Human Follicle-stimulating Hormone Receptor Research Articles

Effects and risk assessment of halogenated bisphenol A derivatives on human follicle stimulating hormone receptor: An interdisciplinary study

Halogenated bisphenol A (BPA) derivatives are produced during disinfection treatment of drinking water or are synthesized as flame retardants (TCBPA or TBBPA). BPA is considered as an endocrine disruptor especially on human follicle-stimulating hormone receptor (FSHR). Using a global experimental approach, we assessed the effect of halogenated BPA derivatives on FSHR activity and estimated the risk of halogenated BPA derivatives to the reproductive health of exposed populations. For the first time, we show that FSHR binds halogenated BPA derivatives, at 10 nM, a concentration lower than those requires to modulate the activity of nuclear receptors and/or steroidogenesis enzymes. Indeed, bioluminescence assays show that FSHR response is lowered up to 42.36 % in the presence of BPA, up to 32.79 % by chlorinated BPA derivatives and up to 27.04 % by brominated BPA derivatives, at non-cytotoxic concentrations and without modification of basal receptor activity. Moreover, molecular docking, molecular dynamics simulations, and site-directed mutagenesis experiments demonstrate that the halogenated BPA derivatives bind the FSHR transmembrane domain reducing the signal transduction efficiency which lowers the cellular cAMP production and in fine disrupts the physiological effect of FSH. The potential reproductive health risk of exposed individuals was estimated by comparing urinary concentrations (through a collection of human biomonitoring data) with the lowest effective concentrations derived from in vitro cell assays. Our results suggest a potentially high concern for the risk of inhibition of the FSHR pathway. This global approach based on FSHR activity could enable the rapid characterization of the toxicity of halogenated BPA derivatives (or other compounds) and assess the associated risk of exposure to these halogenated BPA derivatives.

P-081 A first in class potentiating therapeutic antibody as a new male fertility treatment

Abstract Study question Can we improve spermatogenesis in male by increasing FSH activity with a potentiating antibody? Summary answer IGYXOS developed a first in class potentiating therapeutic antibody that is able to stimulate spermatogenesis better than gonadotropins only as demonstrated in animal models. What is known already Idiopathic male infertility, including oligozoospermia, oligoasthenoteratospermia (OATS) and azoospermia, accounts for at least 75% of infertile men population, and no treatment is available at the moment for them. IGYXOS developed a monoclonal antibody directed against human FSH. IGX12 is a humanized IgG4 and it is able to potentiate FSH activity in vitro and in vivo in several animal models. Study design, size, duration IGX12 was tested in azoospermic rat and mouse models. The first model was created by treating Wistar Han rats with a GnRH antagonist. Gonadotropin treatment started after full inhibition of spermatogenesis. Four rats per group were treated with FSH+hCG regimen alone or in combination with IGX12. The second model was hpg mice that are congenitally hypogonadal because of their inability to synthetize GnRH. Seven mice per group were treated with FSH only or FSH+IGX12. Participants/materials, setting, methods IGX12 was tested in vitro on HEK293 cells and in vivo in Steelman and Pohley bioassay. The potentiating effect was investigated in azoospermic rats, that were treated for 8 weeks with FSH+hCG or FSH+hCG+IGX12. Hpg mice were treated with Gonal-f or Gonal-f+IGX12 for 7 weeks, followed by a treatment with Menopur or Menopur+IGX12 for a further 7 weeks. At the end of treatment, sperm counts in the testes and in the epididymides were assessed. Main results and the role of chance In vitro on HEK 293 cells expressing the human FSH receptor, IGX12 combined to FSH improved both the potency (EC50) and the efficacy (Emax) of FSH by increasing the cyclic AMP production at all tested FSH concentrations. The potentiating activity was confirmed in vivo in the Steelman and Pohley bioassay. Ovaries weight of immature female rats was increased significantly in the group treated with hCG/FSH+IGX12 compared to hCG/FSH only. To further analyze the potential benefit of IGX12 on male fertility, its effect was investigated in azoospermic rodent models. In rats with acquired azoospermia, treatment with FSH+hCG reached ∼50% of spermatogenesis of wild-type (WT) animals. Doubling the dose of gonadotropins did not improve their effect, whereas the addition of IGX12 on top of FSH+hCG treatment during one spermatogenesis cycle only (8 weeks) significantly improved sperm count in the testes and in the epididymides, reaching the level of WT animals. In hpg mice with congenital azoospermia, sperm count in the testis was 3.6 times higher and the sperm count in the epididymides was 6 times higher in the group treated with IGX12+FSH compared with FSH only (p < 0.05). In conclusion, IGX12 combined to a standard treatment stimulated spermatogenesis better than standard gonadotropin treatment. Limitations, reasons for caution After several proofs of concept and regulatory safety and toxicology evaluation in different animal models, IGX12 safety and tolerability is now assessed in a randomized, double blinded clinical phase I study on healthy men and women volunteers. Wider implications of the findings This first in class potentiating antibody IGX12 represents an innovative and promising new therapy to treat male patients with idiopathic infertility, especially with oligozoospermia. Trial registration number not applicable

