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a Université Paris 7—INSERM-INRA U 418, Paris, France
b INSERM-INRA U 418, Hôpital Debrousse, Lyon, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and RARß were detected in the interstitial tissue from 14.5 dpc, with intense staining in the gonocytes from 20.5 dpc to 8 dpp. The nuclei of all cell types stained faintly for RAR
from 8 dpp. Immunoreactivity for RXR was intense in the gonocytes from 13.5 dpc and in the Leydig cells from 16.5 dpc, and persisted throughout the period studied. RXRß was always detected in the Leydig cells and during a short neonatal period in the gonocytes. RXR gave a faint reaction in the nuclei of all cell types from 20.5 dpc. Unexpectedly, immunostaining for all the receptors tested, except RAR and RXR, was detected in the cytoplasmic compartment of the cells of fetal and neonatal testes, while it was found in the nuclei in immature and adult testes. In cultures of dispersed testicular cells from 3 dpp pups, retinoic acid had a dose-dependent deleterious effect on the survival of the gonocytes and, to a lesser extent, of the somatic cells. These results suggest that retinoids act on the testicular development, especially on germ cells, via RARs and/or RXRs.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The action of RA is mediated by nuclear retinoid receptors, of which there are two classes: the RARs (retinoic acid receptors) and the RXRs (retinoid X receptors). Each is encoded by three genes (RAR, ß, ; and RXR, ß, ) that can produce different isoforms [21]. Both all-trans RA and 9-cis RA can bind to RARs, but only 9-cis RA binds to RXRs. RARs and RXRs can form homodimers or heterodimers in vitro and in vivo, and bind to appropriate RA response elements (RAREs) or retinoid X response element (RXREs) [22]. RXRs can also heterodimerize with and enhance the DNA binding efficiency of thyroid hormone receptors, the vitamin D3 receptor, and the peroxisome proliferator-activated receptor [23, 24].
Both vitamin A deficiency and excess are likely to cause embryonic malformations in several mammalian species [25, 26]. Mice bearing single and double RAR mutations suffer from defects belonging to the fetal VAD syndrome (heart and eye defects and cranio-facial, axial, and limb skeletal abnormalities) [27–29], and there is synergy between mutations in RXR and RAR for the generation of these abnormalities [30]. Lastly, RAR [27] and RXRß [31] null mutant mice are also sterile because of abnormal spermatogenesis; RAR [28] null mutant mice are also sterile because of a defect of the genital tract, while RARß null mutant mice are not [32].
The distribution patterns of RARs and RXRs have been studied in a number of tissues. The mRNAs for all three RARs and all three RXRs are present in the adult murine and rat testis, and mRNA for RAR and RAR have been detected in the postnatal rat testis from 5 dpp [31, 33–40]. Only one study has reported RAR protein in the adult rat testis [35] and another has reported RXRs in the adult murine testis [37]. There are no published reports on the RARs and RXRs in the testis before Day 5 pp, but cellular RA binding proteins were recently found in the fetal and neonatal testis [41], suggesting that retinoids are involved in the development of this organ. Another study examined the effect of RA on the development of the fetal and neonatal testis [42] and showed that the morphogenesis of the fetal testis in culture is altered by RA.
These data prompted us to use an immunohistochemical method to investigate the cellular distribution of the retinoid receptors, RARs and RXRs, during the fetal and neonatal development of the testis, from 13.5 dpc to 8 dpp. Immature and adult testes have allowed us to validate these results and to extend previously published data. On the other hand, a system of culture of dispersed cells from neonatal testes was developed to investigate the effect of RA on the survival of gonocytes and somatic cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Collagenase was obtained from Serva (Heidelberg, Germany) and deoxyribonuclease I (DNase) from Sigma (St. Louis, MO). The culture medium was Ham's F-12/Dulbecco's Modified Eagle's medium (1:1) (Gibco, Grand Island, NY) containing 40 µg/ml gentamicin (Gentalline; Schering-Plough, Levallois-Perret, France). All-trans RE and all-trans RA were purchased from Sigma (St. Louis, MO). Stock solutions (10 mM) of RE and RA were made in absolute ethanol and stored at -20°C in the dark for up to 1 wk.
