Mouse Uterus Research Articles

Aquaporin 9 causes recurrent spontaneous abortion by inhibiting trophoblast cell epithelial-mesenchymal transformation and invasion through the PI3K/AKT pathway†.

Invasion of the endometrium by trophoblast cells is a key event during pregnancy, although the underlying mechanism remains unclear. Aquaporin 9 (AQP 9) is expressed in many eukaryotes and is associated with cell invasion. The objective of this study was to evaluate the significance of AQP9 in recurrent spontaneous abortion. We screened the GSE22490 dataset and further differentiated aquaporin 9 expression in villi. AQP9 was evaluated as one of the key factors in abortion by injecting AQP9 overexpressed plasmid into the uterus of CD1 mice. Trophoblast cells were transfected with AQP9-overexpressing plasmid or siAQP9 to measure cell proliferation, migration, invasion, and apoptosis. Western blot was used to measure changes in the expression of invasion, epithelial-mesenchymal transformation process, and PI3K/AKT pathway. Finally, the role of AQP9 in PI3K/AKT signaling pathway was determined using the PI3K/AKT inhibitor, LY294002, and activator, 740Y-P. AQP9 is highly expressed in recurrent spontaneous abortion villus. Intrauterine injections of AQP9-overexpressing plasmid into CD1 mice resulted in atrophy and blackness of the gestational sac and increased the absorption rate, it is the causative factor of abortion. AQP9 upregulation inhibited the proliferation, invasion, migration, and epithelial-mesenchymal transformation process in vitro of trophoblast cells and increased cell apoptosis. The opposite result was observed after silencing AQP9. AQP9 overexpression also inhibited the PI3K/AKT pathway. LY294002 and 740Y-P partially recovered AQP9-induced trophoblast invasion and migration via the PI3K/AKT pathway. AQP9 reduces the invasive ability of trophoblast cells by regulating PI3K/AKT signaling pathway, participating in recurrent spontaneous abortion.

The NR2F2-HAND2 signaling axis regulates progesterone actions in the uterus at early pregnancy.

Endometrial function is dependent on a tight crosstalk between the epithelial and stromal cells of the endometrium. This communication is critical to ensure a fertile uterus and relies on progesterone and estrogen signaling to prepare a receptive uterus for embryo implantation in early pregnancy. One of the key mediators of this crosstalk is the orphan nuclear receptor NR2F2, which regulates uterine epithelial receptivity and stromal cell differentiation. In order to determine the molecular mechanism regulated by NR2F2, RNAseq analysis was conducted on the uterus of PgrCre;Nr2f2f/f mice at Day 3.5 of pregnancy. This transcriptomic analysis demonstrated Nr2f2 ablation in Pgr-expressing cells leads to a reduction of Hand2 expression, increased levels of Hand2 downstream effectors Fgf1 and Fgf18, and a transcriptome manifesting suppressed progesterone signaling with an altered immune baseline. ChIPseq analysis conducted on the Day 3.5 pregnant mouse uterus for NR2F2 demonstrated the majority of NR2F2 occupies genomic regions that have H3K27ac and H3K4me1 histone modifications, including the loci of major uterine transcription regulators Hand2, Egr1, and Zbtb16. Furthermore, functional analysis of an NR2F2 occupying site that is conserved between human and mouse was capable to enhance endogenous HAND2 mRNA expression with the CRISPR activator in human endometrial stroma cells. These data establish the NR2F2 dependent regulation of Hand2 in the stroma and identify a cis-acting element for this action. In summary, our findings reveal a role of the NR2F2-HAND2 regulatory axis that determines the uterine transcriptomic pattern in preparation for the endometrial receptivity.

Identification and Imaging of Prostaglandin Isomers Utilizing MS3 Product Ions and Silver Cationization.

Prostaglandins (PGs) are important lipid mediators involved in physiological processes, such as inflammation and pregnancy. The pleiotropic effects of the PG isomers and their differential expression from cell types impose the necessity for studying individual isomers locally in tissue to understand the molecular mechanisms. Currently, mass spectrometry (MS)-based analytical workflows for determining the PG isomers typically require homogenization of the sample and a separation method, which results in a loss of spatial information. Here, we describe a method exploiting the cationization of PGs with silver ions for enhanced sensitivity and tandem MS to distinguish the biologically relevant PG isomers PGE2, PGD2, and Δ12-PGD2. The developed method utilizes characteristic product ions in MS3 for training prediction models and is compatible with direct infusion approaches. We discuss insights into the fragmentation pathways of Ag+ cationized PGs during collision-induced dissociation and demonstrate the high accuracy and robustness of the model to predict isomeric compositions of PGs. The developed method is applied to mass spectrometry imaging (MSI) of mouse uterus implantation sites using silver-doped pneumatically assisted nanospray desorption electrospray ionization and indicates localization to the antimesometrial pole and the luminal epithelium of all isomers with different abundances. Overall, we demonstrate, for the first time, isomeric imaging of major PG isomers with a simple method that is compatible with liquid-based extraction MSI methods.

