• 제목/요약/키워드: Trophectoderm

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Temporal Aquaporin 11 Expression and Localization during Preimplantation Embryo Development

  • Park, Jae-Won;Cheon, Yong-Pil
    • 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.

체외성숙 시간이 한우 수정란 생산에 미치는 영향 (Effects of Maturation Time on In-vitro Production of Korean Native Cow Embryos)

  • 박용수;최수호;한진철;박흠대;변명대
    • 한국가축번식학회지
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    • 제27권1호
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    • pp.35-44
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    • 2003
  • The present study was performed to investigate the first polar body(PB) extrusion during in-vitro maturation(IVM) and to examine the effect of different maturation time on the embryo development of Korean Native Cows(KNC) with regard to blastocyst(BL) cell numbers and pregnancy rates. PB extrusion did not take place for the first 12 hours(hr) of IVM, and most of KNC oocytes extruded PB from 14 to 20 hr after the onset of maturation. There was no significant difference in cleavage and 8-cell stage rates among the treatment groups, but BL and BL/8-cell rates were significantly higher(P<0.05) in 18 hr maturation group(31.0$\pm$5.7 and 82.0$\pm$5.1%) than 22 and 24 hr maturation group. The proportion of BL formed on day 7 and 8 was significantly higher(P<0.05) in 18 hr maturation group(85%) than in 24 hr maturation group(55%). There was a significant difference(P<0.05) in inner cell mass, trophectoderm and total cell number between day 7 BL produced by in-vivo and IVM 18 hr and day 8 BL produced by IVM 18 hr and 24 hr. Pregnancy rates are also significantly higher(P<0.05) in in-vivo(56.3%) and IVM 18 hr day 7(50.0%) group than day 8 treatment groups(18 hr: 16.7%, 24 hr: 10.5%). These results suggest that KNC oocytes achieve developmental competency within 20 hr of IVM, and "short" IVM (18 hr) is more effective than "long" IVM(24 hr) in embryo development rates, BL cell numbers and pregnancy rates.

Global DNA Methylation of Porcine Embryos during Preimplantation Development

  • Yeo, S.E.;Kang, Y.K.;Koo, D.B.;Han, J.S.;Yu, K.;Kim, C.H.;Park, H.;Chang, W.K.;Lee, K.K.;Han, Y.M.
    • 한국가축번식학회지
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    • 제27권4호
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    • pp.309-315
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    • 2003
  • DNA methylation at CpG sites, which is a epigenetic modification, is associated with gene expression without change of DNA sequences. During early mouse embryogenesis, dynamic changes of DNA methylation occur. In this study, DNA methylation patterns of porcine embryos produced in vivo and in vitro were examined at various developmental stages by the immunocytochemical staining method. Interestingly, active demethylation was not observed on the paternal pronucleus of porcine zygotes. However, differences were detected in the passive demethylation process between in vivo and in vitro embryos. There was no change in the DNA methylation state until the blastocyst stage of in vivo embryos, whereas partial demethylation was observed in several blastomeres from a 4 cell stage to a morula stage of in vitro embryos. The whole genome of inner cell mass (ICM) and trophectoderm (TE) cells in porcine blastocysts were evenly methylated without de novo methylation. Our findings demonstrate that genome-wide demethylation does not occur in pig embryos during preimplantation development unlike murine and bovine embryos. It indicates that the machinery regulating epigenetic reprogramming may be different between species.

