Effect of Vitrification on the MicroRNA Transcriptome in Mouse Blastocysts
April
Effect of Vitrification on the MicroRNA Transcriptome in Mouse Blastocysts
Xueming Zhao 0 1 2
Haisheng Hao 0 1 2
Weihua Du 0 1 2
Huabin Zhu 0 1 2
0 Embryo Biotechnology and Reproduction Laboratory, Institute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences (CAAS) , Beijing 100193 , P. R. China
1 Funding: This work was supported by grants from the National Natural Science Foundation of China (31472100), the Basic Research Fund of IAS, CAAS (2014ywf-yb-2) and The Agricultural Science and Technology Innovation Program (ASTIP-IAS06-2014, ASTIP-IAS-TS-5)
2 Academic Editor: Zeng-Ming Yang, South China Agricultural University , CHINA
Vitrification is commonly used in the cryopreservation of mammalian blastocysts to overcome the temporal and spatial limitations of embryo transfer. Previous studies have shown that the implantation ability of vitrified blastocysts is impaired and that microRNAs (miRNAs) regulate the critical genes for embryo implantation. However, little information is available about the effect of vitrification on the miRNA transcriptome in blastocysts. In the present study, the miRNA transcriptomes in fresh and vitrified mouse blastocysts were analyzed by miRNA Taqman assay based method, and the results were validated using quantitative real-time PCR (qRT-PCR). Then, the differentially expressed miRNAs were assessed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Overall, 760 known mouse miRNAs were detected in the vitrified and fresh mouse blastocysts. Of these, the expression levels of five miRNAs differed significantly: in the vitrified blastocysts, four miRNAs (mmu-miR-199a-5p, mmu-miR-329-3p, mmu-miR-136-5p and mmu-miR-16-1-3p) were upregulated, and one (mmu-miR-212-3p) was downregulated. The expression levels of all miRNAs measured by the miRNA Taqman assay based method and qRT-PCR were consistent. The four upregulated miRNAs were predicted to regulate 877 candidate target genes, and the downregulated miRNA was predicted to regulate 231 genes. The biological analysis further showed that the differentially expressed miRNAs mainly regulated the implantation of embryos. In conclusion, the results of our study showed that vitrification significantly altered the miRNA transcriptome in mouse blastocysts, which may decrease the implantation potential of vitrified blastocysts.
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Competing Interests: The authors have declared
that no competing interests exist.
Blastocyst transfer has been shown to be an effective approach to improve implantation and
pregnancy rates during the transfer of embryos produced in vitro [1]. To overcome the
temporal and spatial limitations of embryo transfer, several types of cryopreservation methods have
been developed, including controlled freezing [2] and vitrification [3]. Many experiments have
shown that vitrification has a higher efficiency of cryopreservation than controlled freezing [4
7], mainly due to the higher rapid cooling rate of vitrification (>20,000C/min) [8].
Vitrification is commonly applied in bovine [8], mouse [9], and human [10] blastocysts.
However, the implantation of vitrified blastocysts is impaired. Therefore, researchers are now
focusing on the possible influence of blastocyst vitrification on factors such as the inner cell
mass number [11], spindle formation [12], fragmented DNA in nuclei [7,13], the expression
levels of important development-related genes [14], and the sex ratio of offspring after transfer
[15]. Recently, our research group reported the effect of vitrification on the promoter
methylation levels and the mRNA expression levels of octamer-binding transcription factor 4 (OCT4),
Nanog homeobox (NANOG), and caudal-type homeobox 2 (CDX2) in mouse blastocysts [16].
MicroRNAs (miRNAs) are a family of small RNAs that are 2125 nucleotides in length and
are involved in the negative regulation of gene expression at the post-transcriptional level
[17,18]. Many miRNA knockout experiments have illustrated the physiological importance of
individual miRNAs in mice [1922]. Several studies have reported that miRNAs are important
regulators of pluripotency and differentiation and, consequently, of early lineage segregation in
embryonic development [23,24] and maternal-to-embryonic transition [25]. MiRNAs also
regulate the critical genes for embryo implantation [26], and the disruption of blastocyst miRNA
expression is associated with human infertility [27]. Nuclear transfer by microinjection has
been found to alter the miRNA profile of enucleated mouse oocytes [28], indicating that
external stimuli may affect the miRNA profiles of mammalian oocytes. Currently, however, little
information is available on the effect of vitrification on the miRNA transcriptome profile in
oocytes and embryos.
In the present study, the TaqMan Array Rodent MicroRNA A+B Cards Set v3.0 with an
Applied Biosystems 7900 HT Fast Real-time PCR system was utilized to detect the miRNA
transcriptome in fresh (...truncated)