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

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 Gßγ 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.

Two human menopausal gonadotrophin (hMG) preparations display different early signaling in vitro.

Commercial hMG drugs are marketed for the treatment of infertility and consist of highly purified hormones acting on receptors expressed in target gonadal cells. Menopur® and Meriofert® are combined preparation of FSH and hCG and are compared in vitro herein. To this purpose, the molecular composition of the two drugs was analyzed by immunoassay. The formation of FSH receptor and LH/hCG receptor (FSHR; LHCGR) heteromer, intracellular Ca2+ and cAMP activation, ß-arrestin 2 recruitment and the synthesis of progesterone and estradiol were evaluated in transfected HEK293 and human primary granulosa lutein cells treated by drugs administered within the pg-mg/ml concentration range. Molecular characterization revealed that Meriofert® has a higher FSH:hCG ratio than Menopur® which, in turn, displays the presence of LH molecules. While both drugs induced similar FSHR-LHCGR heteromeric formations and intracellular Ca2+ increase, Meriofert® had a higher potency than Menopur® in inducing a cAMP increase. Moreover, Meriofert® revealed a higher potency than Menopur® in recruiting ß-arrestin 2, likely due to different FSH content modulating the tridimensional structure of FSHR-LHCGR-ß-arrestin 2 complexes, as evidenced by a decrease in bioluminescence resonance energy transfer signal. This drug-specific activation of intracellular signaling pathways is consistent with the molecular composition of these preparations and impacts downstream progesterone and estradiol production, with Menopur® more potent than Meriofert® in inducing the synthesis of both the steroids. These findings are suggestive of distinct in-vivo activities of these preparations, but require cautious interpretation and further validation from clinical studies.

Enhanced expression of the sphingosine-1-phosphate-receptor-3 causes acute myelogenous leukemia in mice.

Acute myeloid leukemia (AML) carries a 10-100 fold lower mutational burden than other neoplastic entities. Mechanistic explanations for why a low number of mutations suffice to induce leukemogenesis are therefore required. Here we demonstrate that transgenic overexpression of the wild type sphingosine-1-phosphate receptor 3 (S1P3) in murine hematopoietic stem cells is sufficient to induce a transplantable myeloid leukemia. In contrast, S1P3 expression in more mature compartments does not cause malignant transformation. Treatment with the sphingosine phosphate receptor modulator Fingolimod, which prevents receptor signaling, normalized peripheral blood cell counts and reduced spleen sizes in S1P3 expressing mice. Gene expression analyses in AML patients revealed elevated S1P3 expression specifically in two molecular subclasses. Our data suggest a previously unrecognized contribution of wild type S1P3 signaling to leukemogenesis that warrants the exploration of S1P3 antagonists in preclinical AML models.

GnRH Antagonists Produce Differential Modulation of the Signaling Pathways Mediated by GnRH Receptors.

Commercial gonadotropin-releasing hormone (GnRH) antagonists differ by 1-2 amino acids and are used to inhibit gonadotropin production during assisted reproduction technologies (ART). In this study, potencies of three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in inhibiting GnRH-mediated intracellular signaling, were compared in vitro. GnRH receptor (GnRHR)-transfected HEK293 and neuroblastoma-derived SH-SY5Y cell lines, as well as mouse pituitary LßT2 cells endogenously expressing the murine GnRHR, were treated with GnRH in the presence or absence of the antagonist. We evaluated intracellular calcium (Ca2+) and cAMP increases, cAMP-responsive element binding-protein (CREB) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation, ß-catenin activation and mouse luteinizing-hormone ß-encoding gene (Lhb) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca2+ rapid increase revealed dose-response accumulation with potency (EC50) of 23 nM in transfected HEK293 cells, transfected SH-SY5Y and LßT2 cells. Cetrorelix inhibited the 3 × EC50 GnRH-activated calcium signaling at concentrations of 1 nM-1 µM, demonstrating higher potency than Ganirelix and Teverelix, whose inhibitory doses fell within the 100 nM-1 µM range in both transfected HEK293 and SH-SY5Y cells in vitro. In transfected SH-SY5Y, Cetrorelix was also significantly more potent than other antagonists in reducing GnRH-mediated cAMP accumulation. All antagonists inhibited pERK1/2 and pCREB activation at similar doses, in LßT2 and transfected HEK293 cells treated with 100 nM GnRH. Although immunostainings suggested that Teverelix could be less effective than Cetrorelix and Ganirelix in inhibiting 1 µM GnRH-induced ß-catenin activation, Lhb gene expression increase occurring upon LßT2 cell treatment by 1 µM GnRH was similarly inhibited by all antagonists. To conclude, this study has demonstrated Cetrorelix-, Ganirelix- and Teverelix-specific biased effects at the intracellular level, not affecting the efficacy of antagonists in inhibiting Lhb gene transcription.

Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars.

Recombinant follicle-stimulating hormone (FSH) (follitropin alfa) and biosimilar preparations are available for clinical use. They have specific FSH activity and a unique glycosylation profile dependent on source cells. The aim of the study is to compare the originator (reference) follitropin alfa (Gonal-f®)- with biosimilar preparations (Bemfola® and Ovaleap®)-induced cellular responses in vitro. Gonadotropin N-glycosylation profiles were analyzed by ELISA lectin assay, revealing preparation specific-patterns of glycan species (Kruskal-Wallis test; p < 0.05, n = 6) and by glycotope mapping. Increasing concentrations of Gonal-f® or biosimilar (1 × 10-3-1 × 103 ng/ml) were used for treating human primary granulosa lutein cells (hGLC) and FSH receptor (FSHR)-transfected HEK293 cells in vitro. Intracellular cAMP production, Ca2+ increase and ß-arrestin 2 recruitment were evaluated by BRET, CREB, and ERK1/2 phosphorylation by Western blotting. 12-h gene expression, and 8- and 24-h progesterone and estradiol synthesis were measured by real-time PCR and immunoassay, respectively. We found preparation-specific glycosylation patterns by lectin assay (Kruskal-Wallis test; p < 0.001; n = 6), and similar cAMP production and ß-arrestin 2 recruitment in FSHR-transfected HEK293 cells (cAMP EC50 range = 12 ± 0.9-24 ± 1.7 ng/ml; ß-arrestin 2 EC50 range = 140 ± 14.1-313 ± 18.7 ng/ml; Kruskal-Wallis test; p ≥ 0.05; n = 4). Kinetics analysis revealed that intracellular Ca2+ increased upon cell treatment by 4 µg/ml Gonal-f®, while equal concentrations of biosimilars failed to induced a response (Kruskal-Wallis test; p < 0.05; n = 3). All preparations induced both 8 and 24 h-progesterone and estradiol synthesis in hGLC, while no different EC50s were demonstrated (Kruskal-Wallis test; p > 0.05; n = 5). Apart from preparation-specific intracellular Ca2+ increases achieved at supra-physiological hormone doses, all compounds induced similar intracellular responses and steroidogenesis, reflecting similar bioactivity, and overall structural homogeneity.

Inferring biallelism of two FSH receptor mutations associated with spontaneous ovarian hyperstimulation syndrome by evaluating FSH, LH and HCG cross-activity.

