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Laboratoire Estrogènes et Reproduction, EA 2608-USC INRA 2006, Université de Caen, Esplanade de la Paix, 14032 Caen Cedex, France
¹ INRA, UMR1245, INSERM U 418 Hopital Debrousse, 29 rue Sœur Bouvier, 69322 Lyon Cedex 05, France

(Requests for offprints should be addressed to S Carreau; Email: serge.carreau{at}unicaen.fr)

D Silandre is a recipient of a fellowship from Région Basse Normandie

	Abstract

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Aromatase is the key enzyme responsible for the irreversible transformation of androgens into estrogens. It is encoded by the cyp19 gene and is expressed in the mammalian testis under the control of the proximal promoter PII. Since both somatic and germ cells contain a biologically active aromatase, we looked for the existence of other promoters that may direct the expression of aromatase in adult rat germ cells. Besides the promoter II, we have shown the presence of transcripts derived from the brain promoter PI.f in spermatogonia–preleptotene spermatocytes (G–PL), pachytene spermatocytes (PS), and round spermatids (RS). A new aromatase cDNA has been isolated by 5'-rapid amplification of cDNA ends that we named I.tr (testis rat). The I.tr transcripts are found in all the testicular cell populations studied with a greater expression in PS. Because of the utilization of these three promoters in the adult rat testis, we studied their putative involvement according to the age. At 10 days, aromatase expression was very low and then strongly increased between 10 and 20 days, with a preferential activation of PII and PI.tr. Transcripts coming from PI.f were found starting from 20 days onwards. The new promoter PI.tr, localized between promoters PI.f and PII, is devoid of a TATA box but contains a transcriptional initiator (Inr) and putative regulatory sequences. Therefore, the identification of the specific trans-activating factors should bring some enlightenments to understand the regulation of these three promoters in germ cells especially according to their stage of development.

	Introduction

Top Abstract Introduction Materials and methods Results Discussion References

 
The androgens/estrogens balance is essential for normal sexual development and reproduction in mammals. In the testis, maintenance of this balance is under a fine tuning via endocrine and paracrine factors, but is also related to aromatase activity. Aromatase, an enzymatic complex, catalyzes conversion of androgens into estrogens and is localized in the endoplasmic reticulum of various tissues including brain, gonads, placenta, and adipose tissue. Aromatase is composed of two proteins, a ubiquitous reductase and a specific cytochrome P450 aromatase (P450arom). The P450arom is encoded by a single cyp19 gene in most of the species except for the pig in which three distinct genes encode three aromatase isoenzymes (Graddy et al. 2000) and for the fish in which two cyp19 genes (specifically expressed in brain and gonads) have been identified (Kishida & Callard 2001).

The human cyp19 gene is located in the 21.2 region of the long arm of chromosome 15 and is ~123 kb length. This gene contains nine coding exons (exons II–X), and upstream of exon II, a regulatory region is present (Simpson et al. 1994). This 5'-region contains ten promoters that regulate CYP19 gene expression in a tissue-specific manner. In humans, the main promoters used are PI.1, PI.2, and PI.2a in the placenta, PI.3 and I.4 in the adipose tissue, PI.5 in fetal tissues, PI.6 in the bone, PI.7 in endothelial cells, PI.f in the brain, and PII in the ovary and the testis (Bulun et al. 2004). As a consequence, P450arom transcripts present in these various tissues differ in their 5'-UTR giving rise to different mRNAs; nevertheless, the encoded protein is identical with a molecular mass of 55 kDa (Nakajin et al. 1986). In fact, whatever be the promoter used, each untranslated first exon is spliced into a common splice junction localized 39 bp upstream of the translation starting site (adenine, uracil, guanine (AUG); Simpson et al. 1997).

In the mouse, the cyp19 gene is located on chromosome 9, and Golovine et al.(2003) have shown that three promoters control specifically aromatase expression in gonads (Pov and Ptes) and the brain (Pbr).

