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A Regulatory Role of Sulfhydryl Groups in Modulation of Sperm Membrane Conformation by Heavy Metals: Sulfhydryl Groups as Markers for Infertility Assessment
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
247, 716–718 (1998)
RC988841
A Regulatory Role of Sulfhydryl Groups in Modulation of Sperm Membrane Conformation by Heavy Metals: Sulfhydryl Groups as Markers for Infertility Assessment Manish Nivsarkar, Bapu Cherian, and Satish Patel B. V. Patel Pharmaceutical Education and Research Development Centre, Thaltej-Gandhinagar Highway, Ahemadabad-380 054, Gujarat, India
Received May 5, 1998
Sperm membrane sulfhydryl groups, when masked by heavy metals like cobalt and copper at very low concentration (1009) shows inhibition of lipid peroxidation and superoxide dismutase activity. Inhibition of lipid peroxidation is suggested to lead to reduction in membrane fluidity, a prerequisite for normal sperm function. Augmentation of lipid peroxidation by pentoxifylline in oligospermia provides evidence to this hypothesis that membrane sulfhydryls play a regulatory role in membrane modulation. It is therefore suggested that these sulfhydryl groups can be used as a tool for infertility assessment in unexplained male infertility. q 1998 Academic Press
Defective sperm function is the largest single defined cause of human infertility and yet there is a dearth of reliable procedures for diagnosing or treating this condition. This is because of lack of enough fundamental knowledge concerning the etiology of male infertility and the precise nature of the defects in morphology, biochemistry and fertilizing potential of the spermatozoa. In addition to the various known causes of male infertility such as low sperm count, motility, viability, volume, agglutination and defective morphology [1], several intrinsic defects have been reported such as abnormal pyruvate utilization [2], high ATP accumulation [3], low zona-free hamster egg penetration scores [4], overexpression of superoxide dismutase and absence of sperm membrane sulfhydryl groups (in oligospermia) [5]. In addition to known factors involved in infertility, a large number of men are diagnosed as cases of unexplained male infertility. Sulfhydryl groups of the sperm membrane play a very important role in sperm motility and metabolism [6] and evidence is also available for the involvement of these surface thiols in normal sperm functioning [7]. It has been known that divalent cations like cadmium and copper are hazardous to mammalian sper0006-291X/98 $25.00
matozoa [8-9]. The spermeostatic properties of copper have been widely utilized in copper containing intrauterine devices as contraceptive agents. Cobaltous ion have been shown to exercise powerful sperm immobilizing properties at extremely low concentrations, which could be recovered by a sulfhydryl compound, cysteine. The loss of sperm surface thiol groups and the augmented production of superoxide anion radical was stated to be the reason for the loss of motility [10]. The sulfhydryl blocking properties of both copper and cobalt is utilized to study role of sulfhydryl groups in sperm membrane modulation. This paper reports a possible regulatory role of sulfhydryl groups in sperm membrane modulation and as a marker for fertility assessment. MATERIALS AND METHODS Semen Human ejaculates were obtained from the pathological laboratory of a local hospital and after their clinical assessment and estimate of motility, count, normal and abnormal forms were collected in a sterile vial containing 2ml HBSS (Hanks Balanced Salt Solution) (pH 7.2) at room temperature. On the basis of count and motility, samples were classified into 2 groups: normal and oligospermic samples. The semen samples thus obtained were centrifuged at 3500 rpm (500 1 g) for 10 minutes at room temperature, resuspended in 2 ml HBSS and then divided into two equal parts. One part of the normal cases were incubated with 1009 M Cu// and the other part was used as control. Similarly in oligospermic cases one part was incubated with 3.6 mM (final concentration) pentoxifylline and the other part was used as a control for 30 minutes at 377C. Then the volume were made upto 5 ml with HBSS and centrifuged at room temperature for 10 minutes at 3500 rpm. The pellets obtained were resuspended in 2 ml HBSS at 377C and were used in subsequent investigations. Semen samples from the cauda epididymis of mature male albino rats (3-5 months old) maintained under temperature (277C) and light (14hr light : 10 hr dark)- controlled conditions and provided food and water ad libitum were used for these studies. Animals were sacrificed and the semen samples from the cauda region of the epididymis were collected in 2 ml HBSS and processed as above. The samples were then divided into two equal parts one part was treated with 1009 M concentration of Co// or Cu// (final concentration) for 30 minutes
716
Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 8841
/
6956$$$341
06-05-98 22:53:09
bbrcg
AP: BBRC
Vol. 247, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Histographical representation of changes in MDA levels in normal spermatozoa without and with 1009 M copper treatment (a) and (b) is MDA levels in oligospermic samples without and with PTF treatment.
