• 대한치과교정학회지
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• 제26권6호
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• pp.667-676
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• 1996

We have examined the in vitro stage-related chondrogenic potential of human mandibular and limb bud mesenchyme cells using micromass culture. Our results indicate that limb bud mesenchyme cells as early as stage 16 by Carnegie system (37 days), well before the initiation of in vivo chondrogenesis, have chondrogenic potential which is expressed in micromass culture. These results are correlated with stage-related chondrogenic potential of human limb bud in vivo as a result of Alcian blue staining. The proliferation of chondrogenic cells increased in the first 3 days after culture and then decreased. These results were correlated with the cell cycle analysis of which the number of $G_0^1/G_1$ phase increased markedly after 3 days of culture, while the percentage of cells in S phase was decreased. On the other hand, it was rarely differentiated in the mandible. We examined the effects of two PKC modulators such as phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC, and staurosporine (STSN), an inhibitor of PKC. PMA inhibited the chondrogenesis, whereas STSN promoted the chondrogenesis in a dose dependent manner. In addition, PMA exerted no inhibitory effect when the cells were pretreated for 24 h with STSN, implying that the chondrogenic events might be settled at an early step in vitro and FKC may act as a negative modulator. Collectively, these results demonstrate, for the first time, the stage-related chondrogenic potential of human mandibular and limb bud mesenchyme cells and the role of PKC during chondrogenesis in vitro & in vivo.

• Reproductive and Developmental Biology
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• 제33권2호
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• pp.85-91
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• 2009

In this study, in vitro maturation system using fetal bovine serum (FBS) or porcine follicular fluid (pFF) was investigated to produce comparable oocytes to those derived from in vivo. Control group of oocytes was cultured in TCM 199 supplemented with 0.1% polyvinyl alcohol (PVA). Other three groups of oocytes were cultured in TCM 199 supplemented with 10% FBS, 10% pFF or 5% FBS + 5% pFF, respectively. After 44 h maturation, oocytes with the first polar body were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}sec$. Also, matured oocytes of four groups were reconstructed and fused. Reconstructed embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. The oocytes matured in the medium supplemented with FBS or/and pFF showed significantly higher maturation rates (64.0 vs. 73.9 to 85.2%). In PA embryos, cleavage rates (89.7 vs. 77.1 to 86.6%) and blastocysts rates (30.0 vs. 16.2 to 26.2%) were significantly higher in pFF group (p<0.05). In NT embryos, there was no difference among treatments in cleavage rate, but the blastocyst rates (28.5 vs. 15.5 to 24.6%) were significantly higher in pFF group (p<0.05). The apoptosis rate was significantly higher (p<0.05) in the control than other groups (10.8 vs. 4.9 to 8.2% for PA, 3.1 vs. 0.5 to 1.3% for NT). In order to select the comparable oocyte to in vivo oocytes, each group of oocytes was stained with Brilliant cresyl blue (BCB) after 42h maturation. The matured oocytes were separated according to color of cytoplasm; stained group (BCB+) and unstained group (BCB-). The oocytes matured in the presence of FBS or/and pFF showed significantly higher staining rates (70.3 to 72.7 vs. 35.1%) (p<0.05). To verify the fact that the supplementation of FBS or/and pFF can increase the maturation rates, cdc2 kinase activity, the catalytic subunit of MPF, was determined. The cdc2 kinase activity of the oocytes matured in the medium supplemented with FBS or/and pFF was significantly higher than control group (6.7 to 9.3 vs. 3.8). In conclusion, the supplementation of FBS or/and pFF can support in vitro maturation rate of porcine oocytes through the increment of cdc2 kinase activity level in the cytoplasm.

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.319-327
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• 2011

