Progesterone-induced Progesterone Receptor Membrane Component 1 Rise-to- Decline Changes are Essential for Decidualization

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Bielfeld, Tanja Fehm, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3029459/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Feb, 2024 Read the published version in Reproductive Biology and Endocrinology → Version 1 posted 8 You are reading this latest preprint version Abstract Background Decidualization of endometrial cells is the prerequisite for embryo implantation and subsequent placenta formation and is induced by rising progesterone levels following ovulation. One of the hormone receptors contributing to endometrial homeostasis is Progesterone Receptor Membrane Component 1 (PGRMC1), a non-classical membrane-bound progesterone receptor with yet unclear function. In this study, we aimed to investigate how PGRMC1 contributes to human decidualization. Methods To gain insight into PGRMC1-implication in infertility-related diseases, we analyzed its expression profile in RNA-sequencing datasets of endometrial biopsies. To further explore the function of PGRMC1 in human decidualization, we implemented an inducible decidualization system, which is achieved by culturing two human endometrial stromal cell lines in decidualization-inducing medium containing medroxyprogesterone acetate and 8-Br-cAMP. In our system, we measured PGRMC1 expression during hormone induction as well as decidualization status upon PGRMC1 knockdown at different time points. We further conferred proximity ligation assay to identify PGRMC1 interaction partners. Results PGRMC1 expression was altered in patients with infertility-related diseases and impaired decidualization, being significantly downregulated in most datasets. In in vitro experiments, we observed that PGRMC1 expression follows a rise-to-decline pattern, in which its expression level initially increased during the first 6 days after induction (PGRMC1 increasing phase) and decreased in the following days (PGRMC1 decreasing phase). Knockdown of PGRMC1 expression before the induction led to a failed decidualization, while its knockdown after induction did not inhibit decidualization, suggesting that the progestin-induced ‘PGRMC1 increasing phase’ is essential for normal decidualization. Furthermore, we found that the interactions of PHB1 and PHB2 with PGRMC1 were induced upon progestin treatment. Knocking down either PHB individually or both slowed down the decidualization process compared to the control, suggesting that PGRMC1 cooperates with PHBs to regulate the decidualization. Conclusions According to our findings, PGRMC1 expression followed a progestin-induced rise-to-decline expression pattern during human endometrial decidualization process; and the correct execution of this expression program was crucial for successful decidualization. Thereby, the results of our in vitro model explained how PGRMC1 dysregulation in patients with impaired decidualization contributes to the manifestation of their disease. Decidualization Progesterone receptor membrane component 1 (PGRMC1) Endometrium Telomerase-immortalized human endometrial stromal cells (T-HESCs) Infertility Prohibitin-1 (PHB1) Prohibitin-2 (PHB2) AG205 Rise-to-decline pattern in-fertility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Human endometrium tissue is highly dynamic going through proliferative, secretory, and menses phases during a regular menstrual cycle [ 1 – 3 ]. Correspondingly, its functional layer exhibits steroid hormone-dependent proliferation, progesterone-stimulated differentiation, and shedding in the absence of the trophoblast [ 3 ]. After the postovulatory phase, the rising circulating levels of progesterone drive human endometrial stromal cells (HESCs) to differentiate into decidual cells, which is referred to as the decidualization process [ 2 – 5 ]. Decidualization is the morphological transformation of HESCs from a proliferating fibroblastic phenotype to an enlarged and rounded epithelial shape, accompanied by secretion of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), which is required for female fertility [ 2 , 3 , 5 ]. In the presence of a trophoblast, the decidualized endometrium will be maintained through the increased level of progesterone. Otherwise, it will be shed away with a rapid drop of the progesterone level [ 3 ]. A successful decidualization process is an essential prerequisite for embryo implantation and subsequent placenta formation. During decidualization, progesterone (P4) classically affects the endometrium through activation of two major well-characterized progesterone receptor PR-A and PR-B [ 5 ]. Progesterone receptor membrane component 1 (PGRMC1), one of the non-classical progesterone receptors, also rapidly respond to progesterone during decidualization; however, its function in this process is still being elucidated. In the human endometrium, PGRMC1 is abundantly expressed during the proliferative phase of the menstrual cycle in both endometrial and stromal cells. Whereas, in the secretory phase its expression levels dramatically decreased [ 6 ]. Overexpression of PGRMC1 in primary HESCs abrogated decidualization [ 7 ] and reduced PGRMC1 expression observed in multiple gynecological and obstetrics diseases [ 8 – 10 ]. Therefore, PGRMC1 was proposed as a fertility stabilizer to decidualization, whose expression must be finely tuned during the entire decidualization to support female fertility [ 11 ]. How this is achieved remains an enigma. The prohibitin proteins (PHBs), prohibitin-1 (PHB1) and prohibitin-2 (PHB2), are ubiquitously expressed and highly conserved in eukaryotic cells [ 12 ]. PHBs has been reported to act as transcriptional corepressors for ERα in vitro and in vivo [ 13 – 15 ]. Loss of PHBs led to dysfunctional mitochondria, further resulting in male infertility and ovarian aging in females [ 16 , 17 ]. Besides, PHB1 is downregulated in the eutopic and ectopic endometrium of patients with endometriosis compared to women without endometriosis [ 18 ]. An uterus-selective, conditional PHB2 knockout mouse model showed a subfertility phenotype with litters reduced both in number and size [ 19 ]. This implies that appropriate protein levels of PHB1/2 as well as of PGRMC1 are required for optimal uterine function and fertility. In breast cancer cells, progestin-activated PGRMC1 associated with PHBs to stimulate cellular proliferation [ 20 ]. Binding of activated PGRMC1 to PHBs was accompanied by decreased PHBs-ERα-interaction, resulting in elevated expression of ER-dependent genes. Whether the progestin-depended interaction between PHBs and PGRMC1 also occurs during decidualization has never been characterized before. Therefore, the role of their interaction with regards to female fertility remains to be elucidated. In this study, we aimed to explore the functional role of PGRMC1 and PHBs, and their interplay for successful decidualization. Materials and Methods Data sources We collected the associated gene expression profiles in publicly available Gene Expression Omnibus (GEO) database ( https://www.ncbi.nlm.nih.gov/geo/ ). Samples from different menstruation phases (proliferative/PE, early secretory/ESE, mid-secretory/MSE, late secretory/LES) were chosen from GSE4888 and GSE56364 to detect expression of PGRMC1 [ 21 , 22 ]. Other analyzed infertility-related diseases datasets included chronic placental inflammation (CPI) (GSE68474), repeated implantation failure (RIF) (GSE65099, GSE16532), endometriosis (GES120103, GSE51981) [ 23 – 27 ]. All raw data were background-subtracted and normalized. Cell Culture The hTERT-immortalized human endometrial stromal cells (T-HESCs) were purchased from abm (T0533). Both the cell lines T-HESCs and St-T1 were maintained in phenol-red free Dulbecco’s Modified Eagle Medium//Ham’s F12 (DMEM/F12; Gibco, Thermo Fisher Scientific, 11039021) medium supplemented with 10% ( v/v ) charcoal-stripped fetal bovine serum (Thermo Fisher Scientific, 12676029), 100 units/mL penicillin-streptomycin (Thermo Fisher Scientific, 2321118), 50µg/ml gentamycin sulfate (Biowest, L0012), 200µM sodium pyruvate (Biowest, L0624) and 1.5g/L sodium bicarbonate (Biowest, L0680) (hereafter referred to as complete medium) in a humidified incubator at 37 o C in the presence of 5% CO 2 . Cells (passage number < 10) were regularly tested negative for mycoplasma. Chemical Compounds AG205 (Sigma-Aldrich) was diluted in 2% charcoal-stripped FBS complete medium to 15 mM. Medroxyprogesterone acetate (MPA) and 8-Br-cAMP MPA (cAMP) were prepared from a 10 mM and 5 mM stock solution, respectively. Immunofluorescence Staining Cells were seeded and cultured in chamber slides (Nunc Lab-Tek, Thermo Fisher Scientific C7182-1PAK) fixed with 4% formaldehyde (Sigma-Aldrich, 20649296018) for 10 min at room temperature (RT), washed with washing buffer (Dako, Glostrup, Denmark, S3006) (3 x 5 min each). Then, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, T8787) in PBS for 10 min at RT and washed with washing buffer again (3 x 5 min each). DAKO protein block buffer (Dako, X0909) was added and incubated for 1h at RT before incubating with primary antibodies specific for PGRMC1 (Abcam, ab48012), PHB1 (Abcam, ab75766), PHB2 (Cell signaling, 14084S) and Vimentin (Abcam, ab02547) overnight at 4 o C. The next day, cells were washed with washing buffer (3 x 5 min each) and incubated with secondary antibodies (Donkey-anti-goat, Alexa 488: Invitrogen, A11055; Donkey-anti-rabbit, Alexa 488: Invitrogen, A31573) for 1 h at RT in a humidified chamber in the dark. Nucleic acid was stained with DAPI (Thermo Fisher Scientific, 15733122) simultaneously with co-incubated secondary antibodies. After the final wash, the cells were mounted with Fluorescent Mounting Medium (Dako, S3023). Negative controls were prepared for each sample following the same staining procedure with isotype controls instead of primary antibodies. Fluorescence signals were detected with an Axioplan 2 Imaging fluorescence microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Proximity Ligation Assay The in-situ proximity ligation assay (PLA) procedure was performed with the Duolink® PLA Kit (Sigma-Aldrich, DUO92008) and following the manufacturers protocol. The cells were incubated with the primary antibodies i.e., anti-PGRMC1 (Abcam, ab48012) with PHB1 (Abcam, ab75766) and PHB2 (Cell signaling, 14085S) overnight at 4 o C. The slides were washed twice for 5 min with buffer A, followed by incubation with the PLA probes (anti-goat PLUS and anti-rabbit MINUS) in antibody diluent for 60 min at 37 o C. After washing twice for 5 min with buffer A, ligation was performed using ligase diluted in ligation buffer for 30 min at 37 o C. Then the cells were washed with buffer A before incubation for 100 min with amplification stock solution at 37 o C. After washing twice for 10 min with buffer B, nuclear DNA was labeled with DAPI for 10 min and slides were mounted with mounting medium. Negative PLA control was performed using respective isotype control antibodies (isotype goat, Abcam, ab37373; isotype rabbit, Abcam, ab37415). Red fluorescence dots inside the cellular areas representing a single protein-protein interaction were quantified using image J software. MTT Assay and Western Blotting MTT assay and western blotting of PGRMC1 and PHB1, PHB2 were performed as previously described [ 20 ]. Subcellular Protein Fractionation A subcellular protein fractionation kit (Thermo Fisher Scientific) was used to fractionate proteins into cytoplasmic, membrane, and nuclear fractions. Cells were harvested as pellets. The pellet was lysed with cytoplasmic extraction buffer, membrane extraction buffer, and nuclear extraction buffer. Primary antibodies specific for β-actin (Santa Cruz Biotechnology), Calreticulin (Santa Cruz Biotechnology), and Histon H3 (Cell signaling) were used to indicate the purity of the cytoplasmic, membrane, and nuclear fractions, respectively. Co-Immunoprecipitation Co-immunoprecipitation was performed using the Pierce Co-IP kit (Thermo Fisher Scientific). Briefly, the anti-PGRMC1 antibody (Cell signaling) was first immobilized for 2h using AminoLink Plus coupling resin. In parallel, cell pellets were resuspended in ice-cold IP Lysis buffer. An amount of 500 µg protein was incubated with resin at 4 o C overnight. After incubation, the resin was washed, and protein complexes bound to the antibody were eluted using elution buffer. Subsequent western blot analyses were performed as described before. Gene silencing (siRNA Transfection) To knock down PGRMC1 expression in T-HESCs, FlexiTube GeneSolution (Qiagen) was used, containing four siRNA(s) that specifically target human PGRMC1 mRNAs. Cells were transfected with the final concentration of 10 nM PGRMC1 siRNA(s) or negative control siRNA (siCTL) (Thermo Fisher Scientific) using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to recommended procedures. Afterwards, cells were treated with decidualization medium containing either induction cocktail or DMSO, and harvested at different time points for downstream experiments. For PHB1 and PHB2 mRNA expression inhibition (siPHB1, siPHB2: Qiagen), the same siRNAs concentration and method was used above. Quantitative Reverse-Transcription PCR (qRT-PCR) RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s specifications. Reverse transcription of RNA into cDNA was performed with the Omniscript RT kit (Qiagen) according to