Acetylcholine causes an increase of intracellular calcium in human sperm

Transcription

1 Moleculr Humn Reproduction Vol.11, No.12 pp , 25 Advnce Access publiction Jnury 18, 26 doi:1.193/molehr/gh245 Acetylcholine cuses n increse of intrcellulr clcium in humn sperm C.Bry 1, J.-H.Son nd S.Meizel Deprtment of Cell Biology nd Humn Antomy, School of Medicine, University of Cliforni t Dvis, One Shields Avenue, Dvis, CA, USA 1 To whom correspondence should be ddressed t: Deprtment of Cell Biology nd Humn Antomy, School of Medicine, University of Cliforni t Dvis, One Shields Avenue, Dvis, CA, USA. E-mil: cmbry@ucdvis.edu Sperm nicotinic cetylcholine receptors (nachrs) cn influence motility nd the initition of crosome rection (AR). We report tht AR initition by cetylcholine (ACh) in cpcitted humn sperm requires both N + nd C 2+ in the externl medium. Preincubtion with 5 M 3-quinuclidinyl benzilte (QNB) or 5 nm strychnine filed to inhibit the ACh-initited AR, demonstrting tht muscrinic AChRs nd nachrs contining 9 subunits do not medite this event. Choline (2.5, 5 nd 1 mm), highly specific but low potency gonist of the 7 nachr initited AR, with its effect blocked by the nachr ntgonist methyllycconitine (MLA). ACh (5 4 M) stimulted smll trnsient rise in the intrcellulr C 2+ in sperm popultions loded with FURA-2, with 2 M ACh being mximl (146 nm 23 SEM). The nachr ntgonists, -bungrotoxin ( -BTX) nd MLA, reduced the ACh-initited C 2+ rise by 75 nd 78%, respectively, demonstrting the mjority of the rise is medited through nachrs contining 7 or 9 subunits. Single cell imging studies using FLUO-3 resolved two ptterns of ACh-stimulted C 2+ increse in the sperm hed: 94% of responding sperm displyed rise (59.6% 5.7 SEM increse from resting fluorescence intensity), returning to resting levels over period of 2 3 min. The remining sperm (6%) displyed shrp spike of C 2+ ( 1 min; 86% 4.3 SEM chnge in fluorescence intensity), followed by brupt loss of fluorescence, pttern suggestive of AR. A C 2+ influx in the sperm midpiece ppered to ccompny the C 2+ influx seen in the hed. These observtions confirm n ionotropic role for nachrs in sperm function. Key words: clcium/nicotinic cetylcholine receptors/sperm Introduction The crosome is lrge secretory vesicle locted towrds the nterior of the hed of mmmlin sperm. The crosome rection (AR) is n exocytotic event essentil for fertiliztion. Upon binding to zon pellucid (ZP; glycoprotein mtrix surrounding the oocyte), the sperm undergoes the AR (Yngimchi, 1994). During AR initition sperm s plsm membrne fuses with the underlying outer crosoml membrne leding to their fenestrtion nd vesicultion, to relese the crosoml contents nd induce modifictions of the remining sperm hed plsm membrne (Yudin et l., 1988; Yngimchi, 1994). Relese of the crosoml contents, coupled with thrust provided by the hyperctivted beting of its flgellum enble sperm to penetrte the ZP (Yngimchi, 1994). The primry in vivo inititor of AR is believed to be glycoprotein constituent of ZP, designted ZP3 (or ZPC) (Wssrmn, 1999; Kopf, 22), lthough number of endogenous gents including progesterone (Osmn et l., 1989), prostglndin (E 1 ) (Schefer et l., 1998) nd cetylcholine (ACh) (Bry et l., 22b) re cpble of triggering crosoml exocytosis in vitro. The nture of the receptors nd chnnels on the sperm plsm membrne ctivted during ZP-initited AR nd complex-signlling events ssocited with the AR re still under investigtion. The ctions N + nd C 2+ re importnt for ZP-initition of AR in cpcitted mouse, hmster nd bovine sperm (Yoshimtsu nd Yngimchi, 1988; Arnoult et l., 1999). For AR initition in humn sperm by recombinnt humn ZP3, the presence of C 2+ nd N + ions in the extrcellulr medium is essentil (Bry et l., 22) (Jung-Ho Son, unpublished dt). During AR initition, influx of N + nd C 2+ through s yet unidentified poorly selective ction chnnels contributes to membrne depolriztion (Forest et l., 1993; Flormn et l., 1998). Following membrne depolriztion trnsient rise in [C 2+ ] I is triggered through the opening of voltge-operted C 2+ chnnels (VOCCs). This initil spike in intrcellulr C 2+ concentrtion [C 2+ ] I ctivtes phospholipse Cγ (PLCγ) to generte inositol triphosphte (IP 3 ) nd dicylglycerol. IP 3 cts upon IP 3 receptor chnnels locted on the outer crosoml membrne (Wlensky nd Snyder, 1995), to relese clcium ions from the intr-crosoml store (Herrick et l., 25), leding to the opening of store-operted C 2+ chnnels on the plsm membrne (Flormn et l., 1998; Jungnickel et l., 21). The clcium rise ctivtes multiple downstrem-signlling pthwys ultimtely leding the AR (Breitbrt, 22). Mny neuronl receptors, including nicotinic cetylcholine receptors (nachrs) hve been implicted in sperm function (Meizel, 24). In vertebrtes, 1 α, 4 β nd single δ, ε nd γ nachr subunits hve been identified to dte. Muscle type nachrs conform to strict stoichiometry of either (α1) 2 β1 γ δ (expressed in fetl tissue) or (α1) 2 β1 γ ε (dult tissue). Neuronl-type nachrs re composed solely of α nd β subunits, but subunit ssocition is promiscuous, generting high level of functionl diversity in terms of phrmcologicl specificities, chnnel permebility nd kinetics. The mechnisms governing the co-ssocition of different nachr subunits is poorly understood Downloded from t Pennsylvni Stte University on Februry 23, 213 The Author 26. Published by Oxford University Press on behlf of the Europen Society of Humn Reproduction nd Embryology. All rights reserved. For Permissions, plese emil: journls.permissions@oxfordjournls.org 881

