• Reproductive and Developmental Biology
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• 제36권3호
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• pp.173-181
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• 2012

Sphingosine-1-phosphate (S1P) has a many function involved proliferation, differentiation and survival of many cells. In this study, to investigate whether S1P improve the developmental competence of porcine embryos, 50 nM of S1P were supplemented during in vitro maturation (with EGF or without EGF) medium and/or in vitro culture (IVC) medium. Addition of S1P was significantly increased the rate of oocytes reaching metaphase II (MII) compared to the control (83.5 vs. 64.1%) in without EGF medium, but not with EGF medium (89.5 vs. 84.6%). When treated with $1{\mu}M$ of N1N-dimethylsphingosine (DMS), a sphingosine kinase inhibitor which is blocked endogenous generation of S1P, the meiotic progression rates to MII stage (without EGF: 45.2 and with EGF: 66.7%) were significantly decreased and degeneration rates (without EGF: 51.2 and with EGF: 30.1%) were increased in both medium compared to control group during IVM periods. Also, the rates of blastocyst formation was significantly increased in the S1P treated group compared to control group (29.0 vs. 19.2%) of EGF supplemented medium, whereas there were no effect in the EGF free medium (9.0 vs. 10.5%). After 12 h IVM, the phosphorylation of ERK1 and ERK2, which is major signaling pathway of MAP kinase, were increased in the S1P group than that of control or DMS group. When supplemented of S1P during IVC, the rates of blastocyst formation and total cell number (30.2% and 40.6) were significantly increased in S1P-treated group compared with control (20.1% and 32.5), DMS (12.3% and 25.1), and S1P plus DMS group (24.7% and 33.6). The percentage of apoptosis nuclei in the S1P group was significantly decreased than other groups. Also, the rates of blastocyst formation (26.7 vs. 14%) and total cell number (42.8 vs. 32.5) were significantly increased in the S1P group than those of control group when S1P added during the entire IVM and IVC periods. Taken together, our results indicate that S1P supplementation in IVM and/or IVC medium affects beneficial effect of meiotic maturation and subsequent developmental competence of porcine embryos.

• Animal cells and systems
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• 제14권2호
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• pp.83-89
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• 2010

Gangliosides are a ubiquitous component of the membranes of mammalian cells that have been suggested to play important roles in various cell functions such as cell-cell interaction, adhesion, cell differentiation, growth control and signaling. However, the role that gangliosides play in the immune rejection response in xenotransplantation is not yet clearly understood. In this study, differential expression patterns of gangliosides in HEK293 (human embryonic kidney cells), PK15 (porcine kidney cells), NIH-kd (NIH-mini pig kidney cells, primary cultured) and the cortex, medulla and calyx of the NIH-mini pig kidney were investigated by high-performance thin-layer chromatography (HPTLC). The results revealed that HEK293, PK15 and NIH-kd contained GM3, GM2 and GD3 as major gangliosides. Moreover, GM3, which are the gangliosides of NIH-kd, were expressed at higher levels than HEK293 and PK15. Especially, GT1b were expressed in HEK293 and NIH-kd but not in PK15. Finally, GM1 and GD1a were expressed in NIH-kd, but not in HEK293 or PK15. These results suggest that differential expression patterns of gangliosides from HEK293, PK15 and NIH-kd are related to the immune rejection response in xenotransplantation.

• 한국가축번식학회지
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• 제21권2호
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• pp.139-146
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• 1997

