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Quantitative analysis in LC3-II protein in vitro maturation of porcine oocyte

Published online by Cambridge University Press:  12 June 2013

SeungHoon Lee ,
Yuuki Hiradate ,
Yumi Hoshino ,
Kentaro Tanemura  and
Eimei Sato
SeungHoon Lee*
Affiliation:
Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Aobaku, Sendai 981-8555, Japan.
Yuuki Hiradate
Affiliation:
Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aobaku, Sendai 981-8555, Japan.
Yumi Hoshino
Affiliation:
Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aobaku, Sendai 981-8555, Japan.
Kentaro Tanemura
Affiliation:
Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aobaku, Sendai 981-8555, Japan.
Eimei Sato
Affiliation:
Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aobaku, Sendai 981-8555, Japan.
*
All correspondence to: SeungHoon Lee. Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Aobaku, Sendai 981-8555, Japan. Tel:/Fax: +81 22 717 8687. e-mail: b0ad9201@s.tohoku.ac.jp
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Summary

Microtubule-associated protein light chain 3 (LC3)-II is a marker of autophagosome. In this study, LC3-II expression was used to identify autophagy, during the in vitro maturation of porcine oocytes. In a time-course experiment, cumulus–oocyte complexes (COCs) were cultured in NCSU23 medium for 0 h, 14 h, 28 h or 42 h. The cumulus cells were removed and denuded oocytes were processed for western blotting or immunostaining. Western blotting showed that the LC3-II levels changed over time, with maximum levels observed at 14 h and minimum levels at 42 h. Immunostaining of LC3 showed the signals with dot shapes and ring shapes in oocytes at every group that probably represent autophagosomes. To ascertain whether autophagic induction and degradation were occurring, we treated the cultures with autophagic inhibitors. Lysosomal protease inhibitor E64d and pepstatin A increased the LC3-II levels and wortmannin, inhibitor of autophagic induction, decreased the LC3-II levels. Western blotting and immunostaining demonstrated that LC3-II is present in porcine oocytes cultured in vitro. The decreased LC3-II levels after wortmannin treatment suggest that it is newly generated in porcine oocytes, a phenomenon that represents autophagic induction. Furthermore, increased LC3-II levels after E64d and pepstatin A addition imply that LC3-II is degraded by lysosomal proteases, an indication of autophagic degradation. Our results suggest that autophagy, which is a dynamic process whereby autophagosomes are newly generated and subsequently degraded, is probably occurring in porcine oocytes during in vitro maturation.

Keywords

AutophagyAutophagy inhibitorIn vitro maturationLC3Porcine oocyte
Type
Research Article
Information
Zygote , Volume 22 , Issue 3 , August 2014 , pp. 404 - 410
Copyright
Copyright © Cambridge University Press 2013 

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