Molecular and Cellular Endocrinology, 11 (1991) Rl-R5 0 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/91/$03.50
MOLCEL
Rl
02514
Rapid Paper
Progesterone
acts at the plasma membrane Stanley Meizel and Kenneth
Depuiment
of human sperm
0. Turner
of Cell Biology and Human Anatomy, School of Medicine, University of California - Davis, Davis, CA 956168643. (Received
Key words; Progesterone;
Steroid;
Sperm;
Plasma
15 January
1991; accepted
membrane;
Calcium;
18 February
Acrosome
U.S.A.
1991)
reaction;
(Human)
Summary There has been increasing interest in the relationship between rapid effects of steroids and steroidplasma membrane interaction. This laboratory has previously reported that progesterone increases human sperm cytosolic free calcium ([Ca2’li) and thereby initiates the human sperm acrosome reaction (AR) in < 1 min. Herein, to test whether progesterone acts at the sperm plasma membrane, progesterone 3-( O-carboxymethyl)oxime : bovine serum albumin (BSA) conjugate (free of unconjugated progesterone) was added to capacitated human sperm. Furaassays were used to detect < 1 min changes in [Ca2’],, and indirect immunofluorescence was used to assay the AR occurring 1 min after stimulus addition. The conjugate increased [Ca2+li and the AR (though less than did unconjugated progesterone). Enzyme immunoassays demonstrated that the concentrations of unconjugated progesterone in conjugate-treated sperm suspensions did not increase over those of control suspensions. Since the progesterone : BSA conjugate presumably does not cross the sperm plasma membrane, progesterone must act at that membrane to increase [Ca2’], and the AR.
Introduction The mammalian sperm acrosome reaction (AR) is a specialized exocytotic process involving fusion of a membrane of the acrosome (a secretory granule-like organelle in the sperm head) with the overlying sperm plasma membrane (see discussion by Yudin et al., 1988). The mammalian AR is essential for fertilization and occurs, in the presence of an initiator, in sperm that have been previously modified in some way (capacitated) by residence in the female reproductive tract or by in
Address for correspondence: Dr. Stanley Meizel, Department of Cell Biology and Human Anatomy, School of Medicine, University of California - Davis, Davis, CA 95616-8643, U.S.A.
vitro incubation (Yanagimachi, 1988). The human AR can be initiated in vitro by at least two molecules present at the site of fertilization, an egg zona pellucida component (Cross et al., 1988) and progesterone (Osman et al., 1989). Progesterone and several other progestins, but not other steroids tested, rapidly increase cytosolic free calcium ([Ca2+li) in uncapacitated and capacitated human sperm (Thomas and Meizel, 1989; Blackmore et al., 1990; Meizel et al., 1990). Through this increased [Ca’ ‘I,, progesterone initiates the AR in capacitated human sperm within 1 min after addition (Meizel et al., 1990). The lowest effective concentration for AR initiation (under a particular set of in vitro conditions), 250 ng/ml, is much higher than blood levels, but progesterone may be present in pg/ml concentra-
tions at the fertilization site (Osman et al., 1989; Meizel et al.. 1990). There is increasing interest in the relationship between rapid. non-genomic cellular effects of steroids and possible steroid interactions with the plasma membrane (Koenig et al., 1989; Schumacher, 1990). The rapid responses of human sperm to progesterone suggest steroid action at the plasma membrane (Thomas and Meizel, 1989), particularly since the mature sperm lacks the cytoplasmic apparatus for protein synthesis. but the possibility still exists that the steroid must enter the cytoplasm to exert its effects. In the present report, the hypothesis that progesterone acts at the plasma membrane was tested by determining whether a progesterone covalently bound to bovine serum albumin (BSA) would rapidly increase [Ca”], and initiate the AR in capacitated human sperm. Since the progesterone : BSA conjugate presumably would not rapidly diffuse across the sperm plasma membrane. and since sperm are not known to exhibit endocytotic activity, any effects of that conjugate should be due to the steroid’s action at the sperm plasma membrane. Part of these results were presented elsewhere as an abstract (Meizel and Turner. 1990). Materials