2 C.Bry, J.-H.Son nd S.Meizel (LeNovere et l., 22; Drgo et l., 23; Hogg et l., 23). Neuronl nachrs cn be ctegorized into two subtypes: heteromeric pentmers nd homomeric pentmers. Heteromeric pentmers consist of combintion of α2-1 nd β2-4 subunits in n (α) 2 (β) 3 stoichiometry. Homomeric pentmers consist exclusively of α7, α8 or α9 subunits, with α7 nachr being the most bundnt exmple of this subgroup. Homomeric α7 nachrs hve been demonstrted to exist in vivo (Drisdel nd Green, 2). Humn sperm express α3, α5, α7, α9 nd β4 nachr subunits, with expression loclized to the midpiece, neck nd post-crosoml regions (Kumr nd Meizel, 25). Micromolr concentrtions of ACh cn initite the AR in both cpcitted humn nd mouse sperm (Bry et l., 22b; Son nd Meizel, 23). The ACh-initited AR is inhibited by seprte pre-incubtion with α-bungrotoxin (α-btx), α-conotoxin IMI (α-ctx IMI) nd methyllycconitine (MLA) ntgonists tht ll bind to α7 nachrs. These ntgonists lso inhibited AR initition by recombinnt humn ZP3 or solubilized mouse ZP in humn nd mouse sperm, respectively (Bry et l., 22b; Son nd Meizel, 23). Sperm nachrs my ply role in the initition of the ZP-initited AR in vivo. This work further studies the mechnism by which ACh initites AR in cpcitted humn sperm. We investigted the requirement for the presence of C 2+ nd N + ions in the externl medium for the ACh-initited AR. Muscrinic AChRs hve been reported to be present on humn sperm (Bccetti et l., 1995). We investigted the influence of quinuclidinyl benzilte (QNB) (n ntgonist of muscrinic AChRs) upon ACh-initited AR. To determine the potentil contribution of α9 nachrs to the ACh AR we incubted sperm with strychnine, n ntgonist of α9 subunit contining nachrs before AR initition. We demonstrte tht millimolr concentrtions of choline, highly specific but low potency α7 nachr gonist re s potent s 2 μm in inititing the AR. Spectrofluorimetry ws used to investigte whether ACh ddition to cpcitted humn sperm popultions could cuse n increse in intrcellulr clcium concentrtions [C 2+ ] I nd whether such chnges were inhibitble by the bsence of N + ions in the externl medium or pre-incubtion with α-btx or MLA. Single-cell imging studies were used to further resolve the clcium influx into humn sperm upon ACh ddition. Mterils nd methods Mterils Slts nd metbolites used for incubtion nd wsh medi were of regent grde nd purchsed from Fisher Scientific (Pittsburgh, PA, USA), Irvine Scientific (Irvine, CA, USA), Mllinckrodt (Pris, KY, USA) or Sigm (St. Louis, MO, USA). Percoll ws obtined from Amershm Biosciences (Uppsl, Sweden). ACh, choline chloride, N-methyl-D-glucmine, α-btx, MLA, 3- QNB, strychnine chloride, ionomycin nd.1% poly-l-lysine solution were purchsed from Sigm. ConA-fluorescein isothiocynte (FITC) ws purchsed from EY Lbortories (Sn Mteo, CA, USA). Fluoromount mountnt Gurr ws purchsed from Gllrd-Schlesinger Industries (New York, NY, USA), Frction V bovine lbumin (Pentex #81 66 lot 59) ws purchsed from Miles (Knkkee, IL, USA). FURA-2-AM, 1,2 bis(minophenoxy)ethne N, N, N, N tetrcetic cid (tetrsodium slt) (cell imperment) (BAPTA), FLUO-3- AM nd Live/Ded sperm vibility kit were purchsed from Moleculr Probes Inc (Eugene, OR, USA). The deionized wter used in experiments ws purified to 18 MΩ-cm with NANO-pure ion exchnge system (Brnsted/ Thermolyne, Dubuque, IA, USA). Preprtion nd cpcittion of humn sperm Protocols for humn sperm studies were pproved by the Humn Subjects Committee t the University of Cliforni, Dvis. Semen smples were obtined by msturbtion from pool of helthy donors. A popultion of >95% motile sperm ws obtined by centrifugtion of semen smples through discontinuous Percoll grdient nd subsequent wshing in humn sperm medium 882 (HSM), s previously described (Surez et l., 1986). For sperm to respond to AR inititors, they must first undergo series of moleculr chnges collectively termed cpcittion (Yngimchi, 1994; Jiswl nd Eisenbch, 22). Sperm suspensions were prepred by diluting the sperm to /ml in HSM contining 26 mg/ml bovine serum lbumin (BSA) (HSM-26B) nd cpcitted by incubtion of 5 μl liquots for 18 h t 37 C nd 5% CO 2 /95% ir. In previous studies of C 2+ chnges in humn sperm, we utilized 24 h cpcittion period (Bry et l., 22b) but the 18 h cpcittion period resulted in more sperm surviving the subsequent procedures used in this study. For experiments in this study exmining AR initition, cpcittion period of between 18 nd 24 h ws used. AR initition nd ssy Cpcitted humn sperm were trnsferred into HSM-3B (Thoms nd Meizel, 1988) nd plced in microcentrifuge tubes for AR determintion; 2 μl liquots were used for experiments involving ACh. Initil experiments demonstrted tht 2 μm ACh ws optiml for the AR initition following n 18 h cpcittion period nd ws used for ll subsequent studies. Following AR initition, sperm were incubted t 37 C in 5% CO 2 / 95% ir for 25 min. Sperm were fixed in 4% formldehyde in phosphtebuffered sline (PBS), their crosomes stined using ConA-FITC nd mounted in fluoromount. Acrosoml sttus ws ssessed, with minimum of two hundred sperm from ech tretment scored in blind fshion (Meizel nd Turner, 1993). ACh is lbile molecule nd is hydrolysed by cetylcholinesterse (AChE; EC ) nd other cholinesterses. AChE is expressed on the neck, midpiece nd til of humn sperm (Mor et l., 21). To confirm the increse in AR levels of cpcitted sperm observed following incubtion with ACh ws due to the ction of ACh nd not to its brek down products, we exmined the influence of choline nd cette (2 μm) seprtely nd together upon the levels of AR observed. Millimolr concentrtions of choline ct s highly specific but low potency gonist of α7 nachrs (Mndelzys et l., 1995; Ppke et l., 1996). To investigte whether ctivtion of α7 nachrs lone ws sufficient to initite the AR, we incubted liquots cpcitted humn sperm with millimolr concentrtions of choline (2.5, 5 nd 1 mm finl concentrtions; dissolved in PBS) using the conditions described. To confirm tht choline s ction ws through α7 nachrs, we pre-incubted sperm with the nachr ntgonist MLA (1 nm finl concentrtion; in PBS; 1 min) before the ddition of choline nd AR ssessment. As MLA is prepred s citrte slt, we lso determined whether pre-incubtion with this slt lone (1 nm finl concentrtion; in PBS) influenced AR levels. In ll AR studies, liquots of sperm (5 μl) were removed before nd following tretment nd the motility of the smple determined. The percentge of motile sperm ws determined by exmintion using phse contrst microscopy (1 2 sperm per tretment) nd subjective score ws ssigned to the qulity of sperm motility using scle from 1 (twitching, non-progressive motion) to 4 (vigorous forwrd motility) (Thoms nd Meizel, 1988). Investigtion of the requirement for extrcellulr N + nd C 2+ for the ACh-initited AR For N + -free studies, liquots of cpcitted sperm were wshed twice nd resuspended