Osmolar Modulation Drives Reversible Cell Cycle Exit and Human Pluripotent Cell Differentiation via NF-κВ and WNT Signaling.

Terminally differentiated cells are commonly regarded as the most stable cell state in adult organisms, characterized by growth arrest while fulfilling their specialized functions. A better understanding of the mechanisms involved in promoting cell cycle exit will improve the ability to differentiate pluripotent cells into mature tissues for both pharmacological and therapeutic use. Here, it demonstrates that a hyperosmolar environment enforces a protective p53-independent quiescent state in immature hepatoma cells and in pluripotent stem cell-derived models of human hepatocytes and endothelial cells. Prolonged culture in hyperosmolar conditions stimulates changes in gene expression promoting functional cell maturation. Interestingly, hyperosmolar conditions do not only trigger growth arrest and cellular maturation but are also necessary to maintain this maturated state, as switching back to plasma osmolarity reverses the changes in expression of maturation and proliferative markers. Transcriptome analysis revealed sequential stages of osmolarity-regulated growth arrest followed by cell maturation, mediated by activation of NF-κВ, and repression of WNT signaling, respectively. This study reveals that a modulated increase in osmolarity serves as a biochemical signal to promote long-term growth arrest and cellular maturation into different lineages, providing a practical method to generate differentiated hiPSCs that resemble their mature counterpart more closely.

TRPM3 as a novel target to alleviate acute oxaliplatin-induced peripheral neuropathic pain.

ABSTRACT: Chemotherapy-induced peripheral neuropathic pain (CIPNP) is an adverse effect observed in up to 80% of patients of cancer on treatment with cytostatic drugs including paclitaxel and oxaliplatin. Chemotherapy-induced peripheral neuropathic pain can be so severe that it limits dose and choice of chemotherapy and has significant negative consequences on the quality of life of survivors. Current treatment options for CIPNP are limited and unsatisfactory. TRPM3 is a calcium-permeable ion channel functionally expressed in peripheral sensory neurons involved in the detection of thermal stimuli. Here, we focus on the possible involvement of TRPM3 in acute oxaliplatin-induced mechanical allodynia and cold hypersensitivity. In vitro calcium microfluorimetry and whole-cell patch-clamp experiments showed that TRPM3 is functionally upregulated in both heterologous and homologous expression systems after acute (24 hours) oxaliplatin treatment, whereas the direct application of oxaliplatin was without effect. In vivo behavioral studies using an acute oxaliplatin model for CIPNP showed the development of cold and mechano hypersensitivity in control mice, which was lacking in TRPM3 deficient mice. In addition, the levels of protein ERK, a marker for neuronal activity, were significantly reduced in dorsal root ganglion neurons derived from TRPM3 deficient mice compared with control after oxaliplatin administration. Moreover, intraperitoneal injection of a TRPM3 antagonist, isosakuranetin, effectively reduced the oxaliplatin-induced pain behavior in response to cold and mechanical stimulation in mice with an acute form of oxaliplatin-induced peripheral neuropathy. In summary, TRPM3 represents a potential new target for the treatment of neuropathic pain in patients undergoing chemotherapy.

Protease secretions by the invading blastocyst induce calcium oscillations in endometrial epithelial cells via the protease-activated receptor 2.

