• Reproductive and Developmental Biology
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• 제38권4호
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• pp.143-146
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• 2014

Antioxidants, as reactive oxygen species scavengers, are one of the beneficial additives in serum-free defined culture medium. In this study, three separate experiments were performed to determine the effects of 3-hyroxyflavone added to the culture medium on the developmental competence of follicular bovine oocytes during in vitro maturation (IVM) and/or in vitro culture (IVC). The rate of blastocyst developed from oocytes cultured in IVM medium with 3-hyroxyflavone was significantly higher than that from control oocytes (39.0% vs. 26.3%, p<0.001), respectively. However, oocytes cultured in the medium with addition of 3-hyroxyflavone only at IVC period did not show significance in the blastocyst development when compared with control. When 3-hyroxyflavone was added to both IVM and IVC media, the rate of blastocyst formation was even significantly lower (21.1%) than control (26.5%; p<0.05). The present findings suggested that antioxidative activity of 3-hydroxyflavone added to only IVM medium beneficially affected the developmental competence of follicular bovine.

• Development and Reproduction
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• 제16권1호
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• pp.69-75
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• 2012

Hatching occurred in the time dependent manners and strictly controlled. Although, the hatching processes are under the control of muti-embryotrophic factors and the expressed G proteins of cell generate integrated activation, the knowledge which GPCRs are expressed during hatching stage embryos are very limited. In the present study, which G proteins are involved was examined during blastocyst development to the hatching stage. The early-, expanded-, and lobe-stage blastocysts were treated with various $G_{\alpha}$ activators and H series inhibitors, and examined developmental patterns. Pertusis toxin (PTX) improved the hatching rate of the early-stage blastocyst and lobe-formed embryos. Cholera toxin (CTX) suppressed the hatching of the early-stage blastocyst and expanded embryos. The effects of toxins on hatching and embryo development were changed by the H7 and H8. These results mean that PTX mediated GPCRs activation is signaling generator in the nick or pore formation in the ZP. In addition, PTX mediated GPCR activation induces the locomotion of trophectoderm for the escaping. CTX mediate GPCRs activation is the cause of suppression of hatching processes. Based on these data, it is suggested that various GPCRs are expressed in the periimplantation stage embryos and the integration of the multiple signals decoding of various signals in a spatial and temporal manner regulate the hatching process.

• Proceedings of the KSAR Conference
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.11-11
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• 2001

In this study, we examined the developmental potential of reconstructed bovine embryos and the fate of donor mitochondria during their preimplantation development after nuclear transfer. Isolated cumulus cells were used as donor cells in nuclear transfer. Cumulus cells labelled with MitoTracker Green FM fluorochrome were injected into enucleated bovine MII oocytes and cultured in vitro. MitoTracker labelling on donor cells did not have a detrimental effect on blastocyst formation following nuclear transfer. Cleavage rate was about 69%(56/81) and blastocyst formation rate was 6.2% (5/81) at 7 days after nuclear transfer. The labelled mitochondria dispersed to the cytoplasm and became distributed among blastomeres and could be identified up to the 8- to 15-cell stages. Small patches of mitochondria were detected in some 8- to 15-cell stage embryos (5/20). However, donor mitochondria were not detected in embryos at the 16-cell stage and subsequent developmental stages. In the control group, mitochondria could be identified in arrested 1-cell embryos up to 7 days after nuclear transfer These results suggest that donor mitochondria disappear from recipient cytoplasm before 16-cell stage following nuclear transfer in reconstructed bovine embryos.

• Reproductive and Developmental Biology
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• 제28권4호
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• pp.221-226
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• 2004

The present study was performed to investigate the effects of in vitro maturation (IVM) and in vitro fertilization (IVF) duration on the development of Korean Native Cattle embryos. The time of blastocyst formation and the quality of blastocysts based on cell numbers were examined. The cleavage rate increased with the length of IVF duration in the groups of 18-hr IVM, but was constant in the groups of 24-hr IVM. The development rate to the 8-cell stage was significantly higher in the IVM 18: IVF 20 group than in the IVM 24: IVF 24 group. The development rate to the blastocyst stage was highest in the IVM 18: IVF 20 group, significantly different from that of the IVM 18: IVF 16, IVM 24: IVF 20 and IVM 24: IVF 24 group. The time of blastocysts formation tended to be shorter when IVM and IVF duration were decreased. The number of inner cell mass, trophoblast and the total cells were significantly higher in the IVM 18: IVF 16 group than in the IVM 24: IVF 24 group (P<0.05). These results demonstrated that the IVM and IVF duration should be adequate for the efficient production of bovine embryos, and it might particularly be essential to determine the proper combination of IVM and IVF duration.

