Differential Expression Pattern of Goat Uterine Fluids Extracellular Vesicles miRNAs during Peri-Implantation

Abstract

Early pregnancy failure occurs when a mature embryo attaches to an unreceptive endometrium. During the formation of a receptive endometrium, extracellular vesicles (EVs) of the uterine fluids (UFs) deliver regulatory molecules such as small RNAs to mediate intrauterine communication between the embryo and the endometrium. However, profiling of small RNAs in goat UFs' EVs during pregnancy recognition (day 16) has not been carried out. In this study, EVs were isolated from UFs on day 16 of the estrous cycle or gestation. They were isolated by Optiprep™ Density G radient (ODG) and verified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. Immunostaining demonstrated that CD63 was present both in the endometrial epithelium and glandular epithelium, and stain intensity was greater in the pregnant endometrium compared to the non-pregnant endometrium. Small RNA sequencing revealed that UFs' EVs contained numerous sRNA families and a total of 106 differentially expressed miRNAs (DEMs). Additionally, 1867 target genes of the DEMs were obtained, and miRNA-mRNA interaction networks were constructed. GO and KEGG analysis showed that miRNAs were significantly associated with the formation of a receptive endometrium and embryo implantation. In addition, the fluorescence in situ hybridization assay (FISH) showed that chi-miR-451-5p was mainly expressed in stromal cells of the endometrium and a higher level was detected in the endometrial luminal epithelium in pregnant states. Moreover, the dual-luciferase reporter assay showed that chi-miR-451-5p directly binds to PSMB8 and may play an important role in the formation of a receptive endometrium and embryo implantation. In conclusion, these results reveal that UFs' EVs contain various small RNAs that may be vital in the formation of a receptive endometrium and embryo implantation.

Keywords: embryo implantation; endometrium; extracellular vesicles; goat; miRNA; uterine fluids.

Figure 1

Characterization of EVs from porcine UFs. (A) TEM analysis of extracellular vesicles from goat UFs. Scale bar = 100 nm. (B) NTA of the extracellular vesicles from goat UFs. (C) Western blotting detected EV protein markers, TSG101 and HSP70, in the UFs’ EVs fraction and the endometrium. Endoplasmic reticulum membrane marker (Calnexin) was only detected in the endometrium. (D) TEM analysis of endometrial luminal epithelium on day 16 of pregnancy (n = 3). Legend: C16, on day 16 of the estrous cycle; P16, on day 16 of pregnancy. Arrowhead refers to EVs.

Figure 2

CD63 levels in pregnant and non-pregnant endometrium. (A) Expression pattern of CD63 in pregnant and non-pregnant endometrium. Scale bar = 200 μm. (B) Quantitative analysis of CD63 by measuring the average integrated optical density (IOD) in LE and GE of pregnant and non-pregnant endometrium. Data are shown as the mean ± SEM values (n = 3). Legend: C16, on day 16 of the estrous cycle; P16, on day 16 of pregnancy; LE, endometrial luminal epithelium; GE, glandular epithelium. ** p < 0.01 was considered especially significant.

Figure 3

Overall differences of miRNAs in pregnant and non-pregnant state. (A) The Venn diagram shows the differential expression of miRNAs. (B) Heatmap shows the expression patterns of miRNAs between C16 and P16. (C) The volcano plots of the differentially expressed miRNAs on P16 and C16. Legend: C16, on day 16 of the estrous cycle; P16, on day 16 of pregnancy.

Figure 4

Validation of the expression level of miRNAs. The randomly selected DEMs were verified by RT-qPCR and the results showed was consistent with the RNA-seq data. Data are presented as mean ± SEM values (n = 3).

Figure 5

DEMs’ target genes regulatory network. The red circle represents target genes, the rhombus represents DEMs and the yellow rhombus represents chi-miR-1343.

Figure 6

GO and KEGG enrichment analysis of predicted target genes of DEMs. (A) GO analysis of predicted target genes of SEMs. (B) Top 20 KEGG pathways of predicted target genes of DEMs. The x-axis indicates the ratio of the predicted target genes to the annotated genes enriched in this pathway, while the y-axis indicates the KEGG pathway. “Count” means the number of predicted target genes enriched in this pathway. The color represents the degree of enrichment, with red representing significant enrichment. The red font indicates a relationship between the pathways and endometrial development and remodeling. The yellow font indicates a relationship between the pathways and embryo implantation. The blue font indicates a relationship between the pathways and metabolism. The green font indicates a relationship between the pathways and maintenance of pregnancy.

Figure 7

Chi-miR-451-5p expressed in stromal cells of the endometrium. Fluorescence in situ hybridization (FISH) assays for miR-451-5p in the endometrium on C16 and P16 tissue samples (n=3). Scale bar = 100 μm. Legend: C16, on day 16 of the estrous cycle; P16, on day 16 of pregnancy; LE, endometrial luminal epithelium; GE, glandular epithelium; SC, stroma cell.

Figure 8

Chi-miR-451-5p targets the 3′ UTR of PMSB8. (A) The predicted binding site of chi-miR-451-5p in the 3′UTR of PSMB8. (B) The design of luciferase reporter. PSMB8 3′ UTR sequence contains the chi-miR-451-5p binding site; PSMB8 3′ UTR Mut sequence has mutations of the chi-miR-451-5p binding site. (C) 293T cells co-transfected with luciferase reports of PMSB83′ UTR (WT) or PMSB83′ UTR (Mut) and chi-miR-451-5p mimics or negative control (NC) before the luciferase reporter assay. Data are shown as the mean ± SEM values (n = 3). * p < 0.05 was considered statistically significant.