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Defective calcium influx and acrosome reaction (spontaneous and progesterone-induced) in spermatozoa of infertile men with severe teratozoospermia*
FERTILITY AND STERILITY Copyright
©
Vol. 61, No.2, February 1994
1994 The American Fertility Society
Printed on acid-free paper in U. S. A.
Defective calcium influx and acrosome reaction (spontaneous and progesterone-induced) in spermatozoa of infertile men with severe teratozoospermia*
Sergio Oehninger, M.D.t:j: Peter Blackmore, Ph.D.§ Mahmood Morshedi, Ph.D.t
Carlos Sueldo, M.D. II Anibal A. Acosta, M.D. t Nancy J. Alexander, Ph.D.1T
The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia; University of California, San Francisco-Fresno, San Francisco, California; and National Institutes of Health, Bethesda, Maryland
Objective: To evaluate the acrosome reaction and its prerequisite, a calcium influx, in spermatozoa of infertile men with a high incidence of abnormal sperm forms. Design: Prospective, controlled study. Setting: Academic tertiary assisted reproduction center. Patients: Patients (n = 14) were allocated in the study after semen evaluation showed teratozoospermia «14% normal sperm forms) as diagnosed by strict criteria. Interventions: After swim-up separation ofthe motile fraction, acrosome reactions were evaluated using Pisum sativum agglutinin (both spontaneously and exogenously induced with P and the calcium ionophore A23187, both at 10 MM); the intracellular-free [Ca2 +1 i was assessed by the fluorescent fura-2 indicator (basal and after P). Results: Patients did not show the typical P-induced wave of [Ca 2 +1 i that was observed in controls but rather a blunted response, no response at all, or abnormal basal [Ca 2 +1 i levels. The percent of basal acrosome reaction was significantly lower for patients. versus controls postswimup, and at 1 hour and 3 hours. Furthermore, there was a significant difference in the response of acrosome reaction to P both at 1 hour and 3 hours, with patients showing almost no response at all. However, patients' acrosome reaction response to the calcium ionophore was similar to those of fertile men. Conclusion: Infertile patients with a high incidence of abnormal sperm forms as diagnosed by strict criteria have a low incidence of spontaneous acrosome reaction and a diminished P-stimulated acrosome reaction, whereas the nonspecific response to a calcium ionophore is conserved. Parallel abnormalities of [Ca2+L were observed in patients, suggesting that these sperm populations may have a defective nongenomic P sperm receptor and/or abnormalities of other membrane transduction systems. Fertil Steril 1994;61:349-54 Key Words: Teratozoospermia, calcium influx, acrosome reaction, progesterone, calcium ionophore
The presence of severe teratozoospermia (high incidence of abnormal sperm forms) is associated with a poor outcome under IVF conditions. The assessment of sperm morphology using strict criteria Received May 10, 1993; revised and accepted September 23, 1993. * Presented in part at the 18th Annual Meeting of the American Society of Andrology, Tampa, Florida, April 16 to 19, 1993. t The Jones Institute for Reproductive Medicine, DepartVol. 61, No.2, February 1994
ment of Obstetrics and Gynecology, Eastern Virginia Medical School. :j: Reprint requests: Sergio Oehninger, M.D., The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, Virginia 23507. § Department of Pharmacology, Eastern Virginia Medical School. II Department of Obstetrics and Gynecology, University of California, San Francisco-Fresno. 11 National Institutes of Child Health Development, National Institutes of Health.
Oehninger et a1.
Sperm, calcium influx, and acrosome reaction
349
has enhanced objectivity while examining this parameter and has increased the ability to prognosticate fertilization results in the clinical setting (1, 2). Within this context, sperm morphology constitutes a biomarker of sperm dysfunctions crucial to the fertilization process. These dysfunctions may be multiple and can be manifested at the level of motility characteristics (dyskinetic) (3), zona pellucida (ZP)-binding capacity (4), and ability to penetrate zona-free eggs (5). Whether defects of sperm head decondensation and DNA also are present in these abnormal sperm populations still remains to be determined. Sperm capacitation involves a variety of metabolic and functional changes that include, among others, an influx of calcium ions resulting in the acrosome reaction (6). The objective of this prospective study was to investigate the acrosome reaction and its prerequisite, a calcium influx, in spermatozoa of infertile men with a high incidence of abnormal sperm forms.
MATERIALS AND METHODS Subjects
Fourteen infertile men and 15 fertile donors provided semen samples to be used in these experiments. The infertile patients were selected on the basis of the following criteria: teratozoospermia «14% normal forms as evaluated by strict criteria) (1,7), and a sperm concentration of>20 X 106 /mL and progressive motility >30% in the original ejaculate (to minimize the impact of these 2 parameters as confounding factors on sperm responses). Subjects with leukocyte and/or immature germ cell concentrations> 1 X 106 /mL were not included in the study. All patients had a normal physical examination and a normal serum endocrine profile. Their diagnosis was that of infertility (> 1 year duration) assoclated with idiopathic teratozoospermia. Semen samples from fertile men (donors) were used as controls if ejaculates had an original sperm concentration >60 X 106 /mL, progressive motility >60%, and >14% normal forms (strict criteria). Semen Analysis and Preparation
Semen specimens were obtained by masturbation after 2 to 4 days of sexual abstinence and studied after liquefaction was complete «30 minutes) and within 1 hour of collection. Sperm morphology was 350
Oehninger et al.
