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| INSL3 (Insulin-like 3 Peptide ) |
Essential for Mediating the Transabdominal Phase of Testicular Descent during Early Development |
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Scheme to show the relationship between INSL3/RXFP2 system and testosterone as endpoint effectors of the HPG axis within the testis. Arrows are directed only to cells where there are known to be specific cognate receptors. Sertoli cells are the most representative cells of the prepubertal testis. INSL3 is a testicular hormone secreted during fetal/neonatal period and after puberty. INSL3 arises as a biomarker of Leydig cell functionality.
Figure from: Ivell R. et al., Front Endocrinol (Lausanne). 2014 Jan. Insulin-Like Factor 3 and the HPG Axis in the Male.
The testes secrete four hormones (anti-Müllerian hormone, insulin-like peptide 3, Inhibin B and testosterone) from two endocrine cell types. It is unknown whether anti-Müllerian hormone and insulin-like peptide 3 levels have a diurnal variation, and if so, whether they covary during the day with testosterone and InhB. Sera were obtained from 13 men at 00:00, 06:00, 09:00, 12:00, 14:00, 17:00 and 19:00 hours and the levels of their testicular hormones measured by ELISA. A second cohort of 20 men was similarly examined with blood drawn at 19:00 and the following 06:00. Anti-Müllerian hormone levels exhibited a subtle diurnal pattern with a 19:00 peak that was 4.9% higher on average than the 06:00 nadir (p = 0.004). The decrease in anti-Müllerian hormone coincided with a rise in testosterone and InhB, but there was no association between the person-to-person variation in the diurnal patterns of anti-Müllerian hormone and testosterone or Inhibin B. Insulin-like peptide 3 had no diurnal pattern, with only minor sporadic variation between time points being observed in some men. In conclusion, the diurnal and sporadic variation of each testicular hormone is distinct, indicating that the major regulation is at the level of the hormone rather than at the endocrine cell type. Consequently, the balance of the hormones being released by the testes has complex variation during the day. The physiological significance of this will vary depending on which combinations of testicular hormones that the target cells respond to.
This paper used INSL3 Fluorescent EIA kit (Phoenix Pharmaceuticals Cat. # FEK-035-27) for its hormone determination
Chong YH, Pankhurst MW, McLennan IS, PLoS One. 2015 Jul 20;10(7):e0133637. doi: 10.1371/journal.pone.0133637. eCollection 2015.
Prenatal sex hormones can induce abnormalities in the reproductive system and adversely impact on genital development. We investigated whether sex hormones in cord blood influenced the ratio of the second to fourth digit lengths (2D/4D) in school-aged children. Of the 514 children who participated in a prospective cohort study on birth in Sapporo between 2002 and 2005, the following sex hormone levels were measured in 294 stored cord blood samples (135 boys and 159 girls); testosterone (T), estradiol (E), progesterone, LH, FSH, inhibin B, and insulin-like factor 3 (INSL3). A total of 350 children, who were of school age and could be contacted for this survey, were then requested via mail to send black-and-white photocopies of the palms of both the left and right hands. 2D/4D was calculated in 190 children (88 boys and 102 girls) using photocopies and derived from participants with the characteristics of older mothers, a higher annual household income, higher educational level, and fewer smokers among family members. 2D/4D was significantly lower in males than in females (p<0.01). In the 294 stored cord blood samples, T, T/E, LH, FSH, Inhibin B, and INSL3 levels were significantly higher in samples collected from males than those from females. A multivariate regression model revealed that 2D/4D negatively correlated with INSL3 in males and was significantly higher in males with <0.32 ng/mL of INSL3 (p<0.01). No correlations were observed between other hormones and 2D/4D. In conclusion, 2D/4D in school-aged children, which was significantly lower in males than in females, was affected by prenatal Leydig cell function.
The authors used EK-035-27 from Phoenix for their study
Few studies have been undertaken to assess the possible effects of bisphenol A (BPA) on the reproductive hormone balance in animals or humans with often contradictory results. We investigated possible direct endocrine disruption by BPA of the fetal testes of 2 rat strains (14.5-17.5 days post-coitum) and humans (8-12 gestational weeks) and under different culture conditions. BPA concentrations of 10(-8)M and 10(-5)M for 72 h reduced testosterone production by the Sprague-Dawley fetal rat testes, while only 10-5M suppressed it in the Wistar strain. The suppressive effects at 10-5M were seen as early as 24h and 48 h in both strains. BPA at 10(-7)-10(-5)M for 72 h suppressed the levels of fetal rat Leydig cell insulin-like factor 3 (INSL3). BPA exposure at 10(-8)M, 10(-7)M, and 10(-5)M for 72 h inhibited testosterone production in fetal human testes. For the lowest doses, the effects observed occurred only when no gonadotrophin was added to the culture media and were associated with a poorly preserved testicular morphology. We concluded that (i) BPA can display anti-androgenic effects both in rat and human fetal testes; (ii) it is essential to ascertain that the divergent effects of endocrine disruptors between species in vitro do not result from the culture conditions used, and/or the rodent strain selected; (iii) the optimization of each in vitro assay for a given species should be a major objective rather than the search of an hypothetical trans-species consensual model-system, as the organization of the testis is intrinsically different between mammalian species; (iv) due to the uncertainty existing on the internal exposure of the human fetal testis to BPA, and the insufficient number of epidemiological studies on the endocrine disruptive effects of BPA, caution should be taken in the extrapolation of our present results to the human reproductive health after fetal exposure to BPA.
