Significance: The cation channel TRPM3 is highly expressed in the sensory system, where it plays a key role in the detection of noxious heat and the development of inflammatory heat hypersensitivity. Our understanding of the physiological role of TRPM3 in the sensory system and other tissues is hampered by the lack of potent pharmacologic tools, however. This study describes CIM0216, a small-molecule TRPM3 agonist. Our results indicate that CIM0216 is much more potent than established TRPM3 agonists, particularly owing to its ability to open two distinct cation-permeable pores in TRPM3. Using CIM0216 as a pharmacologic tool, we reveal that activation of TRPM3 evokes the release of calcitonin gene-related peptide from sensory nerve terminals and of insulin from pancreatic islets.

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.

Genetic engineering of the mouse genome identified many genes that are essential for embryogenesis. Remarkably, the prevalence of concomitant placental defects in embryonic lethal mutants is highly underestimated and indicates the importance of detailed placental analysis when phenotyping new individual gene knockouts. Here we introduce high-resolution contrast-enhanced microfocus computed tomography (CE-CT) as a nondestructive, high-throughput technique to evaluate the 3D placental morphology. Using a contrast agent, zirconium-substituted Keggin polyoxometalate (Zr-POM), the soft tissue of the placenta (i.e., different layers and cell types and its vasculature) was imaged with a resolution of 3.5 µm voxel size. This approach allowed us to visualize and study early and late stages of placental development. Moreover, CE-CT provides a method to precisely quantify placental parameters (i.e., volumes, volume fraction, ratio of different placental layers, and volumes of specific cell populations) that are crucial for statistical comparison studies. The CE-CT assessment of the 3D morphology of the placentas was validated (i) by comparison with standard histological studies; (ii) by evaluating placentas from 2 different mouse strains, 129S6 and C57BL/6J mice; and (iii) by confirming the placental phenotype of mice lacking phosphoinositol 3-kinase (PI3K)-p110α. Finally, the Zr-POM–based CE-CT allowed for inspection of the vasculature structure in the entire placenta, as well as detecting placental defects in pathologies characterized by embryonic resorption and placental fusion. Taken together, Zr-POM–based CE-CT offers a quantitative 3D methodology to investigate placental development or pathologies. Significance During pregnancy, the placenta functions as surrogate fetal lungs, kidneys, and gut. Proper placental functioning is therefore paramount during embryonic development. Indeed, placental defects are highly prevalent in mouse mutants showing embryonic death. Here we validate a contrast agent that allows development of the intact placenta to be visualized using microfocus computed topography. This technique enables an initial 3D inspection of the overall placental structure and provides the possibility to quantify different compartments in the placenta. Moreover, the nondestructive nature of this contrast agent permits further histological processing of the same sample when defects are observed.

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.

Are members of the transient receptor potential (TRP) channel superfamily functionally expressed in the human endometrial stroma? The Ca²⁺-permeable ion channels TRPV2, TRPV4, TRPC6 and TRPM7 are functionally expressed in primary endometrial stromal cells. Intercellular communication between epithelial and stromal endometrial cells is required to initiate decidualization, a prerequisite for successful implantation. TRP channels are possible candidates as signal transducers involved in cell–cell communication, but no fingerprint is available of the functional distribution of TRP channels in the human endometrium during the luteal phase of the menstrual cycle. Endometrial biopsy samples (previously frozen) from patients of reproductive age with regular menstrual cycles, who were undergoing diagnostic laparoscopic surgery for pain and/or infertility, were analysed. Samples were obtained from the menstrual (Days 1–5, n = 3), follicular (Days 6–14, n = 6), early luteal (Days 15–20, n = 5) and late luteal (Days 21–28, n = 5) phases. In addition, a total of 13 patient samples taken during the luteal phase were used to set up primary cell cultures for further experiments. Quantitative real-time PCR (qRT–PCR), immunocytochemistry, Fura2-based Ca²⁺-microfluorimetry and whole-cell patch clamp experiments were performed to study the functional expression pattern of TRP channels. Specific pharmacological agents, such as Δ⁹-tetrahydrocannabinol, GSK1016790A and 1-oleoyl-2-acetyl-glycerol, were used to functionally assess the expression of TRPV2, TRPV4 and TRPC6, respectively. Expression of TRPV2, TRPV4, TRPC1, TRPC4, TRPC6, TRPM4 and TRPM7 was detected at the mRNA level in endometrial biopsies (n = 19) and in primary endometrial stromal cell cultures obtained from patients during the luteal phase (n = 5) of the menstrual cycle. Messenger RNA levels of TRPV2, TRPC4 and TRPC6 were significantly increased (P < 0.01) in the late luteal phase compared with the early luteal phase. Immunocytochemistry experiments showed a positive staining for TRPV2, TRPV4, TRPC6 and TRPM7 in the plasma membrane and in the cytoplasm of primary endometrial stromal cells. Ca²⁺-microfluorimetry revealed significant increases (P < 0.001) in intracellular Ca²⁺ levels when stromal cells were incubated with specific activators of TRPV2, TRPV4 and TRPC6. Further functional characterization was performed using whole-cell patch clamp experiments. Taken together, these data provide evidence for the functional activity of TRPV2, TRPV4, TRPC6 and TRPM7 channels in primary stromal cell cultures. Although mRNA levels are detected for TRPV6, TRPC1, TRPC4 and TRPM4, the limited supply of specific antibodies and lack of selective pharmacological agents restricted any additional analysis of these ion channels. Embryo implantation is a dynamic developmental process that integrates many signalling molecules into a precisely orchestrated programme. Our findings identified certain members of the TRP superfamily as candidate sensors in the epithelial-stromal crosstalk. These results are very helpful to unravel the signalling cascade required for successful embryo implantation. In addition, this knowledge could lead to new strategies to correct implantation failure and facilitate the development of novel non-hormonal contraceptives. This work was supported by grants from the Research Foundation-Flanders (G.0856.13N to J.V.), the Research Council of the KU Leuven (OT/13/113 to J.V. and T.D. and PF-TRPLe to T.V.) and by the Planckaert-De Waele fund (to J.V.). K.D.C. and K.H. are funded by the FWO Belgium. None of the authors have a conflict of interest.