A SPGY copy homologous to the mouse gene Dazla and the Drosophila gene boule is autosomal and expressed only in the human male gonad
A SPGY copy homologous to the mouse gene Dazla and the Drosophila gene boule is autosomal and expressed only in the human male gonadZhihong Shan1,+, Peter Hirschmann1,+, Thomas Seebacher1, Angela Edelmann1, Anna Jauch1, Jane Morell2, Peter Urbitsch1 and Peter H. Vogt1,*
1Reproductiongenetics in Institute of Human Genetics, University of Heidelberg, D-69120 Heidelberg, Germany and 2Department Reproductionbiology, German Primate Center GmbH, D-37077 Göttingen, Germany
Received August 15, 1996;Revised and Accepted September 20, 1996
We have isolated a series of human testis poly(A) cDNA clones by cross-hybridization to SPGY1, a Y gene homologous to DAZ. Their sequence analysis revealed an identical nucleotide composition in different `full-length' clones, suggesting that all were encoded by the same gene. We mapped this gene to the short arm of chromosome 3 and designated it SPGYLA(SPGYlike autosomal). Comparison of the SPGYLA cDNA sequence with the cDNA sequences of DAZ and SPGY1 revealed two prominent differences. The tandem repetitive structure of 72 bp sequence units (DAZ repeats) is absent. SPGYLA contains only one 72 bp sequence unit. Downstream of it, a specific 130 bp sequence domain is present which is absent in DAZ and SPGY1 but present in the mouse gene Dazla and in the Drosophila gene boule. SPGYLA encodes an RNA binding protein expressed only in the human male gonad. The data presented give strong evidence that not DAZ but SPGYLA is the functional human homologue of Dazla and boule.
In eukaryotes, RNA binding proteins play important roles in various aspects of the splicing process and in the nuclear export of primary transcripts, in cytoplasmic RNA stability and during translation (1 ). One well characterized group of RNA binding proteins contain one or more copies of the so-called RNA recognition motif (RRM) in their N-terminal regions. The RRM domain consists of ~80 amino-acids (2 ). Most RRM proteins are expressed constitutively. However, cell type-specific RRM proteins also exist and seem to be functional in the brain (3 ,4 ), during sex determination (Drosophila; 5 ,6 ) and in the germ line (in Drosophila, for spermatogenesis, 7 ; in the mouse, for spermatogenesis and oogenesis, 8 ,9 ; in human, for spermatogenesis, 10 -12 ). The recent discovery of the Drosophila gene boule with a high homology of its RNA binding domain to that of the human gene DAZ (13 ) suggests that RNA binding proteins in the germ line are highly conserved and might fulfil essential regulative functions during the complex process of spermatogenesis. Gene products of boule function during the male meiotic cell cycle at the G2-M transition, and loss of boule function results in azoospermia (13 ). Therefore, it was argued that DAZ (because of its distinct sequence homology to boule) must have an essential function during the meiotic cell cycle in human spermatogenesis. However, this is inconsistent with the fact that the total deletion of DAZ can be inherited from father to son (14 ) and that deletion of DAZ is not always correlated with azoospermia but also with other sterile phenotypes (14 ,15 ). Therefore, DAZ deletions seem to be compatible with development of motile spermatozoa, and the primary mutation effect in men with deletion of DAZ is subfertility but not azoospermia (14 ,16 ). These results suggested that the functional human homologue of boule had not yet been isolated and that DAZ might not have the same function as boule in spermatogenesis (17 ).
The mouse homologue of boule is located on chromosome 17 (9 ). Because of its distinct sequence homology to DAZ, it was designated Dazla (DAZlike autosomal). Therefore, if a human homologue of boule/Dazla exists, it is expected to be autosomal and located on the human homologue of mouse chromosome 17, chromosome 6 (17 ,18 ).
We have analysed this hypothesis experimentally by screening different cDNA libraries prepared from adult human testis poly(A) RNA with the cDNA probe SPGY1, a Y gene homologous to DAZ (11 ). SPGY1 is member of the SPGY (spermatogenesis gene on the Y) locus in distal Yq11 containing several DAZ-related genes, in addition to DAZ itself (12 ,16 ). Their transcripts are characterized by different numbers of a tandem repetitive 72 bp sequence unit (DAZ repeat). DAZ contains seven such repeats (11 ), SPGY1 contains 12 (12 ,14 ).