the manufacturer’s instructions. Quantitative PCR was performed using QuantiFast SYBR Green PCR Kit (Qiagen) and LightCycler ®480 System (Roche). Primers for PGRMC1 (Qiagen), PRL (Qiagen) and HPRT1 (Qrigene, Rockville, MD, USA). The delta-delta cycle threshold method was used to normalized expression to the reference gene HPRT1 [ 28 , 29 ]. Statistical Analysis A two-tailed paired Student’s t -test was used to analyze experiments comparing two experimental groups or two-way ANOVA for multiple comparisons of more than two groups. A value of p < 0.05 was considered significant. All statistical analyses were performed with GraphPad Prism 9.0. Results were reported as means with standard deviation. Results PGRMC1 expression is dysregulated in patients with impaired decidualization To understand the dynamics of PGRMC1’s expression changes during normal decidualization, we initially investigated its expressional profile by mining publicly available RNA-sequencing data sets from endometrial biopsies (GEO accession numbers: GSE6364 and GSE4888). In a normal menstrual period, PGRMC1 mRNA level gradually decreased from the proliferative phase to the secretory (including early-, mid-, and late-) phase, manifesting the highest level in the proliferation phase and the lowest level in the late-secretory phase (Fig. 1 A-B), consistent with previously reported data [ 9 , 30 ]. This indicates that PGRMC1 may have an important role in regulating cellular proliferation and may not be required for decidualization in the secretory phase as it is consecutively decreased at mRNA level after progesterone stimulation. We hypothesized that the dynamic changes of PGRMC1 have an important role during the menstrual cycle that must be finely tuned. Apart from normal decidualization, expression of PGRMC1 also changes in chronic placental inflammation (CPI) and in infertility-related diseases including repeated implantation failure (RIF), and endometriosis, in which the common pathological factor is impaired decidualization. In most data sets lower levels of PGRMC1 mRNA were frequently observed in patients who suffered from infertility-related disease compared to a control group (Fig. 1 C-E), suggesting a tight connection between impaired decidualization and downregulated PGRMC1 mRNA. However, we unexpectedly found that a higher level of PGRMC1 mRNA was observed in some RIF and endometriosis patients (Fig. 1 F-G), implying that upregulated PGRMC1 expression could also be a cause of decidualization disruption. Thus, we concluded that any kind of dysregulation of PGRMC1 expression may lead to an impaired decidualization. Rise-to-decline trend of PGRMC1 expression during in vitro decidualization To answer the question of how a dysregulation of PGRMC1 expression may impair decidualization, we established a hormone-inducible in vitro decidualization model in T-HESCs based on visualizing its morphological changes and by measuring the expression level of the decidual marker prolactin (PRL) (Fig. 2 A). After being exposed to the decidualization induction cocktail consisting of the P4 analog MPA plus cAMP for 10 days, morphological changes of T-HESCs were inspected by microscopy in bright-field and by immunofluorescent analysis of the cytoskeletal marker vimentin. With this protocol, T-HESCs underwent a transformation from a fibroblast-like shape to a polygonal epithelial-like shape (Fig. 2 B-C) accompanied with a significant increase of PRL mRNA expression compared to non-induced controls (Fig. 2 D). Both the morphological changes and enhanced expression of PRL indicate a successfully established the hormone-induced decidualization model, allowing to investigate the role of PGRMC1 in decidualization. Since dysregulated PGRMC1 expression is associated with decidualization failure in infertility-related diseases, we aimed to determine and modulate the expression level of PGRMC1 in our system to study its impact on the decidualization process. First, we determined the protein expression profile of PGRMC1 during the in vitro decidualization program. Intriguingly, we found that PGRMC1 expression gradually increased from the day the induction cocktail had been added (D0), peaking at day 6 (D6) post-induction, followed by a constant decrease until day 14 (D14) (Fig. 2 E-F). This protein expression change could also be observed in the St-T1 cell line (Additional file 1A). In contrast with the observation of gradually decreased mRNA levels of PGRMC1 during the secretory phase of the normal menstrual cycle (Fig. 1 A-B), our in vitro decidualization model revealed that the promotion of cells into decidualized state comprises a PGRMC1 increasing phase and a PGRMC1 decreasing phase, both on protein and mRNA level (Fig. 2 E-G). This data suggests that PGRMC1 was regulated at transcriptional level during decidualization. We termed this PGRMC1 protein dynamic changes as PGRMC1 ‘rise-to-decline’ changes in decidualization. It is well known that increasing P4 levels initiate decidualization, although the activity of PGRMC1 in decidualization seems to be independent of P4 [ 3 , 5 ]. Consistently, the T-HESC cells can go through the decidualization process treated with either P4, MPA, or cAMP (Additional file 1B-C). Intriguingly, the PGRMC1 expression changes can be observed at each condition, which led us to the conclusion that the PGRMC1 rise-to-decline changes are a universal mechanism within the decidualization program. The rise-to-decline changes of PGRMC1 are required for decidualization. To explore the potential role of the PGRMC1 rise-to-decline changes during the decidualization, we firstly downregulated its expression before hormone induction with an optimized concentration of an siRNA-mix specific for PGRMC1 mRNA (Fig. 3 A). Importantly, PGRMC1 mRNA levels were remained suppressed throughout 10 days post-siRNA-transfection (Fig. 3 B). Likewise, expression of PGRMC1 protein was completely abrogated from day 2 (D2) to day 10 (D10) after siRNA transfection (Additional file 2A-B). T-HESCs with suppressed PGRMC1 expression were further treated with the decidualization induction cocktail. As indicated by the lack of morphological transformation and PRL production over 10 days of hormone treatment period (Fig. 3 C, Additional file 3), these cells did not undergo decidualization. Thus, in the absence of the progestin-induced PGRMC1 increasing phase decidualization failed. These results prompted us to investigate if expression of PGRMC1 is needed for decidualization at the time point of induction – as a kind of a program switch – or later. To this aim, we postponed the transfection of PGRMC1 suppressing siRNAs to after the induction of decidualization. First, we treated T-HESC cells for 2 days with the combination of MPA and cAMP to induce decidualization followed siRNA transfection (Fig. 3 D) and investigated the cells up to day 10 (D10) after induction. As expected, PGRMC1 mRNA levels started to decrease (Fig. 3 E, blue line) after 2 days of transfection of PGRMC1-specific siRNAs and the PGRMC1 mRNA levels stayed below mRNA levels reached during normal induction of decidualization (Fig. 3 E, red line). Interestingly, in addition to morphological changes (Additional file 4), PRL mRNA expression level first dropped, but between D6 and D8 not only recovered to a comparative level to that of normal induction, was even four days earlier compared to the normal induction, indicating a promoted decidualization (Fig. 3 F). We further measured the effects of knocking down PGRMC1 after 4 days of induction with MPA/cAMP on the decidualization program. The results are very similar to the outcome achieved when suppressing PGRMC1 after 2 days of induction (Fig. 3 G-H). The results could be additionally reproduced in the St-T1 cell line (Additional file 5A-D). Taken together, the PGRMC1 rise-to decline changes are required for a proper decidualization. PGRMC1 accumulates outside the nucleus during decidualization It has been reported that PGRMC1 translocates from cytoplasmic membranes to the nucleus during decidualization [ 7 ]. Recently, PGRMC1-mediated proteomic changes have been well characterized after decidualization, suggesting that PGRMC1 binds to proteins involved in translation, ATP generation, protein maturation, glucose transport, and lipid metabolism [ 31 ]. Almost all these proteins locate in the cytoplasm or on membranes, but not in the nucleus. This raises the question of why proteins interacting with PGRMC1 are barely found to be in the nucleus. To better understand the question, we initially assessed PGRMC1 protein subcellular localization by immunofluorescence. Without induction of decidualization PGRMC1 was essentially located in the cytoplasm, but significantly accumulated close to the nucleus after induction (Fig. 4 A). To further verify these observations, we fractionated the cells into soluble parts containing cytoplasm, membrane, and nucleus and detected the PGRMC1 protein by western blot. In line with the immunofluorescence results, PGRMC1 was only observed in the membrane fraction but not in the nucleus (Fig. 4 B). This indicates that PGRMC1 accumulated at the nucleus periphery but not in the nucleus during hormone-induced decidualization. Interactions of PHB1/PHB2 to PGRMC1 mediate decidualization We have recently demonstrated in breast cancer cells, that progestin-activated PGRMC1 interacts with PHB1/PHB2 resulting in enhanced ERα-dependent transcription and cell proliferation [ 32 ]. In analogy, here we found that PGRMC1 colocalized with PHB1 and PHB2 in the cytoplasm and at the nucleus periphery after induction, whereas barely colocalization signals could be observed without induction revealed by immunofluorescence (Additional file 6A-B). This suggests a potential interaction between PHBs and PGRMC1 introduced by progestin treatment. Then, PLA was performed to further explore the associations between PGRMC1 and PHB1/2. Upon induction, a significantly higher PLA signal suggesting the interaction of PGRMC1 to PHB1/2 could be observed compared to the control (Fig. 5 A-B). We next performed the Co-IP analysis confirming that both PHB1 and PHB2 co-precipitate with PGRMC1 (Fig. 5 C-D), which is consistent with the observation made in MIA PaCa-2 cells [ 33 ]. The interactions between PGRMC1 and PHB1 (A) or PHB2 (B) in T-HESCs were analyzed with proximity ligation assay upon decidualization induction. Each red spot represents a single interaction. Nuclear stain: DAPI. Magnification 40×. (C), (D) : PHB1 and PHB2 co-precipitate with PGRMC1. PGRMC1 was immunopurified from native whole-cell lysates of T-HESC using anti-PGRMC1 antibody. Western blot analyses of co-immunoprecipitated PHB1 (C) and PHB2 (D) in T-HESCs with and without decidualization induction. To explore the function of PGRMC1-PHBs interaction during decidualization, we downregulated PHBs via siRNA transfection, reaching expression levels decreased by 50–80% compared to the control for individual PHBs (Fig. 6 A-C). Knocking down either PHBs alone or both PHBs before hormone induction partly impaired the decidualization process (Fig. 6 D-G), but the cells still could achieve morphological transformation (Additional file 7). These effects on decidualization are comparable to the results achieved with suppressed PGRMC1. AG205 does not affect PGRMC1 rise-to-decline changes and decidualization AG205 was reported to be a specific inhibitor of PGRMC1 and was broadly used to explore PGRMC1’s role in decidualization [ 34 , 35 ]. Recent data, however, question the specificity of AG205 for PGRMC1 [ 35 – 37 ]. The PHBs protein expression level on day 2 or day 10 after transfection of T-HESCs with 10 nM siPHB1 (A) , 10 nM siPHB2 (B) , and 10 nM siPHBs mixture (5 nM siPHB1 and 5 nM siPHB2) (C) , respectively, analyzed by western blot. The PRL mRNA expression changes in T-HESCs with (red line) and without (black line) induction (D) . The PRL mRNA expression changes in T-HESCs transfected with 10 nM siPHB1 (E) , 10 nM siPHB2 (F) , 10 nM siPHBs mixture (5 nM siPHB1 and 5 nM siPHB2) (G) upon decidualization induction. Results are shown as the mean ± SEM from three biological replicates. Statistical analysis was performed by two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Taking advantage of the critical role of PGRMC1 rise-to-decline changes for decidualization, we tested the effect of AG205 on PGRMC1 and the decidualization process. Since AG205 concentrations used in previous reports were high enough to impair cellular viability [ 33 , 36 , 38 ], we initially determined the appropriate concentration of AG205 that did not affect cell viability. In the MTT assay (Fig. 7 A), a concentration below 15 µM had no (or a moderate) effect, whereas a concentration higher than 15 µM had a detrimental effect on cellular viability, which is consistent with previously reported [ 35 ]. In addition, decidualization was successfully achieved with T-HESCs treated AG205 with concentrations below 15 µM, as indicated by the increasing expression of PRL and the change in cell morphology (Fig. 7 B-C). Furthermore, AG205 treatment did neither affect PCRMC1 protein level during decidualization, nor its rise-to-decline expression profile (Fig. 7 D). Moreover, the interaction of PGRMC1 to PHBs was not disturbed as confirmed by PLA (Fig. 7 E-F), which is in line with a previous report [ 33 ]. Based on these results, we propose that AG205 (< 15 µM) has no effect on the observed PGRMC1 functions during decidualization. Discussion