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a Institute of Reproductive Medicine of the University, D-48129 Münster, Germany
b Institute for Hormone and Fertility Research, University of Hamburg, D-22529 Hamburg, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 with and without exon 
) were identified, which displayed high sequence identity to mouse and human isoforms. The insertion of exon was observed for the first time in the primate testis. CREM activator expression was confined to the testis, where it was seen in late pachytene spermatocytes and round spermatids in specific spermatogenic stages, as revealed by in situ hybridization. Comparison of the mRNA and the recently described protein expression indicated a lack of translational delay of CREM expression. Comparative analysis of testicular CREM expression by reverse transcription-polymerase chain reaction yielded several transcripts in the rat, mouse, hamster, and marmoset; two transcripts in cynomolgus and rhesus monkeys; and one transcript in men. These findings suggest an evolutionary trend from multiple activator isoforms to a single activator transcript in men.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The CREM gene consists of at least 12 exons [10, 11], and functional domains of the CREM protein are encoded by specific exons [6]. Exons C and G encode glutamine-rich domains that are necessary for transactivation. Exons E and F represent a protein domain rich in phosphorylation sites for several kinases [9, 12]; and exons H and Ia or Ib encode the basic domain and the leucine zipper, respectively. Interestingly, a recent report suggests a phosphorylation-independent mechanism of CREM activation in the testis [13]. The functions of the amino acids encoded by exons B and are not known. Activator isoforms are characterized by the insertion of two exons that encode the kinase-inducible domain and at least one of two exons flanking the kinase-inducible domain and encoding glutamine (Q)-rich domains. These domains are important for protein-protein interaction [6]. The testicular CREM activator/repressor expression pattern is well characterized in the mouse [7], but little is known regarding that of primate species and other rodents.
Access to human testicular tissue is limited, precluding systematic and detailed studies of CREM isoforms in patients with specific spermatogenic lesions. For this reason, we cloned and sequenced testicular CREM activator isoforms, and we analyzed CREM mRNA expression by in situ hybridization in the cynomolgus monkey, a particularly appropriate model for study of the regulation of human spermatogenesis [14]. To further investigate CREM activators, a comparative analysis of transcripts was undertaken in the testes of the rat, mouse, hamster, marmoset, rhesus and cynomolgus monkey, and man.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testicular tissue was obtained from adult, sexually mature, Sprague-Dawley rats, mice (C57BL/6), Djungarian hamsters (Phodopus sungorus), marmosets (Callithrix jacchus), rhesus monkeys (Macaca mulatta), and cynomolgus monkeys (Macaca fascicularis). Monkey organs (uterus, ovary, testis, prostate, seminal vesicle, adrenal, kidney, liver, gallbladder, pancreas, spleen, skeletal muscle, heart, aorta, pituitary, hypothalamus, pineal, cortex, and cerebellum) were collected when the animals were killed. The animals were maintained in accordance with the German Federal Law on the Care and Use of Laboratory Animals. Tissues were fixed in Bouin's solution for histological evaluation or frozen in liquid nitrogen for RNA analysis. Human testis tissue was obtained during orchidectomy from a 70-yr-old prostate carcinoma patient with complete spermatogenesis in most seminiferous tubules. The patient had given informed consent for such investigations to be performed.
Library Screening
A cynomolgus monkey testis cDNA library was constructed using the oligo-dT-primed cDNA synthesis kit (Stratagene, Heidelberg, Germany) employing the Uni-ZAP XR unidirectional phagemid vector. The library had a complexity of 3.75 x 106 plaque forming units and an average insert size of 1–1.5 kilobases. Approximately 1 x 106 recombinant phage clones were screened for the presence of CREM using a [32P]dCTP-labeled human CREM cDNA [15], base pairs (bp) 1214-1522. Plating of recombinant phages and generation of replica filters were performed according to the manufacturer's protocol. The filters were prehybridized in 20-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate), single-strength Denhardt's solution (0.02% w:v Ficoll 400, 0.02% w:v BSA, 0.02% w:v polyvinylpyrrolidone), 1 M NaPO4 (pH 6.8), 20% w:v SDS, and 0.1 µg/ml denatured and sheared salmon sperm DNA at 60°C for 2–4 h. Hybridization conditions were identical to those for the prehybridization except that the labeled cDNA was added to a final concentration of 1 x 106 cpm/ml of hybridization solution. Hybridization was performed at 60°C for 16 h. Filters were washed at a final stringency of single-strength SSC/0.1% w:v SDS at 60°C and air dried. Autoradiographic exposure of the filters was carried out at -80°C for 2 days. Positive clones were purified by plaque isolation and a subsequent secondary screening procedure. Inserts were excised according to the manufacturer's protocol. The resulting phagemid contained the cDNA inserted at the EcoRI site of the pBluescript SK(-). The corresponding cDNAs were sequenced using the LiCOR sequencer (MWG Biotech, Ebersberg, Germany). Both strands of the cDNA were sequenced and analyzed using the DNAsis (Hitachi, Japan) sequence analysis software.