P-654 A first in class potentiating therapeutic antibody as a new treatment for female fertility

Abstract Study question Can we improve controlled ovarian stimulation (COS) outcome in female by increasing FSH activity with a potentiating antibody? Summary answer IGYXOS developed a first in class potentiating antibody that improves COS treatments’ outcome by increasing FSH efficacy in vivo as demonstrated in non-human primates (NHP). What is known already For a COS in IVF cycles, FSH-containing preparations are injected to patients in order to obtain mature and fertilizable oocytes. However, the efficacy of these treatments is still limited and several attempts are needed before reaching a pregnancy and a live birth. One of the parameters that could be improved to avoid several attempts is the number of mature and thus fertilizable oocytes. It was previously demonstrated that breeding animals treated with gonadotropins could develop anti-gonadotropin antibodies that enhance their bioactivity instead of inhibiting it. These females had a high kidding rate and were hyperprolific. Study design, size, duration IGYXOS developed a monoclonal antibody (mAb) IGX12, a humanized IgG4, directed against human FSH. IGX12 was selected based on its ability to increase FSH activity in vitro and in vivo in rat., It was tested in mature cynomolgus female monkeysthat were treated as women undergoing ovarian stimulation for IVF with FSH alone or in combination with IGX12. Participants/materials, setting, methods IGX12 was tested in vitro on HEK 293 cells expressing the human FSH receptor. In vivo, the mAb was tested according to the Steelman and Pohley protocol. The potentiating effect was further investigated in NHP treated with mAb combined to a standard FSH treatment, under an agonist protocol. After hCG injection, oocytes were retrieved and their quality was analyzed. Blood was collected for hormonal assessments. Main results and the role of chance In vitro on HEK 293 cells expressing the human FSH receptor, IGX12 combined to FSH improves both the potency (EC50) and the efficacy (Emax) of FSH by increasing the cyclic AMP production at all tested FSH concentrations. The potentiating activity was confirmed in vivo in the Steelman and Pohley bioassay. Immature female rats received hCG+FSH alone or combined to mAb, twice a day, during 3 days. The animals were sacrificed the fourth day of treatment and the weight of the ovaries was compared. Ovaries weight was increased significantly in the group treated with hCG/FSH+IGX12 compared to hCG/FSH only. To further analyze the potential benefit of a IGX12 on human fertility, its effect was investigated in mature cynomolgus monkeys. IGX12 combined to a standard FSH treatment stimulated the follicular growth better than FSH only, and tripled the number of collected mature oocytes. Improved follicular growth correlated well with increased estradiol secretion during follicular phase and progesterone secretion during luteal phase. Limitations, reasons for caution After several proofs of concept and regulatory safety and toxicology evaluation in different animal models, IGX12 safety and tolerability is now assessed in a randomized, double blinded clinical phase I study on healthy men and women volunteers. Wider implications of the findings This first in class potentiating antibody IGX12 represents an innovative and promising new therapy to better stimulate patients with limited response to classical FSH treatments in order to improve the outcome of COS, and for normo-responders to avoid repetition of treatments. Trial registration number not applicable

An intracellular VHH targeting the Luteinizing Hormone receptor modulates G protein-dependent signaling and steroidogenesis

Luteinizing hormone (LH) is essential for reproduction, controlling ovulation and steroidogenesis. Its receptor (LHR) recruits various transducers leading to the activation of a complex signaling network. We recently identified iPRC1, the first variable fragment from heavy-chain-only antibody (VHH) interacting with intracellular loop 3 (ICL3) of the follicle-stimulating hormone receptor (FSHR). Because of the high sequence similarity of the human FSHR and LHR (LHCGR), here we examined the ability of the iPRC1 intra-VHH to modulate LHCGR activity. In this study, we demonstrated that iPRC1 binds LHCGR, to a greater extent when the receptor was stimulated by the hormone. In addition, it decreased LH-induced cAMP production, cAMP-responsive element-dependent transcription, progesterone and testosterone production. These impairments are not due to Gs nor β-arrestin recruitment to the LHCGR. Consequently, iPRC1 is the first intra-VHH to bind and modulate LHCGR biological activity, including steroidogenesis. It should help further understand signaling mechanisms elicited at this receptor and their outcomes on reproduction.

