Mol. Hum. Reprod. Advance Access originally published online on May 10, 2006
Molecular Human Reproduction 2006 12(6):401-406; doi:10.1093/molehr/gal043
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Insulin-like factor 3 gene mutations in testicular dysgenesis syndrome: clinical and functional characterization
1Department of Histology, Microbiology and Medical Biotechnologies, Centre for Male Gamete Cryopreservation, University of Padova, Padova, Italy and 2Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas, USA
3 To whom correspondence should be addressed at: Department of Histology, Microbiology and Medical Biotechnologies, Centre for Male Gamete Cryopreservation, University of Padova, Via Gabelli 63, 35121 Padova, Italy. E-mail: carlo.foresta{at}unipd.it
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Abstract |
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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.
Key words: cryptorchidism/INSL3/LGR8/male infertility/testicular cancer
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Introduction |
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Insulin-like factor 3 (INSL3), initially named Leydig insulin-like peptide (Ley-I-L) (Adham et al., 1993
) and also known as relaxin-like factor (RLF), is a member of the relaxin-like hormone family. INSL3 is expressed in prenatal and post-natal Leydig cells of the testis, and transcripts of the INSL3 gene represent one of the most abundant mRNAs of these cells.
INSL3 is produced as an immature pre-prohormone, composed of A and B chains connected by a C-peptide. A signal peptide at the N-terminus is then excised to form the prohormone. The connecting C-peptide is removed during the processing of the inactive hormone, and two inter-chain and one intra-chain disulphide bonds are formed in the active hormone. The INSL3 gene is comprised of two exons, with an intron interrupting the C-peptide coding domain, and it is localized in chromosome 19 in the last intron of the Janus kinase 3 (JAK3) gene (Burkhardt et al., 1994; Ivell and Bathgate, 2002; Sadeghian et al., 2005).
Research on INSL3 in humans has expanded in recent years following the identification, in rodents, of a role for this peptide in the transabdominal phase of testicular descent by acting on gubernaculum (Nef and Parada, 1999; Zimmermann et al., 1999). As a consequence, numerous human mutation analyses have sought to elucidate the possible involvement of INSL3 and its specific receptor leucine-rich repeat-containing G-protein-coupled receptor (LGR8) in human cryptorchidism (reviewed in Ferlin and Foresta, 2005). Although the cumulative frequency of mutations in INSL3 is relatively low in patients with undescended testis (1.5–2.0%) and definitive in vitro proof of a causative role for many of these mutations is still lacking (Bogatcheva et al., 2003), they represent the first description in humans of genetic alterations specifically associated with the cryptorchid phenotype.
Additional actions for the INSL3–LGR8 system have been further suggested in adults, on the basis of INSL3 expression in adult-type Leydig cells (Roche et al., 1996; Ivell et al., 1997; Zimmermann et al., 1997; Balvers et al., 1998; Hombach-Klonisch et al., 2004; Kawamura et al., 2004) and the wide distribution of LGR8 expression in many tissues (Overbeek et al., 2001; Hsu et al., 2002; Foresta et al., 2004; Kamat et al., 2004) in combination with a high concentration of INSL3 in adult males (Foresta and Ferlin, 2004; Foresta et al., 2004; Bay et al., 2005). In rats, a paracrine role in testis (prevention of germ cell apoptosis) and ovary (oocyte maturation) has been suggested (Kawamura et al., 2004; Anand-Ivell et al., 2006), but specific actions in humans are unknown. In post-natal life, INSL3 is produced constitutively but in a differentiation-dependent manner by the Leydig cells under the long-term Leydig cell-differentiation effect of LH (Ivell et al., 1997; Foresta et al., 2004; Bay et al., 2005); however, possible endocrine effects of INSL3 on tissues and organs other than gubernaculum remain to be demonstrated.
Cryptorchidism is the major risk factor for testicular cancer and reduced fertility, and it may reflect fetal testicular dysgenesis syndrome (TDS) (Skakkebaek et al., 2001; Kaleva and Toppari, 2005). It has been suggested that this syndrome can result in cryptorchidism, testicular cancer, abnormal spermatogenesis and hypospadias, and these disorders may be associated with each other (Skakkebaek et al., 2001). Both genetic and environmental factors, including endocrine-disrupting chemicals, can contribute to abnormal testicular development. Interestingly, it has been shown that the mechanism by which estrogens and some environmental factors acting as endocrine disruptors affect the development of TDS is related to Leydig cell dysfunction with lower testosterone and INSL3 production (Ivell et al., 2005; McKinnell et al., 2005; Foster, 2006).
