{"paper_id":"326c0f75-0b4c-4b8d-8f1c-d4a42a0b07d3","body_text":"ORIGINAL ARTICLE\nCellular and Molecular Life Sciences          (2025) 82:346 \nhttps://doi.org/10.1007/s00018-025-05857-9\nimaging of solvent-cleared organs\nIRES  internal ribosome entry site\nIV  current-voltage relationship\nKO  knock out\nLH  luteinizing hormone\nMEEC  mouse endometrial epithelial cell\nMESC  mouse endometrial stromal cell\nSOCE  store-operated Ca 2+ entry\nTRPV6  transient receptor potential vanilloid 6\nIntroduction\nIt is well known that Ca2+ plays a major role in many stages \nof the reproductive process, from germ cell maturation \nto placental and embryonic development. However, the \nprecise function and regulation of Ca 2+ during subsequent \nreproductive processes is incompletely understood.\nThe transient receptor potential (TRP) vanilloid 6 \n(TRPV6) channel is highly selective for Ca 2+ and plays a \nAbbreviations\nACE  angiotensin converting enzyme\nCPA  cyclopiazonic acid\nDVF  divalent-free\neR26  enhanced-Rosa26\nFSH  follicle-stimulating hormone\nIC  IRES-Cre\niDISCO  immunolabeling-enabled three-dimensional \nAdela Sota and Andreas Beck contributed equally.\n \r Petra Weissgerber\npetra.weissgerber@uni-saarland.de\n1 Experimental and Clinical Pharmacology and Toxicology, \nCenter for Molecular Signaling (PZMS), Saarland University, \n66421 Homburg, Germany\n2\t Center\tfor\tGender-Specific\tBiology\tand\tMedicine\t(CGBM),\t\nSaarland University, 66421 Homburg, Germany\n3 Institute of Pharmacology, Heidelberg University,  \n69120 Heidelberg, Germany\nAbstract\nThe Ca2+-selective transient receptor potential vanilloid 6 (TRPV6) channel plays a fundamental role in the female and \nmale murine reproductive system. We have previously shown that TRPV6 is essential for male fertility, and necessary \nfor a proper placental Ca 2+\t transport,\t embryonic\t bone\t development\t and\t calcification,\tas\t well\t as\t for\t extracellular\t matrix\t\nformation in the placental labyrinth. Here, we show that lack of functional TRPV6 results in impaired fecundity in female \nmice\t with\t increased\t latency\t to\t first\tpregnancy,\tlonger\t interpregnancy\t intervals\t and\t fewer\t and\t smaller\t litters.\t In\t mouse\t\nendometrium the TRPV6 protein is expressed in epithelial cells (MEECs). Using patch clamp recording and Ca 2+ imaging, \nwe show TRPV6-dependent whole-cell currents and that TRPV6 contributes to cytoplasmic Ca 2+ signaling in MEECs. \nMEECs lacking functional TRPV6 Ca 2+\tchannels\treveal\ta\tsignificantly\treduced\tfrequency\tof\tspontaneous\tcytosolic\tCa2+ \noscillations, shown in isolated cells and in situ in whole mount uterus preparations. Our results reveal a previously \nunknown physiological role for TRPV6 in the regulation of endometrial Ca 2+ homeostasis and its impact on female \nfecundity in mice, providing a molecular and cellular framework for further investigation of reproductive disorders, such \nas those associated with defective Ca 2+ regulation in women.\nKeywords Transient receptor potential vanilloid 6 · Endometrium · Epithelium · Cytosolic Ca 2+ imaging · Whole-cell \npatch clamp\nReceived: 24 April 2025 / Revised: 24 July 2025 / Accepted: 29 July 2025\n© The Author(s) 2025\nTRPV6 channel function is involved in endometrial epithelial cell Ca2+ \nsignaling and female mouse fecundity\nAdela Sota1 · Andreas Beck1 · Philipp Wartenberg1,2 · Anna-Lena Gehl1 · Manuel Winter1 · Ulrich Wissenbach1 · \nMarc Freichel3 · Markus R. Meyer1 · Ulrich Boehm1,2 · Veit Flockerzi1 · Claudia Fecher-Trost1 · Petra Weissgerber1\n1 3\nCellular andM olecular Life Sciences\n\n\nA. Sota et al.\nfundamental role for Ca2+ (re)-uptake [1, 2] and transcellular \nCa2+ transport across epithelial tissue barriers [ 3–5]. \nThus, TRPV6-dependent cytosolic Ca 2+ changes initiate \nand\t coordinate\t different\t signaling\t pathways\t and\t thereby\t\ncellular and systemic physiological and pathophysiological \nprocesses [6]. In the last years, remarkable progress has been \nmade in structural analysis of TRPV6 in both closed and \nopen channel states [ 7, 8]. Structures have been revealed \nin the absence and presence of divers modulators such \nas 2-APB, (4-phenylcyclohexyl) piperazine derivatives \n(PCHPDs) such as cis-22a, ruthenium red and econazole, or \ngenistein [ 9–17]. However, pharmacological tools remain \nlimited\t and\t are\t not\t yet\t specifically\t targeted\t at\t TRPV6\t\nfunction in isolated primary cells, nor do they appear to be \nparticularly\teffective,\tas\twith\tsoricidin\t[18].\nIn mice, TRPV6 is expressed in exocrine pancreas, \nsalivary gland, placenta, small intestine, cecum, prostate \nand epididymis [ 2, 3, 6, 19–22]. Lack of Trpv6 results in \nhypofertility of male mice [1, 2]. A decreasing intraluminal \nCa2+ concentration along the epididymal segments is \nessential to produce mature spermatozoa during the \nepididymal passage. Using Trpv6-deficient\t (Trpv6−/−) \nmice and mice carrying a single-point mutation within \nthe channel pore of TRPV6 (D541A, new nomenclature \nD581A [ 23]; Trpv6mt/mt), leading to a non-functional \nTRPV6 channel [ 2], we showed that TRPV6 proteins \nare essential constituents of the underlying Ca 2+ uptake \nmechanism in the epididymis. In female mice, TRPV6 is \nexpressed in the yolk sac and in labyrinth trophoblasts of \nthe placenta contributing to maternal-fetal Ca 2+ supply of \nthe embryo. The absence of the channel leads to impaired \nbone\t growth\t with\t shorter\t and\tless\t calcified\t femurs\t in\tthe\t\noffspring,\t given\t that\t Trpv6-deficient\t trophoblasts\t absorb\t\nsignificantly\t less\t Ca2+ from the maternal blood [ 3]. In \nhuman, TRPV6 loss-of-function mutations may also \nresult in under-mineralized bones and skeletal dysplasia \nwith postnatal recovery [ 24, 25] and transient neonatal \nhyperparathyroidism [ 26–28].\nEmbryo transfer experiments revealed that both the \nmaternal and fetal parts of the placenta contribute to \nembryonic development and Ca 2+ accumulation in the \nbones [3]. In addition to expression in the fetal structures, \ni.e. placental labyrinth and yolk sac, TRPV6 is also \nexpressed in the maternal part of the placenta, the decidua \n[3]. The decidua is formed by a transformation of the \nmaternal endometrium, a process named decidualization \nand is the essential prerequisite for both the implantation \nof the blastocyst and the maintenance of the pregnancy. \nThis suggests that the Trpv6-deficient\t phenotype\t of\t the\t\nembryo is probably not only caused by the lack of TRPV6 \nin trophoblasts but also depends on its presence and \nprobably channel function in the endometrium.\nA successful pregnancy requires a complex dialogue \nbetween the implanting embryo and the endometrium. \nThe multi-step process of embryo implantation is initiated \nby the hatched blastocyst and followed by adhesion, \nattachment and subsequent invasion of trophoblast cells \nthrough the endometrial epithelium into the stroma [ 29, \n30]. The human endometrium constitutes the inner lining \nof the uterus and undergoes monthly cycles of breakdown \nand repair in preparation for a possible pregnancy. It \nconsists of a single layer of epithelial cells lining the uterine \nlumen and the underlying stroma which varies in thickness \naccording\tto\tfluctuations\tof\tthe\tovarian\thormones\testrogen\t\nand progesterone. While decidualization in humans \nroutinely occurs during the monthly estrous cycle and is \npropagated by the invasion of a blastocyst, the initiation \nof decidualization in mice requires the presence of the \nblastocyst in the uterine lumen [30–32]. However, the exact \nmechanism of decidualization, the exact signaling between \nthe endometrial epithelial cells, which get in contact with \nthe blastocyst, and the underlying endometrial stromal cells, \nwhich\tthen\tproliferate\tand\tdifferentiate\tinto\tdecidual\tcells,\t\nsupporting the implantation of the embryo, remain poorly \nunderstood. The need for Ca 2+\t in\t the\t different\tgestational\t\nprocesses implicates the presence of specialized ion \nchannels to regulate Ca 2+ homeostasis [ 29]. The TRPV6 \nchannel might be the sensor and messenger molecule in the \nendometrial epithelial cells involved in the transformation \nof\textracellular\tstimuli\tinto\tthe\tinflux\tof\tCa2, inducing and \ncoordinating underlying signaling pathways.\nUsing isolated uteri and primary endometrial cells from \nwild-type\tmice,\twe\tidentified\tan\talmost\texclusive\texpression\t\nof TRPV6 in the epithelial cells of the murine endometrium. \nFurthermore, our studies uncovered that TRPV6 channels \ncontribute to Ca2+\tinflux\tand\tspontaneous\tCa2+ oscillations in \nthe wild-type mouse endometrial epithelial cells (MEECs), \nin which we recorded distinct TRPV6-dependent whole-cell \ncurrents. Phenotypically, female Trpv6−/− [1] and Trpv6mt/mt \nmice [2], both lacking functional TRPV6 channels, revealed \nincreased\tlatency\tto\tfirst\tpregnancy,\tlonger\tinterpregnancy\t\nintervals and fewer and smaller litters. Our results suggest, \nthat TRPV6-dependent Ca 2+\t influx\tin\t MEECs\t contributes\t\nto the decidualization process and thus to female fecundity \nin mice.\nMaterials and methods\nMice\nAll animal care and experimental procedures were reviewed \nand approved in accordance with the guidelines and ethical \nregulations established by the animal welfare committee of \n1 3\n  346  Page 2 of 21\n\nTRPV6 channel function is involved in endometrial epithelial cell Ca 2+ signaling and female mouse fecundity\nSaarland University. Adult (8–12 weeks old) female mice \nwere\tkept\tunder\ta\tstandard\tlight/dark\tcycle\t(12\th/12\th)\twith\t\nfood and water ad libitum.\nStudies were performed on wild-type mice of the mixed \n129/SvJ\t×\tC57BI/6\tN\tbackground,\twhich\tis\tthe\tbackground\t\nof the Trpv6−/−, Trpv6mt/mt, Trpv6-IC and Trpv6−/−-IC mice \n(see below). Trpv6−/− mice carry a deletion of about one \nthird of the protein coding region of the Trpv6 gene including \nexons 13, 14 and 15, coding for a part of domain S5, the \nchannel pore, domain S6, and the cytosolic C-terminus \n[1]. The Trpv6mt/mt mice represent a functional TRPV6 \nknock-out, homozygously carrying a single-point mutation \nwithin exon 13, coding for the channel pore (D541A, new \nnomenclature D581A [ 23]), leading to a non-functional \nTRPV6 channel [2].\nTo visualize TRPV6-expressing cells, Trpv6-IRES-Cre \nmice (Trpv6-IC; [3]) were bred to homozygous enhanced-\nRosa26-floxed-stop-reporter\tmice\t(eR26-τGFP;\t[33]). Due \nto\ta\tloxP\tflanked\t(floxed)\tstrong\ttranscriptional\ttermination\t\nsequence,\t the\t eR26-reporter\t allele\t terminates\t τGFP\t\ntranscription prematurely, but when the mice are crossed \nwith Cre-expressing mice, the Cre-mediated excision \nof\t the\t floxed\t termination\t sequence\t leads\t to\t constitutive\t\nτGFP\t expression.