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Biology of Reproduction 66, 500-507 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

RNA Binding Protein Musashi1 Is Expressed in Sertoli Cells in the Rat Testis from Fetal Life to Adulthood

P.T.K. Saunders1,,a, S.M. Maguirea, S. Macphersona, M.C. Fenelona, S. Sakakibarab, and H. Okano2,,b

a MRC Human Reproductive Sciences Unit, Edinburgh EH3 9ET, United Kingdom b Department of Neuroanatomy, Osaka University Graduate School of Medicine, Osaka, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Musashi1 (Msi1) gene identified in mouse is a member of a subfamily of RNA binding proteins that are highly conserved across species. Msi1 expression is highly enriched in proliferative cells within the developing central nervous system. Within the testis, proliferation and differentiation of germ cells takes place within the seminiferous epithelium, where these cells are supported physically and functionally by Sertoli cells that do not themselves proliferate following the onset of puberty. RNA binding proteins expressed in testicular germ cells are essential for normal fertility. Preliminary data suggested the mRNA for Msi1 was present in ovary; therefore, we used an Msi1-specific cRNA and monoclonal antibody to investigate whether Msi1 was expressed in the testis. Msi1 mRNA was expressed in rat testis from birth until adulthood; in situ hybridization revealed silver grains within the seminiferous epithelium. Immunohistochemical studies demonstrated that at all ages examined (from Fetal Day 14.5 until adulthood) Msi1 protein was expressed in Sertoli cells. In fetal and adult rat ovaries, Msi1 was detected in granulosa cells and their precursors. In Sertoli cells, protein was detected in both cytoplasmic and nuclear compartments; in adult testes, the immunointensity of the nuclear staining was stage dependent, with highest levels of expression in Sertoli cells at stages I–VI. In rat gonads, the RNA binding protein Msi1 is expressed in both proliferating and nonproliferating Sertoli and granulosa cells.

Sertoli cells, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis is the process whereby diploid stem cells differentiate and undergo meiosis followed by a series of complex transformations to become the mature haploid spermatozoa. Germ cells develop within the seminiferous epithelium in close association with the somatic Sertoli cells. The Sertoli cells are known to provide germ cells at different stages of their development with factors necessary for their survival and differentiation and with physical support [1, 2]. RNA binding proteins expressed in germ cells are important for mammalian fertility. For example, homologues of the autosomal RNA-binding protein dazl have been identified in a wide range of species, including human [3], mouse [4], Xenopus (xDazl) [5], zebrafish (zDazl) [6], and Drosophila (boule) [7]. In mice, the deletion of dazl results in infertility in both males and females [4], consistent with expression of dazl protein in germ cells in both the ovary and testis [4, 8]. Likewise, mammalian homologues of the Y-box proteins FRGY-1 and FRGY-2, first identified in Xenopus oocytes, are expressed in the testis [9]. The mammalian homologue of the Drosophila melanogaster staufen gene, which encodes an RNA binding protein (Dm Stau) required for the localization and translational repression of specific mRNAs within the Drosophila oocyte, is expressed in the germ cells of the mouse testis and ovary [10].

Musashi1 (Msi1) [11] in mouse is a member of a subfamily of RNA binding proteins that have also been identified in multiple species. The family includes Drosophila Musashi (d-msi) [12], Xenopus laevis nervous system-specific ribonucleoprotein 1 [13], Caenorhabditis elegans MSI1 [14], and human Musashi homologue 1 (MSI1) [15]. A high degree of conservation has been noted between family members; for example, d-msi and MSI1 show 58.8% amino acid identity within their RNA recognition motifs (RRMs) [15] and C. elegans MSI1 has 74% and 76% amino acid identity with human MSI1 within RRM1 and RRM2, respectively [14].

At the time we started this study, the only information available on gonadal expression of Msi1 was that its mRNA had been detected in the ovary by Northern blotting [11]. As part of continuing investigations of the role(s) of RNA binding proteins in male fertility, we decided to use rodent tissues to determine whether Msi1 was expressed in testis. Our expectation was that like the other RNA binding proteins we had examined expression would occur in germ cells. The results obtained have demonstrated expression of Msi1 in testis and ovary but have localized the protein to the somatic cells within follicles and seminiferous tubules rather than germ cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Recovery

Rats were housed under standard animal husbandry conditions and fed ad libitum. Gonads recovered from fetuses of timed-mated females on Days 14.5–20.5 postcoitum (the day on which a plug was observed was counted as Day 0.5), testes from immature animals (Postnatal Days 1, 5, 11, 15, 16, 18, 21, 28, and 35; n = 4–6 rats/group), and ovaries from adults (n = 4) were immersion fixed in Bouin solution for 6 h, stored in 70% ethanol. Tissues were embedded in paraffin wax, and 5-µm sections were cut. Testes from adult rats (Days 70–90) were recovered from perfusion-fixed animals as described previously [16]. Ovaries from adult mice (n = 6) and testes from immature and adult rats (ages as above) were used for extraction of RNA (Tri Reagent; Sigma, St. Louis, MO).

