Antioxidant capacity of the epididymis

R.J. Potts1, T.M. Jefferies and L.J. Notarianni

School of Pharmacy and Pharmacology, University of Bath,Bath, BA2 7AY, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The human epididymis provides an optimal environment for the storage and maturation of spermatozoa. However, the ability of the epididymis to protect spermatozoa from oxidative attack whilst stored at this site, through the local actions of antioxidants, has not thus far been well studied. This study assessed the contribution of the epididymis to seminal plasma antioxidant activity, by comparing the semen of normozoospermic and vasectomized men. Total seminal plasma antioxidant activity was measured, as were concentrations of urate, ascorbate and thiols, antioxidants that are abundant in human semen. Thiobarbituric acid reactive species (TBARS) were measured to indicate lipid peroxidation. Total antioxidant activity and thiol content were significantly lower (P < 0.05) in the plasma from vasectomized men compared with that of normozoospermic donors. Ascorbate and urate were found at similar concentrations in the plasma of both groups. The concentration of TBARS was significantly higher (P < 0.001) in the semen from vasectomized individuals compared with the normozoospermic group. The results indicate that the epididymis contributes to the antioxidant capacity of seminal plasma and possesses region-specific antioxidant activity, which may potentially protect spermatozoa from oxidative attack during storage at this site.

Key words: antioxidants/epididymis/reactive oxygen species/seminal plasma


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reactive oxygen species (ROS) are believed to be important mediators of damage to spermatozoa and have been associated with decreased motility, abnormal morphology and a lowered capacity for sperm–oocyte penetration (Mann et al., 1980Go; Aitken and Fisher, 1994Go; Aitken, 1995Go). In order to counteract the toxic effects of ROS, seminal plasma and spermatozoa possess an array of antioxidant mechanisms. The antioxidant enzymes catalase, superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione reductase (GRD) have all been detected in seminal plasma (Sanocka et al., 1996Go; Alkan et al., 1997Go; Yeung et al., 1998Go). In addition, semen contains high concentrations of thiol groups, ascorbic and uric acid, as well as less substantial amounts of glutathione (GSH) and {alpha}-tocopherol (Li 1975Go; Lewis et al., 1997Go; Ochsendorf et al., 1998Go). Spermatozoa themselves also possess high concentrations of thiol groups, as well as smaller amounts of ascorbic acid, {alpha}-tocopherol, uric acid and GSH (Li, 1975Go; Lewis et al., 1997Go; Ochsendorf et al., 1998Go).

The human epididymis provides an optimal environment for sperm storage and maturation. The capacity of the epididymis, however, to protect spermatozoa from oxidative attack through the local actions of antioxidants during epididymal storage has not thus far been well studied. Similar concentrations of SOD, GPX, GRD and catalase-like enzyme activity in human seminal plasma from normozoospermic and vasectomized donors (with ductal occlusion and hence no epididymal contribution to the ejaculate) were found (Yeung et al., 1998Go). Although the epididymis has been found to synthesize and secrete significant amounts of extracellular SOD, similar concentrations were detected in the semen of men with an intact ductal system and in that of vasectomized men (Williams et al., 1998Go). GSH has been located in rat and bull epididymal homogenates (Agrawal and Vanha-Perttula, 1988a,b), and the enzyme {gamma}-glutamyl transpeptidase, required for the regeneration of oxidized or conjugated GSH, has also been found within the rat epididymal lumen (Hinton et al., 1991Go).

The mRNA distribution of various antioxidants and protective enzymes has also been investigated in the male reproductive tract of several species. Cellular, phospholipid hydroperoxide and epididymal GPX mRNA have been detected in the epididymis (Zini and Schlegel, 1997aGo,bGo). Cytoplasmic copper-zinc and extracellular SOD mRNA have been detected in epididymal epithelia (Nonogaki et al., 1992Go; Zini and Schlegel, 1997aGo), as have low concentrations of catalase mRNA (Zini and Schlegel, 1996Go) and the gene expressing glutathione S-transferase (GST) (Hales et al., 1980Go).

