human sperm plasma membrane progesterone receptor(s) and the

BIOLOGY OF REPRODUCTION 54, 993-1001 (1996)

Human Sperm Plasma Membrane Progesterone Receptor(s) and the Acrosome Reaction'

Khalida Sabeur,3 '4 Dean P. Edwards,5 and Stanley Meizel 2 4

Department of Cell Biology and Human Anatomy, 4 University of California School of MedicineDavis, California 95616-8643

Department of Pathology,5 University of Colorado Health Sciences Center, Denver, Colorado 80262

ABSTRACT

Progesterone initiation of the human sperm acrosome reaction (AR) includes stimulation of a transient Ca2 influx and a transient CIefflux. A role for one or more plasma membrane receptors has been suggested, but, except for evidence supporting the involvement ofa sperm GABAA-like receptor/CI- channel, there is little information about possible receptor identity. Here, we attempt to identify plasmamembrane progesterone receptors in human sperm using a mouse monoclonal antibody (mAb C-262) raised againstthe C-terminal steroid-binding domain of the human intracellular progesterone receptor. C-262 inhibited the progesterone-initiated AR in a dose-dependentmanner. Maximum inhibition was 77% as detected by fluorescein isothiocyanate (FITC)-concanavalin A (ConA). Motility was unaffected.A control mouse mAb (h-151) raised against the human estrogen receptor did not inhibit the progesterone-initiated AR. Western blottingwith C-262, but not with h-151, detected a major sperm protein band of 50-52 kDa. In indirect immunofluorescence localization studies,live and ethanol-fixed uncapacitated sperm and fixed capacitated sperm incubated with C-262, but not with h-151, displayed fluorescenceat the equatorial segment region of the sperm head plasma membrane. In spectrofluorometric studies using capacitated sperm loadedwith the Ca2 probe Fura-2 or the Cl- probe MEQ, C-262 but not h-151 inhibited both Ca2+ influx and ClI efflux. These ion fluxes could bedue to the binding of progesterone to two different receptor/channels or to its binding to one and cross talk with the other. Our resultsstrongly support the involvement of sperm plasma membrane receptors in the progesterone-initiated AR and provide a candidate for onesuch receptor.

INTRODUCTION

The mammalian acrosome reaction (AR) is a modifiedform of exocytosis involving the fusion of a membrane froma sperm head organelle, the acrosome, with the overlyingsperm plasma membrane, followed by vesiculation of fusedmembranes and release of acrosomal contents [1, 2]. TheAR is essential for sperm penetration of the zona pellucida,a glycoprotein coat directly surrounding the egg, and forfusion of the sperm and egg plasma membranes [3].

The zona pellucida can initiate the AR in vitro and isgenerally considered to be the in vivo initiator of the AR [4].However, the mammalian AR can also be initiated in vitroby progesterone [5-9], a steroid secreted by the cumulusoophorus-follicular cells that remain associated with theovulated zona-enclosed egg. Since the cumulus is presentat the time of fertilization, progesterone should also be pres-ent and may have a role in initiating the AR of fertilizingsperm in vivo [5, 6]. Such a role could be a direct and/orsynergistic one [6]. Recent in vitro studies with mouse spermsuggest that previous brief exposure of sperm to progester-one can potentiate the zona-initiated AR [8]. Progesteronealso appears to stimulate capacitation of human and porcine

Accepted December 18, 1995.Received October 24, 1995.'This research was supported by NIH grant HD-23098 and a UC Davis Faculty Research

grant to S.M. and by NIH grant CA 46938 to D.P.E.2Correspondence. FAX: (916) 752-8520; e-mail: [email protected] address: Reproductive Biology, Suber House, University of California School

of Medicine, Davis, CA 95616.

sperm [10, 11]. There has been a great deal of recent evi-dence that nongenomic steroid effects in several cell typesare due to steroid interaction with plasma membrane re-ceptors [12, 13], and the effect of progesterone on mam-malian sperm also appears to involve such receptors. Anextracellular increase in intracellular Ca 2+ ([Ca2 +]i) is es-sential for the AR [14], and some progestins including pro-gesterone, but not other types of steroids, have been shownto initiate a several-fold increase in [Ca2+]i and the AR withinseconds after addition to human sperm [6, 15, 16]. Theseprogesterone effects on sperm are too fast to be due to theclassic steroid mechanism that includes binding of intracel-lular receptor/hormone complex to chromatin and newprotein synthesis. Furthermore, progesterone that is cova-lently bound to BSA (a molecule that does not enter thesperm) also increases human sperm [Ca2 +]i and the AR, sug-gesting that the steroid acts at the level of the plasma mem-brane [17, 18]. Moreover, addition of an antibody againstprogesterone to human sperm, together with suboptimallevels of progesterone, was found to stimulate rapid Ca2 +

influx and the AR, leading to the interpretation that anti-body-mediated aggregation of a progesterone receptorcomplex on the cell surface was responsible for the stimu-lation [19]. A conjugate of fluorescein isothiocyanate (FITC)-labeled BSA-progesterone has been used to localize pro-gesterone receptor sites on the sperm head plasmamembrane of viable human sperm [20, 21]. Even though itappears that the progesterone-induced exocytosis in humansperm is mediated by a receptor localized in the plasma

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membrane, neither the chemical nature of the receptormolecule nor the mode of function of this receptor isknown. While several sperm receptor candidates for thezona protein that initiates the AR have been identified [4],there is little information concerning the nature of putativesperm progesterone receptors. Two human sperm proteins,a 220-kDa surface antigen (SAA1) and a 94-kDa protein thatdisplayed progesterone-mediated tyrosine phosphoryla-tion, have been implicated in the regulation of the proges-terone-initiated AR [22, 23], but whether these proteins areactually the plasma membrane receptors for the ligands inquestion remains to be elucidated.

