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Effect of progesterone on human zona pellucida sperm binding and oocyte penetrating capacity
Vol. 60, No. 1, July 1993
FERTILITY AND STERILITY
Printed on acid-free paper in U. S. A.
Copyright " 1993 The American Fertility Society
Effect of progesterone on human zona pellucida sperm binding and oocyte penetrating capacity
Carlos E. Sueldo, M.D.*t Sergio Oehninger, M.D.:j: Elisabet Subias, Ph.D.* Mary Mahony, Ph.D.:j:
Nancy J. Alexander, Ph.D.§ Lani J. Burkman, Ph.D. I\ Anibal A. Acosta, M.D.:j:
University of California San Francisco-Fresno, Fresno, California; Jones Institute for Reproductive Medicine, Eastern Virginia }!edical School, Norfolk, Virginia; National Institutes of Health, Bethesda, Maryland; and Grand Island Biologic Company (GIBCO) Laboratories, Grand Island, New York
Objective: To determine if sperm exposure to P produces an enhancement in its fertilizing capacity. Design: Sperm from fertile donors were exposed toP at 0.1 and 1.0 ~g/mL for 1 or 24 hours. The effects on hyperactivated (HA) motility at 1 and 4 hours, acrosome reaction (as determined by Pisum sativum agglutinin or T6/antibody techniques), on human zona pellucida binding (by using the hemizona assay), and on the penetrating ability (by using the zona-free hamster ova assay) were evaluated. Results: Exposure to P at 1.0 ~g/mL enhanced HA motility after 1 and 4 hours of P exposure, the acrosome reaction after 24 hours' incubation, the number of sperm bound/hemizona after 1-hour incubation, and the penetration rates in the hamster ova assay at both incubation intervals. Conclusion: Sperm exposure to P enhances its fertilizing capacity in fertile men, and further investigation is warranted as a possible treatment for male factor patients. Fertil Steril 1993;60:137-40 Key Words: Progesterone, acrosome reaction, human zona sperm binding, hamster ova penetration, sperm hyperactive motility
The human sperm acrosome reaction involves fusion of the outer acrosomal membrane with the plasma membrane at several sites, releasing the contents of the acrosome to the extracellular space
Received May 1, 1992; revised and accepted March 4, 1993. Presented at the 40th Annual Meeting of the Pacific Coast Fertility Society, Indian Wells, California, April9 to 12, 1992. *Department of Obstetrics and Gynecology, University of California San Francisco-Fresno. t Reprint requests: Carlos E. Sueldo, M.D., Department of Obstetrics and Gynecology, University of California San Francisco-Fresno, Fresno, California 93703. :j: Jones Institute for Reproductive Medicine, Eastern Virginia Medical School. §Contraceptive Development Branch, National Institutes of Health. II GIBCO Laboratories. Vol. 60, No. 1, July 1993
(1). Although the precise time at which the acrosome reaction occurs during the fertilization process is controversial, it is agreed that it has to occur before the sperm can penetrate the zona pellucida (ZP) and fuse with the oocyte plasma membrane (1). An early event involved in the acrosome reaction is an obligatory increase in free cytosolic calcium (2). It has been reported that preovulatory human follicular fluid (FF) enhances the influx of calcium through the sperm cell membrane (3), the number of acrosome reactions (4-7), and the penetrating capacity in the zona-free hamster ova sperm penetration assay (SPA) (5, 8). Osman et al. (9) purified and identified (P) and 17-hydroxyprogesterone in the fraction of human FF responsible for the increment in acrosome reaction. In the present study we evaluated the effect of Sueldo et al.
P and sperm fertilizing capacity
137
P at different concentrations and lengths of exposure on human sperm acrosome reaction and the impact on hyperactivated (HA) motility, human ZP binding, and hamster ova penetrating capacity.
