Ravinder Anand-Ivell

Expression of the Insulin-Like Peptide 3 (INSL3) Hormone-Receptor (LGR8) System in the Testis

Abstract The new peptide hormone insulin-like peptide 3 (INSL3) is a member of the insulin-relaxin family, yet, unlike insulin, it signals through a new G-protein coupled receptor, LGR8, distantly related to the receptors for LH and FSH. INSL3 is produced in large amounts by the Leydig cells of the testis in both fetal and adult mammals. Using a combination of mRNA analysis by RT-PCR, immunohistochemistry, ligand-binding, and/or bioactivity assays, the distribution of LGR8 expression was assessed in testicular tissues and cells and in the epididymis. There was consistent agreement that LGR8 was expressed in meiotic and particularly postmeiotic germ cells and in Leydig cells, though not in Sertoli or peritubular cells. Leydig cells appear to express only a low level of the LGR8 gene product; other transcripts may be present, representing nonfunctional products. Messenger RNA analysis suggested that LGR8 transcripts in germ cells represented mostly full-length forms. LGR8 mRNA was also expressed in the epididymis, though no function can yet be ascribed to this expression. Therefore, the INSL3/LGR8 system represents a further paracrine hormone-receptor system in the testis, which conveys information about Leydig cell status to germ cells, and possibly as part of an autocrine feedback loop.

Biology of insulin-like factor 3 in human reproduction

BACKGROUND Insulin-like factor 3 (INSL3) is a neohormone that has evolved to address specific mammalian traits, in particular, the first phase of testicular descent towards the scrotum during mid-gestation. METHODS A thorough literature search was made in PubMed using the terms INSL3, as well as the older synonyms RLF and Ley-IL. RESULTS INSL3 is a major secretory product of the testicular Leydig cells in the fetus and in adult men, and in rodent models, reduction in fetal INSL3 expression is an early marker of the testicular dysgenesis syndrome. In women, it is produced in lower amounts by ovarian theca and luteal cells, and circulating levels are increased in women with polycystic ovarian syndrome. During pregnancy, there is evidence for an interaction regulating the feto-placental unit. The presence of INSL3 in amniocentesis samples taken at 12-14 weeks gestation is absolutely specific for male gender, and levels are predictive of subsequent pre-eclampsia and/or birthweight. INSL3 is also involved in adult traits, such as spermatogenesis and bone metabolism. In adult men, INSL3 is constitutively expressed and secreted into the bloodstream at a constant level, reflecting the number and/or functional capacity of the Leydig cells. In complete contrast, testosterone is highly variable within individuals, is acutely responsive to fluctuations in the hypothalamic-pituitary-gonadal axis and appears to have marginal diagnostic value. INSL3 declines consistently with age in adult men. CONCLUSIONS INSL3 promises to become an important new diagnostic tool to characterize those men with late-onset hypogonadism and to add clinical diagnostic value at amniocentesis.

Constitutive regulation of the Insl3 gene in rat Leydig cells.

Insulin-like factor 3 (Insl3) is a major new product of the Leydig cells in all mammalian species so far examined. The rat Insl3 gene is encoded by two exons in close juxtaposition to the Jak3 gene. Using RT-PCR analysis we now show that in the rat testis it is expressed as both major and minor splice variants, the former encoding the normal protein, the latter a truncated peptide comprising a C-terminally extended B-domain. Both transcripts are produced in constant relative amounts uniquely in the Leydig cells of the postnatal testis and in no other testicular cell type. Rat Insl3 protein is also expressed only in Leydig cells after postnatal day 30. Although specific mRNA is present at earlier times, corresponding protein is not detected. Semi-quantitative RT-PCR analysis of Insl3 transcripts in the mouse MA-10 tumour Leydig cell-line under a wide range of stimulation regimes shows that in an acute context, the Insl3 gene is expressed absolutely constitutively. This is confirmed by transfection and electrophoretic mobility shift (EMSA) analysis of the rat Insl3 gene promoter, wherein the importance of three putative SF-1 responsive elements is underscored, although these appear to differ in their relative importance from their counterparts in the mouse Insl3 gene.

