©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
In Male Mouse Germ Cells, Copper-Zinc Superoxide Dismutase Utilizes Alternative Promoters That Produce Multiple Transcripts with Different Translation Potential (*)

(Received for publication, August 2, 1994; and in revised form, October 28, 1994 )

Wei Gu (1) Carlos Morales (2) Norman B. Hecht (1)(§)

From the  (1)Department of Biology, Tufts University, Medford, Massachusetts 02155 and the (2)Department of Anatomy, McGill University, Montreal, Quebec H3A 2B2, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Copper-zinc superoxide dismutase (SOD-1) is an enzyme that is widely expressed in eukaryotic cells and performs a vital role in protecting cells against free radical damage. In mouse testis, three different sizes of SOD-1 mRNAs of about 0.73, 0.80, and 0.93 kilobases (kb) are detected. The 0.73-kb mRNA is found in early stages of male germ cells and in all somatic tissues. The mRNAs of 0.80 and 0.93 kb are exclusively detected in post-meiotic germ cells. RNase H digestions and Northern blot analyses reveal that the three SOD-1 mRNAs are derived from two transcripts, a ubiquitously expressed transcript and a post-meiotic transcript, which differ by 114-120 nucleotides. RNase protection assays demonstrate that the additional nucleotides present in the post-meiotic mRNA are solely in the 5`-untranslated region. Using a probe derived from the 5`-untranslated region of the 0.93-kb SOD-1 mRNA, we have established that it originates from an alternative upstream promoter contiguous with the somatic SOD-1 promoter. Polysomal gradient analysis of the three mouse testis SOD-1 mRNAs reveals that the 0.93-kb SOD-1 mRNA is primarily non-polysomal, while the 0.80- and 0.73-kb SOD-1 mRNAs are mostly polysome associated. A faster migrating form of the 0.93-kb SOD-1 mRNA is present on polysomes as a result of partial deadenylation. In a cell-free translation system, the 0.73-kb SOD-1 mRNA translates about 2-fold more efficiently than the 0.93-kb SOD-1 mRNA. These data demonstrate that male germ cells transcribe two size classes of SOD-1 mRNAs with different translation potential by utilizing two different promoters, post-meiotic SOD-1 mRNAs undergo adenylation changes, and one of the post-meiotic SOD-1 mRNAs is transcribed during mid-spermiogenesis and translated days later in a partially deadenylated form.


INTRODUCTION

Superoxide dismutase (SOD) (^1)is an essential enzyme in the cellular pathway that inactivates free radicals. SOD converts superoxide anion radicals into oxygen and hydrogen peroxide, which in turn are converted by catalase into water. Although several isozymes of SOD have been detected (McCord and Fridovich, 1969; Barra et al., 1984; Marklund, 1982), the copper-zinc SOD (SOD-1) is the primary superoxide dismutase isozyme present in a wide variety of cells (Crapo et al., 1992).

In mammals, the SOD-1 gene is highly conserved, consisting of five exons with similar splicing sites (Kim et al., 1993; Levanon et al., 1985; Benedetto et al., 1991) and encoding a protein of about 16 kDa (Sherman et al., 1984; Hsu et al., 1992). Moreover, the 225 nucleotides flanking the transcriptional start site of the SOD-1 gene are also strongly conserved, showing similarities of 84% between rat and mouse, 56% between rat and human, and 54% between mouse and human (Hsu et al., 1992). Although SOD-1 is encoded by a single copy gene (Levanon et al., 1985), two SOD-1 mRNAs differing by about 200 nucleotides have been found in human tissue culture cells (Lieman-Hurwitz et al., 1982). The two mRNAs have identical 5`-untranslated regions (UTRs) and coding regions, but they differ in their 3`-UTRs (Sherman et al., 1984). In contrast, only one mRNA of about 0.70 kb has been detected in the somatic tissues of rat and mouse (Delabar et al., 1987; Benedetto et al., 1991). Recently, two transcripts of 0.77 and 0.94 kb have been reported in rat testis (Jow et al., 1993).

Since spermatozoa are highly vulnerable to free radical damage and SOD-1 plays an important role in preventing oxidative injury, we have begun to investigate the regulation of genes such as SOD-1 that can protect male germ cells from reactive oxygen species. Here, we present evidence for three size classes of SOD-1 mRNAs in the mouse testis; one, a post-meiotic transcript, is translationally controlled during mouse spermiogenesis. We also demonstrate that haploid germ cells transcribe two different SOD-1 mRNAs by utilizing the somatic SOD-1 promoter and an alternative upstream testis-specific promoter. The extended 5`-UTR of the longest post-meiotic SOD-1 mRNA may fine tune synthesis of SOD-1 in late stages of germ cell development since it is less efficiently translated in vitro and in vivo.


EXPERIMENTAL PROCEDURES

RNA Purification and Northern Blot Hybridization

CD-1 mice were purchased from Charles River Laboratories. Round spermatids and elongating spermatids were isolated by sedimenting a dissociated testicular cell suspension at unity gravity over a linear 2-4% bovine serum albumin gradient in a Staput chamber (Romrell et al., 1976). Total RNA was isolated from adult mouse testis, liver, heart, kidney, brain, the testes of prepuberal mice of 6, 12, 17, 22, and 25 days of age, and isolated germ cell populations by the method of Alcivar et al.(1989). Northern blot hybridizations were performed as previously described (Gu et al., 1994).

