Purified pituitary FSH21/18 and FSH24 (17), equine FSH (eFSH) and dg-eLHt (37) were kindly supplied by Professor George Bousfield, Wichita State University, Wichita, KS, USA. CAGE 552 fluorophore dye was purchased from Abberior. N-terminally hemagglutinin-tagged FSHR (HA-FSHR) encoded plasmid DNA were constructed as previously described (32). Primary antibody HA.11 was purchased from BioLegend^®. Plasmid DNA encoding GloSensor™-20F, cAMP-response element-luciferase reporter gene (cre-luciferase), Renilla luciferase reporter gene (R-luciferase), GloSensor™ reagent stock and Dual-Luciferase^® Reporter Assay System were purchased from Promega.

HEK293 cells were maintained and cultured in 5% CO₂ in air at 37°C in Dulbecco′s Modified Eagle′s Medium (DMEM-6429, Sigma-Aldrich) supplemented with 10% Fetal Bovine Serum (FBS-F9665, Sigma-Aldrich), and 1% Antibiotic-Antimycotic (15240062, ThermoFisher). For PD-PALM experiments, cells were transiently transfected with 3 μg HA-FSHR, and then re-plated 24 hours later onto 8-well 1.5 glass-bottomed chamber slides. For cAMP GloSensor™ and cre-luciferase activity assays, HEK293 cells were co-transiently co-transfected with 3 μg of HA-FSHR and either 1 µg GloSensor™-20F DNA plasmid or 800 ng cre-luciferase and 150 ng R-luciferase DNA plasmids, and cells re-plated the following day onto white clear-bottomed 96-well plates (50,000 cells/well). All transient transfections were carried out in tissue culture-treated 6-well plates (600,000 cells/well) using Lipofectamine 2000^® (Invitrogen) as per manufacturer’s instructions. Cells were assayed 48 hours post-transfection.

To assess FSHR monomer, dimer and oligomer populations at the plasma membrane, PD-PALM experiments were performed as previously described (32, 34, 35). Briefly, HEK293 cells expressing HA-FSHR were pre-incubated for 30 minutes with CAGE 552-labeblled HA.11 antibody at 37°C, protected from light. HA.11 primary antibodies were labeled with CAGE 552 photoswitchable dyes at a 1:1 ratio, as previously described (34, 35) and following manufacturer’s protocols (Abberior). At the end of the pre-incubation, antibodies were removed, and cells were treated with 0 (control), or either 30 or 1 ng/ml of eFSH, FSH21/18, FSH24 or dg-eLHt for 0, 2-, 5- or 15 minutes. Cells were washed with PBS and fixed for 30 minutes with 0.2% glutaraldehyde in 4% PFA at room temperature. Following fixation, cells were subsequently washed, stored and imaged in PBS using an inverted Zeiss Elyra PS.1 microscope with a 100x 1.45 NA objective lens in TIRF-mode at a rate of 10 frames/second over a total of 31,500 frames.

Individual FSHR molecules were resolved as previously described (32, 34, 35). To summarize, cropped non-overlapping 5 x 5 μm areas, within cell boundaries, of fluorescent intensity image frames were analyzed using the QuickPALM Fiji plug-in, generating x-y coordinates of each FSHR molecule. The range of FSHR density basally observed at the cell surface was 10-80 FSHR/μm² across all experiments. For standardization of data analysis and to eliminate receptor density as a variable factor, cells with FSHR expression levels of ~10-40 FSHR/μm² were selected for analysis, as this was the physiological receptor density range previously reported for the FSHR (30) and other native GPCRs (38). To prevent the overestimation of FSHR association, coordinates localized within 15 nm of 15 consecutive frames were filtered using an add-on algorithm JAVA program. To determine the number of associated FSHRs and type of associated forms, from the localized and filtered x-y coordinates, a PD-interpreter JAVA program was employed to perform Getis-Franklin neighborhood analysis with a search radius of 50 nm. Reconstructed heat maps representing the different numbers of associated FSHRs were produced as a result.

