Possible Relevance of Soluble Luteinizing Hormone Receptor during Development and Adulthood in Boys and Men

Abstract

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are agonists for the luteinizing hormone receptor (LHCGR) which regulates male reproductive function. LHCGR may be released into body fluids. We wish to determine whether soluble LHCGR is a marker for gonadal function. Cross-sectional, longitudinal, and intervention studies on 195 healthy boys and men and 396 men with infertility, anorchia, or Klinefelter Syndrome (KS) were used to correlate LHCGR measured in serum, seminal fluid, urine, and hepatic/renal artery and vein with gonadal function. LHCGR was determined in fluids from in vitro and in vivo models of human testicular tissue and cell lines, xenograft mouse models, and human fetal kidney and adrenal glands. Western blot showed LHCGR fragments in serum and gonadal tissue of similar size using three different antibodies. The LHCGR-ELISA had no species cross-reactivity or unspecific reaction in mouse serum even after human xenografting. Instead, sLHCGR was released into the media after the culture of a human fetal kidney and adrenal glands. Serum sLHCGR decreased markedly during puberty in healthy boys (p = 0.0001). In healthy men, serum sLHCGR was inversely associated with the Inhibin B/FSH ratio (β -0.004, p = 0.027). In infertile men, seminal fluid sLHCGR was inversely associated 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). The injection of hCG lowered sLHCGR in serum and urine of healthy men (p < 0.01). In conclusion, sLHCGR is released into body-fluids and linked with pubertal development and gonadal function. Circulating sLHCGR in anorchid men suggests that sLHCGR in serum may originate from and possibly exert actions in non-gonadal tissues. (ClinicalTrials: NTC01411527, NCT01304927, NCT03418896).

Keywords: LH receptor; NTera2; TCAM2; development; extra-gonadal effects; fetal adrenal gland; fetal kidney; gonadotropins; infertility; puberty.

Figure 1

In vitro and baseline analyses of luteinizing hormone receptor (LHCGR). (A). Reverse Transcriptase PCR (RT-PCR) with a primer spanning exon 2–4 of LHCGR in two testis tissue and one ovarian tissue sample. Two bands (red arrows) of approximately 100 and 200 bp were sequenced and identified as LHCGR isoforms. (B) Immunohistochemistry of human adult testis tissue stained with LHR029 antibody from the ELISA kit showing LHCGR expression in Leydig cells (green arrows). Image is from × 40 magnification (C): Western Blot detecting LHCGR with three antibodies targeting different domains examined on testis tissue (T), and in serum from men with high Soluble LHCGR (sLHCGR) level (S1) and low sLHCGR level (S2). (D) Human sLHCGR is unmeasurable in serum from WT nude mice, in xenograft (XG) mice implanted with a human seminoma cell line (TCam2), treated with either vehicle, hCG, or LH. sLHCGR is measurable in low levels in media from TCam2 cells but not in a human embryonic carcinoma cell line, NTera2. (E) sLHCGR present in urine at comparable levels in healthy and hypogonadal men. sLHCGR measurable in serum from 8 men with both testicles removed at comparable levels as in healthy young men. sLHCGR measurable in media from the cultured human fetal adrenal gland and human fetal kidney. (F) Total sLHCGR from arterial- and venous phase across liver, kidney and lower extremity measured in one man and two post-menopausal women shows no significant difference in arterial and venous levels, independent of the organ. (G) SDS-protein gel electrophoresis of human serum samples after Immunoglobulin and albumin removal from left well 1–6: serum from 2 normal men, 1 man with testicular germ cell cancer, seminoma, 1 man with testicular germ cell cancer, non-seminoma, and 2 pregnant women. Four bands of approximately 35, 50, 60, and 75 kDa from the pregnant woman in well number 6 were analyzed by LC-MS/MS.

