Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro.

The signal transduction of the equine lutropin/choriogonadotropin receptor (eLH/CGR) is unclear in naturally occurring activating/inactivating mutants of this receptor, which plays an important role in reproductive physiology. We undertook the present study to determine whether conserved structurally related mutations in eLH/CGR exhibit similar mechanisms of signal transduction. We constructed four constitutively activating mutants (M398T, L457R, D564G, and D578Y) and three inactivating mutants (D405N, R464H, and Y546F); measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary cells; and investigated cell-surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney 293 cells. The eLH/CGR-L457R-, -D564G-, and -D578Y-expressing cells exhibited 16.9-, 16.4-, and 11.2-fold increases in basal cAMP response, respectively. The eLH/CGR-D405N- and R464H-expressing cells presented a completely impaired signal transduction, whereas the Y546F-expressing cells exhibited a small increase in cAMP response. The cell-surface receptor loss was 1.4- to 2.4-fold greater in the activating-mutant-expressing cells than in wild-type eLH/CGR-expressing cells, but was completely impaired in the D405N- and Y546F-expressing cells, despite treatment with a high concentration of agonist. In summary, the state of activation of eLH/CGR influenced agonist-induced cell-surface receptor loss, which was directly related to the signal transduction of constitutively activating mutants.

Structures of full-length glycoprotein hormone receptor signalling complexes.

Luteinizing hormone and chorionic gonadotropin are glycoprotein hormones that are related to follicle-stimulating hormone and thyroid-stimulating hormone1,2. Luteinizing hormone and chorionic gonadotropin are essential to human reproduction and are important therapeutic drugs3-6. They activate the same G-protein-coupled receptor, luteinizing hormone-choriogonadotropin receptor (LHCGR), by binding to the large extracellular domain3. Here we report four cryo-electron microscopy structures of LHCGR: two structures of the wild-type receptor in the inactive and active states; and two structures of the constitutively active mutated receptor. The active structures are bound to chorionic gonadotropin and the stimulatory G protein (Gs), and one of the structures also contains Org43553, an allosteric agonist7. The structures reveal a distinct 'push-and-pull' mechanism of receptor activation, in which the extracellular domain is pushed by the bound hormone and pulled by the extended hinge loop next to the transmembrane domain. A highly conserved 10-residue fragment (P10) from the hinge C-terminal loop at the interface between the extracellular domain and the transmembrane domain functions as a tethered agonist to induce conformational changes in the transmembrane domain and G-protein coupling. Org43553 binds to a pocket of the transmembrane domain and interacts directly with P10, which further stabilizes the active conformation. Together, these structures provide a common model for understanding the signalling of glycoprotein hormone receptors and a basis for drug discovery for endocrine diseases.

Identification of Key Receptor Residues Discriminating Human Chorionic Gonadotropin (hCG)- and Luteinizing Hormone (LH)-Specific Signaling.

(1) The human luteinizing hormone (LH)/chorionic gonadotropin (hCG) receptor (LHCGR) discriminates its two hormone ligands and differs from the murine receptor (Lhr) in amino acid residues potentially involved in qualitative discerning of LH and hCG. The latter gonadotropin is absent in rodents. The aim of the study is to identify LHCGR residues involved in hCG/LH discrimination. (2) Eight LHCGR cDNAs were developed, carrying "murinizing" mutations on aminoacidic residues assumed to interact specifically with LH, hCG, or both. HEK293 cells expressing a mutant or the wild type receptor were treated with LH or hCG and the kinetics of cyclic adenosine monophosphate (cAMP) and phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2) activation was analyzed by bioluminescence resonance energy transfer (BRET). (3) Mutations falling within the receptor leucine reach repeat 9 and 10 (LRR9 and LRR10; K225S +T226I and R247T), of the large extracellular binding domain, are linked to loss of hormone-specific induced cAMP increase, as well as hCG-specific pERK1/2 activation, leading to a Lhr-like modulation of the LHCGR-mediated intracellular signaling pattern. These results support the hypothesis that LHCGR LRR domain is the interaction site of the hormone ß-L2 loop, which differs between LH and hCG, and might be fundamental for inducing gonadotropin-specific signals. (4) Taken together, these data identify LHCGR key residues likely evolved in the human to discriminate LH/hCG specific binding.

