• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2002.11a
• /
• pp.84-84
• /
• 2002

The advantages of the OPS techniques(Vajta G et al, Mol Reprod Dev 51: 53-58,1998) give 1) high survival rates of various types of eggs, 2) quick and simple process, 3) inexpensive equipment and reduced chilling injury. The efficiency of IVM/IVF technique in the porcine species is relatively lower than that obtained in other species such as ruminants. Two experiments were designed to investigate the effects of in-vitro fertilization of porcine oocytes matures using different OPS protocol for chilling and warming of vitrification. Porcine oocytes from ovaries collected at abattoir were matured for 44 hours in TCM199 Earle's salt supplemental with pyruvate, pff, L-cysteine, hormones and gentamycin. Oocytes were denuded and fertilized with frozen boar semen by common method. Porcine embryos produced routinely by in-vitro culture system of NCSU23 medium. The vitrification and the warming were conducted by OPS method with the glass micropipette instead of straw vessels and modified the protocol of G.Vajta(1999). In Exp 1, Chilling/Warming:Holding Medium(HM)+EG+DMSO/HM +sucrose Medium(SM) at 39$^{\circ}C$ warm stage. In Exp 2, : PBS+CS+EG+Ficoll+ Trehalose/PBS+Trehalose at 25$^{\circ}C$ stage. Filling, freezing, packing, thawing out and further culturing were performed to follow the basic protocol of G Vajta. During IVM-lVC and post-warming, fertilization parameter and developmental potential were compared to and statistically analysed. It was not significantly different from Exp 1 and Exp 2 but 25$^{\circ}C$ of stage was slightly higher on the morula/blastocyst forming rate and better atmosphere for worker than that at 39$^{\circ}C$ stage.

• Development and Reproduction
• /
• v.18 no.1
• /
• pp.33-41
• /
• 2014

We investigated the process of yolk absorption in Korean rose bitterling, Rhodeus uyekii, and determined the changes in its morphometric characteristics. The R. uyekii from 1 days post hatching (DPH) to 21 DPH, the eye diameter (ED) was 5.4 at 5 DPH. Thereafter, the ED/total length (TL) ratio increased to 10.7 at 21 DPH (p<0.05). The yolk length (YL) decreased from 95.4 to 1.1 by 21 DPH, and this rate of decrease was greater than that for any other dimension (p<0.05). 12 morphometric dimensions/TL for the R. uyekii were measured at each sampling day from 21 DPH to 170 DPH. At just hatching, the average TL and BW were $6.1{\pm}0.09mm$ and $4.9{\pm}0.07mg$, respectively. At 53 DPH, the average TL was $12.9{\pm}0.28mm$ and the average BW was $14.7{\pm}0.72mg$; the total length growth equation was $TL=5.507e^{0.038t}$ ($R^2=0.916$). Further, the body weight growth equation was $BW=3.3647e^{0.0296t}$ ($R^2=0.9354$). The dimensions with regard to body depth showed the greatest growth rates in the external characteristics of the fish (p<0.05). The patterns of the morphometric characteristics measured in this study can be classified in three ways. The patterns were shown to be increased (y=0.0992x+12.07, $R^2=0.8333$), decreased (y=-0.0569x+42.029, $R^2=0.8395$) or maintained (y=0.005x+18.263, $R^2=0.3678$) from 21 DPH to 170. These results will provide useful indices for the successful rearing of the R. uyekii.

• Development and Reproduction
• /
• v.17 no.1
• /
• pp.63-72
• /
• 2013

A specific inhibitor of RNA polymerase II, ${\alpha}$-amanitin is broadly used to block transcriptional activities in cells. Previous studies showed that ${\alpha}$-amanitin affects in vitro maturation of cumulus-oocyte-complex (COC). In this study, we evaluated the target of ${\alpha}$-amanitin, and whether it affects oocytes or cumulus cells (CCs), or both. We treated ${\alpha}$-amanitin with different time period during in vitro culture of denuded oocytes (DOs) or COCs in comparison, and observed the changes in morphology and maturation status. Although DOs did not show any change in morphology and maturation rates with ${\alpha}$-amanitin treatment, oocytes from COCs were arrested at metaphase I (MI) stage and CCs were more scattered than control groups. To discover causes of meiotic arrest and scattering of CCs, we focused on changes of cumulus expansion, gap junctions, and cellular metabolism which to be the important factors for the successful in vitro maturation of COCs. Expression of genes for cumulus expansion markers (Ptx3, Has2, and Tnfaip6) and gap junctional proteins (Gja1, Gja4, and Gjc1) decreased in ${\alpha}$-amanitin-treated CCs. However, these changes were not observed in oocytes. In addition, expression of genes related to metabolism (Prps1, Rpe, Rpia, Taldo1, and Tkt) decreased in ${\alpha}$-amanitin-treated CCs but not in oocytes. Therefore, we concluded that the transcriptional activities of CCs for supporting suitable transcripts, especially for its metabolic activities and formation of gap junctions among CCs as well as with oocytes, are important for oocytes maturation in COCs.

