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Endocrinology Vol. 143, No. 5 1578-1588
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Atce1: A Novel Mouse Cyclic Adenosine 3',5'-Monophosphate-Responsive Element-Binding Protein-Like Gene Exclusively Expressed in Postmeiotic Spermatids

Gil Stelzer and Jeremy Don

Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel

Address all correspondence and requests for reprints to: Dr. Jeremy Don, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. E-mail: . don{at}mail.biu.ac.il


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Members of the ATF/CREB family of transcription factors are involved in gene activation in various physiological systems ranging from metabolite homeostasis, through regulation of cell cycle, to learning and memory. Two members of this family, cAMP-responsive element binding protein (CREB), and cAMP-responsive element modulator (CREM) are active during mammalian spermatogenesis and are required for this process, as has been shown by knockout and dominant negative experiments. In an effort to identify mouse proteins that interact with the testis-specific protein Tctex2, a mouse testis expression library was screened via the two-hybrid system, using the carboxyl-terminal portion of this protein as bait. A clone containing two overlapping open reading frames, related by a frameshift of one nucleotide, was subsequently isolated. The peptide that interacted with Tctex2 does not initiate from a consensus AUG codon, and it is not clear whether it exists physiologically. However, the other reading frame, initiating from an AUG codon, encodes a 315-amino acid peptide with significant sequence homology to a subfamily of the CREB genes whose prototype is the mouse LZIP peptide. This novel CREB-like peptide, designated Atce1, is specifically expressed in the testis. A developmental study using Northern hybridization and in situ hybridization analyses revealed that Atce1 transcripts begin to accumulate in testes of mice 24 d after birth, reflecting expression in mid/late round spermatids. Interestingly, EMSAs revealed that in vitro translated Atce1 binds specifically to a nuclear factor-{kappa}B-binding element rather then to a CRE element. Potential roles for Atce1 are discussed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
MAMMALIAN SPERMATOGENESIS is a complex multistep process that occurs within the seminiferous tubules. Traditionally, this process is divided into three major stages. The first consists of mitotic proliferation and differentiation of spermatogonial stem cells into diploid primary spermatocytes. The second stage consists of meiotic division of the primary spermatocytes through which the DNA content of the cells is reduced by half, to produce haploid spermatids. The third stage, spermiogenesis, is a maturation step during which histones are replaced by protamines, resulting in an intensive condensation of the chromatin, extrusion of the residual body substantially reduces the cytoplasmic volume, and a tail is formed. Each of these steps requires a particular combination of expression of genes, some of which are testis and cell type specific, some of which are expressed as specific alternatively spliced transcripts, and some of which exhibit a quantitative specific expression pattern (1, 2).

Several somatic cell types participate in the endocrine, paracrine, or autocrine regulation of spermatogenesis (3, 4, 5, 6). The main somatic cell types that affect spermatogenesis are the peritubular myoid cells that constitute the wall of the seminiferous tubules, the Leydig cells, located in between the tubules (interstitial regions), which secrete testosterone in response to an LH signal, and Sertoli cells, located within the tubules, which nurture the differentiating germ cells. Spatial and temporal gene expression as well as cell to cell cross-talk must, therefore, be tightly regulated to assure proper spermatogenesis.

An important molecular regulatory mechanism that has been shown to operate at different stages of spermatogenesis is cAMP-dependent transcriptional regulation. Two predominant transcription factors that take part in this pathway are cAMP-responsive element-binding protein (CREB) and cAMP-responsive element modulator (CREM). They both contain a basic domain/leucine zipper motif (bZIP) through which they bind a specific DNA cis element, designated the cAMP-responsive element (CRE), present in the promoter of target genes. cAMP-dependent phosphorylation of a specific serine residue (S133 and S117 on CREB and CREM, respectively) activates these transcription factors to bind p300/CREB-binding protein (CBP), a coactivator that recruits the basal transcription machinery to enable transcription initiation (for reviews, see Refs. 7 and 8).

During spermatogenesis, FSH binding to Sertoli cells results in elevation of intracellular cAMP levels and activation of CREB, leading to expression of the genes necessary to support germ cell differentiation (9). Moreover, TNF{alpha} secreted by haploid round spermatids intimately associated with Sertoli cells (during spermatogenic stages II–VIII in rat and mouse), promotes nuclear factor-{kappa}B (NF-{kappa}B)-induced expression of CREB and thereby contributes to the CREB-dependent gene expression (10, 11). Expression of an unphosphorylatable dominant negative form of CREB in Sertoli cells in vivo leads to apoptosis of meiotic spermatocytes, demonstrating that germ cell survival depends upon CREB-mediated gene expression (12). One of the peptides whose expression is directly induced by CREB activity in Sertoli cells is a CREM isoform designated ICER (inducible cAMP early repressor). This peptide consists only of the bZIP and hence functions as a potent repressor of cAMP-responsive genes (13). ICER binds to CRE-like elements in the promoter of CREB and thereby down-regulates its level, enabling a new spermatogenic wave to initiate (14).

