| Early embryo loss is one of the main causes of pregnancy failure in mammals.Studies have shown that the majority of embryo loss occurs during the peri-implantation period,which is often seen in high-yielding dairy cows,sows,and other domestic animals,severely reducing the reproductive efficiency.The development of assisted reproduction technology enables in vitro fertilization and embryo transfer;however,the pregnancy rate after embryo transfer has not been significantly improved,mainly due to implantation failure.Therefore,it is imperative to address this issue by investigating the regulatory mechanisms of embryo implantation.The main factors affecting embryo implantation include embryo viability,the establishment of endometrial receptivity and the effective communication between them.Endometrial receptivity refers to a specific physiological state that the endometrium achieves to support embryo attachment within a limited period.It is a prerequisite for implantation initiation and inadequate receptivity remains the major cause of implantation failure,suggesting the dominant role of the mother.A considerable body of evidence indicates that early steps of implantation,including establishing receptivity,have many common aspects across species regardless of their different implantation strategies.However,the specific mechanisms underlying the establishment of endometrial receptivity are not yet clear because of the individual or collective contribution of uterine compartments and the complexity of the molecular regulatory network behind them.MicroRNAs(miRNAs)are small non-coding RNAs that can regulate the expression of multiple genes simultaneously at the post-transcriptional level.They are involved in a variety of biological processes,including embryo implantation.However,their role in establishing receptivity remains unclear.Considering the similarity of the early implantation steps among species,this study used mouse as the experimental subject.Through small RNA sequencing,the expression profile of uterine miRNAs in mice during the pre-receptive,receptive and implantation phase were analyzed,and the miRNAs with significant differential expression and could indeed affect the outcome of implantation were selected as the target miRNA.Then,the expression pattern of the target miRNA in early pregnancy in mice was investigated through RT-qPCR and in situ hybridization,and the factors led to this specific trend were explored in several mouse models.Finally,the molecular mechanisms by which the target miRNA regulates the establishment of endometrial receptivity were investigated.The main results of this study are as follows.(1)A total of 42 miRNAs were differentially expressed during the pre-receptive(Day 1 of pregnancy),receptive(Day 4 of pregnancy),and implantation phase(Day 5 of pregnancy).(|log2(Foldchange)|≥1.5,FDR<0.05).Through RT-qPCR analysis,miR-192-5p was found to be significantly downregulated during the receptive and implantation phase(P<0.001).Transient upregulation of miR-192-5p by uterine injection of miRNA agomirs led to implantation failure,suggesting its role in regulating embryo implantation.(2)miR-192-5p showed a consistent downregulated expression during early pregnancy(Day 1-7 of pregnancy)in mice and its expression remained at low levels during and after the peri-implantation phase.In situ hybridization results revealed that miR-192-5p was mainly expressed in the luminal and glandular epithelium,as the endometrium entered the receptive phase,the expression of miR-192-5p in the luminal epithelium decreased significantly.Analysis of the uterine miR-192-5p expression in mice of pseudopregnancy,delayed and activated implantation,artificial decidualization indicated that the embryonic factors were not the main cause of miR-192-5p inhibition during the peri-implantation period.The detection of miR-192-5p highly expressed in the estrus phase and lowly expressed in the diestrus phase of the estrus cycle suggested that this miRNA is associated with the cyclic changes of the endometrium itself.(3)By treating ovariectomized mice with a different set of steroids,we showed that estrogen(E2)significantly upregulated miR-192-5p expression in the endometrial epithelium.Progesterone(P4)could downregulate miR-192-5p expression when acting alone,and suppress miR-192-5p expression to a significant level when acting together with estrogen.When mimicked the hormonal environment during the receptive phase by constitutive treatment of P4 followed by stimulation with E2,the expression of miR-192-5p was significantly repressed,indicating that the downregulation of miR-192-5p during the peri-implantation phase was caused by the combined effect of E2 and P4.(4)In vivo studies revealed that transit upregulation of miR-192-5p impairs endometrial receptivity,as evidenced by the maintenance of microvilli and loss of pinopods on the apical surface of the luminal epithelium.Moreover,the expression of several receptive markers expressed in the endometrial epithelium were disrupted during the receptive phase in mice,suggesting miR-192-5p interfered the performance of epithelium when establishing receptivity.In vitro studies revealed that miR-192-5p was highly expressed in non-receptive endometrial epithelial cell lines(HEC-1-A cells),and lowly expressed in receptive endometrial epithelial cell lines(Ishikawa and RL95-2 cells).Inhibiting miR-192-5p expression in HEC-1-A cells resulted in altered cell shape,downregulation of junctional proteins(such as E-cadherin,ZO-1),and rearrangement of cytoskeletal structures(e.g.,stress fibers,surface microvilli,etc.).In addition,miR-192-5p inhibition downregulated the expression of anti-adhesion molecule Mucin 1,leading to the enhancement of blastocyst adhesion on the surface of HEC-1-A cells.Further investigation revealed that the transcription factor repressor Zinc finger E box-binding homeobox 2(ZEB2)and the cytoskeleton-associated regulator Rho GTPase-activating protein 19(ARHGAP19)are the target genes of miR-192-5p in both mice and humans.These molecules were upregulated during the receptive uteri and endometrial epithelial cells,and manipulating miR-192-5p levels changed the endogenous expression of ZEB2 and ARHGAP19 in vivo and in vitro.In addition,overexpressing ARHGAP19 in HEC-1-A cells could partially recapitulate the phenotype produced by miR-192-5p inhibition,including the rearrangement of cytoskeletal structure and the decrease of E-cadherin.In addition,overexpression of ARHGAP19 led to the redistribution of E-cadherin in HEC-1-A cells,and the cells exhibited a piling-up tendency.These phenomena were similar to the alterations that occur within the endometrial epithelial cells in establishing receptivity,suggesting ARHGAP19 may contribute to the acquisition of a receptive phenotype.In conclusion,this study unraveled the differential expressions of miRNAs in the endometrium during early pregnancy in mice.Besides,the study conducted further investigations on the role of miR-192-5p,one of the miRNAs explored above,in the establishment of endometrial receptivity during the peri-implantation phase.miR-192-5p was highly expressed in non-receptive endometrial epithelium and was involved in maintaining epithelial polarity and anti-adhesive properties on the cell surface.During early pregnancy in mice,miR-192-5p was significantly suppressed by the combined effect of E2 and P4,resulting in the downregulation of anti-adhesive molecule Mucin 1.Meanwhile,the expression of morphology and cytoskeleton-associated genes,such as ZEB2 and ARHGAP19,were released,prompting the rearrangement of junctional proteins and cytoskeleton structures,which eventually led to the loss of epithelial polarity and the acquisition of receptive phenotypes.These findings help elucidate the mechanisms involved in miRNA-based regulation of uterine physiology in early pregnancy,and provide a theoretical reference for improving pregnancy rate in mammalians.Also,miR-192-5p may serve as a novel biomarker for receptivity evaluation in assisted reproduction. |
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