TYPE Original Research PUBLISHED 25 April 2023 DOI 10.3389/fcell.2023.1147610 Divergent patterns of meiotic OPEN ACCESS double strand breaks and synapsis EDITED BY Ricardo Benavente, Julius Maximilian University of Würzburg, initiation dynamics suggest an Germany REVIEWED BY evolutionary shift in the meiosis Roberta B. Sciurano, National Scientific and Technical Research Council (CONICET), Argentina program between American and Frédéric Baudat, Université de Montpellier, France Australian marsupials *CORRESPONDENCE Jesús Page, jesus.page@uam.es F. Javier Valero-Regalón 1†, Mireia Solé 1,2†, Pablo López-Jiménez 1, † These authors have contributed equally María Valerio-de Arana 1, Marta Martín-Ruiz 1, to this work Roberto de la Fuente 3, Laia Marín-Gual 4,5, Marilyn B. Renfree 6, RECEIVED 18 January 2023 Geoff Shaw 6, Soledad Berríos 7, Raúl Fernández-Donoso 7, ACCEPTED 06 April 2023 PUBLISHED 25 April 2023 Paul D. Waters 8, Aurora Ruiz-Herrera 4,5, Rocío Gómez 1 and CITATION Jesús Page 1* Valero-Regalón FJ, Solé M, 1 López-Jiménez P, Valerio-de Arana M, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain, 2 Martín-Ruiz M, de la Fuente R, Genetics of Male Fertility Group, Unitat de Biologia Cel·lular, Universitat Autònoma de Barcelona, Spain, 3 Marín-Gual L, Renfree MB, Shaw G, Department of Experimental Embryology, Institute of Genetics and Animal Biotechnology of The Polish Berríos S, Fernández-Donoso R, Academy of Sciences, Jastrzębiec, Poland, 4Departament de Biologia Cel·lular, Universitat Autònoma de Waters PD, Ruiz-Herrera A, Gómez R and Barcelona, Barcelona, Spain, 5Genome Integrity and Instability Group, Institut de Biotecnologia i Page J (2023), Divergent patterns of Biomedicina, Barcelona, Spain, 6School of BioSciences, The University of Melbourne, Melbourne, VIC, meiotic double strand breaks and Australia, 7Programa de Genética Humana, Facultad de Medicina, Instituto de Ciencias Biomédicas, synapsis initiation dynamics suggest an Universidad de Chile, Santiago, Chile, 8School of Biotechnology and Biomolecular Science, Faculty of evolutionary shift in the meiosis program Science, University of New South Wales, Sydney, NSW, Australia between American and Australian marsupials. Front. Cell Dev. Biol. 11:1147610. doi: 10.3389/fcell.2023.1147610 In eutherian mammals, hundreds of programmed DNA double-strand breaks COPYRIGHT © 2023 Valero-Regalón, Solé, López- (DSBs) are generated at the onset of meiosis. The DNA damage response is Jiménez, Valerio-de Arana, Martín-Ruiz, then triggered. Although the dynamics of this response is well studied in de la Fuente, Marín-Gual, Renfree, Shaw, eutherian mammals, recent findings have revealed different patterns of DNA Berríos, Fernández-Donoso, Waters, Ruiz-Herrera, Gómez and Page. This is an damage signaling and repair in marsupial mammals. To better characterize open-access article distributed under the these differences, here we analyzed synapsis and the chromosomal distribution terms of the Creative Commons of meiotic DSBs markers in three different marsupial species (Thylamys elegans, Attribution License (CC BY). The use, distribution or reproduction in other Dromiciops gliorides, and Macropus eugenii) that represent South American and forums is permitted, provided the original Australian Orders. Our results revealed inter-specific differences in the author(s) and the copyright owner(s) are chromosomal distribution of DNA damage and repair proteins, which were credited and that the original publication in this journal is cited, in accordance with associated with differing synapsis patterns. In the American species T. elegans accepted academic practice. No use, and D. gliroides, chromosomal ends were conspicuously polarized in a bouquet distribution or reproduction is permitted configuration and synapsis progressed exclusively from the telomeres towards which does not comply with these terms. interstitial regions. This was accompanied by sparse H2AX phosphorylation, mainly accumulating at chromosomal ends. Accordingly, RAD51 and RPA were mainly localized at chromosomal ends throughout prophase I in both American marsupials, likely resulting in reduced recombination rates at interstitial positions. In sharp contrast, synapsis initiated at both interstitial and distal chromosomal regions in the Australian representative M. eugenii, the bouquet polarization was incomplete and ephemeral, γH2AX had a broad nuclear distribution, and RAD51 and RPA foci displayed an even chromosomal distribution. Given the basal evolutionary position of T. elegans, it is likely that Frontiers in Cell and Developmental Biology 01 frontiersin.org Valero-Regalón et al. 10.3389/fcell.2023.1147610 the meiotic features reported in this species represent an ancestral pattern in marsupials and that a shift in the meiotic program occurred after the split of D. gliroides and the Australian marsupial clade. Our results open intriguing questions about the regulation and homeostasis of meiotic DSBs in marsupials. The low recombination rates observed at the interstitial chromosomal regions in American marsupials can result in the formation of large linkage groups, thus having an impact in the evolution of their genomes. KEYWORDS marsupial, meiosis, evolution, synapsis, recombination, Thylamys, Dromiciops, Macropus Introduction distribution of meiotic recombination have a significant impact on genome evolution (Lenormand et al., 2016; Bergero et al., 2021). Meiosis is a complex and highly regulated process, by which In mammals, meiotic studies have been traditionally focused in homologous chromosomes synapse, recombine and segregate. model species, mainly the house mouse and humans. However, Synapsis refers to the tight association of homologs during comparative studies are important to understand if the features meiotic prophase I by a structure called the synaptonemal described in these models are present in other species. For instance, complex (SC). The SC is formed by two axial/lateral elements the organization and composition of the SC seem to be particularly (AE/LEs), one per homologue, held together by transverse well conserved (Fraune et al., 2012). Other features, like the filaments (TFs), which emanate from each of the LEs and frequency of recombination, have also received great attention, overlap in a central region to form the central element (CE) (von though they are more variable between species (Dumont and Wettstein et al., 1984; Page and Hawley, 2004). Recognition of Payseur, 2008; Segura et al., 2013). Additional aspects, like the homologues in mammals (and many other organisms) is mediated regulation of chromosome segregation, remain unexplored in by the formation of hundreds of programmed DNA double-strand most mammals, especially in non-eutherians. This is the case in breaks (DSBs) by the SPO11 protein at the beginning of prophase I marsupials, the sister group of eutherian mammals, which diverged (leptotene stage) (Keeney et al., 2014). The formation of DSBs from each other around 165 million years ago. There are currently triggers a DNA damage response that follows the homologous about 270 marsupial species, distributed in America and Australia. recombination pathway, leading to the molecular interaction of They are grouped into two main clades: Ameridelphia, which chromosomes. The broken DNA molecule uses the intact DNA comprises the Orders Didelphimorphia and Paucituberculata; and sequence of the homologue as a template for DNA repair during Australidelphia, which includes the Australian Orders zygotene. These molecular interactions, in turn, stimulate and Dasyuromorphia, Peramelemorphia, Notoryctemorphia and facilitate the synapsis of homologous chromosomes. In mammals, Diprodontia (Figure 1A) (Duchêne et al., 2017). Intriguingly, most DSBs produced during meiosis are repaired through a process Australidelphia also includes an American sister clade, the Order that leads to gene conversion (non-reciprocal recombination Microbiotheria, only represented by two species of monito del events), whereas some of them result in reciprocal exchange monte (Dromiciops gliroides and D. bozinovici) (D’Elía et al., events that lead to the formation of crossovers (COs) at the end 2016; Feng et al., 2022; Fontúrbel et al., 2022). of pachytene (at least one CO per bivalent) (Cole et al., 2012). These Marsupials are characterized by their unique reproductive COs are visualized cytologically as chiasmata, which hold strategy, in which pregnancy is uniformly short and the altricial recombined homologous chromosomes together until they young are born at an early developmental stage. Development is segregate during anaphase of the first meiotic division (Roeder, usually completed within an abdominal pouch, with the pouch 1997). young dependent on a highly specialized milk (Tyndale-Biscoe Besides a role in ensuring faithful chromosome segregation, it and Renfree, 1987). Marsupials also present a number of genetic is commonly accepted that recombination increases genetic and chromosomal differences compared to eutherians (Graves and variability in natural populations through the generation of new Renfree, 2013). Two of the most relevant are: 1) Their reduced haplotypes, which are later subjected to evolutionary drift and number of chromosomes (Hayman, 1990; Deakin, 2018). Although selection (Barton and Charlesworth, 1998; Otto and Lenormand, chromosome numbers range from 2n = 10 to 2n = 34, they present a 2002). In contrast, suppression of recombination at specific bimodal distribution and most species have either 2n = 14 or 2n = 24 chromosomal regions leads to the genetic isolation of these (Deakin and Potter, 2019; Deakin and O’Neill, 2020). Since the chromosome segments and the formation of large linkage genome size is comparable to that of eutherians, marsupial groups. If allele combinations cannot be reshuffled by chromosomes are usually much larger. 