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International Journal of Morphology

versión On-line ISSN 0717-9502

Int. J. Morphol. v.25 n.2 Temuco jun. 2007

http://dx.doi.org/10.4067/S0717-95022007000200017 

 

Int J. MorphoL, 25(2):341-346,2007.

 

Histological Study on the Male Reproductive Organs of Mouse CF1 Treated With Boron

Estudio Histológico de los Órganos Reproductivos del Ratón Macho CF1 Tratado con Boro

 

**Espinoza-Navarro, O.; *Vilaxa, A.; *Granifo, L.; *Rojas, S. & *Rodríguez, H.

* Laboratorio de Biología Celular, Reproducción y Desarrollo (BCRD). Universidad de Tarapacá, Arica, Chile.
**Laboratorio Biología de la Reproducción, ICBM. Facultad de Medicina. Universidad de Chile, Santiago, Chile.

Dirección para correspondencia


SUMMARY: Boric acid and borate are abundant in the nature and widely used in industrial, agricultural and cosmetology applications. However it is informed as toxic in a high dose. Residents of Arica-Chile with boron-rich geology deposits are regularly exposed to higher levels in drinking water in concentrations of 6. 0 and 12.0 mg/L. The objective of this study was to analyze the damage in the masculine reproductive apparatus of mouse CF1, exposed to boric acid in concentrations of 0.54; 2.0-6.0 and 12.0 mg/L, after 60 days of treatment. Male gonad segments were fixed in Bouin's solution for 6 hours and embedded in paraffin, longitudinal sections (5 mm) were processed for histology using the hematoxyline/eosin method.

The results observed in treated animals indicate a significant increase in the weight, volume and size of testicles and seminal vesicles. The histological analysis of testicle shows in treated an epithelium notoriously altered with vacuohzations, scarce gonias and few spermatids. Gonias present picnoticals nuclei, suggesting apoptosis. In conclusion boron in high dose produces important cytotoxics effects, with degeneration of epithelium of seminiferous tubules, possibly inhibiting the spermiation, causing testicular hypertrophy, altering the masculine reproductive patterns and a reduction in fertility of mice CF1.

KEYWORDS: Boric acid; Spermatogenesis; Testicular atrophy; Mouse.

RESUMEN: El ácido bórico y el borato son abundantes en la naturaleza y con amplio uso industrial, agrícola y cosmetológico. Sin embargo, se informa como tóxico en alta dosis. Residentes de la ciudad de Arica-Chile, están expuestos a altos niveles de boro presente en el agua potable, con concentraciones 6.0 y 12.0 mg/1. El objetivo de este estudio fue analizar el daño en el sistema reproductivo masculino del ratón CF1, expuesto a ácido bórico en concentraciones de 0.54; 2.0-6.0 y 12.0 mg/1, por un período de 60 días de tratamiento. Los segmentos masculinos de las gónadas fueron fijados en solución de Bouin por 6 horas e incluidos en parafina, secciones longitudinales (5 \aa) fueron procesadas para histología usando el método hematoxilina/eosina. Los resultados observados en animales tratados indican un aumento significativo en el peso, el volumen y el tamaño de testículos y de vesículas seminales. El análisis histológico de testículo de individuos tratados, muestra un epitelio alterado con vacuolizaciones, escasas gomas y pocas espermátidas. Gonias presentan núcleos picnóticos, sugiriendo apoptosis. En conclusión el boro en alta dosis produce efectos citotóxicos importantes, con degeneración de epitelio de túbulos seminíferos, posiblemente inhibiendo la espermiación, causando hipertrofia testicular, alterando los patrones reproductivos masculinos y una reducción en la fertilidad de los ratones CF1.

PALABRAS CLAVE: Ácido bórico; Espermatogénesis; Atrofia testicular; Ratón.


INTRODUCTION

In the last decade the city of Arica-Chile, has been affected by the serious problem of environmental impact with boron in the drinking water. This happen because the underground waters or napas of the Valley of Azapa and the Valley of Lluta, transport in its route from the mountains large boron concentrations from the geologic deposits.

The water for the human use of the city of Arica have a variable boron concentrations that fluctuate between 2.0 mg/L, in the northern sector and 12.0 mg/L in the southern sector. The world-wide norms of tolerance for the human health was fixed between 0.2 to 0.6 mg of boron by litter (WHO, 1996; EPA, 2000).

The reproductive patterns are altered by extreme variations in the boron levels. On one hand decrease in the boron levels has been demonstrated that interferes the embryo-foetal development in Xenopus (Fort et al, 1998; Rowe & Eckhert, 1999), also it has been reported its effects in rodents, with alterations at formation level of blastocists and increase of dead embryos (Lanoue et al., 1998).