MATERIALS AND METHODS
Semen from fertile donors was used for all the experiments. After a standard swim-up procedure by double centrifugation and incubation at 37°C in 5% C02 in air, sperm were exposed to P (Rugby Laboratory, Long Island, NY) at concentrations of 0.1 ~tg/mL and 1.0 ~tg/mL (diluted in culture media) for either 1 hour or 24 hours and for 1 and 4 hours to assess HA. Acrosome Reaction
The proportion of sperm that has undergone acrosome reaction was determined by two techniques: the Pisum sativum agglutinin as described by Cross et al. (10), assessing sperm integrity with the use of the supravital stain Hoechst 33258 (10) or the T6/antibody with immunofluorescent staining as described by Coddington et al. (11). Pisum sativum agglutinin was applied to sperm exposed to P at 0.1 and 1.0 Jtg/mL during 1 and 24 hours, whereas T6 was only used to sperm exposed to P at 1.0 ~tg/mL for 1 hour. To determine the proportion of sperm that has undergone acrosome reaction, 200 sperm were evaluated under each experimental condition. Human Sperm Hyperactive Motility
The steps in this assay were done as described by Burkman (12). Semen from seven fertile donors were tested after centrifugation and layering of culture media (Ham's F-10; GIBCO, Grand Island, NY); an aliquot of the supernatant was diluted to 2 X 106 motile/mL in culture media for effective videotaping as described (12). Hyperactive motility was then assessed by transmitting the videotaped images to the monitor of a Hamilton Thorn Research Motility Analyzer (HTM model 2030, version 7.0; Damvers, MA) and identifying motility patterns consistant with hyperactivation (13).
oocytes (unfertilized oocytes from the IVF program) were equally bisected as previously described (14). One hemizona was placed in a droplet with sperm exposed to P, whereas the matching hemizona was placed in a droplet with sperm not exposed toP (control). After 4 hours of coincubation (37°C in 5% C0 2 in air), each hemizona was removed and rinsed using a fine glass pipette in medium to separate loosely attached sperm; then the number of tightly bound sperm was counted in each hemizona. Sperm Penetration Assay
The basic steps in this assay were done as already described in our laboratory (15). After a standard swim-up, sperm were exposed to P at 0.1 or 1.0 ~tg for 1 hour or 24 hours before the coincubation with hamster ova free of ZP. The same experimental conditions were followed with a portion of the same sperm sample not exposed toP (control) before gamete coincubation. A motile sperm population of 5 X 106 /mL was used for the SPA, and the gametes were co incubated for 3 hours. The penetration rates were determined by assessing the number of ova penetrated over the number of ova exposed. Statistical Analysis
Student's t-test for the comparison of the means of two samples was used to analyze the hemizona data. Chi-squared analysis was used to compare percentages of acrosome and SPA results. Hyperactive motility was compared by using Wilcoxon's rank test. A P value < 0.05 was considered statistically significant. RESULTS
The percentage of sperm that underwent acrosome reaction under each experimental condition is shown in Table 1. Because there was no statistical difference between the two techniques used to assess acrosomalloss, results in Table 1 represent at 1.0 Jtg both groups combined. Progesterone at 1.0
Table 1
Effect of P on Sperm Acrosome Reaction*
Hemizona Assay (HZA)
After exposure to P at 1.0 ~tg/mL concentration for either 1 or 24 hours, a 100-~tL droplet of the sperm suspension (0.5 X 106 motile sperm/mL) was placed in a Petri dish under oil. By micromanipulation (Narishige, Tokyo, Japan) salt-stored human 138
Sueldo et al.
P and sperm fertilizing capacity
Control (free of P) (n = 11) Pat 0.1 !Lg/mL (n = 7) P at 1.0 !Lg/mL (n = 11)
1-h incubation
24-h incubation
10 ± 2.1 11 ± 2.0 18 ± 2.9
9 ± L7t 12 ± 3.1 30 ± 2.9t
*Values are means± SD. t p < 0.01.
Fertility and Sterility
Table 2
Effect of P on Sperm HA Motility*
Hyperactive motility
Controlt
P:j:
11.1 ± 6.5 7.2 ± 4.2
15.7 ± 9.0§ 11.2 ± 6.1§
h
1 (n 4 (n
= =
7) 7)
* Values are mean percents ± SD. t Free of P. :j: 1.0 f.lg/mL. § P < 0.05 compared with control.
