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Testis |
a Research Unit Molecular Biology, Research Institute for the Biology of Farm Animals, 18196 Dummerstorf, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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central nervous system, estradiol, gene regulation, ovary, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[1] and ER-ß [2], both of which regulate the expression of a variety of different genes. However, in some tissues as brain, fast-acting nongenomic effects of estrogens have also been observed [3, 4]. In many tissues such as ovary, placenta, brain, and testis, the key genes of estrogen signaling and biosynthesis, ESR1 (ER-), ESR2 (ER-ß), and Cyp19, which encode the enzyme aromatase cytochrome P450, are coexpressed, suggesting that estrogen acts locally as a paracrine or autocrine factor [5–8]. Disruption of these genes in mice demonstrated that estrogen signaling is not only important for the development and differentiation of the reproduction systems in females, but also in males [9–13]. At present, the view has been widely accepted that at least in rodents, the function of male gonads is essentially regulated by estrogens that are locally produced by all testicular somatic and germ cell types [14, 15]. In addition, behavior, bone formation, and fat deposition are significantly influenced by paracrine estrogen actions [16–19]. In mice and rats, ovary, brain, and testis are the organs that produce estrogen [20]. Estrogen biosynthesis has not been observed within the murine placenta [20], although it has been observed in other species such as primates, cattle, sheep, pigs, and rabbits [21–24]. In several mammalian species that have been investigated, tissue-specific expression of Cyp19 is regulated by using different promoter regions and alternative splicing [25]. This results in the generation of Cyp19 transcript variants with different 5' untranslated regions (UTRs) but identical coding sequences. In humans, nine untranslated exons, which encode different 5' UTRs and corresponding promoter regions, have been identified. The different promoter regions and untranslated exons span more than 90 kilobase pairs (kb) of the gene, whereas the coding exons are restricted to 30 kb of genomic sequence [26, 27]. In cattle and sheep, Cyp19 is transcribed and alternatively spliced to seven and six transcript variants, respectively [24, 28, 29]. Cyp19 expression in the placenta is controlled, surprisingly, by different species-specific promoters in humans, cattle, sheep, pigs, and rabbits [22–24, 30], whereas expression in ovary and brain is mainly regulated by promoters, which are conserved between different species: the most proximal, ovarian promoter (promoter II or 2 [28, 31]), and the distally located brain-promoter (promoter 1f or 1.4, [28, 32]). In rodents, transcripts derived from two different promoter regions have been identified so far. In rats and mice, the conserved proximal promoter was found to direct ovarian transcripts, whereas the distally located promoter was found to be preferentially active in the brain [33, 34]. All Cyp19 transcripts of testicular somatic and germ cells in rats are derived from the proximal promoter II [35].
An alternative mechanism of tissue-specific regulation has been described in pigs and fishes. In these species, more than one functional copy of the aromatase encoding gene has been identified [36–38]. Whereas in fish, both Cyp19a and Cyp19b are preferentially expressed in either ovary or brain [39]; in pigs, different aromatase transcripts and corresponding isoenzymes are derived from three different Cyp19 genes in placenta, ovary, and embryonic tissues [36, 40]. Also in cattle, transcripts from a second copy of Cyp19 have been found at low concentrations [41]; however, it was demonstrated that these transcripts do not include open reading frames due to point mutations and aberrant splicing, and thus they could be regarded as transcripts that were derived from a nonfunctional pseudogene (Cyp19
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During the present investigation, Cyp19 transcript variants from mouse gonads and brains were isolated using rapid amplification of cDNA 5' ends (5' RACE). By means of the isolated untranslated exons and the corresponding promoter regions the complete mouse Cyp19 locus could be reconstructed from a recently published bacterial artificial chromosome (BAC) clone. Concentrations of isolated transcripts were estimated in gonad and brain tissues with real time polymerase chain reaction (PCR) during different stages of postnatal development in order to further elucidate tissue-specific regulation of the mouse Cyp19 gene.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tissue Collection and RNA Preparation
Tissues were collected from mice of the outbreed strain FztDu [42] at Postnatal Days P0, P7, and P14, and from adult animals (6 wk of age). Before RNA preparation, all tissues were immediately submerged and stored in RNAlater solution (Qiagen, Hilden, Germany) at 4°C for 1 to 2 days or at -20°C for longer storage. Whole brains were dissected and thalamic areas together with hypothalamic areas were collected. Testes and ovaries were dissected out and freed from adjacent tissues. In young animals (P0 to P14), gonads and brain tissues from five animals were immediately pooled in RNAlater solution. Adult ovaries were collected at proestrus stages.
