BACKGROUND-AIM Leptin (Ob) has been shown to play a role in the regulation of the female reproductive system acting on the gonadotropic axis and on the ovaries. Ob exerts its biological effects by interacting with its receptor (Ob-R) which has been detected in the ovarian cells, including thecal cells, granulosa cells, and oocytes of several species. In the canine species, the expression and localization of Ob-R in oocytes has not been previously documented. In this study we explored the Ob-R protein expression in canine oocytes in two different stages of the oestrus cycle. METHODS Canine cumulus-oocyte complexes (COCs) were recovered from ovaries of adult bitches (n=15) undergoing routine ovariohysterectomy in two different stages of oestrus cycle (anestrus and estrus). After fixation and permeabilization, COCs (n=30 for each stage) were incubated with the primary antibody (1:50, mouse anti Ob-R) overnight at 4°C, followed by incubation with an anti-mouse fluorescein isothiocyanate secondary antibody (1:100). Nuclei were counterstained with Hoechst 33342. COCs were mounted on glass slides and observed using a confocal laser-scanning microscope. The fluorescence intensity of the cytoplasm area of the oocytes was measured at the equatorial plane. The statistical significance of the results was evaluated by the Analysis of Variance (ANOVA). RESULTS Ob-R was detected in cumulus cells (CC) and in the cytoplasm of all oocytes analyzed. Ob-R was localized within all layers of CC. Ob-R was homogeneously distributed throughout the cytoplasm of the oocytes in both stage of the oestrus cycle. The analysis of fluorescence levels revealed that the oocytes collected from bitches at the estrus stage exhibited a significantly higher protein content compared to those collected in the anestrus stage(P<0,001). CONCLUSIONS Leptin receptors are expressed in canine COCs. The presence of an increased expression of Ob-R in oocytes recovered from bitches in estrus indicates that Ob could be involved in oocyte growth and maturation. Further investigations are needed to understand the role of Ob in oocyte in vitro maturation and its potential in enhancing meiotic competence of bitch oocytes.

Immunodetection of leptin receptor in canine oocytes in two different stages of the oestrus cycle / Maltana, A.; Podda, A.; Falchi, L.; Zedda, M. T.; Ledda, M.; Scanu, A.; Bogliolo, L.. - (2022). ((Intervento presentato al convegno 19th International Congress on Animal Reproduction tenutosi a Bologna (Italy) nel 26th-30th JUNE 2022.

Immunodetection of leptin receptor in canine oocytes in two different stages of the oestrus cycle

Maltana A.
Membro del Collaboration Group
;
Podda A.
Membro del Collaboration Group
;
Falchi L.
Membro del Collaboration Group
;
Zedda M. T.
Membro del Collaboration Group
;
Ledda M.
Membro del Collaboration Group
;
Scanu A.
Membro del Collaboration Group
;
Bogliolo L.
2022-01-01

Abstract

BACKGROUND-AIM Leptin (Ob) has been shown to play a role in the regulation of the female reproductive system acting on the gonadotropic axis and on the ovaries. Ob exerts its biological effects by interacting with its receptor (Ob-R) which has been detected in the ovarian cells, including thecal cells, granulosa cells, and oocytes of several species. In the canine species, the expression and localization of Ob-R in oocytes has not been previously documented. In this study we explored the Ob-R protein expression in canine oocytes in two different stages of the oestrus cycle. METHODS Canine cumulus-oocyte complexes (COCs) were recovered from ovaries of adult bitches (n=15) undergoing routine ovariohysterectomy in two different stages of oestrus cycle (anestrus and estrus). After fixation and permeabilization, COCs (n=30 for each stage) were incubated with the primary antibody (1:50, mouse anti Ob-R) overnight at 4°C, followed by incubation with an anti-mouse fluorescein isothiocyanate secondary antibody (1:100). Nuclei were counterstained with Hoechst 33342. COCs were mounted on glass slides and observed using a confocal laser-scanning microscope. The fluorescence intensity of the cytoplasm area of the oocytes was measured at the equatorial plane. The statistical significance of the results was evaluated by the Analysis of Variance (ANOVA). RESULTS Ob-R was detected in cumulus cells (CC) and in the cytoplasm of all oocytes analyzed. Ob-R was localized within all layers of CC. Ob-R was homogeneously distributed throughout the cytoplasm of the oocytes in both stage of the oestrus cycle. The analysis of fluorescence levels revealed that the oocytes collected from bitches at the estrus stage exhibited a significantly higher protein content compared to those collected in the anestrus stage(P<0,001). CONCLUSIONS Leptin receptors are expressed in canine COCs. The presence of an increased expression of Ob-R in oocytes recovered from bitches in estrus indicates that Ob could be involved in oocyte growth and maturation. Further investigations are needed to understand the role of Ob in oocyte in vitro maturation and its potential in enhancing meiotic competence of bitch oocytes.
Immunodetection of leptin receptor in canine oocytes in two different stages of the oestrus cycle / Maltana, A.; Podda, A.; Falchi, L.; Zedda, M. T.; Ledda, M.; Scanu, A.; Bogliolo, L.. - (2022). ((Intervento presentato al convegno 19th International Congress on Animal Reproduction tenutosi a Bologna (Italy) nel 26th-30th JUNE 2022.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11388/301150
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