In the rat, the cyp19 gene is situated on chromosome 8 and up to now two promoters have been described: the promoter PI.f in the brain (Yamada-Mouri et al. 1996) and the promoter PII (Young & McPhaul 1998) which is the main one directing aromatase gene expression in the ovary (Hinshelwood et al. 1993) and the testis (Lanzino et al. 2001). In the rat testis, the cellular expression of aromatase is age dependent: during the fetal and neonatal development, aromatase is expressed in Sertoli cells, whereas in the adult, aromatase has been localized in many cell types including Leydig cells, spermatocytes, spermatids, and spermatozoa (Carreau et al. 2003, 2006). The amount of P450arom mRNA decreases according to the stage of maturation the rat germ cells: it is higher in pachytene spermatocytes (PS) than in round spermatids (RS) and spermatozoa. However, the aromatase activity evolves inversely (Levallet et al. 1998).

In purified rat germ cells (PS and RS), Bourguiba et al. (2003a,b) have shown that numerous factors are involved in the regulation of aromatase gene expression as cyclic AMP (cAMP), cytokines, and steroids. However, it is not known on which promoter(s) these factors act. Indeed, in the adult rat, Lanzino et al.(2001) have demonstrated that the promoter PII is the main one that directs the aromatase gene expression in Sertoli, Leydig, and germ cells, but according to their data other promoters might be involved especially in germ cells. Moreover, only five responsive elements in the proximal promoter PII of the rat aromatase gene have been identified: one steroidogenic factor-1 (SF-1) binding site at –90 bp relative to the starting site of transcription, one GATA binding site at –129 bp, and three CRE-like sequences at –169, –231, and –335 bp (Young & McPhaul 1998, Bouchard et al. 2005). All together, these observations favor the existence of additional promoters that may control the aromatase gene expression in rat testicular cells.

In the present study, we have shown that the promoter PII is not the only one involved in the regulation of aromatase gene expression in mature rat germ cells. Indeed, we have demonstrated that two other promoters are used: PI.f (brain promoter) and a new promoter called PI.tr (testis rat promoter). In addition, we have looked for the expression of the transcripts issued from these different promoters in the whole rat testis according to the age and shown that PII, PI.f, and PI.tr are likely playing an important role between 10 and 30 days, a period corresponding to meiosis completion.

	Materials and methods

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Animals

Sprague–Dawley rats were purchased from the University of Caen, France. They were bred under standard conditions (12 h light:12 h darkness cycle and controlled room temperature) with standard rat food and water ad libitum. All animal procedures were carried out in accordance with the French Government Regulations (Services Vétérinaires de la Santé et de la Production Animale, Ministère de l’Agriculture) and approved by the Local Ethical Committee of the University.

Tissue collection

Animals were killed by decapitation, and then several tissues (testis, ovary, brain, pituitary, liver, and muscle) were dissected, flash-frozen in liquid nitrogen, and stored at –80 °C until RNA extraction.

Purification and incubation of Germ cells

Preparation of an enriched fraction of spermatogonia–preleptotene spermatocytes and PS
Spermatogonia–preleptotene spermatocyte fractions were isolated from 23-day-old Sprague–Dawley rats. Germinal cells were extracted from the testis by mechanical dissociation and a combination of trypsin and DNase according to Vigier et al.(2004), then they were separated by centrifugal elutriation (Bucci et al. 1986). The enriched spermatogonia–preleptotene spermatocytes fraction was further subjected to differential plating (12 h at 33 °C in Ham’s F-12/Dulbecco’s modified Eagle’s medium (DMEM) (PAN-Biotech, Brumath, France) containing 0.2% fetal calf serum (Fisher Bio-block Scientific, Illkirch, France)) in order to eliminate the contaminating somatic cells. Consequently, the Sertoli and myoid cells remained attached to the culture plates, and the spermatogonia–preleptotene spermatocytes in suspension were collected. The purity of that enriched germ cell fraction ranged between 78 and 82%. Preparation and purification of PS by elutriation were performed as described by Vigier et al.(2004).

