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 ß-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.

Functional characterization of follicle-stimulating hormone and luteinizing hormone receptors in cloudy catshark, Scyliorhinus torazame.

The follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in cloudy catshark were cloned, and recombinant FSHR and LHR were expressed for characterization. Ventral lobe extract (VLE) from the pituitary contains homologous FSH and LH, and it stimulated the cAMP signaling of FSHR and LHR dose-dependently. Two transcript variants of LHR (LHR-L with exon 10 and LHR-S without) were identified, and LHR-S was the dominant form with higher basal cAMP activity without VLE stimulation. Among various developmental stages of follicles, FSHR expression was mainly associated with the pre-vitellogenic and early white follicles. When follicles were recruited into vitellogenesis, the expression of FSHR decreased while of LHR was upregulated reciprocally, suggesting that LHR may also be responsible for the control of vitellogenesis in chondrichthyans. The expression of LHR-L was upregulated among maturing follicles before ovulation, indicating LHR-L could have a specific role in receiving the LH surge signal for final maturation. Plasma LH-like activity was transiently increased prior to the progesterone (P4)-surge and testosterone-drop at the beginning of P4-phase, supporting a pituitary control of follicle-maturation via LH signaling in chondrichthyans. The expression of follicular LHR was downregulated during the P4-phase when LH-like activity was high, indicating that the LH-dependent downregulation of LHR is conserved in chondrichthyans as it is in other vertebrate lineages. (213 words).

Glycoprotein-glycoprotein Receptor Binding Detection Using Bioluminescence Resonance Energy Transfer.

The glycoprotein receptors, members of the large G protein-coupled receptor family, are characterized by a large extracellular domains responsible for binding their glycoprotein hormones. Hormone-receptor interactions are traditionally analyzed by ligand-binding assays, most often using radiolabeling but also by thermal shift assays. Despite their high sensitivity, these assays require appropriate laboratory conditions and, often, purified plasma cell membranes, which do not provide information on receptor localization or activity because the assays typically focus on measuring binding only. Here, we apply bioluminescence resonance energy transfer in living cells to determine hormone-receptor interactions between a Gaussia luciferase (Gluc)-luteinizing hormone/chorionic gonadotropin receptor (LHCGR) fusion and its ligands (human chorionic gonadotropin or LH) fused to the enhanced green fluorescent protein. The Gluc-LHCGR, as well as other Gluc-G protein-coupled receptors such as the somatostatin and the C-X-C motif chemokine receptors, is expressed on the plasma membrane, where luminescence activity is equal to membrane receptor expression, and is fully functional. The chimeric enhanced green fluorescent protein-ligands are properly secreted from cells and able to bind and activate the wild-type LHCGR as well as the Gluc-LHCGR. Finally, bioluminescence resonance energy transfer was used to determine the interactions between clinically relevant mutations of the hormones and the LHCGR that show that this bioassay provides a fast and effective, safe, and cost-efficient tool to assist the molecular characterization of mutations in either the receptor or ligand and that it is compatible with downstream cellular assays to determine receptor activation/function.

Therapeutic benefits of central LH receptor agonism in the APP/PS1 AD model involve trophic and immune regulation and are reproductive status dependent.

The mechanisms that underly reproductive hormone effects on cognition, neuronal plasticity, and AD risk, particularly in relation to gonadotropin LH receptor (LHCGR) signaling, remain poorly understood. To address this gap in knowledge and clarify the impact of circulating steroid hormones on the therapeutic effects of CNS LHCGR activation, we delivered the LHCGR agonist human chorionic gonadotropin (hCG) intracerebroventricularly (ICV) and evaluated functional, structural, plasticity-related signaling cascades, Aß pathology, and transcriptome differences in reproductively intact and ovariectomized (OVX) APP/PS1 AD female mice. Here we demonstrate that CNS hCG delivery restored function to wild-type levels only in OVX APP/PS1 mice. Spine density was increased in all hCG treated groups independently of reproductive status. Notably, increases in BDNF signaling and cognition, were selectively upregulated only in the OVX hCG-treated group. RNA sequencing analyses identified a significant increase in peripheral myeloid and pro-inflammatory genes within the hippocampi of the OVX group that were completely reversed by hCG treatment, identifying a potential mechanism underlying the selective therapeutic effect of LHCGR activation. Interestingly, in intact mice, hCG administration mimicked the effects of gonadectomy. Together, our findings indicate that CNS LHCGR agonism in the post-menopausal context is beneficial through trophic and immune mechanisms. Our findings also underscore the presence of a steroid-LHCGR mechanistic interaction that is unexplored yet potentially meaningful to fully understand "post-menopausal" brain function and CNS hormone treatment response.

