Research Article 3137 SPOC1 (PHF13) is required for spermatogonial stem cell differentiation and sustained

Annegret Bo¨ rdlein1,*, Harry Scherthan2,*, Claudia Nelkenbrecher1, Tina Molter1, Michael R. Bo¨sl3, Christine Dippold1, Kerstin Birke4, Sarah Kinkley5, Hannah Staege5, Hans Will5 and Andreas Winterpacht1,` 1Institute of Human Genetics, University Hospital Erlangen, University of Erlangen-Nu¨rnberg, 91054 Erlangen, Germany 2Bundeswehr Institute of Radiobiology affiliated to the University of Ulm, 80937 Munich, Germany 3Max-Planck-Institute for Neurobiology, 82152 Martinsried, Germany 4Institute of Anatomy II, University of Erlangen-Nu¨rnberg, 91054 Erlangen, Germany 5Heinrich-Pette-Institut, Leibniz Institute of Experimental Virology, 20251 Hamburg, Germany *These authors contributed equally to this work `Author for correspondence ([email protected])

Accepted 17 May 2011 Journal Cell Science 124, 3137–3148 2011. Published by The Company of Biologists Ltd doi: 10.1242/jcs.085936

Summary SPOC1 (PHF13) is a recently identified protein that has been shown to dynamically associate with somatic chromatin, to modulate chromatin compaction and to be important for proper cell division. Here, we report on the expression of SPOC1 in promyelocytic leukaemia zinc finger (PLZF)-positive undifferentiated spermatogonial stem cells (SSCs) of the mouse testis. To investigate further the biological function of SPOC1 in germ cells we generated Spoc1 mutant mice from a gene-trap embryonic stem cell clone. Postpubertal homozygous Spoc12/2 animals displayed a pronounced progressive loss of germ cells from an initially normal germ epithelium of the testis tubules leading to testis hypoplasia. This loss first affected non-SSC stages of germ cells and then, at a later time point, the undifferentiated spermatogonia. Remarkably, successive loss of all germ cells (at .20 weeks of age) was preceded by a transient increase in the number of undifferentiated Aaligned (Aal) spermatogonia in younger mice (at .10 weeks of age). The number of primary Spoc12/2 , the proliferation of germ cells, and the initiation and progression of was normal, but we noted a significantly elevated level of apoptosis in the Spoc12/2 testis. Taken together, the data argue that SPOC1 is indispensable for stem cell differentiation in the testis and for sustained spermatogenesis.

Key words: Spermatogonial stem cell, Differentiation, SPOC1 (PHF13), Spermatogenesis Journal Cell Science

Introduction resulting in primary . After completion of first Stem cells are fundamental for the regenerative capacity of organ meiotic prophase, a reductional division (meiosis I) and a systems (Dick, 2008). They are characterized by having the subsequent mitosis-like division (meiosis II) lead to the potential to self-renewal and to differentiate. The mechanisms formation of haploid that develop into spermatozoa regulating the balance between these processes remain poorly (Aponte et al., 2005; de Rooij, 2001; He et al., 2009; Olive and understood. An impressive example for the capacity of self- Cuzin, 2005; Russell et al., 1990). During differentiation, the germ renewal and differentiation of stem cells comes from cells migrate from the basal lamina into the adluminal spermatogenesis, where millions of spermatozoa are produced compartment of the seminiferous epithelium by traversing the each day in the postpubertal male. Thus, spermatogenesis is one blood–testis barrier (BTB) formed by Sertoli cells. This process of the most efficient cell-producing systems in the adult requires extensive interaction between Sertoli cells, as well as mammalian organism (Sharpe et al., 2003), making it an between Sertoli and germ cells. This tight coordination between excellent model system to elucidate the general mechanisms of germ cell movement and differentiation is required in order to stem cell renewal and differentiation. avoid arrest of spermatogenesis and apoptosis during this passage It is suggested that, in non-primate mammals, spermatogonial (reviewed by Mruk and Cheng, 2004). The basal compartment stem cells (SSCs) are single cells (Asingle,As) attached to the provides a niche assuring a specialized microenvironment capable basement membrane of the seminiferous tubules. Depending on of generating the necessary balance between self-renewal and whether the cells undergo self-renewal or differentiation, the As differentiation. This process is regulated by extrinsic niche stimuli spermatogonia either divide into two separate (As) cells or into a secreted by the Sertoli cells, as well as by intrinsic gene expression pair of spermatogonia (Apaired,Apr), respectively. The Apr enters in the SSCs. Although several extrinsic stimuli have been additional rounds of cell divisions after which the spermatogonia described (Chen et al., 2005; Hofmann, 2008; Oatley and form chains and are called Aaligned (Aal) spermatogonia. Apr and Brinster, 2008) little is known about the intrinsic factors that Aal spermatogonia are still considered undifferentiated (Nakagawa regulate SSC self-renewal, proliferation and differentiation. In et al., 2007). The Aal cells then further differentiate into the A1–A4 undifferentiated SSCs (As–Aal), some core transcription factors are spermatogonia, intermediate and B spermatogonia, which undergo known (e.g. OCT4 and SOX2) that are of general importance for a final round of replication and enter the first meiotic prophase, the maintenance of the pluripotency of stem cells (Dann et al., 3138 Journal Cell Science 124 (18)

