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a Department of Genetics and Cell Biology, Department of Biochemistry and Biophysics, Center for Reproductive Biology, Washington State University, Pullman, Washington 99164
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, ß, and 
. These receptors are postulated to dimerize and regulate the transcription of retinoid-responsive genes that are crucial for germ cell development. In this study, we determined the cellular and subcellular localization of six retinoid receptors in the developing rat testis to identify the specific cellular sites and times of receptor expression. Immunohistochemical results revealed the expression of RAR, RARß, RXR, and RXR proteins in somatic and germ cells throughout postnatal development. In contrast, the expression of RAR and RXRß did not increase until 30–35 days of age in somatic cells from the testis. Interestingly, RAR and RXR had a similar subcellular localization pattern in Sertoli cells throughout postnatal testis development, while RAR and RXR were both present in the nucleus of spermatocytes and elongating spermatids. These results suggest that RAR may potentially dimerize with RXR in Sertoli cells and with RXR in germ cells. In addition, we demonstrate that the only RAR in the nucleus of early meiotic germ cells is RAR.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, ß, and . Genetic studies have shown that at least two of these receptors, RAR and RXRß, are critical for spermatogenesis [7, 9]. The testes from RAR knockout mice had a morphology similar to the VAD testis [7], whereas the RXRß knockout mice were sterile because of abnormal spermiogenesis [9]. The few sperm that were present in the epididymis of the RXRß knockout mice had coiled tails, impaired acrosomal attachment, and decreased motility. In addition, as the RXRß mutants aged, the germinal epithelium degenerated, and Sertoli cells accumulated lipid droplets [9]. The RARß, RAR, and RXR mutants were found to have normal spermatogenesis [10–12]. These normal phenotypes may be explained by the high sequence homology between the RARs or RXRs, which could artificially allow for functional redundancies in knockout mice [8, 13]. With respect to the RXR mutant mice, they died in utero between embryonic days 13.5 and 16.4 and therefore did not live long enough to reveal whether RXR is required for postnatal testis development and adult spermatogenesis [14].
The testis is a complex organ containing many cell types, which include the somatic cells such as Sertoli cells, Leydig cells, and peritubular myoid cells, and the germ cells [15]. Furthermore, during postnatal testis development, somatic cells undergo proliferation and differentiation, and gonocytes develop into spermatocytes and spermatids for the first time, preparing for the first spermiation [15]. One way to understand which cells are responsive or when they are responsive to vitamin A is to examine the cellular and temporal localization of the retinoid receptors in the testis. Previously, mRNA for RARß was shown to be present only in Sertoli cells, whereas the other five receptor mRNAs were detected in both somatic and germ cells in the rodent testis [9, 16–20]. However, another group has detected RXRß mRNA only in Sertoli cells [9]. At the protein level, we and others have demonstrated RAR, RXR, RXRß, and RXR expression in the adult testis [9, 19, 20]. However, there are no developmental studies on the protein cellular localization of the RARs and RXRs throughout postnatal testicular development in the current literature.
The retinoid receptors are transcription factors that upon activation regulate the expression of retinoid-responsive genes (for review, see [21, 22]). There are multiple steps in receptor activation such as nuclear localization, ligand binding, and dimerization. The RARs only dimerize with RXRs, whereas the RXRs can homodimerize or dimerize with other nuclear receptors including peroxisome proliferator-activated receptors (PPAR), vitamin D receptor, and thyroid hormone receptors [23]. It has been postulated that in vivo the type of dimers formed specifies which genes are regulated [24, 25]. That is why retinoids may be able to regulate divergent cellular processes such as cell proliferation, differentiation, and apoptosis [25, 26]. Therefore, it is important to investigate the potential heterodimeric partners found in each cell type in the testis.
