Pathogenicity, tissue tropism and potential vertical transmission of SARSr-CoV-2 in Malayan pangolins.

Malayan pangolin SARS-CoV-2-related coronavirus (SARSr-CoV-2) is closely related to SARS-CoV-2. However, little is known about its pathogenicity in pangolins. Using CT scans we show that SARSr-CoV-2 positive Malayan pangolins are characterized by bilateral ground-glass opacities in lungs in a similar manner to COVID-19 patients. Histological examination and blood gas tests are indicative of dyspnea. SARSr-CoV-2 infected multiple organs in pangolins, with the lungs the major target, and histological expression data revealed that ACE2 and TMPRSS2 were co-expressed with viral RNA. Transcriptome analysis indicated that virus-positive pangolins were likely to have inadequate interferon responses, with relative greater cytokine and chemokine activity in the lung and spleen. Notably, both viral RNA and viral proteins were detected in three pangolin fetuses, providing initial evidence for vertical virus transmission. In sum, our study outlines the biological framework of SARSr-CoV-2 in pangolins, revealing striking similarities to COVID-19 in humans.

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.

Up-regulation of inflammatory, oxidative stress, and apoptotic mediators via inflammatory, oxidative stress, and apoptosis-associated pathways in bovine endometritis.

Endometritis is the inflammation of the endothelial lining of the uterine lumen and is multifactorial in etiology. Escherichia (E.) coli is a Gram-negative bacteria, generally considered as a primary causative agent for bovine endometritis. Bovine endometritis is characterized by the activation of Toll-like receptors (TLRs) by E. coli, which in turn triggers inflammation, oxidative stress, and apoptosis. The objective of this study was to investigate the gene expression of inflammatory, oxidative stress, and apoptotic markers related to endometritis in the uteri of cows. Twenty uterine tissues were collected from the abattoir. Histologically, congestion, edema, hyperemia, and hemorrhagic lesions with massive infiltration of neutrophil and cell necrosis were detected markedly (P < 0.05) in infected uterine samples. Additionally, we identify E. coli using the ybbW gene (177 base pairs; E. coli-specific gene) from infected uterine samples. Moreover, qPCR and western blot results indicated that TLR2, TLR4, proinflammatory mediators, and apoptosis-mediated genes upregulated except Bcl-2, which is antiapoptotic, and there were downregulations of oxidative stress-related genes in the infected uterine tissue. The results of our study suggested that different gene expression regimes related to the immune system reflex were activated in infected uteri. This research gives a novel understanding of active immunological response in bovine endometritis.

Aberrant activation of Notch1 signaling in the mouse uterine epithelium promotes hyper-proliferation by increasing estrogen sensitivity.

In mammals, the endometrium undergoes dynamic changes in response to estrogen and progesterone to prepare for blastocyst implantation. Two distinct types of endometrial epithelial cells, the luminal (LE) and glandular (GE) epithelial cells play different functional roles during this physiological process. Previously, we have reported that Notch signaling plays multiple roles in embryo implantation, decidualization, and postpartum repair. Here, using the uterine epithelial-specific Ltf-iCre, we showed that Notch1 signaling over-activation in the endometrial epithelium caused dysfunction of the epithelium during the estrous cycle, resulting in hyper-proliferation. During pregnancy, it further led to dysregulation of estrogen and progesterone signaling, resulting in infertility in these animals. Using 3D organoids, we showed that over-activation of Notch1 signaling increased the proliferative potential of both LE and GE cells and reduced the difference in transcription profiles between them, suggesting disrupted differentiation of the uterine epithelium. In addition, we demonstrated that both canonical and non-canonical Notch signaling contributed to the hyper-proliferation of GE cells, but only the non-canonical pathway was involved with estrogen sensitivity in the GE cells. These findings provided insights into the effects of Notch1 signaling on the proliferation, differentiation, and function of the uterine epithelium. This study demonstrated the important roles of Notch1 signaling in regulating hormone response and differentiation of endometrial epithelial cells and provides an opportunity for future studies in estrogen-dependent diseases, such as endometriosis.

Notch effector recombination signal binding protein for immunoglobulin kappa J signaling is required for the initiation of endometrial stromal cell decidualization†.