and methods
Rernovul of unconjuguted progesterone from the proge.rterone:BSA conjugate. Progesterone 3(O-carboxymethyl)oxime:BSA (31 mol progesterone/mol BSA, Sigma Chemical Co., St. Louis, MO, U.S.A.) was dissolved in phosphate-buffered saline (PBS) (5.5 mg in 5 ml) into which 3 mg Norite and 0.3 mg dextran were then added. The resulting mixture was gently stirred for 24 h at 4 o C and then centrifuged twice at 48,000 X g for 90 min at 2°C. The protein content of the conjugate supernatant was determined using E,, at 279 nm = 6.67 (Janatova et al., 1968). Aliquots were stored at - 21°C. Stock solutions were 1.24 mg and 0.124 mg per ml PBS. Prepurution and cupucitation of sperm. Human semen was obtained by masturbation from healthy donors. A > 95% motile population of sperm was prepared using a discontinuous Percoll gradient (Suarez et al., 1986). Washed sperm were resuspended (6 x 10h sperm/ml) in a modified Tyrodes
solution (Thomas and Meizel. 1988) that included 26 mg BSA/ml (catalogue No. 7030, Sigma Chemical Co., St. Louis. MO, U.S.A.). The resuspended sperm were then capacitated by incubating 200 ~1 aliquots in 15 ml polystyrene conical centrifuge tubes (Sarstedt, Hayward, U.S.A.) at 37 o C for 24 h for AR studies or at 40°C for 6.5 h for furastudies (Thomas and Meizel, 1988) in a humid atmosphere of 5%, CO,-95% air (pH of media, 7.4-7.6). Assu,: of the effect of the progesterone: BSA conjugute on AR initiution. In each experiment. the following were added to duplicate 200 ~1 aliquots of sperm suspensions (concentrations are final in sperm suspensions): 52 1-18 conjugate/ml PBS (equivalent to 6.66 pg of progesterone/ml) or 5.2 pg conjugate/ml PBS (equivalent to 0.66 pg progesterone/ml) or 0.66 pg progesterone/ml progesterone solvent medium or a solvent control. The stock solution of progesterone added consisted of 100 pg/ml progesterone and 10% dimethyl sulfoxide (DMSO) (v/v) dissolved in a Hepes buffered medium (Thomas and Meizel, 1989) containing 26 mg/ml BSA (catalogue No. A7030, Sigma Chemical Co., St. Louis, MO, U.S.A.). The same volume of PBS used for conjugate additions was added to progesterone and solvent control tubes. and the same volume of progesterone solvent medium used for progesterone additions was added to the conjugate and solvent control tubes. In all incubation suspension tubes, the final concentrations of DMSO and A7030 BSA were 0.1% (v/v) and 26 mg/ml respectively. Sperm motility and quality was evaluated by phase contrast microscopy of 10 ~1 of suspensions (Suarez et al., 1986). The remaining 190 ~1 aliquot of each type of treated sperm suspension was fixed without washing, one min after additions and the AR determined by indirect immunofluorescence using mouse monoclonal HS21, a gift of Dr. K. Bechtol (Suarez et al., 1986; Thomas et al., 1988). Duplicate aliquots were used for assay of progesterone (see below). Assq~s for unconjuguted progesterone. Aliquots (200 ~1) of the capacitation medium and the conjugate in that medium or of capacitated sperm suspensions 1 min after conjugate or solvent control additions were extracted with 2 ml of petroleum ether. After solvent evaporation. residues
R3
