Mol. Hum. Reprod. Advance Access originally published online on September 2, 2005
Molecular Human Reproduction 2005 11(8):567-574; doi:10.1093/molehr/gah209
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Combinations of genetic changes in the human cAMP-responsive element modulator gene: a clue towards understanding some forms of male infertility?
nik2
21Institute of Biochemistry, Medical Center for Molecular Biology, 2Clinic for Reproduction of Domestic Animals, University of Ljubljana, 3Department of Obstetrics and Gynecology, Andrology Center, University Medical Center Ljubljana, Ljubljana, Slovenia, 4Institut de Génétique et Biologie Moléculaire et Cellulaire, CNRS, INSERM, Université Louis Pasteur Illkirch-C.U. de Strasbourg, Illkirch, Strasbourg, France and 5Center for Functional Genomics and Bio-Chips, Institute of Biochemistry, University of Ljubljana, Ljubljana, Slovenia
6 To whom correspondence should be addressed at: Center for Functional Genomics and Bio-Chips, Institute of Biochemistry, University of Ljubljana, Zaloka 4, SI-1000 Ljubljana, Slovenia. E-mail: damjana.rozman{at}mf.uni-lj.si
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The cAMP-responsive element modulator (CREM) gene plays a pivotal role in the mouse spermatogenesis, but its role in the human infertility has not been fully established. We performed a mutation screening in 13 Slovenian men with round spermatid arrest and in six controls. Eleven genetic changes have been identified in the human CREM gene, three novel single-nucleotide polymorphisms [within the promoters P1, P3 and intervening sequence 1 (IVS1)], one insertion (IVS2) and one non-sense mutation (exon
). Some infertile patients seem to accumulate potentially harmful genetic changes. We identified a patient with no CREM immunoreactive protein that was homozygous for the nucleotide changes in all promoters, IVS 1, 2, 6, and was heterozygous for the mutation in exon . Interestingly, insertion in IVS2 (IVS2-58_55insT) results in a four-fold decrease in binding of nuclear proteins. Computer predictions suggested the presence of a potential novel CREM promoter, however, random amplification of cDNA ends from the human testis cDNA library was not successful in confirming a novel transcription start site of the CREM gene. Screening of a larger number of patients and controls is required to elucidate whether the observed combinations of genetic changes in the CREM gene can explain some forms of male infertility. Key words: CREM/expression/mutation detection/non-obstructive azoospermia/regulation
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The cAMP-responsive element modulator (CREM) gene encodes both activators and repressors of cAMP-dependent transcription (Sassone-Corsi, 1995
). Products of the CREM gene are not involved in the spermatid formation but are essential for the initiation of spermatid maturation and development (Blendy et al., 1996; Nantel et al., 1996). The CREM gene is regulated differentially during male germ cell development. CREM expression is developmentally switched from antagonist forms in premeiotic germ cells to the activator forms in postmeiotic germ cells and is regulated at multiple levels: at the transcriptional level by the use of four different promoters (Molina et al., 1993; Stehle et al., 1993; Daniel et al., 2000), at the level of transcript splicing (Foulkes et al., 1991, 1992), by the combinatorial usage of exons and by alternative polyadenylation sites (Foulkes et al., 1991, 1992) and at the level of translation initiation (Delmas et al., 1992; Gellersen et al., 2002).
Mice lacking the CREM gene exhibit a specific arrest of round spermatid development, a phenotype similar to some human infertility conditions—the round spermatid maturation arrest (Blendy et al., 1996; Nantel et al., 1996). Circumstantial evidence is available suggesting that CREM deficiency is associated with spermatogenic disorders in men (Peri et al., 1998; Weinbauer et al., 1998). Additionally, an increasing number of genes expressed in testis and involved in spermatogenesis and fertility have been shown to contain cAMP-responsive element (CRE) in their promoter regions (reviewed in Behr and Weinbauer, 2001) which may cause indirect effects in the case of CREM deficiency. Expression of CREM activators thereby seems to be a prerequisite for normal spermatogenesis. If only repressor forms are expressed, this may result in male infertility (Blocher et al., 2003).