at 377C and the other part was used as a control. After incubation the volumes were made upto 5 ml wit HBSS and centrifuged at room temperature for 10 minutes at 3500 rpm. The pellets obtained were resuspended in 2 ml HBSS at 377C and were used in subsequent investigations.
Protocol for Estimation of Lipid Peroxidation Preparation of the sample. The sperm samples in HBSS (pH 7.2) were homogenized at a speed of 5000 rpm, using a Elteck homogenizer NS 3 (3 cycle of 30 sec. each). The homogenates were then centrifuged at 3500 rpm (500 1 g) for 10 minutes. The pellets were resuspended in 1 ml HBSS which was then used for lipid peroxidation assay. Assay for lipid peroxidation. The lipid peroxidation was measured in terms of malonaldehyde (MDA): thiobarbituric acid (TBA) reaction as reported by Okawa et al (1979) [11]. The reaction mixture contained 0.1 ml of tissue homogenate (as described above), 0.2 ml of 8.1% SDS, 1.5 ml of 20% glacial acetic acid and 1.5 ml of 0.8% aqueous solution of TBA. The pH of 20% acetic acid was preadjusted with 1M NaOH at pH 3.5. The mixture was finally made upto 4 ml with distilled water and heated at 957C for 1 hr, on a water bath. After cooling, 1 ml of distilled water and 5ml of a mixture of n-butanol and pyridine (15:1 v/v) was added and the mixture was shaken vigorously on a vortex mixer. Then after centrifugation at 2200 1 g for 5 minutes, the amount of MDA formed was measured by the absorbence of the upper organic layer at 532 nm (extinction coefficient of MDA is 1.45 1 105/min./ cm) using appropriate controls. Measurements were made using a Jasco UV/VIS spectrophotometer.
Assay of Superoxide Dismutase Activity in Spermatozoa Sperm samples obtained from male albino rats were washed with HBSS (pH 7.2) and were treated with cobalt and copper ions (as described above). After incubation these samples were washed with HBSS (pH 7.2) and were resuspended in 4 ml chilled (47C) Tris buffer (50 mM, pH 8.2). These suspensions were homogenized at 47C at a speed of 13000 rpm (3 cycles of 30 sec. each) using Elteck homogenizer NS 3. The homogenates were treated with 1 ml of 1% triton X 100 (v/v) for 20 minutes, at 47C. Homogenates were then centrifuged at a speed of 15000 rpm at 47C using a Remi C30 high speed centrifuge with a fixed angle rotor. The supernatant was used for the assay of superoxide dismutase activity by the method of Marklund and Marklund (1974) [12]. All the calculations were made as per mg of protein.
FIG. 2. Histographical representation of changes in MDA levels in rat caudal spermatozoa with cobalt (a) and copper (b) treatment.
