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Genes to Cells (2007) 12, 721-733. doi:10.1111/j.1365-2443.2007.01088.x
© 2007 Blackwell Publishing or its licensors
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Male-specific sterility caused by the loss of CR16

 Shiro Suetsugu1,2,{dagger}, Yoshibumi Banzai1,{dagger}, Masayoshi Kato1,3,{dagger}, Kiyoko Fukami4, Yuki Kataoka5, Yoshimi Takai6, Nobuaki Yoshida5 and Tadaomi Takenawa1,*

1 Department of Biochemistry, and 5 Division of Gene Expression and Regulation, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
2 PRESTO, JST, 4-1-8, Honcho, Kawaguchi City, Saitama 332-0012, Japan
3 Department of Molecular Biology and Biochemistry, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto, Nagano 390-8621, Japan
4 Laboratory of Genome and Biosignal, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
6 Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The gene encoding the protein known as "corticosteroids and regional expression 16" (CR16) has been shown to be regulated by glucocorticoids. CR16 is a member of the Wiskott–Aldrich syndrome protein (WASP)-interacting protein (WIP) family. It binds to the neural WASP (N-WASP), which activates the Arp2/3 complex to induce actin polymerization. CR16 is highly expressed in the testes, particularly in the Sertoli cells, which harbor sperm progenitors and play an important role in spermatogenesis. We found male-specific sterility in the CR16-knockout mice. The sperms of the CR16-knockout mice had abnormal head morphology, and greatly diminished fertilization ability in in vitro fertilization experiments. CR16 and N-WASP were localized to the actin filaments at the Sertoli cell–spermatid junctions (SspJs). The level of N-WASP but not the transcript was decreased in the testes and Sertoli cells of the CR16-knockout mice. Therefore, CR16 and N-WASP are suggested to play important roles in spermatogenesis.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Corticosteroids and regional expression 16 (CR16) was found to be a gene whose expression is regulated by corticosteroids (Masters et al. 1996). CR16 is a member of the Wiskott–Aldrich syndrome protein (WASP)-interacting protein (WIP) family that includes WIP, CR16, and the WIP- and CR16-homologous protein (WICH/WIRE) (Masters et al. 1996; Ramesh et al. 1997; Ho et al. 2001; Martinez-Quiles et al. 2001; Aspenstrom 2002; Kato et al. 2002). The WIP family proteins are reported to form protein complexes with WASP and neural WASP (N-WASP) (Ramesh et al. 1997; Ho et al. 2001; Martinez-Quiles et al. 2001; Aspenstrom 2002; Kato et al. 2002; Zettl & Way 2002). The expression of WASP is restricted to hematopoietic cells. Initially, N-WASP was referred to as neural WASP because it was abundantly expressed in the neural tissue; however, it is ubiquitously expressed in a variety of tissues.

WASP and N-WASP bind to the actin monomer and the Arp2/3 complex; this causes a burst of actin polymerization through the activation of the Arp2/3 complex for the nucleation of actin polymerization. The Arp2/3 complex activation occurs when other molecules including Cdc42 or proteins with the SH3 domain bind to the Cdc42-Rac-interactive-binding (CRIB) motif or the proline-rich region of WASP and N-WASP (Takenawa & Miki 2001; Pollard & Borisy 2003).

The activation of the Arp2/3 complex by the WIP family proteins does not occur directly. They bind to the WASP homology 1 (WH1) domain of WASP and N-WASP. The associations of WASP or N-WASP with the WIP family proteins can be stable, which may assist in the maintenance of the WASP levels (Krzewski et al. 2006; Sawa & Takenawa 2006). The interaction with the WIP family proteins is believed to suppress the WASP/N-WASP activity (Martinez-Quiles et al. 2001; Hertzog et al. 2004; Ho et al. 2004); however, WIP also serves as a scaffold that links WASP to adaptor proteins such as CrkL and Nck, and it is recruited to the region of vigorous actin polymerization (Frischknecht et al. 1999; Moreau et al. 2000; Sasahara et al. 2002). The WIP or CR16 protein forms a protein complex with N-WASP, and this complex can be activated by Cdc42 and Toca-1 (Masters et al. 1996; Ho et al. 2001; Ho et al. 2001, 2004).

