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Progesterone at the picomolar range is a chemoattractant for mammalian spermatozoa

2006; Elsevier BV; Volume: 86; Issue: 3 Linguagem: Inglês

10.1016/j.fertnstert.2006.02.080

1556-5653

María E. Teves, Flavia Barbano, Héctor Alejandro Guidobaldi, Raúl Sánchez, W. Miska, Laura C. Giojalas,

Olfactory and Sensory Function Studies

By means of a videomicroscopy system and a computer image analysis, we performed chemotaxis assays to detect true chemotaxis in human spermatozoa, in parallel to immunohistochemistry detection of progesterone inside the cumulus cells. Progesterone indeed chemotactically guides mammalian spermatozoa at very low hormone concentrations, and the cumulus oophorus could be a potential place for sperm chemotaxis mediated by progesterone in vivo. By means of a videomicroscopy system and a computer image analysis, we performed chemotaxis assays to detect true chemotaxis in human spermatozoa, in parallel to immunohistochemistry detection of progesterone inside the cumulus cells. Progesterone indeed chemotactically guides mammalian spermatozoa at very low hormone concentrations, and the cumulus oophorus could be a potential place for sperm chemotaxis mediated by progesterone in vivo. Sperm chemotaxis is a cell transport mechanism that guides spermatozoa up an attractant concentration gradient (1Eisenbach M. Chemotaxis. Imperial College Press, London2004Google Scholar). In recent years, by means of approaches that differentiate chemotaxis from other cell-accumulation processes, sperm chemotaxis toward follicular fluid was observed in humans (2Ralt D. Manor M. Cohen-Dayag A. Tur-Kaspa I. Ben-Shlomo I. Makler A. et al.Chemotaxis and chemokinesis of human spermatozoa to follicular factors.Biol Reprod. 1994; 50: 774-785Crossref PubMed Scopus (119) Google Scholar), mice (3Oliveira R.G. Tomasi L. Rovasio R.A. Giojalas L.C. Increased velocity and induction of chemotactic response in mouse spermatozoa by follicular and oviductal fluids.J Reprod Fertil. 1999; 115: 23-27Crossref PubMed Scopus (63) Google Scholar), and rabbits (4Fabro G. Rovasio R.A. Civalero S. Frenkel A. Caplan R. Eisenbach M. et al.Chemotaxis of capacitated rabbit spermatozoa to follicular fluid revealed by a novel directionality-based assay.Biol Reprod. 2002; 67: 1565-1571Crossref PubMed Scopus (90) Google Scholar) and toward oviductal fluid in mice (3Oliveira R.G. Tomasi L. Rovasio R.A. Giojalas L.C. Increased velocity and induction of chemotactic response in mouse spermatozoa by follicular and oviductal fluids.J Reprod Fertil. 1999; 115: 23-27Crossref PubMed Scopus (63) Google Scholar). Further characterization of mammalian sperm chemotaxis showed that the chemotactic response was expressed by a small sperm subpopulation (approximately 10%), consisting of capacitated spermatozoa (4Fabro G. Rovasio R.A. Civalero S. Frenkel A. Caplan R. Eisenbach M. et al.Chemotaxis of capacitated rabbit spermatozoa to follicular fluid revealed by a novel directionality-based assay.Biol Reprod. 2002; 67: 1565-1571Crossref PubMed Scopus (90) Google Scholar, 5Cohen-Dayag A. Tur-Kaspa Y. Dor J. Mashiach S. Eisenbach M. Sperm capacitation in humans is transient and correlates with chemotactic responsiveness to follicular factors.Proc Natl Acad Sci U S A. 1995; 92: 11039-11043Crossref PubMed Scopus (183) Google Scholar, 6Giojalas L.C. Rovasio R.A. Fabro G. Gakamsky A. Eisenbach M. Timing of sperm capacitation appears to be programmed according to egg availability in the female genital tract.Fertil Steril. 