Molecular Human Reproduction, Vol. 6, No. 11, 967-972,
November 2000
© 2000 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
CREM activator and repressor isoforms in human testis: sequence variations and inaccurate splicing during impaired spermatogenesis
Institute of Reproductive Medicine of the University, Domagkstr. 11, D-48129 Münster, Germany
Abstract
cAMP responsive element modulator (CREM) activators are specifically expressed in haploid germ cells prior to cell elongation and are essential for spermatid development in mice. Recent studies indicate that CREM activators are also involved in the process of spermatid maturation in men. Unlike the activators, CREM repressors were suggested to be absent from adult mouse and dog testes. The present work investigates CREM transcripts in human testis with normal (n = 4) and impaired spermatogenesis (n = 2). Two activator transcripts could be identified corresponding to the 
2 isoform with and without exon 
. Interestingly, four CREM repressors could be isolated from specimens with complete spermatogenesis. These were -repressor (exons B, E, F, H, Ib), CREM 
C-F, ß (exons B, G, H, Ib), CREM C-G, ß (exons B, H, Ib), and CREM C-G, 
(exons B, H, Ia, Ib). These isoforms were also present in cynomolgus monkey testes. A novel CREM splice variant (CREM C-H) was detected in a specimen with spermatid maturation defect. Beyond that, inaccurate CREM splicing, giving rise to inactive transcripts, was encountered in a specimen with impaired spermatogenesis. In conclusion, several CREM repressor transcripts are present in adult human testes, and altered transcript splicing is associated with impaired spermatogenesis.
CREM/human/infertility/spermatogenesis/splicing
Introduction
The transcription factor cAMP responsive element modulator (CREM) is an important component of cAMP-mediated signal transduction and couples extracellular signals to gene regulation (Sassone-Corsi, 1998
). Spermatogenesis encompasses the production of haploid spermatozoa from diploid stem cells; during this process, CREM proteins are highly expressed in postmeiotic germ cells of the primate testis (Weinbauer et al., 1998; Behr and Weinbauer, 1999) and the rodent testis (Delmas et al., 1993; Behr and Weinbauer, 1999). Two studies have identified CREM as an indispensable factor for spermatid development in the mouse (Blendy et al., 1996; Nantel et al., 1996). Mice with a homozygously inactivated CREM gene exhibit round spermatid maturation arrest at the early stages of postmeiotic germ cell development. It has been found subsequently that lack of CREM expression at the mRNA and protein level can also be associated with spermatid maturation arrest in infertile patients (Weinbauer et al., 1998; Steger, 1999).
Remarkably, testicular CREM expression switches from repressors to activators during spermatogenic development in mice (Foulkes et al., 1992; Nantel and Sassone-Corsi, 1996). Whereas in the prepubertal testis up to day 13 of age (prior to appearance of round spermatids) only CREM repressors are expressed, CREM activator transcripts are expressed exclusively from day 14 of age onwards. This switch from repressor to activator during mouse spermatogenesis and stabilization of the activator transcript in germ cells has been reported to be dependent on the gonadotrophic hormone FSH (Foulkes et al., 1993) although subsequent research has failed to confirm this observation (Dierich et al., 1998; Behr and Weinbauer, 1999).
A typical feature of the CREM gene is its modular structure (for review see Walker and Habener 1996; Sassone-Corsi, 1998). The gene encodes five different functional domains: two alternatively used 3'-located bZIP DNA-binding domains (DBD), a more 5'-located domain rich in serine residues which can be phosphorylated by several kinases (kinase-inducible domain, KID; de Groot et al., 1993; Delmas et al., 1993) and two glutamine-rich domains flanking the KID. Such glutamine-rich domains are known to mediate protein–protein interactions. All functional domains are encoded by specific exons.