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from the tissues by the Ultraspec method (Biotecx, Houston, TX). The RNA was precipitated with isopropanol at -20°C for 1–2 h, washed with 75% ethanol, and then dissolved in diethyl pyrocarbonate water. Reverse transcription was carried out in a mixture consisting of 250 µM dNTP, 100 U murine Moloney leukemia virus-reverse transcriptase (Promega, Madison, WI), and 100 pmol antisense primer in Promega RT buffer. Reverse transcription was performed at 37°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. After a denaturation period of 2 min of 95°C followed 35 cycles of 95°C for 90 sec, 58°C for 40 sec, and 72°C for 90 sec, PCR was performed with 3 µl cDNA, 100 µM 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. Each reaction (5–10 µl) was run on a 2% w:v agarose gel. Primers used for RT-PCR were 5'-TGGTAAGTTGCCATGTCACC-3' (CREM exon H reverse, position 725–706 of the human cDNA sequence [16]), 5'-ATGACCATGGAAACAGTTGAATC-3' (CREM exon B forward, position 1–23 of the human cDNA sequence [16]). Cynomolgus monkey, mouse, and human cDNA share identical sequences for primer binding sites.
Preparation and Labeling of Riboprobes and In Situ Hybridization
First, pGEM-T vector (Promega, Heidelberg, Germany) containing the cDNA representing CREM exons E, F, G, H, and Ib [10] was linearized using either the restriction enzyme SacI or SacII (Pharmacia, Freiburg, Germany). Complementary RNA synthesis was performed by SP6-RNA polymerase (sense cRNA) for SacII-linearized cDNA or by T7 RNA polymerase (antisense cRNA) for the SacI-linearized plasmid using the digoxigenin (DIG)-RNA labeling kit from Boehringer (Mannheim, Germany). Paraffin-embedded tissues were cut into 3- to 4-µm sections and analyzed by in situ hybridization as described previously [17]. Prehybridization solution contained 50% formamide, 4-strength STE (10 mM Tris-HCl, 10 mM EDTA, 1 mM NaCl), single-strength Denhardt's solution, 125 µg/ml salmon sperm DNA, and 125 µg/ml yeast RNA. Hybridization was performed at 50°C overnight in the presence of 100–150 ng of the DIG-labeled sense and antisense riboprobes. After hybridization, slides were incubated for 30 min in 20 µg/ml RNase A at 37°C. For signal detection, antidigoxygenin-alkaline-phosphatase (Boehringer), nitroblue tetrazolium chloride solution (NBT), and 5-bromo-4-chloro-3-indolyl-phosphate solution (X-phosphate; Boehringer) were used, and slides were counterstained with Mayer's hemalaun.
Histological Evaluation
Stage classification of spermatogenesis was performed as described for the macaque [18, 19] and for the rat [20, 21].
Northern Blot Analysis
For RNA isolation, testes from the various species were homogenized using RNAzol B (Biotecx, Houston, TX) followed by a chloroform extraction. Testicular RNA from a CREM-deficient mouse was included as a control. In these mice the exons H, Ia, and Ib are replaced by a neomycin-lacZ cassette [2]. The RNA was precipitated with isopropanol at 4°C for 2–3 h, washed with 75% ethanol, and then diluted in DEPC water. Northern blotting was carried out as described previously [8]. Filters were prehybridized at 50°C for 60 min in DIG Easy Hyb buffer (Boehringer), and hybridization was performed using 10–20 ng DIG-labeled cDNA/ml hybridization solution. The CREM cDNA probe comprised exons E, F, G, H, and Ib of the cynomolgus monkey. Hybridization was performed at 50°C for 16 h. Detection of the hybridized probe was performed according to the instructions for the DIG labeling and detection kit (Boehringer). Filters were exposed to High Performance film (Amersham Pharmacia Biotech, Piscataway, NJ) for 2 h, and size of the transcripts was determined by comparison with rRNA bands.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two clones were isolated, spanning from the poly(A) tail to exon E [10]. In order to obtain the 5' end of the open reading frame of the cDNA, RT-PCR was performed using primers located at the 5' end of exon B (forward) and in exon H (reverse). Two activator isoforms were amplified, cloned, and sequenced. The complete sequences of the open reading frames were deduced from overlapping DNA fragments (Fig. 1). Analysis of the sequences revealed the presence of exons B, E, F, G, H, and Ib in the shorter splice variant and exons B, E, F, G, , H, and Ib in the larger transcript. Exon C, the first Q-rich domain, located between exons B and E, was absent. The amino acids at positions 29, 30, and 55, which are conserved in primates, have no counterpart in the rodent. The predicted open reading frames of the two cloned monkey activator isoforms were 849 and 885 bp, respectively. The putative proteins consisted of 283 and 295 amino acids.
