• Development and Reproduction
• /
• v.3 no.1
• /
• pp.75-85
• /
• 1999

To investigate the origin and action mechanism of cytoplasmic factors as regulators of morphogenesis, the embryonic development, RNA synthesis and protein phosphorylation were examined in reconstituted embryos. A half of 1-cell mouse embryo with both pronuclei was electrofused with the enucleated cytoplasm of 1- or 2-cell embryos which were cultured for 24 hrs from post 20 hrs hCG in CZB with or without cycloheximide (CHX, an inhibitor of protein synthesis; P+P-CHX group), genistein (Gen, an inhibitor of tyrosine protein kinase; P+2-Gen group) and 6-dimethylaminopurine (6-DMAP, an inhibitor of serine-threonine protein kinase; P＋2-DMAP group), and co-cultured with Vero cells for 5 days. And their development, cell numbers at compaction, [5, 6-$^3$H]-uridine incorporation into RNA and the pattern of protein phosphorylation after labeling of [$^{32}$ P] orthophosphate were compared with that of the reconstituted embryos such as P+2 or P+P (control group). Embryonic development and the time of RNA synthesis in P+P-CHX were similar to those in P+P. But the time and the cell stages of embryonic compaction in P+P-CHX were similar to those in P+2. The compaction was initiated at 4-cell in P+2 and P+2-Gen, but at 8-cell in P+P and P+2-DMAP. On a two-dimensional gel electrophoresis, phosphorylation of 80KD and 110KD proteins were inhibited after 3 hrs of reconstruction in the embryo of P+2-DMAP when compared with that of P+2 and P+2-Gen. These results suggest that protein synthesis between 1- and 2-cell stage affects the timing of embryonic genome activation, and that cytoplasmic factors derived from oocyte or their modification regulates the time schedule of embryonic compaction in mouse. Also, serine-threonine protein kinase has an important role on the regulation of compaction.

• Reproductive and Developmental Biology
• /
• v.39 no.3
• /
• pp.77-81
• /
• 2015

This study was carried out to evaluate the effects of embryonic stage and toxicities of cryoprotectant on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology. Toxicities of two cryoprotectants, dimethyl sulfoxide (DMSO) and ethylene glycol (EG) were investigated using a murine embryo model. Female F-1 mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to either DMSO or EG. Embryo development was evaluated up to the blastocyst stage. The total cell count of blastocysts that were treated with DMSO ($68.1{\pm}24.1$) at the 2-cell stage was significantly lower than that were treated with EG ($81.2{\pm}27.0$) or the control ($99.0{\pm}18.3$) (p<0.001). On comparison of two cryoprotectant treated groups, the DMSO treated group showed a decreased cell count compared with the EG treated group (p<0.05). Both DMSO ($15.4{\pm}1.5$) and EG ($10.2{\pm}1.4$) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control ($6.1{\pm}0.9$, p<0.0001). In addition, the DMSO treated group showed more apoptotic cells than the EG treated group (p<0.001). The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to either DMSO or EG at room temperature. When comparing two cryoprotective agents, EG appeared to be less toxic than DMSO at least in a murine embryo model.

• Korean Journal of Veterinary Research
• /
• v.29 no.2
• /
• pp.123-128
• /
• 1989

Demi-embryos were successfully produced by bisection of ICR mouse embryos at preimplantation stages. They were microsurgically bisected using a microsurgical blade attached to a micromanipulator after pretreatment with 0.5% pronase in PBS for two minutes or not. Embryos with softened zona pellucida were more easily bisected and less damaged than intact embryos. The highest success rate in bisection has been achieved by selecting blastocysts(94.1% in success rate with intact blastocysts and 100% in success rate with zona softened blastocysts). Demi-embryos without zona pellucida were cultured in D-PBS or M-16 medium at $37^{\circ}C$, 5% $CO_2$ in air for 72 hours for 2-cell stage embryos, 48 hours for 4-to 8-cell stage embryos, 24 hours for morula stage embryos and 6~12 hours for blastocyst stage embryos. For the in vitro culture of 2-cell stage embryos, $100{\mu}M$ 2Na-EDTA was added to the media. M-16 medium was better for the in vitro development of mouse embryos than PBS, and PBS is not considered to be suitable for long-term culture of embryos, especially at early stage of cleavage. In M-16 medium, developing rate of demi-embryos of which pair underwent development to form eublastocysts was 15.8% at 2-cell stage, 16.8% at 4-cell stage, 38% at 8-cell stage, 89.6% at morula stage and 94.4% at blastocyst stage, respectively. The more rapid and efficient production of demi-embryos and higher viability after bisection can be expected by softening zona pellucida with pronase and by selecting morulae or blastocysts rather than embryos at early stage of cleavage.

