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Insulin-Like Factor 3 |
Department of Histology, Microbiology, and Medical Biotechnologies, Center for Male Gamete Cryopreservation, University of Padova (C.F., A.B., C.V., P.D., A.G., A.F.), 35121 Padova, Italy; and Clinic of Obstetrics and Gynecology, University of Bologna and S. Orsola-Malpighi Hospital (M.C.M.), 40138 Bologna, Italy
Address all correspondence and requests for reprints to: Dr. Carlo Foresta, Department of Histology, Microbiology, and Medical Biotechnologies, Center for Male Gamete Cryopreservation, University of Padova, Via Gabelli 63, 35121 Padova, Italy. E-mail: carlo.foresta{at}unipd.it.
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Introduction |
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Other than the role in testicular descent and germ cell function, this ligand-receptor system may have additional roles in adult men. In fact, INSL3 is also produced in adult-type Leydig cells (1, 2, 18, 19, 20), and LGR8/GREAT is expressed in several tissues other than gubernaculum, with the highest expression level in testis and brain (6, 9). To analyze this possibility and to better understand the physiology of this novel hormonal system, in this preliminary report we performed the first analysis in humans of INSL3, using a recently developed, INSL3-specific RIA kit to measure the concentration of INSL3 in serum of normal adult subjects, including women at different phases of the menstrual cycle and different groups of men affected by different testicular pathologies. Furthermore, to better elucidate the possible hormonal regulation of INSL3 production, we have also analyzed the production of INSL3 in men with untreated hypogonadotropic hypogonadism (HH) and after human chorionic gonadotropin (hCG) treatment, in normal men treated with cyproterone acetate plus testosterone enanthate as male contraception program (21), and in men treated with a GnRH analog and after recombinant human (r-h) FSH/hCG.
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Subjects and Methods |
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Informed consent was obtained from each subject and the authors’ institutional ethical committee approved the study. We selected the following groups of subjects: 40 normal adult men (age, 29.4 ± 3.4 yr), 10 normal adult women (age, 28.6 ± 3.1 yr) at different times of the menstrual cycle (follicular phase d 3, ovulatory phase 24 h after the urinary LH peak, and luteal phase d 21–22), five untreated orchidectomized men, nine untreated men with 47,XXY Klinefelter’s syndrome and azoospermia, 32 severely infertile men (sperm count <5 million/ml, confirmed on at least two occasions) with high FSH (>10 IU/liter) and severe hypospermatogenesis (reduced number of germ cells, as analyzed by testicular fine needle aspiration cytology) (22), six men with HH under basal conditions at the time of first visit (untreated) and after a 4-wk period of treatment with hCG (2000 IU twice per week, im), 10 normal adult men before and after 16 wk of treatment with cyproterone acetate (CPA; 50 mg twice a day, orally) plus testosterone enanthate (TE; 100 mg/wk, im) as a male contraception program (21), and two infertile men before and 30 d after administration of the GnRH analog leuprolide (3.75 mg, im) and during the following 2 months of therapy with r-hFSH (100 IU daily, im) and hCG (2000 IU twice per week, im).
Hormone determinations
Serum concentrations of INSL3 in all subjects were measured in duplicate by RIA using a novel INSL3 RIA kit (Phoenix Pharmaceuticals, Belmont, CA). The kit uses a rabbit polyclonal anti-INSL3 serum raised against full-length synthetic INSL3 peptide. Analysis is performed on 100 µl diluted (1:4) serum samples. Assay validation was assessed by determining the recovery of expected amounts of INSL3 in samples to which exogenous INSL3 was added. Intra- and interassay coefficients of variations were determined. In all subjects LH, FSH, and testosterone were also measured by immunoradiometric assay with commercial kits (Adaltis Italia, Bologna, Italy).