Total RNA preparations were performed by using the RNeasy mini kit (Qiagen) as recommended by the supplier. The quality of RNA was analyzed after separation on a 1% ethidium bromide stained agarose gel containing 2.2 M formaldehyde. RNA was quantified in a GeneQuant II instrument (Pharmacia, Freiburg, Germany).
5' RACE
To isolate Cyp19 transcripts from different tissues and to determine the start sites of transcription, 5' RACE was carried out with the 5'/3'-RACE Kit (Roche, Mannheim, Germany). Two micrograms of total RNA from each tissue (brain from newborn males, testis and ovary from adult animals) were reverse transcribed with primer E5r (5'-GAGCATGTTAGAGGTGTCCAGCA-3'), which binds to an exon 5 encoded sequence. For the first amplification primer, E3r (5'-GACTCTCATGAATTCTCCATACATCT-3', Fig. 1a) was combined with the dT17-adapter primer provided with the kit. For the second, "nested", amplification the reverse primer E3rr (5'-AATGAGGGGCCCAATTCCCAGA-3', Fig. 1a) was combined with the adapter primer. RACE products were cloned into the pGEM-T Vector (Promega, Mannheim, Germany) and subsequently sequenced.
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Reverse Transcription-Polymerase Chain Reaction
In order to amplify the transcript variants Tov, Ttes, and Tbr that had been isolated by 5' RACE, the sequence information of RACE products was used to design forward primers that bind to the different 5' UTR of these transcripts (Eov, 5'-CAGCCAAACCGCTGGGTTACGT-3'; Ebr, 5'-ATCCGGTTTTTAAACGGCTGCGCATC-3'; and Etes, 5'-AAAGCGATGGGGAAAACCGGAGAG-3'; see Fig. 1a). For cDNA synthesis, 1 µg of total RNA was reverse transcribed in a 25 µl reaction using Moloney murine leukemia virus (MMLV) reverse transcriptase (RT), RNase H Minus, Point Mutant (Promega, Madison, WI). Reverse primer E5r was used if only the 5'region, comprising sequences encoded by exons 1 to 3, needed to be subsequently amplified, whereas the primer E10r (5'-GTGAGGTTCACGCCACCTACTC-3'), which binds to an exon 10 encoded sequence was used if the complete coding region needed to be amplified. For amplifications, 5' UTR specific forward primers (Eov, Ebr, and Etes) were combined with reverse primers E3r or E10r for the first amplification and subsequently with nested primers E3rr or E10rr (5'-CGGATAAGTAATGCCCCAGAGTAG-3') for the second amplification, respectively. The 5' region was amplified with Taq polymerase (Qbiogene, Heidelberg, Germany), longer products with the Expand High Fidelity PCR system (Roche) using 1.25 µl of 25 µl cDNA for the first amplification (30 cycles) and 0.01 µl of PCR product for the second, nested amplification (30 cycles) in both cases. The identity of all different products was checked at least once by cloning and sequencing.
Real Time PCR
Concentrations of different transcript variants (Tbr, Tov, and Ttes) in different sexes, tissues, and developmental stages were measured to estimate the activities of the corresponding promoter regions. One microgram of total RNA was reverse transcribed in a 25-µl reaction volume using MMLV RT, RNase H Minus, Point Mutant (Promega) with primer E5r. The freshly synthesized cDNA was cleaned using the High Pure PCR Product Purification Kit (Roche) and eluted in a 100 µl elution buffer.
