• Clinical and Experimental Reproductive Medicine
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• v.50 no.4
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• pp.244-252
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• 2023

Objective: We evaluated the efficacy of the newly developed optimized in vitro culture (OIVC) dish for cultivating preimplantation mouse embryos. This dish minimizes the need for mineral oil and incorporates microwells, providing a stable culture environment and enabling independent monitoring of individual embryos. Methods: Mouse pronuclear (PN) zygotes and two-cell-stage embryos were collected at 18 and 46 hours after human chorionic gonadotropin injection, respectively. These were cultured for 120 hours using potassium simplex optimized medium (KSOM) to reach the blastocyst stage. The embryos were randomly allocated into three groups, each cultured in one of three dishes: a 60-mm culture dish, a microdrop dish, and an OIVC dish that we developed. Results: The OIVC dish effectively maintained the osmolarity of the KSOM culture medium over a 5-day period using only 2 mL of mineral oil. This contrasts with the significant osmolarity increase observed in the 60-mm culture dish. Additionally, the OIVC dish exhibited higher blastulation rates from two-cell embryos (100%) relative to the other dish types. Moreover, blastocysts derived from both PN zygotes and two-cell embryos in the OIVC dish group demonstrated significantly elevated mean cell numbers. Conclusion: Use of the OIVC dish markedly increased the number of cells in blastocysts derived from the in vitro culture of preimplantation mouse embryos. The capacity of this dish to maintain medium osmolarity with minimal mineral oil usage represents a breakthrough that may advance embryo culture techniques for various mammals, including human in vitro fertilization and embryo transfer programs.

• Korean Journal of Animal Reproduction
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• v.17 no.4
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• pp.305-310
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• 1994

These expriments were carried out to investigate existence of H-Y antibody in the rat serum immunized against H-Y antigen from rat spleen cells and effect of H-Y antiserum on development of mouse male embryos. The results obtained were summerized as follows : 1. When mouse embryos were cultured for 48∼72 hrs in the Ham's F10 containing 16% of FBS(fetal bovine serum) or RNS(rat normal serum), percentages of embryos developed from 2, 4, 8 and 16-cell embryo to morulae were 20, 27, 94 and 100%, respectively, in FBS and 8, 7, 94 and 100%, respectively, in RNS. Eight to 16-cell embryos showed no difference in development rate between FBS adn RNS. 2. When 8∼16-cell mouse embryos were cultured for 24∼48 hrs in the Ham's F10 containing FBS, RNS＋GPC(guinea pig complement) and RAS(rat antiserum)＋GPC, proportions of embryos developed to the expanded blastocyst stage were 100, 82.4 and 52.1∼53.6%(ave.52.9), respectively, so that it was suggested that rat antiserum suppressed development of male embryos. 3. When 8∼16-cell mouse embryos were cultured for 24∼48 hrs in the Ham's F10 containing FBS, RNS, RNS＋GPC and RAS＋GPC, proportions of embryos developed to the expanded blastocyst stage were 94.5, 90.9, 82.3 and 47%, respectively, and the embryos developed in the medium containing RAS＋GPC seemed to be female. These results indicated that the antisera prepared through immunized against H-Y antigen from rat spleen cell, possessed H-Y antibody which supressed development of male embryos.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2004.10a
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• pp.69-69
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• 2004

• 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.

• Korean Journal of Animal Reproduction
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• v.12 no.2
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• pp.77-83
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• 1988

This study was done with mouse embryo to assess effects of freezing media containing sucrose, freezing metods(1-F, 0.3$^{\circ}C$/min;2-F, 3-5$^{\circ}C$/min;3-F, 15$^{\circ}C$min;4-F, LN2 vapour) and cell freezers on the embryo survival determined using the FDA test. The results are summarized as follows. 1. The FDA score obtained with 1, 2, 3 and 4-F was 3.8, 3.6, 3.2 and 3.2, respectively. There was a significant difference(P<0.05) between 1-F, 3-F and 2-F, 4-F. 2. The score at the morular stage(3.8) higher(P<0.005) than the blastocyst stage of embryos(3.2). 3. No difference (P>0.05) was found between the score obtained with a automatic embryo freezer(4.0) and a liquid nitrogen container(3.7).

• Clinical and Experimental Reproductive Medicine
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• v.49 no.1
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• pp.26-32
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• 2022

Objective: We investigated the effect of supplementing fertilization medium and/or culture medium with astaxanthin (AST) on the two phases of in vitro fertilization: gamete fertilization and embryo development. Methods: Mouse cumulus-oocyte complexes were divided into four groups with 5 µM AST added to the fertilization medium (group 3, n=300), culture medium (group 2, n=300), or both media (group 4, n=290). No AST was added to the control group (group 1, n=300). Results: The fertilization rate was significantly higher (p<0.001) in the groups using AST supplemented fertilization medium (group 3, 79.0%; group 4, 81.4%) than those without AST (group 1, 56.3%; group 2, 52.3%). The blastocyst rate calculated from the two-cell stage was significantly lower (p<0.001) in the groups using AST-supplemented embryo culture medium (group 2, 58.0%; group 4, 62.3%) than in those without AST (group 1, 82.8%; group 3, 79.8%). The blastocyst rate calculated from the number of inseminated oocytes was highest in group 3 (189/300, 63.0%) and lowest in group 2 (91/300, 30.3%) with statistical significance compared to other groups (p<0.001). There were significantly higher numbers of cells in the inner cell mass and trophectoderm, as well as significantly higher total blastocyst cell counts, in group 3 than in the control group. Conclusion: An increased blastocyst formation rate and high-quality blastocysts were found only in the fertilization medium that had been supplemented with AST. In contrast, AST supplementation of the embryo culture medium was found to impair embryo development.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.127-137
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• 1994

