• Korean Journal of Animal Reproduction
• /
• v.22 no.1
• /
• pp.43-50
• /
• 1998

The effect of oxygen tension on embryonic development in co-culture was evaluated from the standpoint of the reduction of dissolved oxygen concentration by the oxygen consumption of feeder cells. Three co-culture systems using bovine oviductal epitherial cells (BOEC), African green monkey kidney cells (Vero cells) or buffalo rat liver cells (BRLC) have been compared in terms of development of bovine embryos derived from oocytes matured and fertilized in vitro. Among the co-cultured embryo, Vero cells su, pp.rted the highest developmental rate (29%) and the other two showed the similar rates. When the co-cultures were incubated in three different oxygen tension such as 5, 10, 20% oxygen atmosphere, embryos co-cultured with Vero cells at 10%-O2 resulted in the highest percentage of development. From the measurement of oxygen consumption of feeder cells, BRLC consumed 1.38　10-10 mg-O2/min/cell which was higher than 0.94　10-10 and 0.26　10-10mg-O2/min/cell for Vero cells and BOEC, respectively. Based on the oxygen consumption data, the phenomena of optimum oxygen tension required in embryo development in vitro has been analyzed, and we suggested that gas phase oxygen concentration, oxygen consumption rate of feeder cells and the number of feeder cells should be considered for the design of optimal co-culture system for effective fertilization of embryos in vitro.

• Journal of The Korean Society of Grassland and Forage Science
• /
• v.14 no.3
• /
• pp.201-205
• /
• 1994

The objectives of this research are to determine the seasonal and varietal differences of total digestible nutrients(TDN) and in vitro dry matter digestibility(1VDMD) of tall fescue grown in Takwalyon areas, based on the data of preceding paper(Lee et al. 1994b). The results are may be summarized as follows : 1. The values of total digestible nutrients(TDN) and in vitro dry matter digestibility(1VDMD) in 1st cutting were lower than the values of 2nd and 3rd cutting, and it was due to different stages of growth and seasonal difference of climatic conditions in this areas. 2. The relative yield(RY) had negative significantly correlation with in vitm dry matter digestibility(1VDMD) of 1st cutting. 3. Total digestible nutrients(TDN) indicated high positive significant correlation with in vitro dry matter digestility (IVDMD) in all cuttings. 4. The variety of Johnstone showed a high values of total digestible nutrients(TDN) and in vitm dry matter digestibility(1VDMD) of 2nd and 3rd cutting, but Fuego was lower than the other varieties.

• Journal of Microbiology and Biotechnology
• /
• v.4 no.3
• /
• pp.183-190
• /
• 1994

Cell-free extracts prepared from cultured insect cells, Spodoptera. frugiperda, were analyzed for activation of early gene transcription of an insect baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). The template DNA used for in vitro transcription assays contained promoter sites for the baculovirus genes that have been classified as immediate early ($\alpha$) or early genes. These genes are located in the HindIII-K/Q region of the AcNPV genome. Nuclei isolated from the AcNPV-infected Spodoptera frugiperda cells were also used for in vitro transcription analysis by RNase-mapping the labeled RNA synthesized from in vitro run-on reaction in the isolated nuclei. The genes studied by this technique were p26 and pl0 genes which were classified as delayed early and late gene, respectively. We found that transcription of the genes from the HindIII-K region was accurately initiated and unique in the whole cell extract obtained from uninfected cells, although abundance of the in vitro transcripts was reverse to that of in vivo RNA. With isolated nuclei transcription of the p26 gene was inhibited by $\alpha$-amanitin suggesting that the p26 gene was transcribed by host RNA polymerase II. However, transcription of the pl0 gene in isolated nuclei was not inhibited by $\alpha$-amanitin, but rather stimulated by the inhibitor. We also found that the synthesis of $\alpha$-amanitin-resistant RNA polymerase was begun before 6 hr p.i., the time point at which the onset of viral DNA replication as well as the appearance of a-amanitin-resistant viral transcripts were detected. These studies give us strong evidence to support the previous data that early genes of AcNPV were transcribed by host RNA polymerease III, while transcription of late genes was mediated at least by a novel $\alpha$-amanitin-resistant RNA polymerase.

