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BOR - Papers in Press, published online ahead of print April 16, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.013961
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BIOLOGY OF REPRODUCTION 69, 592–601 (2003)
DOI: 10.1095/biolreprod.102.013961
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Regulation of Aromatase Gene Expression in Purified Germ Cells of Adult Male Rats: Effects of Transforming Growth Factor ß, Tumor Necrosis Factor {alpha}, and Cyclic Adenosine 3',5'-Monosphosphate1

S. Bourguiba3, S. Chater4, C. Delalande3, M. Benahmed4, and Serge Carreau2,3

USC-INRA EA 2608,3 University of Caen, 14032 Caen-Cedex, France INSERM U 407,4 69921 Oullins, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogens are key regulators of sexual differentiation and development in vertebrates. The P450 aromatase (P450arom) is the steroidogenic enzyme responsible for the synthesis of estrogens from androgens. In the adult rat testis, aromatase transcripts and activity have been observed in somatic cells and germ cells, including pachytene spermatocytes (PS) and round spermatids (RS), but little is known concerning regulation of the aromatase gene expression, especially in germ cells. The quality of germ cell preparations was assessed by the absence of androgen-binding protein and stem cell factor transcripts, two specific markers for Sertoli cells. By employing a competitive quantitative reverse transcriptase-polymerase chain reaction technique, we confirmed that germ cells contained P450arom transcripts and demonstrated that the aromatase gene was up-regulated by cAMP. Conversely, transforming growth factor (TGF) ß1 inhibited Cyp19 gene expression in a dose- and a time-dependant manner in both PS and RS. The addition of tumor necrosis factor (TNF) {alpha} to purified germ cells induced an increase of the amount of P450arom mRNA in PS, although an inhibitory effect was observed in RS. When PS were treated with dexamethasone (Dex), a similar enhancement of the aromatase transcript level was observed, whereas an inhibitory effect was recorded for RS. Furthermore, in either TGFß1- or TNF{alpha}-treated germ cells, the addition of Dex stimulated the aromatase gene transcription. Experiments using 5' rapid amplification of cDNA ends suggested that promoter PII is mainly concerned in the regulation of the aromatase gene expression in germ cells of adult male rats; however, the presence of other promoters could not be excluded.

cytokines, estradiol, gene regulation, growth factors, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The testis is a complex organ that serves two crucial functions: synthesis and secretion of steroid hormones and production of spermatozoa. It is well known that normal testicular development and maintenance of spermatogenesis are controlled by gonadotropins and testosterone [1]. Even though the presence of estrogens in the testis is now well documented, their functions in the male reproductive tract are not fully understood and thus are extensively studied [2]. Indeed, aromatase inhibitors reduce spermatid maturation in rats and monkeys [3]. Furthermore, in the male mice deficient in aromatase, the disruption of spermatogenesis appears specifically to affect early round spermatid (RS) differentiation [4]. Taken together, these data, along with the demonstration of the presence of estrogen receptor ß in human [5], mouse [6], and rat [7, 8] germ cells, support a local role for estrogens in spermatogenesis.

The cytochrome P450 aromatase (P450arom), encoded by the single-copy Cyp19 gene (15q21.1), catalyzes the rate-limiting step in estrogen biosynthesis. Human aromatase gene spans approximately 123 kilobases (kb), with a coding region of nine exons (exons II–X) and an untranslated exon I. Cyp19 gene expression is regulated by multiple tissue-specific promoters producing alternate 5'-untranslated regions that are then spliced onto a common 3'-splice acceptor site in the exon II upstream of the translation start site [9]. Transcriptional regulation of Cyp19 is the major, although not the exclusive, mechanism controlling the amount of aromatase.

Expression of P450arom in human placenta is controlled primarily by a promoter I.1 that lies at least 40 kb upstream from the start site of translation [10]. A promoter proximal to the translation start site, called promoter PII, regulates P450arom expression in ovary and testis (for review, see [11, 12]). Human ovary promoter PII contains elements such as cAMP-responsive element (CRE), AP-1, glucocorticoid-responsive element (GRE), a putative transforming growth factor (TGF) ß1-responsive element, SP-1, and steroidogenic factor-1 (SF1) [13]. Similar elements, like SF1 and CRE are present in rat promoter PII [14–16]. In both the ovary and testis, FSH and LH act, via somatic cells, through increasing concentrations of intracellular cAMP to induce expression of P450arom. In germ cells, a high level of soluble adenylyl cyclase that generates second messenger directly at the sites of cAMP action has been reported [17]. Promoter PII activity is regulated by cAMP and requires the transcription factors cAMP response element-binding protein (CREB), cAMP response-element modulator (CREM), and SF1 [18]. In human adipose tissue as well as in fetal liver, the primary promoter I.4 lies approximately 15 kb upstream of the start site of translation [19] and is a TATA-less promoter driven by glucocorticoids and class I cytokines, such as interleukin-6 and tumor necrosis factor (TNF) {alpha} [20].