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Comparison of the CREM amino acid sequences of the cynomolgus monkey with the corresponding human and mouse sequences displayed an overall identity of 92% (Fig. 1). The identity between the monkey and the human was approximately 98% and between the monkey and the mouse more than 94%. However, comparisons of the different functional domains of the CREM sequence from three species revealed striking differences in sequence identity between the domains. Amino acids 1–40 encoded by exon B were conserved only in 26 of 40 positions (65%). In contrast, the other domains were highly conserved. The kinase inducible domain spanning amino acids 41–120 were identical in 73 of 80 positions (> 91%) in all species. The transactivating Q-rich domain consisted of amino acids 121–183, and all amino acids within this domain were identical in all species. Comparison of exon between monkey and mouse revealed identity in 11 of 12 amino acids (92%). The dimerization and DNA binding domain (amino acids 196–295) were also highly conserved (99% identity). The stop codon was at position 296 of the 2 isoform of the monkey.
Tissue-Specific Expression of CREM Activators
The expression of CREM activators was analyzed by RT-PCR in 19 different tissues of the cynomolgus monkey. Detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as internal control. The GAPDH-specific fragment could be amplified in all tested organs, but a CREM-specific fragment could be detected only in the testis. In the remaining organs including the ovary, uterus, organs of the male urogenital tract, pituitary, and additional other neuronal tissues, CREM activator transcript could not be detected (Fig. 2).
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Localization of Testicular CREM mRNA Expression
CREM mRNA expression was detected in the cytoplasm of spermatocytes and spermatids of the monkey and was related to the stage of spermatogenesis (Figs. 3 and 4). Among spermatocytes, CREM mRNA was confined to pachytenes in stages VII–XI and to secondary spermatocytes in stage XII (Fig. 3c). Round spermatids expressed CREM mRNA during stages I–VIII, and expression was highest during stages I–VII but declined sharply thereafter and became undetectable from stage IX onward (Fig. 3, d and e). Rat pachytene spermatocytes expressed CREM mRNA from stage IX onward, secondary spermatocytes in stage XIV, and round spermatids in stages I to VII/VIII (Fig. 3, f–h). Comparison of the CREM mRNA expression with that of the protein [8] revealed a lack of translational delay (Fig. 4).
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Comparison of Testicular CREM Activator Expression in Primate and Rodent Species
A RT-PCR strategy was chosen to detect CREM activator transcripts using primers located in exon B (forward primer) and exon H (reverse primer). Using this primer pair, all previously described activator isoforms could be detected (Figs. 5 and 6). In the human testis only a single isoform was detectable. The fragment size was 578 bp and consisted of exons B, E, F, G, and H. In addition to the fragment amplified from the human testis, the cynomolgus monkey expressed another isoform that contained exon . The rhesus monkey showed an expression pattern identical to that seen in the cynomolgus monkey. In contrast to these findings, two larger isoforms were found in the marmoset monkey. The sizes of the two corresponding PCR fragments were about 650 and 700 bp. The rodent species expressed three activator isoforms. The upper band corresponded to the isoform (761 bp) and the lower band to the 2 isoform (614 bp). The middle band, with an estimated size of about 700 bp, could not be related to known isoforms. In marmosets and rats, the smallest PCR fragment reproducibly showed the highest signal intensity, whereas in the mouse the fragment representing the isoform always showed highest fluorescence.