Work from this laboratory has suggested the involvementin the progesterone-initiated human AR of another type ofreceptor, a unique steroid receptor/C1 channel complexresembling, but not identical to, the GABAA receptor/C1channel from mammalian central nervous system neurons[24]. On the basis of results of spectrofluorometric and ARstudies using antagonists of the central nervous system GA-BAA receptor or using a Cl -deficient medium, it appearsthat the progesterone-initiated AR requires C1- flux but thatthe increase in [Ca2+]i essential to the human sperm AR isindependent of the AR Cl- requirement [25]. Such resultscould be explained by the presence of two plasma mem-brane progesterone receptors--one receptor initiating theincrease in [Ca2 +]i and the other initiating an increased Clflux via activation of a GABAA-like receptor [251. It has alsobeen suggested by others that, in addition to one spermprogesterone receptor involved in Ca2+ influx, there is an-other involved in Na + influx [26].

The goal of the present study was to use, as a probe forhuman sperm progesterone plasma membrane receptor(s)involved in the progesterone-initiated AR, a monoclonal an-tibody (mAb), C-262, which recognizes the C-terminal tailof intracellular progesterone receptors and blocks bindingof progesterone to those intracellular receptors [27]. Theability of C-262 to block progesterone binding, togetherwith the fact that the C-terminal tail is 100% conserved be-tween intracellular progesterone receptors from differentspecies (reviewed in [27]), suggests that the mAb epitopecontains determinants that directly contact progesterone.We hypothesized that although a sperm membrane receptoris not likely to be related to members of the intracellularreceptor family, the two types of proteins may share se-quence homologies in their progesterone-binding sites suchthat mAb C-262 would cross-react with a membrane recep-tor and block its binding to progesterone. Therefore, in thepresent study we have investigated by Western blotting andimmunofluorescence the ability of C-262 to detect a specificprotein in human sperm and to localize that protein to aspecific region in sperm. Additionally we have askedwhether C-262 could specifically block progesterone-me-diated effects at the plasma membrane of human sperm,including Ca2 + influx. Cl efflux, and, of course, the AR.

MATERIALS AND METHODS

Materials

All chemical used were reagent grade. Sodium chloride,potassium chloride, calcium chloride dihydrate, magnesiumchloride sixhydrate, methanol, glacial acetic acid, and bro-mochloroindolylphosphate/nitroblue tetrazolium (BCIP/NBT) were purchased from Fisher Scientific (Fair Lawn, NJ).BSA, glycerol, Percoll, polyvinyl alcohol (PVA), EDTA,EGTA, sodium azide, benzamidine hydrochloride, bacitra-cin, 4-2-aminoethyl benzenesulfonyl fluoride hydrochlo-ride (AEBSF), aprotinin, and pepsatatin were purchasedfrom Sigma Chemical Company (St. Louis, MO). The follow-ing electrophoresis grade chemicals were purchased fromBio-Rad (Hercules, CA): ammonium persulfate, SDS,N,N,N',N'tetramethylethylenediamine, and biotinylated mol-lecular weight standards. A seprAcryl-30 solution containing30:0.8 acrylamide:Bis was purchased from Integrated Sep-aration Systems (Natick, MA). Leupeptin was purchasedfrom Boehringer-Mannheim (Mannheim, Germany), andthe bicinchoninic acid (BCA) protein assay kit from PierceChemical Co. (Rockford, IL). FITC-rabbit anti-mouse IgGwas purchased from Zymed (San Francisco, CA); goat anti-mouse IgG conjugated to alkaline phosphatase, from Caltag(San Francisco, CA); FITC-concanavalin (ConA) lectin, fromEY (San Mateo, CA); and a mouse mAb against human trans-cortin, from Chemicon (Temecula, CA).

Mouse mAbs were raised against synthetic peptides cor-responding to the C-terminal tail steroid-binding site of thehuman intracellular progesterone receptor (C-262) [271 andthe hinge region of human intracellular estrogen receptor(h-151) (D.P. Edwards, unpublished studies), and againstthe native B form of the intracellular progesterone receptorprotein (B-30) [28]. The C-262 mAb recognizes all speciesof the intracellular progesterone receptor examined be-cause the C-terminal tail is highly conserved. The h-151mAb recognizes mouse, rat, and human full-length estrogenreceptors. The B-30 mAb recognizes a region in the uniqueN-terminal segment of the progesterone receptor B form.All mAbs were purified from mouse ascites fluids by am-monium sulfate precipitation and diethylaminoethyl-cellu-lose ion-exchange chromatography and were > 95% pure.Stock mAb solutions contained 0.02% sodium azide beforedilutions in experiments.