2008; Oatley and Brinster, 2008). Furthermore, three factors, by lacZ staining and its sequence can easily be determined by 59 promyelocytic leukaemia zinc-finger (PLZF, also known as rapid amplification of cDNA ends (59 RACE). In the ESC clones ZBTB16 and ZFP145) (Buaas et al., 2004; Costoya et al., 2004; Xb691 and Xa022, the 59 RACE product contained sequences Falender et al., 2005; Hobbs et al., 2010), TATA-box-binding- from exon 1 [nucleotides 303 to 354 of the Spoc1 (Phf13) cDNA protein-associated factor 4B (TAF4B) (Falender et al., 2005) and (GenBank accession number NM_172705)] of the Spoc1 gene, NANOS2 an RNA-binding protein (Sada et al., 2009; Saga, 2010) indicating an insertion of the gene-trap vector into intron 1 have also been shown to be important for SSC maintenance. (Fig. 1A). We further mapped the integration sites to between Whereas TAF4B is involved in SSC formation and proliferation, nucleotides +917 and +918 (Xb691) and +876 and +877 (Xa022) PLZF and NANOS2 are directly involved in SSC self-renewal. of intron 1 relative to the first base of intron 1. On the RNA level PLZF functions both as a transcriptional repressor and as an this insertion leads to the termination of Spoc1 gene transcripts activator of genes implicated in self-renewal of SSCs and in after exon 1, which contains the translation start site. differentiation of undifferentiated A-type (As–Aal) spermatogonia Mutant mice were generated from both ESC clones by (Buaas et al., 2004; Dadoune, 2007; Hobbs et al., 2010). Loss-of- injection into blastocysts from C57BL/6 mice. Subsequent function studies for PLZF and NANOS in the mouse result in intercrosses of heterozygous animals generated a mixed genetic animals with a progressive loss of germ cells that culminate in a background. The phenotypes were identical for both mice strains Sertoli-cell-only phenotype in a variable percentage of tubules and therefore the data are presented only for the mouse strain (Buaas et al., 2004; Costoya et al., 2004; Sada et al., 2009). generated from ESC clone Xb691. For PCR genotyping (Fig. 1B) Recently, we identified the gene SPOC1 (for survival-time- of mutant Spoc1, primers were generated to both the b-gal associated PHD protein in ovarian cancer), also designated cassette and to intron 1, as shown in Fig. 1A. To determine the PHF13, and demonstrated that enhanced SPOC1 RNA levels in effect of the gene-trap insertion on splicing of Spoc1, RT-PCR primary and recurrent epithelial ovarian cancers are correlated was performed on RNA isolated from tail cuts of heterozygous with a shorter survival time in a cohort of patients (Mohrmann and homozygous mice using primers corresponding to the b- et al., 2005). The SPOC1 protein localizes to the nucleus and galactosidase sequence of the gene-trap vector, as well as from contains a plant homeodomain (PHD), which is believed to exon 1 of Spoc1. Sequencing of the resulting RT-PCR products regulate chromatin-specific interactions (Bienz, 2006). In revealed splicing of exon 1 to the splice acceptor site of the gene- agreement with this prediction, we have recently demonstrated trap cassette in homozygous (2/2) and heterozygous (+/2) that SPOC1 is dynamically associated with chromatin and that it animals (data not shown). Heterozygous animals expressed b- plays a role in proper chromosome condensation and cell division galactosidase from the gene-trap construct at a low level in most (Kinkley et al., 2009). SPOC1 RNA is detectable in most tissues tissues, demonstrating splicing and functionality of the gene trap but the highest levels are present in the testis where it exclusively (Fig. 1C). localizes to spermatogonia (Mohrmann et al., 2005). These In order to prove the efficient knockdown of endogenous findings suggest a testis-specific functional role of SPOC1 in SPOC1 in vivo, several approaches were undertaken. First, spermatogonial stem cells. Beyond this, there is very little additional data available on SPOC1 expression and function. In northern blot analysis of testis tissue (mice at 18 weeks of age) Journal Cell Science particular, the role of SPOC1 during development and in was performed with a probe corresponding to the Spoc1 39 carcinogenesis remains unclear. Therefore, in order to gain untranslated region (39-UTR). There was no detectable Spoc1 insight into the in vivo functions of the SPOC1 protein we mRNA in homozygous animals (Fig. 1D), whereas heterozygous created and analyzed mice whose Spoc1 gene has been disrupted animals showed an ,50% reduction of Spoc1 expression in by a gene-trap insertion. comparison to wild-type animals. Here, we report that mice deficient for SPOC1 display a Next, we performed quantitative real-time PCR (qRT-PCR) progressive loss of germ cells, which is accompanied by with a probe corresponding to the junction of exon 2 and 3 of apoptosis of pachytene spermatocytes. We provide evidence wild-type Spoc1 cDNA. Accordingly, RNA was extracted from that this is not caused by defects in meiosis, but rather by a the testes of homozygous mice and their wild-type littermates at disturbed differentiation of SSCs, where SPOC1 is normally various ages (Fig. 1E). We observed a low expression of coexpressed together with PLZF. Thus, SPOC1 represents a new persisting wild-type Spoc1 transcript ranging from 2 to 14 % in intrinsic factor indispensable for sustained spermatogenesis and the homozygous mutant mice compared with wild-type tissue in the differentiation of the SSC pool. the different animals. The residual Spoc1 transcripts are probably due to skipping of the gene-trap construct in some transcripts. Results Nevertheless, western blot analysis of testis tissue from Spoc1 gene disruption by a gene-trap insertion homozygous mice demonstrated that, at the protein level, no Embryonic stem cells (ESCs) carrying a mutant Spoc1 (Phf13) detectable SPOC1 was observed at different ages (5, 10 and 20 locus were obtained from a library of ESC clones generated by weeks). By contrast, a strong signal for SPOC1 was observed in random insertional mutagenesis. The Spoc1 locus in the selected wild-type animals, and an intermediate signal was detected from ESC clone was disrupted by a gene-trap vector containing extracts of heterozygous animals (Fig. 1F). Immunoblotting intronic sequences and a splice acceptor site of the engrailed 2 against SPOC1 was performed using a monoclonal anti-SPOC1 (En2) gene, as well as b-geo, a fusion of b-galactosidase and antibody (Kinkley et al., 2009). These data indicate that mice neomycin phosphotransferase II (BayGenomics, CA). The with a homozygous gene-trap insertion represent a complete resulting insertional mutation leads to a fusion transcript functional Spoc1-knockdown at the protein level and we containing sequences from gene-specific exons upstream of the therefore refer to these animals as Spoc12/2 mice. This insertion joined to the b-geo marker and terminated by a poly(A) conclusion is further strengthened by the immunohistochemical site. As a result, expression of the trapped gene can be detected studies described below (Fig. 2). SPOC1, a new factor essential for spermatogenesis 3139

Fig. 1. Disruption of the Spoc1 gene by gene-trap insertion. (A) Schematic representation of the genomic structure of the Spoc1 gene and the insertion of the b-geo cassette between exon 1 and 2. Insertion of the b-geo cassette leads to a disruption of Spoc1 gene transcription and expression. The arrows indicate the PCR primers used for genotyping. (B) Representative PCR genotype analysis of wild-type (+/+) mice and mice heterozygous (+/2) or homozygous (2/2) for the gene-trap allele. Primers for the wild-type allele (WT-F and WT-R) generate a 151-bp amplicon and primers for the gene trap allele (ko-F, ko-R) a 332-bp amplicon. (C) Splicing and functional capability of the gene trap is indicated by b- galactosidase staining of paraformaldehyde-fixed embryos extracted at E13.5. (D) Spoc1 mRNA expression in mouse testis analyzed by northern blot hybridization with a probe corresponding to the Spoc1 39 UTR. The corresponding size of ribosomal RNAs are indicated. The northern blot was re-probed with Gapdh in order to control for RNA-loading differences. The exposure time is 1 week. (E) Real-time PCR analysis of RNA isolated from the testis of differently aged wild-type (+/+) and homozygous (2/2) mice for the Spoc1 gene-trap allele. Quantification of the gene trap induced knockdown was performed with probes for Spoc1 spanning the exon2– exon3 boundary and the housekeeping gene encoding RPII to normalize for variations in RNA amount and reverse transcription efficacy. Samples were analyzed in triplicate. (F) Western blot showing a ,43 kDa SPOC1 band that was detected in protein lysate from wild-type +/2

Journal Cell Science and Spoc1 testis but not in the corresponding Spoc12/2 tissues in mice at 5, 10 and 20 weeks of age. Actin was used for standardization of protein loading.

