315 EFFICIENCY OF CHEMICAL OR ELECTRICAL ACTIVATION OF BOVINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 265
Author(s):  
M. P. Milazzotto ◽  
W. B. Feitosa ◽  
R. Simões ◽  
C. M. Mendes ◽  
M. E. O. A. Assumpção ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Embryo Quality ◽  
Calcium Influx ◽  
Polar Body ◽  
Chemical Activation ◽  
Electrical Activation ◽  
Activation Process ◽  
Oocyte Activation ◽  
Blastocyst Formation ◽  
First Polar Body

Activation of in vitro matured oocytes is essential for the success of nuclear transfer embryo production. Oocyte activation is promoted by the release of intracellular calcium and influx of extracellular ions, and can be chemically induced by calcium ionophores such as A23187 (CA) or ionomycin (IO). Electrical stimulation (EL) is an essential stage in nuclear transfer protocols for the fusion of enucleated oocytes with the donor's cell nucleus. Moreover, EL can be used as an alternative method to induce calcium influx through the formation of pores in the plasma membrane. This work aimed to evaluate the effect of electrical pulse vs the use of different calcium ionophores (A23187 or ionomycin) as primary agents of bovine oocyte activation, with or without the addition of BSA, on the rate of blastocyst formation and blastocyst quality. BSA was used to quench the activation process after a 5-min exposure to CA or IO. Cumulus-oocyte complexes were matured in TCM-199 medium with FCS and hormones for 18 h at 38.5�C and 5% CO2 in air. After removal of cumulus cells, oocytes presenting the first polar body were selected and maintained in SOFaa medium to complete 24 h of maturation. They were then divided into five treatments groups 1-CA (CA 5 mM, 5 min); 2-CAB (CA 5 mM, 5 min; BSA, 5 min); 3-IO (IO 5 mM, 5 min); 4-IOB (IO 5 mM, 5 min; BSA, 5 min); and 5-EL (EL 1.5 kV/cm, 20 �s, 2 pulses). After treatments, oocytes were kept in 6-dimethylaminopurine for 3 h and cultured in SOFaa medium for 7 days at 38.5�C and 5% CO2 in air. Rates of cleavage and blastocyst were evaluated respectively on Days 2 and 7 of culture. To evaluate embryo quality, Hoechst 33342/propidium iodide staining was used. Data were evaluated by ANOVA and submitted to LSD test for embryo rates and t-test for embryo quality. Four replicates were carried out with a total of 89 oocytes per treatment. There was a difference (P < 0.05) in rate of development to blastocyst between treatments 1-CA (54.4%a), 3-IO (51.4%a), and 5-EL (54.5%a) compared with 4-IOB (18.3%b). Treatment 2-CAB (39.8%ab) did not show any difference from the others. There was no difference (P > 0.05) among treatments in total number of cells: 1-CA (63.1a), 2-CAB (57.2a), 3-IO (60.9a), 4-IOB (72.4a), and 5-EL (58.4a). However, there was a difference (P < 0.01) in the percentage of viable cells between treatments 1-CA (49.9%a), 2-CAB (45.8%a), 3-IO (64.9%a), and 4-IOB (50.9%a) in comparison to 5-EL (82.7%b). In conclusion, BSA, when associated with IO, had a negative effect on embryonic developmental rates. The different calcium ionophores used and the BSA did not improve embryo quality. Although there were no significant differences between electrical and chemical activation on the rate of blastocyst formation, it is important to point out that higher quality embryos were achieved by using electrical activation. This work was supported by FAPESP 03/00156-9.

scholarly journals 303PARTHENOGENETIC DEVELOPMENT OF PIG OOCYTES BY CHEMICAL ACTIVATION

