Molecular Human Reproduction, Vol. 8, No. 1, 8-15,
January 2002
© 2002 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Isolation of the human testatin gene and analysis in patients with abnormal gonadal development
1 Department of Cell and Molecular Biology, Medical Nobel Institute, 2 Center for Genomics Research and 3 Department of Molecular Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden
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We have previously isolated the testatin gene using a modified mRNA differential display method on RNA from developing male and female mouse gonads. This gene is specifically expressed during early testis development, immediately after the onset of the testis-determining gene Sry. The protein encoded by testatin has features that are characteristic for type 2 cystatins, a family of small inhibitors of cystein proteases such as the cathepsins. We have now isolated the human orthologue of this gene. We describe here the sequence, genomic structure, chromosomal location, and expression pattern of the human testatin gene. Like mouse testatin, human testatin is specifically expressed in the testis, suggesting that it has a function in reproduction. We have therefore also investigated whether the human testatin gene plays a role in disorders of gonadal development, by sequencing the gene in patients with gonadal dysgenesis, with true hermaphroditism, and in children with less well-defined intersex conditions. We found no sequence aberrations in these patients apart from an H109P polymorphism which was also found in fertile controls. This is the first genetic analysis of testatin in humans.
cystatin/protease inhibitors/sex determination/testatin/testis development
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Introduction |
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The lysosomal cathepsins and their inhibitors, the cystatins, constitute one of the most important cysteine protease/protease inhibitor systems in the human body. They are involved in a large number of normal processes such as intracellular catabolism of proteins, processing of prohormones and proenzymes, antigen presentation, bone resorption, and implantation (Chapman et al., 1997
; Turk et al., 2000). Disturbed expression of cathepsins/cystatins is connected with a wide variety of pathological processes, including tumorigenesis, micro-organism invasion, viral infections, and inflammatory conditions (Henskens et al., 1996).
Members of the cystatin superfamily function both intracellularly and extracellularly as potent inhibitors of cysteine proteases such as the cathepsins. Based on homology, size and location, members of the cystatin superfamily have been divided into three families; the stefins (type 1), the cystatins (type 2) and the kininogens (type 3). The type 2 cystatins include cystatin C, D, E/M, F, S, SA and SN, of which the most well-known member is cystatin C. These cystatins are primarily found extracellularly and are characterized by two disulphide bridges in the C-terminal region of the ~120 amino acid long protein. Human cystatin C has been found in all biological fluids and tissues examined, with highest concentrations in seminal fluid and brain (Abrahamson et al., 1986; Yasuhara et al., 1993). Abnormal metabolism of cystatin C in the central nervous system is the cause for hereditary cystatin C amyloid angiopathy (HCCAA) and specific point mutations in cystatin B correlate with an autosomal recessive form of epilepsy, EPMI (Henskens et al., 1996; Pennacchio et al., 1996). In contrast to the brain, little is known about the function of the cathepsin/cystatin systems in the testis. Several cathepsins, including cathepsin A, B, C, D, H, L, S and V, are expressed in the testis, suggesting a role for cathepsins/cystatin C in testis differentiation or spermatogenesis (Esnard et al., 1992; Chung et al., 1998). However, mice deficient in cystatin C are fertile and show no gross abnormalities up to 6 months of age (Huh et al., 1999).
We have recently isolated a testis-specific, cystatin-related gene from mouse and named it testatin (Töhönen et al., 1998; Kanno et al., 1999). Its narrow expression pattern contrasts sharply with the broad expression pattern of cystatin C. Testatin expression is mainly confined to the pre-Sertoli cells in the testis, and the onset of expression coincides with early testis development. It has been suggested that testatin, together with cystatin T and the cystatin-related epididymal spermatogenic (CRES) gene belong to a new subfamily within the type 2 cystatins based on the restricted expression pattern of these genes (Cornwall et al., 1992; Shoemaker et al., 2000). The specific expression patterns of these cystatins imply that they perform very specific functions in reproduction.