RNase H Digestions

The method of Nanbu et al.(1991) was used. RNA aliquots (15 µg) were denatured in 80% formamide at 65 °C for 10 min and annealed with 15 µg of oligo(dT) (Pharmacia Biotech Inc.) or with 1 µg of an oligonucleotide complementary to the SOD-1 mRNA (described below) for 90 min at 50 °C in 10 mM Pipes, pH 6.4, 40% formamide, 0.4 M NaCl, and 1 mM EDTA. The hybridized nucleic acids were ethanol precipitated, dried, resuspended in 100 µl of RNase H digestion buffer (20 mM Tris, pH 7.4, 100 mM KCl, 10 mM MgCl(2), and 0.1 mM dithiothreitol), and incubated with 6 units of RNase H for 45 min at 37 °C. The samples were extracted twice with phenol-chloroform, and the cleaved RNAs were precipitated with ethanol. The precipitates were electrophoresed in 1% agarose gels and analyzed by Northern blotting.

Isolation of SOD-1 cDNAs and the 5`-Flanking Region of the SOD-1 Gene

SOD-1 cDNAs were isolated from a mouse testis round spermatid lambda gt11 library (kindly provided by Dr. E. M. Eddy of NIEHS) by plaque hybridization (Maniatis et al., 1982) with a P-labeled cDNA probe encoding 500 bp of the mouse SOD-1 cDNA (kindly provided by Dr. Gordon, Mt. Sinai Medical Center, NY). After screening approximately 1 times 10^6 clones, 10 positive clones were isolated, and the inserts of four of the clones were PCR amplified using lambda gt11 primers. Polymerase chain reactions were performed using a Perkin-Elmer Thermal Cycler (Perkin-Elmer Corp.). 25 cycles (1 min at 94 °C, 1 min at 50 °C, and 1 min at 72 °C) were performed in 50-µl aliquots of 10 mM Tris, pH 8.3, 4 mM MgCl(2), 0.2 mM dATP, dCTP, dGTP, dTTP, 2 mM dithiothreitol, 50 mM KCl, and 1 unit of Taq polymerase (AmpliTaq, Perkin-Elmer Corp.). Reaction products were electrophoresed in 1.2 or 3% agarose gels and gel purified. The PCR products of four clones were subcloned into the TA cloning vector (Invitrogen), and sequence analysis was performed by the Sanger dideoxy chain termination method.

A 0.9-kb mouse genomic DNA fragment that contains the first exon and the 5`-flanking region of the SOD-1 gene was isolated by digestion of a 16-kb fragment of SOD-1 genomic DNA (kindly provided by Dr. Gordon, Mt. Sinai Medical Center) with restriction enzymes BamHI and SmaI. The fragment was subcloned into a pGEM 3Z vector and sequenced by the Sanger dideoxy chain termination method.

Oligonucleotides Complementary to SOD-1

Two oligonucleotides, RH1 and RH2 (purchased from Operon Technologies), were used for RNase H digestions. RH1 (5`-CGCCATGCTTCCCCGGAAGAC-3`) is complementary to nucleotides 91-111 of the somatic SOD-1 mRNA (Benedetto et al., 1991). RH2 (5`-GAGGGTAGCAGTTGAGTCTG-3`) is complementary to nucleotides 596-616 of the somatic SOD-1 mRNA. For the RNase protection assays, a 335-bp DNA fragment was amplified using RH1 and RP1, an oligonucleotide (5`-TCACAGTTAGAAGACAATAG-3`) that is complementary to nucleotides -224 to -205 of the SOD-1 gene. To detect the post-meiotic SOD-1 mRNA, a DNA fragment was amplified by PCR using oligonucleotides TS1 and TS2. TS1 (5`-GCGGCCAGGGAGCTCCAC-3`) contains nucleotide sequence -114 to -97 of the SOD-1 gene whereas TS2 (5`-AACGAGGCCCTGGCGCCA-3`) contains nucleotide sequence -15 to +3 of the SOD-1 gene.

In Situ Hybridization

Tissue preparation, hybridization conditions, and radioautography were as previously described (Morales and Hecht, 1994).

RNase Protection Assays

RNase protection analysis was performed using a modification of Ausubel et al.(1991). A 335-bp fragment (-225 to 110) containing the promoter and the 5`-region of SOD-1 was PCR amplified from the 16-kb mouse genomic SOD-1 fragment using oligonucleotides RP1 and RH1 and subcloned. P-Labeled antisense RNA was transcribed from the linearized 335-bp SOD-1 subclone and isolated by electrophoresis in a 6% acrylamide gel. The radiolabeled RNA was recovered from the acrylamide gel by swirling gel slices in elution buffer containing 300 mM sodium acetate, 0.5% SDS, and 2 mM EDTA for 4 h, followed by precipitation with 2.5 volumes of 100% ethanol. Radiolabeled RNA (2 times 10^5 cpm) was mixed with RNA aliquots (10 µg) in 30 µl of hybridization buffer (40 mM Pipes, pH 6.4, 400 mM NaCl, 1 mM EDTA, and 80% formamide), denatured by heating to 85 °C for 3 min, and annealed overnight at 50 °C. Unhybridized RNAs were removed by incubation at 37 °C for 20 min with 350 µl of 10 mM Tris, pH 7.4, 500 mM NaCl, 5 mM EDTA, 50 µg/ml RNase A, and 2 µg/ml RNase T1. The RNases were subsequently removed by incubating the samples with 50 µg of proteinase K and 20 µl of 10% SDS for 15 min at 37 °C. The protected RNA:probe hybrids were extracted with phenol:chloroform (1:1) and precipitated with 2.5 volumes of 100% ethanol after the addition of yeast carrier tRNA (10 µg). The pellet was resuspended in 10 µl of RNA running buffer (80% formamide, 10 mM EDTA), denatured at 95 °C for 2 min, and analyzed in 6% acrylamide sequencing gels.

