TYPE Original Research PUBLISHED 12 October 2022 DOI 10.3389/fcell.2022.1009776 Meiotic chromosome dynamics OPEN ACCESS and double strand break EDITED BY Mónica Pradillo, Complutense University of Madrid, formation in reptiles Spain REVIEWED BY Laia Marín-Gual 1,2†, Laura González-Rodelas 1,2†, Miguel Angel Brieño-Enriquez, Magee-Womens Research Institute, Maria M. Garcias 1,2, Lukáš Kratochvíl 3, Nicole Valenzuela 4, United States Arthur Georges 5, Paul D. Waters 6 and Aurora Ruiz-Herrera 1,2* Sean Burgess, 1 University of California, United States Departament de Biologia Cel·lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain, 2Genome Integrity and Instability Group, Institut de Biotecnologia i *CORRESPONDENCE Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain, 3Department of Aurora Ruiz-Herrera, Ecology, Faculty of Science, Charles University, Prague, Czech Republic, 4Department of Ecology, aurora.ruizherrera@uab.cat Evolution, and Organismal Biology, Iowa State University, Ames, IA, United States, 5Institute for Applied † These authors contributed equally to Ecology, University of Canberra, Canberra, ACT, Australia, 6School of Biotechnology and Biomolecular this work and share first authorship Sciences, Faculty of Science, UNSW, Sydney, NSW, Australia SPECIALTY SECTION This article was submitted to Nuclear Organization and Dynamics, a section of the journal During meiotic prophase I, tightly regulated processes take place, from pairing Frontiers in Cell and Developmental Biology and synapsis of homologous chromosomes to recombination, which are RECEIVED 02 August 2022 essential for the generation of genetically variable haploid gametes. These ACCEPTED 23 September 2022 processes have canonical meiotic features conserved across different PUBLISHED 12 October 2022 phylogenetic groups. However, the dynamics of meiotic prophase I in non- CITATION mammalian vertebrates are poorly known. Here, we compare four species from Marín-Gual L, González-Rodelas L, Sauropsida to understand the regulation of meiotic prophase I in reptiles: the M. Garcias M, Kratochvíl L, Valenzuela N, Georges A, Waters PD and Australian central bearded dragon (Pogona vitticeps), two geckos (Paroedura Ruiz-Herrera A (2022), Meiotic picta and Coleonyx variegatus) and the painted turtle (Chrysemys picta). We first chromosome dynamics and double strand break formation in reptiles. performed a histological characterization of the spermatogenesis process in Front. Cell Dev. Biol. 10:1009776. both the bearded dragon and the painted turtle. We then analyzed prophase I doi: 10.3389/fcell.2022.1009776 dynamics, including chromosome pairing, synapsis and the formation of double COPYRIGHT strand breaks (DSBs). We show that meiosis progression is highly conserved in © 2022 Marín-Gual, González-Rodelas, M. Garcias, Kratochvíl, Valenzuela, reptiles with telomeres clustering forming the bouquet, which we propose Georges, Waters and Ruiz-Herrera. This promotes homologous pairing and synapsis, along with facilitating the early is an open-access article distributed pairing of micro-chromosomes during prophase I (i.e., early zygotene). under the terms of the Creative Commons Attribution License (CC BY). Moreover, we detected low levels of meiotic DSB formation in all taxa. Our The use, distribution or reproduction in results provide new insights into reptile meiosis. other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the KEYWORDS original publication in this journal is reptile, meiosis, gametogenesis, micro-chromosomes, DSBs, recombination, bouquet cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Introduction Meiosis is used by all sexually reproducing organisms to form haploid gametes (oocytes or sperm) via two consecutive cell divisions preceded by one round of genome replication. This follows a tightly regulated progression of chromosome condensation and folding, coupled with changes to the epigenome and gene expression (Hammoud et al., 2014; Alavattam et al., 2019; Patel et al., 2019; Vara et al., 2019; Vara and Ruiz-Herrera, Frontiers in Cell and Developmental Biology 01 frontiersin.org Marín-Gual et al. 10.3389/fcell.2022.1009776 2022). Meiosis generates genetically variable gametes by Meiotic prophase I is commonly subdivided into four recombination of the two parental chromosomes during different stages (leptotene, zygotene, pachytene and diplotene) prophase I. This involves faithful chromosome synapsis, the based on the dynamics of meiotic chromosomes and their formation of double strand breaks (DSBs) and DNA exchange telomeres (reviewed in Bolcun-Filas and Handel, 2018). The (crossovers, COs) between homologues. pairing of homologous chromosomes begins at leptotene with FIGURE 1 Phylogeny of the reptiles included in the study. (A) Phylogenetic relationships of the four reptilian species included in the study. For each phylogenetic branch, variation in diploid numbers and sex determination system are indicated. (B) Mitotic karyotypes of the four studied species: Pogona vitticeps, Paroedura picta, Chrysemys picta and Coleonyx variegatus. The bearded dragon (P. vitticeps) and the painted turtle (C. picta) karyotypes include macro- and micro-chromosomes, whereas the western banded gecko (C. variegatus) and the ocelot gecko (P. picta) karyotypes include chromosomes of progressively smaller size. Karyotypes correspond to mitotic metaphases from fibroblast primary cell cultures. GSD: genotypic sex determination; TSD: temperature sex determination. Frontiers in Cell and Developmental Biology 02 frontiersin.org Marín-Gual et al. 10.3389/fcell.2022.1009776 