Follicle-Stimulating Hormone Increases Testicular Anti-Müllerian Hormone (AMH) Production through Sertoli Cell Proliferation and a Nonclassical Cyclic Adenosine 5'-Monophosphate-Mediated Activation of the AMH Gene

Céline Lukas-Croisier1, Celina Lasala, Juliette Nicaud, Patricia Bedecarrás, T. Rajendra Kumar, Martin Dutertre, Martin M. Matzuk, Jean-Yves Picard, Nathalie Josso and Rodolfo Rey

Centro de Investigaciones Endocrinológicas (Consejo Nacional de Investigaciones Científicas y Técnicas; C.L., P.B., R.R.), Hospital de Niños "R. Gutiérrez," C1425EFD Buenos Aires, Argentina; Unité de Recherches sur l’Endocrinologie du Développement (Intitut National de la Santé et de la Recherche Médicale; C.L.-C., J.N., M.D., J.-Y.P., N.J., R.R.), Département de Biologie, Ecole Normale Supérieure, 92120 Montrouge, France; and Departments of Pathology and Molecular and Cellular Biology (T.R.K., M.M.M.) and Department of Molecular and Human Genetics (M.M.M.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Dr. Rodolfo Rey, Centro de Investigaciones Endocrinológicas, Hospital de Niños, Gallo 1330, C1425EFD Buenos Aires, Argentina. E-mail: rodolforey{at}cedie.org.ar.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Anti-Müllerian hormone (AMH) production by testicular Sertoli cells is high before puberty and can be further induced by FSH. Our objective was to delineate the mechanisms by which FSH stimulates AMH production. Assay of serum AMH levels and histological morphometric analysis in prepubertal FSH-deficient transgenic mice showed that serum AMH and testicular mass were decreased owing to reduced Sertoli cell number. All parameters resumed normal values in mice treated with recombinant FSH. We also analyzed the ability of FSH and the factors involved in its signaling pathway to activate AMH transcription by transfecting AMH promoter-luc reporter constructs of different lengths in a prepubertal Sertoli cell line. Our results showed that FSH activates AMH transcription via adenylate cyclase, cAMP, and protein kinase A but involving a nonclassical cAMP-response pathway requiring nuclear factor-{kappa}B and activating protein 2 binding sites, which lie more than 1.9 kb upstream of the AMH transcription start site. This is the first report showing the importance of distant sequences in the regulation of AMH expression. We conclude that prepubertal testicular AMH production is increased by FSH stimulation through Sertoli cell proliferation and an enhancement of AMH gene transcription.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ANTI-MÜLLERIAN HORMONE (AMH), also called Müllerian-inhibiting substance (MIS), is a member of the TGF-ß superfamily of growth and differentiation factors, involved in the hormonal control of sex differentiation in the male fetus (reviewed in Refs. 1 and 2). In the testis, AMH is synthesized by Sertoli cells, the somatic component of the seminiferous tubules, from the very onset of fetal testicular differentiation (3). Upon binding to a specific membrane receptor in Müllerian ducts, AMH induces the regression of the anlagen of the uterus and Fallopian tubes in the male fetus (reviewed in Refs. 1 and 2). A growing amount of evidence indicates that AMH also acts as a negative modulator of Leydig cell differentiation and testosterone secretion in the male (reviewed in Refs. 2 and 4).

The ontogeny of AMH expression in the postnatal male is characterized by the existence of high levels until puberty; then, AMH expression is significantly down-regulated (5, 6), which results in a decrease of serum AMH to very low levels (reviewed in Refs. 2 , 7 , and 8). The regulatory mechanisms involved in the activation of AMH expression in the fetal Sertoli cell have been extensively studied (reviewed in Ref. 2), but less is known concerning the regulation of testicular AMH production after birth. Using clinical and experimental approaches, we have shown that pubertal elevation of the androgen concentration within the testis is responsible for the inhibition of AMH production (reviewed in Ref. 7). When the negative effect of androgens is absent owing either to impaired testosterone synthesis (9, 10) or to the lack of androgen receptor expression in Sertoli cells, e.g. in the normal fetus and neonate (6) and in the androgen insensitivity syndrome (6, 11), testicular AMH output can be stimulated by FSH (6). The cellular and molecular mechanisms underlying FSH stimulation of AMH production are not known.

