Effects Of Chi-let-7b-5p Derived From Goat Uterus Exosome On Embryo Attachment

Reproductive rate has a significant impact on the economic benefits of animal production in the breeding of ruminant species like goats.Goats in production confront issues such a poor conception rate and frequent miscarriages.In-depth study of the embryo implantation stage is essential to enhancing animal reproduction since it is a pivotal step in the establishment of mammalian pregnancy.Exosomes have the ability to govern the operation of target cells and act as"messengers"by transferring numerous substances,including proteins,lipids,nucleic acids,and others,that they carry.Exosomes,which play a role in regulating the embryo implantation process,have been found in the uterine fluid of humans and other animals,according to reports.The research on this topic in goats is,however,still in its early stages.In this work,a goat early pregnancy model was developed,and uterine irrigations from goat pregnancies of 5 days（pre-implantation）,15 days（pregnancy recognition period）,and18 days（embryo adhesion period）were collected.The exosome was extracted by ultracentrifugation,and the extracted samples were identified by transmission electron microscopy,nanoparticle diameter tracking analyzer,and Western Blot.High throughput sequencing technology was used to reveal the expression profiles of m RNAs and mi RNAs in the exosomes of goat uterus irrigating fluid during different pregnancy days.GO analysis and KEGG analysis were utilized to identify the key roles and signaling pathways involved in the target genes of mi RNAs and m RNAs that exhibit differential expression;This study discovered differential expression of let-7b-5p in the uterine tissues of goats with various gestational days based on the sequencing data.Let-7b-5p is thought to play a role in controlling the implantation of goat embryos.Detect whether it has an impact on the proliferation of goat endometrial epithelial cells and the expression of genes related to embryo implantation by transfecting mi RNA mimic or inhibitor,find the predicted target genes according to the mi RNA target gene online prediction software,and verify the targeting relationship through dual luciferase reporting experiments;Inject mi RNA agmir or antagmir into the uterine horn of mice on the third day of pregnancy,and verify the effect of let-7b-5p on embryo implantation by counting the number of implantation sites in the mouse uterus.The results are as follows:（1）The sample had a characteristic cup-shaped structure under the transmission electron microscope,and a study of the particle size revealed that the concentration was more than 10¹⁰ particles/m L and that the average particle size ranged from 75-78 nm.The exosomes had been successfully extracted from the goat uterine flushing fluid,according to a positive Western Blot result for the exocrine marker protein.（2）In the 5-day and 15-day gestation groups,the sequencing results revealed 10635differently expressed m RNAs and 26 differentially expressed mi RNAs;the samples from days 5 and 18 of pregnancy included 35 and 7362 differentially expressed mi RNAs and m RNAs,respectively;The samples from days 15 and 18 of pregnancy contained 9220differently expressed m RNAs and 30 differentially expressed mi RNAs.The results of the gene ontology（GO）analysis revealed that the target genes predicted by these differentially expressed m RNAs and mi RNAs are membrane-related organelles with catalytic,binding,and transcriptional regulatory activities,which are involved in processes like metabolism,biological regulation,and development.According to the results of the KEGG analysis,it participates in a number of signal regulation processes,including metabolic pathways,oxidative phosphorylation,the phosphatidylinositol signaling system,the Fox O signaling pathway,adhesive junctions,the TGF-beta signaling pathway,the Rap1 signaling pathway,the PI3K-Akt signaling pathway,the Toll-like receptor signaling pathway,and others.The RT-PCR detection of the differentially expressed mi RNAs（chi mi R-199b-5p,chi mi R-136-5p,and chi mi R-106b-3p）and m RNAs（FTH1,PTGS2,CTNNB1,ACSL4,and GPX4）in the randomly selected sequencing results was consistent with the sequencing results,proving the reliability of the sequencing results.（3）Chi-let-7b-5p expression in goat uterine tissue declined as the number of pregnancy days increased,and it was primarily found in stromal cells,glandular epithelial cells,and endometrial lumen epithelial cells;does not interfere with endometrial epithelial cell growth;Let-7b-5p mimic enhanced the expression of ISG15 and HOXA10 while downregulating the expression of genes linked to embryo implantation,CXCL10 and MUC1.Inhibition of let-7b-5p increased the expression of CXCL10 and MUC1 while decreasing the expression of LIF;According to the dual luciferase report findings,let-7b-5p specifically targeted CXCL10.Compared to NC mimic,chi-let-7b-5p mimic reduced luciferase activity by 63%in the psi CHECK 2-CXCL10 WT group,while in the psi CHECK 2-CXCL10 MUT group,there was no significant difference in luciferase activity between NC mimic and chi-let-7b-5p mimic,and let-7b-5p mimic upregulated the protein expression level of CXCL10,The let-7b-5p inhibitor inhibits the protein expression level of CXCL10.According to the immunohistochemistry findings,CXCL10 was mostly expressed in stromal cells,deep muscle layers,glandular epithelial cells,and endometrial epithelial cells,and the level of m RNA expression rose with the number of pregnancy days.（4）On the third day of pregnancy,let-7b-5p agmir or antagmir was injected into the uterine horn using a mouse early pregnancy model.The findings demonstrated that let-7b-5p overexpression in mice greatly decreased the number of embryonic implantation sites in the uterus.Inhibiting let-7b-5p expression had no impact on the quantity of mouse embryonic implantation sites.Additionally,overexpressing let-7b-5p decreased the m RNA levels of the genes LIF,SPP1,and HOXA10 that are involved in mouse embryo implantation,whereas inhibiting let-7b-5p expression increased LIF m RNA level and SPP1expression.In conclusion,this study used high-throughput sequencing to compare the differential expression of m RNAs and mi RNAs in the secretions of different days of goat early pregnancy uterine fluid.It found that the differentially expressed chi-let-7b-5p can target the expression of embryo implantation factor CXCL10,and overexpression of let-7b-5p significantly inhibits the embryo implantation rate of mice,offering a new theory for the regulation mechanism of domestic animal.