Current Biology 16, 1441–1446, July 25, 2006 ª2006 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2006.06.

024

Report
The Drosophila Calcipressin Sarah
Is Required for Several Aspects
of Egg Activation
Vanessa L. Horner,1 Andreas Czank,2 Janet K. Jang,3 in sarah mutant eggs, indicating that the Drosophila
Navjot Singh,4 Byron C. Williams,1 Jaakko Puro,5 egg’s competence to support male pronuclear matura-
Eric Kubli,2 Steven D. Hanes,4,6 Kim S. McKim,3 tion is acquired during activation.
Mariana F. Wolfner,1 and Michael L. Goldberg1,*
1
Department of Molecular Biology and Genetics Results and Discussion
Cornell University
Ithaca, New York 14853 Mutations in the sarah Gene Cause Female Sterility
2
Institute of Zoology The several independent sarah (sra) alleles described
University of Zürich here were found in genetic screens for sterile Drosophila
Zürich CH-8057 females whose embryos arrest development very soon af-
Switzerland ter fertilization. In the Supplemental Data available with
3
Waksman Institute and this article online, we show that sarah corresponds to
Department of Genetics CG6072, which encodes calcipressin, a highly conserved
Rutgers, the State University of New Jersey inhibitor of the calcium- and calmodulin-dependent phos-
Piscataway New Jersey 08854 phatase, calcineurin [2, 3]. All of these sra mutations are at
4
Molecular Genetics Program a minimum strong hypomorphs (see the Supplemental
Wadsworth Center Results); the accompanying article by Takeo et al. [4] de-
New York State Department of Health scribes an authentic sra null allele whose phenotypic
120 New Scotland Avenue effects are very similar to those we describe here.
Albany, New York 12208 Mutations of sarah affect neither the fertility of males
5
Department of Biology nor the viability of adults of either sex. However, sarah
University of Turku mutations have previously been shown to influence
Turku SF-20500 learning and memory [5], as well as ovulation and court-
Finland ship behaviors [2].
6
Department of Biomedical Sciences
School of Public Health Eggs from sarah Mutant Mothers Arrest
State University of New York in Anaphase I
Albany, New York 12208 Although sarah mutant females lay eggs, no embryos
hatch ([2]; Table S1). To determine the underlying defect,
we examined these eggs and their precursors in more
Summary detail. Oocytes within the ovaries of sra mutant females
appear normal, as determined by staining for DNA and
Activation of mature oocytes initiates development by tubulin. In the wild-type, mature oocytes are arrested
releasing the prior arrest of female meiosis, degrading in metaphase I because the meiotic spindle pulls homol-
certain maternal mRNAs while initiating the translation ogous chromosomes to opposite spindle poles while
of others, and modifying egg coverings. In vertebrates the homologs are held together by chiasmata, generat-
and marine invertebrates, the fertilizing sperm triggers ing tension [6]. This metaphase I arrest also occurred
activation events through a rise in free calcium within in all observed sra oocytes (n = 65), as established by
the egg. In insects, egg activation occurs indepen- a single mass of chromatin at the midpoint of a single
dently of sperm and is instead triggered by passage spindle (Figure S3; see also [7]). Thus, sra mutations
of the egg through the female reproductive tract [1]; it do not affect bivalent formation or metaphase arrest
is unknown whether calcium signaling is involved. prior to ovulation.
We report here that mutations in sarah, which encodes To characterize the developmental defect, we mated
an inhibitor of the calcium-dependent phosphatase heteroallelic sarah females and heterozygous control fe-
calcineurin, disrupt several aspects of egg activation males to wild-type males and allowed them to lay eggs
in Drosophila. Eggs laid by sarah mutant females ar- for 2 hr. Laid eggs were fixed and stained with propidium
rest in anaphase of meiosis I and fail to fully polyadeny- iodide to visualize the chromosomes and with a-tubulin
late and translate bicoid mRNA. Furthermore, sarah antibodies to reveal the spindles. Eggs laid by control fe-
mutant eggs show elevated cyclin B levels, indicating males developed as expected for 0–2 hr embryos; most
a failure to inactivate M-phase promoting factor (MPF). had completed meiosis and were undergoing cleavage
Taken together, these results demonstrate that calcium divisions and gastrulation (data not shown).