The Notch signaling pathway is required for reproductive success. This pathway activates its transcriptional effector, recombination signal binding protein for immunoglobulin kappa J (Rbpj), to induce transcription of its target genes. This signaling pathway is required for successful decidualization, implantation, and uterine repair following parturition. To identify the compartmental specific roles of the Notch signaling pathway in the establishment of pregnancy, we generated epithelial and decidual stromal cell specific knockouts of Rbpj utilizing lactoferrin iCre and Prl8A2 iCre, respectively. Both conditional knockout mouse models were fertile. The Rbpj epithelial knockout mice displayed 27% resorption sites at E15.5, but this did not significantly impact the number of live born pups compared with controls. In addition, the Rbpj epithelial knockout mice displayed increased estrogen signaling in their stromal compartment. Given that both mouse models exhibited fertility comparable to control animals, the epithelial and stromal specific nature of the iCre recombinases utilized, and previously published Rbpj total uterine knockout mouse models, we conclude that Notch effector Rbpj signaling is required at the initiation of pregnancy to support decidualization in stromal cells, but that Rbpj is not required in the epithelial compartment nor is it required for post-implantation pregnancy success.

Zearalenone affects the growth of endometriosis via estrogen signaling and inflammatory pathways.

Endometriosis is a chronic, inflammatory, estrogen-dependent gynecological disease characterized by the growth of endometrial stromal cells and glands outside the uterine cavity in response to hormones, which commonly occurs in reproductive-age women. Zearalenone (ZEA) is a toxic metabolite produced by Fusarium, which acts as estrogen activity because of the similarity of its structure to estrogen. In this study, we used an endometriosis mouse model: 15 days after ovariectomy, endometrial fragments were sutured on the pelvic wall, and exogenous estrogen was supplied using an estrogen-releasing silicone tube embedded subcutaneously. Mice were treated with different doses of ZEA by gavage for 21 days. The results show that ZEA significantly inhibited the growth of ectopic endometrium in a dose-dependent manner. The proliferation of cells decreased while apoptosis increased in the ectopic tissues of ZEA-treated mice compared to the vehicle group. The expression of estrogen receptor-α and its downstream targets MUC1 and p-AKT decreased, indicating an impaired estrogen signaling activity by ZEA treatment. In addition, the decreased expression of pro-inflammatory cytokine Tnf-α, Il-1ß, and Il-6, the lower number of macrophages and neutrophils cells, and the inhibited NF-κB signaling pathway suggest the inflammatory response in the ectopic endometrium was also suppressed by ZEA treatment. However, when the exogenous estrogen supply is removed, ZEA, in turn, plays an estrogen-like role that promotes cell proliferation in the ectopic endometrium. In summary, our data suggest ZEA acts as an antagonist in endometriotic tissue when estrogen is sufficient but turns to estrogenic activity in the absence of estrogen in the development of endometriosis. ZEA also inhibits ectopic tissue growth by inhibiting inflammatory response in the endometriosis model.

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.

[Research progress in process and mechanisms of embryo implantation].

The onset of tight connection between embryo and uterine endometrium terms "embryo implantation", the beginning and a key step of mammalian pregnancy. Defective implantation leads to failure of pregnancy and infertility. In recent years, along with the technological advance, researches on embryo implantation have achieved great advances. This paper reviews the key research achievements that have been reached in the last decade in the field of embryo implantation, focusing on the changes, roles, and underlying mechanisms of both luminal and glandular epithelia during implantation process, as well as their interactions with embryo trophoblast cells and endometrial stromal cells.

Autoimmune Regulator is required in female mice for optimal embryonic development and implantation†.

