• 한국수정란이식학회지
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• 제15권1호
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• pp.15-21
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• 2000

These studies were undertaken to evaluate morphological normality and development competence in vitro of hyman oocytes following vitrificatioin using ethylene glycol and electron microscopic grid. Human immature oocytes retrieved from natural and stimulated cycles was vitrified at 0 or 48 h and 0, 8 to 15 or 24 to 28 h after maturation culture, respectively. In oocytes retrieved from unstimulated cycle, no signifciant differences were found in morphological normality (56 to 63%) and fertilization (31 to 37%) rates between the times of vitrification. In stimulated patients, however, more oocytes were morphologically normal when vitrified at 24 to 28 h than when vitrified at 0 or 8 to 15 h after maturation culture. Regardlesss of the hormonal stimulation, high cleavage rates(83 to 100%) were obtained in all treatment groups but did not differ significantly. Twenty to 43% of cleaved oocytes developed to the blastocyst stage at 6 days after IVF. These results suggest that vitrified oocytes from unstimulated and stimulated cycles could develop to the blastocyst stage, regardless of the stages of vitrification.

• Reproductive and Developmental Biology
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• 제38권4호
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• pp.147-158
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• 2014

Differentiated nuclei can experimentally be returned to an undifferentiated embryonic status after nuclear transfer (NT) to unfertilized metaphase II (MII) oocytes. Nuclear reprogramming is triggered immediately after somatic cell nucleus transfer (SCNT) into recipient cytoplasm and this period is regarded as a key stage for optimizing reprogramming. In a recent study (Dai et al., 2010), use of m-carboxycinnamic acid bishydroxamide (CBHA) as a histone deacetylase inhibitor during the in vitro early culture of murine cloned embryos modifies the acetylation status of somatic nuclei and increases the developmental competence of SCNT embryos. Thus, we examined the effects of CBHA treatment on the in vitro preimplantation development of porcine SCNT embryos and on the acetylated status of histone H3K9 on cloned embryos at the zygote stage. We performed the three groups SCNT: SCNT (NT), CBHA treatment at the porcine fetus fibroblast cells (PFFs) used as donor cells prior to SCNT (CBHA-C) and CBHA treatment at the porcine SCNT embryos during the in vitro early culture after oocyte activation (CBHA-Z). The PFFs were treated with a $15{\mu}M$ of CBHA (8 h) for the early culture and the porcine cloned embryos were treated with a $100{\mu}M$ concentration of CBHA during the in vitro early culture (10 h). Cleavage rates and development to the blastocyst stage were assessed. No significant difference was observed the cleavage rate among the groups (82.6%, 76.4% and 82.2%, respectively). However, the development competence to the blastocyst stage was significantly increased in CBHA-Z embryos (22.7%) as compared to SCNT and CBHA-C embryos (8.6% and 4.1%)(p<0.05). Total cell numbers and viable cell numbers at the blastocyst stage of porcine SCNT embryos were increased in CBHA-Z embryos as compared to those in CBHA-C embryos (p<0.05). Signal level of histone acetylation (H3K9ac) at the zygote stage of SCNT was increased in CBHA-Z embryos as compared to SCNT and CBHA-C embryos. The results of the present study suggested that treatment with CBHA during the in vitro early culture (10 h) had significantly increased the developmental competence and histone acetylation level at the zygote stage.

• 한국가축번식학회지
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• 제25권1호
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• pp.63-69
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• 2001

본 연구는 돼지 미성숙 난포란을 체외에서 성숙, 수정시킨 후, 체외배양액내에 NO Scavenger인 hemoglobin과 억제제인 L-NAME의 첨가 배양이 돼지 체외수정란의 체외발육에 미치는 효과를 검토하였다. 체외배양 44시간에 NCSU 배양액에 hemoglobin을 각각 0, 1 및 5$\mu\textrm{g}$/$m\ell$가 첨가된 처리구에서 상실배기 이상 발육된 수정란의 체외 발육율은 각각 52.4%, 57.6% 및 57.4%로써 hemoglobin 첨가군이 대조군에 비해 다소 높은 발육성적을 나타냈으나 통계적 유의성은 인정되지 않았다 (P〉0.05). 체외배양 96시간에 NCSU23 배양액에 hemoglobin을 0, 1 및 5$\mu\textrm{g}$/$m\ell$ 첨가한 처리구에서 상실배이상 발육된 체외 발육성적은 각각 66.2%, 70.8% 및 62.8%로 1$\mu\textrm{g}$/$m\ell$ hemoglobin을 첨가한 처리구가 다른 처리구보다 다소 높게 나타났지만 통계적으로 유의차는 인정되지 않았다 (P〉0.05). 체외배양 44시간에 NCSU23 배양액에 L-NAME를 각각 0, 10, 50 및 100mM을 첨가한 처리구에서 상실배기 이상 발육된 체외발육율은 각각 65.2%, 73.5%, 70.1% 및 58.8%로 L-NAME를 10mM첨가한 처리구와 50mM 첨가한 구가 무 첨가구와 100mM 첨가구보다 통계적으로 유의하게 높은 성적을 얻었다 (P＜0.05). 각 처리구에서 생산된 배반포기수정란의 세포수에는 커다란 차이가 인정되지 않았다.

