DEVELOPMENTAL DYNAMICS 243:1448–1456, 2014 DOI: 10.1002/DVDY.24166

PATTERNS & PHENOTYPES

Telomere Distribution Pattern and Synapsis Initiation During Spermatogenesis in Zebrafish a

Kenji Saito,1 Chiharu Sakai,1 Toshihiroa Kawasaki,1 and Noriyoshi Sakai1,2* 1

Genetic Strains Research Center, National Institute of Genetics, Mishima, Japan Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Mishima, Japan

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2

Background: Telomeres are located at ends of eukaryotic chromosomes and can affect proper chromosomal positioning. During spermatogenesis, the appropriate dynamics and behavior of chromosomes is crucial to generate haploid cells through meiosis. Here, we describe telomere distribution patterns during spermatogenesis in zebrafish, especially during meiotic prophase I, using fluorescence in situ hybridization. This was combined with synaptonemal complex protein 3 immunostaining, which allows the staging of spermatocytes. Results: During spermatogonial proliferation and the preleptotene stage, telomeres were dispersed throughout the nucleus. During the leptotene stage, telomeres temporarily moved to one pole of the nucleus at which g-tubulin was located, forming the telomere bouquet. The cluster lasted until the onset of zygotene where it coincided with terminal synapsis initiation. They then spread around the periphery of the nucleus during the zygotene to pachytene stages. During postmeiotic stages, telomeres in spermatids and sperm were again dispersed throughout the nuclei. Application of this procedure in meiotic mutants confirmed that meiotic telomere clustering is independent of axial element formation of the synaptonemal complex. Conclusions: These data clearly showed the clustering and distributions of telomeres throughout spermatogenesis in zebrafish. This procedure could be used to screen for mutants that have primary defects in C 2014 Wiley Periodicals, Inc. telomere clustering. Developmental Dynamics 243:1448–1456, 2014. V Key words: telomere bouquet; meiosis; Sycp3; meiotic mutant Submitted 18 December 2013; First Decision 27 June 2014; Accepted 3 July 2014; Published online 16 July 2014

Introduction Meiosis is a specialized cell division process that occurs in all organisms with a sexual life cycle. It differs from mitosis because germ cells divide twice after a single DNA replication event, by first separating homologous chromosomes and then separating sister chromatids. During meiotic prophase I, pairing of homologous chromosomes is associated with intranuclear redistribution of chromosomes and a synaptonemal complex (SC) forms between each pair of homologous chromosomes, which is involved in regulating homologous recombination (Roeder, 1997). Telomeres are specialized structures at the ends of linear eukaryotic chromosomes and are composed of tandem repeats of a hexanucleotide (TTAGGG) sequence in vertebrates. Telomeres are essential for the normal genomic and genetic stability of cells by protecting chromosomes from terminal fusions or degradation that typically occurs during the DNA replication cycle (Greider, 1996; Ducray et al., 1999; Feldser et al., 2003). In addition, studies of mammalian cells have recently revealed that telomeric DNA repeats associate with the six-protein complex shelterin, *Correspondence to: Noriyoshi Sakai, Genetic Strains Research Center, National Institute of Genetics, 1111 Yata, Mishima 411–8540, Japan. E-mail: [email protected] Grant sponsor: Grant-in-Aid from the Ministry of Education, Culture, Sport, Science, and Technology, Japan; Grant numbers: 23013023; 25251034

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which prevents the activation of DNA damage signaling pathways (de Lange, 2009). Telomeres are scattered throughout the nucleus in somatic cells (Vourc’h et al., 1993; Luderus et al., 1996; Scherthan et al., 1996). However, when cells enter meiosis, telomeres relocate to the nuclear envelope and attach to the nucleoplasmic face of the inner nuclear membrane by means of their axial element end (Liebe et al., 2004). During the leptotene-zygotene transition, telomeres embedded in the inner nuclear membrane move into a polarized configuration called the bouquet and cluster around the centrosome (Scherthan, 2001; Harper et al., 2004). Telomere clustering and nuclear movement facilitate the alignment of homologous chromosomes, and promote their pairing and recombination in fission yeast (Ding et al., 2004). Loss of telomere clustering results in reduced homologous recombination and increased missegregation (Cooper, 2000; Yamamoto and Hiraoka, 2001). In mice, targeted disruption of the Sun1 or Terb1 genes, which are required for telomere attachment to the nuclear envelope, prevents telomere clustering (or chromosome movement) and efficient homologue pairing/synapsis in meiosis, which causes deficiencies in growing and mature oocytes and postmeiotic spermatogenic cells (Ding et al., 2007; Shibuya et al., 2014). Thus, telomere clustering is an important initial step in meiotic progression and germ cell development. However, its precise Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy. 24166/abstract C 2014 Wiley Periodicals, Inc. V

