• Asian-Australasian Journal of Animal Sciences
• /
• 제32권8호
• /
• pp.1112-1121
• /
• 2019

Objective: Gram-negative bacteria lipopolysaccharide (LPS) has been reported to be associated with uterine impairment, embryonic resorption, ovarian dysfunction, and follicle retardation. Here, we aimed to investigate the toxic effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. Methods: First, we developed an in vitro model to study the response of bovine cumulusoocyte complexes (COCs) to LPS stress. After incubating germinal vesicle COCs in $10{\mu}g/mL$ of LPS, we analyzed the following three aspects: the expression levels of the LPS receptor toll-like receptor 4 (TLR4) in COCs, activities of intracellular signaling protein p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappa B (NF-${\kappa}B$); and the concentrations of interleukin (IL)-$1{\beta}$, tumor necrosis factor (TNF)-${\alpha}$, and IL-6. Furthermore, we determined the effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. Results: The results revealed that LPS treatment significantly elevated TLR4 mRNA and protein expression levels in COCs. Exposure of COCs to LPS also resulted in a marked increase in activity of the intracellular signaling protein p-p38 MAPK and NF-${\kappa}B$. Furthermore, oocytes cultured in maturation medium containing LPS had significantly higher concentrations of the proinflammatory cytokines IL-$1{\beta}$, TNF-${\alpha}$, and IL-6. LPS exposure significantly decreased the first polar body extrusion rate. The cytoplasmic maturation, characterized by polar body extrusion and distribution of peripheral cortical granules, was significantly impaired in LPS-treated oocytes. Moreover, LPS exposure significantly increased intracellular reactive oxygen species levels and the relative mRNA abundance of the antioxidants thioredoxin (Trx), Trx2, and peroxiredoxin 1 in oocytes. Moreover, the early apoptotic rate and the release of cytochrome C were significantly increased in response to LPS. The cleavage, morula, and blastocyst formation rates were significantly lower in parthenogenetically activated oocytes exposed to LPS, while the incidence of apoptotic nuclei in blastocysts was significantly increased. Conclusion: Together, these results provide an underlying mechanism by which LPS impairs maturation potential in bovine oocytes.

• IMMUNE NETWORK
• /
• 제21권5호
• /
• pp.34.1-34.16
• /
• 2021

Sjögren's syndrome (SS) is an autoimmune disease characterized by dryness of the mouth and eyes. The glandular dysfunction in SS involves not only T cell-mediated destruction of the glands but also autoantibodies against the type 3 muscarinic acetylcholine receptor or aquaporin 5 (AQP5) that interfere with the secretion process. Studies on the breakage of tolerance and induction of autoantibodies to these autoantigens could benefit SS patients. To break tolerance, we utilized a PmE-L peptide derived from the AQP5-homologous aquaporin of Prevotella melaninogenica (PmAqp) that contained both a B cell "E" epitope and a T cell epitope. Repeated subcutaneous immunization of C57BL/6 mice with the PmE-L peptide efficiently induced the production of Abs against the "E" epitope of mouse/human AQP5 (AQP5E), and we aimed to characterize the antigen specificity, the sequences of AQP5E-specific B cell receptors, and salivary gland phenotypes of these mice. Sera containing anti-AQP5E IgG not only stained mouse Aqp5 expressed in the submandibular glands but also detected PmApq and PmE-L by immunoblotting, suggesting molecular mimicry. Characterization of the AQP5E-specific autoantibodies selected from the screening of phage display Ab libraries and mapping of the B cell receptor repertoires revealed that the AQP5E-specific B cells acquired the ability to bind to the Ag through cumulative somatic hypermutation. Importantly, animals with anti-AQP5E Abs had decreased salivary flow rates without immune cell infiltration into the salivary glands. This model will be useful for investigating the role of anti-AQP5 autoantibodies in glandular dysfunction in SS and testing new therapeutics targeting autoantibody production.