A regulatory mechanism of mouse kallikrein 1 gene expression by estrogen

Tissue kallikrein 1 (Klk1) is a serine protease that degrades several proteins including insulin-like growth factor binding protein-3 and extracellular matrix molecules. Klk1 mRNA expression in the mouse uterus was increased by estradiol-17β (E2). The present study aimed to clarify the regulatory mechanism for Klk1 expression by estrogen. The promoter analysis of the 5′-flanking region of Klk1 showed that the minimal promoter of Klk1 existed in the −136/+24 region, and the estrogen-responsive region in the −433/-136 region. Tamoxifen increased Klk1 mRNA expression and the promoter activity, suggesting the involvement of AP-1 sites. Site-directed mutagenesis for the putative AP-1 sites in the −433/-136 region showed that the two putative AP-1 sites were involved in the regulation of Klk1 expression. Binding of estrogen receptor α (ERα) to the −433/-136 region was revealed by Chip assay. These results indicated that ERα bound the two putative AP-1 sites and transactivated Klk1 in the mouse uterus.

The role of mesenchymal estrogen receptor 1 in mouse uterus in response to estrogen

Estrogens play important roles in uterine growth and homeostasis through estrogen receptors (ESR1 and ESR2). To address the role of ESR1-mediated tissue events in the murine uterus, we analyzed mice with a mesenchymal tissue-specific knockout of Esr1. Isl1-driven Cre expression generated Esr1 deletion in the uterine stroma and endometrium (Isl-Esr1KO). We showed that overall structure of the Isl1-Esr1KO mouse uterus developed normally, but estrogen responsiveness and subsequent growth were defective, suggesting that mesenchymal ESR1 is necessary for both epithelial and mesenchymal cell proliferation. Furthermore, RNA-seq analysis revealed that the majority of estrogen-induced genes were regulated by stromal ESR1. In control mice, E2 administration induced 9476 up-regulated differentially expressed genes (DEGs), whereas only 1801 up-regulated DEGs were induced by E2 in Isl1-Esr1KO mice. We further showed that stromal ESR1-regulated genes in the mouse uterus included several growth factors and cytokines, which are potential factors that regulate epithelial and stromal tissue interaction, and also genes involved in lipid homeostasis. Therefore, we infer that stromal ESR1 expression is indispensable for most estrogen actions in the mouse uterus and the current results provide new insights into estrogen-mediated homeostasis in female reproductive organs.

Mechanistic study of copper nanoparticle (CuNP) toxicity on the mouse uterus via apelin signaling.

Copper nanoparticles (CuNPs) have been widely utilized in various applications. Due to its wider application, humans are at risk of its exposure. It has been reported that the exposure of CuNPs can lead to organ accumulation and affect organ toxicity. Recent study suggested that CuNPs can translocate into the uterus and affect uterine injury in rat, whereas uterine toxicity still remains unclear. The uterus is an important female organ which is required to sustain pregnancy. Thus, uterine structure and physiology are important. Therefore, this study hypothesized that CuNPs might have a toxic effect on the uterine features of mice. In this study, we have investigated the potential effects of CuNPs on the uterus of mice both in vivo and in vitro. In in vivo study, two groups of female mice were exposed to 5 and 50 mg/kg/day via oral exposure. In vivo results showed that CuNP treatment decreases the body weight and uterus weight and changes in antioxidant status with low estrogen and progesterone levels. Furthermore, CuNPs up-regulated the expression of caspase3 and down-regulated the expression of apelin receptor (APJ). Immunolocalization of apelin showed low abundance in the CuNP-treated uterus. These results suggest a poor apelin signaling in the uterus after CuNP treatment. The in vivo findings were further supported by the in vitro studies. Firstly, the uterus was cultured with 5 and 40 μg of CuNPs, and in the second in vitro experiment, the uterus was divided into 4 groups: control, 40 μg of CuNPs, 40 μg of CuNPs with apelin, and 40 μg of CuNPs with apelin receptor antagonist (ML221). In vitro study showed that CuNPs could directly induce the oxidative stress and apoptosis as well as changing antioxidant status in the uterus. The in vitro apelin 13 (APLN 13) treatments alleviated the expression of BCL2 and improved the antioxidant markers in CuNP-treated uterus. These results also provided an evidence of apelin-mediated signaling in the CuNP-treated uterus. In summary, our results present evidence that CuNPs can stimulate apoptotic pathways which may lead to uterine impairment due to weak apelin signaling.

