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. 2005 Feb 9;24(3):611-22.
doi: 10.1038/sj.emboj.7600549. Epub 2005 Jan 20.

A type I DnaJ homolog, DjA1, regulates androgen receptor signaling and spermatogenesis

Kazutoyo Terada  1 , Kentaro Yomogida, Tomoaki Imai, Hiroshi Kiyonari, Naoki Takeda, Tsuyoshi Kadomatsu, Masato Yano, Shinichi Aizawa, Masataka Mori
Affiliations

A type I DnaJ homolog, DjA1, regulates androgen receptor signaling and spermatogenesis

Kazutoyo Terada et al. EMBO J. .

Abstract

Two type I DnaJ homologs DjA1 (DNAJA1; dj2, HSDJ/hdj-2, rdj1) and DjA2 (DNAJA2; dj3, rdj2) work similarly as a cochaperone of Hsp70s in protein folding and mitochondrial protein import in vitro. To study the in vivo role of DjA1, we generated DjA1-mutant mice. Surprisingly, loss of DjA1 in mice led to severe defects in spermatogenesis that involve aberrant androgen signaling. Transplantation experiments with green fluorescent protein-labeled spermatogonia into DjA1(-/-) mice revealed a primary defect of Sertoli cells in maintaining spermiogenesis at steps 8 and 9. In Sertoli cells of DjA1(-/-) mice, the androgen receptor markedly accumulated with enhanced transcription of several androgen-responsive genes, including Pem and testin. Disruption of Sertoli-germ cell adherens junctions was also evident in DjA1(-/-) mice. Experiments with DjA1(-/-) fibroblasts and primary Sertoli cells indicated aberrant androgen receptor signaling. These results revealed a critical role of DjA1 in spermiogenesis and suggest that DjA1 and DjA2 are not functionally equivalent in vivo.