PGRMC1 has been demonstrated to play a role in various reproductive tissues, particularly endometrial stromal cells [ 32 , 39 – 41 ]. It influences the decidualization process and female fertility [ 42 ]. This study discovered a connection between dysregulated PGRMC1 mRNA levels and impaired decidualization in infertility-related diseases. We also revealed that PGRMC1 protein exhibits a rise-to-decline pattern after progestin stimulation, essential for normal decidualization (Additional file 8). Additionally, PHB1/2 involved in the decidualization program by interacting with PGRMC1. Despite the unclear mechanisms behind PGRMC1 dysregulation and decidualization failure, PGRMC1 expression levels may serve as a useful fertility indicator. Previous reports focused on PGRMC1 mRNA profile changes during decidualization, with few investigations into protein level dynamics. We measured both mRNA and protein levels of PGRMC1 after inducing decidualization and observed a rise-to-decline pattern. The observed increase and decrease of PGRMC1 protein expression fits into cyclic changes observed in vivo [ 30 ]. The overall dynamic changes of the PGRMC1 protein level during a normal menstrual cycle are composed of two peaks: one occurs in the secretory phase, as revealed in this study and the other one occurs in the proliferative phase as previously reported [ 9 , 30 ]. It resembles estrogen dynamics during the menstrual cycle, suggesting PGRMC1 expression may be regulated by estrogen concentration or a similar mechanism [ 1 – 3 ]. As PGRMC1 overexpression in breast cancer cells leads to higher E2 secretion, T-HESCs E2 production might depend on PGRMC1 activation. Further research is needed to understand the relationship between estrogen and PGRMC1 expression, including the possibility of estrogen receptor-mediated transcription regulation. Knocking down PGRMC1 before hormone treatment inhibited decidualization, highlighting its crucial role as a 'switch' at this stage. Appropriate PGRMC1 protein levels are needed to initiate decidualization upon P4/cAMP stimulation. The PGRMC1 rise-to-decline pattern can be induced by various treatments (Additional file 1), suggesting a common signaling pathway that correlates with decidualization, which requires further investigation. PGRMC1 seems less necessary after decidualization initiation, as knocking it down either does not affect or even facilitates the process. It is unclear why PRL expression initially drops and then increases when PGRMC1 is knocked down after decidualization induction. Downregulating PGRMC1 after progestin treatment doesn't hamper decidualization, indicating its critical role during the increasing phase and induction. This aligns with observations that PGRMC1 downregulation in the secretory phase promotes decidualization [ 34 ]. Overall, PGRMC1 activation by P4 may facilitate the switch from cellular proliferation to decidualization initiation through various biological processes, while the mechanism of how downregulated PGRMC1 promotes decidualization warrants further investigation. PGRMC1 associates with proteins involved in protein biosynthesis, intracellular transport, and mitochondrial activity to promote decidualization [ 31 , 40 ]. However, little is known about how PGRMC1 interacts with these proteins to regulate decidualization. We found that PGRMC1 binds to PHBs at the nucleus periphery after P4 treatment, suggesting it may function as a scaffold protein for decidualization in endometriosis stromal cells. PGRMC1 could be anchored on the membrane of various organelles, co-transporting with them during decidualization-related morphological changes [ 43 ]. PHBs form a super complex in mitochondria, playing roles in lipid biogenesis, ATP generation, and more [ 12 , 44 ]. Knocking down PHBs partially impaired decidualization, similar to PGRMC1 knockdown, suggesting PGRMC1-PHBs interactions may influence decidualization as a complex, requiring further investigation. We speculate that PGRMC1 binding to PHBs may inhibit cellular proliferation and facilitate differentiation, acting as a proliferation-differentiation switch. We found that the small molecule AG205 neither affect PGRMC1-PHBs interaction, nor decidualization in our study (Fig. 7 ). Although AG205 has been shown to interact with PGRMC1 in vitro , its in vivo interaction remains unknown. Our data align with a recent study demonstrating that AG205 concentrations over 15 µM reduce cell proliferation, and concentrations above 30 µM result in cell death in HEC-1A and T-HESC cells [ 35 ]. Furthermore, our findings are consistent with a previous report indicating that a high concentration (50 µM) of AG205 did not affect decidualization [ 36 ]. Finally, a general limitation needs to be taken into account. The in vitro experiments performed in cell lines assured reproducibility within the established system, while primary patient endometrium tissues are highly heterogeneous and, in general, require analysis of a large cohort in order to obtain a statistically significant result. Therefore, in this study, we focused on the analysis of publicly available datasets and two cell lines. Nevertheless, to confirm PGRMC1’ switch-like rise-to-decline expression pattern in vivo, a mouse model with inducible PGRMC1-downregulation could be exploited in future studies. Conclusion Based on the results of our study, we postulate that P4/progestin-induced PGRMC1 rise-to-decline expression is essential to start the decidualization program, but, once decidualization started, PGRMC1 is not needed to drive it. Since PGRMC1 expression was downregulated in most analyzed transcriptome datasets of endometrium biopsies of patients with infertility-related diseases, our PGRMC1-knockdown experiments mirrored this situation in vitro and consequently demonstrated a decidualization failure, potentially leading to infertility. Taken together, we explained how dysregulated PGRMC1 expression could impact endometrial stromal cell differentiation in patients and thereby lead to manifestation of their disease. Declarations Ethics approval and consent to participate: not applicable Consent for publication: not applicable Availability of data and materials: The datasets analyzed in this study are available at Gene Expression Omnibus (GEO) database: https://www.ncbi.nlm.nih.gov/geo/ with the GEO accessions GSE4888, GSE56364, GSE68474, GSE65099, GSE16532, GES120103, GSE51981. Competing interests: The authors declare that they have no competing interests. Funding: This project was funded by the “German Research Foundation (Deutsche Forschungsgemeinschaft, DFG, NE 805/6-1)”, and "The Brigitte and Dr. Konstanze Wegener Foundation (#51, Duesseldorf, Germany)”, and “The Medical Faculty of the Heinrich-Heine University Duesseldorf (#48/2018)”, and “The Shanghai Shenkang Hospital Development Center (SHDC12021113)”. Authors’ contributions: Conceptualization, H.L., H.N.; Investigation, H.L., N.S.; Methodology, H.L., A.P.B. and N.S.; Supervision, T.F., D.N., Z.C., N.S., H.N.; Project administration, T.F., H.N.; Writing-original draft, H.L.; Writing-review and editing, A P.B., A.F., N.S. and H.N. All authors have read and agreed to the published version of the manuscript. Acknowledgements: We are grateful to our colleagues in the Department of Gynecology and Obstetrics, Heinrich-Heine University Duesseldorf for their helpful comments. References Reed BG, Carr BR. In: Feingold, et al. editors. The Normal Menstrual Cycle and the Control of Ovulation. South Dartmouth (MA); 2000. Endotext , K.R.. Jabbour HN, et al. Endocrine regulation of menstruation. Endocr Rev. 2006;27(1):17–46. Mihm M, Gangooly S, Muttukrishna S. The normal menstrual cycle in women. Anim Reprod Sci. 2011;124(3–4):229–36. Gellersen B, Brosens JJ. Cyclic decidualization of the human endometrium in reproductive health and failure. 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The role of progesterone receptor membrane component (PGRMC) in the endometrium. Steroids. 2022;184:109040. Cahill MA, et al. The emerging role of progesterone receptor membrane component 1 (PGRMC1) in cancer biology. Biochim Biophys Acta. 2016;1866(2):339–49. McCallum ML, et al. Conditional Ablation of Progesterone Receptor Membrane Component 1 Results in Subfertility in the Female and Development of Endometrial Cysts. Endocrinology. 2016;157(9):3309–19. Chen YJ, et al. PGRMC1 acts as a size-selective cargo receptor to drive ER-phagic clearance of mutant prohormones. Nat Commun. 2021;12(1):5991. Merkwirth C, Langer T. Prohibitin function within mitochondria: essential roles for cell proliferation and cristae morphogenesis. Biochim Biophys Acta. 2009;1793(1):27–32. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.tif Additional file 1: Rise-to-decline expression pattern of PGRMC1 is linked to the decidualization program. (A) PGRMC1 protein expression changes during 9 days of decidualization were measured by western blot in the St-T1 cell line. (B) PGRMC1 protein expression changes during 10 days of stimulation with MPA, cAMP, and DMSO, respectively, were measured by western blot in T-HESC. (C) PGRMC1 protein expression levels on day 6 and day 10 when cultured with DMSO, nomegestrel (NOM), P4, cAMP, MPA/cAMP (M+A), and MPA, respectively, measured by western blot in T-HESC. Additionalfile2.tif Additional file 2: PGRMC1 is effectively downregulated by siRNA on protein level. (A) The PGRMC1 protein expression on day 2 and day 10 after transfection of T-HESCs with either 10 nM anti-PGRMC1 siRNA (siPGRMC1) or unspecific scrambled-control siRNA (siCTL). (B) A comparison of the PGRMC1 protein expression changes within 10 days after transfection of T-HESCs with either 10 nM siPGRMC1 or 10 nM siCTL. Additionalfile3.tif Additional file 3: PGRMC1-downregulation before decidualization induction impairs morphological remodeling of T-HESC. The cellular morphology changes of the T-HESCs induced with either DMSO (upper panel) or MPA/cAMP (down panel) after 10 days of siRNA treatment (siCTL, left panel; siPGRMC1, right panel). Scale bar: 200µm. Additionalfile4.tif Additional file 4: PGRMC1-downregulation after decidualization induction does not impair morphological remodeling of T-HESC. The cellular morphology changes of the T-HESCs induced with either DMSO (non-induction, column 1) or MPA/cAMP (Induction, columns 2-4). I (column 3) and II (column 4) indicate that siRNA treatment on T-HESCs was conducted on day 2 or day 4 of decidualization induction, respectively. Scale bar: 200µm Additionalfile5.tif Additional file 5: PGRMC1-downregulation after progestin induction does not impair decidualization in the St-T1 cell line. The mRNA expression levels of PGRMC1 (A, C) and PRL (B, D) in St-T1 treated with MPA/cAMP for decidualization induction (red line), and non-induction (black line). The mRNA expression levels of PGRMC1 and PRL in St-T1 cells transfected with 10 nM siPGRMC1 (blue line) on the second (A, B) and fourth day (C, D) after decidualization induction, respectively. Results are shown as the mean ± SEM from three independent biological replicates. Statistical analysis was performed by a two-way ANOVA test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The red * indicates the comparison between the red and black lines. The blue * indicates the comparison between the red and blue lines. Additionalfile6.tif Additional file 6: PGRMC1 and PHB1/PHB2 co-localize in T-HESCs. Double Immunofluorescence staining for PGRMC1 (red) and PHB1 (green) or PHB2 (green) in T-HESCs treated with DMSO (A) as control or MPA/cAMP (B) for decidualization induction. Magnification: 40x. Scale bar: 20 µm. Additionalfile7.tif Additional file 7: Morphological changes during decidualization upon PHB downregulation. The cellular morphology changes of the T-HESCs induced with either DMSO (non-Induction) or MPA/cAMP (Induction) upon either PHBs knockdown alone or both. Scale bar: 200 µm. Additionalfile8.tif Additional file 8: Overview over the role of PGRMC1 in human endometrial decidualization and infertility. Upon stimulation with progesterone or MPA, the PGRMC1 rise-to-decline changes are essential for successful decidualization of the human endometrial cells (upper panel). With downregulated PGRMC1 expression before induction (model for abrogated PGRMC1 in patients from Figure 1), the decidualization program cannot be carried out, leading to decidualization dailure (bottom panel). Cite Share Download PDF Status: Published Journal Publication published 03 Feb, 2024 Read the published version in Reproductive Biology and Endocrinology → Version 1 posted Editorial decision: Major revision 13 Oct, 2023 Reviews received at journal 21 Jul, 2023 Reviewers agreed at journal 13 Jul, 2023 Reviewers agreed at journal 25 Jun, 2023 Reviewers invited by journal 10 Jun, 2023 Editor assigned by journal 07 Jun, 2023 Submission checks completed at journal 07 Jun, 2023 First submitted to journal 06 Jun, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3029459","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":217768574,"identity":"e850cdc1-7ce5-4b84-911a-b65d0fee4dd7","order_by":0,"name":"Hailun Liu","email":"","orcid":"","institution":"University Hospital and Medical Faculty of the Heinrich-Heine University Duesseldorf, Life Science Center","correspondingAuthor":false,"prefix":"","firstName":"Hailun","middleName":"","lastName":"Liu","suffix":""},{"id":217768576,"identity":"479b1c8c-42bb-46d5-93f7-9a25053aa520","order_by":1,"name":"André