More importantly, the WH1 domain is a hot spot for mutations in patients with Wiskott–Aldrich syndrome, thereby suggesting that the WASP/WIP interaction plays important roles in WASP function (Ochs & Notarangelo 2005). Among hematopoietic cells, the WIP-knockout T cells and WASP-knockout T cells were observed to have a similar phenotype with regard to defects such as decreased actin filament formation in response to anti-CD3 antibodies (Snapper et al. 1998; Anton et al. 2002). Taken together, these findings indicate that WIP family proteins are important for WASP function.

In this study, we generated CR16-knockout mice. These mice had defective spermatogenesis, and the CR16-knockout males were sterile. The N-WASP level was decreased in the testes, thereby suggesting the involvement of N-WASP-CR16 protein complex in the process of spermatogenesis.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Generation of CR16-knockout mice

We screened a mouse cDNA library with human CR16 as a probe because a mouse homologue of CR16 has not been reported. The predicted amino acid sequence of the mouse CR16 was well conserved with that of rats (Fig. 1A). The mouse CR16 had a splice variant ({Delta}ins) that lacked a region known to interact with N-WASP, that was coded by exon 6 (Fig. 1A,B) (Ho et al. 2001).


Figure 1
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  		Figure 1  Mouse CR16. (A) Amino acid sequence of CR16. The exons are mapped by underlines and dashed-underlines. The Verprolin homology region and N-WASP binding region are also indicated by boxes. (B) Amino acid alignment of the Verprolin homology region or N-WASP binding region. (C, D) Location of the CR16 gene on mouse chromosome 6. CR16 includes the previously identified RefSeq RNA KM_620 310. The coding region of the CR16 gene is divided into eight exons.

 
To examine the physiological roles of CR16, we generated CR16-knockout mice by targeted homologous recombination. A ß-galactosidase (LacZ) reporter with a nuclear localization signal (NLS) was substituted for the exon 1 containing the ATG (Figs 1C,D and 2A). Thus, LacZ expression could be controlled by the native CR16 promoter. Southern blotting and PCR analyses of the genomic DNA showed that the CR16 gene was disrupted as expected (Fig. 2B,C).


Figure 2
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  		Figure 2  Gene-targeting strategy. (A) Exon 1 of CR16 was replaced by the nuclear-localization-tagged ß-galactosidase (lacZ), xanthine/guanine phosphoribosyl transferase gene (gpt), and the neo gene (neo). The diphtheria toxin A (DTA) gene was inserted at the 3'-end of the CR16 gene in the targeting vector. EcoRI (EI) sites are indicated. (B) Southern blot analysis of mouse genomic DNA. The expected fragments generated by EcoRI are 11.5 kb pairs (kbp) for the wild-type allele, and 9.5 and 4.5 kbp for the mutant alleles. The designations +/+, +/– and –/– indicate CR16+/+, CR16+/– and CR16–/– mice, respectively. (C) PCR analysis of the genomic DNAs from CR16+/+, CR16+/– and CR16–/– mice. (D) Western blot analysis of various mouse tissue lysates with anti-CR16 antibodies. The brain expresses a splice variant of CR16 that lacks the N-WASP-binding region, thereby yielding an additional band. (E) Western blot analysis of the tissue lysates from CR16+/– and CR16–/– mice with anti-CR16, anti-WICH and anti-ß-galactosidase antibodies.

 
Next, we generated an antibody against CR16 and examined the distribution of CR16 in various tissues by Western blotting (Fig. 2D). The splice variant of CR16 without the region that was known to interact with N-WASP was expressed only in the brain, whereas the full-length CR16 was expressed in both the brain and the testes in mice (Fig. 2D), as reported in rats and humans (Masters et al. 1996; Ho et al. 2001). We confirmed that the splice variant of CR16 was not co-immunoprecipitated with N-WASP (data not shown). Although the band of CR16 on testis lysates appeared at the position between the two bands on brain lysate, the immunoprecipitation experiments of brain lysates indicate that full-length CR16 is expressed in testis (Fig. 7A). Western blotting of tissues from the CR16-knockout mice confirmed that CR16 was not expressed in the testes or brain (Fig. 2E).


