• Journal of Veterinary Science
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• 제24권1호
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• pp.4.1-4.16
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• 2023

Background: In vitro culture of preantral follicles is a promising technology for fertility preservation. Objectives: This study aims to investigate an optimized three-dimensional (3D) fetal bovine serum (FBS)-free preantral follicle culture system having a simple and easy operation. Methods: The isolated follicles from mouse ovaries were randomly divided in an ultra-low attachment 96-well plates supplement with FBS or bovine serum albumin (BSA) culture or encapsulated with an alginate supplement with FBS or BSA culture. Meanwhile, estradiol (E₂) concentration was assessed through enzyme-linked immunosorbent assay of culture supernatants. The diameter of follicular growth was measured, and the lumen of the follicle was photographed. Spindle microtubules of oocytes were detected via immunofluorescence. The ability of oocytes to fertilize was assessed using in vitro fertilization. Results: The diameters were larger for the growing secondary follicles cultured in ultra-low attachment 96-well plates than in the alginate gel on days 6, 8, and 10 (p < 0.05). Meanwhile, the E2 concentration in the BSA-supplemented medium was significantly higher in the alginate gel than in the other three groups on days 6 and 8 (p < 0.05), and the oocytes in the FBS-free system could complete meiosis and fertilization in vitro. Conclusions: The present study furnishes insights into the mature oocytes obtained from the 3D culture of the preantral follicle by using ultra-low attachment 96-well plate with an FBS-free system in vitro and supports the clinical practices to achieve competent, mature oocytes for in vitro fertilization.

• Parasites, Hosts and Diseases
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• 제36권2호
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• pp.99-108
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• 1998

베네수엘라분선충의 충란, 자충 띤 성충을 장기간 보존할 수 있는 방안을 마련하기 위하여 여러 가지 배양환경에 대한 연구를 수헝하였던 바, 다음과 같은 결과를 얻었다. 분변 내의 충란은 $4^{\circ}C$ 에서 25일간, 실온에서는 15일간 생존하였으며, 분변에서 분리한 충란은 $4^{\circ}C$의 생리적 식염수에 서 24시간 생존하였으나, 상실배기의 충란은 건조한 공기에 민감하여 12시간 내에 환성을 잃었다. 감염형 자충을 polyvinylbag에 넣어 $20^{\circ}C$에 보관하였을 경우, 분변 물질이 첨가된 0.12% 영양배 지에서는 45일, 영양배지는 28일간 생존하였다 한편, 영양배지에서 배양된 감염 자충은 상수에서 는 32일간, 멸균 식염수에서는 22일간 생존하였다. 인공감염시켜 얻은 성충은 $37^{\circ}C$에서 9일간, 9:1배지 (10% 쥐 혈청을 첨가)와 혈청을 첨가하지 않은 영양배지를 매일 교환하여 주어도 4일동안 밖에 생존하지 못하였다. 성충 암컷은 실험관 내에서 산란하고, 자충으로 부화되었지만 혈청을 첨가한 배지에서도 감염자충으로 발육하지 못하였다.

• 한국치위생학회지
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• 제15권2호
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• pp.325-332
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• 2015

Objectives: The purpose of this study was to investigate tooth whitening maintenance efficacy of several dentifrices containing effective ingredients for tooth whitening. Methods: Hydroxyapatite specimens(HAPs) staining was done by using modified Stookey's methods. HAPs were treated with 2.9% hydrogen peroxide containing strip for whitening, and were shaken with several dentifrice slurry(dentifrice 1 : artificial saliva 2) for 30 minutes. The HAPs were finally dipped in staining solution for an hour. Shaking and dipping were repeated 4 times and lightness values were measured by colorimeter at each step. In clinical test, test 4 dentifrice and control dentifrice were evaluated by 21 subjects for 2 months after receiving institutional review board(IRB) approval. Organoleptic(vita shade guide) and instrumental(SHADEEYE-NCC) evaluation were performed for whiteness change of teeth. Statistical analysis was performed using repeated measures ANOVA, Tukey's post hoc test and ${\chi}^2$-test(p<0.05). Results: All dentifrices showed statistical significance in comparison with control dentifrice containing sodium fluoride and test 4 dentifrice containing sodium pyrophosphate, sodium metaphosphate, candelilla wax, and sodium fluoride showed statistical significance in comparison with other dentifrices by inhibiting staining in vitro(p<0.05). In clinical test, test 4 dentifrice showed better effects than control dentifrice in organoleptic and instrumental evaluation in tooth whitening maintenance efficacy(p<0.05). The awareness toward tooth whitening maintenance efficacy for 2 months use showed that test 4 dentifrice was much better than control dentifrice, but did not show statistically significant(p>0.05). Conclusions: Dentifrice containing sodium pyrophosphate, sodium metaphosphate, candelilla wax and sodium fluoride was more effective in keeping teeth white.