signaling is involved in Drosophila egg activation and In contrast, none of the eggs laid by sarah heteroallelic
suggest a molecular mechanism for the sarah pheno- females progressed beyond meiosis, even though these
type. We also find the conversion of the sperm nucleus females mated avidly with males and produced fertilized
into a functional male pronucleus is compromised eggs (Table S1 and see below). The characteristic phe-
notype was arrest during anaphase of the first meiotic
division (Figures 1A–1D and Table S2). A few eggs
*Correspondence: mlg11@cornell.edu from mothers of certain presumably weaker genotypes
Current Biology
1442
Figure 1. Meiotic Aberrations in Laid Eggs Produced by sarah
Mutant Mothers
(A–D) Anaphase I arrest; (E–H) rare defect in anaphase I; (F and G)
rare mistakes in anaphase II. DNA stained with propidium iodide is
red; tubulin staining is green. (A and B) sra687/Df(3R)sbd45; (C)
sraA108/Df(3R)sbd45; (D) sra619/sra619; (E) sraA426/Df(3R)sbd45;
(F–H) sraA426/sra619. In (H), two polar bodies are seen, but in one
polar body the chromosomes are out of the focal plane. Description
of the various sra alleles may be found in the Supplemental Data.
Scale bars represent 6 mm.
showed other meiotic aberrations, probably due to low
residual sra activity (Figures 1E–1H and Table S2).
Some anaphase I figures were abnormal, with chromo-
somes scattered throughout the spindle (Figure 1E). A
few eggs appeared to be in meiosis II because two spin-
dles were observed. However, these two spindles were
generally parallel to one another instead of end to end as
in wild-type meiosis II [8], and the two spindles were
usually asynchronous, with one in metaphase and the
other in anaphase (Figure 1F). In a few eggs, some pyc-
notic chromosomes appeared to have separated from
the main spindle and nucleated secondary spindles
(Figure 1G). Apparently normal polar bodies formed in
a single egg (Figure 1H), so meiosis may rarely complete
in eggs from mothers with the weakest sra alleles.
To verify that sra eggs usually arrest in anaphase I, we
also examined them by high-resolution Feulgen and
Giemsa staining. Figure 2 contrasts anaphase I progres-
sion in wild-type eggs (panels A–E) with the anaphase I
Figure 2. Feulgen and Giemsa Staining Demonstrating Anaphase I
Arrest in sra Eggs
(A–E) The course of anaphase I in wild-type controls. Late in
anaphase I, ‘‘middle pole’’ material accumulates at the division
equator (arrows in [D] and [E]; see also [9]). (F and G) Anaphase I
arrest in eggs from sra619/sra619 mutant mothers. (H and I) Anaphase
I arrest in eggs produced by homozygous mutant germline clones in
mothers heterozygous for sra1652. Scale bars represent 10 mm.
arrest in sra eggs (panels F–I). In every arrested ana-
phase, three large chromosomes are directed toward
the two poles of the spindle; occasionally, the tiny fourth
chromosome is seen to lead each of the two groups of
chromosomes (Figure 2H). Sister chromatids must
therefore remain attached (at least at their centromeres)
during the anaphase movements. We never observed
the accumulation of ‘‘middle pole’’ material that ordinar-
ily forms late in anaphase I and eventually demarcates
the boundary between the two metaphase II spindles
(arrows in Figures 2D and 2E; see also [9]), indicating
that the arrest occurs early in anaphase I.
Consistent with the fact that egg activation in Dro-
sophila is independent of fertilization [1], the eggs laid
by virgin sra mutant females also arrest at anaphase I
(data not shown).
Meiotic Effects of sarah Mutations Are Germline
Specific
Mutant germline clones were created via the FLP-dom-
inant female sterile technique ([10]; see the Supplemen-
tal Data for details). The eggs produced by mutant germ-
lines were double stained with Feulgen and Giemsa for
high-resolution cytometry of meiotic figures. Anaphase
I arrest was observed in all cases (n = 45); panels H
and I of Figure 2 depict anaphase I arrest in these germ-
line clones. The genotype of the germline therefore de-
termines the sra mutant phenotype in eggs. Transcripts
of sra accumulate to high levels in germline nurse cells
and oocytes during oogenesis [2].
