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*TRANS-RETINOIC ACID
Endocrinology Vol. 139, No. 10 4269-4276
Copyright © 1998 by The Endocrine Society

ARTICLES

The Effect of 9-cis-Retinoic Acid on Proliferation and Differentiation of A Spermatogonia and Retinoid Receptor Gene Expression in the Vitamin A-Deficient Mouse Testis1

Ingrid C. Gaemers, Edwin Sonneveld, Ans M. M. van Pelt, Bianca H. G. J. Schrans, Axel P. N. Themmen, Paul T. van der Saag and Dirk G. de Rooij

Department of Cell Biology (I.C.G., A.M.M.v.P., B.H.G.J.S., D.G.d.R.), Medical School, Utrecht University, Utrecht, The Netherlands; Hubrecht Laboratory (E.S., P.T.v.d.S.), Netherlands Institute for Developmental Biology, Utrecht, The Netherlands; Department of Endocrinology and Reproduction, Erasmus University Rotterdam (A.P.N.T.), Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Dr. I. C. Gaemers, Netherlands Cancer Institute, Section Tumor Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. E-mail: gaemers{at}nki.nl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Retinoid X receptors (RXRs) are key regulators in retinoid signaling. Knowledge about the effects of 9-cis-retinoic acid (9-cis-RA), the natural ligand for the RXRs, may also provide insight in the functions of RXRs. In this study, the effect of 9-cis-RA on spermatogenesis in vitamin A-deficient (VAD) mice was examined. Administration of 9-cis-RA stimulated the differentiation and subsequent proliferation of the growth-arrested A spermatogonia in the testis of VAD mice. However, compared with all-trans-retinoic acid (ATRA), relatively higher doses of 9-cis-RA were necessary. This could not simply be due to a lower or delayed activity of 9-cis-RA, as simultaneous administration of ATRA and 9-cis-RA did not cause a synergistic effect. Instead, the presence of 9-cis-RA diminished the effect of ATRA by approximately one third.

Studies of in vivo transport and metabolism showed that ATRA and 9-cis-RA, after administration to VAD mice, penetrated the testis equally well. However, 9-cis-RA was metabolized much faster than ATRA, and other metabolites were formed. This may account for the above-described differential effects of ATRA and 9-cis-RA on spermatogenesis.

Similar to ATRA, 9-cis-RA transiently induced the messenger RNA expression of the nuclear RA receptor RARß, suggesting a role for this receptor in the effects of retinoids on the differentiation and proliferation of A spermatogonia. In contrast, the messenger RNA expression of the nuclear retinoid receptors RXR{alpha}, -ß, and -{gamma} was not changed significantly by administration of their ligand, 9-cis-RA. Hence, 9-cis-RA does not seem to exert its effect on spermatogenesis through altered expression of the RXRs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RETINOIDS play a role in numerous biological systems. In the testis, vitamin A deficiency (VAD) causes a disruption of the spermatogenic epithelium (1, 2), resulting in the disappearance of germ cells. The only remaining germ cells in the VAD mouse testis are the undifferentiated A spermatogonia (2), probably because of an arrest of these cells just before differentiation into A1 spermatogonia (3, 4). Spermatogenesis can be reinitiated by administration of retinoids, among which are retinol (2, 5, 6, 7), all-trans-retinoic acid (ATRA) (8, 9), and 4-oxo-retinoic acid (4-oxo-RA) (8), resulting in the formation of A1 spermatogonia and, subsequently, a synchronized epithelium (2, 6, 7, 9).

The effects of retinoids are mediated by nuclear retinoid receptors. To date, most studies have focused on the RA receptors (RARs) (10, 11, 12), which can bind the above-mentioned retinoids. In the mouse testis, the presence of RAR{alpha}, -ß, and -{gamma} has been shown (10, 11, 12). Retinoid status and short term administration of ATRA were shown to affect RARß expression in the mouse testis (11). The generation of RAR{alpha} or -{gamma} null mutant mice, which display male sterility among other defects, has indicated the involvement of these receptors in spermatogenesis (13, 14).

