Molecular Human Reproduction, Vol. 10, No. 2, pp. 129-135, 2004
© European Society of Human Reproduction and Embryology 2004
Expression of activator of CREM in the testis (ACT) during normal and impaired spermatogenesis: correlation with CREM expression
1Institute of Veterinary Anatomy, Histology and Embryology, University of Giessen, 2Institute of Anatomy, Developmental Biology, University of Essen and 3Institute of Reproductive Medicine, University of Muenster and 4Covance Laboratories GmbH, Muenster, Germany
5 To whom correspondence should be addressed at: Institute of Veterinary Anatomy, Histology and Embryology, Frankfurter Strasse 98, 35392 Giessen, Germany. e-mail: Klaus.Steger{at}vetmed.uni-giessen.de
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ABSTRACT |
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The cAMP-responsive element modulator (CREM) is involved in regulating gene expression in haploid spermatids. Transcriptional activity of the CREM protein is thought to be regulated by activator of CREM in the testis (ACT). Applying RT–PCR and in situ hybridization, cell-specific gene expression of ACT was demonstrated in man, cynomolgus monkey and mouse. During normal spermatogenesis, RT–PCR revealed a strong signal in all three species. We sequenced monkey ACT cDNA and demonstrated that the putative amino acid sequence is highly conserved between these species. In situ hybridization demonstrated ACT mRNA in mid and late pachytene spermatocytes and in round spermatids. Among four infertile men with round spermatid maturation arrest (RSMA), only one patient revealed a strong signal for ACT, while three patients displayed a weak signal for both RT–PCR and in situ hybridization, although germ cells normally expressing ACT were present in these patients. In addition, CREM knockout mice known to be infertile due to RSMA also exhibited only a weak amplification product for ACT cDNA. ACT mRNA was barely detectable in some round spermatids, but was completely absent in pachytene spermatocytes. Database search revealed two and one CRE within the putative human and mouse ACT promoters respectively. Our findings indicate a conserved function of ACT during the evolution of mammalian spermatogenesis and suggest a role for CREM in ACT transcriptional regulation.
Key words: Key words: ACT/human/monkey/mouse/spermatogenesis
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Introduction |
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Several spermatid-specific genes are known to contain a cAMP-responsive element (CRE) serving as binding site for the transcription factor cAMP-responsive element modulator (CREM) (Sassone-Corsi, 1995). CREM is essential for spermatogenesis, since male mice lacking a functional CREM gene are sterile due to round spermatid maturation arrest (Blendy et al., 1996; Nantel et al., 1996). Infertile men exhibiting round spermatid maturation arrest reveal a substantial reduction or a complete lack at the level of both CREM protein (Weinbauer et al., 1998) and CREM mRNA (Steger et al., 1999). Due to alternative transcriptional start sites, alternative transcript splicing, and alternative translational start sites, the CREM gene gives rise to functionally different proteins with either activating or repressing potential on target gene expression (reviewed in Daniel et al., 2000; Behr and Weinbauer, 2001; Gellersen et al., 2002). The presence of additional and inaccurately spliced CREM repressor isoforms has been reported in patients with impaired spermatogenesis (Behr and Weinbauer, 2000).
CREM was thought to be activated via phosphorylation of Ser-117 by protein kinase A (PKA) (Sassone-Corsi, 1995). Substitution of serine to alanine, however, has been demonstrated not to affect CREM activity in round spermatids implying that, at least in germ cells, phosphorylation of Ser-117 is dispensable for CREM activation (Fimia et al., 1999). In contrast to somatic cells, a phosphorylation-independent activation of CREM by activator of CREM in the testis (ACT) has been suggested (Fimia et al., 1999). While Morgan and Whawell (2000) failed to detect transcripts of human ACT by northern blot analysis, Palermo et al. (2001) confirmed the presence of ACT mRNA in human testis by RT–PCR.
The present study investigates the expression and localization of ACT in normal and infertile testes.
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Testicular tissue
Ethical approval was granted by the ethics committee of the Justus-Liebig-University, Giessen, decision number 75/00. After written informed consent, testicular biopsies obtained from 14 infertile men, aged 22–44 years, were used. In 10 patients with obstructive azoospermia after vasectomy, biopsies were carried out for diagnostic reasons during vasectomy reversal. Histological evaluation revealed normal spermatogenesis. These samples served as normal controls. In four patients with non-obstructive azoospermia, biopsies were carried out for diagnostic reasons. Histological evaluation exhibited impaired spermatogenesis with complete lack of sperm due to a total arrest of spermatogenesis at the level of round spermatids. Monkey testes were available from four adult and mature purpose-bred cynomolgus monkeys (Macaca fascicularis). All animals were healthy at the time of tissue collection. The age range of the animals was 5–10 years. Testes from mice (n = 4) were obtained from a control group of an animal experiment which was carried out in accordance with the international guiding principles for biomedical research involving animals. The production of CREM knockout mice has already been reported (Wistuba et al., 2002). Two testes were used from these mice.