Figure 7
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  		Figure 7  N-WASP expression in the CR16–/– testes. (A) Lysates of the testes of the CR16+/+ or CR16–/– mice were subjected to immunoprecipitation with an anti-N-WASP or anti-CR16 antibody. The Western blots of the precipitates and the original lysates are shown. (B) The amounts of N-WASP, CR16 and actin in the whole testes lysates and Sertoli cell preparations. The Western blots of the total lysates are shown. (C, D) Amount of N-WASP in the whole testis sections (C) or Sertoli cell preparations (D) with equal amounts of the lysate. The level of N-WASP in the testis or Sertoli cells of the CR16+/– mice was set to an arbitrary value of 1, and the level of N-WASP in the CR16–/– mice is presented relative to this value. (E) Reverse transcriptase (RT)-PCR analysis for the quantification of N-WASP mRNA in the testes. The bands for GAPDH, N-WASP and CR16 amplified from 20 or 200 ng of total testes RNA is shown. From the calibration curve, the level of N-WASP mRNA of CR16–/– testes was 1.04 times that of the CR16+/– testes.

 
Abnormal spermatogenesis in CR16–/– mice

CR16+/– mice were healthy and fertile. Crosses of CR16+/– male with CR16+/– female yielded CR16+/+, CR16+/– and CR16–/– offsprings in the predicted Mendelian ratio (Table 1A). Both male and female CR16–/– mice were healthy and had no apparent defects (data not shown). Histological examination of the CR16–/– brain revealed no obvious abnormality (data not shown). Female CR16–/– mice had normal fertility (Table 1A,B); however, the fertility of male CR16–/– mice appears to be impaired (Table 1C). When male CR16+/– mice were mated with wild-type females, ~86% of the wild-type females were impregnated after a 72-h mating period, and their average litter size was 7.8 pups. On the other hand, when male CR16–/– mice were mated with wild-type females, only 13% of the wild-type females were impregnated, and their average litter size was 2.5 pups. The ratio of male and female pups was not different between CR16+/– x CR16–/– and CR16–/– x CR16–/– crossings (Table 1B). Thus, the loss of CR16 primarily affects male fertility.


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  		Table 1  Results of the cross breedings
 
To identify the defects in the sperms of CR16–/– mice, we first examined the sperms from these mice by differential interference contrast (DIC) microscopy. As shown in Fig. 3A, there was an obvious abnormality in the sperm heads of CR16–/– mice. Electron microscopy revealed that the sperm heads of the CR16–/– mice were extremely thin and had abnormal morphology when compared with those of the CR16+/– mice (Fig. 3B). Approximately 22% of the sperms in CR16–/– mice had normal heads whereas 83% of the sperms in CR16+/– mice had normal heads. Using DIC microscopy, we then estimated the percentage of motile sperms to examine their viability. The percentage number of motile sperms was slightly decreased in the CR16–/– mice; however, this decrease was not significant when compared with that observed in the CR16+/– mice (Fig. 3D).


Figure 3
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  		Figure 3  Abnormal sperms of the CR16–/– mice. (A) Differential interference contrast (DIC) micrograph of the sperms of the CR16+/– and CR16–/– mice. The scale bar represents 10 µm. Arrowheads indicate abnormal sperms. (B) Transmission electron micrograph of the lateral (upper) or the longitudinal (lower) sections of the sperms of the CR16+/– and CR16–/– mice. The scale bar represents 1 µm. (C) Sperms that had an abnormal sperm head were counted under a DIC microscope and represented as a percentage of total sperms. (D) Sperms were observed under a DIC microscope, and the number of motile sperms was recorded. The motile sperms are shown as a percentage of total sperms. (E) In vitro fertilization studies with CR16+/+, CR16+/– or CR16–/– sperms and CR16+/+ eggs. The percentage of eggs that proceeded to the two-cell stage (24 h) is indicated.