The effects of high doses have been studied in rats, mice and dogs, having an inhibition in the spermiation and focalized damage. Other effects of high doses in rabbits and mice involve reduction of the foetal weight and abnormalities in the skeleton (Price et al, 1996a,b).

Effects on the fertility patterns were studied in Turkish workers of a population with high boron levels in the consumption water, having values of infertility of 3.4% and 2.7%, those were considered insignificant when comparing them with Turkish regions without contamination with boron (Sayli, 2003).

Huel et al. (2004), affirms that in the Turkish populations exposed to boron there was an increase in the birth of girls, not related to random factors.

Moorman et al. (2000), informs of the impact in the Human Reproductive Health of several chemical substances, concluding that the boric acid occupies the second place of impact. Equal conclusions reaches Cain et al (2004), finding the higher levels of sensitivity to the inhalation to oxide of calcium and to sodic borate.

The present work evaluates the changes produced by boron at different concentrations its impact in the spermatogenesis, and in the germinal line of the testicle of mice CF1.

MATERIAL AND METHOD

Mice CF1, biological model genetically standardized, widely accepted in the scientific community were used (Espinoza et al, 2002), and maintained under standard conditions of feeding, hygiene and cycle of normal light, the provided water was different, for the three initial groups formed; (progenitors) of eight females and eight males each one (n=48), in the following conditions:

A)  To the group control purified water was administered, obtained of the Company Agua Delzar Arica - Chile), which presents a boron concentration of 0.54 mg/L. (Chemical analysis made by the University of Tarapacá, Chile).

B)  Treated group (Tl) with drinking water, obtained of the Company Aguas del Altplano, Arica-Chile, which supposes a variable boron concentration between 2.0 mg/L and 6.0 mg/L (Cornejo & Figueroa, 2002).

C) Treated group (T2) with a known solution of boron of 12 mg/L (H3B03, P.M. 61.83, purity 99.8%, Scharlav).

After 60 days of treatment, the males were scarified to extract testicles and seminal vesicles. The testicles were weighed and process for optical microscopy just like the seminal vesicles.

Testicle and seminal vesicles, were fixed by immersion in Bouin-Hollande solution, and embedded in paraffin following a standard protocol for histology (Durfort, 1977). Longitudinal section of 5^im were processed for histology using the conventional technique of H/E and observed in light microscope, Leitz brand, with incorporated Nikon camera.

The obtained data were analyzed using the statistical package SPSS, version 12.0. The differences between the groups were determined by the variance analysis (ANOVA), with a value of p < 0.05. The differences were determined by the test of Duncan and test of Tuckey.

RESULTS

The photos 1A to 1C show the histology of testicles of mice CF1.

When analyzing the histology of gonads and germinal epithelium of the seminiferous tubules of the control group of mice CF1 are observed that they present a normal cytology in relation to the different phases of the spermatogenesis, showing the cytomorphologics changes from spermatogonia at the formation and liberation of the spermatozoa in the light of tubule (1 A).


  Fig. 1A. Seminiferous tubules of mice CF1 which were administered "agua Delzar" (0.54 mg/L. of boron), during 60 days (control group). Normal histology, with presence of germinal line defined and all the cellular elements are observed, from gonias in the base and spermatozoa at the light of the tube (blue arrow). H/E. 40X.

Germinal epithelium of the seminiferous tubules in treated group with drinking water (2.0 mg/L to 6.0 mg/L of boron) apparently presents a normal epithelium, but in the light of the tubule a diminution of spermatozoa (IB).


  Fig. 1B. Shows seminiferous tubules treated with drinking water in concentrations of boron of 2.0 mg/L to 6.0 mg/L. The height of epitelium is normal, picnotics nuclei are observed (red arrow) and little presence of spermatozoa in the light of the tube (blue arrow). H/E. 100X.

In seminiferous tubules of the exposed group to boron concentrations of 12 mg/L is observed a low epithelium with cellular vacuolization and cells that present picnotics nuclei, which could suggest apoptosis. Total absence of spermatozoa in the lumen is observed (1C).


  Fig. 1C. Seminiferous tubules of mice CF1 treated with 12 mg/L of boron, is observed a great alteration of germinative epithelium and presence of vacuo lizations (black arrow), in the basal zone are observed picnotics nuclei (blue arrow), that suggest apoptosis, in addition, total absence of spermatozoa in the light of seminiferous tubules is observed (blue arrow). H/E, 100X.

The seminal vesicles of control group present a normal pseudo-stratified epithelium constituted by low cylindrical cells in compartmens leaned to the basal line of conjuntive tissue of normal structure (2A).


