scholarly journals 341APOPTOSIS IN SOMATIC CELL CLONED AND EGFP TRANSGENIC BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 290
Author(s):  
S.L. Lee ◽  
S.Y. Choe ◽  
G.J. Rho
Keyword(s):  
Dna Fragmentation ◽  
Technology Development ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Development Project ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cell Stage ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Fragmentation Ratio

The overall success rate achieved by cloning techniques to date is low, mainly due to deficiencies in nuclear reprogramming, gene expression and DNA fragmentation, which result in early and late embryonic losses. This study was carried out to compare the incidences of DNA fragmentation during development of IVF, parthenote (PT), nuclear transfer (NT) and transgenic-cloned (TG) embryos. Terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL) with propidium iodide counterstaining was used for determination of DNA fragmentation and total cell number. Donor cells were fetal fibroblasts with or without transfection with EGFP, and cultured in DMEM+15% FCS until confluent, for up to 5 days. At 19h post maturation (hpm), enucleated oocytes were reconstructed with donor cells and activated at 24hpm with the combination of ionomycin (5μM, 5min) and cycloheximide (10μg/mL, 5h) after electric fusion by a single DC pulse (1.6KV/cm, 60μs) delivered by a BTX 200. Parthenotes were produced by the same activation protocol at 24hpm. The eggs and control IVF embryos were cultured in CR1aa at 39°C in a humidified atmosphere of 5% CO2 in air. Differences among groups were analyzed using one-way ANOVA after arc-sine transformation of proportional data. Embryos at the 8-cell stage in all treatments, IVF, PT, NT and TG, showed DNA fragmentation. The apoptotic cell index (total number of apoptotic nuclei/total number of nuclei) of Day 7 blastocysts was significantly (P<0.05) higher in TG and NT embryos (17/91, 18.6±4.0% and 13/94, 13.8±4.7%, respectively) compared to IVF and PT embryos (9/122, 7.4±3.4% and 8/93, 8.6±2.9%, respectively). TUNEL positive cells were detected in almost all blastocysts at Day 7 and were mainly observed in the ICM. The DNA fragmentation ratio of the ICM in the blastosysts at Day 7 (number of apoptotic nuclei in the ICM/total number of apoptotic nuclei in the blastocyst) was significantly (P<0.05) higher in TG embryos (64.7±21.4%) than in IVF (44.4±28.0%), PT (50.0±18.6%) or NT (53.0±32.5%) embryos. These results indicate a higher occurrence of DNA fragmentation observed in NT and TG embryos when compared to IVF and PT embryos. In addition, ICM of TG blastocysts revealed a high DNA fragmentation ratio, which may be related to early embryonic loss after transfer of the resulting embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 300012-05-3-SB010 and Cho-A Pharm. Co. LTD.]

scholarly journals 32 EFFICIENCY OF FEMALE-DERIVED DONOR CELLS ON HIGH POSTNATAL SURVIVAL IN PIG CLONING

2005 ◽  
Vol 17 (2) ◽  
pp. 166
Author(s):  
S.K. Cho ◽  
M.R. Park ◽  
D.N. Kwon ◽  
E.K. Lee ◽  
S.J. Kang ◽  
...  
Keyword(s):  
Adenosine Monophosphate ◽  
Cell Number ◽  
Developmental Competence ◽  
Cell Stage ◽  
Male And Female ◽  
Electric Pulses ◽  
Donor Cells ◽  
Fetal Fibroblasts

