세포질내 정자주입법(ICSI)에 있어서 정자흡입 및 난자내 주입방법에 관한 연구

이택후,김항진,송건호,김대근,전상식,박윤규,서태광,전병균,류은경,이은숙,문진수,김광철 경북대학교 의학연구소 2000 경북대학교병원의학연구소논문집 Vol.4 No.1

Study on Method of Sperm Aspiration and Injection into an Oocyte in Intracytoplasmic Sperm Injection(ICSI) Immobilization of spermatozoa prior to intracytoplasmic sperm iniection(ICSI) sometimes results in crooked tail and this makes it difficult to aspirate sperm into an injection pipette tail first. Head-first sperm aspiration into an injection pipette avoid this problem due to the bigger size of the sperm head. The effect of head or tail-first sperm injection into an oocyte on fertilization cleavage, percentage of grade I embryos and development to blastocyst stage in ICSI program has been studied. A single living immobilized spermatozoa from oligoasthenozoospermic patient was injected into an oocyte head-first or tail-first according to the treatment. Eighteen hours after microinjection, oocytes ware inspected for survival and fertilization Fertilized oocytes with two pronuclei were cultured in 30μl drop of mHTF supplemented with 10% heat-inactivated follicular fluid(FF) at 37℃. On day 2. embryo transfer was performed with cleaved embryos. The remaining 2-8 cell stage embryos were co-cultured with BRL cells in mHTF + 10% FF for 72 hours and the developmental stage was observed. The data were analyzed by Analysis of Variance. A total of 164 oocytes from 36 cycles were assigned to earth treatment and ICSI was performed(88 head-first, tail-first). The rates of normal fertilization were 81.8% and 76.3% for head-first and tail-first, respectively. Of the fertilized oocytes, the percentage of cleaved embryos and the percentage of grade 1 embryo among cleaved embryos were 88.9% and 68.8%, 93.1% and 74.1% for head-first and tail-first, respectively. Of the 2-8 cell embryos cultured, 44.4%(16/36) and 50.0%(10/20) for head first and tail first, respectively developed to blastocyst stage. There were no differences in fertilization, cleavage, rates of grade 1 embryos, and development to blastocyst stage. In conclusion, head-first or tail-first sperm injection into an oocyte in ICSI program does not affect fertilization and subsequent embryo development to blastocyst stage in vitro.

Characterization of X-linked RNA Transcripts in Matured Bovine Spermatozoa

Jeon, Byeong-Gyun,Kumar, B. Mohana,Rho, Gyu-Jin The Korean Society of Animal Reproduction 2011 Reproductive & developmental biology Vol.35 No.3

Although the function and utility of RNA transcripts derived from matured spermatozoa remains unclear, they might play important roles in the establishment of a paternal genome and subsequently embryo development. Herein, we investigated the expression of X-chromosome linked RNA transcripts in matured bovine spermatozoa. The total RNA was extracted from the matured spermatozoa, and then converted to cDNA. Autosomal genes (ACT-${\beta}$ and H-2A) and X-chromosome linked genes (ANT3, HPRT, MeCP2, RPS4X, XIAP, XIST and ZFX) were analyzed for the characterization of X-chromosome linked RNA transcripts and compared to female fibroblasts by RT-PCR. The transcripts of autosomal genes (ACT-${\beta}$ and H2A) and X-chromosome linked genes (ANT3, HPRT, MeCP2, RPS4X and ZFX) were not detected in spermatozoa. However, XIAP (X-linked inhibitor of apoptosis protein) and XIST (X-chromosome inactive-specific transcript, a kind of paternal imprinted gene) transcripts were detected in spermatozoa, and relative levels of XIAP and XIST transcripts were similar and 0.5-fold lower when compared to female fibroblasts, respectively. Based on the findings, it is summarized that the presence of RNA transcripts of XIAP and XIST in the isolated spermatozoa may imply their role in inhibition of apoptosis and induction of X-chromosome inactivation in embryo development.

Differential cytotoxic effects of sodium meta-arsenite on human cancer cells, dental papilla stem cells and somatic cells correlate with telomeric properties and gene expression.

