The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 8 4146-4148
Copyright © 2004 by The Endocrine Society
X-Linked Sex-Determining Region Y Box 3 (SOX3) Gene Mutations Are Uncommon in Men with Idiopathic Oligoazoospermic Infertility
Gerald Raverot,
Herve Lejeune,
Tom Kotlar,
Michel Pugeat and
J. Larry Jameson
Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University, Feinberg School of Medicine (G.R., T.K., J.L.J.), Chicago, Illinois 60611; Departement de Medecine de la Reproduction, Hospices Civils de Lyon, Hopital E. Herriot and Institut National de la Santé et de la Recherche Médicale-Institut National de la Recherche Agronomique, Unité 418, Hopital Debrousse (H.L.), 69007 Lyon, France; and Fédération d’Endocrinologie du Pôle Est, Hospices Civils de Lyon, Hôpital Neurologique Pierre Wertheimer, and Institut National de la Santé et de la Recherche Médicale, Hopital Debrousse (M.P.), 69394 Lyon, France
Address all correspondence and requests for reprints to: Dr. J. Larry Jameson, Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Feinberg School of Medicine, Galter Pavilion, Suite 3-150, 251 East Huron Street, Chicago, Illinois 60611-2908. E-mail: ljameson{at}northwestern.edu.
 |
Abstract
|
|---|
The X-linked sex-determining region Y box 3 (SOX3) gene is expressed in the developing gonads and brain. Sox3-null mice developed according to genetic sex, but the hemizygous null males were hypogonadal, with extensive Sertoli cell vacuo-lization, loss of germ cells, and reduced sperm count. We hypothesized that SOX3 mutations might occur in a subset of infertility patients. Genomic DNA samples from 56 infertile men with idiopathic oligo-azoospermia were screened for SOX3 mutations. Three nucleotide substitutions (609 T
C, 732 AC, and 978 GA) were identified, none of which altered the amino acid sequence, suggesting that they are polymorphic variants. The 609 TC substitution was in the HMG box, and the two other substitutions were identified within the polyalanine repeat regions. Three patients had 609 TC, 2 patients had 609 TC and 732 AC, and one had 978 GA. These data indicate that mutations in the SOX3 gene are not a common cause of male infertility.
|
Introduction
|
|---|
MALE AND FEMALE factors contribute about equally to infertility, which affects about 10–15% of couples (1). Although many causes (varicocele, cryptorchidism, genital infection, and antisperm antibodies) of male infertility have been identified, about 50% of patients are still assigned a diagnosis of idiopathic infertility. Microdeletions of regions of the Y chromosome required for effective spermatogenesis have been found in approximately 15% of these cases (2). Although mutations in a considerable number of genes can cause pathology in the male reproductive system (3), few have been identified as common causes of male infertility.
The sex-determining region Y box 3 (SOX3) has been hypothesized to play a role in the sex-determining process (4, 5). It is a member of the high mobility group (HMG) family of transcription factors and is located on the X chromosome (6). SOX3 was cloned based on its HMG box homology to sex-determining region Y (Sry) (7), the male-determining gene. In a recent study, SOX3-null mice developed according to genetic sex, but the hemizygous males had small testes and a reduced number of sperm (8). Testicular histology revealed extensive Sertoli cell vacuolization, loss of germ cells, and disruption of the seminiferous tubules. Small testes were also observed in a human 46,XY male with complete loss of SOX3 (6, 9). These findings suggest that SOX3 plays an important role in testis development and possibly sperm maturation. We hypothesized that SOX3 mutations might cause oligoazoospermic male infertility and analyzed the SOX3 gene in 56 male patients with idiopathic oligoazoospermic infertility.
|
Subjects and Methods
|
|---|
Subjects and DNA isolation
Fifty-six unrelated infertile French men with oligo- or azoospermia were included in this study. Oligoazoospermia (sperm count of <20 x 106 sperm/ml or undetectable) was confirmed by at least two independent semen analyses performed according to the World Health Organization 1999 guidelines (10). Subjects were subjected to detailed clinical investigations, including physical examination, cytogenetic analysis, and endocrinological studies. Subjects were not included if they had a plausible cause of infertility (greater than grade 2 varicocele, cryptorchidism, karyotypic abnormality, Y microdeletion, or obstructive infertility). The majority of patients investigated (n = 45) had primary infertility (without any previous pregnancy); 11 patients had secondary infertility (defined by having at least one child, but now unable to conceive after trying for 1 yr or more).
Genomic DNA was extracted from patients’ blood leukocytes for DNA sequence analysis. The study design and procedures were approved by the institutional review board of Northwestern University, and informed consent was obtained from each subject.
Sequence analysis
A total of 1575 bp of the single exon SOX3 gene, comprising 113 bp of the 5'-flanking sequence, 121 bp of the 3'-untranslated sequence, and the entire coding sequence, was determined by direct sequencing of five partially overlapping amplicons obtained by PCR with genomic DNA. The primer sequences and lengths of amplified fragments are listed in Table 1
.
