The Mouse Spo11 Gene Is Required for Meiotic Chromosome Synapsis Peter J Romanienko, R.Daniel Camerini-Otero  Molecular Cell  Volume 6, Issue 5, Pages 975-987 (November 2000) DOI: 10.1016/S1097-2765(00)00097-6

Figure 1 Generation of Spo11-Deficient Mice (A) Gene disruption of the Spo11 locus. The targeting construct and genomic locus of Spo11 are shown. The EcoRI site in intron 1 was disrupted, and another EcoRI site was created at the junction of the neo cassette and exon 1 of Spo11 to facilitate identification of targeted clones. The probe was derived from the Spo11 cDNA encompassing exons 10–13. An EcoRI digest would give a 13 kb band from wild-type genomic DNA and a 16 kb band from a targeted allele. (B) Southern blot analysis of representative genotypes of Spo11 knockout mice. Homozygous knockout, −/−; heterozygous, +/−; wild-type, +/+. The digest was done with EcoRI and probed with Spo11 cDNA, exons 10–13. The arrows indicate positions of bands and their sizes are indicated. (C) PCR genotyping of Spo11 knockout mice by PCR. Mice used in experiments were genotyped by PCR. The same mice were used for Southern blot analysis in (B), indicating the method is consistent with results from Southern blotting. The primer locations are indicated in (A). Targeting at the Spo11 locus removes the binding site for one Spo11-specific primer. (D) Northern analysis of Spo11 knockout mice. Poly(A)+ RNA isolated from testes of mice with the indicated genotypes was probed with the mouse coding sequence cDNA, and the same blot was later probed with the mouse β-actin cDNA. The Spo11 transcript (1.8 kb) was absent in −/− testes and reduced in the heterozygote. The β-actin signal is not relevant to the phenotype and serves as a loading standard. Postmeiotic α-actin was absent in −/− testes. Molecular Cell 2000 6, 975-987DOI: (10.1016/S1097-2765(00)00097-6)

Figure 2 Histological Examination of Adult Testes from Spo11−/− Mice (A) Reduced testicular size in Spo11−/− mice. Wild-type, +/+; heterozygote, +/−; and homozygote knockout, −/− testes were compared from 6-week-old male sibs. (B) Hematoxylin- and eosin-stained cross section of a seminiferous tubule from a wild-type adult mouse. Magnification is 400×. (C) Hematoxylin- and eosin-stained cross sections of seminiferous tubules from a Spo11−/− adult mouse. Magnification is 400×. (D) Fluorescent TUNEL assay in wild-type mouse testis section; labeling was detected in some tubules near the basal lamina (not shown). Magnification is 200×. (E) Fluorescent TUNEL assay in Spo11−/− mouse testis section; labeled cells (green fluorescence within tubules) were detected in about 20% of tubules. Magnification is 200×; the arrow indicates positive tubule. Molecular Cell 2000 6, 975-987DOI: (10.1016/S1097-2765(00)00097-6)

Figure 3 Histological Examination of Adult and Fetal Ovaries from Spo11−/− Mice (A) Hematoxylin- and eosin-stained adult wild-type ovary. Arrow indicates maturing follicle. Eight to twelve follicles were generally seen per section. Magnification is 200×. (B) Hematoxylin- and eosin-stained adult Spo11−/− ovary. Arrow indicates follicle, but note absence of any other follicles. No more than one or two follicles were seen per section. Magnification is 200×. (C) Hematoxylin- and eosin-stained wild-type fetal ovary, 15 dpc. The two female fetal mice were from the same dam. Arrows denote cells with condensed chromatin. Magnification is 1000×. (D) Hematoxylin- and eosin-stained Spo11−/− fetal ovary, 15 dpc. Note fewer cells with condensed chromatin morphology as compared to (C). The number of cells with this morphology was half of those seen in wild type. Magnification is 1000×. Molecular Cell 2000 6, 975-987DOI: (10.1016/S1097-2765(00)00097-6)

Figure 4 Indirect Immunofluorescence of Spo11−/− Spermatocytes (A) Leptotene stage Spo11−/− spermatocyte. Merged image of αScp3 (red) and the centromere antisera CREST (green). All images initially viewed at 1000× magnification. Scale bar, 10 μm. Overlap of red and green signals results in yellow signal. (B) Zygotene-like stage of arrest in Spo11−/− spermatocytes. Merged image of αScp3 (red) and the centromere antisera CREST (green). Three nuclei are shown. (C) Zygotene wild-type spermatocyte; merged image of αScp3 (red) and the αRad51 (green). (D) Zygotene-like Spo11−/− spermatocyte; merged image of αScp3 (red) and the αRad51 (green). (E) Zygotene wild-type spematocyte; merged image of αScp3 (red) and the αDmc1 (green). (F) Zygotene-like Spo11−/− spermatocyte; merged image of αScp3 (red) and the αDmc1 (green). (G) Zygotene-like Spo11−/− spematocyte showing extent of synapsis, indicated by arrows. Stained with αScp3 (red). Molecular Cell 2000 6, 975-987DOI: (10.1016/S1097-2765(00)00097-6)

