Error-prone ZW pairing and no evidence for meiotic sex chromosome inactivation in the chicken germ line - PubMed (original) (raw)

Error-prone ZW pairing and no evidence for meiotic sex chromosome inactivation in the chicken germ line

Silvana Guioli et al. PLoS Genet. 2012.

Abstract

In the male mouse the X and Y chromosomes pair and recombine within the small pseudoautosomal region. Genes located on the unsynapsed segments of the X and Y are transcriptionally silenced at pachytene by Meiotic Sex Chromosome Inactivation (MSCI). The degree to which MSCI is conserved in other vertebrates is currently unclear. In the female chicken the ZW bivalent is thought to undergo a transient phase of full synapsis at pachytene, starting from the homologous ends and spreading through the heterologous regions. It has been proposed that the repair of the ZW DNA double-strand breaks (DSBs) is postponed until diplotene and that the ZW bivalent is subject to MSCI, which is independent of its synaptic status. Here we present a distinct model of meiotic pairing and silencing of the ZW pair during chicken oogenesis. We show that, in most oocytes, DNA DSB foci on the ZW are resolved by the end of pachytene and that the ZW desynapses in broad synchrony with the autosomes. We unexpectedly find that ZW pairing is highly error prone, with many oocytes failing to engage in ZW synapsis and crossover formation. Oocytes with unsynapsed Z and W chromosomes nevertheless progress to the diplotene stage, suggesting that a checkpoint does not operate during pachytene in the chicken germ line. Using a combination of epigenetic profiling and RNA-FISH analysis, we find no evidence for MSCI, associated with neither the asynaptic ZW, as described in mammals, nor the synaptic ZW. The lack of conservation of MSCI in the chicken reopens the debate about the evolution of MSCI and its driving forces.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. RPA localization in chicken oocytes.

Oocytes stained for RPA (red) and SYCP1/SYCP3 (green). RPA and SYCP1 co-staining was performed first (Figure S1). After imaging, the slides were then stained for SYCP3 and new pictures were taken. (A) Zygotene, RPA foci are on synapsed and unsynapsed chromosomes (indicated with arrows and arrowheads, respectively). (B–D) Pachytene; RPA foci on autosomal synaptic axes are still abundant at early pachytene (B), but rapidly decrease and then disappear by late pachytene (C–D). Pachytene cells were classified as late if they contained fewer than five foci on synaptic autosomes. (C) Late pachytene oocyte containing a synapsed ZW bivalent RPA negative. (D) Late pachytene oocyte containing an unsynapsed Z and W carrying chains of RPA foci on the Z. Z and W were identified by hybridisation to Z and W chromosome paints (data not shown). Drawings of ZW synaptic configurations are shown to the right of panel C and D. Scale bar = 10 µm.

Figure 2

Figure 2. Oocytes with unresolved DNA DSBs on the Z chromosome progress to diplotene.

(A–F) 6 dph oocytes stained for RPA (red) and SYCP3 (green). The diplotene cells were classified as early when desynapsis was partial and late when desynapsis was complete. (A–B) Late pachytene; (C–D) early diplotene; (E–F) late diplotene. (A, C, E) Cells RPA negative (RPA−ve), and (B, D, F) cells carrying a chain of RPA foci (RPA+ve). (G) Graph showing the percentage of RPA+ve cells at each stage. Total number of cells analysed: late pachytene, 141; early diplotene, 223; late diplotene, 169. ZW chromosome painting performed after antibody staining showed that the Z chromosome accounted for 85% of the RPA+ve cells identified at pachytene and 74% at early diplotene, making the percentage of late pachytene ZWRPA+ = 21.7% and the percentage of early diplotene ZWRPA+ = 19.6%. The percentage of ZWRPA+ pairs at late diplotene was not calculated as in many of these cells it was not possible to unambiguosly identify the entire Z axis. The quantification was done on ovaries from the chicken line ISA Brown. (H) 14 dph oocytes stained for RPA (red) and SYCP3 (green). 90% of the cells are negative for SYCP3; no RPA was identified in these cells (number of cells analysed: 60). Scale bar = 10 µm.

Figure 3

Figure 3. The late pachytene ZWRPA+ pair has no MLH1 focus.

Oocytes triple stained for SYCP3 (green), RPA (blue) and MLH1 (red). (A) The ZWRPA+ pair carrying a chain of RPA foci on the Z never has a MLH1 focus (white arrowhead indicates the expected location of the focus); total number of oocytes analysed: 62. (B) The fully synapsed ZW bivalent always carries a MLH1 focus at one end (white arrow); Total number of oocytes analysed: 138. Drawings of the ZW pair to the right of ech panel. ZW pairs were identified using ZW chromosome paints (data not shown). Scale bar = 10 µm.

Figure 4

Figure 4. Many ZWRPA+ pairs have PAR-PAR misalignment.

Late pachytene oocytes triple-stained for SYCP3 (green), RPA (blue), MLH1 (red) and subsequently hybridised to Z and W chromosome paints (top right insets: Z, green cloud; W, red cloud). Total number of ZWRPA+ oocytes analysed: 134. (A) Z and W axes are clearly separated; (B) only the q-end of the Z axis is close to the W; (C) both ends of the Z are close to the W (see text for percentages of each class). The ZW bivalents are also schematised to the right (black line = Z; grey line = W; green = Z paint; red = W paint). Scale bar = 10 µm.

Figure 5

Figure 5. ZW bivalent desynapsis occurs in synchrony with autosomal desynapsis.