Leptin increases nitric oxide level via increase in iNOS expression in early pregnant mouse uterus

Leptin increases nitric oxide level via increase in iNOS expression in early pregnant mouse uterus

Pepper fruit (Dennettia tripetala [annonaceae]) seed inhibits uterine contractions via blockade of intracellular calcium release and extracellular calcium influx in ex vivo studies

Dennettia tripetala (DT) is a tree found in rain forest belt of Nigeria, Cameroon and Ivory Coast. The seed is commonly used in preparing food for mothers after delivery because it is believed to play a role in postpartum uterine contraction. There is however no scientific evidence validating this claim. Hence, the study aimed to investigate the effects of DT seed on non-pregnant and pregnant uterus using mouse model. The dried seeds of DT were macerated in methanol and the effects of DT extract were investigated on uterus tissues isolated from non-pregnant and pregnant mice. DT extract was examined on spontaneous uterine contraction, contractions induced by oxytocin, and high potassium chloride (KCl)-induced depolarization. Its effect was also evaluated on calcium ion (Ca2+) release from internal stores. The DT extract suppressed spontaneous uterine contractility in both pregnant and non-pregnant uterus in a concentration-dependent manner. It significantly inhibited uterine contractions elicited by high KCl and oxytocin-induced contractions in both pregnant and non-pregnant uterus. The extract also inhibited uterine contractility induced by oxytocin in the Ca2+-free medium in pregnant and non-pregnant uterus. The study has provided scientific evidence that DT seed extract has uterine relaxing effect in both pregnant and non-pregnant mouse uterus possibly by blocking intracellular Ca2+ released and influx of extracellular Ca2+. The result has shown that DT has no benefit in reducing the risk of post-partum hemorrhage, however, it may have potential benefits in management of dysmenorrhea and preterm labour in non-gravid and gravid uteri respectively.

P-343 The role of Yes-Associated protein (YAP) activity in uterus adenogenesis process

Abstract Study question Could Yes-Associated protein(YAP) be effective in the adenogenesis process that occurs in the mouse uterus in the postnatal period? Summary answer It was observed that the YAP and phospho-YAP levels decreased in mice with inhibited gland development during adenogenesis process when compared to the control groups. What is known already The mouse uterus is composed of undifferentiated mesenchyme surrounded by a single layer of epithelium at birth. On the 5th Postnatal Day (PD), the luminal epithelium begins to bud, and the process called adenogenesis, which is termed as gland development in the uterus, begins. The gland development is evident on the PD 10th day. The uterus shows adult tissue characteristics by PD 15th-20th days. Although it has been stated that various signaling pathways are active in this process, Hippo signaling pathway role in the adenogenesis process have not been clarified yet. Study design, size, duration The uterus with normal gland development and gland development inhibited by Progesterone were evaluated for YAP and phospho-YAP, which is one of the main components of Hippo signaling pathway, by immunohistochemistry, western blotting, and q-RT-PCR methods. For experimental groups, 50 µg/g Progesterone was dissolved in 0.1 mL Sesame oil and for control groups, only 0.1 mL of Sesame oil given subcutaneously on PN 2nd-10th days. Animals were sacrificed on the PN 5th-10th and 15th days. Participants/materials, setting, methods In total of 30 newborn Balb/c female mice were used. They were divided as control (n = 15) and experimental(n = 15) groups. After the injections were finished, mice from both groups at PN5th(n = 5), PN10th(n = 5), and PN15th(n = 5) days were cervical dissociated, and uteri were collected. For immunohistochemical analyses, tissues were taken into buffered formalin, paraffin blocks were made, and the sections were taken. Protein and RNA isolations were performed from uteri for western blotting and q-RT-PCR analyses, respectively. Main results and the role of chance When YAP expression was evaluated by immunohistochemistry, western blotting and q-RT-PCR methods, there was no difference between the groups on the PN 5th day. But there was an active signaling was observed in the control groups on the PN10th day, which is very important for gland formation, and on the PN 15th day, that is effective in the maturation process. On the PN10th and 15th days, it was determined that YAP and phospho-YAP signal expression were decreased significantly in the experimental groups when compared with the control groups in immunohistochemically and protein levels. In addition, these findings were supported by mRNA analysis with YAP. Limitations, reasons for caution The study was performed in vivo mouse model. Further studies can be done with a transgenic animal model. Wider implications of the findings Considering the contribution of uterine gland development to the implantation process, errors in the development of these glands may cause infertility. This study can bring a new perspective to researching the uterus adenogenesis period and causes of infertility and treatment approaches. Trial registration number not applicable

P-361 Is HIF-2α involved in pre-menstrual conditioning of the endometrium to ensure optimal repair and prevent heavy menstrual bleeding?