Comparative Study of the Steroidogenic Effects of Human Chorionic Gonadotropin and Thieno[2,3-D]pyrimidine-Based Allosteric Agonist of Luteinizing Hormone Receptor in Young Adult, Aging and Diabetic Male Rats.

Low-molecular-weight agonists of luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor (LHCGR), which interact with LHCGR transmembrane allosteric site and, in comparison with gonadotropins, more selectively activate intracellular effectors, are currently being developed. Meanwhile, their effects on testicular steroidogenesis have not been studied. The purpose of this work is to perform a comparative study of the effects of 5-amino-N-tert-butyl-4-(3-(1-methylpyrazole-4-carboxamido)phenyl)-2-(methylthio)thieno[2,3-d] pyrimidine-6-carboxamide (TP4/2), a LHCGR allosteric agonist developed by us, and hCG on adenylyl cyclase activity in rat testicular membranes, testosterone levels, testicular steroidogenesis and spermatogenesis in young (four-month-old), aging (18-month-old) and diabetic male Wistar rats. Type 1 diabetes was caused by a single streptozotocin (50 mg/kg) injection. TP4/2 (20 mg/kg/day) and hCG (20 IU/rat/day) were administered for 5 days. TP4/2 was less effective in adenylyl cyclase stimulation and ability to activate steroidogenesis when administered once into rats. On the 3rd-5th day, TP4/2 and hCG steroidogenic effects in young adult, aging and diabetic rats were comparable. Unlike hCG, TP4/2 did not inhibit LHCGR gene expression and did not hyperstimulate the testicular steroidogenesis system, moderately increasing steroidogenic proteins gene expression and testosterone production. In aging and diabetic testes, TP4/2 improved spermatogenesis. Thus, during five-day administration, TP4/2 steadily stimulates testicular steroidogenesis, and can be used to prevent androgen deficiency in aging and diabetes.

Molecular characterization of two Russian sturgeon gonadotropin receptors: Cloning, expression analysis, and functional activity.

Sturgeons are being used in aquaculture because wild populations are now endangered due to overfishing for caviar. A challenge in working with sturgeon as an aquacultured species is its long and slow reproductive development. Reproduction is a hormonally regulated process that involves hierarchical signaling between the brain, pituitary gland, and gonads. In an effort to better understand the hormonal regulation of sturgeon reproduction, we have cloned the Russian sturgeon (st), Acipenser gueldenstaedtii, luteinizing hormone receptor (stLHR) and follicle stimulating hormone receptor (stFSHR) and measured their expression from previtellogenic to mature ovarian follicles. Sturgeon LHR and FSHR expression was elevated in early-vitellogenic and mature follicles compared with pre-vitellogenic and mid-vitellogenic follicles, and only LHR expression increased during late-vitellogenesis. Recombinant sturgeon FSH and LH both activated sturgeon LHR and FSHR in a cAMP reporter assay. Further molecular characterization of these receptors was accomplished by in silico modeling and cAMP reporter assays using heterologous recombinant gonadotropins from human and piscine species. There was no apparent trend in heterologous LH and/or FSH activation of the sturgeon LHR or FSHR. These data suggest that permissive activation of LHR and FSHR are a consequence of some yet undetermined biological characteristic(s) of different piscine species.

Diseases caused by mutations in luteinizing hormone/chorionic gonadotropin receptor.

Accumulating evidence showed that the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) is an essential regulator of sexual development and reproduction from zebrafish to human. Activating and inactivating mutations of LHCGR gene have been identified from patients of different phenotypes. Familial male-limited precocious puberty, Leydig cell hypoplasia, and empty follicle syndrome are caused by LHCGR mutations. More than 50 mutations have been reported from subjects of different ethnic backgrounds. Functional analyses of the mutant LHCGR revealed multiple defects, including cell surface expression, ligand binding, and signaling. The difference of the two native ligands and signaling pathway activated by LHCGR are illustrated. Potential therapeutic implications from the analyses of the naturally occurring LHCGR mutations, such as pharmacological chaperones, are highlighted.

In silico analysis, temporal expression and gonadotropic regulation of receptors for follicle-stimulating hormone and luteinizing hormone in testis of spotted snakehead Channa punctata.