 
We performed in vitro fertilization experiments to establish the inability of the sperms to cause fertilization (Fig. 3E). Twenty-four hours after insemination, ~80% of the eggs were fertilized with the sperms of the CR16+/– mice, whereas only 20% of the eggs were fertilized with the sperms of the CR16–/– mice. These results clearly demonstrated the defects in the sperms of the male CR16–/– mice.

Expression of CR16 in the Sertoli cells

Sperm maturation is known to occur on the Sertoli cells. To examine the possible defect in spermatogenesis, we then observed the sections of seminiferous tubules of CR16+/+, CR16+/– and CR16–/– mice. Hematoxylin–eosin staining revealed that except for the spermatids during the late stages of spermatogenesis, there was no apparent defect in the cells in the seminiferous tubules (Fig. 4A). During the late stages of spermatogenesis, the head of the spermatids appeared thin and abnormal (Fig. 4A,B). Stage VII is characterized by elongated spermatids that move to the luminal aspect of the seminiferous epithelium. Spermatids develop with migration toward the luminal surface of the seminiferous tubules. Spermatids development was characterized as stages and steps. Stages are classified by the appearance of section of seminiferous tubules, whereas steps are classified according to the development of spermatids. Therefore, each stage of seminiferous tubules contains spermatids of two or three steps. Stage VII seminiferous tubules contain spermatids of step 7 and 16. Step 7 spermatids reside in the outer area of the tubules (arrowheads in Fig. 4B') that had no apparent morphological defects. Step 16 spermatids reside at the luminal surface of the tubule, and are ready to detach from the Sertoli cells. The thinner heads of step16 spermatids at the luminal surface of seminiferous tubules were clear in CR16–/– testis; however, the number of spermatids was not significantly different (Fig. 4A,B and B'). Therefore, the defect in sperm development is suggested to occur at the latest step of spermatogenesis.


Figure 4
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  		Figure 4  CR16 expression in the Sertoli cells. (A) Hematoxylin–eosin (HE) staining of the testis sections of CR16+/+, CR16+/– and CR16–/– mice. The colors blue and red indicate the nuclei and cytosol, respectively. The scale bar represents 25 µm. (B) Periodic acid Schiff staining of the testis sections. The testis sections of CR16+/– and CR16–/– mice containing spermatids at stage VII of spermatogenesis were stained. (B') Enlarged images of the insets in (B). Arrows indicate the step 16 spermatids, and arrowheads indicate the step 7 spermatids. (C) ß-Galactosidase activity in the nuclei of Sertoli cells of the CR16+/+ and CR16–/– testis by the nuclear-localized lacZ substituted for CR16 gene (blue). The section was counterstained with eosin (red). (D) The number of Sertoli cells was counted in circular-shaped sections of the seminiferous tubules.

 
We then examined the expression of CR16 in the testis sections to identify the cells that expressed the CR16 protein. The activity of the CR16 promoter in the testes was determined by examining the activity of nuclear-localized ß-galactosidase (Fig. 4C). Clear ß-galactosidase activity was observed in the nuclei of Sertoli cells in the CR16+/– mice but not in those of the wild-type. ß-Galactosidase activity was not observed in the other cell types, including the sperm (Fig. 4C). These data suggested that CR16 expression was restricted to the Sertoli cells.

Subsequently, we measured the number of Sertoli cells in the seminiferous tubule sections by examining their nuclear shapes. The tubules with similar cutting angles were selected for the counting. As shown in Fig. 4D, the number of Sertoli cells was not reduced in the CR16–/– mice.

