미세주입된 소 수정란에서 유전자적중 효율 및 배반포 품질 확인

김민지,박효진,김진우,김세은,박다솜,양슬기,김인수,제갈호근,강만종,김소섭,박정준,구덕본 강원대학교 동물생명과학연구소(구 강원대학교 동물자원공동연구소) 2018 동물자원연구 Vol.29 No.2

This study aimed to produce high-quality blastocysts and establish appropriate microinjection conditions for the introduction of target gene. First, we identified embryo development to the blastocyst stage after microinjection using the CRISPR/Cas9 system on the Cas9 protein or mRNA. As a result, we confirmed that blastocyst development in the Cas9 mRNA injected group significantly increased when compared to the Cas9 protein injected group (p<0.05). However, the blastocyst gene targeting rate increased in the Cas9 protein injected group when compared to the Cas9 mRNA injected group (p<0.05). Next, we treated the injection medium with 10 µg/ml of cytochalasin B (CB), and the microinjected embryos were cultured in CR1-aa medium supplemented with 0.1 µM of melatonin (Mela). Consequently, the blastocyst formation rate significantly increased in the CB treated group (p<0.05). After microinjecting embryos with the CB treated injection medium, we investigated blastocyst formation and quality via Mela treatment. Consequently, the Mela treated group demonstrated significantly increased blastocyst formation rates when compared to the non-treated group (p<0.05). Furthermore, immunofluorescence assay using RAD51 (DNA repair detection protein) and H2AX139ph (DNA damage detection protein) showed an increase in RAD51 positive cells in Mela treated embryos. Therefore, we verified the improvement in knock-in efficiency in microinjected bovine embryos using Cas9 protein. These results also demonstrated that the positive effect of the CB and Mela treatments improved the embryonic developmental competence and blastocyst qualities in genetically-edited bovine embryos.

Cytochalasin B 첨가 용액에서 미세주입된 돼지 수정란의 착상전 발달과 품질에서 melatonin의 효과 : Effects of melatonin on pre-implantation embryonic development and quality of microinjected porcine embryos under the cytochalasin B containing solution

김민지 ․ 구덕본 대구대학교 산업기술연구소 2018 産業技術硏究 Vol.29 No.2

Production of transgenic animals are being used in various fields such as commercial application, human diseases model and drug discovery. One of these animals production method of transgenic animal is direct transfer by pronuclear microinjection. However, the production efficiency of transgenic animals using the pronuclear microinjection is remarkably low. In addition, appropriate conditions such as improving the embryo developmental competence and high quality of blastocysts for the production of transgenic embryos are being required, but studies for these process have not been reported. Therefore, this study investigated the embryonic developmental competence and quality of blastocysts after pronuclear microinjection by using cytochalasin B (CB) or/and melatonin. Cytochalasin B (CB) is important to play role in microinjection by making smoother of the zygote cytoplasm, as well as melatonin is known to improve the embryonic developmental competence and embryos quality. First, we performed the microinjection using injection medium with 10 µg/ml CB and cultured the microinjected zygotes in 0.1 µM melatonin treated within culture medium. Next, we confirmed the DNA damage of microinjected blastocysts using immunofluorescence staining with RAD51 (DNA repairs detection protein) and H2AX139ph (DNA damage detection protein). CB treated group significantly increased blastocyst development rate compared with CB non-treated group (P < 0.05; 25.0 ± 7.2% vs. 17.0 ± 1.3%). After microinjection under the CB treatment, blastocyst development rate and formation of expanded blastocysts were higher in melatonin treated group than those of melatonin non-treated group (29.7 ± 8.3% vs. 24.4 ± 7.7%). In addition, melatonin treated group increased the numbers of RAD51 positive cells compared with non-treated group (P < 0.05). Taken together, these results suggest that CB and melatonin treatment have positive effects for improving the production efficiency of transgenic embryos.