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Fig. 1. FISH using a Cy3-conjugated (CCCTAA)3 PNA telomere probe in metaphase cells and pachytene spermatocytes. A–C: FISH using a telomere probe in metaphase chromosomes prepared from the cells of a 1-day-old zebrafish embryo. Panel A shows FISH signals, panel B shows nuclear staining with TO-PRO-3, and panel C shows a merged image of panels A and B. More than 90% of telomeres were clearly detected. D–F: FISH using a telomere probe in combination with immunocytochemical labeling of Sycp3 in pachytene spermatocyte spreads. Panel D shows FISH signals, panel E shows Sycp3 signals, and panel F shows a merged image of panels D and E. Telomere signals were detected at both ends of Sycp3-positive structures. Scale bars ¼ 10 mm.

function in meiotic progression remains obscure, especially in vertebrates. We recently isolated several zebrafish mutants that have defects in spermatogenesis using an N-ethyl-N-nitrosourea mutagenesis screen and a three-generation mating scheme (Saito et al., 2011). The mutant phenotypes in testicular sections indicated that meiotic defects are the most common abnormalities in these mutants. Identification of mutations that cause abnormalities in telomere dynamics among these mutants would facilitate understanding of the role of telomeres in the pairing of homologous chromosomes in vertebrates. As an initial step to identify mutants with defects in telomere dynamics, we visualized and characterized the distribution of telomeres during spermatogenesis in zebrafish, especially during meiotic prophase I. This was combined with analysis of SC protein 3 (Sycp3) expression patterns, which allows the exact staging of spermatocytes. In addition, we examined telomere behavior in spermatocytes of two previously described mutants, its and imo. Both mutants are sterile: the its mutant arrests at the preleptotene stage of meiotic prophase I, whereas the imo mutant arrests at the zygotene stage (Saito et al., 2011). This analysis showed that telomere clustering occurs independently of the formation of axial elements of the SC, allowing screening for mutants that have primary defects in telomere clustering.

Results Dynamic Behavior of Telomeres During Zebrafish Spermatogenesis We initially examined the specificity of the fluorescence in situ hybridization (FISH) telomere probe using zebrafish cells. Meta-

phase chromosome spreads were prepared from 1-day-old embryonic cells and hybridized with the Cy3-labeled (CCCTAA)3 probe. Fluorescent signals were detected at both ends of the chromosomes in sister chromatids (Fig. 1A–C). More than 90% of the telomeres were observed. In addition to metaphase chromosome spreads, we examined the specificity of this probe in surfacespread spermatocytes isolated from adult zebrafish testis by performing FISH with this probe in combination with immunocytochemical labeling of Sycp3. Using this technique, specific fluorescent signals of the telomere probe were detected at both ends of Sycp3-positive structures in the SC (Fig. 1D–F). Next, we performed FISH using the telomere probe on cryosections of a testis to analyze the distribution of telomeres in germ cells during zebrafish spermatogenesis. The zebrafish testis is composed of cyst-like structures that contain germ cells undergoing spermatogenesis. Within a cyst, spermatogenesis occurs synchronously; therefore, each cyst contains only spermatogonia, spermatocytes, or spermatids. The location of telomere signals in germ cells was different within each cyst (Fig. 2). To identify each developmental stage of germ cells, we performed FISH using the telomere probe in combination with immunostaining for Sycp3 and green fluorescent protein (GFP) in vas::egfp transgenic zebrafish (Krovel and Olsen, 2002). The zebrafish homologue of the Drosophila vasa gene is an informative marker of spermatogonia. Hence, the cytoplasmic localization of GFP in spermatogonia enabled these cells to be identified and distinguished from other germ cells in vas::egfp transgenic zebrafish. Sycp3 is not only a marker of all primary spermatocytes, but is also used as a stage-specific marker based on its localization patterns (Ashley, 2004; Ozaki et al., 2011; Saito et al., 2011). We performed antibody staining following FISH with the telomere probe. Immunostaining for GFP revealed that telomeres were dispersed