were assayed for progesterone with a solid-phase, microtiter plate, enzyme immunoassay procedure (Munro and Stabenfeldt, 1984) by the U.C. Davis Vet. School Clinical Endocrinology Lab. Assuy of the effect of progesterone: BSA conjugute on (Cu’ ‘/ ,. The methods for determining [Ca”], using fura- were essentially those used by Thomas and Meizel (1988, 1989). In brief: capacitated sperm resuspended in a Hepesbuffered medium without glucose were incubated for 30 min with 1 PM of the acetoxymethyl ester of fura(Molecular Probes, Junction City, OR, U.S.A.) and centrifuged through 40% Percoll to remove extracellular fura-2; additions of conjugate, progesterone or solvent control to 1 ml aliquots of fura- loaded sperm suspensions (6 X 10h,/ml) were made in a stirred cuvette at 40 o C; spectrofluorometric assays used a single wave length of excitation (339 nm) with emission monitored at 500 nm; estimation of [Ca2’], was made according to Pollock et al. (1986) assuming a K, of 224 nM for Ca’+/fura-2 (Grynkiewicz et al., 1985). The calibration of the furasignal was modified by substituting the optimal concentration of iononmycin (calcium salt, Calbiochem, La Jolla, CA, U.S.A.), 16 PM (established by titra-
TABLE
tion), fura-
for digitonin in determining Ca2+-saturated fluorescence. Materials. Other chemicals were reagent grade and were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.) or Fisher Scientific Co. (Fair Lawn, NJ, U.S.A.). Results Unconjugated progesterone was detected in untreated conjugate (32 ng/pg) but not in the capacitation medium or in dextran-charcoal treated conjugate. Dextran-charcoal treated conjugate used at the equivalent of 0.66 and 6.6 pg of progesterone/ml significantly increased the AR over the solvent control (Table 1). However, both concentrations of conjugate were less effective than unconjugated progesterone at 0.66 pg/ml (Table 1). The very low levels of unconjugated progesterone present in the conjugate-treated sperm suspensions did not differ significantly from that present in solvent control suspensions during the 1 min incubation of conjugate with sperm (Table 1). The BSA used to prepare the conjugate was Sigma catalogue No. A7888 (Sigma Chemical Co. Technical Service). In preliminary experiments, addition to capacitated human sperm of 5.2 and
1
ACROSOME GESTERONE
REACTION IN SPERM
OF HUMAN SUSPENSIONS
SPERM AND OF THE 1 MIN AFTER ADDITION
CONCENTRATION OF PROGESTERONE:
OF UNCONJUGATED BSA CONJUGATE
Treatment (n = 6) ”
%AR (mean * SD)
ng uncoqugated progesterone/ml sperm suspension (mean f SD)
Progesterone (0.66 pg/ml) Conjugate (52 pg/ml = 6.6 pg/ml progesterone) ’ Conjugate (5.2 pg protein/ml = 0.66 pg/ml progesterone) Solvent control f
44.s3*5.13 h 27.58 + 7.28 ‘.’ 29.32 + 5.03 b.‘ 14.95 _t 4.63
_
’
PRO-
1.16iO.33’ 1.11 kO.33 d 1.37 * 0.45
Sperm motility was 80-9096 in all experiments, and there was no difference in percent motility or the intensity of motility between control and experimental treatments. Significant difference in AR between treatment and solvent control, p < 0.05 by Dunnett r-test for multiple comparisons (percentage data transformed by arcsin fi). Significant difference in AR between conjugate and unconjugated progesterone, p < 0.05 by Dunnett r-test for multiple comparisons (percentage data transformed by arcsin fi). No significant difference between treatment and solvent control, p 2 0.15 by paired r-test with Bonferroni correction for multiple comparisons. Based on 31 mol of progesterone/mol of BSA (Sigma Chemical Co. Technical Service). See Materials and Methods for solvent control.
R4
[Ca2+ ] i
Fig. 1. The effect of the progesterone: BSA conjugate on the [Ca*+ 1, of furaloaded capacitated human sperm. Traces are representative of three similar experiments (sperm motility was 85%): (a) untreated sperm: (b) sperm treated with the dextran-charcoal preparation of BSA (in PBS) used to synthesize conjugate; (c) sperm treated with conjugate (5.2 pg protein/ml = 0.66 pg/ml progesterone) in PBS: (d) sperm treated with 0.66 pg/ml progesterone. Solvent for progesterone (see Materials and Methods) has no effect on the [Ca’+ 1, (Thomas and Meizel. 1989). All traces were corrected for the presence of extracellular fura(Thomas and Meizel. 1988).
