Mol. Hum. Reprod. Advance Access originally published online on April 5, 2006
Molecular Human Reproduction 2006 12(4):263-267; doi:10.1093/molehr/gal020
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The role of the testis-specific gene hTAF7L in the aetiology of male infertility
Research Centre for Reproduction and Genetics, Vrije Universiteit Brussel, Brussels, Belgium
1 To whom correspondence should be addressed at: Research Centre for Reproduction and Genetics, Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium. E-mail: katrien.stouffs{at}az.vub.ac.be
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Abstract |
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The X-linked TAF7L gene is homologous to the autosomal transcription factor TAF7. Together with its testis-specific expression pattern, this might point to an important function in spermatogenesis. In order to analyse the involvement of the hTAF7L gene in the aetiology of male infertility, a total of 25 patients with maturation arrest of spermatogenesis have been analysed for the presence of mutations in this gene. Four alterations of the nucleotide sequence, with concomitant changes in the amino acid sequence, have been observed in 12 patients. All sequence alterations were also found either in a control group consisting of men with proven fertility or in a control group with men with normal spermatogenesis. Therefore, these alterations are probably polymorphisms.
Key words: male infertility/mutations/TAF7L/X chromosome
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Introduction |
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Male infertility is usually diagnosed on the basis of abnormal sperm values observed in conventional semen analysis. Unfortunately, the underlying basis of these abnormalities still remains largely unknown. Two categories of causes may be distinguished: acquired and congenital causes. Special interest into congenital causes of male infertility has particularly arisen since the development of assisted reproductive technology. Patients with very low sperm concentrations are now also able to reproduce by ICSI. Consequently, genetic causes of male infertility might be transmitted to the offspring. Therefore, for adequate counselling of the couple, it is important to gain more insight into the genetic origin of male infertility.
We are especially interested in the sex chromosomes because of their hemizygote exposure in men: mutations in genes located on the X or Y chromosome cannot be compensated for by a second normal gene as is the case for autosomal genes. For proteins with a crucial function during spermatogenesis, such mutations may cause serious fertility problems.
We became attracted by the TATA box Binding Protein (TBP)-Associated Factor 7 Like or TAF7L gene. This gene was first isolated from mouse spermatogonia. In mice and humans, TAF7L is located on the X chromosome and is homologous to the autosomal TAF7 gene (Wang et al., 2001
). Mouse studies have shown expression of the mTAF7L protein in the cytoplasm of spermatogonia and early spermatocytes and in the nucleus of late pachytene spermatocytes and round spermatids. The shift of mTAF7L proteins from the cytoplasm to the nucleus coincides with a decrease of mTAF7 in both the cytoplasm and nucleus. At the same time, an increase in mTBP was observed. Together with TBP, TAF proteins are part of the transcription factor TFIID in mice and humans. In mice, a fraction of nuclear mTAF7L was also shown to interact with mTBP.
Inactivation of yeast and mammalian TAF genes has already been shown to lead to cell cycle arrest. Falender et al. (2005) have shown that the autosomal mTAF4b gene is required in mouse spermatogenesis. In Drosophila melanogaster, mutations in the dTAF5L gene (also called Cannonball) cause male sterility. This autosomal dTAF5L gene is a testis-specific homologue of dTAF5 (Hiller et al., 2004). Likewise, hTAF7L and mTAF7L are testis-specific homologues of the ubiquitously expressed hTAF7 and mTAF7, respectively.
In this study, we set out to investigate the role of the hTAF7L gene in human spermatogenesis. On the basis of the observed expression pattern in mice, we decided to screen patients with a maturation arrest of spermatogenesis for the presence of mutations in the hTAF7L gene. If this gene is important for spermatogenesis, we may expect spermatogenesis to be disturbed, and it is likely that a maturation arrest will be observed.