J.Lg/mL increased the number of acrosome reaction in comparison with control conditions (free of P exposure), yet this difference became significant only after sperm exposure to P during 24 hours, 30 ± 2.9 versus 9.0 ± 1.7 P < 0.01. Progesterone at 0.1 J.Lg did not have any effect on acrosome reaction as compared with control. Results on HA motility are shown in Table 2 and expressed in percentage of sperm evaluated showing hyperactive motility pattern. At 1 and 4 hours of P exposure at 1.0 J.Lg/mL, sperm showed significantly increased hyperactive motility compared with control conditions (free of P) 15.7 ± 9.0 and 11.2 ± 6.1 versus 11.1 ± 6.5 and 7.2 ± 4.2, respectively, P < 0.05. The results of the HZA are expressed by the number of tightly bound sperm per hemizona (mean ± SEM). Progesterone at 1.0 J.Lg/mL during 1-hour incubation significantly increased the number of tightly bound sperm per hemizona in comparison with control conditions (free of P exposure) 52.6 ± 7.2 versus 35.3 ± 5.5, respectively, P < 0.01. On the other hand at 24 hours, both experimental conditions showed a very low binding, but there was no difference between P and control, 4.5 ± 3.2 versus 4.0 ± 1.2, respectively, (not significant). Results of the SPA are expressed by the penetration rates, representing the number of ova penetrated per number of ova exposed (mean ± SD). Progesterone at 1.0 J.Lg/mL significantly increased ova penetration in comparison with control conditions at 1 hour (60% ± 3.6% versus 31% ± 2.0%, P < 0.05) and at 24 hours (66.0% ± 3.6% versus 39.1% ± 2.7% P < 0.05). Progesterone at 0.1 J.Lg/mL did not show any effect on the SPA compared with control conditions.
DISCUSSION
Progesterone in this study was able to increase the number of sperm acrosome reaction, HA sperm, the number of sperm bound to the human ZP, and the penetrating ability in the zona-free hamster ova Vol. 60, No. 1, July 1993
system. These effects were only shown when P was used at 1.0 J.Lg/mL, a concentration previously used by Osman et al. (9), shown to enhance sperm acrosome reaction. These authors demonstrated that the concentration of P within the cumulus matrix is in excess of 1 J.Lg/mL, enough to elicit an increase in cytosolic calcium, an obligatory event leading to the occurrence of acrosome reaction. Blackmore and co-workers (2) demonstrated that P at 1.0 J.Lg/mL produced a maximum response in rapid calcium passage through the plasma membrane by a mechanism that is not yet completely clear. This finding is similar to the calcium passage enhancement produced by human preovulatory FF, rich in P, as reported by Thomas and Meizel (3). There is evidence that during the normal fertilization process the acrosome reaction occurs when the acrosome-in· tact sperm comes in contact with a component of the ZP known as ZP3 (16); whether P interacts synergistically with ZP3 as speculated by Blackmore (2) remains to be determined. In our study, an increment in acrosome reaction by P was only shown after a long incubation (24 hours) in agreement with data reported by Suarez et al. (7) and Stock et al. (17) using FF; this finding might explain the increase SPA rates observed after 24 hours of P incubation. On the other hand, the increased ZP binding and SPA rates after short incubation (1 hour) with P at 1.0 J.Lg/mL is possibly due to the increased HA motility observed in this study. It has been reported that capacitating sperm near the time of fertilization express a distinct motility pattern known as hyperactivation and characterized by vigorous flagellar movements, marked lateral displacement of the sperm head, and a nonprogressive trajectory (18). There is evidence that the incidence of hyperactivation correlates with sperm fertilizing capacity in vitro (19) and sperm binding to the ZP (16). Mbizvo et al. (18) demonstrated that human preovulatory FF enhanced HA motility, whereas this effect was abolished by the use of a FF fraction stripped of steroids. In assessing the sperm motility patterns with and without P exposure in our study, it became evident by computerized sorting with the HTM analyzer as described by Burkman (12), that P significantly enhanced HA motility at a concentration of 1.0 J.Lg/mL. We therefore concluded that HA motility after a short incubation in P was greatly enhanced, and this finding explains the increased tightly bound sperm to the human ZP and the enhanced penetration in the hamster ova system in agreement with Coddington et al. (14) and Jinno et al. (19), respectively. Whether the enhancement in sperm fertilizing capacity after Sueldo et al.