the formation of a protein scaffold along chromosomes and the painted turtle (Chrysemys picta) as a representative of composed of cohesins and proteins specific to the Testudines (turtles). These species are emerging models for synaptonemal complex (SC). This coincides with the thermal and reproductive physiology (Valenzuela, 2009; generation of DSBs by the endonuclease protein SPO11 Starostová et al., 2013; Kubička et al., 2015), as well as (Keeney et al., 1997). Telomeres play an important role developmental biology (Noro et al., 2009). The three lizards during the leptotene-zygotene transition, clustering to form a have genotypic sex determination (GSD). The bearded dragon structure known as the bouquet (Scherthan et al., 1996; Liebe has ZW sex chromosomes (Ezaz et al., 2005; Koubová et al., et al., 2004; Reig-Viader et al., 2013). At zygotene, DSBs are 2014), whereas the western banded gecko and the ocelot gecko repaired, leading to their resolution as either COs or non-COs GSD systems are still unknown (Rovatsos et al., 2019; Keating (NCOs) between sister chromatids. It is not until pachytene that et al., 2022). However, the genetic sex determination of the chromosomes are completely synapsed and COs are resolved as bearded dragon can be overridden by temperature to produce chiasmata (the points where genetic material is actually viable ZZ females (Quinn et al., 2007; Holleley et al., 2015). Most exchanged). The mechanisms underlying meiotic progression turtle species have temperature-dependent sex determination have been extensively studied in several model organisms, (TSD, including C. picta), although XY and ZW systems are including yeast, fruit flies, nematodes, mice and zebrafish also present in different lineages (Bista and Valenzuela, 2020) (Zickler and Kleckner, 2015; Blokhina et al., 2019; Imai et al., (Figure 1). 2021). However, our understanding of the dynamics of meiotic Our study unveils shared features between bearded dragon prophase I and recombination among non-mammalian amniote and painted turtle spermatogenesis. We also observed that all vertebrates (i.e., sauropsids–birds/reptiles) remains incomplete reptiles examined here present an equivalent pattern of prophase (Segura et al., 2013; Marín-Gual et al., 2022). I progression forming the bouquet at early stages, where Amniote vertebrates shared a last common ancestor homologous micro-chromosomes synapse first and cluster approximately 325 mya (Shedlock and Edwards, 2009) together. Remarkably we detected low rates of DSB formation (Figure 1) and are characterized by distinctive chromosome in reptiles when compared to mammals, suggesting that low morphology and evolutionary labile sex determination. recombination rates are a distinctive feature of reptiles. Sauropsids display variation in chromosome number, especially in birds (2n = 40–138), although this is less pronounced in reptiles (2n = 22–68) (Ruiz-Herrera et al., Material and methods 2012; Montiel et al., 2016; Waters et al., 2021). The non-avian sauropsids (reptiles) are composed of Squamata (lizards and Samples snakes), Sphenodontia (tuatara), Crocodilia (crocodiles and alligators), and Testudines (turtles). Reptiles are characterized Male bearded dragons (n = 4, P. vitticeps) were obtained from by the presence of generally well conserved micro- and macro- captive colonies in Canberra (ACT, Australia) at the end of the chromosomes (Waters et al., 2021) and by a high variability in breeding season (February). Male ocelot geckos (n = 3, P. picta) their sex-determining systems (i.e., ZZ/ZW, XX/XY or and male western banded geckos (n = 1, C. variegatus) were temperature sex determination - TSD) (Ezaz et al., 2006) originated from breeding colonies in Charles University in Prague (Figure 1). While meiotic progression in the chicken has been (Czech Republic). Male painted turtles (n = 3, C. picta) were wild- studied and mirrors eutherians (Schoenmakers et al., 2009; caught in Iowa (United States) at the end of the breeding season Guioli et al., 2012), little is known about meiosis in reptiles. under appropriate permits from Iowa’s DNR. The few existing reports focused on CO formation (Lisachov et al., 2017, 2019; Spangenberg et al., 2021) and formation of unreduced eggs in parthenogenetic lineages (Lutes et al., 2010), Primary fibroblast cell culture and but whether meiotic progression in reptiles resembles the process karyotyping described for either mammals or zebrafish (which last shared a common ancestor with amniotes approximately 400 mya) is Four primary fibroblast cell lines were derived from all reptile currently unknown. species studied. Samples of connective tissue were washed in Here we provide a comparative analysis of key features of 1xPBS supplemented with an antibiotic-antimycotic solution spermatogenesis and meiotic prophase I progression in (100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml previously uncharacterised reptile linages, with a focus on gentamicin and 0.25 μg/ml amphotericin B). Cultures were meiotic recombination. We examined the ocelot gecko established by disaggregating tissue with a scalpel blade and (Paroedura picta) and the western-banded gecko (Coleonyx resuspending cells in AmnioMAX. Cell cultures were incubated variegatus) as representatives of Gekkota (geckos), the at 28°C in 5% CO2. Australian central bearded dragon (Pogona vitticeps) as a For karyotyping, cells were arrested in metaphase by adding representative of Iguania (iguanas, agamids and chameleons), 80 μl of Colcemid (10 μg/ml) to 10 ml of medium for 2 h and Frontiers in Cell and Developmental Biology 03 frontiersin.org Marín-Gual et al. 10.3389/fcell.2022.1009776 then trypsinised. Cells were centrifuged down at 600 xg for 5 min counterstained with anti-fade solution (Vectashield) and resuspended in 5 ml of hypotonic solution (0.075M KCl) for containing 8 μg/ml DAPI (4′,6′-diamidino-2-phenylindole). 