• Journal of Plant Biotechnology
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• 제7권1호
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• pp.75-80
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• 2005

Eleutherococcus senticosus (also called Acanthopanax senticosus), belonging to Araliaceae family, has been used as an important medicinal woody plant. Mature seeds of Eleutherococcus senticosus have rudimentary (extremely immature) zygotic embryos and require a long-term stratification for about 18 months to induce germination. Here, through the methods of endosperm removal and other exogenous treatments, we investigated the factors for inducing rudimentary embryos by in vitro culture, Rudimentary zygotic embryos in seeds were at globular to heart-shaped stage at about $250{\mu}m$ in length just after harvest of fruits. When the seeds without testa were cultured on 1/2 MS (Murashige and Skoog 1962) medium, they did not germinate regardless of medium and sucrose concentrations but the removal of endosperm tissue markedly stimulated the growth of rudimentary zygotic embryos. The embryo reached ear-lier maturation, once when the endosperm surrounding the rudimentary embryos was removed. Rudimentary zygotic embryos developed cotyledons within 3 weeks of culture after endosperm emoval. However, post-mature zygotic embryos failed to germinate though they were morphologically normal, indicating another dormancy of embryos. $GA_3\;(2.0\;\cal{mg/L})$ and/or charcoal ($0.2\%$) treatment rapidly enhanced the germination of zygotic embryos. These results suggest that E. senticosus seeds have double dormancy; i. e. morphological rudimentary dormancy influenced by surrounding endosperm and physiological dormancy after post-maturation of zygotic embryos. Based on the above findings, we established the rapid germination of rudimentary zygotic embryos by in vitro culture of excised seeds with endosperm removal and $GA_3$ treatment.

• 한국수정란이식학회지
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• 제24권2호
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• pp.89-95
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• 2009

Successful in vitro embryo production heavily relies on the normal maturation and fertilisation of oocytes. We examined the normal and abnormal fertilisation of zebu cattle oocytes matured in vitro. Immature cumulus oocyte complexes (COCs) from zebu cattle ovaries at slaughter were matured in vitro (IVM) for 24 h. The oocytes were either fixed, stained and examined for nuclear changes or fertilised in vitro (IVF) with Percoll-separated, heparintreated spermatozoa (1.0 ${\times}$ $10^6$/mL) of zebu (n = 7) and crossbred bulls (n = 7). After 18 h of sperm-COCs co-incubation at $39^{\circ}$C with 5% $CO_2$ in humidified air, the presumptive zygotes were fixed, stained and examined for pronuclei. The number of oocytes retrieved per ovary was 5.4 ${\pm}$ 0.7. The percentage of matured oocytes was 73.0. The difference in motility of spermatozoa before and after Percoll seperation was significant (p<0.001). The percentages of normal and abnormal fertilisation (polyspermia and oocytes with one pronucleus) varied significantly depending on individual bulls (p<0.05). A protocol for IVF of IVM oocytes in Bangladeshi zebu cattle is developed. A future study may elucidate the capacity of such IVM-IVF oocytes to develop to the blastocyst stage for transfer to surrogate mother.

• 한국수정란이식학회지
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• 제9권1호
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• pp.1-6
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• 1994

This experiment was investigated the effect of presence of granulosa cells from follicles of different size on bovine oocyte maturation, cleavage and development to late stage. The nuclear and cytoplasmic maturation of oocytes in the IVM-IVF system are critical for subsequent embryo development. Granulosa cells when the co-cultured with oocytes may interact with cumulus-oocytes complexes and influence the development competence of the oocytes. Granulosa cells from medium (2~6 mm) and large(>1O mm) size follicles were recovered by aspiration, washed 3 times by centrifugation at 500 x g for 5 min. and used for co-culture at a concentration of 2~3 x 106 cells/mi. The oocytes were matured in vitro (IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g/ml FSH, 10 $\mu$g/ml LH, 1 $\mu$g/ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro (IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro (I VC) with bovine oviductal epithelial cells for 7 to 9 days. The assessment of maturation revealed that Grade J oocytes showed significantly(P

• 한국수정란이식학회지
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• 제12권1호
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• pp.111-116
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• 1997

