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Expression of Testicular Germ Cell Genes Identified by Differential Display Analysis

NEELAKANTA RAVINDRANATH

MARTIN DYM

From the ^* Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington; and Department of Cell Biology, Georgetown University Medical Center, Washington, District of Columbia.

Correspondence to: Michael D. Griswold, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660 (e-mail: griswold{at}mail.wsu.edu).

Received for publication October 22, 2002; accepted for publication November 22, 2002.

	Abstract

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Using differential display reverse transcriptase–polymerase chain reaction (DDRT-PCR) we identified transcripts encoding for the RNA helicase mDEAH9, Ran binding protein 5 (RanBP5), and 3 novel complementary DNAs designated GC3, GC12, and GC14 in developing testicular germ cells. Sources of RNA for the initial DDRT-PCR screen were purified mouse type A spermatogonia, adult mouse wild-type testis, and W/W^v mutant mouse testis. We identified cDNA fragments for mDEAH9, RanBP5, GC3, GC12, and GC14 in testis and type A spermatogonia samples from wild-type mice, but not in samples from the W/W^v mouse testis. These same transcripts were absent in Northern blots of testis RNA from mice treated with busulfan 30 days prior, but were present in testis RNA from wild-type mice at 5, 15, 25, and 40 days of age. The mDEAH9 gene was expressed in many tissues, whereas RanBP5 and GC12 genes were expressed predominantly in the testis with much lower expression in other tissues. The expression of GC3 and GC14 were limited to the testis as evidenced by Northern blot and RT-PCR analyses. The mDEAH9 transcript was not detected in cultured interstitial cells but was found at low levels in cultured immature Sertoli cells, whereas the RanBP5, GC3, GC12, and GC14 transcripts were not detected in either cultured testicular interstitial cells or cultured Sertoli cells. RT-PCR analyses of isolated spermatogonia, pachytene spermatocytes, and round spermatids revealed that mDEAH9, RanBP5, GC3, GC12, and GC14 genes were expressed in all 3 cellular populations. In situ hybridization analyses of testis samples from 40-day-old mice localized expression of mDEAH9, RanBP5, GC3, GC12, and GC14 to the seminiferous tubules. RanBP5 expression appeared to be regulated during the cycle of the seminiferous epithelium, with the highest expression in stages III through VII. Expression of GC14 was greatest in the meiotic germ cell populations.

Differential display reverse transcriptase–polymerase chain reaction (DDRT-PCR) was developed by Liang and Pardee (1992), and serves as an effective method for identifying differentially expressed genes (Bauer, 1993; Liang, 1993). This PCR-based technique allows for the preparation and identification of a subpopulation of complementary DNAs (cDNAs) from specific cells and tissues. Unlike subtractive and differential hybridization techniques, DDRT-PCR allows direct comparisons between different cell and tissue types, multiple sample comparisons, and easier detection of rare messages. In the present study, genes uniquely expressed in germ cells were identified using DDRT-PCR. In order to identify messenger RNAs (mRNAs) expressed in type A spermatogonia, and which are unique to these testicular germ cells, we compared cDNAs that were derived from isolated type A spermatogonia, wild-type adult testes, and germ cell–deficient W/W^v mutant testes in mice. This procedure resulted in the identification of several transcripts, including an RNA helicase, mDEAH9; Ran-binding protein 5 (RanBP5); and 3 novel cDNAs, GC3, GC12, and GC14.

	Materials and Methods

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Animals

Male W/W^v mutant mice (WCB6F1) and male wild-type litter-mates at 6 weeks of age were purchased from Jackson Laboratory (Bar Harbor, Me). Balb/c mice were purchased from the university stocks at the Eastlick Vivarium at Washington Sate University or at Georgetown University. Male Balb/c mice of 4–6 weeks of age were injected i.p. with 40 mg/kg busulfan (Sigma Chemical Company, St Louis, Mo) as previously described (Viguier-Martinez et al, 1984; Bucci et al, 1987). Testes were harvested 30–35 days later and the total RNA was extracted as described below.

Cell Isolation and RNA Isolation

Sertoli cells and interstitial cells were isolated from 15- to 30day-old male Balb/c mice as previously described (Karl, 1990) and maintained in serum-free Dulbecco modified Eagle medium for 4 days at 35°C. Type A spermatogonial cells were isolated from 7-day-old Balb/c mice, whereas pachytene spermatocytes and round spermatids were isolated from adult mice as described by Bellve and colleagues (1977) with minor modifications (Dym et al, 1995). Total RNA was extracted from various mouse tissues from 40-day-old mice using the acid guanidinium-phenol-chloroform method (Chomczynski, 1987). Total RNA was purified from cultured Sertoli cells, cultured interstitial cells, and isolated germ cell populations using TRIZOL (Gibco BRL, Grand Island, NY) according to the manufacturer's specifications. Poly(A)+ enriched RNA was isolated by a single pass over an oligo(dT) cellulose column.