30 min at 37°C. Chromosomes were then fixed by addition of fixative solution (3:1 methanol/acetic acid) and metaphase spreads were obtained by dropping 15 µl of cell suspension Microscopy and image analysis onto a cleaned dry slide. Slides were baked at 65°C for one hour and kept at −20°C until use. Metaphases were stained PAS-hematoxylin–stained tissue sections were analyzed on an homogenously with DAPI for the karyotype analysis. Olympus CH2 microscope, and images were captured using a Zeiss Axiophot Microscope and Olympus C5060 camera. For fluorescent sample analysis and image capturing, a Zeiss Axioskop fluorescence Histology and testis morphometry microscope connected to a ProgRes Jenoptik camera was used. The image capture software ProgRes CapturePro was employed for Testes from the bearded dragon and the painted turtle were image acquisition and image processing. collected for histological procedures. Briefly, testes were fixed The accumulation of foci in the bouquet was analyzed as the overnight in Bouin’s solution (70% saturated picric acid, 25% percentage of foci per cell located in the bouquet region, formaldehyde and 5% glacial acetic acid). Then, samples were previously delimited as the area where synaptonemal complex dehydrated, cleared and embedded in paraffin using standard (i.e., SYCP3 signal) begins to assemble and SYCP3 intensity is procedures. Sections (7 µm) were stained with PAS-hematoxylin. higher. Only cells with a well-defined bouquet were included in the analysis. Spermatocyte spreads and immunofluorescence Statistical analysis Testicular biopsies were obtained immediately after animal Statistical significance for the DSB analysis as RPA and dissection and processed as previously described (Garcia-Cruz RAD51 foci, and for the analysis of the percentage of DSB et al., 2011) in order to obtain spermatocyte spreads. Briefly, a foci in the bouquet was determined using two-sided Mann- piece of the testicular biopsy was carefully minced on a slide; 1% Whitney U-tests. The critical value for statistical significance Lipsol was added and incubated for 30 min at room was p < 0.05 for all tests. Each plot or its figure legend indicates temperature. Then, a fixative solution containing 4% the statistical methods and corresponding p-values. All boxplots paraformaldehyde was added, and slides were kept in a are represented as centre lines (median), box limits (interquartile humid chamber. After two hours, slides were washed in 1% range; 25th and 75th percentiles) and whiskers (largest and photo-flo solution and further processed for lowest data points inside the first and third quartiles plus immunofluorescence, or frozen at −20°C until use. 1.5 times the interquartile range). Immuno-staining of meiocytes was performed using the following primary antibodies: rabbit antibody against SYCP3 (#ab15093, Abcam, 1:100 dilution), rabbit antibody against Results SYCP1 (#ab15087, Abcam, 1:100 dilution), rabbit antibody against TRF2 (#NB110-57130SS, Novus Biologicals, 1: Spermatogenesis progression in the 100 dilution), mouse antibody against RNA pol II (#5408, bearded dragon and the painted turtle Abcam, 1:400 dilution), rabbit antibody against RAD51 (#PC130, Calbiochem, 1:50 dilution), rabbit antibody against We first characterized spermatogenesis progression in the RPA32/RPA2 (#10359, Abcam, 1:100 dilution), mouse antibody bearded dragon (P. vitticeps) and the painted turtle (C. picta) against MLH1 (#51–1327GR, BD PharmigenTM, 1:100 dilution), (Figure 2), following the mammalian classification of germ cell rabbit antibody against MLH1 (#ab47703, Abcam, 1: morphology (Russell et al., 1993). Both the bearded dragon 100 dilution) and rabbit antibody against γH2AX (#H5912, (Figures 2A,B) and the painted turtle (Figures 2C,D) had a Sigma-Aldrich, 1:100 dilution). histological organisation of germ cells within the seminiferous Fluorochrome-conjugated secondary antibodies were used epithelia (between the basal lamina and the lumen) that was for detection (all from Jackson ImmunoResearch similar to that of eutherian mammals (Russell et al., 1993) and Laboratories). Antibodies were diluted in PBST (Tween other amniotes (Gribbins, 2011). 0.05% in PBS). Primary antibodies were incubated In both species, spermatogonia (A and B) were restricted overnight at 4°C in a humid chamber and secondary to the basal lamina (Figures 2A,C). Type A spermatogonia antibodies for 1 h at 37°C in a humid chamber. After presented a rounded nucleus showing one nucleolus, whereas washing away the excess of secondary antibodies, DNA was type B spermatogonia contained densely stained chromatin Frontiers in Cell and Developmental Biology 04 frontiersin.org Marín-Gual et al. 10.3389/fcell.2022.1009776 FIGURE 2 Testis histology. Histological cross-sections of seminiferous tubules of (A) the bearded dragon and (C) the painted turtle stained with PAS- hematoxylin. Dashed circles and arrowheads point clusters of different cell types. Scale bars: (i) 200 μm, (ii) 100 μm, and (iii, iv) 20 μm. Germ cell types found within the seminiferous epithelium and their progression in (B) the bearded dragon and (D) the painted turtle. Scale bar: 10 μm. Legend type: Spg A, type A spermatogonia; Spg B, type B spermatogonia; L, leptotene