Figure 1.
In D. simulans embryos from incompatible crosses (CI), paternal chromosomes fail to condense and improperly segregate during the first mitosis.
(A,C,E,H) are uninfected controls in white boxes. (B,D,F,G,I,J) are CI embryos. Paternal, but not maternal chromosomes incorporate acetylated histone H4 during de novo nucleosome assembly (green). DNA is detected with propidium iodide (red). (A,B) pronuclear apposition. (C,D) prometaphase. (E,F,G) anaphase A (F) or B (E,G). (H,I) telophase. (J) late telophase/second S phase. Scale bar is 5 µm.
Figure 2.
Protamine incorporation and removal appear normal in D. simulans CI crosses.
(A,B,C) In infected D.simulans transgenic male testis, Protamine-GFP is detected in groups of late spermatid nuclei (arrowheads in A and B) and in sperm nuclei in seminal vesicles (C). (D,E,F,G,H) Confocal sections of embryos from non-infected females crossed with infected, transgenic males. Protamine-GFP is never detected in the male nucleus (arrowhead) as early as the second female meiotic division (D) or at the pronuclear apposition stage (E). (F,G,H,I) Cycle 1 embryos in metaphase (F), anaphase (G) or telophase (H,I). The embryos in G–I display an obvious CI phenotype with lagging paternal chromatids or chromatin bridges (arrows). No Protamine-GFP is detected in the late paternal chromatin. (I) embryo containing a second, non-activated sperm nucleus (asterisk) whose Protamine-GFP has not been removed serving as internal control for Protamine-GFP detection in embryos. (J) Embryo from non-infected females crossed with non-infected transgenic males. Protamine-GFP is never detected in the male nucleus (arrowhead) in this control. DNA is stained with propidium iodide (red) in all panels except B and C. GFP is detected either directly (A,B,C) or with the use of an anti-GFP antibody (green) (D,E,F,G,H,I,J). Scale bar is 50 µm in A and 10 µm in all other panels.
Figure 3.
Histone variant H3.3 deposition is abnormal in CI D. melanogaster / D. simulans hybrid crosses.
(A) Embryos from hybrid control (uninfected D. melanogaster females x uninfected D. simulans males) or CI (uninfected D. melanogaster females x infected D. simulans males) crosses were stained to reveal a tagged H3.3 (green) and DNA (propidium iodide in red), after sperm entry. The two female meiotic products are still in metaphase II, indicating that sperm entry just occurred (in white frame). (A′) H3.3 deposition is undistinguishable between embryos from hybrid control or CI crosses during pronuclear apposition. Note that the male pronucleus is always slightly smaller then the female pronucleus. (B) Acetylated histone H4 colocalizes with H3.3 in perinuclear rings in CI. Magnification of male pronuclei from hybrid crosses, acetylated H4 in purple. Scale bar is 10 µm.
Figure 4.
In D. simulans, replication of the male pronucleus is prolonged in CI embryo.
(A) Embryos from control, rescue, or CI crosses were fixed and stained for PCNA (red), and DNA (propidium iodide, cyan). Scale bars are 10 µm. (B) Synchrony was scored when both apposed pronuclei were PCNA negative. Conversely, asynchrony was established when a pronucleus was PCNA positive whereas its counterpart was negative. (C) In CI embryo, PCNA is present in male pronuclear chromatin after pronuclear envelopes breakdown and spindle assembly. Embryos from control and CI crosses were fixed and stained for PCNA, and with two monoclonal antibodies, the anti-lamin ADL84 and an anti-tubulin to reveal the presence of the pronuclear envelopes and the spindle set up respectively (in green). The asterisk marks the uncondensed male pronucleus. Scale bar is 10 µm. Male pronuclei can be identified according to their smaller size compare to female pronuclei during apposition (A), or because of the chromosome condensation defects in CI (C). (D) % of PCNA positive male pronuclei after NEB in control crosses and CI crosses.
Figure 5.
A schematic of key events in the transformation of sperm to male pronucleus in embryos from normal and CI crosses.
Normal cross: Immediately following fertilization, the specialized nuclear envelope (lacking nuclear pores) of the male pronucleus is removed. Next, the protamines are removed and replaced by maternally supplied histones, including the replication-independent histone H3.3. This event is followed by lamin deposition and formation of a conventional nuclear envelope containing nuclear pores. Next, S-phase is initiated and upon completion, Cdk1 is activated driving nuclear envelope breakdown, chromosome condensation, and spindle assembly. CI cross: At the cytological level, removal of the sperm nuclear envelope and protamines appear normal. Often however, histone H3.3 deposition is abnormal, resulting in a ring of histone H3.3 encompassing the paternal pronucleus. This is the earliest documented CI phenotype in embryos and is similar to that observed for mutants in the chromatin remodeling protein Chd1. Imaging PCNA, a marker for replicating chromosomes, indicates that replication initiates normally in CI embryos, but is prolonged or incomplete. This may be a direct result of the earlier defects in H3.3 deposition. Replication delays activate S-phase checkpoints and thus are likely the cause of the previously described delays in Cdk1 activation and nuclear envelope breakdown.