2004; 82: 247-249Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Follicular and oviductal fluids are mixtures of different molecule types (from peptides to hormones). Therefore, a long-standing question is: Which is/are the chemoattractant molecule(s) in these fluids? Progesterone, the main steroid present in the egg microenvironment by the time of ovulation, has been assayed for human sperm chemotaxis by different groups, giving rise to contradictory results (7Sliwa L. Effect of some sex steroid hormones on human spermatozoa migration in vitro.Eur J Obstet Gynecol Reprod Biol. 1995; 58: 173-175PubMed Google Scholar, 8Villanueva-Diaz C. Arias-Martinez J. Bermejo-Martinez L. Vadillo-Ortega F. Progesterone induces human sperm chemotaxis.Fertil Steril. 1995; 64: 1183-1188Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 9Wang Y. Storeng R. Dale P.O. Abyholm T. Tanbo T. Effects of follicular fluid and steroid hormones on chemotaxis and motility of human spermatozoa in vitro.Gynecol Endocrinol. 2001; 15: 286-292PubMed Google Scholar, 10Jeon B.G. Moon J.S. Kim K.C. Lee H.J. Choe S.Y. Rho G.J. Follicular fluid enhances sperm attraction and its motility in human.J Assist Reprod Gen. 2001; 18: 407-412Crossref PubMed Scopus (22) Google Scholar, 11Jaiswal B.S. Tur-Kaspa I. Dor J. Mashiach S. Eisenbach M. Human sperm chemotaxis: is progesterone a chemoattractant?.Biol Reprod. 1999; 60: 1314-1319Crossref PubMed Scopus (69) Google Scholar). Clarification of this issue would shed light on the mechanism of sperm transportation, with relevant implications in reproductive medicine for the treatment of male/female infertility and/or contraception. In the last decade, four groups reported that P is a chemoattractant for human spermatozoa (7Sliwa L. Effect of some sex steroid hormones on human spermatozoa migration in vitro.Eur J Obstet Gynecol Reprod Biol. 1995; 58: 173-175PubMed Google Scholar, 8Villanueva-Diaz C. Arias-Martinez J. Bermejo-Martinez L. Vadillo-Ortega F. Progesterone induces human sperm chemotaxis.Fertil Steril. 1995; 64: 1183-1188Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 9Wang Y. Storeng R. Dale P.O. Abyholm T. Tanbo T. Effects of follicular fluid and steroid hormones on chemotaxis and motility of human spermatozoa in vitro.Gynecol Endocrinol. 2001; 15: 286-292PubMed Google Scholar, 10Jeon B.G. Moon J.S. Kim K.C. Lee H.J. Choe S.Y. Rho G.J. Follicular fluid enhances sperm attraction and its motility in human.J Assist Reprod Gen. 2001; 18: 407-412Crossref PubMed Scopus (22) Google Scholar), whereas another group suggested that the hormone either is not a sperm chemoattractant or is a weak chemoattractant (11Jaiswal B.S. Tur-Kaspa I. Dor J. Mashiach S. Eisenbach M. Human sperm chemotaxis: is progesterone a chemoattractant?.Biol Reprod. 1999; 60: 1314-1319Crossref PubMed Scopus (69) Google Scholar). All these studies based their conclusions on assays run at high concentration ranges of P (μmol/L to mmol/L). Furthermore, those studies claiming a positive chemotactic response to P (7Sliwa L. Effect of some sex steroid hormones on human spermatozoa migration in vitro.Eur J Obstet Gynecol Reprod Biol. 1995; 58: 173-175PubMed Google Scholar, 8Villanueva-Diaz C. Arias-Martinez J. Bermejo-Martinez L. Vadillo-Ortega F. Progesterone induces human sperm chemotaxis.Fertil Steril. 1995; 64: 1183-1188Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 9Wang Y. Storeng R. Dale P.O. Abyholm T. Tanbo T. Effects of follicular fluid and steroid hormones on chemotaxis and motility of human spermatozoa in vitro.Gynecol Endocrinol. 