The present study was carried out to produce in vitro fertilized embryos with immature follicular oocytes collected by transvaginal aspiration from Holstein cows. A simple aspiration apparatus consists of two stainless steel tubes, an inner tube (needle holder; 1.2cmdiameter, 55cm long) and an outer tube (1.5cm diameter, 4Scm long), and a hand-operated vacuum pump was used. Under epidural anesthesia, the needle guide was passed into the vagina of the cow to a point next to the cervix. An ovary was placed against the wall of the vagina over the end of the aspiration needle by rectal manipulation. As the needlepassed into the ovary, an assistant was asked to apply vacuum(l00mrnHg) and the ovary was manipulated back and forth in all directions over the needle. When all sites of the ovary was aspirated, the needle was withdrawn and the needle guide was moved to the other side of ovary and the procedure was repeated. When the oocyte aspiration procedure was finished, collected fluid was transported to laboratory. Oocytes surrounded with at least 1 layer of cumulus cells were matured, fertilized and cultured in vitro. The results were as follows; Ninety seven oocytes were collected by transvaginal aspiration from seventeen Holstein cows(5.7 /head). The number of oocytes surrounded with at least 1 layer of cumulus cells were 60(61.9%). Following in vitro maturation, fertilization and culture, the cleavage and development rate to morula+blastocyst were 83.3% and 30.0%, respectively. From this study, transferable in vitro fertilized embryos could be produced with imma- ture follicular oocytes collected by transvaginal aspiration from Holstein cows using a simple aspiration apparatus.

• 한국수정란이식학회지
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• 제32권3호
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• pp.111-122
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• 2017

The objective of this experiment was to explore the effects of Roscovitine (Rosco) prior to in vitro maturation (IVM) of immature pig oocyte. Brilliant cresyl blue test has been used to select the good quality of oocyte. Specifically, the effects of Rosco exposure on nuclear and cytoplasmic maturation, diameter, intracellular glutathione (GSH) and reactive oxygen species (ROS), and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT embryos have been measured. Cumulus oocyte complexes (COCs) have been exposed in $75{\mu}M$ of Rosco for 22 and 44 h. The COCs that were matured in the IVM for 44 h without Rosco used as control group. Diameter of matured porcine oocytes 44 h culture with Rosco was significantly lower than 22 h culture with Rosco and control groups. GSH was higher in control group than 22 h and 44 h with Rosco but reduction of ROS in 22 h than 44 h with Rosco. In PA, exposure with Rosco 44 h oocytes group has been significantly lower than 22 h and control group in rates of maturation, cleavage and blastocyst formation. Similarly, in SCNT embryos rates of maturation, cleavage and formation of blastocyst have been also significantly lower in 44 h Rosco treated group than other two groups. SCNT embryos treated with Rosco 22 h showed greater expression levels of POU5F1, DPPA2 and NDP52Il mRNA compared with other two groups. Our results demonstrate that Rosco treatment with 22 h prior to IVM improves the development competence of porcine oocyte.

• Journal of Plant Biotechnology
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• 제45권4호
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• pp.347-356
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• 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.

• 한국수정란이식학회지
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• 제18권1호
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• pp.1-14
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• 2003

본 연구는 미성숙, 성숙 단계의 돼지 난포란이 유리화 동결에 의한 동결보존이 가능한지를 조사 하고자 실시하였다. 난포란은 세포질 내 지방구를 분극시키기 위해 원심분리를 실시하였고, 미세조작기를 이용하여 지방구를 제거하였다. 돼지 난포란을 CB 처리하여 원심분리 후 지방구를 제거한 지방제거구(Delipated), CB 처리 후 원심분리만 하여 지방구를 분극시킨 원심분리구(Centrifuged), 아무처리도 하지 않은 대조구(Control)를 EM grid를 이용한 유리화 동결, 융해 한 후 생존율과 체 외 발생율을 조사하였다. 그 결과를 요약하면 다음과 같다. 1. 미성숙 시기에 지방제거, 원심분리, 무처리 후 유리화 동결을 실시한 돼지 난포란의 생존율은 각각 15.1%, 0%, 0%로 지 방제 거구에서만 유의적으로 높은 생존율을 나타내었다(p<.01). 2. 성숙 시기에 지방제거, 원심분리, 무처리 후 유리화 동결을 실시한 돼지 난포란의 생존율은 각각 12.2%, 0%, 0%로 지방제거구에서만 유의적으로 높은 생존율을 나타내었다(P<.01). 3. 미성숙 시기에 지방제거 후 유리화 동결을 실시하여 생존한 난자치 핵 성숙율은 37.5%로 동결하지 않은 난포란의 성숙율 68.9%보다 유의적으로 낮은 것이었다(P<.01). 4. 미성숙 시기에 지방제거, 원심분리, 무처리 후 유리화 동결을 실시하여 생존한 난자의수정 후 배 발달율은 12.5%, 0%, 0%로 지방 제거구에서만 배 발달을 나타었으나, 세 처리간의 유의차는 없었고, 이것은 동결하지 않은 난포란의 배 발달율 56.1%보다는 유의 적으로 낮은 것이었다(P<.05). 5. 성숙 시기에 지방제거, 원심분리, 무처리 후 유리화 동결을 실시하여 생존한 난자의 수정 후 배 발달율은 25.0%, 0%, 0%로 지방제거구에서만 배 발달을 나타내었으나 세 처리간의 유의차는 없었고, 이것은 동결하지 않은 난포란의 배 발달율 67.9%보다는 유의적으로 낮은 것이었다(P<.05).

