Nongenomic progesterone receptor on human spermatozoa: biochemical aspects and clinical implications

Steroids 64 (1999) 143–148

Rapid actions of progesterone

Nongenomic progesterone receptor on human spermatozoa: biochemical aspects and clinical implications Elisabetta Baldia,*, Michaela Luconia, Lorella Bonaccorsia, M. Maggia, S. Francavillab, A. Gabrieleb, G. Properzib, G. Fortia a

Dipartimento di Fisiopatologia Clinica, Unita` di Andrologia, Universita` di Firenze, Florence, Italy b Dipartimento di Medicina Interna e Sanita` Pubblica, Universita` dell’Aquila, L’Aquila, Italy Manuscript received September 19, 1998; accepted October 30, 1998

Abstract Progesterone (P) is a physiological stimulus of human sperm functions. It is present in high levels at the site of fertilization (cumulus oophorus) and has been described to affect several sperm functions, including motility, capacitation, acrosome reaction, and the ability to bind and to respond to zona proteins. The effects of the steroid are mediated essentially by an increase of intracellular calcium concentrations, stimulation of activity of phospholipases, phosphorylation of proteins, efflux of chloride. These effects are due to activation of a rapid, nongenomic pathway. Whether the effects of progesterone are mediated or not by specific interactions with sperm membrane proteins is questioned. By using an antibody directed against the C-terminal region (P-binding region) of the genomic receptor, we have recently identified two sperm proteins with molecular weights distinct from the classic genomic receptors. In addition, ligand blot analysis with peroxidase-conjugated P demonstrated that P specifically binds these two proteins. Classical ligand binding experiments demonstrated the presence of two specific binding sites with affinity in the nanomolar and in the micromolar range, respectively. The involvement of progesterone in the physiological process leading to fertilization of the oocyte is suggested by several studies. In particular, the demonstration that sperm responsiveness to progesterone is impaired in subfertile patients and that is strictly correlated to the ability of fertilizing the oocyte represents a further indication of the participation of the steroid in this process. In addition, the determination of sperm responsiveness may be predictive of fertilizing ability with a positive predictive value of 90% and can be clinically useful for the preliminary assessment of the male partner to select the appropriate assisted reproductive technique. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Progesterone; Nongenomic; Spermatozoa; Acrosome reaction; Receptor; Phosphorylation

1. Background Progesterone (P) was the first steroid described to have nongenomic effects [1]. In this pioneer study, Seyle [1] reported that P induces, almost immediately after exposure, an anesthetic effect. Later on it was shown that this effect was indeed specific, involving an interaction of P with membrane GABAA receptors [2]. Among the several rapid/ nongenomic pathways of steroid hormones discovered so far are the nongenomic effects of P on spermatozoa (for review, see [3]). These are probably the most studied, in terms of number of published papers, since their discovery. * Corresponding author. Elisabetta Baldi, Dipartimento di Fisiopatologia Clinica, Unita` di Andrologia, Universita` di Firenze, Viale Pieraccini 6, I-50139 Florence, Italy; Tel.: 139-55-4271366; Fax: 139-55-4271-371; E-mail: [email protected]

Sperm life after ejaculation is characterized by two essential events for the process of fertilization, i.e., capacitation and acrosome reaction. Capacitation [4] occurs during sperm transit in the female genital tract and consists of several biochemical and functional processes ultimately leading to increased hyperactivated motility (necessary to cross the cumulus matrix that surrounds the oocyte) as well as sensitivity to physiological agonists of acrosome reaction (AR). AR consists of fusion and fenestration of the outer acrosomal membrane with the plasma membrane and release of acrosomal enzymes that aid the spermatozoon to penetrate the various investments of the oocyte. Evidence for the presence of rapid effects of P on spermatozoa was first reported by Osman et al. [5], who showed that P was responsible for the acrosome reaction inducing activity of the follicular fluid. High concentrations (1–10 mg/ml) of P are indeed present in the cumulus matrix that surrounds the