• 한국수정란이식학회지
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• 제32권4호
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• pp.267-274
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• 2017

Alpha lipoic acid (ALA) is a biological membranes compound. As the antioxidant, it decreases the oxidized forms of other antioxidant substances such as vitamin C, vitamin E, and glutathione (GSH). To examine the effect of ALA on the in vitro maturation (IVM) of porcine oocytes, we investigated intracellular GSH and reactive oxygen species (ROS) levels, and subsequent embryonic development after parthenogenetic activation (PA). Intracellular GSH levels in oocytes treated with 50uM ALA increased significantly (P < 0.05) and exhibited a significant (P < 0.05) decrease in intracellular ROS levels compared with the control group. Oocytes matured with 50 uM of ALA during IVM displayed significantly higher cleavage rates (67.8% vs. 83.4%, respectively), and higher blastocyst formation rates and total cell number of blastocysts after PA (31.6%, 58.49 vs. 46.8%, 68.58, respectively) than the control group. In conclusion, these results suggest that treatment with ALA during IVM improves the cytoplasmic maturation of porcine oocytes by increasing the intracellular GSH levels, thereby decreasing the intracellular ROS levels and subsequent embryonic developmental potential of PA.

• 한국가축번식학회지
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• 제17권3호
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• pp.193-199
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• 1993

Bovine follicular oocytes were matured in two different conditions, TCM 199＋10% FBS with or without hormones (0.01 unit/ml ovine follicle stimulating hormone, 0.01 unit/ml ovine luteinizing hormone and 1$\mu\textrm{g}$/ml $\beta$-estradiol). There was no significant difference in maturation and fertilization rates of the oocytes between two groups. The result indicates that hormonal treatment does not have beneficial effect on in vitro maturation and fertilization of follicular oocytes. IVF-derived cone-cell bovine embryos were injected with foreign DNA (CChcLf) by microinjection method and then co-cultrued with bovine oviductal epithelial cells. Developmental rate of microinjected embryos to blastocyst stage (21%) was similar to that of non-injected embryos(29%). This result represents that microinjected bovine embryos produced in vitro have a potential of development to normal blastocysts.

• Asian-Australasian Journal of Animal Sciences
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• 제23권9호
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• pp.1152-1158
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• 2010

In differentiating human embryonic stem (d-hES) cells there are a number of types of cells which may secrete various nutrients and helpful materials for pre-implantation embryonic development. This study examined whether the d-hES could function as a feeder cell in vitro to support mouse embryonic development. By RT-PCR analysis, the d-hES cells revealed high expression of three germ-layered differentiation markers while having markedly reduced expression of stem cell markers. Also, in d-hES cells, LIF expression in embryo implantation-related material was confirmed at a similar level to undifferentiated ES cells. When mouse 2PN embryos were cultured in control M16 medium, co-culture control CR1aa medium or co-cultured with d-hES cells, their blastocyst development rate at embryonic day 4 (83.9%) were significantly better in the d-hES cell group than in the CR1aa group (66.0%), while not better than in the M16 group (90.7%)(p<0.05). However, at embryonic days 5 and 6, embryo hatching and hatched-out rates of the dhES cell group (53.6 and 48.2%, respectively) were superior to those of the M16 group (40.7 and 40.7%, respectively). At embryonic day 4, blastocysts of the d-hES cell group were transferred into pseudo-pregnant recipients, and pregnancy rate (75.0%) was very high compared to the other groups (M16, 57.1%; CR1aa, 37.5%). In addition, embryo implantation (55.9%) and live fetus rate (38.2%) of the d-hES cell group were also better than those of the other groups (M16, 36.7 and 18.3%, respectively; CR1aa, 23.2 and 8.7%, respectively). These results demonstrated that d-hES cells can be used as a feeder cell for enhancing in vitro and in vivo developmental potential of mouse pre-implantation embryos.

• Reproductive and Developmental Biology
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• 제35권4호
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• pp.469-473
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• 2011