52 pg/ml of a dextran-charcoal treated preparation of A7888 BSA did not initiate the AR (data not shown). Thus, dextran-charcoal treated A7888 BSA was not included in the controls for experiments shown in Table 1. The conjugate (but not dextran-charcoal treated A7888 BSA) stimulated a rapid, transient increase though less than did proin sperm [Cal’],. gesterone (Fig. 1). Discussion
Steroid-synthetic polymer conjugates have been previously used to demonstrate that progesterone agonists can act at the plasma membrane to stimulate maturation of Xenopus luevis oocytes (Ishikawa et al., 1977; Baulieu et al., 1978) a cell which appears to have a plasma membrane progesterone receptor (Sadler and Maller, 1982). Progesterone : BSA conjugates have been used to study putative progesterone receptors in rat brain nerve cell membrane preparations (Ke and Ramirez, 1990). The present studies utilizing a progesterone : BSA conjugate demonstrate that progesterone, even when covalently-bound to a large molecule which presumably does not cross the sperm plasma membrane, will rapidly increase [Ca”], and initiate the AR in capacitated human sperm. It is unlikely that unconjugated progesterone, released in some way from the conjugate, is responsible for the present results be-
cause the very low concentrations of unconjugated progesterone present in conjugate-treated sperm suspensions were not different than those of control suspensions during the 1 min incubations. The lowered effectiveness of the conjugate compared to unconjugated progesterone could be due to the modification of progesterone at the C-3 position in the former or to some of the covalently-bound progesterone being present at sites in the BSA that are not readily accessible to interaction with the sperm membrane. The present results support the hypothesis that progesterone is acting at the sperm plasma membrane rather than having to enter the cytoplasm. Rapid effects and specific plasma membrane binding sites have been reported for various steroids in several cell types (Schumacher, 1990). Plasma membrane binding sites for progesterone in human sperm have been reported (Hyne and Boettcher, 1977; Cheng et al., 1981), and a preliminary report described the same progesterone-binding protein present in both human seminal plasma and sperm (Neulen et al., 1990). However, an integral sperm plasma membrane protein with high specificity and high affinity for progesterone has not yet been demonstrated. Whether progesterone increases the [Ca2’], and the AR of human sperm by interaction with a specific plasma membrane receptor and/or through some other mechanism(s) at the plasma membrane (Meizel et al., 1990) remains to be determined.
R5
Acknowledgements This work was funded by NIH grant HD-23098 and funds from the Dean’s Office, U.C. Davis Medical School. We thank T. Madley and Dr. G. Stabenfeldt for progesterone assays and Dr. M. Pillai for help in preliminary experiments. References Baulieu, E.-E., Godeau, F., Schorderet, M. and SchorderetSlatkine. S. (197X) Nature 275, 593-59X. Blackmore. P.F., Beebe, S.J., Danforth, D.R. and Alexander, N. (1990) J. Biol. Chem. 265, 1376. Cheng, C.Y., Boettcher, B., Rose, R.J., Kay, D.J. and Tinneberg, H.R. (1981) lnt. J. Andrology 4. 1. Grynkiewicz, G., Poenie, M. and Tsien. R.Y. (1985) J. Biol. Chem. 260, 3440-3450. Hyne. R.V. and Boettcher, B. (1977) Contraception 15, 163174. Ishikawa. K.. Hanaoka, Y., Kondo. Y. and Imai. K. (1977) Mol. Cell. Endocrinol. 9, 91-100. Janatova. J.. Fuler, J.K. and Hunter, M.J. (1968) J. Biol. Chem. 243, 3612-3622. Ke, F.-C. and Ramirez, V.D. (1990) J. Neurochem. 54,467-472.