• Korean Journal of Animal Reproduction
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• 제17권4호
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• pp.365-373
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• 1994

To investigate the effect of EDTA on the in vitro development of parthenogenetic eggs of ICR strain mice, those were cultured in 35mm culture dishes containing NaHCO3-BMOC-3 medium supplemented with 10, 50, 100, or 500$\mu$M of EDTA at 37$^{\circ}C$ for 96hrs. under the atmosphere of 5% CO2 and 95% air. EDTA supplementation of 10, 50, or 100$\mu$M to medium significantly(P<0.01) increase morula and blastocyst formation rate compared with controls in haploid(19.8, 25.9, 39.0% vs. 0.0%). And compared with 10, or 50$\mu$M of EDTA supplementation, significantly(P<0.01) higher morula and blastocyst formation rate resulted from EDTA supplementatin of 100$\mu$M. Both the nuclear number and diameter of blastocysts developed from parthenogenetic eggs were not affected by the morphological types when they were cultured, or the supplementary concentrations of EDTA. The nuclear number of blastocysts developed from haploid, diploid, and immediately cleavaged eggs was 44.8$\pm$1.2, 45.2$\pm$1.5, and 45.4$\pm$1.8, respectively. And the diameter of those eggs ranged 104.4$\pm$1.8, 104.3$\pm$1.2, and 103.8 1.3$\mu$m, respectively.

• Clinical and Experimental Reproductive Medicine
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• 제47권2호
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• pp.130-134
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• 2020

Objective: The aim of this study was to compare the effects of conventional insemination (in vitro fertilization [IVF]) and intracytoplasmic sperm injection (ICSI) on the fertilization, developmental competence, implantation potential, and clinical pregnancy rate of embryos derived from in vitro matured oocytes of patients with polycystic ovary syndrome (PCOS). Methods: A prospective study was carried out among 38 PCOS patients who had undergone In vitro maturation (IVM) treatment. In total, 828 immature oocytes were collected from 42 cycles and randomly assigned for insemination by IVF (416 oocytes) or ICSI (412 oocytes). After fertilization, the embryos were cultured until the blastocyst stage and single embryos were transferred after endometrial preparation and under ultrasound guidance. Results: No significant differences were found in the maturation rate (78.1% vs. 72.6% for IVF and ICSI insemination, respectively; p= 0.076), fertilization rate (59.4% vs. 66.9% for IVF and ICSI insemination, respectively; p= 0.063), or the formation of good-quality blastocysts (40.9% vs. 46.5% for IVF and ICSI insemination, respectively; p= 0.314). Implantation and clinical pregnancy also did not show significant differences. Conclusion: There was a comparable yield of in vitro matured oocytes derived from PCOS patients in terms of fertilization, blastocyst formation, implantation rate, and clinical pregnancy between IVF and ICSI insemination. These findings provide valuable insights for choosing assisted reproductive treatment in women with PCOS, as IVM offers promising outcomes and is less invasive and less costly.

• Korean Journal of Animal Reproduction
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• 제27권2호
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• pp.125-133
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• 2003

The present study was carried out to examine the effect of $\beta$-Mercaptoethanol ($\beta$-ME) on in vitro maturation (IVM) of porcine follicular oocytes and oxygen concentration with $\beta$-ME on in vitro development (IVD) of porcine IVM/IVF embryos. The results were summarized as follows. 1. The rates of nuclear maturation, penetrated oocytes, polyspermic oocytes, pronucleus formation and mean numbers of the penetrated sperms were not significantly different using NCSU-23 maturation media for 0, 25, 50 and 100 $\mu$M $\beta$-ME (P>0.05). 2. The rates of blastocyst formation at day 7 after in vitro fertilization were higher in oocytes matured with 25 $\mu$M $\beta$-ME (25.4$\pm$0.9%) than in those matured with 0 (14.5$\pm$1.6%), 50 (17.3$\pm$1.7%) and 100 $\mu$M (12.4$\pm$1.3%) (P<0.05). However, no differences ware found in total cell numbers of blastocyst among the treatments. 3. The rates of blastocyst formation at day 7 after in vitro fertilization were higher in the NCSU-23 Culture medium With 25 $\mu$M $\beta$-ME (23.6$\pm$2.8%) than in those Cultured With 0 (15.4$\pm$4.4%), 12.5 (17.5$\pm$2.3%) and 50 $\mu$M $\beta$-ME (18.6$\pm$2.1%) Under the 5% and 20% $O_2$ Concentrations (P<0.05). However, no differences was found in total cell numbers of blastocyst among the treatments. These results suggested that the addition of 25 $\mu$M $\beta$-ME in the IVM/IVD media were effective on the porcine embryo production. However, the rates of blastocyst formation and total cell numbers of blastocyst at day 7 of porcine IVM/IVF embryos were not significantly different in the NCSU-23 culture medium under 5% and 20% 02 concentrations.