\t All\t Trpv6-IC/eR26-τGFP\t animals\t in\t\nthe F1 generation are heterozygous for the Trpv6-IC and \neR26-τGFP\talleles\tand\texhibit\tτGFP\texclusively\tin\tTrpv6-\nexpressing cells, more precisely in cells where the TRPV6 \npromotor had been active [ 34].\nTo visualize and to analyze cells, in which the \nTRPV6 gene has been knocked-out, we generated a \nnew Trpv6−/−-IRES-Cre ( Trpv6 KO-IC) mouse strain \n(see Fig. S3). For construction of the targeting vector \n(LpmCaTL_88),\t genomic\t DNA\t was\t isolated\t from\t R1\t\nES cells and used as a template for polymerase chain \nreaction\t (PCR)\t amplification\t of\t5′\tand\t3′\thomology\t arms\t\nwith Pfu\t polymerase.\t The\t genomic\t sequence\t of\t the\t 5′\t\nhomology contained exons 6 to 12 of the Trpv6 gene and \n3\t additional\t stop\t codons\t in\t 3\t different\t reading\t frames\t\nand a DTA cassette for negative selection. An internal \nribosome entry site (IRES) sequence followed by a Cre \nrecombinase\t complementary\t DNA\twas\t inserted\t after\t the\t\nfinal\t stop\t codon.\t The\t IRES\t element\t will\t result\t in\t the\t\nproduction\t of\ta\tbicistronic\t messenger\t RNA,\t from\t which\t\nTRPV6 and Cre recombinase are independently translated. \nThis sequence is followed by an FRT (Flp recognition \ntarget)\t sequence-flanked\t pgk-promotor-driven\t neomycin \t\nresistance gene cassette (neo r) and a Flp-ACE cassette, \nwhich\t directs\t self-induced\t deletion\t of\tDNA\tsequences\t as\t\nthey pass through the male germ line [ 35]. The testes-\nspecific\t promoter\t from\t the\t angiotensin-converting \t\nenzyme gene (ACE) was used to drive the expression \nof\t the\t Flp-recombinase\t gene.\t The\t 3′\t homology\t arm\t was\t\ncloned downstream of this cassette. An enhanced GFP \ncassette and the herpes simplex virus thymidine kinase \n(tk) cassette were introduced for negative selection (Fig. \nS3a). ES cell culture was essentially done as described [ 2, \n36]. 10 of 333 double-resistant, GFP-negative colonies \nshowed correct homologous recombination at the Trpv6 \nlocus ( Trpv6L2). GFP-positive cell colonies were \ndiscarded.\t Recombination\t was\t confirmed\t by\t Southern \t\nBlot\t hybridization\t with\t a\t5′\t and\t 3′\t probe\t external\t to\t the\t\ntargeting vector and a neo probe (Fig. S3b). Germline \nchimeras were obtained by injection of 2 selected ES \ncell\t clones\t into\t C57Bl/6\t blastocysts\t and\t subsequently \t\ncrossed\t with\tC57Bl6/N\t mice\tto\tget\tanimals\t heterozygous \t\nfor the Trpv6−/−-IC allele where the neo cassette is \nalready removed (Fig. S3c, d). Trpv6−/−-IC mice were \nkept\t on\ta\tmixed\t (129/SvJ\t ×\tC57BI/6\t N)\tbackground\t and\t\nbred to homozygosity. To visualize cells in which the \nTRPV6 gene has been knocked out, two generations of \nbreeding\t are\tneeded.\tThe\tfirst\tbreeding\t consists\t of\ta\tcross\t\nbetween the Trpv6−/−-IC\t and\t eR26-τGFP \t mice.\t The\t F1\t\nTrpv6−/−-IC/eR26-τGFP \tmice\t are\t heterozygous\t for\t both\t\nTrpv6−/−-IC\t and\t eR26-τGFP \t alleles.\t Now,\t female\t F1\t\nmice are bred with male heterozygous Trpv6−/−-IC mice \nto produce the target genotype (note: homozygous male \nTrpv6−/− mice are hypofertile [ 1]. This drastically lowers \nthe probability of producing the target genotype to only \n6.25%\tof\tthe\toffspring\taccording\t to\tMendel.\tHowever,\tthe\t\nactual frequency of the desired genotype in females was \nonly 5.43%. Taking into account that the average litter \nsize was 6.93 ± 1.12 (81 litters from 23 breeding pairs) \nextensive\t breeding\t efforts\t are\t required\t to\t obtain\t a\tsmall\t\nnumber of female animals with the correct genotype and \nage.\t All\t female\t mice\t finally\t used\t were\t homozygous\t for\t\nthe Trpv6−/−-IC allele and heterozygous for the eR26-\nτGFP\t allele\t and\t express\t τGFP\t in\t cells\t where\t the\t Trpv6 \npromotor had been active.\nTo analyze cytosolic Ca2+ changes in TRPV6-expressing \ncells in isolated uteri in situ, Trpv6-IC mice were crossed with \neR26-GCaMP3 mice, having the calcium indicator GCaMP3 \ninserted into the Rosa26 locus [37].\tA\tloxP-flanked\ttriple\tstop\t\nsignal blocks the expression of GCaMP3. The F1 Trpv6-IC/\neR26-GCaMP3 mice are heterozygous for both Trpv6-IC \nand for eR26-GCaMP3 alleles and exclusively exhibit the \nGCaMP3 Ca2+-sensor protein in Trpv6-expressing cells. To \nanalyze cytosolic Ca 2+ changes in TRPV6 knock-out cells \nin isolated uteri in situ again two generations of breeding are \nneeded.\tThe\tfirst\tbreeding\tconsists\tof\ta\tcross\tbetween\tthe\t\nTrpv6−/−-IC and eR26-GCaMP3 mice. The F1 Trpv6−/−-IC/\neR26-τGFP\t mice\t are\t heterozygous\t for\t both\t Trpv6−/−-IC \nand\teR26-τGFP\talleles\t(Fig.\tS3e).\tNow,\tfemale\tF1\tmice\tare\t\nbred with male heterozygous Trpv6−/−-IC mice to produce \nthe\t target\t genotype.\tAll\t female\t animals\t finally\tused\t were\t\n1 3\nPage 3 of 21   346 \n\nA. Sota et al.\ninstructions\t and\t analyzed\t with\t the\t xPONENT\t software\t\n(Luminex\tCorporation)\taccording\tto\tthe\tprotocol\t(Luminex/\nMILLIPLEX MAP Human Pituitary Magnetic Bead Panel, \nMerck). Samples were pipetted as duplicates and the mean \nwas calculated. Measurements with an intra-assay value \nabove 20% were excluded. In four independent runs the \nquality controls were within 97% of the expected range. 3% \nwere below the minimum expected value.\nPrimary cells isolation\nThe isolation of mouse endometrial epithelial cells \n(MEECs) and stromal cells (MESCs) was performed \naccording to the method described by De Clercq et al. [40]. \nUterine horns were dissected and placed in a dish containing \nHanks’ Balanced Salt Solution (HBSS+, Thermo Fisher \nScientific,\t Waltham,\t MA,\t USA)\t supplemented\t with\t 100\t\nU/mL\tpenicillin\tand\t100\tµg/mL\tstreptomycin.\tAll\tresidual\t\nadipose and connective tissue were removed under the \nstereo microscope (Zeiss Stemi 2000-CS). Uterine horns \nwere cut open longitudinally to expose the uterine lumen \nand transferred to a tube containing 2.5% pancreatin \nand 0.25% trypsin in HBSS+. The tube was incubated \nhorizontally for 60 min at 4 °C on a shaker, 45 min at room \ntemperature (RT, no shaking) and 15 min at 37 °C in a \nwater bath (no shaking). The following MEEC and MESC \nisolation steps were performed in a sterile environment \nunder\ta\tlaminar\tflow\tcabinet.\nFor MEEC isolation, after two hours of incubation, \nthe uteri were transferred into a dish with cold MEEC \nmedium (DMEM, Sigma) containing 10% FBS (Thermo \nFisher\t Scientific),\t 0.5\t µg/mL\t amphotericin\t B\t (Thermo\t\nFisher\t Scientific),\t100\t µg/mL\t gentamicin\t (Thermo\t Fisher\t\nScientific),\t 25%\t MCDB-105\t medium\t (Cell\t Applications\t\nInc,\t San\t Diego,\t CA,\t USA),\t 5\t µg/mL\t insulin\t (Sigma))\t for\t\n5 min to inactivate trypsin activity. The digested tissue was \nthen transferred into a tube containing cold HBSS+. The \ntube was vortexed for 10 s to release the epithelial sheets \nand the tissue was rinsed in a clean Petri dish with 3 mL \nHBSS + and vortexed in two additional tubes, obtaining \na total of three tubes containing epithelial sheets. The \nepithelial sheets were recovered by gently pipetting the \nthree\tcell\tsuspensions\ton\ta\t100\tμm\tnylon\tmesh\tto\tremove\t\ntissue debris. The collected cell suspension was centrifuged \nat 500 x g for 5 min. The pellet was resuspended in 12 mL of \nMEEC medium and mixed well. The solution was put aside \nto settle for 5 min in order to separate remaining MESC \nby gravity sedimentation. After 5 min, the upper 2 mL of \nthe suspension were removed and the cell suspension was \ncentrifuged again at 500 x g for 5 min and resuspended in \n3 mL MEEC medium, depending on the number of isolated \nuteri\tand\tthe\tfinally\tdesired\texperimental\tcell\tdensity.\nhomozygous for the Trpv6−/−-IC allele and heterozygous for \nthe eR26-GCaMP3 allele, expressing GCaMP3 only in cells \nwhere the Trpv6 promotor had been active.\nThe Trpv6- and Trpv6−/−-IC/eR26-τGFP\tmice\twere\tused\t\nto\tanalyze\tthe\tTRPV6\texpression\tprofile\tin\tthe\tendometrium,\t\nand the Trpv6- and Trpv6−/−-IC/eR26-GCaMP3\t mice\t\nserved for the functional analysis (cytosolic Ca 2+ imaging) \nof TRPV6 channels in isolated uteri in situ.\nFecundity analysis\nFecundity\t is\t defined\t as\t the\t ability\t to\t reproduce,\t i.e.\t to\t\nproduce\t offspring,\tin\t contrast\t to\t fertility,\twhich\t indicates\t\nthe ability to conceive. To quantify the fecundity rate of \nwild-type, Trpv6−/− and Trpv6mt/mt mice, we analyzed \nand averaged the litter size and the time interval between \nsubsequent litters for each mating couple over a period of \nup to 12 months and calculated the ratio from both. Trpv6−/− \nand Trpv6mt/mt mating couples comprised of heterozygous \nmale and homozygous female mice.\nVaginal cytology and standardization of estrous \ncycle stage\nEstrous\tcycle\tstages\tof\tthe\tfemale\tmice\twere\tidentified\tby\t\nvaginal cytology [ 38, 39].\tV aginal\tlining\t was\tflushed\t3–5\t\ntimes\t with\t 40\t µL\t NaCl\t and\t the\t final\tcell\t suspension\t was\t\nplaced\t on\t a\tglass\t slide\t and\t examined\t under\t a\tbright\t field\t\nlight microscope using a 10X objective (Zeiss Axio Imager.\nM2, Carl Zeiss, Oberkochen, Germany). Trpv6 expression \nand proliferation of endometrial cells in mice is highest at \nestrus (Fig. 2 and [40]). Thus, only mice that were in estrus, \nidentified\tby\tthe\tdominant\t presence\t of\tcornified\tepithelial\t\ncells and the lack of leukocytes in the vaginal smear, \nhad been used for further experiments. To standardize \nthe estrous cycle stage, adult female mice were injected \nsubcutaneously\t with\t 50\tµL\tof\t17β-estradiol\t (E2)\t solution\t\n(100\t ng/50\t µL\t sesame\t oil;\t Sigma-Aldrich,\t St.\t Louis\t and\t\nBurlington, MA, USA) for three consecutive days prior to \nuterus and cell isolation.\nHormone measurements\nTrunk blood was collected from Trpv6−/− and control mice, \nallowed to clot for 30 min at room temperature, centrifuged \nfor 10 min at 4 °C at 2,000xg, the serum removed and \nsubsequently centrifuged for a further 10 min at 4 °C at \n2,000xg.\t The\t serum\t was\t stored\t at\t −20\t °C\t until\t analyzed.