This study in spotted snakehead Channa punctata was aimed to develop a comprehensive understanding of testicular gonadotropin receptors, from their sequence characterization, temporal expression to gonadotropic regulation, in seasonally breeding teleosts. A single form of follicle-stimulating hormone receptor (cpfshra) and luteinizing hormone/choriogonadotropin receptor (cplhcgr), was identified from testicular transcriptome data of C. punctata. Although deduced full-length protein sequence for cpFshra (694 amino acids) and cpLhcgr (691 amino acids) showed homology with their counterparts of other vertebrates, multiple insertion-deletion-substitution of residues suggest marked alterations in their structure and ligand specificity. The absolute quantification of testicular cpfshra and cplhcgr was estimated along the reproductive cycle following real-time PCR. The temporal expression profile showed highest testicular expression of both the gonadotropin receptors during resting phase. Their expression progressively decreased during preparatory and spawning phases concomitant with spermatogonial proliferation and differentiation and spermiogenesis. However, levels of cpfshra and cplhcgr sharply increased during post-spawning when seminiferous lobules were largely devoid of germ cells. To explore gonadotropic regulation of testicular cpfshra and cplhcgr, one group of fish of resting phase was administered with single dose of human chorionic gonadotropin (hCG; 5,000 IU/kg body mass) on day 0 and sacrificed on day 3 and day 5, while another group receiving two injections of hCG (day 0 and day 7) was sacrificed on day 14. The expression pattern of testicular gonadotropin receptors in hCG-treated fish sacrificed after 3, 5 and 14 days was similar to that of preparatory, spawning and postspawning phases, respectively. Likewise, testicular histology of hCG-treated fish sacrificed on day 3, day 5 and day 14 was comparable with that of preparatory, early spawning and late spawning phases, respectively. In light of the fact that gonadotropin receptors are largely expressed on somatic cells, an apparent decrease in testicular cpfshra and cplhcgr levels during preparatory and spawning phases or after 3 and 5 days from first hCG injection might not be due to downregulation of their expression. Rather, this could be due to dilution of somatic cell mRNA by large amount of germ cell mRNA. To verify this assumption, effect of hCG on plasma level of androgens was investigated employing enzyme-linked immunosorbent assay. A marked increase in plasma level of testosterone and 11-ketotestosterone was observed after hCG treatment in C. punctata. This would have been possible only when hCG upregulated the expression of testicular gonadotropin receptors.

Cloning, expression and functional characterization of a novel luteinizing hormone receptor in the orange-spotted grouper, Epinephelus coioides.

Luteinizing hormone receptor (LHR) plays a critical role in reproduction by mediating LH signaling in the gonad. In this study, we cloned a novel lhr gene from the orange-spotted grouper, named glhr2. The cloned complete open reading frame sequence of glhr2 was 2082 bp in length, encoding a protein of 693 amino acids, sharing approximately 50% amino acid identity with glhr1. glhr1 and glhr2 were primarily expressed in gonad, brain and hypothalamus with low expression in other tissues such as gill, spleen, etc. The expressions of both glhr1 and glhr2 increased during vitellogenesis, while decreased during natural female to male sex change. The two gLHRs both could be activated by equine LH or human chorionic gonadotropin, but not by human follicle stimulating hormone. Both gLHR1 and gLHR2 activation stimulated the expression of cAMP response element driven reporter gene in a dose-dependent manner, while gLHR2 but not gLHR1 also activated serum response element driven reporter gene expression. This was the first study to demonstrate that two active LHRs exist in fish with possible different functional roles.

Luteinizing Hormone/Human Chorionic Gonadotropin Receptor Immunohistochemical Score Associated with Poor Prognosis in Endometrial Cancer Patients.

The aim of this study was to develop a scoring system of the immunohistochemical (IHC) expression of luteinizing hormone/human chorionic gonadotropin receptor (LHCG-R) in endometrial cancer (EC) patients. Nonconsecutive hysterectomy specimens containing EC collected from April 2013 to October 2015 were selected. Hematoxylin-eosin stained sections from each case were reviewed and representative sections from each tumor were selected. IHC staining was performed for the detection of LHCG-R. The percentage of stained cells and the staining intensity were assessed in order to develop an immunohistochemical score. Moreover, we examined the correlation of the score with grading and lymphovascular space invasion (LVSI). There was a statistically significant positive correlation between grading and IHC scoring (p = 0.01) and a statistically significant positive correlation between LVSI and IHC score (p < 0.01). In conclusion, we suggest that the immunohistochemical score presented here could be used as a marker of bad prognosis of EC patients. Nevertheless, further studies are needed in order to validate it. The study was registered in the Careggi Hospital public trials registry with the following number: 2013/0011391.

