Meiotic analyses show adaptations to maintenance of fertility in X1Y1X2Y2X3Y3X4Y4X5Y5 system of amazon frog Leptodactylus pentadactylus (Laurenti, 1768)

Heterozygous chromosomal rearrangements can result in failures during the meiotic cycle and the apoptosis of germline, making carrier individuals infertile. The Amazon frog Leptodactylus pentadactylus has a meiotic multivalent, composed of 12 sex chromosomes. The mechanisms by which this multi-chromosome system maintains fertility in males of this species remain undetermined. In this study we investigated the meiotic behavior of this multivalent to understand how synapse, recombination and epigenetic modifications contribute to maintaining fertility and chromosomal sexual determination in this species. Our sample had 2n = 22, with a ring formed by ten chromosomes in meiosis, indicating a new system of sex determination for this species (X1Y1X2Y2X3Y3X4Y4X5Y5). Synapsis occurs in the homologous terminal portion of the chromosomes, while part of the heterologous interstitial regions performed synaptic adjustment. The multivalent center remains asynaptic until the end of pachytene, with interlocks, gaps and rich-chromatin in histone H2A phosphorylation at serine 139 (γH2AX), suggesting transcriptional silence. In late pachytene, paired regions show repair of double strand-breaks (DSBs) with RAD51 homolog 1 (Rad51). These findings suggest that Rad51 persistence creates positive feedback at the pachytene checkpoint, allowing meiosis I to progress normally. Additionally, histone H3 trimethylation at lysine 27 in the pericentromeric heterochromatin of this anuran can suppress recombination in this region, preventing failed chromosomal segregation. Taken together, these results indicate that these meiotic adaptations are required for maintenance of fertility in L. pentadactylus.


Results
The studied specimens showed a karyotype with 2n = 22 chromosomes. FISH analysis with a telomeric probe was performed in ten diakinesis of each individual and showed marking only on the chromosomal ends of the five bivalents with two terminal chiasms, one bivalent with an interstitial chiasm and a multivalent formed by five chromosomal pairs. No interstitial markings were observed (Fig. 1).
The meiotic behavior of L. pentadactylus chromosomes was investigated by immunodetection of SMC3 that marks the axis of cohesins associated with the complex synaptonemal. Quantitative analysis of the progression of the synapse during the phases of prophase I is shown in Fig. 6a. During leptotene, a short lateral axis of the synaptonemal complex formed and remained asynaptic (Fig. 2a). During early zygotene, homologous pairing started through the chromosomal tips, which were located at a pole of the cell nucleus (Fig. 2b). Formation of interlocks was also observed during this phase (Fig. 2c). In late zygotene, the polarization of chromosomal tips was no longer visible, but interlocks between bivalents and the asynaptic axis, as well as gaps, were observed (Fig. 2d,  www.nature.com/scientificreports/ During pachytene, the synaptonemal complex formed and extended throughout the bivalents; during late pachytene, long asynaptic regions, gaps and interlocks were observed (Fig. 2f,g). Synapsis was incomplete in the center of the multivalent complex, suggesting an absence of homology in this region (Fig. 2h,i). None of the cells in pachytene showed an open configuration of the meiotic ring. The synaptonemal complex became disordered during early diplotene, making it possible to detect asynaptic regions during this phase (Fig. 2j). Five bivalents and the meiotic ring could be distinguished during diplotene and diakinesis (Fig. 2k,l). Moreover, the disorganization of the synaptonemal complex during these phases resulted in the dispersion of SMC3 cohesion throughout the chromosome axis (Fig. 2k,l).
Immunodetection of Synaptonemal Complex Protein 3 (SYCP3), a protein of the lateral element of the synaptonemal complex, demonstrated similar results to SMC3, in relation to the dynamics of pairing, synapse and synaptic adjustment along the prophase I of L. pentadactylus (Fig. 7a,d). Immunolocalization of CREST on SYCP3 axes in early pachytene demonstrated that in this stage centromeric region is synapsed in some axes, while in others was observed process of pairing of kinetochores (Fig. 7a-c). In intermediate/late pachytene, immunodetection of CREST revealed a pattern similar to observed in early pachytene, with presence of centromeres in synapsed/adjusted or asynaptic regions of multivalent ( Fig. 7d-f).