2001; 15: 286-292PubMed Google Scholar, 10Jeon B.G. Moon J.S. Kim K.C. Lee H.J. Choe S.Y. Rho G.J. Follicular fluid enhances sperm attraction and its motility in human.J Assist Reprod Gen. 2001; 18: 407-412Crossref PubMed Scopus (22) Google Scholar) used assays that could not differentiate chemotaxis from other sperm-accumulation processes. To determine unequivocally whether P is a sperm chemoattractant, we chose experimental conditions that allowed us to clearly determine chemotaxis. These conditions were: [1] a chemotactic parameter (% of oriented cells) that determines chemotaxis independently of variations in the sperm speed or in the acquisition of a hyperactivated motility (4Fabro G. Rovasio R.A. Civalero S. Frenkel A. Caplan R. Eisenbach M. et al.Chemotaxis of capacitated rabbit spermatozoa to follicular fluid revealed by a novel directionality-based assay.Biol Reprod. 2002; 67: 1565-1571Crossref PubMed Scopus (90) Google Scholar), because the latter processes can also cause sperm accumulation (1Eisenbach M. Chemotaxis. Imperial College Press, London2004Google Scholar); [2] a high number of analyzed spermatozoa per treatment in order to detect the small chemotactic sperm subpopulation, because in mammals approximately 10% of the cells are involved (1Eisenbach M. Chemotaxis. Imperial College Press, London2004Google Scholar); [3] cell suspensions enriched with capacitated spermatozoa, because only those cells are able of chemotactic response (4Fabro G. Rovasio R.A. Civalero S. Frenkel A. Caplan R. Eisenbach M. et al.Chemotaxis of capacitated rabbit spermatozoa to follicular fluid revealed by a novel directionality-based assay.Biol Reprod. 2002; 67: 1565-1571Crossref PubMed Scopus (90) Google Scholar, 5Cohen-Dayag A. Tur-Kaspa Y. Dor J. Mashiach S. Eisenbach M. Sperm capacitation in humans is transient and correlates with chemotactic responsiveness to follicular factors.Proc Natl Acad Sci U S A. 1995; 92: 11039-11043Crossref PubMed Scopus (183) Google Scholar, 6Giojalas L.C. Rovasio R.A. Fabro G. Gakamsky A. Eisenbach M. Timing of sperm capacitation appears to be programmed according to egg availability in the female genital tract.Fertil Steril. 2004; 82: 247-249Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar); [4] follicular fluid as a well established positive control (1Eisenbach M. Chemotaxis. Imperial College Press, London2004Google Scholar); and [5] a wide range of P concentrations (0.1 pmol/L to 1 mmol/L) not fully investigated in the past. The low level of P ranges was chosen because the human sperm chemotactic response to specific attractants such as burgeonal (12Spehr M. Gisselmann G. Poplawski A. Riffell J.A. Wetzel C.H. Zimmer R.K. Hatt H. Identification of a testicular odorant receptor mediating human sperm chemotaxis.Science. 2003; 299: 2054-2058Crossref PubMed Scopus (580) Google Scholar) and RANTES chemokine (13Isobe T. Minoura H. Tanaka K. Shibahara T. Hayashi N. Toyoda N. The effect of RANTES on human sperm chemotaxis.Hum Reprod. 2002; 17: 1441-1446Crossref PubMed Scopus (55) Google Scholar) was elicited at low attractant concentrations. Therefore, we assumed that, if a P-mediated chemotactic response does occur in spermatozoa, it should be observed at low attractant levels. For comparison with earlier studies that have given rise to the controversy regarding P sperm guidance, we also assayed higher (though nonphysiologic) P concentrations. Human spermatozoa chemotactically responded to a concentration gradient generated from 1 to 100 pmol/L of P. The results, which showed the expected bell-shaped dose-response curve (typical of a chemotactic response of any cellular model [1Eisenbach M. Chemotaxis. Imperial College Press, London2004Google Scholar]), were statistically different from the negative control (P<.001) but not from the positive control, follicular fluid (Fig. 1A). The maximal level of chemotactic spermatozoa was detected at 10 pmol/L of P, which was elicited by a small sperm subpopulation (8% vs. the control group; Fig. 1A), in line with the mean level of capacitated spermatozoa (12%, data not shown). A smaller second chemotactic peak was observed at about 1 μmol/L of P (P<.03; Fig. 1A). Between 