ARTICLE
Received 3 Mar 2016
| Accepted 7 Oct 2016 | Published 23 Nov 2016
Unlocking sperm chromatin at fertilization requires
a dedicated egg thioredoxin in Drosophila
Samantha Tirmarche
1
, Shuhei Kimura
1
, Raphae
¨
lle Dubruille
1
,Be
´
atrice Horard
1
& Benjamin Loppin
1
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.
DOI: 10.1038/ncomms13539
OPEN
1
Laboratoire de Biome
´
trie et Biologie E
´
volutive, Universite
´
de Lyon, Universite
´
Lyon 1, CNRS, UMR 5558, Villeurbanne F-69622, France. Correspondence and
requests for materials should be addressed to B.L. (email: benjamin.loppin@univ-lyon1.fr).
NATURE COMMUNICATIONS | 7:13539 | DOI: 10.1038/ncomms13539 | www.nature.com/naturecommunications 1
I
n sexually reproducing animals, the differentiation of
haploid spermatids into mature spermatozoa involves major
reorganization of the nuclear architecture
1
. Starting as round
nuclei after the second male meiotic division, spermatid nuclei
slowly transform to eventually acquire the final, species-specific
shape of mature sperm nuclei. Extreme compaction of nuclear
DNA generally accompanies the streamlining of spermatids,
resulting in the shutdown of basic nuclear activities, including
transcription and DNA repair
2
. In most species, sperm nuclear
compaction requires the massive replacement of somatic-type
histones with sperm nuclear basic proteins (SNBPs)
3,4
. SNBPs
encompass a heterogeneous group of chromosomal proteins that
are specifically expressed in male germ cells and deposited during
spermiogenesis
3,5
. Protamines, the best characterized SNBPs, are
small (50–60 aa), arginine rich proteins present in vertebrates and
some invertebrates
6,7
. A model based on mammalian protamines
has proposed that these positively charged proteins bind the
major groove of the double helix and form toroid-like structures
containing about 50 kilobases of sperm DNA
7–9
. Protamines in
eutherian mammals and a few other animal groups are also
enriched in cysteine residues, which are otherwise rare in
chromosomal proteins, including histones and most SNBPs
3
.
During sperm maturation in eutherian mammals, oxidation of
the protamine cysteine thiols (–SH) allows the formation of a
tridimensional network of disulfide bridges (–S–S–).
Intermolecular disulfide crosslinks notably participate in the
stabilization of sperm chromatin by connecting adjacent
chromatin fibres
7,8,10,11
. It is actually well established that for
most species of mammals, a thiol reducing agent, such as
dithiothreitol (DTT), is required to elicit sperm nuclear
decondensation in vitro. In addition, it has been alternatively
proposed that in human sperm, protamines thiols are
non-covalently bridged by Zinc (Zn
2 þ
), thereby preventing or
limiting the formation of excess disulfide bonds that could
perturb sperm nuclear decondensation at fertilization
12
.
In this work, we functionally characterized the Drosophila
maternal thioredoxin Deadhead (DHD) and we demonstrate that
DHD is required to unlock sperm chromatin at fertilization.
Thioredoxins are small redox proteins found in all organisms.
They typically reduce disulfide bonds on target proteins using
a pair of cysteine thiols present in their conserved active
CGPC site
13
. Thioredoxins play important metabolic, protective
or signalling functions but the molecular bases of their
target specificity or functional specialization remain poorly
understood
14
. Three classical thioredoxins are found in
Drosophila melanogaster. Trx-2 is a non-essential ubiquitous
protein that participates in the protection against oxidative
stress
15,16
, whereas TrxT and DHD are sex-specific thioredoxins
encoded by a pair of adjacent genes
17,18
. While the role of the
testis-specific TrxT is unknown, DHD is specifically expressed in
the female germline and is essential for embryo development
19,20
.
In this work, we demonstrate that DHD is essential for sperm
nuclear decondensation at fertilization.
Results
deadhead mutant females produce gynohaploid embryos. The
original characterization of the dhd maternal effect phenotype
showed that the vast majority of eggs produced by homozygous
mutant females (hereafter referred to as dhd eggs) were fertilized
but failed to develop
19
. Interestingly, Salz and colleagues
also observed that about 5% of dhd embryos reached late
embryogenesis and showed severe head developmental defects
19
.