Sequence analysis of SPGY1 homologous cDNA clones identified a subclass of clones with a common sequence structure absent in DAZ or SPGY1 but present in boule and Dazla. Two features were most prominent. The 72 bp sequence unit (DAZ repeat) was not present in a tandem repetitive structure but as a single sequence unit like in Dazla and boule. Adjacent to it, a 130 bp sequence domain was analysed which is absent in DAZ and SPGY1 but present in Dazla and boule. Here we present the isolation of the complete cDNA sequence of this SPGY gene and the sequence of its putative translation product. It maps to the short arm of chromosome 3 and therefore is the first SPGY copy mapped to an autosome. We designated it as SPGYLA (SPGYlike autosomal). It encodes RNA binding protein only expressed in the human male gonad. Our results strongly suggest that not DAZ but SPGYLA is the functional human homologue of Dazla and boule in human spermatogenesis.
During analysis of the genomic sequence structure of SPGY genes by DNA blot experiments, we found that not all SPGY genes could be located in the AZFc region in distal Yq11. Azoospermic men with deletion of AZFc retained a cross-hybridizing genome fragment also present in the female genome (12 ). The same was observed in blots with the DAZ probe (11 ). We were able to identify this non-Y gene copy by analysis of a series of cDNA clones isolated by homology to SPGY1. They were characterized by the absence of all but one of the tandem repetitive 72 bp sequence units (DAZ repeats) found in DAZ (11 ) and in SPGY1 (12 ,14 ), and the presence of a specific sequence unit of 130 nucleotides (sequence position 762-891 in full-length cDNA clone CT355 submitted to the GenBank database; accession no. U66726) which is absent in the sequence of DAZ, SPGY1 and other cDNA clones with tandem repetitive 72 bp sequence units.
All cDNA clones containing this sequence structure (one 72 bp sequence unit plus the specific 130 bp sequence unit downstream of it) were identical, so presumably were derived from one SPGY gene. It was mapped by fluorescence in situ hybridization (FISH) analysis to the short arm of chromosome 3 (Fig. 1 ). As other SPGY copies (analysed so far) were mapped to the AZFc region in distal Yq11 (14 ,16 ), the new autosomal copy was designated as SPGYLA (SPGYlike autosomal). We scrutinized the mapping position of SPGYLA by PCR analysis of a panel of human-hamster hybrid cell lines containing overlapping subsets of human chromosomes. With the SPGYLA-specific primer pair (LA1-for/LA1-rev), a PCR product was present in all hybrid cells containing human chromosome 3.
Male-specific genomic DNA fragments cross-hybridizing to the Dazla probe were only observed in the genome of human and gorilla (9 ). As SPGYLA and Dazla sequences are homologous to each other, we expected a similar pattern of cross-hybridization in zoo-DNA blots with the human SPGYLA probe. Consequently, male-specific hybridization fragments were observed in the genome of chimpanzee (Pan troglodytes) and gorilla (Gorilla gorilla) (data not shown). However, in a zoo-DNA blot containing genomic DNA samples of a new world monkey (Platyrrhini: Callithrix jacchus) and of two old world monkeys (Catarrhini: Macaca mulatta, Macaca nemestrina), male-specific SPGYLA cross-hybridizing fragments were only present in the DNA of the old world monkeys but not in the DNA of the new world monkey Callithrix (Fig. 4 ). Cross-hybridization signals in the mouse genome, the cattle genome and the wallaby genome (Macropus eugenii) marked the evolutionary conservation of the SPGYLA sequence structure.