in medi osmoticlly identicl to HSM-3B, designted N-B (N + -contining medium: 126 mm NCl, 25 mm KHCO 3,.36 mm KH 2 PO 4, 2.5 mm CCl 2,.5 mm MgCl 2 2 mm glucose,.25 mm pyruvte, 19 mm lctte.34 streptomycin,.21 mm K + penicillin nd 3 mg/ml of BSA) nd NMDG-B (N + -deficient: 126 mm N-methyl-D-glucmine, 25 mm KHCO 3,.36 mm KH 2 PO 4, 2.5 mm CCl 2,.5 mm MgCl 2 2 mm glucose,.25 mm pyruvte, 19 mm lctte.34 streptomycin,.21 mm K + penicillin nd 3 mg/ ml) (Grci nd Meizel, 1996). The ph nd [Cl ] of the NMDG-B were mde equl to tht of N-B through the ddition of HCl. We lso wished to determine whether the trnsfer of sperm into N + free medium interfered with their bility to undergo AR even when intrcellulr C 2+ ws incresed by mens of C 2+ ionophore. For tht experiment, we ttempted to initite the AR by incubtion of sperm with ionomycin [3 μm;.5% dimethylsulphoxide (DMSO); 1 min] in N + -free medium. The totl number of live sperm ws determined for control nd ionomycin-treted sperm with the Moleculr Probes Live/Ded sperm vibility kit. Downloded from t Pennsylvni Stte University on Februry 23, 213

3 Humn sperm cetylcholine receptors nd clcium influx To remove extrcellulr C 2+, sperm were wshed nd re-suspended in C 2+ -free medium of HSM-3B supplemented with finl concentrtion of 7.5 mm BAPTA (Bry et l., 22b) immeditely before the strt of the experiment. AR initition by 2 μm ACh nd ssessment of AR were performed. Effect of the AChR ntgonists QNB nd Strychnine on AChinitited AR QNB (5 μm) cn ntgonize muscrinic-type AChRs. QNB (5 μm finl concentrtion) dissolved in DMSO (.5% DMSO finl concentrtion) or solvent vehicle ws dded to liquots of cpcitted sperm 1 h before AR initition by ACh (2 μm) nd AR ssessment. Strychnine (5 nm) cn ntgonize nachrs contining α9 subunits (Verbitsky et l., 2). Strychnine (5 nm in PBS) or the solvent vehicle (PBS) ws dded to liquots of cpcitted sperm 1 min before AR initition by 2 μm nd AR ssessment. Investigtion of the ACh-stimulted [C 2+ ] I rise by spectrofluorimetry Cpcitted sperm suspensions in HSM-26B were prepred for cuvette bsed [C 2+ ] I studies by pooling liquots nd loding the cetoxy-methyl ester of FURA-2 (2 μm finl concentrtion; in DMSO.5% finl concentrtion; 4 min; 37 C) nd trnsfer into HSM-3B (Thoms nd Meizel, 1988). Mesurement of [C 2+ ] I ws performed every.5 s using n Hitchi F-2 spectrofluorometer (excittion wvelength of 364/385 nm; emission wvelength of 51 nm nd 5 nm excittion/emission bnd pss) (Grci nd Meizel, 1999). Mesurements were performed on 6 μl liquots of FURA-2 loded sperm. Smples were stirred mgneticlly nd temperture mintined t 37 C. A constnt flow of 5% CO 2 ws pssed over the top of the smple to mintin ph. Following equilibrtion nd the estblishment of stble bseline, dditions were mde using pre-positioned Hmilton syringe. Additions were mde using syringe specific for given regent, t 1/2 v/v. Following ACh ddition chnges in sperm, [C 2+ ] I ws mesured for n dditionl 12 s. Ech experiment ws ended with clibrtion, performed by the sequentil ddition of 2 μm digitonin nd 25 mm Tris EGTA (finl concentrtions). Clcultion of [C 2+ ] I ws performed s described previously (Grynkiewicz et l., 1985), using K d of 224 nm for FURA-2 loded humn sperm t 37 C (Hrper et l., 23). The clibrtion step ws omitted occsionlly, for sperm to be removed for visul ssessment of motility (Thoms nd Meizel, 1988). Effect of ACh on sperm [C 2+ ] I Sperm suspensions were prepred for C 2+ mesurement nd plced in the spectrofluorimeter, nd ACh (5 μm to 2 mm finl concentrtions; dissolved in PBS) ws dded to the cuvette. To limit potentil hydrolysis of ACh, ech solution ws mde immeditely before ddition. Initil experiments demonstrted 2 μm ACh ws the optiml concentrtion to obtin the mximl chnge in [C 2+ ] I (Figure 4B), nd, therefore, this concentrtion ws used for ll subsequent C 2+ studies. Effect of nachr ntgonists on ACh-stimulted [C 2+ ] I rise Sperm were pre-incubted with 1 nm finl concentrtion of α-btx (n ntgonist binding to α1, α7, α8 nd α9 nachr subunits; 45 min pre-incubtion; in PBS) or MLA (n ntgonist binding to α7 nd α9 nachr subunits; 2 min; dissolved in PBS). Concentrtions of the ntgonists used inhibit the onset of the ACh-initited AR in humn sperm (Bry et l., 22b). As MLA is prepred s citrte slt, control experiments were undertken to determine whether pre-incubtion with this slt lone (1 nm finl concentrtion; in PBS) influenced either the bsl level of C 2+ in FURA-2 loded cells or the chnge in [C 2+ ] I (Δ[C 2+ ] I ) by ACh. Anlysis of spectrofluorimetric dt Experiments included in the dt set were selected upon the criteri tht ech mintined stedy bseline over the control period, nd bsl levels of [C 2+ ] I were comprble (within 5 nm of ech other). Bsl clcium levels were clculted from men [C 2+ ] I over the control period before ACh ddition. The pek vlue ws tken s the highest vlue within 2 s following the ddition of ACh (with single pek points excluded). Overll chnge in [C 2+ ] I (Δ[C 2+ ] I ) ws clculted using the formul Δ[C 2+ ] I = [C 2+ ] I (pek) [C 2+ ] I (bsl). In experiments using nachr ntgonists, % inhibition of the Δ[C 2+ ] I ws clculted using the eqution % inhibition = {Δ[C 2+ ] I Ag Δ[C 2+ ] I C / Δ[C 2+ ] I (ACh) Δ[C 2+ ] I C} 1, where Δ[C 2+ ] I ACh denotes the chnge in intrcellulr C 2+ level following the ddition of ACh, Δ[C 2+ ] I Ag denotes the chnge in intrcellulr clcium levels initited by ACh in the presence of n nachr ntgonist nd Δ[C 2+ ] I C chnge in intrcellulr C 2+ levels by the solvent vehicle (PBS). Investigtion of the ACh-initited [C 2+ ] I rise by single-cell imging Following cpcittion, 1 μl liquots of sperm were loded with FLUO-3- AM (5 μm finl concentrtion; in DMSO with.1% F-127 pluronic; finl concentrtion of DMSO.5%) for 3 min t 37 C 5% CO 2. For the finl 15 min of dye loding, sperm were trnsferred to temperture-controlled (37 C) imging chmber (volume 2 μl; Wrner Instruments, Hmden, CT, USA) nd cells dhered to the upper surfce of the ventrl coverslip (coted with poly-llysine;.1% in ddh 2 O nd ir dried). Excess probe nd unttched sperm were removed by wshing fresh HSM-3B through the chmber. Cells were imged on Zeiss/Axiovert S1 microscope fitted with n XF14-ALPHA