assessed after slide staining with the quick stain technique and using strict criteria (1, 2, 7). These slides were read on the same day at a magnification of Xl,OOO and documented after counting 200 cells. Sperm concentration and percent progressive motility were objectively measured with an automated computerized sperm motion analyzer (Cell-Soft Semen Analyzer; Labsoft Division of Cryo Resources, Ltd., New York, NY) using fixed parameter settings as previously described (3). After liquefaction, the semen samples were subjected to a swim-up separation ofthe sperm motile fraction. One milliliter of semen was diluted with 2 mL of medium consisting of Biggers, Whitten, and Whittingham supplemented with bovine serum albumin (0.3%) and centrifuged at 290 X g for 10 minutes. The supernatants were then discarded and a second wash performed. Finally, 1 mL of medium was gently layered over the undisturbed pellets, and specimens were incubated for 1 hour at 37°C in 5% CO 2 in air. The swim-up fractions were removed after incubation and divided into aliquots for evaluation of calcium influx and acrosome reaction. The intracellular-free Ca 2 + concentration [Ca 2 +]i was assessed after sperm were loaded with fura-2 (8). Cells (5 to 10 X 106 cells/mL) were incubated with 4 ,uM fura-2/ AM for 1 hour, then centrifuged at 2,000 X g for 5 minutes, and resuspended in FM3B buffer and kept in the dark at room temperature to prevent photobleaching (8). Aliquots (0.5 mL) of cells were incubated at 37°C in 6 X 50-mm glass test tubes containing a small magnetic stirring bar, in a SPEX ARMC spectrofluorometer (Spex Industries, Edison, NJ). Aliquots of agents (2 to 5 ,uL) were then added to the sperm suspension 10 to 15 seconds after data collection was started. Cells were excited at 340 and 380 nm, respectively, and emission measured at 505 nm. Data were collected between 2 and 5 minutes, depending on the protocol used. Integration time was usually 0.1 second, with a time increment of 0.5 second. On completion of experiments, cells were lysed with 0.01 % (wt/vol) digitonin, and then 10 mM ethyline glycol tetraacetic acid was added to obtain fluorescence values of fura 2 at both wavelengths when it was either saturated or depleted of calcium. Autofluorescence of cells was determined at both wavelengths by adding 2 mM manganese chloride, in the presence of 20 ,uM ionomycin, to fura-2-loaded cells. Autofluorescence values were subtracted from values obtained in the fura-2-loaded cells and levels of [Ca 2 +]; calculated (8). The glass cuvette was washed
Sperm, calcium influx, and acrosome reaction
Fertility and Sterility
with 95% ethanol after each experiment to remove any traces of steroid adhering to the glass (8). Acrosome reaction evaluations were performed using the fluorescent probe fluorescein isothiocyanate-labeled Pisum sativum lectin, following previously described techniques (9). Acrosomereacted sperm were diagnosed when total loss of the acrosomal cap was observed (bar pattern) or no immunofluorescence was seen at all. To exogenously induce acrosome reactions, two agonists were used: P (Sigma Chemical Co., St. Louis, MO) first dissolved in dimethylsulfoxide and then in culture medium, and the calcium ionophore A23187 (Sigma Chemical Co.). Both P and A23187 were tested at a final concentration of 10 ~M.
Donor Patient Normozoospermia Teratozoospermia (n=15) (n=lO)
800
~
600
§
~
"
"0
~ 400 0; "5
'iii
"
--B~asa-I-P~ea-k-B~.~-al -~Pe-ak--
• ":.I-[
Figure 1 Comparison of basal and P-stimulated (peak) [Ca2 +l; in fertile controls (n = 15) versus teratozoospermic patients (n = 10).
Experimental Design
After swim-up separation of the motile fraction, [Ca 2 +t was evaluated as described above (basal). The calcium influx response was subsequently assessed after cells were loaded with the calcium indicator fura-2 and P added at 10 ~M. Similar procedures were used for samples from teratozoospermic infertile patients and fertile donors. Spontaneous acrosome reactions were assessed after swim-up separation (basal) and after 1 hour and 3 hours while sperm droplets were kept incubated in culture medium at 37°C in 5% CO 2 in air. In parallel droplets acrosome reactions were investigated after addition ofP or A23187 (10 ~M) immediately after swim-up separation. Duplicate slides were evaluated for each treatment and time analyzed and assessed blindly by two different experienced observers. At least 200 cells were evaluated per slide. Statistical Analysis
Comparisons of acrosome-reacted sperm in teratozoospermic patients versus donors (basal and stimulated) were performed using Student's t-test. Changes in [Ca2+t after P stimulation (poststimulus minus basal levels) in patients versus donors were also compared using Student's t-test. Because of the distribution of basal [Ca2+t levels for teratozoospermic levels, the F-test was used to evaluate differences between SDs in both groups. Results are expressed as means ± SE. Probability values (P) <0.05 were considered significant. RESULTS
The original semen evaluation of the infertile patients (n = 14) showed a sperm concentration of 75 Vol. 61, No.2, February 1994
± 8 X 1Q6/m L (range, 20 to 136), progressive motility of 48% ± 4% (range, 30% to 71 %), and normal morphology 6% ± 1 % (range, 1 % to 11 %). Terato-
zoospermia was characterized by severe sperm head abnormalities, including defects in the size and shape of the head, nuclear vacuoles, cytoplasmic droplets, and abnormal amount of acrosomal content (severely amorphous) (1,7). Slightly abnormal forms (1, 7) represented <20% of total sperm, whereas neck and tail defects contributed <10% of abnormalities. None of the patients had microcephalia or totally acrosome-less sperm (roundhead syndrome). Basal and Stimulated [Ca2 +]; in Spermatozoa From Teratozoospermic Patients and Fertile Controls
There was no significant difference in the basal [Ca2+t between spermatozoa from teratozoospermic patients (n = 10) versus fertile controls (n = 15) (195 ± 42 versus 140 ± 10 nM, respectively, P> 0.5). Fertile controls had a homogenous level of basal [Ca 2 +t (range, 93 to 226 nM). Conversely, patients with teratozoospermia showed a significant dispersion of [Ca2+t basal levels with much lower and higher values observed (range, 36 to 400 nM). The F-test for significance of the difference between the SDs for patients versus controls (129 versus 40) showed P = 0.0002 (Fig. 1). Progesterone (P)-stimulated [Ca2 +t peak levels were significantly higher in fertile controls (469 ± 24 nM) than in teratozoospermic patients (264 ± 6 nM) (P = 0.0002). Progesterone-stimulated [Ca2+li expressed as the difference between peak and basal levels (change) was also significantly
Oehninger et al.