The present study was conducted to investigate the developmental competence of male and female somatic cell derived nuclear transfer (NT) porcine embryos and also the production and survival efficiency of cloned male and female piglets. Maturation of porcine COCs was accomplished by incubation in NCSU-23 medium supplemented with 0.6 mM cysteine, 10% porcine follicular fluid, 1 mM dibutyryl cyclic adenosine monophosphate, and 0.1 IU/mL human menopausal gonadotrophin for 20 h and then culture without dbcAMP and hMG for another 18 to 24 h. Fetal cells were isolated from a male fetus and two female fetuses, and cultured in ES-DMEM medium containing 10% FCS. Enucleated oocytes were fused with fetal fibroblasts (passage 4 to 15). Reconstructed embryos were cultured in NCSU-23 with 4 mg/mL BSA under mineral oil at 39°C in 5% CO2 in air for up to 6 days. NT eggs that had been activated with electric pulses and cultured for 1 or 2 days were transported to the experimental station in modified NCSU-23 with antibiotics. NT embryos were surgically transferred into the oviducts of recipients between Day 27 and Day 30; pregnancy was determined by ultrasound. The potential of NT embryos to develop into blastocysts was not different among donor cells of different origins. However, the mean cell number of in vivo female and male blastocysts (83.8 ± 46.2 to 99.2 ± 55.7) was higher than in in vitro culture of NT groups (31.4 ± 8.29 to 33.2 ± 10.15). A total of 11,535 NT embryos (1- to 8-cell stage) were surgically transferred into 66 surrogate gilts. Among fourteen pregnant gilts, four recipients aborted during the period of conception. Five pregnant gilts delivered fifteen female piglets, 1.28 ± 0.33 kg (0.48∼1.83 kg) in female piglets and 0.84±0.25 kg (0.45∼1.25 kg) in male piglets. Nine live cloned female (60.0%) and four male piglets (18.2%) were produced. According to these results, survival rates and birth weights of female cloned piglets were higher than those of cloned male piglets (P < 0.05). This study suggests that use of female, compared with male, fetal fibroblast cells as nuclear donors may increase cloning outcomes. This work was supported in part by a grant program from RDA(Biogreen21) and Cho-A, Republic of Korea.

74 EXAMINATION OF ABNORMAL CELL DIVISION AND CHROMOSOME ABERRATION IN PIG PARTHENOTES AND CLONED EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 145
Author(s):  
S. J. Uhm ◽  
M. S. Kim ◽  
M. K. Gupta ◽  
H. Y. Lee ◽  
S. J. Park ◽  
...  
Keyword(s):  
Cell Division ◽  
Chromosome Aberration ◽  
Bovine Serum ◽  
Polar Body ◽  
Cell Number ◽  
Chromosome 1 ◽  
Hybridization Technique ◽  
Cell Stage ◽  
Abnormal Cell ◽  
Fetal Fibroblasts

Blastomere fragmentation is commonly observed in pig embryos and is associated with reduced blastocyst and pregnancy rates. This study examined the effect of the frequency of abnormal cell division and chromosome aberration on the embryonic developmental ability of pig parthenotes and nuclear transferred (NT) embryos. Pig immature oocytes cultured in TCM-199 supplemented with 10% pig follicular fluid, 0.2 mM pyruvate, 10 ng/mL epidermal growth factor (EGF), 5 �g/mL Folltropin V, 1 �g/mL estradiol-17�, and 25 �g/mL gentamycin for 44 h. Cumulus cells from matured oocytes were removed by vortexing for 1 min in TL-HEPES medium containing 0.1% hyarunonidase. Denuded oocytes were enucleated using 20 um micropipette in TCM-HEPES medium containing 7.5 �g/mL cytochalasin B (CB) and 10% fetal bovine serum, and were reconstructed with fetal fibroblasts by electrofusion (two DC pulses of 2.0 kV/cm for 30 �s). For production of parthenotes and reconstructed embryos, denuded oocytes were activated by a DC pulse of 1.0 kV/cm for 30 �s and then cultured for 4 h in NCSU23 with 10 �g/mL CB and 0.4% bovine serum albumin for inhibition of polar body extrusion. Subsequently, these oocytes were cultured in 50 �L of NCSU23 containing 0.4% BSA for 7 days at 39�C in a humidified atmosphere of 5% CO2 in air. The frequency of chromosome aberrations was evaluated using fluorescent in situ hybridization technique with a porcine chromosome-1 submetacentric specific probe. Data were analyzed by Student's t-test and ANOVA using SAS software as appropriate (SAS Institute, Inc., Cary, NC, USA). Parthenotes and NT embryos showed similiar cleavage rates (61.4 and 62.9%), but the blastocyst rate of parthenotes (18.4%) was significantly higher (P < 0.05) than that of NT embryos (10.4%). The frequency of chromosome aberration in NT embryos (39.8%) at the 4-cell stage on Day 3 of culture was significantly higher (P < 0.05) than that of parthenotes (21.9%). The percentage of fragmentation was significantly higher (P < 0.05) in NT embryos (51.7%) than in parthenotes (27.1%). Furthermore, the developmental rates of non-fragmented parthenotes (40.0%) and NT (22.9%) embryos to the blastocyst stage were significantly higher (P < 0.05) than those of fragmented parthenote and NT embryos (17.3 and 5.9% respectively). The total cell number of non-fragmented parthenote and NT embryos (34.4 � 10.0 and 29.7 � 7.5) were significantly higher (P < 0.05) than those of fragmented parthnote and NT embryos (22.3 � 9.6 and 18.4 � 6.2 respectively). Therefore, these results indicate that chromosomal abnormality and embryonic fragmentation could be associated with reduced developmental ability in pig NT embryos. This work was supported by the Research Project on the Production of Bio-organs, Ministry of Agriculture and Forestry, Republic of Korea.

