Expression of BEND2 during spermatogenesis (A) Schematic representation of mouse Gm15262/Bend2 and its novel splice variant 255p1. The exons are shown as purple boxes. The predicted domains are labeled below the exons. SAP130_C: C-terminal domain of histone deacetylase complex subunit SAP130; BEN: BEN domain. A pair of gRNAs (orange arrows) target exon 11 of Bend2 within the first BEN domain to generate the Bend2Δ11 mutation by CRISPR/Cas9. The in-house BEND2 antibody is generated using full-length 255P1 sequence as immunogen. (B) BEND2 localization in wild-type mouse testis. Staging of the seminiferous epithelium is based on the localization of spermatocytes (indicated by the expression and localization of ϒH2AX(green)) and spermatids (indicated by DAPI). The tubule stage is shown in uppercase Roman numerals. Scale bar, 20 µm. (C) Magnification of BEND2-positive cells from testis sections. Expression of BEND2 was characterized in cells along spermatogenesis from spermatogonia to late diplotene spermatocyte. Scale bar, 10 µm. (D) BEND2 localization in SPO11- and DMC1-deficient testis. In these cases, SYCP3 (green) was used to identify spermatocytes. Scale bar, 50 µm. Spg: spermatogonia; B: B-type spermatogonia; PL: pre-leptotene spermatocyte; L: leptotene spermatocyte; Z: zygotene spermatocyte; Z- like: zygotene-like spermatocyte; EP: early pachytene spermatocyte; LP: late pachytene spermatocyte; P-like: pachytene-like spermatocyte; D: diplotene spermatocyte.

Disruption of BEND2 in Bend2Δ11/y mice. (A) Mouse appearance and testis size. Scale bar, 5mm. (B) Bend2 expression in testis by RT-PCR. The expected size of the WT allele was 576bp. Sanger sequencing result showed that the amplified DNA in the mutated allele (372bp, green arrowhead) was from an mRNA that resulted from skipping exon 11 of Bend2. (C) Detection of BEND2 by WB from testis protein extracts. The orange arrowhead indicates the full-length BEND2 protein band. An extra protein band ∼75 kDa (black arrowhead) was also detected in both wild-type and mutant testes. (D) Detection of BEND2 by IF. Testis sections were treated with antigen retrieval using Tris-EDTA buffer before staining against BEND2 (red) and SYCP3 (green). Scale bar, 10 µm.

The full length BEND2 is dispensable to complete spermatogenesis in mice

(A) PAS-H stained mouse testis sections. Scale bar, 20 µm. (B) Apoptosis detection on testis sections by TUNEL assay. Scale bar, 20 µm. (C) Quantification of TUNEL- positive cells. The horizontal lines represent the mean ± SD. N=1125 for Bend2+/y; N=1195 for Bend2Δ11/y, ****p<0.0001 t-test. (D) Classification of TUNEL-positive cells. The columns and error lines indicate the mean ± SD. N=4, p>0.05 One-Way ANOVA.

Bend2Δ11/y males display minor recombination defects. (A) Chromosomal synapsis in spermatocytes. Representative images of SYCP3 and SYCP1 staining in spermatocyte nuclei from the stages shown (left). Meiotic prophase staging of spermatocytes (right). L: leptotene, Z: zygotene, P: pachytene, D: diplotene. The columns and lines indicate the mean and SD. N=4, *p=0.019 One-Way ANOVA. (B) Examination of DSBs in Bend2Δ11/y spermatocytes. Representative images of ϒH2AX staining in spermatocyte nuclei along meiotic prophase (left). Increased ϒH2AX signals are detected during late prophase in Bend2Δ11/y spermatocyte (white arrowhead). Quantification of ϒH2AX patches in spermatocyte nuclei per sub-stages (right). The patches were counted manually using the same method for every pair of control and mutant mice. From left to right, N=76/74, 86/83, 73/75, and 78/81, ***p=0.0001 (late pachytene) and 0.0007 (early diplotene), *p=0.0222 t-test. Analysis of RPA (C) and RAD51(D) foci counts in control and Bend2Δ11/y spermatocytes. RPA and RAD51foci were counted using ImageJ with the same method for every Bend2+/y and Bend2Δ11/y mice pair. From left to right, N=44/43, 51/43, 59/46, 45/39, 81/77, and 48/43, **p=0.0015 for RPA quantification; N=52/43, 42/46, 44/46, 51/45, 77/73, and 45/49, *p=0.0428 and ***p=0.0002 for RAD51 quantification; t-test. C) Examination of CO formation in Bend2Δ11/y spermatocytes. Representative images of MLH1 in control and mutant spermatocyte nuclei (left). Quantification of MLH1 foci in spermatocyte nuclei (right). Only spermatocytes containing ≥ 19 MLH1 foci/nucleus were counted. N=74 for Bend2+/y and 68 for Bend2Δ11/y, p>0.05 Mann-Whitney test. The horizontal lines represent mean ± SD (B-E). Scale bar, 10 µm.

