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Strategies for meiotic sex chromosome dynamics and telomeric elongation in Marsupials

['Laia Marín-Gual', 'Departament De Biologia Cel Lular', 'Fisiologia I Immunologia', 'Universitat Autònoma De Barcelona', 'Cerdanyola Del Vallès', 'Genome Integrity', 'Instability Group', 'Institut De Biotecnologia I Biomedicina', 'Laura González-Rodelas', 'Gala Pujol']

Date: 2022-04

During meiotic prophase I, homologous chromosomes pair, synapse and recombine in a tightly regulated process that ensures the generation of genetically variable haploid gametes. Although the mechanisms underlying meiotic cell division have been well studied in model species, our understanding of the dynamics of meiotic prophase I in non-traditional model mammals remains in its infancy. Here, we reveal key meiotic features in previously uncharacterised marsupial species (the tammar wallaby and the fat-tailed dunnart), plus the fat-tailed mouse opossum, with a focus on sex chromosome pairing strategies, recombination and meiotic telomere homeostasis. We uncovered differences between phylogroups with important functional and evolutionary implications. First, sex chromosomes, which lack a pseudo-autosomal region in marsupials, had species specific pairing and silencing strategies, with implications for sex chromosome evolution. Second, we detected two waves of γH2AX accumulation during prophase I. The first wave was accompanied by low γH2AX levels on autosomes, which correlated with the low recombination rates that distinguish marsupials from eutherian mammals. In the second wave, γH2AX was restricted to sex chromosomes in all three species, which correlated with transcription from the X in tammar wallaby. This suggests non-canonical functions of γH2AX on meiotic sex chromosomes. Finally, we uncover evidence for telomere elongation in primary spermatocytes of the fat-tailed dunnart, a unique strategy within mammals. Our results provide new insights into meiotic progression and telomere homeostasis in marsupials, highlighting the importance of capturing the diversity of meiotic strategies within mammals.

The generation of haploid gametes is a hallmark of sexual reproduction. And this is accomplished by a complex, albeit tightly regulated, reductional cell division called meiosis. Although meiosis has been extensively studied in eutherian mammal model species, our understanding of the mechanisms regulating chromosome synapsis, recombination and segregation during meiosis progression is still incomplete especially in non-eutherian mammals. To fill this gap and capture the diversity of meiotic strategies among mammals, we study previously uncharacterised representative marsupial species, an evolutionary assemblage that last shared a common ancestry more than 80 million years ago. We uncover novel, hence non-canonical, strategies for sex chromosome pairing, DNA repair, recombination and transcription. Most importantly, we reveal the uniqueness of marsupial meiosis, which includes the unprecedented detection of alternative mechanism (ALT) for the paternal control of telomere length during prophase I. Our findings suggest that ALT (previously only associated to cancer cells) could play a role in telomere homeostasis in mammalian germ cells.

Funding: This work was supported by the Ministry of Economy, Industry and Competitiveness (CGL2017-83802-P to A.R-H. and CGL2014-53106-P to J.P.), the Spanish Ministry of Science and Innovation (PID2020-112557GB-I00 to A.R-H.) and the Agència de Gestió d'Ajuts Universitaris i de Recerca, AGAUR (SGR1215 to A.R-H.). L.M.-G. and C.V. were supported by a FPU predoctoral fellowship from the Ministry of Science, Innovation and University (FPU18/03867) and by a FPI predoctoral fellowship from the Ministry of Economy, Industry and Competitiveness (BES-2015-072924), respectively. P.D.W. is supported by Australian Research Council Discovery Projects (DP170101147, DP180100931 and DP210103512). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Marín-Gual et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here we provide new insights into key features of meiotic prophase I progression in previously uncharacterised Australian marsupial linages, including an American taxon for comparison ( Fig 1 ). We examined the tammar wallaby (Macropus eugenii) a representative of Macropodidae, the fat-tailed dunnart (Sminthopsis crassicaudata) a representative of Dasyuridae, and the fat-tailed mouse opossum (Thylamys elegans) a representative of Didelphidae that is endemic to the Americas. We showed sex chromosomes pairing configurations during prophase I that are distinct between species, and described that there were lower levels of γH2AX on autosomes than sex chromosomes, most probably due to low rates of DSB formation. Importantly, we detected that telomeres were actively transcribed and elongated during prophase I in the fat-tailed dunnart, resetting all paternally inherited telomeres so that they are long in sperm.