Post-transfection, cells were pre-equilibrated for 2 hours at 37°C in 88% CO₂-independent media (18045, Gibco) supplemented with 10% FBS and 2% GloSensor™ cAMP reagent stock, as per manufacturer’s instructions. Following this, cells were treated for 30 minutes at 37°C with 0-100 ng/ml of eFSH, FSH21/18, FSH24 or dg-eLHt. Real-time cAMP florescence was measured using a multi-mode plate reader (PHERAstar^® FS, BMG Labtech) using the parameter of 100 flashes per well, with a cycle time of 36 seconds.

Post-transfection, cells were stimulated in serum-free DMEM supplemented and treated with 0-100 ng/ml eFSH, FSH21/18, FSH24 or dg-eLHt for 4-6 hours at 37°C. As an early-response gene, we expected this length of treatment to be sufficient for rapid gene expression induction in concordance with previous work (15). At the end of the incubation period, cells were lysed and treated with the Dual-Luciferase^® Reporter Assay System, as per manufacturer’s instructions. Lysate preps were measured for cre-luciferase and R-luciferase (for internal transfection control) luminescence levels using a multi-mode plate reader (PHERAstar^® FS, BMG Labtech).

For PD-PALM studies, to compare the effect of FSHR ligands on the percentage of total FSHR homomers, ordinary one-way ANOVA, followed by Tukey’s multiple comparisons test were conducted. To compare the effect of different FSHR ligands on the percentage of FSHR homomer subtypes, we performed multiple unpaired t-tests followed by Holm-Šídák’s multiple comparisons. For each experiment a total of 3 individual sections/well were imaged containing 3-4 cells. For each section typically 15 ROIs, within cell borders, were analyzed as previously described (34). For GloSensor™ assays, a baseline read of each well was performed for 10 read cycles prior to FSHR ligand treatment. The average baseline value of each cell was subtracted from its respective FSHR ligand-treatment, from the same wells. The mean cAMP response was plotted over 30 minutes and second order smoothened graph with 10 neighbors was performed. The area under the curve (AUC) measurements at 2-, 5- and 15-minutes were determined by measuring the total area from the number of peaks. All GloSensor™ data were represented as fold change/basal. Comparisons of the AUC between FSHR ligands were carried out using one-way ANOVA, followed by Tukey’s multiple comparisons test. Cre-luciferase luminescence readings were normalized to R-luciferase luminescence readings from the same well, to control for transfection efficiency. All data were represented as fold change/basal. Analysis of concentration-response curve were made using two-way ANOVA, followed by Dunnett’s multiple comparisons test. Comparisons between FSHR ligands at specific concentrations were performed using two-way ANOVA, followed by Dunnett’s multiple comparisons test. For PD-PALM, a minimum of 3 independent experiments were performed, for GloSensor™ and cre-luciferase assays, a minimum of 3 independent experiments in triplicate were conducted. All data presented represent the mean ± SEM. All statistical evaluations were performed using GraphPad Prism V9, and significance was determined as a probability value of p<0.05.

To determine the effects of differentially glycosylated FSHR ligands on cell surface FSHR oligomerization, we utilized the previously described single molecule imaging technique, PD-PALM, which afforded imaging of individual FSHR molecules to <10nm resolution (34). Two concentrations of FSH glycoforms were utilized, based on previous reports showing differential cAMP production evoked by FSH21/18 and FSH24, with ~50% of maximal cAMP production at 30 ng/ml in the concentration ranges assessed, and low-level cAMP production at 1 ng/ml (15).