BACKGROUND: Early embryo implantation is a complex phenomenon characterized by the presence of an implantation-competent blastocyst and a receptive endometrium. Embryo development and endometrial receptivity must be synchronized and an adequate two-way dialogue between them is necessary for maternal recognition and implantation. Proteases have been described as blastocyst-secreted proteins involved in the hatching process and early implantation events. These enzymes stimulate intracellular calcium signaling pathways in endometrial epithelial cells (EEC). However, the exact molecular players underlying protease-induced calcium signaling, the subsequent downstream signaling pathways and the biological impact of its activation remain elusive. METHODS: To identify gene expression of the receptors and ion channels of interest in human and mouse endometrial epithelial cells, RNA sequencing, RT-qPCR and in situ hybridization experiments were conducted. Calcium microfluorimetric experiments were performed to study their functional expression. RESULTS: We showed that trypsin evoked intracellular calcium oscillations in EEC of mouse and human, and identified the protease-activated receptor 2 (PAR2) as the molecular entity initiating protease-induced calcium responses in EEC. In addition, this study unraveled the molecular players involved in the downstream signaling of PAR2 by showing that depletion and re-filling of intracellular calcium stores occurs via PLC, IP3R and the STIM1/Orai1 complex. Finally, in vitro experiments in the presence of a specific PAR2 agonist evoked an upregulation of the 'Window of implantation' markers in human endometrial epithelial cells. CONCLUSIONS: These findings provide new insights into the blastocyst-derived protease signaling and allocate a key role for PAR2 as maternal sensor for signals released by the developing blastocyst.

The Impact of Chronic Endometritis on Infertility: Prevalence, Reproductive Outcomes, and the Role of Hysteroscopy as a Screening Tool.

OBJECTIVES: The objective of this study was to examine the prevalence of chronic endometritis (CE) in infertile women, its impact on reproductive outcomes, and the accuracy of hysteroscopy as a screening tool for CE. DESIGN: This was a prospective observational study. PARTICIPANTS: Participants involved in this study were 514 asymptomatic patients with infertility. SETTING: The review was conducted in a tertiary care center. METHODS: The participants underwent a hysteroscopy and endometrial biopsy (EMB). Antibiotics were given for cases of CE. We investigated the prevalence of CE in patients starting assisted reproductive technologies (ART) as a primary outcome. Secondary outcomes were the clinical pregnancy rate (CPR) in the ART cycle after hysteroscopy, EMB, and antibiotic treatment in cases of CE; the cumulative CPR in the subsequent 2 years after hysteroscopy and EMB; the sensitivity and specificity of hysteroscopy as a screening tool compared to EMB as the "gold standard" for diagnosing CE. RESULTS: CE was identified in 2.8% of patients starting ART (11/393). CPRs did not differ significantly between patients with CE and the entire cohort of patients without CE in the subsequent ART cycle (OR: 0.43; 95% CI: 0.09-2.02) or in the 2 years after EMB (OR: 0.56; 95% CI: 0.16-1.97). In a matched control comparison (with matching for age, basal FSH, and cause of infertility), CPR in patients with CE did not differ in the subsequent ART cycle (OR: 0.39; 95% CI: 0.09-1.61); however, their CPR in the 2 years after EMB was significantly lower (OR: 0.22; 95% CI: 0.13-0.38). The sensitivity and specificity of hysteroscopy as a screening tool for diagnosing CE were 8.3% and 90.1%, respectively. LIMITATIONS: Due to our cohort's low CE prevalence, we could not detect significant differences in CPRs. CONCLUSION: CE is rare in our studied population of asymptomatic patients starting ART. Hysteroscopy cannot replace EMB for diagnosing CE.

Urine-Derived Kidney Progenitor Cells in Cystinosis.

Nephropathic cystinosis is an inherited lysosomal storage disorder caused by pathogenic variants in the cystinosin (CTNS) gene and is characterized by the excessive shedding of proximal tubular epithelial cells (PTECs) and podocytes into urine, development of the renal Fanconi syndrome and end-stage kidney disease (ESKD). We hypothesized that in compensation for epithelial cell losses, cystinosis kidneys undertake a regenerative effort, and searched for the presence of kidney progenitor cells (KPCs) in the urine of cystinosis patients. Urine was cultured in a specific progenitor medium to isolate undifferentiated cells. Of these, clones were characterized by qPCR, subjected to a differentiation protocol to PTECs and podocytes and assessed by qPCR, Western blot, immunostainings and functional assays. Cystinosis patients voided high numbers of undifferentiated cells in urine, of which various clonal cell lines showed a high capacity for self-renewal and expressed kidney progenitor markers, which therefore were assigned as cystinosis urine-derived KPCs (Cys-uKPCs). Cys-uKPC clones showed the capacity to differentiate between functional PTECs and/or podocytes. Gene addition with wild-type CTNS using lentiviral vector technology resulted in significant reductions in cystine levels. We conclude that KPCs present in the urine of cystinosis patients can be isolated, differentiated and complemented with CTNS in vitro, serving as a novel tool for disease modeling.