The aim of the present study was to assess the contribution of the human epididymis to seminal plasma antioxidant activity that may protect spermatozoa from oxidative attack whilst stored at this site, by comparing the semen of normozoospermic and vasectomized men. Total antioxidant activity was determined in the plasma of both groups, in addition to thiol, ascorbic acid and uric acid content, individual antioxidants that are abundant in human semen. Indication of the amounts of lipid peroxidation was also determined according to the amount of thiobarbituric acid reactive species (TBARS) in seminal plasma.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Normozoospermic semen samples were obtained from patients being screened for fertility at the Royal United Hospital, Bath, UK. After liquefaction of the sample at room temperature, semen analysis using World Health Organization criteria (WHO, 1993) was performed. Normozoospermic samples were defined as possessing >=20x106 spermatozoa/ml, motility >=50%, normal morphology >=40% and <1x106 leukocytes/ml. Semen was also obtained from vasectomized individuals, 3–4 months post-operatively, from the Bath Clinic, Bath, UK, and were included for use in the study if the samples were azoospermic and contained <1x106 leukocytes/ml. All samples were obtained by masturbation after 3 days of abstinence. This study had the approval of the Bath District Ethics Committee.

Determination of total antioxidant activity
Total antioxidant activity was determined spectrophotometrically according to the ability of seminal plasma antioxidants to scavenge the 2,2'-azinobis(3-ethylbenzothiazoline 6-sulphonate) (ABTS) radical cation, ABTS+, inhibiting its absorption at 734 nm (Rice-Evans and Miller, 1994Go; Miller and Rice-Evans, 1996Go).

Semen (0.5 ml) was centrifuged at 3000 g for 10 min. An 8.4 µl aliquot of the supernatant was added to a solution containing 2.5 µmol/l metmyoglobin and 150 µmol/l ABTS in phosphate buffered saline (PBS). In order to start the reaction, 375 µmol/l hydrogen peroxide was added to the mixture, producing a final volume of 1 ml. The solution was immediately vortexed, placed in a 30°C incubator and a clock started. After 165 s, the reaction mixture was transferred to a 1 cm cuvette and absorbance read at 734 nm. Calibrations were performed using the antioxidant Trolox dissolved in PBS, in place of seminal plasma.

Determination of total thiol concentration
Total plasma thiol concentration was determined according to the method described by Hu (1994). Semen (1 ml) was centrifuged at 3000 g for 10 min. A 200 µl aliquot of the supernatant was mixed with 600 µl 0.25 mol/l Tris base containing 0.20 mmol/l Na2ETDA, pH 8.2, 40 µl 10 mmol/l 2,2-dithiobisnitrobenzoic acid and 3.16 ml absolute methanol. The solution was vortex mixed and left to stand at room temperature for 20 min. The solution was then centrifuged at 3000 g for 10 min and the absorbance of the clear supernatant measured at 412 nm and subtracted from a blank containing distilled water in place of plasma. Total thiol concentration was then calculated using a standard curve constructed using reduced glutathione (GSH) in distilled water in place of plasma.

Determination of ascorbic acid and uric acid concentration
Total plasma ascorbic and uric acid concentrations were determined according to a modified method (Pappa-Louisi and Pascalidou, 1998Go), using high performance liquid chromatography (HPLC) with UV detection. The liquid chromatography system consisted of a Jasco (Jasco Ltd., Great Dunmow, Essex, UK) PU-980 pump, a Jasco 851-AS autosampler fitted with a 50 µl injection loop, a Hichrom (Hichrom Ltd., Reading, Berkshire, UK) 5 µm 150x4.6 mm C18 column and a Jasco UV-975 detector set at 280 nm. The mobile phase consisted of 0.05 mol/l disodium hydrogen phosphate buffer containing 2 mmol/l Na2EDTA and 5 mmol/l cetyltrimethylammonium bromide, adjusted to pH 4.0 using orthophosphoric acid. The mobile phase was filtered through a 0.45 µm filter under vacuum and degassed using helium before use. All separations were carried out at ambient room temperature, using a flow rate of 0.8 ml/min.