Bicoid mRNA Is Neither Fully Polyadenylated
nor Translated in sarah Mutant Eggs
The translation of Bicoid (Bcd) upon activation organizes
anterior development in embryos. During oogenesis,
Role of Drosophila Calcipressin in Egg Activation
1443

Figure 3. Failure of bicoid mRNA Polyadenylation and Translation in Eggs from sra Mothers
(A) Polyadenylation of bicoid mRNA does not occur fully in sra mutant eggs. (Top) Wild-type fertilized and unfertilized laid eggs extend the poly(A)
tail length of bcd mRNA more than 120 nucleotides further than do unactivated eggs in the ovary. (Bottom) Eggs laid by sra mutant females
extend the poly(A) tail length of bcd no more than approximately 64 nucleotides.
(B) Translation of bicoid mRNA into Bicoid protein does not occur in sra mutant eggs. Western blots of the indicated extracts were probed with
antibody against Bicoid. (Lanes 1, 2, and 3 at left) In the wild-type, Bicoid protein is not synthesized until the eggs are laid and thus activated. (The
high level of Bcd seen in laid unfertilized eggs was an aberration not seen in other experiments.) (Lane 4) bcd12 is a null allele that serves as a neg-
ative control. (Lanes 5–8) Bicoid is not translated in laid eggs from sra mothers, but it is made in laid eggs from control heterozygous females.
(C) Cyclin B levels measured on Western blots are high in unfertilized wild-type eggs and fertilized sra or cortex embryos, but not in control
fertilized eggs.

bcd mRNA is synthesized in nurse cells and transported
to oocytes, where it remains untranslated. Upon acti-
vation, bcd mRNA is rapidly polyadenylated and trans-
lated [11, 12].
To test for effects of sarah on bcd mRNA polyadeny-
lation, we measured the length of the poly(A) tails on
bcd mRNAs by using the PCR poly(A) test (PAT [13]).
In the wild-type, more than 120 A’s are added to these
RNAs within 1 hr of egg laying and activation (Figure 3A),
in agreement with previous reports [14]. In contrast,
eggs laid by sra mutant females add only about 64 nu-
cleotides of poly(A) to bcd mRNA upon egg laying (Fig-
ure 3A), similar to the 80 base extension that occurs in
cortex mutants [14].
The failure of sra mutant eggs to fully polyadenylate
bcd transcripts predicts these eggs will be compro-
mised in their ability to translate Bcd protein. To test
this assumption, we probed lysates from fertilized, laid
eggs of sra mutant females for Bcd on Western blots.
As controls, ovaries from wild-type females as well as
fertilized and unfertilized laid eggs from wild-type and
siblings heterozygous for the same sra alleles were
also examined. In wild-type and heterozygous controls,
Bcd is not observed in mature, unactivated oocytes but
accumulates to high levels upon activation, as ex-
pected. However, we did not observe Bcd translation
in eggs laid by any sra mutants (Figure 3B).
Vitelline Membranes Are Cross-Linked in sarah
Mutant Eggs
To ascertain whether VM (vitelline membrane) cross-
linking occurs in sra eggs, we allowed mated females
to lay eggs for 2 hr timed intervals, and these in vivo ac-
tivated eggs were treated with 50% bleach for two min-
utes. Non-cross-linked eggs lysed within the 2 min incu-
bation, but if VM cross-linking and subsequent eggshell
hardening occurred, eggs became resistant to 50%
bleach [1]. Eggs laid by sra females underwent VM
cross-linking to the same extent as those from heterozy-
gous controls (Table S3). In contrast, all mature stage-14
ovarian oocytes of all genotypes are lysed by bleach
(data not shown).
Although VM cross-linking occurs in sra eggs acti-
vated in vivo by egg laying, results from in vitro activa-
tion suggest that the cross-linking may be slower or
less efficient than in wild-type eggs. If mature stage-
14 oocytes are placed in a hypotonic activation buffer
for 25 min, approximately 50% (n = 88) of sra eggs un-
dergo cross-linking, as opposed to 93% (n = 95) of
controls (p < 0.001). When sra eggs that had been acti-
vated in vitro for 25 min were aged in a physiological
buffer for an additional 90 min, 72% (n = 94) underwent
VM cross-linking. This is significantly higher (p < 0.01)
than 50%, indicating that cross-linking increases in
sra eggs over time.