Animals
Wistar-strain rats were housed and bred as previously described [43]. Briefly, females were caged with the males for the night, and the day following an overnight mating was counted as 0.5 dpc. Pregnant rats from gestational Days 13.5 to 20.5 were anesthetized between 1400 h and 1600 h by an i.p. injection of 4 mg/100 g sodium pentobarbital (Sanofi, Libourne, France), and the fetuses were rapidly removed from the uterus. The sex of the gonad is not yet morphologically recognizable on fetal Day 13.5. The sex of the fetuses was therefore determined with the sex chromatin test performed on the amniotic membrane [44]. Natural birth occurred between 21.5 dpc at 1400 h and 22.5 dpc at 1800 h. For a precise timing of the postnatal development, only pups born between 1900 h on 21.5 dpc and 0800 h on 22.5 dpc (i.e., 60% of the neonates) were kept. Each litter contained 8 pups. Neonates on 3, 5, or 8 dpp, immature rats on 17 dpp, and adult rats (90 dpp) were killed by cervical dislocation, and their testes were immediately removed.
Immunohistochemistry
Immunostaining was performed as previously described [43] with the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) on paraffin-embedded sections of testes previously fixed in Bouin's fluid. Deparaffinized sections were rehydrated, treated with 0.3% H2O2 in methanol to block endogenous peroxidases, and incubated with normal goat serum for the polyclonal antibodies (RAR, RARß, RXR, RXRß, and RXR) or with normal horse serum for the monoclonal antibody (RAR) to block nonspecific protein binding. The sections were subsequently incubated overnight with the primary antibody diluted in 1.5% goat serum or horse serum in a humidified chamber at 4°C. The distributions of the primary antibodies were revealed with a biotinylated goat anti-rabbit secondary antibody for the polyclonal antibodies, or with a biotinylated horse anti-mouse secondary antibody for the monoclonal antibody and an avidin-biotin-peroxidase complex. Peroxidase was visualized with 3,3'-diaminobenzidine tetrahydrochloride (DAB). Sections were rinsed in PBS between each step.
Primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were affinity-purified rabbit polyclonal antibodies raised to peptides from the carboxyl terminus of RAR (residues 443–462 of the human RAR1), RARß (residues 430–447 of the human RARß2), and RXRß (residues 483–502 of the human RXRß1), or the amino terminus of RXR (residues 2–21 of the human RXR) and RXR (residues 2–21 of the mouse RXR). Anti-RAR antibody was a monoclonal antibody raised to full-length human RAR (1–454). The anti-RAR and anti-RXR antibodies used were stated by Santa Cruz Biotechnology to be specific for each receptor and did not cross-react with other RARs or RXRs. The dilutions used were anti-RAR 1:2000 for fetal and neonatal testes and 1:200 for immature and adult testes, anti-RARß 1:400, anti-RAR 1:5, anti-RXR 1:300, anti-RXRß 1:200, and anti-RXR 1:200.
Controls included preadsorption of the polyclonal primary antibody with 0.1 to 1 µg/ml of the antigenic peptide and dilution of the monoclonal primary antibody. For each age, 3–4 tissue blocks were prepared, and 3–6 sections from each block were taken for immunostaining.