\t\nHormone measurements were performed using the Luminex \nxMAP technology (MAGPIX, Luminex Corporation) in \ncombination with the mouse pituitary kit (MPTMAG-49 K, \nMerck Millipore) according to the manufacturer’s \n1 3\n  346  Page 4 of 21\n\nTRPV6 channel function is involved in endometrial epithelial cell Ca 2+ signaling and female mouse fecundity\nseries\tof\tdehydration.\tThe\tcells\twere\tdipped\tin\ttwo\tdifferent\t\nconcentrations of ethanol (70%, 96%), then washed in absolute \nethanol\tfor\t2\t min\tand\tfinally\tincubated\tin\t xylene\t(Applied\t\nBiosystems, Waltham, MA, USA) for clearing. After 3 min, \nthe cells were mounted by using a non-aqueous mounting \nmedium (Depex; Serva, Heidelberg, Germany) and imaged \nwith an automated slide scanner (Zeiss Axio Scan Z1).\nImmunohistochemistry\nMEEC\tand\tMESC\tcultures\twere\twashed\t3\t×\t5\tmin\twith\tPBS\t\non\t a\tshaker,\t fixed\tfor\t 10\t min\t with\t 4%\t paraformaldehyde \t\n(PFA, Sigma; no shaking) and washed three times with \nPBS without Ca2+ and Mg2+\t(Thermo\tFisher\tScientific).\tTo\t\nstain cytokeratin and vimentin, established marker proteins \nof MEECs and MESCs, respectively [ 40, 41], the cells \nwere permeabilized under shaking (50 rpm) with 0.2% \nTriton-X 100 (Carl Roth, Karlsruhe, Germany) for 10 min \nand washed again three times with PBS before they were \nincubated\t for\t2\th\tin\t5%\tnormal\t goat\tserum\t (NGS;\tV ector\t\nLaboratories,\t Newark,\t CA,\t USA)\t in\t PBS\t to\t block\t non-\nspecific\t antibody\t binding.\t Finally,\t MEECs\t and\t MESCs\t\nwere stained with antibodies against established markers, \ncytokeratin (1:1000, Sigma, MEECs) and vimentin (1:500, \nCell Signaling Technology, Danvers, MA, USA, MESCs) \nrespectively at 4 °C on a shaker according to [ 40]. All \nantibodies\t were\t diluted\t in\t PBS\t with\t 0.5%\t NGS.\t After\t\n24 h of incubation in the primary antibody solution, the \ncells\t were\tfirst\twashed\t three\t times\t with\tPBS\ton\ta\tshaker,\t\nthen they were incubated in the dark with secondary \nantibodies (Alexa Fluor 594-conjugated goat anti-mouse \nIgG (Invitrogen) and Alexa Fluor 488-conjugated goat \nanti-rabbit\t IgG\t (Invitrogen);\t 1:1000\t in\t 0.5%\t NGS)\t for\t\n1 h on a shaker. They were washed another three times \nwith PBS on a shaker and then incubated in the dark for \nnuclear staining with Hoechst 33258 (1:1000, Sigma) in \nPBS\t for\t 15\t min.\t After\t a\t final\t triple\t wash\t with\t PBS,\t the\t\ncoverslips were mounted on glass slides (Fluoromount, \nSouthern Biotech). For GFP staining, the cells were \npermeabilized and blocked for 1 h at RT using a blocking \nsolution containing 0.2% Triton X-100 and 5% normal \ndonkey\tserum\t(NDS,\tJackson\tImmuno\tResearch).\tMEECs\t\nand MESCs were then incubated overnight at 4 °C \nwith primary antibodies; MEECs were incubated with \nmonoclonal mouse anti-pancytokeratin (1:1000, Sigma) \nand\tchicken\t anti-GFP\t(1:1000,\t Thermo\t Fisher\t Scientific),\t\nand MESCs were incubated with monoclonal rabbit anti-\nvimentin (1:500, Cell Signaling Technology) and chicken \nanti-GFP. Antibodies were diluted in the blocking solution. \nCells\t were\t washed\t the\t next\t day\t 3\t×\t5\t min\t with\t PBST\t\n(0.05% Tween 20 in PBS) and incubated with secondary \nantibodies for 2 h at RT; MEECs were incubated with \nThe MESCs were then isolated from the same \npreparation according to the protocol [ 40]. Therefore, two \ndigestion\t mixtures\t were\t prepared\t by\t dissolving\t 300\t µL\t\nof\t the\t 1\t mg/mL\tcollagenase\t (Sigma)\t in\t 2.7\t mL\tof\t 0.05%\t\ntrypsin/EDTA\t solution\t (Thermo\t Fisher\t Scientific).\t Three\t\nsmall\tPetri\tdishes\twere\tfilled\twith\tcold\t(4\t°C)\tHBSS\t+\tand\t\n3\t×\t15\tmL\ttubes\twith\t3\tmL\tof\tMESC\tmedium.\tAfter\t30\tmin\t\nof\tincubation\tin\tthe\tfirst\tMESC\tdigestion\tmix,\tthe\tdigested\t\ntissue trypsin solution was shaken gently for 10 s to detach \nthe MESCs from the uterine tissue. The uteri were then \ntransferred\t into\t the\t first\t Petri\t dish\t containing\t 3\t mL\t cold\t\n(4 °C) HBSS + and rinsed well. 3 mL of the MESC medium \nwere added into the MESC digestion mix to inhibit trypsin \nactivity. After rinsing in HBSS+, the uteri were transferred \nto one of the tubes containing 3 mL of MESC medium and \nshaken gently for 10 s. This step was repeated three times \nin total (transferring uteri from HBSS + to MESC medium), \nso the uteri were rinsed and gently shaken for 10 s in each \nof the three tubes. In the end, this protocol results in four \nMESC suspensions: one tube containing MESCs in trypsin \nsolution and MESC medium, and three tubes with MESCs \nin MESC medium. Finally, the uteri were transferred in \nthe second MESC digestion mix and incubated in a water \nbath for 30 min at 37 °C, vertically. Yet, the collected cells \nare an impure collection of mostly stromal cells and some \nepithelial cells. Therefore, another three small Petri dishes \nwere\t prepared\t with\t cold\t (4\t °C)\t HBSS\t+\tand\t 3\t×\t15\t mL\t\ntubes with 3 mL of MESC medium. After the uteri were \nshaken gently for 10 s in four separate tubes, the stromal \ncells were collected by passing the content of the tubes \nthrough\t a\t 40\t μm\t nylon\t mesh.\t The\t mesh\t was\t rinsed\t with\t\nan additional 5 mL of MESC medium. The cell suspension \nwas centrifuged at 500 x g for 7 min and the pellet was \nresuspended in 3 ml MESCs medium.\nFinally, almost pure cultures of MEECs and MESCs from \nwild-type, Trpv6−/− and Trpv6mt/mt mice were obtained and \nplated on 12 mm and 25 mm collagen-coated coverslips \nand incubated at 37 °C with 5% CO 2 in preparation for \nbiochemical and functional experiments. Apparently, both \nprimary cell types, isolated from the three genotypes (wild-\ntype, Trpv6−/− and Trpv6mt/mt), revealed no morphological \ndifferences\tin\tculture.\nHistological staining\n24\th\tafter\tplating,\tthe\tMEECs\tand\tMESCs\twere\tfixed\twith\t\ncold absolute ethanol for 5 to 7 min and then gently rinsed \nin cold tap water to wash and hydrate the cells. After bathing \nin\t hematoxylin\t (Morphisto\t GmbH,\t Offenbach\t am\t Main,\t\nGermany) for 6 min and in warm tap water for 4 min, cells \nwere incubated in eosin (Mephisto GmbH) for another \n6 min and quickly rinsed in tap water before going through a \n1 3\nPage 5 of 21   346 \n\nA. Sota et al.\nthe dark [ 42]. The tissues were imaged using a light-sheet \nmicroscope (UltraMicroscope Blaze™, Miltenyi Biotec, \nBergisch Gladbach, Germany).\nMass spectrometry (MS)\nTRPV6 immunoprecipitations from mouse uterus, MEECs and \nMESCs\t Uteri\tfrom\tdifferent\tgenotypes\tand\testrous\tstages\tor\t\nMEEC\t and\t MESC\t cells\t were\t resuspended\t in\t RIPA\t buffer\t\n(150\tmM\tNaCl,\t50\tmM\tTris\tHCl,\tpH\t8.0,\t5\tmM\tEDTA,\t1%\t\nNonidet\tP40,\t0.1%\tSDS,\t0.5%\tNa-deoxycholate,\tpH\t7.4),\t\nsupplemented with proteinase inhibitors (Roche, Mannheim, \nGermany). Uteri tissue was minzed by ultraturrax treatment \nor MESC and MEEC cell solution was sheared ten times \n(27G gauge needle) on ice and then incubated for 30 min \nat 4 °C on a shaker. After centrifugation at 100,000x g at \n4 °C for 45 min, the supernatant containing the solubilized \nproteins was collected and the protein concentration was \ndetermined by Biochinonic BCA-assay (Thermo Fisher \nScientific,\t Germany).\t 10\t mg\t uterine\t proteins\t or\t 0.6–\n1.2\tmg\tMESC/MEEC\tproteins\twere\tincubated\tfor\t16\th\tat\t\n4\t°C\tin\tthe\tpresence\tof\t10\tµg\tanti-TRPV6\tantibody\t1271\t\n(directed\tagainst\tthe\tN-terminus)\tor\tanti-TRPV6\tantibody\t\n429\t(directed\tagainst\tthe\tC-Terminus)\tcoupled\tto\t50\tµl\tof\t\nDynabeads™Protein G (Invitrogen, Schwerte, Germany). \nThe beads were collected using a magnetic rack, washed \nthree\t times\t with\t 1\t mL\t RIPA\t buffer\tand\t were\t eluted\t with\t\n50\tµL\tdenaturing\tsample\tbuffer\t(final\tconcentration:\t60\tmM\t\nTris HCl, pH 6.8, 4% SDS, 10% glycerol including 0.72 M \nβ-mercaptoethanol).\tThe\telute\twas\tincubated\tfor\t20\tmin\tat\t\n60 °C and analysed by mass spectrometry. The same elutes \nwere used for western blot analysis (Figs. 2 and 3d). For \nTRPV6 detection, membranes were incubated with the \nmonoclonal C-terminal TRPV6 antibody (20C6).\nGel electrophoresis of proteins and sample Preparation for \nmass spectrometry  Proteins elutes from the TRPV6 IPs \nwere\t separated\t on\t NuPAGE®\t 4%−12%\t Bis-Tris\t gradient\t\ngels\t (Thermo\t Fisher\t Scientific,\t Germany),\t fixed\t in\t the\t\npresence of 40% ethanol and 10% acetic acid, incubated 3 \ntimes for 10 min with water and stained with Coomassie \n(0.12%\t (w/v)\t Coomassie\t G-250\t (20%\t (v/v)\t methanol,\t\n10%\t(v/v)\tphosphoric\tacid,\t10%\t(w/v)\tammonium\tsulfate).\t\nStained gel areas were cut in pieces and washed twice \nalternately\t with\t buffer\tA\t (50\t mM\t NH4HCO3)\t and\t buffer\t\nB\t (50\t mM\t NH4HCO3/50%\t (v/v)\t acetonitrile).\t Reduction\t\nof\t disulfide\t bonds\t was\t done\t by\t incubation\t at\t 56\t °C\t for\t\n30 min in the presence of 10 mM dithiothreitol (Applichem, \nGermany)\t in\t buffer\tA,\t followed\t by\t carbamidomethylation\t\nwith\tiodacetamide\t(Thermo\tScientific,\tGermany)\tat\t21\t°C\tin\t\ndarkness for 30 min in the presence of 5 mM iodoacetamide \nin\tbuffer\tA.\tGel\tpieces\twere\twashed\ttwice\talternating\twith\t\nanti-mouse Cy5 (1:1000, company) and Alexa Fluor \n488\t donkey\t anti-chicken\t IgG\t (1:500,\t Jackson\t Immuno\t\nResearch), and MESCs were incubated with anti-rabbit \nCy5\t(1:1000,\tJackson\tImmuno\tResearch)\t and\tAlexa\tFluor\t\n488\t donkey\t anti-chicken\t IgG\t (1:500,\t Jackson\t Immuno\t\nResearch). The secondary antibodies were diluted in PBS. \nThe\tcells\twere\twashed\t3\t×\t5\tmin\twith\tPBST\tand\tincubated\t\nwith Hoechst 33258 (1:1000) in PBS for 10 min in the dark \nat\tRT.\tThe\tcell-containing\t coverslips\t were\tfinally\twashed\t\n2\t×\t5\tmin\twith\tPBST\tand\tmounted\tupside\tdown\ton\ta\tslide\t\nwith Fluoromount (Southern Biotech). All slides were \nimaged\t using\t an\t epifluorescence\t microscope\t (Zeiss\t Axio\t\nImager M2).\nImmunolabeling-enabled three-dimensional \nimaging of solvent-cleared organs (iDISCO)\nAdult (animal number, n = 3 for Trpv6-IC/eR26-τGFP ,\t\nn = 5 for Trpv6−/−-IC/eR26-τGFP)\t female\t mice\t were\t\nanesthetized with a mix of ketamine and xylazine. Mice \nwere transcardially perfused with PBS, followed by 4% \nparaformaldehyde (PFA). The uteri and ovaries were \ndissected\t and\t post-fixed\tin\t 4%\t PFA\t for\t 3\t h\t at\t 4\t °C.\t The\t\nsamples were then slowly dehydrated at room temperature \n(RT) in increasing concentrations of methanol (VWR \nChemicals, Radnor, PA, USA). Dehydration was followed \nby overnight delipidation in a 66% dichloromethane (DCM, \nSigma)/33%\t methanol\t solution\t at\t 4\t °C\t with\t rotation.