Progesterone and RU486: Opposing effects on human sperm (capacitation/hyperactivation/acrosome reaction/intracellular calcium concentration)

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 91, pp , Janary 1994 Medical Sciences Progesterone and RU486: Opposing effects on hman sperm (capacitation/hyperactivation/acrosome reaction/intracelllar calcim ncentration) JING YANG*, CATHERINE SERRES*, DANIEL PHILIBERTt, PAUL ROBELt, ETIENNEEMILE BAULIEUU, AND PIERRE JOUANNET* *Laboratoire d'histologieembryologiecytog6n6tiqe, Faclt6 de M6decine, Universite ParisSd, 9427 KremlinBicetre, France; tcentre de Recherche RosselUCLAF, 9323 Romainville, France; and WInstitt National de la Sante et de la Recherche Medicale, Unit6 33, KremlinBicetre, France Contribted by EtienneEmile Balie, September IS, 1993 ABSTRACT Progesterone indced a rapid inflx of calcim in capacitated hman sperm, followed by a longlasting, dosedependent increase of intracelllar free calcim. Thereafter, progesterone increased the fraction of hyperactivated sperm and the acrosome reaction. On the ntrary, the progesterone antagonist RU486 (mifepristone) indced an immediate and transient, dosedependent decrease of intracelllar free calcim and a drop in the vales of sperm movement parameters related to hyperactivation. Moreover, RU486 nteracted the effects of progesterone on calcim inflx, lateral sperm head displacement, and the acrosome reaction. Therefore, RU486 effects were opposite to those of progesterone. The natre of the membrane receptor(s) involved is nknown. Several steroids bearing 11(phenyl sbstittions, with different pharmalogical profiles, were also investigated. It was nclded that the steroid strctre and chemical grops added to the 11(phenyl inflence effects on calcim inflx. Capacitation leads to fnctional changes ofthe sperm that are characterized by modified movement parameters designated as hyperactivation and by exocytotic events (the acrosome reaction) (13). The acrosome reaction is an essential step in the mplex series of processes implied in Etherian fertilization, necessary for sperm penetration throgh the zona pellcida and fsion with the oocyte plasma membrane. It can be indced by folliclar flid, cmls cells (4), or zona pellcida (5). Progesterone (PROG) was identified as a major mponent of the folliclar flid for indcing the acrosome reaction in hman sperm (6), preceded by an immediate, transient calcim inflx into spermatozoa (7, 8). These rapid biological effects sggest a nongenomic mechanism and a cell srface receptor for PROG like the one responsible for the meiotic matration of Xenops laevis oocyte (9). The antiprogestin RU486 {RU38486, mifepristone, 17( hydroxy11(8[4(dimethylamino)phenyl]17apropynylestra4,9dien3one} binds with high affinity to the intracelllar PROG receptor in most vertebrate species (1). It has been reported that this potent antiprogestin has either a small or negligible inhibitory effect on PROGmediated calcim inflx into hman sperm (11, 12). In this stdy, we evalated the effect of RU486, alone or in mbination with PROG, on intracelllar free calcim ncentration ([Ca2l]i) and the movement of hman sperm and acrosome reaction, two calcimrelated events (3, 13). MATERIALS AND METHODS Materials. PROG, hman serm albmin (HSA) fraction 5, Fra2AM, Flo3AM, Hoechst 33258, floresceinlabeled Pism sativm aggltinin, verapamil, EGTA, Triton X1, sodim pyrvate, and Hepes were prchased from Sigma. The pblication sts of this article were defrayed in part by page charge payment. This article mst therefore be hereby marked "advertisement" in acrdance with 18 U.S.C solely to indicate this fact. 529 RU486, 17phydroxy11p{4[2dimethylamino)ethoxy]phenyl}17apropynylestra4,9dien3one(RU399), 11[4(dimethylamino)phenyl]17apropynylestra1,3,5(1)triene 3,17f3diol (RU41291), 1183{4[2(dimethylamino)ethoxy] phenyl}estra1,3,5(1)triene3,17,diol (RU39411), rtisterone, testosterone, estradiol, and 17amethylnorpregna 4,9diene3,2dione (R52) were kindly provided by RosselUCLAF. Ionomycin (Iono) was prchased from Calbiochem. All other chemicals (e.g., salts for bffers) were prchased from Merck. Sperm Preparation. Hman semen was obtained from healthy donors. Motile spermatozoa were selected by centrifgation throgh a twostep (47.595%) miniperll gradient and resspended in a hypertonic BWW medim (14) ntaining 166 mm NaCl, 5 mm KCl, 1.3 mm CaCl2, 1.2 mm KH2PO4, 1.2 mm MgSO4, 5.5 mm glse,.25 mm sodim pyrvate, 21 mm sodim lactate, 25 mm NaHCO3, 2 mm Hepes, and.8% HSA [BWW/HSA(+)] (41 mosmoles/ liter, ph 7.4 at room temperatre). Measrement of Intracelllar Calcim. Measrements were performed on selected sperm kept at least 2 hr in capacitating medim BWW/HSA(+), nless otherwise stated. Cells (51 x 16 per ml) were incbated with Fra2AM (2,M final ncentration) at 37 C, for 45 min. After washing (6 x g, 1 min) in BWW withot HSA (BWW), 4 x 16 cells per ml were resspended in BWW. Florescence signal was rerded at 37 C sing a spectroflorometer at excitation wavelengths of 34 nm and 38 nm (PTI M21) (Kontron, Zrich) or at excitation wavelengths of 34 nm, 36 nm, and 38 nm (Hitachi F2) (Bran Science Tec, Les Ulis, France). Florescence emission was monitored at 55 nm. Either PROG or the steroid hormone rtisterone, testosterone, or estradiol, the progestin R52, or the 11pphenylsbstitted steroid RU486, RU399, RU41291, or RU39411 dissolved in absolte ethanol was added to the incbation medim at.1% final ethanol ncentration. At the end of each assay, 5,M Iono was added to the sample to measre the maximm signal of florescence; then sperm was permeabilized with.5% Triton X1 and depleted of calcim by the addition of 1 mm EGTA (ph 9.5) to measre the minimm signal of florescence. These vales were sed to calclate [Ca2+l acrding to Grynkiewicz et al. (15). Abbreviations: PROG, progesterone; R52, 17amethylnorpregna 4,9diene3,2dione; RU38486 (RU486, mifepristone), 17,8 hydroxy113[4(dimethylamino)phenyl]17apropynylestra4,9 dien3one; RU399, 173hydroxy11{4[2(dimethylamino)ethoxy]phenyl}17apropynylestra4,9dien3one; RU39411, 11f{4[2 (dimethylamino)ethoxy]phenyllestra1,3,5(1)triene3,17,bdiol; RU41291, 11,B[4(dimethylamino)phenyl]17apropynylestra1,3, 5(1)triene3,17pdiol; [Ca2+]i, intracelllar free calcim ncentration; HSA, hman serm albmin; Iono, ionomycin; ECso, halfmaximally effective ncentration; VSL, straight line velocity; VCL, crvilinear velocity; LIN, linearity; ALH, amplitde of lateral head displacement; MOT, motile cells. To whom reprint reqests shold be addressed.

2 53 Medical Sciences: Yang et al. Movement Analysis. After capacitation in BWW/HSA(+) nder 5%CO2 in air at37 C for 2 hr and incbation at37 C for 2 min with 31.4,M PROG, 23.4,M RU486, or,m 1 Iono, sperm samples were deposited into capillary tbes of 2 A&m inner depth. Sperm movement was analyzed at37 C with a mpterized atomatic system (HT M23) (Hamilton Thorn Research). The rate of frame acqisition was 25 Hz. The parameters, measred acrding to the international terminology (16), on at least 1 motile cells were (i) the straight line velocity (VSL) evalated as the linear distance vered by the sperm dring 1(,m/s); s (ii) the crvilinear velocity (VCL) evalated as the actal distance vered by the head of sperm dring (,m/s); 1 s (iii) the linearity (LIN), which is the VSL/VCL ratio x 1; (iv) the amplitde of the lateral head displacement (ALH) expressed,m; in and (v) the percentage of motile cells (MOT). Finally, the sort fraction was calclated as the percentage of sperm with VCL > 1,m/s, ALH > 6,m, and LIN <6 and nsidered as hyperactivated cells (modified from Robertson et al.) (17). Acrosome Reaction. After capacitation in BWW/HSA(+) at37 C, nder 5% CO2 in air for 22 hr, sperm (6 x 16 per ml) were incbated with steroids at37 C, for 3 min. Iono (1,M) dissolved in dimethyl slfoxide (.5% final ncentration) was sed as positive ntrol. The acrosome stats and viability of sperm were determined acrding to Cross et al. (18). The amont of live sperm that nderwent acrosome reaction (acrosome reacted sperm) was expressed in percent of total sperm nmber. At least 2 sperm were evalated in each experiment. Statistical Analysis. Reslts