• Clinical and Experimental Reproductive Medicine
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• v.31 no.4
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• pp.201-207
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• 2004

Objective: The Id family of helix-loop-helix proteins are thought to affect the balance between cell growth and differentiation by negatively regulating the function of basic-helix-loop-helix (bHLH) transcriptional factors. The aim of this study was to investigate the expression pattern of Ids (Id-1, -2, -3, and -4) in preimplantation mouse embryos at mRNA and protein levels. Methods: Oocytes and preimplantation embryos were collected from reproductive organs of female ICR mice following superovulation. RT-PCR was performed to investigate the mRNA expression patterns of Id genes and their protein were localized by immunofluorescence analysis. Results: Id-1 and Id-3 mRNAs were strongly expressed at the germinal vesicle (GV) oocyte and the blastocyst stages. Id-2 mRNA was expressed throughout preimplantation embryo development, but Id-4 was not expressed. Immunofluorescence showed that Id-1 and Id-2 were predominantly localized in cytoplasmic region, but the immunofluorescence signal of Id-3 was weak throughout preimplantation embryo development. Conclusion: These data show for the first time that Ids are expressed in preimplantation mouse embryos and suggest that Ids may play an important role in early preimplantation embryo development and uterine physiological changes.

• Development and Reproduction
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• v.3 no.1
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• pp.69-74
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• 1999

Insulin-like growth factors (IGF-1 and IGF-2) play an important regulatory role in premplantation embryonic development. To study the role of IGF-1 during premplantation embryonic development in mouse, the presence of mRNA transcripts for IGF-1 and IGF-lR in the oocytes and preimplantation embryos was examined. In this study, the transcripts of IGF-1 was detected in oocytes using primers for IGF-1. The PCR products were identified by Msp I restriction enzyme digest. We revealed that the transcripts of IGF-1 and IGF-1R were presented in the oocytes and preimplantation embryos. The highest mRNA levels in GV stage oocytes were decreased at 4- or 8-cell stage and then reincreased upto blastocyst. The presence of IGF-1 and IGF-lR in GV-oocytes suggests that the transcripts in the early stage embryos were derived from maternal genome. Additionally, the presence of IGF-1 and IGF-lR in the oocytes and preimplantation embryos suggests that IGF-1 plays an autocrine role during preimplantation embryonic development through IGF-lR as a signalling pathway.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.3
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• pp.253-259
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• 2000

Objective: To verify the effect of Matrigel, a ECM complex from Engelbreth-Holm-Swarm (EHS) mouse sarcoma on the preimplantation development and apoptosis of mouse fertilized eggs. Method: Late pronucleus stage eggs were cultured through the blastocyst stage in the presence of Matrigel (0.5%, v/v). Characteristics of apoptosis and cell number assesed by Hoecst staining and TUNEL labeling at the blastocyst stage, respectively. Results: Morphological development, number of cells per embryo was significantly increased but rate and number of TUNEL positive nuclei of the embryo were decreased in the presence of Matrigel. Conclusion: This result suggested that at low concentration of Matrigel improves both viability and morphological development in the preimplantation mouse embryos.

• Development and Reproduction
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• v.2 no.2
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• pp.197-203
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• 1998

DNA methylation seems to play an important regulatory role in gene expression and cell differentiation during postimplantation embryonic development. However, the significance of DNA methylation which is maintained by the DNA MTase during preimplantation embryonic development, is not fully understood. In order to study the role of DNA methylation in the preimplantation embryos, the expression of DNA MTase transcripts was monitored in the oocytes and preimplantation embryos. The mRNA of DNA MTase was detected in the oocytes and pleimplantation embryos. The relative mRNA levels of DNA MTase were high from the stages of GV-oocytes and pronuclear embryos, and thereafter decreased gradually. By the treatment of $\alpha$-amanitin, it was confirmed that the transcripts presented in pronuclear embryos was derived from the maternal genome. The presence of transcripts of DNA MTase in the oocytes and pronuclear embryos suggests that the maintenance of DNA methylation may be necessary and seems to play an important role in gene expression and cell differentiation during preimplantation embryonic develop-ment in mouse.

