© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 4298-4310 doi:10.1242/dev.112888

RESEARCH ARTICLE The zinc-finger basonuclin 2 is required for proper mitotic arrest, prevention of premature meiotic initiation and meiotic progression in mouse male germ cells Amandine Vanhoutteghem1,Sébastien Messiaen2, Françoise Hervé1, Brigitte Delhomme1, Delphine Moison2, Jean-Maurice Petit3, Virginie Rouiller-Fabre2, Gabriel Livera2 and Philippe Djian1,*

ABSTRACT germ cells enter shortly after they reach the fetal gonad, Absence of mitosis and meiosis are distinguishing properties of whereas male germ cells become quiescent in the fetal gonad and male germ cells during late fetal and early neonatal periods. initiate meiosis after birth, when spermatogonia start forming Repressors of male germ cell meiosis have been identified, but spermatocytes. Transplantation studies have shown that bipotential mitotic repressors are largely unknown, and no protein repressing germ cells are intrinsically programmed to differentiate following both meiosis and mitosis is known. We demonstrate here that the the female pathway and to undergo early meiosis (McLaren and zinc-finger protein BNC2 is present in male but not in female germ Southee, 1997). Prevention of meiosis in fetal testis is achieved cells. In testis, BNC2 exists as several spliced isoforms and through synthesis in male Sertoli cells of CYP26B1, an enzyme that presumably binds to DNA. Within the male germ cell lineage, degrades the retinoic acid necessary for meiosis induction (Bowles BNC2 is restricted to prospermatogonia and undifferentiated et al., 2006; Koubova et al., 2006). When the level of testicular spermatogonia. Fetal prospermatogonia that lack BNC2 multiply CYP26B1 decreases, prevention of meiosis is taken over by excessively on embryonic day (E)14.5 and reenter the cell cycle NANOS2, a protein that is synthesized by fetal male germ cells in prematurely. Mutant prospermatogonia also engage in abnormal the absence of retinoic acid signaling (Suzuki and Saga, 2008). meiosis; on E17.5, Bnc2−/− prospermatogonia start synthesizing During spermatogenesis, spermatogonia undergo meiosis, in the synaptonemal protein SYCP3, and by the time of birth, the first division of which homologous pair, many Bnc2−/− prospermatogonia have accumulated large recombine and synapse. Disturbance of any of these processes amounts of nonfilamentous SYCP3, thus appearing to be blocked leads to massive apoptosis-mediated death of the spermatocytes at at leptonema.Bnc2−/− prospermatogonia do not undergo proper a specific check point in stage IV of the seminiferous epithelial male differentiation, as they lack almost all the mRNA for the male- cycle (Hamer et al., 2008). specific methylation protein DNMT3L and have increased levels of Basonuclin 2 (BNC2) is an extremely conserved zinc-finger mRNAs that encode meiotic , including STRA8. Bnc2−/− protein (Romano et al., 2004; Vanhoutteghem and Djian, 2004). It is prospermatogonia can produce spermatogonia, but these enter orthologous to BNC1, a protein that is essential for germ cells meiosis prematurely and undergo massive apoptotic death during (Mahoney et al., 1998; Tseng and Green, 1992; Yang et al., 1997; meiotic . This study identifies BNC2 as a major regulator of Zhang et al., 2012). Mice with a homozygous disruption of Bnc2 die male germ stem cells, which is required for repression of meiosis shortly after birth from a cleft palate (Hervé et al., 2012; and mitosis in prospermatogonia, and for meiosis progression Vanhoutteghem et al., 2011, 2009). during spermatogenesis. In view of the extreme evolutionary Because of its abundance in testis (Vanhoutteghem and Djian, conservation of BNC2, the findings described here are likely to 2004), we thought that BNC2 might have a function in male apply to many species. reproduction in addition to its function in palatal mesenchymal cells. Direct evidence for a function of BNC2 as a DNA-binding KEY WORDS: BNC2, Gonocytes, Mouse, Spermatogonia, protein that regulates meiosis and mitosis in male germ cells is Transcription described below.

INTRODUCTION RESULTS In mammals, primordial germ cells remain sexually bipotential In mouse testis, BNC2 is presumably a DNA-binding protein during their migration across the embryo; they become sexually and exists as several splicing isoforms differentiated upon reaching the fetal gonad. Sexual differentiation In this study, we have used the previously reported Ayu21:18 mouse is correlated with the timing of entry into meiosis. In mice, female line, the Bnc2 of which is disrupted by a gene-trap insertion. The disrupted allele is likely to be null because all major Bnc2 mRNA isoforms are absent from the homozygous mutant 1Laboratoire de physiologie cérébrale, Centre National de la Recherche (Vanhoutteghem et al., 2009). 2 Scientifique, UniversitéParis Descartes, UMR 8118, Paris, France. Laboratoire de We have previously described, in human keratinocytes, an développement des gonades, UniversitéParis Diderot, Sorbonne Paris Cité, INSERM U967, CEA/DSV/iRCM/SCSR, Fontenay-aux-Roses F-92265, France. insoluble form of human BNC2, which colocalizes with the splicing 3Service central de microscopie, Centre Universitaire des Saints-Peres,̀ Université factor SC35 in nuclear speckles and has a presumed function in Paris Descartes, Paris, France. nuclear RNA processing. The apparent molecular mass of insoluble *Author for correspondence ([email protected]) BNC2 is 145 kDa (Vanhoutteghem and Djian, 2006). We also found that, in human keratinocytes, there exists a second BNC2

Received 16 May 2014; Accepted 11 September 2014 isoform with a molecular mass of 160 kDa and a homogenous DEVELOPMENT

4298 RESEARCH ARTICLE Development (2014) 141, 4298-4310 doi:10.1242/dev.112888 distribution throughout the nucleus. The two human isoforms result Because alternative promoters and alternative splicing are from alternative splicing of the transcript. prominent features of the human BNC2 gene, we searched mouse As shown in Fig. 1A, when the testes of newborn mice were testis for alternative Bnc2 promoters and alternatively spliced Bnc2 analyzed by western blotting with an antibody against amino acid transcripts. For promoter mapping, the 5′ end of the mRNA was residues 722-849 of BNC2 (herein referred to as BNC2722-849), amplified by 5′ rapid amplification of cDNA ends (RACE), resolved three protein bands in the range of 120-160 kDa were detected in by using agarose gel electrophoresis and then sequenced. A single Bnc2+/+ and Bnc2+/− testis, but not in Bnc2−/− testis. These proteins transcription start site in exon 1 was found (supplementary material were present in the nuclear extract and absent from the cytoplasm. Fig. S2A,B). Alternative exons were identified by using RT-PCR The major protein ran at ∼160 kDa, and we thought it probable that with primers in either exons 1 and 3, or 4 and 6. The unfractionated it was the mouse equivalent of human soluble BNC2. The identity products were cloned and sequenced. Because a large number of of the 160-kDa protein was confirmed by its staining with another clones were sequenced, we could obtain an estimate of the relative antibody against BNC2 (supplementary material Fig. S1A). abundance of each Bnc2 mRNA isoform. We found a total of 11 Testicular BNC2 was found in the soluble nuclear extract and in exons, of which five were constitutive (exons 1, 3, 4, 5 and 6) and six association with chromatin. No BNC2 was detected in either the were alternative (1a, 2, 2a, 2c, 5cS,5cL and 5b) (supplementary insoluble nuclear fraction or the cytoplasm (Fig. 1B). We conclude material Fig. S2C). The 5′ alternative exons (1a, 2, 2a and 2c) were that in mouse testis, BNC2 is a largely soluble nuclear protein with all in frame with the rest of the coding region and caused variations an apparent molecular mass of ∼160 kDa. in the N-terminal part of the protein, whereas the 3′ alternative exons 722-849 Both of the antibodies against BNC2 and amino acid (5cS,5cL and 5b) resulted in either premature termination and residues 661-782 of BNC2 produced homogenous nuclear staining suppression of the last three zinc-fingers or disruption of finger 4 in newborn testis (supplementary material Fig. S1B-E). Such a with conservation of fingers 5 and 6. As a result, testis contained a diffuse nuclear distribution is in keeping with the presence of family of BNC2 proteins with a constant central sequence that BNC2 in the soluble nuclear extract and its association with included the three N-terminal zinc-fingers and the nuclear chromatin. This suggests that BNC2 is a DNA-binding protein localization signal, but variable N-terminal regions and variable in testis. numbers of C-terminal zinc-fingers (Fig. 1C).