• Korean Journal of Animal Reproduction
• /
• v.26 no.4
• /
• pp.329-338
• /
• 2002

This study was carried out to investigate the developmental rates of embryo reconstructed with different cell type and to estimate correlation of transcriptional level of octamer-binding transcription factor 4 (Oct4) and fibroblast growth factor 4 (FCF4) gene on peri-implantation stage embryos. Donor cells were transferred into perivitelline space of enucleated oocytes. The karyoplast-cytoplast couplets were accom- plished by cell to cell fusion and activated with ionomycin and 6-dimethylaminopurine. Reconstructed embryos were co-cultured with bovine oviduct epithelial cells in CR 1 aa medium. There is no difference in blastocyst formation rate following nuclear transfer UT) with fetal fibroblast cell (16/50; 32.0%), cumulus cell (16/49; 32.6%) and ear cell (17/52; 32.6%). The expression level of Oct4 and FCF4 in peri-implantation bovine embryo derived from in vitro fertilization (IVF) and NT were determined by reverse-transcription polymerase chain reaction (RT-PCR) technique. In peri-implantation of IVF result in a transient increased of FCF4 paralleled by an increased expression of Oct4. However, Oct4 gene was highly expressed in hatching blastocysts derived from NT compared to IVF. Also, FGF4 expression level in hatching blastocysts and outgrowth stage derived from NT was lower than that of IVF. In conclusion, it is suggested that the different transcription patterns observed in nuclear transfer embryos may lead to a lower rate of embryo development, implantation and pregnancy.

• Reproductive and Developmental Biology
• /
• v.30 no.4
• /
• pp.279-285
• /
• 2006

Vitrification has been suggested to be an effective method for the cryopreservation of human ES cells. However, the efficiency of vitrification with different vehicles remains a matter of ongoing controversy. The objective of this study was to assess the efficiency of cryopreservation in human ES cells by vitrification using different vehicles. A human ES cell line and a variety of vehicles, including micro-droplet (MD), open-pulled straw (OPS) and electron microscopic grid (EM-grid), were employed in an attempt to assess vitrification efficiency. In order to evaluate the survivability and the undifferentiated state of the post-vitrified human ES cells, we conducted alkaline phosphatase staining and characterization via both RT-PCR and immunofluorescence assays. The survival rates of the post-vitrified human ES cells using MD, OPS and EM-grid were determined to be 61.5%, 66.6% and 53.8%, respectively. There also exist significant differences between slow-freezing and vitrification (p<0.01). However, no significant differences were detected between the vehicle types. Finally, the pluripotency of human ES cells after thawing was verified by teratoma formation. Cryopreservation using vitrification is more effective than slow-freezing, and the efficiency of vehicles proved effective with regard to the preservation of human ES cells.

• Reproductive and Developmental Biology
• /
• v.30 no.4
• /
• pp.287-291
• /
• 2006

We have developed a new passaging technique for the expansion of human embryonic stem cells (hESCs) that involves simply pipetting portions of hESCs acquired from colonies, reducing the laborious and time-consuming steps in the expansion of hESCs. Compared to general mechanical methods of passaging, our pipetting method allowed hESCs colonies to be broken into small fragments, which showed significantly higher attachment rates onto feeder cell layers. This technique produced three times the number of hESCs colonies than conventional mechanical methods. In addition, this pipetting method allowed us to distinguish differentiated hESCs from undifferentiated hESCs during hESCs colony pipetting. The hESCs cultured by pipetting method displayed normal human chromosomes for over 60 passages. According to RT-PCR and immunohistochemical analysis, the hESCs successfully maintained their undifferentiated state and pluripotency which was also confirmed by teratoma formation in viva Therefore, the pipetting method described in this study is a useful tool to efficiently and quickly expand hESCs on a large scale without enzyme treatment.

• Reproductive and Developmental Biology
• /
• v.32 no.3
• /
• pp.147-152
• /
• 2008