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Materials and methods |
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Patient selection
DNA of a total of 25 patients with maturation arrest of spermatogenesis was analysed for the presence of mutations in the hTAF7L gene. For all patients, testicular sperm extraction (TESE) was performed, during which a testicular biopsy was also taken for histological examination within the frame of their fertility treatment (Tournaye et al., 1997
). Histological examination of testicular tissues was performed in the pathology department of our university hospital. The final description of the testicular phenotype reported in this study is based on the most advanced stage of spermatogenesis observed in the histological sample, combined with the absence or presence of spermatozoa in testicular tissue or in the ejaculate. When a heterogeneous testicular phenotype was observed, patients were categorized according to the most advanced stage of spermatogenesis found in testicular tissues. For example, when the histology showed tubuli with Sertoli cell-only syndrome as well as with maturation arrest, the histology of this sample was designated as maturation arrest. Moreover, if sperm was found at TESE and the histology was Sertoli cell-only syndrome, these samples were referred to as having incomplete Sertoli cell-only syndrome. Taking these data into consideration, we classified eight patients having complete maturation arrest (cMA) as (cMA1–8) and 17 patients having an incomplete maturation arrest (iMA) as (iMA1–17). When spermatozoa were found in the ejaculate, the concentration was <0.3 x 106 spermatozoa/ml. DNA samples from two control groups were used: the first group consisted of men with proven fertility, whereas in the second group, men with normal spermatogenesis, as determined by a testicular biopsy, were included.
Polymerase chain reaction
DNA was isolated from peripheral blood using Qiagen’s ‘QIamp Blood Maxi Kit Protocol’ (Qiagen, Leusden, The Netherlands). Primers were designed to be able to amplify and sequence the entire coding region and parts of the flanking introns of the hTAF7L gene (Table I) and were synthesized by Eurogentec (Seraing, Belgium).
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A single PCR was set up for exons 5 and 6 and exons 10 and 11. PCR reactions were performed in a 100 µl mix containing 500 ng of DNA, 1 x PCR Buffer II (Applied Biosystems, Lennik, Belgium), 2 mmol/l of MgCl2 (Applied Biosystems), 0.2 mmol/l of each dNTP (GE Healthcare, Diegem, Belgium), 1 µmol/l of each primer and 1.25 units of Taq polymerase (Applied Biosystems). Thermocycling conditions consisted of an initial denaturation of 5 min at 94°C, 30 or 35 cycles of 1 min at 94°C, 1 min at a variable annealing temperature (see Table I), 2 min at 72°C and a final extension of 7 min at 72°C. PCR products were analysed on a 2% agarose gel.
After purification, all samples were sequenced with primers used for amplification. For exon 5 and 6, an additional internal primer (5'-CACAAGGAATTAGGATTCAGC-3') was used. All samples were run on the ABI3130 or ABI3130xl Genetic Analyser (Applied Biosystems).
Restriction analysis
In order to be able to rapidly analyse control samples for the modifications observed in exons 1 and 9, we set up a restriction reaction. A fragment for exon 1 was amplified with the following primers: 5'-GGACAGCTCCCCATTTCTTC-3' (forward) and 5'-GTTTCTCCGAAAGAAGGTCCTC-3' (reverse). Thermocycling conditions consisted of an initial denaturation of 5 min at 94°C, 30 cycles of 1 min at 94°C, 1 min at 60°C and 2 min at 72°C and a final extension of 7 min at 72°C. The restriction reaction was performed with MnlI. For the PCR of exon 9, primers described in Table I were used, after which the digestion was performed with AciI.
Fragment analysis
Primers for fragment analysis of exon 10 are located around a repeat structure present in exon 10. Primers were 5'-CGGGAGATGTTCAGTGATTC-3' (forward) and 5'-AGCTGCCTTTCCAGATACTC-3' (reverse). The reverse primer was labelled with Fam. The thermocycling conditions included 20 cycles of 1 min at 94°C, 1 min at 62°C and 2 min at 72°C. The difference in length of the fragments was analysed on the ABI3130.
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Results |
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A total of 25 patients showing maturation arrest of spermatogenesis were included in this study. For most patients, spermatogenesis was still going on in a few tubuli. Only for eight of them were no spermatozoa found either in a biopsy sent for histological examination or in multiple testicular samples analysed during TESE by wet preparation.
Sequencing of the complete open reading frame of the hTAF7L gene was performed for all 25 patients. The sequence from Genbank (BC043391 [GenBank] ) was used as the reference sequence. The ‘A’ of the ATG translation initiation signal, at position 13 in BC043391 [GenBank] , was numbered +1 (Figure 1).
