Verification of autophagy in porcine oocytes and investigation of its roles

Seunghoon Lee,Seokho Kim,Hyeon Yang,Hayeon Wi,Yongjin Jo,Haeyun Jeong,Ji-Youn Kim,Sun Keun Jung,Sung June Byun 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

Autophagy is well-conserved cellular recycling process. However, autophagy in in-vitro porcine oocyte maturation have not been discovered yet. In this study, we confirmed autophagosome in-vitro culturing porcine oocytes by detection of LC3 which is promised marker protein of autophagosome. Then, we also examine the relationship between autophagy and oocyte maturation by treatment of autophagic inhibitors or nuclear maturation inhibitors. Autophagy activation was regarded to amount of autophagosome in this study. To autophagosome detection in porcine oocytes, Immunochemistry and westernblotting using LC3 antibody was conducted by culturing time of 0 h, 14 h, 28 h, and 42 h. Then, we measured LC3-II levels using western blotting after inhibiting nuclear maturation of oocytes via cAMP treatment in an in vitro culture, to clarify whether nuclear maturation affects autophagy. To clarify whether autophagy affects nuclear maturation, we also counted mature oocytes after inhibiting autophagy by treating with wortmannin or a mixture of E64d and pepstatin A. Both groups of cAMP, which were applied for different treatment times, showed the same levels of LC3-II, while their maturation rate was approximately four times higher in 22h treatment than that of the cAMP 42h treatment group. This indicated that neither cAMP nor nuclear status affected autophagy. Autophagy inhibition during in vitro maturation with wortmannin treatment decreased oocyte maturation rates by approximately half, but autophagy inhibition in the E64d/pepstatin A mixture treatment did not significantly affect the oocyte maturation rate. Thus, wortmannin itself, or the autophagy induction step, but not the degradation step, is involved in the oocyte maturation of porcine oocytes. Overall, we confirmed autophagic dynamic during porcine in-vitro maturation. Moreover, we suggest that oocyte maturation does not exist upstream of autophagy, but autophagy may exist upstream of oocyte maturation.

Effect of Wnt signaling pathway activation on the efficient generation of bovine intestinal organoids

Kang Won Park,Hyeon Yang,Hayeon Wi,Sun A Ock,Poongyeon Lee,In-Sul Hwang,Bo Ram Lee 한국동물생명공학회(구 한국동물번식학회) 2022 Journal of Animal Reproduction and Biotechnology Vol.37 No.2

Recent progress has been made to establish intestinal organoids for an in vitro model as a potential alternative to an in vivo system in animals. We previously reported a reliable method for the isolation of intestinal crypts from the small intestine and robust three-dimensional (3D) expansion of intestinal organoids (basal-out) in adult bovines. The present study aimed to establish next-generation intestinal organoids for practical applications in disease modeling-based host-pathogen interactions and feed efficiency measurements. In this study, we developed a rapid and convenient method for the efficient generation of intestinal organoids through the modulation of the Wnt signaling pathway and continuous apical-out intestinal organoids. Remarkably, the intestinal epithelium only takes 3-4 days to undergo CHIR (1 µM) treatment as a Wnt activator, which is much shorter than that required for spontaneous differentiation (7 days). Subsequently, we successfully established an apical-out bovine intestinal organoid culture system through suspension culture without Matrigel matrix, indicating an apical-out membrane on the surface. Collectively, these results demonstrate the efficient generation and next-generation of bovine intestinal organoids and will facilitate their potential use for various purposes, such as disease modeling, in the field of animal biotechnology.

Beneficial effect of EDTA for canine parthenote development during in vitro culture

Haeyun Jeong,Jin-Gu No,Whi-Cheul Lee,Hayeon Wi,Sun A Ock,Jae-Seok Woo,Seunghoon Lee 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

In vitro culture (IVC) can be used for a variety of assisted reproductive technologies. However, IVC in dog has been low efficient compared to other mammalian. It is believed that an embryo developmental block in IVC embryos is cause of low production efficiency. There is no study of embryo developmental block in dog yet. In this study, we attempted to estimate the beneficial role of EDTA in canine parthenogenic (PA) embryos development to overcome embryo developmental block. The PA embryos were divided into EDTA treated and non-treated groups. Embryo developmental efficiency was measured by activating chemically parthenote. After EDTA induction, PA embryos were evaluated for embryonic development, ROS activity, mitochondrial integrity, ATP production and genomic activation. The EDTA treated PA embryos showed significantly higher survival rate and improved cavity formation compared to non-treated. Moreover, cytoplasmic ROS level was mitigated and mitochondrial membrane potential was found significantly higher in EDTA treated group followed by higher ATP production. Furthermore, major embryonic genomic activation specific markers/factors were also elevated in EDTA treated group. Conclusively, these results indicated that EDTA showed substantially positive effect to overcome embryo developmental block in canine.

