• 제목/요약/키워드: Frozen

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Development of an aequorin-based assay for the screening of corticotropin-releasing factor receptor antagonists (CRF1 길항제 스크리닝을 위한 에쿼린 기반 세포실험 개발연구)

  • Noh, Hyojin;Lee, Sunghou
    • Journal of the Korea Academia-Industrial cooperation Society
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    • v.16 no.11
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    • pp.7575-7581
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    • 2015
  • Corticotropin-releasing factor(CRF), one of the stress driven neuropeptides, was widely proposed to influence hair loss and re-growth. For the development of receptor antagonists, the screening system based on intracellular calcium signal process was developed and optimized. The aequorin parental cells were transfected with CRF1 receptor and alpha 16 promiscuous G protein cDNA to establish HEK293a16/hCRF1, a stable cell line for the human CRF1 receptor. In HEK293a16/hCRF1 cells, the range of sauvagine dose response was 12-fold higher($EC_{50}:15.21{\pm}1.83nM$) than in the transiently expressed cells, hence essential conditions for the antagonist screening experiments such as the robust signals and high solvent tolerance were secured. The standard antagonists for the CRF1 receptor, antalarmin and CP154526, resulted $IC_{50}$ values of $414.1{\pm}5.5$ and $290.7{\pm}1.9nM$, respectively. Similar results were presented with frozen HEK293a16/hCRF1 cells. Finally, our HEK293a16/hCRF1 cells with the aequorin based cellular functional assay can be a model system for the development of functional cosmetics and modulators that can have a clinical efficacy on hair re-growth.

Geomorphic Features of ${\check{O}}rumkol$(Frozen Valley) Area (Kyungnam Province, South Korea) - Mainly about Talus - (경남 밀양 얼음골 일대의 지형적 특성 -Talus를 중심으로-)

  • Jeon, Young-Gweon
    • Journal of the Korean association of regional geographers
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    • v.3 no.1
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    • pp.165-182
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    • 1997
  • The aim of this paper is to clarify geomorphic features on talus within ${\check{O}}rumkol$ and the origin of ${\check{O}}rumkol$. ${\check{O}}rumkol$ is located in Milyang of Kyungnam province, in South Korea. ${\check{O}}rumkol$ is good area to study talus. because it is characterized by following three geomorphic landscapes : free face surrounding ${\check{O}}rumkol$ ; ${\check{O}}rumkol$ with deep and wide valley floor ; lots of taluses typically developing within ${\check{O}}rumkol$. The main results can be summarized as follows: 1) The origin of ${\check{O}}rumkol$ may be suggested two assumptions : one is that its origin have been resulted from intrusion structure(intrusive rock might capture less resistant rock as tuff) ; the other is that its origin have been resulted from volcanic depression after intrusion or eruption. But these assumptions are not obvious. therefore more geological evidences will be supplemented after this 2) The characteristics of ${\check{O}}rumkol$ talus (1) Pattern ${\check{O}}rumkol$ taluses are tongue-shaped or cone-shaped in appearance. They are $50{\sim}200m$ in length and the range of the maximum width from 25 to 115m and one of their mean slope gradient from 32 to $36^{\circ}$ (2) Origin ${\check{O}}rumkol$ taluses have been formed under periglacial environment in the last glacial age and they are classified into rock fall talus type, considering in conjunction with the shape, hardness, sorting, weathering conditions of constituent debris. (3) The stage of landform development ${\check{O}}rumkol$ talus slope profiles are mainly concave slope. This concave slope type was eventually caused by talus creep at the lower end of the talus. That means new additions of debris from the free face have virtually ceased and there is no evidence of recent motion in the deposit. Now it is predominant that vegetation cover is gradually increasingly. Therefore ${\check{O}}rumkol$ taluses appear to be relict form stage. at present.

