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Regular Article |
a Université Paris 7 and INSERM-INRA U 418, Tour 33/43, case 7126, 75251 Paris Cedex 05, France
ABSTRACT
We have previously shown that retinoic acid (RA) is able to act on the development of Leydig, Sertoli, and germ cells in the testis in culture (Livera et al., Biol Reprod 2000; 62:1303–1314). To identify which receptors mediate these effects, we have now added selective agonists and antagonists of retinoic acid receptors (RARs) or retinoid X receptors (RXRs) in the same organotypic culture system. The RAR
agonist mimicked most of the effects of RA on the cultured fetal or neonatal testis, whereas the RAR ß, 
, and pan RXR agonists did not. The RAR agonist decreased the testosterone production, the number of gonocytes, and the cAMP response to FSH of fetal testis explanted at 14.5 days postconception (dpc). The RAR agonist disorganized the cords of the 14.5-dpc cultured testis and increased the cord diameter in cultured 3-days-postpartum (dpp) testis in the same way as RA. All these RA effects could be reversed by an RAR antagonist and were unchanged by an RAR ß/ antagonist. The RAR ß agonist, however, increased Sertoli cell proliferation in the 3-dpp testis in the same way as RA, and this effect was blocked by an RAR ß antagonist. The RAR and the pan RXR agonists had no selective effect. These results suggest that all the effects of RA on development of the fetal and neonatal testis are mediated via RAR , except for its effect on Sertoli cell proliferation, which involves RAR ß.
developmental biology, Leydig cells, Sertoli cells, spermatogenesis, testosterone
INTRODUCTION
Retinoic acid (RA) and its precursor, vitamin A or retinol, are essential for the maintenance of normal reproductive functions in adults rats [1], and RA is an important regulator of the development of many organs [2, 3]. However, few data are available on the role of RA in development of the fetal and neonatal testis. Adding RA to 14.5-day-old fetal testis in culture affects the deposition of laminin and fibronectin in the basement membrane [4]. This finding was recently confirmed and extended by studies showing that RA inhibits formation of the seminiferous cords in cultured 13.5- and 14.5-days-postconception (dpc) testis [5, 6]. Using cultures of dispersed testicular cells from neonates, it was shown that RA decreases the number of gonocytes after 3 days [7] and inhibits the incorporation of tritiated thymidine after 24 h [6]. Recently, we used organotypic cultures to show that retinoids have multiple effects on the development and/or function of the three main cell types in fetal and neonatal testes [5]. Most of these effects changed with the developmental stage of the testis. All-trans RA reversed organization of the Sertoli cells into seminiferous cords only at the beginning of their formation (i.e., 14.5 dpc). Thereafter, RA increased the volume of the Sertoli cells without (18.5 dpc) or with (3-days-postpartum [dpp]) enhancing their mitotic activity. The cAMP produced in response to FSH was significantly lower in RA-treated fetal and neonatal testes than in controls. All-trans RA dramatically reduced the number of gonocytes per testis at 14.5 dpc (when gonocytes proliferate). Lastly, retinoids (all-trans RA and retinol) reduced both basal and acute LH-stimulated testosterone secretions by the Leydig cells in cultured 14.5-dpc testis (when Leydig cells differentiate and testosterone production per testis increases).
The retinoid receptors mediating these effects remain unknown. Two families of retinoid receptors have been identified: the RA receptors (RARs) that recognize both all-trans and 9-cis RA stereoisomers, and the retinoid X receptors (RXRs) that recognize only 9-cis RA. Each family contains three types of receptors: , ß, and . The RARs and RXRs can form heterodimers and homodimers in vitro and in vivo, and they can bind to appropriate RA response elements or retinoid X response elements [8].
Recent studies using reverse transcription-polymerase chain reaction and/or immunohistochemistry have demonstrated that RARs (, ß, and ) and RXRs (, ß, and ) are expressed in all cell types in the fetal and neonatal testis [6, 7, 9]. The appearance, the abundance, and/or the distribution of each receptor change according to a specific developmental pattern. This makes predicting which homodimer and heterodimer are involved in each RA effect very difficult.
Therefore, to identify which receptors mediate the RA effect on the development of fetal and neonatal testicular functions, we developed a pharmacological approach. Using various RAR-selective or RXR-selective agonists and antagonists, we have identified the agonists that can mimic and the antagonists that can block the RA effects on the cellular and functional development of Leydig, germ, and Sertoli cells in organotypic culture.
