperm fertilizing apaity and male fertility Wen Ming Xu, Qi Xian Shi, Wen Ying Chen, Chen Xi Zhou, Ya Ni, Dewi Kenneth Rowlands, Guo Yi Liu, Hu Zhu, Ze Gang Ma, Xiao Fei Wang, Zhang Hui Chen, Si Chang Zhou, Hong Shan Dong, Xiao Hu Zhang, Yiu Wa Chung, Yu Ying Yuan, Wan Xi Yang, and Hsiao Chang Chan Epithelial Cell iology Researh Center, Li Ka Shing Institute of Health Sienes, Department of Physiology, Faulty of Mediine, Chinese University of Hong Kong, Shatin, NT, Hong Kong, China; Zhejiang Aademy of Medial Sienes, Hangzhou 3113, China; Zhejiang University Shool of Mediine, College of Life Sienes, Hangzhou 3158, China; and Department of Physiology, Harin Medial University, Harin 1586, China Edited y Ryuzo Yanagimahi, University of Hawaii, Honolulu, HI, and approved April 16, 27 (reeived for review Otoer 23, 26) Cysti firosis transmemrane ondutane regulator (CFTR) is an anion hannel, mutations of whih ause ysti firosis, a disease haraterized y defetive Cl and HCO 3 transport. Although >95% of all CF male patients are infertile eause of ongenital ilateral asene of the vas deferens (CAVD), the question whether CFTR mutations are involved in other forms of male infertility is under intense deates. Here we report that CFTR is deteted in oth human and mouse sperm. CFTR inhiitor or antiody signifiantly redues the sperm apaitation, and the assoiated HCO 3 -dependent events, inluding inreases in intraellular ph, AMP prodution and memrane hyperpolarization. The fertilizing apaity of the sperm otained from heterozygous CFTR mutant mie is also signifiantly lower ompared with that of the wild-type. These results suggest that CFTR in sperm may e involved in the transport of HCO 3 important for sperm apaitation and that CFTR mutations with impaired CFTR funtion may lead to redued sperm fertilizing apaity and male infertility other than CAVD. iaronate CFTR sperm apaitation Cysti firosis (CF) is a ommon hereditary disease aused y mutations of the gene enoding ysti firosis transmemrane ondutane regulator (CFTR), a AMP-ativated anion hannel, with linial manifestations of progressive lung disease, panreati insuffiieny, and infertility in oth sexes (1, 2). CF male patients are infertile mostly due to ongenital ilateral asene of the vas deferens (CAVD) and ostrutive azoospermia. The asene of sperm in the ejaulatory dut of CF males has led to the overlook of possile involvement of CFTR in normal sperm funtion. Studies have reported a higher prevalene of CFTR gene mutations in otherwise healthy men presenting with redued sperm quality ompared with ontrols with normal sperm parameters (3). Interestingly, a reent study showed that in infertile males, the frequeny of CFTR heterozygosity is 2-fold higher than in the general population (4), whih suggests possile involvement of CFTR mutations in other forms of male infertility. However, the exat role of CFTR in this ontext remains osure. Mammalian sperm are unale to fertilize eggs until they undergo an ativation proess, known as apaitation, in the female reprodutive trat (5, 6). Capaitation is known to e assoiated with elevation of intraellular ph (7) and hyperpolarization of the sperm plasma memrane (8) and these events have een shown to depend on extraellular HCO 3 (9, 1). The regulatory role of HCO 3 in sperm apaitation has een attriuted to its diret ativation of a HCO 3 -sensitive solule form of adenylyl ylase (11) that in turns ativates AMP prodution and various downstream ellular events, suh as protein tyrosine phosphorylation, leading to apaitation (12 14). Despite the mounting evidene indiating the key importane of HCO 3 in sperm apaitation, the transporting mehanism responsile for the entry of HCO 3 into sperm remains poorly understood. CFTR is a hannel protein known to ondut oth Cl and HCO 3, defets of whih due to its gene mutations ause CF, although the exat mehanisms underlying most of the linial manifestations remain elusive (15 18). Reent studies have indiated a signifiant role of CFTR, either diret or indiret, in HCO 3 