DDRT-PCR

Differential display was performed using the Hieroglyph Kit (Genomyx, Fullerton, Calif). Reverse transcriptase reactions were performed on mouse tissues using 200 ng of total RNA isolated from W/W^v testes, wild-type testes, and isolated type A spermatogonial cells. RT reactions (20 µL) were performed in duplicate at 42°C for 60 minutes using two different RT enzymes, avian myeloblastosis virus (AMV), and Superscript II (both from Gibco BRL, with an oligo(dT) primer (200 nM); 5'-ACGACTCACTATAGGGC(dT₁₂)MN-3' (where M = A, G, or C; and N = A, T, G, or C) in a single-strength buffer according to the manufacturer's specifications for each RT enzyme. The underlined sequence of the oligo(dT) primer is half of the T7 promoter sequence. Complementary DNA samples (2 µL) were amplified by PCR in duplicate 20-µL reactions containing single-strength buffer: 1.5 mM MgCl₂, 20 µM deoxynucleotide triphosphates (dNTPs), the 3' oligo(dT) primer (0.2 µm), and one arbitrary 5' primer (0.2 µm; listed below as ARP1–4), 0.125 µCi (alpha-³³P)deoxyadenosine triphosphate (dATP), and 1 U AmpliTaqR (Perkin Elmer, Boston, Mass). Reactions were carried out at 95°C for 2 minutes, then 4 cycles at 94°C for 30 seconds, 46°C for 30 seconds, and 72°C for 2 minutes; followed by 26 cycles at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 2 minutes; and a final extension at 72°C for 7 minutes. Negative controls were included in which RNA, RT, or cDNA was replaced with deionized water. Samples were analyzed by denaturing polyacrylamide (4.5% LR-Optimized HR-1000, Genomyx) gel electrophoresis using a GenomyxLR GX100 DNA Sequencer (Genomyx). Complementary DNA fragments were visualized with autoradiography using Kodak Biomax film (VWR, Seattle, Wash). Putative germ cellspecific cDNA fragments were excised from the gel and incubated in 100 µL of deionized water overnight at room temperature. Complementary DNA fragments were reamplified by PCR in 100- µL reactions containing single-strength buffer (20 µM dNTPs, 1.5 mM MgCl₂, 0.2 µM full-length T7 primer, 0.2 µM full-length M13 reverse [M13r], 0.5 U Taq DNA Polymerase [Gibco BRL], and 40 µL cDNA) for 30 cycles at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 2 minutes, and a final extension at 72°C for 7 minutes. PCR products were then cloned into p-Gem-T Easy Vector (Promega, Madison, Wis) using the standard protocol. The arbitrary primers used in the DDRT-PCR were as follows: ARP1, 5'-ACAATTTCACACAGGACGACTCCAAG-3'; ARP2, 5'-ACAATTTCACACAGGAGCTAGCATGG-3'; ARP3, 5'-ACAATTTCACACAGGAGACCATTGCA-3'; and ARP4, 5'-ACAATTTCACACAGGAGCTAGCAGAC-3'. The underlined sequences in the arbitrary primer is half of the M13r (-48) sequence.

DNA Sequencing and Synthesis

Oligonucleotide primers SP6END (5'-AGCTATGCATCGAACGCGTT-3') and T7END (5'-TTGGACCCGACGTCGCA-3') were used to sequence the DDRT-PCR clones. The amplified differential display fragments contain the T7 and M13r sequences, therefore primers SP6END and T7END, were designed that bind to the polylinker region between the SP6 and T7 promoters of the p-Gem-T Easy Vector, respectively. DNA sequencing analysis was carried out by the Laboratory of Bioanalysis and Biotechnology I (LBBI) at Washington State University. Sequences of cloned DDRT-PCR fragments were analyzed using the GenBank/European Molecular Biology Laboratory database using the basic local alignment search tool (BLAST) for homology identification. Full-length cDNAs were submitted to GenBank using the Bankit internet program at the National Center for Biotechnology Information.

Northern Blot Analysis

Poly(A)+ enriched RNA samples (6–8 µg) from specific organs, cultured Sertoli cells, and cultured interstitial cells were fractioned in a 1% agarose/formaldehyde gel and transferred to a nylon membrane (Hybond-N, Amersham Pharmacia, Arlington Heights, Ill) and UV cross-linked with the UV Stratagene 1800 (Stratagene, La Jolla, Calif). The DDRT-PCR cDNA fragments were radiolabeled with (alpha-³²P)dATP using a Rad Prime DNA Labeling Kit (Gibco BRL). Northern blots were hybridized overnight at 42°C with labeled cDNA probes in 50% formamide, 50 mM NaH₂PO₄, 5x Denhart solution, 5x saline-sodium citrate (SSC), 0.1% sodium dodecyl sulfate (SDS), 2% dextran, and 1 mM ethylenediamine tetraacetic acid (EDTA). Following hybridization, blots were washed in 2x SSC/0.1% SDS for 10 minutes at room temperature, 1x SSC/0.1% SDS for 30 minutes at 65°C, and 0.1% SSC/0.1% SDS for 30 minutes at 65°C. After blots were washed, they were placed in a phosphor screen cassette (Molecular Dynamics, Sunnyvale, Calif) and allowed to expose a phosphor screen for 8 to 12 hours. The signals were detected using a Molecular Dynamics PhosphorImager 445 SI and ImageQuant software (Molecular Dynamics). Ribosomal protein S2 cDNA probe was used as a control for loading RNA (Mukherjee et al, 1996).