filter set (Omeg Opticl Inc, Brttlebro, VT, USA), fst DX-1 opticl switching system (Solmere technology group, UT, USA) nd illuminted with 75-W xenon source. Imge cpture ws controlled by Openlb softwre (Improvision, Coventry, UK) running on G4 Mcintosh computer. Imge cpture ws performed every 1 s for totl of 6 s, using either 4 nd 63 mgnifiction through XR GEN III+ ICCD cmer (Stnford Photonics, CA, USA) nd digitlized (CG-7 color frme grbber; Scion Corportion, MD, USA). ACh ws dissolved in HSM-3B immeditely before the strt of ech experiment nd injected into the chmber following control period of 2 cpture cycles (2. μl 1/1 v/v). Addition of ACh ws performed using Hmilton syringe connected to remote ir piston t the edge of the chmber, over period of 1 s, llowing gentle diffusion of ACh over the dherent sperm. An outflow port on the imging chmber ws kept open to ensure the dded volume cused no chnge in internl pressure. Imges were cptured for further 4 s (4 cycles). Control experiments demonstrted tht injection of HSM-3B cused no chnge in clcium levels of the imged sperm (dt not shown). Processing of single-cell dt Dt from single-cell experiments ws processed using Openlb softwre (Improvision). Following bckground subtrction, regions of interest (ROIs) were drwn round ech sperm hed within the field of view nd eight-bit greyscle mesurements were extrcted. For sperm dhered to the cover slip with the midpiece firmly ttched nd mesurements of C 2+ chnges in the midpiece were mde. Any cell tht moved out of its ROI during the course of the experiment ws excluded from the nlysis. Rw intensity vlues were imported into n Excel spredsheet (Microsoft, Settle, WT, USA), converted from greyscle vlues nd normlized using the eqution R = [(F F rest )/F rest ] 1% (Kirkmn- Brown et l., 2), where R is the normlized fluorescence intensity, F is the fluorescence intensity t given time point nd F rest is the men fluorescence intensity derived from the 2 time points mesured during the control period. Sttisticl nlysis All AR percentge dt were trnsformed to the rcsine of their squre roots (Sokl nd Rohlf, 1981). The Duncn s new multiple rnge test (SAS, 1994) ws used for the comprison of group men differences. Sttisticl significnce ws set s P.5. Results Extrcellulr N + nd C 2+ ions re required for the AChinitited AR Removl of N + ions from the externl medium significntly inhibited stimultion of the AR by 2 μm ACh (Figure 1, ). From this dt it ws clculted tht the level of ACh-initited AR observed in N + free medium represents 89% inhibition of AR when compred to tht seen in N + -contining medium. No difference ws observed between untreted cells nd solvent controls, indicting the trnsfer of the cells into N + -deficient medium or the solvent vehicle did not influence 883 Downloded from t Pennsylvni Stte University on Februry 23, 213

4 C.Bry, J.-H.Son nd S.Meizel () (b) Figure 1. The cetylcholine (ACh)-initited crosome rection (AR) in cpcitted humn sperm is dependent on the presence of both extrcellulr N + nd C 2+ ions. Cpcitted sperm (6 1 6 /ml) were incubted with ACh (2 μm) for 3 min in medium contining or deficient in either N + () or C 2+ (b) ions. The level of AR ws then ssessed s described in Mterils nd methods. Ech column represents the men vlue from five different experiments (); brs on ech column represent the ±SEM of the men: different letter superscripts denote significnt difference between tretments (P.5). bsl AR levels (Figure 1, ). Sperm in N + -free medium were still ble to undergo ionomycin-initited AR [n = 3; ionomycin (3 μm) ±1.3%]. The removl of extrcellulr C 2+ from the externl medium inhibited AR initition by ACh (Figure 1, b). This dt demonstrtes the complete inhibition of AR initition by ACh (2 μm) in C 2+ -free medium when compred to levels chieved in C 2+ -contining medium. No difference ws observed in AR between untreted cells (C 2+ present) nd solvent controls, indicting removl of extrcellulr C 2+ nd the solvent vehicle did not influence the bsl AR (Figure 1, b). ACh is lbile molecule. We confirmed the increse in the levels of AR observed ws due to the ction of ACh nd not its brekdown products choline nd cette, s incubtion of sperm with 2 μm concentrtions of either compound seprtely or both compounds together produced no significnt stimultion in AR compred to their solvent vehicle (PBS). The following results were obtined (n = 4, % AR ± SEM, P.5 for choline, cette nd choline + cette tretments compred to PBS nd P.5 for ACh compred to PBS): PBS 1. ± 1.4%; ACh (2 μm) 22.2 ± 1.4%; choline (2 μm) 13. ± 1.6%; cette (2 μm) 1.7 ± 1.4%; cette (2 μm) + choline (2 μm) 13.3 ± 1.4%. QNB nd strychnine do not influence the ACh-initited AR The incubtion of cpcitted sperm with QNB (5 μm) followed by the ddition of ACh (2 μm) did not significntly inhibit AR initition when compred to its solvent vehicle (.5% DMSO) nd ACh. The following dt were obtined (n = 3, % AR ± SEM; P.5 for ll results compred to PBS + DMSO nd P.5 for QNB + ACh b N + free Untreted Solvent ACh Untreted Solvent ACh Untreted Solvent ACh 2uM μm Untreted Solvent ACh 2uM μm 2 Tretment 18 b C 2+ fre e 16 Untreted Solvent ACh Untreted Solvent ACh Untreted Solvent ACh 2uM μm Untreted Solvent ACh2uM μm Tretment Tretment results compred to DMSO + ACh):.5% DMSO + PBS 7.9 ±.8%;.5% DMSO + ACh 2 μm 16.9 ±.9%; QNB 5 μm + ACh 2 μm 15.3 ±.2%. The incubtion of cpcitted sperm with 5 nm strychnine before the ddition of ACh (2 μm) did not significntly inhibit AR initition when compred to its solvent vehicle (PBS). The following dt were obtined (n = 3, % AR ± SEM; P.5 for ll results compred to PBS + PBS): PBS + PBS 13.2 ± 2.5%; strychnine (5 nm) + PBS 11.5 ± 2.1%; PBS + ACh (2 μm) 27.6 ± 3.2; strychnine (5 nm) + ACh (2 μm) 25.4 ± 2.9%. Choline cn initite the AR t mm concentrtions The ddition of 2.5, 5 nd 1 mm concentrtions of choline to sperm cpcitted for between 18 nd 24 h resulted in significnt increse in AR, when compred to AR levels in the PBS solvent control (Figure 2). To confirm the ction of choline ws through α7 nachrs, we preincubted sperm with the nachr ntgonist MLA (1 nm finl concentrtion; in PBS) before the ddition of choline nd AR ssessment. MLA is prepred