We noticed that these defects were reminiscent of the incomplete
head involution typically observed in haploid Drosophila
embryos
21–23
. In addition, early mention of sperm nuclear
decondensation defects in dhd eggs (see ref. 24) prompted us
to reinvestigate the dhd phenotype in detail.
The original dhd
J5
allele is a 1.4 kb deficiency on the X
chromosome that disrupts dhd and the immediately adjacent,
paralogous gene Trx-T.AsTrx-T is strictly expressed in the male
germline
18
, the dhd
J5
allele can be used to specifically address the
maternal function of dhd
19
. In addition, the dhd maternal effect
embryonic lethal phenotype is fully rescued by a genomic
transgene containing dhd but not Trx-T
19
(Fig. 1a; Table 1). To
test the possibility that late dhd embryos could represent haploid
escapers of the early arrest phenotype, we crossed dhd
J5
mutant
females with males homozygous for a cid-GFP transgene.
In contrast to embryos from a control cross that zygotically
expressed the centromeric marker CID::GFP in all cells, none of
Df(1)J5
P[dhd
XhoI/XhoI
] genomic transgene
TrxT dhd CG4198
CG15930
1 kbp
DNA
CID-GFP
DNA
CID-GFP
CID-GFP
DNA
CID-GFP
DNA
CID-GFP
CID-GFP
PB
PB
DNA
DJ-GFP
Meiosis II Cycle 1 Cycle 2
DNA
H3K27me2
2n 2n
n
n
w
1118
dhd
J5
w
1118
X cid-GFP
dhd
J5
X cid-GFP
w
1118
dhd
J5
a
b
c
d
Figure 1 | DHD is required for sperm nuclear decondensation. (a )Thedhd
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 mm. (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 mm. (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 mm.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13539
2 NATURE COMMUNICATIONS | 7:13539 | DOI: 10.1038/ncomms13539 | www.nature.com/naturecommunications
the dhd embryos that reached gastrulation expressed the paternal
marker (Fig. 1b). Direct observations of mitotic figures in dhd
embryos were also indicative of haploid development
(Supplementary Fig. 1). We conclude that rare dhd embryos
escaping early arrest develop as gynohaploids after the loss of
paternal chromosomes.
dhd affects sperm nuclear decondensation. We then examined
fertilization and zygote formation in dhd eggs to follow the fate of
paternal chromosomes. As for other insects, fertilization is
internal in Drosophila and the needle-shaped, extremely compact
sperm nucleus immediately initiates decondensation after its
delivery in the egg cytoplasm
25
. Accordingly, during the second
female meiotic division (the earliest stage that can be practically
observed), the sperm nucleus in wild-type eggs typically appears
roundish or at least partially decondensed. In striking contrast,
the sperm nucleus systematically retained its original needle-
shape in fertilized dhd eggs, showing no sign of decondensation
(100%, n ¼ 66; Fig. 1c). Observation of early syncytial divisions in
dhd embryos confirmed that the paternal genome, still compacted
in the sperm nucleus, does not participate in development, thus
providing an explanation for the origin of gynohaploid embryos
(Fig. 1d).
The female pronucleus often fails to migrate in dhd eggs.In
Drosophila, female meiosis resumes at egg activation, which
occurs shortly after ovulation and independently of fertilization.
With its two spindles organized in tandem, the second meiotic
division generates four meiotic products typically aligned
orthogonally to the antero-dorsal egg surface. In fertilized eggs,
the innermost meiotic product then migrates towards the male
nucleus and becomes the female pronucleus. Pronuclear
migration occurs along microtubules of the giant sperm aster,
which is nucleated from the paternally-inherited centrioles
25,26
.