MATERIALS AND METHODS
Animals
Female Sprague-Dawley rats from Charles River (Saint Aubin les Elbeuf, France) were housed under a controlled photoperiod (lights-on 0600 to 2000 h) and fed a commercial diet (U.A.R., Villemoisson sur Orge, France) with tap water ad libitum. Males were caged with the females overnight. Because the estimated time of ovulation and fertilization was 0200 h, the day following an overnight mating was counted as 0.5 dpc. Pregnant rats were anesthetized by an i.p. injection of 4 mg/ml of sodium pentobarbital (Sanofi, Libourne, France) on 14.5 dpc, and the testes were removed aseptically from the fetuses under a binocular microscope and immediately explanted in vitro. Natural birth occurred between Day 21.5 at 1300 h and Day 22.5 at 1800 h. Because precise timing of postnatal development was desired, only pups born between 1800 h on 21.5 dpc and 0800 h on 22.5 dpc were kept. Fetal Day 22.5 was counted as Day 0 dpp for all newborns. The number of neonates in each litter was standardized at eight pups. Neonates were killed by cervical dislocation on 3 dpp, and their testes were immediately removed.
Chemicals and Solutions
The culture medium was Ham F12/Dulbecco modified Eagle medium (1:1 v:v; Gibco, Grand Island, NY) containing 0.35% glutamine (Flow Laboratories, Rockville, MD) and 80 µg/ml of gentamicin (Gentalline; Schering-Plough, Levallois-Perret, France). Ovine (o) LH (NIH.LH S19; 1.01 NIH.LH.S1 U/mg) was a gift from Dr. A.F. Parlow (NIDDK, Bethesda, MD). Recombinant hFSH (12 000 IU/mg) was a gift from Dr. B. Mannaerts (Organon International, Oss, The Netherlands). All-trans and 9-cis RA were purchased from Sigma (St. Louis, MO). Anti-vimentin monoclonal antibody was purchased from Dako (Trappes, France). Anti-cAMP antibody was generously provided by Dr. J.M. Saez (INSERM U 369, Lyon, France). The RAR (CD336, also named Am580), RAR ß (CD2314), RAR (CD666), and pan RXR (CD3640) agonists as well as RAR (CD2503, also named Ro 41-5253), RAR ß/ (CD2665), and pan RXR (CD3700) antagonists were generously provided by Dr. Uwe Reichert (Galderma R&D, Sophia Antipolis, France). The specificity of binding of these analogues was studied using nuclear extracts from COS-7 cells transfected with each of the RARs and RXRs ([10] for CD336, CD666, CD2503, and CD 2665; [11] for CD 2314; U. Reichert and S. Michel, personal communication, for CD3640 and CD3700). Each analogue binds to its specific receptor(s) with a Kd that is at least 20-fold lower than that obtained with the other receptors. The specificity of the biological effect of each analogue has been assessed using different cell types in culture by Galderma Laboratory and others on many occasions. As an example, using these analogues, it has been shown that the activation of RAR induces apoptosis, whereas the coactivation of RAR and RAR inhibits the RA-induced apoptosis in cultured thymocytes [10, 12], and that RAR ß activation is necessary for the retinoid growth inhibition of neuroblastoma cells in culture [11]. Lastly, transactivation experiments showed that CD3640 is a specific agonist for the RXRs, and that CD3700 antagonizes this activity (U. Reichert and S. Michel, personal communication).
Organ Cultures
Testes were cultured on Millipore (Bedford, MA) filters (pore size, 0.45 µm) as described elsewhere [13]. Briefly, intact, 14.5-dpc fetal testes were placed on filters. Testes from 3-dpp neonates were cut into 18 pieces, and the pieces were placed on a filter. The filter bearing the pieces of testis was floated on culture medium in tissue-culture dishes and incubated at 37°C in a humidified atmosphere containing 95% air/5% CO2 for 3 days. The culture was performed for 72 h with or without retinoids, and the medium was changed every 24 h. Synthetic analogues were diluted in dimethyl sulfoxide (DMSO), and control medium was added with DMSO alone. The antagonists were always added 30 min before the agonist or RA.
At the end of the culture period, 100 ng/ml of oLH, 200 mIU/ml of recombinant hFSH, or 1 mM 5-bromo-2'-deoxyuridine (BrdU; Amersham, Bucks, UK) was added to all media for the last 3 h of the culture. The explanted testis was fixed for 2 h at 4°C in Bouin fluid, embedded in paraffin, and then cut into 5-µm sections.
