Caffeine and dithiothreitol delay ovine oocyte ageing

2010 ◽  
Vol 22 (8) ◽  
pp. 1254 ◽  
Author(s):  
Xiao-Fang Ye ◽  
Shi-Bin Chen ◽  
Li-Qin Wang ◽  
Yun-Cheng Zhao ◽  
Xue-Feng Lv ◽  
...  
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Limited Extent ◽  
Blastocyst Formation ◽  
Parthenogenetic Activation ◽  
Cell Numbers ◽  
Reconstructed Embryos ◽  
Intracellular Glutathione ◽  
Post Onset

The intracellular glutathione levels and developmental competence of aged oocytes after parthenogenetic activation, somatic cell nuclear transfer and intracytoplasmic sperm injection in the presence or absence of caffeine or dithiothreitol (DTT) were examined. The following results were found: (1) ovine oocytes were fully aged 30 h post-onset of maturation culture; (2) the appropriate concentrations of caffeine and DTT for oocyte culture were 5 mM and 1 mM, respectively; (3) when nuclear transfer-reconstructed embryos were treated with caffeine or DTT following fusion, no increase in the frequency of development to blastocyst was observed (P > 0.05), but the cell numbers of blastocysts increased (P < 0.05); (4) both caffeine and DTT increased the blastocyst formation rates of intracytoplasmic sperm-injected embryos (P < 0.05); (5) caffeine increased the glutathione content of aged oocytes (P < 0.05). The glutathione content of DTT-treated aged oocytes was higher than that of oocytes matured for 36 h (P < 0.05). In conclusion, caffeine and dithiothreitol delay oocyte ageing but only to a limited extent.

Parthenogenetic activation and somatic cell nuclear transfer of porcine oocytes activated by an electric pulse and AZD5438 treatment

Zygote ◽  
2017 ◽  
Vol 25 (4) ◽  
pp. 453-461 ◽  
Author(s):  
Xiao-Chen Li ◽  
Qing Guo ◽  
Hai-Ying Zhu ◽  
Long Jin ◽  
Yu-Chen Zhang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Electric Pulse ◽  
Formation Rate ◽  
Developmental Competence ◽  
Oocyte Activation ◽  
Blastocyst Formation ◽  
Parthenogenetic Activation

SummaryWe examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P < 0.05). The blastocyst formation rate was higher in the group treated with AZD5438 for 4 h than in the groups treated with AZD5438 for 2 or 6 h (42.8% vs. 38.6% and 37.2%, respectively; P > 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.

Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

2018 ◽  
Vol 30 (10) ◽  
pp. 1342 ◽  
Author(s):  
Zhao-Bo Luo ◽  
Long Jin ◽  
Qing Guo ◽  
Jun-Xia Wang ◽  
Xiao-Xu Xing ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Abnormal Development ◽  
Control Group ◽  
Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

41 STUDY ON THE ESTABLISHMENT OF SOMATIC CELL NUCLEAR TRANSFER USING IN VIVO-MATURED OOCYTES IN DOGS

2006 ◽  
Vol 18 (2) ◽  
pp. 129 ◽  
Author(s):  
G. Jang ◽  
M. Kim ◽  
H. J. Oh ◽  
F. Y. Heru ◽  
M. S. Hossein ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Chemical Activation ◽  
Developmental Competence ◽  
Perivitelline Space ◽  
Parthenogenetic Activation ◽  
Vaginal Cytology