Preparation of Msi1 cDNA

A cDNA clone encoding Msi1 was amplified by polymerase chain reaction (PCR) [17] from a pool of mouse ovarian cDNAs prepared by random priming. Msi1-specific primers were selected to amplify bases 129–540 of the published sequence of Msi1 (accession no. D49654) [11]: 5' primer, 5'-GTTCATCGGAGGACTCAG-3'; 3' primer, 5'-GCTCTCAAACGTGACAAAT-3'. A single 413-base pair cDNA strand was amplified and cloned into the pCRII-TOPO vector (Invitrogen, NV Leek, The Netherlands) to create plasmid Msi1CRII. The identity of the cDNA was confirmed by sequence analysis.

RNA Extraction and Northern Blots

Total RNA (10 µg/lane) was separated on 1.5% denaturing agarose gels using standard conditions [18], transferred onto Hybond-N membranes (Amersham, Slough, Berkshire, UK) by capillary blotting using 10x saline-sodium citrate (SSC), and fixed to the membrane by ultraviolet light (Spectrolinker XL1000; Spectronics Co., Westbury, NY). Msi1 cDNA was labeled with 32P-dCTP using the Amersham Rediprime kit, and membranes were hybridized overnight at 60°C in 0.2 M phosphate buffer (pH 7.2) containing 1% (w/v) BSA (Sigma), 7% (w/v) SDS (Sigma), and 15% (v/v) deionized formamide, washed twice for 30 min each time in 40 mM phosphate buffer containing 1% SDS, then placed in stormscanner cassettes containing storage phospho screens (Molecular Dynamics, Sunnydale, CA) for 2 days. To determine RNA loading/transfer efficiency for each sample, membranes were stripped and rehybridized to a cDNA specific for 18S rRNA [19]. The relative optical densities of the Msi1 and 18S RNAs for each sample were determined on a Storm optical scanner using the ImageQuant program (Molecular Dynamics); the experiment was repeated twice.

In Situ Hybridization

DNA templates for riboprobe synthesis were prepared by digestion of Msi1CRII with XhoI or BamHI; antisense and sense riboprobes labeled with 35S-UTP were prepared by in vitro transcription using SP6 or T7 RNA polymerase, respectively, for 1–2 h at 37°C [10]. Following a brief incubation of sections with prehybridization buffer (50% deionized formamide, 4x STE [1x STE = 150 mM NaCl, 2.5 mM Tris, 0.25 mM EDTA], 1x Denhardt solution, 125 µg/ml salmon sperm DNA, 125 µg/ml yeast RNA, 10 mM dithiothreitol), hybridization was performed overnight at 50°C in 40 µl hybridization buffer (prehybridization buffer with 10% dextran sulfate) containing 106 cpm 35S-labeled cRNA. Excess probe was removed by washing in 4x SSC at room temperature. Sections were treated with ribonuclease A at 37°C (20 µg/ml for 30 min) and incubated first with with RNase buffer alone for 30 min and then with 2x SSC at room temperature and at 45°C for 30 min at each temperature. A final wash of 0.5x SSC at room temperature for 30 min was carried out before tissues were dehydrated, air dried, developed using NTB 2 (Kodak, Rochester, NY) emulsion for 3–8 wk, and photographed on a microscope (Olympus Optical Co., London, UK) using dark-field illumination.

Antibodies

The anti-Msi1 monoclonal antibody was produced in rats using a GST-mouse-Msi1 fusion protein as described in detail by Kaneko et al. [20]. Western blotting has been used to demonstrate that this antibody is specific for Msi1 and does not cross-react with Musashi2 [20]. Its application for the immunolocalization of Msi/MSI proteins in neuronal cells has already been described by Okano and collaborators [11, 20, 21]. A mouse monoclonal antibody specific for vimentin was obtained from DAKO (catalog no. M0725; Glostrup, Denmark); no specific staining was observed when primary antibodies were replaced with normal mouse serum (data not shown).

Fluorescent Coimmunolocalization of Msi1 and Vimentin

Sections were subjected to antigen retrieval by pressure cooking [22] for 5 min in 0.01 M citrate buffer, pH 6, allowed to stand undisturbed for a further 20 min, and washed in cold tap water and then in 1% (w/v) sodium borohydride for 10 min on a rocker platform. After a brief rinse in tap water, sections were washed twice in PBS, pH 7.4 (Sigma) for 5 min each; identical washes in PBS were carried out between all subsequent incubation steps. Sections were blocked with 1 part normal rabbit serum (NRS; Diagnostics Scotland, Lanark, Scotland, UK) and 4 parts PBS containing 5% BSA for 30 min at room temperature, incubated in the same buffer containing anti-Msi1 (1:5000) for 2 h at room temperature, and then incubated in biotinylated rabbit anti-rat IgG (Vector Laboratories, Peterborough, UK) diluted 1:500 in NRS/PBS/BSA (30 min). After incubation at room temperature for 2 h with avidin alexafluor-546 (Molecular Probes, Eugene, OR) diluted 1:200 in PBS, the fluorescence of the alexafluor-546 was enhanced by incubation with biotin (4 drops/ml PBS) for 2 min. Excess biotin was removed, and sections were reblocked for 30 min in NRS/PBS/BSA, incubated overnight with mouse anti-vimentin (DAKO) (1:200) at 4°C, rewashed, and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (1:20, Sigma) for 2 h. Sections were mounted in Permafluro (Immunotech-Coulter, High Wycomb, Bucks, UK) and viewed using a Provis epifluorescece microscope (Olympus). To view the alexafluor-546, excitation filters at 510–550 nm and emission filters at 590–800 nm were used. To view the FITC, excitation filters at 460–490 nm and emission filters at 515–550 nm were used.