This study was carried out to investigate the inhibiting or promoting effect of fetal bovine serum fractionated by the molecular weight and to examine the effect of reconstruction of serum fractions on the development of 1- and 2-cell mouse embryos fertilized in vitro (IVEE) . The serum was separated by ultrafiltration or gel filtration methods and added in m-KRB medium for culture of IVFE. The developemental ability(cavitation and hatching) of embryos following culture of day 4 and 6 was compared among fractions. Small molecular weight fraction( <3 kDa) significantly inhibited the development of 1-and 2-cell IVFE to the blastocyst stages, compared with other fractions. One-cell IVFE were more sensitively damaged than 2-cell embryos by that fraction and arrested mainly at 2~4 cell stages. Moreover, small amount(<3%,v /v) of the inhibiting fraction acted even with protein rich fraction(100~30 kDa) and arrested the embryonic development. On the other hand, 100~30 kDa fraction promoted the embryonic development and no inhibiting effect was observed at the level of 50%(v /v) in culture medium In the experiment of gel filtraton, =30 kDa fraction showed the highest promoting effect on the embryonic development, but <4 kDa fraction inhibited significantly the development. These results suggest that serum contains not only small molecular weight inhibitory component(s) but also promoting one rather than albumin on embryonic development. And serum can be more effectively used in the IVF program after removal of inhibitory component(s) by one of above separation methods.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.1-4
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• 2008

This study was performed to investigate the effect of laser-assisted hole in the zona pellucida (ZP) of frozen-thawed ICR mouse embryos on the process of hatching that is critical for expanded blastocysts to implant into endometrium, Vitrification medium, composed of ethylene glycol and sucrose supplemented with 7.5% (w/v) PVP, was used to freeze $2{\sim}4$ cell stage embryos recovered from oviducts of superovulated and mated female mice before storing them in $LN_2$. Right after thawing them, a laser beam was shot to make a hole in ZP followed by culturing in KSOM for $96{\sim}120\;hr$ and examining development to blastocyst and hatching every 12 hr. Laser-treated embryos showed significantly higher hatching rate compared to control (92.9% vs. 22.1%, p<0.05). From around Day 4, blastocysts developed from laser-treated embryos started hatching while the blastocysts of control group failed to hatch showing a lot of shrinkage. This study shows that a laser-assisted hole in ZP improves the hatching rate of blastocysts developed from frozen-thawed, in vitro cultured ICR mouse embryos.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.277-282
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• 2015

Cryopreservation affects osmotic tolerance and intracellular ion concentration through changes in expression levels of water and ion channels. Control of these changes is important for cell survival after cryopreservation. Relatively little is known about changes in $K^+$ channel expression compared to water channel expression. This study was performed to investigate changes in TASK-2 channel (KCNK5: potassium channel, subfamily K, member 5), a member of two-pore domain $K^+$ channel family, in cryopreserved mouse ovaries. Cryopreservation increased TASK-2 mRNA expression in mouse ovaries. In addition, TASK-2 protein expression was upregulated in vitrified and slowly frozen ovaries. TASK-2 protein was expressed in all area of granulosa cells that surround the oocyte within the follicle, except nucleus. Viability of cells overexpressed with TASK-2 was higher than that of vector-transfected cells. Our results found that TASK-2 expression was increased by cryopreservation and overexpression of TASK-2 decreased cryopreservation-induced cell death. These results suggest that TASK-2 upregulation might reduce cryodamage.

• Clinical and Experimental Reproductive Medicine
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• v.47 no.2
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• pp.114-121
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• 2020

Objective: Despite extensive research on implantation failure, little is known about the molecular mechanisms underlying the crosstalk between the embryo and the maternal endometrium, which is critical for successful pregnancy. Profilin 1 (PFN1), which is expressed both in the embryo and in the endometrial epithelium, acts as a potent regulator of actin polymerization and the cytoskeletal network. In this study, we identified the specific role of endometrial PFN1 during embryo implantation. Methods: Morphological alterations depending on the status of PFN1 expression were assessed in PFN1-depleted or control cells grown on Matrigel-coated cover glass. Day-5 mouse embryos were cocultured with Ishikawa cells. Comparisons of the rates of F-actin formation and embryo attachment were performed by measuring the stability of the attached embryo onto PFN1-depleted or control cells. Results: Depletion of PFN1 in endometrial epithelial cells induced a significant reduction in cell-cell adhesion displaying less formation of colonies and a more circular cell shape. Mouse embryos co-cultured with PFN1-depleted cells failed to form actin cytoskeletal networks, whereas more F-actin formation in the direction of surrounding PFN1-intact endometrial epithelial cells was detected. Furthermore, significantly lower embryo attachment stability was observed in PFN1-depleted cells than in control cells. This may have been due to reduced endometrial receptivity caused by impaired actin cytoskeletal networks associated with PFN1 deficiency. Conclusion: These observations definitively demonstrate an important role of PFN1 in mediating cell-cell adhesion during the initial stage of embryo implantation and suggest a potential therapeutic target or novel biomarker for patients suffering from implantation failure.