This study investigated whether the addition of porcine sperm cytosolic factor (SCF) at fusion/activation affects in vitro development of porcine parthenogenetic(PA) and nuclear transfer (NT) embryos. To determine the optimum concentration of SCF, control group of oocytes was activated with 0.3M mannitol (1.0 mM $CaCl_2{\cdot}2H_2O$), other three groups of oocytes were parthenogentically activated with the fusion medium (0.1mM $CaCl_2{\cdot}2H_2O$) supplemented with 100, 200 or 300 ${\mu}$g/ml SCF, respectively. Matured oocytes were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}$sec. The activated embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. Oocytes activated in the presence of SCF showed a significantly higher blastocyst rate than control (p<0.05). Apoptosis rate was significantly lower in 100 ${\mu}$g/ml SCF group than other groups (p<0.05). Cdc2 kinase activity in control and SCF treatment group of oocytes was determined using MESACUP cdc2 kinase assay kit at 1, 5, 10, 15, 30, 45 and 60 min after activation. Cdc2 kinase activity was significantly decreased (p<0.05) in SCF group than MII oocytes or control within 5 min. For NT embryo production, reconstructed oocytes were fused in the fusion medium supplemented with 0.1 mM $CaCl_2{\cdot}2H_2O$ (T1), 1.0 mM $CaCl_2{\cdot}2H_2O$ (T2) and 0.1 mM $CaCl_2{\cdot}2H_2O$ with 100 ${\mu}$g/ml SCF (T3). Fused embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. Developmental rate to blastocyst stage was significantly higher in T3 than other groups (23.0% vs. 13.5 to 15.2%) (p<0.05). Apoptosis rate was significantly lower in T3 than T1 or T2 (p<0.05). The relative abundance of Bax-${\alpha}$/Bcl-xl was significantly lower in in vivo or SCF group than that of control (p<0.05). Moreover, the expression of p53 and caspase3 mRNA was significantly lower in in vivo or SCF group than that of control (p<0.05). These results indicate that the addition of SCF at fusion/activation might improve in vitro development of porcine NT embryos through regulating cdc2 kinase level and expression of apoptosis related genes.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.118-118
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• 2002

Programmed cell death (PCD) is thought as a well-controlled process by which unwanted cells are selectively eliminated. During the last decade many researches have elucidated molecules and their interactions involved in cell death by using largely in vitro induction of cell death or survival signals in a more defined manner, While these critical information and novel findings provide us with clearer understanding of mechanisms underlying cell death, it does by no means explain how PCD occurs and which cells or tissues are affected during normal embryonic development in vivo. In this study, we used zebrafish to examine whether the PCD is occurring selectively or randomly in developing embryos by whole mount in situ TUNEL analysis with specific markers for neural cells. The result revealed that the degree and distribution of TUNEL staining varied considerably throughout gastrulation stage, and there was also a number of TUNEL-negative embryos. Most of TUNEL-positive cells were scattered randomly throughout the blastoderm. During the gastrulation stage about 75 % of the embryos analyzed exhibited more than 5 TUNEL-positive cells. As the dorsal epiblast begins to thicken rather abruptly near the end of gastrulation, TUNEL-positive cells were mainly located along the dorsal side. Although there were some variations in TUNEL staining during segmentation and pharyngeal stages, TUNEL staining continued to be localized to the central nervous system, and was also detected in the sensory organs, trigeminal ganglions, and the primary sensory neurons. High levels of the cell death in developing brain between 20-somite and prim-6 stages are thought to play a role in the morphogenesis and organization of the brain. At prim-16 stage, cell death is considerably reduced in the brain region. Dying cells are mainly localized to the prospective brain region where ectodermal cells are about to initiate neurogenesis. As development progressed, high levels and more reproducible patterns of cell death were observed in the developing nervous system. Intensive TUNEL staining was restricted to the trigeminal ganglions, the primary sensory neurons, and sensory organs, such as olfactory pits and otic vesicles. Thus, PCD patterning in zebrafish embryos occurs randomly at early stages and becomes restricted to certain region of the embryos. The spatio-temporal pattern of PCD during the early embryonic development in zebrafish will provide basic information for further studies to elucidate genes involved in. regulation of PCD largely unknown in vivo during vertebrate embryogenesis.

• 한국동물학회지
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• 제15권1호
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• pp.25-33
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• 1972

배아의 발생에 대한 연구는 in vivo 나 in vitro의 방법을 통해 근래 비?적 활발하게 진행되고 있다. 이들의 연구결과에 따르자면, 성분이나 함량 혹은 물리학적 성질이 혈청따위의 체액과는 다른 전방수가 들어차 있는 안전방의 환경은 배아의 발생에 극히 부적당한 곳이라 할 수 있다. 그럼에도 불구하고 본실험의 결과를 보면 초기배아의 발생은 극히 정상적으로 진행되고 있다. 이런 점에 비추어 우리는 두 가지 가능성을 추론할 수 있다. 첫째는, 배아의 어느 한정된 범위에서는 그의 환경에 쉽게 적응할 수 있는 능력을 가지고 있을 것이라는 생각이다. 배아세포가 미분화의 상태에 있다는 점과 대사의 정도가 분화된 다른 세포들에 비해 비교적 낮다는 점을 고려할 때 이들 배아는 안전방내의 환경과 같은 특수한 곳에서도 능히 생존할 수가 있는 것이다. 둘째는, 배아를 받아들인 후 전방수의 특성이 달라질 것이라는 점이다. 토끼에서는 일단 안전방내의 전방수가 유출되고 나면 혈청과 비슷한 체액이 약8시간 동안 안전방내에 형성되고 그뒤 차차로 본래의 전방수로 대치된다. 쥐에서도 이러한 체액이 안전방내에 형성된다면 배아를 받아들인 안전방내의 전방수의 성질이 혈청과 비슷해지므로 배아의 발생이 가능해질 수가 있다. 단지 그러한 체액이 전방수의 유출 후 120시간까지 그대로 남아있을 것인가에 대한 의문은 그대로 남아있지만 위의 두가지 가능성에 대하여는 배아의 생태나 행동과 관련시켜 앞으로 더 연구해야 할 것이다.