Ben Maamar M, Lesn¨¦ L, Desdoits-Lethimonier C et al., PLoS One. 2015 Feb 23;10(2):e0117226. doi: 10.1371/journal.pone.0117226. eCollection 2015.
Relaxin-like factor (RLF), also called insulin-like peptide 3 (INSL3), is a member of the insulin/relaxin gene family and is produced by testicular Leydig cells. While the understanding of its effects is growing, very little is known about the structural and functional properties of native INSL3. Here, we demonstrate that native INSL3 isolated from goat testes is a single-chain structure with full biological activity, and is constitutively expressed and secreted by Leydig cells. Using a series of chromatography steps, native INSL3 was highly purified as a single 12-kDa peak as revealed by SDS-PAGE. MS/MS analysis provided 81% sequence coverage and revealed a distinct single-chain structure consisting of the B-, C-, and A-domains deduced previously from the INSL3 cDNA sequence. Moreover, the N-terminal peptide was six amino acid residues longer than predicted. Native INSL3 exhibited full bioactivity in HEK-293 cells expressing the receptor for INSL3. Immunoelectron microscopy and Western blot analysis revealed that INSL3 was secreted by Leydig cells through the constitutive pathway into blood and body fluids. We conclude, therefore, thatgoat INSL3 is constitutively secreted from Leydig cells as a B-C-A single-chain structure with full biological activity.
Siqin, Minagawa I, Okuno M et al., Biol Chem. 2013 Sep;394(9):1181-94. doi: 10.1515/hsz-2012-0357.
STUDY QUESTION: How does insulin-like factor 3 (INSL3) concentration in blood vary across the menstrual cycle in women?
SUMMARY ANSWER: INSL3 is secreted by the theca interna cells of growing antral follicles and is phasic in its expression.
WHAT IS KNOWN ALREADY: The relaxin-like hormone INSL3 is known to be expressed in follicles of several mammal species, and was recently shown in cows to be specifically secreted into the bloodstream by growing antral follicles, corresponding to follicular waves. In males INSL3 is known to be acutely independent of the hormones of the hypothalamic-pituitary-gonadal axis, suggesting that in women INSL3 might be a novel biomarkerfor antral follicle recruitment and development.
STUDY DESIGN, SIZE, DURATION: Two cohorts of women were studied. First, 18 healthy women of reproductive age were followed longitudinally for one and a half cycles, with blood sampling and hormone measurement every 2-3 days. A second cohort comprised a cross-sectional study of 909 women attending an infertility clinic, with a single blood sample taken at entry, together with other clinical and hormonal parameters.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples from both retrospective cohorts were analyzed for INSL3 using a highly sensitive time-resolved fluorescent immunoassay, and data were analyzed in comparison with other clinical and hormonal parameters.
MAIN RESULT AND THE ROLE OF CHANCE: For young healthy women of reproductive age, we showed a phasic expression of INSL3corresponding to antral follicle growth in both the follicular and luteal phases of the cycle, which was significantly (P < 0.05) elevated compared with that during menses. For women attending an infertility clinic, those with diagnosed polycystic ovarian syndrome indicated significantly (P < 0.0005) greater circulating INSL3 levels and those with low ovarian reserve showed significantly (P < 0.002) decreased INSL3 values.
LIMITATIONS, REASONS FOR CAUTION: These were retrospective studies and the results were obtained from natural cycles only, with their inherent variability.
WIDER IMPLICATIONS OF THE FINDINGS: We show for the first time that INSL3 in women does vary across the menstrual cycle, and appears to reflect the number of growing antral follicles recruited within both follicular and luteal phases.
STUDY FUNDING/COMPETING INTEREST(S): The present retrospective study was largely supported by departmental funds. There were no competing interests.
Anand-Ivell R, Tremellen K, Dai Y et al., Hum Reprod. 2013 Nov;28(11):3093-102. doi: 10.1093/humrep/det349. Epub 2013 Sep 5
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.
Ivell R, Wade JD, Anand-Ivell R Biol Reprod. 2013 Jun 13;88(6):147. doi: 10.1095/biolreprod.113.108969. Print 2013 Jun.
BACKGROUND: Cryptorchidism, incomplete pubertal development, and low testosterone are manifestations of hypogonadism in Prader-Willi syndrome (PWS). Insulin-like peptide-3 (INSL3) facilitates testicular descent in the fetus and reflects Leydig cell number in adults. INSL3 levels in PWS have not been previously reported.
OBJECTIVES: The objectives of the study were to characterize the age-related changes in INSL3 in PWS males and correlate INSL3 with unilateral vs. bilateral cryptorchidism, body mass index, gonadotropins, testosterone, anti-mullerian hormone (AMH), and inhibin B.
STUDY DESIGN AND PARTICIPANTS: We measured INSL3, LH, FSH, testosterone, AMH, and inhibin B in 40 PWS males (23 deletion, 17 uniparental disomy) aged 2 months to 36 yr. Control samples for INSL3 were obtained from 365 normal males, aged 1 d to 36 yr.
