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Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) (N.V., C.D., C.R.-E., M.O.-A., P.C., N.B.G., M.M.), and Institut Clinique de la Souris (ICS) (P.C., M.M.), Centre National de la Recherche Scientifique (CNRS)/Institut National de la Santé et de la Recherche Médicale (INSERM)/Université Louis Pasteur de Strasbourg (ULP)/Collège de France, 67404 Illkirch Cedex, Communauté Urbaine de Strasbourg, France
Address all correspondence and requests for reprints to: Manuel Mark, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut Clinique de la Souris (ICS), Centre National de la Recherche Scientifique (CNRS)/Institut National de la Santé et de la Recherche Médicale (INSERM)/Université Louis Pasteur de Strasbourg (ULP)/Collège de France, BP10142, 67404 Illkirch Cedex, Communauté Urbaine de Strasbourg, France. E-mail: marek{at}igbmc.u-strasbg.fr.
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Abstract |
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and RXRß are confined to Sertoli cells, whereas RAR
is expressed in spermatogonia and RARß, RXR, and RXR are colocalized in step 7–8 spermatids. Correlating these expression patterns with the pathological phenotypes generated in response to RAR and RXR mutations and to postnatal vitamin A deficiency suggests that spermiation requires RXRß/RAR heterodimers in Sertoli cells, whereas spermatogonia proliferation involves, independently of RXR, two distinct RAR-mediated signaling pathways in both Sertoli cells and spermatogonia. Our data also suggest that the involvement of RA in testis development starts when primary spermatogonia first appear. |
Introduction |
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Systemic administration of retinoic acid (RA), the active metabolite of vitamin A, can restore spermatogenesis from growth-arrested A spermatogonia in vitamin A-deficient (VAD) testis (8). However, under physiological conditions, less than 1% of the RA present in the testis originates from the circulation (9). Instead, testicular RA is synthesized in situ (10). It is predominantly produced from retinol (ROL) through a two-step metabolic pathway (reviewed in Ref.11), ROL being provided either by the blood where it circulates bound to the ROL-binding protein (RBP) (12) or by the local stores of retinyl esters (reviewed in Ref.13). The first step, oxidation of ROL into retinaldehyde, involves alcohol dehydrogenases, of which three are expressed in the mouse testis: the ubiquitous ADH3, ADH1 in Sertoli cells, and ADH4 in late spermatids (14, 15). The second step, oxidation of retinaldehyde into RA, is catalyzed by four retinaldehyde dehydrogenases (RALDH1–4, encoded by the Aldh1a1 to Aldh1a3 and Aldh8a1 genes, respectively) (11, 16), of which Aldh1a1 and Aldh1a2 are expressed in the rodent testis (17, 18). Alternatively, RA can be produced from ß-carotenes carried along into chylomicron remnants (19), through their conversion into retinaldehyde by the ß-carotene 15,15'-monoxygenase, which is expressed in testis (reviewed in Ref.20). Aside from synthesis, degradation of RA is also an important balancing mechanism that protects cells from inadequate RA stimulation (21). It is catalyzed by at least three cytochrome P450 hydroxylases (CYP26A1, CYP26B1, and CYP26C1), which repeatedly hydroxylate RA and its metabolites into increasingly water-soluble products that are less active and readily excretable (Ref.22 and references therein). Whether they are expressed in testis is not known.
Inside cells, ROL and RA are bound to cellular binding proteins (23). The ROL-binding proteins, CRBP1 and CRBP2, are both involved in ROL storage in vivo (2, 24). In the adult rat testis, CRBP1 is localized mainly in Sertoli cells (25, 26), whereas CRBP2 is not expressed (27, 28). As for RA-binding proteins, CRABP1 and CRABP2, their actual functions remain controversial (23, 29, 30). In the rat testis, CRABP1 is detected in spermatogonia (31) and CRABP2 in developing Sertoli and Leydig cells (32).
In the nucleus, RA binds to two types of nuclear receptors, the RA receptors (RAR, -ß, and - isotypes that bind both all-trans and 9-cis RA stereoisomers) and the rexinoid receptors (RXR, -ß, and - isotypes that bind 9-cis RA only). Although RARs and RXRs may function as homodimers in vitro (33), RXR/RAR heterodimers have been clearly shown to be the functional units transducing the RA signal in the mouse, both during development and in adult tissues (34, 35), where they act as regulators of transcription by binding to RA response elements located in the regulatory regions of target genes (33). Unliganded RAR/RXR heterodimers, bound to RA response elements, recruit transcriptional corepressor complexes associated with histone deacetylase activity, resulting in chromatin condensation and transcriptional silencing. Upon binding of RA, corepressors are released, and the recruitment of coactivator complexes exhibiting histone transacetylase activity results in chromatin decondensation and activation of target gene transcription (36). In addition, transcriptional activation by retinoids might extend beyond RXR/RAR heterodimers, because RXR is also a heterodimeric partner of a number of other nuclear receptors (e.g. peroxisome proliferator-activated receptors, liver X receptors, farnesoid X receptor, and orphan receptors) (reviewed in Ref.37). It is finally worth noting that liganded RAR can alternatively modulate, independently from RXR, the expression of genes through repressing activity of activator protein 1 (AP-1) transcription complexes (heterodimers between fos- and jun-related proteins) (Ref.38 and references therein).