Jeon, Byeong-Gyun,Kumar, B Mohana,Kang, Eun-Ju,Maeng, Geun-Ho,Lee, Yeon-Mi,Hah, Young-Sool,Ock, Sun-A,Kwack, Dae-Oh,Park, Bong-Wook,Rho, Gyu-Jin Potamitis Press 2011 Anticancer research Vol.31 No.12

<P>We investigated the effects of sodium meta-arsenite (NaAsO(2)) on human cancer cells (MDA-MB-231, MCF-7 and U-87 MG), dental papilla tissue stem cells (DPSCs) and somatic cells [MRC-5 fetal fibroblasts and adult muscle cells (MCs)] by examining telomeric properties, endogenous reverse transcriptase (RT) activity and the expression of tumorigenesis-linked genes. Half maximal inhibitory concentration (IC(50)) values were higher in DPSCs and MCs, possessing longer telomere lengths when compared to cancer cells. Levels of telomerase and RT activity, and the expression of protein 53 (p53), B-cell lymphoma 2 (BCL2), nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB), transforming growth factor beta (TGFβ) and vascular endothelial growth factor (VEGF) were significantly lower in cancer cells following sodium meta-arsenite treatment, whereas the effect was absent or marginally detected in DPSCs and somatic cells. Collectively, sodium meta-arsenite effectively induced cellular cytotoxicity by inhibiting telomerase and RT activity, and down-regulating transcript levels in cancer cells with shorter telomere lengths, whereas more tolerance was evident in DPSCs and somatic cells possessing longer telomere lengths.</P>

Variation of telomerase activity and morphology in porcine mesenchymal stem cells and fibroblasts during prolonged in vitro culture.

Jeon, Byeong-Gyun,Kwack, Dae-Oh,Rho, Gyu-Jin Marcel Dekker 2011 Animal biotechnology Vol.22 No.4

<P>The purpose of this study was to examine the telomerase activity, population doubling time (PDT), morphological alterations, and the cell cycle status with activity of senescence-associated-s-galactosidase in porcine mesenchymal stem cells (MSCs) and fibroblasts during an extended in vitro culture. MSCs and fibroblasts were isolated from bone marrow and ear skin of a miniature pig, respectively, and cultured up to 20 passages. The analysis was carried out in MSCs and fibroblasts at 1, 5, 10, 15, and 20 passages. Relative telomerase activity (RTA) levels were significantly (P?<?0.05) higher in MSCs than in fibroblasts at all the passages. The PDT and cellular size slightly increased in MSCs at later passages. In contrast, fibroblasts had significantly (P?<?0.05) increased PDT and cellular size, and the morphology revealed senescent-like abnormal type after passage 10. Further, the high incidence of s-galactosidase stained cells was observed in fibroblasts compared to that of MSCs at passage 15, and cell cycle stage at G0?/?G1 phase was significantly (P?<?0.05) increased in the fibroblasts at 15 and 20 passages compared to that of MSCs. Based on these observations, we concluded that porcine MSCs possessed more tolerance against senescence and aging compared to fibroblasts following prolonged in vitro culture.</P>

Comparisons of Development Potential in Bovine SCNT Embryos using Donor Cells treated with Different Demethylating Inhibitors

Byeong-Gyun Jeon,Gie-Joon Jeong,Gyu-Jin Rho 한국수정란이식학회 2015 한국동물생명공학회지 Vol.30 No.3

To improve the developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos, this study compared the developmental rates to blastocyst stage in the SCNT embryos using donor fibroblasts treated with 5-azacytidine (5AC) and S-adenosylhomocysteine (SAH) at different concentrations. Their reprogramming efficiency level was investigated with level of telomerase activity. Donor fibroblasts isolated from adult ear skin of a cow were exposed to 5AC and SAH at different concentrations during 2 passages. After nuclear transfer into enucleated recipient oocytes, the cleavage and developmental rates were significantly (p<0.05) decreased in the SCNT embryos using 5AC-treated fibroblasts (5AC-SCNT embryos), compared with those of non-treated control (control-SCNT embryos) and SAH-treated fibroblasts (SAH-SCNT embryos). The developmental rates to blastocyst stage tended to be slightly increased in the SAH-SCNT embryos at each of the concentrations, and especially, the developmental rates in the SCNT embryos using 1.0 mM SAH-treated fibroblasts were significantly (p<0.05) higher than that of control SCNT embryos. The mean numbers of total and ICM cell in blastocysts were also significantly (p<0.05) decreased in the 5AC-SCNT embryos, compared with those of other SCNT blastocysts. Further, the level of telomerase activity was also significantly (p< 0.05) decreased in the 5AC-SCNT embryos than those of control and SAH-SCNT embryos. Whereas, a significantly (p<0.05) up-regulated telomerase activity was observed in SAH-SCNT embryos, compare with that of control-SCNT embryos. In conclusion, SCNT embryos using hypomethylated donor cells with SAH, not 5AC, may improve the developmental potential and reprogramming efficiency.

