• Applied Microscopy
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• v.26 no.3
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• pp.329-348
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• 1996

The prenatal development of lateral motor columns in the lumbar spinal cord was studied by electron microscopy in human embryos and fetuses ranging from 9 mm to 260 mm crown-rump length ($5{\sim}30$ weeks of gestational age). At 9 mm embryo, the lateral motor column were developed from ventro-lateral projection into the marginal layer and composed of primitive neuroblasts. At 20 mm embryo the primitive motor neurons were packed closely together and could readly be distinguished from primitive glioblasts by a presence of large nuclei. The primitive multipolar neurons were observed in lateral motor column at 40 mm fetus. At 80 mm fetus multipolar neurons were characterized by their many dendrites and axons in the vicinity of motor neuron perikarya. At 260 mm fetus, the motor neurons were large and contained all intracytoplasmic structures in the cytoplasm which were also found in mature motor neuron in lateral motor column. The first axo-dendritic synapses found at 40 mm fetus and increased in number throughout fetal development. Axo-somatic synapses with spherical vesicles were first observed at 80 mm fetus. A few axo-somatic synapses were found at next prenatal stages. Axo-dendritic and axo-somatic synapses contained mixed populations of spherical and flattened vesicles by 120 mm fetus. These findings indicate that axo-dendritic synapses develop prior to axo-somatic synapses in the spinal cord during neurogenesis.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.3
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• pp.111-117
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• 2015

Objective: In order to increase the number of mature oocytes usable for intracytoplasmic sperm injection (ICSI), we aimed to investigate the effect of co-culturing granulosa cells (GCs) on human oocyte maturation in vitro, the fertilization rate, and embryo development. Methods: A total of 133 immature oocytes were retrieved and were randomly divided into two groups; oocytes that were cultured with GCs (group A) and oocytes that were cultured without GCs (group B). After in vitro maturation, only oocytes that displayed metaphase II (MII) underwent the ICSI procedure. The maturation and fertilization rates were analyzed, as well as the frequency of embryo development. Results: The mean age of the patients, their basal levels of follicle-stimulating hormone, and the number of oocytes recovered from the patients were all comparable between the two study groups. The number of oocytes that reached MII (mature oocytes) was 59 out of 70 (84.28%) in group A, compared to 41 out of 63 (65.07%) in group B (p=0.011). No significant difference between fertilization rates was found between the two study groups (p=0.702). The embryo development rate was higher in group A (33/59, 75%) than in group B (12/41, 42.85%; p=0.006). The proportion of highest-quality embryos and the blastocyst formation rate were significantly lower in group B than in group A (p=0.003 and p<0.001, respectively). Conclusion: The findings of the current study demonstrate that culturing immature human oocytes with GCs prior to ICSI improves the maturation rate and the likelihood of embryo development.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2001.11a
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• pp.40-46
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• 2001

The use of pluripotent stem cells has tremendous advantages for various purposes but these cell lines with proven germ-line transmission have been completely established only in the mouse. Embryonic germ (EG) cell lines are also pluripotent and undifferentiated stem cells established from primordial germ cells (PGCs). This study was conducted to establish and characterize the chicken EG cells derived from gonadal primordial germ cells. We isolated gonadal PGCs from 5.5-day-old (stage 28) White leghorn (WL) embryos and established chicken EG cells lines with EG culture medium supplemented with human stem cell factor (hSCF), murine leukemia inhibitory factor (mLIF), bovine basic fibroblast growth factor (bFGF), human interleukin-11 (hIL-11), and human insulin-like growth factor-I (hIGF-I). These cells grew continuously for 4 months (10 passages) on a feeder layer of mitotically active chicken embryonic fibroblasts. These cells were characterized by screening with the Periodic acid-Shiff＇s reaction, anti-SSEA-1 antibody, and a proliferation assay after several passages. As the results, the chicken EG cells maintained characteristics of undifferentiated stem cells as well as that of gonadal PGCs. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types when re-seeded onto culture dish. The chicken EG cells were injected into blastodermal layer at stage X and dorsal aorta of recipient embryo at stage 14 (incubation of 53hrs) and produced chimeric chickens with various differentiated tissues derived from the EG cells. The germline chimeras were also successfully induced by using EG cells. Thus, Chicken EG cells will be useful for the production of transgenic chickena and for studies of germ cell differentiation and genomic imprinting.

