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. 2024 Feb 3;22(1):20.
doi: 10.1186/s12958-024-01188-9.

Progesterone-induced progesterone receptor membrane component 1 rise-to-decline changes are essential for decidualization

Hailun Liu  1 , André Franken  1 , Alexandra P Bielfeld  2 , Tanja Fehm  1 , Dieter Niederacher  1 , Zhongping Cheng  3   4 , Hans Neubauer #  5 , Nadia Stamm #  6
Affiliations

Progesterone-induced progesterone receptor membrane component 1 rise-to-decline changes are essential for decidualization

Hailun Liu et al. Reprod Biol Endocrinol. .

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: We first analyzed PGRMC1 expression profile during a regular menstrual cycle in RNA-sequencing datasets. 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: In a regular menstrual cycle, PGRMC1 mRNA expression is gradually decreased from the proliferative phase to the secretory phase. 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 prohibitin 1 and prohibitin 2 with PGRMC1 were induced upon progestin treatment. Knocking down each of the prohibitins slowed down the decidualization process compared to the control, suggesting that PGRMC1 cooperates with prohibitins to regulate 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 during decidualization may present a new perspective on infertility-related diseases.

Keywords: AG205; Decidualization; Endometrium; Infertility; Progesterone receptor membrane component 1 (PGRMC1); Prohibitin-1 (PHB1); Prohibitin-2 (PHB2); Rise-to-decline pattern; Telomerase-immortalized human endometrial stromal cells (T-HESCs).

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
PGRMC1 expression profile during menstrual cycle. Relative transcript scores of PGRMC1 expression in different stages of a regular menstrual cycle (GSE6364) (A) and (GSE4888) (B). Relative transcript scores of PGRMC1 expression level are shown as mean ± SEM. Statistical analysis was performed by two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig. 2
Fig. 2
A rise-to-decline expression pattern of PGRMC1 was revealed by in vitro decidualization. A Schematic representation of in vitro decidualization system. The cellular morphology changes of T-HESCs on day 0 and day 10 were imaged with microscopy in bright filed (B) or immunofluorescence staining (C). PGRMC1 was stained by Alexa Fluor-488 (green), and the nucleus was stained by DAPI in blue. Scale bar: 200 µm. The mRNA expression levels of PRL 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 (D). The dynamic changes of PGRMC1 protein expression from 1 to 14 days induction and non-induction control (on Day 14) were measured by western blot (E) and the bar plot with the relative densitometric analysis of the corresponding PGRMC1 protein level (p value calculation based on ‘D0’) (F). β-actin was used as a loading control. The mRNA expression levels of PGRMC1 in T-HESCs during decidualization (G). 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
Fig. 3
Fig. 3
The rise-to-decline changes of PGRMC1 are required for decidualization. (A): Schematic representation of in vitro decidualization system after PGRMC1 downregulation by siRNA. qRT-PCR analysis of PGRMC1 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 (B). The PRL mRNA expression level in T-HESCs after MPA/cAMP-induced decidualization upon transfection with 10 nM siPGRMC1 or siCTL, analyzed with qRT-PCR (C). The workflow for PGRMC1 downregulation after decidualization induction (D). mRNA expression levels of PGRMC1 (E, G) and PRL (F, H) in T-HESCs treated with MPA/cAMP for decidualization induction (red line) and non-induction (black line). Blue lines indicate the mRNA levels of PGRMC1 and PRL when transfected with 10 nM siPGRMC1 on the second (E, F) and fourth (G, H) day after decidualization induction, respectively. The statistical analysis of mRNA levels of PGRMC1 (and PRL) 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 < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig. 4
Fig. 4
Prei-nuclear PGRMC1-signal increased during decidualization. (A): Immunofluorescence staining of PGRMC1 in T-HESCs treated with DMSO (left) as control or MPA/cAMP (right) for 10 days decidualization induction. PGRMC1 shows in red and the nucleus was stained with DAPI in blue. Scale bar: 200 µm. (B) Analysis 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 (55 kDa), β-actin (47 kDa), and Histon H3 (17 kDa) were used as loading controls for the membrane, cytoplasmic, and nuclear fractions, respectively
Fig. 5
Fig. 5
PGRMC1 interacts with PHBs during decidualization. The interactions between PGRMC1 and PHB1 (A) or PHB2 (B) in T-HESCs were analyzed with proximity ligation assay upon decidualization induction from day 2 to day 10. ‘Day 0’ indicates the induction day. Each red spot represents a single interaction. Nuclear stain: DAPI. Magnification 40 ×
Fig. 6
Fig. 6
Downregulation of PHBs partly impairs decidualization. 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), respectively, was analyzed by western blot. Densitometric analysis was performed with imagej and values were normalized to β-actin. The PRL mRNA expression changes in T-HESCs with (red line) and without (black line) induction was determined by qPCR and normalized to HPRT1 as a reference gene (C). The PRL mRNA expression changes in T-HESCs transfected with 10 nM siPHB1 (D), 10 nM siPHB2 (E) upon decidualization induction were determined by qPCR. 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
Fig. 7
Fig. 7
AG205 does not affect decidualization. The 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 (A). The absorbance values for cultures with AG205 were compared to the DMSO control (0 µM). The PRL mRNA expression levels were analyzed after cells were cultured with (black line) or without (red line) 15 µM AG205 (B). Results are shown as the mean ± SEM from three independent biological replicates. Statistical analysis was performed by two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 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 (C). 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 (D). β-actin was used as a loading control. The interactions between PGRMC1 and PHB1 (E) or PHB2 (F) in T-HESCs were analyzed by proximity 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 ×
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