• 한국가축번식학회지
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• 제20권4호
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• pp.467-472
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• 1997

본 연구는 체내생산 공핵배 핵이식의 최적 통전전압을 결정하기 위하여 그리고 공핵배의 질이 핵이식난자의 융합과 체외발달에 미치는 영향을 조사하기 위하여 수행되었다. 수핵난자는 체외성숙후 25∼27시간에 제핵하였고 난자의 추가성숙을 위해서 융합전에 18∼20시간 더 배양하였다. 상실배시기의 공핵배는 다배란 처녀우에서 채란한 후 질에 따라 양질과 저질로 구분하여 공시하였다. 공핵배의 핵이식은 체외성숙후 42∼44시간에 행하였고, 융합은 0.75kV/cm 또는 1.0kV/cm DC 전압으로 체외성숙 후 43∼45시간에 실시하였다. 융합율은 두 전안갑에 차이가 없었으나 난할율과 M＋B 발달율은 19.0%와 29.4%였다. 공핵배의 질은 융합율과 난할율에 크게 영향이 없었으나 저질공핵배의 핵이식으로 부터는 상실배의 발달이 없었다. 본 결과에서 체내생산 공핵배 핵이식의 최적전압은 1.0kV/cm DC이었으며 공핵배의 질은 핵이식배 발달에 영향하는 중요 요인 중에 하나이었다.

• 한국임상수의학회지
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• 제9권1호
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• pp.323-332
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• 1992

The present study was carried out to examine the effect of oviduct epithelium and its conditioned medium on e development of early bovine embryos in vitro. Oocytes obtained from ovarian follicles of slaughtered cows were cultured in TCM199 with 10% fetal calf serum for 22-24hrs and then fertillzed in vitro using frozen-thawed semen treated with BO-caffein, BO-BSA(20mM heparin added). Oviduct epithelium was collected in each stage of the estrus cycle and conditioned medium was the medium in which oviduct epithelium in early luteal stage was cultured. In vitro fertilized bovine embryos of 1～2 cell were co-cultured with oviduct epithelium from different estrus cycles, cultured in conditioned medium, and cultured in rabbit oviduct. The cleavage rates of in vitro fertilized early bovine embryos co-cultured with oviduct epithelial cell from early luteal, luteal and follicular phase of estrus cycle(67.2～70.8%) and cultured in conditioned medium(56.7%) were significantly(p<0.05) higher than that of the control(44.2%) The rate of development to morula or blastocyst stage in oviduct epithelial cell co-culture(15.3～32.5%) from three phase of estrus cycles and conditioned medium(14.5%) were significantly(p<0.05) higher than that of the control(5.2%). The oviduct epithelial cell from early luteal phase gave a significantly( p<0.05) higher rate of development to morula or blastocyst stage than both luteal and follicular phase. The results of in vivo culture in rabbit oviduct of early bovine embryos were 52.1% for the cleavage rate and 26.7% for the rate of development to morula or blastocyst stage.

• Plant Biotechnology Reports
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• 제3권4호
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• pp.333-340
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• 2009

