Molecular Human Reproduction, Vol. 6, No. 4, 298-302,
April 2000
© 2000 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Absence of mutations involving the INSL3 gene in human idiopathic cryptorchidism
1 Immunogénétique Humaine, INSERM U276, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France, 2 Andrology Unit, University of Florence, Florence, Italy, and 3 Laboratoire d'Histologie, Biologie de la Reproduction et Cytogenetique, Hôpital Tenon, Paris, France
Abstract
The aetiology of cryptorchidism is for the most part unknown and appears to be multifactorial. Recently, a product of Leydig cells termed Leydig insulin-like hormone (INSL3) has been proposed as a putative trophic hormone of the first part of descent. Absence of Insl3 in male mice results in bilateral cryptorchidism and mutations involving this gene may be a cause of cryptorchidism in man. We sequenced both exons of the human INSL3 gene in 31 men who presented with idiopathic unilateral or bilateral cryptorchidism. The only sequence variant was an amino acid substitution in the C-peptide of the molecule. This change was also found in a control group of normal fertile men indicating that it is a polymorphism unrelated to the phenotype. These results suggest that mutations involving the human INSL3 gene are not a common cause of cryptorchidism in man.
idiopathic cryptorchidism/INSL3/Leydig insulin-like hormone/polymorphism
Introduction
During male embryogenesis, the descent of the testis is a direct result of two functional phases. In man the first phase, termed transabdominal migration of the testis, occurs at 10–15 weeks gestation, and the second phase, termed inguinoscrotal migration, occurs at 26–35 weeks gestation. Two structures which originate from the genital mesentery, the cranial suspensory ligament (CSL) and the caudal genital ligament (gubernaculum) play a crucial role in this process. In male mice the outgrowth of the gubernaculum and regression of the CSL result in the transabdominal movement of the testis into the inguinal region. In the second phase the gubernaculum has to regress in order to allow the testis to descend from the inguinal region to the scrotum. Failure of this process results in cryptorchidism. In Western countries almost 3% of boys are operated on for cryptorchidism. International trends suggest that the rate of cryptorchidism, together with other anomalies of male development such as hypospadias and testicular cancer, may be increasing (J.Radcliff Hospital Cryptorchidism Study Group, 1960; Giwercmann et al., 1993). Cryptorchidism is the most significant risk factor for testicular cancer increasing the risk 2.5–11-fold (Benson et al., 1991
). The mechanism of descent is complex and appears to require the interaction of hormonal and mechanical or anatomical factors. Various studies indicate both environmental and genetic contributions. The aetiology of cryptorchidism is for the most part unknown and appears to be multifactorial.
It is widely accepted that the inguinoscrotal migration requires androgens. For the first part of the descent a putative trophic hormone `gubernaculotrophin' has been proposed (Visser and Heyns, 1995). Recently a product of Leydig cells termed Leydig insulin-like hormone (INSL3) has been shown to have many characteristics of a gubernaculotrophin (Nef and Parada, 1999; Zimmermann et al., 1999). INSL3 is a member of the insulin hormone superfamily, is specifically expressed in Leydig cells of the fetal and postnatal testis and in theca cells of the post-natal ovary (Zimmermann et al., 1997). It was originally termed Ley I-L and later referred to as relaxin-like factor (RLF) (Adham et al., 1993; Bullesbach and Schwabe, 1995). Like other members of the insulin-like hormone superfamily, the protein is synthesized as a 131 amino acid preproprotein. The preproprotein contains a 24 amino acid signal peptide, a B chain (31 amino acids at the N-terminus) and an A chain (26 amino acids at the C-terminus) connected by a C-peptide, which mediates correct folding of the molecule and the formation of three disulphide bridges in the active hormone. Following proteolytic processing the active peptide is composed of the B and A chains (Adham et al., 1993; Burkhardt et al., 1994a,b). The gene consists of two small exons and has been localized to 19p13.3-p12 (Burkhardt et al., 1994a). Co-transfection of a steroidogenic factor 1 (SF-1) -containing expression vector together with an INSL3 promoter–chloramphenicol transferase (CAT) construct into HeLa cells, which lack the endogenous SF-1 protein, resulted in CAT gene transcription, which indicates that SF-1 may control INSL3 transcription (Zimmermannn et al., 1998).