were expressed as mean ± SEM. A paired t test was sed for statistical evalation. RESULTS Effects of Steroids on [Ca2+]J. In a first set of experiments, we observed that PROG indced a transient increase of [Ca2+],, followed by a send phase when the level of [Ca2+], remained slightly elevated above the basal level within the sal observation time (1 min) (Fig.1A) and even after 3 min. The effect of PROG on [Ca2+], was dose dependent (Fig. 1B). The maximm response was obtained with 1,M PROG, and the halfmaximally effective ncentration (EC5) was abot 3 nm. The increase of [Ca2+]i indced by PROG (1 AM) was inhibited when sperm were preincbated with 1 mm verapamil for 2 min (Fig. 1B). Under the same experimental nditions, the steroid hormones rtisterone, testosterone, and estradiol and the synthetic progestin R52 added at 1,M ncentration also indced transient increases of [Ca2+]i of 63.3% ± 5.3%, 46.3% ± 8.8%, 43.6% ± 6.3%, and 3.4% ± 4.1%, respectively, of the increase prodced by PROG (mean ± SEM, n = 5). PROG (1 M) added 2 min after these steroids prodced a frther transient increase of [Ca2+]i, althogh it was smaller than that prodced by PROG alone. Conversely, there was no frther increase in [Ca2+] when either PROG or the other steroids (1,M) were added 2 min after PROG (1,M) (data not shown). When RU486 was added to the sperm sspensions nder the same experimental nditions, the florescence signal decreased at 34 nm, increased at 38 nm, and did not change at 36 nm (Fig. 2). Acrding to Grynkiewicz et al. (15), sch florescence changes characterize a decrease in [Ca2+]i. The florescence signal of 1 AM nesterified Fra2 added to a ntrol soltion, mimicking internal medim and ntaining no calcim, was not modified by RU486 (data not shown). The decrease of the florescence signal was maximm 15 s after addition of RU486 (Fig. 3A) and was dosedependent (Fig. 3B). The maximm decrease of [Ca2+], prodced by RU486 (1,M) was 57.5% ± 3.8% (mean ± SEM, P <.1, n = 11) of the basal level. With the 1,LM RU486 dose, a parasite effect, increasing the florescence signal at 34 nm and 38 nm, was Q r 6 A 5oof 4[ A iproc(1lm) / I1 Time, min C Log molar PROG FIG. 1. PROG increases [Ca2+]i in hman sperm. The sperm were loaded with Fra2 (see text). PROG was dissolved in absolte ethanol and added to the incbation medim (.1% fmal ethanol ncentration);.1% ethanol has no effect on [Ca2+]1i. The florescence signal was measred with a PTI M21 spectroflorometer. (A) Time rse of the effect of 1,M PROG on [Ca2+]i. (B) Doseresponse crves of [Ca2+]i to PROG [C, ntrol level (1%6) in absence of PROG]. The peak of [Ca2+]i (o) and the calcim level after 1 min (o) were rerded (mean ± SEM, n = 3). After preincbation with verapamil (1 mm) for 2 min, the peak of [Ca2+]i prodced by 1,M PROG was abolished (A). sperimposed on the signal provided by the decrease of [Ca2+]j. At later times, [Ca2+]i retrned to the basal level for dosec 1,M, whereas it remained nder basal level at doses > 1,M within the 1min observation time (Fig. 3A). The decrease of [Ca2+]j indced by RU486 was nfirmed in hman sperm loaded with another calcim indicator, 1,M Flo3 AM, at 56 nm excitation wavelength and 526 nm emission wavelength (Hitachi, F2). Preliminary reslts were also obtained with three analogs of RU486. RU399 differs from RU486 only by the tamoxifenlike side chain at position 4 of the llphenyl. It is also an antiglrtisteroid and a potential antiprogestin (Table 1) (19). RU41291 has the same 11 3 and 17a side chains as RU486 bt is a phenol steroid like estrogen. It is a potential antiglrtisteroid c a Proc. Natl. Acad Sci. USA 91 (1994) I I 34nm 36 nm 38nm v4 cd I RU486 1 min FIG. 2. Characterization of [Ca2+]i changes indced by RU486 in hman sperm. Spermatozoa (4 x 16 per ml) were capacitated in BWW/HSA(+) and then resspended in BWW and loaded with Fra2AM at 2,M final ncentration. The florescence signal was measred with excitation wavelengths at 34 nm, 36 nm, and 38 nm and emission wavelength at 55 nm (Hitachi F2). The final ncentration of RU486 was 2.3,M in.1% ethanol.