Culture of Dispersed Testicular Cells
Neonatal testicular cells were cultured essentially as previously described [8, 45]. The testes from 10 newborns on 3 dpp were decapsulated and incubated for 10 min at 37°C in 5 ml Dulbecco's PBS without calcium and magnesium (Gibco) containing 0.2 mg/ml collagenase and 0.01 mg/ml DNase, with gentle shaking. Enzymatic digestion was combined with mechanical disruption by repeated pipetting at 5 and 10 min. The suspension was then diluted with 30 ml Dulbecco's PBS and allowed to sediment under gravity for 12 min. The interstitial cells in the supernatant were discarded, and the pellet containing the undigested cord fragments was again digested for 20 min with collagenase/DNase combined with repeated pipetting at 7, 14, and 20 min. Thirty milliliters of Dulbecco's PBS was added, and the remaining undigested fragments were eliminated by settling for 5 min under gravity. The supernatant containing single cells was collected and centrifuged for 15 min at 100 x g, and the pellet was suspended in a defined volume of culture medium. The cells were counted in a hemocytometer, and the gonocytes (2–3%) were recognized by their large size and smooth shape. The somatic cells were mainly Sertoli cells. There were few peritubular myoid cells (less than 2%) as determined by their alkaline phosphatase activity [46], and few Leydig cells (less than 3%) as determined by their 3ß-hydroxy steroid dehydrogenase activity. The cells were plated in 96-multiwell culture dishes (0.32-cm2 area; Falcon, Grenoble, France) at 75 000 cells/100 µl per well and cultured at 37°C in a humidified atmosphere of 5% CO2 in air. They were allowed to adhere for the first 24 h (Day 0) in the culture medium without any supplementation. RA or RE was then added at the indicated concentration, and incubation continued for 3 days. At the end of Day 3, 50% of the medium was replaced by fresh medium, fresh factors were added, and the culture was continued until the end of Day 6.
Evaluation of Cell Number and Mitotic Index in Cell Culture
The changes in the numbers of the somatic and germ cells during culture were obtained by trypsinization (trypsin-EDTA; Gibco) and counting the cells in a hemocytometer. Toluidine blue was added (0.001%) to the cell suspension before counting to facilitate identification of the gonocytes. The somatic cells became blue with hints of purple, while the gonocytes were light blue without any purple. The gonocytes were also smooth and had one or two clearly visible nucleoli.
The mitotic index of the gonocytes was measured on dispersed testicular cells cultured in a Lab-Tek (Nunc, Roskilde, Denmark) chamber. The 5-bromo-2'-deoxyuridine (BrdU) incorporated into proliferating cells was detected by immunocytochemistry at the end of Day 3 [47]. Briefly, cultured cells were labeled by adding BrdU and 5-fluoro-2'-deoxyuridine (labeling reagent diluted 1:100 according to the manufacturer's instructions; Amersham, Bucks, UK) for 3 h. Cells were fixed in Bouin's fluid and incubated with 0.3% H2O2 in methanol for 30 min at 20°C and then with a mouse anti-BrdU monoclonal antibody (cell proliferation kit; Amersham) for 1 h at 20°C. The antibody bound to the nuclei was detected by a peroxidase-linked anti-mouse IgG and DAB, and the cells were counterstained by brief immersion in hematoxylin to identify the gonocytes. The slides were rinsed in PBS between each step. The mitotic index was evaluated as the percentage of gonocytes showing a BrdU-positive immunoreaction; at least 300 gonocytes were analyzed.
Statistics
All values are means ± SEM. The statistical significance of the difference between the mean values for the treated and untreated cultures was evaluated using Student's t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RAR (Figs. 1 and 2)
RAR immunoreactivity was detected in rare spots of the male gonadal anlage at 13.5 dpc, and staining in the mesenchyme surrounding the developing tubules was evident at 14.5 dpc. This cytoplasmic staining was more intense in cells morphologically resembling Leydig cells from 16.5 dpc, and it persisted during the whole fetal period. A few gonocytes contained immunoreactivity at 18.5 dpc, and all of them were stained at 20.5 dpc, but the intensity of the signal was not uniform; it was faint in some cells and dark in others. The gonocytes were very strongly stained around 3–5 dpp, but there were always a few unstained gonocytes. Staining was, unexpectedly, in the cytoplasm as in the Leydig cells. The seminiferous tubules had become elongated at 8 dpp because of the proliferation of Sertoli cells, and few germ cells still showed immunoreactivity. The immature testis (17 dpp) contained germ cells that had entered meiosis and were unstained or had very faint nuclear staining. The germ cells in the adult testis were immunoreactive, and the intensity of the staining decreased from spermatogonia to early round spermatids. The Sertoli cells were faintly stained or unstained.