The human CREM gene has been mapped to chromosome 10p11.1-12.1 in 1993 (Masquilier et al., 1993). Its exon/intron organization was at that time predicted in accordance to the splice variant cDNAs isolated from various tissues and predicted similarities with the cAMP-responsive element-binding protein (CREB) gene (Entrez S68271
[GenBank]
; Masquilier et al., 1993). It was postulated that CREM has two alternative DNA-binding domains, that are incorporated into the main open-reading frame by alternative splicing, and 10 exons encoding different functional domains (Foulkes et al., 1991). At least 14 exons have been identified to date, three of them being non-translated. This nomenclature of the exonic structure is based on the nomenclature provided by Walker and Habener (1996) and extended by Gellersen and Daniel (Daniel et al., 2000; Gellersen et al., 2002).
The structure of the CREM gene is complex. The CREM transcripts can be generated from four different promoters. The ‘conventional’ CREM promoter P1 exhibits housekeeping characteristics. It is located upstream of exon B, is GC rich and not inducible by cAMP (Sassone-Corsi, 1995). The CREM
2 activator isoform is transcribed from promoter P1 and represents one of the most abundant transcripts in the human testis (Blocher et al., 2003). Also all other CREM mRNAs that contain exon B are transcribed from promoter P1. Exon B carries the first translation initiation codon, whereas exons A0 and II (A) seem not to be translated. A second promoter, P2, is located within the 10 kb intron between exons G and (Molina et al., 1993; Stehle et al., 1993). This promoter is strongly inducible by cAMP and contains two pairs of CRE elements and is responsible for the transcription of the cAMP-inducible CREM isoform inducible cAMP early repressor (ICER). Two additional promoters P3 and P4 have been described in the rat CREM gene sequence, being located between exons B and C (Daniel et al., 2000).
The aim of this study was to evaluate the role of nucleotide changes in the CREM gene in male infertility, by screening for potential mutations and/or polymorphisms in DNA samples of Slovenian men with round spermatid maturation arrest, in comparison with controls. As the beginning of our study dates before the completion of the human genome sequence, the exon/intron boundaries and some intervening sequences (IVS) of the human CREM gene had to be determined to design primers for mutation screening. Two selected human bacterial artificial chromosome (BAC) clones have been sequenced (GenBank AH009287
[GenBank]
) to determine exons and corresponding boundaries that were predicted by the originally published cDNA sequence (Entrez S68271
[GenBank]
; Masquilier et al., 1993).
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Patients, hormones and tissue
Twenty-three infertile men (mean age = 37.2 years) attending the Infertility Out-patient Clinic at the Department of Obstetrics and Gynecology, Andrology Center, Ljubljana, Slovenia and affected by azoospermia were enrolled consecutively after informed consent. All men underwent an andrological examination and were tested for serum FSH levels. FSH levels were measured by Microparticle Enzyme Immunoassay (AxSYM, Abbott Laboratories, Abbot Park, IL, USA). According to anamnesis, clinical examination (testicular volume) and FSH level, men were affected either by non-obstructive azoospermia (n = 17) or by obstructive azoospermia (n = 6). Testicular biopsy was performed to assess testicular histology and to recover sperm to be used in further intracytoplasmic sperm-injection procedure. Tissue samples were collected during routine biopsy procedures, fixed in 4% buffered paraformaldehyde and embedded in paraffin. After staining with haematoxylin eosin, tissue sections were examined by the same observer (J..). The diagnoses were as follows: normal spermatogenesis, maturation arrest, hypospermatogenesis and Sertoli cell-only (SCO) syndrome. Additionally, cytological examination of the samples was performed to search for sperm cells. In the absence sperm, testicular samples were prepared to observe spermatids. Immunohistochemical staining using anti-CREM-specific antibody (Santa Cruz, Santa Cruz, CA, USA) was performed on the same samples. According to histological and cytological diagnosis and results of immunohistochemical staining of testis samples, three groups of patients were selected for mutation screening. As positive controls, patients with normal spermatogenesis (obstructive azoospermia) were used.