SPOC1 is coexpressed with PLZF in undifferentiated intrinsic factor involved in SSC self-renewal (Buaas et al., 2004; spermatogonia (As–Aal) Costoya et al., 2004; Payne and Braun, 2006). We observed the SPOC1 expression and localization was investigated by X-gal expression of both proteins in the nuclei of gonocytes, the staining of Spoc1+/2 testis, which expresses b-galactosidase under progenitors of SSCs, in testis 3 days post partum (dpp) (Fig. 2C), the control of the Spoc1 promoter (Fig. 2A). X-gal staining as well as in spermatogonia of 10-week-old mice (Fig. 2D,E), showed a granular signal in a few peritubular basal cells in most demonstrating that SPOC1 is expressed in undifferentiated A- tubules, consistent with the position of spermatogonia (Buaas et al., type spermatogonia. Quantitative analysis over a developmental 2004; Costoya et al., 2004; Dadoune, 2007). A similar window [postnatal day (P) P1 to 10 weeks] demonstrated that spermatogonia-specific pattern was observed when testes there was coexpression of PLZF in 77–100% of SPOC1-positive sections were stained with the monoclonal rat anti-SPOC1 cells, whereas SPOC1 was found to be coexpressed in 97–100% antibody (Fig. 2B). Spoc2/2 gonocytes were PLZF positive of PLZF-positive spermatogonia (supplementary material Table (staining not shown) but were negative for SPOC1 (Fig. 2C), S1 and Fig. S1). Furthermore, whole-mount immunofluorescence demonstrating the specificity of the antibody, as well as the confirmed SPOC1 localization in seminiferous tubules of adult knockdown of SPOC1, in the mouse model. The higher number of mice (12 weeks of age) and revealed that SPOC1 was expressed b-galactosidase-positive cells in comparison with cells stained in single (As), paired (Apr) and aligned (Aal) spermatogonia with anti-SPOC1 antibody might be due to a greater sensitivity of (supplementary material Fig. S2). Taken together these results the enzyme-linked b-galactosidase staining, to post-transcriptional demonstrate that SPOC1 is expressed, in a manner similar to degradation of Spoc1 in some Spoc1-RNA-containing cells or to PLZF, in undifferentiated spermatogonial stem cells. diffusion of X-gal, and remains to be investigated. To demonstrate further that SPOC1 is expressed in early Spoc12/2 mice have smaller testes and a reduced number spermatogonia, we evaluated immunohistochemically the of germ cells coexpression of SPOC1 with PLZF. PLZF is a marker of Spoc1+/2 mice were indistinguishable from wild-type littermates undifferentiated A-type spermatogonia (As–Aal) and is a known on the basis of appearance and breeding capacity. Intercrosses of 3140 Journal Cell Science 124 (18)

Fig. 2. SPOC1 expression in testis is restricted to

undifferentiated A-type spermatogonia (As–Aal). (A) b- galactosidase expression (x-gal) under the control of the Spoc1 promoter in testis from Spoc1+/2 animals (at 10 weeks of age). (B) Immunohistochemical detection of SPOC1 in testis from 10-week-old mice. SPOC1-positive cells show a brown signal (DAB staining) and are localized in the periphery of the seminiferous tubule (arrows). (C) Immunofluorescence staining of SPOC1 (green) and PLZF (red) protein in different testis sections from 3 dpp wild-type (+/+) and knockout (2/2) animals. SPOC1 is expressed in gonocytes (*), as is PLZF (red). No SPOC1 protein was detected in the knockout (2/2) gonocytes. Nuclei were stained with DAPI (grey). The two proteins were stained in different colours and different sections, to avoid crossreaction of the secondary antibodies to rat and mouse primary antibodies. (D) Immunofluorescence localization of SPOC1 (green) protein in adult wild-type testis

Journal Cell Science (at 10 weeks of age). SPOC1 is coexpressed with PLZF [red; a

marker for undifferentiated A-type spermatogonia (As–Aal)]. (E) The same image detail as in D at higher magnification. Nuclei were stained with DAPI. Scale bars: 50 mm (A,B); 10 mm (C–E)

the heterozygous animals produced ,14% Spoc12/2 animals, from aged mice (59 weeks) revealed a complete germ cell loss in which deviates from the expected Mendelian ratio of 25% Spoc12/2 animals, whereas heterozygous (Spoc1+/2) and wild- (supplementary material Fig. S3). Preliminary observations type mice appeared normal (Fig. 3D). Together, these data indicate intrauterine death [at embryonic day (E) E13.5–E15.5] indicate a vast breakdown of spermatogenesis, which explains the as well as peri- and early postnatal lethality, explaining the early infertility of the Spoc12/2 mice. divergent Mendelian ratio. The observed phenotype is solely caused by the absence of Gross phenotypic examination of Spoc12/2 mice revealed that Spoc1 and not by additional non-specific gene-trap integrations. there was initially no visible abnormalities. However, This can be concluded from the following observations: (i) mice homozygous males rapidly (at 5 weeks and older) developed a generated from two independent ESC clones (Xb691 and Xa022) reproductive defect and required a substantially prolonged period show similar phenotypes; (ii) the b-galactosidase expression of time to impregnate the females, whereas heterozygous animals pattern in Spoc1+/2 mice matches the expression of Spoc1,as bred normally (supplementary material Fig. S4). A more detailed determined by RNA in situ hybridzation and imunohistochemical examination of Spoc12/2 mice at different ages revealed a detection; and (iii) in the mixed genetic background we observed substantially reduced testis size (Fig. 3A). Already at 5 weeks of a 100% penetrance between the Spoc12/2 genotype and the age the relative testis weight (testis weight to body weight ratio, observed phenotypic changes. GSI) was dramatically reduced (56%) compared with wild-type animals (Fig. 3B). Correspondingly, we found a substantial Abnormal spermatogenesis in Spoc12/2 mice reduction in the cross-sectional area of seminiferous tubules in In contrast to Spoc1+/2 testis, which appeared phenotypically Spoc12/2 mice (Fig. 3C). number was decreased to 28.6% normal and indistinguishable from those in wild-type mice even (5 weeks), 9.3% (10 weeks) and 10.2% (20 weeks) of the wild- after .12 months (Fig. 3D), histological examination of type number, respectively. Histological investigation of testis Spoc12/2 testes revealed severe testis atrophy with profoundly SPOC1, a new factor essential for spermatogenesis 3141

Fig. 3. Testis hypotrophy in Spoc12/2 mice. (A) Anatomy of the reproductive tract in 9-week-old Spoc1+/+ and Spoc12/2 mice. Testes (T) and seminal vesicles (SV) are indicated. (B) Variation in gonadosomatic index [GSI5(testis weight divided by body weight) 6 100] between Spoc12/2 and Spoc1+/+ mice. (C) Reduced cross- sectional area of the seminiferous tubuli in Spoc12/2 mice. The cross-sectional area was measured using the AxioVision 4.8 software (Carl Zeiss MicroImaging). Only tubuli with a Feret diameter .0.5 were assessed [n5487 for wild type and 554 for knockout (at 5 weeks), 788 and 1324 (at 10 weeks), and 313 and 317 (at 20 weeks)].

