Part Ⅰ：Possible Relevance Of Soluble Luteinizing Hormone Receptor During Development And Adulthood in Boys And Men
Apr 06, 2023
Simple Summary
The reproductive hormones luteinizing hormone (LH) and human chorionic gonadotropin (hCG), both agonists of the luteinizing hormone receptor (LHCGR), are essential for male reproduction during development and adulthood. lHCGR is expressed and stimulates testosterone production by testicular Leydig cells. In this study, we demonstrate that soluble LHCGR is present in the blood, urine, and semen of healthy boys and men, as well as in patients with sex chromosome aberrations. We show how circulating levels of sLHCGR correlate with pubertal development, testicular function, and semen quality, and demonstrate that LHCGR is released from fetal human non-gonadal tissue. HCG is released into serum via the testes and other organs, suggesting a possible extragonadal role for LH or hCG in boys and men.
Abstract
Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are agonists of the luteinizing hormone receptor (LHCGR), which regulates male reproductive function. lHCGR may be released into body fluids. We wanted to determine whether soluble LHCGR is a marker of gonadal function. Cross-sectional, longitudinal, and interventional studies were performed on 195 healthy boys and men and 396 men with infertility, deficiency, or Klinefelter syndrome (KS) to link LHCGR measured in serum, semen, urine, and liver/kidney arteries to gonadal function. lHCGR was measured in vitro and in vivo models of human testicular tissue and cell lines, xenograft mouse models, and human Western blot results showed that LHCGR fragments were detected in similar-sized serum and gonadal tissues using three different antibodies. Even after human xenografts, the LHCGR-ELISA showed no interspecies cross-reactivity or non-specific reactions in mouse serum. In contrast, sLHCGR was released into the culture medium after human fetal kidney and adrenal gland culture. Serum sLHCGR was significantly lower in healthy boys at puberty (p = 0.0001). In healthy males, serum sLHCGR was negatively correlated with the inhibin B/FSH ratio (β-0.004,p = 0.027). In infertile men, semen sLHCGR was negatively correlated with serum FSH (β 0.006, p = 0.009), sperm concentration (β-3.5, p = 0.003), and total sperm count (β-3.2, p = 0.007). Injection of hCG reduced sLHCGR in the serum and urine of healthy men (p <0.01). In conclusion, sLHCGR is released into body fluids and is associated with pubertal development and gonadal function. The circulating sLHCGR in orchid males suggests that sLHCGR in serum may originate in non-gonadal tissues and may function in non-gonadal tissues. (Clinical Trials: NTC01411527, NCT01304927, NCT03418896).
Keywords
LH receptor; puberty; development; infertility; Cistanche benefits; extra-gonadal effects; gonadotropins; fetal kidney; fetal adrenal gland; TCAM2; NTera2
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Introduction
Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are both agonists of the luteinizing hormone receptor (LHCGR), which is essential for male reproduction during development and adulthood [1]. HCG is a G protein-coupled receptor known to be expressed in Leydig, granular lutein, and membrane cells and is a potent regulator of sex steroid production [1]. hCG is a potent and persistent agonist but is only physiologically important during fetal development [2]. Except for some germ cell cancers and pancreatic cancers, hCG is not produced in adulthood, although an earlier study found low pulsatile secretion of pituitary-hCG in adult males and females. hCG is used to treat women to induce ovulation during fertility treatment, for men to stimulate testosterone synthesis and potential spermatogenesis in hypogonadal men, or as a diagnostic test for endogenous testosterone production [4]. Ablation of the mouse LH receptor (LHCGR) leads to a dramatic decrease in testosterone and semen quality [5], and several research groups have identified mutations and SNP in LHCGR, demonstrating its importance for human reproductive function [6-8].