0 and 1 μmol/L P there was no significant increase in the sperm velocity nor in the induction of the hyperactivated pattern of movement (Fig. 1B). Similar results were observed in the rabbit, although the maximum chemotactic response was at 100 pmol/L of P (data not shown). On the other hand, at high P concentration (10 μmol/L to 1 mmol/L), where no chemotactic signal was detected, a significant increase in the level of human hyperactivated spermatozoa was observed (P<.001; Fig. 1B), in agreement with other reports (14Calogero A.E. Burrello N. Palermo I. Grasso U. D'Agata R. Effects of progesterone on sperm function: mechanisms of action.Hum Reprod. 2000; 15: 128-145Crossref Scopus (75) Google Scholar). Earlier studies that reported a positive chemotactic response of human spermatozoa toward P (7Sliwa L. Effect of some sex steroid hormones on human spermatozoa migration in vitro.Eur J Obstet Gynecol Reprod Biol. 1995; 58: 173-175PubMed Google Scholar, 8Villanueva-Diaz C. Arias-Martinez J. Bermejo-Martinez L. Vadillo-Ortega F. Progesterone induces human sperm chemotaxis.Fertil Steril. 1995; 64: 1183-1188Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 9Wang Y. Storeng R. Dale P.O. Abyholm T. Tanbo T. Effects of follicular fluid and steroid hormones on chemotaxis and motility of human spermatozoa in vitro.Gynecol Endocrinol. 2001; 15: 286-292PubMed Google Scholar, 10Jeon B.G. Moon J.S. Kim K.C. Lee H.J. Choe S.Y. Rho G.J. Follicular fluid enhances sperm attraction and its motility in human.J Assist Reprod Gen. 2001; 18: 407-412Crossref PubMed Scopus (22) Google Scholar) used variants of an accumulation assay in which chemotaxis cannot be successfully distinguished from chemokinesis and hyperactivation. In the present study, the hyperactivation effect of P was observed at high hormone concentrations in the absence of a sperm chemotactic response. Therefore, it is probable that the P-stimulated cell accumulation, observed in some of the earlier studies at the μmol/L–mmol/L range, and which was attributed to chemotaxis (7Sliwa L. Effect of some sex steroid hormones on human spermatozoa migration in vitro.Eur J Obstet Gynecol Reprod Biol. 1995; 58: 173-175PubMed Google Scholar, 8Villanueva-Diaz C. Arias-Martinez J. Bermejo-Martinez L. Vadillo-Ortega F. Progesterone induces human sperm chemotaxis.Fertil Steril. 1995; 64: 1183-1188Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 9Wang Y. Storeng R. Dale P.O. Abyholm T. Tanbo T. Effects of follicular fluid and steroid hormones on chemotaxis and motility of human spermatozoa in vitro.Gynecol Endocrinol. 2001; 15: 286-292PubMed Google Scholar, 10Jeon B.G. Moon J.S. Kim K.C. Lee H.J. Choe S.Y. Rho G.J. Follicular fluid enhances sperm attraction and its motility in human.J Assist Reprod Gen. 2001; 18: 407-412Crossref PubMed Scopus (22) Google Scholar), was mainly due to trapping caused by hyperactivation, in line with the suggestion of Jaiswal et al. (11Jaiswal B.S. Tur-Kaspa I. Dor J. Mashiach S. Eisenbach M. Human sperm chemotaxis: is progesterone a chemoattractant?.Biol Reprod. 1999; 60: 1314-1319Crossref PubMed Scopus (69) Google Scholar). Although Villanueva-Diaz et al. (8Villanueva-Diaz C. Arias-Martinez J. Bermejo-Martinez L. Vadillo-Ortega F. Progesterone induces human sperm chemotaxis.Fertil Steril. 1995; 64: 1183-1188Abstract Full Text PDF PubMed Scopus (68) Google Scholar) assayed almost as low chemoattractant concentrations as those at which we found the highest chemotactic activity, they probably missed the chemotactic signal, because they apparently did not use capacitated spermatozoa in their experiments, which is essential for the detection of sperm chemotaxis (4Fabro G. Rovasio R.A. Civalero S. Frenkel A. Caplan R. Eisenbach M. et al.Chemotaxis of capacitated rabbit spermatozoa to follicular fluid revealed by a novel directionality-based assay.Biol Reprod. 