Purification of pachytene spermatocytes and round spermatids
Testicular mixed germ cells of 90-day-old rats were obtained by trypsin–DNase treatment (Bourguiba et al. 2003a); the germ cell suspension was washed in PBS supplemented with 6 mM glucose (Merck) and 10 mM pyruvic acid (Sigma). The germ cells were filtered through fine nylon mesh to hold Sertoli cells and then through glass wool to remove spermatozoa. The separation of the different cell types was realized by unit gravity sedimentation through a BSA (Roche Diagnostics) gradient (0.2–2.75%) in a Sta-Put apparatus (Bellve et al. 1977). The fractions enriched with PS and RS were identified and washed with the above PBS buffer. The cells (2.5x10⁶ PS/ml and 4.5x10⁶ RS/ml) were incubated for 4 h in Ham’s F-12/DMEM (Sigma) with 2% Ultroser, serum substitutes without steroids (Ciphergen, Le Raincy, France), containing NaHCO₃ (2.44 g/l), Hepes (3.57 g/l), streptomycin (100 mg/l), penicillin (100 000 U/l), and fungizone (250 µg/l), and supplemented with 10 mM pyruvic acid (Sigma) and 6 mM glucose (Merck). Then the cells were further incubated in a fresh medium devoid of serum for 12 h at 32 °C under a humidified atmosphere of 5% CO₂ and 95% air.

To estimate the contamination of the germ cell fractions by Leydig cell, a 3ß-hydroxysteroid dehydrogenase histochemical staining (Klinefelter et al. 1987), a specific marker of Leydig cells, was realized, showing < 1% contamination. Moreover, germ cells were incubated for 4 h in Ham’s F-12/DMEM in the presence of 2% Ultroser SF (Ciphergen), which quickly improves the adherence of Sertoli cells (De et al. 1993). For both germ cell preparations, the purity was higher than 95% (the major contamination was by other germ cells).

Purification and incubation of Sertoli cells

Sertoli cells were isolated from testes of 20-day-old Sprague–Dawley rats by sequential enzymatic digestion according to the method described by Dorrington et al.(1975). Cells were seeded at a density of 250 000 cells/ cm² and cultured for 48 h in Ham’s F12/DMEM supplemented with 2% Ultroser SF in a humidified atmosphere of 5% CO₂ and 95% air at 32 °C. On day 3, germ cells were removed by hypotonic treatment using 20 mM Tris–HCl (pH 7.2), and 5 days after plating, Sertoli cells were incubated for 24 h with or without db cAMP (0.5 mM) in medium devoid of Ultroser SF.

RNA extraction

The RNAgents kit (Promega) was used to extract RNAs from several tissues and from the various purified testicular germ cells. The purity and integrity of the RNAs were checked spectroscopically at 260 and 280 nm respectively and by electrophoresis on agarose gel (1.5%) stained with ethidium bromide (Sigma).

RT-PCR assay

Total RNA (2 µg) was reverse transcribed for 1 h at 42 °C with 200 IU Moloney murine leukemia virus reverse transcriptase (Promega), 20 IU RNasin, 0.2 µg oligo dT, and 500 µM dNTP in a total volume of 40 µl. An aliquot of the cDNA obtained was used for PCR; amplification was performed with 1.5 IU Taq DNA polymerase (Promega) in PCR buffer containing 200 µM dNTP, 1.5 mM MgCl₂, and 25 pmoles of each primer (Eurobio, Les Ulis, France) in a total volume of 50 µl. The PCR primers and the size of the resulting PCR product are shown in Table 1. The different cycle profiles used are summarized in Table 2 with the number of PCR cycles for each transcript. In order to quantify the different transcripts, we determined the optimal conditions for PCR, and the L19 ribosomal protein mRNA was used as a standard to assess the relative mRNA levels as described by Tena-Sempere et al.(2002).

The resulting PCR products were analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide (Sigma). Gels were photographed using photoprint Vilbert Lourmat system and analyzed with NIH image computer program (http://rsb.info.nih.gov/nih-image).

