Membrane Estrogen Receptor (GPER) and Follicle-Stimulating Hormone Receptor (FSHR) Heteromeric Complexes Promote Human Ovarian Follicle Survival

Abstract

Classically, follicle-stimulating hormone receptor (FSHR)-driven cAMP-mediated signaling boosts human ovarian follicle growth and oocyte maturation. However, contradicting in vitro data suggest a different view on physiological significance of FSHR-mediated cAMP signaling. We found that the G-protein-coupled estrogen receptor (GPER) heteromerizes with FSHR, reprogramming cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with preferential Gαs protein/cAMP-pathway coupling and FSH responsiveness of patients undergoing controlled ovarian stimulation. In contrast, FSHR/GPER heteromers triggered anti-apoptotic/proliferative FSH signaling delivered via the Gβγ dimer, whereas impairment of heteromer formation or GPER knockdown enhanced the FSH-dependent cell death and steroidogenesis. Therefore, our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation.

Keywords: Endocrine Regulation; Female Reproductive Endocrinology; Molecular Biology.

Conflict of interest statement

ML and GO are Merck Serono SpA employees without any conflict of interest.

© 2020 The Author(s).

Figures

Graphical abstract

Figure 1

Excessive FSHR Expression Levels Negatively Impact HEK293 Cell Viability (A) Association of FSHR to the Gαs protein increases with higher receptor expression levels was assessed in HEK293 cells co-expressing the FSHR/rluc- (donor) and the Gαs protein/venus-tagged BRET biosensor. Data were interpolated by non-linear regression and compared by Kruskal Wallis test and Dunn's post-test (100 and 200 ng/well of FSHR-encoding plasmid versus the 25 ng/well condition; p = 0.0014; mean ± SEM; n = 5). (B–E) Comparison of constitutive FSHR coupling to Gαs, Gαq, and Gαi with increasing receptor expression levels, in HEK293 cells transfected using 25 (B), 50 (C), 100 (D), and 200 (E) ng/well of FSHR/rluc-encoding plasmid. Receptor level-dependent increase in affinity occurs with Gαs but not with Gαq and Gαi proteins (Kruskal Wallis test and Dunn's post-test; p 2 co-treatment inhibits the decrease in cell viability induced by the cAMP analog (∗ = significantly different versus 0.0 8-br-cAMP-treated samples; Kruskal Wallis test and Dunn's post-test; p < 0.05; n = 8). (J) Cell viability is lower in HEK293/FSHR transfected cells stimulated with FSH compared with vehicle (Mann-Whitney's U-test; p = 0.0003; n = 8). (K) Summary: FSHR coupling to the Gαs protein/cAMP-pathway, occurring at high receptor expression levels and in the absence of E2, decreases cell viability. This action is counteracted by estrogen.

Figure 2

The FSHR Forms Heteromers with GPER (A) Predicted structural model of the heterodimer between FSHR (green) and GPER (violet) seen in directions perpendicular (left) and parallel (right) to the bundle main axis. In this dimer, H6 of FSHR interacts with H7 of GPER and H6 of GPER interacts with H7 of FSHR. (B) Western blotting for FSHR and GPER transient expression and co-expression in HEK293 cells, using validated receptor-specific antibodies (Figure S6). β-ACTIN was used as loading control. (C–E) Representative confocal microscopy image of tagged-GPER and FSHR co-localization by immunofluorescence, in HEK293 cells transiently transfected with FSHR and GPER. A specific primary antibody was used for GPER followed by a TRITC-labelled secondary antibody, whereas nuclei were blue-stained by DAPI. FSHR was visualized by the venus tag. Bar = 25 μm. (F) Formation of FSHR/rluc- and GPER/venus-tagged heteromers in transfected HEK293 cells. BRET ratio values resulting from molecular interactions were represented as mean ± SEM. Specific association is indicated by data interpolation using non-linear regression, which results in BRET saturation curve (n = 4). (G and H) FSHR-GPER associations at the single-molecule level were visualized and quantitated by photo-activated localization microscopy with photo-activatable dyes (PD-PALM) in HEK293 cells. (I) Representative reconstructed PD-PALM images of detected FLAG-GPER and HA-FSHR molecule at the plasma membrane. Images are reconstructed from 2-2 μm2 areas after x-y coordinate localization using QuickPALM followed by a 50 nm radius neighborhood analysis of receptor molecules. Scale bar represents 0.3 μm. (J) Quantitative analysis of hetero-, homo-, and monomeric forms of FSHR and GPER when concomitantly expressed in HEK 293, using dual channel PD-PALM; mean ± SEM, n = 5. (K) Quantitative evaluation of the types of FSHR and GPER homomers. Data are expressed as percentage of total receptor forms, including monomers; mean ± SEM, n = 10. (L) Quantitative analysis of heteromeric assemblies between FSHR and GPER using dual channel PD-PALM reveals diverse heterodimeric complexes; mean ± SEM, n = 8 cells. (M) Analysis of individual protomer composition within heterotrimers and heterotetramers demonstrates the preferential occurrence of FSHR protomers versus GPER protomers within these individual multimers; mean ± SEM, n = 8.

