Microtubule distribution during meiosis in wild type and <i>rsw4</i>.

<p>Fluorescent micrographs of meiocytes showing microtubules (green) and DNA (blue) for (A–D, I–L) wild type and (E–H, M–P) <i>rsw4</i> exposed to 30°C for 2 days. (A, E) Metaphase I. (B, C, F, G) anaphase I. (D, H) Telophase I. (I, M) Metaphase II. (J, N) Late anaphase II. (K, O) Telophase II. (L, P) Pre-cytokenisis. Bar  =  10 µm.</p

Female gametophyte development in wild type and <i>rsw4</i>.

<p>Confocal fluorescence micrographs of ovules of (A–D) wild type, (E–H) <i>rsw4</i> exposed to 30°C for 2 days, and (I –L) <i>AtESP-RNAi</i> plants. Images are the result of autofluorescence. Gametophyte development was staged according to Christensen et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019459#pone.0019459-Christensen1" target="_blank">[37]</a>. (A) FG1 embryo sac with a functional megaspore (arrow). (B) FG3 embryo sac. (C) FG 4 embryo sac. (D). FG7 embryo sac. (E) Stage FG1 in <i>rsw4</i> showing a degraded functional megaspore (arrow). (F) FG2 embryo sac showing degenerated megaspore nuclei (arrow). (G) An abnormal FG3 embryo sac with degraded nuclei (arrow). (H) A normal FG7 embryo sac. (I) FG5 embryo sac in <i>AtESP</i>-RNAi. (J) FG6 embryo sac. (K) FG7 stage embryo sac. (L) Ovule with a degraded embryo sac. Bars  =  5 µm.</p

Centromere behavior during meiosis in wild type and <i>rsw4</i>.

<p>Fluorescent micrographs of chromosome spreads stained for DNA (red) and centromeres by means of in situ hybridization (yellow) of (A–F) wild type and (G–L) <i>rsw4</i> exposed to 30°C for 2 days. (A, G) Leptotene. (B, H) Pachytene. (C, I) Metaphase I to Anaphase I transition. (D, J) Telophase I. (E, K) Metaphase II. (F, L) Telophase II. Note the reduction of centromeric signal during meiosis II in <i>rsw4</i>, indicating the nondisjunction of the bivalents. Bar  =  10 µm.</p

Chromosome positioning during meiosis in wild type and <i>rsw4</i>.

<p>Fluorescent micrographs of DAPI-stained chromosome spreads for wild type (A–C, G–I) and <i>rsw4</i> (D–F, J–O) exposed to 30°C for 2 days. (A, D) Pachytene. (B, E) Prometaphase I. (C, F) Early anaphase I. (G, J) Anaphase I. (H, K) Early anaphase II. (I, L) Telophase II. (M–O) Images of (M) Anaphase I. (N) Telophase I. (O) Telophase II in rsw4. Bar  =  10 µm.</p

Dynamics of Response to Asynapsis and Meiotic Silencing in Spermatocytes from Robertsonian Translocation Carriers

<div><p>Failure of homologous synapsis during meiotic prophase triggers transcriptional repression. Asynapsis of the X and Y chromosomes and their consequent silencing is essential for spermatogenesis. However, asynapsis of portions of autosomes in heterozygous translocation carriers may be detrimental for meiotic progression. In fact, a wide range of phenotypic outcomes from meiotic arrest to normal spermatogenesis have been described and the causes of such a variation remain elusive. To better understand the consequences of asynapsis in male carriers of Robertsonian translocations, we focused on the dynamics of recruitment of markers of asynapsis and meiotic silencing at unsynapsed autosomal trivalents in the spermatocytes of Robertsonian translocation carrier mice. Here we report that the enrichment of breast cancer 1 (BRCA1) and histone γH2AX at unsynapsed trivalents declines during the pachytene stage of meiosis and differs from that observed in the sex body. Furthermore, histone variant H3.3S31, which associates with the sex chromosomes in metaphase I/anaphase I spermatocytes, localizes to autosomes in 12% and 31% of nuclei from carriers of one and three translocations, respectively. These data suggest that the proportion of spermatocytes with markers of meiotic silencing of unsynapsed chromatin (MSUC) at trivalents depends on both, the stage of meiosis and the number of translocations. This may explain some of the variability in phenotypic outcomes associated with Robertsonian translocations. In addition our data suggest that the dynamics of response to asynapsis in Robertsonian translocations differs from the response to sex chromosomal asynapsis in the male germ line.</p> </div

Exclusion of the inactive chromatin mark histone H3K27me3 from unsynapsed chromosomal regions is limited to the XY-bivalent in single translocation carriers.

<p>Z - zygotene, EP – early pachytene, LP – late pachytene, D – diplotene spermatocytes. Arrows point to the XY bivalents. Arrowheads indicate unsynapsed trivalents. <b>A</b> – Zygotene spermatocyte is enriched for H3K27me3; <b>B-F</b> - the H3K27me3 histone mark is excluded from the XY-bivalent in early (<b>B</b>) mid (<b>C</b>), late (<b>D</b>, <b>E</b>) pachytene and diplotene (<b>F</b>) spermatocytes. However, H3K27me3 is not excluded from the unsynapsed trivalent (<b>B</b>, <b>C</b> and <b>E</b>). <b>D</b> - Only one of 15 late pachytene spermatocytes with unsynapsed trivalent showed H3K27me3 exclusion.</p

Histone H3.3 marks in metaphase/anaphase I spermatocytes from carriers of one or three Robertsonian translocations.