In germ cells at early meiotic stages of prophase I, antagonist isoforms of CREB ({gamma} and {alpha}{gamma}, lacking the bZIP domain) as well as of CREM ({alpha}, ß, and {gamma}, lacking the trans-activating domain) are present (15, 16). As the primary spermatocytes complete the meiotic division and enter the haploid phase, a prominent switch occurs to the activating isoforms of CREM ({tau} and {theta}) (16, 17 ; reviewed in Ref. 18). These CREM activators are indispensable for spermiogenesis, as CREM knockout mice were shown to be completely sterile exhibiting postmeiotic arrest at the first step of spermiogenesis (19, 20). Several genes functioning at the haploid stage were shown to be activated by CREM, including that encoding transition protein-1, which plays a role in chromatin condensation (reviewed in Refs. 18, 21 , and 22). An interesting feature of CREM activity in haploid round spermatids is its phosphorylation independence. It has been shown, at least for the CREM{tau} transcription factors, that they interact with ACT (activator of CREM in testis), which functions as a coactivator, thus bypassing the requirement for the phosphorylation-dependent binding to CBP (23).

In this study we report the identification and characterization of a novel CREB-like mouse gene that is expressed exclusively in the haploid phase of spermatogenesis and thus might be an additional link in the regulatory system that orchestrates the complex process of spermiogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Two-hybrid screen
Commercial HF7c yeast cells (prototrophic for Uracil, CLONTECH Laboratories, Inc., Palo Alto, CA) were transformed with a construct encoding 142 amino acids, constituting the carboxyl terminus of Tctex2, fused to the Gal4 DNA-binding domain (BD-Tctex2, using the pBD-GAL4 phagemid vector; Stratagene, La Jolla, CA). A mouse testis expression library contained within a promoter activation domain (AD)-GAL4 shuttle vector (AD-cDNA, Leu+) was prepared according to the manufacturer’s instructions using the HybriZAP-Two Hybrid Predigested Vector Kit (Stratagene). BD-Tctex2-transformed yeast cells were grown on selective Ura-, Trp- SD medium. While in the logarithmic growth stage, these cells were transformed with the AD-cDNA library and grown on Ura-, Trp-, Leu-, His- SD agar plates. Positive clones were transferred to fresh plates with the same selective medium and later grown for 2 cycles on Leu- plates to allow for loss of the BD-Tctex2 vector. Subsequently growing colonies were replicated onto Trp- plates to enable selection of colonies that had lost the BD-Tctex2 vector. The AD-cDNA vector was isolated from the resulting yeast colonies by transfecting Escherichia coli cells (HB-101) with DNA extracted from these yeast cells, and selection for ampicillin resistance. Purified AD-cDNA plasmids were then used to retransform HF7c cells, containing the BD-Tctex2, and positive colonies were reselected by testing for the reporter genes His (plating again on Ura-, Trp-, Leu-, His- SD agar plates) and lacZ (blue colonies in ß-galactosidase assay). cDNAs from clones positive for the reporter genes were subcloned into the Bluescript plasmid and sequenced.

Obtaining full-length cDNA by cDNA library screening
A mouse testis cDNA library (Stratagene) was plated (750,000 plaque-forming units) on NZY agar plates, and plaques were transferred to nitrocellulose membranes (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH), denatured, neutralized, washed, and baked at 80 C under vacuum for 2 h. An Atce1 (see Results)-specific PCR product, 343 bp long (forward primer, 5'-CCCCCGGATCCATACACTGCTCAGAAACATC-3'; reverse primer, 5'-CCCCCCAAGCTTTGCTTTTCTTCATCTGTCAGG-3'), was labeled with [{alpha}-32P]dCTP (Multiprime DNA Labeling System kit; Amersham Pharmacia Biotech, Arlington Heights, IL) and cleaned by gel filtration to be used as a probe. Prehybridization and hybridization were followed by high stringency washes, the last of which was in 0.1x SSC for 25 min at 65 C. Filters were exposed to X-OMAT AR film (Kodak, Rochester, NY) for 15 h at -80 C, and phagemids from positive plaques were extracted. Three rounds of plaque purification were performed to verify clone specificity. After in vivo excision, the resulting cDNA was subsequently sequenced.

The GCG (Genetics Computer Group, Madison, WI) sequence analysis software package (version 9.0) as well as Blast tools were used to search for homologous sequences in GenBank, SwissProt, and other databases and to identify potential functional domains.

