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1 Department of Haematology & Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
2 Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan
3 Department of Pathology, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan
4 Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 3-10 Kanda-Surugadai 2-Chome, Chiyoda-ku, Tokyo 101-0062, Japan
5 Laboratory for Vertebrate Body Plan, RIKEN Centre for Developmental Biology, 2-2-3 Minatojima-MinamiMachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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CIZ (Cas-interacting zinc finger protein) was originally cloned by Far-Western screening using the SH3 domain of p130Cas as a probe (Nakamoto et al. 2000). CIZ encodes multiple isoforms, which include an N-terminal serine/threonine-rich region, a proline-rich sequence responsible for binding to p130Cas SH3 domain, a putative nuclear localization signal, 5, 6 or 8 Krüppel-type zinc fingers, a glutamine-alanine repeat and a poly glutamine sequence (Nakamoto et al. 2000). The expression of CIZ is ubiquitous, but higher expression is observed in testis, heart and brain (Nakamoto et al. 2000; Thunyakitpisal et al. 2001). CIZ is a nucleo-cytoplasmic shuttling protein found in the nucleus, and, together with Cas, at focal adhesions (Nakamoto et al. 2000). In the nucleus, CIZ is a transcription factor that binds to its CIZ-consensus poly (dA) or poly (dT) sequences (Nakamoto et al. 2000; Thunyakitpisal et al. 2001) and regulates transcription from matrix methalloproteinase (MMP-1, MMP-3, MMP-7) promoters and from the type-I collagen promoter (Furuya et al. 2000; Nakamoto et al. 2000). Meanwhile, another group cloned Nmp4 by its binding to type-I collagen promoter and found that some of the alternative forms of Nmp4 are identical to CIZ (Thunyakitpisal et al. 2001).
Recently, we reported the role of CIZ/Nmp4 in BMP (Bone mophogenetic protein) signalling (Shen et al. 2002). In osteoblastic MC3T3E1 cells, CIZ/Nmp4 over-expression suppressed the BMP2-enhanced expression of osteoblastic differentiation markers such as alkaline phosphatase, osteocalcin and type-I collagen, as well as the upstream transcription factor Cbfa1. Furthermore, CIZ/Nmp4 also blocked the BMP-induced Smad 1 and Smad 5 activation of transcription from BMP-specific promoter. On the other hand, BMP stimulation maintains the expression of CIZ/Nmp4 in osteoblast culture (Shen et al. 2002). Thus CIZ/Nmp4 is an inhibitor of BMP/Smad signalling.
Drosophila Rotund (St Pierre et al. 2002), Drosophila Squeeze (Allan et al. 2003), C. elegans Lin-29 (Rougvie & Ambros 1995) and mammalian CIZ/Nmp4 form a conserved subfamily of zinc finger proteins, showing high homologies in the zinc finger region and poly glutamine sequences in the C-terminal domain. The Drosophila rotund (rn) phenotype is male and female sterility as well as defects in several adult body structures (Cavener et al. 1986). Squeeze protein plays a role in axon pathfinding of Tv-neurone and acts in cooperation with Apterous and retrograde BMP signal, to activate the expression of FMRFa neuropeptide (Allan et al. 2003). Lin-29 is critical for the larva/adult switch and regulates the transcription of adult-specific and larva-specific collagen genes, by binding to the promoter sequences that are very similar to the CIZ/Nmp4 binding region (Furuya et al. 2000; Nakamoto et al. 2000; Rougvie & Ambros 1995; Thunyakitpisal et al. 2001). Murine CIZ/Nmp4 is located at chromosome 6 band F1 (nmp4 locus) (Alvarez et al. 2001). ZNF384, a human homolog of CIZ/Nmp4, is located at 12p13 (Margolis et al. 1997; Pawlak et al. 1998).
In this study, we disrupted the CIZ/Nmp4 gene of mouse, generated CIZ/Nmp4-deficient mice and found testicular degeneration and increased apoptosis of spermatogenic cells.
