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Endocrinology Vol. 139, No. 3 1213-1220
Copyright © 1998 by The Endocrine Society

ARTICLES

Effect of Anti-Mullerian Hormone on Sertoli and Leydig Cell Functions in Fetal and Immature Rats1

V. Rouiller-Fabre, S. Carmona, R. Abou Merhi, R. Cate, R. Habert and B. Vigier

Institut National de la Santé et de la Recherche Medicale-Institut National de Recherches Agronomiques U-418, Equipe de Différenciation Cellulaire des Gonades, Université Paris 7 (V.R.-F., R.H.), and URA-CNRS 1449, Laboratoire de Physiologie de la Reproduction, Equipe de Différenciation de la Gonade (S.C., B.V.), 75251 Paris; and Institut National de Recherches Agronomiques, Laboratoire de Biologie Cellulaire et Moléculaire, Bâtiment des Biotechnologies (R.A.M.), 78352 Jouy en Josas, France; and Biogen, Inc. (R.C.), Cambridge, Massachusetts 02142

Address all correspondence and requests for reprints to: Dr. B. Vigier, Unité INRA "Differenciation Cellulaire et Moléculaire" Bâtiment des Biotechnologies, 78352 Jouy en Josas, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Anti-Mullerian hormone (AMH) is mainly involved in the regression of Mullerian ducts in male fetuses, but it may have other functions linked to gonadal development. The present study examines the effect of AMH on steroidogenesis by Sertoli and Leydig cells in fetal and immature rats during the period where AMH is physiologically produced in the testis.

The basal aromatase activity of Sertoli cells in primary culture was strongly stimulated (77–91%) by cAMP. AMH (35 nM) reduced cAMP-stimulated aromatase activity by 49–69% as early as fetal day 16 and until postnatal day 20. This effect was dose dependent and was seen after 48 h in culture. AMH also blocked the Sertoli cell aromatase activity stimulated by FSH, but LH did not stimulate this activity, confirming that the aromatase activity effectively resulted from Sertoli cells and not from contaminating Leydig cells. RT-PCR analysis showed that AMH reduced aromatase activity by decreasing the amount of aromatase messenger RNA.

AMH also inhibited the LH-stimulated testosterone production by dispersed fetal Leydig cells in culture in a dose-dependent manner. The inhibitory effect of AMH did not depend on the fetal stage studied (16 or 20 days postconception) and resulted from a drop in the steroidogenic activity of each Leydig cell without affecting the number of 3ß-hydroxysteroid dehydrogenase-positive cells.

These data provide the first evidence that AMH, like other members of the transforming growth factor-ß family, has an autocrine/paracrine effect on testicular steroidogenic function during the fetal and prepubertal periods.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANTI-MULLERIAN hormone (AMH), or Mullerian inhibiting substance (MIS) or factor (MIF), is responsible for the regression of Mullerian ducts in male fetuses (1). It is a 140-kDa glycoprotein homodimer belonging to the transforming growth factor-ß (TGFß) family (for review, see Refs. 2–4). AMH is produced by Sertoli cells from the time when the testicular seminiferous cords differentiate until pubertal maturation (5, 6, 7, 8, 9, 10) and also by postnatal granulosa cells (11). Both cell types express the AMH type II receptor (12, 13, 14), suggesting that AMH has an autocrine role.

It has been shown that AMH reverses the steroidogenic sex pattern of ovine, rabbit, and rat fetal ovaries in vitro by blocking the synthesis of aromatase (cytochrome P450 aromatase) (15, 16). AMH also decreases the amount of aromatase in granulosa cells from postnatal rats and immature pigs. Lastly, AMH decreases the amount of LH receptor messenger RNA (mRNA) in postnatal rat granulosa cells (17). These effects of AMH all suggest that it is implicated in the negative control of estrogen production and ovarian follicle maturation.