Journal Cell Science (D) Testis histology in aged Spoc12/2 mouse. Histological staining of testis from Spoc1+/+, Spoc1+/2 and Spoc12/2 mice with hematoxylin and eosin (HE, upper panel) and Toluidine Blue (lower panel) at 59 weeks of age. Complete germ cell loss is apparent in Spoc12/2 testis, whereas Spoc1+/+ and Spoc1+/2 testes look normal. Scale bars: 100 mm.

altered spermatogenesis in a large number of tubules. Detailed Sertoli-cell-only phenotype (Fig. 4A,B and Fig. 5B). A gross histological investigation of Spoc12/2 testes from mice at 5, 10 quantification of these changes is depicted in supplementary and 20 weeks of age revealed an increase in the number of material Fig. S5. The Spoc12/2 Sertoli cells were similar in number seminiferous tubules that showed a reduced number of germ cells and size to those of wild type (Fig. 4) and expressed the Sertoli- with progressing age. In 5-week-old animals spermatogenesis specific protein GATA1 (Fig. 4C). When we determined the number appeared normal despite a reduced cross-sectional area of of GATA1-positive cells per seminiferous tubule in sections from seminiferous tubules (Fig. 3C, Fig. 4A), whereas tubules from 10-week-old animals, there were no significant differences between 10-week-old animals were disorganized (Fig. 4A). In 20-week-old wild-type and Spoc12/2 testes (data not shown). animals the number of tubules with abnormal spermatogenic In summary, our histological data demonstrate a normal onset of epithelium had further increased and the testis became completely spermatogenesis in juvenile animals followed by a successive loss atrophic. Unfortunately, inter-individual variation prevented an of spermatogenetic cells from the germ cell epithelium after 5- exact quantification of this phenotype. Nevertheless, tubules with a weeks post partum. The loss of germ cells in early developmental normal germinal epithelium were rarely present at this age. stages, but the presence of later developmental stages of germ Remarkably, several tubules (per testis) predominantly contained cells, as was observed in older animals, indicates an ongoing Sertoli cells and a few spermatozoa but no developmentally process of depletion of germ cells possible owing to a failure in younger germ cells, whereas other tubules contained Sertoli cells stem cell renewal. Accumulation of Aal spermatogonia in the together with chains of Aal spermatogonia (Fig. 7B) and still absence of more progressed germ cell stages might arise through a other tubules (25% of tubules at 20 weeks of age) showed a failure in their differentiation and/or their entry into meiosis. 3142 Journal Cell Science 124 (18)

Fig. 4. Spermatogonial depletion and Sertoli-cell-only phenotype in Spoc12/2 mice. (A) Histological staining of testis from Spoc1+/+ and Spoc12/2 mice with Toluidine Blue at different ages. At 5 weeks, tubules and germ epithelium from Spoc12/2 mice look normal in comparison with wild-type animals, whereas a progressive germ cell loss is apparent at older ages. (B) Sertoli-cell-only phenotype in Spoc12/2 testis. Sertoli cells (DAPI stain, color inverted) are characterized by one or two chromocenters (black dots) in the nucleus. (C) Immunohistochemical staining of GATA-1 in 10-week-old Spoc1+/+ and Spoc12/2 testis. The persistence of Sertoli cells in Spoc12/2 tubules is indicated by GATA1 expression. Scale bars: 50 mm (A,C); 10 mm (B).

Normal numbers of gonocytes and Sertoli cells are present were stained with antibodies against PCNA and Ki67, two widely Journal Cell Science in the gonadal anlage of Spoc12/2 mice used proliferation markers that are expressed in a subset of A-, To exclude that the phenotypes observed are simply caused by a In-, and B-spermatogonia, as well as in prophase spermatocytes reduced number of gonocytes before the formation of SSCs, (Costoya et al., 2004; Wrobel et al., 1996). PCNA staining for histological evaluation of Spoc12/2 and wild-type testes (3 dpp) mice at 5, 10 and 20 weeks of age revealed a slightly reduced was performed and these testes were stained for PLZF and appearance in the mutant being significant only at 20 weeks SPOC1, respectively. These experiments demonstrated that (P50.034) (supplementary material Fig. S7). By contrast, Ki67 Spoc12/2 and wild-type animals displayed similar numbers of staining revealed no significant difference (P.0.16) between gonocytes (Fig. 2C), strongly arguing that the observed loss of Spoc12/2 and Spoc1+/+ mice at 5, 10 and 20 weeks of age germ cells in the postpubertal testis is a result of impaired SSC (supplementary material Fig. S8). At 1 week of age no significant self-renewal and/or differentiation. difference in the number of PCNA- or Ki67-positive cells was apparent (data not shown). These results strongly indicate that the Spoc12/2 testes show increased apoptosis and normal demise of spermatogenesis is driven by enhanced apoptosis in the proliferation of germ cells knockout. The observed reduction in testis size and seminiferous tubule Arrest of spermatogenesis can be elicited by the absence of cross-sectional area indicate either a reduced proliferation of numerous factors, some of which are required for passage germ cells or increased apoptosis. To address these possibilities through prophase I (Cooke and Saunders, 2002; Scherthan, we performed TUNEL assays and immunohistochemical 2003). To determine whether Spoc1 deficiency causes defects in stainings of apoptosis and proliferation markers. The TUNEL development, we studied the course of prophase I assays revealed a substantially increased overall rate of apoptosis by following meiotic differentiation through markers for double- in tubules from 5-week- and 20-week-old Spoc12/2 animals strand break (DSB) formation and repair (phosphorylated H2AX, (Fig. 5A–C), which was confirmed by staining of activated known as cH2AX) (Mahadevaiah et al., 2001; Meyer-Ficca et al., caspase 3 (supplementary material Fig. S6). Apoptosis was 2005) and the homologous recombination repair protein MRE11 strongly increased in prophase I cells, especially in the pachytene that is expressed at high levels in meiotic cells (Eijpe et al., stage at seminiferous epithelial stages IV to VII (Fig. 5D–G). 2000). Determination of testes tubule stages was performed as Stage X tubuli served as internal positive controls for the assay described previously (Russell et al., 1990). because these tubuli always contain TUNEL-positive and Normal patterns of cH2AX formation and localization were TUNEL-negative cells (Voet et al., 2003). Next, testis sections detected in prophase I cells of testis sections from 10-week-old SPOC1, a new factor essential for spermatogenesis 3143 Journal Cell Science

Fig. 5. Increased apoptosis in Spoc12/2 testis. (A,B) Apoptosis (TUNEL, green) in testis section of Spoc12/2 and wild-type mice at 20 weeks of age. Many Sertoli-cell-only tubules are visible (*). Nuclei are stained with DAPI (grey, colour-inverted from blue). Leydig cells between tubules display fixation-induced unspecific green autofluorescence. TUNEL-stained wild-type testis sections at the same age display only a few apoptotic cells. (C) Overall number of apoptotic cells per tubule in wild-type and knockout mice at 5, 10 and 20 weeks of age. (D) Frequency of apoptotic cells per Spoc12/2 and wild-type testis cross-section (20 weeks of age). Metaphase I cells were identified in stage XII tubules, pachytene cells in stage I–X, and ‘others’ represents cell death at the tubule periphery (i.e. spermatogonia or spermatocytes near the periphery). Usually this category includes cells with advanced apoptosis, complicating assessment of cell type. Cell death was more abundant in Spoc12/2 cells in pachytene and ‘other cells’ located at the tubule periphery. (E–G) Details of apoptosis in knockout tubules. (E) Detail of a stage IV tubule with apoptotic nuclei (TUNEL, bright green) and pachytene nuclei denoted by a red dot (XY body) revealed by anti-cH2AX antibody (red) immunostaining. Nuclei are revealed by colour-inverted DAPI, grey. (F) Detail of stage VI tubule displaying a pachytene spermatocyte that is TUNEL+ (yellowish-green, *) and displays an XY body signal (cH2AX, faint red dot). Strong red dots mark the XY body in healthy pachytene nuclei (DAPI, colour-inverted to grey). Apoptotic nuclei are stained green (TUNEL). The tubulus periphery is marked by the elongated horizontal nuclei above the nuclei (large faint nuclei with a strong dark chromocenter). (G) Detail of stage VII tubule displaying preleptotene nuclei (PL) with patchy cH2AX and an A-type gonium (A). Numerous apoptotic bodies (green) at different stages of condensation are seen. P, pachytene spermatocyte with XY body signal (cH2AX, red dot). Nuclei are stained with DAPI (colour-inverted to grey). The tubule periphery runs along the right border of the images. Scale bars: 50 mm (A,B); 10 mm (E–G).