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Results |
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To establish CIZ/Nmp4-targeted mice, we isolated genomic clones of the murine CIZ/Nmp4 (nmp4) locus by screening a 129/Sv mouse genomic library with a murine CIZ cDNA probe. Five overlapping clones which spanned the CIZ/Nmp4 locus were obtained. The CIZ/Nmp4 targeting construct pBS-CIZLacZneo was constructed to introduce ß-galactosidase fused to CIZ-N-terminus and neo resistance gene into the second exon of CIZ/Nmp4 (Fig. 1A), which is utilized by all the fully sequenced alternative forms of CIZ/Nmp4 (Nakamoto et al. 2000; Thunyakitpisal et al. 2001).
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The disapperance of three bands were seen in CIZ–/– cells by Western blotting on immunoprecipitates of the primary embryonic fibroblasts with anti-CIZ antibody (Fig. 1C). To further confirm the disappearance of CIZ/Nmp4 in CIZ–/– cells, we performed immunofluorescence antibody staining. With anti-CIZ antibody, the nuclear staining observed in CIZ+/+ cells clearly declined in CIZ–/– cells (Fig. 1D,E). On the other hand, the cytoplasmic staining, which we assumed to be nonspecific in the previous report (Nakamoto et al. 2000), was not so different (Fig. 1D,E). The focal adhesion staining that we observed in rat 3Y1 cells was not obviously apparent in the primary embryonic fibroblasts nor in NIH3T3 cells (data not shown).
Smaller body size, impaired spermatogenesis and male fertility defects
CIZ–/– mice (genetic background 25% CBA and 75% C57/BL6) were born at the expected Mendelian ratio and were viable until adulthood. However, CIZ–/– mice tended to be smaller when compared to CIZ+/+ and CIZ+/– littermates (Fig. 1F). This smaller body size appears to be retained throughout adulthood in both male and female mice (Fig. 1G). Although CIZ/Nmp4 is reported to play a role in osteoblasts (Furuya et al. 2000; Thunyakitpisal et al. 2001), X-ray analyses of CIZ–/– mice revealed no macroscopic changes in bone structure (data not shown).
The pathological analysis of organs including bones revealed no obvious abnormalities, except in the testis. Blood cell analysis also indicated normal numbers of peripheral blood cells (data not shown). In the testis of CIZ–/– mice, some of the seminiferous tubules strikingly lacked all spermatogenic cell types and were occupied by Sertoli cells only (indicated by asterisks in Fig. 2B,D,F), while other seminiferous tubules looked intact and contained spermatogenic cells in all stages of spermatogenesis (indicated by the symbol ‘+’ in Fig. 2B,D), as in the CIZ+/+ mice (Fig. 2A,C,E). In contrast, we found no obvious changes in Leydig cells and Serotoli cells (respectively indicated by arrows and arrowheads in Fig. 2E,F) nor in the epididymis (data not shown). This pathological degeneration resembled that of the human disease, ‘Germinal-cell aplasia with focal spermatogenesis’ (Levin 1989). However, a large variability in the degree of testicular degeneration was observed among CIZ–/– mice: some animals even showed no obvious abnormality in the testes at the age of 8 weeks or later (data not shown). We also noticed a large variability in the age on onset of sterility among CIZ–/– mice. Some mice were initially fertile but became sterile later while others were sterile even when young. We mated 4-10 months-old CIZ–/– or CIZ+/+ males with 6 week-old wild-type B6 female mice for 30 days. CIZ+/+ males gave rise to a pregnancy rate of 8/10 resulting in 50 pups, while CIZ–/– males gave a pregnancy rate of 4/10 resulting in 27 pups. This result indicates that CIZ–/– males were partially infertile.
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Although the average testis weight of CIZ+/– male was between that of CIZ+/+ and of CIZ–/– mice (data not shown), we could not detect any histological defect in their testes nor any sign of reduced fertility. CIZ–/– female mice bore pups when they were mated with CIZ+/+ mice.
