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The objective of this paper is to present a synoptic view of the environmental performance of non-financial Brazilian firms during the period from 2007 to 2012. The seven indicators (environmental management, adopted procedures, information and relationship policy, investments, use of natural resources, waste treatment and additional environmental action), cover a broad environmental spectrum. A major element of the study is to allow, from aggregated values, an overview of the evolution of the indicators in the market and in the sector. The originality of this work is ensured by three factors: 1) the database method, which was developed by the research team; 2) the creation of a benchmark, which made the prospective analysis of highly correlated indicators possible; 3) the possibility of analysis of the firms individually, using the seven indicators to highlight the outcomes of the environmental decisions underway.

Steroid hormones like testosterone and estradiol are primary produced in the testes. Innumerous studies suggest the role of estrogens in the male reproduction via their specific receptors named estrogen receptor α (ERα) and estrogen receptor β(ERβ). In adult stallions, it have been found the presence of high levels of plasma estrogens, supporting the importance of the estrogen receptors and also of cytochrome P450 aromatase, the enzyme responsible for aromatization of androgens into estradiol. The present study aimed to confirm the presence of both ERs and cytochrome P450 aromatase enzyme via immunohistochemistry in adult stallion testes showing that this tissue is responsive to estrogen. Tissue specimens were collected after castration, washed in saline, placed in plastic cassettes, fixed in 10% buffered formalin for 24h and stored in 70% ethanol until they were embedded in paraffin. 4µm-tissue-section slides were deparaffinized with xylene, rehydrated in graded alcohol and then washed in tap water for 10 minutes. For antigen retrieval, sections were either microwaved for 3 periods of 5 minutes (ERβ) or in a water bath at 96°C for 30 minutes (cytochrome P450 aromatase) in sodium citrate solution 10mM (pH=6.0). Slides were allowed to cool for 20 minutes and then washed ten times in tap water. Endogenous peroxidase activity was quenched with 8% peroxidase solution for 20 minutes followed by 10 baths in tap water and incubated with a 3% milk solution for 1 hour at room temperature. Slides were washed in TRIS buffered solution 10 mM (pH=7.4), encircled using DakoPen (Dako, USA) and incubated with the primary mouse anti-human ERβ monoclonal antibody (clone PPG5/10, Dako, USA), diluted 1:100 in antibody diluent (Dako, USA) or rabbit anti-human cytochrome P450 aromatase (Hauptman Woodward Medical Research Institute Inc., USA), diluted 1:1000. Both were incubated in a humidified chamber for 18h at 4°C. Then, slides were washed in TRIS buffered solution and incubated with the secondary antibody (Advance, Dako, USA). Tissue sections were washed in TRIS buffered solution and DAB was added as chromogen staining substrate for 5 minutes. Tissue sections were rinsed in TRIS buffered solution, counterstained with Mayers hematoxylin for 30 seconds, dehydrated and preserved using Permount mounting medium. For negative controls, another section was incubated with either mouse or rabbit immunoglobulin (Dako, USA) instead of primary antibody. For the evaluation of immunoreactivity, stained sections were observed and photographed using a Leica DML optic microscope (DMLB, Germany) at 400x magnification. All slides were evaluated for positively stained nuclei (ERβ) or cytopmasmatic immunostainning (aromatase). It could be observed a strong immunostainning for cytochrome P450 aromatase in almost all interstitial area, demonstrating that the leydig cells' cytoplasm were positive for this enzyme. However, the nuclei of the cells remained negative. It was also observed for the nuclei of leydig, sertoli and germ cells an ERβ immunostainning. However, for leydig cells it could be visualized a mixed pattern, with some positive and other negative cell regions. The majority of sertoli cells were positive with strong immunostainning. The majority of round germ cells were also positive. However, elongated germ cells were negative. At the present moment we failed to find immunostainning for ERα and new protocols are yet to be performed. Research supported by Fapesp.(poster)