Abstract PR-006: A novel bispecific NK cell engager targeting FSHR and Siglec-7 displays potent anti-tumor immunity against ovarian cancer

Abstract Ovarian cancer (OC) is the deadliest gynecologic malignancy, with the highest mortality among cancers of the female reproductive system. Because of the close interaction between the OC cells and tumor microenvironment (TME), development of approaches which not only target tumor cells but also can maintain their anti-tumor function in this TME is of high importance. Uniquely, Follicle stimulating hormone receptor (FSHR) represents such an important target with specificity for OC and ovarian granulosa cells. We have recently developed monoclonal antibodies (mAbs) that specifically recognize human FSHR on the surface of multiple OC. We characterized them, selected potent anti-FSHR mAb: D2AP11 for detailed investigation and immune engineering in the form of a bispecific T cell engager (D2AP11-TCE) (Bordoloi et al., JCI Insight, 2022). Since most OCs are extremely aggressive and have restricted treatment choices, engaging additional immune effector components may further help. We have developed mAbs against PAN NK marker; Siglec 7 in humanized mice. We identified several Siglec-7 mAbs through high throughput screening and down-selected the pertinent clones. By using a highly potent anti-Siglec-7 clone DB7.2, we have developed a novel NK cell engager (NKCE): DB7.2xD2AP11 NKCE, by linking two antibody binding fragments (scFVs) arranged to bind FSHR, the OC antigen and hman Siglec-7, the NK marker. The NKCE was efficiently expressed in vitro and showed potent binding to both FSHR overexpressing K562 cells as well as Siglec-7 overexpressed HEK293T cells. This novel NKCE resulted in robust and specific killing of multiple OC targets in vitro in the presence of human PBMCs or NK cells, from multiple donors. The OC targets evaluated for killing by the NKCE include high grade serous adenocarcinoma, ovarian clear cell adenocarcinoma and ovarian cystadenocarcinoma lines which carry different mutations; BRCA1&2, AKT, TP53, PIK3CA, BRAF etc. and exhibit resistance against a wide array of cancer drug targets; PARP, HSP90, HDAC, MTORC, DNA alkylating agents, EGFR, PI3K, and WEE1, among others. DB7.2xD2AP11 NKCE displayed killing with EC50-142.87 pM and 236.6 pM against target OVISE-FSHR and OVCAR3 cells respectively, indicating its potency. Further, DNA encoded DB7.2xD2AP11 NKCE significantly attenuated the tumor burden in OVCAR3-FSHR and OVISE challenged humanized mice model. Additionally, this novel NKCE significantly enhanced the median survival of OVCAR3-FSHR challenged mice, suggesting in vivo potency. These data support further research on this novel NKCE as crucial tools for treating OC as well as targeted therapies for other malignancies. Of note, it is the first study of the development of Siglec-7 or any Siglec family member as a bispecific immune engager for targeted immunotherapy. Citation Format: Devivasha Bordoloi, Abhijeet J. Kulkarni, Opeyemi S. Adeniji, Pratik S. Bhojnagarwala, Shushu Zhao, Candice Ionescu, Mohamed bdel-Mohsen, David B. Weiner. A novel bispecific NK cell engager targeting FSHR and Siglec-7 displays potent anti-tumor immunity against ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr PR-006.

Abstract 1803 Investigating the Role of Caveolin in Human FSH Receptor Activity

Abstract 1803 Investigating the Role of Caveolin in Human FSH Receptor Activity

Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHR.

Developing modulatory antibodies against G protein-coupled receptors is challenging. In this study, we targeted the follicle-stimulating hormone receptor (FSHR), a significant regulator of reproduction, with variable domains of heavy chain-only antibodies (VHHs). We built two immune VHH libraries and submitted them to multiplexed phage display approaches. We used next-generation sequencing to identify 34 clusters of specifically enriched sequences that were functionally assessed in a primary screen based on a cAMP response element (CRE)-dependent reporter gene assay. In this assay, 23 VHHs displayed negative or positive modulation of FSH-induced responses, suggesting a high success rate of the multiplexed strategy. We then focused on the largest cluster identified (i.e., PRC1) that displayed positive modulation of FSH action. We demonstrated that PRC1 specifically binds to the human FSHR and human FSHR/FSH complex while potentiating FSH-induced cAMP production and Gs recruitment. We conclude that the improved selection strategy reported here is effective for rapidly identifying functionally active VHHs and could be adapted to target other challenging membrane receptors. This study also led to the identification of PRC1, the first potential positive modulator VHH reported for the human FSHR.