Germ cells are potent targets for growth factors and cytokines. Indeed, pachytene spermatocytes (PS) and RS produce TGFß and TNF{alpha}, but only TGFß receptors are present in germ cells [21]. In the testis, the TGFß superfamily, including TGFß1, TGFß2, TGFß3, inhibins, activins, and bone morphogenetic proteins (BMPs), have been reported to affect testicular function as well as maintenance of spermatogenesis and Leydig cell steroidogenesis [22–24]. Studies regarding the testicular activity of TGFß ligands and related peptides have revealed very little information regarding their signaling pathways in this tissue. The TGFß signals through the sequential activation of two cell surface serine/threonine kinase receptors, known as type I and type II receptors, which then activate the downstream signal transduction cascade. The type I kinase receptor transduces intracellular signals by activation of various proteins, including Smad proteins. The signal is propagated primarily through protein-protein interactions between Smad proteins, which are hetero-oligomeric, and between Smads and transcription factors. Specifically, the phosphorylated Smad hetero-oligomerizes with the ubiquitous Smad 4, translocates into the nucleus, and activates the transcription of various target genes. Smads 1, 5, and 8 mediate the action of BMPs, whereas Smads 2 and 3 are specific for TGFß and activin signaling. Smad 4 is a common mediator, because it forms hetero-oligomeric complexes with other activated Smads. Smads 6 and 7 negatively regulate other Smad proteins by preventing their phosphorylation (for review, see [25]). Smad proteins have been shown to interact with DNA-binding proteins, such as human FAST1 [26], mouse FAST2 [27], and p300/CREB-binding protein [28], and also to bind directly to specific DNA sequences [29]. The information available regarding the distribution and function of Smad proteins in the testis is extremely limited. The mRNAs for Smads 1–7 have been identified in cultured porcine Leydig cells, indicating the presence of a functional pathway [30].

The localization of aromatase within the testis has been a subject of considerable interest for a number of years. Past efforts to localize the source of testicular estrogens have led to the assertion that Leydig cells synthesize estrogen in adults. Sertoli cells are the major source in immature animals [31–33], however, and the aromatase has been immunolocalized in the Leydig cells of vertebrates [34]. Even so, striking species differences exist, because in mouse [35], rat [36, 37], bank vole [38], and brown bear [39], the aromatase is present not only in Leydig and Sertoli cells but also in germ cells, including PS, RS, and spermatozoa [36, 40, 41]. Little information is available, however, concerning regulation of the testicular P450arom, especially in germ cells; thus, study of the control of Cyp19 gene expression is fundamental to bring enlightenment regarding the regulation of the testicular balance of androgens and estrogens.

In the present study, which was conducted with purified PS and RS from adult male rat, we took advantage of a highly specific quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) method [36, 42] to study the effect of TGFß, TNF{alpha}, and cAMP treatments on the aromatase gene expression and, at the same time, measured the estradiol output. We show that the Cyp19 gene expression and enzyme activity in both PS and RS are under the control of TGFß and TNF{alpha}. We also report that cAMP up-regulates aromatase gene expression in these cells. Furthermore, an increase of Cyp19 mRNA by TGFß or TNF{alpha} has been observed in dexamethasone (Dex)-treated germ cells. In addition, we demonstrate that adult rat germ cells express the specific mRNAs for Smads 1–7, indicating the presence of a functional aspect of TGFß receptors in these cells. Our experiments using 5' rapid amplification of cDNA ends (RACE) suggested that promoter PII drives the aromatase transcription in PS and RS; nevertheless, we could not exclude the presence of other transcripts derived from other promoters.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Germ Cell Purification

Testicular mixed germ cell preparation was obtained by the trypsin-DNase procedure with some modifications [36, 43]. Briefly, testes were removed from adult Sprague-Dawley rats (95 ± 10 days), decapsulated, and submitted to mechanical disruption in PBS (pH 7.3). The interstitial cells were removed by four successive decantations. Tubules were treated with 0.025% trypsin plus 0.1% DNase (Sigma, St. Quentin Fallavier, France) in PBS for 45 min at 32°C. After incubation, the cell suspension was washed in PBS supplemented with 3.3 mM glucose and 6 mM pyruvate (Sigma). The resulting suspension was filtered first through fine nylon mesh to remove Sertoli cell aggregates and then through glass wool to minimize spermatozoa content in the preparation. Further separation of testicular cell types was accomplished by unit gravity sedimentation through a BSA (Roche, Mannheim, Germany) gradient (0.2–2.75%) in a Sta-Put apparatus [44]. The PS- and RS-enriched fractions were collected, washed at least three times with PBS, and subsequently used for incubation and microsomal extraction. Purity of fractionated germ cells was routinely monitored under a phase-contrast microscope. 3ß-Hydroxysteroid dehydrogenase (3ß-HSD) histochemical staining, a specific marker of Leydig cells, was used to analyze the purity of germ cell fractions. To estimate the Sertoli cell contamination of the germ cell preparations, a histochemical staining with Oil Red O (Sigma) [45] was performed. Moreover, RT-PCR was applied to detect whether two-specific markers of Sertoli cells—andogen-binding protein (ABP) and stem cell factor (SCF) mRNAs [46, 47]—were present in germ cell preparations. The viability of germ cells before and after incubation was evaluated using the Trypan blue exclusion test (Sigma); the stained cells were considered to be dead.

