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. 2023 Apr 15;21(1):37.
doi: 10.1186/s12958-023-01085-7.

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

Aurélie Hennes #  1   2 , Johanna Devroe #  1   2   3 , Katrien De Clercq  1   2 , Martina Ciprietti  1   2 , Katharina Held  1   2 , Katrien Luyten  1 , Nele Van Ranst  2 , Nina Maenhoudt  4 , Karen Peeraer  1   3 , Hugo Vankelecom  4 , Thomas Voets  2 , Joris Vriens  5   6
Affiliations

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

Aurélie Hennes et al. Reprod Biol Endocrinol. .

Abstract

Background: Early embryo implantation is a complex phenomenon characterized by the presence of an implantation-competent blastocyst and a receptive endometrium. Embryo development and endometrial receptivity must be synchronized and an adequate two-way dialogue between them is necessary for maternal recognition and implantation. Proteases have been described as blastocyst-secreted proteins involved in the hatching process and early implantation events. These enzymes stimulate intracellular calcium signaling pathways in endometrial epithelial cells (EEC). However, the exact molecular players underlying protease-induced calcium signaling, the subsequent downstream signaling pathways and the biological impact of its activation remain elusive.

Methods: To identify gene expression of the receptors and ion channels of interest in human and mouse endometrial epithelial cells, RNA sequencing, RT-qPCR and in situ hybridization experiments were conducted. Calcium microfluorimetric experiments were performed to study their functional expression.

Results: We showed that trypsin evoked intracellular calcium oscillations in EEC of mouse and human, and identified the protease-activated receptor 2 (PAR2) as the molecular entity initiating protease-induced calcium responses in EEC. In addition, this study unraveled the molecular players involved in the downstream signaling of PAR2 by showing that depletion and re-filling of intracellular calcium stores occurs via PLC, IP3R and the STIM1/Orai1 complex. Finally, in vitro experiments in the presence of a specific PAR2 agonist evoked an upregulation of the 'Window of implantation' markers in human endometrial epithelial cells.

Conclusions: These findings provide new insights into the blastocyst-derived protease signaling and allocate a key role for PAR2 as maternal sensor for signals released by the developing blastocyst.