To verify the distribution of DSBs performed by Spo11 and the occurrence of MSUC during prophase I of L. pentadactylus, we performed immunodetection of γH2AX. Incubation with antibody to γH2AX revealed that this histone variant was diffusely present during leptotene (Fig. 3a-c) but was strongly present at the chromosomal ends, the start of synapses, in zygotene cells (Fig. 3d-f). γH2AX was strongly expressed throughout the chromatin during the transition from zygotene to pachytene (Fig. 3g-i). During intermediate pachytene, this marker was weakly expressed throughout the chromatin, but was strongly expressed in synapsed regions ( Fig. 3j-l). During diplotene, γH2AX signals were observed in the chromosomal chain and the bivalent ( Fig. 3m-o). γH2AX signals, however, were progressively reduced during diakinesis ( Fig. 3p-r).
The repair dynamics of DSBs in the early stages of L. pentadactylus prophase I was investigated through the immunodetection of Rad51. This recombinase was slightly expressed during leptotene (Figs. 4a-c, 6b). In early zygotene, Rad51 show polarized expression in the nucleus, predominantly on non-synapsed SMC3 axes in an average 48 (Fig. 4m-o). ANOVA test demonstrated that the difference in the amount of Rad51 foci in the pachytene is significant compared to the zygotene (p < 0.001). Rad51 expression, however, was not observed during diplotene. Analysis of the colocalization of Rad51 and telomeres showed that this association is more frequent during the early and late zygotene, with a statistically significant decrease during the transition to the pachytene (Kruskal-Wallis p < 0.001) (Fig. 6c).
Immunodetection of H3K27me3 was performed to investigate the role of this epigenetic modification in transcriptional inactivation of chromatin during L. pentadactylus prophase I. H3K27me3 was observed in isolated regions of chromatin during leptotene (Fig. 5a,b), with more intense expression in an isolated region also observed during zygotene (Fig. 5c,d). During pachytene and diplotene, this epigenetic marker was observed on the synaptic or asynaptic axis of chromosomes ( Fig. 5e-h). This pattern of H3K27me3 distribution was also observed during diakinesis/metaphase I, with additional weak signals throughout other chromosomal regions ( Fig. 5i,j). In metaphase II, H3K27me3 was observed only in the pericentromeric heterochromatin ( Fig. 5k,l).

Discussion
The present results confirm that 2n = 22 is conserved in the Amazon frog L. pentadactylus. However, the number of meiotic ring components was found to vary intraspecifically, with ten chromosomes observed in the present study and 12 in a previous study of this species 34 . These cytotypes differ in relation to a large bivalent observed in our sample. In specimens from the state of Mato Grosso, this bivalent was regarded as likely being a component of the multivalent complex [32][33][34] . Meiotic chains, whether associated with autosomal or sex chromosomes, are unstable and can bind other karyotype-associated elements, depending on the occurrence of new rearrangements during the evolutionary process 36 . In this way, we describe a new multiple sex chromosome system, X1Y1X2Y2X3Y3X4Y4X5Y5, in the Amazon frog L. pentadactylus. The cytotype described here may be distinct from those of other populations of L. pentadactylus. Similarly, a phylogenetic analysis based on mitochondrial (16S and COI) and nuclear (BDNF and C-myc) genes showed high genetic divergence among populations of L. pentadactylus in the Amazon basin, suggesting that chromosomal rearrangements could explain this large degree of differentiation of this populations 34 .
The mechanisms by which sex chromosomes pair are highly variable, with some pairings involving short terminal homologous regions of allosomes, called pseudoautosomal regions. In the platypus, for example, the sex chromosome pairing involves five pseudoautosomal regions 37 . BrdU banding indicated that the meiotic ring in L. pentadactylus originated from multiple translocations among terminal portions of the chromosomes involved 32 . The present study showed that these short rearranged regions are sufficiently homologous to enable meiotic pairing in these species to start regularly at chromosomal ends during zygotene. Movements of telomeres and their associations with nuclear membranes in bouquet may also contribute to the correct co-alignment of the meiotic ring components of L. pentadactylus 38,39 . Moreover, the presence of γH2AX and Rad51 at non-synapsed chromosomal tips in leptotene, and their subsequently becoming synapsed in zygotene (Fig. 6c), indicate that double strand-breaks (DSBs) during early prophase I are important for meiotic pairing in this species (see section "Introduction"). The recombinases Rad51 and Dmc1 repair these breaks using the homologous chromosome as a template 40 . In Danio rerio, for example, the activation of these enzymes next to telomeres is almost completely Scientific RepoRtS | (2020) 10:16327 | https://doi.org/10.1038/s41598-020-72867-x www.nature.com/scientificreports/ homologous for initiation of local synapses 39 . This event may be needed to recognize homologous sequences and start the organization of synaptonemal complexes in