Localization of CR16 in Sertoli cells

Trans-interaction between nectin-2{alpha} and nectin-3{alpha} mediates the formation of Sertoli cell–spermatid junctions (SspJs). Therefore, the localization of nectin-2{alpha} indicates the presence of SspJs (Bouchard et al. 2000; Ozaki-Kuroda et al. 2002). We obtained the specific antibody against CR16 (Fig. 5A). To examine the possible functions of CR16 in the Sertoli cells, the testis sections were stained with phalloidin to visualize the actin filaments and with antibodies against nectin-2{alpha} and CR16. In the SspJs of the CR16+/– mice, CR16 and nectin-2{alpha} were co-localized along with the actin filaments (Fig. 5B). In the SspJs of the CR16–/– mice, the CR16 signals were almost absent, thereby indicating the specific signals of the anti-CR16 antibody. However, this result also indicates that the signals observed at the rim of the seminiferous tubules in the CR16+/– mice are non-specific ones, because they still remain in the CR16–/–mice (Fig. 5B). Without primary antibodies including anti-CR16, no signals appeared at the SspJs (Fig. 5C).


Figure 5
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  		Figure 5  Localization of N-WASP and CR16 at Sertoli-cell-spermatid junctions (SspJs). (A) CR16 antibody. Western blotting of the total testis lysate was performed with rabbit polyclonal CR16 antibody. (B–F) Cryosections of the seminiferous tubules of the CR16+/– (upper) and CR16–/– (lower) mice were stained (green in "merged" image) with antibodies for (B) CR16 (rabbit polyclonal, green), nectin-2{alpha} (rat monoclonal, red), and actin filaments (phalloidin, blue), (C) secondary antibody for rabbit immunogloblin only (green), nectin-2{alpha} (rat monoclonal, red), and actin filaments (phalloidin, blue), (D) N-WASP (rabbit polyclonal, green), nectin-2{alpha} (rat monoclonal, red), and actin filaments (phalloidin, blue) and (E) phosphotyrosine (mouse monoclonal, green), actin filaments (phalloidin, red) and DNA (TO-PRO3, blue). The scale bar represents 20 µm. Enlarged images marked with boxes are shown as insets in (B) and (D). Enlarged images for (E) are shown in (F). In (B), the anti-CR16 antibody stained the rim of CR16–/– seminiferous tubules, as indicated by the non-specific signal at the rim of the seminiferous tubules.

 
CR16 is known to bind to N-WASP. Therefore, we examined the localization of N-WASP in the testis sections. N-WASP was co-localized with nectin-2{alpha} in the testes of the CR16+/– mice and more importantly in the testes of the CR16–/– mice (Fig. 5D), thereby suggesting that although CR16 is co-localized with N-WASP, CR16 is not involved in the localization of N-WASP in the testes. Despite the possible involvement of CR16 in the actin cytoskeleton, phalloidin staining did not detect significant changes in the actin filaments at the SspJs (Fig.  5B,D).

It is reported that tyrosine phosphorylation was observed at the cell–cell junctions between the Sertoli cells and other cells including spermatids (Maekawa et al. 2002). We stained the seminiferous tubule of the CR16+/– and the CR16–/– mice sections by an anti-phosphotyrosine antibody to examine whether the Sertoli cells in the CR16–/– mice had normal morphology. There was no apparent difference in the distribution of phosphotyrosine staining in the testis sections of the CR16+/– and CR16–/– mice (Fig. 5E,F), thereby suggesting that there was no apparent defect in the Sertoli cell morphology.

Subsequently, SspJs were examined by electron microscopy (Fig. 6). Examination of the organization of SspJs revealed that the actin filament bundles were aligned at the SspJs in the testes sections of the CR16+/+ and CR16+/– mice (Fig. 6A and data not shown). However, although the actin bundles were observed in the testes of the CR16–/– mice, these bundles were slightly sparse (Fig. 6B). However, there was no abnormal cavity or discontinuity in the junctions between the Sertoli cells and spermatids (Fig. 6B), thereby suggesting that the SspJs were formed in the CR16–/– mice as well.


