• Journal of Plant Biotechnology
• /
• v.45 no.4
• /
• pp.347-356
• /
• 2018

Chionanthus retusus is a small deciduous tree that is widely used in landscaping due to its beautiful white spring flowers and ornamental value. Conventional propagation through seeds requires one to two years of breaking dormancy. The objective of this study was to determine the conditions of in vitro germination in C. retusus. In vitro embryo culture was carried out to investigate the effects of six factors: basal media (McCown Woody Plant Medium (WPM) and Murashige and Skoog (MS)); plant growth regulators (different combinations and concentrations of naphthaleneacetic acid (NAA), 6-Benzylaminopurine (BA), and gibberellic acid ($GA_3$)); embryo age (collected weekly beginning 36 days after fruit setting); low temperature pretreatment (storing $4^{\circ}C$ for 1, 2, 3, and 4 weeks); coconut additives (100, 200, and $300ml{\cdot}L^{-1}$); and genotype (grouping plants depending on their flowering nature). The basal medium used in this study was WPM with $2mg{\cdot}L^{-1-1}\;GA_3$, $20g{\cdot}L^{-1}$ sucrose, and $6g{\cdot}L^{-1}$ Agar. WPM medium mixed with $GA_3$, resulted in higher germination rate as compared to when using a combination of auxin and cytokinin. $GA_3$ at $2mg{\cdot}L^{-1}$ was the most effective of all combinations and concentrations of PGRs. WPM medium with $2mg{\cdot}L^{-1}GA_3$ resulted in better and faster germination (75.93%). Embryos collected at 57 days after fruit setting had the highest percent of germinated seeds (87.04%) while low-temperature pretreatment of fruits at $4^{\circ}C$ for two weeks produced the highest germination (95.37%). These results of this study could be an open ground for development of an efficient protocol for commercial production of the ornamental tree.

• Clinical and Experimental Reproductive Medicine
• /
• v.50 no.3
• /
• pp.177-184
• /
• 2023

Objective: Reconstructed oocytes after polar body genome transfer constitute a potential therapeutic option for patients with a history of embryo fragmentation and advanced maternal age. However, the rescue of genetic material from the first polar body (PB1) through introduction into the donor cytoplasm is not yet ready for clinical application. Methods: Eighty-five oocytes were obtained following in vitro maturation (IVM) and divided into two groups: PB1 nuclear transfer (PB1NT; n=54) and control (n=31). Following enucleation and PB1 genomic transfer, PB1 fusion was assessed. Subsequently, all fused oocytes underwent intracytoplasmic sperm injection (ICSI) and were cultured in an incubator under a time-lapse monitoring system to evaluate fertilization, embryonic morphokinetic parameters, and cleavage patterns. Results: Following enucleation and fusion, 77.14% of oocytes survived, and 92.59% of polar bodies (PBs) fused. However, the normal fertilization rate was lower in the PB1NT group than in the control group (56.41% vs. 92%, p=0.002). No significant differences were observed in embryo kinetics between the groups, but a significant difference was detected in embryo developmental arrest after the four-cell stage, along with abnormal cleavage division in the PB1NT group. This was followed by significant between-group differences in the implantation potential rate and euploidy status. Most embryos in the PB1NT group had at least one abnormal cleavage division (93.3%, p=0.001). Conclusion: Fresh PB1NT oocytes successfully produced normal zygotes following PB fusion and ICSI in IVM oocytes. However, this was accompanied by low efficiency in developing into cleavage embryos, along with an increase in abnormal cleavage patterns.