3 Medical Sciences: Yang et al. t II41 I C " log molar RU486 FIG. 3. RU486 decreases [Ca2+]i in hman sperm. RU486 was added to Fra2loaded spermatozoa. The florescence signal was measred with a PTI M21 spectroflorometer. (A) Time rse of the effect of RU486 (1 AM) on [Ca2+]i. (B) Doseresponse crve of [Ca2+]i to RU486 [C, ntrol level (1lo) in absence of RU486]. The peak of [Ca2+]i (e) and the calcim level after 1 min (o) were rerded (mean + SEM, n = 4). and antiprogestin. RU39411 has the steroid strctre of estradiol with the 11j side chain of RU399. It is an antiestrogen with slight estrogen agonist activity (222). At 1,M ncentration, RU399 and RU39411 decreased [Ca2+]1 to 47.3% ± 4.6% and 57.7% ± 5.7%, respectively, of basal level, whereas RU41291 increased [Ca2+]i to 248.3% 36.1% of basal level (mean ± SEM, n = 3). The effect of PROG was again tested after a 2min preincbation interval with RU486. At the eqimolar ncentration of 1,M, RU486 significantly inhibited the PROG response by 59.7% ± 11.5% (mean ± SEM, P <.2, n = 7) (Table 2). After a 1min preincbation, RU486 at ncentrations of.1,.1, and 1,M did not nteract the stimlation prodced by PROG when it was incbated at.1,m ncentration, which already increases [Ca2+]j by 3fold as shown in Fig. 1B. Only at 1,M did RU486 significantly decrease the effect of.1,m PROG by 69.% ± 13.8% Table 1. Relative binding affinities (%) of RU38486, RU399, RU41291, and RU39411 for intracelllar steroid hormone receptors Compond PR GR MR AR ER RU <.1 25 <.1 RU <.1 RU < <.1 RU < The relative binding affinities of test mponds were determined for the following cytosol receptors: progesterone receptor (PR) of rabbit ters, glrtisteroid receptor (GR) of rat thyms, mineralortisteroid receptor (MR) of rat kidney, androgen receptor (AR) of rat ventral prostate, and estrogen receptor (ER) of rat ters. The radioactive reference steroids were PROG, dexamethasone, aldosterone, testosterone, and estradiol. Their affinity for the rresponding receptor was taken as 1%7. Cytosol samples were incbated with the relevant radioligand in the presence of increasing ncentrations of nonradioactive reference or test steroids, at C for 24 hr (except in the case of ER, which was incbated at 25 C for 5 hr). B 5 Proc. Natl. Acad. Sci. USA 91 (1994) 531 Table 2. Effects of PROG and RU486, alone or mbined, on [Ca2+]1i in hman sperm Peak [Ca2li, % Treatment of basal level PROG 546. ± 28.3* RU ± 1.8* + PROG ± 14.3t* The steroids were added (1 IAtM ncentration) to Fra2loaded spermatozoa. The peak response was measred and expressed as % of basal level. For mpetition experiments, the sperm sspensions were preincbated with RU486 for 2 min before the addition of PROG. The vales are mean ± SEM of seven separate experiments. *, P <.1 verss basal; t, P <.1 verss basal; *, P <.2 verss PROG. (mean ± SEM, P <.5, n = 4) (Fig. 4). We ld not test the inhibitory effect of 1 AM RU486 becase of its parasite effect. RU486 was also tested in mbination with the other steroid hormones and progestin. Preincbation with 1 pm RU486 for 2 min also inhibited the increase of [Ca2+]1 prodced by 1,M rtisterone, testosterone, estradiol, orr52 by 55.% ± 11.9%, 26.3% ± 11.2%, 66.3% ± 25.