Melatonin Protects Rabbit Somatic Cell Nuclear Transfer (SCNT) Embryos from Electrofusion Damage

Pengxiang Qu,Fang Hu,Yue Du,Shuaiqingying Guo,Shiwei Luo,Sixin Fu,Yue Dong,Hongyu Qin,Chong Shen,Ying Xue,Enqi Liu

doi:10.1038/s41598-020-59161-6

Pengxiang Qu, Fang Hu + Show 9 more

Open Access

https://doi.org/10.1038/s41598-020-59161-6

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Journal: Scientific Reports	Publication Date: Feb 10, 2020
Citations: 16	License type: open-access

Affiliation: Xi'an Jiaotong University, University of Oxford

Abstract

The study’s objectives were to examine the effects of electrofusion on rabbit somatic cell nuclear transfer (SCNT) embryos, and to test melatonin as a protective agent against electrofusion damage to SCNT embryos. The levels of reactive oxygen species (ROS), the epigenetic state (H3K9me3), and the content of endoplasmic reticulum (ER) stress-associated transcripts (IRE-1 and CHOP) were measured. Melatonin was added during the preimplantation development period. The total blastocyst cell numbers were counted, and the fragmentation rate and apoptotic index were determined and used to assess embryonic development. Electrofusion increased (1) ROS levels at the 1-, 2-, 4-, and 8-cell stages; (2) H3K9me3 levels at the 2-, 4-, and 8-cell stage; and (3) the expression of IRE-1 and CHOP at the 8-cell, 16-cell, morula, and blastocyst stages. The treatment of SCNT embryos with melatonin significantly reduced the level of ROS and H3K9me3, and the expression levels of IRE-1 and CHOP. This treatment also significantly reduced the fragmentation rate and apoptotic index of blastocysts and increased their total cell number. In conclusion, the electrofusion of rabbit SCNT embryos induced oxidative stress, disturbed the epigenetic state, and caused ER stress, while melatonin reduced this damage. Our findings are of signal importance for improving the efficiency of SCNT and for optimizing the application of electrical stimulation in other biomedical areas.

Highlights

Previous studies reported that electrostimulation induced reactive oxygen species (ROS) in the embryos of rats, mice and pigs but only a few studies focused on somatic cell nuclear transfer (SCNT) embryos[12,13]
The results showed that ROS levels in electrically stimulated SCNT embryos at the 1, 2, 4, and 8-cell stages were significantly higher than those in the embryos in the non-electrofusion group (P < 0.05) (Fig. 1B)
We determined that the cleavage and blastocyst rates in the non-electrofusion group were significantly lower than in the electrofusion groups, indicating that the electrofusion method was better than the piezo method in terms of activating SCNT embryos

Summary

Introduction

Previous studies reported that electrostimulation induced ROS in the embryos of rats, mice and pigs but only a few studies focused on SCNT embryos[12,13]. SCNT embryos can manifest higher ROS levels, and this can cause damage to mitochondria, cell membranes, and DNA, and abnormal gene expression[5,14,15]. Abnormal epigenetic reprogramming occurs in a large proportion of cloned embryos, and this impedes improvement in SCNT efficiency[1,9]. Electrostimulation regulates the epigenetic state in somatic cells; there are few reports on the effect of electrostimulation on the epigenetic state of embryos[16,17]. We optimized the electrofusion procedure, and studied its effects on SCNT embryos, oxidative stress, epigenetic state and ER stress. The developmental competence of SCNT embryos was assessed in terms of their fragmentation rate, apoptotic index, blastocyst rate, and total cell number in blastocysts

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Conclusion