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tact one-year-old females have been decrease than these of -month-old females. In CASTmales, the testosterone levels two months following castration had been beneath detection limits. Western Blot To obtain myocardial nuclear fractions, LV samples have been homogenized in ice-cold lysis buffer containing a protease inhibitor cocktail. Lysates have been centrifugated and the resulting pellet was resuspended in nuclear buffer, incubated min in ice and centrifugated once again. The supernatants containing the nuclear fraction have been harvested. Thirty mg of nuclear protein extracts were resolved on sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidene difluoride membranes. The membranes have been incubated with principal antibodies to phospho-Smad and to ELK. The membranes have been incubated with peroxidase conjugated secondary antibodies. Immunoreactivity was detected with Advanced ECL detection reagents. Two weeks right after TAC, LV geometrical and functional deterioration was extra severe in intact males than in females Collagen content by light microscopy Hearts have been fixed in paraformaldehyde, cryopreserved with sucrose and frozen at uC whilst embedded in OCT compound for cryosectioning. Coronal sections in the level of the papillary muscle tissues were stained with picrosirius red and viewed with polarized light below dark-field optics to detect birefringence of mature collagen fibers. Every single section was 163768-50-1 web photographed. 5 fields were randomly chosen from each of 3 sections per animal. Within each field, the proportion of labeled area was calculated by densitometric analysis with all the ImageJ application. Histological assessment of myocardial fibrosis Hearts were fixed in paraformaldehyde for h and, then, embedded in paraffin. The degree of fibrosis was determined in mm sections stained with Masson's trichrome. Experiments with cell lines NIH-T fibroblasts and HC cardiomyocytes were cultivated in Dulbecco's modified Eagle's medium supplemented with heat-inactivated fetal bovine serum, unitsml penicillin and mgml streptomycin at uC in CO. Cells were incubated together with the nonaromatizable androgenic steroid dihydrotestosterone for h. Total mRNA was obtained and retrotranscribed to execute qPCR for TGF-bs as described above. All assays were performed in duplicate and repeated on three separate occasions. Androgens and LV Remodeling in Stress Overload TAC-induced myocardial overexpression of remodelingrelated genes was more pronounced in males than in females The myocardial mRNA levels of TGF-b, TGF-b and TGFb also because the nuclear protein levels of phosphorylated Smad elevated drastically just after TAC only in intact male mice. In females, neither the nuclear levels of phosphoSmad nor the TGF-b mRNA expression have been drastically modified by the stress overload situation. In parallel, extracellular matrix genes showed a important upregulation in TAC-males, reaching larger expression values than in TAC-females. The changes observed inside the mRNA expression of collagen have been confirmed by picrosirius red histological staining. The percentage of the location stained by picrosirius red was .. fold larger in TAC-males than in TAC-females. The levels of a-MHC mRNA have been significantly lowered only in TAC-males, whereas b-MHC mRNA was elevated right after TAC in each sexes to a related extent. TAC-induced myocardial fibrosis was prevented in males by systemic inhibition of TGF-b A series of TAC-male mice were treated through the follow-up period using a neutralizing monoclonal antibody anti