oligospermic sperm samples with and without treatment. The MDA concentration in normal (control) (Fig. 1a) samples were significantly inhibited (Põ0.0001) after 1009 M (final concentration) of Cu// treatment as compared to control. However, there was a sharp increase in MDA levels after pentoxifylline treatment in oligospermic samples (Põ0.00001) (Fig. 1b). Figure 2 shows the changes in amount of MDA formed in rat caudal spermatozoa with 1009 M cobalt and copper (figure 2a and 2b respectively). The MDA levels were significantly decreased (Põ0.001) when the spermatozoa were treated with 1009 M copper. The MDA levels also decreased significantly (Põ0.00001) after 1009 M cobalt treatment. Superoxide dismutase levels were also inhibited significantly (Põ0.00001) in the presence of 1009M cobalt and copper (Figs. 3a and 3b, respectively). Membrane integrity and its proper functioning are the basic characteristics of the sperm membrane including cellular recognition and information transduction during cell cell interaction. Changes in fatty acid composition of membranes as well as the amount of individual sterols account for the change in fluidity. Normal spermatozoa are reported to show high membrane fluidity [12]. Therefore it is evident that membrane fluidity is an important factor for sperm functions. Lipid peroxidation plays a crucial role in inducing membrane fluidity [13]. The results presented here shows that blocking of sperm membrane sulfhydryl groups both in human and rat caudal sperm inhibits lipid peroxidation and superoxide dismutase activity.
RESULTS AND DISCUSSION Figure 1 shows the change in the amount of MDA formed as a result of lipid peroxidation in normal and
FIG. 3. Histographical representation of changes in the levels of SOD in rat caudal spermatozoa with cobalt (a) and copper (b) treatment.
717
AID
BBRC 8841
/
6956$$$342
06-05-98 22:53:09
bbrcg
AP: BBRC
Vol. 247, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
It has been reported earlier that copper containing antifertility devices are believed to act via production of toxic oxygen radical species including hydroxyl and superoxide anion radical [14] and these radical species are known to induce membrane fluidity via lipid peroxidation. But, contrary to the above statement we found that blocking of sulfhydryl groups by copper and cobalt inhibits lipid peroxidation in normal spermatozoa and treatment of oligospermic samples with pentoxifylline increases lipid peroxidation. However, this is a unique phenomenon which remains to be elucidated. But it is quite evident that membrane sulfhydryl groups play an important role in sperm membrane modulation by reducing membrane fluidity. Decrease in motility and loss of sperm function in unexplained male infertility can be attributed to these sulfhydryl groups of the sperm membrane and is suggested that in such cases of infertility the active sulfhydryl groups are masked which results in loss of sperm function. Thus, it is suggested that sperm membrane sulfhydryl groups are important entities of the membrane and can be used as a tool for infertility assessment in unexplained male infertility and can be targeted for contraceptive research.
REFERENCES 1. Gopalkrishna, K. (1995) Current Science 68(4), 353–362. 2. Meaker, S. R. (1934) in Human Sterility. The Williams and Wilkins Co., Baltimore. 3. Dolci, S., Greco, E., Piccione, E., Banchone, C., and Manna, C. (1987) Patol. Clin. Utet Ginecol. 15(1), 1. 4. Aitken, R. J. (1982) in Clinical Practices in Urology—Male Infertility (Hargreave, T., Ed.), Springer Verlag Berlin, Heidelberg, New York. 5. Sinha, S., Kumar, G. P., Laloraya, M., and Warikoo, D. (1991) Biochem. Biophys. Res. Commun. 174(2), 510–517. 6. Mann, T., and Lutwak-Mann, C. (1981) in Male Reproductive Function and Semen p. 337, Springer Verlag, Berlin. 7. Sinha, S., Kumar, G. P., and Laloraya, M. (1994) Biochem. Biophys. Res. Commun. 198(1), 266–273. 8. Parizek, J. (1956) Nature 117, 1036–1037. 9. Mann, T. (1958) Proc. Soc. Study Fertil. 9, 3–10. 10. Kumar, G. P., Laloraya, M., and Laloraya, M. M. (1990) Contraception 41(6), 633–639. 11. Okawa, H., Ohishi, N., and Yogi, K. (1979) Anal. Biochem. 95, 35. 12. Marklund, S., and Marklund, G. (1974) Eur. J. Biochem. 47, 469–474. 13. Jain, S., Thomas, M., Kumar, G. P., and Laloraya, M. (1993) Biochem. Biophys. Res. Commun. 195(2), 574–580. 14. Mann, T., and Leone, E. (1953) Biochem. J. 53, 110–115.