\tThe\t\nuteri were then washed in methanol in RT, chilled at 4 °C for \n2 h, and bleached in 5% hydrogen peroxide (H2O2, Sigma). \nEach sample was rehydrated the next day in a series of \ndecreasing concentrations of methanol. This was followed \nby incubation in a blocking and permeabilizing solution \n(PBSGT: 1X PBS, 0.2% Gelatin (VWR Chemicals), 1% \nTriton X-100 and 0.02% sodium azide (Sigma) against \nmicrobial contamination) for 4 days with rotation at RT. All \nthe following antibody incubation steps were performed at \n37\t°C\tto\tincrease\tantibody\tpenetration.\tSamples\twere\tfirst\t\nincubated with primary antibodies (rabbit anti-GFP, 1:5000, \nInvitrogen) in PBSGT for 2 weeks with rotation. After the \nincubation, they were washed several times during the course \nof the day as well as overnight. Samples were incubated \nwith secondary antibodies (donkey anti-rabbit Cy5, 1:1000, \nJackson\t ImmunoResearch\t Inc.,\t West\tBaltimore\t Pike,\t PA,\t\nUSA) in PBSGT for 1 week with rotation. All steps following \nincubation with secondary antibodies were performed in \ndark conditions. They were further incubated in increasing \nconcentrations of methanol and then delipidized overnight. \nNext,\tthe\tuteri\twere\tincubated\tin\t100%\tDCM\twith\trotation\t\nuntil they sank at the bottom of the container, then they were \ntransferred in 100% benzyl ether (DBE; Sigma). After 2 h \nof clearing, samples were stored in a new DBE solution in \n1 3\n  346  Page 6 of 21\n\nTRPV6 channel function is involved in endometrial epithelial cell Ca 2+ signaling and female mouse fecundity\nand\t Proteome\t Discoverer\t 1.4\t (Thermo\t Fisher\t Scientific,\t\nGermany) software or Peaks Studio10.6 (Bioinformatic \nSolutions Inc. Canada). Peptides were matched to tandem \nmass spectra by Mascot version 2.4.0 (Matrix Science) by \nsearching an SwissProt database (version 2018_05, number \nof protein sequences 557.992 containing 16.992 mus \nmusculus sequences) against mouse proteins. MS 2 spectra \nwere matched with a mass tolerance of 7 ppm for precursor \nmasses and 0.5 Da for peptide fragment ions. Tryptic digest, \ntwo missed cleavage sites, cysteine carbamidomethylation \nas\t a\t fixed\t modification\t and\t deamidation\t of\t asparagine\t\nand glutamine, acetylation of lysine and oxidation of \nmethionine\t as\t variable\t modifications\t were\t used\t for\t the\t\nsearch.\t The\t MASCOT\t output\t files\t were\t loaded\t in\t the\t\nsoftware\t Scaffold\t(V ersion\t4.8.8,\t Proteome\t Software\t Inc.,\t\nPortland,\t OR).\t To\tensure\t significant\tprotein\t identification\t\nthe\t protein\t probability\t filter\twas\t set\t to\t protein\t FDR:\t 5%\t\npeptide FDR:1% decoy. Protein probabilities were assigned \nby the Protein Prophet algorithm [ 43]. Proteins that \ncontained\t similar\t peptides\t and\t could\t not\t be\t differentiated\t\nbased\t on\t MS/MS\t analysis\t alone\t were\t grouped\t to\t satisfy\t\nthe\tprinciples\tof\tparsimony.\tRaw\tdata\tfrom\tMEEC/MESC\t\nimmunoprecipitations were analyzed by Peaks Studio 10.6. \nTherefore, spectra were searched against a Swiss Prot mouse \ndatabase (version 2024, including 21708 entries). MS 1 and \nMS2 spectra were matched with a mass tolerance of 10 ppm \nfor precursor masses and 0.7 Da for peptide fragment ions. \nTryptic digest and up to three missed cleavage sites were \nallowed, carbamidomethylation on cysteine were used as \na\t fixed\tmodification\tfor\t database\t search\t and\t deamidation\t\nof asparagine and glutamine, acetylation of lysine and \noxidation\tof\tmethionine\twere\tused\tas\tvariable\tmodifications.\nCa2+ imaging in isolated MEECs\nMEECs from Trpv6−/−, Trpv6mt/mt and wild-type mice, \nplated on 25 mm collagen-coated coverslips, were loaded \nwith\t 5\t µM\t Fura-2\t AM\t (Invitrogen)\t for\t 30\t min\t at\t RT\t in\t\nRinger’s\tsolution\tcontaining\t115\tmM\tNaCl,\t5\tmM\tKCl,\t2\t\nmM CaCl2, 2 mM MgCl2, 10 mM HEPES, 10 mM glucose, \npH 7.4. Subsequently, the coverslips were placed in a \nbath chamber, washed three times with Ringer´s solution \nand\t mounted\t with\t a\t volume\t of\t 300\t µL\t of\t nominal\t Ca2+-\nfree or Ca 2+-containing Ringer´s solution (see start of \nexperiments in Fig. 4, with or without ORAI channel \nblocker GSK7975A or BTP2 (both Merck)) on the stage \nof a Zeiss AxioVert S100 inverted microscope equipped \nwith\t a\t Fluar-20x/0.75\tobjective\t (Zeiss),\t a\t monochromator\t\n(Polychrom V , TILL Photonics) and a charge-coupled \ndevice camera (Clara CCD, Andor Technology). Changes \nin intracellular Ca 2+ concentration were recorded at 1 Hz \nas\t fluorescence\t (>\t440\t nm)\t ratio\t (F340/F380),\t calculated\t\nbuffer\t A\t and\t B\t and\t then\t dried\t in\t a\t vacuum\t centrifuge.\t\nFor in-gel digestion, the gel pieces were incubated in the \npresence\tof\t15\tµL\tof\tporcine\ttrypsin\t(10\tng/µl,\tPromega)\tin\t\nbuffer\tA\tat\t37\t°C\tovernight.\tTryptic\tpeptides\twere\textracted\t\ntwice\twith\t50\tµL\textraction\tbuffer\t(2.5%\tformic\tacid/50%\t\nacetonitrile) in an ultrasonic bath. Both supernatants were \ncombined and concentrated in a vacuum centrifuge and \nresuspended\tfinally\tin\t21\tµL\tof\t0.1%\tformic\tacid.\nNano ESI-LC-MS 2 measurements  Tryptic peptides were \nanalysed\t by\t nanoflow\t LC-HR-MS/MS\t (Ultimate\t 3000\t\nRSLC nano UHPLC-system coupled to an LTQ Orbitrap \nVelos Pro or an Eclipse Tribrid mass spectrometer (all \nThermo\t Fisher\t Scientific,\t Germany).\t Peptides\t analysed\t\nby\tthe\tOrbitrap\tV elos\tsetup\twere\tfirst\ttrapped\ton\ta\tcolumn\t\n(100\tμm\tx\t2\tcm,\tAcclaim\tPepMap100C18,\t 5\tμm,\tThermo\t\nFisher\t Scientific)\t and\t separated\t on\t a\t reversed\t phase\t C18\t\ncolumn\t (Acclaim\t PepMap\t capillary\t column,\t C18;\t 2\t μm;\t\n75\t μm\t x\t 25\t cm,\t Thermo\t Fisher\t Scientific)\t at\t a\t flow\trate\t\nof\t200\tnL/min\tduring\ta\t120\tmin\tgradient\tbuild\twith\tbuffer\t\nA (water and 0.1% formic acid) and B (90% acetonitrile \nand 0.1% formic acid). Eluted peptides were directly \nsprayed into the mass spectrometer through a coated \nsilica\t electrospray\t emitter\t (PicoTipEmitter,\t 30\t μm,\t New\t\nObjective) and ionized at 2.2 kV . MS spectra were acquired \nin\ta\tdata-dependent\tmode.\tFull\tscan\tMS\tspectra\t(m/z\t300–\n1700) were acquired in the Orbitrap analyser using a target \nvalue of 10e6. The 10 most intense peptide ions with charge \nstates\t>\t+\t2\twere\tfragmented\tin\tthe\thigh-pressure\tlinear\tion\t\ntrap by low-energy CID (35% normalized collision energy). \nPeptides analysed with an Eclipse Tribrid mass spectrometer \n(Thermo\tScientific,\tGermany)\twere\tfirst\ttrapped\ton\ta\tC18\t\ntrap\t column\t (75\t μm\t ×\t 2\t cm,\t Acclaim\t PepMap100C18,\t\n3\tμm,\tnano\tviper)\tand\tseparated\ton\ta\treverse\tphase\tcolumn\t\n(nano\tviper\tAcclaim\tPepMap\tcolumn,\tC18;\t2\tμm;\t75\tμm\t×\t\n50 cm). Peptides were separated for 120 min by a gradient, \ngenerated\t with\tbuffer\tA\tand\tbuffer\tB\tat\ta\tflow\trate\tof\t300\t\nnl/min.\t The\t effluent\twas\t sprayed\t into\t an\tOrbitrap\t Eclipse\t\nTribrid\t mass\t spectrometer\t (Thermo\t Scientific,\t Germany)\t\nusing\t a\t coated\t emitter\t (PicoTipEmitter,\t 30\t μm,\t New\t\nObjective, Woburn, MA, USA, ionization energy: 2.4 keV) \nand measured in data dependent mode. MS1 peptide spectra \nwere acquired using the Orbitrap analyzer ( R = 120k, RF \nlens\t=\t30%\t m/z\t=\t375–1500,\t MaxIT:\t auto,\t profile\t data,\t\nintensity threshold of 10e 4). Dynamic exclusion of the \n10 most abundant peptides was performed for 60 s. MS 2 \nspectra were collected in the linear ion trap (isolation mode: \nquadrupole, isolation window: 1.2, activation: HCD, HCD \ncollision energy: 30%, scan rate: fast, data type: centroid).\nRaw LC-MS2 data analysis \t Tryptic\tpeptides\twere\tidentified\t\nby analysing raw data with the MASCOT algorithm \n1 3\nPage 7 of 21   346 \n\nA. Sota et al.\nby a patch pipette with a slightly broken tip. Osmolarity of \nall solutions ranged between 285 and 305 mOsm. V oltage \nramps of 50 ms duration spanning a voltage range from \n−100\t to\t+100\t mV\twere\t applied\t at\t0.5\t Hz\tfrom\t a\tholding\t\npotential (Vh) of 0 mV over a period of up to 360 s using the \nPatchMaster 2.90 software (HEKA, Reutlingen, Germany). \nAll voltages were corrected for a 10 mV liquid junction \npotential.\t Currents\t were\t filter\tat\t 2.9\t kHz\t and\t digitized\t at\t\n100\tµs\tintervals.\t Capacitive\t currents\t and\tseries\tresistance\t\nwere determined and corrected before each voltage ramp \nusing the automatic capacitance compensation of the \nEPC-9. Inward and outward currents were extracted from \neach individual ramp current recording by measuring \nthe\t current\t amplitudes\t at\t −80\t and\t +80\t mV ,\trespectively,\t\nand plotted versus time. Representative current-voltage \nrelationships (IVs) were extracted at indicated time points. \nTo obtain voltage relationships of net currents developing \nduring application of DVF saline and 20 s after its removal \n(Fig. 6b and e), currents before DVF condition were \nsubtracted.\t Net\t current\t voltage\t relationships\t of\tmTRPV6\t\ncurrents in HEK-293 cells (Fig. 6d) were obtained by \nsubtracting the basic current after break-in. All currents \nwere\tnormalized\tto\tthe\tcell\tsize\t(pA/pF).\nConfocal Ca2+ imaging on isolated uteri in situ\nUterine horns of adult female Trpv6-IC/eR26-GCaMP3\tand\t\nTrpv6−/−-IC/eR26-GCaMP3\tmice\tin\tthe\testrus\tphase\tof\tthe\t\ncycle were dissected and placed in an ice-cold Ringer’s \nsolution\t containing\t 115\t mM\t NaCl,\t 5\t mM\t KCl,\t 2\t mM\t\nMgCl2, 2 mM CaCl2, 10 mM HEPES, 10 mM glucose, pH \n7.4. Residual connective and adipose tissue were removed, \nand the uteri were dissected and opened longitudinally \nto expose the endometrium. The epithelial layer of the \nendometrium\twas\tintactly\tscraped\toff\twith\ta\tscalpel\tblade\t\nto detach it from the smooth muscle layer of the uterus. \nCytosolic Ca2+ imaging experiments were performed using \nan upright confocal microscope (Zeiss, LSM 710) equipped \nwith a multi-line argon laser and a water immersion 20x \nobjective (Zeiss, Plan-Apochromat). Ca 2+-dependent \nGCaMP3\t fluorescence\t(493–598\t nm,\t excitation\t 488\t nm),\t\nappearing exclusively in MEECs, was recorded at 2 Hz at 2 \nmM and 0.5 mM extracellular Ca2+ (see start of experiments \nin Fig. 5). Ca2+-free solution containing 0.5 mM EGTA and \n10\tµM\tCPA\twere\tapplied\tas\tindicated.