Expanding the phenotypic spectrum of LHCGR signal peptide insertion variant: novel clinical and allelic findings causing Leydig cell hypoplasia type II.

PURPOSE: Leydig cell hypoplasia (LCH) type II is a rare disease with only a few cases reported. Patients presented with hypospadias, micropenis, undescended testes, or infertility. In this study, we report a new patient with compound heterozygous variants in the LHCGR gene and LCH type II phenotype. METHODS: Whole exome sequencing (WES) was performed followed by Sanger sequencing to confirm the detected variants in the patient and his parents. RESULTS: A novel missense variant (p.Phe444Cys) was identified in a highly conserved site and is verified to be in trans with the signal peptide's 33-bases insertion variant. CONCLUSION: Our research provides a more comprehensive clinical and genetic spectrum of Leydig cell hypoplasia type II. It highlighted the importance of WES in the diagnosis of this uncommon genetic disorder as well as the expansion of the genotype of LCH type II.

Fetal Leydig cells: What we know and what we don't.

During male fetal development, testosterone plays an essential role in the differentiation and maturation of the male reproductive system. Deficient fetal testosterone production can result in variations of sex differentiation that may cause infertility and even increased tumor incidence later in life. Fetal Leydig cells in the fetal testis are the major androgen source in mammals. Although fetal and adult Leydig cells are similar in their functions, they are two distinct cell types, and therefore, the knowledge of adult Leydig cells cannot be directly applied to understanding fetal Leydig cells. This review summarizes our current knowledge of fetal Leydig cells regarding their cell biology, developmental biology, and androgen production regulation in rodents and human. Fetal Leydig cells are present in basement membrane-enclosed clusters in between testis cords. They originate from the mesonephros mesenchyme and the coelomic epithelium and start to differentiate upon receiving a Desert Hedgehog signal from Sertoli cells or being released from a NOTCH signal from endothelial cells. Mature fetal Leydig cells produce androgens. Human fetal Leydig cell steroidogenesis is LHCGR (Luteinizing Hormone Chronic Gonadotropin Receptor) dependent, while rodents are not, although other Gαs -protein coupled receptors might be involved in rodent steroidogenesis regulation. Fetal steroidogenesis ceases after sex differentiation is completed, and some fetal Leydig cells dedifferentiate to serve as stem cells for adult testicular cell types. Significant gaps are acknowledged: (1) Why are adult and fetal Leydig cells different? (2) What are bona fide progenitor and fetal Leydig cell markers? (3) Which signaling pathways and transcription factors regulate fetal Leydig cell steroidogenesis? It is critical to discover answers to these questions so that we can understand vulnerable targets in fetal Leydig cells and the mechanisms for androgen production that when disrupted, leads to variations in sex differentiation that range from subtle to complete sex reversal.

Effect of Gonadotropin-Releasing Hormone Antagonist on the Expression Patterns of Insulin-Like Growth Factor 1, Androgen Receptor, and Luteinizing Hormone Receptor in an Experimental Rat Model of Functional Ovarian Cysts.

We studied the expression of insulin-like growth factor 1 (IGF-1), androgen receptor (AR) and luteinizing hormone receptor (LHR) in the ovaries under the conditions of the modeling and subsequent treatment of functional ovarian cysts with gonadotropin-releasing hormone antagonist (ant-GnRH). The intensity of IGF-1, LHR, and AR expression in the generative elements of rat ovaries changed under conditions of functional ovarian cysts simulation, as well as during treatment with ant-GnRH. In both experimental groups, the expression levels of the studied markers in preantral follicles and epithelial lining of cysts were found to be related to the number of growing follicles and cysts. A divergence of LHR and AR expression indices and a more pronounced decrease in the number of cystic cavities were observed in the group receiving ant-GnRH. These changes demonstrate a positive effect of ant-GnRH on intra-ovarian regulatory factors and a therapeutic effect in functional ovarian cysts.

Elevated luteinizing hormone receptor signaling or selenium treatment leads to comparable changes in adrenal cortex histology and androgen-AR/ZIP9 signaling.

