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

Neohormones in milk

Neohormone systems evolved specifically to regulate those mammalian traits, such as internal fertilization, pregnancy and lactation, which have proved to be central to the success, environmental independence, and adaptability of mammals as a vertebrate group. Neohormones such as oxytocin or relaxin are not only involved in the regulation of mammary gland development and function, but are also significant components of milk itself. Particularly for the latter hormone, it has been shown for the pig that relaxin in the first milk is taken up by the gastrointestinal tract of the offspring, enters the neonatal circulation and can have specific physiological and epigenetic effects on target organs such as the female reproductive system. Nevertheless, there are large gaps in our knowledge and understanding of such lactocrine systems especially in regard to other neohormones, species, and neonatal organ systems

The Physiology of Reproduction – Quo vadis?

The reproductive system in males and females reflects a highly dynamic underlying physiology. Yet our current understanding of this system is still largely based upon relatively simplistic snapshots of individual component cells and tissues. Gamete production as well as gonadal hormone synthesis and its influence are the manifestations of dynamic and redundant informational networks and processes, whose qualitative and quantitative dimensions, especially through development from embryo through puberty and adulthood into ageing, are still largely uncharted. Whilst the recent huge advances in molecular science have helped to describe the components of the reproductive system in ever greater detail, how these interact and function in space and time dimensions is still largely obscure. Recent developments in microfluidics, stem cell biology, and the integration of single-cell transcriptomics with tissue dynamics are offering possible methodological solutions to this issue. Such knowledge is essential if we are to understand not only the normal healthy functioning of this system, but also how and why it is affected in disease or by external impacts such as those from environmental endocrine disruptors, or in ageing. Moreover, operating within a complex network of other physiological systems, its integrational capacity is much more than the generation of male and female gametes and their roles in fertility and infertility; rather, it represents the underpinning support for health and well-being across the lifespan, through pregnancy, puberty, and adulthood, into old age

Insulin-like factor 3 as a monitor of endocrine disruption

Insulin-like factor 3 (INSL3) is generated and secreted by differentiated interstitial Leydig cells of the testes in both fetal and adult males of all mammalian species so far analyzed. All evidence to date suggests that it is produced constitutively, independently of acute regulation by the hypothalamo-pituitary–gonadal (HPG) axis, in amounts which reflect the numbers and differentiation status of the Leydig cells. This Leydig cell functional capacity is otherwise monitored only by androgen output, which, however, is massively confounded by acute regulation from the HPG axis and other factors leading to substantial and irregular short-term variation. Leydig cells are a primary target of endocrine-disrupting agents in the context of the testicular dysgenesis syndrome in the fetal male, as well as in the adult. In the male fetus, INSL3 is responsible for the first phase of testicular descent, and hence is directly linked to the etiology of cryptorchidism. In this study, by measuring INSL3 production, for example, during fetal life via amniotic fluid, or as secretions from fetal testis explants, or in adult peripheral blood, we and others have shown that INSL3 represents a useful quantitative and sensitive endpoint for assessing the impact of endocrine-disrupting agents and their mechanisms of action

Amplified RNA degradation in T7-amplification methods results in biased microarray hybridizations

BACKGROUND: The amplification of RNA with the T7-System is a widely used technique for obtaining increased amounts of RNA starting from limited material. The amplified RNA (aRNA) can subsequently be used for microarray hybridizations, warranting sufficient signal for image analysis. We describe here an amplification-time dependent degradation of aRNA in prolonged standard T7 amplification protocols, that results in lower average size aRNA and decreased yields. RESULTS: A time-dependent degradation of amplified RNA (aRNA) could be observed when using the classical "Eberwine" T7-Amplification method. When the amplification was conducted for more than 4 hours, the resulting aRNA showed a significantly smaller size distribution on gel electrophoresis and a concomitant reduction of aRNA yield. The degradation of aRNA could be correlated to the presence of the T7 RNA Polymerase in the amplification cocktail. The aRNA degradation resulted in a strong bias in microarray hybridizations with a high coefficient of variation and a significant reduction of signals of certain transcripts, that seem to be susceptible to this RNA degrading activity. The time-dependent degradation of these transcripts was verified by a real-time PCR approach. CONCLUSIONS: It is important to perform amplifications not longer than 4 hours as there is a characteristic 'quality vs. yield' situation for longer amplification times. When conducting microarray hybridizations it is important not to compare results obtained with aRNA from different amplification times