One part of the testicular tissue was snap-frozen in liquid nitrogen, the other part was fixed by immersion in Bouin’s fixative and embedded in paraffin using standard techniques. For histological evaluation, 5 µm paraffin sections were stained with haematoxylin.
Amino acid alignment
The monkey ACT sequence was obtained by sequencing both strands of an RT–PCR product amplified with the primers ACT5' (5'-ATGACAACTGCTC ACTTTTAC-3') and ACT3' (5'-CTAGATGTCAGTGTCCATTC-3'). The primers were derived from the human ACT sequence (Accession BC021723) and span the entire open reading frame. The start and stop codons of translation are shown in italics. If the monkey ACT sequence deviates from the human ACT sequence within the region covered by the primers, we were not able to detect such a difference by the strategy applied. However, the region corresponding to the primers does not contribute to the LIM domains. The monkey ACT cDNA sequence was translated into a protein sequence using the ExPASy translate tool and aligned to the mouse (Accession NM_021318) and human (Accession BC021723) ACT amino acid sequences by Clustal W analysis.
Search for CRE in the putative mouse and human promoters
In order to analyse the ACT gene promoter, we searched the databases for the human and mouse ACT transcripts with the longest 5'-untranslated region, presumably indicating the start of transcription of the ACT gene. The resulting sequences were then aligned with the corresponding genomic contigs and the 500 bp upstream of the 5'-end of the mRNA was checked for the presence of CRE.
RNA extraction, cDNA synthesis and PCR amplification
Total RNA from testicular tissue was extracted with Trizol® reagent, according to the manufacturer’s protocol (Life Technologies, Germany).
First strand cDNA synthesis of 2 µg RNA was performed using superscript II reverse transcriptase, according to the manufacturer’s protocol (Promega, Germany).
The 346 bp amplification product of the ACT gene (Accession XM_004274) was generated using PCR with 5'-GAGTGGCAATTATTGTGTGCCATG-3' as forward primer (bp 438–461) and 5'-ACCCACCAAGGAGACAGAG CATT-3' as reverse primer (bp 783–761). Four microlitres of cDNA were added to 5 µl 10xPCR-buffer II (including 1.5 mmol/l MgCl2), 1 µl dNTP (5 mmol/l each), 1 µl of each primer (10 µmol/l), 0.5 µl polymerase (Promega), and DEPC–H2O to a final volume of 50 µl. PCR conditions were 1x94°C for 2 min, 40x94°C for 45 s, 58°C for 45 s, 72°C for 45 s, 1x72 °C for 8 min.
As internal control, a 202 bp amplification product of the ß-actin gene (Accession NM_001101) was generated using PCR with 5'-CCTTCCTG GGCATGGAGTCCTG-3' as forward primer (bp 867–888) and 5'-GGAG CAATGATCTTGATCTTC-3' as reverse primer (bp 1068–1048). All primers were purchased from MWG-Biotech (Germany).
Digoxigenin (DIG)-labelled cRNA probes
The 346 nt PCR product of the human ACT gene was subcloned in pGEM-T (Promega). Plasmids were transformed in the XL1-Blue E. coli strain (Stratagene, Germany) and extracted by column purification (Qiagen, Germany). DNA-sequencing of the plasmid was performed by Qiagen. In vitro transcription of the DIG-labelled ACT cRNA was performed using the 10xRNA-DIG Labeling-Mix (Boehringer Mannheim, Germany) and RNA polymerases T3 and SP6. Vectors containing the ACT inserts were digested with NcoI and NotI (New England Biolabs, Germany) for the production of sense cRNA (NcoI) and antisense cRNA (NotI) respectively.
