title: The regulation of autophagy in porcine blastocysts: Regulation of PARylation-mediated autophagy via mammalian target of rapamycin complex 1 (mTORC1) signaling.
abstract: Poly(ADP-ribosyl)ation (PARylation) acts as a modulator of selective autophagic degradation of ubiquitinated aggregates for cellular quality control, functioning in pro-survival role. It was reported previously that the inhibition of PARylation resulted in autophagy defects leading accumulation of ubiquitinated aggregates SQSTM1/p62 and apoptosis in porcine blastocysts. Thus, this study aims to investigate the mechanism between PARylation and autophagy in porcine blastocysts. In vitro produced (IVP) embryos were treated with 3-aminobenzamide (3ABA, poly (ADP-ribose) polymerase inhibitor) and/or rapamycin (RAPA, an mTORC1 inhibitor) during blastocyst formation. Then, these treated blastocysts were analyzed by real-time PCR, immunocytochemistry and TUNEL Assay. We found that the 3ABA treatment increased mTORC1 downstream target, phosphorylation of thr389 p70S6K (p-p70S6K-thr389), suggesting an increase in mTORC1 activity. Co-treatment with rapamycin (RAPA), mTORC1 inhibitor, restored the 3ABA-induced autophagy defects to those of the controls by normalizing mTORC1 activity. Moreover, autophagy induction, with only RAPA treatment, increased the rate of blastocyst development (70.05 ± 0.93 vs. 50.61 ± 3.49%), total cell number (58.48 ± 2.94 vs. 49.58 ± 2.43) and blastomere survival, but decreased the accumulation of SQSTM1/p62 aggregates. In summary, mTORC1 signaling is a key mechanism of PARylation-autophagy and its inhibition improved developmental ability and embryo quality by promoting selective autophagic degradation of ubiquitinated aggregates in porcine blastocysts. Therefore, these findings have significant implications for understanding the importance of autophagy regulation for successful in vitro production of porcine embryos.
authors.affiliation: Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Seoul, South Korea.
authors.forename: Hye Ran
authors.initials: HR
authors.lastname: Lee
authors.affiliation: Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Seoul, South Korea.
authors.forename: Duk Hyoun
authors.initials: DH
authors.lastname: Kim
authors.affiliation: Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Seoul, South Korea.
authors.forename: Min Gyeong
authors.initials: MG
authors.lastname: Kim
authors.affiliation: Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Seoul, South Korea.
authors.forename: Jun Sung
authors.initials: JS
authors.lastname: Lee
authors.affiliation: Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Seoul, South Korea; Korea Institute of Toxicology, 30, Baekak 1-gil, Jeongeup-si, Jeollabuk-do, South Korea.
authors.forename: Jeong Ho
authors.initials: JH
authors.lastname: Hwang
authors.affiliation: Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Seoul, South Korea. Electronic address: htl3675@konkuk.ac.kr.
authors.forename: Hoon Taek
authors.initials: HT
authors.lastname: Lee
mesh_terms: D000818:Animals; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D001549:Benzamides / Q000494:pharmacology; D001755:Blastocyst / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology*; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology; D013552:Swine; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D001549:Benzamides; D046912:Multiprotein Complexes; C025160:3-aminobenzamide; D011065:Poly(ADP-ribose) Polymerases; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus
keywords: Autophagy; Blastocyst; PARylation; Pig; Poly(ADP-ribosyl)ation; mTOR