International Journal of Andrology, 1992, 15, pages 229-237
Effect of zinc on human sperm motility and the acrosome reaction M. RIFFO, S . LEIVA and J . ASTUDILLO University ofChile, Faculty of Medicine, Department of Cell Biology and Genetics, Santiago, Chile
Summary This study has assessed the effect of zinc on human sperm motility and the acrosome reaction in vitro. Progressively motile human sperm were selected by swim-up and by glass bead columns and then incubated in a medium in which capacitation happened in an asynchronous way. Different doses of zinc (1, 10, 100 and 1000 VM) were added for periods of 2 , 4 or 6 h. Other samples were incubated with zinc (1000 y ~ ) and , after 1 h incubation, the zinc was removed. Aliquots of each culture were used to evaluate progressive motility and the acrosome reaction using a triple-stain technique. Sperm motility was reduced when the amount of zinc added was 3 100 VM, and these doses also caused a significant reduction in the % of sperm undergoing the acrosome reaction. After removal of zinc and further incubation in zinc-free medium for 1 h, an increase in the percentage of motile and acrosome-reacted sperm was observed. However, the increase in acrosome reaction did not reach the values observed in controls. Results suggest that extracellular zinc acts as an inhibitor of human sperm motility and the acrosome reaction (and/or capacitation and the acrosome reaction). This inhibitory effect is reversible and occurs in a dose-dependent fashion. The probable mechanisms involved are discussed. Keywords: acrosome reaction, human sperm motility, zinc. Introduction The presence of zinc in seminal plasma was reported for the first time in 1921 (Bertrand & Vladesco, 1921). Later studies demonstrated that prostatic fluid was the principal source of zinc in seminal plasma (Mawson & Fischer, 1952), and that sperm were also rich in zinc (Mann & Lutwak-Mann, 1981). Zinc seems to be incorporated into the mammalian spermatozoon during the later stages of sperm differentiation (Kruczynski et al., 1985), and also at ejaculation (Bjorndahl et al., 1986). Clinical studies of the role of seminal zinc content on seminal parameters are miscellaneous. Some authors found that low seminal zinc levels were correlated with a decrease in fertilizing ability of sperm (Pandy er al., 1983). Other authors Correspondence: Dr S. Leiva, Department of Cell Biology and Genetics, University of Chile, Faculty of Medicine. P.O. Box 70061-7, Santiago, Chile.
229
230 M . Rijfo, S.Leiva and J . Astudillo found that zinc content in seminal plasma was correlated negatively with progressive sperm motility (Stegmayr & Ronquest, 1982; Carreras & Mendoza, 1990). The discrepancy between these studies could be because zinc is acting through different mechanisms on sperm motility and fertilizing ability. When ejaculation occurs, sperm are released together with the acidic prostatic fluid (rich in zinc, citric acid and acid phosphatase) in the first fraction, whereas the following fractions contain fewer sperm, and consist mainly of an alkaline vesicular fluid, rich in fructose and zinc bound to high molecular-weight ligands of seminal vesicular origin (Arver, 1982; Bjorndahl er al., 1991). These ligands may block incorporation of zinc into the sperm nucleus, and in situations in which the seminal vesicles are absent, incorporation of zinc is increased (Kvist er al., 1990). On the other hand, when vesicular fluid was added to medium containing sperm, their zinc content decreased (Bjorndahl & Kvist, 1990). It has long been known that zinc is an integral part of several enzymes and can be important for the stability of some macromolecules (i.e. proteins, lipoproteins). Zinc is a component of various biological membranes, and it binds to proteins and lipoproteins (Chvapil, 1973; Bettger & O'Dell, 1981). High levels of extracellular zinc stabilizes membranes and may interfere with ion movement across membranes (Bettger & O'Dell, 1981). For these reasons, both effects may be related to some pre-fertilization membrane phenomena, such as capacitation and the acrosome reaction. However, the involvement of zinc in these phenomena has not been examined. In order to establish the role of zinc in sperm motility and the stability of the sperm plasma membrane, we have studied the in-vitro effects of different concentrations of zinc on sperm motility and the incidence of the acrosome reaction.