5' Rapid Amplification of cDNA Ends

5' Rapid amplification of cDNA ends (RACE) was performed by using the 5' RACE System version 2.0 (18374-058; Gibco BRL). Briefly, first-strand cDNAs were synthesized in 25-µL reactions containing 20 mM Tris pH 8.4, 50 mM KCl, 2.5 mM MgCl₂, 10 mM dithiothreiotol (DTT), 100 nM gene-specific primer (GSP, see below), 400 µM dNTPs, 3 µg of total testis RNA from 40-day-old mice, and 200 U Superscript II RT incubated at 50°C for 60 minutes. Following the 60-minute incubation, the cDNA reactions were heated to 70°C for 10 minutes then cooled to 37°C. One microliter of RNase mix was added to the reaction, and then incubated for 30 minutes at 37°C. The cDNAs were then purified on GlassMax spin columns by adding 120 µL of binding buffer to the cDNA reaction mixtures, and then transferred to the GlassMax spin columns and centrifuged at 13 000 x g for 20 seconds. The cartridges were washed 4 times with 0.4 mL of cold wash buffer and twice with cold 70% ethanol. Complementary DNA was eluted from the cartridge with 50 µL of distilled water preheated to 65°C. Purified cDNAs were then tailed with terminal deoxynucleotidyl transferase (TdT) in a reaction containing 10 mM Tris pH 8.4, 25 mM KCl, 1.5 mM MgCl₂, 200 µM dCTP, 10 µL purified cDNA, and 1 µL TdT for 10 minutes at 37°C. The cDNA fragments were amplified by PCR using 20 mM Tris pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 200 µM dNTP, 400 nM nested GSP, 400 nM abridged anchored primer, 5 µL tailed cDNA, and 0.5 U Taq for 35 cycles at 94°C for 30 seconds, 55°C for 1 minute, 72°C for 4 minutes, and a final extension at 72°C for 10 minutes.

PCR products were purified and visualized on a 1% agarose gel, cloned into p-Gem-T Easy Vector, and sequenced. The procedure was repeated with new primers until the full-length cDNA was sequenced.

In order to verify the sequence, reverse primers (sense) were generated following the same protocol that was used in the RT-PCR process. Antisense primers for 72A were as follows: 72A31, 5'-TTAAAACATGAAAGGAATCAACGACA-3'; 72A32, 5'-CTCATCAGTTACTGCTCTGAGGTT-3'; 72A33, 5'-CAAGTCATAGTAGGGGACAAACTT-3'; 72A34, 5'-GGTCTTCCATGGTCTGAAGCAAA-3'; 72A35, 5'-GAGGAATCGTCTGTGCGATGA-3'; 72A36, 5'-GTTTCCTCCGCCTGT-3'; and 72A37, 5'-GTTTCCGCACCACATTGTCGG-3'. Sense primers for 72A were 72A51, 5'-TCATAGACCTGCAGAAACGTCAGGA-3'; 72A52, 5'-TACGGAGGGTCGAGGAGTTTC-3'; 72A53, 5'-TCGTCGGTGAACCCTGTTAGAAGT-3'; and 72A54, 5'-CTTTGATTCAGTCAGCTCAC-3'.

Antisense primers for GC3 were GC3R, 5'-CAGCTAGTTGTACCTCCCCAG-3'; GC3R1, 5'-ATGTTGGCTGCAGCCTGCAG-3'; GC3R2, 5'-CCGGTCTGTAACGTTCACGTC-3'; GC3R3, 5'-TGTACCCAGTGAGGTACCTGG-3'; and GC3R4, 5'-GCTCAGACTGTGGCCCTTCACC-3'. Sense primers for GC3 were GC3F1, 5'-CTGCCAACTGCCTAAGAGGTGGAG-3'; GC3F2, 5'-CACCACCATAGCCGCCAAG-3'; GC3F3, 5'-GGATGGCCTTGTACCATGTTCAC-3'; and GC3F4, 5'-CACAGATGCAGTTGACGAGCTG-3'.

Antisense primers for GC12 were GC12R, 5'-GAGATGGTGAGTCTGCTTCATAC-3'; GC12R1, 5'-GTGACAGACACTGGAGGTTGCTC-3'; GC12R2, 5'-CTCTCACTACTGCAACCTTGTGGC-3'; GC12R3, 5'-CGATCTTGTTCTGGTCCGTGAC-3'; and GC12R4, 5'-CGTTTCCACGTGACTTCTTCTTGG-3'. Sense primers for GC12 were GC12F, 5'-CCGAGAAGTAACCAGTGCATC-3'; GC12F1, 5'-GTGACCCGAGAGTGAGCGAAGC-3'; GC12F2, 5'-GAGTAGACTTGTCGGATGAGGAG-3'; and GC- 12F3, 5'-GCTTCCTCAGTCTGATAGCAGTG-3'.

Antisense primers for GC14 were GC14R, 5'-GTACAGTTCACACAGGTCGATG-3'; GC14R1, 5'-CCAGAGCTTCAAGGCTTCTTAGC-3'; GC14R2, 5'-GTCTTGCCAACAGCACAGCAG-3'; and GC14R3, 5'-CGCTGTCATGGTCCCTCCAGC-3'.