Incubation Procedure

The cells were incubated (2.5 x 106 PS/ml and 4.5 x 106 RS/ml) at 32°C under a humidified atmosphere of 5% CO2 and 95% air in Ham F-12/DME medium (Seromed, Berlin, Germany) containing NaHCO3 (1.2 g/L), Hepes (3.57 g/L), streptomycin (150 µg/ml), penicillin (30 µg/ml), and fungizone (0.25 µg/ml) (Sigma) and supplemented with 10 mM pyruvate and 6 mM glucose. Germ cells were cultured for various times (0–18 h) with or without human recombinant TGFß1 or TNF{alpha} (Preprotech, Rocky Hill, NJ), cAMP or Dex (both from Sigma).

RNA extraction

The RNAgents total RNA isolation system (Promega, Charbonnieres, France) was used to extract RNAs from PS and RS. This reagent is an improvement of the single-step RNA isolation method developed by Chomczynski and Sacchi [48]. The purity and integrity of the RNAs were checked spectroscopically and by gel electrophoresis before use.

RT-PCR Assay

Single-strand cDNAs were obtained from RT of 500 ng of total RNAs as follows: 1 h at 37°C with 200 U of Moloney murine leukemia virus-reverse transcriptase, 500 µM dNTPs, 0.2 µg of oligo-dT, and 24 U of RNasin (Promega) in a final volume of 10 µl. The cDNAs obtained were further amplified by PCR using selected oligonucleotides. The PCR was performed for 35 cycles (30 sec at 94°C, 30 sec at 60°C, and 1 min at 72°C, with a 2-sec delay at each cycle) in the presence of 200 µM dNTPs, 50 pmol primer set, and 1.5 U of Taq polymerase (Promega) in a final volume of 50 µl. The P450arom primers were selected to amplify a highly conserved sequence (289 base pairs [bp]), including helical and aromatic regions of the P450arom gene [36] (Table 1). The amplified products were subjected to electrophoresis on a 2% agarose gel stained with ethidium bromide. To estimate the Sertoli cell contamination of the germ cell preparations, two sets of ABP and SCF gene-specific primers were synthesized (Table 1)


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  		TABLE 1. Oligonucleotides primer used for RT-PCR

3H2O Assay for P450arom Activity

Microsomes were isolated from homogenates of purified germ cells by differential centrifugation as previously described [49]. Aromatase activity was assayed by measuring the rate of incorporation of 3H from [3H]androstenedione to [3H]H2O according to the method of Thompson and Siiteri [50] with minor modifications. Microsomes were incubated with 1ß-3H-androst-4-ene-3,17-dione (1ß-3H-A; 26.64 x 103 dpm/pmol; New England Nuclear, Les Ulis, France) in Tris-maleate buffer (pH 7.4) for 1 h at 34°C in a shaking water bath. The reaction was started by the addition of the cofactor NADPH (10 mM) to a final volume of 0.5 ml and was stopped by adding 1 ml of chloroform. After centrifugation (2700 x g, 5 min, 4°C), 500-µl aqueous phases were treated with an equal volume of charcoal (7%)-dextran (1.5%) to remove unconverted 1ß-3H-A. After 10 min, the charcoal was separated from the aqueous phase by centrifugation (2700 x g, 10 min, 4°C). The supernatant was removed, and the radioactivity was counted. Microsomal fractions from human placenta and rat leg muscle were used as positive and negative controls, respectively. Blank values were obtained from identical incubations in the absence of microsomes. The P450arom activity was expressed as fmol mg protein-1 h-1; protein concentration in microsomal fractions was determined with the Bradford assay using BSA as standard [51].

Quantitative Competitive RT-PCR assay

Using a highly specific quantitative RT-PCR method [36], the amount of P450arom mRNA present in the total RNA extracted from PS and RS was assessed. Briefly, after RT and PCR with the P450arom-specific set of primers (Table 1), the amplified products were run on a 4% agarose gel stained with ethidium bromide, then photographed and submitted to densitometric scanning analysis (Bio-Profil System; Vibert Lourmat, Marne La Valleé, France). The intensity and surface of each band were calculated and the sample:standard ratio plotted against the standard concentration, and then the quantity of P450arom mRNA present in the total RNA was determined [42].