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Fig. 2. Overall image of FISH signals using a telomere probe in zebrafish testis. A–C: FISH using a telomere probe in cryosections of a sexually mature testis. Panel A shows FISH signals, panel B shows nuclear staining with TO-PRO-3, and panel C shows a merged image of panels A and B. D: Same area viewed in panels A–C stained with periodic acid–Schiff (PAS) and hematoxylin. Zebrafish germ cells develop synchronously within each cyst (outlined in panel B). These cysts are formed by Sertoli cells in the tubular basement membrane, which is stained by PAS. Telomeres in the germ cells exhibit different patterns in each cyst. Scale bar ¼ 50 mm.

throughout the nuclei in spermatogonia (Fig. 3A–C). Sycp3 was first detected in spermatocytes at the preleptotene stage, which were identified by the presence of one or more intranuclear Sycp3 aggregates (Fig. 3D). Such aggregates mark the onset of prophase in spermatocytes in mice and cattle (Scherthan et al., 1996; Pfeifer et al., 2001, 2003). Similar to spermatogonia, telomeres were dispersed throughout the nuclei of preleptotene spermatocytes (Fig. 3E,F). In spermatocytes at the leptotene or the early zygotene stage, Sycp3 was detected as short fine threads, which localized at one pole of the nucleus (Fig. 3G). Unlike in spermatogonia and preleptotene spermatocytes, telomeres in leptotene spermatocytes were observed at the pole of the nucleus at which Sycp 3-positive short threads were localized (Fig. 3H,I). Later in meiosis, during the pachytene stage, telomeres relocated over the entire nuclear periphery (Fig. 3J–L). In spermatids, telomeres were scattered throughout the nucleus again (Fig. 3M–O), similar to their localization in sperm (Fig. 3P–R).

Clustering and Redistribution of Telomeres in Meiotic Prophase in Zebrafish Telomeres had a dynamic distribution pattern during meiotic prophase in cryosections of testis; therefore, we confirmed the

distribution of telomeres by performing FISH using a telomere probe and immunocytochemical labeling of Sycp3 in chromosome spreads of spermatocytes. As was the case in cryosections, telomeres were scattered throughout the nuclei in preleptotene spermatocytes (Fig. 4A). In early leptotene spermatocyte nuclei, which were identified by the presence of intranuclear Sycp3 aggregates and the accumulation of Sycp3 at one pole of the nucleus, some telomeres were localized at the nuclear periphery together with Sycp3, while other telomeres were still intranuclear and were not localized in any particular region (Fig. 4B). This indicates that telomeres clustering initiates during the leptotene stage in zebrafish. In spermatocytes at the early zygotene stage, which were identified by the presence of thick Sycp3positive threads at the bouquet base, telomere were clustered over a slightly wider area and associated with these Sycp3positive lateral elements (Fig. 4C). Spermatocytes at the zygotene stage were identified by the appearance of distinct elongated threads of Sycp3-positive lateral elements (Fig. 4D). Some telomeres remained clustered at a small region of the nuclear periphery, while others were dispersed over the nuclear periphery (Fig. 4D). Spermatocytes at the pachytene stage were distinguished from spermatocytes at earlier stages by the appearance

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To further confirm the localizations of telomeres, we obtained lateral images of spermatocytes by performing three-dimensional (3D) reconstruction of serial sections obtained using confocal laser microscopy. Such 3D reconstruction along the vertical axis provided an image of the outline of the nucleus and the FISH signals of the telomere probe. In spermatocytes at the leptotene stage, telomeres were clustered at one region of the nuclear periphery, giving rise to a bouquet configuration (Fig. 4F), while in spermatocytes at the zygotene stage, telomeres were scattered over the nuclear periphery (Fig. 4G).