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Figures

Figure 1
Figure 1
Genomic structure of mouse DjA1 and generation of DjA1-mutant mouse. (A) Schematic diagram of DjA1 gene from C57BL/6 mouse. Exons are indicated by rectangles. An arrow in exon 2 indicates the initial methionine. Arrowheads in exon 9 represent positions of putative poly(A) signal sequences. Relative positions of phage clones (EMBL-0205 and -0207) and plasmid clones (pGT22 and pGTE0202) are indicated by lines (accession # AB183426). (B) RNA blot analysis for DjA1. A filter with mouse embryonic stage poly(A)+ RNAs (Clontech) was hybridized, using as probes 32P-labeled mouse DjA1 cDNA. (C) Targeting vector for DjA1 mutation. Exons 1–6 are indicated by filled rectangles. The predicted sizes of normal and targeted alleles and the location of D2 probe used in Southern blot analysis (D) are shown. B, BsaBI; E, EcoRI; N, NotI; V, EcoRV; X, BstXI. (D) Southern blot analysis by EcoRV digestion of genomic DNAs (10 μg each). (E) Northern blot analysis of DjA1 from testis. Total RNA samples were analyzed using digoxigenin-labeled cRNA probes. Integrity of the RNA samples was verified using ethidium bromide staining. (F) Western blot analysis of DjA1 from testis.
Figure 2
Figure 2
Phenotypic abnormalities of DjA−/− male mice. (A, B) Growth curves for male (A) and female (B) DjA1-mutant mice (n=15 for each genotype of each sex). Littermates of the DjA1+/+ mice showed virtually identical curves to DjA1+/− mice. (C) Gross anatomy of male genital tracts from DjA1+/− and DjA1−/− mice (4 months of age). Adipose tissues were removed from the tracts. (D) Testis weight of each phenotype at 4 months of age. (E, F) Spermatozoa from cauda epididymis of the DjA1+/− (E) and DjA1−/− (F) mice. (G, H) Hematoxylin–eosin-stained sections of DjA1+/− (G) and DjA1−/− (H) testes. Atrophied tubules are marked with asterisks. (I, J) Apoptotic cells in DjA1+/− (I) and DjA1−/− (J) testes. Apoptotic cells were labeled in situ using the TUNEL method and visualized using 3,3′-diaminobenzidine as a substrate. Sections were counterstained with methyl green. Tubules with TUNEL-positive cells are marked with asterisks. Arrowheads indicate apoptotic cells that lost contact with Sertoli cells. Bar=50 μm (E–J).
Figure 3
Figure 3
High expression of type I DnaJs in testis and increased expression of DjA2 in DjA1−/− mice. (A, B) Sections of DjA1+/− and DjA1−/− testes were mounted on the same slide glass, and decorated with a mAb against DjA1. Peroxidase activity was visualized using 3,3′-diaminobenzidine as a chromogen. Sections were counterstained with hematoxylin. Signals of DjA1 in primary and secondary spermatocytes are indicated with P and S, respectively (A). Nonspecific signals in the interstitial area are indicated with asterisks (A, B). (C, D) In situ hybridization of DjA1 mRNA from DjA1+/− and DjA1−/− testes was carried out using digoxigenin-labeled DjA1 probe. Alkaline phosphatase activity was visualized using 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium as a chromogen. Sections were counterstained with nuclear fast red. (E) Total RNA samples from fractionated testicular cells and mouse tissues (5 μg each) were subjected to Northern blot analysis, using digoxigenin-labeled DjA1 and DjA2 cRNAs as probes. Arrowheads indicate positions of 2.4 and 1.6 kb bands (DjA1) and 2.1, 2.2, 3.1 and 4.0 kb bands (DjA2). Protein samples of testicular cells (F) or tissues (G) were subjected to Western blot analysis using a monoclonal anti-DjA1, or polyclonal anti-DjA2 or anti-DjB1 antibodies (0.4 μg/ml each), or antiserum against DjA4 (1/1000 dilution). An arrowhead indicates position of expected size of DjA4. EP and LP, early and late stage pachytene spermatocytes; RS and ES, round and elongated spermatids. Bar=50 μm (A–D).
Figure 4
Figure 4
Defect of spermiogenesis in DjA1−/− seminiferous tubules. GFP-labeled spermatogonia from GFP-transgenic mice were transplanted into seminiferous tubules of DjA1+/− and DjA1−/− mice (n=2 and 4, respectively). (A, B) Observation of whole testes under a fluorescent stereomicroscope at 2 months after transplantation. Representative sections under fluorescent microscope (C, D) and the same sections stained with hematoxylin (E, F). Bar=1 mm (A, B) and 75 μm (C–F).
Figure 5
Figure 5
Aberrant functions of Sertoli cell as revealed by dislocated nuclei, accumulation of AR protein without mRNA increase, and induction of testosterone-responsive genes. Sections of DjA1+/− (A, C) and DjA1−/− (B, D) testes on the same slide glass were probed with AR antibody. Signals were visualized using 3,3′-diaminobenzidine as a chromogen. Sections were counterstained with hematoxylin. Some AR-positive cells in (A, B) are marked with capital letters. Strong AR signals in Sertoli cell nuclei are indicated with arrowheads (D). B, smooth muscle cell surrounding blood vessel; M, myoid cell; L, Leydig cell; S, Sertoli cell. (E) Western blot analysis of total protein from testis and prostate. Protein samples were prepared under denaturing conditions from two pairs of littermates (3 months of age). The respective pairs of mice were derived from independent ES strains. (F) Semiquantitative PCR analysis. Total RNAs were isolated from the testes of littermates (4 months of age). (G) Real-time PCR analysis. Values were normalized for levels of β-actin expression (n=5 pairs of littermates), and mean value for respective gene expression for DjA1+/− mice was set as 100%. (H) Northern blot analysis of Pem and testin mRNAs. Total RNA samples were analyzed using 32P-labeled probes. Bar=100 μm (A, B) and 50 μm (C, D).
Figure 6
Figure 6
Repression of AR transactivation by DjA1 and decreased androgen binding to the receptor. (A) Luciferase reporter assay in DjA1+/− and DjA1−/− MEFs. The MMTV-luciferase activities were normalized for internal reporter activities and are represented as values relative to the activity of DjA1+/− cells in the presence of testosterone. Internal reporter activities for DjA1+/− and DjA1−/− MEFs were 100±13 and 34±1.9 U, respectively. Values are shown as mean±s.d. of three experiments. DHT, 5α-dihydrotestosterone. (B) Saturation curve and Scatchard plot (inset) of [3H]R1881 binding to primary Sertoli cells.
Figure 7
Figure 7
Expression of nectin-2 and -3 in testis of DjA1+/− and DjA1−/− mice. Frozen testis sections (7 μm) of DjA1+/− (A, C) and DjA1−/− (B, D) mice were stained with rat mAb 502-57 (green) against nectin-2 (A, B) and with rat mAb 103-A1 (green) against nectin-3 (C, D). Nuclei were visualized with Hoechst 33258 DNA stain (blue), and co-immunofluorescence and phase contrast images were combined (A–D). Nectin-2-positive signals within basal compartment are indicated by arrowheads (A, B). (E) Northern blot analysis of nectin-2 and -3 mRNAs. Total RNA samples were analyzed using 32P-labeled probes. Bar=50 μm (A–D).
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