Southern Blot Analysis of the SOD-1 Gene

High molecular weight genomic DNA was isolated from the spleens of CD-1 mice and digested with restriction enzymes. The digested DNA fragments were resolved in 0.7% agarose gels, transferred to nylon membranes (DuPont NEN), and hybridized to P-labeled SOD-1 cDNAs.

Polysomal Gradients

Fractionation of post-mitochondrial extracts over sucrose gradients was performed as previously described (Kleene et al., 1984; Hake et al., 1990). Post-mitochondrial supernatants were prepared from adult testes, and 4 ml were loaded over a 35-ml 10%-35% sucrose gradient over a 2-ml 60% sucrose cushion. After centrifugation at 28,000 rpm for 3 h, the gradient was fractionated into six tubes, and RNA was purified for Northern blot analysis as described above.

Cell-free Translations

Two EcoRI-AvaI cDNA fragments, a 645-bp somatic type SOD-1 sequence, and a 759-bp testis-specific SOD-1 sequence were PCR generated from cDNAs and subcloned into pGEM 3Z (Promega). The two cDNA fragments have identical 5`-UTR, coding, and 3`-UTR sequences, and the testis-specific SOD-1 cDNA has an additional 114 nucleotides at its 5`-end. Capped SOD-1 transcripts were transcribed from the AvaI-linearized pGEM 3Z SOD-1 constructs using T7 RNA polymerase. Trace amounts of [^3H]CTP were incorporated into the mRNAs for quantitation, and the integrity and sizes of the transcripts were verified in 1% formaldehyde-agarose gels. Equimolar amounts ranging from 1 to 8 pmol of the SOD-1 mRNAs were translated in rabbit reticulocyte lysates in the presence of [S]methionine (Amersham Corp.), and aliquots of the translated proteins were analyzed by electrophoresis in 12.5% polyacrylamide gels, followed by fluorography.


RESULTS

Three Sizes of SOD-1 mRNAs Are Expressed during Male Germ Cell Differentiation

The amounts and sizes of SOD-1 mRNAs in mouse liver, brain, spleen, heart, and kidney and in prepuberal and sexually mature mouse testes were examined by Northern blotting. A SOD-1 mRNA of approximately 0.73 kb was observed in all somatic tissues and in the RNAs from prepuberal and adult testes (Fig. 1A, lanes1-11). In addition, a developmentally increasing amount of two additional SOD-1 transcripts of about 0.80 and 0.93 kb was detected in the testes of 22-day-old, 25-day-old, and adult mice (Fig. 1A, lanes9-11).


Figure 1: Northern blot of SOD-1 mRNAs in mouse somatic tissues and prepuberal and adult mouse testes. A, RNA aliquots (10 µg) from adult mouse liver (lane1), brain (lane2), spleen (lane3), heart (lane4), and kidney (lane5) and testes from 6-day-old (lane6), 12-day-old (lane7), 17-day-old (lane8), 22-day-old (lane9), 25-day-old (lane10) and adult mice (lane11) were electrophoresed in 1% agarose gels and transferred to nylon membranes. The membranes were hybridized with a P-labeled cDNA-encoding mouse SOD-1. The arrows indicate the positions of the three sizes of SOD-1 mRNAs. B, RNA aliquots (10 µg) from testes from 17-day-old mice (lane1), adult mice (lane2), round spermatids (lane3), and elongating spermatids (lane4) were analyzed as in A.



Since the 0.80- and 0.93-kb SOD-1 mRNAs first appear in the testes of prepuberal mice at the time of differentiation when post-meiotic spermatids are seen, we analyzed enriched populations of cells to better define the cellular locations of the two large SOD-1 transcripts. RNAs isolated from enriched populations of round and elongating spermatids were hybridized to the SOD-1 cDNA. The developing spermatids predominantly contain SOD-1 mRNAs of 0.80 and 0.93 kb and little, if any, of the 0.73-kb SOD-1 mRNA (Fig. 1B, lanes3 and 4). Round spermatids contain predominantly the 0.93-kb mRNA and a smaller amount of the 0.80-kb mRNA. In elongating spermatids, most of the SOD-1 mRNA migrates as a heterogenous band faster than the 0.93-kb SOD-1 mRNA seen in round spermatids. Testes of 17-day-old and adult mice show their expected one and three SOD-1 mRNAs, respectively (Fig. 1B, lanes1 and 2). We conclude that in place of the ubiquitously expressed 0.73-kb somatic SOD-1 transcript, post-meiotic male germ cells contain two additional SOD-1 mRNAs with slower electrophoretic mobilities.

The Post-meiotic SOD-1 mRNAs Contain Additional Nucleotides at the 5` Termini

Since superoxide dismutase 1 is a single copy gene and three different sizes of SOD-1 mRNAs are detected in mouse testis, we have utilized RNase H digestion assays to define structural differences among the mRNAs (Fig. 2). A single band of deadenylated SOD-1 mRNAs of about 0.63 kb is detected in liver (Fig. 2A, lane2), whereas two deadenylated mRNA bands of about 0.63 and 0.75 kb are seen in adult testis (Fig. 2A, lane4). The faster migrating testicular RNA comigrates with the 0.63-kb somatic SOD-1 mRNA. After deadenylation, the slower migrating testis-specific SOD-1 mRNA appears to be approximately 120 nucleotides longer than the 0.63-kb SOD-1 mRNA. These data suggest that in adult mouse testis, the three SOD-1 mRNAs are derived from two transcripts, one of which is about 120 nucleotides longer than the somatic SOD-1 mRNA.