• Molecules and Cells
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• 제40권2호
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• pp.117-122
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• 2017

Despite the fact that porcine embryonic stem cells (ESCs) are a practical study tool, in vitro long-term maintenance of these cells is difficult in a two-dimensional (2D) microenvironment using cellular niche or extracellular matrix proteins. However, a three-dimensional (3D) microenvironment, similar to that enclosing the inner cell mass of the blastocyst, may improve in vitro maintenance of self-renewal. Accordingly, as a first step toward constructing a 3D microenvironment optimized to maintain porcine ESC self-renewal, we investigated different culture dimensions for porcine ICM-derived cells to enhance the maintenance of self-renewal. Porcine ICM-derived cells were cultured in agarose-based 3D hydrogel with self-renewal-friendly mechanics and in 2D culture plates with or without feeder cells. Subsequently, the effects of the 3D microenvironment on maintenance of self-renewal were identified by analyzing colony formation and morphology, alkaline phosphatase (AP) activity, and transcriptional and translational regulation of self-renewal-related genes. The 3D microenvironment using a 1.5% (w/v) agarose-based 3D hydrogel resulted in significantly more colonies with stereoscopic morphology, significantly improved AP activity, and increased protein expression of self-renewal-related genes compared to those in the 2D microenvironment. These results demonstrate that self-renewal of porcine ICM-derived cells can be maintained more effectively in a 3D microenvironment than in a 2D microenvironment. These results will help develop novel culture systems for ICM-derived cells derived from diverse species, which will contribute to stimulating basic and applicable studies related to ESCs.

• Parasites, Hosts and Diseases
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• 제50권4호
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• pp.309-315
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• 2012

Clonorchis sinensis is a biological carcinogen inducing human cholangiocarcinoma, and clonorchiasis is one of the important endemic infectious diseases in East Asia. The present study investigated survival longevity of C. sinensis adult worms in various in vitro conditions to find the best way of keeping the worms longer. The worms were maintained in 0.85% NaCl, 1${\times}$PBS, 1${\times}$Locke's solution, RPMI-1640, DMEM, and IMDM media, and in 1${\times}$Locke's solution with different supplements. All of the worms died within 3 and 7 days in 0.85% NaCl and 1${\times}$PBS, respectively, but survived up to 57 days in 1${\times}$Locke's solution. The worms lived for 106 days in DMEM, and 114 days in both RPMI-1640 and IMDM media. The survival rate in RPMI-1640 medium was the highest (50%) compared to that in DMEM ($20{\pm}10%$) and in IMDM ($33.3{\pm}25.2%$) after 3 months. The 1${\times}$Locke's solution with 0.005% bovine bile supplement showed increased duration of maximum survival from 42 days to 70 days. Higher concentration of bile supplements than 0.005% or addition of glucose were disadvantageous for the worm survival. The worms died rapidly in solutions containing L-aspartic acid, L-glutamic acid, and adenine compared to L-arginine, L-serine, and L-tryptophan. In conclusion, the 1${\times}$Locke's solution best supports the worms alive among inorganic solutions for 57 days, and the RPMI-1640 medium maintains living C. sinensis adults better and longer up to 114 days in vitro than other media.

• Clinical and Experimental Reproductive Medicine
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• 제39권1호
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• pp.10-14
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• 2012

The ovarian follicles develop initially from primordial follicles. The majority of ovarian primordial follicles are maintained quiescently as a reserve for the reproductive life span. Only a few of them are activated and develop to an advanced follicular stage. The maintenance of dormancy and activation of primordial follicles are controlled by coordinated actions of a suppressor/activator with close communications with somatic cells and intra-oocyte signaling pathways. Many growth factors and signaling pathways have been identified and the transforming growth factor-beta superfamily plays important roles in early folliculogenesis. However, the mechanism of maintaining the dormancy and survival of primordial follicles has remained unknown for decades. Recently, since the first finding that all primordial follicles are activated prematurely in mice deficient forkhead box O3a, phosphatidylinositol 3 kinase/phosphatase and tensin homolog (PTEN) signaling pathway was reported to be important in the regulation of dormancy and initial follicular activation. With these informations on early folliculogenesis, clinical application can be expected such as in vitro maturation of immature oocytes or in vitro activation of follicles by PTEN inhibitor in cryopreserved ovarian cortical tissues for fertility preservation.