2004 ◽  
Vol 16 (2) ◽  
pp. 271
Author(s):  
C.S. Park ◽  
D.I. Jin ◽  
M.Y. Kim ◽  
Y.J. Chang ◽  
Y.J. Yi
Keyword(s):  
Cytochalasin B ◽  
Polar Body ◽  
Chemical Activation ◽  
Formation Rate ◽  
Penicillin G ◽  
Oocyte Activation ◽  
Blastocyst Formation ◽  
Parthenogenetic Development ◽  
First Polar Body

Efficient activation is essential for the success of animal cloning by nuclear transfer. The aim of this study was to investigate the effects of chemical activation agents on parthenogenetic development of pig oocytes matured in vitro. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Cumulus-free oocytes showing first polar body were selected for activation. Oocytes were activated as follows. First, all oocytes were activated with 25mM HEPES buffered NCSU-23 medium containing 8% ethanol for 10min. After that, in treatment 1, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B for 3h. In treatment 2, oocytes were incubated in the NCSU-23 medium supplemented with 10μgmL−1 cycloheximide for 3h. In treatment 3, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B for 1.5h, and then were incubated in the NCSU-23 medium supplemented with 10μgmL−1 cycloheximide for 1.5h. In treatment 4, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B plus 10μgmL−1 cycloheximide for 3h. Following activation, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA for further culture for 20 and 144h. Activated oocytes were fixed and stained for evaluation of activation rate, cleaved oocytes, blastocyst formation rate and cell numbers per blastocyst. Data were analysed by ANOVA and Duncan’s multiple range test using the SAS program. The rate of oocyte activation was higher in treatment 4 (62.1%) than in treatment 1, 2 and 3 (52.0, 49.6 and 58.0%, respectively). The percentage of cleaved oocytes was lower in treatment 1 and 2 (56.9 and 55.2%) than in treatment 3 and 4 (68.8 and 68.5%). The rate of blastocyst formation from the cleaved oocytes was higher in treatment 3 and 4 (19.8 and 22.0%) than in treatment 1 and 2 (12.1 and 11.7%). Mean cells per blastocyst were lowest in treatment 2 (21.2±0.9) compared to treatment 1, 3 and 4 (27.3±2.2, 30.4±3.8 and 30.9±3.4, respectively). In conclusion, cytochalasin B combined with cycloheximide was more efficient for parthenogenetic development of pig oocytes matured in vitro.

scholarly journals 304INFLUENCE OF ELECTRICAL STIMULUS ON ACTIVATION OF PIG OOCYTES MATURED IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 271
Author(s):  
Chang Sik Park ◽  
Dong Il Jin ◽  
Young June Chang ◽  
Moon Young Kim ◽  
Young Joo Yi
Keyword(s):  
Acetic Acid ◽  
Polar Body ◽  
Stimulus Frequency ◽  
Electrical Stimulus ◽  
Electrical Activation ◽  
Penicillin G ◽  
Oocyte Activation ◽  
Activation Rate ◽  
First Polar Body

Electrically induced activation of pig oocytes deserves particular attention for research on parthenogenesis. The aim of this study was to improve electrical activation of in vitro matured pig oocyte. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Cumulus-free oocytes involving first polar body were selected for activation. For electrical activation, oocytes were rinsed twice in 0.3M mannitol solution supplemented with 0.1mM CaCl2, 0.2mMMgCl2, 0.5mM HEPES and 0.01% BSA, and transferred to a chamber consisting of two electrodes 1mm apart which were overlaid with the same activation solution. Experiment 1 was conducted to investigate the effect of electrical pulse on oocyte activation. Oocytes were activated with DC pulses of 1.0, 1.5, 2.0 and 2.5kVcm−1 for 30, 60 and 90μs, respectively. Experiment 2 was carried out to investigate the effect of electrical stimulus frequency on oocyte activation. Oocytes were activated one, two and three times, with a DC pulse of 1.0kVcm−1 for 60μs. After activation, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA and cultured for 20h. Activated oocytes were fixed for 48h in 25% acetic acid (v:v) in ethanol at room temperature, and stained with 1% orcein (w:v) in 45% acetic acid (v:v) to examine pronucleus formation. Data were analyzed by ANOVA and Duncan’s multiple range test using the SAS program. The rate of activation was highest in the DC pulse of 1.0kVcm−1 for 60μs (75.1%) compared with the other durations and strengths (62.5–63.1%). Activation rate by electrical stimulus frequency was highest (76.0%) when oocytes were activated by a one-time pulse. In conclusion, the results suggested that electrical stimulus with a single DC pulse of 1.0kVcm−1 for 60μs might be more efficient than other strengths and durations for activation of pig oocytes.