P and sperm fertilizing capacity
139
exposure to P shown in this study will be manifested in the presence of poor quality semen remains to be determined, and studies are currently underway to evaluate the possible role of P in the treatment of male factor patients. REFERENCES 1. Yanagimachi T. Physiology of reproduction. In: Knobil E, Neill JD, editors. Vol. 1. New York: Raven Press, 1988:13585. 2. Blackmore P, Beebe S, Danforth D, Alexander N. Progesterone and 17 hydroxyprogesterone, novel stimulators of calcium influx in human sperm. J Bioi Chern 1990;265:137680. 3. Thomas P, Meizel S. An influx of extracellular calcium is required for initiation of the human sperm acrosome reaction induced by human follicular fluid. Gamete Res 1988;20:397-411. 4. Siiteri J, Gottlieb W, Meizel S. Partial characterization of a fraction from human follicular fluid that initiates the human sperm acrosome reaction in vitro. Gamete Res 1988;20:25-42. 5. Siegel MS, Paulson RJ, Graczykowski JW. The influence of human follicular fluid on the acrosome reaction, fertilizing capacity and proteinase activity of human spermatozoa. Hum Reprod 1990;5:975-80. 6. Tesarik J. Comparison of acrosome reaction inducing activities of human cumulus oophorus, follicular fluid and ionophore A23187 in human sperm populations of proven fertilizing ability in vitro. J Reprod Fertil1985;74:383-8. 7. Suarez S, Wolf DP, Meizel S. Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid. Gamete Res 1986;14:107-21. 8. Yee B, Cummings LM. Modification of the sperm penetration assay using human follicular fluid to minimize false negative results. Fertil Steril 1988;50:123-8.
140
Sueldo et al.
P and sperm fertilizing capacity
9. Osman RA, Andria ML, Jones AD, Meizel S. Steroid induced exocytosis: the human sperm acrosome reaction. Biochem Biophys Res Commun 1989;160:828-33. 10. Cross NL, Morales P, Overstreet JW, Hanson FW. Two simple methods for detecting acrosome reacted human sperm. Gamete Res 1986;15:213-26. 11. Coddington CC, Fulgham DL, Alexander NJ, Johnson D, Herr JC, Hodgen GD. Sperm bound to zona pellucida in hemizona assay demonstrate acrosome reaction when stained with T-6 antibody. Fertil Steril1990;54:504-8. 12. Burkman LJ. Discrimination between nonhyperactivated and clasical hyperactivated motility patterns in human spermatozoa using computerized analysis. Fertil Steril 1991;55:363-71. 13. Burkman LJ. Characterization of hyperactivated motility by human spermatozoa during capacitation: comparison of fertile and oligozoospermic sperm population. Arch Androl 1984;13:153-65. 14. Coddington CC, Franken DR, Burkman LJ, Oosthjizen WT, Kruger T, Hodgen GD. Functional aspects of human sperm binding to the zona pellucida using the hemizona assay. J Androl1991;12:1-8. 15. Gocke S, LambertH, Swanson J, Sueldo CE. Comparison of the penetrating capacity of human spermatozoa into human cervical mucus and zona free hamster eggs. Infertility 1986;9:37-47. 16. Vasquez MH, Phillips DM, Wassarman PM. Interaction of mouse sperm with purified sperm receptors covalently linked to silica beads. J Cell Sci 1989;92:713-22. 17. Stock CE, Bates R, Lindsay KS, Edmonds DK, Fraser LR. Extended exposure to follicular fluid is required for significant stimulation of the acrosome reaction in human spermatozoa. J Reprod Fertil1989;86:401-11. 18. Mbizvo MT, Burkman LJ, Alexander NJ. Human follicular fluid stimulates hyperactivated motility in human sperm. Fertil Steril1990;54:708-12. 19. Jinno M, Burkman LJ, Coddington CC. Human sperm hyperactivated motility (HA) and egg penetration. Bioi Reprod 1987;36 Suppl 1:53.
Fertility and Sterility
FERTILITY AND STERILITY
Printed on acid-free paper in U. S. A.