Novel compound heterozygous variants in the LHCGR gene identified in a subject with Leydig cell hypoplasia type 1.

BACKGROUND: Leydig cell hypoplasia (LCH) is a rare disease and one of the causes of male disorder of sexual differentiation (DSD). Inactivating mutations in the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) gene account for the underlying LCH pathogenicity. This study aimed to analyze the clinical presentation and diagnosis as well as highlight the molecular characteristics of a subject with LCH type 1. CASE PRESENTATION: Clinical data were collected from the subject and analyzed. Next generation sequencing of the immediate family pedigree using peripheral blood genomic DNA was performed, and the relevant mutations were verified with Sanger sequencing. We describe the case of a 5-year-old patient with DSD, presenting with a lateral inguinal hernia accompanied by abnormal hormone tests. The genetic analysis revealed novel compound heterozygous variants in the LHCGR gene, including a splice site mutation (c.681-1 G>A) and a frameshift variant (c.1582_1585del ATAT, p.Ile528*). CONCLUSIONS: We identified novel compound heterozygous variants in the LHCGR gene, and expanded the genotype-phenotype correlation spectrum of LHCGR variants.

[Familial male-limited precocious puberty due to Asp578His mutations in the LHCGR gene: clinical characteristics and gene analysis in an infant].

The aim of the study was to provide a descriptive analysis of familial male-limited precocious puberty (FMPP), which is a rare inherited disease caused by heterozygous constitutively activating mutations of the luteinizing hormone/choriogonadotropin receptor gene (LHCGR). The patient was a ten-month-old boy, presenting with penile enlargement, pubic hair formation, and spontaneous erections. Based on the clinical manifestations and laboratory data, including sexual characteristics, serum testosterone levels, GnRH stimulation test, and bone age, this boy was diagnosed with peripheral precocious puberty. Subsequently the precocious puberty-related genes were analyzed by direct DNA sequencing of amplified PCR products from the patient and his parents. Genetic analysis revealed a novel heterozygous missense mutation c.1732G>C (Asp578His) of the LHCGR gene exon11 in the patient, which had never been reported. His parents had no mutations. After combined treatment with aromatase inhibitor letrozole and anti-androgen spironolactone for six months, the patient's symptoms were controlled. The findings in this study expand the mutation spectrum of the LHCGR gene, and provide molecular evidence for the etiologic diagnosis as well as for the genetic counseling and prenatal diagnosis in the family.

Genetic evidence of 'genuine' empty follicle syndrome: a novel effective mutation in the LHCGR gene and review of the literature.

Empty follicle syndrome (EFS) is a reproductive disorder in which no oocytes are retrieved during IVF. The existence of genuine EFS (GEFS) is still controversial, and to date, only one missense mutation of Luteinizing Hormone/Choriogonadotropin Receptor (LHCGR) has been reported to be associated with this disease. Here, we describe a GEFS patient in a non-consanguineous family from China. A 27-year-old woman presented with a 5-year history of primary infertility and LH resistance-like ovaries of unequal sizes, but with normal levels of circulating LH. In spite of a satisfactory ovarian reserve and response, no oocytes were retrieved after two cycles of IVF. Her condition did not appear to be failure of the hCG injection. It is more likely to be a genetic cause. A novel homozygous mutation in LHCGR gene, c.1345G>A (p.Ala449Thr), was detected in this patient. Each of her parents is heterozygous for this change, and the change was absent from 407 control subjects. Alanine at this amino acid position was highly conserved and replacement of threonine was predicted to disrupt the third transmembrane helix of the rhodopsin-like G protein-coupled receptor domain. Protein localization studies revealed that a portion of the mutant LHCGR protein molecules was retained intracellularly. Signalling studies demonstrated that this mutation had differing effects on the response of LHCGR to hCG or LH at different concentrations. Specifically, at a concentration <1 IU/ml, the mutant was activated by hCG stimulation but partially resistant to LH stimulation; at a higher concentration (>1 IU/ml), the mutant was activated by both hCG and LH. These data suggest that screening for mutations in the LHCGR gene may assist in the diagnosis of patients with GEFS. The literature describing the relationship between phenotype and genotypes in females is reviewed, and possible aetiologies and treatment options for this disease are proposed based on our and other studies.

Assessing reproductive toxicity and antioxidant enzymes on beta asarone induced male Wistar albino rats: In vivo and computational analysis.