59 HIGHER BLASTOCYST FORMATION OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DOES NOT GUARANTEE BETTER PREGNANCY IN PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 147
Author(s):  
E. Lee ◽  
K. Song ◽  
Y. Jeong ◽  
S. Hyun
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Skin Fibroblasts ◽  
Miniature Pig ◽  
Donor Cells ◽  
Fetal Fibroblasts

Generally, blastocyst (BL) formation and embryo cell number are used as main parameters to evaluate the viability and quality of in vitro-produced somatic cell nuclear transfer (SCNT) embryos. We investigated whether in vitro development of SCNT pig embryos correlates with in vivo viability after transfer to surrogates. For SCNT, cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with follicular fluid, hormones, EGF, cysteine, and insulin for the first 22 h and in a hormone-free medium for 18 h. Three sources of pig skin cells were used as nuclear donor: (1) skin fibroblasts of a cloned piglet that were produced by SCNT of fetal fibroblasts from a Landrace × Yorkshire × Duroc F1 hybrid (LYD), (2) skin fibroblasts of a miniature pig having the human decay accelerating factor gene (hDAF-MP), and (3) skin fibroblasts of a miniature pig with a different strain (MP). MII oocytes were enucleated, subjected to nuclear transfer from a donor cell, electrically fused, and activated 1 h after fusion. SCNT embryos were cultured in a modified NCSU-23 (Park Y et al. 2005 Zygote 13, 269–275) for 6 days or surgically transferred (110–150 fused embryos) into the oviduct of a surrogate that showed standing estrus on the same day as SCNT. Embryos were examined for cleavage and BL formation on Days 2 and 6, respectively (Day 0 = the day of SCNT). BLs were examined for their cell number after staining with Hoechst 33342. Pregnancy was diagnosed by ultrasound 30 and 60 days after embryo transfer. Embryo cleavage was not affected by donor cells (82, 81, and 72% for LYD, hDAF-MP, and MP, respectively), but BL formation was higher (P &lt; 0.05) in hDAF-MP (16%) than in LYD (9%) and MP (6%). MP showed higher (P &lt; 0.05) BL cell number (46 cells/BL) than hDAF-MP (34 cells) but did not show a difference from LYD (37 cells). LYD and MP showed higher pregnancy rates (Table 1) on Days 30 and 60, even though they showed lower BL formation in vitro. Due to a relatively small number of embryo transfers through a limited period, we could not exclude any possible effects by seasonal or operational differences. These results indicated that pregnancy did not correlate with in vitro BL formation of SCNT pig embryos but rather were affected by the source of donor cells. Table 1.In vivo development of somatic cell nuclear transfer pig embryos derived from different sources of donor cells This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.

Sperm-borne small RNAs improve the developmental competence of pre-implantation cloned embryos in rabbit

Zygote ◽  
2021 ◽  
pp. 1-6
Author(s):  
Hongyu Qin ◽  
Pengxiang Qu ◽  
Huizhong Hu ◽  
Wenbin Cao ◽  
Hengchao Liu ◽  
...  
Keyword(s):  
Small Rnas ◽  
Apoptotic Cell ◽  
Epigenetic Modification ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Total Cell Number ◽  
Cell Stage ◽  
Micro Rnas