Materials and methods
Patients and semen samples Sperm samples were obtained from 40 patients aged between 18 and 35 years, who consulted for couple-infertility at the Unit of Reproductive Biology, Faculty of Medicine, University of Chile. The subjects selected had semen samples with progressive sperm motility > 70%. The samples were collected by masturbation after 4 days of abstinence. After liquefaction, ejaculates were assessed for seminal volume, sperm count, total sperm per ejaculate, sperm motility, sperm morphology, seminal fructose and seminal zinc according to the guidelines recommended by W.H.O. (WHO, 1987). Ejaculates were divided into two aliquots. One was used for the determination of zinc in seminal plasma by a colorimetric method (Lampugnani & Maccheroni, 1984). This method is based on the reaction of zinc with 4-(2-pyridylazo) resorcinol in a buffered solution at pH 9.5. The other fraction was used to select motile sperm by a swim-up technique. After recovery, sperm were washed and passed through glass bead (75-150 pm) columns, to obtain sperm suspensions with >95% forward progressive motility, as described previously by Daya & Awatkin (1987).
Zinc and sperm function 231 Effect of zinc The sperm selected by the methods described above were capacitated in Ham’s-F10 medium supplemented with 1.2 mM lactate; 23.8 mM bicarbonate, 160 IU ml-’ penicillin and 3 mg ml-’ bovine serum albumin (fraction V, Sigma Chemical Co., St. Louis, Mo, USA). Sperm were incubated in either the presence or absence of zinc, at 37°C in 5% COz; 95% humidified air for periods of 2, 4 or 6 h. The p H of sperm suspensions were maintained at 7.4-7.6 throughout the experiments. To each tube containing an initial volume of 190 pl sperm suspension in Ham’s-F10, was added 10 p1 PBS or 10 p1 increasing amount of ZnS04 in PBS. The final zinc content was 1, 10, 100 or 1000 p ~At. the end of each period of incubation, aliquots (50 1.11) were removed for assessment of: (a) the percent of progressively motile sperm, (b) the acrosome reaction (150 pl) using the triple-stain technique described by Talbot & Chacon (1981). The staining technique was performed with some modifications: Trypan blue was replaced by Direct blue (2%) (Sigma Co., St. Louis, MO, USA), glutaraldehyde was used at 4% instead of 396, Rose Bengale was used at pH 6, and stained sperm were air-dried instead of ethanol-dried. Several fields were observed randomly and all sperm in each field were examined until 400 had been scored. In other experiments, sperm suspensions (190 pl) were incubated with 1 mM zinc (10 1.11) and after 2 or 4 h they were washed with Ham’s F-10 medium and centrifugated at 600 g for 5 min. Aliquots of 50 p1 were then removed for assessment of sperm motility and the remaining 150 pl sperm suspension was then incubated in free-zinc Ham’s-F10 medium for 1 h. At the end of the period of incubation, forward progressive motility and the acrosome reaction were assessed. Statistical analysis The percentage of progressively motile sperm and the 5% of viable sperm undergoing the acrosome reaction after the different experiments were analysed by analysis of variance. Results
Zinc concentration in seminal plasma The zinc concentration in seminal plasma ranged from 1.4 to 9.7 mg 100 ml-’ (approximately 1 mM). Determination of the seminal plasma levels of zinc was important to ascertain the quantity of zinc to be added to the culture medium in order to mimic the conditions observed in the ejaculate. Dose-response effects of increasing concentrations of zinc on sperm motility and the acrosome reaction During the period of incubation a decrease in forward progressive sperm motility was observed. This reduction was evident by 6 h (Fig. 1). Addition of increasing concentrations of zinc resulted in a further reduction in sperm motility only at the two highest concentrations (100 p~ and 1 mM). This effect was evident after 2 h incubation (Fig. 1). Samples incubated with 0.1 or 10 pM zinc showed similar
232 M . Riffo, S. Leiva and J . Astudillo a
4 Incubation time (h)
a
mOpM
lpM
0l O p M
mImpM
B l m M
Fig. 1. Effect o f increasing zinc concentrations on forward progressive sperm motility after different periods o f incubation. Data are means f SEM. ( I , I': P
