Oviduct fluid is essential for the fertilization and subsequent preimplantation development. Glycine is abundant in oviduct fluid and is reported to be critical for preimplantation development of fertilized eggs in mammals. However, the mechanism by which glycine exerts its action on fertilized eggs is yet to be understood. Here we show that glycine regulates the preimplantation development of mouse fertilized eggs via glycine receptors. Among them, the alpha-4 subunit (Glra4) and the β subunit are expressed in mouse fertilized eggs, and lacking Glra4 inhibits embryonic development to the blastocyst stage, decreases the number of cells in the blastocysts and the litter size. Thus, we identify a novel function of the glycine receptor, which is considered to act mainly as a neurotransmitter receptor, as a regulator of embryonic development and our data provide new insights into the interactions between oviduct milieu and mammalian fertilized egg.
Supplemental Table S1. The data of animal number used in the study.
Supplemental Table S2. The crRNA and primers used in the study.
Supplemental Figure S1. While Glrb is expressed in various tissues, all alpha subunits are expressed in limited tissues, especially in the brain. Expression atlas data of mouse were obtained from Söllner et al. (Söllner et al. 2017; Array Express accession number E-MTAB-6081). Red circle indicates the expression level calculated Log2 (TPM).
Supplemental Figure S2. Heat map of reanalysis of RNA-seq data from public database. RNA-seq datasets from bovine (GSE59186), and human (GSE101571) embryos from the NCBI GEO database were used. Human RNA-seq data was aligned against the corresponding NCBI version 38 genomes with annotation files provided by the UCSC computer program STAR (version 2.6.0) (Dobin et al., 2013). The number of sequences aligned to each gene was counted using featureCount (version 1.6.0) (Liao et al., 2014). Since bovine sequences were obtained using ABI SOLiD equipment, the output format of which is not supported by the software, short reads were aligned against bovine mRNA sequences provided by the Bovine Genome Database (http://bovinegenome.org/) (Elsik et al., 2016) using Bowtie (version 1.1.2) (Langmead et al., 2009), and the number of mapped reads was counted using an in-house program.
Supplemental Figure S3. Predicted 3D structure of the wild type and mutant GLRA4 protein using I-TASSER software.