Fig. 1. BNC2 exists as multiple isoforms, the major of which is soluble and associated with chromatin. (A,B) Western blot analysis of newborn testis. − (A) BNC2 runs as a 160-kDa protein in nuclear extracts (NE) of Bnc2+/+ and Bnc2+/ testes (arrows). The protein is neither found in cytoplasm (Cyt) nor in the mutant. Two weaker bands with an electrophoretic mobility of ∼140 and ∼120 kDa might correspond to splicing or degradation products of BNC2. Proteins with an − − electrophoretic mobility of 100 kDa or less are stained nonspecifically, as they are found in Bnc2 / testis. (B) Subnuclear fractionation. BNC2 (arrows) is found in nuclear (NE) and chromatin (Chr) extracts; it is absent from the insoluble nuclear fraction (Nins) and cytoplasm (Cyt). (C) Map of the Bnc2 gene and the splicing isoforms detected by using RT-PCR. Exons are represented by green boxes and designated by Arabic numerals. Intron sizes (kb) are shown in between the green boxes. The arrowhead indicates the most abundant RNA isoform. BNC2 protein isoforms resulting from alternative splicing are shown in the schematic to the right. Black and red vertical bars represent zinc-fingers and nuclear localization signal, respectively. Protein isoforms vary by their N-terminal regions and/or number of C-terminal zinc-fingers. (D) Agarose gel electrophoresis of RT-PCR products. Using primers in exons 1 and 5 (1+5), the most abundant isoform contains exons 1, 2, 2a, 3, 4 and 5. Using primers in exons 5 and 6 (5+6), the most abundant isoform contains exon 6 directly spliced to exon 5. Therefore, the major isoform contains exons 1, 2, 2a and 3-6. (E) Western blot analysis of nuclear extract from HeLa cells that had been transduced with lentivirus encoding the 1, 2, 2a, 3-6 isoform of BNC2 fused to c-myc. In transduced cells that had been stained with an antibody against myc (+LVmyc), myc-tagged BNC2 runs at − 160 kDa (arrow). It is not detected in nontransduced cells ( LVmyc). Staining of transduced cells with an antibody against amino acid residues 1-34 of

BNC2 (+LVBNC2) confirms that the 160-kDa protein is BNC2 (arrow). DEVELOPMENT

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The most abundant RNA isoform was composed of exons 1, 2, these interstitial cells were Leydig cells, we double-stained 2a, 3-6 (Fig. 1D) and encoded a 1099-amino acid protein. All other testisforBNC2andfor3-β-hydroxysteroid dehydrogenase isoforms were much less abundant. The RNA comprising exons 1, (Majdic et al., 1998). No BNC2 was detected in Leydig cells 2a and 3-6, which is fairly abundant in human keratinocytes (Fig. 2C). On E18.5, BNC2 was found in the nuclei of (Vanhoutteghem and Djian, 2007) and encodes the speckle- prospermatogonia at a lower level than that on E14.5. There was associated BNC2, was not detected, thus explaining the absence also some protein in interstitial cells. Neither Sertoli nor Leydig of speckle-associated BNC2 in mouse testis. Lentiviral expression cells possessed detectable BNC2 (Fig. 2E,F). in HeLa cells of the 1, 2, 2a, 3-6 isoform fused to c-myc resulted in Postnatal distribution of BNC2 was investigated by indirect the production of soluble BNC2 that had an apparent molecular immunofluorescence staining. At birth, BNC2 was found to occur in mass undistinguishable from that of the testicular protein (Fig. 1E). prospermatogonia and interstitial cells (Fig. 3A) but was absent from both Sertoli (Fig. 3B) and Leydig cells (Fig. 3C). On P8, BNC2 is found in prospermatogonia and undifferentiated prospermatogonia had been replaced by spermatogonia, located in spermatogonia. It is absent from Sertoli and Leydig cells the basal layer of the tubular epithelium, whereas spermatocytes To define the cell types expressing Bnc2 in fetal testis, had started to form suprabasally. BNC2 was confined to some paraffin-embedded sections of Bnc2+/+ testes were stained basal cells, identified as undifferentiated spermatogonia by the by using immunohistochemistry and the antibody against presence of the promyelocytic leukemia zinc-finger protein (PLZF; BNC2722-849 (subsequently used in all immunofluorescence ZBTB16 – Mouse Genome Informatics), a marker of spermatogonial and immunohistochemistry analyses). We first examined testes progenitor cells (Buaas et al., 2004; Costoya et al., 2004). BNC2 was on embryonic day (E)14.5, when germ cells undergo male absent from both differentiated spermatogonia and spermatocytes differentiation. As shown in Fig. 2A, abundant nuclear BNC2 (Fig. 3D-F). On postnatal day 30, BNC2 was found in a few cells was detected in prospermatogonia, identified by their central within the tubules and in some cells outside of the tubules. Within the position within the testicular cords and by the presence of tubules, BNC2-positive cells were invariably basal; they were VASA (DDX4 – Mouse Genome Informatics) (Castrillon et al., identified as undifferentiated spermatogonia by double staining for 2000). Sertoli cells, which entirely form the tubular epithelium BNC2 and PLZF; all tubular BNC2-containing cells possessed PLZF until postnatal day (P)6, did not possess detectable levels of and vice versa. Differentiating spermatogonia, spermatocytes and BNC2. This was confirmed by double staining for BNC2 and spermatids lacked both BNC2 and PLZF (Fig. 3G-I). The BNC2- anti-Müllerian hormone. Some interstitial cells possessed containing cells located outside of the tubules (cells stained red in smaller amounts of BNC2 (Fig. 2B). To determine whether Fig. 3G,I) probably corresponded to the nonsteroidogenic interstitial