Sertoli cells express the FSH receptor, a seven-transmembrane-domain receptor belonging to the superfamily of G protein-coupled receptors. The best characterized pathway of FSH action in Sertoli cells is mediated through a Gs heterotrimeric protein, formed by {alpha}-, ß-, and {gamma}-subunits. The activated receptor catalyzes the exchange of GTP for GDP and the dissociation of the G protein heterotrimer. The GTP-bound Gs{alpha} protein activates adenylate cyclase resulting in increased intracellular cAMP levels, responsible for the stimulation of protein kinase A (PKA). In turn, PKA phosphorylates a class of transcription factors binding to cAMP-responsive elements (CREs) in FSH-target genes (reviewed in Ref. 12). More recently, alternative pathways have been described for FSH signaling, involving phosphatidylinositol 3-kinase (PI3-K) and protein kinase B (PKB; Refs. 13 and 14). In the prepubertal Sertoli cell, FSH has also been shown to increase DNA and protein synthesis resulting in cell proliferation and protein secretion (reviewed in Ref. 15). The FSH-dependent elevation of serum AMH we have previously reported (6) could be explained by an increased AMH expression in individual Sertoli cells or by an increase in the number of Sertoli cells, or both. The purpose of this work was to delineate the mechanisms by which FSH stimulates testicular AMH output. Furthermore, because no consensus CRE sequences have been found in the 5'-flanking sequences of the AMH gene studied to present, we investigated other potentially involved molecular pathways.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Serum AMH Is a Marker of Sertoli Cell Number in the Prepubertal Male
To evaluate the effect of FSH on testicular AMH output and its correlation with Sertoli cell number before puberty, we used prepubertal FSH-deficient (FSH-/-) transgenic male mice bearing a targeted deletion of exons 1, 2, and most of exon 3 of the gene encoding the FSH ß-subunit (16). Results in Fig. 1AGo show that serum AMH was moderately but significantly lower in prepubertal FSH-/- mice than in normal littermates (FSH+/-). Prepubertal FSH-/- mice also had lower testicular mass, owing to a lesser number of Sertoli cells (Fig. 1BGo). Both serum AMH (Fig. 1AGo) and Sertoli cell number (Fig. 1BGo) attained normal values in FSH-/- males treated with recombinant FSH for 6 d. These results lead us to the conclusion that, in prepubertal male mice, serum AMH levels are regulated by FSH and may reflect the total number of functional Sertoli cells, whose proliferation is FSH stimulated. Both the lack of FSH and its exogenous administration had a greater effect on testicular volume than on Sertoli cell number (Fig. 1BGo), which can be explained by the fact that FSH also regulates germ cell number (17).



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Figure 1. Effect of FSH on Testicular AMH Production and Sertoli Cell Number

FSH-deficient (FSH-/-) transgenic male mice and normal littermates (FSH+/-) were killed at 7 d of age, serum AMH was assayed by ELISA (A), and morphometric studies of the testes were performed (B). FSH-deficient mice were divided into two groups: one of them received 0.5 U of recombinant FSH, and the other received the same volume of saline (sal.) every day from birth to d 6. Results were analyzed using a paired t test. *, P < 0.05 when compared with FSH+/-.

 
AMH Promoter Activity Is Enhanced after Activation of the FSH Signaling Pathway in SMAT1 Cells
To test whether FSH is also capable of activating AMH gene transcription in prepubertal Sertoli cells, we examined its ability to enhance the transcription of human AMH promoter-luciferase reporter constructs [5'human AMH (hAMH)-luc] transfected into SMAT1 cells, a clonal, immortalized Sertoli cell line, derived from a prepubertal mouse testis and expressing the transcription factors needed for basal AMH production (18). The longest 5'hAMH-luc construct contained 3068 bp of the hAMH gene 5'-flanking region. Because SMAT1 cells do not express the FSH receptor (FSH-R), to test FSH action on 5'hAMH-luc activity, we cotransfected an FSH-R expression vector into SMAT1 cells and incubated them with or without 2 U/ml of recombinant human FSH. We observed a modest but consistent enhancement of the activity of -3068-5'hAMH-luc after 36 h of incubation with FSH (Fig. 2AGo). At 2 h, the enhancement was not uniformly observed, which explains the higher variability and lack of statistically significant difference when compared with the basal condition. An activation of the 5'AMH-3068-luc construct was also obtained when SMAT1 cells devoid of FSH-R expression were cotransfected with a Gs{alpha}-protein expression vector (Fig. 2BGo), when adenylate cyclase was directly stimulated with cholera toxin, or when cells were incubated with dibutyryl cAMP (dbcAMP; Fig. 2CGo). The effect induced by the addition of dbcAMP was significant at 2-h incubation and was maximal at 24 h. Altogether, these results indicate that the FSH-R-dependent G protein adenylate cyclase pathway can be activated in SMAT1 cells and that it results in an increased transcriptional activity of a 3-kb hAMH promoter. For practical reasons, we performed most of the subsequent experiments using only dbcAMP for 24 h.



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Figure 2. AMH Promoter Activity Is Enhanced after Activation of the FSH Signaling Pathway at Various Levels

SMAT1 cells were transfected with 1 µg of the -3068-5'hAMH-luc construct and luc activity was assessed 48 h later. A, One microgram of an empty pcDNA or of a pcDNA-FSH receptor expression vector was cotransfected and cells were incubated, during the last 2 or 36 h, in medium with or without 2 U/ml of recombinant FSH. B, One microgram of an empty pcDNA or of a pcDNA-Gs{alpha} expression vector was cotransfected. C, Cells were incubated in basal medium or in medium containing 1 mM dbcAMP for 2, 6, or 24 h, or 1 µg/ml of cholera toxin for 24 h. In all cases, results (mean ± SEM of four experiments) were analyzed using a paired t test. *, P < 0.05 FSH vs. basal in pcDNA-FSH-R transfected cells, or pcDNA-Gs{alpha} vs. pcDNA, or dbcAMP or cholera toxin vs. basal.