In recent years it has become clear that retinoid X receptors (RXRs) (15) play a key role in retinoid signaling, where they can act as either homodimers or heterodimeric partners to other nuclear receptors, including RARs (reviewed in Ref. 16). It was shown that the heterodimers, which can bind to response elements in the promoters of some genes, are the active elements in the RAR pathway. The RARß gene was the first gene found to contain a RA response elements (17, 18, 19). Binding of ligands to the receptors affects the formation of RAR/RXR heterodimers and RXR homodimers (16). Dependent on the 5'- or 3'-position of RXR in the heterodimer, the RAR-RXR complex can act as an activator or an inhibitor of transcription of retinoid-responsive genes (20). In the mouse testis RXR{alpha}, -ß, and -{gamma} are present (11, 21, 22). Although their expression does not seem to change upon ATRA administration (11), the importance of these receptors in spermatogenesis was established through RXRß null mutant mice, in which spermatogenesis was found to be abnormal (22).

9-cis-RA is a stereoisomer of ATRA that can be formed in vivo by isomerization of ATRA (23) or by oxidation of 9-cis-retinal (24). Although chemically related, a major difference between ATRA and 9-cis-RA is the fact that 9-cis-RA can bind to both RARs and RXRs (15), whereas ATRA can only bind to RARs. Therefore, the action of 9-cis-RA may be more versatile than that of ATRA. Other differences between ATRA and 9-cis-RA may originate at the level of transport and metabolism. ATRA metabolism has been studied extensively, and it may provide another level of regulation of retinoid action (25, 26, 27). ATRA can be metabolized to more polar metabolites, such as 4-hydroxy-RA and 4-oxo-RA. Originally considered to be inactive, 4-oxo-RA was recently proven to be at least as active as ATRA itself (11, 26). The enzymes involved in the oxidation of retinoids are probably cytochrome P450s, e.g. CYP26 (27, 28). Unfortunately, little is known about in vivo transport and metabolism of 9-cis-RA (29, 30, 31).

At present, the roles of RXRs and their natural ligand, 9-cis-RA, in spermatogenesis are not clear. In this study, we examined the effect of 9-cis-RA on the reinitiation of spermatogenesis in VAD mice. To validate the biological relevance of these results, in vivo transport and metabolism of 9-cis-RA (and ATRA) in mice were studied by HPLC analysis. Finally, the messenger RNA (mRNA) expression of RARß and the RXRs in the testis was studied after administration of 9-cis-RA to VAD mice to assess possible changes in the expression of these receptors upon 9-cis-RA exposure.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments
Breeding pairs of Nc/Cpb-U mice (Central Laboratory Animal Facility, Utrecht University, The Netherlands) were fed a vitamin A-deficient diet (Teklad Trucking, Madison, WI) for at least 4 weeks. The born male mice also received this diet until they became VAD. Animals were used at the age of 14–16 weeks, when their body weights started to decrease and they showed symptoms of deficiency (upstanding hair, squinting eyes).

ATRA was purchased from Sigma (St. Louis, MO). 9-cis-RA was purchased from Biomol (Plymouth Meeting, PA). The purity of the retinoids was confirmed by HPLC and proved to be more than 99%. Both retinoids were dissolved at 10-1 M in dimethylsulfoxide (DMSO) and stored in the dark at -20 C.

In Exp 1A, different amounts of 9-cis-RA or ATRA (in 16% DMSO-H2O, except for 1-mg quantities, which were dissolved in 32% DMSO-H2O) were administered by ip injection to VAD mice (four animals per group). In Exp 1B, VAD mice (four animals per group) received different amounts of 9-cis-RA and ATRA (0.25 mg of each in 16% DMSO-H2O or 0.5 mg of each in 32% DMSO-H2O). Control VAD animals received solvent alone. Animals were killed 24 h after retinoid administration by CO2. All animals in Exp 1A and 1B received an ip injection of 5-bromodeoxyuridine (BrdU; 100 mg/kg BW; Sigma) 2 h before they were killed.

In (independent) Exp 2A and 2B (extraction of retinoids and HPLC analyses), five groups of two VAD mice each were injected ip with, respectively, 0.25 mg 9-cis-RA (two groups), 0.25 mg ATRA (two groups), or solvent only (16% DMSO-H2O). The groups that had received retinoids were killed by CO2 either 1 or 2.5 h after injection. From all animals, serum, liver, and both testes were removed, snap-frozen in liquid nitrogen (with the exception of a small part of the testes for histological examination of the VAD status), and stored at -80 C.

