JCS01328 Supplemental Material

Experimental Design

Mouse oocytes, fertilized eggs, pronuclear stage zygotes, two-cell, four-cell, eight-cell, morula and blastocyst were collected, stained with the panel of anti-modified histone antibodies and prepared for indirect immunofluorescence analysis using scanning confocal microscopy. Every panel shown is a representation of a minimum of three repeated treatments with at least three individuals selected, per stage, per treatment. For fertilization, zona-free mouse eggs were incubated with capacitated sperm for ~45 minutes prior to fixation. Therefore, most sperm seen in these images have bound to eggs but have not fused. Chromatin staining is stronger in sperm nuclei than that in decondensing sperm chromatin within the egg cytoplasm, and can therefore be more easily visualized. For simplification purposes, only representative examples of single optical sections of selected developmental stages are shown for each of the modifications tested.

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Anti-histone modification antibodies are not reactive with sperm proteins. Capacitated mouse caudal epididymal sperm were air dried on to a microscope slide, fixed and permeablized in a manner similar to that for eggs and embryos. Indirect immunofluorescence was then performed using a panel of rabbit anti-histone modification polyclonal antibodies and a monoclonal antibody (1:100 dilution) generated against protamine, a known tissue-specific protein that localizes to murine sperm chromatin. The protamine control was included to ensure that the permeabilization protocol was sufficient to allow for antibody access to chromatin. Indirect immunofluorescent (IIF: B, D, F, H, J, L, N) and transmitted light (Light: A, C, E, G, I, K. M) images of modified histones were then captured at ´230 using a Zeiss fluorescent microscope. The anti-protamine antibody reacted with mouse sperm heads (B) while all anti-histone modification antibodies (D, F, H, J, L, N) were not seen to react with mouse sperm proteins.

• Supplemental Figure 2 -

  Fig. S2. Absorption of anti-histone modification antibodies with modified peptides blocks antibody reactivity with immature oocyte chromatin. Anti-histone modification antibodies were either pre-absorbed with saturating amounts of cognate histone modification peptide or were not. The antibodies were then used to probe fixed and permeabilized immature mouse oocytes and indirect immunofluorescence was performed in order to evaluate antibody specificity. Images were captured using a Zeiss fluorescent microscope (´230). When the anti-histone modification antibodies were not pre-absorbed with cognate peptide, reactivity with nuclear chromatin was observed using all antibodies tested. (A) histone H3 arginine 17-methyl [Me(Arg17)H3], (B) histone H4 arginine 3-methyl [Me(Arg3)H4], (C) histone H3 lysine 4 methyl [Me(Lys4)H3], (D) histone H3 lysine 9-methyl [Me(Lys9)H3], (E) hyperacetylated H4 and (F) phosphorylated serine 1 on histone H4/H2A [Ph(Ser1)H4/H2A]. (G-L) Respective light images for A-F. However, when the anti-histone modification antibodies were pre-absorbed with their cognate modified peptide, reactivity with nuclear chromatin was not observed (M-R). (S-X) Respective light images for (M-R). A limited amount of non-specific reactivity with cytoplasmic proteins was observed in all cases.