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Revista chilena de anatomía

versión impresa ISSN 0716-9868

Rev. chil. anat. v.18 n.1 Temuco  2000

http://dx.doi.org/10.4067/S0716-98682000000100008 

ACUTE EFFECT OF PARATHION ON THE SEMINIFEROUS EPITHELIUM OF
IMMATURE MICE

EFECTO AGUDO DEL PARATHION SOBRE EL EPITELIO SEMINÍFERO DE
RATONES INMADUROS

* Cristián Sobarzo
** Eduardo Bustos-Obregón

* Faculty of Basic Sciences, Universidad Católica de Temuco, Chile.
** Biology of Reproduction Unit, Program of Morphology, ICBM, Faculty of Medicine, Universidad de Chile, Chile.
Partially financed by EDID99/014 - DID, Universidad de Chile.

SUMMARY: The present work aims at establishing the toxic effects on spermatogenesis of a single injection of the organophosphorous agropesticide Parathion, administered to immature male mice. Seven days old animals were injected intraperitoneally with 1/3 the LD50.

Body and testicular weights were measured. Quantitative as well as qualitative analysis of seminiferous tubules and germ cell populations were done for control and injected mice 8, 16, 28 and 50 days after injection.

The cytotoxic effect of the pesticide is substantiated by a decrease of body and testis weight and an early damage of germ cells in treated mice. The effect is reversible and recovers at longer intervals.

KEY WORDS: 1. Agropesticides; 2. Spermatogenic; 3. Mice.

INTRODUCTION

In the adult, the seminiferous epithelium is a renewing tissue that produces millions of spermatozoa daily. Germ cell proliferation (mitosis and meiosis) and differentiation (spermiogenesis) of spermatids generate characteristic germ cell associations in space and time (RUSELL et al., 1990).

In the mouse, spermatogenesis starts soon after birth, when the seminiferous tubules contain Sertoli cells and gonocytes. Thereafter, spermatogonial cells arise (type A, In and B). By one week of age, meiosis starts so that at 14 days pachytene spermatocytes and at 22-24 days the first spermatids appear (BELLVÉ et al., 1977) and at 28 days, spermatogenesis is qualitatively though not quantitatively completed. In the adult, the duration of the spermatogenic cycle in the mouse is 34-35 days (OAKBERG, 1958; CLERMONT & TROTT, 1969).

The numerous and complicated cell events of spermatogenesis may be damaged by testicular noxa of different nature (Björge et al., 1996).

Testicular toxicants may affect any of the gonadal cells, either somatic (Sertoli, Leydig) or germ cells, but because of the close interdependence of all these cell types, the damage will be extensive to all of them (Brinkworth & Handelsam, 1997).

Most studies concerning pesticides have analyzed spermatogenesis in adult animals in terms of cytotoxic or genotoxic effects (FLESSEL et al., 1993; RÍOS et al., 1995).

ParathionR is a widely used organophosphorous agropesticide known to be a potent testicular toxicant in rodents. However, its effect upon the installation of spermatogenesis in immature animals, which is the aim of this work, has not yet been explored.

MATERIAL AND METHOD

Immature male CF-1 mice were used. They were kept in the animal room of the Unit, at 18-20ºC, with 12:12 hs light/darkness regimen and fed commercial pellet, with water ad-libitum.

ParathionR (99.2% w/v) (0,0 diethyl O-p-nitrophenylmonophosphate (Sigma, USA) diluted in 0,85% NaCl solution, was intraperitoneally injected, using a single dose (1/3 the LD50 dose, determined to be 20 mg/kg body weight). Controls were injected with the same volume of 0,85% NaCl solution. Treated animals and their controls were sacrificed in groups of 5 at 8, 16, 28 and 50 days after injection.

Body and testis weigthts were registered. Mice were injected when they were 7 days old (age at which the spermatogenic line is represented only by spermatogonia).

The testes were dissected out, fixed in Bouin's fluid and processed for routine light microscopy and stained with P.A. Schiff-hematoxylin.

Fifty cross sections of seminiferous tubules per mouse were inspected under 400X. Tubular diameter and epithelial height were measured using digitalized images with the help of a public software (Osiris, version 2,5), created by the University Hospital of Geneve (Switzerland).

Germ cell populations were quantified in the different stages of the cycle, identified by the criteria of Oakberg and expressed as mean number of cells (± SEM) per tubular cross section.

Anova and Student's t tests were used for statistical analysis of the results of control vs treated mice, p < 0,05 being considered significant.

RESULTS

Table I shows the mean value of body and testis weights for control and treated mice for the 4 periods after a single injection of parathion. There is an increase in body and testes weights with age, but the weights of treated mice are consistently and significantly lower than their controls.