Detection of Smad mRNAs in Germ Cells and Determination of Their Levels by Semiquantitative RT-PCR

For RT-PCR, 500 ng of total RNA were reverse transcribed as described above. Four microliters of RT reaction mixture from each sample were amplified with either Smads or ß-actin primers (50 pmol) (Table 1), 200 µM dNTPs, 1.5 mM MgCl2, and 1.5 U of Taq polymerase (Promega) in a final volume of 50 µl. The number of PCR cycles was determined to ensure that the amplification products to be analyzed were obtained during the linear phase of the reaction; thus, the number of PCR cycles was fixed at 35 and 22 for Smads and ß-actin, respectively. To amplify Smads 1, 2, 3,and 5, PCR was carried out, after preheating at 94°C for 2 min, through 35 cycles of 94°C for 1 min, 64°C for 1 min, and 72°C for 2 min, followed by a final extension at 72°C for 10 min. The PCR for Smads 4, 6, and 7 was performed as follows: 35 cycles of 1 min at 94°C, 1 min at 59°C, and 2 min at 72°C, followed by a final step of elongation at 72°C for 10 min. The PCR conditions used to study the internal control (ß-actin) were 22 cycles of 94°C for 1 min., 55°C for 1 min, and 72°C for 2 min, followed by a final extension at 72°C for 10 min. The amplified products were subjected to electrophoresis on a 2% agarose gel stained with ethidium bromide and then photographed. The intensities of the signal corresponding to Smads were analyzed using the BioProfil system. The relative amounts of Smad mRNAs in germ cells (in the presence and the absence of TGFß) were normalized to ß-actin.

Estradiol Determinations

In germ cell culture media, 17ß-estradiol was measured by RIA according to a previously reported method [52]. Briefly, after incubation, germ cells were pelleted and used for RNA extraction; steroids were then extracted from culture media by 10 volumes of diethyl ether. A highly specific antibody was obtained from P.A.R.I.S. (Compiègne, France) and labeled 17ß-estradiol from NEN (Life Science Products, Les Ullis, France). The average recoveries were between 88% and 98%, and the detection limit of the RIA was 6 pg/ml. The intraassay coefficient of variation was 8%. An inter-assay coefficient of variation was not applicable, because all the samples were run in a single assay.

Amplification of the 5' End of P450arom Transcripts

The 5' RACE PCR technique [53] was used for the characterization of sequences located at the 5' end of the aromatase transcripts. The first step, RT, was performed using 2 µg of total RNA, an oligonucleotide primer located in exon V of the aromatase gene (Ex Vrev 5'-cccaggaagagcgtgttaga-3'), the AMV reverse transcriptase, and dNTPs solution according to the manufacturer's instructions (Roche). The first cDNA was then purified from nonincorporated nucleotides and primers by the High Pure PCR Product Purification Kit (Roche), in which the elution buffer was replaced by 10 mM Tris-HCl (pH 8). Terminal transferase was then used to add a homopolymeric A-tail to the 3' end of the cDNA. The tailed cDNA was amplified by PCR using a second primer located in the exon III of Cyp 18 (Ex IIIrev 5'-gaaatgagaggcccgattcc-3') and an oligo-dT anchor primer provided by the kit. For the second amplification, "nested-PCR," the reverse primer located in exon II of the Cyp19 gene (Ex IIrev 5'-aatcaggaggaggaggccca-3') was combined with the anchor primer provided by the manufacturer. The resulting PCR product was electrophoresed on a 2% agarose gel and then purified. Sequencing of the cDNAs was performed using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Courtaboeuf, France)

Statistical Analysis

All data are expressed as the mean ± SEM of triplicate determinations performed in at least three independent experiments carried out with different cell preparations. Statistical analysis was performed using ANOVA, and means were compared using the Tukey-Kramer test. Statistical significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of P450 Aromatase mRNA in Adult Rat Germ Cells by RT-PCR

The P450arom mRNA was detected in adult rat germ cells by using RT-PCR, and the amplified cDNA fragment evidenced the correct predicted size of 289 bp expected from the P450arom-specific primers used (Fig. 1A). Ovarian RNA generated RT-PCR product of the same size, whereas no signal was detected in adult Sertoli cells. When water was added instead of RNA and then treated in the same conditions, no amplified signal was observed (negative control). After Sta-Put, the purity of germ cell fractions was checked under a microscope, and the cell preparations were submitted to 3ß-HSD histochemical staining. In both germ preparations, the purity was higher than 95% (the major contamination was by other germ cells), and no positive 3ß-HSD cells were observed. The specific Sertoli cell coloration by Oil Red O was negative (not shown); in addition, we could not detect any SCF mRNA in PS and RS fractions (Fig. 1B). Concerning ABP, a weak signal was visible in PS (Fig. 1C).