RESEARCH QUESTION: What is the cumulative effect of two follicle-stimulating hormone receptor (FSHR) mutations in spontaneous ovarian hyperstimulation syndrome (sOHSS) pathogenesis? Are these mutations in the mono- or biallelic state? DESIGN: Two FSHR mutations were found in a pregnant patient affected by sOHSS with no predisposing conditions. While the p.Asn106His mutation is novel, the p.Ser128Tyr mutation has been associated with sOHSS previously. The patient's FSHR gene was analysed by Sanger sequencing, and FSHR cDNAs carrying a single or both point mutations were created by mutagenesis in vitro. cAMP activation by recombinant FSH, luteinizing hormone (LH), human chorionic gonadotropin (HCG) and thyroid-stimulating hormone (TSH) was evaluated in transfected HEK293 cells by bioluminescence resonance energy transfer. RESULTS: All mutations decreased the 50% effective concentration of FSH calculated for cAMP (P < 0.05, n = 6), resulting in two- to 10-fold lower ligand potency. TSH failed to induce an FSHR-mediated increase in intracellular cAMP, while LH was approximately four-fold more potent than HCG in p.Ser128Tyr FSHR-expressing HEK293 cells despite lower cAMP plateau levels (P < 0.05, n = 5). The p.Ser128Tyr FSHR mutation was found to be responsible for an LH-/HCG-induced increase in cAMP when it was in the biallelic heterozygous state with p.Asn106His, but no increase in cAMP was induced in the monoallelic state. CONCLUSION: In-vitro data support that, in pregnant patients with sOHSS, the two FSHR mutations have an opposing effect on the pathogenesis of sOHSS and are in the biallelic heterozygous form, allowing HCG to induce a p.Ser128Tyr FSHR-mediated increase in cAMP.

Abacavir, nevirapine, and ritonavir modulate intracellular calcium levels without affecting GHRH-mediated growth hormone secretion in somatotropic cells in vitro.

Growth Hormone (GH) deficiency is frequent in HIV-infected patients treated with antiretroviral therapy. We treated GH3 cells with antiretrovirals (nevirapine, ritonavir or abacavir sulfate; 100 pM-1 mM range), after transfection with human growth hormone releasing hormone (GHRH) receptor cDNA. Cells viability, intracellular cAMP, phosphorylation of CREB and calcium increase, GH production and secretion were evaluated both in basal condition and after GHRH, using MTT, bioluminescence resonance energy transfer, western blotting and ELISA. Antiretroviral treatment did not affect GHRH 50% effective dose (EC50) calculated for 30-min intracellular cAMP increase (Mann-Whitney's U test; p ≥ 0.05; n = 4) nor 15-min CREB phosphorylation. The kinetics of GHRH-mediated, rapid intracellular calcium increase was perturbed by pre-incubation with drugs, while GHRH failed to induce the ion increase in ritonavir pre-treated cells (ANOVA; p < 0.05; n = 3). Antiretrovirals did not impact 24-h intracellular and extracellular GH levels (ANOVA; p ≥ 0.05; n = 3). We demonstrated the association between antiretrovirals and intracellular calcium increase, without consequences on somatotrope cells viability and GH synthesis. Overall, these results suggest that antiretrovirals may not directly impact on GH axis in HIV-infected patients.

Probing the Effect of Sildenafil on Progesterone and Testosterone Production by an Intracellular FRET/BRET Combined Approach.

Forster resonance energy transfer (FRET)-based biosensors have been recently applied to the study of biological pathways. In this study, a new biosensor was validated for the first time in live HEK293 and steroidogenic MLTC-1 cell lines for studying the effect of the PDE5 inhibitor on the hCG/LH-induced steroidogenic pathway. The sensor improves FRET between a donor (D), the fluorescein-like diarsenical probe that can covalently bind a tetracysteine motif fused to the PDE5 catalytic domain, and an acceptor (A), the rhodamine probe conjugated to the pseudosubstrate cGMPS. Affinity constant ( Kd) values of 5.6 ± 3.2 and 13.7 ± 0.8 µM were obtained with HEK293 and MLTC-1 cells, respectively. The detection was based on the competitive displacement of the cGMPS-rhodamine conjugate by sildenafil; the Ki values were 3.6 ± 0.3 nM (IC50 = 2.3 nM) in HEK293 cells and 10 ± 1.0 nM (IC50 = 3.9 nM) in MLTC-1 cells. The monitoring of both cAMP and cGMP by bioluminescence resonance energy transfer allowed the exploitation of the effects of PDE5i on steroidogenesis, indicating that sildenafil enhanced the gonadotropin-induced progesterone-to-testosterone conversion in a cAMP-independent manner.