0039-128X/99/$–see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 9 - 1 2 8 X ( 9 8 ) 0 0 1 0 0 - 7

144

E. Baldi et al./Steroids 64 (1999) 143–148

Fig. 1. Effect of L-type calcium channel inhibitor verapamil on sperm intracellular calcium waves induced by progesterone (P). Human spermatozoa were capacitated for 24 h and loaded with fura 2/AM. [Ca21]i was measured with a spectrofluorimeter (LS 50B, Perkin Elmer). The initial peak of Ca21 increase stimulated by the steroid is followed by a sustained plateau that may last several minutes (control). Two different concentrations of verapamil did not modify the response.

oocyte, which must be necessarily crossed by the sperm to reach the zona pellucida and penetrate it. These concentrations are similar to those determining the biological effects of the steroid in spermatozoa [3]. Later on, these effects were confirmed in other studies and the signal transduction pathways activated by the steroid characterized. It is now clear that the effects of P on human spermatozoa are mediated by a pathway quite distinct from the classic genomic one. Indeed, most of its effects occurs within seconds following addition of the agonist, and can be induced also by a nonpermeable P analogue [6,7] and thus cannot be attributed to a genomic effect. In addition, sperm cells are devoid of nuclear P receptors [8,9]. Whether the effects of P on spermatozoa are important for stimulation of capacitation [10], onset of hyperactivated motility [11], induction of AR [12] or “priming” of the cells to the action of or binding to zona pellucida proteins [13,14] is still a matter of discussion.

2. Is a specific receptor(s) involved? The effects of P on spermatozoa are mediated essentially by three signaling pathways (reviewed in [3]): rapid increase of intracellular [Ca21]i [15] and tyrosine phosphor-

ylation of sperm proteins [16 –18], efflux of Cl2 [19] and stimulation of phospholipase C [20]. Cross-talks among these pathways have been also demonstrated [21–23]. In particular, it has been demonstrated that tyrosine kinase activation by P is important for the secondary phase of calcium increase [21,23] and for the efflux of chloride [22] stimulated by the steroid. Recently, the involvement of protein kinase C (PKC) [24,25], extracellular-signal regulated kinases (ERKs) [26,27] and the product of the oncogene shc [28] have also been demonstrated. The effect of P on [Ca21]i increase (Fig. 1) is due to influx from extracellular medium, since it is eliminated by the presence of EGTA and lantanium in the external medium [3,15]. Recent evidence indicates the existence of intracellular calcium stores at the level of the acrosome in spermatozoa [29,30], however, whether release of calcium from the acrosome is important for the effect of P is debated [3]. The involvement of L-type voltage-sensitive calcium channels in the action of P is also unlike [3]. Fig. 1 clearly show that verapamil at two different concentrations does not modify [Ca21]i waves generated by P. Similar results were also obtained with nifedipine and with different T-type current antagonists, such as amiloride, pimozide, and mibefradil (Bonaccorsi and Baldi, unpublished results). Although perturbing effects of P on membrane fluidity

E. Baldi et al./Steroids 64 (1999) 143–148 Table 1 Concentration (B max 5 sites/cell) and affinity constant (K d) of the two predicted binding sites (R1 and R2) for the indicated ligands

Bmax Agonists Progesterone 11bOH-progesterone 17aOH-progesterone RU486 GABA DHT Norgestrel

R1

R2

274 6 87 K d (nM) 0.58 6 0.42 ND ND ND ND ND ND

39 6 12 3 106 K d (mM) 26 6 4 81 6 40 273 6 70 7.6 6 2.8 .10000 .10000 .10000

Values 6 SEM were derived from computer modeling of 10 families of competitive curves for progesterone-11a-glucuronide-[125I]iodotyramine [9]. ND, not detectable; RU486, mifepristone; GABA, g-aminobutyric acid; DHT, dihydrotestosterone.