<p>Panels on the left show H3.3S31 immunostaining merged with DAPI staining. Panels on the right show DAPI staining alone. Arrowheads indicate chromosomal trivalents. <b>A</b> – a single H3.3S31-enrichment domain in a spermatocyte from a single translocation carrier corresponds to the sex body; <b>B</b> - two H3.3S31-enrichment domains correspond to the X and Y univalents; <b>C, D</b> - three H3.3S31-enrichment domains correspond to XY (D) or X and Y separately (C) and autosomal centromeric regions. <b>E</b> - a single H3.3S31-enrichment domain in a spermatocyte from a carrier of three translocations corresponds to the sex body. <b>F-G</b> - FISH for chromosomes 8 and 12 (red) shows presence (<b>F</b>) or absence (<b>G</b>) of co-localization of the Rb(8;12) trivalent with the autosomal H3.3S31 enriched domain in carriers of three translocations. <b>H</b> – distribution of spermatocytes with one, two or more than two H3.3S31 enrichment domains in wild type congenic mice without translocations, heterozygous Rb(8;12) carriers and heterozygous (Rb(1;3), Rb(8;12) and Rb(9;14) carriers. <b>I</b> – percent spermatocytes with autosomal H3.3S31 enrichment in heterozygous carriers of translocations compared to wild type congenic males.</p

Localization of the heterochromatin mark, histone H3K9me3, at unsynapsed trivalents in meiotic prophase I spermatocytes from carriers of a single or three translocations.

<div><p>Z - zygotene, EP – early pachytene, LP – late pachytene, D – diplotene spermatocytes. Arrows point to the XY bivalents. Arrowheads indicate unsynapsed trivalents. The bottom sets of panels show only SC immunostaining to facilitate the identification of unsynapsed regions and the XY-bivalent.</p> <p><b>A-D</b> - spermatocytes from wild type mice; <b>E-H</b> - spermatocytes from carriers of one translocation; <b>I- L</b> - spermatocytes from carriers of three translocations. <b>A</b>, E and I –zygotene spermatocytes with H3K9me3 enrichment throughout the nucleus. In wild type mice, H3K9me3 is enriched at the sex body in early pachytene (<b>B</b>), is lost in mid and late (<b>C</b>) pachytene and reappears in diplotene (<b>D</b>) spermatocytes. <b>F</b>- an unsynapsed autosomal trivalent in early pachytene spermatocytes from a single translocation carrier shows enrichment with H3K9me3 when associated with or in close proximity to the sex body. <b>G</b> – an unsynapsed trivalent in a mid pachytene spermatocyte from a single translocation carrier shows distinct H3K9me3 localization at centromeres, but not the rest of the unsynapsed region. The XY-bivalent in the same nucleus shows H3K9me3 enrichment only at the centromere of the X-chromosome. <b>H</b> – diplotene spermatocyte from a single translocation carrier with H3K9me3 enrichment at the sex body. <b>J</b> – in carriers of three translocations, unsynapsed autosomal trivalents in early pachytene spermatocytes show enrichment with H3K9me3 when associated with or in close proximity to the sex body. <b>K</b> –synapsed trivalents in a late pachytene spermatocyte from a carrier of three translocations show H3K9me3 localization at centromeres. The XY-bivalent in the same nucleus shows H3K9me3 enrichment only at the centromere of the X-chromosome. <b>L</b> – XY-body enrichment with H3K9me3 in diplotene spermatocytes from carriers of three translocations.</p></div

Dynamics of γH2AX localization to the chromosomal trivalents during the pachytene stage differs from the enrichment of this marker at the XY bivalent in spermatocytes of single translocation carriers.

<p>EP- early pachytene, MP – mid pachytene; LP – late pachytene spermatocytes. Arrowheads indicate trivalents. <b>A</b> - Distribution of γH2AX-positive and negative trivalents in 172 pachytene spermatocytes (from 5 mice). The y axis shows the percent spermatocytes with different γH2AX enrichment at different stages. <b>B</b> - Example of a γH2AX-negative unsynapsed trivalent in an early pachytene spermatocytes (asyn/ no γH2AX); <b>C</b> - Example of a γH2AX enrichment of a synapsing trivalent in early pachytene spermatocytes (syn/γH2AX).</p

Identification of the Rb(8;12) trivalent in spermatocytes by FISH.

<p><b>A</b> –pachytene spermatocyte with a FISH signal corresponding to the Rb(8;12) trivalent, no H3.3 localization is detected; <b>B</b> –metaphase/anaphase I spermatocyte with H3.3 enrichment at the sex body; FISH shows the Rb(8;12) trivalent in the center of the nucleus; <b>C</b> –metaphase II spermatocyte with H3.3 enrichment at a sex chromosome and only one FISH-positive spot corresponding to Rb(8;12); <b>D</b> – structure of the trivalent in a metaphase/anaphase I spermatocyte. The picture has been magnified to show detail. A diagram with the inferred structure is shown on the right.</p