There is still no firm evidence that AMH is also a local regulator of steroidogenesis in the testis. AMH could inhibit the aromatase in the Sertoli cells, as these cells have the same embryological origin as the granulosa cells and are the major site of aromatase activity in fetal and immature testis (18, 19). AMH could also act locally to control steroidogenesis in Leydig cells. It has recently been shown that the number and function of Leydig cells in the adult are reduced in transgenic male mice in which the AMH gene is overexpressed and increased in mice in which the gene is inactivated (20, 21, 22).

As AMH is produced in the testis only during fetal and prepubertal life, the present study focuses on a possible effect of AMH during this period. Primary cultures of rat testicular cells were used to investigate the effect of AMH on the aromatase activity of fetal and postnatal rat Sertoli cells and its effect on the steroidogenic activity of fetal Leydig cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Wistar strain rats were mated overnight, and the following day was counted as day 0 postconception (pc). The day of birth was counted as day 0 postpartum (pp). Testes were removed from fetal day 16 to postnatal day 20.

Chemicals and solutions
Human recombinant AMH was purified from the medium of human AMH-transfected CHO cells by immunochromatography and quantified by reading optical density at 280 nm, using an extinction coefficient of 1 (23). Sertoli cell were cultured in CMRL 1066 (Eurobio, Les Ulis, France) supplemented with 10% female FCS (Sodexar, Rouen, France), 6 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Dispersed Leydig cells were grown in Ham’s F-12-DMEM (1:1; Life Technologies, Grand Island, NY) containing 5 µg/ml transferrin and 40 µg/ml gentamicin (Gentalline, Schering-Plough, Levallois-Perret, France). Ovine LH (NIH LH S19; 1.01 NIH LH S1 units/mg) and ovine FSH (NIDDK oFSH) were gifts from the NIDDK, NIH (Bethesda, MD). (Bu)2cAMP, soybean trypsin inhibitor, deoxyribonuclease I (DNase), nitroblue tetrazolium, transferrin, NAD, insulin, and isobutylmethylxanthine (IBMX) were purchased from Sigma Chemical Co. (St. Louis, MO). Collagenase was obtained from Serva (Heidelberg, Germany) and trypsin from (Eurobio, Les Ulis France).

Culture of Sertoli cells
The seminiferous tubules were dissected out from the testes of rats (fetal day 16 to postnatal day 20) under a microscope and incubated for 30 min at 37 C in 1 mg/ml collagenase in PBS, without calcium or magnesium, containing 0.5 mg/ml BSA and then sedimented at unit gravity. The interstitial tissue and peritubular cells in the supernatant were discarded. The pellet containing the seminiferous tubules was incubated with 0.1% trypsin-1 mM EDTA for 15–30 min. The cells were collected by centrifugation (100 x g), suspended in CMRL 1066 and 10% female FCS, plated in 24-well microplates at 7.5 x 105 cells in 1 ml/well, and incubated at 37 C in a controlled humidified atmosphere of 95% air-5% CO2 for 48 h. The cells were then placed in fresh medium containing 0.1 mM IBMX.

Sertoli cell purity was assayed on an aliquot of cell suspension plated in a glass Lab-Tek chamber (Nunc, Naperville, IL), fixed in Bouin’s fluid, and stained with hematoxylin-eosin or in a plastic chamber slide for immunohistochemical detection of transferrin using a rabbit polyclonal antibody against rat transferrin (gift from Dr. Guillou, INRA, Nouzilly, France). Specific binding was detected with an antirabbit fluorescein-linked secondary antibody (Amersham, Arlington Heights, IL). The appearance of the Sertoli cell monolayer from postnatal day 5 testes (5 dpp Sertoli cells) is shown in Fig. 1AGo. Nearly all cells had a typical epithelial appearance with rounded nuclei and wide clear spread-out cytoplasm that gave a positive immunological reaction for transferrin, a typical product of Sertoli cells (Fig. 1BGo). Specificity of staining for transferrin was checked by replacing the antibody with IgG from non-immune serum. These morphological and immunological criteria indicated that the Sertoli cell cultures from fetal and postnatal testes (16 dpc to 20 dpp) were more than 90% pure.