• Korean Journal of Animal Reproduction
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• v.16 no.4
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• pp.341-346
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• 1993

The development of isolated blastomeres from mammalian preimplantation embryos has been basically studied for the multiplication of embryos from superior animals. Therefore, this study was investigated the effect of co-culture with mouse fetal fibroblast cells(MFFC) on in vitro development of blastomeres from mouse preimplantation embryos. Mature female ICR mice were treated with hormone to induce superovulation and embryos were collected at each 2, 4, and 8-cell stage. Then, after removing zona pellucida with protease, blastomeres were isolated by micropipetting, or reconstituted with different stage blastomere, and incubated for 72 hrs either in T6 or TCM199 or on the monolayer of MFFC, which was prepared with fibroblast cells from 14∼14 day mouse fetus. After incubation, we examined their development rates every day and the nuclei numbers of each blastocyst by Hoechst-33342 staining. In the development rates of blastomeres, there were no significant differences between media but the higher rateswere found in the monolayer of MFFC, regardless of reconsititution. In addition, blastomeres cultured with MFFC had slightly greater number of nuclei than those cultured in single media. Generally, the higher development rates of blastomeres were found from earlier stage embryos than the later ones, regardless of culture conditions. Reconsitituted blastomeres had more nuclei but did not show the higher development rates, compared to the single blastomeres. Taken together, our results suggest that co-culture with MFFC have a beneficial effect on the in vitro development of blastomeres from mouse embryos.

• Molecules and Cells
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• v.20 no.3
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• pp.423-428
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• 2005

Immediately after fertilization, a chromatin remodeling process in the oocyte cytoplasm extracts protamine molecules from the sperm-derived DNA and loads histones onto it. We examined how the histone H3-lysine 9 methylation system is established on the remodeled sperm chromatin in mice. We found that the paternal pronucleus was not stained for dimethylated H3-K9 (H3-$m_2K9$) during pronucleus development, while the maternal genome stained intensively. Such H3-$m_2K9$ asymmetry between the parental pronuclei was independent of $HP1{\beta}$ localization and, much like DNA methylation, was preserved to the two-cell stage when the nucleus appeared to be compartmentalized for H3-$m_2K9$. A conspicuous increase in H3-$m_2K9$ level was observed at the four-cell stage, and then the level was maintained without a visible change up to the blastocyst stage. The behavior of H3-$m_2K9$ was very similar, but not identical, to that of 5-methylcytosine during preimplantation development, suggesting that there is some connection between methylation of histone and of DNA in early mouse development.

• Development and Reproduction
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• v.18 no.3
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• pp.153-160
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• 2014

Adaptive development of early stage embryo is well established and recently it is explored that the mammalian embryos also have adaptive ability to the stressful environment. However, the mechanisms are largely unknown. In this study, to evaluate the possible role of aquaporin in early embryo developmental adaptation, the expression of aquaporin (AQP) 5 gene which is detected during early development were examined by the environmental condition. To compare expression patterns between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP5 by whole mount immunofluorescence. At in vivo condition, Aqp5 expressed in oocyte and in all the stages of preimplantation embryo. It showed peak at 2-cell stage and decreased continuously until morula stage. At in vitro condition, Aqp5 expression pattern was similar with in vivo embryos. It expressed both at embryonic genome activation phase and second mid-preimplantation gene activation phase, but the fold changes were modified between in vivo embryos and in vitro embryos. During in vivo development, AQP5 was mainly localized in apical membrane of blastomeres of 4-cell and 8-cell stage embryos, and then it was localized in cytoplasm. However, the main localization area of AQP5 was dramatically shifted after 8-cell stage from cytoplasm to nucleus by in vitro development. Those results explore the modification of Aqp5 expression levels and location of its final products by in vitro culture. It suggests that expression of Aqp5 and the roles of AQP5 in homeostasis can be modulated by in vitro culture, and that early stage embryos can develop successfully by themselves adapting to their condition through modulation of the specific gene expression and localization.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.289-298
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• 2015