RESULTS: INSL3 levels (mean and range) for PWS age groups younger than 6 months, 0.5-10.0 yr, 10.1-19.0 yr, and older than 19.0 yr were 217 (68-380), 42 (16-112), 390 (16-1028), and 642 (290-964) pg/ml, respectively, and did not differ significantly from values for normal males. In seven of 14 boys aged 10.1-19 yr, INSL3, testosterone, and LH were low (37.4 ± 19.4 pg/ml, 1.44 ± 0.46 nmol/liter, 0.3 ± 0.6 IU/liter). The other seven with higher INSL3, testosterone, and LH (693.1 ± 305.8 pg/ml, 5.91 ± 2.77 nmol/liter, 2.7 ±1.9 IU/liter) had more advanced pubertal development. INSL3was normal in seven of nine males aged older than 19 yr, despite low testosterone in six. After controlling for age, INSL3 correlated with LH (P = 0.005) and testosterone (P < 0.001) but not with FSH, AMH, or inhibin B.
CONCLUSIONS: Most PWS males have normal INSL3 levels. By contrast, testosterone levels after infancy are low. These findings suggest a specific defect in Leydig cell function.
The authors used ELISA and INSL peptide from Phoenix for their study
Hirsch HJ, Eldar-Geva T, Gross-Tsur V et al., J Clin Endocrinol Metab. 2013 Jan;98(1):E135-43. doi: 10.1210/jc.2012-2171. Epub 2012 Nov 12.
OBJECTIVES: Testicular function declines with obesity as a result of central and peripheral mechanisms, including a primary dysfunction of the Leydig cells. The levels of insulin-like factor 3 (INSL3), a sensitive marker of Leydig cell impairment, have never been evaluated in obese men. To better evaluate the hormonal function of the testis in obese men, we analysed their INSL3 plasma levels and compared them with the obesity status and the other reproductive hormones.
DESIGN: Cross-sectional study.
PATIENTS: Thirty-one obese men [body mass index (BMI) >30 kg/m(2)) aged 22-49 years and 64 age-matched nonobese men.
MEASUREMENTS: Plasma concentrations of INSL3, testosterone (T), sex hormone-binding globulin (SHBG), oestradiol (E(2)), LH, FSH. Free testosterone (FT) levels were calculated.
RESULTS: Obese men had significantly lower plasma concentrations of total T, SHBG, FT and INSL3, and higher levels of E(2) with respect to nonobese men. LH and FSH values were not different from controls. In obese men, we found a significant negative correlation between BMI andINSL3, and a positive correlation between INSL3 and T. Only one (1/31, 3.2%) obese man had subnormal T levels. On the contrary, 10/31 (32.3%) obese men had low INSL3 values.
CONCLUSIONS: This study showed for the first time that INSL3 levels decrease with obesity, probably as a result of a primary dysfunction of the Leydig cells. INSL3 is a reliable marker of Leydig cell general impairment, whereas T mainly reflects the steroidogenic activity of these cells.
Foresta C, Di Mambro A, Pagano C et al., Clin Endocrinol (Oxf). 2009 Nov;71(5):722-6. doi: 10.1111/j.1365-2265.2009.03549.x. Epub 2009 Feb 18.
PURPOSE OF REVIEW: Biomarkers of prepubertal testicular function have become widely available only in recent years. The aim of this review is to update the knowledge on key biomarkers used to assess hypogonadism in boys.
RECENT FINDINGS: Sertoli cells are the most representative cells of the prepubertal testis. Anti-Müllerian hormone and inhibin B are essential biomarkers of Sertoli cell function. Also, INSL3 arises as an additional marker of Leydig cell dysfunction.
SUMMARY: The widespread use of these biomarkers has enhanced our knowledge on the pathophysiology and diagnosis of prepubertal male hypogonadism. Beyond their well known germ-cell toxicity, oncologic treatments may also affect Sertoli cell function. Pathophysiology is not the same in all aneuploidies leading to infertility: while hypogonadism is not evident until mid-puberty in Klinefelter syndrome, it is established in early infancy in Down syndrome. In Noonan syndrome, the occurrence of primary hypogonadism depends on the existence of cryptorchidism, and Prader-Willi syndrome may present with either primary or combined forms of hypogonadism. Prepubertal testicular markers have also provided insights into the effects of environmental disruptors on gonadal function from early life, and helped dissipate concerns about testicular function in boys born preterm or small for gestational age or conceived by assisted reproductive technique procedures
Valeri C, Schteingart HF, Rey RA., Curr Opin Endocrinol Diabetes Obes. 2013 Jun;20(3):224-33. doi: 10.1097/MED.0b013e328360be2c.
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 normalbiomarker levels as well as their perturbations in a wide range of reproductive pathologies
Anand-Ivell R, Dai Y, Ivell R. Fertil Steril. 2013 Mar 15;99(4):1153-60. doi: 10.1016/j.fertnstert.2012.12.023. Epub 2013 Jan 18.