Autoimmune Regulator (AIRE) regulates central immune tolerance by inducing expression of tissue-restricted antigens in thymic medullary epithelial cells, thereby ensuring elimination of autoreactive T cells. Aire mutations in humans and targeted Aire deletion in mice result in multiorgan autoimmune disease, known in humans as autoimmune polyglandular syndrome type 1 (APS-1). APS-1 is characterized by the presence of adrenal insufficiency, chronic mucosal candidiasis, and/or hypoparathyroidism. Additionally, females often present with gonadal insufficiency and infertility. Aire-deficiency (KO) in mice results in oophoritis and age-dependent depletion of follicular reserves. Here, we found that while the majority of young 6-week-old Aire-KO females had normal follicular reserves, mating behavior, and ovulation rates, 50% of females experienced embryonic loss between gestation day (GD) 5.5 and 7.5 that could not be attributed to insufficient progesterone production or decidualization. The quality of GD0.5 embryos recovered from Aire KO mice was reduced, and when cultured in vitro, embryos displayed limited developmental capacity in comparison to those recovered from wild-type (WT) mice. Further, embryos flushed from Aire KO dams at GD3.5 were developmentally delayed in comparison to WT controls and had reduced trophoblastic outgrowth in vitro. We conclude that AIRE does not play a direct role in uterine decidualization. Rather, reduced fertility of Aire-deficient females is likely due to multiple factors, including oophoritis, delayed preimplantation development, and compromised implantation. These effects may be explained by autoimmune targeting of the ovary, embryo, or both. Alternatively, altered embryonic development could be due to a direct role for AIRE in early embryogenesis.

RBPJ mediates uterine repair in the mouse and is reduced in women with recurrent pregnancy loss.

Unexplained recurrent pregnancy loss (uRPL) is associated with repeated embryo loss and endometrial repair with elevated endometrial expression of inflammatory cytokines, including IFN-γ. Notch signaling through its transcription factor recombination signal binding protein Jκ (RBPJ) regulates mechanisms including the immune response and repair after tissue injury. Initially, null mutation of RBPJ in the mouse uterus ( Pgrcre/+Rbpjf/f; Rbpj c-KO) results in subfertility, but we have found that these mice become infertile after pregnancy as a result of dysfunctional postpartum uterine repair, including delayed endometrial epithelial and myometrial regeneration. RNA sequencing of postpartum uterine repair sites revealed global up-regulation of inflammatory pathways, including IFN signaling. Consistent with elevated IFN-γ, macrophages were recruited and polarized toward an M1-cytotoxic phenotype, which is associated with preventing repair and promoting further tissue injury. Through embryo transfer experiments, we show that dysfunctional postpartum repair directly impairs future embryo implantation in Rbpj c-KO mice. Last, we clinically correlated our findings from the Rbpj c-KO mouse in women diagnosed with uRPL. Reduced RBPJ in women with uRPL was associated with increased levels of IFN-γ. The data, taken together, indicate that RBPJ regulates inflammation during endometrial repair, which is essential for future pregnancy potential, and its dysregulation may serve as an unidentified contributor to uRPL in women.-Strug, M. R., Su, R.-W., Kim, T. H., Mauriello, A., Ticconi, C., Lessey, B. A., Young, S. L., Lim, J. M., Jeong, J.-W., Fazleabas, A. T. RBPJ mediates uterine repair in the mouse and is reduced in women with recurrent pregnancy loss.

Aberrant activation of canonical Notch1 signaling in the mouse uterus decreases progesterone receptor by hypermethylation and leads to infertility.

In mammalian reproduction, implantation is one of the most critical events. Failure of implantation and the subsequent decidualization contribute to more than 75% of pregnancy losses in women. Our laboratory has previously reported that inhibition of Notch signaling results in impaired decidualization in both women and a transgenic mouse model. In this study, we generated a Notch gain-of-function transgenic mouse by conditionally overexpressing the Notch1 intracellular domain (N1ICD) in the reproductive tract driven by a progesterone receptor (Pgr) -Cre. We show that the overexpression of N1ICD in the uterus results in complete infertility as a consequence of multiple developmental and physiological defects, including the absence of uterine glands and dysregulation of progesterone and estrogen signaling by a Recombination Signal Binding Protein Jκ-dependent signaling mechanism. We further show that the inhibition of progesterone signaling is caused by hypermethylation of its receptor Pgr by Notch1 overexpression through the transcription factor PU.1 and DNA methyltransferase 3b (Dnmt3b). We have generated a mouse model to study the consequence of increased Notch signaling in female reproduction and provide the first evidence, to our knowledge, that Notch signaling can regulate epigenetic modification of the Pgr.

