The role and regulation of insulin-like peptide 3 (INSL3) in the female reproductive system

The peptide hormone Insulin-like peptide 3 (INSL3) shows important roles in the reproductive system. In the male, the testicular Leydig cell peptide INSL3 is involved in fetal testis descent and sperm maturation during the adulthood. In females of reproductive age INSL3 is mainly produced by the ovarian follicular theca interna cells. The current project aims to explore the detailed actions of INSL3 in the female reproductive system. Firstly, the secretion pattern of the INSL3 peptide in the serum of young women and women attending an infertility clinic was assessed. The serum levels of INSL3 peptide appeared to increase during the follicular phase, decrease during the luteal phase, and drop to a nadir at menses. The secretion pattern corresponded to the growth of antral follicles within a follicular wave. Pathological conditions leading to an alteration of the antral follicle count, such as polycystic ovarian syndrome and low ovarian reserve, were accompanied by an increase or decrease in the serum INSL3 level, respectively. The bovine system was adopted and validated as a model for the human in regard to the follicular production of INSL3, as the secretion pattern for INSL3 in bovine serum through estrous cycles was similar to that in women. Primary bovine theca interna cells were then used to study the regulation of the INSL3 gene and its secreted peptide products. At both peptide and mRNA levels INSL3 production could be up-regulated by progesterone receptor signalling. Together with the analysis of the transcriptional activity of the bovine INSL3 gene promoter, it could be shown that estradiol (E2) – estrogen receptor alpha (ERα) signalling stimulated and E2-ERß signalling decreased INSL3 production. Dihydrotestosterone (DHT) acting through the androgen receptor (AR) and androstenedione (A4) probably acting through ERα and/or ERß both contribute to regulation of INSL3 production. Moreover, luteinising hormone (LH) from the anterior pituitary also influenced the INSL3 production, with the downstream pathway from low level LH stimulation probably involving synergy between cAMP-PKA and ER signalling. The potential target of INSL3 was also identified in the female bovine reproductive system; full-length transcripts for the INSL3 receptor, RXFP2, were detectable in ovarian theca interna cells, in oocytes, as well as in the myometrium. Meanwhile, full-length transcripts for the closely related receptor, RXFP1, were detected in theca interna cells, endometrium and in myometrium, although the gene encoding the ligand for this receptor, relaxin, has been deleted during ruminant evolution. When transfecting DNA expression plasmids encoding the individual RXFP2 and RXFP1 receptors into HEK293T cells, the expressed bovine RXFP2 could be activated by bovine and human INSL3 (EC50 = 0.7 & 0.6nM) and porcine and human relaxin (EC50 = 10.5 & 27.3nM). The expressed bovine RXFP1 could be activated by porcine and human relaxin (EC50 = 10.8 & 48.7nM) and human relaxin 3 (EC50 = 97.9nM). Functional analysis showed that bovine myometrial cells were able to respond to both exogenous relaxin and INSL3, whereas theca interna cells could respond to INSL3 only. Together, however, these experiments support the view that endogenous RXFP2 and RXFP1 are both fully functional in the bovine even though the primary ligand for the latter is missing. The results showed that for the female INSL3 is mainly produced by the theca interna cells of antral follicles, and is thus a potential biomarker of ovarian functionality. The expression of INSL3 can be regulated by sex steroid hormones in an auto/paracrine manner, as well as by LH in an endocrine manner. In conclusion, the INSL3-RXFP2 system acts in follicles and probably also the uterus to enable and orchestrate female reproductive physiology

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

INSL3 Variation in Dogs Following Suppression and Recovery of the HPG Axis

Insulin-like peptide 3 (INSL3) is a constitutive product of mature, adult-type Leydig cells of the testes and consequently in most mammals is an ideal biomarker with which to monitor pubertal development. A new heterologous time-resolved fluorescence immunoassay was developed and validated to measure circulating INSL3 in the blood of adult male dogs. Compared to other species, INSL3 concentration is low with marked variation between individuals, which appears to be independent of breed, age, or weight. A model system was then used in which a cohort of beagle dogs was subject to a GnRH-agonist implant to suppress the HPG axis and spermatogenesis, followed by implant removal and recovery. Unlike testosterone, INSL3 levels were not fully suppressed in all animals by the GnRH agonist, nor was the recovery of Leydig cell function following implant removal uniform or complete, even after several weeks. In dogs, and dissimilar from other species (including humans), Leydig-cell INSL3 appears to be quite variable between individual dogs and only weakly connected to the physiology of the HPG axis after its suppression by a GnRH-agonist implant and recovery. Consequently, INSL3 may be less useful in this species for the assessment of testis function

Neohormones as biomarkers of reproductive health

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

The role and regulation of insulin-like peptide 3 (INSL3) in the female reproductive system