Semen (1 ml) was centrifuged at 3000 g for 10 min. A 300 µl aliquot of the supernatant was admixed with an equal volume of cold 10 % w/v metaphosphoric acid containing 2 mmol/l Na2EDTA. A 10 µl aliquot of 3,4-dihydroxybenzylamine (5 mg/ml) was added to the acidified plasma solution, which acted as an internal standard for the quantification of ascorbic acid. Unlike ascorbic acid, uric acid is stable in sample and standard solutions and was quantified using an external uric acid calibration. The mixture was vortexed and centrifuged at 3000 g for 10 min at 4°C. A 100 µl aliquot of the supernatant was added to 50 µl 0.5 mol/l Tris buffer containing 10 mmol/l dithiothreitol, pH 9.0, in order to reduce oxidised ascorbic acid (dehydro-L-ascorbic acid) to ascorbic acid. After 5 min at 25°C, the reaction was quenched by addition of 50 µl 0.2 mol/l H2SO4. The volume was made up to 0.5 ml with 0.05 mol/l disodium hydrogen phosphate buffer containing 2 mmol/l Na2EDTA, pH 4.0, and filtered through a disposable 0.2 µm filter into a capped vial. Prior to injection, the sample was stored on ice in the dark. An injection volume of 50 µl was used for the analyses. All standards were prepared in mobile phase without the addition of cetyltrimethylammonium bromide.

Determination of TBARS concentration
Lipid peroxidation was measured according to the concentration of thiobarbituric acid reactive species (Buege and Aust, 1978Go). Seminal plasma was separated from spermatozoa by centrifugation of semen (1.5 ml) for 10 min at 3000 g. To 1 ml of the resulting supernatant was added 2 ml thiobarbituric acid reagent (15 % w/v trichloroaectic acid, 0.375% w/v thiobarbituric acid and 0.25 N HCl). The solution was then heated at 100°C for 15 min. After cooling, the precipitate was removed by centrifugation at 1000 g for 10 min, and the absorbance of the supernatant determined at 535 nm against a blank containing distilled water instead of biological sample. The concentration of TBARS in the samples were calculated using the molar extinction coefficient of malondialdehyde (1.56x105 mol/l/cm).

Statistical analysis
An Anderson-Darling test for normality was carried out on each data group to be examined statistically. Depending on the outcome, either an unpaired t test or a Mann-Whitney U test was employed to test differences between samples from normozoospermic donors and from vasectomized donors. A P-value of < 0.05 was considered statistically significant. All statistical analyses were carried out using Minitab 11.12 (Minitab Inc., State College, PA, USA).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Measurement of total antioxidant capacity
The total antioxidant capacity of seminal plasma in normozoospermic samples ranged from 473.69 to 887.84 µmol/l, and from 316.84 to 825.83 µmol/l in samples acquired from men with ductal occlusion (Table IGo). The normozoospermic group possessed significantly higher total antioxidant activity than that obtained using plasma from vasectomized individuals (684.17 ± 103.94 and 606.69 ± 104.86 µmol/l respectively; P < 0.02; t-test). In order to assess whether the total antioxidant capacity of seminal plasma changed during time, semen was obtained from five normozoospermic and five vasectomized individuals every fortnight over a period of 2 months and the antioxidant activity in these samples was determined. It was found that the antioxidant capacity varied by <5% of the mean activity in the same individual during this time (mean ± SD 3.92 ± 0.74%). Thus, it would seem that over short periods at least, the total seminal plasma antioxidant activity of a given man is reasonably stable.


View this table:
[in this window]
[in a new window]
 
Table I. Concentrations of antioxidants and thiobarbituric reactive species (TBARS) in the seminal plasma of vasectomized and normozoospermic men. Values shown are mean ± SD
 
Measurement of thiol concentration
Plasma thiol content ranged from 76.93 to 261.71 µmol/l in normozoospermic men and 59.91 to 227.68 µmol/l in vasectomized men (Table IGo). Thiol concentration was significantly higher in the plasma of normozoospermic men compared with that of vasectomized individuals (128.60 ± 45.81 and 101.55 ± 40.09 µmol/l respectively; P < 0.05; Mann-Whitney U test).