Sense primers for GC14 were GC14F, 5'-GAACCCATACCTTCTAAGACAT-3'; GC14F1, 5'-CTTCTGCTGGAGGGACCATGA-3'; GC14F2, 5'-GCTCAAGAGAAGACAGAAGCTCCAGC-3'; and GC14F3, 5'-CGCTGTCATGGTCCCTCCAGC-3'.

In Situ Hybridization Analysis

Riboprobes for in situ hybridization analyses were generated from specific cDNA sequences subcloned into the p-Gem-T Easy Vector. The RanBP5 cDNA fragment was generated from the 5' RACE PCR corresponding to nucleotides 535–2400. The cDNA fragment of mDEAH9 was a PCR product generated using gene-specific primers 104A31 and 104A52 (5'-GGGCGTACACATCCTGTTGAGAT-3') corresponding to nucleotides 1400–3200. The cDNA fragment of GC3 was a PCR product generated using gene-specific primers for GC3F1 and GC3R, corresponding to nucleotides 7–1170. The cDNA fragment of GC12 was a PCR product generated using gene-specific primers GC12F1 and GC12R4, corresponding to nucleotides 3–1662. The cDNA fragment of GC14 was a PCR product generated using gene-specific primers GC14F1 and GC14R, corresponding to nucleotides 668–1256. The cloned cDNA inserts were amplified by PCR from the vector using conditions described above with 400 nM SP6 promoter primer, 400 nM T7 promoter primer, and 0.5 ng of plasmid to produce a template for in vitro transcription. The PCR mixture was treated with 1 µL of 20 mg/mL proteinase K for 10 minutes at room temperature, extracted with 1 volume of phenol, then precipitated in 0.1 volume of 3 M sodium acetate and 2.5 volumes of 100% ethanol. Antisense and sense probes were produced in reactions consisting of 10 µL of (alpha-³³P)UTP (NEG 607H; NEN, Boston, Mass); 1 µL (20 units) of RNAsin (Promega); 50 units T7 or SP6 RNA polymerase (Gibco BRL); 1x transcription buffer; 10 mM DTT; 2.5 mM each rATP, rGTP, and r CTP; and 25 ng of template according to the manufacturer's specifications. In situ hybridization analyses were performed on testis samples from 40-day-old mice as described by Lok et al (2000). Slides were hybridized overnight at 52°C, dipped in Kodak emulsion NTB-2 (VWR), and exposed for 7 to 21 days at room temperature.

RT-PCR Analysis

For RT-PCR, 1 µg each of total RNA from 40-day-old mice testis, spleen, skeletal muscle, brain, kidney, heart, liver, lung, ovary, isolated type A spermatogonia, pachytene spermatocytes, round spermatids, and W/W^v mutant testes were reverse transcribed in a 20-µL reaction at 42°C for 60 minutes using 0.2 units of Superscript II (Gibco BRL) and 50 ng of oligo(dT) primer in single-strength cDNA buffer according to the manufacturer's specifications. These products were then amplified by PCR in a reaction volume of 50 µL containing 1 µL of the RT reaction, single-strength buffer, 20 µM dNTPs, 1.5 mM MgCl₂, 400 nM antisense gene-specific primer, 400 nM sense gene-specific primer, and 0.5 unit of Taq DNA polymerase (Gibco BRL) for 30 cycles at 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute, and a final extension at 72°C for 7 minutes. Gene-specific primers for mDEAH9 were 104A5, 5'-CATTATTGCCAAACTTCAATCCA-3' and 104A3, 5'-AGTCAGTGCCTCTGTATGGA-3'; for RanBP5 the primers were 72A51 and 72A31; for GC3 they were GC3R and GC3F2; for GC12 they were GC12R and GC12F; for GC14 they were GC14R and GC14F; and for ubiquitin they were UB-1, 5'-AGTCCACCCTGCACCTGGTTCTCCG-3' and UB-2, 5'-CCTCAAGCGCAGGACCAAGTGCAGAG-3'. PCR products (10 µL) were separated on a 1.0% agarose gel/0.5% TAE buffer and stained with ethidium bromide for visualization. Amplified cDNA fragments were extracted from the gel, cloned into a p-Gem-T Easy Vector, and sequenced for verification of correct sequence.