For real time PCR, 2- and 4-µl samples of the purified cDNA were amplified with the LightCycler-FastStart DNA Master SYBR Green I Kit (Roche) in 10 µl of total reaction volume. Amplification and quantification of generated products were performed in a LightCycler instrument (Roche) under the following cycling conditions: preincubation at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 10 sec, extension at 72°C for 10 sec, and a single point fluorescence acquisition at 83°C for 6 sec.
For real time PCR reactions the reverse primer E3rr was either combined with different 5' UTR-specific forward primers to estimate concentrations of transcript variants Tbr, Tov, and Ttes, or in order to assess concentrations of all variants (Tall), with forward primer E2 (5'-GGCCAAATAGCGCAAGATGTTCTT-3'), which binds to a sequence included in all transcript variants (Fig. 1a). PCR products that were generated with different primer combinations were cloned into pGEM-T vector and analyzed by sequencing. During real time PCR the sizes of generated products were monitored from randomly selected samples by agarose gel electrophoresis analysis (3% agarose, ethidium bromide staining). The melting peak (mp) of each sample was routinely determined by melting curve analysis in order to ascertain that only the expected products had been generated. Tov specific products were generated with primer combination Eov/E3rr (275 base pairs [bp]; mp, 86.1°C), Tbr products with primers Ebr/E3rr (279 bp; mp, 85.6°C), Ttes products with primers Etes/E3rr (250 bp; mp, 86.5°C) and Tall with primers E2/E3rr (196 bp; mp, 86.0°C).
To generate external standard curves for each run, different concentrations (2 x 10-17 to 2 x 10-13 µg DNA/µl) of Tov PCR product cloned into pGEM-T plasmid vector were coamplified with primers E2/E3rr. Fluorescence signals, which were recorded on-line during amplification, were subsequently analyzed using the Second Derivative Maximum method of the LightCycler Data Analysis software (Roche). Copy numbers were calculated relative to the amount of total RNA.
Tissue samples of all developmental stages were analyzed as pools of five animals, which were either pooled before (stages P0, P7, and P14) or after RNA preparation (adult tissues). Each pool was measured at least three times (three independent RT reactions) and the means and SEM were calculated.
Isolation of the Genomic Region Flanking Exontes
In order to isolate and characterize the presumable promoter Ptes, which directs expression of the newly identified transcript variant Ttes, the sequence upstream from the start site of transcription of the corresponding untranslated exon (exontes) was isolated by the PCR based vectorette system, Universal GenomeWalker Kit (BD Clontech, Heidelberg, Germany) according to the user manual. Briefly, high molecular weight genomic DNA from a mouse that had been prepared by standard procedures using proteinase K (Roche) digestion and phenol/chloroform extraction [43] was completely digested with the restriction enzymes DraI, EcoRV, PvuII, ScaI, and StuI. The blunt ended genomic fragments were ligated to adapters that included two primer binding sites. Subsequently, aliquots from all five ligated samples were used as templates for PCR. Two successive PCR reactions using the Expand High Fidelity PCR System (Roche) were performed by combining two different adapter primers provided with the kit with two nested, gene-specific reverse primers Gr and Grr (5'-CGTTCCCCATGGTAACCCGCGGGCAT-3' and 5'-ATGCCTCTCCGGTTTTCCCCATCGCT-3', see Fig. 1a) derived from the 5' UTR of Ttes. For analysis, PCR products were cloned into the pGEM-T vector system (Promega) and sequenced.
DNA Sequencing and Database Search
Sequencing was performed with the automated sequencing systems ABI PRISM 310 Genetic Analyzer using the ABI PRISM BigDye Kit (both from PE Applied Biosystems, Weiterstadt, Germany) or Li-COR 4200 Series DNA Analysis Systems (MWG, Heidelberg, Germany) using the Thermo Sequenase fluorescent-labeled primer cycle sequencing kit (Amersham Pharmacia, Freiburg, Germany).