Moreover, dasyurids are characterised by an extreme telomere length dimorphism between homologous chromosomes, which evolved before the dasyurid radiation at least 50 Mya [ 33 , 34 ]. Initial observations in male dasyurids showing that Y chromosomes had long telomeres and X chromosomes had short telomeres, suggested that telomere length dimorphism was due to a parental-of-origin effect, with long telomeres inherited from the paternal germline and short telomeres from the maternal germline [ 33 ]. This begs for description of a novel strategy of telomere length homeostasis in the parental germline. When and where in the male germline telomeres are elongated needs experimental validation.

(A) Phylogenetic relationships of the three marsupial species included in the study, with representation of sex chromosome structure for each species. Human sex chromosomes are included for comparison. Variation in diploid numbers is indicated for each phylogenetic branch. All marsupials lack a pseudo-autosomal region (PAR). The tammar (M. eugenii) X chromosome is large compared to the fat-tailed dunnart (S. crassicaudata) and the fat-tailed mouse opossum (T. elegans). Moreover, the tammar X chromosome bears NOR sequences in the centromeric region and the p-arm contains a region of shared DNA repeats with the q-arm of the Y chromosome [ 31 ]. (B) Meiotic karyotypes of the species included in the study: M. eugenii, S. crassicaudata and T. elegans. Karyotypes correspond to primary spermatocytes at pachytene labelled with antibodies against SYCP3 (green) and centromeres (red). The tammar sex chromosomes form a highly condensed dense plate at pachytene.

A salient feature of sex chromosomes is their transcriptional silencing during prophase I–a phenomenon called meiotic sex chromosome inactivation (MSCI) [ 19 , 20 ]. MSCI is a specialization of the MSUC (meiotic silencing of unsynapsed chromatin) process and it is characterised by accumulation of chromatin modifications in response to asynapsed chromatin during prophase I, including the phosphorylation of histone H2AX on serine 139 (γH2AX) [ 21 – 24 ]. MSCI is restricted to the heterogametic sex in species with heteromorphic sex chromosomes, and is a conserved epigenetic silencing programme in therian mammals [ 25 – 28 ]. It is a meiotic checkpoint that detects the presence of partial or completely unsynapsed homologous chromosomes, which results in the inactivation of ‘pachytene-lethal’ genes on the Y chromosome [ 29 , 30 ]. But, not all marsupials have the same sex chromosome structure [ 18 ], suggesting that there could be requirement for different meiotic pairing and silencing strategies. The tammar wallaby (and other macropods) are characterised by the X chromosome bearing a nucleolus organising region (NOR) near the centromere, along with a recently acquired satellite repeat region on the p-arm that is shared with the q-arm of the Y chromosome ( Fig 1 ) [ 18 , 31 ]. This is considered a derived state [ 31 , 32 ] as dasyurid marsupials (e.g., Tasmanian devil, quolls and dunnarts) have a conserved X chromosome structure that is shared with American marsupials [ 15 ].

One exceptional property of marsupial sex chromosomes is that, unlike eutherian sex chromosomes, the X and Y chromosomes do not share a homologous region within which recombination occurs (i.e., pseudo-autosomal region or PAR) [ 16 ]. As a consequence, sex chromosomes associate during prophase I via a marsupial specific structure called the dense plate (DP) [ 13 , 17 , 18 ], which is rich in synaptonemal complex proteins and ensures faithful segregation in the absence of synapsis and recombination.