	Results

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DDRT-PCR

Differential display was used to identify genes expressed specifically in germ cells in the testis. We identified genes that were expressed in type A spermatogonia but not in somatic cells in the testis by comparing a cDNA subpopulation from isolated type A spermatogonia with those from W/W^v mouse testes. The W/W^v mouse testis has been characterized as germ cell-deficient (Mintz, 1957; Nocka, 1990). Genes expressed in both type A spermatogonia and wild-type testis samples but not expressed in the W/W^v testis samples represented putative germ cell-expressed mRNAs. The cDNA fragments from the DDRT-PCR gel were selected for further analysis if they were absent in the W/W^v testis samples and present in type A spermatogonia and wild-type testes samples; and consistent in both AMV and Superscript II RT reactions, duplicate PCRs, and multiple samples. Representative autoradiograms of DDRT-PCR gels are illustrated in Figure 1A and B. Figure 1A depicts a standard autoradiogram with duplicated AMV (A1 and A2) and Superscript II (S1 and S2) samples across the gel. Complementary DNA fragments range in size from a few hundred base pair (bp) at the bottom of the gel to more than 3000 bp at the top of the gel. Figure 1B is an enlarged area of the A1 and S1 regions of 2 fragments, mDEAH9 and RanBP5, which represent the expression of putative germ cell-specific genes within the testis. Only mDEAH9 and RanBP5 autoradiographs are illustrated because GC3, GC12, and GC14 autoradiographs could not be resolved in the digital images. Complementary DNA fragments were excised from the spermatogonia and wild-type testis sample lanes, reamplified using PCR, and then cloned and sequenced. The cDNA inserts were used to search the GenBank database using BLAST. Two of the cloned cDNA inserts showed identical or high similarities to several reported sequences in the GenBank database, including a 481 bp sequence that matched the mouse RNA helicase m-DEAH9 (accession number AF017153), a 1524 bp sequence that had 93% similarity to the human RanBP5 sequence (accession number Y08890). In addition, three novel cDNAs (GC3, 479 bp; GC12, 790 bp; and GC14, 338 bp) matched no identified gene sequences reported in GenBank, but they did match several reported expressed sequence tags (ESTs) from the testis.

View larger version (66K): [in this window] [in a new window]   Figure 1. Illustration of DDRT-PCR autoradiograms. (A) Illustration of a typical full-length autoradiogram of the DDRT-PCR gel. Samples from each duplicate RT reaction are labeled A1 and A2 for AMV, and S1 and S2 for Superscript II. (B) Illustration of two enlarged regions representing cDNA candidates RNA helicase and RanBP5. Each lane was loaded with 7 µL of the PCR reaction mixture from samples. W indicates W/Wv testis; A, purified spermatogonia; N, adult wild-type testis. Arrow indicates location of the RNA helicase and the RanBP5 cDNAs.

5' RACE

To determine the full-length sequence of the cDNAs reported herein, 5' RACE PCR was performed by designing primers from the original DDRT-PCR fragment. The mouse RanBP5 mRNA (accession number AF294327) is 5013 bases in length excluding the poly(A) tail. The resulting RanBP5 mRNA encodes a protein of 1098 amino acids. The deduced protein sequence of the mouse RanBP5 has 98% sequence similarity to human RanBP5.

GC3 (accession number AF294328) is 1388 bp excluding the poly(A) tail and contains an open reading frame (ORF) encoding a 297 amino acid fragment that has 44% similarity to a human hypothetical protein (accession number AK001137) that contains ankyrin repeat motifs. GC12 (accession number AF294329) is 1728 bp excluding the poly(A) tail and contains an ORF encoding a 462 amino acid fragment, which has 98% nucleotide and 76% amino acid sequence similarity to a human hypothetical protein (accession number D26018). GC14 (accession number AY026045) is 1266 bp in length excluding the poly(A) tail and contains an ORF encoding a 166 amino acid fragment. Neither the nucleotide nor the fragment sequences for GC14 have homology to any sequence in the GenBank database.

Northern Blots

To verify that the cDNA fragments isolated from the DDRT-PCR gel were expressed only in germ cells and not in somatic cells in the testis, Northern blot analyses were carried out on poly(A)+-enriched RNA from total testes of 5-, 15-, 25-, and 40-day-old wild-type mice and 60-day-old mice that had been treated with busulfan 30 days previously. DDRT-PCR cDNA inserts for RNA helicase, RanBP5, GC3, GC12, and GC14 were used to generate random-primed cDNA probes for Northern blot analyses as described in "Materials and Methods." Total testis RNA from 5-day-old mice were used as a source of poly(A)+-enriched RNA to verify the expression of the DDRT-PCR clones in spermatogonia. The testis sample from 15-day-old mice coincides with the first meiotic division during spermatogenesis. The testis samples from 25-day-old mice coincides with the formation of haploid spermatids, and the 40-day-old testis sample represents sexually mature adult mice (McCarrey, 1993). Likewise, to verify that the mDEAH9, RanBP5, GC3, GC12, and GC14 mRNAs were not expressed in testicular somatic cells, total testis RNA from mice that were treated with busulfan 30 days previously were used as a germ cell-deficient model (Viguier-Martinez, 1984; Bucci, 1987). Northern blot analyses showed the mDEAH9, RanBP5, GC3, GC12, and GC14 mRNAs were present in the testis samples from 5-, 15-, 25-, and 40-day-old mice, and were clearly absent in busulfan-treated mouse testes (Fig. 2). The strong signal in the testis samples from 5-, 15-, 25-, and 40-day-old mice suggests that the mDEAH9 and RanBP5 transcripts were expressed in the spermatogonia and more advanced germ cells. The GC3 and GC12 transcripts were expressed at low levels in the testes from 5-day-old mice and at much higher levels in testes from 15-, 25-, and 40-day-old mice (Fig. 2). This suggests that GC3 and GC12 are expressed at higher levels in postmitotic germ cells. Conversely, GC14 was expressed at its highest levels in testes of 15-day-old mice with lower levels in testes from 5-, 25-, and 40-day-old mice (Fig. 2), which suggests that GC14 may be predominantly expressed in mitotic germ cells.