Sperm, calcium influx, and acrosome reaction
351
Panel B
Panel A ~ 500
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: /
.;I:/:..
~:'
o .
.:".: ......
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normozoospermia "\'"
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120
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Figure 2 Effect of P (10 ILM) on (Ca 2 +)i in human sperm. (A), Patient with teratozoospermia showing a blunted response. (B), Patients with teratozoospermia (n = 2) showing almost no response at all.
higher in controls than in patients (328 ± 19 versus 73 ± 24 nM, respectively, P < 0.0001). Figure 2 depicts a comparison of P effects on [Ca2 +t in sperm from donor samples (all donors gave a similar response) and from three representative teratozoospermic samples. Patients with teratozoospermia did not show the typical P-induced wave of [Ca2 +]i that was observed in controls but, rather, a blunted response or almost no response at all. P and Calcium Ionophore-Stimulated Acrosome Reaction Responses From Teratozoospermic Patients and Fertile Controls
The percentage of sperm that spontaneously underwent acrosome reaction was significantly higher in fertile donors (n = 15) than in teratozoospermic patients (n = 14) at the three time points studied (basal or postswim-up, 1 hour, and 3 hours) (Table 1). Furthermore, there was a significant difference in the response to P (10 ~M) both at 1 hour (11 % ± 3% versus 4% ± 0.5%, P < 0.001) and 3 hours (23% ± 8% versus 3% ± 0.5%, P < 0.001) for controls versus patients. Although teratozoospermic patients showed negligible response to P at 1 hour
and 3 hours, their responses to A23187 were similar to those of fertile donors (19% ± 3% versus 26% ± 6% at 1 hour and 34% ± 5% versus 44% ± 10% at 3 hours, respectively, for patients and controls, P > 0.5). Donors showed a significant enhancement of acrosome-reacted sperm after P stimulation both at 1 hour (7% ± 2% versus 11 % ± 3%, P < 0.02) and 3 hours (12 ± 2 versus 23 ± 8, P < 0.05), but teratozoospermic patients had no response to P (1 hour: 3 ± 0.5 versus 4 ± 0.5, P> 0.5; 3 hours: 4 ± 1 versus 3 ± 0.5, P> 0.5) (Table 1).
DISCUSSION
Infertility associated with teratozoospermia is probably due to a concomitance of defective sperm functions. We and others have reported that teratozoospermia is a frequent finding in infertile men consulting for infertility (10, 11). The assessment of morphology using strict criteria enhances objectivity while examining this sperm feature. Here we have demonstrated that a group of men with severe teratozoospermia (characterized by severe sperm head defects) have a reduced ability to initiate the cascade of events that lead to the acrosome reaction, both in basal, capacitating conditions, and in response to a physiological stimulus such as P. Therefore, these dysfunctions associated with motion abnormalities (3), a poor ZP binding capacity (4), and a decreased fusogenic ability in zona-free eggs (5) may be responsible for the reduced IVF rates observed III the clinical setting. Whether those defective sperm functions are concomitant within a given population of teratozoospermic sperm or occur in an isolated fashion remains to be elucidated. In cases of oligozoospermia, it has been documented that the following enzymatic deficiencies are present: an increased production of reactive
Table 1 Acrosome-Reacted Sperm in Basal and Stimulated Conditions in Teratozoospermic Patients and Normozoospermic Fertile Controls Acrosome-reacted sperm (%)
Ih
Basal
Teratozoospermia patients (n Fertile donors (n = 15)
=
14)
Postswim-up
Spontaneous
P
A23187
Spontaneous
P
A23187
3 ± 0.5* 6 ± 0.5
3 ± 0.5*:j:§ 7 ± 2§
4 ± 0.5* 11 ± 3
19 ± 3t 26. ± 6
4 ± 1 *:j:§ 12 ± 2§
3 ± 0.5* 23 ± 8
34 ± 5t 44 ± 10
* P < 0.001 patients compared with donors. t P > 0.5 patients compared with donors.
352
Oehninger et al.
3h
:j: P > 0.5 spontaneous versus P. § P < 0.05 spontaneous versus P; spontaneous versus A23187.
