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.
Highlights
Goats are valuable livestock breeds as they provide fiber, meat, milk, and other byproducts [1], and have promising importance in biomedicine and the transgenic production of pharmaceutical drugs such as human enzymes [2]
Uteri were obtained from goats slaughtered on day 16, and uterine fluids’ (UFs) was collected after uteri removal by flushing with 30 mL of Dulbecco’s phosphate-buffered saline
UFs’ extracellular vesicles (EVs) appeared as cup-shaped vesicular structures (Figure 1A) that were mainly detected in OptiprepTM Density G radient (ODG) fractions 9, 10, and 11
Summary
Goats are valuable livestock breeds as they provide fiber, meat, milk, and other byproducts [1], and have promising importance in biomedicine and the transgenic production of pharmaceutical drugs such as human enzymes [2]. Pregnancy failure occurs when a mature embryo attaches to a non-receptive endometrium, hindering reproduction and herd development [3]. The endometrium undergoes remarkable changes during the formation of a receptive state, including the proliferation of endometrial stromal cells (ESCs), differentiation of endometrium epithelial cells (EECs), and the acquisition of adhesion properties that allow the embryo to attach and subsequently invade [4,5,6]. Numerous studies have shown that a non-receptive endometrium can lead to transplantation failure of the in vitro fertilization embryo [7,8]. Further research is needed to explore the specific regulatory mechanisms of UFs’ EVs underlying the endometrial changes during the formation of a receptive state to improve animal husbandry development and embryo transplantation success rate
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