• Clinical and Experimental Reproductive Medicine
• /
• v.30 no.1
• /
• pp.5-14
• /
• 2003

Objective: Present study was aimed to verify the effect of granulocyte macrophage-colony stimulating factor (GM-CSF) in the preimplantation development of mouse embryos and the involvement of the mitogen activated protein kiase (MAPK) in the GM-CSF signaling. Methods: Two-cell embryos were cultured for 96 h in the presence or absence of GM-CSF (0, 0.4, 2, 10 ng/ml) and PD98059, a MEK inhibitor (10 ${\mu}M$). Morphological development, cell number per blastocyst, and apoptotic nuclei, were eamined. MAPK activity of embryonic immunoprecipitate by MAPK (ERK1/2) antibody was measured by in vitro phosphorylation of myelin basic protein. Results: At post hCG 122 h the embryonic development among the experimental groups was significantly different (p=0.018). The rate of blastocyst development and cell number per embryo were the highest in 2 ng/ml GM-CSF treatment group. The percent of apoptotic cells of the GM-CSF-treated embryos was the lowest among the group. In blastocysts, GM-CSF treatment transiently increased MAPK activity. PD098059 attenuated the effect of GM-CSF on the morphological development, increase in cell number per blastocyst, down regulation of apoptosis, and upregulation of MAPK activity, suggesting that activation of MAPK cascade possibly mediated the embryotropic effect of GM-CSF. Conclusion: This result suggested that GM-CSF potentiated the development of preimplantation mouse embryos by activation of MAPK.

• Korean Journal of Animal Reproduction
• /
• v.19 no.2
• /
• pp.81-88
• /
• 1995

Culture medium (ASF-301) of tHUE-2 cell, human endothelial cell line, and culture medium of these cells (conditioned medium : CM) which affect embryonic development of in vivo fertilized 1-cell embryos of mouse were examined. Two-cell stage block of mouse embryos was overicomed in ASF-301 and CM without EDTA, which usually added in basic medium (modified Whitten Medium: MWM, control) to overcome the 2-cell stage block. The developmental rates of embryos to the blastocyst stage were significantly increased in MWM containing 12.5% of growth factors added to ASF-301 (10mg/ $\ell$ transferrin, 1mg/$\ell$ insulin, 0.01mg/$\ell$ EGF) than those of 100% addition and control, 78.0% vs 20.8 and 52.3%, respectively (P<0.05), but the growth factors was not affected the hatching rate of blastocyst. Using ASF-301 or CM which was not treated, embryonic development into the blastocyst and hatched blastocyst stages were not affected. However, proportions of embryonic development into the blastocyst and hatched blastocyst stages were significantly higher in dilution (ASF-301 1:10; CM 1:3~1:6) than those in control (P,0.05). In ASF-301 dialyzed M.W.<10000 dialysis membrane, the developmental rate upto the hatched blastocyst stage was significantly increased, compared to ASF-301 which was not dialyzed (P<0.05), and hatching rate of blastocyst of these group was singnificantly increased than those in MWM (P<0.05). Compared to CM which was not dialyzed, however, in dialyzed CM was significantly decreased, compared to untreated CM (P<0.05), especially any hatched blastocyst was not appeared. As a result of these experiments indicated that a kind or porper treatment such as a dilution of complex synthetic cell culture medium and conditioned medium, and that a optimal concentration of growth factors are usuful for embryo cultrue in vitro.

• Proceedings of the Korean Society of Developmental Biology Conference
• /
• 2003.10a
• /
• pp.7-8
• /
• 2003

Since it was first reported in 1997, somatic cell cloning has been demonstrated in several other mammalian species. On the mouse, it can be cloned from embryonic stem (ES) cells, fetus-derived cells, and adult-derived cells, both male and female. While cloning efficiencies range from 0 to 20%, rates of just 1-2% are typical (i.e. one or two live offspring per one hundred initial embryos). Recently, abnormalities in mice cloned from somatic cells have been reported, such as abnormal gene expression in embryo (Boiani et al., 2001, Bortvin et al., 2003), abnormal placenta (Wakayama and Yanagimachi 1999), obesity (Tamashiro et ai, 2000, 2002) or early death (Ogonuki et al., 2002). Such abnormalities notwithstanding, success in generating cloned offspring has opened new avenues of investigation and provides a valuable tool that basic research scientists have employed to study complex processes such as genomic reprogramming, imprinting and embryonic development. On the other hand, mouse ES cell lines can also be generated from adult somatic cells via nuclear transfer. These 'ntES cells' are capable of differentiation into an extensive variety of cell types in vitro, as well assperm and oocytes in vivo. Interestingly, the establish rate of ntES cell line from cloned blastocyst is much higher than the success rate of cloned mouse. It is also possible to make cloned mice from ntES cell nuclei as donor, but this serial nuclear transfer method could not improved the cloning efficiency. Might be ntES cell has both character between ES cell and somatic cell. A number of potential agricultural and clinical applications are also are being explored, including the reproductive cloning of farm animals and therapeutic cloning for human cell, tissue, and organ replacement. This talk seeks to describe both the relationship between nucleus donor cell type and cloning success rate, and methods for establishing ntES cell lines. (중략)