Previous immunohistochemical (IHC) and in situ hybridization (ISH) studies have shown the distributions of RARs, RXRs, ROL- and RA-metabolizing enzymes, and ROL- and RA-binding proteins in testis (25, 26, 31, 32, 39, 40, 41, 42, 43, 44) as well as in Sertoli cell lines (45). However, most of these studies were performed in rats, and many IHC experiments lacked the proper controls (46). Because targeted mutagenesis makes the mouse a unique model to study gene function, we decided to thoroughly investigate the cellular localization of RA-metabolizing enzymes, ROL- and RA-binding proteins, RARs, and RXRs in the mouse testis, at different stages of postnatal development and in adults. Our results show that the previous IHC data on RAR and RXR distribution need to be revised. They further reveal that RA is likely to act, in the mouse seminiferous epithelium, as a paracrine signal whose level is tightly controlled and varies according to the stage of the seminiferous epithelium cycle. This study provides the solid basis that is required to undertake the somatic mutagenesis experiments aimed at determining the mechanisms of action and the cellular and molecular targets of RA in male reproduction.
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Materials and Methods |
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Staging of the seminiferous epithelium
The cycle of the seminiferous epithelium is divided into 12 stages, each defined by a specific association of germ cells (47). These epithelial stages are precisely delineated on periodic acid-Schiff-stained histological sections from Bouin-fixed testes, but not necessarily on sections destined for IHC and ISH analyses. In these instances, we identified an epithelial stage or a cohort of consecutive stages using one or two hallmark(s) such as numerous mitotic figures at stage XII; condensed and elongated spermatids, scattered throughout the thickness of the epithelium, at stages II–VI; and preleptotene spermatocytes at stages VII–VIII. Stage I was defined as a segment containing small round spermatids continuous with a segment at stage XII. Stages IX, X, and XI were distinguished from one another by the degree of elongation of spermatid nuclei.
Histology
Tissues were fixed in Bouin’s fluid and embedded in paraffin. Five-micrometer-thick sections were stained with periodic acid-Schiff or Mallory’s trichrome (see supplemental data published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).
IHC
Antibodies used in IHC experiments are listed in supplemental Table 1
. Five different fixation protocols were tested (supplemental Table 1, and see supplemental data for additional details on fixations), and each experiment was repeated twice on at least three different mice per age group. After rinsing in PBS containing 0.1% (vol/vol) Tween 20 (three times for 5 min each at room temperature), sections were incubated with the affinity-purified primary antibodies for 16 h at 4 C in a humidified chamber. Detection of the bound primary antibodies was achieved for 45 min at room temperature in a humidified chamber, using either a Cy3-conjugated goat antirabbit IgG (Biomol Immuno Research Laboratories, Exeter, UK), a Cy3-conjugated goat antiguinea pig IgG (Euromedex, Souffelweyersheim, France), or a biotinylated antimouse antibody (Vectastain; Vector Laboratories, Burlingame, CA), depending upon the origin of the primary antibody (supplemental Table 1). Nuclei were counterstained with 4',6-diamidino-2-phenyl-indole (DAPI) (Roche Diagnostics, Meylan, France) diluted in the mounting medium at 10 µg/ml (Vectashield; Vector).
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The plasmids containing full-length Rara (1.6 kb), Rarb (1.7 kb), Rarg (1.9 kb), Rxra (1.4 kb), Rxrb (1.3 kb), Rxrg (1.4 kb), Cyp26a1 (1,7 kb), Rbp1 (650 bp), Crabp1 (760 bp), and Crabp2 (830 bp) cDNAs or parts of Cyp26b1 (680 bp; exons 2–5), Cyp26c1 (470 bp; exons 4–5), Adfp (800 bp; exons 3–7), Aldh1a1 (1 kb; exons 8–13), Aldh1a2 (468 bp, exons 2–6), Aldh1a3 (500 bp; exons 6–9), Bcmo1 (420 bp; exons 9–11), and Lrat (406 bp; exons 1–2) cDNA were linearized and used as templates for the synthesis of the sense or antisense riboprobes.
