Unlocking sperm chromatin at fertilization requires a dedicated egg thioredoxin in Drosophila

Abstract

In most animals, the extreme compaction of sperm DNA is achieved after the massive replacement of histones with sperm nuclear basic proteins (SNBPs), such as protamines. In some species, the ultracompact sperm chromatin is stabilized by a network of disulfide bonds connecting cysteine residues present in SNBPs. Studies in mammals have established that the reduction of these disulfide crosslinks at fertilization is required for sperm nuclear decondensation and the formation of the male pronucleus. Here, we show that the Drosophila maternal thioredoxin Deadhead (DHD) is specifically required to unlock sperm chromatin at fertilization. In dhd mutant eggs, the sperm nucleus fails to decondense and the replacement of SNBPs with maternally-provided histones is severely delayed, thus preventing the participation of paternal chromosomes in embryo development. We demonstrate that DHD localizes to the sperm nucleus to reduce its disulfide targets and is then rapidly degraded after fertilization.

Figure 1. DHD is required for sperm nuclear decondensation.

(a) The dhd genomic region (4F4, X chromosome) showing the genomic P[dhd^XhoI/XhoI] rescue transgene and the Df(1)J5 deletion that disrupts dhd and TrxT. (b) Zygotic expression of the paternally-inherited cid-GFP transgene, which expresses a GFP-tagged centromeric histone (insets), is detected in late control embryos but not in dhd embryos. Scale bar, 50 μm. (c) At fertilization, the sperm nucleus (inset) fails to decondense in dhd eggs. The sperm flagellum is stained with the Don Juan(DJ)-GFP marker (green). DNA is in red. Note that the four female meiotic products are visible in both eggs. Scale bar, 10 μm. (d) The sperm nucleus does not participate in the early zygotic mitoses in dhd embryos. Maternal chromosomes are visualized with anti-H3K27me2 staining (green) and appear yellow. PB: polar bodies. Scale bar, 5 μm.

Figure 2. Sperm aster formation and pronuclear migration are affected in dhd eggs.

(a) Left: a dhd egg at pronuclear apposition. The three polar bodies (PB) are visible. Right: a dhd egg in which the female pronucleus failed to migrate and remained associated with the polar bodies. The respective percentage of each phenotype is indicated (n=586). Sperm nuclei were visualized using the paternal Mst35Ba-GFP transgene (green). DNA is stained with propidium iodide (red). (b) Top panel: a w¹¹¹⁸ egg during meiosis II showing the male nucleus and the sperm acrosome (arrowhead). Bottom: a dhd eggs during meiosis II with the acrosome still associated to the anterior extremity of the sperm nucleus (arrowhead). The acrosome is visualized with the paternal Snky-GFP marker. (c) Eggs in telophase of meiosis II from w¹¹¹⁸ and dhd^J5 females stained for DNA (white), α-tubulin (red) and γ-tubulin (green) to reveal nuclei, the sperm aster and the centrosomes, respectively (the female meiotic products are not visible on these confocal sections). In w¹¹¹⁸ eggs (upper panels), the anti-γ-tubulin stains the zygotic centrosome (which contains a pair of sperm centrioles) at proximity of the male pronucleus (100%, n=25; arrowhead, inset). In dhd eggs (lower panels), the centrosome appears diffused and almost systematically associated (98%, n=53) to the compacted sperm nucleus (arrowhead, inset). The brackets indicate the extent of the sperm aster in w¹¹¹⁸ and dhd^J5eggs. (d) Left: a dhd egg in telophase of meiosis II. Right: a dhd embryo after the end of cycle 1. The centrosomes (arrowheads, inset) have duplicated and detached from the male nucleus. The rosette of polar body (PB) chromosomes is visible. Green: γ-tubulin, red: DNA. Scale bars, 10 μm.

Figure 3. DHD is required for the timely removal of SNBPs at fertilization.

(a) Pronuclear apposition in eggs from control (w¹¹¹⁸) or dhd^J5 females mated with transgenic Mst35Ba-GFP males. The sperm nucleus (arrowhead) in dhd eggs still contains Mst35Ba-GFP (green). (b) Eggs or cycle 1 embryos from w¹¹¹⁸, dhd^J5 or dhd^J5; P[dhd^XhoI/XhoI] (rescue) females mated with Mst35Ba-GFP males. The replacement of SNBPs with histones is severely delayed in dhd eggs and the phenotype is fully rescued by the genomic transgene. (c) Dynamics of SNBP/histone replacement in dhd eggs fertilized by control (Mst77F-GFP) or mutant (ΔMst35B ; Mst77F-GFP) sperm. Distribution of phenotypic classes is shown for each stage. ‘> Cycle 1' indicates embryos with the polar body condensed into a rosette of chromosomes. Scale bars, 5 μm.

Figure 4. DTT induces SNBP eviction and sperm nuclear decondensation in vitro.

Mst35Ba-GFP sperm dissected from seminal vesicles were incubated with DTT for the indicated time and stained with propidium iodide (PI, red) and mBrB (blue). (a–c) Quantification of PI (a), native GFP (b) and mBrB (c) fluorescent signals in representative experiments (Mann–Whitney test. ***P<0.001. ****P<0.0001). Error bars indicate SD calculated for 29 analysed nuclei for each time point. (d) Confocal images of Mst35Ba-GFP sperm nuclei incubated with DTT for the indicated time. Arrowheads indicate condensed nuclear regions positive for both mBrB and Mst35Ba-GFP. Scale bar: 5 μm.

Figure 5. Expression and distribution of maternally-expressed DHD protein.

(a–b) Western blot analyses of DHD expression in adult tissues (a) or embryos collected during the indicated time windows after egg laying (b). α-tubulin was used as loading control. Ov: ovaries, T: testis, C: carcasses. See also Supplementary Fig. 6. (c) Confocal images of eggs or embryos laid by w¹¹¹⁸ or dhd^J5 females and stained for DHD (left) and DNA (right). A close-up of the region containing the nuclei is shown on the right (arrows: male and female nuclei; PB: polar bodies). Scale bar, 10 μm

Figure 6. DHD is specifically targeted to the sperm nucleus at fertilization.

(a) Mechanism of disulfide bond reduction on target proteins by wild-type DHD. (b) Strategy for trapping DHD on its targets: the replacement of the resolving cysteine with a serine (C34S) in the WCGPCK motif is predicted to stabilize the mixed-disulfide between DHD and its target. (c) Sperm nuclei in eggs laid by females of the indicated genotypes stained for DNA and with the anti-DHD antibody. The number of DHD-positive sperm nuclei is indicated for each genotype. See also Supplementary Fig. 8. Scale bar, 5 μm.

Figure 7. DHD protein is sufficient to decondense sperm nuclei in vitro.

(a) Confocal images of Mst35Ba-GFP sperm nuclei incubated with recombinant thioredoxins. The DHD^WT recombinant thioredoxin induces nuclear decondensation and Mst35Ba-GFP removal in a way similar to DTT treatment. The sperm nuclei incubated with DHD^C34S or Trx-2 recombinant proteins appear similar to sperm nuclei incubated with control buffer. Green: Mst35Ba-GFP, Blue: mBrB, Red: DNA. (b,c) Quantification of native GFP signal (b) or mBrB fluorescence (c) from sperm nuclei incubated with the recombinant thioredoxins. Error bars indicate SD calculated for 30 analysed nuclei for each experiment. Scale bar, 5 μm.