• Journal of Animal Reproduction and Biotechnology
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• v.34 no.4
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• pp.292-299
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• 2019

Interferon tau (IFNT) regulation, an anti-luteolytic factor produced by conceptuses of the ruminant ungulates, is essential for the maintenance of early pregnancy, but a definitive mechanism for its temporal transcription has not been elucidated. We and others have observed the T-box protein eomesodermin (EOMES) exhibited high mRNA expression in the ovine embryonic trophectoderm; thus, both caudal-relatedhomeobox-2 (CDX2) and EOMES coexist during the early stages of conceptus development. Objective of this study was to examine the effect of EOMES on ovine IFNT gene transcription when evaluated with CDX2, ETS2 and AP1 transcription factors implicated in the control of cell differentiation in the trophectoderm. In this study, quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis between ovine trophoblast cells was initially performed, finding that transcription factors CDX2 and 'EOMES transcription factor mRNAs' were specific to trophectoderm cells. These mRNAs were also found in days 15, 17, and 21 ovine conceptuses. Furthermore, human choriocarcinoma JEG3 cells (trophoblast cell line) were cotransfected with an ovine IFNT (-654bp)-luciferase reporter (-654-oIFNT-Luc) construct and several transcription factor expression plasmids. Cotransfection of the reporter construct with CDX2, ETS2 and AP1 increased transcription of -654-oIFNT-Luc by about 11-fold compared with transfection of the construct alone. When cells were initially transfected with EOMES followed by transfection with CDX2, ETS2 and/or AP1, the expression of -654-oIFNT-Luc was decreased. Also, EOMES factor inhibited the stimulatory activity of CDX2 alone. These results suggest that when conceptuses attach to the uterine epithelium, ovine IFNT gene transcription is down-regulated by an increase of EOMES factor expression in the attached ovine trophoblast cells.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.12
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• pp.1708-1714
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• 2005

Keratinocyte growth factor (KGF) functions in epithelial growth and differentiation in many tissues and organs. KGF is expressed in the uterine endometrial epithelial cells during the estrous cycle and pregnancy in pigs, and receptors for KGF (KGFR) are expressed by conceptus trophectoderm and endometrial epithelia. KGF has been shown to stimulate the proliferation and differentiation of conceptus trophectoderm. However, the role of KGF on the endometrial epithelial cells has not been determined. Therefore, this study determined the effect of KGF on proliferation and differentiation of endometrial epithelial cells in vitro and in vivo using an immortalized porcine luminal epithelial (pLE) cell line and KGF infusion into the uterine lumen of pigs between Days 9 and 12 of estrous cycle. Results showed that KGF did not stimulate proliferation of uterine endometrial epithelial cells in vitro and in vivo determined by the $^3$H]thymidine incorporation assay and the proliferating cell nuclear antigen staining, respectively. Effects of KGF on expression of several markers for epithelial cell differentiation, including integrin receptor subunits $\alpha$4, $\alpha$5 and $\beta$1, plasmin/trypsin inhibitor, uteroferrin and retinol-binding protein were determined by RT-PCR, Northern and slot blot analyses, and immunohistochemisty, and KGF did not affect epithelial cell differentiation in vitro and in vivo. These results show that KGF does not induce epithelial cell proliferation and differentiation, suggesting that KGF produced by endometrial epithelial cells acts on conceptus trophectoderm in a paracrine manner rather than on endometrial epithelial cells in an autocrine manner.

• Reproductive and Developmental Biology
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• v.38 no.1
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• pp.21-34
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• 2014

The majority of early embryonic mortality in pregnancy occurs during the peri-implantation stage, suggesting that this period is important for conceptus viability and the establishment of pregnancy. Successful establishment of pregnancy in all mammalian species depends on the orchestrated molecular events that transpire at the conceptus-uterine interface during the peri-implantation period. This maternal-conceptus interaction is especially crucial in pigs because in them non-invasive epitheliochorial placentation occurs, in which the pre-implantation phase is prolonged. During the pre-implantation period, conceptus survival and the establishment of pregnancy are known to depend on the developing conceptus receiving an adequate supply of histotroph, which contains a wide range of nutrients and growth factors. Evidence links growth factors including epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), vascular endothelial growth factor (VEGF), and colony-stimulating factor 2 (CSF2) to embryogenesis or implantation in various mammalian species; however, in the case of pig, little is known about such functions of these growth factors, especially their regulatory mechanisms at the maternal-conceptus interface. Our research group has presented evidence for promising growth factors affecting cellular activities of primary porcine trophectoderm (pTr) cells, and we have identified potential intracellular signaling pathways responsible for the activities induced by these factors. Therefore, this review focuses on promising growth factors at the maternal-conceptus interface regulating the development of the porcine conceptus and playing pivotal roles in implantation events during early pregnancy in pigs.