VAD animals in Exp 3A each received an ip injection of 0.5 mg 9-cis-RA (in 16% DMSO-H2O). Control VAD animals received solvent alone. Control non-VAD mice were killed without any treatment. At several time intervals after retinoid administration, animals were killed by CO2 (two animals per time point, which were processed separately). In Exp 3B, three groups of three VAD mice each, were injected ip with, respectively, 0.5 mg 9-cis-RA, 0.5 mg ATRA, or solvent only (16% DMSO-H2O) and killed by CO2 6 h after injection. From all animals both testes were removed, snap-frozen in liquid nitrogen, and stored at -80 C for RNA isolation, with the exception of a small part for histological examination.

VAD animals remained on the VAD diet during all experiments.

Labeling index and immunohistochemistry
Testes of the animals in Exp 1 were fixed in Carnoy’s fixative and embedded in Technovit 7100 (Kulzer, Wehrheim, Germany) to determine the BrdU labeling index. Three-micron sections were cut and processed immunohistochemically using the BrdU-immunogold-silver staining method as previously described (32). The percentage of BrdU-labeled A spermatogonia was determined using epipolarization microscopy. At least 200 A spermatogonia were counted/testis.

Statistics
After testing for homoscedasticity, using Bartlett’s test for homogeneity of variances, variances proved to be homogeneous, and the Tuckey-Kramer method was used. Data are expressed as the mean ± SD.

Histology
To check the VAD status of the animals used, small parts of testes from animals in Exp 2 were fixed in Bouin’s fluid and embedded in glycol methacrylate Technovit 7100. Three-micron sections were cut and stained with periodic acid-Schiff and Gill’s hematoxylin no. 3. All sections were examined using light microscopy.

Extraction of retinoids and HPLC
Testes and livers were homogenized in 0.8 ml water with a Polytron (Janke and Kunkel, IKA-Labor Technik, Staufen, Germany); 0.4 ml serum was mixed with 0.4 ml water. Retinoids were extracted as described previously (33). In short, 3 ml methanol-dichloromethane (2:1) were added to 0.8 ml homogenized testis, liver, or serum, and the mixtures were vortexed for 1 min and filtered through a glass sinter. The residues were washed once with 3 ml methanol-dichloromethane (2:1) and once with 5 ml dichloromethane. Filtrates and washes were combined, and 1.75 ml 0.9% (wt/vol) NaCl was added. One microliter of [11,12-3H]RA (50 Ci/mmol; New England Nuclear Life Science Products, Boston, MA) was added to serve as an internal control for retinoid extraction efficiency. The mixtures were vortexed for 1 min, and upper layers were removed. Bottom layers were evaporated using a stream of nitrogen, and the dry residues were dissolved in 125 µl methanol-60 mM ammonium acetate (9:1), pH 5.75. Detection of the retinoids was performed using HPLC. Samples were transferred to a small glass tube and centrifuged for 15 sec at 1000 x g. The upper layers were injected into a reverse phase HPLC system containing a Spherisorb S50DS2 column (25 x 0.46 cm; Phase Separations, Norwalk, CT). Retinoids were separated using gradient elution with solvent A (60 mM ammonium acetate, pH 5.75) and solvent B (methanol). The gradient program with a flow rate of 1 ml/min was 5 min isocratic at 65% solvent B, followed by a convex gradient (no. 4, Waters Associates, Brussels, Belgium) to 85% solvent B in 15 min, a linear gradient to 99% solvent B in 10 min, and another 10 min isocratic at 99% solvent B. Retinoids were detected by measuring absorbance at 350 nm in a model UV flow spectrometer (Waters Associates). The radiolabeled internal standard was detected on-line with an L8506 radiochromatography monitor (Berthold, Bad Wildbad, Germany) equipped with a Z-1000 flow cell and a scintillant flow rate of 2 ml/min. Radioinert retinoids were run as standards to determine specific retinoid retention times: 4-oxo-RA, 12.87 min; 4-OH-RA, 13.79 min; 13-cis-RA, 23.90 min; 9-cis-RA, 25.03 min; ATRA, 25.82 min; all-trans-retinol, 32.55 min; and all-trans-retinal, 33.27 min. 4-Oxo-RA and 4-OH-RA were provided by Drs. M. C. Hsu and L. H. Foley (Hoffmann-La Roche Laboratories, Nutley, NJ). All other retinoids (except 9-cis-RA; Biomol) were obtained from Sigma. Single retinoid peaks were quantified by integration of the peak areas using the Borwin chromatography software program Winflow version 1.2.1 (Borwin Software, JMBS Developments, Le Fontanil, France).