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  		Figure 1. Fetal and postnatal rat testicular cells in culture. A and B, Sertoli cells from postnatal day 5 testes (5 dpp Sertoli cells) were cultured in CMRL 1066 medium containing 10% female FCS for 4 days. A, Appearance of cells fixed in Bouin’s fluid and stained with hematoxylin-eosin; the arrows show phagocytic vesicles. B, Immunocytochemical detection of transferrin using a double antibody method with a rabbit antitransferrin primary antibody and a secondary fluorescein-linked sheep antirabbit IgG antibody. C, 3ßHSD-positive cells in culture. Testicular cells from 20-day-old fetuses were cultured for 1 day in DMEM-Ham’s F-12 medium containing 0.4% FCS or insulin (5 µg/ml) and then for 3 days (D1 to D3) in medium without serum and insulin, but containing LH (100 ng/ml). Cells were reacted with 3ßHSD, fixed in formol-ethanol (9:1), and counterstained with eosin. Leydig cells (3ßHSD-positive cells) are shown by arrows. The bars correspond to 20 µm.

 
Culture of dispersed fetal testicular cells
The technique used was essentially that described by Habert and Brignaschi (24) with minor modifications (25). Briefly, at least 40 testes from 16- and 20-day fetuses were carefully minced and incubated for 30 min at 32 C with gentle shaking in 5 ml Dulbecco’s PBS without calcium or magnesium (Life Technologies) containing 0.2 mg/ml collagenase, 0.05 mg/ml soybean trypsin inhibitor, 0.01 mg/ml DNase, and 40 µg/ml gentamicin. This enzymatic digestion was combined with mechanical disruption by repeated pipetting at 15 and 25 min. The cells were washed twice with 30 ml Dulbecco’s PBS and sedimented at unit gravity for 12 min. The sedimented material was digested with trypsin, plated as described for postnatal Sertoli cells, and used for dispersed fetal Sertoli cell cultures. The unsedimented fraction was centrifuged at 100 x g for 10 min, resuspended in DMEM-Ham’s F-12 (1:1), and used for dispersed fetal Leydig cell culture. Cell viability was assessed by trypan blue (0.04%) exclusion, and over 90% of the cells were viable. The dispersed fetal Leydig cells were plated in 96-well culture dishes at 1.3 x 105 to 1.8 x 105 cells/100 µl/well and cultured in a controlled humidified atmosphere of 95% air-5% CO2. The medium was supplemented with 0.4% FCS and 5 µg/ml insulin for the first day in culture (D0). For the following days (D1 to D3), the cells were cultured with or without AMH in medium containing 100 ng/ml LH to prevent the rapid drop in testosterone production that occurs in its absence. The media were changed every 24 h.

At the end of the culture, Leydig cells were identified by the cytochemical detection of 3ß-hydroxysteroid dehydrogenase (3ßHSD) activity and counted (Fig. 1CGo). Briefly, attached cells in their culture medium were frozen and stored at -80 C. Immediately before use, they were thawed and incubated for 4 h at 37 C with 0.25 mg/ml nitro blue tetrazolium, 0.28 mg/ml nicotinamide, 0.60 mg/ml NAD, and 0.05 mg/ml dehydroepiandrosterone. The dark blue positive cells (3ßHSD+) in each well were counted under an inverted microscope. In other wells, the cells were removed with trypsin-EDTA, and the total numbers of cells were counted in a hemocytometer. The ratio between the number of 3ßHSD-positive cells and total cells was about 1/100.

Testosterone RIA
The amounts of testosterone secreted in the medium were measured in duplicate after each culture period by RIA (26, 27) without prior extraction or chromatography, because 17ß-hydroxy-5{alpha}-androstane-3-one, which is the only steroid that cross-reacts significantly in the testosterone RIA (64%), is secreted in minute amounts by the fetal rat testis (28, 29).