2) The Y chromosome is recombination, beneficial alleles are likely to be lost by either generally tiny and does not share a pseudoautosomal region (PAR) background selection or random drift (Graves, 1995; with the X due to extreme degeneration of the former over Charlesworth et al., 2005; Bachtrog, 2013). Finally, both gene evolutionary time (Graves et al., 1998). In fact, the Y conversion and CO formation can alter the GC content of chromosome can be lost in some somatic tissues, as reported in genomic regions where these events accumulate (called males of the family Peramelidae (Watson et al., 1998). hotspots) by a process known as GC-biased gene conversion The special features of marsupial chromosomes also have an (gBGC) (Duret and Galtier, 2009). Therefore, the frequency and impact on their behavior during meiosis. The most noticeable Frontiers in Cell and Developmental Biology 02 frontiersin.org Valero-Regalón et al. 10.3389/fcell.2023.1147610 FIGURE 1 (A). Phylogenetic relationships of extant marsupial orders. The arrangement presented is based on the phylogeny published by Duchêne and coworkers (Duchêne et al., 2017). Although the topology of the tree is still controversial, Microbiotheria is grouped to Australidelphia in all the trees consulted. The Orders included in this study are highlighted in red. (B–D). Meiotic karyotypes of the three studied species: SYCP3 in green and centromeres in red. Bivalents are ordered by size, according to previous reports. In Thylamys elegans (B) the position of the NOR (Nu) on the short arm of bivalent 6 was detected using anti-fibrillarin antibody (pink). The position of the NOR on the X chromosome of Macropus eugenii was detected by an accumulation of SYCP3. Scale bar in red: 10 µm. feature is the behavior of sex chromosomes. The absence of a PAR 2008; Wang et al., 2011). Moreover, marsupial males seem to be on the XY pair precludes their reciprocal synapsis and more recombinogenic than females, as opposed to the higher recombination in male meiosis, thus challenging the usual way recombination rates in most female eutherian mammals (Bennett by which homologous chromosomes ensure their segregation et al., 1986; Samollow et al., 2004; Samollow et al., 2007). Many during first meiotic division. Instead, sex chromosomes in factors seem to regulate the genome-wide rate of recombination in marsupials present an alternative mode of association, which eukaryotes. These include chromosome number, length of the SC relies on the formation of a specific structure called the dense and of chromatin loops (Segura et al., 2013; Mercier et al., 2015; plate (DP) that maintains the sex chromosome association from Ruiz-Herrera et al., 2017; Wang et al., 2019). Other factors like prophase I (Solari and Bianchi, 1975; Sharp, 1982; Seluja et al., 1987; genetic background and sex are also relevant (Gruhn et al., 2013; Page et al., 2003) until they segregate at anaphase-I (Page et al., Baier et al., 2014). In particular, we have recently proposed that the 2006). Although small differences between species have been found low recombination rates observed in marsupial males might result regarding the timing of DP formation (Marín-Gual et al., 2022b), the from the induction of fewer DSBs during prophase I, potentially development and dynamics of the DP are well conserved (Sharp, leading to the formation of fewer COs (Marín-Gual et al., 2022b). 1982; Page et al., 2005; Fernández-Donoso et al., 2010), indicating Our previous study revealed that in three species of phylogenetically that the DP represents a feature that originated before the radiation distant marsupials, the overall number of DSBs was significantly of marsupials (Page et al., 2005). The emergence of this alternative lower than in eutherian mammals (i.e., mice and humans), mechanism of segregation allowed for the proper transmission of sex concomitant with low γH2AX levels on autosomes. chromosomes after their complete differentiation. Interestingly, In addition to overall recombination rates, the distribution of analogous mechanisms of sex chromosome segregation have recombination events along chromosome is a field of intense independently appeared in eutherian species with completely research. Among eutherians, DSBs have been reported to appear differentiated sex chromosomes (de la Fuente et al., 2007; de la fairly evenly distributed in mice, whereas COs tend to accumulate Fuente et al., 2012; Gil-Fernández et al., 2020; Gil-Fernández et al., towards the distal regions of chromosomes in this species (Froenicke 2021). et al., 2002; de Boer et al., 2006; Grey et al., 2009; Brick et al., 2018; Li The striking behavior of sex chromosomes may have obscured et al., 2019). In contrast, both DSBs and COs clearly accumulate at other meiotic differences in marsupials. This includes distal regions in humans (Oliver-Bonet et al., 2007; Pratto et al., recombination rates, i.e., the number of COs per cell, which are 2014). However, studies in non-model mammals are scarce. In this lower in marsupials compared to eutherians (Zenger et al., 2002; regard, in our previous study on marsupials we detected inter- Samollow et al., 2004; Samollow et al., 2007; Dumont and Payseur, specific differences in the pattern of DSBs distribution along Frontiers in Cell and Developmental Biology 03 frontiersin.org Valero-Regalón et al. 10.3389/fcell.2023.1147610 chromosomes (Marín-Gual et al., 2022b). Here we test whether DSB formaldehyde in PBS for 10 min and then squashed on a slide. occurrence is evenly distributed along chromosomes in marsupials. Coverslip was removed after freezing in liquid nitrogen and slides To achieve this, we analyzed the localization of proteins related to were rehydrated in PBS until use. DNA damage response and repair (γH2AX, RAD51, and RPA), along with SC components (SYCP1 and SYCP3) and telomeric DNA sequences, during meiosis in species that capture the deepest Immunofluorescence divergences within marsupials: the American species Thylamys elegans and D. gliroides, and the Australian species Macropus Slides were incubated overnight at 4 °C with the following eugenii. Our results uncover remarkable differences in the antibodies diluted in PBS: rabbit anti-SYCP3 (ab15093, Abcam, initiation and progression of synapsis between homologous 1:200 dilution), rabbit anti-SYCP1 (ab15087, Abcam, 1: chromosomes, as well as in the distribution pattern of DNA 200 dilution), mouse anti-γH2AX (05-636, Upstate, 1: repair markers, with American species showing an extreme 1000 dilution), rabbit anti-RAD51 (PC130, Calbiochem, 1: polarization towards chromosomal ends. This behavior may have 50 dilution), rabbit anti-RPA2 (ab10359, Abcam, 1: important consequences for recombination rates and distribution, 50 dilution), mouse anti-fibrillarin (ab4566, Abcam; 1: which in turn could impact genome evolution. 