s citrte slt; we hve previously determined tht pre-incubtion of sperm with this slt lone (sodium citrte 1 nm finl concentrtion; in PBS) hs no influence on AR levels (Bry et l., 22b). ACh ddition cuses rise in intrcellulr clcium in humn sperm popultions Of the experiments tht showed mesurble chnge in [C 2+ ] I upon ACh ddition, only those tht mintined stble initil bseline throughout the subsequent series of experiments nd exhibited comprble bseline levels of [C 2+ ] I were included in the dt set described here (2/52 experiments). The men clculted bsl [C 2+ ] I from ll experiments ws 21 ± 7 nm (n = 47). Bsl levels of [C 2+ ] I were comprble to tht reported previously for humn sperm following n equivlent cpcittion period (Ptrt et l., 2). ACh ddition cused rise in [C 2+ ] I ; Figure 3A shows typicl experimentl trce. Time tken to rech the pek vlue ws 8 15 s following ddition, fter which the trnsient decyed over 6 s. All concentrtions of ACh (5, 1, 2 nd 4 μm) tested cused significnt increse in [C 2+ ] I when compred to the PBS control (Figure 3B) (Duncn s new multiple rnge test; P.5) (Figure 3). Amplitude of Δ[C 2+ ] I ws Figure 2. Millimolr concentrtions of choline cn initite the crosome rection (AR) in cpcitted humn sperm through nicotinic cetylcholine receptors (nachrs). Cpcitted sperm (6 1 6 /ml) were incubted with cetylcholine (ACh) (2 μm) or choline (Cho) mm in the bsence nd presence of methyllycconitine (MLA; 1 nm). The level of AR ws then ssessed s described in Mterils nd methods. Ech column represents the men vlue of three different experiments; brs on ech column represent the ±SEM of the men; different letter superscripts denote significnt difference between tretments (P.5). Downloded from t Pennsylvni Stte University on Februry 23, 213

5 Humn sperm cetylcholine receptors nd clcium influx Figure 3. Acetylcholine (ACh)-medited elevtion of intrcellulr C 2+ levels in cpcitted humn. (A) Representtive trce from humn sperm loded with FURA-2 demonstrting rise in [C 2+ ] I following ACh (2 μm) ddition. Cpcitted sperm were loded with FURA-2 s described in Mterils nd methods; ddition of 2 μm ACh (or PBS) is denoted by the br. (B) Chnges in intrcellulr clcium levels fter ACh stimultion re dose dependent. Ech group represents the men vlue of Δ[C 2+ ] I derived from different experiments; the number of experiments (n) is denoted on the x xis; brs on ech column represent the ±SEM of the men; different letter superscripts denote significnt difference between tretments (P.5). A C ACh Time (seconds) ACh 5 1 T ime (seconds) B D uM ACh BTX+2uM ACh 2 μ M ACh Tretment α -BTX + 2 μm ACh Tretment 2uM ACh MLA+2uM ACh 2 μ M ACh MLA + 2 μm ACh Tretment Tretment b b Downloded from t Pennsylvni Stte University on Februry 23, 213 Figure 4. Nicotinic cetylcholine receptor (nachr) ntgonists inhibit cetylcholine (ACh)-medited Δ[C 2+ ] I in cpcitted humn sperm. (A) Trce demonstrting tht α-bungrotoxin(α-btx) reduces the ACh-initited elevtion of C 2+ in FURA-2 loded humn sperm. Sperm were pre-incubted with α-btx (1 nm; light grey) or PBS for 45 min before the ddition of 2 μm ACh (denoted by the br). (B) α-btx reduces the ACh-stimulted C 2+ influx. Ech column represents the men vlue derived from three different experiments; brs on ech column represent the ±SEM of the men; different letter superscripts denote significnt difference between tretments (P.5). (C) Representtive trce demonstrting the bility of methyllycconitine (MLA) to reduce the elevtion of C 2+ initited by ACh in FURA-2 loded humn sperm cells. Sperm were s pre-incubted with MLA (1 nm) (light grey) or solvent vehicle (drk grey) for 45 min before the ddition of ACh (2 μm; denoted by the br). (D) MLA reduces the ACh-stimulted C 2+ influx. Ech group represents the men ± SEM of three different experiments: different letter superscripts denote significnt difference between tretments (P.5). dose dependent, 2 μm cusing chnge of 146 ± 23 nm (n = 8). 4 μm ACh cused smller rise, with higher concentrtions of 2 nd 2 mm ACh-inhibiting C 2+ chnges (dt not shown). Addition of solvent vehicle (PBS) cused smll but sttisticlly insignificnt increse in [C 2+ ] I : PBS Δ[C 2+ ] I 13.3 ± 2.5 nm (n = 1). Addition of ACh (4 μm) to sperm lone cused no chnge in utofluorescence. 885

6 C.Bry, J.-H.Son nd S.Meizel -BTX nd MLA cn reduce the ACh-stimulted C 2+ rise Pre-incubtion of FURA-2 loded sperm with either α-btx (1 nm) or MLA (1 nm), significntly reduced the size of the trnsient C 2+ influx seen upon ddition of 2 μm ACh (Figure 4A nd C). By subtrcting the men vlue of Δ[C 2+ ] I from prllel experiments in which PBS lone ws dded, we were ble to remove the contribution of the solvent vehicle from the overll C 2+ chnge nd clculte the efficiency of ech ntgonist in blocking the C 2+ influx initited by ACh. The presence of α-btx effected 75% reduction of the men pek vlue of the C 2+ influx initited by ACh (Figure 4B). Pre-incubtion with MLA led to 78% reduction of the men pek vlue of the C 2+ influx initited by ACh (Figure 4D). Single-cell imging of the ACh-stimulted C 2+ rise Addition of the solvent vehicle (HSM-3B) hd no effect on the FLUO-3 fluorescence. From 19 experiments, using cells from six different experimentl donors we mesured increses in [C 2+ ] I in 125 sperm [18% of totl of 685 cells exmined with 2.2% (±6.4 SEM) cells responding per experiment] upon the ddition of ACh (2 μm). The chnges in C 2+ in responding sperm occurred immeditely or shortly fter (within 1 min) ddition of ACh into the imging chmber. The vrition in onset of the response my reflect heterogeneous ctivtion sttes of the sperm within the experimentl popultion nd inherent vrition in the sensitivity of given cell bility to respond to ACh. ACh ddition initited two distinct ptterns of increse in [C 2+ ] I in the sperm hed (Figure 4). The most frequently observed pttern of C 2+ influx into the hed following ACh ddition ws rise to pek nd decline bck to bsl levels (94% of responding cells; n = 117 cells). From initition this pttern of C 2+ typiclly took 1 2 s to rech pek nd ws followed by decline phse lsting between 1 nd 2 s (Figure 4A). The men mplitude of the chnge of [C 2+ ] I ws 59.6% (±5.7 SEM) increse in