These study was carried out to investigate the effects of the supplementation with sodium nitroprusside (SN) and nitric oxide (NO) of canine oocytes on IVM rates. Oocytes were incubated in TCM-199 supplement with at 0.03~0.10 mM SN and 0.3~1.0 mM NO for 48 hrs. Oocytes were transferred to 50 ul drops of maturation medium covered mineral oil and cultured in a $CO_2$ incubator (5% $CO_2$, 95% air, $38^{\circ}C$). The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03, 0.05, 0.07, 0.10 mM SN were $25.9{\pm}3.5%$, $36.4{\pm}3.2%$, $33.3{\pm}3.5%$, $28.8{\m}3.2%$, respectively. The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03~0.07 mM SN were significantly increased compare to the control ($26.0{\pm}2.2%$). The in vitro maturation rates of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.3, 0.5, 0.7, 1.0 mM NO were $28.0{\pm}4.2%$, $36.5{\pm}3.6%$, $30.0{\pm}3.8%$, $19.2{\pm}3.5%$, respectively. The in vitro maturation rate of oocytes in TCM-199 medium supplemented with 0.3 and 0.5 mM NO were significantly increased compare to the control ($26.0{\pm}2.2%$). The in vitro maturation rates of oocytes cultured for 12~48 hrs in TCM-199 medium supplement with 0.05 mM SN were $26.0{\pm}3.2%$, $28.0{\pm}3.4%$, $38.0{\pm}3.2%$, respectively. The in vitro maturation rate of oocytes cultured for 12~48 hrs in TCM-199 medium supplement with 0.5 mM NO were $22.0{\pm}3.0%$, $30.0{\pm}3.8%$, $36.0{\pm}4.2%$, respectively. These result was significantly increased compare to the control.

• 한국가축번식학회지
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• 제21권4호
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• pp.345-353
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• 1997

Antioxidants and antioxidants with somatic cell co-culture, bovine oviduct epithelial cells(BOEC) and buffalo rat liver cells(BRLC), were studied as a mean of increasing the development and intracellular glutathione(GSH) concnetrations of bovine embryos derived from in vitro matured(IVM) and in vitro fertilized(IVF) oocytes. Cell numbers and intracellular GSH concentrations of blastocysts were also counted. The developmental rate beyond morula stages in CRlaa containing taurine(2.5mM), superoxide dismutase(SOD, 600U) and catalase(250U) were 1%, 75.0%, 64.8% and 62.3%, respectively. The developmental rate in antioxidant groups was significantly higher than in control(P<0.05). The intracellular GSH concentrations of blastocysts cultured in 0, 2.5mM taurine, 600U SOD and 250U catalase were 33.8pM, 39.3pM, 42.3pM and 54.8pM, respectively. This result indicated that the developmental rates and intracellular GSH concentrations of catalase group was significantly higher than any other groups(P<0.05). The developmental capacity in CRlaa plus various antioxidants co-cultured with BOEC were 55.3%(control), 74.1%(2.5mM taurine), 66.7%(600U SOD) and 70.7%(250U catalase) and in CRlaa plus various antioxidants co-cultured with BRLC in control, 2.5mM taurine, 600U SOD and 250U catalase were 63.8%, 75.5%, 71.0% and 73.5%, respectveily, the intracellular GSH concentrations of blastocyst embryos co-cultured with BOEC and BRLC in CRlaa with 0.25mM taurine, 600U SOD and 250U catalase were 73.4pM and 64.4pM, 79.9pM and 67.5pM, 82.3pM and 71.7pM, and 83.0pM and 80.0pM, respectively. Cell numbers of blastocysts were not difference in all experimental groups. These studies indicate that andtioxidants and antioxidant with somatic cell co-culture can increase the proportion of embryo that developed into morula and blastocysts, and the intracellular GSH concentrations of blastocyst embryos.

• 한국가축번식학회지
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• 제18권1호
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• pp.31-37
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• 1994

This study carried out to investigate the effects of cryoprotectants in the medium on the survival rate of rapidly frozen porcine bisected demi-embryos. The porcine bisected demi-embryos following dehydration by cryoprotectants containing sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water bath. Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows : 1. The survival rates of without-zona pellucida embryos and 2 blastomeres porcine embryos were 10.0 and 7.1%, respectively. The rate of unfrozen blastomeres (20.00%) was significantly higher than that of non-frozen oocytes. 2. The survival rates of with and without-zona pellucida of bisected porcine embryos by micromanipulator were 20.0 and 14.3%, respectively. 3. The developmental rates of with and without-zona pellucida of bisected poricine embryos by micromanipulator were 13.3 and 7.1%, respectively.

• 한국수정란이식학회지
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• 제30권4호
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• pp.315-317
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• 2015

In vitro culture of murine embryos is an important step for in vitro production systems including in vitro fertilization and generations of genetically engineered mice. M16 is widely used commercialized culture media for the murine embryos. Compared to other media such as potassium simplex optimization medium, commercial M16 (Sigma) media lacks of amino acid, glutamine and antibiotics. In the present study, we optimized M16 based embryo culture system using commercialized antibiotics-glutamine or amino acids supplements. In vivo derived murine zygote were M16 media were supplemented with commercial Penicillin-Streptomycin-Glutamine solution (PSG; Gibco) or MEM Non-Essential Amino Acids solution (NEAA; Gibco) as experimental design. Addition of PSG did not improved cleavage and blastocyst rates. On the other hand, cleavage rate is not different between control and NEAA treated group, however, blastocyst formation is significantly (P<0.05) improved in NEAA treated group. Developmental competence between PSG and NEAA treated groups were also compared. Between two groups, cleavage rate was similar. However, blastocyst formation rate is significantly improved in NEAA treated group. Taken together, beneficial effect of NEAA on murine embryos development was confirmed. Effect of antibiotics and glutamine addition to M16 media is still not clear in the study.