185 EXPRESSION AND LOCALIZATION OF µ-OPIOID RECEPTOR IN CANINE OOCYTES

Endogenous opioid peptides (EOP), through G-protein-coupled receptors, control metabolism and many physiological and pathological conditions. Once EOP are linked to their receptors, above all µ-opioid receptor (MOR), a block of the Ca2+ channel occurs (Sciorsci et al. 2000 Immunopharm. Immunotox. 22, 575–626). The disruption of Ca2+ homeostasis interferes with many Ca2+-mediated/dependent actions. Our previous studies demonstrated the presence of MOR in human, bovine, and equine oocytes, in sperm cells of several species (equine, canine, etc.), in mare's tube, in ovine, bovine and mouse embryos. The presence of MOR on the male canine gamete lets us hypothesize its presence on the female gamete, too. In this study we demonstrated the presence of MOR on canine oocytes by immunofluorescence (IF) and western blot (WB) analysis, and we speculate on its possible functional role. Canine ovaries were obtained from healthy bitches randomly chosen among those arriving at our veterinary hospital for surgical ovariectomy without considering the period of their reproductive cycle. Oocytes were collected by ovary slicing and tested to check for the presence of MOR. For IF, oocytes were washed in 100 mm glycine in PBS and incubated for 30 min in PBS-1% BSA. Control oocytes were incubated with primary rabbit polyclonal antibody against the rat 3rd extracellular loop of MOR (Chemicon, Temecula, CA, USA). All oocytes were incubated for 2 h at room temperature with a FITC-conjugated anti-rabbit IgG-secondary antibody diluted 1:200 in Evans blue/PBS, washed, and visualized by laser scanning confocal microscope. For the WB, crude plasma membranes were obtained from pools of oocytes. They were lysed in Laemli buffer and loaded on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels. After electrophoresis, proteins were electrotransferred (semi-dry apparatus, BioRad, Milano, IT) to Immobilon-P membranes (Millipore, Bedford, MA, USA). Filters were blocked for 1 h and blotted overnight at 4�C against the same primary antibody used for IF, diluted 1:7500 in blocking buffer. After washing, membranes were incubated with a 1:10 000 dilution of peroxidase-conjugated goat anti-rabbit IgG secondary antibody for 2 h at room temperature. Reactive bands were visualized by Supersignal West Pico Chemiluminescent substrate (Pierce, Milano, IT). A negative control was performed. The IF highlighted, by clear brilliant green, the MOR's localization on canine oocytes. The negative control did not present any fluorescent region or spotted coloring. The WB revealed the presence of one immunoreactive band of approximately 65 kDa, thus confirming the results obtained by IF. No reactivity was evident when the primary antibody was adsorbed with an excess of immunizing peptide. The presence of MOR on canine oocytes indicates its possible role in the modulation of oocyte metabolism. These data strongly confirm previous evidence from our research unit on the involvement of the opioidergic system during gamete development and interaction, thus allowing us to speculate on a primary role of MOR in controlling key events of the reproductive activity.