O-104 The Impact of application of CRISPR /dCas9 systems for increasing the expression of FSH receptor in human granulosa cells

Abstract Study question What are the molecular and physiological effects on human granulosa cells, when CRISPR/dCas9 epigenome edition technology is efficiently used to increase FSH receptor (FSHR) activity? Summary answer The significant increase in expression of FSHR maintained by dCAS9 systems has effects on several fundamental cellular events involving steroidogenic, life-sustaining,cell death related signaling sub-pathways. What is known already Manipulation of the expression of FSHR can result in biological events that include fundamental activities of FSH as regulation of folliculogenesis, production of steroidogenic hormones. FSHR can be activated in vitro by FSH analogues, and signaling networks activate ERK1/2 pathway through p38 mitogen-activated protein kinases. CRISPR/dCAS9 can be used to activate or inhibit gene action without silencing the gene. In spite of being used in many systems, interference of FSHR expression by epigenome edition is very limited as well as the use of in vitro tools to increase FSHR expression for investigating IVM, folliculogenesis, PCOS, FSH hyper/hyposensitivity, cancer biology. Study design, size, duration In-vitro cultured HGrC1 (human granulosa cell line) cells were treated by follitropin-alfa (Gonal-f). The experimental groups were designed in triplicates with cells i. without any genome edition and cells that have i. non-targeting gRNA, ii.dCAS9 activated FSHR, iii. dCAS9 activated FSHR and FSH treatment for 5 min, 15 min, or 1 hr; iv. only FSH treatment for 5 min, 15 min or 1 hr. FSHR related gene and protein expressions and estrogen production were evaluated. Participants/materials, setting, methods For human FSHR gene, two separate gRNAs were designed. Annealing, digestion, ligation, transformation, colony formation, plasmid isolation, Sanger sequencing analysis, MOI were accomplished for dCAS9/SAM system. HGrC1 (immortalized human granulosa cell line) cells were treated with 75 IU/ml Gonal-f in related groups. After treatment, in triplicates, gene/protein expression for MTOR, FSHR, AKT, MAPK8, MAPK1, TP 53, CASP 3, ESR1, CYP 19A1 were evaluated by qPCR, WB and IF. Estradiol was measured by chemiluminescence immunoassay. Main results and the role of chance FSHR has increased significantly in dCas9, dac9 + 5min Gonal-f, dCas9 + 15min Gonal-f, dCas9 + 1h Gonal-f; mostly in 5 min compared to groups without dCas9. The system plasmids did not show any non-specific effects.MAPK1, MAPK8, ESR1, MTOR, AKT, P53 and CASP3 gene expressions showed significant increase in dcas9 system groups compared to control and NT, whereas in all time-dependent treatments, a significant increase was seen in groups that have dcas9+Gonal-f compared to the ones treated with only Gonal-f. As time dependent manner has been investigated, expressions of MAPK1, MAPK8 and ESR1 were highest at min 5, expressions of CASP and AKT were highest at min 15 and the expression of MTOR was highest at hour 1, whereas FSHR and p53were strongly expressed at all time points (p < 0.05 for all mentioned results). Gene expression profiles were also confirmed for P-38 MAPK, AKT, FSHR by WB. IF stainings have revealed higher expression of AKT and MAPK in both dCas9 and dcas9+Gonal-f 5 min compared to the control group, however the intensity of the staining was not satisfactory enough. Interestingly, estrodiol(E2) levels were found to be higher in the 5 minutes only Gonal-f treatment and dcas9 with Gonal-f treatment, while the other groups were similar to control. Limitations, reasons for caution -This study has been conducted in cell line, rather than primary granulosa cells which are not easy targets for epigenome edition. -No testosterone derivatives were used as substrate for aromatase. This has been reflected in the results as significant increase in FSHR and related genes but non-significant increase in E2 production. Wider implications of the findings -dcas9/SAM system can be used efficiently increase other FSH-receptors, which is a potent way to reveal molecular sub-pathways in a temporal manner;eg. in male reproductive system or in other cells of female RS. -FSH related conditions as PCOS can be examined in vitro. -FHSR unresponsive conditions can be modeled in vitro. Trial registration number not applicable