• Genomics & Informatics
• /
• v.10 no.1
• /
• pp.16-22
• /
• 2012

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of multiple fluid-filled cysts that expand over time and destroy renal architecture. The proteins encoded by the $PKD1$ and $PKD2$ genes, mutations in which account for nearly all cases of ADPKD, may help guard against cystogenesis. Previously developed mouse models of $PKD1$ and $PKD2$ demonstrated an embryonic lethal phenotype and massive cyst formation in the kidney, indicating that $PKD1$ and $PKD2$ probably play important roles during normal renal tubular development. However, their precise role in development and the cellular mechanisms of cyst formation induced by $PKD1$ and $PKD2$ mutations are not fully understood. To address this question, we presently created $Pkd2$ knockout and $PKD2$ transgenic mouse embryo fibroblasts. We used a mouse oligonucleotide microarray to identify messenger RNAs whose expression was altered by the overexpression of the $PKD2$ or knockout of the $Pkd2$. The majority of identified mutations was involved in critical biological processes, such as metabolism, transcription, cell adhesion, cell cycle, and signal transduction. Herein, we confirmed differential expressions of several genes including aquaporin-1, according to different $PKD2$ expression levels in ADPKD mouse models, through microarray analysis. These data may be helpful in $PKD2$-related mechanisms of ADPKD pathogenesis.

• Development and Reproduction
• /
• v.4 no.1
• /
• pp.29-35
• /
• 2000

A phosphatidylinositol 3-kinase (PI3K) is a upstream component of insulin signaling by which protein synthesis can be stimulated in many systems. To elucidate involvement of PI3K and its downstream mammalian target of rapamycin (mTOR) in the insulin signaling in pleimplantation mouse embryos, 8-cell embryos were cultured to blastocysts in the presence or absence of insulin and／or inhibitor drugs. The number of blastomeres per blastocyst, protein synthesis, and protein phosphorylation were examined. There was significant difference in embryonic development to blastocyst stage and hatching was potentiated by the insulin supplementation. The increase in the mean celt numbers per blastocyst was apparent in the insulin culture. Wortmannin, a PI3K inhibitor and rapamycin, an inhibitor of mTOR abolished the stimulatory effect of insulin on morphological development mitosis and protein synthesis. In autoradiography, phosphoproteins pp22 and pp30 which undergo phosphorylation in response to insulin were identified. Taken together, it can be suggested that PI3K and mTOR engaged in insulin signaling in the mouse embryo 8-cell onward and mediate embryotropic offset of insulin.

• Korean Journal of Animal Reproduction
• /
• v.16 no.4
• /
• pp.317-323
• /
• 1993

This study was carried out to study effects of the addition level of acetamide and non-permeable cryoprotectants(Ficoll, sucrose) in VS(20% glycerol＋10% ethyleneglycol) and equilibration time on the survival of vitrified mouse morulae. The results are summarized as follows: 1. When 10, 15 and 20% of acetamide were added to the new vitrification solution(20G 10E), FDA-scores of embryos were 4.4(control), 4.4(10%), 3.6(15, 20%), respectively. The addition of acetamide did not affect the survival of forzen-thawed morulae(P<0.05). 2. The survival rate betwen 5 min(3.5) and 10 min(4.6), 10 min(4.6) and 20 min(3.2) of equilibration in 10% sucrose, and 20 min(3.2) and 5 min(4.0), or 10 min(4.3) in 20% sucrose were significantly different(P<0.05). The highest survival(4.6) rate was obtained in mouse morulae equilibrated in VS(20G 10E) containing 10% sucrose for 10 minutes. 3. FDA-score of morulae frozen in the new vitrification solution containing 0, 10, 20 and 30% Ficoll was 4.5, 4.2, 4.4 and 4.6, respectively and had no significant effect among concentrations of Ficoll(P>0.05). The development rate after culture(24h) was 89%(20% Ficoll) and 93%(30% Ficoll), respectively.

• Development and Reproduction
• /
• v.9 no.1
• /
• pp.33-41
• /
• 2005

Identification of differentially expressed genes at blastocyst stage embryos would provide insights into early development and differentiation. Here, we applied a new differential display reverse transcription polymerase chain reaction(DD RT-PCR) technology, called annealing control primers(ACP) system to identify the genes that are specifically or prominently expressed in mouse blastocysts compared to embryonic stem(ES) cells. Using 100 ACPs, 26 clones were perceived as differentially expressed genes in mouse blastocysts. A BLAST search revealed that cloned genes had significant sequence similarities with known genes in the GenBank/EMBL data base. Among them, 15 genes were selected and conformed by RT-PCR. This analysis suggests that the ACP system is a practical method for the identification of stage-specific genes using small numbers of mouse embryos.