Theca cell INSL3 and steroids together orchestrate the growing bovine antral follicle

Insulin-like peptide 3 (INSL3) and its specific receptor RXFP2 are both expressed by theca interna cells of the growing antral follicle where they form an essential regulatory element in the production of the steroid precursor androstenedione. Using primary cultures of bovine theca cells from the mid follicular phase together with steroid agonists and antagonists we have examined how ovarian steroids modulate INSL3 expression. Transcript analysis shows that these cells express estrogen receptors ? and ?, androgen and progesterone receptors, besides the orphan nuclear receptors SF1 and nur77. Whereas, exogenous androgens have little or no effect, the androgen antagonist bicalutamide stimulates INSL3 production. In contrast, estrogen receptor agonists, as also progesterone, are stimulatory. Importantly, estrogen receptor signaling is convergent with the protein kinase A signaling pathway activated by LH, such that the estrogen receptor antagonist can inhibit the mild stimulatory effect of LH, and vice versa the PKA antagonist H89 blocks stimulation by estradiol. A significant finding is that the major steroid metabolite androstenedione appears to act predominantly as an estrogen and not an androgen in this system. Transfection of INSL3 gene promoter-reporter constructs together with various steroid receptor expression plasmids supports these findings and shows that steroid action uses non-classical pathways not requiring canonical steroid-responsive elements in the proximal promoter region. Together, the results indicate that increasing estrogens in the follicular phase stimulate a feedforward loop driving INSL3 signaling and thereby promoting steroidogenesis in the growing antral follicle until the LH surge which effectively switches off INSL3 expression

Non-classical mechanisms of steroid sensing in the ovary: lessons from the bovine oxytocin model

Steroidogenic tissues such as the ovary, testes or adrenal glands are paradoxical in that they often indicate actions of steroid hormones within a dynamic range of ligand concentration in a high nanomolar or even micromolar level, i.e. at the natural concentrations existing within those organs. Yet ligand-activated nuclear steroid receptors act classically by direct interaction with DNA in the picomolar or low nanomolar range. Moreover, global genomic studies suggest that less than 40% of steroid-regulated genes involve classical responsive elements in gene promoter regions. The bovine oxytocin gene is a key element in the maternal recognition of pregnancy in ruminants and is regulated via an SF1 site in its proximal promoter. This gene is also regulated by steroids acting in a non-classical manner, involving nuclear receptors which do not interact directly with DNA. Dose-response relationships for these actions are in the high nanomolar range. Similar ‘steroid sensing’ mechanisms may prevail for other SF1-regulated genes and predict alternative pathways by which environmental endocrine disruptors might influence the functioning of steroid-producing organs and hence indirectly the steroid-dependent control of physiology and development

Physiology and evolution of the INSL3/RXFP2 hormone/receptor system in higher vertebrates