Male mice, mutant for Leydig insulin-like hormone (Insl3) gene, exhibit bilateral cryptorchidism (Nef and Parada, 1999; Zimmermann et al., 1999). Insl3–/– mutant males are infertile, with a normal penis but the absence of visible testes. Internal examination revealed bilateral abdominal cryptorchidism. Testis size of the homozygotes was decreased. In adult testis spermatids and mature spermatozoa were absent and the number of primary spermatocytes were reduced. It is not clear whether the complete absence of germ cells was an indirect result of increased environmental temperature in cryptorchid testis or if it was a direct result of the absence of Insl3, which may be required for germ cell development or maintenance. The urogenital tract of Insl3 null mutant mice was normal with the exception of the gubernaculae which failed to develop normally. In Insl3 heterozygous mutants (Insl3+/–) Nef and Prada (1999) found partial cryptorchidism, probably due to delayed gubernacular regression, while Zimmermann et al. (1999) reported no delay in testicular descent. The different findings in the two papers are probably due to the analysis of the phenotype at different developmental stages. The phenotype of Insl3–/– does not appear to be a consequence of androgen deficiency, since sexual behaviour of the null mutant mice was not altered, male accessory organs developed normally and circulating concentrations of testosterone were similar to wild-type mice. These observations suggest that INSL3 is an excellent candidate gene for human cryptorchidism. Insl3 may have a role in the regulation of the oestrous cycle and follicular development, since null mutant female mice had an oestrous cycle twice as long as that of wild-type females, which resulted in a reduced fecundity (Nef and Parada, 1999). However, Zimmermann et al. (1999) found the oestrous cycle length within the normal range.
On the basis of these data we decided to perform a mutation screen of the human INSL3 gene in 31 cases of idiopathic unilateral or bilateral cryptorchidism. This analysis indicated the presence of an alanine to theronine substitution in the connecting C-peptide. This change was also present in a collection of control fertile males with no history of cryptorchidism. These data indicate that mutations of the INSL3 gene are unlikely to be a common cause of cryptorchidism in man.
Materials and methods
Molecular analysis
Sequences corresponding to human INSL3 exons 1 and 2 were amplified using the primers F2/R1 or F5/R5 for exon 1 and F4/R4 for exon 2. F2: 5'-AAA GAC TCG TTG CCC AGT GCT CCC T-3'; R1 5'-GCA TCT GCG CCT ACG TGC AC-3'; F5: 5'-CTC TGG GAG AAG TAC ATC CAA G-3; R5: 5'-CAC TCC TGG CTA ACG GCT CTG G-3'. F4: 5'-CTG GAG AGA CGA CAT CTG CT-3'; R4: 5'-GTG AGC ACC CAT CCC AGG AGG TAA TC-3'. The polymerase chain reaction (PCR) conditions for the amplification of exon 1 with primers F2/R1 were 95°C for 5 min followed by 33 cycles of 95°C 40 s, 61°C 35 s, 72°C 45 s. The PCR conditions using the primers F5/R5 were 95°C 5 min, followed by 33 cycles of 95°C 45 s, 60°C 35 s and 72°C 45 s. The PCR conditions for amplification of exon 2 were 95°C for 5 min followed by 35 cycles of 95°C 1 min, 60°C 45 s and 72°C for 45 s. DNA sequence analysis was performed using at least 200 ng of purified DNA, 20 ng of primer, and fluorescently labelled Taq DyeDeoxy terminator reaction mix (applied biosystems) according to the manufacturer's instructions. DNA sequence was determined using a 373A automated DNA sequencer (Applied Biosystems). Restriction enzyme digestion of PCR products was performed using the EagI restriction enzyme (New England Biolabs) according to the manufacturer's conditions.
Patients
A total of 31 cases of idiopathic bilateral or unilateral cryptorchidism, of European origin, were recruited for the study. The clinical details are summarized in Table I. All cases presented with primary infertility. European men of known fertility and absence of a clinical history of cryptorchidism (n = 9) were used as a control.