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RARß (Figs. 1 and 3) Immunoreactivity for RARß was detectable in the interstitial tissue at 14.5 dpc, and staining intensified slightly in the Leydig cells during fetal and neonatal development. Faint staining persisted in the cytoplasm of the Leydig cells in the immature testis. Immunostaining was perinuclear in the adult testis. The cytoplasm of some gonocytes was also immunostained from 18.5 dpc, and its intensity increased at 3–5 dpp and disappeared at 8 dpp. Meiotic germ cells in the immature testis had immunostaining in the nuclei, which persisted in the adult in the spermatocytes and in the round and the elongating spermatids. Weak staining was also detected in the Sertoli cells of adults at some stages of the seminiferous epithelium (VII and XIV).
RAR (Figs. 1 and 4)
No RAR immunoreactivity was detected in the fetal testis. There was weak nuclear immunostaining in the Leydig cells at 3 dpp and faint nuclear staining in all cell types at 8 dpp. This staining persisted in the immature testis at 17 dpp. The nuclei of the Leydig cells in the adult testis were immunostained, as were the Sertoli cells and the germ cells until they became round spermatids.
RXR (Figs. 1 and 5)
Staining for RXR was detected in the gonocytes as early as 13.5 dpc. This cytoplasmic crescent-shaped staining intensified during fetal life and was very intense at 20.5 dpc. The signal remained intense until 3–5 dpp and extended to the whole cytoplasm. It became fainter at 8 dpp. There was faint staining in the nuclei of the meiotic germ cells of the immature testis of 17 dpp, which persisted in the adult testis until the round spermatid stage. Reactivity was also detectable at 14.5 dpc in some cells of the interstitial tissue, which were morphologically identified as Leydig cells from 16.5 dpc, and was very intense in the fetal, neonatal, and immature testis. This staining was in the cytoplasm but extended to the nucleus from 3 dpp. Leydig cells were still slightly immunoreactive in the adult testis. Sertoli cells first showed weak nuclear staining in the neonate at 8 dpp, and the staining persisted until the adult stage.
RXRß (Figs. 1 and 6) No RXRß immunoreactivity was detected in the fetal testis until 18–20 dpc, when weak staining appeared in the cytoplasm and nucleus of Leydig cells. This immunoreactivity persisted throughout neonatal life. Leydig cells were more intensely stained in the adult, and staining was perinuclear. Weak cytoplasmic staining also appeared in the germ cells at 18–20 dpc; it intensified slightly at 3–5 dpp and disappeared at 8 dpp. The germ cells entering meiosis had nuclear immunoreactivity at 17 dpp. The spermatogonia, spermatocytes, and elongated spermatids of the adult testis were faintly stained. The nuclei of Sertoli cells were stained in the adult testis of all stages of the seminiferous epithelium cycle.
RXR (Figs. 1 and 7)
Testes were not immunostained from 13.5 to 16.5 dpc. Weak staining appeared in the germ cells and Leydig cells at 18.5 dpc and was located in the nuclei at 20.5 dpc. The nuclei of Sertoli cells were also stained, and all cell types therefore had nuclear staining. This ubiquitous staining persisted during neonatal life (3 and 8 dpp), intensified in the immature testis (17 dpp), and persisted in the adult. The immunoreactivity was very intense in the spermatogonia and in the spermatocytes, was less pronounced in round spermatids and was absent from later stages of spermatogenesis.
Controls No immunostaining with polyclonal antibodies was observed when rabbit serum was used instead of primary antibody or after preabsorption of the anti-RAR or anti-RXR antibodies with the appropriate peptide. Staining for RARs and RXRs was therefore specific, including the cytoplasmic localization.
Effect of Retinoids on the Number of Somatic and Germ Cells in Culture (Fig. 8)
Cells were counted on Day 0, Day 3, and Day 6. There were significant decreases (P < 0.05) in the numbers of both somatic cells (from 47.0 ± 5.8 x 103 to 37.8 ± 5.9 x 103 cells/well; n = 7) and germ cells (from 647 ± 97 to 426 ± 80 cells/well; n = 7) from Day 0 to Day 3, but the numbers of both cell types significantly increased later (49.1 ± 8.2 x 103 somatic cells/well and 715 ± 167 germ cells/well on Day 6; n = 7; P < 0.05 compared to Day 3).