Preparation and Capacitation of Sperm

Human semen was obtained by masturbation fromhealthy donors. Liquefied semen samples were centrifugedat 300 X g for 20 min on a two-step Percoll gradient (80%/40%), and the sperm pellet was washed as described byThomas and Meizel [14]. This procedure yields a populationof > 95% motile sperm. The medium used for the Percollgradient and for the subsequent washing step was a mod-ified Tyrode's solution that included 25 mM NaHCO, 3 mg/

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ml BSA, metabolites, streptomycin sulfate, and penicillin G.The sperm pellets from the Percoll gradient were resus-pended and diluted to a concentration of 6 x 106 sperm/ml in the medium described above except with 26 mg/mlBSA. Mammalian sperm usually respond to AR initiatorsonly after changes, occurring in vitro or in vivo, collectivelyknown as capacitation [3]. Under conditions similar to thoseused here, progesterone initiates the AR only in capacitatedsperm [6]. To capacitate resuspended sperm, 200-pl aliquotswere incubated in 15-ml polypropylene conical centrifugetubes (Falcon Blue Max; Becton Dickinson Labware, Lin-coln Park, NJ) at 37°C for 24 h in a humid atmosphere of5% CO2:95% air (pH of medium, 7.4-7.6).

Sperm AR Studies

Sperm were preincubated at various dilutions for 5 minwith mAb C-262 (1:10, 1:100, or 1:500; final concentrationsat 150 pig/ml, 15 g/ml, and 3 ig/ml), mAb h-151 (1:10,final concentration 400 pg/ml), or mAb B-30 (150 g/ml)before a 5-min incubation with the AR initiator, progester-one (3.18 gM, final concentration) in dimethylsulfoxide(DMSO; 0.1% final concentration). Other aliquots of spermwere preincubated for 5 min with mAb diluent (PBS) beforea 5-min incubation with progesterone or its solvent control(0.1% DMSO). Aliquots (3 p.1) of sperm suspensions wereremoved for determination of the percentage of motilesperm and for subjective estimates of the quality of spermmotility [141. The rest of the sperm were then fixed, and thepercentage of acrosome-reacted sperm was determined bythe FITC-ConA method (all as previously described [291).Two hundred sperm were counted in each sample to de-termine the percentage of sperm that had acrosome-re-acted. Arcsine-transformed AR percentage data were ana-lyzed through use of the one-tailed Dunnett t-test formultiple comparisons. Values of p - 0.05 were consideredsignificantly different.

In some studies, we investigated the effect of 5-minpreincubation of human sperm with mAb C-262 (150 gg/ml) on the AR initiated by the calcium ionophore ionomy-cin. Monoclonal antibody C-262-treated and solvent con-trol-treated capacitated sperm were acrosome-reacted aspreviously described [30].

Positive Control Preparation

A cell pellet of Sf9 insect cells containing a high level ofthe A form of human progesterone receptor expressed frombaculovirus was prepared as previously described [31]. Thefrozen pellet was homogenized gently at 4°C in a Douncehomogenizer in 4 ml of a lysis buffer consisting of 10 mMTris (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, 0.4 M NaCl,10% glycerol, and a cocktail of protease inhibitors (leupep-tin, bacitracin, aprotinin, and pepsatin). The homogenatewas centrifuged at 100 000 x g for 30 min at 40C to yield asoluble supernatant that was immediately frozen at - 80°C.

SDS-Gel Electrophoresis and Western Blot Analysis

Uncapacitated human sperm were freed from seminalplasma and cellular debris by Percoll gradient centrifugationas previously described [14] except that BSA was replacedby PVA (1 mg/ml). The Percoll gradient medium also con-tained a cocktail of protease inhibitors: 1 mM benzamidine,1 mM AEBSF, 25 gM leupeptin, 1 mM EDTA, and 1 mMEGTA. Sperm (180 million) were resuspended in capacita-tion medium containing 1.33-strength Percoll gradient in-hibitor cocktail and PVA instead of BSA. Aliquots (0.75 ml)were solubilized with 0.25 ml of 4-strength SDS sample buf-fer consisting of Tris, SDS, glycerol, B3-mercaptoethanol, andbromophenol blue (final concentrations 0.33 M, 3.3%, 15%,5%, and 0.03%, respectively) and boiled for 5 min. An ali-quot was dissolved in SDS sample buffer with no [3-mer-captoethanol or bromophenyl blue for protein determina-tion by BCA kit.

Electrophoresis by SDS-PAGE under reducing conditions[32] was carried out with a mini-gel electrophoresis unit(Hoefer Scientific, San Fransisco, CA). Each lane was loadedwith 35 jig protein on 12% acrylamide gel slabs. Biotinylatedprotein standards of 6500-200 000 kDa were electropho-resed in parallel with the sperm extract. The whole cell ex-tract of Sf9 insect cells, containing very high levels of bacu-lovirus-expressed A form of human progesterone receptor,was also loaded on the gel as a positive control [31].

The separated proteins were electrophoretically trans-ferred to Immobilon-P (Millipore, Bedford, MA) membranes[331. After electrophoretic transfer, strips of membrane werecut and incubated for 5 h in a blocking solution, i.e, 3% BSATris-buffered saline-Tween 20 (TBST) solution consisting of10 mM Tris, 150 mM NaCl, and 0.05% Tween 20 (pH 7.4),and then for 20 h at room temperature with mAb C-262 or,as a negative control, with mAb h-151. The antibodies werediluted to 10 lg/ml in the blocking solution. Another gelloaded with sperm extract and serum (as a source of trans-cortin) was transferred, and the membrane was incubatedwith an antibody against transcortin as another control.