In this study, we describe the protein cellular and subcellular localization of all six retinoid receptors throughout postnatal testis development to elucidate the sites of vitamin A action in the testis both spatially and temporally. The retinoid receptor localization patterns indicate that vitamin A is important in specific germ cells and somatic cells throughout postnatal testis development. These patterns suggest that RAR may dimerize with RXR in Sertoli cells and with RXR in germ cells. We also demonstrate that RAR is the only RAR present in the nucleus of early spermatocytes. This suggests that RAR male knockout mice are sterile because there are no other RARs in the early spermatocytes to compensate for RAR deficiency.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Sprague-Dawley rats were obtained from an in-house vivarium. All rats were fed ad libitum. Animal experimentation was conducted in accordance with the highest standards of humane animal care as outlined in the NIH Guide for the Care and Use of Laboratory Animals.
Primary Sertoli Cells and Germ Cells
Sertoli cells were enzymatically dissociated from 40 testes from 20-day-old rats, plated on 150-mm Petri dishes, and maintained at 32°C in serum-free F-12 medium containing 5 µg/ml insulin and 200 ng/ml testosterone for 4–6 days as previously described [27, 28].
Germ cells from four adult male rats (52–67 days of age) were isolated using collagenase and trypsin as previously described [29]. The percentage of germ cells in each preparation was determined. The cells were smeared onto a slide, fixed with Bouin's solution for 1 h, and stained with hematoxylin, and the number of germ cells was determined. More than 200 cells per preparation were counted. The average percentage of germ cells in three separate germ cell preparations was 88.6 ± 1.7 (mean ± SD).
Antibodies
All anti-RAR and -RXR antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-RAR antibody used was raised against a human RAR peptide, consisting of amino acids 443–462 (CSPSLSPSSNRSSPATHSP). The anti-RARß antibody used was against a human RARß peptide, consisting of amino acids 430–447 (SISPSSVENSGVSQSPLVQ). The anti-RAR antibody was against a human RAR peptide, amino acids 436–454 (CSSEDEVPGGQGKGGLKSPA). The anti-RXR antibody was against a human RXR peptide, consisting of amino acids 2–21 (DTKHFLPLDFSTQVNSSLTS). The anti-RXR antibody was against amino acids 2–21 (YGNYSHFMKFPTGFGGSPGH) of mouse RXR. Two different RXRß antibodies were used. The first antibody was an anti-RXRß-N antibody raised against the N-terminal amino acids 2–21 (LGPDSRSPDSSSPNPLSQGI) of mouse RXRß2. The second antibody was an anti-RXRß1 antibody raised against a human RXRß peptide, consisting of amino acids 2–21 (SWAARPPFLPQRHAEGSVGR).
Western Blot Analysis
Soluble proteins from testes of three adult rats, isolated germ cells, and cultured Sertoli cells were collected as previously described [19]. Briefly, the testes or cells were homogenized in lysis buffer (50 mM Tris-HCl [pH 7.5], 250 mM NaCl, 0.1% Triton X-100, 50 mM NaF, 5 mM EDTA) containing a cocktail of proteinase inhibitors (100 µg/ml PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin). Protein concentration was determined by the Bradford assay [30] with BSA as the standard.
Proteins from testes, isolated germ cells, and cultured Sertoli cells were loaded onto 10% SDS-polyacrylamide gels and subjected to electrophoresis. The proteins were transferred to an Immobilon-P membrane (Millipore Co., Bedford, MA), blocked with 5% blotto (Carnation, Los Angeles, CA) in Tris-buffered saline (TBS) for 1 h at room temperature, and incubated with primary antibody in TBS/Tween-20 for 1 h. This was followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Amersham Life Science Inc., Arlington Heights, IL) in TBS/Tween-20 for 30 min. The proteins were detected by the Enhanced Chemiluminescence Western blotting system (Amersham Life Science Inc.) as described in the protocols supplied by the manufacturer.
Immunohistochemistry
Testes from four adult rats and at least three rats at 5, 10, 15, 20, 25, 30, 35, and 40 days of age were fixed in Bouin's solution for 6 h, embedded in paraffin, cut into 3-µm-thick sections, and mounted onto 2% aminopropyltriethoxysilane-coated slides. The immunohistochemical reactions were performed as described previously [19, 31]. As negative controls, serial sections were put through the same procedure without any primary antibody or incubated with primary antibody preabsorbed with a 50-fold excess of synthetic immunizing peptide (Santa Cruz Biotechnology). The tissue sections were not counterstained with hematoxylin and eosin.