The group of mainly CREM-negative patients (n = 5), where no or only individual cells have been stained, and the group of CREM-positive patients (n = 8) with hypospermatogenesis were selected for mutation screening together with controls. The four patients with SCO syndrome were excluded. The research was approved by the Slovenian Medical Ethics Committee. It relies on the principles of the Helsinki Declaration, the Oviedo Convention and the provisions of the Draft Protocol to the Oviedo Convention, on biomedical research. The confidentiality of the personal medical data as well as other personal data relating to the individual have been assured. Groups of patients are described in Table I.
|
Immunocytochemistry
Sections (five microns) were mounted on slides coated with 3-aminopropyl triethoxy-silane (TESPA, Sigma, Taufkirchen, Germany) and dried overnight at 50°C. Before incubation with primary antibody, sections were dewaxed, rehydrated in graded ethanols, washed in water and Tris-buffered saline (TBS) (0.05 M Tris–HCl, pH 7.4, 0.85% NaCl), followed by blocking endogenous peroxidase by incubating the section for 30 min in 1% H2O2 in TBS. Sections were subjected to antigen retrieval by microwaving in 0.01 M citrate buffer (pH 6.0) on 700 W for 20 min and, thereafter, left standing for 20 min without disturbance. Sections were then washed for 5 min in TBS and blocked using normal rabbit serum (Dako, Glostrup, Denmark) diluted 1:5 in TBS. Purified polyclonal antibodies directed against CREM (Santa Cruz) were used at a dilution of 1:100 in TBS containing 20% normal goat serum. Sections were incubated with primary antibodies overnight at 4°C in humid chamber. The following day coverslips were removed, sections washed twice in TBS (5 min each wash), incubated for 30 min with goat anti-rabbit immunoglobulins (Dako) diluted 1:100 in TBS and, then, washed again in TBS (twice for 5 min). For the detection of bound antibodies, sections were first incubated with rabbit peroxidase–antiperoxidase complex (Dako) for 30 min and washed twice in TBS (5 min each). Colour reaction product was developed by incubating sections in a mixture of 0.05% (w/v) 3,3'-diaminobenzidine (DAB) tetrahydrochloride (Sigma) in 0.05 M Tris–HCl, pH 7.4, and 0.01% hydrogen peroxide. After 5–15 min, sections were washed in distilled water, counterstained with hematoxylin, dehydrated in graded ethanols, cleared in xylene and covered with a coverslip using Pertex-mounting medium (CellPath plc, Hemel Hempstead, UK). Specificity of the antibodies was controlled by using non-immune rabbit serum instead of primary antibodies.
Tissue and image analysis
Sections were analysed, and images captured into a PC computer with Lucia image analysis system (Nikon, Melville, NY, USA) using a Nikon Optophot microscope with Sony CCD camera.
Screening of human genomic BAC library and isolation of BAC DNA
Human genomic BAC library was screened by two PCR-generated human CREM-specific probes, spanning exons F and Ia–Ib. Six positive BAC clones were isolated. Two of them (134A8 and 186A15) were used as templates for automated sequencing procedure using gene-specific CREM primers. For DNA isolation, clones were grown on LB + chloramphenicol agar plates. A starter culture was inoculated by a single colony, picked from a freshly streaked selective plate. Culturing and isolation were done according to the very low-copy plasmid/very low-copy cosmid purification protocol using Plasmid Midi kit 25 (Qiagen, La Jolla, CA, USA).
Automated sequencing and mutation screening
Automated sequencing was performed by the ABI Prism 310 Genetic Analyser using gene-specific CREM primers and ABI Prism® BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA). PCRs were performed on a Perkin Elmer 9600 thermocycler with 25–50 ng of genomic DNA. PCR primers for each of the exons were selected according to the determined intronic sequences (Entrez AH009287
[GenBank]
). PCRs for exons B, C, E, F, G, H, Ia and Ib were performed with 25–50 ng of genomic DNA, 50 mM KCl, 10 mM Tris–HCl (pH 8.3), 0.4 mM dNTPs and 0.5 U of AmpliTaqTM in a total volume of 25 µL. Primer sequences and MgCl2 concentrations are given in Table II. After initial denaturation of 5 min at 95°C, the fragments were amplified by 35 cycles of denaturation at 95°C for 30 s, annealing at temperatures as designated in Table II for 30 s, extension at 72°C for 30–40 s, followed by the final extension step of 10 min at 72°C. Amplifying and sequencing the promoter regions (P1, P2, P3 and P4) and exons II, 
1, 2, ICER and were performed with previously described primers (Domschke et al., 2003). The most distal part of the P1 promoter was not amplified.
|
Preparation of nuclear extracts and gel-shift analysis
Because additional human testis tissue was not available, we chose the routinely used human choriocarcinoma cell line JEG-3 to isolate nuclear extracts for initial gel-shift analyses. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% bovine serum and 1% L-glutamine in humidified incubator at 37°C, and 5% CO2 and were split once per week 1:6. Nuclear extracts were isolated from four 60 mm culture dishes containing cells at 70% confluency according to the previously published protocol (Deryckere and Gannon, 1994). Protein concentrations were determined by the Bio-Rad dye-binding assay, according to the instructions of the manufacturer (BIO-RAD, Hercules, CA, USA).