Franken","email":"","orcid":"","institution":"University Hospital and Medical Faculty of the Heinrich-Heine University Duesseldorf, Life Science Center","correspondingAuthor":false,"prefix":"","firstName":"André","middleName":"","lastName":"Franken","suffix":""},{"id":217768578,"identity":"00c923ba-cf74-4b4b-9be5-398659881a6e","order_by":2,"name":"Alexandra P. Bielfeld","email":"","orcid":"","institution":"University Hospital, Heinrich Heine University Duesseldorf","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"P.","lastName":"Bielfeld","suffix":""},{"id":217768580,"identity":"0dd8391e-6d03-452c-a01a-508c71bfa536","order_by":3,"name":"Tanja Fehm","email":"","orcid":"","institution":"University Hospital, Heinrich Heine University Duesseldorf","correspondingAuthor":false,"prefix":"","firstName":"Tanja","middleName":"","lastName":"Fehm","suffix":""},{"id":217768582,"identity":"6b2b4af5-48ff-43fe-9a54-42daeea6dca7","order_by":4,"name":"Dieter Niederacher","email":"","orcid":"","institution":"University Hospital and Medical Faculty of the Heinrich-Heine University Duesseldorf, Life Science Center","correspondingAuthor":false,"prefix":"","firstName":"Dieter","middleName":"","lastName":"Niederacher","suffix":""},{"id":217768583,"identity":"e1ed38da-01a9-4e34-9173-1947fb588dfe","order_by":5,"name":"Zhongping Cheng","email":"","orcid":"","institution":"Tongji University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhongping","middleName":"","lastName":"Cheng","suffix":""},{"id":217768585,"identity":"eb090919-4777-4dfb-bbf4-f7714035f698","order_by":6,"name":"Hans Neubauer","email":"data:image/png;base64,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","orcid":"","institution":"University Hospital and Medical Faculty of the Heinrich-Heine University Duesseldorf, Life Science Center","correspondingAuthor":true,"prefix":"","firstName":"Hans","middleName":"","lastName":"Neubauer","suffix":""},{"id":217768588,"identity":"de990491-9f02-4a8e-84b6-d232315871e2","order_by":7,"name":"Nadia Stamm","email":"","orcid":"","institution":"University Hospital and Medical Faculty of the Heinrich-Heine University Duesseldorf, Life Science Center","correspondingAuthor":false,"prefix":"","firstName":"Nadia","middleName":"","lastName":"Stamm","suffix":""}],"badges":[],"createdAt":"2023-06-06 12:59:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3029459/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3029459/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12958-024-01188-9","type":"published","date":"2024-02-03T15:01:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":40148989,"identity":"3d758689-859f-4b9e-a36f-d724949b68f8","added_by":"auto","created_at":"2023-07-17 16:59:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":448792,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePGRMC1 is dysregulated in infertility-associated diseases.\u003c/strong\u003e Relative transcript scores of PGRMC1 expression in different stages of a normal menstrual cycle (GSE6364) \u003cstrong\u003e(A) \u003c/strong\u003eand (GSE4888) \u003cstrong\u003e(B)\u003c/strong\u003e. The comparison of relative transcript scores of PGRMC1 expression between healthy females and chronic placental inflammation patients (GSE68474) (\u003cstrong\u003eC\u003c/strong\u003e), repeated implantation failure patients (GSE65099)\u003cstrong\u003e(D), \u003c/strong\u003e(GSE16532) \u003cstrong\u003e(F)\u003c/strong\u003e, endometriosis patients (GSE120103) \u003cstrong\u003e(E), \u003c/strong\u003e(GSE51981) \u003cstrong\u003e(G), \u003c/strong\u003erespectively. Relative transcript scores of PGRMC1 expression level are shown as mean ± SEM. Statistical analysis was performed by two-way ANOVA. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/182e0277c98f179a6f2e39a3.png"},{"id":40148471,"identity":"b5508b6c-e0f3-4bbb-801b-19a4366e3a59","added_by":"auto","created_at":"2023-07-17 16:51:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3246524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA rise-to-decline expression pattern of PGRMC1\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ewas\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003erevealed by\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edecidualization.\u003c/strong\u003e \u003cstrong\u003e(A):\u003c/strong\u003e Schematic representation of \u003cem\u003ein vitro\u003c/em\u003e decidualization system.\u003cstrong\u003e \u003c/strong\u003eThe cellular morphology changes of T-HESCs on day 0 and day 10 were imaged with microscopy in bright filed \u003cstrong\u003e(B)\u003c/strong\u003e or immunofluorescence staining \u003cstrong\u003e(C). \u003c/strong\u003ePGRMC1 was stained by Alexa Fluor-488 (green), and the nucleus was stained by DAPI in blue. Scale bar: 200 µm. The\u003cstrong\u003e \u003c/strong\u003emRNA expression levels of \u003cem\u003ePRL\u003c/em\u003e in T-HESCs were analyzed with qRT-PCR when cells were cultured with MPA/cAMP (red line) for decidualization or DMSO (black line) as control \u003cstrong\u003e(D)\u003c/strong\u003e. The dynamic changes of PGRMC1 protein expression during the 14 days of decidualization were measured by western blot \u003cstrong\u003e(E) \u003c/strong\u003ewith the relative densitometric analysis of the corresponding PGRMC1 protein level \u003cstrong\u003e(F)\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eβ-actin was used as a loading control. The\u003cstrong\u003e \u003c/strong\u003emRNA expression levels of \u003cem\u003ePGRMC1\u003c/em\u003e in T-HESCs\u003cstrong\u003e \u003c/strong\u003eduring decidualization\u003cstrong\u003e(G)\u003c/strong\u003e. Results are shown as the mean ± SEM from three biological replicates. Statistical analysis was performed by two-way ANOVA. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/8f2776fde1d3d95c07abeb14.png"},{"id":40148475,"identity":"769dd843-b74d-426e-a408-5f699d31764f","added_by":"auto","created_at":"2023-07-17 16:51:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":445096,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe rise-to-decline changes of PGRMC1 are required for decidualization. (A):\u003c/strong\u003e Schematic representation of \u003cem\u003ein vitro\u003c/em\u003e decidualization system after PGRMC1 downregulation by siRNA. qRT-PCR analysis of \u003cem\u003ePGRMC1\u003c/em\u003e mRNA expression changes in T-HESCs transfected with either 10 nM of siRNA against PGRMC1 (siPGRMC1) or 10 nM control siRNA (siCTL) for up to 10 days \u003cstrong\u003e(B)\u003c/strong\u003e. The \u003cem\u003ePRL\u003c/em\u003e mRNA expression level in T-HESCs after MPA/cAMP-induced decidualization upon transfection with 10 nM siPGRMC1 or siCTL, analyzed with qRT-PCR \u003cstrong\u003e(C)\u003c/strong\u003e. The workflow for PGRMC1 downregulation after decidualization induction \u003cstrong\u003e(D)\u003c/strong\u003e. mRNA expression levels of \u003cem\u003ePGRMC1\u003c/em\u003e \u003cstrong\u003e(E, G)\u003c/strong\u003e and \u003cem\u003ePRL\u003c/em\u003e \u003cstrong\u003e(F, H)\u003c/strong\u003e in T-HESCs treated with MPA/cAMP for decidualization induction (red line) and non-induction (black line). Blue lines indicate the mRNA levels of \u003cem\u003ePGRMC1\u003c/em\u003e and \u003cem\u003ePRL\u003c/em\u003e when transfected with 10nM siPGRMC1 on the second (\u003cstrong\u003eE, F\u003c/strong\u003e) and fourth (\u003cstrong\u003eG, H\u003c/strong\u003e) day after decidualization induction, respectively. The statistical analysis of mRNA levels of \u003cem\u003ePGRMC1\u003c/em\u003e (and \u003cem\u003ePRL\u003c/em\u003e) between cells with non-induction and induction indicated by red stars, or cells with PGRMC1 knockdown after induction indicated by blue stars. Results are shown as the mean ± SEM from three independent biological replicates. Statistical analysis was performed by two-way ANOVA. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/24298c77fdb5da9d8cb0e451.png"},{"id":40148990,"identity":"5a12b0a3-271f-4ac8-ae45-e85f43f9140d","added_by":"auto","created_at":"2023-07-17 16:59:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1958061,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePGRMC1 accumulates outside the nucleus and co-localizes with PHBs during decidualization. (A)\u003c/strong\u003e Immunofluorescence staining of PGRMC1 in T-HESCs treated with DMSO (left) as control or MPA/cAMP (right) for decidualization induction. PGRMC1 shows in red and the nucleus was stained with DAPI in blue. Scale bar: 200µm. \u003cstrong\u003e(B) \u003c/strong\u003eAnalysis of PGRMC1 localization by subcellular fractionation in T-HESCs treated with DMSO (left) or MPA/cAMP (right), measured by western blot. PGRMC1 was immunoblotted in equal amounts of cytoplasmic (CE), membrane (ME), and nuclear (NE) biomaterial. Compartment-specific markers: Calreticulin (55kDa), β-actin (47kDa), and Histon H3 (17kDa) were used as loading controls for the membrane, cytoplasmic, and nuclear fractions, respectively.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/e19416e41afb2200e599d5e5.png"},{"id":40148470,"identity":"9ae61e59-9a34-4bb6-9171-6677886c87aa","added_by":"auto","created_at":"2023-07-17 16:51:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1781387,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePGRMC1 interacts with PHBs during decidualization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe interactions between PGRMC1 and PHB1 \u003cstrong\u003e(A)\u003c/strong\u003e or PHB2 \u003cstrong\u003e(B)\u003c/strong\u003e in T-HESCs were analyzed with proximity ligation assay upon decidualization induction. Each red spot represents a single interaction. Nuclear stain: DAPI. Magnification 40×. \u003cstrong\u003e(C), (D):\u003c/strong\u003e PHB1 and PHB2 co-precipitate with PGRMC1. PGRMC1 was immunopurified from native whole-cell lysates of T-HESC using anti-PGRMC1 antibody. Western blot analyses of co-immunoprecipitated PHB1 \u003cstrong\u003e(C)\u003c/strong\u003e and PHB2 \u003cstrong\u003e(D)\u003c/strong\u003e in T-HESCs with and without decidualization induction.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/7af4050a34efb7d13668ebb4.png"},{"id":40148472,"identity":"562afec2-9366-4dd6-b6e8-009d248445dd","added_by":"auto","created_at":"2023-07-17 16:51:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":311959,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDownregulation of PHBs impairs decidualization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PHBs protein expression level on day 2 or day 10 after transfection of T-HESCs with 10 nM siPHB1 \u003cstrong\u003e(A)\u003c/strong\u003e, 10 nM siPHB2 \u003cstrong\u003e(B)\u003c/strong\u003e, and 10 nM siPHBs mixture (5 nM siPHB1 and 5 nM siPHB2) \u003cstrong\u003e(C)\u003c/strong\u003e, respectively, analyzed by western blot. The \u003cem\u003ePRL\u003c/em\u003e mRNA expression changes in T-HESCs with (red line) and without (black line) induction \u003cstrong\u003e(D)\u003c/strong\u003e. The \u003cem\u003ePRL\u003c/em\u003e mRNA expression changes in T-HESCs transfected with 10 nM siPHB1 \u003cstrong\u003e(E)\u003c/strong\u003e, 10 nM siPHB2 \u003cstrong\u003e(F)\u003c/strong\u003e, 10 nM siPHBs mixture (5 nM siPHB1 and 5 nM siPHB2) \u003cstrong\u003e(G) \u003c/strong\u003eupon decidualization induction. Results are shown as the mean ± SEM from three biological replicates. Statistical analysis was performed by two-way ANOVA. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/c77e65dc1ca7ccd50f67cdab.png"},{"id":40148991,"identity":"67be05ba-5571-4d71-8faf-880254d96032","added_by":"auto","created_at":"2023-07-17 16:59:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2332331,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAG205 does not affect decidualization.\u003c/strong\u003eThe influence of AG205 on T-HESCs viability was performed after the cells were incubated with indicated concentrations of AG025 for 10 days and analyzed with colorimetric assay \u003cstrong\u003e(A)\u003c/strong\u003e. The absorbance values for cultures with AG205 were compared to the DMSO control (0 µM). The \u003cem\u003ePRL\u003c/em\u003e mRNA expression levels were analyzed after cells were cultured with (black line) or without (red line) 15 µM AG205 \u003cstrong\u003e(B)\u003c/strong\u003e. Results are shown as the mean ± SEM from three independent biological replicates. Statistical analysis was performed by two-way ANOVA. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001. The cellular morphology changes of T-HESCs were imaged with microscopy in the bright field when cells were cultured without (upper panel) or with (down panel) MPA/cAMP upon 15 µM AG205 treatment \u003cstrong\u003e(C)\u003c/strong\u003e. Scale bar: 200 µm. The PGRMC1 protein expression changes in T-HESCs were analyzed by western blot when cells were treated with DMSO (left panel) or 15 µM AG205 (right panel) upon decidualization induction \u003cstrong\u003e(D)\u003c/strong\u003e. β-actin was used as a loading control. The interactions between PGRMC1 and PHB1 \u003cstrong\u003e(E)\u003c/strong\u003e or PHB2 \u003cstrong\u003e(F) \u003c/strong\u003ein T-HESCs were analyzed by\u003cstrong\u003e \u003c/strong\u003eproximity ligation assay when cells were cultured with 15 µM AG205 upon decidualization induction. Each red spot represents a single interaction. Nuclear stain: DAPI. Magnification 40×.