• Clinical and Experimental Reproductive Medicine
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• 제44권1호
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• pp.40-46
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• 2017

Objective: To describe in vitro development of human embryos derived from an individual with a homozygous pathogenic variant in NLRP7 (19q13.42) and recurrent hydatidiform mole (HM), an autosomal recessive condition thought to occur secondary to an oocyte defect. Methods: A patient with five consecutive HM pregnancies was genomically evaluated via next generation sequencing followed by controlled ovarian hyperstimulation, in vitro fertilization (IVF) with intracytoplasmic sperm injection, embryo culture, and preimplantation genetic screening. Findings in NLRP7 were recorded and embryo culture and biopsy data were tabulated as a function of parental origin for any identified ploidy error. Results: The patient was found to have a pathogenic variant in NLRP7 (c.2810+2T>G) in a homozygous state. Fifteen oocytes were retrieved and 10 embryos were available after fertilization via intracytoplasmic sperm injection. Developmental arrest was noted for all 10 embryos after 144 hours in culture, thus no transfer was possible. These non-viable embryos were evaluated by karyomapping and all were diploid biparental; two were euploid and eight had various aneuploidies all of maternal origin. Conclusion: This is the first report of early human embryo development from a patient with any NLRP7 mutation. The pathogenic variant identified here resulted in global developmental arrest at or before blastocyst stage. Standard IVF should therefore be discouraged for such patients, who instead need to consider oocyte (or embryo) donation with IVF as preferred clinical methods to treat infertility.

• 한국가축번식학회지
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• 제23권4호
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• pp.281-291
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• 1999

본 실험은 체외 생산된 한우 배반포기배에 적합한 동결 / 융해 방법을 찾고자 실시하였다. 체외배양 7일째에 생산된 배반포기배는 동해제 EFS40(40% ethylene glycol, 18% ficoll, 0.3 M sucrose 그리고 10% FBS가 첨가된 m-DPBS)과 embryo container인 EM grid (V-G) 또는 straw(V-S)를 이용해서 초자화 동결하였다. 동결과 융해는 두 방법 모두 2 단계로 실시하였으며, 처리시간은 V-G 방법이 2 분과 3분, V-S 방법이 3.5분과 10분 각각 소요되었다. 체외 생존능 평가는 융해 후 24시간째의 재팽창율과 48 시간째의 부화율로 조사하였다. 본 실험에서 얻어진 결과는 다음과 같다. 팽창 배반포기배를 이용하여 동결액 노출과 동결과정의 냉해가 배의 생존에 미치는 영향을 조사하였던 바, 융해 후 24시간째, 동결액 노출군 (100.0%)의 결과는 대조군 (100.0%)과 차이가 없었으며, 두 동결군 (V-G: 87.8%, V-S: 77.8%)의 생존율과 비교해 볼 때 유의하게 높았다 (P＜0.00l). 그러나, 융해 후 48시간째 각 처리군의 부화율을 조사하였던 바, V-G 군 (67.8%)은 V-S 군 (53.3%)보다 유의하게 높게 나타났으며 (P＜0.05), 동결액 노출군 (73.3%)과도 유의한 차이를 나타내지 않았다. 또한, 배발달단계 (초기, 팽창, 부화초기 배반포)와 동결에 사용된 embryo container(EM grid, straw)가 체외 생존율에 미치는 영향을 동시에 비교하였던 바, embryo container 에 상관없이 빠르게 발달된 배반포기배가 느리게 발달하는 난자군보다 유의하게 높은 생존율을 나타내었다 (초기 : 57.1, 24.4%; 팽창 : 84.7, 60.6%; 부화초기 : 91.7, 80.0%)(P＜0.001). 특히, 팽창 배반포기배와 부화초기 배반포기배에서, 융해 후 48시간째, V-G군(67.8, 95.0%)의 부화율이 V-S군(53.0, 65.0%)보다 유의하게 높게 나타나 동결시 EM grid 의 유용성을 확인할 수 있었다 (P＜0.05, P＜0.001). 따라서, 한우 배반포기배는 EM grid를 사용하는 초자화 동결방법으로 간편하면서도 효율적이고 성공적으로 동결보존 할 수 있다는 것을 알았다.

• 한국수정란이식학회지
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• 제30권4호
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• pp.319-325
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• 2015