have been described in several cells, including ejaculated spermatozoa [31] it is now believed that the vast majority of the biological effects of P on spermatozoa are mediated by specific cell surface receptor(s). This conclusion is based on several pieces of evidence: 1) the signal transduction pathways stimulated by P (see above) are similar to those activated by classic ligand-membrane receptor interaction in somatic cells; 2) response to P undergoes homologous and heterologous desensitization [9,32], a feature that characterizes receptor-mediated events; 3) responsiveness to P in ejaculated sperm develops only after a latency period (i.e., capacitation), probably involving exposition of the P receptors on the surface [12,32,33]; 4) the high steroid specificity of the response, as shown by elegant structure-efficiency experiments by Blackmore et al. [34]. Demonstration of existence of specific sperm membrane receptors by “classic” ligand binding experiments is difficult due to the high permeability of P and the low specific activity of available radioactive tracers. Using [3H]P, Neulen et al. [35] performed ligand binding experiments in spermatozoa and seminal fluid, identifying a single binding site with a Kd of about 10 nM. We used a nonpermeable iodinated P derivative (P-11a-glucuronide-[125I]iodotyramine), which specifically binds to capacitated spermatozoa in a time- and temperature-dependent way [9]. This tool identified two membrane binding sites with different affinity and capacity for P and a certain degree of steroid specificity (Table 1). In particular, P was the only compound, among those tested, able to bind to the high affinity site (Table 1). Functional studies revealed that the dose-dependent curve of P-induced [Ca21]i increase in capacitated sperm was biphasic, with a first component with an EC50 in the nanomolar range and a second component in the micromolar one [9]. Conversely, other steroids did not show such a biphasic dose-response curve. More importantly, a significant correlation exists between pK and log EC50 for the steroid tested (r 5 0.94, p , 0.05). Further characterization of sperm membrane progesterone receptor was performed by ligand and Western

145

blot experiments [9]. Ligand blot experiments were performed by using peroxidase-conjugated-P, while Western blot was obtained with an antibody directed against the C-terminal domain of the genomic P receptor (a-c262 monoclonal antibody). This antibody was shown previously to detect one-two unusual proteins in sperm lysates and to inhibit the biological response of the steroid [36]. With both tools, we identified two specific progesterone-binding proteins with molecular weights of '54 and 57 kDa (Fig. 2). These results were not dissimilar to those reported previously by Sauber et al. [36] using a-c262 antibody and by Benoff et al. (37) using peroxidase-conjugated P, who also showed the presence of a protein in the 52–58 kDa range [36,37]. We do not know, at present, whether the two protein bands identified with ligand and Western blots [9] represent the two binding sites identified with ligand binding technique, however, the inhibitory activity of a-c262 on P-induced calcium influx [36] and AR [9,36,9] makes this possibility very attractive. To induce AR, sperm receptor for the stimulating agonist must be appropriately located on the acrosome. By using FITC-BSA-conjugated P, Tesarik et al. [38,39] demonstrated the surface localization of sperm binding site for this probe. In addition, these authors demonstrated a migration of the binding site at the level of the equatorial segment after occurrence of AR [39]. Similar results were obtained by Benoff et al. [37]. In contrast, Sauber et al. [36] using a-c262 antibody demonstrated localization of the sperm P receptor at the equatorial segment in acrosome-intact spermatozoa. In addition, using the same probe as Tesarik et al. [38,39], Aitken et al. [32] demonstrated selective localization at the acrosomal region in a small proportion of live spermatozoa, however, the pattern was quite similar to that observed with BSA-FITC preparation (free of P) and did not correlate with any of the biological effects of the steroid. Our preliminary results using FITC-BSA– conjugated P to localize the P receptor in human sperm demonstrated no differences between binding of the steroid-containing probe and the BSA-FITC one, although redistribution of the binding to the equatorial segment in acrosome reacted sperm was observed (Francavilla et al., unpublished results). Similar results were obtained recently by Medrano et al. [40]. It appears that FITC-BSA– conjugated P is not the appropriate tool to study the localization of the sperm P receptor, and we probably have to wait for sequencing, cloning, and development of a specific antibody to answer this important question.