The effect of artificial shrinkage and assisted hatching on the development of mouse blastocysts and cell number after vitrification

  • Kim, Hye Jin;Lee, Ki Hwan;Park, Sung Baek;Choi, Young Bae;Yang, Jung Bo
    • Clinical and Experimental Reproductive Medicine
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    • 제42권3호
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    • pp.94-100
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    • 2015
  • Objective: The goal of this study was to ascertain optimal assisted hatching (AH) method in frozen embryo transfer. We compared the effect of depending on whether mechanical or laser-AH was performed before or after the vitrification of embryo development rate and blastocyst cell numbers. Methods: In order to induce superovulation, pregnant mare's serum gonadotropin followed by human chorionic gonadotropin were injected into 4- to 5-week-old female mice. 2-cell embryos were then collected by flushing out the oviducts. The Expanded blastocysts were recovered after the collected embryos were incubated for 48 hours, and were then subjected to artificial shrinkage (AS) and cross-mechanical AH (cMAH) or quarter-laser zona thinning-AH (qLZT-AH) were carried out using the expanded blastocysts before or after vitrification. After 48 hours of incubation, followed by vitrification and thawing (V-T), and blastocysts were fluorescence stained and observed. Results: The rate of formation of hatched blastocysts after 24 and 72 hours of incubation was significantly higher in the AS/qLZT-AH/V-T group than in the other groups (p<0.05). The cell number of the inner cell mass was higher in AS/V-T/non-AH and AS/V-T/cMAH groups than those of others (p<0.05). In the control group, the number of trophectoderm and the total cell number were higher than in the AS-AH group (p<0.05). Conclusion: The above results suggest that AS and AH in vitrification of expanded blastocysts lead to the more efficient formation of hatched blastocysts in mice.

The effect of various assisted hatching techniques on the mouse early embryo development

  • Park, Sung Baek;Kim, Hye Jin;Choi, Young Bae;Ahn, Kwang Hwa;Lee, Kee Hwan;Yang, Jung Bo;Yu, Chang Seok;Seo, Byoung Boo
    • Clinical and Experimental Reproductive Medicine
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    • 제41권2호
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    • pp.68-74
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    • 2014
  • Objective: In search of an ideal method of assisted hatching (AH), we compared the effects of conventional micropipette-AH and laser-AH on the blastocyst formation rate (BFR) and blastocyst cell numbers. Methods: Four-to five-week-old ICR female mice were paired with male mice after superovulation using Pregnant mare's serum gonadotropin (PMSG) and hCG. The two-cell embryos were flushed from the oviducts of female mice. The retrieved two-cell embryos underwent one of five AH procedures: single mechanical assisted hatching (sMAH); cross mechanical assisted hatching (cMAH); single laser assisted hatching (sLAH); quarter laser assisted hatching (qLAH); and quarter laser zona thinning assisted hatching (qLZT-AH). After 72 hours incubation, double immunofluorescence staining was performed. Results: Following a 72 hours incubation, a higher hatching BFR was observed in the control, sMAH, cMAH, and sLAH groups, compared to those in the qLAH and qLZT-AH groups (p<0.05). The hatched BFR was significantly higher in the qLAH and qLZT-AH groups than in the others (p<0.05 for each group). The inner cell mass (ICM) was higher in the control and sMAH group (p<0.05). The trophectoderm cell number was higher in the cMAH and qLAH groups (p<0.05). Conclusion: Our results showed that the hatched BFR was higher in groups exposed the the qLAH and qLZT-AH methods compared to groups exposed to other AH methods. In the qLAH group, although the total cell number was significantly higher than in controls, the ICM ratio was significantly lower in than controls.

체외수정과 단위발생된 돼지 난자의 체외발달 양상의 비교 (In Vitro Developments of Porcine Embryos Produced by In Vitro Fertilization and Parthenogenetic Activation)

  • 엄상준;김남형;김은영;김묘경;차수경;윤산현;박세필;정길생;임진호
    • Clinical and Experimental Reproductive Medicine
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    • 제24권1호
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    • pp.43-49
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    • 1997
  • 본 연구는 단위발생과 체외수정으로 생산된 돼지 난자의 발달양상과 inner cell mass(ICM) 그리고 trophectoderm (TE)의 세포배열을 조사하기 위하여 실시하였다. 단위발생은 ethanol 단독처리(haploid) 혹은 ethanol과 cytochalasin B을 공동처리(diploid)하였던바, 단위발생란은 체외수정란에 비하여 배반포까지의 발달이 저조하였지만, 단위발생에 있어서 ethanol과 cytochalasin B을 공동처리한 군이 ethanol 단독처리한 군보다 배반포까지 발달이 촉진되었다. 또한 단위발생란의 경우 total 세포수와 ICM 수에 있어서 체외수정란에 비하여 현저하게 감소되었지만, ethanol과 cytochalasin B을 공동처리한 단위발생란이 ethanol 처리된 단위발생란보다는 현저하게 높은 total 세포수와 ICM 수가 조사되었다. 이상의 결과로, 돼지의 착상전 배발달 양상과 ICM와 TE의 세포배열에 있어서 ploidy가 영향을 미친다는 것을 알수 있었다.