 
FSH Activates AMH Transcription through a Nonclassical PKA-Dependent Mechanism
Because the AMH promoter lacks a canonical CRE (2, 19), the classical pathway involving PKA-mediated phosphorylation of CRE-binding protein and subsequent binding to AMH 5'-flanking sequences seemed unlikely. Therefore, we searched for alternative molecular mechanisms that could explain the response to FSH. Signaling pathways recently described as intracellular targets of cAMP include PKB phosphorylation mediated by PI3-K, which can be blocked by LY294002, and activation of p38 MAPK and ERK cascades, which can be blocked by SB203580 and PD98059 respectively (Fig. 3AGo; Refs. 13 and 14). We incubated SMAT1 cells with dbcAMP in presence or absence of an inhibitor of PKA, H89, or of LY294002, SB203580, or PD98059, and measured AMH in the culture medium by ELISA (Fig. 3BGo). Only H89 was able to completely block cAMP-mediated AMH production. Similar results were observed in luc assays after transfection of -3068-5'hAMH-luc to SMAT1 cells (Fig. 3CGo). These results indicate that cAMP-mediated activation of the AMH promoter in the immature Sertoli cell line SMAT1 involves PKA and not PKB, ERK, or p38 MAPK.



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Figure 3. cAMP-Dependent AMH Promoter Activation Is Blocked by an Inhibitor of PKA

A, FSH-mediated up-regulation of target genes via cAMP may involve PKA or more recently described signaling pathways involving cAMP-regulated guanine nucleotide exchange factors. Different inhibitors can be used to specifically block each of the various pathways (13 14 ). B, SMAT1 cells were incubated for 24 h in basal medium or in medium containing 1 mM dbcAMP alone or with either 10 µM H89 (PKA inhibitor), 25 µM LY294002 (PI3-K inhibitor), 20 µM SB203580 (p38 kinase inhibitor), or 10 µM PD98059 (MAPK inhibitor), and murine AMH concentration was determined by ELISA in the culture medium. Cells were detached by mild trypsinization and counted. C, SMAT1 cells were transfected with -3068-5'hAMH-luc (1 µg), and luc activity was assessed 48 h later. In the last 24 h, cells were exposed to the same treatments as in B. Results are given as fold induction, considering as 1 the activity in the well with basal medium. In all cases, results (mean ± SEM of four experiments) were analyzed using Student’s t test, in which all columns were compared with the dbcAMP column. *, P < 0.05.

 
Sequences Lying More Than 1.9 kb Upstream of the AMH Transcription Start Site Are Involved in the Response to cAMP
The proximal promoter of the murine AMH gene has been extensively studied, and response elements for SOX-9 (20, 21), steroidogenic factor 1 (SF-1; Refs. 22, 23, 24), and GATA factors (24, 25, 26) have been shown to play important roles in the regulation of AMH expression. Sequence analysis of the 5'-flanking region of the hAMH gene revealed that GATA, SF-1, and SOX-9 binding sites are present within 220 bp of the transcriptional start site, in the same 5'-to-3' order as in the mouse (Fig. 4Go). To map the sequences responsible for cAMP responsiveness in the hAMH 5'-flanking region, we generated several luc reporter constructs containing fragments of various lengths of the hAMH promoter (Fig. 4Go) that were used for transfections into SMAT1 cells. First, we observed that in basal conditions the 3068-bp promoter was the most active, with a clearly higher trans-activating capacity than the 423- and the 202-bp promoters (Fig. 5AGo). The 83-bp promoter had no trans-activating ability, as compared with the empty pGL2B luc plasmid. Second, cAMP enhanced the activity of the 3068 bp but not that of the shorter promoters. Furthermore, FSH increased the trans-activation of the 3068-bp promoter, but not that of the 423-bp promoter (Fig. 5BGo). These results strongly suggested that sequences of the AMH promoter involved in the response to FSH lie between positions -428 and -3068 relative to the transcription initiation site.



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Figure 4. Schematic Representation of the Mouse AMH (mAMH) and hAMH Gene 5'-Flanking Sequences and of 5'hAMH-luc Constructs Used in this Study

Localization of binding sites for SF-1, GATA-4, and SOX-9 in the mAMH promoter have been described (20 22 24 25 ). Nucleotide positions of binding sites in the human promoter are according to the sequence published by Guerrier et al. (42 ). Localization of the human SAP62 gene is indicated according to Dresser et al. (44 ).

 


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Figure 5. FSH and cAMP Enhance the Activity of the 3068-bp AMH Promoter

SMAT1 cells were transfected with 1 µg of an empty pGL2B plasmid or of pGL2B containing different lengths of the hAMH 5'-flanking sequences, and luc activity was assessed 48 h later. Cells were incubated with or without 1 mM dbcAMP for 24 h (A) or 2 U/ml of recombinant FSH for 36 h (B). Results (mean ± SEM of seven experiments in A and four in B) were analyzed using Student’s t test. *, P < 0.05; **, P < 0.01, dbcAMP or FSH vs. basal.

 
To test this hypothesis, we generated two short 5'AMH-luc constructs lacking the proximal promoter sequences: one spanning from -3068 to -1921 and a shorter one spanning from -2198 to -1921 (Fig. 4Go). As seen in Fig. 6Go, both the -3068–1921-5'hAMH-luc and the full-length -3068-5'hAMH-luc constructs, with intact nuclear factor (NF)-{kappa}B and activating protein 2 (AP2) sites, showed a similar response to cAMP. The response of the -2198–1921-5'hAMH-luc construct was somewhat lower but still significant. Altogether, these data indicate that sequences lying upstream of position -1921 contribute to, and are sufficient for, cAMP enhancement of the AMH promoter activity. Furthermore, relevant sequences must exist between -2198 and -3068.