spermatocyte; Z, zygotene spermatocyte; P, pachytene spermatocyte; D, diplotene spermatocyte; MI, metaphase I; Sp II, secondary spermatocyte; RS, round spermatid; ES, elongating spermatid; Sp Z, spermatozoa. and more than two nucleoli (between 2-4 nucleoli) (Figures clumps of condensed filamentous chromatin within the 2B,D). Large populations of cells subsequently progress nucleus. Pachytene spermatocytes displayed an open through meiosis towards the centre of the seminiferous nucleoplasm and their nuclei contained thick chromatin tubule. Meiotic cells were characterized by an increase in fibres. Finally, diplotene spermatocytes had chromatin fibres size and condensed chromatin. This included recognizable in a tight circle and degenerating nuclear membranes. We also stages of prophase I: leptotene, zygotene, pachytene and distinguished meiotic cells with the chromosomes fully diplotene (Figure 2). Both first and second meiotic divisions condensed and aligned at the metaphase plate. During the and secondary spermatocyte stages occurred rapidly, as all second meiotic division, secondary spermatocytes contained three phases were found in low proportions in cross-sections randomly dispersed chromatin fibres (Figures 2B,D). of seminiferous tubules (Figures 2A,C). Leptotene Spermiogenesis (the differentiation and maturation of spermatocytes were distinguished by dense filamentous sperm) encompasses a longer period than previous stages as chromatin at the nuclei. Zygotene spermatocytes exhibited large populations of round and elongating spermatids were Frontiers in Cell and Developmental Biology 05 frontiersin.org Marín-Gual et al. 10.3389/fcell.2022.1009776 FIGURE 3 Synapsis dynamics during prophase I. Spermatocyte spreads labelled with antibodies against SYCP3 (green) and SYCP1 (red), counterstaining the DNA with DAPI (blue) for (A) the bearded dragon and (B) the ocelot gecko. Scale bar: 10 μm. White dashed circle: cluster of micro-chromosomes. Frontiers in Cell and Developmental Biology 06 frontiersin.org Marín-Gual et al. 10.3389/fcell.2022.1009776 FIGURE 4 Telomere dynamics during prophase I. Spermatocyte spreads labelled with antibodies against SYCP3 (green) and TRF2 (red), counterstaining the DNA with DAPI (blue) for (A) the bearded dragon, (B) the ocelot gecko and (C) the western banded gecko. Scale bar: 10 μm and 2 μm (insets). White arrowheads: telomeres from which SC is beginning to assemble. Yellow arrowheads: completely associated micro-chromosomes (i.e., lateral elements of the SC completely assembled between both telomeric ends). Frontiers in Cell and Developmental Biology 07 frontiersin.org Marín-Gual et al. 10.3389/fcell.2022.1009776 observed (Figure 2). Spermiogenic cells were divided into three Moreover, phosphorylated RNA polymerase II (the active different stages: i) round spermatids, the smallest cell type, form of RNA pol II) was detected in spermatocytes of all four rounded with fully condensed chromatin; ii) elongating species, with increasing signal intensity through prophase I spermatids with their round nuclei and condensed chromatin (Supplementary Figure S3), mirroring therian mammals (Page becoming elongated; iii) mature sperm after the completion of et al., 2012; Marín-Gual et al., 2022) and insects (Viera et al., spermiogenesis and the elongation process was finalised. 2017). The absence of transcriptional repression of any specific pair of chromosomes during pachytene (no antibodies against γH2AX yielded any positive staining) suggested that meiotic sex Micro-chromosomes pair earlier during chromosome inactivation (MSCI) was absent in the four reptilian prophase I than macro-chromosomes species, contrasting male mammals (Turner et al., 2005; Ruiz- Herrera and Waters, 2022). The absence of MSCI is not We then analyzed the meiotic chromosome pairing strategies surprising because our reptile models either do not have sex in all four species. Chromosome pairs in all four reptile species chromosomes (the painted turtle, Valenzuela et al., 2014), or largely differ in size (Figure 1). The chromosome complement of because males are the homogametic sex (the bearded dragon, the reptiles herein varied: 2n = 32 chromosomes in the bearded Ezaz et al., 2005), or because sex chromosomes are likely poorly dragon (6 pairs of macro chromosomes and 10 pairs of micros, differentiated (the ocelot gecko and the western banded gecko, including the sex chromosomes) (Young et al., 2013), 2n = 32 in Keating et al., 2022; Rovatsos et al., 2019). the western-banded gecko (16 pairs of acrocentric chromosomes with continuous decreasing of size from large to small) (Pokorná et al., 2010), 2n = 36 chromosomes continuously decreasing in DSBs dynamics in reptiles size in the ocelot gecko (Main et al., 2012; Koubová et al., 2014) and 2n = 50 in the painted turtle (13 pairs of macro We then analyzed the dynamics of DSB formation by chromosomes and 12 pairs of micros) (Badenhorst et al., immunodetection of the recombination proteins RPA 2015) (Figure 1B). (Replication Protein A) and RAD51 (Radiation Sensitive Axial elements of the synaptonemal complex labelled with 51) (Figure 5; Supplementary Figures S4, S5) as no anti-SYCP3 were used to classify spermatocytes into the different antibodies against MLH1 yielded any positive staining in prophase I stages (leptotene, early zygotene, late zygotene and reptile spermatocytes (data non shown). RPA binds to the pachytene; Figure 3 and Supplementary Figure S1) as previously 3’ DNA strand following DSBs formation, and is subsequently described (Alavattam et al., 2018). The proportion of thick replaced