• Clinical and Experimental Reproductive Medicine
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• 제49권4호
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• pp.248-258
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• 2022

Objective: This research investigated the effects of human chorionic gonadotropin (HCG)-producing peripheral blood mononuclear cells (PBMCs) on the implantation rate and embryo attachment in mice. Methods: In this experimental study, a DNA fragment of the HCG gene was cloned into an expression vector, which was transfected into PBMCs. The concentration of the produced HCG was measured using enzyme-linked immunosorbent assay. Embryo attachment was investigated on the co-cultured endometrial cells and PBMCs in vitro. As an in vivo experiment, intrauterine administration of PBMCs was done in plaque-positive female mice. Studied mice were distributed into five groups: control, embryo implantation dysfunction (EID), EID with produced HCG, EID with PBMCs, and EID with HCG-producing PBMCs. Uterine horns were excised to characterize the number of implantation sites and pregnancy rate on day 7.5 post-coitum. During an implantation window, the mRNA expression of genes was evaluated using real-time polymerase chain reaction. Results: DNA fragments were cloned between the BamHI and EcoRI sites in the vector. About 465 pg/mL of HCG was produced in the transfected PBMCs. The attachment rate, pregnancy rate, and the number of implantation sites were substantially higher in the HCG-producing PBMCs group than in the other groups. Significantly elevated expression of the target genes was observed in the EID with HCG-producing PBMCs group. Conclusion: Alterations in gene expression following the intrauterine injection of HCG-producing PBMCs, could be considered a possible cause of increased embryo attachment rate, pregnancy rate, and the number of implantation sites.

• Clinical and Experimental Reproductive Medicine
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• 제47권2호
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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.

• 한국가축번식학회지
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• 제18권2호
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• pp.141-150
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• 1994

전자현미경에 의해 자궁부착 전후의 돼지 수정란의 형태형성 및 분화에 따른 배발생 과정을 검토하였다. 돼지 초기배는 자궁이주후 균일하게 자궁에 배분되기전 약 2~3일간은 자궁각의 proximal portion에 존재하며, 임신 4일째에 할구와 할구의 경계를 상실하는 tight한 gap junction을 가진 상실배로 발달한다. 배반포를 형성하는 시기에 estradiol 17$\beta$는 compact한 상실배를 cavitated blastocyst로 발달을 촉진시키면서, steroid hormone이 이후의 배발생을 지배한다. Hatching의 시기는 교배후 6~7일경 zona pellucida을 둘러사고 있는 glycoprotein의 thinning과 lysis에 의해 이루워지는데, hatching 과정은 embryo의 세포수와 무관하였으며, 이때의 embryo의 직경은 0.5~1.0mm인 것을 본 실험에서 확인하였다. 12일경부터는 embryo는 prostaglandins, IGF-binding protein, retinol binding protein, plasminogen activator등의 단백질이 풍부해 이들 인자가 elongation 개시 후보로 고려될 수 있었다. 또한 이 시기의 embryo는 embryonic disc로 발달시 progesterone과 estrogen을 estradiol 17$\beta$로 전활할 수 있으며, 이러한 변화와 함께 spherical stage로부터 tubular 혹은 filamentous form으로 변형되었다. Estrogen이 임신을 통해 prostagladins의 분비를 uterine lumen에 지시하는지는 알 수 없으나 13일 경을 전후해 conceptus estrogen이 uterine arterial blood flow, uterine vasular permeability을 증가시키는 것으로 나타났으며, 자궁에서 protein과 calcium, PGF2$\alpha$, plasminogen inhibitor를 증가시키는 것으로 나타났다. 이 시기의 자궁 변화와 함께 embryo의 attachment는 trophoblast와 uterine membrane사이의 느슨한 결합에 의해 개시되었으며, 18일경 uterine과 trophoblastic microvili의 interdigitation에 의해 완성된다. 이 시기에 conceptus attachment를 위해 필요한 uterine microvili에서의 glycocalyx의 형성과 endometrial epithelium의 erosion을 야기하기 위해 plasminogen activator을 분비하였으며, 반면 자궁에서 plasminogen 역할을 하는 것은 estrogen이며, blastocyst cell 표면의 lectin binding이 attachment에 중요한 역할을 한다. 이상과 같은 일련의 과정을 거친 초기배는 성공적인 임신으로 유도된다고 본다. 따라서, 본 연구는 이상과 같이 착상을 전후한 시기의 배를 전자현미경에 의해 형태형성의 변화를 특히 착상을 전후해 배 취사율이 높은 시기를 대상으로 분석하였다. 이 분석 시기중 성공적인 착상성공율은 56%(71/126)였다.

