Figure 1.
DNA degradation by divalent cations in vas deferens sperm is more severe and less reversible than in epididymal sperm.
Spermatozoa from the epididymides (lanes 3–5) or the vas deferens (lanes 6–8) were embedded in agarose plugs, and incubated in mHCZB supplemented with MnCl2 and CaCl2 as described in Methods, for 1.5 hrs at RT. They were then treated without EDTA (lanes 4 and 7) or with (lanes 5 and 8) to reverse TOP2B-induced breaks. The plugs were then electrophoresed by FIGE. Control lanes (3 and 6) were untreated spermatozoa. Lines 1 and 2 are markers.
Figure 2.
DNA fragmentation induced in SCF spermatozoa is not detectable by TUNEL after fertilization.
(a–d1) A Vas-SCF zygote stained with TUNEL and counterstained with DAPI 5 hrs after sperm injection, and visualized by confocal microscopy. (e–h1) A control zygote that was fixed and treated with DNAse I 10 mins after fertilization, and then stained by TUNEL. (i–l1) A control zygote that was treated with DNAse I 5 hrs after fertilization. Double columns in each panel present the same embryo at different focal planes. S – sperm, Mc – maternal chromatin, M – maternal pronucleus, P – paternal pronucleus, Pb – polar body. All images are shown at the same magnification (bars = 20 µm).
Figure 3.
γH2AX is detectable in the paternal pronuclei of Vas-SCF zygotes before DNA synthesis.
(A) Vas-SCF zygotes showing three different patterns of γH2AX staining. (Aa) Fluid expression of γH2AX in the paternal pronucleus (P) and no visible expression in the maternal pronucleus; (Ab), Punctate γH2AX expression in the paternal pronucleus; (Ac), Punctate expression in both pronuclei. (B) Four groups of embryos were analyzed and scored for γH2AX staining in each pronuclei (n = 30 to 47 zygotes in each group).
Figure 4.
DNA replication is delayed in Vas-SCF zygotes.
(A) Three different patterns of EdU staining in Vas-SCF zygotes during S-phase. Each set of four images represents the same zygote at different focal planes to view the paternal (right) and maternal (left) pronuclei. (Aa-d) In most cases during early S-phase, EdU staining was only visible in the maternal pronucleus. (Ae-h) EdU staining was much more intense in the maternal pronucleus, but was it was also visible in the paternal. (Ai-l) Edu staining was visible at equal intensity in both pronuclei. All images are shown at the same magnification, bar = 20 µm. (B) Zygotes with any EdU staining in the paternal pronucleus, whether it was much less or equal to that of the maternal pronucleus, were scored as positive. (C) Only zygotes that had paternal pronuclei with EdU staining intensities equal to that of the maternal pronuclei in the same zygote were scored only as positive. Error bars represent standard deviations, and points on the graphs for which the error bars are not visible had standard deviation values below the size that is covered by the point drawn to indicate the value. For at 50 Vas-SCF zygotes were scored for each time point, and 30 Vas-Ctrl zygotes were scored. In all zygotes at 8 hrs and beyond, maternal pronuclei had strong EdU staining. All images are shown at the same magnification, bar = 20 µm.
Figure 5.
Chromosomal analysis of Vas-SCF and Epi-SCF zygotes.
(A) Vas-Ctrl; normal maternal (M) and paternal (P) chromosome complements, n = 20 chromosomes each. Maternal and paternal chromosome plates can be distinguished based on chromosome morphology; maternal chromosomes are usually shorter and thicker. (B) Epi-SCF; normal maternal chromosome complement (M, n = 20) and paternal complement (P) with multiple chromosome aberrations. Some of the chromosome fragments are shown with arrowheads. (C) Vas-SCF, normal maternal complement (M, n = 20 chromosomes) and paternal chromatin seen as early stage of prematurely condensed chromosomes (PCC). (D-F) Vas-SCF; examples of paternal chromatin at different levels of premature chromosome condensation (PCC). Note the increasing chromatin condensation and formation of few separate chromosomes (arrows), from D to F, as well as presence of chromosome fragments (arrowheads). Scale = 20 µm.
Table 1.
Paternal Chromosomal analysis of zygotes obtained after ICSI with SCF-induced sperm.
Figure 6.
Developmental arrest in embryos with damaged DNA.
Embryos produced by injection of Epi-Ctrl, Vas-Ctrl, Epi-SCF and VAS-SCF were closely followed for four days in culture, and scored for their stage of development. (A) Diagram of the early (first cycle) stages of development that were scored. Quantitative data showing the developmental progress at 8 (B), 19 (C), 24 (D), and 48 (E) hrs after fertilization. Fertilization was achieved by ICSI with injections timed not to exceed 15 min per group. For explanation of developmental stages see text. The experiment was repeated twice with at least 45 embryos examined for each group. Error bars represent standard deviations.
Figure 7.
Zygotic response to severe paternal DNA damage.
Oocytes fertilized with sperm that have damaged DNA (A) form pronuclei within the normal time, but phosphorylate H2AX in the paternal pronuclei (B). DNA replication is delayed in the paternal but not the maternal pronucleus, and this causes both pronuclei to delay progression to metaphase (C). The pronuclear membranes break down synchronously, though late (D). Some embryos do not progress past the G2/M border, but those that do die or arrest before they progress to blastocysts (F).