Oogenesis is altered in Bend2 mutant females (A) Fertility evaluation of Bend2Δ11/Δ11 females. Two-month-old Bend2Δ11/Δ11 females were crossed with wild-type males for 5 months; litter size data of 4 animals per genotype were collected for analysis. *p=0.0124 t-test. (B) PAS-H stained histological ovary sections from females at different ages. Scale bar, 0.5 mm. Quantification (C) and classification (D-G) of analysed follicles from ovaries at age of 1 week (D), 3 weeks (C), 15-20 weeks (F) and 40 weeks (G). From 1 week to 40 weeks, the number of ovaries per genotype is 4/4/8/4. The columns and lines indicate the mean and SD. **p=0.0012 and 0.0040 (C); ***p=0.0007 (D); **p=0.0024, ****p<0.0001 (F); t-test.

Synapsis and recombination in Bend2Δ11/Δ11 females (A) Chromosomal synapsis in oocytes. Representative images of SYCP3 and SYCP1 staining in 18 dpc and 1 dpp oocyte nuclei are shown (left). Meiotic prophase staging of 18 dpc and 1dpp oocytes (right). L: leptotene, Z: zygotene, P: pachytene, D: diplotene. The columns and lines indicate the mean and SD. The number of animals analyzed per genotype, 2 for 18 dpc and 3 for 1 dpp. P>0.5 for all the comparisons One-Way ANOVA. (B) Examination of DSBs in Bend2Δ11/Δ11 oocytes. Representative images of ϒH2AX staining in 18 dpc and 1 dpp oocyte nuclei at the pachytene and diplotene stage (left). Quantification of ϒH2AX patches in oocyte nuclei at sub-stages (right). The mean intensity of ϒH2AX staining in 18 dpc oocyte nuclei was measured by Image J. The patches of ϒH2AX in 1 dpp oocyte nuclei were counted manually. The horizontal lines represent the mean ± SD. From left to right, the number of analyzed nuclei per genotype: 40/30, 64/48, 81/27, and 57/28. *p=0.0288, ****p<0.0001 t-test. (C) Analysis of RPA (C) and RAD5 (D) foci in control and Bend2Δ11/Δ11 oocytes. Representative images of RPA staining in 18 dpc and 1 dpp oocyte nuclei from zygotene to diplotene stage (left). Quantification of RPA foci present in oocyte nuclei at sub-stages (right). Zygotene and early pachytene nuclei analyzed were from 18 dpc ovaries, and late pachytene and diplotene nuclei analyzed were from 1 dpp ovaries. Foci were counted manually. The horizontal lines represent the mean ± SD. From left to right, number of analyzed nuclei: 12/13, 27/17, 41/42, and 65/49 for RPA, and 8/11, 27/23, 38/46, and 62/55 for RAD51. p>0.5 for all the comparisons t-test. (E) Examination of CO formation in control and Bend2Δ11/Δ11 oocytes. Representative images of MLH1 in 1 dpp oocyte nuclei (left). Quantification of MLH1 foci in 1 dpp oocyte nuclei (right). Only oocytes containing ≥20 MLH1 foci/nucleus were counted. The horizontal lines represent the mean ± SD. N=120 for Bend2+/+, 95 for Bend2Δ11/Δ11; **p=0.0057 Mann-Whitney test. Scale bar, 10 µm.

Expression and localization of Bend2 (A) Expression of 255P1 in mouse gonads and somatic tissues by RT-PCR. Actin- 400bp; 255P1-576bp. (B) Expression pattern of EGFP-255P1 in mouse testis. Representative images of spermatocyte squash and spread stained against SYCP3 and GFP. DNA is counterstained with DAPI (blue). Squash: patch-like signals at the heterochromatic region. Spread 1: cell exhibiting clear foci signals at telomeres along with patch-like signals. Spread 2: cell exhibiting foci signals at the chromosomal telomeres and axes. Signal accumulation at XY chromosomes (white square). Scale bar, 10 µm. (C) BEND2 localization in 16 dpc mouse ovary. 16 dpc ovary sections were treated with antigen retrieval using Tris-EDTA buffer and then stained with BEND2 (red) antibody and ϒH2AX (green) antibody. Strong BEND2 signals were occasionally found in early meiotic prophase oocyte (upper) and oogonia (lower) before DSBs appear. Scale bar, 20 µm.

Examination of the NHEJ pathway activity in Bend2Δ11/y mice (A) Representative images of Ku70 expression pattern in wild type and Bend2Δ11/y testis. Scale bar, 40 μm. (B) Quantification of Ku70 foci in pachytene and diplotene spermatocytes. Foci colocalizing on SYCP3 were counted manually from at least 40 cells per animal. The columns and lines indicate the mean and SD (N=2). (C) Western blot analysis of Ku70 and LINE-1 proteins. (D) Quantification of relative Ku70 protein levels by WB. The columns and lines indicate the mean and SD (N=3).

Analysis of LINE-1 retrotransposon expression in Bend2Δ11/y mice (A) Representative images of LINE-1 expression pattern in wild type and Bend2Δ11//y testis. Scale bar, 40 μm. Quantification of LINE-1 positive tubules (B) and relative protein levels in Bend2Δ11/y animals from Figure S2 (C). The columns and lines indicate the mean and SD (N=3). *p=0.046 (B) and *p=0.02 (C) t-test.