3. Are GABAA receptors involved in the action of P? As mentioned above, at least in the central nervous system, nongenomic effects of P are mediated by interaction with GABAA receptors [2]. In search for a possible interaction of P with GABAA receptors in human sperm, we tested the effects of GABA, GABAA receptors agonists, and

146

E. Baldi et al./Steroids 64 (1999) 143–148

Fig. 2. (A) Ligand blot analysis of whole human sperm lysates using peroxidase-conjugated progesterone to reveal P-binding proteins. (B,C) Western blot analysis of sperm proteins with antibodies directed respectively against the ligand binding region (a-c262, B) and N-Terminal Binding Region (aPR, C) of the progesterone genomic receptor. Note the identification of the same two proteins (indicated by the arrows) in A and B. From Luconi M, Bonaccorsi L, Maggi M, Pecchioli P, Krausz C, Forti G, Baldi E. Identification and characterization of functional nongenomic progesterone receptors on human sperm membranes. J Clin Endocrinol Metab 1998;83:877–90. Reprinted with permission.

antagonists on sperm [Ca21]i increase stimulated by P [12]. We found a little ('20%) inhibition of P response with bicuculline and picrotoxin, two GABAA receptors antagonists, whereas GABA or the pharmacological GABAA agonist muscimol were not able to increase sperm [Ca21]i [9,12] or to stimulate acrosomal exocytosis [9,32]. A wide range of GABA concentrations was also tested for the ability to displace P-11a-glucuronide-[125I]iodotyramine binding in human sperm [9], but no displacement was obtained at any concentration. Other authors, however, reported effects of GABA or GABA agonists on AR and motility or GABAA antagonists on calcium waves induced by P [41– 44]. In addition, GABAA receptor/chloride channel has been found in human sperm [19,41]. It is possible that this channel is somehow unusual with respect to the neuronal one [42]; indeed, it requires long capacitation time to be activated in order to became responsive to GABA and shows quite different kinetic parameters (it is activated only by low GABA concentrations, 0.5–3 mM). However, in our hands, GABA was not able to stimulate AR or calcium influx even in these conditions ([9], unpublished results). Also, it has been shown that GABA-induced AR is dependent on influx of calcium [42]. It is worth noting, however, that so far nobody has shown sperm calcium waves in response to this agonist [9,12,45], even by using single cell

[Ca21]i evaluation [32]. Thus, we conclude that GABAA receptor/chloride channels may be involved in chloride efflux stimulated by P (an effect that does not appear to be mediated by the two P binding sites found in our experiments—see above), while calcium influx is mediated by pathways that do not involve GABA receptors.

4. Clinical implications of nongenomic effects of P on spermatozoa The clinical significance of the rapid actions of P on spermatozoa has been investigated in several studies. It was initially found by our and other groups that sperm responsiveness to P is reduced in oligozoospermic and infertile patients [46 – 48], suggesting a possible involvement of the sperm progesterone receptor in oocyte fertilization. We investigated whether responsiveness to progesterone could represent a reliable test of sperm fertilizing ability by studying nearly 100 unselected couples undergoing in vitro fertilization. We found that both intracellular calcium increase and AR in response to P (ARPC) were significantly correlated to sperm fertilizing ability [49] and revealed to be predictive of fertilization outcome at determined cut-off values [50]. In particular, we obtained positive predictive

E. Baldi et al./Steroids 64 (1999) 143–148

values .90% for both intracellular calcium increase 1.2fold and AR inducibility .7%. Both tests were highly sensitive, moderately specific, and highly reproducible [50]. Positive predictive values may rise .95% when the two tests are combined. Recently, these data have been confirmed by others authors [51–53]. Since the analysis of sperm responsiveness to P is quite simple and highly specific, it does not require expensive equipment and, for the evaluation of [Ca21]i is totally objective, we believe that assessment of sperm responsiveness to P may be of clinical usefulness for the preliminary assessment of the male partner to select the appropriate assisted reproductive technique.