Copyright " 1993 The American Fertility Society
Effect of progesterone on human zona pellucida sperm binding and oocyte penetrating capacity
Carlos E. Sueldo, M.D.*t Sergio Oehninger, M.D.:j: Elisabet Subias, Ph.D.* Mary Mahony, Ph.D.:j:
Nancy J. Alexander, Ph.D.§ Lani J. Burkman, Ph.D. I\ Anibal A. Acosta, M.D.:j:
University of California San Francisco-Fresno, Fresno, California; Jones Institute for Reproductive Medicine, Eastern Virginia }!edical School, Norfolk, Virginia; National Institutes of Health, Bethesda, Maryland; and Grand Island Biologic Company (GIBCO) Laboratories, Grand Island, New York
Objective: To determine if sperm exposure to P produces an enhancement in its fertilizing capacity. Design: Sperm from fertile donors were exposed toP at 0.1 and 1.0 ~g/mL for 1 or 24 hours. The effects on hyperactivated (HA) motility at 1 and 4 hours, acrosome reaction (as determined by Pisum sativum agglutinin or T6/antibody techniques), on human zona pellucida binding (by using the hemizona assay), and on the penetrating ability (by using the zona-free hamster ova assay) were evaluated. Results: Exposure to P at 1.0 ~g/mL enhanced HA motility after 1 and 4 hours of P exposure, the acrosome reaction after 24 hours' incubation, the number of sperm bound/hemizona after 1-hour incubation, and the penetration rates in the hamster ova assay at both incubation intervals. Conclusion: Sperm exposure to P enhances its fertilizing capacity in fertile men, and further investigation is warranted as a possible treatment for male factor patients. Fertil Steril 1993;60:137-40 Key Words: Progesterone, acrosome reaction, human zona sperm binding, hamster ova penetration, sperm hyperactive motility
The human sperm acrosome reaction involves fusion of the outer acrosomal membrane with the plasma membrane at several sites, releasing the contents of the acrosome to the extracellular space
Received May 1, 1992; revised and accepted March 4, 1993. Presented at the 40th Annual Meeting of the Pacific Coast Fertility Society, Indian Wells, California, April9 to 12, 1992. *Department of Obstetrics and Gynecology, University of California San Francisco-Fresno. t Reprint requests: Carlos E. Sueldo, M.D., Department of Obstetrics and Gynecology, University of California San Francisco-Fresno, Fresno, California 93703. :j: Jones Institute for Reproductive Medicine, Eastern Virginia Medical School. §Contraceptive Development Branch, National Institutes of Health. II GIBCO Laboratories. Vol. 60, No. 1, July 1993
(1). Although the precise time at which the acrosome reaction occurs during the fertilization process is controversial, it is agreed that it has to occur before the sperm can penetrate the zona pellucida (ZP) and fuse with the oocyte plasma membrane (1). An early event involved in the acrosome reaction is an obligatory increase in free cytosolic calcium (2). It has been reported that preovulatory human follicular fluid (FF) enhances the influx of calcium through the sperm cell membrane (3), the number of acrosome reactions (4-7), and the penetrating capacity in the zona-free hamster ova sperm penetration assay (SPA) (5, 8). Osman et al. (9) purified and identified (P) and 17-hydroxyprogesterone in the fraction of human FF responsible for the increment in acrosome reaction. In the present study we evaluated the effect of Sueldo et al.
P and sperm fertilizing capacity
137
P at different concentrations and lengths of exposure on human sperm acrosome reaction and the impact on hyperactivated (HA) motility, human ZP binding, and hamster ova penetrating capacity.
MATERIALS AND METHODS
Semen from fertile donors was used for all the experiments. After a standard swim-up procedure by double centrifugation and incubation at 37°C in 5% C02 in air, sperm were exposed to P (Rugby Laboratory, Long Island, NY) at concentrations of 0.1 ~tg/mL and 1.0 ~tg/mL (diluted in culture media) for either 1 hour or 24 hours and for 1 and 4 hours to assess HA. Acrosome Reaction
The proportion of sperm that has undergone acrosome reaction was determined by two techniques: the Pisum sativum agglutinin as described by Cross et al. (10), assessing sperm integrity with the use of the supravital stain Hoechst 33258 (10) or the T6/antibody with immunofluorescent staining as described by Coddington et al. (11). Pisum sativum agglutinin was applied to sperm exposed to P at 0.1 and 1.0 Jtg/mL during 1 and 24 hours, whereas T6 was only used to sperm exposed to P at 1.0 ~tg/mL for 1 hour. To determine the proportion of sperm that has undergone acrosome reaction, 200 sperm were evaluated under each experimental condition. Human Sperm Hyperactive Motility
The steps in this assay were done as described by Burkman (12). Semen from seven fertile donors were tested after centrifugation and layering of culture media (Ham's F-10; GIBCO, Grand Island, NY); an aliquot of the supernatant was diluted to 2 X 106 motile/mL in culture media for effective videotaping as described (12). Hyperactive motility was then assessed by transmitting the videotaped images to the monitor of a Hamilton Thorn Research Motility Analyzer (HTM model 2030, version 7.0; Damvers, MA) and identifying motility patterns consistant with hyperactivation (13).