The early fetal mammalian gonad has the potential to develop into either an ovary or a testis. Several syndromes exist in which this development is abnormal. The gonadal developmental abnormalities can be classified into gonadal dysgenesis in 46,XX or 46,XY individuals, testicular development with sex reversal in 46,XX individuals (XX males), and true hermaphroditism, with development of both ovarian and testicular tissue in one individual. Normally, the initial sex-determining switch depends on the presence or absence of the SRY gene on the Y chromosome. The SRY gene encodes the testis-determining factor, a transcription factor whose expression induces testis differentiation (Berta et al., 1990; Gubbay et al., 1990; Jager et al., 1990; Sinclair et al., 1990; Koopman et al., 1991). Deletion or mutations of SRY can be found in 15–20% of patients with 46,XY gonadal dysgenesis, and ~85% of all 46,XX males have SRY in their genomes, often translocated to the X chromosome. A few additional genes have been identified as being involved in gonad development. These include the SRY-related SOX9 gene (Tommerup et al., 1993; Wagner et al., 1994; Schafer et al., 1996), the Wilms' tumour 1 gene (WT1) (Call et al., 1990) and the genes for the orphan nuclear receptors DAX1 (Bardoni et al., 1994; Muscatelli et al., 1994; Zanaria et al., 1994) and SF1 (Luo et al., 1994; Taketo et al., 1995). However, as yet no target genes that are directly regulated by SRY have been pinpointed. Also, no underlying molecular explanation can be found in the vast majority of all patients with abnormal gonadal development.
Due to its specific expression in pre-Sertoli cells of the testis and its onset of expression immediately after the peak of SRY expression, testatin is a candidate for involvement in early testis development. Here we report the cloning of the human testatin gene, its expression pattern, chromosomal location, and analysis in patients with disturbed gonadal development.
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Materials and methods |
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Screening of adult testis cDNA library
The mouse testatin cDNA was used as a probe to screen an adult human testis cDNA library (BD Biosciences Clontech, Palo Alto, USA). The screening procedure was performed according to the manual for the kit. Recombinants (4x106) were screened and sequence analysis of positive clones was performed by an in-house core facility at Center for Genomics Research (CGR), Karolinska Institutet.
RNA expression
A human RNA dot blot, containing polyA+ RNA from 50 different adult and fetal human tissues (Clontech), was hybridized with [
32P]dATP-labelled PCR fragment spanning exons 1–3 of human testatin cDNA. The 340 bp long probe was produced with primers HT5'94 (5'-CACGAGCAAAGGGACTGTGATG-3') and HT 3' (5'-CAAGCAGGTCTTGTTCAG-3'), as earlier described (Tohonen et al., 1998). The hybridization was performed in ExpressHyb (Clontech) at 65°C overnight. The filter was washed twice for 20 min in 2xstandard saline citrate (SSC) and 1% sodium dodecyl sulphate (SDS) at 65°C then twice for 20 min in 0.1xSSC and 0.5% SDS at 55°C.
Chromosome localization
A panel of 83 radiation hybrid (hamster/human) cell lines was used to establish the chromosome localization of the human testatin gene (radiation hybrid panel G3; Research Genetics, Huntsville, AL, USA). The somatic hybrid cell lines retaining individual human chromosomes were screened by PCR. Two different PCR reactions were used. A 169 bp fragment covering exon 3 was produced by HT5'366 (5'-CTTCTTCACCATCAGCACCAGG-3') and HT3'533 (5'-GCTCAGCCACTGAAGAGTCCTC-3'). A 1000 bp fragment covering intron 2 was produced by HT5'325 (5'-CCTTTCCAAGAAAGCACAGAGC-3') and HT3'87 (5'-CCTGGTGCTGATGGTGAAGAAG-3'). PCR amplifications were carried out under the following conditions: 92°C for 2 min, then 35 cycles of 92°C for 1 min, 55°C for 1 min and 72°C for 1 min (exon 3) or 1 min 20 s (intron 2). The positive clones were analysed by Stanford Human Genome Center where a putative chromosome localization was given.
Isolation of the genomic testatin clone
Filters with RPCI1,3–5 human pac library-clones (Roswell Park Cancer Institute, New York, NY, USA) were used to screen for the human testatin gene. A 340 bp PCR fragment spanning exons 1–3 of human testatin cDNA was produced with primers HT5'94 (5'-CACGAGCAAAGGGACTGTGATG-3') and HT 3' (5'-CAAGCAGGTCTTGTTCAG-3') and used as a probe. Hybridization was performed overnight at 65°C in a solution containing 5xSSC, 5xDenhardt's, 0.5% SDS, ssDNA. The filters were then washed twice for 10 min in 2xSSC, 0.5% SDS at room temperature, twice for 20 min in 0.1xSSC, 0.5% SDS at 60°C, then twice for 20 min at 50°C in the same wash buffer, followed by exposure on film overnight.