Oligonucleotides used to generate double-stranded fragments containing non-mutated and mutated sequence around insertion of T into IVS2-58-55 upstream of exon B (–58 to –55 insT) were CREM-3T sense (5'-TCAACCTGTATTTCCAAATCATT-3') and antisense (5'-AAGTATTTGGAAATACAGGTTGA-3') and CREM-4T sense (5'-TCAACCTGTATTTTCCAAATACTT-3') and antisense (5'-AAGTATTTGGAAAATACAGGTTGA-3'). Gel-shift experiments were performed, as described previously (Rozman et al., 1999), and were repeated multiple times with identical results. Autoradiograms were scanned and quantified by densitometry, using Un-Scan-ITM, version 5.1 (Silk Scientific, Orem, UT, USA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Detection of the CREM-immunoreactive protein in testis of infertile men
Immunocytochemical staining resulted in a strong positive staining of round spermatids in all testicular tubules in men with normal spermatogenesis (Figure 1a). In patients with SCO, there was no immunopositive staining, as expected (Figure 1d). Based on the intensity of staining and the absolute presence or absence of positive cells in different testicular tubules of the same individual, patients with spermatogenic arrest were divided into two groups. One group represented patients where immunoreactive CREM protein had been observed, although not necessarily in all tubules (Figure 1b). The second group was constituted by CREM-immunonegative patients (Figure 1c), although in some cases rare individual immunopositive germ cells have been observed.
|
Genomic analysis of the human CREM gene from the BAC clones
The exon–intron boundaries of the entire human CREM gene have been determined by sequencing clones 134A8 and 186A15 from the human BAC library. Data have been deposited into GenBank (Entrez AH009287
[GenBank]
) before the release of the human genome sequence. Comparison of exonic sequences to the originally published CREM cDNA sequences (Entrez S68271
[GenBank]
, S68134
[GenBank]
and NM001881, Masquilier et al., 1993) showed some differences: three silent changes in exons B and Ib, an amino acid substitution in exon B, an insertion of C in untranslated region between the two alternative DNA-binding domains, four substitutions in the 3'-untranslated region and deviation in the first 13 bp at the beginning of exon C. The human genome sequence at the beginning of exon C (5'-GTTTCTGTGGCTGGA-3') is homologous to the equivalent cat (Entrez X99115
[GenBank]
.1), rat (Z15158
[GenBank]
.1) and mouse (AK016156
[GenBank]
.1 and AK015641
[GenBank]
.1) testis CREM mRNA sequences. In 2000, Daniel et al. (2000) suggested an existence of a polymorphism in the human CREM gene, resulting from an insertion of a partial Alu element at 5' end of the exon C. This insertion has not been detected in any of our patients or controls. Observed changes are summarized in Table III.
|
Screening for mutations in the human CREM gene in the Slovenian infertile patients
The screening of exons II (A), B, 1, 2, C, E, F, G, ICER, , H, Ia and Ib, and of the four promoters has been completed in 13 patients and six controls. Altogether 11 different changes have been detected (Figure 2). In intron 1 (patients 31, 29, 40, 24, 39, 41 and 35; controls 23, 43 and 44), a single-nucleotide change A>G was found 9 bp upstream of the exon–intron boundary (IVS1-9 A>G). Two patients (31 and 39) and one control (43) were homozygous for this change, whereas the others were heterozygous. An insertion of T 58–55 bp upstream of exon B (IVS2-58_55insT) and an intronic change upstream of exon C (IVS6-3C>T) were detected in four patients and two controls. The CREM-negative patient 31 was homozygous for both changes, whereas the other patients (29, 41 and 35) and CREM-positive controls (23 and 43) were heterozygous. We used NNSPLICE 0.9 version of the splice-site predictor program (Reese et al., 1997) to assess whether intronic changes might have an effect on splicing sites. Computer predictions indicate a high probability (score 0.82) for the creation of a novel donor splice site as a consequence of IVS6-3C>T. On the contrary, changes in IVS1 and IVS2 seem not to have a significant influence.
|
Among seven polymorphisms that have been detected in the promoter regions of the CREM gene (Figure 2), two single-nucleotide changes (–248A>C in promoter P1 and –439G>A in promoter P3) are novel, whereas others have been described previously (Domschke et al., 2003; Hamilton et al., 2004), SNP database (http://www.ncbi.nlm.nih.gov/SNP). Genotypes and phenotypes of the patients and controls are summarized in Table I. The deletion of the ATT base pair triplet in promoter P2 (Domschke et al., 2003) was also found in a homozygous form only in patient 31. In exon of patient 31 and control 23, a non-sense mutation (33G>A) was identified.
