METTL3 is essential for normal progesterone signaling during embryo implantation via m6A-mediated translation control of progesterone receptor.

Embryo implantation, a crucial step in human reproduction, is tightly controlled by estrogen and progesterone (P4) via estrogen receptor alpha and progesterone receptor (PGR), respectively. Here, we report that N6-methyladenosine (m6A), the most abundant mRNA modification in eukaryotes, plays an essential role in embryo implantation through the maintenance of P4 signaling. Conditional deletion of methyltransferase-like 3 (Mettl3), encoding the m6A writer METTL3, in the female reproductive tract using a Cre mouse line with Pgr promoter (Pgr-Cre) resulted in complete implantation failure due to pre-implantation embryo loss and defective uterine receptivity. Moreover, the uterus of Mettl3 null mice failed to respond to artificial decidualization. We further found that Mettl3 deletion was accompanied by a marked decrease in PGR protein expression. Mechanistically, we found that Pgr mRNA is a direct target for METTL3-mediated m6A modification. A luciferase assay revealed that the m6A modification in the 5' untranslated region (5'-UTR) of Pgr mRNA enhances PGR protein translation efficiency in a YTHDF1-dependent manner. Finally, we demonstrated that METTL3 is required for human endometrial stromal cell decidualization in vitro and that the METTL3-PGR axis is conserved between mice and humans. In summary, this study provides evidence that METTL3 is essential for normal P4 signaling during embryo implantation via m6A-mediated translation control of Pgr mRNA.

Aberrant activated Notch1 promotes prostate enlargement driven by androgen signaling via disrupting mitochondrial function in mouse.

The prostate is a vital accessory gonad in the mammalian male reproductive system. With the ever-increasing proportion of the population over 60 years of age worldwide, the incidence of prostate diseases, such as benign prostatic hyperplasia (BPH) and prostate cancer (PCa), is on the rise and is gradually becoming a significant medical problem globally. The notch signaling pathway is essential in regulating prostate early development. However, the potential regulatory mechanism of Notch signaling in prostatic enlargement and hyperplasia remains unclear. In this study, we proved that overactivation of Notch1 signaling in mouse prostatic epithelial cells (OEx) led to prostatic enlargement via enhancing proliferation and inhibiting apoptosis of prostatic epithelial cells. Further study showed that N1ICD/RBPJ directly up-regulated the androgen receptor (AR) and enhanced prostatic sensitivity to androgens. Hyper-proliferation was not found in orchidectomized OEx mice without androgen supply but was observed after Dihydrotestosterone (DHT) supplementation. Our data showed that the number of mitochondrion in prostatic epithelial cells of OEx mice was increased, but the mitochondrial function was impaired, and the essential activity of the mitochondrial respiratory electron transport chain was significantly weakened. Disordered mitochondrial number and metabolic function further resulted in excessive accumulation of reactive oxygen species (ROS). Importantly, anti-oxidant N-Acetyl-L-Cysteine (NAC) therapy could alleviate prostatic hyperplasia caused by the over-activation of Notch1 signaling. Furthermore, we observed the incremental Notch signaling activity in progenitor-like club cells in the scRNA-seq data set of human BPH patients. Moreover, the increased number of TROP2+ progenitors and Club cells was also confirmed in our OEx mice. In conclusion, our study revealed that over-activated Notch1 signaling induces prostatic enlargement by increasing androgen receptor sensitivity, disrupting cellular mitochondrial metabolism, increasing ROS, and a higher number of progenitor cells, all of which can be effectively rescued by NAC treatment.

Comparative transcriptome analysis of experimental cryptorchidism: of mice and cynomolgus monkeys.

In most mammalian species, the testis descends from the abdomen into the scrotum during fetal or neonatal life. The failure of testicular descent, a pathological condition known as cryptorchidism, has long been the subject of scientific interest in a wide range of fields, including medicine, developmental biology, and evolutionary biology. In this study, we analyzed global gene expression changes associated with experimental cryptorchidism in mice by using RNA-seq. A total of 453 differentially expressed genes were identified, of which 236 genes were upregulated, and 217 genes were downregulated. Gene ontology, pathway, and gene network analysis highlighted the activation of inflammatory response in experimental cryptorchidism. By examining the promoter regions of differentially expressed genes, we identified 12 causal transcription factors. In addition, we also induced experimental cryptorchidism in two cynomolgus monkeys and performed RNA-seq. A cross-species comparison was performed at the gene expression level. Our study provides a valuable resource for further understanding the molecular mechanisms of cryptorchidism in mammals.

