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Genes to Cells (2008) 13, 623-633. doi:10.1111/j.1365-2443.2008.01193.x
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Spermatogenesis-specific association of SMCY and MSH5

 Chihiro Akimoto1, Hirochika Kitagawa1, Takahiro Matsumoto1,2 and Shigeaki Kato1,2,*

1 The Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
2 ERATO, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The status of chromatin during spermatogenesis is dynamically regulated by specific histone codes or stage-specific histone changes. The functional links between such epigenetic regulation and proteins regulating meiosis are largely unknown. In mammals, genes encoded on the Y chromosome are thought to possess male-specific biological functions. While genes located within the azoospermia factor region (AZF) are known to be involved in spermatogenesis, the physiological function of individual genes is not known. SMCY is a gene mapped to the AZF, and in this report, we analyzed the function of SMCY protein during spermatogenesis. Biochemical identification of the proteins with which it interacted showed that SMCY formed a distinct complex with MSH5, a critical meiosis-regulatory protein in the human testicular germ cell line, NEC8. As anticipated, histone H3K4 demethylase activity was detected. Immunohistochemical analysis revealed the co-localization of SMCY with MSH5 at a specific stage of meiotic prophase progression during murine spermatogenesis. Our results suggest that SMCY may have a male-specific function as a histone H3K4 demethylase by recruiting a meiosis-regulatory protein to condensed DNA.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
In the male reproductive system, differentiation from the male genital primordium into testis cord is accompanied by transiently expressed intra-cellular signaling cascades and differentiation factors. Among them, Sry, encoded on the Y chromosome, is the key sex-determining factor. Following sex determination by transient expression of SRY and the downstream cascade of the transcription factors, such as SOX9 and SF1 (Jimenez & Burgos 1998; Komatsu et al. 2004; Zubair et al. 2006), testicular development is subsequently regulated by exposure to androgen, a sex steroid hormone stimulated by humoral factors such as LH and FSH. The phenotypes of mice lacking sex steroid hormone receptors suggest that both androgen and estrogen are required for testicular development and germ cell development (Eddy et al. 1996; Hess et al. 1997; Sato et al. 2003). In addition, a region on the long arm of the human Y chromosome, named azoospermia factor a–d (AZF a–d), appears to be critical for normal regulation of spermatogenesis (Vogt 2004, 2005). Unlike the human, the short arm of the Y chromosome, designated Spy, is required for murine spermatogenesis (Mazeyrat et al. 1998). Although Eif2s3y encoded in the Spy region has been identified as a key mouse-specific regulator of spermatogonia proliferation and differentiation (Mazeyrat et al. 2001), the function of the other genes, including Smcy, conserved from mouse Spy to human AZF, have not been well documented.

Male-specific meiosis in mammalian spermatogenic germ cells is regulated by unique factors, including meiosis-specific recombination factors and testis-specific transcription factors which are distinct from somatic mitosis. After differentiating from spermatogonia, spermatocytes remain in a long prophase accompanied by morphologic alteration of nuclei and chromosomes before the first division. This prophase is divided into five stages: preleptotene, leptotene, zygotene, pachytene and diplotene. Following DNA condensation, chromatin fibers are formed during the leptotene and zygotene stages, followed by homologous chromosome pairing and recombination mediated by DNA repair-related factors, such as MSH5, DMC1, and MLH1, which are expressed specifically during meiosis (Edelmann et al. 1996; Pittman et al. 1998; de Vries et al. 1999; Edelmann et al. 1999). Chromosomes condense and pair together, forming the synaptonemal complex (SC) in zygotene cells and mature in pachytene cells. During this stage, X and Y chromosomes are inactivated (male sex chromosome inactivation) and form the XY body (Handel 2004).

Spermatogenesis is also regulated through male-specific chromatin reorganization via histone modifiers and chromatin remodeling factors. Accompanying the replacement of testis-specific histone variants and protamines from somatic histones, dynamic regulation of chromatin status and meiosis-specific histone modification has been reported (Kimmins & Sassone-Corsi 2005). For example, methylation of the N-terminal tail of histone H3 is now considered a marker for active transcription (H3K4) or heterochromatinization (H3K9) (Hayashi et al. 2005; Ruthenburg et al. 2007; Tachibana et al. 2007). However, the links between the regulation of chromatin structure and the functions of meiosis-regulating factors at each stage during spermatogenesis are largely unknown.