Measurement of ascorbic acid and uric acid concentration
Figure 1Go displays a typical chromatogram of a seminal plasma extract, obtained as described under Materials and methods. Under the conditions employed, all peaks of interest were well resolved from interfering peaks, and eluted in <12 min. Total seminal plasma ascorbic acid content ranged from 214.81 to 523.40 µmol/l in all individuals (Table IGo). No significant difference in plasma ascorbic acid content was found between normozoospermic and vasectomized men (384.88 ± 122.23 and 396.48 ± 87.20 µmol/l respectively; P > 0.05; t- test). Concentrations of uric acid in seminal plasma ranged from 119.73 to 312.02 µmol/l in all individuals (Table IGo). There was also no significant difference in uric acid content between the normozoospermic and post-vasectomy data groups (197.62 ± 66.73 and 189.51 ± 53.34 µmol/l respectively; P > 0.05; t-test).



View larger version (16K):
[in this window]
[in a new window]
 
Figure 1. Typical chromatogram of a seminal plasma extract executed as described in text. The mobile phase (pH 4.0) consisting of 0.05 mol/l disodium hydrogen phosphate, 2 mmol/l Na2EDTA and 5 mmol/l cetyltrimethylammonium bromide, produced elution times of 6.69 min for uric acid, 9.15 min for ascorbic acid, and 3.20 min for the ascorbate internal standard, 3,4-dihydroxybenzylamine.

 
Measurement of TBARS concentration
Plasma concentrations of TBARS ranged from 2.71 to 9.11 nmol/ml in the normozoospermic group and 4.59 to 10.00 nmol/ml in the vasectomized group (Table IGo). The concentration of TBARS was significantly higher in the seminal plasma of samples obtained from vasectomized donors compared to normozoospermic donors (7.98 ± 1.36 and 5.66 ± 1.38 nmol/ml respectively; P < 0.001; t-test).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study investigated whether the human epididymis is a region-specific source of antioxidants that are measurable in the ejaculate, by comparing the semen of normozoospermic and vasectomized men. Total antioxidant activity was measured, as well as concentrations of the individual antioxidants, ascorbate, urate and thiol groups. In addition, concentrations of TBARS, indicative of concentrations of lipid peroxidation, were determined. The study is the first, to our knowledge, to demonstrate that seminal plasma from men with an intact ductal system possesses higher total antioxidant capacity and greater thiol group concentrations, compared with plasma from vasectomized individuals. Additionally, concentrations of peroxidized lipids and their breakdown products were lower in the seminal plasma of normozoospermic men compared with specimens from donors post-vasectomy, which may be due in part, to the greater antioxidant capacity that was demonstrated.

The generation of ROS is an essential prerequisite for the normal functioning of many cells; however, excessive ROS formation can lead to cellular pathology. Over-exposure of spermatozoa to ROS is associated with decreased motility, abnormal morphology and a lowered capacity for oocyte penetration. Several studies have also demonstrated that high levels of certain antioxidants are positively correlated with semen quality (Fraga et al., 1991Go; Kobayashi et al., 1991Go; Fraga et al., 1996Go; Suleiman et al., 1996Go). The antioxidant capacity of spermatozoa and the environs within which they occur would seem, therefore, to be a significant factor in determining the aetiology of male infertility.

Ascorbate, urate and thiols are the major individual antioxidants present in human semen (Lewis et al., 1997Go), and were therefore selected for measurement in this study. Thiol content in normozoospermic semen was significantly higher than in samples obtained from vasectomized men, suggesting the accumulation of thiol-containing compounds such as GSH in the epididymal lumen. Ascorbic acid and uric acid were found at similar concentrations in the seminal plasma from both normozoospermic and vasectomized men and do not, therefore, appear to be key antioxidants in the epididymis.

The total antioxidant capacity of seminal plasma, calculated in this study according to the ability of antioxidants to scavenge the radical cation, ABTS+., was determined to be 684.17 ± 103.94 µmol/l for the normozoospermic group. This figure is under half the total antioxidant capacity determined using the same method for adult human blood plasma (mean 1.46 ± 0.14 mmol/l; Rice-Evans and Miller, 1994), but was significantly higher than that found in the plasma of men with ductal occlusion. The elevated TBARS concentrations found in the seminal plasma of vasectomized individuals concurs with the lower antioxidant capacity that was demonstrated in this group, in that reduced antioxidant concentrations would provide less protection against the deleterious effects of ROS, such as generation of lipid peroxidation. Overall, the data appears to indicate that certain antioxidants, such as thiol-containing compounds but not urate and ascorbate, accumulate in the epididymal lumen, where they may then protect against the adverse effects of ROS.

