The presence of the u-opioid receptor in the isthmus of mare oviduct

Abstract

The presence of the μ-opioid receptor and the type of glycosylation in the third extra-cellular loop of this receptor was investigated in the isthmus of mare oviduct during oestrus by means of immunoblotting and immunohistochemistry combined with enzymatic (Nglycosidase F and O-glycosidase) and chemical (ßelimination) treatments. Immunoblotting analysis showed that the μ -opioid receptor consists of two peptides with molecular weights of around 65 and 50 kDa. After N-deglycosylation with N-glycosidase F an additional immunoreactive peptide was observed at around 30 KDa. The cleavage of O-glycans by Oglycosidase failed in immunoblotting as well as in immunohistochemistry investigations, revealing that the third extra-cellular loop of the μ -opioid receptor expressed in mare isthmus oviduct contains some modifications of the Galß(1-3)GalNAc core binding to serine or threonine. Immunohistochemistry revealed the μ-opioid receptor in the mucosal epithelium, some stromal cells, muscle cells and blood vessels. In ciliated cells the μ-opioid receptor showed N-linked glycans, since the immunoreactivity was abolished after Nglycosidase F treatment, whereas it was preserved in the apical region after ß-elimination. Most non-ciliated cells expressed the μ-opioid receptor with both N- and Olinked oligosaccharides, as revealed by the abolition of immunostaining after N-glycosidase F and ßelimination. Stromal cells, endothelial and muscle cells of blood vessels expressed the μ -opioid receptor containing both N- and O-linked oligosaccharides. Myosalpinx myocytes expressed the μ-opioid receptor with O-linked oligosaccharides. The immunopositive myocytes formed a circular coat in the intrinsic musculature, whereas they were arranged in some isolated, oblique bundles in the extrinsic musculature. In conclusion, the μ-opioid receptor could have a role in the production and the movement of isthmus lumen content that contributes to ensuring the effective condition of the sperm in the mare oviduct.