AIM: Beta asarone is the major constituent of oil obtained from Acorus calamus, the Indian traditional medicine plant. Several studies have shown that beta asarone causes liver and cardiac damages but the reproductive toxicity is not well understood. The present study was initiated to investigate whether beta asarone has the potential to cause reproductive toxicity by inducing oxidative stress in the testis of male Wistar albino rats. MATERIALS AND METHODS: For this study, the animals were divided into six groups: Group I was treated with saline (normal saline), Group II with DMSO (vehicle control) and Group III with cisplatin (10mg/kgb.wt.). Group IV, V and VI animals were administrated at three dose levels of beta asarone 12.5, 25 and 50mg/kgb.wt. The treatment was carried out for 14days and animals were sacrificed on 29th day and processed for sperm analysis, hormone assay, histopathological, and antioxidant enzymatic assays. We also used molecular docking studies to predict the binding nature of beta asarone with luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR). KEY FINDINGS: Beta asarone administered at a dose of 50mg/kgb.wt. was responsible for inducing certain noticeable degenerative changes in histopathological analysis of the tissue. This was supported by altered sperm morphology and hormonal variations when compared to the control groups. Antioxidant enzyme levels were also found to be decreased. This was further validated by molecular docking studies. SIGNIFICANCE: The present study provides evidence that beta asarone administered at a dose of 50mg/kg b.wt. is capable enough in bringing about moderate amount of degenerative changes in rat testis and altered antioxidant status. Therefore provides a suitable evidence to prove that beta asarone causes reproductive toxicity.

THE RELATIONSHIP BETWEEN PHYSICAL-CHEMICAL CHARACTERISTICS AND BIOLOGICAL ACTIVITY OF PEPTIDE 562­572 OF LUTEINIZING HORMONE RECEPTOR MODIFIED BY DECANOYL RADICALS AT THE N- AND C-TERMINI.

Lipophilic derivatives of peptides corresponding to the cytoplasmic regions associated with the G-protein coupled receptors (GPCRs) are capable of functioning as an intracellular agonist. Previously, we have shown that peptides corresponding to region 562­572 of luteinizing hormone receptor (LHR) and modified by decanoate and palmitate at the C-terminus activate adenylyl cyclase (AC) in the testes of rats. The stimulating effect of peptide 562­572 modified by decanoates at the N- and C-termini (IV) peaked at a concentration of 10(­5) M and then subsequently decreased with increasing concentration. We hypothesized that this may be due to ability of the peptide IV to micelle formation. To test this suggestion, we examined the relationship between biological activity, hydrophobicity and ability to micelle formation for peptide IV and other acylated derivatives of peptide 562­572 including the derivatives containing C-terminal decanoate (III) and palmitate (VI). It has been shown that the stimulating effect of peptide IV at a concentration of 10(­5) M on AC activity in the plasma membranes of rat testes and ovaries is only slightly inferior to that of peptide VI and superior to the corresponding effect of peptide III. The effect of peptide IV at a concentration of 10­3 M was reduced by 20­27 % and amounted to 50­51 and 87­88 % of that of peptides VI and III, respectively. Despite the high hydrophobicity, the peptide IV had abnormally low retention time in reverse-phase HPLC when it was eluted from the Nucleosil C8 column, even lower than that of unmodified peptide 562­572. However, with increasing concentration of trifluoroacetic acid in the eluent from 0.1 to 0.5 % causing the destruction of micelle-like structures, the retention time of the peptide IV was significantly increased, whereas it remained unchanged in the case of the other peptides. Surface tension of aqueous solution of peptide IV insignificantly decreased with the increase of its concentration, but then, at peptide concentration of 710(­6) M, the sharp decline and the plateau were found, which indicates the beginning of the formation of micelles. Thus, at concentration of 10(­5) M and higher the peptide IV forms micelles which prevents its interaction with the receptor. The ability of GPCR-peptides to self-aggregation and micelle formation should be taken into account when developing their membrane-active analogues.

Loss-of-Function Mutations in the Human Luteinizing Hormone Receptor Predominantly Cause Intracellular Retention.