RNA expression analysis of LGR8/GREAT
We used specific primers to evaluate LGR8/GREAT expression (5'-TGCACAGAGAGCACAGCAGAATGGCTC-3' and 5'-GGACAGTGCAACCCGATGTGAAAGACC-3') (23), amplifying a 241-bp specific fragment. cDNAs subjected to PCR were obtained from Human Pituitary Gland Marathon-Ready cDNA (BD Clontech, Palo Alto, CA) and Human Multiple Tissue cDNA (MTC) Panel (BD Clontech). Amplification conditions (using GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA) included an initial denaturation step of 4 min at 94 C, followed by 35 cycles of 30-sec denaturation at 94 C, 1.5-min annealing at 68 C, 1.5-min extension at 72 C, and a final elongation step at 72 C for 4 min. PCR amplification was performed in a 25-µl reaction volume containing 2.5 µl buffer 10x [15 mM MgCl2, 670 mM Tris-HCl (pH 8.8), 160 mM (NH4)2-SO4, and 0.1% Tween 20], 2.5 µl dexoy-NTPs (2 mM of each deoxy-NTP), 0.4 µl Taq DNA polymerase (5 U/µl), 1 µl of each primer (0.1 µg/µl), and 2 µl dimethylsulfoxide. PCR products were separated on a 2% agarose gel (Roche, Mannheim, Germany) by electrophoresis in 1x Tris-acetate/EDTA buffer. All PCR products were also directly sequenced on both strands. The additional fragment of about 550 bp seen in the lane corresponding to the hypophysis was extracted from agarose gel with the Sephaglas BandPrep Kit (Amersham Biosciences, Uppsala, Sweden) and directly sequenced.
Statistical analysis
Comparisons of means of hormonal concentrations among groups were analyzed by the Wilcoxon rank-sum test. The results are expressed in the text as the mean ± SD. Comparisons of hormonal concentrations at baseline and after therapy in HH men, CPA/TE-treated men, and infertile men treated with GnRH analog plus r-hFSH/hCG were calculated with the Wilcoxon signed ranks test for matched pairs. Relations between INSL3 and the other hormones were tested by linear regression analysis. The analysis was carried out with the open-source statistical software R (http://cran.r-project.org). P values (two-sided) of less than 0.05 and less than 0.01 were regarded as significant and highly significant, respectively.
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Results |
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shows the standard curve and parallelism analysis on pooled diluted serum samples. Assay linearity was confirmed using dilutions (1:1 and 1:4) of 15 different serum samples added with different doses of exogenous INSL3 (125, 250, and 500 pg), and linearity was established to be roughly 25–420 pg/ml with a 50% inhibitory concentration of about 91.2 pg/ml. The mean recovery of INSL3 ranged from 81.5–110.0%. Intra- and interassay coefficients of variation were less than 5% and less than 10% respectively (10 determinations/sample at concentrations of 125, 250, and 500 pg/ml). No cross-reactivity was found, in particular with insulin, INSL4, INSL5, INSL6, INSL7, relaxin, C peptide, testosterone, inhibin B, and LH, tested at concentrations from 2.56 pg/ml to 25,600 ng/ml. Figure 2 shows the INSL3 serum concentrations in the different groups. In normal men the mean serum concentration of INSL3 was 562.3 ± 155.4 pg/ml. The INSL3 mean serum level was significantly lower in normal women (99.5 ± 21.7 pg/ml; P < 0.001), confirming that this hormone is produced mainly in a male-specific manner. Untreated orchidectomized men showed a very low INSL3 serum concentration (69.5 ± 26.3 pg/ml; P < 0.001 vs. normal adult men), demonstrating that the testis is the quite exclusive source of this hormone. Patients with Klinefelter’s syndrome had significantly lower INSL3 serum concentrations (157.5 ± 77.1 pg/ml; P < 0.001 vs. normal adult men), suggesting that these patients have severe testicular damage also involving Leydig cells. Infertile men with severe hypospermatogenesis show significantly lower INSL3 levels compared with normal adult men (289.0 ± 69.0 pg/ml; P < 0.05), but significantly higher levels compared with patients with Klinefelter’s syndrome (P < 0.05), suggesting a less severe impairment of Leydig cell function. In severely infertile men the concentrations of testosterone were not different from those in normal men (452.8 ± 157.1.9 vs. 538.5 ± 154.0 ng/dl), whereas in Klinefelter’s subjects the reduced Leydig cell function was evidenced both by significantly lower than normal testosterone concentrations (394.7 ± 118.7 vs. 538.5 ± 154.0 ng/dl; P < 0.05) with higher LH concentrations (13.8 ± 4.6 vs. 2.9 ± 0.9 IU/liter; P < 0.001) and significantly lower than normal INSL3 concentrations. LH concentrations in men with severe hypospermatogenesis were not statistically different from control values (5.7 ± 3.1 vs. 2.9 ± 0.9 IU/liter).