소와 돼지의 난관 상피세포가 마우스 초기배의 발달 에 미치는 영향과 체외배양에 있어 최적의 배양조건을 알아보기 위하여 ICR 계통의 마우스에 PMSG 7.5 IU와 hCG 7.5 IU를 각각 복강주사하고 자연교미하여 48시간 경과 후에 난관에서 2-세포 초기배를 D-PBS로 관류하여 회수하였다. 회수된 배아는 소와 돼지의 난관 상피세포와 공배양하여 그 효사를 배반포 발달율과 핵의 수를 조사하였다. 또한 생체내와 실험 관내의 발육상태를 비교하기 위하여 hCG접종후 120 시간동안 생체내에서 발육시킨 신선 배반포배를 자궁 에서 채취하여 그 핵수를 계산하였다. 마우스 초기배는 TCM 199, Ham's F-10, Medicult IVF 배양액에서 소 난관 상피세포 또는 돼지 난관 상피세포와 공배양할 경우 91-97%의 높은 배반포 발달율을 보였으며 난관 상피세포간의 차이는 나타 나지 않았다. 각 배양조건에 따라 배양된 배반포의 핵수는 체내에서 자란 배반포에 비해 체외에서 배양한 배반포에서 유의적으로 적었다. 체외배양중 핵수는 공배양하지 않은 TCM 199, Ham's F-10, Medicult IVF medium 에서 각각 68.1${\pm}$6.00, 67.3${\pm}$4.49, 66.4${\pm}$5.64개로 나타났으며 BOEC와 공배양하였을 경우에는 94.3${\pm}$8.61, 92.5${\pm}$7.60, 92.1${\pm}$6.107B, POEC와 공배양하였을 때는 93.3${\pm}$5.80, 92.9${\pm}$6.53, 92.3${\pm}$7.35개로 체내에서 배양된 배반포의 경우의 107.2${\pm}$7.43개보다 적었다. 이상의 결과로 난관 상피세포인 BOEC와 POEC는 마우스 초기배야와의 체외공배양시 배아의 발달과 분화에 이로운 영향을 주어 발달율과 부화율를 향상시키나 핵수 증가에서는 체내조건보다 미홉한 것으로 사료된다.

• Reproductive and Developmental Biology
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• 제32권2호
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• pp.135-140
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• 2008

Recent studies on nuclear transfer and induced pluripotent stem cells have demonstrated that differentiated somatic cells can be returned to the undifferentiated state by reversing their developmental process. These epigenetically reprogrammed somatic cells may again be differentiated into various cell types, and used for cell replacement therapies through autologous transplantation to treat many degenerative diseases. To date, however, reprogramming of somatic cells into undifferentiated cells has been extremely inefficient. Hence, reliable markers to identify the event of reprogramming would assist effective selection of reprogrammed cells. In this study, a transgene construct encoding enhanced green fluorescent protein (EGFP) under the regulation of human Oct4 promoter was developed as a reporter for the reprogramming of somatic cells. Microinjection of the transgene construct into pronuclei of fertilized mouse eggs resulted in the emission of green fluorescence, suggesting that the undifferentiated cytoplasmic environment provided by fertilized eggs induces the expression of EGFP. Next, the transgene construct was introduced into human embryonic fibroblasts, and the nuclei from these cells were transferred into enucleated porcine oocytes. Along with their in vitro development, nuclear transfer embryos emitted green fluorescence, suggesting the reprogramming of donor nuclei in nuclear transfer embryos. The results of the present study demonstrate that expression of the transgene under the regulation of human Oct4 promoter coincides with epigenetic reprogramming, and may be used as a convenient marker that non-invasively reflects reprogramming of somatic cells.

• Animal Bioscience
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• 제36권8호
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• pp.1180-1189
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• 2023

Objective: Discovering the mechanism of cell specification is important to manipulate cellular lineages. To obtain lineage-specific cell lines, the target lineage needs to be promoted, and counterpart lineages should be suppressed. Embryos in the early blastocyst stage possess two different cell populations, the inner cell mass (ICM) and trophectoderm. Then, cells in the ICM segregate into epiblasts (Epi) and primitive endoderm (PrE). PrE cells in embryos show specific expression of platelet-derived growth factor (PDGF) and its receptor, PDGF receptor A (PDGFRA). In this study, we suppressed PDGF signaling using two methods (CRISPR/Cas9 injection and inhibitor treatment) to provide insight into the segregation of embryonic lineages. Methods: CRISPR/Cas9 RNAs were injected into parthenogenetically activated and in vitro fertilized embryos. The PDGF receptor inhibitor AG1296 was treated at 0, 5, 10, and 20 µM concentration. The developmental competence of the embryos and the number of cells expressing marker proteins (SOX2 for ICM and SOX17 for PrE) were measured after the treatments. The expression levels of the marker genes with the inhibitor were examined during embryo development. Results: Microinjection targeting the PDGF receptor (PDGFR) A reduced the number of SOX17-positive cell populations in a subset of day 7 blastocysts (n = 9/12). However, microinjection accompanied diminution of Epi cells in the blastocyst. The PDGF receptor inhibitor AG1296 (5 µM) suppressed SOX17-positive cells without reducing SOX2-positive cells in both parthenogenetic activated and in vitro fertilized embryos. Within the transcriptional target of PDGF signaling, the inhibitor significantly upregulated the Txnip gene in embryos. Conclusion: We identified that PDGF signaling is important to sustain the PrE population in porcine blastocysts. Additionally, treatment with inhibitors was a better method to suppress PrE cells than CRISPR/Cas9 microinjection of anti-PDGF receptor α gene, because microinjection suppressed number of Epi cells. The PDGF receptor might control the number of PrE cells by repressing the proapoptotic gene Txnip. Our results can help to isolate Epi-specific cell lines from blastocysts.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.68-68
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• 2001