Intrauterine human chorionic gonadotropin infusion in oocyte donors promotes endometrial synchrony and induction of early decidual markers for stromal survival: a randomized clinical trial.

STUDY QUESTION: Does a single intrauterine infusion of human chorionic gonadotropin (hCG) at the time corresponding to a Day 3 embryo transfer in oocyte donors induce favorable molecular changes in the endometrium for embryo implantation? SUMMARY ANSWER: Intrauterine hCG was associated with endometrial synchronization between endometrial glands and stroma following ovarian stimulation and the induction of early decidual markers associated with stromal cell survival. WHAT IS KNOWN ALREADY: The clinical potential for increasing IVF success rates using an intrauterine hCG infusion prior to embryo transfer remains unclear based on previously reported positive and non-significant findings. However, infusion of CG in the non-human primate increases the expression of pro-survival early decidual markers important for endometrial receptivity, including α-smooth muscle actin (α-SMA) and NOTCH1. STUDY DESIGN, SIZE, DURATION: Oocyte donors (n=15) were randomly assigned to receive an intrauterine infusion of 500 IU hCG (n=7) or embryo culture media vehicle (n=8) 3 days following oocyte retrieval during their donor stimulation cycle. Endometrial biopsies were performed 2 days later, followed by either RNA isolation or tissue fixation in formalin and paraffin embedding. PARTICIPANTS/MATERIALS, SETTING, METHODS: Reverse transcription of total RNA from endometrial biopsies generated cDNA, which was used for analysis in the endometrial receptivity array (ERA; n = 5/group) or quantitative RT-PCR to determine relative expression of ESR1, PGR, C3 and NOTCH1. Tissue sections were stained with hematoxylin and eosin followed by blinded staging analysis for dating of endometrial glands and stroma. Immunostaining for ESR1, PGR, α-SMA, C3 and NOTCH1 was performed to determine their tissue localization. MAIN RESULTS AND THE ROLE OF CHANCE: Intrauterine hCG infusion was associated with endometrial synchrony and reprograming of stromal development following ovarian stimulation. ESR1 and PGR were significantly elevated in the endometrium of hCG-treated patients, consistent with earlier staging. The ERA did not predict an overall positive impact of intrauterine hCG on endometrial receptivity. However, ACTA2, encoding α-SMA was significantly increased in response to intrauterine hCG. Similar to the hCG-treated non-human primate, sub-epithelial and peri-vascular α-SMA expression was induced in women following hCG infusion. Other known targets of hCG in the baboon were also found to be increased, including C3 and NOTCH1, which have known roles in endometrial receptivity. LIMITATIONS, REASONS FOR CAUTION: This study differs from our previous work in the hCG-treated non-human primate along with clinical studies in infertile patients. Specifically, we performed a single intrauterine infusion in oocyte donors instead of either continuous hCG via an osmotic mini-pump in the baboon or infusion followed by blastocyst-derived hCG in infertile women undergoing embryo transfer. Therefore, the full impact of intrauterine hCG in promoting endometrial receptivity may not have been evident. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest a potential clinical benefit for intrauterine hCG prior to embryo transfer on Day 3 in counteracting endometrial dyssynchrony from ovarian stimulation and promoting expression of markers important for stromal survival. Finally, there were no obvious negative effects of intrauterine hCG treatment. STUDY FUNDING/COMPETING INTERESTS: Funding for this work was provided by NICHD R01 HD042280 (A.T.F.) and NICHD F30 HD082951 (M.R.S.). C.S. and P.D.-G are co-inventors of the patented ERA, which is owned by IGENOMIX SL and was used in this study, and C.S. is a shareholder in IGENOMIX SL. M.R.-A. is employed by IGENOMIX SL. No other authors have any conflicts of interest to report. TRIAL REGISTRATION NUMBER: This study was registered with ClinicalTrials.gov (NCT01786252). TRIAL REGISTRATION DATE: 5 February 2013. DATE OF FIRST PATIENT'S ENROLLMENT: 10 May 2013.

Implantation and Establishment of Pregnancy in Human and Nonhuman Primates.

