MOLECULAR REPRODUCTION A N D DEVELOPMENT 30173-181 (1991)

Analysis of a Murine Male Germ Cell-Specific Transcript That Encodes a Putative Zinc Finger Protein CHRISTER HOOG,' MARTIN SCHALLING? EVA GRUNDER-BRUNDELL,l AND BERTIL DANEHOLT' Departments of Molecular Genetics and 'Histology, Karolinska Institutet, Stockholm, Sweden

ABSTRACT A cDNA species, corresponding to a gene with testis-specificexpression (TSGA),was isolated from a testis cDNA library. The temporal and spatial expression of TSGA was studied by in situ hybridization as well as RNA filter hybridization. In tissue sections,the TSGA sequence was confined to cells within the seminiferous tubules. For filter hybridization, RNA was isolated from testis of prepubertal rats of different ages as well as from enriched populations of various germ cell types. It was found that TSGA is expressed only in male germ cells and that the steady-state level of TSGA transcripts reaches a maximum during the meiotic and the postmeiotic stages of germ cell development, suggesting a meiotic or postmeiotic function for the encoded protein. TSGA encodes a putative protein having 1,214 amino acids and contains a zinc finger, a structure that previously has been shown to mediate binding to nucleic acids. Key Words: Spermatogenesis, In situ hybridization, Temporal expression

INTRODUCTION Male germ cells in mammals undergo a number of precise structural and functional changes during maturation (Hecht, 1986; Bellve, 1979). The developmental process is initiated as groups of stem cells start to differentiate into spermatocytes. Following meiotic prophase, the germ cells develop into spermatids and spermatozoa. This progressive germ cell maturation process occurs in intimate contact with a somatic cell type, the Sertoli cells, within the seminiferous tubules (Parvinen e t al., 1986). The precise regulation of male germ cell differentiation requires a strict program of stage- and cell-specific gene expression in germ cells a s well as in surrounding somatic cell types. A few testisspecific genes, showing temporally distinct expression patterns during germ cell differentiation, have been characterized and recently reviewed (Willison and Ashworth, 1987; Propst et al., 1988). In addition to these genes, a number of cDNA clones with a germ cellspecific pattern of expression have been described (Thomas et al., 1989). To understand such basic processes as gamete production, genetic recombination, and chromosomal imprinting, it will be important to

0 1991 WILEY-LISS, INC.

reveal the nature of many more of the genes involved as well as the functions and interactions of the corresponding proteins. We describe here the isolation and characterization of a gene, testis-specific gene A (TSGA), th a t is expressed only in male germ cells. This gene is transcribed in meiotic prophase cells as well as in postmeiotic cell types. We show that the protein sequence deduced from a TSGA cDNA clone contains a wellknown sequence motif, a zinc finger, a protein motif that is known to mediate DNA and RNA binding (Miller et al., 1985; Berg, 1988; Evans, 1988).

MATERIALS AND METHODS Isolation of RNA and DNA Total RNA and poly-A+ RNA from various Sprague Dawley rat tissues were isolated a s described by Sambrook et al. (1989). Lambda and plasmid DNA preparations and manipulations were carried out with standard methods (Sambrook et al., 1989). RNAs from specific testicular cell types were obtained according to the following procedure: Testis from 33-42-day-old male rats were decapsulated, and cell suspensions were obtained by trypsinization of the seminiferous tubules (Heyting et al., 1985, 1988). In each cell preparation, 5 x 10' cells were loaded into a n elutriation rotor (Beckman model J-GMIE), provided with a 5 ml elutriation chamber, to yield cell fractions that were sequentially purified in a self-generating Percoll density gradient (Meistrich et al., 1981). The homogeneity of each fraction was determined microscopically. The pachytene spermatocytes and the round haploid cell types were routinely more than 95% homogenous. The Sertoli cell fraction was about 80% pure, the contamination consisting of pachytene spermatocytes and spermatogonia. Total RNA was isolated from these cell fractions as described above.

Received May 1, 1991; accepted June 17, 1991. Martin Schalling's present address is Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 12139. Address reprint requests to Christer Hoog, Department of Molecular Genetics, Doktorsringen ZA, CMB, Karolinska Institutet, Box 60 400, 10401 Stockholm, Sweden.