Fig. 2. Cellular distribution of BNC2 in fetal testis. Paraffin sections of fetal testis double-stained for BNC2 and markers of prospermatogonia, Sertoli and Leydig cells. (A-C) E14.5. (A) Prospermatogonia identified by the presence of cytosolic VASA contain an abundant amount of nuclear BNC2. Sertoli ‘S’ cells (arrows) lack BNC2. (B) Double staining for BNC2 and anti- Müllerian hormone (AMH), a Sertoli cell marker. Sertoli cells contain abundant AMH but no BNC2. Some interstitial cells contain BNC2 at a lower level than that in prospermatogonia. (C) Double staining for BNC2 and Leydig cell-specific 3-β- hydroxysteroid dehydrogenase (3βHSD). Leydig cells lack BNC2 (arrowheads). Prospermatogonia contain BNC2, but Sertoli cells do not. (D-F) E18.5. Results are similar to those at E14.5, except that BNC2 level in prospermatogonia is lower. In B,C,E,F, whole testis is shown in the upper images, a higher magnification is shown in the lower images. i, nonsteroidogenic interstitial cells; p, prospermatogonia. Scale bars: 25 μm. DEVELOPMENT

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Fig. 3. Postnatal BNC2 testicular distribution. Frozen sections double-stained by using indirect immunofluorescence for BNC2 and various germ and somatic cell markers. DNA is stained blue with DAPI. (A-C) BNC2 distribution on P0. Insets show details of main frames. (A) BNC2 and VASA double staining. BNC2 is present in VASA-containing prospermatogonia (arrowheads) and interstitial cells, and absent from Sertoli cells ‘S’. (B) BNC2 and AMH staining. Sertoli cells contain AMH but no BNC2 (arrowheads). (C) BNC2 and 3βHSD staining. BNC2 is absent from Leydig cells that were identified by the presence of 3βHSD (arrowheads). (D-F) BNC2 and PLZF staining at P8 to assess BNC2 distribution during incipient spermatogenesis. BNC2 is strictly colocalized with PLZF; therefore, it is specific to undifferentiated spermatogonia and absent from differentiated spermatogonia and spermatocytes. D,E show the single stains, F shows the merged images. (G-I) BNC2 and PLZF staining at P30. BNC2 and PLZF are colocalized in undifferentiated spermatogonia (arrowheads), which are very sparse at this age. Neither BNC2 nor PLZF is detected in the progeny of undifferentiated spermatogonia. Some interstitial cells contain BNC2 (arrow). G,H show the single stains, I shows the merged images. i, nonsteroidogenic interstitial cells; p, prospermatogonia. Scale bars: 50 μm (A-C); 25 μm (D-I).

cells that had been identified at earlier ages. Therefore, in the germ chromatin; no mitotic prospermatogonia were seen (Fig. 4H). cell lineage, BNC2 was restricted to the most primitive cells – In Bnc2−/− mice, many prospermatogonia were similar to those of prospermatogonia and undifferentiated spermatogonia. wild-type littermates, but 20-30% showed condensed chromosomes and appeared engaged in either mitosis or meiosis (Fig. 4I). To Fetal Bnc2−/− prospermatogonia multiply excessively on determine whether prospermatogonia with condensed chromosomes E14.5 and reenter the cell cycle prematurely were engaged in mitosis, we double-stained newborn testes for Ki-67 In view of the abundance of BNC2 in prospermatogonia, we thought and VASA. In the wild type, no prospermatogonia stained positively that the protein might have a function in these cells. The fact that for Ki-67; all cells containing Ki-67 were somatic cells, as they BNC2 has been implicated in cell multiplication (Vanhoutteghem lacked VASA (Fig. 4J). In the Bnc2−/− newborns, approximately one- et al., 2009) prompted us to compare the mitotic status of the third of the prospermatogonia contained Ki-67 and therefore were prospermatogonia of the Bnc2−/− mice with that of their wild-type engaged in the mitotic cycle (Fig. 4K). littermates. During the fetal period, prospermatogonia divide actively from around E12 and then gradually become quiescent Bnc2−/− prospermatogonia are engaged in an incomplete between E14 and E16, depending on the mouse strain (Vergouwen form of meiosis et al., 1991). We analyzed fetal germ cell proliferation by using We then tested whether Bnc2−/− prospermatogonia could also be immunohistochemical staining of testicular sections for VASA and engaged in meiosis. Testes were stained for Ki-67 (MKI67 – Mouse Genome Informatics), a nuclear marker of protein 3 (SYCP3), a marker of early meiosis (Lammers et al., multiplying cells (Gerdes et al., 1983). On E14.5, ∼40% of Bnc2+/+ 1994). On E17.5, wild-type testis did not contain any SYCP3- germ cells contained Ki-67, whereas almost all Bnc2−/− germ cells positive cells (Fig. 5A), whereas in the Bnc2−/− littermates, ∼10% expressed Ki-67 (Fig. 4A-C). Real-time quantitative PCR (RT- of the prospermatogonia contained foci of SYCP3 (Fig. 5B). By P0, qPCR) analyses have indicated that Lefty and Nodal, two Bnc2+/+ prospermatogonia still stained negatively for SYCP3 known to be expressed in mitotically active prospermatogonia (Fig. 5C), whereas in the Bnc2−/− testis, the proportion of (Souquet et al., 2012), are overexpressed in Bnc2−/− testis at E14.5. SYCP3-positive prospermatogonia had increased to 25-30%. Expression of neither Nanos2, a marker of male differentiation, nor SYCP3 was mostly in the form of large aggregates (Fig. 5D) but Oct4 (Pou5f1 – Mouse Genome Informatics), a germ cell marker, sometimes in threads typical of zygotene (Fig. 5E). The presence of was affected (supplementary material Fig. S3). On both E17.5 and SYCP3 in Bnc2−/− prospermatogonia was confirmed using another E18.5, all prospermatogonia in both Bnc2−/− and Bnc2+/+ testes antibody against SYCP3. Because a nonfilamentous distribution of lacked Ki-67 (Fig. 4D-G). Therefore, we conclude that fetal SYCP3 is typical of cells in the preleptotene and leptotene stages prospermatogonia lacking BNC2 multiply excessively on E14.5 that have not yet formed synaptonemal complexes, we hypothesized but do reach mitotic quiescence by E17.5. that most Bnc2−/− prospermatogonia that had entered meiosis had We then examined newborn testes by staining paraffin-embedded remained thereafter blocked in preleptotene or leptotene. The sections with hematoxylin & eosin (HE). In Bnc2+/+ newborns, presence of the premeiotic-specific protein stimulated by retinoic prospermatogonia were evident at the center of the cords as large acid 8 (STRA8) (Koubova et al., 2006; Mark et al., 2008; Oulad- −/− round cells with a spherical nucleus and dispersed homogenous Abdelghani et al., 1996) confirmed the engagement of Bnc2 DEVELOPMENT