We have previously shown that this organotypic culture of 14.5 dpc in synthetic medium is a suitable model with which to study development of the germ, Sertoli, and Leydig cells, because the number and functions of these cell types considerably increase in control cultures [5, 13].
Identification and Counting of Gonocytes
All the serial sections from one 14.5-dpc cultured testis were mounted on slides, deparaffinized, rehydrated, and stained with hematoxylin-eosin. The gonocytes were identified by their large, spherical, lightly stained nuclei containing fine chromatin granules and two or more globular nucleoli and by a clearly visible cytoplasmic membrane [5]. All the gonocytes in three different sections (at one-third, one-half, and two-thirds through the gonad) of each testis were counted and the number divided by the corresponding area as measured by a computerized video densitometer (Biocom, les Ulis, France) to determine the average gonocyte density per unit surface area. This density was then multiplied by the cumulative areas of all the sections from the testis to obtain the total number of gonocytes in the whole testis (CC). The Abercrombie formula was used to correct for any double-counting due to the appearance of a single cell in two successive sections:
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/4 to correct for the over-representation of smaller profiles in sections through spherical particles. Here, DM was measured on each testis studied by at least 100 random determinations using a micrometer eye piece previously calibrated with a micrometer on the microscope plate. All countings and measurements were done blind. Measurement of BrdU Incorporation Index
Measurement of the BrdU incorporation index (i.e., % of cells showing a clear, positive immunoreaction to BrdU) was performed as described elsewhere [15]. Cultured 3-dpp testes were labeled with BrdU (diluted 1:100 according to the manufacturer's instructions) during the last 3 h of culture. Incorporation of BrdU into proliferating cells was detected by immunocytochemistry according to the manufacturer's recommendations. Briefly, randomly chosen sections were mounted and incubated with 0.3% H2O2 in methanol at 20°C for 30 min to inactivate endogenous peroxidases and then with a mouse anti-BrdU monoclonal antibody at 20°C for 1 h. The antibody bound to the nuclei was detected by a peroxidase-linked anti-mouse IgG. Finally, slides were stained with 3,3'-diaminobenzidine (DAB; Sigma). The BrdU incorporation index was obtained by a blind counting of all the gonocyte or Sertoli cell nuclei on the sections.
Vimentin Immunocytochemistry
Cords were visualized by immunocytochemical detection of vimentin with the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Deparaffinized sections were rehydrated and rinsed in PBS and endogenous peroxidases. Nonspecific protein binding was blocked by incubation in hydrogen peroxide followed by incubation in normal goat serum. The sections were then incubated overnight in an anti-vimentin antibody (1:50) in a humidified chamber at 4°C. The distribution of this primary antibody was revealed with a biotinylated goat anti-mouse secondary antibody and the avidin-biotin-peroxidase complex. Peroxidase was visualized with DAB. Sections were rinsed in PBS between each step. The specificity of vimentin staining was checked by replacing the antibody with nonimmune IgG.
Testosterone RIA
The testosterone secreted into the medium was measured daily in duplicate by RIA as described elsewhere [16]. No extraction or chromatography was performed, because 17ß-hydroxy-5-androstan-3-one, the only steroid that cross-reacts (64%) significantly with testosterone, is secreted in minute amounts by the fetal rat testis [16].
Briefly, diluted culture media or testosterone standards (100 µl) were incubated at 4°C with anti-testosterone antibody (a generous gift from Dr. Meusy-Dessolle, INRA, Jouy en Josas-78, France) diluted 1:25 000. Then, [3H]testosterone tracer was 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 of the testosterone RIA, calculated as the ratio between the standard deviations and the mean values of 15 determinations of the same solution containing 1 ng/ml of testosterone, were 3% and 10% respectively.
cAMP RIA
After 72 h in culture, the media were replaced by fresh medium containing 1 mM IBMX (3-isobutyl-1-methylxanthine) plus 200 mIU/ml of recombinant hFSH. Three hours later, media were collected, and their cAMP concentration were evaluated as described elsewhere [17].
