Verma1, K.C. Kanwar
of Biophysics, Panjab University, Chandigarh 160014, India
Asian J Androl 1999 Sep; 1: 151-154
reactive oxygen species; vitamin E; sperm motility; lipid peroxidation
oxygen species (ROS) such as the superoxide anion (O2·-),
the hydroxyl radical (·OH)
and hypochlorite radical (·OHCl) produced by the spermatozoa and the
contaminating leucocytes in the seminal fluid adversely affect sperm motility[1,2]
and also impair their fertilizability.
damage by ROS gets exacerbated when sperm are washed and freed of seminal plasma
since ROS constantly produced by the spermatozoa and the contaminating
leucocytes are not neutralized by the antioxidants present in the seminal plasma[4,5].
2 Materials and methods
sperm samples (n=8) from healthy donors were obtained from the local
hospital in accordance with the Helsinki Declaration and only those having
motility higher than 60% and count over 20 millions/mL were used for the present
studies. Each human ejaculate was split into four equal fractions. The split
seminal fractions were centrifuged at 1000×g and the supernatent (plasma)
discarded. Thereafter, these were reconstituted in Ringer-Tyrode (NaCl 0.8 g,
KCl 0.02 g, CaCl2 0.02 g, NaHCO3 0.1 g, NaH2PO4
0.005 g, MgCl2 0.01 g, glucose 0.1 g, Hepes 0.5 g, and 100 mL
twice-distilled water). Ringer-Tyrode (RT fraction without vitamin E) served as
control. The experimental fractions comprised RT supplemented with three
different concentrations of vitamin E (alpha-tocopherol acetate, E. Merck,
India, 0.1, 1.0 and 2.0 mmol/L).Vitamin E was dissolved in ethanol and an
emulsion was formed by vortex mixing in RT medium before addition to the
spermatozoa. The final ethanol
concentration was <2% by volume.
number of motile and immotile spermatozoa from control as well as
experimental fractions were counted at room temperature (30℃±2℃)
at different time intervals ranging from 0.5 h to 6 h. The viability of
spermatozoa was also evaluated using eosin.
compared with the control, a significant dose-dependent improvement in sperm motility
was noticed from 1 h onwards in the experimental fractions supplemented with
1.0 mmol/L and 2.0 mmol/L concentrations of vitamin E; sperm motility was
maximum after a 6-h
incubation in 2.0 mmol/L vitamin E where it approximated
52.9% (P<0.01) higher in comparison with the control (Figure
Likewise percent spermatozoal viability also improved significantly from 2 h onwards in 1.0 and 2.0 mmol/L vitamin E. Increase in viability vs the control was the highest at 2.0 mmol/L concentration where it approximated 62.6% (P<0.01) following a 6-h incubation (Figure 1B).
compared with the control, statistically significant decrease in MDA formation
was noticeable only after 4 h and 6 h of incubation of the experimental sperm
fraction supplemented with 2.0 mmol/L vitamin E. MDA formation was maximally
reduced (30%; P<0.01) following 4 h of incubation in 2.0 mmol/L
vitamin E sperm fraction (Figure 1C).
gradual decrease in spermatozoal motility and viability with concomitant
increase in MDA in RT from 0.5 h to 6 h observed during the course of present
study have been attributed to oxidative stress to which spermatozoa are
subjected to during storage[13,14]. Human spermatozoa contain little
antioxidant enzymes (viz. catalase, glutathione peroxidase, and superoxide
dismutase) to counteract fully the oxidative stress. Oxygen free
radicals generated by the spermatozoa and the contaminating leucocytes produce a
fall in intracellular ATP levels which adversely affect the sperm motility and
also initiate lipid peroxidation in the polyunsaturated fatty acid rich sperm
plasma membrane[13,16,17] culminating in increased cell permeability,
enzyme inactivation and production of spermicidal end products[14,18,19].
Lipid peroxidation also impairs fertilizing potential of sperm owing to loss of
membrane fluidity or to selective inactivation of some of the
biochemical pathways leading to acrosomal reaction. The present
observations record a significant improvement in sperm motility and
viability following vitamin E treatment in vitro. Higher MDA levels
(representative of lipid peroxidation) are recorded in spermatozoal fractions
suspended in RT only.
Following vitamin E treatment, MDA levels in the experimental fractions are
lowered. These findings are supported by earlier workers who have reported
improved testicular histoarchitecture and sperm quality following vitamin E
dietary supplementation in the different animal species[8,22,23].
Vitamin E, a chain breaking antioxidant, not only scavenges oxygen radicals from
within the membrane but also intercepts peroxyl and alkoxyl radicals which are
generated during the conversion of lipid hydroperoxides that fuel the
peroxidative chain reaction thereby preventing this damaging process from
propagating through plasma
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to Dr. K.C. Kanwar.
Tel: +91-172-54 1441, ext 1364 Fax: +91-172-77 8269
1Present address: Department of Biophysics, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India.
Received 1999-05-19 Accepted 1999-08-19