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. 2019 May 10;101(2):347–359. doi: 10.1093/biolre/ioz089

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© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.

This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

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Figure 4. — Assessment of DSB formation and repair in Spo11^P306T/P306T mutants throughout meiotic prophase I. (A) Meiotic chromosome surface spreads immunolabeled with RPA2 and SYCP3 at different Prophase I substages. E, early. Here and in panel “b,” pachytene substage (early vs late) was determined on the basis of H1t staining (only late pachynema is positive for H1t; staining not included in these images). Size bar = 20μm. Quantification of RPA2 foci throughout Prophase I is plotted on the right. Actual numbers are as follows. Leptonema: +/+ (n = 3 mice, cells = 22, avg = 164 ± 3.8), Spo11^P306T/P306T (n = 3, cells = 27, avg = 164 ± 3.5), Spo11^P306T/⁻ (n = 2, cells = 23, avg = 39 ± 6.2), Spo11^+/- (n = 2, cells = 20, avg = 175 ± 6.6); Zygonema: +/+ (n = 3, cells = 36, avg = 93 ± 5.9), Spo11^P306T/P306T (n = 3, cells = 27, average = 81 ± 3.8), Spo11^P306T/⁻ (n = 2, cells = 28, avg = 37 ± 4.4), Spo11^+/- (n = 2, cells = 29, avg = 92 ± 4.7); Early pachynema: +/+ (n = 3, cells = 87, avg = 37 ± 2.4), Spo11^P306T/P306T (n = 3, cells = 72, avg = 35 ± 2.7), Spo11^P306T/⁻ (n = 2, cells = 29, avg = 24 ± 4.5), Spo11^+/- (n = 2, cells = 24, avg = 20 ± 2.2). (B) Meiotic chromosome spreads immunolabeled for RAD51 foci during indicated prophase substages. Size bar = 20μm. (C) Quantification of RAD51 foci at prophase I substages. Actual numbers are as follows: leptonema: +/+ (n = 4, cells = 26, avg = 220 ± 4.1), Spo11^P306T/P306T (n = 5, cells = 24, avg = 211 ± 7.8), Spo11^P306T/⁻(n = 5, cells = 23, avg = 70 ± 7.9), Spo11^+/⁻ (n = 2, cells = 17, avg = 193 ± 4.3); zygonema: +/+ (n = 4, cells = 43, avg = 122 ± 2.8), Spo11^P306T/P306T (n = 5, cells = 52, avg = 115 ± 2.6), Spo11^P306T/⁻ (n = 5, cells = 59, avg = 30 ± 3.6), Spo11^+/- (n = 2, cells = 25, avg = 101 ± 4.7); early pachynema: +/+ (n = 4, cells = 23, avg = 14.2 ± 2.6), Spo11^P306T/P306T (n = 5, cells = 70, avg 40.8 ± 3.4), Spo11^P306T/⁻ (n = 5, cells = 80, avg = 15 ± 1.2), Spo11^+/- (n = 2, cells = 37, avg = 8.7 ± 1.5). (D) Quantification of DMC1 levels at prophase I substages. Leptonema: WT (n = 3, cells = 32, avg = 194 ± 3.3) vs mutant (n = 3, cells = 32, avg = 189 ± 3.6); zygonema: WT (n = 3, cells = 33, avg = 99 ± 4.6) vs mutant (n = 3, n = 33, avg = 92 ± 2.8); early pachynema: WT (n = 3, cells = 47, avg = 8.9 ± 2.0) vs mutant (n = 3, cells = 32, avg = 27 ± 4.1, P = 2.6 × 10⁻⁵); Late pachynema: WT (n = 3, cells = 20, avg = 2.2 ± 0.89) vs mutant (n = 3, cells = 20, avg = 1.6 ± 0.51). All statistics were done using unpaired Student T-test. Avg ± SEM.