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First published online 10 August 2005
doi: 10.1242/dev.02003

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RNF17, a component of the mammalian germ cell nuage, is essential for spermiogenesis

¹ Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
² Howard Hughes Medical Institute, Whitehead Institute, and Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142, USA
³ Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan

View larger version (42K): [in a new window]   Fig. 1. Identification of two Rnf17 isoforms. (A) Northern blot analysis of Rnf17 expression. Two Rnf17 transcripts (5.2 kb and 3.5 kb) were detected in the testis. ß-Actin served as a loading control. (B) Schematic diagram of two RNF17 protein isoforms. (C) Western blot analysis of RNF17 proteins. Testicular protein extracts (30 µg) were resolved by SDS-PAGE. Anti-serum 1774 recognized both RNF17L and RNF17S. Anti-serum GP8 was specific to RNF17L. (D) RNF17 is largely cytoplasmic. Cytoplasmic and nuclear fractions of adult testis were prepared and subjected to western blot analysis. MVH (Mouse Vasa homolog) is a cytoplasmic protein control (Toyooka et al., 2000); SYCP3 (synaptonemal complex protein 3) is a nuclear protein control (Chuma and Nakatsuji, 2001); ß-actin served as a loading control. C, cytoplasmic fractions; N, nuclear fractions.

View larger version (88K): [in a new window]   Fig. 2. Distribution of RNF17 in male germ cells. Sections of adult testis were immunostained with anti-RNF17 antibodies (anti-serum 1774, red). For purposes of staging, testis sections were also stained with anti-SP-10 antibodies (green) and counterstained with DAPI (blue). Only part of a seminiferous tubule is shown in each panel. Anti-RNF17 staining alone is shown in A,C,E,G,I,K. The merged image of anti-RNF17, anti-SP10 and DAPI is shown in B,D,F,H,J,L. The stage of each seminiferous tubule is indicated in the center. Representative RNF17 granules are indicated by arrows in pachytene or diplotene spermatocytes, or by arrowheads in elongating spermatids. Immunostaining of Rnf17-null testes with antiserum 1774 was negative (data not shown). Control experiments without primary antibodies were also negative (data not shown). Scale bar: 20 µm.

View larger version (79K): [in a new window]   Fig. 3. Differential distribution of RNF17 protein isoforms. Adult testis sections were immunostained with anti-sera 1774 in red (A) and GP8 in green (B). The seminiferous tubule shown is in stage X. In the merged image (C), selected RNF17 granules are indicated by arrows in the spermatocytes, and by arrowheads in the spermatids. Scale bar: 10 µm.

View larger version (90K): [in a new window]   Fig. 4. RNF17 granules are distinct organelles in the testis. Double immunostaining of adult testis sections was performed with antiserum 1774 (red) and anti-TDRD1 antibodies (green) or anti-LAMP2 antibodies (green). RNF17 granules (red) are prominent in spermatocytes (A,C) and elongating spermatids (B,D). Dynamic changes of chromatoid bodies (green) are seen in spermatocytes (A) and spermatids (B). Lysosomes (green) are not present in spermatocytes (C) but are abundant in spermatids (D). Scale bar: 10 µm.

View larger version (116K): [in a new window]   Fig. 5. Ultrastructural analysis of RNF17 granules by immuno-EM. Affinity purified anti-RNF17 antibodies (anti-serum 1774) were used as primary antibodies. Secondary antibodies were labeled with 10 nm gold particles. (A) The RNF17 granule in a spermatocyte. (B) The RNF17 granule in a spermatid. (C) The RNF17 granule in a cytoplasmic residual body. Arrows indicate the accumulation of gold particles in the 500 nm RNF17 granules. Immuno-EM without the primary antibody was negative (data not shown). cb, chromatoid body; m, mitochondria; n, nucleus; v, vacuole.

View larger version (41K): [in a new window]   Fig. 6. RNF17 binds to itself both in vivo and in vitro. (A) Identification of the RNF17 granule formation domain. Truncated RNF17 polypeptides were expressed as GFP fusion proteins in NIH 3T3 cells. Formation of granules was assayed by fluorescence microscopy. +, formation of GFP-RNF17 granules; -, diffuse GFP-RNF17 distribution. The numbers adjacent to the endpoints designate the corresponding amino acid positions. (B) Distribution of GFP-fusion proteins expressed in NIH 3T3 cells. Punctate granules were present in cells expressing RNF17L (1-1640), RNF17S (1-1130), RNF17 (1-626), RNF17 (1-287) or RNF17 (243-287). RNF17 (286-626) or GFP alone (data not shown) was diffusely distributed. (C) Co-immunoprecipitation of RNF17L and RNF17S from testis. Immunoprecipitation (IP) using testis protein extracts was carried out with RNF17L-specific antibodies (anti-serum GP8). Immunoprecipitated proteins were subjected to western blot analysis with anti-serum 1774. IP with pre-bleed serum served as a control. (D) GST-pulldown assay. GST-RNF17 (1-287) was expressed in E. coli and purified with glutathione beads. RNF17 (1-626) was in vitro translated in the presence of [35S]methionine. GST alone served as a control.

View larger version (38K): [in a new window]   Fig. 7. Targeted disruption of the Rnf17 gene. (A) Schematic diagram of the wild-type allele, the targeting vector and the targeted allele. PGK-Neo: neomycin-resistance gene driven by the PGK1 promoter. PCR primers (P1, P2, P3 and P4) used for genotyping are indicated. (B) PCR analysis of genomic DNA from Rnf17 mice. +/+, wild type; +/-, heterozygote; -/-, homozygote. All four primers were included in each PCR assay. WT, wild-type allele; MT, mutant allele. (C) Absence of RNF17 proteins in the Rnf17-deficient testes. Western blot analysis was done with both anti-sera 1774 (as shown) and GP8 (data not shown).

View larger version (116K): [in a new window]   Fig. 8. Spermiogenesis defects in Rnf17-deficient mice. (A,B) Histological analysis of seminiferous tubules in adult wild-type (A) and Rnf17-/- (B) mice at low magnifications. (C,D) Histology of wild-type and Rnf17-/- seminiferous tubules at high magnifications. White arrows, round spermatids; black arrow, an abnormal multinucleated cell. (E) Wild-type epididymal tubules (arrow) are filled with sperm. (F) Epididymal tubules (arrow) from Rnf17-/- mice lack sperm but contain spermatid-like germ cells. (G,H) Acrosomal morphology in wild-type (Stage IX) and Rnf17-deficient seminiferous tubules. Testis sections were immunostained with anti-SP10 antibodies (green) and DNA was stained with DAPI (blue). Scale bars: 25 µm.

View larger version (77K): [in a new window]   Fig. 9. Analysis of gene expression in Rnf17-/- testes. ß-Actin was included as a control for RNA loading. (A) Northern blots of testis-specific genes in adult testes. +/+, wild type; +/-, heterozygote; -/-, homozygote. (B) Gene expression during juvenile testis development. Five testis-specific genes (Act, Tp1, Prm1, Prm2 and Ldh3) and four key regulators of spermiogenesis (Crem, Trf2, Miwi and Tpap) were examined. The age of mice (in days after birth) is indicated. +/+, wild type; -/-, Rnf17 deficient.