An insertion of T 58–55 bp upstream of exon B was the first change that was identified. As exons Ao and II (A) seem not to be translated, we hypothesized that this change located just upstream of the exon B, which carries the first translation initiation codon, might have some effect on the promoter-binding properties. Therefore we analysed the sequence with the program MatInspector (Quandt et al., 1995). At the spot of the insertion, three potential binding sites for transcription factors CCAAT/enhancer-binding proteins_01 (C/EBPB_01), C/EBP_C and NFAT_Q6 were identified. Comparative analysis of the sequence with and without the insertion indicated that the insertion possibly affects binding to the DNA element. As only the CREM-negative patient 31 was homozygous, although the two patients (35 and 41) classified to group 2 (CREM positive with reduced level of CREM protein and hypospermatogenesis) and two positive controls (23 and 43) were heterozygous for the insertion, we wanted to establish whether the change in the homozygous form affects binding of regulatory proteins to the potential transcription factor-binding site. Results of the gel shifts show that CREM-4T primer pair which represents the sequence with the insertion binds JEG-3 nuclear proteins with a four-times lower affinity compared with the primer pair CREM-3T (Figure 3, compare columns 1 and 3 with 2 and 4). Nuclear proteins that bind to this element are still under investigation.
|
5' random amplification of cDNA ends (RACE) analysis by CREM-specific antisense primers from exon B and the sense-linker primer did not result in specific amplification of a CREM-related fragment from the human testis cDNA library (data not shown). Several independent-cloned PCR products have been sequenced but were not related to the CREM gene.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
We completed the screening of all known exons (except exon
) and the four promoters of the human CREM gene. Eleven genetic changes have been identified in 19 screened patients and CREM-positive controls. A linkage disequilibrium between the different changes has been observed, as all of the changes were detected in several subsets of patients or controls. Five haplotypes can be deduced from combinations in patients and controls (later in the text referred to as haplotypes A, B, C, D and E). We attempted to assess a possible influence of the combination of changes on phenotypes even if our study groups were too small for any statistical analysis. Haplotypes of the patients and controls are shown in Figure 4. From all haplotypes, haplotype A is the most common (25 of 38 chromosomes) and thereby seems to be the original one, whereas haplotypes B (one chromosome), C (five chromosomes), D (five chromosomes) and E (two chromosomes) have probably evolved at a later time.
|
Haplotype C in a homozygous form was found only in patient 39 that was classified in group 2 (CREM-positive patients with reduced level of CREM protein and hypospermatogenesis; Table I). This patient is homozygous for the promoter P1 (–246A>G, –333C>G), P3 and P4 polymorphisms as well as for intronic change in IVS1. The combination of promoter polymorphisms might be the cause for a weaker signal of the CREM protein in the testis. The genotype of the CREM-negative patient 31 (E/D) is rather complex, therefore it is difficult to assess the possible effects of all additional changes that he carries in comparison with patient 39 (C/C). Patient 31 is homozygous for all changes within promoters P1, P2, P3 and P4 and also for intronic changes in IVS1, IVS2 and IVS6 and is heterozygous for the non-sense mutation in exon . Recent data show that haploinsufficiency of some genes might lead to a weak mutator phenotype (Fodde and Smits, 2002). In the case of patient 31, the combination of several homozygous changes might affect the transcription of the CREM gene and also the stability of mRNA because of its altered secondary structure. Additional polymorphism in P1 in combination with other genetic changes could be responsible for the reduced transcription of the major activator isoform 2 and other isoforms that have been characterized in human testis by Behr and Weinbauer (2000), Behr et al. (2000) and Blocher et al. (2003). The deletion of ATT in promoter P2 might also have an effect, although this promoter is usually not active in male germ cells (Daniel et al., 2000). Even if exon is rarely detected in human CREM transcripts (Blocher et al., 2003), an additional activator isoform CREM 2 with exon has been isolated from a testis exhibiting round spermatid maturation arrest (Behr and Weinbauer, 2000; Blocher et al., 2003). The function of the protein domain encoded by exon is not known, but the sequence seems to be highly conserved between mouse and monkey (Behr et al., 2000). As a consequence of IVS6-3C>T, the computer prediction showed a high probability (score 0.82) for the creation of a novel donor splice site just a few bp upstream of the usual acceptor splice site of exon C, which may lead to splicing of an alternative exon with the usage of the cryptic acceptor splice site on position –115 bp from the beginning of exon C. This can potentiate the inclusion of a new 111 bp exon in the mRNA transcript of the CREM gene. Exon C is usually absent in the most common isoforms in the human testis; however, larger isoforms containing exon C have recently been identified (Gellersen et al., 2002).
