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Insulin-like peptide 3 (INSL3) is a small peptide hormone of the insulin-relaxin family which is produced and secreted by the fetal Leydig cells in the testes only. It appears to be undetectable in female fetuses. In the human fetus INSL3 synthesis begins immediately following gonadal sex determination at weeks 7 to 8 post coitum and the peptide can be detected in amniotic fluid 1 to 2 weeks later. INSL3 acts through a unique G-protein-coupled receptor, called RelaXin-like Family Peptide receptor 2 (RXFP2), which is expressed by the mesenchymal cells of the gubernacular ligament linking the testes to the inguinal wall. The role of INSL3 in the male fetus is to cause a thickening of the gubernaculum which then retains the testes in the inguinal region, while the remainder of the abdominal organs grow away in an antero-dorsal direction. This represents the first phase of testis descent and is followed later in pregnancy by the second inguino-scrotal phase whereby the testes pass into the scrotum through the inguinal canal. INSL3 acts as a significant biomarker for Leydig cell differentiation in the fetus and may be reduced by maternal exposure to endocrine disrupting chemicals, such as xenoestrogens or phthalates, leading to cryptorchidism. INSL3 may have other roles within the fetus, but as a Leydig cell biomarker its reduction acts also as a surrogate for anti-androgen action., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ivell, Mamsen, Andersen and Anand-Ivell.)

The relaxin family of peptide hormones and their cognate GPCRs are becoming physiologically well-characterized in the cardiovascular system and particularly in female reproductive processes. Much less is known about the physiology and pharmacology of these peptides in male reproduction, particularly as regards humans. H2-relaxin is involved in prostate function and growth, while insulin-like peptide 3 (INSL3) is a major product of the testicular Leydig cells and, in the adult, appears to modulate steroidogenesis and germ cell survival. In the fetus, INSL3 is a key hormone expressed shortly after sex determination and is responsible for the first transabdominal phase of testicular descent. Importantly, INSL3 is becoming a very useful constitutive biomarker reflecting both fetal and post-natal development. Nothing is known about roles for INSL4 in male reproduction and only very little about relaxin-3, which is mostly considered as a brain peptide, or INSL5. The former is expressed at very low levels in the testes, but has no known physiology there, whereas the INSL5 knockout mouse does exhibit a testicular phenotype with mild effects on spermatogenesis, probably due to a disruption of glucose homeostasis. INSL6 is a major product of male germ cells, although it is relatively unexplored with regard to its physiology or pharmacology, except that in mice disruption of the INSL6 gene leads to a disruption of spermatogenesis. Clinically, relaxin analogues may be useful in the control of prostate cancer, and both relaxin and INSL3 have been considered as sperm adjuvants for in vitro fertilization., Linked Articles: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc., (© 2016 The British Pharmacological Society.)

The relaxin family of peptide hormones are structurally closely related to one another sharing a heterodimeric A-B structure, like that of insulin. They may also be active as unprocessed B-C-A pro-forms. Relaxin has been shown to pay a key role within the ovary, being involved in follicle growth, and ovulation. Relaxin is produced in large amounts also by the corpus luteum where it acts as an endocrine hormone positively affecting implantation, placentation and vascularization during the all-important first trimester phase of pregnancy establishment. Relaxin exerts its functions via the receptor RXFP1. Insulin-like peptide 3 (INSL3) in contrast acts through the related receptor RXFP2, and plays an essential role in the production of androgens within growing antral follicles. INSL3 is also produced in large amounts by the male fetus shortly after sex determination, where it controls the first transabdominal phase of testicular descent. However, this fetal INSL3 is also able to influence placental and maternal physiology, indicating associations with later preeclampsia and/or fetal growth restriction. Other members of this relaxin-like family of peptides, such as INSL4, INSL5 and INSL6 are less well studied, though all suggest modulatory roles in ovarian and/or placental function., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

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.

In myometrium of pigs and rats, though not humans, relaxin appears to mediate an inhibition of spontaneous and oxytocin-induced contractility, presumably acting through a G-protein coupled receptor (RXFP1) to generate cAMP. In humans, circulating relaxin is highest in the first trimester, including the time of implantation, when transitory uterine quiescence could help a blastocyst to implant. We investigated whether relaxin can activate adenylate cyclase in primary human myometrial cells from non-pregnant tissue, and we show that relaxin is able to stimulate the generation of cAMP in a manner, which is dependent upon a tyrosine phosphorylation activity, as in the endometrium. We identified transcripts for the relaxin receptor RXFP1 as full-length variants, though a minor splice variant missing exon 2 was also present in low amounts. These cells also express transcripts encoding RXFP2, the receptor for the closely related hormone, INSL3. Although able to respond to relaxin at high concentrations, this receptor does not appear to function by contributing to the cAMP production in human myometrial cells, nor does INSL3 act as a functional agonist or antagonist of relaxin action. In conclusion, the inability of relaxin to inhibit contractility in human myometrial cells would appear to be due to events downstream of simple cAMP generation.