seretion and the possile involvement of defetive CFTRmediated HCO 3 seretion in the pathogenesis of CF (19). Our study in ref. 2 demonstrated a diret role of CFTR in mediating uterine HCO 3 seretion, impairment of whih leads to redued sperm apaitation and the fertilizing apaity of sperm. The demonstrated dependene of sperm apaitation on the HCO 3 ontent of the uterine trat, the aility of CFTR to ondut HCO 3, and the reported higher prevalene of CFTR gene mutations in otherwise healthy men presenting with redued sperm quality ompared with that otained from normal sujets, have prompted us to hypothesize that CFTR may also e expressed in sperm and play a role in mediating the HCO 3 entry important for the fertilizing apaity of sperm. Results and Disussion Expression and Loalization of CFTR in Human and Mouse Spermatozoa. Although CFTR expression has een shown in the testis of rodents (21) and rat germ ells (22), its expression and funtion in mature sperm have not een demonstrated. We first examined the expression of CFTR in oth human and mouse sperm, using immunofluoresene staining and Western lot analysis. Sperm were either donated y volunteers with proven fertility or olleted from the auda epididymides of adult ICR (an outred Institute for Caner Researh strain) mie. Using onfoal imaging, CFTR was immunoloalized to the equatorial segment of human and mouse sperm (Fig. 1 a and ). In Western lot analysis, a mouse antihuman CFTR monolonal antiody (C-terminal; R&D Systems, Minneapolis, MN) deteted a and of 16 kda in human sperm, and a polylonal rait antiody [Alomone Las (Jerusalem, Israel); atalog no. ACL-6] raised against the C-terminal of mouse CFTR also revealed the presene of CFTR in mouse sperm (Fig. 1 and d). Involvement of CFTR in Sperm Capaitation-Related and iaronate- Dependent Proesses. We then addressed the question whether CFTR is involved in mediating the HCO 3 entry important for sperm apaitation. Mouse sperm were olleted from the auda Author ontriutions: W.M.X. and Q.X.S. ontriuted equally to this work; W.M.X., Q.X.S., and H.C.C. designed researh; W.M.X., Q.X.S., W.Y.C., C.X.Z., Y.N., D.K.R., G.Y.L., Z.G.M., X.F.W., and H.S.D. performed researh; H.Z., Z.H.C., S.C.Z., X.H.Z., Y.W.C., Y.Y.Y., and W.X.Y. ontriuted new reagents/analyti tools; W.M.X., Q.X.S., W.Y.C., and H.C.C. analyzed data; and H.C.C. wrote the paper. The authors delare no onflit of interest. This artile is a PNAS Diret Sumission. Areviations: WW, iggers, Whitten, and Whittingham; CAVD, ongenital ilateral asene of the vas deferens; CF, ysti firosis; CFTR, ysti firosis transmemrane ondutane regulator; CTC, hlortetrayline. To whom orrespondene should e addressed. hsiaohan@uhk.edu.hk. 27 y The National Aademy of Sienes of the USA PNAS June 5, 27 vol. 14 no. 23

3 a 25 kd 16 kd 15 kd epididymis and inuated in iggers, Whitten, and Whittingham (WW) medium for indution of apaitation. CFTR inhiitor- 172, a speifi CFTR hannel loker (23), was added during inuation, and its effet on sperm apaitation was assessed y hlortetrayline (CTC) staining, a onventional method for d 1 µm 16 kd 15 kd Fig. 1. Expression and loalization of CFTR in human and mouse spermatozoa. (a) Immunostaining of CFTR in human sperm, using a monolonal human CFTR antiody (1:5; Aam) and the ound antiody deteted with Alex 488- onjugated seondary antiody, and onfoal imaging with images of the same field (enlargement in Insets): differential interferene ontrast image (Upper Center); sperm nulei stained with DAPI (Upper Right); immunostaining of CFTR showing positive staining in the equatorial segment (Lower Center); superimposed image of DAPI and CFTR staining (Lower Right and Inset; arrow indiates CFTR loalization). Upper Left image is the negative immunostaining ontrol of CFTR with the mouse isotype speifi IgM, whereas Lower Left is the orresponding differential interferene ontrast image. (Sale ar, 5 m.) () Immunofluoresene of CFTR in mouse sperm with orresponding images as depited in a, showing CFTR loalization in the equatorial segment of mouse sperm. (Sale ar, 1 m.) () Western lotting analysis of human sperm CFTR protein proed y monolonal CFTR antiody (1:1,; R&D Systems) (left lane) or with normal mouse IgG ontrol (Santa Cruz iotehnology; atalog no. S-2762) (right lane). (d) Identifiation of CFTR in mouse sperm y Western lot analysis proed y polylonal CFTR antiody (1:5) (Alomone Las; atalog no. ACL-6). This antiody reognizes a and at expeted mouse CFTR MW 16 kda (left lane), whih is speifially loked y the preinuation of immunizing CFTR peptides (Alomone Las; atalog no. ACL-6) (right lane). 