718
AID
BBRC 8841
/
6956$$$342
06-05-98 22:53:09
bbrcg
AP: BBRC
247, 716–718 (1998)
RC988841
A Regulatory Role of Sulfhydryl Groups in Modulation of Sperm Membrane Conformation by Heavy Metals: Sulfhydryl Groups as Markers for Infertility Assessment Manish Nivsarkar, Bapu Cherian, and Satish Patel B. V. Patel Pharmaceutical Education and Research Development Centre, Thaltej-Gandhinagar Highway, Ahemadabad-380 054, Gujarat, India
Received May 5, 1998
Sperm membrane sulfhydryl groups, when masked by heavy metals like cobalt and copper at very low concentration (1009) shows inhibition of lipid peroxidation and superoxide dismutase activity. Inhibition of lipid peroxidation is suggested to lead to reduction in membrane fluidity, a prerequisite for normal sperm function. Augmentation of lipid peroxidation by pentoxifylline in oligospermia provides evidence to this hypothesis that membrane sulfhydryls play a regulatory role in membrane modulation. It is therefore suggested that these sulfhydryl groups can be used as a tool for infertility assessment in unexplained male infertility. q 1998 Academic Press
Defective sperm function is the largest single defined cause of human infertility and yet there is a dearth of reliable procedures for diagnosing or treating this condition. This is because of lack of enough fundamental knowledge concerning the etiology of male infertility and the precise nature of the defects in morphology, biochemistry and fertilizing potential of the spermatozoa. In addition to the various known causes of male infertility such as low sperm count, motility, viability, volume, agglutination and defective morphology [1], several intrinsic defects have been reported such as abnormal pyruvate utilization [2], high ATP accumulation [3], low zona-free hamster egg penetration scores [4], overexpression of superoxide dismutase and absence of sperm membrane sulfhydryl groups (in oligospermia) [5]. In addition to known factors involved in infertility, a large number of men are diagnosed as cases of unexplained male infertility. Sulfhydryl groups of the sperm membrane play a very important role in sperm motility and metabolism [6] and evidence is also available for the involvement of these surface thiols in normal sperm functioning [7]. It has been known that divalent cations like cadmium and copper are hazardous to mammalian sper0006-291X/98 $25.00
matozoa [8-9]. The spermeostatic properties of copper have been widely utilized in copper containing intrauterine devices as contraceptive agents. Cobaltous ion have been shown to exercise powerful sperm immobilizing properties at extremely low concentrations, which could be recovered by a sulfhydryl compound, cysteine. The loss of sperm surface thiol groups and the augmented production of superoxide anion radical was stated to be the reason for the loss of motility [10]. The sulfhydryl blocking properties of both copper and cobalt is utilized to study role of sulfhydryl groups in sperm membrane modulation. This paper reports a possible regulatory role of sulfhydryl groups in sperm membrane modulation and as a marker for fertility assessment. MATERIALS AND METHODS Semen Human ejaculates were obtained from the pathological laboratory of a local hospital and after their clinical assessment and estimate of motility, count, normal and abnormal forms were collected in a sterile vial containing 2ml HBSS (Hanks Balanced Salt Solution) (pH 7.2) at room temperature. On the basis of count and motility, samples were classified into 2 groups: normal and oligospermic samples. The semen samples thus obtained were centrifuged at 3500 rpm (500 1 g) for 10 minutes at room temperature, resuspended in 2 ml HBSS and then divided into two equal parts. One part of the normal cases were incubated with 1009 M Cu// and the other part was used as control. Similarly in oligospermic cases one part was incubated with 3.6 mM (final concentration) pentoxifylline and the other part was used as a control for 30 minutes at 377C. Then the volume were made upto 5 ml with HBSS and centrifuged at room temperature for 10 minutes at 3500 rpm. The pellets obtained were resuspended in 2 ml HBSS at 377C and were used in subsequent investigations. Semen samples from the cauda epididymis of mature male albino rats (3-5 months old) maintained under temperature (277C) and light (14hr light : 10 hr dark)- controlled conditions and provided food and water ad libitum were used for these studies. Animals were sacrificed and the semen samples from the cauda region of the epididymis were collected in 2 ml HBSS and processed as above. The samples were then divided into two equal parts one part was treated with 1009 M concentration of Co// or Cu// (final concentration) for 30 minutes
716
Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 8841
/
6956$$$341
06-05-98 22:53:09
bbrcg
AP: BBRC
Vol. 247, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Histographical representation of changes in MDA levels in normal spermatozoa without and with 1009 M copper treatment (a) and (b) is MDA levels in oligospermic samples without and with PTF treatment.