218 AN EVALUATION OF IN VITRO MATURATION OOCYTE ACTIVATION METHODS FOR IMPROVING OVINE INTRACYTOPLASMIC SPERM INJECTION EFFICIENCY

2016 ◽  
Vol 28 (2) ◽  
pp. 240
Author(s):  
J. E. Hernández ◽  
Y. Ducolomb ◽  
S. Romo ◽  
R. Fierro ◽  
M. E. Kjelland ◽  
...  
Keyword(s):  
Polyvinyl Alcohol ◽  
Follicular Fluid ◽  
Polar Body ◽  
Chemical Activation ◽  
Calcium Ionophore ◽  
Oocyte Activation ◽  
Sperm Suspension ◽  
Maturation Medium ◽  
Frozen Semen ◽  
First Polar Body

Given previous low sperm decondensation rates and poor oocyte activation in sheep ICSI (10–20%), we evaluated activation techniques for IVM/ICSI. Incubations were performed in a 5% CO2 cell incubator at 38.5°C and saturated humidity. Sheep ovaries were collected at an abattoir and transported <3 h to the laboratory. Follicular fluid was aspirated from 2–8 mm follicles using an 18-gauge needle and syringe with 1 mL of modified Tyrode’s medium supplemented with 10 mM sodium lactate, 10 mM HEPES, and 0.1% polyvinyl alcohol (TL-HEPES-PVA, 7.3–7.4 pH), with 200 IU mL–1 heparin. Cumulus-oocyte complexes (COC) with compact cumulus mass and uniform cytoplasm were selected from the follicular fluid and washed 3× in 500-µL drops of maturation medium (TCM 199) with Earle’s salts and 26.2 mM sodium bicarbonate and l-glutamine with 0.1% polyvinyl alcohol, 0.91 mM sodium pyruvate, 3.05 mM d-glucose, 0.57 mM cysteine, and 10 ng mL–1 epidermal growth factor. Next, 500 µL of maturation medium with 0.5 μg mL–1 LH, 0.5 μg mL–1 FSH, and 10% (vol/vol) of FCS was placed in sterile 4-well plates with 20 to 30 COC/well and with mineral oil for 24 h incubation. The COC were placed in a 500-µL drop of TCM 199-HEPES (TCM 199-H) with 300 IU of hyaluronidase for 3 min and washed (3×) in TCM 199-H. Next, 20 to 30 oocytes were placed in 250-µL droplets of TCM 199-H under a microscope to identify the first polar body (PB). Oocytes with PB were placed in 100-µL droplets of modified Tris-buffered medium (mTBM) for 1 to 4 h of incubation. The groups formed were (1) control: oocytes manipulated as in ICSI but no injection, (2) false injection: oocyte pierced but no sperm insertion, (3) chemical activation (c-a): 7% ethanol (7%Et) × 5 min, (4) c-a: 50 µM calcium ionophore (CaI) × 10 min, (5) c-a: 5 µM ionomicine (Io) × 5 min, (6) ICSI, and (7) 7%Et × 5 min + ICSI. For ICSI, 2 straws of frozen semen from a proven ram were thawed and diluted 1 : 10 with TCM 199-H and 3 mg mL–1 BSA, and then centrifuged 3 min at 200 × g. The sperm pellet was diluted with 100 µL of TCM 199-H, and 2 mL of TCM 199-H was added to a 45° bent tube for a 1-h swim-up. Next, 500 µL of supernatant was diluted to 1 × 106 sperm mL–1 and 10 µL added to 10 µL of 10% polyvinylpyrrolidone (PVP). Five oocytes at a time were placed in a Petri dish with a 10-µL drop of TCM-199-H, with 1% gentamycin, 2% serum, and one 2-µL drop of sperm suspension-PVP. Groups of 10 to 20 oocytes were activated in 100-mL drops of respective chemical in TCM 199-H at 20 to 22°C. Oocytes were washed (3×) in mTBM and set in 200 mL of mTBM for 18 to 20 h of incubation. Oocytes were stained with 10 μg mL–1 Hoechst 33258 for 15 min to assess pronucleus formation. Pearson χ2 tests showed statistical differences (α = 0.05) among the groups (χ2 = 123.165, P < 0.001); for example, groups 1 and 7 (χ2 = 68.179, P < 0.001) and 6 and 7 (χ2 = 42.842, P < 0.001). Results (oocytes, percentage activated) for each group were (1) n = 151, 13.2%, (2) n = 78, 32%, (3) n = 393, 53.6%, (4) n = 350, 46.8%, (5) n = 78, 42.3%, (6) n = 200, 24.5%, and (7) n = 123, 60.9%. The highest percentage of oocyte activation was achieved using 7%Et × 5 min + ICSI.