Abstract Study question Does HIF-2α play a key role in pre-menstrual conditioning of the non-pregnant endometrium to optimise menstrual breakdown/repair and prevent heavy menstrual bleeding (HMB)? Summary answer HIF-2 was higher in secretory endometrium of women with HMB versus controls, but manipulation of HIF-2α in the mouse model did not impact endometrial breakdown/repair. What is known already Hypoxia inducible factor (HIF) is the master regulator of the cellular response to hypoxia and has two common alpha isoforms (HIF-1α/HIF-2α) with overlapping but distinct target genes. We revealed that menstrual endometrial HIF-1α is necessary for normal menstrual endometrial repair. We detected HIF-2α in human endometrium exclusively during the secretory phase. The role of HIF-2α in the non-pregnant endometrium remains undetermined but was important for successful uterine implantation and lung vascular remodelling in mice. We hypothesised that endometrial HIF-2 is required for optimising the endometrial vasculature pre-menstrually to ensure appropriate vasoconstriction and endometrial repair during menstruation. Study design, size, duration HIF-2 was examined in human endometrial tissue and a mouse model of simulated menstruation. Hif2α +/- mice provided genetic reduction of HIF-2 during simulated menses versus wild-type littermates. HIF-2α was also pharmacologically inhibited during the secretory phase (PT2385 vs vehicle controls). To increase HIF-2, Hif1α +/- mice were treated with a HIF stabiliser (DMOG) during the secretory phase to increase HIF-2α followed by decreased HIF-1α at menses –mimicking human endometrial findings in women with HMB. Participants/materials, setting, methods Human secretory endometrium was collected (ethically approved and consented) from women with objectively measured HMB (n = 5) and controls (n = 4) and HIF-2 detected by Western blot. Mouse uterus was collected at the time of progesterone withdrawal (T0, decidualisation) and 8h (T8, endometrial breakdown) and 24h (T24, endometrial repair) after progesterone withdrawal. Histological endometrial repair and menstrual blood loss (MBL) were quantified. Decidualisation markers were examined by RT-qPCR. Endometrial vessel maturity and hypoxia were assessed using immunohistochemistry/immunofluorescence. Main results and the role of chance Human secretory endometrial HIF-2α protein levels were higher in those with HMB compared to controls (P < 0.05). Genetic reduction of HIF-2α in our mouse model of menses (Hif2α +/-) revealed no significant differences in endometrial breakdown (T8) or repair grade (T24) and no difference in MBL at T8 versus controls. There were no significant differences in expression of decidualisation markers (Prlr/Prl3c1) and vessel maturity at T0 (CD31/α-SMA immunofluorescence staining), or the presence of hypoxia at T8 (pimonidazole immunohistochemistry staining). Pharmacological inhibition of HIF-2 at the time of decidualisation also showed no significant difference in endometrial repair at T24 versus controls. Increasing pre-menstrual HIF-2 via pharmacological stabilisation of HIF-2α in Hif1α+/- mice similarly did not significantly affect endometrial breakdown (T8) or repair (T24) or MBL at T8 versus vehicle treated controls, in contrast to our findings in women with HMB. Limitations, reasons for caution Histological scoring of endometrial breakdown/repair in the menstruation mouse model may not be sensitive enough to detect subtle endometrial changes with increased HIF-2α. As HIF-1α delays endometrial repair, an additional timepoint 36h after progesterone withdrawal may better capture any delay in endometrial repair as a result of increased secretory HIF-2. Wider implications of the findings HIF-2α may play a greater role in implantation than pre-menstrual conditioning of the endometrium to optimise menstrual breakdown/repair and limit menstrual blood loss. Although endometrial breakdown was not significantly affected by HIF-2, the impact on repair warrants further investigation to fully delineate the role of HIF-2 in the non-pregnant endometrium. Trial registration number Not applicable

P-517 Downregulated INHBB in endometrial tissue of recurrent implantation failure patients impeded decidualization through the ADCY1/cAMP signalling pathway

