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International Journal of Morphology

versión On-line ISSN 0717-9502

Int. J. Morphol. v.21 n.2 Temuco  2003 

Int. J. Morphol., 21(2):155-159, 2003.



Eduardo Bustos-Obregón & Patricio González-Hormazábal 

SUMMARY: Organophosphorous pesticides are a healh problem worldwide, mostly for agroworkers, which are around 2600 millions of person in the year 2001. Little is known about male reproductive damage elicited by these chemicals.

The acute effects of malathion (single intraperitoneal injection), 1/12 the LD50 to mice were analyzed at 1, 8, 16, 35 and 40 days after injection, in terms of spermatogenic cell proliferation and apoptosis and of Sertoli cell compromise as revealed by immunocytochemical detection of CK-18 (cytokeratin). The micronuclei test was done to assess for genotoxic activity of the agropesticide.

The results showed decreased germ cell number followed by compensatory spermatogonial proliferation by 16 days (as shown by thymidine-H3 testicular uptake), increased apoptotic rate, mainly of spermatogonia, and preleptotene spermatocytes. Sertoli cell reactivity to CK-18, denoting alteration of them, probably due to germ cell damage, was seen in treated mice. Genotoxicity for somatic cells was demonstrated by the micronuclei test.

Therefore, it is assumed that Sertoli as well as Leydig cells are affected together with spermatogenesis; recuperation of testicular morphology is seen by longer time intervals.

KEY WORDS : 1. Spermatogenesis; 2. Organophosphorous agropesticides; 3. Proliferation; 4. Apoptosis. 


Organophosphorous agropesticides are used worldwide, so that the WHO estimates that 5% of the World population of agroworkers are directly exposed to pesticides.In the year 2001 this population was calculated in 2.600 millions of persons.

Intoxication by phosphorated pesticides is due to the inhibition of the enzyme acetylcholinesterase in exposed organisms (Taylor, 1996).

The reproductive effects of these chemicals are scarcely known and more emphasis has been put in the role of chemical pollution as endocrine disrupter (Bustos-Obregón, 2001). Kristensen et al. (1997) reported malformations in newborns in Norway due to the exposure of their parents to pesticides. The teratogenic effects of malathion (a widely used organophosphorous pesticide) are debatable (Karlow & Marton, 1961; Khera et al., 1978).

Therefore, analysis of the male reproductive function after exposure to malathion was found of interest and is addressed mainly in regard to cell proliferation and differentiation in mouse testis.


Animals: 30 male CF1 mouse (10-12 weeks old) were intraperitoneally (ip) injected with 1/12 of the LD50 (240 mg/Kg body weight) of commercial Malathion (95,2% w/v); Anasac, Santiago, Chile [CAS 16.34 - 78-2 S-1,2-bis etoxycarbonil ethyl 0,0- dimethyl phosphoroditioate]. Groups of 6 animals were sacrificed 1, 8, 16, 35 and 40 days post injection (pi). One control group was ip. injected with the vehicle (corn oil) and sacrificed 40 days pi.

Testicular histopathology: The right testis was fixed in alcoholic Bouin for 8 hours and embedded in paraffin.

Testicular sections of control and experimental mice were treated by the method of TUNEL, to evaluate apoptosis, according to Sasso-Cerri & Miraglia (2002) and with immunohistochemical reaction for cytokeratin CK-18, as stated by Steger et al. (1999).

Micronuclei test: used to evaluate genotoxic effect of a chemical using bone marrow red cells, was performed after exposing a group of 6 mice to malathion for 24 hs, following the techniques published by Heddle (1973).

Thymidine-H3 testicular uptake: one hour before sacrifice, mice were intraperitoneally injected with 2,5 µC/g, body weight of thymidine-H3 (Sigma, St. Louis, MO), specific activity 5,0 Ci/mM. Testis were extracted and processed as detailed in Bustos-Obregón & Ramírez (1997) to estimate thymidine-H3 uptake into testicular DNA as a marker of cell proliferation as revealed by scintillation counting (Thumann & Bustos, 1978).

Statistical analysis are expressed as means ± standard error of the mean. Differences for treated vs controls in the micronuclei test was analyzed by Mann-Whitney and thymidine uptake by the Student's "t" test.