scholarly journals First polar body morphology before ICSI is not related to embryo quality or pregnancy rate

2004 ◽  
Vol 19 (10) ◽  
pp. 2334-2339 ◽  
Author(s):  
P.M. Ciotti ◽  
L. Notarangelo ◽  
A.M. Morselli-Labate ◽  
V. Felletti ◽  
E. Porcu ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Embryo Quality ◽  
Polar Body ◽  
Body Morphology ◽  
First Polar Body

98 BLASTOCYST DEVELOPMENT RATE OF CLONED CAT EMBRYOS USING SERIAL CLONING

2007 ◽  
Vol 19 (1) ◽  
pp. 166
Author(s):  
X. J. Yin ◽  
H. S. Lee ◽  
E. G. Choi ◽  
X. F. Yu ◽  
B. H. Choi ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Second Generation ◽  
Assisted Reproductive Technologies ◽  
Polar Body ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Blastocyst Development ◽  
First Polar Body

Domestic cats are a useful research model to develop assisted reproductive technologies for the conservation of endangered felids. Previously, we produced cloned offspring derived from somatic cell nuclear transfer of ear skin fibroblasts obtained from a deaf, odd-eyed, male Turkish Angora. The aim of this study was to assess the cloning efficiency of the fibroblasts derived from a cloned cat. Fibroblast cell lines were established from 6-mm skin biopsies taken from a deaf, odd-eyed, male Turkish Angora and his clone. The protocol for nuclear transfer was described previously (Yin et al. 2005 Reproduction 129, 245–249). Briefly, cumulus cells were removed from the ova by gently pipetting them into TCM-199 supplemented with 0.1% hyaluronidase. The denuded oocytes were then cultured in TCM-199 supplemented with 0.2 �g mL-1 demecolcine for 1 h and placed into TCM-199 containing 5 �g mL-1 cytochalasin B and 0.2 �g mL-1 demecolcine. The first polar body and protruded chromatin plate were removed with a beveled micropipette. Micromanipulation was used to place a single donor cell nucleus into the perivitelline space of enucleated ova. The ovum-cell couplets were fused and pulse activated. The activated couplets were cultured in 500 �L of CRI medium supplemented with 0.3% BSA for 2 days. The cleaved embryos were cultured in CRII medium supplemented with 10% FBS for 5 days. The cleavage and blastocyst development rates were 38.5% and 3.5% for second generation cloned embryos. A total of 310 second generation cloned embryos were transplanted to 9 surrogates, and 2 pregnancies at 30 days were determined by ultrasonography. One pregnancy was aborted at 40 days of gestation; the second pregnancy continued. These results indicate that the serial cloning of a cat can be generated efficiently up until pregnancy. This work was supported by KOSEF (grant #M10525010001-05N2501-00110).