In situ hybridization
In situ hybridization was performed as described previously (Steger et al., 1998). Briefly, 5 µm paraffin sections were dewaxed, partially digested with proteinase K, and postfixed in 4% paraformaldehyde. After prehybridization in 20% glycerol, sections were covered with the DIG-labelled sense or antisense cRNA probes. Both cRNA were used at a dilution of 1:100 in hybridization buffer containing 50% deionized formamide, 10% dextran sulphate, 2xstandard saline citrate (SSC), 1xDenhardt’s solution, 10 µg/ml salmon sperm DNA, and 10 µg/ml yeast t-RNA. Hybridization was performed overnight at 37°C in a humidified chamber containing 50% formamide in 2xSSC. Post-hybridization washes were performed, according to Lewis and Wells (1992). After blocking with 3% bovine serum albumin, sections were incubated with the anti-DIG Fab-antibody conjugated to alkaline phosphatase (Boehringer Mannheim) overnight at 4°C. Staining was visualized by developing sections with nitroblue-tetrazolium/5-bromo-4-chloro-3-indolylphosphate in a humidified chamber protected from light. For each test, negative controls were performed using DIG-labelled cRNA sense probes.
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In man, monkey and mouse, the ACT protein consists of 284 putative amino acids (Figure 1), indicating a high conservation of the tertiary structure of the protein. When human and mouse primary structure were compared, the identity was
86% (243 out of 284 amino acids). By contrast, the putative amino acid sequence was almost completely conserved between man and monkey. Merely at two sites (positions 28 and 150), tyrosine residues (in one-letter-code Y) were replaced by structurally very similar phenylalanine residues (F) in monkey compared with man. In the mouse, there were tyrosines (Y) at positions 28 and 150 suggesting a rather late nucleotide exchange in evolution. Concerning the five LIM domains, it is the second one that is best conserved in the ACT protein.
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In testes exhibiting normal spermatogenesis, RT–PCR revealed a 346 bp amplification product for man, cynomolgus monkey and mouse (Figure 2, lanes 1, 6 and 8). Among infertile men with round spermatid maturation arrest, the intensity of the PCR signals is heterogeneous (Figure 2, lanes 2–5). In mice lacking a functional CREM gene, ACT mRNA was almost undetectable by RT–PCR (Figure 2, lanes 9 and 10). Amplification of ß-actin was used as internal standard displaying a constant signal at 202 bp. Negative controls without reverse transcriptase revealed no amplification products (Figure 2, lane 7).
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Applying the ACT cRNA antisense probe, in situ hybridization in testes from man, cynomolgus monkey and mouse exhibiting normal spermatogenesis revealed a strong signal in primary spermatocytes of the mid and late pachytene stage and in round spermatids (Figure 3A, E and H, Figure 4, Figure 5). In man (Figure 5 A), ACT mRNA resulted in strong signals in pachytene spermatocytes (stages I–VI) and weak signals in step 1 and 2 round spermatids (stages I and II). In the cynomolgus monkey (Figure 5B), ACT mRNA was present in pachytene spermatocytes from stage V to stage XII and in step 1–7 round spermatids (stages I–VII). In the mouse (Figure 4, Figure 5C), ACT mRNA could be demonstrated in pachytene spermatocytes from stage V to stage XII and in step 1–8 round spermatids (stages I–VIII). Negative controls involving the ACT cRNA sense probe resulted in no signals (Figure 3B, F and G). In men with round spermatid maturation arrest, only a small number of round spermatids was present within the seminiferous tubules and many of these cells were multinucleated, whereas spermatocytes, which normally express ACT mRNA, were present in these samples in significant number. However, expression of ACT mRNA was either drastically reduced (Figure 3C) or completely absent (Figure 3D). This heterogeneous expression pattern in patients with round spermatid maturation arrest is in keeping with data obtained by RT–PCR (Figure 2, lanes 2–5). In mice lacking a functional CREM gene, only a few round spermatids, but no pachytene spermatocytes, revealed a very weak signal for ACT mRNA compared with the wild-type control testis (Figure 3I and H).
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When analysing the putative promoter region of the human and mouse ACT gene, we found two CRE in the human 5'-flanking region at positions –31 and –62 (relative to the putative start of transcription, which was designated +1) and one in the mouse 5'-flanking region at position –41 (Figure 6).
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ACT has been reported to represent a phosphorylation-independent and, thus, new pathway for transcriptional activation by CREM in germ cells (Fimia et al., 1999). It has been demonstrated that it is the third LIM domain which interacts with the kinase-inducible domain of CREM. However, as has been shown in the present study, not the third but the second LIM domain is the best conserved domain during mammalian evolution. This suggests that the second domain serves as an important interface for the interaction of ACT with other proteins of the transcriptional machinery in germ cells. Since ACT is germ cell-specific, it is tempting to speculate that the second LIM domain of ACT may interact with other germ cell-specific transcription factors, such as TRF2 (Zhang et al., 2001a,b).