• Clinical and Experimental Reproductive Medicine
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• 제27권1호
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• pp.47-57
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• 2000

Objective: The effects of cryopreservation with or without coculture on the in vitro development of blastomere-biopsied 8-cell mouse embryos were investigated. This experimental study was originally designed for the setup of a preclinical mouse model for the preimplantation genetic diagnosis (PGD) in human. Methods: Eight-cell embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BL(표현불가)/CBA(표현불가)). Using micromanipulation, one to four blastomeres were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acid Tyrode's solution (ATS). A slow-freezing and rapid-thawing protocol with 1.5M dimethyl sulfoxide (DMSO) and 0.1M sucrose as cryoprotectant was used for the cryopreservation of blastomere- biopsied 8-cell mouse embryos. After thawing, embryos were cultured for 110 hours in Ham's F-10 supplemented with 0.4% bovine serum albumin (BSA). In the coculture group, embryos were cultured for 110 hours on the monolayer of Vero cells in the same medium. The blastocyst formation was recorded, and the embryos developed beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. Results: The survival rate of embryos after cryopreservation was significantly lower in the blastomere-biopsied (7/8, 6/8, 5/8, and 4/8 embryos) groups than in the non-biopsied, zona intact (ZI) group. Without the coculture, the blastocyst formation rate of embryos after cryopreservation was not significantly different among ZI, the zona drilling only (ZD), and the balstomere-biopsied groups, but it was significantly lower than in the non-cryopreserved control group. The mean number of cells in embryos beyond blastocyst stage was significantly higher in the control group ($50.2{\pm}14.0$) than in 6/8 ($26.5{\pm}6.2$), 5/8 ($25.0{\pm}5.5$), and 4/8 ($17.8{\pm}7.8$) groups. With the coculture using Vero cells, the blastocyst formation rate of embryos after cryopreservation was significantly lower in 5/8 and 4/8 groups, compared with the control, 7/8, and 6/8 groups. The mean number of cells in embryos beyond blastocyst stage was also significantly lower in 4/8 group ($25.9{\pm}10.2$), compared with the control ($50.2{\pm}14.0$), 7/8 ($56.0{\pm}22.2$), and 6/8 ($55.3{\pm}25.5$) groups. Conclusion: After cryopreservation, blastomere-biopsied mouse embryos have a significantly impaired developmental competence in vitro, but this detrimental effect might be prevented by the coculture with Vero cells in 8-cell mouse embryos biopsied one or two blastomeres. Biopsy of mouse embryos after ZD with ATS is a safe and highly efficient preclinical model for PGD of human embryos.

• Clinical and Experimental Reproductive Medicine
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• 제26권1호
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• pp.9-20
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• 1999

The genetic defects in human gametes and embryos can cause adverse effects on overall reproductive events. Biopsy of embryos for preimplantation genetic diagnosis (PGD) offers a new possibility of having children free of the genetic disease. In addition, advanced embryo culture method may enhance the effectiveness of embryo biopsy for the practical application of PGD. This experimental study was undertaken to evaluate the effects of coculture on the development in vitro of biopsied mouse embryos as a preclinical model for PGD of human embryos. Embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BLfemale/CBAmale). Using micromanipulation, 1, 2, 3 or 4 blastomeres of 8-cell stage embryos were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acidic Tyrode's solution (ATS). After biopsy of blastomeres, embryos were cultured in vitro for 110 hours in Ham's F-10 supplemented with 0.4% BSA or cocultured on the monolayer of Vero cells in the same medium. The frequence of blastocyst formation were recorded, and the embryos beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. There was no significant difference in the blastocyst formation between the zona intact control group and the zona drilling (ZD) only, or biopsied groups. The hatching rate of all the treatment groups except 4/8 group was significantly higher than that of control group. In all the treatment groups, there was a significant reduction in the mean cell number of embryos beyond blastocyst stage ($50.2{\pm}14.0$ in control group vs. $41.2{\pm}7.9$ in ZD, $39.3{\pm}8.8$ in 7/8, $29.7{\pm}6.4$ in 6/8, $25.1{\pm}5.7$ in 5/8, and $22.1{\pm}4.3$ in 4/8 groups, p<0.05). When the same treatments were followed by coculture with Vero cells, a similar pattern was seen in the blastocyst formation and the hatching rate. However, in all the treatment groups, there was a significant increase in the mean cell number of embryos beyond blastocyst stage with coculture, compared with the parallel groups without coculture. In the cleavage rate of biopsied blastomeres cultured for 110 hours after IVF, there was no significant difference between coculture and non-coculture groups (87.2% vs. 78.7%). However, the mean cell number of embryos developed from the biopsied blastomeres was significantly higher in coculture group ($11.5{\pm}4.7\;vs.\;5.9{\pm}1.9$, p<0.05). In conclusion, biopsy of mouse embryos after ZD with ATS is a safe and highly efficient method for PGD, and coculture with Vero cells showed a positive effect on the development in vitro of biopsied mouse embryos and blastomeres as a preclinical model for PGD of human embryos.

• Journal of Embryo Transfer
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• 제16권1호
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• pp.41-46
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• 2001

This study was carried out to evaluate the effect of culture methods on development of embryos with each developmental stage after heat shock in bovine oocytes. The results obtained were as fellows. 1. The culture method after heat shock on development of embryos was better drop-culture than co-culture. 2. The medium without amino acids were not effect of heat sock on development of embryos but it was in need of amino acid during formation of blastocyst.