Koenig. H.. Fan, C.-C., Goldstone. A.D.. Lu, C.Y. and Trout, J.J. (1989) Circ. Res. 64. 415-426. Meizel, S. and Turner. K.O. (1990) J. Cell Biol. 111. 89a. Meizel. S., Pillai, M.C.. Diaz-Perez, E. and Thomas. P. (1990) in Fertilization in Mammals (Bavister. B.D.. Cummins. J. and Roldan. E.R.S.. eds.), pp. 205-222. Serono Symposia USA. Norwell. MA. Munro. C. and Stabenfeldt, G. (1984) J. Endocrinol. 101, 41-49. Neulen. J.. Blackmore. P. and Beebe. S. (1990) Biol. Reprod. 42, Suppl. 1, 132. Osman. R.A., Andria. M.L., Jones. D.A. and Meizel, S. (1989) Biochem. Biophys. Res. Commun. 160. X28-833. Pollock. W.K.. Rink. T.J. and Irvine, R.F. (1986) Biochem. J. 235, X69-877. Sadler, S.E. and Maller, J.L. (1982) J. Biol. Chem. 257. 3555361. Schumacher, M. (1990) Trends Neurosci. 13. 359-362. Suarez. S.S., Wolf. D.P. and Meizel, S. (1986) Gamete Res. 14. 107-121. Thomas. P. and Meizel. S. (1988) Gamete Res. 20. 397-411. Thomas, P. and Meizel, S. (1989) Biochem. J. 264. 539-546. Yanagimachi. R. (1985) in Physiology of Reproduction (Knobil. E. and Neill. J.D.. eds.). pp. 135-185. Raven Press, New York. Yudin. A.1.. Gottlieb, W. and Meizel. S. (1988) Gamete Res. 20, 11-24.
MOLCEL
Rl
02514
Rapid Paper
Progesterone
acts at the plasma membrane Stanley Meizel and Kenneth
Depuiment
of human sperm
0. Turner
of Cell Biology and Human Anatomy, School of Medicine, University of California - Davis, Davis, CA 956168643. (Received
Key words; Progesterone;
Steroid;
Sperm;
Plasma
15 January
1991; accepted
membrane;
Calcium;
18 February
Acrosome
U.S.A.
1991)
reaction;
(Human)
Summary There has been increasing interest in the relationship between rapid effects of steroids and steroidplasma membrane interaction. This laboratory has previously reported that progesterone increases human sperm cytosolic free calcium ([Ca2’li) and thereby initiates the human sperm acrosome reaction (AR) in < 1 min. Herein, to test whether progesterone acts at the sperm plasma membrane, progesterone 3-( O-carboxymethyl)oxime : bovine serum albumin (BSA) conjugate (free of unconjugated progesterone) was added to capacitated human sperm. Furaassays were used to detect < 1 min changes in [Ca2’],, and indirect immunofluorescence was used to assay the AR occurring 1 min after stimulus addition. The conjugate increased [Ca2+li and the AR (though less than did unconjugated progesterone). Enzyme immunoassays demonstrated that the concentrations of unconjugated progesterone in conjugate-treated sperm suspensions did not increase over those of control suspensions. Since the progesterone : BSA conjugate presumably does not cross the sperm plasma membrane, progesterone must act at that membrane to increase [Ca2’], and the AR.
Introduction The mammalian sperm acrosome reaction (AR) is a specialized exocytotic process involving fusion of a membrane of the acrosome (a secretory granule-like organelle in the sperm head) with the overlying sperm plasma membrane (see discussion by Yudin et al., 1988). The mammalian AR is essential for fertilization and occurs, in the presence of an initiator, in sperm that have been previously modified in some way (capacitated) by residence in the female reproductive tract or by in
Address for correspondence: Dr. Stanley Meizel, Department of Cell Biology and Human Anatomy, School of Medicine, University of California - Davis, Davis, CA 95616-8643, U.S.A.