European Molecular Biology Laboratory (EMBL) and GenBank databases were searched using FASTA software, which is part of the WWW2HUSAR software package from the German Cancer Research Center (Heidelberg, Germany).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Altogether, 20 RACE clones were isolated and could be unambiguously identified as Cyp19 transcripts because of sequence similarities with a published mouse transcript [44] (EMBL/GenBank accession number D00659). Five clones had truncated or no 5' UTR and were therefore considered as artifacts due to the premature stopping of the reverse transcription reaction. Analysis of the remaining 15 clones revealed that from three different tissues (ovary, testis, and brain), three different transcript variants had been amplified (Fig. 1a). The 5' UTR of all 10 ovarian transcripts (Tov) were identical to that of ovary aromatase cDNA described by Honda et al. [33]. Three of these clones also indicated an identical start site of transcription (position 221 of EMBL/GenBank accession number D67046), whereas the remaining seven clones started one nucleotide farther downstream. This position was considered as the major transcription start site. Three transcripts from newborn brain (Tbr) could be identified as brain aromatase cDNA [33]. Start sites of transcription were at position 431 of EMBL/GenBank accession number D67045, which is two nucleotides downstream from that described by Honda et al. [33]. The 5' UTR of both clones isolated from adult testis (Ttes) did not show sequence similarities to any known Cyp19 transcript.
Isolation of Exontes Flanking Sequences and Reconstruction of the Mouse Cyp19 Locus
By using a PCR based vectorette technique with primers that had been derived from the 5' UTR of the newly identified testis transcript Ttes, the sequence flanking the corresponding untranslated exon, which presumably harbors promoter Ptes, could be derived from two overlapping PCR products of different lengths (1598 and 740 bp), which had been generated from the StuI and DraI digested libraries, respectively (see Materials and Methods). The combined sequence was submitted to the EMBL database as accession number AJ437576.
A database search using the isolated genomic sequence AJ437576 as a key and sequences of various public databases as targets eventually identified a recently submitted mouse BAC (RP23-126K16, EMBL/GenBank accession number AC113972) because of its high sequence similarity (99.6% identity). In addition, the complete cDNA [44], alternative exons, promoters [33], and partial intronic sequences (unpublished) of the mouse Cyp19 gene could also be mapped to this BAC. After rearranging relevant sequence contigs of BAC RP23-126K16 the complete Cyp19 locus of the mouse could be reconstructed from 6 of the 14 contigs (Fig. 1b). According to this preliminary map, the mouse Cyp19 locus comprises about 60 kb. The three noncoding exons (br, tes, and ov) and their flanking promoter regions cover more than half of the locus, whereas all nine coding exons are restricted to about 29 kb. Promoter Pbr is located about 31 kb upstream, and Ptes about 10 kb upstream from the start site of translation that is located within exon 2. As in other species, Pov is the most proximal promoter.
Expression of Cyp19 Transcript Variants in Gonads and Brain
By combining 5' UTR specific primers Eov, Ebr, and Etes with reverse primers E10r for the first amplification and E10rr for the second, nested amplification, specific products of all transcript variants could be generated by RT-PCR (Tov, 1646 bp; Tbr, 1650 bp; and Ttes, 1621 bp). However, as shown in Figure 2a, products of Ttes were present only in testis. To the contrary, short amplicons from the 5' region of Ttes could be detected in all tissues (Fig. 2b).
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Real time PCR was performed by amplifying the 5' regions of transcript variants encoded by the first two to three exons. Therefore, quantification included full-length as well as possibly 3' truncated Cyp19 messengers.
To ascertain that data derived from pooled tissue samples are similar to those from individual samples, testes and brains from five adult males were analyzed for Cyp19 transcript concentrations (primer combination E2/E3rr) as pooled and individual RNA samples. Pooled tissues were measured five times following five independent RT reactions, and individual tissues were reverse transcribed and measured once each. Real time PCR revealed that mean transcript concentrations determined from individual samples (brain, 8.2 ± 1.2 x 103 copies/µg RNA; testis, 19.9 ± 1.1 x 103 copies/µg RNA) were similar to those determined from pooled samples (Fig. 3, a and b), however, showed relatively large standard errors due to variations between individual samples.