Colocalization of Msi1 and Ethidium Homodimer

Sections were processed, subjected to antigen retrieval, washed successively in borohydride and PBS, and blocked in NRS as described above; sections were washed with PBS (twice for 5 min each time) between all subsequent incubations. Sections were incubated in anti-Msi1 diluted 1:2000 in NRS/PBS for 2 h at room temperature and were reincubated sequentially with biotinylated rabbit anti-rat Ig diluted 1:500 in NRS/PBS for 30 min and with avidin alexafluor-488 (Molecular Probes) diluted 1:200 in PBS (2 h, room temperature); the fluorescence of the alexafluor-488 was enhanced by incubation with biotin (Vector Laboratories; 4 drops/ml PBS) for 2 min. Excess biotin was removed by washing and all nuclei were stained red by incubation with ethidium homodimer (2.5 µl/ml of PBS; Molecular Probes) for 1 min, excess stain was removed, and sections were mounted in Permafluor. Fluorescent images were captured on a Zeiss LSM 510 laser scanning microscope (Carl Zeiss Inc., Thornwood, NY). The alexafluor-488 was detected using the krypton/argon laser (excitation beam, 488 nm) and an emission band-pass filter BP of 505–530 nm. The eithidium homodimer was detected with the helium/neon laser (excitation beam, 543 nm) and an emission long-pass filter LP560.

Immunohistochemistry for Msi1

Sections were dewaxed and rehydrated, and endogenous peroxidase was blocked by incubation in 3% (v/v) hydrogen peroxide in methanol for 30 min. After washing in Tris-buffered saline (TBS; 0.05 M Tris, pH 7.4, 0.85% NaCl), sections were subjected to antigen retrieval (see above) before washing in TBS for 5 min. Sections were blocked for 30 min at room temperature with 1 part NRS and 4 parts TBS containing 5% (w/v) BSA. Anti-Msi1 antibody was diluted 1:20 000 in blocking buffer and incubated on sections under coverslips overnight at 4°C. After washing twice for 5 min each time in TBS, sections were incubated with biotinylated rabbit anti-rat immunoglobulins (Vector), diluted 1:500 in blocking buffer for 30 min, washed twice for 5 min each time in TBS, and incubated for 30 min with avidin-biotin-horseradish peroxidase complex (DAKO). After further washes in TBS (twice for 5 min each time), bound antibodies were visualized using 0.05% (w/v) 3,3'-diaminobenzidene tetra-hydrochloride (Sigma) in 0.05 M Tris-HCl, pH 7.4, and 0.01% hydrogen peroxide. Sections were then washed in water, counterstained with hematoxlin, dehydrated, and mounted. Specificity of immunostaining was checked using normal rat serum in place of primary antibody or using secondary antibody alone. Images were captured onto a DC330 camera and assembled using Photoshop 5.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Msi1 mRNA in Rat Testis

Northern blot analysis of total testicular RNA resulted in the detection of 1 major Msi1 mRNA transcript of approximately 3.1 kilobases consistent with previously published results [11]. In 2 separate experiments, highest levels of expression of Msi1 mRNA relative to total testicular RNA appeared to be present on and around Day 18 of postnatal life (results not shown).

Hybridization of Msi1 cRNA to sections of immature rat testes (Postnatal Days 6–16) resulted in localization of silver grains over the seminiferous epithelium (Fig. 1a, arrows); the level of expression of Msi1 mRNA did not appeared to vary among individual tubules. Following puberty (e.g., on Days 28 and 35) when the diameter of the seminiferous tubule had expanded, the silver grains were localized around the base of the tubule and appeared to differ in intensity among tubules (Fig. 1c, arrows).



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  		FIG. 1. In situ hybridization with Msi1 cRNA. Silver grains (arrows) are concentrated over the seminiferous tubules in rats at all ages examined, including Day 11 (a and b) and Day 35 (c and d). In older animals, the highest concentration of silver grains is at the periphery of the tubule, consistent with expression in Sertoli cells or spermatogonia (c, white arrows; d, black arrows). Exposure 33 days, magnification x20; b and d are light-field views of the same sections shown in a and c, respectively. Inset (c) shows sense strand control. Bar = 100 µm

Localization of Msi1 Protein to the Cytoplasm and Nuclear Compartments of Sertoli Cells

To determine the cellular site(s) of Msi1 expression in the rat testis, the protein was colocalized with vimentin because previous studies have shown that this structural protein is found in intermediate filaments of somatic cells within the mammalian testis throughout life. In the Sertoli cells of adult rats, vimentin shows stage-dependent changes in distribution and is concentrated in the perinuclear region [23]. In the present study, the pattern of vimentin staining in the adult (Fig. 2d) was identical to that reported previously with clear staining of perinuclear cytoplasm (arrows). In immature animals (Fig. 2, a–c), immunostaining for vimentin was concentrated around the periphery of the seminiferous epithelium in Sertoli cell cytoplasm, which appeared as fine projections into the center of the tubule (see Day 16, Fig. 2b). The subcellular distribution of Msi1 (Fig. 2, red staining) was distinct from that of vimentin, although it was clearly not present in germ cells (Fig. 2, a and c). At Day 16, the red immunopositive signal was present in the center of the tubule (Fig. 2b, asterisk), but this signal was lost once the tubule lumen expanded (Fig. 2c), consistent with protein expression in Sertoli cell cytoplasm. An immunopositive signal was also detected in Sertoli cell nuclei (Fig. 2, b and c, small arrows).