In this study, we further tried to clarify the role of INSL3 in human testicular descent and cryptorchidism and explored the hypothesis that alterations in INSL3 function may represent a key event in the development of TDS. To achieve this, we screened for INSL3 mutations in many subjects with one or more signs of TDS, namely cryptorchidism, infertility and testicular cancer.
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Materials and methods |
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Subjects and mutation analysis of INSL3
During the last 5 years, we recruited 967 subjects with a history of maldescended testes and/or infertility and/or testicular cancer (Table I). Initial screening was performed in men with a history of maldescended testes (540 subjects, age 20–47): 203 had bilateral cryptorchidism and 337 had unilateral cryptorchidism (age at orchidopexy 1–27 years). One hundred and thirty-five of these 540 subjects are part of previously published papers looking at INSL3–LGR8 gene mutations in cryptorchidism (Ferlin et al., 2003
; Foresta and Ferlin, 2004). After semen and clinical analyses (see next paragraph), patients with a history of maldescended testes were further subgrouped into isolated maldescent (group 1, 130 men), maldescent plus infertility (sperm count <20 x 106/ml) (group 2, 370 men) and maldescent plus infertility and testicular cancer (seminoma) (group 3, 40 men). We then selected 290 infertile men (age 24–45 years) with a sperm count <20 x 106/ml, without a history of maldescended testes and without testicular cancer (group 4). Finally, we selected 137 men with testicular cancer (stage I seminoma) who were referred to our centre for semen cryopreservation before orchiectomy and/or chemotherapy (age 18–38 years). Of them, 65 had only seminoma (group 5) and 72 had seminoma plus a sperm count <20 x 106/ml (group 6).
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A careful history and physical examination were performed in all patients. At least two semen analyses were performed, following WHO recommendations (World Health Organization, 1999), and reproductive hormone (FSH, LH and testosterone) concentrations were determined. The presence of neoplasm was evaluated by testicular ultrasound examination and confirmed by surgical exploration and histological analysis.
Karyotype and Y chromosome micro deletion analyses (Ferlin et al., 2005) were performed in men with a sperm count <10 x 106/ml. Mutations in cystic fibrosis transmembrane regulator (CFTR) gene was performed in men with obstructive azoospermia, as demonstrated by testicular cytological or histological analysis (Foresta et al., 1992) and/or by the absence of vas deferens. Four hundred and fifty men without a history of maldescended testes or retractile testes, without testicular cancer and with a normal sperm count were used as controls.
All patients and controls were Caucasian from different regions of Italy. Informed consent was obtained from each subject, and the authors’ institutional ethical committee approved the study.
Genomic DNA was extracted from peripheral blood of each subject. Both exons of INSL3 were PCR amplified and subjected to direct sequencing, as previously described (Ferlin et al., 2003).
Functional analysis of INSL3 mutations
To produce and analyse the mutant INSL3 hormones, we used a previously described approach (Bogatcheva et al., 2003). To produce targeted mutations in the wild-type INSL3 cDNA, we employed conventional site-directed mutagenesis strategy using a set of mutated sense/antisense primers, DNA amplification, DpnI restriction and clone selection. Resultant cDNA constructs were verified by sequencing of both DNA strands and re-cloned into pcDNA3.1/myc-HisB vector. Plasmids were purified using the high-purity plasmid midiprep kit (Marligen Bioscience, Ijamsville, MD, USA).