5 µm 1 µm assessing sperm apaitation ased on alteration in staining patterns of the sperm head during the proess, with F pattern indiating inapaitated, pattern for apaitated, and for apaitated and arosome-reated sperm (Fig. 2a). As shown in Fig. 2, the CFTR inhiitor onentration dependently redued the perentage of apaitated sperm, as evidened y the inreased perentage of sperm exhiiting F pattern and the redued perentage with pattern, indiating the involvement of CFTR in sperm apaitation. eause sperm apaitation depends on HCO 3 entry, the effet CFTR inhiitor has on sperm apaitation ould e due to its loking of the hannel for HCO 3 entry. We then tested whether the CFTR inhiitor or antiody ould inhiit the apaitation-assoiated intraellular ph inrease that is due to HCO 3 entry. After inuated with apaitation-induing media for 2 h in the presene of the CFTR inhiitor (1 M), sperm were loaded with a ph-sensitive dye, 2,7-is-2(2-arosyethyl)-5-(and-6)-aroxyfluoresene, aetoxymethyl ester, for measurement of intraellular ph. As shown in Fig. 2, although this inhiitor had no signifiant effet on the intraellular ph of sperm inuated in HCO 3 -free medium, it signifiantly inhiited the HCO 3 -indued intraellular ph inrease during the apaitation proess (P.1). To further onfirm that CFTR is involved in mediating HCO 3 influx in sperm, we also performed single ell imaging experiment, in whih the HCO 3 -indued hange in intraellular ph ould e monitored in real time. The result (Fig. 2d) showed that addition of HCO 3 produed an inrease in ph i that was inhiited when sperm were preinuated with CFTRinh-172 (P.5). These data onfirmed that CFTRinh-172 was indeed ale to inhiit the HCO 3 -indued ph inrease in sperm. A monolonal CFTR antiody (1:5 dilution; Alexis iohemials, San Diego, CA; atalog no. ALX ), whih had een shown to inhiit CFTR funtion (24), was also found to have signifiant inhiitory effet on sperm intraellular ph inrease (P.5; Fig. 2e), onsistent with the involvement of CFTR in mediating HCO 3 entry during apaitation. eause HCO 3 is negatively harged, its entry into sperm ould indue memrane hyperpolarization, a proess known to e assoiated with sperm apaitation (1). We then examined the effet of the CFTR inhiitor on HCO 3 -indued memrane hyperpolarization y monitoring sperm memrane potential hanges with a memrane potential-sensitive fluoresent dye DiSC 3 (5). Sperm were originally inuated in HCO 3 -free solution, and upon addition of extraellular HCO 3 (5 mm), a memrane hyperpolarization in the sperm ould e oserved, whih ould e reversed y the CFTR inhiitor in a onentration-dependent manner (Fig. 2f), onfirming the involvement of CFTR in mediating HCO 3 entry into sperm. It is now estalished that the regulatory effet of HCO 3 on apaitation is mediated y its diret ativation of a solule adenylyl ylase in sperm (11). Numerous studies have shown that the stimulation of solule adenylyl ylase ativity in sperm y HCO 3 leads to an inrease in AMP prodution and phosphorylation of tyrosine kinase, one of the main downstream targets of AMP, leading to sperm apaitation (12). In the present study, the ELISA measurement indeed showed that a 6-fold inrease in sperm AMP prodution ould e indued y 25 mm HCO 3 ompared with that inuated in HCO 3 -free medium (Fig. 2g). Pretreatment with CFTR inhiitor (5 M) in the asene of HCO 3 did not signifiantly affet the AMP prodution ut signifiantly redued the HCO 3 -indued AMP inrease (P.5; Fig. 2g), suggesting that this proess, whih is vital to sperm apaitation, depends on CFTR. The oservations that CFTR inhiitor or antiody ould inhiit sperm apaitation as well as a numer of HCO 3 - dependent and apaitation-assoiated events are onsistent with a role of CFTR in mediating HCO 3 entry important for sperm apaitation. MEDICAL SCIENCES Xu et al. PNAS June 5, 27 vol. 14 no