Briefly, diluted culture media or testosterone standards (100 µl) were incubated at 4 C with 100 µl anti-testosterone antibody (a generous gift from Dr. Meusy-Dessolle) diluted 1:25,000. Then, 100 µl [3H]testosterone tracer were added, and the incubation was continued for 2 h at 4 C. Bound and free hormone fractions were separated with dextran-charcoal, and the bound hormone was counted in a scintillation solution. The minimum concentration of testosterone detectable in the medium was 70 pg/ml. The intra- and interassay variations in the testosterone RIA, determined as the ratio between the SDs and the mean values of 15 determinations of the same solution containing 1 ng/ml testosterone, were 3% and 10% respectively.

RT-PCR assays
Total RNA was extracted from Sertoli cell cultures using the RNA/DNA/protein isolation solvent (TRI Insta Pure, Eurogenetec, Belgium) and digested for 2 h at 37 C with 2 U/µg ribonuclease (RNase)-free DNase (Boehringer Mannheim, Mannheim, Germany). RNA (5 µg) were reverse transcribed at 42 C for 50 min with 200 U Superscript II (Life Technologies) in a final volume of 20 µl containing 7.5 µM random hexamers (Pharmacia, Uppsala, Sweden) and 20 U RNase inhibitor (Boehringer Mannheim). Negative controls were prepared by omitting reverse transcriptase.

An aliquot (1:10) of this reaction was amplified in a Perkin-Elmer apparatus (Norwalk, CT) in a final volume of 100 µl containing 0.2 mM of each deoxy-NTP (Ultrapure Solution, Pharmacia), 150 µM of each specific primer, 2 U Taq polymerase (Perkin-Elmer/Cetus), 50 mM KCl, 10 mM Tris-HCl (pH 9.3), 2.5 mM MgCl2, and 0.1 mg/ml gelatin (Sigma). One tenth of the PCR product was loaded on 2% agarose gels for electrophoresis, and the separated bands were visualized with ethidium bromide. Specific primer sequences (ARO1: 5'-CTGTCGTGGACTTGGTCATG-3'; ARO2: 5'-GGGGCCCAAAGCCAAATGGC-3') corresponding to the pig aromatase gene and partially covering exons 9 and 10 were used under the following PCR conditions: 94 C for 1 min, 58 C for 1 min, and 72 C for 1 min for 35 cycles. The amplification buffer contained 5% formamide. The amplified band was 196 bp long.

Expression of the ß-actin gene was used as a positive control to verify that complementary DNA samples contained equivalent amounts of material. PCR conditions were 94 C for 1 min, 55 C for 1 min, and 72 C for 1 min for 30 cycles. Primer sequences corresponding to exons 2 and 3 of the rat ß-actin gene were used to amplify a 421-bp specific band (RßAG: 5'-CCAACCGTGAAAAGATGACC-3'; RßA2: 5'-CGCTCA-TTGCCGATAGTGAT-3').

Aromatase assay
Aromatase activity in cultured Sertoli cells was assayed by the tritiated water (3H2O) technique (30) that was validated previously for estrogen (estradiol and estrone) production upon fetal ovine gonads and human placental microsomes (15, 17). The culture medium was removed, and the cells were incubated at 37 C for 5 h with 0.5 mM 1ß-[3H]androstenedione (27 Ci/mM; New England Nuclear-DuPont de Nemours, Les Ulis, France) in a controlled humidified atmosphere of 95% air-5% CO2. The tritiated steroid was then removed from the medium by chloroform extraction and dextran-charcoal adsorption. The remaining radioactivity, representing the tritiated water formed during aromatization, was measured in a scintillation counter, and the results are expressed as the amount of precursor metabolized per mg protein.

Protein assay
Tissue samples were solubilized by incubation in 0.4 M NaOH for 20 h at 37 C and neutralized with 0.4 M HCl, and proteins were measured by the Bradford technique using the Bio-Rad protein assay kit (Bio-Rad, Ivry, France).

Statistical analysis
All values are the mean ± SEM. The significance of the differences between mean values for treated and corresponding untreated controls was evaluated by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Antiaromatase effect of AMH in fetal and immature Sertoli cells
The basal aromatase activity of fetal Sertoli cells in vitro was very low. It increased slightly in cells from newborns but remained low (Fig. 2Go). This basal aromatase activity was increased 77–91% by (Bu)2cAMP. As early as fetal day 16 and until postnatal day 20, AMH significantly reduced by 49–69% the in vitro cAMP-stimulated aromatase activity.