The LHCGR gene is located on chromosome 2 and consists of 11 exons, of which exons 1-10 encode extracellular structural domains and exon 11 encodes 7 transmembrane and intracellular structural domains [9]. There are several isoforms of LHCGR, one of which is thought to be secreted because it lacks a transmembrane anchor [10]. Another possibility is that the release of LHCGR into body fluids is dependent on the enzymatic cleavage of proteins on the cell membrane, such as ADAMs, MMPs, and other known transmembrane proteins cleaved. Indeed, hCG has been shown to specifically upregulate MMP-2 and MMP-9 [11,12], and several studies have shown post-transcriptional regulation of LHCGR [13,14]. Soluble LHCGR (LHC) has been measured in female serum and follicular fluid by the ELISA platform [15,16]. The specificity of the antibodies used in the ELISA assay was validated by expressing different LHCGR fragments in CHO cells for epitope localization and detecting expression in the human placenta [15-17]. sLHCGR was detected in serum by LC-MS/MS [18]. serum concentrations of sLHCGR in women were associated with fertility treatment, preeclampsia, preterm birth, and pregnancy in children carrying Down syndrome implantation rates [15,16], but have not been validated by other research groups to date. To our knowledge, sLHCGR has never been described in the body fluids of healthy males, developing adolescent boys, or males with sex chromosome aberrations. sLHCGR may function in the male circulation as a marker of gonadal function through high-affinity binding transport or alteration of LH or hCG activity.
Materials and Methods
1. Cohorts
Young Men from the General Population
In Denmark, all healthy young men over the age of 18 must undergo a physical examination to determine their suitability for military service. Since 1996, approximately 300 conscripts, representing the general population, have been examined for semen quality each year. They all provided semen samples, completed a questionnaire, had ultrasound examinations of their testicles, and had blood samples drawn. Serum and semen samples from the biobank, as well as ultrasound assessments and information on BMI and age from 148 men surveyed in 2013 and 2014, were included in the current study (Ethics Committee approval: H-KF-289428).
Pre- and Post-Pubertal Healthy Boys
The Copenhagen Adolescence Study is a combined cross-sectional and longitudinal study of healthy Danish children [20]. From randomly selected public schools, 6203 children were invited to participate. The total number of participants was 2020 children. Longitudinal follow-up of 209 children (101 boys) was conducted every 6 months and included physical examination, Tanner assessment of pubertal stages, and blood and urine samples. Thirty-six boys with longitudinal follow-up were included in this study to assess sLHCGR during puberty (clinical trial: NTC01411527).
Infertile Men without Serious Comorbidity
The Copenhagen-Bone-Gonad Study is a cohort study of 307 infertile men without severe comorbidities who attended our male clinic from 2011 to 2015, including a total of 1427 men [21,22]. All men were referred to our clinic due to low semen quality and desire for parenthood. All underwent a physical examination, including testicular ultrasound, had blood samples drawn, and provided semen samples. A specialist obtained their detailed medical histories. The men were followed for 150 days by the researchers and took vitamin D or a placebo as previously described. All data provided in this study were from the initial pre-intervention visit and all included variables were predefined (clinical trial: NCT01304927).
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Men and Boys with Klinefelter Syndrome and Patients with Anorchia
We identified 64 boys and men with Klinefelter syndrome (KS), 47 post-pubertal and 16 pre-pubertal, defined according to Tanner staging, from our male clinic. We had exact chromosomal aberrations, medical history, pubertal onset status, and serum information available for all. 7 boys underwent longitudinal follow-up with regular clinical evaluation and blood sampling before and during puberty onset.
We also included 8 males with normal chromosome count who had bilateral testes removed for previous conditions: testicular germ cell carcinoma (n = 4), diffuse prostate cancer (n = 1), bilateral cryptorchidism, and pediatric orchiectomy (n = 1), bilateral germ cell neoplasia in situ (n = 1), and advanced testicular cancer after removal of the remaining testes due to trauma (n = 1).
Adults Undergoing Splanchnicus Flow Measurement
We included three men and three postmenopausal women with normal liver function who were admitted for suspected mesenteric ischemia and referred for measurement of visceral blood flow but showed no mesenteric ischemia. Blood samples were collected simultaneously from the liver, kidney, femoral vein, and the corresponding artery (Ethics Committee approval: H.18048245)
Intervention Study: Healthy Men Exposed to Human Chorion Gonadotropin
Eleven healthy men were recruited to participate in the clinical intervention through a Danish Rigshospitalet announcement. Volunteers were treated with 5000 IU human chorionic gonadotropin and their maximum production of endogenous testosterone was measured by comparing baseline blood and urine samples with follow-up samples collected 8, 24, and 72 hours after hCG injection (clinical trial: NCT03418896).
2. Mice, Human Tissue, and Cell Lines
Two strains of human testicular cancer cells NTera-2 and TCam-2, representing embryonal carcinoma and seminoma, respectively, were used and cultured as previously described [23].