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A Randomized Comparative Study of a Standard Anterior Capsular Release versus Inferior Extended Release for the Treatment of Shoulder Stiffness

  • Alzeyadi, Ahmed Abdullah;Kim, Yang-Soo;Lee, Hyo-Jin;Park, Sung-Ryeoll;Sung, Gwang Young;Kim, Dong-Jin;Jung, Ji-Hwan;Kim, Jong-Ho
    • Clinics in Shoulder and Elbow
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    • v.20 no.3
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    • pp.117-125
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    • 2017
  • Background: To compare the clinical outcomes of arthroscopic capsular release in patients with and without inferior capsular release for shoulder stiffness. Methods: Between January 2010 and December 2015, 39 patients who underwent arthroscopic capsular release for shoulder stiffness were enrolled and randomized into two groups. In group I, 19 patients underwent arthroscopic capsular release of the rotator interval and anterior capsule. In group II, 20 patients underwent arthroscopic capsular release of the anterior to inferior capsule, including the rotator interval. The American Shoulder and Elbow Surgeons score, Constant scoring system, Simple Shoulder Test, visual analogue scale for pain, and range of motion (ROM) were used for evaluation before surgery, at 3, 6, and 12 months after surgery and on the last follow-up. Results: Preoperative demographic data revealed no significant differences (p>0.05). The average follow-up was 16.07 months. Both groups showed significantly increased ROM at the last follow-up compared with preoperative (p<0.05). At the last follow-up, no statistical differences were found (p>0.05) between groups I and II in functional scores and ROM (forward flexion, p=0.91; side external rotation, p=0.17; abduction external rotation, p=0.72; internal rotation, p=0.61). But we found that group II gained more flexion compared to group I at 3 months and 6 months (p<0.05) after the surgery. Conclusions: Both techniques of capsular release are effective for stiffness shoulder. However, the extended inferior capsular release shows superiority in forward flexion over anterior capsular release alone during 6 months of follows-up (level of evidence: Level I, therapeutic randomized controlled trial).

Studies on the Effects of Co-culture of Cumulus Cell, Oviduct Epithelial Cell and Hormones and Freezing on !fl Vitro Developmental Rates of Bovine Embryos (소 수정란의 난구세포, 난관 상피세포, 호르몬과의 공배양 및 동결이 체외발생에 미치는 영향에 관한 연구)

  • 이종진;이명헌;김상근
    • Journal of Embryo Transfer
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    • v.12 no.1
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    • pp.27-36
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    • 1997
  • The studies were carried out to investigate the effects of co-culture with cumulus cells and oviduct epithelial cells on the in vitro fertilization and cleavage rate of bovine follicular cocytes and to determine the optimum thawing temperature and equilibration time on in vitro developmental rate of frozen bovine embryos. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes were cultured in TGM-199 medium containing 10 IU /ml의 PM SG, 10 IU /ml의 hCG, ip g/ml의 $\beta$-estradiol and 10% FCS for 24~48 hrs in incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. The bovine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquld nitrogen and thawed in 3$0^{\circ}C$ water. Survival rate was defined as developmental rate on in vitro culture or FDA-test. The results are sunanarized as followes :1. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with cumulus cells in TCM499 medium were 75.0~76.8% and 17.3~27.6%, respect-ively. And in-vitro fertilization rates of cumulus-enclosed oocytes(55.4%)were significantly(p<0.05) higher than cumulus-denuded oocytes (23.1%). 2. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with l$\times$ l04cells /ml, 1 x l06cells /ml, lx l08cells /ml and 1 x l015cells /ml oviduct epithelial cells in TCM-199 medium were 74.5~77.8% and 15.7~21.20 respectively.3. The in-vitro fertilization and in vitro developmental rates of bovine oocytes cocultured in '1CM-199 media containing PMSG, hCG, PMSG+hCG. PMSG+$\beta$-estradiol, hCG+$\beta$-estradiol 0 to 40 hrs after insemination were 74.0~77.4% and l8.9~23.l%, re-spectiv ely.4.The survival rates of bovine embryos thawed after rapid freezing in the freezing medium containing a various concentration of sucrose added 1.5M and 2.OM glycerol,DMSO and propanediol were 23.5~31.4% and 20.6~34.l%, respectively. 5. The temperature thawed at 3$0^{\circ}C$ after rapid freezing of bovine embryos resulted in a significantly higher embryos survival rate than did at 2$0^{\circ}C$ and 35$^{\circ}C$.6. The equilibration time on the survival rates of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (10~20min.). (Key words : bovine embryos, co-culture, freezing, in vitro development)

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Cryo-Ability of Boar Sperm sorted by Percoll Containing of Antioxidative Enzyme (항산화 효소가 첨가된 Percoll에 의해 분리한 돼지 정액의 동결-융해 능력)