Contributions of nonconventional intramolecular hydrogen bond on the bioactivity of a series of substituted 6-amino, 4-phenyl, tetrahydroquinoline derivatives

Although intramolecular hydrogen bond formation/disruption is a critical tool in the drug absorption process, it is difficult to be determined due to its group-atom nature. We proposed a proper descriptor to reasonably correlate pIC50 of a series of 19 substituted 6-amino, 4-phenyl, tetrahydroquinoline (6A4PTHQ) derivatives with their non-conventional intramolecular hydrogen bond (nCIHB) using quantum chemical calculations at B3LYP/6-31++G (d,p) level of theory. Effects of the protic and aprotic solvents and water on the nCIHB were studied. Solvents, especially protic solvents, weaken nCIHB in all 6A4PTHQ derivatives. It was found that the nCIHB is preserved within the 6A4PTHQ derivatives when docked in the human follicle-stimulating hormone (FSH) receptor. However, Molecular dynamic simulations showed that nCIHB disruption occurs in the aqueous compartment of the two-layered membrane, which enhances the absorption process via entropy increasing mechanism. Our approach may lead to the clarification of the complex role of nCIHB in drug transport and absorption processes in further research.

P-677 Increasing the efficacy of FSH with a potentiating monoclonal antibody

Abstract Study question Can we potentiate FSH activity with antibodies to improve ovarian stimulation? Summary answer IGYXOS developed a potentiating monoclonal antibody that improves FSH potency and efficacy in vitro and in vivo as demonstrated in different animal models. What is known already FSH plays a key role in reproduction. For a controlled ovarian stimulation in IVF cycles, FSH-containing preparations are injected to patients in order to obtain mature and fertilizable oocytes. However, the efficacy of these treatments is frequently limited and sometimes several attempts are needed before reaching a pregnancy and a live birth. It was previously demonstrated that breeding animals treated with gonadotropins could develop anti-gonadotropin antibodies that enhance their bioactivity instead of inhibiting it. These females had a high kidding rate and sometimes were hyperprolific. Study design, size, duration IGYXOS developed a monoclonal antibody (mAb) directed against human FSH. The mAb CF12 was selected based on its ability to increase FSH activity in vitro and in vivo in female rat. Its potentiating activity was tested on different human FSH preparations. The potentiating effect of CF12 was further investigated in ewe treated with mAb alone or FSH alone. Participants/materials, setting, methods CF12 was tested in vitro on HEK 293 cells expressing the human FSH receptor and a cell reporter system for cAMP. In vivo, the mAb was tested according to the Steelman and Pohley protocol on different human FSH preparations. Its potentiating effect was also investigated in an ovulation induction protocol on mature ewes. Main results and the role of chance In HEK 293 cells expressing the human FSH receptor, CF12 combined to FSH improved both the potency (EC50) and the efficacy (Emax) of FSH by increasing the cyclic AMP production at all tested FSH concentrations. The potentiating activity was confirmed in immature female rats treated with hCG+FSH alone or combined to mAb, twice a day, during 3 days. The animals were sacrificed the fourth day of treatment and the weight of the ovaries was compared. Ovaries weight was significantly increased in the group treated with hCG/FSH+CF12 compared to hCG/FSH only. To further analyze the effect of CF12 on ovulation induction, it was investigated in ewe as a breeding animal model. Animals were first synchronized with a progestagen and then treated either with FSH alone or with CF12 alone. The number of ovulations was investigated by endoscopy by counting corpora lutea and the progesterone secretion was measured over the luteal cycle. All females treated with CF12 ovulated (100%) by contrast 40% ovulated with FSH treatment. The ovulation rate was 1.8 fold higher in group treated with CF12 compared to FSH group. Moreover, progesterone secretion during luteal phase was increased in animals treated with CF12 suggesting a better quality of corpora lutea. Limitations, reasons for caution After several proofs of concept obtained in different animal models, the compound is currently in the humanization phase before starting the CMC process and then preclinical studies. Wider implications of the findings The potentiating antibody CF12 is a new concept. It represents an interesting strategy in reproductive health, particularly to improve the IVF outcome. CF12 is currently developed for human application by our company and could be a game changer in the field of ovarian stimulation by enhancing the efficacy of FSH. Trial registration number not applicable

Lipid Raft Disruption Alters Human Follicle Stimulating Hormone Receptor Signaling

