help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tomboc, M.
Right arrow Articles by Witchel, S. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tomboc, M.
Right arrow Articles by Witchel, S. F.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*OMIM
*Protein*UniGene
*Compound via MeSH
*Substance via MeSH
Medline Plus Health Information
*Testicular Disorders
The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 11 4013-4018
Copyright © 2000 by The Endocrine Society

From the Clinical Research Centers

Insulin-like 3/Relaxin-Like Factor Gene Mutations Are Associated with Cryptorchidism1

Marlah Tomboc, Peter A. Lee, Mohamed F. Mitwally, Francis X. Schneck, Mark Bellinger and Selma F. Witchel

Division of Pediatric Endocrinology (M.T., M.F.M. S.F.W.), Children’s Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, Pennsylvania 15213; Department of Pediatrics (P.A.L.), Pennsylvania State University School of Medicine, Hershey, Pennsylvania; and Department of Urological Surgery (F.S., M.B.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Address all correspondence and requests for reprints to: Selma F. Witchel, M.D., Division of Pediatric Endocrinology, Children’s Hospital of Pittsburgh, University of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, Pennsylvania 15213. E-mail: witches{at}chplink.chp.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cryptorchidism is a common anomaly of male sexual differentiation. Two phases of testicular descent are recognized, transabdominal and inguinoscrotal. With evidence that androgens and Müllerian 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NORMAL MALE SEXUAL differentiation is a complex process requiring testicular development, stabilization of the Wolffian ducts, regression of the Müllerian duct derivatives, and testicular descent into the scrotum. Although many of the genetic and hormonal factors involved in the process of sexual differentiation have been established (1), the molecular mechanism underlying the sexually dimorphic position of the gonads remains unknown. Incomplete testicular descent, cryptorchidism, is a common anomaly of male sexual differentiation affecting approximately 3% of human males at birth. By 1 yr of age, the prevalence rate declines to 1% because of spontaneous testicular descent (2). Consequences of cryptorchidism include the need for surgical intervention and increased risks to develop oligo/azoospermia and testicular tumors (3).

Two phases of testicular descent have been recognized (4). In the first (or transabdominal) phase, the testes descend to the inguinal region. During this phase, rapid gubernacular cell proliferation occurs concurrently with regression of the cranial suspensory ligament. This first phase of testicular descent occurs between 10–15 weeks gestation in humans and around 15.5–17.5 days post coitum in mice. During the second (or inguinal-scrotal) phase, the testes descend from the inguinal region into the scrotum by regression of the gubernaculum. This second phase occurs at 28–35 weeks gestation in humans and 2–3 weeks after birth in mice (5).

Thus, testicular descent requires a series of dynamic changes in the gubernaculum and the cranial suspensory ligament. Hormones such as androgens and Müllerian inhibitory hormone (MIH) have been investigated as factors that control testicular descent (6, 7). Current evidence suggests that transabdominal descent is not androgen-dependent (8). Although cryptorchidism is the most common presenting feature for persistent Müllerian duct syndrome, the aberrant positioning of the testes in this disorder has been attributed to anatomic obstruction, because the testes are typically attached to the Müllerian duct derivatives with variable positioning ranging from intraabdominal to transverse testicular ectopia. Persistent Müllerian duct syndrome, caused by either MIH or MIH type II receptor mutations, is a rare genetic cause of cryptorchidism (9, 10, 11). However, normal testicular descent in MIH-deficient knockout mice suggests that, at least in mice, the role of MIH in testicular descent is minimal (12).

Hence, existence of a third testicular hormone was postulated. Using an in vitro system, a low-weight molecular substance, known as descendin, derived from testicular extracts was found to stimulate porcine gubernacular cell proliferation; whereas ovarian extracts, testosterone, dihydrotestosterone, MIH, and inhibin, did not (13, 14). Recently another testicular hormone, insulin-like 3 (INSL3), also known as Leydig insulin-like protein (LEY I-L) or relaxin-like factor (RLF) (15) has been identified. The gene and its protein product have been characterized in several species, including man, pig, sheep, and marmoset monkey (16, 17, 18, 19). Similar to insulin and relaxin, it consists of a B chain, a connecting peptide region, and an A chain. As anticipated for a hormone involved in testicular descent, sexually dimorphic expression was observed in mice, with expression of Insl3 first noted at postcoital day 13.5 in male embryos and not until postnatal day 6 in females (20).