Abstract Study question To identify the mechanism of Inhibin Subunit Beta B (INHBB) in the regulation of human endometrial stromal cells (HESCs) decidualization in recurrent implantation failure (RIF). Summary answer INHBB was decreased in endometrial stromal cells of women with RIF, which attenuated decidualization in vitro by suppressing ADCY1-induced cAMP production and cAMP-mediated signalling. What is known already INHBB encodes a preprotein subunit of inhibin/ activin, functional cytokines belonging to the transforming growth factor-β (TGF-β) family. INHBB mRNAs were expressed in human decidualized endometrium from the first trimester of pregnancy and increased as the gestation progressed. Furthermore, INHBB expression increases in the mouse uterus in areas undergoing decidualization. These studies indicated that INHBB might play an essential role in endometrial decidualization. Study design, size, duration The Medical Ethics Committee of Nanjing Drum Tower Hospital approved this study (No. 2013-081-01). Written informed consent was obtained from each participant. Between January 2020 and November 2021, 28 infertile and 18 fertile control women aged 20-40 years with normal and regular menstrual cycles (25-35 days) and no history of steroid hormone medication in the last 3 months were included in the study. Participants/materials, setting, methods RNA-seq was conducted to identify the differentially expressed genes in the endometria from control and RIF patients. RT-qPCR, WB, and immunohistochemistry were performed to analyse the expression levels of INHBB in decidualised HESCs. RT-qPCR and immunofluorescence were used to detect changes in decidualization after knockdown INHBB. Then, RNA-seq was used to dig out the mechanism of INHBB regulating decidualisation. Main results and the role of chance Our results showed significantly reduced expression of INHBB in endometrial stromal cells of women with RIF. In addition, INHBB was increased in the endometrium of the secretory phase and significantly induced in in-vitro decidualization of HESCs. Notably, with RNA-seq and siRNA-mediated knockdown approaches, we demonstrated that the INHBB-ADCY1-mediated cAMP signalling pathway regulates the reduction of decidualization. We found a positive association between the expression of INHBB and ADCY1 in endometria with RIF (R2= 0.3785, P = 0.0005). Limitations, reasons for caution Our results indicated that INHBB regulates cAMP level by inducing the expression of ADCY1, while the regulation mechanism between INHBB and ADCY1 in endometrial decidualization requires further exploration. Wider implications of the findings The decline of INHBB in HESCs suppressed ADCY1-induced cAMP production and cAMP-mediated signalling, which attenuated decidualization in RIF patients, indicating that INHBB is an essential component in the decidualization process. Trial registration number not applicable

Expressions of Notch signalling pathway members during early pregnancy in mice.

Although pregnancy is initiated and maintained through highly complex mechanisms, it is essential to understand the events that occur before and during early pregnancy to understand a healthy implantation process. The Notch signal, thought to be involved in this process, is frequently the subject of research with its different aspects. To better understand the role of Notch signaling in the peri-implantation period of the mouse uterus, we investigated the state of expression and localization of Notch 3, Notch 4, Rbp-J, Hes1, Hes7, Hey2, HeyL, and Fbw7 in the uterus and implantation sites in early pregnancy. Balb/C mice were divided into groups D1, D4, D5, D6, and D8. For D5 and D6 groups, implantation sites were identified by intravenous injection of Chicago blue. IHC, WB, and QRT-PCR methods were used. Notch 3 was very strong positive on the 4th day of pregnancy. Notch 4 was highly expressed on days 4, 5, 6, and 8 of pregnancy when P4 levels were high. Hes 1 level was at the lowest on the 4th day of pregnancy. Hes 7 protein expression gradually increased from D1 to D8 in the uteri and implantation sites. Hey 2 expression was at the highest level on the 1st and 4th days. Hey L expression was on the apical of the glands. Fbxw7 that expression was high on the 1st and 4th days of pregnancy. Notch signaling may play an essential role in regulating endometrial receptivity. In addition, our Hes7 results are new to the literature.