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  		Figure 2. Ontogeny of the antiaromatase effect of AMH on cAMP-stimulated rat Sertoli cells in culture. Sertoli cells at different stages of development were cultured for 1 day in medium plus 10% female FCS. The culture medium was then replaced by the same medium containing IBMX (0.1 mM; control), or IBMX and (Bu)2cAMP (1 mM) without AMH (cAMP) or with AMH (35 nM; cAMP+AMH), and the culture was continued for 3 days. These conditions of treatment with AMH produced the maximal antiaromatase effect (Figs. 3Go and 4Go). The aromatase activity was assayed by the 3H2O technique; cells were incubated for 5 h with 1ß-[3H]androstenedione (0.5 mM; 27 Ci/mM). Values are the mean ± SEM of 3–14 determinations, as indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (statistical comparison between cAMP and cAMP plus AMH).

 
The characteristics of the effect of AMH on cAMP-stimulated aromatase activity were then studied with Sertoli cells from postnatal day 5 testes (5 dpp Sertoli cells). The effect of AMH was similar when female FCS was omitted from the culture medium (data not shown). The effect of AMH was dose dependent (Fig. 3Go). Inhibition was statistically significant from 7 nM, and the maximal effect occurred around 30 nM with an aromatase activity equal to 30% of the control value. The ED50 was 14 nM. The time course of the antiaromatase effect of AMH (Fig. 4Go) showed that aromatase activity of the cells was stimulated by incubation with cAMP for 24 h, whereas AMH gave statistically significant (P < 0.01) inhibition only after incubation for 2 days.



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  		Figure 3. Dose-response curve for the antiaromatase effect of AMH on cAMP-stimulated 5 dpp Sertoli cell cultures. Sertoli cells (5 dpp) were cultured with or without different concentrations of AMH, and the aromatase activity was assayed as described in Fig. 2Go. Results are expressed as the percent reduction in the aromatase activity of AMH-treated cells compared with that of the corresponding control cells. Values are the mean ± SEM of three determinations.

 


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  		Figure 4. Time course of the effect of cAMP and AMH on the aromatase activity of 5 dpp Sertoli cell cultures. Cells were cultured and treated, and the aromatase was assayed as described in Fig. 2Go, except that the aromatase was assayed after several periods in culture. Values are the mean ± SEM of 6–14 determinations, as indicated. **, P < 0.01; ***, P < 0.001 (statistical comparison between cAMP and cAMP plus AMH).

 
We confirmed that the target cells of AMH were the Sertoli cells by measuring the effect of AMH on the aromatase activity of 5 dpp Sertoli cells under different conditions (Fig. 5Go). Neither AMH nor LH, alone or together, modified the basal aromatase activity, suggesting that any contaminating Leydig cells did not account for the aromatase activity. In contrast, FSH stimulated the aromatase activity, but less effectively than cAMP. The FSH-stimulated aromatase activity was significantly reduced by AMH.



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  		Figure 5. In vitro effect of AMH on aromatase activity stimulated by different factors. Sertoli cells (5 dpp) were cultured for 1 day in medium containing 10% female FCS and then for 3 days with or without (Bu)2cAMP (1 mM), FSH (0.2 U/ml), or LH (100 ng/ml) with or without AMH (35 nM). Cell culture and the aromatase assay were performed as described in Fig. 2Go. Values are the mean ± SEM of three to nine determinations, as indicated. **, P < 0.01 (statistical comparison with the corresponding control values).

 
The effect of AMH on cAMP-stimulated aromatase activity was also investigated by RT-PCR analysis. The amount of aromatase mRNA was clearly increased by cAMP, and this effect was markedly reduced by AMH (Fig. 6Go).