Summary The low efficiency of somatic cell nuclear transfer (SCNT) greatly limits its application. Compared with the fertilized embryo, cloned embryos display abnormal epigenetic modification and other inferior developmental properties. In this study, small RNAs were isolated, and miR-34c and miR-125b were quantified by real-time PCR; results showed that these micro-RNAs were highly expressed in sperm. The test sample was divided into three groups: one was the fertilized group, one was the SCNT control group (NT-C group), and the third group consisted of SCNT embryos injected with sperm-borne small RNA (NT-T group). The level of tri-methylation of lysine 9 on histone H3 (H3K9me3) at the 8-cell stage was determined by immunofluorescence staining, and the cleavage ratio, blastocyst ratio, apoptotic cell index of the blastocyst and total cell number of blastocysts in each group were analyzed. Results showed that the H3K9me3 level was significantly higher in the NT-C group than in the fertilized group and the NT-T group. The apoptosis index of blastocysts in the NT-C group was significantly higher than that in the fertilized group and the NT-T group. The total cell number of SCNT embryos was significantly lower than that of fertilized embryos, and injecting sperm-borne small RNAs could significantly increase the total cell number of SCNT blastocysts. Our study not only demonstrates that sperm-borne small RNAs have an important role in embryo development, but also provides a new strategy for improving the efficiency of SCNT in rabbit.

40 EFFECT OF CHEMICAL ACTIVATION ON DEVELOPMENT AND APOPTOSIS OF PREIMPLANTATION PORCINE EMBRYOS DERIVED FROM NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 129
Author(s):  
G.-S. Im ◽  
J.-S. Seo ◽  
I.-S. Hwang ◽  
S.-W. Kim ◽  
H.-S. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Electric Pulse ◽  
Chemical Activation ◽  
Cell Mass ◽  
Cell Number ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Donor Cells ◽  
Significant Difference ◽  
Blastocyst Rate

Activation is one of key factors for improving developmental ability of pre-implantation nuclear transfer (NT) embryos. This study investigated the effect of chemical activation following fusion/activation on the development and apoptosis of pre-implantation porcine embryos derived from NT. Oocytes were aspirated from ovaries collected from a local abattoir, and then matured in TCM-199 for 42 to 44 h. Donor cells were prepared from a 35-day-old porcine fetus. Matured oocytes were enucleated and donor cells were introduced into the perivitelline space. Fusion/activation was conducted with two electric pulse of 1.2 kV/cm for 30 �s. Fused embryos were divided into four groups. The first one was the control without chemical activation; the other three groups were treated with thimerosal (0.2 mM for 10 min; T) and then with dithiothreitol (8 mM for 30 min; DTT), 6-dimethylaminopurine (2 mM for 3 h; 6-DMAP), or cycloheximide (10 �g/mL for 6 h; CH). Treated embryos were cultured in porcine zygote medium-3 (PZM-3) at 38.5�C under 5% CO2 in air for 6 days. Cleavage and blastocyst rate were determined on Days 3 and 6, respectively. Apoptosis was analyzed with a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay from day 1 to 7. Embryos treated with chemicals following fusion/activation showed significantly higher blastocyst rates compared to control embryos fused/activated by electric pulse alone (12.6% for control vs. 21.1% for DTT, 20.8% for 6-DMAP, 20.6% for CH; P < 0.05). Although total cell number of blastocysts showed no significant difference, the ratio of inner cell mass to trophectoderm was significantly higher (P < 0.05) in embryos with chemical activation than in those without it (11.9 vs. 19.4, 18.1, and 24.1%; P < 0.05). Occurrence of apoptosis was first observed on Day 3, but there was no significant difference among treatments until Day 6. It was significantly increased in embryos with chemical activation on Day 7 compared to control embryos (5.1 vs. 7.1, 7.8, and 7.8%; P < 0.05). These results indicate that chemical activation following fusion/activation could support significantly a higher blastocyst rate for pre-implantation porcine embryos derived from nuclear transfer; however, it can increase occurrence of apoptotic cells at blastocyst stage.