Transgenic mice with targeted deletion of the Insl3 gene showed bilateral cryptorchidism, normal virilization of the external genitalia, and normal androgen-dependent behavior. Morphological evaluation of the homozygous knockout males revealed developmental abnormalities of the gubernaculum (21, 22). Development of the Wolffian duct structures was normal, and Müllerian duct structures were absent, indicating appropriate testosterone and MIH secretion. Thus, these findings excluded the possibility that cryptorchidism in the Insl3 knockout mice was secondary to androgen deficiency or the loss of the MIH-mediated activity.

Of interest, the phenotype of heterozygous male mice differed according to the age at which the animals were examined. At birth, approximately 75% of transgenic mice heterozygous for Insl3 deletions had partial unilateral or bilateral undescended testes. But, all adult heterozygotes showed full testicular descent. Thus, the phenotype of the Insl3 heterozygotes is similar to the human situation in which spontaneous resolution of cryptorchidism often occurs. With this implicit evidence that Insl3 mediates testicular descent in mice, we sought to determine whether mutations in INSL3/RLF could be associated with human cryptorchidism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects

Blood samples for extraction of genomic DNA were obtained from 145 males who had previously undergone surgical correction for cryptorchidism and from 36 adult male controls. Ages of the subjects ranged from 1–54 yr; and for the controls, 26–54 yr. This protocol is approved by the Human Rights Committee of the Children’s Hospital of Pittsburgh. Written informed consent was obtained from all adult participants and from parents of all children.

Mutation detection studies

Genomic DNA was extracted from peripheral blood leukocytes. Mutation detection studies were performed using single-strand conformational polymorphism (SSCP) analysis. Exon 1 was amplified using 5'-tgggagaaaggctctggcac-3' and 5'-ctgggctcatgcatgcaaac-3' primers (Fig. 1Go). For exon 2, the sequences of the primers used were 5'-tgcatgcatgagtgtttggtggg-3' and 5'-atcagtagggacagagggtc-3'. The PCR reactions consisted of 0.125 µg genomic DNA, 0.15 µL of each of 20 pmol primers, 1.25 µL 10x PCR buffer, 0.07 µL Taq polymerase (Display TAQ FL), 0.09 µL 32P deoxy-ATP (10 mCi/mL, NEN Life Science Products, Boston, MA), and 8.3 µL sterile water in a total vol of 12.5 µL. PCR thermocycler conditions for exons 1 and 2 were 94 C for 30 sec, 64 C for 30 sec, and 72 C for 1 min, for 30 cycles, with a final extension at 72 C for 7 min. Three distinct gel conditions were used for each PCR product: 1) MDE gel (FMC BioProducts, Rockland, ME) with 0.6x Tris-Boric Acid-0.5 M EDTA (TBE) buffer at 7 W, at room temperature for 18 h (exon 1) and 15 h (exon 2); 2) 5% acrylamide with 0.5x TBE buffer at 30 W, at 4 C for 4.5 h (exon 1) and 4 h (exon 2); and 3) 5% acrylamide with 5% glycerol and 0.5x TBE at 20 W, at room temperature for 6 h. Using these three different gel conditions, we have typically detected 85–90% of sequence variants (23).



View larger version (12K):
[in this window]
[in a new window]
  		Figure 1. Cartoon of INSL3/RLF gene.

 
Unique conformers were excised from the dried gels and eluted into 100 µL 0.1x Tris-HCl-EDTA. A 5-µL aliquot of each eluted conformer was reamplified using the original primer pair and thermocycling conditions. The PCR products were sequenced in both sense and antisense orientations, either manually using the Sequitherm Excel II DNA sequencing kit (Epicentre Technologies, Madison, WI) or using an ABI automated sequencer 377R according to the manufacturer’s protocol. When nucleotide alterations altered restriction sites, restriction fragment digestion of PCR-amplified genomic DNA was performed to confirm the presence of the sequence variant (see Results).