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Estimates of Cyp19 transcript numbers by real time PCR revealed that in gonads at stage P0, concentrations are similar in male and female neonates (Fig. 3a). However, whereas Cyp19 expression was transiently reduced in immature testes between P7 and P14, ovarian expression increased dramatically from stage P14. Expression in adult ovaries exceeded that of neonatal ovaries by about 100 times.
5' UTR specific PCR revealed that all three transcript variants were present in gonads and brains of both sexes throughout postnatal life, but at very different concentrations. The arithmetically added concentrations of the three transcript variants (Tov, Ttes, and Tbr) were between 83% (adult testis) and 127% (P0 testis) of the experimentally determined concentrations of all transcripts (Tall).
In testes of all developmental stages investigated, Ttes contributed the majority of Cyp19 transcripts. In neonatal ovaries, Tov only slightly exceeded concentrations of alternative transcript variants. But Tov contributed more than 97% of Cyp19 transcripts at P14, and almost 100% in adult ovaries.
Brain tissues of both sexes showed similar Cyp19 transcript levels at P0 (Fig. 3b). At P7, expression was unchanged in males but it dropped to about half in females, which resulted in remarkable differences between both sexes. At P14 in male brains, Cyp19 expression also dropped to adult levels. Relative concentrations of different transcript variants were similar in both sexes at all stages investigated. Tbr clearly predominated in brain by contributing more than 85% of all Cyp19 transcripts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been shown that Tov or homologous transcripts of other species, which are derived from the most proximal promoter of Cyp19 (Pov in mice, PII in humans, or P2 in cattle and sheep), is the principal transcript variant in the mature ovary. This confirms earlier investigations in species such as mice [33], rats [45], rabbits [30], humans [31], cattle, and sheep [24]. However, Tbr and Ttes could be detected at low but similar levels throughout ovarian development. In adult ovaries, both transcripts contributed less than 1% compared to total Cyp19 transcript numbers. The dramatic increase of ovarian Cyp19 expression from Day P14 therefore must be due to increased activity of Pov only.
This proximal promoter was also described as the major promoter in the rat testis [35]. As in other species, therefore, this promoter was described as the major gonadal promoter [46]. Contrary to these observations, the mouse testis does not express Tov as the major Cyp19 transcript. RACE experiments and 5' UTR specific real time PCR performed during this study demonstrated instead that the novel transcript variant Ttes is the major testicular transcript in mice. This suggests that the newly defined promoter region, Ptes, is the principal mouse testis promoter. It may be suggested that this promoter is identical to the newly isolated sequence flanking the untranslated exontes, the transcription start site of which has been defined by 5' RACE experiments. However, RACE products may have been artificially truncated, and the start site could therefore be farther upstream.
Between P7 and P14, transcript concentrations were remarkably reduced compared with those in P0 and adult testes. Experiments of the present study do not allow speculations on mechanisms or physiological functions of this transient decline. The fact that the relative proportions of transcript variants was not remarkably changed at P14, however, suggests that all promoters, including Ptes, show transient and proportional reductions of their activity.
In rat testis and ovary, Cyp19 transcripts with alternatively spliced coding sequences have been found [47, 48]. These aberrant transcripts may encode nonfunctional enzymes and may be involved in controlling aromatase expression and thus modulation of estrogen production in testis [48]. A similar observation was also described for the rat brain. In this tissue, a truncated Cyp19 transcript was observed, the presence of which, however, was not associated with aromatase activity [49]. Experiments in the present study demonstrate that only in testis do all three transcript variants, Tov, Tbr, and Ttes, include full-length reading frames, although PCR products of the 5' region of each transcript could be generated from all tissues. This suggests that within ovary and brain, only truncated species of Ttes are present, which are unlikely to encode fully active aromatase enzymes, but instead may be the result of a posttranscriptional regulatory mechanism.