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). These data suggest that the production of INSL3 by the Leydig cells is related to LH, the main regulator of Leydig cell function. This hypothesis is corroborated by the finding that serum INSL3 concentrations in untreated men with HH were very low compared with those in normal adult men (63.2 ± 6.7 pg/ml; P < 0.05; Fig. 2
). To better explore this hypothesis, which has been recently supported in vivo also in rats (18), we used different pharmacological approaches and human models. First, we treated 10 normal men with CPA and testosterone (that causes a reduction in gonadotropins below the sensitivity of the RIA assay after 16 wk of treatment) (21). This treatment caused a significant reduction of circulating INSL3 (from 693.3 ± 131.8 to 139.8 ± 64.9 pg/ml; P < 0.001; Fig. 4A). According to these data, similar results (although not statistically significant due to the low number of patients studied), were obtained by treatment with the GnRH analog leuprolide (that reduces the production of LH and FSH from the pituitary gland) in two infertile men, who show a reduction in INSL3 serum concentrations from 328.6 ± 125.8 to 52.7 ± 23.1 pg/ml. Subsequent treatment of these patients with r-hFSH and hCG restored the normal INSL3 concentrations (and testosterone concentrations), confirming that INSL3 production by Leydig cells is under the control of gonadotropins, in particular of LH (Fig. 4B). Treatment of six men with HH with hCG for 1 month also supports the hypothesis that INSL3 production is related to LH. In fact, at the end of the treatment, INSL3 serum concentrations doubled from 55.2 ± 7.9 to 111.4 ± 7.6 pg/ml (P < 0.05; Fig. 5) in a manner similar to testosterone (from 67.4 ± 7.6 to 109.1 ± 7.1 ng/dl). Furthermore, to better understand ovarian secretion of INSL3 and its relation to LH, we studied 10 normal women at different phases of the cycle (follicular, ovulatory, and luteal phases). No difference in serum INSL3 concentrations was observed (Fig. 6).
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). In particular, PCR showed the expected band in positive control tissues (testis and kidney) and hypophysis, and its absence in negative control tissues (liver and prostate). In the hypophysis we obtained an additional unexpected fragment of about 550 bp. To verify the possible occurrence of an alternative splicing of LGR8/GREAT only in this tissue, we sequenced this PCR fragment and found that it represents a nonspecific product corresponding to a gene called Acinus (24). We can therefore conclude that specific expression of LGR8/GREAT is present at the pituitary level.
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Discussion |
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We found that INSL3 is measurable in serum of normal adult men at relatively high concentrations (562.3 ± 155.4 pg/ml; range, 310.1–908.5 pg/ml). Such concentrations are 1/100th of testosterone concentrations, but are in the same range and even higher than those of the most important Sertoli cell hormone, inhibin B. INSL3 serum concentrations in women are very low, confirming that this hormone is produced mainly in a male-specific manner. Therefore, although INSL3 may contribute to oocyte maturation acting in a paracrine manner, ovarian secretion of this hormone is less than one fifth of testis secretion. Results in untreated orchidectomized men showed that INSL3 is almost exclusively of testicular origin, confirming expression analysis that indicated the Leydig cells as the most important source of this hormone (1, 2, 3, 25), with low expression level in the ovary (1, 2). Important findings were obtained when we compared INSL3 serum concentrations of men with Klinefelter’s syndrome and men with infertility due to severe hypospermatogenesis. Patients with Klinefelter’s syndrome are affected by severe testicular damage involving both the spermatogenic and the endocrine component of the testis, as evidenced by lower testosterone and higher LH concentrations compared with controls. The low INSL3 serum levels found in these subjects can therefore be considered an additional marker of the severe testicular damage also involving Leydig cells. In other words, the reduced production by the Leydig cells of testosterone and INSL3 both represent a marker of Leydig cell dysfunction. On the contrary, patients with severe hypospermatogenesis had normal testosterone and LH levels, whereas INSL3 concentrations were reduced compared with control values and were higher compared with levels in Klinefelter’s subjects. We have interpreted these findings as suggesting mild Leydig cell dysfunction. In other words, in patients with severe infertility, Leydig cell dysfunction is evidenced only by a significantly reduced production of INSL3, whereas the LH-testosterone axis is still apparently normal. INSL3 concentrations could therefore represent a more sensitive marker of Leydig cell function than testosterone itself.