Analysis of the human testatin gene for mutations
Genomic DNA was prepared from frozen blood following standard procedures. PCR conditions were 92°C for 2 min, then 35 cycles of 92°C for 1 min, 58°C for 1 min and 72°C for 1 min, followed by a final extension step of 72°C for 10 min. Primers HT+5'C (5'-ATCCTGTCCTTCCTGTCCTG-3') and HT intronI-3'd (5'-ACCCCACTCCCATTTTTCAC-3') produced a 480 bp fragment covering exon 1. Primers HT intronI-5'a (5'-GACATCTTCACCAGCAGCTAC-3') and HT intronII-3'a (5'-CTTTGTCAGTTGCACCTGTGC-3') produced a 271 bp fragment covering exon 2. Primers HT intronII-5'a (5'-CTTGAGCCATGAGCTTCCTG-3') and HT-3'533 (5'-GCTCAGCCACTGAAGAGTCCTC-3') produced a 243 bp fragment covering exon 3. Sequence analysis of PCR products was performed by an in-house core facility at CGR, Karolinska Institutet.
Allele-specific PCR for typing of the H109P polymorphism was performed from 100 ng genomic DNA, using two reactions for each subject. Each reaction contained one primer specific for the His or Pro allele (Test 3 and Test 4, respectively), together with an upstream and a downstream primer (Test 1 and Test 2). PCR conditions were established to generate a short, allele-specific band in the presence of the matching sequence, and only a long control fragment in its absence. The sequences of the primers were: Test 1 (5'-CATCTTCTGGAAATGCAGCTG-3'); Test 2 (5'-CACCCAAGAGCAGCAGGAG-3'); Test 3 (5'-CTCTGTGCTTTCTTGGAAAG-3'); Test 4 (5'-CTCTGTGCTTTCTTGGAAAT-3').
Patients
A group of patients with abnormal gonadal develoment was analysed. Mutations in the SRY gene of these patients had been excluded. Five patients had 46,XY gonadal dysgenesis (complete dysgenesis in three cases, partial in two), whereas six were true hermaphrodites (five with a 46,XY karyotype, one with 46,XX). We also analysed an additional 10 patients all of whom had a 46,XY karyotype and severe undermasculinization (perineoscrotal hypospadias and a bifid scrotum); for these patients we did not have detailed clinical information regarding the status of the gonads. The androgen receptor and SRY genes had been sequenced in these cases and found to be normal. Controls were unselected Swedish men and women with proven fertility (with at least one child).
Databases
BLAST searches were performed with mouse testatin cDNA sequences in the following databases: GenBank + EMBL + DDBJ + PDB included in the University of Wisconsin GCG package, Celera, UniGene (htpp://www.ncbi.nlm.nih.gov/UniGene) and GeneLynx (http://www.genelynx.org) (Altschul et al., 1997; Venter et al., 2001).
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Isolation of human testatin
The human orthologue of testatin was found by screening a human adult testis cDNA library using mouse testatin cDNA as a probe. Two overlapping clones, derived from the same gene, were isolated. One open reading frame (ORF) that potentially encoded a protein of 147 amino acids (Figure 1A
) was identified. The N-terminal part of the ORF had the properties of a signal peptide and computer predictions indicated a possible signal peptide cleavage site between amino acids Ala28 and Trp29. The full-length amino acid sequence derived from the ORF was similar to type 2 cystatins, with highest homology to mouse testatin (Table I). We therefore named the newly isolated cDNA human testatin. As in mouse testatin, there is a predicted SOX9 binding site within exon 1 (Figure 1A) and this is conserved within these genes.
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The evolutionary relationship between human and mouse testatin was also validated by comparing their amino acid sequences with those of mouse cystatin T, human and mouse cystatin C, and CRES. The alignment showed that human and mouse testatin segregate together and are distinct from cystatin C, CRES and cystatin T (Figure 2A
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We also performed BLAST searches in different databases by screening with mouse testatin cDNA and protein sequences. In all searches, mouse testatin exhibited highest homology to human testatin (data not shown).
The deduced human testatin amino acid sequence displays several of the characteristic features of the type 2 cystatins. The type 2 cystatins, cystatin C, D, E/M, F, S, SA and SN, are small, secreted proteins of ~120 amino acid residues. They have four characteristic cysteines forming two disulphide bridges in the C-terminal domain and three short conserved regions that form the inhibitory site of the protein. Both human and mouse testatin have the signal peptide and the four conserved cysteines that are found in type 2 cystatins. However, the testatin proteins contain only one of the three regions necessary for the protease inhibitory function executed by type 2 cystatins, the PW domain (Figure 2B
). The most conserved parts of testatin do not correspond to regions involved in cathepsin inhibition. Instead high homologies are found between amino acids 50 and 69, and amino acids 108 and 122.