2002; 67: 1565-1571Crossref PubMed Scopus (90) Google Scholar, 5Cohen-Dayag A. Tur-Kaspa Y. Dor J. Mashiach S. Eisenbach M. Sperm capacitation in humans is transient and correlates with chemotactic responsiveness to follicular factors.Proc Natl Acad Sci U S A. 1995; 92: 11039-11043Crossref PubMed Scopus (183) Google Scholar, 6Giojalas L.C. Rovasio R.A. Fabro G. Gakamsky A. Eisenbach M. Timing of sperm capacitation appears to be programmed according to egg availability in the female genital tract.Fertil Steril. 2004; 82: 247-249Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) (Figs. 1C and 1D). In contrast, Jaiswal et al. (11Jaiswal B.S. Tur-Kaspa I. Dor J. Mashiach S. Eisenbach M. Human sperm chemotaxis: is progesterone a chemoattractant?.Biol Reprod. 1999; 60: 1314-1319Crossref PubMed Scopus (69) Google Scholar) concluded that P either is not a sperm chemoattractant or is a weak one. Although those authors performed the chemotaxis assays with capacitated spermatozoa and determined chemotaxis with strict criteria, they studied those P concentrations claimed in earlier studies (7Sliwa L. Effect of some sex steroid hormones on human spermatozoa migration in vitro.Eur J Obstet Gynecol Reprod Biol. 1995; 58: 173-175PubMed Google Scholar, 8Villanueva-Diaz C. Arias-Martinez J. Bermejo-Martinez L. Vadillo-Ortega F. Progesterone induces human sperm chemotaxis.Fertil Steril. 1995; 64: 1183-1188Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 9Wang Y. Storeng R. Dale P.O. Abyholm T. Tanbo T. Effects of follicular fluid and steroid hormones on chemotaxis and motility of human spermatozoa in vitro.Gynecol Endocrinol. 2001; 15: 286-292PubMed Google Scholar, 10Jeon B.G. Moon J.S. Kim K.C. Lee H.J. Choe S.Y. Rho G.J. Follicular fluid enhances sperm attraction and its motility in human.J Assist Reprod Gen. 2001; 18: 407-412Crossref PubMed Scopus (22) Google Scholar) to cause sperm chemotaxis (1–100 μg/mL); however, the concentration range assayed by those authors is orders of magnitude higher than the P chemotactic dose found in the present study (Fig. 1A). In summary, sperm chemotaxis toward P was not definitely demonstrated in the past, because extremely low concentrations of the hormone were not assayed in a system that allowed clear chemotaxis identification. Thus, these two different sperm processes (chemotaxis and hyperactivation) are both stimulated by P but at poles-apart concentrations, low for chemotaxis and high for hyperactivation, in agreement with the presence of high- and low-affinity P receptors on the sperm surface (15Luconi M. Bonaccorsi L. Maggi M. Pecchioli P. Krausz C. Forti G. et al.Identification and characterization of functional nongenomic progesterone receptors on humans sperm membrane.J Clin Endocrilnol Metab. 1998; 83: 877-885Crossref PubMed Scopus (147) Google Scholar). Humans and rabbits are two mammalian species that show different reproductive strategies: periodic ovulation in humans and mating-induced ovulation in the rabbit. These differences are reflected in the kinetics of capacitation, which seems to be programmed according to the differential timing of their own egg availability (6Giojalas L.C. Rovasio R.A. Fabro G. Gakamsky A. Eisenbach M. Timing of sperm capacitation appears to be programmed according to egg availability in the female genital tract.Fertil Steril. 