The most interesting genetic change observed was the insertion of T in IVS2, that was also detected in a homozygous form only in patient 31 (4T/4T). A comparative analysis of nucleotide change IVS2-58_55insT with the program MatInspector showed that 3T–4T change might result in lowering the binding capacity of transcription factor C/
EBP_C, suggesting also that this IVS might harbour some promoter-like activities. To evaluate which nuclear proteins bind to this IVS, we performed gel-shift analyses. Surprisingly, a four-fold diminished binding capacity of nuclear proteins to the IVS2 region with the insertion (4T/4T), compared with the sequence without the insertion (3T/3T), has been observed. To further establish whether this region contains some promoter-like characteristics, we performed computer prediction analyses using the AceView database (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly) (Lander et al., 2001) and blast (Altschul et al., 1990). About 5 kb regions of 5' from the beginning of different CREM variants have been aligned with the genomic CREM sequence. The upstream region of two CREM cDNA variants (aDec03 1030 bp AM_1023 bp and bDec03 1432 bp AM_1424 bp) corresponds exactly to the IVS2 region upstream of exon B (Figure 5), suggesting the presence of an additional promoter in IVS2. Regions of all other variants correspond to the locations of already-known CREM promoters (P1–P4) and the region upstream of exon II (A). Extensive RACE analyses with two different CREM-specific primers did so far fail to support the existence of a functional promoter upstream of exon B. However, RACE analyses cannot exclude the possibility that the promoter at that region of the CREM gene does exist. However, the testis transcript resulting from the potential P5 promoter may be so rare that it might be detected only by screening hundreds of PCR clones of undefined length. In any case, the mutation of the C/EBP site in IVS2 of the human CREM gene should not be ignored. The binding site for the transcription factor C/EBP is also an enchancer element (Lekstrom-Himes and Xanthopoulos, 1998) that could work in large distances and could affect transcription from other CREM promoters (Blackwood and Kadonaga, 1998; Lee and Young, 2000). Consequently, we cannot exclude the possibility that the homozygous change in patient 31 might also contribute to the diminished CREM mRNA expression resulting in the absence of the CREM protein.
|
Besides the genetic changes, differences in FSH level have also been observed. The two infertile patients (32 form group 1 and 20 from group 2) with highly elevated FSH levels (204 and 30 mIU/ml) exhibited no nucleotide changes in the CREM gene. High levels of FSH (above 8.5 mIU/ml) usually indicate patients with non-obstructive azoospermia. However, according to the recent findings, this diagnostic criterion wrongly excludes many patients with maturation arrest; in some cases, no increases in FSH levels were observed (Ezeh, 2000). Our data suggest that in some patients high levels of FSH combined with no nucleotide changes in the CREM gene might contribute to male infertility, in contrast to other patients, where infertility might depend on an aberrant CREM gene, with FSH levels that remain normal. This is interesting since FSH regulates CREM function in testis at the level of post-transcriptional processing (Foulkes et al., 1993). If the first pathway (elevated FSH) is relevant, some other genetic and/or epigenetic changes that are involved in arresting the maturation process of spermatids are likely. Monaco et al. (1995) showed that FSH specifically induced expression of the ICER isoform lacking exon in Sertoli cells, thus leading to the down-regulation of cAMP-induced transcription (Monaco et al., 1995).