Northern blot analysis
Total RNA was isolated from various mouse tissues using Tri-reagent (Sigma, St. Louis, MO). For Northern blotting, 20 µg/sample of total RNA was electrophoresed on denaturing 1% agarose-2.2 M formaldehyde gel, followed by transfer to a Nytran Plus membrane (Schleicher \|[amp ]\| Schuell, Inc.) and UV cross-linking. Probe was labeled as described for the cDNA library screening. Prehybridization and hybridization were followed by high stringency washes, the last of which was in 0.1x SSC for 25 min at 65 C. The membrane was then exposed to X-OMAT AR film (Kodak) for 20 h at -80 C and developed thereafter.

In situ hybridization
Tissues were fixed in 4% paraformaldehyde (in PBS, pH 7.4) at 4 C for 24 h, washed in PBS, and thereafter kept in 70% ethanol at 4 C until embedding. For paraffin embedding, samples were dehydrated by passing through increasing ethanol concentrations (85%, 95%, and 2 x 100%, 30 min each), followed by incubation in ethanol/toluene (1:1), 100% toluene, toluene/dissolved paraffin (1:1), and 3x dissolved paraffin at 60 C (30 min each). After solidification, blocks were cut into 4-µm sections and placed on Tespa (3-aminopropyltriethoxysilane, Sigma)-treated slides, which were kept with desiccant at room temperature until used. Before hybridization, sections were deparaffinized by two xylene washes (10 min each) and subsequently treated as follows: hydration in decreasing ethanol concentrations (2 x 100%, 95%, 85%, 75%, 50%, and 30%, 2 min each), postfixation in 4% paraformaldehyde (20 min), PBS washes (two, 5 min each), proteinase K treatment for 8 min (20 µg/ml in 5 mM EDTA and 50 mM Tris-HCl, pH 7.5), PBS wash (5 min), refixation in 4% paraformaldehyde (5 min), incubation in 0.1 M triethanolamine (pH 8.0) with acetic anhydride added to 0.25% (10 min), PBS and 0.85% saline washes (5 min each), and dehydration in increasing ethanol concentrations (opposite procedure of hydration). Prehybridization and hybridization were performed according to the procedure described by Jaffe et al. (24). Slides were exposed to photographic emulsion for 2 wk before being developed, counterstained with hematoxylin and eosin, and viewed on an Olympus Corp. microscope (Melville, NY) under bright- or darkfield optics.

Chromosomal mapping
Step 1.
Eight micrograms of mouse genomic DNA from B (C57 Black/6J) and D (DBA/2J) strains were each digested with a panel of restriction enzymes and analyzed by Southern blot analysis using an Atce1-specific probe to search for a restriction enzyme that would yield a polymorphic pattern. After electrophoresis, samples were transferred and cross-linked to a Nytran membrane and hybridized with an [{alpha}-32P]dCTP-labeled Atce1 probe. Prehybridization, hybridization, and membrane washes were carried out as described above for the cDNA library screening.

Step 2.
Eight micrograms of genomic DNA from the B and D strains along with 35 genetically defined hybrids of these strains (The Jackson Laboratory, Bar Harbor, ME) were digested with the selected enzyme (BglI) and analyzed as described above. The approximate chromosomal location of Atce1 was deduced by comparing the overall B or D signal pattern of the hybrid strains with that of hundreds of genetic markers spread over the entire mouse genome, as supplied by The Jackson Laboratory.

