Additional file 1 of Rat PRDM9 shapes recombination landscapes, duration of meiosis, gametogenesis, and age of fertility
Creators: name: Mihola, Ondrej
name: Landa, Vladimir
name: Pratto, Florencia
name: Brick, Kevin
name: Kobets, Tatyana
name: Kusari, Fitore
name: Srdjan Gasic
name: Smagulova, Fatima
name: Grey, Corinne
name: Flachs, Petr
name: Vaclav Gergelits
name: Tresnak, Karel
name: Silhavy, Jan
name: Mlejnek, Petr
name: R. Daniel Camerini-Otero
name: Pravenec, Michal
name: Petukhova, Galina V.
name: Trachtulec, Zdenek
Publisher: name: figshare
Year: 2021
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: Additional file 1: Fig. S1. Translations of mRNAs from rat Prdm9 mutants (red) described in Fig. 1 aligned in the SET domain with homologs from other species. G278, N320 and Y341 are amino acids essential for H3K4 trimethylation activity of PRDM9 [12, 27]. Fig. S2. Semiquantitative qRT-PCR of adult rat testicular RNAs detects no mutant mRNA degradation. The expression values were normalized with Actb primers. Primer pair “Exon 1–3” detected the KRAB-encoding exons, pair “Exon 9–10” the last SET/PR-exon plus the Zn-finger-exon. Two pairs (“20 a.a. deletion”, “Intron 8”) were specific for the new mRNAs of the mutant with the largest deletion (KO516-U, KO516-L, respectively), as we did not detect them in the wild-type testes (wt/wt) or in the testes hetero- or homozygous for other Prdm9 deletions (KO/wt, KO/KO). Fig. S3. Alternative views of male (a) and female (b) rat fertility parameters measured via dissections. For legend, see Figs. 2 and 3. Fig. S4. Impaired spermiation efficiency in Prdm9-deficient SHR rat. Representative images of PAS-H-stained tubular stages VIII, IX and X of Prdm9-deficient and control rats. Step 19 (S19) mature spermatids aligned near the tubule lumen for spermiation in stage VIII. However, some S19 spermatids were present beyond stage VIII in Prdm9KO/KO, indicating failed spermiation - note the abnormal retention of S19 in mutant stages IX and X. Fig. S5. Spermatogenic arrests in tubular stages IV and XII of Prdm9-deficient PWD mouse. Testicular histology using PAS-H staining. Examples of tubular stages IV and XII of Prdm9-deficient and control PWD males (108 dpp). Red arrows point to degenerated spermatocytes in mutant seminiferous tubules. See Fig. 4 for abbreviations. Fig. S6. Meiotic synapsis and DSB repair in pachytene spermatocytes of adult Prdm9-deficient rats detected by immunostaining of chromosome spreads- representative images. a-f, Staining of synaptonemal complex (SYCP3), asynapsed chromosomal regions (HORMAD2), and sites under repair (γH2AX). Completely synapsed autosomes were observed in normal cells (a-d). Sex chromosomes synapsed within the pseudo-autosomal region (a-c) became asynapsed during late pachynema (d). Aberrant spermatocytes harbored completely (e) and partially (f) asynapsed autosome pairs. g-l Labelling of SYCP1, SYCP3, and γH2AX. In normal early pachynema, all chromosomes synapsed (g-i); asynapsed X and Y were present at late pachynema (j). Abnormal spermatocytes carrying both autosomal and X/Y asynapses (k,l) were also detected. m-r, Immunostaining for SYCP3, centromeric proteins (CENT), and early stages of DSB repair (RAD51/DMC1). In normal spermatocytes, a few RAD51/DMC1 foci were observed on autosomes and multiple foci on sex chromosomes from early pachynema (m-p). All RAD51/DMC1 signal disappeared in normal late pachynema (q). Abnormal cells showed many strong RAD51/DMC1-stained foci on asynapsed chromosomes (r). Fig. S7. Correlations at hotspots in different rat strains. Hotspot strength is compared among all strains. Only autosomal hotspots that were called in both strains are considered. Each dot represents one hotspot and hotspot density is shown as a gray (low) to blue (high) gradient. The Spearman correlation coefficient (R2) is indicated for each comparison. Fig. S8. DSB hotspots are stronger on chromosome X than on autosomes. Note that since there is only one copy of chromosome X in males, hotspot strength is scaled by a factor of two.