• Development and Reproduction
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• v.5 no.1
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• pp.17-21
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• 2001

This study was conducted to investigate the expression pattern of Trypsin-like enzyme and the effect of a trypsin inhibitor(benzimidine) on hatching process during in-vitro culture of mouse preimplantation embryos. The Trypsin-like enzyme was identified by rhodamine-conjugated Trypsin substrate probe. The expression of trypsin-like enzyme was firstly detected at the late morula stage, and the enzyme was uniformly localized in the trophectoderm of late blastocysts. Especially, intense fluorescence was observed in the blebbing area of hatching blastocysts. Bisbenzamidine, contained in culture media, did not alter embryonic development from 4-cell stage to the expanded blastocyst but decrease the hatching rate in ImM concentration (15.8% vs 89.7%, p<0.02). In the treatment of bisbenzimidine (5mM) for 12 hours according to the embryonic stage of mouse, the hatching rate of control (83.0%) and treatment in late blastocysts (8.7%) were significantly (p<0.01) different. From these results, we suggested that the hatching enzyme having trypsin-like activity was localized from the late morula stage, and the hatching process by this enzyme was activated in the late blastocyst stage of mouse embryos.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.35-41
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• 1998

This study was carried out to investigate the development rates of human embryos co-cultured with cumulus cells to each blastocyst stage. Human zygotes were co-cultured on cumulus cell monolayer in YS medium supplemented with 20% hFF. On day 2, if patient had four or more "good" embryos (regular blastomeres without fragmentation), embryos were further cultured for 72hrs. Blastocysts on day 5 were classified into early blastocyst (ErB), early expanding blastocyst (EEB), middle expanding Blastocyst (MEB), and expanded blastocyst (EdB) on the basis of their morphological aspects of trophectoderm cells and blastocoele. Subsequently, maximum 3 of best blastocysts were transferred in 486 cycles. The results in this study were as follows: Patients who had four or more "good" embryos on day 2 were 498 persons, but patients whose embryos could not be transferred due to failure in development to the blastocyst stage on day 5 were 12 persons (2.4%). The development rate of embryos to the blastocyst stage was 58.2% (2,885/4,957) on day 5, and the rates that developed to the ErB, EEB, MEB, and EdB stage were 15.0% (743/4,957), 14.9% (739/4,957), 14.4% (714/ 4,957), and 13.9% (689/4,957), respectively. Total 1366 blastocysts were transferred in 486 cycles (mean number=2.81). The implantation rate and the ongoing implantation rate obtained by observing the number of G-sac and FHB were 29.9% (409/1,366) and 22.5% (308/1,366), respectively. The clinical pregnancy rate was 51.2% (249/486), and the ongoing pregnancy rate' was 39.1% (190/486). Among women showing ongoing pregnancy, women with singleton were 50% (95/190), women with twin were 37.9% (72/190), and women with triplet were 12.1% (23/190). Although triplet pregnancy rate in this study was high such as 12.1%, because many blastocysts with high viability were produced in our co-culture system using cumulus cells on day 5, we really believe that a multiple pregnancy except twin should not occur by selecting good embryos for maximum two blastocyst transfer. These results demonstrate that autologous cumulus cells may be used for the production of blastocysts with high developmental competence, and the use of autologous cumulus cells to be collected easily, and to be treated conveniently at OPU must be an effective means for obtaining high implantation and pregnancy rate.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.3
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• pp.181-189
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• 2010

The placenta, which is a temporary organ derived from the fetus during pregnancy, is critical to support fetus development via optimal regulation between mother and fetus. Trophoblast as a major cell population of the placenta is one of the earliest to differentiate and shows an extensive proliferation or/and differentiation up to the formation of the placenta. The role of the trophoblast show dynamic changes from early embryo implantation to placentation during pregnancy. Implantation of the blastocyst into the endometrium of the maternal uterus is mediated by invasion of the differentiated trophoblast (e.g. syncytiotrophoblast) from the trophectoderm. During pregnancy, the unique role of the trophoblast is to invasion, eroding, and metastasizing in the placenta as well as to ensure appropriate bidirectional nutrient or waste flow required for growth and maturation of the embryo. The dysfunction of the trophoblast during pregnancy can result in several gynecological diseases including preeclampsia and congenital malformation in neonatal medicine. Therefore, trophoblasts act as a conclusive factor in placental and fetal development. This brief review outlines the classification of trophoblast and its function in the placenta during pregnancy. Also, we introduce the latest research in trophoblast for implantation and the placenta development, and the application potential of trophoblast for infertility and obstetrical diseases.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.19-25
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• 2016