ACT is absent or at least drastically reduced in mice lacking a functional CREM gene and in men with round spermatid maturation arrest, in which CREM has been demonstrated to be only barely detectable or completely absent (Steger et al., 1999). This suggests that CREM plays an important role in ACT transcriptional regulation. The presence of the well-conserved CRE in the putative ACT promoter regions of mouse and man, in addition, suggests a direct effect of CREM on ACT transcriptional regulation. However, it does not answer the question how CREM can regulate the ACT promoter, if CREM-dependent transcriptional activation in germ cells itself depends on ACT as a co-factor. Nevertheless, our data provide evidence that CREM represents an essential factor for ACT expression and highlights the role of CREM as a key regulator of spermiogenesis.
In the present study, ACT cDNA was amplified from human testis homogenates, corroborating data from Palermo et al. (2001). In addition, ACT mRNA was localized to pachytene spermatocytes (stages I–VI) and step 1 and 2 round spermatids applying in situ hybridization. CREM mRNA has been demonstrated in pachytene spermatocytes (stages IV–VI) and step 1–3 round spermatids (Steger et al., 1999), while the CREM protein was reported to be present only in step 1–3 round spermatids (Weinbauer et al., 1998; Steger et al., 1999).
In the cynomolgus monkey, CREM protein has been reported to be present in step 3–7 round spermatids (Behr and Weinbauer, 1999). In the present study, ACT mRNA has been demonstrated in pachytene spermatocytes (stages V–XII) and in step 1–7 round spermatids, suggesting an interaction between CREM and ACT in this preclinical animal model.
In the mouse, both CREM mRNA and CREM protein is present in pachytene spermatocytes and round spermatids (Delmas et al., 1993; Behr and Weinbauer, 1999, 2001). Transcripts of ACT were demonstrated in pachytene spermatocytes (stages V–XII) and step 1–8 round spermatids (this study). The corresponding protein has been reported to be solely present in round spermatids, exhibiting a nuclear signal in step 1–7 spermatids and a cytoplasmic signal in step 8–10 spermatids (Fimia et al., 1999; Macho et al., 2002), suggesting a temporal uncoupling of transcription and translation.
Infertile men with round spermatid maturation arrest revealed a heterogeneous ACT mRNA expression. ACT mRNA is absent in pachytene spermatocytes and in most round spermatids where ACT is normally expressed, although these cells are present. Applying RT–PCR, one patient revealed a strong amplification product for ACT cDNA, while three other patients displayed only a weak signal. These results correspond with data obtained by in situ hybridization (this study) and data from Steger et al. (1999). In the latter study, CREM has been demonstrated to be only barely detectable or completely absent in men with round spermatid maturation arrest. In addition, Behr and Weinbauer (2000) reported the presence of additional, unusual and inaccurately spliced CREM transcripts in patients with impaired spermatogenesis.
Male mice lacking a functional CREM gene have been demonstrated to be infertile due to round spermatid maturation arrest (Blendy et al., 1996; Nantel et al., 1996). In the present study, as in infertile men exhibiting round spermatid maturation arrest, CREM knockout mice have also been demonstrated to exhibit only a weak signal for ACT cDNA. In situ hybridization, in addition, revealed weak staining in some round spermatids, but no staining in pachytene spermatocytes, suggesting a delayed and/or incomplete expression of the ACT gene resulting in a block of spermatid differentiation followed by infertility.
Both the stage- and cell-specific gene expression of ACT during normal spermatogenesis of various species and alterations of these expression patterns in men with impaired spermatogenesis and CREM-deficient mice will contribute to our understanding of the complex regulation of CREM-dependent gene expression in haploid spermatids. In conclusion, our observations point to a key role of ACT in primate and rodent spermatogenesis and suggest that testicular CREM can regulate ACT expression.
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Acknowledgements |
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We are grateful to Prof. Dr W.Weidner, Urological Clinic of the University, Giessen, and Dr S.Kliesch, Urological Clinic of the University, Muenster, for providing the human testicular biopsies, as well as Prof. Dr E.Nieschlag, Institute of Reproductive Medicine of the University, Muenster, for providing the testes of the CREM knockout mice. The skilful technical assistance of J.Dern-Wieloch, G.Erhardt, A.Hax, A.Hild, I.Kromberg and R.Sandhowe is gratefully acknowledged. Funding of this research programme was provided by DFG grants STE 892/1-3 and BE 2296/4-1.
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Submitted on September 26, 2003; accepted on October 7, 2003.
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