Implantation and the establishment of pregnancy are critical for the propagation of the species, but yet remain the limiting steps in human and primate reproduction. Successful implantation requires a competent blastocyst and a receptive endometrium during a specific window of time during the menstrual cycle to initiate the bilateral communication required for the establishment of a successful pregnancy. This chapter provides an overview of these processes and discusses the molecular mechanisms associated with implantation of the blastocyst and decidualization of the uterus in primates.

Therapeutic administration of Luteolin protects against Escherichia coli-derived Lipopolysaccharide-triggered inflammatory response and oxidative injury.

Endometritis reduces reproductive effectiveness and leads to significant financial losses in the dairy sector. Luteolin is a natural phyto-flavonoid compound with many biological activities. However, the therapeutic effect of Luteolin against lipopolysaccharides (LPS)-induced endometritis has not yet been explored. A total of eighty female Kunming mice were randomly assigned into four treatment groups (n = 20). Following a successful initiation of the endometritis model by LPS, Luteolin was intraperitoneally administered three times, at six-hour intervals between each injection in the Luteolin groups. The histopathological findings revealed that Luteolin significantly alleviated uterine injury induced by LPS. Moreover, Luteolin suppressed the synthesis of pro-inflammatory mediators [interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α] while promoting the synthesis of an anti-inflammatory mediator (IL-10) altered by LPS. Furthermore, Luteolin significantly mitigated the LPS-induced oxidative stress by scavenging malondialdehyde (MDA) and reactive oxygen species (ROS), accumulation and boosting the capacity of antioxidant enzyme activities such as superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione peroxidase 1 (Gpx1) in the uterine tissue of mice. Additionally, injection of Luteolin markedly increased the expression of Toll-like receptors (TLR) 4 both at mRNA and protein levels under LPS stimulation. Western blotting and ELISA findings demonstrated that Luteolin suppressed the activation of the NF-κB pathway in response to LPS exposure in the uterine tissue of mice. Notably, Luteolin enhanced the anti-oxidant defense system by activating the Nrf2 signaling pathway under LPS exposure in the uterine tissue of mice. Conclusively, our findings demonstrated that Luteolin effectively alleviated LPS-induced endometritis via modulation of TLR4-associated Nrf2 and NF-κB signaling pathways.

Combined analysis of microRNome and 3'-UTRome reveals a species-specific regulation of progesterone receptor expression in the endometrium of rhesus monkey.

The establishment of endometrial receptivity is a prerequisite for successful pregnancy, which is controlled by a complex mechanism. MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as important regulators of gene expression. However, the contribution of miRNAs in endometrial receptivity is still unknown. Here we used rhesus monkey as an animal model and compared the endometrial miRNA expression profiles during early-secretory (pre-receptive) phase and mid-secretory (receptive) phase by deep sequencing. A set of differentially expressed miRNAs were identified, 8 of which were selected and validated using quantitative RT-PCR. To facilitate the prediction of their target genes, the 3'-UTRome was also determined using tag sequencing of mRNA 3'-termini. Surprisingly, about 50% of the 10,677 genes expressed in the rhesus monkey endometrium exhibited alternative 3'-UTRs. Of special interest, the progesterone receptor (PGR) gene, which is necessary for endometrial receptivity, processes an ultra long 3'-UTR (~10 kb) along with a short variant (~2.5 kb). Evolutionary analysis showed that the 3'-UTR sequences of PGR are poorly conserved between primates and rodents, suggesting a species-biased miRNA binding pattern. We further demonstrated that PGR is a valid target of miR-96 in rhesus monkey and human but not in rodents, whereas the regulation of PGR by miR-375 is rhesus monkey-specific. Additionally, we found that miR-219-5p regulates PGR expression through a primate-specific long non-coding RNA immediately downstream of the PGR locus. Our study provides new insights into the molecular mechanisms underlying endometrial receptivity and presents intriguing species-specific regulatory roles of miRNAs.

Progesterone and DNA damage encourage uterine cell proliferation and decidualization through up-regulating ribonucleotide reductase 2 expression during early pregnancy in mice.