Current Biology
1444
Figure 4. State of the Sperm Nucleus after Fertilization of sra Mutant
Eggs by Wild-Type Sperm
DNA stained with propidium iodide is red in all panels. (A) sra687/
Df(3R)sbd45 with sperm tail in green; the arrowhead shows the
sperm nucleus, and the arrow points to the female meiotic nucleus.
(B) sraA108/sra619 with tubulin in green. The inset shows apposed,
fully decondensed wild-type male and female pronuclei just prior
to the first mitotic division.
(C and D) sra687/Df(3R)sbd45 with histone H1 in green. The H1 stain-
ing unexpectedly surrounds the chromosomes not only in the sperm
nucleus but also in the anaphase-arrested female nucleus that
serves as a positive control in these eggs.
(E and F) sra687/Df(3R)sbd45 with phosphohistone H3 (Ser-10) stain-
ing in green.
The inset boxes in panels (C)–(F) display the sperm nucleus found in
the same embryo as the female meiotic nucleus in the same panel.
Scale bars represent 20 mm in (A) and 6 mm in other panels.
The Male Pronucleus Does Not Mature in sarah
Mutant Eggs
Mutations in sra do not prevent the fertilization of eggs
by sperm; using an antibody that recognizes the sperm
tail (see Supplemental Experimental Procedures), we
observed the sperm tail in the majority of the eggs pro-
duced by mated sra mothers (Figure 4A). By examining
the sperm nucleus in fertilized sra eggs, we could thus
investigate the interaction between egg activation and
maturation of the sperm nucleus into the male pronu-
cleus. In fertilized sra eggs, the sperm nucleus was
round (Figures 4A and 4B), but its diameter is only 1.7
6 0.04 mm (n = 12), much smaller than that of fully decon-
densed male pronuclei seen just prior to pronuclear
fusion (w5 mm; insert in Figure 4B; see also [15]).
To determine if histone exchange occurs in the wild-
type sperm nucleus of sra mutant eggs, fertilized laid
eggs were stained with propidium iodide for DNA and
an antibody that recognizes histone H1, which is not
present in mature sperm before fertilization [16]. The
sperm nucleus in these eggs is consistently associated
with histone H1 (n = 13; Figures 4C and 4D). Thus, pater-
nal chromosomes associate with histone H1 from mater-
nal stores in sra mutant eggs; we presume that H1 target-
ing requires previous replacement of sperm protamines
with the core histones on paternal chromatin.
The very first steps of sperm nuclear envelope break-
down, chromosome decondensation, and the replace-
ment of protamines with histones therefore occur in
sarah mutant eggs. Sperm produced by males homozy-
gous for the male-sterile gene sneaky cannot undergo
these steps even in wild-type eggs. As a result, the
DNA of sneaky sperm nuclei in eggs cannot be stained
by the membrane-impermeable dye propidium iodide
and retains an elongated needle-shaped configuration
[17]. In contrast, the sperm nucleus in sarah eggs can
be stained by propidium iodide and assumes a rounded
shape. Mutations in the sesame gene, which encodes the
maternal factor Hira, a histone chaperone required for
nucleosome assembly, result in a failure of core histones
to incorporate into the sperm nucleus [18]. Sarah-medi-
ated activation events thus appear to be required for nei-
ther sneaky nor sesame function; nor do they appear to
be required for protamine/histone exchange in general.
However, further development of the sperm nucleus
does not occur in sra eggs. To determine if the sperm
nucleus replicates its genome, we stained laid, fertilized
sra mutant eggs for proliferating cell nuclear antigen
(PCNA). PCNA functions as a processivity factor of
DNA polymerase d during DNA replication; in early divid-
ing Drosophila embryos, it colocalizes with nuclei during
interphase and occasionally in late anaphase or telo-
phase, but never in metaphase [19]. PCNA was never
detected on either the sperm nucleus (n = 24) or the ana-
phase-arrested female nucleus (n = 24). In wild-type
controls, staining was observed on the male and female
apposed pronuclei 69.6% of the time (n = 23). Sperm nu-
clei prior to apposition did not stain with PCNA in the
wild-type controls (n = 15).