• Journal of Plant Biotechnology
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• 제47권1호
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• pp.26-39
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• 2020

In maize, immature embryos (IEs) are highly regenerative explants most suitable for producing high frequencies of plantlet regeneration in vitro. Apart from media, explants, and hormones, genotypic variation also influences in vitro characters to a great extent. In the present study, IEs were used to study the distinctive effect of variation of size/stage and hormones in different genotypes on five in vitro characters viz., frequency of callus induction, growth rate of total callus, frequency of E. callus induction, and volume and number of regenerated plantlets. LS medium with different concentrations of 2,4-D (0.5, 1.5, 2.5, 4.0 and 5.0 mg/L) were used to study the former four in vitro characters, and medium with 6-benzylaminopurine and kinetin (0.5 mg/L, each) was used for plantlet regeneration. IEs of 1.0, 1.5, 2.0, 2.5 and 3.0 mm in size were isolated from four inbred lines viz., NM74C, NM81A, NM5883 and NM5884. Two-way ANOVA revealed that explant size and genotypes, as well as hormonal concentrations showed significant effects on in vitro characters. Two millimeter IEs were found to be suitable for in vitro cultures. LS medium with 1.5 mg/L 2,4-D and LS with BAP and Kn (0.5 mg/L, each) were found to be the best hormonal concentrations for callus induction, maintenance, and regeneration, respectively. Among the four genotypes, NM81A and NM5883 yielded more non-embryogenic and Type I E. calli. In contrast, NM74C and NM5884 yielded more highly regenerative Type II calli. Inbred line NM5884 was found to be the best among these four genotypes.

• Asian-Australasian Journal of Animal Sciences
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• 제21권3호
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• pp.340-345
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• 2008

Oxidative stress is one of the major causes of failure of in vitro storage of boar semen. Reactive oxygen species (ROS) are one of the important mediators of oxidative stress during in vitro storage of boar semen. Our study examined the effects of taurine on sperm characteristic and on in vitro developmental embryos during in vitro storage of boar semen for 7 days. Semen was randomly aliquoted into 3 centrifuge tubes and treated with different concentrations of taurine (25-100 mM). The characteristics of boar sperm were analyzed for motility by light microscopy, viability by using a Makler counting chamber and membrane integrity by a hypoosmotic swelling test (HOST). The percentages of motile spermatozoa in taurine groups after 5 days were significantly higher compared to the control. Sperm viability in the control was lower than in taurine groups after 7 days irrespective of different taurine concentration. In the hyoosmotic swelling test (HOST), significantly higher results were obtained in taurine groups after 3 days. Also, the developmental rates of IVM/IVF porcine embryos from semen treated with pyruvate and taurine were significantly increased when compared with the control (p<0.05). These results indicate that supplementation of taurine as an antioxidant in boar semen extender can improve the semen quality.

• Asian-Australasian Journal of Animal Sciences
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• 제29권10호
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• pp.1398-1406
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• 2016

In general, the seminiferous tubule basement membrane (STBM), comprising laminin, collagen IV, perlecan, and entactin, plays an important role in self-renewal and spermatogenesis of spermatogonial stem cells (SSCs) in the testis. However, among the diverse extracellular matrix (ECM) proteins constituting the STBM, the mechanism by which each regulates SSC fate has yet to be revealed. Accordingly, we investigated the effects of various ECM proteins on the maintenance of the undifferentiated state of SSCs in pigs. First, an extracellular signaling-free culture system was optimized, and alkaline phosphatase (AP) activity and transcriptional regulation of SSC-specific genes were analyzed in porcine SSCs (pSSCs) cultured for 1, 3, and 5 days on non-, laminin- and collagen IV-coated Petri dishes in the optimized culture system. The microenvironment consisting of glial cell-derived neurotrophic factor (GDNF)-supplemented mouse embryonic stem cell culture medium (mESCCM) (GDNF-mESCCM) demonstrated the highest efficiency in the maintenance of AP activity. Moreover, under the established extracellular signaling-free microenvironment, effective maintenance of AP activity and SSC-specific gene expression was detected in pSSCs experiencing laminin-derived signaling. From these results, we believe that laminin can serve as an extracellular niche factor required for the in vitro maintenance of undifferentiated pSSCs in the establishment of the pSSC culture system.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.1
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• pp.199.2-200
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• 2003

Choline is an important membrane phospholipid constituent and a neurotransmitter precursor that is minimally synthesized in brain. The long-term maintenance of brain choline concentration is dependent on choline transport from plasma, which occurs via saturable transport system at the blood-brain barrier. In the present study, we examined to elucidate the characteristics of transport of cationic amines, especially choline which is one of cationic amines, to BBS using conditionally immortalized rat brain capillary endothelial cell line (TR-BBB) in vitro. (omitted)