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/568cd244a0137a030a7cea51.png"},{"id":50674082,"identity":"da342d1b-744a-4506-84b4-51d62a75d297","added_by":"auto","created_at":"2024-02-05 15:08:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3802303,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/3ea70616-caa6-4921-b766-78b12d44fc1b.pdf"},{"id":40148480,"identity":"08d1a8f2-1601-4896-a09b-61e4f2f858d4","added_by":"auto","created_at":"2023-07-17 16:51:35","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23936896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: Rise-to-decline expression pattern of PGRMC1 is linked to the decidualization program. (A) \u003c/strong\u003ePGRMC1 protein expression changes during 9 days of decidualization were measured by western blot in the St-T1 cell line.\u003cstrong\u003e (B) \u003c/strong\u003ePGRMC1 protein expression changes during 10 days of stimulation with MPA, cAMP, and DMSO, respectively, were measured by western blot in T-HESC. \u003cstrong\u003e(C) \u003c/strong\u003ePGRMC1 protein expression levels on day 6 and day 10 when cultured with DMSO, nomegestrel (NOM), P4, cAMP, MPA/cAMP (M+A), and MPA, respectively, measured by western blot in T-HESC.\u003c/p\u003e","description":"","filename":"Additionalfile1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/3c3eb2d21359e4eec589fa58.tif"},{"id":40148468,"identity":"54174d67-3c89-4670-8557-c7687793578c","added_by":"auto","created_at":"2023-07-17 16:51:34","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1173230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2: PGRMC1 is effectively downregulated by siRNA on protein level. (A) \u003c/strong\u003eThe PGRMC1 protein expression on day 2 and day 10 after transfection of T-HESCs with either 10 nM anti-PGRMC1 siRNA (siPGRMC1) or unspecific scrambled-control siRNA (siCTL). \u003cstrong\u003e(B)\u003c/strong\u003e A comparison of the PGRMC1 protein expression changes within 10 days after transfection of T-HESCs with either 10 nM siPGRMC1 or 10 nM siCTL.\u003c/p\u003e","description":"","filename":"Additionalfile2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/fa1ef4d46f565e25dcee7d82.tif"},{"id":40148479,"identity":"91a32f14-15f1-4066-9089-6ddcd2cee6b0","added_by":"auto","created_at":"2023-07-17 16:51:35","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3236810,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 3: PGRMC1-downregulation before decidualization induction impairs morphological remodeling of T-HESC. \u003c/strong\u003eThe cellular morphology changes of the T-HESCs induced with either DMSO (upper panel) or MPA/cAMP (down panel) after 10 days of siRNA treatment (siCTL, left panel; siPGRMC1, right panel). Scale bar: 200µm.\u003c/p\u003e","description":"","filename":"Additionalfile3.tif","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/2f2999d788fd5c66e689ab55.tif"},{"id":40148482,"identity":"a12f1f37-c1ec-4783-b8c6-ad28f54686e5","added_by":"auto","created_at":"2023-07-17 16:51:37","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":50114376,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 4: PGRMC1-downregulation after decidualization induction does not impair morphological remodeling of T-HESC. \u003c/strong\u003eThe cellular morphology changes of the T-HESCs induced with either DMSO (non-induction, column 1) or MPA/cAMP (Induction, columns 2-4). I (column 3) and II (column 4) indicate that siRNA treatment on T-HESCs was conducted on day 2 or day 4 of decidualization induction, respectively. Scale bar: 200µm\u003c/p\u003e","description":"","filename":"Additionalfile4.tif","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/cd83433ac3bf40ef5967487e.tif"},{"id":40148992,"identity":"b92e38ce-074b-420c-844a-504c17bfb512","added_by":"auto","created_at":"2023-07-17 16:59:34","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":4155800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 5: PGRMC1-downregulation after progestin induction does not impair decidualization in the St-T1 cell line.\u003c/strong\u003e The mRNA expression levels of \u003cem\u003ePGRMC1\u003c/em\u003e \u003cstrong\u003e(A, C)\u003c/strong\u003e and \u003cem\u003ePRL\u003c/em\u003e \u003cstrong\u003e(B, D)\u003c/strong\u003e in St-T1 treated with MPA/cAMP for decidualization induction (red line), and non-induction (black line). The mRNA expression levels of \u003cem\u003ePGRMC1 \u003c/em\u003eand \u003cem\u003ePRL\u003c/em\u003e in St-T1 cells transfected with 10 nM siPGRMC1 (blue line) on the second (\u003cstrong\u003eA, B\u003c/strong\u003e) and fourth day (\u003cstrong\u003eC, D\u003c/strong\u003e) after decidualization induction, respectively. Results are shown as the mean ± SEM from three independent biological replicates. Statistical analysis was performed by a two-way ANOVA test. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001. The red * indicates the comparison between the red and black lines. The blue * indicates the comparison between the red and blue lines.\u003c/p\u003e","description":"","filename":"Additionalfile5.tif","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/2aed0eafe6ed791f8f5269c0.tif"},{"id":40148478,"identity":"0df257a1-7656-4e4f-bf35-19d5748a21c5","added_by":"auto","created_at":"2023-07-17 16:51:34","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":3557656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 6: PGRMC1 and PHB1/PHB2 co-localize in T-HESCs. \u003c/strong\u003eDouble Immunofluorescence staining for PGRMC1 (red) and PHB1 (green) or PHB2 (green) in T-HESCs treated with DMSO \u003cstrong\u003e(A\u003c/strong\u003e) as control or MPA/cAMP \u003cstrong\u003e(B) \u003c/strong\u003efor decidualization induction. Magnification: 40x. Scale bar: 20 µm.\u003c/p\u003e","description":"","filename":"Additionalfile6.tif","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/562653aaf8fcac7680f6f1b3.tif"},{"id":40148481,"identity":"a52de662-d944-47b1-90e9-f93e025b5053","added_by":"auto","created_at":"2023-07-17 16:51:37","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":49297944,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 7: Morphological changes during decidualization upon PHB downregulation. \u003c/strong\u003eThe cellular morphology changes of the T-HESCs induced with either DMSO (non-Induction) or MPA/cAMP (Induction) upon either PHBs knockdown alone or both. Scale bar: 200 µm.\u003c/p\u003e","description":"","filename":"Additionalfile7.tif","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/47d45379cfbe645170f4af29.tif"},{"id":40148476,"identity":"22907882-2646-47e8-811e-adad0bedcf0f","added_by":"auto","created_at":"2023-07-17 16:51:34","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":228148,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 8: Overview over the role of PGRMC1 in human endometrial decidualization and infertility.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUpon stimulation with progesterone or MPA, the PGRMC1 rise-to-decline changes are essential for successful decidualization of the human endometrial cells (upper panel). With downregulated PGRMC1 expression before induction (model for abrogated PGRMC1 in patients from Figure 1), the decidualization program cannot be carried out, leading to decidualization dailure (bottom panel).\u003c/p\u003e","description":"","filename":"Additionalfile8.tif","url":"https://assets-eu.researchsquare.com/files/rs-3029459/v1/2cf07a3531ebea46aae04118.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Progesterone-induced Progesterone Receptor Membrane Component 1 Rise-to- Decline Changes are Essential for Decidualization","fulltext":[{"header":"Background","content":"\u003cp\u003eHuman endometrium tissue is highly dynamic going through proliferative, secretory, and menses phases during a regular menstrual cycle [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Correspondingly, its functional layer exhibits steroid hormone-dependent proliferation, progesterone-stimulated differentiation, and shedding in the absence of the trophoblast [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. After the postovulatory phase, the rising circulating levels of progesterone drive human endometrial stromal cells (HESCs) to differentiate into decidual cells, which is referred to as the decidualization process [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Decidualization is the morphological transformation of HESCs from a proliferating fibroblastic phenotype to an enlarged and rounded epithelial shape, accompanied by secretion of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), which is required for female fertility [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In the presence of a trophoblast, the decidualized endometrium will be maintained through the increased level of progesterone. Otherwise, it will be shed away with a rapid drop of the progesterone level [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A successful decidualization process is an essential prerequisite for embryo implantation and subsequent placenta formation.\u003c/p\u003e \u003cp\u003eDuring decidualization, progesterone (P4) classically affects the endometrium through activation of two major well-characterized progesterone receptor PR-A and PR-B [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Progesterone receptor membrane component 1 (PGRMC1), one of the non-classical progesterone receptors, also rapidly respond to progesterone during decidualization; however, its function in this process is still being elucidated. In the human endometrium, PGRMC1 is abundantly expressed during the proliferative phase of the menstrual cycle in both endometrial and stromal cells. Whereas, in the secretory phase its expression levels dramatically decreased [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Overexpression of PGRMC1 in primary HESCs abrogated decidualization [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and reduced PGRMC1 expression observed in multiple gynecological and obstetrics diseases [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, PGRMC1 was proposed as a fertility stabilizer to decidualization, whose expression must be finely tuned during the entire decidualization to support female fertility [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. How this is achieved remains an enigma.\u003c/p\u003e \u003cp\u003eThe prohibitin proteins (PHBs), prohibitin-1 (PHB1) and prohibitin-2 (PHB2), are ubiquitously expressed and highly conserved in eukaryotic cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. PHBs has been reported to act as transcriptional corepressors for ERα \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Loss of PHBs led to dysfunctional mitochondria, further resulting in male infertility and ovarian aging in females [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Besides, PHB1 is downregulated in the eutopic and ectopic endometrium of patients with endometriosis compared to women without endometriosis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. An uterus-selective, conditional PHB2 knockout mouse model showed a subfertility phenotype with litters reduced both in number and size [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This implies that appropriate protein levels of PHB1/2 as well as of PGRMC1 are required for optimal uterine function and fertility. In breast cancer cells, progestin-activated PGRMC1 associated with PHBs to stimulate cellular proliferation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Binding of activated PGRMC1 to PHBs was accompanied by decreased PHBs-ERα-interaction, resulting in elevated expression of ER-dependent genes. Whether the progestin-depended interaction between PHBs and PGRMC1 also occurs during decidualization has never been characterized before. Therefore, the role of their interaction with regards to female fertility remains to be elucidated.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to explore the functional role of PGRMC1 and PHBs, and their interplay for successful decidualization.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData sources\u003c/h2\u003e \u003cp\u003eWe collected the associated gene expression profiles in publicly available Gene Expression Omnibus (GEO) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Samples from different menstruation phases (proliferative/PE, early secretory/ESE, mid-secretory/MSE, late secretory/LES) were chosen from GSE4888 and GSE56364 to detect expression of PGRMC1 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Other analyzed infertility-related diseases datasets included chronic placental inflammation (CPI) (GSE68474), repeated implantation failure (RIF) (GSE65099, GSE16532), endometriosis (GES120103, GSE51981) [\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. All raw data were background-subtracted and normalized.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eThe hTERT-immortalized human endometrial stromal cells (T-HESCs) were purchased from abm (T0533). Both the cell lines T-HESCs and St-T1 were maintained in phenol-red free Dulbecco\u0026rsquo;s Modified Eagle Medium//Ham\u0026rsquo;s F12 (DMEM/F12; Gibco, Thermo Fisher Scientific, 11039021) medium supplemented with 10% (\u003cem\u003ev/v\u003c/em\u003e) charcoal-stripped fetal bovine serum (Thermo Fisher Scientific, 12676029), 100 units/mL penicillin-streptomycin (Thermo Fisher Scientific, 2321118), 50\u0026micro;g/ml gentamycin sulfate (Biowest, L0012), 200\u0026micro;M sodium pyruvate (Biowest, L0624) and 1.5g/L sodium bicarbonate (Biowest, L0680) (hereafter referred to as complete medium) in a humidified incubator at 37\u003csup\u003eo\u003c/sup\u003eC in the presence of 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells (passage number\u0026thinsp;\u0026lt;\u0026thinsp;10) were regularly tested negative for mycoplasma.