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Fig. 1. Spo11-/- spermatocytes do not phosphorylate H2AX in the sex chromatin and fail to form a sex body. (A) Zygotene-like Spo11-/- spermatocytes display a wide range of 
H2AX staining patterns. Analysis of Spo11-/- surface-spread preparations stained for SCP3, SCP1 and H2AX revealed that a proportion of zygotene-like spermatocytes is devoid of H2AX (i), whereas most nuclei contain one or more localized H2AX chromatin domains. Examples are shown of nuclei containing one (ii), two (iii) or more (iv) H2AX signals. (B) Quantitative analysis of H2AX staining patterns observed in Spo11-/- zygotene-like spermatocytes. 350 nuclei were scored and classified according to their stage (based on SCP3 staining) and H2AX staining pattern. Zygotene-like spermatocytes (n=290 nuclei, two animals) were classified as nuclei devoid of H2AX (i) or nuclei displaying one or more localized H2AX signals (ii-iv). These zygotene-like spermatocytes containing one or more localized H2AX signals (n=197 nuclei) were further classified according to the number of H2AX signals: one signal (ii), two signals (iii) and more than two signals (iv). Examples of each of these nuclei are displayed in Fig. 1Ai-iv. (C) The H2AX signals frequently observed in zygotene-like spermatocytes are not on the sex chromatin. Combined FISH (whole-chromosome paint probes against the X and Y chromosomes are shown in green) and immunostaining (antibodies against SCP3 are shown in red and those against H2AX in blue) performed on structurally preserved spermatocytes from wild-type (a-c) and Spo11-/- (d,f) mice (two sets of littermates). In wild-type spermatocytes, the X-Y chromatin contains H2AX (c). In SPO11-deficient zygotene-like spermatocytes, even in those nuclei displaying a single H2AX signal, this does not overlap with the X and Y chromatin. None of the 50 Spo11-/- nuclei analysed contained a H2AX signal overlapping with either the X or the Y chromatin. Notice that the X chromosome appears extended and does not synapse with the Y chromosome. (D) Spo11-/- spermatocytes do not form a sex body. Combined SCP3 immunostaining and FISH (using differently labeled whole-chromosome probes recognizing the X and Y chromatin) was performed on methanol-acid-fixed Spo11-/- spermatocytes. 123 zygotene-like nuclei (two animals) were scored and classified according to the relative positions of the sex chromosomes as apart (a) or close (b). Almost 80% of SPO11-deficient zygotene-like nuclei displayed the X and Y chromosomes widely separated, whereas 21% contained the sex chromosomes close to one another. Nevertheless, in those nuclei containing the sex chromosomes in proximity, the X is always extended and barely contacting the Y on one end. This disposition clearly differs from the spatial configuration usually adopted by the X-Y chromosomes in the sex body in wild-type spermatocytes, providing further cytological evidence for the lack of sex body in SPO11-deficient spermatocytes.
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