Spoc12/2 mice, with strong cH2AX signals being present in labels condensing mitotic and meiotic chromosomes (Cobb et al., leptotene and zygotene nuclei and at the XY body of pachytene- 1999; Hendzel et al., 1997). Meiotic metaphase I and II cells diplotene spermatocytes (Fig. 6A,B). A physiological chromatin (found in the lumen of stage XII tubules; Fig. 6C) and mitotic signal (Meyer-Ficca et al., 2005) was also seen in spermatids of metaphases (observed at the periphery of testis tubules in most Spoc12/2 and wild-type animals (Fig. 6A,B). Strong expression stages (data not shown) were similar in wild-type and Spoc12/2 of the DNA DSB repair protein Mre11 was present in early meiotic testis, indicating that cell division is not affected. Furthermore, cells and at the XY body of pachytene spermatocytes of knockout the normal patterns of cH2AX and Mre11 localization from (Fig. 6C) and wild-type (not shown) testes, which is consistent leptotene to pachytene stages and the faithful XY-body formation with previous studies on wild-type mice (Eijpe et al., 2000). suggest a normal course of DNA repair and chromosome pairing Next, we investigated meiotic and mitotic cell division by during prophase I in Spoc1-deficient meiocytes. In agreement, immunostaining for phosphorylated histone H3 serine 10, which meiotic chromosome pairing and sister chromatid (SC) 3144 Journal Cell Science 124 (18)

Fig. 6. Normal cH2AX, Mre11, phosphorylated H3 staining and normal chromosome pairing in Spoc12/2 testis. (A–C) Immunohistochemical staining of testis sections from 9- to 10-week-old Spoc12/2 mice. (A,B) cH2AX in Spoc1+/+ and Spoc12/2 in stage X–XI tubules. Elongating spermatids show a cH2AX signal (green) due to endogenous DSBs during chromatin remodelling. Zygotene spermatocytes (row of peripheral cells at the top of the image) display large strong and patchy cH2AX signals, while spermatids below show a diffusely green nuclear cH2AX signal. In the wild-type tubule, these are below the focus plane. (C) A stage XII Spoc12/2 tubule showing expression of Mre11 (red) a marker for homologous recombination and DNA repair (Eijpe et al., 2000) in peripheral zygotene spermatocytes, whereas the green chromatin staining marks prometa- and metaphase I and II cells after immunofluorescence staining for phosphorylated histone H3 serine 10 (p-H3) (green). The pattern observed matches the staining in wild-type testis (not shown). Nuclei in all panels are stained with DAPI (blue). (D) Immunofluorescence images of chromosome spreads stained with anti-cH2AX, anti-SYCP3 antibody and DAPI (for DNA). Surface spreads of spermatocytes were stained for the axial element protein SYCP3 (red) and the DSB marker cH2AX (green). DNA was counterstained with DAPI (blue). Axial element formation and chromosome pairing, as deduced by growing axial elements in leptotene, zygotene and fully formed SCs in pachytene cells, as well as the concomitant regression of the ubiquitous cH2AX signal in leptotene to the XY body in pachytene spermatocytes was the same in wild-type and mutant cells, indicating normal homologue pairing and DSB repair in Spoc12/2 spermatocytes.

Journal Cell Science formation, was found to be normal by cH2AX and SYCP3 (Liebe average number of PLZF-positive cells per tubule remained et al., 2006) staining of spreads of wild-type and Spoc12/2 unchanged in both neonatal and adult testis (Fig. 7D). This result spermatocytes (Fig. 6D). Together, these findings suggest that can be attributed to tubules exclusively present in Spoc12/2 testis DNA repair and chromosome pairing progress normally in that showed a strong increase in the number of PLZF-positive Spoc12/2 mice. cells (.10) clustered in the periphery of a few mutant tubules (Fig. 7B). Quantification revealed 3.9% of tubules with .10 The spatial distribution of PLZF-positive undifferentiated PLZF-positive cells in Spoc12/2 and 0.1% in wild-type testis in spermatogonia is significantly altered in Spoc12/2 mice mice at 5 weeks of age At 10 weeks of age, the values were even Because the histological data indicate a defect in SSC self- more divergent, with 6.7% in Spoc12/2 compared with 0.1% in renewal or differentiation, we specifically investigated the wild-type testis. This accumulation of cells represents Aal distribution of undifferentiated spermatogonia over time in the spermatogonia and can be found in tubules that in addition Spoc2/2 testes. PLZF is a marker for undifferentiated contain only Sertoli cells. 2/2 spermatogonia (As,Apr and Aal) and is co-expressed with the Because Spoc1 mice with a mixed 129 and C57BL/6 stem cell markers OCT4 (also known as POU5F1) and GDNF genetic background were used throughout most of the family receptor a1 (GFRa1) (Buaas et al., 2004; Costoya et al., experiments, we sought to verify this unexpected result in a 2004; Payne and Braun, 2006). Using a mouse anti-PLZF congenic gene-trap line in order to exclude genetic background antibody, we examined testis sections from wild-type and effects. We therefore generated a congenic mouse strain by Spoc12/2 mice at P0 (neonates) and 20 weeks of age. Tubules successive backcrossing of Spoc1+/2 animals with C57BL/6 without PLZF-positive cells [measured according to Buaas et al. wild-type animals. Testes from knockout mice of generation N12 (Buaas et al., 2004)] and PLZF-positive cells per tubulus were were investigated. The testis showed a comparable, but slightly counted, respectively. In the adult testis, only strongly PLZF- more severe phenotype than in the mixed genetic background expressing cells at the periphery of the tubules (thus representing (data not shown). Again, PLZF-positive cells per tubule were As–Aal spermatogonia) were scored (Fig. 7). The data obtained counted in testis sections from mice at 5, 10 and 20 weeks of age clearly demonstrated that the number of tubules with PLZF- (Fig. 8). The result clearly demonstrated an almost complete loss positive cells was unchanged between neonatal (P0) Spoc1+/+ and of PLZF-positive spermatogonia at 20 weeks of age in Spoc12/2 Spoc12/2 mice, whereas there was a highly significant reduction mice (Fig. 8C). Remarkably, this was preceded by an increase in (P,0.0001) in adult mice (Fig. 7A,C). Unexpectedly, the the number of tubules with extended chains of spermatogonia and SPOC1, a new factor essential for spermatogenesis 3145

