Home | Archive | AJA @ Nature | Online Submission | News & Events | Subscribe | APFA | Society | Links | Contact Us | 中文版 |
 |
Status
of vitamin E and reduced glutathione in semen of oligozoospermic and azoospermic
patients
A.
Bhardwaj, A. Verma, S. Majumdar, K. L. Khanduja
Departments
of Biophysics and Experimental Medicine, Postgraduate Institute of Medical
Education & Research, Chandigarh, India
Asian J Androl 2000 Sep; 2: 225-228
Keywords:
oligospermia;
glutathione; vitamin E; semen
Abstract
Aim: To investigate the status of seminal plasma reduced glutathione (GSH) and vitamin E in three different conditions of spermatogenesis: azoospermia, oligozoospermia and normospermia. Methods: Reduced glutathione was measured in the seminal plasma by the method of Moron et al (1979), and vitamin E estimation was performed by the method of Taylor et al (1976). Results: Vitamin E levels in seminal plasma of oligospermic and azoospermic samples were significantly decreased to 65.54% and 66.04% respectively as compared to the normospermic group. Levels of reduced glutathione were also significantly decreased in oligospermic and azoospermic group, and the reduction in azoospermic group (76.73%) was more pronounced than oligozoospermic group (62.07%). Conclusion: The decrease in reduced glutathione, an endogenous antioxidant, levels in azoospermic and oligozoospermic conditions may cause disruption in the membrane integrity of spermatozoa as a consequence of increased oxidative stress.1 Introduction
Oligozoospermia
(<20106 spermatozoa/mL) and azoospermia are the major
causes of male infertility[1].
Several investigators have demonstrated that damaged or defective
spermatozoa produce higher levels of reactive oxygen species (ROS). The
possible relationship between the prevalence of ROS and infertility has
been investigated in recent years. As many as 20% semen samples from an
unselected population of men attending an infertility clinic produced
significant levels of ROS[2,3]. Furthermore, there is an inverse
correlation between the percentage of motile
spermatozoa and level of ROS[2]. Generation of ROS in semen
is negatively associated with both the outcome of sperm-oocyte fusion
assay and fertility in vivo[4]. ROS (superoxide anion,
hydrogen peroxide, and hydroxyl radicals), as well
as the fatty acid peroxide generated by ROS attack on cell membrane phospholipids,
are associated with loss of mammalian sperm motility and decreased capacity
for sperm-oocyte fusion[5-8]. In addition, the degradation
products of these lipid peroxides such as hydroxyalkenals and malonaldehyde
are also highly toxic to spermatozoa and cause an irreversible loss of
motility.
2 Materials and methods
2.1
Collection of Semen
Human
semen samples, 12 each of normospermic, oligospermic and azoospermic subjects,
were obtained from men attending the infertility clinic of the institute
after taking the informed consent and clearance from the ethical
committee of the institute. Semen samples were divided into the 3 categories
on the basis of number of spermatozoa (n) present/mL of the semen, normospermic,
n20106/mL,
motility 40% or more with forward progression, oligospermic, n20106/mL
and azoospermic, n=0. Seminal plasma was separated from semen by
centrifugation at 1000g.
The supernatant was used for the vitamin E and reduced glutathione
(GSH) estimation.
2.2
Estimation of vitamin E
Vitamin
E was measured spectrofluorometrically by the method of Taylor et al[17].
Saponification and extraction of vitamin E from the seminal plasma was
done by the method of Desai et al[18].
Briefly, to 1 mL of seminal plasma, 2 mL of 1% ascorbic acid was
added. After thorough mixing, the tubes were incubated for 30 min at 70.
Then the tubes were cooled on ice and 1 mL of distilled water and 4 mL
of hexane were added. The samples were vigorously vortexed and centrifuged
at 1,600g for 10 min. The upper hexane layer was removed and treated
with 60% H2SO4
for 30 sec to remove vitamin A. Vitamin E was estimated in this layer
with spectrofluorometer at excitation wavelength of 286 nm and emission
wavelength of 330 nm.
2.3
Estimation of reduced glutathione
Reduced
glutathione was measured according to the method of Moron et al[19].