References [1] Seyle H. Anhestetic effects of steroids hormones. Proc Soc Biol Med 1941;46:116 – 8. [2] Lan NC, Bolger MB, Gee KW. Identification and characterization of a pregnane steroid recognition site that is functionally coupled to an expressed GABAA receptor. Neurochem Res 1991;16:347–56. [3] Revelli A, Massobrio M, Tesarik J.Nongenomic actions of steroid hormones in reproducive tissues. Endocrine Rev 1998;19:3–17. [4] Baldi E, Luconi M, Bonaccorsi L, Krausz C, Forti G. Human sperm activation during capacitation and acrosome reaction: role of calcium, protein phosphorylation and lipid remodelling pathways. Front Biosci 1996;1:d189 –205. [5] Osman RA, Andria ML, Jones AD, Meizel S. Steroid induced exocytosis: the human sperm acrosome reaction. Biochem Biophys Res Commun 1989;160:828 –33. [6] Blackmore PF, Lattanzio F. Cell surface localization of a novel non-genomic progesterone receptor on the head of human sperm. Biochem Biophys Res Commun 1991;181:31– 6. [7] Balckmore PF, Neulen J, Lattanzio F, Beebe SJ. Cell surface binding sites for progesterone mediate calcium uptake in human sperm. J Biol Chem 1991;266:18655–9. [8] Castilla JA, Gil T, Molina J, Hortas ML, Rodriguez F, Torres-Mun˜oz J, Vergara F, Herruzo AJ. Undetectable expression of genomic progesterone receptor in human spermatozoa. Hum Reprod 1995;10: 1757–1760,. [9] Luconi M, Bonaccorsi L, Maggi M, Pecchioli P, Krausz C, Forti G, Baldi E. Identification and characterization of functional nongenomic progesterone receptors on human sperm membranes. J Clin Endocrinol Metab 1998;83:877–90. [10] De Lamirande E, Harakat A, Gagnon C. Human sperm capacitation induced by biological fluids and progesterone, but not by NADH or NADPH, is associated with the production of superoxide anion. J Androl 1998;19:215–25. [11] Uhler ML, Leung A, Chan SYW, Wang C. Direct effects of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction. Fertil Steril 1992;58:1191– 8. [12] Baldi E, Casano R, Falsetti C, Krausz Cs, Maggi M, Forti G). Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa. J Androl 1991;12:323–30. [13] Roldan R S, Murase T, Shi Q-X. Exocytosis in spermatozoa in response to progesterone and zona pellucida. Science 1994;266:1578 – 81. [14] Cheng FP, Fazeli AR, Voorhour WF, Tremoleda JL, Bevers MM, Colenbrander B. Progesterone in mare follicular fluid induces the acrosome reaction in stallion spermatozoa and enhances in vitro binding to the zona pellucida. Int J Androl 1998;21:57– 66. [15] Blackmore PF, Beebe SJ, Danforth DR, Alexander N). Progesterone and 17-hydroxyprogesterone. Novel stimulators of calcium influx in human sperm. J Biol Chem 1990;265:1376 – 80.