View larger version (91K): [in this window] [in a new window]   Figure 2. Northern blot analysis of mouse testis mRNA probed with RNA helicase mDEAH9, RanBP, GC3, GC12, GC14, and ChoB cDNA. Each lane was loaded with approximately 6 µg of poly(A)+ enriched testis RNA isolated from mouse testes at 5–8 days (shown as 5), and at 15, 25, and 40 days of age, and testes of mice treated with busulfan 30 days previously (Bus).

The expression of mDEAH9, RanBP5, GC3, GC12, and GC14 were further analyzed in the isolated cell types from testis and other reproductive tissues. Northern blot analyses were performed on poly(A)+ enriched RNA isolated from cultured Sertoli cells and cultured interstitial cells from 15- to 30-day-old mice and ovaries, uterus, epididymis, and whole testes from 40-day-old mice. The mDEAH9 transcript was detected in the cultured immature Sertoli cell samples but not in the interstitial cell samples (Fig. 3). In addition, the mDEAH9 transcript was detected in the tissues from ovary and uterus, but not the epididymis (Fig 3). Conversely, RanBP5, GC3, GC12, and GC14 mRNAs were not detected in the cultured immature Sertoli cells or interstitial cells; or in tissues from ovary, uterus, or the epididymis (Fig. 3). The absence of RanBP5, GC3, GC12, and GC14 mRNAs in the cultured immature Sertoli cell and interstitial cell samples further demonstrates the putative germ cell specificity of these three transcripts within the testis. The low expression of mDEAH9 in the cultured Sertoli cells suggests that this transcript may be germ cell-enriched in the testis but the expression may not be limited to germ cells.

View larger version (56K): [in this window] [in a new window]   Figure 3. Northern blot analysis of mouse reproductive tissues probed with RNA helicase mDEAH9, RanBP5, GC3, GC12, GC14, and ChoB cDNA. Each lane was loaded with approximately 8 µg of poly(A)+ enriched RNA isolated from mouse cultured Sertoli cells and cultured interstitial cells (Inter) from 15-to 30-day-old mice and testes (Testis), epididymis (Epid), uterus, and ovary tissues isolated from 40-day-old mice.

Finally, we examined the gene expression of mDEAH9, RanBP5, GC3, GC12, and GC14 in other mouse tissues in the testis. Northern blot analyses were performed on poly(A)+ enriched RNA isolated from spleen, skeletal muscle, brain, kidney, heart, liver, lung, and testis of 40-day-old mice. The mDEAH9 and RanBP5 mRNAs were present in all tissues analyzed (Fig. 4). Although expression of RanBP5 mRNA was detected in tissues other than testis, expression was greatest in the testis compared with other tissues analyzed (Fig. 4). GC3, GC12, and GC14 were detected only in the testis samples (Fig. 4).

View larger version (62K): [in this window] [in a new window]   Figure 4. Northern blot analysis of mouse organ tissues probed with RNA helicase mDEAH9, RanBP5, GC3, GC12, GC14, and ChoB cDNA. Each lane was loaded with approximately 8 µg of poly(A)+ enriched RNA isolated from 40-day-old mice spleen, skeletal muscle (Skel. Mus.), brain, kidney, heart, liver, lung, and testes (testis).

In Situ Hybridization

In situ hybridization analyses were performed in order to localize the expression of mDEAH9, RanBP5, GC3, GC12, and GC14 genes in the testis. The expression of mDEAH9, GC3, GC12, and GC14 transcripts were detected within the seminiferous tubules and did not appear to be regulated by the stages of the cycle of the seminiferous epithelium (Fig. 5A, B, Fig. 5G–L). Gene expression of RanBP5 was localized primarily to advanced germ cells in the seminiferous tubules, with highest expression in pachytene spermatocytes and round spermatids (Fig. 5C through F). RanBP5 mRNA was detected in all tubules, however, expression of the RanBP5 transcript varied considerably among different stages of the seminiferous epithelium cycle. The highest level of expression was observed in stages III through VII. GC14 expression was greatest in premeiotic germ cell populations (Fig. 5K and L). Expression of mDEAH9, RanBP5, GC3, GC12, and GC14 transcripts were not detected in interstitial cell populations, suggesting that within the testis the expression is absent or very low in these cell types.

View larger version (318K): [in this window] [in a new window]   Figure 5. In situ hybridization for RNA helicase mDEAH9 (A, B), RanBP5 (C–F), GC3 (G, H), GC12 (I, J), and GC14 (K, L) in 40-day-old mouse testes. Paraffin-embedded sections (10 µm) were hybridized with 33P-labeled sense and antisense riboprobes as described in "Materials and Methods." Darkfield micrograph of a cross section hybridized with anstisense (A) and sense (B) RNA helicase cRNA at 125x magnification. Darkfield micrograph of a cross section hybridized with anstisense (C–E) and sense (F) RanBP5 cRNA at (C) 50x, (D) 100x, and (E, F) 125x magnification. Darkfield micrograph of a cross section hybridized with anstisense (G) and sense (H) GC3 cRNA at 125x magnification. Darkfield micrograph of a cross section hybridized with anstisense (I) and sense (J) GC12 cRNA at 125x magnification. Darkfield micrograph of a cross section hybridized with anstisense (K) and sense (L) GC14 cRNA at 125x magnification. Photographs were taken with an Olympus OLY-200 digital camera using MagnaFire software version 1.0 (Optronics, Goleta, Calif).