This study was conducted to evaluate the effects of type of culture media (BM, G2, OS, TCM, and MEM) on B6D2F1 mice oogenesis. In the present study, B6D2F1/CrljOri $F_1$ mice were utilized in order to maximize oogenesis. Also we used TCM-199, Dulbecco's medified Eagle's medium (DMEM), embryo culture medium (Fertilization medium, Cleavage medium, Blastocyst medium), G series medium and One step medium. In vitro maturation was highest in BM followed by the order of OS, MEM, TCM and G2 ($90{\pm}2.8%>88{\pm}3.2%>85{\pm}4.9%>78{\pm}10.2%>64{\pm}7.7%$, respectively). To note, the G2 group was statistically different compared to other groups (p<0.05). On the other hand the fertilization rate was highest in the G2 group followed by BM, OS, TCM, and MEM ($87{\pm}7.2%>85{\pm}6.9%>74{\pm}14.0%>71{\pm}13.8%>2{\pm}1.4%$, respectively). The MEM group was significantly lower compared to other groups (p<0.05). The developmental rate was highest in the OS group followed by the G2 group and the BM group albeit no statistical significance was noted ($73{\pm}11.6%>71{\pm}9.2%>66{\pm}10.4%$). Of note, all cells of the TCM and MEM groups were died during embryonic development. The zona hatched rate ($51{\pm}9.8%$ vs. $50{\pm}9.1%$ vs. $47{\pm}7.2%$ for BM, G2, and OS respectively) and attached rate ($45{\pm}12.3%$ vs. $38{\pm}16.1%$ vs. $37{\pm}11.5%$ for BM, G2, and OS respectively) were not different amongst groups. No difference was found in total cell numbers ($74{\pm}13.9$ vs. $64{\pm}9.2$ vs. $76{\pm}6.7$ for BM, G2, and OS respectively), ICM cell numbers ($20{\pm}1.9$ vs. $14{\pm}1.8$ vs. $15{\pm}2.1$), TE cell numbers ($55{\pm}12.5$ vs. $49{\pm}10.7$ vs. $61{\pm}5.9$), % ICM ($30{\pm}2.8%$ vs. $24{\pm}7.0%$ vs. $22.8{\pm}2.2%$) and ICM:TE ratio ($1:2{\pm}0.5$ vs. $1:3.1{\pm}0.8$ vs. $1:3.1{\pm}0.5$) amongst groups. In summary, these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.279-288
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• 1996

The objective of this study was to determine the effect of EGF on the development of IVM/IVF bovine embryos and their ICM and TE cell number. In addition, we examined the combined effect of EGF and coculture to the bovine embryo development and the expression of EGF-R protein on bovine embryos by indirect immunofluorescence. The results obtained in these experiments were summarized as follows: When the IVM/IVF 4- to 8-cell embryos were treated at 0, 1, 10, 100 ng/ml of EGF, EGF treatment group showed improved development to blastocyst and increased pattern of ICM and TE cell number compared with control, although there is not significantly different. The stimulating effect of EGF (10 ng/ml) to the develop ment level of IVM/IVF bovine embryos significantly increased development rate to blastocyst after 8-cell stage (p<0.05), although there is no significant effect to the increase of ICM and TE cell numbers. Also, expression of EGF-R on the bovine embryonic stage by indirect immunofluor escence presents after 4-cell stage and the intensity of the EGF-R staining was variable with the development progression. On the other hand, embryos cultured in coculture group added either with or without EGF commonly indicated the significant difference in development rate to blastocyst and Total cell number compared with control. These results suggest that the a addition of EGF to the coculture may stimulate the coculture effect between IVM/IVF bovineembryos and cumulus cells. Therefore, EGF could promote preimplantation bovine embryo development by binding with expressed EGF~R after 4-cell stage, and stimulate the production of embryotrophic factors from the coculture environment. Also, the present study showed that there was no significant effect of EGF to the increase of ICM and TE cell number although the rate of blastocyst significantly increased when treated with EGF after 8-cell stage (p<0.05).

