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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
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.    

The present study deals with the effect of a single injection of parathion in immature CF-1 mice, in order to analyze its toxic action at the beginning of spermatogenesis.

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

All observations were quantified and analyzed by the Chi-square test or t-test, and the significance of difference was set at P<0.05.

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).

Figure 3.   Thermal stability of chromatin: metachromatic nuclei (%) 28 days (A) and 50 days (B) after parathion. cP<0.01 and dP<0.001, Chi-square test, vs Control group.

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.


[1] Bellv A, Cavicchia J, Clarke Millette C, O'Brien D, Bhatnagar Y. Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. J Cell Biol 1977;  74: 68-85.
[2] Malkov M, Fisher Y, Don J. Developmental schedule of the postnatal rat testis determined by flow cytometry. Biol Reprod 1998; 59: 84-92.
[3] Bjorge C, Brunborg G, Wiger R, Holme J, Scholz T, Dybing E. et al. A comparative study of chemically induced DNA damage in isolated human and rat testicular cells. Reprod Toxicol 1996; 10: 509-19.
[4] Pandey N, Gundevia F, Prem A, Ray P. Studies on the genotoxicity of endosulfan, an organochlorine insecticide, in mammalian germ cells. Mutat Res 1990; 242: 1-7.
[5] Rios AC, Salvadori DM, Oliveira SV, Ribeiro LR. The action of the herbicide Paraquat on somatic and germ cells of mice. Mutat Res 1995; 328: 113-8.
[6] Yousef MI, Bertheussen K, Ibrahim HZ, Helmi S, Seehy MA. Salem MH. A sensitive sperm-motility test for the assessment of cytotoxic effect of pesticides. J Environ Sci Health 1996; 31: 99-115.
[7] Russo A,  Levi A. Detection of aneuploidy in male germ cells of mice by means of a meiotic micronucleus assay. Mutat Res 1992; 281: 187-91.
[8] Allard E,  Hall S,  Boekelheide K. Stem cell kinetics in rat testis after irreversible injury induced by 2,5-hexanedione. Biol Reprod 1995; 52: 186-92.
[9] Bustos-Obregn E, Valenzuela M, Rojas M. Agropesticides and testicular damage. In: F Martnez-Garca, J Regadera, editors. Male Reproduction. A multidisciplinary overview, Chapter 20. Madrid: Churchill Comunications; 1998.
[10] El Nahas SM, De Hondt HA, Abdov HE. Chromosome aberrations in spermatogonia and sperm abnormalities in Curacron-treated mice. Mutat Res 1989; 222: 409-14.
[11] Ray A, Chatterjee S, Ghosh S, Bhattacharya K, Pakrashi A, Ded C. Quinalphos-induced suppresion of spermatogenesis, plasma gonadotrophins, testicular testosterone production, and secretion in adults rats. Environ Res 1992; 57: 181-9.
[12] Mathew G, Vijayalaxmi K, Rahiman A. Methyl parathion-induced sperm shape abnormalities in mouse. Mutat Res 1992;  280: 169-73.
[13] Vigil P, Bustos-Obregn E. Alkylating agents and mouse spermatogenesis: effects of a single dose of cyclophosphamide.  Androloga 1985; 17: 276-82.
[14] 14.   Wyrobek A, Bruce W. Chemical induction of sperm abnormalities in mice. Proc Natl Acad Sci USA 1975; 72: 4425-9.
[15] Tejada  R, Mitchell C, Norman A, Marik J,  Friedman S. A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil Steril 1984;  42: 87-91.
[16] Janca  F, Jost L, Evenson D. Mouse testicular and sperm cell development characterized from birth to adulthood by dual parameter flow cytometry. Biol Reprod 1986; 34: 613-23.
[17] Krzanowska H. Sperm head abnormalities in relation to the age and strain of mice. J Reprod  Fert 1981; 62: 385-92.
[18] Albert M, Roussel C. Changes from puberty to adulthood in the concentration, motility, and morphology of mouse epididymal spermatozoa. Int J Androl 1983; 6: 446-60.
[19] Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 1980; 240: 1131-3.
[20] Evenson  DP, Janca FC, Baer RK, Jost LK, Karabinus DS. Effect of 1,3
-dinitrobenzene on prepubertal, pubertal, and adult mouse spermatogenesis. J Toxicol Environ Health 1989; 28: 67-80.
[21] Evenson DP, Jost LK,  Baer RK. Effects of methyl methasulfonate on mouse sperm chromatin structure and testicular cells kinetics. Environ Mol Mutag 1993; 21: 144-53.
[22] Meistrich  ML. Quantitative correlation between testicular stem cell survival, sperm production, and fertility in the mouse after treatment with different cytotoxic agents. J Androl 1982; 3: 58-68.
[23] Rodriguez H,  Bustos-Obregn E. An in vitro model to evaluate the effect of  an organophosphoric agropesticide on cell proliferation in mouse seminiferous tubules.  Androloga  2000; 32: 1-5.

24] Lyon M. Sensitivity of various germ-cell stages to environmental mutagens. M
utat  Res 1981; 87: 323-45.


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