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eChemical Compounds\u003c/h2\u003e \u003cp\u003eAG205 (Sigma-Aldrich) was diluted in 2% charcoal-stripped FBS complete medium to 15 mM. Medroxyprogesterone acetate (MPA) and 8-Br-cAMP MPA (cAMP) were prepared from a 10 mM and 5 mM stock solution, respectively.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eImmunofluorescence Staining\u003c/h2\u003e \u003cp\u003eCells were seeded and cultured in chamber slides (Nunc Lab-Tek, Thermo Fisher Scientific C7182-1PAK) fixed with 4% formaldehyde (Sigma-Aldrich, 20649296018) for 10 min at room temperature (RT), washed with washing buffer (Dako, Glostrup, Denmark, S3006) (3 x 5 min each). Then, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, T8787) in PBS for 10 min at RT and washed with washing buffer again (3 x 5 min each). DAKO protein block buffer (Dako, X0909) was added and incubated for 1h at RT before incubating with primary antibodies specific for PGRMC1 (Abcam, ab48012), PHB1 (Abcam, ab75766), PHB2 (Cell signaling, 14084S) and Vimentin (Abcam, ab02547) overnight at 4\u003csup\u003eo\u003c/sup\u003eC. The next day, cells were washed with washing buffer (3 x 5 min each) and incubated with secondary antibodies (Donkey-anti-goat, Alexa 488: Invitrogen, A11055; Donkey-anti-rabbit, Alexa 488: Invitrogen, A31573) for 1 h at RT in a humidified chamber in the dark. Nucleic acid was stained with DAPI (Thermo Fisher Scientific, 15733122) simultaneously with co-incubated secondary antibodies. After the final wash, the cells were mounted with Fluorescent Mounting Medium (Dako, S3023). Negative controls were prepared for each sample following the same staining procedure with isotype controls instead of primary antibodies. Fluorescence signals were detected with an Axioplan 2 Imaging fluorescence microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eProximity Ligation Assay\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein-situ\u003c/em\u003e proximity ligation assay (PLA) procedure was performed with the Duolink\u0026reg; PLA Kit (Sigma-Aldrich, DUO92008) and following the manufacturers protocol. The cells were incubated with the primary antibodies i.e., anti-PGRMC1 (Abcam, ab48012) with PHB1 (Abcam, ab75766) and PHB2 (Cell signaling, 14085S) overnight at 4\u003csup\u003eo\u003c/sup\u003eC. The slides were washed twice for 5 min with buffer A, followed by incubation with the PLA probes (anti-goat PLUS and anti-rabbit MINUS) in antibody diluent for 60 min at 37\u003csup\u003eo\u003c/sup\u003eC. After washing twice for 5 min with buffer A, ligation was performed using ligase diluted in ligation buffer for 30 min at 37\u003csup\u003eo\u003c/sup\u003eC. Then the cells were washed with buffer A before incubation for 100 min with amplification stock solution at 37\u003csup\u003eo\u003c/sup\u003eC. After washing twice for 10 min with buffer B, nuclear DNA was labeled with DAPI for 10 min and slides were mounted with mounting medium. Negative PLA control was performed using respective isotype control antibodies (isotype goat, Abcam, ab37373; isotype rabbit, Abcam, ab37415). Red fluorescence dots inside the cellular areas representing a single protein-protein interaction were quantified using image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMTT Assay and Western Blotting\u003c/h2\u003e \u003cp\u003eMTT assay and western blotting of PGRMC1 and PHB1, PHB2 were performed as previously described [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSubcellular Protein Fractionation\u003c/h2\u003e \u003cp\u003eA subcellular protein fractionation kit (Thermo Fisher Scientific) was used to fractionate proteins into cytoplasmic, membrane, and nuclear fractions. Cells were harvested as pellets. The pellet was lysed with cytoplasmic extraction buffer, membrane extraction buffer, and nuclear extraction buffer. Primary antibodies specific for β-actin (Santa Cruz Biotechnology), Calreticulin (Santa Cruz Biotechnology), and Histon H3 (Cell signaling) were used to indicate the purity of the cytoplasmic, membrane, and nuclear fractions, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCo-Immunoprecipitation\u003c/h2\u003e \u003cp\u003eCo-immunoprecipitation was performed using the Pierce Co-IP kit (Thermo Fisher Scientific). Briefly, the anti-PGRMC1 antibody (Cell signaling) was first immobilized for 2h using AminoLink Plus coupling resin. In parallel, cell pellets were resuspended in ice-cold IP Lysis buffer. An amount of 500 \u0026micro;g protein was incubated with resin at 4 \u003csup\u003eo\u003c/sup\u003eC overnight. After incubation, the resin was washed, and protein complexes bound to the antibody were eluted using elution buffer. Subsequent western blot analyses were performed as described before.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGene silencing (siRNA Transfection)\u003c/h2\u003e \u003cp\u003eTo knock down PGRMC1 expression in T-HESCs, FlexiTube GeneSolution (Qiagen) was used, containing four siRNA(s) that specifically target human PGRMC1 mRNAs. Cells were transfected with the final concentration of 10 nM \u003cem\u003ePGRMC1\u003c/em\u003e siRNA(s) or negative control siRNA (siCTL) (Thermo Fisher Scientific) using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to recommended procedures. Afterwards, cells were treated with decidualization medium containing either induction cocktail or DMSO, and harvested at different time points for downstream experiments. For PHB1 and PHB2 mRNA expression inhibition (siPHB1, siPHB2: Qiagen), the same siRNAs concentration and method was used above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Reverse-Transcription PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eRNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer\u0026rsquo;s specifications. Reverse transcription of RNA into cDNA was performed with the Omniscript RT kit (Qiagen) according to the manufacturer\u0026rsquo;s instructions. Quantitative PCR was performed using QuantiFast SYBR Green PCR Kit (Qiagen) and LightCycler \u0026reg;480 System (Roche). Primers for \u003cem\u003ePGRMC1\u003c/em\u003e (Qiagen), \u003cem\u003ePRL\u003c/em\u003e (Qiagen) and \u003cem\u003eHPRT1\u003c/em\u003e (Qrigene, Rockville, MD, USA). The delta-delta cycle threshold method was used to normalized expression to the reference gene HPRT1 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eA two-tailed paired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used to analyze experiments comparing two experimental groups or two-way ANOVA for multiple comparisons of more than two groups. A value of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant. All statistical analyses were performed with GraphPad Prism 9.0. Results were reported as means with standard deviation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePGRMC1 expression is dysregulated in patients with impaired decidualization\u003c/h2\u003e \u003cp\u003eTo understand the dynamics of PGRMC1\u0026rsquo;s expression changes during normal decidualization, we initially investigated its expressional profile by mining publicly available RNA-sequencing data sets from endometrial biopsies (GEO accession numbers: GSE6364 and GSE4888). In a normal menstrual period, PGRMC1 mRNA level gradually decreased from the proliferative phase to the secretory (including early-, mid-, and late-) phase, manifesting the highest level in the proliferation phase and the lowest level in the late-secretory phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B), consistent with previously reported data [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This indicates that PGRMC1 may have an important role in regulating cellular proliferation and may not be required for decidualization in the secretory phase as it is consecutively decreased at mRNA level after progesterone stimulation. We hypothesized that the dynamic changes of PGRMC1 have an important role during the menstrual cycle that must be finely tuned.\u003c/p\u003e \u003cp\u003eApart from normal decidualization, expression of PGRMC1 also changes in chronic placental inflammation (CPI) and in infertility-related diseases including repeated implantation failure (RIF), and endometriosis, in which the common pathological factor is impaired decidualization. In most data sets lower levels of PGRMC1 mRNA were frequently observed in patients who suffered from infertility-related disease compared to a control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E), suggesting a tight connection between impaired decidualization and downregulated PGRMC1 mRNA. However, we unexpectedly found that a higher level of PGRMC1 mRNA was observed in some RIF and endometriosis patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-G), implying that upregulated PGRMC1 expression could also be a cause of decidualization disruption. Thus, we concluded that any kind of dysregulation of PGRMC1 expression may lead to an impaired decidualization.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRise-to-decline trend of PGRMC1 expression during\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003edecidualization\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo answer the question of how a dysregulation of PGRMC1 expression may impair decidualization, we established a hormone-inducible \u003cem\u003ein vitro\u003c/em\u003e decidualization model in T-HESCs based on visualizing its morphological changes and by measuring the expression level of the decidual marker prolactin (PRL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). After being exposed to the decidualization induction cocktail consisting of the P4 analog MPA plus cAMP for 10 days, morphological changes of T-HESCs were inspected by microscopy in bright-field and by immunofluorescent analysis of the cytoskeletal marker vimentin. With this protocol, T-HESCs underwent a transformation from a fibroblast-like shape to a polygonal epithelial-like shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C) accompanied with a significant increase of PRL mRNA expression compared to non-induced controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Both the morphological changes and enhanced expression of PRL indicate a successfully established the hormone-induced decidualization model, allowing to investigate the role of PGRMC1 in decidualization.\u003c/p\u003e \u003cp\u003eSince dysregulated PGRMC1 expression is associated with decidualization failure in infertility-related diseases, we aimed to determine and modulate the expression level of PGRMC1 in our system to study its impact on the decidualization process. First, we determined the protein expression profile of PGRMC1 during the \u003cem\u003ein vitro\u003c/em\u003e decidualization program. Intriguingly, we found that PGRMC1 expression gradually increased from the day the induction cocktail had been added (D0), peaking at day 6 (D6) post-induction, followed by a constant decrease until day 14 (D14) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F). This protein expression change could also be observed in the St-T1 cell line (Additional file 1A). In contrast with the observation of gradually decreased mRNA levels of PGRMC1 during the secretory phase of the normal menstrual cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B), our \u003cem\u003ein vitro\u003c/em\u003e decidualization model revealed that the promotion of cells into decidualized state comprises a PGRMC1 increasing phase and a PGRMC1 decreasing phase, both on protein and mRNA level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-G). This data suggests that PGRMC1 was regulated at transcriptional level during decidualization. We termed this PGRMC1 protein dynamic changes as PGRMC1 \u0026lsquo;rise-to-decline\u0026rsquo; changes in decidualization.\u003c/p\u003e \u003cp\u003eIt is well known that increasing P4 levels initiate decidualization, although the activity of PGRMC1 in decidualization seems to be independent of P4 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Consistently, the T-HESC cells can go through the decidualization process treated with either P4, MPA, or cAMP (Additional file 1B-C). Intriguingly, the PGRMC1 expression changes can be observed at each condition, which led us to the conclusion that the PGRMC1 rise-to-decline changes are a universal mechanism within the decidualization program.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe rise-to-decline changes of PGRMC1 are required for decidualization.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the potential role of the PGRMC1 rise-to-decline changes during the decidualization, we firstly downregulated its expression before hormone induction with an optimized concentration of an siRNA-mix specific for PGRMC1 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Importantly, PGRMC1 mRNA levels were remained suppressed throughout 10 days post-siRNA-transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Likewise, expression of PGRMC1 protein was completely abrogated from day 2 (D2) to day 10 (D10) after siRNA transfection (Additional file 2A-B).