Analysis of ß-galactosidase activity
Unfixed testes were frozen in embedding medium. Ten-micrometer-thick sections were air dried, hydrated in PBS, fixed in 1% (wt/vol) paraformaldehyde, 0.1% (wt/vol) glutaraldehyde in PBS containing 1% (vol/vol) Tween 20 for 1 min at room temperature, washed in PBS, and then incubated in PBS containing 2 mM MgCl2, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 1 mg/ml 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal) at 30 C for 20 h. Sections were counterstained with DAPI diluted in the mounting medium at 10 µg/ml (Vectashield; Vector).
Gold chloride method for detection of retinyl esters
Mice were perfusion fixed (see supplemental data) with 0.05% (wt/vol) chromic acid in water, under dim light. After dissection, organs were cut in 5 x 10 x 10-mm slices, fixed for 30 min in the perfusion solution, and immediately embedded in 0.8–4% (wt/vol) melting agar according to the stiffness of the sample. Fifty-micrometer-thick vibratome sections were immersed for 10 min in 0.05% chromic acid before staining in gold chloride solution overnight at room temperature. Sections were washed three times in water and mounted in 70% (vol/vol) glycerol in PBS.
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Results |
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, A and E) (see also Ref.39). Rarb and Rxra transcripts were colocalized in the round spermatid layer of tubule sections from stages VII and VIII of the seminiferous epithelium cycle (asterisks and double asterisks in Fig. 1, B and D). Low levels of Rarg transcripts were unevenly distributed at the periphery of all tubule sections (Fig. 1C). Rxrg transcripts were detected in some tubule sections from epithelial stages VII and VIII, but only in part of the circumference of the round spermatid layer (double asterisks in Fig. 1F, compare with B and D). Nonradioactive ISH combined with a DAPI nuclear counterstain allowed accurate identification of Rxra- and Rxrg-expressing cells: all steps 7 and 8 round spermatids expressed Rxra (R, Fig. 1G), whereas only a subset of these spermatids expressed Rxrg transcripts, at the time of sperm release (R, Fig. 1H). Low levels of Rxra transcripts were also detected in steps 2, 3, 4, 5, and/or 6 spermatids (which can hardly be distinguished from one another on histological sections processed for ISH; see Materials and Methods), as well as in step 9 spermatids (not shown). Therefore, Rara, Rarg, and Rxrb are expressed in all tubules irrespective of the epithelial stages, in contrast to Rarb, Rxra, and Rxrg, which are expressed mainly or exclusively in steps 7 and 8 spermatids (Table 1
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by IHC: RP(F), Ab9(F), and sc-551 (supplemental Table 1). In wild-type (WT) males, the RP(F) antibody yielded a strong signal in the nuclei of all Sertoli cells (S, Fig. 2, A and B), in agreement with previous observations using a different protocol (44). RAR was undetectable in germ cells (E, P, and R, Fig. 2B) as well as in peritubular cells (M, Fig. 2B). Importantly, the RP(F) antibody yielded no nuclear signal in Rara-null testis (50), demonstrating that it was actually specific for RAR (Fig. 2, C and D). In contrast, the weak cytoplasmic signal exhibited by Leydig cells (LY, Fig. 2, A–D) was unrelated to RAR, because it was detected in WT (Fig. 2, A and B) and Rara-null testis (Fig. 2, C and D). In WT testis, sc-551 yielded a strong cytoplasmic signal in Sertoli cells (S, Fig. 2, E and F), whereas Ab9(F) gave a strong nuclear signal in Sertoli and germ cells (supplemental Fig. 1 and supplemental Table 1). However, irrespective of the IHC protocol that was employed, identical signals were observed in Rara-null testis (S, Fig. 2, G and H, and supplemental Table 1), indicating that neither sc-551 nor Ab9(F) did specifically recognize RAR. Altogether, our results show that RAR is essentially, if not only, present in the nuclei of Sertoli cell in the adult mouse testis (Table 1).