In this report, we characterized the function of Smcy, a gene located within the region responsible for spermatogenesis both in the human and the mouse (AZF and Spy, respectively). Consistent with a previous report, histone H3K4 demethylase activity was detected in SMCY in a human testicular germ cell line. Biochemical approaches showed that SMCY formed a distinct complex with MSH5 which seemed to function together at a specific developmental stage before the initiation of meiosis. This observation suggests a male-specific function of a gene on the Y chromosome is achieved during spermatogenesis by forming a distinct protein complex containing a meiosis-regulatory protein in a spatio-temporal manner.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
SMCY is a member of the JARID subfamily within the JmjC proteins. The histone demethylase activity of SMCY in HEK293 cells and in H1299 cells is similar to other JARID proteins (Lee et al. 2007a), but the biological functions of this protein in specific tissues are still unknown. While Smcy is included in the Spy region, its specific biological functions during spermatogenesis remain to be studied. To address the biological impact of SMCY during spermatogenesis, we first determined whether human SMCY has histone demethylase activity in testicular germ cells. Using an in vitro demethylation assay with calf thymus histones as a substrate, immunoprecipitated SMCY from NEC8 cells (stably expressing SMCY) was found to possess a demethylase activity specific for di- and tri-methylated histone H3K4 (Fig. 1A). This result was further confirmed by an in vivo demethylation status assay using a chromatin fraction of NEC8 cells with or without over-expressed SMCY (Fig. 1B). Tri-methylated H3K9 level was not affected as in either assay (Fig. 1A,B). The demethylase activity of SMCY was not detected when SMCY was knocked down by siRNA (Fig. 1A,B). To further confirm the correlation between the expression of SMCY and the observed methylated status of histones, we analyzed the level of histone methylation in each cell using confocal fluorescence microscopy. As demonstrated by in vivo histone demethylation assays, di- and tri-methylated H3K4 staining was reduced in cells over-expressing SMCY (Fig. 1C upper panels). In contrast, the intensities of mono-methylated H3K4 and tri-methylated H3K9 were almost unchanged (Fig. 1C lower panels). From these results, we conclude that SMCY possesses active enzymatic activity in vitro and in vivo and that it serves as a histone demethylase which is specific for H3K4 in testicular germ cells.


Figure 1
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  		Figure 1  SMCY functions as a histone H3K4 demethylase in the human testicular germ cell line NEC8. (A) H3K4-specific demethylase activity of SMCY in vitro. An SMCY immunoprecipitate from NEC8 cells stably expressing SMCY with or without transfection of SMCY siRNA was used for the in vitro demethylation assay. Histones were subjected to immunoblot by methylation-specific H3 antibodies after incubation with an immunoprecipitate of SMCY in demethylation buffer. (B) In vivo H3K4-specific demethylase activity of SMCY in NEC8 cells. NEC8 cells with or without transfection of human SMCY expression vector and SMCY siRNA were used for an in vivo demethylation assay. Chromatin fractions were prepared from cell lysates and were subjected to Western blot analysis using antibodies against histones (with residue-specific methylation) and FLAG for detection of SMCY. (C) Over-expression of SMCY resulted in reduction of H3K4me2/3 signals in NEC8 cells. An expression plasmid carrying GFP-SMCY was transiently transfected into NEC8 cells and stained by anti-GFP (green) and appropriate antibodies against methylated histones (H3K4me1, H3K4me2, H3K4me3 or H3K9me3; red). Nuclei were counterstained with DAPI.

