MicroRNA-34c Expression in Donor Cells Influences the Early Development of Somatic Cell Nuclear Transfer Bovine Embryos

Abstract

The essence of the reprogramming activity of somatic cell nuclear transfer (SCNT) embryos is to produce normal fertilized embryos. However, reprogramming of somatic cells is not as efficient as the reprogramming of sperm. In this report, we describe the effect of an inducible, specific miR-34 microRNA expression in donor cells that enables a similar level of sperm:transgene expression on the early development of SCNT embryos. Our results showed that donor cells with doxycycline (dox)-induced miR-34c expression for the preparation of SCNT embryos resulted in altered developmental rates, histone modification (H3K9ac and H3K4me3), and extent of apoptosis. The cleavage rate and blastocyst formation of the induced nuclear transfer (NT) group were significantly increased. The immunofluorescence signal of H3K9ac in embryos in the induced NT group significantly increased in two-cell-and eight-cell-stage embryos; that of H3K4me3 increased significantly in eight-cell-stage embryos. Although significant differences in staining signals of apoptosis were not detected between groups, lower apoptosis levels were observed in the induced NT group. In conclusion, miR-34c expression induced by dox treatment enhances the developmental potential of SCNT embryos, modifies the epigenetic status, and changes blastocyst quality.

Sperm-borne miR-449b influences cleavage, epigenetic reprogramming and apoptosis of SCNT embryos in bovine

“…Previous study has shown that injection of sperm-borne miR-34c inhibitor into zygotes could inhibit DNA synthesis and significantly suppress the first cleavage division in mouse 13 . In bovine, we also observed that high expression of miR-34c in somatic cell donor cells affected cleavage and development of SCNT embryos 28 . As part of the same family, miR-34c and miR-449b share the same seed sequence and many target genes 14 , 35 .…”

“…Bovine fetal fibroblast cells were cultured as described previously 28 . The head and viscera of 35-day-old fetuse were discarded.…”

“…The transfection of Tet-On 3G expression vector into cells was performed according to previous description 28 . Briefly, cells were transfered into 4-mm cuvette gap with 10 μg of miR-449b-pTRE3G-BI-eGFP and 5 μg of pEF1α-Tet3G, and transfected using the BTX Electro Cell Manipulator ECM2001 with three pulses of 1-msec at 510 V. Then transfected cells were transferred to a 90-mm dish for culture.…”

“…Numerous studies have shown inducible Tet-On system is a convenient tool for reversible and very tightly controlled conditional gene expression in zebrafish, drosophila, murine, porcine and bovine models 23 – 27 . In our previous study, we successfully used the Tet-On system to induce expression of miR-34c in bovine fibroblasts 28 .…”

Effect of ezrin regulation by sperm-borne miR-183 on the formation of microvilli and early development of bovine embryos.

“…Previously, it was thought that only oocytes contained regulators of embryonic development. However, a large body of evidence suggests that sperm also contains signi cant amounts of proteins, mRNAs, tRNAs, tsRNAs, PIWI-interacting RNAs, and miRNAs, which have recently been shown to have important effects on embryonic development [2,3,4,5]. Many post-transcriptional regulatory factors, such as miRNAs, can be delivered into oocytes during fertilization.…”