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.29-36
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• 2004

The objective of this study was to investigate the effects of amino acid supplementation of oocyte maturation medium on 1st polar body(PB) extrusion, embryo development and blastocsyt cell number. In experiment 1, Cumulus oocyte complexes(COCs) were matured in in vitro maturation(IVM) medium supplemented with 1, 2, or 4-fold of 10 $\mu$l/ml MEM non-essential amino acid(NEAA) and 20 Park, $\mu$ l/ml BME essential amino acid(EAA). The PB extrusion rate of oocytes matured in 1-fold amino acid group was significantly higher than that matured in medium without amino acid (p＜0.05), but it was decreased by the increase of the dosage of amino acid. There were no difference in the percentage of embryos reaching 2-cell, 8-cell and blastocyst in all treatments. The number of trophectoderm(TE) cells and total cell number of blastocysts were highest in 2-fold amino acid group, and the number of inner cell mass(ICM) cells was increased by the increase of the dosage of amino acid. In experiment 2, COCs were matured in IVM medium with 1, 5, or 10 mg/ml lactalbumin hydrolysate(LAH). The PB extrusion rate of oocytes matured in medium with 5 mg LAH was significantly higher than that matured in medium with 1 mg LAH (p＜0.05). The development rate to the blastocyst stage was significantly higher in non-supplement and 1 mg LAH group than in 5 mg and 10 mg LAH group (p＜0.05). The number of TE cells and total cell number did not differ among treatment groups, but the number of ICM cells was increased by the increase of LAH supplement. These results suggested that the supplement of certain group of amino acid in IVM medium effective on the quality of blastocyst, and further studies will be accompany with the search of new sources of amino acid used for the use of in vitro embryo production.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.233-239
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• 1997

Embryos of most mammalian species grown in vitro would undergo developmental arrest at the approximate time of genomic activation. Stage-specific cell block and the resulting rapid loss of embryo viability in conventional culture media have limited the duration for which embryos may be cultured prior to transfer. As a result, embryos are usually transferred to the uterus at the 4-to 8-cell stage to avoid the loss of viability associated with long-term in vitro culture. Early transfer has led to asynchrony of the endometrium-trophectoderm interaction at the time of implantation and a resultant reduction in the rate of implantation. To overcome these problems, a variety of co-culture systems has been devised in which embryos can develop for a longer period prior to embryo transfer. Vero cells, derived from African green monkey kidney, share a common embryologic origin with cells from the genital tract. In addition, they are potentially safe to use, since they are highly controlled for viruses and other contaminants. Therefore, co-culture using Vero cells has been widely utilized to enhance embryo viability and development, although not without controversies. We thus designed a series of experiments to demonstrate whether Vero cells do indeed enhance mouse embryo development as well as to compare the efficacy of co-culturing mouse 1-cell embryos on Vero cell monolayer in both Ham's F-10 and human tubal fluid (HTF) culture media. 1-cell stage ICR mouse embryos were cultured either in the presence of Vero cells (Group A) or in conventional culture medium alone (Group B). In Ham's F-10 significantly more 3-to-8cell embryos developed in group A than group B (59.8 versus 10.0%; p<0.01). In contrast, there was no significant difference in embryonic development both group A and group B in HTF. However, significant differences were noted only in later embryonic stage (13 and 0%; p<0.05 of group A and B respectively, hatching or hatched). In Ham's F-10, we also could observe the beneficial effect of Vero cell on hatching process (70.7 and 42.1%; p<0.05 of group A and group B respectively).

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.195-203
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• 2018

Interferon-tau (IFNT) is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants. Also, multiple interferon genes exist in cattle, However, molecular mechanisms of these bovine IFNT (bIFNT) genes whose expressions are limited have not been characterized. We and others have observed that expression levels of bovine subtype IFNT genes in the tissues of ruminants; thus, bIFNT1 and other new type I (bIFNTc1/c2/c3) gene co-exist during the early stages of conceptus development and non-trophoblast cells. Its genes transcription could be regulated through CDX2 and ETS2 and JUN and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Bovine ear-derived fibroblast cells, were co-transfected with luciferase reporter constructs carrying upstream (positions -1000 to +51) regions of bIFNT1 and other new type I gene and various transcription factor expression plasmids. Compared to each - 1kb-bIFNT1/c1/c2/c3-Luc increased when this constructs were co-transfected with CDX2, ETS2, JUN and/or CREBBP. Also, Its genes was had very effect on activity by CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. However, the degree of transcriptional activation of the bIFNTc1 gene was not similar to that bIFNT1/c2/c3 gene by expression plasmid. Furthermore, Sequence analyses also revealed that the expression levels of bIFNT1/c2/c3 gene mRNAs expression were highest on day 17, 20 and 22 trophoblast and, Madin-Darby bovine kidney (MDBK), Bovine ear-derived fibroblast (EF), and endometrium (Endo) non-trophoblast cells. But, bIFNTc1 mRNA had not same expression level, bIFNTc1 lowest levels than those of IFNT1/c2/c3 gene in both trophoblast and non-trophoblast cells. These results demonstrate that bovine subtype bIFNT genes display differential, in the trophoblast and non-trophoblast cells.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.68-68
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• 2001