\u003c/p\u003e\u003cp\u003eT-HESCs with suppressed PGRMC1 expression were further treated with the decidualization induction cocktail. As indicated by the lack of morphological transformation and PRL production over 10 days of hormone treatment period (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Additional file 3), these cells did not undergo decidualization. Thus, in the absence of the progestin-induced PGRMC1 increasing phase decidualization failed.\u003c/p\u003e \u003cp\u003eThese results prompted us to investigate if expression of PGRMC1 is needed for decidualization at the time point of induction \u0026ndash; as a kind of a program switch \u0026ndash; or later. To this aim, we postponed the transfection of PGRMC1 suppressing siRNAs to after the induction of decidualization. First, we treated T-HESC cells for 2 days with the combination of MPA and cAMP to induce decidualization followed siRNA transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and investigated the cells up to day 10 (D10) after induction. As expected, PGRMC1 mRNA levels started to decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, blue line) after 2 days of transfection of PGRMC1-specific siRNAs and the PGRMC1 mRNA levels stayed below mRNA levels reached during normal induction of decidualization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, red line). Interestingly, in addition to morphological changes (Additional file 4), PRL mRNA expression level first dropped, but between D6 and D8 not only recovered to a comparative level to that of normal induction, was even four days earlier compared to the normal induction, indicating a promoted decidualization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). We further measured the effects of knocking down PGRMC1 after 4 days of induction with MPA/cAMP on the decidualization program. The results are very similar to the outcome achieved when suppressing PGRMC1 after 2 days of induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H). The results could be additionally reproduced in the St-T1 cell line (Additional file 5A-D). Taken together, the PGRMC1 rise-to decline changes are required for a proper decidualization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePGRMC1 accumulates outside the nucleus during decidualization\u003c/h2\u003e \u003cp\u003eIt has been reported that PGRMC1 translocates from cytoplasmic membranes to the nucleus during decidualization [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Recently, PGRMC1-mediated proteomic changes have been well characterized after decidualization, suggesting that PGRMC1 binds to proteins involved in translation, ATP generation, protein maturation, glucose transport, and lipid metabolism [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Almost all these proteins locate in the cytoplasm or on membranes, but not in the nucleus. This raises the question of why proteins interacting with PGRMC1 are barely found to be in the nucleus.\u003c/p\u003e\u003cp\u003eTo better understand the question, we initially assessed PGRMC1 protein subcellular localization by immunofluorescence. Without induction of decidualization PGRMC1 was essentially located in the cytoplasm, but significantly accumulated close to the nucleus after induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To further verify these observations, we fractionated the cells into soluble parts containing cytoplasm, membrane, and nucleus and detected the PGRMC1 protein by western blot. In line with the immunofluorescence results, PGRMC1 was only observed in the membrane fraction but not in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This indicates that PGRMC1 accumulated at the nucleus periphery but not in the nucleus during hormone-induced decidualization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eInteractions of PHB1/PHB2 to PGRMC1 mediate decidualization\u003c/h2\u003e \u003cp\u003eWe have recently demonstrated in breast cancer cells, that progestin-activated PGRMC1 interacts with PHB1/PHB2 resulting in enhanced ERα-dependent transcription and cell proliferation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In analogy, here we found that PGRMC1 colocalized with PHB1 and PHB2 in the cytoplasm and at the nucleus periphery after induction, whereas barely colocalization signals could be observed without induction revealed by immunofluorescence (Additional file 6A-B). This suggests a potential interaction between PHBs and PGRMC1 introduced by progestin treatment. Then, PLA was performed to further explore the associations between PGRMC1 and PHB1/2. Upon induction, a significantly higher PLA signal suggesting the interaction of PGRMC1 to PHB1/2 could be observed compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). We next performed the Co-IP analysis confirming that both PHB1 and PHB2 co-precipitate with PGRMC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D), which is consistent with the observation made in MIA PaCa-2 cells [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe interactions between PGRMC1 and PHB1 \u003cb\u003e(A)\u003c/b\u003e or PHB2 \u003cb\u003e(B)\u003c/b\u003e in T-HESCs were analyzed with proximity ligation assay upon decidualization induction. Each red spot represents a single interaction. Nuclear stain: DAPI. Magnification 40\u0026times;. \u003cb\u003e(C), (D)\u003c/b\u003e: PHB1 and PHB2 co-precipitate with PGRMC1. PGRMC1 was immunopurified from native whole-cell lysates of T-HESC using anti-PGRMC1 antibody. Western blot analyses of co-immunoprecipitated PHB1 \u003cb\u003e(C)\u003c/b\u003e and PHB2 \u003cb\u003e(D)\u003c/b\u003e in T-HESCs with and without decidualization induction.\u003c/p\u003e \u003cp\u003eTo explore the function of PGRMC1-PHBs interaction during decidualization, we downregulated PHBs via siRNA transfection, reaching expression levels decreased by 50\u0026ndash;80% compared to the control for individual PHBs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). Knocking down either PHBs alone or both PHBs before hormone induction partly impaired the decidualization process (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-G), but the cells still could achieve morphological transformation (Additional file 7). These effects on decidualization are comparable to the results achieved with suppressed PGRMC1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAG205 does not affect PGRMC1 rise-to-decline changes and decidualization\u003c/h2\u003e \u003cp\u003eAG205 was reported to be a specific inhibitor of PGRMC1 and was broadly used to explore PGRMC1\u0026rsquo;s role in decidualization [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Recent data, however, question the specificity of AG205 for PGRMC1 [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe PHBs protein expression level on day 2 or day 10 after transfection of T-HESCs with 10 nM siPHB1 \u003cb\u003e(A)\u003c/b\u003e, 10 nM siPHB2 \u003cb\u003e(B)\u003c/b\u003e, and 10 nM siPHBs mixture (5 nM siPHB1 and 5 nM siPHB2) \u003cb\u003e(C)\u003c/b\u003e, respectively, analyzed by western blot. The \u003cem\u003ePRL\u003c/em\u003e mRNA expression changes in T-HESCs with (red line) and without (black line) induction \u003cb\u003e(D)\u003c/b\u003e. The \u003cem\u003ePRL\u003c/em\u003e mRNA expression changes in T-HESCs transfected with 10 nM siPHB1 \u003cb\u003e(E)\u003c/b\u003e, 10 nM siPHB2 \u003cb\u003e(F)\u003c/b\u003e, 10 nM siPHBs mixture (5 nM siPHB1 and 5 nM siPHB2) \u003cb\u003e(G)\u003c/b\u003e upon decidualization induction. Results are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from three biological replicates. Statistical analysis was performed by two-way ANOVA. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003cp\u003eTaking advantage of the critical role of PGRMC1 rise-to-decline changes for decidualization, we tested the effect of AG205 on PGRMC1 and the decidualization process. Since AG205 concentrations used in previous reports were high enough to impair cellular viability [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], we initially determined the appropriate concentration of AG205 that did not affect cell viability. In the MTT assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), a concentration below 15 \u0026micro;M had no (or a moderate) effect, whereas a concentration higher than 15 \u0026micro;M had a detrimental effect on cellular viability, which is consistent with previously reported [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In addition, decidualization was successfully achieved with T-HESCs treated AG205 with concentrations below 15 \u0026micro;M, as indicated by the increasing expression of PRL and the change in cell morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C). Furthermore, AG205 treatment did neither affect PCRMC1 protein level during decidualization, nor its rise-to-decline expression profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Moreover, the interaction of PGRMC1 to PHBs was not disturbed as confirmed by PLA (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-F), which is in line with a previous report [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Based on these results, we propose that AG205 (\u0026lt;\u0026thinsp;15 \u0026micro;M) has no effect on the observed PGRMC1 functions during decidualization.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePGRMC1 has been demonstrated to play a role in various reproductive tissues, particularly endometrial stromal cells [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. It influences the decidualization process and female fertility [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This study discovered a connection between dysregulated PGRMC1 mRNA levels and impaired decidualization in infertility-related diseases. We also revealed that PGRMC1 protein exhibits a rise-to-decline pattern after progestin stimulation, essential for normal decidualization (Additional file 8). Additionally, PHB1/2 involved in the decidualization program by interacting with PGRMC1. Despite the unclear mechanisms behind PGRMC1 dysregulation and decidualization failure, PGRMC1 expression levels may serve as a useful fertility indicator.\u003c/p\u003e \u003cp\u003ePrevious reports focused on PGRMC1 mRNA profile changes during decidualization, with few investigations into protein level dynamics. We measured both mRNA and protein levels of PGRMC1 after inducing decidualization and observed a rise-to-decline pattern. The observed increase and decrease of PGRMC1 protein expression fits into cyclic changes observed \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The overall dynamic changes of the PGRMC1 protein level during a normal menstrual cycle are composed of two peaks: one occurs in the secretory phase, as revealed in this study and the other one occurs in the proliferative phase as previously reported [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It resembles estrogen dynamics during the menstrual cycle, suggesting PGRMC1 expression may be regulated by estrogen concentration or a similar mechanism [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. As PGRMC1 overexpression in breast cancer cells leads to higher E2 secretion, T-HESCs E2 production might depend on PGRMC1 activation. Further research is needed to understand the relationship between estrogen and PGRMC1 expression, including the possibility of estrogen receptor-mediated transcription regulation.\u003c/p\u003e \u003cp\u003eKnocking down PGRMC1 before hormone treatment inhibited decidualization, highlighting its crucial role as a 'switch' at this stage. Appropriate PGRMC1 protein levels are needed to initiate decidualization upon P4/cAMP stimulation. The PGRMC1 rise-to-decline pattern can be induced by various treatments (Additional file 1), suggesting a common signaling pathway that correlates with decidualization, which requires further investigation. PGRMC1 seems less necessary after decidualization initiation, as knocking it down either does not affect or even facilitates the process. It is unclear why PRL expression initially drops and then increases when PGRMC1 is knocked down after decidualization induction. Downregulating PGRMC1 after progestin treatment doesn't hamper decidualization, indicating its critical role during the increasing phase and induction. This aligns with observations that PGRMC1 downregulation in the secretory phase promotes decidualization [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Overall, PGRMC1 activation by P4 may facilitate the switch from cellular proliferation to decidualization initiation through various biological processes, while the mechanism of how downregulated PGRMC1 promotes decidualization warrants further investigation.\u003c/p\u003e \u003cp\u003ePGRMC1 associates with proteins involved in protein biosynthesis, intracellular transport, and mitochondrial activity to promote decidualization [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, little is known about how PGRMC1 interacts with these proteins to regulate decidualization. We found that PGRMC1 binds to PHBs at the nucleus periphery after P4 treatment, suggesting it may function as a scaffold protein for decidualization in endometriosis stromal cells. PGRMC1 could be anchored on the membrane of various organelles, co-transporting with them during decidualization-related morphological changes [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. PHBs form a super complex in mitochondria, playing roles in lipid biogenesis, ATP generation, and more [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Knocking down PHBs partially impaired decidualization, similar to PGRMC1 knockdown, suggesting PGRMC1-PHBs interactions may influence decidualization as a complex, requiring further investigation. We speculate that PGRMC1 binding to PHBs may inhibit cellular proliferation and facilitate differentiation, acting as a proliferation-differentiation switch.