Mito-TEMPO에 의한 미토콘드리아 유래 초과산화물의 감소가 돼지 난모세포 성숙에 미치는 영향

양슬기,박효진,이상민,김진우,김민지,김인수,제갈호근,구덕본 한국동물생명공학회(구 한국동물번식학회) 2019 Journal of Animal Reproduction and Biotechnology Vol.34 No.1

Morphology of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage as one of the evaluation criteria for oocyte maturation quality after in vitro maturation (IVM) plays important roles on the meiotic maturation, fertilization and early embryonic development in pigs. When cumulus cells of COCs are insufficient, which is induced the low oocyte maturation rate by the increasing of reactive oxygen species (ROS) in porcine oocyte during IVM. The ROS are known to generate including superoxide and hydrogen peroxide from electron transport system of mitochondria during oocyte maturation in pigs. To regulate the ROS production, the cumulus cells is secreted the various antioxidant enzymes during IVM of porcine oocyte. Our previous study showed that Mito-TEMPO, superoxide specific scavenger, improves the embryonic developmental competence and blastocyst formation rate by regulating of mitochondria functions in pigs. However, the effects of Mito-TEMPO as a superoxide scavenger to help the anti-oxidant functions from cumulus cells of COCs on meiotic maturation during porcine oocyte IVM has not been reported. Here, we categorized experimental groups into two groups (Grade 1: G1; high cumulus cells and Grade 2: G2; low cumulus cells) by using hemocytometer. The meiotic maturation rate from G2 was significantly (p < 0.05) decreased (G1: 79.9 ± 3.8% vs G2: 57.5 ± 4.6%) compared to G1. To investigate the production of mitochondria derived superoxide, we used the mitochondrial superoxide dye, Mito-SOX. Red fluorescence of Mito-SOX detected superoxide was significantly (p < 0.05) increased in COCs of G2 compared with G1. And, we examined expression levels of genes associated with mitochondrial antioxidant such as SOD1, SOD2 and PRDX3 using a RT-PCR in porcine COCs at 44 h of IVM. The mRNA levels of three antioxidant enzymes expression in COCs from G2 were significantly (p < 0.05) lower than COCs of G1. In addition, we investigated the anti-oxidative effects of Mito-TEMPO on meiotic maturation of porcine oocyte from G1 and G2. Meiotic maturation and mRNA levels of antioxidant enzymes were significantly (p < 0.05) recovered in G2 by Mito-TEMPO (0.1 μM, MT) treatment (G2: 68.4 ± 3.2% vs G2 + MT: 73.9 ± 1.4%). Therefore, our results suggest that reduction of mitochondria derived superoxide by Mito-TEMPO may improves the meiotic maturation in IVM of porcine oocyte.

한우 번식우 사양관리 형태 변화에 따른 혈중 MPT와 인공수정 수태율과의 비교 분석

박정준,유한준,최하영,김민지,신종서,구덕본 한국동물생명공학회(구 한국동물번식학회) 2018 발생공학 국제심포지엄 및 학술대회 Vol.2018 No.06