5'-Rapid Amplification of cDNA ends (RACE)

RACE was performed to identify the different transcripts of P450arom. In brief, according to the manufacturer’s protocol, 1 µg testis total RNA was used for 5'-RACE cDNA synthesis (BD SMART RACE cDNA Amplification Kit, BD Biosciences Clontech, Erembodegem, Belgium). Then two PCRs were performed: the first one with Universal primer A Mix (UMP, BD Biosciences Clontech) and a specific antisense primer, AS1 (5'₇₅₂-AGCCAGGACCTGG-TATGGAAGATGAGCTCT-3') located between exons II and III under the following conditions: 5 cycles of 94 °C for 30 s and 72 °C for 3 min, 5 cycles of 94 °C for 30 s, 70 °C for 30 s, and 72 °C for 3 min, and 27 cycles of 94 °C for 30 s, 68 °C for 30 s, and 72 °C for 3 min. Then a nested PCR was realized with Nested Universal Primer A (NUP, BD Biosciences Clontech) and antisense primer, AS2 (5'₇₁₂-AATCAGGAGGAGGAGGAGGCC-CAT-3') located in exon II under following conditions: 20 cycles of 94 °C for 30 s, 68 °C for 30 s, and 72 °C for 3 min. These two specific primers of aromatase (AS1 and AS2) have been described by Lanzino et al.(2001). Each product obtained by nested PCR was ligated to pGEM-T easy Vector (Promega), followed by transformation in JM109 bacteria. About 60 clones were picked up randomly and subjected to colony PCR using pGEM-T easy Vector-specific primers T7 and SP6 (T7: 5'-TAATACGACTCACTATAGGG, SP6: ATTTAG-GTGACACTATAGAA).

Sequence analyses

For each cDNA amplified, the identity was verified by sequencing (GENOME express, Meylan, France). Then the sequences were subjected to BLAST homology search (Altschul et al. 1990) (http://www.ncbi.nlm.nih.gov/BLAST).

The TFSearch (http://www.cbrc.jp/research/db/TFSEARCH.html) and Nsite (http://www.softberry.com/cgi-bin/programs/promoter/nsite.pl) programs were used to identify the potential cis-acting elements in the 5'-flanking region of the new exon.

Statistical analysis

Results are means±S.E.M. Statistical analysis was performed using ANOVA (SigmaStat for Windows, Version 3.1; SPSS Inc., Chicago, IL, USA), and means were compared using Dunnett’s method. Statistical significance was accepted at P<0.05.

	Results

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Analysis of aromatase transcripts in germ cells of the adult rat

Aromatase transcripts were detected in all the germ cell populations studied: spermatogonia and preleptotene spermatocyte-enriched fraction (G–PL), PS, and RS when using specific primers (Table 1) for total aromatase (Fig. 1A). In order to compare P450arom expression between these three germ cell populations, we performed semiquantitative RT-PCRs. The intensity of the aromatase signal was statistically different between G–PL and PS: the level of P450arom transcripts was threefold higher in PS when compared with the mixture of G–PL. In addition, the amount of P450arom mRNA in RS was slightly lower than in PS, but 2.3-fold greater than in G–PL though these differences were not statistically significant (Fig. 1B).

View larger version (24K): [in this window] [in a new window]   Figure 1 Aromatase expression in four different populations of rat germ cells: total germ cells (TG), spermatogonia and preleptotene spermatocytes (G–PL), pachytene spermatocytes (PS), and round spermatids (RS). (A) Visualization of aromatase transcripts from TG, G–PL, PS, and RS on 2% agarose gel after RT-PCR. RNA was replaced with water for negative control (nc). (B) Results are expressed as the ratio of aromatase to L19 signal intensities, and TGs are considered to be 100%. The bar chart represents mean± S.E.M. (n=3). Histograms without common letters are significantly different (P<0.05).

In order to amplify only aromatase transcripts derived from PII, we used a forward primer specific for the untranslated exon II (5'PII; Table 1) and a reverse primer specific for exon III (3'PII; Table 1). We obtained a fragment of 241 bp length in germ cells (PS and RS) of 90-day-old rat (Fig. 2). The sequence analysis of this PCR product from pachytene spermatocyte cDNA showed an identity of 99% with the rat ovary aromatase sequence published by Hickey et al.(1990).

