Wee1B depletion promotes nuclear maturation of canine oocytes

Abstract

Most mammalian oocytes are arrested at the germinal vesicle stage by activation of Wee1B. Meiotic resumption is regulated by inactivation of Wee1B and activation of cell division cycle 25B. The aim of this study was to determine whether treatment with Wee1B-targeting small interfering RNA (Wee1B-siRNA) promotes nuclear maturation of canine oocytes from germinal vesicle stage to metaphase II (MII) stage. In experiment 1, the percentage of canine oocytes that matured to MII stage was higher (P < 0.05) among oocytes cultured in vitro for 72 hours than among those cultured for 24 and 48 hours (5.4 ± 2.5% vs. 0.0 ± 0.0% and 1.4 ± 1.0%, respectively). Furthermore, the percentage of oocytes that matured to metaphase I (MI) stage was higher (P < 0.05) among oocytes cultured for 48 and 72 hours than among those cultured for 24 hours (14.9 ± 10.0% and 22.4 ± 8.1%, respectively, vs. 5.7 ± 6.0%). In experiment 2, canine oocytes were intracytoplasmically microinjected with Wee1B-siRNA (50 μM) at various culture time points (0, 24, 48, or 72 hours). The nuclear configuration of the exception of oocytes in the 72-hour group was examined after 84 hours of culture. The percentage of oocytes that matured to the MII stage was higher (P < 0.05) among those treated with Wee1B-siRNA at 0 hours than among control oocytes and those injected at 72 hours (18.0 ± 1.7% vs. 2.1 ± 2.8% and 0.0 ± 0.0%, respectively). Moreover, the percentage of oocytes that matured to the MI stage was higher (P < 0.05) among those injected at 0 hours than among control oocytes and those injected at 24 and 72 hours (45.9 ± 6.8% vs. 22.1 ± 3.5%, 22.8 ± 10.0%, and 10.0 ± 4.4%, respectively). In experiment 3, oocytes were intracytoplasmically microinjected with Wee1B-siRNA at 0 hours of IVM and cultured for 0, 24, 48, or 72 hours. Thereafter, maturation-related gene expression was analyzed by quantitative real-time polymerase chain reaction. Messenger RNA expression of cAMP and cell division cycle 25B was lower (P < 0.05) in oocytes injected at 48 hours than in the other groups. Messenger RNA expression of cAMP was lower (P < 0.05) in oocytes injected at 0 hours than in control oocytes and those injected at 72 hours. Messenger RNA expression of mitogen-activated protein kinase 1 and mitogen-activated protein kinase 3 was higher (P < 0.05) in oocytes injected at 72 hours than in the other groups. In conclusion, we confirmed that Wee1B-siRNA microinjection enhances the percentages of canine oocytes that reach the MI and MII stages. These data suggest that Wee1B-siRNA microinjection could be a useful strategy to obtain mature canine oocytes for research and assisted canine reproduction.

Keywords: Dog; IVM; Oocyte; Wee1B-siRNA; cAMP.