Briefly, diluted culture media or cAMP standards (500 µl) were added with 10 µl of triethylamine and 5 µl of acetic anhydride. Acetylated medium and standards were incubated at 4°C overnight with anti-cAMP antibody (a generous gift from Dr. J.M. Saez) diluted 1:2000 and [125I]cAMP tracer. Bound and free hormone fractions were separated with propanol. The minimum concentration of cAMP detectable in the medium was 3.5 pg/ml. The intra- and interassay variations of the cAMP RIA were 7% and 9%, respectively.
Statistical Analysis
All values are presented as mean ± SEM. The significance of the differences between the mean values for the two testes from the same fetus were evaluated using the Student paired t-test. Other means were compared using one-way ANOVA (Fisher's test).
RESULTS
Effect of Retinoids on Fetal Testicular Testosterone Production
Basal testosterone secretion of testes from 14.5-day-old fetuses increased during the culture in control medium (Fig. 1) with a pattern that mimicked the one previously described in vivo [13]. Basal and LH-stimulated testosterone productions were dose-dependently decreased by all-trans or 9-cis RA (Fig. 1). To compare the efficiency of these isoforms, one testis was cultured with 3 x 10-8 M or 10-6 M all-trans RA, and the other testis from the same fetus was cultured with the same concentration of 9-cis RA. No difference was observed in the testosterone production of the paired testes (data not shown).
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The RAR , ß, , and pan RXR agonists, when used at the highest dose (3 x 10-6 M), all decreased both basal and acute LH-stimulated testosterone production (Fig. 2), indicating a lack of specificity of the agonists at this dose, as has been previously described in other cell types [18]. Only the RAR agonist strongly inhibited testosterone secretion at 10-8 M, whereas the RAR ß and pan RXR agonists had no effect and the RAR agonist had only a slight negative effect (P < 0.05, n = 4). The RAR agonist was the only agonist that changed testosterone production at 10-9 M. This agonist decreased testosterone secretion in a dose-dependent manner from 10-9 to 10-12 M (P < 0.001, ANOVA) and was effective at doses as low as 10-11 M. Because the dose-dependent range of RA bioactivity extended from 10-6 to 10-8 M (Fig. 1) [5], the RAR agonist was at least 1000-fold more potent than RA. This difference is probably due to a difference of affinity (the Kd of RA for RAR is 19.5 nM and that of the RAR agonist is 8 nM), but it may also result from a longer half-life of the agonist than of the RA. As RAR and RXR agonists may act in synergy [18–21], the effect of the pan RXR agonist (10-10 M) together with 10-10 M RAR agonist was compared with that of RAR agonist alone. No apparent difference was found in basal and acute LH-stimulated testosterone secretions between these two treatments (data not shown). However, surprisingly, addition of higher concentration of the pan RXR agonist (10-9 M) reduced the inhibitory effect of 10-6 M RA by 25% (P < 0.05) (data not shown).
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Lastly, the RAR antagonist (3 x 10-6 M) alone had no effect on either the basal or the LH-stimulated testosterone production, but it suppressed the inhibitory effect of the RAR agonist and of the all-trans RA (Fig. 3). The RAR antagonist also suppressed the slight negative effect of the RAR agonist, suggesting that this effect was not specific to RAR . Furthermore, the RAR ß/ antagonist did not change the negative effect of all-trans RA. Surprisingly, the pan RXR antagonist inhibited the effect of RA (Fig. 3).
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Effect of Retinoids on the Number of Gonocytes
All-trans RA (10-6 M) considerably reduced the number of gonocytes (Fig. 4). Of the agonists tested (10-10 M), only the RAR agonist mimicked the effect of RA. Higher concentrations of RAR agonist (10-9 M) caused an almost complete loss of all the gonocytes after 3 days in culture (4% of the control values). Lastly, the effect of all-trans RA was reversed by 3 x 10-6 M RAR antagonist.
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Effects of Retinoids on Morphogenesis of Fetal and Neonatal Testes
At the time of explantation and after culture for 3 days with or without RAR , ß, , and pan RXR agonists, vimentin was immunodetected to visualize the Sertoli cells. On 14.5 dpc, the vimentin immunostaining was detected in the basal part of the Sertoli cells and weakly in the interstitium, as described by others [22, 23]. After culture in control medium or RA treatment, the staining was still present in both Sertoli and Leydig cells (Figs. 5 and 6).