Thermal cycling was performed with an initial predenaturation at 94 C for 4 min, followed by 32 cycles of denaturation at 94 C for 1 min, primer annealing at 56 C for 1 min, and primer extension at 72 C for 1 min. The final extension was carried out at 72 C for 15 min. Buffer conditions have been described previously (11). Amplicons were purified on a 1.5% agarose gel using a QIAquick gel extraction kit (Qiagen, Valencia, CA), then subsequently sequenced in forward and reverse directions using dRhodamine Terminator Cycle Ready Reaction kits (Applied Biosystems, Foster City, CA) and automated sequencers ABI PRISM 3100 genetic analyzer (Applied Biosystems). The sequences obtained were compared with the reference sequence (GenBank accession no. NM_005634). The A of the ATG translation initiation codon was designated +1 (12).
|
Results
|
|---|
Sequencing of the single exon, 113 bp of the 5'-flanking sequence and 121 bp of the 3'-untranslated sequence of SOX3 did not reveal any mutations in the 56 patients studied. A nucleotide substitution was detected at three sites in six patients, none of which altered the predicted amino acid sequence: 609 TC (five patients, 8.9%), 732 AC (two patients, 3.6%), and 978 GA (one patient, 1.8%). The first variant (609 TC), located in the HMG box, did not change the tyrosine residue. The two other variants (732 AC, 978 GA), located in two different polyalanine tracts, did not change the alanine residue (Fig. 1). The first two variants have been described previously (13). The third variant, 978 GA, has not been previously described and was found in only one patient. We cannot exclude the possibility that these silent mutations might alter DNA methylation, RNA stability, or other features that regulate SOX3 expression. However, there was no correlation between these nucleotide variants and the clinical manifestations (Table 2).
|
Discussion
|
|---|
Despite advances in assisted reproductive technologies, infertility is a major health problem. Approximately 15% of couples are unable to conceive within 1 yr of unprotected intercourse. A plausible etiology is found in about 50% of cases. Few genetic causes of infertility have been identified in humans (14); nevertheless, genetic etiologies are thought to underlie many cases of idiopathic infertility. As described by Wang and co-workers (15) in a systematic search for genes expressed in mouse spermatogonia, but not in somatic tissue, the X chromosome has a high concentration of spermatogenesis genes.
SOX3, located on the X chromosome, is expressed in the urogenital ridge (7), from which the gonads develop, and it continues to be expressed in the adult gonads of both sexes (8, 16). SOX3 is present in gonads lacking germ cells (7), consistent with expression in somatic cells. However, in chickens, SOX3 is detected in the primordial germs cells of both sexes (17), and expression in germ cells cannot be excluded. In the adult, SOX3 is still expressed in the testis.
SOX3-null mice developed according to genetic sex. However, female SOX3-null mice displayed high rates of follicular atresia and severely reduced fertility. Male SOX3-null mice have Sertoli cell dysfunction with extensive vacuolization, loss of germ cells, and reduced sperm count.
The pattern of SOX3 expression and its location on the X chromosome make it a plausible gene candidate for various pathologies, such as sex reversal (13), primary ovarian failure (18), or Rett syndrome (19). However, no SOX3 mutations were found in these disorders.
In humans, a large deletion (including SOX3) of the X chromosome has been described in men with varying degrees of mental retardation (9, 20, 21). In at least two cases, small testes were associated with these deletions (9, 20). In a different model of gonadal dysgenesis, deletion of Ahch (Dax1) caused a severe testicular phenotype in mice (22) that was only subsequently confirmed in humans (23).
The current study indicates that SOX3 mutations are not a common cause (<2%) of idiopathic male infertility. It is possible, however, that SOX3 mutations might be associated with specific testis pathologies or with mental retardation. Thus, SOX3 should still be considered in future studies of male infertility until other more commonly affected genes are identified.
|
Acknowledgments
|
|---|
We thank Wen Xia Gu and Gokhan Ozisik for valuable assistance, and Eun-Jig Lee for critical reading and helpful discussion.
|
Footnotes
|
|---|
This work was supported in part by NIH Grant HD-044801 and by a grant from the French Society of Endocrinology and Novo-Nordisk (to G.R.).
Abbreviations: HMG, High mobility group; SOX3, X-linked sex-determining region Y box 3; Sry, sex-determining region Y.
Received February 9, 2004.