Oocytes double-stained for SYCP3 (green) and RPA (blue) and subsequently hybridised to Z and W chromosome paints (green and red clouds respectively in top right insets); only the late pachytene and early diplotene cells negative for RPA were considered. (A–C) Late pachytene cells (total number analysed: 134); (A) ZW is fully synapsed; (B) ZW is partially desynapsed; (C) Z and W are fully desynapsed. (D–F) Early diplotene cells (total number analysed: 54); (D) ZW is fully synapsed; (E) ZW is partially desynapsed; (F) Z and W are fully desynapsed. Relative percentages are indicated in the insets at bottom right corner of each panel. To the side of each panel is a schematic of the ZW pair (Thick black line, synapsed ZW; black and grey thin lines, desynapsed Z W, respectively). In most pachytene cells the ZW pair is synapsed, in most early diplotene cells ZW is partially desynapsed. Scale bar = 10 µm.

Figure 6

Figure 6. Chick oocytes undergo a wave of γH2AFX coincident with the beginning of meiosis.

(A–B) Ovarian section from E14 embryos injected with BrdU two hours before dissection and double-stained for BrdU (green) and γH2AFX (red). Many germ cells are positive for both markers (e.g. white arrows). (C–D) Oocyte nuclei from E14 ovarian spreads double-stained for γH2AFX (red) and SYCP3 (green). SYCP3 is only present in some of the γH2AFX positive cells. (E–H) γH2AFX timecourse on ovarian spreads. The cells were double-stained with SYCP3 for staging. γH2AFX is downregulated by mid-pachytene. (I–J), Pachytene cells triple-stained for SYCP3 (green), RPA (green) and γH2AFX (red): γH2AFX is only present in the cell containing asynaptic RPA+ve chromosomes (left nucleus), where it colocalises with some RPA foci (white arrows). Scale bar = 10 µm.

Figure 7

Figure 7. Epigenetic status of Z and W chromosomes at pachytene.

(A–F) Oocyte nuclei double stained for SYCP3 (green) and H3K9me3 (A–C), or CBX1 (D–F) (red). Z and W have been identified by chromosome paint (data not shown). Asynapsed Z and W have been pseudo-coloured white and blue, respectively; synapsed ZW has been pseudo-coloured turquoise. Both markers stain the W chromatin, and the heterochromatic q end of the Z (indicated with a white arrow). (G–H) Ovarian somatic cells stained for H3K9me3 (red) (G), and subsequently hybridised to Z (green) and W (red) chromosome paints (H). Z and W chromatin are always H3K9me3 positive (green and, respectively, white arrows in G); total number of cells analysed: 67. (I–J) Oocyte nuclei double-stained for SYCP3 (green) and H3K9me3 (red), and subsequently processed for DNA-FISH using BAC CH261-125H16 (Z5) as a probe. This BAC contains ∼200KB of Z DNA, including the gene Dmrt1, located at 27 Mb on the chicken Z physical map (

www.ensemble.org

). The signal (turquoise) is indicated by a white arrow. The probe was found to localise outside the H3K9me3 domain (I) or to the domain border (J) in 89% of the synapsed ZW pairs analysed; total number of cells analysed: 40. Scale bar = 10 µm.

Figure 8

Figure 8. A global downregulation of transcription occurs at leptotene-zygotene and is maintained into pachytene and early diplotene.

(A) Schematic of the Z chromosome showing the location of the Z probes used in the analysis. (B) RNA-FISH analysis using Z (Z1–Z9) and autosomal (A1–A3) probes on oocytes from E14 (see Materials and Methods for probe identity). The oocytes were identified by subsequent staining for γH2AFX. The bars represent the percentage of expressing oocytes. (C) (Top) Timecourse results using the probes screened at E14and subsequently analysed at E19, 1 dph (D1), 6 dph (D6); the oocytes were identified by staining for SYCP3. The Y axis indicates the percentage of expressing oocytes. (C) (Bottom) Charts representing the percentage of leptotene/zygotene (L/Z), pachytene (P) and diplotene (D) cells in the ovaries analysed at E19, D1 and D6; the counting was carried out on spreads generated from the cell dispersions used for RNA-FISH, after staining for SYCP3 and RPA. (D) Oocyte nuclei from ovarian spreads subject to RNA-FISH using the probe Z5 (cyan) and stained for SYCP3 (red) and RPA (green); (Top) Positive oocyte containing ZWRPA+; (Bottom) Positive oocyte containing a fully synapsed ZW. The positive signal (cyan) is also indicated by a white arrow. Scale bar = 10 µm. (E) Plots summarising the results from the RNA-FISH on spreads using probes Z4, Z5, Z7, Z8. (Top) Relative percentages of synaptic and asynaptic ZW within the positive late pachytene oocytes. (Bottom) Chart representing the relative percentages of synaptic and asynaptic ZW within the total population of late pachytene oocytes. The activity of the pachytene ZW pair is not biased by its synaptic status.

Figure 9

Figure 9. The chick sex chromosome meiotic synaptic behaviour: a new model.

Schematic representing the dynamics of synapsis and DSB repair of the ZW pair during early meiosis. Autosome axes in black; Z in green; W in red. RPA foci are shown as blue dots, γH2AX as circles. In about 80% of the oocytes that reach pachytene ZW undergoes synapsis and DSB repair by mid-late pachytene and desynapses at diplotene. In 20% of oocytes ZW remains asynapsed and maintains unrepaired DSBs, nevertheless the cells go to diplotene. Different shades of blu represent different levels of transcriptional activity (dark blue: high, light blue: low). The oocytes undergo a global downregulation of transcriptional activity at leptotene/zygotene. At pachytene, no Z specific wave of silencing is evident in any of its configurations. Autosomes and Z chromosomes behave alike; they maintain a low level of transcription at least into early diplotene. See Discussion for more details.

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