(53) Controls of Nuclear and Cytoplasm Maturation in Bovine Oocytes Mediated by Gap Junctions During in Vitro Maturation Process

Controls of nuclear and cytoplasm maturation in bovine oocytes mediated by gap junctions during in vitro maturation process

Hinano Kozuka¹; Shunki Ono¹; Kota Okubo¹; Takashi Fujii¹; Ken Sawai¹

1. Department of Animal Science, Faculty of Agriculture, Iwate University, Iwate, Japan

Abstract Text:

In vitro production of embryos (IVP), consisting of in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF) and in vitro culture (IVC) of embryos, are widely used in livestock breeding and genetic improvement. Synchronization of nuclear and cytoplasmic maturation status in IVM strongly influences the production of high-quality embryos for embryo transfer. However, the artificial isolation of the cumulus-oocyte complex (COC) from the follicle results in a spontaneous resumption of meiosis and insufficient cytoplasmic maturation of the oocyte at the end of IVM period. This problem is closely related to intercellular communication through gap junctions between granulosa cells (GC) and cumulus cells (CC), or CC and oocyte. This gap junctions are constituted by connexons consisting of connexin (CX) hexamers, which are crucial for the transport of substances involved in oocyte cytoplasmic and nuclear maturation. CX43 is involved in communication between the GC and CC, and CX37 between the CC and oocyte. However, roles of CX43 and CX37 for nuclear and cytoplasmic maturation in bovine IVM oocytes are unknown. Therefore, the aim of this study was to determine changes in the expression levels and localization of CX43 and CX37 during the bovine IVM process. Immature oocytes collected from small follicles (2 -8 mm) of the ovaries collected at a slaughterhouse were cultured in the IVM medium and nuclear phase were observed at 0, 3, 6, 12 and 20 h after the initiation of IVM. The rate of oocytes at germinal vesicle breakdown (GVBD) stage was increased from 3 h (15.0%) to 6 h (94.5%) after the initiation of IVM (P< 0.05). The percentages of metaphase-Ⅰ (M-I) and metaphase-II (M-II) also increased significantly (P < 0.05) from 6 h (24.0%) to 12 h (55.6%), and 12 h (0.5%) to 20 h (81.3%), respectively. Gene expression levels analysis of GJA1 (CX43) and GJA4 (CX37) was performed using quantitative real-time PCR. The results showed that the expression levels of GJA1 in COCs and CC, and GJA4 in oocytes were significantly decreased from 6 h to 12 h of after the initiation of IVM (P< 0.05). The expression levels of GJA4 in COCs were significantly (P < 0.05) reduced from 0 h to 3 h. These findings suggest that gap junctions are closely related to meiotic resumption and meiotic progression.