L. pentadactylus. www.nature.com/scientificreports/ Once started, synapsis covers the homologous terminal portion of chromosomes, and proceeds throughout most of the length of the ring components. Because of rearrangements 32 , these non-terminal regions may lack homology, suggesting extensive heterosynapsis in this anuran species. This type of synaptic adjustment is widely found in mammals [41][42][43] , as it prevents the dangerous effects of transcriptional inactivation and of the action of www.nature.com/scientificreports/ meiotic checkpoint proteins 44 . Heterosinapses between centromeres, as observed in L. pentadactylus (Fig. 7), are facilitated by the fact that such regions do not need homology to perform synapse, due to their structural heterogeneity 18 . The presence of Rad51 in these heterosynapsed regions during pachytene of L. pentadactylus suggests that this protein can help stabilize heterosynapses after formation of the SYCP3 axis 39,43 . The multivalent center remains asynaptic until the beginning of the diplotene, suggesting that heterosynapse is not fully performed in this species. This result differs from findings in some pigs, which are characterized by complete synaptic adjustment, overtaking the breakpoint limits and not permitting central asynapsis 45 . In L. pentadactylus, the asynapsis in the center of the multivalent complex can also contribute to the suppression of crossing-over in this region, avoiding segregation problems during anaphase I. The maximum degree of heterosynapsis of multivalent complexes occurs later than that of regular bivalents, which, during later pachytene, are completely synapsed or in an initial stage of disorganization of synaptonemal complexes. This asynchrony may be due to the lack of homology and by spatial arrangements of chromosomes involved in meiotic multiplexes inside the cell nucleus 46,47 . Interlocks, as observed in zygotene and pachytene of L. pentadactylus, are often present in individual carriers of heterozygous translocations and can delay synaptic processes 19 . Chromosomal movements associated with depolymerization and subsequent reorganization of components of synaptonemal complexes can resolve interlocks 48 . These findings suggest that some asynaptic regions and gaps registered in pachytene of L. pentadactylus may be associated with this mechanism of action.
At the start of meiosis I, the protein H2AX is phosphorylated by the kinase ATM, promoting structural modifications at sites of DSBs that can assist in DNA repair 49 . These findings suggest that the immunolocalization pattern of γH2AX in leptotene/zygotene of L. pentadactylus corresponds to regions of programmed DNA breaks, which are involved in the recombination process and contribute to homologous pairing. The results of the present study also indicate that these epigenetic modifications are highly dispersed throughout cellular chromatin during the zygotene to pachytene transition. A second wave of γH2AX formation may involve ATR kinase rather than ATM, typical of the prophase I stage 23 . Similar dynamics have been observed in eutherian mammals; www.nature.com/scientificreports/ however, the progression of pachytene tends to reduce the quantity of γH2AX, with the latter located only in sex chromosomes 50,51 . In contrast, γH2AX in L. pentadactylus persists until diplotene, albeit in reduced quantities, on all chromatins of the ring and the bivalents. The fact, that γH2AX is expressed in asynaptic regions of meiotic multiples 43,52 , may be due to the delay associated with synapsis and in DSBs repair, as they recruit silencing factors such as ATR for these regions 22 . Proto-XY has been identified in meiotic rings of L. pentadactylus 34 . The association of multivalents with XY sex bodies results in the spreading of γH2AX, increasing the signals of this histone variant in asynaptic regions containing the multivalents 53 . Thus, we suggest that asynaptic regions of the meiotic multivalent of L. pentadactylus are transcriptionally inactivated during pachytene by the formation of γH2AX. The extensive asynapsis in L. pentadactylus may promote infertility in males of these species. Normally, failures in the synaptic process block the meiotic cycle, induce apoptosis, and eliminate spermatocytes, reducing the fertility of that individual 41 . Transcriptional silence is required for progression of the meiotic cycle. However, in cases of high asynapsis, spermatocytes can present errors in MSUC, inactivating genes crucial for meiosis and for the survival of spermatids, as well as initiating apoptosis 54 . Although a checkpoint in pachytene is sensitive to synapse and repair of DSBs 55 , this checkpoint can tolerate a certain degree of asynapsis 7,56 . Moreover, the stop in the meiotic cycle is dependent on the genes that are activated and inactivated because of the defect in MSUC 57 . Although the central region of the multivalent complex remains asynaptic during late/intermediate pachytene of L. pentadactylus, some heterosynapsed regions were positive for foci of Rad51 and γH2AX. This may be sufficient to send positive feedback to the pachytene checkpoint, allowing the normal progression of prophase I 56 . Similar results were observed in mice 58 .