\tAll\tsolutions\twere\t\ngravity applied through a valve controller system (Warner \nInstruments, VC-6 Valve Controller and TTL switch, \nCED Micro1401-3). Regions of interest were marked \nwith\tthe\tZEN\tBlack\tsoftware\t(Zeiss)\tand\tthe\tfluorescence\t\nrecordings\twere\tanalyzed\twith\tImageJ\tand\tplotted\tas\tF/F0 \n(fluorescence\tintensity\tF\tdivided\tby\tthe\tbasic\tfluorescence\t\nat the beginning of the experiment F0) versus time.\nduring 50 ms excitation at 340 and 380 nm after subtraction \nof the background. Individual cells were selected as regions \nof interest (ROI) with Live Acquisition (LA) software \n(TILL\t Photonics)\t and\t F340/F380\t was\t plotted\t versus\t\ntime. The SERCA inhibitor cyclopiazonic acid (CPA) and \nCaCl2\twere\tadded\tto\tthe\tbath\tto\treach\tfinal\tconcentrations\t\nas indicated. For analysis, peak amplitude and area under \nthe curve of Ca 2+ release and Ca 2+\t influx\twere\t calculated\t\nafter subtraction of the baseline right before store depletion \n(CPA) and Ca 2+\t re-addition\t as\t DF340/F380\t and\t DF340/\nF380 x s, respectively.\nHEK-293 and COS-7 cell culture and transfection\nHEK-293 cells (ATCC, CRL 1573) and COS-7 cells (ATCC, \nCRL-1651), obtained from the American Type Culture \nCollection (ATCC, Manassas, V A), were cultured in 75 \ncm2\tflasks\tin\tminimal\tessential\tmedium\t(MEM\tHEK-293)\t\nand\tDulbecco’s\tmodified\teagles\tmedium\t(DMEM\tCOS-7)\t\n(both Life Technologies, Carlsbad, USA) containing 10% \nfetal calf serum (FCS; Life Technologies) at 37 °C and 5% \nCO2. For transient transfection, cells were grown in 3 cm \ndiameter\t culture\t dishes\t until\t 80%\t confluence\t and\t then\t\ntransiently\t transfected\t with\t 4\t µg\t of\t pCAGGS-mTRPV6-\nIRES-GFP\t cDNA\t in\t 5\t µL\t of\t the\t PolyFect® reagent \n(Qiagen, Hilden, Germany). Dishes were trypsinized and \ntransfected cells were plated on 10 mm PLL-coated glass \ncoverslips for patch clamp experiments, performed on \nGFP-expressing cells 48 h after transfection. For western \nblot analysis 24 h after transfection cells were resuspended \nwith\t150\tµL\tdenaturing\tsample\tbuffer\t(final\tconcentration:\t\n60 mM Tris HCl, pH 6.8, 4% SDS, 10% glycerol including \n0.72\t M\t β-mercaptoethanol)\t and\t incubated\t at\t 60\t °C\t for\t\n20 min before applying on SDS-PAGE [23].\nElectrophysiological recordings\nWhole cell currents of MEECs and HEK-293 cells were \nrecorded\t in\tthe\ttight\tseal\tpatch\tclamp\tconfiguration\t using\t\nan\t EPC-9\t amplifier\t (HEKA\t Electronics,\t Lambrecht,\t\nGermany). Patch pipettes were pulled from glass capillaries \nGB150T-8P (Science Products, Hofheim, Germany) at a \nPC-10\t micropipette\t puller\t (Narishige,\t Tokyo,\tJapan)\t and\t\nhad\t resistances\t between\t 3\t and\t 5\t MΩ\t when\t filled\t with\t\ninternal\t solution\t (in\t mM):\t 120\t Cs-Glutamate,\t 8\t NaCl,\t 1\t\nMgCl2, 10 HEPES, 10 Cs-BAPTA, pH adjusted to 7.2 \nwith CsOH. Standard external solution contained (in mM): \n140\t NaCl,\t 10\t CaCl2, 10 CsCl, 2 MgCl 2, 10 HEPES, 10 \nglucose,\t pH\tadjusted\t to\t7.2\twith\t NaOH.\tWhere\t indicated\t\ndivalent-free (DVF) saline, based on standard external \nsolution without Ca 2+ and Mg 2+ but with 10 mM EGTA, \nwas pressure applied directly onto the patch clamped cell \n1 3\n  346  Page 8 of 21\n\nTRPV6 channel function is involved in endometrial epithelial cell Ca 2+ signaling and female mouse fecundity\noutliers beyond the whiskers are depicted as dots. The \nsignificance\t of\t the\t non-parametric\t data\t was\t evaluated\t by\t\nMann-Whitney U tests (two groups) or Kruskal Wallis with \nDunn’s multiple comparison tests (more than two groups). \nP values of less than 0.05 were considered statistically \nsignificant.\t Final\t figures\t were\t prepared\t with\t CorelDraw\t\n(Alludo,\tOttawa,\tON,\tCanada).\nResults\nTRPV6-deficiency impairs fecundity in female mice\nAs previously shown [3], TRPV6 is expressed in the decidua \nduring pregnancy. The decidua is the essential prerequisite \nfor perception and maintenance of the embryo. Homozygous \nTrpv6−/−-deficient\tfemale\tmice\tor\tfemale\tmice\thomozygously\t\ncarrying a single-point mutation within the channel pore \nof TRPV6 (D581A), leading to a non-functional TRPV6 \nchannel ( Trpv6mt/mt),\t conceived\t their\t very\t first\t litter\t after\t\nthe\tfirst\tmating\tin\taverage\tabout\t5\tdays\tlater\tas\tcompared\t\nto the respective wild-type mice (Fig. 1a, open circles). In \nStatistics\nCa2+ imaging data were analyzed using OriginPro 2021b \n(OriginLab\tCorporation,\tNorthampton,\tMA,\tUSA)\tand\tIgor\t\nPro 6.31 (WaveMetrics, Portland, OR, USA). Fitmaster 2.90 \nsoftware (HEKA, Reutlingen, Germany) and GraphPad \nPrism 9 (GraphPad software, Boston, MA, USA) were used \nto analyze and plot patch clamp data, as well as for statistical \ntesting and graphing.\nCurrent traces vs. time are shown as means ± standard \nerror of the mean (SEM). The normality of data \ndistribution was tested using the Shapiro-Wilk test. Data \nof normal distribution (parametric) were plotted as bar \ngraphs showing means ± standard deviation (SD) and \nthe\t significance\t was\t assessed\t either\t by\t unpaired\t two-\ntailed\t Student’s\t t-test\t (two\t groups)\t or\t one-way\t ANOV A\t\nwith Tukey’s multiple comparison test (more than two \ngroups).\t Non-parametric\t data\t are\t shown\t as\t Tukey’s\t box\t\nand whiskers with median and boxes, which extend from \nthe 25th to the 75th percentile (interquartile range [IQR]). \nWhiskers are extended to the most extreme data point that \nis no more than 1.5 x IQR from the edge of the box, and \nFig. 1 \t Trpv6-deficiency\t impairs\t fecundity\t in\t female\t mice.\t Waiting\t\ntime\t until\t first\tpregnancy\t (a), interval between litters ( b), litter size \n(c), fecundity rate ( d), blood concentration of FSH (follicle-stimulat -\ning hormone) and LH (luteinizing hormone; e), estrous cycle length \n(f), number (no.) of estrus stages ( g) and time spent in estrus during \na period of 35 days ( h) in female wild-type, Trpv6−/− and Trpv6mt/mt \nmice.\tNon-parametric\tand\tparametric\tdata\tare\tpresented\tas\tTukey\tbox\t\n(interquartile range (IQR) from 25th to the 75th percentile) and whis -\nkers (extended to the most extreme data point no more than 1.5 x IQR \nfrom edge of the box) with median (a, b, f-h) and bar graphs with \nmean ± SD (c-e), respectively, and are statistically analyzed by Krus -\nkal-Wallis\t(a,\tb,\tf-h),\tone-way\tANOV A\t(c,\td)\tand\tStudent´s\tt-test\t(e).\t\nNumbers\ton\ttop\tof\tthe\tgraphs\trepresent\tthe\tP\tvalues.\tDots\tand\tcircles\t\nin a, b and f-h represent single and mean values, respectively. Circles \nin d and e represent single values. The numbers of analyzed matings \n(a-c) and individual mice (d-h) are indicated below the boxes and bars\n \n1 3\nPage 9 of 21   346 \n\nA. Sota et al.\nin wild-type mice was high at estrus, we focused on a \npossible functional expression of TRPV6 in the uterus of \nmice at that cycle stage.\nTo substantiate the presence of TRPV6 in the uterus, \nwe isolated uteri from adult female Trpv6-IC/eR26-τGFP\t\nmice at estrus and visualized GFP-positive cells in the \nendometrial cell layers using immunolabeling-enabled \nthree-dimensional imaging of solvent-cleared organs \n(iDISCO [44]), (Fig. 3a). The mouse endometrium consists \nof two major cell types, epithelial and stromal cells. \nTRPV6-positive cells apparently appeared in the epithelial \ncell layer. To identify the TRPV6-expressing cells, we \nisolated the uteri of adult female Trpv6-IC/eR26-τGFP\t\nmice at estrus and cultured the mouse endometrial epithelial \ncells (MEECs) and stromal cells (MESCs), each separately. \nMEECs\tand\tMESCs\twere\tidentified\tby\ta\tpositive\tstaining\t\nof cytokeratin and vimentin, respectively (Fig. 3b). While \nthe MESC culture was devoid of GFP-positive cells, about \n10% of the MEECs were GFP-positive, i.e. the TRPV6 \npromotor had been active. For further experiments we \nprepared MEEC and MESC cultures from wild-type, \nTrpv6−/− and Trpv6mt/mt mice. Figure 3c and Supplementary \nFig. 2 demonstrate highly enriched MEEC and MESC \ncultures,\t verified\t by\t positive\t staining\t against\t cytokeratin\t\nand\t vimentin,\t which\t did\t not\t differ\t between\t the\t three\t\ngenotypes (Fig. 3c and Fig. S2). Western blot and mass \nspectrometric analysis of TRPV6 immunoprecipitations \nfrom\t wild-type\t cells\t show\t a\t>\t100-fold\t higher\t expression\t\nof TRPV6 in MEECs compared to MESCs (Fig. 3d-f). In \naddition, the time between consecutive litters was increased \nby more than 20% and the average litter size was decreased \nfrom 8.1 pups in wild-type to 6.7 and 6.0 pups in Trpv6−/− \nand Trpv6mt/mt mice, respectively (Fig. 1b, c). Calculated \nfrom these parameters (see methods) the fecundity rate was \nsignificantly\t reduced\t in\t mice\t lacking\t functional\t TRPV6\t\nchannels (Fig. 1d).\nIn estrus, TRPV6 is present in MEECs\nWhile in Trpv6mt/mt mice the blood level for LH but not for \nFSH\t was\t significantly\t increased\t (Fig.\t 1e) and the estrus \ncycle length decreased (Fig. 1f), neither the number of estrus \nstages (Fig. 1g) nor the time in estrus (Fig. 1h; Fig. S1) was \ndifferent\t between\t the\t two\t functional\t TRPV6-KO\t models\t\n(Trpv6−/− and Trpv6mt/mt) and wild-type mice.\nWe\t recently\t identified\t TRPV6\t in\t a\t subset\t of\t\nendometrial cells in the uterus of pregnant mice ([ 21], \nsee also Fig. 3a). Western Blot and mass spectrometry \nanalysis of TRPV6 antibody-enriched protein lysates of \nisolated uteri from wild-type and Trpv6mt/mt mice revealed \nthe presence of TRPV6 protein at proestrus, estrus and \nmetestrus but not at diestrus, demonstrating a cycle-\ndependent expression of TRPV6 (Fig. 2). The MS results \nalso show that TRPV6 abundance in the uterus of wild-\ntype and Trpv6mt/mt mice is comparable, as the number \nof\t TRPV6\t peptides\t identified\t across\t all\t cycle\t stages\t is\t\nsimilar (see Fig. 2, lower panel). Uteri from Trpv6−/− \nmice lack the TRPV6 protein. Since protein expression \nFig. 2 Estrous cycle-dependent expression of TRPV6 in the mouse \nuterus. Upper panel: Western blot detection of TRPV6 protein in \nlysates\t of\t HEK-293\t cells\t transfected\t with\t mTRPV6\t cDNA\t or\t in\t\nelutes after immunoprecipitation (IP) of TRPV6 from pregnant uteri \n(E12.5) and during all four stages of the estrous cycle from wild-type, \nTrpv6−/− and Trpv6mt/mt uteri. For IP an antibody directed against the \nN-terminus\t and\t for\t detection\t an\t antibody\t against\t the\t C-terminus\t of\t\nTRPV6 was used. The green lines indicate mono- and multimers of \nTRPV6 proteins (also seen as smeared bands in elutes from Trpv6mt/mt \nuteri), while the asterisk indicates IgG multimers. Lower panel: total \nnumber of tryptic TRPV6 peptide spectra (PSM) detected by mass \nspectrometry\tafter\tTRPV6-specific\tIP\tfrom\tmurine\tuteri\t(n\t=\t2).