\u003c/p\u003e \u003cp\u003eWe found that the small molecule AG205 neither affect PGRMC1-PHBs interaction, nor decidualization in our study (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Although AG205 has been shown to interact with PGRMC1 \u003cem\u003ein vitro\u003c/em\u003e, its \u003cem\u003ein vivo\u003c/em\u003e interaction remains unknown. Our data align with a recent study demonstrating that AG205 concentrations over 15 \u0026micro;M reduce cell proliferation, and concentrations above 30 \u0026micro;M result in cell death in HEC-1A and T-HESC cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, our findings are consistent with a previous report indicating that a high concentration (50 \u0026micro;M) of AG205 did not affect decidualization [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFinally, a general limitation needs to be taken into account. The \u003cem\u003ein vitro\u003c/em\u003e experiments performed in cell lines assured reproducibility within the established system, while primary patient endometrium tissues are highly heterogeneous and, in general, require analysis of a large cohort in order to obtain a statistically significant result. Therefore, in this study, we focused on the analysis of publicly available datasets and two cell lines. Nevertheless, to confirm PGRMC1\u0026rsquo; switch-like rise-to-decline expression pattern in vivo, a mouse model with inducible PGRMC1-downregulation could be exploited in future studies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBased on the results of our study, we postulate that P4/progestin-induced PGRMC1 rise-to-decline expression is essential to start the decidualization program, but, once decidualization started, PGRMC1 is not needed to drive it. Since PGRMC1 expression was downregulated in most analyzed transcriptome datasets of endometrium biopsies of patients with infertility-related diseases, our PGRMC1-knockdown experiments mirrored this situation in vitro and consequently demonstrated a decidualization failure, potentially leading to infertility. Taken together, we explained how dysregulated PGRMC1 expression could impact endometrial stromal cell differentiation in patients and thereby lead to manifestation of their disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe datasets analyzed in this study are available at Gene Expression Omnibus (GEO) database: https://www.ncbi.nlm.nih.gov/geo/ with the GEO accessions GSE4888, GSE56364, GSE68474, GSE65099, GSE16532, GES120103, GSE51981.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This project was funded by the \u0026ldquo;German Research Foundation (Deutsche Forschungsgemeinschaft, DFG, NE 805/6-1)\u0026rdquo;, and \u0026quot;The Brigitte and Dr. Konstanze Wegener Foundation (#51, Duesseldorf, Germany)\u0026rdquo;, and \u0026ldquo;The Medical Faculty of the Heinrich-Heine University Duesseldorf (#48/2018)\u0026rdquo;, and \u0026ldquo;The Shanghai Shenkang Hospital Development Center (SHDC12021113)\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026rsquo; contributions: Conceptualization, H.L., H.N.; Investigation, H.L., N.S.; Methodology, H.L., A.P.B. and N.S.; Supervision, T.F., D.N., Z.C., N.S., H.N.; Project administration, T.F., H.N.; Writing-original draft, H.L.; Writing-review and editing, A P.B., A.F., N.S. and H.N. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eWe are grateful to our colleagues in the Department of Gynecology and Obstetrics, Heinrich-Heine University Duesseldorf for their helpful comments.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eReed BG, Carr BR. In: Feingold, et al. editors. The Normal Menstrual Cycle and the Control of Ovulation. South Dartmouth (MA); 2000. \u003cem\u003eEndotext\u003c/em\u003e, K.R..\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJabbour HN, et al. Endocrine regulation of menstruation. Endocr Rev. 2006;27(1):17\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMihm M, Gangooly S, Muttukrishna S. The normal menstrual cycle in women. 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Endocrinology. 2011;152(3):1047\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBai Y et al. \u003cem\u003ePGRMC1 Promotes Progestin-Dependent Proliferation of Breast Cancer Cells by Binding Prohibitins Resulting in Activation of ERalpha Signaling.\u003c/em\u003e Cancers (Basel), 2021. 13(22).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalbi S, et al. Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women. Endocrinology. 2006;147(3):1097\u0026ndash;121.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurney RO, et al. Gene expression analysis of endometrium reveals progesterone resistance and candidate susceptibility genes in women with endometriosis. Endocrinology. 2007;148(8):3814\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaman K, et al. 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Novel nonclassic progesterone receptor PGRMC1 pulldown-precipitated proteins reveal a key role during human decidualization. Fertil Steril. 2020;113(5):1050\u0026ndash;1066e7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeluso JJ, Pru JK. \u003cem\u003eProgesterone Receptor Membrane Component (PGRMC)1 and PGRMC2 and Their Roles in Ovarian and Endometrial Cancer\u003c/em\u003e. Cancers (Basel), 2021. 13(23).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeakel SL, et al. Protein complexes including PGRMC1 and actin-associated proteins are disrupted by AG-205. Biochem Biophys Res Commun. 2020;524(1):64\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsuru A et al. \u003cem\u003ePGRMC1 Regulates Cellular Senescence via Modulating FOXO1 Expression in Decidualizing Endometrial Stromal Cells\u003c/em\u003e. Biomolecules, 2022. 12(8).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThieffry C et al. \u003cem\u003eAG-205 Upregulates Enzymes Involved in Cholesterol Biosynthesis and Steroidogenesis in Human Endometrial Cells Independently of PGRMC1 and Related MAPR Proteins\u003c/em\u003e. Biomolecules, 2021. 11(10).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalsano S, et al. Deciphering the Role of PGRMC1 During Human Decidualization Using an In Vitro Approach. J Clin Endocrinol Metab. 2021;106(8):2313\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang-Eckhardt L, Eckhardt M. A progesterone receptor membrane component 1 antagonist induces large vesicles independent of progesterone receptor membrane component 1 expression. Biol Chem. 2020;401(9):1093\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmed IS, et al. Progesterone receptor membrane component 1 (Pgrmc1): a heme-1 domain protein that promotes tumorigenesis and is inhibited by a small molecule. J Pharmacol Exp Ther. 2010;333(2):564\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCahill MA. Progesterone receptor membrane component 1: an integrative review. J Steroid Biochem Mol Biol. 2007;105(1\u0026ndash;5):16\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVelazquez Hernandez DM, Vazquez-Martinez ER, Camacho-Arroyo I. The role of progesterone receptor membrane component (PGRMC) in the endometrium. Steroids. 2022;184:109040.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCahill MA, et al. The emerging role of progesterone receptor membrane component 1 (PGRMC1) in cancer biology. Biochim Biophys Acta. 2016;1866(2):339\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcCallum ML, et al. Conditional Ablation of Progesterone Receptor Membrane Component 1 Results in Subfertility in the Female and Development of Endometrial Cysts. Endocrinology. 2016;157(9):3309\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen YJ, et al. PGRMC1 acts as a size-selective cargo receptor to drive ER-phagic clearance of mutant prohormones. Nat Commun. 2021;12(1):5991.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerkwirth C, Langer T. Prohibitin function within mitochondria: essential roles for cell proliferation and cristae morphogenesis. Biochim Biophys Acta. 2009;1793(1):27\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Decidualization, Progesterone receptor membrane component 1 (PGRMC1), Endometrium, Telomerase-immortalized human endometrial stromal cells (T-HESCs), Infertility, Prohibitin-1 (PHB1), Prohibitin-2 (PHB2), AG205, Rise-to-decline pattern, in-fertility","lastPublishedDoi":"10.21203/rs.3.rs-3029459/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3029459/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eDecidualization of endometrial cells is the prerequisite for embryo implantation and subsequent placenta formation and is induced by rising progesterone levels following ovulation. One of the hormone receptors contributing to endometrial homeostasis is Progesterone Receptor Membrane Component 1 (PGRMC1), a non-classical membrane-bound progesterone receptor with yet unclear function. In this study, we aimed to investigate how PGRMC1 contributes to human decidualization.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo gain insight into PGRMC1-implication in infertility-related diseases, we analyzed its expression profile in RNA-sequencing datasets of endometrial biopsies. To further explore the function of PGRMC1 in human decidualization, we implemented an inducible decidualization system, which is achieved by culturing two human endometrial stromal cell lines in decidualization-inducing medium containing medroxyprogesterone acetate and 8-Br-cAMP. In our system, we measured PGRMC1 expression during hormone induction as well as decidualization status upon PGRMC1 knockdown at different time points. We further conferred proximity ligation assay to identify PGRMC1 interaction partners.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePGRMC1 expression was altered in patients with infertility-related diseases and impaired decidualization, being significantly downregulated in most datasets. In \u003cem\u003ein vitro\u003c/em\u003e experiments, we observed that PGRMC1 expression follows a rise-to-decline pattern, in which its expression level initially increased during the first 6 days after induction (PGRMC1 increasing phase) and decreased in the following days (PGRMC1 decreasing phase). Knockdown of PGRMC1 expression before the induction led to a failed decidualization, while its knockdown after induction did not inhibit decidualization, suggesting that the progestin-induced \u0026lsquo;PGRMC1 increasing phase\u0026rsquo; is essential for normal decidualization. Furthermore, we found that the interactions of PHB1 and PHB2 with PGRMC1 were induced upon progestin treatment. Knocking down either PHB individually or both slowed down the decidualization process compared to the control, suggesting that PGRMC1 cooperates with PHBs to regulate the decidualization.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eAccording to our findings, PGRMC1 expression followed a progestin-induced rise-to-decline expression pattern during human endometrial decidualization process; and the correct execution of this expression program was crucial for successful decidualization. Thereby, the results of our \u003cem\u003ein vitro\u003c/em\u003e model explained how PGRMC1 dysregulation in patients with impaired decidualization contributes to the manifestation of their disease.\u003c/p\u003e","manuscriptTitle":"Progesterone-induced Progesterone Receptor Membrane Component 1 Rise-to- Decline Changes are Essential for Decidualization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-07-17 16:51:29","doi":"10.21203/rs.3.rs-3029459/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-10-13T07:41:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-07-21T22:51:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"873f978f-466d-4b06-9d39-89f4552469a5","date":"2023-07-13T11:53:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7ea60f4b-cf79-49e5-88e2-74344006d949","date":"2023-06-25T13:04:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-06-10T20:57:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-06-07T08:37:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-06-07T08:37:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Reproductive Biology and Endocrinology","date":"2023-06-06T12:54:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6a676c01-c2df-4803-8bfa-659184e2eb00","owner":[],"postedDate":"July 17th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-02-05T15:04:30+00:00","versionOfRecord":{"articleIdentity":"rs-3029459","link":"https://doi.org/10.1186/s12958-024-01188-9","journal":{"identity":"reproductive-biology-and-endocrinology","isVorOnly":false,"title":"Reproductive Biology and Endocrinology"},"publishedOn":"2024-02-03 15:01:27","publishedOnDateReadable":"February 3rd, 2024"},"versionCreatedAt":"2023-07-17 16:51:29","video":"","vorDoi":"10.1186/s12958-024-01188-9","vorDoiUrl":"https://doi.org/10.1186/s12958-024-01188-9","workflowStages":[]},"version":"v1","identity":"rs-3029459","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3029459","identity":"rs-3029459","version":["v1"]},"buildId":"KpppsY2wPL7CB4kaJOidf","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}