Photography
Images were digitized using a Vistascan flatbed scanner (Umax Technologies, Inc., Fremont, CA) for Western blots or a Sprintscan 35/LE slide scanner (Polaroid, Cambridge, MA) for immunohistochemical photomicrographs. Figures were assembled using Adobe Photoshop (Adobe Photoshop Inc., Mountain View, CA). Figures were printed with a Tektronix Phaser 440 dye sublimation printer (Tektronix, Inc., Wilsonville, OR).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, RARß, and RAR in Testes of Developing Rats
To characterize the anti-RAR antibodies in rat testicular cell extracts before their use on testicular tissue sections by immunohistochemical methods, Western blot analyses were performed with soluble proteins isolated from testes, germ cells, and cultured primary Sertoli cells from rats. The three RARs detected in the rat testicular cell lysates were all in the range of expected sizes (45–55 kDa). Four protein isoforms of RAR, representing RAR1 and RAR2, were detected in germ cell lysates (Fig. 1A). Previously, we have reported the same four isoforms in total testicular extracts [19]. In Sertoli cells, only three isoforms were observed. With regard to RARß, three isoforms were found in the extracts of adult testes and Sertoli cells, whereas only one major isoform was detected in germ cell lysates (Fig. 1B). As for RAR, one major isoform was revealed in all three cell extracts as well as two minor isoforms (Fig. 1C). These results were similar to previously reported RAR isoforms detected from the extracts of COS-1 cells that were transfected with mouse RAR, RARß or RAR expression vectors [7, 32, 33].
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Using these characterized antibodies, immunohistochemistry was performed on testicular sections collected from adult rats or rats at 5, 10, 15, 20, 25, 30, 35, and 40 days of age to examine the protein cellular localization of the RARs in testes of developing rats. RAR was present in Sertoli cells throughout development. While the Sertoli cell expression was detected throughout the cytoplasm, there was a darker stain in Sertoli cells along the basal side of the seminiferous epithelium in rats at 5–15 days of age (Fig. 2, A–C). The level of RAR decreased in rats at 20 days of age, but a low level was distinctively present, localized to both the nucleus and cytoplasm (Fig. 2, I–K). RAR was also present in the early meiotic spermatocytes and pachytene spermatocytes in rats starting at 20 days of age (Fig. 2, D–G and I–K), and in round and elongating spermatids starting at 30 and 35 days of age (Fig. 2J; 35-day data not shown). This expression in germ cells continued in the adults and had the same stage-specific expression pattern as reported previously (Fig. 2, F, G, and K) [19]. Although the significance is unknown, an occasional Golgi/acrosome stain was observed in pachytene spermatocytes and round spermatids (Fig. 2, E and J).
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RARß was detected in Sertoli cells from rats at 15 days of age to adulthood (Fig. 3). The receptor was predominantly cytoplasmic in the Sertoli cells in rats at 15 days of age, but it was detected in the nucleus and the cytoplasm of Sertoli cells as animals matured (Fig. 3). RARß was also localized to the cytoplasm of gonocytes (Fig. 3A), the cytoplasm of spermatogonia from rats at 15 days of age (Fig. 3, C and G), and the cytoplasm of early meiotic and pachytene spermatocytes (Fig. 3, D, E and H). The RARß stain was darker in germ cells and Sertoli cells along the basal side of seminiferous epithelium. In addition, RARß was observed in the interstitium throughout postnatal development (Fig. 3).
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RAR was present in the nucleus and cytoplasm of both Sertoli cells and Leydig cells in rats starting at 30 days of age (Fig. 4, D–G and I–K). No expression was detected in germ cells. The apparent stain on the Golgi apparatus of pachytene spermatocytes and round spermatids (Fig. 4, E, F, and J) was still observed in the negative control (Fig. 4H) obtained using antibody preabsorbed with immunizing peptide.
