• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.25-32
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• 1996

This work has been carried out to examine the number of Total, ICM and TE cells of F1 mouse blastcysts at day 4 after IVF by differential labelling of the nuclei with polynucleotide-specific fluorochromes and to obtain a fundamental information of preimplantation mouse embryo development. Blastocysts produced by superovulated B6CBA F1(C57BL/${\times}$CBA) eggs were inseminated with $1{\times}10^6$spermatozoa/ml and cultured in M16 medium at $37^{\circ}C$, 5% $CO_2$ incubator for 95hrs. Blastocysts were classified as early, middle, expanded and hatching stage according to the developmental morphology; blastocoel expansion and zona thickness. The results obtained in these experiments were summarized as follows; 1) The development rate of blastocysts at 95hrs after IVF was 86.7% and classified blastocysts to early, middle, expanded and hatching were 16.3%, 18.9%, 10.5% and 40.9%, respectively. 2) The numbers of total blastomere using bisbenzimide in the classified blastocysts to early, middle, expanded and hatching were 35.6${\pm}$1O.4, 49.4${\pm}$8.6, 60.8${\pm}$1O.7 and 62.7${\pm}$13.9, respectively. 3) In ICM and TE cell number by using differential labelling with polynucleotide-specific fluorochrome in the classified blastocysts to early, middle, expanded and hatching; ICM numbers were 9.6${\pm}$3.0, 13.6${\pm}$3.9, 16.0${\pm}$3.3 and 19.5${\pm}$4.6, respectively and TE cell numbers were 30.6${\pm}$5.1, 39.9${\pm}$5.8, 42.2${\pm}$8.1 and 43.7${\pm}$11.1, respectively. These results showed the same increase pattern according to development advance level. Also, when compared with the results of total count were obtained between bisbenzimide only and differential labelling, both of them showed the same increase pattern according to development level and at the same time their cell numbers were almost the same. So, rapid and simple cell count method using differential labelling can be used for the examination of later preimplantation development or as an indicator of embryo quality according to the variables of culture conditions.

• Development and Reproduction
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• v.6 no.2
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• pp.97-104
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• 2002

In most mammals, mature oocyte-cumulus complexer(OCCs) ovulate into the oviduct where fertilization by sperm takes place. However, the complex that fail to fertilize eventually undergoes degeneration while they reside in the oviduct. Yet there is no blown mechanism how both oocyte and cumulus cells degenerate. Using human follicular fluid (hFF), bovine oviductal tissue extract (BOX) and mouse OCC, the present study aimed to find how the oviduct influence the viability of the oocyte and cumulus cells in vitro. There was no difference of oocyte maturation rate between the control and BOX-treated groups. However, there was a significant difference in the survival of cumulus cells between two groups. Cumulus cells cultured in the presence of hFF alone underwent initially expansion and then they formed monolayer in the culture dish. Even after 72 hr, they proliferated well and showed fibroblast-like morphology. Cumulus cells cultured in the presence of both hFF and BOX also expanded after 24 hr, however, after 72 hr culture, they eventually detached and degenerated. Cumulus cells cultured in the BOX alone gave a similar drastic result. When the cumulus cells cultured in the presence of BOX were stained with DAPI, their nuclei showed partial condensation and fragmentation. After detailed analysis of these cells by TUNEL assay, many nuclei of them exhibited well stained spots indicating the signs of apoptosis. Based upon these observations, it is suggested that BOX might possess a factor that leads mouse cumulus cells to undergo apoptosis in vitro.

• Development and Reproduction
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• v.12 no.1
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• pp.31-39
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• 2008

Neural tissue has limited intrinsic capacity of repair after injury, and the identification of alternate sources of neural stem cells has broad clinical potential. We isolated mesechymal-like stem cells from human adipose tissues (AT-MSCs), and studied on transdifferentiation-promoting conditions in neural cells. Dopaminergic and cholinergic neuron induction of AT-MSCs was also studied. Neural differentiation was induced by adding bFGF, EGF, dimethyl sulphoxide (DMSO) and butylated hydroxyanisole(BHA) in N2 Medium and N2 supplement. The immunoreactive cells for $\beta$-tubulin III, a neuron-specific marker, GFAP, an astrocyte marker, or Gal-C, an oligodendrocyte marker, were found. AT-MSCs treated with bFGF, SHH and FGF8 were differentiatied into dopaminergic neurons that were immunopositive for TH antibody. Differentiation of MSCs to cholinergic neurons was induced by combined treatment with basic fibroblast growth factor (bFGF), retinoic acid (RA) and sonic hedgehog (Shh). AT-MSCs treated with DMSO and BHA rapidly assumed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including neuro D1, $\beta$-tubulin III, GFAP and nestinwas markedly elevated during this acute differentiation. While the stem cell markers such as SCF, C-kit, and Stat-3 were not expressed after preinduction medium culture, we confirmed the differentiation of dopaminergic and cholinergic neurons by TH/$\beta$-tubulin III or ChAT/ $\beta$-tubulin III positive cells. Conclusively, AT-MSCs can be differentiated into dopaminergic and cholinergic neuronsand these findings suggest that AT-MSCs are alternative cell source of treatment for neurodegenerative diseases.