  • Lee, Kyung-Jin;Lee, Sang-Hee;Joo, Seon-Ho;Kim, Yu-Jin;Yang, Jin-Woo;Lee, Yeon-Ju;Hwangbo, Yong;Lee, Seunghyung;Lee, Seung Tae;Lee, Eunsong;Park, Choon-Keun
    • Journal of Embryo Transfer
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    • v.30 no.3
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    • pp.121-128
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    • 2015
  • The objective of this study was to evaluate the efficiency of sperm cryosurvival in boar sperm separated by Percoll containing antioxidant enzymes. The boar semen was collected into a pre-warmed ($37^{\circ}C$) thermos bottle by gloved-hand method and was separated by 65% Percoll with superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) before freezing. The frozen sperm was thawed at $38.5^{\circ}C$ for 45 sec in water-bath for sperm characteristic analysis. The sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction, Rhodamine123/PI double staining for mitochondrial integrity and were analyzed using flow cytometry. In results, sperm viability, acrosome reaction and mitochondrial integrity were improved in separated sperm groups compared with unseparated sperm by Percoll (UP) group. Especially, viability was significantly higher in sperm separated by Percoll containing 400 IU CAT group compared with other groups (P<0.05). And acrosome reaction was decreased in sperm separated by Percoll with 300 IU SOD, 400 IU CAT and 0.5 mM GSH groups compared with other groups, however, there were no significantly difference mitochondrial integrity among sperm separated by Percoll with antioxidant enzymes. In conclusion, we suggest that use of Percoll containing antioxidant enzymes for sperm separation will be beneficial for sperm cryopreservation in pigs.

Effect of Supplementation of Trehalose, Glycerol on Conventional Freezing and Vitrification of Boar Sperm

  • Choi, Sun-Ho;Lee, Mi-Jin;Lee, Kyung-Mi;Sa, Soo-Jin;Kim, Hyun-Jong;Jin, Hyun-Ju;Song, Yong-Sup;Park, Jun-Cheol
    • Journal of Embryo Transfer
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    • v.29 no.4
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    • pp.397-401
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    • 2014
  • The boar sperm has more lipid droplets and specialty of seminal plasma compared with other species, causing difficulties of freezing sperm and decreases for the utilization of frozen semen into the artificial insemination. However, several studies reported significant results for the recovery of sperm motility and reproductive by addition of cryoprotectants and seminal plasma after thawing. This study was designed to investigate the effects of supplementation of trehalose or glycerol in the LEY (lactose and egg yolk in BTS) solution for the conventional freezing and vitrification process. Two boars aged 16 months were used to collect semen for 2 times in a week. The samples were allotted to 3 freezing solutions (LEY + glycerol 10.5% + OEP 1.5%, LEY + trehalose 1M + OEP 1.5%, and sucrose 1.5M + trehalose 1 M + OEP 1.5%) after centrifugation at 800 g for 10 minutes. Semen was equilibrated in freezing solutions for 10 minutes and injected into plastic straws with 2~3 air bubbles to minimize freezing damages. Vitrification was performed to locate sperm in 5 cm above $LN_2$ for 5 minutes, and the conventional freezing was conducted with an automatic freezer. Motility and survival rates were measured by CASA (Computer assisted sperm an alyzing system) and FITC (Fluorescein isothiocyanate), respectively after thawing semen at $50^{\circ}C$ for 12 seconds. The results were analyzed by ANOVA with STATVIEW statistical program. The vitrificatioin solution (LEY + 10.5% glycerol + 1.5% OEP) presented higher motility (20.9%) than other solutions while the solution (LEY + 1M trehalose + 1.5% OEP) showed the lowest (motility : 5.2%). However, survival rates of vitrified sperms detected by FITC showed 1~4% live sperms in almost of dead sperms at all vitrification solutions' groups, but survival rate of freezing solution of LEY + 1M trehalose + 1.5% OEP LEY and LEY + 10.5% glycerol + 1.5% OEP were showed 49%, and 79%, respectively. There were differences (P<0.05) survival rate of conventional freezing in LEY + 10.5% glycerol + 1.5% OEP and LEY + 1M trehalose + 1.5% OEP and the remaining showed no differences. The results suggested that vitrified boar semen was not enough to be utilized for the artificial insemination, but it showed possibility to utilize for ICSI and conventional freezing with glycerol would be useful method for artificial insemination in pig while we choose the outstanding semen against tolerance to freezing damages.