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  		Figure 6. RT-PCR analysis of aromatase mRNA production in vitro in 5 dpp Sertoli cells. Sertoli cells were cultured for 2 days in medium containing (Bu)2cAMP with or without AMH as described in Fig. 2Go. At the end of the culture, Sertoli cell total RNA (5 µg) was reverse transcribed. An aliquot (1:10) of this reaction was amplified, and 1/10th of the PCR product was electrophoresed on 2% agarose gel and visualized with ethidium bromide and UV transillumination. Expression of the ß-actin gene amplified in the same RT-PCR conditions was used as a quantitative control of the amount of RNA amplified by RT-PCR.

 
Effect of AMH on the testosterone produced by fetal Leydig cells in culture
Dispersed Leydig cells from fetal day 20 testis were cultured in AMH (0.035–35 nM) for 3 days. Testosterone production was inhibited by AMH in a dose-dependent manner (Fig. 7Go). The maximum effect resulted in a production of 60% of the control value. The ED50 (i.e. the AMH concentration giving a testosterone production 80% of the control value) was 4.5 nM.



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  		Figure 7. Dose-response curve for the effect of AMH on the rate of testosterone secreted by dispersed fetal Leydig cells in vitro. Testicular cells from 20 day fetal testis were cultured for 1 day in medium containing 0.4% FCS and (5 µg/ml) insulin and then for 3 days (D1 to D3) in medium without serum or insulin but in the presence of (100 ng/ml) LH or LH plus AMH (35 nM). The medium was changed every day, and the testosterone secreted into the medium on D3 was radioimmunoassayed. Results are expressed as the percentage of testosterone secreted by the AMH-treated cells on D3 compared with that secreted by the corresponding control. The total amount of testosterone secreted by control fetal Leydig cells was 6.7 ± 0.6 ng/well (n = 6). Values are the mean ± SEM of six determinations.

 
The effect of AMH was monitored as a function of the age of the fetal Leydig cells and as a function of the exposure time (Fig. 8Go). In control culture of dispersed Leydig cells from fetal day 16 and 20 testes (16 and 20 dpc cells), LH-stimulated testosterone production increased throughout the culture, but the increase was more pronounced on day 3 for 16 dpc cells than for 20 dpc cells. AMH did not affect the LH-stimulated testosterone production during the first 24 h, but inhibited it after 48 and 72 h. AMH had the same inhibitory action on fetal cells of all ages, as the amounts of testosterone secreted after incubation for 3 days with AMH were 38.4 ± 3.2% and 47.5 ± 7.4% (n = 5) of the corresponding control values for 16 and 20 dpc cells, respectively. The difference between the two ages was not statistically significant.



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  		Figure 8. Time course of AMH action on testosterone secretion by dispersed fetal Leydig cells. Testicular cells from 16 and 20 day fetal testis (16 and 20 dpc cells) were cultured as described in Fig. 7Go with or without AMH (35 nM). The LH-stimulated testosterone secreted into the medium on each day of culture was radioimmunoassayed. The results are the mean ± SEM of 9–12 determinations. **, P < 0.01; ***, P < 0.001 (statistical comparison with the corresponding control values).

 
Testosterone secreted into the medium on day 3 was compared with the number of 3ßHSD-positive cells present in the same well to determine whether the inhibitory effect of AMH caused a drop in the steroidogenic activity of the fetal Leydig cells and/or reduced the number of Leydig cells. As for the number of total cultured testicular cells (data not shown), AMH did not alter the number of 3ßHSD-positive cells in either 16 and 20 dpc cell culture (Fig. 9AGo). Consequently, AMH reduced the steroidogenic activity of the fetal Leydig cells (Fig. 9BGo).



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  		Figure 9. Effect of AMH on dispersed Leydig cells in culture. A, Effect on the number of 3ßHSD-positive cells. B, Effect on LH-stimulated testosterone production by each 3ßHSD-positive cell. Sixteen and 20 dpc testicular cells were cultured as described in Fig. 7Go with or without AMH (35 nM). The medium was changed every day. The testosterone in the medium on D3 was radioimmunoassayed and expressed as nanograms per 3ßHSD-positive cells counted at the end of this day. The results are the mean ± SEM of n determinations, as indicated. ***, P < 0.001 (statistical comparison with the corresponding control values).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
AMH is produced as early as the beginning of Sertoli cell differentiation in male fetuses and continues to be synthesized throughout fetal life and after birth until puberty (5, 6, 8, 9, 10, 31). This has led to speculation that AMH plays a local role during gonad differentiation in addition to its function during Mullerian duct regression. We looked for this physiological role of AMH by examining its effect on Sertoli and Leydig cell function during the period of testicular AMH production.

