Xiaorui Liu

Characterization of the Transcriptional Complexity of the Receptive and Pre-receptive Endometria of Dairy Goats.

Endometrium receptivity is essential for successful embryo implantation in mammals. However, the lack of genetic information remains an obstacle to understanding the mechanisms underlying the development of a receptive endometrium from the pre-receptive phase in dairy goats. In this study, more than 4 billion high-quality reads were generated and de novo assembled into 102,441 unigenes; these unigenes were annotated using published databases. A total of 3,255 unigenes that were differentially expressed (DEGs) between the PE and RE were discovered in this study (P-values < 0.05). In addition, 76,729–77,102 putative SNPs and 12,837 SSRs were discovered in this study. Bioinformatics analysis of the DEGs revealed a number of biological processes and pathways that are potentially involved in the establishment of the RE, notably including the GO terms proteolysis, apoptosis, and cell adhesion and the KEGG pathways Cell cycle and extracellular matrix (ECM)-receptor interaction. We speculated that ADCY8, VCAN, SPOCK1, THBS1, and THBS2 may play important roles in the development of endometrial receptivity. The de novo assembly provided a good starting point and will serve as a valuable resource for further investigations into endometrium receptivity in dairy goats and future studies on the genomes of goats and other related mammals.

Analysis of Differentially Expressed Genes in Ovaries of Polytocous versus Monotocous Dairy Goats Using Suppressive Subtractive Hybridization

In this study, suppressive subtractive hybridization (SSH) was performed to screen differentially expressed genes in ovarian tissues between polytocous and monotocous goats. From the SSH cDNA library, we obtained 29 differentially expressed sequence tags (ESTs) that have high similarity with known genes in the public database, which were involved in signal transduction, transcriptional regulation, cellular molecular dynamics, cytoskeleton, metabolism and oxidation-reduction. In addition, one novel EST that has no similarity with known genes in the public database was obtained. Eight ESTs, TIMP1, NUCB1, OAZ1, CXXC5, CAPNS2, ATP5A1, Z and RPL5, were further examined for the reproducibility of the SSH data by the real-time quantitative PCR. The results confirmed an increased expression of respective mRNA in ovarian tissues of polytocous goats compared with those of monotocous goats. The study has identified several genes (known or unknown) that may have effect on follicular development, ovulation and egg activation in goats.

MiR-26a promoted endometrial epithelium cells (EECs) proliferation and induced stromal cells (ESCs) apoptosis via the PTEN-PI3K/AKT pathway in dairy goats†

Changes in endometrial cell morphology and function are absolutely necessary for successful embryo implantation. In this study, miR‐26a was widely expressed in dairy goats, and was found to be regulated by β‐estradiol (E2) and progesterone (P4) in endometrial epithelium cells (EECs) as well as stromal cells (ESCs). Furthermore, miR‐26a played a role in the regulation of cells proliferation and apoptosis by directly regulating PTEN and indirectly regulating the PI3K/AKT pathway in EECs but not in ESCs of dairy goats in vitro. In addition, miR‐26a regulated the expression of osteopontin (OPN), vascular endothelial growth factor (VEGF), Cyclooxygenase‐2 (COX‐2), and prolactin (PRL) in endometrial cells. Therefore, we could get a conclusion that miR‐26a had very complex and diverse functions in the endometrial cells during the development of endometrial receptivity in dairy goats. This study provided an efficient platform for studying the regulatory effect of miR‐26a on endometrial cells during the development of endometrial receptivity in dairy goats.

The developmental transcriptome landscape of receptive endometrium during embryo implantation in dairy goats

Under natural conditions, some embryos cannot implant successfully because of the dysfunction of receptive endometrium (RE). Thus, it is imperative for us to study the molecular mechanisms involved in the formation of the RE from pre-receptive endometrium (PE). In this study, the endometrium from gestational day 5 (D5, PE) and gestational day 15 (D15, RE) dairy goats were selected to systematically analyze the transcriptome using strand-specific Ribo-Zero RNA-Seq, >120 million high-quality paired-end reads were generated and 47,616 transcripts were identified in the endometrium of dairy goats. A total of 810 mRNAs were differentially expressed genes (DEGs) between the RE and PE meeting the criteria of P-values<0.05. Bioinformatics analysis of the DEGs revealed that a number of biological processes and pathways were potentially involved in the establishment of the RE, notably energy metabolism and amino acid metabolism. Furthermore, we speculated that CXCL14, IGFBP3, and LGALS15 potentially participated in the development of endometrium. Whats more, putative SNPs, InDels and AS events were identified and analyzed in the endometrium. In a word, this resulting view of the transcriptome greatly enhances the comprehensive transcript catalog and uncovers the global trends in gene expression during the formation of receptive endometrium in dairy goats.

Testin was regulated by circRNA3175-miR182 and inhibited endometrial epithelial cell apoptosis in pre-receptive endometrium of dairy goats

Circular RNAs (circRNAs) in various tissues and cell types from mammalian sources have been studied. However, present knowledge on circRNAs in the development of pre‐receptive endometrium (PE) in dairy goats is limited. In the pre‐receptive endometrium of dairy goats, higher circRNA3175 (ciR3175) levels, lower miR‐182 levels and higher Testin (TES) levels were detected. And ciR3175 could decreased the miR‐182 levels by acting as a miRNA sponge, and miR‐182 could down‐regulated the expression level of TES via the predicted target site in endometrial epithelial cells (EECs) in vitro. Via this way, ciR3175 functioned as a competing endogenous RNAs (ceRNA) that sequestered miR‐182, thereby protecting TES transcripts from miR‐182‐mediated suppression in EECs in vitro. Further, TES inhibited EECs apoptosis by decreasing the expression level of BCL‐2/BAX via the MAPK pathway. Thus, a ciR3175‐miR182‐TES pathway in the endometrium was identified in EECs, and the modulation of which could emerge as a potential target in regulating the pre‐receptive endometrium development in dairy goats.