To obtain the gene structure for human testatin, the human testatin cDNA was used to screen filters with RPCI1,3–5 human pac library clones. Two genomic clones were isolated, containing the entire human testatin gene including the three coding exons. The gene organization for the human and mouse testatin genes are similar, with three exons of approximately the same length and two introns (Figure 1B).
The human testatin gene maps to 20p11.2
The genomic localization of human testatin was determined by screening a panel of 83 radiation hybrids by PCR, using two different primer pairs. Computer analysis at the Stanford Human Genome Centre gave us a significant lod score of 14.49 for linkage to the marker for cystatin C, CST3, that has been mapped to 20p11.2 (Figure 3). Within this region of chromosome 20, five additional cystatin type 2 genes have been mapped, cystatin D, F, SN, SA and S (Figure 3) (Morita et al., 2000). This region of human chromosome 20 is syntenic to the region of 81.4 cM on chromosome 2 in mouse, where cystatin C, cystatin T and the CRES genes are located.
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The human testatin is predominantly expressed in the testis
Several members of the cystatin type 2 family are expressed in a wide variety of tissues. In contrast, mouse testatin has so far only been found to be expressed in fetal and adult testis. To investigate if the human testatin mRNA also shows a restricted distribution, a human RNA master blot containing mRNA from a panel of different fetal and adult human tissues was screened using the human testatin cDNA as a probe. The result shows that the human testatin gene is strongly expressed in adult testis (Figure 4
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Screening for testatin gene mutations in disturbed gonadal development
Mouse testatin is expressed in pre-Sertoli cells immediately after the peak of Sry expression. Due to this timing and to the site of testatin expression in the male fetal gonad, it is reasonable to speculate that it plays a role in testis differentiation. Mutations in this gene could thus lead to abnormal sexual development.
We sequenced the exons and exon/intron borders of the human testatin gene in 11 patients with abnormal development of the gonads, and in an additional 10 karyotypic males who were born with ambiguous genitalia and for whom more detailed clinical information was lacking. We found a C to A transversion at nucleotide 336 in exon 2, causing a proline to histidine change at amino acid 109 (H109P, Figure 1, Table II). Apart from this base substitution, no deviations from the original reference sequence were found. The H109P variant was also found in normal controls. To evaluate the frequencies of the two alleles, we typed 50 normal fertile Swedish controls (25 men and 25 women) for the H109P polymorphisms by allele-specific PCR. Allele frequencies were: His: 0.58 (0.54 for men, 0.62 for women), Pro: 0.42 (0.46 for men, 0.38 for women). For the female controls, the distribution of genotypes did not seem to be in Hardy–Weinberg equilibrium (Table II). However, this is probably due to the small number of individuals genotyped.
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Discussion |
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We describe here the isolation and characterization of the human testatin gene. Several observations provide clear evidence that the gene we have isolated is the human orthologue to mouse testatin: (i) the isolated gene was the only gene found when a human adult testis library was screened using a mouse testatin cDNA probe, (ii) the isolated human gene was also the best human testatin candidate in in-silico screens when mouse testatin was used as a probe, (iii) the human and mouse testatin proteins grouped together, separately from the cystatin C and CRES homologues, in phylogenetic studies, and (iv) the newly isolated gene showed the same restricted expression pattern as mouse testatin, with expression predominantly in the testis.
The type 2 cystatin genes have undergone a surprisingly high rate of evolutionary change. As an example, mouse cystatin C protein shares only 71.7% identity (75.8% similarity) to the human orthologue. CRES has also diverged rapidly, and exhibits 58.5% identity (65.0.% similarity) between the mouse and human proteins. The overall amino acid divergence between mouse and human testatin is even higher, 57.1% identity (66.7% similarity). Thus, testatin and CRES, which both belong to a subgroup within type 2 cystatins and are both selectively expressed, are evolving more rapidly than cystatin C, which is ubiquitously expressed. Rapid sequence divergence has mainly been shown for genes involved in intergenomic conflict, as occurs between a host and pathogen (Whitfield et al., 1993), and specifically for genes involved in male-driven evolution. For example, the sex-determining Y chromosome gene, SRY, exhibits a remarkably high level of divergence (O'Neill and O'Neill, 1999). This might be explained by the higher substitution rate that has been shown in Y chromosome genes compared with autosomal or X chromosome genes (Shimmin et al., 1993; Huang et al., 1997). However, other genes that are specifically expressed in reproductive tissues evolve rapidly; these include the homeobox gene Pem located on the X chromosome (Maiti et al., 1996) and the zona pellucida glycoproteins ZP2 and ZP3, and oviductal glycoprotein, OGP (Swanson et al., 2001). As the rate of sequence divergence is assumed to be faster in genes affecting sexual function and fertility (Singh and Kulathinal, 2000), it is interesting that the testis-specific testatin has undergone a high rate of evolutionary change as reported here.