Mammals (represented by monotremes, marsupials and eutherians) last shared a common ancestor approximately 185 million years ago (Mya) [ 4 ] and are characterised by distinctive genome plasticity [ 5 , 6 ]. Despite genome reshuffling, canonical features of the meiotic programme are well conserved in eutherian mammals (i.e., human, non-human primates, rodents and bovids) [ 3 , 7 – 12 ]. In contrast, detailed immunofluorescence studies on chromosome pairing during prophase I in marsupials are scarce and restricted to a handful of American species [ 13 , 14 ]. Due to their distant relationship with eutherian mammals, and that Australian and American species shared a common ancestor 80 Mya [ 15 ], marsupials offer a unique opportunity to explore previously uncharacterised meiotic features. This includes unique sex chromosome pairing strategies, recombination and meiotic telomeric homeostasis.

A hallmark of sexual reproduction is the generation of haploid gametes with half the chromosome complement of progenitor cells by a complex, albeit tightly regulated, reductional cell division called meiosis. Meiosis generates genetically variable gametes by homologous recombination, which involves faithful chromosome synapsis and DNA exchange between homologous chromosomes during meiotic prophase I. The mechanisms underlying meiotic progression have been extensively studied in model organisms, including yeast, fruit flies, nematodes, mice and, more recently, zebrafish [ 1 , 2 ]. This has revealed canonical features that are conserved across large evolutionary time scales, including fundamental events such as the formation of double strand breaks (DSBs—essential for meiotic recombination), homologous chromosome pairing and synapsis, and the formation of the telomeric bouquet. However, important differences between taxa have been noted, highlighting that our understanding of mammal meiotic prophase I is still incomplete, especially in non-traditional model species [ 3 ].

Results

Sex chromosome meiotic pairing strategies in marsupials The chromosome complement of tammar wallaby is 2n = 16, whereas both fat-tailed mouse opossum and fat-tailed dunnart are characterised by 2n = 14 (Fig 1). Differences in diploid numbers are mainly due to lineage-specific chromosome rearrangements in macropods [35], highlighting the derivative state of this clade of Australian marsupials. Anti-SYCP3 antibody labelled axial elements of the synaptonemal complex were used to classify spermatocytes into the different prophase I stages, following previous observations in marsupials [13,14] (Fig 2). At leptotene, short filaments of SYCP3 were observed in the three species, representing the forming axial elements (S1 Fig). Chromosome ends appear clustered in a bouquet configuration (S1A Fig). Axial elements become larger at zygotene, when synapsis between homologous chromosomes takes place, as revealed by SYCP3 and SYCP1 labelling (Fig 2A). The distinction between early and late zygotene was based on the relatively length of discontinuous axial structures. At pachytene, autosomes have completed synapsis. Spermatocytes at pachytene were divided into three sub-stages (early, mid and late) based on the previously described structure and behaviour of sex chromosomes [13]. Briefly, at early pachytene sex chromosomes were separated with thick axial element labelling (SYCP3 signal). At mid pachytene sex chromosomes became associated, had thinner axial elements than autosomes and the DP was beginning to form. Late pachytene was distinguished by the presence of a fully developed DP. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Pairing dynamics during prophase I. (A) Spermatocyte spreads in prophase-I labelled with antibodies against SYCP3 (green) and SYCP1 (red) for the tammar wallaby (M. eugenii), the fat-tailed dunnart (S. crassicaudata), and the fat-tailed mouse opossum (T. elegans). Scale bar = 10μm. (B) Sex chromosomes pairing configurations during prophase I (separated, associated, X ring or DP) for tammar wallaby, fat-tailed dunnart, and fat-tailed mouse opossum. In tammar wallaby the DP can adopt two different configurations: the early DP has an open configuration, whereas the late DP is compacted. Scale bar = 2μm. (C) Percentage of cells with different sex chromosomes configurations for primary spermatocytes from tammar wallaby (N = 41 cells in zygotene, N = 19 cells in early pachytene, N = 49 cells in mid pachytene and N = 81 cells in late pachytene), fat-tailed dunnart (N = 22 cells in zygotene, N = 19 cells in early pachytene, N = 19 cells in mid pachytene and N = 28 cells in late pachytene), and fat-tailed mouse opossum (N = 23 cells in zygotene, N = 22 cells in early pachytene, N = 161 cells in mid pachytene and N = 168 cells in late pachytene). Cell type legend: Z, zygotene; EP, early pachytene; MP, mid pachytene; LP, late pachytene. https://doi.org/10.1371/journal.pgen.1010040.g002 The general trend in all three marsupials was for the X and Y to associate after autosomes had paired (Figs 2 and S1B–S1D). During mid pachytene the sex chromosomes approach each other to form the DP, adopting four possible configurations that were classified as the following: (i) separated–sex chromosomes not in contact, (ii) associated–sex chromosomes in contact but DP not formed, (iii) ‘X ring’–the X chromosome forms a ring while approaching the Y, along with thickening of their axes without forming the DP, and (iv) DP–sex chromosomes come together and the DP forms (Fig 2B). In tammar, the DP adopted two further configurations: an early DP with an open configuration, and a late DP with a more compacted structure (Fig 2B). Although these four sex chromosomes pairing configurations were present in all three marsupials, differences in structure were apparent in early pachytene. Separated X and Y chromosomes in tammar wallaby showed thicker axial axes (SYCP3 labelling) than the fat-tailed mouse opossum and dunnart (Fig 2A and 2B). As sex chromosomes approach each other in early pachytene, the tammar X chromosome remain in a ‘stretched’ configuration. This contrasted the fat-tailed mouse opossum and dunnart, in which telomeres of the X chromosome were close to each other (Fig 2B). Whereas the fat-tailed mouse opossum and dunnart formed a neat and clear ring, the tammar X associated in a large, intensely stained irregular structure. This was not resolved until formation of the DP, which was also different in configuration when compared to the non macropod species (Fig 2B). These results mirror early electron microscopy observations [18], and are consistent with the pattern detected in T. elegans and other American species [14]. Moreover, we found differences in the proportions of pairing configurations as prophase I progressed, particularly during mid pachytene (Fig 2C). The fat-tailed mouse opossum (the American marsupial representative) presented very few cells with X chromosomes forming rings in mid pachytene (3.7% cells). This contrasted both Australian representatives (tammar wallaby and fat-tailed dunnart), where 34.7% and 36.8% of cells, respectively, presented X rings. Collectively, these results suggest differences in the timing of sex chromosomes association and DP formation.