We first analyzed the effect of human recombinant AMH on the aromatase activity of cultures of Sertoli cells from fetal day 16 to postnatal day 20 rat testes. The characteristic epithelial appearance of the Sertoli cells in vitro and their transferrin production indicated that these cultures contained less than 10% contaminating peritubular or interstitial cells. The fetal and immature rat Sertoli cells aromatized androstenedione as early as fetal day 16 until the oldest stage studied (postnatal day 20). Aromatase activity was very low during fetal life, in agreement with our previous data showing that the intratesticular estradiol concentration is minute (29). The basal aromatase activity of the Sertoli cells increased until postnatal day 12 and decreased thereafter. This agrees well with the change in the cellular distribution of aromatase, which shifts from Sertoli to Leydig cells around postnatal day 15. (32, 33). The aromatase activity of the Sertoli cells was stimulated by (Bu)2cAMP, and the cells became more responsive with age. This result extends previous findings showing that (Bu)2cAMP stimulates testicular aromatase activity as early as fetal day 18 (34).

The stimulating effect of cAMP in cells of all ages examined was greatly reduced (49–69%) by AMH. These data are the first evidence that AMH inhibits the aromatase activity of fetal and immature rat Sertoli cells. The effect of AMH was dose dependent, with an ED50 (14 nM) comparable to that observed in vitro for the rat fetal ovary and immature granulosa cells (16, 17). An identical delay (2 days) was required to block the aromatase activity of the cAMP-stimulated fetal and immature Sertoli cells and the LH-stimulated production of testosterone by fetal Leydig cells as for all the other previously described biological effects of AMH (for general review, see Ref.4).

AMH is secreted specifically by Sertoli cells (35, 36). AMH receptors, which are homologous to the type II receptor of the TGFß family, have been recently detected in the fetal and postnatal Sertoli cells (12, 13, 37). Thus, the inhibition of aromatase activity by AMH is probably an autocrine action.

AMH also reduced the Sertoli cell aromatase activity stimulated by FSH. The effect of FSH on the aromatase activity of 5 dpp rat Sertoli cells is in agreement with published data showing the presence of FSH receptors on these cells as early as day 15 or 16 of fetal life (38, 39) and with the stimulatory effect of FSH on testicular aromatase activity as early as fetal day 18 (34). In contrast to FSH, LH did not stimulate aromatase activity in these cells. This demonstrates that the aromatase activity effectively resulted from Sertoli cells and not from contaminating Leydig cells. The inhibition of cAMP-stimulated aromatase activity in 16 and 20 dpc Sertoli cells by human recombinant AMH seems in disagreement with the absence of the same effect reported previously with a 2-fold higher concentration of bovine native AMH (75 nM) on the cAMP-stimulated aromatase activity of rat testis explanted at the same fetal age (16). This could be because testes in organ cultures are insensitive to exogenous AMH, as the receptors at the surface of the Sertoli cells are saturated by a local high concentration of endogenous AMH, whereas in Sertoli cells in culture, AMH secretion is directly diluted in the culture medium. Furthermore, AMH production by Sertoli cells in culture falls very rapidly with time (40).

Lastly, the RT-PCR analysis of aromatase mRNA in 5 dpp Sertoli cells in culture showed that the antiaromatase effect of AMH resulted from a decrease in the amount of aromatase mRNA due to decreased gene transcription or to reduced mRNA stability. In the same way, Northern blot analysis showed that AMH also decreases aromatase mRNA in immature rat granulosa cells (17).