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Figure 6. cAMP Enhancement of the AMH Promoter Activity Involves NF{kappa}B and AP2 Binding Sites Lying More than 1921 bp Upstream of the Transcription Start Site

SMAT1 cells were transfected with 1 µg of the full-length -3068-5'hAMH-luc construct or of constructs consisting of sequences between -3068 and -1921 or between -2198 and -1921 with wild-type (open boxes) or mutated (crossed boxes) AP2 and/or NF{kappa}B sites, and luc activity was assessed 48 h later. A, Basal activity of the different constructs. B, Effect of the incubation with 1 mM dbcAMP during the last 24 h. Results are given as fold induction (mean ± SEM of four experiments), considering as 1 for each construct (dotted line) the luc activity without dbcAMP. Results were analyzed using a one-sample t test, in which the mean for each construct was compared with 1, which means no activation after dbcAMP addition. *, P < 0.05; **, P < 0.01.

 
An NF{kappa}B Binding Site at Position -2208 and an AP2 Binding Site at Position -1936 Are Required for the Enhancement of the AMH Promoter Activity by cAMP
A detailed analysis of the distal sequences between -1921 and -3068 upstream of the hAMH start initiation site revealed the existence of a consensus NF{kappa}B binding site at -2208 and a consensus AP2 site at -1936 (Fig. 4Go). We performed targeted mutagenesis of these sites in both the full-length 3068-5'hAMH-luc and the -3068–1921-5'hAMH-luc constructs. Mutations of NF{kappa}B and/or AP2 sites did not significantly affect the basal activity of the full-length promoter (Fig. 6AGo). Mutation of the NF{kappa}B site resulted in a dramatic decrease in, although not a complete abolition of, the response to cAMP (Fig. 6BGo). Note that deleting the -3068–2198 region, which encompasses the NF{kappa}B site, had the same effect. Mutation of the AP2 site also impaired the responsiveness to cAMP, although in the context of the full-length promoter this effect seemed less pronounced. A complete abrogation of cAMP responsiveness could not be obtained by mutating both NF{kappa}B and AP2 sites.

To further investigate the possible involvement of these two factors in AMH response to cAMP-mediated FSH stimulation in Sertoli cells, we investigated NF{kappa}B and AP2 expression in prepubertal mouse testes and in SMAT1 cells by immunocytochemistry (Fig. 7Go). In paraffin sections of 6-d-old mouse testes, NF{kappa}B reaction was intense in Sertoli, germ, peritubular, and interstitial cells, whereas AP2 reaction was faint in the basal cytoplasm of Sertoli cells and more intense in peritubular and interstitial cells. SMAT1 cells showed cytoplasmic expression of both factors in basal conditions. After incubation with dbcAMP, the immunoreactivity was concentrated in the perinuclear cytoplasm and extended into the nucleus.



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Figure 7. AP2 and NF{kappa}B Are Expressed in Prepubertal Sertoli Cells and in the SMAT1 Cell Line

Paraffin-embedded testicular tissue obtained from 6-d-old mice (A–C), and SMAT1 cells (D–I) were submitted to immunohistochemical staining using antibodies for NF{kappa}B p65 (A, D, G) or AP2 (B, E, H) and AMH (C, F), used as a specific marker of Sertoli cells (3 ), or noninmune serum (I), as a negative control. NF{kappa}B (A) was positive in Sertoli cells (asterisks), germ cells (large arrowhead), and interstitial cells (arrow). AP2 (B) was slightly positive in the basal area of Sertoli cell cytoplasm (arrow), and more intensely positive in peritubular and interstitial cells. AMH (C) was positive in the cytoplasm of Sertoli cells (asterisks) but negative in the interstitial tissue (Int.). SMAT1 cells were cultured in basal medium, except for panels G and H, where 1 mM dbcAMP was added for 24 h.

 
Finally, we analyzed the effect of NF{kappa}B or AP2 overexpression on the activity of the 3-kb AMH promoter with wild-type or mutated NF{kappa}B or AP2 sites (Fig. 8Go). Overexpression of AP2 or NF{kappa}B resulted in an enhanced activity of the wild-type AMH promoter, whereas cotransfection of an excess of the inhibitor of {kappa}B (I{kappa}B) abrogated the effect induced by NF{kappa}B. Moreover, AP2 and NF{kappa}B were unable to increase the basal activity of the AMH promoter when their respective binding sites were mutated. Altogether, our results show that cAMP induction of the hAMH gene promoter activity in the prepubertal Sertoli cell line SMAT1 involves far upstream sequences (i.e. -3068/-1921) and requires NF{kappa}B and AP2 binding sites located in this region, although other sequences may also be involved (Fig. 9Go).



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Figure 8. AP2 and NF{kappa}B Factors Enhance the Activity of the 3068-bp AMH Promoter

SMAT1 cells were cotransfected with 0.5 µg of AP2 or NF{kappa}B expression plasmids, or their respective empty vectors, and 1 µg of a -3068-5'hAMH-luc construct carrying either wild-type (open boxes) or mutated (crossed boxes) AP2 or NF{kappa}B sites, and luc activity was assessed 48 h later. An I{kappa}B expression plasmid (2 µg) was cotransfected with NF{kappa}B (0.5 µg) where indicated. Total amount of transfected DNA per well was completed to 3.5 µg using pCMV vector in all cases. An empty pGL2B vector was used a negative control for -3068-5'hAMH-luc construct, and luc constructs with proven response elements for either AP2 or NF{kappa}B were used to monitor trans-activation efficacy.