Fig. 7. PLZF-positive cells in testis from wild-type and Spoc12/2 mice. (A) Comparison of immunohistochemical staining of PLZF-positive cells in testis sections from neonatal (P0) and 20-week-old animals. PLZF-staining (brown DAB signal) marks the gonocytes (P0) and undifferentiated A-type

spermatogonia (As–Aal) (adult), respectively. The tissue was counterstained with Mayers Hemalaun. (B) Tubules of adult Spoc12/2 animals. A few Spoc12/2 tubules (*) contain an abnormally high number of peritubular PLZF-positive cells. This phenomenon was not observed in Spoc1+/+ testes. (C) Graph showing the percentage of tubules without PLZF-positive cells as counted in neonates (P0) and 20-week-old mice (adult) Spoc12/2 and Spoc1+/+.(D) Graph showing the average number of PLZF-positive cells per tubule (calculation based on the total number of tubules analyzed). For C and D, a total number of 853 and 719 tubules from adult wild-type and knockout testis, and 737 and 626 tubules in P0 wild-type and knockout testis were scored, respectively. P-values were calculated using the x2 test. A value of P,0.05 was considered as significant. Scale bars: 50 mm.

thus an elevated number of Aal spermatogonia, which was more apoptotic germ cells overall, but specifically in the already visible at 5 weeks of age (Fig. 8A,B). This result pachytene stage of Spoc12/2 testis, whereas there was no Journal Cell Science confirms our previous findings and demonstrates that although significant difference in the number of PCNA- or Ki67-positive further differentiated germ cells are almost completely lost, (proliferating) cells or the number of primary gonocytes. undifferentiated spermatogonia still proliferate and accumulate, Immunostainings of molecular markers for initiation and generating extended chains of Aal spermatogonia. With progression of meiosis were also normal. These results indicate progressive aging these cells also eventually disappear, leading that the seminiferous epithelium gradually loses its ability to start to a Sertoli-cell-only phenotype. The results in the congenic and/or complete new waves of differentiation, whereas the strain strongly indicate a profound defect in differentiation mutant epithelium has a compromised capacity to support the (and not in proliferation or self-renewal) of the SSCs, leading transit of spermatocytes I through the pachytene stage. to the observed transient accumulation of undifferentiated Overall, our findings strongly indicate that SPOC1 represents a spermatogonia in mutant tubules of older mice. new intrinsic regulating factor in undifferentiated and PLZF- positive spermatogonia (As–Aal) that is responsible for Discussion maintaining proper and sustained differentiation of cells from Here, we have shown that SPOC1 is essential for the maintenance the self-renewing stem cell pool. This conclusion is based on of spermatogenesis in the postpubertal testis and is a prerequisite several observations. First, the initial colonization of the for sustained spermatogenesis and normal fertility in adult mice. seminiferous tubules by germ cells appears to be normal, given Spoc12/2 mice displayed a wild-type number of gonocytes and a that the number of gonocytes after birth does not differ between normal first wave of spermatogenesis in juvenile animals, but a the seminiferous tubules of Spoc12/2 and wild-type animals. progressive loss of germ cells was initiated several weeks after Thus, factors that regulate primordial germ cell migration and puberty. This loss first affected non-SSC stages of germ cells and early stages of proliferation appear intact. Second, morphological then, at a later time point, the undifferentiated spermatogonia, and histological analysis, as well as immunohistochemical which finally resulted in a large number of Sertoli-cell-only staining indicates that the observed testis atrophy is not caused seminiferous tubules and a nearly complete atrophy of the testes by a failure in supporting cells or in hormone status because in animals older than 20 weeks. The testes tubules of younger Sertoli cell number and seminal vesicles were normal. Third, the Spoc12/2 animals displayed an altered distribution of biologically active SSCs first appear in male mice of ,3–4 dpp undifferentiated spermatogonia in tubules, with a transient (McLean et al., 2003). It has been suggested that two different increase in Aal spermatogonia and a successive loss of all germ populations of gonocytes are present in the postnatal period. In cells with increasing age. Furthermore, there were substantially the first postnatal week gonocytes directly develop into Kit- 3146 Journal Cell Science 124 (18)

spermatogenesis is unaffected and the ensuing meiosis and spermiogenesis can proceed normally. Consequently, our data suggest a specific defect in the differentiation of spermatogonia descending from the second subpopulation of gonocytes, which is characterized by a self-renewing capability. Forth, we observed a consecutive loss of spermatogenic substages, with a loss of spermatocytes I preceding the loss of spermatids, and finally spermatozoa, eventually leading to Sertoli-cell-only tubules in animals .20 weeks of age. This sequential abrogation of spermatogenic stages, together with the transient increase in Aal spermatogonia and the subsequent loss of all undifferentiated spermatogonia, strongly suggests a failure of SSCs to transit to differentiating spermatogonia. Hyperproliferation of undifferentiated spermatogonia leading to a transient alignment of undifferentiated spermatogonia is not probable because PCNA- and Ki67-positive cells were not increased in Spoc12/2 testis. It is tempting to speculate that the observed transient clustering of PLZF-positive spermatogonia along the periphery in some mutant tubules indicates an abrogated differentiation and migration of spermagonia, given that both processes are tightly coupled (reviewed by Lie et al., 2009; Mruk and Cheng, 2004). Finally, we noted an increased apoptotic rate in germ cells in the pachytene stage during stages IV to VII of the seminiferous epithelial cycle and in spermatogonia. However, most of the spermatocytes managed to complete prophase I and spermatogenesis. This agrees with the normal expression and localization of Mre11 and cH2AX (prophase I recombination markers), phosphorylated histone H3 (a meiotic and somatic metaphase marker) and the presence of a normal XY-body and normal chromosome pairing during prophase I in the Spoc12/2 and wild-type mice. These data suggest that a pachytene (recombination) checkpoint at stage IV of the seminiferous cycle (Ashley et al., 2004; Barchi et al., 2005) is not responsible for the increased cell death of Spoc12/2 spermatocytes. Because, Journal Cell Science in mitotic cells, SPOC1 is important for prophase condensation and chromosome alignment in metaphase (Kinkley et al., 2009), and given that prophase I of meiotic cells is equivalent to the G2 phase of mitotic cells and involves chromosome condensation into pachytene, it seems probable that SPOC1 could also play a role in meiotic prophase I chromatin function during pachytene chromosome condensation. Hence, the absence of SPOC1 in the knockout might underlie the elevated levels of pachytene Fig. 8. PLZF-positive cells in testis from wild-type and Spoc12/2 mice apoptosis. The surviving spermatocytes do not seem to have with a congenic C57BL/6 background. Number of tubules without PLZF- defects in haploid () development because spermatids positive cells from 5- (A), 10- (B) and 20-week-old (C) animals (wild-type, and spermatozoa are the last form to occur before Sertoli-cell- grey; Spoc12/2, black). In total, 697 (+/+) and 552 (2/2) for A; 774 (+/+) only tubules form. The overall increase of apoptotic cells in and 912 (2/2) for B; and well as 758 (+/+) and 933 (2/2) for C tubules were Spoc12/2 testis by 5 weeks of age probably explains the reduced scored, respectively. testis size and tubular volume. Taken together with the differentiation defect of SSCs, this might explain the fast expressing and, therefore, differentiating spermatogonia. These demise of spermatogenesis in the knockout. cells are the starting point of a first round of spermatogenesis. An alternative explanation for the observed phenotype would They do not undergo self-renewal and are therefore rapidly be an altered differentiation and migration process in mutant depleted (de Rooij, 1998; de Rooij and Russell, 2000; Yoshida spermatogonia and carry over of alterations that cause cell death et al., 2007). By contrast, a second subpopulation of gonocytes in some pachytene spermatocytes. It is known that even the develops into a pool of spermatogonia, which undergo self- slightest deregulation affecting either the ability of the germ cell renewal and thus provides a stem cell population for all to traverse through the BTB or its migration through the subsequent rounds of spermatogenesis (de Rooij, 1998; de adluminal compartment can cause arrest of spermatogenesis Rooij and Russell, 2000; Yoshida et al., 2007). The fact that leading to germ cell degeneration and apoptosis (reviewed by Lie the testes of 5-week-old Spoc12/2 mice show seminiferous et al., 2009; Mruk and Cheng, 2004). Such a scenario in the tubules with almost unaffected spermatogenesis (although Spoc12/2 testes could feasibly result in a deregulation of the reduced in diameter) indicates that the first round of differentiation processes responsible for proper spermatogenesis SPOC1, a new factor essential for spermatogenesis 3147