Over 6.7 million people struggle with infertility each year. Studying signaling by reproductive hormones in fertility can allow us to gain a better understanding of the signaling pathways that must function correctly for proper fertility. Some infertility is due to incorrect human follicle stimulating hormone receptor (hFSHR) function. When follicle stimulating hormone (FSH) binds to hFSHR this begins a signaling cascade where the end product is the maturation of sperm by Sertoli cells in men, and egg development and production of estrogen through stimulation of granulosa cells in women. It has been determined that hFSHR is localized to microdomains of the cell membrane called lipid rafts which are characterized by higher concentrations of sphingolipids and cholesterol. This composition and structure makes them less fluid than non‐raft membranes, and it is believed that lipid rafts regulate signaling of many proteins that reside in the rafts, including hFSHR. It was hypothesized that if the lipid rafts in cell membranes were removed then hFSHR signaling would be altered. To remove lipid rafts HEK293 cells stably expressing hFSHR were treated with sphingolipid synthesis inhibitors (Myriocin or Fumonisin B1) or a cholesterol withdrawing drug (Methyl‐β‐cyclodextrin). Removing either the sphingolipids or cholesterol results in disruption of the lipid rafts. hFSHR signaling from the cells with disrupted lipid rafts were compared to wild type hFSHR signaling by western blot. Two pathways were investigated: cAMP production and the activation of p44/42 MAP kinase (ERK1/2). cAMP production was measured indirectly by detecting phosphorylated cAMP Response Element Binding protein (p‐CREB) and p44/42 MAPK signaling was measured by directly detecting the phosphorylation of the kinase (p‐p44/42). It was found that treatment with the lipid raft disrupting agents resulted in increased basal cAMP production (as measured by pCREB activation). However, time‐dependent hFSH stimulation was decreased compared to cells with intact lipid rafts. In contrast, p‐p44 signaling in the drug treated cells was not altered compared to untreated cells. These results suggest that G protein mediated activation of adenylyl cyclase to produce cAMP is lipid raft dependent while p44/42 MAPK activation is not. Further understanding of how lipid raft residency allows for functional hFSHR signaling would allow for the development of new treatments and pharmaceuticals for men and women struggling with infertility.

Investigating the Role of CRAC in FSH Receptor Function and Structure

Human infertility is a complex disorder that can often be attributed to a dysfunction of the endocrine system. Follicle‐stimulating hormone (FSH) is one of many hormones that participate in a complex process in both women and men to regulate normal reproduction. The dysfunction of this hormone and its receptor are some of the many causes of infertility. FSH is secreted by the anterior pituitary and, in women, initiates a cascade of biological events that enable ovulation. FSH carries out its function by binding and activating specific receptors. The FSH receptor (FSHR) is a G protein‐coupled receptor (GPCR) that is located in the cell membrane of target cells in the ovaries and testes. GPCRs often interact with a variety of molecules beyond their ligands including, in some cases, cholesterol in the plasma membrane. The Cholesterol Recognition/Interaction Amino Acid Consensus Sequence (CRAC) is one such binding domain and is represented by the sequence LV‐XXXX‐Y‐XXXXX‐K/R. The human FSHR contains a sequence consistent with the CRAC motif in the first intracellular loop L‐TTLQ‐Y‐KLTVP‐R. We hypothesized that mutating this domain will negatively affect the signaling of the receptor upon ligand activation. To test this hypothesis tyrosine 375 was mutated to phenylalanine (Y375F) which should render the CRAC inactive. After establishing a cell line stably expressing the mutant receptor, activation of the p44/42 (ERK1/2) pathway was measured. Preliminary data suggest that signaling of the hFSHR‐Y375F mutant is decreased relative to the wild‐type receptor. As we learn more about this receptor, we continue to gain knowledge about conditions such as infertility. This information can facilitate the development of innovative treatments which can help many families who struggle to conceive.

Functional characterization of naturally-occurring constitutively activating/inactivating mutations in equine follicle-stimulating hormone receptor.