158 ASTAXANTHIN EFFECTS ON MATURATION, FERTILIZATION, AND DEVELOPMENT OF PORCINE OOCYTES CULTURED UNDER HEAT STRESS

2014 ◽  
Vol 26 (1) ◽  
pp. 193 ◽  
Author(s):  
L. T. K. Do ◽  
V. V. Luu ◽  
Y. Sato ◽  
M. Taniguchi ◽  
T. Otoi
Keyword(s):  
Heat Stress ◽  
Dna Fragmentation ◽  
Cell Number ◽  
Total Cell ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Control Group ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Maturation Medium ◽  
Significant Difference

Heat stress can engender various disorders in reproductive functions such as impairment of oocyte maturation, fertilization, and embryonic development. Astaxanthin, an extremely common carotenoid, is a typical fat-soluble antioxidant that scavenges ROS and blocks lipid peroxidation. Moreover, astaxanthin has been shown to improve the development of embryos exposed to heat stress by a reduction in stress-inducible genes. This study was conducted to investigate the effects of astaxanthin supplementation on the meiotic competence, fertilization, and development of porcine oocytes exposed to high temperature (41°C) during maturation culture. Cumulus–oocyte complexes (COC) collected from ovaries were transferred into maturation medium supplemented with astaxanthin (0, 0.25, 0.5, or 1.0 ppm) and were then cultured for 46 h at 41°C or 38.5°C. After maturation culture, the COC were subjected to IVF and embryo culture to evaluate the fertility and development of oocytes. The total cell number and DNA fragmentation in the blastocysts were assessed using terminal deoxynucleotidyl transferase dUTP nick end labelling and Hoechst 33342 staining. The total numbers of oocytes matured at 41°C and 38.5°C in each treatment group were 432 to 470 and 426 to 444, respectively. Data were analysed using ANOVA, followed by Fisher's protected least significant difference test. Exposure to elevated temperatures during maturation culture significantly reduced the proportions of oocytes that reached metaphase II. When the COC were cultured in the maturation medium supplemented with 0.5 and 1.0 ppm of astaxanthin under heat stress conditions (41°C), the supplementation of astaxanthin significantly improved the proportions of maturation, fertilization, and blastocyst formation compared with the control group (0 ppm) (50–52%, 45–55%, and 11–12% v. 17, 25, and 6%, respectively). The supplementation of the maturation medium with 0.25 ppm of astaxanthin improved only blastocyst formation (9.6%). Similarly, the supplementation of astaxanthin at 1.0 ppm improved the proportions of maturation, fertilization, and blastocyst formation of oocytes matured at 38.5°C s compared with the control group (67, 57, and 18% v. 48, 33, and 12%, respectively). However, no beneficial effect of astaxanthin supplementation was found in the total cell number or DNA fragmentation in the blastocysts, irrespective of culture temperature. Our findings show that the supplementation of astaxanthin to maturation medium improves maturation, fertilization, and embryo development of porcine oocytes exposed to heat stress during maturation culture.

scholarly journals Analysis of DNA fragmentation in bovine somatic nuclear transfer embryos using TUNEL

Reproduction ◽  
2002 ◽  
pp. 813-819 ◽  
Author(s):  
M Fahrudin ◽  
T Otoi ◽  
NW Karja ◽  
M Mori ◽  
M Murakami ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Fragmented Nucleus ◽  
Number Of Cells

The production of cloned animals is an inefficient process because of early or late embryonic losses. This study focused on the DNA fragmentation that occurs during embryonic development. The occurrence of DNA fragmentation was examined in bovine embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (NT) using the terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL). IVF and NT embryos at the two-cell to blastocyst stage were stained by TUNEL for the analysis of DNA-fragmented nuclei and with propidium iodide for determination of the total number of cells. DNA fragmentation was first detected in NT embryos at the four-cell stage, but in IVF embryos at the six- to eight-cell stage. The percentage of embryos with at least one DNA-fragmented nucleus increased with the advance of the developmental stage of embryos in both IVF and NT groups. The DNA-fragmented nucleus index in NT embryos that developed beyond the four-cell stage was significantly higher (P<0.01) than that of IVF embryos at the same stage. In the both IVF and NT groups, TUNEL-labelled cells were detected in almost all blastocysts and were mainly observed in presumptive inner cell mass (ICM) cells of embryos. The DNA-fragmented nucleus index was negatively correlated with the total number of cells in NT blastocysts, but this relationship was not observed in IVF blastocysts. These results suggest that the high occurrence of DNA fragmentation observed in NT embryos may be related to early embryonic loss after transfer.