 
Recently, many histone demethylases were characterized as components of distinct protein complexes possessing specific biological functions (Lee et al. 2007b). To decipher the functional role of SMCY in spermatogenesis, we examined functional interactants of SMCY using nuclear extracts prepared from NEC8 cells and an affinity-purification method (Fig. 2A). Recombinant FLAG-tagged SMCY (expressed by a baculoviral system) was used as functional bait (Lee et al. 2007a) (Fig. 2B) and followed a standard anti-FLAG affinity-purification procedure as previously conducted in this laboratory (Yanagisawa et al. 2002; Kitagawa et al. 2003; Fukuda et al. 2007). After size fractionation on a glycerol density gradient (Tagami et al. 2004) (Fig. 2C), we analyzed the protein bands contained in the fractions forming multi-protein complexes with SMCY (pools 2 and 3 in Fig. 2D) by peptide mass fingerprinting. Among the identified proteins, we focused on MSH5 in pools 2 and 3 (Fig. 2D: indicated as arrow). MSH5 is a member of the MutS homologue family of proteins and is essential for meiosis (Borner et al. 2004). To verify the association between SMCY and MSH5 in vivo, we performed a co-immunoprecipitation assay with NEC8 cells stably expressing FLAG-tagged-SMCY (Fig. 2E). We thus identified MSH5, a meiosis-regulatory protein, as a candidate functional partner of SMCY in a testicular germ cell-specific multi-protein complex. However, deficiency of MSH5 by the knockdown did not affect the histone demethylase activity of SMCY in vitro or in vivo (data not shown).


Figure 2
Figure 2
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  		Figure 2  Identification of MSH5 in the SMCY complex. (A) Schematic representation of purification protocols. Nuclear SMCY interactants were affinity-purified using recombinant FLAG-SMCY protein conjugated with anti-FLAG beads using NEC8 cells. (B) Recombinant FLAG-tagged SMCY protein was used as a bait for purification. Recombinant full-length SMCY isolated from Sf9 insect cells was analyzed by Coomassie brilliant blue staining. A cell lysate of Sf9 cells lacking the expression vector was used as a mock control. (C) Silver staining of SMCY complex-forming fractions separated on a glycerol gradient. FLAG peptide eluates were separated on a 10%–40% glycerol density gradient by ultracentrifugation. Fractions were collected from the top of the gradient, and an aliquot of each fraction (Fr), as well as the input was analyzed by SDS-PAGE. Arrowhead indicates the bait. (D) Silver staining of the pooled fractions. The fractions collected from the glycerol gradient were pooled as indicated on the top of the panels in Fig. 2C. Each pool was TCA precipitated. Arrow (approximately 90 kDa band in pools 2 plus 3) indicates MSH5. (E) Interaction of SMCY and MSH5 was detected by immunoprecipitation analysis in NEC8 cells. A cell lysate of NEC8 cells stably expressing FLAG-SMCY was immunoprecipitated by an anti-FLAG antibody and bound proteins were subjected to Western blot analysis with antibody indicated on the left side of the panel.

 
Next we tried to explore the functional relevance between SMCY and MSH5 during spermatogenesis. MSH5 is known to be expressed in a germ cell-specific manner and MSH5 null mice showed defects in spermatogenesis due to meiotic arrest around zygotene stage (de Vries et al. 1999; Edelmann et al. 1999). However, detailed expression analysis of MSH5 protein during spermatogenesis has not been performed. Given the formation of a complex between SMCY and MSH5, we speculated that those proteins probably co-operated at a specific stage of spermatogenesis. Thus, we examined the expression profile of murine SMCY and MSH5 during testis development as well as the methylation status of histone H3 by immunohistochemical analysis (Fig. 3A). To determine the developmental stage of each cell, we used SCP3 as a marker for the specific stage of meiotic prophase spermatocytes and CDH1 for a marker of undifferentiated spermatogonia (Yuan et al. 2000; Tokuda et al. 2007). Spermatogenesis-specific histone codes are regulated by various histone modifiers such as Meisetz (identified as a meiosis-specific histone H3K4 methyltransferase) and G9a (an H3K9 mono- and di-methyltransferase) (Hayashi et al. 2005; Tachibana et al. 2007). As expected, the intensities of mono-, di- and tri-methylated H3K4 histones were greatly altered during all stages of murine testis development. The expression of SMCY was detectable from early premeiotic spermatocyte to spermatid, but increased from the leptotene stage of the spermatocyte with the highest expression levels at the pachytene stage as indicated from p15 and p28 sections (Fig. 3B,C). Strong staining of MSH5, particularly in the outer layer cells, was seen in section p28, reflecting the high expression of MSH5 at early preleptotene to zygotene stages and the lower expression in the pachytene stage in spermatocytes (Fig. 3B,C). From this observation, we tentatively concluded that SMCY associates with MSH5 in meiotic prophase spermatocytes (from the preleptotene to pachytene stage).