Figure 2: RNase H analysis of SOD-1 mRNAs. Aliquots (15 µg) of adult total testis RNA were hybridized to oligo(dT) and oligonucleotides RH1 or RH2. The hybrids were then digested with RNase H, electrophoresed in 1% agarose gels, blotted onto nylon membranes, and hybridized with a P-labeled SOD-1 cDNA probe. A, control and RNase H digested liver and testis SOD-1 RNAs. Lanes1 and 3, undigested RNA samples of adult mouse liver and testis, respectively; lanes2 and 4, RNase H digested RNA samples of adult mouse liver and testis RNA, respectively. B, control and RNase H digested testis SOD-1 mRNAs after hybridization to complementary oligonucleotides and oligo(dT). Lane1, Undigested adult testis RNA; lane2, RNase H treated adult testis RNA sample after hybridization to RH2; lane3, RNase H treated adult testis RNA sample after hybridization to oligo RH1 and oligo(dT).



To determine where the additional nucleotides are located in the post-meiotic SOD-1 transcripts, SOD-1 mRNAs were hybridized with specific oligonucleotides, split into 5`- and 3`-regions by RNase H digestion, and then analyzed by Northern blotting. When the 5`-UTR and poly(A) tails were detached from the SOD-1 coding region, one major mRNA of about 0.6 kb and a small fragment were detected with a probe for the coding region of mouse SOD-1 (Fig. 2B, lane3). Separation of the 3`-UTR from the coding region of SOD-1 mRNA yielded two major bands of RNA with a size difference of about 120 nucleotides (Fig. 2B, lane2). This suggests that the additional sequence present in the post-meiotic SOD-1 transcripts is at the 5` terminus.

The Mouse SOD-1 Gene Is a Single Copy Gene That Utilizes Two Promoters in Mouse Testis

To more precisely characterize the post-meiotic SOD-1 mRNAs, we isolated and sequenced SOD-1 cDNAs from a mouse round spermatid cDNA library. Two populations of SOD-1 cDNAs were obtained (Fig. 3). One group of cDNAs contained a sequence identical to the coding and 3`-UTR sequences of the somatic cell SOD-1 cDNAs previously described by Bewley(1988) and Benedetto et al.(1991). The second group of SOD-1 cDNAs contained the identical sequence present in the shorter SOD-1 cDNAs and an additional 114 nucleotides at their 5`-ends (denoted by underlinedsequences between the arrows in Fig. 3). This suggests that the post-meiotic SOD-1 mRNAs and the somatic SOD-1 mRNA are derived from the same single copy gene.


Figure 3: Sequence analysis of mouse testicular SOD-1 cDNAs and gene. The underlining between the arrows indicates the additional RNA segment located at the 5` terminus of the testis-specific SOD-1 mRNA. The largesolidarrow and +1 indicate the primary somatic transcription start site. The smallopen and solidarrows represent the primary transcription start sites of the post-meiotic 0.93-kb SOD-1 mRNA. The ATG codon representing the start of translation of SOD-1 is boxed. Additional conserved sequences from a mouse genomic fragment of SOD-1 gene are included. The abbreviations of consensus elements are C, CAAT box; SP1, transcription factor element; CRE, cAMP-responsive element; AP-3, PEA3-RS, and NF-E(1), transcription factor elements; mP1-C, mP1-G, mP1-E, and mP1-F, the conserved sequences present in the promoters of mouse protamine 1, protamine 2, and transition protein 1 (Johnson et al., 1988).



To better establish the structural relationship of the SOD-1 cDNAs, a 0.90-kb fragment containing 750 nucleotides of the 5`-flanking region of the SOD-1 gene was isolated and sequenced. Computer analysis of 630 nucleotides 5` to the predicted transcription start site of the post-meiotic SOD-1 mRNA reveals a group of sequence elements known to be transcription factor binding sites as well as conserved sequences present in other post-meiotically expressed genes (Fig. 3). Among the most notable protein binding sites are a CAAT box present at -210, two Sp1 binding sites located at -133 and -529, and two cAMP-responsive element modulator (CREM) binding sites located at -228 and -606. In addition, several conserved sequence elements previously seen in the promoters of other testis-specific genes by Johnson et al. (1988) are denoted with mP1-C, mP1-E, mP1-F, and P1-G (Fig. 3).

Sequence analysis of the SOD-1 cDNAs and genomic DNA suggests that the additional sequence present in the longer SOD-1 cDNAs and in the 0.93-kb SOD-1 mRNAs is contiguous with the somatic SOD-1 promoter. Two different approaches, Southern blotting and RNase protection assays, were used to confirm this and also to monitor for alternative splicing. Restriction digested mouse genomic DNA was analyzed by Southern blotting with a coding region probe of SOD-1 and with a probe specific to the 114 nucleotide 5`-UTR sequence of the post-meiotic SOD-1 transcript (Fig. 4). When DNA digested with the enzymes BamHI, EcoRI, HindIII, or SacI was hybridized with the SOD-1 coding region cDNA, two or three bands of DNA with sizes ranging from about 5-11 kb were seen (Fig. 4A, lanes1-4). Rehybridization of the same blot with a SOD-1 probe specific for the 5` terminus of the post-meiotic SOD-1 transcript detected in each digestion one DNA fragment previously seen with the SOD-1 coding region cDNA probe (Fig. 4B, lanes1-4). This establishes that the 5`-UTR sequence of the post-meiotic SOD-1 mRNA is present in the same genomic DNA fragment that encodes the somatic SOD-1 mRNA.