4 a F F Perentage (%) F Control 2nM 1µM 5µM 25µM phi 7.5 Control CFTRi HCO3- + HCO3- d Inrease in phi Control CFTRi e Inrease in phi Control IgG CFTR A f voltage hange ontrol) Memrane (% of Control F i( 2nM) C TR CFTRi( 1µM) CF TRi( 5µ M) g (pmol/ml) AMP Con. 1. Control CFTRi HCO HCO Fig. 2. Involvement of CFTR in sperm apaitation-related and iaronate-dependent proesses. (a) Distint CTC fluoresene staining patterns in sperm (with arrowheads indiating different patterns). () The perentage of inapaitated (F pattern) and apaitated sperm ( pattern) enhaned and attenuated, respetively, y the CFTR inh-172 (CFTRi) in a onentration-dependant manner., P.5;, P.1 when ompared with respetive ontrols. () CFTRi (1 M) attenuated intraellular ph inrease, measured with 2,7-is-2(2-arosyethyl)-5-(and-6)-aroxyfluoresene, aetoxymethyl ester in sperm inuated for 2hinHCO 3 -ontaining apaitating medium (HCO 3 )(P.5) ut without effet in HCO 3 -free medium (HCO 3 ). (d and e) Pretreatment with CFTRi (1 M) (d) and monolonal CFTR antiody (1:5 dilution) (e) for15 min attenuated HCO 3 (5 mm)-indued inrease in intraellular ph measured y real-time single ell imaging experiments. (f) Conentration-dependent effet of the CFTR inhiitor on the amplitude of HCO 3 -indued hyperpolarization. (g) Effet of CFTR inhiitor on the HCO 3 -stimulated AMP prodution in sperm. Spermatozoa were preinuated in WW media (HCO 3 ) with or without 5 M CFTRi for 1 min and then hallenged with the same volume of 25 mm HCO 3 (or same volume of HCO 3 -free medium) for 1 min efore AMP measurement. In eah experiment, the ontrol group was hallenged with the same volume/onentration of vehile (Me 2 SO) or ontrol antiody as the experimental group. Normal mouse IgG ontrol (Santa Cruz iotehnology, Santa Cruz, CA; atalog no. S-2762) was used as a ontrol for CFTR antiody. For all statisti figures, data are shown as mean SEM; n 3;, P.5;, P.1;, P.1 vs. ontrol). Demonstration of Redued HCO 3 Transport in Sperm of Heterozygous Cftr / Mie. To further onfirm the role of CFTR in sperm apaitation and thus the fertilizing apaity of sperm, we examined sperm apaitation and fertility in a CF mouse model, CFTR tm1un, with a phenotype similar to the CF disease-ausing F58 mutation in humans (25). eause the homozygous mutant mie show CF phenotypes and often die efore puerty, it is not feasile to use the homozygous mutant mie. Instead, heterozygous mutant mie ould e used, eause the heterozygous CFTR mutant mie (Cftr / ) have een shown to have impaired CFTR funtion, i.e., redued Cl and fluid seretion (26) or fewer CFTR-mediated transloated Salmonella typhi into the gastrointestinal sumuosa (24) ompared with the wild-type (Cftr / ) mie. A reent study also showed that in infertile males, the frequeny of CFTR heterozygosity is 2-fold higher than that in the general population, indiating possile dosage effet of CFTR on the fertility (4). We therefore examined whether the sperm from the Cftr / mie show redued aility to undergo apaitation ompared with those from wild-type littermates (C57L/6J akground). The results showed that after 2-h inuation in the apaitation-induing medium, the perentage of apaitated sperm ( pattern), as demonstrated y CTC staining, of the heterozygous mutant mie was signifiantly lower than that of the wild-type ontrol (P.1; Fig. 3a). We then tested whether the redued apaitation was due to a defet in transporting HCO 3 in sperm of Cftr / mie y measuring HCO 3 -indued memrane hyperpolarization. The magnitude of the HCO 3 -indued memrane hyperpolarization in Cftr / sperm was indeed signifiantly redued ompared with the wild-type ontrol (P.5; Fig. 3), suggesting