Effect of zinc on human sperm motility and the acrosome reaction M. RIFFO, S . LEIVA and J . ASTUDILLO University ofChile, Faculty of Medicine, Department of Cell Biology and Genetics, Santiago, Chile
Summary This study has assessed the effect of zinc on human sperm motility and the acrosome reaction in vitro. Progressively motile human sperm were selected by swim-up and by glass bead columns and then incubated in a medium in which capacitation happened in an asynchronous way. Different doses of zinc (1, 10, 100 and 1000 VM) were added for periods of 2 , 4 or 6 h. Other samples were incubated with zinc (1000 y ~ ) and , after 1 h incubation, the zinc was removed. Aliquots of each culture were used to evaluate progressive motility and the acrosome reaction using a triple-stain technique. Sperm motility was reduced when the amount of zinc added was 3 100 VM, and these doses also caused a significant reduction in the % of sperm undergoing the acrosome reaction. After removal of zinc and further incubation in zinc-free medium for 1 h, an increase in the percentage of motile and acrosome-reacted sperm was observed. However, the increase in acrosome reaction did not reach the values observed in controls. Results suggest that extracellular zinc acts as an inhibitor of human sperm motility and the acrosome reaction (and/or capacitation and the acrosome reaction). This inhibitory effect is reversible and occurs in a dose-dependent fashion. The probable mechanisms involved are discussed. Keywords: acrosome reaction, human sperm motility, zinc. Introduction The presence of zinc in seminal plasma was reported for the first time in 1921 (Bertrand & Vladesco, 1921). Later studies demonstrated that prostatic fluid was the principal source of zinc in seminal plasma (Mawson & Fischer, 1952), and that sperm were also rich in zinc (Mann & Lutwak-Mann, 1981). Zinc seems to be incorporated into the mammalian spermatozoon during the later stages of sperm differentiation (Kruczynski et al., 1985), and also at ejaculation (Bjorndahl et al., 1986). Clinical studies of the role of seminal zinc content on seminal parameters are miscellaneous. Some authors found that low seminal zinc levels were correlated with a decrease in fertilizing ability of sperm (Pandy er al., 1983). Other authors Correspondence: Dr S. Leiva, Department of Cell Biology and Genetics, University of Chile, Faculty of Medicine. P.O. Box 70061-7, Santiago, Chile.
229
230 M . Rijfo, S.Leiva and J . Astudillo found that zinc content in seminal plasma was correlated negatively with progressive sperm motility (Stegmayr & Ronquest, 1982; Carreras & Mendoza, 1990). The discrepancy between these studies could be because zinc is acting through different mechanisms on sperm motility and fertilizing ability. When ejaculation occurs, sperm are released together with the acidic prostatic fluid (rich in zinc, citric acid and acid phosphatase) in the first fraction, whereas the following fractions contain fewer sperm, and consist mainly of an alkaline vesicular fluid, rich in fructose and zinc bound to high molecular-weight ligands of seminal vesicular origin (Arver, 1982; Bjorndahl er al., 1991). These ligands may block incorporation of zinc into the sperm nucleus, and in situations in which the seminal vesicles are absent, incorporation of zinc is increased (Kvist er al., 1990). On the other hand, when vesicular fluid was added to medium containing sperm, their zinc content decreased (Bjorndahl & Kvist, 1990). It has long been known that zinc is an integral part of several enzymes and can be important for the stability of some macromolecules (i.e. proteins, lipoproteins). Zinc is a component of various biological membranes, and it binds to proteins and lipoproteins (Chvapil, 1973; Bettger & O'Dell, 1981). High levels of extracellular zinc stabilizes membranes and may interfere with ion movement across membranes (Bettger & O'Dell, 1981). For these reasons, both effects may be related to some pre-fertilization membrane phenomena, such as capacitation and the acrosome reaction. However, the involvement of zinc in these phenomena has not been examined. In order to establish the role of zinc in sperm motility and the stability of the sperm plasma membrane, we have studied the in-vitro effects of different concentrations of zinc on sperm motility and the incidence of the acrosome reaction.