Optimization of In Vivo Embryo Production and Pregnancy following Embryo Transfer in Hanwoo Cattle

  • Jeon, Soon-Hong;Jung, Kyoung Sub;Choi, Jae-Won;Heo, Young-Tae;Xu, Yong-Nan;Kim, Nam-Hyung
    • Journal of Embryo Transfer
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    • v.28 no.4
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    • pp.307-314
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    • 2013
  • Embryos formed in vivo were collected from 171 donors housed in Chung Cheong Buk-Do Institute of Livestock and Veterinary Research of the Chungbuk community during the years 2009~2012. We evaluated annual embryo collection, effect of follicle stimulating hormone (FSH), controlled internal drug release (CIDR) and prostaglandin (PG) administration to the donor for superovulation and controlling the estrus cycle, seasonal effects of embryo collection and compared the number of embryos recovered as per the collection days and pregnancy rate. In all, 1,243 embryos were collected from 118 donors with an average of $7.31{\pm}5.35$ embryos per donor, out of which 69.4% were transferable. Dosages of FSH required for inducing superovulation in various donors were compared. Average number of embryos collected from donors administered with 30 AU of FSH ($7.13{\pm}5.74$ per donor) was not significantly different from that of donors who were given an injection of 24 AU of FSH ($7.53{\pm}4.91$ per donor). However, the percentage of transferable embryos in the 30AU FSH-administered group (63.2 %, 449 of 711) was higher than that in the 24AU FSH-administered group (77.8%, 414 of 532). In the group of donors under a natural estrus cycle, the FSH dose administered did not influence the number of transferable embryos produced ($7.49{\pm}6.25$ per donor for 30 AU of FSH vs $7.49{\pm}4.92$ per donor for 24 AU of FSH). However, in donors administered with CIDR and PG for controlling the estrus cycle, the FSH dose affected the average number of transferable embryos collected ($4.25{\pm}2.87$ per donor for 30 AU of FSH vs $8.50{\pm}6.36$ per donor for 24 AU of FSH). We collected embryos from donors 6, 7 or 8 days after artificial insemination (AI). Results showed that the percentage of transferable embryos among those collected 8 days after AI was significantly higher than that among embryos collected 6 or 7 days after AI. Seasonal variations did not affect number of recovered embryos and pregnancy rates in natural estrus cycle and CIDR treatment groups (48.28% and 42.55%) but higher than pregnancy rate of frozen embryos (19.63%). These results indicated that administration of FSH beyond a threshold dose (at least 24 AU) has no beneficial effect on the production embryos and that collection of embryos 7~8 days after AI is optimal for embryo recovery. CIDR treatment induced superovulation in short term and had no influence on the natural estrus cycle. Finally, although good-quality embryos were transferred, freezing significantly reduced the pregnancy rates after transfer.

Effect of ${\beta}-Mercaptoethanol$ Supplement during In Vitro Maturation on IVM, IVF and Glutathione Level in Bovine Oocytes (소 미성숙 난포란의 체외성숙시 ${\beta}-Mercaptoethanol$의 첨가가 체외성숙, 체외수정 및 Glutathione 수준에 미치는 영향)

  • Oh, Shin-Ae;Kim, Chang-Keun;Chung, Yung-Chai;Pang, Myung-Geol
    • Development and Reproduction
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    • v.10 no.4
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    • pp.239-245
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    • 2006
  • Experiments were conducted to determine the effects of beta-mercaptoethanol(${\beta}-ME$) supplements to the maturation medium on in vitro fertilization(IVF) and intracellular glutathione(GSH) concentration. Bovine cumulus-intact oocytes were matured in TCM-199 medium containing FBS, hormonal supplements, and ${\beta}-ME$(0, 25 and $50\;{\mu}M$) for 12h and 24 h. After culture, cumulus-free matured oocytes were co-incubated with frozen-thawed spermatozoa for 24h. Maturation rate increased(p<0.05) in ${\beta}-ME$ treatment group, but no significant differences among treatment groups. Also, increases(p<0.05) in intracellular GSH concentration before and after fertilization were observed in $50\;{\mu}M\;{\beta}-ME$ supplements to the maturation medium. Male pronuclear formations after IVF was increased(p<0.05) in ${\beta}-ME$ treatment group, but no significant difference among treatment groups. In conclusion, supplementing ${\beta}-ME$ into the maturation medium increased maturation rates, fertilization rates, and intracellular GSH concentrations.