The importance and regulation of adrenal androgen production and signaling are not completely understood and are scarcely studied. In addition, there is still a search for appropriate animal models and experimental systems for the investigation of adrenal physiology and disease. Therefore, the main objective of the study was to evaluate the effect of luteinizing hormone (LH) signaling and selenium (Se2+) exposure on androgen adrenal signaling via canonical androgen receptor (AR), and membrane androgen receptor acting as zinc transporter (zinc- and iron-like protein 9; ZIP9). For herein evaluations, adrenals isolated from transgenic mice with elevated LH receptor signaling (KiLHRD582G) and adrenals obtained from rabbits used for ex vivo adenal cortex culture and exposure to Se2+ were utilized. Tissues were assessed for morphological, morphometric, and Western blot analyses and testosterone and zinc level measurements.Comparison of adrenal cortex histology and morphometric analysis in KiLHRD582G mice and Se2+-treated rabbits revealed cell hypertrophy. No changes in the expression of proliferating cell nuclear antigen (PCNA) were found. In addition, AR expression was decreased (p < 0.001) in both KiLHRD582G mouse and Se2+-treated rabbit adrenal cortex while expression of ZIP9 showed diverse changes. Its expression was increased (P < 0.001) in KiLHRD582G mice and decreased (P < 0.001) in Se2+-treated rabbits but only at the dose 10 ug/100 mg/ tissue. Moreover, increased testosterone levels (P < 0.05) and zinc levels were detected in the adrenal cortex of KiLHRD582G mice whereas in rabbit adrenal cortex treated with Se2+, the effect was the opposite (P < 0.001).

The mystery of transient pregnancy-induced cushing's syndrome: a case report and literature review highlighting GNAS somatic mutations and LHCGR overexpression.

PURPOSE: Transient pregnancy-induced Cushing's syndrome is a rare condition characterized by the manifestation of symptoms solely during pregnancy, which typically resolve spontaneously following delivery or miscarriage. While it has been established that GNAS is associated with adrenal tumors, its specific role in the pathogenesis of pregnancy-induced Cushing's syndrome remains uncertain.This work aims to examine the association between GNAS mutation and pregnancy-induced Cushing's syndrome. METHODS: DNA was extracted from patients' peripheral blood and tumor tissues for whole-exome sequencing (WES) and Sanger sequencing. We used AlphaFold to predict the protein structure of wild-type and mutant GNAS and to make functional predictions, and immunohistochemistry was used to detect disease-associated protein expression. A review and summary of reported cases of transient pregnancy-induced Cushing's syndrome induced by pregnancy was conducted. RESULTS: Using WES, we identified a somatic mutation in GNAS (NM_000516, c.C601T, p.R201C) that was predicted to have a deleterious effect using computational methods, such as AlphaFold. Human chorionic gonadotropin (hCG) stimulation tests had weakly positive results, and immunohistochemical staining of adrenal adenoma tissue also revealed positivity for luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and cytochrome P450 family 11 subfamily B member 1 (CYP11B1). We reviewed 15 published cases of transient Cushing's syndrome induced by pregnancy. Among these cases, immunohistochemical staining of the adrenal gland showed positive LHCGR expression in 3 case reports, similar to our findings. CONCLUSION: Transient pregnancy-induced Cushing's syndrome may be associated with somatic GNAS mutations and altered adrenal pathology due to abnormal activation of LHCGR.

Luteinizing hormone stimulates ingression of mural granulosa cells within the mouse preovulatory follicle†.

Luteinizing hormone (LH) induces ovulation by acting on its receptors in the mural granulosa cells that surround a mammalian oocyte in an ovarian follicle. However, much remains unknown about how activation of the LH receptor modifies the structure of the follicle such that the oocyte is released and the follicle remnants are transformed into the corpus luteum. The present study shows that the preovulatory surge of LH stimulates LH receptor-expressing granulosa cells, initially located almost entirely in the outer layers of the mural granulosa, to rapidly extend inwards, intercalating between other cells. The cellular ingression begins within 30 min of the peak of the LH surge, and the proportion of LH receptor-expressing cell bodies in the inner half of the mural granulosa layer increases until the time of ovulation, which occurs at about 10 h after the LH peak. During this time, many of the initially flask-shaped cells appear to detach from the basal lamina, acquiring a rounder shape with multiple filipodia. Starting at about 4 h after the LH peak, the mural granulosa layer at the apical surface of the follicle where ovulation will occur begins to thin, and the basolateral surface develops invaginations and constrictions. Our findings raise the question of whether LH stimulation of granulosa cell ingression may contribute to these changes in the follicular structure that enable ovulation.