• Korean Journal of Animal Reproduction
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• v.21 no.1
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• pp.39-46
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• 1997

본 연구는 체외생산된 소 배반포기배의 inner cell mass (ICM) 세포에서 epidermal growth factor-receptor (EGF-R)의 발현 유무를 immunosurgery와 indirect immunofluorescence (간접 면역 형광방법)을 이용하여 조사하고자 실시하였다. 본 실험에 사용된 ICM 세포는 체외수정 후 7∼8일째에 회수된 소 배반포기배로부터 immunosurgery 방법을 실시하여 얻어졌으며, 회수된 ICM세포는 live/dead 염색방법을 통한 생사 유무와 EGF-R 발현 유무 조사에 공시되었다. 특히, 배반포기배에 대한 immunosurgery를 위해 trophectoderm 세포에 대한 rabbit anti-bovine trophectoderm cell antibody (RABTE)를 제조하여 사용하였다. 결과를 요약하면 다음과 같다. ICM세포의 회수율은 RABTE와 guinea pig serum (complement)에 각각 15∼30분과 15∼60분동안 처리했을 경우 16.7∼74.2%였으며, 또한 처리시간이 각각 30분과 30분일 때 가장 높은 회수율(74.2%)을 얻었다. Immunosurgery 후 얻어진 ICM세포의 생존 유무를 조사하기 위해 live/dead 염색 방법을 이용하였던바, ICM세포의 생존율은 complement가 60분 처리된 군(69.3%)을 제외한 모든 처리군에서 84.0∼91.6%의 높은 생존율을 나타냈다. 또한, 회수된 ICM세포에 대한 EGF-R의 존재를 확인하였다. 따라서, ICM세포에서의 EGF-R의 발현은 인위적으로 첨가된 EGF의 이용 가능성을 높임으로서 체외에서의 착상전 배 발달을 증진시킬 수 있을 것으로 사료된다.

• 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.

• 대한생식의학회:학술대회논문집
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• 2001.11a
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• pp.117-117
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• 2001

• Clinical and Experimental Reproductive Medicine
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• v.44 no.4
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• pp.193-200
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• 2017

Objective: This study was conducted to investigate the efficacy of laser-assisted hatching (LAH) and various vitrification times for embryonic development and blastocyst cell numbers. Methods: First, 2-cell and 8-cell embryos were collected by flushing out the oviducts. In the control groups, they were vitrified for 8 or 10 minutes without LAH. The LAH groups underwent quarter laser zona thinning-assisted hatching before vitrification (4, 6, and 8 minutes or 4, 7, and 10 minutes, respectively). After incubation, double-immunofluorescence staining was performed. Results: The hatched blastocyst rate 72 hours after the 2-cell embryos were thawed was significantly higher in the 2LAH-ES8 group (33.3%) than in the other groups (p< 0.05). In the control-8 group ($22.1{\pm}4.6$), the cell number of the inner cell mass was higher than in the LAH groups (p< 0.05). The number of trophectoderm cells was higher in the 2LAH-ES6 group ($92.8{\pm}8.9$) than in the others (p< 0.05). The hatched blastocyst rate 48 hours after the 8-cell embryos were thawed was higher in the 8LAH-ES4 group (45.5%) than in the other groups, but not significantly. The inner cell mass cell number was highest in the 8LAH-ES7 group ($19.5{\pm}5.1$, p< 0.05). The number of trophectoderm cells was higher in the 8LAH-ES10 group ($73.2{\pm}12.1$) than in the other groups, but without statistical significance. Conclusion: When LAH was performed, 2-cell embryos with large blastomeres had a lower hatched blastocyst rate when the exposure to vitrification solution was shorter. Conversely, 8-cell embryos with small blastomere had a higher hatched blastocyst rate when the exposure to vitrification solution was shorter.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.37-41
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• 2005