\tNote\t\nthat\t TRPV6-specific\t tryptic\t peptides\t are\t detected\t in\t wild-type and \nTrpv6mt/mt (pore mutant) but not in Trpv6−/− (global knockout) uteri \n(n.d. = no detection)\n \n1 3\n  346  Page 10 of 21\n\nTRPV6 channel function is involved in endometrial epithelial cell Ca 2+ signaling and female mouse fecundity\nFig. 3 TRPV6 is expressed in MEECs, but not in MESCs. (a) iDISCO \ncleared uterus of a Trpv6-IC/eR26-τGFP\t mouse.\t 2D\t projection\t of\t a\t\n3D image stack from uterus with ovaries (top) and 2D image of the \nendometrium\t at\t higher\t magnification\t (bottom).\t GFP-expressing\t\ncells (green) are mainly observed in the epithelial cell layer of the \nendometrium. ( b) Immunostaining of eGFP (green) in endometrial \nepithelial\t (MEEC)\t and\t stromal\t (MESC)\t cells,\t identified\tby\t positive\t\nstaining against cytokeratin and vimentin (red), cultured from uteri of \nTrpv6-IC/eR26-τGFP\tmice.\t(c) Staining against cytokeratin (red) and \nvimentin (green) in MEEC and MESC cultures, isolated from wild-type \nmice uteri. In b and c Hoechst was used to stain nuclei (blue; top: single \npictures; bottom: merged pictures). ( d) Western blot of lysates from \nTRPV6\t cDNA-transfected\t and\t non-transfected\t COS-7\t (COS)\t cells\t\nand elutes from MEEC and MESC cultures after immunoprecipitation \n(IP)\t with\t TRPV6\t antibodies\t (N-terminal)\t incubated\t with\t the\t\nmonoclonal C-terminal TRPV6 antibody (20C6). (e) Protein sequence \nof\tmouse\tTRPV6\t(SwissProt:\tQ91WD2)\twith\tamino\tacids\tidentified\t\nby\tMS/MS\tfragmentation\tin\tMEECs\thighlighted\t(bold\tletters,\tgrey),\t\nwhich\t cover\t 33%\t of\t the\t sequence\t (3\t technical\t replicates).\t Notably,\t\none peptide detected by mass spectrometry is located upstream of the \ninitially annotated initiation methionine [ 22], which is shown in red. \n(f) Comparison of the semiquantitative TRPV6 protein abundance \ndetected by MS2 analysis in the elutes after immunoprecipitation from \nMEECs and MESCs. The protein abundance is shown as sum of all \ndetected parent TRPV6 peptide areas (n = 3 unpaired t-test, bar graphs \nwith mean + SD)\n \n1 3\nPage 11 of 21   346 \n\nA. Sota et al.\nTRPV6 contributes to spontaneous Ca2+ oscillations \nin MEECs in situ\nTo study cytosolic Ca 2+ signaling in TRPV6-expressing \nand TRPV6 knock-out cells in the endometrium in \nsitu, we prepared uteri from adult female Trpv6-IC/\neR26-GCaMP3 and Trpv6−/−-IC/eR26-GCaMP3\t mice\t\nat estrus, which express the endogenous Ca 2+-indicator \nGCaMP3 in a Cre-dependent manner in Trpv6+/+-Cre \nand Trpv6−/−-Cre cells, i.e. in the MEECs (Fig. 5a). For \nCa2+ imaging, the uterus tube was cut open and spread \nflat\tinto\t the\t bath\t chamber\t with\t the\t endometrial\t layer\t on\t\ntop. 30–50% of the cells in the endometrial layer of the \nuteri prepared from the Trpv6- and Trpv6−/−-IC/eR26-\nGCaMP3\t mice\t expressed\t the\t fluorescent\t Ca2+ indicator. \nThe uteri of both genotypes revealed regular cytosolic \nCa2+ oscillations in the GCaMP3-positive MEECs, which \ndisappeared over time in the absence of extracellular Ca 2+ \n(Fig. 5b, c) and after depletion of the intracellular Ca 2+ \nstores by CPA (Fig. 5d; all traces represent Ca 2+ signals \nfrom single cells). While the reduction of extracellular \nCa2+ generally resulted in less spontaneous Ca 2+ signals \nin the MEECs of both genotypes, Trpv6-deficient\t cells\t\nrevealed\t significantly\t reduced\t frequencies\t of\t basic\t Ca2+ \noscillations as compared to Trpv6 wild-type cells in both \n2 mM and 0.5 mM extracellular Ca 2+ (Fig. 5e, f). The data \nsuggest that both Ca 2+ release and Ca 2+\tinflux\tcontribute \t\nto the spontaneous Ca 2+ oscillations in MEECs in situ, \nand\tthat\tTRPV6\tchannel\t activity\t is\tsignificantly\t involved \t\nin this process.\nthe later only few and low abundant TRPV6 peptide spectra \nwere\tidentified,\twhich\tcould\tbe\tdue\tto\tlow\tcontamination\t\nwith\t MEECs.\t Notably,\t one\t peptide\t detected\t by\t mass\t\nspectrometry is located upstream of the initially published \nstarting methionine (Fig. 3e, red). For positive and negative \ncontrol\tof\tthe\tWestern\tblot,\tTRPV6-cDNA-transfected\t and\t\nnon-transfected COS cells were used (Fig. 3d).\nTRPV6 contributes to Ca2+ influx in MEECs\nTo prove a possible contribution of TRPV6 in cytosolic \nCa2+ signaling in the mouse endometrial epithelial cells, \nwe isolated MEECs from adult wild-type, Trpv6−/− and \nTrpv6mt/mt female mice at estrus and performed Ca2+ imaging \nexperiments using the Ca 2+-sensitive\t fluorophore\t Fura-2.\t\nNo\t apparent\t morphological\t alterations\t were\t observed\t in\t\nthe isolated uteri of the three genotypes. About 45% of the \ncultured MEECs revealed spontaneous Ca2+ signals (Fig. 4a, \nb; traces in a represent Ca2+ signals from single cells). While \nthe\tpercentage\tof\tspontaneously\tactive\tMEECs\tdid\tnot\tdiffer\t\nbetween the genotypes, MEECs from wild-type mice revealed \nin average two Ca 2+ peaks and MEECs from Trpv6−/− and \nTrpv6mt/mt mice only one Ca2+ peak within 5 min (Fig. 4b, c). \nIn addition, the basic Ca2+ level in wild-type cells was slightly \nbut\tsignificantly\thigher\tthan\tin\tTrpv6−/− and Trpv6mt/mt cells \n(Fig. 4d). To prove whether TRPV6 contributes to Ca 2+ \ninflux\tin\t MEECs,\twe\t depleted\tthe\t intracellular\tCa2+ stores \nby the inhibition of Ca2+ re-uptake into the stores using the \nSERCA inhibitor cyclopiazonic acid (CPA) in the absence of \nextracellular Ca2+ and subsequently re-added Ca2+. In Trpv6−/− \nand Trpv6mt/mt MEECs the peak amplitude of the Ca2+\tinflux\t\nafter Ca2+\tre-addition\twas\tsignificantly,\tand\tits\tarea\tunder\tthe\t\ncurve tendentially reduced as compared to wild-type cells \n(Fig. 4e; traces represent Ca2+ signals from single cells).\nCa2+ store depletion activates store-operated Ca 2+ \nchannels, especially ORAI1-3 [ 45],\t which\t significantly\t\ncontribute to the Ca 2+\t influx\tafter\t Ca2+ re-addition. While \nin the presence of GSK7975A and BTP2 (YM-58483), \ntwo well established inhibitors of store-operated Ca 2+ \nchannels [ 46, 47], Ca 2+ entry upon Ca 2+ re-addition after \nstore depletion in Trpv6−/− and Trpv6mt/mt cells almost \ncompletely disappeared, wild-type MEECs still revealed \na\t significant\tCa2+\t influx\t(Fig.\t 4f, g; traces represent Ca 2+ \nsignals from single cells). The data suggests that TRPV6 \nessentially contributes to Ca 2+\tinflux\tin\twild-type\tMEECs.\t\nHowever, the CPA-mediated Ca 2+ release in the absence \nand presence of GSK7975A and BTP2 was increased in \nTrpv6−/− and Trpv6mt/mt MEECs as compared to the wild-\ntype cells (Fig. 4e-g). Apparently, the functional ablation of \nTRPV6 leads to a higher Ca2+ content of the CPA-sensitive \nintracellular Ca2+ stores.\nFig. 4 TRPV6 contributes to Ca 2+\tinflux\tin\tcultured\tMEECs.\tRepre-\nsentative cytosolic Ca2+\trecordings\t(F340/F380)\tfrom\tMEECs\tisolated\t\nfrom wild-type (black), Trpv6−/− (red) and Trpv6mt/mt (blue) mice in the \npresence of 2 mM extracellular Ca2+ (a) and in the nominal absence of \nextracellular Ca2+ (0 Ca2+)\twith\tsubsequent\tstore-depletion\tby\t10\tµM\t\ncyclopiazonic acid (CPA) and 2 mM Ca2+ re-addition (e-g, left panels) \nin the absence (e) and presence (f, g) of ORAI inhibitors GSK7975A \n(10\tµM,\tf)\tand\tBTP2\t(3\tµM,\tg).\t(b-d) Percentage of oscillating cells \n(b), number (no.) of cytosolic Ca 2+ peaks per active cell within 5 min \n(c) and basic Ca 2+ levels (d) analysed from experiments as shown in \na. (e-g, right panels) Peak amplitude and area under the curve of the \nbaseline-subtracted Ca 2+\t signals\t (ΔF340/F380)\t after\t CPA-induced\t\nstore depletion (release) and Ca 2+\tre-addition\t(influx)\tanalysed\tfrom\t\nexperiments\t as\t shown\t in\t the\t respective\t left\t panels.\t Non-parametric\t\nand parametric data are presented as Tukey box (interquartile range \n(IQR) from 25th to the 75th percentile) and whiskers (extended to the \nmost extreme data point no more than 1.5 x IQR from edge of the \nbox) with median (c-g) and bar graphs with mean ± SD (b), respec -\ntively, and are statistically analyzed by Kruskal-Wallis (c-g) and one-\nway\tANOV A\ttests\t(b).\tDots\tin\tb\trepresent\tsingle\tvalues\tand\tdots\tand\t\ncircles in c-g represent single values beyond the whiskers and mean \nvalues,\trespectively.\tNumbers\ton\ttop\tof\tthe\tgraphs\trepresent\tthe\tP\tval-\nues. The numbers in b represent the number of measured dishes, and \nin\tc-f\t(n/x/y/z)\tthe\tnumber\tof\tanalyzed\tcells\t(n),\trecorded\tin\t(w)\tdishes\t\nfrom (y) independent cultures, prepared from (z) mice\n1 3\n  346  Page 12 of 21\n\nTRPV6 channel function is involved in endometrial epithelial cell Ca 2+ signaling and female mouse fecundity\nbeen recorded in any primary cell type, i.e. cells acutely \nprepared from living tissue expressing Trpv6, controlled \nby the corresponding cell type from Trpv6 knockouts. \nWe recorded whole-cell currents in MEECs isolated from \nwild-type, Trpv6−/− and Trpv6mt/mt mice, but a TRPV6-like \nTRPV6-dependent whole-cell currents in wild-type \nMEECs\nTo the best of our knowledge, no distinct endogenous \nTRPV6-dependent whole-cell Ca 2+ current has yet \n \n1 3\nPage 13 of 21   346 \n\nA. Sota et al.\nFig. 5  Trpv6-deficiency\t lowers\t frequency\t of\t Ca2+ oscillations in \nMEECs in situ . ( a)\t GCaMP3\t fluorescence\t in\t isolated\t uteri\t from\t\nTrpv6-IC/eR26-GCaMP\t and\t Trpv6−/−-IC/eR26-GCaMP\t mice,\t i.e.\t in\t\ncells where the TRPV6 promotor had been active. ( b-d) Representa-\ntive cytosolic Ca 2+\t changes\t (F/F0) detected in GCaMP3-expressing \ncells from uteri of Trpv6- (Trpv6 (wt), black) and Trpv6−/−-IC/eR26-\nGCaMP3 mice (Trpv6−/−, red) at 2 mM (b, d) and 0.5 mM (c) extracel-\nlular Ca2+. At the indicated time points Ca 2+-free saline with 0.5 mM \nEGTA\t(b,\tc;\tblue)\tor\t10\tµM\tcyclopiazonic\tacid\t(CPA;\td)\twere\tapplied.