CIZ/Nmp4 is expressed in spermatogenic cells in testis
In order to get more insight into the role of CIZ/Nmp4 in testis, we determined cell type specificity in the expression of CIZ/Nmp4. To do so, spermatogenic cells and in Sertoli cells were isolated from primary cultures of rat testicular cells: we used rats because separation of these two cell types of the mouse was difficult. The result of Northern blotting with a CIZ probe showed that CIZ/Nmp4 is expressed predominantly in spermatogenic cells and little, if any, expression was observed in Sertoli cells (Fig. 3A). The perinuclear blue products of ß-galactosidase in the frozen section of testes of CIZ+/– mice confirmed the expression of CIZ/Nmp4 in spermatogenic cells (Fig. 3C). However, the possibility that CIZ/Nmp4 is faintly expressed in Sertoli cells could not be ruled out. We next checked the changes in CIZ/Nmp4 expression with age by Northern blotting. As shown in Fig. 3D, the expression of CIZ/Nmp4 in mouse testes increased with age, suggesting a larger role of CIZ/Nmp4 in adult spermatogenesis than in adolescence. Finally, RNA from testes of CIZ–/– mice did not give signals (Fig. 3D) and proteins from testes of CIZ–/– mice lacked a signal corresponding to CIZ in IP-Western blotting with anti-CIZ antibody (Fig. 3E), confirming loss of CIZ expression in the testis.
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Testicular degeneration found in CIZ–/– mice suggested that spermatogenic cell apoptosis is enhanced in the CIZ–/– testis. We therefore used a terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay to detect apoptosis in the testis of CIZ–/– and CIZ+/+ mice. At the age of 8 weeks, the seminiferous tubules in the testes of CIZ–/– adult mice contained on average 0.85 TUNEL-positive cells compared to 0.23 TUNEL-positive cell in wild-type testes (Fig. 4A–D,G). Histologically, most TUNEL-positive cells were classified as dividing secondary spermatocytes (Fig. 4D) and smaller numbers of TUNEL-positive cells were classified as primary spermatocytes (data not shown). The time course of TUNEL-positive cell counts in the seminiferous tubules indicated that CIZ–/– mice had higher rates of apoptosis than CIZ+/+ mice after 4 weeks of age (Fig. 4G). The larger standard deviation reflects the variability in the degree of degeneration among individuals, but the difference between CIZ–/– and CIZ+/+ were statistically significant (Fig. 4G). Surprisingly, 2 week-old CIZ–/– testes contained fewer apoptotic spermatogenic cells than 2 week-old wild-type testes, although this could be attributed to a reduction in spermatogenic cell number in the tubules of CIZ–/– animals (Fig. 4E,F).
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We next examined apoptosis of primary cultured spermatogenic cells. Testicular cells prepared from either CIZ+/+ or CIZ–/– mice were cultured, and the mode of apoptosis was compared between the two spermatogenic cell populations. As shown in Fig. 5, the progress of apoptosis did not significantly differ in spermatogenic cells with or without CIZ/Nmp4 expression. The extent of phosphatidylserine externalization similarly increased (Fig. 5A), desialylation did not seem to occur in both cell populations (Fig. 5B), and distribution of spermatogenic cell populations with ploidy of 1n, 2n, and 4n remained almost the same during primary culture (Fig. 5C). These results indicate that there is no significant difference in the mode of apoptosis of testicular spermatogenic cells in primary culture irrespective of CIZ/Nmp4 expression.