Gangliosides exist in glycosphingolipid-enriched domains on the cell membrane and regulate various functions such as adhesion, differentiation, and receptor signaling. Ganglioside GM3 by ST3GAL5 enzyme provides an essential function in the biosynthesis of more complex ganglio-series gangliosides. However, the role of gangliosides GM3 in porcine oocytes during in vitro maturation and early embryo development stage has not yet understood clear. Therefore, we examined ganglioside GM3 expression patterns under apoptosis stress during maturation and preimplantation development of porcine oocytes and embryos. First, porcine oocytes cultured in the NCSU-23 medium for 44 h after $H_2O_2$ treated groups (0.01, 0.1, 1 mM). After completion of meiotic maturation, the proportion MII (44 h) was significantly different among control and the H2O2 treated groups ($76.8{\pm}0.3$ vs $69.1{\pm}0.4$; 0.01 mM, $55.7{\pm}1.0$; 0.1 mM, $38.2{\pm}1.6%$; 1 mM, P<0.05). The expressions of ST3GAL5 in $H_2O_2$ treated groups were gradually decreased compared with control group. Next, changes of ST3GAL5 expression patterns were detected by using immunofluorescene (IF) staining during preimplantation development until blastocyst. As a result, we confirmed that the expressions of ST3GAL5 in cleaving embryos were gradually decreased (P<0.05) according to the early embryo development progress. Based on these results, we suggest that the ganglioside GM3 was used to the marker as pro-apoptotic factor in porcine oocyte of maturation and early embryo production in vitro, respectively. Furthermore, our findings will be helpful for better understanding the basic mechanism of gangliosides GM3 regulating in oocyte maturation and early embryonic development of porcine in vitro.

• BMB Reports
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• 제49권10호
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• pp.572-577
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• 2016

The short gastrulation (sog) shadow enhancer directs early and late sog expression in the neurogenic ectoderm and the ventral midline of the developing Drosophila embryo, respectively. Here, evidence is presented that the sog primary enhancer also has both activities, with the late enhancer activity dependent on the early activity. Computational analyses showed that the sog primary enhancer contains five Dorsal (Dl)-, four Zelda (Zld)-, three Bicoid (Bcd)-, and no Single-minded (Sim)-binding sites. In contrast to many ventral midline enhancers, the primary enhancer can direct lacZ expression in the ventral midline as well as in the neurogenic ectoderm without a canonical Simbinding site. Intriguingly, the impaired transcriptional synergy between Dl and either Zld or Bcd led to aberrant and abolished lacZ expression in the neurogenic ectoderm and in the ventral midline, respectively. These findings suggest that the two enhancer activities of the sog primary enhancer are functionally consolidated and geographically inseparable.

• 한국임상수의학회지
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• 제16권1호
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• pp.128-137
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• 1999

Plasma progesterone (P$_4$) concentrations were measured for confirming the estrus observation and for the early pregnancy diagnosis in 130 cows of small farmers. Ultrasonographic examinations were performed from day 30 after artificial insemination to establish the characteristic ultrasonographic appearances of gestational structures in each pregnant stages. Of the 130 cows inseminated, 111 cows (85.4%) were an ovulatory estrus, 12 cows (9.2%) were an unovulatory estrus, and 7 cows (5.4%) were the error of estrus detection, respectively. The accuracy for early pregnancy diagnosis in 111 ovulatory estrus cows achieved when the discriminatory concentration at day 21 after artificial insemination was placed at 3.0 ng-/ml in plasma, was 86.7 % for positive diagnosis and 100% for negative diagnosis, respectively. Pregnancy diagnosis by ultrasonography were performed to evaluate gestational structures from day 30 after artificial insemination in 83 cows. Pregnant cows were 72 of 83 cows. The characteristic ultrasonography of gestational structures in each gestational stages was as follows. The embryo proper was observed within anechoic fetal fluid between 28 and 40 days after insemination, and amnion and embryonic heartbeat was also detected in this period. Between days 41 and 50, embryo proper was detected as an discriminated from head and body, and forelimb buds and hindlimb buds were also observed in this period. Between days 51 and 60, an embryo proper was clearly discriminated from head and body, and fetal movement, forelimb buds and hindlimb buds were observed in this period. Between days 61 and 70, fetus was completely developed, and fetal skeleton, organs and cotyledon were observed. After day 71, each organs of fetus were rapidly developed and a fetus was partially observed in screen because fetus was too big and larger, These results indicate that plasma P$_4$ determination at days 0,6 and 21 after artificial insemination can be utilized for confirming the estrus observation and for early pregnancy diagnosis. Also, ultrasonography was reliable method for early pregnancy diagnosis at day 30 after artificial insemination.

• 한국발생생물학회지:발생과생식
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• 제23권1호
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• pp.63-72
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• 2019

The head gap gene buttonhead (btd) is required for the patterning of head segments in the early Drosophila embryo. Mutant phenotypes of btd display a gap-like phenotype in which antennal, intercalary, mandibular and the anterior portion of the maxillary segments are eliminated. In agreement with the phenotypes, btd is expressed in a stripe covering the head segments at the blastoderm stage. During the early phase of the germband extension, btd is expressed in stripes with single segmental periodicity, which is required for the formation of the peripheral nervous system. In contrast to the key role of btd in Drosophila embryonic development, it has been suggested that Tribolium ortholog of btd (Tc-btd) is dispensable for embryonic head development. In order for better understanding of the requirement of Tc-btd in the early Tribolium embryo, we re-analyzed the expression patterns and functions of Tc-btd during embryonic segmentation. Tc-btd is expressed in segmental stripes at the stages of blastoderm and germband elongation. Up to 28.3% of embryos in which Tc-btd is knocked down displays the loss of antennal, mandibular and the pregnathal regions in the head, with abdominal segments being disrupted in the trunk. Our findings suggest that Tc-btd is required for the head and trunk development in the early Tribolium embryo.