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  		FIG. 1. A) Detection of P450arom mRNA in purified adult rat germ cells by RT-PCR. Mixed germ cells before purification (GC), PS RNA (PS), RS RNA (RS), and seminiferous tubules RNA (ST) generated RT-PCR products of the predicted size of 289 bp. Ovarian RNA (Ov) was used as positive control and also generated RT-PCR product of the predicted size (289 bp). Water was used instead of RNA as negative control (nc) and adult Sertoli cells (S90d) as control of contamination. M corresponds to the DNA ladder. B) Coamplification of P450arom mRNA and SCF mRNA: mixed germ cells before purification (GC, lane2), PS (lane 3), RS (lane 4), adult Sertoli cells (S90d, lane 5), seminiferous tubules RNA (ST, lane 6), ovary (Ov, lane 7), and nc (lane 8, water was used instead of RNA). C) Coamplification of P450arom mRNA and ABP mRNA: mixed germ cells before purification (GC, lane2), PS (lane 3), RS (lane 4), adult Sertoli cells (S90d, lane 5), seminiferous tubules RNA (ST, lane 6), ovary (Ov, lane 7), and negative control (lane 8, water was used instead of RNA)

P450arom Activity in Purified Rat Germ Cells

To confirm that the specific transcript for aromatase in germ cells was fully translated in a functional protein, the conversion rate of androstenedione to estrone was measured in germ cells of adult rat testes as well as in human placenta and rat leg muscle microsomes (Fig. 2). The P450arom activities (fmol mg protein-1 h-1) in germ cells were as follows: mixed germ cells, 220 ± 13; PS, 89 ± 21; and RS, 158 ± 10. Human placenta microsomes used as a positive control converted 1ß-3H-A to estrone at a rate of 66 960 ± 1546 fmol mg protein-1 h-1. It is noteworthy that in muscle, a residual aromatase activity was detectable.



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  		FIG. 2. P450arom activity in adult rat germ cells: mixed germ cells (GC), PS, and RS. Muscle and human placenta were used as negative and positive controls, respectively. The data are represented as the mean ± SEM (n = 3). Histograms without common letters are statistically different (P < 0.05)

Effect of TGFß1 on P450arom mRNA Level and Aromatase Activity in Purified Germ Cells

The TGFß1 inhibited Cyp19 expression in germ cells in a time- and dose-dependant manners (Fig. 3). The TGFß1 exerted an inhibitory effect on P450arom mRNA level in PS both at 12 and 18 h (P < 0.05) (Fig. 3A). The maximal effect was observed with 1 ng/ml of TGFß1 (65% decrease when compared to control, P < 0.05) (Fig. 3B). A significant inhibitory effect of TGFß1 on the amount of P450arom mRNA was observed when RS were incubated for 18 h (P < 0.05) (Fig. 3C). It is noteworthy that after 12 h, a decrease (not significant) of the P450arom mRNA level was already observed in RS. In addition, we have treated RS with various amounts of TGFß1 and observed a dose-dependent diminution in the amount of specific aromatase transcripts, with a maximum achieved with 1 ng/ml (65% decreases when compared to control) (Fig. 3D).



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  		FIG. 3. Effects of TGFß1 on Cyp19 mRNA expression in PS and RS. Both PS and RS were incubated (A and C) for different times (6–18 h) in the absence or presence of TGFß1 (1 ng/ml) and for 18 h (B and D) with increasing concentrations of TGFß1 (0.1–5 ng/ml). Cyp19 mRNA was measured by quantitative competitive RT-PCR. The data are represented as the mean ± SEM. Histograms without common letters are statistically different (P < 0.05)

The inhibitory effect of TGFß1 on germ cell aromatase gene transcription was confirmed by the estradiol measurement. As expected, TGFß1 induced a decrease of estradiol production in both germ cell populations. The diminutions were of 38% and 47% compared to untreated cells (P < 0.05) when PS and RS, respectively, were incubated for 18 h with 1 ng/ml of TGFß1.

Identification of Smads, the Mediators of the TGFß Signal

As shown in Figure 4A, mRNAs for Smads 1–7 were detected in both PS and RS of adult rat. A semiquantitative RT-PCR, with ß-actin as internal control, was performed to analyze the effect of TGFß on the expression of Smad mRNAs in these germ cells. Expression of Smad 1, 2, 3, 4, 5, and 7 mRNAs did not change significantly between PS and RS when these cells were incubated for 18 h without TGFß (Fig. 4B). Conversely, Smad 6 mRNA was significantly more abundant in PS than in RS (P < 0.05) (Fig. 4B). After treatment with 1 ng/ml of TGFß, an increase of Smad 2 mRNA expression was recorded in RS but not in PS, whereas Smad 3 mRNA level increased in PS but not in RS. Furthermore, Smad 7 mRNA expression was significantly enhanced (P < 0.05) in both germ cells after TGFß treatment. The mRNA expression of the remaining Smads (1, 4, 5, and 6) did not change significantly in PS and RS when compared to untreated cells (Fig. 4B).