Recombinant wild-type and mutant INSL3 peptides were obtained by transfecting pancreatic HIT cells grown in a T-25 flask with 5 µg of the expression construct encoding wild-type or mutant INSL3 peptide. The exact concentration of INSL3 peptides in transfected cell-conditioned media was assessed with INSL3 radioimmunoassay kit (Phoenix Pharmaceuticals, Belmont, CA, USA). Different amounts of conditioned media were used for the stimulation of 293T cells transiently transfected with LGR8 construct (Bogatcheva et al., 2003). The treatment was performed in the presence of 250 µM 3-isobutyl-1-methylxanthine (IBMX) for 20 min. cAMP level in 293T cell lysates was detected using Amersham enzyme immunoassay system (Amersham Pharmacia Biotech, Piscataway, NJ, USA). cAMP concentration in each well was measured in duplicate. The efficiency of LGR8 transfection was normalized by the analysis of the secreted alkaline phosphatase (AP) activity (pAPtag-5 vector from GenHunter, Nashville, TN, USA, was used for co-transfection) with p-Nitrophenyl Phosphate (pNPP) as a substrate (Sigma, St. Louis, MO, USA). All experiments were repeated at least three times using cells from independent transfections.
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Apart from common polymorphisms found in both patients and controls (A24G, V43L and A60T), six nucleotide substitutions leading to amino acid changes were found in heterozygous condition in patients only (18 subjects) (Table II and Figure 1). These mutations were not present in 450 controls.
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The prevalence of INSL3 mutations in TDS patients was therefore 1.9% (18/967) (Table I) (P < 0.005 versus controls by Fisher’s exact test) and was similar in men presenting isolated maldescent (2/130, 1.5%), isolated infertility (3/290, 1.0%), isolated seminoma (2/65, 3.1%), maldescent and infertility (9/370, 2.4%), infertility and seminoma (1/72, 1.4%), maldescent and infertility and seminoma (1/40, 2.5%). The cumulative prevalence of INSL3 mutations in patients with alterations of testis descent is therefore 2.2% (12/540), in patients with infertility is 1.8% (14/772) and in patients with seminoma is 2.3% (4/177). In men with a history of testicular maldescent, the prevalence of INSL3 mutations was similar in men with bilateral (4/203, 1.9%) and unilateral cryptorchidism (8/337, 2.4%).
Of the six mutations, three (P93L, R102C and R102H) have been previously reported (Tomboc et al., 2000; Marin et al., 2001; Ferlin et al., 2003; Foresta and Ferlin, 2004), whereas three represent novel findings. The first of these novel mutations was found in exon 1 (11 G
A), and it causes an arginine to histidine change in the fourth amino acid of the signal peptide (R4H). The other two mutations were in exon 2 and caused amino acid changes in the C-peptide: 205 TC causes a tryptophane to arginine change in amino acid 69 (W69R) and 215 GA causes an arginine to lysine change in amino acid 72 (R72K).
To establish whether INSL3 mutations, especially the new R4H, W69R and R72K mutations, are pathogenetic for cryptorchidism, male infertility and/or testicular cancer, first we carefully analysed clinical data of mutated patients (Table III) and then functionally characterized the novel mutations (Figure 2).
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R4H was found in one patient affected by severe oligozoospermia with a testicular histological picture of severe hypospermatogenesis and without alterations in testicular descent. His karyotype was normal, but Y chromosome micro deletion analysis showed a deletion in the AZFc region (absence of markers sY254 and sY255). W69R was found in three patients. Two had a history of testicular maldescent (one bilateral and one unilateral cryptorchidism) and were affected by severe infertility. One of them was also affected by seminoma in the ex-cryptorchid testis. The third patient was affected by seminoma with normal semen analysis and without a history of cryptorchidism. R72K was found in three patients without a history of testicular maldescent. One of them was azoospermic and was treated at the age of 17 years with chemotherapy for Hodgkin disease. The other two patients had seminoma: one showed normozoospermia and one severe oligozoospermia. P93L was found in five patients, and four of them had a history of maldescent (three unilateral cryptorchidism and one bilateral cryptorchidism). Seminal analysis in these patients ranged from severe oligozoospermia to normozoospermia. One of them also had an inversion of the long arm of chromosome 11, of unclear significance. The fifth patient was affected by obstructive azoospermia associated with the 5T allele in the CFTR gene. Six mutations led to change in amino acid 102: R102C in one case and R102H in five cases. All six patients reported alterations in testis descent. Spermatogenesis was reduced in all cases but two which were affected by obstructive azoospermia.
In the five cases (one W69R, two P93L, one R102C and one R102H) in which parent’s DNA was available, we found that the mutation was transmitted from the mothers, who have no phenotypic abnormalities.