© 2020 Elsevier Inc. Although the insulin-like peptide hormone INSL3 and its cognate receptor RXFP2 (relaxin-family peptide receptor 2) have existed throughout chordate evolution, their physiological diversification appears to be linked closely with mammalian emergence and radiation. In contrast, they have been lost in birds and reptiles. Both hormone and receptor are expressed from autosomal genes which have maintained their synteny across vertebrate evolution. Whereas the INSL3 gene comprises only two exons closely linked to the JAK3 gene, RXFP2 is normally encoded by 18 exons. Both genes, however, are subject to alternative splicing to yield a variety of possibly inactive or antagonistic molecules. In mammals, the INSL3-RXFP2 dyad has maintained a probably primitive association with gametogenesis, seen also in fish, whereby INSL3 promotes the survival, growth and differentiation of male germ cells in the testis and follicle development in the ovary. In addition, however, the INSL3/RXFP2 system has adopted a typical ‘neohormone’ profile, essential for the promotion of internal fertilisation and viviparity; fetal INSL3 is essential for the first phase of testicular descent into a scrotum, and also appears to be associated with male phenotype, in particular horn and skeletal growth. Circulating INSL3 is produced exclusively by the mature testicular Leydig cells in male mammals and acts as a potent biomarker for testis development during fetal and pubertal development as well as in ageing. As such it can be used also to monitor seasonally breeding animals as well as to investigate environmental or lifestyle conditions affecting development. Nevertheless, most information about INSL3 and RXFP2 comes from a very limited selection of species; it will be especially useful to gain further information from a more diverse range of animals, especially those whose evolution has led them to express unusual reproductive phenotypes

Influence on the adult male Leydig cell biomarker insulin‐like peptide 3 of maternal exposure to estrogenic and anti‐androgenic endocrine disrupting compound mixtures: A retrospective study

Insulin-like peptide 3 (INSL3) is a peptide biomarker secreted specifically by the mature Leydig cells of the testes. It is constitutive, has low within-individual variance, and effectively measures the functional capacity of Leydig cells to make testosterone. In young adult men there is a large 10-fold range of serum INSL3 concentration, persisting into old age, and implying that later hypogonadal status might be programmed in early life. To determine whether maternal exposure to environmental endocrine disrupting compounds (EDCs) influences adult serum INSL3 concentration, using a retrospective paradigm, INSL3 was measured in young adult male rats (80–90 days) from the F1 generation of females maternally exposed to varied doses of bisphenol A (BPA), butylparaben, epoxiconazole, and fludioxonil as single compounds, as well as estrogenic and anti-androgenic mixtures of BPA and butylparaben, and di(2-ethylhexyl) phthalate and procymidone respectively. A mixture of BPA and butylparaben significantly reduced circulating INSL3 concentration in adult male progeny. The remaining compounds or mixtures tested, though sufficient to induce other effects in the F1 generation were without significant effect. Maternal exposure to low concentrations of some EDCs may be a contributing factor to the variation in the Leydig cell biomarker INSL3 in young adulthood, though caution is warranted translating results from rats to humans

Male seminal parameters are not associated with Leydig cell functional capacity in men

Background: Insulin-like peptide 3 (INSL3) is a constitutive, secreted peptide produced in the male uniquely by the Leydig cells of the testes. It is a biomarker for Leydig cell functional capacity, which is a measure of the numbers and differentiation status of these steroidogenic cells and lacks the biological and technical variance of the steroid testosterone. This retrospective study was carried out to examine the relationship between seminal parameters and the Leydig cell compartment, and secondarily to assess other factors responsible for determining Leydig cell functional capacity. Methods: INSL3 was assessed together with seminal, anthropometric, and hormonal parameters in a Swedish cohort of 18-year-old men, representing the average population, and in a smaller, more heterogeneous cohort of men visiting an Australian infertility clinic. Results and discussion: Average INSL3 concentration at 18years is greater than that reported at younger or older ages and indicated a large 10-fold variation. In neither cohort was there a relationship between INSL3 concentration and any semen parameter. For the larger, more uniform Swedish cohort of young men, there was a significant negative relationship between INSL3 and BMI, supporting the idea that adult Leydig cell functional capacity may be established during puberty. In both cohorts, there was a significant relationship between INSL3 and FSH, but not LH concentration. No relationship was found between INSL3 and androgen receptor trinucleotide repeat polymorphisms, reinforcing the notion that Leydig cell functional capacity is unlikely to be determined by androgen influence alone. Nor did INSL3 correlate with the T/LH ratio, an alternative measure of Leydig cell functional capacity, supporting the view that these are independent measures of Leydig cell function