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Cellular Localization of RXR, RXRß, and RXR in Testes of Developing Rats
To characterize anti-RXR antibodies in rat testicular cell extracts before their use on the testicular tissue sections by immunohistochemical methods, Western blot analyses were performed with cellular extracts from testes, isolated germ cells, and cultured primary Sertoli cells. Three RXR protein isoforms were detected in germ cell extracts, whereas only one major isoform was observed in the extracts of Sertoli cells and testes (Fig. 5A). Although similar isoforms were seen, the sizes of RXR in rats were slightly smaller than those of mouse RXR detected in the COS-1 cells transfected with mouse RXR expression vector [34]. With regard to RXRß, the anti-RXRß-N antibody detected two protein isoforms in testes (Fig. 5B). The smaller form was found in the Sertoli cells and the larger form in the germ cells. The anti-RXRß1 antibody reacted only with the larger RXRß isoform (data not shown), as expected from a previous report indicating that RXRß1 is 72 amino acids larger than RXRß2 [35]. In addition, anti-RXR antibody reacted with two protein isoforms in Sertoli cells and one predominant isoform in adult testes and germ cells (Fig. 5C). These rat isoforms detected by antibodies for RXRß and RXR were similar in size to the mouse isoforms previously detected in extracts from transfected COS-1 cells overexpressing mouse RXRß or RXR [34].
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Using these characterized antibodies, immunohistochemistry was performed on testicular tissue sections from adult rats and rats at 5, 10, 15, 20, 25, 30, 35, and 40 days of age to examine the protein cellular localization of RXRs throughout postnatal development. Although RXR protein was present in Sertoli cells at all times (Fig. 6), the protein was localized to the Sertoli cell cytoplasm in the young animals (Fig. 6, A–C) and to both the nucleus and cytoplasm as the animals matured (Fig. 6, I–K). This expression pattern was similar to the expression pattern for RAR in Sertoli cells shown in Figure 2. In addition, RXR was present in germ cells and interstitial cells throughout the times studied (Fig. 6). The germ cell expression was mostly cytoplasmic in gonocytes, spermatogonia, early and late spermatocytes, and round spermatids (Fig. 6).
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Immunohistochemical analysis of RXRß was performed using two different RXRß antibodies: RXRß-N and RXRß1. The RXRß-N antibody is expected to recognize both RXRß1 and RXRß2, whereas the RXRß1 antibody is expected to recognize only RXRß1 [35]. The RXRß-N antibody detected RXRß in Sertoli cells of rats at 35 days of age (Fig. 7, E and I). This delayed expression was both nuclear and cytoplasmic (Fig. 7, I–K) and increased to higher levels in the adult Sertoli cells (Fig. 7, G and K). In contrast, RXRß was not detected in germ cells (Fig. 7). In the interstitium, the immunopositive cells for RXRß were seen in a distinct population of interstitial cells but not in blood vessels from rats at 5–30 days of age (Fig. 7). This was different from the RXRß1-positive cells in the interstitium. The anti-RXRß1 antibody only detected RXRß1 in the interstitial cells in rats starting at 30 days of age, and it increased as the animals matured (Fig. 8, C–E and G).
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RXR protein was present in Sertoli cells, interstitial cells, and peritubular myoid cells throughout development (Fig. 9). In germ cells, RXR was observed in gonocytes, spermatogonia, early and late spermatocytes, and round and elongating spermatids (Fig. 9). The expression was predominately nuclear in both somatic and germ cells. In addition, a spermatogenic stage-specific pattern of expression was seen (Fig. 9, G and H). RXR was high in the early pachytene spermatocytes (Fig. 9G) and decreased as the spermatocytes progressed through meiosis (Fig. 9H). The protein reappeared in the round spermatids at stages I–V (Fig. 9G, upper tubule), decreased at stages VI–VIII (Fig. 9G, lower tubule), was high in the elongating spermatids at stage X (Fig. 9H), and was not detected in the elongated spermatids (Fig. 9K). This spermatogenic stage-specific expression pattern for RXR in germ cells was similar to that for RAR, as previously reported [19].
