Antiviral drugs are often nucleoside analogues
that are known to have potential teratogenic, embryotoxic,
carcinogenic and antiproliferative activities
ACV [9-(2-hydroxyethoxymethyl) guanine] is
a synthetic purine nucleoside analogue that was
introduced as the fifth anivirals drug commonly
used in the early 1980’s. ACV has been reported
to be very effective against the treatment of herpes
simplex and varicella zoster infections and
it also protects immune-suppressed patients that
receive transplants from cytomegalovirus (
The androgen production and spermatogenesis
are two main testicular functions. The interstitial
Leydig cells produce testosterone which is a kind of
androgen. Spermatogenesis occurs in seminiferous
tubules. Gonadotropins affect normal spermatogenesis
qualitatively and quantitatively (
Spermatogonia are sensitive to toxins interfering
with DNA replication because these cells go
through several mitotic divisions (
In this study forty male Wistar rats (220 ± 20 g) were obtained from animal house of Faculty of Science, Urmia University and kept under specific conditions on a constant 12-hour light/dark cycle and at a controlled temperature of 22 ± 2˚C. Standard pellet food and tap water were available ad libitum. Animals were allowed to acclimatise for one week before experimental use. It should be noted that this study was an experimental study accordance with the Guidance of Ethical Committee for research on Laboratory Animals of Urmia University.
ACV (MYLAN, France) was used at three dose
levels, 4, 16 and 48 mg/kg based on previous studies
Animals were segregated into 5 groups of eight each. Group 1 served as control, normal and apparently healthy rats that did not receive any type of treatment. Group 2 served as sham control and received distilled water (i.p. injection) for 15 consecutive days. Groups 3, 4 and 5 (the drug treated groups) were administered respectively 4, 16 and 48 mg/kg/day ACV (i.p. injection) for 15 consecutive days.
18 days after the last injection 4 animals from each group were sacrificed by CO2 inhalation. The blood samples were collected from jugular vein andsubsequently the serumwas harvested and frozen. The testes were removed surgically. Total experimental duration was 33 days.
Left epididymal sperms were collected by slicing the epididymides in 5 ml of human tubal fluid (HTF) +4 mg/ml bovine serum albumin (BSA) and incubating for 5 min at 37˚C in an atmosphere of 5% CO2 to allow sperm to swim out of the epididymal tubules.
The epididymal sperm count was determined
by hemocytometry (Neubauer chamber) and the
method described in the WHO manual (1999)
A part of sperm suspension was used for preparing
smears to evaluate the sperm shape abnormalities
Sperm viability was evaluated as follows. A 20
μl of 0.5% eosin Y and nigrosin were added into
an equal volume of the sperm suspension. After
2 min of incubation at room temperature, slides
were viewed by light microscope with magnification
of 400. Dead sperms appeared to be pink and
live sperms were not stained. In each sample 400
sperms were counted and viability percentages
were calculated (
The spermatozoa were divided as motile or immotile.
Motility of the spermatozoa was evaluated
under a light microscope (Olympus Co., Tokyo,
Japan). One drop of sperm suspension was
placed on a glass slide, covered with a coverslip,
and 10 random fields of view were examined at
400× magnification. The number of motile and
nonmotile sperm was counted. Motility was then
expressed as the percentage of motile sperm to the
total number of sperm (
Male infertility and abnormal spermatogenesis
have close relation with sperm DNA damage (
Histones are replaced by transition proteins
and then by protamines during the later stages
of spermatogenesis, spermatid nuclear changing
and condensing. The DNA strands are tightly
wrapped around the protamine molecules to create
toroidal structures (
After blood sampling the serum was separated using a centrifuge and kept at -70˚C until analysis of testosterone hormone. Serum testosterone concentrations were measured by using a testosterone Electrochemiluminescence Kit (Roche Diagnostics, Germany, Limit of Detection: 0.025 ng/ml).
The data are presented as the mean ± SEM. Differences between groups were analyzed by One Way Analysis of Variance (ANOVA) followed by Tukey test using SPSS package, version 16 and level of significance was taken as p<0.05.