RNA isolation
Total RNA was isolated using the LiCl-urea method (34). Briefly, frozen testes were lysed and homogenized in 3 M LiCl-6 M urea with a Polytron (Brinkmann Instruments, Westbuty, NY) and left on ice for several hours. The RNA was pelleted by ultracentrifugation (30,000 rpm, 25 min; TLA 100.3 rotor, Beckman, Palo Alto, CA). The pellet was dissolved in 0.1% SDS, and proteins were removed by repeated phenol extractions. After ethanol precipitation, the amount of RNA was determined by measuring the OD260 with a spectrophotometer and was dissolved at 5 µg/µl.

Ribonuclease (RNase) protection assay probes
Complementary [{alpha}-32P]UTP-labeled (Amersham, Arlington Heights, IL; 3000 Ci/mmol) RNA probes were made by transcription of pBluescript IISK- plasmids containing the following murine complementary DNA fragments: RARß, 116 bp (nucleotides 1226–1342); RXR{alpha}, 275 bp (nucleotides 1500–1775); RXRß, 176 bp (nucleotides 2007–2183); RXR{gamma}, 222 bp (nucleotides 1488–1710); glyceraldehyde-3-phosphate dehydrogenase (GAPD), 58 bp [nucleotides 346–404; numbering according to Zelent et al. (10) (RARß) and Leid et al. (35) (RXRs)]. Plasmid constructs were described by Jonk (36). Before transcription with T3 or T7 polymerase, the plasmids were linearized with restriction enzymes. RNA probes were purified by electrophoresis on a polyacrylamide-urea sequencing gel.

RNase protection assay
Equal amounts of total RNA each were hybridized overnight at 47 C with 5 x 104 cpm RAR or RXR probe and 1 x 104 cpm GAPD probe (labeled to a 3-fold lower specific activity) by adding this mix (5 µl volume) to 25 µl 80% formamide, 400 mM NaCl, 40 mM PIPES (pH 6.4), and 1 mM EDTA. As controls for the hybridization specificity, samples were included in which the total RNA was replaced by yeast transfer RNA. After hybridization, nonhybridized RNA was digested by adding 350 µl Tris-HCl (pH 7.5), 300 mM NaCl, 1 mM EDTA, and 1–2 mg/ml RNase T1 (1300 U/mg; Life Technologies, Gaithersburg, MD) for 60 min at 37 C. RNA was purified by proteinase K digestion, phenol/chloroform extraction, ethanol precipitation, and electrophoresis on 6% polyacrylamide-7 M urea-TBE gels (1 x TBE = 50 mM Tris, 50 mM boric acid, 1 mM EDTA). Gels were fixed, dried, and exposed to a phosphorscreen (Molecular Dynamics, Sunnyvale, CA), followed by exposure to x-ray film (Fuji). Quantification of the protected bands was performed using a Molecular Dynamics PhosphorImager with ImageQuant software. All data were normalized against GAPD values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of 9-cis-RA on the proliferation of A spermatogonia
To determine whether 9-cis-RA has an effect on the proliferation and differentiation of growth-arrested A spermatogonia in the VAD mouse testis, different amounts of 9-cis-RA were injected, and the BrdU labeling index of the A spermatogonia was determined. It was found that 9-cis-RA is indeed capable of stimulating the proliferation of A spermatogonia in the VAD mouse testis (Fig. 1AGo), although only at the two highest doses used (0.5 and 1 mg 9-cis-RA) was the labeling index of the A spermatogonia in the VAD testis significantly (P < 0.01) higher than the VAD value (17.0 ± 1.3% and 45.0 ± 3.2%, respectively; mean ± SD). At both lower doses of 9-cis-RA (0.13 and 0.25 mg), labeling indexes comparable to those obtained in nontreated VAD animals were observed (5.9 ± 1.0% and 7.7 ± 1.9%, respectively, vs. 5.4 ± 0.5% for VAD animals). At all concentrations tested, the percentage of BrdU-labeled A spermatogonia in 9-cis-RA-treated animals was significantly different from the corresponding ATRA values (P < 0.01). Compared with ATRA (Fig. 1AGo), the dose-response curve of 9-cis-RA has a similar slope, but the curve seems to be shifted to a higher concentration. In the ATRA curve a maximum occurs, but the occurrence of such a maximum in the 9-cis-RA curve could not be verified because doses of 9-cis-RA higher than 1 mg could not be tested due to the limited solubility of this retinoid in DMSO.