View larger version (43K): [in this window] [in a new window]   Figure 2 Expression of P450arom transcripts issuing from promoter PII in pachytene spermatocytes (PS) and round spermatids (RS) from adult rats. Visualization of transcripts II from PS and RS on 2% agarose gel after RT-PCR. RNA was replaced with water for negative control (nc). M corresponds to DNA ladder (100 bp ladder).

Likewise, with a forward primer specific for exon I.f (5'PI.f; Table 1) and a reverse primer specific for the aromatase coding region (3'PI.f; Table 1), we showed for the first time the presence of a specific transcript of 147 bp length issuing from PI.f in both PS and RS (Fig. 3A). Sequence analysis of this PCR product from pachytene spermatocyte cDNA showed an identity of 98% with the rat brain aromatase sequence (Fig. 3B) published by Kato et al.(1997).

View larger version (30K): [in this window] [in a new window]   Figure 3 Expression of P450arom transcripts originating from promoter PI.f (brain promoter) in pachytene spermatocytes (PS) and round spermatids (RS) from adult rats. (A) Visualization of transcripts I.f from PS and RS on 2% agarose gel after RT-PCR. RNA was replaced with water for negative control (nc). M corresponds to DNA ladder (100 bp ladder). (B) Nucleotide sequence alignment of the rat pachytene spermatocyte exon I.f of P450arom cDNA amplified by PCR with P450arom cDNA of the rat brain in italics (Kato et al. 1997). Indicates the splicing site between exon I.f (underlined) and exon II. This alignment was realized by BLAST.

After the colony PCR, different products of amplification were obtained from 60 selected clones. Of the clones, 50% did not have fragment inserted in pGEM-T easy Vector, 24% of them had genomic DNA, and 13% had a fragment of 172 bp length with a sequence similar to the sequence of transcript II described by Hickey et al.(1990). At last, 13% had an insert corresponding at a transcript of 413 pb with a homology of 96% with a fragment of the nucleotide sequence of the rat aromatase promoter published by Young & McPhaul (1998) and the region downstream the splicing site was completely identical to the sequence published by Hickey et al.(1990) (Fig. 4). No aromatase transcript I.f has been found, showing that they are in smaller proportion when compared with aromatase transcripts II and I.tr. Therefore, we selected new primers (Table 1) to assess the presence of this new transcripts I.tr in PS and RS. Indeed, according to the conditions reported in Table 2, RT-PCRs gave a signal at the expected size (479 bp) corresponding to the aromatase transcript specific for the new promoter in both types of purified germ cells (Fig. 5A).

View larger version (27K): [in this window] [in a new window]   Figure 4 Nucleotide sequence of the 5'-RACE-amplified transcript encoding putative exon I.tr and its alignment with the nucleotide sequence of the rat aromatase (+1/+413) published by Young & McPhaul (1998) and Hickey et al.(1990) in italics. Indicates the splicing site between exon I.tr (underlined) and exon II. (+1) Denotes the site of transcription initiation, and the site of translation initiation is represented by the ATG (in bold). This alignment was realized by BLAST. This sequence has been reported on GenBank under accession number EF110566.

View larger version (30K): [in this window] [in a new window]   Figure 5 (A) Detection of P450arom transcripts issuing from promoter PI.tr by RT-PCR in pachytene spermatocytes (PS) and round spermatids (RS) of 90-day-old rats. RNA was replaced with water for negative control (nc). M corresponds to DNA ladder (100 bp ladder). (B) Regulation of transcripts I.tr by db cAMP (0.5 mM) in Sertoli cells of 20-day-old rats. Ratios exon I.tr/L19 are expressed as mean± S.E.M. (n=3). Histograms without common letters are significantly different (P<0.01). (C) Sequence of promoter I.tr (–82/–1) with its exon (+1/+246). The putative site of a transcriptional initiator (Inr) has been underlined and the putative regulatory sites have been boxed. Upper case letters indicate exon I.tr sequence that was obtained from testis.