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As CIZ/Nmp4 is known to be involved in the regulation of collagen expression (Furuya et al. 2000; Thunyakitpisal et al. 2001), we studied the expression of several collagens in the testis of wild-type and CIZ mutant mice. As shown in Fig. 6A, the expression of procollagen type 1 alpha 1 (COL1A1) was almost restricted to Sertoli cells, where no expression of CIZ/Nmp4 was observed (Fig. 3A). The COL1A1 signal in Northern blot was higher in the testis from 8-week-old CIZ–/– mice than in that of CIZ+/+ mice (Fig. 6C), but this difference is likely due to the difference in Sertoli cell/spermatogenic cell ratio between CIZ–/– and CIZ+/+ animals. The expression of both procollagen type 3 alpha1 (COL3A1) and procollagen type 4 alpha3 (COL4A3) was also higher in CIZ–/– mice (Fig. 6C), but both were mainly expressed in Sertoli cells (Fig. 6B). As a screening for other collagens, we checked the expression of collagen 2 and collagen 10 by RT-PCR. Although we detected their expression in the brain, we could not detect their expression in the testis (data not shown). Therefore, the absence of co-localization between CIZ and collagen expression suggests that the defect in spermatogenesis seen in CIZ–/– mice is unlikely due to a change in collagen expression.
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Possible involvement of BMP signalling in the testis phenotype
CIZ/Nmp4 inhibits BMP2-induced up-regulation of alkaline phosphatase, osteocalcin, type-I collagen as well as Cbfa1 (Runx2/AML3/Osf2) (Shen et al. 2002). The expression of Cbfa1 in the testis is reported, although its regulation and function in the testis are unknown (Ogawa et al. 2000). Cbfa1 was expressed in both spermatogenic cells and Sertoli cells (Fig. 6B). However the expression of Cbfa1 was not significantly different between CIZ+/+ and CIZ–/– testes (Fig. 6C).
CIZ/Nmp4 is also reported to interfere with Smad1 and Smad5 in BMP2 signalling in osteoblast-like cells (Shen et al. 2002). In testis, Smad1 (Madr1) is specifically expressed in spermatogenic cells from the pachytene spermatocyte stage to the round spermatid stage (Zhao & Hogan 1997), although the role of Smad1 in spermatogenesis is not known. The study by immunofluorescence of CIZ/Nmp4 and Smad1 expression revealed that CIZ/Nmp4 appeared more concentrated in regions where Smad1 was also expressed (Fig. 7A–C). In higher magnification, CIZ/Nmp4 was co-localized with Smad1 within cells (Fig. 7D–F). In contrast, in CIZ–/– testis, the nuclear staining of CIZ/Nmp4 was not obvious, although the cytoplasmic staining was observed (Fig. 7H,K), whereas Smad1 remained expressed in the nucleus (Fig. 7G,J).
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Discussion |
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CIZ–/– male mice were partially infertile and the weight of testes of old CIZ–/– mice was smaller than those of CIZ+/+ mice. Histologically, some of the seminiferous tubules totally lacked spermatogenic cells and contained only Sertoli cells (Fig. 2). Apoptosis of spermatogenic cells was increased in the testis of 4 week, 8 week and 5 month old CIZ–/– males (Fig. 4). Considering the fact that some of the tubules of the youngest CIZ–/– mice had already fewer spermatogenic cells than normal, the real increase in apoptosis in CIZ–/– testes could be higher than detected. However, some CIZ–/– male mice remained fertile even after 6 months of age, illustrating the variability in phenotype among mutant animals. Furthermore, the frequency of apoptosis in primary spermatogenic cell culture from CIZ–/– testes was not different from that of CIZ+/+ testes. The variability among individuals and the discrepancy between in vivo and in vitro studies suggest that the apoptosis is caused by subtle changes of microenvironment in the testis.
Cell-cell interactions between spermatogenic cells and Sertoli cells are known to play pivotal roles in spermatogenesis. Sertoli cells produce many factors and surface molecules that support the survival of spermatogenic cells as well as elicit apoptosis. Sertoli cells also phagocytose spermatogenic cells that are undergoing apoptosis. Therefore, the increased apoptosis of spermatogenic cells described above might be attributed to an abnormality of Sertoli cells. However, we regard this improbable, because CIZ/Nmp4 expression in testis was mainly localized in spermatogenic cells, and was undetectable in Sertoli cells (Fig. 3). In addition, the phagocytic activity (Shiratsuchi et al. 1997) of Sertoli cells from CIZ–/– mice assayed against apoptotic rat spermatogenic cells in primary culture or against apoptotic murine thymocytes after dexamethasone treatment was not different from that of CIZ+/+ mice (data not shown).