After four subsequent washes (15 min each) in 50 ml ofTBST solution without antibody, the membranes were in-cubated for 2 h with alkaline phosphatase-conjugated goatmAb against mouse IgG (used at 1:3000 in the 3% BSA-TBSTsolution). After four more rinses in TBST without antibody,the membrane blots were incubated in alkaline phospha-tase buffer and then visualized with freshly prepared BCIP /NBT; finally, the reaction was blocked with EDTA and themembranes were rinsed with water [34]. The biotinylatedstandard proteins were blocked in 1% gelatin-TBST and de-tected with avidin-alkaline phosphatase.

Human sperm extracts were also blotted in the presenceof an mAb antibody against the corticosteroid-binding glob-ulin (CBG), also known as transcortin, a protein with anapparent molecular mass of 52 kDa [28]. Procedures used

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were the same as those described for mAb C-262. No CBGimmunoactivity was detected in sperm preparations, butsuch immunoactivity was detected in the human serum pos-itive control (data not shown).

Immunofluorescence Studies

Indirect immunofluorescence localization of the proges-terone human sperm receptor was carried out with mAb C-262 and, as a control, mAb h-151. For these localizationstudies, both mAbs were used at 150 jig/ml sperm (finalconcentrations).

For experiments with fixed sperm, 200-pl aliquots of un-capacitated or capacitated sperm were suspended in 5 mlof the capacitation medium without BSA and centrifuged at300 x g for 10 min, and after removal of supernatant thesperm were fixed and permeabilized with 95% ethanol for15 min at 4°C. After fixation, sperm were centrifuged at 300X g for 10 min; after removal of supernatant, they wereresuspended in 200 pl1 of 10 mM Tris/150 mM NaCl (pH 7.4)containing 0.1% (v:v) Triton X-100 and 3 mg/ml BSA andincubated at room temperature for 2 h with mAb C-262 orcontrol antibody. For experiments with live sperm, unca-pacitated human sperm were resuspended (2 00 -pl aliquotsin 15-ml polypropylene centrifuge tubes) in the capacitationmedium except that the BSA concentration was 3 mg/ml.Sperm suspensions were then incubated with mAb C-262or control antibody for 24 h at 370C in a humidified 95%air:5% CO 2 atmosphere.

Live or fixed sperm were then washed with 5 ml of 10mM Tris/150 mM NaCl (pH 7.4) by centrifugation (300 x gfor 10 min) to remove excess primary antibody and wereincubated in the presence of FITC-labeled rabbit anti-mouseIgG (diluted 1:100 in PBS containing 3 mg/ml BSA to a finalconcentration of 3.75 pg/ml) for 2 h at 37°C (live) or at roomtemperature (fixed). As another control, duplicate spermpreparations were also incubated with the FITC-labeled sec-ond antibody only. After a final resuspension in 5 ml of 10mM Tris/150 mM NaC (pH 7.4) and centrifugation at 300 Xg for 10 min to remove excess secondary antibody, fixed orlive sperm samples were adhered onto a 10-well Teflonmasked microscope slide coated with cell-Tak (CollaborativeBiomedical Products, Bedford, MA). A small amount of FITC-Guard was added prior to fluorescence microscopy. Cellswere observed at 1250 x magnification with immersion oilby means of an Olympus BH-RFL-W fluorescence micro-scope (Olympus Corporation of America, Burlingame, CA).

Ca 2t Studies

Ca2 + was assayed according to the procedures of Thomasand Meizel [14] as modified by Meizel and Turner [29]. Briefly,sperm were capacitated in 500-pl aliquots at 6 million cells/ml for 24 h at 37°C in a humidified 95% air:50% CO2 atmo-sphere in the capacitation medium containing 26 mg/mlBSA. Aliquots of capacitated sperm were pooled (2 ml /tube).

Fura-2/Am was then added to the capacitated sperm sus-pension at a concentration of 1 pM, and the sperm were in-cubated for an additional 30 min at 37°C. Each 2-ml samplewas then centrifuged through 40%0/ Percoll at 300 x g for 20min, then washed and resuspended in a Hepes-buffered me-dium containing 10 mM HCO 3 and equilibrated in a fluo-rometric cuvette at 370 C. Monoclonal antibody C-262 or con-trol mAb h-151 was added to the suspension (finalconcentrations of 150 and 400 pg/ml), and the cells wereincubated for an additional 5 min at 37°C before the additionof progesterone. Before Ca2 + measurements, the percentageof motile sperm was determined and the quality of motilityrated by means of subjective estimates [30].

Fluorescence caused by the binding of Ca2+ to Fura-2was monitored by means of an Hitachi F-2000 (Hitachi In-struments Inc., San Jose, CA) spectrofluorometer with ex-citation wavelengths of 340 nm and 380 nm and an emissionwavelength of 510 nm [29]. Excitation and emission band-pass widths were each 10 nm. The data were corrected forautofluorescence [16]. [Ca2'+] was computed as previouslydescribed [29] using a Kc of 285 nM for Fura-2 at 37°C. Datawere analyzed through use of the one-tailed Dunnett t-testfor multiple comparisons. Values of p - 0.05 were consid-ered significantly different.