• Development and Reproduction
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• v.14 no.4
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• pp.269-279
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• 2010

Some characteristics of germ cell differntiations and the function of accessory cells during spermatogenesis, and mature sperm ultrastructure in male Protothaca (N.) jedoensis were investigated by transmission electron microscope observations. The morphology of the spermatozoa of this species has a primitive type and is similar to those of other species in the subclass Heterodonta. Accessory cells, which are connected to adjacent germ cells, are involved in the supplying of the nutrients for germ cell development. The morphologies of the sperm nucleus and the acrosome of this species are the cylindrical type and cap shape, respectively. Spermatozoa are approximately $46{\sim}50{\mu}m$ in length including a long sperm nucleus (about $2.44{\mu}m$ in length), an acrosome (about $0.45{\mu}m$ in length), and tail flagellum (about $42{\sim}46{\mu}m$). The axoneme of the sperm tail shows a 9+2 structure. As some characteristics of the acrosomal vesicle structures, the basal and lateral parts of basal rings show electron opaque part (region), while the anterior apex part of the acrosomal vesicle shows electron lucent part (region). These characteristics of the acrosomal vesicle were found in the family Veneridae and other several families in the subclass Heterodonta. These common characteristics of the acrosomal vesicle in the subclass Heterodonta can be used for phylogenetic and systematic analysis as a taxonomic key or a significant tool. The number of mitochondria in the midpiece of the sperm of this species are four, as one of common characteristics appear in most species in the family Veneridae and other families in the subclass Heterodonta. However, exceptionally, only three species in Veneridae of the subclass Heterodonta contain 5 mitochondria. The number of mitochondria in the sperm midpiece can be used for the taxonomic analysis of the family or superfamily levels as a systematic key or an important tool.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.27-34
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• 1996

The objective of this study was to examine the cell number of Total, ICM and TE cells of bovine blastocysts according to development progression cultured in CR1 medium, which was reported as successfully supporting medium for preimplantaion bovine embryo development to the blastocyst stage, by differential labelling of the nuclei with immunosurgery and polynucleot-ide-specific fluorochromes. Blastocysts were obtained at day 8 after in vitro fertilization and classified to early, middle, expanded stage according to the developmental morphology; blastocoel expansion and zona thickness. Also, bias tocysts in the same category were divided into two parts to check the Total cell number by using bisbenzimide only and ICM, TE and Total cell number by using immunosurgery and two polynucleotide-specific fluorochromes. 1) The development rate of blastocysts at day 8 after in vitro fertilization was 29.3% and classified bIas tocysts to early, middle, expanded and hatching stage were 8.7, 9.9, 7.6 and 3.1%, respectively. 2) The numbers of total blastomere using bisbenzimide in the classified blastocysts to early, middie and expanded were 46.9${\pm}$8.6, 66.2${\pm}$12.5 and 122.8 ${\pm}$ 14.4, respectively. This indicated that CR1 is a appropriate culture medium for bovine embryo development. 3) The count of ICM and TE cell number by using differential labelling with immunosurgery and polynucleotide-specific fluorochromes in the classified blastocysts to early, middle and expanded; ICM cell numbers of were 12.8${\pm}$5.9, 26.3${\pm}$8.4 and 35.5${\pm}$15.0, respectively and TE cell numbers were 30.5${\pm}$5.0, 4 41.3${\pm}$8.2 and 81.1${\pm}$13.4, respectively. These results presented that the increase of ICM and TE cell numbers averaged two and three doublings between early and expanded blastocyst stage and also total cell number counted from ICM nuclei and TE nuclei by using differential label-ling showed the increase pattern with development advance level and the results were similar to total cell number obtained from bisbenzimide treatment only. Therefore, the differential labelling of ICM and TE nuclei in situ is a very useful technique to evaluate embryo qualities and can be used as an indicator on study of preim-plantation embryo development.