Edaravone (Eda) is a potent scavenger of inhibiting free radicals including hydroxyl radicals ($H_2O_2$). Reactive oxygen species (ROS) such as $H_2O_2$ can alter most kinds of cellular molecules such as lipids, proteins and nucleic acids, cellular apoptosis. In addition, oxidative stress from over-production of ROS is involved in the defective embryo development of porcine. Previous study reported that Eda has protective effects against oxidative stress-like cellular damage. However, the effect of Eda on the preimplantation porcine embryos development under oxidative stress is unclear. Therefore, in this study, the effects of Eda on blastocyst development, expression levels of ROS, and apoptotic index were first investigated in preimplantation porcine embryos. After in vitro fertilization, porcine embryos were cultured for 6 days in PZM medium with Eda ($10{\mu}M$), $H_2O_2$ ($200{\mu}M$), and Eda+$H_2O_2$ treated group, respectively. Rate of blastocyst development was significantly increased (P<0.05) in the Eda treated group compared with only $H_2O_2$ treated group. And, we measured intracellular levels of ROS by DCF-DA staining methods and investigated numbers of apoptotic nuclei by TUNEL assay analysis is in porcine blastocyst, respectively. Both intracellular ROS levels and the numbers of apoptotic nucleic were significantly decreased (P<0.05) in porcine blastocysts cultured with Eda ($10{\mu}M$). More over, the total cell number of blastocysts were significantly increased (P<0.05) in the Eda-treated group compared with untreated group and the only $H_2O_2$ treated group. Based on the results, Eda was related to regulate as antioxidant-like function according to the reducing ROS levels during preimplantation periods. Also, Eda is beneficial for developmental competence and preimplantation quality of porcine embryos. Therefore, we concluded that Eda has protective effect to ROS derived apoptotic stress in preimplantation porcine embryos.

• Development and Reproduction
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• v.6 no.1
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• pp.25-30
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• 2002

Endocrine disruptors have been reported to adversely affect reproduction and embryonic development in wild animals. One of the major abnormalities observed during early embryonic development is cellular fragmentation. In this study, we exposed mouse preimplantation embryos to PCB, BPA and DDT in vivo or in vitro. Embryos exposed to endocrine disrupter showed a variety of morphological abnormalities such as fragmentation, irregular blastomeres and cracked empty zonae pellucidae. To investigate the levels of gene expression related which genes contribute to apoptosis in preimplantation mouse embryos, we carried out the reverse transcription polymerase chain reaction to assess mRNA levels far apoptotic gene. Bcl-2, bad and bax expression levels were compared between control group and endocrine disrupter treated group. Expression level of bcl-2 gene tended to be lower in the treated group than control while expression levels of bad and bax genes were higher in the treated group. Results of this study may provide a useful tool for rapidly screening developmental toxicants in preimplantation embryos exposed to endocrine disruptors in vivo or in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.1
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• pp.1-7
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• 2015

Stress coping mechanisms are critical to minimize or overcome damage caused by ever changing environmental conditions. They are designed to promote cell survival. The unfolded protein response (UPR) pathway is mobilized in response to the accumulation of unfolded proteins, ultimately in order to regain endoplasmic reticulum (ER) homeostasis. Various elements of coping responses to ER stress including Perk, Ask1, Bip, Chop, Gadd34, Ire1, Atf4, Atf6, and Xbp1 have been identified and were found to be inducible in oocytes and preimplantation embryos, suggesting that, as a normal part of the cellular adaptive mechanism, these coping responses, including the UPR, play a pivotal role in the development of preimplantation embryos. As such, the UPR-associated molecules and pathways may become useful markers for the potential diagnosis of stress conditions for preimplantation embryos. After implantation, ER stress-induced coping responses become physiologically important for a normal decidual response, placentation, and early organogenesis. Attenuation of ER stress coping responses by tauroursodeoxycholate and salubrinal was effective for prevention of cell death of cultured embryos. Further elucidation of new and relevant ER stress coping responses in periimplantation embryos might contribute to a comprehensive understanding of the regulation of normal development of embryonic development and potentiation of embryonic development in vitro.