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.

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.

Sensitivity of time‐resolved diffraction data to changes in internuclear distances and atomic positions

Jeong Haeyun,Ki Hosung,Kim Jong Goo,Kim Jungmin,Lee Yunbeom,Ihee Hyotcherl 대한화학회 2022 Bulletin of the Korean Chemical Society Vol.43 No.3

Time-resolved x-ray liquidography (TRXL) is a powerful technique to study molecular structural dynamics in the solution phase. Typically, a TRXL experiment is conducted during limited beamtime at a beamline of a synchrotron or an x-ray free-electron laser, demanding a proper design and careful planning. In this regard, the optimal q range needs to be determined to find the optimal x-ray energy and sample-to-detector distance. For such purpose, here, we present effective ways to quantify the sensitivity of the TRXL data as a function of q to various factors such as the atomic positions, internuclear distances, solvent cage, and bulk solvent. The developed approaches are also applicable to other types of time-resolved diffraction, such as ultrafast electron diffraction.

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.

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.