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quenced on both strands using subclones generated by a n exonuclease/Sl deletion kit (Promega Biotech). DNA fragments with deletions of the appropriate sizes were identified by separation on agarose gels and were then used for dideoxysequencing (Sanger et al., 1977) using Sequenase I1 (USB) after alkali denaturation of the plasmid DNA (Mierendorf and Pfeiffer, 1987). Gaps or ambiguities in the DNA sequence were resolved by using internal sequencing primers. The sequence data were compiled using the GCG program package (Devereux et al., 1984). 5‘-ATGAG(NG)ATGTGGAA(A/G)TT(CiT)CC(NG/T/C) AAG-3’ (positions 1209-1232 in the HOPl sequence), RNA Filter Hybridization whereas the second oligo-nucleotide (OL2) has the sequence 3’-TT(G/A)GT(G/A)CT(T/C)AT(G/A)TT(T/C) Purified total RNA (15 pg) or poly-A+ RNA (0.7 p.8) ACCGT(G/A)AA(G/A)-5’ (positions 1659-1682 in the was denatured and electrophoresed on 2.2 M HOPl sequence). formaldehyde/agarose gels in MOPS buffer according to OL1 was mixed with poly-A+ RNA from either testis Sambrook et al. (1989). The RNA was transfered to or liver and used as a primer for AMV reverse tran- Hybond C-extra filters (Amersham) using a vacuoblot scriptase (Boehringer Mannheim) in a first strand system (Pharmacia). The filters were baked for 2 h r and cDNA reaction, using a commercially available cDNA hybridized overnight at 37°C in a standard hybridizakit (Promega Biotec) according to the manufacturers tion solution (Sambrook e t al., 1990) containing 50% recommendations. After completion of the reaction, formamide and 32Prandom prime-labeled DNA probes 1/50 of the cDNA was amplified (Saiki et al., 1988) with (Feinberg and Vogelstein, 1983). The filters were the two oligonucleotides, OL1 and OL2, as primers, washed twice with 2 x SSC and 0.2% SDS for 15 min at using a gene amplification kit (Perkin Elmer Cetus). room temperature and twice with 0.2 x SSC and 0.5% The mixture was incubated a t 95°C for 5 min and SDS for 30 min at 60°C and then exposed to autoradslowly cooled to 30°C; AmpliTaq DNA polymerase iographic film at -70°C with a n intensifying screen. (Perkin Elmer Cetus) was then added, and the temperature cycling procedure was initiated according to the In Situ Hybridization manufacturer’s recommendations in a Perkin Elmer One sense oligonucleotide (HP4-S, 5’-ACAGTGAACetus DNA Thermal cycler. The initial annealing temperature was slowly raised from 37°C in the first CAAGAGGAAGAAGTCCTTAGAACCATCCAAGATtwo cycles to 45°C in the following two cycles and GGAGATTC-3’, positions 3371-3419 in the TSGA finally to 55°C during the last 30 cycles. The resulting DNA sequence) and one antisense oligonucleotide reaction products were electrophoretically separated on (HP4-AS, 3’-CTGTCACTTGTTCTCCTTCTTCAGGAa 2% agarose gel and a 500-bp-long testis-specific band ATCTTGGTAGGTTCTACCTC-TAA-5’,positions 33703418 in the TSGA DNA sequence) were synthesized was eluted from the gel. The DNA fragment was kinased using polynucle- and then purified on a 12% urea-polyacrylamide gel. otide kinase (New England Biolabs), ligated into a n Both oligonucleotides were labeled at their 3’-end Smal cut pBluescript SK- vector (Stratagene) and using terminal deoxynucleotidyl transferase (IBI) and transformed into X1 lBlue bacterial cells (Stratagene). 35S-dATP(New England Nuclear) to a specific activity Plasmid DNA was prepared using a Quiagen plasmid of -1 x lo9 cpm/pg. The preparation of rat testis sections, hybridization of labeled oligonucleotides to preparation column. The cloned polymerase chain reaction (PCR) insert sections and posthybridization steps were performed as was labeled using a random prime kit (Promega Bio- described previously (Schalling et al., 1988). tech) and used to screen a commercially available rat testis cDNA library in the lambda ZapII vector (StratRESULTS agene), made from poly-A+ RNA isolated from 6Amplification of a Testis-Specific cDNA week-old Sprague Dawley males. A 2.2-kb-long cDNA Sequence clone was isolated (TSGA-3’1,and a 340-bp-long EcoRl In a n attempt to isolate a rat homologue to a yeast fragment from the 5‘-end of this cDNA clone was used meiotic gene, HOPl (Hollingworth et al., 1990), two to rescreen the same library. This resulted in the degenerate oligonucleotide mixtures were used as isolation of a 2.6 kb long partially overlapping cDNA primers in amplification reactions from cDNA (see clone (TSGA-5’). All lambda ZapII clones were conMaterials and Methods). Rat liver and testis mRNA verted into pBluescript SK- clones prior to analysis was amplified separately with the help of the PCR according to the manufacturers recommendations technique (Saiki et al., 1988) a t reduced stringency (Stratagene). conditions. The resulting amplified material was anaDNA Sequencing lyzed on a 2% agarose gel, and showed a 500 bp-long The PCR fragment cloned into a pBSK vector and the amplified DNA band present in testis cDNA but not in two cDNA clones, TSGA-3’ and TSGA-5’, were se- liver cDNA (data not shown).