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Adding RA or RE to the culture medium decreased the number of germ cells after 3 days of culture. The effect of RA was dose-dependent and highly significant at 10-7 M. RE was less effective than RA. This inhibiting effect of retinoids was maintained or slightly increased on Day 6 in culture and reached more than 85% with 10-5 M RE and 10-6 M RA. Although RA reduced the number of gonocytes after 3 days of culture, it did not change the percentage of gonocytes with a positive immunohistochemical reaction for BrdU on this day (18.8 ± 2.0% in control vs. 22.4 ± 3.5% with 10-7 M RA; n = 4).
The number of somatic cells was not affected by any of the concentrations of RA or RE studied on Day 3, but it was reduced by 40–50% by 10-5 M RE and 10-6 M RA on Day 6. Lower concentrations of RA had no significant effect.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in the adult rat [35] and of RXR, RXRß, and RXR in the mouse testes [38] and provided new data on the distributions of RARs and RXRs in the adult rat testis. All three RAR proteins were detected in the adult testis. They were detected in the Sertoli cells, in accordance with the report of the presence of the three mRNA transcripts in these cells [38, 39]. RARß and RAR were also detected in the Leydig cells but RAR was not, in agreement with a previous report [35]. RAR protein gave the most intense signal in the somatic cells. All three RARs were detected in the germ cells throughout the prophase of meiosis, as reported for RAR [35]. RXRs were present generally at the same sites as described in the mouse testis [38] except that RXR was found in the Leydig cells and not in the mast cells. All three RXR proteins were present in the Leydig cells and Sertoli cells, and in the germ cells during the prophase of meiosis.
Most of the RARs and RXRs were detected in all cell types in the fetal and neonatal testis. However, the appearance or abundance of each receptor corresponded to a specific developmental pattern during spermatogenesis. RXR was detected in the germ cells from 13.5 dpc and RAR and RARß from 14.5 dpc in cells that were probably Leydig cell precursors. Other receptors were detected later, from 18–20 dpc for RXRß and RXR; but RAR was observed only in the neonate from 8 dpp. It has been postulated that RAR/RXR heterodimers are the major functional units responsible for transducing the retinoid signal during development [30]. However, our results do not allow us to speculate on the combination of heterodimers likely to transduce the RA signal in the fetal testicular cells in vivo. All six receptors were present more or less early in the Leydig cells and in the germ cells, while RXR was found at a low level in the fetal and neonatal Sertoli cells from 3 dpp and RAR and RXR from 8 dpp. These last three receptors were also the only receptors in the nuclei during fetal or neonatal life. Although immunohistochemistry does not provide quantitative estimates, RAR and RXR are probably the most abundant retinoid receptors in the fetal and neonatal testis, since they gave most intense staining, and RAR was revealed in the fetal testis with a very high dilution of antibody, which showed no signal in the adult.