• Journal of Embryo Transfer
• /
• v.30 no.2
• /
• pp.83-89
• /
• 2015

The sexual maturation occurred by the changes of steroid hormones was known to sex-dependently and/or age-dependently regulate the lipid metabolism in various animal species. Our current study demonstrates that lipid and its functional fatty acids can be changed depending on the status of sexual maturation. Of the functional fatty acids, ${\gamma}$-linolenic acid (GLA; 18:3n-6) is an important factor for maintaining human health. The purpose of our study was to investigate the level of GLA in mice with different stages of sexual maturation. To this end, the longissimus muscle (LM) of immature (3-week-old) and mature (7-week-old) female mice was analysed for the fatty acid composition by gas chromatography. Furthermore, both gene and protein level of ${\Delta}6$ desaturase (FADS2) which is involved in GLA metabolism by real time PCR and Western blotting, respectively. Mature females showed greater (P<0.05) serum $17{\beta}$-estradiol (E2) level and LM GLA contents than immature group. The mRNA and protein levels of FADS2, which converts precursor linoleic acid into GLA, were higher (P<0.05) in mature female mice than in immature mice. In conclusion, these results show that sexual maturation of female mice induces GLA and FADS2 contents in LM.

• Journal of Embryo Transfer
• /
• v.17 no.3
• /
• pp.195-201
• /
• 2002

This study was to assess the developmental capacity of oocytes matured in vitro after 20, 15, 10, 5 and 0 days of organ culture when ovaries were isolated from juvenile mice at 0-, 5-, 10-,15- and 20-day old, respectively, and to develop in vitro culture system that observed a view to morphology of ovaries and nucleus maturation of oocytes. The size of ovaries decreased 35.9%, 8.7%, 1.2% and 14.4% after 20, 15, 10, 5 days of organ culture when the ovaries were isolated from 0-, 5-, 10 and 15-day old mice, respectively. After organ culture, the recovery rates, diameters of oocytes and the number of oocytes progressed from GV to MII were increased as increasing age of mice.

• Journal of Korean Academy of Oral and Maxillofacial Radiology
• /
• v.6 no.1
• /
• pp.45-50
• /
• 1976

The author observed morphological change in palate development of rat embryo after irradiation of x-ray on the one side of the duplex uterus. The time-matings occured between 6 p.m. and 8 p.m. and all females with copulation plugs at 8 a.m. were isolated and properly marked for evidence of copulation. The lower left abdomen of mothers were exposed to x-radiation on the 7 1/2th, 9 1/2th, 11 1/2th day of gestation, respectively 150, 250, 350, 500rads. At 18 1/2th day of post-conception, the pregnant females were dissected and the contents of the two uteri examined. The translucent sample by Alizarin red S stain were prepared. The results were as follows; 1. The result that groups irradiated by 250rads and 350rads made marked difference in comparison with the control group suggests the x-ray to be a inducing factor of cleft palate. 2. At 11 1/2th day of gestation, incidence of cleft palate induced by x-irradiation was highest. 3. Mortality showed the highest frequency at 7 1/2th day of gestation and tended to decrease in according to increasing of age. 4. Morphology of cleft palate induced by x-irradiation showed similarity in comparison with those induced by other factors having reported ever.

• Clinical and Experimental Reproductive Medicine
• /
• v.47 no.2
• /
• pp.94-100
• /
• 2020

The inverse correlation between maternal age and pregnancy rate represents a major challenge for reproductive endocrinology. The high embryo ploidy error rate in failed in vitro fertilization (IVF) cycles reflects genetic misfires accumulated by older oocytes over time. Despite the application of different follicular recruitment protocols during IVF, gonadotropin modifications are generally futile in addressing such damage. Even when additional oocytes are retrieved, quality is frequently poor. Older oocytes with serious cytoplasmic and/or chromosomal errors are often harvested from poorly perfused follicles, and ovarian vascularity and follicular oxygenation impact embryonic chromosomal competency. Because stimulation regimens exert their effects briefly and immediately before ovulation, gonadotropins alone are an ineffective antidote to long-term hypoxic pathology. In contrast, the tissue repair properties (and particularly the angiogenic effects) of platelet-rich plasma (PRP) are well known, with applications in other clinical contexts. Injection of conventional PRP and/or its components (e.g., isolated platelet-derived growth factors as a cell-free substrate) into ovarian tissue prior to IVF has been reported to improve reproductive outcomes. Any derivative neovascularity may modulate oocyte competence by increasing cellular oxygenation and/or lowering concentrations of intraovarian reactive oxygen species. We propose a mechanism to support intrastromal angiogenesis, improved follicular perfusion, and, crucially, embryo ploidy rescue. This last effect may be explained by mRNA upregulation coordinated by PRP-associated molecular signaling, as in other tissue systems. Additionally, we outline an intraovarian injection technique for platelet-derived growth factors and present this method to help minimize reliance on donor oocytes and conventional hormone replacement therapy.