Statistical analysis

AbSTAT statistical software (Release 1.94, Anderson-Bell, Boulder, CO) was used to perform {chi}-square analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exon 1

SSCP analysis of exon 1 showed two distinct conformer patterns (Fig. 2Go). The conformer patterns were identified both among subjects with cryptorchidism and among controls. Conformers were excised from subjects who were homozygous for each pattern. On sequence analysis, eluted bands from one conformer pattern showed the normal sequence. Sequence analysis of eluted bands from the second conformer pattern showed two nucleotide changes. The first change was G->A at nucleotide position 1266, which mapped to codon 18 of the B chain of exon 1. This variant did not change the amino acid, leucine. The second change was G->A at nucleotide position 1318. The predicted consequence of this variation is to change codon 36 from alanine to threonine in the C peptide region of the protein. Because the conformer pattern showed homozygosity, both nucleotide changes were shown to be located on the same allele.



View larger version (131K):
[in this window]
[in a new window]
  		Figure 2. Autoradiograph of exon 1. Two conformer patterns were detected in formerly cryptorchid patients and control subjects.

 
Among the patients treated for cryptorchidism (Table 1Go), 69 were homozygous for the normal allele, 19 were homozygous for two nucleotide changes, 1266G->A and 1318G->A. Fifty-seven subjects were heterozygous; 1 allele carried the normal sequence and the other allele carried both nucleotide changes. Among the control subjects, 19 men were homozygous for the normal allele, 4 men showed only the variant allele, and 13 men were heterozygous. Allelic frequency was comparable among patients and controls (P > 0.05).


View this table:
[in this window]
[in a new window]
  		Table 1. Exon 1 variant. Distribution of the variants, identified by SSCP analysis of exon 1, is listed below

 
The sequence variations at positions 1266 and 1318 generate conservative amino acid changes and were found in comparable distribution in both affected subjects and controls. Thus, these variants most likely represent neutral linked polymorphisms and do not influence INSL3/RLF activity.

Exon 2

SSCP analysis of exon 2 revealed four different conformer patterns (A, B, C, and D). The majority of subjects (142 patients and 36 control subjects) showed the same pattern. Sequence analysis of conformer pattern A showed the normal sequence. Three different conformers (B, C, and D) were identified in 3 formerly cryptorchid subjects (Fig. 3Go). All three individuals were heterozygous for a sequence variant with the presence of a unique conformer in addition to the common conformer pattern noted in the majority of patients and control subjects.



View larger version (34K):
[in this window]
[in a new window]
  		Figure 3. Autoradiograph of exon 2 conformers. A, Conformer which showed 2450C->T variant; B, conformer which showed intron variant; C, conformer which showed 2511C->T variant.

 
The propositus from whom conformer B was isolated presented with an incarcerated right inguinal hernia at 6 weeks of age and, at that time, was found to have a right undescended testis. He subsequently underwent right inguinal herniorrhaphy and orchiopexy. Sequence analysis of conformer B showed a C->T mutation at nucleotide 2450. The consequence of this mutation is that arginine at codon 49 is changed to a termination codon (R49X). This mutation also caused the loss of a Bsma1 site. Restriction fragment digestion confirmed the results of the SSCP analysis that this patient was heterozygous for the 2450C->T mutation. Restriction fragment digests of genomic DNA samples obtained from the maternal grandparents, mother, half-brother, and half-sister of the propositus were also examined (Fig. 4Go). The propositus, his mother, and maternal grandfather were heterozygous for the R49X mutation. Because this patient was African-American, 90 genomic DNA samples (180 chromosomes) from healthy control African-American males were studied. None showed the 2450 C->T variant.



View larger version (44K):
[in this window]
[in a new window]
  		Figure 4. Restriction fragment length polymorphism analysis. Upper portion, Diagram of family pedigree; lower portion, results of BsmaI digest. The 2450C->T variant causes the loss of a BsmaI site. Carrier status is indicated by the shaded markers in the pedigree diagram.

 
The patient from whom conformer C was isolated presented at 8 months of age with a nonpalpable right testis. At the time of surgery, his right testis was located intraabdominally. Sequence analysis of unique conformer C showed a C->T change at nucleotide 2511. The consequence of this mutation is that codon 69 is changed from proline to leucine (P69L). This variation did not alter restriction sites. None of the 36 healthy control subjects show this variant. Genomic DNA samples from an additional 69 males were evaluated; none were found to carry the 2511 C->T variant.