a defet in HCO 3 transport in Cftr / sperm. The AMP inrease in response to HCO 3 in Cftr / sperm was also redued ompared with that in the wild-type ontrol (P.5; Fig. 3), onfirming that a defet in CFTR affets the HCO 3 -dependent events important for sperm apaitation. Demonstration of Redued Fertility in Vitro and in Vivo in Heterozygous Cftr / Mie. eause apaitation is a neessary proess of sperm ativation efore fertilization, impaired sperm apaitation eause of defetive CFTR is expeted to affet fertility outome oth in vitro and in vivo. We performed in vitro fertilization to ompare the fertilizing apaity of heterozygous Cftr / sperm to Xu et al.

5 a Perentage (%) 7 F V (mv ) /+ +/+ HCO 3 - HCO 3 - +/+ +/- +/- +/- AMP inrease (pmol) /+ +/- Fig. 3. Demonstration of redued HCO 3 transport in sperm of heterozygous Cftr / mie. (a) CTC staining results showing signifiantly lower perentage of apaitated sperm (exhiiting pattern) in Cftr / ompared with the wild-type sperm. () Cftr / sperm exhiit a redued HCO 3 -indued memrane hyperpolarization ompared with wild-type ontrol. The memrane voltage hanges after the addition of 5 mm HCO 3 were ompared among the wild-type (n 4) and heterozygous sperm (n 4). (Right) Shows the orresponding representative traing (sale ar indiate 1 min). () Sperm of Cftr / mie (n 3) show redued HCO 3 -indued AMP inrease ompared with wild-type ontrol (n 3). Sperm inuated with HCO 3 -free medium were hallenged with 25 mm HCO 3 for 3 s efore AMP measurement. AMP inrease were ompared etween Cftr / mie and wild-type ontrol. Errors ars are SEM., P.5 and, P.1 vs. ontrol. that of Cftr / sperm. Ooytes were otained from wild-type female mie and inuated with the Cftr / sperm. The in vitro fertility rate was determined as the ratio of two-ell emryos otained to the total numer of the ooytes used. The results showed that the perentage of fertilized eggs when inuated with Cftr / sperm (13 of 73, 16%) was signifiantly lower than that with Cftr / sperm (28 of 6, 48.5%) (P.1; Fig. 4a). The impaired fertilizing apaity in Cftr / sperm ould also e evidened y their redued inding and penetration of zona pelluida-free eggs ompared with the wild-type sperm (Fig. 4). To further demonstrate the role of CFTR in male fertility in a physiologial ontext, we examined the fertility rate of male Cftr / mie through natural mating. Cftr / female mie housed overnight with Cftr / males showed normal numer of vaginal plugs the following morning. However, only five of the nine Cftr / males tested had offspring, ompared with 1% of the age mathed wild-type males having offspring, and the averaged litter size in females mated with the Cftr / males was also redued (Tale 1). It should e noted that apart from apaitation, other sperm funtions, suh as sperm motility, might also e impaired in Cftr / mie, ontriuting to the redued fertility oserved. Indeed, we also oserved a signifiant redution in sperm motility with ompromised forward movement parameters in Cftr / mie ompared with wild-type ontrol (Fig. 4 and Tale 2). These findings are also in line with a role of CFTR in transporting HCO 3, eause HCO 3 has also een linked to sperm motility (27, 28). Taken together, these results have onfirmed that defetive CFTR in sperm leads to ompromised fertilizing apaity of sperm. The redued fertility in Cftr / mie may stem from less funtional CFTR in the heterozygous mie, eause it has een reported y others (29) that less (ut a detetale amount) wild-type CFTR mrna was present in multiple tissues of heterozygote CFTR tm1un mie. Although CFTR has long een thought to funtion as a hloride hannel, emerging evidene indiates that it an also transport HCO 3 diretly or indiretly (19). The role of CFTR in transporting HCO 3 has een mainly assoiated with epithelial seretion, therey regulating the luminal ph thought to e important for the physiologial funtion of a numer of organs, inluding the nasal epithelial (3) and airways (31). Here, we demonstrate the involvement of CFTR in transporting HCO 3 in nonsomati ells, namely