Differential MicroRNA Expression Involved in Endometrial Receptivity of Goats

Abstract

Endometrial receptivity represents one of the leading factors affecting the successful implantation of embryos during early pregnancy. However, the mechanism of microRNAs (miRNAs) to establish goat endometrial receptivity remains unclear. This study was intended to identify potential miRNAs and regulatory mechanisms associated with establishing endometrial receptivity through integrating bioinformatics analysis and experimental verification. MiRNA expression profiles were obtained by high-throughput sequencing, resulting in the detection of 33 differentially expressed miRNAs (DEMs), followed by their validation through quantitative RT-PCR. Furthermore, 10 potential transcription factors (TFs) and 1316 target genes of these DEMs were obtained, and the TF-miRNA and miRNA-mRNA interaction networks were constructed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that these miRNAs were significantly linked to establishing endometrial receptivity. Moreover, the fluorescence in situ hybridization (FISH) analysis, dual-luciferase report assay, and immunohistochemistry (IHC) analysis corroborated that chi-miR-483 could directly bind to deltex E3 ubiquitin ligase 3L (DTX3L) to reduce its expression level. In conclusion, our findings contribute to a better understanding of molecular mechanisms regulating the endometrial receptivity of goats, and they provide a reference for improving embryo implantation efficiency.

Keywords: DTX3L; chi-miR-483; endometrium receptivity; goat; high-throughput sequencing; implantation.

Figure 1

Overview of the sequences generated by miRNA sequencing (miRNA-Seq). (A) Sequence length distribution of the sequences generated by miRNA-Seq of the two sets of libraries. The length distribution peaked at 22 nt, i.e., the desired miRNA length. Blue and red represent the results from total sequences obtained from C16 and P16 endometrial samples, respectively. (B) Classification of small RNA sequences obtained from individual C16 and P16 endometrial samples. (C) The density distribution of miRNA expression. The abscissa represents the value of log₁₀ transcripts per million (TPM), and the ordinate represents the corresponding density.

Figure 2

Differentially expressed miRNAs (DEMs) in goat endometrium. (A) Venn diagrams of known miRNAs. (B) Clustering analysis of DEMs. The color scale is from −2.0 (blue, lower miRNA expression level) to 2.0 (red, higher miRNA expression level). Each row represents one miRNA, and each column represents one sample. (C) Volcano plots of DEMs. Each point represents one miRNA. The abscissa represents the value of log₂ fold-change; the ordinate value represents −log₁₀ q-value.

Figure 3

Validation of the expression of miRNAs using qRT-PCR. The relative expression level of miRNA was quantified relative to the expression level of U6 using the comparative cycle threshold (2^−ΔΔCt) method. Data are displayed as the mean ± standard error of the mean (SEM) values (n = 3).

Figure 4

Predicted transcription factors of DEMs. (A) The top 10 transcription factors (TFs) with the most TF binding sites (TFBSs). The circles in blue represent DEMs, and the squares in green represent TFs. The dotted line represents the relationship between TFs and miRNAs, and a darker color of the red arrow indicates more TFBSs between the miRNA and TF. (B) Binding motifs between the 10 TFs and chi-miR-483.

Figure 5

DEM–target gene regulatory network. The circles in red represent target genes and the chevrons in blue represent DEMs.

Figure 6

Functional analysis of DEMs. (A) Gene Ontology (GO) enrichment analysis of the target genes of DEMs, including biological process, cellular component, and molecular function. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the target genes of DEMs.

Figure 7

In situ hybridization analysis of chi-miR-483 in the C16 and P16 uterus. The chi-miR-483 was abundantly expressed in the uterine luminal epithelium or glandular epithelium of C16, while it was slightly expressed in P16. The section stained with hybridization buffer without probe was used as the negative control (NC; (Figure S2, Supplementary Materials). Legend: LE, endometrial luminal epithelium; GE, glandular epithelium. Scale bar = 100 μm.

Figure 8

Chi-miR-483 targets the 3′-untranslated region (UTR) of deltex E3 ubiquitin ligase 3L (DTX3L). (A) The predicted binding site of chi-miR-483 in the 3′UTR of DTX3L according to bioinformatics analysis. (B) Design of the luciferase reporter. WT, the wildtype sequence of DTX3L-3′UTR contains the chi-miR-483 binding site; Mut, the sequence of DTX3L-3′UTR with a mutation in the chi-miR-483 binding site. (C) 293T cells were co-transfected with wildtype (WT) or mutant (Mut) luciferase reports of DTX3L 3′UTR with chi-miR-483 mimics or negative control (NC) mimics. The luciferase reporter assay demonstrated that chi-miR-483 significantly decreased the luciferase activity of DTX3L WT in 293T cells. Data are shown as the mean ± SEM values (n = 3, ** p < 0.01, Student’s t-test).

Figure 9

Immunohistochemical analysis of DTX3L in the C16 and P16 uterus. (A) Images stained with DTX3L antibodies. The positive signal of DTX3L was distinctly detected in the uterine luminal epithelium or glandular epithelium in P16. The section stained with nonrelevant immunoglobulin G served as the negative control (NC). (B) Quantitative analysis of DTX3L by measuring the average integrated optical density (IOD) in the endometrium. Asterisks indicate significant differences (mean ± SEM) between C16 and P16 (*** p < 0.001); the p-value was determined by Student’s t-test. Legend: LE, endometrial luminal epithelium; GE, glandular epithelium. Scale bar = 100 μm.