The African-American subject from whom conformer D was isolated presented with unilateral cryptorchidism. At the time of surgical correction, his right testis was found to be within the right inguinal canal. Sequence analysis of unique conformer D showed a C->T nucleotide change at nucleotide 1259, which is located in the intron. No consensus splicing signals are altered. This conformer was identified in 16 African-American control subjects and in 0 of the 105 Caucasian control subjects.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Consequences of cryptorchidism include impaired spermatogenesis and testicular neoplasms, conditions that may influence reproductive competence. It could, thus, be anticipated that the process of testicular descent involves multiple genes, some with redundant function, to ensure proper testicular descent and fertility. Nevertheless, cryptorchidism is a common disorder affecting 3% of male infants at birth. Familial cryptorchidism has been described, but seems to be uncommon, ranging from 1–6% (24). Among patients with cryptorchidism, testicular position shows phenotypic heterogeneity with locations ranging from intraabdominal to the inguinal canal (25, 26). In some cases of unilateral cryptorchidism, abnormalities can occur in the contralateral scrotal testis, suggesting the possibility that an intrinsic testicular abnormality may be present in some affected individuals (27).

Based on the phenotype of the knockout mouse, one gene that might be associated with cryptorchidism in humans is INSL3/RLF. INSL3/RLF, a member of the insulin-like hormone superfamily, is expressed predominantly in the Leydig cells of the fetal and adult testes and in the theca cells of the postnatal ovaries and seems to be a marker of Leydig cell differentiation status (28, 29, 30). Transcription of the INSL3/RLF gene is mediated by steroidogenic factor-1, an orphan nuclear receptor essential for fetal pituitary, adrenal, and gonadal differentiation (31, 32, 33). Recently, an INSL3/RLF receptor has been identified (34).

Using SSCP analysis, we have identified three variants involving the coding region and one involving the intron in the INSL3/RLF gene. The 2450C->T mutation that changes codon 49 from arginine to a termination codon is clearly a deleterious mutation because it leads to a truncated protein product lacking the A chain. The 2511 C->T change that changes codon 69 from proline to leucine is likely deleterious because it leads to a nonconservative amino acid change, changes a highly conserved residue, and occurs in less than 1% of the population (Fig. 5Go). Both mutations occur in the connecting peptide region of the INSL3/RLF protein. The intron variant seems to be a rare polymorphism occurring preferentially in African-Americans. The linked L18L;A36T variant seems to be a common polymorphism because the frequency of the less common allele is high and is similar among the patients and control subjects (32.8% and 29.2%, respectively). This exon 1 variant was previously described (35, 36).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 5. Comparison of protein sequences. Amino acid residues are indicated by the single universal letter code. The R residue at codon 49 and the P residue at position 69 are underlined.

 
The propositus with the R49X mutation presented with incarcerated right inguinal hernia at 6 weeks of age and, at that time, was found to have a right undescended testis. He subsequently underwent right inguinal herniorrhaphy and orchiopexy. The maternal grandfather, who has the same mutation, does not recall being told that he had cryptorchidism and never required surgical repair of cryptorchidism. As to the grandfather’s reported phenotype, we cannot exclude the possibility that he had cryptorchidism with spontaneous resolution during childhood or that the presence of an incarcerated inguinal hernia altered the natural history of cryptorchidism for the proband. Phenotypic heterogeneity, perhaps secondary to modifier loci, could account for the different clinical features. The mother of the propositus reported no fertility problems.

To the best of our knowledge, these are the first reported human mutations identified in INSL3/RLF that are associated with cryptorchidism. Finding mutations in 2 patients with cryptorchidism, 1 of whom had an intraabdominal testis, provides additional circumstantial evidence that INSL3/RLF is involved in testicular descent in humans. Both mutations were unique and were located in the connecting peptide region of the protein. Because both patients were heterozygous for INSL3/RLF mutations, haploinsufficiency is the likely mechanism. The frequency of INSL3/RLF mutations as a cause of cryptorchidism seems to be low because we only found 2 of 145 (1.4%) formerly cryptorchid subjects to have INSL3/RLF mutations. This low frequency of INSL3/RLF mutations may account for the negative findings reported in a cohort of 30 boys with cryptorchidism (36) and 31 men with cryptorchidism (37). Other candidate loci include the INSL3/RLF receptor, other downstream signaling intermediates, or HOXA10 (38, 39, 40, 41).