• Journal of the Korean Academy of Child and Adolescent Psychiatry
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• v.12 no.2
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• pp.256-262
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• 2001

Objective：The enlargement of the lateral ventricle is well established finding in adult-onset schizophrenia, but in the patients with early-onset psychotic disorder(EOPD) is still controversal. To identify structural abnormalities of the lateral ventricle in patients with EOPD and difference of the brain developmental course in EOPD group, planimetrical assessment of the lateral ventricle was conducted by means of retrospective quantitative neuroimaging. Methods：The brain MR images of 14 patients with EOPD(mean age $14.0{\pm}2.1$ years) who met the DSM-III-R diagnostic criteria for schizophrenia and bipolar disorder and 24 control subjects with simple headache(mean age $10.0{\pm}2.1$ years) were collected. Single best view for the lateral ventricle, one axial slice was selected for analysis. MR images were redigitalized with flatbed scanner and data were analyzed with NIH IMAGE software. Results：In the patients with EOPD, there were significant correlations between aging and lateral ventricular sizes and between aging and ventricular brain ratio(VBR). The statistical significance of normal asymmetry of the lateral ventricle was not observed for the EOPD group. The EOPD group had larger lateral ventricular sizes and VBR than control group, but these results were not statistically significant. Conclusion：These findings suggest that the brain abnormalities in patients with EOPD progress from the earlier course of the disorders and the course of brain development in EOPD group is different from control group.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.1
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• pp.1-12
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• 2002

Objective: In order to acquire the technique for the establishment of human embryonic stem cells (ESe) derived from the human frozen-thawed embryos produced in IVF-ET program, this study was performed to establish mouse ESC derived from the in vitro fertilized embryos. Materials and Methods: After Fl hybrid (C57BL female $\times$ CBA mael) female mice were superovulated with PMSG and hCG treatment, their oocytes were retrieved and inseminated, and the fertilized embryos were cultured for 96-120 hours until the expected stages of blastocysts were obtained. To isolate the inner cell mass (ICM), either the blastocysts were treated with immunosurgery, or the whole embryos were cultured for 4 days. Isolated ICMs were then cultured onto STO feeder cell layer, and the resultant ICM colonies were subcultured with trypsin-EDTA treatment. During the subculture process, ESC-like cell colonies were observed with phase contrast microscopy. To identify ESC in the subcultured ESC-like cell colonies, alkaline phosphatase activity and Oct-4 (octamer-binding transcription factor-4) expression were examined by immunohistochemistry and RT-PCR, respectively. To examine the spontaneous differentiation, ESC-like cell colonies were cultured without STO feeder cell layer and leukemia inhibitory factor (LIF). Results: Seven ESC-like cell lines were established from ICMs isolated from the in vitro fertilized embryos. According to the developmental stage, the growth of ICMs isolated from the expanded blastocysts was significantly better than that of ICMs isolated from the hatched blastocysts (80.3% vs. 58.7%, p<0.05). ESC-like cell colonies were only obtained from ICMs of expanded blastocysts. However, the ICMs isolated from the embryos treated with immunosurgery were poorly grown and frequently differentiated during the culture process. The established ESC-like cell colonies were positively stained with alkaline phosphatase and expressed Oct-4, and their morphology resembled that observed in the previously reported mouse ESC. In addition, following the extended in vitro culture process, they maintained their expression of cell surface markers characteristic of the pluripotent stem cells such as alkaline phosphatase and Oct-4. When cultured without STO feeder cell layer and LIF, they were spontaneously differentiated into the various types of cells. Conclusion: The findings of this study suggest that the establishment of mouse ESC can be successfully derived from the in vitro fertilized embryos. The established ESC-like cells expressed the cell surface markers characteristic of the pluripotent stem cells and spontaneously differentiated into the various types of cells.