Interacting with luteinizing hormone receptor provides a new elucidation of the mechanism of anti-androgenicity of bisphenol S.

Bisphenol S (BPS) exhibited inhibitory effects on androgen synthesis, but its target of action remains unclear. We investigated the effects of BPS exposure at environmentally relevant concentrations (1 µg/L, 10 µg/L and 100 µg/L) for 48 h on androgen synthesis in rat ovarian theca cells and explored the underlying mechanisms, target site and target molecule. The results showed that BPS exposure inhibited the transcript levels of steroidogenic genes and reduced the contents of androgen precursors, testosterone and dihydrotestosterone. BPS exposure decreased the phosphorylation levels of extracellular signal-related kinase 1/2 (ERK1/2), and the inhibitory effects of BPS on testosterone content and steroidogenic gene expression were blocked by ERK1/2 agonist LY2828360, suggesting that ERK1/2 signaling pathway mediates the inhibitory effects of BPS on androgen synthesis. BPS mainly accumulated on the cell membrane, impermeable BPS-bovine serum albumin exposure still inhibited androgen synthesis, BPS interacted with rat luteinizing hormone receptor (LHR) via formation of hydrogen bonds in the transmembrane region, and the inhibitory effects of BPS on ERK1/2 phosphorylation were blocked by luteinizing hormone (the natural agonist of LHR), indicating that LHR located on the cell membrane is the target of action of BPS. This paper provides a new elucidation of the mechanism of anti-androgenicity of BPS, especially for the non-genomic pathways.

Comparison of Steroidogenic and Ovulation-Inducing Effects of Orthosteric and Allosteric Agonists of Luteinizing Hormone/Chorionic Gonadotropin Receptor in Immature Female Rats.

Gonadotropins, including human chorionic gonadotropin (hCG), are used to induce ovulation, but they have a number of side effects, including ovarian hyperstimulation syndrome (OHSS). A possible alternative is allosteric luteinizing hormone (LH)/hCG receptor agonists, including the compound TP4/2 we developed, which remains active when administered orally. The aim was to study the effectiveness of TP4/2 (orally, 40 mg/kg) as an ovulation inducer in FSH-stimulated immature female rats, compared with hCG (s.c., 15 IU/rat). TP4/2 stimulated progesterone production and corpus luteum formation; time-dependently increased the ovarian expression of steroidogenic genes (Star, Cyp11a1, Cyp17a1) and genes involved in ovulation regulation (Adamts-1, Cox-2, Egr-1, Mt-1); and increased the content of metalloproteinase ADAMTS-1 in the ovaries. These effects were similar to those of hCG, although in some cases they were less pronounced. TP4/2, in contrast to hCG, maintained normal LH levels and increased the ovarian expression of the LH/hCG receptor gene, indicating preservation of ovarian sensitivity to LH, and did not cause a sustained increase in expression of vascular endothelial growth factor-A involved in OHSS. Thus, TP4/2 is an effective ovulation inducer that, unlike hCG, has a lower risk of OHSS and ovarian LH resistance due to its moderate stimulating effect on steroidogenesis.

Gonadotropin independent sexual precocity in a Pakistani male infant from an activating mutation in LHCGR gene.

A male child, aged seven months, visited the out patients clinic of the National Institute of Child Health, Karachi, in May 2020 with the features of iso-sexual puberty. After ruling out the more common causes of early puberty, like congenital adrenal hyperplasia and tumours secreting chorionic gonadotropin hormone, hormonal assessment indicated raised testosterone independent of gonadotropin. The volume of the testicles was symmetric and testicular ultrasonography revealed no mass. Genetic analysis for the LHCGR gene was performed for confirmation which revealed activating heterozygous missense pathogenic mutation in c.1732G>T (p.Asp578Tyr). This is the first reported case of testotoxicosis (FMPP) from Pakistan which was genetically confirmed.

Precise Correction of Lhcgr Mutation in Stem Leydig Cells by Prime Editing Rescues Hereditary Primary Hypogonadism in Mice.