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  		FIG. 4. Identification of Smads in adult rat PS and RS by RT-PCR. The cDNAs obtained from RT of total RNAs were amplified by PCR with specific primers. A) PCR was performed in the absence of cDNAs. B) Effect of TGFß1 on Smad mRNAs expression in PS and RS. The data are represented as the mean ± SEM. Histograms without common letters are statistically different (P < 0.05). nc, Negative control

Effect of TNF{alpha} on P450arom mRNA Level and Aromatase Activity in Purified Germ Cells

The TNF{alpha} induced an increase of the amount of P450arom mRNA in PS after 12 h (nonsignificant effect) and 18 h (P < 0.05) of treatment (Fig. 5A). In our conditions, the maximal stimulatory effect of the cytokine was observed with 20 ng/ml (72% increase of the level of P450arom transcripts when compared with untreated cells, P < 0.05) (Fig. 5B). Conversely, in RS, TNF{alpha} treatment was without effect during the first 12 h of incubation, although a decrease of the P450arom mRNA level was observed after 18 h of treatment (P < 0.05) (Fig. 5C). This inhibitory effect was maximal with 20 ng/ml of TNF{alpha} (48% decrease when compared with control cells, P < 0.05) (Fig. 5D). In TNF{alpha}-treated PS (20 ng/ml, 18 h), estradiol output was enhanced significantly (82% vs. control, P < 0.05); in contrast, this cytokine had no effect on estradiol production in haploid germ cells.



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  		FIG. 5. Effects of TNF{alpha} on Cyp19 mRNA expression in PS and RS. Both PS and RS were incubated (A and C) for different times (6–18 h) in the absence or presence of TNF{alpha} (20 ng/ml) and for 18 h (B and D) with increasing concentrations of TNF{alpha} (1–20 ng/ml). The data are represented as the mean ± SEM. Histograms without common letters are statistically different (P < 0.05)

Effect of cAMP on P450arom mRNA

As indicated previously, aromatase is expressed in the rat testes by means of a promoter proximal to the start site of translation (PII) [11]; in addition, it was shown that cAMP induced the expression of aromatase transcripts for the proximal promoter PII [15, 19]. To examine whether the expression of aromatase gene is controlled by cAMP in germ cells, PS and RS were treated with 1 mM cAMP analogue for 18 h. In PS, the Cyp19 mRNA level was enhanced 2-fold by cAMP (P < 0.05) (Fig. 6) when compared with untreated cells. Conversely, a slight increase (nonsignificant) in aromatase gene expression was noted in RS incubated with cAMP (Fig. 6).



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  		FIG. 6. Effects of cAMP (1 mM, 18 h) on Cyp19 mRNA expression in PS and RS. The data are represented as the mean ± SEM. Histograms without common letters are statistically different (P < 0.05). contr, Control

Effect of Dexamethasone on Aromatase Gene Expression

We have shown previously that TNF{alpha} regulated aromatase gene expression in both PS and RS. It was found that TNF{alpha} stimulated aromatase expression in adipose stromal cells in the presence of Dex and that the action of TNF{alpha} involved two distinct sites, a GRE and an AP1-binding site in promoter PI.4 [54]. To determine whether Dex plays a role on aromatase gene regulation in germ cells, PS and RS were incubated with 100 nM Dex [55] for 18 h. In PS, Dex alone caused a 2-fold increase in Cyp19 mRNA level (P < 0.05) (Fig. 7, A and B); furthermore, the combination of Dex and TNF{alpha} further increased the amount of P450arom transcripts (3-fold increase, P < 0.05 when compared with untreated cells) (Fig. 7A). Moreover, when PS were treated with TGFß and Dex, the Cyp19 mRNA level was enhanced 5.5-fold (P < 0.001 when compared with TGFß-treated cells) (Fig. 7B). On the other hand, Dex alone induced a nonsignificant decrease of P450arom mRNA in RS (30% diminution when compared with control cells, P < 0.05) (Fig. 7, C and D). In contrast, the combination of Dex and TNF{alpha} improved the P450arom gene transcription (3-fold increase, P < 0.05 when compared with TNF{alpha}-treated cells) (Fig. 7C). As in PS, Dex together with TGFß induced a positive effect on the amount of Cyp19 mRNA in RS (3-fold increase, P < 0.05 when compared with TGFß-treated cells) (Fig. 7D).



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  		FIG. 7. Effects of Dex on Cyp19 mRNA expression in PS (A and B) and RS (C and D). Influence of Dex on Cyp19 mRNA in PS in the presence or in the absence of TGFß1 or TNF{alpha} was measured by quantitative competitive RT-PCR. The data are represented as the mean ± SEM. Histograms without common letters are statistically different (P < 0.05)

Isolation of Cyp19 Transcripts from Germ Cells by 5' RACE

To isolate Cyp19 transcripts from germ cells incubated with different factors and to determine the start sites of transcription, 5' RACE experiments were carried out. All isolated transcripts were identified as Cyp19 transcripts as a result of sequence similarities with the published rat sequence [14]. The majority of transcripts share a common region of 40 nucleotides upstream of the initiator methionine, which was previously positioned at 59 nucleotides [14] and revised at position 97 nucleotides downstream of the transcription start site [16] (Fig. 8). The greater part of transcripts had truncated 5' untranslated region because of premature cessation of the RT reaction. Isolated transcripts from cAMP treated-germ cells (PS and RS) showed an almost full-length 5' untranslated region, suggesting that these transcripts are derived from promoter PII as described by Lanzino et al. [11]. The remaining RACE products may have been artificially truncated, however, and the start site could therefore be further upstream.