CREM expression is regulated at multiple levels (for review see Sassone-Corsi, 1998): at transcriptional level by the use of two different promoters (Molina et al., 1993; Stehle et al., 1993), at the level of transcript splicing (Foulkes et al., 1991, 1992) and at the level of translational initiation (Delmas et al., 1992; Gellersen et al., 1997). This gives rise to functionally different proteins with either activating or repressing potential on target gene expression. A full length activator transcript in the mouse testis consists of eight or nine exons (see Figure 2A for details; nomenclature based upon Walker and Habener, 1996) depending on the alternative usage of the two DBD. Exons C and G encode the glutamine-rich transactivating domains. Exons E and F represent the protein domain rich in phosphorylation sites for several kinases and exons H, and Ia or Ib encode the basic domain and the leucine zipper, respectively, constituting the DBD. A recent report suggests a phosphorylation-independent mechanism of CREM activation in the testis by the transcriptional activator of CREM in the testis (ACT; Fimia et al., 1999). However, a more recent study failed to detect the mRNA for ACT in the human testis by Northern blotting (Morgan and Whawell, 2000). The functions of the amino acids encoded by the 5'-located exon B and the 12 amino acids encoding exon are not known. Activator isoforms are characterized by the insertion of the two exons which encode the kinase-inducible domain and at least one of two exons flanking the kinase-inducible domain and encoding glutamine-rich transactivation domains.
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and CREM repressor/activator expression is well characterized in the mouse testes (Foulkes et al., 1992
, 1993; Delmas et al., 1993) but much less is known for the primate testis. Recent characterization of CREM expression in non-human primates with complete spermatogenesis demonstrated the presence of the activator isoform 2 with and without the insertion of exon (Behr et al., 2000). Comparative analysis of testicular CREM expression revealed a trend from multiple activator isoforms in rodent and marmoset testis to two and one activator transcripts in Old World monkeys and men respectively. CREM repressors are present in a variety of organs and structures (Gellersen et al., 1997; Uyttersprot and Miot, 1997), but they have been reported to be absent from the adult mouse and dog testis. The present work aims at clarifying the CREM transcript pattern and protein expression in human testes with complete spermatogenesis, in testis with impaired spermatogenesis, and in a patient with round spermatid maturation arrest.
Materials and methods
Testicular tissue
Five testes were obtained during orchidectomy from prostate cancer patients. None of the patients had received hormonal therapy prior to surgery. Two biopsy specimens (left and right) were collected from a patient undergoing testicular sperm extraction (TESE). Tissues were frozen on dry ice or in liquid nitrogen and stored at –80°C for RNA analyses. A sample of fresh tissue was fixed in Bouin's solution for analysis of spermatogenesis and CREM expression. The patients had given informed consent for such investigations to be performed. Frozen testis material from intact adult cynomolgus monkeys (Macaca fascicularis) was also included in the analysis.
Spermatogenic status of testes used for CREM transcript analysis
Spermatogenesis was intact and complete in four testes. In one testis (obtained from a cancer patient), spermatogenesis was complete but with markedly reduced abundance of spermatids. Histological analysis of the biopsy specimens obtained from the patient undergoing TESE revealed round spermatid maturation arrest. Frozen samples from all testes were analysed by subsequent cloning and sequencing of the polymerase chain reaction (PCR) products.
RNA extraction and cDNA synthesis
Total RNA was extracted from the tissues by the Ultraspec method (Biotecx, Houston, TX, USA). The RNA was precipitated with isopropanol at –20°C for 1–2 h, washed with 75% ethanol, and then dissolved in diethyl pyrocarbonate-water. RNA concentrations were determined spectrophotometrically at 260 nm. Reverse transcription (RT) was carried out in a mixture consisting of 250 µmol/l dNTP, 100 U MMLV-RT (Promega, Heidelberg, Germany), and 100 pmol antisense primer (Poly dT) in Promega RT buffer. RT was performed at 42°C for 60 min and stopped by heating for 5 min at 95°C. The resulting cDNA templates were stored at –20°C or directly used for PCR.