Hereditary primary hypogonadism (HPH), caused by gene mutation related to testosterone synthesis in Leydig cells, usually impairs male sexual development and spermatogenesis. Genetically corrected stem Leydig cells (SLCs) transplantation may provide a new approach for treating HPH. Here, a novel nonsense-point-mutation mouse model (LhcgrW495X ) is first generated based on a gene mutation relative to HPH patients. To verify the efficacy and feasibility of SLCs transplantation in treating HPH, wild-type SLCs are transplanted into LhcgrW495X mice, in which SLCs obviously rescue HPH phenotypes. Through comparing several editing strategies, optimized PE2 protein (PEmax) system is identified as an efficient and precise approach to correct the pathogenic point mutation in Lhcgr. Furthermore, delivering intein-split PEmax system via lentivirus successfully corrects the mutation in SLCs from LhcgrW495X mice ex vivo. Gene-corrected SLCs from LhcgrW495X mice exert ability to differentiate into functional Leydig cells in vitro. Notably, the transplantation of gene-corrected SLCs effectively regenerates Leydig cells, recovers testosterone production, restarts sexual development, rescues spermatogenesis, and produces fertile offspring in LhcgrW495X mice. Altogether, these results suggest that PE-based gene editing in SLCs ex vivo is a promising strategy for HPH therapy and is potentially leveraged to address more hereditary diseases in reproductive system.

Post-trigger luteinizing hormone concentration to positively predict oocyte yield in the antagonist protocol and its association with genetic variants of LHCGR.

BACKGROUND: The concentration of human chorionic gonadotropin (hCG)/ luteinizing hormone (LH) after triggering is generally accepted as a predictor of the normal ovarian response to the trigger, but few studies have explored the distribution model of concentration and its impact on oocyte yield. Genetic variations in LHCGR, known as a receptor for hCG and LH, also play a role in oocyte maturation and retrieval. The objective of the study was to investigate the impact of concentrations of hCG/LH after triggering on oocyte yield and its association with genetic variants of LHCGR. METHODS: A retrospective cohort study including 372 antagonist IVF cycles, in which 205 received the recombinant hCG trigger and 167 received the gonadotropin-releasing hormone agonist (GnRH-a) trigger, was conducted. The post-trigger concentrations of hCG/LH and the LHCGR N312S (rs2293275) genotype were evaluated in patients to analyse the impact of these factors on oocyte yield. RESULTS: The oocyte retrieval rate (ORR) increased significantly among the low-, medium- and high-hCG-concentration groups (0.91 ± 0.25, 0.99 ± 0.23 and 1.08 ± 0.19, P < 0.001) and among the low-, medium- and high-LH-concentration groups (0.80 ± 0.29, 0.95 ± 0.21 and 1.07 ± 0.19, P < 0.001). The Pearson correlation coefficient between the post-trigger hCG concentration and ORR was 0.242 (P < 0.001), and that between the LH concentration and ORR was 0.454 (P < 0.001). After adjustment for confounding factors, high post-trigger LH concentrations remained associated with the significantly higher ORRs (adjusted R2 = 0.541, P < 0.001). Patients with the AG genotype of LHCGR N312S were more likely to have low post-trigger LH concentrations (46.10 IU/L versus 60.91 IU/L, P < 0.001) and a significantly lower ORR (0.85 versus 0.96, P = 0.042) than patients with the GG genotype after the GnRH-a trigger. CONCLUSIONS: The post-trigger LH concentration can positively predict oocyte yield in antagonist IVF cycles, and patients with the AG genotype of LHCGR rs2293275 could have a suboptimal oocyte yield using the GnRH-a trigger.

Highly-Sensitive In Vitro Bioassays for FSH, TSH, PTH, Kp, and OT in Addition to LH in Mouse Leydig Tumor Cell.

We demonstrate here that highly sensitive in vitro bioassays for FSH, TSH, and PTH can be set up in mouse Leydig Tumor Cells (mLTC), in addition to the normal LH/CG bioassay, after they were transfected with expression vectors encoding the corresponding Gs Protein-Coupled Receptors (GsPCR), such as FSHR, TSHR, or PTHR. Although the ß2 adrenergic receptor is also a GsPCR, its expression in mLTC led to a significant but very low cAMP response compared to those observed with FSH, TSH, or PTH. Similarly, after transfection of the GiPCR MT1 melatonin receptor, we did not observe any inhibitory effect by melatonin of the LH or hCG stimulation. Interestingly, after transfection of mLTC with the human kisspeptin receptor (hKpR), which is a GqPCR, we observed a dose-dependent synergy of 10-12-10-7 M kisspeptin variants with a fixed concentration of 0.3 nM LH or hCG. Without any exogenous receptor transfection, a 2 h preincubation with OT or AVP led to a dose-dependent cAMP response to a fixed dose of LH or hCG. Therefore, highly sensitive in vitro bioassays for various hormones and other GPCR ligands can be set up in mLTC to measure circulating concentrations in only 3-10 µL of blood or other body fluids. Nevertheless, the development of an LHRKO mLTC cell line will be mandatory to obtain strict specificity for these bioassays to eliminate potential cross-reaction with LH or CG.