In contrast to the conclusion of the original report
19
, we observed
that female meiosis resumed normally (100%, n ¼ 17) in dhd
eggs, with the four meiotic products aligned in a way
indistinguishable to wild-type eggs (Fig. 1c). We however
noticed that, subsequently, the female pronucleus frequently
(78%, n ¼ 586) failed to migrate toward the sperm nucleus and
instead remained at the egg periphery with the three other
products, as in wild-type unfertilized eggs (Fig. 2a). Pronuclear
migration also fails in eggs fertilized by sperm from paternal
effect male sterile mutants affecting sperm plasma membrane
(SPM) breakdown, such as sneaky (snky), misfire (mfr) and
wasted (wst)
27–29
. SPM breakdown is indeed not only critical for
sperm nuclear decondensation but is also required for the release
of paternal centrioles in the egg cytoplasm and the formation of
the sperm aster. To determine if persistence of the plasma
membrane on the sperm nucleus could account for the dhd
phenotype, we crossed control and dhd mutant females with
males expressing Snky-GFP
30
. Snky-GFP is a specific marker of
the sperm acrosome, a membrane-bound vesicular structure at
the anterior tip of the sperm nucleus. After fertilization in
Drosophila, the acrosome normally detaches from the sperm
nucleus after the removal of SPM and remains at close proximity
in the egg cytoplasm
30
. Interestingly, in dhd eggs, the acrosome
remained connected to the anterior extremity of the sperm
nucleus, opening the possibility that SPM breakdown was not
completed in dhd eggs (Fig. 2b). Anti-a-tubulin immunostaining
however revealed that a sperm aster was systematically present in
dhd eggs, in sharp contrast to eggs fertilized with snky mutant
sperm where this structure is never observed (ref. 27 and
Supplementary Fig. 2). Although this result demonstrates that
SPM breakdown is at least initiated in dhd eggs, we noted that the
sperm aster appeared abnormally small compared with wild-type
eggs observed at the same stage (Fig. 2c). Additionally, we
observed that the zygotic centrosome was poorly defined in dhd
eggs and remained associated to the needle-shape nucleus
(Fig. 2d). In early zygotes, however, the centrosome duplicated
and eventually detached from the sperm nucleus (Fig. 2d). These
observations suggest that the release of paternal centrioles is
delayed in dhd eggs, which in turn likely affects the recruitment of
pericentriolar material and the timely growth of the sperm aster.
Replacement of SNBPs with histones is delayed in dhd eggs.
Drosophila melanogaster sperm nuclei almost entirely lack
histones and sperm DNA is instead packaged with at least four
SNBPs: the almost identical Mst35Ba and Mst35Bb paralogous
proteins (also known as ProtA and ProtB, respectively), Mst77F
and the recently identified Prtl99C
31,32
. In contrast to
protamines, Drosophila SNBPs are substantially larger proteins
(144–215 aa) that contain equivalent levels of arginine and lysine
basic residues. In addition, these protamine-like proteins all
possess a motif related to the High Mobility Group (HMG) box
found in insects but not in mammals
32,33
. At fertilization,
Drosophila SNBPs are rapidly removed from the sperm nucleus
and immediately replaced with maternally-provided histones
in a process that requires the nucleosome assembly complex
HIRA
34–36
. To analyse the replacement of SNBPs with histones in
dhd eggs, we crossed mutant females with males expressing GFP-
tagged versions of the fly SNBPs (refs 31,37). Strikingly, we
observed that the sperm nucleus systematically retained SNBPs in
dhd eggs (Fig. 2a) and failed to incorporate histones (100%,
n ¼ 33) (Fig. 3a,b). Importantly, we observed an identical and
equally penetrant phenotype in eggs from dhd
J5
/Df(1)C70
females, where Df(1)C70 is a chromosomal deletion that
encompasses the dhd genomic region (Supplementary Fig. 3;
Table 1). Furthermore, this phenotype was fully rescued by a
transgene containing the genomic dhd sequence (Fig. 3b and
Table 1), thus demonstrating that the observed phenotype is
caused by the loss of dhd function.
Table 1 | Embryo hatching rates.
Genotype of females Genotype of males No. of eggs Hatch. rate (%)
w
1118
w
1118
/Y 264 90.9
w
*
, Df(1)dhd
J5
/w
*
, Df(1)dhd
J5
w
1118
/Y 577 0
w
*
, Df(1)dhd
J5
/ Df(1)JC70 w
1118
/Y 489 0
w
*
, Df(1)dhd
J5
/w
*
, Df(1)dhd
J5
; CyO, P[dhd
XhoI/XhoI
,w
þ
]/ þ w
1118
/Y 136 80.1
w
*
, Df(1)dhd
J5
/w
*
, Df(1)dhd
J5
w
1118
/Y ; DMst35B/DMst35B 328 0
w
*
, Df(1)dhd
J5/
w
*
, Df(1)dhd
J5
; P[dhd
WT
,w
þ
]/P[dhd
WT
,w
þ
]w
1118
/Y 251 80.5
w
*
, Df(1)dhd
J5
/w
*
, Df(1)dhd
J5
; P[dhd
C34S
,w
þ
]/P[dhd
C34S
,w
þ
]w
1118
/Y 955 0.2
The Df(1)dhd
J5
deficiency and the P[dhd
XhoI/XhoI
] genomic rescue transgene are described in Fig. 1a. Df(1)JC70 is a large deficiency uncovering the dhd locus. DMst35B mutation is described in ref. 39.