The cAMP/PKA pathway: steroidogenesis of the antral follicular stage.

Pituitary gonadotropins, follicle-stimulating (FSH) and luteinizing hormone (LH) promote follicular recruitment and support antral follicle growth, maturation and selection, resulting in ovulation of the dominant follicle. FSH and LH biological functions are mediated by G protein-coupled receptors, FSHR and LHCGR, resulting in the activation of a number of signaling cascades, such as the cyclic AMP/protein kinase A (cAMP/PKA) pathway. Some in-vitro data are consistent with the dual, proliferative and pro-apoptotic role of cAMP, leaving unanswered questions on how cAMP/PKA signaling is linked to the follicle fate. Progression of the antral stage is characterized by the presence of dynamic serum gonadotropin and estrogen levels, accompanying proliferation and steroidogenesis of growing as well as apoptosis of atretic follicles. These events are parallel to changes of FSHR and LHCGR density at the cell surface occurring throughout the antral stage, reasonably modulating the cAMP/PKA activation pattern, cell metabolism and functions. Understanding whether gonadotropins and receptor expression levels impact on the steroidogenic pathway and play a role in determining the follicular fate, may put new light on molecular mechanisms regulating human reproduction. The aim of the present review is to update the role of major players modulating the cAMP/PKA pathway and regulating the balance between proliferative, differentiating and pro-apoptotic signals.

Heterogeneous hCG and hMG commercial preparations result in different intracellular signalling but induce a similar long-term progesterone response in vitro.

STUDY QUESTION: Are four urinary hCG/menotropin (hMG) and one recombinant preparation characterized by different molecular features and do they mediate specific intracellular signaling and steroidogenesis? SUMMARY ANSWER: hCG and hMG preparations have heterogeneous compositions and mediate preparation-specific cell signaling and early steroidogenesis, although similar progesterone plateau levels are achieved in 24 h-treated human primary granulosa cells in vitro. WHAT IS KNOWN ALREADY: hCG is the pregnancy hormone marketed as a drug for ARTs to induce final oocyte maturation and ovulation, and to support FSH action. Several hCG formulations are commercially available, differing in source, purification methods and biochemical composition. STUDY DESIGN, SIZE, DURATION: Commercial hCG preparations for ART or research purposes were compared in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: The different preparations were quantified by immunoassay with calibration against the hCG standard (Fifth IS; NIBSC 07/364). Immunoreactivity patterns, isoelectric points and oligosaccharide contents of hCGs were evaluated using reducing and non-reducing Western blotting, capillary isoelectric-focusing immunoassay and lectin-ELISA, respectively. Functional studies were performed in order to evaluate intracellular and total cAMP, progesterone production and ß-arrestin 2 recruitment by ELISA and BRET, in both human primary granulosa lutein cells (hGLC) and luteinizing hormone (LH)/hCG receptor (LHCGR)-transfected HEK293 cells, stimulated by increasing hormone concentrations. Statistical analysis was performed using two-way ANOVA and Bonferroni post-test or Mann-Whitney's U-test as appropriate. MAIN RESULTS AND THE ROLE OF CHANCE: Heterogeneous profiles were found among preparations, revealing specific molecular weight patterns (20-75 KDa range), isoelectric points (4.0-9.0 pI range) and lectin binding (P < 0.05; n = 7-10). These drug-specific compositions were linked to different potencies on cAMP production (EC50 1.0-400.0 ng/ml range) and ß-arrestin 2 recruitment (EC50 0.03-2.0 µg/ml) in hGLC and transfected HEK293 cells (P < 0.05; n = 3-5). In hGLC, these differences were reflected by preparation-specific 8-h progesterone production although similar plateau levels of progesterone were acheived by 24-h treatment (P ≥ 0.05; n = 3). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The biological activity of commercial hCG/hMG preparations is provided in International Units (IU) by in-vivo bioassay and calibration against an International Standard, although it is an unsuitable unit of measure for in-vitro studies. The re-calibration against recombinant hCG,quantified in grams, is based on the assumption that all of the isoforms and glycosylation variants have similar immunoreactivity. WIDER IMPLICATIONS OF THE FINDINGS: hCG/hMG preparation-specific cell responses in vitro may be proposed to ART patients affected by peculiar ovarian response, such as that caused by polycystic ovary syndrome. Otherwise, all the preparations available for ART may provide a similar clinical outcome in healthy women. STUDY FUNDING AND COMPETING INTEREST(S): This study was supported by a grant of the Italian Ministry of Education, University and Research (PRIN 2015XCR88M). The authors have no conflict of interest.