Notch1 is crucial for decidualization and maintaining the first pregnancy in the mouse†.

The endometrium undergoes a pregnancy-delivery-repair cycle multiple times during the reproductive lifespan in females. Decidualization is one of the critical events for the success of this essential process. We have previously reported that Notch1 is essential for artificial decidualization in mice. However, in a natural pregnancy, the deletion of Notch1 (PgrCre/+Notch1f/f, or Notch1d/d) only affects female fertility in the first 30 days of a 6-month fertility test, but not the later stages. In the present study, we undertook a closer evaluation at the first pregnancy of these mice to attempt to understand this puzzling phenomenon. We observed a large number of pregnancy losses in Notch1d/d mice in their first pregnancy, which led to the subfertility observed in the first 30 days of the fertility test. We then demonstrated that the initial pregnancy loss is a consequence of impaired decidualization. Furthermore, we identified a group of genes that contribute to Notch1 regulated decidualization in a natural pregnancy. Gene ontogeny analysis showed that these differentially expressed genes in the natural pregnancy are involved in cell-cell and cell-matrix interactions, different from genes that have been previously identified from the artificial decidualization model, which contribute to cell proliferation and apoptosis. In summary, we determined that Notch1 is essential for normal decidualization in the mouse uterus only in the first pregnancy but not in subsequent ones.

Cell-Cell Communication at the Embryo Implantation Site of Mouse Uterus Revealed by Single-Cell Analysis.

As a crucial step for human reproduction, embryo implantation is a low-efficiency process. Despite rapid advances in recent years, the molecular mechanism underlying embryo implantation remains poorly understood. Here, we used the mouse as an animal model and generated a single-cell transcriptomic atlas of embryo implantation sites. By analyzing inter-implantation sites of the uterus as control, we were able to identify global gene expression changes associated with embryo implantation in each cell type. Additionally, we predicted signaling interactions between uterine luminal epithelial cells and mural trophectoderm of blastocysts, which represent the key mechanism of embryo implantation. We also predicted signaling interactions between uterine epithelial-stromal crosstalk at implantation sites, which are crucial for post-implantation development. Our data provide a valuable resource for deciphering the molecular mechanism underlying embryo implantation.

Identification of Intercellular Crosstalk between Decidual Cells and Niche Cells in Mice.

Decidualization is a crucial step for human reproduction, which is a prerequisite for embryo implantation, placentation and pregnancy maintenance. Despite rapid advances over recent years, the molecular mechanism underlying decidualization remains poorly understood. Here, we used the mouse as an animal model and generated a single-cell transcriptomic atlas of a mouse uterus during decidualization. By analyzing the undecidualized inter-implantation site of the uterus as a control, we were able to identify global gene expression changes associated with decidualization in each cell type. Additionally, we identified intercellular crosstalk between decidual cells and niche cells, including immune cells, endothelial cells and trophoblast cells. Our data provide a valuable resource for deciphering the molecular mechanism underlying decidualization.

Pooling analysis reveals that galectin-1 is a reliable prognostic biomarker in various cancers.

Galectin-1 is reported to be upregulated in various human cancers. However, the relationship between galectin-1 expression and cancer prognosis has not been systematically assessed. In this study, we searched PubMed, Web of Science, and Embase to collect all relevant studies and a meta-analysis was performed. We found that increased galectin-1 expression was associated with tumor size (odds ratio [OR] = 1.75; 95% confidence interval [CI]: 1.06-2.89; p = 0.029), clinical stage (OR = 3.89; 95% CI: 2.40-6.31; p < 0.001), and poorer differentiation (OR = 1.39; 95% CI: 1.14-1.69; p = 0.001), but not with age (OR = 1.07; 95% CI: 0.82-1.39; p = 0.597), sex (OR = 0.89; 95% CI: 0.74-1.07; p = 0.202), or lymph node metastasis (OR = 2.57; 95% CI: 0.98-6.78; p = 0.056). In addition, we found that high galectin-1 expression levels were associated with poor overall survival (HR = 2.12; 95% CI: 1.71-2.64; p < 0.001). The results were further validated using The Cancer Genome Atlas data set. Moreover, high galectin-1 expression was significantly associated with disease-free survival (hazard ratio [HR] = 1.60; 95% CI: 1.17-2.19; p = 0.003), progression-free survival (HR = 1.93; 95% CI: 1.65-2.25; p < 0.001), and cancer-specific survival (HR = 1.82; 95% CI: 1.30-2.55; p < 0.001). Our meta-analysis demonstrated that galectin-1 might be a useful common biomarker for predicting prognosis in patients with cancer.