%, and 29.7% ± 2.6%, respectively (mean ± SEM, n = 3). At the same 1,M ncentration as RU486, RU399, RU41291, and RU39411 inhibited the stimlation of [Ca2+] prodced by PROG to abot 75% of ntrol level with PROG alone (n = 3). Effect of Steroids on Sperm Movement. Since the variations in [Ca2+]1 mediated by steroids occrred in a few sends, the effects of steroids on sperm movement parameters were tested after a 2min incbation interval (Table 3). After 2 hr of capacitation, MOT, VSL, and LIN were nchanged in the presence of 31.4,M PROG, whereas VCL, ALH, and the sort fraction were significantly increased. In ntrast, MOT, ALH, and the sort fraction were significantly decreased in the presence of 23.4,M RU486. Moreover, in separate experiments, the increased vales of sperm movement parameters indced by 31.4,M PROG were partially inhibited by preincbation with 23.4,M RU486 for 1 min, althogh the only statistically significant decrease was the one of ALH (6. ±.3,m with PROG alone verss 5.5 ±.2,m with RU486 pls PROG; mean ± SEM, P <.2, n = 7). Indction of the Acrosome Reaction by Steroids. Hman sperm were capacitated for 22 hr and then incbated at 37 C for 3 min with increasing ncentrations of PROG. The percentages of live acrosome reacted sperm were significantly increased by PROG at 3.1,M (7.9% ±.8%) and 31.4,M (11.7% ± 2.5%) ncentrations, mpared to ntrol (4.% ± 1.1%) (mean ± SEM, P <.5, n = 3). The difference between both doses of PROG was not statistically c 4C rq 2( 1( )o { 3 1 A RU486 PROG (l1m) (.1 M) 1 1 B RU486 PROG ( pm) (.TmM) l /t 1 Ic FIG. 4. Effects of PROG on [Ca2+]i in hman sperm preincbated with RU486. PROG (.1,M) was added to Fra2loaded sperm after preincbation with 1,M RU486 (A) or 1,M RU486 (B) for 1 min. The florescence signal was measred with a PTI M21 spectroflorometer. An experiment representative of for is shown. JIAw

4 532 Medical Sciences: Yang et al. Proc. Natl. Acad Sci. USA 91 (1994) Table 3. Capacitated sperm movement parameters: Modlation by PROG or RU486 Parameter Control PROG (31.4,M) RU486 (23.4,M) Iono$ (1 M) MOT, % 73.6 ± ± ± 3.1t 7. ± 3.2 VCL,,m/s 81.9 ± ± 2.6t 77.2 ± ± 4.t VSL,,m/s 53.6 ± ± ± ± 3.1 LIN, % 65.3 ± ± ± ± 2.2 ALH, Am 5.6 ± ±.2* 5.1 ±.2* 6.7 ±.3t Sort fraction, % 9.3 ± ± 2.t 6.6 ± 1.t 18.7 ± 3.4i Steroids with 1% ethanol final ncentration were added to 1 x 16 spermatozoa per ml sspended in BWW/HSA(+) after 2 hr of capacitation; 1% ethanol was also added to the ntrol incbation. After 2 min at 37 C, the sperm movements were rerded, and the different parameters were atomatically mpted. The vales are means ± SEM. *, P <.5, t, P <.2, i, P <.1 verss ntrol. n = 11 experiments. $n = 7 experiments. significant and 62.8,M PROG did not frther increase the nmbers of acrosome reacted sperm. When PROG was replaced by 23.4,M RU486, no increase of live acrosome reacted sperm occrred. However, the stimlatory effect of 31.4 AM PROG was significantly nteracted by preincbation with 23.4,M RU486 for 2 min (P <.2, n = 6) (Fig. 5). DISCUSSION We report that PROG indces a rapid transient increase of [Ca2+]i in hman sperm, followed by a longlasting phase when [Ca2+]i stays above basal level (7, 8, 12). Under or experimental nditions, PROG was definitely more active than previosly described (8, 12), since a halfmaximal increase of [Ca2+]i was attained at PROG ncentrations in the nanomolar range. The peak and platea of [Ca2+]i are PROG dose dependent. We nfirm that three other steroid hormonesrtisterone, testosterone, and estradioland the synthetic progestin R52 also stimlate calcim ptake, althogh at lower levels than PROG (8). A send application ofprog 2 min after a first incbation with a maximally active ncentration of PROG (1,M) does not affect [Ca2+]i any more. This refractoriness to a send application of steroid also occrs with the for other mponds tested, ths sggesting a mmon reglatory mechanism. Several sperm movement parameters, VCL, ALH, and sort fraction, which