Testicular descent is a complex multistep embryonic
process requiring the interaction between anatomical and hormonal
factors. Failure in any of these steps results in cryptorchidism,
the most frequent congenital anomaly of the urogenital tract in
human males. Evidence for a genetic cause for cryptorchidism is
numerous and supported by animal models. In particular, INSL3 and
LGR8/GREAT proteins seem to act as ligand and receptor,
respectively, and to have a role in gubernaculum development
involved in testicular descent. In a cohort of 87 ex-cryptorchid
patients and 80 controls, we looked for mutations in INSL3 and
LGR8/GREAT genes by sequencing. Patients were classified on the
basis of seminal, hormonal, and testicular cytological analyses. We
found three mutations in the INSL3 gene in four patients and one
LGR8/GREAT mutation in four patients (8 of 87, 9.2%). The eight
patients show different phenotypes, ranging from normozoospermia to
complete azoospermia, and from bilateral cryptorchidism to
retractile testes. Furthermore, the endocrine function of the testis
appears normal in all subjects. The findings of our study
demonstrate that INSL3-LGR8/GREAT mutations are frequently
associated with human cryptorchidism and are maternally inherited.
The only clinical consequence of alterations of the INSL3-LGR8/GREAT
system seems to be failure of the testis to normally descend in the
scrotum during embryonic development, without affecting the
spermatogenic and endocrine components of the testis
itself.
Ferlin A, et al. J Clin Endocrinol Metab. 2003 Sep;88(9):4273-9
Cryptorchidism is a common anomaly of
male sexual differentiation. Two phases of testicular descent are
recognized, transabdominal and inguinoscrotal. With evidence that
androgens and Mullerian inhibitory hormone were not completely
responsible for testicular descent, the existence of a third
testicular hormone mediating testicular descent was postulated.
Insulin-like 3 (INSL3) [also known as relaxin-like factor (RLF) and
Leydig insulin-like protein (LEY I-L)] is a member of the
insulin/relaxin hormone superfamily that is highly expressed in
Leydig cells. The phenotype of transgenic mice with targeted
deletion of the Insl3 gene was bilateral cryptorchidism with
morphological evidence of abnormal gubernacular development. With
this implicit evidence that Insl3 mediates testicular descent in
mice, we performed mutation detection analysis of the coding regions
of the 2 exon INSL3 gene in genomic DNA samples obtained from 145
formerly cryptorchid patients and 36 adult male controls.
Single-strand conformational polymorphism analysis was used for the
mutation detection studies. Two mutations, R49X and P69L, and
several polymorphisms were identified. Both mutations were located
in the connecting peptide region of the protein. The frequency of
INSL3/RLF gene mutations as a cause of cryptorchidism is low,
because only 2 of 145 (1.4%) formerly cryptorchid patients were
found to have mutations.
Restriction fragment length polymorphism analysis. Upper portion, Diagram of family pedigree; lower
portion, results of BsmaI digest. The 2450CT variant
causes the loss of a BsmaI site. Carrier status is indicated
by the shaded markers in the pedigree diagram.
Tomboc M, et al. J Clin Endocrinol Metab. 2000 Nov;85(11):4013-8
Two independent studies demonstrated that
transgenic mice with a targeted deletion of the insulin-like 3 (
INSL3) gene presented bilateral cryptorchidism. Studies in humans
have investigated the possibility that mutations in the INSL3 gene
are the cause of cryptorchidism. In the present study, genomic DNA
was obtained from 150 patients with idiopathic cryptorchidism. DNA
was amplified and the polymerase chain reaction products of both
exons were sequenced. A previously unidentified missense mutation
was found in only one of the patients studied. In exon 2, a
heterozygous C/G substitution at nucleotide 2560, which turned
asparagine into lysine at codon 86, was documented. The familial
study revealed that the mother was a heterozygous carrier of the
mutation and the father was a homozygote wild type. We also found
three polymorphic changes, previously reported in exon 1. The
Asn-into-Lys change is likely deleterious because it leads to a
nonconservative amino acid substitution, changing a highly conserved
residue. This mutation, located in the A-chain of the INSL3 protein,
is the first mutation reported in this region. This finding provides
new evidence that INSL3 is involved in testicular descent in humans;
however, mutations of this gene are not a frequent cause of
cryptorchidism.
Canto P, et al. J Hum Genet. 2003;48(2):86-90
Testicular descent is a unique physiological adaptation found in therian mammals allowing optimal spermatogenesis below core body temperature. Recent studies show that INSL3, produced by Leydig cells, and its receptor LGR8 (RXFP2) are essential for mediating the transabdominal phase of testicular descent during early development. However, the origin and genetic basis for this physiological adaptation is not clear. Using syntenic mapping and the functional characterization of contemporary and resurrected relaxin family hormones, we show that derivation of INSL3-mediated testicular descent involved the duplication of an ancestral RLN3-like gene that encodes an indiscriminate ligand for LGR7 (RXFP1) and LGR8. This event was followed by acquisition of the LGR7-selective characteristics by a daughter gene (RLN3) prior to the evolution of the common ancestor of monotremes, marsupials, and placentals. A subsequent mutation of the other daughter gene (INSL3) occurred before the emergence of therian mammals, which then led to the derivation of the reciprocal LGR8-specific characteristics of INSL3. The stepwise evolution of these independent signaling pathways through gene duplication and subsequent divergence is consistent with Darwinian theory of selection and adaptation, and the temporal proximity suggests an association between these genetic events and the concurrent evolution of testicular descent in ancestral therian mammals.