• 한국수정란이식학회지
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• 제27권3호
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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.

• 한국발생생물학회지:발생과생식
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• 제13권1호
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• pp.1-11
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• 2009

착상은 배아측과 모체측의 내분비적, 측분비적 및 자가분비적 인자들의 상호 작용에 의하여 조절된다. 착상의 적기는 $2{\sim}4$일로서 이 특수한 시간대 안에서 signaling, appositioning, attachment 및 invasion을 통하여 순차적으로 배아의 착상이 이루어지는데, 이는 자궁내막과 배아로부터 여러 사이토카인, 성장인자, 부착인자와 같은 다수의 생화학 인자의 생성과 분비를 포함하며 이로 인하여 수용적인 자궁내막이 형성된다. LIF, CSF-1, IL-1과 같은 사이토카인들은 착상을 이끄는 연속적인 사건에서 중요한 역할을 수행하며 integrin, L-selectin ligands, glycodelin, mucin-1, HB-EGF, pinopodes는 appositioning과 attachment에 관여한다. 배아 또한 사이토카인과 성장인자 및 LIF, CSF-1, IGF, HB-EGF에 대한 수용체들을 분비하여 능동적으로 대처한다. 자궁내막의 수용성을 평가하고 자연주기 또는 보조생식술을 이용한 임신의 예후를 예측하는데, 이와 같은 인자들이 유용한지는 앞으로 더 연구되어야 한다. 현재로서는 내막조직으로부터 채취한 integrins, pinopodes, glycodelin, LIF가, 자궁강내 세척액에서는 glycodelin과 LIF가 유망한 인자로 떠오르고 있다. 혈액내 마커로서는 VEGF, glycodelin, CSF가 약간의 연관성을 보여주고 있다. 이러한 인자들이 보조생식술 전후로 자궁내막의 기능과 임신의 예후를 평가하는 선별검사로 이용될 수 있는지를 증명하기 위해서는 향후 불임 여성과 대조군인 가임 여성을 대상으로 한 대규모의 연구가 필요할 것이다. 인간의 착상에서 이들 인자들의 기능을 충분히 이해해야 치료적 기법으로 연결되어 보조생식술에서도 더 높은 성공률을 이루어 낼 것으로 사료된다.

• 한국수정란이식학회지
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• 제28권2호
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• pp.141-147
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• 2013

The purpose of this study was attempted to new methods in mammalian embryos vitrification. This method was affected to increase of the embryo vitrification efficiency and it would be applied to the field of embryo transfer to recipient by modified loading method of embryo into 0.25 ml plastic straw. The frozen mouse embryos were carried out warmed from two different cell stages (8-cell and blastocyst, respectively) by attachment of an embryo in the vitrification straw (aV) method. All groups were cultured in M-16 medium to determine the development and survivability for 24 h, respectively. Results shown that, the survivability of two different groups were significantly different (94.8% vs. 70.9%). Total cell number was not significantly different the non-frozen blastocyst ($99.7{\pm}12.4$) compared to the post-thaw blastocyst ($94.8{\pm}15.1$). From the 8-cell embryo, total cell number of frozen blastocysts were significantly lower than others groups ($74.7{\pm}14.6$, p<0.05). In the case of cell death analysis, the blastocysts from non-frozen and frozen-thawed 8-cell group were not different ($0.0{\pm}0.0$ vs. $1.9{\pm}3.1$, p>0.05). However, the apoptotic nuclei of blastocyst were significantly observed the frozen-thawed group ($5.4{\pm}4.4$) compared to non-frozen group (p<0.05). Therefore, this new method of embryos using in-straw dilution and direct transfer into other species would be more simple procedure of embryo transfer rather than step-wise dilution method and cryopreservation vessels, so we can be applied in animal as well as human embryo cryopreservation in further.