by RAD51 and/or DMC1 by early prophase I (He (i.e., synapsis) and thin (i.e., unsynapsis) SYCP3 filaments et al., 1995; Keeney et al., 1997). As such, the number of RPA were used to distinguish between earlier and later stages of and RAD51 sites in early prophase is a proxy for the number of zygotene spermatocytes. Zygotene spermatocytes with ≤ 50% DSBs, as previously described for various mammalian taxa of thick SYCP3 filaments (i.e., synapsis) were classified as “early (Segura et al., 2013; Ruiz-Herrera et al., 2017; Marín-Gual stage”, whereas zygotene spermatocytes with > 50% of synapsis, et al., 2022). were classified as “late stage” (Alavattam et al., 2018). We successfully detected RPA foci in spermatocyte spreads In all four species we observed short filaments of SYCP3 at of the western banded gecko, the ocelot gecko, and the bearded leptotene, which represented the forming axial elements dragon (Figures 5A,B; Supplementary Figure S4; Table 1). (Figure 3; Supplementary Figure S1). The general trend in all Both geckos had equivalent numbers of RPA foci at four species was for the chromosomes to start pairing at one pole leptotene and early zygotene (Mann-Whitney test, p ≥ 0.05, of the cell at leptotene, forming the bouquet, from which SYCP3- Table 1). However, they had different RPA replacement positive filaments assembled from telomeres (Figure 4; dynamics, because the mean number of RPA foci was Supplementary Figure S2). As prophase I progressed, axial higher in the western banded gecko compared to the ocelot elements become larger at zygotene, when synapsis between gecko by late zygotene (Mann-Whitney test, p < 0.001) and homologous chromosomes takes place, as revealed by pachytene (Mann-Whitney test, p < 0.01) (Table 1). SYCP3 and SYCP1 labelling (Figure 3; Supplementary Figure Furthermore, the RPA loading and replacement dynamics S1) (Alavattam et al., 2018). At pachytene, autosomes had in the bearded dragon differed from both geckos, with completed synapsis. Remarkably, we found that micro- lower RPA foci per cell at early zygotene (Mann-Whitney chromosomes completed synapsis earlier than macro- test, p < 0.001), intermediate values at late zygotene (Mann- chromosomes, forming a discrete cluster (Figure 3A). This Whitney test, p < 0.001) and higher RPA foci at pachytene previously undescribed pattern was also highlighted by compared to the ocelot gecko (Mann-Whitney test, p < 0.05) TRF2 immunostaining of telomeres, which revealed that some (Figure 5B; Table 1). micro-chromosomes were fully synapsed in early zygotene Similar dynamics (i.e., a decreasing numbers of foci as (Figure 4; Supplementary Figure S2). prophase I progressed) were detected for RAD51, which we Frontiers in Cell and Developmental Biology 08 frontiersin.org Marín-Gual et al. 10.3389/fcell.2022.1009776 FIGURE 5 Double strand break formation dynamics during reptilian prophase I. (A) Late zygotene spermatocyte spreads labelled with antibodies against SYCP3 (green) and RPA (red), counterstaining the DNA with DAPI (blue) for the western banded gecko, the ocelot gecko and the bearded dragon. Scale bar: 10 μm. (B) Plot representing the number of RPA foci per cell detected at leptotene, early zygotene, late zygotene and pachytene. (C) Late zygotene spermatocyte spreads labelled with antibodies against SYCP3 (green) and RAD51 (red), counterstaining the DNA with DAPI (blue) for the painted turtle, the ocelot gecko and the bearded dragon. Scale bar: 10 μm. (D) Plot representing the number of RAD51 foci per cell detected at leptotene, early zygotene, late zygotene and pachytene. (E) Early zygotene spermatocyte spread labelled with SYCP3 (green) and RPA (red), counterstaining the DNA with DAPI (blue) in the bearded dragon. Dashed yellow circle: RPA foci detected in the bouquet. Blue circle: nuclei perimeter. (F) Plots representing the percentage of (i) RPA and (ii) RAD51 foci detected in the bouquet for the western banded gecko, the painted turtle, the ocelot gecko and the bearded dragon. Only cells with a well-defined bouquet were included in the analysis. The number of cells examined per species per stage, illustrated in panels (B,D,F), are listed in Table 1. Mann-Whitney test (*p < 0.05, **p < 0.01 and ***p < 0.001). Legend: L, leptotene; EZ, early zygotene; LZ, late zygotene; P, pachytene. Frontiers in Cell and Developmental Biology 09 frontiersin.org Marín-Gual et al. 10.3389/fcell.2022.1009776 TABLE 1 Dynamics of DSB formation during prophase I in the four reptiles included in the study. Values indicate the average number of RPA or RAD51 foci per cell immunodetected in leptotene, early zygotene, late zygotene, and pachytene, as well as the fraction of the total foci per cell detected in the bouquet. Values in parenthesis indicate the number of cells analyzed in each case. Leptotene Early zygotene Late zygotene Pachytene % foci in bouquet Mean RPA foci/cell Western-banded gecko 115 (29) 95 (20) 82 (24) 49 (17) 69 (28) Ocelot gecko 115 (30) 94 (25) 61 (27) 36 (11) 69 (29) Bearded dragon – 85 (32) 70 (32) 44 (41) 70 (32) Mean RAD51 foci/cell Painted turtle 103 (29) 75 (37) 56 (37) 22 (17) 70 (26) Ocelot gecko 65 (31) 44 (37) 31 (35) 19 (25) 66 (24) Bearded dragon 61 (17) 53 (36) 40 (24) 21 (34) 75 (15) detected in spermatocyte spreads of the painted turtle, the ocelot primarily composed of Sertoli cells and germ cells. Then, gecko and the bearded dragon (Figures 5C,D; Supplementary germ cell differentiation takes place in a continuous Figure S5; Table 1). Both lizards had equivalent values of manner with a species-specific time interval (e.g., 8.6 days RAD51 foci per cell at leptotene (Mann-Whitney test, p ≥ in mice and 16 days in humans, Russell et al., 1993). 