Figure 3
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  		Figure 3  Expression profile of SMCY and MSH5 during murine spermatogenesis. (A) Both SMCY and MSH5 were detected along with the appearance of spermatocytes. Immunohistochemical analysis of serial section of mouse testes collected on each postnatal day (PND) indicated on the top of the panel. Purple staining indicates positive reactivity of each serial section. CDH1 or SCP3 were used to identify undifferentiated spermatogonia or spermatocytes. (B) Higher magnification figures of immunostainings for SMCY and MSH5 from p9, p15, p28 mouse testes were shown. L: leptotene, Z: zygotene, P: pachytene, ST: spermatid. (C) A scheme for stage-dependent expression of SMCY and MSH5 in murine spermatogenesis. Green arrows represent each PNDs. The corresponding developmental stages are shown below. Expression levels of SMCY and MSH5 at each spermatogenic stage were schematically represented by the numbers of plus marks.

 
Cell suspensions from adult mouse testes comprise cells at every stage of sperm differentiation. Therefore, we tested the possibility of co-localization of SMCY and MSH5 by double stained immunofluorescence analysis with surface-spread testicular cells (Fig. 4A). In the early leptotene stage, we detected cells expressing MSH5 alone, but MSH5 could not be found on condensed DNA (left panel in Fig. 4A). Cells co-expressing SMCY and MSH5 were detected only from the leptotene stage to the zygotene stage and co-localization of SMCY and MSH5 was detected on condensed DNA in those cells (middle panel in Fig. 4A). After the pachytene stage, MSH5 was barely detectable, whereas SMCY was localized on condensed DNA (right panel in Fig. 4A). Next, we examined the correlation of H3K4 methylation status and expression of SMCY and MSH5 from leptotene to zygotene stages in each cell (Fig. 4B). In the cells where both SMCY and MSH5 were expressed, di- and tri-methylated H3K4 appeared demethylated compared to the intensity of mono-methylated H3K4 (Fig. 4B, indicated by arrowhead). Together, these results suggest that SMCY forms a distinct complex with MSH5 during spermatogenesis followed by the initiation of meiosis.


Figure 4
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  		Figure 4  Functional correlation between SMCY and MSH5 in meiotic prophase spermatocyte of murine spermatogenesis. (A) SMCY and MSH5 were partially co-localized on condensed DNA in leptotene/zygotene spermatocytes. Confocal laser scanning analysis localizing SMCY and MSH5 on staged spermatocytes (leptotene, leptotene/zygotene and pachytene from left to right). SCP3 stainings of each stage were shown in lower panels. (B) The level of H3K4 demethylation is inversely-correlated with the expression of both SMCY and MSH5 in leptotene/zygotene spermatocytes. Immunohistochemical analysis of p28 mouse testis. Layered structure of developing spermatogenic cells in the section is indicated in the right panel. Arrowheads indicate the cells expressing both SMCY and MSH5. L: leptotene, Z: zygotene, P: pachytene, ST: spermatid. (C) Schematic model for tissue-specific complex formation of SMCY. In testicular germ cells, SMCY forms a distinct protein complex containing MSH5 and likely promotes the initiation of meiosis. SMCY reportedly forms another complex containing Ring6a in kidney cells (Lee et al. 2007a) and functions as a transcriptional co-repressor.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Many histone modifier complexes have been identified by biochemical purification. Each possesses a tissue-specific biological function determined by its unique combination of components. In the case of SMCY, the complex works as a transcriptional co-repressor in HEK293 kidney cells (Lee et al. 2007a). Contrary to that report, we identified another functional complex containing MSH5 in testicular germ cells. Given its stage-specific co-localization on condensed DNA, we speculated that the function was related to the initiation of meiosis via chromatin condensation, and not transcription. These results suggest that SMCY forms a specific complex in a temporally specific manner and that the associated proteins might define the biological function of the SMCY complexes. Since the genes encoded by the Y chromosome are male-specific, the biological significance of SMCY may arise as a result of formation of a male-specific complex during spermatogenesis (Fig. 4C).