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  		FIG. 2. Double immunofluroescence. a–d) Double immunostaining for Msi1 (red) and vimentin (green). On Day 5 (a), vimentin is localized at the periphery of the rat seminiferous tubule, and Msi1 (red )is present within the seminiferous cord and absent from germ cells (g) and the intersitium (i). At older ages (Day 16, b; Day 21, c) in addition to positive staining within Sertoli cell cytoplasm (e.g., asterisk, b) staining of nuclei is visible (short white arrows). In the Sertoli cells of the adult rat, vimentin is associated with intermediate filaments and concentrated around the periphery of the nucleus (long arrows), whereas Msi1 immunopositive staining is more widely expressed within the cell. e and f) Double immunostaining for Msi1 (green) and ethidium homodimer (red). In the adult testis, nuclei of all classes of germ cells are stained intensely with the ethidium homodimer, staining of Sertoli cell nuclei is significantly less intense than that of the germ cells, but the periphery of the nuclei and the nucleoli are easily identified (e.g., arrows, e). Msi1 protein is clearly visible in the Sertoli cell cytoplasm between the germ cells (asterisks, e) and within the Sertoli cell nuclei (arrows, f) outlined by the ethidium homodimer staining. Magnification x100 for a–c, x200 for d. Bars = 50 µm (a), 25 µm (d)

Staining of fixed sections of adult testicular tissue with ethidium homodimer allowed the identification of nuclei of all cell types within the seminiferous epithelium (Fig. 2, e and f); the intensity of ethidium staining was far greater in the germ cells than in Sertoli cells. Sertoli cell nuclei stain poorly with other nuclear stains when tissue is embedded in paraffin (unpublished observations). Double staining with anti-Msi1 antibodies confirmed that the protein was present in Sertoli cell cytoplasm (Fig. 2, e and f, green staining, asterisk) but was also present within the Sertoli cell nuclei (Fig. 2, e and f, arrows) and provided confirmation of the results obtained with vimentin.

Developmental Expression of Msi1 Protein in Sertoli Cells

Msi1 protein was expressed in the rat testis from Fetal Day 14.5, the earliest day on which we can distinguish testis and ovary morphologically [24], up to and including adulthood (Days 70–90). In fetal testes from Days 14.5 (Fig. 3a) and 17.5 (Fig. 3c), Sertoli cells within the seminiferous cords were immunopositive (arrows), whereas germ cells (g) were clearly immunonegative. In immature testes, for example on Day 11 (Fig. 3e), prior to the formation of a lumen within the seminiferous tubule, Sertoli cell cytoplasm in the center of the tubule (asterisk) was immunopositive and Sertoli cell nuclei were clearly stained. At later ages (Day 19, Fig. 3f; Day 35, Fig. 3g), immunopositive staining was maintained in Sertoli cells, where it was detected in both nuclear and cytoplasmic compartments, and variations in intensity of staining between individual tubule cross sections were detected. To examine whether the early onset of expression of Msi1 in the fetal testis was associated with organization of somatic cells into seminiferous cords, fetal ovaries of similar ages were immunostained (Day 14.5, Fig. 3b; Day 18.5, Fig. 3d). At all ages examined, some somatic cells within the fetal ovary were immunopositive for Msi1 (arrowheads), and germ cells were immunonegative. Msi1 was also detected in both nuclear and cytoplasmic compartments of granulosa cells in preantral and antral follicles in adulthood (Fig. 3h).



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  		FIG. 3. Expression of Msi1 protein in testes and ovaries from rats. In testes, Msi1 protein is expressed in rat Sertoli cells (SC, arrows) at all ages examined including Fetal Days 14.5 (a) and 17.5 (c); protein is present in both SC cytoplasm and nucleus. In most samples, the intensity of the immunoreaction is higher in the nucleus (e.g., Day 11, e; Day 19, f). In the immature testis (Day 11, e), the cytoplasm of the SC, which occupies the center of the tubule, is clearly immunopositive (asterisks). On Day 35 (g), intensity of Msi1 expression in SC nuclei is variable among tubules. In ovaries, Msi1 is present in somatic cells during fetal life (Day 14.5, b; Day 18.5, d), and granulosa cells in both small (sm) and large (Gc) follicles are immunopositive in adult ovaries (h). In, Interstitium; g, gonocyte (immunonegative); lu, lumen. Magnification x40. Bar (a) = 50 µm (applies to all images)

Stage-Dependent Expression of Msi1 Protein in Adult Sertoli Cells

In the adult rat testis, Msi1 was expressed in Sertoli cell nuclei and cytoplasm as during other stages of development. However, although Sertoli cell cytoplasm was immunopositive in cells at all stages of the spermatogenic cycle a marked variation, which appeared to be stage dependent, was noted in the intensity of nuclear staining among individual Sertoli cells (Fig. 4), with highest levels of immunoexpression apparent in nuclei during the first half of the cycle (stages I–VI, Fig. 4, a, b, and d). Examination of sections on a confocal microscope confirmed that the Msi1 protein was not colocalized with vimentin in intermediate filaments (data not shown).