EMSA
Atce1 and luciferase proteins were in vitro translated using the TNT Coupled Reticulocyte Lysate Systems kit (Promega Corp.) and according to the manufacturer’s specifications. Synthesized complementary (forward and reverse) oligonucleotides corresponding to specific binding elements were annealed in annealing buffer [20 mM Tris (pH 8.0), 1 mM EDTA pH-8.0, 50 mM NaCl] starting at 65 C for 2 min and then gradually lowering the temperature by 0.5 C each 30 sec to 25 C. Ten picomoles of annealed oligonucleotides were end labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase (Roche) and then purified by a Sephadex G-25 column. Thirty microliters of TNT reaction mixtures that were preincubated at room temperature for 10 min were mixed with the labeled oligonucleotides (4 µl) and further incubated at room temperature for 25 min in binding buffer (total volume, 50 µl) containing 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 0.25 µg/µl poly(deoxyinosine-deoxycytidine). Electrophoresis was performed for 4 h at 20 mA with an SE-600 series electrophoresis apparatus containing a nondenaturing 5% polyacrylamide gel and a 1x TBE buffer. The gels were dried and then exposed to X-OMAT AR film (Kodak) for 20 h at -80 C. The oligonucleotide sequences used in these assays were as follows: 5'-TCG AGC TCG GAT GGC TGA CGT CAG AGA TTA CTC-3' and 5'-CGA GAG TAA TCT CTG ACG TCA GCC ATC CGA GCT-3' for the CRE element of the somatostatin promoter, 5'-GAT CTG TAT GTA GTG ACG TCA CAA GAG AGC G-3' and 5'-GAT CCG CTC TCT TGT GAC GTC ACT ACA TAC A-3' for the CRE element of the transition protein 1 promoter, 5'-TCG AGC TCG GAT GAT TTT GTA ATG GGG TTA CTC-3' and 5'-CGA GAG TAA CCC CAT TAC AAA ATC ATC CGA GCT-3' for the C/EBP element of the albumin promoter, 5'-TCG AGC TCG GAT CAA AGT TTA GTC AAT TAC TC-3' and 5'-CGA GAG TAA TTG ACT AAA CTT TGA TCC GAG CT-3' for the activating protein-1 (AP-1) element of the c-jun promoter, and 5'-CGA GAG TAA TTG ACT AAA CTT TGA TCC GAG CT-3' and 5'-CGA GAG TAA CCC GGG AGA TTC CCC TCC GAG CT-3' for the NF-{kappa}B-binding element of the IL-2 receptor-{alpha} promoter (25, 26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Cloning of Atce1
Based on the evolutionary conservation of the meiotic process, we recently attempted to identify mouse genes that would functionally complement a mutation in the key gene, IME1, which activates meiosis in the budding yeast Saccharomyces cerevisiae. Several mouse clones that were able to activate reporter genes through specific interaction with promoters of yeast early meiotic genes were isolated (in preparation). One of these clones was shown to encode the previously reported gene, Tctex2, which confers male sterility upon mutation (27). To identify mouse genes that could interact with Tctex2, 2-hybrid analysis was performed using the 142 carboxyl terminal amino acids of Tctex2 fused to the Gal4-BD as a bait. This domain of Tctex2 interacted specifically with promoters of the yeast early meiotic genes. For the screen we used a mouse testis expression library in which the testicular proteins are fused to the Gal4-AD. Two colonies representing two different mouse genes, were positive for expression of the reporter genes (see Materials and Methods for selection procedure) and hence used for further characterization. This report focuses on one of these clones that contained a 550-bp insert. Northern analysis of total mouse testis RNA using this insert as a probe yielded an apparent signal of 1.35 kb (not shown), suggesting that the clone did not contain the full-length cDNA. Using our insert as a probe, we performed a high stringency screening of a mouse testis cDNA library (Stratagene) from which we isolated a cDNA clone, the sequence of which is presented in Fig. 1Go. This clone contained two overlapping open reading frames related by a frameshift of one nucleotide. The reading frame encoding the peptide that interacted with Tctex2 (nucleotides 477–869) does not initiate from a consensus AUG codon, and it is not clear yet whether it exists physiologically. However, with a frameshift of 1 nucleotide, this cDNA clone contained a much longer open reading frame of 315 amino acids (Fig. 1Go). This reading frame, which starts with a canonical start codon, is characterized by a basic domain (amino acids 140–162, gray background) followed by a leucine zipper motif. The 6 C-terminal amino acids of the marked basic domain (dark gray background, Fig. 1Go) constitute a putative nuclear localization signal. In addition, two glutamine-rich regions can be noted. The first is located at the amino-terminal region of the peptide, and the second included in the leucine zipper region and extends beyond it toward the carboxyl end of the peptide. A hydrophobic stretch of 19 amino acids (amino acids 219–237) is evident just C-terminal to the leucine zipper. A search through the various databases revealed that this peptide shows significant homology to members of the CREB family of transcription factors. More specifically, it showed the most significant homology to a CREB subfamily prototyped by the mouse LZIP-1 and LZIP-2 genes (28), in which the bZIP domain that constitutes the CRE-binding domain is located in the central region of the peptide (Fig. 2AGo). This new gene was designated Atce1 (attaching to CRE-like 1; GenBank accession no. AF287260). Atce1 exhibits 43% identity and 62% similarity over 128 amino acids to LZIP-1 and LZIP-2 (Fig. 2BGo). In addition, 48% identity and 65% similarity over 112 amino acids and 45% identity and 62% similarity over 155 amino acids were demonstrated with human luman (CREB3; Ref. 29) and human CREB-H (25), respectively (Fig. 2BGo). In all cases the homology was confined to the bZIP domain including the hydrophobic stretch.



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  		Figure 1. cDNA sequence of Atce1. The Atce1 open reading frame starts with a putative start codon encoding a 315-amino acid peptide. A basic DNA-binding domain appears in a gray background (amino acids 140–162), enclosing within it a putative nuclear localization signal appearing in dark gray. The basic domain is followed by a leucine zipper motif (leucine residues spaced 7 amino acids apart are indicated with a black background). A glutamine-rich region appears in the N-terminal region of the peptide, and an additional one begins within the leucine zipper motif continuing onward toward the C terminus. A hydrophobic stretch of 19 amino acids (amino acids 219–237) is located just C-terminal to the leucine zipper.

 


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  		Figure 2. Structural similarity between Atce1, LZIP, and CREB proteins. A, Schematic representation of the CREB, LZIP, and Atce1 proteins exhibiting similarity with respect to their DNA-binding and leucine zipper domains. Atce1 also has two glutamine-rich domains, designated Q1 (near the N terminus of the protein) and Q2 (beginning within the leucine zipper domain). Potential PKA phosphorylation sites are illustrated as dotted gray arrows. The location of a putative transmembrane domain encoded by the hydrophobic stretch is shown as a wavy gray box. B, Amino acid sequence similarity among Atce1, LZIP1/2, luman (CREB3), and CREB-H (ranges from 62–65%), all of which are mammalian members of the CREB subfamily prototyped by LZIP. Similar amino acids appear in bold within boxes, whereas identical amino acids have a black background.

 
Expression characterization of Atce1
To determine the tissue distribution of Atce1 expression in the mouse, RNA was isolated from nine adult tissues, including testis, epididymis, ovary, kidney, liver, lung, heart, brain, and spleen, and analyzed by Northern blot hybridization. A fragment encoding the N-terminal region of Atce1 that does not show significant homology to any sequence in GenBank was used as a specific probe. A single transcript of about 1.3 kb was detected exclusively in testis RNA (Fig. 3AGo). No signal could be detected in any other tissue.



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  		Figure 3. Northern blot analysis of Atce1. Samples of mouse total RNA (20 µg) were electrophoresed in denaturing 1% agarose-2.2 M formaldehyde gel, blotted onto a Nytran membrane, and hybridized with a probe specific for Atce1 (consisting of the 5'-region of the clone) under stringent conditions. A, RNA was extracted from the following tissues: testis (T), epididymis (Ep), ovary (O), kidney (K), liver (Li), lung (Lu), heart (H), brain (B), and spleen (Sp). The observed signal exhibited specificity of expression of Atce1 to testis. B, A developmental blot using total RNA (20 µg) from mouse testes at pn d 7, 10, 12, 14, 17, 21, 24, and 27 and adult testes. Results show that Atce1 transcripts begin to appear on pn d 24, whereas the major part of Atce1 accumulation occurs later, from d 24 onward. This pattern corresponds to the appearance of mid/late round spermatids in the testis.

 
To define the spermatogenic stages at which Atce1 is expressed within the testis, we took advantage of the established developmental schedule of mouse testis (30, 31). It has been shown that testes of d 7 postnatal (pn) mice contain somatic cells and spermatogonia cells only. By pn d 10 leptotene spermatocytes appear, by pn d 12 zygotene spermatocytes are present, and by pn d 14 and 17 early and late pachytene spermatocytes, respectively, are seen. Haploid round spermatids first appear at pn d 21 and elongating spermatids around pn d 24–25. By d 30 elongated spermatozoa can be found. Total RNA was isolated from testes of pups at different postnatal ages and analyzed by Northern blot hybridization using the Atce1-specific probe. As shown in Fig. 3BGo, a faint signal first appears at pn d 24, whereas at pn d 27 the signal is significantly intensified. These results suggest that not only is Atce1 specific to the haploid stage of spermatogenesis, but that it accumulates rather late during mid/late round spermatid stages.

To further define the cell specificity of Atce1 expression within the testis, in situ hybridization experiments on testis sections from normal mature as well as from newborn animals was performed. Antisense and sense RNA probes derived from the Atce1-specific region were used for experimental and control hybridizations, respectively. Prominent signal was observed in some, but not all, tubules of the mature testis (Fig. 4Go, A and B). A careful examination of the cell associations within the different tubules revealed that tubules significantly expressing Atce1 are at stages V–VIII of the spermatogenic cycle, whereas tubules at stages I–IV hardly express Atce1 if at all (not shown). A higher magnification examination demonstrated that round spermatids are the predominant cell population that express Atce1 (Fig. 4Go, C and D). No signal above background could be detected in primary spermatocytes or over interstitial regions (Fig. 4Go) or when the sense probe was used in control experiments (not shown). These results suggest, in agreement with the Northern analysis, that Atce1 transcripts accumulate rather late during the round spermatid stage. Moreover, when sections from postnatal testes were analyzed by in situ hybridization, no signal above background could be detected until pn d 24. No signal was detected on pn d 21, when haploid round spermatids start to appear (Fig. 5Go). This also strengthens our conclusion that significant amounts of Atce1 are present only in mid/late round spermatids.