Specificity of aromatase transcript I.tr

To verify whether this new transcript is testis specific, its presence has been searched in several tissues as ovary, brain, pituitary, and adrenal by RT-PCR. For this experiment, new primers (5'PI.tr:ATCTGCCATCGG-AAAATGAT and 3'PI.tr:TCTCCTCTCCA CTGATC-CAGA) have been designed. Indeed, some non-specific amplifications were observed in these tissues with primers 5'PI.tr and 3'PI.tr described in Table 1. As shown in Fig. 6, aromatase transcript I.tr (511 pb) was only present in testis contrary to aromatase transcripts II and 1.f, which were found in testis, ovary, and brain (data not shown).

View larger version (42K): [in this window] [in a new window]   Figure 6 Expression of total aromatase transcripts and transcripts I.tr in testis (T), ovary (O), brain (B), pituitary (P), and adrenal gland (A). L19 transcript expression was verified in order to show the good efficiency of the RT. RNA was replaced with water for negative control (nc). M corresponds to DNA ladder (100 bp ladder).

In the elutriated G–PL population, the levels of the different aromatase transcripts were lower than in PS (Fig. 7A). However, a significant difference between PS and G–PL was observed only for the total aromatase transcripts (threefold less) and the transcript I.tr (fivefold less). As for the transcript from PI.f, it was found in all samples of PS but the presence of this transcript in G–PL was observed only in one sample among three.

View larger version (15K): [in this window] [in a new window]   Figure 7 Expression of total aromatase transcripts and transcripts II, I.f, and I.tr in different populations of rat germ cells. (A) Obtained by elutriation, a mixture of spermatogonia–preleptotene spermatocytes (G–PL) and pachytene spermatocytes (PS), and (B) purified by gravity sedimentation, pachytene spermatocytes (PS) and round spermatids (RS). Total RNA was extracted, and cDNA obtained by RT was amplified by PCR. The resulting amplification products were analyzed as described in Materials and Methods. Results are expressed as the ratio of total aromatase transcript, transcripts coming from PII, PI.f, and PI.tr to L19 signal intensities and PS are considered to be 100%. The bar chart represents mean± S.E.M. (n=3). Histograms without common letters are significantly different (P<0.05).

Age-related changes in the amount of the various aromatase transcripts in the whole rat testis

In order to determine whether one promoter was more implicated than the other ones in aromatase gene expression according to the age of the rat, we quantified total aromatase transcripts, and transcripts II, I.f, and I.tr by semiquantitative RT-PCR (Fig. 8A) in whole testis. The primers used for PCR and conditions of amplification for the different products are reported in Tables 1 and 2. We observed that total aromatase gene expression changed according to the age (between 10 and 90 days). At 10 days, gene expression was low and then it increased at 20 days (sevenfold) and remained stable until 90 days. We checked also for the expression of each specific transcript (transcripts II, I.f, and I.tr) according to the age. The age-related changes of transcripts II and I.tr were similar to those of the transcripts of total aromatase with an increase between 10 and 20 days. As for PI.f-specific transcripts, they were very low until 20 days, then increased sharply between 20 and 30 days (fourfold) and remained stable until 90 days (Fig. 8B).

View larger version (26K): [in this window] [in a new window]   Figure 8 Expression of various P450arom transcripts in testes of 10-, 20-, 30-, 60-, and 90-day-old rats. (A) Visualization of total aromatase transcripts and transcripts II, I.f, and I.tr in rat testis on 2% agarose gels after RT-PCR. RNA was extracted from rat testis of age 10 (lane 1), 20 (lane 2), 30 (lane 3), 60 (lane 4), and 90 days (lane 5). In lane 6, RNA was replaced with water. M corresponds to DNA ladder (100 bp ladder). (B) Age-related ratios of total aromatase/L19, Exon II/L19, Exon I.f/L19, and exon I.tr/L19 in the rat testis at different ages (10, 20, 30, 60, and 90 days). The resulting amplification products were analyzed as described in Materials and methods. Data are expressed as means in relation to 30-day-old rats with a mean arbitrarily fixed at 1, and S.E.M. are indicated as bars (n=5 for each age). Data without common letters are significantly different (P<0.05).