Phenotypic spectrum of the recurrent TRPM3 p.(Val837Met) substitution in seven individuals with global developmental delay and hypotonia.

TRPM3 encodes a transient receptor potential cation channel of the melastatin family, expressed in the central nervous system and in peripheral sensory neurons of the dorsal root ganglia. The recurrent substitution in TRPM3: c.2509G>A, p.(Val837Met) has been associated with syndromic intellectual disability and seizures. In this report, we present the clinical and molecular features of seven previously unreported individuals, identified by exome sequencing, with the recurrent p.(Val837Met) variant and global developmental delay. Other shared clinical features included congenital hypotonia, dysmorphic facial features (broad forehead, deep-set eyes, and down turned mouth), exotropia, and musculoskeletal issues (hip dysplasia, hip dislocation, scoliosis). Seizures were observed in two of seven individuals (febrile seizure in one and generalized tonic-clonic seizures with atonic drops in another), and epileptiform activity was observed in an additional two individuals. This report extends the number of affected individuals to 16 who are heterozygous for the de novo recurrent substitution p.(Val837Met). In contrast with the initial report, epilepsy was not a mandatory feature observed in this series. TRPM3 pathogenic variation should be considered in individuals with global developmental delays, moderate-severe intellectual disability with, or without, childhood-onset epilepsy.

TRP channel expression correlates with the epithelial-mesenchymal transition and high-risk endometrial carcinoma.

Transient receptor potential (TRP) channels excel in cellular sensing as they allow rapid ion influx across the plasma membrane in response to a variety of extracellular cues. Recently, a distinct TRP mRNA expression signature was observed in stromal cells (ESC) and epithelial cells (EEC) of the endometrium, a tissue in which cell phenotypic plasticity is essential for normal functioning. However, it is unknown whether TRP channel mRNA expression is subject to the phenotypic switching that occurs during epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET), and whether TRP channel mRNA expression is associated with aggressive phenotypes in endometrial cancer (EC). Here, we induced EMT and MET in vitro using in primary EEC and ESC, respectively, and analyzed expression and functionality of TRP channels using RT-qPCR and intracellular Ca2+ imaging. The outcome of these experiments showed a strong association between TRPV2 and TRPC1 mRNA expression and the mesenchymal phenotype, whereas TRPM4 mRNA expression correlated with the epithelial phenotype. In line herewith, increased TRPV2 and TRPC1 mRNA expression levels were observed in both primary and metastatic EC biopsies and in primary EC cells with a high EMT status, indicating an association with an aggressive tumor phenotype. Remarkably, TRPV2 mRNA expression in primary EC biopsies was associated with tumor invasiveness and cancer stage. In contrast, increased TRPM4 mRNA expression was observed in EC biopsies with a low EMT status and less aggressive tumor phenotypes. Taken together, this dataset proved for the first time that TRP channel mRNA expression is strongly linked to cellular phenotypes of the endometrium, and that phenotypic transitions caused by either experimental manipulation or malignancy could alter this expression in a predictable manner. These results implicate that TRP channels are viable biomarkers to identify high-risk EC, and potential targets for EC treatment.

Partial Agonistic Actions of Sex Hormone Steroids on TRPM3 Function.