Sera were collected from tumor-free nude mice (WT) and TCam-2 xenograft nude mice treated with vector, LH, or hCG, respectively (Animal Experimentation Inspectorate, Copenhagen, Denmark, license number: 2012-15-2934-00051).
First-trimester human fetal tissues were collected from donated women's tissues after planned abortions. Fetal kidney and fetal adrenal tissue were cultured in suspension drop models as described previously and the medium was collected for analysis [24,25] (Ethics Committee approval: H-1-2012-007).
Adult testicular specimens were obtained from ovariectomized men with testicular cancer (Ethics Committee approval: H-1-2012-007). Peritumoral tissue containing non-malignant areas was stored at - 80◦ C or fixed overnight at 4◦ C in formalin or modified Stieve 's-fixative (200 mL 37% formaldehyde, 40 mL acetic acid in 1 L 0.05 M phosphate buffer, pH 7.4).
3. Biochemical Analyses
Detection of total sLHCGR in serum, semen, urine, and various culture media was performed in a validation process using ELISA kits from the New Dutch Biomarker Catalyst Laboratory (NBCL) using the antibody LHR029 against human LHCGR and measuring all of these repeatedly. the ELISA assay had a detection limit of 0.01 pmol/mL and a maximum level of 15.55 Six men from the general population with sLHCGR levels above the upper limit of detection had their samples diluted and remeasured, showing levels up to 30 pmol/mL. the performance of the ELISA platform has been described in detail previously [15-17].
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We also set up two different in-house ELISA, set up by our laboratory technicians, and all further reagents were provided by Origin Biomarkers Ltd, UK, according to their suggested setup [26]. Firstly, circulating sLHCGR bound to hCG was detected using sandwich ELISA kits with antibodies 5A10C9 (ProMab) and am00904pun (OriGene) directed against LHCGR and hCG, respectively. Secondly, we similarly used in-house ELISA to determine total liver, kidney, and lower limb arterial and venous samples from six adults sLHCGR, where we used the same antibody LHR029 (NBCL Holland) against sLHCGR. Two different protocols were tested. All sLHCGR measurements described in this paper were performed using NBCL ELISA, except for the hCG-sLHCGR complex and arterial/venous samples from the organs described above. In healthy boys, testosterone was analyzed using the DPC-coated- a count RIA kit (Diagnostic Products, Los Angeles, CA) with a limit of detection (LOD) of 0.23 nmol/L and a CV of 8.6% using time-resolved immunofluorescence (Delfia; Wallac, Turku, Finland ), the detection limit was 0.20 nmol/l with a CV of 6.4%. In adults, testosterone and sex hormone binding globulin (SHBG) were analyzed by chemiluminescence immunoassay (Access, Beckman Coulter, USA) with LODs of 0.35 nmol/L and 0.33 nmol/L, respectively. Serum and urine FSH and LH levels were measured using time-resolved immunofluorescence (Delfia; Wallac, Turku, Finland) with detection limits of 0.06 and 0.05 IU/L, respectively, and CV and lt; both at 5%. Inhibin B assay was performed using a specific bilateral enzyme immunoassay (inhibin B genII, Beckman Coulter, USA) CV <11%. For AMH, we used the Beckman Coulter enzyme immunoassay (Immunotech, Beckman Coulter, Marseilles, France) and a CV <8%. Finally, IGF1 and IGF-BP3 were determined using the chemiluminescence assay of the IDS-iSYS immunodiagnostic system with CVs of 10% and 9%, respectively. Free estradiol was calculated using Mazer's constant and free testosterone (FT) was calculated using Vermeulen's formula [27,28].
Semen Analysis
Healthy young men and infertile adult participants provided semen samples and obtained self-reported information on the duration of ejaculatory abstinence. Semen analysis was performed by trained technicians as previously described [19]. Semen volume was estimated by weighing. For sperm viability assessment, 10 μ L of well-mixed semen replicates were placed on slides and examined under a × 400x microscope at a heated section of 37◦ C. Sperm were classified as either advanced viable (WHO class a + B), non-progressive viable (class C) or inactive (class D). The average of the two viability assessments was used. To assess sperm concentration, samples were diluted in a distilled water solution of 0.6 moL/L NaHCO3 and 0.4% (v/v) formaldehyde and then evaluated using a Bürker-Türk hemocytometer and the average of the assessments was used. Only spermatozoa with tails were counted. Finally, smears were prepared, and stained with Pap stain, and sperm morphology was evaluated strictly according to the criteria of [29].