Briefly, to 200 L of seminal plasma 800L distilled
water and then 2 mL of sodium phosphate-EDTA buffer, containing 0.6 mol/L
DTNB 5,5'dithiobis-2 (nitrobenzoic acid) were added. Optical density
of the yellow colored complex developed by the reaction of GSH and DTNB
(Ellman's reagent) was measured at 405 nM.
2.4
Statistical analysis
3
Results
3.1
Levels of vitamin E
Figure
1 shows the levels of vitamin E in the seminal plasma of normospermic,
oligozoospermic and azoospermic subjects. Values (meanSD) for the three
groups were 11.82.70, 7.840.71 and 7.800.73 g/mL respectively.
The concentration of this vitamin in oligospermic and azoospermic samples
were significantly decreased in comparison to the normospermic group (P<0.01).
Figure
1. Levels (means)
of vitamin E (g
/ mL) in seminal plasma of normospermic, oligospermic,
and azoospermic subjects.
cP<0.01 in comparison to normospermic.
3.2
Levels of reduced glutathione
The
contents of reduced glutathione (Figure
2) were significantly (P<0.01) decreased from 58.014.0
to 22.05.0 and 13.54.0 g/mL in oligospermic and azoospermic groups
respectively.
Though
the contents of vitamin E in oligospermic and azoospermic samples were
similar, the levels of reduced glutathione in azoospermic group were significantly
(P<0.01) decreased in comparison to the oligospermic group.
Figure 2. Levels (means) of reduced glutahione (g of GSH/mL) in seminal plasma of normospermic, oligospermic, and azoospermic subjects. cP<0.01 in comparison to normospermic. fP<0.01 in comparison to oligospermic.
4
Discussion
Although
the importance of seminal plasma in the protection of spermatozoa
against ROS is well established, only few studies have investigated
its antioxidative properties, and the possible relationship between infertility
and plasma antioxidant defense systems. It has been reported that -tocopherol
concentration, superoxide dismutase and glutathione peroxidase activities
of spermatozoa were significantly elevated in leukocytospermia[20].
It has further been suggested that -tocopherol might play a role[15],
in association with antioxidant
enzymes, for preserving the functional competence of spermatozoa subjected
to an oxidative stress[21].
References
[1]
World Health Organisation. WHO Laboratory manual for the examination of
human semen and semen-cervical
mucus interaction. 2nd Edition. Cambridge: The press syndicate of the
University of Cambridge, 1987: 27.
[2] Iwasaki A, Gagnon C. Formation of reactive oxygen species in spermatozoa
of infertile patients. Fertil Steril 1992; 57: 409-16.
[3] Zini A, de Lamirande E, Gagnon C. Reactive oxygen species in semen
of infertile patients: levels
of superoxide dismutase and catalase like activities in seminal plasma.
Int J Androl 1993; 16: 183-8.
[4] Aitken JR, Irvine DS, Wu FC. Prospective analysis of sperm-oocyte
fusion and reactive
oxygen species generation as criteria for the diagnosis of infertility. Am
J Obstst Gynecol 1991; 164: 542-51.
[5] Jones R, Mann T, Sherins R. Peroxidative breakdown of phospholipids
in human spermatozoa, spermicidal properties of fatty acid peroxides and
protective action of seminal plasma. Fertil Steril 1979; 31: 531-7.
[6] Aitken JR, Clarkson JS. Cellular
basis of defective sperm function and its association with the genesis
of reactive oxygen species by human spermatozoa.
J Reprod Fertil 1987; 81:
459-69.
[7] Alvarez JG, Touchtone JC, Blasco L, Storey B.
Spontaneous lipid peroxidation and
production of hydrogen peroxide and superoxide in human spermatozoa:
superoxide dismutase as a major enzyme protectant against oxygen
toxicity. J Androl
1987; 8: 338-48.
[8] Aitken JR, Clarkson JS, Fishel S.
Generation of reactive oxygen species, lipid peroxidation and human
sperm function. Biol Reprod 1989; 40:
183-97.
[9] Nissen HP, Kreysel HW. Superoxide
dismutase in human semen.
Klinische Wochenschrift 1983; 61:
63-5.
[10] Alvarez JG, Storey BT. Role
of glutathione peroxidase in protecting mammalian spermatozoa from loss
of motility caused by spontaneous lipid peroxidation. Gam Res 1989; 23:
77-90.