Effect of EDTA on canine parthenote development during in vitro culture

Haeyun Jeong,Minghui Zhao,Jin-Gu No,Imran Ullah,Whi-Cheul Lee,Hayeon Wi,Sun A Ock,Jae-Seok Woo,Tai-young Hur,Gi-sun Im,Jong-Gug Kim,Seunghoon Lee 한국수정란이식학회 2018 한국동물생명공학회지 Vol.33 No.3

Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for animal cloning. Until now, SCNT has been inefficient, especially in dog. It is believed that an embryo developmental block in SCNT embryos is cause of low production efficiency. However, no studies have been performed on canines for embryo developmental block. In this study, we attempted to evaluate the beneficial role of EDTA in canine parthenogenic (PA) embryos development to overcome embryo developmental block. The PA embryos were divided into 0.01 mM EDTA treated and non-treated groups. Embryo developmental efficiency was measured by activating chemically parthenote. After EDTA induction, PA embryos were evaluated for embryonic development, Reactive Oxygen Species (ROS) activity, mitochondrial integrity, ATP production and genomic activation. The EDTA treated PA embryos showed significantly higher survival rate and improved cavity formation compared to non-treated. Furthermore, cytoplasmic ROS level was mitigated and mitochondrial membrane potential was found significantly higher in EDTA treated group followed by higher ATP production. Moreover, major embryonic genomic activation specific markers/factors were also elevated in EDTA treated group. Conclusively, we elucidated that EDTA showed substantially positive effect to overcome embryo developmental block in canine.

Prediction method for obtaining matured canine oocytes by hormone analysis

Seunghoon Lee,Mi-Ryung Park,In-Sul Hwang,Min guk Lee,Keon Bong Oh,Sun A Ock,Hayeon Wi,Tai-Young Hur,Bong-Whan Choi,Sung June Byun 한국실험동물학회 2020 한국실험동물학회 학술발표대회 논문집 Vol.2020 No.7

개 parthenote in vitro culture시 EDTA 첨가에 의한 발달율 향상

정해윤,노진구,이휘철,위하연,옥선아,우제석,허태영,임기순,김종국,이승훈,Jeong, Haeyun,Zhao, Minghui,No, Jin-Gu,Ullah, Imran,Lee, Whi-Cheul,Wi, Hayeon,Ock, Sun A,Hur, Tai-young,Woo, Jae-Seok,Im, Gi-sun,Kim, Jong-Gug,Lee, Seunghoon 한국수정란이식학회 2018 한국동물생명공학회지 Vol.33 No.3

Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for animal cloning. Until now, SCNT has been inefficient, especially in dog. It is believed that an embryo developmental block in SCNT embryos is cause of low production efficiency. However, no studies have been performed on canines for embryo developmental block. In this study, we attempted to evaluate the beneficial role of EDTA in canine parthenogenic (PA) embryos development to overcome embryo developmental block. The PA embryos were divided into 0.01 mM EDTA treated and non-treated groups. Embryo developmental efficiency was measured by activating chemically parthenote. After EDTA induction, PA embryos were evaluated for embryonic development, Reactive Oxygen Species (ROS) activity, mitochondrial integrity, ATP production and genomic activation. The EDTA treated PA embryos showed significantly higher survival rate and improved cavity formation compared to non-treated. Furthermore, cytoplasmic ROS level was mitigated and mitochondrial membrane potential was found significantly higher in EDTA treated group followed by higher ATP production. Moreover, major embryonic genomic activation specific markers/factors were also elevated in EDTA treated group. Conclusively, we elucidated that EDTA showed substantially positive effect to overcome embryo developmental block in canine.