• 한국발생생물학회지:발생과생식
• /
• 제11권3호
• /
• pp.263-272
• /
• 2007

생쥐의 난자는 특별한 첨가물이나 난구세포 없이 체외 배양해도 성숙율이 높은 반면 돼지 난자의 체외성숙율은 매우 낮다. 본 연구는 이러한 차이의 원인을 연구하기 위하여 생쥐와 돼지 난자의 에너지 생성에 관여하는 유전자인 malate dehydrogenase(Mor2)의 기능을 RNAi를 이용하여 비교 분석하였다. 생쥐와 돼지 각각의 Mor2 double-stranded RNA(dsRNA)를 제작하고, 생쥐는 난구세포를 제거한 미성숙(GV) 난자의 세포질에 mMor2 dsRNA를 미세 주입한 후 16시간 동안 배양하였고, 돼지는 난구세포를 제거하거나(DO) 혹은 그대로인 상태(COCs)로 pMor2 dsRNA를 미세 주입한 후 48시간 동안 배양하였다. 사용한 배양액은 M199에 10%의 porcine follicular fluid, pyruvate, p-FSH, EGF, cystein, estradiol-$17{\beta}$ 등이 첨가된 배양액을 사용하였고, 배양 후 난자의 형태학적 변화를 관찰하고, Mor2 mRNA양의 변화를 RT-PCR로 확인하여 RNAi 결과, 염기서열 특이적으로 Mor2 발현이 억제됨을 확인하였다. 돼지 난자의 pMor2 RNAi 결과, DO 난자에서는 약 58%의 난자가 MI에서 성숙이 억제되었으나, COCs에선 84.4%가 MII로 성숙하는 것을 볼 수 있었다. 이는 돼지의 난구세포가 난자 성숙에 매우 중요하며, 특히 malate 공급에 관여할 것임을 시사한다. 선행 연구 Mor2 RNAi 결과, 생쥐는 GV에서, 본 연구에서 돼지는 MI에서 성숙이 정지되었기 때문에 이러한 차이가 배양액의 차이인지 다시 mMor2 RNAi 후 생쥐 난자를 M199 배양액에 16시간 동안 배양한 후 성숙율을 관찰하였더니 MII로의 성숙율은 62%로 대조군과 비교하여 큰 차이가 없었다(non-injected: 76.8%, buffer-injected: 73.3%). 이는 mMor2 RNAi 결과가 배양액의 조성에 의해 극복될 수 있음을 보여준 결과다. 생쥐와 돼지에서 서로 다른 대사과정을 갖고 있음은 바로 이 두 종에서의 체외성숙율 차이의 원인이 될 수 있을 것이며, 앞으로 두 종에서의 난자와 난구세포간의 상호작용 및 대사 경로 등에 관한 심도 깊은 비교 분석 연구를 통하여 돼지의 체외성숙율을 증가시킬 수 있는 배양 시스템의 개발이 가능할 것으로 기대된다.

• Clinical and Experimental Pediatrics
• /
• 제50권11호
• /
• pp.1067-1071
• /
• 2007

목 적 : 초극소저출생체중아의 생존율 향상에 따라 장기 신경발달 장애가 감소했는지 여부와 이들의 장기 신경발달의 예후 인자에 대해 알아보고자 하였다. 방 법 : 1994년 11월부터 2004년 7월까지 삼성서울병원 신생아중환자실에서 입원 치료 받은 초극소저출생체중아 중 교정나이 18개월에 외래에서 추적관찰이 가능하였던 134명을 대상으로 하였으며 외래 방문 시 진찰소견과 의무기록을 후향적으로 분석하였다. 대상 환아를 1994년 11월부터 1999년 12월까지인 제 I기와 2000년 1월부터 2004년 7월까지인 제 II기로 나누었으며 각각 36명과 98명이 해당되었다. 결 과 : 제 I기에 비하여 제 II기에 재태연령과 출생체중이 낮았지만 생존율은 향상되었으며(제 I기: 60.0%, 제 II기: 74.7%) 뇌성마비는 감소하였고(제 I기: 22.2%, 제 II기: 8.2%) 따라잡기 성장은 향상되었다(제 I기: 25.0%, 제 II기: 51.0%). 뇌실주위 백질연화증, 패혈증과 기관지폐 이형성증의 이환율은 제 II기에 감소하였다. 뇌성마비의 가장 큰 위험요인은 3도 이상의 고도 뇌실내출혈, 따라잡기 성장의 실패와 뇌실주위 백질연화증이었다. 결 론 : 초극소저출생체중아의 생존율 향상은 장기적인 예후의 향상과 관련되어 있으며 신생아 관리의 질향상과 관련된 뇌실주위 백질연화증의 감소, 고도 뇌실내출혈의 감소, 더 나은 영양공급이 장기적인 예후의 향상과 관련된 것으로 보인다.