Circ-8073 regulates CEP55 by sponging miR-449a to promote caprine endometrial epithelial cells proliferation via the PI3K/AKT/mTOR pathway

Circular RNAs (circRNAs) are a large class of endogenous non-coding RNAs that function as regulators in various cells and tissues. Here, the function and mechanism of circRNA8073 (Circ-8073) on endometrial epithelial cells (EECs) and the development of endometrial receptivity were investigated in dairy goats. Circ-8073 could bind to and inhibit miR-449a activity. Circ-8073 binding to the target site of miR-449a had a negative feedback relationship. Centrosomal protein55 (CEP55) was a direct target gene of miR-449a, and Circ-8073 could increase the expression levels of CEP55 by sponging miR-449a in EECs in vitro. Circ-8073/miR-449a/CEP55 could promote EECs proliferation via the PI3K/AKT/mTOR pathway. In addition, CEP55 could regulate the expression levels of vascular endothelial growth factor (VEGF) and forkhead box M1 (FOXM1) in EECs, which contributed to the development of endometrial receptivity. These findings showed that Circ-8073 regulated CEP55 by sponging miR-449a to promote EEC proliferation via the PI3K/AKT/mTOR pathway, suggesting that it could function as a regulator in the development of endometrial receptivity in dairy goats.

MiR-449a regulates caprine endometrial stromal cell apoptosis and endometrial receptivity

In this study, an RT-qPCR analysis showed that the expression levels of miR-449a in the pre-receptive endometrium were lower compared to the receptive endometrium, which is consistent with previous sequencing data (previous investigations). To detect the role of miR-449a in endometrial receptivity, we transfected caprine endometrial stromal cells (ESCs) cultured in vitro with miR-449a mimics. The results revealed that miR-449a decreased the mRNA and protein levels of LGR4 by targeting its 3′-untranslated region. The miR-449a mimics significantly reduced the G1 cell population from 52.56% (mimic NC) to 42.19% with a concordant increase in the G2 and S cell populations from 47.44% (mimic NC) to 57.81%, suggesting that miR-449a caused ESC cell cycle arrest. In addition, the number of apoptotic cells was significantly higher in ESCs transfected with miR-449a mimics (P < 0.05) than in ESCs transfected with mimic NC. In vivo, rich pinopodes were observed on the endometrial surface in the miR-449a agomir group compared with the miR-449a antagomir group. The results of hematoxylin-eosin staining showed that endometrial thickness was significantly increased in the miR-449a agomir group compared with the miR-449a antagomir group. These results suggest that miR-449a could enhance endometrial receptivity.

miR-182 aids in receptive endometrium development in dairy goats by down-regulating PTN expression

Increasing evidence has shown that miRNAs play important roles in endometrium development during the menstrual cycle in humans and many other animals. Our previous data indicated that miR-182 levels increase 15.55-fold and pleiotrophin (PTN) levels decrease 20.97-fold in the receptive endometrium (RE, D15) compared with the pre-receptive endometrium (PE, D5) in dairy goats. The present study shows that miR-182 is widely expressed in different tissues of dairy goats and that its expression levels are regulated by E2 and P4 in endometrial epithelium cells (EECs). We confirmed that PTN is a target of miR-182 and that miR-182 regulates the protein levels of AKT, Bcl-2, FAS, MAPK, Caspase-3 and SP1 in EECs. Furthermore, miR-182 up-regulates or maintains the expression levels of osteopontin (OPN), cyclooxygenase-2 (COX-2) and prolactin receptor (PRLR) in EECs, suggesting that miR-182 is an important regulatory factor in the construction of endometrial receptivity in dairy goats. In conclusion, miR-182 participates in the development of endometrial receptivity by down-regulating PTN and affecting the expression of select apoptosis-related genes and increasing or maintaining the expression levels of OPN, COX-2 and PRLR in the EECs of dairy goats.

LncRNA882 regulates leukemia inhibitory factor (LIF) by sponging miR-15b in the endometrial epithelium cells of dairy goat: ZHANG et al.

Despite the fact that long noncoding RNAs (lncRNAs) play roles in almost all biological processes, little is known about their biological function in the endometrium during the formation of endometrial receptivity. In this study, a comprehensive analysis of lncRNAs in goat endometrial tissues on Day 5 (prereceptive endometrium, PE) and Day 15 (receptive endometrium, RE) of pregnancy was performed by using RNA‐Seq. As a result, 668 differentially expressed lncRNAs (DELs) were found between the PE and RE. Further study showed that lncRNA882, regulated by estrogen (E2) and progestin (P4), could act as competing endogenous RNAs (ceRNAs) for miR‐15b, which inhibited the expression of transforming growth factor‐b‐activated kinase 1 binding protein 3 (TAB3) and then indirectly regulated the level of leukemia inhibitory factor (LIF). This was helpful for the formation of endometrial receptivity in dairy goats. In conclusion, we elucidated the endometrium lncRNA profiles of PE and RE in dairy goats; lncRNA882 acted as a ceRNA for miR‐15b and then indirectly regulated the level of LIF in goat endometrial epithelium cells. Thus, this study helped us to better understand the molecular regulation of endometrial receptivity in dairy goats.