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

In the rat testis, P450arom expression is known to be mainly regulated via the proximal promoter PII (Carreau et al. 2004). However, the study conducted by Lanzino et al.(2001) has shown that other promoters could be implicated to direct aromatase gene expression in testicular cells. In fact, our work demonstrates the use of additional promoters, and for the first time to our knowledge, three aromatase transcripts have been observed in the germ cells studied (G–PL, PS, and RS): one is derived from promoter PII, and another one is from promoter PI.f, which is the main promoter responsible for aromatase expression in the brain (Yamada-Mouri et al. 1996, Kato et al. 1997). Although some transcripts issuing from PI.f have been detected in the testis of monkey (Yamada-Mouri et al. 1995), rat (Yamada-Mouri et al. 1996), and mouse (Golovine et al. 2003), it is the first time that they are described in rat germ cells; they are more expressed in PS and RS than in a mixture of spermatogonia and preleptotene spermatocytes. Indeed, some samples of G–PL have very low levels of specific transcripts issuing from PI.f, whereas other samples do not express these transcripts. The third type of transcript derives from a new promoter, that we named PI.tr, which has been identified in the three germ cell fractions (G–PL, PS and RS). Knowing that during testicular development, the proportion of germ cells increases and that the stages of germ cell maturation are dynamic, we studied the use of each promoter in the whole rat testis between 10 and 90 days of age. In this study, we have observed that at 10 days, aromatase expression is very low and, consequently, the levels of the three transcripts (transcripts II, I.f, and I.tr) are almost undetectable. At 10 days, seminiferous tubules contain only somatic cells and spermatogonia. Between days 10 and 20, the number of spermatogonia increases, and the first spermatocytes (preleptotene, leptotene, zygotene, and pachytene) appears (Zhengwei et al. 1990, Malkov et al. 1998). During that period of intense cell proliferation and maturation, aromatase gene expression increases sevenfold to reach its maximum at 20 days. It appears that the use of PII and of PI.tr is privileged and, indeed, the transcripts II and I.tr are present in the G–PL fraction. Although aromatase expression does not vary between 20 and 30 days, the level of the transcripts issuing from PI.f strongly increases. This period corresponds to the enhancement of the number of spermatocytes and to the apparition of RS, which are first observed at 25 days (Zhengwei et al. 1990, Malkov et al. 1998). Now these two germ cell populations (PS and RS) use the three promoters PII, PI.tr, and PI.f to control aromatase expression. The number of various germ cells remains unchanged after the 40th day (Zhengwei et al. 1990), which might explain why no significant variation of the total aromatase transcript level is observed in our study between days 30 and 90. The proportion of the different transcripts (II, I.f, and I.tr) is modified in the various germ cells, which may explain the aromatase changes in the whole rat testis according to the age. Indeed, these aromatase changes cannot be due to Leydig cells because the level of P450arom mRNA is stable between immature and adult rats (Levallet & Carreau 1997). Moreover, it is known that in the rat Sertoli cells, the amount of P450arom mRNA falls of 75% between 20 and 40 days (Levallet & Carreau 1997), and in the mature rat, the low level of P450arom gene expression in Sertoli cells may be related to the well-established inhibition of aromatase activity by the neighboring germ cells (Boitani et al. 1981). Consequently, we can conclude that changes of aromatase expression, between 20 and 90 days, are explained by germ cells.

In the mouse testis, Golovine et al.(2003) have also underlined the use of three different promoters: Pov, Pbr, and Ptes. Pov and Pbr correspond to promoters PII and PI.f of the rat respectively. However, PI.tr does not have any homology with another promoter, and it does not correspond to the specific testis promoter (Ptes) of the mouse. All together, these data suggest that in the mouse and the rat, at least three promoters actively regulate cyp19 expression in the testis but only two promoters are similar (Pov-PII and Pbr-PI.f). In the testis of Bos taurus, four different transcripts containing exons 2, 1.1, 1.3, or 1.4 have been found (Furbass et al. 1997). In the human testes, only two types of transcripts have been described: exon I.6- and exon II-specific transcripts, and their levels are increased in testicular tumor (Shozu et al. 1998).