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  		Figure 4. In situ hybridization of adult mouse testis with Atce1 showing expression specific to the round spermatid stage. Four-micron-thick paraffin sections of adult mouse testes were hybridized with an Atce1-specific antisense riboprobe. Sections were exposed to the emulsion for approximately 2 wk and then were stained with eosin-hematoxylin. A and B, Low magnification shows an overview of the section in which signal seems to correspond to the stage of the spermatogenic wave of the tubules. Bar, 100 µm. C and D, High magnification reveals that the signal is specific to cells at the round spermatid (RS) stage. The signal seems to characterize tubules at stages V–VIII of the spermatogenic cycle. Bar, 10 µm. A and C, Brightfield view; B and D, darkfield view. PS, Primary spermatocytes; Ly, Leydig cells.

 


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  		Figure 5. In situ hybridization of pn d 21 and 24 mouse testis with Atce1. Four-micron-thick paraffin sections of 21- and 24-d-old mouse testes were hybridized with an antisense riboprobe specific to Atce1. Sections were exposed to the emulsion for approximately 2 wk, and then stained with eosin-hematoxylin. A and B, Low magnification of 21-d-old mouse testis exhibits only a background signal. E and F, Low magnification of 24-d-old mouse testis demonstrating that most of the tubules express high levels of Atce1. Bar, 100 µm. C and D, High magnification of 21-d-old mouse testis confirming that even though round spermatids are present, only background signal is apparent. G and H, High magnification of 24-d-old pup testes reveals that cells at the round spermatid stage display specific Atce1 signal. Bar, 10 µm. A, C, E, and G, Brightfield view; B, D, F, and H, darkfield view.

 
Chromosomal localization of Atce1
With the goal of identifying known mutations that may map to the same chromosomal location as Atce1, we determined the chromosomal location of Atce1, using the hybrid polymorphism method. First, we performed Southern analysis of genomic DNA of two distantly related mouse strains, DBA/2J (D) and C57 Black/6J (B), in an attempt to identify a restriction enzyme that would result in polymorphism when hybridized with the Atce1 probe. Then, using the same restriction enzyme (BglI in our case), we analyzed 35 hybrid lines derived from these 2 inbred strains (The Jackson Laboratory). Comparing the obtained pattern of polymorphism (Fig. 6AGo) to The Jackson Laboratory’s database of genomic markers enabled us to map Atce1 to the mouse chromosome 3, about 41.7–43.6 centimorgans from the centromere (Fig. 6BGo). Only 1 mutation (soc) has been mapped to this region, and the phenotype of homozygote mutants (hair growth abnormalities) suggests that it is irrelevant to spermatogenesis.



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  		Figure 6. Atce1 maps to mouse chromosome 3. A, Eight micrograms of mouse genomic DNA from the B (C57 Black/6J) and D (DBA/2J) strains as well as from 35 genetically defined hybrids of these two strains were digested with BglI. After electrophoresis and transfer to a membrane, the DNA was hybridized with the Atce1-specific probe. Each hybrid genomic DNA was then assigned to B or D pattern type with respect to the Atce1 signal. B, The overall hybrid pattern was then compared with tables supplied by The Jackson Laboratory in which the B/D pattern of hundreds of genetic markers spread over the entire genome are given. This comparison enabled us to assign the Atce1 locus to approximately 41.7–43.6 centimorgans from the centromere of chromosome 3.

 
DNA binding specificity of ATCE1
To determine the DNA binding specificity of the Atce1 protein, gel mobility shift assays were performed. Synthesized complementary (forward and reverse) oligonucleotides, corresponding to the CRE element of the somatostatin promoter, the C/EBP element of the albumin promoter, the AP-1 element of the c-jun promoter, and the NF-{kappa}B binding element of the IL-2 receptor-{alpha} promoter, were annealed, end labeled with {gamma}-32P, and incubated with the full-length in vitro translated Atce1. Following electrophoresis, the dried gels were exposed to film and analyzed for supershift of protein-oligonucleotide complexes. To our surprise, Atce1 showed specific binding to the NF-{kappa}B rather then to the CRE element itself (Fig. 7AGo). To confirm these results we repeated the experiment with oligonucleotides, corresponding to the CRE element of the transition protein 1 promoter, an element that has been shown to be specifically activated by CREM{tau} during the haploid phase of spermatogenesis (26). The results obtained in this set of experiments were in complete agreement with those obtained with the CRE element of the somatostatin promoter. No binding to the CRE element was apparent, whereas prominent binding to the NF-{kappa}B-binding element was evident (not shown). The binding specificity was further confirmed by competition analysis between 32P-labeled NF-{kappa}B-binding element and increasing concentrations of cold (unlabeled) NF-{kappa}B or CRE element sequences (Fig. 7BGo). One picomole of cold NF-{kappa}B-binding sequence was sufficient to eliminate Atce1 binding to the labeled NF-{kappa}B-binding element, whereas even 10 pmol cold CRE sequences did not reduce the binding intensity of Atce1 to the labeled NF-{kappa}B-binding element. In vitro translated luciferase as well as the naive reticulocyte lysate were used as negative controls. We concluded that Atce1 binds specifically to the NF-{kappa}B-binding element and not to the CRE element.