\t\n(e, f) The box (interquartile range (IQR) from 25th to the 75th percen-\ntile) and whiskers (extended to the most extreme data point no more \nthan 1.5 x IQR from edge of the box) blots with median depict the \nnumber of distinct Ca2+ peaks (oscillations) within 5 min in 2 mM (e) \nand 0.5 mM (f) external Ca 2+. The data was statistically analyzed by \nMann-Whitney’s tests. P values are shown on top of the plots. Dots and \ncircles in e and f represent single values beyond the whiskers and mean \nvalues, respectively. Measurements were obtained from 7 preparations \n(uteri), i.e. 7 animals per genotype for each experimental condition and \ninclude the number of cells as shown in brackets\n \n1 3\n  346  Page 14 of 21\n\nTRPV6 channel function is involved in endometrial epithelial cell Ca 2+ signaling and female mouse fecundity\nendometrium\t with\t the\t highest\t mRNA\t level\t at\t proestrus\t\nand estrus in mouse [53], diestrus in rat [54] and during the \nproliferative phase in human endometrial tissue [ 55]. We \nidentified\tTRPV6-positive\tcells\tin\tthe\tdecidua\tof\tpregnant\t\nTrpv6-IC/eR26-tGFP\tmice\t[3] and the endometrial layer of \nuteri in Trpv6-IC/eR26-tGFP\tand\tTrpv6-IC/eR26-GCaMP3\t\nmice at estrus (Figs. 3a and 5a). In addition, and in agreement \nwith De Clercq et al. [ 53], we found the highest levels for \nthe TRPV6 protein at proestrus and estrus (Fig. 2). The \nexpression studies suggest that TRPV6 is especially needed \nfor proper endometrial function during the proliferative \nphase of the estrous cycle and during pregnancy.\nThe mouse endometrium consists of epithelial cells \n(MEECs) and stromal cells (MESCs). The present study \nrevealed\ta\tsignificant\texpression\tof\tTRPV6\tproteins\tmainly\t\nin MEECs (Fig. 3). Again, this is in good agreement with De \nClercq\tet\tal.\twho\treported\ton\tthe\tdistinct\tmRNA\texpression\t\nof TRPV4, TRPV6 and TRPM6 in mouse and human \nendometrial epithelial cells, while endometrial stromal cells \nmainly\texpress\tTRPV2,\tTRPC1/4\tand\tTRPC6\tmRNA\t[53].\nDuring the estrous cycle, the human endometrium trans -\nforms into the decidua, which enables the implantation of \nthe blastocyst and a successful pregnancy. In mice, decidu -\nalization has been shown to depend on the presence of a \nblastocyst in the uterine lumen [30–32]. Thus, the blastocyst \nmust send a signal to the MEECs, which further signal to \nthe\tunderlying\tstromal\tcells,\tto\tfinally\ttrigger\tand\tmaintain\t\ndecidualization. Pre-implantation blastocysts are shown \nto secrete diverse hormones, growth factors and proteases \nwhich might serve as signaling molecules in this process \n[56–59]. As part of the sensing and signaling pathway in \nthe MEECs Ca 2+\t influx\t had\t been\t suggested,\t initiated\t by\t\nactivation\t of\t the\t epithelial\t sodium\t channel\t (ENaC)\t and\t\nmediated either by a depolarization-dependent activation of \nvoltage-gated Ca2+ channels (VGCC) or a reverse mode of \nthe\t sodium\t calcium\t exchanger\t (NCX)\t [60, 61]. Cytosolic \nCa2+ signaling in the MEECs has already been shown to be \nimportant for the adhesion between the embryonic tropho -\nblasts and the endometrial epithelial cells [29, 62]. However, \nHennes\tet\tal.\tdid\tnot\tfind\tany\tevidence\tfor\tthe\tinvolvement\t\nof\tENaC,\tVGCC\tand\tNCX\tto\tCa2+\tinflux\tin\tMEECs\t[63]. \nInstead,\t they\t identified\t the\t protease-activated\t receptor\t 2\t\n(PAR-2) as the molecular entity initiating cytosolic Ca 2+ \noscillations in MEECs which depend on the phospholipase \nC\t (PLC)/inositol-1,4,5-trisphospate\t receptor\t (IP3R)/store-\noperated Ca2+ entry (SOCE) pathway upon secretion of pro-\nteases from the invading blastocyst. In their experiments, \nCa2+ oscillations in primary MEECs had been induced by \ntrypsin. SOCE and Orai1 expression had also been shown \nin Ishikawa cells, a human endometrial adenocarcinoma \ncell line with characteristics of luminal endometrial epithe -\nlial cells [64]. In addition, Piezo1, a mechanosensitive Ca2+ \nCa2+ current was not detectable either (Fig. 6a). It has been \nshown, that in the absence of extracellular divalent cations \nthe Ca 2+-selective pore of TRPV6 becomes permeable \nto\t monovalent\t cations,\t resulting\t in\t a\t significant\t boost\t of\t\nthe inward current [ 22, 23, 48]. Upon removal of divalent \ncations, i.e. during the application of divalent-free saline \n(DVF),\t a\t significant\t whole-cell\t current\t appeared\t in\t the\t\nMEECs of all three genotypes (Fig. 6a, top panel; current \nvoltage\t relationships\t at\tdifferent\ttime\tpoints\t are\tshown\tin\t\nthe lower panel). However, the net DVF-mediated inward \ncurrent (but not the outward current) in wild-type MEECs \nwas\t significantly\t larger\t than\t in\t Trpv6−/− and Trpv6mt/mt \ncells (Fig. 6b). While this (net) extra current in the presence \nof functional TRPV6 ion channels is very small (in average \nless\tthan\t2\tpA/pF),\tits\tcurrent-voltage\trelationship\t(Fig.\t6c, \nDVF-mediated net current in wild-type minus Trpv6−/− \n(red) or minus Trpv6mt/mt (blue)) reveals exactly the same \ncharacteristic course as at divalent-free conditions for \nhuman TRPV6 transiently expressed in HEK-293 cells \n(Fig. 6d). The other part of the DVF-mediated current \nand the transient, mainly outwardly directed current after \nre-addition of divalent-containing bath solution reveal \nsimilar shapes and amplitudes in all three genotypes \n(Fig. 6a, b and e) and apparently do not depend on TRPV6.\nDiscussion\nThe TRPV6 ion channel is crucial for male mouse fertility \n[1, 2]\tand,\tin\tpregnant\tfemale\tmice,\tfor\ta\tsufficient\tsupply\tof\t\nCa2+ to the embryo, which is important for fetal growth, bone \ncalcification\tand\tbone\tdevelopment\t[3]. In addition, TRPV6 \ncontrols extracellular matrix structure of the placental \nlabyrinth [49]. The present study shows that mice lacking \nfunctional\tTRPV6\tchannels\thave\tproblems\twith\ttheir\tfirst\t\npregnancy, have a longer latency between pregnancies and \nproduce\tfewer\toffspring,\ta\tphenomenon\tknown\tas\timpaired\t\nfecundity (Fig. 1).\nEmbryo transfer experiments revealed that TRPV6 in \nboth the fetal tissue (labyrinth trophoblasts, yok sac) and \nthe maternal tissue (decidua) of the placenta contributes to \nmurine embryonic development and bone mineralization \n[3]. In that previous study, we investigated the function of \nTRPV6 in labyrinth trophoblasts, i.e. in the fetal part of \nthe placenta. Here we show the functional expression of \nTRPV6 in the maternal tissue, namely in epithelial cells of \nthe endometrium, the inner layer of the uterus.\nTRPV6\t has\t already\t been\t identified\t in\t pig\t luminal\t\nendometrial cells and bovine endometrium during \npregnancy [50, 51] and in sheep endometrial tissue during \nthe proliferative phase [ 52]. A cycle-dependent expression \nof TRPV6 has been shown for mouse, rat and human \n1 3\nPage 15 of 21   346 \n\nA. Sota et al.\nFig. 6 MEECs reveal a TRPV6-dependent whole-cell current at DVF \nconditions. (a)\tIn-\tand\toutward\tcurrents\trecorded\tat\t−80\tand\t80\tmV\t\nduring\t voltage\t ramps\t spanning\t from\t −\t100\t to\t 100\t mV ,\tapplied\t at\t\n0.5 Hz, and plotted versus time in MEECs isolated from wild-type (wt), \nTrpv6−/− and Trpv6mt/mt mice (top panels). Bars indicate the applica -\ntion of divalent-free saline (DVF). Current-voltage-relationships (IVs) \nat the time points as indicated by the colored lines are shown in the \nlower panels. Black traces are behind the green traces. ( b) IVs (left) \nand\tamplitudes\tat\t−80\tmV\t(middle)\tand\t80\tmV\t(right)\tof\tthe\tnet\tDVF-\nmediated currents (currents just before DVF had been subtracted). ( c) \nIV of the net current appearing in DVF saline only in wild-type but \nnot in Trpv6−/− and Trpv6mt/mt MEECs (calculated from traces in b: \nwt minus Trpv6−/− (red) and wt minus Trpv6mt/mt (blue)). ( d) IVs of \nthe net whole-cell currents before (in Ca 2+, light blue) and in DVF \nsaline\t(black)\tin\tHEK-293\tcells\ttransfected\twith\tmTRPV6\tcDNA.\t(e) \nIVs of the net current 20 s after DVF in wt, Trpv6−/− and Trpv6mt/mt \nMEECs.\tAll\tcurrents\twere\tnormalized\tto\tthe\tcell\tsize\t(pA/pF).\tData\t\nshow means ± SEM (a, top), means ± SD (bar graphs in b, circles rep -\nresent single values) or just means (all IVs). The parametric data in b \nwere\tanalyzed\tby\tone-way\tANOV A\ttests,\twith\tP\tvalues\tbelow\tor\ton\t\ntop of the bars. The numbers of analyzed cells is shown in brackets\n \n1 3\n  346  Page 16 of 21\n\nTRPV6 channel function is involved in endometrial epithelial cell Ca 2+ signaling and female mouse fecundity\nstill unclear. Yet, we have no direct experimental evidence \nthat the reduced fecundity of Trpv6 KO mice is due to the \naltered Ca2+ oscillations in endometrial cells. However, the \nTRPV6 channel could possibly be a sensor and messenger \nmolecule in the EECs involved in the transformation of \nextracellular\tstimuli\t into\t the\t influx\tof\t Ca2+, inducing and \ncoordinating\t underlying\t signaling\t pathways.\t Our\t findings\t\nthat Trpv6-deficient\tfemales\thave\treduced\tCa2+ oscillations \nin endometrial cells support this hypothesis.\nIt is still unclear whether TRPV6 plays a direct role as \nmaternal endometrial sensor for the blastocyst signaling \nbut considering that lack of functional TRPV6 channels \nresults in impaired fecundity, it might be involved in the \nearly implantation process. The loss of TRPV6-mediated \nCa2+ entry could lead to a higher incidence of implantation \nfailure, which would require multiple attempts to reach \na successful mating, and thus to a longer latency to the \nfirst\t and\t subsequent\t pregnancies.\t Furthermore,\t Ca2+ \ninflux\tvia\tTRPV6\t might\t be\t involved\t in\t the\t signaling\t that\t\ntriggers decidualization of the underlying stromal cells. \nDecidual cells are important for embryo implantation and \nthe maintenance of pregnancy. The observed smaller litter \nsizes from homozygous Trpv6-deficient\t mothers\t might\t\nbe the result of implantation failure of some but not all \nembryos.\t Implantation\t could\t also\t be\t affected\tby\t the\t lack\t\nof TRPV6 in the trophoblasts of the blastocyst, which form \nan altered extracellular matrix [ 49]. Trpv6\t deletion\t affects\t\nCa2+ signaling in MEECs and female fecundity to the same \nextend as the Trpv6 pore mutant. Thus, as in the epididymis \n[1]\tthe\teffects\tare\tmediated\tby\tthe\tmissing\tchannel\tfunction\t\nand not by other functions of the protein.\nFemale mice expressing a non-functional TRPV6 \nchannel protein ( Trpv6mt/mt) revealed increased LH blood \nlevels (Fig. 1e). This might represent a compensatory \neffect\t to\t a\t reduced\t TRPV6-dependent\t Ca2+ signaling. In \nthis respect, estrogen has been shown to upregulate TRPV6 \nin MEECs [ 55]. However, LH directly increases the \nprogesterone level, which might be the reason for a shorter \nestrous cycle as observed in the Trpv6mt/mt mice (Fig. 1f). \nWe found that although the time spent in estrus stage by the \nTrpv6mt/mt\tfemales\twas\tnot\tsignificantly\tdifferent\tfrom\tthat\t\nof the wild-type mice, there was a clear tendency for the \nTrpv6mt/mt females to have a shorter estrus. We observed \nthat while in Trpv6mt/mt mice the estrous stage occurred \nalmost exclusively during nighttime, wild-type mice were \nstill in the estrous phase the next morning. A shorter estrus \nleads to a smaller time window for the receptivity of the \nuterus and for the implantation of the embryo and thus to \na lower fecundity.