249 INTRACYTOPLASMIC SPERM INJECTION OF ELAND (TAUROTRAGUS ORYX) AND BONGO (TRAGELAPHUS EURYCERUS)ANTELOPE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 241 ◽  
Author(s):  
G. Wirtu ◽  
C. E. Pope ◽  
M. C. Gomez ◽  
A. Cole ◽  
D. L. Paccamonti ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Intracytoplasmic Sperm Injection ◽  
Transvaginal Ultrasound ◽  
Polar Body ◽  
Chemical Activation ◽  
Cumulus Cell ◽  
Calcium Ionophore ◽  
Male Gamete ◽  
Assisted Reproductive Technique ◽  
First Polar Body

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique applicable in cases of limited male gamete availability. Moreover, it bypasses barriers of the oocyte, thus avoiding poorly understood species-specific capacitation events affecting sperm–egg interaction. In the present study, we evaluated the application of conventional and piezo drill-assisted ICSI and whether subsequent chemical activation is required for initiating embryonic development in eland (Taurotragus oryx) and bongo (Tragelaphus eurycerus) oocytes. Oocytes were collected using transvaginal ultrasound-guided follicular aspiration after gonadotropin-induced ovarian stimulation and incubated in modified TCM-199 medium (Gomez et al. 2000 Reprod. Fertil. Dev. 12, 423) containing 10% FBS. After 3 to 24 h, the cumulus cell layers were removed either by repeated mouth-pipetting and/or by using hyaluronidase. Oocytes with an extruded first polar body were used for ICSI and the other oocytes were returned to culture and evaluated every six hours Piezo drill-assisted (Kimura and Yanagimachi 1995 Biol. Reprod. 52, 709) or conventional (Gomez et al.) ICSI were done as described previously using glass pipettes with internal tip diameters of 9–10 µm. We used frozen–thawed or freshly collected spermatozoa that were kept in HEPES-buffered Tyrode's medium (Gomez et al.) for up to 24 h. Four to 6 h after ICSI, 3 activation treatments were examined: (1) none; (2) 7% ethanol, 5 min; or (3) calcium ionophore (5 µM, 5 min) followed by DMAP (2 mM, 4 h). Then we cultured oocytes in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 at 38.5°C in one of 3 media: SOF, α-MEM, or CR1aa containing essential and nonessential amino acids and FBS. Fifty-three of 70 (76%) eland oocytes survived after piezo-ICSI, and 13 of 16 (81%) survived after conventional ICSI. For bongo oocytes, 27 of 30 (90%) survived piezo-ICSI and all (n = 8) survived after conventional ICSI. Table 1 outlines cleavage data on Day 2. Generally, embryonic development was arrested at about 10 cells. In summary, eland and bongo oocytes can survive both conventional and piezo drill-assisted ICSI. Activation treatments do not appear to be a prerequisite for initiating cleavage after ICSI in eland and bongo antelope oocytes. Table 1.Cleavage of eland and bongo antelope oocytes after conventional or piezo-ICSI and three activation treatments

40 TRICHOSTATIN A TREATMENT EFFECTS ON IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER CAT EMBRYOS DEPEND ON RECIPIENT CYTOPLASM SPECIES

2015 ◽  
Vol 27 (1) ◽  
pp. 113
Author(s):  
L. T. K. Do ◽  
Y. Sato ◽  
M. Taniguchi ◽  
T. Otoi
Keyword(s):  
Nuclear Transfer ◽  
Treatment Effects ◽  
Trichostatin A ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Control Group ◽  
Fluid Medium ◽  
First Polar Body ◽  
Bovine Oocytes