Figure 2

Longitudinal measurements of sLHCGR in serum and urine. (A): Repeated measurements of serum sLHCGR in adult Klinefelter men (timespan 4–36 months). (B): Repeated measurements of serum sLHCGR in 23 orchiectomized men. (C): Change in serum sLHCGR compared with baseline in 11 healthy men injected with 5000 IU hCG. (D): Change in urine sLHCGR in 11 healthy men injected with 5000 IU hCG. (E): Longitudinal measurements of sLHCGR in healthy boys during puberty. (F): Longitudinal measurements of sLHCGR in boys with Klinefelter syndrome (KS) during puberty.

Figure 3

Association between sLHCGR in serum and reproductive hormones, age and BMI in adult men. (A): sLHCGR and gonadal function in 148 young healthy men. A1: Linear regression shows an inverse association between sLHCGR and Inhibin B/FSH-ratio. A2: Serum sLHCGR stratified into quartiles and inhibin B/FSH-ratio, bar shows mean, p-values adjusted for multiple comparisons using Dunn-Bonferroni post hoc test. (B–D): Serum sLHCGR in a pooled analysis of all men with normal sex-chromosomes after pubertal onset. B1: Linear regression showing an inverse association between sLHCGR and estradiol. B2: sLHCGR in all men stratified into three groups based on baseline estradiol, bar shows mean, p-value from Kruskal Wallis. C1: Linear regression showing an inverse association between sLHCGR and age. C2: sLHCGR in all men stratified into five groups based on age, bar shows mean, p-value from Kruskal Wallis. D1: Linear regression showing an inverse association between sLHCGR and BMI. D2: sLHCGR in all men stratified into four groups based on BMI, bar shows mean, p-value from Kruskal Wallis.

Figure 4

Seminal sLHCGR levels in normal and infertile men. (A): Seminal levels of sLHCGR in healthy and infertile men bar shows mean. (B): Serum vs. seminal level of sLHCGR in healthy and infertile men, bar shows mean and groups are compared using Wilcoxon Paired Test. (C): Linear regression showing associations between seminal and serum sLHCGR in infertile men. (D): Linear regression showing associations between seminal sLHCGR and serum FSH in infertile men. (E): Linear regression showing association between seminal sLHCGR and serum Inhibin B.

Figure 5

Association between sLHCGR in seminal fluid and semen quality. (A): Serum and seminal LHCGR concentration and semen quality in 61 men. A1: Linear regression between total sperm count and seminal sLHCGR-level. A2: Total sperm count cut-off value for low vs. normal (< 40 mill), bar shows mean. A3: Linear regression between sperm concentration and seminal sLHCGR-level. A4: Borderline significantly lower sLHCGR with decreased sperm concentration: cut-off value for low vs. normal (<15 mill/mL). A5: Significantly lower sLHCGR with decreased sperm concentration: cut-off for severe oligospermia (<5mill/mL). A6: Significantly lower sLHCGR with decreased sperm concentration: cut-off of 1 mill/mL, bar shows mean. (B): Pooled analyses of seminal sLHCGR data from both healthy and infertile men. B1: Linear regression between seminal/serum sLHCGR-ratio and total sperm count. B2: Significantly lower sLHCGR with decreased sperm concentration: cut-off for low vs. normal total sperm count (<40 mill), bar shows mean. B3: Linear regression between seminal/serum sLHCGR-ratio and sperm concentration. B4: Significantly lower sLHCGR with decreased sperm concentration: cut-off for low vs. normal total sperm concentration (<15 mill/mL). All seminal analyses adjusted for time of abstinence.

Figure 6

Sandwich-ELISA measuring hCG bound to sLHCGR in serum with and without intervention and proteins detectable in serum at the correct size. (A): The bound fraction hCG-sLHCGR before and after injection with 5000 IU hCG in 8 healthy men. (B): Measurement of the hCG-sLHCGR complex in a serum sample (blue: baseline), after spiking with 2 commercially available hCG products in different concentrations (green and red, respectively) (C): Level of the hCG-sLHCGR complex in serum from 8 infertile men after spiking with hCG (Pregnyl) in 2 concentrations.