2004; 82: 247-249Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In both species, the follicular-fluid chemotactic ability is acquired during the incubation time for capacitation (1Eisenbach M. Chemotaxis. Imperial College Press, London2004Google Scholar). Therefore, we investigated the correlation between the percentage of induced acrosome-reacted spermatozoa and those that chemotactically respond to P along the incubation time. The cells were confronted with the higher chemotactic concentration of P (10 pmol/L and 100 pmol/L for humans and rabbits, respectively) at different time points during sperm capacitation, and the level of A23187-induced acrosome-reacted cells was determined in parallel. A strong significant correlation between the net proportions of both chemotactic and acrosome-reacted spermatozoa was observed along the time (humans: r2 = 0.98; P<.001; rabbits: r2 = 0.81; P<.01; Figs. 1C and 1D). Therefore, in both species, only capacitated spermatozoa are able to chemotactically respond to P, a sperm guidance mechanism that seems to be rather conserved in mammals. Our in vitro experiments show that P guides mammalian spermatozoa by chemotaxis. However, this conclusion cannot be extended to what may be happening in vivo. By the time of ovulation, P is almost everywhere in the egg microenvironment, but a stable P concentration gradient should be formed for chemotaxis to occur. Recently, it was suggested that if chemotaxis exists in vivo, the most probable site of its occurrence should be along the cumulus oophorus (16Bahat A. Tur-Kaspa I. Gakamsky A. Giojalas L.C. Breitbart H. Eisenbach M. Thermotaxis of mammalian sperm cells: a potential navigation mechanism in the female genital tract.Nat Med. 2003; 9: 149-150Crossref PubMed Scopus (178) Google Scholar), whose particular viscosity and organization could keep a stable chemoattractant gradient. Following ovulation, the cumulus cells synthesize and secrete P (17Yamashita Y. Shimada M. Okazaki T. Maeda T. Terada T. Production of Progesterone from de novo-synthesized cholesterol in cumulus cells and its physiological role during meiotic resumption of porcine oocytes.Biol Reprod. 2003; 68: 1193-1198Crossref PubMed Scopus (93) Google Scholar) and its carrier protein (18Baltes P. Sánchez R. Peña P. Villegas J. Turley H. Miska W. Evidence for the synthesis and secretion of a CBG-like serpin by human cumulus oophorus and fallopian tubes.Andrologia. 1998; 30: 249-253Crossref PubMed Scopus (11) Google Scholar) which makes P soluble once out of the cell. We showed that the cumulus cells producing P are radially distributed (Figs. 1E and 1F), suggesting that, when the hormone is excreted, such cell distribution favors a P gradient formation from the center to the periphery of the cumulus cellular mass. These results raise the possibility that the cumulus oophorus could be a potential place of P-mediated sperm chemotaxis in vivo. In summary, a possible mechanism of action of P in vivo could be as follows: In the vicinity of the cumulus, the low levels of P activate in the spermatozoon the high-affinity P receptors, and, as a consequence, the spermatozoon swims toward the cumulus mass by chemotaxis. Once inside the cumulus, where the P concentration is higher, the low-affinity P receptors are stimulated in the spermatozoon, leading to the acquisition of a hyperactivated pattern of movement, which helps it to pass across the cumulus mass and the zona pellucida. In parallel, an unknown attractant probably secreted by the egg (19Sun F. Bahat A. Girsh E. Katz N. Giojalas L.C. Tur-Kaspa I. et al.Human sperm chemotaxis: both the egg and its surrounding cumulus cells secrete sperm chemoattractants.Hum Reprod. 2005; 20: 761-767Crossref PubMed Scopus (101) Google Scholar), could guide the spermatozoon to the egg surface. The authors thank Diego Uñates (Ph.D. student, UNC-Argentina) for performing some of the rabbit chemotaxis experiments, Peter Koenig (Ph.D., Institute of Anatomy, University of Giessen, Germany) for confocal images, and Roberto A. Rovasio (Ph.D., UNC-Argentina) for critically reading the manuscript.