Ke-Ren Cheng1, Xiang-Wei Fu1, Rui-Na Zhang1, Gong-Xue Jia1, Yun-Peng Hou2, Shi-En Zhu3. 1. Key Laboratory of Animal Genetics, Breeding and Reproduction, Ministry of Agriculture and National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, People's Republic of China. 2. State Key Laboratory for Agro Biotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China. 3. Key Laboratory of Animal Genetics, Breeding and Reproduction, Ministry of Agriculture and National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, People's Republic of China. Electronic address: zhushien@cau.edu.cn.
Abstract
OBJECTIVE: To examine whether mouse oocytes vitrification could alter the deoxyribonucleic acid (DNA) methylation of differentially methylated regions (DMRs) of three imprinted genes in in vitro fertilized blastocysts. DESIGN: In vitro experiments using murine model. SETTING: State key laboratory and university research laboratory. ANIMAL(S): Kunming white mice. INTERVENTION(S): The mouse metaphase II oocytes were vitrified. After thawing, the surviving oocytes were fertilized in vitro to produce blastocysts. The blastocysts derived in vitro from fresh oocytes were used as a control. The DNA methylation patterns of the DMRs of imprinted genes in oocytes and blastocysts and the relative expression of DNMTs (Dnmt1, Dnmt3a, Dnmt3b, and Dnmt3l) in oocytes and blastocysts were detected. MAIN OUTCOME MEASURE(S): Methylation patterns of DMRs of H19, Peg3, and Snrpn analyzed by bisulfite mutagenesis and sequencing. Expression levels of messenger ribonucleic acid as measured by real-time reverse-transcriptase polymerase chain reaction. RESULT(S): After oocytes vitrification, the methylation levels at H19, Peg3, and Snrpn DMRs in blastocysts were decreased. However, there was no significant difference in the percentage of hypermethylated strands at Peg3 DMRs between the vitrified and control groups. DNMTs expression in vitrified oocytes and the expression of Dnmt3b in blastocysts derived from vitrified oocytes were significantly reduced. CONCLUSION(S): Oocytes vitrification could lead to the loss of DNA methylation of imprinted genes (H19, Peg3, and Snrpn) in mouse blastocysts, which is mainly caused by the reductions of DNMTs after vitrification of oocytes.
OBJECTIVE: To examine whether mouse oocytes vitrification could alter the deoxyribonucleic acid (DNA) methylation of differentially methylated regions (DMRs) of three imprinted genes in in vitro fertilized blastocysts. DESIGN: In vitro experiments using murine model. SETTING: State key laboratory and university research laboratory. ANIMAL(S): Kunming white mice. INTERVENTION(S): The mouse metaphase II oocytes were vitrified. After thawing, the surviving oocytes were fertilized in vitro to produce blastocysts. The blastocysts derived in vitro from fresh oocytes were used as a control. The DNA methylation patterns of the DMRs of imprinted genes in oocytes and blastocysts and the relative expression of DNMTs (Dnmt1, Dnmt3a, Dnmt3b, and Dnmt3l) in oocytes and blastocysts were detected. MAIN OUTCOME MEASURE(S): Methylation patterns of DMRs of H19, Peg3, and Snrpn analyzed by bisulfite mutagenesis and sequencing. Expression levels of messenger ribonucleic acid as measured by real-time reverse-transcriptase polymerase chain reaction. RESULT(S): After oocytes vitrification, the methylation levels at H19, Peg3, and Snrpn DMRs in blastocysts were decreased. However, there was no significant difference in the percentage of hypermethylated strands at Peg3 DMRs between the vitrified and control groups. DNMTs expression in vitrified oocytes and the expression of Dnmt3b in blastocysts derived from vitrified oocytes were significantly reduced. CONCLUSION(S): Oocytes vitrification could lead to the loss of DNA methylation of imprinted genes (H19, Peg3, and Snrpn) in mouseblastocysts, which is mainly caused by the reductions of DNMTs after vitrification of oocytes.
Authors: Clementina Cantatore; Jenny S George; Raffaella Depalo; Giuseppe D'Amato; Molly Moravek; Gary D Smith Journal: J Assist Reprod Genet Date: 2021-05-22 Impact factor: 3.357