Accepted April 16, 2004.
|
References
|
|---|
- Hull MG, Glazener CM, Kelly NJ, Conway DI, Foster PA, Hinton RA, Coulson C, Lambert PA, Watt EM, Desai KM 1985 Population study of causes, treatment, and outcome of infertility. Br Med J 291:1693–1697
- Krausz C, Quintana-Murci L, McElreavey K 2000 Prognostic value of Y deletion analysis: what is the clinical prognostic value of Y chromosome microdeletion analysis? Hum Reprod 15:1431–1434[Abstract/Free Full Text]
- Achermann JC, Ozisik G, Meeks JJ, Jameson JL 2002 Genetic causes of human reproductive disease. J Clin Endocrinol Metab 87:2447–2454[Free Full Text]
- Graves JA 1998 Interactions between SRY and SOX genes in mammalian sex determination. Bioessays 20:264–269[CrossRef][Medline]
- Graves JA 1998 Evolution of the mammalian Y chromosome and sex-determining genes. J Exp Zool 281:472–481[CrossRef][Medline]
- Stevanovic M, Lovell-Badge R, Collignon J, Goodfellow PN 1993 SOX3 is an X-linked gene related to SRY. Hum Mol Genet 2:2013–2018[Abstract/Free Full Text]
- Collignon J, Sockanathan S, Hacker A, Cohen-Tannoudji M, Norris D, Rastan S, Stevanovic M, Goodfellow PN, Lovell-Badge R 1996 A comparison of the properties of Sox-3 with Sry and two related genes, Sox-1 and Sox-2. Development 122:509–520[Abstract]
- Weiss J, Meeks JJ, Hurley L, Raverot G, Frassetto A, Jameson JL 2003 Sox3 is required for gonadal function, but not sex determination, in males and females. Mol Cell Biol 23:8084–8091[Abstract/Free Full Text]
- Wolff DJ, Gustashaw KM, Zurcher V, Ko L, White W, Weiss L, Van Dyke DL, Schwartz S, Willard HF 1997 Deletions in Xq26.3-q27.3 including FMR1 result in a severe phenotype in a male and variable phenotypes in females depending upon the X inactivation pattern. Hum Genet 100:256–261[CrossRef][Medline]
- World Health Organization 1999 WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction, 4th Ed. Cambridge: Cambridge University Press
- Ito M, Mori Y, Oiso Y, Saito H 1991 A single base substitution in the coding region for neurophysin II associated with familial central diabetes insipidus. J Clin Invest 87:725–728
- den Dunnen JT, Antonarakis SE 2000 Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat 15:7–12[CrossRef][Medline]
- Lim HN, Berkovitz GD, Hughes IA, Hawkins JR 2000 Mutation analysis of subjects with 46, XX sex reversal and 46, XY gonadal dysgenesis does not support the involvement of SOX3 in testis determination. Hum Genet 107:650–652[CrossRef][Medline]
- Matzuk MM, Lamb DJ 2002 Genetic dissection of mammalian fertility pathways. Nat Cell Biol 4(Suppl):s41–s49
- Wang PJ, McCarrey JR, Yang F, Page DC 2001 An abundance of X-linked genes expressed in spermatogonia. Nat Genet 27:422–426[CrossRef][Medline]
- Shen JH, Ingraham HA 2002 Regulation of the orphan nuclear receptor steroidogenic factor 1 by Sox proteins. Mol Endocrinol 16:529–540[Abstract/Free Full Text]
- Uchikawa M, Kamachi Y, Kondoh H 1999 Two distinct subgroups of Group B Sox genes for transcriptional activators and repressors: their expression during embryonic organogenesis of the chicken. Mech Dev 84:103–120[CrossRef][Medline]
- Davison RM, Davis CJ, Conway GS 1999 The X chromosome and ovarian failure. Clin Endocrinol (Oxf) 51:673–679[CrossRef][Medline]
- Xiang F, Buervenich S, Nicolao P, Bailey ME, Zhang Z, Anvret M 2000 Mutation screening in Rett syndrome patients. J Med Genet 37:250–255[Abstract/Free Full Text]
- Raynaud M, Ronce N, Ayrault AD, Francannet C, Malpuech G, Moraine C 1998 X-linked mental retardation with isolated growth hormone deficiency is mapped to Xq22-Xq27.2 in one family. Am J Med Genet 76:255–261[CrossRef][Medline]
- Hamel BC, Smits AP, Otten BJ, van den Helm B, Ropers HH, Mariman EC 1996 Familial X-linked mental retardation and isolated growth hormone deficiency: clinical and molecular findings. Am J Med Genet 64:35–41[CrossRef][Medline]
- Yu RN, Ito M, Saunders TL, Camper SA, Jameson JL 1998 Role of Ahch in gonadal development and gametogenesis. Nat Genet 20:353–357[CrossRef][Medline]
- Seminara SB, Achermann JC, Genel M, Jameson JL, Crowley Jr WF 1999 X-Linked adrenal hypoplasia congenita: a mutation in DAX1 expands the phenotypic spectrum in males and females. J Clin Endocrinol Metab 84:4501–4509[Abstract/Free Full Text]
Top
Abstract
Introduction
