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Journal of Venomous Animals and Toxins

Print version ISSN 0104-7930On-line version ISSN 1678-4936

J. Venom. Anim. Toxins vol.6 n.2 Botucatu  2000 

Histopathological changes in albino mouse testis induced by the administration of Tityus n. sp. venom



1 Universidad de Oriente, Escuela de Medicina, Departamento de Ciencias Fisiológicas; 2 Departamento de Microbiología y Parasitología; 3 Departamento de Ciencias Morfológicas; 4 Laboratorio de Alacranología, Ciudad Bolívar, Venezuela.



ABSTRACT. Scorpion envenoming is a public health concern in northeastern Venezuela. Specimens of the genus Tityus are responsible for most of these. In experimental animals, Tityus venom produces histopathological changes in the skeletal muscle and pancreas, but its toxicity to the reproductive system has not been studied. The aim of this work is to describe the histopathological changes in testis and epididymis of albino mice induced by the administration of Tityus n. sp. venom. Sub-lethal doses of venom (3.75 mg/g mouse) were administered intramuscularly (IM) daily for 4 days. On the fifth day, the animals were sacrificed and the testes and epididymes were quickly removed and processed for light microscopy. The venom induced alterations in spermatogenesis. Sertoli cell vacuolation, immature germ cell shedding, spermatocyte arrest, and low sperm volume were observed in seminiferous tubules. Leydig cells were hardly affected. Vascular dilation and congestion were detected in the interstitial tissue. Immature germ cells were found in epididymal tubule lumina, but no abnormalities were observed in epididymal epithelial cells. These results show that Tityus n. sp. venom causes changes in mouse seminiferous epithelium, probably due to indirect action through the Sertoli cell.
 KEY WORDS: Testis, Sertoli cell, epididymis, scorpion, venom, mouse, histopathology.




Scorpion envenoming is a public health hazard in Venezuela, especially in the northeast where it is hyperendemic (1). Species belonging to the genus Tityus n. sp. are said to be responsible for envenomings in this area (1,2,11).

Tityus venom induces damage to the skeletal muscle and pancreas of experimental animals(3), but this has not been studied in the reproductive system.

Worldwide, the only other study about the effect of scorpion venom on male gonad has been with Leiurus quinquestriatus of the Buthidae family. Hafeiz et al. (6) has reported that a single dose of fraction VII from L. quinquestriatus venom given to rats is capable of inducing degenerative changes in seminiferous tubules, resulting in necrozoospermia

Due to the lack of information on the effect of scorpion venoms on the male gonad, the aim of this research was to study the effect of Tityus n. sp. venom on the testis and epididymis morphology in mice.



SCORPION VENOM. Tityus n. sp. venom was obtained by electrical stimulation of the terminal telsum of female specimens in the Laboratory of Alacranology of the School of Medicine - Universidad de Oriente (Ciudad Bolívar - Venezuela). The venom was freeze-dried for 8 hours, weighed, and stored at 5ºC until use, when it was resuspended in sterile bidestilled water.

ANIMALS. Twenty NMRI-IVIC male albino mice weighing 20-30g, purchased from Instituto Venezolano de Investigaciones Cientificas, were randomly divided into 2 equal groups and housed in stainless steel cages under standard animal house conditions. They were fed a pellet and water diet ad libitum.

VENOM ADMINISTRATION. Mice were parenterally treated with Tityus n. sp venom as described by Nawar et al. (8) with modifications. The experimental group was injected IM daily with sublethal doses (3.75 mg/g body weight) for 4 days and the control group was injected only with sterile bidestilled water in equivalent volumes, according to body weight.

TISSUE PROCESSING. All the animals were sacrificed on the fifth day by cervical dislocation and their testes and epididymes quickly removed. These organs were immersed in Bouin's fixative for 1 hour at 4ºC, then cut in 2 mm thick slices and again immersed in the same fixative for 24 hours. Tissues were then dehydrated with graded alcohols and embedded in Paraplastâ. Paired sections of each block were cut into 3-5 mm, stained with hematoxylin-eosin and iron hematoxylin, and examined under a Zeiss light microscope.

Qualitative analysis based on mouse spermatogenic stage was performed by using the criteria developed by Oakberg (10). The window of exposure or window of effect was determined by using the knowledge of spermatogenesis kinectics. The most advanced phase of cell development in the window was considered as the first cell type affected by the venom and the least advanced the last cell type affected.



TESTIS. All venom-treated mice showed, to a varied extent, some evidence of disruption of spermatogenesis, but no abnormalities were detected in control group testes (Figure 1).



The germinal epithelium of affected tubules appeared disorganized, disrupted and showed shedding of germ cells into the lumina (Figure 2). A reduced volume of mature spermatozoa was noticeable. The tubules in stages IX, X, XI, and XII were more frequently affected.