The heterodimeric peptide hormone relaxin acts through the novel G-protein coupled receptor LGR7 to elicit the production of cAMP in the human monocyte cell line THP-1. The very small number of receptors on the cell surface, and the lack of response in cell membranes imply the involvement of a cytoplasmic signal amplification process. Here we show that this process comprises a novel and specific tyrosine kinase activity close to the receptor, and involves neither protein kinase A, mitogen-activated protein kinase, nor phosphoinositide-3 kinase activities as major upstream components. Furthermore, this novel involvement of a tyrosine kinase activity is cell-type dependent, being largely absent from LGR7-transfected HEK293T cells, and receptor-dependent; vasoactive intestinal peptide or isoproterenol signalling in the same cells does not require this tyrosine kinase activity.

The small peptide hormone relaxin is a member of a rapidly evolving family of hormones and growth factors, whose mode of action appears to be particularly adapted to purely mammalian physiology. It is representative of a new category of hormones, referred to as neohormones, which appear to have evolved specifically to accommodate the needs of viviparity, lactation and wound repair. The mechanism of receptor signalling has also evolved in this family, with older members using receptor tyrosine kinases and new members such as relaxin adopting 7-transmembrane G-protein coupled receptors. Although relaxin primarily generates cAMP as second messenger, studies of relaxin signalling show that this does not conform to a classic G-protein dependent activation of adenylate cyclase: it requires additional cytoplasmic components, it can involve further coupling to PI3-kinase and PKCzeta and it is absolutely dependent on a tyrosine kinase activity linked closely to the relaxin receptor. Relaxin may also independently activate glucocorticoid receptors. This diversity of signalling leads to a broad range of possible downstream transcriptional effects. Finally, in tissues where relaxin is known to be effective, there is often also local relaxin induction, amplifying the effects of the endocrine hormone.

The heterodimeric peptide hormone relaxin in most cells appears to signal through a G-protein-coupled receptor, LGR7. Whereas in artificial cell systems, made by transfection of receptor-expressing gene constructs into cells normally not presenting the receptor, classic activation of adenylate cyclase appears to be mediated by Gs, in cells naturally expressing the receptor, this type of coupling appears to be very weak. Instead, there is good evidence of other intermediate steps involving cytoplasmic components and tyrosine kinase activity. Part of the complexity of relaxin signaling is also manifest in the variable time course of cAMP production evident in the THP-1 cell line, which appears to depend on passage number and, hence, presumably on differentiation status. It is therefore important to distinguish between immediate early effects, short to mid-term responses, and long-term responses likely the consequences of specific gene upregulation.

Background: Insulin-like peptide 3 (INSL3) is a member of the relaxin family of neohormones which has evolved to address specifically mammalian aspects of reproduction related to viviparity and internal fertilization. It was originally identified as a major product of testicular Leydig cells and has proved to be an important biomarker of Leydig cell functional capacity. However, INSL3 is also produced by theca interna cells of growing antral follicles and is secreted into the bloodstream in phases corresponding to the number and health of the follicles. Moreover, gene silencing experiments have shown that INSL3 is essentially required for androstenedione synthesis, which is the major steroid precursor for the granulosa cells of antral follicles to produce oestrogens. Knockout studies in mice confirm that loss of INSL3 or its receptor in females leads to partial infertility, with reduced follicle numbers, ovulations and litter size. Circulating INSL3 concentration corresponds to the reproductive lifespan, beginning with puberty and declining at the menopause, and thus may contribute to the physiology of other organ systems, particularly those relevant for hormone replacement strategies.Search Methods: A literature review was carried out by exhaustive searching of literature databases (PubMed and Google Scholar) with the search terms INSL3, RLF, Ley-IL and RXFP2.Objective and Rationale: We present the first comprehensive review of INSL3 and its specific receptor RXFP2, and their roles in the context of female reproductive physiology. Moreover, we highlight the potential involvement of INSL3 in female reproductive pathology, such as PCOS, its clinical application as a valuable biomarker of reproductive processes, and its potential for therapeutic interventions.Outcomes: In the female mammal, INSL3 is largely produced by the theca interna cells of growing antral follicles during the follicular phase of the menstrual (oestrous) cycle. Within the follicle, INSL3 acts via its G-protein-coupled receptor, RXFP2, in an autocrine/paracrine manner to orchestrate and drive the production of the major steroid precursor androstenedione and its conversion by granulosa cells into oestrogens. These in turn create a positive feedback loop promoting the expression of more theca cell INSL3. This is countered by the follicular production of bone morphogenetic proteins and by the LH surge. Thus, the activity of the theca cell INSL3-RXFP2 system effectively determines the production of estradiol within an antral follicle through the follicular phase. INSL3 is also secreted into the circulation where it acts as a valuable biomarker to monitor the growth of antral follicles; it is consequently increased in PCOS and decreased in women with premature ovarian failure (POF). As an endocrine factor, INSL3 may also influence bone metabolism and kidney function. Additionally, INSL3 or its analogues may prove valuable as an adjunct in hormone replacement therapy or to monitor or influence IVF protocols.Wider Implications: The INSL3-RXFP2 system represents a new regulatory pathway essential for the proper functioning of growing antral follicles. We still know very little about its involvement in pathologies such as PCOS or POF, and its role as a new biomarker of female function needs to be explored more widely to improve diagnosis and treatment of ovarian dysfunction. We need to examine how INSL3 might be used to improve IVF protocols and outcomes. Opportunities should also be investigated in regard to the systemic application of INSL3 as a rejuvenant therapy, with positive effects on bone or kidney function, and possibly also for fertility regulation. Most research to date has involved animal models; this now needs to be extended to include more human studies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The hormone relaxin is important in female reproduction for embryo implantation, cardiovascular function, and during labor and lactation. Relaxin is also synthesized in males by organs of themale tract.We hypothesized that relaxinmight be one component of seminal plasma responsible for eliciting the female cytokine response induced in the uterus at mating. When recombinant relaxin was injected into the uterus of wild-type (Rln+/+) mice at estrus, it evoked the production of Cxcl1 mRNA and its secreted protein product CXCL1 in four of eight animals. Mating experiments were then conducted using mice with a null mutation in the relaxin gene (Rln-/- mice). qRT-PCR analysis of mRNA expression in wild-type females showed diminished uterine expression of several cytokine and chemokine genes in the absence of male relaxin. Similar differences were also noted comparing Rln-/- and Rln+/+ females mated to wild-type males. Quantification of uterine luminal fluid cytokine content confirmed thatmale relaxin provokes the production of CXCL10 and CSF3 in Rln+/+ females. Differences were also seen comparing Rln-/- and Rln+/+ females mated with Rln-/- males for CXCL1, CSF3, and CCL5, implying that endogenous relaxin in females might prime the uterus to respond appropriately to seminal fluid at coitus. Finally, pan-leukocyte CD45 mRNA was increased in wild-type matings compared to other combinations, implying that male and female relaxin may trigger leukocyte expansion in the uterus. We conclude that male and/or female relaxin may be important in activating the uterine cytokine/chemokine network required to initiate maternal immune adaptation to pregnancy. [ABSTRACT FROM AUTHOR]

