• Development and Reproduction
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• v.22 no.3
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• pp.263-273
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• 2018

Aquaporin (AQP) 3, a facilitated transporter of water and glycerol, expresses in placenta and fetal membranes, but the detailed localization and function of AQP3 in placenta remain unclear. To elucidate a role of AQP3 in placenta, we defined the expression and cellular localization of AQP3 in placenta and fetal membranes, and investigated the structural and functional differences between wild-type and AQP3 null mice. Gestational sacs were removed during mid-gestational period and amniotic fluid was aspirated for measurements of volume and composition. Fetuses with attached placenta and fetal membranes were weighed and processed for histological assessment. AQP3 strongly expressed in basolateral membrane of visceral yolk sac cells of fetal membrane, the syncytiotrophoblasts of the labyrinthine placenta and fetal nucleated red blood cell membrane. Mice lacking AQP3 did not exhibit a significant defect in differentiation of trophoblast stem cells and normal placentation. However, AQP3 null fetuses were smaller than their control litter mates in spite of a decrease in litter size. The total amniotic fluid volume per gestational sac was reduced, but the amniotic fluid-to-fetal weight ratio was increased in AQP3 null mice compared with wild-type mice. Glycerol, free fatty acid and triglyceride levels in amniotic fluid of AQP3 null mice were significantly reduced, whereas lactate level increased when compared to those of wild-type mice. These results suggest a role for AQP3 in supplying nutrients from yolk sac and maternal blood to developing fetus by facilitating transport of glycerol in addition to water, and its implication for the fetal growth in utero.

• 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.

• Clinical and Experimental Reproductive Medicine
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• v.40 no.1
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• pp.33-37
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• 2013

Autophagy is a degradation process that acts in response to environmental stressors. Recently, autophagy has been detected in normal term, preeclamptic and intrauterine growth-restricted placentas. The object of this work was to investigate the presence of autophagy in first trimester voluntary interruption of pregnancy placental villi by the expression of autophagy-related proteins, light chain 3 (LC3), and Beclin-1. In first trimester placental villi laser scanning confocal microscopy (LSCM) analysis revealed LC3 and Beclin-1 immunoreactivity prevalently located in villous cytotrophoblasts. Using LSCM, LC3, and Beclin-1 were localized to the cytoplasm of the trophoblast layer in human full-term placentas. Beclin-1 expression and LC3 activation were confirmed by western blotting. These data emphasize that autophagy activation is different among cytotrophoblasts and syncytiotrophoblasts depending on the gestational age and thus we speculate that autophagy might play a prosurvival role throughout human pregnancy.

• 대한생식의학회:학술대회논문집
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• 1997.11a
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• pp.57-57
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• 1997

• Clinical and Experimental Reproductive Medicine
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• v.46 no.4
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• pp.189-196
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• 2019

Objective: We aimed to evaluate the effects of different oxygen conditions (20% [high O₂], 5% [low O₂] and 5% decreased to 2% [dynamic O₂]) on mouse pre- and peri-implantation development using a novel double-channel gas supply (DCGS) incubator (CNC Biotech Inc.) to alter the oxygen concentration during in vitro culture. Methods: The high-O₂ and low-O₂ groups were cultured from the one-cell to the blastocyst stage under 20% and 5% oxygen concentrations, respectively. In the dynamic-O₂ group, mouse embryos were cultured from the one-cell to the morula stage under 5% O₂ for 3 days, followed by culture under 2% O₂ to the blastocyst stage. To evaluate peri-implantation development, the blastocysts from the three groups were individually transferred to a fibronectin-coated dish and cultured to the outgrowth stage in droplets. Results: The blastocyst formation rate was significantly higher in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. The total cell number was significantly higher in the dynamic-O₂ group than in the low-O₂ and high-O₂ groups. Additionally, the apoptotic index was significantly lower in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. The trophoblast outgrowth rate and spread area were significantly higher in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. Conclusion: Our results showed that a dynamic oxygen concentration (decreasing from 5% to 2%) had beneficial effects on mouse pre- and peri-implantation development. Optimized, dynamic changing of oxygen concentrations using the novel DCGS incubator could improve the developmental competence of in vitro cultured embryos in a human in vitro fertilization and embryo transfer program.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.79-79
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• 2002

