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

Abstract

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.

Figure 1

Meiotic multivalent in L. pentadactylus. (a) Diakinesis showing ring (ten chromosomes) and six regular bivalents. (b) FISH with telomeric probe (green) in diakinesis cell of L. pentadactylus. (c) Schematic representation of cell in (“b”); arrow = meiotic ring, arrowheads = bivalent with an interstitial chiasma, asterisks = bivalent with two terminal chiasmas. Barr = 10 μm.

Figure 2

Temporal dynamics of synaptonemal complex in L. pentadactylus through SMC3 (red). (a) Leptotene. (b) Early zygotene; the arrows indicate interlocks, arrowheads show regions of homologous early pairing. (c) Schematic representation of interlocks observed in (“b”). (d) Late zygotene: arrow indicates tips of bivalents realizing synapsis. (e) Schematic representation of bivalent evidenced in “d”, highlighting interlocks along of non-synapsed region. (f) Early pachytene; asterisk demonstrate gaps, arrowhead shows interlock, and arrow indicates bivalent non-synapsed. (g) Intermediate pachytene. (h) Late pachytene; the arrows indicate interlocks between bivalent and asynaptic regions of multivalent. (i) Schematic representation of non-homologous synapsis and interlock evidenciated in (h). (j) Early diplotene (arrows show asynaptic regions). (k) Late diplotene. (l) Diakinesis. Barr = 10 μm.

Figure 3

Distribution of γH2AX epigenetic modification along the synaptic process in L. pentadactylus. The cohesin SMC3 is shown in green, and γH2AX in red. In the right column, overlay both images. (a–c) Leptotene. (d-f) Zygotene; note the strongly marked terminal regions. (g–i) Late zygotene. (j–l) Intermediate/late pachytene. (m–o) Diplotene. (p–r) Diakinesis. Barr = 10 μm.

Figure 4

Immunolocation of Rad51 in L. pentadactylus prophase I. The cohesin SMC3 is shown in green, and Rad51 in red. In the right column, overlay both images. (a–c) Leptotene. (d–f) Leptotene/zygotene transition. (g–i) Zygotene. (j–l) Initial pachytene. (m–o) Late Pachytene. Barr = 10 μm.

Figure 5

Distribution of H3K27me3 in meiosis I of L. pentadactylus. The H3K27me3 marking is shown in green; chromosomes were counterstained with DAPI. (a,b) Leptotene. (c,d) Zygotene. (e,f) Pachytene. (g,h) Diplotene. (i,j) Diakinesis/metaphase I. (k,l) Metaphase II. Barr = 10 μm.

Figure 6

Quantitative analysis of synapsis and Rad51 foci in meiosis I of L. pentadactylus. (a) Synapsis progression during L. pentadactylus prophase I, based on the average of the synapsed/adjusted SMC3 axis lengths. (b) Distribution of Rad51 on synapsed/adjusted and asynaptic SMC3 axes; differences between the means of foci Rad51 is highly significant between zygotene and pachytene (p < 0.001). (c) Dot plot demonstrating the distribution of Rad51 foci in telomeric regions during L. pentadactylus prophase I. Each dot represents a cell (n = 88). Significant differences are observed between the means of foci of this recombinase between zygotene and pachytene (Kruskal–Wallis test, p < 0.001).

Figure 7

Immunodetection with SYCP3 (red) and CREST (green) showing distribution of centromeres on synapsed/adjusted and asynaptic axes of synaptonemal complex. (a–c) Early pachytene. (d–f) Late pachytene. The arrows indicate kinetochores in synapsed/adjusted regions; arrowheads indicate kinetochores in asynaptic regions. The centromere markings in asynaptic regions are highlighted in the box. Barr = 10 μm.