• 대한수의학회지
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• 제15권2호
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• pp.177-180
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• 1975

This study was undertaken to obtain more detailed knowledge of the oxygen consumption rate of chick embryos, of the relationship between the increasing rate of embryo weight and that of oxygen consumption, of the daily increase of oxygen consumption by that of embryo weight, and of the metabolic rate of the White Leghorn eggs. The results obtained are summarized as follows. 1. The average oxygen consumption rate of the chicken embryo at the 3rd day of incubation is 0.8ml/h, STPD. It is strongly suggested that this value can used as a physiological criterion to classify the fertilized and unfertilized eggs, on the other hand oxygen consumption rate of the fertilized eggs reaches a plateau between the 18th and 20th days. 2. There exists a parallel relationship between the increasing rate of embryo weight and that of oxygen consumption rate during the incubation period. 3. There are not exist a parallel relationship between the daily increase of embryo weight and that of oxygen consumption during the whole incubation period. 4. The metabolic rate of chicken embryo(ml/h/g) is highest in the early stage of incubation and it started to decrease sharply until the 8th day follow by slow decrease thereafter.

• 한국발생생물학회지:발생과생식
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• 제7권2호
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• pp.95-103
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• 2003

세포내 칼슘은 다양한 세포에서 중요한 생리적 반응을 일으키며, ruthenium red와 ryanodine은 중요한 칼슘 조절자로 작용한다. Ruthenium red는 세포내 칼슘 저장고에서의 calcium induced calcium release(CICR)를 저해한다. Ryanodine은 ryanodine 통로를 통한 칼슘 방출을 촉진한다. 본 실험은 두 조절자가 생쥐 난자와 초기배아의 세포내 칼슘이온 농도에 영향을 미치는지 여부와 그 유효농도를 알아보고자 수행하였다 난자 및 초기배아내 칼슘이온 함량 변화는 Fluo-3/AM을 이용하여 공초점 레이저주사 현미경을 사용하여 실시간으로 측정하였다. Ruthenium red는 고농도(30$\mu$M, 300$\mu$M)에서 난자와 초기배아의 세포내 칼슘이온 농도를 저하시켰고, ryandoine은 저농도(0.01$\mu$M)에서 세포내 칼슘이온 농도를 증가시켰지만 고농도(10$\mu$M)에서는 세포내 칼슘이온 농도를 감소시켰다. 본 실험결과를 보면, ruthenium red와 ryanodine은 생쥐의 난자 및 초기배아에서도 세포내 칼슘이온 농도에 영향을 미쳤고, 그 유효농도는 근세포를 포함한 체세포와는 차이가 있었다.

• 한국발생생물학회지:발생과생식
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• 제19권1호
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• pp.53-60
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• 2015

Environmental conditions during early mammalian embryo development are critical and some adaptational phenomena are observed. However, the mechanisms underlying them remain largely masked. Previously, we reported that AQP5 expression is modified by the environmental condition without losing the developmental potency. In this study, AQP11 was examined instead. To compare expression pattern between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP11 by whole mount immunofluorescence. When the fertilized embryos were developed in the maternal tracts, the level of Aqp11 transcripts was decreased dramatically until 2-cell stage. Its level increased after 2-cell stage and peaked at 4-cell stage, but decreased again dramatically until morula stage. Its transcript level increased again at blastocyst stage. In contrast, the levels of Aqp11 transcript in embryos cultured in vitro were as follows. The patterns of expression were similar but the overall levels were low compared with those of embryos grown in the maternal tracts. AQP11 proteins were localized in submembrane cytoplasm of embryos collected from maternal reproductive tracts. The immune-reactive signals were detected in both trophectoderm and inner cell mass. However, its localization was altered in in vitro culture condition. It was localized mainly in the plasma membrane of the blastocysts contacting with external environment. The present study suggests that early stage embryo can develop successfully by themselves adapting to their environmental condition through modulation of the expression level and localization of specific genes like AQP11.

• 한국수정란이식학회지
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• 제24권1호
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• pp.71-76
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• 2009

It is not easy for porcine embryos produced by in vitro systems to develop into blastocysts with high quality. To solve this problem, many researchers have developed novel culture methods. However, the formation of blastocysts with high quality is still low. In this study, we aimed to produce piglet following transfer of in vitro produced early embryos ($2{\sim}4$ cell stage embryos) or morula and blastocyst. The $2{\sim}4$ cell stage embryos were transferred to five estrus-synchronized recipients (200 embryos per recipient). One of the five sows farrowed three piglets, which contain two live piglets and one dead piglet, 114 days after embryo transfer. However, two recipients transferred with morula and blastocysts did not farrow. Microsatellite analysis confirmed that the genomic DNA of two live piglets were not genetically identical to that of the recipient. These results indicate that it is possible to obtain piglets by transfer of early embryos produced by in vitro production (IVP) systems.