Sperm, calcium influx, and acrosome reaction
Fertility and Sterility
oxygen species (12), increased creatine-N-phosphotransferase activity (13), and increased lactate dehydrogenase activity (14). Moreover, in cases of severe oligozoospermia, no acrosome-reacted spermatozoa were detected in response to follicular fluid (FF) (15), and lower in vitro penetration rates of zona-free hamster oocytes have been reported (16). Recently, it has been shown that oligozoospermic patients (17) and infertile men with normal semen parameters, as evaluated by World Health Organization (WHO) (18) standards, also have a decreased responsiveness to P in terms of calcium influx and acrosome reaction (17). We speculate that in this latter group of patients, morphology abnormalities might have been identified if strict criteria had been used (as we have shown in a previous comparison of WHO and strict criteria in patients with failed fertilization) (19). In the present study, patients were selected on the basis of teratozoospermia with acceptable sperm concentration and progressive motility. This was performed to have a homogenous group of patients and to reduce the impact of other sperm deficiencies as confounding factors. We therefore believe that defective acrosome reactions are present in patients with a high incidence of abnormal sperm morphology, as well as in patients with "pure" oligozoospermia (20). Furthermore, patients with "pure" teratozoospermia, as shown here, have abnormal basal [Ca2+L levels (very low to very high), a finding not previously reported for oligozoospermia patients. Because multiple mechanisms are determinants for basal levels of [Ca2+L, we still cannot provide a specific dysfunction responsible for this abnormality. Progesterone is present in high levels in FF and has been indicated as a physiological stimulus for initiation of the acrosome reaction in human spermatozoa. Progesterone induces an increase in [Ca2 +L in capacitated and capacitating human sperm (8), which is the first event of the acrosome reaction. Furthermore, P increases sperm hyperactivated motility, ZP binding, and zona-free oocyte penetration in fertile men (donors) (21). A distinct nongenomic cell surface receptor for P exists in human sperm (22), and a defective function of this receptor has been reported to be present in infertile men with "unexplained" infertility (17). We speculate that in teratozoospermic patients, there may be a defective nongenomic P sperm receptor and/or abnormalities of other membranetransduction systems. The fact that basal [Ca2+L levels were unusually low or high in some patients Vol. 61, No.2, February 1994
with teratozoospermia (significant differences between SDs in patients versus controls) might indicate either an abnormal permeability of the sperm plasma membrane to calcium ions (calcium channels) or deficiencies in the intracellular mechanism(s) that regulate free [Ca2+L, that is, calcium pumps. These findings differ from those reported for oligozoospermic patients (20). Here, the response to the calcium ionophore A23187 was similar in both groups, suggesting that this may not be due primarily to a plasma membrane permeability defect. A relationship between sperm cell size, head shape, and creatine kinase content in the motile fractions of human sperm has been reported (23). This can be a reflection of an increase of enzyme contents (cytoplasmic retention) accompanied or not by a true biochemical alteration ofthe spermatozoa in cases of oligozoospermia. This type of defect might also be present in sperm populations with teratozoospermia, compromising the capacity to bind to the ZP (24) (Huszar A, Vigue L, Oehinger S, abstract). In conclusion, infertile patients with a high incidence of abnormal sperm forms as diagnosed by strict criteria have a low incidence of basal (spontaneous) acrosome reaction and a diminished P-stimulated [Ca 2 +L and acrosome reaction, whereas the nonspecific response to a calcium ionophore is conserved. Acknowledgments. The authors acknowledge the following staff members of The Jones Institute for Reproductive Medicine, Norfolk, Virginia: Cheryl Oscar, M.S., for her technical assistance, and Ms. Pauline M. Clynes for her editorial assistance in the preparation of this manuscript.
REFERENCES
RJ, Matta JF, Oehninger S. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril1988;49:112-7. Oehninger S, Acosta AA, Morshedi M, Veeck L, Swanson RJ, Simmons K, et al. Corrective measures and pregnancy outcome in in vitro fertilization in patients with severe sperm morphology abnormalities. Fertil SteriI1988;50:2837. Oehninger S, Acosta R, Morshedi M, Philput C, Swanson RJ, Acosta AA. Relationship between morphology and motion characteristics of human spermatozoa in semen and in the swim-up sperm fractions. J AndroI1990;11:446-52. Oehninger S, Toner JP, Muasher SJ, Coddington CC, Acosta AA, Hodgen GD. Prediction of fertilization in vitro with human gametes: is there a litmus test? Am J Obstet Gynecol 1992;167:1760-7. Kruger TF, Swanson RJ, Hamilton M, Simmons K, Acosta AA, Matta JF, et al. Abnormal sperm morphology and other
1. Kruger TF, Acosta AA, Simmons KF, Swanson
2.
3.
4.
5.
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Sperm, calcium influx, and acrosome reaction
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6.
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semen parameters related to the outcome of the hamster oocyte human sperm penetration assay. Int J Androl 1988;11:107-13. Yanagimachi R. Mechanisms of fertilization in mammals. In: L Mastroianni, JD Biggers, editors. Fertilization and embryonic development in vitro. New York: Plenum Press, 1981:81-187. Kruger TF, Menkveld R, Stander FSH, Lombard CJ, Van der Merwe JP, van Zyl JA, et al. Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril1986;46:1118-23. Blackmore PF, Beebe SJ, Danforth DR, Alexander NJ. Progesterone and 17a-hydroxyprogesterone. Novel stimulators of calcium influx in human sperm. J Bioi Chern 1990;265:1376-80. Cross NL, Morales P, Overstreet JW, Hanson FW. Induction of the acrosome reaction by the human zona pellucida. Bioi Reprod 1988;38:235-44. Oehninger S, Franken D, Alexander N, Hodgen GD. Hemizona assay and its impact on the identification and treatment of human sperm dysfunctions. Andrologia 1992;24: 307-21. Menkveld R, Stander FSH, Kotze TJ, Kruger TF, van Zyl JA. The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum Reprod 1990;5:586-92. Aitken RJ, Clarkson JS, Hargreave T, Richardson D, Best F. Analysis of the relationship between defective sperm function and generation of reactive oxygen species in cases of oligozoospermia. J Androl 1989;10:214-20. Huszar G, Corrales M, Vique L. Correlation between sperm creatine phosphokinase activity concentration in normospermic and oligospermic men. Gamete Res 1988;19:67-75. Casano R, Orlando C, Serio M, Forti G. LDH and LDH-X activity in sperm from normospermic and oligozoospermic men. Int J AndroI1991;14:257-63.