Other growth factors belonging to the TGFß superfamily also modulate the stimulation of aromatase activity in these cells by FSH. Like AMH, activin and TGFß decrease the FSH-stimulated aromatase activity (41, 42) in immature rat (activin) and porcine (TGFß) Sertoli cells, but they increase the stimulating effect of the gonadotropin on the granulosa cells (43). TGFß has been shown to decrease the concentration of cAMP in porcine Sertoli cells (42).

There is no clear evidence that aromatase and estrogens are involved in gonadal differentiation. Excess estrogens inhibit Mullerian duct regression in male fetuses (7, 44) and induce testicular feminization in many vertebrate species, including some eutherian mammals, such as the marsupial opossum (45, 46). AMH could play a local role in normal testicular development by negatively controlling the synthesis of aromatase and the production of estrogens. AMH also decreases the aromatase activity in cultures of fetal ovaries and causes a morphological masculinization of the gonads (15, 47). However, inherited or experimentally induced defects in AMH biosynthesis or its receptors do not have a major effect on testicular differentiation (21, 48). One possible explanation for this apparent discrepancy could be a compensatory action of other factors of the TGFß family.

The present study also demonstrated a dose-dependent inhibitory effect of AMH on the LH-stimulated testosterone production by dispersed fetal Leydig cells in vitro. Our culture system allowed the study of the fetal-type Leydig cells, which differ from adult-type Leydig cells in many morphological and physiological characteristics (49, 50, 51), as testicular cells were collected on fetal day 16 or 20 and cultured only for 4 days, whereas the first progenitors of adult-type Leydig cells do not appear in vivo before postnatal day 14 (52). Consequently, these results are the first evidence suggesting that AMH is involved in the control of fetal Leydig cell function.

This action is due to reduced steroidogenic activity and not to cell destruction, as the number of cultured 3ßHSD-positive cells was not affected by AMH. It has been shown that the number of adult Leydig cells was increased in mice in which the AMH system had been knocked out (21, 48), whereas it was decreased in transgenic mice with overexpression of AMH (53), but no data are available on the development of the fetal Leydig cell population in these animals.

The present study shows that the inhibition of Leydig cell function by AMH does not depend on the fetal stage and results from a drop in the steroidogenic activity of each 3ßHSD-positive cell. These results are in accordance with the observation that some male transgenic mice chronically overproducing human AMH show feminization of the external genitalia and have impaired Wolffian duct development (20). Similarly, adult transgenic males overproducing AMH have greatly reduced serum testosterone concentrations (22) resulting from the decreased expression of acute steroidogenic regulatory protein (StAR), P450-SCC, 3ßHSD, and P450–17{alpha} (54). The greatest drop is in the P450–17{alpha} mRNA in these animals. These data can be linked to our recent finding showing that TGFß strongly inhibits this enzymatic activity in fetal Leydig cells (25).

In situ hybridization and Northern blotting both showed AMH receptor type II transcripts in the fetal seminiferous tubules and in immature Sertoli cells, but they were not found in fetal or adult Leydig cells (12, 13, 14). However, a recent abstract by Racine et al. (53) reported the detection of AMH mRNA receptor type II in adult mouse Leydig cells using RT-PCR technology. This can be linked to our recent immunohistochemical study showing that both type I and type II TGFß receptors are present in the fetal Leydig cells (55). Thus, it is possible that AMH acts directly on both Sertoli cells and Leydig cells to control steroidogenic enzyme activities, perhaps by inhibiting the cAMP pathway.

In conclusion, AMH, which can masculinize fetal ovaries in vitro and in vivo (15, 20, 22, 47), also blocks the stimulatory effects of LH on androgen production by fetal Leydig cells and the stimulation of aromatization by FSH or cAMP in Sertoli cells as early as fetal day 16. These data suggest that AMH is involved in the autocrine and paracrine control of the steroidogenesis and its sexual orientation in the fetal gonads, like other factors of the TGFß family.


   Acknowledgments
 
We thank S. Perlman and F. Louis for technical assistance, and Dr. J. M. Saez for helpful advice and discussion.


   Footnotes
 
1 This work was supported by INSERM, INRA, and Université Paris 7. English was revised by Dr. Owen Parkes. Back

Received August 15, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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