Manuscript title POSTNATAL EXPRESSION AND ANDROGEN REGULATION OF HOXBES2 HOMEOPROTEIN IN RAT EPIDIDYMIS Short running head: ANDROGEN REGULATION OF HOXBES2 HOMEOPROTEIN

25 The multifunctional and androgen regulated epididymis with distinct delineation of specific segments and a constellation of region-specific gene expression is well known to provide a conducive microenvironment for the maturation and storage of mature spermatozoa. HOXBES2, an epididymis-specific molecule first reported by our group, exhibits cell-and region-specific expression. It was found to be localized in the cytoplasm of the principal epithelial cells with 30 maximum expression in the distal segments of the rat epididymis. Immunoreactivity to HOXBES2 was visualized on the luminal spermatozoa and on the stereocilia. The present study was undertaken to determine whether HOXBES2 expression is regulated by androgens and postnatal epididymal development. Towards this, the epididymis was disallowed an access to circulating androgens either by chemical or biological castration. In bilaterally orchidectomized 35 animals, the levels of immunoreactive HOXBES2 protein declined to approximately <5 % of those seen in sham operated animals. Exogenous DHT supplementation (250μg/kg body weight) for 7 days restored the expression levels to ≥ 90 % of that observed in intact animals. Ethylene dimethane sulfonate (EDS) administration completely abolished HOXBES2 expression in the epididymis and supplementation with DHT or DHT + E2 combined for 10 days almost re40 established HOXBES2 expression to near normalcy. However, in the ‘estradiol alone supplemented’ EDS treated group, HOXBES2 localization remained undetectable. Levels of immunoreactive HOXBES2 remained unaltered following unilateral efferent duct ligation suggested that Hoxbes2 expression is not critically dependent on testicular factors. During postnatal development, the protein expression in the epididymis begins to appear from day 40 and 45 50 and increased from day 60 to 70 onwards coinciding with the mature levels of circulating androgens and the well differentiated epididymis. Thus, the data obtained from this study suggests that the expression of HOXBES2 homeoprotein could be regulated by androgens and its expression level is closely associated with the postnatal development of the epididymis.