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15. Calvo L, Vantman D, Banks SM, Tezon J, Koukoulis GN, Dennison L, et al. Follicular fluid-induced acrosome reaction distinguishes a subgroup of men with unexplained infertility not identified by semen analysis. Fertil Steril 1989;52:1048-54. 16. Aitken RJ, Ross A, Hargreave T, Richardson D, Best F. Analysis of human sperm function following exposure to the ionophore A23187. J AndroI1984;5:321-9. 17. 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. 18. World Health Organization. WHO Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 2nd ed. Cambridge: The Press Syndicate of the University of Cambridge, 1987:43-5. 19. Oehninger S, Acosta AA, Kruger T, Veeck LL, Flood J, Jones HW Jr. Failure offertilization in in vitro fertilization: the "occult" male factor. J In Vitro Fert Embryo Transf 1988;5:181-7. 20. Falsetti C, Baldi E, Kravsz C, Casano R, Failli P, Forti A. Decreased responsiveness to progesterone of spermatozoa in oligozoospermic patients. J Androl 1993;14:17-22. 21. Sueldo CE, Oehninger S, Subias E, Mahony M, Alexander NJ, Burkman L, et al. Effect of progesterone on human zona pellucida sperm binding and oocyte penetrating capacity. Fertil Steril 1993;60:137-40. 22. Blackmore PF, Neulen J, Lattanzio F, Beebe S. Cell surface-binding sites for progesterone mediate calcium uptake in human sperm. J Bioi Chern 1991;266:18655-9. 23. Huszar G, Vigue L. Incomplete development of human spermatozoa is associated with increased creatine phosphokinase concentration and abnormal head morphology. Mol Reprod Dev 1993;34:292-8. 24. Menkveld R, Franken D, Kruger TF, Oehninger S. Sperm selection capacity of the human zona pellucida. Mol Dev 1991;30:346-52.
Sperm, calcium influx, and acrosome reaction
Fertility and Sterility
©
Vol. 61, No.2, February 1994
1994 The American Fertility Society
Printed on acid-free paper in U. S. A.
Defective calcium influx and acrosome reaction (spontaneous and progesterone-induced) in spermatozoa of infertile men with severe teratozoospermia*
Sergio Oehninger, M.D.t:j: Peter Blackmore, Ph.D.§ Mahmood Morshedi, Ph.D.t
Carlos Sueldo, M.D. II Anibal A. Acosta, M.D. t Nancy J. Alexander, Ph.D.1T
The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia; University of California, San Francisco-Fresno, San Francisco, California; and National Institutes of Health, Bethesda, Maryland
Objective: To evaluate the acrosome reaction and its prerequisite, a calcium influx, in spermatozoa of infertile men with a high incidence of abnormal sperm forms. Design: Prospective, controlled study. Setting: Academic tertiary assisted reproduction center. Patients: Patients (n = 14) were allocated in the study after semen evaluation showed teratozoospermia «14% normal sperm forms) as diagnosed by strict criteria. Interventions: After swim-up separation ofthe motile fraction, acrosome reactions were evaluated using Pisum sativum agglutinin (both spontaneously and exogenously induced with P and the calcium ionophore A23187, both at 10 MM); the intracellular-free [Ca2 +1 i was assessed by the fluorescent fura-2 indicator (basal and after P). Results: Patients did not show the typical P-induced wave of [Ca 2 +1 i that was observed in controls but rather a blunted response, no response at all, or abnormal basal [Ca 2 +1 i levels. The percent of basal acrosome reaction was significantly lower for patients. versus controls postswimup, and at 1 hour and 3 hours. Furthermore, there was a significant difference in the response of acrosome reaction to P both at 1 hour and 3 hours, with patients showing almost no response at all. However, patients' acrosome reaction response to the calcium ionophore was similar to those of fertile men. Conclusion: Infertile patients with a high incidence of abnormal sperm forms as diagnosed by strict criteria have a low incidence of spontaneous acrosome reaction and a diminished P-stimulated acrosome reaction, whereas the nonspecific response to a calcium ionophore is conserved. Parallel abnormalities of [Ca2+L were observed in patients, suggesting that these sperm populations may have a defective nongenomic P sperm receptor and/or abnormalities of other membrane transduction systems. Fertil Steril 1994;61:349-54 Key Words: Teratozoospermia, calcium influx, acrosome reaction, progesterone, calcium ionophore
The presence of severe teratozoospermia (high incidence of abnormal sperm forms) is associated with a poor outcome under IVF conditions. The assessment of sperm morphology using strict criteria Received May 10, 1993; revised and accepted September 23, 1993. * Presented in part at the 18th Annual Meeting of the American Society of Andrology, Tampa, Florida, April 16 to 19, 1993. t The Jones Institute for Reproductive Medicine, DepartVol. 61, No.2, February 1994
ment of Obstetrics and Gynecology, Eastern Virginia Medical School. :j: Reprint requests: Sergio Oehninger, M.D., The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, Virginia 23507. § Department of Pharmacology, Eastern Virginia Medical School. II Department of Obstetrics and Gynecology, University of California, San Francisco-Fresno. 11 National Institutes of Child Health Development, National Institutes of Health.
Oehninger et a1.
Sperm, calcium influx, and acrosome reaction
349
has enhanced objectivity while examining this parameter and has increased the ability to prognosticate fertilization results in the clinical setting (1, 2). Within this context, sperm morphology constitutes a biomarker of sperm dysfunctions crucial to the fertilization process. These dysfunctions may be multiple and can be manifested at the level of motility characteristics (dyskinetic) (3), zona pellucida (ZP)-binding capacity (4), and ability to penetrate zona-free eggs (5). Whether defects of sperm head decondensation and DNA also are present in these abnormal sperm populations still remains to be determined. Sperm capacitation involves a variety of metabolic and functional changes that include, among others, an influx of calcium ions resulting in the acrosome reaction (6). The objective of this prospective study was to investigate the acrosome reaction and its prerequisite, a calcium influx, in spermatozoa of infertile men with a high incidence of abnormal sperm forms.