and thus trigger a subsequent apoptosis during or right after For Spoc1 detection, an Assays-on-Demand system (Applied Biosystems) (1 ml 206 probe assay, 10 ml26 qPCR mastermix and 100 ng cDNA) was used. To traverse through the BTB or migration through the adluminal normalize for variations in RNA amount and reverse transcription efficacy, compartment. Of note, this process would also not affect the first transcript levels encoding the housekeeping RNA-polymerase II (RPII) were wave of spermatogenesis because the BTB forms later during determined. The following primers and probes were used: RpII Fw (59- GAATCCGCATCATGAACAGTGAT-39), Rev (59-CATCCATTTTATCCAC puberty (reviewed by Cheng et al., 2009). This explanation would CACCTCTT-39), probe (59-CTCCTCTTGCATCTTGT-39;59-FAM + 39- be consistent with the expression of the SPOC1 protein in TAMRA), Spoc1 probe (59-AAAGGAGGAGCTCCCCCTGAGGAGC-39). undifferentiated, PLZF-positive spermatogonia and with the Analysis was performed with ABI PRISM 7900 HT SDS V2.1.1-Software. altered (clustered) distribution of these cells even in tubules For northern blot analysis total RNA from testis was isolated using TRIzol Reagent (Invitrogen), separated by formaldehyde agarose gel electrophoresis and with an otherwise Sertoli-cell-only phenotype. blotted onto a nylon membrane (Amersham) as previously described (Tagariello PLZF is a known intrinsic transcription factor important for the et al., 2008). As a hybridization probe we used a mixture of two different PCR- self-renewal of SSCs. Interestingly, PLZF and SPOC1 are products, amplified with the primers Spoc-mus-Fw1 (59-GTGAGCAGATCAAA- CCGTGAGAG-39) and Spoc-mus-Rev1 (59-GTGGAAAGGCGGCTACACATT- coexpressed in undifferentiated SSCs and the phenotype of the GTC-39), as well as mSPOC-Fw-5 (59-GCCACTGTTACACTGGGTGTC-39) 2/2 2/2 Spoc1 mice is very similar to the testis phenotype of Plzf and mSPOC-Rv-5 (59-GAAAGGTGACACACTGGCCAC-39), respectively. The (luxoid) mice (Buaas et al., 2004; Costoya et al., 2004). PLZF probes are located in the Spoc1 39 UTR (exon 4). belongs to the BTB–POZ-ZF (for broad complex, tramtrack, bric- a`-brac or poxvirus and zinc finger) family of transcription factors. Western analysis Protein extracts were prepared from the ovary and testis of 18-week-old mice by It has been suggested that this group of proteins represents homogenizing in RIPA buffer (,300 ml per 5 mg tissue; 50 mM Tris-HCl pH 8, transcriptional repressors that act by epigenetically modifying 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS; freshly chromatin (Kelly and Daniel, 2006) and that PLZF probably prepared with Complete Mini Protease Inhibitor from Roche). The proteins were immunoblotted and probed with a rat monoclonal anti-SPOC1 antibody (Kinkley influences the epigenetic program of spermatogonial cells (Buaas et al., 2009) at a concentration of 1:1000 and a rabbit polyclonal anti-actin antibody et al., 2004). Similarly, SPOC1 is a PHD-finger-containing (sc-1616, Santa Cruz Biotechnology) at a dilution of 1:3000. The membranes were protein that is predominantly chromatin-associated and is further probed with secondary horseradish peroxidase (HRP)-conjugated rabbit anti- rat-IgG antibody (1:15,000) (P0450, Dako) or swine anti-rabbit-IgG antibody involved in regulating chromatin structure and in mitotic (1:15,000) (P0399, Dako) antibodies, respectively. The protein bands were chromosome condensation (Kinkley et al., 2009). Therefore, visualized using ECL reagents (Pierce) as described by the manufacturer. owing to the similarities between SPOC1 and PLZF, and because PHD-containing proteins are repeatedly being identified as new Histological processing epigenetic readers and modifiers of chromatin, it is tempting to After perfusion, the testis were fixed in Ito-solution for at least 12 hours, washed in cacodylic buffer for 3 hours and incubated for 2 hours in 1% osmium tetroxide in speculate that SPOC1 also affects stem cell differentiation in a cacodylic buffer. After washing in cacodylic buffer, the testis were dehydrated manner similar to PLZF by altering the epigenetic program. in ascending ethanol concentrations (70, 80, 90 and 100%) and embedded through Interestingly, another PHD finger protein (PYGO2) has recently acetone: 30 minutes in ethanol and acetone (1:1), 30 minutes in acetone, 2 hours in acetone and epon (1:1) and then epon for 2 hours. The epon was hardened for been shown to modulate chromatin and to be involved in 24 hours at 60˚C and then 48 hours at 90˚C. The testis was sagittally cut using an spermiogenesis (Nair et al., 2008). Of note, the reduced number Ultracut microtome (Reichert-Jung, Germany) assembled with a diamond knife of Spoc12/2 offspring (compared with the expected Mendelian (Drukker, Germany). The sections were stained with Toluidine Blue for 1 minute at 50˚C, washed for 10 minutes in Xylene and mounted with Entellan (Merck, Journal Cell Science ratio) indicates a profound effect on viability with reduced Germany). Sections were analyzed with an Olympus IX 70 microscope. penetrance, possibly due to the variation of epigenetic factors. In summary, we have identified a new intrinsic factor in Immunohistochemistry undifferentiated PLZF-positive spermatogonia that is required for Testes were fixed in 10% formalin overnight, washed with tap water for 24 hours, the differentiation of self-renewing spermatogonial stem cells embedded in paraffin and sectioned at 5-mm thickness. Immunohistochemical staining was performed on deparaffinized and rehydrated sections subjected to into spermatocytes, possibly through a chromatin-mediated effect antigen unmasking using 10 mM citrate buffer 1 hour at 95˚C. After inactivation of on differentiation-inducing genes. A further study will thus be endogenous peroxidase (30 minutes in 0.3% H2O2, 10% methanol in PBS at room conducted to identify the effect of SPOC1 disruption at the temperature) and three 5-minute washing steps in PBS, a permeabilization step (20 minutes in 0.5% Triton X-100 in PBS) was performed. After an additional expression level. three 10-minute washing steps in PBS, the further steps are executed with Vectastain Elite ABC Kit (Vector Laboratories). Materials and Methods Sections were incubated with rat monoclonal anti-SPOC1 antibody (1:10) Generation of Spoc1 gene-trap mutant mice (Kinkley et al., 2009) rabbit polyclonal anti-Mre11 antibody (1:200, Novus All animal experiments were performed according to approved guidelines. To Biologicals, Littleton, CO), rabbit polyclonal anti-H2AX antibody (1:500, Biomol, develop a Spoc1-deficient mouse strain we used ESC lines, Xb691 and Xa022, which Hamburg), rat monoclonal anti-GATA1 antibody (1:200, Santa Cruz have a gene-trap insertion in the Spoc1 gene between exon 1 and 2 (Bay Genomics, Biotechnology, California, USA), anti-phosphorylated histone H3 (serine 10) San Francisco, CA; http://www.genetrap.org). According to JAX convention the antibody, mouse polyclonal anti-PLZF antibody (1:250, Santa Cruz gene-trap allele was designated as Phf13Gt(XB691)Byg and Phf13Gt(XA022)Byg but is here Biotechnology), mouse monoclonal anti-PCNA antibody (PC10 Calbiochem) or abbreviated to Spoc12.TheSpoc1 gene-trapped ESCs were microinjected into rabbit polyclonal anti-Ki67 antibody (Bethyl Laboratoris) overnight at 4˚C. C57BL/6 host blastocysts to generate germline chimeras. The resulting Spoc12 line Fluorescence detection of primary antibodies was performed as described was maintained as heterozygotes and intercrossed to obtain homozygotes (Spoc12/2). (Barrionuevo et al., 2009). Nuclei were counterstained with hemalaun or DAPI. The genotypes were determined by PCR. The wild-type Spoc1 allele PCR was Sections were analyzed using Olympus IX 70 (peroxidase staining) or a Zeiss detected with primers in intron 1, WT-F (59-CCTGGTGGAGCAAGTTGGAAC-39) Axioplan 2 microscope, equipped with fluorescence filters for red, green and blue and WT-R (59-CTCACCTTGTCGCTACCCCAAC-39), which produced a 151-bp excitation (Chroma) (fluorescent staining). amplicon. To detect the gene-trap allele, the primers ko-F (59-GGGTGCGT- TGGTTGTGGATAAG-39) and ko-R (59-CATTCAGGCTGCGCAACTGTTGGG- TUNEL assays 39) were used which produced a 332-bp amplicon. TUNEL assays were performed using the In Situ Cell Death Detection Kit and Fluorescein (Roche) on paraffin-embedded tissue following the instructions of the Expression studies manufacturer. Quantification of relative transcript levels of Spoc1 was performed with the QuantiTect Probe RT-PCR kit (Qiagen) in an ABI PRISM 7900 HT sequence Preparation of meiotic chromosome spreads detection system (Applied Biosystems). The assays were performed in triplicate in Preparation of spermatocyte spreads was performed as described previously 384-well optical plates (Applied Biosystems) with a final volume of 20 ml each. (Scherthan et al., 2011). 3148 Journal Cell Science 124 (18)