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  		FIG. 4. Expression of Msi1 protein in adult rat testes. Expression in Sertoli cell nuclei (arrowheads) is stage dependent, with a more intense immunohistochemical reaction in tubules during the first half of the cycle, i.e., stages II (a), III (a and d), and IV (b), than during stages VII (b and c), VIII (not shown), IX (c), and X (d). Msi1 protein is present in the cytoplasm of the Sertoli cells throughout the cycle (arrows). Magnification x40. Bar (a) = 50 µm (applies to all images)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we demonstrated for the first time that the RNA binding protein Msi1 is expressed by Sertoli cells within the seminiferous epithelium of the rat testis. Msi1 is present in the Sertoli cells from the earliest time at which we can detect morphological differentiation of the fetal testis (Day 14.5) [24], throughout the development of the first wave of spermatogenesis, and into adulthood. Similar results were obtained when sections of mouse testes were immunostained (unpublished observations). The expression of Msi1 by somatic cells was a surprise; we had expected it to be present in the germ cells, which express several RNA binding proteins that play important roles in regulating protein expression [25, 26]. Preliminary studies using ovarian tissues revealed that Msi1 is expressed by ovarian somatic cells during fetal life and within the granulosa cells of the follicles during adulthood. Sertoli cells and granulosa cells both differentiate from somatic cell precursors that are present in the genital ridge prior to sex determination (reviewed in [27]). Numerous studies have demonstrated that similarities in the pattern of gene expression exist between these 2 cell types, probably reflecting the essential role they play in supporting germ cell differentiation and maturation in males and females, respectively. For example, both Sertoli and granulosa cells express FSH receptor [28], estrogen receptor beta [29], androgen receptor [30, 31], inhibin [32], and GATA transcription factors [33]. Msi1 expression would therefore appear to represent another example of a protein that may be important for somatic cell function within both male and female gonads.

The apparent variability in the level of Msi1 protein detected in adult Sertoli cell nuclei at different stages of the spermatogenic cycle may be a reflection of their response to different functional demands placed upon them by the germ cells they are supporting [2, 34]. For example, cyclical variation in the expression of receptors to FSH [35] and androgen [30] and in FSH-stimulated cAMP levels [36] have all been used as evidence that the hormonal regulation of spermatogenesis may be mediated via stage-specific responsiveness of Sertoli cells [2]. The most intense immunohistochemical staining for Msi1 was present in Sertoli cell nuclei at stages I–V; at these stages, Sertoli cells also express the highest levels of FSH receptor mRNA [35], the phosphodiesterase PDEIVd subtype [37], and the cAMP binding protein CREB, which mediates FSH-induced changes in gene expression [38]. Additional studies such as Western blot analysis of staged fragments will be needed to provide independent evidence of stage-dependent variation and to allow more exact measurement of the total amount of Msi1 protein present at each stage. Unfortunately, our attempts to investigate whether stage-dependent expression of Msi1 could be due to hormonal regulation of the gene have been hampered by our inability to maintain expression in Sertoli cells in primary cultures (unpublished observations). A similar downregulation in expression of the androgen-regulated homeobox gene PEM has been reported to occur in vitro even though the protein was readily detectable in Sertoli cell nuclei on tissue sections [39]. We speculate that the loss of Msi1 and PEM reflects the need for Sertoli cells to maintain functional links to each other and for a close association with germ cells to maintain differentiated cell function.

In the absence of a suitable cell culture system, we can consider the role(s) played by other RNA binding proteins within the testis and data newly emerging from studies on Msi1 in neural cells in considering how Msi1 may influence Sertoli cell function. Our understanding of the role(s) of RNA binding proteins in testicular cells has been enhanced by consideration of their pattern of expression. For example, RBM1 (RNA-binding motif, formerly YRRM [40]) is expressed exclusively within the nucleus of germ cells and is associated with regions of the cell where splicing of RNA is taking place [41]. In contrast, the mouse homologue of the human single-stranded DNA binding protein translin was first identified as an RNA binding protein involved in translational regulation in testicular germ cells [42] and has been shown to dimerize and to associate with microtubules in the cytoplasm [43, 44]. In mouse testis, translin is detected in the cytoplasm of premeiotic spermatogonia, but as germ cells enter meiosis significant amounts of protein are detected in association with nuclear chromatin. At later stages of germ cell development, the protein is in the nuclei and cytoplasm of spermatocytes, whereas in haploid germ cells the protein is largely found in the cytoplasm [45]. These data suggest a role for translin in mRNA transport from nucleus to cytoplasm and the sharing of mRNAs between germ cells [46]. Another RNA binding protein, Rbm3, has recently been identified in mouse Sertoli cells and in parts of the brain, intestine, uterus, and spleen [47]. In the figures published by Danno et al. [47], Rbm3 was most abundant in Sertoli cell nuclei, but some immunopositive staining appeared to be present in the cytoplasm, although in adulthood expression was not stage dependent. No data are yet available on the functional activity of Rbm3.