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  		FIG. 8. Schematic representation of promoter PII and nucleotides of the isolated RACE transcripts from cultured rat germ cells (with or without treatments). The promoter PII of the rat aromatase gene is represented with the start transcription site indicated as +1. The potential splice acceptor site is indicated as +58. Arrow shows the position of the third primer used to perform RACE reactions. A schematic representation of transcripts isolated from cultured PS and RS is shown


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
From these in vitro studies performed on purified PS and RS from adult rats, we have shown that these cells express P450arom transcripts and that the regulation of the aromatase gene expression is under the control of various hormones (glucocorticoid, growth factor, cytokine, and cAMP). The P450arom mRNA level is 5-fold higher in premeiotic germ cells than in haploid cells, as reported earlier [36]. Furthermore, PS and RS contain active P450arom and therefore should be considered as a new source of estrogens in testicular tissue, as claimed in previous reports [35, 37]. The regulation of the Cyp19 gene expression was analyzed using a very sensitive quantitative competitive RT-PCR method [36, 42]. Because of the high sensitivity of this assay, the cellular fractions should be highly purified; accordingly, ABP and SCF mRNAs were used as specific markers of Sertoli cell contamination of the germ cell preparations. The coamplification (based on a multiplex PCR) of ABP and P450arom mRNAs syntheses in one hand and of SCF and P450arom mRNAs on the other supports the germ cell origin of the P450arom in adult rat.

In rodents, the regulation of aromatase gene expression, especially in germ cells, is not understood. Both LH and cAMP control the regulation of aromatase gene expression in rat Leydig cells [42], probably via the CRE localized in the promoter PII of the aromatase gene [16]. Our data show a significant increase of P450arom mRNA expression in PS treated with cAMP analogue, whereas in RS, a nonsignificant increase was observed in the presence of cAMP. It has, however, been reported that CREM repressor isoforms are expressed at low levels in spermatogonia, whereas during meiosis, the CREM activator transcript is abundantly expressed in PS and stabilized under the influence of FSH. In postmeiotic germ cells, only activator forms of CREM are present [56]. Furthermore, Shell et al. [57] demonstrated that in adult rat seminiferous tubules, the activator form of CREB is limited to those cells containing Sp1 (transcription factor), including Sertoli cells and primary spermatocytes from preleptotene through midpachytene stages of development. The transcription factors CREM and CREB bind as dimers to CREs within gene promoters, and when phosphorylated on specific serine, the proteins interact with the CREB-binding protein coactivator. The addition of CREB-binding protein contributes to the induction of gene transcription by facilitating the recruitment of general transcription factors and RNA polymerase to the promoter [57]. Therefore, the presence of both CREM and CREB in spermatocytes explains the high sensitivity of these cells to cAMP analogue. Our data are in agreement with those obtained in CREM knockout mice showing a decrease of aromatase gene expression when compared to wild-type mice (data not shown). In addition, 5' RACE experiments indicated that aromatase transcripts in cAMP-treated PS and RS are derived from the proximal promoter PII of the aromatase gene, which has been described as the major promoter in the rat testis [11].

Germ cells synthesize TGFß and are a target for the peptide; the expression of TGFß peptides and TGFß receptors are regulated according to the stage of spermatogenesis [58, 59]. Moreover, it has been reported that TGFß1 inhibits the FSH-induced aromatase activity in Sertoli cells [60] and enhances Dex-induced aromatase activity in human osteoblast-like cells and THP-1 cells [61]. In adult rat purified germ cells, we found that TGFß1 exerts an inhibitory effect on aromatase gene expression in PS and RS. Although the intracellular mechanism(s) by which TGFß1 exerts its inhibitory effect on aromatase gene expression in germ cells remains to be established, we report here the presence of Smad 1–7 mRNAs in both PS and RS. In these cells, TGFß1 stimulates Smad 2, 3, and 7 mRNAs, whereas the levels of the other Smad mRNAs were not modified. These data suggest that Smads 2, 3, and 7 play an important role in the molecular mechanism of TGFß action during spermatogenesis. We may propose that the signaling response to TGFß is mediated by nuclear proteins such as c-fos or Smads, which have been reported to mediate TGFß1 inhibition of gene expression [62, 63]. Also, TGFß1 could operate through TGFß1 response element; such a consensus element is present in the human promoter PII of the aromatase gene [13]. It remains to investigate whether such cis-acting elements responsible for TGFß1 action are efficient to reduce Cyp19 mRNA in germ cells.