Assessment of Embryo-Induced Transcriptomic Changes in Hamster Uterus Using RNA-Seq.

BACKGROUND/AIMS: The mouse is widely used as an animal model for studying human embryo implantation. However, the mouse is unique in that both ovarian progesterone and estrogen are critical to implantation, whereas in the majority of species (e.g. human and hamster) implantation can occur in the presence of progesterone alone. METHODS: In this study, we analyzed embryo-induced transcriptomic changes in the hamster uterus during embryo implantation by using RNA-seq. Differentially expressed genes were characterized by bioinformatic analysis. RESULTS: We identified a total of 781 differentially expressed genes, of which 367 genes were up-regulated and 414 genes were down-regulated at the implantation site compared to the inter-implantation site. Functional clustering and gene network analysis highlighted the cell cycle process in uterus upon embryo implantation. By examining of the promoter regions of differentially expressed genes, we identified 7 causal transcription factors. Additionally, through connectivity map (CMap) analysis, multiple compounds were identified to have potential anti-implantation effects due to their ability to reverse embryo-induced transcriptomic changes. CONCLUSION: Our study provides a valuable resource for in-depth understanding of the mechanism underlying embryo implantation.

Single-cell analysis of mouse uterus at the invasion phase of embryo implantation.

BACKGROUND: Embryo implantation into the uterus is a crucial step for human reproduction. A hypothesis has been proposed that the molecular circuit invented by trophoblasts for invasive embryo implantation during evolution might be misused by cancer cells to promote malignancy. Unfortunately, our current understanding of the molecular mechanism underlying embryo implantation is far from complete. RESULTS: Here we used the mouse as an animal model and generated a single-cell transcriptomic atlas of the embryo implantation site of mouse uterus at the invasion phase of embryo implantation on gestational day 6. We revealed 23 distinct cell clusters, including 5 stromal cell clusters, 2 epithelial cell clusters, 1 smooth muscle cell cluster, 2 pericyte clusters, 4 endothelial cell clusters, and 9 immune cell clusters. Through data analysis, we identified differentially expression changes in all uterine cell types upon embryo implantation. By integrated with single-cell RNA-seq data from E5.5 embryos, we predicted cell-cell crosstalk between trophoblasts and uterine cell types. CONCLUSIONS: Our study provides a valuable resource for understanding of the molecular mechanism of embryo implantation.

Revisiting the Transcriptome Landscape of Pig Embryo Implantation Site at Single-Cell Resolution.

Litter size is one of the most economically important traits in commercial pig farming. It has been estimated that approximately 30% of porcine embryos are lost during the peri-implantation period. Despite rapid advances over recent years, the molecular mechanism underlying embryo implantation in pigs remains poorly understood. In this study, the conceptus together with a small amount of its surrounding endometrial tissues at the implantation site was collected and subjected to single-cell RNA-seq using the 10x platform. Because embryo and maternal endometrium were genetically different, we successfully dissected embryonic cells from maternal endometrial cells in the data according to single nucleotide polymorphism information captured by single-cell RNA-seq. Undoubtedly, the interaction between trophoblast cells and uterine epithelial cells represents the key mechanism of embryo implantation. Using the CellChat tool, we revealed cell-cell communications between these 2 cell types in terms of secreted signaling, ECM-receptor interaction and cell-cell contact. Additionally, by analyzing the non-pregnant endometrium as control, we were able to identify global gene expression changes associated with embryo implantation in each cell type. Our data provide a valuable resource for deciphering the molecular mechanism of embryo implantation in pigs.

Discovery of Novel 3-Amino-4-alkoxyphenylketones as PDE4 Inhibitors with Improved Oral Bioavailability and Safety against Spatial Memory Impairments.