It is well known that genes expressed in different tissues create testis-specific RNAs by alternative splicing. Moreover, genes expressed in testis and transcribed pre-meiotically might be translated only post-meiotically (20 -22 ). This indicates that in the male germ line, the availability of functional gene products might be regulated primarily by post-transcriptional processes. Testis-specific RNA binding proteins are strong candidates to be involved in such processes. They were described for Drosophila, (7 ,13 ), mouse (8 ,9 ,23 ) and human (10 -12 ). All contain at least one copy of the highly conserved RRM in the N-terminal part of their protein structure. RRM domains consist of ~80 amino acids. Two so-called RNP boxes (RNP-1, an octapeptide; RNP-2, a hexapeptide) can be matched in RRM domains of RNA binding proteins in yeast as in human. This suggests that RRM domains have a very ancient origin and that the mode of their interaction with specific RNAs is highly conserved. The specificity of the RNA-protein binding reaction is thought to be achieved by auxiliary sequence blocks in the RRM domain and the C-terminal part of the RRM protein structure (2 ,24 ). By these criteria, we suppose that the protein of SPGYLA presented here is the functional human homologue of the protein encoded by the mouse gene Dazla and most likely also of the protein encoded by the Drosophila gene boule.
SPGYLA- and Dazla-encoded proteins have similar molecular weights (SPGYLA, 33 178 Da; Dazla, 33 312 Da) and the sequences are 89% identical (93% similar) along their total length (Fig. 2 a). In contrast to this, the DAZ-encoded protein (with one consensus 72 bp sequence unit encoding 24 amino acids) has a higher molecular weight (44 300 Da) and is homologous to the Dazla-encoded protein only along 201 amino acids. Their C-terminal peptides are different because of the presence of the 130 bp sequence unit in the protein-coding region of SPGYLA and Dazla and its absence in DAZ. Therefore, our supposition is that not DAZ but SPGYLA is the functional human homologue of the mouse gene Dazla.
A functional homology between the boule- and SPGYLA-encoded proteins is also most likely but, due to the large evolutionary distance between Drosophila and human, it is difficult to judge it by sequence comparison. Both are RNA binding proteins with different conserved domains including the RRM domain (Fig. 2 b). Moreover, the cDNA structure of boule is homologous to the cDNA structure of Dazla and SPGYLA. All three contain one homologous 72 bp sequence unit and the 130 bp sequence unit downstream of it. A functional homology of all three proteins is therefore most likely.
Mapping of human genes homologous to mouse genes of mouse chromosome 17 show that they are clustered on the short and long arm of human chromosome 6 (18 ). Therefore, it was suggested recently that the functional human homologue of Dazla/boule should also be located on chromosome 6 (17 ). However, FISH analysis of the location of SPGYLA on human chromosomes does not support this prediction. A distinct hybridization signal was only observed on the short arm of chromosome 3 (Fig. 1 ). No additional FISH signals were observed on chromosome 6. This result was confirmed by mapping of SPGYLA on a human-hamster hybrid cell panel. It is intriguing to note that, despite its sequence homology to SPGY genes in distal Yq11, the SPGYLA probe does not hybridize on the Y chromosome. We explain this result by the presence of a 1.9 kb long 3' UTR domain in the SPGYLA cDNA structure. This sequence region is specific for this gene and represented 66% of the length of the SPGYLA FISH probe used.
No evidence exists for the presence of a male fertility locus on chromosome 3 by genetic or molecular linkage analysis (25 ). Comparative mapping of DNA loci of human chromosome 3 to mouse chromosomes indicates that this human chromosome is composed of chromatin domains of different mouse chromosomes (3, 6, 9, 14 and 16 ) (25 ), and now also of 17.
SPGYLA is only expressed in the human male gonad. Its transcription product is polyadenylated and has a length of 2.9 kb. The second weak cross-hybridizing RNA signal (2.1 kb) observed in the testis RNA blot is difficult to explain at the moment. Cross-hybridization to testis transcripts of other Y chromosomal SPGY genes was excluded by using a SPGYLA-specific 3' UTR probe. Moreover, DAZ and SPGY1 hybridized to testis RNA of 3.5 kb (11 ,12 ). It is interesting to note that the mouse homologue Dazla also hybridized to two different testis RNA populations (9 ). Our result might therefore reflect the use of an alternative polyadenylation site in SPGYLA transcripts. A second possibility may be that a divergent SPGYLA copy is present on the same or another autosome or on the Y chromosome as discussed below.