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  		Figure 1. A, Effects of administration of different doses of 9-cis-RA or ATRA on the proliferation of growth-arrested A spermatogonia in the VAD mouse testis (mean ± SD; n = 4). 1, Not significantly different from VAD. All other points are significantly (P < 0.01) different from VAD and each other. B, Effect of simultaneous administration of 9-cis-RA and ATRA on the proliferation of growth-arrested A spermatogonia in the VAD mouse testis (mean ± SD; n = 4). I, Mice received 0.25 mg ATRA, 0.25 mg 9-cis-RA, or 0.25 mg ATRA plus 0.25 mg 9-cis-RA; II, mice received 0.50 mg ATRA, 0.50 mg 9-cis-RA, or 0.50 mg ATRA plus 0.50 mg 9-cis-RA. *, Significantly different from VAD and single retinoid values (P < 0.01).

 
Surprisingly, when combinations of equal amounts of 9-cis-RA and ATRA were given to VAD mice, the effect was not cumulative. Instead, labeling indexes were approximately 30% less than those after administration of ATRA alone (Fig. 1BGo). The labeling index was 21.5 ± 3.0% with both 0.25 mg ATRA and 0.25 mg 9-cis-RA, whereas with 0.25 mg ATRA or 0.25 mg 9-cis-RA alone the labeling indexes were 32 ± 1.5% and 7.7 ± 1.9%, respectively (Fig. 1BGo). With 0.5-mg retinoid doses, these values were 34.6 ± 1.8%, 48.6 ± 1.8%, and 17.0 ± 1.3%, respectively (Fig. 1BGo). At all concentrations tested, the values found with the combination of retinoids were significantly different from the corresponding values found with only one retinoid (P < 0.01).

In vivo transport and metabolism of 9-cis-RA or ATRA in VAD mice
To determine whether 9-cis-RA or ATRA in fact reach target tissues (e.g. the testis) in VAD mice, the in vivo transport and metabolism of these retinoids were examined by HPLC analysis. The results of a retinoid analysis of serum, liver, and testes isolated from VAD mice that had received ATRA or 9-cis-RA for 1 or 2.5 h are shown in Table 1Go. Figure 2Go shows HPLC profiles of retinoids extracted from testes of these mice. As shown in Fig. 2BGo, VAD testes did not contain measurable levels of retinoids. Both ATRA and 9-cis-RA reached the mouse testis after ip injection (Table 1Go and Fig. 2Go). They did so equally well, as at short times after the administration of either ATRA or 9-cis-RA, comparable amounts of ATRA or 9-cis-RA, respectively, were found (results not shown). However, from Table 1Go it is clear that the amount of 9-cis-RA had greatly decreased within 1–2.5 h after injection, whereas the amount of ATRA was still high. This was reflected both quantitatively and qualitatively in the metabolites found; the metabolites of ATRA were small amounts of 9-cis-RA and 13-cis-RA, whereas more polar metabolites were found after 9-cis-RA injection. In the liver, 9-cis-RA was also rapidly metabolized, but, surprisingly, ATRA metabolism also had started within 2.5 h after injection. In serum and liver extracts of VAD mice, no retinoids were detectable (comparable to the data found with VAD testes; results not shown).


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  		Table 1. Analysis of retinoid metabolites in VAD mice treated with ATRA or 9-cis-RA

 


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  		Figure 2. HPLC analysis of retinoid metabolism in testes of VAD mice treated with ATRA or 9-cis-RA. A, Retinoid standards (1–7; see below); B, VAD mice killed untreated; C, VAD mice killed 1 h after ip injection of 0.25 mg ATRA; D, VAD mice killed 2.5 h after ip injection of 0.25 mg ATRA; E, VAD mice killed 1 h after ip injection of 0.25 mg 9-cis-RA; F, VAD mice killed 2.5 h after ip injection of 0.25 mg 9-cis-RA. 1, 4-Oxo-RA; 2, 4-hydroxy-RA; 3, 13-cis-RA; 4, 9-cis-RA; 5, ATRA; 6, all-trans-retinol; 7, all-trans-retinal; 8, unknown, probably 9-cis-4-oxo-RA.