fluorescence intensity reltive to resting levels. This men vlue ws derived from 2 rndomly picked cells within the smple. A second, less frequently observed pttern of C 2+ influx in the sperm hed following ACh ddition ws rpid rise to spike, followed by brupt loss of fluorescence to below the initil resting level (Figure 4B) (6% of totl cells responding; n = 8 cells). The men mplitude of the spike ws 86% (±4.3 SEM; n = 8 cells) increse in fluorescence intensity reltive to resting levels. Previous imging studies during AR initition in humn sperm hve reported similr events, with the sudden loss of fluorescence ttributed to the dispersl of fluorophore from the sperm hed s the plsm membrne nd outer crosoml membrnes fuse, indicting the onset of the AR (Tesrik et l., 1996; Meizel et l., 1997). Most sperm demonstrting ACh-initited C 2+ influx retined flgellr motility when dhered to the poly-l-lysine cover slip, which ws pprent by movements of the midpiece region throughout ech experiment. However, of the few sperm with both dherent heds nd midpiece tht responded to ACh (n = 4 cells), we observed with rise in [C 2+ ] I in the midpiece, concurrent to tht seen in sperm hed. Figure 5 shows n ACh-initited C 2+ rise in the midpiece, concurrent to the spike nd loss of fluorescence pttern in the sperm hed indictive of AR. Sperm motility The percentge of motile sperm in ll experimentl smples except ionomycin dditions ws the sme s the controls (rnge ws 7 8% in different experiments). No differences were found between the qulity of motility in experimentl nd control tubes in ny single experiment. Motility decresed fter ionomycin dditions but vibility 886 A B Figure 5. Chnges in C 2+ levels re observed in the imged heds of cpcitted humn sperm loded with the C 2+ sensitive probe FLUO-3, following ddition of cetylcholine (ACh; presence indicted by the grey br). Two ptterns of [C 2+ ] I increse in the humn sperm hed were observed following the ddition of ACh. (A) Four representtive trces from seprte experiments represent the dynmics of the most frequently observed pttern of C 2+ chnge into hed. (B) Four representtive trces from seprte experiments showing the rise nd sudden loss of fluorescent pttern of C 2+ in sperm hed following the ddition of ACh, pttern suggestive of crosoml exocytosis. stining (with SYBR14 nd propidium iodide dyes: Moleculr Probes Inc.) indicted comprble levels of live nd ded sperm in experimentl nd control tretments [n = 3, % live sperm ± SEM; P.5 (DMSO solvent control 74.1 ±.3%; ionomycin 69.2 ± 1.1%)]. Discussion Micromolr concentrtions of ACh cn initite AR in cpcitted humn nd mouse sperm, nd nachr ntgonists inhibit the ACh-initited AR (Bry et l., 22b; Son nd Meizel, 23). Expression of nachrs hs been demonstrted on both mouse nd humn sperm (Bry et l., 25; Kumr nd Meizel, 25). The signlling molecule ACh cn interct with two different types of receptors: nicotinic nd muscrinic. Immunocytochemicl studies hve suggested the presence of muscrinic AChRs on humn sperm (Bccetti et l., 1995). An ntgonist of muscrinic AChRs, QNB, shown to inhibit the ZP-initited AR in mouse sperm t 5 μm (Flormn nd Storey, 1981), hd no influence on the AR initited by ACh in humn sperm, suggesting muscrinic type AChRs do not ply n role 3 Time (seconds) 3 Time (seconds) ACh 4 ACh Downloded from t Pennsylvni Stte University on Februry 23, 213

7 in the ACh-initited AR. Both α-btx nd MLA block AR (Bry et l., 22b) nd substntil portion of the C 2+ influx initited by ACh. α-btx binds the α1, α7, α8 nd α9 nachr subunits; however, the α1 subunit is only found in muscle nd the α8 is not mmmlin subunit (Hogg et l., 23; Fucile, 24). MLA is n ntgonist of both α7 nd α9 nachrs. Humn sperm express both α7 nd α9 nachr subunits, loclized to the posterior sperm hed nd the midpiece, respectively (Kumr nd Meizel, 25). Our previous work hs shown tht the humn sperm α7 nachr is involved in the AR initited by ACh or by recombinnt humn ZP3 (Bry et l., 22b). Here, we demonstrte tht strychnine, n ntgonist of α9 subunit contining nachrs t the concentrtion used here (Verbitsky et l., 2), hs no influence on the level of AR initited by ACh, suggesting ctivtion of the α7 nachr is solely responsible. As choline, specific gonist of α7 nachrs initites the AR in cpcitted humn sperm to levels equivlent to those chieved with ACh, nd the ction of choline is blocked by MLA, we hve confirmed the ACh initited AR is medited through α7 nachrs. The requirement for C 2+ influx during AR initition hs been demonstrted in sperm from mny species (Yngimchi, 1994). Previous histochemicl studies hve reported micromolr concentrtions of ACh, nd nicotine induce C 2+ uptke in hypotoniclly wshed rm (Stewrt nd Forrester, 1979) nd bovine sperm cells (Nelson et l., 1982). In this study, AR initition by ionomycin ws not blocked by α-btx, demonstrting nachr functioning is upstrem of C 2+ influx. The ACh-initited AR is dependent on the presence of extrcellulr N + nd C 2+ ions. We hve demonstrted tht ACh initites rpid clcium influx into cpcitted humn sperm. Both α-btx nd MLA block AR (Bry et l., 22b) nd substntil portion of the C 2+ influx initited by ACh. MLA is n ntgonist of both α7 nd α9 nachrs (Verbitsky et l., 2). Humn sperm express both α7 nd α9 nachr subunits (Kumr nd Meizel, 25); it is likely ech ntgonist could exert their influence on sperm function reported in this study through α7 nachrs, α9 nachrs or both. As humn sperm lso express α3, α5 nd β4 nachr subunits (Kumr nd Meizel, 25), which form heteromeric nachrs in vivo (in either α3 (2) α5 β4 (2) or α3 (3) β4 (2) stoichiometry) nd re insensitive to both α-btx nd MLA (Hogg et l., 23), the α-btx nd MLA resistnt component of the ACh-initited rise in [C 2+ ] I my be due to ctivtion of these nachrs. Additionlly, s the ctivtion of muscrinic AChRs cn lso ctivte cellulr C 2+ influx pthwys (Akermn et l., 24), we cnnot discount tht such receptors potentilly present on sperm lso contribute in prt to the observed C 2+ rise, lthough we cn discount them from significnt role in the Ach-initited AR. Humn sperm α7 nachrs re loclized to the neck nd post-crosoml regions (Kumr nd Meizel, 25); ctivtion of these receptors might led to C 2+ increses