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Two patients were found with the same alteration of the sequence in exon 1 (c.181G > A), which causes the change of a glutamic acid into a lysine (E61K) at the amino acid level. These two patients had an iMA (Tables II and III). In exon 9, the change c.922A > G was found in a total of eight patients. At the amino acid level, this substitution predicts the change of a serine into a glycine (S308G). These eight patients included three men with a cMA and five men with an iMA (Tables II and III). A deletion of six base pairs (GGATGA), located in exon 10, at cDNA positions 1047–1052, was found in the DNA of two patients; one featured cMA and the other an iMA (Tables II and III). This deletion was present in a repeat structure normally consisting of (GAN)28 with N = A, C, G or T and in patients thus being (GAN)26. The deletion of six base pairs causes the absence of a glutamic acid and an aspartic acid at positions 350 and 351 (p.D350_E351del). Instead of 14 glutamic acid and 14 aspartic acid residues, for the patients with the six base pair deletion, now only 13 are present [D3(ED)2D(ED)6/7 KE5D]. Furthermore, we observed two patients (iMA1 and iMA5), with alterations of the DNA sequence in exon 13 (c.1373G > A) of the hTAF7L gene. At the amino acid level, an arginine is replaced by a histidine (R458H). These two patients were the same as those with the changes detected in exon 1.
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A total of 12 patients were thus detected as having a hTAF7L gene with differences in their sequence compared with the published hTAF7L sequence. Four of these patients had cMA, whereas for eight of them some spermatozoa were found (Tables II and III).
In order to assess whether these alterations of the hTAF7L gene are mutations, we analysed DNA samples from normal fertile controls (Table III). Furthermore, the amino acid sequences of mouse and human TAF7L were compared as well as the cDNA sequences from mice, chimpanzees and humans (Figures 1 and 2). For exons 1 and 9, a restriction reaction was set up in order to investigate control samples for the presence or absence of the alterations observed in these exons. For exon 1, a total of 100 men with proven fertility were analysed. For a single patient, the alteration c.181G > A was observed, indicating that this change probably represents an uncommon polymorphism. When comparing the TAF7L protein from mouse and human, this alteration seems to be located in a fragment that is absent in the mouse protein (Figure 2). Also in humans, it remains to be elucidated where the start codon is positioned. Indeed, recent protein sequences published at the NCBI website placed the start codon at position 258 (Figure 1). Therefore, the observed change in exon 1 probably represents a polymorphism. Furthermore, the start codon in the hTAF7 gene also appears to be located after the alteration observed in exon 1.
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The alteration in exon 9 is located outside the conserved TAFII55 domain but seems to be conserved in mouse and human TAF7L and human TAF7 (Figure 2). For this substitution, i.e. the change of an A to a G, a total of 60 men from the control group were screened. Seven of these men featured this alteration.
To detect the six base pair deletion in exon 10, primers were developed to be able to observe the difference in length between the normal and the shorter copy by fragment analysis. A shorter version of exon 10 was detected in 10 out of 40 controls. In mice, only 11 glutamic acid and 11 aspartic acid residues are present, indicating that the deletion of a glutamic acid and an aspartic acid residue probably will not have functional consequences (Figure 2). In addition, the hTAF7 gene contains only two aspartic acid residues and three glutamic acid residues (Figure 2).
Exon 13 was sequenced for 100 patients with proven fertility. In this group of men, no differences with the reference sequence were observed. Unlike the two infertile men with the substitution in exon 1 as well as in exon 13, the single fertile man who had the substitution c.181G > A in exon 1 did not have the substitution in exon 13. As the two patients with this alteration were of Arabic origin, a second control group consisting of 23 Arabic men with normal spermatogenesis was screened. One of these Arabic men had the alteration c.1373G > A, indicating that this alteration does not disturb spermatogenesis. We also tested this man for the presence of the alteration in exon 1 and found that the change c.181G > A was not present. Furthermore, the alteration R458H, corresponding to the alteration in exon 13, is located in the C-terminal region of the TAF7L protein. This region does not contain a conserved functional domain (Figure 2).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Infertility affects about 15% of couples seeking to have children. Many causes of male infertility are known, including endocrine disorders or infections. In the last two decades, it has become obvious that genetic factors also play a pivotal role in the aetiology of male infertility. However, the group of men for whom the cause of their fertility problems cannot be explained remains large.
Mouse studies have shown that >2000 genes are involved in spermatogenesis. Also in humans, the origin of fertility problems in a large number of patients is likely to be caused by defects in genes involved in spermatogenesis. Because the number of genes involved in spermatogenesis is huge, we decided to focus on genes located on the sex chromosomes. As men only have a single X and Y chromosome, defects in either of these genes might have an immediate impact on spermatogenesis. In a previous study, the USP26 gene was screened for the presence of mutations in patients with a Sertoli cell-only syndrome (Stouffs et al., 2005). Eight patients were found with three sequence alterations of the hUSP26 gene. The presence of these alterations in infertile men has also been described in a recent American study (Paduch et al., 2005).