147

[16] Tesarik J, Moos J, Mendoza C. Stimulation of protein tyrosine phosphorylation by a progesterone receptor on the cell surface of human sperm. Endocrinol 1993;133:328 –35. [17] Luconi M, Bonaccorsi L, Krausz Cs, Gervasi G, Forti G, Baldi E. Stimulation of protein tyrosine phosphorylation by platelet-activating factor and progesterone in human spermatozoa. Mol Cell Endocrinol 1995;108:35– 42. [18] Emiliozzi C, Cordonier H, Guerin JE, Ciapa B, Benchaib M, Fenichel P. Effects of progesterone on human spermatozoa prepared for invitro fertilization. Int J Androl 1996;19:39 – 47. [19] Meizel S. Amino acid neurotransmitter receptor/chloride channels of mammalian sperm and the acrosome reaction. Biol Reprod 1997;56: 569 –74. [20] Murase T, Roldan ERS. Progesterone and zona pellucida activate different trasducing pathways in the sequence of events leading to diacylglicerol generation during mouse sperm acrosomal exocytosis. Biochem J 1996;320:1017–23. [21] Bonaccorsi L, Luconi M, Forti G, Baldi E. Tyrosine kinase inhibition attenuates the plateau phase of calcium increase in response to progesterone in human spermatozoa. FEBS Lett 1995;364: 83– 6. [22] Meizel S, Turner KO. Chloride efflux during the progesterone-initiated human sperm acrosome reaction is inhibited by lavendustin A, a tyrosine kinase inhibitor. J Androl 1996;17:327–33. [23] Tesarik J, Carreras A, Mendoza C. Single cell analysis of tyrosine kinase dependent and independent Ca21 fluxes in progesterone induced acrosome reaction. Mol Hum Reprod 1996;2:225–32. [24] O’Toole CMB, Roldan ERS, Fraser LR. Protein kinase C activation during progesterone-stimulated acrosomal exocytosis in human spermatozoa. Mol Hum Reprod 1996;2:921–7. [25] Bonaccorsi L, Krausz C, Pecchioli P, Forti G, Baldi E. Progesteronestimulated intracellular calcium increase in human spermatozoa is calcium-independent. Mol Hum Reprod 1998;4:259 – 68. [26] Luconi M, Krausz C, Barni T, Vannelli GB, Forti G, Baldi E. Progesterone stimulates p42 extracellular-signal regulated kinase (p42ERK) in human spermatozoa. Mol Hum Reprod 1998;4:251– 8. [27] Luconi M, Barni T, Vannelli GB, Krausz C, Marra F, Benedetti PA, Evangelista V, Francavilla S, Properzi G, Forti G, Baldi E. Extracellular-signal regulated kinases modulate capacitation of human spermatozoa. Biol Reprod 1998;58:1476 – 89. [28] Morte C, Iborra A, Martinez P. Phosphorylation of Shc proteins in human sperm in response to capacitation and progesterone treatment. Mol Reprod Dev 1998;50:113–20. [29] Blackmore PF. Thapsigargin elevates and potentiates the ability of progesterone to increase intracellular free calcium in human sperm: possible role of perinuclear calcium. Cell Calcium 1992;14:53– 60. [30] Walensky LD, Snyder SH. Inositol 1,4,5-trisphosphate receptors selectively localized to the acrosomes of mammalian sperm. J Cell Biol 1995;130:857– 69. [31] Shivaji S, Jagannadham MV. Steroid-induced perturbations of membranes and its relevance to sperm acrosome reaction. Biochim Biophys Acta 1992;1108:99 –109. [32] Aitken RJ, Buckingham DW, Irvine DS. The extragenomic action of progesterone on human spermatozoa: evidence for ubiquitous response that is rapidly down-regulated. Endocrinol 1996;137:3999 – 4009. [33] Aitken RJ, Harkiss D, Knox W, Patersonand M, Irvine S. On the cellular mechanism by which the bicarbonate ion mediates the extragenomic action of progesterone on human spermatozoa. Biol Reprod 1998;58:186 –96. [34] Blackmore PF, Fisher JF, Spilman CH, Bleasdale JE. Unusual steroid specificity of the cell surface progesterone receptor on human sperm. Mol Pharmacol 1996;49:727–39. [35] Neulen J, Ishikawa K, Blackmore PF, Beebe SJ. Identification and characterization of progesterone binding proteins in human semen. Endocrine J 1993;1:397– 404.