Recent studies have shed light on the actions of Msi1 in neural cells in several species. In Drosphila, dmsi is essential for asymmetric cell divisions that occur during formation of external sensory organs [12]. In contrast, in C. elegans MSI1 is not expressed in proliferating neural progenitors but in postmitiotic neurones and is essential for male mating behavior [14]. In the mouse, msi1 has been localized to the ventricular zone of the neural tube during embryogenesis [11]. The high level of conservation of sequence among Msi1 proteins in different species has allowed a comparison of the immunoexpression of the proteins in the brains of Xenopus, chick, mouse [20] and human [21]. In all 4 species, the proteins were immunolocalized to proliferating cells, and in the mouse brain immunoreactivity was almost completely confined to areas of active neurogenesis and gliogenesis. In cultured neural cells, Msi1 protein has been detected in the cytoplasm or the nucleus or in both compartments [20] and was present in mitotic and immature postmitotic neurons but not in mature neurons with long, thick processes. In the rat testis, active proliferation of Sertoli cells is highest during fetal life, diminishes after birth, and stops on or about Day 15 [48]; therefore, expression of Msi1 in the testis is not confined to proliferating cells.

Studies to identify which RNA sequences Msi1 regulates have shown to that it can bind poly G and poly U in vitro [11] and can bind to UG-rich sequences ([G/A]UnAGU; n = 1–3) on SELEX [49]. The localization of Msi1 to both the cytoplasm and nucleus in Sertoli cells is similar to that of heterogeneous nuclear ribnucleoprotein A1 (hnRNPA1), with which it shares some sequence homology within the RNA recognition motifs [11, 50] Heterogeneous nRNPA1 has also been shown to bind to UG-rich sequences that are present in splice sites of pre-mRNAs [51]. A number of hnRNPs, including hnRNPA1, have been shown to shuttle between the nucleus and cytoplasm, and Msi1 may function within the Sertoli cell in a similar way by binding to nascent mRNAs within the nucleus and, once these mRNAs are processed, facilitating their export into the cytoplasm. Alternatively, Msi1 may act like translin does [42] to regulate expression of specific mRNAs. In Drosophila, dmsi represses translation of tramtrack, which codes for a protein involved in regulation of the Notch signaling pathway [52]. The consensus binding site for Msi1 has recently been identified within the 3' untranslated region (UTR) of the mRNA mammalian numb (m-numb), which encodes a membrane-associated antagonist of Notch signaling [49]. Reporter gene assays using the 3' UTR of m-numb have shown that Msi1 could regulate m-numb expression at the translational level [49]. Additional studies are needed to identify mRNA targets in Sertoli and granulosa cells, the translation of which could be regulated by mammalian Msi1, but mammalian homologues of the Notch signaling pathway may be potential targets.

The RNA binding protein Msi1 is expressed in the cytoplasm and nucleus of proliferating and nonproliferating Sertoli cells. However, the functional significance of gonadal Msi1 remains to be determined.


   FOOTNOTES
 
First decision: 29 May 2001.

1 Correspondence: Philippa T.K. Saunders, MRC Human Reproductive Sciences Unit, 37 Chalmers Street, Edinburgh EH3 9ET, UK. FAX: 44 131 228 5571; p.saunders{at}ed.ac.uk Back

2 Current address: Department of Physiology, Keio University School of Medicine, Tokyo, Japan. Back

Accepted: September 28, 2001.