• BMB Reports
• /
• v.34 no.2
• /
• pp.102-108
• /
• 2001

A time-dependent folding process was used to determine whether or not protein disulfide isomerase (PDI) plays an important role in the maturation of nascent lactoferrin polypeptides. Interaction between lactoferrin and PDI was analyzed according to the co-immunoprecipitation of the two proteins. The results indicate that lactoferrin folding requires a significant interaction with PDI and its binding is relatively brief compared to other nascent polypeptides. The amount of lactoferrin interacting with PDI increases up to half a minute and sharply decreases beyond this time point. During the refolding process that follows reduction by DTT, lactoferrin polypeptides heavily interact with PDI and the interaction period was extended compared to the normal folding process. In terms of the temperature effect on PDI-lactoferrin interaction, PDI binds to lactoferrin polypeptides longer at a lower temperature (here, $25^{\circ}C$) than $37^{\circ}C$. The lactoferrin-PDI interaction was also studied in vitro. According to the in vitro experiment data, PDI was still functional in cell lysates assisting lactoferrin folding into the mature form. PDI interacts with lactoferrin polypeptides for an extended period during the folding in vitro. During the refolding process in vitro, intermolecular aggregates and refolding oligomers matured into a functional form after PDI binds to the lactoferrin. These results suggest that PDI provides a prolonged chaperoning activity in the refolding processes and that there appears to be a greater requirement for PDI chaperone activity in the refolding of lactoferrin polypeptides.

• Journal of Plant Biotechnology
• /
• v.40 no.1
• /
• pp.43-48
• /
• 2013

Oreorchis coreana Finet is threatened globally by over-collection from its natural habitats for horticultural purposes. Its rarity in nature makes this plant one of the most endangered species in Korea. In this study, we investigated the effects of sodium hypochlorite (NaOCl) on orchid seed viability and seed germination. An in vitro bioassay swelling test using immature seeds was compared with a standard chemical procedure using triphenyl tetrazolium chloride (TTC) to test seed viability. In general, the bioassay was more appropriate for estimating embryo viability after a prolonged pre-treatment (more than 1 h) in 1% NaOCl, a surface sterilant often used to enhance germination of seeds of terrestrial plants. Therefore, an efficient method for investigating in vitro swelling of immature seeds is urgently needed. We established a method for determining the viability and swelling of O. coreana seeds via in vitro examination of immature seeds. Treatment of immature seeds with 1% NaOCl for 10 min greatly enhanced the extent of swelling of immature zygote embryos when compared to untreated seeds. These data obtained here appear to be comparable to viability and swelling that occurs in O. coreana seeds via asymbiotic germination.

• Korean Journal of Plant Resources
• /
• v.11 no.1
• /
• pp.106-110
• /
• 1998

The present study has been undertake to obtain the fundatmental data of optimum germination condition and to establish storage time for artificial pollinationin Codonopsis lancelata pollen. In vitro condition for germination of freshly collected and stored pollen were examined. The optium temperature for germination of fresh pollen was $25^{\circ}C$. The optium sucrose concentration in the medium ranged from 30 to 40 % and optium pH 6.0% for pollen germination. The rate of pollen germination accelerated considerably in the medium with 1% agar. 30% sucrose, and pH 6. C. lanceolata pollen remained viable for 15 days when stored at 5$^{\circ}C$ with silica gel as desiccant.