• Journal of Embryo Transfer
• /
• v.14 no.3
• /
• pp.163-169
• /
• 1999

This study was carried out to investigate the effect of co-culture system(bovine oviduct epithelial cells; BOEC) and defined culture system(modified TALP ; mTALP) on the development of IVM-IVF embryos, and survival of in vitro produced blastocysts after freezing and thawing. Occytes from the slaugheterhous ovaries were matured and fertilized using general protocol. The results obtained were as the following: 1. Survival rates of frozen-thawed blastocysts using 10% glycerol as cryoprotectant was higher in day 7 blastocysts than in Day 8 and 9 blastocysts from co-cultrue system, but survival rate of frozen-thawed blastocysts was higher in Day 10 blastocysts than in day 8 and 9 blastocysts from defined culture system. Regardless of their age, survival rate of frozen-thawed blastocysts was significantly higher (p<0.05) in co-culture system than in defined culture system. 2. The cell number of blastocysts was significanlty higher (p<0.05) in Day 7 blasotcysts than in Day 8 and 9 blastocysts from co-cultures, but the cell number of blsstocysts was significantly higher (p<0.05) in Day 10 blastocysts than in Day 8 and 9 blastocysts from defined culture system. Regardless of the culture system, blastocysts with higher cell number showed higher survival rates after freezing and thawing.

• Clinical and Experimental Reproductive Medicine
• /
• v.26 no.1
• /
• pp.75-81
• /
• 1999

The beneficial effect of glucose and phosphate ions in culture medium on the development of human embryos in vitro has not been fully elucidated. The purpose of this study was to evaluate the influence of fertilization and culture of embryos in glucose/phosphate-free m-TALP medium on pregnancy rates in IVF-ET program. The patients in 244 IVF-ET cycles received GnRH agonist + HMG regimens. A does of 10,000 IU HCG was administered when two or more dominent follicles reached 18mm in diameter. Thirty-six hours after HCG, oocytes were recovered transvaginally using ultrasound guidance. Aspirated oocytes were matured for 4 to 6 h in TCM-199 supplemented with 10% follicular fluid (FF). Insemination was carried out with 50,000 motile spermatozoa in TCM-199 + 10% FF or m-TALP + 5% FF + 5% fetal cord serum (FCS) according to experimental design. After 6 h, oocytes were washed 3 to 4 times and cultured in each fresh medium. After 20 h, oocytes were freed from cumulus/corona cells and examined for the presence of pronuclei. Fertilized oocytes were transferred into each co-culture drops and cultured for further incubation. On day 3, embryo transfer was performed with grade 1 and 2 embryos. Monolayers for co-culture of embryos were prepared by plating $1{\times}10^5$ cumulus cells/ml in 10ul drop of TCM-199 + 10% FF or m-TALP + 5% FF + 5% FCS media 24 h prior to the onset of co-culture. Development to 4 to 16 cell stage was observed at 70x magnification following two days of incubation. Pregnancy was confirmed by detecting increasing serum ${\beta}$-hCG concentrations for 11 days following embryo transfer. Data were analyzed by ${\chi}^2$-test. Oocytes from 244 IVF-ET cycles were randomized. The number of cycles and mean age of patients were 97 and 147, 31.3 yrs and 31.2 yrs for TCM-199 (control) and m-TALP groups, respectively. The mean number of retrieved oocytes/cycle, fertilization rates, number of embryos transferred/ET and pregnancy rates were 11.1 and 10.3, 65.1% and 67.3%, 4.1 and 4.7, 28.9% and 43.8% for TCM-199 and m-TALP groups, respectively. Differences in the pregnancy rates were found between control and m-TALP groups (p<0.05). The pregnancy rate of patients divided according to maternal age groups of ${\leq}30$, 31-35, $36{\leq}$ were 44.4% and 49.0%, 26.1% and 41.3%, 29.2% and 41.2% for control and m-TALP groups, respectively. These data indicate that culture of human embryos in glucose/phosphate-free m-TALP medium improves pregnancy rates.