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후성 유전학적 리프로그래밍과 클로닝 (Epigenetic Reprogramming and Cloning)

  • 한용만;강용국;구덕본;이경광
    • 한국발생생물학회지:발생과생식
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    • 제7권2호
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    • pp.61-68
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    • 2003
  • 포유동물의 초기 발생과정 중 접합체가 전능성이나 다능성을 가지기 위해서는 전반적인 DNA 메틸화를 포함하는 후성 유전학적 리프로그래밍의 복잡한 과정을 거쳐야만 한다. 본 연구팀에서는 공여핵의 후성 유전학적 리프로그래밍 과정을 조사하기 위하여 소 복제수정란에서 메틸화 양상을 분석하였다. 복제수정란의 비정상적인 메틸화 양상이 다양한 반복염기서열에서 관찰되었지만 single-copy유전자들의 염기서열은 정상적인 메틸화 양상을 보여주었다. 전반적으로 복제수정란의 전반적인 메틸화 상태는 정상수정란과 완전히 다른 양상을 보여주었다. 또한 복제 배반포의 영양외배엽세포에서 특이적으로 높은 메틸화 수준은 현 복제동물에서 빈번히 나타나는 불완전한 태반형성에 작용할 수 있을 것이다. 결론적으로 복제수정란의 비정상적 발생은 공여핵의 불완전한 후성 유전학적 리프로그래밍에 기인할 수 있다는 사실을 제시하게 되었다. 이러한 공여핵의 후성 유전학적 과정의 이해는 복제수정란의 비정상적 발생을 보다 분명히 밝힐 수 있을 것이다.

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Regulation of Pluripotency-related Genes and Differentiation in Mouse Embryonic Stem Cells by Direct Delivery of Cell-penetrating Peptide-conjugated CARM1 Recombinant Protein

  • Choi, Sara;Jo, Junghyun;Seol, Dong-Won;Cha, Soo Kyung;Lee, Jeoung Eun;Lee, Dong Ryul
    • 한국발생생물학회지:발생과생식
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    • 제17권1호
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    • pp.9-16
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    • 2013
  • Coactivator-associated arginine methyltransferase 1 (CARM1) is included in the protein arginine methyltransferase (PRMT) family, which methylates histone arginine residues through posttranslational modification. It has been proposed that CARM1 may up-regulate the expression of pluripotency-related genes through the alteration of the chromatin structure. Mouse embryonic stem cells (mESCs) are pluripotent and have the ability to self-renew. The cells are mainly used to study the genetic function of novel genes, because the cells facilitate the transmission of the manipulated genes into target mice. Since the up-regulated methylation levels of histone arginine residue lead to the maintenance of pluripotency in embryos and stem cells, it may be suggested that CARM1 overexpressing mESCs elevate the expression of pluripotency-related genes in reconstituted embryos for transgenic mice and may resist the differentiation into trophectoderm (TE). We constructed a fusion protein by connecting CARM1 and 7X-arginine (R7). As a cell-penetrating peptide (CPP), can translocate CARM1 protein into mESCs. CPP-CARM1 protein was detected in the nuclei of the mESCs after a treatment of 24 hours. Accordingly, the expression of pluripotency-related genes was up-regulated in CPP-CARM1-treated mESCs. In addition, CPP-CARM1-treated mESC-derived embryoid bodies (EBs) showed an elevated expression of pluripotency-related genes and delayed spontaneous differentiation. This result suggests that the treatment of recombinant CPP-CARM1 protein elevates the expression of pluripotency-related genes of mESCs by epigenetic modification, and this protein-delivery system could be used to modify embryonic fate in reconstituted embryos with mESCs.