본 연구에서는 in vitro에서 성숙된 난자의 핵성숙(Polar Body extrusion)에 소요되는 시간과 배반포 단계로의 발달능력 사이의 관계를 비교하여 조기에 발달능력을 가진 embryo를 선발할 수 있는 IVP 체계를 개발하고자 하였으며 in vitro maturation(IVM)에 따른 first polar body(PB) 형성, IVM과 IVF 시간이 oocyte의 발달에 미치는 영향과 생산된 배반포의 세포수를 평가하였다. IVM은 TCM199 배양액을 사용하였고 in vitro fertilization(IVF)은 Fer -TALP용액을 사용하였으며 in vitro culture(IVC)는 CRlaa 배양액을 사용하여 2일까지는 0.3% BSA를 3일 부터는 10%FBS와 bovine oviduct epithelial cell을 첨가하여 배양하였다. IVM 시간에 따른 PB의 출현율은 0hr(0%), 6hr(0%), 12hr(0%), 14hr(8.7%), 16hr(40.5%), 18hr(48.0%), 20hr(65%), 22(68%) 그리고 24hr(74.5%)을 보였으며 IVM 시간에 따른 cleavage 및 8cell 발달율 사이에는 유의적인 차이가 없었으나 배반포(BL) 및 8cell에서 배반포로 발달률은 18시간(BL 31$\pm$6, BL/8cell 82 $\pm$5%)에서 가장 높게 나타났으며 24시간(BL 17$\pm$2, BL/8cell 60$\pm$8%)과 유의적인 차이를 보였다(P<0.05). IVC 7일째 배반포의 총세포수와 trophoblast(TE) 세포수는 IVM 18시간(mean$\pm$S.E.; total: 131.1$\pm$34.0, TE: 97.6$\pm$29.6)에서 24시간(total: 112.2$\pm$17.5, TE: 80.1$\pm$15.6)보다 유의하게 많은 것으로 나왔으나(P<0.05) 7일째의 inner cell mass(ICM) 숫자(18hr 33.5$\pm$12.8 vs 24hr 32.1$\pm$12.0)와 8일째 ICM, TE 그리고 총 세포수에는 유의성 있는 차이가 없었다. IVM 18시간에서 PB 형성과 8cell 발달률 사이에 높은 상관성을 보였고 배반포 및 8cell에서 배반포 단계로 높은 발달률을 보였으며 생산된 배반포의 TE 숫자와 총 세포수가 유의하게 많은 것으로 나타났다. 따라서 IVM 18시간 실시하였을 경우 보다 많은 세포수를 가진 배반포 발달 가능성이 높은 embryo를 조기에 선발 가능할 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.2
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• pp.174-180
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• 2009

This study was carried out to establish an appropriate condition for the efficient cryopreservation of the mouse pronuclear embryo. In vitro cryopreservation of pronuclear embryos was carried out by slow freezing or vitrification methods and development rate of 2-cell, blastocyst and hatched blastocyst was measured as well as survival rate of the thawed pronuclear embryo. After slow freezing, vitrification and thawing of mouse pronuclear embryos, the survival rate and blastocyst development rate for the vitrification group was 97.3 and 53.4%, respectively, which was significantly higher as compared to the slow freezing group with 88.6 and 23.9%, respectively (p<0.05). Blastocyst developmental rate in each experimental group was significantly higher for 21 h in the post-hCG group at 40.5-57.0% than the 24 h post-hCG group at 40.5% (p<0.05). ICM (Inner cell mass) cell numbers of blastocyst-stage embryos during the different stages of mouse pronuclear embryos, slow freezing and vitrification period in the control and vitrification groups were 22.1${\pm}$2.7 and 17.0${\pm}$3.1-22.0${\pm}$3.2, respectively; hence, the slow freezing group (10.2${\pm}$2.0) had significantly higher cell numbers than those of the other two groups (p<0.05). Trophoblast (TE) cell number in the control group, 65.8${\pm}$12.6, was significantly higher than in the slow freezing group, 41.6${\pm}$11.1 (p<0.05). The total cell numbers in the control group and 21 h post hCG group were 87.9${\pm}$13.6 and 81.8${\pm}$14.1, respectively, and were significantly higher than for the slow freezing group (51.8${\pm}$12.6; p<0.05).