HEK293 cells transiently expressing HA-FSHR were treated ± 30 ng/ml of FSH21/18 and FSH24 for 2-, 5- and 15 minutes. As a positive control, cells were also stimulated with the potent FSHR activator, eFSH, which is a naturally occurring analog of hypo-glycosylated FSH21/18. Conversely, as a negative control for cAMP activation, we utilized the FSHR β-arrestin biased agonist, dg-eLHt, which has been previously shown to display minimal cAMP production, with biased activation of β-arrestin (36, 37). Representative images (top panels) and heat maps depicting the number of associated molecules (bottom panels) were generated. Analysis of the basal number of associated FSHR showed that 30.2 ± 1.8% of FSHR were associated as dimers and oligomers ( Figure 1 ), with ~70% as FSHR monomers. Analysis of the basal composition of associated cell surface FSHR showed 15.5 ± 0.8% resided as dimers and 5.5 ± 0.5% as trimers ( Figure 1 ), suggesting that basally the majority of FSHR reside as lower-order homomers and monomers. Acute 2-minute treatment of HEK293 cells expressing FSHR with either eFSH or FSH21/18 significantly decreased the overall percentage of associated FSHR, with 20.0 ± 1.3% and 17.5 ± 1.6% associated as dimers and oligomers, respectively ( Figure 1Aii ). A decrease was observed in almost all FSHR homomeric subtypes (dimers, trimers, pentamers and 6-8 oligomers) ( Figure 1Aiii ). In contrast, treatment with FSH24 had no effect on the total percentage of associated FSHR, however modulation in the type of FSHR homomeric complexes was observed with a modest increase in ≥9 complexes and a decrease in dimers. Surprisingly, 2-minute treatment with dg-eLHt showed a trend for increasing FSHR association with 38.7 ± 3.8% of FSHR molecules associated ( Figure 1Aii ), with 15.9 ± 2.9% FSHR as trimers ( Figure 1Aiii ).

A 5-minute stimulation with eFSH treatment showed the percentage of FSHR associations to resemble basal ( Figure 1Bi ), suggesting a rapid re-organization of FSHR monomers into FSHR homomers. The documented fast binding kinetics of eFSH may explain this (17). FSH21/18, however, maintained a sustained reduction in FSHR homomers ( Figure 1Bi ). Interestingly, 5-minute treatment with FSH24 resulted in FSHR dissociation ( Figure 1Bi ), with a decrease in dimeric and trimeric FSHR homomers (p< 0.001) ( Figure 1Bii ). A 5-minute dg-eLHt treatment sustained the increase in FSHR association observed at 2 minute-treatment ( Figure 1Bi ), suggesting that different FSH ligands have distinct effects on FSHR oligomerization.

A more chronic 15-minute treatment with either eFSH, FSH21/18 or FSH24 resulted in FSHR total homomeric complex percentages resembling those of basal levels ( Figure 1Ci ). However, dg-eLHt-treated cells continued to show increased FSHR association (49.7 ± 6.4%) with increases observed in trimers, tetramers to ≥9 complexes ( Figure 1Cii ), further supporting the proposition that different FSH ligands can differentially modulate FSHR association.

Since FSH concentrations and glycosylation patterns are differentially regulated across the menstrual cycle (39) and have also been shown to change with age (13), we sought to determine the effects of FSH ligand concentration on FSHR association. As previously, HEK293 cells expressing FSHR were treated ± eFSH, FSH21/18, FSH24 or dg-eLHt for 2-, 5- or 15-minutes, but using 1 ng/ml of each. Representative reconstructed images of FSHR localizations and heat maps showing associated molecules following 2-minute treatment were generated ( Figure 2Ai ). Assessment of FSHR association following 2-minute treatment with all ligands revealed no significant changes in the total percentage of FSHR homomers ( Figure 2Aii ), nor the type of FSHR homomeric complexes observed ( Figure 2Aiii ), suggesting that lower concentrations of FSHR ligands had little effect on FSHR association at this acute time-point.

Similarly, 5-minute treatment with 1 ng/ml eFSH, FSH21/18 and dg-eLHt had no effect on FSHR association ( Figure 2Bi ). Interestingly, FSH24 induced a significant increase in FSHR association, with an increase in the formation of pentamers (6.1 ± 2.6%) ( Figure 2Bii ), contrasting to the dissociation of FSHR homomers observed with 30 ng/ml FSH24 shown above. Intriguingly, 15-minute treatment with FSH21/18 also induced FSHR association ( Figure 2Ci ), with an increase in FSHR tetramers (9.0 ± 1.8%) and ≥9 oligomers (18.2 ± 4.1%) ( Figure 2Cii ). FSH24-treated cells appeared to show FSHRs return to basal configuration ( Figure 2C ). Taken together, these data suggest that different FSHR ligands specify distinct re-organization of FSHR monomer, dimer, and oligomer populations in both a concentration- and time-dependent manner.