Embryo implantation into the maternal uterus is a crucial step for the successful establishment of mammalian pregnancy. Following the attachment of embryo to the uterine luminal epithelium, uterine stromal cells undergo steroid hormone-dependent decidualization, which is characterized by stromal cell proliferation and differentiation. The mechanisms underlying steroid hormone-induced stromal cell proliferation and differentiation during decidualization are still poorly understood. Ribonucleotide reductase, consisting of two subunits (RRM1 and RRM2), is a rate-limiting enzyme in deoxynucleotide production for DNA synthesis and plays an important role in cell proliferation and tumorgenicity. Based on our microarray analysis, Rrm2 expression was significantly higher at implantation sites compared with interimplantation sites in mouse uterus. However, the expression, regulation, and function of RRM2 in mouse uterus during embryo implantation and decidualization are still unknown. Here we show that although both RRM1 and RRM2 expression are markedly induced in mouse uterine stromal cells undergoing decidualization, only RRM2 is regulated by progesterone, a key regulator of decidualization. Further studies showed that the induction of progesterone on RRM2 expression in stromal cells is mediated by the AKT/c-MYC pathway. RRM2 can also be induced by replication stress and DNA damage during decidualization through the ATR/ATM-CHK1-E2F1 pathway. The weight of implantation sites and deciduoma was effectively reduced by specific inhibitors for RRM2. The expression of decidual/trophoblast prolactin-related protein (Dtprp), a reliable marker for decidualization in mice, was significantly reduced in deciduoma and steroid-induced decidual cells after HU treatment. Therefore, RRM2 may be an important effector of progesterone signaling to induce cell proliferation and decidualization in mouse uterus.

Distinguish Characters of Luminal and Glandular Epithelium from Mouse Uterus Using a Novel Enzyme-Based Separation Method.

The uterine luminal epithelium, glandular epithelium, and stromal cells are vital for the establishment of pregnancy. Previously studies have shown various methods to isolate mouse uterine epithelium and stromal cells, including laser capture microdissection (LCM), enzyme digestion, and immunomagnetic beads. Despite the importance of the endometrial epithelium as the site of implantation and nutritional support for the conceptus, there is no isolated method to separate the luminal epithelium and glandular epithelium. Here, we establish a novel enzyme-based way to separate two types of epithelium and keep their viability. In this article, we analyzed their purity by mRNA level, immunostaining, and transcriptome analysis. Our isolation method revealed several unstudied luminal and glandular epithelial markers in transcriptome analysis. We further demonstrated the viability of the isolated epithelium by 2D and 3D cultures. The results showed that we successfully separated the endometrial luminal epithelium and glandular epithelium. We also provided an experimental model for the following study of the physiological function of the different parts of the uterus and related diseases.

Vanillin prevents the growth of endometriotic lesions through anti-inflammatory and antioxidant pathways in a mouse model.

Endometriosis is an estrogen-dependent chronic inflammatory gynecological disease defined by the presence of endometrial glands and mesenchyme outside the uterine cavity, named ectopic endometrium. Recent studies showed that endometriosis is associated with hormone imbalance, inflammation and oxidative stress. As the main component of vanilla bean extract, vanillin is widely used as a flavoring agent in the food, pharmaceutical, and cosmetic industries. It is known for its anti-inflammatory, antibacterial, and antitumor properties, but its therapeutic efficacy in endometriosis has not been studied. In this study, we evaluated the roles of vanillin in this disease using an induced endometriotic mouse model. The results showed that vanillin significantly inhibited the growth of endometrial lesions. Compared with the control group, the weight and volume of lesions were reduced considerably in the vanillin-treated group, showing its fantastic ability to inhibit cell proliferation and promote apoptosis. In addition, in the treatment group, mRNA expression of the pro-inflammatory cytokines Tnfa, Infg, Il1b, and Il6 was reduced, the number of macrophages and neutrophils was decreased, and the NF-κB signaling pathway was inhibited, indicating that vanillin suppressed the inflammatory response in the ectopic endometrium. Besides, we found that the intensity of tissue reactive oxygen species (ROS) was significantly lower, and mitochondrial complex IV expression was reduced in the vanillin-treated group. Meanwhile, treatment of the immortalized human endometriotic epithelial cell line (11Z) with vanillin resulted in the downregulation of cyclin genes that drive the cell proliferation process, inhibited cell proliferation, promoted apoptosis, and downregulated the expression of LPS-induced inflammatory cytokines. Most importantly, our data showed that the vanillin treatment had only minimal effects on the eutopic endometrium with respect to the pregnancy process, indicating its safety to be used in treating endometriosis in adults. In conclusion, our data suggest that vanillin has potential therapeutic properties for endometriosis as a regulatory molecule of cell proliferation, apoptosis, inflammation, and oxidative stress.

