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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 19, 2006 as doi:10.1096/fj.04-3314fje. |
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,1,
,1
,||,2
* Institut National de la Santé et de la Recherche Médicale U589, Toulouse, France; Université Paul Sabatier, Toulouse, France;
MilleGen, Prologue Biotech, Labège, France;
Laboratoire "Oncogenèse, Différenciation et Transduction du Signal,’ Villejuif, France; and
|| Centre Hospitalier Universitaire de Rangueil, Toulouse, France
2Correspondence: E-mail: pratsac{at}toulouse.inserm.fr
SPECIFIC AIMS
Fibroblast growth factor 2 (FGF-2) is involved in both development and maintenance of testis, especially in gametes production and steroids synthesis. Previous studies have shown that FGF-2 expression is mostly regulated at the translational level, resulting in synthesis of several isoforms with different localizations and functions. The FGF-2 mRNA translation in controlled by an IRES, a structural element in the mRNA leader region allowing translation to occur by a nonclassical mechanism. Few data are available about IRES pathophysiological function, as most studies of IRES regulation have been performed in vitro.
Here we have studied in vivo the physiological role of the FGF-2 IRES in the control of FGF-2 expression during testis development and maintenance of testis function.
PRINCIPAL FINDINGS
1. FGF-2 IRES is age dependently regulated in mouse testis germinal cells
We have developed a unique transgenic animal model to study tissue specificity and regulation of IRES-dependent translation in vivo. This is based on use of a bicistronic vector expressing Renilla luciferase (LucR) and firefly luciferase (LucF), encoded by the same mRNA (Fig. 1
A). The upstream cistron (LucR) is translated via the classical cap-dependent scanning mechanism, whereas translation of the second cistron (LucF) is IRES-driven.
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Sexually immature (3 wk postpartum) or mature (6, 12, or 60 wk postpartum) transgenic mice carrying bicistronic transgenes with EMCV, FGF-2, or c-myc IRES were analyzed for IRES activity in testis. The FGF-2 IRES, low at 3 wk, increased up to 6-fold at 6 wk (Fig. 1B
). In contrast, EMCV and c-myc IRES activities remained stable, suggesting that testis maturation is accompanied by a specific activation of the FGF-2 IRES.
To determine which testis cells express LucF in vivo, sections of fresh testes were analyzed by immunohistofluorescent staining (Fig. 1C
) or immunohistochemistry (Fig. 1D
). With both techniques, staining appeared at 6 wk in cells morphologically identified as type A spermatogonias, stem cells of the germinal line, identified by two spermatogonia markers, hnRNPAI and Oct 3/4. Analysis of endogenous FGF-2 revealed specific labeling in spermatogonia at 6 wk; labeling at 3 wk was mostly localized in the interstitial compartment, presumably in Leydig cells. EMCV IRES had a strong activity in blood vessels and interstitial cells in young and adult testis, demonstrating that the LucF expression in adult spermatogonia is due to a physiological control specific to the FGF-2 IRES. Thus, the IRES-dependent mechanism allows a germinal cell-specific activation of FGF-2 expression.
2. FGF-2 IRES activation in adult mice spermatogonia is testosterone-dependent
In immature testis, spermatogenesis is driven by FSH; in the adult this process is controlled by testosterone produced by Leydig cells (Fig. 2
). Transgenic mice were treated by testosterone at 3 wk postpartum. This resulted in a 5-fold increase of IRES activity, reaching the level observed in mature testis (Fig. 3
A). We also tried to block the testosterone action by using a siRNA directed against the androgen receptor (AR). Results showed a slight but significant decrease of FGF-2 IRES but not of EMCV IRES activity (Fig. 3B
, left). FGF-2 expression was also down-regulated in mice treated with AR-siRNA. High molecular weight isoforms known for their intracrine mode of action are specifically concerned by this decrease (Fig. 3B
, right).
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3. Testosterone induces protein binding to the FGF-2 mRNA 5' UTR
Testicular extracts of 3 and 6 wk-old mice were used for cross-linking experiments with RNA probe corresponding to the FGF-2 5' UTR RNA (containing the IRES), and analyzed by 2-dimensional gel electrophoresis (Fig. 2C
). A different profile of bound testicular proteins was observed at 3 or 6 wk. Extracts of testosterone-treated immature mice testis showed the same profile as that observed in adults. Such a correlation between changes of protein binding and FGF-2 IRES activity increase asks the question of the involvement of specific ITAFs (IRES-trans-acting factors) in the IRES-dependent control of FGF-2 expression at the start of spermatogenesis. In fact, hnRNAPI was recently shown as an activating ITAF of the FGF-2 IRES.
CONCLUSIONS AND SIGNIFICANCE
The present report describes one of the first IRES-mediated regulations of gene expression in a physiological process.
In mature testis, FGF-2 expression is transcriptionally down-regulated. Here we show that in such conditions the IRES-dependent translational mechanism allows specific expression of FGF-2 in spermatogonia. As FGF-2 is crucial at the start of spermatogenesis, the IRES-dependent mechanism appears as an alternative, in the absence of FGF-2 produced by the paracrine way, to trigger spermatogonia proliferation and/or differentiation toward the spermatogenesis process.
Another novel finding is the hormonal control of the FGF-2 IRES activity. Adult spermatogenesis is controlled by Leydig cells-produced testosterone. We show that testosterone triggers activation of the FGF-2 IRES, in a period coinciding with emergence of Leydig cell function. This testosterone-dependent regulation is fully consistent with the IRES activation observed in adult but not in immature spermatogonia. It has been shown that FGF-2 expressed by germ line cells induces testosterone production by Leydig cells. Thus, the IRES would be implied in the emergence of a paracrine loop leading to spermatogenesis in mature testis (Fig. 2B
).
AR depletion using siRNA was able to partially suppress IRES activation. This partial effect can be explained by the 70% efficiency of AR depletion. AR, although pivotal for adult spermatogenesis, is not expressed by spermatogonia but by Sertoli cells. Thus, testosterone presumably acts indirectly, through cell-cell communication between Sertoli cells and spermatogonia. An alternative hypothesis is that activation of FGF-2 expression might be mediated by nongenomic effects of testosterone involving a membrane receptor.
Another feature of FGF-2 expression in spermatogonia is the synthesis of FGF-2 high molecular weight isoforms. Such isoforms have been described in germ cells, and we show here that AR siRNA blocks expression of the high molecular weight isoforms. Thus, expression of HMW nuclear FGF-2 might have a role in the start of spermatogenesis by an intracrine process (Fig. 2B
).
Regulation of the FGF-2 IRES activity involves participation of regulatory proteins, IRES trans-acting factors (ITAFs), acting as repressors or activators: we have recently shown that this IRES is negatively regulated by p53 while activated by HnRNPA1. The proteins bound to the FGF-2 IRES are putative ITAFs. These ITAFs would be induced, activated, or translocated in response to a stimulus coming from Sertoli cells, leading to formation of an RNA-protein complex called "IRESome," which would activate the IRES and increase FGF-2 expression (Fig. 2C
). Translational control plays a central role in the meiotic stages of spermatogenesis, but little is known about translational control during mitosis occurring in the pre-meiotic stages. In fact, FGF-2 may not only act as a mitogenic factor but also as a differentiation factor.
The finding of IRES-dependent regulation in a crucial process such as spermatogenesis highlights the relevance of cellular mRNA IRESs in the control of gene expression in various pathophysiological processes.
FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3314fje;
This article has been cited by other articles:
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