Jae-Il Park, et al., Genome Res. 2008 June; 18(6): 974–985.
The relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the relaxin family peptide (RXFP) receptors 1-4, respectively. Although key binding residues have been identified in the B-chain of the relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human relaxin-2 and -3 (H2 and H3) relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 relaxin. In contrast, A-chain-truncated H3 relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. Our results provide new insights into the action of relaxins and demonstrate that the role of the A-chain for relaxin activity is both peptide- and receptor-dependent.
Hossain MA, Rosengren KJ, et al., J Biol Chem. 2008 Jun 20;283(25):17287-97. Epub 2008 Apr 22.
Serum samples for immunoreactive INSL3 concentration analysis were assayed in duplicate by using a commercial RIA (Phoenix Pharmaceutical, Belmont, CA). The assay is based on a polyclonal rabbit antiserum raised against full-length human INSL3 and on 125I-labeled INSL3 as the tracer. The assay performance was checked before analysis by verifying specificity, sensitivity, precision, and accuracy. To detect the specificity, a cross-reactivity test was conducted via a binding assay with decreasing concentrations of human insulin, INSL4, INSL5, INSL6, and INSL7. The sensitivity was determined as the lower detection limit (28). The precision was checked by using replicates of a serum pool control to measure intraassay and interassay variability. Finally, to detect the accuracy of the method, linearity of dilution was determined by serially diluting serum pool control, and recovery was evaluated by measuring pooled serum samples spiked with increasing standard human INSL3 concentrations before analysis in the RIA. Cross-reactivity with human insulin, INSL4, INSL5, INSL6, and INSL7, used to evaluate the specificity of the INSL3 assay, was 0%. The sensitivity determined as the lower detection limit was 1.8 pg/ml. The intraassay and interassay coefficients of variation were 3.1 and 6.8%, respectively. Suitability of the assay to measure INSL3 accurately was demonstrated by results of linearity and recovery (slope of 1; mean of recovery of 104%), which demonstrated the absence of bias.
Alessandra Gambineri, et al. The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 6 2066-2073
The administration of testosterone plus a progestogen functions as a male contraceptive by inhibiting the release of pituitary gonadotropins. After 3 to 4 months of treatment, most men are azoospermic or severely oligospermic (1 million sperm/mL). However, 10% to 20% of men have persistent sperm production despite profound gonadotropin suppression. Since insulin-like factor 3 (INSL3) has been shown to prevent germ cell apoptosis in mice, we hypothesized that INSL3 might be higher in men with persistent spermatogenesis during treatment with male hormonal contraceptives. In a retrospective analysis, we measured serum INSL3 in 107 men from 3 recent male hormonal contraceptive studies and determined the relationship between suppression of spermatogenesis and serum INSL3. At the end of treatment 63 men (59%) were azoospermic and 44 men (41%) had detectable sperm in their ejaculates. Baseline INSL3 did not predict azoospermia; however, end of treatment serum INSL3 was significantly higher in nonazoospermic men compared with those with azoospermia (median [interquartile range]: 95 [73–127] pg/mL vs 80 [67–101] pg/mL; P = .03). Furthermore, serum INSL3 was positively correlated with sperm concentration (r = .25; P = .009) at the end of treatment and was significantly associated with nonazoospermia by multivariate logistic regression (P = .03). After 6 months of treatment with a hormonal male contraceptive regimen, higher serum INSL3 concentrations were associated with persistent sperm production. INSL3 may play a role in preventing complete suppression of spermatogenesis in some men on hormonal contraceptive regimens. This finding suggests that INSL3 may be a potential target for male contraceptive development.
Serum insulin-like factor 3 at the end of treatment by study and combined in subjects with azoospermia (A shaded) compared with those with nonazoospermia (A top) and in those with severe oligospermia (SO shaded) compared with those with nonsevere oligospermia (B bottom) by study and combined. Boxes represent median and interquartile ranges, with outliers dscepicted as circles. P for the comparisons between groups is included above the line above the box and whiskers plot.
Measurements:
Serum INSL3 was measured by a radioimmunoassay (RIA) (Phoenix Pharmaceuticals Inc, Belmont, Calif). The normal range was 291 to 1132 pg/mL. The lower limit of detection was 16 pg/mL; the intraassay coefficients of variation were 7.6%, 5.2%, and 5.7% and the interassay coefficients of variation were 30%, 13%, and 8.1% for low, mid, and high pools, respectively.
JOHN K. AMORY et al. Journal of Andrology, Vol. 28, No. 4, July/August 2007
Ablation of LGR7-activation activity of AncRFLC and restoration of lost LGR7-activation activity in human INSL3 by point mutation. (A–C) LGR7 (RXFP1)- and LGR8 (RXFP2)-activation activity of AncRFLC RB12H, zRFLC1 RB12H, and zRFLC2 RB12H peptides (A), opossum INSL3 peptide (oINSL3) (B), and INSL3 HB12R and INSL3 HB12A peptides (C). Mean ± SEM, N = 4.
Jae-Il Park, Sheau Yu Teddy Hsu, et al. Genome Res. 2008 June; 18(6): 974–985.