   Acknowledgments
 
We gratefully acknowledge the nursing assistance of Amy Gilliland and Janet Bell. We thank David Finegold, M.D.; Robert Ferrell, Ph.D.; Mark A. Sperling, M.D.; Ram Menon, M.D.; and John Kasik, M.D., for helpful discussions.


   Footnotes
 
1 Supported in part by grants from the NIH [R29-HD-34808 (to S.F.W.) and 5M-01-RR-00084 (to the General Clinical Research Center)], from the Genentech, Inc. Foundation (to S.F.W. and to P.A.L.), and from the American Heart Association (to S.F.W.). Back

Received May 15, 2000.

Revised July 5, 2000.

Accepted July 14, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. McElreavey K, Fellous M. 1999 Sex determination and the Y chromosome. Am J Med Genet (Semin Med Genet) 89:176–185.
  2. Berkovitz GS, Lapinski RH, Dolgin SE, Gazella JG, Bodiam CA, Holzman IR. 1993 Prevalence and natural history of cryptorchidism. Pediatrics. 92:44–49.[Abstract/Free Full Text]
  3. Giwercman A, Bruun E, Frimodtmoller C, Skakkebaek NE. 1989 Prevalence of carcinoma in situ and other histopathological abnormalities in testes of men with a history of cryptorchidism. J Urol. 142:998–1002.
  4. Husmann DA, Levy JB. 1995 Current concepts in the pathophysiology of testicular undescent. Urology. 46:267–276.[CrossRef][Medline]
  5. Hutson MH, Hasthorpe S, Heyns CF. 1997 Anatomical and functional aspects of testicular descent and cryptorchidism. Endocr Rev. 18:259–280.[Abstract/Free Full Text]
  6. McMahon DR, Kramer SA, Husmann DA. 1995 Antiandrogen induced cryptorchidism in the pig is associated with failed gubernacular regression and epididymal malformations. J Urol. 154:553–557.[CrossRef][Medline]
  7. Hutson JM, Donahoe PK. 1986 The hormonal control of testicular descent. Endocr Rev. 7:270–283.[Abstract/Free Full Text]
  8. Ahmed SF, Cheng A, Dovey L, et al. 2000 Phenotypic features, androgen receptor binding, and mutational analysis in 278 clinical cases reported as androgen insensitivity syndrome. J Clin Endocrinol Metab. 85:658–665.[Abstract/Free Full Text]
  9. Imbeaud S, Belvill C, Messika-Zeitoun L, et al. 1996 A 27 base-pair deletion of the anti-Müllerian type II receptor gene is the most common cause of the persistent Müllerian duct syndrome. Hum Mol Genet. 5:1269–1277.[Abstract/Free Full Text]
  10. Josso N, Picard JY, Imbeaud S, Carré-Eusèbe D. Zeller J, Adamsbaum C. 1993 The persistent Müllerian duct syndrome: a rare cause of cryptorchidism. Eur J Pediatr. 152(Suppl 2):S76–S78.
  11. Knebelmann B, Boussin L, Guerrier D, et al. 1991 Anti-Müllerian hormone Bruxelles: a nonsense mutation associated with the persistent Müllerian duct syndrome. Proc Natl Acad Sci USA. 88:3767–3771.[Abstract/Free Full Text]
  12. Behringer RR, Finegold MJ, Cate RL. 1994 Mullerian-inhibiting substance function during mammalian sexual development. Cell. 79:415–425.[CrossRef][Medline]
  13. Fentener-van Vlissingen JM, van Zoelen EJJ, Ursem PJF, Wensing CJG. 1988 In vitro model of the first phase of testicular descent: identification of a low molecular weight factor from fetal testis involved in proliferation of gubernaculum testis cells and distinct from specified polypeptide growth factors and fetal gonadal hormones. Endocrinology. 123:2868–2877.[Abstract/Free Full Text]
  14. Visser JH, Heyns CF. 1995 Proliferation of gubernaculum cells induced by a substance of low molecular mass obtained from fetal pig testes. J Urol. 153:516–520.[CrossRef][Medline]
  15. Adham IM, Emmen JMA, Engel W. 2000 The role of the testicular factor INSL3 in establishing the gonadal position. Mol Cell Endocrinol. 160:11–16.[CrossRef][Medline]