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−/− Fig. 4. Mitotic cycling of Bnc2 prospermatogonia in fetal and − − neonatal testis. Sections of Bnc2+/+ and Bnc2 / testis double- stained for VASA and Ki-67 or with HE. (A,B) E14.5. Paraffin- embedded sections stained by using immunohistochemistry. (A) In a substantial number of cords of wild-type testis, prospermatogonia identified by the presence of VASA do not contain Ki-67. − − (B) In Bnc2 / testis, nearly all prospermatogonia possess Ki-67. (C) The number of prospermatogonia engaged in the cell cycle is significantly higher in homozygous mutant than in wild-type embryos. At least 200 cells counted per testis. n=3, ***P<0.001, unpaired Student’s t-test. Error bars show mean±s.d. (D,E) E17.5. Frozen sections of whole testis stained by using indirect − − immunofluorescence. All Bnc2+/+ and Bnc2 / prospermatogonia lack Ki-67 and therefore are quiescent. Many somatic cells contain Ki-67. Single prospermatogonia lacking Ki-67 are shown to the right at higher magnification. (F,G) Similar results at E18.5 were found on paraffin sections stained by using immunohistochemistry. (H,I) HE-stained paraffin sections of newborn testes. Single prospermatogonia are shown to the right at higher magnification. (H) In wild-type mouse, numerous prospermatogonia are present in the center of testicular cords; they possess round homogenous nuclei (arrowheads). (I) In mutant testis, many prospermatogonia are engaged in cell division as shown by the lack of nuclear membrane, condensation and symmetrical distribution of chromosomes (arrowheads). (J,K) Frozen sections of newborn testis examined by immunofluorescence staining. (J) In wild-type testis, all prospermatogonia lack Ki-67 and therefore are not engaged in mitosis. Only Ki-67-positive cells are somatic cells (arrowheads). Prospermatogonia lacking Ki-67 and a somatic cell lacking VASA but containing Ki-67 are shown at higher − − magnification to the right. (K) In Bnc2 / testis, numerous prospermatogonia contain Ki-67 (arrowheads) and possess condensed chromosomes typical of mitosis. Prospermatogonia containing Ki-67 and engaged in mitosis are shown at higher magnification to the right. Scale bars: 25 μm.

prospermatogonia in meiosis. STRA8 was not detectable in wild- for RNA polymerase I (Iuchi and Green, 1999; Tian et al., 2001). type testis (Fig. 5F,G). In order to determine whether the The first pair of zinc-fingers of BNC2 is very similar to that of prospermatogonia that had abnormally engaged in meiosis were BNC1 and has been shown to bind to the rRNA gene promoter undergoing apoptosis, we stained testes at E17.5, E18.5 and P0 for in vitro (Romano et al., 2004). We did not detect any decrease in the activated caspase 3, which is associated with apoptotic cell death number of 18S and 28S RNA molecules in the Bnc2−/− testes. (Nicholson et al., 1995). Almost no cleaved caspase 3-containing On the contrary, we observed a slight increase (supplementary cells were detected in either the Bnc2−/− mice or their wild-type material Table S1). We conclude that BNC2 is not a transcription littermates (supplementary material Fig. S4A,B). Double staining factor for RNA polymerase I. for SYCP3 and cleaved caspase 3 was performed on P0 testis; none A list of genes specifically expressed in each testicular cell of the numerous SYCP3-containing prospermatogonia stained type is given in the supplementary material Table S2. The positive for the caspase (supplementary material Fig. S4C,D). expression of the genes encoding DNMT3L and albumin showed We finally sought to determine whether the prospermatogonia the greatest changes. Both genes are associated with male germ engaged in the mitotic cycle and containing Ki-67 (Fig. 4K) cell differentiation and showed over a tenfold decrease in were the same ones that aberrantly produced SYCP3. We triple- expression. DNMT3L is a zinc-finger protein that resembles stained newborn Bnc2−/− testes for VASA, SYCP3 and Ki-67; cytosine-5-methyltransferase 3, but lacks its catalytic domains. prospermatogonia contained either Ki-67 or SYCP3, but never the Dnmt3l is expressed from E14.5 until the perinatal period in two proteins together (Fig. 5H,I). prospermatogonia, but not in oocytes; it is required for retrotransposon inactivation (Aapola et al., 2000; Bourc’his and BNC2 is required for expression of Dnmt3l and repression of Bestor, 2004; Bourc’his et al., 2001; Shovlin et al., 2007). The meiotic proteins in prospermatogonia albumin gene is specifically expressed in prospermatogonia of To identify the genes of which the expression was affected by a lack newborn testis and is not expressed in the ovary (Gelly et al., of BNC2, we used massive parallel sequencing (RNA-Seq). To 1994; McLeod and Cooke, 1989). We also observed an increase achieve this, we prepared total RNA from the testes of six Bnc2−/− in the premeiotic-specific mRNAs encoding STRA8, MSX1 and and four Bnc2+/+ newborn mice. We first examined the ribosomal MSX2 (Le Bouffant et al., 2011), all of which are normally

(r)RNA gene transcripts. BNC1 is known to be a transcription factor specific to female germ cells during fetal life. DEVELOPMENT

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−/− Fig. 5. Abnormal meiosis in fetal and neonatal Bnc2 prospermatogonia. Immunofluorescence staining of Bnc2+/+ − − and Bnc2 / testis for the indicated combinations of VASA and the meiotic proteins SYCP3, STRA8 and Ki-67. DNA is stained with DAPI (blue). (A,B) E.17.5. (A) Wild-type prospermatogonia contain VASA and no SYCP3. (B) Some − − Bnc2 / prospermatogonia contain SYCP3, which forms nuclear foci (arrowheads). (C-I) P0. (C) Wild-type prospermatogonia do − − not contain SYCP3. (D) 25-30% of Bnc2 / prospermatogonia − − contain aggregated SYCP3 (arrowheads). (E) A few Bnc2 / prospermatogonia contain threads of SYCP3, typical of zygotene (arrowheads). (F) Neither STRA8 nor SYCP3 is detected in wild- − − type testis. (G) Both proteins coexist in some Bnc2 / prospermatogonia (arrowhead). (H) Black and white DAPI staining, nuclei of prospermatogonia are clearly recognizable − − (arrow and arrowheads). (I) Bnc2 / prospermatogonia contain VASA and either SYCP3 (arrow) or Ki-67 (arrowheads), but not SYCP3 and Ki-67 together. Scale bars: 25 μm (A-D,F-I); 12.5 μm (E).