Received: May 4, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Griswold MD. Interactions between germ cells and Sertoli cells in the testis. Biol Reprod 1995; 52:211-216[Abstract]
  2. Sharpe RM. Regulation of spermatogenesis. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, 2nd ed. New York: Raven Press; 1994: 1363–1434
  3. Saxena R, Brown LG, Hawkins T, Alagappan RK, Skaletsky H, Reeve MP, Reijo R, Rozen S, Dinulos MB, Disteche CM, Page DC. The DAZ gene cluster on the human Y chromosome arose from an autosomal gene that was transposed, repeatedly amplified and pruned. Nat Genet 1996; 14:292-299[CrossRef][Medline]
  4. Ruggiu M, Speed R, Taggart M, McKay SJ, Kilanowski F, Saunders P, Dorin J, Cooke HJ. The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis. Nature 1997; 389:73-77[CrossRef][Medline]
  5. Houston DW, Zhang J, Maines JZ, Wasserman SA, King ML. A Xenopus DAZ-like gene encodes an RNA component of germ plasm and is a functional homologue of Drosophila boule. Development 1998; 125:171-180[Abstract]
  6. Maegawa S, Yasuda K, Inoue K. Maternal mRNA localization of zebrafish DAZ-like gene. Mech Dev 1999; 81:223-226[CrossRef][Medline]
  7. Eberhart CG, Maines JZ, Wasserman SA. Meiotic cell cycle requirement for a fly homologue of human deleted in azoospermia. Nature 1996; 381:783-785[CrossRef][Medline]
  8. Ruggiu M, Saunders PTK, Cooke HJ. Dynamic subcellular distribution of the DAZL protein is confined to primate male germ cells. J Androl 2000; 21:470-477[Abstract]
  9. Kwon YK, Murray MT, Hecht NB. Proteins homologous to the Xenopus germ cell-specific RNA-binding proteins p54/p56 are temporally expressed in mouse germ cells. Dev Biol 1993; 158:90-100[CrossRef]
  10. Saunders PTK, Pathirana S, Maguire SM, Doyle M, Wood T, Bownes M. The mouse staufen gene is expressed in germ cells during oogenesis and spermatogenesis. Hum Mol Reprod 2000; 6:983-991
  11. Sakakibara S, Imai T, Hamaguchi K, Okabe M, Aruga J, Nakajima K, Yasutomi D, Nagata T, Kurihara Y, Uesugi S, Miyata T, Ogawa M, Mikoshiba K, Okano H. Mouse-Musashi-1, a neural RNA-binding protein highly enriched in the mammalian CNS stem cell. Dev Biol 1996; 176:230-242[CrossRef][Medline]
  12. Nakamura M, Okano H, Blendy J, Montell C. Musashi, a neural RNA-binding protein required for Drosphila adult sensory organ development. Neuron 1994; 13:67-81[CrossRef][Medline]
  13. Richter K, Good P, Dawid I. A developmentally regulated, nervous system specific gene in Xenopus encodes a putative RNA binding protein. New Biol 1990; 2:556-565[Medline]
  14. Yoda A, Sawa H, Okano O. MSI-1, a neural RNA-binding protein, is involved in male mating behaviour in Caenorhabditis elegans. Genes Cells 2000; 5:885-895[Abstract]
  15. Good P, Yoda A, Sakakibara S, Yamamoto A, Imai T, Sawa H, Ikeuchi T, Tsuji S, Satoh H, Okano H. The human Musashi homolog 1 (MSI1) gene encoding the homologue of Musashi/Nrp-1, a neural RNA-binding protein putatively expressed in CNS stem cells and neural progenitor cells. Genomics 1998; 52:382-384[CrossRef][Medline]
  16. Millar MR, Sharpe RM, Maguire SM, Saunders PTK. Cellular localisation of messenger RNAs in rat testis: application of digoxigenin labelled probes to embedded tissue. Cell Tissue Res 1993; 273:269-277[CrossRef][Medline]
  17. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988; 239:224-230
  18. Davis LG, Dibner MD, Battey JF. Basic Methods in Molecular Biology. New York: Elsevier; 1986
  19. Turner KJ, Morley M, Macpherson S, Millar MR, Wilson JA, Sharpe RM, Saunders PTK. Modulation of gene expression by androgen and oestrogens in the testis and prostate of the adult rat following androgen withdrawal. Mol Cell Endocrinol 2001; 178:73-87[CrossRef][Medline]
  20. Kaneko Y, Sakakibara S, Imai T, Suzuki A, Nakamura Y, Sawamoto K, Ogawa Y, Toyama Y, Miyata T, Okano H. Musashi1: an evolutionally conserved marker for CNA progenitor cells including neural stem cells. Dev Neurosci 2000; 22:139-153[CrossRef][Medline]
  21. Pincus DW, Keyoung HM, Harrison-Restelli C, Goodman RR, Fraser RA, Edgar M, Sakakibara S, Okano H, Nedergaard M, Goldman SA. Fibroblast growth factor-2/brain derived neurotrophic factor-associated maturation of new neurons generated from adult human subependymal cells. Ann Neurobiol 1998; 43:576-585
  22. Norton AJ, Jordan S, Yeomans P. Brief, high-temperature heat denaturation (pressure cooking): a simple and effective method of antigen retrieval for routinely processed tissues. J Pathol 1994; 173:371-379[CrossRef][Medline]
  23. Zhu LJ, Zong SD, Phillips DM, Moo-Young AJ, Bardin CW. Changes in the distribution of intermediate filaments in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development. Anat Rec 1997; 248:391-405[CrossRef][Medline]
  24. Majdic G, Millar MR, Saunders PTK. Immunolocalisation of androgen receptor to interstitial cells in fetal rat testes and to mesenchymal and epithelial cells of associated ducts. J Endocrinol 1995; 147:285-293[Medline]
  25. Elliott DJ, Cooke HJ. The molecular genetics of male infertility. Bioessays 1997; 19:801-809[CrossRef][Medline]
  26. Kwon Y, Hecht N. Binding of a phosphoprotein to the 3' untranslated region of the mouse protamine 2 mRNA temporally represses its translation. Mol Cell Biol 1993; 13:6547-6557[Abstract/Free Full Text]
  27. Swain A, Lovell-Badge R. Mammalian sex determination: a molecular drama. Genes Dev 1999; 13:755-767[Free Full Text]
  28. Dierich A, Sairam MR, Monaco L, Fimia GM, Gansmuer A, LeMeur M, Sassone-Corsi P. Impairing follicle-stimulating hormone (FSH) signaling in vivo: targeted disruption of the FSH balance leads to aberrant gametogenesis and hormonal imbalance. Proc Natl Acad Sci U S A 1998; 95:13612-13617[Abstract/Free Full Text]