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The Sertoli cells in newly explanted, 14.5-dpc testes were almost all aggregated in cords (Fig. 5A). The seminiferous cords correctly completed their formation in control medium (Fig. 5B). However, those cultured in 10-9 M RAR agonist had altered organization, and most were disrupted (Fig. 5C), as were those cultured in 10-6 M all-trans RA [5]. This effect was also observed with 10-10 M RAR agonist in more restricted areas. The RAR ß and and RXR agonist had no effect (data not shown) even at 10-8 M. The RAR -selective antagonist (3 x 10-6 M) reversed the effect of RA in most parts of the testis (data not shown).
The seminiferous cords of explants of 3-dpp testes were not disrupted by the RAR agonist (Fig. 6). However, the RAR agonist mimicked the positive effect of all-trans RA on the diameter of the cords (Fig. 6) that we have previously described [5]. None of the other agonists tested modified the diameter of the cords (Table 1).
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Effect of Retinoids on Sertoli Cell Proliferation and Function
Because the in vitro effect of RA on Sertoli cell proliferation was previously observed with 3-dpp testis and not with 14.5-dpc testis [5], we used 3-dpp testes to identify the receptor pathway involved in this effect. Incubation of 3-dpp testes for 3 days with all-trans RA (10-7 M) increased the mitosis of Sertoli cells (36%) (Fig. 7). The RAR and and pan RXR agonists (10-9 M) had no effect on BrdU incorporation into Sertoli cells, but the RAR ß agonist mimicked the effect of RA (44% increase). Furthermore, RAR ß/ antagonist (3 x 10-6 M) totally suppressed the stimulatory effect of RA, whereas the RAR antagonist had no effect.
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The differentiated functions of the Sertoli cells in 3-dpp testes were assayed by measuring the cAMP produced in response to acute stimulation by FSH (3 h) after 3 days of culture in the presence or absence of retinoids (Fig. 8). The FSH-induced cAMP secretion was strikingly reduced by all-trans RA (10-6 M). This effect was reproduced by RAR agonist (10-9 M), whereas RAR ß, , and pan RXR agonists had no effect. Furthermore, the RAR antagonist totally suppressed the effect of all-trans RA, but the RAR ß/ antagonist did not. The RAR agonist also decreased the amount of cAMP secreted by 14.5-dpc testes in response to FSH after 3 days in culture (data not shown).
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DISCUSSION
We used RAR- and RXR-selective agonists and antagonists to investigate the nature of the receptors involved in the effects of RA on the differentiation and function of Leydig, germ, and Sertoli cells in an organotypic culture system. We observed that all-trans RA decreased both basal and LH-stimulated testosterone secretions of 14.5-dpc testis, confirming our previous results [5]. An RXR agonist did not perturb testosterone secretion, suggesting that RXR does not act as an homodimer in the control of testicular steroidogenesis. The RA effect probably involves an RAR -specific pathway, because it was reproduced exclusively by an RAR agonist and completely inhibited by an RAR antagonist. Furthermore, we report two findings suggesting that RXR and RAR do not act in synergy in our model, as they do in other cell types [18–21]. First, all-trans RA, which binds to RARs, was as efficient as 9-cis RA, which binds to both RARs and RXRs. Second, the inhibition of testosterone secretion by low concentrations of RAR agonist was unchanged by addition of low concentrations of RXR agonist. Surprisingly, we observed that high concentrations of RXR antagonist or agonist can reverse the effect of all-trans RA. Inhibition by RXR antagonist of the RAR/RXR heterodimer activation due to an RAR agonist has also been reported in HL 60 cells [24]. The explanation offered was that an active RAR/RXR heterodimer must be formed by an RAR bound to its ligand and an RXR unbound to its ligand. These heterodimers are active on one type of RA response element, the direct repeat 5 response element [8]. All things considered, we believe that RAR is responsible for decreasing testosterone secretion and may act by heterodimerization with RXR, which must remain unbound to RA in the active heterodimer RAR/RXR. This is in accordance with the facts that RAR mRNA was detected in the testis [6] and that RAR and RXR proteins were immunolocalized in the testicular interstitium on 14.5 dpc [7].