50 dilution), human anti-centromere (441-10BK-50, Antibodies Incorporated, 1:50 dilution). In addition, many antibodies were used against DMC1, MLH1, MLH3, and other Materials and methods proteins associated with COs (PRR19, CNTD1, CDK2) that yielded no positive labeling. After incubation, slides were Animals washed three times in PBS and subsequently incubated for 1 hour at room temperature with secondary antibodies Two T. elegans (Didelphidae) and two D. gliroides conjugated with Alexafluor 350, Alexafluor 488, Cy3 or Cy5 (Microbiotheriidae) males were collected in central and Southern (Jackson ImmunoResearch Laboratories) all of them diluted 1: Chile, respectively, from natural populations under permission of 100 in PBS. After three washes in PBS slides were stained with Corporación Nacional Forestal (Conaf). Handling of animals was 10 μg/ml DAPI, washed in PBS and mounted with Vectashield. performed according to the ethical rules stablished by the University of Chile. Two M. eugenii (Macropodidae) males were collected from wild populations originating on Kangaroo Island (South Australia) Fluorescence In Situ hybridization for that were later held in a breeding colony in Melbourne (Victoria, telomeric DNA repeats Australia). Sampling was conducted under ethics approval from the University of Melbourne Animal Experimentation Ethics FISH was conducted as previously described (de la Fuente et al., Committees and followed the Australian National Health and 2014). After immunofluorescence, slides were rinsed in PBS, fixed in Medical Research (2013) guidelines. The karyotypes of these 4% formaldehyde in PBS for 10 min, dehydrated in an ethanol series species are as follows: T. elegans 2n = 14; D. gliroides 2n = 14; (70%, 90%, and 100%) for 5 min each and air dried. Hybridization M. eugenii 2n = 16. The meiotic karyotypes of the three species were mixture containing 70% deionized formamide (Sigma), 10 μM arranged according to length and centromere position of each FITC-labelled (C3TA2)3 peptide-nucleic acid (PNA) probe bivalent (Figures 1B–D), in agreement with previous reports (Applied Biosystems), and 2.1 mM MgCl2 buffer (pH 7.0) in (Page et al., 2003; Fernández-Donoso et al., 2010; Marín-Gual 8 mM Tris (pH 7.2) was added to each slide. DNA was et al., 2022b). denatured for 3 min at 80°C. Hybridization was performed for 2 h at room temperature. Slides were then washed twice for 15 min each with 70% formamide in distilled water containing Spermatocyte spreads and squashes 10 mM Tris (pH 7.2) and 10% BSA, and then three times with TBS (1 M Tris, 1.5 M NaCl (pH 7.5) containing 0.005% Tween- Testicular samples were obtained and subsequently processed. 20) for 5 min each. Slides were then dehydrated in an ethanol series, For spreads, we used the protocol previously described by Peters and air-dried, stained with 10 μg/ml DAPI and mounted with coworkers (Peters et al., 1997), with slight modifications for Vectashield. marsupial samples (Page et al., 2005). Briefly, a cell suspension was incubated in 10 mM sucrose solution in distilled water for 15 min. The suspension was spread onto a slide dipped in 1% Microscopy and image processing formaldehyde in distilled water (pH 9.5), containing 100 mM sodium tetraborate and 0.15% Triton-X100. Cells were left to Observations were made on an Olympus BX61 microscope settle for 1.5 h in a humid chamber and subsequently washed equipped with appropriate fluorescence filters and an Olympus with 0.4% Photoflo (Kodak) in distilled water. Slides were air DP72 digital camera. The images were processed using the public dried at room temperature and then rehydrated in phosphate domain software ImageJ (National Institutes of Health, saline buffered (PBS: NaCl 137 mM, KCl 2.7 mM, Na2HPO4 United States; http://rsb.info.nih.gov/ij) and Adobe Photoshop 10,1 mM, KH2PO4 1.7 mM, pH 7.4) before immunostaining. For 7.0 (Adobe). Spread images were taken as single-plane pictures, squashes, we used a previously described method (Page et al., 1998; whereas squashed spermatocytes were photographed at 0.2 μm Page et al., 2003). Seminiferous tubules were fixed in 2% intervals and the resulting stack images processed in ImageJ. Frontiers in Cell and Developmental Biology 04 frontiersin.org Valero-Regalón et al. 10.3389/fcell.2023.1147610 Quantitative analysis of RPA distribution of chromosomes were still polarized in a bouquet conformation at this stage. SYCP1 was observed as continuous lines that regularly For the analysis of RPA foci chromosomal distribution, only expanded from the ends towards the centers of the chromosomes, early pachytene spermatocytes in which bivalents could be clearly and there was no interstitial initiation of synapsis. This feature was discerned from each other were chosen from the overall cell still observable at late zygotene (Figure 2C). The only exception to population study. Bivalents were identified according to their synapsis beginning from telomeres was for the chromosome pair length and centromere position. In the case of T. elegans, the bearing the nucleolar organizing region (NOR) (see Figure 4C). The location of fibrillarin signal associated to the short arm allowed NOR is located near the telomere of the short arm (Figure 1) and it the identification of bivalent 6. Each bivalent was measured using the had delayed synapsis. Even though synapsis in the autosomes was Free Hand tool in ImageJ. The distance of centromeres and RPA foci completed by pachytene, the sex chromosomes remained from the tip of the short arm of the bivalents was assessed as follows: unsynapsed at this stage (Figure 2D). In the other American each focus was manually drawn as an intersection line with the species, D. gliroides, the pattern of AE formation and synapsis outline of the SC, yielding the longitudinal position of the focus. progression was almost identical, including the conspicuous Then, each bivalent was divided into 10 different segments, being bouquet configuration, the thickening of the distal regions of the segment 1 the distal portion of the shortest arm. Finally, the position LEs at early zygotene, the distal initiation of synapsis, and its of each RPA focus was ascribed to a specific segment (from 1 to 10). subsequent progression to interstitial regions (Figures 2E–H). A minimum of 15 spermatocytes were recorded for each individual Remarkably, AE formation and SC assembly in M. eugenii (2 T. elegans and 2 M. eugenii males). contrasted the pattern in American species. Overall, we observed three main differences. First, at the leptotene-zygotene transition, the AEs appeared as short fragments or dots evenly distributed, Statistical analyses instead of accumulated in the region where fragments of SYCP1 signal were observed (Figure 2I). Second, Quantitative data were analyzed using Prism GraphPad 7.0. The SYCP1 fragments did not adopt a markedly polarized distribution of RPA foci along chromosomes was compared to a distribution, indicating that the bouquet is not as evident as in random distribution by a χ2 goodness of fit test with 9 degrees of the American species. Third, at early zygotene synapsis initiated freedom. Statistical significance was considered for p < 0.05. The both at the distal and interstitial regions of each chromosome relationship between RPA foci number and SC length was evaluated (Figure 2J), a feature that was still detectable at late zygotene by Spearman correlation coefficient (r). (Figure 2K). At pachytene, synapsis of the autosomes was complete, whereas sex chromosomes remained unsynapsed (Figure 2L). Results In order to better characterize the differences in the formation and dynamics of the bouquet polarization, we combined the Chromosome synapsis dynamics immunolabeling of SYCP3 protein with the localization of telomeric DNA sequences by FISH (Figure 3). In T. elegans, We first studied the synaptic behavior of chromosomes during telomeres appeared clearly polarized in all spermatocytes at the meiosis in the selected species via the immunolocalization of the transition between leptotene and zygotene (Figure 3A) and also at proteins SYCP3 and SYCP1, the main components of the axial/ early zygotene (Figure 3B). This polarization was subsequently lost lateral elements (AE/LEs) and transverse filaments of the SC, with zygotene progression, but some telomeres occasionally respectively. The localization patterns of these proteins were used remained associated with each other at late zygotene (Figure 3C) to classify spermatocytes into the different prophase I stages, and even at early pachytene (Figure 3D). In contrast, chromosomal following previous observations in marsupials (Page et al., 2003; ends were more dispersed in M. eugenii. We observed that in 50% of Page et al., 2005; Marín-Gual et al., 2022b). spermatocytes at the leptotene-zygotene transition (n = 84) In T. elegans, during early prophase I SYCP3 was usually telomeres did not form clusters, although many times they were accompanied by the appearance of a SYCP1 signal (Figure 2A), observed preferentially distributed in one-half of the nucleus making it difficult to discriminate between leptotene and zygotene. (Figure 3E). The remaining spermatocytes showed one or two This suggests that the formation of the AEs was concurrent with the (sometimes more) telomere clusters, but these groups usually did initiation of synapsis early in prophase I in this species. Thus, at early not incorporate all chromosome ends (Figure 3F). At early zygotene, stages of prophase I the AEs were just partially formed, appearing clusters were usually dissolved, and telomeres were dispersed with dotted signal along most of the chromosome, but forming short (Figure 3G). These results suggest that in M. eugenii the bouquet lines at the regions where two AEs associate (Figure 2A). These polarization is incomplete and more ephemeral than in the synapsed segments were mostly grouped in a small region (i.e., a American species. bouquet configuration). In addition, SYCP3 revealed a thickening at FISH against telomeric DNA repeats also revealed the presence the ends of the AEs. Therefore, we refer to this stage as the leptotene- of interstitial telomeric sequences in the largest bivalent of T. elegans zygotene transition. At a subsequent stage, early zygotene (Figure 3D), as previously described (Page et al., 2006). Intriguingly, (Figure 2B), the