PMC

(a,b) Pronuclear formation and the first zygotic mitosis. (i–x) Confocal images of eggs or embryos at the indicated stages stained for histones to reveal nuclei. Left panels views were reconstituted by fusing two confocal images of the anterior and the posterior regions. Right panels are magnifications of the nuclei (the insets in (i–iii) show the male pronucleus). Male pronuclei are indicated (arrows). PB, polar bodies. Scale bars, 10 µm.

NE dynamics during zygote formation. Confocal images of embryos stained for Lamin Dm0 (green), acetylated histone H4 (red) and histones (blue). (a) Top panels show fertilized eggs at the indicated stages. Below are magnifications of the male pronucleus for each egg (insets). At fertilization, Lamin Dm0 is detected for the first time on the male pronucleus at the onset of pronuclear migration. (b–d) First zygotic cycle at the indicated stage. Paternal chromosomes are enriched in acetylated histone H4. Male and female pronuclei appose without fusing their NE and divide separately in mitosis 1. In anaphase, Lamin Dm0 is weakly detected on chromosomes. (e) Prophase of cycle 2. Scale bars, 10 µm.

HIRA complex is essential for paternal chromatin assembly at fertilization. (a) Confocal images of fertilized eggs in meiosis II stained for DNA (red) and HIRA or histone H3.3 variant (green). In wild-type embryos (wt), the maternal histone H3.3 chaperone HIRA specifically accumulates in the male pronucleus at fertilization, where it promotes de novo assembly of nucleosomes independently of DNA synthesis. Newly assembled paternal chromatin is specifically enriched in H3.3. HIRA cooperates with YEM for de novo assembly of paternal chromatin after the removal of SNBPs. In eggs laid by Hira^ssm or yem²/Df(3R)3450 females, H3.3 is not deposited in the male pronucleus. (b) Pronuclear apposition in wt and Hira^ssm eggs. The male pronucleus appears abnormally condensed in mutant eggs. (c) A gynohaploid embryo laid by a yem²/Df(3R)3450 female in prophase of the second mitosis. The male pronucleus is visible between the two haploid nuclei. Scale bars, 10 µm.

Sperm and fertilization in D. melanogaster. (a) Isolated D. melanogaster spermatozoa from transgenic flies expressing a Don Juan-GFP fusion protein (Dj : GFP) [] that stains the flagellum and a ProtamineB-RFP fusion protein (ProtB : RFP) []. Note that only a fraction of the flagella is visible on this picture. Scale bar, 10 µm. (b) In D. melanogaster, spermatozoa including the whole flagellum penetrate the egg. A confocal image of a freshly laid egg, with its chorion and dorsal appendages. The flagellum of a Dj : GFP fertilizing spermatozoon is visible in the cytoplasm (arrow). Scale bar, 100 µm. (c) A confocal section of a dechorionated egg in metaphase of meiosis II stained for DNA. The vitelline envelope has not been removed and the protruding micropyle is visible at the anterior tip of the egg (arrow). The male pronucleus and female meiotic chromosomes are indicated with symbols. (d) An egg at pronuclear apposition stained for DNA (red). The Dj : GFP sperm flagellum (green) is coiled in the anterior region of the egg. PB, polar bodies. (e) A blastoderm embryo stained as in (d). The flagellum is still detected in the anterior region.

Sperm aster formation, pronuclear migration and organization of the gonomeric spindle. (a–h) Schematic of zygote formation in D. melanogaster. (a) At fertilization, maternal chromosomes are in metaphase of meiosis I. The spermatozoon enters the egg through the micropyle (arrow). The needle-shaped sperm nucleus is still packaged with SNBPs (green) and two centrioles are visible at the junction with the sperm tail: a GC and a centriole precursor, called the PCL (represented in blue). (b) Metaphase of meiosis II. The two meiotic spindles are connected by an aster of microtubules, the central spindle pole body (arrow). SNBPs have been replaced by histones and the male pronucleus has begun to decondense. The paternal centrioles recruit PCM and initiate the formation of the sperm aster. (c) Pronuclear migration. By the end of female meiosis, the sperm aster has increased considerably in size and captures the innermost female meiotic product, which becomes the female pronucleus. (d) Pronuclear apposition. The centrosomes have duplicated and are positioned around the male pronucleus (arrow). All nuclei are in S phase. The three polar body nuclei remain at the egg periphery. (e) Metaphase of first mitosis. Each set of parental chromosomes occupies one half of the gonomeric spindle. The polar bodies have condensed into two rosettes of metaphase-like chromosomes (n and 2n). (f) Anaphase of first mitosis. (g) Telophase of first mitosis and karyogamy. The centrosomes have duplicated. (h) Interphase of second mitosis. (i–k) Confocal images of eggs stained for α-Tubulin (green) and DNA (red). (i) Metaphase of meiosis II. (j) Pronuclear apposition. (k) Metaphase of first mitosis. Scale bars, 10 µm.