MATERIALS AND METHODS Subjects
Fourteen infertile men and 15 fertile donors provided semen samples to be used in these experiments. The infertile patients were selected on the basis of the following criteria: teratozoospermia «14% normal forms as evaluated by strict criteria) (1,7), and a sperm concentration of>20 X 106 /mL and progressive motility >30% in the original ejaculate (to minimize the impact of these 2 parameters as confounding factors on sperm responses). Subjects with leukocyte and/or immature germ cell concentrations> 1 X 106 /mL were not included in the study. All patients had a normal physical examination and a normal serum endocrine profile. Their diagnosis was that of infertility (> 1 year duration) assoclated with idiopathic teratozoospermia. Semen samples from fertile men (donors) were used as controls if ejaculates had an original sperm concentration >60 X 106 /mL, progressive motility >60%, and >14% normal forms (strict criteria). Semen Analysis and Preparation
Semen specimens were obtained by masturbation after 2 to 4 days of sexual abstinence and studied after liquefaction was complete «30 minutes) and within 1 hour of collection. Sperm morphology was 350
Oehninger et al.
assessed after slide staining with the quick stain technique and using strict criteria (1, 2, 7). These slides were read on the same day at a magnification of Xl,OOO and documented after counting 200 cells. Sperm concentration and percent progressive motility were objectively measured with an automated computerized sperm motion analyzer (Cell-Soft Semen Analyzer; Labsoft Division of Cryo Resources, Ltd., New York, NY) using fixed parameter settings as previously described (3). After liquefaction, the semen samples were subjected to a swim-up separation ofthe sperm motile fraction. One milliliter of semen was diluted with 2 mL of medium consisting of Biggers, Whitten, and Whittingham supplemented with bovine serum albumin (0.3%) and centrifuged at 290 X g for 10 minutes. The supernatants were then discarded and a second wash performed. Finally, 1 mL of medium was gently layered over the undisturbed pellets, and specimens were incubated for 1 hour at 37°C in 5% CO 2 in air. The swim-up fractions were removed after incubation and divided into aliquots for evaluation of calcium influx and acrosome reaction. The intracellular-free Ca 2 + concentration [Ca 2 +]i was assessed after sperm were loaded with fura-2 (8). Cells (5 to 10 X 106 cells/mL) were incubated with 4 ,uM fura-2/ AM for 1 hour, then centrifuged at 2,000 X g for 5 minutes, and resuspended in FM3B buffer and kept in the dark at room temperature to prevent photobleaching (8). Aliquots (0.5 mL) of cells were incubated at 37°C in 6 X 50-mm glass test tubes containing a small magnetic stirring bar, in a SPEX ARMC spectrofluorometer (Spex Industries, Edison, NJ). Aliquots of agents (2 to 5 ,uL) were then added to the sperm suspension 10 to 15 seconds after data collection was started. Cells were excited at 340 and 380 nm, respectively, and emission measured at 505 nm. Data were collected between 2 and 5 minutes, depending on the protocol used. Integration time was usually 0.1 second, with a time increment of 0.5 second. On completion of experiments, cells were lysed with 0.01 % (wt/vol) digitonin, and then 10 mM ethyline glycol tetraacetic acid was added to obtain fluorescence values of fura 2 at both wavelengths when it was either saturated or depleted of calcium. Autofluorescence of cells was determined at both wavelengths by adding 2 mM manganese chloride, in the presence of 20 ,uM ionomycin, to fura-2-loaded cells. Autofluorescence values were subtracted from values obtained in the fura-2-loaded cells and levels of [Ca 2 +]; calculated (8). The glass cuvette was washed
Sperm, calcium influx, and acrosome reaction
Fertility and Sterility
with 95% ethanol after each experiment to remove any traces of steroid adhering to the glass (8). Acrosome reaction evaluations were performed using the fluorescent probe fluorescein isothiocyanate-labeled Pisum sativum lectin, following previously described techniques (9). Acrosomereacted sperm were diagnosed when total loss of the acrosomal cap was observed (bar pattern) or no immunofluorescence was seen at all. To exogenously induce acrosome reactions, two agonists were used: P (Sigma Chemical Co., St. Louis, MO) first dissolved in dimethylsulfoxide and then in culture medium, and the calcium ionophore A23187 (Sigma Chemical Co.). Both P and A23187 were tested at a final concentration of 10 ~M.
Donor Patient Normozoospermia Teratozoospermia (n=15) (n=lO)
800
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Figure 1 Comparison of basal and P-stimulated (peak) [Ca2 +l; in fertile controls (n = 15) versus teratozoospermic patients (n = 10).