\nOur experiments revealed that the Ca 2+ content of the \nCPA-sensitive intracellular Ca2+ stores was increased upon \nfunctional ablation of TRPV6. We did not study this in more \npermeable\t cation\t channel,\t functionally\t identified\t in\t mice\t\nand human endometrial epithelial cells, has also been sug -\ngested as potential signaling transducer between the blasto-\ncyst and the endometrium [65].\nHowever,\there\twe\tidentified\tfor\tthe\tfirst\ttime\tfunctional\t\nTRPV6 channels and their contribution to Ca 2+\tinflux\tand\t\nspontaneous Ca2+ signaling in living MEECs in vitro and \nin situ (Figs. 4, 5 and 6). Ablation of functional TRPV6 \nchannels did not abolish the spontaneous Ca 2+ oscillations, \nwhich depend on both Ca 2+ release and Ca 2+\t influx,\t but\t\nsignificantly\t reduced\t their\t frequency\t in\t isolated\t primary\t\nMEECs (Fig. 4) as well as in complete uterine tissue (Fig. 5). \nHennes et al. also observed spontaneous Ca 2+ oscillations \nin MEECs, which they explained by the induction of \nmechanical stimulation of the tissue during experimental \nhandlings\tprior\tto\tthe\tfluorescent\tmeasurements\t[63]. While \nthis might be a possible reason for Ca2+ oscillations seen in \nfreshly isolated uterine tissue, it does not reasonably explain \nspontaneous Ca2+ signals in the isolated primary MEECs. \nThe slightly higher basic level of cytosolic Ca2+ in wildtype \nMEECs (Fig. 4d), which might be due to a small Ca2+\tinflux\t\nvia TRPV6, might maintain the frequency of cytosolic Ca2+ \noscillations. These Ca 2+ oscillations may be triggered by \nthe cytosolic Ca2+ itself, e.g. via Ca2+-induced Ca2+ release \nand inositol 1,4,5-trisphosphate (InsP 3) receptors [ 66]. We \ndid not study the source of Ca 2+ during the spontaneous \ncytosolic Ca 2+ oscillations in more detail and the precise \nmechanism of such Ca 2+ oscillations, especially in non-\nexcitable cells, such as MEECs, is still not completely \nunderstood. However, our data suggest that both Ca2+ infux \nand Ca 2+ release are contributing and functional TRPV6 \nchannels are involved.\nThe embryo-maternal communication is of the utmost \nimportance for the coordination of the implantation process: \nearly embryo implantation is a complex event that requires \nan implantation-competent blastocyst and a receptive \nendometrium. Prior to implantation, the endometrial cells \n(EECs) sense the presence of the invading embryo and initiate \na number of intracellular signals, including Ca2+ oscillations \n[63]. These oscillations are essential for preparing the uterine \nlining\tfor\tembryo\timplantation\tby\tinfluencing\tgene\texpression,\t\ncell adhesion and the secretion of factors that support \nimplantation. In particular, the EECs signal the underlying \nendometrial stromal cells to undergo decidualization. \nDuring\t this\t process,\t stromal\t cells\t differentiate\tinto\t round,\t\nsecretory, pseudo-epithelial cells that provide nutrients for \nthe invading blastocyst and alter local immunity to allow \nfor proper implantation [67, 68]. Dysregulation of the Ca 2+ \noscillations might lead to impaired embryo implantation and \nreduced fecundity. The exact molecular players underlying \nCa2+ oscillations, the subsequent downstream signaling \npathways\t and\t the\t biological\teffects\tof\t their\t activation\tare\t\n1 3\nPage 17 of 21   346 \n\nA. Sota et al.\nthe blastocyst and the endometrium, and thus in the \nimplantation process itself is not yet known and requires \nfurther investigation. In this regard, an endometrial-\nspecific\tdeletion\tof\tthe\tTrpv6 gene would help to study the \nfunction of TRPV6 in endometrial epithelial cells in more \ndetail. However, the results could lead to novel approaches \nto improve the treatment of women with reproductive \ndisorders, such as those associated with defective Ca 2+ \nregulation, either in vivo or for in vitro fertilization.\nSupplementary Information The online version contains supplementary \nmaterial available at \th\tt\tt\tp\t\ts\t:\t/\t\t/\td\to\ti\t\t.\to\t\tr\tg\t/\t\t1\t0\t.\t1\t\t0\t0\t7\t\t/\ts\t0\t\t0\t0\t1\t8\t-\t0\t2\t5\t-\t0\t5\t8\t5\t7\t-\t9.\nAcknowledgements We thank Martin Simon-Thomas, Kathrin \nSchetting for technical support and Tanja Maurer, Mirjam Göttel, \nJohannes\t Stegner,\tAlina\t V elten,\tOliver\t Glaser,\t Jacqueline\t Schmidt,\t\nSandra Dittgen and Heidi Löhr for mouse colony management and \nsupport on cell culture, hormone injection, vaginal smear analysis, \nmouse\t preparation\t and\t microscopy.\tWe\talso\t thank\t Joris\tVriens\t(KU\t\nLeuven) for his help in establishing the culture of MEECs and MESCs.\nAuthor contributions Study design and conception: PW, CFT, AB. \nExperimental implementation: AS, AB, PWa, CFT, ALG, MW and PW. \nData analysis: AS, CFT, AB, PWa, MW, and PW. Data interpretation: \nall authors. MRM, UB, UW, MF and VF provided instrumentations, \nmice,\tplasmids\tand\tantibodies.\tThe\tfirst\tdraft\tof\tthe\tmanuscript\twas\t\nwritten by AS, AB and PW. All authors commented on previous \nversions of the manuscript. All authors have revised the manuscript \ncontent\tand\tapproved\tthe\tfinal\tversion.\nFunding Open Access funding enabled and organized by Projekt \nDEAL. This work was funded by the Deutsche Forschungsgemein -\nschaft\t(DFG,\tGerman\tResearch\tFoundation)\tWE4866/1–1\t(to\tPW)\tand\t\nFE629/2\t−\t1\t (to\t CFT),\t State\t Chancellery\t Saarland\t (INST\t 256/551-1\t\nFUGB (to M.R.M), by the Homburg Forschungsförderungsprogramm \n[HOMFOR] (to AB) and by the Forschungsausschuss der Universität \ndes Saarlandes (to AB).\nData availability The mass spectrometry proteomics data have been \ndeposited to the ProteomeXchange Consortium via the PRIDE [ 72] \npartner\trepository\twith\tthe\tdataset\tidentifier\tPXD060418\tand\t\th\tt\tt\tp\t\ts\t:\t/\t\t/\td\t\no\ti\t\t.\to\t\tr\tg\t/\t\t1\t0\t.\t6\t\t0\t1\t9\t\t/\tP\tX\t\tD\t0\t6\t0\t4\t1\t8. The data reported in this paper is  a v a i l a b l \ne from the corresponding author upon reasonable request.\nDeclarations\nCompeting interests\t The\t authors\t have\t no\t relevant\t financial\tor\t non-\nfinancial\tinterests\tto\tdisclose.\nEthics approval Our studies did not include human participants, human \ndata, or human tissue. All animal care and experimental procedures \nwere reviewed and approved in accordance with the guidelines and \nethical regulations established by the animal welfare committee of \nSaarland University.\nOpen Access   This article is licensed under a Creative Commons \nAttribution 4.0 International License, which permits use, sharing, \nadaptation, distribution and reproduction in any medium or format, \nas long as you give appropriate credit to the original author(s) and the \nsource, provide a link to the Creative Commons licence, and indicate \nif changes were made. The images or other third party material in this \ndetail, and we have no explanation for that yet. Maybe TRPV6 \nis expressed in intracellular organelles and contributes to \na consistent leakage from these stores. However, since \nthe frequency of Ca 2+ oscillations, which also depend on \nintracellular Ca2+ release (see Fig. 5), are reduced in Trpv6-\ndeficient\tcells,\tmore\tCa2+ might remain in the stores.\nSince the pharmacological tools, including cis-22a \nwhich\tis\tthe\tmost\teffective\tantagonist\tavailable,\thave\tonly\t\nbeen\t proven\t to\t affect\tCa2+\tinflux\tand\t membrane\t currents\t\nof\toverexpressed\t TRPV6\trather\tthan\tspecifically\ttargeting\t\nnative TRPV6 function, we used MEECs from Trpv6−/− \nand Trpv6mt/mt animals as controls [ 1, 2], especially for \nelectrophysiological measurements, rather than pursuing \na pharmacological strategy. Years ago, we developed the \nTrpv6-deficient\t mouse\t models\t Trpv6−/− and Trpv6mt/mt as \nvalid controls for wild-type animals, and we have used \nthem ever since as controls for wild-type animals, as well \nas for organs and cells isolated from them. In our opinion, \nusing a genetic approach to eliminate TRPV6 activity \nin order to control its cellular function in the wild-type \nmay\treduce\tthe\toff-target\teffects\ttypically\t associated\t with\t\npharmacological approaches.\nA low number of endometrial cells positive for GFP \nmay\t reflect\tthe\t rapid\t turnover\t of\t endometrial\t cells\t in\t the\t\nbrief murine estrus cycle and the cycle-dependent TRPV6 \nexpression. The genetic approach to label these cells is \nbinary, requiring Cre-mediated recombination to switch \non GFP expression. Thus, it may take up to 24 hours after \ninitial TRPV6 expression to detect GFP. The Rosa26 locus \nis a genetic region used in many mouse lines to integrate \ntransgenes and achieve consistent gene expression across \ndifferent\t cell\t types\t and\t developmental\t stages.\t Although\t\nthis Rosa26 locus is generally considered to be expressed \nubiquitously, there are reports of reduced expression \nin certain cell types and variation in expression levels \ndepending\ton\tthe\tspecific\tinserted\tgenes\t[69, 70]. In addition \nto\t the\t cell\t type,\t specific\t transgene\t constructs\t or\t other\t\nregulatory\tinfluences\tcould\tcontribute\tto\tthis\tphenomenon.\t\nThe genetic background of the Rosa26 reporter mouse \nstrains\t(C57BL/6J\tfor\teR26-τGFP\tand\tC57BL/6\tN\tfor\teR26-\nGCaMP3)\tcould\talso\tresult\tin\tdifferences\tbetween\tMEECs\t\npositive for GCaMP3, which are isolated from Trpv6-IC/\neR26-GCaMP3 animals, and MEECs positive for GFP, \nwhich are isolated from Trpv6-IC/eR26-τGFP\t animals.\t It\t\nis also known that the 3’ coding sequence is expressed at \nsignificantly\tlower\tlevels\tthan\tthe\t5’\tcoding\tsequence\t[71], \nbut this hardly applies to the GFP and GCaMP3 reporter \nconstructs used here.\nOur\tstudy\tprovides\tthe\tfirst\tinsight\tinto\tthe\tphysiological\t\nfunction of TRPV6 channels in the mouse uterus and their \npotential importance for female fecundity and fertility. \nWhether TRPV6 is directly involved in signaling between \n1 3\n  346  Page 18 of 21\n\nTRPV6 channel function is involved in endometrial epithelial cell Ca 2+ signaling and female mouse fecundity\nof a selective TRPV6 calcium channel inhibitor. 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