The aim of this study was to improve efficiency and quality of the production of Korean Native Cow embryos. We examined effects of ovarian morphology and maturation vessel on the development and cell number of blastocysts. The development rates to the 2-cell embryos from oocytes collected from the ovaries of different morphological statues were similar ranging between 70.3 and 84.1%. The development rate to the 8 cell- and blastocyst-stage embryos was the highest in the group without both corpus luteum (CL) and follicle. The inner cell mass (ICM), trophectoderm (TE) and total cell number (TCN) were significantly higher in the groups of follicular cyst and regressive CL than other treatment groups, and the same pattern was observed in the ICM/TCN ratio. The development rate to the 2-cell stage was significantly higher in 0.5-㎖ straw group than 0.25-㎖ straw group. However, the development rates to the blastocyst stage were similar between the dish and the straw group. There were no differences in the number of ICM and TE cells, TCN and ICM/TCN ratio of blastocysts from oocytes matured in the different vessels.

• Reproductive and Developmental Biology
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• v.38 no.1
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• pp.9-19
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• 2014

For successful embryo implantation, the communication of the maternal endometrium with the conceptus trophectoderm is required essentially. In pigs, conceptuses undergo morphological change in length to enlarge the physical contact area with the maternal endometrium and secrete estrogen to induce the maternal recognition of pregnancy during the peri-implantation period. Conceptus-derived estrogen prevents luteolysis by conversion in direction of $PGF_{2{\alpha}}$ secretion from the uterine vasculature to the uterine lumen as well as it affects on expression of the uterine endometrial genes. In addition to estrogen, conceptuses release various signaling molecules, including cytokines, growth factors, and proteases, and, in response to these signaling molecules, the maternal uterine endometrium also synthesizes many signaling molecules, including hormones, cytokines, growth factors, lipid molecules, and utilizes ions such as calcium ion by calcium regulatory molecules. These reciprocal interactions of the conceptus trophectoderm with the maternal uterine endometrium make development and successful implantation of embryos possible. Thus, signaling molecules at the maternal-conceptus interface may play an important role in the implantation process. This review summarized syntheses and functions of signaling molecules at the maternal-conceptus interface to further understand mechanisms of the embryo implantation process in pigs.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.4
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• pp.337-346
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• 2005

Objective: The oligosaccharide moieties of glycoproteins and proteoglycans have a vital function in blastocyst differentiation. Concanavalin (ConA), a lectin, is known to bind on the preimplantation embryos, especially on blastocyst. In this study, we investigated whether ConA can modulate the trophoblast development and about the regulating mediator. Also, we investigated whether expansion is enough for hatching procession of the mouse blastocyst. Method: Embryos were collected at 72 h post hCG injection and chemicals were treated after 24 h (96 hr post hCG injection). ConA or calcium ionophore A23187 were exposed to blastocyst and than analysis the developmental process for 48 hr. Intracellular free-$Ca^{2+}$ concentration in trophectoderm was measured with confocal laser microscope after exposing to ConA or calcium ionophore A23187. ConA-pretreated blastocyst exposed to the calcium ionophore A23187 and then analyzed the developmental process. Otherwise ouabain was treated to the blastocyst to block the $Na^+/K^+$-ATPase activity. Results: In contrast to the control blastocyst, the ConA-exposed blastocysts developed beyond the expansion stage with significantly high rate (90.4%) at 12 h post administration. ConA induced an increase the intracellular $Ca^{2+}$ concentration in trophectoderm. Calcium ionophore A23187 also stimulated expansion of blastocyst. Most of the control blastocysts developed to the hatching stage at 144 h post hCG injection. However, strongly 65% of the ConA-exposed embryos were arrested at expanded stage at same time point. The developmental progression rates to hatching stage of both ConA- and calcium ionophore A23187-expose blastocysts were significantly lower than that of the control. However ConA-pretreated embryos developed to the hatching stage like control embryos. Ouabain showed a tendency to delayed the progress to expansion stage but did not inhibit the development to the hatching stage. Conclusion: ConA-mediated expansion is the result of the increase of intracellular free-calcium in blastocyst stage embryo. It is suspected that expansion of the blasocyst is a essential indirect factor in hatching and the calcium may triggering the cellular mechanisms for the both expansion and hatching progression.