The developmental ability of interspecies somatic cell nuclear transfer (iSCNT) embryos decreases as the taxonomic distance between the donor and recipient species increases. Treatment of cat iSCNT embryos using bovine oocytes with 50 nM of trichostatin A (TSA) improves in vitro embryonic development (Wittayarat et al. 2013 Cell. Reprogram. 15, 301–308). This study investigated whether the TSA treatment effects differ between the development of cat iSCNT embryos reconstructed with porcine and bovine oocytes. Porcine and bovine cumulus-oocyte complexes were in vitro matured for 44 h and 24 h, respectively. After cumulus cell removal, enucleation was performed by aspiration of the metaphase II plate and the first polar body using a piezo-driven pipette. A cat fibroblast cell was then injected into cytoplasm of successfully enucleated oocyte. Reconstructed cybrids were electrically activated by a single 1.5 kV cm–1 pulse for 100 µs (pig-cat embryos), or a 2.3 kV cm–1 pulse for 30 µs (cow-cat embryos). Pig-cat and cow-cat embryos were cultured in porcine zygote medium (PZM)-5 and modified synthetic oviducal fluid medium (mSOF), respectively. After electrical activation, pig-cat and cow-cat embryos were cultured in medium supplemented with 5 µg mL–1 cytochalasin B + 50 nM TSA (TSA group) or without TSA (control group), and the cow-cat embryo medium was also supplemented with 10 µg mL–1 cycloheximide. After 2 h, TSA-treated pig-cat and cow-cat embryos were incubated in medium supplemented with TSA for 22 h, followed by 48 h incubation without TSA. Pig-cat and cow-cat control embryos were cultured in medium without TSA for 70 h after activation. Then, all pig-cat and cow-cat embryos were cultured in porcine blastocyst medium (PBM) or mSOF medium supplemented with 5% fetal bovine serum, respectively, for 5 additional days. Four to seven replicates were performed for each experiment. Data were analysed using Student's t-test. For pig-cat embryos, no difference was observed in cleavage rates between both groups, but development to the blastocyst stage was higher in the pig control group (n = 147, 8.0%) than that of pig TSA group (n = 131, 0.7%; P < 0.05). In contrast, development to the blastocyst stage in cow-cat embryos was not observed in the cow control group (n = 125, 0%), but it was observed in cow TSA group (n = 136, 3.7%). These results indicate that TSA treatment effects are species-specific, but those effects remain to be clarified.

scholarly journals Luteolin Orchestrates Porcine Oocyte Meiotic Progression by Maintaining Organelle Dynamics Under Oxidative Stress

2021 ◽  
Vol 9 ◽  
Author(s):  
Soo-Hyun Park ◽  
Pil-Soo Jeong ◽  
Ye Eun Joo ◽  
Hyo-Gu Kang ◽  
Min Ju Kim ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Blastocyst Formation ◽  
Cortical Granules ◽  
Expression Levels ◽  
Meiotic Progression ◽  
First Polar Body ◽  
Underlying Mechanisms

Increasing evidence has demonstrated that oxidative stress impairs oocyte maturation, but the underlying mechanisms remain largely unknown. Here, for the first time, we examined the antioxidant role of luteolin in meiotic progression and the underlying mechanisms. Supplementation of 5 μM luteolin increased the rates of first polar body extrusion and blastocyst formation after parthenogenetic activation, and the expression levels of oocyte competence (BMP15 and GDF9)-, mitogen-activated protein kinase (MOS)-, and maturation promoting factor (CDK1 and Cyclin B)-related genes were also improved. Luteolin supplementation decreased intracellular reactive oxygen species levels and increased the expression levels of oxidative stress-related genes (SOD1, SOD2, and CAT). Interestingly, luteolin alleviated defects in cell organelles, including actin filaments, the spindle, mitochondria, the endoplasmic reticulum, and cortical granules, caused by H2O2 exposure. Moreover, luteolin significantly improved the developmental competence of in vitro-fertilized embryos in terms of the cleavage rate, blastocyst formation rate, cell number, cellular survival rate, and gene expression and markedly restored the competencies decreased by H2O2 treatment. These findings revealed that luteolin supplementation during in vitro maturation improves porcine meiotic progression and subsequent embryonic development by protecting various organelle dynamics against oxidative stress, potentially increasing our understanding of the underlying mechanisms governing the relationship between oxidative stress and the meiotic events required for successful oocyte maturation.