Table I: Value averages (x ± SEM) of the body weigth, testicles and epididymes of mouse males CF1 prepubers, controls and treated with a dose of Parathion, 20 mg/ kg b.w., at 8, 16, 28 50 days post-inyection.
  Body weigth (gr) Testicles weigth (mg) Epididymes weigth (mg)

Period Post-inyection Control Treated Control Treated Control Treated
8 11.93 ± 0.33 08.88 ± 0.67** 19.97 ± 0.69 13.63 ± 0.31*** 04.82 ± 0.31 3.7 ± 1.88**0
16 18.74 ± 0.76 14.45 ± 0.64** 48.86 ± 0.74 40.45 ± 1.45*** 10.57 ± 0.23 08.7 ± 0.45***
28 29.64 ± 1.1- 25.24 ± 0.76** 83.05 ± 1.86 77.17 ± 2.12*** 21.32 ± 0.96 16.31± 0.37***
50 30.5 -± 0.66 27.26 ± 1.09** 89.55 ± 2.13 81.76 ± 2.25**0 28.21 ± 0.20 25.67± 0.68**0

** p<0.01 *** p<0.001

Morphometric analysis of seminiferous tubules: Mean tubular diameter (all stages of the cycle) (Fig. 1) increases from day 8 post injection (pi) to day 50 both in the control (107,8 ± 0,96 up to 202,1 ± 1,02 µm, respectively) and in the treated mice (100,14 ± 0,66 and 180,0 ± 0,51 µm).



Fig. 1. Mean tubular diameter (µm) (x ± SD) all stages of the cycle in testicles, control and treated prepuberal mouse CF1 with a dosis intraperitoneal of Parathion: 20 mg/ kg b.w. , 8 , 16, 28 and 50 days post-injection.

Treated animals show values significantly lower (p < 0,001) at all intervals examined.

In Fig. 2, mean tubular diameters by stages of the cycle are shown at 8 and 16 days pi. There is a significant increase between both intervals for both groups by about 28%. However, values are lower for treated animals.


Fig. 2. Mean tubular diameters (µm) by stages of the cycle spermatogenic in testicles, controls and treated prepuberal mouse CF1 with a dosis intraperitoneal of Parathion: 20 mg/kg b.w., 8 and 16 days post-injection.

Tubular diameters increase also between 28 and 50 days pi by 13% for controls and by 8% for the treated mice (Fig. 3). Differences between stages are more relevant by 50 days pi, probably due to the fact of a more accurate identification of the stage at this interval when all generations of advanced spermatids are present near the tubular lumen.


Fig. 3. Mean tubular diameters (µm) by stages of the cycle spermatogenic in testicles, controls and treated prepuberal mouse CF1 with a dosis intraperitoneal of Parathion: 20 mg/kg b.w.,28 y 50 days post-injection.

Height of the seminiferous epithelium. These values are shown in Fig. 4. In both groups the epithelial height increases with the age of the animals. In the control; there is a 50% increase between 8 and 50 days pi. In the treated animals, the increment is only 36%. The difference is highly significant (p < 0,001).


Fig. 4. Mean (x ± SD) of the heigth of the seminiferous epithelium, controls and treated prepuberal mouse CF1 with a dosis intraperitoneal of Parathion: 20 mg/kg b.w. at 8, 16, 28 and 50 days post-injection.

Quantitative analysis of germ cell populations. In the Table II it can be seen that the germ cell populations underwent a significant decrease in number in the treated mice at 8 and 16 days p.i., affecting mostly spermatogonia and primary spermatocytes. By 28 days, this difference was relevant for zygotene spermatocytes and for spermatid round at 50 days pi.

Table II. Semicuantitative evaluation of the different spermatogenic populations of the germinal epithelium in CF1 mice, treated with a dose of Parathion: 20 mg/ kg b. w. at 8, 16, 28 and 50 days post-inyection.
Interval   Cell type  

8 days Sptg. B ê PL ê P (m-f) ê0000
16 days Sptg. B ê PL ê L ê Z ê P ê
  Sp. R ê    
28 days Z ê C2° ê Sp. R ê0000
50 days Sp. R ê    

Spg. B: spermatogonium type B
PL: preleptotene spermatocite.
  L: leptotene spermatocite.
Z: zigotene spermatocite.
P: paquitene spermatocite.
C2°: secundary Sp spermatocite.
R: round spermatid.
   

Qualitative analysis of tubular morphology. Testicular histological pictures of control and treated mice at the 4 intervals observed are shown in Figs. 5 y 6 (a, b, c, and d).






Fig. 5. Microphotographs of mouse testicular sections (control and treated animals). The bar corresponds to 50 µm. Seminiferous tubules of mice, 8 days after injection. a) control; b) parathion treated. Germ cell populations are qualitatively similar in both cases. The most advanced spermatogenic cells correspond to pachytene spermatocytes (® ). At 16 days p.i. there are no marked differences. However, treated mice (d) have tubules of a smaller diameter and very scarce lumen (*) as compared to controls (c). The most advanced germ cells by this period are round spermatids (®).





Fig. 6. Microphotographs of mouse testicular sections (control and treated animals). The bar corresponds to 50 µm. Seminiferous tubules of mice 28 days after injection. a) control; b) parathion treated. In both groups the most advanced germ cell class are the elongated spermatids (Spt). By 50 days p.i. tubular morphology is similar in control (c) and treated (d) mice and corresponds to the normal seminiferous epithelium of adult animals. Germ cell population is qualitatively completed, from spermatogonia (Spg) up to the so-called testicular spermatozoa (Sptz) near the tubular lumen.