The present study was performed to collect in vivo matured canine oocytes for somatic cell nuclear transfer (SCNT) and to investigate the developmental competence of canine parthenogenetic and SCNT embryos as the preliminary research for producing cloned dog. The day of ovulation as described by Hase et al. (2000 J. Vet. Med. Sci. 62, 243-248) was determined by serum progesterone levels and at that time vaginal cytology was performed to assess the cornified index. In vivo-matured oocytes were recovered by retrograde flushing of the oviducts at around 48 h (n = 20) or 72 h (n = 25) after the estimated time of ovulation. Overall size of each oocyte, as well as ooplasmic diameter, zona pellucida thickness, and perivitelline space width, was determined after removing the cumulus cells by pipetting (Exp. 1). To determine activation protocols, two treatments, (1) chemical activation (10 �M Ca ionophore for 4 min, followed by incubation for 4 h with 1.9 mM 6-dimethylaminopurine) and (2) electrical stimulation (3.1?3.4 kV/cm in 0.25M mannitol solution), were evaluated to induce parthenogenetic activation of oocytes (Exp. 2). Donor cells were obtained from the primary cell culture of a canine ear skin biopsy, and SCNT was performed according to our laboratory procedures (Jang et al. 2004 Theriogenology 62, 512-521). Three voltages (1.7?2.0 kV/cm, 2.1-2.4 kV/cm, and 3.1-3.4 kV/cm) were tested for fusion. The fused couplets were subjected to chemical or electrical stimulation as in parthenogenetic activation and in vitro developmental competence was monitored (Exp. 3). As a result, more in vivo-matured canine oocytes were obtained at 72 h (92%) than at 48 h (15%) after ovulation; the 72-h occytes had progesterone concentrations of 4-8 ng/mL and a cornified index (vaginal cytology) of 83.34. The average number of oocytes recovered was 12 and sizes of ooplasmic diameter, cytoplasm, zona pellucida, and perivitelline space in in vivo canine-matured oocytes (n = 120) were 178.8 � 9.3 �m, 125.0 � 8.2 �m, 21.7 � 3.7 �m, and 12.7 � 3.5 �m, respectively. Parthenogenetically activated oocytes developed to the 16-cell and morula stages, but failed to develop to the blastocyst stage. Among the three voltages, in the highest voltage (75.2%) the number of fused couplets was increased compared to either of the other voltages (33.3% and 44.0%). Cleavage rates (60.9% vs. 58.0%) of cloned embryos were not significantly affected by method of activation. In terms of in vitro developmental competence, cloned embryos developed to the 16-cell or morula stage in vitro after electrical or chemical activation, respectively. In conclusion, in the present study we demonstrated that measurement of progesterone levels, in combination with evaluation of vaginal cytology, can be used to determine the estimated time of ovulation in bitches. In addition, we determined fusion/activation protocols that resulted in in vitro development of a portion of parthenogenetically activated and cloned embryos to the 16-cell and morula stages. This study was supported by grants from the Biogreen 21-1000520030100000.

Selection of pre- versus postpubertal pig oocytes for parthenogenetic activation and somatic cell nuclear transfer

2015 ◽  
Vol 27 (3) ◽  
pp. 544 ◽  
Author(s):  
H. S. Pedersen ◽  
Y. Liu ◽  
R. Li ◽  
S. Purup ◽  
P. Løvendahl ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Oocyte Growth ◽  
Parthenogenetic Activation ◽  
Selection Of

Pig oocytes have been used increasingly for in vitro production techniques in recent years. The slaughterhouse-derived oocytes that are often used are mostly of prepubertal origin. The aims of the present study were to compare the developmental competence between pre- and postpubertal pig oocytes, and to develop a simple and practical method for the selection of prepubertal pig oocytes for parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) based on oocyte morphology after IVM and oocyte inside zona pellucida (ZP) diameter (‘small’ ≤110 µm; ‘medium’ >110 µm; ‘large’ ≥120 µm). Meiotic competence and blastocyst rates after PA and SCNT of prepubertal oocytes increased with oocyte size, with the large prepubertal oocytes reaching a level similar to postpubertal oocytes after SCNT. Blastocyst cell number was not related to oocyte inside ZP diameter and oocyte donor to the same extent as blastocyst rate. Very low blastocyst rates were obtained after PA of morphologically bad pre- and postpubertal oocytes. In conclusion, measurement of inside ZP diameter combined with morphological selection is useful to remove incompetent oocytes. Further studies are needed to clarify the relative importance of cytoplasmic volume and stage in oocyte growth phase.