Figure 4: Southern blot analysis of the SOD-1 gene. Aliquots (10 µg) of CD-1 mouse genomic DNA were digested with restriction enzymes, fractionated through 0.7% agarose gel, and transferred to a nylon membrane. A, the membrane was hybridized to a P-labeled mouse SOD-1 coding region cDNA probe. Lane1, digestion with BamHI; lane2, digestion with EcoRI; lane3, digestion with HindIII; lane4, digestion with SacI. B, after removal of the SOD-1 cDNA probe, the blot was reprobed with the 5`-UTR-specific SOD-1 probe of the 0.93-kb mRNA.



To determine if the post-meiotic mRNAs are produced by alternative splicing, RNase protection assays were performed. Based upon the cDNA and genomic sequences of the mouse SOD-1 gene (Benedetto et al., 1991), P-labeled antisense RNA from -224 to +112 of the SOD-1 gene was synthesized (Fig. 5). The antisense RNA was hybridized to liver RNA (lane1), from testis RNA of l7-day-old mice (lane2), and to testis RNA from adult mice (lane3). Following RNase digestion, a group of RNase-protected bands (lowerbracket) with sizes of about 105-112 nucleotides was detected in the samples from liver, prepuberal testes, and adult testes, suggesting that they correspond to the 5`-start sites of the ubiquitous SOD-1 mRNA. An additional more slowly migrating group of two distinct RNA bands with sizes of about 226-232 nucleotides (upperbracket) was detected solely with adult mouse testis RNA (lane3), suggesting that these bands represent the start sites of the post-meiotic SOD-1 mRNA. The size differences of 114-120 nucleotides between the slower and faster migrating bands in Fig. 5are in agreement with the RNase H digestion (Fig. 2) and cDNA sequence data (Fig. 3). The multiple RNase-protected bands in each group suggest that several transcription start sites are used. Since there is size heterogeneity among the SOD-1 transcripts, we arbitrarily use 114 nucleotides, the size difference of the two groups of testicular SOD-1 cDNAs we have isolated, when we compare the size of the transcripts from the somatic and post-meiotic promoters. These data establish that the transcription start sites of the 0.93 and 0.73-kb SOD-1 mRNAs are contiguous.


Figure 5: RNase protection assay of testicular SOD-1 mRNAs. Aliquots (15 µg) of RNA were annealed to 2 times 10^5 cpm of P-labeled single-stranded antisense SOD-1 RNA and digested with RNases A and T(1). The protected RNA fragments were analyzed in a 6% acrylamide sequencing gel. Lane1, liver RNA; lane2, testis RNA from 17-day-old mice; lane3, adult testis RNA; lane4, antisense RNA probe only. The protected RNA regions are indicated in brackets. The lanes labeled GATC are DNA sequencing ladders used to calculate the sizes of the protected RNA fragments.



Cellular Localization of SOD-1 mRNAs from the Two Testis SOD-1 Promoters

To localize precisely the cellular sites of expression of SOD-1 transcripts from the two SOD-1 promoters utilized in the mouse testis, two ^3H-labeled probes were hybridized in situ and visualized radioautographically. The first probe, an antisense RNA to the coding region of SOD-1, yielded a radioautographic reaction in all cells of the seminiferous epithelium including the Sertoli cells (Fig. 6, upperpanel; Fig. 7, lowerpanel). No major differences in hybridization intensity were seen among the different stages of the cycle. A moderate reaction was also observed over the cells present in the interstitial space. The second probe, specific to the 5`-untranslated region of the post-meiotic SOD-1 mRNA, was predominantly detected in spermatids from step 6 to step 16 (Fig. 6, middlepanel; Fig. 7, twoupperpanels). In early steps 1-5, the cytoplasm of these round spermatids remained unreactive (Fig. 7, upperrightpanel), suggesting that transcription of postmeiotic SOD-1 mRNA initiates at a detectable level at the beginning of step 6 spermatids. Control RNA sense probes did not yield any radioautographic reaction, indicating that the reactions generated by the antisense probes were specific (Fig. 6, lowerpanel).


Figure 6: In situ hybridization of mouse seminiferous tubules. Upperpanel, cross section of mouse seminiferous epithelium hybridized in situ with a ^3H-labeled RNA probe to the SOD-1 coding region and visualized by radioautography. Silver grains uniformly overlay all cells of the seminiferous tubules (stageIX) (times400). Middlepanel, cross section of mouse seminiferous epithelium hybridized in situ with a ^3H-labeled RNA probe specific to the 5`-UTR of the post-meiotic SOD-1 mRNA. The probe generated silver grains on spermatids (step9) of the seminiferous tubule (stageIX) (times400). Lowerpanel, cross section of mouse seminiferous epithelium hybridized in situ with a ^3H-labeled sense RNA probe to the SOD-1 coding region. This probe did not yield any radioautographic reaction (times400).




Figure 7: In situ hybridization of mouse seminiferous tubules. Upperpanels, cross section of mouse seminiferous epithelium at stage XI hybridized in situ with a ^3H-labeled RNA probe specific to the 5`-UTR of the post-meiotic SOD-1 mRNA. The probe generated silver grains over the cytoplasm of steps 11 and 13 elongated spermatids but not over the cytoplasm of step 1 spermatids. Upperrightpanel, cross section of a seminiferous tubule at stage I hybridized as described for leftupperpanel (times400). Lowerpanel, cross section of mouse seminiferous epithelium hybridized in situ with a ^3H-labeled RNA probe to the coding region of SOD-1. Silver grains uniformly overlay all cells of the seminiferous tubule (stageIV) (times400).



