Impaired gametogenesis in mice that overexpress the RNA-binding protein HuR - PubMed (original) (raw)

Impaired gametogenesis in mice that overexpress the RNA-binding protein HuR

Marilyne Levadoux-Martin et al. EMBO Rep. 2003 Apr.

Abstract

A series of experiments, using cell culture models or in vitro assays, has shown that the RNA-binding protein HuR increases the half-life of some messenger RNAs that contain adenylate/uridylate-rich decay elements. However, its function in an integrated system has not yet been investigated. Here, using a mouse model, we report that misregulation of HuR, due to expression of an HuR transgene, prevents the production of fully functional gametes. This work provides the first evidence for a physiological function of HuR, and highlights its involvement in spermatogenesis.

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Figures

Figure 1

Figure 1

Transgene structure. Schematic representation of the β-actin–HuR (BA–HuR) (A), β-actin–loxP–GFP–loxP–HuR (BA–GH) (B) and BA–HGdel (C) transgenes. GFP, green fluorescent protein.

Figure 2

Figure 2

Analysis of transgene expression. (A) Western blot analysis of Myc–HuR expression using the 9E10 anti-Myc antibody on samples from 293 cells (either untransfected (lane 1), or transfected with the β-actin–HuR (BA–HuR) construct (lane 2), and from various organs, as indicated, from wild-type (lanes 3, 6, 10, 13, 18 and 21), BA–HuR line 18 (lanes 4, 5, 7, 8, 11, 15, 17, 20 and 23) or BA–HuR line 6 (lanes 9, 12, 14, 16, 19 and 22) mice. (B) Comparison of the methylation status of β-actin regulatory sequences in BA–HuR and β-actin–Auf1 mice. A Southern blot was carried out on DNA that was extracted from brain, digested with the indicated restriction enzymes, and hybridized with a β-actin probe. The structure of the β-actin–HuR transgene and the location of the _Msp_I and _Hpa_II sites are shown in the bottom panel. (C) Western blot analysis of transgene expression in BA–GH line 21 using anti-green-fluorescent-protein (anti-GFP) and anti-Myc antibodies to analyse samples from spleen, testis, brain, thymus and heart. C+ indicates a C1 mouse from the BA–HGdel line 21 different to that used for the first five lanes. (D). Myc–HuR (transgenic) and endogenous HuR expression, as detected by using the anti-Myc or anti-HuR antibodies on testes from BA–HGdel lines 21, 27 and 31 (from the C1 and C2 generations) and from wild-type mice. (E) Northern blot analysis was carried out on RNA extracted from muscle, lung and testis from BA–HGdel line 21 and line 27 mice, and the blot was hybridized with an HuR or GAPDH probe. BA–Auf1, β-actin–Auf1; SV40, simian virus 40; WT, wild type.

Figure 3

Figure 3

Analysis of transgene configuration in BA–HGdel transgenic lines. (A) Southern blot analysis was carried out on DNA extracted from BA–HGdel mice after Cre excision in vivo and digestion with _Bam_HI. When several copies of the BA–GH transgene are located in a concatameric array in the mouse genome, Cre-mediated recombination between LoxP sites with the same orientation removes the intervening DNA, leaving one copy of the BA–HGdel transgene at the original chromosomal location (indicated by asterisks). (B) Interpretation of the number of copies of the transgene and their orientation in each BA–HGdel line. The arrows indicate the location of the _Bam_HI restriction sites; the numbers (in kilobases) indicate the sizes of the fragments recognized by the different probes. L, 1-kb ladder; Endo, endogenous HuR.

Figure 4

Figure 4

Analysis of endogenous and transgenic HuR expression at the cellular level. (A–C) Immunohistochemical analysis; testis sections (3–5 μm) from β-actin–HuR (BA–HuR) lines 19 (A) and 18 ((B) and (C)) were incubated with the 9E10 antibody to detect the HuR transgene ((A) and (B)), or with the 19F12 antibody to detect endogenous HuR (C). (D–J) Confocal analysis. Testis sections (∼60 μm) from BA–HGdel lines 21 (D–G) and 31 (H–J) were incubated with chromomycin (green), which specifically stains nuclei (D, F, H), or with 9E10 (red), which detects transgenic HuR (G, I). Double labelling gave an orange staining, which was mainly restricted to the round spermatid population; this was only visible in BA–HGdel line 21 sections (E), and not in BA–HGdel line 31 sections (J). ES, elongated spermatids; PS, primary spermatocytes; RS, round spermatids; S, spermatogonia.

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