The least advanced cell type affected by venom action was spermatocyte. The tubules in stages IX to XII showed necrotic spermatocytes with either karyolysis or karyorrexis, loss of cell cohesion and germ cell shedding at the adluminal compartment (Figure 3). Asynchronization of cell association was observed in this lineage. Spermatocytes lining the seminiferous epithelium appeared to be arrested in their development, and there was absence of spermatids and spermatozoa (Figure 4).





Spermatids were the most advanced phase of cell development affected by the venom, but they were not as frequently degenerated as spermatocytes. In some tubules eosinophilic giant multinucleated cells were present in the lumina (Figure 5). Other tubules showed a loss of spermatid formation with spermatocyte degeneration or arrest.



Spermatogonia did not show histopathological changes by light microscopy.

Mainly the Sertoli cells showed large basal vacuoles in their cytoplasm (Figure 6).



Leydig cells were not as affected as germinal cells, but in some cases they showed nuclear distortion, such as karyolysis and karyorrexis.

Blood vessels of the interstitial tissue appeared occasionally dilated and congested in venom-treated animal testes (Figure 7).



EPIDIDYMIS. The epididymal epithelia of the experimental group appeared similar to those of the control group. However, the lumen of venom-treated animals showed lower sperm density and substantial quantities of exfoliated germ cells and cytoplasmic debris (Figure 8).




Sub-lethal doses of Tityus n. sp. venom were capable of inducing damage to mouse seminiferous epithelium, altering spermatogenesis.

The presence of vacuoles in the cytoplasm of the Sertoli cell shows direct damage to this cell. Russell and Griswold (13) have reported that these lesions are the early morphological sign of testicular injury and are considered as the main Sertoli cell response to many xenobiotics. Nevertheless, in this study, these lesions were not the only change indicating Sertoli cell damage. Spermatogenic arrest together with other features, such as the presence of giant cells corresponding to spermatids with incomplete cytokinesis during meiosis, epithelial disorganization, and the appearance of immature germinal cells in the epididymis indicate a primary alteration in the Sertoli cells (9,13).

Loss of contact with germinal cells and their presence in the epididymis, and luminal shedding observed in Tityus n. sp. venom-treated mice are possible consequences of the alteration of specialized intercellular junctions between Sertoli and germinal cells. Ultrastructural studies on testicular xenobiotics have reported that ectoplasmic unions and tubule-bulbar complexes between the Sertoli and germ cells were seriously damaged, causing spermatogenic cell sloughing (13).

In our study, significant damage was observed in spermatocytes and spermatids that are located in the adluminal compartment above the inter-Sertoli cell stretch unions, suggesting that Tityus n. sp. venom alters these structures, since this type of lesion is also observed with xenobiotics, producing a selective loss of germinal cells located in the adluminal compartment (13).

The presence of seriously affected tubules in the last stages of spermatogenesis (IX-XII) surrounded by other seemingly normal tubules indicates that mouse seminiferous epithelium is susceptible to the action of Tityus n. sp. venom. In these stages, the most frequently found cells are spermatogonia A, primary spermatocytes, and spermatids. In this case, specific lesions in the spermiogenesis stages coincided with nuclear shape remodeling and acrosome arrangement (10). El-Asmar et al. (5) reported that primary spermatocytes and spermatids are the most affected spermatogenic cells in rats treated with a spermicidal fraction of Leiurus quinquestriatus. They report degenerated spermatids at cap phase with damage to the acrosome vesicle.

Spermatogenic arrest at spermatocyte level and immature spermatid shedding probably contributed to the low spermatic density observed in both seminiferous and epididymal tubules in Tityus n. sp. venom-treated mice.

Spermatogonia were not affected by the venom at light microscopy level. Some authors have reported that spermatogonia seem to be the most resistant spermatogenic population to the secondary damage in acute exposure to a xenobiotic for the Sertoli cell. However, a sustained exposure can cause cellular depletion over a long time (9,13).

Leydig cells were not altered by Tityus n. sp. venom. This feature is in agreement with that of El-Asmar et al. (5), who reported minimal ultrastructural damage to Leydig cells in rats treated with spermicidal fraction of Leiurus quinquestriatus, indicating cell resistance to scorpion venom action.

Vascular dilation and congestion found in the interstitial tissue have been described in organs of experimental venom-treated animals. These changes could be a consequence of the action of vasodilator substances like serotonin and histamine present in most scorpion venoms (4,7,8,12,14,15).

To our knowledge, this is the first time that the effect of scorpion venom on experimental animal epididymes has been studied. Although epididymal epithelium of Tityus n. sp. venom-treated mice did not show histopathological changes at light microscopy, the luminal content reflected many of the abnormalities occurring in the testis.

Histopathological features found in this study suggest that Tityus n. sp. venom induced indirect damage to mouse germinal cells and spermatogenesis through its action on the Sertoli cells. However, further studies in this field are required to identify the precise underlying mechanisms.



We are grateful to Mr. Luis Gallardo (Instituto Venezolano de Investigaciones Científicas) for his technical assistance.



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Received 19 April 1998
Accepted 06 July 1999

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