Blastocyst formation, consisting of the inner cell mass (ICM) and trophectoderm (TE), is the first differentiation process during embryonic development in mammals. It has been hypothesized that the proportion of ICM to TE in the blastocyst may be crucial for subsequent developmental competence of early embryos, which it may be expressed as a sensitive indicator for evaluating in vitro systems. In this study ICM/total cell ratio of nuclear transfer (NT) embryos was compared with IVF-derived and in vivo embryos. Somatic cell nuclei obtained from a fetus at Day 40 of gestation were transferred into the enucleated oocyte and then cultured in NCSU 23 medium for 6 days as previously described (Koo et al., Biol. Reprod. 2000; 63:986-992). ICM and TE cells of blastocysts were determined by using a differential staining method (Han et al., Biol. Reprod. 1999; 60:1110-1113). Development rate (9.8$\pm$2.5%, 23/225) to the blastocyst stage of NT embryos was lower than IVF embryos (23.8$\pm$2.7%, 53/223). Thus, a difference was detected in the in vitro developmental rate to blastocyst stage between NT and IVF-derived embryos (P<0.05). In the next experiment, we investigated ICM and TE nuclei to assess the quality of blastocysts that produced by NT, IVF and in vivo, respectively. NT blastocysts (27.6$\pm$8.3) showed a smaller total cell number than IVF-derived (42.6$\pm$17.4) and in vivo embryos (283.9$\pm$103.5) (P<0.05). Ratios of ICM/total cells in NT, IVF and in vivo blastocysts were 15.1$\pm$ 18.6% (n=56), 12.3$\pm$9.2% (n=57) and 30.4$\pm$6.8% (n=40), respectively. Individual blastocysts for the ratio of ICM/total cells were assigned to 3 groups (I; <20%, II; 20 to 40% and III;>40%). As the results, most in vivo blastocysts (97.5%, 39/40) were distributed into group II while most NT (78.6%, 44/56) and IVF-derived blastocysts (82.5%, 47/57) were allocated to group I. Thus, our data show that NT or IVF-derived embryos have aberrant morphology during early development in vitro systems, suggesting that these anomalies may result in developmental failures of the NT embryos to term.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.4
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• pp.292-297
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• 2022

Direct injection of CRISPR/Cas9 into zygotes enables the production of genetically modified nonhuman primates (NHPs) essential for modeling specific human diseases, such as Usher syndrome, and for developing novel therapeutic strategies. Usher syndrome is a rare genetic disease that causes loss of hearing, retinal degeneration, and problems with balance, and is attributed to a mutation in MYO7A, a gene that encodes an uncommon myosin motor protein expressed in the inner ear and retinal photoreceptors. To produce an Usher syndrome type 1B (USH1B) rhesus macaque model, we disrupted the MYO7A gene in developing zygotes. Identification of appropriately edited MYO7A embryos for knockout embryo transfer requires sequence analysis of material recovered from a trophectoderm (TE) cell biopsy. However, the TE biopsy procedure is labor intensive and could adversely impact embryo development. Recent studies have reported using cell-free DNA (cfDNA) from embryo culture media to detect aneuploid embryos in human in vitro fertilization (IVF) clinics. The cfDNA is released from the embryo during cell division or cell death, suggesting that cfDNA may be a viable resource for sequence analysis. Moreover, cfDNA collection is not invasive to the embryo and does not require special tools or expertise. We hypothesized that selection of appropriate edited embryos could be performed by analyzing cfDNA for MYO7A editing in embryo culture medium, and that this method would be advantageous for the subsequent generation of genetically modified NHPs. The purpose of this experiment is to determine whether cfDNA can be used to identify the target gene mutation of CRISPR/Cas9 injected embryos. In this study, we were able to obtain and utilize cfDNA to confirm the mutagenesis of MYO7A, but the method will require further optimization to obtain better accuracy before it can replace the TE biopsy approach.