In this study, we have investigated another gene located on the X chromosome: the hTAF7L gene, a gene that is homologous to the autosomal hTAF7 gene.
From mouse studies, it is known that this gene is expressed during spermatogenesis, from the stage of spermatogonia till the stage of round spermatids. During meiosis, a shift in protein expression from the cytoplasm to the nucleus is observed. At the same time, the concentration of mTAF7 protein decreases in the nucleus. These observations might point to an important function of the mTAF7L gene in spermatocytes and/or in round spermatids. We hypothesized that if the expression in men is similar, defects in this gene will cause a maturation arrest at the level of meiosis or early spermatid differentiation. Therefore, we analysed patients showing a maturation arrest of spermatogenesis for the presence of mutations in the hTAF7L gene. A total of 25 patients were analysed. For 12 patients, a total of four different alterations of the DNA sequence of the hTAF7L gene were observed. None of these alterations was already described at the NCBI website.
Two patients showed the same two sequence modifications: one located in exon 1 and another in exon 13. This might indicate that the two changes are linked. However, in the group of men with proven fertility, we detected the c.181G > A alteration in exon 1 in one man, but not the change in exon 13 (c.1373G > A). Furthermore, one man with normal spermatogenesis had the alteration in exon 13, but not in exon 1. Altogether, we may conclude that these two alterations of the hTAF7L sequence are probably not linked. Because the alterations observed in exon 1 and exon 13 were found in one man with proven fertility and one man with normal spermatogenesis, respectively, we may conclude that these modifications are not causing the maturation arrest as observed in the patients.
For exon 9, eight patients had the alteration c.922A > G in hTAF7L. Also in the control group, seven out of 60 patients had this modification, indicating that this alteration is not linked to male infertility.
Furthermore, an additional two patients were found to have a deletion of six base pairs of exon 10. This deletion was located in a repeat structure, indicating that it probably represents a polymorphism. We analysed 40 fertile controls for the presence of this deletion, and indeed 10 of them were found to carry the deletion.
Altogether, we may thus conclude that none of the observed alterations was exclusively present in infertile men. Although these data indicate that mutations in the hTAF7L gene are probably not a common cause of male infertility, this does not indicate that hTAF7L has no role in spermatogenesis. More research is necessary to identify the exact function of the hTAF7L gene in spermatogenesis. To this end, the exact localization of the proteins in human testicular tissues should first be determined, and secondly, knockout mice lacking this gene might provide a clue in recognizing the consequences of the absence of the hTAF7L gene.
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Acknowledgements |
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K.Stouffs is a research assistant of the Fund for Scientific Research Flanders (Belgium) (Fonds voor Wetenschappelijk Onderzoek-Vlaanderen).
We thank the laboratory, clinical and paramedical staff of the centres for Medical Genetics, Reproductive Medicine and the Department of Pathology for their assistance. Special thanks to Deborah Vandermaelen and Bart Saerens for their technical help. We also thank Michael Withburn of the Language Education Centre for proofreading the manuscript. The work was supported by grants from the Fund for Scientific Research (FWO-Vlaanderen) and from the Research Council and a Concerted Action of the Free University of Brussels (Vrije Universiteit Brussel).
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References |
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Hiller M, Chen X, Pringle MJ, Suchorolski M, Sancak Y, Viswanathan S, Bolival B, Lin TY, Marino S and Fuller MT (2004) Testis-specific TAF homologs collaborate to control a tissue-specific transcription program. Development 131,5297–5308.
Paduch DA, Mielnik A and Schlegel PN (2005) Novel mutations in testis-specific ubiquitin protease 26 gene may cause male infertility and hypogonadism. Reprod Biomed Online 10,747–754.[ISI][Medline]
Stouffs K, Lissens W, Tournaye H, Van Steirteghem A and Liebaers I (2005) Possible role of USP26 in patients with severely impaired spermatogenesis. Eur J Hum Genet 13,336–340.[CrossRef][ISI][Medline]
Tournaye H, Verheyen G, Nagy P, Ubaldi F, Goossens A, Silber S, Van Steirteghem AC and Devroey P (1997) Are there any predictive factors for successful testicular sperm recovery in azoospermic patients? Hum Reprod 12,80–86.
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Submitted on January 6, 2006; accepted on January 25, 2006.
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