PCR and analysis of PCR products
After a denaturation period of 2 min at 95°C followed by 35 cycles at 95°C for 40 s, at 56°C for 40 s, and 72°C for 50 s, PCR was performed using 3 µl cDNA, 100 µmol/l of each dNTP, 1.25 U Taq polymerase (Promega), and 50 pmol of both sense and antisense primers in PCR buffer (Promega). The final volume of each reaction was 25 µl. 10 µl of each reaction were run on a 2% w/v agarose gel and stained with ethidium bromide. Primers used for RT-PCR were: 5'-ATGACCATGGAAACAGTTGAATC-3' (CREM exon B forward, position 1–23 of the human cDNA sequence) (Masquilier et al., 1993) and 5'-CTGTAATCAGTTCATAGTTAAATATTTCTA-3' (CREM 3' UTR reverese, position 1429–1400 of the human cDNA sequence) (Masquilier et al., 1993). Thus, the forward primer is located at the 5' end of exon B and the reverse primer located at the most 5' end of the untranslated region downstream of exon Ib. The nomenclature used for the exonic organization of the CREM is based upon that provided by Walker and Habener (1996) and is depicted in Figure 2A.
Cloning and sequencing of PCR products
PCR products were cloned directly into the pGEM-T easy vector (Promega) according to the manufacturer's instructions, or PCR products of interest were excised from the gel using a scalpel, eluted using a PCR product purification kit (Boehringer Mannheim, Germany) and cloned into the vector. The resulting constructs were transformed into the E.coli XL 1-blue strain according to standard methods. Transformed clones were selected on ampicillin-containing agar plates. Plasmids for sequencing were isolated from 3ml of culture medium of the selected clones. Sequencing was done using a sequencing kit (RPN 2438; Amersham Pharmacia Biotech, Little Chalfont, UK) and SP6 and T7 primers. The resulting products were analysed by the LI-COR sequencer (MWG Biotech, Ebersberg, Germany).
Results
CREM transcripts in testes with complete spermatogenesis
RT-PCR yielded five distinct bands (Figure 1, right lane). Cloning and sequencing revealed one activator transcript and four repressor transcripts (Figure 2B). The activator transcript corresponded to the 2 isoform without exon . The repressor isoforms were the -repressor consisting of exons B, E, F, H, Ib, CREM C-F, ß (exons B, G, H, Ib), CREM C-G, ß (exons B, H, Ib), and CREM C-G, (exons B, H, Ia, Ib). All five CREM transcripts could also be detected in the adult cynomolgus monkey testis (not shown).
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Unusual CREM transcripts present in testes with arrested spermatogenesis
A
2 activator including exon was identified for the first time in the human testis (Figure 1, middle lane and Figure 2C). This was isolated from the testes with spermatid maturation arrest. Furthermore, a novel CREM splice variant (CREM C-H) comprising only exons B and Ib was detected in this specimen. Since the splice junction was not in frame of the normal ORF, a nonsense splice variant was yielded. This splice variant could not be seen as a distinct band on agarose gels, indicating a very low abundance. Furthermore, three additional transcripts were present in the testes with round spermatid maturation arrest (Figure 1) but were hardly visible in testes with complete spermatogenesis.
Inaccurate splicing in the testis with impaired spermatogenesis
Three basically activating transcripts with inaccurate splicing were detected in the testis with reduced spermatogenesis. One transcript corresponded to the 2 isoform lacking the 5'-located 98 bp of the KID. This deletion resulted in a frame shift followed by the introduction of an early stop codon (Figure 3a). Another deletion was found in exon G of an activating transcript. This clone lacked the 84 3'-located bp of exon G (Figure 3b). Due to the exclusion of exactly 28 codons, this transcript remained in frame and might rather function as a repressor. A 5 bp deletion was also discovered in a 2 transcript at the junction of exon H with exon Ib (Figure 3c). This deletion resulted in an early stop codon and loss of the leucine zipper encoded by the non-mutated exon Ib.