SOD-1 mRNAs Are Differentially Adenylated in Post-meiotic Germ Cells

In sexually mature testes, a third SOD-1 mRNA of about 0.80 kb is seen (Fig. 1B, lane2). To determine whether the 0.80-kb SOD-1 transcript is derived from the 0.93- or the 0.73-kb SOD-1 mRNAs, we combined RNase H digestions with hybridization to probes that can differentiate between the two SOD-1 mRNAs (Fig. 8). When blots containing RNAs from prepuberal and adult testes and enriched populations of round spermatids and elongating spermatids were hybridized to the post-meiotic 5` terminus probe (Fig. 8A), the 0.93-kb mRNA was detected in adult mouse testes (lane3) and round spermatids (lane5) but not in the testes of 17-day-old mice (lane1). In elongating spermatids, the 5` terminus probe hybridized to a heterogenous population of SOD-1 mRNAs, migrating faster than the 0.93-kb SOD-1 mRNA (Fig. 8A, lane7). The 5` terminus probe did not hybridize to the 0.73-kb SOD-1 mRNA in any RNA sample we have examined. Following deadenylation of the SOD-1 mRNAs, the post-meiotic 5` terminus cDNA probe detected one SOD-1 mRNA of about 0.75 kb in adult testes, round spermatids, and elongating spermatids (Fig. 8A, lanes4, 6, and 8). This suggests that the heterogenous population of SOD-1 mRNA detected by the 5` terminus probe in elongating spermatids are deadenylated forms of the 0.93-kb transcript.


Figure 8: Identification of post-meiotic SOD-1 mRNAs. Aliquots (10 µg) of RNA from testes of 17-day-old mice, adult testes, round spermatids, and elongating spermatids were annealed to oligo(dT) and then digested with RNase H. The RNAs were separated in a 1% agarose gel, transferred to nylon membranes, and hybridized with a P-labeled SOD-1 probe specific for the 5`-UTR of the 0.93-kb SOD-1 mRNA (A); or, after removal of the original probe, the blot was rehybridized with a P-labeled SOD-1 coding region cDNA (B). Lanes1, 3, 5 and 7, undigested RNAs of testes of 17-day-old mice, adult testes, round spermatids, and elongating spermatids, respectively. Lanes2, 4, 6, and 8, RNase H digested RNA samples of testes of 17-day-old mice, adult testes, round spermatids, and elongating spermatids, respectively.



To determine whether lengthened transcripts of the 0.73-kb SOD-1 mRNA also contribute to the intermediate size SOD-1 mRNAs, the same filter was rehybridized with the SOD-1 cDNA probe (Fig. 8B). Three size classes of SOD-1 mRNAs were detected in adult testis (Fig. 8B, lane3), and two transcripts of sizes about 0.80 and 0.93 kb were seen in round spermatids (Fig. 8B, lane5), whereas only the 0.73-kb transcript was seen in testis RNA from 17-day-old mice (Fig. 8B, lane1). Following deadenylation, two RNA bands were detected in the adult testis, round spermatid and elongating spermatid RNAs (Fig. 8B, lanes4, 6, and 8), demonstrating that in spermatids SOD-1 mRNAs of about 0.80 kb are derived from the 0.73-kb somatic SOD-1 transcript.

The 0.93-kb SOD-1 mRNA Is Primarily Non-polysomal, whereas the 0.80- and 0.73-kb SOD-1 mRNAs Are Polysome Associated

To determine the relative translational utilization of each of the three SOD-1 mRNAs in mouse testis, post-mitochondrial extracts from total testes were fractionated by sucrose gradient sedimentation, and purified RNAs were hybridized to SOD-1 cDNA probes (Fig. 9). Most of the 0.80- and 0.73-kb SOD-1 transcripts were present in the polysomal fractions (Fig. 9A, lanes5 and 6), whereas the majority of the 0.93-kb SOD-1 transcript was detected in the non-polysomal fractions (Fig. 9A, lanes2 and 3).


Figure 9: Distribution of SOD-1 mRNAs in a fractionated post-mitochondrial testicular extract. Fraction1 represents the top of the gradient. LaneT, unfractionated adult testis RNA control. Equal volumes of RNA from each fraction were electrophoresed in 1% agarose gels and blotted on nylon membranes. For each autoradiogram, the mRNA sizes are noted at the right. A, autoradiogram obtained after hybridization with the SOD-1 coding region cDNA. B, after the SOD-1 probe was removed, the blot was rehybridized with the probe specific to the 5`-UTR of the 0.93-kb SOD-1 mRNA. C, to confirm proper resolution of polysomal and non-polysomal mRNAs in the sucrose gradient, the same blot was rehybridized, after the SOD-1 probes were removed, with a cDNA probe encoding mouse protamine 2. The presence of the 830-nucleotide mP2 mRNA in the non-polysomal fractions and the 700-nucleotide mP2 in the polysome fractions confirms the polysomal or non-polysomal positions of the SOD-1 mRNAs in the gradient (Kleene, et al., 1984).



To better quantitate the distribution of transcripts derived from the 0.93-kb SOD-1 mRNA, the blots were rehybridized with the 5` terminus probe after the SOD-1 cDNA probe was removed (Fig. 9B). Although most of the 0.93-kb SOD-1 mRNA is found in the non-polysomal fractions, the 5` terminus probe detects a population of heterogeneous and shortened SOD-1 mRNAs on polysomes, suggesting that some of the 0.93-kb SOD-1 mRNA is translated in a partially deadenylated form. Based upon our findings of a distinct band of 0.93-kb SOD-1 mRNA in round spermatids but a heterogeneous and shortened population of SOD-1 mRNAs derived from the 0.93-kb transcript in elongating spermatids (Fig. 8), we conclude that shortening of the 0.93-kb SOD-1 mRNA occurs concomitant with translation in elongating spermatids. Northern blot analysis of deadenylated aliquots of non-polysomal SOD-1 mRNA (a deadenylated aliquot of lane3 from Fig. 9) and polysomal SOD-1 mRNA (a deadenylated aliquot of lane6 from Fig. 9) reveals two SOD-1 transcripts with similar electrophoretic mobilities in the non-polysomal and polysomal fractions (data not shown). Quantitation of the polysomal and non-polysomal amounts of the two SOD-1 mRNAs confirms the distribution differences seen in Fig. 9with the majority of the 0.73-kb transcript on polysomes and the majority of the 0.93-kb transcript in the non-polysomal fraction. Since the two SOD-1 transcripts only differ in their 5`-UTRs, this suggests that the lengthened 5`-UTR may reduce the efficiency of polysome loading of SOD-1 mRNAs in post-meiotic cells.

