Fig 2: Rbm46 −/− arrested spermatocytes have shortened AEs and display partial synapsis. (A) Chromosome spreads of PD35 Rbm46+/− and Rbm46−/− spermatocytes costained with the chromatid central element marker SYCP1 (green) and the AE marker SYCP3 (red), showing that Rbm46−/− spermatocytes arrest at the leptotene stage and present “zygotene-like” spermatocytes. The scale bar is 5 µm. (B) Chromosome spread of Rbm46+/− and Rbm46−/− mice spermatocytes costained with SYCP3 (red) and cohesin subunits RAD21 and SMC3 in leptotene and zygotene or zygotene-like spermatocytes. The scale bar is 5 µm. (C) Chromosome spread of Rbm46+/− and Rbm46−/− mice spermatocytes costained with SYCP3 (red) and γ-H2AX as indicated for DSB signal; DMC1 and RAD51 as indicated for DSB-repair foci. The scale bar is 5 µm. (D) Model of RBM46 functional mechanism in meiosis. Abnormal distribution of cohesin subunit proteins was observed in Rbm46−/− mice spermatocytes, and chromosomes showed shortened AEs and partial synapsis, resulting in failure of meiosis to progress to the zygotene stage.

Fig 3: Transcription dynamics during prophase I in the tammar wallaby.(A) Representative images of tammar wallaby (M. eugenii) spermatocyte spreads in prophase-I labelled with antibodies against SYCP3 (green) and RNA pol II (red). Scale bar = 10μm. (v-vi) Active transcription of the X chromosome with an open configuration. Scale bar = 2μm. White dashed circles: RNA pol II signal on the X chromosome. (B) (i) Percentage of cells with different transcription patterns for spermatocytes in leptotene (N = 9 cells), early zygotene (N = 24 cells), late zygotene (N = 29 cells), early pachytene (N = 19 cells), mid pachytene (N = 49 cells) and late pachytene (N = 81 cells). (ii) Percentage of cells with different transcription patterns for spermatocytes with different sex chromosome configurations: separated (N = 58), associated (N = 10 cells), X ring (N = 17 cells), early DP (N = 24 cells) and late DP (N = 81 cells). (C) Transcription along the X chromosome. (i) Spermatocyte spread labelled with antibodies against SYCP3 (green), RNA pol II (red) and centromeres (blue). White dashed circles: X chromosome. Scale bar = 10μm. (ii) Distribution of the RNApol II signal along X chromosomal arms. The X axis represents the positions on the chromosomal axes from the centromeric end (black dot) to p-arm and q-arm telomeres. The Y axis indicates the frequency of RNA pol II signal detected for each 10% interval of chromosomal length (N = 30 cells). Black dot: centromere. (D) Co-localization of RNA pol II and γH2AX. Spermatocyte spreads labelled with antibodies against SYCP3 (green), RNA pol II (red) and γH2AX (blue). White dashed circles: sex chromosomes. Scale bar = 10μm.

Fig 4: 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.

Fig 5: Failure to repair DNA damage and form crossovers in Ino80cKO spermatocytes. A, B) Distribution of crossover marker MLH1 (green), costained against SCP3 (red) in pachytene-staged spermatocyte spreads from wild-type (A) and Ino80cKO (B) spermatocytes. C, D) Distribution of ssDNA binding factor RPA in the same populations. Insets show magnified view of a representative autosomal chromosome corresponding to the marked area from the respective spermatocyte spread.