  16. Adham IM, Burkhardt E, Benahmed M, Engel W. 1993 Cloning of a cDNA for a novel insulin-like peptide of the testicular Leydig cells. J Biol Chem. 268:26668–26672.[Abstract/Free Full Text]
  17. Burkhardt E, Adham IM, Brosig B, Gastmann A, Mattei M-G, 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][Medline]
  18. Roche PJ, Butkus A, Wintour EM, Tregar G. 1996 Structure and expression of Leydig insulin-like peptide mRNA in the sheep. Mol Cell Endocrinol. 121:171–177.[CrossRef][Medline]
  19. Zarreh-Hoshyari-Khah MR, Einspanier A. Ivell R. 1999 Differential splicing and expression of the relaxin-like factor gene in reproductive tissues of the marmoset monkey (Callithrix jacchus). Biol Reprod. 60:445–454.[Abstract/Free Full Text]
  20. Zimmerman S, Schöttler P, Engel W, Adham IM. 1997 Mouse Leydig-like (LEY I-L) gene: structure and expression during testis and ovary development. Mol Reprod Dev. 47:30–38.[CrossRef][Medline]
  21. Zimmerman S, Steding G, Emmen JM. 1999 Targeted disruption of the Insl3 gene causes bilateral cryptorchidism. Mol Endocrinol. 13:681–691.[Abstract/Free Full Text]
  22. Nef S, Parada L. 1999 Cryptorchidism in mice mutant for Insl3. Nat Genet. 22:295–299.[CrossRef][Medline]
  23. Nayak S, Lee PA, Witchel SF. 1998 Variants of the type II 3ß-hydroxysteroid dehydrogenase gene in children with premature pubic hair and hyperandrogenic adolescents. Mol Genet Metab. 64:184–192.[CrossRef][Medline]
  24. Czeizel A, Erodi E, Toth J. 1981 Genetics of undescended testis. J Urol. 126:528–529.[Medline]
  25. Hutcheson JC, Snyder3rd HM , Zuniga ZV, et al. 2000 Ectopic and undescended testes: 2 variants of a single congenital anomaly? J Urol. 163:961–963.[CrossRef][Medline]
  26. Herzog B, Steigert M, Hadziselimovic F. 1992 Is a testis located at the superficial inguinal pouch (Denis Browne pouch) comparable to a true cryptorchid testis? J Urol. 148:622–623.[Medline]
  27. Von der Maase H, Rorth M, Walbom-Jorgensen S, et al. 1986 Carcinoma in situ of the undescended testis in patients with testicular germ cell cancer: study of 27 cases in 500 patients. BMJ. 293:1398–1401.
  28. Balvers M, Speiss AN, Domagalski R, et al. 1998 Relaxin-like factor expression as a marker of differentiation in the mouse testis and ovary. Endocrinology. 139:2960–2970.[Abstract/Free Full Text]
  29. Klonisch T, Ivell R, Balvers M, et al. 1999 Expression of relaxin-like factor is down-regulated in human testicular Leydig cell neoplasia. Mol Hum Reprod. 5:104–108.[Abstract/Free Full Text]
  30. Bamberger AM, Ivell R, Balvers M, et al. 1999 Relaxin-like factor (RLF): a new specific marker for Leydig cells in the ovary. Int J Gynecol Pathol. 18:163–168.[Medline]
  31. Zimmermann S, Schwärzler A, Buth S, Engel W, Adham IM. 1998 Transcription of the Leydig insulin-like gene is mediated by steroidogenic factor-1. Mol Endocrinol. 12:706–713.[Abstract/Free Full Text]
  32. Luo X, Ikeda Y, Parker KL. 1994 A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell. 77:481–490.[CrossRef][Medline]
  33. Ingraham HA, Lala DS, Ikeda Y, et al. 1994 The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev. 8:2302–2312.[Abstract/Free Full Text]
  34. Büllesbach EE, Schwabe C. 1999 Specific, high affinity relaxin-like receptors. J Biol Chem. 274:22354–22358.[Abstract/Free Full Text]
  35. Tashima LS, Hieber AD, Greenwood FC, Bryant-Greenwood GD. 1995 The human Leydig insulin-like (hLEY I-L) gene is expressed in the corpus luteum and trophoblast. J Clin Endocrinol Metab. 80:707–710.[Abstract]