As in mouse gonads, all three transcript variants were detected in the brain areas investigated. The relative proportions of the different transcripts were similar between the sexes, showing clear predominance of Tbr at both stages investigated (P0 and adult). This confirms investigations by others who also demonstrated in mice and rats that transcript variants homologous to Tbr, which were derived from an alternative, distally located promoter region (Pbr in mice, P1.f in humans, P1.4 in cattle and sheep), are preferentially expressed in the brain [33, 50]. Contrary to the relative distribution of transcript variants, concentrations of Tall are different in male and female brains, particularly around P7. Highest expression was found in neonates of both sexes, which corresponds to earlier investigations demonstrating that Cyp19 expression and aromatase activity peaks during early postnatal life in various brain areas [8, 51, 52]. However, at P7, expression had declined to about adult levels in females but was still unchanged in males. This agrees with the observation by MacLusky et al. [51], who found that the aromatase activity declines earlier during development in the hypothalamic-preoptic area in females than it does in males. It is interesting that the first week after birth was found to be crucial for establishing sexual differences in the brain [53]. It is possible that the differences in Cyp19 expression during that time might be functionally associated with the establishment of sexual brain dimorphisms in rodents. The reason for different levels of Cyp19 expression in both sexes is not clear. Several investigations have demonstrated that expression of Cyp19 is stimulated by androgen actions [52, 54, 55]. In fact, plasma testosterone levels are higher in male than in female rat pups. However, the most pronounced differences were found during late fetal development and in the first hours after birth [56, 57]. This does not directly correspond to the observed differences of Cyp19 transcript concentrations between the sexes, which are not apparent in neonates, but are particularly pronounced several days later during postnatal life.
The reconstruction of the Cyp19 locus from BAC sequence contigs also revealed that in mice, regulatory regions represented by untranslated exons and their flanking promoters cover more of the locus than all nine coding exons together. This emphasizes the complex mechanisms of transcriptional regulation of this gene. The most distal promoter, Pbr, is located at least 31 kb from the first coding exon. Although the precise distances could not be determined because of still uncertain gaps between the sequence contigs, this distance corresponds well to that estimated for the homologous human brain-specific promoter 1.f [26], suggesting the conserved character of this genomic region. In the human gene, the most distally located promoter region, which is responsible for strong placenta-specific expression (PI.1), was found more than 90 kb upstream from the start site of translation. According to our data, the mouse Cyp19 locus comprises at least 60 kb, and according to investigations by others [58], it is located on chromosome 9.
Experiments presented during this study demonstrate the expression of Cyp19 transcripts with three different 5' UTR variants. This suggests that in mice, at least three different promoters actively regulate Cyp19 expression in gonads and brain. Additional transcript variants may exist at low concentrations but they were not detected with 5' RACE, and thus were not included in our quantification. The data available confirmed the view that Cyp19 regulation in ovary and brain is generally conserved among different species. Only in the horse ovary a switch between the ovary and brain promoters has been described during ovarian cycling [59]. Whereas ovarian transcripts are derived almost exclusively from the most proximal promoter, brain derived aromatase expression is mainly regulated by activity of the distally located brain promoter.
In the mouse testis, however, a third, so far unknown transcript variant was found to predominate. This suggests that the corresponding promoter Ptes decisively regulates testicular Cyp19 expression in this species. In order to understand the complex Cyp19 regulation in the mouse testis, further studies are necessary to elucidate the activity of this promoter in different testicular cell types and to define regulatory elements and binding transcription factors. Comparative analysis in rat that shows activity of a completely different promoter region in testis [35] may also help to unravel testicular Cyp19 regulation in both species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Jens Vanselow, Research Unit Molecular Biology, Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany. FAX: 49 38208 68702; e-mail: vanselow{at}fbn-dummerstorf.de
Received: 4 June 2002.
First decision: 2 July 2002.
Accepted: 2 October 2002.
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