Introduction: Insulin-like factor 3 (INSL3) is a member of the
relaxin-insulin family, and it is expressed in
pre- and postnatal Leydig cells of the testis.This peptide affects testicular descent during
embryonic development. Studies from our group
and others showed a clear association between
mutations in INSL3 gene or its receptor
LGR8/GREAT and cryptorchidism also in humans.
Interestingly, these mutations seem not to
directly affect spermatogenesis or endocrine
function of the testis. The expression of
LGR8/GREAT in different tissues and the
production of INSL3 also by adult-type Leydig
cells, suggest additional roles of this hormonal
system in adulthood.
Materials and Methods: In
this preliminary report, we performed the first
analysis in humans of INSL3 using a novel
radioimmunoassay kit to measure INSL3
concentrations in serum of normal men (n=30) and
women (n=10), and with different testicular
pathologies, including, hypogonadotropic
hypogonadism. (n=2), subjects with untreated
orchiectomized men (n=5), Klinefelter subjects
(n=9), severely infertile men (32), and in men
treated with different kinds of hormonal regimen
of the hyopthalamic-pituitarygonadal axis (such
as ciproterone acetate and testosterone, as male
contraception program (n=10), and different
stimulation tests (such as with GnRH analogue
and FSH plus hCG). Analysis of the expression of
LGR8 in different tissues was performed by
RT-PCR.
Results and Conclusions: The results show that INSL3 is circulating in
adult men, and it is of almost exclusive testis
origin. Serum concentrations are 566.3+/-163.5
pg/mL.and therefore at the same range of inhibin
B. Subjects with severe testicular damage, such
as men with severe infertility, produce low
amount of INSL3, and the concentrations of this
hormone seem to reflect the functional status of
the Leydig cells. In particular, INSL3
concentrations may even be a more sensitive
marker of Leydig cell function than testosterone
itself. INSL3 serum concentrations are
positively correlated with LH and with
testosterone concentrations. Analysis of men
treated with different combinations of hormones
of the hypothalamic-pituitary-testis axis
suggests that the production of INSL3 is related
LH, in a manner similar to that of the
LH-testosterone axis. We found a specific
expression of LRG8 mRNA at the pituitary level
and this finding supports the hypothesis of a
possible pituitary-Leydig axis involving LH and
INSL3, even if further studies are in progress
to confirm these results.
Standard curve (F; triplicate measurements)
and parallelism analysis (f) of pooled diluted
serum samples.
Individual determinations of
INSL3 concentrations in the different groups of
subjects. Continuous and dashed lines indicate
the mean value and the 95% confidence intervals,
respectively. The mean± SD are as follows:
normal adult men, 562.3 ± 155.4 pg/ml (range,
310.1–908.5 pg/ml; 95% confidence interval,
514.2–610.1 pg/ml); normal adult women in the
follicular phase, 99.5 ± 21.7 pg/ml (range,
69.0–130.4 pg/ml; 95% confidence interval,
86.1–112.9 pg/ml); untreated orchidectomized
men, 69.5 ± 26.3 pg/ml (range, 52.5–108.0 pg/ml;
95% confidence interval, 27.7–111.3 pg/ml);
untreated Klinefelter’s syndrome, 157.5 ± 77.1
pg/ml (range, 28.2–237.5 pg/ml; 95% confidence
interval, 98.2–216.7 pg/ml); severely infertile
men, 289.0 ± 69.0 pg/ml (range, 142.0–425.0
pg/ml; 95% confidence interval, 265.1–312.9
pg/ml); and hypogonadotropic hypogonadism, 63.2
± 6.7 pg/ml (range, 58.5–68.0 pg/ml; 95%
confidence interval, 2.9–123.6 pg/ml).
°, P <
0.05 vs. normal adult men;
*, P < 0.001 vs.
normal adult men;
#, P < 0.05 vs. Klinefelter’s
syndrome, untreated orchidectomized men,
hypogonadotropic hypogonadism, and normal adult
women.
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Positive
correlations between INSL3 and LH (r = 0.6; P <
0.001; A) and between INSL3 and testosterone (r
= 0.7; P < 0.001; B), and absence of relation
between INSL3 and FSH (r = 0.1; P < 0.42; C) in
normal males.
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A, Individual determinations of INSL3
concentrations in 10 normal men at baseline and
after 16 wk of therapy with CPA (50 mg twice a
day, orally) plus TE (100 mg/wk, im). Mean
concentrations ± SD are as follows: 693.3 ± 131.8 pg/ml (range, 519.5–908.5; 95% confidence
interval, 599.0–787.6 pg/ml) at baseline and
139.8 ± 64.9 pg/ml (range, 68.0–281.5 pg/ml; 95%
confidence interval, 93.4–186.2 pg/ml) after
therapy (P 0.001). B, Individual
determinations of INSL3 concentrations in two
infertile men at baseline, 30 d after
administration of the GnRH analog leuprolide
(3.75 mg, im), and during the following 2-month
therapy with r-hFSH (100 IU every day, im) and
hCG (2000 IU twice per week, im). Mean
concentrations ± SD are as follows: 328.6 ± 125.8 pg/ml at baseline, 52.7 ± 23.1 pg/ml after
the GnRH analog, 307.5 ± 123.7 pg/ml after 1
month of therapy with gonadotropins, and 339.5 ± 120.9 pg/ml after 2 months of therapy with
gonadotropins.