Sex hormone steroidal drugs were reported to have modulating actions on the ion channel TRPM3. Pregnenolone sulphate (PS) presents the most potent known endogenous chemical agonist of TRPM3 and affects several gating modes of the channel. These includes a synergistic action of PS and high temperatures on channel opening and the PS-induced opening of a noncanonical pore in the presence of other TRPM3 modulators. Moreover, human TRPM3 variants associated with neurodevelopmental disease exhibit an increased sensitivity for PS. However, other steroidal sex hormones were reported to influence TRPM3 functions with activating or inhibiting capacity. Here, we aimed to answer how DHEAS, estradiol, progesterone and testosterone act on the various modes of TRPM3 function in the wild-type channel and two-channel variants associated with human disease. By means of calcium imaging and whole-cell patch clamp experiments, we revealed that all four drugs are weak TRPM3 agonists that share a common steroidal interaction site. Furthermore, they exhibit increased activity on TRPM3 at physiological temperatures and in channels that carry disease-associated mutations. Finally, all steroids are able to open the noncanonical pore in wild-type and DHEAS also in mutant TRPM3. Collectively, our data provide new valuable insights in TRPM3 gating, structure-function relationships and ligand sensitivity.

Transient Receptor Potential Channels in the Epithelial-to-Mesenchymal Transition.

The epithelial-to-mesenchymal transition (EMT) is a strictly regulated process that is indispensable for normal development, but it can result in fibrosis and cancer progression. It encompasses a complete alteration of the cellular transcriptomic profile, promoting the expression of genes involved in cellular migration, invasion and proliferation. Extracellular signaling factors driving the EMT process require secondary messengers to convey their effects to their targets. Due to its remarkable properties, calcium represents an ideal candidate to translate molecular messages from receptor to effector. Therefore, calcium-permeable ion channels that facilitate the influx of extracellular calcium into the cytosol can exert major influences on cellular phenotype. Transient receptor potential (TRP) channels represent a superfamily of non-selective cation channels that decode physical and chemical stimuli into cellular behavior. Their role as cellular sensors renders them interesting proteins to study in the context of phenotypic transitions, such as EMT. In this review, we elaborate on the current knowledge regarding TRP channel expression and activity in cellular phenotype and EMT.

Transient receptor potential channel regulation by growth factors.

Calcium (Ca2+) is one of the most universal secondary messengers, owing its success to the immense concentration gradient across the plasma membrane. Dysregulation of Ca2+ homeostasis can result in severe cell dysfunction, thereby initiating several pathologies like tumorigenesis and fibrosis. Transient receptor potential (TRP) channels represent a superfamily of Ca2+-permeable ion channels that convey diverse physical and chemical stimuli into a physiological signal. Their broad expression pattern and gating promiscuity support their potential involvement in the cellular response to an altering environment. Growth factors (GF) are essential biochemical messengers that contribute to these environmental changes. Since Ca2+ is essential in GF signaling, altering TRP channel expression or function could be a valid strategy for GF to exert their effect onto their target. In this review, a comprehensive understanding of the current knowledge regarding the activation and/or modulation of TRP channels by GF is presented.

Mimicking Sampson's Retrograde Menstrual Theory in Rats: A New Rat Model for Ongoing Endometriosis-Associated Pain.

Endometriosis is a prevalent gynecologic disease, defined by dysfunctional endometrium-like lesions outside of the uterine cavity. These lesions are presumably established via retrograde menstruation, i.e., endometrial tissue that flows backwards during menses into the abdomen and deposits on the organs. As ongoing pain is one of the main pain symptoms of patients, an animal model that illuminates this problem is highly anticipated. In the present study, we developed and validated a rat model for ongoing endometriosis-associated pain. First, menstrual endometrial tissue was successfully generated in donor rats, as validated by gross examination, histology and qPCR. Next, endometriosis was induced in recipient animals by intraperitoneal injection of menstrual tissue. This resulted in neuro-angiogenesis as well as established endometriosis lesions, which were similar to their human counterparts, since epithelial and stromal cells were observed. Furthermore, significant differences were noted between control and endometriosis animals concerning bodyweight and posture changes, indicating the presence of ongoing pain in animals with endometriosis. In summary, a rat model for endometriosis was established that reliably mimics the human pathophysiology of endometriosis and in which signs of ongoing pain were detected, thus providing a new research tool for therapy development.