ObjectiveFollicle-stimulating hormone (FSH) is the central hormone involved in mammalian reproduction, maturation at puberty, and gamete production that mediates its function by control of follicle growth and function. The present study investigated the mutations involved in the regulation of FSH receptor (FSHR) activation.MethodsWe analyzed seven naturally-occurring mutations that were previously reported in human FSHR (hFSHR), in the context of equine FSHR (eFSHR); these include one constitutively activation variant, one allelic variant, and five inactivating variants. These mutations were introduced into wild-type eFSHR (eFSHR-wt) sequence to generate mutants that were designated as eFSHR-D566G, -A306T, -A189V, -N191I, -R572C, -A574V, and -R633H. Mutants were transfected into PathHunter EA-parental CHO-K1 cells expressing β-arrestin. The biological function of mutants was analyzed by quantitating cAMP accumulation in cells incubated with increasing concentrations of FSH.ResultsCells expressing eFSHR-D566G exhibited an 8.6-fold increase in basal cAMP response, as compared to that in eFSHR-wt. The allelic variation mutant eFSHR-A306T was not found to affect the basal cAMP response or half maximal effective concentration (EC50) levels. On the other hand, eFSHR-D566G and eFSHR-A306T displayed a 1.5- and 1.4-fold increase in the maximal response, respectively. Signal transduction was found to be completely impaired in case of the inactivating mutants eFSHR-A189V, -R572C, and -A574V. When compared with eFSHR-wt, eFSHR-N191I displayed a 5.4-fold decrease in the EC50 levels (3,910 ng/mL) and a 2.3-fold decrease in the maximal response. In contrast, cells expressing eFSHR-R633H displayed in a similar manner to that of the cells expressing the eFSHR-wt on signal transduction and maximal response.ConclusionThe activating mutant eFSHR-D566G greatly enhanced the signal transduction in response to FSH, in the absence of agonist treatment. We suggest that the state of activation of the eFSHR can modulate its basal cAMP accumulation.

Fshr: a fish sex-determining locus shows variable incomplete penetrance across flathead grey mullet populations.

SummaryWhole-genome sequencing data were produced from a single flathead grey mullet female and assembled into a draft genome sequence, whereas publicly available sequence data were used to obtain a male draft sequence. Two pools, each consisting of 60 unrelated individuals, respectively, of male and female fish were analyzed using Pool-Sequencing. Mapping and analysis of Pool-Seq data against the draft genome(s) revealed >30 loci potentially associated with sex, the most promising locus of which, encoding the follicle-stimulating hormone receptor (fshr) and harboring two missense variants, was genotyped on 245 fish from four Mediterranean populations. Genotype data showed that fshr represents a previously unknown sex-determining locus, although the incomplete association pattern between fshr genotype and sex-phenotype, the variability of such pattern across different populations, and the presence of other candidate loci reveal that a greater complexity underlies sex determination in the flathead grey mullet.

Investigating Human Follicle Stimulating Hormone Receptor and its Partners Using the APEX Assay

Follicle stimulating hormone (FSH) and its receptor (FSHR) play important roles in reproduction and fertility in both men and women. Fertility problems arise when FSH, FSHR, or any downstream signaling don’t function properly. FSHR is a G‐protein coupled receptor (GPCR) found on the cell surface of granulosa cells in women and Sertoli cells in men. When activated by FSH, FSHR initiates a signaling cascade that can result in different downstream results such as ovarian follicular development and estrogen production in women and sperm production and regulation in men. To better understand how the receptor functions, the interactions between FSH, FSHR and other effector proteins involved in signaling must be identified and understood. One hypothesized mechanism of signaling is through the receptor localization in lipid rafts. The lipid rafts act as a signaling complex where FSHR and other effector proteins colocalize to make for efficient signaling of FSHR. This colocalization in lipid rafts is believed to play an important role and we plan to study the interactions between FSHR, effector proteins, and lipid rafts using the APEX Assay. The APEX Assay has been developed to study these interactions using different GPCRs. This assay utilizes an engineered ascorbate peroxidase (APEX) which is attached to the C‐terminus of FSHR. APEX modifies proteins using biotin phenol and hydrogen peroxide, which creates a biotin‐radical, labeling proteins within a small radius of FSHR because of the short lived nature of the radical. Using mass spectrometry, the biotinylated proteins can be quantitatively analyzed to give a better understanding of the proteins associated with FSHR and the changes in the proteins over time after hormone treatment, as well as lipid raft localization. Using HEK293 cells, we are working on creating a stable cell line expressing an FSHR‐APEX construct. Once the cell line is created, the FSHR‐APEX receptor can be used in the APEX Assay to analyze the proteins that are involved in FSHR signaling. This research will give us a better insight as to what effector proteins FSHR interacts with, when these interactions occur, and if they occur in lipid rafts. This will provide us with a better understanding as to how the receptor signals and initiates downstream signaling and how this might affect fertility in humans.