X-gal staining He, Z., Kokkinaki, M. and Dym, M. (2009). Signaling molecules and pathways Tissue was fixed in 4% paraformaldehyde for 2 hours at 4˚C. After three washing regulating the fate of spermatogonial stem cells. Microsc. Res. Tech. 72, 586-595. steps in PBS (4˚C), tissue was incubated in 30% sucrose in PBS at 4˚C with slight Hendzel, M. J., Wei, Y., Mancini, M. A., Van Hooser, A., Ranalli, T., Brinkley, B. R., agitation. For cryosections, the tissue was embedded in Tissue-Tek (Sakura, Bazett-Jones, D. P. and Allis, C. D. (1997). Mitosis-specific phosphorylation of Staufen, Germany). Sections were washed in detergent solution (16 PBS, 2 mM histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. MgCl2, 0.02 % NP-40 and 0.01% Na-deoxycholate in water) for 15 minutes before incubation with staining solution [16 PBS, 20 mM Tris HCl, pH 7.5, 2 mM Chromosoma 106, 348-360. MgCl , 0.02% NP-40, 0.01% Na-deoxycholate, 5 mM K Fe(CN) ,5mM Hobbs, R. M., Seandel, M., Falciatori, I., Rafii, S. and Pandolfi, P. P. (2010). Plzf 2 3 6 regulates germline progenitor self-renewal by opposing mTORC1. Cell 142, 468-479. K4Fe(CN)6, 0.75 mg/ml X-Gal in DMSO] under optical control. To stop the staining reaction, sections were washed three times in post-washing solution (16 Hofmann, M. C. (2008). Gdnf signaling pathways within the mammalian spermato- PBS, 5 mM EDTA, pH 8 in water). Sections were mounted with Crystal Mount gonial stem cell niche. Mol. Cell. Endocrinol. 288, 95-103. Kelly, K. F. and Daniel, J. M. (2006). POZ for effect-POZ-ZF transcription factors in (Sigma, Taufkirchen, Germany) and Clarion (Biomeda). Sections were analyzed cancer and development. Trends Cell Biol. 16, 578-587. with the Olympus IX 70 microscope. Kinkley, S., Staege, H., Mohrmann, G., Rohaly, G., Schaub, T., Kremmer, E., Winterpacht, A. and Will, H. (2009). SPOC1: a novel PHD-containing protein H. Scherthan acknowledges the technical assistance of D. Gassen, modulating chromatin structure and mitotic chromosome condensation. J. Cell Sci. A.W. that of S. Richter, and H.W. that of K. Reumann, U. Matschl 122, 2946-2956. and N. Lohrengel. The work of H.W., H. Staege and S.K. was Lie, P. P., Cheng, C. Y. and Mruk, D. D. (2009). Coordinating cellular events during spermatogenesis: a biochemical model. Trends Biochem. Sci. 34, 366-373. supported by a grant from the Deutsche Krebshilfe and Mu¨ggenburg- Liebe, B., Petukhova, G., Barchi, M., Bellani, M., Braselmann, H., Nakano, T., Stiftung. H. Scherthan received support from the DFG (SCHE350/ Pandita, T. K., Jasin, M., Fornace, A., Meistrich, M. L. et al. (2006). Mutations 10-1, SPP1384), The Heinrich-Pette Institut is supported by the that affect meiosis in male mice influence the dynamics of the mid-preleptotene and Germany Federal Ministry of Health and the Freie und Hansestadt bouquet stages. 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