Determination of the Zygotene Stage According to the Expression Pattern of Sycp1

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We used the patterns of Sycp3-positive lateral elements in the SC to stage meiotic prophase I. Therefore, we examined the expression pattern of Sycp1, a component of transverse filaments, to determine the zygotene stage. In chromosome spreads of spermatocytes, Sycp1 signals were not detected in the preleptotene (Fig. 5A) or early leptotene (Fig. 5B) stages. In similar spermatocytes in which Sycp3-positive threads at the bouquet base were thick as shown in Figure 4C, weak signals of Sycp1 were detected (Fig. 5C,H,N,R). Thus, we confirmed that the early zygotene stage is shown in Figure 4C. At the zygotene and pachytene stages, Sycp1 signals completely co-localized with Sycp3-positive threads (Fig. 5D,E,I,J,M,O).

Cytoplasmic Localizations of a-Tubulin and g-Tubulin During Meiotic Prophase To better understand where telomeres cluster in spermatocytes during the leptotene stage, the localizations of the microtubule proteins a-tubulin and g-tubulin were investigated. g-Tubulin is a conserved component of the centrosome (Stearns et al., 1991) and a-tubulin is assembled into microtubules. g-Tubulin was observed at the site of telomere clustering in leptotene spermatocytes (Fig. 6A). a-Tubulin was also restricted to a small section upon formation of the bouquet configuration in leptotene spermatocytes (Fig. 6B,C). In zygotene spermatocytes, cytoplasmic atubulin spread over the surface of the nucleus (Fig. 6D). These results suggest that telomeres cluster near to the centrosome in zebrafish spermatocytes. Fig. 3. Distribution and assembly of telomeres in zebrafish spermatogenic cells. A–C: Telomeres in spermatogonia. Spermatogonia were identified by EGFP expression in vas::EGFP transgenic zebrafish testis. Panel A shows anti-GFP antibody staining (green), panel B shows FISH signals using a telomere probe (magenta), and panel C shows a merged image of panels A and B. D–L: Telomeres in meiotic spermatocytes. Spermatocytes at preleptotene (D–F), leptotene or early zygotene (G–I), and pachytene (J–L) stages were identified according to their Sycp3 expression patterns. Panels D, G, and J show anti-Sycp3 antibody staining (green); panels E, H, and K show FISH signals using a telomere probe (magenta); and panels F, I, and L show merged images. Nuclei were stained with TO-PRO-3 and are displayed in blue (A–L). M–R: Distributions of telomeres in spermatids (M–O) and sperm (P–R). Panels M and P show nuclear staining with TO-PRO-3, panels N and Q show FISH signals using a telomere probe, and panels O and R show merged images (TO-PRO-3, green; FISH signal, magenta). Scale bar ¼ 10 mm.

of short and thick threads of Sycp3-positive lateral elements (Fig. 4E). In this stage, telomeres were dispersed over the nuclear periphery and did not cluster.

Behavior of Telomere Clustering in the Zebrafish Meiotic Mutants, Its and imo Two meiotic mutants of zebrafish have been isolated, namely, its, which has a defect in the preleptotene stage, and imo, which has a defect in the zygotene stage (Saito et al., 2011). We have not identified the mutated genes in these mutants or the functional consequences of these mutations. Thus, we analyzed patterns of telomere clustering and distribution in these mutants. Staging of spermatocytes in these mutants was achieved by immunostaining for Sycp3. In its spermatocytes in Figure 7, Sycp3 staining showed the pattern at the preleptotene stage as shown in Figure 4A and that described previously (Saito et al. 2011). In some spermatocytes of this mutant, telomeres were scattered throughout the nucleus, which is the same pattern as that observed at the preleptotene stage (Fig. 7A,B), while in other spermatocytes, telomeres were clustered at the nuclear pole, which is the same pattern as that observed at the leptotene

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stage (Fig. 7C,D). In these spermatocytes of the its mutant (Fig. 7C,D), telomere clustering occurred before axial element formation of the SC, suggesting that this mutant does not have a primary defect in telomere clustering and that telomere clustering occurs independently of the formation of axial elements of the

SC. In spermatocytes of the imo mutant, the patterns of telomere clustering and distribution and Sycp3 staining were the same as in wild-type spermatocytes, except that spermatocytes of this mutant did not reach the later stages of pachytene (Fig. 8). Therefore, it is likely that the machinery involved in the clustering and distribution of telomeres operates normally until the zygotene-pachytene transition in the imo mutant.