Fertility analysis of L. pentadactylus should also consider chromosomal segregation in anaphase I. A terminal or subterminal crossing-over pattern is required for multivalents to form a zig-zag configuration during the www.nature.com/scientificreports/ metaphase I/anaphase I transition 36 . Only a small fraction of DSBs produced during leptotene/zygotene are repaired during cross-over 18 . The pattern of Rad51 expression during early meiosis I in the present study suggested that homologous recombination occurs in synapsed regions of the L. pentadactylus multivalent, adjacent to chromosomal ends, which were shown to be homologous 36 . The absence of chiasma from the interstitial regions of meiotic rings of L. pentadactylus is consistent with the terminal recombination observed in this amphibian species 32 . Moreover, the alternating anaphasic segregation of multivalent components of L. pentadactylus 32 , indicates that the meiotic cycle follows regularly, ensuring the formation of balanced gametes and contributing to the maintenance of fertility in this species. The distribution of H3K27me3 in metaphase II observed in the present study suggested that this histone modification is an indicator of pericentromeric heterochromatin in this species. Moreover, this is the first study to show the presence of an epigenetic marker in the centromere of an anuran. In other species, H3K27me3 is generally related to facultative heterochromatin, promoting the reversible inactivation of (i) genes presents on the X chromosome 59 ; (ii) repetitive DNA associated with synaptonemal complex organization 60,61 ; (iii) the nonsynapsed autosomal region 24 ; and (iv) the ZW chromatin of birds 26 . DSBs tend not to form in repetitive regions, as they may promote crossing-over in non-allelic regions, generating genomic instability, harmful rearrangements to chromosomal segregation 62,63 . In most organisms, DNA sequences located in pericentromeric regions are generally silenced by H3K9me3 62,64 . The simultaneous occurrence of H3K27me3 and other epigenetic markers at some genomic loci results in the simultaneous regulation of several genetic functions, including the inactivation of transposable elements [65][66][67] . Moreover, despite the absence of H3K9me3, the formation and stability of pericentromeric heterochromatin are maintained due to allocation of H3K27me3 to this region 68 . These findings suggest that the presence of H3K27me3 in the pericentromere during meiosis of L. pentadactylus may be necessary to avoid recombination adjacent to the centromere, preventing problems with chromosomal segregation, especially of elements involved in sex ring.
The present study demonstrated that the multiple sex chromosome system in L. pentadactylus can present interpopulation variation and suggest that a set of meiotic mechanisms intrinsic to this species (including partial synaptic adjustment, pericentromeric sequence inactivation by H3K27me3, MSUC and persistence of Rad51 foci) help regulate the meiotic cycle and ensure the maintenance of fertility in this anuran. These findings demonstrate the high functional plasticity of meiotic proteins, and allow us to understand the way in which cells of

Methods
Sample.  Fluorescence in situ hybridization (FISH). Testes were incubated in hypotonic solution, consisting of 0.075 M KCl for 30 min at 37 ºC, and fixed in Carnoy's solution composed of methanol and glacial acetic acid (3:1). Slides containing meiotic preparations were treated with 1% pepsin for 20 min at 37 ºC and washed in 2 × SSC solution. The slides were subsequently dehydrated in 70%, 90% and 100% alcohol for 5 min each. The telomeric probe was denatured by incubation at 100 ºC and the solution added to the slides. The slides were then covered with cover slips, and the DNA was denatured by incubation at 70 ºC. The slides were subsequently washed in 2 × SSC and 4 × SSC-Tween 20 at 40 ºC, followed by incubation with FITC-conjugated antibody to digoxigenin. The samples were subsequently counterstained with DAPI containing antifading Vectashield.
Statistical analysis. An average of 30 cells was analyzed to define each meiotic stage. Measurements of the bivalent and multivalent synapsed/adjusted region were obtained from 52 spermatocytes (between leptotene and pachytene) using the Drawid software 69 . The Rad51 foci count was performed on 88 spermatocytes (between zygotene and pachytene). Graphical and statistical analyzes were generated using the GraphPad 6.0 software. The normality of the data was verified using the Shapiro-Wilk test. Only foci present at the ends of the synapsed synaptonemal complex were considered, to determine the fraction of Rad51 colocated with telomeres, and the averages obtained were compared using the Kruskal-Wallis non-parametric test. Rad51 averages on synapsed/adjusted, and asynaptic axes were compared using the ANOVA and Turkey test. In all statistical analyzes, the level of significance was p = 0.005.

Data availability
All relevant data are within the paper. Data can be requested from the corresponding author.