RT-PCR

To determine whether mDEAH9, RanBP5, GC3, GC12, and GC14 transcripts were present in specific germ cell populations, isolated type A spermatogonia, pachytene spermatocytes, and round spermatids were analyzed by RT-PCR. Gene-specific primers for mDEAH9, RanBP5, GC3, GC12, and GC14 were used to amplify each cDNA (Fig. 6). Each of the isolated germ cell populations expressed mDEAH9, RanBP5, GC3, GC12, and GC14 mRNAs. In addition, RT-PCR analyses of W/W^v testis samples also indicated the presence of the transcripts for mDEAH9, RanBP5, GC3, GC12, and GC14. Most likely, this low positive signal arises from the few spermatogonia that are present in the W/W^v testis (Parreira et al, 1998). These data suggest that the genes for mDEAH9, RanBP5, GC3, GC12, and GC14 were present at some level at all stages of spermatogenesis.

View larger version (82K): [in this window] [in a new window]   Figure 6. RT-PCR analyses of RNA helicase mDEAH9, RanBP5, GC3, GC12, GC14, and ubiquitin from isolated germ cells and testis samples. RT-PCR products (10 µL) were separated on a 1% agarose/ 0.5% TAE gel followed by ethidium bromide staining. W indicates W/Wv testis; A, type A spermatogonia; P, pachytene spermatocytes; R, rounds spermatids; N, wild-type adult testis; -cDNA, cDNA was replaced with water.

Northern blot analyses shown in Figure 4 suggest that GC3, GC12, and GC14 were expressed only in testis. RT-PCR analyses were performed on the same RNA samples used in Figure 4 and including the ovary in order to verify the testis specificity of the expressed genes. GC3 and GC14 were detected only in the testis samples, whereas GC12 was detected in the heart, brain, liver, kidney, spleen, ovary, skeletal muscle, and testis but not in the lung (Fig. 7).

View larger version (36K): [in this window] [in a new window]   Figure 7. RT-PCR analyses of GC3, GC12, GC14, and ubiquitin from specific tissues. RT-PCR products (10 µL) were separated on a 1% agarose/0.5% TAE gel followed by ethidium bromide staining. Total RNA was isolated from spleen, skeletal muscle (Skel. Mus.), brain, kidney, heart, liver, lung, and testes (testis) of 40-day-old mice for RT-PCR analyses. -cDNA indicates that cDNA was replaced with water.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The development of the DDRT-PCR procedure has led to the identification of many genes that are differentially regulated in various cell and tissue types. Here we report the use of DDRT-PCR to identify genes that are expressed in isolated testicular type A spermatogonia and in more advanced germ cells. The technique allowed us to analyze the gene expression profile of type A spermatogonia and to simultaneously compare it with neighboring somatic cell types in the testis, such as the W/W^v mutant testis.

We used an additional control for the cDNA reaction during the DDRT-PCR procedure by comparing results for the first-strand cDNA synthesis reaction, from 2 different RT enzymes, AMV and Superscript II. Putative germ cell-specific cDNA fragments were selected from the DDRT-PCR gel only if their presence and absence were consistent between the RT reactions using 2 different enzymes, duplicate PCR reactions, and multiple samples. These controls greatly reduced the selection of false positives in the reaction.

RNA helicases comprise a large family of proteins that have been identified in many biological systems (Luking, 1998). They are members of the conserved "DEAD/ DEAH box" proteins and play a central role wherever RNA is present. RNA helicases are involved in cellular processes such as nuclear and mitochrondrial splicing, RNA editing, ribosomal RNA processing, translation initiation, nuclear mRNA export, and mRNA degradation.

The RNA helicase mDEAH9 has strong amino acid sequence homology (>65% identity) with the yeast splicing variant Prp43 (Gee, 1997). In yeast, Prp43 is responsible for late pre-mRNA splicing, which facilitates spliceosome disassembly (Arenas, 1997; Imamura, 1998). Alterations in the prp43 allele result in splicing complexes that retain intron lariats (Arenas, 1997). These Prp proteins in yeast have been shown to be associated with Cdc28, and their function is required for cell cycle progression (Imamura, 1998). Although a direct functional role in cell cycle progression in mammalian cells has not been identified for mDEAH9, it is reasonable to speculate that the mitotic and meiotic germ cells that express this gene would have a similar functional requirement for mRNA splicing and cell cycle progression. The high level of mitosis and meiosis that occurs in testicular germ cells may explain the why elevated expression levels of the mDEAH9 mRNA occur in the testis compared with those in most other tissues (Figs. 3 and 4). However, mDEAH9 was also detected in cultured immature Sertoli cells, but not in the testis of busulfan-treated mice. Either MDEAH9 may have a developmental role in the Sertoli cell, or the level of Sertoli cell expression in the testis sample from busulfan-treated mice was too low to detect.