Using the antioxidant data obtained from vasectomized and normozoospermic men, the antioxidant capacity of the epididymis can be estimated by taking into account the proportion of the ejaculation volume that is contributed by the epididymis. It has been calculated (Jouannet and David, 1978Go) that the epididymis voids ~0.7 ml in the average ejaculate. The difference in antioxidant capacity determined between individuals with an intact ductal system and vasectomized men in the present study is 77.48 µmol/l (684.17–606.69 µmol/l). If we assume that the volume the epididymis contributes to the ejaculation is 0.7 ml, then the estimated antioxidant capacity of the epididymal fluid is 54.24 nmol.

Previous studies have reported lower total antioxidant activity in the semen of infertile men compared with that from normozoospermic donors (Lewis et al., 1995Go; Smith et al., 1996Go), in addition to higher concentrations of lipid peroxidation (Sanocka et al., 1996Go) and ROS (Alkan et al., 1997Go). The abundance of antioxidants and oxidative stress in the epididymis, therefore, may be an important determinant in male fertility. Further studies are required to determine if low antioxidant activity and high oxidative stress in the epididymis itself are associated with poor fertility.

We report here that seminal plasma from vasectomized men contains less total antioxidant capacity, lower thiol group concentrations and higher amounts of lipid peroxidation, compared with men with an intact ductal system.


    Acknowledgments
 
The authors thank the staff of the Bath Clinic and Royal United Hospital, Bath, UK, and D.Bamford, former consultant gynaecologist at the Royal United Hospital, for help in several aspects of the study. This work was supported by a grant from the University of Bath.


    Notes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Agarwal, Y.P. and Vanha-Perttula, T. (1988a) Gamma-glutamyl-transferase transpeptidase, glutathione, and L-glutamic acid in the rat epididymis during postnatal development. Biol. Reprod., 38, 996–1000.[Abstract]

Agarwal, Y.P. and Vanha-Perttula, T. (1988b) Glutathione, L-glutamic acid and gamma-glutamyl-transferase transpeptidase in the bull reproductive tissues. Int. J. Androl., 11, 123–131.[ISI][Medline]

Aitken, R.J. (1995) Free radicals, lipid peroxidation and sperm function. Reprod. Fertil. Dev., 7, 659–668.[ISI]

Aitken, R.J. and Fisher, H. (1994) Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioassays, 16, 259–267.[ISI][Medline]

Alkan, I., Simsek, F., Haklar, G. et al. (1997) Reactive oxygen species production by the spermatozoa of patients with idiopathic infertility: Relationship to seminal plasma antioxidants. J. Urol., 157, 140–143.[ISI][Medline]

Buege, J.A. and Aust, S.D. (1978) Microsomal lipid peroxidation. Methods Enzymol., 52, 302–310.[Medline]

Fraga, C.G., Motchnik, P.A., Shigenaga, M.K. et al. (1991) Ascorbic acid protects against endogenous oxidative DNA damage in human sperm. Proc. Natl. Acad. Sci. USA, 88, 11003–11006.[Abstract]

Fraga, C.G., Motchnik, P.A., Wyrobek, A.J. et al. (1996) Smoking and low antioxidant levels increase oxidative damage to sperm DNA. Mutat. Res., 351, 199–203.[ISI][Medline]

Hales, B.F., Hachey, C. and Robaire, B. (1980) The presence and longitudinal distribution of the glutathione-S-tranferases in rat epididymis and vas deferens. Biochem. J., 189, 135–142.[ISI][Medline]

Hinton, B.T., Palladino, M.A., Mattmueller, D.R. et al. (1991) Expression and activity of gamma-glutamyl transpeptidase in the rat epididymis. Mol. Reprod. Dev., 28, 40–46.[ISI][Medline]

Hu, M.L. (1994) Measurement of protein thiol groups and glutathione in plasma. Methods Enzymol., 233, 380–385.[ISI][Medline]

Jouannet, P. and David, G. (1978) Evolution of the properties of semen immediately following vasectomy. Fertil. Steril., 29, 435–441.[ISI][Medline]