Blastocyst formation, consisting of the inner cell mass (ICM) and trophectoderm (TE), is the first differentiation process during embryonic development in mammals. It has been hypothesized that the proportion of ICM to TE in the blastocyst may be crucial for subsequent developmental competence of early embryos, which it may be expressed as a sensitive indicator for evaluating in vitro systems. In this study ICM/total cell ratio of nuclear transfer (NT) embryos was compared with IVF-derived and in vivo embryos. Somatic cell nuclei obtained from a fetus at Day 40 of gestation were transferred into the enucleated oocyte and then cultured in NCSU 23 medium for 6 days as previously described (Koo et al., Biol. Reprod. 2000; 63:986-992). ICM and TE cells of blastocysts were determined by using a differential staining method (Han et al., Biol. Reprod. 1999; 60:1110-1113). Development rate (9.8$\pm$2.5%, 23/225) to the blastocyst stage of NT embryos was lower than IVF embryos (23.8$\pm$2.7%, 53/223). Thus, a difference was detected in the in vitro developmental rate to blastocyst stage between NT and IVF-derived embryos (P<0.05). In the next experiment, we investigated ICM and TE nuclei to assess the quality of blastocysts that produced by NT, IVF and in vivo, respectively. NT blastocysts (27.6$\pm$8.3) showed a smaller total cell number than IVF-derived (42.6$\pm$17.4) and in vivo embryos (283.9$\pm$103.5) (P<0.05). Ratios of ICM/total cells in NT, IVF and in vivo blastocysts were 15.1$\pm$ 18.6% (n=56), 12.3$\pm$9.2% (n=57) and 30.4$\pm$6.8% (n=40), respectively. Individual blastocysts for the ratio of ICM/total cells were assigned to 3 groups (I; <20%, II; 20 to 40% and III;>40%). As the results, most in vivo blastocysts (97.5%, 39/40) were distributed into group II while most NT (78.6%, 44/56) and IVF-derived blastocysts (82.5%, 47/57) were allocated to group I. Thus, our data show that NT or IVF-derived embryos have aberrant morphology during early development in vitro systems, suggesting that these anomalies may result in developmental failures of the NT embryos to term.

• Reproductive and Developmental Biology
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• 제39권3호
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• pp.89-95
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• 2015

체외 배양액에 성장호르몬 및 사이토카인의 첨가는 초기배 발육 및 생산된 배반포의 질에 영향을 미칠 수 있다. 본 연구는 돼지 유도만능줄기세포(porcine induced pluripotent stem cell, piPSC)의 조정배지(conditioned medium, CM)가 돼지 난자의 체외성숙 및 단위발생 후 초기배 발육에 미치는 영향을 검토하기 위하여 수행하였다. 난자-난구세포 복합체(cumulus-oocyte complex, COC)는 0(control), 25, or 50%의 줄기세포 배양액(stem cell medium, SM) 또는 CM이 첨가된 체외성숙 배양액으로 배양하였으며, 성숙된 난자는 활성화 유도 후 같은 농도의 SM 또는 CM을 첨가한 체외배양액에서 배양하였다. 체외 성숙율은 CM-25% 그룹에서 대조구보다 유의적으로 높았으나 (p<0.05), 다른 SM 또는 CM 처리구와는 차이가 없었다. 배반포 형성율은 CM-25% 그룹(29.2%)에서 대조구(20.7%), SM-50%(19.6%) 및 CM-50%(23.66%) 처리구보다 유의적으로 높았다(p<0.05). 배반포에서의 세포수 및 세포사 비율은 SM-25% 그룹이 대조구에 비하여 유의적인 차이가 나타났다(p<0.05). 난자의 질과 연관되어 있는 유전자들(Oct4, Klf4, Tert 및 Zfp42)의 발현은 CM-25% 그룹에서 대조구보다 유의적으로 증가되었다(p<0.05). 따라서 본 실험의 결과 체외성숙(IVM) 및 체외발달(IVC) 배양액에 25% 수준의 CM의 첨가는 돼지 단위발생 난자의 배발달과 난자의 질적 향상에 기여하는 것으로 사료된다.