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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: [email protected]
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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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References

Abeydeera, L.R. (2002). In vitro production of embryos in swine. Theriogenology 57, 256–273.CrossRefGoogle ScholarPubMed
Backer, J.M. (2008). The regulation and function of class III PI3Ks: novel roles for Vps34. Biochem. J. 410, 1–17.CrossRefGoogle ScholarPubMed
Blommaart, E.F., Krause, U., Schellens, J.P., Vreeling-Sindelarova, H. & Meijer, A.J. (1997). The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur. J. Biochem. 243, 240–6.CrossRefGoogle ScholarPubMed
Choi, J.Y., Jo, M.W., Lee, E.Y., Yoon, B.K. & Choi, D.S. (2010). The role of autophagy in follicular development and atresia in rat granulosa cells. Fertil. Steril. 93, 2532–7.CrossRefGoogle ScholarPubMed
Dunn, W.A., Jr. (1990). Studies on the mechanisms of autophagy: maturation of the autophagic vacuole. J. Cell Biol. 110, 1935–45.CrossRefGoogle ScholarPubMed
Eskelinen, E.L. (2005). Doctor Jekyll and Mister Hyde: autophagy can promote both cell survival and cell death. Cell Death Differ. 12 (Suppl 2), 1468–72.CrossRefGoogle ScholarPubMed
Faerge, I., Strejcek, F., Laurincik, J., Rath, D., Niemann, H., Schellander, K., Rosenkranz, C., Hyttel, P.M. & Grondahl, C. (2006). The effect of FF-MAS on porcine cumulus–oocyte complex maturation, fertilization and pronucleus formation in vitro . Zygote 14, 189–99.CrossRefGoogle ScholarPubMed
Funahashi, H., Koike, T. & Sakai, R. (2008). Effect of glucose and pyruvate on nuclear and cytoplasmic maturation of porcine oocytes in a chemically defined medium. Theriogenology 70, 1041–7.CrossRefGoogle Scholar
Gozuacik, D. & Kimchi, A. (2004). Autophagy as a cell death and tumor suppressor mechanism. Oncogene 23, 2891–906.CrossRefGoogle ScholarPubMed
Han, J., Pan, X.Y., Xu, Y., Xiao, Y., An, Y., Tie, L., Pan, Y. & Li, X.J. (2012). Curcumin induces autophagy to protect vascular endothelial cell survival from oxidative stress damage. Autophagy 8, 12–25.CrossRefGoogle ScholarPubMed
Hemelaar, J., Lelyveld, V.S., Kessler, B.M. & Ploegh, H.L. (2003). A single protease, Apg4B, is specific for the autophagy-related ubiquitin-like proteins GATE-16, MAP1-LC3, GABARAP, and Apg8L. J. Biol. Chem. 278, 51841–50.CrossRefGoogle ScholarPubMed
Hoshino, Y., Yokoo, M., Yoshida, N., Sasada, H., Matsumoto, H. & Sato, E. (2004). Phosphatidylinositol 3-kinase and Akt participate in the FSH-induced meiotic maturation of mouse oocytes. Mol. Reprod. Dev. 69, 77–86.CrossRefGoogle ScholarPubMed
Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y. & Yoshimori, T. (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, 5720–8.CrossRefGoogle ScholarPubMed
Lee, M.S., Kang, S.K., Lee, B.C. & Hwang, W.S. (2005). The beneficial effects of insulin and metformin on in vitro developmental potential of porcine oocytes and embryos. Biol. Reprod. 73, 1264–8.CrossRefGoogle ScholarPubMed
Mizushima, N. (2007). Autophagy: process and function. Genes Dev. 21, 2861–73.CrossRefGoogle ScholarPubMed
Mizushima, N. & Yoshimori, T. (2007). How to interpret LC3 immunoblotting. Autophagy 3, 542–5.CrossRefGoogle ScholarPubMed
Mortimore, G.E. & Poso, A.R. (1987). Intracellular protein catabolism and its control during nutrient deprivation and supply. Annu. Rev. Nutr. 7, 539–64.CrossRefGoogle ScholarPubMed
Mortimore, G.E. & Schworer, C.M. (1977). Induction of autophagy by amino-acid deprivation in perfused rat liver. Nature 270, 174–6.CrossRefGoogle ScholarPubMed
Nagashima, H., Grupen, C.G., Ashman, R.J. & Nottle, M.B. (1996). Developmental competence of in vivo and in vitro matured porcine oocytes after subzonal sperm injection. Mol. Reprod. Dev. 45, 359–63.3.0.CO;2-U>CrossRefGoogle ScholarPubMed
Noguchi, M., Yoshioka, K., Kaneko, H., Iwamura, S., Takahashi, T., Suzuki, C., Arai, S., Wada, Y. & Itoh, S. (2007). Measurement of porcine luteinizing hormone concentration in blood by time-resolved fluoroimmunoassay. J. Vet. Med. Sci. 69, 1291–4.CrossRefGoogle ScholarPubMed
Petters, R.M. & Wells, K.D. (1993). Culture of pig embryos. J. Reprod. Fertil. Suppl. 48, 61–73.Google ScholarPubMed
Quinn, P., Barros, C. & Whittingham, D.G. (1982). Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. J. Reprod. Fertil. 66, 161–8.CrossRefGoogle ScholarPubMed
Ratky, J., Rath, D. & Brussow, K.P. (2003). In vitro fertilization of in vivo matured porcine oocytes obtained from prepuberal gilts at different time intervals after hCG injection. Acta Vet. Hung. 51, 95–101.CrossRefGoogle ScholarPubMed
Shintani, T. & Klionsky, D.J. (2004). Autophagy in health and disease: a double-edged sword. Science 306, 990–5.CrossRefGoogle ScholarPubMed
Tanida, I., Minematsu-Ikeguchi, N., Ueno, T. & Kominami, E. (2005). Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy 1, 84–91.CrossRefGoogle Scholar
Tsukamoto, S., Kuma, A. & Mizushima, N. (2008). The role of autophagy during the oocyte-to-embryo transition. Autophagy 4, 1076–8.CrossRefGoogle ScholarPubMed
Wu, Y.T., Tan, H.L., Shui, G., Bauvy, C., Huang, Q., Wenk, M.R., Ong, C.N., Codogno, P. & Shen, H.M. (2010). Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. J. Biol. Chem. 285, 10850–61.CrossRefGoogle Scholar
Xie, Z. & Klionsky, D.J. (2007). Autophagosome formation: core machinery and adaptations. Nature Cell Biol. 9, 1102–9.CrossRefGoogle ScholarPubMed
Xu, Y.N., Cui, X.S., Sun, S.C., Lee, S.E., Li, Y.H., Kwon, J.S., Lee, S.H., Hwang, K.C. & Kim, N.H. (2011). Mitochondrial dysfunction influences apoptosis and autophagy in porcine parthenotes developing in vitro . J. Reprod. Dev. 57, 143–50.CrossRefGoogle ScholarPubMed
Yano, T., Mita, S., Ohmori, H., Oshima, Y., Fujimoto, Y., Ueda, R., Takada, H., Goldman, W.E., Fukase, K., Silverman, N., Yoshimori, T. & Kurata, S. (2008). Autophagic control of Listeria through intracellular innate immune recognition in Drosophila . Nat. Immunol. 9, 908–16.CrossRefGoogle ScholarPubMed
Yorimitsu, T. & Klionsky, D.J. (2005). Autophagy: molecular machinery for self-eating. Cell Death Differ. 12 (Suppl 2), 1542–52.CrossRefGoogle ScholarPubMed