We also stained fertilized eggs for the mitotic marker
phosphohistone H3 (Serine 10), which is normally de-
posited on chromosomes during prophase [20]. Mater-
nally derived chromosomes stained intensely, as ex-
pected given their cell-cycle arrest, but the majority of
sperm nuclei (n = 8/9) in the same embryos did not stain
for phosphohistone H3 (Figures 4E and 4F). Together,
these findings indicate that the sperm nuclei are ar-
rested in a pre-S phase, premitotic state in the absence
of maternal sra function. Although it may appear para-
doxical that the male and female genomes in these
eggs are arrested at very different stages of the cell
cycle, precedents in sea urchin zygotes indicate that
the cell cycles of male and female pronuclei are indepen-
dently regulated, probably by the differential accumula-
tion of active MPF in the respective nuclei [21].
In Figure 4B, we stained fertilized sra eggs for tubulin
as well as for DNA to determine whether the sperm nu-
cleus could elaborate the prominent sperm aster along
which the female pronucleus migrates to allow pronu-
clear apposition. The sperm aster is first visible in wild-
type zygotes when the female genome is undergoing
anaphase II and then grows enormously during telo-
phase II [15]. However, we never observed a sperm as-
ter, nor indeed any organized microtubule system asso-
ciated with sperm nuclei, in sra eggs.
Role of Drosophila Calcipressin in Egg Activation
1445
Sperm nuclei in sra mutant eggs thus become ar-
rested prior to DNA replication, prophase of the first mi-
totic division, formation of the sperm aster, and nuclear
apposition. Our results taken together indicate that
sperm nuclear-envelope breakdown, initial chromatin
decondensation, and histone exchange are dependent
on male-supplied factors such as sneaky and maternal
factors including sesame, but these early steps are inde-
pendent of sarah-mediated egg activation. Later steps
in sperm activation do require Sarah and concomitant
egg activation.
Cyclin B Is Elevated in sra Mutant Eggs
Because injection of nondegradable cyclin B causes
early anaphase arrest of syncytial blastoderm mitoses
[22], cyclin B elevation might explain the anaphase I ar-
rest seen in sra mutant embryos. We thus probed ly-
sates from fertilized, laid eggs of sra mutant females
for cyclin B on Western blots (Figure 3C). As controls,
eggs from wild-type females (fertilized and unfertilized)
and sra heterozygotes were also tested, as were acti-
vated eggs from cortex mutant mothers that were termi-
nally arrested in metaphase II [23]. Eggs from sra
mothers showed an elevation of cyclin B when they
were compared to those from sra heterozyogotes and
wild-type fertilized eggs (Figure 3C). We presume that
the low levels of cyclin B in the latter embryos result
from asynchronous development of the population dur-
ing the 2 hr collection, so that only a small proportion of
embryos are in mitosis. Wild-type unfertilized eggs and
cortex eggs showed elevated cyclin B levels similar to
those seen in sra mutants, consistent with meiotic arrest
(Figure 3C). As discussed below, these results likely re-
flect a role for Sarah as a regulator of M phase-promot-
ing factor (MPF) during egg activation.
A Model for the Role of Sarah in Egg Activation
In vertebrates and marine invertebrates, fertilization trig-
gers a transient rise in free Ca2+, and this rise is respon-
sible for subsequent activation events such as modifi-
cation of the eggshell, prevention of polyspermy, and
cell-cycle resumption (reviewed in [24]). We show here
that a protein involved in a calcium signal-transduction
pathway is necessary for several egg-activation events
in Drosophila. The sarah gene product is a calcipressin;
the human calcipressin DSCR1 can directly bind to and
inhibit calcineurin, the only known phosphatase that is
dependent on both calcium and calmodulin [3]. The
accompanying paper by Takeo et al. [4] verifies that the
fly Sarah protein shares these biochemical properties.
Our present understanding of egg activation in Xeno-
pus and the activities of calcipressin make strong pre-
dictions for the role of sra in meiotic reactivation
(Figure S4). Upon fertilization of frog eggs, Ca2+ activates
calmodulin-dependent protein kinase II (CaMKII), which
in turn directs the inactivation of Anaphase Promoting
Complex (APC) inhibitors such as Erp1/Emi2 [25, 26].
