Aberrant Expression Patterns of Genes Involved in Segregation of Inner Cell Mass and Trophectoderm Lineages in Bovine Embryos Derived from Somatic Cell Nuclear Transfer

Despite advances in the production of somatic cell nuclear transfer (SCNT) embryos, significant embryo losses are persistent, particularly around implantation. Malformations of the placenta and in a variety of organs are seen, and have been linked to deviant epigenetic reprogramming. The aim of the present study was to examine the formation of germ layers, which are prerequisites for formation of the embryo proper and placenta, in invivo-derived (in vivo), partly in vitro-cultured (IVC), and SCNT ovine embryos. Embryos were derived as follows: In vivo embryos (n = 27) were flushed from the uterus on Days 7, 9, 11, and 13. For IVC embryos (n = 22) in vivo zygotes were flushed, followed by culture in the presence of 20% human serum, transfer to the uterus on Day 6, and flushing as in vivo embryos. SCNT embryos (n = 41) were produced by fusion of serum starved granulosa cells with enucleated oocytes, followed by activation, culture in SOF, transfer to the uterus on Day 6, and flushing as described for in vivo embryos. Recovered embryos were processed for light microscopy (LM) and transmission electron microscopy (TEM), and paraffin sections were immunohistochemically labelled for the germ layers: alpha-1-fetoprotein for potential endoderm, cytokeratin-8 for potential ectoderm, and vimentin for potential mesoderm. A consistent delay of the IVC and particularly the SCNT embryos was noted throughout all time points: On Days 7 and 9, differentiation of the inner cell mass into hypoblast and epiblast was evident in 7 out of 12 in vivo embryos, whereas this phenomenon was less prominent or absent in 9 out of 13 IVC and 13 out of 15 SCNT embryos. Furthermore, 6 of the IVC and 12 of the SCNT embryos lacked an identifiable embryonic disc. On Day 11, half of the in vivo embryos had initiated gastrulation, evidenced by localization of endoderm and mesoderm precursor cells between the hypoblast and the epiblast. This feature was noted in only a single IVC and in none of the SCNT embryos. On Day 13, all in vivo embryos had completed gastrulation including the formation of somatic and visceral mesoderm. This feature was noted in only 1 out of 3 IVC and in none of the SCNT embryos. Likewise, amniotic folds were seen in one third of the in vivo embryos at this stage, but not observed in any IVC or SCNT embryos. The immunohistochemical markers displayed the same cell lineage localization in all three groups of embryos, but a developmental delay in the IVC and in particular the SCNT embryos was evident. In conclusion, ovine IVC and SCNT embryos develop at a slower rate than in vivo embryos at least up until Day 13 of gestation.

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Effects of treatment with a microRNA mimic or inhibitor on the developmental competence, quality, epigenetic status and gene expression of buffalo (Bubalus bubalis) somatic cell nuclear transfer embryos

Reproduction Fertility and Development ◽

10.1071/rd19084 ◽

2020 ◽

Vol 32 (5) ◽

pp. 508

Author(s):

S. Sah ◽

A. K. Sharma ◽

S. K. Singla ◽

M. K. Singh ◽

M. S. Chauhan ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Inner Cell Mass ◽

Cell Mass ◽

Bubalus Bubalis ◽

Cell Number ◽

Developmental Competence ◽

Cell Stage ◽

Morula Stage

Expression levels of 13 microRNAs (miRNAs) were compared between buffalo blastocysts produced by somatic cell nuclear transfer through hand-made cloning and IVF to improve cloning efficiency. Expression of miR-22, miR-145, miR-374a and miR-30c was higher, whereas that of miR-29b, miR-101, miR-302b, miR-34a, miR-21 and miR-25 was lower, in nuclear transferred (NT) than IVF embryos; the expression of miR-200b, miR-26a and miR-128 was similar between the two groups. Based on these, miR-145, which is involved in the regulation of pluripotency, was selected for further investigation of NT embryos. miR-145 expression was lowest at the 2-cell stage, increased through the 4-cell stage and was highest at the 8-cell or morula stage in a pattern that was similar between NT and IVF embryos. miR-145 expression was higher in NT than IVF embryos at all stages examined. Treatment of reconstructed embryos 1h after electrofusion with an inhibitor of miR-145 for 1h decreased the apoptotic index and increased the blastocyst rate, total cell number, ratio of cells in the inner cell mass to trophectoderm, global levels of acetylation of histone 3 at lysine 18 and expression of Krueppel-like factor 4 (KLF4), octamer-binding transcription factor 4 (OCT4) and SRY (sex determining region Y)-box 2 (SOX2) in blastocysts. Treatment with an miR-145 mimic had the opposite effects. In conclusion, treatment of NT embryos with an miR-145 inhibitor improves the developmental competence and quality, and increases histone acetylation and expression of pluripotency-related genes.