  36. Koskimies P, Virtanen H, Lindström M, et al. 2000 A common polymorphism in the human relaxin-like factor (RLF) gene: no relationship with cryptorchidism. Pediatr Res. 47:538–541.[Medline]
  37. Krausz C, Quitana-Murci L, Fellous M, Siffroi JP, McElreavey K. 2000 Absence of mutations involving the INSL3 gene in human idiopathic cryptorchidism. Mol Hum Reprod. 6:298–302.[Abstract/Free Full Text]
  38. Palejwala S, Stein D, Wojtczuk A, Weiss G, Goldsmith LT. 1998 Demonstration of a relaxin receptor and relaxin-stimulated tyrosine phosphorylation in human lower uterine segment fibroblasts. Endocrinology. 139:1208–1212.[Abstract/Free Full Text]
  39. Rijli FM, Matyas R, Pellegrini M, et al. 1995 Cryptorchidism and homeotic transformations of spinal nerves and vertebrae in Hoxa-10 mutant mice. Proc Natl Acad Sci USA. 92:8185–8189.[Abstract/Free Full Text]
  40. Satokata I, Benson G, Maas R. 1995 Sexually dimorphic sterility phenotypes in Hoxa10-deficient mice. Nature. 374:460–463.[CrossRef][Medline]
  41. Kolon TF, Wiener JS, Lewitton M, Roth DR, Gonzales Jr ET, Lamb DJ. 1999 Analysis of homeobox gene HOXA10 mutations in cryptorchidism. J Urol. 161:275–280.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
Y. Lue, C. Wang, Y. Cui, X. Wang, J. Sha, Z. Zhou, J. Xu, C. Wang, A. P. Sinha Hikim, and R. S. Swerdloff
Levonorgestrel Enhances Spermatogenesis Suppression by Testosterone with Greater Alteration in Testicular Gene Expression in Men
Biol Reprod, March 1, 2009; 80(3): 484 - 492.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
C. Foresta, D. Zuccarello, A. Garolla, and A. Ferlin
Role of Hormones, Genes, and Environment in Human Cryptorchidism
Endocr. Rev., August 1, 2008; 29(5): 560 - 580.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
H.E. Virtanen and J. Toppari
Epidemiology and pathogenesis of cryptorchidism
Hum. Reprod. Update, January 1, 2008; 14(1): 49 - 58.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
S M L C Mendis-Handagama, H B S Ariyaratne, L Mrkonjich, and R Ivell
Expression of insulin-like peptide 3 in the postnatal rat Leydig cell lineage: timing and effects of triiodothyronine-treatment
Reproduction, February 1, 2007; 133(2): 479 - 485.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
A. Ferlin, N.V. Bogatcheva, L. Gianesello, A. Pepe, C. Vinanzi, A.I. Agoulnik, and C. Foresta
Insulin-like factor 3 gene mutations in testicular dysgenesis syndrome: clinical and functional characterization
Mol. Hum. Reprod., June 1, 2006; 12(6): 401 - 406.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
J. D. Veldhuis, J. N. Roemmich, E. J. Richmond, and C. Y. Bowers
Somatotropic and Gonadotropic Axes Linkages in Infancy, Childhood, and the Puberty-Adult Transition
Endocr. Rev., April 1, 2006; 27(2): 101 - 140.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
R. Yoshida, M. Fukami, I. Sasagawa, T. Hasegawa, N. Kamatani, and T. Ogata
Association of Cryptorchidism with a Specific Haplotype of the Estrogen Receptor {alpha} Gene: Implication for the Susceptibility to Estrogenic Environmental Endocrine Disruptors
J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4716 - 4721.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
K. Bay, S. Hartung, R. Ivell, M. Schumacher, D. Jurgensen, N. Jorgensen, M. Holm, N. E. Skakkebaek, and A.-M. Andersson
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., June 1, 2005; 90(6): 3410 - 3418.
[Abstract] [Full Text] [PDF]