To realize PDE4 inhibitors with good developmental potentiality for the treatment of dementia, structure-based optimizations of lead compound FCPR03 resulted in novel aminophenylketones 9c and 9H with low nanomolar potency, which displayed comparable activity to rolipram, satisfactory bioavailability (F% = 36.92 and 42.96% respectively), and good blood-brain barrier (BBB) permeability switching from the cyclopropyl methoxy group to the cyclopropyl methylamine and the amide group to the corresponding ketone. Emetogenicity evaluation on a combined ketamine/xylazine anesthesia mice alternative model demonstrated that 9H displays no emetogenicity even at an oral dose of 5 mg/kg. In contrast, rolipram and roflumilast displayed emetogenicity at an oral dose of 0.5 mg/kg. In acute toxicological evaluation, 9H showed no obvious toxicological effect on mice when administered at oral doses below 625 mg/kg. Further investigations revealed that 9H improves the memory and cognitive impairment of Alzheimer's disease (AD) model mice induced by Aß25-35.

The Pre-Implantation Embryo Induces Uterine Inflammatory Reaction in Mice.

It has been well established that uterine function during the peri-implantation period is precisely regulated by ovarian estrogen and progesterone. The embryo enters the uterine cavity before implantation. However, the impact of pre-implantation embryo on uterine function is largely unknown. In the present study, we performed RNA-seq analysis of mouse uterus on day 4 morning of natural pregnancy (with embryos in the uterus) and pseudo-pregnancy (without embryos in the uterus). We found that 146 genes were upregulated, and 77 genes were downregulated by the pre-implantation embryo. Gene ontology and gene network analysis highlighted the activation of inflammatory reaction in the uterus. By examining the promoter region of differentially expressed genes, we found that NF-kappaB was a causal transcription factor. Finally, we validated 4 inflammation-related genes by quantitative RT-PCR. These 4 genes are likely the main mediators of the inflammatory reaction in the uterus triggered by the pre-implantation embryo. Our data indicated that the pre-implantation embryo causes uterine inflammatory reaction, which in turn might contribute to the establishment of uterine receptivity and embryo implantation.

Comparative Analysis of Mouse Decidualization Models at the Molecular Level.

The mouse is widely used to study decidualization and there are three well-established mouse models of decidualization, namely natural pregnancy decidualization (NPD), artificial decidualization (AD), and in vitro decidualization (IVD). However, the extent of similarity and difference between these models at the molecular level remains largely unknown. Here, we performed a comparative analysis using the RNA-seq approach. In the NPD model, which is thought to be the golden standard of mouse decidualization, we found a total of 5277 differentially expressed genes, with 3158 genes being up-regulated and 2119 genes being down-regulated. A total of 4294 differentially expressed genes were identified in the AD model: 1127 up-regulated genes and 3167 down-regulated genes. In comparison to NPD, 1977 genes were consistently expressed, whereas only 217 genes were inconsistently expressed, indicating that AD is a reliable model for mouse decidualization. In the IVD model, RNA-seq analysis revealed that 513 genes were up-regulated and 988 genes were down-regulated. Compared to NPD, 310 genes were consistently expressed, whereas 456 genes were inconsistently expressed. Moreover, although the decidualization marker Prl8a2 (prolactin family 8 subfamily a member 2) was up-regulated, the widely-used marker Alpl (alkaline phosphatase liver/bone/kidney) was down-regulated in the IVD model. Therefore, we suggest that the IVD model should be optimized to mimic NPD at the transcriptomic level. Our study contributes to an increase in the knowledge about mouse models of decidualization.

Identification of Gene Expression Changes Associated With Uterine Receptivity in Mice.

The mouse is a widely used animal model for studying human reproduction. Although global gene expression changes associated with human uterine receptivity have been determined by independent groups, the same studies in the mouse are scarce. The extent of similarities/differences between mice and humans on uterine receptivity at the molecular level remains to be determined. In the present study, we analyzed global gene expression changes in receptive uterus on day 4 of pregnancy compared to non-receptive uterus on day 3 of pregnancy in mice. A total of 541 differentially expressed genes were identified, of which 316 genes were up-regulated and 225 genes were down-regulated in receptive uterus compared to non-receptive uterus. Gene ontology and gene network analysis highlighted the activation of inflammatory response in the receptive uterus. By analyzing the promoter sequences of differentially expressed genes, we identified 12 causal transcription factors. Through connectivity map (CMap) analysis, we revealed several compounds with potential anti-receptivity activity. Finally, we performed a cross-species comparison against human uterine receptivity from a published dataset. Our study provides a valuable resource for understanding the molecular mechanism underlying uterine receptivity in mice.