the sperm, therey eliiting the AMP-dependent signaling pathway on whih sperm apaitation depends. CFTR in sperm memrane may at as a HCO 3 -onduting hannel, as demonstrated in other tissues, inluding the endometrial epithelium (2). Interestingly, the apaitation-assoiated and HCO 3 -dependent events a rate(ivf) Fertility / + +/- +/- +/+ (% ) m otility wild type +/- MEDICAL SCIENCES Fig. 4. Demonstration of redued fertility in vitro and in vivo and dereased motility in heterozygous Cftr / mie. (a) In vitro fertilization result showing redued perentage of ooyte fertilized with sperm from Cftr / mie ompared with wild-type sperm. The total numer of eggs tested was 6 (inuated with wild-type sperm) and 73 (inuated with Cftr / sperm), respetively., P.1. () Representative phase-ontrast images of zona pelluida-free ooytes after sperm-egg inuation for 4 h, showing redued inding or penetration y Cftr / sperm (Right) ompared with wild-type sperm (Left). () Comparison of sperm motility etween wild-type and heterozygous CF mie sperm. After 1 min inuation in omplete medium, the sperm were washed with sperm washing medium, and the motile and total sperm were ounted. For eah mouse, at least 2 sperm were sored. The derease in motility was onfirmed y using omputer-assisted sperm analysis (n 3). Xu et al. PNAS June 5, 27 vol. 14 no

6 Tale 1. Comparison of in vivo fertility outome etween Cftr / and Cftr / mie Mie Total pups Pups per litter SEM Fertility, %(n) Cftr / (n 9) % (9) Cftr / (n 9) % (5), P.1. were not signifiantly augmented y forskolin, a AMP-evoking agent shown to ativate CFTR (data not shown). This suggests that the CFTR hannel in sperm, as shown in the airways (32), is open at asal level, allowing the entry of HCO 3 when sperm enounter the HCO 3 -rih uterine fluid. Alternatively, CFTR may failitate HCO 3 transport y ating as a Cl hannel that either affets memrane voltage, therey providing the driving fore for the eletrogeni Na/ HCO 3 otransporter (1), or provides a Cl reyling pathway required for the operation of a Cl / HCO 3 exhanger (33), as in the ase of panreati HCO 3 seretion (34). Interestingly, defetive CFTR has een shown to affet the expression and funtion of these HCO 3 transporter in the traheal epithelial ells resulting in defetive HCO 3 seretion (35). Nevertheless, whether it is involved in HCO 3 transport diretly or indiretly in sperm, CFTR is important for the fertilizing apaity of sperm, as demonstrated in the present study. The presently demonstrated and previously unsuspeted role of CFTR in sperm funtion may unravel the mysteries of many unexplained ases of male infertility, eause 1,2 mutations in CFTR have een identified sine its disovery with various defets (2). These mutations may affet CFTR funtion to different extents, whih may explain the oservation that ertain CFTR mutations are assoiated with redued sperm quality in men who do not present CF phenotype. CFTR may also play different roles in sperm funtion other than HCO 3 transport for sperm apaitation, eause CFTR is also known to regulate an array of other proteins and ellular proesses. Together with the reported involvement of CFTR in HCO 3 seretion y the female reprodutive trat (2), the present finding of CFTR involvement in sperm apaitation indiates that CFTR may have far-reahing effet on reprodution of oth sexes. Experimental Proedures Immunofluoresene Straining of CFTR in Human and Mouse Sperm. For indiret immunofluoresene studies of CFTR loalization, sperm were either donated y volunteers with proven fertility or olleted from the auda epididymides of fertile adult ICR mie and washed y entrifugation although a three-layer gradient of Peroll. Sperm were fixed in 4% paraformaldehyde (we also applied other fixation onentrations from.2% to 4%) overnight at 4 C. Sperm were smeared on slide after washing with PS for three times, air dried, and susequently loked with 1% SA at room temperature for 1 h. After two more washings in PS (5 min eah), sperm were inuated with primary CFTR Tale 