The RNA-Seq results for Dnmt3l and Stra8 were confirmed by similar to those seen in Bnc2−/− prospermatogonia (see Fig. 5B,D). using RT-qPCR. On E18.5, the level of the methylase-like RNA We thought it probable that cells with large aggregates had was approximately fourfold lower in Bnc2−/− testis than in that of accumulated SYCP3 that could not disperse in synaptonemal wild type. This difference reached tenfold on P0. On E18.5 and P0, complexes because the cells had not progressed to zygotene. These the levels of the Stra8 mRNA were ∼15- and 3.5-fold higher in spermatocytes did not contain activated caspase 3 and therefore Bnc2−/− embryos than in wild-type embryos, respectively, were not apoptotic (supplementary material Fig. S4E,F). We (supplementary material Fig. S5). Therefore, BNC2 is necessary conclude that in the 9-day-old Bnc2−/− mouse, the entry of for proper male differentiation. spermatogonia into meiosis is greatly accelerated, but the cells are then blocked or delayed at the leptotene-zygotene transition. Large numbers of leptotene-blocked spermatocytes accumulate in prepubertal Bnc2−/− mice BNC2 is required for meiotic progression of spermatocytes Even though the Bnc2 mutation is overwhelmingly lethal We then compared the Bnc2−/− and Bnc2+/− testes of 1-month-old (Vanhoutteghem et al., 2009), we obtained in the course of this mice. Staining with HE showed that all Bnc2+/− tubules contained study six Bnc2−/− males that survived the perinatal period because basal spermatogonia, several layers of spermatocytes and groups of their palates closed. Because BNC2 is found in undifferentiated spermatids towards the lumen (Fig. 7A). By contrast, the Bnc2−/− spermatogonia, we thought that it might have a function in tubules could be divided into type I tubules that contained only spermatogenesis. basal cells on most of their circumference (Fig. 7B), and type II We first examined 9-day-old males. At this age, the germ cell tubules that were stratified and sometimes contained typical population comprises spermatogonia and spermatocytes at the pachytene spermatocytes, often located in the center of the tubule initial stages of meiosis (Bellvé et al., 1977; Goetz et al., 1984). instead of in their normal peripheral location (Fig. 7C,D). No Undifferentiated spermatogonia were identified by double staining spermatids were seen in any of the Bnc2−/− tubules. Type I tubules for BNC2 and PLZF, and spermatocytes by the presence of SYCP3. represented 46% of total tubes (n=100). In 9-day-old wild-type mice, numerous PLZF-containing In order to clarify the nature of the cells present in Bnc2−/− undifferentiated spermatogonia were visible in the basal layer of tubules, we first characterized undifferentiated spermatogonia by the tubules (Fig. 6A-C). The number of these spermatogonia was using PLZF staining. Heterozygous testes contained an average of not markedly different in the Bnc2−/− littermates (Fig. 6D-F). In 1.3 PLZF-positive spermatogonia per tubule (Fig. 7E); a similar contrast to the similarity of the PLZF staining, the SYCP3 staining number was found in Bnc2−/− tubules (Fig. 7F). We then identified differed between Bnc2−/− and wild-type testis. In the latter, only mitotic and meiotic cells by double staining for Ki-67 and SYCP3. ∼12% of the tubules had started producing spermatocytes, in which In the Bnc2+/− testis, tubules generally possessed a small number of SYCP3 had mostly formed threads typical of the zygotene stage basal cells that were positive for Ki-67 and a suprabasal layer of (Scherthan et al., 1996) (Fig. 6G). In Bnc2−/− testes, over 80% of the meiotic cells with filamentous SYCP3 (Fig. 7G). In the Bnc2−/− tubules contained vast numbers of SYCP3-positive cells, most of testis, tubules were smaller and could be divided into two types, which showed large aggregates of SYCP3; no zygotene which were identified by using HE staining. Type I tubules spermatocytes were observed (Fig. 6H). These aggregates were contained only basal cells, virtually all of which had nuclear Ki-67 DEVELOPMENT

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(Fig. 7K), a marker of differentiating spermatogonia (Yoshinaga et al., 1991). The differentiating Bnc2−/− spermatogonia were nearly all engaged in the mitotic cycle as they contained both nuclear Ki-67 and histone H3 phosphorylated on serine residue 10, a protein that is strictly associated with the chromosomal condensation that occurs during mitosis and meiosis (Goto et al., 1999; Gurley et al., 1978) (Fig. 7L). Bnc2−/− type II tubules showed intense staining of SYCP3 and γ-H2AX over several suprabasal layers. Both SYCP3 and γ-H2AX had a distribution similar to that of leptotene-zygotene spermatocytes, except that the staining was much more intense (compare Bnc2−/− spermatocytes in Fig. 7M,N with normal leptotene-zygotene spermatocytes indicated by the arrowhead in Fig. 7I). We presumed that these cells were abnormal spermatocytes blocked in leptotene-zygotene (Liebe et al., 2006; Mahadevaiah et al., 2001). In other tubules, the cells in the luminal region showed filamentous SYCP3 and XY bodies typical of pachytene spermatocytes (Fig. 7N,O). We were puzzled by the existence of two kinds of tubules in the mutant testis, and we questioned whether, in type I tubules, spermatogonia had been unable to form spermatocytes or whether they had formed spermatocytes that subsequently underwent apoptotic death. To investigate apoptosis, TdT-mediated dUTP- biotin end labeling (TUNEL) was used. A low level of apoptotic death was observed in heterozygous testis (approximately one cell per five tubules; see Fig. 8A). By contrast, ∼15% of the mutant littermate tubules contained many apoptotic cells (Fig. 8B) that accumulated in the suprabasal layers (Fig. 8C). Staining for activated caspase 3 confirmed the results of the TUNEL assay Bnc2−/− Fig. 6. Prepubertal mice possess spermatogonial stem cells but (Fig. 8D,E). We interpret the findings as follows: during accumulate large numbers of abnormal leptotene spermatocytes. −/− − − Immunofluorescence staining of testes of 9-day-old Bnc2+/+ and Bnc2 / mice spermatogenesis in Bnc2 mice, spermatocytes enter meiosis for BNC2, PLZF and SYCP3. DNA is stained with DAPI (blue). (A-F) Double but undergo apoptotic death sometime during meiotic prophase. exposure of BNC2 and PLZF. (A-C) In wild-type testis, undifferentiated Death of the spermatocytes during stages I-V of the cycle of the spermatogonia contain both BNC2 and PLZF (arrowheads). A,B show single seminiferous epithelium (Oakberg, 1956) results in type I tubules, − − colors, C shows the merged image. (D-F) In Bnc2 / testis, undifferentiated which contain only spermatogonia until stage VI, when new spermatogonia contain PLZF (arrowheads) but no BNC2. Their number is not preleptotene spermatocytes are formed that enter leptotene in stage obviously different from that of wild-type testis. D,E show single colors, F VIII (Fig. 7M) and pachytene at the end of stage XII (Fig. 7N), thus shows the merged image. (G,H) Staining for SYCP3. (G) In wild-type testis, a few tubules have formed meiotic cells, identified by the presence of SYCP3. generating type II tubules. The situation observed here is analogous − − The inset shows that most spermatocytes are in zygotene. (H) Bnc2 / testis to that of the SYCP3 mutant, in which tubules containing only is smaller than wild-type testis (in keeping with the reduced overall size of Sertoli cells and spermatogonia form through massive apoptotic − − Bnc2 / mice) and contains a large numbers of cells with staining of SYCP3, death during meiosis prophase (Yuan et al., 2000). mostly in the form of large aggregates (inset), presumably spermatocytes μ arrested at leptonema. Scale bars: 25 m. BNC2 is necessary to maintain the pool of PLZF-positive spermatogonia and no SYCP3 and therefore were dividing. The situation was Finally, we examined 2-month-old Bnc2−/− mice. In the wild-type reversed for type II Bnc2−/− tubules, which contained very few Ki- testis, all tubules contained densely packed germ cells at all stages of 67-positive cells, but numerous basal and suprabasal SYCP3- differentiation (Fig. 9A). Undifferentiated spermatogonia were positive cells (Fig. 7H). detected by the presence of PLZF (Fig. 9B). In the Bnc2−/− testis, The stage of meiosis of Bnc2+/− and Bnc2−/− cells was some seminiferous tubules lacked germ cells altogether, whereas determined by double staining for SYCP3 and phosphorylated others retained a homogenous population of cells that appeared to be histone H2AX (γ-H2AX). Phosphorylation of H2AX is required for germ cells. Neither spermatocytes nor spermatids were observed the formation of γ-H2AX foci at meiotic double-strand breaks (Fig. 9C). No PLZF-positive cells were detected among the 300 (Mahadevaiah et al., 2001). γ-H2AX produces a weak diffuse Bnc2−/− tubules that we examined (Fig. 9D). The presumed germ nuclear staining in type A spermatogonia (Hamer et al., 2003), cells of Bnc2−/− mice did not look like tumor cells, and how forms multiple foci in leptotene and zygotene spermatocytes and these cells could have persisted in the absence of PLZF-positive concentrates in a unique XY body in pachytene spermatocytes. spermatogonia in 2-month-old mice is unclear. Younger Bnc2−/− Bnc2+/− testis contained numerous pachytene spermatocytes, mice still retain spermatogonial stem cells, and the presumed located suprabasally; in some tubules, leptotene and zygotene Bnc2−/− germ cells may have been produced from these earlier spermatocytes were detected in the basal layer (Fig. 7I). In type I spermatogonial stem cells before their pool had been exhausted. tubules of Bnc2−/− testes, most of the cells showed weak diffuse nuclear γ-H2AX staining and no SYCP3 staining, and therefore Testicular somatic cell development does not require BNC2 were type A spermatogonia (Fig. 7J). The majority of these Because BNC2 was found in nonsteroidogenic interstitial cells –/– spermatogonia were differentiating because they contained c-KIT (Fig. 3C), we examined these cells in the Bnc2 mice. We detected DEVELOPMENT