In conclusion, even if no direct connection between the detected nucleotide changes in the CREM gene and lack of its expression has been observed in our groups of infertile men, individual patterns of homozygous and heterozygous alternations could exert pathological changes that render these patients infertile. A phenomenon, called ‘genetic load factor’ (Vogt, 2004), proposes that multiple somatic genetic disorders, namely nucleotide changes in several other genes, may cause male infertility, through a specific pattern of usually heterozygous nucleotide changes that affect an apparently remote cell system.
|
Acknowledgements |
|---|
We thank Dr. Jennifer Skaug from MRC Genome Resource Facility, Department of Genetics, The Hospital for Sick Children, Toronto, Ontario, for screening the human genomic BAC library. We also thank Dr. Jasna
inkovec for the interpretation of the clinical data and Nina Hojnik for her contribution to the work on IVS2-58_55insT variants. |
Notes |
|---|
* The authors equally contributed to this work.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Altschul SF, Gish W, Miller W, Myers EW and Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215,403–410.[CrossRef][ISI][Medline]
Behr R and Weinbauer GF (2000) CREM activator and repressor isoforms in human testis: sequence variations and inaccurate splicing during impaired spermatogenesis. Mol Hum Reprod 6,967–972.
Behr R and Weinbauer GF (2001) cAMP response element modulator (CREM): an essential factor for spermatogenesis in primates? Int J Androl 24,126–135.[CrossRef][ISI][Medline]
Behr R, Hunt N, Ivell R, Wessels J and Weinbauer GF (2000) Cloning and expression analysis of testis-specific cyclic-3',5'-adenosine monophosphate-responsive element modulator activators in the nonhuman primate (Macaca fascicularis): comparison with other primate rodent species. Biol Reprod 62,1344–1351.
Blackwood EM and Kadonaga JT (1998) Going the distance: a current view of enhancer action. Science 281,61–63.[CrossRef]
Blendy JA, Kaestner KH, Weinbauer GF, Nieschlag E and Schutz G (1996) Severe impairment of spermatogenesis in mice lacking the CREM gene. Nature 380,162–165.[CrossRef][Medline]
Blocher S, Behr R, Weinbauer GF, Bergmann M and Steger K (2003) Expression of CREM isoforms in human and equine testis with normal and impaired spermatogenesis. Andrologia 35,3–4.
Daniel PB, Rohrbach L and Habener JF (2000) Novel cyclic adenosine 3',5'-monophosphate (cAMP) response element modulator theta isoforms expressed by two newly identified cAMP-responsive promoters active in the testis. Endocrinology 141,3923–3930.
Delmas V, Laoide BM, Masquilier D, de Groot RP, Foulkes NS and Sassone-Corsi P (1992) Alternative usage of initiation codons in mRNA encoding the cAMP-responsive-element modulator generates regulators with opposite functions. Proc Natl Acad Sci USA 89,4226–4230.
Deryckere F and Gannon F (1994) A one-hour minipreparation technique for extraction of DNA-binding proteins from animal tissues. Biotechniques 16,405.
Domschke K, Kuhlenbaumer G, Schirmacher A, Lorenzi C, Armengol L, DiBella D, Gratacos M, Garritsen HS, Nothen MM, Franke P et al. (2003) Human nuclear transcription factor gene CREM: genomic organization, mutation screening, and association analysis in panic disorder. Am J Med Genet B Neuropsychiatr Genet 117,70–78.[Medline]
Ezeh UI (2000) Beyond the clinical classification of azoospermia. Hum Reprod 15,2356–2359.
Fodde R and Smits R (2002) Cancer biology. A matter of dosage. Science 298,761–763.
Foulkes NS, Borelli E and Sasone-Corsi P (1991) CREM gene: use of alternative DNA-binding domains generates multiple antagonists of cAMP-induced transcription. Cell 64,739–749.[CrossRef][ISI][Medline]
Foulkes NS, Mellstrom B, Benusiglio E and Sassone-Corsi P (1992) Developmental switch of CREM function during spermatogenesis: from antagonist to activator. Nature 355,80–84.[CrossRef][Medline]
Foulkes NS, Schlotter F, Pévet P and Sassone-Corsi P (1993) Pituitary hormone FSH directs the CREM functional switch during spermatogenesis. Nature 362,264–267.[CrossRef][Medline]
Gellersen B, Kempf R, Sandhowe R, Weinbauer GF and Behr R (2002) Novel leader exons of the cyclic adenosine 3',5'-monophosphate response element modulator (CREM) gene, transcribed from promoters P3 and P4, are highly testis-specific in primates. Mol Hum Reprod 8,965–976.