2. Movement parameters of sperm reovered from wild-type and Cftr / mie Parameters Wild type Cftr / VAP( m s-1) VSL( m s-1) VCL( m s-1) n 3. Data were otained y using omputer-assisted sperm analysis and are expressed as mean SEM., P.5 ompared with orresponding values (unpaired t test). VAP, averaged path veloity; VSL, straight line veloity; VCL, urvilinear veloity. monolonal antiody (1:5; Aam, Camridge, U.K.; atalog no. a2784) or an equivalent onentration of mouse purified IgM ontrol (gift from M. Gwrish, Department of Immunology, righam and Women s Hospital, Harvard Medial Shool, oston, MA) at 4 C overnight. Sperm were washed with PS three times and inuated with seondary antiody (1:5, Alex 488-onjugated goat-anti-mouse IgG; Moleular Proes, Eugene, OR; atalog no. A-1117) in dark room for 1hatroom temperature. Unounded antiody was removed y washing with PS three times for 5 min eah and then ounterstained with DAPI (Sigma, St. Louis, MO; atalog no. D9564) for 5 min. Finally, sperm were washed with PS and deionized water one. The slides were stored in dark ox efore visualization. Sperm Preparation. The proedure for retrieving sperm is as follows: Cauda epididymides were disseted from adult ICR mie and mined, then plaed in a modified human tual fluid medium (Irvine Sientifi, Santa Ana, CA) to allow dispersion of sperm. After 1 min, sperm in the suspension were washed in 1 ml of the same medium y entrifugation at 8 g for 1 min at room temperature (24 C). Sperm were then resuspended to a final onentration of ells per ml and diluted in the appropriate medium depending on the experiment performed. Assessment of Sperm Capaitation and Fertilization. Sperm apaitation in mie was deteted y CTC staining as desried in ref. 2. A total of at least 2 sperm were ounted to assess the different CTC staining patterns reognized previously. and patterns were taken to indiate apaitated sperm: The pattern was apaitated and arosome-intat sperm; the pattern was apaitated and arosome-reated sperm. The methods for in vitro fertilization in mie followed those desried in ref. 2. Measurement of Intraellular ph in Spermatozoa. Caudal epididymal spermatozoa were otained from adult (8 14 week) ICR mie. Spermatozoa were olleted and adjusted to ells/ml with omplete WW medium (95 mm NaCl/44 M sodium latate/25 mm NaHCO 3 /2 mm Hepes/5.6 mm D-gluose/4.6 mm KCl/1.7 mm CaCl 2 /1.2 mm KH 2 PO 4 /1.2 mm MgSO 4 /.27 mm sodium pyruvate/.3% wt/vol SA, 5 units per ml peniillin/5 g/ml streptomyin, ph 7.4). Cells were then inuated in a 5% CO 2 inuator at 37 C with the appropriate inhiitor added. When needed, 5 M 2,7-is-2(2-arosyethyl)-5-(and-6)-aroxyfluoresene, aetoxymethyl ester was added, and the inuation ontinued for a further 3 min. Afterward, the ells were pelleted and washed twie to remove free dye and adjust to ells per ml. To determine the intraellular ph (phi), the fluoresene signal was reorded y a LS-5 luminesent spetrometer (PerkinElmer Optoeletronis, Fremont, CA). A radiometri analysis of fluoresene data, using exitation wavelengths of 49/44 nm and an emission of 53 nm (8-nm exitation/emission andpass), was performed. Caliration was performed as desried in ref. 1. Single-Cell Intraellular ph Measurement. The hamers for imaging were prepared y oating overslips with 5 g/ml polyd-lysine, shaking off exess and allowing to air-dry. Sperm (2 1 7 ells per ml) were loaded with 3 M 2,7-is-2(2-arosyethyl)-5-(and-6)- aroxyfluoresene, aetoxymethyl ester (Moleular Proes) for 15 min at 37 C. The dye-loaded sperm were then entrifuged for 5 min at 1, g and resuspended in the original volume of medium. Laeled sperm were diluted 1:2 in the HCO 3 -free medium, plaed in the hamer immediately, and left for 1 3 min, after whih the unattahed sperm were removed y washing with HCO 3 -free medium. The hamer was then mounted on the mirosope (IX7; Olympus, Tokyo, Japan) with 4 fluor ojetive, and fluoresene hanges were reorded on a CCD amera used in ontinuous aquisition mode. The ratio (49:44) of two signals is diretly Xu et al.