Isolation of TSGA cDNA Clones Two oligonucleotides were synthesized for the subsequent cloning experiment. They represent two parts of the coding region of the HOPl gene in yeast (Hollingsworth et al., 1990), but the DNA sequences were adjusted to fit the codon choice usage found in rats (Aota et al., 1988). To account for wobbling in the third base position of a codon, the two 24-mers were synthesized as mixtures on a Pharmacia DNA synthesizer. The sequence of the first oligonucleotide (OL1) is

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Fig. 1. Tissue-specificexpression of TSGA. mRNA isolated from a variety of rat (A) or mice (B) tissues were electrophoresed, transfered to nitrocellulose filters, and hybridized with a labelled TSGA probe (4 days exposure; upper panel). The same filters were washed and rehybridized with a probe for p-actin as a positive control (6 hr exposure; lower panel). The sizes of the TSGA and the p-actin transcripts were deduced from a comparison with a n RNA size standard (BRL).

Tissue Distribution of TSGA mRNA To analyze further this tissue-specific amplification pattern, the PCR DNA fragment was purified by gel electrophoresis and cloned into a pBluescript SK (Stratagene) vector. This cloned PCR fragment was then labeled and used to hybridize a rat tissue RNA blot. As can be seen in Figure lA, the probe detects a 4.5 kb poly-A+ RNA species only in testis. After prolonged exposure of the autoradiogram a trace of mRNA of the same size was also detected in several other tissues. A similar profile of tissue-specific expression has been reported for other testis-specific transcripts, although the significance of this low level of expression in other tissues is not understood (Rappold et al., 1987; Wolgemuth et al., 1987). Lacking any formal classification system, we designated the identified gene TSGA (for testis-specific gene A). To establish whether this testisspecific expression pattern is evolutionarily conserved, a mouse RNA blot was hybridized with the same TSGA probe. It can be seen in Figure 1B that a similar testis-specific expression is observed for the TSGA sequence also in mice. Temporal and Spatial Distribution of TSGA mRNA in Testis To understand the role of the testis-specific gene A and its transcript during spermatogenesis, it is impor-

tant to define the spatial and temporal expression pattern of the TSGA transcript in testis. Spermatogenesis is initiated synchronously in all tubules in the seminiferous epithelium 4 days after birth in rats, when a subpopulation of spermatogonial stem cells starts to differentiate synchronously (Clermont and Perey, 1957). It is therefore possible, by isolating RNA from testis of prepubertal rats of different ages, to determine the earliest stage at which transcription occurs. It can be seen in Figure 2 that the number of RNA molecules detected by the TSGA probe reach a maximum in testis 22 days after birth. This coincides with the accumulation of late meiotic cells (pachytene spermatocytes), which first appear at about day 15 after birth in rat testis (Clermont and Perey, 1957). Most cell types within testis represent germ cells in various stages of development, i.e., spermatogonia, spermatocytes or spermatids. It is, however, important to demonstrate directly that the transcription observed takes place in the germ cells and not in somatic cells in testis, e.g., in the Sertoli cells, in Leydig cells, or in various peritubular cells. To define the cellular localization of the TSGA mRNA within testis, two techniques were used, cellular fractionation and in situ hybridization. The result of the cellular fractionation experiment is shown in Figure 3. Various testicular cell types were purified according to size and density by a