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−/− − − − Fig. 7. Abnormal meiotic progression in 1-month-old Bnc2 males. (A-D) HE-stained sections of Bnc2+/ and Bnc2 / testis. (A) A typical seminiferous − − − tubule in Bnc2+/ mouse, spermatogonia, primary spermatocytes and spermatids are visible. (B-D) Tubules of Bnc2 / testis. (B) Type I tubule with mostly basal cells. (C) Stratified type II tubule with no typical meiotic spermatocytes. (D) Stratified tubule with pachytene spermatocytes in the luminal space instead of in their − − normal suprabasal position. Spermatids are absent from all Bnc2 / tubules. (E-O) Immunofluorescence staining. (E,F) Staining for PLZF. Undifferentiated − − − − spermatogonia (arrowheads) are present in similar numbers in Bnc2+/ (E) and Bnc2 / tubules (F). (G,H) Double staining for SYCP3 and Ki-67. (G) In Bnc2+/ − − testis, tubules generally contain a small number of cycling cells in basal layer (Ki-67), and suprabasal meiotic cells (SYCP3). (H) In Bnc2 / mouse, tubules are smaller. Type I tubules contain a large proportion of Ki-67-positive cycling cells and no cells with SYCP3, whereas type II tubules possess very few cycling − cells and many meiotic cells. (I) Bnc2+/ testis stained for SYCP3 and γ-H2AX showing a typical tubule with suprabasal pachytene spermatocytes, recognized by the presence of filamentous SYCP3 and the concentration of γ-H2AX in XY bodies; part of an adjacent tubule with leptotene spermatocytes in the basal layer is − − − − visible (arrowhead). (J-O) Bnc2 / tubules. (J-L) Type I Bnc2 / tubules. (J) Cells do not contain SYCP3 and show weak diffuse nuclear staining for γ-H2AX, characteristic of spermatogonia. (K) Such spermatogonia are identified as differentiating by the presence of c-KIT. (L) Nearly all differentiating spermatogonia − − are multiplying as they contain Ki-67 and histone H3 phosphorylated on serine residue 10. DNA is not stained in J-L. (M-O) Type II Bnc2 / tubules. (M) In − − most type II tubules, an abundance of diffuse SYCP3 and γ-H2AX identifies cells as spermatocytes that are blocked in leptotene. (N) Other type II Bnc2 / tubules have a ring of abnormal leptotene spermatocytes and a luminal space containing pachytene spermatocytes. (O) Some type II tubules possess simple epithelium comprising spermatogonia surrounding pachytene spermatocytes. Scale bars: 25 μm (A-F,I-O); 100 μm (G,H). no abnormalities of interstitial cells in either Bnc2−/− fetuses or Bnc2–/– mice. The mRNA for the androgen receptor was not newborns. We also examined Sertoli cells by using both significantly affected by the absence of BNC2 (supplementary immunostaining of anti-Müllerian hormone in paraffin-embedded material Table S2). Therefore, lack of BNC2 appeared to affect sections and RT-qPCR analysis of the Sertoli cell markers Cyp26b1, predominantly, if not exclusively, the germ cell lineage. Fgf9 and Sox9. We detected no overt defect in Bnc2−/− Sertoli cells on either E14.5 (Fig. 10A-C) or E18.5 (Fig. 10D-F). We found no Germ cell BNC2 is male specific abnormalities in the number or the morphology of Leydig cells that We finally asked whether Bnc2 was expressed in female germ cells. we identified by histological staining for 3-β-hydroxysteroid We first studied postmigratory germ cells at the time of their sexual dehydrogenase. RT-qPCR analysis of Leydig cell markers determination. Male and female primordial germ cells were purified detected a decrease in insulin-like 3 mRNA on E14.5 and inhibin on E12.5 and E13.5 by using magnetic-activated cell sorting and βA mRNA on E18.5. The other Leydig cell-specific mRNAs tested stage-specific embryonic antigen 1 (FUT4 – Mouse Genome (Cyp11a1, Star and Notch) were unaffected (Fig. 10G-L). Informatics) (Solter and Knowles, 1978). Microarray analysis In the RNA-Seq analysis, markers specific for Sertoli cells showed that the Bnc2 mRNA was much more abundant in male showed almost no difference between Bnc2−/− and Bnc2+/+ newborn than in female germ cells. The difference in Bnc2 expression was mice. Myoid cell markers were also relatively unaffected, except for comparable to that of Nanos2 (Fig. 11A), a marker of male actin α2. Leydig cell-specific mRNAs tended to be increased in the primordial germ cells (Tsuda et al., 2003). DEVELOPMENT

