Abstract

The main goal of this study was to evaluate expression and distribution of P450arom in the hypothalamus of sheep at different stages of fetal development (E30, E40, E50, E80, E100, E115). Fetal heads were removed and hypothalami dissected and fixed with ice-cold 4% paraformaldehyde for 3-4 hrs and then cryoprotected with 30% sucrose. Coronal cryotome sections of the head at the hypothalamic level, 10μm thick, were obtained and underwent immunoperoxidase technique (streptavidin biotin complex) employing two polyclonal antibody: anti-P450arom and anti-androgen receptor (AR), and a monoclonal antibody, anti-βIII. With the aim of evaluating P450arom gene expression, samples from E30 and E50 the earliest stage among those examined, to be processed with in situ hybridization technique using a biotinylated cDNA probe. Finally, blot analyses were performed in order to verify the antibody specificity, as well as quantify possible differences in P450arom amounts between sexes. For sexing, fetal samples at E35 and E55 underwent DNA extraction and PCR amplification of the sex determining region of the Y chromosome using primers (SRYA) specific to the sheep. Moreover double immunofluorescences were carried out using four antibody: a polyclonal antibody: anti-P450arom and three monoclonal antibody: anti-GFAP, anti-PCNA and anti-βIII tubuline.

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