 
RARß gene expression in VAD mouse testis after 9-cis-RA administration
Previously we have shown (11), that the effect of ATRA on the proliferation of A spermatogonia in the VAD mouse testis correlates with an increased mRNA expression of the RARß gene. Besides being the ligand for RXRs, 9-cis-RA also binds to RARs. Therefore, we determined the effect of 9-cis-RA on the mRNA expression of RARß. Very low expression of RARß was detected in VAD mouse testes (Fig. 3AGo), but RARß mRNA was rapidly induced by 9-cis-RA administration. RARß mRNA expression was induced maximally about 4- to 5-fold compared with VAD values 6 h after 9-cis-RA injection (Fig. 3BGo). This was identical to the effect of ATRA on RARß expression (Fig. 3CGo). Twenty-four hours after 9-cis-RA administration, expression had returned to levels close to those in nontreated VAD mice (Fig. 3Go, A and B).



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  		Figure 3. A, RARß mRNA expression in testes of 9-cis-RA-treated VAD mice. RNase protection assay was performed with 15 µg total testicular RNA, using RARß and GAPD probes. The positions of RARß- and GAPD-specific protected fragments are indicated. *, Nonspecific signal. Numbers indicate hours after the administration of 9-cis-RA to VAD mice. Each lane represents one animal. T, Hybridization of RARß and GAPD probes with 15 µg transfer RNA, followed by digestion with RNase T1. Signals in this lane represent nonspecific signals. N, Testis of a normal mouse (control). B, Quantitative analysis of the RARß mRNA expression shown in Fig. 2AGo (mean ± range; n = 2). The transfer RNA lane was used for background values. Data were normalized against GAPD values. Values of VAD mouse testes were set at 1.0. C, RARß mRNA expression in testes of VAD mice that had received no retinoids, ATRA, or 9-cis-RA for 6 h. The RNase protection assay was performed with 15 µg total testicular RNA, using RARß and GAPD probes. The positions of RARß- and GAPD-specific protected fragments are indicated. *, Nonspecific signal. Each lane represents one animal.

 
RXR gene expression in VAD mouse testis after 9-cis-RA administration
Because 9-cis-RA is the only known ligand for RXRs, we investigated whether RXR mRNA expression in the VAD mouse testis is influenced by the administration of 9-cis-RA. Using RNase protection assays, mRNA expression of RXR{alpha} and -ß was shown to occur at high and equivalent levels. A representative autoradiograph is shown in Fig. 4Go. Within 24 h after the injection of 9-cis-RA, no change in their mRNA expressions could be observed.



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  		Figure 4. Testicular mRNA expression of RXR{alpha} after the administration of 0.5 mg 9-cis-RA to VAD mice. Total testicular RNA (7.5 µg) was hybridized to RXR{alpha} and GAPD probes and digested with RNase T1 (2600 U/ml). The positions of RXR{alpha}- and GAPD-specific protected fragments are indicated. Numbers indicate hours after the administration of 9-cis-RA to VAD mice. Each lane represents one animal. T, 7.5 µg transfer RNA, control lane; N, testis of normal mouse (controls). The absence of the RXR{alpha} signal in lane 13 is probably caused by irregularities in the gel lane. In other experiments, a different expression of RXR{alpha} at 24 h was never observed. B, Testicular mRNA expression of RXRß after the administration of 0.5 mg 9-cis-RA to VAD mice. Total testicular RNA (7.5 µg) was hybridized to RXRß and GAPD probes and digested with RNase T1 (1300 U/ml). The positions of RXRß- and GAPD-specific protected fragments are indicated. Numbers indicate hours after the administration of 9-cis-RA to VAD mice. Each lane represents one animal. N, Testis of a normal mouse; T, 7.5 µg transfer RNA (control lanes).

