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. 2018 Jul 5;9:2621. doi: 10.1038/s41467-018-04850-0

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — A conditional strategy for efficient depletion of ATR during male mouse meiosis. P30 testis and body weights (a), testis western blots (b), and periodic acid–Schiff and hemotoxylin/eosin-stained stage IV testis sections (c) in Atr^fl/+ Stra8-Cre males (n = 9 males), Atr^fl/– Stra8-Cre males (n = 10 males), Atr^fl/+ Ngn3-Cre males (n = 13 males) and Atr^fl/– Ngn3-Cre males (n = 13 males; means and p values for a indicated; unpaired t-test). Testis weights in Atr^fl/+ Stra8-Cre and Atr^fl/+ Ngn3-Cre males are not significantly different from those in Atr^fl/+ Cre negative males derived from the same crosses (n = 17 Atr^fl/+ Cre negative males from the Stra8-Cre cross, p = 0.37, n = 11 Atr^fl/+ Cre negative males from the Ngn3-Cre cross, p = 0.06). Inset in c shows presence of elongating spermatids in some tubules from Atr^fl/– Stra8-Cre males. d, e ATR (magenta) and SYCP3 (green) staining in Atr^fl/+ Ngn3-Cre (denoted Atr^fl/+) and Atr^fl/– Ngn3-Cre (denoted Atr^fl/–) males. In Atr^fl/+ males (n = 2 males) ATR is observed as foci (see insets) in 85% of leptotene cells (n = 20 cells) and 82% of zygotene cells (n = 28 cells), and on the asynapsed region of the XY bivalent in 100% of pachytene cells (n = 30 cells). In Atr^fl/– males (n = 2 males) ATR is observed in no cells at these three stages (n = 21, 30 and 32 cells at leptonema, zygonema and pachynema, respectively). f Validation of ATR depletion in Atr^fl/– by analysis of MSCI. In Atr^fl/+ males, all pachytene cells show coating of the X and Y chromosome (arrowheads) but not the pseuodautosomal regions (PAR, arrowhead) by γH2AX (magenta; left panels; XY bivalent in box magnified in smaller panels). This coating causes silencing of the X-chromosome gene Scml2 (magenta; right panels) and compartmentalization of the XY bivalent (labeled with HORMAD2; green) in the sex body (n = one male, 29 cells; magnified in smaller panels). g In Atr^fl/– males, XY chromosome γH2AX coating and sex body compartmentalization do not occur. As a result, Scml2 expression (arrow) persists in all early pachytene cells (n = one male; 30 cells). Scale bar in (c) 20 µm, other scale bars 10 µm