  Fig. 2A. Seminal vesicle of control (0.54 mg/L of boron). A normal pseudo stratified epithelium of low cylindrical cells is identified clearly in basal line of conjuntive tissue. 40X, H/E.

In the group treated with drinking water it appears a thickening of the basal line of epitelium. To the interior of the vesicle a secretion exists that behaves as basofile according to dye with H/E; this would be abnormal with respect to the cytomorphology of this organ (2B).


  Fig. 2B. Seminal vesicle of animal treated with drinking water (2.0 mg/L to 6.0 mg/L of boron), is observed a thickening in basal epithelium of conjuntive tissue (blue arrow) and a basofile secretion between the epithelial cells in reaction at the dying of H/E (black arrow). 100X.

The group treated with concentration of boron of 12 mg/L shows to a considerable thickening of the conjuntive tissue with few epithelial prolongations and great increase of calcareous secretions that invade almost all the light of the seminal vesicles (2C).


  Fig. 2C. Seminal vesicle of mouse treated with 12 mg/L of boron. It is observed a considerable thickening of the conjuntive tissue (blue arrow), few epithelial prolongations and increase of the calcareous secretion that occupy almost all the light of the organ (black arrow). 100X, H/E.

In table I, it shows the changes in the weight and the size of testicles and seminal vesicles treated with different boron concentrations (p<0.001). A significant increase in the weight of testicles and seminal vesicles is observed, similarly an increase significant in the size of testicles in the groups treated with drinking water (0,54 mg/1 of boron) and in the group treated with 12,0 mg/1 of boron, T1 and T2 respectively. These results express the effect of high doses of boron with hypertrophy in these male reproductive organs.


DISCUSSION

The boron is recognized as an essential micronutrient in the cellular metabolism, nevertheless, studies performed reveal that the high doses and deficiency cause morphologic and physiological alterations in most of the living beings.

Moorman et al. and Cain et al., concluding that the boric acid occupies the second place of impact in environmental study, and therefore its study must be prioritize.

The data collected show that exist an increase of testicular weight with a dose-dependent effect, nevertheless Fukuda et al. (2000), observed a diminution of the weight of the masculine gonads. We can attribute this variation of weight to an increase of the lumen of the atrophied seminiferous tubule due to the increase of cellular secretions and to a higher period of exposition of this element in study. Along with the increase of the testicular weight we observed an increase of the testicular size that is related directly to the concentrations of administered doses being this highlighted in the group treated with the concentrations of 12 mg/L of boron.

The histological study of the seminiferous tubule and seminal vesicles show significant cellular changes in the analyzed samples of treatedgroups, which presented atrophied seminiferous tubules due to an alteration marked of the germinal epitelium and cells at level of the basal line with pycnotics nuclei that suggest apoptosis, similar to the founding made by Ku et al. (1993), which determined an inhibition of the spermiation which total absence of spermatozoa in the lumen of the atrophied tubule. Similar alterations were observed in seminal vesicles that presented a thickening in the basal line of the epitelium and a secretion of abnormal calcareous type for this gland, that invades in a high percentage the light of the seminal vesicle.

The present result show a significant increases of weight of testicles and seminal vesicle exposed to high concentrations of boron with a dose-dependent effect, altering the spermatogenesis and therefore the reproduction, which confirms the studies made by Lee et al. (1978); Chapin et al. (1994); Price et al. (1996a,b); and Fukuda et al.

CONCLUSIONS

Treatment with high concentration of boron in the drinking water, affects the male reproductive patterns of the mouse CF1:

- Altering the basal epithelium of the seminal tubules of the testicles, with an increase in the lumen and an absence of spermatozoids. Great presence of vacuolizations in the germinative epithelium and increase of testicular size and weight.
- Causing changes in seminal vesicles, showing a larger size, histologically it is observed a great accumulation of calcareous secretion inside it.

The present investigation contributes scientific information on the effects of the high concentrations of boron and to adopt the necessary measures to the problem of contamination of this element in the drinking water that consumes the population of the city of Arica-Chile, and others problems of environmental contamination. (Espinoza-Navarro & Bustos-Obregón, 2005).

AKNOWLEDGEMENTS

Research supported by Universidad de Tarapacá, Project N° 4767 and Project N°4783.

 

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Received: 02-03-2007 Accepted: 12-04-2007

Correspondence to:

Dr. Omar Espinoza-Navarro
Laboratorio de Biología Celular, Reproducción y Desarrollo
Universidad de Tarapacá
General Velásquez 1775
Casilla 7-D.
Arica - CHILE

E-mail: oespinoz@uta.cl

 

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