AEs were almost completely formed. Synapsis the interstitial telomeric signals of the two homologous began at chromosomal ends, which was evidenced by the chromosomes often appeared displaced along that bivalent, which presence of SYCP1 in the region where the AEs (now called LEs) is concurrent with a displacement of the centromeric signals (see of homologous chromosomes were associated. Moreover, the ends detail in Figure 3D). This displacement suggests the presence of a Frontiers in Cell and Developmental Biology 05 frontiersin.org Valero-Regalón et al. 10.3389/fcell.2023.1147610 FIGURE 2 Synapsis progression during prophase (I). Spread spermatocytes labeled with antibodies against SYCP3 (red) and SYCP1 (green). (A–D). Thylamys elegans. Short filaments of SYCP1 are seen between AEs at the leptotene-zygotene transition (A). These filaments appear mostly polarized to a specific nuclear region, the bouquet area (asterisk). Synapsis progresses during early (B) and late zygotene (C). Polarization of chromosomal ends is still observed (asterisks). Sex chromosomes (X, Y) lie in the bouquet region. Synapsis is complete at pachytene (D) except for the sex chromosomes. (E–H). D. gliroides. Chromosomal ends are polarized to the bouquet area (asterisks) at leptotene-zygotene transition (E) and early zygotene (F). Synapsis progresses during late zygotene (G) and is complete at pachytene (H). Sex chromosomes remain separated. (I–L) Macropus eugenii. AEs appear as short fragments in the whole nucleus during the leptotene-zygotene transition (I). At early zygotene (J) synapsis is initiated both at the chromosomes ends and at interstitial regions (arrows). This is also observed at late zygotene (K). Synapsis is complete at pachytene (L), with sex chromosomes lying separately. Bar: 10 μm. synaptic mismatch in the central region of the bivalent. We also corresponding with chromosomal ends polarized in the bouquet revealed the presence of interstitial telomeric repeats in M. eugenii configuration. At early zygotene, γH2AX labeling was mostly bivalents (Figure 3H), as previously described (Bender et al., 2012; associated with the chromosomal regions where synapsis was Marín-Gual et al., 2022b). No displacement of centromeres or initiated, whereas the rest of the nucleus remained devoid of telomeric repeats was detected in this species (detail in Figure 3H). γH2AX (Figure 4B). At this stage, the bouquet polarization was still observed. At late zygotene, an increase of γH2AX signal was observed, localized mainly over the regions of autosomes that had Distribution of DSBs during prophase I not completed synapsis, as well as over the chromatin around the AEs of the sex chromosomes (Figure 4C). At pachytene, once In mammals, synapsis initiation is dependent on the occurrence autosomes had completed full synapsis, γH2AX signal was only of DNA DSBs at the beginning of meiosis (Baudat et al., 2000; detectable over the sex chromosomes (Figure 4D). The distribution Romanienko and Camerini-Otero, 2000). To assess if the differences of γH2AX during meiosis in D. gliroides was similar, albeit not detected in the progression of synapsis could be linked to a completely identical, to that of T. elegans. γH2AX was mostly differential distribution of DSBs, we studied the localization of detected at the chromosomal ends at leptotene and early the phosphorylated form of histone H2AX (γH2AX), a widely zygotene (Figures 4E,F) and accumulated at unsynapsed used marker of DNA damage during meiosis (Mahadevaiah chromosomes in late zygotene, where the signal seemed to be et al., 2001; Turner et al., 2004). Mirroring previous observation more intense than in T. elegans. (Figure 4G). During pachytene (Marín-Gual et al., 2022b) we found that in T. elegans only a few γH2AX labeling remained only on sex chromosomes (Figure 4H). small foci of γH2AX became detectable at leptotene-zygotene on the Crucially, in M. eugenii the γH2AX signal was different. At chromatin around the AEs formation (Figure 4A). This location the leptotene-zygotene transition (Figure 4I) the signal was followed the pattern of chromosome synapsis described above, distributed over all chromosomes, with no specific Frontiers in Cell and Developmental Biology 06 frontiersin.org Valero-Regalón et al. 10.3389/fcell.2023.1147610 FIGURE 3 Localization of telomeric sequences in Thylamys elegans and Macropus eugenii. Spread spermatocytes were labeled with antibodies against SYCP3 (red) and telomeric sequences revealed by FISH (green) in (A–C) and (E–G), and SYCP3 (blue), centromeric proteins (red) and telomeric sequences (green) in (D) and (H) (A–D). Thylamys elegans. Telomeres are clustered (asterisks) at the lepto-zygotene transition (A) and early zygotene (B). At late zygotene (C) a slight polarization is still evident (asterisk), and some telomeres appear associated (arrows). At pachytene (D), some telomeric associations (arrows) are occasionally observed. Displacement of interstitial telomeric and centromere signals is evident in the largest bivalent (arrowhead), enlarged in the squared image. (E–H). Macropus eugenii. At the leptotene-zygotene transition (E,F) telomeric signals (small dots) may appear greatly dispersed (E) or forming several clusters (asterisks in F). Some large FISH signals are also observed (arrows). Chromosomal ends do not show any specific clustering at early zygotene (G) or at pachytene (H). Immunolabeling of centromeric proteins (H) reveal that the large FISH signals correspond to interstitial telomeric sequences located at centromeres (arrows) or near centromeres (arrowhead). No displacement of the centromeres or telomeric signal was observed (enlarged imaged in H). Bar: 10 μm. accumulations at chromosomal ends or any other region. This recombinases that mediate the invasion of an intact DNA broad distribution was also observed at early zygotene template to repair the DSBs, and RPA, which protects single (Figure 4J). At late zygotene, γH2AX tends to disappear from stranded DNA molecules generated during homologous synapsed chromosomes but an intense signal was detected over recombination (Brown and Bishop, 2015). Here we report the the still unsynapsed autosomal regions and over the sex chromosomal distribution of RAD51 and RPA in the species chromosomes (Figure 4K). At pachytene, γH2AX signal studied. Unfortunately, DMC1 did not yield a positive result. remained only over the sex chromosomes (Figure 4L). We first analyzed the distribution of RAD51 in squashed The striking differences in the intensity and distribution of spermatocytes. At the leptotene-zygotene transition, a few γH2AX between marsupial species lead us to test whether the RAD51 foci were observed in T. elegans, mainly located at faint signal observed in T. elegans could be due to an artifact of chromosomal ends and grouped in the bouquet configuration the spreading technique. Thus, we evaluated in this species the (Figure 6A). Some additional foci were observed scattered over distribution of γH2AX in spermatocyte squashes, which maintained the nucleus. At early zygotene (Figure 6B), foci remained localized the three-dimensional organization of the nucleus and provided mostly close to the chromosomal ends. At late zygotene (Figure 6C), better preservation of the chromatin (Figure 5). This confirmed the the bouquet configuration was lost, and some RAD51 foci were patterns detected in spermatocyte spreads; that is, the almost localized interstitially along bivalents. The X chromosome complete absence of γH2AX at leptotene and early zygotene was accumulated numerous RAD51 foci at late zygotene and also at a bona fide feature of T. elegans (Figures 5A–C). The accumulation early pachytene (Figure 6D). The number of RAD51 foci decreased of γH2AX at the unsynapsed regions started when synapsis had with pachytene progression and the protein was completely absent greatly progressed on the autosomes (Figures 5D,E). During by late pachytene (not shown). A similar trend was observed for D. pachytene, γH2AX was only present on the sex chromosomes, gliroides in spermatocyte spreads (Figures 6E–H). Most foci were either before they paired (Figures 5F,G) or after they completed associated with the short SYCP3 filaments at leptotene (Figure 6E). their pairing and the formation of the dense plate (DP) (Figure 5H). However, in this species, some RAD51 foci appeared on interstitial regions of chromosomes along with synapsis progression (Figures 6F,G). This suggested progressive incorporation of RAD51 along Nuclear distribution of DNA repair proteins chromosomes during zygotene. Some foci were still detectable at early pachytene (Figure 6H). Similar to T. elegans, the X The induction of DSBs triggers the activation of the homologous chromosome presented abundant RAD51 foci (Figure 6H). recombination repair pathway and the incorporation of proteins In sharp contrast, RAD51 foci appeared evenly distributed over involved in this process, such as RAD51 and DMC1, the the nucleus in M. eugenii spermatocyte spreads. At the leptotene- Frontiers