• Clinical and Experimental Reproductive Medicine
• /
• v.32 no.2
• /
• pp.91-99
• /
• 2005

Objective: To define an appropriate vitrification condition of preantral follicle that yields high survival and to evaluate growth and ovulation rate of mouse follicles during in vitro culture after vitrification. Methods: Preantral follicles were isolated mechanically from mouse ovaries that were surgically recovered from mice aged 14 days. Retrieved preantral follicles were placed in EG (Ethylene Glycol) for 2, 5, 10 minutes and transferred to EFS-40 (40% EG, 18% Ficoll-70, 0.5 M sucrose) for 0.5, 1, 2 minutes. And then, preantral follicles were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing was carried out at room temperature. After defining the most appropriate vitrification condition that yields high survival, in vitro growth and ovulation rate of follicles were evaluated. Results: Appropriate vitrification condition that yield high survival rate ($83.2{\pm}2.1%$) of preantral follicle was EG for 5 minutes and EFS-40 for 0.5 minutes. In vitro survival rate of the vitrified preantral follicles were $85.5{\pm}0.5%$, $67.9{\pm}0.8%$ and $40.2{\pm}0.5%$ on day 2, 6 and 10. And in vitro growth of the vitrified preantral follicles were $107.1{\pm}16.1{\mu}m$, $117.1{\pm}18.4{\mu}m$, $178.4{\pm}45.6{\mu}m$ and $325.4{\pm}54.4{\mu}m$ on day 0, 2, 6 and 10. Although in vitro survival rate and growth of vitrified preantral follicles were lower than that of non-vitrified preantral follicles, the patterns of survival and growth were similar in vitrified and non-vitrified preantral follicles. The ovulation rate of antral follicles that was grown from vitrified preantral follicles was $32.6{\pm}1.2%$. Conclusion: Vitrified preantral follicles could be grown to antral sizes, and mature oocytes that can be used for IVF-ET programs were produced successfully. These data suggest that cryopreservation of preantral follicle by vitrification can be used for the preservation of the fertility.

• Clinical and Experimental Reproductive Medicine
• /
• v.44 no.1
• /
• pp.40-46
• /
• 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.

• International Journal of Oral Biology
• /
• v.30 no.4
• /
• pp.131-134
• /
• 2005

In the present study, performances of several in vitro maturation (IVM) systems for porcine follicular oocytes were evaluated, and an efficient chemically defined IVM system for porcine oocytes was proposed. The proposed one-step culture system supplemented with polyvinylalcohol (PVA) gave competitive efficiencies in terms of oocyte maturation and blastocyst development after parthenogenetic activation and in vitro culture, compared with the conventional two-step culture system by a supplementation of porcine follicular fluid (pFF). Additionally, it is identified that the proposed chemically defined one-step culture system yielded the comparable level of blastocyst production to the conventional maturation system in porcine somatic cell nuclear transfer (SCNT). Therefore, one can eliminate un-expected effects accompanied by supplementation of pFF. No medium replacement during whole maturation period is an additional benefit by applying this new system. Thus, these data support that the developed PVA supplemented chemically defined one-step IVM system for porcine follicular oocyte might be used in porcine SCNT program.

• Korean Journal of Agricultural Science
• /
• v.45 no.3
• /
• pp.419-427
• /
• 2018

The present study was conducted to investigate the effect of different levels of Korean corn grain on in vitro ruminal fermentation with total mixed ration (TMR) as a basal feed. Three ruminal cannulated Holstein steers (Body Weight $479{\pm}33.0kg$) were used as rumen fluid donors. Treatments for in vitro fermentation were TMR only (control, 3.0 g), TMR substituted partially with high level (HC, TMR 1.5 and corn 1.5 g), and with low level of Korean corn grain (LC, TMR 2.25 and corn 0.75 g), respectively. To measure in vitro ruminal pH, gas production, ammonia N and volatile fatty acids (VFA), the in vitro fermentation incubation was triplicated at $39^{\circ}C$, 120 rpm for 0, 1, 3, 6, 12, 24 and 48 h, respectively. Mean ruminal pH was significantly lower (p < 0.05) for HC than control. Changes in rumen pH was rather similar between the groups till 6 h after incubation, but the lowest pH for HC (pH 5.10) appeared at 48 h compared with control and LC. Total gas production was tended (p < 0.09) to be higher and ammonia N was significantly lower (p < 0.05) for HC than control and LC. Total VFA was higher (p < 0.05) for HC and LC than control but no differences appeared between HC and LC. Overall, the present data indicate that feeding different levels of Korean domestic corn grain may lead to high and sustainable starch degradation in the rumen.