Mallory stained micrographs, showing the different thickness of the tunica albuginea in the control group (A) compared to the Boron treated group (B). Bar = 100 μ m. 

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... for the production of prostaglandins produced during inflammation. Previous studies have indicated that COX-2 is involved in the reduction of steroid hormones (Zirkin, 2005). It has also been suggested that cyclooxygenase-2 may play an important role in the testis when spermatogenesis is impaired (Frungieri et al. , 2002; Wang et al., 2003). Ecotoxicology studies show that different elements found naturally in our environment are toxic to our body, especially to the reproductive system. In the case of organophosphorus insecticides and some heavy metals like lead, which causes decreased sperm counts and disequilibrium between pro-and antioxidant factors, eliciting oxidative damage to cells (Bustos-Obregon & Hartley, 2008). The effect of trace elements in the environment is an important public health problem. There is considerable literature concerning the general toxicology of these elements. However little attention has been given to the adverse effect of these elements in male reproduction, although the information exists, stressing the continued de- cline in semen quality worldwide (Dindyal, 2004). This may be due to urbanization and industrialization. In the male infertility clinic, growing evidence suggests that some environmental trace elements may be associated with a subtle and chronic exposure to environmental chemicals in the air, water and food. The purpose of this study is to evaluate the testis following administration of high doses of boron, in an animal model similar to humans inhabiting the north of Chile, that for many years were affected by a high intake of boron in drinking water. Experimental design . Twenty sexually mature male CF-1 mice Mus domesticus (30-40 g) were used. One group (ten mice) drank Santiago’s tap water (0.6 mg Boron/L), and the other ten mice were exposed to drinking water containing 12 mg Boron/L for 42 days (one complete spermatogenic cycle of 33.2 days, plus one cycle of 8.5 days), from 85 days of age until 127 days old, simulating Arica’s tap water human intake. Animals were kept under standard animal room conditions, and sacrifice was done according to the bioethical norms of the Medical School. Histological Technique . The testes were removed and fixed in alcoholic Bouin's fluid, and then processed for paraffine embedding and staining with HE, Mallory, immunohistochemistry for COX-2, or Picrosirious Red (polarized light). Histopathology. Testicular sections HE stained were examined at 1000x, recording plugging tubular epithelial vacuolization, epithelial desquamation and percentage ratio of interstitium/tubules. Morphometry . Digitalized testicular sections, photographed at 400x, were used to measure epithelial height ( μ m) tubular diameter ( μ m), tubular lumen diameter (mm) and interstitial area (%), counting 100 tubules per mouse. Images were processed for analysis using Image Tool 3.0. Immunohistochemistry . Testicular sections were processed according to Corominas (1997), and the technical recommendations of the primary antibody anti-COX-2 manufacturer (Santa Cruz Biotechnology), contrasted with light hematoxylin. Evaluation of Tunica Albuginea . In Mallory stained testicular sections, thickness of the tunica albuginea was measured (using Image Tool 3.0). Testicular sections stained with Picrosirius Red were examined by polarized light microscopy (Zeiss Axiostar Plus Mod), according to Junqueira et al. (1979). Collagen type I, form thick and tight fibers, with an intense yellow to red birefringence. Type III collagen fibers form thin fibers, composed of thin fibrils, freely arranged, with a weak birefringence of a greenish color (Junqueira et al. ). To quantify this different birefringence of collagen fibers, Image Pro 6.0 Software was used, obtaining a percentage of the different colors on a photograph captured in the polarized light microscope. A similar configuration of the software to Meruane et al. (2011) was used. It quantifies the yellow and red as collagen I and green as collagen III, to establish the percentage among them. Statistic . The results were expressed in % or microns ( m) ± SD, using the t-Student statistical test to find significance (p <0.05) in the results. Effect of Boron treatment on testicular histopathology . Histopathological analysis revealed a significant increase in abnormalities of the seminiferous tubules in the Boron group compared to the control group (% SD, p<0.05) (Table I). Effect of Boron tratment on morphometric parameters . Morphometric evaluation of tubular diameter, luminal diameter, epithelial height and interstitial area, reveal significant differences (p<0.05) between the control and Boron group (Table II). Inmunohistochemical COX-2 Expression. Showed a significantly greater percentage of immunoreactive tubules (p <0.05) in the Boron exposed group compared to control group (Table III). (Mallory