• 한국발생생물학회지:발생과생식
• /
• 제20권3호
• /
• pp.219-225
• /
• 2016

Most of the commercial devices for vitrification are directly immersed into the warming solution (WS) for increasing of warming rate. However, the previous modified cut standard straw (MCS) which has reported is difficult to immerse into the WS. The aim of this study was to investigate whether the long cut straw (LCS) could be useful as a stable tool for vitrified-warmed human blastocysts. A total of 138 vitrified-warmed cycles were performed between November 2013 and November 2014 (exclusion criteria: women ${\geq}38$ years old, poor responder, surgical retrieval sperm, and severe male factor). The artificial shrinkage was conducted using 29-gauge needles. Ethylene glycol and dimethyl sulfoxide (7.5% and 15% (v/v)) were used as cryoprotectants. Freezing and warming were conducted using the LCS tool. The cap of LCS was removed using the forceps in the liquid nitrogen ($LN_2$) and then directly immersed into the first WS for 1 min at $37^{\circ}C$ (1 M sucrose). Only re-expanded blastocysts were transferred after it was cultured in sequential media for 18-20 h. A total of 294 blastocysts were warmed, and all were recovered (100%). Two hundred eighty-five embryos were survived (96.9%). The vitrified-warmed blastocysts of all patients were transferred without any cancellation. We were able to achieve a reasonable implantation (24.2%), following by clinical pregnancy (36.2%), which then continued to ongoing pregnancy (36.2%), and live birth (31.2%). Using LCS is achieved the acceptable rates of survival, pregnancy and live birth. Therefore, the LCS could be considered as a stable and simple tool for human embryo vitrificaton.

• Clinical and Experimental Reproductive Medicine
• /
• 제31권1호
• /
• pp.9-17
• /
• 2004

Objective: The aim of this study were to compare the effects of EG and PROH on cryopreservation of mouse and human embryos, and to find the optimal protocol for embryo freezing. Methods: Human embryos derived from fertilized eggs showing 3 pronuclei (PN) and mouse embryos were divided into two groups respectively: dehydrated with 1.5 M EG + 0.2 M sucrose or 1.5 M PROH + 0.2 M sucrose using the slow freezing method. Moreover mouse embryos were controlled the exposure time of cryoprotectant during dehydration or rehydration steps. Results: The survival rates of human embryos were 79.2% (84/106) in EG group and 77.9% (88/113) in PROH group. In mouse embryos, the survival and development rates up to blastocyst were 70.6% (245/347), 44.1% (123/279) in EG group and 62.1% (198/319), 45.1% (123/279) in PROH group, respectively. However, in EG group, partially damaged embryos after thawing were decreased compared to PROH group. In combination group, when the exposure time during dehydration and rehydration were reduced, the survival and embryonic developments were increased slightly, but not significant. Conclusion: Cryopreservation of mouse and human embryos at cleavage stage by using EG or PROH exhibited no statistical difference in the survival rate and/or developmental rate to blastocyst. However, the use of EG for cryopreservation of embryos might reduce the exposure time of the cryoprotectant because of a high permeation of EG and result in lessen its toxic effects.

• 한국발생생물학회지:발생과생식
• /
• 제11권2호
• /
• pp.105-109
• /
• 2007

이 연구는 실내 사육수조에서 자연산란 후 수정된 난을 대상으로 수온에 따른 난 발생속도와 부화율을 조사하였다. 부화에 이르기까지 각 수온조건에서 소요된 시간은 $15^{\circ}C$에서 상실기 이후 발생이 진행되지 않았고, 18, 21, 24, $27^{\circ}C$에서 각각 70시간 30분, 44시간 10분, 29시간 10분 그리고 24시간 30분이 소요되었다. 부화율은 $15^{\circ}C$에서 0%였고, 18, 21, 24 그리고 $27^{\circ}C$에서 각각 $8.3{\pm}1.2%$, $18.0{\pm}6.2%$, $24.0{\pm}4.0%$ 그리고 $17.0{\pm}7.2%$로 뚜렷한 차이 없이 $24^{\circ}C$에서 다소 높았고 21와 $27^{\circ}C$에서 비슷한 경향이었으며 $18^{\circ}C$에서 가장 낮게 나타났다. 이러한 결과에서 자바리의 난발생 수온범위는 $18{\sim}27^{\circ}C$로 제안된다.

• Journal of Preventive Medicine and Public Health
• /
• 제51권3호
• /
• pp.163-168
• /
• 2018