본 연구는 사양관리 변경에 따른 혈중 주요성분 분석과 수태율과의 상관관계를 분석하고 자 수행되었다. 연구자가 관리하고 있는 횡성 관내의 1 농가를 대상으로 한우 암소 사양관 리 변화와 인공수정 수태율을 조사하면서 얻은 자료를 동일기간 내 번식우의 혈액으로터 분석한 혈중 MPT 결과와 비교분석하였다. 분석에 이용된 농가는 연구자가 5년 이상 인공 수정 업무와 사양관리 컨설팅을 수행하고 있는 농가로 최근 3년간 평균 이상의 인공수정 수태율을 나타내었지만 2017년 중반기 사양관리 형태의 변경으로 인한 급격한 인공수정 수태율 저하를 겪은 농가이다. 조사기간은 2017년 1월부터 2017년 11월까지이며 최근 3년 간 동일한 사양관리 형태를 유지하였던 2017년 5월까지의 기간 중 2016년부터 2017년 5 월까지의 조사결과를 대조군(67두)으로 사용하였으며, 사양관리 형태를 변경하였던 2017년 6월부터 2017년 11월까지의 결과를 실험군(25두)으로 사용하였다. 조사항목으로 수태율 관 련 인자는 평균 인공수정횟수, 평균 공태기간, 평균 발정주기, 인공수정 수태율 자료를 조 사하였고 혈중 MPT 분석은 AST, GGT, Glucose, Cholesterol, BUN, NEFA 항목을 조사하 였으며 급여되는 사료의 섭취량 또한 조사하였다. 실험결과, 사양관리는 대조군 농후사료 2~3 kg+건초 무제한+사일리지 2~3 kg/일, 실험군 농후사료 3 kg+오차드글라스 3 kg+혼 합건초 3 kg/일의 형태로 이루어졌다. 대조군과 실험군의 혈중 MPT 분석결과 NEFA 160.9±12 및 75.3±39 uEq/L, Glucose 60.1±8.2 및 89.4±10.7 mg/dL, Cholesterol 151.6± 21.1 및 101.9±21.2 mg/dL, BUN 12.8±3.5 및 10.0±6.2 mg/dL, AST 69.6±24.0 및 86.2±12.5 U/L, GGT 19.6±8.0 및 17.7±7.1 U/L로 확인되었다. 또한 건물, TDN 및 단백질 섭취량은 건물 6.87 및 8.05 kg, TDN 3.83 및 5.59 kg, 단백질 0.67 및 0.9 kg으로 조사되 었다. 인공수정 수태율 조사결과 대조군과 실험군에서 87.5±7.9 및 75.1±8.4로 조사되었으 며 수태율 관련인자는 평균인공수정횟수 1.2±0.5 및 2.5±0.4, 평균공태기간(일) 71±8.4 및 90.3±6.5, 평균발정주기(일) 21.4±0.5 및 22.5±0.6으로 조사되었다. 결론적으로 사양관리 형 태의 변경으로 인한 수태율 저하의 원인을 탐색하고자 본 연구가 수행되었으며 사료섭취에 따른 혈중 MPT 분석결과, NEFA의 농도에서 큰 변화가 관찰되었다. 본 연구에서 분석한 농가의 사양관리 형태 변화는 건초의 급여방법이 바뀐 것이 주요한 점으로써, 양질의 조사 료 공급원을 다양하게 유지하여 사양관리의 효율을 높이고자 현장의 여러 농가에서 최근 적용되고 있는 방법이다. 하지만 조사료의 선택과 급여량은 검증된 방법으로 고려되어야 한다. 본 연구결과는 실험두수가 부족하므로 추가적으로 다양한 농가 사양관리 형태의 조 건에서 동일한 실험결과와의 비교분석이 필요하다고 생각된다.

Mito-TEMPO에 의한 미토콘드리아 유래 초과산화물의 감소가 돼지 난모세포 성숙에 미치는 영향

양슬기,박효진,이상민,김진우,김민지,김인수,제갈호근,구덕본 사단법인 한국동물생명공학회 2019 한국동물생명공학회지 Vol.34 No.1

Morphology of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage as one of the evaluation criteria for oocyte maturation quality after in vitro maturation (IVM) plays important roles on the meiotic maturation, fertilization and early embryonic development in pigs. When cumulus cells of COCs are insufficient, which is induced the low oocyte maturation rate by the increasing of reactive oxygen species (ROS) in porcine oocyte during IVM. The ROS are known to generate including superoxide and hydrogen peroxide from electron transport system of mitochondria during oocyte maturation in pigs. To regulate the ROS production, the cumulus cells is secreted the various antioxidant enzymes during IVM of porcine oocyte. Our previous study showed that Mito-TEMPO, superoxide specific scavenger, improves the embryonic developmental competence and blastocyst formation rate by regulating of mitochondria functions in pigs. However, the effects of Mito-TEMPO as a superoxide scavenger to help the anti-oxidant functions from cumulus cells of COCs on meiotic maturation during porcine oocyte IVM has not been reported. Here, we categorized experimental groups into two groups (Grade 1: G1; high cumulus cells and Grade 2: G2; low cumulus cells) by using hemocytometer. The meiotic maturation rate from G2 was significantly (p < 0.05) decreased (G1: 79.9 ± 3.8% vs G2: 57.5 ± 4.6%) compared to G1. To investigate the production of mitochondria derived superoxide, we used the mitochondrial superoxide dye, Mito-SOX. Red fluorescence of Mito-SOX detected superoxide was significantly (p < 0.05) increased in COCs of G2 compared with G1. And, we examined expression levels of genes associated with mitochondrial antioxidant such as SOD1, SOD2 and PRDX3 using a RT-PCR in porcine COCs at 44 h of IVM. The mRNA levels of three antioxidant enzymes expression in COCs from G2 were significantly (p < 0.05) lower than COCs of G1. In addition, we investigated the anti-oxidative effects of Mito-TEMPO on meiotic maturation of porcine oocyte from G1 and G2. Meiotic maturation and mRNA levels of antioxidant enzymes were significantly (p < 0.05) recovered in G2 by Mito-TEMPO (0.1 μM, MT) treatment (G2: 68.4 ± 3.2% vs G2 + MT: 73.9 ± 1.4%). Therefore, our results suggest that reduction of mitochondria derived superoxide by Mito-TEMPO may improves the meiotic maturation in IVM of porcine oocyte.