Nuclear Modeling and Developmental Potential of Bovine Somatic Nuclear Transfer Embryos Cloned by Two Different Activation Methods

Jeon, Byeong-Gyun,Rho, Gyu-Jin The Korean Society of Animal Reproduction 2011 Reproductive & developmental biology Vol.35 No.1

The present study investigated the nuclear remodeling, development potential with telomerase activity and transcription level of X-linked genes (ANT3, HPRT, MeCP2, RPS4X, XIAP, XIST and ZFX) in the bovine somatic cell nuclear transfer (SCNT) embryos using two different fusion and activation methods. Female adult fibroblasts were injected into perivitelline space of in vitro matured oocytes. The oocyte-nucleus complexes were fused and followed by immediately either activated (Group 1), or activated at 1 h post-fusion (hpf) (Group 2), respectively. The incidence of normal premature chromosome condensation (PCC) at 1 hpf was slightly increased in the Group 2, compared to those of Group 1, but there was no significant (p<0.05) difference. The incidence of normal pronucleus (PN) and chromosome spread at 5 and 18 hpf were significantly (p<0.05) higher in the Group 2 than those of Group 1. The cleavage rate to 2-cell stage, developmental rate to blastocyst stage, and the mean number of total and ICM cell numbers were significantly (p<0.05) higher in the Group 2, compared to those of Group 1. Level of telomerase activity was significantly (p<0.05) higher in the SCNT blastocysts of Group 2, compared to those of Group 1. Transcript levels of HPRT, MeCP2 and XIST were not significantly (p<0.05) different between blastocysts of Group 1 and 2. However, transcript level of ANT3, RPS4X, XIAP and ZFX were significantly (p<0.05) up-regulated in the SCNT blastocysts of Group 2, compared to those of Group 1. Taken together, it is concluded that oocyte activation at 1 hpf induces the enhanced developmental potential by efficient nuclear remodeling and subsequent facilitation of the nuclear reprogramming of bovine SCNT embryos.

Variation of Transcribed X-linked Genes in Bovine Embryos Cloned with Fibroblasts at Different Age and Cell Cycle

Jeon, Byeong-Gyun,Rho, Gyu-Jin The Korean Society of Animal Reproduction 2011 Reproductive & developmental biology Vol.35 No.2

The present study compared the developmental potential, telomerase activity and transcript levels of X-linked genes (ANT3, HPRT, MeCP2, RPS4X, XIAP, XIST and ZFX) in the bovine somatic cell nuclear transfer (SCNT) embryos derived from different age and cell cycle of female donor nucleus. In experiment 1, the fusion rate, cleavage rate to 2-cell stage, developmental rate to blastocyst stage, and the mean number of total and ICM cells was slightly increased in embryos cloned with fetal fibroblasts compared to those with adult fibroblasts, but there was no significantly (p<0.05) differences. Telomerase activity was also similar in blastocysts cloned with fetal and adult fibroblasts. Up-regulated RPS4X and down-regulated MeCP2, XIAP, and XIST transcript level were observed in blastocysts cloned with adult fibroblasts, compared to those with fetal fibroblasts. In experiment 2, the fusion rate, cleavage rate to 2-cell stage, developmental rate to blastocyst stage, and the mean number of total and ICM cells was significantly (p<0.05) increased in embryos cloned with fetal fibroblasts at early G1 phase of the cell cycle, compared to those of fetal fibroblasts at late G1 phase. DNMT1 transcript was observed to significantly (p<0.05) increased in the fetal fibroblasts at 3 hrs after trypsin treatment of confluent culture. Further, level of telomerase activity and transcribed X-linked genes was also significantly (p<0.05) higher in the early G1 SCNT blastocysts than those of late G1. The results imply that fetal fibroblasts at early G1 phase induces the enhanced developmental potential and up-regulated telomerase activity and X-linked gene, but aberrant transcript pattern of X-linked genes may be displayed in the SCNT embryos.