oocytes (unfertilized oocytes from the IVF program) were equally bisected as previously described (14). One hemizona was placed in a droplet with sperm exposed to P, whereas the matching hemizona was placed in a droplet with sperm not exposed toP (control). After 4 hours of coincubation (37°C in 5% C0 2 in air), each hemizona was removed and rinsed using a fine glass pipette in medium to separate loosely attached sperm; then the number of tightly bound sperm was counted in each hemizona. Sperm Penetration Assay
The basic steps in this assay were done as already described in our laboratory (15). After a standard swim-up, sperm were exposed to P at 0.1 or 1.0 ~tg for 1 hour or 24 hours before the coincubation with hamster ova free of ZP. The same experimental conditions were followed with a portion of the same sperm sample not exposed toP (control) before gamete coincubation. A motile sperm population of 5 X 106 /mL was used for the SPA, and the gametes were co incubated for 3 hours. The penetration rates were determined by assessing the number of ova penetrated over the number of ova exposed. Statistical Analysis
Student's t-test for the comparison of the means of two samples was used to analyze the hemizona data. Chi-squared analysis was used to compare percentages of acrosome and SPA results. Hyperactive motility was compared by using Wilcoxon's rank test. A P value < 0.05 was considered statistically significant. RESULTS
The percentage of sperm that underwent acrosome reaction under each experimental condition is shown in Table 1. Because there was no statistical difference between the two techniques used to assess acrosomalloss, results in Table 1 represent at 1.0 Jtg both groups combined. Progesterone at 1.0
Table 1
Effect of P on Sperm Acrosome Reaction*
Hemizona Assay (HZA)
After exposure to P at 1.0 ~tg/mL concentration for either 1 or 24 hours, a 100-~tL droplet of the sperm suspension (0.5 X 106 motile sperm/mL) was placed in a Petri dish under oil. By micromanipulation (Narishige, Tokyo, Japan) salt-stored human 138
Sueldo et al.
P and sperm fertilizing capacity
Control (free of P) (n = 11) Pat 0.1 !Lg/mL (n = 7) P at 1.0 !Lg/mL (n = 11)
1-h incubation
24-h incubation
10 ± 2.1 11 ± 2.0 18 ± 2.9
9 ± L7t 12 ± 3.1 30 ± 2.9t
*Values are means± SD. t p < 0.01.
Fertility and Sterility
Table 2
Effect of P on Sperm HA Motility*
Hyperactive motility
Controlt
P:j:
11.1 ± 6.5 7.2 ± 4.2
15.7 ± 9.0§ 11.2 ± 6.1§
h
1 (n 4 (n
= =
7) 7)
* Values are mean percents ± SD. t Free of P. :j: 1.0 f.lg/mL. § P < 0.05 compared with control.
J.Lg/mL increased the number of acrosome reaction in comparison with control conditions (free of P exposure), yet this difference became significant only after sperm exposure to P during 24 hours, 30 ± 2.9 versus 9.0 ± 1.7 P < 0.01. Progesterone at 0.1 J.Lg did not have any effect on acrosome reaction as compared with control. Results on HA motility are shown in Table 2 and expressed in percentage of sperm evaluated showing hyperactive motility pattern. At 1 and 4 hours of P exposure at 1.0 J.Lg/mL, sperm showed significantly increased hyperactive motility compared with control conditions (free of P) 15.7 ± 9.0 and 11.2 ± 6.1 versus 11.1 ± 6.5 and 7.2 ± 4.2, respectively, P < 0.05. The results of the HZA are expressed by the number of tightly bound sperm per hemizona (mean ± SEM). Progesterone at 1.0 J.Lg/mL during 1-hour incubation significantly increased the number of tightly bound sperm per hemizona in comparison with control conditions (free of P exposure) 52.6 ± 7.2 versus 35.3 ± 5.5, respectively, P < 0.01. On the other hand at 24 hours, both experimental conditions showed a very low binding, but there was no difference between P and control, 4.5 ± 3.2 versus 4.0 ± 1.2, respectively, (not significant). Results of the SPA are expressed by the penetration rates, representing the number of ova penetrated per number of ova exposed (mean ± SD). Progesterone at 1.0 J.Lg/mL significantly increased ova penetration in comparison with control conditions at 1 hour (60% ± 3.6% versus 31% ± 2.0%, P < 0.05) and at 24 hours (66.0% ± 3.6% versus 39.1% ± 2.7% P < 0.05). Progesterone at 0.1 J.Lg/mL did not show any effect on the SPA compared with control conditions.