Ligand-dependent modulation of the related luteinizing hormone receptor homomers and LHR/FSHR and FSH/GPER heteromers has been shown to regulate signal amplitude and specificity (32–34) To investigate if the time- and concentration-dependent changes in FSHR monomers and homomers observed at the plasma membrane correlated with modulation in cAMP signals in our cell system, we employed the cAMP GloSensor™ reporter, which afforded real-time monitoring of cAMP production. HEK293 cells expressing FSHR were treated ± 0-100 ng/ml of eFSH, FSH21/18, FSH24 or dg-eLHt. Full cAMP concentration-response curves showing the AUC and maximal response of cAMP accumulation were recorded ( Supplementary Figure S1 ), and the 30- and 1 ng/ml data extrapolated for further analysis at the 2-, 5-, and 15-minute time points, for correlation with the time points utilized for PD-PALM experiments. The mean cAMP accumulated over 30 minutes following a 30 ng/ml treatment with all ligands were plotted ( Figure 3Ai ). A 2-minute treatment with either eFSH and FSH21/18 induced a significant increase in cAMP production of 8.6 ± 2.6- and 6.7 ± 0.8-fold change/basal, respectively ( Figure 3Aii ). There were no significant effects of either FSH24 or dg-eLHt, on cAMP production at this time point ( Figure 3Aii ). When compared and correlated with PD-PALM data, a trend was observed for 2-minute 30 ng/ml eFSH and FSH21/18 promoting dissociation of FSHR homomers into predominantly monomers ( Figures 1Aii, iii ), suggesting that dissociation of FSHR oligomers into monomers and re-organization of FSHR oligomeric complexes may, at least in part, promote acute cAMP production. Moreover, that no change or enhancement of FSHR oligomerization may facilitate low level production of cAMP.

Treatment for 5-minutes with FSH24 significantly increased cAMP ( Figure 3Aiii ). When compared to observations with PD-PALM data, a decrease in FSHR association at 5-minutes FSH24 treatment was observed ( Figure 1B ). This provided further support that FSHR dissociation into monomers may promote cAMP production. Differences in the magnitude of cAMP accumulation between eFSH, FSH21/18 and FSH24 were observed ( Supplementary Figure S1 ) and may result from the temporal differences in the kinetics of FSHR homomeric complex dissociation and profile of FSHR homomers favored by different FSHR ligands. We observed predominantly eFSH- and FSH21/18-dependent pentamer dissociation with acute treatment ( Figures 1Aiii, Bii ) compared to FSH24-dependent FSHR dimer and trimer dissociation ( Figure 1Bii ). The dg-eLHt preparation failed to significantly stimulate cAMP production ( Figure 3Aii ), as compared to PD-PALM data, which showed increased FSHR oligomerization ( Figure 1 ).

A 15-minute stimulation with either eFSH, FSH21/18 or FSH24 continued to significantly increase cAMP production ( Figure 3Aii ). However, from the maximal cAMP concentration-response curves ( Supplementary Figure S1Cii ), steady-state levels appeared to have been reached. Interestingly, at this time point, FSHR homomer arrangements predominantly resembled basal conditions in all treatment groups ( Figure 1C ), suggesting that this receptor configuration may be important in initiating FSHR signal activation, with other mechanisms such as receptor internalization important in maintaining cAMP production thereafter. As anticipated, dg-eLHt was unable to induce significant cAMP production at any time point analyzed ( Figure 3Aii ). PD-PALM data at the corresponding time point showing preferential re-arrangement of FSHR into higher order oligomers ( Figure 1 ), suggesting that low level cAMP production (and potential β-arrestin recruitment and subsequent signaling) may be mediated, at least in part, by FSHR oligomer formation.