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Individual determinations of INSL3
concentrations in six men withHHat baseline and
after 1 month of therapy withhCG(2000 IU twice
per week, im). Mean concentrations ± SD are as
follows: 55.2 ± 7.9 pg/ml (range, 46.8–68.0; 95%
confidence interval, 48.9 – 61.5 pg/ml) at
baseline and 111.4 ± 7.6 pg/ml (range,
100.0–115.1; 95% confidence interval,
105.3–117.5 pg/ml) after therapy (P <
0.05).
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Foresta C, et al., J Clin Endocrinol Metab. 2004 Dec;89(12):5952-8
Context Insulin-like factor 3
(INSL3) is produced by the Leydig cells and in
adults its secretion is dependent on the state
of differentiation of these cells, which, in
turn, is dependent on LH. However, the secretion
and regulation of INSL3 during puberty is
unknown. Objective To evaluate INSL3
concentrations during normal male puberty and
its relation to LH, FSH and testosterone. Design
Cross-sectional study conducted from January to
December 2005. Setting Academic clinics Patients
75 healthy male subjects aged 9.5-17.5 yr,
homogeneously distributed into five pubertal
groups of 15 according to Tanner stages.
Interventions None Main outcome measures Mean
testicular volume and LH, FSH, testosterone and
INSL3 concentrations in relation to age and
pubertal stage. Results We observed an increase
of INSL3 and LH levels from Tanner stage 2 to 4,
and an increase of FSH from stage 2 to 3.
Testosterone levels increased from stage 3 to 4.
No differences were seen for all measured
hormones between stage 4 and 5. The increase in
INSL3 seems therefore to anticipate the increase
in testosterone. However, INSL3 plasma
concentrations at pubertal stages 4 and 5 are
about one fourth of adult levels, whereas FSH,
LH and testosterone reached adult levels by
stage 4. Positive significant correlations were
found between INSL3 and LH for all pubertal
stages. Conclusions This study provides
information on the physiological dynamics of
INSL3 showing that the serum concentrations of
this hormone increased progressively throughout
puberty, under the differentiating action of LH
on Leydig cells. INSL3 is therefore confirmed to
represent a marker of Leydig cell
differentiation and function. However, a
prolonged exposition to LH seems to be necessary
to reach INSL3 concentrations of adults. A
possible use of INSL3 in puberty disorders is
promising.
Dr. Ferlin
has declared that he does not have a financial
relationship with any manufacturer/s of any
commercial product/s.
Ferlin A, et al. J Clin Endocrinol Metab. 2006 Jun 27; [Epub ahead of print]
Insulin-like factor 3 (INSL3)
plays a crucial role in testicular descent.
Genetic ablation of Insl3 or its G
protein-coupled receptor, leucine-rich
repeat-containing G-protein-coupled receptor
(Lgr8), causes cryptorchidism in mice. Mutation
analyses of INSL3 in humans showed an
association with cryptorchidism but led to
non-conclusive data about a causative role. In
this study, we explored the hypothesis that
mutations in INSL3 may be associated with the
signs of testicular dysgenesis syndrome (TDS).
We screened for mutations in INSL3 gene in 967
subjects with a history of maldescended testes
and/or infertility and/or testicular cancer and
in 450 controls. Furthermore, we carried out in
vitro functional analysis of three novel
mutations by analysis of INSL3-dependent cAMP
increase in cells expressing LGR8. We found six
INSL3 mutations in 18 of 967 patients (1.9%) and
no mutations in controls. Prevalence of
mutations was similar in the different groups of
patients (cryptorchidism and/or infertility
and/testicular cancer). Three mutations were
novel findings (R4H, W69R, and R72K); however,
their analysis showed normal cAMP increase after
the activation of LGR8 receptor. In conclusion,
we found a significant association of INSL3 gene
mutations in men presenting one or more signs of
TDS syndrome. However, a causative role for some
of these mutations is not clearly supported by
functional analyses. Although a role for
mutations of INSL3 and LGR8 genes in
cryptorchidism is reasonable, additional studies
are needed to establish an association between
the disruption of INSL3 pathway and higher risk
of infertility or testicular
cancer.
Ferlin A, et al. Mol Hum Reprod. 2006 Jun;12(6):401-6. Epub 2006 May 10
CONTEXT: Gonadotropic
regulation of the testicular Leydig cell hormone
insulin-like factor 3 (INSL3) is incompletely
characterized.
OBJECTIVE: The objective of this
study was to assess the effects of gonadotropin
suppression and induced or spontaneous recovery
on serum INSL3.
DESIGN AND PARTICIPANTS: Serum
samples from 15 men enrolled in a short-term
study of gonadotropin stimulation, suppression,
and recovery and 11 men in a long-term study of
gonadotropin suppression and spontaneous
recovery were analyzed for INSL3. Intervention:
Gonadotropins were suppressed by exogenous
testosterone and progestin. Recovery was
spontaneous or induced with exogenous
gonadotropins. OUTCOME MEASURE: The outcome
measure was serum INSL3 in relation to other
reproductive hormones.