Different findings of our study combined with recent data in animals (18, 19) support the hypothesis that the production of INSL3 is related to LH and that a pituitary-Leydig axis exists for this hormone. First, we found a significant positive correlation between INSL3 and LH concentrations very similar to that found between testosterone and LH. Second, patients with HH have very low INSL3 concentrations, comparable to those found in women, and treatment with hCG produced an increase in INSL3 levels. Treatment of normal men with CPA/TE or treatment of infertile men with a GnRH analog to induce a hypogonadotropic state caused a fall in INSL3 serum concentrations. In this second group of patients, treatment with r-hFSH/hCG restored INSL3 levels. All of these findings are consistent with previous studies reporting that LH/hCG is essential to induce INSL3 expression in adult Leydig cells of the mouse (20). In fact, the hpg mouse, which has a defective gonadal axis caused by a deletion in the hypothalamically expressed gene for GnRH with consequent gonadotropin deficiency (26), exhibits very low expression of Insl3 (20). Furthermore, in the mouse, Insl3 becomes up-regulated in the testis during puberty concomitant with establishment of the pituitary-gonadal axis (20). These data perfectly agree with ours in men with hypogonadotropic hypogonadism and in men treated with GnRH analog and then gonadotropins. More recent data obtained in rats showed that LH stimulates INSL3 transcripts in ovarian thecal and testicular Leydig cells (18), and seasonal up-regulation of INSL3 production in the roe deer was shown to be related to LH-dependent differentiation of Leydig cells (19). In women we did not observe any modification of INSL3 serum concentration during the menstrual cycle. Although additional investigations on this topic are needed, it seems that the LH-dependent increase in INSL3 production in the ovary observed in rats (18) is not associated with an increase in INSL3 secretion in the blood.
Taken together, these data suggested that a pituitary-testicular axis involving LH and INSL3 may exist, where INSL3 production by Leydig cells is stimulated by LH. A possible negative feedback at the pituitary level of INSL3 is supported by the finding of a specific LGR8/GREAT expression in the hypophysis. Although additional studies are needed to understand the possible relation between LH and INSL3, the finding of INSL3 receptor at the pituitary level is intriguing, because it suggests a systemic role for this novel hormonal system and a novel pituitary-testicular axis. However, because INSL3 levels are correlated with circulating testosterone, it is not clear to date whether possible effects at the pituitary level are mediated by androgens or directly by INSL3.
In conclusion, we report for the first time that INSL3 is a male-specific peptide produced in a differentiation-dependent fashion within adult-type Leydig cells other than in the fetal population of Leydig cells. INSL3 represents a circulating hormone in adult men, probably produced in a LH-dependent manner. The INSL3-LGR8/GREAT ligand-receptor pair is involved in testis descent, but additional roles of this hormonal system in adult men are plausible, with it acting both as a paracrine mediator on germ cells and as an endocrine factor elsewhere. Importantly, it seems that INSL3 concentrations may reflect the functional status of Leydig cells possibly better than testosterone concentrations.
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Acknowledgments |
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Footnotes |
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Abbreviations: CPA, Cyproterone acetate; GREAT, G protein-coupled receptor affecting testicular descent; hCG, human chorionic gonadotropin; HH, hypogonadotropic hypogonadism; INSL3, insulin-like factor 3; LGR8, leucine-rich repeat-containing G protein-coupled receptor 8; r-h, recombinant human; TE, testosterone enanthate.
Received March 29, 2004.
Accepted September 8, 2004.
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) of pooled diluted serum samples.