 


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Figure 9. Schematic Model to Explain the Increase of Testicular AMH Production after FSH Stimulation

In basal conditions, AMH expression depends on the trans-activating potential of several nuclear factors, SOX-9, GATA-4, SF-1, and WT-1 (20 21 22 23 24 25 ). FSH binds to a seven-transmembrane-domain G protein-coupled receptor; the {alpha}-subunit of the heterotrimeric Gs protein induces adenylate cyclase (AC) activity resulting in an increase of cAMP concentration and PKA activation. PKA is capable of phosphorylating AP2 (47 48 ) and I{kappa}B (49 50 ), which releases NF{kappa}B. Subsequently, the phosphorylated AP2 and NF{kappa}B translocate to the nucleus and enhance AMH transcription. FSH-dependent PKA activity also induces cell proliferation. Together, the increase in Sertoli cell number and the enhancement of AMH transcription in each Sertoli cell results in an increase of testicular AMH production and secretion to the circulation.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In fetal and postnatal life, testicular AMH production is hormonally regulated by androgens and FSH. Elevated intratesticular testosterone concentration has a potent inhibitory effect on AMH at puberty (6, 27), but in the absence of androgen action on Sertoli cells (9, 10, 11) FSH up-regulates testicular AMH production (6, 28). In this work, we provide new insight into the cellular and molecular mechanisms that explain the elevation of serum AMH after FSH stimulation. Based on results obtained from a clinical study on pediatric patients with various testicular disorders, we previously proposed that serum AMH levels are highly indicative of the number of functional Sertoli cells (9). Here, we provide further experimental evidence that serum AMH levels are correlated with the size of the Sertoli cell population; we also show that FSH is capable of enhancing AMH gene transcription. We can therefore conclude that the positive effect of FSH on testicular AMH production is due both to the proliferation of Sertoli cells and to the increase in AMH transcriptional activity per Sertoli cell. Furthermore, we show that sequences lying more than 1.9 kb upstream from the AMH transcription start site are involved in the activation of AMH gene expression in response to FSH.

Testicular function has classically been associated with androgen and sperm production. Because these two functions are quiescent before pubertal onset, the testis has been considered to be an inactive organ during childhood. Owing to the lack of an experimentally proven and clinically widely accepted marker of FSH action on prepubertal Sertoli cells, evaluation of the existence and functional potential of testicular tissue in boys has exclusively relied on Leydig cell testosterone secretion in response to human chorionic gonadotropin administration. However, Sertoli cells are very active before puberty: they proliferate and have an intense protein synthesis activity (reviewed in Refs. 29 and 30), including the secretion of high amounts of AMH. Serum AMH has been shown to be an extremely useful marker of prepubertal testicular Sertoli cell function, with no need for stimulation tests (31, 32). The fact that the number of spermatozoa produced by the adult testis is conditioned by the FSH-dependent, prepubertally attained size of the Sertoli cell population (33, 34, 35, 36) reinforces the importance of Sertoli cell proliferation in fetal and early postnatal life in response to FSH. Previous work showed that testes of adult mice with a knockout of the FSHß gene (16, 17) or of the FSH receptor gene (37, 38) were smaller in size owing to low germ cell number resulting from diminished Sertoli cell number and their capacity to nurture germ cells. Using the FSHß knockout model, here we show that if no FSH is exogenously supplied the number of Sertoli cells per testis is already decreased at the onset of puberty, i.e. the age at which Sertoli cells stop dividing, providing further evidence of the importance of FSH in early life for the achievement of quantitatively normal sperm production and testicular size in adulthood. Furthermore, our data indicate that AMH is a marker of both Sertoli cell proliferation and protein synthesis activity in response to FSH before puberty and also support the hypothesis that serum AMH might be an useful marker of FSH action in the assessment of testicular function in prepubertal boys.

The activation and maintenance of basal levels of AMH expression in fetal life, experimentally studied by several groups (21, 22, 23, 24, 25, 26, 39), are mostly independent of gonadotropins, as also shown by clinical observations in patients with hypogonadotropic hypogonadism (40) and by our present results in FSHß gene knockout mice. Indeed, testicular AMH production is not abolished in the absence of FSH action. However, as we show here, it is moderately but significantly reduced, and resumes normal levels after exogenous FSH administration.