RESULTS: Serum INSL3 was
not acutely sensitive to gonadotropins. In both
studies, INSL3 declined markedly with
gonadotropin suppression (6-13.5% of baseline; P
< 0.05). In the short-term study, human
chorionic gonadotropin partially restored
suppressed serum INSL3 within 4 d of
administration (from 7.5 to 38.3% baseline; P
< 0.05); the increase correlated with the
corresponding increase in serum pro-alphaC (r =
0.82; P < 0.01). FSH did not stimulate the
suppressed INSL3. In the long-term study, serum
testosterone recovered significantly better (80%
baseline) compared with serum INSL3 (38.9%
baseline; P < 0.01) in the presence of fully
recovered serum LH.
CONCLUSIONS: INSL3 is not
sensitive to gonadotropin stimulation in normal
men, but declines markedly in response to
gonadotropin deprivation. After suppression,
INSL3 was responsive to hCG 4 d after
administration. After long-term suppression,
INSL3 did not recover to the same degree as
testosterone, suggesting that INSL3 is more
sensitive to Leydig cell impairment than
testosterone.
Bay K, et al. J Clin Endocrinol Metab. 2006 Mar;91(3):1108-11. Epub 2006 Jan 4.
Insulin-like 3 (INSL3) is a
hormone produced by testicular Leydig cells
throughout life. During embryonic life it
regulates an essential step of testicular
descent, whereas in adults it acts as a male
germ cell survival factor. Despite the
importance of INSL3 for male sex differentiation
and function, very little is known regarding the
molecular mechanisms that regulate its
expression. So far, the nuclear receptor SF-1 is
the only transcription factor known to regulate
the mouse Insl3 promoter in Leydig cells. In
order to further our understanding of the
transcriptional regulation of INSL3 expression,
we have isolated the human INSL3 promoter and
tested the effects of the nuclear receptors
SF-1, LRH-1, and Nur77 on its activity in Leydig
cells. In transfections assays, all three
nuclear receptors activated the human INSL3
promoter but especially Nur77, which acted
through a novel regulatory element. Thus, the
human INSL3 promoter constitutes a novel target
for the orphan nuclear receptor
Nur77.
Tremblay JJ, Robert NM. Ann N Y Acad Sci. 2005 Dec;1061:183-9
The human prostate gland is a
well known source of H1 and H2 relaxin but
information is lacking on the expression and
potential role of the INSL3 peptide hormone
within the prostate gland. In the present study
we have investigated the expression of human
INSL3 in patients with benign prostate
hyperplasia (BPH), prostate intraepithelial
neoplasia (PIN) and prostate carcinoma tissues.
Of the prostate epithelial cells, strongest
INSL3 expression was detected in the basal
epithelial cell compartment. Weaker INSL3 mRNA
expression and immunoreactive INSL3 production
were observed in secretory epithelial cells and
in interstitial smooth muscle cells. Prostate
epithelial cells were also a source for
transcripts encoding the INSL3 receptor LGR8
suggesting the presence of an
autocrine/paracrine INSL3-LGR8 ligand-receptor
system within the human prostate. Three human
prostate carcinoma cell lines displayed
differential gene activity for INSL3 and LGR8.
While LNCaP was devoid of INSL3, the
androgen-insensitive PC-3 and the stromal
prostate cell line hPCP co-expressed INSL3 and
LGR8 transcripts. In addition to expressing
INSL3 mRNA, the LGR8-negative DU-145 also
expressed an INSL3 splice form formerly
demonstrated in thyroid carcinoma cells. When
incubated with recombinant INSL3, PC-3 cells
showed significantly increased intracellular
cAMP levels indicating functional LGR8
receptors. INSL3 did not alter the proliferative
or metabolic activity of PC-3 carcinoma cells.
Instead, PC-3 responded to INSL3 with
significantly enhanced tumor cell motility and a
transcriptional down-regulation of ErbB
receptors and EGF. All-trans-retinoic acid was
demonstrated in PC-3 to up-regulate LGR8 gene
activity in a dose- and time-dependent manner
while having no effect on INSL3 gene activity.
In conclusion, we have identified a functional
INSL3-LGR8 ligand-receptor system in human
prostate carcinoma cells.
Klonisch T, et al. Int J Oncol. 2005 Aug;27(2):307-15
Median and 95% confidence intervals of the median of measurements of age, INSL3, testosterone, LH, FSH, and mean testicular volume at each stage of puberty. Values for adults (15) are reported for comparison
Correlation of INSL3 with LH
in all subjects (n: 75). The regression line
(solid line) and 95% confidence intervals
(dotted lines) are indicated. r: 0.92; 95%
confidence intervals:
0.87-0.95;
P<0.001.
Individual serum concentrations of
INSL3 and mean testicular volume in 75 healthy
boys as a function of age.
Scatter diagram of individual
serum concentrations of LH, FSH, testosterone,
and INSL3 at each stage of puberty (15 subjects
per stage). The median is indicated by solid
lines. Significance is indicated when the median
is statistically different from the median of
the previous stage of puberty. *P<0.001;
**P<0.005; §P<0.05. Values for adults (15)
are reported for comparison.
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Relaxin;Relaxin-2;INSL3_Kit;INSL4;INSL5;INSL6;INSL7;LGR Relaxin_1
%INSL 3%
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