By 8 days pi, the seminiferous tubules already have a clear lumen and a compact epithelium with no signs of depletion, vacuolization or cell sloughing. Pachytene spermatocytes is the most advanced germ cell generation, near the lumen. At the following intervals, tubular diameter and epithelial height increase and there is a small luminal reduction. By 16 days pi (23 days of age) the most advanced germ cells are round spermatids. By 28 days (35 days pi 57 days of age), intratesticular spermatozoa are seen in the tubular lumen. Differences in cell populations between control and treated mice are not easily appreciated, though they can be certified by cell counting, as previously described.

DISCUSSION

This paper analyzes the acute effect of a single dose of the organophosphorous agropesticide Parathion upon the onset and installation of spermatogenesis in immature CF 1 mice.

Testicular weight decreases in treated animals at the 4 intervals examined (at the same rate than body weight). There is a tendency to recovery by 50 days. In adult mice, no such a difference has been reported (Mathew et al., 1992; Jagetia et al., 1996). In immature rats, evaluation of the same reproductive parameters after administration of p.p.-DDE did not result in differences between control and treated rats (ROMERO, 1992). EVENSON et al. (1989) using 1, 3-dinitro benzene (m-DNB) in prepuberal, puberal and adult mice, found differences only in the adult animals. According to our results, Parathion is a more potent cytotoxic compound for very young (7 days old) mice, resulting in body and testis decrease in weight.

The morphometric analysis is consistent with work in the rat by Wing & Christensen (1982). Tubular diameter is stage dependent. During the first intervals (8 and 16 days pi), differences are minimal between control and treated mice since not all cell associations are completed as yet. Differences are more evident at 28 and 50 days pi. From these observations, it can be concluded that a single injection of Parathion alters the normal growth and cell association development of the stages of the cycle at early periods of spermatogenesis and this effect can be still recognized at longer intervals (ie. 50 days pi).

The qualitative histological analysis of the testis indicated that the spermatogenic development of immature CF1 mice was similar to the reports of Bellvé et al. and Malkow et al. (1998), so that the first spermations take place at 35 days of age.

The establishment of the cell associations was not different in control and treated mice. No obvious changes in testicular histology, such as tubular depletion, sloughing or blockade could be detected.

Other pesticides, however, such as quinalphos (RAY et al., 1992) or aldrin (Chatterjee et al., 1998) provoke massive germ cell degeneration. In adult mice exposed to parathion, we have observed tubular depletion and blockade 8 days pi (Bustos-Obregón et al., 1998).

In the present experiments at 8 days pi, treated mice exhibit increased height of the seminiferous epithelium with no enlargement of the tubular diameter and a consequent narrowing of the tubular lumen. The latter change has been described in cases of decreased tubular fluid secretion by Sertoli cells (Russell et al.), which may be damaged by Parathion.

The more sensitive germ cells affected by Parathion are progressively in time type In and B spermatogonia, and early primary spermatocytes at 8 and 16 days pi. At later intervals (28 days pi) zygotene spermatocytes are compromised. This is the same pattern observed in rats treated with another organophosphorous pesticide, curacron (El Nahas et al., 1989) or quinalphos (RAY et al.). Cytotoxic effects on germ cells have also been demonstrated for malathion (Contreras & Bustos-Obregón, 1999).

Testicular toxicity seems to be present early after injection (8 days), a fact observed also with other cytotoxic agents (Matsui et al., 1995; SPANO et al., 1996). The effect of pure parathion is reversible and of a short period of action, since 28 days pi a recovery of spermatogonial and early spermatocytes counts can be observed.

In summary, our results point out to a cytotoxic, reversible effect of a single, moderate, dose of parathion that interferes with spermatogenic cell proliferation and differentiation in the immature mice testis.

RESUMEN: En el presente se estudió el efecto tóxico para la espermatogenesis, de una inyección única del agropesticida organofosforado parathion, administrada a ratones inmaduros. Animales de 7 días de edad fueron inyectados por vía intraperitoneal con 1/3 de la LD50.

Fue medido el peso corporal y testicular. Se efectuó un análisis cuali y cuantitativo de los túbulos seminíferos y de las poblaciones de células germinales en ratones controles y tratados, a los 8, 16, 28 y 50 días post-inyección.

El efecto tóxico del pesticida se demuestra por una disminución del peso corporal y testicular y un daño temprano de las células germinales de los animales tratados. El efecto es reversible y la recuperación ocurre en los intervalos largos post-inyección.

PALABRAS CLAVE: 1. Agropesticidas; 2. Espermatogénesis; 3. Ratón.

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Correspondence to :
Prof. Cristián Sobarzo A.
Depto. Ciencias Biológicas y Físicas
Facultad de Ciencias
Universidad Católica de Temuco
Casilla 15-D
Temuco - CHILE
Phone : 56-45-205 657; Fax : 56-45-211 034
e-mail : crsob@uctem.cl

Recibido : 27-3-2000
Aceptado: 29-04-200

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