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Results
The results of the DNA sequencing are indicated in Table I
. Two silent polymorphisms were observed in exon 1, in both cryptorchid and normal men. These are a G
A transversion at position 27 and at position 126 (position 1 is taken as the first A of the initiation codon). In 16 cryptorchid men a GA transversion was observed at position 178 in the region encoding the C-peptide. This nucleic acid substitution is predicted to result in a conservative amino acid change of an alanine to threonine residue. Of the 31 cases analysed, one was observed to be homozygous for this change and 15 were heterozygous (Figure 1). This base pair substitution results in the removal of an EagI restriction enzyme site. To determine if this change is associated with the cryptorchid phenotype or if it is a common polymorphism present in the general population, we digested PCR-amplified DNA fragment corresponding to exon I with the enzyme EagI in DNA samples from the normal control group. Enzymatic digestion indicated that in the control group of nine individuals, two were homozygotes and three were heterozygous for this base pair change (Figure 2). This result indicates that the predicted amino acid substitution is present at a high frequency in the general population and is probably not associated with the phenotype.
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Discussion
We have demonstrated the presence of an amino acid change at position 178 (exon 1) in the connecting C-peptide of INSL3. This change was present in both cryptorchid males and in a control population of fertile males with no clinical history of cryptorchidism. This indicates that the polymorphism is unrelated to the phenotype and our data suggest that mutations of the Insl3 gene are not a frequent cause of cryptorchidism. There may be a number of reasons for this. The phenotype of the null mutant mouse is bilateral abdominal cryptorchidism, whereas in man, intra-abdominal testis is a very rare finding (5–10% of cases). Therefore, analysis of a much greater number of patients and especially the analysis of cases with abdominal bilateral cryptorchidism may identify mutations in the INSL3 gene. Mutations may be present in upstream activators or downstream targets of INSL3. Mutations or sequence variants in transcriptional regulatory elements either 5' or 3' to the gene may be associated with the phenotype. However we failed to detect mutations in the PCR product that included exon 1 from each patient of this study. This fragment included the transcription initiation site, the TATA box, a CAAT-like and an SP1 consensus sequence, all of which are conserved between man and mice (Burkhardt et al., 1994a).
If INSL3 is not a common cause of human cryptorchidism what are the other candidates? The commonest cause of cryptorchidism is believed to be a defect in prenatal androgen secretion secondary to an insufficient pituitary gonadotrophin secretion or a low production of gonadotrophin by the placenta (Levy and Husmann, 1995; Hosie et al., 1999). Defects in androgen synthesis are associated with isolated cryptorchidism, but this is an uncommon phenomena (Rajfer and Walsh, 1977). Other rare genetic causes include mutations of the X-linked androgen receptor gene, but mutations of this molecule are often associated with other urogenital anomalies such as hypospadias, or complete feminization (Wiener et al., 1998; Nordenskjold et al., 1999). Migration of the gubernaculum beyond the inguinal region is absent in patients with complete androgen resistence (Hutson, 1986) and in these patients the gubernaculum remains enlarged with a failure of regression (Hutson et al., 1997). These data indicate that androgen insufficiency-related cryptorchidism is a different entity from that of the first phase cryptorchidism. Isolated cryptorchidism has been described in some families (Duvie et al., 1990). Pardo-Mindan et al. reported two families with a mode of transmission that suggested autosomal dominant or Y-linked inheritance (Pardo-Mindan et al., 1975). However, microdeletions of the long arm of the human Y chromosome are a frequent cause of male infertility but are only rarely associated with cryptorchidism (Fagerli et al., 1999). Other pedigrees have been described where multiple generations have been affected (Corbus and O'Conor, 1922; Perrett and O'Rourke, 1969). In many cases cryptorchidism is associated with other urogenital anomalies. Familial clustering of cryptorchidism has also been described. Czeizel et al. (1981) confirmed undescended testis in 1.5–4.0% of fathers and 6.2% of brothers. More recently absence of the HoxA10 gene in male mouse is associated with cryptorchidism, however, a screen of 45 cryptorchid boys detected a 24 bp deletion in one case that may have been associated with the phenotype (Kolon et al., 1999). Other cases of cryptorchidism are associated with deletions of 10q26.1–26.3 (Suzuki et al., 1998; Mutoh et al., 1999). These data suggest that the genetic causes of human cryptorchidism are likely to be heterogenous.
Acknowledgments
The authors are grateful for the financial support of the Institut National sur la Recherche Médicale (INSERM), of the Telethon, Italy (grant n281/b), of the Université Paris VII, Fondation pour la Recherche Médicale (FRM) and Association pour le Recherche sur la Cancer (ARC).
Notes
4 To whom correspondence should be addressed 
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Submitted on September 8, 1999; accepted on January 10, 2000.
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