Specific Signal Transduction of Constitutively Activating (D576G) and Inactivating (R476H) Mutants of Agonist-Stimulated Luteinizing Hormone Receptor in Eel.

We investigated the mechanism of signal transduction using inactivating (R476H) and activating (D576G) mutants of luteinizing hormone receptor (LHR) of eel at the conserved regions of intracellular loops II and III, respectively, naturally occurring in mammalian LHR. The expression of D576G and R476H mutants was approximately 58% and 59%, respectively, on the cell surface compared to those of eel LHR-wild type (wt). In eel LHR-wt, cAMP production increased upon agonist stimulation. Cells expressing eel LHR-D576G, a highly conserved aspartic acid residue, exhibited a 5.8-fold increase in basal cAMP response; however, the maximal cAMP response by high-agonist stimulation was approximately 0.62-fold. Mutation of a highly conserved arginine residue in the second intracellular loop of eel LHR (LHR-R476H) completely impaired the cAMP response. The rate of loss in cell-surface expression of eel LHR-wt and D576G mutant was similar to the agonist recombinant (rec)-eel LH after 30 min. However, the mutants presented rates of loss higher than eel LHR-wt did upon rec-eCG treatment. Therefore, the activating mutant constitutively induced cAMP signaling. The inactivating mutation resulted in the loss of LHR expression on the cell surface and no cAMP signaling. These data provide valuable information regarding the structure-function relationship of LHR-LH complexes.

Luman regulates the activity of the LHCGR promoter.

Testosterone in male mammals is mainly secreted by testicular Leydig cells, and its secretion process is regulated by the hypothalamic-pituitary-gonadal axis. After receiving the luteinizing hormone (LH) stimulus signal, the lutropin/choriogonadotropin receptor (LHCGR) on the Leydig cell membrane transfers the signal into the cell and finally increases the secretion of testosterone by upregulating the expression of steroid hormone synthase. In previous experiments, we found that interfering with the expression of the Luman protein can significantly increase testosterone secretion in MLTC-1 cells. In this experiment, we found that knockdown of Luman in MLTC-1 cells significantly increased the concentration of cAMP and upregulated the expression of AC and LHCGR. Moreover, an analysis of the activity of the LHCGR promoter by a dual luciferase reporter system showed that knockdown of Luman increased the activity of the LHCGR promoter. Therefore, we believe that knockdown of Luman increased the activity of the LHCGR promoter and upregulated the expression of LHCGR, thereby increasing the concentration of intracellular cAMP and ultimately leading to an increase of testosterone secretion by MLTC-1 cells.

LGR4: A New Receptor Member in Endocrine and Metabolic Diseases.

Classic hormone membrane receptors, such as leucine-rich repeat-containing G protein-coupled receptor (LGR) 1 (follicle-stimulating hormone receptor), LGR2 (luteinizing hormone receptor), and LGR3 (thyrotropin receptor), are crucial in endocrinology and metabolism, and the identification of new receptors can advance this field. LGR4 is a new member of this G protein-coupled receptor family and shows ways of expression and function similar to those of LGR1/2/3. Several recent studies have reported that, unlike LGR5/6, LGR4 plays essential roles in endocrine and metabolic diseases, including hypothalamic-gonadal axis defects, mammary gland dysplasia, osteoporosis, cardiometabolic diseases, and obesity. An inactivating mutation p.R126X in LGR4 leads to osteoporosis, electrolyte disturbance, abnormal sex hormone levels, and weight loss, whereas an activating mutation p.A750T is associated with bone mineral density, insulin resistance, and adiposity. Though several paracrine ligands are known to act on LGR4, the endocrine ligands of LGR4 remain poorly defined. In this review, we highlight LGR4 dysfunction in clinical diseases, animal models, and pathophysiological changes, discuss their known ligands and downstream signaling pathways, and identify unresolved questions and future perspectives of this new receptor.