Immunofluorescence localization of mu-opioid receptors on buffalo (Bubalis Bubalis) primary and in vitro matured oocytes.

Opioids exert their activity via the interaction with multiple G-protein coupled receptors, namely mu, delta and kappa-opioid receptors and have been related with many reproductive events. In our previous studies we showed expression, localization and functional roles of the mu-opioid receptor (MOR) in bovine oocytes. The objective was to determine, by immunoflorescence (IF) detection, whether MOR is present in buffalo immature and in vitro matured (IVM) oocytes. Five oocytes, at the germinal vesicle (GV) stage and five IVM oocytes, were quickly denuded and kept in 4% (v/v) paraformaldehyde until examination. On the day of the test, all oocytes were washed in 100mM glycine in PBS and incubated for 30 min in PBS-1%BSA. Control oocytes were incubated in PBS-1% BSA for 90 min, while a 1:2500 dilution of the primary rabbit antibody against the third extracellular loop of MOR, was applied to test oocytes and allowed to react. All oocytes were washed in PBS, then incubated with a FITC-conjugated anti rabbit IgG-secondary antibody diluted 1:200 in Evans Blue/PBS 1x to counterstain negative cells. Oocytes were visualized by a laser scanning confocal microscope. The IF highlighted, by intense brilliant green, the localization of MOR’s on buffalo GV and IVM oocytes. The negative control did not show any fluorescent region or spotted colouring. We suggest that opioid receptors in immature and in vitro matured oocytes may interact with opioid peptides locally produced within the female reproductive tract.