At 1 and 8 days pi there is a significant decrease in plasma acetylcholinesterase activity with a recuperation after 16 days (not shown). This activity was used to ascertain the degree of intoxication of the animals and is reported elsewhere.

Concerning testicular histology, we have reported previously a decrease in seminiferous epithelial height and tubular diameter from 8 days pi onwards. These values tend to normalize by 35 and 40 days pi (González-Hormazabal &Bustos-Obregón, 2003).

Thymidine-H3 uptake into testicular DNA tends to increase by 16 days pi (Fig. 1).

Fig. 1. Thymidine-H3 uptake into the testicular DNA expressed as cpm/µg DNA (mean ± SEM), at different days after treatment.

The method of TUNEL denotes no apoptotic germ cells in control animals. Apoptotic nuclei are seen in treated mice 1 day pi (A XII spermatogonia) (Fig. 2, arrows); B spermatogonia 8 days pi (stage VI) (Fig. 3) and type A at stage I, (Fig. 4, arrow). Apoptosis of preleptotene spermatocytes (arrows) and step (9) spermatids in stage IX are seen in Fig. 5.

CK-18 cytokeratin was positive in peritubular and Leydig cells of control mice (Fig. 6). In treated mice, a strong reaction was seen in the Sertoli cell expansions associated to spermatids in different steps of the acrosomic and maturation phases, (Fig. 7) by 1 day. A similar picture is seen by 8 days (Fig. 8), but Sertoli cell expansions become CK-18 (-) by that interval at stage IX at the moment of spermiation (Fig. 9). By 40 days, most CK-18 reactivity in Sertoli cells and late spermatids (spt) is lost (Fig. 10).

Fig. 2. Apoptotic type A spermatogonial nuclei (arrows) at stage XII of the cycle (TUNEL method). Bar: 5 µm.
Fig. 3. Apoptotic nuclei of typr B of spermatogonia eight days after pi. (TUNEL method). Bar: 5 µm.
Fig. 4. Apoptotic nuclei of type A spermatogonia at stage I (arrow). (TUNEL method). Bar: 5 µm.
Fig. 5. Apoptotic P.L. spermatocyte nuclei (arrows) and step 9 spermatids (9). (TUNEL method). Bar: 20 µm.
Fig. 6. CK-18 control (non treated animal) showing positive reaction on peritubular and Leydig cells. Bar: 50 µm.
Fig. 7. Positive CK-18 Sertoli cell expanssions associated to elongated spermatids by 1 day pi. Bar: 20 µm.
Fig. 8. Positive CK-18 Sertoli cell expanssions associated to elongated spermatids by 8 days pi. Bar: 20 µm.
Fig. 9. CK-18 negative Sertoli cells (S) by 8 days pi. at stage IX, during spermiation, denoted by the presence of residual bodies (CR). Bar 20 µm.
Fig. 10. CK-18 absence of reactivity in Sertoli cells (s) and late spermatids (spt) by 40 days pi. Bar: 20 µm.

The micronuclei test showed a significative difference in the frequency of micronuclei in polychromatic erythrocytes in treated vs control mice (10,0 ± 2,5 vs 3,4 ± 1,2), respectively (p < 0.01).


Plasma acetylcholinesterase levels is a useful clinical method to evaluate exposure to organophosphoric pesticide (Nigg & Knaak, 2000). A decrease to 59% 1 day after malathion injection implies that there was an acute effect in mice under our experimental conditions (Bustos-Obregón & González-Hormazábal, 2003). Therefore, the dose used (1/12 LD50) is useful to evaluate organic damage. The testis has been reported to be damaged by commercial malathion according to Wenda-Rózewicka (1983); the response is similar to that observed after treatment with busulphan (van Keulen & De Rooij, 1973) or X-rays (Dym & Clermont, 1970). These noxa affect the renewing type A spermatogonia (A1 to A4), which normally have a high mitotic rate (Clermont & Bustos-Obregón, 1968). Krause et al. (1976) in immature rats reported that malathion elicits a decrease in the number of renewing spermatogonia.

This response has been analyzed by van Keulen & De Rooij, who reported that after a single ip administration of busulphan (a DNA alkylating agent) to rats, renewing spermatogonia diminish in number 8 to 14 days pi but start proliferating to reach a clear peak precisely 16 days pi, matching very closely our observations on thymidine-H3 uptake.