62 DIFFERENT EFFECT OF TRICHOSTATIN A ON IN VITRO DEVELOPMENT OF PORCINE TRANSGENIC SOMATIC CELL NUCLEAR TRANSFER AND PARTHENOGENETICALLY ACTIVATED EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 112 ◽  
Author(s):  
H. X. Wei ◽  
K. Zhang ◽  
Y. F. Ma ◽  
Y. Li ◽  
Q. Y. Li ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Polar Body ◽  
Cell Number ◽  
Cell Numbers ◽  
First Polar Body ◽  
Electrical Fusion

Accumulating evidence suggests that trichostatin A (TSA), a histone deacetylase inhibitor, can increase the success rate of somatic cloning. The objective of this study was to investigate the effect of 50 nm TSA treatment on the development of porcine somatic cell nuclear transfer (SCNT) and parthenogenically activated (PA) embryos. Cumulus-oocyte complexes were matured in vitro. The oocytes with the first polar body (PB1) were chosen for SCNT, and the rest with PB1 or good morphology were selected for PA by a single 100-μs direct current pulse of 1.6 kV cm–1, the same parameter as for electrical fusion. GFP transgenic fetal fibroblast cells were used as nuclear donors. Data were analyzed using SPSS (13.0; SPSS, Inc., Chicago, IL, USA) with one-way ANOVA. In Experiment 1, immediately after electrical fusion and activation, the reconstructed embryos were randomly cultured in porcine zygote medium 3 (PZM3) with 10 μg mL–1 cytochalasin B (CB) and 10 μg mL–1 cycloheximide (CHX), with either 0 nm (control) or 50 nm TSA for the first 4 h, before being cultured for another 20 h in PZM3 without CB and CHX. After being washed, the embryos were cultured in PZM3 medium without TSA until Day 6 at 39.0°C, 5% CO2, 5%O2, 90% N2, and 100% humidity. The same experimental design was used for PA embryos concurrently. The results showed that there were no significant differences in blastocyst rates for SCNT or PA between control and TSA groups (23.0 ± 6.1% v. 27.9 ± 6.3%; 21.0 ± 1.0% v. 17.5 ± 3.2%, respectively). Neither were there differences in the cell numbers of blastocysts (38.3 ± 5.7 v. 32.2 ± 3.4; 42.2 ± 3.5 v. 39.0 ± 1.9, respectively). In Experiment 2, TSA treatment was prolonged to either 36 or 40 h. The blastocyst rates of SCNT were increased (7.3 ± 1.2% (0 h), 13.3 ± 2.6% (36 h), and 20.0 ± 3.3% (40 h)), whereas those of PA were decreased (46.7 ± 5.0% (0 h), 27.7 ± 6.5% (36 h), and 30.8 ± 6.3% (40 h)). The cell numbers of blastocysts from either SCNT or PA were also decreased (SCNT: 47.5 ± 3.8, 37.5 ± 2.0, and 37.1 ± 3.3; PA: 46.1 ± 1.9, 37.5 ± 1.9, and 39.3 ± 2.2; P < 0.05). In Experiment 3, the cell number and the apoptotic index of Day 5, 6, and 7 PA blastocysts treated with 0 or 50 nm TSA were determined by the terminal deoxynucleotide-mediated nick end labeling (TUNEL) assay (Table 1). The results suggested that TSA treatment probably delayed embryo development, which may be one of the reasons for the lower cell numbers in the TSA-treated group. Table 1. Cell apoptosis of PA blastocyst by TUNEL

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