Effects of vitrification of cumulus-enclosed porcine oocytes at the germinal vesicle stage on cumulus expansion, nuclear progression and cytoplasmic maturation
Ruth Appeltant A , Tamás Somfai B D , Elisa C. S. Santos B , Thanh Quang Dang-Nguyen A , Takashi Nagai C and Kazuhiro Kikuchi A
+ Author Affiliations
- Author Affiliations
A Division of Animal Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organisation, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan.
B Animal Breeding and Reproduction Research Division, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organisation, Ikenodai 2, Tsukuba, Ibaraki 305-0901, Japan.
C Department of Research Planning and Coordination, National Agriculture and Food Research Organisation, Ikenodai 2, Tsukuba, Ibaraki 305-0901, Japan.
D Corresponding author. Email: somfai@affrc.go.jp
Reproduction, Fertility and Development 29(12) 2419-2429 https://doi.org/10.1071/RD16386
Abstract
Although offspring have been produced from porcine oocytes vitrified at the germinal vesicle (GV) stage, the rate of embryo development remains low. In the present study, nuclear morphology and progression, cumulus expansion, transzonal projections (TZPs), ATP and glutathione (GSH) levels were compared between vitrified cumulus–oocyte complexes (COCs) and control COCs (no cryoprotectant treatment and no cooling), as well as a toxicity control (no cooling). Vitrification was performed with 17.5% (v/v) ethylene glycol and 17.5% (v/v) propylene glycol. Vitrification at the GV stage caused premature meiotic progression, reflected by earlier GV breakdown and untimely attainment of the MII stage. However, cytoplasmic maturation, investigated by measurement of ATP and GSH levels, as well as cumulus expansion, proceeded normally despite detectable damage to TZPs in vitrified COCs. Moreover, treatment with cryoprotectants caused fragmentation of nucleolus precursor bodies and morphological changes in F-actin from which oocytes were able to recover during subsequent IVM culture. Reduced developmental competence may be explained by premature nuclear maturation leading to oocyte aging, although other mechanisms, such as initiation of apoptosis and reduction of cytoplasmic mRNA, can also be considered. Further research will be required to clarify the presence and effects of these phenomena during the vitrification of immature COCs.
Additional keywords: cryopreservation, cumulus cell, cytoskeleton, gamete.
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