Cr(V) involvement in the toxicity pathway of testicular damage

M. L. Pereira¹, T. M. Santos², R. Pires das Neves¹, F. G. Costa³, J. Pedrosa de Jesus²

Asian J Androl  2002 Jun; 4:  153-155             

Keywords: Cr(V); blood-testis barrier; integrity; spermatogenesis; horseradish peroxidase

Abstract

Aim: The functional integrity of the blood-testis barrier (BTB) in male mice exposed to Cr(V) was studied in order to clarify the mechanism underlying testicular injury. Methods: Adult male mice were subcutaneously injected repeated doses of 8.02 mmol (0.5 mL) of Cr/mouse^.day for 5 days. Animals receiving a similar volume of bis(hydroxyethyl)-aminotris(hydroxymethyl)methane buffer (BT) were used as controls. The animals were sacrificed on day 6 and small fragments of seminiferous tubules, approximately 8-10 mm length, were incised and sutured at both ends. They were exposed in vitro to horseradish peroxidase-containing culture medium for 10 minutes. Tissues were then fixed and processed for ultrastructural studies. Results: Controls and Cr(V)-treated group resulted in the uptake of the tracer by Sertoli cells. However, the major finding consisted in the permeability of the BTB only in the Cr(V)-group, as evidenced by the presence of the tracer within the junctions between the neighbouring Sertoli cells. Conclusion: The BTB is disrupted in mice submitted to Cr(V). The permeability of the BTB is a crucial feature to be investigated for the understanding of lesions within the seminiferous tubule.

1 Introduction

Chromium(VI) is a well-recognized environmental pollutant that may bring about adverse effects on both humans and animals [1-2]. Additionally, there are potential hazards for industrial workers [3-4]. Cr(V), generated as a metabolic intermediate from the intracellular reduction of Cr(VI), is one of the major candidates for the carcinogenic forms of the chromium compounds [5-7]. Recently, it was indicated that Cr(VI) also produce undesirable effects on male reproductive physiology [8-11]. We have just demonstrated that a Cr(V) complex, [CrV-BT]^2-, was able to induce testicular lesions in the mouse [12]. To our knowledge, no other references are so far available in the literature concerning the effects of Cr(V) on male reproduction.

The present work describes for the first time the effects of the new Cr(V) compound on the functional integrity of the blood-testis barrier (BTB), which plays a significant role on spermatogenesis.

2 Materials and methods

2.1 Animals

Experiments were conducted in 60 days old male Charles River mice (30 g) kept under laboratory conditions for small rodents (12h light/12h dark cycle; 60 % humidity; 222). Food and water were available ad libitum. Twenty animals were randomly divided in two groups, the Cr(V)-treated and the controls of 10 animals each.

[Cr^V-BT]^2- [BT is bis(hydroxyethyl)-aminotris(hydroxymethyl)methane buffer] was obtained in situ in aqueous solution, by full ligand substitution (30 min) from the precursor complex Na[Cr^VO(ehba)²], sodium bis(2-ethyl-2-hydroxybutanoato)oxochromate(V) [6,13].

2.3 Treatment of animals

Mice of the Treated group were subcutaneously administered 8.02 mmol (0.5 mL) of Cr/mouse^.day for 5 days [14]. The Control group was injected with a similar volume of a physiological solution (BT) for the same period of time. Mice were sacrificed on day 6 under ether anaesthesia and the testis was removed.

2.4 In vitro incubation with horseradish peroxidase

The seminiferous tubules were isolated from the control and the treated mice and around sixty small fragments of the tubules (each about 8-10 cm in length) were incised and sutured at both ends with a cotton thread. Tubules were then incubated in vitro for 10 min at 37 with Eagle's medium (Biomrieux) containing 5 mg/mL of horseradish peroxidase (HRP Type II; Sigma) supplemented with 10 % fetal calf serum (GIBCO) as described [15].

2.5 Ultrastructural studies

The tubular fragments were then fixed with 2.5 % glutaraldehyde in 0.1 M caccodilate buffer and placed for 45 min in 10 mL of Tris buffer solution with 5 mg of diaminobenzidine and 0.1 mL of 1 % H₂O₂. Tissues were postfixed in 2 % buffered osmium tetroxide, dehydrated and embedded in Epon 812. Thin sections prepared with a diamond knife were double stained with uranyl acetate and lead citrate. At least 2 or 3 sections were made in each tubule. Observations were conducted using a Hitachi transmission electron microscope.

The seminiferous epithelium fragments of the Cr(V)-injected mice, in particular the Sertoli cells, were able to uptake the HRP added to the culture medium. The uptake of this macromolecule was accomplished by small vesicles, which moved into the intercellular space where the releasing of the tracer took place. HRP also permeated the intercellular junctions between the neighbouring Sertoli cells, reaching the adlumenal compartment of the seminiferous tubules (Figure 1). The intercellular spaces around the Sertoli cells were widened, and the tight contacts with the neighbouring cells were disrupted in all the Cr(V)-treated animals. In the controls, the Sertoli cells were also able to uptake the tracer, but the BTB was not damaged (inset).

Figure 1. Ultrastructural aspect of the intercellular junctions between Sertoli cells in Cr(V)-injected mouse filled with the tracer (arrows); S -Sertoli cell; V -Vacuoles; inset -The impenetrable inter-Sertoli cell tight junctions of a control mouse (arrows). Bar = 1 mm.

This work was conducted to shed light on the possible mechanisms of Cr(V)-induced testis injury and demonstrated for the first time that the BTB is the potential target site. Consequently, exfoliation of immature germ cells within the lumen of the seminiferous tubules, as described in our previous paper [12], may result from the failure of the BTB integrity. It is well known that Sertoli cells provide, select and/or transport from the systemic circulation the required molecules for germ cell development. Thus, substances from the interstitial tissue must pass through or between Sertoli cells in order to reach the adlumenal compartment of the seminiferous tubules. However, the presence of tight junctions between these somatic cells, prevent most substances from reaching the adluminal compartment, unless they pass through the Sertoli cell cytoplasm [16,17]. These findings support the conclusion that Cr(V) is implicated in the bio-toxic pathway of testis injury, by disrupting the BTB.

Other investigations have already demonstrated damages on male reproductive physiology induced by Cr(VI) compounds [8-11]. Comparative studies between Cr(VI) and Cr(V) effects on mice testis are in progress in our lab in order to obtain a better understanding of the mecha-nisms underlying chromium in vivo toxic effects.

Acknowledgements

This study was supported in part by grants from the Research Centre on Inorganic Chemistry and Materials, from Aveiro University (Portugal).

References

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Correspondence to: Dr. M. L. Pereira, Department of Biology, University of Aveiro, 3810-193, Aveiro, Portugal.
Fax: +351-234-426 408
E-mail: lpereira@bio.ua.pt
Received 2002-03-05      Accepted 2002-05-10