Experimental Design
After swim-up separation of the motile fraction, [Ca 2 +t was evaluated as described above (basal). The calcium influx response was subsequently assessed after cells were loaded with the calcium indicator fura-2 and P added at 10 ~M. Similar procedures were used for samples from teratozoospermic infertile patients and fertile donors. Spontaneous acrosome reactions were assessed after swim-up separation (basal) and after 1 hour and 3 hours while sperm droplets were kept incubated in culture medium at 37°C in 5% CO 2 in air. In parallel droplets acrosome reactions were investigated after addition ofP or A23187 (10 ~M) immediately after swim-up separation. Duplicate slides were evaluated for each treatment and time analyzed and assessed blindly by two different experienced observers. At least 200 cells were evaluated per slide. Statistical Analysis
Comparisons of acrosome-reacted sperm in teratozoospermic patients versus donors (basal and stimulated) were performed using Student's t-test. Changes in [Ca2+t after P stimulation (poststimulus minus basal levels) in patients versus donors were also compared using Student's t-test. Because of the distribution of basal [Ca2+t levels for teratozoospermic levels, the F-test was used to evaluate differences between SDs in both groups. Results are expressed as means ± SE. Probability values (P) <0.05 were considered significant. RESULTS
The original semen evaluation of the infertile patients (n = 14) showed a sperm concentration of 75 Vol. 61, No.2, February 1994
± 8 X 1Q6/m L (range, 20 to 136), progressive motility of 48% ± 4% (range, 30% to 71 %), and normal morphology 6% ± 1 % (range, 1 % to 11 %). Terato-
zoospermia was characterized by severe sperm head abnormalities, including defects in the size and shape of the head, nuclear vacuoles, cytoplasmic droplets, and abnormal amount of acrosomal content (severely amorphous) (1,7). Slightly abnormal forms (1, 7) represented <20% of total sperm, whereas neck and tail defects contributed <10% of abnormalities. None of the patients had microcephalia or totally acrosome-less sperm (roundhead syndrome). Basal and Stimulated [Ca2 +]; in Spermatozoa From Teratozoospermic Patients and Fertile Controls
There was no significant difference in the basal [Ca2+t between spermatozoa from teratozoospermic patients (n = 10) versus fertile controls (n = 15) (195 ± 42 versus 140 ± 10 nM, respectively, P> 0.5). Fertile controls had a homogenous level of basal [Ca 2 +t (range, 93 to 226 nM). Conversely, patients with teratozoospermia showed a significant dispersion of [Ca2+t basal levels with much lower and higher values observed (range, 36 to 400 nM). The F-test for significance of the difference between the SDs for patients versus controls (129 versus 40) showed P = 0.0002 (Fig. 1). Progesterone (P)-stimulated [Ca2 +t peak levels were significantly higher in fertile controls (469 ± 24 nM) than in teratozoospermic patients (264 ± 6 nM) (P = 0.0002). Progesterone-stimulated [Ca2+li expressed as the difference between peak and basal levels (change) was also significantly
Oehninger et al.
Sperm, calcium influx, and acrosome reaction
351
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Figure 2 Effect of P (10 ILM) on (Ca 2 +)i in human sperm. (A), Patient with teratozoospermia showing a blunted response. (B), Patients with teratozoospermia (n = 2) showing almost no response at all.
higher in controls than in patients (328 ± 19 versus 73 ± 24 nM, respectively, P < 0.0001). Figure 2 depicts a comparison of P effects on [Ca2 +t in sperm from donor samples (all donors gave a similar response) and from three representative teratozoospermic samples. Patients with teratozoospermia did not show the typical P-induced wave of [Ca2 +]i that was observed in controls but, rather, a blunted response or almost no response at all. P and Calcium Ionophore-Stimulated Acrosome Reaction Responses From Teratozoospermic Patients and Fertile Controls
The percentage of sperm that spontaneously underwent acrosome reaction was significantly higher in fertile donors (n = 15) than in teratozoospermic patients (n = 14) at the three time points studied (basal or postswim-up, 1 hour, and 3 hours) (Table 1). Furthermore, there was a significant difference in the response to P (10 ~M) both at 1 hour (11 % ± 3% versus 4% ± 0.5%, P < 0.001) and 3 hours (23% ± 8% versus 3% ± 0.5%, P < 0.001) for controls versus patients. Although teratozoospermic patients showed negligible response to P at 1 hour
and 3 hours, their responses to A23187 were similar to those of fertile donors (19% ± 3% versus 26% ± 6% at 1 hour and 34% ± 5% versus 44% ± 10% at 3 hours, respectively, for patients and controls, P > 0.5). Donors showed a significant enhancement of acrosome-reacted sperm after P stimulation both at 1 hour (7% ± 2% versus 11 % ± 3%, P < 0.02) and 3 hours (12 ± 2 versus 23 ± 8, P < 0.05), but teratozoospermic patients had no response to P (1 hour: 3 ± 0.5 versus 4 ± 0.5, P> 0.5; 3 hours: 4 ± 1 versus 3 ± 0.5, P> 0.5) (Table 1).
DISCUSSION
Infertility associated with teratozoospermia is probably due to a concomitance of defective sperm functions. We and others have reported that teratozoospermia is a frequent finding in infertile men consulting for infertility (10, 11). The assessment of morphology using strict criteria enhances objectivity while examining this sperm feature. Here we have demonstrated that a group of men with severe teratozoospermia (characterized by severe sperm head defects) have a reduced ability to initiate the cascade of events that lead to the acrosome reaction, both in basal, capacitating conditions, and in response to a physiological stimulus such as P. Therefore, these dysfunctions associated with motion abnormalities (3), a poor ZP binding capacity (4), and a decreased fusogenic ability in zona-free eggs (5) may be responsible for the reduced IVF rates observed III the clinical setting. Whether those defective sperm functions are concomitant within a given population of teratozoospermic sperm or occur in an isolated fashion remains to be elucidated. In cases of oligozoospermia, it has been documented that the following enzymatic deficiencies are present: an increased production of reactive
Table 1 Acrosome-Reacted Sperm in Basal and Stimulated Conditions in Teratozoospermic Patients and Normozoospermic Fertile Controls Acrosome-reacted sperm (%)
Ih
Basal
Teratozoospermia patients (n Fertile donors (n = 15)
=
14)
Postswim-up
Spontaneous
P
A23187
Spontaneous
P
A23187
3 ± 0.5* 6 ± 0.5
3 ± 0.5*:j:§ 7 ± 2§
4 ± 0.5* 11 ± 3
19 ± 3t 26. ± 6
4 ± 1 *:j:§ 12 ± 2§
3 ± 0.5* 23 ± 8
34 ± 5t 44 ± 10
* P < 0.001 patients compared with donors. t P > 0.5 patients compared with donors.
352
Oehninger et al.