Figure 6
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  		Figure 6  Actin bundles at the Sertoli cell-spermatid junctions (SspJs). Spermatids in the seminiferous tubules from the testes of the (A) CR16+/+ and (B) CR16–/– mice were observed by transmission electron microscopy. Enlarged images for (A) and (B) are shown in (a), (b) and (c), (d), respectively. The scale bars represent 2 or 0.125 µm. A, acrosome; N, nuclei of the elongated spermatids; S, Sertoli cell. The arrows indicate actin filament bundles at SspJs.

 
Decreased amount of N-WASP in the testes and Sertoli cells of the CR16–/– mice

We then examined the association of N-WASP with CR16 in the testes. Immunoprecipitation of testes lysates with the anti-N-WASP antibody or anti-CR16 antibody revealed that there is an interaction between N-WASP and CR16 in the testes (Fig. 7A). In the CR16–/– mice, immunoprecipitation with the anti-CR16 antibody did not result in the co-precipitation of N-WASP from the testes lysates (Fig. 7A).

A comparison of the amount of N-WASP in the whole testis or isolated Sertoli cells revealed that the amount of N-WASP was reduced in the CR16–/– mice (Fig. 7B). Quantitative Western blotting showed that there was a 50% reduction in the N-WASP levels in the whole testis and Sertoli cells in the CR16–/– mice (Fig. 7C). To examine the mechanism by which CR16 regulates the amount of N-WASP, the molar ratio of CR16 :  N-WASP was examined in the whole testis or in the cultured Sertoli cells (Fig. 7D). In the Sertoli cells, the number of CR16 molecules was ~90% of that of the N-WASP molecules (Fig. 7D). In contrast, the number of CR16 molecules was ~30% that of the N-WASP molecules in the whole testis (Fig. 7D).

Quantitative PCR analysis using the GAPDH gene as a control revealed that the expression of N-WASP mRNA was not decreased (Fig. 7E). The level of N-WASP mRNA in the CR16–/– testes was 1.04 times that in the CR16+/– testes. Thus, it is suggested that CR16 maintain the N-WASP levels in the Sertoli cells.

Serum testosterone and follicle-stimulating hormone (FSH) concentrations of the CR16–/– mice

We measured the serum concentrations of testosterone and FSH to obviate the epigenetic effects of CR16-knockout. As shown in Fig. 8, there were no obvious differences in the hormone concentrations of the CR16+/– and the CR16–/– sera.


Figure 8
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  		Figure 8  Testosterone and follicle-stimulating hormone (FSH) in the sera of CR16+/– and CR16–/– mice. (A) The serum testosterone concentration was analyzed by electrochemiluminescence immunoassay. (B) Serum FSH concentration was analyzed by radioimmunoassay. The concentration is plotted over day instars.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this study, we generated CR16-knockout mice. We observed that the loss of this gene caused defects in spermatogenesis. More importantly, CR16 and N-WASP form a complex in the Sertoli cells. The molar ratio of CR16 : N-WASP was ~0.9, thereby indicating the presence of the N-WASP–CR16 protein complex in the Sertoli cells.

Although CR16 was expressed in both the brain and the testes, the defect was observed only during spermatogenesis. There are three WIP family proteins, including WIP, WICH/WIRE and CR16. Since WIP and WICH are expressed in the brain, it is possible that they compensate for the absence of CR16 in the brain.

Although N-WASP and CR16 interact in the testes and Sertoli cells, their exact mechanisms of actions at SspJs, particularly that of CR16, are not clear. One possibility is that CR16 is important for maintaining the level of N-WASP, which plays an important role in SspJ formation. In this case, the decreased level of N-WASP resulted in a defect in SspJ formation. The equivalent amounts of CR16 and N-WASP in the Sertoli cells is suggestive of this possibility. The second possibility is that CR16 has functions that are independent of N-WASP. Although the function of CR16 alone is not yet clear, it is possible that free CR16 plays a role in SspJ formation.

N-WASP stability is regulated by proteasome-mediated degradation, and the treatment of cells with a proteasome inhibitor (MG-132) increases the level of N-WASP (Suetsugu et al. 2002; Park et al. 2005). Thus, it is possible that the association between CR16 and N-WASP is involved in the proteasome-mediated degradation of N-WASP.