vitro incubation (Yanagimachi, 1988). The human AR can be initiated in vitro by at least two molecules present at the site of fertilization, an egg zona pellucida component (Cross et al., 1988) and progesterone (Osman et al., 1989). Progesterone and several other progestins, but not other steroids tested, rapidly increase cytosolic free calcium ([Ca2+li) in uncapacitated and capacitated human sperm (Thomas and Meizel, 1989; Blackmore et al., 1990; Meizel et al., 1990). Through this increased [Ca’ ‘I,, progesterone initiates the AR in capacitated human sperm within 1 min after addition (Meizel et al., 1990). The lowest effective concentration for AR initiation (under a particular set of in vitro conditions), 250 ng/ml, is much higher than blood levels, but progesterone may be present in pg/ml concentra-
tions at the fertilization site (Osman et al., 1989; Meizel et al.. 1990). There is increasing interest in the relationship between rapid. non-genomic cellular effects of steroids and possible steroid interactions with the plasma membrane (Koenig et al., 1989; Schumacher, 1990). The rapid responses of human sperm to progesterone suggest steroid action at the plasma membrane (Thomas and Meizel, 1989), particularly since the mature sperm lacks the cytoplasmic apparatus for protein synthesis. but the possibility still exists that the steroid must enter the cytoplasm to exert its effects. In the present report, the hypothesis that progesterone acts at the plasma membrane was tested by determining whether a progesterone covalently bound to bovine serum albumin (BSA) would rapidly increase [Ca”], and initiate the AR in capacitated human sperm. Since the progesterone : BSA conjugate presumably would not rapidly diffuse across the sperm plasma membrane. and since sperm are not known to exhibit endocytotic activity, any effects of that conjugate should be due to the steroid’s action at the sperm plasma membrane. Part of these results were presented elsewhere as an abstract (Meizel and Turner. 1990). Materials
and methods
Rernovul of unconjuguted progesterone from the proge.rterone:BSA conjugate. Progesterone 3(O-carboxymethyl)oxime:BSA (31 mol progesterone/mol BSA, Sigma Chemical Co., St. Louis, MO, U.S.A.) was dissolved in phosphate-buffered saline (PBS) (5.5 mg in 5 ml) into which 3 mg Norite and 0.3 mg dextran were then added. The resulting mixture was gently stirred for 24 h at 4 o C and then centrifuged twice at 48,000 X g for 90 min at 2°C. The protein content of the conjugate supernatant was determined using E,, at 279 nm = 6.67 (Janatova et al., 1968). Aliquots were stored at - 21°C. Stock solutions were 1.24 mg and 0.124 mg per ml PBS. Prepurution and cupucitation of sperm. Human semen was obtained by masturbation from healthy donors. A > 95% motile population of sperm was prepared using a discontinuous Percoll gradient (Suarez et al., 1986). Washed sperm were resuspended (6 x 10h sperm/ml) in a modified Tyrodes
solution (Thomas and Meizel. 1988) that included 26 mg BSA/ml (catalogue No. 7030, Sigma Chemical Co., St. Louis. MO, U.S.A.). The resuspended sperm were then capacitated by incubating 200 ~1 aliquots in 15 ml polystyrene conical centrifuge tubes (Sarstedt, Hayward, U.S.A.) at 37 o C for 24 h for AR studies or at 40°C for 6.5 h for furastudies (Thomas and Meizel, 1988) in a humid atmosphere of 5%, CO,-95% air (pH of media, 7.4-7.6). Assu,: of the effect of the progesterone: BSA conjugute on AR initiution. In each experiment. the following were added to duplicate 200 ~1 aliquots of sperm suspensions (concentrations are final in sperm suspensions): 52 1-18 conjugate/ml PBS (equivalent to 6.66 pg of progesterone/ml) or 5.2 pg conjugate/ml PBS (equivalent to 0.66 pg progesterone/ml) or 0.66 pg progesterone/ml progesterone solvent medium or a solvent control. The stock solution of progesterone added consisted of 100 pg/ml progesterone and 10% dimethyl sulfoxide (DMSO) (v/v) dissolved in a Hepes buffered medium (Thomas and Meizel, 1989) containing 26 mg/ml BSA (catalogue No. A7030, Sigma Chemical Co., St. Louis, MO, U.S.A.). The same volume of PBS used for conjugate additions was added to progesterone and solvent control tubes. and the same volume of progesterone solvent medium used for progesterone additions was added to the conjugate and solvent control tubes. In all incubation suspension tubes, the final concentrations of