Home page
 Eur J EndocrinolHome page
A. Ferlin, A. Garolla, A. Bettella, L. Bartoloni, C. Vinanzi, A. Roverato, and C. Foresta
Androgen receptor gene CAG and GGC repeat lengths in cryptorchidism
Eur. J. Endocrinol., March 1, 2005; 152(3): 419 - 425.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
C. Foresta, A. Bettella, C. Vinanzi, P. Dabrilli, M. C. Meriggiola, A. Garolla, and A. Ferlin
A Novel Circulating Hormone of Testis Origin in Humans
J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 5952 - 5958.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
G. Vinci, M.-N. Anjot, C. Trivin, H. Lottmann, R. Brauner, and K. McElreavey
An Analysis of the Genetic Factors Involved in Testicular Descent in a Cohort of 14 Male Patients with Anorchia
J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6282 - 6285.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
J. Gianotten, M.P. Lombardi, A.H. Zwinderman, R. J. Lilford, and F. van der Veen
Idiopathic impaired spermatogenesis: genetic epidemiology is unlikely to provide a short-cut to better understanding
Hum. Reprod. Update, November 1, 2004; 10(6): 533 - 539.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
N. V. Bogatcheva, A. Truong, S. Feng, W. Engel, I. M. Adham, and A. I. Agoulnik
GREAT/LGR8 Is the Only Receptor for Insulin-Like 3 Peptide
Mol. Endocrinol., December 1, 2003; 17(12): 2639 - 2646.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
A. Truong, N. V. Bogatcheva, C. Schelling, G. Dolf, and A. I. Agoulnik
Isolation and Expression Analysis of the Canine Insulin-Like Factor 3 Gene
Biol Reprod, November 1, 2003; 69(5): 1658 - 1664.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
A. Ferlin, M. Simonato, L. Bartoloni, G. Rizzo, A. Bettella, T. Dottorini, B. Dallapiccola, and C. Foresta
The INSL3-LGR8/GREAT Ligand-Receptor Pair in Human Cryptorchidism
J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4273 - 4279.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
T. Klonisch, K. Steger, A. Kehlen, W. R. Allen, C. Froehlich, J. Kauffold, M. Bergmann, and S. Hombach-Klonisch
INSL3 Ligand-Receptor System in the Equine Testis
Biol Reprod, June 1, 2003; 68(6): 1975 - 1981.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
Y. Kubota, C. Temelcos, R.A.D. Bathgate, K.J. Smith, D. Scott, C. Zhao, and J.M. Hutson
The role of insulin 3, testosterone, Mullerian inhibiting substance and relaxin in rat gubernacular growth
Mol. Hum. Reprod., October 1, 2002; 8(10): 900 - 905.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
R. Ivell and R. A.D. Bathgate
Reproductive Biology of the Relaxin-Like Factor (RLF/INSL3)
Biol Reprod, September 1, 2002; 67(3): 699 - 705.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Kumagai, S. Y. Hsu, H. Matsumi, J.-S. Roh, P. Fu, J. D. Wade, R. A. D. Bathgate, and A. J. W. Hsueh
INSL3/Leydig Insulin-like Peptide Activates the LGR8 Receptor Important in Testis Descent
J. Biol. Chem., August 23, 2002; 277(35): 31283 - 31286.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
J. C. Achermann, G. Ozisik, J. J. Meeks, and J. L. Jameson
Genetic Causes of Human Reproductive Disease
J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2447 - 2454.
[Full Text] [PDF]

Home page
 EndocrinologyHome page
I. A. Hughes
Minireview: Sex Differentiation
Endocrinology, August 1, 2001; 142(8): 3281 - 3287.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tomboc, M.
Right arrow Articles by Witchel, S. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tomboc, M.
Right arrow Articles by Witchel, S. F.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*OMIM
*Protein*UniGene
*Compound via MeSH
*Substance via MeSH
Medline Plus Health Information
*Testicular Disorders

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals