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Originally published In Press as doi:10.1074/jbc.M404467200 on September 28, 2004

J. Biol. Chem., Vol. 279, Issue 50, 52493-52499, December 10, 2004
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Germ Cell Nuclear Factor Relieves cAMP-response Element Modulator {tau}-mediated Activation of the Testis-specific Promoter of Human Mitochondrial Glycerol-3-phosphate Dehydrogenase*

Mirjana Rajkovic{ddagger}§, Ralf Middendorff¶||, Marianne G. Wetzel{ddagger}, Danijel Frkovic{ddagger}, Sebastian Damerow{ddagger}, Hans J. Seitz{ddagger}, and Joachim M. Weitzel{ddagger}**

From the {ddagger}Institut für Biochemie und Molekularbiologie and Institut für Anatomie, Zentrum für Experimentelle Medizin, Universitätsklinikum Hamburg-Eppendorf, 20246 Hamburg, Germany, §Institute of Biochemistry, Faculty of Medicine, 11000 Belgrade, Serbia and Montenegro, and ||Institut für Anatomie und Zellbiologie, Justus-Liebig-Universität Giessen, 35385 Giessen, Germany

Received for publication, April 22, 2004 , and in revised form, August 30, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is an essential component of the glycerol phosphate shuttle that transfers reduction equivalents from the cytosol into the mitochondrion. Within the testis, immunohistological analysis localized human mGPDH to late spermatids and to the midpiece of spermatozoa. The expression of human mGPDH is regulated by two somatic promoters, and here, we describe a third testis-specific promoter of human mGPDH. The usage of this testis-specific promoter correlates with the expression of a shortened mGPDH transcript of ~2.4 kb in length, which is solely detectable from testicular RNA. Within the testis-specific promoter, we detected a cAMP-response element (CRE) site at -51, which binds the testis-specific transcriptional activator CRE modulator {tau} (CREM{tau}) in electrophoretic mobility shift assays. This recognition site overlaps with a nuclear receptor binding half-site at -49, which binds the testis-specific transcriptional repressor germ cell nuclear factor (GCNF). Both factors compete for binding to the same DNA response element. Ectopic expression of CREM{tau} in HepG2 cells activated a promoter-driven luciferase construct in transient transfection experiments. Additional cotransfection of GCNF relieved this activity, suggesting a down-regulation of CREM{tau}-mediated activation by GCNF. This effect was preserved by introducing the CRE/nuclear receptor-binding element into a heterologous promoter context. Our data suggest a down-regulation of CREM{tau}-mediated gene expression by GCNF, which might be a general regulation mechanism for several postmeiotically expressed genes with a temporal expression peak during early spermatid development.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH)1 is an essential component of the glycerol phosphate shuttle. In conjunction with the cytosolic glycerol-3-phosphate dehydrogenase, this shuttle uses the interconversion of glycerol-3-phosphate to dihydroxyacetone phosphate to oxidize cytosolic NADH by transferring reduction equivalents from the cytosol to the mitochondrion (1, 2). The expression of human and rat mGPDH is regulated by two somatic promoters in a tissue-specific manner (3–7), but rat mGPDH is additionally regulated by a third testis-specific promoter (8). The usage of the testis-specific promoter of mGPDH results in a shortened transcript length of ~2.4 kb containing an alternate first exon at its 5' end and a shortened 3'-untranslated region; however, the open reading frame remains unaffected (3, 8). In rat testis, mGPDH transcripts have been detected in postmeiotic germ cells during early spermatid development, whereas mGPDH proteins have been detected during late spermatid development (8). The knockout of mGPDH in transgenic mice leads to reduced fertility (9, 10).

Within the testis, spermatogenesis is a complex developmental process that includes the mitotic proliferation of spermatogonial stem cells, meiotic prophase, division of spermatocytes, and morphological changes of haploid spermatids to highly specialized spermatozoa (reviewed in Ref. 11). The developmental program of spermatogenesis is regulated by several testis-specific transcription factors, e.g. the cAMP-response element modulator {tau} (CREM{tau}) or the germ cell nuclear factor (GCNF).

CREM{tau}, a testis-specific transcriptional activator, is an alternative splice product of the CREM gene belonging to a family of proteins, which are regulated by cAMP and bind to cAMP DNA-response elements (CREs) (consensus sequence: 5'-TGACGTCA-3') (12, 13). Target gene disruption of the CREM gene (including CREM{tau}) leads to infertility in transgenic mice (14, 15). Moreover, CRE-binding sites have frequently been observed in testicular expressed genes implicated in spermatogenesis and fertility (8, 16, 17), suggesting a critical role for CREM{tau} in proper regulation of these genes during spermatogenesis.

Germ cell nuclear factor (also known as retinoid receptor-related testis-associated receptor) is a member of the nuclear receptor superfamily of ligand-activated transcription factors (18, 19); however, a ligand for GCNF is currently unknown. Target gene disruption of GCNF leads to embryonic lethality (20). In vitro studies suggested that GCNF acts as a transcriptional repressor and may inhibit transcriptional activation mediated by other nuclear receptors (21, 22). GCNF can specifically bind to direct repeats of nuclear receptor half-site (5'-AGGTCA-3') with zero base pair spacing between the half-sites or to extended single half-sites (23, 24). The binding sites for GCNF have been described e.g. within the protamine gene promoters that are expressed from postmeiotic male germ cells (16, 23, 25).

In this study, we reported the identification and initial characterization of a testis-specific promoter of human mGPDH. mGPDH contributes to the aerobic metabolism of sperms powering its motility, which is required for fertilization. The testicular expression of mGPDH is regulated by a balanced activity between CREM{tau} and GCNF, which might be a general regulation mechanism for several postmeiotically expressed target genes implicated in sperm motility and fertility.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and Characterization of DNA Sequences—A human promoter C fragment was amplified by PCR using primers hum1 (5'-ACTGTGTTGTATATAACTTCC-3') and hum2 (5'-GAAGACAGAGAATAAAGTCC-3') and human genomic DNA as template. The resulting 211-bp PCR fragments (corresponding to nucleotide positions 101880–102090; GenBankTM accession number AC092686 [GenBank] ) were gel-purified, ligated into pGEM-T Easy (Promega, Mannheim, Germany), and sequenced. This promoter fragment was further subcloned into pGL3-Basic (Promega) upstream of the luciferase gene, generating the construct hu(-106/+105)-Luc.

Human promoter C sequence from -57 to -38 (5'-CCTTTGTGAGGTCATGAATG-3', see Fig. 1) was designed as double-stranded oligonucleotides and ligated upstream of the minimal rat prolactin promoter (from -38 to +36) into pGL3-Basic (26), generating the construct CREwt-rPRL-Luc. A mutant variant of this construct was similarly designed using oligonucleotides containing the sequence 5'-CCTTTGTCTGGAGATGAATG-3' (the italic residues indicated the mutated one compared with wild type), resulting in the construct CREmut-rPRL-Luc.



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  		FIG. 1.
Alignment of human (Hu) and rat (Rat) mGPDH genomic sequences. The human sequence corresponds to nucleotides 101904–102062 (GenBankTM accession number AC092686 [GenBank] ), and the rat sequence corresponds to nucleotides 2948–3113 (GenBankTM accession number AJ495842 [GenBank] ). Uppercase letters indicate sequences of exon 1c. Lowercase letters indicate non-transcribed genomic sequences. Boldface arrows and fine arrows specify major and minor transcriptional start sites, respectively. The CRE- and NR-binding sites (see "Results") are indicated by boxes.

 
To characterize the 5' end of human testicular mGPDH mRNA, we used a rapid amplification of cDNA ends (RACE) technique, which allows only amplification from full-length transcripts with an intact 5'-cap structure (GeneRacer, Invitrogen) as described previously (8). RNA was isolated from testis tissue of two patients aged older than 50 years who were undergoing orchiectomy as the primary treatment of prostatic carcinoma. RNA was reverse-transcribed with the mGPDH-specific primer WL274 (5'-CAGATATCTCACACCACCATGG-3'). The 5' end amplification was performed with the GeneRacer 5' primer and the mGPDH-specific primer WL273 (5'-TGAGCTTCTCTGGAAGGAGG-3') using the PCR setup and cycling parameters suggested by the supplier's protocol. These first amplification products were subjected to a nested PCR using GeneRacer 5'-nested primer and the gene-specific nested primer WL301 (5'-ACAGTTGCAAGAGCACCTCC-3'). The amplification products were gel-purified, ligated into pGEM-T Easy, and sequenced.

mRNA Expression Analysis—Multiple human tissue Northern blots were obtained from Clontech (Heidelberg, Germany). A human mGPDH cDNA probe was prepared with [{alpha}-32P]dCTP by random prime labeling (Roche Applied Science). Prehybridization, hybridization, and washing were performed under high stringency conditions according to the manufacturer's instructions. To control for the relative amount of RNA in each lane, after hybridization with mGPDH cDNA, the blots were stripped by boiling in 0.1% SDS for 1 min and reprobed with a human glyceraldehyde-3-phosphate dehydrogenase cDNA.

Quantitative "real-time" PCR was conducted using the LC-DNA Master SYBR Green kit in a LightCycler (Roche Applied Science) as described previously (27). Testicular RNA was prepared as described above and reverse-transcribed with a mixture of random hexamer and oligo(dT) primers (Amersham Biosciences). Exon 1-specific PCR reactions (4) were performed using the common human mGPDH-specific reverse primer WL273 and the following human exon 1-specific forward primers: exon 1a, WL285 (5'-GAGTAGGAGAAGCCAGATCC-3'); exon 1b, WL304 (5'-GCCGAGGCTCTGATTCTGG-3'); and exon 1c, WL192 (5'-ATCAGTCACAACACTCATATCC-3'). Rat mGPDH-specific primers were as described previously (4). The crossing points of target gene amplification products were normalized to the crossing points of the "housekeeping" gene ubiquitin using the standard adjustments recommended by the supplier as described previously (27).

Electrophoretic Mobility Shift Assay—In vitro translated CREM{tau}, C-terminally fused with the FLAG epitope (26), and GCNF, N-terminally fused with the hemagglutinin (HA) epitope (28), were synthesized by a transcription/translation-coupled reticulocyte lysate system (Promega) according to the manufacturer's instructions. 1 µl of in vitro translated CREM{tau} or GCNF was incubated with 10 fmol of radioactively labeled double-stranded oligonucleotide probe hu(-62/-32) (5'-GGTATCCTTTGTGAGGTCAACAATGACATTA-3') in the presence of 1 µg of poly(dA-dT)·poly(dA-dT) as described previously (8). For competition experiments, a 100-fold molar excess of double-stranded oligonucleotides wild-type hu(-62/-32), mutated hu(-62/-32) (5'-GGTATCCTTTGTCTGGAGAACAATGACATTA-3') (the italic residues indicated the mutated one compared with wild type), or wild-type hu(-106/+105) was added to the binding reaction. For supershift experiments, 1 µl of rabbit polyclonal anti-CREM (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-HA (Cell Signaling Technology, Frankfurt am Main, Germany), or mouse monoclonal anti-FLAG (M2) (Sigma) antibodies were added to the binding reaction mixtures and preincubated for 30 or 60 min at room temperature. The shifted DNA bands were separated on 5% polyacrylamide gels and visualized by autoradiography on Kodak X-Omat AR films (Eastman Kodak Co.).

Transient Transfection Assay—Human hepatocarcinoma HepG2 cells were cultured under standard conditions in Dulbecco's modified Eagle's medium plus Glutamax (Invitrogen) and 10% fetal calf serum as described previously (4, 7). Transient transfection experiments were performed using a modified calcium phosphate technique. For each 9.6-cm2 dish, 1.4 µg of the promoter containing pGL3-Basic luciferase reporter plasmid (for constructs, see above) was mixed with 0.8 µg of CREM and 0.8, 1.6, or 2.4 µg of GCNF expression plasmids or the corresponding amount of unspecific salmon sperm DNA in 250 mM CaCl2. This solution was mixed with the same volume of 280 mM NaCl, 3 mM Na2HPO4, and 50 mM HEPES, pH 7.2, and incubated for 30 min. 230 µl of the DNA/calcium phosphate mixture was added drop by drop to the culture medium containing ~7 x 105 cells/well, and the cells were harvested after an 18-h incubation. Expression plasmids were as follows: CREM{tau}; CREM-10/FLAG and CREM{alpha}; CREM-22/FLAG in pRc/cytomegalovirus (all gifts of Birgit Gellersen) (26); and GCNF in pCMX (a gift of Uwe Borgmeyer) (24). Luciferase activities were determined as described previously (4, 7) and normalized to the total protein concentration of the samples, which were determined by the Bradford method (Bio-Rad). Luciferase assays were carried out in duplicate, and each construct was tested in at least five independent transfections with three culture dishes/experiment ± S.D. Significant values were estimated by Student's t test.

Immunohistochemistry—Immunohistochemistry experiments of human testis tissue using an anti-mGPDH-specific antibody (directed against amino acids 42–206 of mGPDH) were performed as described previously (8). Partial testes tissues were obtained from fertile patients aged from 20 to 40 years with intact spermatogenesis who were investigated to exclude a testicular tumor. For immunohistological analysis of mGPDH in spermatozoa, sperm suspensions were centrifuged (649 x g, 1 min), the supernatant was discarded, and the pellet was fixed by the administration of 4% paraformaldehyde in phosphate-buffered saline (PBS) (136 mM NaCl, 50 mM Na2HPO4, pH 7.4) followed by application to BD Falcon culture slides. After sedimentation (40 min, 4 °C), the supernatant was discarded and chambers were washed with PBS followed by drying of spermatozoa at 36 °C to improve adhesion. Subsequently, chambers were washed with PBS again and incubated with 2% normal swine serum (DAKO, Hamburg, Germany) in PBS containing 0.5% Triton X-100 for 30 min. Normal swine serum was replaced by primary antibody solution (0.2% bovine serum albumin, 0.1% NaN3, 0.5% Triton X-100 in PBS) containing rabbit polyclonal anti-mGPDH (1:600). Spermatozoa were incubated with the primary antibody at 4 °C overnight followed by three washing cycles with PBS. Secondary antibody (anti-rabbit IgG (Alexa 488 nm) (1:1000), Molecular Probes, Lexington, KY) was administered in PBS containing 0.5% Triton X-100 for 60 min in a dark chamber. Slides were washed with PBS three times and covered for fluorescence microscopy (Zeiss Axioskop, filter set 09, Zeiss, Oberkochen, Germany). For negative controls, the primary antibody was omitted or preimmune serum (1:600) was used instead of the antibody.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Human mGPDH Promoter C—To test the hypothesis that human mGPDH might be regulated by a testis-specific promoter, we performed a BLAST search using the rat promoter C sequence (GenBankTM accession number AJ495842 [GenBank] ) as query sequence against the entire GenBankTM data base entries. We were able to detect a human sequence with a sequence identity of 87% (see Fig. 1). A detailed inspection located this sequence 36.9 kb downstream of exon 1b and 2.3 kb upstream of exon 2 within the human mGPDH gene on chromosome 2q24.1 (GenBankTM accession number AC092686 [GenBank] ), suggesting a human orthologous sequence of promoter C.

The usage of promoter C in rat correlates with an alternative transcript variant, which contains the sequence of the non-translated exon 1c at its 5' end (3, 4). To verify that exon 1c-containing transcripts are also detectable from human testicular RNA, we amplified the 5' ends of mGPDH transcripts by RACE-PCR. Indeed, we successfully amplified exon 1c-containing transcripts from human testicular RNA. Because this RACE-PCR technique amplifies only those cDNA molecules, which have been generated from full-length transcripts (see "Experimental Procedures"), we simultaneously mapped the major transcriptional start site (Fig. 1, designated as +1, boldface arrow) and identified four additional alternate start sites (Fig. 1, fine arrows) of these transcripts.

Northern blot analysis from rodent RNA detected a short mGPDH transcript of ~2.4 kb solely in testis, whereas most somatic tissues contain a longer transcript of ~6.5 kb (3, 29, 30). To test whether this testis-specific transcript might also be detectable from human tissues, we performed Northern blot analysis from 23 human tissue samples. As shown in Fig. 2A,a short transcript of ~2.4 kb was solely detectable from testicular RNA. For quantification of the concentration of exon 1b- and exon 1c-containing transcripts in human and rat testes, we conducted a quantitative real-time PCR assay. As shown in Fig. 2B, exon 1c-containing transcripts were 2.6-fold as much expressed as exon 1b-containing transcripts in human, whereas exon 1c-containing transcripts were 23-fold higher expressed than exon 1b-containing transcripts in rat. This correlates well with the ratio of 6.5 to 2.4 kb transcript in human (Fig. 2A), rat, and mouse testes (3, 29) and may also correlate with the different spermatogenesis efficiencies in rodents and man.



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  		FIG. 2.
A, expression analyses of mGPDH in 23 human tissues. Multiple tissue Northern blots were hybridized using radioactively labeled probes from mGPDH and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. Transcript lengths are indicated in kb. B, relative abundance of exon 1-containing mGPDH transcripts in human and rat testicular RNA. Human and rat testicular RNA were reverse-transcribed and adjusted to a quantitative real-time PCR amplification using different exon 1-specific forward and common reverse primers. Expression levels of exon 1c-containing transcripts (1c) were normalized to the expression levels of exon 1b-containing transcripts (1b).

 
Transient Transfection Experiments—To examine the regulation of human mGPDH promoter C in testis, we introduced a sequence of the promoter from -106 to +105 into a luciferase reporter construct (hu(-106/+105)-luc). This sequence portion includes the highly conserved region between rat and human (Fig. 1) and has previously been identified to be the important sequence for promoter C activity in rat (8). After transfection of the construct hu(-106/+105)-luc into HepG2 cells, the promoter activity was barely detectable. In contrast, additional cotransfection of the transcriptional activator CREM{tau} increased the hu(-106/+105)-Luc activity 9-fold above background level (Fig. 3A). This activation was completely blocked after additional cotransfection with increasing amounts of the transcriptional repressor GCNF (Fig. 3A).



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  		FIG. 3.
Transcriptional modulatory function of CREM isoforms and GCNF on the activity of human promoter C fragments. Transient transfection experiments were performed in human hepatocarcinoma HepG2 cells. The expression vectors for CREM{alpha} and CREM{tau} and increasing amounts of GCNF were cotransfected with a luciferase reporter vector carrying the human promoter C fragment from -106 to +105 (A) or from -57 to -38 in front of the minimal rat prolactin promoter (CREwt-rPRL-Luc) or a mutated version (CREmut-rPRL-Luc) (B). Promoter activities are presented as a percentage of hu(-106/+105)-Luc or CREwt-rPRL-Luc activities, respectively, normalized to the total protein concentration of the cell extract ± S.D. Each construct was tested in at least five independent transfections with three culture dishes/experiment. Significant values estimated by Student's t test are given (*, p < 0.05; #, p < 0.01).

 
Because sequence inspections identified a putative CRE site from -51 to -44 (Fig. 1), we introduced a sequence portion from -57 to -38 into a reporter plasmid upstream of the minimal rat prolactin promoter, generating the heterologous promoter construct CREwt-rPRL-Luc. As shown in Fig. 3B, the induction of CREwt-rPRL-Luc after cotransfection of CREM{tau} into HepG2 cells was well preserved within this region. Cotransfection of CREM{alpha} (lacking the activator domain of CREM{tau}) had no significant influence on the promoter activity. Furthermore, the activation of CREM{tau} depended on the presence of an intact CRE site because mutation of this site (CRE-mut-rPRL-Luc) prevented activation (Fig. 3B).

The CRE site contains the sequence 5'-TGAGGTCA-3' (Fig. 1, -51/-44), thus differing from the sequence of a typical CRE site (5'-TGACGTCA-3') by one nucleotide. Interestingly, this nucleotide exchange generates a putative nuclear receptor (NR)-binding half-site 5'-AGGTCA-3' (Fig. 1, -49/-44). To test that this site might be a functional binding site for nuclear receptors, we cotransfected the testis-specific transcriptional repressor GCNF together with the reporter construct CREwt-rPRL-Luc and CREM{tau} into HepG2 cells. As shown in Fig. 3B, GCNF was able to relieve the CREM{tau}-mediated activation of CREwt-rPRL-Luc, suggesting an interference of CREM{tau} and GCNF signal pathways.

Electrophoretic Mobility Shift Assay—We next tested whether an in vitro translated FLAG-tagged CREM{tau} is able to bind to the CRE/NR site. Incubation of CREM{tau} with a radioactively labeled DNA fragment containing the CRE/NR site (-62/-32) generated slower migrating bands in electrophoretic mobility shift assay experiments (Fig. 4A, arrows). These CREM·DNA complexes were abolished by the addition of a 100-fold molar excess of unlabeled wild-type CRE/NR fragment but not by the addition of a 100-fold molar excess of unlabeled mutated CRE/NR site-containing fragment. Incubation with an anti-CREM antibody abolished the formation of the CREM{tau}·DNA complexes, whereas an anti-FLAG antibody supershifted this complex to lower mobility (Fig. 4A, arrowhead).