Immediately after the [Ca2+]i assay, aliquots (200 pl) ofthe remaining Fura-2-loaded sperm were used for AR stud-ies. Fura-2-loaded sperm were preincubated with proges-terone receptor mAb C262, estrogen receptor mAb H151,or the solvent control for 5 min; then progesterone or thesolvent control was added for 5 min. Cells were fixed andassayed for the AR as described above.

Cl Studies

The membrane-permeable Cl -sensitive probe, 6-meth-oxy-N-ethyl-1-2 dihydroquinoline (dihydro-MEQ), was syn-thesized from MEQ according to a method developed byBiwersi and Verkman [35] and modified by Turner and Meizel[36]. The dihydro-MEQ was diluted to 50 mM with DMSO.Capacitated sperm were loaded with dihydro-MEQ (50 pM)by incubation for 1 h in a Hepes-buffered medium containing10 mM HCO 3 and Mg 2 + and Ca '2 as gluconate salts. Spermwere then washed by Percoll gradient centrifugation. Aftercentrifugation, sperm were counted, diluted to 10 million/ml with the Hepes-buffered medium, protected from ex-posure to light, and equilibrated in a fluorometric cuvette.Monoclonal antibody C-262 or control mAb h-151 was addedto the suspension (final concentrations of 150 and 400 pg/ml, respectively), and the cells were incubated for an ad-ditional 5 min before addition of progesterone.

After loading, dihydro-MEQ is oxidized to MEQ by intra-cellular enzymes [35]. MEQ exhibits minimum compartmen-talization in capacitated human sperm (approximately 8%,[36]). The fluorescence of MEQ is quenched by a collisional

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mechanism with C1- [351, and therefore, decreased intra-cellular Cl- would be indicated by increased fluorescence.Data were analyzed through use of the one-tailed Dunnettt-test for multiple comparisons. Values of p - 0.05 wereconsidered significantly different.

Immediately after the Cl- assay, sperm capacitation wastested by determining the ability of the remaining MEQ-loaded sperm to undergo the AR. Aliquots (200 I1) of theremaining MEQ-loaded sperm were preincubated with mAbC-262 or control mAb h-151 or with the solvent control; thenprogesterone or the solvent control was added for 5 min.Cells were fixed with 4% formaldehyde and assayed for theAR with FITC-labeled ConA as described above. Aliquots ofsperm suspension were evaluated for motility and quality.

RESULTS

Effect of mAb C-262 on the AR

The results presented in Figure 1 show that mAb C-262inhibited the progesterone-initiated AR in a dose-dependentmanner. Maximum inhibition was 77% at the highest con-centration used (1:10, final concentration 150 pg/ml). Onthe other hand, the estrogen receptor mAb h-151 did notinhibit the progesterone-initiated AR. In addition, mAb C-262 had no effect on the AR when added in the absence ofprogesterone (Fig. 1). Moreover, another mAb, B-30, whichrecognizes an epitope in the N-terminus of the intracellularprogesterone receptor and does not affect progesteronebinding, was tested in AR experiments but had no effect onthe human sperm AR in the absence or the presence ofprogesterone (data not shown). Thus, inhibition of the pro-gesterone-initiated AR was detected only with the mAb (C-262) that binds to the C-terminal tail of the intracellular pro-gesterone receptor and is known to block progesteronebinding in that receptor. Furthermore, mAb C-262 did notinhibit the ionomycin-induced human sperm AR (data notshown). In all these AR experiments, motility was 70-80%after additions, and there was no difference between con-trol and experimental motility (percentage AR and subjec-tive quality estimate). Thus, we can assume that differencesin AR were not due to sperm death.

Effect of mAb C-262 on Ca2+ Influx

Figure 2 is representative of three experiments showingthat progesterone stimulated a rapid, transient increase inhuman sperm Ca 2+ , as reported previously [15], and thatpreincubation of the sperm with mAb C-262 but not mAbh-151 inhibited that increase. Table 1 summarizes the resultsof all the Ca + studies. When the sperm were preincubatedwith mAb C-262 before addition of progesterone, there wasa 56% inhibition of the progesterone-mediated Ca 2+ influx.At the same time, the AR was inhibited by 65% (Table 1).On the other hand, the control mAb h-151 did not signifi-

FIG. 1. Effect of mAbs against steroid receptors on AR initiation by progesterone.Human sperm were incubated for 5 min with progesterone receptor mAb C-262 atdifferent dilutions (1:10, 1:100, or 1:500; final concentrations 150, 15, and 3 gig/ml)or with estrogen receptor mAb h-151 (diluted 1:10, final concentration 400 g/ml)prior to addition of progesterone (P, 3.18 pM), or with solvent control (SC). *, Sig-nificantly different from progesterone, p - 0.01 (n = 3). Sperm motility, determinedat the end of the experiments, was 70-80% but was not different between treat-ments and control in any single experiment.