Discussion

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In this study, we investigated the distribution of telomeres during spermatogenesis in zebrafish by performing FISH using a telomere probe, in combination with monitoring GFP expression patterns in vas::egfp transgenic zebrafish, which provides a marker of spermatogonia, and Sycp3 expression patterns, which allows the exact staging of spermatocytes. The results clearly showed the clustering and distributions of telomeres throughout spermatogenesis in zebrafish, from the mitotic phases of spermatogonia to sperm. The movement of telomeres during spermatogenesis observed in this study is in agreement with that observed in previous histological studies of mammals, apart for the timing of telomere clustering and telomere distributions during postmeiotic stages. In addition, we investigated the localizations of a-tubulin and g-tubulin in zebrafish spermatocytes to observe the centrosome. The results suggest that telomere clustering in zebrafish meiosis occurs close to the centrosome, as it does in mammals (Tomita and Cooper, 2006). In spermatocytes at the preleptotene stage, telomeres were dispersed throughout nuclei similar to their distribution in mitotic spermatogonia, as has been reported in mammals (Scherthan et al., 1996; Pfeifer et al., 2001, 2003). During early leptotene, some telomeres moved into the region of the nuclear periphery at which short Sycp3-positive threads were gathered. Thereafter, telomeres remained clustered until the initiation of synapsis in the early zygotene stage, during which Sycp1-positive transversal elements were detected. These results suggest that telomere clustering in zebrafish is established during the leptotene stage. Telomere clustering (bouquet formation) in mammalian spermatocytes occurs a little later, where it coincides with

Fig. 4.

Fig. 4. Analysis of the dynamics of meiotic telomeres in zebrafish by performing FISH using a telomere probe in combination with immunocytochemical labeling of Sycp3. Telomeres were analyzed in chromosome spreads of spermatocytes isolated from adult testis. A–E: Immunocytochemical labeling of Sycp3 in combination with FISH using a telomere probe in spermatocytes at preleptotene (A), early leptotene (B), early zygotene (C), zygotene (D), and pachytene (E) stages. Left panels show Sycp3 staining (green), middle panels show FISH signals (magenta), and right panels show merged images of the left and middle panels. Nuclei were stained with TO-PRO-3 (blue). F,G: Threedimensional analysis of meiotic cells. Serial optical sections were obtained using confocal laser microscopy, and 3D images were reconstructed using software attached to the microscope after double staining of Sycp3 and telomeres in spermatocytes at leptotene (F) and zygotene (G) stages. Left panels show representative examples of single plane images obtained from the serial sections. Sycp3 and telomere signals are displayed in green and magenta, respectively. Middle and right panels show axial and lateral views, respectively, of the 3D reconstructed images. Telomere signals and nuclear staining are displayed in magenta and green, respectively. In the right panels, the surface of the glass slide is represented by a white dashed line. Bars ¼ 10 mm.

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Fig. 5. Immunocytochemical analyses of Sycp1 and Sycp3 in primary spermatocytes. Localization of Sycp1 (A–E), Sycp3 (F–J), merged Sycp1 and Sycp3 staining (K–O), and merged Sycp1, Sycp3, and nuclear staining with TO-PRO-3 (P–T) are shown at the preleptotene (A,F,K,P), early leptotene (B,G,L,Q), early zygotene (C,H,N,R), zygotene (D,I,M,S), and pachytene (E,J,O,T) stages. Each developmental stage of spermatocytes was identified according to their synaptonemal complex (SC) structures. Bar ¼ 10 mm.