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Fig. 2. Developmental expression of TSGA. Total RNA (15kg) from prepubertal and mature rats were isolated at the indicated day after birth, electrophoresed, transfered to nitrocellulose filters, and hybridized with a labeled TSGA probe. Total RNA (15 kg) from liver was isolated from rats at 42 days of age. The integrity of the RNA was verified before transfer by visualizing the ribosomal RNA content with ethidium bromide (data not shown).

combination of both centrifugal elutriation and Percoll gradient sedimentation (see Materials and Methods), and the RNA from each cell type was prepared. As is seen in Figure 3, the TSGA message is abundant in meiotic (pachytene spermatocytes) and in postmeiotic (round haploid spermatids) cell types. No expression of the TSGA message was observed in Sertoli cells, in epididymis, or in liver (the weak signal observed in the Sertoli lane most likely originate from the pachytene spermatocytes that contaminate this cell fraction). In situ hybridization has the advantage over the cell fractionation procedure th at the cellular interactions within the seminiferous epithelium are not destroyed. Hybridization signals can therefore be directly monitored within the appropriate histological context. When rat germ cells differentiate in the seminiferous tubules, they move from the periphery of the tubule towards the lumen. As a result, a series of concentric rings of germ cells of the same developmental stage will appear in each tubular cross section. To analyze the spatial distribution of the TSGA RNA, a sense and a n antisense oligonucleotide probe were made, based on the TSGA DNA sequence (see below). Both oligonucleotides were purified by gelelectrophoresis, labeled using 35S-dATP and applied to rat testis tissue sections. As can be seen in Figure 4C and D, no specific hybridization signal is detected with the sense TSGA oligonucleotide probe. However, a strong and reproducible signal is seen within the tubules using the antisense TSGA oligonucleotide probe (Fig. 4A, B). When the section viewed by bright-field illumination (Fig. 4A) is carefully compared with the same section viewed by dark-field illumination (Fig. 4B), it can be seen that the concentric rings of label within the tubules represent early and late meiotic cell types as well a s post-

Fig. 3. Expression of TSGA in populations of isolated male adult rat germ cells. Total RNA (15 kg) from Sertoli cells (Sertoli), pachytene spermatocytes (pachytene), round haploid cell types (haploid), as well as from three different tissues, were electrophoresed, transfered to nitrocellulose filters, and hybridized with a labeled TSGA probe. The integrity of the ribosomal RNA content was verified before transfer (data not shown).

meiotic germ cell types. No signal is apparent in peritubular cell types, Sertoli cells or in the spermatogonial cell types closest to the basal lamina of the tubule.

Sequence Analysis of the TSGA cDNA Clone The PCR-derived TSGA clone was used to screen a rat testis cDNA library and a 2.2-kb-long cDNA clone (TSGA-3’) was isolated. To extend this sequence, a 340-bp-long EcoRl fragment from the 5‘-end of this cDNA clone was prepared, labeled, and used to rescreen the same cDNA library. A 2.6-kb-long cDNA clone was now isolated (TSGA-5’) and shown to contain a 340bp-long overlap at its 3’-end with the TSGA-3’ clone. Both cDNA clones were completely DNA sequenced, and the assembled DNA sequence, containing 4505 bp, probably represents a full-length cDNA clone, as this size closely matches the size of the TSGA mRNA molecules detected in RNA blots. The original PCRderived TSGA clone is localized in between positions 3346 and 3845 in this cDNA sequence. A single long open reading frame was observed within the TSGA cDNA sequence and the complete 1,214-amino-acids-long sequence is shown in Figure 5, along with the corresponding nucleotide sequence. This open reading frame was preceded by a 299-bp-long 5’-untranslated region, having stop codons in all three reading frames and followed by a 564-bp-long 3‘untranslated region (see legend to Fig. 5). The predicted TSGA amino acid sequence corresponds to a protein with a calculated molecular mass of 135kD . An