SspJ is an adherens junction (AJ). The AJs (Takeichi 1991; Gumbiner 1996; Takai & Nakanishi 2003) between the Sertoli cells and spermatids (SspJs) are not typical. Among the SspJs, the existence of the cadherin–catenin system has remained unclear (Grove & Vogl 1989; Vogl et al. 2000; Kierszenbaum & Tres 2004). Rather, the hetero-trans-interaction between nectin-2 and nectin-3, immunoglobulin domain-adhesive proteins, mediates the adhesion at SspJs (Ozaki-Kuroda et al. 2002). Nectin-2-deficient mice exhibit male-specific sterility and have defects in the later stages of sperm maturation (Bouchard et al. 2000; Ozaki-Kuroda et al. 2002). Actin bundles are formed at the nectin interaction sites in SspJs, and the defects in the actin bundle formation are observed in nectin-2-deficient mice (Ozaki-Kuroda et al. 2002). However, the regulation of the actin cytoskeleton downstream of nectins is poorly understood. When nectins interact with each other, Cdc42 and Rac are activated (Kawakatsu et al. 2002). Therefore, it is possible that Cdc42 in turn activates N-WASP or the N-WASP-CR16 complex for actin filament organization at SspJs. However, the mechanisms of CR16 and/or N-WASP function at SspJs remains to be elucidated.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Targeted disruption of the CR16 gene

A mouse cDNA library was screened for the CR16 cDNA with human CR16 as a probe. A mouse 129 Sv/J genomic library was screened for CR16 with mouse CR16 as a probe, and several fragments containing exons that included the ATG were isolated. In our targeting vector (Fig. 2), a 10-kb pair DNA fragment encompassing the start codon of the CR16-coding sequence was deleted and replaced with NLS-LacZ and a neomycin-resistance minigene. The linearized targeting vector was electroporated into 129/Ola embryonic stem (ES) cells. Two independent heterozygous ES cell clones were used to generate chimeric mice by a blastocyst injection, and the mutant animals were bred on a mixed 129/Ola x C57BL/6J background. Genotyping was performed by Southern blotting and polymerase chain reaction (PCR) assays (Fig. 1). For Southern blot analysis, the genomic DNA was digested with EcoRI and hybridized with 5' and 3' probes. The primers for PCR analysis were 5'-CCCTTTTCTCTTTTGCAGACC-3', 5'-GTTTTCCCAGTCACGACGTT-3' and 5'-CAGCAAACTCCTCCTTTTCAAG-3'.

Observation of sperms and in vitro fertilization assay

Ovulation was induced with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG); subsequently, the eggs were collected from the ampulla of the oviduct. Sperms were collected from the caudal portion of the epididymis and then cultured in modified Krebs–Ringer (TYH) medium for 90 min for capacitation. After a 24-h insemination with the same number of sperms, the number of fertilized eggs at the two-cell stage was counted. For sperm examination, cauda epididymal sperm were obtained from 6- to 12-week-old mice and capacitated for 1 h in HS medium (135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 30 mM Hepes, 10 mM glucose, 10 mM lactic acid, 1 mM pyruvic acid, 5 mg/mL BSA, 15 mM NaCO3, pH 7.4) at 37 °C under CO2 (Fukami et al. 2003).

Antibodies

Anti-N-WASP and anti-CR16 antibodies were generated by immunizing rabbits with the verprolin-cofilin-acidic (VCA) region of N-WASP and amino acids 111–218 of CR16 as antigens, respectively. Anti-phosphotyrosine (PY100) antibody was obtained from Cell Signaling Technology.

Quantitative Western blotting
Lysates and sequentially diluted purified proteins or antigens (0.1 ng to 1 µg) of N-WASP, CR16 and WICH were subjected to Western blotting. Subsequently, densitometric analysis determined the levels of protein in the lysates.

Quantitative PCR analysis
Total RNA was extracted from testes using RNA Spin Mini Kit (GE healthcare). PCR was performed with One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) (TAKARA) and ABI7900HT (Applied Biosystems).