scholarly journals 75 RABBIT NUCLEAR TRANSFER WITH CULTURED SOMATIC CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 187 ◽  
Author(s):  
F. Yang ◽  
B. Kessler ◽  
S. Ewerling ◽  
E. Wolf ◽  
V. Zakhartchenko
Keyword(s):  
Nuclear Transfer ◽  
Cultured Cells ◽  
Somatic Cells ◽  
Cumulus Cells ◽  
Cell Stage ◽  
Electric Pulses ◽  
Donor Cells ◽  
Fetal Fibroblasts

Cloned rabbits have been obtained by somatic cell nuclear transfer (SCNT) only with fresh, non-cultured cumulus cells (Chesne et al. 2002 Nat. Biotechnol. 20, 366–369). For the purpose of generating transgenic animals by SCNT, donor cells must be cultured and modified prior to use as nuclear donors. The objective of this study was to optimize the SCNT procedure using cultured cumulus or fibroblast cells. MII oocytes were harvested from superovulated Zika rabbits, and maternal chromosomes were removed by demecolcine-assisted enucleation (Yin et al. 2002 Biol. Reprod. 67, 442–446). Two types of somatic cells originating from Ali/Bass rabbits were used as nuclear donors: cumulus cells collected from in vivo-matured oocytes and cultured for 1–5 passages, and primary fetal fibroblasts obtained from Day 16 fetuses and grown to confluence or starved for 4–5 days. Somatic donor cells and recipient cytoplasts were fused with 2 electric pulses (1.95 kV/cm, 25 μs each, 1 s interval). Twenty to 40 min after fusion, cloned embryos were activated first with the same electropulses as for fusion, and then immediately followed by 1 h incubation in 2 mM 6-dimethylaminopurine and 5 μg/mL cytochalasin B in culture medium (B2 medium supplemented with 10% FCS). Cloned embryos were either transferred at the 2- and 4-cell stage to asynchronized recipients or cultured in vitro for 6 days. Data were compared using chi-square test, and differences were considered significant when P < 0.05. Our results demonstrate that cloned rabbits can be produced by SCNT with cultured cells but the efficiency of this technique is still very low irrespective of the type of donor cells. Table 1. Development of cloned embryos derived from somatic cells This research was supported by the Therapeutic Human Polyclonals, Inc.

scholarly journals De NovoDemonstration and Co-localization of Free-Radical Production and Apoptosis Formation in Rat Kidney Subjected to Ischemia/Reperfusion

2001 ◽  
Vol 12 (5) ◽  
pp. 973-982 ◽  
Author(s):  
CHIANG-TING CHIEN ◽  
PO-HUANG LEE ◽  
CHAU-FONG CHEN ◽  
MING-CHIEH MA ◽  
MING-KUEN LAI ◽  
...  
Keyword(s):  
Oxidative Damage ◽  
Dna Fragmentation ◽  
De Novo ◽  
Ischemia Reperfusion Injury ◽  
Apoptotic Cell ◽  
Rat Kidney ◽  
Ischemia Reperfusion ◽  
Cell Number ◽  
Dependent Manner ◽  
Hypoxia Reoxygenation

Abstract. Ischemia-induced oxidative damage to the reperfused kidney was examined. A modified chemiluminescence method, anin situnitro blue tetrazolium perfusion technique, and a DNA fragmentation/apoptosis-related protein assay were adapted for demonstrationde novoand co-localization of reactive oxygen species (ROS) production and apoptosis formation in rat kidneys subjected to ischemia/reperfusion injury. The results showed that prolonged ischemia potentiated proapoptotic mechanisms, including increases in the Bax/Bcl-2 ratio, CPP32 expression, and poly-(ADP-ribose)-polymerase fragments, and subsequently resulted in severe apoptosis, including increases in DNA fragmentation and apoptotic cell number in renal proximal tubules (PT) and distal tubules (DT) in a time-dependent manner. The increased level of ROS detected on the renal surface was correlated with that in blood and was intensified by a prolonged interval of ischemia. The main source of ROS synthesis was the PT epithelial cells. The ROS and apoptotic nuclei detected in the PT cells can be ameliorated by superoxide dismutase (SOD) treatment before reperfusion. However, the apoptotic nuclei remained in DT in the SOD-treated rats, indicating that formation of apoptosis in DT was not influenced by the small amounts of ROS produced. In PT and DT cell cultures, significant increases in apoptotic cells and ROS were evident in PT cells after hypoxia/reoxygenation insult. Furthermore, the oxidative damage in PT, but not in DT, can be alleviated by ROS scavengers SOD and hexa(sulfobutyl)fullerene, confirming that PT are vulnerable to ROS. These results lead us to conclude that ROS produced in significant amounts in PT epithelium under ischemia/reperfusion or hypoxia/reoxygenation conditions may be responsible for the apoptotic death of these cells.