CFP1 governs uterine epigenetic landscapes to intervene in progesterone responses for uterine physiology and suppression of endometriosis

Progesterone (P4) is required for the preparation of the endometrium for a successful pregnancy. P4 resistance is a leading cause of the pathogenesis of endometrial disorders like endometriosis, often leading to infertility; however, the underlying epigenetic cause remains unclear. Here we demonstrate that CFP1, a regulator of H3K4me3, is required for maintaining epigenetic landscapes of P4-progesterone receptor (PGR) signaling networks in the mouse uterus. Cfp1f/f;Pgr-Cre (Cfp1d/d) mice showed impaired P4 responses, leading to complete failure of embryo implantation. mRNA and chromatin immunoprecipitation sequencing analyses showed that CFP1 regulates uterine mRNA profiles not only in H3K4me3-dependent but also in H3K4me3-independent manners. CFP1 directly regulates important P4 response genes, including Gata2, Sox17, and Ihh, which activate smoothened signaling pathway in the uterus. In a mouse model of endometriosis, Cfp1d/d ectopic lesions showed P4 resistance, which was rescued by a smoothened agonist. In human endometriosis, CFP1 was significantly downregulated, and expression levels between CFP1 and these P4 targets are positively related regardless of PGR levels. In brief, our study provides that CFP1 intervenes in the P4-epigenome-transcriptome networks for uterine receptivity for embryo implantation and the pathogenesis of endometriosis.

Oroxylin A relieves intrauterine adhesion in mice through inhibiting macrophage pyroptosis via SIRT3-SOD2-ROS pathway.

Intrauterine adhesion (IUA) is manifested by endometrial fibrosis and inflammation, which seriously affects female reproductive health. Macrophages are mainly inflammatory cells and have been reported to participate in the fibrosis of IUA. Oroxylin A (OA), a kind of flavonoid compounds, was showed to possess the inhibitory effects on inflammation and fibrosis. However, the role of OA in IUA remains unclear. In the present study, we found that OA effectively alleviated the level of inflammation and uterine fibrosis in IUA mice. OA also decreased the macrophage pyroptosis which increased in uteri of IUA mice. Pyroptosis is a programmed cell death accompanied by an inflammatory response. Moreover, OA repressed the mediators of pyroptosis including the expression of NOD-like receptor family pyrin domain containing 3 (NLRP3), caspase-1 and Gasdermin D (GSDMD) and the release of IL-1β, IL-18 and cleaved-caspase-1 in J774A.1 cells induced by LPS/ATP in vitro. Mechanistically, the alleviation of OA on uterine fibrosis is achieved by inhibiting macrophage pyroptosis via SIRT3-SOD2-ROS pathway. Our data indicate that OA may serve as an effective agent for the treatment of the endometrial fibrosis with IUA.

139 Cyclic changes in uterine epithelial sodium channel in mares

Epithelial sodium channel (ENaC) has been localized within many organs of various species, including the uterus of mice, rats, and humans. Additionally, ENaC expression within the rodent uterus was differentially influenced by 17β-estradiol (E2) and progesterone (P4), and has been proposed to be the major mechanism regulating uterine fluid absorption and secretion. In the equine, ENaC has been observed in the trachea, hooves, and endometrium; however, the potential relationship between E2, P4, and ENaC in the mare endometrium has yet to be elucidated. The aim of this study was to investigate possible interactions between E2, P4, and ENaC from cycling mares using immunohistochemistry (IHC). Estrus was synchronized for all mares (n = 13) by daily administration of oral altrenogest (ALT) for 15 consecutive days. On the last day of ALT treatment, all mares received 5 mg of dinoprost IM. Following synchronization, all mares were teased daily with a stallion. Transrectal ultrasonography (US) for observation of ovarian activity and day of ovulation (OV; d0) were completed every other day until OV. On d10, US, blood collection via jugular venipuncture, and a uterine biopsy sample were collected. Biopsy samples were fixed to later complete ENaC evaluation by IHC. A uterine biopsysample and blood collection had also been similarly collected and processed for each mare during estrus when she simultaneously had a ≥ 30mm Graafian follicle present and was displaying estrual behaviors. For statistical comparison, a paired t-test was applied. Mares averaged an estrus duration of 4.85 ± 1.86 d. Plasma E2 was 2.27 ± 0.45 pg/mL on d10 and 3.58 ± 0.48 pg/mL on estrus sample day. Plasma P4 concentration was significantly higher (P < 0.01) on d10 when compared with estrus sample day (8.53 ± 1.17 ng/mL vs 0.04 ± 0.02 ng/mL, respectively). The percentage of positively stained ENaC cells was significantly higher (P < 0.01) on d10 when compared with estrus sample day (81.27 ± 3.19% vs 58.84 ± 3.23%). In conclusion, our findings indicate that sex steroids, specifically P4, may regulate ENaC within the mare endometrium, with ENaC being increased during diestrus and decreased during estrus. Collectively, the changes of ENaC concentrations during the estrous cycle might impact the critically sensitive fluid environment within the mare uterus.

Endocannabinoid System Components of the Female Mouse Reproductive Tract Are Modulated during Reproductive Aging.

