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Sperm
quality in mice acutely treated with parathion
Cristian
Sobarzo, Eduardo Bustos-Obregn
Faculty
of Basic Sciences, Catholic University of Temuco and Biology of Reproduction
Unit, Program of Morphology, ICBM, Faculty of Medicine, University of
Chile. Santiago 7 , Chile.
Asian J Androl 2000 Jun; 2: 147-150
Keywords:
epididymis;
spermatozoa; organophosphorous compounds; agropesticides; spermatogenesis
Abstract
Aim: To investigate the toxic effect of a single injection of the organophosphorous agropesticide, parathion, on spermatogenesis in immature male mice. Methods: Seven-day old mice received a single injection of parathion intraperitoneally at a dose of 1/3 LD50. The epididymal sperm count, sperm morphology and chromatin thermal stability were analyzed 28 and 50 days after injection. Results: Sperm counts were decreased and teratozoospermia and thermal denaturation of DNA increased after parathion injection. Sperm parameters were changed to a greater extent in younger animals, denoting a higher lability of spermatogenic process at its beginning. The damages could recover a long time after parathion administration. Conclusion: Organophosphorous agropesticides are testicular toxicants, eliciting reversible cytotoxic and cytogenetic alterations in germ cells.1 Introduction
In
the mouse, spermatogenesis starts soon after birth. During the prepubertal period the seminiferous epithelium shows only
Sertoli cells and spermatogonia; the latter
gives rise to primary spermatocytes, thus entering into the first
meiotic prophase. By days 22-24, the first spermatids appear (stages I-VII)
and around day 28, spermatogenesis is qualitatively, though not yet quantitatively,
completed[1,2].
All
these cellular transformations are events that may be damaged by environmental
toxicants[3,4], including agropesticides[5-12]. Bustos-Obregn et al[9] have specifically reported
the testicular toxicity of an organophosphorous agropesticide, parathion.
2 Materials and methods
2.1
Animals
Immature
male CF-1 mice were used. They were kept in an animal house, at 18-20,
with 12/12 hours light/darkness regimen and were fed commercial pellet
and water ad-libitum.
Parathion®0,0-diethyl-O-p-nitrophenyl-mono-phosphate
(Sigma, USA)diluted in
0.85% saline solution (99.2%
w/v), was injected intraperitoneally at a single dose of 20 mg/kg body
weight (1/3 LD50). Controls were injected the same volume of
the saline. Mice were injected
when they were 7 days old (when the spermatogenic line is represented
only by spermato-gonia). Animals were sacrificed in groups
of 5 on day 28 and day 50 after injection and the epididymides were dissected
for the determination of sperm count, morphology and chromatin stability.
2.2
Sperm count
The
cauda epididymidis was cut and weighed. A cell suspension was prepared
by macerating the cauda in 2.0 mL of 0.85% saline. The cell suspension
was kept for 24 hours at 4; it was then filtered through a double gauze
layer and an aliquot
of the sample was used for sperm count in a Neubauer chamber.
2.3
Sperm morphology
From
the cauda maceration, sperm samples were obtained, fixed in neutral formalin,
smeared, air dried, stained with hematoxylin/eosin, and inspected under
a light microscope (1000).
Teratozoospermic forms were classified according to Vigil and Bustos-Obregn[13].
2.4
Sperm chromatin stability
The
thermal pattern of DNA denaturation was tested by incubating sperm samples
at 90
for 0, 2, 6 and 10 minutes. The sperm smear was then stained with acridine
orange according to Tejada et al[14]. Normal chromatin
stains orthochromatic, while denatured nuclei stain metachromatic under
a fluorescence microscope (Zeiss MO1) at 524 nm.
2.5
Statistical analysis
3
Results
3.1
Sperm count
Figure
1 shows the caudal sperm count 28 and 50 days after injection. There was
a significant decrease (P<0.05) in the count in treated mice
on day 28, and an insignificant
decrease on day 50.
Figure
1. Total sperm count in cauda epididymis (106/mg epididymal
weight) 28 and 50 days after a single intraperitoneal injection of parathion
(20 mg/kg). bP<0.05 Students' t test vs
Control group.
3.2
Sperm morphology
The
percentages of teratozoospermia are shown in
Figure 2. A higher percentage
of teratozoospermia of the head and flagellum was seen in the control
group on day 28 (73.4%) than on day 50 (12.2%).
On day 28 the treated group showed a lower flagellar anomaly compared
with the controls (P<0.05). On day 50 there was
a marked decrease (P<0.01) in normal formed sperm in the treated
group as a result of a high teratozoospermia of the head and flagellum
(P<0.01 and 0.001,
respectively).
Figure
2. Teratozoospermia (head and flagellar anomalies) 28 days (A) and
50 days (B) after parathion. bP<0.05 and dP<0.001
Students' t test vs Control group.
3.3
Thermal stability of DNA
Results
are shown in Figure 3 as percentage
of metachromatic sperm nuclei (red fluorescence).
It can be seen that in both groups all the values were higher on
day 28 (even before the thermal shock) compared with those on day 50;
besides, they were significantly higher in the treated than in the corresponding
control groups (P<0.01-0.001). On day 50, the values were negligible
in both groups at 0 minute, but were increased at 2 and 6 minutes with
even higher values in the treated group (P<0.001).
4
Conclusions
It
is interesting to note that teratozoospermia was high on day 28 (35 days
of age) in the control mice, which is in agreement with the data of Janca
et al[15],
who reported a high incidence of sperm abnormalities in immature mice
of 32 and 34 days old; by day 57, the percentage of head anomalies was 3.3%,
which lay within the range (1.2-3.4%) reported for normal adult animals by
Wyrobek and Bruce[16].
In
our observations, parathion significantly influenced the sperm morphology
on day 28 or 50 post injection. Similar
results have been seen in adult mice with
a single dose of parathion[9].
Other organophosphorous as well as chlorinated
pesticides have been observed to have a similar effect[17,18].
Increased
teratozoospermia and diminished resistance of chromatin to thermal shock
in younger animals (even in the controls) may be an evidence of faulty
spermatid differentiation in the first waves of the spermatogenic process[19,20].
There
is a high correlation between metachromasia with acridine orange and sperm
head morphology, both being good indicators for the effect of testicular
toxicants[15,21,22].
Decreased
sperm counts in treated animals denote the lability of spermatogonial
proliferation in mice exposed to parathion.
This pesticide has been found to interfere with the uptake of3H-thymidine
in mouse seminiferous tubules in vitro[23].
The
damage was recoverable only a long time after exposure to the agropesticide,
which is in agreement with the potential reparative ability of spermatogonia
and spermatocytes demonstrated in immature mice after irradiation or exposure
to alkylating agents[24].
In
conclusion, parathion affects spermatogenesis at the very initiation of
the process, interfering with spermatogenic cell differentiation and proliferation.
These effects appear to be reversible a long time after a single exposure to
the agropesticide, provided that a moderate dose has been used.
5
Acknowledgment
The
work was supported by Grant EDID99/014,
University of Chile.
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Correspondence
to: Dr. Eduardo Bustos-Obregn, Biology of Reproduction Unit, Program
of Morphology, ICBM, Faculty of Medicine, University of Chile.
P.O. Box 70061, Santiago 7, Chile.
Tel: +56-2-678 6450. Fax: +56-2-737 3158.
e-mail:
ebustos@machi.med.uchile.cl
Received
2000-02-02 Accepted 2000-04-11