Dynamics of INSL3 Peptide Expression in the Rodent Testis

The Leydig cell-specific factor insulin-like peptide 3 (INSL3) is involved in testicular descent during embryo development, and has been suggested to regulate spermatogenesis and bone metabolism in the adult. Using a new, sensitive assay specific for rodent INSL3, we have mapped the secretion of INSL3 into peripheral blood in mice and during postnatal male rat development (in female rats, circulating INSL3 is at the level of detection). Maximum INSL3 is measured at Postnatal Day (PD) 40 in the rat and decreases to a significantly lower, stable value by PD60, indicating an “overshoot” effect in the establishment of Leydig cell functionality during the first wave of spermatogenesis. Aging rats (∼24 mo) have markedly reduced circulating INSL3 levels, as do humans. Treatment of young adult rats with ethane dimethylsulfonate (EDS) leads to loss of mature Leydig cells and no detectable INSL3 in peripheral blood. INSL3 can be detected first at Day 27 after EDS treatment, returning to near normal levels by Day 37. Both primary rat Leydig cells and the mouse MA-10 tumor cell line secrete substantial amounts of INSL3 into the culture media in a constitutive manner, unregulated by common effectors, including hCG. Analysis of different testicular fluid compartments shows highest INSL3 concentration in the interstitial fluid (391.4 ± 47.8 ng/ml). However, INSL3 evidently traverses the blood-testis barrier to enter the seminiferous compartment, rete testis, and epididymis in sufficient concentration to be able to address the specific INSL3 receptors (RXFP2) on post-meiotic germ cells and in the epididymis.

INSL3 as a Biomarker of Leydig Cell Functionality

ABSTRACT Insulin-like factor 3 (INSL3) is a small peptide hormone made and secreted uniquely by mature Leydig cells in the testes of all mammals. Importantly, this expression and secretion appears to be constitutive and therefore reflects the differentiation status and number of the Leydig cells present, differing thereby from testosterone, which is acutely and homeostatically regulated by the hormones of the hypothalamic-pituitary-gonadal axis. As a consequence, the measurement of INSL3 either as mRNA in the testis or as secreted peptide circulating in the blood provides an excellent assessment of Leydig cell differentiation, for example, during fetal development, puberty, or aging or following exposure to endocrine-disrupting agents. Whereas INSL3 is proving increasingly useful as a biomarker for testis status, less is known about its functions, particularly in the adult male. Current evidence points to autocrine, paracrine, and endocrine roles, acting through the G-protein-coupled receptor called RXFP2, although more research is required to characterize these functions in detail.

Phthalates and Perfluorooctanesulfonic Acid in Human Amniotic Fluid: Temporal Trends and Timing of Amniocentesis in Pregnancy

Background: Measures of prenatal environmental exposures are important, and amniotic fluid levels may directly reflect fetal exposures during hypothesized windows of vulnerability. Objectives: We aimed to detect various phthalate metabolites and perfluorooctanesulfonic acid (PFOS) in human amniotic fluid, to study temporal exposure trends, and to estimate potential associations with gestational week of amniocentesis and maternal age and parity at amniocentesis. Methods: We studied 300 randomly selected second-trimester amniotic fluid samples from a Danish pregnancy-screening biobank covering 1980 through 1996. We used only samples from male offspring pregnancies. We assayed the environmental pollutants by liquid chromatography/triple quadrupole mass spectrometry and analyzed data using generalized linear regression models. Results: We detected the di(2-ethylhexyl) phthalate (DEHP) metabolite mono(2-ethyl-5-carboxypentyl) phthalate (5cx-MEPP) at a median concentration of 0.27 ng/mL [interquartile range (IQR): 0.20–0.37 ng/mL], the diisononyl phthalate (DiNP) metabolite mono(4-methyl-7-carboxyheptyl) phthalate (7cx-MMeHP) at 0.07 ng/mL (IQR: 0.05–0.11 ng/mL), and PFOS at 1.1 ng/mL (IQR: 0.66–1.60 ng/mL). An increase of 1 calendar year was associated with 3.5% lower [95% confidence interval (CI): –4.8%, –2.1%] 5cx-MEPP levels and with 7.1% higher (95% CI: 5.3%, 9.0%) 7cx-MMeHP levels. For each later gestational week of amniocentesis, 5cx-MEPP was 9.9% higher (95% CI: 4.8%, 15.2%), 7cx-MMeHP was 8.6% higher (95: CI: 2.7%, 14.9%), and PFOS was 9.4% higher (95: CI: 3.3%, 15.9%). We observed no associations with maternal age or parity. Conclusions: Measured metabolite levels appeared to parallel decreasing DEHP exposure and increasing DiNP exposure during the study period. The environmental pollutant levels were positively associated with later gestational age at amniocentesis during pregnancy weeks 12–22.