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−/− Fig. 9. Absence of spermatogonial stem cells in 2-month-old Bnc2 mice. (A,C) HE-stained sections. (B,D) Fluorescence staining for PLZF and DNA. (A) Tubules in wild-type mouse contain spermatogonia, spermatocytes, round spermatids and elongated spermatids. (B) Immunostaining for PLZF identifies undifferentiated spermatogonia in the basal layer (arrowheads). − − (C) Bnc2 / tubules contain a homogenous population of germ cells with intermediate-size nuclei and chromatin granules. (D) No PLZF-containing −/− −/− spermatogonia are detected in Bnc2 mice. Scale bars: 25 μm. Fig. 8. Apoptosis of spermatocytes in the testis of 1-month-old Bnc2 mice. (A-C) Immunofluorescent staining using the TUNEL assay. (A) Very little apoptotic death is seen in heterozygous mouse. (B) A mutant littermate possesses a much smaller testis with massive apoptotic death in some zinc-fingers, but whether BNC2 is a transcription factor remains to seminiferous tubules. (C) TUNEL-positive cells are suprabasal. (D,E) Staining be determined. for activated caspase 3. (D) No caspase 3-containing cells are detected in − − heterozygous mice. (E) Apoptotic cells accumulate in ∼15% of Bnc2 / Does BNC2 act directly on male germ cells? tubules. Scale bars: 50 μm. In testis, BNC2 is synthesized by germ cells and nonsteroidogenic interstitial cells. This raises the question of whether BNC2 acts on We then examined fetal ovaries by immunostaining for BNC2 germ cells directly or through interstitial cells. We did not find any and VASA. On E14.5, BNC2 was undetectable in ovarian germ evidence that the number or morphology of interstitial cells was cells (Fig. 11B), whereas it was abundant in male germ cells markedly affected by a lack of BNC2. Two somatic cell types that (Fig. 11C). On P0, double staining for BNC2 and SYCP3 showed could affect germ cells are Leydig and Sertoli cells, but BNC2 was that oocytes did not contain detectable levels of BNC2 and that undetectable in both. Neither of the two main sertolian regulators of entry into meiosis had not been affected by lack of the protein germ cell commitment, Fgf9 and Cyp26b1, was affected by the lackof (Fig. 11D-H). BNC2, and RNA-Seq analysis did not detect meaningful alterations in the number of Leydig or Sertoli cell-specific transcripts in mice DISCUSSION that lacked BNC2. The very strong expression of Bnc2 in BNC2 as a DNA-binding protein with variable numbers of prospermatogonia, particularly at the time of sexual differentiation zinc-fingers on E14.5, and the precise regulation of its expression during The mouse Bnc2 transcript is subject to alternative splicing. As a spermatogenesis further support the notion of a cell-autonomous result, the mouse, like the human (Vanhoutteghem and Djian, phenotype. 2007), possesses a major BNC2 isoform with six zinc-fingers, and The nature of the nonsteroidogenic interstitial cells that less abundant isoforms with different N-terminal sequences and synthesize BNC2 is not clear. Nonsteroidogenic interstitial cells variable numbers of zinc-fingers. Alternative-splicing events include macrophages, myoid cells and mesenchymal cells (Skinner, conserved between human and mouse are likely to be of primary 1991). Because BNC2 has been found in several kinds of biological importance (Yeo et al., 2005). Therefore, both the more mesenchymal cells, it is possible that BNC2 also resides in and the less abundant mouse BNC2 isoforms that are shared with mesenchymal cells in the testis, where it might have a function the human are likely to be functionally important. The in cell multiplication, as it does in the developing palate and urethra pleiomorphism of the BNC2 effect might be explained by (Bhoj et al., 2011; Vanhoutteghem et al., 2009). the existence of multiple isoforms of the protein, as well as by the presence of multiple zinc-finger pairs that each potentially binds to a The function of BNC2 in meiosis and mitosis of male germ different target sequence. In mouse testis, BNC2 is found in the cells soluble nuclear extract and associated with chromatin. Such a BNC2 appears to have functions in both prospermatogonia and distribution certainly suggests that BNC2 binds to DNA through its spermatogonia, and these functions are all related to mitosis DEVELOPMENT

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Fig. 10. Absence of an alteration of Sertoli and Leydig −/− cells in Bnc2 fetal testis. Immunohistochemistry (A-E,G-K) and RT-qPCR analyses (C,F,I,L) of fetal testes for somatic cell markers. (A-F) Sertoli cells. (G-L) Leydig cells. (A-C,G-I) E14.5, (D-F,J-L) E18.5. (A) On E14.5, wild-type testis shows an abundance of BNC2 in prospermatogonia but no BNC2 in Sertoli cells that were − − identified by the presence of AMH. (B) At E14.5, Bnc2 / testis shows prospermatogonia with no BNC2. Sertoli cells are not visibly affected. (C) RT-qPCR analysis of Sertoli cell markers shows no alteration in the expression of these − − markers in Bnc2 / testes. (D-F) Similar results are obtained at E18.5. (G) Wild-type Leydig cells possess − − 3βHSD but no BNC2. (H) Bnc2 / Leydig cells are not obviously affected. (I) The level of insulin-like 3 (Insl3) − − mRNA is decreased in Bnc2 / Leydig cells but not those of inhibin βA(Inhba), Star or Notch RNAs. (J-L) Similar results are obtained at E18.5, except that Inhba instead − − of Insl3 mRNA is decreased in Bnc2 / Leydig cells. Error bars show means±s.d., aP<0.06; *P<0.05. Scale bars: 25 μm.

and meiosis. In contrast to Bnc2−/− spermatocytes, Bnc2−/− meiotic the presence of the indicated antibody and then overnight at 4°C. prospermatogonia do not show evidence of apoptotic cell death, Membranes were washed and incubated for 1 h at room temperature with possibly because prospermatogonia are not part of a cycling a horseradish peroxidase-conjugated secondary antibody. The membranes seminiferous epithelium. Apoptosis of abnormal meiotic cells is were then washed and the blots developed by using chemiluminescence thought to result from a stage IV paracrine signal that is derived from (SuperSignal West Femto Maximum Sensitivity Substrate, Pierce). Further detail is given in the supplementary material methods and antibodies are Sertoli cells. Such a signal is unlikely to exist in the non-cycling listed in supplementary material Table S4. epithelium of fetal and neonatal testis. Many defects in either DNA repair or meiotic pairing cause apoptosis at a checkpoint in stage IV RT-PCR of the epithelial cycle (Hamer et al., 2008). This raises the question For RNA preparation, testes were pulverized under liquid nitrogen. RNA of a possible function of BNC2 in meiotic pairing, recombination or was extracted using the Tripure isolation reagent (Roche). Reverse synapsis. Such a function would have to be indirect because BNC2 transcriptase (RT)-PCR was performed using the superscript first-strand is absent from meiotic cells. synthesis system for RT-PCR (Life Technologies). First strand synthesis was initiated from total RNA (5 μg) with either oligo(dT) or a Bnc2-specific MATERIALS AND METHODS primer (supplementary material Table S3). The cDNAs were then subjected Subcellular fractionation and western blotting to 30 cycles of amplification (95°C for 1 min, 57°C for 1 min and 72°C for Testes were frozen in liquid nitrogen and homogenized on ice in a tissue 1 min) using various combinations of primers (supplementary material grinder using ten strokes. Cytoplasmic and nuclear extracts were prepared Table S3). Sequencing was performed by Eurofins MWG Operon. RT-PCR using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce). We was also used to generate the lentiviral expression vectors, detailed used the Subcellular Protein Fractionation Kit for Tissues (Pierce) for information is given in the supplementary material methods. subcellular fractionation. The residual pellet was thoroughly mixed with Laemmli buffer (Laemmli, 1970). The protein concentration was RT-qPCR determined using a Bio-Rad protein assay. Proteins were then resolved by Gonads were frozen in RLT buffer (Qiagen), and total RNA was extracted electrophoresis and electroblotted onto nitrocellulose membranes. The using the RNeasy Mini-Kit (Qiagen). RNA (200 ng) was reverse transcribed membranes were blocked for 1 h at room temperature, incubated for 1 h in using the High Capacity cDNA Reverse Transcription Kit (Applied DEVELOPMENT