Next, we determined the effects of lower FSHR ligand concentrations that differentially modulated FSHR homomerization, on cAMP production. As with our previous PD-PALM experiments, we utilized 1 ng/ml of eFSH, FSH21/18, FSH24 or dg-eLHt and measured the mean cAMP accumulated over 30 minutes ( Figure 3Bi ). An acute, 2-minute treatment with all ligands, except eFSH, showed minimal increases in cAMP production in comparison to basal ( Figure 3Bii ). When compared to the PD-PALM data ( Figure 2 ), these data correlated with a lack of effect on FSHR oligomerization at 2 minutes, following 1 ng/ml treatment with any FSHR ligand ( Figures 2Aii, iii ).

At 5- and 15 minutes, although we observed an eFSH-dependent increase in cAMP production of 5.8 ± 1.1- and 8.1 ± 1.1-fold, respectively ( Figure 3Bii ), when correlated to the PD-PALM data at these time points, no changes in the total percentage of FSHR homomers at the plasma membrane were observed ( Figure 2 ). This suggests that there may be a dose-dependent threshold for different FSH glycosylated ligands to modulate FSHR homomerization. Small changes were observed in FSHR homomer subtypes, which may be important for modulating the magnitude of cAMP signaling, however this remains to be demonstrated. In contrast, FSH24 treatment at 5- and 15-minutes had no significant effect on cAMP production ( Figure 3Bii ). Interestingly, when correlating PD-PALM analysis, an increase in FSHR oligomerization was observed, predominately from enhanced formation of pentamers ( Figure 2Bii ), which may indicate that low level cAMP production may favor FSHR association. Supporting this observation, we observed increases in the total percentage of FSHR homomers with FSH21/18 treatment at 15 minutes ( Figure 2Ci ), correlating with low level cAMP production at the same time ( Figure 3Bii ). As anticipated, no significant changes in cAMP were observed following 2-, 5-, or 15-minute treatment with dg-eLHt ( Figure 3Bii ).

As the principal pathway activated by FSH/FSHR is Gαs/cAMP/PKA, which is physiologically important for regulating the expression of cre-response genes, including CYP19 essential for estradiol production (16, 19, 40–42), we went on to determine the effects of FSHR ligands on cre-luciferase reporter gene activity. HEK293 cells transiently expressing the HA-FSHR were co-transfected with cre-luciferase and R-luciferase (transfection efficiency control) and treated with ± 0-100 ng/ml of eFSH, FSH21/18, FSH24 and dg-eLHt for 4-6 hours. In line with the GloSensor™ data, eFSH and FSH21/18 were most potent at activating cre-luciferase for all concentrations ≥1 ng/ml when compared to basal in contrast to FSH24-treated cells ( Figure 4A ). As anticipated, dg-eLHt was unable to induce any changes in cre-luciferase activity at lower concentrations. However, at the higher concentrations of 30- and 100 ng/ml, dg-eLHt appeared to act as a weak agonist at the FSHR ( Figure 4A ), in corroboration with previous reports (36).

Comparison of 30 ng/ml treatments with FSHR ligands showed ligand-dependent differences in cre-luciferase activation, with eFSH and FSH21/18 significantly stimulating cre-luciferase activity by 19.3 ± 2.6- and 17.8 ± 0.7-fold increase over basal, respectively, in comparison to an 11.8 ± 0.8-fold increase for FSH24-treated cells (p<0.0001) ( Figure 4B ). This reflects similar differences observed between eFSH, FSH21/18 and FSH24 in 30 ng/ml GloSensor™ cAMP data ( Figure 3Aii ). Furthermore, when compared to FSH ligands eliciting changes in FSH homomerization, it suggests that the changes observed in FSHR complexes at the plasma membrane may contribute to modulating the magnitude of cre-responsive gene activation.

Comparison of cre-luciferase responses following 1 ng/ml treatment with eFSH, FSH21/18, FSH24 and dg-eLHt revealed both eFSH and FSH21/18 induced 7-fold increases in cre-luciferase activation (p<0.001) ( Figure 4B ). Additionally, FSH24 induced a comparable increase (6.1 ± 0.2-fold over basal) in cre-luciferase activity. This was interesting as differential regulation of FSHR homomeric forms and cAMP production was observed at this concentration. As predicted, dg-eLHt failed to significantly induce any increase in cre-luciferase activity ( Figure 4B ), further supporting its β-arrestin biased agonist activity.