define the hyperactivated state of hman spermatozoa (17), are significantly increased by PROG, as previosly reported (23). The percentage of sperm achieving r. 4 e 1 I+ TT CONT PROG RU486 RU486 IONO +PROG FIG. 5. RU486 inhibits the acrosome reaction of capacitated sperm prodced by PROG. Sperm were incbated for 3 min either with 31.4 A.M PROG, 23.4,M RU486, or both steroids. RU486 was added 2 min before PROG. The acrosome stats was evalated as described in the text (mean ± SEM, n = 6). *, P <.5, and +, P <.1 verss ntrol. t, P <.2 verss PROG. CONT, ntrol; IONO, incbation with 1,M Iono. mplete acrosome reaction is also significantly increased after 3 min of incbation with PROG. The precise moleclar mechanism of PROG action is not known, bt its rapid effects on [Ca2+]i (7, 8, 12), sperm movement, and onset of acrosome reaction (24) sggest an action at the membrane level. It has been proposed that voltagedependent calcim channels (VDCCs) are responsible for the increase of [Ca2+]i broght abot by forskolin in porcine sperm (25) and for the acrosomal reaction prodced by mbined elevation of ph and membrane depolarization in ram and bll sperm (26). Similarly, we have fond that the PROG effect on [Ca2+], was inhibited by the VDCC antagonist verapamil, at a ncentration of 1 mm, sggesting that calcim channels may be involved in the effects of PROG in hman sperm. RU486 inhibits the ptake of calcim in sperm and nteracts the stimlation prodced by PROG. The inhibitory action of RU486 per se, to or knowledge, has not been previosly reported. Under or experimental nditions, this effect of RU486 on [Ca2+], was definite and was fond in 11 of 12 experiments on different sperm samples. The PROG antagonist activity of RU486 on calcim ptake in hman sperm is ntroversial and was not fond in a previos report (12). RU486 has high affinity for the intracelllar PROG receptor and displays strong mpetitive antagonism of the genomic effects of PROG (1). The sitation is mpletely different in the case of sperm membrane: we have fond that RU486 is a weak antagonist ofprog, as previosly indicated (11). Indeed, the increase of [Ca2+]i prodced by.1,m PROG was only partially inhibited by a 1fold excess of RU486, whereas an eqimolar ncentration of RU486 markedly nteracts the genomic effects of PROG (1). These reslts are nsistent with the nclsion, previosly proposed on the basis of different ligand affinities, that the sperm membrane receptor of PROG, as that of Xenops oocyte membrane (27), is distinct from the classical intracelllar PROG receptor. Althogh RU486 behaves like an inverse agonist of PROG, it cannot be determined whether the same membrane receptor is involved in the opposite effects of PROG and RU486 on calcim ptake in sperm and sbseqent events. The present reslts are more in favor of distinct sites of action at the membrane level. This nclsion is based on the fact that the inhibitory effect of RU486 on [Ca2+], occrs with a threshold in the micromolar range, whatever the ncentration of PROG. This is spported by the RU486like activity of RU399 and RU39411, the steroid strctres of which are related to PROG and to estradiol, respectively, and which display mpletely different pharmalogical activities (2, 21), althogh they have the same llp side chain. However, RU41291, which has the same side chain as RU486, bt has a 17apropynyl estradiol steroid strctre, behaves as a weak PROG agonist in sperm, as estradiol does. Therefore, the 11, phenyl side chain and the

