Maintenance of meiotic prophase arrest in vertebrate oocytes by a Gs protein-mediated pathway.

Abstract

Maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on an elevated level of cAMP in the oocyte. To investigate how the cAMP level is regulated, we examined whether the activity of an oocyte G protein of the family that stimulates adenylyl cyclase, Gs, is required to maintain meiotic arrest. Microinjection of a dominant negative form of Gs into Xenopus and mouse oocytes, or microinjection of an antibody that inhibits the Gs G protein into zebrafish oocytes, caused meiosis to resume. Together with previous studies, these results support the conclusion that Gs-regulated generation of cAMP by the oocyte is a common mechanism for maintaining meiotic prophase arrest in vertebrate oocytes.

Full text

Page 1

Maintenance of meiotic prophase arrest in vertebrate oocytes by a
Gsprotein-mediated pathway
$
Rebecca R. Kalinowski,aCatherine H. Berlot,bTeresa L.Z. Jones,cLavinia F. Ross,a
Laurinda A. Jaffe,a,*and Lisa M. Mehlmanna,*
aDepartment of Cell Biology, University of Connecticut Health Center, Farmington, CT 06032, USA
bWeis Center for Research, Geisinger Clinic, Danville, PA 17822, USA
cMetabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
Received for publication 10 October 2003, revised 10 November 2003, accepted 12 November 2003
Abstract
Maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on an elevated level of cAMP in the oocyte. To
investigate how the cAMP level is regulated, we examined whether the activity of an oocyte G protein of the family that stimulates adenylyl
cyclase, Gs, is required to maintain meiotic arrest. Microinjection of a dominant negative form of Gsinto Xenopus and mouse oocytes, or
microinjection of an antibody that inhibits the GsG protein into zebrafish oocytes, caused meiosis to resume. Together with previous studies,
these results support the conclusion that Gs-regulated generation of cAMP by the oocyte is a common mechanism for maintaining meiotic
prophase arrest in vertebrate oocytes.
D 2003 Elsevier Inc. All rights reserved.
Keywords: Meiotic prophase arrest; Oocyte maturation; Heterotrimeric G proteins; Zebrafish; Xenopus; Mouse
Introduction
Fully grown oocytes of mammals, frogs and fish remain
arrested in meiotic prophase within the ovarian follicle until
luteinizing hormone (LH) acts on the follicular cells to cause
meiosis to resume (see Masui and Clarke, 1979). In mam-
mals, maintenance of the prophase arrest depends on the
presence of ovarian follicle cells, but in frogs, oocytes
remain arrested even in the absence of follicle cells. Nev-
ertheless, evidence indicates that in both of these vertebrate
groups, the activity of a GsG protein is required to maintain
the arrest. This has been determined by injecting oocytes
with an inhibitory antibody made against the 10 C-terminal
amino acids of the a subunit of Gs(Gallo et al., 1995;
Mehlmann et al., 2002). Because Gsstimulates adenylyl
cyclase, it acts to elevate cAMP. Thus, a requirement for Gs
in maintaining meiotic arrest fits well with other evidence
indicating a requirement for cAMP and adenylyl cyclase in
the oocyte to maintain arrest (Eppig, 1991; Eppig et al.,
2004; Horner et al., 2003; Maller and Krebs, 1977). The a
subunit and/or hg subunit complex of Gs could be the
activator of adenylyl cyclase (Hanoune and Defer, 2001;
Simonds, 1999; Sheng et al., 2001). It is not fully under-
stood how cAMP controls the activation state of the cyclin-
dependent kinase/cyclin B complex (CDK1/CYB) that
determines whether the cell progresses from prophase to
metaphase; however, recent work has identified the CDC25
phosphatase, which directly regulates the activity of CDK1,
as at least one substrate of the cAMP-dependent kinase,
protein kinase A (Ferrell, 1999; Duckworth et al., 2002;
Lincoln et al., 2002; Kishimoto, 2003).
A role for Gsactivity in maintaining meiotic arrest is
consistent with several other previous studies. Under some
experimental conditions, the Gsactivator cholera toxin can
have an inhibitory effect on spontaneous nuclear envelope
or ‘‘germinal vesicle’’ breakdown (GVBD) in isolated
mouse oocytes (Downs et al., 1992; Vivarelli et al., 1983).
0012-1606/$ - see front matter D 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2003.11.011
$Supplementary data associated with this article can be found, in the
online version, doi:10.1016/j.ydbio.2003.11.011.
* Corresponding authors. Department of Cell Biology, University of
Connecticut Health Center, 263 Farmington Avenue, Farmington, CT
06032. Fax: +1-860-679-1661.
E-mail addresses: bkalinowski@neuron.uchc.edu (R.R. Kalinowski),
chberlot@geisinger.edu (C.H. Berlot), tlzj@helix.nih.gov (T.L.Z. Jones),
lross@neuron.uchc.edu (L.F. Ross), ljaffe@neuron.uchc.edu (L.A. Jaffe),
lmehlman@neuron.uchc.edu (L.M. Mehlmann).
www.elsevier.com/locate/ydbio
Developmental Biology 267 (2004) 1–13