• 한국수정란이식학회지
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• 제11권3호
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• pp.233-240
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• 1996

This study was carried out to determine the effect of cumulus cell attachment and various factors on in vitro maturation of pig foflicular oocytes. Oocytes with various configuration of cumulus cell mass were collected ftom ovaries of mature gilts by asperating with syringe equipped with needles of different gauges, follicle size and with or without cumulus cells. They were cultured in TCM-199 mediun containing FGS(fetal calf serum) for 30~48 hours in incubator with air containing 5% $CO_2$ at 38.5$^{\circ}C$. Mter orcein staining at in vitro maturation condition, GV, GVBD, anaphase, telophase and M II were observed. Results are surumarized as follows: 1. Recovery rates were 55.8, 55.5 and 34.4% when the cumulus-compacted oocytes were collected with 18, 21, 26 gauge needles of syringes, respectively. 2. 79% of oocytes with compacted cumulus cells were at GV stage and most of the oocytes with partially denuded and denuded cumulus cells were from GVBD to M- II stages. 3. Percentage of mature oocytes among those which are follicular diameter of 1~2, 3~6 and over 6 mm was 42.6, 53.2 and 60.8%, respectively. 4. Percentage of mature oocytes among those which are compacted, partially denuded and denuded was 60.5, 46.2 and 35.4% respectively. 5. Percentage of mature oocytes in co-cultured with monolayers of cumulus cells was higher (57.1%) than that found with oocytes cultured alone (53.4%).

• Environmental Analysis Health and Toxicology
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• 제25권2호
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• pp.161-168
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• 2010

The observed the toxic effect of WSF (Water Soluble Fraction) on development time, development rate, attachment rate, survival rate and growth of the larva during the early stage of the Pacific oyster, Crassostrea gigas. The time required early juvenile from the fertilized eggs exposed to crude oil (WSF) was about 13 hours in control group. But that of the crude oil dose group takes 12 hours, with shorter compared to control group. Development and attachment rate of the oyster significantly higher in 0.4 and 0.8 mg/L WSF compared to control group (P<0.05) but significantly lower more than 1.6 mg/L WSF (P<0.05). Survival rate of the oyster larva significantly higher in 0.4 and 0.8 mg/L WSF compared to control group (P<0.05) but significantly lower more than 2.4 mg/L WSF (P<0.05). Survival rate of spot showed no significantly difference compared to control group (P>0.05). Shell growth of the oyster were significantly lower more than 1.6 mg/L WSF compared control group (P<0.05). The effect concentration of crude oil showed that more 0.4 mg/L in development and attachment rate and more 1.6 mg/L in growth. Also, chronic exposure of crude oil even effect also in low concentration.

• Clinical and Experimental Reproductive Medicine
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• 제23권1호
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• pp.95-102
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• 1996

This study was performed to investigate the effect of human follicular fluid (HFF) on development of mouse embryos, for evaluating the suitability of HFF as a substitutive material of human fetal cord serum in ART program. The various concentrations of HFF were added into the culture medium and the effects of HFF concentrations were examined to identify the optimal concentration of HFF for embryo development. The potency of HFF in improving embryo development was compared to that of other protein supplement. Collected HFFs were classified with the maturity of the containing oocytes; mature, immature, atretic, and then the effects of the classified HFFs on embryo development were examined. Also, HFF was separated into the low (<30,000 Da) and high (>30,000 Da) molecular weight fractions and the effects of the fractions on embryo development were investigated. The highest development rate was found in culture medium supplemented with 20% HFF, bnt this rate was reversely reduced at the concentrations of HFF higher than 20%. The development rates to the blastocyst, hatching blastocyst, attachment and outgrowth cultured in mature HFF was significantly higher than those in immature and atretic HFF, and mean cell number in blastocyst was higher in mature HFF than in immature and atretic HFF. The development rates of mouse embryos according to protein sources were significantly higher in HFF than in fetal cord serum (FCS), maternal serum (MS) and bovine serum albumin (BSA), and mean cell number in blastocyst cultured in HFF was higher than that in FCS, MS and BSA. The development rates of embryo and mean cell number in blastocyst cultured in high molecular weight fraction of HFF were higher than those in low molecular weight fraction, but the results of high molecular weight fraction were lower than those of whole HFF. Therefore, these results indicated that human mature follicular fluid was useful for improving the development of mouse embryos, which suggests a possibility that HFF also may be used efficiently for improving the culture condition in human ART program as a protein supplement.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2001년도 후기 제12차 학술대회 논문집
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• pp.23-25
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• 2001

1. Leptin itself was not expressed in mouse uterine tissues. 2. Leptin receptors were not expressed in nonpregnant and little expressed in 3.5 day of pregnant uterine tissues. However, there was a signal in 4.5 and 5.5 day of tissues. 3. The expression level of leptin receptor variants in the implantation sites at around the time of initial embryo attachment (day 4.5 of pregnancy) and during the actual implantation period (day 5.5 of pregnancy) was much lower than that in the interimplantation 4. Finding of the differential expression of leptin receptors in implantation sites compared to interimplantation sites suggests that leptin - leptin receptor system may be one of the delicate regulators in the molecular mechanism of the implantation process.