Histochemistry and immunohistochemistry

For hematoxylin–eosin (HE) and periodic acid–Schiff (PAS) staining, the testis sections were fixed in phosphate-buffered saline (PBS) containing 4% formaldehyde and embedded in paraffin. Spermatogenesis is locally synchronized; therefore, each cross-section of the seminiferous tubules can be classified into one of the 12 developmental stages. Each stage of the tubule includes spermatids at one or two step(s) (e.g., a stage-VI tubule contains step-6 and step-15 spermatids) (Russell et al. 1990). For the ß-galactosidase assay, testes were frozen in optimal cutting temperature (OCT) compound with liquid nitrogen. The cryosections were then examined for LacZ activity and counterstained with eosin Y. For immunofluorescence, the testes were frozen in OCT compound with liquid nitrogen. The cryosections (10-µm thick) were fixed with 3.7% formaldehyde in PBS for 5 min, permeabilized with 0.2% sodium dodecyl sulfate (SDS) in PBS/1% bovine serum albumin (BSA) for 5 min and further blocked with PBS/1% BSA for 1 h. Anti-CR16 rabbit polyclonal antibody, anti-N-WASP rabbit polyclonal antibody and/or anti-Nectin-2{alpha} rat monoclonal antibody (Ozaki-Kuroda et al. 2002) were used as primary antibodies. Alexa Fluor 48 anti-rabbit IgG and Alexa Fluor 563 anti-rat IgG (Molecular Probes) were used as secondary antibodies. Sections were counterstained with Alexa Fluor 633 phalloidin (Molecular Probes). DNA was stained with TO-PRO3 (Molecular Probes). The stained sections were examined and photographed using a confocal microscope (Radiance 2000, Bio-Rad Japan, Tokyo, Japan).

Transmission electron microscopy

Testes from the adult mice (8–12 postnatal weeks) were fixed with 2% glutaraldehyde in 0.1 M cacodylate followed by 2% osmium tetroxide in water for 3 h on ice. Ultrathin sections (70–80 nm) placed on copper grids were treated with 2% uranyl acetate and examined with a JEOL 2000EX electron microscope (JEOL).

Immunoprecipitation
The testes were homogenized in a lysis buffer containing 20 mM Tris–HCl (pH 7.5), 5 mM ethylene diamine tetraacetic acid (EDTA), 150 mM NaCl, 5 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/mL aprotinin, 10 µg/mL leupeptin, 2% Triton X-100 and 10% glycerol. For immunoprecipitation analysis, the lysates were centrifuged at 20 000 x g for 15 min at 4 °C. Subsequently, they were incubated with primary antibodies followed by protein G-magnetic beads (NEB). After washing, the precipitates were examined by Western blotting.

Preparation of Sertoli cells

The whole testes were treated with collagenase and hyaluronidase for 30 min at 32 °C. The cells were then treated with trypsin for 30 min, washed with DMEM supplemented with 10% fetal calf serum (FCS) and cultured in DMEM at 32 °C in 5% CO2. After culturing for 2 days, the cells were harvested for Western blot analysis. Sertoli cells occupied ~50% of total cells by nuclear morphology of DAPI staining.

Serum testosterone and FSH measurements
For testosterone, an electrochemiluminescence immunoassay was applied (ECLusys testosterone; Roche Diagnostics) and radioimmunoassay was used for FSH (GE healthcare, Rat, Biotrak Assay, RPA550).


   Acknowledgements
 
We thank Dr Kamiya, Dr Eto and Dr Nakauchi at the Laboratory of Stem Cell Therapy, Center for Experimental Medicine, The Institute of Medical Science, University of Tokyo, Tokyo, Japan for their helpful discussions and support for the quantitative PCR analysis. This work was also supported by grants-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan and from the Japan Science and Technology Corporation (JST) (T.T. and S.S.).


   Footnotes
 
{dagger}These three authors contributed equally to this work. Back

Communicated by: Eisuke Nishida

* Correspondence: E-mail: takenawa{at}ims.u-tokyo.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Accepted: 20 February 2007





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