synapsis initiation at the leptotene-zygotene transition (Scherthan et al., 1996; Pfeifer et al., 2001, 2003). Medaka exhibits the same distribution of Sycp3 as that reported here, in which Sycp3 foci are detected as short fine threads that emanate from one pole of the nucleus (Iwai et al., 2006). Telomere bouquet formation may start at the leptotene stage in teleosts. In spermatids and sperm of zebrafish, telomere signals showed a scattered distribution throughout the nucleus (Fig. 3). These observations are in agreement with the telomere distribution patterns in rat spermatids (Meyer-Ficca et al., 1998). By contrast, in mice, most telomeres assemble around the nucleolus in the center of the nucleus at the round spermatid stage, and then disperse throughout the nuclei after the elongating spermatid stage (Tanemura et al., 2005). We are unable to explain this inconsistency at present, although it may be owing to species-specific differences. Furthermore, Zalenskaya and Zalensky (2002) reported that telomeres are positioned at the nuclear periphery in sperm in mammals, which is inconsistent with the telomere distribution in zebrafish sperm. We observed telomere signals in thin (8-mmthick) sections of the testis. It is possible that this underlies why signals were detected at different positions in the sperm nuclear envelope in the current study. Further detailed studies and those

using different species would help to better understand this difference and its biological significance. The present study analyzed telomere clustering in two meiotic mutants as a pilot experiment to screen for mutants that have primary defects in telomere clustering. Telomere clustering occurred before formation of axial elements of the SC in the its mutant, which arrests at the preleptotene stage, suggesting that this mutant does not have a primary defect in telomere clustering. Axial element formation of the SC is dispensable for the attachment of meiotic telomeres to the nuclear envelope (for review, see Scherthan, 2007). Thus, FISH using a telomere probe in combination with analysis of Sycp3 expression patterns will help to screen for mutants with defects in the clustering and redistribution of telomeres. We have already isolated many meiotic mutants of zebrafish by using N-ethyl-Nnitrosourea-mutagenesis screening (Saito et al., 2011). Screening of these mutants using the aforementioned FISH approach could identify mutants with defects in telomere dynamics, thereby helping to dissect the pathways that regulate meiotic telomeres. Many molecules that affect telomere clustering have been identified, mainly from mutants of fission yeast (Schizosaccharomyces pombe) and budding yeast (Saccharomyces cerevisiae) that have telomere clustering defects. For example, Taz1 (Cooper

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Fig. 6. Localizations of a-tubulin and g-tubulin in spermatocytes. A: FISH using a telomere probe in combination with immunocytochemical labeling of g-tubulin. B: FISH using a telomere probe in combination with immunocytochemical labeling of a-tubulin. C,D: Double immunocytochemical labeling of a-tubulin and Sycp3 at the early zygotene (C) and zygotene (D) stages. a-Tubulin staining expands during the zygotene stage. Left panels show telomere or Sycp3 staining (magenta), middle panels show g-tubulin or a-tubulin staining (green), and right panels show merged images of the left and middle panels. Nuclei were stained with TO-PRO-3 (blue). Scale bar ¼ 10 mm.

et al., 1998), Rap1 (Chikashige and Hiraoka, 2001; Kanoh and Ishikawa, 2001), Kms1 (Shimanuki et al., 1997), Bqt1, Bqt2 (Chikashige et al., 2006), and Ndj1 (Trelles-Sticken et al., 2000) are required for the tethering of telomeres. Bouquet formation is a widespread phenomenon among eukaryotes; however, only homologues of Taz1 (termed Trf1 and Trf2) and Rap1 (termed Rap1) were identified in a blast search of zebrafish sequences, whereas homologues of Kms1, Bqt1, Bqt2, and Ndj1 were not identified. There is a large phylogenetic distance between yeast and vertebrates; therefore, proteins that have a low amino acid sequence homology to these yeast proteins might perform the same functions in telomere clustering. In addition, analyses of Rap1-deficient mice revealed that the role of Rap1 in meiosis is not conserved between fission yeast and mammals, suggesting that telomeres are tethered by alternative means in mammals (Scherthan et al., 2011). Phenotype-driven screening for zebrafish mutants that have meiotic prophase I defects will help to identify molecules that function in telomere clustering, as well as molecules that have a low sequence similarity to these yeast proteins but which perform the same functions.