In the testes, TNF{alpha} is produced by interstitial macrophages and germ cells (RS and PS), and TNF{alpha} receptors (membrane and soluble) are present in rat and porcine Sertoli cells [1]. A multifunctional cytokine, TNF{alpha} elicits numerous cellular functions depending on the cellular context. This cytokine stimulates germ cell development and reduces FSH stimulated-aromatase activity and inhibin secretion in cultured porcine Sertoli cells [60]. It also stimulates aromatase gene expression in adipose tissue [54]. Pentikäinen et al. [64] found that TNF{alpha} inhibits in vitro-induced apoptosis of human testicular germ cells. Our investigation regarding the putative effect of TNF{alpha} on Cyp19 gene expression in purified rat germ cells shows that this cytokine exerts a stimulatory effect on aromatase gene transcription in PS, whereas an inhibitory effect is observed in RS, therefore suggesting the presence of specific receptors for that cytokine in germ cells. General agreement now exists that TNF{alpha}, on binding to its receptors, activates different signaling pathways, which results in activation of the transcriptional factor NF-{kappa}B. This TNF{alpha}-activated NF-{kappa}B could therefore displace other trans-regulator elements, such AP1 [65]. Moreover, our data show that TNF{alpha} increases the estradiol output in PS, whereas this cytokine has no effect on estradiol production in RS. Thus, in PS, the transcription rate of P450arom is always up-regulated, whereas an opposite effect is observed in RS. In addition, it is noteworthy that truncated P450arom mRNAs are present in a large amount in PS and, to a lesser extent, in RS [66]. It is tempting to speculate that the enhancement of Cyp19 gene expression in PS by TNF{alpha} is necessary to increase estrogen synthesis and then to allow immature cells to progress toward in the developmental pathway.

It has been shown that TNF{alpha} stimulates aromatase expression in human adipose stromal cells in the presence of Dex. The TNF{alpha} action involves two distinct sites, a GRE and an AP1-binding site in promoter PI.4 [54]. Similar elements are present on the human promoter PII [13]. In germ cells, we found that Dex alone regulates Cyp19 expression positively in PS and negatively in RS. Furthermore, our results show a synergistic effect of Dex and TNF{alpha} on P450arom mRNA expression in PS, whereas an additive effect is observed in RS. Therefore, we may propose two hypothetic pathways for TNF{alpha} in germ cells. First, we suggest the presence of putative AP1 site(s) and GRE on promoter PII of rat aromatase gene, as described on human PII. Our 5' RACE experiments show that PII is the major promoter driving aromatase transcripts in rat germ cells. Consequently, the aromatase promoter sequence published by Young and MacPhaul [16] was analyzed using different softwares (SIGSCAN 4.5 with the databases Transfac and TFD according to the method of Prestridge [67], TFSEARSH 1.3, and RGSite Scan Program); all of them showed several putative AP1-binding sites and GRE on rat aromatase PII. Our data could not exclude the presence of other transcripts derived from other promoters that could mediate cytokines and glucocorticoid effects on aromatase gene expression in rat germ cells. Recently, Golovine et al. [68] have demonstrated that the expression of Cyp19 in mouse testis is specifically directed by a promoter called Ptes.

It is noteworthy that TGFß1 in combination with Dex stimulates P450arom gene transcription in both PS and RS. This result suggest that TGFß1 is a potent, multifunctional growth factor that also may act through the GRE and AP1, like TNF{alpha}, to stimulate aromatase gene expression [61].

In summary, by using an in vitro model of mature rat PS and RS, we have shown that several factors direct the expression of the aromatase gene in these cells. Although the promoter PII is probably the major promoter used to regulate this expression, we could not exclude that other promoters could be concerned. Recently, the published rat genome project data (http://www.ncbi.nlm.nih.gov) allowed us to locate the aromatase gene on chromosome 8 (NW_044137), but the promoter regions could not be determined because of a still-uncertain gap between the sequence contigs. Therefore, further investigations of the mechanisms controlling the expression of P450arom gene promoters in the testes are required, especially investigations that focus on the developmental aspects.


   ACKNOWLEDGMENTS
 
The authors thank Drs. Jerome Levallet and Hervé Mittre for constructive comments. We appreciate the excellent technical assistance of Mrs. Colette Edine. We would like to thank Magali Maire for her help in the Smad study and Dr. Paolo Sassone-Corsi for providing CREM knockout mice testes.


   FOOTNOTES
 
1 S.B. was supported by a grant from Institut de Recherches Internationales Servier (IRIS) and from the Comité Régional Biologie et AgroBioindustries (CRAB). Parts of this work were presented at the 12th European Testis Workshop, April 6–10, 2002 (Doorwerth, The Netherlands), and at the Sixth International Aromatase Conference, October 26–30, 2002 (Kyoto, Japan). Back

2 Correspondence: S. Carreau, Biochimie-IRBA, Esplanade de la Paix, 14032 Caen-Cedex, France. FAX: 33 2 31 56 53 20; carreau{at}ibba.unicaen.fr Back

Received: 3 December 2002.

First decision: 7 January 2003.

Accepted: 3 April 2003.


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