3h
:j: P > 0.5 spontaneous versus P. § P < 0.05 spontaneous versus P; spontaneous versus A23187.
Sperm, calcium influx, and acrosome reaction
Fertility and Sterility
oxygen species (12), increased creatine-N-phosphotransferase activity (13), and increased lactate dehydrogenase activity (14). Moreover, in cases of severe oligozoospermia, no acrosome-reacted spermatozoa were detected in response to follicular fluid (FF) (15), and lower in vitro penetration rates of zona-free hamster oocytes have been reported (16). Recently, it has been shown that oligozoospermic patients (17) and infertile men with normal semen parameters, as evaluated by World Health Organization (WHO) (18) standards, also have a decreased responsiveness to P in terms of calcium influx and acrosome reaction (17). We speculate that in this latter group of patients, morphology abnormalities might have been identified if strict criteria had been used (as we have shown in a previous comparison of WHO and strict criteria in patients with failed fertilization) (19). In the present study, patients were selected on the basis of teratozoospermia with acceptable sperm concentration and progressive motility. This was performed to have a homogenous group of patients and to reduce the impact of other sperm deficiencies as confounding factors. We therefore believe that defective acrosome reactions are present in patients with a high incidence of abnormal sperm morphology, as well as in patients with "pure" oligozoospermia (20). Furthermore, patients with "pure" teratozoospermia, as shown here, have abnormal basal [Ca2+L levels (very low to very high), a finding not previously reported for oligozoospermia patients. Because multiple mechanisms are determinants for basal levels of [Ca2+L, we still cannot provide a specific dysfunction responsible for this abnormality. Progesterone is present in high levels in FF and has been indicated as a physiological stimulus for initiation of the acrosome reaction in human spermatozoa. Progesterone induces an increase in [Ca2 +L in capacitated and capacitating human sperm (8), which is the first event of the acrosome reaction. Furthermore, P increases sperm hyperactivated motility, ZP binding, and zona-free oocyte penetration in fertile men (donors) (21). A distinct nongenomic cell surface receptor for P exists in human sperm (22), and a defective function of this receptor has been reported to be present in infertile men with "unexplained" infertility (17). We speculate that in teratozoospermic patients, there may be a defective nongenomic P sperm receptor and/or abnormalities of other membranetransduction systems. The fact that basal [Ca2+L levels were unusually low or high in some patients Vol. 61, No.2, February 1994
with teratozoospermia (significant differences between SDs in patients versus controls) might indicate either an abnormal permeability of the sperm plasma membrane to calcium ions (calcium channels) or deficiencies in the intracellular mechanism(s) that regulate free [Ca2+L, that is, calcium pumps. These findings differ from those reported for oligozoospermic patients (20). Here, the response to the calcium ionophore A23187 was similar in both groups, suggesting that this may not be due primarily to a plasma membrane permeability defect. A relationship between sperm cell size, head shape, and creatine kinase content in the motile fractions of human sperm has been reported (23). This can be a reflection of an increase of enzyme contents (cytoplasmic retention) accompanied or not by a true biochemical alteration ofthe spermatozoa in cases of oligozoospermia. This type of defect might also be present in sperm populations with teratozoospermia, compromising the capacity to bind to the ZP (24) (Huszar A, Vigue L, Oehinger S, abstract). In conclusion, infertile patients with a high incidence of abnormal sperm forms as diagnosed by strict criteria have a low incidence of basal (spontaneous) acrosome reaction and a diminished P-stimulated [Ca 2 +L and acrosome reaction, whereas the nonspecific response to a calcium ionophore is conserved. Acknowledgments. The authors acknowledge the following staff members of The Jones Institute for Reproductive Medicine, Norfolk, Virginia: Cheryl Oscar, M.S., for her technical assistance, and Ms. Pauline M. Clynes for her editorial assistance in the preparation of this manuscript.
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15. Calvo L, Vantman D, Banks SM, Tezon J, Koukoulis GN, Dennison L, et al. Follicular fluid-induced acrosome reaction distinguishes a subgroup of men with unexplained infertility not identified by semen analysis. Fertil Steril 1989;52:1048-54. 16. Aitken RJ, Ross A, Hargreave T, Richardson D, Best F. Analysis of human sperm function following exposure to the ionophore A23187. J AndroI1984;5:321-9. 17. 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. 18. World Health Organization. WHO Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 2nd ed. Cambridge: The Press Syndicate of the University of Cambridge, 1987:43-5. 19. Oehninger S, Acosta AA, Kruger T, Veeck LL, Flood J, Jones HW Jr. Failure offertilization in in vitro fertilization: the "occult" male factor. J In Vitro Fert Embryo Transf 1988;5:181-7. 20. Falsetti C, Baldi E, Kravsz C, Casano R, Failli P, Forti A. Decreased responsiveness to progesterone of spermatozoa in oligozoospermic patients. J Androl 1993;14:17-22. 21. Sueldo CE, Oehninger S, Subias E, Mahony M, Alexander NJ, Burkman L, et al. Effect of progesterone on human zona pellucida sperm binding and oocyte penetrating capacity. Fertil Steril 1993;60:137-40. 22. Blackmore PF, Neulen J, Lattanzio F, Beebe S. Cell surface-binding sites for progesterone mediate calcium uptake in human sperm. J Bioi Chern 1991;266:18655-9. 23. Huszar G, Vigue L. Incomplete development of human spermatozoa is associated with increased creatine phosphokinase concentration and abnormal head morphology. Mol Reprod Dev 1993;34:292-8. 24. Menkveld R, Franken D, Kruger TF, Oehninger S. Sperm selection capacity of the human zona pellucida. Mol Dev 1991;30:346-52.
Sperm, calcium influx, and acrosome reaction
Fertility and Sterility