Patient-derived organoids from endometrial disease capture clinical heterogeneity and are amenable to drug screening.

Endometrial disorders represent a major gynaecological burden. Current research models fail to recapitulate the nature and heterogeneity of these diseases, thereby hampering scientific and clinical progress. Here we developed long-term expandable organoids from a broad spectrum of endometrial pathologies. Organoids from endometriosis show disease-associated traits and cancer-linked mutations. Endometrial cancer-derived organoids accurately capture cancer subtypes, replicate the mutational landscape of the tumours and display patient-specific drug responses. Organoids were also established from precancerous pathologies encompassing endometrial hyperplasia and Lynch syndrome, and inherited gene mutations were maintained. Endometrial disease organoids reproduced the original lesion when transplanted in vivo. In summary, we developed multiple organoid models that capture endometrial disease diversity and will provide powerful research models and drug screening and discovery tools.

Functional expression of the mechanosensitive PIEZO1 channel in primary endometrial epithelial cells and endometrial organoids.

Successful pregnancy requires the establishment of a complex dialogue between the implanting embryo and the endometrium. Knowledge regarding molecular candidates involved in this early communication process is inadequate due to limited access to primary human endometrial epithelial cells (EEC). Since pseudo-pregnancy in rodents can be induced by mechanical scratching of an appropriately primed uterus, this study aimed to investigate the expression of mechanosensitive ion channels in EEC. Poking of EEC provoked a robust calcium influx and induced an increase in current densities, which could be blocked by an inhibitor of mechanosensitive ion channels. Interestingly, RNA expression studies showed high expression of PIEZO1 in EEC of mouse and human. Additional analysis provided further evidence for the functional expression of PIEZO1 since stimulation with Yoda1, a chemical agonist of PIEZO1, induced increases in intracellular calcium concentrations and current densities in EEC. Moreover, the ion channel profile of human endometrial organoids (EMO) was validated as a representative model for endometrial epithelial cells. Mechanical and chemical stimulation of EMO induced strong calcium responses supporting the hypothesis of mechanosensitive ion channel expression in endometrial epithelial cells. In conclusion, EEC and EMO functionally express the mechanosensitive PIEZO1 channel that could act as a potential target for the development of novel treatments to further improve successful implantation processes.

Functional Expression of TRP Ion Channels in Endometrial Stromal Cells of Endometriosis Patients.

Endometriosis is a common gynecological disease that is characterized by the presence of functional endometrial-like lesions in the abdominal cavity. Aside from epithelial cells, these lesions consist of stromal cells that have the capacity to migrate, adhere, proliferate, and induce neuro- and lymphangiogenesis, which allows them to survive at ectopic locations. However, the exact underlying mechanisms that regulate these changes are yet to be elucidated. The common ground of these processes, however, is the second messenger, calcium. In this regard, members of the superfamily of transient receptor potential (TRP) ion channels, which are known to be calcium-permeable and expressed in the endometrium, have emerged as key regulators. Here, we assessed the molecular and functional expression of TRP channels in stromal cells isolated from the eutopic endometrium of endometriosis patients and controls. Using RT-qPCR, high mRNA levels of TRPV2, TRPV4, TRPM4, TRPM7, TRPC1, TRPC3, TRPC4, and TRPC6 were observed in the whole endometrium throughout the menstrual cycle. Additionally, and in line with previous reports of control patients, TRPV2, TRPV4, TRPC1/4, and TRPC6 were present in human endometrial stromal cells (hESC) from endometriosis patients both at the molecular and functional level. Moreover, proliferation and migration assays illustrated that these parameters were not affected in stromal cells from endometriosis patients. Furthermore, comparison between eutopic and ectopic endometrial samples revealed that the RNA expression pattern of TRP channels did not differ significantly. Collectively, although a functional expression of specific ion channels in hESCs was found, their expression did not correlate with endometriosis.