Owing to its temporal correlation with fetal testicular differentiation, AMH expression was first thought to be directly regulated by SRY (41). However, subsequent experiments have ruled out a direct trans-activating potential of SRY on AMH and shown that the combined action of multiple transcription factors is required to activate and maintain AMH expression in the fetal testis. The SRY-related protein SOX-9 (20, 21), SF-1 (22, 23, 24), and GATA factors (24, 25, 26) have been shown to bind to specific response elements present in the 5'-flanking region of the AMH gene and regulate its transcription. Although more than 3 kb of 5'-flanking sequences of the hAMH gene have been known for several years (42), up to now all functional studies have investigated less than 400 bp of the AMH proximal promoter region. A recent study shows that a 370-bp promoter is not sufficient to maintain AMH expression postnatally (43), clearly indicating that other factors binding to DNA elements lying far upstream are also involved in the regulation of AMH expression. In the present work, we have extended the investigation of the regulatory potential of 5'-flanking sequences of the AMH gene to 3 kb upstream of the translation start site. The fact that the 3' end of the gene coding for SAP62/SF3A2, a ubiquitously expressed spliceosome protein, is just 789 bp upstream from the ATG codon in the hAMH gene and 433 bp in the mouse (44) has nourished the hypothesis that 5' regulatory sequences of the AMH gene are very short. However, we show here that a 3-kb AMH promoter has a more potent activating capacity than smaller promoters, at least in the prepubertal Sertoli cell line SMAT1. We also show that the sequences between 1.9 and 3 kb upstream of the hAMH transcription site can up-regulate the activity of the AMH promoter. Owing to their position, these sequences could also be involved in the regulation of SAP62. Because it was beyond the scope of our study, we did not analyze SAP62 expression in SMAT1 cells. Nonetheless, the luc gene was placed at the AMH -not the SAP62-gene position and its activity is, thus, indicative of the AMH gene transcription levels that could be expected. Therefore, we can conclude that, in the present experimental conditions, AMH expression is regulated by distant sequences. Other genes, like IL-8, have already been shown to be transcriptionally activated through distant NF{kappa}B and AP2 binding sites (45).

In this study, we clearly demonstrate that the AMH promoter activity can be enhanced by a classical FSH-regulated signaling cascade involving a G protein, adenylate cyclase and PKA (Fig. 9Go). The best known FSH signal transduction pathway to the nucleus involves PKA phosphorylation of transcription factors binding to CREs in the promoter of target genes. The hAMH promoter lacks a canonical CRE (2, 19); we therefore proposed that FSH action was either indirect or involved a different pathway (19). Here we show that the effect of dbcAMP addition to SMAT1 cells results in an increase of the AMH promoter activity at 2 h, which may indicate that the pathway involved is quite direct. However, an even higher activity was observed at 24 h. Although this may result from the accumulation of luc protein—whose half-life is between 3 and 4 h (46)—in SMAT1 cells, an additive, indirect effect through regulation of an upstream gene cannot be ruled out. Our present results suggest that AP2 and NF{kappa}B binding sites at positions -1936 and -2208, respectively, are, at least in part, responsible for the enhancement of AMH expression induced by FSH via cAMP-PKA. PKA-dependent phosphorylation of AP2 in response to the elevation of intracellular cAMP concentration has been shown to mediate trans-activation of target genes (47, 48) and could explain in part FSH action on AMH (Fig. 8Go). AP2 binding to consensus elements in the human chorionic gonadotropin ß promoter is increased by cAMP only after 24 h (48); a similar time dependency in our model could be an alternative explanation for the latency observed to reach the maximal response in FSH- or cAMP-stimulated conditions.

The mechanism involving NF{kappa}B in FSH-dependent AMH activation is less clear. The transcriptional activity of NF{kappa}B may be regulated at multiple steps in response to more than 150 different stimuli (49, 50). NF{kappa}B is normally retained in the cytoplasm owing to its association with the inhibitory molecule I{kappa}B. Phosphorylation and subsequent proteolysis of I{kappa}B allow NF{kappa}B translocation to the nucleus, where NF{kappa}B induces transcription once associated with the transcriptional cofactor CRE binding protein-binding protein (50). It has also been shown that the stimulation of Sertoli cells with activators of the PKA signaling pathway, such as forskolin or FSH, increases NF{kappa}B DNA binding activity to enhancer motifs within the promoter of cAMP-regulated genes (51, 52). Further studies will be necessary to completely understand the possible mechanisms by which NF{kappa}B may be involved in the regulation of AMH gene expression.

Interestingly, not only FSH but testosterone and AMH itself can regulate NF{kappa}B signaling. AMH has been shown to regulate NF{kappa}B in the mammary gland (53) and the prostate (54). Because Sertoli cells express the specific AMH receptor type II (18, 55, 56) and the elements involved in its signaling pathway (57), it might be possible that AMH regulates its own expression through NF{kappa}B signaling. The interaction between testosterone and FSH in AMH regulation is intricate: whereas testosterone down-regulates AMH production, FSH induces AMH if the negative effect of androgens is absent (6). Mechanisms of physical interaction have been described between NF{kappa}B and nuclear steroid receptors, which explain their mutual antagonism (58, 59). Furthermore, the ligand-bound androgen receptor is capable of decreasing NF{kappa}B activity in the absence of classical androgen responsive elements in the promoters of target genes (60, 61). Because there is no direct binding site for the androgen receptor in the AMH promoter (19), the antagonism between FSH and testosterone action on AMH expression in pubertal Sertoli cells could be due to a cross-talk between the androgen receptor and NF{kappa}B.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
FSH-deficient (FSH-/-) transgenic male mice bearing a homozygous (fshbm1/fshbm1) deletion of exons 1, 2, and most of exon 3 of the gene coding for the ß-subunit of FSH, generated by homologous recombination (16), and heterozygous FSH+/- littermates (fshbm1/+) were obtained by intercrossing homozygous males with heterozygous females. Genotyping was performed by PCR screening of genomic DNA using two primers (FSH 663S: 5'-GGACGTAGCTGTTTACTTCCC and FSH 848A: 5'-AGTGTAGCAGTAGCCCGCACA) allowing the amplification of the second exon of the mouse FSHß gene (62). Agarose gel electrophoresis of the resulting PCR products were analyzed to distinguish between FSH-/- (homozygous for the deletion involving exon 2) and FSH+/- littermates (one deleted and one normal allele). In the same reaction, PCR amplification of 286 bp of the Sry gene (primers: MSRY1: 5'-TAGAGAGCATGGAGGGCCAT and MSRY2: 5'-TGCCACTCCTCTGTGACACT) was performed as control of all male DNAs. Heterozygous males have a normal gonadal function (16). All animal studies were conducted in compliance with European Community guidelines.