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Studies on the Effects of the Follicle Size, Hormone Supplementation, Semen Type and Capacitation Method on In Vitro Maturation and Fertilization Rate of Bovine Follicular Oocytes (난포의 크기, 호르몬의 첨가, 정액의 형태 및 수정능획득 방법 등이 소 난포란의 체외성숙 및 체외수정율에 미치는 영향에 관한 연구)

  • 김상근;이만휘;이봉구;박항균
    • Korean Journal of Animal Reproduction
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    • v.14 no.4
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    • pp.237-244
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    • 1990
  • These studies were carried out to investigate the effects of the follicles size, hormone supplementation, semen types and capacitation methods on in vitro maturation and fertilization of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean Native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3~5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, ECS, BFF and MCC for 24~48hrs. in a incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 18~20hrs. with motile capacitated sperm in the TCF(Tyroide calcium-free) solution containing 200$\mu\textrm{g}$/ml of heparin. The results obtained in these experiments were summarized as follows : 1. The oocytes classified as "A, B, C, D and Degenerative" depending morphological integrity and those were 61.4%, 12.1%, 19.2%, 4.2% and 3.0% of the total oocytes recovered, respectively. The maturation and fertilization rate of the A, B, C class follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS were 89.1%, 78.0%, 52.6% and 78.1%, 66.1%, 33.3%, respectively. 2. The average number of the follicular oocytes recovered from follicles size, 1~2mm, 3~5mm and above 5mm in dimeter were 67, 98 and 63, respectively. The maturation and fertilization rate of the follicular oocytes, cultured in the TCM-199 medium were 56.7%, 82.5%, 46.0% and 44.8%, 71.4%, 28.6%, respectively. 3. The maturation and fertilization rate of follicular oocytes, cultured in the TCM-199 medium supplemented with 5%, 10%, 15%, 20% FCS and hCG, HCG, $\beta$-estradiol were 76.0%~82.3% and 26.2%~70.0%, and those values were higher the supplementation of the hormone than the non-supplementation. 4. The fertilization and cleavage rate of the follicular oocytes, inseminated with spermatozoas of epididymis cauda, neat and frozen semen were 63.3%, 73.3%, 70.0% and 32.7%, 37.8%, 38.3, respectively. 5. The fertilization and cleavage rate of follicular oocytes, fertilized with capacitated spermatozoas by heparin, BFF and HIS methods were 70.0%, 53.8%, 34.2% and 38.3%, 23.1%, 17.1%, respectively. And the fertilization and cleavage rate were higher method of heparin than other methods.r methods.

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Effects of Equilibration Time, Precooling and Straw Loading Method on Survival of Mouse Embryos Frozen by Vitrification (생쥐 수정란의 유리화 동결보존에 있어서 동결전 처리방법에 관한 연구)

  • Gong, Il-Geun;Lee, Eun-Bong;Gang, Dae-Jin;Park, Chung-Saeng
    • Korean Journal of Animal Reproduction
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    • v.15 no.1
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    • pp.49-57
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    • 1991
  • This study was carried out to investigate the effect of equilibration time, precooling and straw loading method on the post-thaw survival rates of mouse embryos cryopreserved by vitrification method. The effect of the vitrification procedure on the damage of the embryos was also examined by the straining of nuclei using Hoechst 33342. The results obtained were summarized as follows; 1. The equilibration in Medium-1 for 10 minutes was considered to be the optimum equilibration time. Embryos equilibrated in Medium-1 for 10 minutes(81.0%) showed significantly(P<0.05) higher survival rates than those equilibrated for 5 minutes(40.0%) or 15 minutes(74.1%). 2. The survival rate of embryos cryopreserved using the double Medium-2 column(81.0%) was significantly(P<0.01) higher than that using the single Medium-2 column, whish was considered to be due to the double Medium-2 column method being more reliable for preventing contamination of diluent solution of 1M sucrose. 3. The survival rate of compacted morula stage embryos cryopreserved with the precooled and non-precooled Medium-2 was not significantly(P<0.05) different. 4. The number of blastomeres of embryos at late blastocyst stage after culture of mouse morulae for 24 to 28hours was significantly(P<0.05) decreased by freezing embryos using vitrification(53.3${\pm}$1.6 vs 41.4${\pm}$1.5), which was considered to be due to the damage of embryos during vitrification and the delay of embryo development by handling in vitro. 5. The vitrification procedure is considered to be a very simple and efficient method for cryopreservation of embryos at early developmental stage.

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