in Cell and Developmental Biology 07 frontiersin.org Valero-Regalón et al. 10.3389/fcell.2023.1147610 FIGURE 4 Localization of DNA damage-related proteins. Spread spermatocytes labeled with antibodies against SYCP3 (green) and γH2AX (red). (A–D). Thylamys elegans. γH2AX is observed as small foci at the leptotene-zygotene transition (A) and early zygotene (B), associated to the synapsing chromosomal ends polarized to the bouquet (asterisks). A more intense γH2AX labeling is observed at late zygotene (C) associated to the AEs of unsynapsed autosomal regions and sex chromosomes (X, Y). The proximal end of chromosome 6 also remains unsynapsed (arrow). At pachytene (D) γH2AX is only observed on the chromatin of sex chromosomes. (E–H). D. gliroides. The pattern of γH2AX is almost identical to the one described for Thylamys elegans, except for a more intense labeling of γH2AX in the unsynapsed regions of chromosomes at zygotene. (I–L) Macropus eugenii. At the leptotene to zygotene transition (I) γH2AX is spread in the whole nucleus. As zygotene proceeds (J,K) γH2AX labeling is reduced in the nucleus and concentrates around the AEs of unsynapsed autosomes and the X chromosome, but not on the Y chromosome. Both sex chromosomes exhibit signal of the antibody at pachytene (L). Bars in A-H and I–L: 10 μm. zygotene transition, foci were associated with the short fragments of pachytene. Because autosomes have completed synapsis at this the forming AEs (Figure 6I). Similarly, from early to late zygotene, stage, every bivalent could be identified thanks to the differences RAD51 foci were distributed all along the synapsing bivalents in length, centromere position and location of the NOR (Figures (Figures 6J,K). Even at early pachytene (Figure 6L), RAD51 foci 1B,D and Supplementary Figure S1). This permitted a did not concentrate at any particular chromosomal region, even quantitative study of the distribution of RPA along each though the number of such foci was prominently reduced. chromosome in both T. elegans and M. eugenii. We analyzed As for RPA, we only obtained a reliable signal of the antibody at least 15 pachytene spermatocytes in two individuals from each in T. elegans and M. eugenii. The dynamics of RPA foci were species. Each bivalent was measured, divided into 10 segments similar to that of RAD51. In T. elegans, most foci were localized to and the position of each RPA focus was then scored along the the chromosomal ends at the leptotene-zygotene transition and bivalent and assigned to a segment. The same methodology was early zygotene (Figures 7A,B). Then, foci also appeared at applied to the X chromosome for both species. interstitial regions during late zygotene and early pachytene We detected that in T. elegans RPA foci accumulated towards (Figures 7C,D) but remained visibly concentrated at the chromosomal ends in all bivalents, particularly in the four chromosomal ends. In contrast, RPA foci in M. eugenii were largest, in which the two distal segments concentrated near or evenly distributed along chromosomes throughout prophase I above 50% of all RPA foci (Figure 8; Table 1). The distribution of (Figures 7E–H). RPA foci increased symmetrically in both chromosomal arms, with just a reduction around centromeres. This was especially relevant for bivalent 6, which bears the NOR on the short arm. Chromosomal distribution of RPA This region accumulated fewer RPA foci (11.41%) compared to the opposite chromosomal end (20.16%) (Table 1). The X A remarkable feature observed regarding RPA dynamics was chromosome, which remained as univalent, also showed a that the number of foci remained high even during early non-random distribution of RPA. The X centromere seemed Frontiers in Cell and Developmental Biology 08 frontiersin.org Valero-Regalón et al. 10.3389/fcell.2023.1147610 FIGURE 5 Localization of DNA damage in Thylamys elegans spermatocytes preserving the 3-dimensional topology of chromosomes. Squashed spermatocytes labeled with antibodies against SYCP3 (green) and γH2AX (red) and DAPI (blue). (A). Leptotene. No γH2AX labeling is observed. AEs appear polarized in a bouquet configuration (asterisk). (B). Early zygotene. A few small γH2AX signals are observed at the region where synapsis is initiating. The bouquet polarization is still evident. (C). Mid zygotene. Synapsis has progressed. A few γH2AX foci are scattered within the nucleus. (D). Mid zygotene. γH2AX labeling starts to be observed at the unsynapsed regions of autosomes and sex chromosomes (X, Y). (E). Late zygotene. γH2AX signal increases on unsynapsed chromosomes. Sex chromosomes are detected at opposite nuclear spaces and the γH2AX labeling extends from their AEs to the surrounding chromatin. (F). Early pachytene. γH2AX is only detected on the chromatin of sex chromosomes, clearly separated in the nucleus. (G). Early-mid pachytene. Sex chromosomes approach and associate to each other. (H). Mid pachytene. Sex chromosomes pair and form the dense plate (arrowhead). γH2AX labels the whole sex body. Bar: 5 μm. to have an effect, with a reduced number of RPA in the flanking Discussion segments. The Y chromosome could not be analyzed due to its small size. Meiotic studies in non-eutherian mammalian species are scarce. In contrast, the distribution of RPA foci in M. eugenii was Only a few reports were devoted to monotremes (Daish et al., 2015; quite homogeneous along bivalents. A χ2 test showed that on most Casey et al., 2017). In marsupials, most studies have focused on the chromosomes RPA location did not significantly depart from a unique behavior of sex chromosomes (Solari and Bianchi, 1975; random distribution (Table 1). The only exceptions were Sharp, 1982; Roche et al., 1986; Seluja et al., 1987; Page et al., 2003; chromosomes 1, 7 and X, on which RPA foci were reduced Page et al., 2005; Fernández-Donoso et al., 2010; Marín-Gual et al., around the centromere. The NOR, which is located in the 2022b). Our recent work on marsupials revealed divergent strategies short arm of the X chromosome (Figure 1D) did not have and for meiotic DNA repair, recombination and transcription (Marín- apparent effect on accumulation of RPA foci. In fact, RPA Gual et al., 2022b). Here we extend these observations and report distribution was quite similar on the X chromosome in both previously uncharacterized features of marsupial meiosis: bouquet species. formation, synapsis initiation and chromosomal distribution of The quantitative analysis of RPA also allowed us to assess a DSBs. Remarkably, our observations suggest an evolutionary shift potential correlation between the number of foci accumulated in the meiosis program between American and Australian on every chromosome and their respective length. Because the X marsupials. In the context of recently published reports on fish chromosome was a univalent and the Y chromosome was too and reptile meiosis (Blokhina et al., 2019; Marín-Gual et al., 2022a), small, we only considered autosomes. We found that in T. our results reveal the persistence of ancestral vertebrate meiotic elegans, RPA foci were underrepresented in bivalents 1 to 3, features in marsupials. This highlights the relevance of comparative and conversely overrepresented in bivalents 4 to 6 (Table 2). studies to fully understand the causes and consequences of meiosis Accordingly, a Spearman correlation analysis of RPA foci evolution. number and SC length showed low correlation (r = 0.41, p < 0.0001) (Figure 9). In contrast, all M. eugenii bivalents presented an increased correlation between chromosome length and RPA The conspicuous bouquet conformation proportion (Spearman correlation analysis r = 0.88, p < 0.0001) could be an ancient feature of vertebrate (Table 2; Figure 9). This reinforces the hypothesis that RPA meiosis distribution in M. eugenii is not dependent on specific features of chromosomes. Their location was equiprobable on any The polarization of telomeres at the beginning of meiosis has chromosome and at any chromosomal region. been described in a wide range of species, from fungi to plants and Frontiers in Cell and Developmental Biology 09 frontiersin.org Valero-Regalón et al. 10.3389/fcell.2023.1147610 FIGURE 6 Localization of homologous recombination repair. Spermatocytes labeled with antibodies against SYCP3 (red) and RAD51 (green). (A–D). Thylamys elegans. Squashed spermatocytes. RAD51 foci are scarce and localized in the bouquet (asterisk) during the leptotene-zygotene transition (A) and also at early zygotene (B). At late zygotene (C) some RAD51 foci are seen at interstitial regions and coat abundantly the X and Y chromosomes (X, Y). At early pachytene (D) most RAD51 foci concentrate in the sex chromosomes. (E–H). D. gliroides. RAD51 foci associate to the short stretches of SYCP3 at the leptotene-zygotene transition (E). At early zygotene (F), most foci are localized in the already synapsed distal regions. At late zygotene (G) discrete foci are also detected over the interstitial regions of autosomes and mostly over the X chromosome. At early pachytene (H), a few foci are still associated to autosomes. (I–L) Macropus eugenii. Spread spermatocytes. At the leptotene-zygotene transition (I) RAD51 foci appear in the whole nucleus. As zygotene proceeds (J,K) RAD51 is clearly observed all along the bivalents. At early pachytene (L), RAD51 foci number has decreased but they are observed all along autosomal bivalents and the sex chromosomes. Bars: 5 μm in (A–D) and 10 μm in (E–L). animals (Zickler and Kleckner, 1998). However, the presence and species of reptiles (Marín-Gual et al., 2022a), these seem to be the extent of this polarization changes from taxa to taxa, and even ancient features of the vertebrate meiotic program, which have between sexes. Formation of the bouquet has been considered a been subsequently maintained in a wide range of groups. crucial factor in chromosome pairing and synapsis initiation (Liebe However, this chromosomal polarization has suffered et al., 2006; Reig-Viader et al., 2016). However, the study of different regulatory modifications in different linages. In eutherian mutants has provided evidence that such a feature is not an absolute mammals, the house mouse displays a visible polarization at requirement, and some model systems like Drosophila melanogaster the beginning of meiosis (Berrios et al., 2010; Berrios et al., 2014), and Caenorhabditis elegans are known to lack a chromosomal but this is brief and often incomplete (Scherthan et al., 1996; bouquet during meiosis (Harper et al., 2004). Lopez-Jimenez et al., 2022). Moreover, while synapsis can start at Our results show the presence of a marked and long-lasting the chromosomal ends in this species, different reports indicated bouquet polarization in two of the three marsupial species that synapsis initiation is mostly interstitial (Boateng et al., 2013; analyzed. This was correlated with the initiation of synapsis, Gruhn et al., 2016). In humans, a striking sexual dimorphism is which was clearly terminal in T. elegans and D. gliroides. Synapsis observed, with males displaying a brief bouquet but initiating in these two species extended from the telomeres to the synapsis almost exclusively at chromosomal ends and females interstitial regions in a zipper-like manner. Similar showing a more persistent bouquet but initiating synapsis at observations were reported in the South American marsupial interstitial regions (Roig et al., 2004; Gruhn et al., 2016). Thus, Rhyncholestes raphanurus, belonging to the Paucituberculata the relevance of the bouquet to drive synapsis initiation seems to Order (Page et al., 2005). Thus, this feature seems to be an have been attenuated in eutherian mammals. In the present old character among marsupials. Moreover, according to study, we found that the Australian marsupial species recent reports in zebrafish (Blokhina et al., 2019) and several analyzed, M. eugenii, mimics the pattern described in mouse, Frontiers in Cell and Developmental Biology 10 frontiersin.org Valero-Regalón et al. 10.3389/fcell.2023.1147610 FIGURE 7 Localization of RPA. Spread spermatocytes labeled with antibodies against SYCP3 (red) and RPA (green). (A–D). Thylamys elegans. Most RPA foci concentrate in the distal regions of autosomes from leptotene to pachytene. From late zygotene to pachytene, RPA foci also accumulate on the sex chromosomes (X, Y). Asterisk indicates the polarization of chromosomal ends. Sex chromosomes. (E–H). Macropus eugenii. RPA foci associate to forming AEs at the leptotene to zygotene transition. From early zygotene onwards, foci are observed along the entire length of the bivalents. Bars in (A–D) and (E–H): 10 μm. FIGURE 8 Chromosomal distribution of RPA foci distribution along bivalents and the X chromosome in Thylamys elegans and Macropus eugenii. Chromosomes have been divided into 10 segments of equal size between telomeres (proximal to distal). Y-axes in the graphs indicate the percentage of RPA foci in each segment. Each bivalent (BV) has been depicted below the corresponding graph. Pink bars indicate the centromere position, yellow bars indicate the NOR. We found a prominent polarization of RPA foci towards the chromosome ends in Thylamys elegans. In contrast, in Macropus eugenii detection of RPA foci uncovered a relatively homogeneous distribution along the entire length of the chromosomes. with a loose short-lived bouquet and synapsis initiating both near and distal synapsis initiation seems to have occurred independently telomeres and interstitially. several times in the evolution of mammals (i.e., Australian marsupial Considering the phylogenetic relationships between the marsupials species and mouse). What could cause this change in chromosome studied here (Figure 1) (Duchêne et al., 2017), and previous reports in behavior? The determinants of bouquet polarization include the binding other vertebrates (Blokhina et al., 2019; Marín-Gual et al., 2022a), the of telomeres to the nuclear envelope and their interaction with most parsimonious explanation is that bouquet polarization and distal cytoskeleton components via transmembrane proteins of the nuclear synapsis initiation are ancestral characters in marsupials, and most envelope (Scherthan, 2007). It is unlikely that these dynamics have been probably in all vertebrates. Then, the loosening of telomere lost in eutherian mammals or in Australian marsupials, but they could be polarization at the beginning of meiosis (becoming more ephemeral regulated differently. Interestingly, a recent report revealed that the and/or less conspicuous), as well as loss of correlation between bouquet formation of a primary cilium in spermatocytes is a crucial factor in Frontiers in Cell and Developmental Biology 11 frontiersin.org Valero-Regalón et al. 10.3389/fcell.2023.1147610 TABLE 1 Percentage of RPA foci per chromosomal segment in the different bivalents (BV) and the X chromosome (X) of Thylamys elegans and Macropus eugenii. A χ2 of goodness of fit test (9 degrees of freedom) was performed to assess the deviation from a random distribution along chromosomes. Significance was considered when p ≤ 0.05. n: number of bivalents analyzed; f = number of foci. Thylamys elegans (% RPA) Segment BV1 (n = 31) BV2 (n = 35) BV3 (n = 33) BV4 (n = 35) BV5 (n = 33) BV6 (n = 35) X (n = 28) (f = 596) (f = 540) (f = 457) (f = 550) (f = 498) (f = 377) (f = 302) 1 26.51 21.85 29.10 27.64 16.87 11.41 14.57 2 9.90 9.07 7.88 10.00 10.24 8.49 9.60 3 5.03 6.30 6.13 5.45 7.43 7.16 9.93 4 2.52 3.15 3.72 6.00 9.44 7.69 2.98 5 5.54 4.81 4.81 3.09 6.22 5.31 3.64 6 4.87 7.22 2.19 3.82 6.02 3.98 8.28 7 3.36 5.56 3.06 4.18 6.83 8.49 10.26 8 6.21 8.15 4.81 6.91 5.62 10.34 10.60 9 11.74 9.26 13.13 8.55 13.45 16.98 12.91 10 24.33 24.63 25.16 24.36 17.87 20.16 17.22 χ2 397.19 256.15 377.73 377.02 93.28 88.79 52.76 p value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0004 Macropus eugenii (% RPA) Segment BV1 (n = 43) BV2 (n = 44) BV3 (n = 44) BV4 (n = 44) BV5 (n = 43) BV6 (n = 43) BV7 (n = 42) X (n = 31) (f = 1525) (f = 1051) (f = 1001) (f = 910) (f = 808) (f = 600) (f = 305) (f = 408) 1 9.18 9.80 9.59 10.11 10.02 10.17 7.87 10.29 2 7.54 8.56 10.49 9.67 10.02 11.50 12.79 8.82 3 11.54 9.13 9.99 9.56 10.27 10.17 10.49 3.19 4 11.08 10.37 9.99 7.69 8.42 8.83 10.16 6.13 5 9.97 10.75 10.49 10.66 10.27 7.33 4.59 9.56 6 11.02 11.04 9.79 11.43 11.01 10.50 6.23 10.54 7 10.30 11.04 10.99 10.11 10.52 9.83 13.11 11.76 8 11.28 10.75 9.79 10.88 11.51 11.17 11.80 10.78 9 9.38 10.56 10.59 11.32 10.27 11.33 15.08 14.95 10 8.72 7.99 8.29 8.57 7.67 9.17 7.87 13.97 χ2 22.94 11.16 4.98 11.54 9.47 8.93 30.31 43.81 p value 0.006 0.2649 0.8357 0.2406 0.3946 0.4435 0.0004 <0.0001 the formation of the bouquet in zebrafish (Mytlis et al., 2022). This et al., 2001; Turner et al., 2004), which appears as scattered foci structure is formed in spermatocytes at leptotene-zygotene, and its at early leptotene and then extends to occupy the whole nucleus removal disrupts bouquet formation, as well as synapsis and during late leptotene (Enguita-Marruedo et al., 2019). In eutherian recombination (Mytlis et al., 2022; Xie et al., 2022). Intriguingly, the mammals (i.e., mouse) the presence of γH2AX is then reduced as formation of the primary cilium in mouse seems to be differently prophase I progresses, and DNA is repaired but remains during late regulated, because this structure is formed in a reduced fraction of stages of prophase I in regions that do not achieve synapsis. This has spermatocytes at the leptotene-zygote transition (Lopez-Jimenez et al., been found to occur on both autosomes, as a feature related to the 2022). This seems to correlate with the absence of a conspicuous bouquet meiotic silencing of unsynapsed chromatin (MSUC) (Baarends et al., in the mouse, although a causative relationship has not yet been 2005; Turner et al., 2005; Manterola et al., 2009), and on the sex demonstrated. Further exploration of mammalian species could chromosomes where it contributes to meiotic sex chromosome provide new insight into the role that cilia play in bouquet formation. inactivation (MSCI) (Turner et al., 2004; Page et al., 2012). Early reports on the localization of γH2AX in marsupials indicated that MSCI also operates in this group (Franco et al., 2007; Hornecker Induction of DSBs and synapsis initiation and et al., 2007; Namekawa et al., 2007). However, other aspects of the progression localization of γH2AX in relation to DNA damage in marsupials have remained unexplored until recently. A characteristic hallmark of meiotic DNA damage is the We have previously shown in marsupials that there are two localization of phosphorylated H2AX (γH2AX) (Mahadevaiah waves of γH2AX accumulation during prophase I, along with lower Frontiers in Cell and Developmental Biology 12 frontiersin.org Valero-Regalón et al. 10.3389/fcell.2023.1147610 TABLE 2 Proportion of RPA foci and SC length of each bivalent at early pachytene, calculated over the number of foci and SC length of autosomes. Only cells in which all bivalents could be recorded have been included. Values are presented as mean ± standard deviation. n: number of cells analyzed; f = number of foci. Thylamys elegans (n = 34; f = 2958) BV1 BV2 BV3 BV4 BV5 BV6 % RPA foci 19.57 ± 4.7 17.91 ± 6.7 15.98 ± 4.9 18.52 ± 7.0 15.20 ± 4.3 12.81 ± 4.9 % SC length 23.89 ± 1.7 21.89 ± 1.7 19.70 ± 1.9 15.12 ± 2.1 9.82 ± 1.3 8.80 ± 1.0 Macropus eugenii (n=41; f= 6968) BV1 BV2 BV3 BV4 BV5 BV6 BV7 % RPA foci 23.84 ± 3.9 16.81 ± 3.6 15.75 ± 3.1 14.50 ± 2.8 13.68 ± 3.1 10.49 ± 2.5 4.93 ± 2.0 % SC length 22.91± 1.9 16.96 ± 2.0 15.37 ± 1.5 14.47 ± 2.6 13.89 ± 1.4 10.93 ± 1.8 5.47 ± 0.9 FIGURE 9 Compared proportion of RPA foci number and SC length chromosome in Thylamys elegans and Macropus eugenii. Each spot represents a bivalent in a cell. Black lines represent the calculated regression line. r = Spearman correlation coefficient. levels of γH2AX on autosomes when compared to eutherians linked to the silencing of unsynapsed regions, i.e., the MSUC/MSCI (Marín-Gual et al., 2022b). Here we extend these initial processes. In contrast, the widespread generation of DSBs in M. observations and report previously uncharacterized differences eugenii is correlated to synapsis initiation at different regions along between marsupial species. In T. elegans and D. gliroides γH2AX the chromosomes, not only chromosomal ends. Thus, the synapsis signal is scarce and mostly restricted to the regions where pattern of homologous chromosomes seems to be conditioned by homologous chromosomes initiate their synapsis, whereas in M. the way DNA damage is produced during prophase I. The pattern eugenii γH2AX is distributed across the whole nucleus. We suggest observed in T. elegans and D. gliroides seems to be ancestral, and that there is a relationship between this finding and the observed even shared by other non-mammalian vertebrates (Blokhina et al., patterns of synapsis initiation and progression. Thus, in the two 2019; Marín-Gual et al., 2022a). American species a low induction of DSBs would occur in the Finally, we highlight the possibility that a part of the DNA chromosomal regions polarized to the bouquet area, triggering the damage occurring in T. elegans and D. gliroides was not initiation of synapsis. In the absence of further (or abundant) DSBs accompanied by H2AX phosphorylation. Although the restricted along interstitial regions of chromosomes, synapsis would progress localization of γH2AX at the bouquet area correlates with from chromosomal ends towards the center of chromosomes, accumulation of RPA and RAD51 in these two species, some probably owing to the self-assembly capabilities of the SC RPA and RAD51 foci appeared outside the areas of γH2AX components. Therefore, the few DSBs scattered along interstitial accumulation. Previous reports in monotreme mammals (Daish regions do not seem to promote SC assembly. Interestingly, these et al., 2015) and some insects (Viera et al., 2017) have indicated that interstitial DSBs do not trigger a conspicuous H2AX γH2AX is not necessarily a marker of all DNA damage during phosphorylation either. Only later, during late zygotene, was prophase I. Our own observations indicate that γH2AX is not γH2AX observed at interstitial regions of the unsynapsed detected during prophase I in some reptiles (Marín-Gual et al., autosomes, as well as on the sex chromosomes. This could be 2022a) (Page, unpublished). Therefore, it seems that some aspects of interpreted as an indication of late DNA damage events DNA damage signaling during meiosis in mammals and other produced exclusively in those regions. Alternatively, it might be vertebrates are yet to be properly characterized. Frontiers in Cell and Developmental Biology 13 frontiersin.org Valero-Regalón et al. 10.3389/fcell.2023.1147610 FIGURE 10 Schematic representation of the two different patterns of SC assembly, synapsis progression and DNA repair observed in the marsupial species. Green lines represent AE/LEs, red clouds represent γH2AX and blue spots represent DNA repair proteins (RAD51/RPA). In each nucleus a bivalent and both sex chromosomes (X, Y) are represented. In the top row, the putative ancestral pattern involves: conspicuous bouquet polarization, AE assembly and synapsis progression from chromosomal ends and preferential localization of DNA damage and repair towards chromosomal distal regions. In the bottom row, the emergent pattern observed in Australian marsupials: loosened bouquet, AEs assembly and synapsis progression at any chromosomal position, and an even distribution of DNA damage and repair events along chromosomes. Differential chromosomal distribution of due to the lack of reactivity of many different antibodies against meiotic DSBs in marsupials MLH1 and other CO markers) in marsupials. In T. elegans RPA foci accumulated towards the chromosomal ends. This pattern resembles Perhaps the most striking finding of this study is the extreme the one characterized in humans, although it is much more difference in the distribution of DNA damage along chromosomes in prominent in the marsupial. In contrast, M. eugenii shows a the species analyzed. Several studies have focused on the overall frequency remarkably even distribution of DSBs along the chromosomes, of recombination across mammals or even eukaryotes (Dumont and resembling the pattern reported in mouse. Moreover, the number Payseur, 2008; Segura et al., 2013; Stapley et al., 2017). In eutherian of DSBs per chromosome has a high correlation with SC (Table 2). mammals, previous reports have found that early diverging linages had On the contrary, large chromosomes appear to accumulate less DSB lower recombination rates (Segura et al., 2013). Furthermore, marsupials than expected in T. elegans, a feature that has been described in show an even lower rate of recombination when compared to eutherians other species, from budding yeast to humans (Kaback, 1996; (Zenger et al., 2002; Samollow et al., 2004; Samollow et al., 2007; Dumont Subramanian et al., 2019). It remains to be determined whether and Payseur, 2008; Wang et al., 2011), which has been attributed to the D. gliroides adheres to one of these patterns, or a different one. induction of fewer DSBs during early stages of meiotic prophase I (Marín- We can only speculate on the mechanisms and consequences of Gual et al., 2022b). the differential chromosomal distribution of DSBs detected in While many studies have stressed the evolutionary relevance of marsupials. In mammals, and many other organisms, DSBs are the recombination rate on chromosomal evolution and populations produced preferentially at recurrent sites referred to as hotspots dynamics (Farré et al., 2012; Capilla et al., 2014; Ullastres et al., 2014; (Paigen and Petkov, 2010; Baudat et al., 2013). Two main types of Dapper and Payseur, 2017; Ritz et al., 2017), the genomic hotspots are usually recognized. The first are placed in promoter implications of the uneven distribution of recombination along regions of genes, which supposedly present an open chromatin chromosomes have received less attention. Initial reports in configuration that makes them accessible to the DSBs producing mouse and human showed that COs tend to locate towards the complexes. The second type is determined by the action of the telomeres (Barlow and Hultén, 1998; Froenicke et al., 2002). histone methyl transferase PRDM9, which tri-methylates histone Likewise, studies on the localization of DSBs by means of DNA H3 at lysine 4 (H3K4me), thus transforming these sites into repair markers like RPA and DMC1 reported an accumulation of preferential spots for breakage by the protein SPO11 (Baudat breaks towards chromosome ends in humans (Oliver-Bonet et al., et al., 2010; Parvanov et al., 2010; Brick et al., 2012). Whereas 2007; Pratto et al., 2014), which is not so evident in mouse (de Boer the first kind of hotspots are conserved within and between species, et al., 2006). Here we reveal striking inter-specific differences in the the ones depending on PRDM9 are more variable, owing to the fast- pattern of RPA distribution (CO distribution could not be analyzed evolving