scholarly journals 41 EFFECT OF CELL TYPES AND PASSAGES ON DEVELOPMENT AND APOPTOSIS OF PORCINE CLONED EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 170
Author(s):  
J.-G. Kim ◽  
Y.-S. Lee ◽  
S.-L. Lee ◽  
S.-A. Ock ◽  
C.-S. Park ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Cell Types ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Female Fetus ◽  
Cell Numbers ◽  
Donor Cells ◽  
Fetal Fibroblasts

The purpose of this study was to improve the efficiency of somatic cell nuclear transfer (SCNT) in pigs by assessing the development, cell numbers and apoptosis when using different cell types as nuclear donors and different numbers of passages. Primary cultures of the donor cells, porcine fetal fibroblasts (pFFF) from a female fetus at ∼30 days of gestation and adult female ear skin cells (pAESC), were established in DMEM + 15% FCS. For nuclear donor, cells at different passages were cultured for 5 days until confluent. Cumulus-oocyte complexes were matured and fertilized in vitro as controls by the following methods (2000 Theriogenology 54, 787–797). Following enucleation, oocytes were reconstructed by transfer of donor cells and fusion with two DC pulses (1.4 kV/cm, 50 μs) in 0.28 M mannitol containing 0.01 mM CaCl2 and MgCl2. Eggs were then cultured in NCSU23 + 1.9 mM 6-dimethylaminopurine for 3 h. SCNT and IVF embryos were cultured in NCSU23 for 54 h and subsequently in the same medium with 5.55 mM glucose for 90 h at 38.5°C in 5% CO2 in air. In Experiment 1, when the rates of development between IVF and SCNT embryos constituted with cells at 5–7 passages were compared, no significant (P < 0.05) differences were observed in the cleavage rates. The rates of blastocyst formation were significantly (P < 0.05) higher in IVF than in SCNT embryos with pFFF and pAESC (21% vs. 15% and 10%), but it did not differ between SCNT embryos. Total cell numbers in IVF blastocysts (35.4 ± 12) were significantly (P < 0.05) higher than in SCNT blastocysts with pFFF and pAESC (28.4 ± 8 and 26.2 ± 10, respectively). The apoptosis signal by TUNEL was initiated at Day 3 in IVF and SCNT embryos. Apoptosis rates in SCNT blastocysts with pFFF and pAESC (13.1 ± 2.5 and 16.6 ± 4.3, respectively) were significantly (P < 0.05) higher than in IVF embryos (3.6 ± 1.4). As the embryos developed, the rates of apoptosis were increased. On Day 6, the rates of apoptosis in IVF (4.8%) were significantly (P < 0.05) lower than those in SCNT embryos with pFFF (13.1%) and pAESC (16.6%). However, both total cell number and apoptosis in SCNT embryos with pFFF and pAESC revealed no significant differences. In Experiment 2, SCNT embryos with pFFF in different cell passages were compared for the development and apoptosis. No significant (P < 0.05) differences were observed in the cleavage rates of SCNT embryos among different cell passages. The rates of blastocyst formation were significantly (P < 0.05) higher in SCNT embryos with 5–7 passages than those with other numbers of passages (14% vs. 6–8%, respectively). Although total cell numbers of SCNT blastocysts did not differ among different cell passages, apoptosis rates were significantly (P < 0.05) higher when the number of cell passages was increased. These results suggest that fetal fibroblasts at 5–7 passages are ideal nuclear donor cells for obtaining high-quality porcine SCNT embryos. This work was supported by grant No. 1000520040020000 from Biogreen 21, Republic of Korea.

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