Progesterone

1500 r -C-262+Progesterone1500 r-

1000

+

50

0

I

4,

0 20 40 60 80 100 120

Time (sec)

FIG. 2. Effect of mAbs against steroid receptors on progesterone-mediated Ca2*

influx. Fura-2-loaded capacitated human sperm were preincubated for 5 min withsolvent control or with progesterone receptor mAb C-262 (150 pg/ml), and proges-terone or solvent control was then added. Traces are from one experiment repre-sentative of three similar separate experiments (results are summarized in Table1). Sperm motility, determined at the end of the experiments, was 65-85% and wasnot different between treatments and control in any single experiment. Sampleswere incubated at 37°C, and sperm concentration was 10 x 106 sperm/mi. Arrowindicates time of progesterone addition.

-

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450

a 400

t 350

U 300

250

200

X 150

100

Progesterone......... C-262+Progesterone-- Solvent Control I

10 15 20 25 30 35 40Time (sec)

FIG. 3. Effect of mAbs against steroid receptors on progesterone-mediated CIefflux in human sperm. Cl quenches MEQ fluorescence, so an increase in fluores-cence means a decrease in cytosolic CI . Capacitated MEQ-loaded sperm werepreincubated for 5 min with solvent control or progesterone receptor mAb C-262(150 pg/ml), and then progesterone or solvent control was added. Fluorescence (inrelative units) was determined at 37°C, and sperm concentration was 10 x 106sperm/ml. Data are representative of three separate experiments. Sperm motility,determined at the end of the experiments, was 60-80% and was not different be-tween treatments and control in any single experiment. Arrow indicates time ofprogesterone addition.

cantly reduce the progesterone-mediated Ca2 + increase (Ta-ble 1). Motility was 65-85% but was the same for all sampleswithin a single experiment.

Effect of mAb C-262 on CI- Flux

The results shown in Figure 3 are representative of threeexperiments concerned with the qualitative cytosolic Clchanges observed when progesterone was added to MEQ-loaded capacitated sperm. The results confirm earlier stud-ies [36] showing that progesterone (3.18 M) stimulates a

TABLE 1. Effects of mAbs against steroid receptors on progesterone-initiated in-creases in [Ca2+]i and the AR in human sperm.

Treatments a'b Mean peak [Ca 2l]i (nM)+SEM Mean % AR+ SEM

Solvent Control 200+36e 10.2+0.9e(0.05% DMSO)Progesterone 1000+_112 24.9 2.4(3.18 M)mAb C-262 551 + 18e 15.3 +

1.3e

mAb h-151d 849+ 11 21.9 + 1.5

an=Three different experiments with capacitated human sperm.bFura-2-loaded sperm were fixed at the end of the experiment, and the AR wasassayed with FITC-ConA. Sperm motility was 65-85% and was the same for all sam-ples within a single experiment.CMonoclonal antibody against progesterone-binding region of human, chicken, rab-bit, and mouse intracellular progesterone receptor.dMonoclonal antibody against the peptide hinge region of human and mouse in-tracellular estrogen receptor.eValues are significantly different from value for progesterone, p < 0.05.

FIG. 4. Western immunoblot of human uncapacitated sperm extract after SDS-gelelectrophoresis. Lane a: Biotinylated protein standards (stained with avidin-alkalinephosphatase); lane b: extract of uncapacitated human sperm (35 ig/protein perlane) blotted with the progesterone receptor mAb C-262 (10 pg/ml) and a goat anti-mouse IgG mAb conjugated to alkaline phosphatase; lane c: extract of Sf9 insectcells containing baculovirus-expressed A form of the intracellular human proges-terone receptor blotted with progesterone receptor mAb C-262 and a goat anti-mouse IgG mAb conjugated to alkaline phosphatase. Numbers refer to molecularmass values (kDa) based on protein standards.

rapid transient decrease in cytosolic Cl (an increase in flu-orescence means a decrease in cytosolic Cl as discussedin Materials and Methods). Moreover, the results in Figure3 show that 5-min preincubation of capacitated sperm withmAb C-262 inhibited the progesterone-mediated cytosolicC1- decrease by 95%. However, there was some inhibitionof the C1- efflux (30%) in the presence of control mAb h-151 (data not shown). Motility was 60-80% but was thesame for all samples within a single experiment.

Western Immunoblot

Western immunostaining after SDS-gel electrophoresiswas carried out with human sperm extracts in order to de-termine the apparent molecular masses of the immunoreac-tive polypeptides detected by mAb C-262. The Western blotimmunostaining (Fig. 4) revealed a major band with an ap-parent molecular mass of 50-52 kDa when the proteaseinhibitors were added as early as in the Percoll gradient. Aminor band of 46 kDa was also present. We also detectedanother band of lower molecular mass (27 kDa) when theprotease inhibitors were not present during Percoll gradientcentrifugation but were added only prior to sperm extrac-tion. No stained bands appeared in controls when thesperm extracts were blotted with control mAb h-151 orwhen the blots were incubated with the alkaline phospha-tase-conjugated antibody only (data not shown).

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As a positive control for mAb C-262, this mAb was usedfor Western immunoblotting of a whole cell extract of Sf9insect cells containing a high level of baculovirus-expressedA form of the intracellular human progesterone receptor.Figure 4 shows C-262 immunoreactivity with a single 94-kDa band that corresponds to the A form of the intracellularprogesterone receptor [31]. Thus, mAb C-262 both recog-nizes the authentic A form of the intracellular progesteronereceptor and cross-reacts with a human sperm protein of 52kDa.