The 0.73-kb SOD-1 mRNA Is More Efficiently Translated than the 0.93-kb SOD-1 mRNA in a Cell-free Translation System

To evaluate the influence of the additional 5`-UTR sequence of the testis-specific SOD-1 mRNA on its translation, we synthesized in vitro somatic type and testis-specific SOD-1 transcripts and compared their translational efficiencies in a rabbit reticulocyte lysate. To ensure that identical molar amounts of mRNAs were used, the template RNAs were synthesized in the presence of low levels of [^3H]CTP, and after normalization to compensate for the different mRNA sizes, 4 pmol of each template were used. The somatic type 0.73-kb SOD-1 mRNA translates about 2-fold more efficiently than the testis-specific 0.93-kb SOD-1 transcript (Fig. 10). Identical results were obtained in titration experiments with RNA amounts ranging from 1 to 8 pmol. These data support the reduced polysome loading of the 0.93-kb SOD-1 mRNA we find in vivo and suggests that the additional 114 nucleotides in the 5`-UTR reduces the translational efficiency of SOD-1 mRNA.


Figure 10: Translation of the 0.73- and 0.93-kb SOD-1 mRNAs in a reticulocyte lysate. Equal amounts (4 pmol) of transcripts from the 0.93-kb SOD-1 mRNA (lane1) or the 0.73-kb SOD-1 mRNA (lane2) were translated. SOD-1 protein synthesis was monitored by [S]methionine incorporation. Lane3 represents a cell-free translation incubation without SOD-1 mRNA. The SOD-1 protein migrates at the expected size of 16 kDa (Levanon et al., 1985). Similar results were obtained in six different experiments using six different preparations of template mRNA.




DISCUSSION

We have identified in mouse testis three SOD-1 mRNAs that code for the same protein. One SOD-1 mRNA of 0.73 kb is expressed in all mouse somatic tissues as well as prepuberal and sexually mature testes. We only detected the other two SOD-1 mRNAs, transcripts of 0.80 and 0.93 kb in the testis, where their expression appears to be stage-specific during male germ cell differentiation. The 0.93- and 0.80-kb mRNAs are only found in the testes of mice where round spermatids have differentiated. The 0.93-kb mRNA, which is mostly in the non-polysomal fractions, is polysome bound in elongating spermatids in a partially deadenylated form. Based upon SOD-1 cDNA and genomic sequence data, RNase protection assays, and Southern blots, we conclude that in the mouse the 0.93-kb SOD-1 mRNA contains 114-120 additional nucleotides in its 5`-untranslated region compared with the ubiquitous SOD-1 mRNA and is transcribed from alternative transcription start sites that are contiguous with the somatic SOD-1 promoter.

Since the 0.93-kb SOD-1 mRNA is first transcribed in haploid germ cells (Fig. 1, Fig. 6, and Fig. 7) and SOD-1 is a single copy gene in the mouse, we can compare sequences conserved in the promoters of post-meiotically expressed testis-specific genes with the promoter used for SOD-1 in spermatids. Genes such as protamines 1 and 2 (Kleene et al., 1983; Hecht, 1993), transition proteins 1 and 2, and a selenium containing sperm mitochondrial protein have been shown to be solely transcribed in haploid germ cells (Heidaran and Kistler, 1987; Kleene and Flynn, 1987; Kleene, 1989). Sequence analyses of the 5`-flanking region of the testis-specific SOD-1 gene reveal it lacks a TATA box, which may explain why we detect heterogeneity in SOD-1 transcript size from this promoter (Fig. 5). Defining the 5` terminus of the longer SOD-1 cDNAs as +1, we detect a CAAT box 96 nucleotides upstream of the alternative transcription initiation site and 210 nucleotides upstream of the somatic promoter transcription start site (Fig. 3). Binding sites for transcription factors Sp1 and CREM and several other conserved sequence elements present in the promoters of mP1, mP2, and mTP1 are found in the 5`-flanking region of the post-meiotic SOD-1 transcription start site. Several studies have demonstrated that a variant of CREM plays a major role in controlling post-meiotic gene transcription in round spermatids (Foulkes et al., 1992; Delmas et al., 1993), and high levels of Sp1 have been detected in spermatids (Saffer et al., 1991). Thus, although the sequence elements for Sp1 and CREM and other highly conserved regions in testis-specific genes could regulate temporal expression of the post-meiotic SOD-1 mRNA in developing spermatids, which sequence elements are essential remains to be established.

The reason why transcription initiates from a novel upstream promoter site for SOD-1 during spermiogenesis needs to be addressed. The post-meiotic SOD-1 mRNA is initially transcribed in round spermatids, a cell type that contains little, if any, of the 0.73-kb SOD-1 transcripts ( Fig. 6and Fig. 8). The down-regulation of SOD-1 mRNAs that are derived from the somatic SOD-1 promoter toward the end of spermatogenesis may result from 1) changes in availability of specific transcription factors required by the somatic SOD-1 promoter, 2) reduced accessibility to essential cis-acting promoter elements in the ubiquitously expressed SOD-1 gene due to chromatin structure changes, or 3) the expression of the novel SOD-1 mRNA with an extended 5`-UTR possibly being required for the storage and utilization of SOD-1 mRNA during spermiogenesis.