To characterize the role of the components of the SMCY complex, we tested the SMCY activity in the absence of MSH5. Contrary to Ring6a as previously reported (Lee et al. 2007a), a knockdown of MSH5 did not affect the demethylase activity of SMCY complex (data not shown). Together with our observation that SMCY co-localized on condensed DNA with MSH5 at leptotene/zygotene stage alone (Fig. 4A middle panel), we presume that MSH5 serves as a DNA recognition factor to accommodate the SMCY complex on a chromosomal locus. On the other hand, MSH5 did not localize on condensed DNA in the absence of SMCY (Fig. 4A left panel), suggesting that complex formation of MSH5 with SMCY might be indispensable for the recruitment of MSH5 to condensed DNA. To test this idea, further analysis of the unidentified components of this SMCY/MSH5 containing complex is clearly needed.

During spermatogenesis, dynamic chromatin remodeling includes replacement of histones and alteration of histone modifications. Our observations revealed that SMCY demethylates di- and tri-methylated H3K4, known as a transcriptionally active chromatin marker (Hayashi & Matsui 2006; Ruthenburg et al. 2007), suggesting that SMCY may be involved partly in the inactivation (condensation) of chromosomes before entering meiosis in normal spermatogenesis. This hypothesis is also supported by the co-localization of SMCY on condensed DNA in leptotene to pachytene spermatocytes (Fig. 4A). The level of global H3K4 methylation is a critical stage determinant and is supposed to be strictly regulated by specific histone modifiers. For example, gene-specific null mice of Meisetz could not maintain normal spermatogenesis from the zygotene to the pachytene stage (Hayashi et al. 2005). Considering the expression profile of SMCY during the meiotic prophase, the demethylase activity of SMCY might also regulate a crucial step of spermatogenesis or uniquely regulate the level of chromatin condensation in a fashion different from the generally accepted model of transcriptional regulation attributed to Meisetz or G9a (Hayashi et al. 2005; Tachibana et al. 2007). Though the controls of epigenetic modifications including histone methylation during spermatogenesis appear complicated, we could reveal a novel stage-specific role of a histone demethylase, SMCY, by finding a functional partner at a specific stage using a biochemical approach. As SMCY does express in the meiotic prophase spermatocyte or later, SMCY might possess another role via forming other stage-specific complexes.

Tissue-specific regulation of gene expression encoded on the Y chromosome is almost unknown. Murine Eif2s3y transgene could not rescue late spermatocyte progression found in Spy-deleted mice. Thus, some of the other genes located within the Spy region are responsible for the completion of spermatogenesis after the second meiotic division (Mazeyrat et al. 2001). Considering that the expression of SMCY could be observed from leptotene spermatocytes to spermatid, Smcy might be another essential Y-chromosomal gene for progression of spermatogenesis. Thus, generating gene-specific null mice for Smcy will provide definitive evidence for the significance of genes located on the Y chromosome in male-specific biological functions, including those of spermatogenesis.


   Experimental procedures
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Plasmids and antibodies

A mammalian expression vector encoding FLAG-tagged Smcy and GFP-tagged Smcy was cloned from a human testis cDNA library (BD Bioscience, San Jose, CA) and inserted into a pcDNA3 vector (Invitrogen, Carlsbad, CA). A baculoviral-expression vector encoding FLAG-tagged Smcy was constructed using pFastBac vector (Invitrogen).

siRNA pool for SMCY (1299003) was purchased from Invitrogen. Nonspecific control siRNA (D-001216-13-20) was purchased from Dharmacon (Lafayette, CO).