148

E. Baldi et al./Steroids 64 (1999) 143–148

[36] Sauber K, Edwards DP, Meizel S. Human sperm plasma membrane progesterone receptor(s) and the acrosome reaction. Biol Reprod 1996;54:993–1001. [37] Benoff S, Rushbrook JI, Hurley IR, Mandel F, Barcia M, Cooper GW, Hershlag A. Co-expression of mannose-ligand and non-nuclear progesterone receptors on motile human sperm identifies and acrosome-reaction inducible subpopulation. Am J Reprod Immunol 1995; 34:100 –15. [38] Tesarik J, Mandoza C, Moos J, Carreras A. Selective expression of a progesterone receptor on the human sperm surface. Fertil Steril 1992; 58:784 –92. [39] Tesarik J, Menodza C. Insights into the function of a sperm-surface progesterone receptor:evidence of ligand-induced receptor aggregation and the implication of proteolysis. Exp Cell Res 1993;205:111–7. [40] Medrano A, Holt WV, Watson PF. Localization of progesteroneBSA-FITC and BSA-FITC probes on the pig sperm plasma membrane during incubation at 39°C and cooling at 5°C. Eighth International Symposium on Spermatology, Montreal, Canada, August 17– 22, 1993, Abstract book. p. 102. [41] Wistrom CA, Meizel S. Evidence suggesting the involvement of a unique human sperm steroid receptor/Cl2 channel complex in the progesterone-initiated acrosome reaction. Dev Biol 1993;159:679 – 90. [42] Shi Q-X, Yuan Y-Y, Roldan ERS. g-Aminobutyric acid (GABA) induces the acrosome reaction in human spermatozoa. Mol Hum Reprod 1997;3:677– 83. [43] Meizel S, Turner KO, Nuccitelli R. Progesterone triggers a wave of increased free calcium during the human sperm acrosome reaction. Dev Biol 1997;182:67–75. [44] Calogero AE, Hall J, Fishel S, Green S, Hunter A, D’Agata R. Effects of gamma-aminobutyric acid on human sperm motility and hyperactivation. Mol Hum Reprod 1996;2:733– 8. [45] Blackmore PF, Im WB, Bleasdale JE. The cell surface progesterone receptor which stimulates calcium influx in human sperm is unlike the

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

A ring reduced steroid site on the GABAA receptor/chloride channel. Mol Cell Endocrinol 1994;104:237– 43. Falsetti C, Baldi E, Krausz C, Casano R, Failli P, Forti G. Decreased responsiveness to progesterone in oligozoospermic patients. J Androl 1993;14:17–22. Tesarik J, Mendoza C. Defective function of a nongenomic progesterone receptor as a sole sperm anomaly in infertile patients. Fertil Steril 1992;58:793–7. Oehninger S, Blackmore PF, Morshedi M, Sueldo C, Acosta A, Alexander NJ. Defective calcium influx and acrosome reaction (spontaneous and progesterone-induced) in spermatozoa of infertile men with severe teratozoospermias. Fertil Steril 1994;61:349 –54. Krausz C, Bonaccorsi L, Luconi M, Fuzzi B, Criscuoli L, Pellegrini S, Forti G, Baldi E. Intracellular calcium increase and acrosome reaction in response to progesterone in human spermatozoa is correlated with in vitro fertilization. Hum Reprod 1995;10:120 – 4. Krausz C, Bonaccorsi L, Maggio P, Luconi M, Fuzzi B, Criscuoli L, Pellegrini S, Forti G, Baldi E. Intracellular calcium increase and acrosome reaction in response to progesterone in human spermatozoa is correlated with in vitro fertilization. Hum Reprod 1996;11:1161–7. Benoff S, Hurley IR, Mandel FS, Paine T, Jacob A, Cooper GW, Hershlag A. Use of mannose ligands in IVF screens to mimic zona pellucida-induced acrosome reactions and predict fertilization success. Mol Hum Reprod 1997;3:839 – 46. Misao R, Miwa K, Morishita S, Fujimoto J, Nakanishi Y, Tamaya T. Immunohistochemical detection of estrogen and progesterone receptors in spermatozoa of infertile men. Int J Fertil Womens Med 1997;42:421–5. Jacob A, Hurley I, Mandel FS, Hershlag A, Cooper GW, Benoff S. Human sperm non-nuclear progesterone receptor expression is a novel marker of fertilization outcome. Mol Hum Reprod 1998;4:533– 542.