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, RARß, RXR, and RXR were expressed in Sertoli cells throughout postnatal development, whereas RAR and RXRß first appeared in Sertoli cells in rats at 30–35 days of age. This delayed temporal expression pattern for RXRß is consistent with a role for this receptor in spermiogenesis, as suggested by the phenotype of the RXRß knockout mice [9].
With respect to cellular localization, RAR and RXRß were present only in somatic cells, while the other receptors were detected in both somatic cells and germ cells (see Tables 1 and 2). Two anti-RXRß antibodies were used because there is inconsistency in the current literature. The anti-RXRß-N antibody, which recognizes both RXRß1 and RXRß2, and the anti-RXRß1 antibody, which recognizes only RXRß1, did not detect RXRß in germ cells. The results indicate that RXRß2 is expressed in Sertoli cells and both RXRß1 and RXRß2 are expressed in the interstitial cells. Our results are consistent with a report that demonstrated detection of RXRß only in Sertoli cells of adult mice [9]. However, another group reported RXRß expression in germ cells of the adult testis using an anti-RXRß antibody raised against the C-terminal amino acids 483–502 (LEHLFFFKLIGDTPIDTFL) of human RXRß [20], which are identical in all three rat RXRs [36–38]. Thus, the discrepancy may have occurred because their antibody recognized all three RXRs.
We also present results that retinoid receptors are not always found in the nucleus in testicular cells (see Tables 1 and 2). In germ cells, RARß and RXR were both localized to the cytoplasm of germ cells throughout development, whereas RAR and RXR were both present in the nucleus of early and late spermatocytes and elongating spermatids. These results suggest that the functional receptors in germ cells may be RAR and RXR. In Sertoli cells, RAR, RXRß, and RXR were predominantly found in the nucleus. In contrast, RAR, RARß, and RXR were primarily localized to the Sertoli cell cytoplasm in the young animals and then appeared to partially translocate into the nucleus in the mature animals.
Since the retinoid receptors are transcription factors that must be localized to the nucleus to be functional [21, 22], the cytoplasmic localization of some of the receptors was an unexpected result. It is also a surprise because, in the field of steroid/thyroid hormone receptors, it is thought that retinoid receptors belong to the class of receptors (thyroid hormone receptors, retinoid receptors, PPAR, etc.) that are found constitutively in the nucleus whether the ligand is bound or not. This has been supported by an immunolocalization study of the retinoid receptors to the nucleus of skin cells [39] and a cell fractionation study which demonstrated that 95% of RAR was localized to the nucleus of HL-60 cells [40].
However, other studies suggest that the subcellular localization of RAR may be regulated by retinoic acid and protein kinase C [41–43]. Retinoic acid has been shown to cause translocation of the promyelocyte-RAR fusion oncoprotein, PML-RAR, from the cytoplasm to the nucleus [41]. In reverse, we have demonstrated that depletion of vitamin A leads to a change in the localization of RAR in the germ cells from the nucleus to the cytoplasm [42]. Moreover, down-regulation of protein kinase C, a molecule that is not a ligand for the receptors, was still able to increase the cytoplasmic localization of RAR in COS-7 cells [43]. Equally important to note, it has been shown recently that the nuclear localization of thyroid hormone receptor, which is a nuclear receptor in the same class as the retinoid receptors, is dependent on the presence of ligand [44], and PPAR is also regulated at the level of nuclear localization [45]. These studies together suggest that the regulation of nuclear localization may not be so unusual for retinoid receptors in certain cells, and testicular cells may be one of those cell types. The regulatory mechanisms and the significance of the cytoplasmic localization of retinoid receptors in testicular cells during postnatal development is not known and requires further investigation. Special attention is needed to determine the role of retinoic acid, the ligand, on the nuclear localization of receptors in testicular cells.
Because of the unexpected pattern of subcellular localization, it is important to point out that the cytoplasmic localization is probably not due to poor fixation, as suggested by a previous report [46]. Our results show that RXR is found predominantly in the nucleus of Sertoli and germ cells, suggesting that proteins are efficiently retained in the nucleus by the fixation method. Similarly supportive, cultured cells prepared with another fixation method also revealed the same cytoplasmic localization for RAR, which can also be changed not only by retinoic acid but also by other factors (data not shown). Furthermore, in living cells without any fixation method, thyroid hormone has been shown to regulate the nuclear trafficking of thyroid hormone receptor fused with a green fluorescent protein [44].