• Journal of Embryo Transfer
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• v.27 no.3
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• pp.189-192
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• 2012

Although embryonic stem cells (ESCs) or ES-like cells are reported from many mammalian species other than the mouse, the culture system for murine ESCs may not be suitable to the other species. Previously many other research groups have modified either human or mouse ESC culture systems for bovine ESC culture. In this study, we compared three different culture mediums consisting of DMEM, ${\alpha}$-MEM or KnockOut$^{TM}$-DMEM (KO), which are modified from human or mouse ESC culture system, for the generation of bovine ESCs. In this study, some pre-requisite events which are important for establishment and long-term propagation of ESCs such as inner cell mass (ICM) attachment on feeder cells, primary colony formation and sustainability after passaging. Once the ICM clumps attached on feeder cells, this was designated as passage 0. In regards to the rate of ICM attachment, ${\alpha}$-MEM was superior to the other systems. For primary colony formation, there was no difference between DMEM and ${\alpha}$-MEM whereas KO showed lower formation rate than the other groups. For passaging, the colonies were split into 2~4 pieces and passed every 5~6 days. From passage 1 to passage 3, DMEM system seemed to be appropriate for maintaining putative bovine ESCs. On the other hand, ${\alpha}$-MEM tended to be more suitable after passage 6. Although ${\alpha}$-MEM support to maintain a ES-like cell progenies to passage 15, all three culture systems which are modified from human or mouse ESC culture media failed to retain the propagation and long-term culture of putative bovine ESCs. Our findings imply that more optimized alternative culture system is required for establishing bovine ESC lines.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.1
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• pp.53-60
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• 2011

Objective: This study was carried out to compare the clinical outcome of elective single cleavage-embryo transfer (eSCET) to that of elective single blastocyst-embryo transfer (eSBET) in human IVF-ET. Methods: This study was a retrospective study which analyzed for 614 women who visited the Daegu Maria Clinic from August 2008 to December 2009. All were under 37 years old and had more than 8 mm of endometrial thickness on the day of hCG administration and at least one good quality embryo on day 3. The eSCETs were performed on day 3 (n=450) and the eSBETs were conducted on day 5 (n=164). Results: The numbers of retrieved oocytes, fertilized oocytes, and day 3 good quality embryos were significantly lower in the eSCET group (12.1${\pm}$6.0, 8.2${\pm}$4.6, and 4.2${\pm}$3.1, respectively) compared to the eSBET group (16.7${\pm}$7.2, 12.1${\pm}$5.0, and 8.5${\pm}$4.5, respectively; p<0.001). However, the clinical pregnancy, implantation, on-going pregnancy, and live birth rates of the eSCET group (46.7, 46.9, 40.0, and 36.7%, respectively) were not statistically different from those of the eSBET group (51.2, 51.8, 45.1, and 43.9%, respectively; p=0.318, 0.278, 0.254, and 0.103, respectively). Conclusion: These results suggested that elective single embryo transfer should be performed regardless of the developmental stage to women less than 37 years old who had more than 8 mm of endometrial thickness on the hCG administration day and at least one good quality embryo on day 3 in order to reduce the twin pregnancy rate without reducing the whole pregnancy rate.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.1
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• pp.33-39
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• 2010

Objective: Birth sex ratio (BSR) with human IVF-ET program is an interesting subject of social and scientific issue but very little information is available in Korea. This study was performed to assess the BSR with IVF-ET and to suggest the effective factors on the BSR. Methods: The national data from 1991~2008 were obtained from governmental Statistics Korea and the delivery data of human IVF-ET program on 2007 and 2008 were provided from the Ministry for Health, Welfare and Family Affairs. The BSR were statistically analyzed according to methods of IVF and to transferred embryos from fresh or frozen-thawed cycles. Results: The BSRs of Korean populations were over 1.10 up to 2002, and then it declined and maintained to 1.06 as balance BSR on 2007 and 2008. In human IVF-ET program, the BSRs were 1.07 on 2007 and 1.06 on 2008, respectively. Conventional IVF on 2008 showed the highest BSR as 1.10, and ICSI the lowest on 2008 as 1.01. There was no significant difference of BSRs related to the methods of in vitro fertilization and the feature of transferred embryos. Conclusion: The BSR of Korea showed 1.06~1.07 as normal and balanced state on 2007 and 2008 both general populations and human IVF-ET program. To the best of our knowledge, this is the first study to evaluate the BSR of human IVF-ET in Korea. There is a need to expand the further studies for national statistics and influencing factors on the BSR with IVF-ET.