Materials and methods
Patients and semen samples Sperm samples were obtained from 40 patients aged between 18 and 35 years, who consulted for couple-infertility at the Unit of Reproductive Biology, Faculty of Medicine, University of Chile. The subjects selected had semen samples with progressive sperm motility > 70%. The samples were collected by masturbation after 4 days of abstinence. After liquefaction, ejaculates were assessed for seminal volume, sperm count, total sperm per ejaculate, sperm motility, sperm morphology, seminal fructose and seminal zinc according to the guidelines recommended by W.H.O. (WHO, 1987). Ejaculates were divided into two aliquots. One was used for the determination of zinc in seminal plasma by a colorimetric method (Lampugnani & Maccheroni, 1984). This method is based on the reaction of zinc with 4-(2-pyridylazo) resorcinol in a buffered solution at pH 9.5. The other fraction was used to select motile sperm by a swim-up technique. After recovery, sperm were washed and passed through glass bead (75-150 pm) columns, to obtain sperm suspensions with >95% forward progressive motility, as described previously by Daya & Awatkin (1987).
Zinc and sperm function 231 Effect of zinc The sperm selected by the methods described above were capacitated in Ham’s-F10 medium supplemented with 1.2 mM lactate; 23.8 mM bicarbonate, 160 IU ml-’ penicillin and 3 mg ml-’ bovine serum albumin (fraction V, Sigma Chemical Co., St. Louis, Mo, USA). Sperm were incubated in either the presence or absence of zinc, at 37°C in 5% COz; 95% humidified air for periods of 2, 4 or 6 h. The p H of sperm suspensions were maintained at 7.4-7.6 throughout the experiments. To each tube containing an initial volume of 190 pl sperm suspension in Ham’s-F10, was added 10 p1 PBS or 10 p1 increasing amount of ZnS04 in PBS. The final zinc content was 1, 10, 100 or 1000 p ~At. the end of each period of incubation, aliquots (50 1.11) were removed for assessment of: (a) the percent of progressively motile sperm, (b) the acrosome reaction (150 pl) using the triple-stain technique described by Talbot & Chacon (1981). The staining technique was performed with some modifications: Trypan blue was replaced by Direct blue (2%) (Sigma Co., St. Louis, MO, USA), glutaraldehyde was used at 4% instead of 396, Rose Bengale was used at pH 6, and stained sperm were air-dried instead of ethanol-dried. Several fields were observed randomly and all sperm in each field were examined until 400 had been scored. In other experiments, sperm suspensions (190 pl) were incubated with 1 mM zinc (10 1.11) and after 2 or 4 h they were washed with Ham’s F-10 medium and centrifugated at 600 g for 5 min. Aliquots of 50 p1 were then removed for assessment of sperm motility and the remaining 150 pl sperm suspension was then incubated in free-zinc Ham’s-F10 medium for 1 h. At the end of the period of incubation, forward progressive motility and the acrosome reaction were assessed. Statistical analysis The percentage of progressively motile sperm and the 5% of viable sperm undergoing the acrosome reaction after the different experiments were analysed by analysis of variance. Results
Zinc concentration in seminal plasma The zinc concentration in seminal plasma ranged from 1.4 to 9.7 mg 100 ml-’ (approximately 1 mM). Determination of the seminal plasma levels of zinc was important to ascertain the quantity of zinc to be added to the culture medium in order to mimic the conditions observed in the ejaculate. Dose-response effects of increasing concentrations of zinc on sperm motility and the acrosome reaction During the period of incubation a decrease in forward progressive sperm motility was observed. This reduction was evident by 6 h (Fig. 1). Addition of increasing concentrations of zinc resulted in a further reduction in sperm motility only at the two highest concentrations (100 p~ and 1 mM). This effect was evident after 2 h incubation (Fig. 1). Samples incubated with 0.1 or 10 pM zinc showed similar
232 M . Riffo, S. Leiva and J . Astudillo a
4 Incubation time (h)
a
mOpM
lpM
0l O p M
mImpM
B l m M
Fig. 1. Effect o f increasing zinc concentrations on forward progressive sperm motility after different periods o f incubation. Data are means f SEM. ( I , I': P