The endocannabinoid (eCB) system has gained ground as a key modulator of several female fertility-related processes, under physiological/pathological conditions. Nevertheless, its modulation during reproductive aging remains unclear. This study aimed to investigate the expression levels of the main receptors (cannabinoid receptor 1,CB1; cannabinoid receptor 2, CB2; G-protein coupled receptor, GPR55; and transient receptor potential vanilloid type 1 channel, TRPV1) and metabolic enzymes (N-acylphosphatidylethanolamine phospholipase D, NAPE-PLD; fatty acid amide hydrolase, FAAH; monoacylglycerol lipase, MAGL; and diacylglycerol lipase, DAGL-α and -β) of this system in the ovaries, oviducts, and uteri of mice at prepubertal, adult, late reproductive, and post-reproductive stages through quantitative ELISA and immunohistochemistry. The ELISA showed that among the receptors, TRPV1 had the highest expression and significantly increased during aging. Among the enzymes, NAPE-PLD, FAAH, and DAGL-β were the most expressed in these organs at all ages, and increased age-dependently. Immunohistochemistry revealed that, regardless of age, NAPE-PLD and FAAH were mainly found in the epithelial cells facing the lumen of the oviduct and uteri. Moreover, in ovaries, NAPE-PLD was predominant in the granulosa cells, while FAAH was sparse in the stromal compartment. Of note, the age-dependent increase in TRPV1 and DAGL-β could be indicative of increased inflammation, while that of NAPE-PLD and FAAH could suggest the need to tightly control the levels of the eCB anandamide at late reproductive age. These findings offer new insights into the role of the eCB system in female reproduction, with potential for therapeutic exploitation.

TXA2 mediates LPA1-stimulated uterine contraction in late pregnant mouse

Lysophosphatidic acid (LPA) is known to increase uterine contraction in the estrus cycle and early pregnancy, however, the effect of LPA in late pregnant uterus and its mechanisms are not clear. In the present study, we show the LPA receptor subtypes expressed and the mechanism of LPA-induced contractions in late pregnant mouse uterus. We determined the relative mRNA expression of LPA receptor genes by quantitative PCR and elicited log concentration-response curves to oleoyl-L-α-LPA by performing tension experiments in the presence and absence of nonselective and selective receptor antagonists and inhibitors of the TXA2 pathway. LPA1 was the most highly expressed receptor subtype in the late pregnant mouse uterus and LPA1/2/3 agonist (Oleoyl-L-α LPA) elicited increased contractions in this tissue that had lesser efficacy compared to oxytocin. LPA1/3 antagonist, Ki-16425, and a potent LPA1 antagonist (AM-095) significantly inhibited the LPA-induced contractions. Further, the nonselective COX inhibitor, indomethacin, and potent thromboxane A2 synthase inhibitor, furegrelate significantly impaired LPA-induced contractions. Moreover, selective thromboxane receptor (TP) antagonist, SQ-29548, and Rho kinase inhibitor, Y-27632 almost eliminated LPA-induced uterine contractions. LPA1 stimulation elicits contractions in the late pregnant mouse uterus using the contractile prostanoid, TXA2 and may be targeted to induce labor in uterine dysfunctions/ dystocia.

Redundant roles of AKT1 and AKT2 in fertility, estrous cyclicity and endometrial gland development.

Apoptosis and cell survival regulation are crucial processes during the estrous cycle to prepare a receptive uterus during implantation for successful recognition of pregnancy. PI3K/AKT signaling has a crucial role during gestation and AKT isoforms (1, 2 or 3) are regulated differently in the endometrium during the estrous cycle and embryo implantation. However, the specific roles of these isoforms are still unclear. We have previously shown that AKT isoforms expression during the rat estrous cycle and gestation are differently regulated. The present study aimed to establish the specific role of AKT isoforms in the mouse uterus. The hypothesis is that a dysregulation of AKT isoforms expression could cause fertility-related issues in an isoform-specific manner. With four different mouse models and in-house crossbreeding, all isoforms KO combinations (single, double and triple) were obtained in PGR expressing tissues. The results demonstrated that in absence of one or more AKT isoforms, female fertility was decreased. Mainly, we have observed smaller litter size, specifically in Akt1-2 KO mice. Additionally, we have found Akt1-2-3 KO mice to be fully infertile. Estrous cyclicity was also disrupted in Akt1-2 KO mice with longer diestrus stage. Moreover, the number of endometrial glands was decreased throughout the estrous cycle suggesting an important role in gland development for AKT1 and AKT2. Our results suggest specific roles between each isoform, but also a partially redundant function of AKT1 and AKT2 in litter size, estrous cyclicity and endometrial gland development. This highlights the importance of AKT in the physiological regulation of mouse fertility.