 
RXR{gamma} could always be detected in VAD mouse testes before and after the administration of 9-cis-RA, but similar to the results obtained with ATRA (11), this receptor was expressed at an extremely low level (results not shown). Even with large amounts of RNA (25 µg) and long exposure times, the signal was difficult to detect in RNase protection assays, and quantification of the signal proved very difficult. For this reason we were not able to assess whether RXR{gamma} mRNA expression is regulated by 9-cis-RA, but based on these results, a substantial increase in the mRNA expression of this gene by 9-cis-RA can be excluded (results not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Retinoids have been proven to be crucial for an accurate spermatogenesis (1, 2). Nuclear retinoid receptors are the mediators of the effects of retinoids (10, 11, 12), and among these receptors, RXRs play a key regulatory role (15, 16). In the present study, it was shown for the first time that administration of 9-cis-RA, the only known ligand of the RXRs, has pronounced biological effects on spermatogenesis in the mouse. 9-cis-RA is able to induce differentiation and proliferation of growth-arrested A spermatogonia in the VAD mouse testis. However, the dose-response curve for the percentage of A spermatogonia in S phase, 24 h after 9-cis-RA administration, is shifted to higher concentrations compared with that of ATRA. This indicates that 9-cis-RA is less efficient than ATRA. Interestingly, when combinations of equal amounts of ATRA and 9-cis-RA are administered to VAD mice, the effect of the retinoids is not cumulative, ruling out the possibility that the effect of 9-cis-RA, compared with that of ATRA, is simply less or delayed. Instead, the labeling indexes found are lower than those seen after the administration of ATRA alone. Several phenomena may account for these observations. First, the combined amounts of ATRA and 9-cis-RA may mimic the effect of a higher dose of a single retinoid, in which case the optimum percentage of BrdU-labeled cells is shifted to an earlier time point, with concomitant lower labeling indexes at a later time point (8). However, the labeling index found with the combination of 0.25 mg ATRA and 0.25 mg 9-cis-RA is less than that found with 0.5 mg ATRA alone. This indicates that the lower labeling indexes found with the retinoid combinations are not caused by a shift of the maximal BrdU labeling to an earlier time point.

Secondly, 9-cis-RA may inhibit the effect of ATRA on the differentiation and proliferation of A spermatogonia in the VAD testis through binding and activation of RXRs. Several RXR hetero- or homodimers may be formed dependent on the amount of 9-cis-RA and RXR present, all of which may interfere with the effects of RAR-RXR dimers on retinoid target genes (11, 16, 35, 37, 38, 39). To our knowledge, in vivo experiments in which 9-cis-RA and ATRA were administered simultaneously have not been reported previously, but some in vitro experiments have been described. Similar to our results for cell proliferation, 9-cis-RA can diminish the effect of ATRA in F9 cells, in this case on the mRNA expression of RXRs (40) and the activin type II receptor (41). In contrast, the administration of various combinations of synthetic retinoids specific for RARs or RXRs appears to lead to synergistic activation of RA-responsive genes, among them RARß, in P19 cells (42). It is not clear what causes the differences between in vivo and in vitro experiments or the apparent cell-specific differences in in vitro experiments.

Thirdly, differences between ATRA and 9-cis-RA may originate at the level of transport and metabolism. In this study it is shown that ATRA and 9-cis-RA, after administration to VAD mice, can penetrate the testis equally well. Therefore, transport can be excluded to be responsible for the observed differences between ATRA and 9-cis-RA. In contrast, we have shown that the in vivo metabolism of ATRA and that of 9-cis-RA differ considerably. 9-cis-RA is metabolized much faster than ATRA and other metabolites are formed. This suggests that different, retinoid-specific, enzymes are involved in ATRA and 9-cis-RA metabolism. In addition, tissue-specific differences also occur, as ATRA metabolism in the liver occurs faster than that in the testis. The fast metabolism of 9-cis-RA may be caused by a constitutively active enzyme (e.g. involved in ß-oxidative chain shortening) (30) or by a fast 9-cis-RA-specific cytochrome P450 (30). Cytochromes P450s are involved in the oxidation of retinoids to more polar metabolites. Recently, a P450 (CYP26) has been identified that is highly specific for the hydroxylation of ATRA, but that does not recognize 9-cis-RA (27, 28, 43). In the liver, CYP26 is induced 2.5 h after ATRA administration (43), which is in agreement with our results. CYP26 is also induced by 9-cis-RA, even though it is not involved in the metabolism of 9-cis-RA (28). This implies that simultaneous administration of 9-cis-RA with ATRA may result in lower amounts of active ATRA. We, then, would also observe an inhibiting effect of 9-cis-RA on the reinitiation of spermatogenesis by ATRA. Additional experiments are necessary to discriminate between the two remaining possibilities [binding and activation of RXRs by (metabolites of) 9-cis-RA or altered metabolism] that may account for the effect of 9-cis-RA on spermatogenesis.

