Comparison of the effects of using standard and simple portable CO2 incubators on the in vitro developmental competence of bovine embryos reconstituted by somatic cell nuclear transfer

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.

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Overexpression of MicroRNA-29b Decreases Expression of DNA Methyltransferases and Improves Quality of the Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle

Microscopy and Microanalysis ◽

10.1017/s1431927618000016 ◽

2018 ◽

Vol 24 (1) ◽

pp. 29-37 ◽

Cited By ~ 4

Author(s):

Shuang Liang ◽

Zheng-Wen Nie ◽

Jing Guo ◽

Ying-Jie Niu ◽

Kyung-Tae Shin ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Cellular Proliferation ◽

Dna Methyltransferases ◽

Blastocyst Stage ◽

Developmental Competence ◽

Somatic Cell Reprogramming

AbstractMicroRNA (miR)-29b plays a crucial role during somatic cell reprogramming. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos, as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared within vitrofertilized embryos. In addition, miR-29b regulates the expression of DNA methyltransferases (Dnmt3a/3bandDnmt1) in bovine SCNT embryos. We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency and downregulation inhibits developmental potency. Nevertheless, the quality of bovine SCNT embryos at the blastocyst stage improved significantly. The expression of pluripotency factors and cellular proliferation were significantly higher in blastocysts from the miR-29b overexpression group than the control and downregulation groups. In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and downregulation groups. Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.

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Developmental Competence of Somatic Cell Nuclear Transfer Embryos Reconstructed from Oocytes Matured In Vitro with Follicle Shells in Miniature Pig

Cloning and Stem Cells ◽

10.1089/clo.2005.7.17 ◽

2005 ◽

Vol 7 (1) ◽

pp. 17-26 ◽

Cited By ~ 23

Author(s):

Yoichiro Hoshino ◽

Masaki Uchida ◽

Yoshiki Shimatsu ◽

Masashi Miyake ◽

Yasumitsu Nagao ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Developmental Competence ◽

Miniature Pig

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

Reproduction Fertility and Development ◽

10.1071/rd13283 ◽

2015 ◽

Vol 27 (3) ◽

pp. 544 ◽

Cited By ~ 6

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.

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Effect of glycosaminoglycans on the preimplantation development of embryos derived from in vitro fertilization and somatic cell nuclear transfer

Reproduction Fertility and Development ◽

10.1071/rd02054 ◽

2003 ◽

Vol 15 (3) ◽

pp. 179 ◽

Cited By ~ 25

Author(s):

Goo Jang ◽

Byeong Chun Lee ◽

Sung Keun Kang ◽

Woo Suk Hwang

Keyword(s):

Hyaluronic Acid ◽

In Vitro Fertilization ◽

Chondroitin Sulfate ◽

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Cell Number ◽

Developmental Competence ◽

Vitro Fertilization

The purpose of this study was to evaluate the effect of glycosaminoglycans (GAGs) added to the culture medium on the developmental competence of bovine embryos derived from in vitro fertilization (IVF) and from somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were either inseminated with 1 × 106 spermatozoa mL−1 or enucleated and reconstructed with bovine adult ear fibroblasts by SCNT. The embryos were then cultured in modified synthetic oviduct fluid (mSOF) containing 8 mg mL−1 bovine serum albumin (BSA) (control mSOF) or control mSOF supplemented with various GAGs (hyaluronic acid, heparin or chondroitin sulfate) in a dose-dependent manner (0.1, 0.5 or 1.0 mg mL−1). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos, 8–16-cell embryos and blastocysts. The mean cell number of flattened blastocysts stained with 5 μ M bisbenzimide on Day 8 was counted. The percentage of blastocyst formation (IVF and SCNT embryos) from cleaved embryos was significantly higher (P < 0.05) in control mSOF supplemented with 0.5 mg mL−1 hyaluronic acid (45% and 47%), heparin (40% and 47%) or chondroitin sulfate (38% and 44%) compared with control mSOF (30–31% and 30–33%). When compared with the efficacy of 0.5 mg mL−1 GAGs, no significant differences were observed in the developmental competence of both IVF and SCNT embryos. Supplementing control mSOF with 0.5 mg mL−1 GAGs had no effect on the cell number of IVF embryos. In contrast, supplementing 0.5 mg mL−1 of hyaluronic acid, heparin or chondroitin sulfate to control mSOF significantly (P < 0.05) increased the numbers of total cells (93–98 v. 88 cells) and trophectoderm (TE) cells (64–66 v. 55 cells), and decreased the inner cell mass (ICM) to TE cell ratio (48.2–49.8 v. 61.3) in SCNT blastocysts compared with embryos in control mSOF. In conclusion, supplementation of culture media with GAGs may improve the development of bovine IVM–IVF and SCNT embryos to the blastocyst stage. The GAGs increased the quality of blastocysts by increasing total cell numbers in the SCNT embryos.