Gonocytes were the second testicular cell type affected by selective agonists and antagonists in our organ culture system. The RAR agonist decreased the number of gonocytes in cultured 14.5-dpc testis. This effect is correlated with our previous observations regarding all-trans RA [5], which acts by increasing apoptosis of these germ cells in the same model. Additionally, the same receptor is implicated in the RA-induced decrease in the number of germ cells in the fetal ovary [25]. In the adult, RAR also plays an important role in the control of spermatogenesis, because mice with invalidation of the RAR gene are sterile due to defective spermatogenesis [26]. Curiously, RAR was not found by immunodetection in fetal male gonocytes at 14.5 dpc [7]. This could be due to the limited sensitivity of the method used. Alternatively, RA may act indirectly on germ cells. However, because testosterone does not alter the number of gonocytes in 14.5-dpc testes (unpublished data), RA probably does not act by decreasing testosterone. Cupp et al. [6] have shown that RA increases the secretion of transforming growth factor (TGF)-ß in testicular cells in culture, whereas the TGF-ß1 and -ß2 reduce the number of fetal gonocytes in organ culture [15]. Thus, whereas RA may not act directly, it could act on the fetal gametogenesis via TGF-ß.
Sertoli cells were the third testicular cell type affected by selective agonists and antagonists in our organ culture system. It had been established that all-trans RA [4–6], 9-cis RA (unpublished data), and a specific RAR agonist [6] disrupted cords in cultured 13.5- or 14.5-dpc testis. We have now extended these data by showing that the RAR agonist had the same effect, and that the RAR antagonist blocked the RA-induced cord disorganization. In neonatal testes, the RAR agonist mimicked the positive effect, which we have previously described [5], of all-trans RA on the diameter of the cords. Thus, the effects of RA on the morphology of Sertoli cells involve an RAR pathway at both stages.
The RAR agonist inhibited the response of Sertoli cells to FSH in cultured fetal and neonatal testis, as did all-trans RA [5]. This effect was reversed by an RAR antagonist. The RAR was never detected by immunohistochemistry in the fetal Sertoli cells [6, 7], but the protein and its transcripts were detected in neonatal Sertoli cells by immunohistochemistry [9] and by in situ hybridization [27]. Therefore, either RA acts directly on neonatal Sertoli cells and indirectly on fetal Sertoli cells or, again, immunohistochemistry was not sensitive enough to detect the protein in fetal Sertoli cells. It is interesting to link our results with the fact that RA increases the expression of RAR and its activity in Sertoli cell lines, and with the fact that this effect is blocked by FSH [28]. Thus, FSH and RA may act reciprocally on their transduction pathway, with FSH inhibiting the response to RAR and RA inhibiting the response to FSH.
Interestingly, the RAR ß agonist increased the proliferation of neonatal Sertoli cells, as did RA, and this was also reversed by a selective antagonist. Therefore, the proliferative and morphogenic effects of RA use different pathways and can be dissociated. Once again, no [7, 9] or little [6] RAR ß was found in neonatal Sertoli cells by immunohistochemistry. However, this small amount may be sufficient to indicate that RAR ß is active in Sertoli cells. That RA uses two different pathways for acting on mitosis and on FSH responsiveness in Sertoli cells can explain why RA acts positively on mitosis and negatively on responsiveness to FSH, which is a mitogenic factor [29].
In conclusion, RA has negative effects on the formation of seminiferous cords, the Sertoli cell function, the germ cell number, and the Leydig cell function in the cultured fetal testis. These effects seem to be mediated by the same receptor, RAR , and some of them may be related to each other. In contrast, the positive effect of RA on Sertoli cell proliferation seems to be mediated by an RAR ß pathway. Lastly, the activation of RXR seems to have no role, thus excluding a possible role of RXR homodimer, but it may be necessary to permit the action of RAR . The physiological relevance of the involvement of RAR in development of the fetal testis is under current investigation in our laboratory using mice with a targeted disruption of the RAR gene.
ACKNOWLEDGMENTS
We thank Dr. U. Reichert, Dr. S. Michel (Galderma Sophia-Antipolis), and Dr. J.M. Saez (INSERM U 369) for fruitful discussions; M. Faro for technical assistance; C. Pairault for helpful technical advice; and J.J. Pairault for help in English revision.
FOOTNOTES
First decision: 12 September 2000.
1 Supported by INSERM, INRA, and Université Paris 7. G.L. is the recipient of a fellowship from the Ministère de l'Education Nationale de la Recherche et de la Technologie. 
2 Correspondence: René Habert, INSERM U 418–Université Paris 7, Tour 33/43, 2 Place Jussieu, 75251 Paris Cedex 05, France. FAX: 33 1 44 27 56 11; habert{at}paris7.jussieu.fr
Accepted: November 29, 2000.
Received: August 9, 2000.
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