Comparisons of Development Potential in Bovine SCNT Embryos using Donor Cells treated with Different Demethylating Inhibitors

Jeon, Byeong-Gyun,Jeong, Gie-Joon,Rho, Gyu-Jin The Korean Society of Embryo Transfer 2015 한국동물생명공학회지 Vol.30 No.3

To improve the developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos, this study compared the developmental rates to blastocyst stage in the SCNT embryos using donor fibroblasts treated with 5-azacytidine (5AC) and S-adenosylhomocysteine (SAH) at different concentrations. Their reprogramming efficiency level was investigated with level of telomerase activity. Donor fibroblasts isolated from adult ear skin of a cow were exposed to 5AC and SAH at different concentrations during 2 passages. After nuclear transfer into enucleated recipient oocytes, the cleavage and developmental rates were significantly (p<0.05) decreased in the SCNT embryos using 5AC-treated fibroblasts (5AC-SCNT embryos), compared with those of non-treated control (control-SCNT embryos) and SAH-treated fibroblasts (SAH-SCNT embryos). The developmental rates to blastocyst stage tended to be slightly increased in the SAH-SCNT embryos at each of the concentrations, and especially, the developmental rates in the SCNT embryos using 1.0 mM SAH-treated fibroblasts were significantly (p<0.05) higher than that of control SCNT embryos. The mean numbers of total and ICM cell in blastocysts were also significantly (p<0.05) decreased in the 5AC-SCNT embryos, compared with those of other SCNT blastocysts. Further, the level of telomerase activity was also significantly (p<0.05) decreased in the 5AC-SCNT embryos than those of control and SAH-SCNT embryos. Whereas, a significantly (p<0.05) up-regulated telomerase activity was observed in SAH-SCNT embryos, compare with that of control-SCNT embryos. In conclusion, SCNT embryos using hypomethylated donor cells with SAH, not 5AC, may improve the developmental potential and reprogramming efficiency.

Characterization and comparison of telomere length, telomerase and reverse transcriptase activity and gene expression in human mesenchymal stem cells and cancer cells of various origins.

Jeon, Byeong-Gyun,Kumar, Basavarajappa Mohana,Kang, Eun-Ju,Ock, Sun-A,Lee, Sung-Lim,Kwack, Dae-Oh,Byun, June-Ho,Park, Bong-Wook,Rho, Gyu-Jin Springer 2011 Cell and tissue research Vol.345 No.1

<P>We have characterized and compared the telomere length, telomerase, reverse transcriptase (RT) activity and expression of genes implicated in cancer and in pluripotency, in human mesenchymal stem cells (MSCs) derived from dental papilla tissue, umbilical cord matrix and adipose tissue and in cancer cells (MDA-MB-231, U-87 MG, and MCF-7). MRC-5 fetal fibroblasts and adult muscle cells were used as somatic cell controls. Telomere length was significantly (P<0.05) higher in MSCs and somatic cells (7.2-9.3 kb) than in cancer cell lines (3.9-6 kb). However, the relative telomerase activity (RTA) in the cancer cell lines was significantly (P<0.05) higher than that of MSCs and somatic cells. RTA tended to be slightly higher in MSCs but no significant differences were observed between some cancer cells and MSCs. However, RTA was not detected in somatic cells. Although differentially displayed, the expression of genes related to cancer (BCL-2, p53, NF-κB, TGF-β, VEGF) and transcription and pluripotency (OCT4, NANOG, STAT3, REX1) were commonly observed in MSCs and cancer cells. Thus, endogenous non-telomerase RTA might be a potential biological marker or regulator among MSCs and cancer cells. Further, by sharing the biological and molecular markers of self-renewal and proliferation with cancer cells, MSCs might play a contributory role as tissue resident stem cells in tumor development.</P>

Characterization of X-chromosome linked RNA transcripts in matured bovine spermatozoa

Byeong-Gyun Jeon,Gyu-Jin Rho 한국동물생명공학회(구 한국동물번식학회) 2007 발생공학 국제심포지엄 및 학술대회 Vol.2007 No.1