Therefore, the increased thymidine-H3 uptake 16 days pi may correspond to compensatory reserve A spermatogonia proliferating two cycles after the chemical insult.

In vitro, we have found that parathion and its metabolite, paraoxon, decreases thymidine-H3 uptake into mice testicular DNA in cultured isolated mice seminiferous tubules (Rodriguez & Bustos-Obregón, 2000).

Malathion could interact also with DNA of somatic cells as shown by the micronuclei test. In fact, Imamura & Talcott (1985) have demonstrated in vitro the alkylating properties of malathion.

Thumann & Bustos-Obregón (1978) postulated that this response is mediated by the spermatogonial chalones, an inhibitor of spermatogonial mitosis produced by spermatogenic differentiated cells.

These results are in keeping with the observation that the majority of apoptotic cells revealed by the TUNEL technique corresponds to spermatogonia (and also preleptotene spermatocytes). Similar observations have been reported for parathion (Bustos-Obregón et al., 2001), suggesting that indeed the phosphoric pesticides interact with the DNA of germ cells.

Testicular toxicant effects of these pesticides goes beyond genotoxic damage to compromise testosterone production by altering Leydig cell steroidogenesis (Ellis et al., 1998). Low testosterone levels depress peritubular cell function, adding a decreased activity of Sertoli cells, due to low P-Mod-S paracrine estimulation and hipoandrogenism. Sertoli cell damage was evident according to our observations on CK-18 reactivity observed at early periods after malathion treatment. Similar observations of CK-18 reactivity in Sertoli cells have been reported in cases of damage of the seminiferous epithelium in different pathologies of human testis (Steger et al., 1996, 1999). This situation cooperates for germ cell damage at later intervals, in view of the important role played by Sertoli cells in supporting the spermatogenic process.

In conclusion, a single dose of malathion elicits a toxic effect on germinal and somatic cells of the testis. There is a depletion of the population of renewing spermatogonia, followed by spermatogonial proliferation. The basal apoptotic rate of the spermatogenic cells is increased by organophosphoric pesticides.

The damage of Sertoli and Leydig cells add an additional compromise of spermatogenesis, in view of the close interrelationships of somatic and germ cells in the testis. 

RESUMEN: Los pesticidas organofosforados son un problema de salud mundial, especialmente para los trabajadores agrícolas, calculados en alrededor de 2.600 millones de personas en el año 2001. Se conoce muy poco sobre el daño reproductivo en el varón provocado por estos agentes.

El efecto agudo de Malathion en inyección intraperitoneal única, de 1/12 la LD50 en ratón fue analizado 1,8,16,35 y 40 días después de la inyección, evaluando proliferación y apoptosis de las células espermatogénicas y daño de las células de Sertoli, estudiados por la detección inmunocitoquímica de citoqueratina (usando CK-18).

Los resultados muestran disminución del número de células germinales, seguida de una proliferación compensatoria de espermatogonias al día 16 (como lo muestra la incorporación de timidina H3 al testículo), aumento de la tasa de apoptosis (especialmente de espermatogonias y espermatocitos en preleptoteno), la reactividad a CK-18 de las células de Sertoli que denota alteración de ellas, probablemente asociada a daño de la línea germinal que se observa en los animales tratados a intervalos cortos. El malathion es genotóxico para las células somáticas como lo demuestra el test de micronúcleo.

En consecuencia, se postula que junto al proceso de espermatogénesis, son afectadas las células de Sertoli y de Leydig. La recuperación morfológica del testículo se observa sólo a intervalos largos.

PALABRAS CLAVE: 1. Espermatogénesis; 2. Agropesticidas organofosforados; 3. Proliferación; 4. Apoptosis. 


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Correspondence to:
Prof. Dr. Eduardo Bustos-Obregón
Program of Morphology, ICBM,
University of Chile Medical School
P .O. Box 70061 - Santiago 7 - CHILE

Fax and Phone : 56-2-2225710

Email :

Received : 07-04-2003

Accepted : 10-05-2003 

Biology of Reproduction Unit, Program of Morphology, ICBM, Faculty of Medicine, University of Chile, Santiago, Chile.
This work was partially financed by Banco Santander - Central Hispano, Madrid, España

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