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Fig. 11. Sexual dimorphism of Bnc2 expression in fetal germ cells, and the absence of an ovarian phenotype in −/− Bnc2 newborn mice. (A) The relative Bnc2 and Nanos2 mRNA levels in male and female germ cells of wild-type mice during the period of germ cell determination. Sorted germ cells were analyzed by using microarray hybridization. White and black columns correspond to female and male germ cells, respectively. Data are expressed in arbitrary units (A.U.) as a function of a common external reference composed of male and female gonads (equal to zero). Bnc2 is differentially expressed in male germ cells on E12.5, and this differential expression becomes more pronounced on E13.5. Differential Bnc2 expression is comparable to that of Nanos2. Error bars show means±s.d., n=4, **P<0.01, ***P<0.001, Student’s t-test. (B) Double staining of wild-type paraffin sections of ovary and testis at E14.5 for BNC2 and VASA shows an absence of detectable BNC2 in oocytes. (C) Abundant nuclear BNC2 protein is detected in prospermatogonia. (D-F) Double staining of wild-type newborn ovary for BNC2 and SYCP3 demonstrates that primordial oocytes, identified by the presence of filamentous SYCP3, do not possess BNC2. Only somatic cells contain BNC2. D,E show single colors, F shows the merged image. − − (G,H) Staining of wild-type and Bnc2 / newborn ovary for SYCP3. (G) Wild-type ovary possesses numerous peripheral oocytes with filamentous SYCP3 that is typical − − of pachytene. (H) Bnc2 / oocytes have also engaged normally in meiosis. Scale bars: 25 μm.

Biosystems) and amplified by using RT-qPCR as previously described except for the anti-γ-H2AX antibody, which was incubated for 2 h at room (Souquet et al., 2012). Either β-actin or Vasa mRNA were included as an temperature. Secondary antibodies were incubated for 1 h at room endogenous reporter. Results are presented as a fraction of the maximum. temperature. The final concentration of all primers was 400 nM. Samples were run in Immunohistochemical analyses were performed as previously described duplicate. (Messiaen et al., 2013). Antibodies are listed in supplementary material Table S4. RNA-Seq In situ analysis of DNA fragmentation was performed using the ApopTag Total RNA was prepared as described under RT-PCR. Sequencing was Fluorescein In Situ Apoptosis Detection Kit (Chemicon International), carried out by Eurofins MWG Operon. according to the manufacturer’s recommendations. Acquisition of images by using microscopy is described in the Histological analysis supplementary material methods. For HE staining, samples were either embedded in paraffin or frozen in optimal cutting temperature (OCT) compound. For paraffin embedding, Acknowledgements We are very grateful to one of the reviewers for insights into the interpretation of the samples were fixed in 4% paraformaldehyde, washed in PBS, embedded in − − different types of tubules found in the adult Bnc2 / mice. paraffin and sectioned at 7 µm. For frozen sections, samples were successively incubated in 4% paraformaldehyde for 1 h, PBS containing Competing interests 15% sucrose for 3 h and PBS containing 30% sucrose overnight, and snap- The authors declare no competing financial interests. frozen in OCT (Miles). Indirect immunofluorescence was performed as previously described Author contributions (Vanhoutteghem and Djian, 2007) with some modifications, described in μ A.V., S.M., F.H., B.D., D.M. and P.D. performed experiments. A.V., F.H., V.R.-F., G.L. the supplementary material methods. Briefly, frozen sections (6 m) were and P.D. prepared the manuscript. J.-M.P. prepared the figures. fixed in 4% formaldehyde for 5 min on ice and permeabilized using 0.2% Triton X-100, or fixed in acetone without permeabilization. For primary Funding polyclonal antibodies, 5% BSA in PBS was used for blocking. For mouse This study was funded by the Association pour la Recherche sur le Cancer, the monoclonal antibodies, MOM (Vector Laboratories) was used for blocking. Ligue contre le Cancer, Centre national de la recherche scientifique (CNRS) and

Incubations with primary antibodies were performed overnight at 4°C, Institut national de la santéet de la recherche médicale (INSERM). DEVELOPMENT

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Supplementary material P. S. (2001). Recombinational DNA double-strand breaks in mice precede Supplementary material available online at synapsis. Nat. Genet. 27, 271-276. http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.112888/-/DC1 Mahoney, M. G., Tang, W., Xiang, M. M., Moss, S. B., Gerton, G. L., Stanley, J. R. and Tseng, H. (1998). Translocation of the zinc finger protein basonuclin from the References mouse germ cell nucleus to the midpiece of the spermatozoon during spermiogenesis. Biol. Reprod. 59, 388-394. Aapola, U., Shibuya, K., Scott, H. S., Ollila, J., Vihinen, M., Heino, M., Shintani, Majdic, G., Saunders, P. T. and Teerds, K. J. (1998). Immunoexpression of the A., Kawasaki, K., Minoshima, S., Krohn, K. et al. (2000). Isolation and initial steroidogenic enzymes 3-beta hydroxysteroid dehydrogenase and 17 alpha- characterization of a novel zinc finger gene, DNMT3L, on 21q22.3, related to the hydroxylase, C17,20 lyase and the receptor for luteinizing hormone (LH) in the cytosine-5-methyltransferase 3 gene family. Genomics 65, 293-298. fetal rat testis suggests that the onset of Leydig cell steroid production is Bellvé, A. R., Cavicchia, J. C., Millette, C. F., O’Brien, D. A., Bhatnagar, Y. M. and independent of LH action. Biol. Reprod. 58, 520-525. Dym, M. (1977). Spermatogenic cells of the prepuberal mouse: isolation and Mark, M., Jacobs, H., Oulad-Abdelghani, M., Dennefeld, C., Féret, B., Vernet, N., morphological characterization. J. Cell Biol. 74, 68-85. Codreanu, C.-A., Chambon, P. and Ghyselinck, N. B. (2008). STRA8-deficient ̈ Bhoj, E. J., Ramos, P., Baker, L. A., Cost, N., Nordenskjold, A., Elder, F. F., Bleyl, spermatocytes initiate, but fail to complete, meiosis and undergo premature S. 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