0.05), whereas the mean was higher in the bearded dragon Both the histological and cytological characterization compared to the ocelot gecko by early zygotene (Mann- presented here for reptiles revealed that spermatogenesis Whitney test, p < 0.001) and late zygotene (p < 0.001) progression is noncystic and highly conserved with respect to (Table 1). In contrast, the painted turtle showed higher mean cell morphology and distribution. Analysis of the histological values of RAD51 foci per cell compared to both lizards at all distribution of different germ cell types within the seminiferous stages of prophase I (Man-Whitney test, p < 0.001) except in epithelia revealed that both the bearded dragon and the painted pachytene (Mann-Whitney test, p ≥ 0.05) (Figure 5D; Table 1). turtle showed similar patters to those described in eutherian Remarkably, RPA and RAD51 loading followed similar mammals (Russell et al., 1993) and other reptiles (Gribbins, 2011; dynamics, with both proteins accumulated in the bouquet Sousa et al., 2014). In fact, all amniotes described to date present region at early stages of prophase I (Figure 5E). Despite some similar testis structure and organization, although differences variation among reptile species, between 66% and 75% of the have been reported in terms of reproductive strategy and total RPA and RAD51 foci per cell were detected in the bouquet behaviour, including both continuous and seasonal breeding (Figure 5F), indicating that DSB formation initiates at telomeres. (Gribbins et al., 2003). In temperate and subtropical lizards, the testicular cycle is divided in two phases: (i) the regenerative phase, which occurs Discussion in the spring, and (ii) the degenerative phase, that begins in summer (Mayhew and Wright, 1970; Amey and Whittier, Our work represents a comparative study of the dynamics of 2000a). So, there is a cycle of hypertrophy and regression of the spermatogenic cycle and prophase I progression in reptiles, reproductive organs. Previous studies in the eastern with an emphasis on chromosome pairing and the formation and bearded dragon Pogona barbata classified testis as (i) repair of meiotic DSBs. regressed (only spermatogonia present), (ii) developing (spermatocytes or spermatids present), and (iii) spermiogenic (spermatogenesis and mature sperm present) Continuous spermatogenic cycle in the (Amey and Whittier, 2000a). Consistently, the observations bearded dragon and the painted turtle made here for the central bearded dragon agree with the observation of maximum spermatogenic activity in spring, Spermatogenesis in vertebrates follows two main followed by the cessation of spermatogenesis directly after the arrangements in the seminiferous epithelia: (i) cystic, in breeding period and testicular recrudescence in February (late which developing germ cell syncytia are individually summer). Both bearded dragons and the painted turtle are encapsulated by Sertoli cells as observed in fish and seasonal breeders (Gibbons, 1968; Amey and Whittier, 2000b) amphibians, and (ii) noncystic, where spermatogenesis takes and since samples were collected at the end of the mating place in seminiferous tubules (reptiles, birds, and mammals) season for both species, our results showed that germ cells (Schulz et al., 2010; Sousa et al., 2014). In species with enter the spermatogenic cell cycle continually during the noncystic spermatogenesis, the seminiferous epithelium is reproductive season, as all cell stages were found in the the building block of seminiferous tubules, which are seminiferous epithelium. Frontiers in Cell and Developmental Biology 10 frontiersin.org Marín-Gual et al. 10.3389/fcell.2022.1009776 Meiosis progression is highly conserved in Low meiotic DSB rates in reptiles reptiles Remarkably, reptiles showed lower levels of DSBs than We found that the progression of prophase I was highly eutherian mammals. Although variable among species, conserved among the reptiles examined. In most species, between 200 and 300 DSBs per cell (mean values) occur chromosomes were organized into chromosome axes but genome-wide during leptotene in eutherian mammals were not yet synapsed at leptotene. This was coupled with (Segura et al., 2013). Marsupials show the lowest the formation of DSBs (Zickler and Kleckner, 2015). In our recombination rates in mammals with less than 150 RPA target reptiles, chromosome axes were detected as short foci per cell in zygotene (Zenger et al., 2002; Samollow stretches of SYCP3 and SYCP1 signal adjacent to the et al., 2004; Marín-Gual et al., 2022). This contrasts with telomes, mirroring the patterns previously described in our results where fewer RPA foci (from 95 to 85 per cell) zebrafish (Blokhina et al., 2019; Imai et al., 2021). and RAD51 foci (from 75 to 44 per cell) (a proxy for DSBs) Moreover, telomeres clustered to one side of the nucleus were observed in early zygotene in reptiles. A closer inspection forming the bouquet, presumably promoting of the data revealed that squamates (geckos and the bearded homologous pairing and synapsis. The bouquet first dragon) showed equivalent values of DSBs in early stages of appeared from leptotene to late zygotene, depending on prophase I, and lower than turtles, the sister taxon to the species. This feature is shared with zebrafish, suggesting archosaurs (birds plus crocodilians). Two biological, non- that early telomere clustering is ancestral and has mutually exclusive alternatives could explain these been