The peptide hormone Insulin-like peptide 3 (INSL3) shows important roles in the reproductive system. In the male, the testicular Leydig cell peptide INSL3 is involved in fetal testis descent and sperm maturation during the adulthood. In females of reproductive age INSL3 is mainly produced by the ovarian follicular theca interna cells. The current project aims to explore the detailed actions of INSL3 in the female reproductive system. Firstly, the secretion pattern of the INSL3 peptide in the serum of young women and women attending an infertility clinic was assessed. The serum levels of INSL3 peptide appeared to increase during the follicular phase, decrease during the luteal phase, and drop to a nadir at menses. The secretion pattern corresponded to the growth of antral follicles within a follicular wave. Pathological conditions leading to an alteration of the antral follicle count, such as polycystic ovarian syndrome and low ovarian reserve, were accompanied by an increase or decrease in the serum INSL3 level, respectively. The bovine system was adopted and validated as a model for the human in regard to the follicular production of INSL3, as the secretion pattern for INSL3 in bovine serum through estrous cycles was similar to that in women. Primary bovine theca interna cells were then used to study the regulation of the INSL3 gene and its secreted peptide products. At both peptide and mRNA levels INSL3 production could be up-regulated by progesterone receptor signalling. Together with the analysis of the transcriptional activity of the bovine INSL3 gene promoter, it could be shown that estradiol (E2) – estrogen receptor alpha (ERα) signalling stimulated and E2-ERß signalling decreased INSL3 production. Dihydrotestosterone (DHT) acting through the androgen receptor (AR) and androstenedione (A4) probably acting through ERα and/or ERß both contribute to regulation of INSL3 production. Moreover, luteinising hormone (LH) from the anterior pituitary also influenced the INSL3 production, with the downstream pathway from low level LH stimulation probably involving synergy between cAMP-PKA and ER signalling. The potential target of INSL3 was also identified in the female bovine reproductive system; full-length transcripts for the INSL3 receptor, RXFP2, were detectable in ovarian theca interna cells, in oocytes, as well as in the myometrium. Meanwhile, full-length transcripts for the closely related receptor, RXFP1, were detected in theca interna cells, endometrium and in myometrium, although the gene encoding the ligand for this receptor, relaxin, has been deleted during ruminant evolution. When transfecting DNA expression plasmids encoding the individual RXFP2 and RXFP1 receptors into HEK293T cells, the expressed bovine RXFP2 could be activated by bovine and human INSL3 (EC50 = 0.7 & 0.6nM) and porcine and human relaxin (EC50 = 10.5 & 27.3nM). The expressed bovine RXFP1 could be activated by porcine and human relaxin (EC50 = 10.8 & 48.7nM) and human relaxin 3 (EC50 = 97.9nM). Functional analysis showed that bovine myometrial cells were able to respond to both exogenous relaxin and INSL3, whereas theca interna cells could respond to INSL3 only. Together, however, these experiments support the view that endogenous RXFP2 and RXFP1 are both fully functional in the bovine even though the primary ligand for the latter is missing. The results showed that for the female INSL3 is mainly produced by the theca interna cells of antral follicles, and is thus a potential biomarker of ovarian functionality. The expression of INSL3 can be regulated by sex steroid hormones in an auto/paracrine manner, as well as by LH in an endocrine manner. In conclusion, the INSL3-RXFP2 system acts in follicles and probably also the uterus to enable and orchestrate female reproductive physiology

INSL3 Variation in Dogs Following Suppression and Recovery of the HPG Axis

Insulin-like peptide 3 (INSL3) is a constitutive product of mature, adult-type Leydig cells of the testes and consequently in most mammals is an ideal biomarker with which to monitor pubertal development. A new heterologous time-resolved fluorescence immunoassay was developed and validated to measure circulating INSL3 in the blood of adult male dogs. Compared to other species, INSL3 concentration is low with marked variation between individuals, which appears to be independent of breed, age, or weight. A model system was then used in which a cohort of beagle dogs was subject to a GnRH-agonist implant to suppress the HPG axis and spermatogenesis, followed by implant removal and recovery. Unlike testosterone, INSL3 levels were not fully suppressed in all animals by the GnRH agonist, nor was the recovery of Leydig cell function following implant removal uniform or complete, even after several weeks. In dogs, and dissimilar from other species (including humans), Leydig-cell INSL3 appears to be quite variable between individual dogs and only weakly connected to the physiology of the HPG axis after its suppression by a GnRH-agonist implant and recovery. Consequently, INSL3 may be less useful in this species for the assessment of testis function

Ovarian expression of insulin-like peptide 3 (INSL3) and its receptor (RXFP2) during development of bovine antral follicles and corpora lutea and measurement of circulating INSL3 levels during synchronized estrous cycles

Insulin-like peptide 3 (INSL3), a major product of testicular Leydig cells, is also expressed by the ovary but its functional role remains poorly understood. Here, we quantified expression of INSL3 and its receptor RXFP2 in theca interna (TIC) and granulosa (GC) compartments of developing bovine antral follicles and in corpora lutea (CL). INSL3 and RXFP2 mRNA levels were much higher in TIC than GC and increased progressively during follicle maturation with INSL3 peaking in large (11-18mm) estrogen-active follicles and RXFP2 peaking in 9-10mm follicles before declining in larger (11-18mm) follicles. Expression of both INSL3 and RXFP2 in CL was much lower than in TIC. In situ hybridization and immunohistochemistry confirmed abundant expression of INSL3 mRNA and protein in TIC. These observations indicate follicular TIC rather than CL as the primary site of both INSL3 production and action, implying a predominantly auto-/paracrine role in TIC. To corroborate the above findings, we showed that in vitro exposure of TIC to a luteinizing concentration of LH greatly attenuated expression of both INSL3 and its receptor while increasing progesterone secretion and expression of STAR and CYP11A1. Moreover, in vivo, a significant cyclic variation in plasma INSL3 was observed during synchronized estrous cycles. INSL3 and estradiol-17β followed a similar pattern, both increasing after luteolysis, before falling sharply after the LH surge. Thus, theca-derived INSL3, likely from the dominant pre-ovulatory follicle, is detectable in peripheral blood of cattle and expression is down-regulated during luteinisation induced by the pre-ovulatory LH surge. Collectively, these findings underscore the likely role of INSL3 as an important intrafollicular modulator of TIC function/steroidogenesis, whilst raising doubts about its potential contribution to CL function