Development of a robust reporter gene-based assay for the bioactivity determination of recombinant human follicle stimulating hormone (rhFSH) pharmaceutical products

FSH plays a key role in the function of the reproductive system of human beings and is widely used both diagnostically and therapeutically in reproductive medicine. With the growing incidence of infertility, the demand for FSH pharmaceutical products is increasing. For this reason, the quality control process for FSH products is becoming more stringent. An accurate determination of bioactivity is crucial for the safety and efficacy of recombinant human follicle stimulating hormone (rhFSH). Up to now, in-vivo bioassay based on FSH-induced increases in rat ovarian weight has been the only method widely accepted by different pharmacopoeias. However this method has such drawbacks as the complex procedures, long assay period and high variability. Here, we established a reporter gene assay (RGA) based on the CHO-K1-FSHR-CRE-Luc cell line that stably expresses human follicle stimulating hormone receptor (hFSHR), as well as a luciferase reporter under the control of cyclic adenosine monophosphate (cAMP) response elements (CRES). Our study showed that our new assay not only has good dose-dependent responsiveness to rhFSH, but it also performs excellently in terms of specificity, precision, linearity, and simplicity compared with in-vivo rat bioassays. These results implied that this robust reporter gene assay may be a viable supplement to the animal in-vivo bioassay and may be employed in potency determination of rhFSH pharmaceutical products.

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 β-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 β-arrestin 2 recruitment in FSHR-transfected HEK293 cells (cAMP EC50 range = 12 ± 0.9–24 ± 1.7 ng/ml; β-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.

Halogenous derivatives of Bisphenol A inhibit the human FSH receptor activity

Halogenous derivatives of Bisphenol A inhibit the human FSH receptor activity

Characterization of Membrane Microdomains on a Human Granulosa Cell Line by Atomic Force Microscopy

Signaling via the Hypothalamic‐Pituitary‐Gonadal (HPG) axis regulates fertility in both men and women. Follicle stimulating hormone (FSH), secreted from the anterior pituitary, binds to a G protein coupled receptor (GPCR) on ovarian granulosa cells. The activation of this receptor triggers a cellular signaling pathway that leads to the promotion of follicle development and estrogen secretion. Our lab has previously demonstrated that the human FSH receptor (hFSHR) shows an affinity for residing in lipid rafts of HEK293 cells which may play a pivotal role in its signaling. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids, with higher stiffnesses than bulk membrane domains. To characterize lipid rafts on the human granulosa cell line hGrC1 we used atomic force microscopy. Atomic force microscopy is a technique used to produce nanoscale resolution topographical images. It can also be used for determining mechanical characteristics, such as stiffness and strain. Here, using contact and force‐volume mode atomic force microscopy, we successfully generated topographical maps of hGrC1s and recorded membrane stiffness measurements. Areas of the granulosa cell membrane showed variable stiffness on a scale of 58.998 nm / pixel, providing the first direct evidence of lipid rafts in human granulosa cell membranes. Demonstration of lipid rafts contributes to the implications of the role of lipid rafts hFSH signalling. By understanding membrane dynamics on granulosa cells we hope to gain insight into how to manipulate hFSHR function.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Discovery of small molecule binders of human FSHR(TMD) with novel structural scaffolds by integrating structural bioinformatics and machine learning algorithms

BackgroundThe activation of follicle stimulating hormone receptor (FSHR) by FSH and the consequent downstream signaling activities are crucial for reproductive health. The role of FSHR in tumor progression as well as osteoporosis advancement has also been well established. Currently, steroid preparations of estrogen and progesterone are being used for managing fertility, in spite of the harmful side effects, as there has not been much success in identification of effective FSHR modulators. Structure-based drug design initiatives for identification of potent and specific FSHR modulators have been impeded due to the non-availability of the complete crystal structure of hFSHR complexed with FSH. MethodsIn this study, we have modeled the 3D structure of transmembrane domain (TMD) of hFSHR and identified molecules that demonstrate good binding affinity by virtual screening of drug-like library of compounds. The 3D structural and pharmacophoric features of the binders and non-binders obtained from virtual screening were further used to develop Support Vector Machine based classifier for TMD binding. Based on the observations from docking and SVM classification, a small molecule was identified for extensive MD simulations and in vitro assays for FSHR modulatory activity. ResultsThe molecule selected based on docking score and SVM prediction was found to inhibit FSH-induced cAMP activity by 80% at 300 μM concentration. ConclusionThe study proposes 1,3-diphenyl-1H-pyrazole-5-carboxylate as a promising scaffold for the design of new and potent FSHR allosteric inhibitors.