IMMUNOCYTOCHEMICAL LOCALIZATION AND REGULATION OF CONNEXIN43 IN THE ADULT-RAT EPIDIDYMIS

Connexin43 (Cx43) is one of the most predominant gap junction proteins found in vivo, and although present in the testis, it has not been ex amined in the epididymis, Immunocytochemistry using an anti-Cx43 antib ody revealed a punctate immunoperoxidase reaction at the apical margin s between adjacent epithelial cells of the efferent ducts. In the epid idymis, punctate reactive sites were observed at the base of the epith elium between basal and principal cells. There was no staining between adjacent principal cells at their apical or lateral margins. Cx43 imm unostaining was also seen between myoid cells surrounding the tubules but only in the cauda epididymidis. Using a Cx43 cDNA probe, Northern blot analysis of total cellular RNA revealed a single hybridizing band of approximately 3.0 kb in all regions of the epididymis. Throughout the epididymis of bilaterally orchidectomized rats at 7 or 14 days, an immunoperoxidase reaction for Cx43 persisted at the base of the epith elium between principal and basal cells. However, in the initial segme nt only, immunolocalization of Cx43 was also observed apically between adjacent principal cells. This apical staining was lost in rats that received testosterone replacement. Myoid cells in the cauda epididymid is of control rats expressed Cx43, however, orchidectomized rats did n ot express Cx43 in this cell layer. Western blots revealed the presenc e of a major protein band at 42 kDa corresponding to unphosphorylated Cx43 as well as Cx43 species at 44 and 46 kDa, which were more promine nt in orchidectomized rats. Together these data represent one of the f irst examples of Cx43 gap junctions between heterologous cell types (i .e. principal and basal cells). Moreover, Cx43 expression in myoid cel ls of the cauda epididymidis is androgen-dependent and in the initial segment of the epididymis only, the intracellular targeting of Cx43 to wards the principal-principal cell interface under normal conditions i s regulated by androgens.