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.177-186
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• 1998

While tubal pregnancy is frequently observed in human, it has been reported to rarely occur in other mammals. To investigate the reason of the absence of tubal pregnancy in other mammals, the ability of bovine tubal(oviductal) fluid to su, pp.rt the outgrowth of mouse embryos waw examined by using an in vitro model system wherein the trophoblast cells of hatched mouse blastocysts attach to and outgrow on tissue culture plates coated with FBS. When mouse blastocysts grwon in vitro from 2-cell embryos were cultrued in the dishes coated with FBS, human follicular fluid(hFF) and bovine follicular fluid(bFF), respectively, underwent outgrowth by spreading onto the plastic dishes during 48 hr. In contrast, none of the embryos cultured in the dishes coated with BSA or bovine obiductal fluid(bOF) did outgrow but remained as late blastocysts. Since addition of bOF at 5mg/ml or higher conc. to the culture medium resulted in degeneration of all embryos during 48 hr culture, 10mM conc. of glutathione(GSH) was added to the bOF-containing medium to circumvent the toxicity of bOF. In addition, bOF was heated $65^{\circ}C$ for 30 min(hbOF) to get rid of its precipitating properties and then added to the culture medium. When blastocysts were cultured in the presence of both hbOF and GSH 45.4% of embryos attached to the culture dishes. However, none of these embryos underwent outgrowth. Fially embryos were cultured in the presence of both hbOF and GSH but in the dishes coated with FBS. When they were examined after 48 hr, all of the blastocysts exhibited well-developed outgrwoth. Based upon these results, it is concluded that bovine oviductal fluid is capable of su, pp.rting the attchment of mouse blastocysts onto the culture plaste whereas it cannot promote the outgrwoth of mouse blastocysts in vitro, probably due to the lack of outgrwoth factor.

• Molecules and Cells
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• v.22 no.2
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• pp.168-174
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• 2006

Enhanced apoptosis has been observed in the placentas of women with preeclampsia, but few studies have examined changes at the molecular level. This study was designed to detect genes specifically expressed in full-term preeclamptic placentas. Tissue samples were collected immediately after cesarean delivery from 11 normal and 8 preeclamptic placentas at 35-40 weeks of gestation. Total RNAs were extracted and hybridized to a cDNA microarray. Results were confirmed by reverse-transcription polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. Hematoxylin and eosin and TUNEL staining were also performed to confirm apoptosis in preeclamptic placentas. Among 205 genes, three were up- or downregulated in preeclamptic placentas. The expression of caspase-10 and death receptor 3 (DR-3) was significantly increased, whereas insulin-like growth factor binding protein-3 (IGFBP-3) was strongly downregulated. RT-PCR analysis and Western blotting confirmed these effects. Immunohistochemical analysis showed that the DR-3, caspase-10 and IGFBP-3 proteins were localized in the syncytial membrane. Apoptosis in the trophoblast was also increased in term placentas from women with pregnancies complicated by preeclampsia. These results suggest that caspase-10, DR-3 and IGFBP-3 are involved in apoptosis in the preeclamptic placenta.

• IMMUNE NETWORK
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• v.15 no.1
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• pp.16-26
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• 2015

The female reproductive tract has two main functions: protection against microbial challenge and maintenance of pregnancy to term. The upper reproductive tract comprises the fallopian tubes and the uterus, including the endocervix, and the lower tract consists of the ectocervix and the vagina. Immune cells residing in the reproductive tract play contradictory roles: they maintain immunity against vaginal pathogens in the lower tract and establish immune tolerance for sperm and an embryo/fetus in the upper tract. The immune system is significantly influenced by sex steroid hormones, although leukocytes in the reproductive tract lack receptors for estrogen and progesterone. The leukocytes in the reproductive tract are distributed in either an aggregated or a dispersed form in the epithelial layer, lamina propria, and stroma. Even though immune cells are differentially distributed in each organ of the reproductive tract, the predominant immune cells are T cells, macrophages/dendritic cells, natural killer (NK) cells, neutrophils, and mast cells. B cells are rare in the female reproductive tract. NK cells in the endometrium significantly expand in the late secretory phase and further increase their number during early pregnancy. It is evident that NK cells and regulatory T (Treg) cells are extremely important in decidual angiogenesis, trophoblast migration, and immune tolerance during pregnancy. Dysregulation of endometrial/decidual immune cells is strongly related to infertility, miscarriage, and other obstetric complications. Understanding the immune system of the female reproductive tract will significantly contribute to women's health and to success in pregnancy.