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Is Required for Spermatogonial Stem Cell Differentiation and Sustained Spermatogenesis

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Is Required for Spermatogonial Stem Cell Differentiation and Sustained Spermatogenesis Research Article 3137 SPOC1 (PHF13) is required for spermatogonial stem cell differentiation and sustained spermatogenesis 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 ß 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 gonocytes, the proliferation of germ cells, and the initiation and progression of meiosis 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 spermatocytes. 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 spermatids 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
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