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(mF) antibody, recognizing the RAR isotype (supplemental Table 1), yielded a nuclear signal within a few cells that were identified as A spermatogonia based on their distribution at the periphery of all seminiferous tubules (A, Fig. 2, K and L) and their cytological features (oval nuclei lying in parallel with the basement membrane and containing little heterochromatin; A, Fig. 2, M–P). The RP(mF) antibody also yielded a faint positive nuclear signal in pachytene spermatocytes (white arrowheads, Fig. 2, M and O). In RargZL–/ZL– mutants, carrying null alleles of Rarg knocked-in with LacZ reporter genes (51), no immunostaining could be detected in spermatogonia, but spermatocytes exhibited a faint signal identical to that of WT spermatocytes (Fig. 2, Q and R, and not shown). Importantly in RargZL–/ZL– testis, A spermatogonia were the only cells to display the ß-galactosidase activity reporting the cells where Rarg was expressed before site-directed mutagenesis (A, Fig. 2, S and T). We conclude that RAR is essentially, if not only, present in the nuclei of A spermatogonia in the adult mouse testis (Table 1).
RXR was detected using the RPRX- and 4RX3A2 antibodies (supplemental Table 1). Because of the embryonic lethality of Rxra-null mutants (34), testes lacking RXR are not available. However, skin of mice lacking Rxra in all epidermal keratinocytes (Rxraep–/–) was used as a negative control of IHC (52). Importantly, the immunostaining generated by RPRX- was indistinguishable in WT and Rxraep–/– skin samples, indicating that RPRX- did not specifically recognize RXR in IHC experiments. Therefore, the antigens revealed by this antibody in the nuclei of several somatic and germ cell types are likely to be unrelated to RXR (not shown). In contrast, the 4RX3A2 antibody yielded no nuclear signal in the keratinocytes of Rxraep–/– mice, demonstrating its specificity for RXR (not shown; see Ref.52). In the adult testis, 4RX3A2 specifically labeled steps 7 and 8 round spermatid nuclei (R, Fig. 2I, compare with J), in accordance with our ISH data (Fig. 1, D and G). Therefore, RXR is essentially, if not only, present in the nuclei of steps 7 and 8 round spermatids in the adult mouse testis (Table 1).
We have reported elsewhere the immunolocalization of RXRß in Sertoli cells, using testis from Rxrb-null mice as negative controls (39). Here, we found that the sc-831 antibody (supplemental Table 1) always gave identical, nonspecific, signals on testis sections from WT and Rxrb-null mice, even though five different IHC protocols were used (supplemental Table 1).
Finally, RARß and RXR could not be detected in adult testis using the antibodies RPß(F)2 and sc-555 (supplemental Table 1), respectively. These antibodies efficiently and specifically recognize RARß and RXR in mouse tissues (53, 54). Therefore, RARß and RXR are either absent from testicular cells or expressed at levels below the detection limits of IHC (Table 1). Altogether, the present data emphasize the importance of tissues from null mutants as the only valid controls to assess the specificity of IHC experiments (46).
Expression of retinoid receptors in the developing mouse testis
Expression of the RARs and RXRs was studied at postnatal d 1 (P1), at P5 (when gonocytes differentiate into primitive spermatogonia), at P10 (i.e. at the onset of meiosis), and at P20 (when postmeiotic cells first appear) (55). RAR was confined to the nuclei of Sertoli cell precursors at P1 and P5 (S, Fig. 3, A–D) and subsequently to those of immature Sertoli cells at P10 and P20 (S, Fig. 3, E–H). Germ cells (G, A, PR, P), as well as precursors of peritubular myoid (M) and Leydig (LY) cells, were not immunostained by the RP(F) antibody (Fig. 3, A–H). RAR was first detected at P5 in primitive spermatogonia (A, Fig. 3, K and L) and in A spermatogonia at P10 and P20 (A, Fig. 3, M–P). Earlier spermatogonia precursors, namely gonocytes, did not express RAR (G, Fig. 3, I and J).
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and not shown). In contrast, Rxrb transcripts were detected within the developing seminiferous tubules at P5, P10, and P20. As exemplified at P5, Rxrb transcripts were confined to the central portion of the tubule sections, which corresponds to the cytoplasm of Sertoli precursor cells (T, Fig. 3R). As expected from their localization in late round spermatids in the adult testis, Rarb and Rxrg transcripts were undetectable in the P1–20 developing testis (data not shown).
Altogether, these data indicate that Rara, Rarg, and Rxrb are expressed in given testicular cell types throughout postnatal development and adult life. They also suggest that RXR can perform distinct functions in prepubertal vs. adult testis.
Expression of RA-synthesizing and RA-degrading enzymes in the adult and developing testis
In adult testis, Leydig and Sertoli cells expressed high and low levels of Aldh1a1 transcripts, respectively (Fig. 4, A and B). Aldh1a2 transcripts were detected in germ cells. They were most abundant in late pachytene spermatocytes and at epithelial stages VII–X (P, Fig. 4, E–H) and in diplotene spermatocytes at stage XI (not shown). They were also present in round spermatids (R2–6) at epithelial stages I (not shown) and II–VI (Fig. 4, E and F). In contrast, they were undetectable in spermatogonia, preleptotene (PR), leptotene, zygotene, and early pachytene spermatocytes as well as in late, steps 7 and 8, round spermatids (R7–8) and in elongating spermatids (e.g. E16) (Fig. 4, E–H, and not shown). Low levels of Aldh1a3 transcripts were detected in adult Leydig cells (data not shown).
