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      Vitrification, in vitro fertilization, and development of Atg7 deficient mouse oocytes

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          Autophagy contributes to the clearance and recycling of macromolecules and organelles in response to stress. We previously reported that vitrified mouse oocytes show acute increases in autophagy during warming. Herein, we investigate the potential role of Atg7 in oocyte vitrification by using an oocyte-specific deletion model of the Atg7 gene, a crucial upstream gene in the autophagic pathway.


          Oocyte-specific Atg7 deficient mice were generated by crossing Atg7 floxed mice and Zp3-Cre transgenic mice. The oocytes were vitrified-warmed and then subjected to in vitro fertilization and development. The rates of survival, fertilization, and development were assessed in the Atg7 deficient oocytes in comparison with the wildtype oocytes. Light chain 3 (LC3) immunofluorescence staining was performed to determine whether this method effectively evaluates the autophagy status of oocytes.


          The survival rate of vitrified-warmed Atg7 f/f ;Zp3-Cre ( Atg7 d/d ) metaphase II (MII) oocytes was not significantly different from that of the wildtype ( Atg7 f/f ) oocytes. Fertilization and development in the Atg7 d/d oocytes were significantly lower than the Atg7 f/f oocytes, comparable to the Atg5 d/d oocytes previously described. Notably, the developmental rate improved slightly in vitrified-warmed Atg7 d/d MII oocytes when compared to fresh Atg7 d/d oocytes. LC3 immunofluorescence staining showed that this method can be reliably used to assess autophagic activation in oocytes.


          We confirmed that the LC3-positive signal is nearly absent in Atg7 d/d oocytes. While autophagy is induced during the warming process after vitrification of MII oocytes, the Atg7 gene is not essential for survival of vitrified-warmed oocytes. Thus, induction of autophagy during warming of vitrified MII oocytes seems to be a natural response to manage cold or other cellular stresses.

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          Guidelines for the use and interpretation of assays for monitoring autophagy.

          In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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            Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice

            Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.
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              In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker.

              Macroautophagy mediates the bulk degradation of cytoplasmic components. It accounts for the degradation of most long-lived proteins: cytoplasmic constituents, including organelles, are sequestered into autophagosomes, which subsequently fuse with lysosomes, where degradation occurs. Although the possible involvement of autophagy in homeostasis, development, cell death, and pathogenesis has been repeatedly pointed out, systematic in vivo analysis has not been performed in mammals, mainly because of a limitation of monitoring methods. To understand where and when autophagy occurs in vivo, we have generated transgenic mice systemically expressing GFP fused to LC3, which is a mammalian homologue of yeast Atg8 (Aut7/Apg8) and serves as a marker protein for autophagosomes. Fluorescence microscopic analyses revealed that autophagy is differently induced by nutrient starvation in most tissues. In some tissues, autophagy even occurs actively without starvation treatments. Our results suggest that the regulation of autophagy is organ dependent and the role of autophagy is not restricted to the starvation response. This transgenic mouse model is a useful tool to study mammalian autophagy.

                Author and article information

                [1 ]Department of Biomedical Science and Technology, Institute of Biomedical Science and Technology, Konkuk University, Seoul, Korea.
                [2 ]Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam, Korea.
                [3 ]Department of Veterinary Medicine, Konkuk University, Seoul, Korea.
                Author notes
                Corresponding author: Hyunjung Jade Lim. Department of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea. Tel: +82-2-450-4087, Fax: +82-2-2030-7809, hlim@

                *These authors contributed equally to this study.

                Clin Exp Reprod Med
                Clin Exp Reprod Med
                Clinical and Experimental Reproductive Medicine
                The Korean Society for Reproductive Medicine
                March 2016
                31 March 2016
                : 43
                : 1
                : 9-14
                Copyright © 2016. The Korean Society for Reproductive Medicine

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Funded by: Ministry of Health and Welfare, CrossRef;
                Award ID: HI12C0055
                Original Article

                Obstetrics & Gynecology

                vitrification, atg7, autophagy, oocytes


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