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Effect of season on the in-vitro maturation and developmental competence of buffalo oocytes after somatic cell nuclear transfer

Environmental Science and Pollution Research ◽

10.1007/s11356-019-07470-3 ◽

2020 ◽

Vol 27 (7) ◽

pp. 7729-7735 ◽

Cited By ~ 3

Author(s):

Hai-Ying Zheng ◽

Chun-Yan Yang ◽

Nong-Qi Yu ◽

Jia-Xiang Huang ◽

Wei Zheng ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

In Vitro Maturation ◽

Developmental Competence

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126 EFFECTS OF THE REPRODUCTIVE STATUS ON DEVELOPMENTAL COMPETENCE OF RECIPIENT OOCYTES AFTER SOMATIC CELL NUCLEAR TRANSFER IN CAT

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab126 ◽

2005 ◽

Vol 17 (2) ◽

pp. 213

Author(s):

T. Otoi ◽

N.W.K. Karja ◽

M. Fahrudin ◽

B. Agung ◽

P. Wongsrikeao

Keyword(s):

Somatic Cell ◽

Reproductive Cycle ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Calf Serum ◽

Reproductive Status ◽

Developmental Competence ◽

Ionophore A23187 ◽

Short Period

The reproductive status of donor cat has been suggested to influence developmental competence of the oocytes after IVM/IVF (Karja et al. 2002 Theriogenology 57, 2289–2298). This study was conducted to examine the effect of the reproductive cycle stage of cat ovaries supplying recipient oocytes for nuclear transfer (NT) on the developmental competence of the oocytes after somatic cell nuclear transfer. Cat ovaries were collected at local veterinary clinics and stored at 35°C for a short period (1–6 h). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into the inactive, follicular or luteal stages. Cumulus-oocyte complexes (COCs) were obtained from ovaries at each stage of the reproductive cycle by mincing/dissection and matured in vitro for 24 h, as previously described (Karja et al. 2002 Theriogenology 57, 2289–2298). In vitro matured oocytes from ovaries at the inactive (n = 114), follicular (n = 124), and luteal (n = 126) stages were mechanically enucleated in PBS supplemented with 5 μL mL−1 of cytochalasin B and 3 mg mL−1 BSA, and reconstructed with fibroblast cells derived from uterus tissue. The couplets were fused in Zimmerman medium with a single DC pulse of 1.5 kV cm−1 for 50 μs. The successfully fused couplets were activated by a 5-min exposure to 10 μg mL−1 calcium ionophore A23187 in MK1 medium (Kanda et al. 1998 J. Vet. Med. Sci. 60, 423–431) followed by 5 h of incubation in MK1 medium supplemented with 10 μg mL−1 cycloheximide. The NT embryos were cultured in MK1 medium supplemented with 4 mg mL−1 BSA at 38.0°C in a humidified atmosphere of 5% CO2 in air. At 72 h of culture, all cleaved NT embryos were transferred to fresh MK1 medium supplemented with 5% fetal calf serum for an additional 4 days to evaluate their ability of development to the blastocyst stage. Data were analyzed by ANOVA. There were no significant differences (P > 0.05) among the fused oocytes derived from ovaries at the inactive, follicular, and luteal stages with respect to the percentages of cleavage (64.4%, 69.4%, and 74.5%, respectively) and blastocyst formation (17.4%, 21.0%, and 12.0%, respectively). These results indicate that the reproductive cycle stage of cat ovaries has no apparent effect on the development at competence of recipient oocytes after somatic cell nuclear transfer.

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

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab79 ◽

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.

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

Zygote ◽

10.1017/s0967199417000272 ◽

2017 ◽

Vol 25 (4) ◽

pp. 453-461 ◽

Cited By ~ 5

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.

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