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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.
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Abstract |
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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.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1A
. 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. 1B). 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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At the end of the culture, Leydig cells were identified by the
cytochemical detection of 3ß-hydroxysteroid dehydrogenase (3ßHSD)
activity and counted (Fig. 1C
). 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
-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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). 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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).
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. 4) 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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). 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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).
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). 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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). 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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). Consequently, AMH reduced the
steroidogenic activity of the fetal Leydig cells (Fig. 9B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 (54). The greatest drop is in the
P450–17 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.
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Acknowledgments |
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Footnotes |
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Received August 15, 1997.
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J. Teixeira, E. Fynn-Thompson, A. H. Payne, and P. K. Donahoe Mullerian-Inhibiting Substance Regulates Androgen Synthesis at the Transcriptional Level Endocrinology, October 1, 1999; 140(10): 4732 - 4738. [Abstract] [Full Text]
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M. M. Lee, C. C. Seah, P. T. Masiakos, C. M. Sottas, F. I. Preffer, P. K. Donahoe, D. T. MacLaughlin, and M. P. Hardy Mullerian-Inhibiting Substance Type II Receptor Expression and Function in Purified Rat Leydig Cells Endocrinology, June 1, 1999; 140(6): 2819 - 2827. [Abstract] [Full Text]
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Y. Mishina, D. J. Whitworth, C. Racine, and R. R. Behringer High Specificity of Mullerian-Inhibiting Substance Signaling in Vivo Endocrinology, May 1, 1999; 140(5): 2084 - 2088. [Abstract] [Full Text]
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N. K. Arambepola, D. Bunick, and P. S. Cooke Thyroid Hormone and Follicle-Stimulating Hormone Regulate Mullerian-Inhibiting Substance Messenger Ribonucleic Acid Expression in Cultured Neonatal Rat Sertoli Cells Endocrinology, November 1, 1998; 139(11): 4489 - 4495. [Abstract] [Full Text] [PDF]
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 L. Gouedard, Y.-G. Chen, L. Thevenet, C. Racine, S. Borie, I. Lamarre, N. Josso, J. Massague, and N. di Clemente Engagement of Bone Morphogenetic Protein Type IB Receptor and Smad1 Signaling by Anti-Mullerian Hormone and Its Type II Receptor J. Biol. Chem., September 1, 2000; 275(36): 27973 - 27978. [Abstract] [Full Text] [PDF]
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A. M. Trbovich, P. M. Sluss, V. M. Laurich, F. H. O'Neill, D. T. MacLaughlin, P. K. Donahoe, and J. Teixeira Mullerian Inhibiting Substance lowers testosterone in luteinizing hormone-stimulated rodents PNAS, March 13, 2001; 98(6): 3393 - 3397. [Abstract] [Full Text] [PDF]
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