in the sperm hed nd midpiece. Two distinct ptterns of C 2+ increse were observed in the hed following ACh ddition: The most frequent ws rise to pek nd decline bck to bsl levels. The less frequent pttern is chrcterized by rpid rise nd brupt loss of fluorescence to below the initil resting level. This spike pttern is probbly due to dispersl of fluorophore from the sperm hed s the plsm membrne nd outer crosoml membrnes fuse nd fenestrte, t the onset of the AR. An increse in the [C 2+ ] I of the sperm midpiece ws detected following ACh ddition (Figure 6). Due to the intervl of smpling we could not resolve whether the C 2+ chnges seen in both the hed nd midpiece were concurrent or one event preceded the other. Adherence of the sperm midpiece nd loss of flgellr beting my indicte the pproch of senescence nd this dt my hve been obtined from moribund cells. We hve not s yet further chrcterized this event or its reltionship to sperm function; however, s nachrs re present upon the midpiece of humn sperm, we felt this observtion ws worthy of inclusion in this study. Humn sperm cetylcholine receptors nd clcium influx Figure 6. Pseudocolour imge series of the reltive chnges in [C 2+ ] I initited by 2 μm cetylcholine (ACh) (time of ddition mrked by rrow) into the midpiece (M) nd hed (H) of cpcitted humn sperm ( 63) loded with clcium sensing probe FLUO-3. Three greyscle imges of sperm re included from the 2, +3 nd +8 s timepoints. The sudden loss of fluorescent in hed pttern of C 2+ in sperm hed following the ddition of ACh is indictive of crosoml exocytosis. Homomeric nachrs heterologously expressed in vitro disply high permebility to C 2+ ions when compred to other nachrs, with flux through these chnnels per se being enough to elevte intrcellulr clcium levels in vivo (Fucile, 24). However, number of recent studies hve demonstrted tht the C 2+ permebility of α7 nachrs is much lower in vivo nd their ctivity is regulted in cell-specific mnner (Fyuk nd Ykul, 25). In ddition, the rpid dectivtion time of nachr chnnels ( 1 ms for α7 nachrs; Fyuk nd Ykel, 25) is irreconcilble with the temporl kinetics of the C 2+ chnges we observed in both our fluorimetric nd single-cell studies. One of the best chrcterized functions of neuronl nachrs hs been the post-synptic propgtion of ction potentils by N + /C 2+ flux, leding to membrne depolriztion (Ashcroft, 2). If the current ctivted through sperm nachrs is sufficient to depolrize the plsm membrne, recruitment of VOCCs my led to further C 2+ influx. Such coupling mechnisms hve been described in other cell types (Feurbch et l., 25). Recruitment of VOCCs following membrne depolriztion during AR initition by ZP is widely ccepted model (Flormn et l., 1998; Kirkmn-Brown et l., 22). A role for α7 nachr signlling in the pthwys ctivted during ZP-initited AR hs been suggested (Bry et l., 22; Son nd Meizel, 23). Humn sperm α7 nachr subunits, most likely ssembled s homomeric α7 nachrs re loclized to the neck nd post-crosoml regions of the sperm hed (Kumr nd Meizel, 25); the VOCCs responsible for the C 2+ wve ssocited with the onset of AR re lso loclized to post-crosoml region (Goodwin et l., 1997). Sperm α7 nachrs my shre identity with the non-specific ction chnnels responsible in prt for membrne depolriztion during AR-initition by ZP nd progesterone (Forest et l., 1993; Flormn et l., 1998). The portion of the chnge in sperm [C 2+ ] I initited by ACh nd inhibited by MLA nd α-btx my be due to the dditive influx of C 2+ through both nachrs nd VOCCs. Although the requirement for extrcellulr N + in the ACh initited AR is suggestive of membrne depolriztion component in this pthwy, further experiments using membrne potentil dyes nd clcium chnnel blockers need to be undertken to further resolve the sequence of events ssocited with the observed C 2+ rise. Activtion of sperm α7 nachrs lone by ACh or choline is enough to trigger AR in our in vitro culture system. Our previous work hs shown tht tht the α7 nachr is essentil to the mouse AR initited by mouse ZP (Meizel nd Son, 25) nd the humn AR initited by humn recombinnt ZP3 (Bry et l., 22b). However, it is importnt to stress tht we do not believe nachrs re the sole rbiters of the AR in vivo. This in vitro study serves to demonstrte nachr-signlling pthwys exists in sperm which potentilly contributes to the ionotropic regultion of sperm function in vivo. Two other sperm neurotrnsmitter receptor/ion chnnels re lso involved in the AR: the glycine receptor/ chloride chnnel in the ZP-initited but not the progesterone-initited AR (Melendrez nd Meizel, 1995; Sto et l., 2; Bry et l., 22) nd the GABA A receptor/chloride chnnel (Wistrom nd Meizel, 1993; 887 Downloded from t Pennsylvni Stte University on Februry 23, 213

8 C.Bry, J.-H.Son nd S.Meizel Shi nd Roldn, 1995) in the progesterone-initited but not the ZP-initited AR. It hs been speculted tht these neurotrnsmitter receptor/ion chnnels ply roles in membrne potentil chnges ssocited with the onset of the AR in vivo (Meizel et l., 1997; Bry et l., 22). The ZP-initited AR, requires ctivtion of both α7 nachr nd glycine receptor (Meizel, 24). Their compound nd simultneous opening my generte sufficient ionic fluxes to bring bout the membrne potentil chnges tht herld the onset of the AR. The concentrtion of ACh used in this study nd the mode in which it ws presented to sperm is unlikely to be representtive of how sperm encounter endogenous cholinergic signls within the femle reproductive trct. ACh cn be synthesized by the enzymes choline cetyltrnsferse (ChAT) nd crnitine cetyltrnsferse. ACh synthesis, ChAT, nd crnitine cetyltrnsferse hve been reported in humn sperm (Goodmn et l., 1984; Ibnez et l., 1991). Within the femle reproductive trct ACh, secretion hs been detected from cells of the vginl mucos, grnulos cells of the cumulus oophorus nd the ovry (Myerhofer et l., 23; Wessler et l., 23). ACh generted by sperm or the tissues of the femle reproductive trct my influence sperm behviour in prcrine or utocrine mnner, potentilly cting to provide low level tonic stimultion of cholinergic receptors on sperm. Sperm do, however, encounter levels of choline equivlent to those used in this study within the