retained over almost 400 million years of vertebrate observations: differences in DSBs per cell observed between evolution. squamates and turtles are due to (i) contrasting chromosome Interestingly, micro-chromosomes completed synapsis number and size between the taxa examined, or (ii) these earlier than macro-chromosomes, forming discrete lineages differ in the genetic determinants of DSBs induction. clusters concurrent with the bouquet. This suggests that The first alternative agrees with previous cytological data. the bouquet facilitates SYCP3 loading and synapsis of Specifically, studies in disparate taxa show that the total number homologous chromosomes (both macro and micro- and distribution of COs (and also initial meiotic DSBs) on a chromosomes) from the telomeres towards the central specific chromosome depends on several factors, such as regions of the chromosomes, at the same time as micro- chromosome size, and an individual’s sex and age (Pardo- chromosome synapsis. Complete homologous synapsis of Manuel De Villena and Sapienza, 2001; Lynn et al., 2004; micro-chromosomes was observed from early zygotene to Garcia-Cruz et al., 2011; Ruiz-Herrera et al., 2017; Wang pachytene. This was coupled with a polarization of DSBs et al., 2019). Larger chromosomes tend to accumulate more towards telomeres, a notable difference compared to some COs (but see recent data in yeast, Murakami et al., 2021), and mammal species where DSBs are distributed more each chromosome arm generally presents at least one CO (the homogenously across the genome as the SC is being obligatory chiasmata) (Sun et al., 2017). Thus, because both assembled (Ruiz-Herrera et al., 2017; Marín-Gual et al., geckos and the bearded dragon have lower diploid numbers 2022), although DSBs and COs have been reported to be (2n = 32–36) than the painted turtle (2n = 50), differences in enriched at sub-telomeric regions in human males (Barlow meiotic DSBs (COs were not reported in this study) are expected and Hultén, 1998; Khil and Camerini-Otero, 2010; Pratto among groups. This rationale would also apply to birds, which et al., 2014). possess high chromosome numbers (typically 2n = 80, Ruiz- The clustering of telomeres during early prophase I Herrera et al., 2012; Waters et al., 2021). In birds, cytological extends previous cytological and genomic studies where analyses of COs (meiotic DSBs are unreported) are restricted to micro-chromosomes in reptiles tend to clump centrally in species from the domesticated groups Galloanserae and mitotic and meiotic metaphase I (Waters et al., 2021), Passeriformes, in which recombination rates are higher (from resulting in higher inter-chromosomal genomic interactions 1.8 cM/Mb to 2.6 cM/Mb; del Priore and Pigozzi, 2020) than between micro-chromosomes than for macro-chromosomes those reported for mammals (from 0.18 cM/Mb to 1.78 cM/Mb; (Waters et al., 2021). This results in micro-chromosomes Segura et al., 2013). Moreover, birds show little variation in forming a structural and functional domain that is recombination rates between groups (del Priore and Pigozzi, maintained in germ cells, probably facilitating homology 2020), and thus are not lineage-dependent (unlike mammals, search and DSBs formation. As most sauropsids are Segura et al., 2013), perhaps related to high genome conservation characterized by very conservative genomes (with few (Waters et al., 2021). macro-chromosomes and up to many micro-chromosomes) The second hypothesis derives from the observation that (Valenzuela and Adams, 2011; Waters et al., 2021), we recombination rates may vary by the presence of different genetic hypothesize that meiotic patterns described herein will determinants of DSBs induction, such as PRDM9 (Mihola et al., apply widely in this clade. 2009; Myers et al., 2010; Grey et al., 2011; Vara et al., 2019). Frontiers in Cell and Developmental Biology 11 frontiersin.org Marín-Gual et al. 10.3389/fcell.2022.1009776 PRDM9 is a meiotic-specific histone (H3) methyltransferase with across vertebrates will further determine the genomic basis of a C-terminal tandem repeat zinc finger (ZnF) domain that adds biodiversity, and how it may be affected by ecotoxicological and H3K4me3 marks at nucleosomes close to DSBs in early meiosis. other environmental changes. This process genetically determines recombination hotspots (Mihola et al., 2009; Baudat et al., 2010; Grey et al., 2011). Experimental work has shown that PRDM9 provokes changes Data availability statement in local chromatin structure that tend to position nucleosomes in ways that increased overall accessibility (Yamada et al., 2020). The original contributions presented in the study are Moreover, in the absence of PRDM9, DSBs tend to form in gene included in the article/Supplementary Material, further promoter regions (Brick et al., 2012; Baker et al., 2015; Lange inquiries can be directed to the corresponding author. et al., 2016). PRDM9 is present in most mammals (with the exception of canids, Muñoz-Fuentes et al., 2011), but substantial allelic variability was described in natural populations, especially Ethics statement in rodents (Buard et al., 2014; Capilla et al., 2014; Vara et al., 2019) contributing to speciation (Smagulova et al., 2016). Most All animals were treated, and tissues collected under importantly, PRDM9 gene was lost at least 13 times appropriate permits, and experiments approved by each independently in vertebrates, including in birds, some snakes, Universities Animal Experimentation Ethics Committees and lizards (Cavassim et al., 2022). Although bearded dragons (University