The Influence of the Prolactins on the Development of the Uterus in Neonatal Mice.

The endometrial gland is one of the most important components of the mammalian uterus. However, few studies have been conducted on the regulatory mechanisms of adenogenesis during the development of endometrium. In the present study, we detected the genes expression of 35 different prolactin family members (PRLs) together with the prolactin receptor (PRL-R) in the endometrium of neonatal mice along with the adenogenesis process, to address which prolactin-like genes play a key role during gland development in mice. We found that: (1) The expression of Prl1a1, Prl3d1, Prl5a1, Prl7a1, Prl7a2, Prl7d1, Prl8a6, Prl8a8, and Prl8a9 genes were significantly increased along with the development of uterine glands. Prl7c1 and Prl8a1 were observably up-regulated on Postnatal day 5 (PND5) when the uterine glandular bud invagination begins. Prl3a1, Prl3b1, and Prl7b1 suddenly increased significantly on PND9. But, Prl3c1 and Prl8a2 were markedly down-regulated on PND5 and the expression of Prl6a1 and Prlr were stable extremely. (2) After continuous injection of Progesterone (P4), a well-known method to suppress the endometrial adenogenesis, the expression of Prl1a1, Prl3d1, Prl5a1, Prl7a1, Prl7a2, Prl7d1, Prl8a6, Prl8a8, Prl8a9, and Prlr were suppressed on PND7. And on PND9, Prl1a1, Prl3d1, Prl8a6, Prl8a8, and Prl8a9 were significantly inhibited. (3) Further analysis of the epithelial and stroma showed that these PRLs were mainly expressed in the endometrial stroma of neonatal mice. Our results indicate that multiple PRLs are involved in uterine development and endometrial adenogenesis. Continued progesterone therapy may alter the expression pattern of these PRLs in endometrial stromal cells, thereby altering the interaction and communication between stroma and epithelium, and ultimately leading to complete suppression of endometrial adenogenesis.

Notch1 signaling enhances collagen expression and fibrosis in mouse uterus.

Fibrosis is a pathological process characterized by abnormal activation of fibroblasts with increased synthesis of extracellular matrix components, including collagens. It may lead to loss of proper tissue architecture and organ function in clinical diseases such as systemic sclerosis and liver fibrosis. Excess accumulation of collagens is considered the primary indicator of fibrosis. Notch signaling has been reported to be involved in the fibrosis of many different organs, including the liver. Our previous study showed that the uterine-specific over-activation of canonical Notch1 signaling in the mouse uterus (Pgrcre/+ Rosa26N1ICD/+ , OEx) results in complete infertility as a consequence of multiple developmental and physiological defects, together with increased collagen accumulation evidenced by Masson's staining. In this study, we further detected expressions of all 44 collagen genes in these Notch1 gain-of-function transgenic mice and found that 18 collagens have been largely affected. In another aspect, using an intrauterine adhesion model (IUA), we mimicked fibrosis in the mouse uterine. The results suggested that Notch receptors were upregulated only 3 days after induction, and most of the fibril-forming collagen began to upregulate 6 days after the surgery. Furthermore, when induced IUA in the N1ICD-OEx mice, the expression of collagens and fibrosis levels were significantly enhanced. At last, as a Notch signaling inhibitor, the γ-secretase inhibitor N-[N-(3,5-difl uorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) pretreatment could alleviate the expression of collagens and the symptoms of fibrosis. These results demonstrate that Notch signaling may play a role in upregulating collagens expression in endometrial fibrosis and might be a potential target of fibrosis therapy in the endometrium.