The in vitro apoptosis assays showed no difference between CIZ–/– and CIZ+/+ testicular cells. In these assays, the spermatogenic cells were cultured with Sertoli cells. Therefore, the discrepancy in the TUNEL assay of the section could not be caused by the direct interaction with Sertoli cells. The difference might be attributed to the microenvironment or the local factors that work in the testis.
Genetically engineered mice with similar phenotypes
Deregulated apoptosis in spermatogenic cells and impaired spermatogenesis have been reported in mice upon loss of expression of particular proteins, including apoptosis-related proteins Bcl-6, Bcl-w and Bax (Knudson et al. 1995; Print et al. 1998; Ross et al. 1998; Kojima et al. 2001), the cytoskeleton-associated proteins, LIM-kinase 2 (Limk2) (Takahashi et al. 2002), the signaling molecule, Akt1 (Chen et al. 2001), and proteins with other functions such as, Huntingtin-interacting protein 1, testis-specific cytochrome c, Egr4, aromatase (cyp 19) and TIF1ß (Robertson et al. 1999; Tourtellotte et al. 1999; Rao et al. 2001; Narisawa et al. 2002; Weber et al. 2002). In addition, BMP8b–/– mice show impaired spermatogenesis at early puberty, increased apoptosis in spermatogenic cells and male sterity (Zhao et al. 1996). Involvement of another BMP, BMP8a in spermatogenesis has also been reported (Zhao et al. 1998). Among mice mentioned above, the phenotype observed in CIZ–/– mice as to spermatogenesis and male fertility is most similar to that in BMP8b–/– mice. The signalling pathway mediated by BMP4/ALK3/Smad5 is involved in the differentiation of spermatogonia (Pellegrini et al. 2003). On the other hand, CIZ/Nmp4 seems to be involved in the BMP signalling: it inhibits the signalling pathway mediated by BMP2/Smad1/Smad5 signalling (Shen et al. 2002). We found in this study that CIZ/Nmp4 is co-localized with Smad1 in nuclei of spermatogenic cells (Fig. 7). These facts collectively suggest that disruption of the BMP signalling is causative, at least partly, of impaired spermatogenesis in CIZ–/– mice.
Other signalling pathways that might be involved in the testicular phenotype of CIZ–/– mice
CIZ/Nmp4 was originally cloned as a p130Cas binding protein, and was reported to regulate the expression of MMPs (Nakamoto et al. 2000). Other groups also reported that the over-expression of CIZ/Nmp4 enhances the transcription from the MMP7 promoter (Martini et al. 2002; Torrungruang et al. 2002). Although we checked the expression of several MMPs in the testis, we could not detect any positive signal. It is therefore improbable that the phenotype is caused by a disregulation of MMPs in the absence of CIZ/Nmp4. Collagen is also reported to be regulated by CIZ/Nmp4 (Furuya et al. 2000; Thunyakitpisal et al. 2001). However, as shown in Fig. 6, the expression of collagens is mostly restricted to Sertoli cells, where CIZ/Nmp4 is not expressed (Fig. 3). Therefore, deregulation of collagen expression does not seem to be involved in the phenotype of CIZ–/– testis. p130Cas, which is highly expressed in testis (Sakai et al. 1994), is a focal adhesion protein that is involved in integrin signalling (Nojima et al. 1995) and integrins are expressed in the spermatogenic stem cells (Shinohara et al. 1999), suggesting the possibility that integrin signalling might be involved in the phenotype of CIZ–/– testis. However, the binding of p130Cas and CIZ/Nmp4 in spermatogenic cells could not be shown with our antibodies.