The most striking observation was that the RAR and RXR proteins were frequently detected in the cytoplasm of the germ cells and in the nucleus and cytoplasm of the Leydig cells in the fetal and neonatal testis, except for RAR and RXR. In contrast, all six receptors were almost exclusively found in the nuclei of all cell types in the adult testis, and in the nuclei of the meiotic germ cells of immature rats. All the immunostaining was specific, since there were no reactions with the antisera preabsorbed with the cognate peptides. Nuclear receptors have been found in the cytoplasm in a number of cases: DAX-1 in interstitial cells of the fetal rat testis [48] and androgen receptors in elongated spermatids and residual bodies in the adult testis [49]. RAR protein was also found in the cytoplasm of cells of the initial segment of the epididymis in the rat [50], and nuclear and cytoplasmic immunostaining for RXRß was found in rat pituitary cells [51]. The change in the subcellular distribution of most of the RAR and RXR proteins from the cytoplasm to the nucleus occurred during the period from 8 to 17 dpp, when meiosis begins. It coincides with a peak in the level of the two mRNAs encoding RAR (2.7 and 3.4 kilobases [kb]), which have been located in the seminiferous tubules from 10 dpp [35], and of the mRNA encoding RAR (3.4 kb) [34, 35, 39]. On the other hand, the spermatogenic cells of the adult testis also contain larger transcripts for RAR (4.0 and 7.0 kb) and a smaller one for RAR (2.0 kb). The presence of these various mRNAs for RAR and RAR in Sertoli and spermatogenic cells suggests that isoforms of RAR and RAR are also present in these cells and that the processing of mRNA is unique to the germ cells [34]. Thus, it is likely that the production of the RARs and RXRs changes greatly during the initiation of meiosis. RAR has also been found to shift from the nucleus to the cytoplasm in the meiotic germ cells of the adult testis during the development of VAD conditions in the rat [36]. It has been suggested that this change is regulated by phosphorylations, an increase in the phosphorylation sites being in favour of a nuclear location [52]. The significance of the shift of RAR and RXR staining from the cytoplasm to the nucleus at the onset of meiosis is not clear. It could reflect alterations in the translation or in the maturation of the proteins, but further investigations are needed.
The second aim of this study was to determine whether the presence of the immunoreactive receptors in the fetal and neonatal testis is related to a biological effect despite their frequent cytoplasmic location. There are no published data available on the effect of RA in the gonocytes. We found that RA causes a dramatic dose-dependent reduction in the number of gonocytes in a dispersed testicular cell culture system. We believe that this is the first report of a negative effect of RA on the germ cell lineage. This effect was probably due to an increase in apoptosis, since the mitotic index was unchanged, and since retinoids are known to cause apoptosis in many other cell types or lines [53, 54]. This effect was unexpected, since vitamin A and RA are required to maintain the proliferation of spermatogonia in adults [20, 55], and since RA increases the number of primordial germ cells in culture [56]. Thus, RA seems to be able to increase both mitosis and apoptosis in germ cells, and the predominance of one or the other of these processes may depend on the developmental stage and on the experimental conditions.
The molecular mechanism of RA action on the gonocytes may involve all the receptors except RAR, which was not detectable. Further studies are therefore required to determine the relative importance of each receptor, perhaps using specific agonists or antagonists of the RARs and RXRs. There may be an indirect effect of RA on the gonocytes via the Sertoli cells in our culture system, but it would not result from a reduction in the number of Sertoli cells, which occurred only on Day 6, i.e., after the reduction in the number of gonocytes. Whether RA acts directly or via autocrine/paracrine factors also remains to be investigated. Transforming growth factor ß (TGFß) could be one of these factors, since we have recently observed that it enhances the apoptosis of gonocytes [4] and RA has been reported to stimulate TGFß secretion and activation [57–59]. The RA-induced dose-dependent decrease in the number of somatic cells, which are over 95% Sertoli cells in our culture system, suggests that the Sertoli cell is also a target for RA. This effect of RA was weak and delayed, and could be transduced by RXR, which was the only receptor detected in this cell type at 3 dpp. The effect of RA on Sertoli cell proliferation or survival may also depend on the developmental stage, since it is positive in the immature pig [60]. Whether retinoids can affect fetal Leydig cell function, as suggested by the presence of all the retinoid receptors in this cell type, is now under investigation. Lastly, the observation that retinol was less potent than RA but was also able to reduce the number of cells in our culture system suggests that the neonatal testicular cells have the same ability as the adult ones [18] to convert retinol into RA.
These results therefore suggest that retinoids exert specific effects via RARs and/or RXRs on all the testicular cell types during fetal and neonatal development, but especially on the gonocytes and the Leydig cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: C. Levacher, INSERM-INRA U 418/Université Paris 7, Case 7126, 2 Place Jussieu, 75251 Paris Cedex 05, France. FAX: 33 1 44 27 56 11; levacher{at}paris7.jussieu.fr
Accepted: August 10, 1999.
Received: April 30, 1999.
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