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Comparison of Testicular CREM Expression by Northern Blotting
Northern blot hybridization of testicular RNA using the monkey cDNA as probe revealed CREM transcripts in the cynomolgus monkey, marmoset, hamster, rat, and mouse (Fig. 7). Testicular RNA from a CREM-deficient mouse testis was included as a control. In this testis only a weak signal was visible, which was shifted to a larger transcript size compared with the other signals. This size difference was due to the insertion of the neomycin-lacZ cassette into the CREM gene, resulting in a transcript larger than that of the normal gene. This finding also demonstrated that the CREM probe did not cross-react with the closely related CREB mRNA, which is also expressed in the testis [10]. In the mouse, rat, and hamster lanes, only one major signal was visible. While the prominent band in the mouse lane comigrated with the 18S RNA, the signal in the rat and hamster lanes indicated a slightly smaller transcript size of the major CREM transcripts in these species. The only signal in the marmoset lane also indicated a smaller transcript size compared with those of the rat and hamster. The most prominent band in the cynomolgus monkey lane migrated similarly to that of the marmoset; and in contrast to observations in the latter species, two additional larger transcripts were present in the cynomolgus monkey. CREM expression seemed discordant between RT-PCR and Northern blotting analyses in the cynomolgus monkey. However, the multiple transcript sizes detected by Northern blotting are most probably due to alternative usage of different polyadenylation sites in activator transcripts that are not detected by RT-PCR (unpublished observations).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 with and without exon ) were cloned and sequenced; the shorter of the two was found to correspond to the human CREM isoform [16]. In the mouse, amino acids 29, 30, and 55 of the human CREM protein were missing [16] but were present in the monkey. The stop codon differed between the two primate species: codon 296 is the stop codon in the monkey and mouse, whereas in the human this codon encodes tyrosine followed by the stop codon at position 297. Hence, this divergence seems to be a very late event during primate evolution. In general, however, the present work and earlier studies reveal a remarkable conservation of CREM amino acid sequence during mammalian evolution [7, 16, 24]. CREM activator expression was further analyzed by RT-PCR using primers located in exon B and exon H. The amplified fragments contained the transactivation domain present in all CREM activators. We evaluated 19 organs and observed that CREM activator expression in the primate was confined to the testis. These results were confirmed for the testis and 9 other organs by Northern blots (not shown). Our findings are at variance from those reported for the dog [24], in which CREM activator transcripts have been found in several organs including liver, heart, kidneys, cortex, and cerebellum. These organs were also investigated in the present study, but activator transcripts could not be detected. These discrepant observations might reflect species differences or different methodology, since the work with dog organs [24] used RNase protection assays. In mice, targeted elimination of all CREM isoforms affected only testicular function and had no discernible effects on the development and health of the animals [2, 3]. The potential physiological role of CREM activators in canine organs, if any, remains to be seen. Basal transcription without physiological relevance is also conceivable [25]. For example, spermatid-specific transcripts were detected by RT-PCR in female peripheral blood cells [26]. The present study demonstrated testis- and germ cell-specific CREM activator expression in the primate, and it is conceivable that CREM activator(s) play a pivotal role only in the testis.
CREM mRNA expression analysis revealed specific signals in stage VII pachytene spermatocytes up to stage IX round spermatids in monkeys, and in stage IX pachytene spermatocytes up to stage IX round spermatids in the rat. This CREM expression pattern is nearly identical to what we reported recently for the CREM protein in these two species ([8] and Fig. 4). The similarity between CREM protein and mRNA expression demonstrates a lack of a translational delay. Translational regulation of gene expression is an important control mechanism occurring during mammalian germ cell development [1, 27] to ensure timely protein synthesis during the late stages of spermatid maturation since advanced spermatids lack transcriptional activity. A striking example of translational delay is provided by the expression pattern of transition proteins and protamines [1, 27] that are targets for CREM. Delayed translation has also been discussed for CREM expression in the mouse testis [9]. However, we did not obtain evidence for translational control of testicular CREM expression in the rat or monkey models. Indeed, there is no obvious need for translational delay of CREM expression, since the expression of CREM protein occurs in the early stages of spermiogenesis [8].
We could not distinguish between activator and repressor isoforms of CREM by in situ hybridization. To gain an insight into the expression of the differing activating CREM isoforms in the rodent and primate testis, this issue was studied by RT-PCR analysis. Mouse, rat, and hamster testes expressed three different CREM activators. In the marmoset, the CREM expression pattern was comparable to that of rodents, but transcript size was smaller. In the macaques, only two CREM activators could be detected; and in the human, only the CREM 2 transcript was present. These findings highlight an evolutionary trend toward a reduction in the number of CREM activator transcripts resulting in only one isoform in the human. Testicular CREM RNA processing appears substantially different between species. The significance of this observation, however, is not clear at present. CREM regulates genes encoding structural proteins [9, 28] and is necessary for the morphological maturation of spermatids [2, 3]. Unlike those of rodents and macaques, human sperm frequently display abnormal head shapes [29]; and it could be argued that the number of CREM activators in spermatids is related to the precision and efficiency of the coordinated maturation and differentiation of sperm.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Deutsche Forschungsgemeinschaft, Confocal Research Group Hamburg/Münster: The Male Gamete: Production, Maturation, Function. 
2 Correspondence: G.F. Weinbauer, Institute of Reproductive Medicine of the University, Domagkstr. 11, D-48129 Münster, Germany. FAX: 49 251 8356093; weinbau{at}uni-muenster.de
Accepted: December 31, 1999.
Received: July 28, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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