Possible phenotypes in stimulation
We and others have reported that CIZ/Nmp4 is involved in BMP2 signalling (Shen et al. 2002) and in the regulation of collagen and MMP expression in osteoblasts (Bidwell et al. 2001; Furuya et al. 2000; Thunyakitpisal et al. 2001; Torrungruang et al. 2002). CIZ–/– mice showed shorter stature than wild-type animals but their bone showed no abnormality in X-ray and in tissue section microscopy. No abnormality in the bones is apparent in CIZ–/– mice in physiological conditions, but it is possible that some abnormalities would arise if CIZ–/– mice were stimulated or subjected to stress, or in the context of a pathological state. Investigation of changes in the bone of CIZ–/– mice in pathological states or under stimulation is in progress (Morinobu, M., Noda, M and others; unpublished observation).
The role of CIZ/Nmp4 in human disease has been reported as EWSR1- or TAF15-CIZ/NMP4 fusion proteins are implicated in leukaemogenesis (Martini et al. 2002). Furthermore, polymorphism of a CAG repeat in the CIZ/Nmp4 gene (CAG-H1) is found in patients with testicular cancer (Ono et al. 2001), and the overrepresentation of the short arm of chromosome 12, where CIZ/Nmp4 is located, is correlated to invasive growth of human testicular tumours (Rosenberg et al. 2000). In this paper, we report impaired spermatogenesis and partial infertility as the phenotype of CIZ–/– mice. This suggests that CIZ/Nmp4 plays a role in testis and that CIZ/Nmp4 can be a candidate for some human testicular diseases including the infertility disease that shows the histology of ‘Germinal-cell aplasia with focal spermatogenesis’ or some of the testicular tumours. Future research is aimed at addressing the role of CIZ/Nmp4 in these diseases.
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Experimental procedures |
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Partial cDNA for mouse CIZ/Nmp4 was cloned by screening a mouse embryo cDNA library (Clontech) with the full-length rat CIZ cDNA (Nakamoto et al. 2000). A 8.9 kb-DNA fragment containing CIZ/Nmp4 (nmp4) gene was isolated from 129/Sv genomic DNA library (Stratagene) by hybridization with the RsaI fragment (263-510 nucleotides) of mouse CIZ cDNA. ß-galactosidase derived from pCMV-ßgal (TROPIX), followed by neo-cassette derived from pMCneopolyA (Stratagene) in reverse direction, was inserted into the NarI site of the 2nd coding exon, so that the N-terminal portion of CIZ is fused to ß-galactosidase. DTA (diphtheria toxin A gene) from pMC1DTApolyA (Stratagene) was added to the 5' end of this fragment for negative selection. This fragment was inserted into pBluescriptSK(-) vector to make the targeting construct.
Twenty micrograms of the targeting construct, linearized with ApaI, was electroporated into 1 x 107 B6 x CBA F1 ES cells, TT2, and the targeted clones were selected for 7 days with 320 µg/mL Geneticin (Invitrogen). The drug resistant colonies were then screened by Southern blot analysis as described below. The correctly targeted clones were aggregated with CD1 mouse embryos at the morula stage. Established male chimeric mice were bred with C57BL/6 (B6) female mice, resulting in germ line transmission.
CD1 and B6 mice were purchased from Clea Japan Inc., and all the animals were maintained and bred at the animal facility in our institute under specific pathogen-free conditions.
Southern blotting and genomic PCR
For genotyping ES clones, approximately 10 µg of genomic DNA extracted from ES cells was digested with BamHI, separated in 0.8% agarose gels, transferred to Hybond-N membranes (Amersham Biosciences) and hybridized with the 5'-probe depicted in Fig. 1. The 5'-probe was made by PCR using a genomic clone as template and the following primers: 5'-GGATCCAAACTCCAGTTCTC and 5'-ATCAAGGAACACACTCCAAC. The probe was labelled by a Multiprime DNA Labeling System (Amersham Biosciences). Hybridization was carried out at 42 °C in a solution containing 50% formamide, 5x SSC, 5x Denhardt's solution, 0.5% SDS, denatured salmon sperm DNA (20 µg/mL) and a 32P-labelled probe. Hybridized filters were washed twice in 2X SSC/0.1% SDS for 20 min at 55 °C, followed by exposure to a BAS-III imaging plate (Fuji Film).