Hamilton SP, Slager SL, Mayo D, Heiman GA, Klein DF, Hodge SE, Fyer AJ, Weissman MM and Knowles JA. (2004) Investigation of polymorphisms in the CREM gene in panic disorder. Am J Med Genet B Neuropsychiatr Genet 126,111–115.[Medline]
Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W et al. (2001) Initial sequencing and analysis of the human genome. Nature 409,860–921.[CrossRef][Medline]
Lee TI and Young RA (2000) Transcription of eukaryotic protein-coding genes. Annu Rev Genet 34,77–137.[CrossRef][ISI][Medline]
Lekstrom-Himes J and Xanthopoulos KG (1998) Biological role of the CCAAT/enhancer-binding protein family of transcription factors. J Biol Chem 273,28545–22858.
Masquilier D, Foulkes NS, Mattei M and Sasone-Corsi P (1993) Human CREM gene: evolutionary conservation, chromosal localization, and indicibility of the transcript. Cell Growth Differnt 4,931–937.
Molina CA, Foulces NS, Lalli E and Sassone-Corsi P (1993) Inducibility and negative autoregulation of CREM: an alternative promoter directs the expression of ICER, an early repressor. Cell 75,875–886.[CrossRef][ISI][Medline]
Monaco L, Foulkes NS and Sassone-Corsi P (1995) Pituitary follicle-stimulating hormone (FSH) induces CREM gene expression in Sertoli cells: involvement in long-term desensitization of the FSH receptor. Proc Natl Acad Sci USA 92,10673–10677.
Nantel F, Monaco L, Foulkes NS, Masquilier D, LeMeur M, Henriksen K, Dierich A, Parvinen M and Sassone-Corsi P. (1996) Spermiogenesis deficiency and germ-cell apoptosis in CREM-mutant mice. Nature 380,159–162.[CrossRef][Medline]
Peri A, Krausz C, Cioppi F, Granchi S, Forti G, Francavilla S and Serio M (1998) Cyclyc adenosine 3',5'-monophosphate-responsive element modulator gene expression in germ cells of normo- and oligoazoospermic men. J Clin Endocrinol Metab 83,3722–3726.
Quandt K, Frech K, Karas H, Wingender E and Werner T (1995) MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res 23,4878–4884.
Reese MG, Eeckman FH, Kulp D and Haussler D (1997) Improved splice site detection in genie. J Comput Biol 4,311–323.[ISI][Medline]
Rozman D, Fink MG-MF, Sassone-Corsi P and Waterman MR (1999) Cyclic adenosine 3',5'-monophosphate (cAMP)/cAMP-responsive element modulator (CREM)-dependent regulation of cholesterogenic lanosterol 14a-demethylase (CYP51) in spermatids. Mol Endocrinol 13,1951–1962.
Sassone-Corsi P (1995) Transcription factors responsive to cAMP. Annu Rev Cell Dev Biol 11,355–377.[CrossRef][ISI][Medline]
Stehle JH, Foulkes NS, Molina CA, Simonneaux V, Pevet P and Sassone-Corsi P (1993) Adrenergic signals direct rhythmic expression of transcriptional repressor CREM in the pineal gland. Nature 365,314–320.[CrossRef][Medline]
Vogt PH (2004) Molecular genetic of human male infertility: from genes to new therapeutic perspectives. Curr Pharm Des 10,471–500.[CrossRef][ISI][Medline]
Walker WH and Habener JF (1996) Role of transcription factors CREB and CREM in cAMP-regulated transcription during spermatogenesis. TEM 7,133–138.
Weinbauer GF, Behr R, Bergmann M and Nieschlag E (1998) Testicular cAMP responsive element modulator (CREM) protein is expressed in round spermatids but is absent in men with round spermatid maturation arrest. Hum Mol Reprod 4,9–15.
Submitted on July 6, 2005; accepted on July 19, 2005.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J.J. Galan, M. De Felici, B. Buch, M.C. Rivero, A. Segura, J.L. Royo, N. Cruz, L.M. Real, and A. Ruiz Association of genetic markers within the KIT and KITLG genes with human male infertility Hum. Reprod., December 1, 2006; 21(12): 3185 - 3192. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||