Fig. 7. Analysis of the distribution of telomeres by performing FISH using a telomere probe in combination with immunocytochemical labeling of Sycp3 in spermatocytes of the its zebrafish mutant. A: Nucleus of a preleptotene spermatocyte from a wild-type zebrafish exhibits one intranuclear Sycp3 aggregate. B: Telomeres are dispersed throughout the nucleus of a preleptotene spermatocyte from an its mutant zebrafish, similar to the wild-type preleptotene spermatocyte (A). C,D: Another spermatocyte from an its mutant zebrafish exhibits aggregated Sycp3 and is most likely at a preleptotene-equivalent stage, while telomeres are restricted to a small region of the nuclear periphery. In panel C, telomeres are close to Sycp3, whereas in panel D, they are not. Left panels show Sycp3 staining (green), middle panels show FISH signals (magenta), and right panels show merged images of the left and middle panels. Nuclei were stained with TO-PRO-3 (blue). Scale bar ¼ 10 mm. These images are representative of 20 nuclei analyzed at each stage per specimen (n ¼ 3).

Experimental Procedures Fish Maintenance Zebrafish were bred and maintained according to standard procedures (Westerfield, 1995). Wild-type experimental fish were the India strain. The transgenic vas::EGFP zebrafish line expresses enhanced green fluorescent protein (EGFP) under the control of the zebrafish vas promoter (Krovel and Olsen, 2002). In the testis of these transgenic fish, EGFP is highly expressed in spermatogonia and its expression decreases as spermatogenesis progresses. The use of zebrafish for experimental purposes was conducted in accordance with the guidelines of the National Institute of Genetics, and all procedures were approved by the Committee for Animal Care and Use of the National Institute of Genetics (An approval number; 24-12).

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filtered through a 40 mm nylon mesh to obtain a suspension of single cells. The cells were placed onto Matsunami adhesivecoated slides (Matsunami Glass, Osaka, Japan) and fixed in PBS containing 1% paraformaldehyde for 10 min at room temperature. Slides were then incubated in PBS containing 0.1% Tween 20 for 10 min and dehydrated in an alcohol series before analysis. For surface-spread spermatocytes, slides were treated with 85 mM NaCl for 30 min before fixation.

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FISH Analysis

Fig. 8. Analysis of the distribution of telomeres by performing FISH using a telomere probe in combination with immunocytochemical labeling of Sycp3 in spermatocytes of the imo zebrafish mutant. FISH and labeling of Sycp3 were performed in the imo spermatocytes at leptotene (A), early zygotene (B), and zygotene (C) stages. A: Telomeres localize within a small region of the nuclear periphery and form a bouquet configuration at the leptotene stage. B: At the early zygotene stage, telomeres start to move over the nuclear periphery. C: Telomeres disperse over the nuclear periphery at the zygotene stage. Left panels show Sycp3 staining (green), middle panels show FISH signals (magenta), and right panels show merged images of the left and middle panels. Nuclei were stained with TO-PRO-3 (blue). Scale bar ¼ 10 mm. These images are representative of 20 nuclei analyzed at each stage per specimen (n ¼ 3).

Sample Preparation For metaphase chromosome spreads, cells were prepared from 1day-old zebrafish embryos. Dechorionated embryos were dissociated with 0.05% trypsin-EDTA solution (Gibco). After dissociation, the cells were exposed to demecolcine (0.2 mg/ml) in zebrafish growth medium (Hashiguchi et al., 2008) for 1 hr to increase the proportion of cells in metaphase. Cells were fixed and slides were prepared as described previously (Kimmel et al., 1995). For FISH using a telomere probe and immunohistochemical staining of fresh-frozen sections, the testis were directly embedded in optimal cutting temperature compound (Sakura Finetechnical, Tokyo, Japan) and frozen in liquid nitrogen. Samples were then sectioned (8 mm thick) and fixed with phosphate buffer saline (PBS) containing 1% paraformaldehyde for 10 min at room temperature. Slides were incubated in PBS containing 0.1% Tween 20 for 10 min and dehydrated in an alcohol series before analysis. For FISH using the telomere probe in combination with immunocytochemistry, or for immunocytochemistry with two antibodies, samples were prepared as previously described (Iwai et al., 2006) with some modifications. Testes were minced with scissors and dissociated into single cells by incubating in Leibovitz’s L-15 medium (Gibco) containing 500 U/ml of collagenase (Sigma) for  30 min at 28 C with pipetting every 10 min. The cells were then