[11] Jeulin C, Soufir JC, Weber P, Laval-Martin D, Calvayrac R.
Catalase activity in human spermatozoa and seminal plasma. Gam
Res 1981; 24: 185-96.
[12] Halliwell B, Gutteridge JMC.
Free radicals in biology and medicine. 2nd ed. Oxford:
Clarendon Press; 1989.p 126-31, 152-7, 253-9.
[13] de Lamirande E, Gagnon C.
Reactive oxygen species and human spermatozoa:
II. Depletion of
adenosine-triphosphate (ATP) plays an important role in the inhibition
of sperm motility. J Androl
1992b; 13: 379-86.
[14] Alvarez JG, Storey BT. Taurine,
hypotaurine, epinephrine and albumin inhibit lipid peroxidation in rabbit
spermatozoa and protect against loss of motility.
Biol Reprod 1983; 29:
548-55.
[15] Verma A, Kanwar KC. Effect of vitamin E on human sperm motility and
lipid peroxidation in vitro. Asian J Androl 1999;1: 151-4.
[16] Niki E. Action of
ascorbic acid as a scavenger of active and stable oxygen radicals.
Am J Clin Nutr 1991; 54:
1119S-1124S.
[17] Taylor SL, Landen MP, Tappel AL.
Sensitive fluorimetric method for tissue tocopherol analysis. Lipids
1976; 11: 530-8.
[18] Desai ID. Vitamin
E analysis methods for animal tissues.
Method Enzymol 1984; 105:
138-42.
[19] Moron MS, Dipierre JW, Mannervik B.
Levels of glutathione reductase and glutathione-S-transferase activities
in rat lung and liver. Biochem Biophy Acta 1979; 582: 67-8.
[20] Therond P, Auger J, Legrand A, Jouannet P.
Alpha-tocopherol in human spermatozoa and seminal plasma:
relationships with motility, antioxidant enzymes and leukocytes.
Mol Hum Reprod 1996; 2: 739-44.
[21] Alkan I, Simsek F, Haklar G, Kervancioglu E, Ozveri H, Yalcin S,
et al. Reactive
oxygen species production
by the spermatozoa of patients with idiopathic infertility:
relationship to seminal plasma antioxidants.
J Urol 1997; 157: 140-3.
[22] Oehsendorf FR, Buhl R, Bastlein A, Beschmann H.
Glutathione spermatozoa and seminal
plasma of infertile men. Hum Reprod 1998; 13:
353-9.
[23] Burk RF. Glutathione-dependent
protection by rat liver microsomal protein against lipid peroxidation.
Biochem Biophys Acta 1983; 757: 21-8.
[24] Hill HE, Burk RF. Influence
of vitamin E and selenium on glutathione dependent protection against
microsomal lipid peroxidation. Biochem Pharmacol 1984; 33:
1065-8.
[25] Miester A, Anderson L. Glutathione.
Ann Rev Biochem 1983; 52: 71-90.
[26] Bjorneboe A, Bjorneboe GEAA, Drevon CA.
Absorption, transport and distribution
of vitamin E. J Nutr 1990;
120: 233-42.
[27] Fukuzawa K, Fujii T. Peroxide
dependent and independent lipid peroxidation: site-specific
mechanisms of initiation by chelated iron and inhibition by -tocopherol.
lipids 1992; 27: 222-33.
[28] Meydani SN, Barklund MP, Liu S, Meydani M, Miller RA, Cannon
JG, et al. Vitamin
E supplementation enhances
cell-mediated immunity in healthy elderly subjects.
Am J Clin Nutr. 1990a; 52: 557-63.
[29] Meydani SN, Barklund MP, Liu S, Meydani M, Miller RA, Cannon JG,
et al. Vitamin E supplementation enhances immune response in elderly
subject. Nutr Res
1990b; 50: 85-7.
Correspondence
to: Prof. K. L. Khanduja, Department of Biophysics, Postgraduate Institute
of Medical Education &
Research, Chandigarh-160
012, India.
Fax: +91-172-744 401 +91-172-745 078
e-mail: medinst@pgi.chd.nic.in
Received
2000-07-10 Accepted 2000-08-28