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CREM nucleotide sequence variations
Altogether 58 human CREM transcripts were cloned and sequenced. Many transcripts corresponded to full length CREM
2 activators with complete sequence identity among different transcripts. Some nucleotide sequence variations were found when compared to the sequence published earlier (Masquilier et al., 1993) (Figure 4). All sequences contained a C instead of a G at position 93 and an A instead of a T at position 98 in exon B. Whereas the first variation did not affect the amino acid sequence (GGG and GGC encode glycine), the second variation (ATT to AAT) caused an amino acid exchange from isoleucine to asparagine (Figure 4a). The published sequence for the second DBD contains a C at position 756 and a T at position 846 (Masquilier et al., 1993). In this work, a T at position 756 and an A at position 846 were found. None of these variations in the DBD caused an amino acid substitution. For the nucleotide sequence sites, cynomolgus monkeys exhibited identical nucleotide sequences to those present in the human CREM sequence (data not shown). In men, codon 284 (TAC) of the 2 isoform (Masquilier et al., 1993; Behr et al., 2000) represents the last protein coding triplet and codon 285 encodes the stop signal. In monkeys and mice codon 284 contains the TAG sequence for the translational stop (Behr et al., 2000). Interestingly, one out of 28 CREM transcripts analysed from the testes with round spermatid maturation arrest also contained the TAG stop codon at position 284 (Figure 4b).
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C) and 98 (TDiscussion
CREM activators are specifically expressed in haploid germ cells prior to spermatid elongation (Delmas et al., 1993; Behr and Weinbauer, 1999) and are essential for spermatid development in the mouse (Blendy et al., 1996; Nantel et al., 1996). More recently, several studies have suggested that CREM is also involved in the process of spermatid maturation in men (Lin et al., 1998; Peri et al., 1998; Weinbauer et al., 1998; Steger et al., 1999). In the human testis, a single CREM activator has been identified so far, i.e. the CREM 2 isoform without exon (Behr et al., 2000; Masquilier et al., 1993). In the present work, we also detected a CREM 2 isoform that contained exon . This isoform has recently been isolated also from testes of macaque monkeys (Behr et al., 2000). Unexpectedly, this isoform was isolated from the testes with round spermatid maturation arrest, whereas in all tissues with complete spermatogenesis, only the CREM 2 could be identified. Possibly this indicates less stringent control of mRNA splicing in this patient. Overall, the testicular CREM activator expression pattern appears more similar for macaque and human testis than for rodent and marmoset testis, as the latter express three or more CREM activator transcripts (Behr et al., 2000). Studies in dogs revealed the presence of several testicular CREM activator transcripts (Uyttersprot and Miot, 1997).
CREM transcript analysis in the developing mouse testis (Foulkes et al., 1992) and in dog testis (Uyttersprot and Miot, 1997) indicated that only CREM activators are expressed in the adult testis with ongoing spermatogenesis. For the mouse, the prepubertal testes only contained CREM repressors at low levels, and high expression of CREM activators specifically occurred in adult testis (Foulkes et al., 1992). Our results obtained during transcript analysis of human (and cynomolgus monkey) testes are at variance with these findings. In fact, we succeeded in the isolation of four CREM repressor transcripts from adult primate testis with ongoing spermatogenesis. The functional relevance of these repressor isoforms remains to be clarified, as does as the cellular localization of testicular CREM repressor expression. Selective in-situ hybridization analysis to distinguish between activators and repressors is unfortunately precluded by the fact that no exon is exclusively confined to either CREM activator or repressor isoforms.