Functional analysis of R4H, W69R and R72K recombinant INSL3 peptides showed that these substitutions did not change the ability of the hormone to induce cAMP increase in the cells expressing LGR8 (Figure 2).
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Discussion |
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A specific association of mutations in INSL3 and LGR8 genes with human cryptorchidism has been described in recent years (reviewed in Adham and Agoulnik, 2003
; Ferlin and Foresta, 2005). Definitive data are not currently available, and the prevalence of mutations is varying in the different studies probably as a result of both ethnic differences and patient selection criteria. However, a cumulative frequency of INSL3–LGR8 mutations of 4–5% is plausible. The phenotype of men with INSL3/LGR8 mutations described in the literature is not uniform, ranging from severe forms of bilateral intra-abdominal testes to milder forms of unilateral inguinal retained testis and eventual spontaneous descent. Spermatogenesis seems not to be directly damaged from such mutations, and the phenotype in terms of sperm production may be the result of testis malposition and age of orchidopexy. However, only a few studies analysed spermatogenesis in men with mutations. Furthermore, a possible role for the INSL3/LGR8 system in testicular cancer has not yet been analysed; however, INSL3 has been implicated in tumorigenesis in other organs (Klonisch et al., 2005). In this study, we tried to clarify the role of INSL3 in human testicular descent and explored the hypothesis that the mutations in INSL3 gene may play a role in the development of TDS. We found a significant association of mutations in INSL3 with TDS, with a prevalence of 1.9%, and no mutations in 450 controls. Mutations were found with similar frequency in men with maldescent of the testes and/or infertility and/or seminoma. Three mutations have been previously described (P93L, R102C and R102H), whereas three were novel findings (R4H, W69R and R72K) and were therefore studied for functional activity in vitro. Phenotype characterization of patients with mutations and functional analysis of INSL3 mutated proteins allowed us to evaluate the possible contribution of INSL3 mutations in such phenotypes.
R4H was found in one patient with severe hypospermatogenesis because of a deletion in the AZFc region of the Y chromosome, and in vitro analysis showed a normal functional activity of the mutated protein. In this mutant, a processing of the pre-prohormone could be affected, because R4H is located in the signal peptide that is excised during protein maturation. Arginine in the fourth position of INSL3 pre-propeptide is well conserved among many species; however, histidine is present in pig sequence. The testiculopathy observed in the patient carrying R4H mutation is therefore better explained by the AZFc deletion rather than the INSL3 mutation. Therefore, conclusive data on R4H mutation cannot be obtained; it seems to represent a rare polymorphism without clinical relevance.
Tryptophane at position 69 is highly conserved in all mammalian INSL3 peptides. The W69R substitution was found only in the patient population, which is consistent with the causative role of the disruption of the INSL3 pathway, at least for testis maldescent. Additionally, a possible role of W69R mutation in the development of seminoma can be hypothesized. However, in vitro analysis showed a normal functional activity of the recombinant protein. It has to be noted, however, that like all other mutations in the C-peptide, W69R mutation might affect hormone processing in vivo and hence decrease the amount of active hormone. Thus, a possible pathogenic role of W69R mutation in cryptorchidism, infertility and seminoma, although plausible, remains to be elucidated.
Amino acid 72 is poorly conserved, with some species including humans having arginine and others having glycine or glutamine at this site. Functional activity of R72K recombinant INSL3 protein was normal. Based on the phenotypes of patients with this mutation, a role of R72K in cryptorchidism and spermatogenic impairment can be excluded. However, additional studies should be undertaken to clarify the possible association of this mutation with a higher risk of seminoma.
More convincing data, although not definitive, were obtained with the P93L mutation. P93L was detected in several patients but not in controls. Four of the five patients with this mutation had alteration in testis descent, and three of them had spermatogenic impairment. Therefore an effect on the descent of the testes could be conceivable. Amino acid at position 93 is well conserved among species; only in the rat peptide, a leucine is found at the homologous site. Proline 93 is part of a conserved sequence, QPLPQ, of the C-peptide; previous protein modelling analysis showed evident secondary structure rearrangement with loss of an entire 
-helix which might lead to protein instability (Ferlin et al., 2003). However, previous analysis showed that P93L substitution does not alter the ability of INSL3 to induce cAMP increase in the cells expressing LGR8 (Bogatcheva et al., 2003).