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70 FIBROBLAST GROWTH FACTOR 4 PROMOTES THE DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN MICE

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab70 ◽

2010 ◽

Vol 22 (1) ◽

pp. 193

Author(s):

T. Mitani ◽

M. Morita ◽

M. Anzai ◽

Y. Nishiyama ◽

K. Moriki ◽

...

Keyword(s):

Growth Factor ◽

Somatic Cell ◽

Fibroblast Growth Factor ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Inner Cell Mass ◽

Cell Mass ◽

Ectopic Expression ◽

Preimplantation Embryos ◽

Fibroblast Growth

Somatic cell nuclear transfer (SCNT) embryos can develop during the preimplantation period; however, most of these die after implantation period. A transcription factor, Cdx2, promotes differentiation of extraembryonic tissues and appears to be involved in the segregation of inner cell mass (ICM) and trophectoderm (TE) in preimplantation embryos. So far, we have demonstrated that the expression of Cdx2 in mouse SCNT embryos is delayed and its expression level is significantly lower than that in intracytoplasmic sperm injection (ICSI) embryos. Moreover, the ectopic expression of Oct-3/4 was observed in the TE tissues of SCNT blastocysts, but not in ICSI blastocysts. Fibroblast growth factor (FGF) receptor 2 (FGFR2) is specifically expressed in 8-cell to morula-stage embryos and trophectoderm (TE) and is essential for implantation; however, FGFR2 expression in SCNT embryos significantly decreases compared with IVF embryos. Therefore, it is likely that abnormality of differentiation that is controlled in development of pre-implantation in SCNT embryos leads to a rapid decrease of subsequent developmental ability. Then, we investigated the effects of FGF4 on development of SCNT embryos. Mouse SCNT embryos were produced according to the method reported previously (Wakayama et al. 1998). B6D2F1 and B6C3F1 female mice were used for the collection of recipient oocytes and donor cells, respectively. Data were analyzed by Student’s t-test. First, the timing to start adding FGF4 was decided by FGFR2 expression time about 54 h after cell injection and treated for 3, 6, 12, 24, and 42 h thereafter. In the case of FGF4 concentration at 25 ng mL-1 with treating time of 6 h from the 4- to 8-cell stages, SCNT embryos significantly promoted the development to morula and blastocyst stages (91 and 45%, respectively) compared with IVF embryos (80 and 30%, respectively; P < 0.05). However, longer treatment of 42 h with FGF4 made their morphology considerably worse. Then, concentrations of FGF4 at 5, 25, 50, 250, and 500 ng mL-1 with treating time of 6 h was examined. In case of FGF4 concentration at 25 and 50 ng mL-1, SCNT embryos significantly promoted the development to morula and blastocyst stages (P < 0.05). Immunohistochemical analysis showed segregation of the expression of Oct-3/4 and Cdx2 in ICM and TE, respectively, in FGF4-treated SCNT embryos, unlike in the case of nontreated SCNT embryos, which showed an ectopic expression of Oct-3/4 in TE tissues. Furthermore, after the transplantation of SCNT embryos treated with FGF4 at 50 ng mL-1 and the treating time of 6 h to recipient mice, most of the transferred embryos implanted and cloned mice were successfully produced as well as nontreated SCNT embryos. Therefore, FGF4 facilitates the development of SCNT embryos especially to the morula and blastocyst stages. This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

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