79 DEVELOPMENTAL COMPETENCE OF OVINE OOCYTES VITRIFIED AT GERMINAL VESICLE STAGE: IN VITRO FERTILIZATION, PARTHENOGENETIC ACTIVATION, AND SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 145
Author(s):  
A. R. Moawad ◽  
I. Choi ◽  
J. Zhu ◽  
K. H. S. Campbell
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Control Groups ◽  
High Frequencies ◽  
Parthenogenetic Activation ◽  
And Control

Oocyte cryopreservation represents an important development in the field of assisted reproductive technologies. This study investigated the effects of vitrification on spindle morphology following subsequent in vitro maturation (IVM), cleavage, and development following IVF and parthenogenetic activation. The developmental competence of ovine oocytes vitrified at the germinal vesicle (GV) stage, matured, and used as cytoplast recipients for somatic cell nuclear transfer (SCNT) was also determined. Cumulus–oocyte complexes obtained at slaughter were divided into 3 groups: 1) untreated (control), 2) toxicity (exposed to vitrification solutions without freezing), and 3) vitrified (2008 Reprod. Fertil. Dev. 20, 122). At 24 hpm (hours post onset of maturation), oocytes were subjected to 1) immunostaining, 2) IVF, or 3) activation by 2 different protocols [calcium ionophore, cycloheximide, and cytochalasin B (CA+CHX/CB), or strontium and CB (Sr/CB)]. The SCNT was performed as previously described (2010 Reprod. Fertil. Dev. 22, 1000–1014). Presumptive zygotes were cultured in vitro for 7 days. No significant differences (P > 0.05; chi-square) were observed in the frequencies of oocytes with normal spindle configuration between vitrified, toxicity, and control groups (50.0, 54.9, and 70.4%, respectively). Cleavage 24, 48 hpi, and morula development (5 days pi) were significantly decreased (P < 0.01) in the vitrified group (17.3, 42.9, and 36.4%) compared with toxicity (47.0, 85.3, and 60.7%) and control (68.9, 89.7, and 62.6%) groups. Blastocyst development significantly decreased (P < 0.01) in the vitrified group (12.3%) compared with toxicity (42.7%) and control (40.4%) groups. Based on cleaved embryos, no significant difference was observed between vitrified and control groups (29.4 v. 45.1%). Post-activation, cleavage 24 hpa (hours post-activation, 6.2 v. 3.8%) and 48 hpa (28.4 v. 27.5%) was significantly lower (P < 0.05) in vitrified oocytes activated by (CA+CHX/CB and Sr/CB) than other groups. No blastocyst developed from vitrified oocytes activated by CA+CHX/CB; however, 3.8% developed from Sr/CB oocytes. This was significantly (P < 0.05) lower than toxicity and control (20.0 and 27.3%) groups. Following SCNT, high frequencies of enucleation (99%) and fusion (98%) were achieved in vitrified and control groups. Cleavage 24 and 48 hpa significantly decreased (P < 0.05) in the vitrified group (31.0 and 48.0%) compared with the control (55.1 and 85.0%). No significant differences were observed in morula (38.0 v. 46.7%) and blastocyst (13.0 v. 23.4%) development. The proportion of cleaved embryos that developed to blastocyst stages was similar in both groups (27.0%). No significant differences (t-test) were observed in total cell numbers, apoptotic nuclei, and proportion of diploid embryos. In conclusion, ovine oocytes vitrified at GV stage can be matured, fertilized, and develop in vitro with high developmental potential. Strontium can be used effectively for activation of vitrified/thawed ovine oocytes. Vitrified/thawed ovine oocytes were used successfully for the first time as recipient cytoplasts for SCNT and produced high frequencies of good-quality blastocyst stage embryos.

61 EFFECT OF ROOM TEMPERATURE HOLDING PROCEDURE ON ABILITY OF OOCYTES TO MATURE AND DEVELOP IN VITRO AFTER EQUINE SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 136 ◽  
Author(s):  
K. Song ◽  
J. Lee ◽  
J. Park ◽  
W. Lee ◽  
Y. Chun ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Room Temperature ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Maturation Rate ◽  
Vitro Development