Correlation between Sestrin-2 and PERK Signaling in Matured Porcine Oocytes according to ER-stress during In Vitro Maturation

박효진,김인수,김진우,양슬기,김민지,구덕본 사단법인 한국동물생명공학회 2019 한국동물생명공학회지 Vol.34 No.3

Sestrin-2 (SESN2) as a stress-metabolic protein is known for its anti-oxidative effects as a downstream factor of PERK pathways in mammalian cells. However, the expression patterns of SESN2 in conjunction with the UPR signaling against to ER stress on porcine oocyte maturation in vitro, have not been reported. Therefore, we confirmed the expression pattern of SESN2 protein, for which to examine the relationship between PERK signaling and SESN2 in porcine oocyte during IVM. We investigated the SESN2 expression patterns using Western blot analysis in denuded oocytes (DOs), cumulus cells (CCs), and cumulus-oocyte complexes (COCs) at 22 and 44 h of IVM. As expected, the SESN2 protein level significantly increased (p < 0.01) in porcine COCs during 44 h of IVM. We investigated the meiotic maturation after applying ER stress inhibitor in various concentration (50, 100 and 200 μM) of tauroursodeoxycholic acid (TUDCA). We confirmed significant increase (p < 0.05) of meiotic maturation rate in TUDCA 200 μM treated COCs for 44 h of IVM. Finally, we confirmed the protein level of SESN2 and meiotic maturation via regulating ER-stress by only tunicamycin (Tm), only TUDCA, and Tm + TUDCA treatment in porcine COCs. As a result, treatment of the TUDCA following Tm pre-treatment reduced SESN2 protein level in porcine COCs. In addition, SESN2 protein level significantly reduced in only TUDCA treated porcine COCs. Our results suggest that the SESN2 expression is related to the stress mediator response to ER stress through the PERK signaling pathways in porcine oocyte maturation.

Reversible Effects of Exogenous GM3 on Meiotic Maturation and Cumulus Cells Expansion of Porcine Cumulus-oocyte Complexes

김진우,박효진,정재민,양슬기,김민지,김인수,제갈호근,구덕본 사단법인 한국동물생명공학회 2018 한국동물생명공학회지 Vol.33 No.4

Ganglioside GM3 is known as an inhibition factor of cell differentiation and proliferation via inhibition of epidermal growth factor receptor (EGFR) phosphorylation. Our previous study showed that the exogenous ganglioside GM3 reduced the meiotic maturation of porcine oocytes and induced apoptosis at 44 h of in vitro maturation (IVM). However, the role of ganglioside GM3 in the relationship between EGFR signaling and apoptosis during porcine oocyte maturation has not yet been studied. First, porcine cumulus-oocyte complexes (COCs) were cultured in the NCSU-23 medium with exogenous ganglioside GM3 according to maturation periods (non-treated, only IVM I: 0 - 22 h, only IVM II: 22 - 44 h and IVM I & II: 0 - 44 h). We confirmed that the proportion of germinal vesicle breakdown (GVBD) increased significantly in the IVM I treated group than in the control group. We also confirmed that the meiotic maturation until M II stage and polar body formation decreased significantly in the only IVM I treated group. Cumulus cell expansion and mRNA levels of the expansion-related factors (HAS2, TNFAIP6 and PTX3) decreased significantly in the IVM I treated group than in the control group. Protein levels of EGFR, p-EGFR, ERK1/2, and p-ERK1/2 decreased significantly in the GM3-treated groups, during the IVM I period. In addition, cellular apoptosis, determined using TUNEL assay, and protein levels of Cleaved caspase 3, were increased significantly in the GM3-treated COCs during the IVM I period. Based on these results, ganglioside GM3 exposure of porcine COCs during the IVM I period reduced meiotic maturation and cumulus cell expansion via inhibition of EGFR activity in pigs.