Treatment, Histological Analysis, and Serum AMH Determination
FSH-/- male mice were separated at birth in two groups: one group was injected sc with 0.5 U recombinant FSH (Gonal-F-75, Serono, Geneva, Switzerland) every day from postnatal d 1–6; the second group, serving as control, was injected with saline solution. All animals were killed on d 7; serum was extracted and stored at -20 C until assayed. Testes were weighed and either used for DNA extraction, or immersion-fixed in Bouin’s fixative overnight, dehydrated in a graded series of ethanol, and finally embedded in paraffin wax.

Morphometric analysis was performed on one testis of seven FSH-/- mice (three treated with FSH and four nontreated) and of three FSH+/- littermates as previously described (63), with slight modifications. The number of Sertoli cells per testis was estimated by counting the number of nucleoli in a known area (10 fields representing 911,879 µm2) using a 20x objective on a Reichert Diastar Microscope, and the images obtained with a Javelin Chromaspin-V CDD video camera (Javelin Electronics, Los Angeles, CA) were digitalized and analyzed using Optimas 4.0 for Windows (Bioscan, Inc., Edmonds, WA). As testicular specific gravity is approximately 1, testicular weight was used to estimate testicular volume. The total Sertoli cell number per testis was obtained by applying the Floderus equation as described (63). The person who performed Sertoli cell counts was blinded to the treatment status and genotype of the mice. Results were subjected to a paired t test.

AMH concentration was measured in the serum obtained from the same animals whose gonads were studied by histological morphometric analysis. Serum AMH was determined using an ELISA as previously described (6).

Plasmids
A 3078-bp fragment of the 5'-flanking region of the hAMH gene (position -3068/+10 relative to the major transcriptional initiation site; Ref. 42) was obtained from pGAMH1 construct (18) by restriction enzyme digestion using SacI and AvrII. AvrII site was rendered blunt using the Klenow fragment of DNA polymerase I, and the resulting construct was subcloned between SacI and BglII sites of luc vectors pGL2B and pGL2P (Promega Corp., Madison, WI) to obtain -3068-5'hAMH-luc plasmids. Four other deletion 5'hAMH-luc constructs, respectively, containing 1921, 423, 202, and 83 bp of the hAMH promoter, were also generated. A chimeric construct including a DraIII-SmaI fragment of pGL2B and a SmaI-NheI (-2198/-1921) fragment of 5'hAMH was subcloned between DraIII and NheI sites of -1921-5'hAMH-luc to obtain -2198-5'hAMH-luc. A similar construct was obtained using pGL2P. A NheI-AvrII (-1921/+10) fragment of pGAMH1 was subcloned in pGL2B linearized by NheI to obtain -1921-5'hAMH-luc. An EcoRV-AvrII (-423/+10) fragment of pGAMH1 was subcloned between SmaI and NheI sites of pGL2B to obtain -423-5'hAMH-luc. A 240-bp fragment of the proximal hAMH promoter was amplified by PCR using 5'-CAGCATGTTGACACATCAG and 5'-CTTTTTGCAAAAGCCTAGGC primers. The amplified fragment was subsequently digested either by HincII and AvrII and subcloned between SmaI and NheI sites of pGL2B to obtain -202-5'hAMH-luc, or by BstUI and AvrII and subcloned between SmaI and NheI sites of pGL2B to obtain -83-5'hAMH-luc. The -3068-5'hAMH-luc and the -2198-5'hAMH-luc constructs subcloned in pGL2P were digested by NheI and HindIII. The resulting longer fragments, respectively, containing -3068-1921-5'hAMH-luc and -2198-1921-5'hAMH-luc, were rendered blunt using the Klenow fragment of DNA polymerase I, and circularized by incubating with T4 DNA ligase.

The FSH receptor expression vector, pcDNA—FSH-R (64), and its control, pcDNA—CAT, were kindly provided by Drs. W. Tribley and M. Griswold (Pullman, WA). The Gs{alpha} protein expression vector, {alpha}s-WT (no. 63315; Ref. 65), was purchased from ATCC (Manassas, VA). The AP2 expression vector (66) and a proven AP2-responsive luc reporter, CGß promoter-luc (67), were kindly provided by Drs. T. Williams (Denver, CO) and T. Kotlar and J. L. Jameson (Chicago, IL). Expression vectors for NF{kappa}B p65 and I{kappa}B (68) and a proven NF{kappa}B-responsive luc reporter plasmid (69) were kindly provided by Drs. A. Israël (Paris, France) and S. Maheswaran (Boston, MA).

Targeted Mutagenesis
Plasmids with mutations in binding sites for AP2 or NF{kappa}B of the hAMH promoter were generated using the pALTER1 site-directed mutagenesis system (Promega Corp.). One AP2 site was mutated (GGGAGGGG -> TGGTACCG) at position -1936, and one NF{kappa}B site was mutated (GGGGTCCCC -> ATGGTACCA) at position -2208.