Six patients had a mutation at position 102 (one R102C and five R102H). All six men presented with testis maldescent and spermatogenesis was damaged in four of them. The arginine at position 102 is the fourth residue before the last amino acid of the C-peptide; it belongs to a stretch of positively charged amino acids centred precisely at R102. Although the native structure of the human INSL3 peptide has not been determined, data on the bovine INSL3 suggest a critical role of this site for protein processing. Because this stretch is located near the endopeptidase cleavage site between C-peptide and A chain, it is possible that the change in the amino acid sequence surrounding the cleavage site might alter the processing of C-peptide that will not be excised (Ferlin et al., 2003). Several mammalian INSL3 peptides contain histidine at this position, and substitution of arginine 102 with histidine does not alter the secondary structure prediction (Ferlin et al., 2003). On the contrary, substitution with a cysteine significantly changes the charge of the region with possible effect on C-peptide processing, and in accordance with this, the functional analysis showed a slight reduction of LGR8 activation by R102C peptide, whereas R102H mutation did not change the ability of the hormone to induce cAMP increase (Bogatcheva et al., 2003). Therefore, although both R102C and R102H are fully compatible with the phenotypes observed, only for R102C do we have some indication of the pathogenetic role.
Taken together, our results suggest that many mutations in INSL3 gene seem to be specifically associated with signs of TDS and especially with alterations in testicular descent. On the contrary, a cause–effect relation is not obvious for many of them based on in vitro analysis. It should be noted that functional studies utilized in this and previously published studies (Bogatcheva et al., 2003) look only at the ability of the recombinant INSL3 proteins to induce cAMP increase in cells expressing LGR8 receptor. Other characteristics of the mutated INSL3 peptide, such as cell-specific efficiency of transcription, translation, processing of the mature protein or its stability, have not been analysed. This is particularly important for mutations located within C-peptide. Therefore, additional studies are warranted, above all for the mutations that seem to be associated with suggestive phenotypes. It should be noted that even if INSL3 gene mutations actually represent a cause of cryptorchidism, infertility and/or testicular cancer, the prevalence of these mutations in the patient population is relatively low. Analysis of the literature shows that 939 men with cryptorchidism have been studied for INSL3 gene mutations, with 1.6% cumulative frequency of mutations (15/939) (reviewed in Ferlin and Foresta, 2005), a number consistent with this study. However, by adding the frequency of LGR8 gene mutations (9/319, 2.8%) (reviewed in Ferlin and Foresta, 2005), for which a clear causative role has been shown (Gorlov et al., 2002), a figure of 4–5% of alterations in the INSL3/LGR8 pathway is plausible in cryptorchid men. In addition to 450 controls of this study, 907 non-cryptorchid men analysed for INSL3 gene and 587 men analysed for LGR8 gene in previous studies displayed none of the aforementioned mutations.
The TDS theory suggests a defect in the differentiation of Sertoli and Leydig cells induced by genetic and/or environmental factors during early fetal development as primary alterations. As a consequence, germ cell proliferation and testosterone production will be impaired (Skakkebaek et al., 2001). In this context, Leydig cell impairment may also produce INSL3 deficiency, thus leading to cryptorchidism. LGR8 has been detected in germ cells and Leydig cells (Anand-Ivell et al., 2006), and complex autocrine/paracrine interactions of the INSL3 system in the testis have been suggested (Kawamura et al., 2004; Anand-Ivell et al., 2006). Therefore, INSL3 and LGR8 gene mutations could result also in germ cell impairment and thus in spermatogenic damage and testicular cancer.
In conclusion, we found a significant association of INSL3 gene mutations in men presenting one or more signs of TDS. However, a causative role for some of these mutations is not obvious and not clearly supported by functional analyses. Although a role for mutations of INSL3 and LGR8 genes in cryptorchidism is reasonable, additional studies should be undertaken to establish an association between the disruption of INSL3 pathway and higher risk of infertility or testicular cancer.
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Acknowledgements |
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We thank Anne Truong for the excellent technical assistance. This work was supported by the R01 HD37067 grant from the National Institute of Health to A.I.A. and by the University of Padova grant to A.F.
