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Oppioidi e funzione riproduttiva nel maschio: localizzazione del recettore mu sugli spermatozoi
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Effetti in vitro di beta-endorfina e naloxone sulla concentrazione intracellulare di calcio in cellule della granulosa murale equina
No full textExpression and localization of mu-opioid receptor in canine oocytes
No full textEndogenous 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 Ca channel occurs
(Sciorsci et al. 2000 Immunopharm. Immunotox. 22, 575–626). The disruption of Ca
homeostasis interferes with many Ca -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
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