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, C and D), but weak at P10 and P20, and started in Leydig cells around P20 (not shown). As in the adult testis, Aldh1a1 transcripts were absent from germ cells (not shown). Low levels of Aldh1a2 transcripts were detected at P5 in Sertoli cells (not shown) and strong expression was observed at P20, concomitantly with the appearance of late pachytene spermatocytes (P, Fig. 4, I and J). Aldh1a3 transcripts were undetectable in the developing testis (not shown).
The Bcmo1 transcripts, encoding the ß-carotene cleaving enzyme, are present in whole-testis extracts (20). They were restricted to germ cells during the late stages of spermiogenesis (Fig. 4, K and L), namely round spermatids at steps 7 and 8 (R7–8), elongating spermatids at steps 9–12 (E10, Fig. 4, K and L), and to a lesser extent, elongated and condensed spermatids at steps 13–15 (E13–15). Bcmo1 transcripts were absent from developing testes analyzed from P1–20 (not shown).
The Cyp26a1, Cyp26b1, and Cyp26c1 transcripts, encoding RA-hydroxylating enzymes, were all restricted to the peritubular myoepithelial cells (M) both in the adult and the developing testis (Fig. 5, A–H, and not shown).
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).
Expression of the cellular RA-binding protein in the adult and developing testis
In the adult mouse testis, Crabp1 transcripts were detected exclusively in A and B spermatogonia. During development, Crabp1 expression was absent at P1 and was very robust in apparently all the spermatogonia at P5, P10, and P20. Other germ cell types as well as the testicular somatic cells did not express Crabp1 (Table 1, supplemental Fig. 2, and not shown). These results indicate that the pattern of Crabp1 expression has been conserved between the rat (31, 32) and the mouse. Contrary to the rat testis (32), the mouse testis did not express Crabp2 during development, and Crabp2 transcripts were also absent from the adult organ. Note that we checked that the probe used in our ISH assays gave a strong and specific signal in mouse embryonic tissues known to express Crabp2 (not shown).
Vitamin A storage in the adult mouse testis
Using the gold chloride method (56), retinyl esters were detected in the lipid droplets located in Sertoli cells (L1, Fig. 6A). Minute positive droplets were also occasionally detected at the luminal side of the seminiferous epithelium (L2, Fig. 6A). Because the technique preserves germ cells only poorly, it is unclear whether these small droplets belonged to spermatids or to Sertoli cells. In contrast, the lipid-containing Leydig cells did not react with the gold chloride, indicating that the staining was specific (LY, Fig. 6A). Along these lines, the vitamin A-storing liver stellate cells (56) and the lipid- but not vitamin A-storing brown adipocytes served, respectively, as positive and negative controls of the histochemical reaction (supplemental Fig. 3).
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, B and C), whereas the Rbp1 transcripts were detected mostly in Leydig cells (LY, Fig. 6, D–F) and at much lower levels in Sertoli cells, at epithelial stages X–XI (Fig. 6, E and F). Note that we did not check the expression of Rbp2 (encoding CRBP2) because this gene is expressed only in the adult intestine in the rat (27, 28).
The adipose differentiation-related protein (ADFP), which is localized at the surface of lipid droplets in many tissues (reviewed in Ref.61), including Sertoli cells (62), stimulates lipid droplet formation (63). To investigate the formation of retinyl ester-containing lipid droplets in Sertoli cells, we analyzed the distribution of ADFP and Adfp transcripts in the adult mouse seminiferous epithelium. ADFP was associated with 1) minute lipid droplets in elongated spermatids (L2, Fig. 6, G and H), 2) residual bodies that detach from these mature spermatids and are phagocytosed by Sertoli cells at spermiation (not shown), and 3) lipid droplets at the base of Sertoli cells (L1, Fig. 6, G and H). In contrast, Adfp transcripts were absent from Sertoli cells but expressed in round spermatids at steps 2–6 and at the beginning of step 7 (Fig. 6, I and J, and not shown).
Altogether, these data suggest that 1) retinyl ester synthesis by LRAT may not involve CRBP1 in the mouse testis, because the latter is expressed in Sertoli cells, whereas the former is expressed in early spermatids (Table 1), and 2) the vitamin A-containing droplets located in Sertoli cells arise, at least in part, from those present in spermatids.