Developmental anomalies in the plumule meristem of peanut (Arachis hypogaea L.) somatic embryos resulted in poor shoot differentiation and reduced plant recovery. Existing meristems with caulogenic potential have never been tested for embryogenesis in peanut. The present experiment was designed to test the mature zygotic embryo axis derived plumule with three meristems for somatic embryogenesis. Embryogenic masses and embryos developed from the caulogenic meristems in the axils. Exposure of 2 weeks in primary medium with $90.5{\mu}M$ 2,4-D suppressed the shoot tip differentiation temporarily which then regained the ability to form the shoot on withdrawal of 2,4-D. Exposure of 4 weeks in primary medium with $90.5{\mu}M$ 2,4-D suppressed the shoot tip differentiation irreversibly. No shoot formation was noted from the tips in any of the cultures which were in secondary medium with $13.6{\mu}M$ 2,4-D. Development of somatic embryos directly from axillary meristems was confirmed histologically. Conversion frequency of these embryos was 11%. Thus, in this report, we describe a method to obtain somatic embryos from the determined organogenic buds of the axillary meristem, by culturing the nodal explant vertically on embryo induction medium. It also displays the possibility of obtaining both embryogenic and organogenic potential in two parts of the same explant simultaneously. The possibility of extending this approach for genetic transformation in in vivo system through direct DNA delivery or Agrobacterium injection in meristems can also be explored. Using Agrobacterium rhizogenes, we have demonstrated the possibility of gene transfer in the axillary meristems of seed-derived plumule explant.

• 한국수정란이식학회지
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• 제25권3호
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• pp.141-144
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• 2010

In vivo embryo produced from Hanwoo donor cows were collected and transferred to Hanwoo recipients. Cows, at random stages of the estrous cycle, received Progesterone Releasing Intravaginal Device (CIDR-plus, InterAg, New Zealand) together with injection of 1 mg estradiol benzoate and 50 mg progesterone, and gonadotropin treatment began 4 day later. For superovulation, a total of 28 mg FSH was intramuscularly injected twice a day in the way of decreasing doses 4 day (5, 5, 4, 4, 3, 3, 2 and 2 mg). Twenty one Hanwoo donor cows were flushed on day 7 of estrus cycle with same FSH and artificial insemination by the same technicians. Embryos were recovered 7 days after the second insemination by flushing the uterus with Embryo Collection Medium. The results obtained were as follows: The rates of transferable embryos were 50.3%, and 78 fresh embryos at morulae and blastocysts stage were transferred into Hanwoo recipients on day 7 of estrus cycle. The pregnancy rates were first embryo transfer 55.6%, 2nd 62.9% and 3rd 57.9%, respectively. In conclusion, These results suggest that CIDR-based superovulation protocol may be effectively used for production of superior Hanwoo embryos. Also, since it seems the condition of recipient cows greatly affect pregnancy rate, it is very important to evaluate recipient for effective cattle production.

• 한국수정란이식학회지
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• 제26권3호
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• pp.153-158
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• 2011

This study was performed in order to simplify the operation and minimize stress of donor and be readily available in the field with low cost and high quality embryos using the Direct Embryo Collection (DEC). Donors, at random stages of the estrous cycle, received a CIDR. 7 days later, 200 mg FSH was treated with 40, 30, 20, 10 mg FSH levels in declining doses twice daily by intramuscular injection for 4 days. On the 3rd day administration of FSH, 25 mg $PGF_2{\alpha}$ was administered and CIDR was withdrawn. After FSH injections were complete, donors were artificially inseminated twice at 12 hr intervals. The donor cattle received 250 ${\mu}g$ GnRH at time of 1 st insemination and embryos were recovered 8 days after the 1st insemination. Embryo collection from superovulated donors was performed to flushing by non-surgical methods of 3-way, 2-way and DEC (l-way). The average number of recovered embryos were 11.25${\pm}$0.63, 12.5${\pm}$0.65 and 11.75${\pm}$0.48 from operations of 3-way, 2-way and DEC methods, respectively. There were no significant differences among the embryo collection methods. Also, The average number of transferable embryos were 6.25${\pm}$0.48, 7.25${\pm}$0.48 and 7.25${\pm}$0.63 from each embryo collection procedures. The number of transferable embryos was no differences among the 3-way, 2-way and DEC methods, respectively. Meanwhile, the ratio of transferable embryos for all recovered embryos from DEC methods was higher as 61.7 % than 55.6 %, 58 % from methods of 3-way, 2-way. And the flushing solution required for recovering embryos by DEC method was significantly lower as 0.28${\pm}$0.32 1 than 1.8${\pm}$0.12 1, 1.75${\pm}$0.10 1 from 3-way, 2-way methods (p<0.05). Also, the time required for recovering embryos by DEC methods was significantly lower as 27${\pm}$2 min than 51${\pm}$3, 45${\pm}$2 min, respectively (p<0.05). In conclusion, these results suggest that DEC method for embryo collection may be effectively used for production of in vivo embryos using less flushing solution and, it might be effectively available in the field compared to conventional embryo recovery methods using 3-way or 2-way balloon catheter.