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  		Figure 7. EMSA of Atce1. A, Synthesized complementary oligonucleotides, representing CRE, C/EBP, AP-1, and NF-{kappa}B elements (derived from various promoters; see Materials and Methods) were annealed, end labeled with [{alpha}-32P]ATP, and incubated with in vitro translated Atce1 protein. In vitro translated Luciferase (Luc) protein as well as lysate not supplemented with DNA for transcription and translation (Lst) were used as negative controls. A specific signal representing DNA binding of Atce1 was observed only with the labeled NF-{kappa}B element (arrow). B, Competition assay for the binding of Atce1 to the NF-{kappa}B element. This assay was performed by adding increasing amounts of unlabeled (cold) NF-{kappa}B or CRE oligonucleotides (0.1, 1, or 10 pmol) to the binding reaction in addition to the labeled NF-{kappa}B element. In vitro translated luciferase (Luc) protein was used as a negative control. A decrease in signal intensity was observed only when unlabeled NF-{kappa}B elements were added. No reduction in signal intensity could be observed when unlabeled CRE elements were added.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
The importance of the CREB/CREM family of transcription factors during mammalian spermatogenesis is apparent from both an evolutionary and a functional perspective. From an evolutionary point of view, the fact that two members of this family, CREB and CREM, operate at various stages of spermatogenesis (with several alternatively spliced forms each) together with the fact that structural and functional homologs of these genes were found to play similar roles in spermatogenesis in other mammals, including rodents, primates, and man (32, 33, 34, 35), reflect the importance of this gene family for proper mammalian spermatogenesis. Several reports have demonstrated that the regulatory effect of CREB and CREM on spermatogenesis involves molecular cross-talk mainly between Sertoli cells and the differentiating germ cells (10, 36). Such evolutionary conservation has been demonstrated by rat spermatogenesis in mouse seminiferous tubules (and vice versa) after rat spermatogonia stem cells transplantation (36, 37). In these experiments rat germ cells correctly interact with mouse Sertoli cells. From a functional point of view, it has been shown that after the injection of a dominant negative CREB form (i.e. an unphosphorylatable form in which Ser133 was replaced by Ala) into rat seminiferous tubules, the mutant molecules found their way into Sertoli cells and consequently caused primary spermatocytes to undergo massive apoptosis (12). This demonstrated the dependence of germ cell viability on Sertoli-derived factors activated by functional CREB. As to CREM, gene-targeting experiments showed that in knockout mice spermatogenesis is arrested immediately after meiosis has been completed. In these mutant mice spermiogenesis does not take place, resulting in total depletion of mature sperm cells (19, 20). Moreover, cases of haploid arrest in human spermatogenesis were also associated with the absence of CREM expression (34). In this study we have identified a new member of the CREB family, Atce1, which exhibits a unique tissue, cell type, and spermatogenic stage specificity. Expression of Atce1 is restricted to testis, and within the testis it is expressed only in haploid spermatids, mainly mid/late stages of round spermatids. Given the key roles that genes from the CREB family play during mammalian spermatogenesis and given the unique expression pattern of Atce1, we propose that this new member of the family might also be a significant link in spermiogenesis.