Spermatogenesis is not altered in Rarb-, Rarg-, Rrxg-, Aldh1a1-, Aldh1a3-, Crabp1-, and Crabp2-null mutants
The Rara- and Rxrb-null males are sterile because of testicular defects, namely degeneration and/or abnormal spermiation (39, 50, 62). In contrast, young Rarb-, Rrxg-, and Aldh1a1-null males are fertile (53, 64, 65). At 12 months of age, Rarb-, Rxrg-, and Aldh1a1-null males (n = 4 for each genotype) were also fertile but occasionally displayed one to three degenerating seminiferous tubules. This abnormality was, however, not related to the mutations, because it was observed with the same penetrance in age-matched WT littermates (not shown). Moreover, failure of spermiation was never observed in these mutant mice. Likewise, the testis from 12-month-old Aldh1a3-null mutants (n = 2), whose lethal malformations were rescued by maternal administration of RA (66), were histologically normal (not shown). Accordingly, these Aldh1a3-null males were fertile. Crabp1- and Crabp2-null mutants as well as Crabp1/Crabp2 double-null mutants (Ref.29 and references therein) have been bred for years in the homozygous state in our animal facility. Histological analysis of the testes from two of each of these mutants at 6 months of age did not reveal any abnormality (not shown).
The sterility of Rarg-null males has been ascribed to the squamous metaplasia of their prostate and seminal vesicle epithelia (67). Accordingly, the testes and epididymides of young (i.e. 2- to 6-month-old) mutants were histologically normal (Fig. 7, A and B). In contrast, the testes from 12-month-old Rarg-null mutants (n = 5) consistently displayed signs of degeneration manifested by a thinning of the seminiferous epithelium and by a loss of germ cells occasionally yielding tubules with only Sertoli cells (D, Fig. 7C). These lesions were associated with accumulations of spermatozoa within the lumen of some seminiferous tubules (SZ, Fig. 7C) and with an extensive keratinization of the caudal epididymis epithelium (bracket, Fig. 7D). These testicular lesions were mimicked in WT males upon ligature of the efferent ductules (Fig. 7E) and resembled those of Esr1-null (formerly estrogen receptor-, ER-null) males in which testicular fluid absorption by the epithelial cells lining the efferent ductules is inhibited (68). In contrast, the epididymal epithelium did not keratinize in efferectomized males (Fig. 7F), indicating that squamous metaplasia in Rarg-null males is actually related to the loss of RAR.
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). It should finally be noted that spermatogenesis in Rxra-, Aldh1a2-, Cyp26a1-, and Cyp26b1-null mutants could not be analyzed, because all of these mutants die in utero or at birth (Refs.21 , 34 , and 69 and references therein). |
Discussion |
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(i.e. sc-551), RXR (i.e. RPRX-), and RXRß (i.e. sc-831) (supplemental Table 1). Likewise, our data demonstrating a nuclear-restricted localization of RAR do not support the proposal that shifts between the cytoplasm and nucleus may regulate the functions of this receptor in Sertoli cells (43, 45). As for RA-synthesizing and RA-degrading enzymes, most of their expression patterns were not studied.
The present work indicates that in the mouse testis during postnatal development and adulthood, RAR and RXR are expressed in well defined cell populations (Table 1 and Fig. 8): Rara and Rxrb in Sertoli cells, Rarb, Rxra, and Rxrg in steps 7 and 8 spermatids, and Rarg in spermatogonia. Spermatocytes do not express RARs. As for RA-synthesizing enzymes, Aldh1a1 is mainly expressed in immature Sertoli cells and in adult Leydig cells and Aldh1a2 in pachytene spermatocytes and round spermatids, Aldh1a3 is faintly expressed in Leydig cells of adults only, and Bcmo1 is restricted to late round and elongating spermatids. As for RA-degrading enzymes, all three Cyp26 genes are confined to peritubular myoid cells, both in the adult and the developing testis. Lastly, our data indicate that Rbp1 is expressed mainly in Leydig cells and at much lower levels in Sertoli cells, and Lrat is expressed in early spermatids, whereas retinyl ester droplets are located in Sertoli cells.
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/RARß and RXR/RARß heterodimers could be functional in these cells. However, Rarb-, Rxrg-, and Rarb/Rxrg-null mice do not display reproductive defects, indicating that both RARß and RXR are dispensable for spermatogenesis in mice fed a standard breeding diet (Refs.53 and 64 and present report). On the other hand, the coexpression of Rara and Rxrb in Sertoli cells and the phenotypes resulting from Rara and Rxrb ablations suggest that RXRß/RAR heterodimers may control spermiation (39, 70).