When transcript sizes of genes expressed in somatic cells and in testis are compared, testis-specific transcripts are often detected. The genes for c-abl, angiotensin-converting enzyme, cytochrome c(S), and beta1,4 galactosyltransferase all utilize testis-specific promoters to produce mRNAs that differ in size from their somatic counterparts (Hake et al., 1990; Shaper et al., 1990; Langford et al., 1991; Wolgemuth and Watrin, 1991; Hecht, 1993). The testicular variant of proenkephalin in rat and mouse is about 300 nucleotides longer than the somatic proenkephalin mRNA because of alternative splicing at its 5` terminus (Kilpatrick et al., 1990). The use of different polyadenylation sites produces cytochrome c(S) transcripts of 0.5-1.3 kb, whereas the use of different promoter start sites results in cytochrome c(T) mRNAs that differ by about 60 nucleotides (Hake et al., 1990; Yiu, et al., 1995). Truncated forms of mRNAs encoding the proto-oncogene fer (Fischman et al., 1990) and transferrin (Griswold et al., 1988) have also been reported. As seen with other testis-specific mRNAs that contain extended 5`-UTRs and are translationally less active than their somatic transcripts (Hake and Hecht, 1993; Rao and Howells, 1993), the additional sequences in the 5`-UTR of the 0.93-kb SOD-1 mRNA may also modulate translation in vivo.

This question also arises: why do male germ cells need multiple SOD-1 mRNAs? SOD-1 plays a vital role in local defense against tissue damage by free radicals in the male genital tract and during the differentiation of germ cells in the testis (Nonogaki et al., 1992). Although SOD-1 activity levels in somatic tissues and cultured cells are often controlled at the transcriptional level (Delabar et al., 1987), in mouse testis, both transcriptional and translational regulation appear to be important. Many mRNAs expressed during spermiogenesis are under translational control (Hecht, 1990, 1993) and specific RNA-protein interactions have been shown to suppress translation of mRNAs encoding protamine 2 for up to 7 days (Kwon and Hecht, 1993). The synthesis of the post-meiotic less efficiently translated 0.93-kb SOD-1 mRNA suggests that in round spermatids, either the mRNA is stored for later utilization or a specific mechanism designed to prevent overexpression of SOD-1 protein is operating. In transgenic mice, overexpression of SOD-1 protein has been shown to cause a pathological state similar to that seen in Down's Syndrome (Sinet, 1982; Avraham et al., 1988; Cehallos-Picot et al., 1992). We do not know whether round and elongating spermatids are exquisitely sensitive to overproduction of SOD-1.

Although the vast majority of the 0.73- and 0.80-kb SOD-1 mRNAs are on polysomes, the opposite is true for the 0.93-kb SOD-1 mRNAs. Moreover, polysome bound transcripts derived from the 0.93-kb SOD-1 mRNAs are heterogenous and migrate more rapidly in a gel as a result of poly(A) tail shortening. We observed that translation of the post-meiotic SOD-1 mRNA is delayed, i.e. the mRNA is stored in round spermatids, and partial deadenylation occurs concomitant with its translation in elongating spermatids. This deadenylation process appears identical to that occurring with many other transcripts expressed in post-meiotic germ cells (Kleene, 1989). In addition to the deadenylation of the 0.93-kb SOD-1 mRNA, adenylation of the 0.73-kb SOD-1 mRNA also occurs. To our knowledge, this is the first report of both deadenylation and adenylation of transcripts from the same gene in the same organ.

The 5`-UTRs of mRNAs perform important roles in regulating their translation. Translation is inhibited by 96% by placing a stem-loop structure in the 5`-UTRs of yeast mRNAs (Laso et al., 1993). Certain testicular mRNAs such as the 1.7-kb cytochrome c(S) and proenkephalin mRNAs contain additional sequences in their 5`-UTRs and are not translationally active (Garrett et al., 1989; Hake and Hecht, 1993). The translational inefficiency of proenkephalin transcripts is directly related to an additional open reading frame in the 5`-UTR (Rao and Howells, 1993). Since the 0.73-kb SOD-1 mRNA is preferentially translated over the 0.93-kb SOD-1 mRNA in vitro, the 0.93-kb SOD-1 mRNA may serve as a backup system to synthesize SOD-1 when late stages of spermatids are in free radical stress. When the predominantly polysomal distribution of the 0.73-kb SOD-1 mRNA is compared with the non-polysomal location of the 0.93-kb SOD-1 mRNA (Fig. 9), the modest in vitro translation differences between the two mRNAs suggests additional factors such as RNA binding proteins may be involved in vivo. The relatively short 5`-UTRs of the multiple SOD-1 mRNAs provide an ideal system to analyze how the 5`-UTR sequences modulate translation. We propose that the 0.93-kb SOD-1 mRNA offers a unique means to post-transcriptionally regulate a crucial protein needed to balance oxidative load and antioxidant reserves in late stages of male germ cell differentiation.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HD 28832. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 617-627-3192; Fax: 617-627-3805.

(^1)
The abbreviations used are: SOD, superoxide dismutase; SOD-1, copper-zinc superoxide dismutase; kb, kilobase(s); Pipes, 1,4-piperazinediethanesulfonic acid; CREM, cAMP-responsive element modulator; UTR, untranslated region; bp, base pair(s); PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Julie Codemo and Agnes Henebury for excellent secretarial assistance.


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