39 Efficacy of roscovitine and dibutyryl cAMP to block premature meiosis in porcine oocytes vitrified at the germinal vesicle stage and their effect on subsequent embryo development
N. Hiep A B , T. Somfai A , Y. Hirao C , T. Dang-Nguyen A , N. Men A , N. Linh B , B. Nguyen B , J. Noguchi A , H. Kaneko A and K. Kikuchi AA Institute of Agrobiological Sciences, NARO, Tsukuba, Ibaraki, Japan
B Institute of Biotechnology, Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam
C Institute of Livestock and Grassland Science, NARO, Tsukuba, Ibaraki, Japan
Reproduction, Fertility and Development 34(2) 254-254 https://doi.org/10.1071/RDv34n2Ab39
Published: 7 December 2021
© 2022 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
Vitrification induces premature meiosis in immature porcine oocytes (Appeltant et al. 2017 Reprod Fertil Dev. 29, 2419-2429). The present study compared the efficacies of the cAMP analogue dibutyryl cAMP (dbcAMP) and the metaphase promoting factor inhibitor roscovitine (ROSC) to block premature meiosis in vitrified oocytes and assayed their effect on subsequent embryo development. Cumulus–oocyte complexes (COCs) of slaughtered gilts were vitrified using Cryotop in a combination of 17.5% propylene glycol, 17.5% ethylene glycol, and 0.3 M sucrose and then warmed in 0.4 M sucrose. Then, the COCs underwent IVM for 22 h in medium supplemented (1) without dbcAMP and ROSC (dbcAMP− ROSC−), (2) with 1 mM dbcAMP (dbcAMP+ ROSC−), (3) with 25 µM ROSC (dbcAMP− ROSC+), or (4) with both 1 mM dbcAMP and 25 µM ROSC (dbcAMP+ ROSC+). In each group, the COCs were subsequently matured for an additional 24 h without dbcAMP and ROSC. The IVM medium was porcine oocyte medium supplemented with 10 ng mL−1 epidermal growth factor, 10 IU mL−1 equine chorionic gonadotrophin, and 10 IU mL−1 human chorionic gonadotrophin. In experiment 1, the nuclear status of membrane intact oocytes was compared among groups after 22 h of IVM by orcein staining, in five replicates. In experiment 2, after 46 h of total IVM, matured oocytes with the first polar body were subjected to parthenogenetic activation with 5 mM ionomycin for 5 min followed by treatment for 3 h with 2 mM 6-dimethylamino purine and 7.5 µL mL−1 cytochalasin B. Then the oocytes were subsequently cultured in porcine zygote medium-5 for 7 days to assay blastocyst formation. Three replicates were performed. Data were analysed by one-way ANOVA and Tukey’s multiple comparison test. After 22 h of IVM, the percentage of oocytes remaining at the germinal vesicle (GV) stage in the dbcAMP− ROSC− group (17.4 ± 4.4%) was significantly lower than that in the other 3 groups (83.5 ± 3.5% to 98.7 ± 0.8%). The percentages of oocytes at the GV stage in the dbcAMP− ROSC+ and dbcAMP+ ROSC+ groups were similar to one another and higher (P < 0.05) than that in the dbcAMP+ ROSC− group (98.7 ± 0.8% and 96.6 ± 1.8% vs. 83.5 ± 3.5%, respectively). In experiment 2, there was no significant difference in the percentage of matured oocytes among the four groups (96.1 ± 0.8% to 97.0 ± 1.7%). The percentage of blastocysts in the dbcAMP+ ROSC− group was tendentiously (P = 0.055) but not significantly higher than that in the dbcAMP− ROSC− group (30.4 ± 3.5% vs. 18.4 ± 2.6%, respectively), and both were significantly higher (P < 0.05) than those in the dbcAMP− ROSC+ and dbcAMP+ ROSC+ groups (4.2 ± 1.1% and 1.0 ± 1.0%, respectively). In conclusion, roscovitine prevented premature meiosis in vitrified oocytes more effectively than dbcAMP; however, it reduced subsequent embryo development without affecting the maturation ability of oocytes.
This research was supported by the aXis project from JST/Japan.