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Fig. 4. In situ hybridization to adult rat testis sections. Antisense (A, B) or sense (C, D)oligonucleotide probes devised from the TSGA cDNA sequence were labeled with 35S-dATPand applied to rat testis sections. Sections were viewed by bright-field (A, C) or dark-field (B, D) illumination. Bar = 50 km.

analysis of the hydrophobicity of the predicted protein sequence shows no sign of long hydrophobic stretches, indicating the existence of leader sequences or of transmembrane domains. A comparison of the TSGA cDNA and amino acid sequence to the EMBL DNA database (release nr. 24.0) and to the Swissprot amino acid sequence database (release nr. 15) did, however, reveal one short protein sequence motif that TSGA shared with several proteins

included in the Swissprot database. This amino acid sequence, found at positions 546-571 in the TSGA amino acid sequence, contains two pairs of cysteines, resembling the zinc finger motif known to mediate nucleic acid binding (Miller et al., 1985; Berg, 1988; Evans, 1988). The putative zinc finger motif in the TSGA cDNA clone has been aligned in Figure 6 with similar motifs found in DNA- and RNA-binding proteins (see Discussion).

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CLONING OF THE TESTIS-SPECIFIC GENE A DISCUSSION Spermatogenesis is a complex and time-consuming process, which lasts for about 40 days in rats (Clermont and Perey, 1957) and for as long as 63 days in humans (Willison and Ashworth, 1987). During this period, the initially appearing spermatogonia develops into spermatocytes and then further into spermatids and mature sperms, each cell type being functionally and morphologically distinct. Apart from all the structural components involved, regulatory molecules and signals have to operate during the male germ cell development to secure a temporal control of this complex process. Genes that respond to such temporal control mechanisms have been found and recently reviewed (Erickson, 1990). We describe here a gene, designated TSGA, which is under spatial as well as temporal gene control. TSGA is expressed in a male germ cell-specific manner in both rats and mice, predominantly during the meiotic and the postmeiotic stages of germ cell development. A similar expression pattern has been reported for a group of genes specifically expressed during male germ cell development. This group include a putative transcription factor (Wolgemuth et al., 1987), a putative translational initiation factor (Leroy et al., 19891, as well as testis-specific forms of lactate dehydrogenase (Thomas et al., 1990), tubulin (Villasante et al., 1986), cytochrome c (Hake et al., 19901, phosphoglycerate kinase 2 (Gold et al., 1983), and histone H2B (Kim et al., 1987). The proteins encoded by this group of genes have all been suggested to be involved in activities related to the late meiotic prophase cells or to haploid cell types; e.g. the testis-specific histone variant has been shown to replace histone H2B during middle pachytene in spermatocytes (Meistrich et al., 19851, whereas the testis-specific PGK-2 protein does not appear until in round haploid spermatids (Gold et al., 1983). Based on the similarities in RNA expression

Fig. 5. Nucleotide sequence and deduced amino acid sequence from TSGA cDNA clones TSGA-5’ and TSGA-3’. TSGA-5’ starts at position 1 and ends at position 2620, whereas TSGA-3‘ starts a t position 2280 and continues to position 4505. The complete open reading frame found is shown, starting at the first in-frame methionine. No other significant open reading frame has been found. The 5’-untranslated sequence (299 bp) and the 3’-untranslated sequence (564 bp) are not included, but the complete DNA sequence has been submitted to the EMBL DNA databank (accession number X59993). The cysteines within the cysteine rich domain, described in the text as part of a potential zinc finger, are encircled. The PCR-derived cDNA sequence starts at position 3346 and ends at position 3845. The OL1 primer makes up the first 24 bases of this PCR-derived cDNA sequence, whereas OL2 makes up the last 24 bases of this sequence. The two oligonucleotides, OL1 and OL2 (see Materials and Methods), was used in the cDNA amplification reaction, originally in a n attempt to isolate a yeast meiotic gene, HOPl (Hollingworth et al., 1990). No homology at the protein level, however, is found between the TSGA and the HOPl genes. The OL1 24-mer starts at the first position of a methionine codon in the HOPl sequence, whereas the OL1 sequence, as part of the TSGA PCR sequence, starts a t the second position of a methionine codon (position 3346). Thus the TSGA gene is not likely to be functionally related to the HOP1 gene.