  29. Saunders PTK, Maguire SM, Gaughan J, Millar MR. Expression of oestrogen receptor beta (ERbeta) in multiple rat tissues visualised by immunohistochemistry. J Endocrinol 1997; 154:R13-R16[Abstract]
  30. Bremner WJ, Millar MR, Sharpe RM, Saunders PTK. Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens. Endocrinology 1994; 135:1227-1234[Abstract]
  31. Tesuka M, Whitlaw PF, Bremner WJ, Millar MR, Symth CD, Hillier SG. Developmental regulation of androgen receptor in rat ovary. J Endocrinol 1995; 145:535-543[Medline]
  32. Ying SY. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev 1988;; 9:267-293[Abstract]
  33. Tremblay JJ, Viger RS. GATA factors differentially activate multiple gonadal promotors through conserved GATA regulatory elements. Endocrinology 2001; 142:977-986[Abstract/Free Full Text]
  34. Parvinen M. Cyclic function of Sertoli cells. In: Russell LD, Griswold MD (eds.), The Sertoli Cell. Clearwater, FL: Cache River Press; 1993: 331–347
  35. Rannikko A, Penttila TL, Zhang FP, Toppari J, Parvinen M, Huhtaniemi I. Stage-specific expression of the FSH receptor gene in the prepubertal and adult rat seminiferous epithelium. J Endocrinol 1996; 151:29-35[CrossRef][Medline]
  36. Kangasniemi M, Kaipia A, Toppari J, Mali P, Huhtaniemi IP, Parvinen M. Cellular regulation of basal and FSH-stimulated cyclic AMP production in irradiated rat testes. Anat Rec 1990; 227:32-36[CrossRef][Medline]
  37. Morena AR, Biotani C, de Grossi S, Stefanini M, Conti M. Stage and cell-specific expression of the adenosine 3',5' monophosphate-phosphodiesterase genes in the rat seminiferous epithelium. Endocrinology 1995; 136:687-695[Abstract]
  38. Suire S, Fontaine I, Guillou F. Follicle stimulating hormone (FSH) stimulates transferrin gene transcription in rat Sertoli cells: cis and trans-acting elements involved in FSH action via cyclic adenosine 3',5'-monophosphate on the transferrin gene. Mol Endocrinol 1995; 9:756-766[Abstract]
  39. Sutton K, Maiti S, Tribley W, Lindsey J, Meistrich M, Bucana C, Sanborn B, Joseph D, Griswold M, Cornwall G, Wilkinson M. Androgen regulation of the Pem homeodomain gene in mice and rat Sertoli and epididymal cells. J Androl 1998; 19:21-30[Abstract/Free Full Text]
  40. Ma K, Inglis J, Sharkey A, Bickmore W, Hill R, Prosser E, Speed R, Thomson E, Jobling M, Taylor K, Wolfe J, Cooke H, Hargreave T, Chandley A. A Y chromosome gene family with RNA binding protein homology: candidates for the azoospermia factor AZF controlling human spermatogenesis. Cell 1993; 75:1287-1295[CrossRef][Medline]
  41. Elliott DJ, Oghene K, Makarov G, Makarova O, Hargreave TB, Chandley AC, Eperon IC, Cooke HJ. Dynamic changes in the subnuclear organisation of pre-mRNA splicing proteins and RBM during human germ cell development. J Cell Sci 1998; 111:1255-1265[Abstract]
  42. Kwon Y, Hecht N. Cytoplasmic protein binding to highly conserved sequences in the 3' untranslated region of mouse protamine 2 mRNA, a translationally regulated gene of male germ cells. Proc Natl Acad Sci U S A 1991; 88:3584-3588[Abstract/Free Full Text]
  43. Han JR, Yui GK, Hecht NB. Testis/brain RNA-binding protein attaches translationally repressed and transported mRNAs to microtubules. Proc Natl Acad Sci U S A 1995; 92:9550-9554[Abstract/Free Full Text]
  44. Wu XQ, Xu L, Hecht NB. Dimerization of the testis brain RNA-binding protein (translin) is mediated through its C-terminus and is required for DNA- and RNA-binding. Nucleic Acids Res 1998; 26::1675-1680[Abstract/Free Full Text]
  45. Morales CR, Wu XQ, Hecht NB. The DNA/RNA-binding protein, TB-RBP, moves from the nucleus to the cytoplasm and through intercellular bridges in male germ cells. Dev Biol 1998; 201:113-123[CrossRef][Medline]
  46. Hecht NB. Intracellular and intercellular transport of many germ cell mRNAs is mediated by the DNA- and RNA-binding protein, testis-brain-RNA-binding protein (TB-RBP). Mol Reprod Dev 2000; 56::252-253[CrossRef][Medline]
  47. Danno S, Itoh K, Matsuda T, Fujita J. Decreased expression of mouse Rbm3, a cold-shock protein, in Sertoli cells of the cryptorchid testis. Am J Pathol 2000; 156:1685-1692[Abstract/Free Full Text]
  48. van Haaster LH, De Jong FH, Docter R, de Rooij DG. The effect of hypothyroidism on Sertoli cell proliferation and differentiation and hormone levels during testicular development in the rat. Endocrinology 1992; 131:1574-1576[Abstract]
  49. Imai T, Tokunaga A, Yoshida T, Hasimoto M, Mikoshiba K, Weinmaster G, Nakafuku M, Okano H. The neural RNA-binding protein Musashi1 translationally regulates mammalian numb gene expression by interacting with its mRNA. Mol Cell Biol 2001; 21:3888-3900[Abstract/Free Full Text]
  50. Garrett DS, Lodi PJ, Shamoo Y, Williams KR, Clore GM, Gronenborn AM. Determination of the secondary structure and folding topology of an RNA binding domain of mammalian hnRNP A1 protein using three-dimensional heteronuclear magnetic resonance spectroscopy. Biochemistry 1994; 15:2852-2858
  51. Burd CG, Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science 1994; 265:615-621[Abstract/Free Full Text]
  52. Okabe M, Imai T, Kursu M, Hiromi Y, Okano H. Translational repression determines a neuronal potential in Drosophila asymmetric cell division. Nature 2001; 411:94-98[CrossRef][Medline]



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P J O'Shaughnessy, L Hu, and P J Baker
Effect of germ cell depletion on levels of specific mRNA transcripts in mouse Sertoli cells and Leydig cells
Reproduction, June 1, 2008; 135(6): 839 - 850.
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