Testosterone regulates FGF‐2 expression during testis maturation by an IRES‐dependent translational mechanism

Abstract

Spermatogenesis is a complex process involving cell proliferation, differentiation, and apoptosis. Fibroblast growth factor 2 (FGF-2) is involved in testicular function, but its role in spermatogenesis has not been fully documented. The control of FGF-2 expression particularly occurs at the translational level, by an internal ribosome entry site (IRES)-dependent mechanism driving the use of alternative initiation codons. To study IRES activity regulation in vivo, we have developed transgenic mice expressing a bicistronic construct coding for two luciferase genes. Here, we show that the FGF-2 IRES is age-dependently activated in mouse testis, whereas EMCV and c-myc IRESs are not. Real-time PCR confirms that this regulation is translational. By using immunohistological techniques, we demonstrate that FGF-2 IRES stimulation occurs in adult, but not in immature, type-A spermatogonias. This is correlated with activation of endogenous FGF-2 expression in spermatogonia; whereas FGF-2 mRNA transcription is known to decrease in adult testis. Interestingly, the FGF-2 IRES activation is triggered by testosterone and is partially inhibited by siRNA directed against the androgen receptor. Two-dimensional analysis of proteins bound to the FGF-2 mRNA 5'UTR after UV cross-linking reveals that testosterone treatment correlates with the binding of several proteins. These data suggest a paracrine loop where IRES-dependent FGF-2 expression, stimulated by Sertoli cells in response to testosterone produced by Leydig cells, would in turn activate Leydig function and testosterone production. In addition, nuclear FGF-2 isoforms could be involved in an intracrine function of FGF-2 in the start of spermatogenesis, mitosis, or meiosis initiation. This report demonstrates that mRNA translation regulation by an IRES-dependent mechanism participates in a physiological process.

Unexpected requirement for a binding partner of the syntaxin family in phagocytosis by murine testicular Sertoli cells

“…injection during 10 d (Figure 3a), according to a previously validated protocol. 15 Txlna depletion was confirmed by qRT-PCR (Figure 3b), western blotting ( Figure 3c) and immunohistochemistry ( Figure 3d). An~40% decrease of Txlna/TXLNA was detected at the end of 25 d after siRNA treatment.…”

“…In vivo siRNA for ablation of endogenous Txlna in testis was carried out according to the protocol established previously. 15 Animals were injected i.p. with 100 μl solution of siRNA daily for 10 d in isotonic saline solution (3 μg per mouse), followed by a 14-d break.…”

Reversible induction of translational isoforms of p53 in glucose deprivation

“…12,35 p53 is also known to have RNA re-annealing properties, binding to the 5′UTR of its own mRNA, 34 as well as of Cdk4 46 and FGF2. 47,48 Recent reports showed that SMAR1 forms a ternary complex with p53-MDM2, in which MDM2 binds to residues 17-26 of p53 and SMAR1 binds to residues 14-16 of p53, with a simultaneous interaction of SMAR1 and MDM2. 37 We report that SMAR1 is able to bind to p53 (1-251) IRES directly and specifically in the in vitro studies; ex vivo .…”

Small RNA molecules in the regulation of spermatogenesis

“…In one in vivo study, DNA vectors expressing shRNAs were introduced into mouse testes via DNA injection and electroporation, and more than 80% of mRNA reduction in the reporter gene EGFP and DsRed2 was observed in spermatogonia, spermatocytes, and round spermatids (Shoji et al 2005), suggesting that the electroporation approach is effective for silencing gene expression of spermatogenic cells in vivo. In another in vivo study, the FGF2-transgenic mice were injected intraperitoneously daily with 100 ml of solution of siRNAs against androgen receptor in isotonic saline solution, and 70% efficiency of androgen receptor protein depletion was obtained as assayed by western blots (Gonzalez-Herrera et al 2006), which illustrates that RNAi is feasible for suppressing gene expression of somatic cells in vivo. The major obstacle to achieving RNAi with longer dsRNAs in mammalian cells is that they cause non-specific mRNA degradation and a general shutdown of host cell protein synthesis.…”

“…Suppression of beta1-integrin by RNAi in Sertoli cells results in occludin redistribution at the Sertoli-Sertoli cell interface and BTB destabilizing (Yan et al 2008). In an in vivo study, mice treated with siRNA targeted against androgen receptor resulted in reduced expression of FGF2 and Small RNAs controlling spermatogenesis established a role for testosterone in the regulation of FGF2 (Gonzalez-Herrera et al 2006). Collectively, these studies illustrate that RNAi technology using siRNAs is an effective approach in exploring the function of specific genes in the regulation of spermatogenesis.…”