The aim of this study was to investigate what components of porcine epididymal fluid (pEF) influences the nuclear maturation of porcine germinal vesicle oocytes. Porcine cumulus-oocytes complexes from follicles were cultured in TCM 199 containing pEF. After 48 h cultures, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. Maturation rate of oocytes was significantly increased in media supplemented with 10% pEF during in vitro maturation (IVM) than in those without pEF. When lipid component of pEF was removed by treating n-heptane, no significant difference was observed in maturation of oocytes between n-heptane treatrment and intact pEF group. However, the proportion of oocytes reaching at metaphase II (M II) was significantly (p<0.05) decreased in the oocytes cultured in media containing trypsin-treated pEF compared to those in media with intact pEF. When porcine GV oocytes were matured in the medium supplemented with intact pEF or pEF heated at $56^{\circ}C$ and $97^{\circ}C$, rates of oocytes remained at GV stage were 11.7%, 29.4% and 42.0%, respectively. However, there were no difference in proportion of oocytes reaching at MII stage among intact pEF group and $56^{\circ}C$ group. Present study suggests that 1) pEF contains an enhancing component(s) for nuclear maturation in vitro of oocytes, 2) protein(s) of pEF may be capable to promote nuclear maturation in vitro, and 3) enhancing component for nuclear maturation may consist of two factors, which are responsible for germinal vesicle breakdown (GVBD) and promotion of MII stage.

• Reproductive and Developmental Biology
• /
• v.32 no.1
• /
• pp.45-49
• /
• 2008

We have previously produced transgenic (TG) mice expressing the human lactoferrin (hLF), interleukin-10 (hIL-10), and thrombopoietin (hTPO) proteins in the milk. In this study, we examined whether simple crossbreeding between two kids of a single transgenic mouse can produce double transgenics co-expressing two human proteins.. The hLF male, and the hIL-10 male were crossbred with the hIL-10 and hTPO females, and the hTPO female, respectively. PCR analysis for genotyping showed 32%, 23% and 24% double transgenic rates for hLF/hIL-10, hLF/hTPO, and hIL-10/hTPO transgenes, respectively. We analyzed the expression levels of the human proteins from double transgenic mice and compared those with their single transgenic siblings. All double transgenic co-expressed two human proteins at comparable levels to singles', unless hTPO was not co-expressed: for hLF, 1.1 mg/ml in hLF/hIL-10, whereas 0.5 mg/ml in hLF/hTPO; for hIL-10, 4.1 mg/ml in hIL-10/hLF, whereas 1.4 mg/ml in hIL-10/hTPO. Ihe downregulation of hTPO to half level of singles' was observed in double transgenic mice. The possible reason why hTPO co-expressed might lead to down-regulation of another human protein was discussed. These results suggested that double transgenic generated by crossbreeding between two singles' could be useful system for bioreactor.

• Reproductive and Developmental Biology
• /
• v.32 no.1
• /
• pp.39-43
• /
• 2008

Most studies on transgenic bioreactors have focused on expression levels of interest genes. In this study we examined whether transgenic bioreactors would inherit expression level of the Oansgene to long-term generations independently of transgene sources. We employed three transgenic mice, which were separately reported, carrying different transgenes and copy numbers, 27 kb of hLF and 22 kb of hIL-10 genomic sequences, and 1.3 kb of hTPO cDNA, respectively. Three females of the transgenic lineages crossbred with a wild-type male up to 20 generations to test transgenic frequencies of their progenies and to determine expression levels of the transgenes. Ultimately, transmission rates of kLF, hIL-10, and hTPO were $64.3{\pm}7.0$, $59.3{\pm}9.8$, and $56.1{\pm}9.7$, respectively, appeared following Mendelian pattern of inheritance. Notably, we found that levels of expressions of hLF, hIL-10, and hTPO in milk were sustained to high numbers of generations. No transgene silencing of expression was observed in every generations of all transgenic mice. In conclusion, we suggest that once established animal bioreactors could consistently transmit the transgene to continual generations, without loss of expressional activity, independently of transgene sources.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.51 no.1
• /
• pp.31-41
• /
• 2018

Seaweeds are composed of a variety of bioactive substances, including polysaccharides, pigments, minerals, peptides, and polyphenols. Among these substances, the arsenic content of seaweeds has been a significant cause for concern. The present study evaluated the toxicity of arsenic from three species of seaweed using a zebrafish Danio rerio model. The arsenic-rich extracts were obtained from Ecklonia cava (ECAE), Undaria pinnatifida (UPAE) and Hizikia fusiformis (HFAE) using a solvent of 50% methanol and 1% $HNO_3$. We investigated the toxicity of the arsenic-rich extracts in zebrafish embryos through survival rate, heart rate, yolk sac edema size, cell death, reactive oxygen species (ROS) production and real-time polymerase chain reaction (PCR). The hepatotoxicity of arsenic-rich extracts was assessed in the liver of adult zebrafish through real-time PCR and histopathology. The survival rates of embryos and adult zebrafish showed no significant changes at any concentration. At 100 ppm, embryos did not exhibit significant differences in heart rate, yolk sac edema size, cell death or ROS production. In addition, apoptosis-related genes in larvae and liver tissue were unaffected by treatment with arsenic-rich extracts. These data will help clarify that developmental changes, hepatic oxidative stress, and apoptosis are not associated with toxicity from arsenic-rich seaweed extracts in a zebrafish model.