• Journal of Life Science
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• v.27 no.6
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• pp.617-622
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• 2017

In this study, we sought to develop an effective cloning system by which human cytochrome $b_5$ (cyt $b_5$) is introduced and expressed in zebrafish. First, the 414 bp human cyt $b_5$ gene was amplified from RNA extracts of HeLa cells using RT-PCR, and the amplicon was subsequently sequenced to confirm that it was intact. Next, cyt $b_5$ was cloned into the pEGFP-N3 vector, which also encodes a fluorescent gene. One-cell stage zebrafish embryos were microinjected with the recombinant vector containing the cyt $b_5$ gene. Fluorescence microscopy confirmed high expression of the fluorescent gene in the injected fry compared to the non-fluorescent control fry. Finally, we extracted RNA from the injected fry and performed RT-PCR to determine whether the human cyt $b_5$ gene is expressed in the transgenic zebrafish. Sequencing analysis further confirmed that the cloned human cyt $b_5$ gene was intact. The transgenic zebrafish model produced in this study will be a useful tool to study therapeutic approaches to cure various diseases related to the deficiency of functional human cyt $b_5$ as well as tools for cloning useful genes in fish.

• Journal of Yeungnam Medical Science
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• v.12 no.1
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• pp.124-134
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• 1995

It is the most important to select optimal culture conditions to promote safe embryo growth in the technique of human in vitro fertilization and embryo transfer. It has been shown that the addition of biologic fluids, such as blood serum, of various origins, improved fertilization and early cleavage rates in numerous species. The purpose of this study is to attempt to measure developmental potential of mouse eggs fertilized and cleaved in Ham's F10 culture medium containing a chelating agent, EDTA and fetal cord serum. In this study, we selected 40 female mice and 20 male mice, and investigated optimal serum concentrations for mouse embryo growth. Two cell stage mouse embryos were cultured in Ham's F-10 medium, Ham's F-10 medium with various concentrations of EDTA, or Ham's F-10 medium with EDTA and 10% human cord serum. Developmental ratios to morula in Ham's F-10 medium containing various concentrations of EDTA and/or 10% fetal cord serum were significantly higher than in unsupplemented Ham's F-10 medium (p<0.05). Developmental ratios to blastocyst in Ham's F-10 containing 10% fetal cord serum and $50{\mu}M$ or $100{\mu}M$ EDTA were significanltly higher than in unsupplemented Ham's F-10 medium (p<0.05). Developmental ratios to morula in Ham's F-10 containing 10% fetal cord serum and $100{\mu}M$ EDTA were significanltly higher than in Ham's F-10 with 10% fetal cord serum used commonly in many human IVF centers(p<0.05). Developmental ratio to blastocyst in Ham's F-10 containing 10% fetal cord serum and $100{\mu}M$ EDTA was significanlty higher than in Ham's F-10 with $200{\mu}M$ EDTA(P<0.05). In summary, embryo development to morula and blastocyst was significanlty higher in the presence of human cord serum or EDTA than in the unsupplemented medium. The most significanly development to morula and blastocyst was obtained at Ham's F-10 medium with $100{\mu}M$ concentration of EDTA and 10% fetal cord serum. These results suggest that Ham's F-10 medium containing 10% fetal cord serum and optimal concentrations of EDTA significantly promoted early cleavage of mouse zygotes, and these will be useful as basic data for the selection of culture medium in human in vitro fertilization.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.159-166
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• 2008

We report here the generation of transgenic chickens that produce human Thrombopoietin (hTPO) using replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G). For the retrovirus vectors, we used hCMV (human Cytomegalovirus) internal promoter to drive the hTPO gene. After confirming the expression of the hTPO gene in various target cells, the concentrated solution of recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). The biological activity of the recombinant hTPO in target cell was significantly higher than its commercially available counterpart. Out of 132 injected eggs, 11 chicks hatched after 21 days of incubation and 4 hatched chicks were found to express vector-encoded hTPO gene. However, 3 out of the 4 transgenics died within one month of hatching. The major significance of this study is that it is one of the very few successful reports on the production of transgenic chickens as bioreactors aiming mass production of commercially valuable and biological active human cytokine proteins.

• Clinical and Experimental Reproductive Medicine
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• v.17 no.1
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• pp.57-63
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• 1990

The purpose of this study is to improve fertilization rate in IVF-ET program of patients with male infertility used micromanipulation technique, partial zona dissection (PZD) or micro-insemination by sperm transfer (MIST). The results were as follows 1. The fertilization rate of non-micromanipulated oocytes and micromanipulated (PZD) oocytes were 12.5% (n=2) and 42.2% (n=19), respectively, and showed significant differences between two groups (p<0.05). 2. The fertilization rate of micromanipulated (MIST) oocytes was 30% (n=27). 3. The damage rate of Group 1 (PZD) and Group 2 (MIST) were 15.7% (3/19) and 29.6% (8/27), respectively. 4. One pregnancy resulted following replacement of micromanipulated (MIST) embryos in 4 patients.