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Fig. 6. Comparison of the putative zinc finger in the TSGA amino acid sequence to other zinc finger domains. The regularly spaced cysteines in the TSGA amino acid sequence were aligned with similar domains in Gal4 (Laughon and Gesteland, 1984), Pprl (Kammerer et al., 19841, Leu3 (Friden and Schimmel, 1987), XPAC (Tanaka et al., 1990), uvrA (Husain et al., 1986), HOPl (Hollingsworth et al., 19901, elF-2p in yeast (Donahue et al., 1988), and the elF-2p protein in humans (Pathak et al., 1988).

pattern, it seems likely that the TSGA-encoded protein also performs a cell function related to the late meiotic or postmeiotic events. The TSGA-encoded protein comprises 1,214 amino acids and contains a well-known amino acid sequence motif, a cluster of regularly spaced pairs of cysteines, forming a potential zinc finger (Evans, 1988). It has been shown that a single zinc finger will bind zinc (Parraga et al., 1988) and mediate DNA binding (Johnston and Dover, 1987) as well as RNA binding (Donahue et al., 1988). In Figure 6, the TSGA-encoded zinc finger motif is compared with DNA- and RNAbinding proteins having only one such zinc finger. Gal4 (Laughon and Gesteland, 1984), Pprl (Kammerer et al., 1984), and Leu3 (Friden and Schimmel, 1987) are examples of three yeast transcription factors that bind to DNA, whereas the uvrA (Husain et al., 1986)and the XPAC (Tanaka et al., 1990) proteins, represent two DNA repair enzymes. The yeast HOPl gene product probably constitutes a structural protein in the synaptonemal complex, which is known to mediate pairing of homologous chromosomes during meiosis (Hollingsworth et al., 1990). It was shown that a single amino acid mutation in the HOPl protein, replacing one cysteine within the zinc finger with a serine, completely abolishes the formation of the synaptonemal complex and DNA binding. Two RNA-binding translational initiation factors, the human (Pathak et al., 1988) and the yeast elF-2P (Donahue et al., 1988), also contain a single zinc finger. In the yeast elF-26 protein, the zinc finger has been shown to be of crucial importance for correct initiation of translation, suggesting an interaction with RNA (Donahue et al., 1988). We conclude, based on these structural comparisons, that the TSGA protein could interact with DNA or RNA and have a structural, enzymatic, or regulatory activity during late meiotic prophase or during spermiogenesis. One obvious suggestion would be that the TSGAencoded protein represents a murine analogue to the structural protein encoded by HOP1, as the cloning strategy used should favor the isolation of a HOP1-type

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gene. However, the TSGA sequence as a whole does not show a similarity to the HOPl gene. In addition, and more importantly, the DNA sequences corresponding to the primers used are not read in the same reading frame in HOPl and TSGA, resulting in quite different amino acid sequences also when these nucleic acid sequences are being translated (see legend to Fig. 6). Therefore, although TSGA and HOPl display local DNA sequence similarities and both encode zinc finger motifs, they are probably functionally unrelated. We have noted that TSGA shows a temporal expression pattern almost identical to those of two other testis-specific genes, Hox-1.4, a regulator of transcription (Wolgemuth et al., 1987), and DlPasl, a gene encoding a protein homologous to a component of the mammalian translation initiation machinery, elF-4A (Leroy et al., 1989). It has been suggested that the DlPasl protein might be part of the temporal translation control system that exists in male germ cells (Leroy et al., 1989). In this context it is interesting to recall that the two translation initiation factors mentioned above, the human and the yeast elF-2p protein, each contains a single zinc finger (Pathak et al., 1988; Donahue et al., 1988). The expression pattern of the TSGA gene and the presence of a zinc finger in the putative gene product suggest the possibility that the TSGA protein could represent a component of a germ cell-specific transcriptional or translation control apparatus.

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