Indirect Immunofluorescence Localization

The localization in human sperm of immunoreactivityagainst mAb C-262 was carried out by indirect immunoflu-orescence. Live sperm incubated for 24 h with the antibody,ethanol-fixed uncapacitated sperm, and ethanol-fixed ca-pacitated sperm exhibited the same labeling pattern (Fig. 5shows the pattern with a fixed capacitated sperm). Many(45 - 50%) of the sperm displayed a distinct fluorescent bandat the same site in the sperm head at the equatorial segmentregion of the acrosome (arrow, Fig. 5). No fluorescent bandor other localized staining was detected with fixed or livesperm when sperm were incubated with the control mAbh-151 or with the FITC-labeled second antibody only. Someof the fixed or live sperm displaying equatorial segmentfluorescence with C-262 also displayed fluorescence in themost posterior region of the head. However, the latter wasalso detected in some sperm that had been incubated withcontrol mAb h-151 and probably represents nonspecific an-tibody binding. Most of the fixed or live sperm in theselocalization experiments were acrosome-intact (90% whenassayed with ConA).

DISCUSSION

The results presented here strongly support the involve-ment of a sperm plasma membrane receptor in the proges-terone-initiated AR. We were able to show that mAb C-262against the C-terminal steroid-binding domain of the mam-malian intracellular progesterone receptor inhibited theprogesterone-initiated AR of human sperm. Such an anti-body would not enter the cells, since sperm do not exhibitendocytosis, and thus must be acting on a receptor on thesperm plasma membrane. The existence of a human spermplasma membrane progesterone receptor was supported bythe following earlier findings: 1) progesterone that was co-valently bound to BSA, a molecule that does not enter thesperm, initiated the AR and an increase in human spermCa2+ [17, 18]; 2) the addition of a mAb against progesteroneafter addition of suboptimal levels of progesterone stimu-lated rapid Ca 2+ influx and the AR, as a result of receptor-mediated receptor aggregation [19]; 3) FITC-labeled BSA-progesterone labeled sites in the heads of some viablehuman sperm [20, 21].

FIG. 5. Indirect immunofluorescence localization of the progesterone receptors)in human sperm. Capacitated sperm previously fixed in 95% ethanol for 15 min at4C were incubated for 2 h with 150 pg/ml of progesterone receptor mAb C-262.Sperm were then incubated with FITC-labeled rabbit anti-mouse IgG for 2 h. Many(45-50%) sperm displayed immunoreactivity as a band on the sperm head (arrow).x 2500.

Our Western immunoblot studies under reducing con-ditions with Mab C-262 detected a single major immunoblotband of 50-52 kDa, a minor band at 46-48 kDa, and severalbarely visible bands of lower apparent molecular masswhen protease inhibitors were added to the uncapacitatedsperm extract as early as in the Percoll gradient. When theprotease inhibitors were added at a later time or in a lowerconcentration, we also detected a lower-molecular-massband of 27 kDa. This band probably represents proteolysisof the 50-52-kDa protein. The apparent molecular mass ofthe 50-52-kDa protein is different from that of the A and Bisoforms of the human intracellular progesterone receptor,94 and 120 kDa, respectively [27, 28, 31], and from that ofa human seminal plasma progesterone-binding protein(90-100 kDa) found associated with sperm [37]. Indeed, ourWestern blot results confirm recent immunofluorescencestudies indicating that ejaculated human sperm do not con-tain the intracellular progesterone receptor [38]. If our hy-pothesis is correct, we would not expect the membrane re-ceptor to be structurally related to the intracellular receptorexcept for the progesterone-binding site.

We suggest that the 50-52-kDa protein detected withmAb C-262 is a sperm plasma membrane progesterone re-ceptor. The 46-48-kDa minor band may be a degradationproduct of the 50-52-kDa molecule or a second type ofplasma membrane progesterone receptor. While the West-ern immunoblot studies were carried out with extracts fromthe entire sperm, the proteins detected with mAb C-262were very probably membrane proteins. Two other results

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obtained with this membrane-impermeable antibody sup-port this conclusion. First, immunofluorescence localizationwith the antibody were the same whether live and intact orfixed and permeabilized sperm were used. Second, the an-tibody did act at the membrane to inhibit the AR.

The 50-52-kDa protein detected by mAb C-262 has anapparent molecular mass quite different from those of twoother human sperm proteins implicated in the progester-one-initiated AR, a 220-kDa sperm surface antigen [22] anda 94-kDa protein [23]. A mAb against the 220-kI)a surfaceantigen blocks the progesterone-initiated AR and proges-terone-mediated Ca 2+ influx [22, 39]. The 94-kDa proteindisplays progesterone-mediated tyrosine phosphorylation[23]. It has not been determined whether either the 220-kDaor the 94-kDa protein is a progesterone receptor.

CBG, a serum glycoprotein of 52 kDa that binds proges-terone as well as cortisol with high affinity [28], is also pres-ent in human seminal plasma [40]. However, since we didnot detect any immunoreactivity with the mAb against CBGin washed sperm, the 50-52-kDa band detected in suchsperm by mAb C-262 is not CBG.