staining) . Tunica albuginea thickness showed a significant decrease (p <0.05) in Boron treated mice (Table IV; Fig. 1). by pricrosirius red staining. The evaluation of the collagen fibers of the connective tissue of the tunica albuginea, by Picrosirius staining, observed in polarized light microscopy, revealed a significant decrease of collagen type I fibers (p<0.05), and a significant increase in collagen type III fibers (p <0.05), in the group exposed to boron (Table V; Fig. 2). Color sample analysis, using Image Pro 6.0, of the Picrosirius stained sections, examined by polarized light microscopy, permitting collagen area measurement. It was found that Control animal had 13.35% of image area for collagen I, and 0.019% for collagen III, while Boron exposed animal had 11.57% of image area for collagen I, and 2.10% for collagen III. Analyzing the situation of people in northern Chile some years ago, morphometric histological findings were consistent with previous studies in rats by Dixon et al . (1976). In addition, due to a previous study showing histopathological effects of boron on mouse liver, at a dose of 12 mg/L in drinking water affects liver connective tissue, and the relationship collagen I/III fibers, Mallory and Picrosirius Red staining was used to evaluate tunica albuginea damage. Morphometric and Histopathological analysis . A significant damage to the testes of animals exposed to 12 mg of boron/L, is evidenced by a decrease in epithelial height of the tubules. These changes can be explained by reduced proliferation of spermatogenic cells. In turn, it depends on Interstitial cells paracrine function through the action of testosterone on sustentacular cells and germ cells. All this alterations were seen as a disruption of androgen status (decrease in circulating testosterone levels), associated with a lower epithelial height, disorganization of the epithelium and germ cell detachment in rats exposed to boric acid (Chapin & Ku, 1994; Naghii et al ., 2011). The decrease in epithelial height in the group exposed to boron may also be due to increased apoptotic index (Blanco-Rodriguez & Martinez-García, 1998), which can be related to the presence of vacuolization found, suggesting loss of specific junctions between sustentaculars cells and germ cells development. This cell loss may be directly related to the number of tubules showing desquamation and subsequent tamponade. Atrophy was observed in more than half of the mice intoxicated with boric acid, thus explaining the relative increase of interstitial area in boron treated animals, which may be due to the activity of prostaglandins in a possible inflammatory process characterized by edema, which is consistent with studies done in rats exposed to Boron, and other metalloids such as arsenic. Spermatogenesis in mammals requires the action of a number of peptides, steroids and hormones. These hor- monal messengers are critical not only for the regulation of germ cell development but also for the proliferation and somatic cell function and thus for the proper development of the testis. Therefore, studies are needed to assess the androgenic status in animals treated with boron, to understand better the disruption of the blood-testis barrier, and its androgen dependency (Meng et al. , 2005). Since the testes is the nest for spermatogonial development, cells that maintain the epithelium in physiological condition (sustentacular cells and interstitial cells), when affected directly or indirectly, cause alteration in the spermatogenic process, with a decrease in quality of the ejaculate. Further studies are needed to assess whether histopathological alterations and changes in morphometric parameters observed by chronic exposure to critical levels of boron, affect long-term fertility. Immunohistochemistry . Boron elicited overexpression of COX-2, which is found in epithelial cells of the distal part of cauda epididymis and vas deferens. COX-2 is the isoform that is expressed in some tissues, only in pathological processes, such as inflammation and cellular stress, which can lead to cell death (Mandelker, 2011). Inflammation causes low sperm quality and could even lead to infertility in animals (Kanokwan, 2011). Overexpression of COX-2, together with increase of the intersticial area, could be taken as an inflammatory process, although in tissue sections of animals exposed to boron no inflammatory leukocyte infiltration was found. May be chemical damage due to boron, through free radical production, as in the case of other metalloids such as arsenic, exerts oxidative stress in cells causing DNA damage through the production of superoxide and hydrogen peroxide (Patrick, 2003), and producing an overexpression of COX-2, which may be due to decreased intracellular levels of arachidonic acid caused by its destruction by the action of free radicals, and increases the production of PGs. In turn, it has been shown that testosterone synthesis is profoundly affected by the production of prostaglandin F2a (PGF2) whose synthesis is catalyzed by the enzyme cyclooxygenase-2 ...