The type 2 cystatin genes include cystatin C, D, F, S, SN and SA. They are located within a cystatin gene cluster on chromosome 20 (Schnittger et al., 1993; Morita et al., 2000). Human testatin is located close to the marker for cystatin C (CST3), which was mapped to 20p11.2. This genomic location corresponds to the region of 81.4 cM on mouse chromosome 2. To our knowledge, neither of these chromosome locations has been associated with disorders in sexual development. Interestingly, a gene reported to be responsible for sex reversal in C57BL/6J-YPOS mice is located in the central region of mouse chromosome 2 (Eicher et al., 1996). However, the exact location of this gene has not been defined.
The structure of type 2 cystatins has so far been based on information from chicken cystatin (Dieckmann et al., 1993). X-ray and NMR analyses show that this cystatin consists of a flexible N-terminal segment, a straight five-turn -helix, and a five-stranded anti-parallel ß-sheet that is twisted and wrapped around the -helix. The cathepsin inhibitory site includes an N-terminal segment, a central loop and a second C-terminal loop. Together they form a tripartite wedge-shaped structure that is complementary to the active site of the cathepsins (Figure 5). A second cystatin domain has recently been published. This domain is responsible for the inhibition of legumain, a protease with an active cleft similar to caspases (Chen et al., 1998; Alvarez-Fernandez et al., 1999). This domain is located on the opposite side to the cathepsin-binding surface (Figure 5). Comparing human and mouse testatin, two regions are particularly well-conserved, amino acids 50–69, and amino acids 108–122. Based on the three-dimensional structure of chicken cystatin, these domains are located in the N-terminal -helix and in the flexible region respectively. Although this type of comparison should be viewed with some caution, it suggests that testatin has important functions in domains opposite to the cathapsin inhibitory sites, but on the same side as the legumain-binding domain in cystatin C. It will be interesting to see if testatin has any inhibitory functions with respect to a specific cathepsin. However, this seems less likely due to the lack of a conserved cathepsin-inhibitory site. Another possibility is that the protein is involved in more caspase-like inhibitory activities.
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Since testatin is specifically expressed in pre-Sertoli cells of the testis starting immediately after the peak of SRY expression, we believe that it plays a role in early testis development. Mutations in this gene could thus lead to abnormal sexual differentiation. The most straightforward hypothesis is that loss of the function of testatin would interfere with normal testis development, and that inactivating mutations would be found in a subset of patients with 46,XY gonadal dysgenesis. However, it is not possible to predict the phenotypic consequences of mutations in this gene, and it is feasible that inactivating versus activating mutations would give different phenotypes in both karyotypic males and females. Therefore, we sequenced the testatin gene in patients with different kinds of developmental disorders of the gonads. Our only finding was a His to Pro polymorphism, where both alleles occur at high frequencies in the general population (0.58 versus 0.42). Since all genotypes (heterozygotes and homozygotes) were found in the patients, this amino acid substitution probably has no significance for gonadal development.
In conclusion, mutations in the human testatin gene are not a common cause of abnormal gonadal development. It remains possible, however, that disease-causing mutations will be found when additional patients with these rare disorders are analysed. We have screened all exons and exon/intron borders for mutations, and it also remains possible that there are mutations that affect the promoter or other regulatory parts of the gene. Another approach for the evaluation of phenotypic consequences of testatin gene mutations is the creation of mice with targeted disruption of this gene. These experiments are underway.
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We are grateful to Magnus Abrahamson for discussions and help with Figure 5
, Catharina Larsson for sharing her knowledge, the radiation hybrid panel and the pac library. This work was supported by Märta and Gunnar V.Philipsons Foundation, Karolinska Institutet Funds and the Swedish Natural Science Research Council to K.N. and by MFR project no. 121 98 and Novo Nordisk Foundation to A.W. |
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4 Present address: Molecular Sciences, AstraZeneca R&D Södertälje, SE-151 85 Södertälje, Sweden
5 To whom correspondence should be addressed. E-mail: Katarina.Nordqvist{at}cmb.ki.se
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Submitted on May 14, 2001;
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