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  		FIG. 4.
Binding of CREM{tau} and GCNF to the CRE/NR response element in electrophoretic mobility shift assays. In vitro translated FLAG-tagged CREM{tau} (A) or in vitro translated HA-tagged GCNF (B) was incubated with the labeled CRE/NR site (-62/-32). For competition experiments, a 100-fold molar excess of wild type CRE/NR site, mutated CRE/NR site, or promoter C fragment hu(-106/+105) was added. Antibodies used for complex interference experiment are indicated. Specific protein-DNA complexes are indicated by arrows, non-specific complexes are indicated by asterisks, and supershift complexes are indicated by arrowheads.

 
To evaluate that this DNA element is also able to bind to the nuclear receptor GCNF, we incubated the same CRE/NR site-containing DNA fragment (-62/-32) with an in vitro translated HA-tagged GCNF. As shown in Fig. 4B, GCNF binds to the CRE/NR site, which includes the putative nuclear receptor half-site (Fig. 4B, arrow). The GCNF·DNA complex was abolished by the addition of a 100-fold molar excess of unlabeled CRE/NR wild-type fragment or an excess of fragment -106/+105 but not by addition of a 100-fold molar excess of CRE/NR-mutated fragment, which simultaneously mutated the nuclear receptor half-site. After incubation with an anti-HA antibody, the GCNF·DNA complex was supershifted to lower mobility (Fig. 4B, arrowhead). Thus, CREM{tau} and GCNF compete for binding to the same DNA element in which a CRE site overlaps with a NR-binding site.

Immunohistochemistry—To analyze the cellular localization of mGPDH within human testicular tissue, we incubated human testicular cross-sections with an anti-mGPDH-specific antibody. As shown in Fig. 5A, arrow, we observed an mGPDH immunostaining during spermatid differentiation of postmeiotic germ cells. Spermatid-specific staining was absent when preimmune serum was used instead of the antibody (Fig. 5B).



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  		FIG. 5.
Immunohistochemical staining of mGPDH in the human testis with an anti-mGPDH polyclonal antibody (a) or with preimmune rabbit serum (b). Immunopositive signals were detected in late spermatids in seminiferous tubule cross-sections (arrow).

 
To further investigate the localization of mGPDH within mature spermatozoa, we incubated spermatozoa with the anti-mGPDH antibody. As shown in Fig. 6, A and B, arrows, the midpiece of spermatozoa showed a strong immunoreactivity. Using the preimmune serum, the midpiece of spermatozoa remained unstained (Fig. 6, C and D, arrows), whereas no immunostaining was observed by omitting the first antibody incubation (Fig. 6E).



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  		FIG. 6.
Immunohistochemical staining of mGPDH in human spermatozoa with an anti-mGPDH polyclonal antibody (a and b) with preimmune rabbit serum (c and d) or without first antibody incubation (e). The arrows indicate a positive staining of mGPDH in the mitochondria-rich midpiece of spermatozoa using the anti-mGPDH antibody (a and b) but not with preimmune serum incubation (c and d).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A Testis-specific Promoter of Human mGPDH—Several lines of evidence suggested that human mGPDH is, beside the two somatic promoters A and B, additionally regulated by a third testis-specific promoter C. First, a RACE-PCR amplification approach amplified mGPDH transcripts with the sequence of exon 1c directly adjacent to exon 2, solely from testicular RNA. The expression of these transcripts is regulated by promoter C, which is located upstream of exon 1c of the mGPDH gene (Fig. 1). Second, the usage of promoter C in rat correlates with a shortened transcript length of ~2.4 kb (3, 29). Among 23 human tissues, an mGPDH transcript of ~2.4 kb was solely detected from testis tissue, suggesting a very similar expression and splicing regulation in human testicular tissue (Fig. 2A). Third, an alignment of the rat promoter C sequence with human genomic sequences identified a 48-bp human sequence portion, which shows an 87% sequence identity (Fig. 1). This sequence is located downstream of exon 1b and upstream of exon 2 sequences of the human mGPDH gene at roughly the same position compared with the rat orthologous mGPDH gene (3).