of Canberra, Charles University in Prague and Iowa have a complete PRDM9 (Cavassim et al., 2022), little is known State University) in accordance with animal ethics guidelines. regarding geckos and turtles. It is tempting to speculate the existence of yet to be discovered genetic determinants of recombination across vertebrates, and that one (or more) of Author contributions these might be responsible for reduced recombination rates observed in the species herein. Further research is needed to AR-H conceived and devised the study. LM-G, LG-R, PDW, fully test these hypotheses. and AR-H designed experiments and analysis. LM-G, LG-R, and MMG performed experiments and analyzed the data. LK, NV, AG, PDW, and AR-H contributed to reagents and data Limitations of the study collection. LM-G, LG-R, PDW, and AR-H wrote the first draft of the manuscript with inputs from all authors. All authors read As the use of non-model species can be challenging, future and approved the final version of the manuscript. studies with a larger number of animals per species will be desirable to capture inter-individual variability in recombination rates. Additionally, here we report results Funding obtained with two early markers of meiotic DSBs (RAD51 and RPA) in four different reptile species, but the This work was supported by the Ministry of Economy, use of direct measures of COs were precluded. Thus, future Industry and Competitiveness (CGL 2017–83802-P to AR-H), research focussed on MLH1 foci (a marker of COs) together the Spanish Ministry of Science and Innovation (PID with the analysis of chiasmata in metaphase I will provide a 2020–112557GGB-I00 to AR-H) and the Agència de Gestió comprehensive view of the recombination dynamics in d’Ajuts Universitaris i de Recerca, AGAUR (SGR1215 to AR- reptiles. H). LM-G was supported by an FPU predoctoral fellowship from the Ministry of Science, Innovation and University (FPU18/03867). LK was supported by Czech Science Conclusion Foundation (no. 20–27236J). A component of this research was supported by the Australian Research Council Overall, our findings provide new insights into meiotic (DP170101147 and DP220101429 to AG and PDW), and by chromosome dynamics and double strand break formation the National Science Foundation of the United States (NSF during reptile spermatogenesis. Shared histological patterns IOS 2127995 to NV). observed between squamates and turtles suggest that they represent the ancestral state. However, future research across more species is warranted to asses conservation of this ancestral Acknowledgments pattern across other sauropsids. Understanding the intricacies of the mechanisms regulating chromosome synapsis, The authors are grateful to Thea B. Gessler and Nicholas E. recombination and segregation during meiosis progression Topping for their assistance during sample collection. Frontiers in Cell and Developmental Biology 12 frontiersin.org Marín-Gual et al. 10.3389/fcell.2022.1009776 Conflict of interest DNA with DAPI (blue) for (A) the western-banded gecko and (B) the painted turtle. Scale bar: 10 μm. The authors declare that the research was conducted in the SUPPLEMENTARY FIGURE 2: absence of any commercial or financial relationships that could Telomere dynamics during prophase I. Spermatocyte spreads labelled be construed as a potential conflict of interest. with antibodies against SYCP3 (green), SYCP1 (red) and TRF2 (blue) for the bearded dragon. Scale bar: 10 μm and 2 μm (insets). White arrowheads: telomeres from which SC is beginning to assemble. Yellow arrowheads: completely synapsed micro-chromosomes (i.e., lateral and central elements of the SC completely assembled between both Publisher’s note telomeric ends). Insets (bottom row panels) show micro-chromosomes completely assembled. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, SUPPLEMENTARY FIGURE 3: Transcriptional dynamics during prophase I. Spermatocyte spreads or those of the publisher, the editors and the reviewers. Any product labelled with antibodies against SYCP3 (green) and RNApol II (red) that may be evaluated in this article, or claim that may be made by its for (A) the bearded dragon, (B) the ocelot gecko, (C) the western banded gecko and (D) the painted turtle. Scale bar: 10 μm. manufacturer, is not guaranteed or endorsed by the publisher. SUPPLEMENTARY FIGURE 4: RPA dynamics during reptilian prophase I. Spermatocyte spreads Supplementary material labelled with antibodies against SYCP3 (green) and RPA (red), counterstaining the DNA with DAPI (blue) for (A) the western banded gecko, (B) the ocelot gecko and (C) the bearded dragon. The Supplementary Material for this article can be found Scale bar: 10 μm. online at: https://www.frontiersin.org/articles/10.3389/fcell.2022. SUPPLEMENTARY FIGURE 5: 1009776/full#supplementary-material. RAD51 dynamics during reptilian prophase I. Spermatocyte spreads labelled with antibodies against SYCP3 (green) and RAD51 (red), SUPPLEMENTARY FIGURE 1: counterstaining the DNA with DAPI (blue) for (A) the painted Synapsis dynamics during prophase I. Spermatocyte spreads labelled with turtle, (B) the ocelot gecko and (C) the bearded dragon. Scale bar: antibodies against SYCP3 (green) and SYCP1 (red), counterstaining the 10 μm. References Alavattam, K. G., Abe, H., Sakashita, A., and Namekawa, S. H. (2018). homolog juxtaposition are coordinated in the zebrafish, Danio rerio. PLoS Genet. 15 Chromosome spread analyses of meiotic sex chromosome inactivation. Methods (1), e1007730. doi:10.1371/journal.pgen.1007730 Mol. 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