In addition, our results clearly indicate that the cytoplasmic localization is not due to absence of ligand, as suggested by a previous report [46]. For example, RXR is detected in the cytoplasm of Sertoli and germ cells at the same time that RXR is found in the nucleus of the same cells. Since these receptors have the same ligand, 9-cis RA, this suggests that absence of ligand is unlikely to be the reason some receptors are found in the cytoplasm. This suggests that there are probably other factors involved in keeping some receptors in the cytoplasm.
The study of receptor heterodimerization in testicular cells is important because different retinoid receptor pairs are postulated to regulate specific retinoid-responsive genes [8, 13]. In F9 embryonal carcinoma cells, the specific heterodimer of RAR and RXR, and not RAR and RXR, regulates the expression of P450RAI, which is an enzyme involved in retinoic acid metabolism [47]. Our subcellular and temporal results indicate potential heterodimeric partners for RAR in different cells in the rat testis. In germ cells, RAR is a likely partner for RXR since both are present in the nucleus of spermatocytes and spermatids with the same temporal expression pattern. RAR and RXR may have a role in meiosis and spermiogenesis. In Sertoli cells, RAR may heterodimerize with RXR, as they were both detected in Sertoli cells with the same subcellular localization pattern throughout development. Specifically, they were localized to the cytoplasm in the pubertal rats and then appeared to translocate to the nucleus in the adult. This receptor pair may be involved in regulation of genes for Sertoli cell differentiation and maintenance, as retinoid receptors have been implicated previously in regulating genes for differentiation of F9 and P19 embryonal carcinoma cells [26, 48].
Our results also suggest that RXRß may heterodimerize with RAR since both receptors were found in Sertoli cells with the same temporal expression pattern. However, it has been suggested that RXRß heterodimerizes with PPAR [9]. This is because Sertoli cells in the RXRß knockout mice accumulated lipid droplets as mice aged [9] and PPAR are thought to regulate genes involved in lipid metabolism [8]. Our results do not rule out this possibility; RXRß may heterodimerize with both RAR and PPAR in Sertoli cells. Further investigation is necessary to establish the physical interaction of heterodimeric partners in the testicular cells.
The RAR knockout mouse study indicates that RAR has a critical role in spermatogenesis [7]. The morphology of testes from the knockout mice resembled that from the VAD rats in that germ cells are stalled at the preleptotene stage of meiosis [6]. Interestingly, we found that the only RAR expressed in the nucleus of early meiotic spermatocytes, which includes preleptotene spermatocytes, is RAR. In contrast, RARß was in the cytoplasm of germ cells, while RAR was not even detected in germ cells at any time throughout development. These results together suggest that the RAR knockout mice are sterile because RAR may be the only functional RAR in early meiotic spermatocytes and other RARs cannot compensate for the loss of RAR.
In conclusion, we have shown the cellular and subcellular localization of all six retinoid receptors throughout postnatal testicular development. We found that some retinoid receptors are not localized constitutively to the nucleus as previously thought. Our results suggest that specific pairs of retinoid receptors may be functional as transcriptional factors in the nucleus at specific times of postnatal testicular development. RAR may heterodimerize with RXR in Sertoli cells and with RXR in germ cells after 20 days of age. In addition, RXRß may heterodimerize with RAR after 30–35 days of age. Furthermore, we found that the RAR knockout mice may be sterile because other RARs are not present in the nucleus of the preleptotene spermatocytes and cannot replace the function of RAR. These findings are important to set the stage for future studies in retinoid receptor interaction, regulation, and function during spermatogenesis and testis development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Kwan Hee Kim, Department of Genetics and Cell Biology, Washington State University, Pullman, WA 99164-4234. FAX: 509 335 1907; khkim{at}mail.wsu.edu
Accepted: June 22, 1999.
Received: March 11, 1999.
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