Localisation of γH2AX on sex chromosomes and transcription are not mutually exclusive in tammar wallaby We then studied the dynamics of γH2AX on sex chromosomes, as it is known to be associated with MSCI in eutherians [25]. During leptotene and zygotene we detected scarce and faint γH2AX signals in the whole nucleus in all three marsupial species (Fig 5). As sex chromosomes approached each other during late zygotene and pachytene, the γH2AX signal was restricted to both the X and Y chromosomes, forming discrete chromatin domains, even if the chromosomes were located at opposite poles of the cell (Fig 5). Gamma H2AX displayed a stronger signal in late pachytene, concomitant with the formation of the DP in all three species (Fig 5), mirroring previous observations [26,28]. PPT PowerPoint slide

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TIFF original image Download: Fig 5. γH2AX progression during prophase-I in marsupials. Spermatocyte spreads labelled with antibodies against SYCP3 (green) and γH2AX (red) for (A) tammar wallaby (M. eugenii), (B) fat-tailed dunnart (S. crassicaudata) and (C) fat-tailed mouse opossum (T. elegans). Scale bar = 10μm. https://doi.org/10.1371/journal.pgen.1010040.g005 Crucially, in tammar wallaby the γH2AX signal collocated with the phosphorylated RNA pol II signal on the X chromosome during zygotene (mainly late zygotene) and early/mid pachytene, before depletion of RNA pol II at late pachytene (Fig 3D) and formation of a closed chromatin domain (Fig 5). Therefore, γH2AX signal was detected on sex chromosomes even though they were physically separated and the X chromosome transcriptionally active (Figs 3 and 5). It was not until formation of the DP in late pachytene, when sex chromosomes were devoid of RNA pol II signal and the γH2AX signal was more intense. This suggests that RNA (either coding or non-coding/repetitive regions) transcribed from the X chromosome (p- and q-arm) escape silencing in tammar until DP formation and MSCI initiation in late pachytene.

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