DISCUSSION
Progesterone in this study was able to increase the number of sperm acrosome reaction, HA sperm, the number of sperm bound to the human ZP, and the penetrating ability in the zona-free hamster ova Vol. 60, No. 1, July 1993
system. These effects were only shown when P was used at 1.0 J.Lg/mL, a concentration previously used by Osman et al. (9), shown to enhance sperm acrosome reaction. These authors demonstrated that the concentration of P within the cumulus matrix is in excess of 1 J.Lg/mL, enough to elicit an increase in cytosolic calcium, an obligatory event leading to the occurrence of acrosome reaction. Blackmore and co-workers (2) demonstrated that P at 1.0 J.Lg/mL produced a maximum response in rapid calcium passage through the plasma membrane by a mechanism that is not yet completely clear. This finding is similar to the calcium passage enhancement produced by human preovulatory FF, rich in P, as reported by Thomas and Meizel (3). There is evidence that during the normal fertilization process the acrosome reaction occurs when the acrosome-in· tact sperm comes in contact with a component of the ZP known as ZP3 (16); whether P interacts synergistically with ZP3 as speculated by Blackmore (2) remains to be determined. In our study, an increment in acrosome reaction by P was only shown after a long incubation (24 hours) in agreement with data reported by Suarez et al. (7) and Stock et al. (17) using FF; this finding might explain the increase SPA rates observed after 24 hours of P incubation. On the other hand, the increased ZP binding and SPA rates after short incubation (1 hour) with P at 1.0 J.Lg/mL is possibly due to the increased HA motility observed in this study. It has been reported that capacitating sperm near the time of fertilization express a distinct motility pattern known as hyperactivation and characterized by vigorous flagellar movements, marked lateral displacement of the sperm head, and a nonprogressive trajectory (18). There is evidence that the incidence of hyperactivation correlates with sperm fertilizing capacity in vitro (19) and sperm binding to the ZP (16). Mbizvo et al. (18) demonstrated that human preovulatory FF enhanced HA motility, whereas this effect was abolished by the use of a FF fraction stripped of steroids. In assessing the sperm motility patterns with and without P exposure in our study, it became evident by computerized sorting with the HTM analyzer as described by Burkman (12), that P significantly enhanced HA motility at a concentration of 1.0 J.Lg/mL. We therefore concluded that HA motility after a short incubation in P was greatly enhanced, and this finding explains the increased tightly bound sperm to the human ZP and the enhanced penetration in the hamster ova system in agreement with Coddington et al. (14) and Jinno et al. (19), respectively. Whether the enhancement in sperm fertilizing capacity after Sueldo et al.
P and sperm fertilizing capacity
139
exposure to P shown in this study will be manifested in the presence of poor quality semen remains to be determined, and studies are currently underway to evaluate the possible role of P in the treatment of male factor patients. REFERENCES 1. Yanagimachi T. Physiology of reproduction. In: Knobil E, Neill JD, editors. Vol. 1. New York: Raven Press, 1988:13585. 2. Blackmore P, Beebe S, Danforth D, Alexander N. Progesterone and 17 hydroxyprogesterone, novel stimulators of calcium influx in human sperm. J Bioi Chern 1990;265:137680. 3. Thomas P, Meizel S. An influx of extracellular calcium is required for initiation of the human sperm acrosome reaction induced by human follicular fluid. Gamete Res 1988;20:397-411. 4. Siiteri J, Gottlieb W, Meizel S. Partial characterization of a fraction from human follicular fluid that initiates the human sperm acrosome reaction in vitro. Gamete Res 1988;20:25-42. 5. Siegel MS, Paulson RJ, Graczykowski JW. The influence of human follicular fluid on the acrosome reaction, fertilizing capacity and proteinase activity of human spermatozoa. Hum Reprod 1990;5:975-80. 6. Tesarik J. Comparison of acrosome reaction inducing activities of human cumulus oophorus, follicular fluid and ionophore A23187 in human sperm populations of proven fertilizing ability in vitro. J Reprod Fertil1985;74:383-8. 7. Suarez S, Wolf DP, Meizel S. Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid. Gamete Res 1986;14:107-21. 8. Yee B, Cummings LM. Modification of the sperm penetration assay using human follicular fluid to minimize false negative results. Fertil Steril 1988;50:123-8.
140
Sueldo et al.
P and sperm fertilizing capacity
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