Previously we have shown that reinitiation of spermatogenesis in the VAD mouse testis by ATRA correlates with an increased mRNA expression of RARß (11). To our surprise, 9-cis-RA proved to be equally efficient in inducing RARß transcription, even though the effect of 9-cis-RA on the reinitiation of spermatogenesis is substantially less than that of ATRA. On the basis of the two options presented above, this would suggest that 1) (metabolites of) 9-cis-RA may stimulate spermatogenesis through trans-activation of a certain set of retinoid-responsive genes (e.g. RARß) when bound to RAR-RXR while at the same time reinitiation of spermatogenesis is inhibited, possibly through other (RXR-liganded) RXR dimers; or 2) metabolites of 9-cis-RA are capable of inducing liganded RARß expression while being unable or less able to reinitiate spermatogenesis. The latter implies that RARß is not directly linked to reinitiation of spermatogenesis, which is in agreement with the fact that RARß knockout mice are fertile (44). Alternatively, RARß may not be available (or may be partly available) for reinitiation of spermatogenesis. Other genes (e.g. CYP26) may be induced by RARß (28), thereby competing for the binding of RARß. Similar to our results, ATRA and 9-cis-RA can equally well restore RARß expression in the tibia of VAD rats (45). In vitro, however, 9-cis-RA seems to be more efficient than ATRA in inducing RARß gene expression in S91 melanoma cells (46) and P19 cells (42), whereas the reverse is true in F9 cells (40, 42).

For the first time, the effect of 9-cis-RA on the gene expression of the RXRs in the (mouse) testis has been examined. Similarly to ATRA (11), the administration of 9-cis-RA does not affect mRNA expression of the RXRs in the testis. Therefore, 9-cis-RA does not seem to exert its effect on spermatogenesis through an altered gene expression of RXRs. Binding of 9-cis-RA to the RXRs, however, may still be responsible for the negative effect of 9-cis-RA on the reinitiation of spermatogenesis (see above). Little is known about a possible influence of 9-cis-RA on the expression of the RXRs. Similar to our in vivo results, the expression of RXR{alpha} and/or RXRß seems unchanged after the administration of ATRA or 9-cis-RA to human neuroblastoma, breast cancer, and Ntera2 cells and rabbit HIG82 cells (47, 48, 49, 50, 51). Altered expression of RXR{alpha} and/or -{gamma} mRNA has been found upon 9-cis-RA administration to murine F9 cells (40) or human HL60 cells (51).

In summary, 9-cis-RA seems able to influence the differentiation of A spermatogonia, both in a positive way, possibly via RARß induction, and in a negative way, which probably involves liganded RXRs or altered retinoid metabolism. Schrader et al. (52) reported on the existence of RXR-dependent and RXR-independent pathways of ATRA action in mammalian cells, where the presence or absence of 9-cis-RA determines by which pathway a response to ATRA is mediated. It may be that in the testis such a balance between ATRA and 9-cis-RA also determines the response. Finally, it is clear that several different retinoids could play roles in spermatogenesis. In a previous study we have shown that 4-oxo-RA, an irreversible metabolite of ATRA, is also able to reinitiate spermatogenesis (8), even more efficiently than ATRA itself. In view of the present results, it is tempting to suggest that 4-oxo-RA is more efficient because it cannot be converted to 9-cis-RA and therefore will not activate RXRs (53). On the other hand, ATRA does not need to be metabolized to 4-oxo-RA to exert its action, as coadministration of liarozole, a retinoid metabolism inhibitor, does not inhibit ATRA action in the VAD testis (11). Clearly, we are faced with a complex system, comprising different cell types, positive and negative retinoid action, and many different receptors, including orphan receptors. However, through the administration of different (receptor-specific) retinoids to VAD mice, a more detailed idea may be obtained of which retinoid signaling pathways are playing a decisive role in spermatogenesis.


   Acknowledgments
 
The authors thank Mrs. H. Roepers and H. J. G. Van de Kant for technical assistance, and T. van Rijn, R. Scriwanek, and M. Niekerk for photography.


   Footnotes
 
1 This work was supported by the Netherlands Organization for Scientific Research through GB-MW Grant 900-544-101 (to A.M.M.v.P.). Back

Received February 27, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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