Effects of Various Addition and Exclusion Time of Glucose on Development of Mouse Two-Cell Embryos

  • Park S. B.;Park K S.;Lee T. H.;Chun S. S.;Kim K S.;Song H. B.
    • Reproductive and Developmental Biology
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    • 제28권4호
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    • pp.227-233
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    • 2004
  • This study was conducted to investigate the effect of various addition and exclusion time of glucose (Control: no addition, A: 24~72 h, B: 24~48 h, C: 48~72 h, D: 0~72 h, E: 0~48 h, F: 0~24 h and 48~72 h, G: 0~24 h) on embryonic developmental capacity of 2-cell embryos in mice. Developed blastocysts were assessed for mean cell number by differential staining. The zona-intact blastocyst (ZiB) rates were higher (p<0.05) in group B than control. However, the zona-escape blastocyst (ZeB) rates were not significantly different in all groups. At 72 h, total blastocyst (ZiB + ZeB) formation rates were not significantly different in all groups. The mean cell number was not significantly different among all groups. The inner cell mass (ICM) cell number was higher (p<0.05) in group F than control, group A, B and G. The trophectoderm (TE) cell number was higher (p<0.05) in control than group A and D. The %ICM was higher (p<0.05) in group C, D and F than control. The ICM : TE ratio was not significantly different in all groups. Between control and glucose group, no significant difference was observed in the total blastocysts (ZiB + ZeB) formation rates. Also, no significant difference was observed in the mean cell number, ICM cell number and ICM : TE ratio. However the TE cell number was higher (p<0.05) in control than glucose group and %ICM was higher (p<0.05) in glucose group than control. In conclusion, glucose added in culture medium was not inhibitory on blastocyst formation but glucose added for 48 ~72 h in culture medium increases %ICM of blastocysts in mice.

A retrospective study of single frozen-thawed blastocyst transfer

  • Hur, Yong Soo;Ryu, Eun Kyung;Song, Seung Hyun;Yoon, San Hyun;Lim, Kyung Sil;Lee, Won Don;Lim, Jin Ho
    • Clinical and Experimental Reproductive Medicine
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    • 제43권2호
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    • pp.106-111
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    • 2016
  • Objective: To study the clinical outcomes of single frozen-thawed blastocyst transfer cycles according to the hatching status of frozen-thawed blastocysts. Methods: Frozen-thawed blastocysts were divided into three groups according to their hatching status as follows: less-than-expanded blastocyst (${\leq}EdB$), hatching blastocyst (HgB), and hatched blastocyst (HdB). The female age and infertility factors of each group were evaluated. The quality of the single frozen-thawed blastocyst was also graded as grade A, tightly packed inner cell mass (ICM) and many cells organized in the trophectoderm epithelium (TE); grade B, several and loose ICM and TE; and grade C, very few ICM and a few cells in the TE. The clinical pregnancy and implantation rate were compared between each group. The data were analyzed by either t-test or chi-square analysis. Results: There were no statistically significant differences in average female ages, infertility factors, or the distribution of blastocyst grades A, B, and C in each group. There was no significant difference in the clinical pregnancy and implantation rate of each group according to their blastocyst grade. However, there was a significant difference in the clinical pregnancy and implantation rate between each group. In the HdB group, the clinical pregnancy and implantation rate were similar regardless of the blastocyst quality. Conclusion: There was an effect on the clinical outcomes depending on whether the blastocyst hatched during single frozen-thawed blastocyst transfer. When performing single frozen-thawed blastocyst transfer, the hatching status of the frozen-thawed blastocyst may be a more important parameter for clinical outcomes than the quality of the frozen-thawed blastocyst.