P[dhd
WT
] and P[dhd
C34S
] transgenes are described in the text. w* indicates an unspecified mutant allele of the white gene.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13539 ARTICLE
NATURE COMMUNICATIONS | 7:13539 | DOI: 10.1038/ncomms13539 | www.nature.com/naturecommunications 3
Interestingly, in dhd embryos that had reached or completed
the first zygotic cycle, histones were eventually detected in the
sperm nucleus despite the persistence of SNBPs (Fig. 3b and
Supplementary Fig. 3). To confirm that this histone staining
reflected de novo assembly of nucleosomes on paternal chroma-
tin, we performed anti-histone immunostainings on embryos
produced by dhd Hira double mutant females. We used the
Hira
ssm
point substitution allele (R225K), which does not affect
female viability or fecundity but strongly impacts de novo
nucleosome assembly in the male pronucleus
34
.Indhd embryos
observed during nuclear cycle 1 or beyond, histones were almost
systematically detected in the needle-shaped sperm nucleus
(95.3%, n ¼ 65). In clear contrast, the sperm nucleus did not
contain detectable levels of histones in a majority of dhd Hira
double mutant embryos observed during the same developmental
time window (74.2%, n ¼ 31), the rest of embryos showing only a
very weak staining in the sperm nucleus that we attribute to
residual Hira activity
34
(Supplementary Fig. 4). These results
confirmed that SNBP/histone replacement is dramatically delayed
in dhd eggs, thus preventing male pronuclear formation.
To more directly test the possibility that the dhd nuclear
phenotype was indeed caused by a delay in SNBP removal, we
took advantage of the fact that deletion alleles of both Mst35Ba/b
protamine-like genes do not prevent the production of
fertilization-competent sperm
38,39
. We reasoned that the
previously reported impact of Mst35Ba/b deletion on sperm
chromatin compaction
32,38
could facilitate the SNBP/histone
replacement at fertilization in dhd mutant eggs. Crossing dhd
mutant females with males homozygous for a knock-out of both
Mst35B copies (DMst35B) (ref. 39) did not restore embryo
viability (Table 1). However, we observed a partial but clear
rescue of sperm chromatin remodelling dynamics, as histone
deposition was detected before the end of female meiosis in
this context (Fig. 3c). In a majority of cycle 1 embryos, sperm
nuclei appeared partially decondensed and the SNBP marker
Mst77F-GFP was replaced with histones. Finally, in later
embryos, the male nucleus frequently (76%, n ¼ 81) appeared as
a mass of chromosome-like structures, a phenotype relatively rare
(7%, n ¼ 91) in control dhd embryos collected for the same period
of time (Fig. 3c). We conclude that DHD is critically required for
the rapid removal of SNBPs from the sperm nucleus at
fertilization.
Drosophila sperm chromatin is locked by disulfide bonds.
Previous work demonstrated that the typical WCGPCK redox
catalytic motif of DHD was essential for its function
20
. We thus
Migration (22 %) No migration (78%)
PB
Mst35Ba-GFP
Meiosis > Cycle 1
PB
γ-Tubulin
DNA
α-Tubulin
γ-Tubulin
DNA
α-Tubulin
α-Tubulin
γ-Tubulin
DNA
γ-Tubulin
DNA
α-Tubulin
γ-Tubulin
DNA
α-Tubulin γ-Tubulin
DNA
γ-Tubulin
DNA
α-Tubulin
DNA
snky-GFP
DNA
PB
dhd
J5
dhd
J5
w
1118
w
1118
dhd
J5
dhd
J5
dhd
J5
dhd
J5
abc
d
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 ¼ 586). Sperm nuclei were visualized using the paternal Mst35Ba-GFP transgene (green). DNA is stained with propidium
iodide (red). (b) Top panel: a w
1118
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 w
1118
and dhd
J5
females stained for DNA (white), a-tubulin (red) and g-tubulin (green) to reveal
nuclei, the sperm aster and the centrosomes, respectively (the female meiotic products are not visible on these confocal sections). In w
1118
eggs (upper
panels), the anti-g-tubulin stains the zygotic centrosome (which contains a pair of sperm centrioles) at proximity of the male pronucleus (100%, n ¼ 25;
arrowhead, inset). In dhd eggs (lower panels), the centrosome appears diffused and almost systematically associated (98%, n ¼ 53) to the compacted
sperm nucleus (arrowhead, inset). The brackets indicate the extent of the sperm aster in w
1118
and dhd
J5
eggs. (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: g-tubulin, red: DNA. Scale bars, 10 mm.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13539
4 NATURE COMMUNICATIONS | 7:13539 | DOI: 10.1038/ncomms13539 | www.nature.com/naturecommunications
wondered whether DHD was involved in the reduction of
putative disulfide bonds present on sperm chromatin.