7 proportional to the ph, and the images were aptured y using MetaFluor from Universal Imaging (Washington, DC). Evaluation of Sperm Plasma Memrane Potential Changes. Sperm plasma memrane hanges were monitored y using the potentialsensitive fluoresent dye DiSC 3 (5) (Moleular Proes) as desried in ref. 1. We use DiSC 3 (5) instead of other memrane voltage dyes eause it allows for internal aliration and has een used in monitoring sperm voltage hange suessfully (1). riefly, ells per ml spermatozoa isolated as desried aove were plaed in a thermostatially ontrolled uvette at 37 C in WW medium. Eight minutes efore the measurement, 1 M DiSC 3 (5) (final onentration) was added to the sperm suspension and further inuated for 5 min, and when used, 1 M aronylyanide m-hlorophenylhydrazone (final onentration) was added, and the sperm were inuated for an additional 2 min. After this period, 3 ml of the suspension was transferred to a gently stirred uvette at 37 C, and the fluoresene (62-/67-nm exitation/emission) was reorded ontinuously. After reahing a steady fluoresene, aliration was performed as desried in ref. 1. AMP Measurement. Sperm were inuated in WW medium (HCO 3 -free) for 1 min with or without the CFTR inhiitor (5 M), then ounted, adjusted to 1 7 ells per ml, and divided into various treatment groups. Fifty miroliters of sperm suspension was added to an equal volume of medium with or without 25 mm HCO 3. Inuations were ended y the addition of 5 vol of ie-old 1 mm HCl in 1% ethanol. Samples were kept on ie for 15 min, then lyophilized and assayed for AMP (Assay Designs, Ann Aror, MI) following the manufaturer s protool for aetylated samples. Sperm Motility Analysis. For the motility analysis, we used an HTM-IVOS system (version 1.8, Hamilton Thorne Researh, everly, MA) with the following settings: ojetive, 4; minimum ell size, five pixels; minimum ontrast, 56; low VAP ut-off, 5.4; low VSL ut-off, 6.2; threshold straightness, 8%; stati size limits, ; stati head intensity,.41.93; magnifiation, Sixty frames were aquired at a frame rate of 6 Hz. At least 2 traks were measured for eah speimen at 37 C. The playak funtion of the system was used to hek its auray. Statistis. For two groups of data, two-tail Student s t tests were used. For three or more groups, data were analyzed y one-way ANOVA and Dunnett s post ho test. A proaility of P.5 was onsidered to e statistially signifiant. We thank Dr. Xu Peng for his help in preparing for onfoal imaging and Dr. M Gwrish (Department of Immunology, righam and Women s Hospital, Harvard Medial Shool, oston, MA) for the kind gift of mouse IgM. This work was supported y Li Ka Shing Institute of Health Sienes of the Chinese University of Hong Kong, National 973 Program Grant 26C542, National Natural Sienes Foundation of China Grants and 36217, Researh Grants Counil of Hong Kong Grant CUHK4534/5M, and the ollaoration projet Grant 1925 from South China National Researh Center. H.C.C. reeived a Crouher Foundation Senior Researh Fellowship. 1. 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