DMSO and A7030 BSA were 0.1% (v/v) and 26 mg/ml respectively. Sperm motility and quality was evaluated by phase contrast microscopy of 10 ~1 of suspensions (Suarez et al., 1986). The remaining 190 ~1 aliquot of each type of treated sperm suspension was fixed without washing, one min after additions and the AR determined by indirect immunofluorescence using mouse monoclonal HS21, a gift of Dr. K. Bechtol (Suarez et al., 1986; Thomas et al., 1988). Duplicate aliquots were used for assay of progesterone (see below). Assq~s for unconjuguted progesterone. Aliquots (200 ~1) of the capacitation medium and the conjugate in that medium or of capacitated sperm suspensions 1 min after conjugate or solvent control additions were extracted with 2 ml of petroleum ether. After solvent evaporation. residues
R3
were assayed for progesterone with a solid-phase, microtiter plate, enzyme immunoassay procedure (Munro and Stabenfeldt, 1984) by the U.C. Davis Vet. School Clinical Endocrinology Lab. Assuy of the effect of progesterone: BSA conjugute on (Cu’ ‘/ ,. The methods for determining [Ca”], using fura- were essentially those used by Thomas and Meizel (1988, 1989). In brief: capacitated sperm resuspended in a Hepesbuffered medium without glucose were incubated for 30 min with 1 PM of the acetoxymethyl ester of fura(Molecular Probes, Junction City, OR, U.S.A.) and centrifuged through 40% Percoll to remove extracellular fura-2; additions of conjugate, progesterone or solvent control to 1 ml aliquots of fura- loaded sperm suspensions (6 X 10h,/ml) were made in a stirred cuvette at 40 o C; spectrofluorometric assays used a single wave length of excitation (339 nm) with emission monitored at 500 nm; estimation of [Ca2’], was made according to Pollock et al. (1986) assuming a K, of 224 nM for Ca’+/fura-2 (Grynkiewicz et al., 1985). The calibration of the furasignal was modified by substituting the optimal concentration of iononmycin (calcium salt, Calbiochem, La Jolla, CA, U.S.A.), 16 PM (established by titra-
TABLE
tion), fura-
for digitonin in determining Ca2+-saturated fluorescence. Materials. Other chemicals were reagent grade and were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.) or Fisher Scientific Co. (Fair Lawn, NJ, U.S.A.). Results Unconjugated progesterone was detected in untreated conjugate (32 ng/pg) but not in the capacitation medium or in dextran-charcoal treated conjugate. Dextran-charcoal treated conjugate used at the equivalent of 0.66 and 6.6 pg of progesterone/ml significantly increased the AR over the solvent control (Table 1). However, both concentrations of conjugate were less effective than unconjugated progesterone at 0.66 pg/ml (Table 1). The very low levels of unconjugated progesterone present in the conjugate-treated sperm suspensions did not differ significantly from that present in solvent control suspensions during the 1 min incubation of conjugate with sperm (Table 1). The BSA used to prepare the conjugate was Sigma catalogue No. A7888 (Sigma Chemical Co. Technical Service). In preliminary experiments, addition to capacitated human sperm of 5.2 and
1
ACROSOME GESTERONE
REACTION IN SPERM
OF HUMAN SUSPENSIONS
SPERM AND OF THE 1 MIN AFTER ADDITION
CONCENTRATION OF PROGESTERONE:
OF UNCONJUGATED BSA CONJUGATE
Treatment (n = 6) ”
%AR (mean * SD)
ng uncoqugated progesterone/ml sperm suspension (mean f SD)
Progesterone (0.66 pg/ml) Conjugate (52 pg/ml = 6.6 pg/ml progesterone) ’ Conjugate (5.2 pg protein/ml = 0.66 pg/ml progesterone) Solvent control f
44.s3*5.13 h 27.58 + 7.28 ‘.’ 29.32 + 5.03 b.‘ 14.95 _t 4.63
_
’
PRO-
1.16iO.33’ 1.11 kO.33 d 1.37 * 0.45
Sperm motility was 80-9096 in all experiments, and there was no difference in percent motility or the intensity of motility between control and experimental treatments. Significant difference in AR between treatment and solvent control, p < 0.05 by Dunnett r-test for multiple comparisons (percentage data transformed by arcsin fi). Significant difference in AR between conjugate and unconjugated progesterone, p < 0.05 by Dunnett r-test for multiple comparisons (percentage data transformed by arcsin fi). No significant difference between treatment and solvent control, p 2 0.15 by paired r-test with Bonferroni correction for multiple comparisons. Based on 31 mol of progesterone/mol of BSA (Sigma Chemical Co. Technical Service). See Materials and Methods for solvent control.