In Korea, it takes time to transport the ovaries of mares to the laboratory because horses are slaughtered only on Jeju island. Also, initiation of in vitro maturation (IVM) may be a little more delayed because of the oocyte collection by scraping of the follicular wall. It was reported that holding procedure of equine oocytes before IVM did not affect the developmental competence after intracytoplasmic sperm injection (Choi et al. 2006 Theriogenology 66, 955–963). The aims of present study were 1) to investigate the meiotic competence of equine oocytes held before IVM according to the type of oocytes, and 2) to examine the in vitro development after somatic cell nuclear transfer (SCNT). Cumulus–oocyte complexes (COCs) were recovered by scraping and washing of the follicular wall with Dulbecco’s modified Eagle medium (D-MEM) supplemented with 0.05% PVA, and classified as compact (Cp) or expended (Ex) depending on the expansion of cumulus or granulosa cells. 2 types of IVM procedures were compared: 1) COCs were matured immediately in IVM medium (TCM-199 supplemented with 5 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS) at 38.5°C under 5% CO2 in air for 24 to 27 h, and then held in holding medium (40% TCM-199 with Earle’s salts, 40% TCM-199 with Hanks’ salts, and 20% FBS) at room temperature for 6 to 7 h (control); or 2) COCs were initially held in holding medium for 6 to 7 h, and then matured in IVM medium for 24 to 27 h (holding). For SCNT, matured oocytes (pooled) were enucleated and electrically fused with equine skin fibroblasts (2.25 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 2 mM 6-DMAP, and cultured in D-MEM supplemented with 10% FBS and 50 ng mL–1 EGF at 38.5°C under 5% CO2, 5% O2, and 90% N2 for 7 to 9 days. Cleavage and blastocyst formation were evaluated on Days 2 and 8, respectively. All analyses were performed using SAS (version 9.1; SAS Institute Inc., Cary, NC, USA). 4 replicates were conducted from May to June 2010. In Ex oocytes, the maturation rate of the holding group (71.4%; 10/16) was not different from that of the control (65.6%; 44/73), and the rate of degenerated oocytes (4.8%; 1/16) in the holding group was not different from that in the control (5.6%; 5/73). However, in Cp oocytes, the degeneration rate of the holding group (65.0%; 31/49) was higher (P < 0.001) than that of the control (28.4%; 23/83), and the maturation rate of the holding group (20.6%; 12/49) was slightly lower (P = 0.07) than that of the control (46.0%; 38/83). After SCNT, the cleavage rate of the holding group (66.7%; 8/9) was not different from that of the control (60.8%; 14/25), and the rates of blastocyst formation of the control and the holding group were 8.1% (2/25) and 16.7% (2/9), respectively. Although the holding procedure may influence to the degeneration of Cp oocytes, it is considered that the developmental competence of equine oocytes held before IVM is not affected after SCNT.

60 EFFECTS OF REPETITIVE IONOMYCIN TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER (SCNT) EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 188
Author(s):  
S. H. Jeong ◽  
S. Kim ◽  
M. K. Choi ◽  
J. M. Na ◽  
J. Choi ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Essential Element ◽  
Developmental Competence ◽  
P Value ◽  
Oocyte Activation ◽  
Reconstructed Embryos ◽  
Group 3

In bovine somatic cell nuclear transfer (SCNT), oocyte activation is an essential element for the successful development of cloned embryos. Chemical treatment with ionomycin induces transient levels of Ca2+, and this has been used in the activation of reconstructed embryos. In vitro-fertilized oocytes are naturally activated by sperm following several Ca2+ transients known collectively as Ca2+ oscillations. We aimed to improve the developmental efficiency of embryos produced by SCNT by mimicking this Ca2+ oscillation artificially. SCNT was performed as follows; bovine calf skin fibroblast cells were transferred into the perivitelline space of IVM oocytes, and then these NT couplets were treated with electrical fusion (2 pulses, 1.75 kV cm-1, 15 μs). Reconstructed embryos were subsequently cultured in SOF medium (5% CO2, 5% O2, and 38°C). To mimic Ca2+ oscillation, we carried out 3 different repetitive ionomycin (10 μM) treatments at 1-h intervals. There were 3 groups: group 1 (4 min, 1 time), group 2 (30 s, 4 times), and group 3 (1 min, 4 times). The difference in embryo development amongst these experimental groups was then analyzed using the one-way ANOVA test after arcsine transformation to maintain homogeneity of variance. All analyses were performed using SAS (version 8.1, SAS Institute, Cary, NC, USA). Significant differences among the treatments were determined when the P-value was <0.05. In experiment 1, to assess developmental efficiency, the cleavage rate was investigated on Day 2 and the formation rate of blastocysts (BL) was examined on Day 7. In group 3, a significant increase in BL formation was observed [47/263 (17.8%), 50/259 (19.3%), and 67/258 (26.0%), respectively]. In experiment 2, culturing each group of embryos with different ionomycin treatments caused no significant differences among the groups in terms of the total number of BL (164.3, 158.5, and 145.1, respectively). In experiment 3, expression of apoptosis-related genes in each group was evaluated by real-time PCR and the TUNEL assay. The 3 BL within each group were analyzed for the expression of apotosis-related genes. Expression of the anti-apoptotic Bcl-2 gene was increased in group 3, whereas the expression of the pro-apoptotic Bax was decreased. A decrease in the number of apoptotic nuclei was also observed in group 3. In conclusion, the present study demonstrated that repetitive ionomycin treatment is an improved activation method that can increase the developmental competence of SCNT embryos by decreasing the incidence of apoptosis.