The expression pattern of SPGYLA is different from the pattern of its mouse homologue Dazla which is expressed also in the female gonad (9 ). We have examined the expression of SPGYLA in human ovaries of different ages (all before menopause) in order to exclude a possible age-dependent inactivation of the SPGYLA gene in the female gonad. However, we were not able to detect any SPGYLA transcripts in any ovary tissue by RT-PCR. We cannot exclude expression of SPGYLA in certain ovarian stages not available from human females for medical reasons. We also cannot exclude the possibility that the ovarian tissues analysed do not show SPGYLA transcription because of their pathological aetiology.
The function of Dazla in the male and female gonad is not yet known. The function of boule most likely occurs during the male meiotic cell cycle at the G2-M transition (13 ). Loss of boule results in azoospermia. Therefore, if SPGYLA is the functional homologue of boule, its function is expected to be at human male meiosis, and SPGYLA mutations should cause azoospermia.
It is attractive to speculate that Y genes of the SPGY locus in distal Yq11 containing a repetitive 72 bp sequence unit (like DAZ and SPGY1) might have evolved from an functional ancestor SPGYLA copy on the Y chromosome with one 72 bp sequence unit. We were able to show that each 72 bp sequence unit is a separate exon unit in the SPGY locus (Vogt et al., in preparation). However, molecular analysis of all SPGY genes in distal Yq11 is an essential prerequisite to distinguish between SPGY gene structures containing repetitive 72 bp exon units and SPGY genes containing only one 72 bp exon unit like SPGYLA. This work is in progress.
Our analysis of the presence of SPGYLA in different primate species suggests that a copy of SPGYLA was translocated to the Y chromosome at a time interval between 36 and 55 million years ago, after divergence of Platyrrhini and before divergence of Catarrhini. Although, we have not yet confirmed the presence of SPGYLA on the Y chromosome of the macaques and baboon by FISH analysis, the male-specific fragments of their genomic DNA blot patterns can be explained most easily by the presence of at least one SPGYLA copy on their Y chromosomes. The presence of a SPGYLA copy on the human Y chromosome, therefore, also cannot yet be excluded. However, because we detect no FISH signal with the SPGYLA probe on the human Y chromosome, its sequence structure is expected to be divergent from that of the autosomal SPGYLA gene described here.
Hybridization of filter discs (duplicates) prepared from plated testis poly(A) cDNA libraries (Clontech: HL1010b; HL3024b) with the probe SPGY1 was performed as described by Quertermous (26 ). Sequence analyses of selected cDNA clones were performed on the Pharmacia A.L.F. express using the Sanger dideoxy chain termination method (Autoread kit, Pharmacia) and the strategy of primer walking on overlapping subfragments from both DNA strands. The sequence of the full-length clone CT355 was submitted to the GenBank database; (accession no. U66726). For sequence alignment analyses, we used the PRETTYBOX and PRETTYPLOT software tools written by Peter Rice, Informatics Division, The Sanger Centre, Cambridge, UK, and part of the HUSAR software package of the German Cancer Institute, Heidelberg.
Genomic DNA samples from peripheral blood lymphocytes of human individuals and animals, and DNA samples of hybrid cell lines were prepared using standard protocols. Conditions used for restriction, blotting, probe labelling and hybridization were the same as described previously (14 ). Genomic PCR for analysis of the specific 130 bp sequence region in CT355 (SPGYLA) was performed with 200 ng of DNA and the primer pair: LA-1for (5'-ATGCCACCACAGTGGCCTGTTG-3') and LA-1rev (5'-aacacccggtaaaggtctccca-3') using the Expandtm Long Template PCR system (Boehringer). Conditions for amplification were: 10* 1 min at 97oC, 1 min at 65oC, 5 min at 68oC followed by 15 cycles of 1 min at 94oC, 1 min at 65oC, 5 min at 68oC. The final elongation time was 5 min at 68oC. The template was denatured for 5 min at 98oC.