It has been suggested that progesterone may bind di-rectly to a unique steroid receptor/C1- channel complex inhuman sperm that resembles, but is not identical to, theneuronal GABAA receptor/C1- channel, thereby activatingthe channel [24]. A GABAA-like receptor/C1- channel hasalso been reported to be involved in the mouse sperm ARinitiated by progesterone [8, 41]. Thus, it is of interest thatmAb C-262 detected a 50-52-kD)a protein and that a proteinof similar apparent molecular mass was also detected inimmunoblots of human sperm with use of an antibodyagainst the bovine cerebral cortex GABAA a subunit [24].Whether the 50-52-kDa protein detected by mAb C-262and that detected by the mAb against the GABAA a subunitare identical remains to be determined.

Our immunofluorescence studies with mAb C-262 local-ized immunoreactivity in a narrow region in the spermhead, presumably overlying the equatorial segment, in 45-50% of live and fixed capacitated or uncapacitated sperm.Some fixatives do not always prevent antigen diffusion orantibody-induced aggregation of antigen [42], but it is un-likely that either possibility is the explanation for the loca-tion of the immunoreactivity seen with mAb C-262, sincethe same results were obtained with live sperm as withsperm fixed in ethanol at 4°C.

I)etection of the same immunoactive sites with bothfixed and unfixed sperm suggests that those sites representplasma membrane location of the sperm progesterone re-ceptor(s). Interestingly, its localization (i.e., apparently theplasma membrane overlying the equatorial segment) is thesame as that previously reported for human sperm with useof an antibody against the bovine cerebral cortex GABAA asubunit [24].

It is not clear why only 45-50% of the sperm displayedimmunoreactivity with mAb C-262, but these percentages

of labeled sperm are greater than those reported by otherlaboratories using a conjugate of FITC-labeled BSA-proges-terone to localize progesterone receptor sites in the humansperm head plasma membrane. Blackmore and Lattanzio[20] reported that FITC-labeled BSA-progesterone conjugatelabeled the entire head plasma membrane or the periacro-somal plasma membrane region in 30% of live, presumablyuncapacitated sperm. Mendoza and Tesarik [43] reportedthat the FITC-labeled conjugate labeled the periacrosomalplasma membrane of 10% of live and intact capacitatedsperm and the plasma membrane over the equatorial seg-ment in acrosome-reacted sperm. In our studies, at least90% of the sperm were acrosome-intact. It has been sug-gested that the low number of sperm that bound FITC-la-beled BSA-progesterone may be due to the possibility thatonly capacitated sperm display the receptor [43]. However,in our studies, the percentages of uncapacitated and capac-itated sperm with immunoreactivity to mAb C-262 were thesame.

Presumably mAb C-262 inhibited the AR by blocking thebinding of progesterone to the receptor(s), and we there-fore wished to determine whether or not that inhibition isassociated with an inhibition of Ca2' and/or Cl flux. Anextracellular Ca2+-dependent increase in [Ca2+]i is requiredfor the initiation of the AR by physiological factors includingprogesterone [14, 15, 44]. Recently, Turner and Meizel [36]used the fluorescent intracellular C1 probe, MEQ, to di-rectly detect qualitative changes in sperm cytosolic Cl- dur-ing the progesterone-initiated AR. In those studies, it wasshown that the steroid mediated a rapid transient decreasein human sperm cytosolic C1 and since that decrease wasinhibited by the GABAA receptor/C1 channel blocker pic-rotoxin, it was assumed that this decrease was due to (1efflux via a GABAA-like receptor/Cl channel. The rapidtime courses of the progesterone-mediated transient in-crease in [Ca'2+] and the decrease in cytosolic Cl shown inFigures 2 and 3 (both < 30 sec) agree with those reportedearlier for the former [6, 15, 16] and the latter [361 and alsowith that reported for AR initiation by progesterone [6]. Thepresent results demonstrate that mAb C-262 inhibited by50% the progesterone-mediated rise in Ca required for theAR while the mAb against the estrogen receptor inhibitedthe rise by only 15% (apparently a nonspecific effect prob-ably attributable to the high concentration of mAb h-151used). In addition, mAb C-262 inhibited the progesterone-stimulated C efflux by 90%, while the control antibodyinhibited it by only 30% (again, an apparently nonspecificeffect). It should be noted that those lower inhibitions ofCa 2 ' and Cl flux by the control mAb did not result in ARinhibition. The larger inhibitions of ion flux by mAb C-262support the view that these events are mediated by spermplasma membrane receptors.

Our present results do not allow us to distinguish be-tween the binding of progesterone to more than one typeof sperm receptor and binding to only one type followed

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by putative receptor cross talk. Nevertheless, the presentstudies provide compelling evidence for the existence ofhuman sperm plasma membrane progesterone receptorsand have detected at least one strong candidate, a 50-52kDa-protein, for such a receptor. Isolation and further char-acterization of the candidate receptor protein detected bymAb C-262 will be carried out in this laboratory.

NOTE ADDED IN PROOF

After acceptance of this paper, we learned of the results of Benoff et al. (Am J ReprodImmunol 1995; 34:100-115). Those authors reported that ... sera of immune infertilitypatients ... inhibited the progesterone-initiated AR of capacitated human sperm and thatprobing with horseradish peroxidase-labeled progesterone detected 70.4 + 1.2 kDa and58.5 + 2.7 kDa proteins in Western blots of human sperm preparations.

ACKNOWLEDGMENTS

We thank Ms. Jodie Casselman, Dr. John Hess, and Dr. Paul FitzGerald for their help inthe Western blot experiments.

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