In most mammals the peptide hormone relaxin is a key physiological component regulating early pregnancy and birth. However, synteny analysis shows that the gene encoding ovarian relaxin-2 is deleted in cows and sheep. While, these ruminants appear to exhibit a relaxin-like physiology, as in other mammals, until now a molecular understanding of this has been lacking. Cloning and expression analysis of the cognate bovine receptor for relaxin, RXFP1, as well as of the structurally related receptor, RXFP2, in female tissues, shows that these are expressed in a similar way to other mammals. RXFP1 transcripts are found in ovarian theca cells, endometrium, and myometrium, whereas RXFP2 transcripts are expressed in ovarian theca cells, oocytes, as well as in myometrium. Transfection of receptor-expressing gene constructs into HEK293 cells indicates that bovine RXFP1 has a greater EC50 at 10-50 nM for porcine or human relaxin, compared to human RXFP1. For bovine RXFP2, in contrast, the EC50 is <1 nM for its cognate ligand, bovine INSL3, but also 10-30 nM for porcine or human relaxin. Functional analysis shows that bovine myometrial cells are able to respond to exogenous relaxin and INSL3 with a significant increase in cAMP. Although expressing mRNA for both RXFP1 and RXFP2, bovine follicular theca cells only respond to INSL3 with a dose-dependent increase in cAMP. Altogether the results suggest that the cow is able to compensate for the missing hormone, and moreover imply that relaxin analogs could offer an important therapeutic option in treating female ruminant infertility. [ABSTRACT FROM AUTHOR]

Neohormone systems are defined as evolutionarily new endocrine or paracrine adaptations that supplement basic physiologic functions and define mammalian success. The relaxin family of peptide hormones are typical neohormones. Because they define the specific mammalian aspects of reproductive physiology, such as viviparity with implantation and placentation, lactation, or in the male the necessary adaptations to sperm needed for successful internal fertilization, they offer excellent biomarkers for characterizing reproductive health and disease. For example, ovarian H2-relaxin aids implantation and the establishment of the placenta, and circulating levels are significantly altered in early miscarriage. In the fetus, testicular INSL3 is responsible for the first phase of testicular descent and may be disrupted in cryptorchidism. In the adult, INSL3 is believed to be involved as an antiapoptotic factor in germ cell survival (male) and follicle selection (female) and acts as an excellent measure of Leydig cell functional capacity, particularly in the aging male. INSL5 and INSL6 appear also to be involved in the maintenance of adequate spermatogenesis. With the development of robust immunoassays for various relaxin family members, we are progressively gathering baseline information about normal biomarker levels as well as their perturbations in a wide range of reproductive pathologies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]