With exception of spermiation defects, Rara- and Rxrb-null testes exhibit distinct sets of abnormalities. Those observed in Rxrb-null testes can be ascribed to a loss of the RXRß/liver X receptor-ß-mediated signal transduction (62). On the other hand, because no RXR other than RXRß is apparently expressed in Sertoli cells, the early testicular degeneration observed in Rara-null (50) but not in Rxrb-null mutants (39, 62) may highlight functions of RAR independent of RXR, for example through a RAR-mediated AP-1 transrepression. Likewise, because no RXR is detectable in spermatogonia, a RAR-mediated AP-1 transrepression may play a role in the mitotic phase of spermatogenesis (71).
Some RA effects on the seminiferous epithelium are apparently not dependent on RAR
The testicular degenerations resulting from Rara ablation and induced by VAD share similar features that strongly support the view that a liganded RAR is instrumental for the adhesion of germ cells to Sertoli cells (Ref.50 and our unpublished results). Another hallmark of VAD, namely the arrest of spermatogonia differentiation (7, 8), is not seen in the Rara-null testis (50). As shown here, RAR is a suitable candidate for mediating RA actions in spermatogonia. However, Rarg ablation has only an indirect impact on testicular histology. This apparent dispensability of RAR for spermatogonia differentiation may be accounted for by a functional redundancy between RARs, because low levels of RAR and/or RARß in spermatogonia, below the detection limits of IHC and ISH, could possibly compensate for the loss of RAR. Alternatively, the arrest of spermatogonia differentiation observed upon dietary VAD could result from a block of RA signal transduction occurring simultaneously in spermatogonia and Sertoli cells. This hypothesis will be tested through somatic mutagenesis of the RAR genes (72), using available transgenic lines expressing the Cre recombinase either in Sertoli cells (73) or in spermatogonia (74).
The peritubular cell layer functions as a catabolic barrier preventing RA from entering the seminiferous tubules
Because the testicular RA is not derived from the circulation (9), it has to be synthesized locally. We found two potential sites of RA synthesis in the adult mouse testis, one extratubular and the other intratubular. The former source relies on the expression of Aldh1a1 in Leydig cells. Although RALDH1 has only a weak retinaldehyde oxidizing activity in vitro, it actually synthesizes RA in vivo (65, 75). Thus, its high level of expression may permit conversion of retinaldehyde into RA in Leydig cells (Ref.18 and present report). That Aldh1a1-null mutants do not display reproductive defects possibly reflects the existence of a functional compensation by RALDH3, which is detected at low levels in Leydig cells.
Any RA made outside of the seminiferous tubules is nevertheless unlikely to reach Sertoli or germ cells, because of the expression, in peritubular myoid cells, of CYP26 (A1, B1, and C1) RA-degrading enzymes. The existence of this catabolic barrier provides the explanation for the inability of low doses of RA to restore spermatogenesis in VAD rodents (8, 9). In this context, it is noteworthy that although pharmacological doses of 4-oxo-RA are able to exert biological activities in the mouse testis (76), most of the oxidized RA metabolites produced by CYP26 enzymes (including 4-oxo-RA) cannot be detected in vivo (10) and are unlikely to exert physiological functions in the mouse (21).
The sources of RA synthesis and the loci of RA signal transduction are distinct in the mouse germ cells
In the testis, both ROL and ß-carotene represent potential substrates for RA synthesis (19, 77). Late round, elongating, and mature spermatids (steps 7–15) have the capacity to generate retinaldehyde from ROL and ß-carotenes through their expression of Adh4 (14) and Bcmo1 (present report), respectively. However, they cannot synthesize RA because they are devoid of RALDH (Fig. 8). Retinaldehyde originating from these spermatids may diffuse toward the closest germ cell population present within the same cell association, namely late pachytene spermatocytes, diplotene spermatocytes, and early (steps 1–6) spermatids, where it can be converted into RA by RALDH2 (Fig. 8). These three germ cell populations represent therefore essential sites of RA synthesis in the seminiferous epithelium. Variations in the expressions levels of Aldh1a2 and Bcmo1 during the seminiferous epithelium cycle may provide a means to modulate local RA concentrations and thus to control specific events of spermatogenesis.