For genotyping mice, genomic DNA extracted from mouse tails was used as template for PCR with the following primer sets. For wild-type allele: WT-1 5'-ACCCGTACTTCTGGCCTTCT, WT-2 5'-CTGAGGCAGGGACAGTTAGC, for recombinant allele: REC1 5'-GATCAGCTGTTGCCAGAGAA, REC2 5'-GGCCTCTTCGCTATTACGC. In some cases, the Southern blotting described above was also used for confirmation.
Immunoprecipitation and western blotting
CIZ+/– male mice were mated with CIZ+/– female mice and E12.5 embryos were collected by caesarian section. Head and inner organ tissues were used for genotyping. Primary embryonic fibroblast-like cells were isolated from the remaining of embryos by trypsin digestion, and cultured in DMEM with 10% foetal bovine serum, penicillin (100 U/mL) and Streptomycin (100 µg/mL). For immunoprecipitation, the primary fibroblast-like cells were lysed with RIPA buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 10 U/mL aprotinin, 2 mM phenylmethylsulphonyl fluoride (PMSF), 1 mM Na3VO4). Cell lysates (1 mg protein) were incubated with 5 µL of anti-CIZ antibody (Nakamoto et al. 2000) for 1 h at 4 °C and subsequently incubated for 1 h at 4 °C with protein-A-Sepharose (Sigma). The beads were washed with RIPA buffer 4 times and boiled in Sample buffer. SDS-PAGE and Western blotting were performed as described using anti-CIZ antibody (Nakamoto et al. 2000).
Immunofluorescence
Immunofluorescence staining of primary embryonic fibroblast-like cells with anti-CIZ antibody was performed as described before (Nakamoto et al. 2000). The anti-Nmp4 (anti-AgA) antibody was a generous gift from Dr Bidwell (Indiana University). Immunofluorescence with anti-Nmp4 antibody was performed as described (Feister et al. 2000). The anti-Smad1 antibody (sc-7965) was purchased from Santa Cruz.
Northern blotting and RT-PCR
Sertoli cells and spermatogenic cells were isolated with a purity of at least 80% from testes of 20-day-old rats as previously described (Shiratsuchi et al. 1997). Total RNAs were extracted from these cell populations or from murine testis as described (Chomczynski & Sacchi 1987). Total RNAs were separated by electrophoresis on a 1% agarose gel, transferred to a Hybond-N membrane (Amersham Biosciences), and hybridized as mentioned above with the CIZ, procollagen type I alpha 1 or procollagen type III alpha 1 probes previously described (Nakamoto et al. 2000, 2002). For RT-PCR, 100 ng of total RNA was mixed with primers and one-step RT-PCR was performed using SuperScript One-Step RT-PCR with Platinum. Taq (Invitrogen). The primers for mouse collagen 2 and mouse collagen 10 (van der Kraan et al. 1998); mouse collagen 4 (Moridaira et al. 2003); rat collagen 2 and rat GAPDH (Khatib et al. 2002); rat collagen 3 and rat MMP13 (Wang et al. 2002); rat collagen 4, rat MMP2 and rat MMP9 (Siu et al. 2003); mouse MMP2 and mouse MMP9 (El Fahime et al. 2000); MMP3 and MMP7 (Haro et al. 2000); rat MMP8 (Sasano et al. 2002) and Cbfa1 (Gilbert et al. 2002) are described elsewhere. The primers used for mouse GAPDH are 5'-ACCACAGTCCATGCCATCAC and 5'-TCCACCACCCTGTTGCTGTA.