RNA samples were extracted from the different tissues indicated in the text using the standard guanidinium isothiocyanate procedure (27 ) or ordered from Clontech (male tissues: brain, cat. no. 64020; heart, cat. no. 64025-1; spleen, cat. no. 64034-1; prostate, cat. no. 64038; female tissues: kidney, cat. no. 63047; liver, cat. no. 64022-1; lung, cat. no. 64023). Poly(A) RNA was purified on oligo(dT) columns (Pharmacia). For RNA blot experiments, RNA samples (20 [mu]g) were electrophoresized on 1% agarose gels containing 2.2 M formaldehyde as denaturing agent. Before blotting, RNAs in the gel were partially hydrolysed in 50 mM NaOH/10 mM NaCl and neutralized in 0.1 M Tris/10* SSC buffer (pH 7.5). Blots were pre-hybridized (5 h) and hybridized (overnight) in a 50% formamide buffer (5* SSPE, 10* Denhardt blocking solution, 2%SDS); 20* SSPE is: 3 M NaCl, 0.2 M NaH2PO4, 0.2 M EDTA (pH 7.4). For the RNA blot experiment we used a SPGYLA-specific 3' UTR probe in order to exclude potential cross-hybridizations to testis transcripts of other SPGY genes. The 3' UTR probe was prepared by amplifying the last 1644 nucleotides of CT355 (positions 1277-2921) by PCR using the CT355 forward primer 355P1 (5'-GAAACTAAGATTTATCTGCAAGG-3') and a vector primer ([lambda]-gt11rev: 5'-TTGACACCAGACCAACTGGTAATG-3'). Cycling conditions: 25* 1 min at 94oC, 1 min at 55oC, 5 min at 72oC. Final extension was for 10 min at 72oC. Denaturation was for 4 min at 94oC. After hybridization, filters were washed under stringent conditions (2* SSC, 0.1% SDS, 65oC). RT-PCR experiments were performed from cDNA aliquots prepared with the SUPERSCRIPTtm II system of Gibco-BRL from all RNA samples indicated in the text. The LA-1for/LA-1rev primer pair was used for analysis of RNA tissue specificity with the following cycling conditions: 30* 1 min at 94oC, 1 min at 65oC, 1.5 min at 72oC. The final extension was for 5 min at 72oC. Denaturation was for 5 min at 94oC. The length of the RNA amplification product is 161 bp (see Fig. 2 b). As a positive control in all RT-PCR experiments, we used actin-forward (5'-ATCCTGCGTCTGGACCTG-3') and actin-reverse (5'-gctgatccacatctgctg-3'), which are two primers of a human [beta]-actin gene (28 ), under the same cycling conditions. They are expected to amplify an RNA product of 525 bp in each tissue analysed.
Fluorescence in situ hybridization with CT355 on human male metaphase chromosomes was carried out as described by Köhler and Vogt (29 ). Both probes were biotinylated by nick translation after a limited DNase I fragmentation. Hybridization signals were amplified once using a a biotinylated anti-avidin antibody (Vector Laboratories). Fluorescent signals were analysed in a Zeiss Axiophot microscope with an aligned filter set for DAPI (LP450-490, BP365, FT395) and FITC (LP515-565, BP450-490, FT510). The Axiophot was coupled to a cooled charge-coupled device (CCD) camera for further image processing using the software package `Gene JOIN' as described by Ried et al. (30 ).
We thank Weiyun Chen at the Biocomputing Unit of the German Cancer Institute for generous support in sequence analyses with the HUSAR software package. Margaret Delbridge (La Trobe University, Bundoora, Australia) is thanked for the wallaby DNA sample. Luda Diatchenko (Clontech Laboratories, Inc, Palo Alto, CA) is thanked for valuable RNA samples of human male and female tissues. Volker Wetzel (Karlsruhe, Germany) is thanked for valuable ovarian tissue samples. We are indebted to Karl-Heinz Grzeschik (University of Marburg, Germany) for providing us with DNA samples of his human-hamster hybrid cell panel. Wolfgang Hennig (PUDONG, Kranenburg, Germany) is thanked for support in automated sequence analysis. Annemarie Wiegenstein is thanked for excellent photographic assistance and Christine Clayton for critically improving our English grammar and expression. Zhihong Shan is a recipient of a Ph.D. fellowship of the C.C. Wu Cultural & Education Foundation Fund, Hong Kong. This work was supported by a grant from the DLR (Bundesministerium für Bildung und Forschung) to P.H.V. (01KY95074).
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+These authors contributed equally to this work
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