Anti-monomethyl H3K4 (07-436), anti-dimethyl H3K4 (07-030) and anti-trimethyl H3K4 (07-473) were purchased from Upstate/Millipore (Billerica, MA). Anti-trimethyl H3K9 (ab8898-100), anti-H3 (ab1791), anti-SMCY/JARID1D (ab35492) and anti-SCP3 (ab15092) were purchased from Abcam (Cambridge, UK). Anti-CDH1 (610 181) was purchased from BD Bioscience. Anti-MSH5 (H4439-M08) was purchased from Abnova (Taipei City, Taiwan). Anti-FLAG antibodies (F-7425, F-3165) and anti-FLAG M2 agarose (A2220) were obtained from Sigma (St Louis, MO). Anti-GFP was purchased from Roche Applied Science (Basel, Switzerland).

In vitro demethylation assay

Cell lysates of NEC8 cells and NEC8 cells stably expressing FLAG-SMCY with or without transfection of SMCY siRNA were immunoprecipitated by anti-FLAG M2 resin, washed, and eluted with FLAG peptide. Eluates were incubated with 5 µg of calf thymus histones (Sigma) in the demethylation buffer (20 mM Tris–HCl [pH 7.5], 150 mM NaCl, 50 µM Fe(NH4)2(SO4)26H2O, 1 mM {alpha}-ketoglutarate, and 2 mM ascorbic acid) for 4 h at 37 °C (Iwase et al. 2007). Reaction mixtures were analyzed by Western blotting using specific antibodies.

In vivo demethylation assay

NEC8 cells transfected with or without FLAG-SMCY expression vector and SMCY siRNA were lysed, homogenized and centrifuged for 30 min at 4 °C at 30 000 g. Pellets were resuspended in lysis buffer (10 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40), sonicated, and were used as the chromatin fraction for Western blotting. The protein concentration of each samples was measured and standardized. Samples were boiled with sample buffer and loaded on SDS-PAGE gels.

Immunofluorescence microscopy

NEC8 cells were plated in slide-chambers and the GFP-SMCY expression plasmid was transfected with Fugene 6 (Roche) according to the manufacturer's instructions. After 48 h, cells were fixed with 4% paraformaldehyde, washed with PBS, and permeabilized for 20 min in 1% Triton X-100 in PBS. Cells were subsequently washed with PBS and blocked with 5% BSA in PBS for 1 h. Cells were incubated with primary antibody in a humidified chamber overnight at 4 °C using histone-modification antibodies and anti-GFP mouse monoclonal antibody at a dilution of 1 : 100. Chromosome spreads were prepared as previously described (Peters et al. 1997) with some modifications. Slides were washed with 1% Tween 20 in PBS (PBST) and incubated with primary antibody overnight at 4 °C. After primary antibody incubation, cells were washed 3 times with PBST and incubated with FITC- or Cy3-conjugated secondary antibodies and DAPI. To obtain higher sensitivity for each antibody (SMCY or MSH5), incubation with biotinylated second antibody (Dako, Glostrup, Denmark) was performed after primary antibody incubation. Avidin-FITC or avidin-rhodamine (Roche) was incubated with fluorescence-conjugated second antibodies. Cells were washed 4 times with PBS and placed in mounting medium for fluorescence analysis (Vector Labs., Inc, Burlingame, CA). Slides were analyzed on a Zeiss confocal laser scanning system 510 and captured images were processed with Adobe Photoshop 7.0 (Ohtake et al. 2007).

Purification of recombinant protein

Full-length human SMCY protein was produced using the Bac-to-Bac system (Invitrogen) (Kitagawa et al. 2002). Sf9 cells infected with the virus encoding SMCY were harvested and resuspended in lysis buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 0.1% Nonidet P-40, 10% glycerol) with protease inhibitors. Cells were lysed with rotation at 4 °C and the cell debris was removed by centrifugation at 15 000 g for 30 min at 4 °C. The supernatants were incubated with anti-FLAG affinity resin (Sigma) for 3 h at 4 °C, washed 4 times with wash buffer (20 mM Tris–HCl [pH 7.5], 10% glycerol) containing 750 mM KCl, and washed 2 times with wash buffer containing 150 mM KCl before contact with the nuclear extract.