features of this enzyme (Grey et al., 2018). Most mammals, Frontiers in Cell and Developmental Biology 14 frontiersin.org Valero-Regalón et al. 10.3389/fcell.2023.1147610 including marsupials have a copy of the Prdm9 gene, but it has been Moreover, the fact that most DSBs are repaired as gene partially or completely lost in the platypus and canids (Cavassim conversion events (Cole et al., 2012; Baudat et al., 2013) does et al., 2022). Therefore, it seems unlikely that the different patterns of not preclude these breaks from being innocuous for the evolution DSB distribution we observe could be attributable to the absence of of some genome features, like GC content. Both reciprocal PRDM9. Instead, it is possible that a differential distribution, usage, recombination and gene conversion induce a shift to the or regulation of the different types of hotspots could be responsible accumulation of GC in the repaired strand, a phenomenon for such differences. One possibility is that T. elegans relies more in known as GC-biased gene conversion (gBGC) (Duret and the use of promoter-related hotspots, with genes concentrated near Galtier, 2009). This mechanism has been detected from yeast chromosomal ends. to mammals and has been proposed to impact the evolution of M. eugenii could be using more PRDM9-dependent hotspots, genomes (Mugal et al., 2015). For instance, it was suggested that which would be expected to result in a more uniform distribution of the enrichment of GC-rich isochores in mammalian genomes DSBs along chromosomes. This even distribution in M. eugenii could be in part a consequence of gBGC (Duret and Galtier, could be also related to the extensive genomic reorganizations 2009). The accumulation of GC content due to gBGC requires the experienced in the family Macropodidae (Deakin, 2018; Deakin recurrent use of recombination hotspots. Given the differential and O’Neill, 2020; Álvarez-González et al., 2022). In fact, recent use of these hotspots across species, different rates of GC reports have shown that lineage-specific evolutionary genomic accumulation are expected. This could partially explain why in reshuffling can influence patterns of higher-order chromatin humans, with a rapid turnover of PRDM9-dependent hotspot, organization (Farré et al., 2015; Álvarez-González et al., 2022), GC accumulation is spread in the whole genome, whereas in birds, and that chromosomal reorganizations can have an impact on with more conserved recombination hotspots, the increase of GC the three-dimensional genome folding and recombination in the content is much more localized to specific genomic regions (Mugal germ line (Vara et al., 2021; Álvarez-González et al., 2022). Thus, et al., 2015). Thus, given the distribution of DSBs in the marsupial genome reshuffling in macropodids could have led to a more even species studied, we foresee an accumulation of GC content due to gBGC distribution of recombination hotspots genome-wide. Interestingly, at the distal regions of chromosomes in T. elegans, compared to M. we found that the X chromosome behaves similarly in the species eugenii. compared here. It is possible that the X chromosome escaped this hotspot reorganization. Further analyses in the study of marsupial genomes and the use of ChIP-Seq approaches to map recombination Concluding remarks hotspots could yield insightful information about this possibility. Our results suggest that marsupials experienced a major shift in some of the key processes of meiosis, such as SC assembly, Genomic and evolutionary consequences of synapsis progression and DSB distribution (Figure 10). Many of divergent recombinogenic patterns these changes seems to have occurred after the split of Microbiotheria and the Australian marsupials, about 60 million The dissimilar pattern of DSBs chromosomal distribution years ago (Feng et al., 2022), although it remains to be may have consequences at the genomic level in marsupials. characterized if some features could have already been present Although not all the DSBs produced during prophase I result in the common ancestor of these two groups. Likewise, further into COs, it seems reasonable to assume that COs could be evenly research is required to determine to what extent the features distributed along chromosomes in M. eugenii. This would observed in M. eugenii are shared by other Australian facilitate the recurrent recombination of allele combinations, marsupials. Moreover, the features observed in T. elegans, thus breaking haplotypes. In the case of T. elegans, however, clearly basal to the rest of the marsupial groups, could have the accumulation of DSBs towards chromosomal ends would been shared with the ancestor of the eutherian mammals before reduce the possibilities of recombination at the interstitial they split apart about 165 million years ago. In view of recent regions of chromosomes. Supporting this view, a previous reports, these features could be even dated back to the appearance study reported that chiasmata are conspicuously terminal in T. of early vertebrates (Blokhina et al., 2019; Marín-Gual et al., elegans (Page et al., 2006). Since T. elegans has a very low 2022a). Expansion of meiosis studies to uncharacterized chromosome number, this would mean the formation of few mammals, including eutherians, marsupials and monotremes, as and large regions of linkage disequilibrium. Such a strategy could well as to other vertebrates (i.e., reptiles, amphibians or fishes), will be beneficial in a very stable environment, as long as allelic shed light on the evolution of meiosis across taxa. Moreover, these combinations at different loci had achieved an optimum studies will undoubtedly have a deep impact in our understanding (Stapley et al., 2017; Wang et al., 2019). In contrast, with of genome evolution. recombination spread all over chromosomes, the resulting generation of new genome-wide allele combinations could have provided some marsupial groups with a higher capacity Data availability statement to adapt to new environments. It is tempting to speculate that this factor could have had an influence in the diversification of The original contributions presented in the study are included in marsupials in Australasia after they diverged from the article/Supplementary Material, further inquiries can be directed Microbiotheria. to the corresponding author. Frontiers in Cell and Developmental Biology 15 frontiersin.org Valero-Regalón et al. 10.3389/fcell.2023.1147610 Ethics statement antibodies against crossover markers. We thank Corinne van Den Hoek and members of the wallaby research group (Walgroup) for The animal study was reviewed and approved by the UAM assistance with the wallabies. We also thank the two reviewers for Ethics Committee for Research and Animal Welfare. their positive and constructive comments. We dedicate this work to the loving memory of Prof. Francisco Bozinovic, for his enthusiasm and motivation in the study of Chilean fauna. Author contributions JP, RG, and PL-J conceived the study. JP, RG, AR-H, FV-R, Conflict of interest MS, and PL-J designed experiments. FV-R, MS, PL-J, MA, MM- R, RF, RG, and JP processed samples and performed the The authors declare that the research was conducted in the experiments. MR, GS, SB, and RF-D contributed materials and absence of any commercial or financial relationships that could be samples. FV-R, MS, PL-J, MA, MM-R, RF, LM-G, PW, AR-H, construed as a potential conflict of interest. RG, and JP and analyzed the data. JP wrote the manuscript and integrated inputs from all authors. All authors read and approved the final version of the manuscript. Publisher’s note All claims expressed in this article are solely those of the authors Funding and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the This work was supported by grants CGL 2014-53106-P to JP reviewers. Any product that may be evaluated in this article, or (Ministerio de Ecomonía y Competitividad, Spain), BIOUAM02- claim that may be made by its manufacturer, is not guaranteed or 2020 to JP and RG (Departamento de Biología, Universidad endorsed by the publisher. Autónoma de Madrid), PID 2020-112557 GB-I00 to AR-H. (Ministerio de Ciencia e Innovación, Spain) and from the Australian Research Council to MBR, GS, and PDW. Supplementary material (DP21103512 and DP220101429). PW is also supported by the NHMRC (APP1182667 and APP2021172). LM-G was supported by The Supplementary Material for this article can be found online a FPU predoctoral fellowship from the Ministry of Science, at: https://www.frontiersin.org/articles/10.3389/fcell.2023.1147610/ Innovation and University (FPU18/03867). full#supplementary-material SUPPLEMENTARY FIGURE S1 Identification of bivalents. Spread spermatocytes at pachytene labeled with Acknowledgments antibodies against SYCP3 (red), RPA (green), centromere proteins (blue) and fibrillarin (pink). (A). T. elegans. (B). M. eugenii. The length and centromere position allowed for the identification of each bivalent. 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