Mutations in G protein-coupled receptors (GPCRs) have been identified for many endocrine hormone signaling deficiencies. Inactivating mutations can impair ligand binding, receptor activation/coupling to signaling pathways, or can cause receptor misfolding and consequent impaired expression at the cell membrane. Here we examine the cell surface expression, ligand binding, and signaling of a range of mutant human luteinizing hormone receptors (LHRs) identified as causing reproductive dysfunction in human patients. The data obtained reveal how mutations in GPCRs can have diverse and severely deleterious effects on receptor function. Furthermore, it was found that impaired functionality of the majority of the mutant LHRs was due to reduced expression at the cell surface (14/20) while only two mutations caused impaired binding affinity and two impaired in signaling. An additional two mutations were found to cause no impairment of receptor function. These data demonstrate that the majority of LHR mutations lead to intracellular retention and highlight the potential for novel pharmacological chaperone therapeutics that can "rescue" expression/function of retained mutant GPCRs.

Dissecting the structural and functional features of the Luteinizing hormone receptor using receptor specific single chain fragment variables.

The Luteinizing hormone receptor (LHR) has a large extracellular domain (amino acid residues, a.a.1-355) and a transmembrane domain (TMD; a.a. 356-699), essential for hormone binding and signaling, respectively. The LHR hinge region (a.a. 256-355) connects the two domains and acts as an activating switch for the receptor by an unknown mechanism. LHR hinge-specific Single chain fragment variables (ScFv) stimulated cAMP production by the stable and transiently transfected cell lines expressing LHR in a hormone-independent manner and the C-terminal region of LHR hinge (a.a. 313-349) was identified as the probable epitope for one agonistic ScFv. This epitope attained a helical conformation upon agonistic ScFv binding and the activity of the ScFv was dependent on Y331 sulfation. ScFv was also able to activate TMD mutants, D578Y and A593P, reemphasizing the role of TM helix VI in LHR activation.

LH (Trp8Arg/Ile15Thr), LHR (insLQ) and FSHR (Asn680Ser) polymorphisms genotypic prevalence in women with endometriosis and infertility.

PURPOSE: To verify if polymorphisms of LH (Trp8Arg/Ile15Thr), LH receptor (insLQ), and FSH receptor (Asn680Ser) are associated with endometriosis and infertility. METHODS: This is a prospective case-control study. Sixty-seven patients with endometriosis and infertility (study group) and 65 healthy fertile patients (control group) were enrolled in the study between July 2010 and July 2013. All patients had their endometriosis diagnosis made or excluded by laparoscopic surgery; study group was submitted to the surgery for infertility investigation and control group for tubal ligation. Day-3 serum hormones were collected from all patients. Analysis of nucleotide mutations for LH polymorphisms (Trp8Arg and Ile15Thr), LHR polymorphism (insLQ), and FSHR polymorphism (Asn680Ser) were performed by PCR. RESULTS: Day-3 FSH, estradiol and LH serum levels were not different between the groups, while CA-125 was higher in patients with endometriosis and infertility. All polymorphisms studied were in Hardy-Weinberg equilibrium. The prevalence of insLQ was significantly higher in patients with endometriosis and infertility (P = 0.005). Allele occurrence in control group was 0.10 versus 0.25 in infertile endometriosis group (P = 0.001). There was no difference regarding Trp8Arg/Ile15Thr (P > 0.05) and Asn680Ser (P > 0.05) prevalence between groups. CONCLUSION: This is the first time that prevalence of insLQ was shown to be higher in patients with endometriosis and infertility than in healthy fertile patients. There was no difference in LH and FSHR polymorphisms' prevalence between groups.

Targeted single molecule sequencing methodology for ovarian hyperstimulation syndrome.

BACKGROUND: One of the most significant issues surrounding next generation sequencing is the cost and the difficulty assembling short read lengths. Targeted capture enrichment of longer fragments using single molecule sequencing (SMS) is expected to improve both sequence assembly and base-call accuracy but, at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. We developed a targeted single molecule sequencing (T-SMS) panel for genes implicated in ovarian response to controlled ovarian hyperstimulation (COH) for infertility. RESULTS: Target enrichment was carried out using droplet-base multiplex polymerase chain reaction (PCR) technology (RainDance®) designed to yield amplicons averaging 1 kb fragment size from candidate 44 loci (99.8% unique base-pair coverage). The total targeted sequence was 3.18 Mb per sample. SMS was carried out using single molecule, real-time DNA sequencing (SMRT® Pacific Biosciences®), average raw read length = 1178 nucleotides, 5% of the amplicons >6000 nucleotides). After filtering with circular consensus (CCS) reads, the mean read length was 3200 nucleotides (97% CCS accuracy). Primary data analyses, alignment and filtering utilized the Pacific Biosciences® SMRT portal. Secondary analysis was conducted using the Genome Analysis Toolkit for SNP discovery l and wANNOVAR for functional analysis of variants. Filtered functional variants 18 of 19 (94.7%) were further confirmed using conventional Sanger sequencing. CCS reads were able to accurately detect zygosity. Coverage within GC rich regions (i.e.VEGFR; 72% GC rich) was achieved by capturing long genomic DNA (gDNA) fragments and reading into regions that flank the capture regions. As proof of concept, a non-synonymous LHCGR variant captured in two severe OHSS cases, and verified by conventional sequencing. CONCLUSIONS: Combining emulsion PCR-generated 1 kb amplicons and SMRT DNA sequencing permitted greater depth of coverage for T-SMS and facilitated easier sequence assembly. To the best of our knowledge, this is the first report combining emulsion PCR and T-SMS for long reads using human DNA samples, and NGS panel designed for biomarker discovery in OHSS.