Optimization of Endometrial Decidualization in the Menstruating Mouse Model for Preclinical Endometriosis Research.

BACKGROUND: To induce endometrial decidualization in rodents, an intrauterine oil stimulus can be delivered via the nontraumatic vagina or via the traumatic laparotomy. However, there is considerable variation in amount of decidualization using these inducing methods. Therefore, we studied which oil delivery route could achieve the highest rate of endometrial decidualization along the full length of both uterine horns. METHODS: To induce decidualization, ovariectomized C57Bl/6J mice were injected with estrogen (100 ng/day; 3 days). A progesterone pellet (5 mg) was implanted subcutaneously, followed by estrogen injections (5 ng/day; 3 days). Oil (20 µL/horn) was injected in the uterus via laparotomy, laparoscopy, or vagina. Four days later, the pellet was removed, followed by hysterectomy after 4 to 6 hours. Endometrial decidualization was evaluated macroscopically and microscopically using hematoxylin and eosin and desmin staining. Furthermore, uterine weight and hormone levels were measured. RESULTS: The proportion of animals with macroscopic bicornuate decidualization was higher after laparoscopic (83%) and laparotomic (89%) injection than after sham injection (11%). Furthermore, macroscopic bicornuate decidualization was significantly higher after laparotomic injection (89%) compared to the vaginal injection (38%). Uterine weight and endometrial surface area were significantly higher in both laparotomy and laparoscopy groups compared to the sham group, while the relative desmin-positive endometrial surface area was only significantly different between the laparotomy and the sham animals. CONCLUSION: Methods using laparoscopic and laparotomic intrauterine oil injection resulted in a higher amount of decidualized endometrium compared to sham injection, although further optimization is needed to reach full bicornuate decidualization.

Of Mice and Women: A Laparoscopic Mouse Model for Endometriosis.

STUDY OBJECTIVE: To demonstrate how a novel laparoscopic approach allows the development of a mouse model for endometriosis after seeding menstrual endometrium from donor mice into the abdominal cavity of syngeneic recipient mice. DESIGN: A step-by-step video description of the techniques used to adapt the estrous cycle of mice towards a menstrual cycle and to subsequently induce endometriosis via laparoscopic seeding of menstrual endometrium. SETTING: University research institute. ETHICS: All animal experiments were ethically approved by KU Leuven, Belgium (ethical approval number: P031/2013). INTERVENTIONS, MEASUREMENTS, AND MAIN RESULTS: Oophorectomized female C57BL/6JRj mice received a series of estrogen injections. Next, a progesterone pellet was administered, together with a second series of estrogen injections. In addition, decidualization of the endometrium was induced with an intrauterine sesame oil stimulus. Four days later the progesterone pellet was removed and menstruation started [1]. Five hours after the progesterone pellet was removal the uterus was harvested, and the menstrual endometrium was dissected and seeded into the abdominal cavity of syngeneic recipient mice to induce endometriosis [2] using a laparoscopic approach [3]. Uterus and lesions were removed from the recipient mice 1 week after induction, and tissues were immunohistochemically stained for H&E, vimentin, and cytokeratin. CONCLUSION: In this video we show a novel methodology to induce endometriosis in mice using laparoscopic inoculation of syngeneic menstrual endometrium, mimicking Sampson's theory of retrograde menstruation [4]. Compared with currently available rodent models, our model offers a less invasive and more physiologic way for fundamental and preclinical endometriosis research, with a high endometriosis incidence and lesion take rate.

Establishing life is a calcium-dependent TRiP: Transient receptor potential channels in reproduction.