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References |
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Adham IM and Agoulnik AI (2003) Insulin-like 3 signalling in testicular descent. Int J Androl 27,257–265.
Adham IM, Burkhardt E, Benahmed M and Engel W (1993) Cloning of a cDNA for a novel insulin-like peptide of the testicular Leydig cells. J Biol Chem 268,26668–26672.
Anand-Ivell RJ, Relan V, Balvers M, Coiffec-Dorval I, Fritsch M, Bathgate RA and Ivell R (2006) Expression of the insulin-like peptide 3 (INSL3) hormone-receptor (LGR8) system in the testis. Biol Reprod March 7 [Epub ahead of print].
Balvers M, Spiess AN, Domagalski R, Hunt N, Kilic E, Mukhopadhyay AK, Hanks E, Charlton HM and Ivell R (1998) Relaxin-like factor expression as a marker of differentiation in the mouse testis and ovary. Endocrinology 139,2960–2970.
Bay K, Hartung S, Ivell R, Schumacher M, Jurgensen D, Jorgensen N, Holm M, Skakkebaek NE and Andersson AM (2005) Insulin-like factor 3 serum levels in 135 normal men and 85 men with testicular disorders: relationship to the luteinizing hormone-testosterone axis. J Clin Endocrinol Metab 90,3410–3418.
Bogatcheva NV, Truong A, Feng S, Engel W, Adham IM and Agoulnik AI (2003) GREAT/LGR8 is the only receptor for insulin-like 3 peptide. Mol Endocrinol 17,2639–2646.
Burkhardt E, Adham IM, Brosig B, Gastmann A, Mattei MG and Engel W (1994) Structural organization of the porcine and human genes coding for a Leydig cell-specific insulin-like peptide (LEY I-L) and chromosomal localization of the human gene (INSL3). Genomics 20,13–19.[CrossRef][Web of Science][Medline]
Ferlin A and Foresta C (2005) The INSL3-LGR8 hormonal system in humans: testicular descent, cryptorchidism and testicular functions. Curr Med Chem: Immunol Endocr Metab Agents 5,421–429.[CrossRef]
Ferlin A, Simonato M, Bartoloni L, Rizzo G, Bettella A, Dottorini T, Dallapiccola B and Foresta C (2003) The INSL3-LGR8/GREAT ligand-receptor pair in human cryptorchidism. J Clin Endocrinol Metab 88,4273–4279.
Ferlin A, Tessari A, Ganz F, Marchina E, Barlati S, Garolla A, Engl B and Foresta C (2005) Association of partial AZFc region deletions with spermatogenic impairment and male infertility. J Med Genet 42,209–213.
Foresta C and Ferlin A (2004) Role of INSL3 and LGR8 in cryptorchidism and testicular functions. Reprod Biomed Online 9,294–298.[Web of Science][Medline]
Foresta C, Varotto A and Scandellari C (1992) Assessment of testicular cytology by fine needle aspiration as a diagnostic parameter in the evaluation of the azoospermic subject. Fertil Steril 57,858–865.[Web of Science][Medline]
Foresta C, Bettella A, Vinanzi C, Dabrilli P, Meriggiola MC, Garolla A and Ferlin A (2004) INSL3: a novel circulating hormone of testis origin in humans. J Clin Endocrinol Metab 89,5952–5258.
Foster PM (2006) Disruption of reproductive development in male rat offspring following in utero exposure to phthalate esters. Int J Androl 29,140–147.[CrossRef][Web of Science][Medline]
Gorlov IP, Kamat A, Bogatcheva NV, Jones E, Lamb DJ, Truong A, Bishop CE, McElreavey K and Agoulnik AI (2002) Mutations of the GREAT gene cause cryptorchidism. Hum Mol Genet 11,2309–2318.
Hombach-Klonisch S, Schon J, Kehlen A, Blottner S and Klonisch T (2004) Seasonal expression of INSL3 and Lgr8/Insl3 receptor transcripts indicates variable differentiation of Leydig cells in the roe deer testis. Biol Reprod 71,1079–1087.
Hsu SY, Nakabayashi K, Nishi S, Kumagai J, Kudo M, Sherwood OD and Hsueh AJ (2002) Activation of orphan receptors by the hormone relaxin. Science 295,671–674.