Affinity purification of SMCY-containing complexes

Affinity purification was performed as previously described (Yanagisawa et al. 2002; Kitagawa et al. 2003; Fukuda et al. 2007) with some modifications. Nuclear extracts from NEC8 cells were incubated with recombinant FLAG-tagged SMCY conjugated to anti-FLAG M2 affinity resin (Sigma), and rinsed twice on the column with washing buffer (20 mM Tris–HCl [pH 7.5], 0.2 mM EDTA, 0.05% Nonidet P-40, 10% glycerol, 0.5 mM PMSF and protease inhibitor cocktails) containing 500 mM KCl and with washing buffer containing 150 mM KCl. Bound proteins were eluted from the column by incubation with 400 µg/mL FLAG peptide in washing buffer for 30 min at room temperature. Fractionation on glycerol gradients was performed as previously described (Fukuda et al. 2007). Each fraction was applied to a Multi GEL II Mini 2%–15% gradient gel (Daiichi Pure Chemicals Co., Ltd, Tokyo, Japan). Mass spectrometry was done as we previously described (Yanagisawa et al. 2002; Kitagawa et al. 2003; Takezawa et al. 2007).

Immunoprecipitation and Western blotting

Immunoprecipitation was performed as previously described (Kitagawa et al. 2003; Fujiki et al. 2005). NEC8 cells stably expressing SMCY were produced by geneticin selection. Cells were washed and resuspended in ice-cold TNE buffer (10 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40), and rotated at 4 °C for 30 min. Cells were homogenized and supernatant was collected after centrifugation. Supernatants were immunoprecipitated with anti-FLAG affinity resin and then Western blotted with anti-FLAG, anti-SMCY and anti-MSH5 antibody.

Immunohistochemical analysis

All tissues were obtained from C57BL/6J mice (Clea Japan Inc., Tokyo, Japan). Testes were collected from each developmental stage and fixed with 4% PFA and then embedded in paraffin. Immunostaining was performed as previously described (Miyamoto et al. 2007; Nakamura et al. 2007). Serial sections of testes (4–5 µm thick) were deparaffinized, rehydrated and washed in PBS. Sections were incubated in sodium citrate buffer [pH 6.0], boiled for 10 min, followed by cooling to room temperature to retrieve antigen. The sections were incubated in 3% H2O2 for 10 min to quench endogenous peroxidase and washed with 1% Tween 20 in PBS for 5 min. To block nonspecific antibody binding, sections were incubated in 5% goat serum in PBS for 1 h. Sections were then incubated with primary antibodies in 2% goat serum/PBS overnight at 4 °C. Staining was then performed using the EnVision+ HRP System (Dako) and 3, 3'-diaminobendizine tetrahydrochloride substrate (Sigma), dehydrated through an ethanol series and xylene, before mounting.


   Acknowledgements
 
We thank Dr Y. Kanai and S. Matoba for helpful discussion. We also thank all the members of our laboratory for their help and discussion. We thank K. Motoi and M. Yamaki for manuscript handling. This work was supported in part by priority areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H.K., S.K.). This work was also supported by JSPS Research Fellowships for Young Scientists (to C.A.).


   Footnotes
 
Communicated by: Kohei Miyazono

* Correspondence: Email: uskato{at}mail.ecc.u-tokyo.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Edelmann, W., Cohen, P.E., Kneitz, B., Winand, N., Lia, M., Heyer, J., Kolodner, R., Pollard, J.W. & Kucherlapati, R. (1999) Mammalian MutS homologue 5 is required for chromosome pairing in meiosis. Nat. Genet. 21, 123–127.[CrossRef][Medline]

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Fukuda, T., Yamagata, K., Fujiyama, S., et al. (2007) DEAD-box RNA helicase subunits of the Drosha complex are required for processing of rRNA and a subset of microRNAs. Nat. Cell Biol. 9, 604–611.[CrossRef][Medline]

Handel, M.A. (2004) The XY body: a specialized meiotic chromatin domain. Exp. Cell Res. 296, 57–63.[CrossRef][Medline]

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Received: 17 December 2007
Accepted: 17 March 2008




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