The APC in turn inactivates MPF through the destruction
of cyclin, relieving the meiotic block and initiating other
egg-activation events [27]. Our results suggest that
essentially the same pathway operates during the acti-
vation of Drosophila eggs. We hypothesize that Sarah
acts early in the pathway by mediating the antagonistic
relationship between calcineurin and CaMKII; that is,
CaMKII activity upon egg activation requires the inhibi-
tion of calcineurin by Sarah (Figure S4) [28].
Consistent with this model is the similarity of the sarah
phenotype to that associated with mutations in another
Drosophila gene called cortex. The Cortex protein is
a member of the Cdc20 protein family, whose members
serve as specificity factors and activators for the APC
[29]. Meiosis arrests normally at metaphase I in cortex
oocytes and resumes when the eggs are laid but then
soon arrests again at metaphase II [30]. The variation in
the phase of the meiotic arrest (metaphase II versus
anaphase I) being an exception, other aspects of egg
activation are similarly affected by mutations in cortex
and sra [23].
The meiotic arrest in both sra and cortex eggs is most
simply explained by the failure of the APC to target cer-
tain molecules for degradation upon egg laying. In cor-
tex eggs, cyclin A fails to be degraded [30]. We show
here that the cyclin B component of MPF remains unde-
graded in sra eggs. These results are consistent with the
mitotic-arrest phenotypes seen in early Drosophila em-
bryos expressing nondegradable cyclin A (metaphase
arrest) or nondegradable cyclin B (early anaphase ar-
rest) [22]. Different APC targets could remain unde-
graded in sra and cortex eggs because there are at least
two Cdc20-like proteins (Cortex and Fzy) in Drosophila
eggs. APCCortex and APCFzy may have different sub-
strate specificities [30] and may be differently regulated
by APC inhibitors downstream of CaMKII (Figure S4).
The failure of the eggs laid by sra mutant mothers to
translate the maternal bcd mRNA (Figure 3B) can also
be understood in the same theoretical framework. The
translation of bcd requires the polyadenylation of its
mRNA; the enzyme poly(A) polymerase that catalyzes
polyadenylation is phosphorylated and thus negatively
regulated by MPF [31]. Failure of APC activation in sra
mutant eggs would therefore prevent both MPF inacti-
vation and bcd mRNA translation (Figure S4).
Although meiosis and bcd translation are both disrup-
ted in sra eggs, vitelline membrane (VM) cross-linking is
apparently normal. VM cross-linking also occurs in other
mutants that block female meiotic progression; such
mutants include cortex, grauzone, prage, and wispy
[23, 32, 33]. In fact, VM reorganization can occur inde-
pendently of every other known egg-activation event, in-
cluding bcd mRNA translation, the degradation of other
maternal mRNAs, and microtubule depolymerization [7,
23, 33, 34]. If there is only a single trigger for egg activa-
tion, it must therefore be able to activate at least two au-
tonomous downstream pathways (one for VM cross-
linking and a second for other events). Because in vitro
activated eggs are defective in several aspects of em-
bryonic development [23, 33], it is difficult to interpret
our finding of delayed VM modification in sra eggs
upon in vitro activation. Although sra function is not for-
mally required for VM organization, sra-dependent pro-
cesses might nonetheless impinge on its efficiency.
In summary, our results indicate that despite its inde-
pendence from a sperm trigger, egg activation in Dro-
sophila involves calcium-mediated pathways that are
likely to be analogous to those in other animals. It is
intriguing that among these downstream events is the
acquisition of the egg’s competence to remodel the
sperm nucleus into the male pronucleus.
Current Biology
1446
Supplemental Data
Supplemental Experimental Procedures, Results, figures, and tables
are available with this article online at http://www.current-biology.
com/cgi/content/full/16/14/1441/DC1/.
Acknowledgements
This work was supported by grants from the National Institutes of
Health (GM48430 to M.L.G., GM44659 to M.W., and GM67142 to
K.M.), the American Cancer Society (DB128 and RSG-9508506-
DDC to S.D.H.), and the Swiss National Science Foundation (to
E.K.). We thank Tim Karr, Ellen Gottlieb, Wael Tadros, Paul Fisher,
and Howard Lipshitz for antibodies.
Received: December 14, 2005
Revised: June 1, 2006
Accepted: June 2, 2006
Published: July 24, 2006
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