For developmental competence of oocyte derived from small follicle (~3 mm in diameter SF), pre-IVM system was developed for in vitro culturing SF. The purpose of this study is to establish the optimal phase and concentration for exogenous addition of pituitary adenylate cyclase-activating peptide (PACAP) on pre-IVM. To establish the appropriate phase for pre-IVM, we assessed nuclear status according to culture duration. The result of the nuclear stage assessment of the COCs (cumulus oocyte complex) from SF are as follow: metaphase I (MI) stage of 0 h (0%), 6 h (0.5%), 12 h (4.8%), 18 h (9.6%) and 24 h (13.4%). The rate of germinal vesicle breakdown (GVBD) and germinal vesicle (GV) in groups between 12 h and 18h groups was no statistically significant difference. Nevertheless, the result of MI stage compared with 0 h and 6 h group showed that the 18 h group accelerated significantly meiotic resumption (P<0.05). PACAP was treated on pre-IVM according to concentration. After 18 h, The 10 μM group showed a significantly (P<0.05) the highest rate on meiotic arrest of COCs: GV stage of control (60.5%), 500 fM (64.%), 1 nM (74.4%), 100 nM (69.9%) and 10 μM (82.1%). COCs obtained from follicles 46 mm in diameter (MF control) and SF and subjected to IVM for 42 h. In the pre-IVM group, COCs obtained from SF and matured with non treatment (Pre-SF(−)PACAP) and 10 μM PACAP (Pre-SF(+)PACAP) for 60 h. After IVM, Pre-SF(+)PACAP group (91.7%) showed significantly increased nuclear maturation than control and Pre-SF(−)PACAP group (81.7% and 81.7%) (P<0.05). The Pre-SF(+)PACAP group showed significantly (P<0.05) increased GSH levels compared with SF and Pre-SF(−)PACAP groups. These results indicated that PACAP is able to delay oocyte meiotic maturation during pre-IVM and consequently improve nuclear and cytoplasmic maturation after IVM. This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Advanced Production Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant number: 115103-02), Republic of Korea.
11 Jul 2016 - 11 Jul 2016