Histological analysis and TUNEL assay
Organs from mice were fixed with 3.7% formaldehyde, embedded with paraffin, serially sectioned and stained with haematoxylin and eosin (H&E). The examined organs were brain, heart, lung, thymus, liver, spleen, kidney, extremities including bones, calvaria, skin including nipples, ovary and testis. For TUNEL assay, the apoptosis in situ detection kit (WAKO) was applied to paraffin-sections from testis, following the manufacturer's instruction. Sections were counterstained with Nuclear Fast Red (Vector Laboratories).
Colorimetric analysis of ß-galactosidase
Testes from mice were fixed with 0.5% glutaraldehyde/phosphate buffered saline (PBS)/2 mM MgCl2 for 1 h, soaked in 30% sucrose/PBS/2 mM MgCl2 overnight, frozen in OCT compound in liquid nitrogen and serially sectioned. The sections were postfixed with 1% formaldehyde in PBS for 5 min, washed twice with PBS and soaked overnight in Lac Z staining solution (5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 0.01% SDS, 0.02% NP-40, 1 mg/mL X-gal). The next day, the sections were washed with water, briefly counterstained with Nuclear Fast Red (Vector Laboratories), washed with water and dehydrated with ethanol and Lemosol (WAKO).
Primary culture of dispersed mouse testicular cells
Preparation and culture of cells present in mouse seminiferous tubules were done essentially according to the procedure for primary culture of rat testicular cells (Nagao 1989; Shiratsuchi et al. 1997). In brief, seminiferous tubules of 7–8-month-old CIZ–/– or CIZ+/+ mice were obtained by treating testes with collagenase and incubated in the presence of trypsin for cells present in the tubules to be released. The dispersed cells were maintained with F12-L15 medium containing 10% preheated foetal bovine serum and 1 µg/mL norepinephrine at 32.5 °C.
Phosphatidylserine externalization assay
Translocation of phosphatidylserine from the cytoplasmic to the exoplasmic leaflet of the plasma membrane was assessed by flow cytometry using annexin V, which specifically binds to phosphatidylserine, as previously described (Martin et al. 1996). In brief, primary cultured mouse testicular cells were treated with 5-carboxyfluorescein-labelled annexin V and propidium iodide and analysed in a flow cytometer. The cells that were less intensely stained with propidium iodide and thus impermeable to annexin V were gated and analysed for the amount of bound annexin V.
Lectin binding assay
Primary cultured mouse testicular cells were suspended in PBS containing fluorescein isothiocyanate-labelled wheat germ agglutinin (Honen) 20–30 mg/mL and propidium iodide, and incubated at room temperature for 30 min. The samples were then washed with PBS and analysed by flow cytometry. The cells less intensely stained with propidium iodide were gated and analysed for the amount of bound lectin.
DNA flow cytometry
Primary cultured mouse testicular cells were treated with PBS containing 35% ethanol and incubated with deoxyribonuclease-free ribonuclease A for 20 min at 37 °C. The cells were then stained with propidium iodide and analysed by flow cytometry as previously described (Shiratsuchi et al. 1999).
Statistics
Values are expressed as means and standard errors. For each parameter, statistical significance of differences between groups was determined by Student's t-test and significance was rejected at P > 0.05.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: nakamoto{at}scripps.edu
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References |
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Received: 9 January 2004
Accepted: 22 March 2004
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, middle curve) and CIZ–/– (
, lower curve) mice were blotted against their age. (G) Body weight in old mice (10–13 months old) of CIZ+/+ male (n = 9, bar 1), CIZ–/– male (n = 13, bar 2), CIZ+/+ female (n = 10, bar 3) and CIZ+/+ female (n = 6, bar 4). The differences of weight between CIZ+/+ and CIZ–/– mice were statistically significant *P < 0.001 (male), P < 0.05 (female).
CIZ+/+ mice. 