Several RNA helicases have been identified in germ cells in the testis, including mDEAH9. RNA helicases PL10 (Leroy, 1989), P68 (Lemaire, 1993), and more recently, GRTH (Tang, 1999), have also been shown to be expressed specifically in late pachytene spermatocytes and round spermatids.

Ran is a family of well-conserved small GTPases whose function has been implicated in both cell cycle progression through mitosis (Belhumeur, 1993; Dasso, 1993), and in nuclear transport of RNA and protein (Rush, 1996; Sazar, 1996; Mattaj, 1998; Kaffman, 1999). Ran is essential for nuclear localization signal (NLS)-dependent protein import (Moore, 1993) by supplying the energy for the transport process (Weis, 1996). The activity of Ran depends on its interaction with several proteins, including nuclear RCC1 (the major nucleotide exchange factor of Ran), cytoplasmic RanGTPase-activating protein (RanGAP1; Bischoff, 1991; Matunis, 1996), and Ran-binding proteins (RanBPs). These RanBPs interact with RanGTP to inhibit (via RanBP1; Bischoff, 1995) or facilitate (via RanBP2; Wu, 1995) the action of Ran transport. The binding of RanBP5 to RanGTP inhibits RanGAP1-induced hydrolysis and nucleotide exchange, and may aid in recruiting Ran to a nuclear pore complex (NPC) (Deane, 1997), or it may bind directly to the NPC in Ran-independent nuclear transportation(Jakel, 1998).

RanBP5 is the mammalian homolog of the yeast Pse1p (Gorlich, 1997). Sequence analysis of human and mouse RanBP5 proteins show they have, respectively, 46% and 51% sequence similarity to Saccharomyces cerevisiae Pse1p. Deletion of the PSE1 gene was lethal to yeast, and growth studies with yeast demonstrated that protein synthesis, cellular growth, and ribosomal protein transportation were Pse1p-dependent (Coutavas, 1993; Rout, 1997). It is reasonable to speculate that RanBP5 may have a function in mammalian cellular growth and protein synthesis similar to that reported with Psep1 in yeast. The testis had the highest level of RanBP5 gene expression of any tissue examined (Figs. 3 and 4), and in situ hybridization analysis showed that the expression of the RanBP5 was regulated during the cycle of the seminiferous epithelium.

GC3 was a novel cDNA identified in our analyses. The GC3 cDNA sequence had no homology with any known gene sequences in GenBank, but it did match several ESTs reported from mouse testis (accession number AI596411). In addition, the GC3 cDNA sequence contained a putative ORF of 297 amino acids, which is homologous to a human hypothetical protein (accession number AK001137). GC3 amino acids 69–183 had 61% similarity with the ankryin repeat motif. The ankryin repeat motif is a common protein sequence motif found in protein-protein interactions and is found in transcription factors, cell differentiation proteins, and structural proteins expressed in a variety of tissues and species (Michaely, 1993; Lokeshwar, 1994; Sedgwick, 1999). To our knowledge, this is the first protein containing an ankryin motif to be expressed in a testis-specific manner. GC3 was detected in testicular germ cells only Northern blot and RT-PCR analyses (Fig. 3, 4, and 7). Spermatogonial cells, pachytene spermatocytes, and round spermatids all appear to express the GC3 gene (Fig. 7).

The GC12 sequence, which was expressed in many tissues and in all germ cell populations, had 98% nucleotide and 76% amino acid similarity to a human gene, KIAA0039, cloned from male myeloblast cell line (accession number D26018). The cDNA sequence contained a putative ORF of 462 amino acids. Computer searches through GenBank and PROSITE internet databases did not identify a common amino acid motif within the putative peptide sequence. However, GC12 amino acids 170–346 had 39% similarity that of rat neurofilament triplet M protein (accession number P12839) and 38% similarity to rat nucleolus-cytoplasm shuttle phosphoprotein (accession number B42680). The testis had the highest level of expression of GC12, suggesting that the putative protein product has a function during spermatogenesis.

The last novel cDNA we identified was GC14. This cDNA was 1272 bp in length and contained a putative ORF of 166 amino acids. Although the GC14 nucleotide sequence is similar to that of GC12, it did not have homology with any known sequences in the GenBank database and the putative ORF did not contain a common amino acid motif when analyzed with GenBank and PROSITE internet databases. However, the GC14 transcript was of special interest because it was detected only in the testis and because it appeared to be present primarily within a limited germ cell population. Of these, pachytene spermatocytes appeared to have the highest level of expression

In summary, we report the use of differential display to identify genes expressed specifically in germ cells in the testis. The identification of germ cell transcripts will increase our understanding of germ cell development and permits the design of more focused questions regarding the regulation of the cellular events during spermatogenesis.

	Acknowledgments

 
We thank Alice Karl and Debra Mitchell for the Sertoli cell and interstitial cell preparations and cultures, and Derek Pouchnik and Gerhart Munske for the DNA sequences and oligonucleotide synthesis.

	Footnotes

 
This work was supported by National Institutes of Health grants R37 HD10808 to M.D.G. and R01 HD33728 to M.D.

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