Calcium plays a key role in many different steps of the reproduction process, from germ cell maturation to placental development. However, the exact function and regulation of calcium throughout subsequent reproductive events remains rather enigmatic. Successful pregnancy requires the establishment of a complex dialogue between the implanting embryo and the endometrium. On the one hand, endometrial cell will undergo massive changes to support an implanting embryo, including stromal cell decidualization. On the other hand, trophoblast cells from the trophectoderm surrounding the inner cell mass will differentiate and acquire new functions such as hormone secretion, invasion and migration. The need for calcium in the different gestational processes implicates the presence of specialized ion channels to regulate calcium homeostasis. The superfamily of transient receptor potential (TRP) channels is a class of calcium permeable ion channels that is involved in the transformation of extracellular stimuli into the influx of calcium, inducing and coordinating underlying signaling pathways. Although the necessity of calcium throughout reproduction cannot be negated, the expression and functionality of TRP channels throughout gestation remains elusive. This review provides an overview of the current evidence regarding the expression and function of TRP channels in reproduction.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization.

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

The functional expression of transient receptor potential channels in the mouse endometrium.

STUDY QUESTION: Does mouse endometrial epithelial cells and stromal cells have a similar transient receptor potential (TRP)-channel expression profile and to that found in the human endometrium? SUMMARY ANSWER: Mouse endometrial epithelial and stromal cells have a distinct TRP channel expression profile analogous to what has been found in human endometrium, and hence suggests the mouse a good model to investigate the role of TRP channels in reproduction. WHAT IS KNOWN ALREADY: An optimal intercellular communication between epithelial and stromal endometrial cells is crucial for successful reproduction. Members of the TRP family were recently described in the human endometrial stroma; however their functional expression in murine endometrium remains unspecified. Furthermore, epithelial and stromal cells have distinct functions in the reproductive process, implying the possibility for a different expression profile. However, knowledge about the functional expression pattern of TRP channels in either epithelial or stromal cells is not available. STUDY DESIGN, SIZE, DURATION: In this study, the expression pattern of TRP channels in the murine (C57BL/6 J strain) endometrium was investigated and compared to the human expression pattern. Therefore, expression was examined in uterine tissue isolated during the natural estrous cycle (n = 16) or during an induced menstrual cycle using the menstruating mouse model (n = 28). Next, the functional expression of TRP channels was assessed separately in endometrial epithelial and stromal cell populations. PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative RT-PCR was used to evaluate the relative mRNA expression of TRP channels in murine uterine tissue and cells. To further assess the functional expression in epithelial or stromal cells, primary endometrial cell cultures and Fura2-based calcium-microfluorimetry experiments were performed. MAIN RESULTS AND THE ROLE OF CHANCE: The expression pattern of TRP channels during the natural estrous cycle or the induced menstrual cycle is analog to what has been shown in human samples. Furthermore, a very distinct expression pattern was observed in epithelial cells compared to stromal cells. Expression of TRPV4, TRPV6 and TRPM6 was significantly higher in epithelial cells whereas TRPV2, TRPC1/4 and TRPC6 were almost exclusively expressed in stromal cells. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although relevant mRNA levels are detected for TRPV6 and TRPM6, and TRPM4, lack of selective, available pharmacology restricted functional analysis of these ion channels. WIDER IMPLICATIONS OF THE FINDINGS: Successful reproduction, and more specifically embryo implantation, is a dynamic developmental process that integrates many signaling molecules into a precisely orchestrated program. Here, we describe the expression pattern of TRP channels in mouse endometrium that is similar to human tissue and their restricted functionality in either stromal cells or epithelial cells, suggesting a role in the epithelial-stromal crosstalk. These results will be very helpful to identify key players involved in the signaling cascades required for successful embryo implantation. In addition, these results illustrate that mouse endometrium is a valid representative for human endometrium to investigate TRP channels in the field of reproduction. STUDY FUNDING/COMPETING INTEREST(S): The Research Foundation-Flanders (G.0856.13 N to J.V.); the Research Council of the Katholieke Universiteit Leuven (OT/13/113 to J.V. and PF-TRPLe to T.V.); the Planckaert-De Waele fund (to J.V.);  Fonds Wetenschappelijk Onderzoek Belgium (to K.D.C. and A.H.). None of the authors have a conflict of interest.