The expression of mGPDH is high in those tissues with a high ATP turnover rate (3, 29), suggesting an important role of mGPDH for appropriate energy production of these cells. Furthermore, human mGPDH was localized to late spermatids and to the mitochondria-rich midpiece of spermatozoa (Figs. 5 and 6) and, moreover, mGPDH knock-out mice showed a reduced fertility (10). Thus, a tight regulation of the testis-specific promoter may responsible for proper fertility, both in man and mice. In this context, the dysfunction of mGPDH may result in reduced motility of spermatozoa and/or alterations of appropriate spermatid development. Further investigation of mGPDH knock-out mice should address these questions.

Regulation of the Testis-specific Promoter by CREM{tau} and GCNF—A sequence inspection of the testis-specific promoter of human mGPDH identified the sequence 5'-TGAGGTCA-3' at position -51 to -44 (Fig. 1). This sequence differs from a typical CRE consensus sequence (5'-TGACGTCA-3') by one nucleotide, thus generating a nuclear receptor-binding site (5'-AGGTCA-3' at -49 to -44). Interestingly, both the testis-specific transcriptional activator CREM{tau} (Fig. 4A) and the testis-specific transcriptional repressor GCNF (Fig. 4B) bind to this sequence motif in electrophoretic mobility shift assays.

Cell experiments demonstrated that CREM{tau} is able to activate a promoter C-containing reporter construct. This activation capacity is preserved within the CRE/NR motif as indicated by introducing this response element into a heterologous reporter construct. Moreover, the CREM{tau}-mediated activation is relieved after additional cotransfection of GCNF (Fig. 3, A and B).

Down-regulation of CREM{tau}-mediated Activation by GCNF— Interestingly, the expression of rat mGPDH has been detected in postmeiotic germ cells restricted from round spermatid (step 2) up to early elongating spermatid (step 11) in a temporal expression peak (8). Since CREM{tau} protein is highly detectable in round spermatids (31), it may serve as a critical regulator for mGPDH up-regulation in round spermatids. GCNF expression reached maximal levels in stage VI–VIII spermatids (32, 33), and GCNF protein was detected at least until spermatids began to elongate (34, 35).

Therefore, it is tempting to speculate that CREM{tau} may be responsible for mGPDH up-regulation in round spermatids, whereas GCNF is responsible for down-regulation during elongating spermatid development. However, other regulation mechanisms such as posttranslational modifications (36) or competing for transcriptional cofactors (22, 37) may contribute to appropriate regulation of mGPDH.

Furthermore, an identical CRE/NR sequence motif, 5'-TGAGGTCA-3', has also been described within the testis-specific promoter of the angiotensin-converting enzyme. This site has been located from -55 to -48 relative to the transcriptional start site and has been shown to be a binding site for CREM{tau} (17). A small portion of the angiotensin-converting enzyme promoter from -91 to +17 can efficiently target a reporter construct to step 4–14 spermatids (38, 39).

Thus, mGPDH and the testis-specific promoter of the angiotensin-converting enzyme have been shown to have the same critical response element and both proteins show similar temporal expression peaks during spermatid development. This expression pattern may be regulated by competing of the transcriptional activator CREM{tau} with the transcriptional repressor GCNF to the same binding site. Further investigations of CREM{tau}- and GCNF-regulated target genes should clarify whether this counteractive regulation might be a general regulation mechanism during spermatid development.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AC092686 [GenBank] and AF311325 [GenBank] .

* This work was supported by grants from the Deutsche Forschungs-gemeinschaft (GRK336 and WE2458/3-1) (to R. M., H. J. S., and J. M. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: Institut für Biochemie und Molekularbiologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. Tel.: 49-40-42803-4526; Fax: 49-40-42803-4862; E-mail: weitzel{at}uke.uni-hamburg.de.

1 The abbreviations used are: mGPDH, mitochondrial glycerol-3-phosphate dehydrogenase; CREM, cAMP-response element modulator; Luc, luciferase; GCNF, germ cell nuclear factor; CRE, cAMP-response element; wt, wild type; mut, mutated; RACE, rapid amplification of cDNA ends; HA, hemagglutinin; PBS, phosphate-buffered saline; NR, nuclear receptor; rPRL, rat prolactin. Back


   ACKNOWLEDGMENTS
 
We are indebted to Birgit Gellersen and Uwe Borgmeyer for the gifts of plasmid DNA and helpful comments on the paper.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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