Ivell R and Bathgate RA (2002) Reproductive biology of the relaxin-like factor (RLF/INSL3). Biol Reprod 67,699–705.
Ivell R, Balvers M, Domagalski R, Ungefroren H, Hunt N and Schulze W (1997) Relaxin-like factor: a highly specific and constitutive new marker for Leydig cells in the human testis. Mol Hum Reprod 3,459–466.
Ivell R, Hartung S and Anand-Ivell R (2005) Insulin-like factor 3: where are we now? Ann N Y Acad Sci 1041,486–496.[CrossRef][Web of Science][Medline]
Kaleva M and Toppari J (2005) Cryptorchidism: an indicator of testicular dysgenesis? Cell Tissue Res 322,167–172.[CrossRef][Web of Science][Medline]
Kamat AA, Feng S, Bogatcheva NV, Truong A, Bishop CE and Agoulnik AI (2004) Genetic targeting of relaxin and insulin-like factor 3 receptors in mice. Endocrinology 145,4712–4720.
Kawamura K, Kumagai J, Sudo S, Chun SY, Pisarska M, Morita H, Toppari J, Fu P, Wade JD, Bathgate RA et al. (2004) Paracrine regulation of mammalian oocyte maturation and male germ cell survival. Proc Natl Acad Sci USA 101,7323–7328.
Klonisch T, Hoang-Vu C and Hombach-Klonisch S (2005) Relaxin-like peptides in neoplastic lesions. Curr Med Chem: Immunol Endocr Metab Agents 5,431–438.[CrossRef]
Marin P, Ferlin A, Moro E, Rossi A, Bartoloni L, Rossato M and Foresta C (2001) Novel insulin-like 3 (INSL3) gene mutation associated with human cryptorchidism. Am J Med Genet 103,348–349.[CrossRef][Web of Science][Medline]
McKinnell C, Sharpe RM, Mahood K, Hallmark N, Scott H, Ivell R, Staub C, Jegou B, Haag F, Koch-Nolte F et al. (2005) Expression of insulin-like factor 3 (Insl3) protein in the rat testis during fetal and postnatal development and in relation to cryptorchidism induced by in utero exposure to Di (n-butyl) phthalate. Endocrinology 146,4536–4544.
Nef S and Parada LF (1999) Cryptorchidism in mice mutant for Insl3. Nat Genet 22,295–299.[CrossRef][Web of Science][Medline]
Overbeek PA, Gorlov IP, Sutherland RW, Houston JB, Harrison WR, Boettger-Tong HL, Bishop CE and Agoulnik AI (2001) A transgenic insertion causing cryptorchidism in mice. Genesis 30,26–35.[CrossRef][Web of Science][Medline]
Roche PJ, Butkus A, Wintour EM and Tregear G (1996) Structure and expression of Leydig insulin-like peptide mRNA in the sheep. Mol Cell Endocrinol 121,171–177.[CrossRef][Web of Science][Medline]
Sadeghian H, Anand-Ivell R, Balvers M, Relan V and Ivell R (2005) Constitutive regulation of the Insl3 gene in rat Leydig cells. Mol Cell Endocrinol 241,10–20.[CrossRef][Web of Science][Medline]
Skakkebaek NE, Rajpert-De Meyts E and Main KM (2001) Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum Reprod 16,972–978.
Tomboc M, Lee PA, Mitwally MF, Schneck FX, Bellinger M and Witchel SF (2000) Insulin-like 3/relaxin-like factor gene mutations are associated with cryptorchidism. J Clin Endocrinol Metab 85,4013–4018.
World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction. Cambridge University Press, Cambridge, UK.
Zimmermann S, Schottler P, Engel W and Adham IM (1997) Mouse Leydig insulin-like (Ley I-L) gene: structure and expression during testis and ovary development. Mol Reprod Dev 47,30–38.[CrossRef][Web of Science][Medline]
Zimmermann S, Steding G, Emmen JM, Brinkmann AO, Nayernia K, Holstein AF, Engel W and Adham IM (1999) Targeted disruption of the Insl3 gene causes bilateral cryptorchidism. Mol Endocrinol 13,681–691.
Submitted on March 14, 2006; accepted on March 31, 2006.
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