Genetics of gonadotropins and their receptors as markers of ovarian reserve and response in controlled ovarian stimulation.

Several controlled ovarian stimulation (COS) protocols have been developed to increase the yield of mature oocytes retrieved in assisted reproductive techniques (ARTs). The ovarian reserve (OR) influences the COS response, and it represents the main parameter that helps clinicians in refining clinical treatments in the perspective of a "personalized" ART. This approach is even more needed in particular conditions such as poor OR or polycystic ovary syndrome. Follicle-stimulating hormone, luteinizing hormone, and human chorionic gonadotropin are currently used in COS at different combinations and with different efficacies, even if the best approach definition is controversial. Differences in individual-specific ovarian response to gonadotropin stimulation can be due to alterations of genes encoding for hormones or their receptors. In particular, FSHB c.-211G>T, FSHR p.Asn680Ser, and c.-29G>A SNP allelic combinations may be used as OR and COS response markers. The purpose of this review is to highlight the evidence-based relevance of mutations and polymorphisms in gonadotropins and their receptor genes as predictive markers of OR and COS response to achieve fine-tuned therapeutic regimens.

Estrogen Modulates Specific Life and Death Signals Induced by LH and hCG in Human Primary Granulosa Cells In Vitro.

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are glycoprotein hormones used for assisted reproduction acting on the same receptor (LHCGR) and mediating different intracellular signaling. We evaluated the pro- and anti-apoptotic effect of 100 pM LH or hCG, in the presence or in the absence of 200 pg/mL 17ß-estradiol, in long-term, serum-starved human primary granulosa cells (hGLC) and a transfected granulosa cell line overexpressing LHCGR (hGL5/LHCGR). To this purpose, phospho-extracellular-regulated kinase 1/2 (pERK1/2), protein kinase B (pAKT), cAMP-responsive element binding protein (pCREB) activation and procaspase 3 cleavage were evaluated over three days by Western blotting, along with the expression of target genes by real-time PCR and cell viability by colorimetric assay. We found that LH induced predominant pERK1/2 and pAKT activation STARD1, CCND2 and anti-apoptotic XIAP gene expression, while hCG mediated more potent CREB phosphorylation, expression of CYP19A1 and procaspase 3 cleavage than LH. Cell treatment by LH is accompanied by increased (serum-starved) cell viability, while hCG decreased the number of viable cells. The hCG-specific, pro-apoptotic effect was blocked by a physiological dose of 17ß-estradiol, resulting in pAKT activation, lack of procaspase 3 cleavage and increased cell viability. These results confirm that relatively high levels of steroidogenic pathway activation are linked to pro-apoptotic signals in vitro, which may be counteracted by other factors, i.e., estrogens.

Human Luteinizing Hormone and Chorionic Gonadotropin Display Biased Agonism at the LH and LH/CG Receptors.

Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) have been considered biologically equivalent because of their structural similarities and their binding to the same receptor; the LH/CGR. However, accumulating evidence suggest that LH/CGR differentially responds to the two hormones triggering differential intracellular signaling and steroidogenesis. The mechanistic basis of such differential responses remains mostly unknown. Here, we compared the abilities of recombinant rhLH and rhCG to elicit cAMP, ß-arrestin 2 activation, and steroidogenesis in HEK293 cells and mouse Leydig tumor cells (mLTC-1). For this, BRET and FRET technologies were used allowing quantitative analyses of hormone activities in real-time and in living cells. Our data indicate that rhLH and rhCG differentially promote cell responses mediated by LH/CGR revealing interesting divergences in their potencies, efficacies and kinetics: rhCG was more potent than rhLH in both HEK293 and mLTC-1 cells. Interestingly, partial effects of rhLH were found on ß-arrestin recruitment and on progesterone production compared to rhCG. Such a link was further supported by knockdown experiments. These pharmacological differences demonstrate that rhLH and rhCG act as natural biased agonists. The discovery of novel mechanisms associated with gonadotropin-specific action may ultimately help improve and personalize assisted reproduction technologies.

Human LH and hCG stimulate differently the early signalling pathways but result in equal testosterone synthesis in mouse Leydig cells in vitro.

BACKGROUND: Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) are glycoprotein hormones regulating development and reproductive functions by acting on the same receptor (LHCGR). We compared the LH and hCG activity in gonadal cells from male mouse in vitro, i.e. primary Leydig cells, which is a common tool used for gonadotropin bioassay. Murine Leydig cells are naturally expressing the murine LH receptor (mLhr), which binds human LH/hCG. METHODS: Cultured Leydig cells were treated by increasing doses of recombinant LH and hCG, and cell signaling, gene expression and steroid synthesis were evaluated. RESULTS: We found that hCG is about 10-fold more potent than LH in cAMP recruitment, and slightly but significantly more potent on cAMP-dependent Erk1/2 phosphorylation. However, no significant differences occur between LH and hCG treatments, measured as activation of downstream signals, such as Creb phosphorylation, Stard1 gene expression and testosterone synthesis. CONCLUSIONS: These data demonstrate that the responses to human LH/hCG are only quantitatively and not qualitatively different in murine cells, at least in terms of cAMP and Erk1/2 activation, and equal in activating downstream steroidogenic events. This is at odds with what we previously described in human primary granulosa cells, where LHCGR mediates a different pattern of signaling cascades, depending on the natural ligand. This finding is relevant for gonadotropin quantification used in the official pharmacopoeia, which are based on murine, in vivo bioassay and rely on the evaluation of long-term, testosterone-dependent effects mediated by rodent receptor.

Follicle-stimulating hormone potentiates the steroidogenic activity of chorionic gonadotropin and the anti-apoptotic activity of luteinizing hormone in human granulosa-lutein cells in vitro.

Luteinizing hormone (LH) and choriogonadotropin (hCG) are glycoprotein hormones regulating ovarian function and pregnancy, respectively. Since these molecules act on the same receptor (LHCGR), they were traditionally assumed as equivalent in assisted reproduction techniques (ART), although differences between LH and hCG were demonstrated at molecular and physiological level. In this study, we demonstrated for the first time that co-treatment with a follicle-stimulating hormone (FSH) dose in the ART therapeutic range potentiates different LH- and hCG-dependent responses in vitro, measured in terms of cAMP, phospho-CREB, -ERK1/2 and -AKT activation, gene expression, progesterone and estradiol production in human granulosa-lutein cells (hGLC). We show that in the presence of FSH, hCG biopotency is about 5-fold increased, in the presence of FSH, in terms of cAMP activation. Accordingly, CREB phosphorylation and steroid production is increased under hCG and FSH co-treatment. LH effects, evaluated as steroidogenic cAMP/PKA pathway activation, do not change in the presence of FSH, which, however, increases LH-dependent ERK1/2 and AKT, but not CREB phosphorylation, resulting in anti-apoptotic effects. The different modulatory activity of FSH on LH and hCG action in vitro corresponds to their different physiological functions, reflecting proliferative effects exerted by LH during the follicular phase and before trophoblast development, and the high steroidogenic potential of hCG requested to sustain pregnancy from the luteal phase onwards.