Figure 5 Spo11 Localization in Meiotic Chromosome Spreads (A) Spo11−/− spermatocyte; merged image of αScp3 (red) and the αSpo11 (green). Note absence of Spo11 signal. All images were initially viewed at 1000× magnification. Scale bar, 10 μm. Overlap of red and green signals results in yellow signal. (B) Leptotene spermatocyte; merged image of αScp3 (red) and the αSpo11 (green). (C) Zygotene spermatocyte; merged image of αScp3 (red) and the αSpo11 (green). Arrows indicate regions of synapsis. (D) Pachytene spermatocyte; merged image of αScp3 (red) and the αSpo11 (green). Arrow indicates the pseudoautosomal region (PAR). (E) Magnification of (C); arrow indicates PAR. (F) Pachytene spermatocyte; merged image of αScp1 (red) and the αSpo11 (green). Arrow indicates PAR. (G) Magnification of (E); arrow indicates PAR. Molecular Cell 2000 6, 975-987DOI: (10.1016/S1097-2765(00)00097-6)

Figure 6 Spo11 Localization and Expression in Mouse Meiotic Mutants (A) RT-PCR analysis of Spo11 expression in testes of mutant mice. The two forms of Spo11 (α and β) are indicated by arrows in the top panel, and the bottom panel shows control amplification of Aop2 (Pittman et al. 1998). The mutants analyzed are listed across the top. 13.5 dpp (days postpartum), amplification from testis cDNA from a juvenile mouse; H20, water control in amplification reaction. α and β forms of Spo11 are shown with primer location relative to exon 2, active site region, and Toprim domain. The sizes of putative proteins are indicated on the right. aa, amino acids. (B) Localization of Spo11 in Dmc1−/− spermatocyte. Merged image of αScp3 (red) and αSpo11 (green). Arrowhead indicates Spo11 staining in synapsed region, arrows indicate ends of axial elements of homologous chromosome participating in homologous synapsis, and asterisks indicate synapsis between nonhomologs. Images were initially viewed at 1000× magnification. The scale bar is 10 μm. Overlap of red and green signals results in yellow signal. (C) Localization of Spo11 in mei1 spermatocytes. Merged image of αScp3 (red) and αSpo11 (green). Arrow indicates Spo11 staining of fully synapsed homologs, and asterisks indicate synapsis between nonhomologs. Two nuclei are shown. Molecular Cell 2000 6, 975-987DOI: (10.1016/S1097-2765(00)00097-6)

Figure 7 Partial Rescue of Meiotic Arrest in Spo11−/− Spermatocytes (A) Induction of Rad51 foci in Spo11−/− spermatocytes 3 days after cisplatin administration. Merged image of αScp3 (red) and αRad51 (green). Cells were in zygotene-like stage. The images were initially viewed at 1000× magnification. Four nuclei are shown; the scale bar is 10 μm. Overlap of red and green signals results in yellow signal. (B) Induction of Dmc1 foci in Spo11−/− spermatocytes 3 days after cisplatin administration. Merged image of αScp3 (red) and αDmc1 (green). Cells were in zygotene-like stage; six nuclei are shown. (C) Colocalization of Rad51 and Dmc1 foci in Spo11−/− spermatocyte 3 days after cisplatin administration. Merged image of αRad51 (red) and αDmc1 (green). (D) Graphic representation of increased synapsis seen in Spo11−/− spermatocytes after cisplatin administration. One −/− mouse was treated with cisplatin for 3 days, the other was injected with saline. The mice were from the same litter and ≥5 weeks old when injected. One hundred nuclei were analyzed for each sample, and the number of CREST signals (centromeres) were recorded. (E) Colocalization of Scp1 and centromeres (CREST antisera) in Spo11−/− spermatocytes 3 days after cisplatin administration. Merged image of αScp1 (red) and CREST (green). Three nuclei are shown, (a) has 33 centromeres; (b), 30, and (c), 28. In many instances, synapsis extends away from the centromere. Note more αScp1 staining coincident with fewer centromeres. Molecular Cell 2000 6, 975-987DOI: (10.1016/S1097-2765(00)00097-6)