Objectives: Due to their developmental characteristics, adolescents have a higher probability than other age groups of experiencing injuries caused by accidents, violence, and intentional self-harm. The severity and characteristics of injuries vary by the intentionality and mechanism of injury; therefore, there is a need for a national-level estimate of the scale and the severity of injuries in adolescents that takes these factors into account. Methods: By using data from the Emergency Department-based Injury In-depth Surveillance Data, National Emergency Department Information System, the Korean National Hospital Discharge In-depth Injury Survey, and cause of death statistics, we calculated the emergency department (ED) visit rate, hospitalization rate, and death rate of injuries per 100 000 adolescents for each injury mechanism. The calculated rates were used to generate the injury pyramid ratio (ratio of death rate to hospitalization rate to ED visit rate) to visualize the scale and the severity of the injury. Results: The mortality rate in adolescents due to injury was 10/100 000; the corresponding rates for hospitalization and ED visits were 1623 and 4923, respectively, resulting in an injury pyramid ratio with the general pyramid form, with a 1:162:492 ratio of deaths to hospitalizations to ED visits. The mortality rate due to suicide/intentional self-harm was 5/100 000, while 35 were hospitalized for this reason and 74 made ED visits. The pyramid ratio of 1:7:15 for intentional self-harm/suicide showed a steep pyramidal form, indicating considerable lethality. The mortality rate due to motor vehicle collisions (MVCs) was 3/100 000; 586 were hospitalized for this reason, while 1023 made ED visits. The pyramid ratio of 1:195:341 for MVCs showed a gradual pyramid form, indicating that the lethality was low and the scale of injury was high. Conclusions: The main categories of injuries in adolescents were visualized in pyramid form, contributing to an understanding of the scale of each injury by mechanism in terms of levels of death, hospitalization, and ED visits. These findings will be helpful for understanding how to prioritize injuries in adolescents.

• 한국가축번식학회지
• /
• 제18권2호
• /
• pp.87-94
• /
• 1994

본 연구는 생쥐 초기배 및 부화후 체외신장에 미치는 인간 양수의 첨가효과를 조사하고자 실시하였다. 생쥐 수정란의 체외발달에 미치는 인간 양수의 적정농도를 확인하고자 기본배양액 (Whitten's medium)에 16주령의 양수 농도를 각각 달리하여 체외배양을 실시한 결과, 20%를 첨가한 구에서 가장 높은 배반포로의 발달율과 부화율을 나타내었으며, 그 이상의 농도에서는 배발달율이 저하되었다. 포유동물 수정란 체외배양시 가장 많이 사용되는 첨가제인 fetal calf serum (FCS)과 bovine serum albumin(BSA)을 첨가한 구화의 배발달 성적을 비교한 결과, 임신중기 양수를 20% 첨가한 구의 배반포로의 발달율 (92.8%)과 부화율 (75.7%)은 기본배양액에서 배양된 것보다 배반포로의 발달율 (82.8%)과 부화율 (31.3%)은 높았으나 0.3% BSA (90.5%, 70.8%)나 10% FCS 첨가구 (94.3%, 74.3%)와는 유의한 차이가 인정되지 않았다. 임신주령에 따른 양수의 첨가효과를 조사한 결과 20%의 임신말기 양수를 첨가한 구의 배반포로의 발달율 (71.9%)과 부화율 (57.3%)은 20% 임신중기 양수를 첨가한 구보다 낮았으며, 부화후 배의 체외신장은 임신중기 양수와 FCS이 첨가된 구에서는 유기되었으나, 임신말기 양수와 BSA가 첨가된 구에서는 배의 체외신장이 유기되지 않았다. 이상의 본 연구결과를 통해 임신중기 양수 내에는 배발달 촉진인자와 배의 체외신장을 유기시키는 물질이 함유되어 있어 포유동물 배의 체외배양에 상당한 영향을 미치고 있다고 사료된다.

• Reproductive and Developmental Biology
• /
• 제35권3호
• /
• pp.385-390
• /
• 2011

The purpose of this study was undertaken to evaluate of cryopreservation efficiency in ${\alpha}$ 1,3-galactosyltransferase knock-out(GalT KO) cloned miniature pig sperm. To compare ability of frozen-thawed sperm characteristics, three different pig strains (GalT KO) cloned miniature pig, PWG miniature pig and Duroc were used. The ejaculated semen from the three pig species was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen ($LN_2$) vapours for 10 min before placing them into $LN_2$ for cryopreservation. After thawing, the sperm ability were assessed for viability (SYBR-14/PI staining), abnormality (Rose Bengal staining), and acrosome status (intactness, intensity and capacitation) (chlorotetracycline, CTC staining). The viability of frozen-thawed GalT KO pig sperm had no significant difference as compared with Duroc and PWG miniature pig sperm. However, The CTC pattern of frozen-thawed GalT KO cloned miniature pig spermatozoa showed significantly lower rates in F pattern and AR pattern (p<0.05) and significantly higher rates in B pattern than Duroc and PWG miniature pig (p<0.05). The abnormality of GalT KO cloned miniature pig sperm was significantly lower as compared to Duroc and PWG miniature pig sperm (p<0.05). In conclusion, GalT KO cloned miniature pig semen can be cryopreserved successfully and used for artificial insemination reasonably.