Research Article: The effects of kinase modulation on in vitro maturation according to different cumulus-oocyte complex morphologies

Date Published: October 11, 2018

Publisher: Public Library of Science

Author(s): Bong-Seok Song, Pil-Soo Jeong, Jong-Hee Lee, Moon-Hyung Lee, Hae-Jun Yang, Seon-A Choi, Hwal-Yong Lee, Seung-Bin Yoon, Young-Ho Park, Kang-Jin Jeong, Young-Hyun Kim, Yeung Bae Jin, Ji-Su Kim, Bo-Woong Sim, Jae-Won Huh, Sang-Rae Lee, Deog-Bon Koo, Kyu-Tae Chang, Sun-Uk Kim, Claude Prigent.


Successful production of transgenic pigs requires oocytes with a high developmental competence. However, cumulus–oocyte complexes (COCs) obtained from antral follicles have a heterogeneous morphology. COCs can be classified into one of two classes: class I, with five or more layers of cumulus cells; and class II, with one or two layers of cumulus cells. Activator [e.g., epidermal growth factor (EGF)] or inhibitors (e.g., wortmannin and U0126) are added to modulate kinases in oocytes during meiosis. In the present study, we investigated the effects of kinase modulation on nuclear and cytoplasmic maturation in COCs. Class I COCs showed a significantly higher developmental competence than class II COCs. Moreover, the expression of two kinases, AKT and ERK, differed between class I and class II COCs during in vitro maturation (IVM). Initially, inhibition of the PI3K/AKT signaling pathway in class I COCs during early IVM (0–22 h) decreased developmental parameters, such as blastocyst formation rate, blastomere number, and cell survival. Conversely, EGF-mediated AKT activation in class II COCs enhanced developmental capacity. Regarding the MAPK signaling pathway, inhibition of ERK by U0126 in class II COCs during early IVM impaired developmental competence. However, transient treatment with U0126 in class II COCs increased oocyte maturation and AKT activity, improving embryonic development. Additionally, western blotting showed that inhibition of ERK activity negatively regulated the AKT signaling pathway, indicative of a relationship between AKT and MAPK signaling in the process underlying meiotic progression in pigs. These findings may help increase the developmental competence and utilization rate of pig COCs with regard to the production of transgenic pigs and improve our understanding of kinase-associated meiosis events.

Partial Text

Transgenic pigs are regarded as potentially good animal models for biomedical research. However, to produce transgenic pigs, porcine cumulus–oocyte complexes (COCs) with high developmental competence are needed because of the low development rate of in vitro-produced (IVP) blastocysts compared with embryos produced in vivo. Many laboratories have cultured COCs of heterogonous morphology in vitro to produce mature oocytes upon fertilization or parthenogenesis (PA) [1, 2]. Moreover, many researchers have cultured COCs to produce IVP embryos according to the number of cumulus cell layers [3, 4]. Several studies have reported on the relationship between follicle diameter and oocyte developmental capacity and on the correlation between oocyte size and developmental competence [1, 5, 6]. Furthermore, researchers have focused on enhancing developmental competence via treatment with supplements, such as growth factors, hormones, and inhibitors, to alter the activity of proteins in COCs [7, 8]. However, there is limited information on the improvement of maturation competence via the regulation of proteins in oocytes according to differences in COC morphology.

Transgenic pigs are required for biomedical and regenerative medicine research due to their anatomical and physiological similarities to humans. Because the efficiency rate of producing transgenic animals is very low, oocytes with high developmental competence are needed to produce transgenic pigs. Thus, additional studies will be necessary to achieve a better understanding of the molecular mechanisms that govern the meiotic process underlying the development of oocytes, to yield more efficient methods of oocyte maturation. The present study was the first to demonstrate the distinct activities of two signaling pathways, PI3K/AKT and MAPK, between class I and II COCs in the pig after 20 h during IVM. It was shown that modulation of these pathways resulted in lower developmental competence in class II COCs compared to class I COCs. In particular, activation of the PI3K/AKT pathway was critical for oocyte maturation, while the MAPK/ERK pathway negatively regulated meiotic progression by impeding the AKT cascade. Additionally, EGF was identified as a positive regulator of oocyte competence in class II COCs following AKT activation, which suggests that the regulation of signaling pathways such as MAPK/ERK and PI3K/AKT influences the behavior of porcine oocytes. Therefore, the present results will further current understanding of the molecular mechanisms underlying the meiotic process in the pig oocyte. To determine the developmental capacity of COCs, porcine COCs were classified according to the number of cumulus cell layers and were cultured in IVM medium. In a previous study, most COCs obtained from ovaries consisted of less than two layers of cumulus cells, and the proportion of COCs with five or more layers of cumulus cells was below 10% [2]. The present study identified distinct types of developmental competence and kinase activity between oocytes derived from class I and class II COCs, and also provided possible novel methods for increasing the competence of oocytes and early embryonic development via the regulation of signaling pathways.




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