46 VITRIFICATION AT THE GERMINAL VESICLE STAGE TRIGGERS PRECOCIOUS MEIOTIC RESUMPTION BUT DOES NOT AFFECT CYTOPLASMIC MATURATION IN CUMULUS-ENCLOSED PORCINE OOCYTES DURING IN VITRO MATURATION

Abstract

Previously we have reported a vitrification protocol that allows preservation of immature porcine oocytes in large numbers (Somfai et al. 2014 PLoS One 9, e97731). However, despite high survival rates, embryo development rates have remained low. The aim of our current research is to reveal factors potentially responsible for reduced developmental competence of vitrified oocytes. As a first step, we investigated the effects of vitrification at the germinal vesicle stage on subsequent nuclear progression and the normality of cytoplasmic functions during in vitro maturation (IVM). Cumulus-enclosed porcine oocytes were vitrified in microdrops, stored, and then warmed by our method (Somfai et al. 2015 Reprod. Fertil. Dev. 27, 124). Then the oocytes were subjected to IVM for 46 h in a chemically defined porcine oocyte medium. During the first 22 h of IVM, the medium was supplemented with 1 mM dibutyryl cyclic adenosine monophosphate, 10 IU mL–1 of eCG, and 10 IU mL–1 of hCG. The following 24 h of IVM was performed in porcine oocyte medium without any supplementation. We compared vitrified/warmed oocytes (vitrified group) with freshly collected immature oocytes (control group) in terms of (1) nuclear progression, (2) intracellular glutathione (GSH), and (3) adenosine triphosphate levels throughout IVM. Each experiment was replicated at least 3 times. Results were analysed by one-way ANOVA and Tukey’s multiple comparison test. A total of 510 oocytes were vitrified of which 422 (82.3%) survived. Only live oocytes were subjected to subsequent assays. Orcein staining revealed that after 22 h of IVM, a significantly higher percentage (P < 0.05) of vitrified oocytes showed germinal vesicle breakdown compared with the control group (22.0 v. 0.9%, respectively). In a similar fashion, after 30 h IVM, a significantly higher (P < 0.05) percentage of oocytes reached the metaphase-II (MII) stage in the vitrified group than in the control group (21.8 v. 0%, respectively). After 46 h of IVM, there was no difference between the vitrified and control groups in terms of the percentage of MII stage oocytes (93.9 and 86.3%, respectively). Analysis of GSH levels in oocytes by the 5,5′-dithio-bis-2-nitrobenzoic acid-glutathione disulfide reductase recycling assay showed no significant difference between the vitrified and control groups at 0 h (6.7 and 7.0 pmol, respectively), 22 h (5.5 and 5.5 pmol, respectively), and 46 h (6.9 and 7.9 pmol, respectively) of IVM. Adenosine triphosphate assay (FL-ASC; Sigma-Aldrich Co., St. Louis, MO) revealed similar adenosine triphosphate contents in the oocytes of the vitrified and control groups at 0 h (1.53 and 1.61 pmol, respectively), 22 h (1.67 and 1.70 pmol, respectively), and 46 h (1.65 and 1.83 pmol, respectively) of IVM. In conclusion, vitrification triggered precocious nuclear maturation even in the presence of dibutyryl cyclic adenosine monophosphate; however, it did not affect GSH levels and overall metabolism. This work was supported by JSPS KAKENHI (Grant Number: 26870839) and JST/JICA SATREPS.