Different alternatively spliced forms of CREB and CREM function during spermatogenesis as either transcriptional activators or repressors. In Sertoli cells, CREB acts as an activator of various factors needed to support germ cell viability and differentiation. ICER, an alternatively spliced form of CREM, on the other hand, acts as a potent repressor of the very same genes activated by CREB, including CREB itself (14). This regulatory loop contributes to the cyclic nature of spermatogenesis. In germ cells there is a sharp switch between antagonist forms of CREM (not ICER) that are expressed (although in low levels) in meiotic spermatocytes, to the {tau} and {theta} activating isoforms that are abundantly expressed in haploid spermatids (17, 18). A common feature of the activating forms is a complete bZIP domain, required for dimerization and DNA binding, and at least one Q (glutamine-rich) domain important for interaction with transcription machinery. In the case of CREB, active forms must also contain a PKA phosphorylation site, S133, which needs to be phosphorylated before binding to CBP and recruiting the basal transcription machinery. All of the suppressive forms are missing at least one of the above-mentioned elements. Atce1 contains a complete bZIP domain, including a putative nuclear localization signal, two Q regions, and two potential PKA phosphorylation residues (S13 and S91). These features favor the idea that Atce1 functions as an activator. This assumption could be further supported by the fact that other members of the LZIP subfamily of CREB [classification suggested by Omori et al. (25)] were shown to function as activators. BBF-2, a Drosophila homolog of this subfamily (38, 39), was reported to act as a tissue specific transcription activator of alcohol dehydrogenase in the Drosophila fat body (equivalent to mammalian liver). The human CREB-H, a liver-specific gene, was shown to activate the luciferase reporter gene in transient transfection assays in vitro (25).

Given the homology of Atce1 to other CRE binding LZIP proteins, especially in the basic domain that is involved in DNA binding, it was a surprise to find out that Atce1 binds to the NF-{kappa}B element and not to the CRE element. There are reports that members of this family of transcription factors bind sequences other then CRE. This is the case with LZIP-1 that binds to the AP-1 element (28) and CREB-H that binds to the B-Box element (25). Another ATF family member, ATFa, was shown to be a component of the NF-ELAM1 complex that is involved in activation of the E-selectin promoter and to bind to the NF-ELAM1 element, which differs from the consensus CRE element by a single nucleotide substitution (40). However, in all of these cases the ATF/CREB members bind these sequences in addition to their ability to bind CRE itself, or alternatively, the sequence is closely related to CRE. Atce1 seems to be a unique case where an ATF/CREB family member binds to the NF-{kappa}B element and does not bind to the CRE element.

If indeed Atce1 acts as an activator, what genes can it activate based on the rather late accumulation of its transcripts during spermiogenesis and its DNA binding specificity? One possibility is that it can activate late haploid genes containing the NF-{kappa}B element in their promoter. This might enable the cell to graduate the expression of the haploid genes in a way that early haploid genes will be activated by CREM, whereas late haploid genes will be activated by Atce1. This seems more reasonable then having two different transcription factors that are both expressed at the haploid phase of spermatogenesis and both recognize the same regulatory element. A second possibility is that there are genes containing both the CRE and the NF-{kappa}B elements in their promoter such that both CREM and Atce1 are involved in regulating their expression. This might enable fine-tuning of the expression. This is how CREB is regulated in Sertoli cells (10, 11), this is how the P-selectin gene is regulated in murine endothelial cells (41), and this is part of the regulatory mechanism of the mouse major histocompatibility class I genes (42). A third possibility is that Atce1, as a member of the ATF/CREB family, can heterodimerize with CREM via their ZIP domain and hence regulate CREM binding to DNA. Potentially it is possible that this latter possibility can be executed in parallel to the former two, performing a very efficient regulatory system. An additional intriguing speculation is that the Atce1 protein is stored in the sperm head to be delivered by fertilization to the zygote, where it might function as a zygotic or early embryonic activator.

It should, however, be stressed that as we do not yet have experimental data to prove that Atce1 indeed acts as an activator, we cannot rule out the possibility that it functions as a repressor that shuts down expression of early haploid genes.


   Conclusion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
We have isolated a new member of the LZIP subfamily of the CREB transcription factors. This new gene, Atce1, is testis specific, and its transcripts start to accumulate exclusively in mid/late round spermatids, a differentiative stage at which few genes are transcriptionally active. Despite being a member of the CREB family that typically bind to CRE elements, Atce1 was found to bind the NF-{kappa}B element rather then the CRE element. It might function as an activator of late haploid genes or as a suppressor of early haploid genes. An alternative possibility is that it might function as a paternal protein in the zygote or in early embryonic developmental stages.


   Acknowledgments
 
The authors are grateful to Prof. Shula Michaeli for her constructive advice, and to Dr. Shelley Schwarzbaum for reviewing the manuscript. This work was performed by Gil Stelzer as part of his Ph.D. research in the Faculty of Life Sciences at Bar-Ilan University.


   Footnotes
 
This work was supported by Grant 97-00318 from the United States-Israel Binational Science Foundation (Jerusalem, Israel).

Abbreviations: AD, Activation domain; AP-1, activating protein-1; BD, binding domain; bZIP, basic domain/leucine zipper motif; CBP, CREB-binding protein; CRE, cAMP-responsive element; CREB, cAMP-responsive element-binding protein; CREM, cAMP-responsive element modulator; NF-{kappa}B, nuclear factor-{kappa}B; pn, postnatal.

Received September 17, 2001.

Accepted for publication January 28, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

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