Kobayashi, T., Miyazaki, T., Natori, M. et al. (1991) Protective role of superoxide dismutase in human sperm motility – superoxide-dismutase activity and lipid peroxide in human seminal plasma and spermatozoa. Hum. Reprod., 6, 987–991.[Abstract]

Lewis, S.E.M., Boyle, P.M., McKinney, K.A. et al. (1995) Total antioxidant capacity of seminal plasma is different in fertile and infertile men. Fertil. Steril., 64, 868–870.[ISI][Medline]

Lewis, S.E.M., Sterling, E.S.L., Young, I.S. et al. (1997) Comparison of individual antioxidants of sperm and seminal plasma in fertile and infertile men. Fertil. Steril., 67, 142–147.[ISI][Medline]

Li, T.-K. (1975) The glutathione and thiol content of mammalian spermatozoa and seminal plasma. Biol. Reprod., 12, 641–646.[ISI][Medline]

Mann, T., Jones, R. and Sherins, R. (1980) Oxygen damage, lipid peroxidation, and motility of spermatozoa. In Steinberger, A. and Steinberger, E. (eds), Testicular Development, Structure and Function. Raven Press, New York, USA, pp. 497–501.

Miller, N.J. and Rice-Evans, C.A. (1996) Spectrophotometric determination of antioxidant activity. Redox Report, 2, 161–171.[ISI]

Nonogaki, T., Noda, Y., Narimoto, K., et al. (1992) Localization of CuZn-superoxide dismutase in the human male genital organs. Hum. Reprod., 7, 81–85.[Abstract]

Ochsendorf, F.R., Buhl, R., Bastlein, A. et al. (1998) Glutathione in spermatozoa and seminal plasma of infertile men. Hum. Reprod., 13, 353–359.[ISI][Medline]

Pappa-Louisi, A. and Pascalidou, S. (1998) Optimal conditions for the simultaneous ion-pairing HPLC determination of L-ascorbic, dehydro-L-ascorbic, D-ascorbic, and uric acids with on-line ultraviolet absorbance and electrochemical detection. Anal. Biochem., 263, 176–182.[ISI][Medline]

Rice-Evans, C. and Miller, N.J. (1994) Total antioxidant status in plasma and body fluids. Methods Enzymol., 234, 279–293.[ISI][Medline]

Sanocka, D., Miesel, R., Jedrzejczak, P. et al. (1996) Oxidative stress and male infertility. J. Androl., 17, 449–454.[Abstract/Free Full Text]

Smith, R., Vantman, D., Ponce, J. et al. (1996) Total antioxidant capacity of human seminal plasma. Hum. Reprod., 11, 1655–1660.[Abstract]

Suleiman, S.A., Elamin Ali, M., Zaki, Z.M.S. et al. (1996) Lipid peroxidation and human sperm motility: protective role of vitamin E. J. Androl., 17, 530–537.[Abstract/Free Full Text]

Williams, K., Frayne, J., McLaughlin, E.A. et al. (1998) Expression of extracellular superoxide dismutase in the human male reproductive tract, detected using antisera raised against a recombinant protein. Mol. Hum. Reprod., 4, 235–242.[Abstract]

World Health Organisation (1993) WHO Laboratory Manual for the Examination of Human Semen and Sperm–cervical Mucus Interaction, 3rd edn. Cambridge University Press, Cambridge, UK.

Yeung, C.H., Cooper, T.G., DeGeyter, M. et al. (1998) Studies on the origin of redox enzymes in seminal plasma and their relationship with results of in-vitro fertilization. Mol. Hum. Reprod., 4, 835–839.[Abstract]

Zini, A. and Schlegel, P.N. (1996) Catalase mRNA expression in the male rat reproductive tract. J. Androl., 17, 473–480.[Abstract/Free Full Text]

Zini, A. and Schlegel, P.N. (1997a) Expression of glutathione peroxidases in the adult male rat reproductive tract. Fertil. Steril., 68, 689–695.[ISI][Medline]

Zini, A. and Schlegel, P.N. (1997b) Identification and characterization of antioxidant enzyme mRNA in the rat epididymis. Int. J. Androl., 20, 86–91.[ISI][Medline]

Submitted on February 19, 1999; accepted on June 25, 1999.