• Applied Microscopy
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• 제42권3호
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• pp.142-146
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• 2012

Doublecortin (DCX) is a microtuble-associated protein that is required for the migration of immature neuroblasts within the chick and mammalian brain. Although it is generally thought that DCX is expressed only in the neuroblasts, some mature neurons maintain DCX expression; for example, horizontal cells in adult rat retina. In this study, we demonstrate that retinal neural progenitors in the early embryonic stage of the chick also expressed DCX, as do developing ganglion cells and horizontal cells in later stages of development. These findings raise the possibility of a role for DCX in retinal neural progenitors, before they become specialized into neuroblasts in the chick.

• 한국작물학회지
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• 제35권4호
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• pp.334-341
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• 1990

인삼종자의 개갑처리과정중 종자단백질 및 몇가지 효소의 전기영동변이를 처리단계별로 조사하여 배의 분화 및 발육과의 관계를 검토하였다. 1. 종자단백질에서는 처리종료시까지 8개의 band를 보였는데 Rf 치가 0.12, 0.24, 0.66 및 0.72의 band는 농염인채 거의 변화가 없었으나 나머지 4개의 band는 처리기간이 길어짐에 따라 옅어지는 경향이었다. 한편 처리후 75일부터 분리채취한 배에서는 Rf 치가 0.76인 band를 확인할 수 없었다. 2. Peroxidase는 처리기간이 길어짐에 따라 band의 수가 증가하여 처리후 45일에는 4개의 band를 보였고 그들의 색조는 처리종료시까지 짙어지는 경향이었으며 배에서도 비슷한 양상으로 나타났다. 3. Esterase는 모두 4개의 band를 보였는데 처리일수가 증가함에 따라 Rf치 0.36의 band는 점차 짙어지는 경향이었고 Rf치 0.56의 band는 반대로 점차 옅어지는 경향이었으며 처리후 95일에는 종자(배분이전의 것)에서 소멸되었던 Rf치 0.20의 band가 배에서 선명하게 나타났다. 4. Glutamate dehydrogenase는 처리후 60일에만 나타난 Rf치 0.16의 band와 전조사기간에 걸쳐 나타난 Rf치 0.14의 band만이 확인되어 단조로운 양상을 보였으며 배에서는 후자만이 나타났다. 5. Glutamate-oxaloacetate transaminase는 분리채취한 배를 포함하여 채종시부터 처리종료시까지 Rf 치 0.36의 band가 유일한 것으로서 매우 단조로웠다.

• Clinical and Experimental Reproductive Medicine
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• 제46권4호
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• pp.166-172
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• 2019

Objective: In vitro maturation (IVM) of immature oocytes can be useful for some infertile patients. In IVM programs, the rates of embryo formation and pregnancy are low. Therefore, it is essential to recognize the main factors involved in regulating oocyte maturation in vitro. The purpose of this study was to investigate the effects of growth differentiation factor 9 (GDF9) and cumulus cell (CC) supplementation in IVM medium on the rates of embryo formation and viability of human blastocysts. Methods: A total of 80 germinal vesicle oocytes from stimulated cycles underwent an IVM program. The oocytes were divided into four groups, where group I consisted of IVM media only and served as the control, group II consisted of IVM+CCs, group III consisted of IVM+GDF9 (200 ng/mL), and group IV consisted of IVM+CCs+GDF9 (200 ng/mL). Intracytoplasmic sperm injection was performed on the IVM oocytes, and the cleavage embryos that were generated were vitrified. Following thawing, the embryos were cultured for 3 additional days, and the viability rates of the developed blastocysts were determined. Results: The maturation rate of the oocytes did not differ significantly across the four groups. The fertilization rate in group II was significantly higher than that in the control group (76.5% vs. 46.2%). Embryo formation was significantly more frequent in all experimental groups than in the control group, while blastocyst formation did not show significant differences in the three experimental groups compared to the control. The mean viability rates in groups II, III, and IV were 58.16%, 55.91%, and 55.95%, respectively, versus 37.78% in the control group (p< 0.05). Conclusion: Supplementation of IVM culture media with GDF9 and CCs enhanced the fertilization, embryo formation, and viability rates of blastocysts generated from vitrified cleavage embryos.