Insulin-like factor 3 levels in amniotic fluid of human male fetuses

BACKGROUND Rodent studies suggest that the peptide hormone insulin-like factor 3 (Insl3) made by the fetal testis is responsible for the first transabdominal phase of testicular descent, and may be affected by xenobiotics to disrupt male reproductive tract development. To date, there is very little information on the production of INSL3 by the human fetus during gestation. The objective of the present study was to determine the concentrations and time course during pregnancy of INSL3 and testosterone production in human fetuses and their associations with maternal characteristics, pregnancy complications and outcome. METHODS This is a retrospective cohort study in which women who contributed amniotic fluid specimens to a bank from 2003-2006 were followed to determine their pregnancy complications and pregnancy outcome. Amniotic fluid specimens were collected from the Reproductive Genetics Laboratory of the Hospital of the University of Pennsylvania subsequent to routine amniocentesis. INSL3 and total testosterone levels were measured in amniotic fluid (from n = 50 female, n = 237 male fetuses) by validated immunoassays and correlated with maternal characteristics, pregnancy complications and outcomes. RESULTS INSL3 was only detectable in amniotic fluid from male fetuses, and highest levels occurred from weeks 15-17 of gestation. INSL3 concentration was positively associated with increased birth weight, the occurrence of pre-eclampsia and advanced maternal age, but not with testosterone levels. CONCLUSIONS INSL3 concentration in human amniotic fluid is potentially predictive of fetal sex and pre-eclampsia, and presumably reflects the functioning of the fetal Leydig cell population.

Insulin-Like Factor 3 and the HPG Axis in the Male

The hypothalamic–pituitary–gonadal (HPG) axis comprises pulsatile GnRH from the hypothalamus impacting on the anterior pituitary to induce expression and release of both LH and FSH into the circulation. These in turn stimulate receptors on testicular Leydig and Sertoli cells, respectively, to promote steroidogenesis and spermatogenesis. Both Leydig and Sertoli cells exhibit negative feedback to the pituitary and/or hypothalamus via their products testosterone and inhibin B, respectively, thereby allowing tight regulation of the HPG axis. In particular, LH exerts both acute control on Leydig cells by influencing steroidogenic enzyme activity, as well as chronic control by impacting on Leydig cell differentiation and gene expression. Insulin-like peptide 3 (INSL3) represents an additional and different endpoint of the HPG axis. This Leydig cell hormone interacts with specific receptors, called RXFP2, on Leydig cells themselves to modulate steroidogenesis, and on male germ cells, probably to synergize with androgen-dependent Sertoli cell products to support spermatogenesis. Unlike testosterone, INSL3 is not acutely regulated by the HPG axis, but is a constitutive product of Leydig cells, which reflects their number and/or differentiation status and their ability therefore to produce various factors including steroids, together this is referred to as Leydig cell functional capacity. Because INSL3 is not subject to the acute episodic fluctuations inherent in the HPG axis itself, it serves as an excellent marker for Leydig cell differentiation and functional capacity, as in puberty, or in monitoring the treatment of hypogonadal patients, and at the same time buffering the HPG output.

Relaxin family peptides in the male reproductive system—a critical appraisal

The human genome project has identified, besides ovarian relaxin (RLN), six other relaxin-like molecules (RLN3, H1-RLN, INSL3-6), most of which appear to be expressed in the testis and/or male reproductive system, together with four different G-protein-coupled receptors responsive to one or other of these peptides. Earlier work on relaxin in the male assumed the simplistic hypothesis of only a single relaxin-like entity. This review systematically examines the expression and physiology of relaxin-like molecules in the male reproductive system in order to reappraise the importance of this hormone system for male reproductive function. Although there are important species differences, only INSL3 and INSL6 appear to be generally expressed at a moderately high level within the testis, whereas ovarian RLN is consistently a major secretory product of the prostate epithelium. However, all members of this relaxin-like family appear to be expressed also at a low level in different organs of the male reproductive system, suggesting possible autocrine/paracrine effects. The four receptors (RXFP1-4) for these peptides are also expressed to differing levels in both somatic and seminiferous compartments of the testis and in the prostate, supporting relevant functions for most members of this interesting peptide family. Recent studies of relaxin family peptides in prostate pathology highlight their functional importance in the clinical context as potential causative, diagnostic and therapeutic agents and warrant more specific and detailed studies of their roles also in regard to male fertility and other aspects of male reproductive function.

Insulin-like factor 3: where are we now?

Abstract: Insulin‐like factor 3 (INSL3), previously known as the relaxin‐like factor (RLF), is a major peptide hormone secreted from the testicular Leydig cells of adult men and circulating in the blood at a concentration of approximately 1 ng/mL. Women also produce INSL3 in the theca interna cells of ovarian follicles, but circulating levels remain below 100 pg/mL. INSL3 is structurally related to relaxin and insulin, but unlike the latter, signals through a novel G‐protein‐coupled receptor, LGR8. Ablation of the gene for INSL3 leads primarily to cryptorchidism because of a defect in the first, transabdominal phase of testicular descent. In the adult knockout mouse, a mild phenotype is evident in the testis and ovary. We have developed a panel of antibodies specific for INSL3 from various species, which are suitable for immunohistochemistry and, more recently, for immunoassays. INSL3 is an important marker for the mature Leydig cell phenotype, where it appears to be expressed constitutively, once the mature differentiation state is achieved. It is also an indicator of differentiation status not only for Leydig cells but also for the theca interna cells of the ovary.