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. 2020 Aug 3;9:e53392. doi: 10.7554/eLife.53392

Figure 2. Expression of ZCWPW1 across meiosis prophase I in mouse testis.

Nuclear spreads from 9 to 10 weeks old WT mice were immunostained with antibodies against ZCWPW1 (red) and the synaptonemal complex protein SYCP3 (green) which labels the chromosome axis, and counterstained with DAPI (blue) to visualise nuclei. Developmental stages are indicated above and below. Yellow arrows point to ZCWPW1 foci clearly visible at both ends of the synaptonemal complex in mid-late pachytene. Additional evidence is provided in Figure 2—figure supplement 2. The dashed circle shows staining in the XY body. Images for the individual channels are provided in Figure 3—figure supplement 1. These images are representative of the results obtained in three mice. Scale bar: 10 μm.

Figure 2—source data 1. Immuno-FISH analysis of ZCWPW1 foci localisation at the synaptonemal complex ends in WT testis (mid-Pachytene to Late Diplotene cells).
Only chromosomes clearly identifiable were included in the analysis. X and Y were excluded as they are covered in ZCWPW1 signal (which strong labels the XY body). Stages: P, Pachytene; D, Diplotene. Tel: Telomeric probe. Cen: Centromeric probe.

Figure 2.

Figure 2—figure supplement 1. ZCWPW1 antibody generation and validation.

Figure 2—figure supplement 1.

(A) Expression and purification of full-length recombinant mouse ZCWPW1 (mZCWPW1) in E. Coli. Left panel: SDS-PAGE analysis and Coomassie blue staining of bacterial lysates before (pre-IPTG) and after (post-IPTG) induction of protein expression with IPTG, the soluble protein fraction (after cell sonication) used for purification, the flow through (incompletely depleted from the target protein) after incubating the soluble fraction with Talon resin beads to bind His-tagged mZCWPW1, the wash containing 5mM imidazole, the protein eluate from the beads using 300mM imidazole, and the purified recombinant protein after further purification from low molecular weight (MW) contaminants by size exclusion. Right panel: Western blot detection of purified His-tagged mZCWPW1 using an anti-His and a mouse polyclonal antibody raised against the human protein (previously tested positively against mouse ZCWPW1 overexpressed in HEK293T cells). *Indicates degradation fragments (likely C-terminal). The purified protein was used to immunise rabbits and produce an antiserum against mZCWPW1. (B-E) Validation of the rabbit ZCWPW1 antiserum by immunofluorescence staining (IF), immunoprecipitation (IP) and western blotting (WB) in B6 testis (B,C) and transfected HEK293T cells (D,E). (B) Testis nuclear spreads from 10 weeks old B6 mice were immunostained with the ZCWPW1 antiserum or the pre-immune serum (red), and chromosome axes were labeled with SYCP3 (green). Representative mid-zygotene and early pachytene cells are shown. No signal is detected by the pre-immune serum. Scale bar: 10μm. (C) IP-WB detection of mZCWPW1 from 10 weeks old B6 mouse testis. Left panel: Lane 1, 100 µg protein extract; Lanes 2–3, IP from 2.6 mg protein extract using ZCWPW1 antiserum (lane 2) or the pre-immune serum (lane 3). The ZCWPW1 antibody detects a unique protein band within the expected MW range (predicted at 70.5 KDa) both by direct WB (lane 1) and IP-WB (lane 2). No signal is detected by the pre-immune serum (lane 3). Right panel: the testis protein extract was resolved on a higher (4–20%) SDS-PAGE gel; no detection of ZCWPW2 is observed by WB at the 38KDa MW range predicted for mouse ZCWPW2, only a single band is present within the expected MW range for ZCWPW1. The images in (B–C) are representative of the results obtained in two mice. (D) Specific IP-WB and WB detection of FLAG-tagged mZCWPW1 over ZCWPW2 from transfected HEK293T cells. Lanes 1,3: protein extracts from untransfected cells; Lanes 2,4: protein extracts from cells transfected with mZCWPW1-FLAG (lane 2) or mZCWPW2-FLAG (lane 4). Lanes 1–2, 5 µg extracts; lanes 3–4: 50 µg extracts. The ZCWPW1 antiserum detects the same protein band as the anti-FLAG antibody in the expected MW range (74 KDa) both by direct WB and by IP-WB, but does not show any reactivity against mZCWPW2-FLAG. The preimmune serum does not detect the mZCWPW1-FLAG protein band. Detection of beta-actin serves as a loading control. The asterisk indicates degradation fragments typically observed, as in (A). (E) Specific IF detection of FLAG-tagged mZCWPW1 over ZCWPW2 from transfected HEK293T cells. Cells were co-immunostained with ZCWPW1 antiserum or the pre-immune serum (green), and a FLAG antibody (red). The ZCWPW1 antiserum detects mZCWPW1-FLAG (precisely overlapping the signal detected by the FLAG antibody results in yellow fluorescence in the merged image), but not mZCWPW2-FLAG protein. No signal is detected with the pre-immune serum.
Figure 2—figure supplement 2. ZCWPW1 localises to both subtelomeric and subcentromeric regions of chromosomes in pachytene cells.

Figure 2—figure supplement 2.

(A) Testis chromosome spreads from 10-week-old WT mice were immunostained for SYCP3 and ZCWPW1, and hybridised by FISH with distal telomeric (Tel) and proximal centromeric (Cen) probes. To aid the visualisation of each signal, the bottom right merged image was decomposed into multiple combinations of two to three individual channels, as indicated. Note that the ZCWPW1 foci do not exactly co-localize with the FISH signals, and generally lie more internally (subtelomerically and subcentromerically) on the chromosome axis. These images are representative of the results obtained for three mice. Scale bar: 10 μm. (B) The localisation of ZCWPW1 foci to either Tel or Cen end, or both ends, of the synaptonemal complex was quantified in mid-Pachytene to late-Diplotene cells. Chromosomes that did not show any ZCWPW1 foci were recorded as ‘not labeled’. Error bars represent 95% confidence intervals using the Wilson method. n = 3–25 cells of each stage from one mouse. Raw data in Figure 2—source data 1.