femle reproductive trct; the men concentrtion of choline in humn cervicl mucus hs been reported to be 5.25 mm (Shrbcher et l., 22). The bility of cervicl mucus to increse hyperctivted motility (Zhu et l., 1992) might be due in prt to choline cting through α7 nachrs. The distinct regionliztion of different nachr subtypes on humn sperm suggests cholinergic signlling my influence dditionl spects of sperm function other thn tht described in this study. C 2+ signlling plys role in mny spects of sperm function, including motility, cpcittion, hyperctivtion nd AR (Breitbrt, 22; Jiswl nd Eisenbch, 22; Surez nd Ho, 23). Segregtion of the complex C 2+ signls ssocited with these phenomen is chieved in prt by comprtmentliztion nd the close sptil nchoring of downstrem-signlling elements to sources of C 2+ flux. It ws previously reported tht nnomolr nd low micromolr concentrtions of ACh stimulte sperm motility through nachrs in number of species, including humn (Dwivedi nd Long, 1989). Recently, we demonstrted tht mice sperm express α7 nachrs on their midpiece nd flgell nd tht mouse deficient in the α7 nachr produce sperm with impired motility (Bry et l., 25). The influence of nachrs upon these diverse spects of sperm function would depend on their functionl chrcteristics, distribution on the plsm membrne nd the subcellulr comprtments into which they exert their ionotrophic effect. Acknowledgements The uthors thnk Dr J. Kirkmn-Brown (University of Birminghm, UK) for dvice on single-cell imging nd Alex Bry for help with the Openlb utomtions. Supported by NIH grnt HD33368 to SM nd Llor Foundtion Fellowship to CB. References Akermn K, Shritmdri R, Krjukov J, Lrsson KP, Courtney MJ nd Kukkonen JP (24) C 2+ -dependent potentition of muscrinic receptormedited C 2+ elevtion. Cell Clcium 36, Arnoult C, Kzm IG, Visconti PE, Kopf GS, Villz M nd Flormn HM (1999) Control of the low voltge-ctivted clcium chnnel of mouse sperm by egg ZP3 nd by membrne hyperpolriztion during cpcittion. Proc Ntl Acd Sci USA 96, Ashcroft FM (2) Ion Chnnels nd Disese. Acdemic Press, London. 888 Bccetti B, Burrini AG, Collodel G, Flugi C, Moretti E nd Piomboni P (1995) Loclistion of two clsses of cetylcholine receptor-like molecules in sperms of different niml species. Zygote 3, Bry C, Son JH nd Meizel S (22) A nicotinic cetylcholine receptor is involved in the crosome rection of humn sperm initited by recombinnt humn ZP3. Biol Reprod 67, Bry C, Son J-H, Kumr P nd Meizel S (22b) A role for the humn sperm glycine receptor/cl chnnel in the crosome rection initited by recombinnt ZP3. Biol Reprod 66, Bry C, Son J-H nd Meizel S (25) Mice deficient in the α7 subunit of the nicotinic cetylcholine receptor produce sperm with impired motility. Biol Reprod 73, Breitbrt H (22) Role nd regultion of intrcellulr clcium in crosoml exocytosis. J Reprod Immunol 53, Drgo J, McColl C, Horne M, Finkelstein D nd Ross S (23) Neuronl nicotinic receptors: insights gined from gene knockout nd knockin mutnt mice. Cell Mol Life Sci 6, Drisdel R nd Green W (2) Neuronl lph-bungrotoxin receptors re lph7 subunit homomers. J Neurosci 2, Dwivedi C nd Long N (1989) Effect of cholinergic gents on humn spermtozo motility. Biochem Med Metb Biol 42,66 7. Fyuk D nd Ykul J (25) C 2+ permebility of nicotinic cetylcholine receptors in rt hippocmpl CA1 interneurons. J Physiol 566, Feurbch D, Lingenhohl K, Dobbins P, Mosbcher J, Corbett N, Nozulk J nd Hoyer D (25) Coupling of humn nicotinic cetylcholine receptors lph 7 to clcium chnnels in GH3 cells. Neurophrmcology 48, Flormn HM nd Storey BT (1981) Inhibition of in vitro fertiliztion of mouse eggs: 3-quinuclidinyl benzilte specificlly blocks penetrtion of zone pellucide by mouse spermtozo. J Exp Zool 216, Flormn HM, Arnoult C, Kzm IG, Li C nd O Toole CM (1998) A perspective on the control of mmmlin fertiliztion by egg-ctivted ion chnnels in sperm: tle of two chnnels. Biol Reprod 59, Forest C, Rossto M nd Di Virgilio F (1993) Ion fluxes through the progesterone-ctivted chnnel of the sperm plsm membrne. Biochem J 294, Fucile S (24) Clcium permebility of nicotinic cetycholine receptors. Cell Clcium 35,1 8. Grci MA nd Meizel S (1996) Importnce of sodium ion to the progesteroneinitited crosome rection in humn sperm. Mol Reprod Dev 45, Grci MA nd Meizel S (1999) The progesterone medited clcium influx nd crosome rection of humn spermtozo: phrmcologicl investigtion of T-type clcium chnnels. Biol Reprod 6, Goodmn D, Adtsi F nd Hrbison R (1984) Evidence for the extreme overestimtion of choline cetyltrnsferse in humn sperm, humn seminl plsm nd rt hert: cse of mistking crnitine cetyltrnsferse for choline cetyltrnsferse. Chem Biol Interct 49, Goodwin LO, Leeds NB, Hurley I, Mndel FS, Pergolizzi RG nd Benoff S (1997) Isoltion nd chrcteriztion of the primry structure of testisspecific L-type clcium chnnel: implictions for contrception. Mol Hum Reprod 3, Grynkiewicz G, Poenie M nd Tsien RY (1985) A new genertion of C 2+ indictors with gretly improved fluorescence properties. J Biol Chem 26, Hrper C, Kirkmn-Brown JC, Brrtt CL nd Publicover SJ (23) Encoding of progesterone stimulus intensity by intrcellulr [C 2+ ] ([C 2+ ] i ) in humn spermtozo. Biochem J 372, Herrick S, Schweissinger D, Kim S, Byn K, Mnn S nd Crdullo R (25) The crosoml vesicle of mouse sperm is clcium store. J Cell Physiol 22, Hogg R, Rggenbss M nd Bertnd D (23) Nicotinic cetylcholine receptors: from structure to brin function. Rev Physiol Biochem Phrmcol 147,1 46. Ibnez CF, Pelto-Huikko M, Soder O, Ritzen E, Hersh L, Hokfelt T nd Persson H (1991) Expression of choline cetyltrnsferse mrna in spermtogenic cells results in n ccumultion of the enzyme in the postcrosoml region of mture spermtozo. Proc Ntl Acd Sci USA 88, Jiswl BS nd Eisenbch M (22) Cpcittion. In Hrdy DM (ed.), Fertiliztion. Acdemic Press, Sn Diego, CA, pp Jungnickel MK, Mrrero H, Birnbumer L, Lemos JR nd Flormn HM (21) Trp2 regultes entry of C 2+ into mouse sperm triggered by egg ZP3. Nt Cell Biol 3, Kirkmn-Brown JC, Bry C, Stewrt PM, Brrtt CL nd Publicover SJ (2) Biphsic elevtion of [C 2+ ] i in individul humn spermtozo exposed to progesterone. Dev Biol 222, Downloded from t Pennsylvni Stte University on Februry 23, 213