5 Medical Sciences: Yang et al. steroid ring strctre seem to be involved in the modlation of calcim ptake. Binding sites for PROGcarboxymethyloximebovine serm albmin njgates have been observed on a sbpoplation of hman sperm (28, 29), sggesting that only a small proportion of capacitated spermatozoa can respond to PROG. The percentage of live acrosome reacted sperm after PROG application in or experiments is in acrdance with the proportion of cells binding the PROGbovine serm albmin njgates (29). Frther work shold determine whether the same cells do bind PROG, show an increased [W+]i, are hyperactivated, and ndergo exocytotic events. Althogh nlikely, it might be that different cells respond to PROG and to RU486. Or reslts are nsistent with the recently described (3) decrease of the fertilization rate observed after RU486 exposre of mose sperm and oocytes, and not reverted by PROG. We are gratefl to B. Berthon and L. Combettes (Institt National de la Sante et de la Recherche Medicale, Unite 274) for their helpfl advice and for the se ofthe Hitachi F2 spectroflorometer. This work was spported by Grant 166 from Direction de la Recherche et des Etdes Doctorales (D.R.E.D.), Ministere de l'edcation Nationale. 1. Chang, M. C. (1984) J. Androl. 5, Astin, C. R. (1952) Natre (London) 17, Yanagimachi, R. (1988) in The Physiology of Reprodction, eds. Knobil, E. & Neill, J. D. (Raven, New York), Vol. 1, pp Tesarik, J. (1985) J. Reprod. Fertil. 74, Cross, N. L., Morales, P., Overstreet, J. W. & Hanson, F. W. (1988) Biol. Reprod. 38, Osman, R. A., Andria, M. L., Jones, A. D. & Meizel, S. (1989) Biochem. Biophys. Res. Commn. 16, Thomas, P. & Meizel, S. (1989) Biochem. J. 264, Blackmore, P. F., Beebe, S. J., Danforth, D. R. & Alexander, N. (199) J. Biol. Chem. 26, Balie, E. E., SchorderetSlatkine, S., Le Goasgne, C. & Blondea, J. P. (1985) Dev. Growth Differ. 27, Balie, E. E. (1989) Science 245, Proc. Natl. Acad. Sci. USA 91 (1994) Blackmore, P. F., Nelen, J., Lattanzio, F. & Beebe, S. J. (1991) J. Biol. Chem. 266, Baldi, E., Casano, R., Falsetti, C., Maggi, M. & Forti, G. (1991) J. Androl. 12, Tash, J. S. & Means, A. R. (1983) Biol. Reprod. 28, Biggers, J. D., Whitten, W. K. & Wittingham, D. G. (1971) in Methods in Mammalian Embryology, ed. Daniel, J. C. (Freeman, San Francis), pp Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985) J. Biol. Chem. 26, World Health Organization (1992) WHO Laboratory Manal for the Examination of Hman Semen and SpermCervical Mcs Interaction (Cambridge Univ. Press, Cambridge, U.K.), pp Robertson, L., Wolf, D. P. & Tash, T. S. (1988) Biol. Reprod. 39, Cross, N. L., Morales, P., Overstreet, J. W. & Hanson, F. W. (1986) Gamete Res. 15, Ojasoo, T. & Raynad, J. P. (1978) CancerRes. 38, Tetsch, G. (1985) in The Antiprogestin Steroid RU486 and Hman Fertility Control, eds. Balie, E. E. & Segal, S. J. (Plenm, New York), pp Classner, A., Nedelec, L., Niqe, F., Philibert, D., Tetsch, G. & Van de Velde, P. (1992) J. Steroid Biochem. Mol. Biol. 41, Tetsch, G., GaillardMogilewsky, M., Lemoine, G., Niqe, F. & Philibert, D. (1981) Biochem. Soc. Trans. 19, Uhler, M. L., Leng, A., Chan, S. Y. W. & Wang, C. (1992) Fertil. Steril. 58, Meizel, S. & Trner, K.. (1991) Mol. Cell. Endocrinol. 11, R1R Okamra, N., Tanba, M., Fkda, A., Sgita, Y. & Nagai, T. (1993) FEBS Lett. 316, Florman, H. M., Corron, M. E., Kim, T. D.H. & Babck, D. F. (1992) Dev. Biol. 152, Blondea, J. P. & Balie, E. E. (1984) Biochem. J. 219, Blackmore, T. F. & Lattanzio, F. A. (1991) Biochem. Biophys. Res. Commn. 181, Tesarik, J., Mendoza, C., Moos, J. & Carreras, A. (1992) Fertil. Steril. 58, Jneja, S. C. & Dodson, M. G. (199) Am. J. Obstet. Gynel. 163,