• Parasites, Hosts and Diseases
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• v.24 no.2
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• pp.109-114
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• 1986

In order to observe the growth and development of Fibricola seoulensis metacercariae, the tadpoles of Rana nigromaculata were experimentally infected with the cercariae. The meta cercariae of various developmental stages were recovered from the tadpoles after 2 to 65 days of infection. They were prepared for morphological observation, and were given orally to mice to observe their infectivity. The following results were obtained. 1. All of the tadpoles exposed to the cercariae were observed to harbour the larvae in their abdominal cavity. 2. The young metacercariae of 2 days after infection were $121.1{\mu}m$ long and $63.3{\mu}m$ wide. They grew linearly for the first 14 days to be $262.0{\mu}m$ long and $166.4{\mu}m$ wide. Thereafter, no more growth recognized until 65 days. 3. The larvae of 2 days old were similar with cercarial body and had 2 suckers, a pharynx, 2 ceca and a primordium of germ cells but no tribocytic organ. On the 8th day, they had tribocytic organ, and their morphology resembled that of mature metacercariae. 4. The metacercariae younger than 10 days could not infect the mice. Only the metacercariae older than 14 days had infectivity. The recovery rates increased by the age of metacercariae from 19.0% in 14 days old to 70.0% in 40 days old. Above findings indicate that the tadpole is indispensable for metacercarial development and it needs at least 2 weeks for maturation. The tadpole is a pivotal host in the life cycle of F. seoulensis for connection between the snail and the frog.

• Development and Reproduction
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• v.11 no.3
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• pp.235-243
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• 2007

Human umbilical cord blood(HUCB) contains a rich source of hematopoietic stem cells, mesenchymal stem cells and endothelial cell precursors. Mesenchymal stem cells(MSCs) in HUCB are multipotent stem cells, differ from hematopoietic stem cells and can be differentiated into neural cells. We studied on transdifferentiation-promoting conditions in neural cells and cholinergic neuron induction of HUCB-derived MSCs. Neural differentiation was induced by addingdimethyl sulphoxide(DMSO) and butylated hydroxyanisole(BHA) in Dulbeco's Modified Essential Medium(DMEM) and fetal bovine serum(FBS). Differentiation of MSCs to cholinergic neurons was induced by combined treatment with basic fibroblast growth factor(bFGF), retinoic acid(RA) and sonic hedgehog(Shh). MSCs treated with DMSO and BHA rapidly assumed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including $\beta$-tubulin III, GFAP and MBP, was markedly elevated during this acute differentiation. The differentiation rate was about $32.3{\pm}2.9%$ for $\beta$-tubulin III-positive cells, $11.0{\pm}0.9%$ for GFAP, and $9.4{\pm}1.0%$ for Gal-C. HUCB-MSCs treated combinatorially with bFGF, RA and Shh were differentiated into cholinergic neurons. After cholinergic neuronal differentiation, the $\beta$-tubulin III-positive cell population of total cells was $31.3{\pm}3.2%$ and of differentiated neuronal population, $70.0{\pm}7.8%$ was ChAT-positive showing 3 folds higher in cholinergic population than neural induction. Conclusively, HUCB-derived MSCs can be differentiated into neural and cholinergic neurons and these findings suggest that HUCB are alternative cell source of treatment for neurodegenerative diseases such as Alzheimer's disease.

• Development and Reproduction
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• v.11 no.3
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• pp.263-272
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• 2007

Contrast to mouse where its in vitro maturation rates are high without specific supplements or presence of the cumulus cells, there are some species, such as porcine, where its in vitro oocyte maturation rates are still very low. This comparative study was conducted to investigate the role of malate dehydrogenase(Mor2) during oocyte maturation by RNAi in the mouse and porcine. The Mor2 double-stranded RNA(dsRNA) was prepared speciesspecifically and microinjected into the cytoplasm of denuded germinal vesicle(GV) oocytes. Oocytes were cultured for 48 h(porcine) and 16 h(mouse) in M199 with 10% porcine follicular fluid, pyruvate, p-FSH, EGF, cystein, and estradiol-$17{\beta}$. We measured changes in oocyte morphology, maturation rates and mRNA levels after Mor2 RNAi. We confirmed gene sequence-specific knock down of Mor2 mRNA in both species after Mor2 RNAi. In contrast to our previous finding that mMor2 RNAi resulted in GV arrest in the mouse, we found that pMor2 RNAi resulted in MI arrest in denuded porcine oocytes(58%), but developed to MII(84.4%) in COCs. To determine whether this difference between mouse and porcine RNAi is due to differences in culture media, we cultured mouse oocytes in the M199 media for 16 h after mMor2 RNAi. Mouse oocytes were developed to MII stage(62%) and there was no statistical difference compared to that of non-injected(76.8%) and buffer-injected(73.3%) control groups. Therefore, we concluded that the mouse and porcine oocytes are having different metabolic systems in relation to malate dehydrogenase for oocyte maturation. This could be a basis for differences in maturation rates in vitro in two species. Further scrutinized studies on the metabolic pathways would led us in finding better culture system to improve oocyte maturation rates in vitro, especially in more challenging species like the porcine.