R4
[Ca2+ ] i
Fig. 1. The effect of the progesterone: BSA conjugate on the [Ca*+ 1, of furaloaded capacitated human sperm. Traces are representative of three similar experiments (sperm motility was 85%): (a) untreated sperm: (b) sperm treated with the dextran-charcoal preparation of BSA (in PBS) used to synthesize conjugate; (c) sperm treated with conjugate (5.2 pg protein/ml = 0.66 pg/ml progesterone) in PBS: (d) sperm treated with 0.66 pg/ml progesterone. Solvent for progesterone (see Materials and Methods) has no effect on the [Ca’+ 1, (Thomas and Meizel. 1989). All traces were corrected for the presence of extracellular fura(Thomas and Meizel. 1988).
52 pg/ml of a dextran-charcoal treated preparation of A7888 BSA did not initiate the AR (data not shown). Thus, dextran-charcoal treated A7888 BSA was not included in the controls for experiments shown in Table 1. The conjugate (but not dextran-charcoal treated A7888 BSA) stimulated a rapid, transient increase though less than did proin sperm [Cal’],. gesterone (Fig. 1). Discussion
Steroid-synthetic polymer conjugates have been previously used to demonstrate that progesterone agonists can act at the plasma membrane to stimulate maturation of Xenopus luevis oocytes (Ishikawa et al., 1977; Baulieu et al., 1978) a cell which appears to have a plasma membrane progesterone receptor (Sadler and Maller, 1982). Progesterone : BSA conjugates have been used to study putative progesterone receptors in rat brain nerve cell membrane preparations (Ke and Ramirez, 1990). The present studies utilizing a progesterone : BSA conjugate demonstrate that progesterone, even when covalently-bound to a large molecule which presumably does not cross the sperm plasma membrane, will rapidly increase [Ca”], and initiate the AR in capacitated human sperm. It is unlikely that unconjugated progesterone, released in some way from the conjugate, is responsible for the present results be-
cause the very low concentrations of unconjugated progesterone present in conjugate-treated sperm suspensions were not different than those of control suspensions during the 1 min incubations. The lowered effectiveness of the conjugate compared to unconjugated progesterone could be due to the modification of progesterone at the C-3 position in the former or to some of the covalently-bound progesterone being present at sites in the BSA that are not readily accessible to interaction with the sperm membrane. The present results support the hypothesis that progesterone is acting at the sperm plasma membrane rather than having to enter the cytoplasm. Rapid effects and specific plasma membrane binding sites have been reported for various steroids in several cell types (Schumacher, 1990). Plasma membrane binding sites for progesterone in human sperm have been reported (Hyne and Boettcher, 1977; Cheng et al., 1981), and a preliminary report described the same progesterone-binding protein present in both human seminal plasma and sperm (Neulen et al., 1990). However, an integral sperm plasma membrane protein with high specificity and high affinity for progesterone has not yet been demonstrated. Whether progesterone increases the [Ca2’], and the AR of human sperm by interaction with a specific plasma membrane receptor and/or through some other mechanism(s) at the plasma membrane (Meizel et al., 1990) remains to be determined.
R5
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