• Clinical and Experimental Reproductive Medicine
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• v.44 no.1
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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.

• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.281-291
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• 1999

The objective of this study was to optimize the freezing/thawing method of in vitro produced Hanwoo blastocysts. Day 7 blastocysts after IVF were vitrified using EFS40 (40% ethylene glycol, 18% ficoll, 0.3 M sucrose and 10% FBS added m-DPBS) as a freezing solution and electron microscope (EM) grid (V-G) or straw (V-S) as an embryo container. In both method, freezing/thawing were treated by 2-step, treatment time was required in V-G method and V-S method, for 2 min / 3 min and 3.5 min / 10 min, respectively. Embryo survival was assessed as re-expanded and hatched rates at 24 h and 48 h after warming, respectively. The results obtained in these experiments were summarized as follows: when the effect of exposure in vitrification solution and chilling injury from freezing procedure on in vitro produced expanded blastocysts were examined, at 24 h after warming, embryo survival in exposure group (100.0%) was not different compared to that in control group (100.0%), although those results were significantly different with two vitrified groups (V-G: 87.8, V-S: 77.8%) (P＜0.001). However, at 48 h after warming, hatched rates of V-G group (67.8%) were significantly higher than those of V-S group (53.3%) (P＜0.05). In addition, this hatched rate in V-G group was not different with that in exposure group (73.3%). When the effects of embryo developmental stage (early, expanded and early hatching blastocysts) and embryo container (EM grid and straw) to the in vitro survival of vitrified-warmed day 7 Hanwoo blastocysts were simultaneously examined, fast developed embryos were indicated the better resistance to freezing than delayed developed one, irrespective of embryo containers (early; 57.1 ＆ 24.4%, expanded; 84.7 ＆ 60.6%, early hatching; 91.7 ＆ 80.0%) (P＜0.001). Especially, in expanded and early hatching blastocysts, embryo survival of V-G group (67.8, 95.0%) was significantly higher than those of V-S group (53.0, 65.0%) at 48 h post warming, respectively (P＜0.05, P＜0.001). Therefore, this study indicates that Hanwoo blastocysts can be cryopreserved more simple, efficient and successful by vitrification method using EM grid.

• Journal of Embryo Transfer
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• v.30 no.4
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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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• v.49 no.10
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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.

• Journal of Veterinary Clinics
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• v.16 no.1
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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.

• Development and Reproduction
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• v.23 no.1
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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.

• Korean Journal of Veterinary Research
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• v.15 no.2
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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.

• Development and Reproduction
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• v.7 no.2
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• pp.95-103
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• 2003

Intracellular calcium is an important physiological factor in most cells, and ruthenium red and ryanodine play an important role as calcium modulators. Ruthenium red inhibits calcium-induced calcium release(CICR) from the intracellular calcium store. Ryanodine activates calcium release through ryanodine channel. The present experiment was performed to investigate the effects of two modulators on calcium ion metabolism and to determine their dose-dependency in oocyte and early embryo of mouse. Intracellular calcium ion concentration was measured in realtime by using confocal laser scanning microscope(CLSM) after loading of Fluo-3/AM in mouse oocytes and early embryos. Ruthenium red decreased intracellular calcium ion concentration in oocytes and early embryos at its high concentration(30, 300 $\mu$M). Ryanodine increased intracellular calcium ion concentration in oocytes and early embryos in low concentration(0.01 $\mu$M) but decreased that at higher concentrations(1, 10 $\mu$M). These results indicate that two modulators affected calcium ion metabolism in oocyte and early embryo of mouse, and their dose-dependency was different from somatic cell including myocytes.

• Development and Reproduction
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• v.19 no.1
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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.

• Journal of Embryo Transfer
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• v.24 no.1
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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.