67 PIG OOCYTES WITH A LARGE PERIVITELLINE SPACE MATURED IN VITRO HAVE GREATER DEVELOPMENTAL COMPETENCE AFTER PARTHENOGENESIS AND SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 139
Author(s):  
J. You ◽  
N. Kim ◽  
S. Kang ◽  
E. Lee
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Separation Procedure ◽  
Perivitelline Space ◽  
Blastocyst Formation ◽  
Significant Difference

The size of perivitelline space (PVS) is closely related with the frequency of polyspermic fertilization in pig oocytes. It has been reported that enlargement of PVS is attributed to accumulation of glycoproteins synthesised and secreted from cumulus cells and that culture of immature oocytes in low-salt medium enlarges PVS in pigs. This study examined the developmental competence of pig oocytes after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in relation to the size of the PVS of oocytes matured in vitro (IVM). Cumulus–oocyte complexes were matured in medium 199 (Experiment 1) or porcine zygote medium (PZM)-3 (Experiment 2) supplemented with pig follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then cultured in hormone-free medium for an additional 22 h. IVM oocytes were activated electrically for PA or used as recipient cytoplasts for SCNT. PA and SCNT embryos were cultured for 7 days in PZM-3 medium supplemented with bovine serum albumin. The intracellular glutathione (GSH) level in IVM oocytes was determined by analysing the fluorescence intensity of oocytes after staining with CellTracker Blue CMF2HC. The expression of CDK1, PCNA, and ERK2 mRNA in IVM oocytes was analysed by RT-PCR. Data were analysed using a general linear model procedure followed by the least significant difference mean separation procedure when the treatments differed at P < 0.05. In Experiment 1, oocytes with a larger PVS had higher (P < 0.05) levels of intracellular GSH (1.0 pixels/oocyte v. 0.6 pixels/oocyte) and blastocyst formation (54% v. 37%) after PA than oocytes with smaller PVS. In Experiment 2, maturation culture of oocytes in PZM-3 with reduced (61.6 mM) NaCl concentration significantly increased (P < 0.05) the size of the PVS (5.2 μM v. 3.3 μM) compared with control oocytes that were matured in PZM-3 containing 108 mM NaCl, although the treatment did not alter the nuclear maturation. Moreover, oocytes with increased PVS expressed more CDK1, PCNA, and ERK2 mRNA and had higher (P < 0.05) intracellular GSH levels (1.6 pixels/oocyte v. 1.2 pixels/oocyte) and increased blastocyst formation after PA (52% v. 41%) and SCNT (32% v. 18%) compared with control oocytes. Our results demonstrate that pig oocytes with a large PVS have greater developmental competence after PA and SCNT, which is attributed to improved cytoplasmic maturation resulting from the enhanced GSH level and transcription factor expression and that enlargement of PVS by the culture in low-NaCl medium also improves developmental competence of pig oocytes. This work was supported by grants (#20070301034040 and #20080401034072) from the BioGreen 21 Program (Rural Development Administration, Republic of Korea).