Result showed that i.p. injection of ACV did not
cause significant changes in total cauda epididymal
sperm count as compared to control and sham
control groups (
ACV significantly decreased serum testosterone
level at 16 and 48 mg/kg dose-levels (p<0.01) in a
dose dependent manner (
Effects of ACV on sperm parameters in adult male rats
|Total sperm/cauda epididymis (106)||Motile sperm (%)||Abnormal sperm (%)||Live sperm (%)|
|205.2 ± 1.314||67.25 ± 2.174||11.5 ± 0.866||69.50 ± 0.866|
|205 ± 1.04||67 ± 2.121||11.25 ± 1.25||68.25 ± 1.376|
|230 ± 1.354||55.75 ± 2.393||18.25 ± 1.6||59.50 ± 2.179|
|185 ± 1.755||41 ± 2.041ab**c*||19.5 ± 4.051||58.25 ± 2.25ab*|
|172.5 ± 1.937||37 ± 6.069abc**||24.5 ± 2.901ab*||41.25 ± 4.366abc**|
Data are presented as mean ± SEM from 4 animals per group. a; significant compared with control, b; significant compared with sham control and c; significant compared with 4mg/kg ACV.
*; P<0.05 and **; P<0.01.
Effects of ACV on DNA damage and Chromatin abnormalities of sperm in adult male rats
|AO+ (%)||AB+ (%)|
|11.5 ± 1.258||10 ± 1.08|
|9 ± 1.471||7.5 ± 1.707|
|19 ± 0.816||27.55 ± 2.561 ab**|
|32.5 ± 2.217 ab**c*||25 ± 2.345 ab**|
|37.75 ± 5.498 abc**||35.75 ± 2.675 ab**d*|
Data are presented as mean ± SEM from 4 animals per group. AO+; Acridine-orange positive, AB+; Aniline blue positive, a; significant compared with control , b; significant compared with sham control, c; significant compared with 4 mg/kg ACV,
d; significant compared with 16mg/kg ACV, *; P<0.05 and **; P<0.01.
Dead sperm appear pink and live sperm are not stained, Eosin/Nigrosinstaining technique (×2000).
A. Sperm with damaged DNA (yellow), B. Sperm with normal DNA (green). Acridine-orange stainingtechnique (×2000).
Sperm head containing immature nuclear chromatin is dark blue and sperm head with mature nuclei is light blue. Aniline blue staining technique (×2000).
Effects of ACV on serum testosterone concentrations in adult male rats. 4 rats from each group were analysedin this experiment. Error bars indicate the standard error of the mean. *; p<0.05 and **; p<0.01.
a; Significant compared with control, b; Significant compared with sham control and c; Significant compared with 4 mg/kg ACV.
In the current study, it was investigated whether
a purine nucleoside analogue-acyclovir has any reproductive
toxic effects in adult male rats or not.
ACV was administrated to rats at doses of 4, 16
and 48 mg/kg body weight (b.w.) These doses were
chosen according to a preparative study which investigated
the effect of doses of 4, 16, 32 and 48
mg/kg b.w of ACV on male reproductive system
in mice (
ACV is reported to inhibit thymidine kinase in
the viral defected DNA and also in non-infected
Sperm count reduction is an important indicator
of male infertility (
Sperm motility often indicates chemical-induced
testicular toxicity (
It was evident that different factors can induce
DNA damage in male germ cells that can result
in adverse effects in offspring (
Furthermore,according to previous studies, to
package DNA properly during spermatogenesis,
protamines are required (
It is well known that in the adult, testosterone
supports spermatogenesis, sperm maturation and
sexual function. Therefore, disruption of testosterone
biosynthesis in Leydig cells can adversely
affect male fertility (
In conclusion, the present study shows that ACV plays negative roles on the reproductive system and function in sexually mature male rats by its adverse effects on the sperm parameters and testosterone production in these animals. Considering the human germ cells have the same sensitivity as that of rat germ cells, cytotoxicity and gonadotoxicity of ACV in humans can be expected. Whether the adverse effects of ACV on male rat fertility are temporary, it remains for further investigation.