Regulation of the Endoplasmic Reticulum Stress by BIP/GRP78 is involved in Meiotic Maturation of Porcine Oocytes In Vitro

박효진,박재영,김진우,양슬기,정재민,김민지,박정준,구덕본 한국발생생물학회 2017 발생과 생식 Vol.21 No.4

In the present study, we investigated the role of binding immunoglobulin protein/glucose-regulated protein, 78-kDa (BIP/GRP78)-regulated endoplasmic reticulum (ER)-stress on meiotic maturation and cumulus cells expansion in porcine cumulus-oocyte complexes (COCs). Previously, it has been demonstrated that unfolded protein response (UPR)-related genes, such as molecules involved in ER-stress defense mechanisms, were expressed in matured oocytes and cumulus cells during in vitro maturation (IVM) of porcine oocytes. However, BIP/GRP78-mediated regulation of ER stress in porcine oocytes has not been reported. Firstly, we observed the effects of knockdown of BIP/GRP78 (an UPR initiation marker) using porcine-specific siRNAs (#909, #693, and #1570) on oocyte maturation. Among all siRNAs, siRNA #693 significantly re-duced the protein levels of UPR marker proteins (BIP/GRP78, ATF4, and P90ATF6) in porcine COCs observed by Western blotting and immunofluorescence analysis. We also observed that the reduction of BIP/GRP78 levels by siRNA#693 signifi-cantly inhibited the meiotic maturation of oocytes (siRNA #693: 32.5±10.1% vs control: 77.8±5.3%). In addition, we also checked the effect of ER-stress inhibitors, tauroursodeoxycholic acid (TUDCA, 200 μM) and melatonin (0.1 μM), in BIP/ GRP78-knockdown oocytes. TUDCA and melatonin treatment could restore the expression levels of ER-stress marker pro-teins (BIP/GRP78, p-eIF2α, eIF2α, ATF4, and P90ATF6) in siRNA #693-transfected matured COCs. In conclusion, these re-sults demonstrated that BIP/GRP78-mediated regulation of UPR signaling and ER stress plays an important role in in vitro maturation of porcine oocytes.

Ganglioside GD1a Activates the Phosphorylation of EGFR in Porcine Oocytes Maturation in vitro

박효진,김진우,박재영,양슬기,정재민,김민지,구덕본 사단법인 한국동물생명공학회 2017 한국동물생명공학회지 Vol.32 No.1

Ganglioside GD1a is specifically formed by the addition of sialic acid to ganglioside GM1a by ST3 - galactoside  -2,3-sialyltransferase 2 (ST3GAL2). Above all, GD1a are known to be related with the functional regulation of several growth factor receptors, including activation and dimerization of epidermal growth factor receptor (EGFR) in tumor cells. The activity of EGF and EGFR is known to be a very important factor for meiotic and cytoplasmic maturation during in vitro maturation (IVM) of mammalian oocytes. However, the role of gangliosides GD1a for EGFR-related signaling pathways in porcine oocyte is not yet clearly understood. Here, we investigated that the effect of ST3GAL2 as synthesizing enzyme GD1a for EGFR activation and phosphorylation during meiotic maturation. To investigate the expression of ST3GAL2 according to the EGF treatment (0, 10 and 50 ng/ml), we observed the patterns of ST3GAL2 genes expression by immunofluorescence staining in denuded oocyte (DO) and cumulus cell-oocyte-complex (COC) during IVM process (22 and 44 h), respectively. Expression levels of ST3GAL2 significantly decreased (p<0.01) in an EGF concentration (10 and 50 ng/ml) dependent manner. And fluorescence expression of ST3GAL2 increased (p<0.01) in the matured COCs for 44 h. Under high EGF concentration (50 ng/ml), ST3GAL2 protein levels was decreased (p<0.01), and their shown opposite expression pattern of phosphorylation-EGFR in COCs of 44 h. Phosphorylation of EGFR significantly increased (p<0.01) in matured COCs treated with GD1a for 44 h. In addition, ST3GAL2 protein levels significantly decreased (p<0.01) in GD1a (10 μM) treated COCs without reference to EGF pre-treatment. These results suggest that treatment of exogenous ganglioside GD1a may play an important role such as EGF in EGFR-related activation and phosphorylation in porcine oocyte maturation of in vitro.