An earlier investigation reported on the CREM transcripts in human testis (Peri et al., 1998). These investigators observed CREM activators in ejaculated spermatozoa, and CREM repressors in oligozoo-/azoospermic men. However, primers were selected flanking the first Q-rich domain and failed to detect this exon in some infertile patients by RT-PCR and subsequent southern hybridization. It must be noted that exon C [first glutamine (Q)-rich domain] has not been demonstrated in human testicular CREM transcripts so far (Masquilier et al., 1993; Behr et al., 2000; present study). Furthermore, the primers chosen did not permit differentiation of CREM-repressing isoforms and the highly abundant CREM 2 activator (Masquilier et al., 1993; Behr et al., 2000; present study). Therefore, the possibility remains that the CREM repressor expression described by Peri and colleagues for adult human testis (Peri et al., 1998) might also represent the CREM 2 activator.
We obtained evidence for misregulation of CREM expression in the testes with reduced and arrested spermatogenesis. These observations comprised additional, unusual, and inaccurately spliced transcripts. Interestingly, functional CREM transcripts were also encountered. Some of the splicing defects resulted in inactive CREM isoforms. Inaccurate transcript splicing might be due to impaired function of splicing factors and/or other RNA binding proteins. There are many examples of testis-specific splicing events and of numerous nuclear RNA binding proteins either essential for spermatogenesis or expressed specifically in male germ cells (Venables and Eperon, 1999). One might speculate that altered RNA binding proteins and/or splicing factors trigger uncoordinated events at the molecular level during the complex process of spermatogenesis and spermatid development.
The stop codon for the human 2 transcript is encoded by TAG at amino acid position 285 (Masquilier et al., 1993; Behr et al., 2000). Codon 284 (TAC) of the human transcript encodes tyrosine. In contrast, in the cynomolgus monkey and in the mouse, a TAG stop signal of translation is encoded at position 284 (Behr et al., 2000). It was suggested, therefore, that that transition from TAG (stop) to TAC (tyrosine) at position 284 could be a late event during primate evolution. One out of 28 transcripts analysed from a testis with reduced spermatogenesis contained a TAG stop codon at position 284 of the CREM 2 isoform rather than the TAC encoding tyrosine. All other transcripts analysed from this testis revealed the normal human sequence. Therefore, the TAG codon at position 284 in the human testis could serve as an allele-specific marker. Interestingly, this marker was also present in a transcript with a 84 bp deletion in exon G and containing exon in addition. Hence, a rarely present marker of one allele is associated with an unusual CREM transcript. It is conceivable that transcripts from this one allele are frequently incorrectly processed and therefore rapidly degraded so that the transcripts from the other allele are heavily underrepresented in the transcript population of this testis.
In conclusion, the present work demonstrated (i) the presence of several CREM repressor transcripts in the adult human testes, and (ii) some altered transcript splicing in testes with impaired spermatogenesis.
Acknowledgments
We are grateful to Dr E.Nieschlag, for his continued support, to Dr S.von Eckardstein for providing access to the TESE specimen, and to Dr J.Gromoll (all at the Institute of Reproductive Medicine, Münster) and Dr R.Ivell (Institute of Hormone and Fertility Research, Hamburg) for helpful discussions and critical comments. The expert technical assistance of Reinhild Sandhowe and Lisa Pekel is gratefully acknowledged. Testicular tissue from prostate cancer patients was kindly provided by Dr H.Schulze, Urology Clinic of the University of Bochum, Marienhospital Herne, and Dr S.Wienholt, Klinikum GmbH, Wuppertal, Germany. This work was supported by the Deutsche Forschungsgemeinschaft, Confocal Research Group Hamburg/Münster: The Male Gamete: Production, Maturation, Function.
Notes
1 Present addresses: University of Pennsylvania School of Medicine, Department of Genetics, Clinical Research Building, 415 Curie Boulevard, Philadelphia, PA 19104-6145, USA, 
2 Covance Laboratories GmbH, Kesselfeld 29, D-48163 Münster, Germany
3 To whom correspondence should be addressed. E-mail: gerhard.weinbauer{at}covance.com
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Submitted on April 27, 2000; accepted on August 17, 2000.
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