Cell Culture, Transfections, Luc Assays, and AMH Determination in Culture Medium
SMAT1 cells, an immortalized immature Sertoli cell line (18), were cultured in DMEM (Life Technologies, Inc., Rockville, MD) supplemented with 10% fetal calf serum, 1x amino acid mix (Eurobio, Les Ulis, France), 100 U/ml penicillin, and 100 µg/ml streptomycin (Eurobio). Cells were plated at 2 x 105 cells/well in six-well plates and transiently transfected with 1 µg of luc reporter plasmids, or at 0.5 x 105 cells/well in 24-well plates and transiently transfected with 0.5 µg of DNA using the LipofectAMINE PLUS Reagent package (Life Technologies, Inc.) according to the manufacturer’s instructions. In brief, 1 d after initial plating, DMEM with fetal calf serum was changed for DMEM without serum. Twenty-four hours later, transfections were performed in DMEM without serum. dbcAMP (Sigma, St. Louis, MO) or cholera toxin (Sigma) was added at 1 mM or 1 µg/ml, respectively. Human recombinant FSH (Gonal-F-75, Serono) was used at 2 U/ml. Kinase inhibitors H89, LY294002, SB203580, or PD98059 (all from Calbiochem, San Diego, CA) were used at 10 µM, 25 µM, 20 µM, or 10 µM, respectively. Luc activity was determined with the luc reporter gene assay kit (Roche Diagnostics, Indianapolis, IN) using a Lumat LB95507 luminometer (EG&G Berthold, Bad Wildbad, Germany) and normalized for protein concentration (all experiments) and for ß-galactosidase activity where the activity of different reporter plasmids were compared (Fig. 5Go). Protein concentration was measured using the bicinchoninic acid kit for protein determination (Sigma), and ß-galactosidase activity was estimated by spectrophotometry at 420 nm, using orthonitrophenyl-ß-D-galactoside as substrate.

To determine the effect of the various kinase inhibitors on AMH secretion to the culture medium, SMAT1 cells were plated and treated as described and AMH was assayed using the ELISA described above. Cells were detached from the plate by gentle trypsinization and counted in a Malassez chamber.

Immunocytochemistry
Paraffin-embedded sections of testicular tissue fixed in paraformaldehyde (4% in PBS) from 6-d-old mice were used for immunocytochemistry using the antigen retrieval technique as previously described (6), with slight modifications. SMAT1 cells, cultured on four-chamber Lab-Tek Permanox slides (Nunc, Naperville, IL) at 2–5 x 104 cells per chamber, were fixed in 4% paraformaldehyde and submitted to immunocytochemistry without antigen retrieval. Primary antibodies were: rabbit polyclonal antirecombinant hAMH L40 (1 µg/ml), rabbit polyclonal anti-NF{kappa}B p65 sc-109 (2 µg/ml, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and rabbit polyclonal anti-AP2 sc-184 (2 µg/ml, Santa Cruz Biotechnology, Inc.). A negative control reaction was performed by replacing the primary antibody with nonimmune rabbit serum. The reaction was revealed using peroxidase Vectastain Elite ABC kit PK-6101 (Vector Laboratories, Inc., Burlingame, CA) and DAB Plus Reagent K-3467 (DAKO Corp., Copenhagen, Denmark).


    ACKNOWLEDGMENTS
 
Recombinant FSH (Gonal-F-75) was a kind gift of Serono International S.A. (Geneva, Switzerland). We are grateful to Drs. W. Tribley and M. Griswold (Pullman, WA), T. Williams (Denver, CO), T. Kotlar and J. L. Jameson (Chicago, IL), A. Israël (Paris, France), and S. Maheswaran (Boston, MA) for plasmids.


    FOOTNOTES
 
This work was financed by the Ministerio de Salud de la Nación, Argentina [Beca Oñativia-Carrillo (to R.R. and C.L.)]; the Consejo Nacional de Investigaciones Científicas y Técnicas [CONICET, Argentina, Grant PIP 0574/98 (to R.R.)]; the Institut National de la Santé et de la Recherche Médicale (Paris, France); the Ministère de l’Education Nationale, de la Recherche et de la Technologie, France (Grant Procédure PAST); the Moran Foundation [Department of Pathology, Baylor College of Medicine, Houston, TX (to T.R.K.)]; and the NIH [Grant CA-60651 (to M.M.M.)]. R.R. is an established researcher with CONICET and an invited professor at the Département de Biologie, Ecole Normale Supérieure (supported by Procédure PAST of the Ministère de l’Education Nationale, de la Recherche et la Technologie).

1 Present address: Service d’Endocrinologie et Maladies Métaboliques, Centre Hospitalier Universitaire, Reims, France. Back

Abbreviations: AMH, Anti-Müllerian hormone; AP2, activating protein 2; dbcAMP, dibutyryl cAMP; CRE, cAMP-responsive element; FSH-R, FSH receptor; I{kappa}B, inhibitor of {kappa}B; luc, luciferase; MIS, Müllerian-inhibiting substance; NF{kappa}B, nuclear factor-{kappa}B; PI3-K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKB, protein kinase B; SF-1, steroidogenic factor 1; SOX9, SRY-related HMG-box gene 9; SRY, sex-determining region Y.

Received for publication May 21, 2002. Accepted for publication December 19, 2002.


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