Figure 3

FSHR-GPER Crosstalk Promotes FSH-Stimulated Cell Viability via Gβγ Dimers (A) GPER transiently expressed in HEK 293 cells simulates E2-induced intracellular Ca2+ increase (Kruskal-Wallis test; p < 0.0001; n = 8; mean ± SEM). Signals were captured by BRET over 150 s, in the presence of the calcium-biosensor. PBS (vehicle)-treated cells provided basal levels. Cells lysed by Triton X- were positive controls and only a 43-s time window is representatively shown. Compounds were added at the 21-s time point. (B) GPER/rluc-coupling to the Gαs protein/venus-tagged was demonstrated by BRET. Values are mean ± SEM, and the logarithmic curve was obtained after interpolation using non-linear regression (n = 8). (C) 10-nM FSH induced cAMP increase in HEK293 cells expressing either one or both FSHR and GPER. 50 pg/ml E2 were added as indicated, whereas mock-transfected and forskolin-treated cells served as basal and positive control, respectively. cAMP values are indicated as induced BRET changes over vehicle-treated mocks (∗ = significantly different versus FSH-treated mocks; two-way ANOVA with Dunnett's correction for multiple tests; p 3 ng/well FSHR-encoding ± 1 × 103 ng/well GPER-encoding plasmid; ∗ = different versus mock; Kruskal-Wallis test; p < 0.0001; n = 8; mean ± SEM).

Figure 4

Decrease of Cell Viability by Disruption of FSHR-GPER Heteromers (A) Side view, in a direction perpendicular to the bundle main axis, of the GPER structural model. The receptor regions are colored as follows: H1, H2, H3, H4, H5, H6, H7, and H8 are, respectively, blue, orange, green, pink, yellow, aquamarine, violet, and red; I1 and E1 are slate; I2 and E2 are gray; and I3 and E3 are magenta. The receptor amino acids participating in the interface with FSHR and subjected to alanine replacement are represented as spheres centered on the Cα-atom. They include Q255, R259, L262, L266, V267, V270, and V277 in H6, L304, T305, I308, L319, I323, and F326, and L327 in H7, and E329 in H8. To obtain a GPER(mut) molecule unable to form heteromers, interacting residues indicated in the box were changed to alanine by de novo DNA synthesis. (B) Demonstration of GPER(mut) functionality by BRET using the calcium-biosensor, in transiently transfected HEK293 cells. The mutant receptor mediates E2-induced intracellular Ca2+ increase compared with vehicle, over 150 s (two-way ANOVA; p < 0.0001; n = 8; mean ± SEM). Compounds were injected at the 21-s time point. (C) FSHR/rluc- and GPER(mut)/venus-tagged proteins do not form heteromers. BRET ratio values resulting from molecular interactions are represented as mean ± SEM, together with data from non-mutant GPER (Figure 2F). Specific association is indicated by data interpolation using linear regression (n = 4). (D–I) Confirmation of GPER(mut) membrane localization and lack of heteromerization with FSHR. (D–F) Representative reconstructed PD-PALM images and heatmap of associations following localization and neighborhood analysis. Scale bar = 0.3 μm (G) Similar number of GPER and GPER(mut) receptors were expressed at the cell membrane (p = 0.06; Mann-Whitney's U-test). (H) Quantitative analysis of hetero-, homo-, and monomeric forms of GPER(mut) when co-expressed with FSHR in HEK293, using dual channel PD-PALM (∗ = significantly different versus monomers; unpaired t test; p U-test; p = 0.0003; n = 8; means ± SEM). (L) Proposed model showing the inability of GPER(mut) to inhibit the activation of FSH-stimulated cAMP production with ensuing decrease of cell viability due to disruption of heteromerization.