Telomeres were labeled with a Cy3-conjugated (CCCTAA)3 peptide nucleic acid (PNA) probe (Panagene, Daejeon, Korea). After rehydration of sections or cells in an alcohol series, the hybridization solution (70% formamide, 20 mM Tris-HCl [pH 7.2], and 0.5% bovine serum albumin) containing the Cy3-conjugated PNA telomere probe (0.2 mg/ml) was added. Samples were dena tured at 82 C for 10 min using a heat block, and then incubated  overnight at 37 C in the dark. After incubation, samples were washed three times in 70% formamide and 20 mM Tris-HCl (pH 7.2) for 15 min, and then washed twice more in PBS. When both FISH and immunostaining were performed, FISH was performed before antibody detection. Samples were counterstained with TOPRO-3 (Invitrogen, Carlsbad, CA) and mounted using Vectashield (Vector Laboratories, Burlingame, CA). FISH images and immunostaining images were obtained using an LSM-510 confocal laser microscope (Carl Zeiss, Germany) by sequential scanning, and analyzed using software attached to this microscope. Threedimensional reconstruction of the images was performed using the same software.

Immunostaining Nonspecific protein-binding sites were blocked by incubating samples in PBS containing 1% bovine serum albumin for 30 min at room temperature. After incubation, samples were incubated over night at 4 C in a moist chamber with the following primary antibodies that had been diluted in blocking solution: rabbit polyclonal anti-zebrafish Sycp3 antibody at 1:300 dilution (Ozaki et al., 2011), rabbit polyclonal anti-GFP antibody at 1:200 dilution (598; MBL, Nagoya, Japan), mouse monoclonal anti-a-tubulin antibody clone DM 1A at 1:500 dilution (T9026; Sigma-Aldrich, St. Louis, MO), and mouse monoclonal anti-g-tubulin antibody clone TU-32 at 1:2,000 dilution (11–543; EXBIO Praha, Vestec u Prahy, Czech Republic). A rat polyclonal anti-zebrafish Sycp1 antibody was prepared using recombinant proteins of zebrafish sycp1 cDNA fragments that were amplified with specific primers (50 TTAGGATCCATGCAAAAAGCATTCAACTT-30 and 50 - TATGGTACCGGTAACTTCTATTTCTGCAT-30 ) as previously reported (Ozaki et al., 2011). After purification and detection of its reactivity by Western blot analysis, the antibody was used at a dilution of 1:300. Samples were washed three times in PBS, and then incubated for 1 hr at room temperature with goat anti-rabbit-IgG conjugated to Cy3 (Jackson Laboratories, ME) and donkey anti-rabbitIgG conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, OR), which had been diluted 1:500 in PBS. All antibodies were tested in individual staining reactions to verify their specificities and performance. No staining was detected in controls samples in which the primary antibodies were omitted (unpublished results). Samples were counterstained with TO-PRO-3 (Invitrogen, Carlsbad, CA) and mounted using Vectashield (Vector Laboratories,

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Burlingame, CA). Immunostained images were obtained using an LSM-510 confocal laser microscope (Carl Zeiss, Germany) by sequential scanning and analyzed using software attached to this microscope. Some sections were stained with periodic acid–Schiff and hematoxylin after telomere fluorescence signals had been examined, and this staining was observed using an Olympus BX51 microscope (Olympus, Tokyo, Japan).

Acknowledgments We thank Ms. E. Ashikawa, N. Kimura, M. Kojima, and N. Suzuki for maintaining the zebrafish stocks.

DEVELOPMENTAL DYNAMICS

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