at 377C and the other part was used as a control. After incubation the volumes were made upto 5 ml wit HBSS and centrifuged at room temperature for 10 minutes at 3500 rpm. The pellets obtained were resuspended in 2 ml HBSS at 377C and were used in subsequent investigations.
Protocol for Estimation of Lipid Peroxidation Preparation of the sample. The sperm samples in HBSS (pH 7.2) were homogenized at a speed of 5000 rpm, using a Elteck homogenizer NS 3 (3 cycle of 30 sec. each). The homogenates were then centrifuged at 3500 rpm (500 1 g) for 10 minutes. The pellets were resuspended in 1 ml HBSS which was then used for lipid peroxidation assay. Assay for lipid peroxidation. The lipid peroxidation was measured in terms of malonaldehyde (MDA): thiobarbituric acid (TBA) reaction as reported by Okawa et al (1979) [11]. The reaction mixture contained 0.1 ml of tissue homogenate (as described above), 0.2 ml of 8.1% SDS, 1.5 ml of 20% glacial acetic acid and 1.5 ml of 0.8% aqueous solution of TBA. The pH of 20% acetic acid was preadjusted with 1M NaOH at pH 3.5. The mixture was finally made upto 4 ml with distilled water and heated at 957C for 1 hr, on a water bath. After cooling, 1 ml of distilled water and 5ml of a mixture of n-butanol and pyridine (15:1 v/v) was added and the mixture was shaken vigorously on a vortex mixer. Then after centrifugation at 2200 1 g for 5 minutes, the amount of MDA formed was measured by the absorbence of the upper organic layer at 532 nm (extinction coefficient of MDA is 1.45 1 105/min./ cm) using appropriate controls. Measurements were made using a Jasco UV/VIS spectrophotometer.
Assay of Superoxide Dismutase Activity in Spermatozoa Sperm samples obtained from male albino rats were washed with HBSS (pH 7.2) and were treated with cobalt and copper ions (as described above). After incubation these samples were washed with HBSS (pH 7.2) and were resuspended in 4 ml chilled (47C) Tris buffer (50 mM, pH 8.2). These suspensions were homogenized at 47C at a speed of 13000 rpm (3 cycles of 30 sec. each) using Elteck homogenizer NS 3. The homogenates were treated with 1 ml of 1% triton X 100 (v/v) for 20 minutes, at 47C. Homogenates were then centrifuged at a speed of 15000 rpm at 47C using a Remi C30 high speed centrifuge with a fixed angle rotor. The supernatant was used for the assay of superoxide dismutase activity by the method of Marklund and Marklund (1974) [12]. All the calculations were made as per mg of protein.
FIG. 2. Histographical representation of changes in MDA levels in rat caudal spermatozoa with cobalt (a) and copper (b) treatment.