Western Blotting
Human testicular tissue was homogenized in lysis buffer, diluted in SDS loading buffer, and then heated at 95◦ C for 5 min. Samples were loaded onto 4-20% precast polyacrylamide gels (BioRad, cat# 456-8096) and run at 100 V for 1 h to separate proteins. To detect sLHCGR in serum, albumin, and IgG were removed using the Pierce albumin /IgG removal kit (Thermo Scientific, Waltham, MA, USA, cat# 89875) before loading samples onto the gel. after gel electrophoresis, proteins were transferred to polyvinylidene difluoride membranes using a wet blotter (BioRad). Membranes were closed in tris-buffered saline and 5% skimmed dry milk for 1 hr, then incubated overnight at 4◦ C with primary antibodies and for 1 hr at RT with secondary antibodies. Membranes were washed with enhanced chemiluminescence (Thermo Scientific Super Signal West Femto Maximum Sensitive Substrate, Cat# 34095) and photodetector with a chemiDoc MP imaging system (BioRad, Cat# 17001402). Three different antibodies were used to detect LHCGR in tissue and serum: Aviva System Biology (OASG04237); NBCL Holland, the same antibody as in the serum ELISA kit (LHR029) and Santa Cruz Biotechnology (Dallas, TX, USA, SC- 26341). β-2 microglobulin was used as a loading control.
Immunohistochemistry
Immunohistochemistry was performed as previously described [25]. Briefly, sections were fixed with modified Stiff fixative, dewaxed, and rehydrated. Antigen extraction was performed in an autoclave containing citrate extraction buffer (10 mM, pH 6.0). Endogenous peroxidase was blocked with 3% (v/v) H2O2 in methanol for 30 min. Between each step, sections were washed in TBS. Sections were incubated with 0.5% BSA horse serum for 30 minutes, followed by overnight incubation with two different primary antibodies for 1 hour at room temperature (LHR029 for WB/ELISA, Santa Cruz Biotechnology SC- 25828). Sections were incubated with secondary antibodies for 30 minutes. Images were visualized with aminoethyl carbazole (AEC, Invitrogen, Life Technologies/Thermo Fisher Scientific, Waltham, MA, USA). Negative controls were without primary antibodies. None of the negative controls were stained. Counterstaining was performed with Mayer's hematoxylin and mounting was performed with Aquatex. Sections were evaluated on a Nikon Microphot-FXA microscope (Nikon Corporation, Melville, NY, USA). The final analysis was performed using the software NDPview version 1.2.36 (Hamamatsu Photonics, Iwata City, Japan.
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Li Juel Mortensen 1, Mette Lorenzen 1, Anne Jørgensen 2, Jakob Albrethsen 2, Niels Jørgensen 2,Søren Møller 3,4, Anna-Maria Andersson 2, Anders Juul 2,4 and Martin Blomberg Jensen 1,5
1. Group of Skeletal, Mineral and Gonadal Endocrinology, University Department of Growth and Reproduction, Rigshospitalet, 2100 Copenhagen, Denmark; li.juel.mortensen@regionh.dk (L.J.M.); mette.lorenzen.01@regionh.dk (M.L.)
2. Department of Growth and Reproduction and International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, University of Copenhagen, Blegdamsvej 9, 2100 Copenhagen, Denmark; Anne.Joergensen.02@regionh.dk (A.J.); Jakob.Christian.Albrethsen@regionh.dk (J.A.); Niels.Joergensen@regionh.dk (N.J.); Anna-Maria.Andersson@regionh.dk (A.-M.A.); Anders.Juul@regionh.dk (A.J.)
3. Center for Functional and Diagnostic Imaging and Research, Department of Clinical Physiology and Nuclear Medicine 260, Hvidovre Hospital, 2650 Copenhagen, Denmark; Soeren.Moeller@regionh.dk
4. Department of Clinical Medicine, Faculty of Health Sciences, Copenhagen University, 2200 Copenhagen, Denmark
5. Division of Bone and Mineral Research, Harvard School of Dental Medicine/Harvard Medical School, Boston, MA 02115, USA