Protective effect of the total flavonoids from Clinopodium chinense against LPS-induced mice endometritis by inhibiting NLRP3 inflammasome-mediated pyroptosis

Ethnopharmacological relevanceClinopodium chinense (Benth.) O. Kuntze (C. chinense) is a Chinese herbal medicine used in treating gynecological hemorrhagic diseases for hundreds of years. Flavonoids are one kind of the major components in C. chinense. The flavonoids of C. chinense (TFC) have a vital role in treating endometritis but the underlying therapeutic mechanisms of TFC against endometritis have been rarely reported. Aim of the studyTo elucidate the therapeutic effect and possible mechanisms of TFC against lipopolysaccharide (LPS)-induced endometritis in vivo and LPS-induced primary mouse endometrial epithelial cells (MEECs) injury in vitro. Materials and methodsThe holistic phytochemicals of the TFC and TFC-contained serum were screened and identified using UPLC-Q-TOF-MS. The model of endometritis was established by intrauterine injection of LPS (5 mg/mL) into female BALB/c mice, and the model mice were treated with TFC for 7 days. The value of MPO was measured by Myeloperoxidase assay kit, the pathological changes in the endometrium were evaluated using H&E staining and transmission electron microscope (TEM), the secretions of IL-18, IL-1β and TNF-α were determined by ELISA kits, the mRNA expressions of IL-18, IL-1β and TNF-α were determined by RT-PCR assay, and the protein levels of TLR4, IKBα, p-IKBα, p65, p-p65, caspase-1, ASC, NLRP3 and GSDMD were measured by Western blot. Subsequently, MEECs were isolated from the uterus of pregnant female mice, injured by LPS for 24 h and incubated with the TFC-contained serum. Finally, cell viability, LDH release, hoechst 33342/PI staining, immunofluorescence staining, scanning electron microscope observation, ELISA assay, RT-PCR detection and Western blot analysis were carried out to further validate the therapeutic effect and the underlying mechanisms of TFC. ResultsA total of 6 compounds in the plasma of mice after being intragastric administrated of TFC were identified. The results in vivo showed that TFC significantly reduced MPO value and alleviated pathological injury of the endometrium. Furthermore, TFC significantly decreased the serum IL-18, IL-1β and TNF-α levels, and the mRNA levels of IL-18, IL-1β and TNF-α. TFC also inhibited the expressions of TLR4, p-IKBα, p-p65, caspase-1, ASC, NLRP3 and GSDMD. Besides, compared with the model group in MEECs cells, TFC-contained serum prevented pyroptosis, decreased the levels of IL-18 and IL-1β, and inhibited the mRNA expressions of IL-18, IL-1β and GSDMD. TFC-contained serum also reversed the activation of NLRP3 inflammasome caused by nigericin, and restrainted the translocation of NF-κB into nuclear. ConclusionsTFC protects mice endometritis from the injury of LPS via suppressing the activation of NLRP3 inflammasome and pyroptosis, the underlying mechanisms of which were related to restraining the TLR4/NF-κB/NLRP3 pathway activation.

The Inactivation of Hippo Signaling Pathway Promotes the Development of Adenomyosis by Regulating EMT, Proliferation, and Apoptosis of Cells.

Adenomyosis is a benign gynecological disease. The pathogenesis of adenomyosis is still unclear. The Hippo signaling pathway is highly conserved in vivo and associated with endometriosis and various cancers. Our objective was to study the expression of Hippo signaling pathway-related proteins in the uterus of mice with and without adenomyosis. We also sought to determine the relationship between the Hippo signaling pathway and cell migration, invasion, proliferation, and apoptosis in adenomyosis. The inactivation of Hippo signaling pathway and abnormal expression of EMT-related proteins were observed in mice with adenomyosis. In vitro, the YAP inhibitor verteporfin can inhibit the proliferation and migration of Ishikawa cells and promote apoptosis, while inhibiting the EMT process. In addition, intraperitoneal injection of verteporfin inhibits EMT process and proliferation and promotes apoptosis of cells in the uterus of adenomyosis mice. It suggests that the Hippo signaling pathway participates in the EMT, proliferation, and apoptosis of cells in adenomyosis. In conclusion, these results suggest that Hippo signaling pathway may be involved in the development of adenomyosis by regulating EMT, proliferation, and apoptosis of cells, which provide a potential target for the treatment of adenomyosis.