Remarkably, all four Drosophila SNBPs identified so far show
conserved cysteine residues, thus opening the possibility that
they could be involved in the formation of disulfide bonds in a
way similar to mammalian protamines (refs 31,32,40,41;
Supplementary Fig. 5). We thus tested the effect of the disulfide
reducing agent DTT on the dynamics of sperm nuclear
decondensation in vitro. Mature Mst35Ba-GFP spermatozoa
obtained from dissected seminal vesicles remained basically
inert in a non-reducing control buffer, showing no evidence of
nuclear decompaction after 30 min of incubation. Strikingly,
incubation of sperm nuclei in the presence of 2 mM DTT induced
dramatic nuclear decondensation and SNBP removal (Fig. 4).
Nuclear swelling appeared maximal in the central region of the
nuclei, which also showed the strongest reduction of Mst35Ba-
GFP fluorescence. In contrast, both extremities of DTT-treated
nuclei remained relatively compacted with a higher density of
SNBP (Fig. 4d). Incubation with DTT also increased the amount
of free thiol groups in sperm nuclei after 10 min, as measured
using the fluorescent marker monobromobimane (mBrB)
(Fig. 4c). After 30 min exposure to DTT, however, mBrB
fluorescence decreased with the exception of nuclear extremities
that still contained higher levels of SNBP. These observations
suggest that after the 10 min treatment, mBrB is crosslinked to
free thiol groups on SNBPs generated by the reduction of
disulfide bonds. A 30 min DTT treatment was however required
to induce significant sperm nuclear decompaction and SNBP
removal. The eventual decrease of mBrB fluorescence observed
after 30 min incubation presumably reflects the eviction of
SNBPs. Taken together, our results strongly suggest that
Drosophila sperm chromatin is stabilized with disulfide bridges,
thus opening the possibility that DHD could be required for their
reduction at fertilization.
DHD directly targets sperm chromatin. Any direct implication
of DHD in the reduction of SNBP disulfide crosslinks would
Pronuclear apposition Meiosis II > Cycle 1
DNA
Mst35Ba-GFP
DNA
Mst35Ba-GFP
♀ ♀
♂ ♂
DNA
Histones
Mst35Ba-GFP
Mst35Ba-GFP
Histones
Meiosis II > Cycle 1Cycle 1
Mst77F-GFP
DNA Histones
Mst77F-GFP
Histones
100 100
n =35
n =31
0 0
= needle-shaped, = decondensed,
= chromosome-like
n =86
n =34
n =91
n =81
%
0 0
100
50
0
100
41
59
0 00
%
%
69
0
24
7
24
76
w
1118
dhd
J5
w
1118
dhd
J5
rescue dhd
J5
∆Mst35B
Control
∆Mst35B
Control
∆Mst35B
Control
dhd
J5
dhd
J5
control X
ΔMst35B X
ab
c
Figure 3 | DHD is required for the timely removal of SNBPs at fertilization. (a) Pronuclear apposition in eggs from control (w
1118
)ordhd
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 w
1118
, 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 (DMst35B ; Mst77F-GFP) sperm. Distribution of phenotypic classes is shown for each stage. ‘4 Cycle 1’ indicates embryos with the
polar body condensed into a rosette of chromosomes. Scale bars, 5 mm.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13539 ARTICLE
NATURE COMMUNICATIONS | 7:13539 | DOI: 10.1038/ncomms13539 | www.nature.com/naturecommunications 5