Keywords: Calcium microfluorimetry; Early embryo implantation; Embryo-uterine crosstalk; Endometrium; Protease-activated receptor 2; Serin protease; Trypsin.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Trypsin induces calcium oscillations in mouse endometrial epithelial cells. A Representative traces of mEEC stimulated with trypsin (2 µg/ml) in the presence or B absence of shear stress (SS) C Percentage of oscillating cells (≥ 2 intracellular Ca2+ peaks). The presence ( +) or absence (-) of Aprotinin, shear stress and extracellular Ca2+ is shown. Data are visualized as mean ± SEM. D depicts trypsin responses in the presence (2 mM Ca2+) or absence (0 mM Ca2+) of extracellular Ca2+. In E trypsin responses were challenged with aprotinin (10 µg/ml). F Representative Ca2+ traces of mEEC upon stimulation with elastase (3U/ml). Ionomycin (2 µM) was added at the end of each experiment as a positive control. n = at least 3 independent experiments with a total minimum of 200 cells per condition. Iono = ionomycin, Tryp = trypsin, Apr = aprotinin, SS = shear stress
Fig. 2
Fig. 2
Expression of PAR2, ENaC and VDCC in mEEC. A Single cells RNA sequencing data on two different mouse endometrial cells cultures obtained from public datasets [26]. Uniform manifold approximation and projection of cell populations identified in the merged endometrial mouse datasets. B Dot plot representing the gene counts in the merged datasets for the genes F2r (Par1), F2rl1 (Par2), F2rl2 (Par3), Cacna1c (Cav1.2), Cacna1f (Cav 1.4), Scnn1a (ENaC) and Cdh1 (E-cadherin). C Expression of Par2 in Uniform manifold approximation and projection (UMAP) space of the subpopulation mouse endometrial epithelial Sox9 positive cells are shown. D, E mRNA expression of Scnn1a, Cacnca1c, Cacn1d,cacna1f and F2rl1 in isolated mEEC (D) and mESC (E). Expression is relatively quantified compared to geometric mean of the housekeeping genes Gapdh. (Data is shown as mean ± SEM. N = 3.) F In situ hybridization RNAscope images of isolated mEEC (left panels) and uterine tissue sections (right panels). Positive signals were detected for Par2 and the epithelial markers E-cadherin and Trpv6. Scale bar: 50 µm
Fig. 3
Fig. 3
No role of ENaC and VDCC in the Ca2+ response. A-B Ca2+ microfluorimetry. Representative traces of mEEC stimulated with trypsin (20 µg/ml) challenged with (A) amiloride (10 µM), B nifedipine (10 µM). C Displays the percentage of responding cells to stimulation with either amiloride (Ami), nifedipine (Nif) and aprotinin (Apr, (10 µg/ml). Data is represented as mean ± SEM. ** p < 0.01, using one way ANOVA corrected from multiple testing with Dunnett’s multiple comparison test and compared to the trypsin condition. D Representative traces of mEEC stimulated with amiloride (10 µM) + trypsin (20 µg/ml). E–F Representative traces for trypsin stimulation of primary mEEC in Na+-free and Ca2+-free extracellular solution. Ionomycin (2 µM) was applied at the end of each protocol as positive control. N = at least 3 different experiments on a total minimum of 300 cells. G Whole-cell patch-clamp experiments of mEEC applying a voltage step protocol ranging from -120 mV holding to + 80 mV in + 20 mV steps in control (Ctrl) condition and in the presence of nifedipine (3 µM), an inhibitor of VDCC. H Current (I) Voltage (V) relationship for currents extracted from steady-state currents in. Insert represents the difference in current amplitude between control (Ctrl) and nifedipine (Nif) condition. Tryp = trypsin, Am = amiloride, Nif = nifedipine, Apr = aprotinin, Iono = ionomycin
Fig. 4
Fig. 4
Validation of the functional PAR2 expression in mEEC. Ca2+ microfluorimetry. A Representative traces of 2-furoyl-LIGRLO-NH2 (5 µM), the PAR2 agonist, stimulation of mEEC. B Percentage of oscillating cells in the responding cell population to trypsin (2 µg/ml), 2-furoyl-LIGRLO-NH2 (5 µM) and the simultaneous application of either trypsin or 2-furoyl-LIGRLO-NH2 with the specific PAR2 inhibitor I-191 (100 nM). Data is shown as mean ± SEM. *** p < 0.001 compared to the trypsin application. Statistical significance was analyzed with a one-way ANOVA corrected for multiple comparisons with Dunnett’s multiple comparison test. C-D Trypsin or 2-furoyl-LIGRLO-NH2 responses were challenged with the inhibitor of PAR2, I-191 (100 nM). Ionomycin was added at the end of each experiment as a positive control. N = at least 3 different experiments with a total minimum of 300 cells
Fig. 5
Fig. 5
Calcium oscillations are dependent on PLC pathway and CRAC channels. Ca2+ microfluorimetry. Representative traces of simultaneous stimulation of mEEC with trypsin (2 µg/ml) and either the PLC inhibitor U73122 (5 µM) (A), the PLC negative control compound U73343 (5 µM) (B), the IP3R inhibitor Caffeine (C) and the CRAC inhibitor YM58483 (10 µM) (D). Ionomycin (2 µM) was added as a positive control. In E the percentage of oscillating cells within the responding cell population is shown for mEEC stimulation with trypsin (2 µg/ml), U73122 (5 µM), U73343 (5 µM), caffeine (60 mM), thapsigargin (10 µM), and YM58483 (10 µM). Experiments with thapsigargin were carried out in 0 mM extracellular Ca2+. Data is shown as mean ± SEM. *** p < 0.001 compared to the trypsin condition, using one-way ANOVA corrected for multiple comparisons with Holm-Sidak. For the PLC inhibitor and thapsigargin data, a two-sample t-test was performed. N = at least 3 independent experiments with a total minimum of 100 cells. Iono = ionomycin, tryp = trypsin, Thapsi = thapsigargin. F Schematic overview of PAR2 activated pathways and it’s modulator (figure created with BioRender.com)
Fig. 6
Fig. 6
Immunofluorescent staining of human blastocysts 5 and 6 days post fertilization. A Trypsin immunostaining on intact human blastocysts with primary Anti-Trypsin (D-1) antibody (sc-137077). B Blastocysts were stained with the same staining procedure but the primary antibody was omitted. Scale bar = 100 µm. TE = trophectoderm, ICM = inner cell mass
Fig. 7
Fig. 7
PAR2 expression in different subsets of human uterine epithelial cells. A Uniform manifold approximation and projection of cell populations in the human endometrium, as described by [5]. B Dot plot representing the average expression and expression percentages of genes in endometrium cell types. C UMAP of the distinct epithelial populations within the human endometrium, D Dot plot representing the average expression and expression percentage of several genes in the epithelial subpopulations. E Expression of the individual PAR-members in UMAP space. F UMAP representation of the epithelial subsets throughout the different stages of the menstrual cycle. G Dot plot showing expression levels for the genes provided in (b) in the different stages of the human cycle
Fig. 8
Fig. 8
hEMO display functional PAR2 expression. A In situ hybridization images of hEMO in 2D and 3D conformation. Signals were detected for PAR2 (F2rl1) and the epithelial marker E-cadherin (Cdh1). Scale bar: 50 µm. B mRNA expression of PAR2, ORAI1 and STIM1 in hEMO. Expression is relatively quantified compared to geometric mean of the housekeeping genes HPRT1 and PGK1. Data is shown as mean ± SEM. C-G Ca2+ microfluorimetry. In C and D representative traces of 2D hEMO stimulation with trypsin (1 µg/ml) or 2-furoyl-LIGRLO-NH2 (250 nM) are shown. In E trypsin responses were challenged with the specific PAR2 inhibitor I-191 (100 nM). F 2D hEMO treated with vehicle, without shear stress. Ionomycin (2 µM) was added at the end of each experiment as positive control. G Percentage of oscillating cells in response to trypsin (2 µg/ml), 2-furoyl-LIGRLO-NH2 (5 µM), simultaneous application of trypsin with the specific PAR2 inhibitor I-191 (100 nM) and vehicle. The presence ( +) or absence (-) of shear stress (SS) is shown. NR: no responding cells. Data is shown as mean ± SEM. N = at least 3 independent experiments on hEMO obtained from minimum two different patients, with a total minimum of at least 130 cells. H mRNA expression of decidualization markers PAEP, SPP1, CXCL14, LIF and GPX3 in hEMO. Expression is relatively quantified compared to the housekeeping gene GAPDH. Data is shown as mean ± SEM. N = 3 independent experiments. Two-way ANOVA with Dunnett’s multiple comparison test. * p < 0.05, ** p < 0.01 compared to EPC condition. Tryp = trypsin, 2-fu = 2-furoyl-LIGRLO-NH2, Iono = ionomycin. SS = shear stress
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