51 SCRIPTAID AND MG132 IMPROVE DEVELOPMENT OF PORCINE EMBRYOS RECONSTRUCTED BY SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 131
Author(s):  
J. Mao ◽  
L. Tracy ◽  
J. Zhao ◽  
K. M. Whitworth ◽  
L. Spate ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
National Institutes Of Health ◽  
Developmental Competence ◽  
Large Animal ◽  
Blastocyst Formation ◽  
Treatment Groups ◽  
Deacetylase Inhibitor

Nuclear transfer efficiency in pigs and other large animal species is low. Previous studies have shown that histone deacetylase inhibitor (Scriptaid) and proteasomal inhibitor (MG132) treatment of somatic cell nuclear transfer (SCNT) pig embryos enhances blastocyst formation and pregnancy. The current experiment was carried out to determine the effects of combined MG132 and Scriptaid treatment on early development of cloned pig embryos reconstituted by SCNT. A total of 328 sow oocytes procured from ART (Madison, WI, USA) were reconstructed using α-1,3-Galactosyltransferase knockout hDAF transgenic pig fetal-derived fibroblast cells. Immediately after electrofusion and activation, SCNT oocytes were treated with 0, 1, or 10 μM MG132 for 2 h and then treated with 500 nM Scriptaid for another 16 h. The SCNT embryos were washed and cultured in Porcine Zygote Medium 3 for 7 days. Percent cleavage was determined on Day 2, and blastocyst formation and cell number were determined on Day 7. The experiment was repreated 8 times. There was no difference (P > 0.05) in percent cleavage (57.9 to 66.7%), or cell number (25.5 to 30.6) among the 3 groups. Interestingly, while there was no difference in the percent blastocyst between the 1 μM and 0 μM MG132 treatment groups, more oocytes from the 1 μM MG132 group developed into blastocysts than in the 10 μM MG132 group (25.1 ± 4.6% v. 12.9 ± 3.3%; P = 0.045). Further research will be conducted to transfer these embryos to surrogate gilts to determine the true developmental competence of these embryos. Supported by the National Institutes of Health National Center for Research Resources (RR018877 and RR013438), and Food for the 21st Century.

In vitro oocyte maturation in a medium containing reduced sodium chloride improves the developmental competence of pig oocytes after parthenogenesis and somatic cell nuclear transfer

2017 ◽  
Vol 29 (8) ◽  
pp. 1625
Author(s):  
Joohyeong Lee ◽  
Hanna Lee ◽  
Yongjin Lee ◽  
Bola Park ◽  
Fazle Elahi ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Oocyte Maturation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Reduced Glutathione ◽  
Developmental Competence ◽  
Hypotonic Medium ◽  
Blastocyst Formation ◽  
Nuclear Maturation

The present study investigated the effects of IVM in hypotonic medium containing reduced (61.6 mM) NaCl compared with isotonic medium containing 108.0 mM NaCl (designated L and N respectively) on oocyte maturation and embryonic development in pigs. IVM culture was divided into four periods at 11-h intervals. Oocytes cultured in N for 33 h and then in L for 11 h of IVM (N-N-N-L) showed significantly improved (P < 0.05) nuclear maturation of oocytes (75.4–79.0% vs 60.2–85.8%) and blastocyst formation (61.5–66.1% vs 45.2–67.5%) after parthenogenesis (PA) compared with other treatments (L-L-L-L, L-L-L-N, L-L-N-L, N-N-L-L, N-N-L-N, L-L-N-L, L-N-N-L and N-L-N-L). Oocytes matured in L-L-L-L and N-N-N-L had an increased (P < 0.05) perivitelline space (11.0–12.5 vs 5.5 µm) and intraoocyte reduced glutathione (GSH) content (1.39–1.41 vs 1.00 pixels per oocyte) relative to oocytes matured in N-N-N-N. Somatic cell nuclear transfer (SCNT) embryos derived from the N-N-N-L treatment had significantly (P < 0.05) higher blastocyst formation (53.5%) than embryos derived from Medium-199 (37.4%) and N-N-N-N (41.8%) treatments. Overall, the results demonstrate that maturation of pig oocytes in hypotonic medium with reduced NaCl during the last 11 h of IVM increases the developmental competence of oocytes after PA and SCNT by improving the cytoplasmic microenvironment, including an increased GSH content in IVM oocytes.

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