Figure 5

Cell Viability Decreases upon Disrupting the GPER-Associated MAGUK/AKAP5 Molecular Inhibitory Complex (A) Model depicting the development of the AKAP5-KO HEK293 cell line by CRISPR/Cas9. The absence of AKAP5 leads to disruption of the GPER-associated inhibitory machinery without impairing FSHR-GPER heteromer formation. (B) FSHR/rluc-GPER/venus-tagged heteromer formation evaluated by BRET, in AKAP5-KO HEK293 cells. Values are expressed as mean ± SEM and interpolated by non-linear regression (n = 4). (C) Intracellular cAMP increase following 10 nM FSH-treatment of AKAP5-KO HEK293 cells, transiently expressing FSHR and/or GPER. Fifty pg/ml E2 were added where indicated and signals acquired by BRET. ∗ = significantly different versus FSH-treated mocks; two-way ANOVA with Tukey's post-hoc test; p < 0.0001; n = 6; means ± SEM. (D) Viability of FSHR- and/or GPER-expressing AKAP5-KO HEK293 cells in the presence and in the absence of 10 nM FSH (∗ = different versus mock; two-way ANOVA with Dunnett's post-hoc test; p 

Figure 6

Link Between FSHR-GPER Co-expression and Follicular Growth Response to FSH (A and B) Representative determination of FSHR and GPER expression in granulosa cells at the antral follicular stage, by immunohistochemistry. Ovarian sections were treated by specific anti-FSHR or -GPER primary antibodies, over hematoxylin background staining (bar = 200 μm). (C) Western blotting demonstrating the presence of both FSHR and GPER in primary human granulosa cell lysates and the inhibition of expression of GPER by 48-h siRNA. HEK293 transiently transfected with FSHR- and GPER-encoding plasmids were used as controls. The efficacy of GPER siRNA is shown and compared with control siRNA (mock)-treated granulosa cells. β-ACTIN was used as a loading control. (D) Correlation between FSHR and GPER gene expression levels in granulosa cells collected from donor normo- (n = 61) and poor-responder (n = 30) women undergoing FSH stimulation for assisted reproduction. Each patient is represented by a point, and mRNA levels were measured by real-time PCR, normalized over the RPS7 housekeeping gene and interpolated by linear regression. Immunohistochemistry and uncropped western blotting for antibody validation are provided as supplemental materials (Figure S6). (E–J) Representative images of GPER and FSHR co-localization in 48-h mock- (E-G) and GPER siRNA-treated (H-J) human granulosa cells, detected by immunofluorescence. Specific primary antibody was used for FSHR and GPER binding, as well as TRITC- (E, H) and FITC-labelled (F, I) secondary antibodies, respectively. Nuclei (blue) were stained by DAPI (G, J). Bar = 25 μm. (K) Intracellular cAMP levels measured in control and GPER siRNA-treated granulosa cells by ELISA, in the presence or absence of 10 nM FSH. Data are represented by box and whiskers plots (∗ = different versus vehicle/mock-treated cells; two-way ANOVA and Sidak's multiple comparisons test; p ≤ 0.0074; n = 8). (L) Granulosa cell viability after 48-h treatment with control/GPER siRNA. Effects of 10-nM FSH were also assessed 24 h before measurements (∗ = different versus vehicle/mock-treated cells; Kruskal-Wallis with Dunn's correction for multiple tests; p = 0.0002; n = 12). (M) Evaluation of procaspase 3 cleavage in granulosa cells under 48-h GPER depletion by siRNA. 10-nM FSH was added 24 h before analysis, as indicated, while total ERK was the loading control. (N) Progesterone levels measured in media of control and GPER siRNA-treated granulosa cells, maintained 24 h in the presence or in the absence of 10 nM FSH, by immunoassay. (∗ = different versus vehicle/mock-treated cells; two-way ANOVA and Fisher's test; p ≤ 0.001; n = 6). (O) Model describing the FSHR/Gαs protein-dependent activation of the steroidogenic/apoptotic pathway, under GPER depletion by siRNA. (P) Correlation between oocyte number and the ratio between E2 serum levels and cumulative FSH dose of normo- (n = 61) and poor-responder (n = 30) women. Patients are represented by points, and data were interpolated using linear regression.