No RAR is expressed in the RA-synthesizing spermatocytes (Fig. 8). Conversely, no RALDH is detected in germ cells that express RAR, namely spermatogonia and steps 7 and 8 spermatids. Despite expression of the alcohol dehydrogenase Adh1 (14) and of Aldh1a1 (present report), the capacity of Sertoli cells to convert ROL into RA is probably limited, because RALDH1 is present at low levels (Table 1) and has a weak retinaldehyde-oxidizing activity (see above). It appears therefore likely that steps 7 and 8 spermatids use RA synthesized by RALDH2 in their immediate precursors (i.e. step 6 spermatids). As for spermatogonia, they may receive most of their RA from the nearest Aldh1a2-expressing cells, i.e. the late pachytene and diplotene spermatocytes (from which they are separated only by slender Sertoli cell processes; Fig. 8), at least under physiological conditions. Interestingly, this compartmentalization of RA synthesis within the seminiferous epithelium may be used to modulate, through a paracrine mechanism, the synchronization between the mitotic and meiotic phases of spermatogenesis. On the other hand, it is well established that spermatogonial differentiation can resume normally under various conditions where more advanced germ cells are absent, notably during recovery from irradiation, during repopulation after transplantation, after replacement of cryptorchid testes in the scrotum, and once VAD mice are put again on a vitamin A-containing diet. Therefore, spermatogonia should also receive some RA synthesized by the neighboring Sertoli cells.
RA signaling appears dispensable for prenatal but not for postnatal testis development
At P1, there is no intratubular source of RA, and RARs are not expressed in gonocytes. Accordingly, fetuses lacking Rara and Rarb, Rara and Rarg, or Rarb and Rarg do not display testicular defects (Ref.53 and references therein). In addition, the CYP26-mediated catabolic barrier that prevents RA from reaching the seminiferous epithelium is present in peritubular myoid cell precursors at P1 (present report) and most probably also at fetal stages (78). Additionally, because the survival of cultured mouse gonocytes and Sertoli cell precursors is dramatically reduced by RA (43), it is possible that RA must be excluded from the fetal testis.
At P5, the seminiferous epithelium is still insulated from exogenous sources of RA through the Cyp26-expressing peritubular myoid cells. However, at this time, the Sertoli cell precursors display a robust expression of Aldh1a1 that may provide a ligand to activate RAR in these cells and RAR in spermatogonia. These observations suggest that, at P5, a RA signal may play important functions in the differentiation of Sertoli cell precursors and primitive spermatogonia.
Retinyl esters are stored in Sertoli cells and may originate from spermatids
We have shown here that mouse Sertoli cells and spermatids contain retinyl esters similarly to their rat counterparts (79). LRAT is the most potent ROL-esterifying enzyme, whose efficiency is enhanced by CRBP1 (58, 59). Rat Sertoli cells accordingly coexpress Lrat and Rbp1, whereas spermatids express Lrat but not Rbp1 (26, 60). In the mouse, we show here that Sertoli cells also express Rbp1 but not Lrat, whereas spermatids express Lrat but not Rbp1. Altogether, these observations suggest that 1) in the testis of both species the main site of Lrat expression is the spermatid population, where CRBPI is absent, and 2) as previously observed in cultured cells (80), ROL esterification by LRAT may not necessarily require CRBP. Importantly, many of the Lrat-null mouse males are sterile (81), suggesting that expression of Lrat in spermatids plays an important role in fertility. On the other hand, our data do not allow us to rule out the possibility that an acyl CoA:retinol acyltransferase activity may catalyze the esterification of ROL in Sertoli cells (82).
ADFP, which is localized at the surface of lipid droplets (reviewed in Ref.61), stimulates their formation (63). Interestingly, the Lrat-expressing spermatids are the only cells of the seminiferous epithelium that contain Adfp transcripts, which are translated into proteins in elongating spermatids. We therefore propose that coexpression of Lrat and Adfp couples retinyl ester synthesis with their packaging in lipid droplets. Our IHC observations also support the possibility that Sertoli cell retinyl ester stores are derived from spermatid lipid droplets that are delivered to the Sertoli cells after spermiation.
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First Published Online October 6, 2005
Abbreviations: ADFP, Adipose differentiation-related protein; AP-1, activator protein 1; CRABP, cellular retinoic acid-binding protein; CRBP, cellular retinol-binding protein; CYP, cytochrome P450 hydroxylase; DAPI, 4',6-diamidino-2-phenyl-indole; IHC, immunohistochemistry; ISH, in situ hybridization; LRAT, lecithin:retinol acyltransferase; P1, postnatal d 1; RA, retinoic acid; RALDH, retinaldehyde dehydrogenase; RAR, RA receptor; RBP, retinol-binding protein; ROL, retinol; RXR, rexinoid receptor; VAD, vitamin A-deficient; WT, wild type.
Received July 27, 2005.
Accepted for publication September 22, 2005.
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