The use of a specific promoter is directed by the binding of specific factors on its regulatory elements. The activity of the proximal promoter PII of rat cyp19 gene is stimulated by (Bu)₂cAMP via CLS. Moreover, it has been shown that the SF-1 motif is necessary for the activity of the PII aromatase promoter (Young & McPhaul 1998, Catalano et al. 2003). Indeed, Pezzi et al.(2004) have shown that liver receptor homolog (LRH)-1 mRNA and protein are expressed not only in Leydig cells but also in germ cells. This factor recognizes the same DNA binding domain as SF-1 and plays an important role in the regulation of aromatase expression (Pezzi et al. 2004). Recently, Bouchard et al.(2005) have identified a conserved GATA binding element on the PII of numerous mammalian species (human, bovine, equine, porcine, rabbit, mouse, and rat). This site is able to activate the transcription of the human aromatase gene via the binding of GATA 3 or 4. The GATA family is composed of six members and recognizes the consensus GATA site: WGATAR (Evans et al. 1988). In the rat testis, GATA 6 is localized in both germ cells and somatic cells, whereas GATA 4 is specific for somatic cells (Lavoie et al. 2004), suggesting that GATA factors can regulate the aromatase gene expression in gonadal cells. The promoter PI.tr that does not correspond to another promoter previously described also possesses a putative GATA site. However, the two nucleotides on both sides of GATA are different from the consensus sequence. The TATA less promoter PI.tr contains a putative transcriptional initiator site (Inr). The human promoter PI.7 of aromatase that also lacks a TATA box has an Inr, which is sufficient to determine the start site location (Sebastian et al. 2002). Other putative cis-acting regulatory elements have been predicted by sequences analysis programs notably a CAAT box and a cAMP responsive element. This last one corresponds to the CLS already described in PII by Young & McPhaul (1998) and, therefore, could be implicated in the activation of promoter PI.tr by cAMP in Sertoli cells, and may also be concerned in the regulation of aromatase expression in rat germ cells (Bourguiba et al. 2003a,b). However, we cannot exclude that other cis-regulatory sequences could be implicated in the activity of the promoter PI.tr localized in the beginning of the sequence published in Young & McPhaul (1998). In fact, until now, the non-coding sequence of aromatase in the genome database of the rat is not available.

In conclusion, we have shown for the first time the existence of a new promoter (PI.tr), which controls, together with promoters PII and PI.f, the aromatase gene expression in rat germ cells. This new promoter is localized between PI.f and PII on the rat genome and the distances separating the different specific exons of these promoters are shown in Fig. 9. Moreover, these three transcripts are more expressed in PS and, to a lesser extent, in RS than in spermatogonia and preleptotene spermatocytes showing also a regulation of aromatase expression according to the spermatogenetic progression. Thus, it will be necessary to examine the mechanisms of regulation of each promoter in the germ cells according to their degree of maturation.

View larger version (5K): [in this window] [in a new window]   Figure 9 Schematic of promoter PII, promoter PI.f, and promoter PI.tr of the rat cyp19 gene. These promoters are represented with the start site of transcription indicated as +1. The site of splicing is placed at 20 nucleotide residues after the PII initiation transcription site. This representation was realized according to the sequence published by Hickey et al.(1990), Yamada-Mouri et al.(1996) and Young & McPhaul (1998).

	Acknowledgements

 
We thank Michèle Vigier and Hélène Bouraïma-Lelong for the preparation of some samples of rat testicular cells, Colette Edine for the technical assistance, and Guillaume Galmiche for his help in the study of specific exon I.f transcripts. This work is supported in part by the Région Basse-Normandie, by the National Institute of Research in Agronomy, and by the French Ministry of Education and Research. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. Parts of this work were presented at the 14th European Testis Workshop, April 22–26, 2006 (Bad Aibling, Germany).

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Received in final form 7 June 2007
Accepted 20 June 2007
Made available online as an Accepted Preprint 21 June 2007

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