oligospermic sperm samples with and without treatment. The MDA concentration in normal (control) (Fig. 1a) samples were significantly inhibited (Põ0.0001) after 1009 M (final concentration) of Cu// treatment as compared to control. However, there was a sharp increase in MDA levels after pentoxifylline treatment in oligospermic samples (Põ0.00001) (Fig. 1b). Figure 2 shows the changes in amount of MDA formed in rat caudal spermatozoa with 1009 M cobalt and copper (figure 2a and 2b respectively). The MDA levels were significantly decreased (Põ0.001) when the spermatozoa were treated with 1009 M copper. The MDA levels also decreased significantly (Põ0.00001) after 1009 M cobalt treatment. Superoxide dismutase levels were also inhibited significantly (Põ0.00001) in the presence of 1009M cobalt and copper (Figs. 3a and 3b, respectively). Membrane integrity and its proper functioning are the basic characteristics of the sperm membrane including cellular recognition and information transduction during cell cell interaction. Changes in fatty acid composition of membranes as well as the amount of individual sterols account for the change in fluidity. Normal spermatozoa are reported to show high membrane fluidity [12]. Therefore it is evident that membrane fluidity is an important factor for sperm functions. Lipid peroxidation plays a crucial role in inducing membrane fluidity [13]. The results presented here shows that blocking of sperm membrane sulfhydryl groups both in human and rat caudal sperm inhibits lipid peroxidation and superoxide dismutase activity.
RESULTS AND DISCUSSION Figure 1 shows the change in the amount of MDA formed as a result of lipid peroxidation in normal and
FIG. 3. Histographical representation of changes in the levels of SOD in rat caudal spermatozoa with cobalt (a) and copper (b) treatment.
717
AID
BBRC 8841
/
6956$$$342
06-05-98 22:53:09
bbrcg
AP: BBRC
Vol. 247, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
It has been reported earlier that copper containing antifertility devices are believed to act via production of toxic oxygen radical species including hydroxyl and superoxide anion radical [14] and these radical species are known to induce membrane fluidity via lipid peroxidation. But, contrary to the above statement we found that blocking of sulfhydryl groups by copper and cobalt inhibits lipid peroxidation in normal spermatozoa and treatment of oligospermic samples with pentoxifylline increases lipid peroxidation. However, this is a unique phenomenon which remains to be elucidated. But it is quite evident that membrane sulfhydryl groups play an important role in sperm membrane modulation by reducing membrane fluidity. Decrease in motility and loss of sperm function in unexplained male infertility can be attributed to these sulfhydryl groups of the sperm membrane and is suggested that in such cases of infertility the active sulfhydryl groups are masked which results in loss of sperm function. Thus, it is suggested that sperm membrane sulfhydryl groups are important entities of the membrane and can be used as a tool for infertility assessment in unexplained male infertility and can be targeted for contraceptive research.
REFERENCES 1. Gopalkrishna, K. (1995) Current Science 68(4), 353–362. 2. Meaker, S. R. (1934) in Human Sterility. The Williams and Wilkins Co., Baltimore. 3. Dolci, S., Greco, E., Piccione, E., Banchone, C., and Manna, C. (1987) Patol. Clin. Utet Ginecol. 15(1), 1. 4. Aitken, R. J. (1982) in Clinical Practices in Urology—Male Infertility (Hargreave, T., Ed.), Springer Verlag Berlin, Heidelberg, New York. 5. Sinha, S., Kumar, G. P., Laloraya, M., and Warikoo, D. (1991) Biochem. Biophys. Res. Commun. 174(2), 510–517. 6. Mann, T., and Lutwak-Mann, C. (1981) in Male Reproductive Function and Semen p. 337, Springer Verlag, Berlin. 7. Sinha, S., Kumar, G. P., and Laloraya, M. (1994) Biochem. Biophys. Res. Commun. 198(1), 266–273. 8. Parizek, J. (1956) Nature 117, 1036–1037. 9. Mann, T. (1958) Proc. Soc. Study Fertil. 9, 3–10. 10. Kumar, G. P., Laloraya, M., and Laloraya, M. M. (1990) Contraception 41(6), 633–639. 11. Okawa, H., Ohishi, N., and Yogi, K. (1979) Anal. Biochem. 95, 35. 12. Marklund, S., and Marklund, G. (1974) Eur. J. Biochem. 47, 469–474. 13. Jain, S., Thomas, M., Kumar, G. P., and Laloraya, M. (1993) Biochem. Biophys. Res. Commun. 195(2), 574–580. 14. Mann, T., and Leone, E. (1953) Biochem. J. 53, 110–115.
718
AID
BBRC 8841
/
6956$$$342
06-05-98 22:53:09
bbrcg
AP: BBRC