Single-molecule analyses of fully functional fluorescent protein-tagged follitropin receptor reveal homodimerization and specific heterodimerization with lutropin receptor.

We have previously shown that the carboxyl terminus (cT) of human follicle-stimulating hormone (FSH, follitropin) receptor (FSHR) is clipped before insertion into the plasma membrane. Surprisingly, several different constructs of FSHR fluorescent fusion proteins (FSHR-FPs) failed to traffic to the plasma membrane. Subsequently, we discovered that substituting the extreme cT of luteinizing hormone (LH) receptor (LHR) to create an FSHR-LHRcT chimera has no effect on FSHR functionality. Therefore, we used this approach to create an FSHR-LHRcT-FP fusion. We found this chimeric FSHR-LHRcT-FP was expressed in HEK293 cells at levels similar to reported values for FSHR in human granulosa cells, bound FSH with high affinity, and transduced FSH binding to produce cAMP. Quantitative fluorescence resonance energy transfer (FRET) analysis of FSHR-LHRcT-YFP/FSHR-LHRcT-mCherry pairs revealed an average FRET efficiency of 12.9 ± 5.7. Advanced methods in single-molecule analyses were applied in order to ascertain the oligomerization state of the FSHR-LHRcT. Fluorescence correlation spectroscopy coupled with photon-counting histogram analyses demonstrated that the FSHR-LHRcT-FP fusion protein exists as a freely diffusing homodimer in the plasma membrane. A central question is whether LHR could oligomerize with FSHR, because both receptors are coexpressed in differentiated granulosa cells. Indeed, FRET analysis revealed an average FRET efficiency of 14.4 ± 7.5 when the FSHR-LHR cT-mCherry was coexpressed with LHR-YFP. In contrast, coexpression of a 5-HT2cVSV-YFP with FSHR-LHR cT-mCherry showed only 5.6 ± 3.2 average FRET efficiency, a value indistinguishable from the detection limit using intensity-based FRET methods. These data demonstrate that coexpression of FSHR and LHR can lead to heterodimerization, and we hypothesize that it is possible for this to occur during granulosa cell differentiation.

Single molecule analysis of functionally asymmetric G protein-coupled receptor (GPCR) oligomers reveals diverse spatial and structural assemblies.

Formation of G protein-coupled receptors (GPCRs) into dimers and higher order oligomers represents a key mechanism in pleiotropic signaling, yet how individual protomers function within oligomers remains poorly understood. We present a super-resolution imaging approach, resolving single GPCR molecules to ∼ 8 nm resolution in functional asymmetric dimers and oligomers using dual-color photoactivatable dyes and localization microscopy (PD-PALM). PD-PALM of two functionally defined mutant luteinizing hormone receptors (LHRs), a ligand-binding deficient receptor (LHR(B-)) and a signaling-deficient (LHR(S-)) receptor, which only function via intermolecular cooperation, favored oligomeric over dimeric formation. PD-PALM imaging of trimers and tetramers revealed specific spatial organizations of individual protomers in complexes where the ratiometric composition of LHR(B-) to LHR(S-) modulated ligand-induced signal sensitivity. Structural modeling of asymmetric LHR oligomers strongly aligned with PD-PALM-imaged spatial arrangements, identifying multiple possible helix interfaces mediating inter-protomer associations. Our findings reveal that diverse spatial and structural assemblies mediating GPCR oligomerization may acutely fine-tune the cellular signaling profile.