• Current Research on Agriculture and Life Sciences
• /
• v.4
• /
• pp.109-113
• /
• 1986

In order to study the changes of pregnancy rates according to the factors, age, season, repeated utilization, synchrony, of recipients (29 holstein heifers, one Korean native heifer and 3 holstein cows) were transfered one embryo (from morulae stage to advanced blastocyst) to each, and the results are as follows ; 1. The effect of age of recipient on pregnancy rate in cows and 18-24months of aged group are 100% and 78%, respectively. Under 14months of aged group recorded the worst, 67%. 2. In winter (Nov.-Jan.) and spring (Feb.-Apr.), the pregnancy rate were better than others 100%, 83%, respectively. In the autumn (Aug.-Oct.), it was only 50%, the worst. 3. Among 31 recipients which were utilized at the first, 25 were pregnanted (80.7%), but in the second utilization after the failure of the first transfering, 2 heifers, none of pregnancy was obtained. 4. Pregnancy rates related to synchronization of estrus between donor and recipients are;-(before donor)12hrs. (100%), -6hrs. (86%), +( after donor)6hrs. (67%), + 12hrs. (79%), and +over 12hrs. (50%), respectively. 5. Differences in the pregnancy rate of the recipients +over 12hrs. were significantly large, so in order to ger good pregnancy rate in embryo transfer, the differences of the synchronization must be reduced inner ${\pm}12hrs$. In general, recipients estrus before donor showed better pregnancy rate than estrus after donor.

• Journal of Embryo Transfer
• /
• v.10 no.2
• /
• pp.105-113
• /
• 1995

This study was conducted to examine the efficiency of enucleation and blastomere isolation from recipient oocytes and donor embryos, respectively and to determine the effect of oocyte age and electric voltage on the fusion rate and in vitro development of the fused oocytes in rabbit nuclear transplantation. Immature oocytes collected from ovarian follicles were matured in vivo for 12 h in TCM-199 containing FCS and hormones and in vivo matured oocytes were collected 17 to 18 h post-HCG. The fresh and frozen donor embryos of 8- to 16-cell stage were collected from the oviduct of superovulated does. The proportion of successfully enucleated oocytes was greatly lower in in vitro matured oocytes (42.3%) than that (62.7%) in in vivo matured oocytes The level of cytochalasin B for in vivo matured oocytes did not affect the efficiency of enuleation, but 7.5 $\mu$g /mL cytochalasin B for in vitro matured oocytes showed a high enucleation rate significantly. The isolation efficiency of a single blastomere nucleus did not differ between 8- and 16-cell stage embryos. The percentage of single blastomeres isolated from 16-cell stage fresh embryos after 0.5% pronase treatment was greatly higher at 16-min treatment (94.4%) than at 8-min(78. 1%) and the blastomeres(61.5%) isolated from frozen-thawed embryos after 16-min pronase were significantly fewer than those of fresh embryos. The age of recipient oocytes affected nuclear fusion rate. The reconstituted oocytes fused at 24-h age showed slightly higher fusion rate (77.8%) than those (65.0%)fused at 18-h age. The fusion rate of in vitro and in vivo matured oocytes inserted with fresh blastomere did not differ among electric voltages, but the cleavage rate and development to morula-blastocysts of in vitro matured oocytes was more higher under 0.6 kV/cm than under 0.8 to 1.2 kV/cm, while the cleavage rate and development of in vivo matured oocytes was higher under 0.8 to 1.0 kV/cm than under 1.2 kV/cm. The fusion and cleavage rate fol1owing insertion with frozen-thawed blastomere was not different between the in vitro and in vivo matured oocytes and was similar to those from fresh blastomere insertion.