Page 2

Conversely, general inhibition of G protein function by
sequestration of G protein hg subunits has been reported
to cause GVBD in Xenopus oocytes (Sheng et al., 2001; see
Discussion), as does general inhibition of G protein-coupled
receptors (GPCRs) by G protein receptor kinases or h-
arrestin (Wang and Liu, 2003). However, G protein receptor
kinases can phosphorylate proteins other than GPCRs (see
Wang and Liu, 2003) and h-arrestin also interacts with
proteins other than GPCRs (Chen et al., 2003; Pierce and
Lefkowitz, 2001). In addition, injection of Xenopus oocytes
with Gs antisense oligonucleotides has been reported to
cause an increase in MAPK activity like that seen in
response to maturation-inducing steroids, although the effect
of the antisense oligonucleotides on meiotic resumption was
not examined (Romo et al., 2002).
All of the G protein modifiers used to date could have
nonspecific targets; even the antibody made against Gs,
which is quite specific in its binding to the Gsprotein in
lysates of mouse and Xenopus oocytes (Mehlmann et al.,
2002, supplementary material), could in principle interact
with other proteins within the cytoplasm of a living oocyte.
Since the concept that oocyte cAMP is generated by Gs
activity and adenylyl cyclase activity in the oocyte itself
differs from the long standing paradigm that meiotic arrest is
maintained in mammalian oocytes due to transfer of cAMP
from follicle cells via gap junctions (see Anderson and
Albertini, 1976; Eppig et al., 2004; Webb et al., 2002), we
felt that it was important to test the Gsrequirement for
maintaining meiotic arrest using an independent method.
We also wished to examine the possible role of other
heterotrimeric G proteins in maintaining meiotic arrest,
particularly for frog, since a previous study suggested this
possibility (Sheng et al., 2001).
To inhibit Gsfunction, we used a dominant negative form
of Gs, in which the sequence of the a subunit of rat Gswas
point-mutated at multiple positions, resulting in a protein
that blocks signaling by Gs-linked receptors (as(a3h5/
G226A/A366S); Berlot, 2002). The effectiveness of this
dominant negative Gs, which we refer to as GsDN, was
demonstrated by transfecting it into tissue culture cells that
also expressed the LH receptor. The cAMP response to
agonist addition in the GsDN-transfected cells was reduced
by 97% relative to the response in control cells without
GsDN (Berlot, 2002). The mutations in this dominant
negative asmutant increase receptor affinity and decrease
receptor-mediated activation (substitutions in the a3h5 loop
region), prevent an activating conformational change re-
quired for dissociation of a from hg (G226A), and decrease
affinity for GDP (A366S). Xenopus and rat Gassubunits are
92% identical in amino acid sequence, and although they
may not be functionally identical in all respects (see
Antonelli et al., 1994), their amino acid sequences are
100% identical in the positions that were modified in GsDN.
Therefore, it is likely that GsDN should behave as a Gs
dominant negative in Xenopus oocytes as it does in mam-
malian cells. We injected RNA encoding GsDN into Xen-
opus and mouse oocytes to determine if it caused meiosis to
resume. To examine the generality of the Gsrequirement for
maintaining meiotic arrest in vertebrate oocytes, we also
investigated the effect of Gsinhibition on meiotic arrest in
zebrafish oocytes.
Materials and methods
Gsconstructs, in vitro transcription
The Gsdominant negative DNA (GsDN) consisted of the
a subunit of rat Gswith point mutations at seven positions:
G226A, N271K, K274D, R280K, T284D, I285T, A366S;
this construct was originally named as(a3h5/G226A/
A366S) (Berlot, 2002). GsDN and the control constructs,
R280K (Berlot, 2002) and G226A (Iiri et al., 1999), were in
the vector pcDNAI/Amp (Invitrogen, Carlsbad, CA). The
plasmids were linearized using XbaI and RNA was tran-
scribed in vitro using T7 polymerase.
Antibodies and other reagents
Affinity-purified antibodies against the Gs a-subunit
(RM) and against the Gqa-subunit (QL) were provided
by Allen Spiegel (NIH, Bethesda, MD). These antibodies
were made against 10-amino-acid peptides corresponding
to the C-termini of Xenopus and mouse Gas and Gaq
(Gallo et al., 1996; Shenker et al., 1991; Simonds et al.,
1989). Non-immune rabbit IgG was obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). The antibodies
were concentrated in PBS as described in Gallo et al.
(1995). DHP (Steraloids, Newport, RI) was dissolved in
EtOH (10 mg/ml) before diluting in the frog or fish oocyte
culture medium (see below). Hypoxanthine (Sigma) was
made as a 200-mM stock in 1 N NaOH, diluted to 4 mM
in the mouse oocyte culture medium, MEM (see below),
and the pH was adjusted to 7.2 with HCl. Pertussis toxin
was obtained from List Biological Laboratories (Campbell,
CA) and activated by incubation in the presence of 10 mM
DTT and 0.1 mM ATP at 37jC for 15 min before
microinjection.
Culture and microinjection of follicle-free Xenopus oocytes
Frogs (Xenopus laevis) were purchased from Nasco
(Fort Atkinson, WI) and were used without gonadotropin
priming. Stage VI follicle-free oocytes (approximately
1200–1300 Am diameter) were obtained by treating pieces
of ovary with collagenase (Duesbery and Masui, 1993;
Gallo et al., 1995). The oocytes were cultured at 18–20jC
in 50% Leibovitz’s L-15 medium, 15 mM HEPES, pH 7.8,
100 Ag/ml gentamicin (all components from Invitrogen) on
agarose-coated dishes (2% Sigma type V, high gelling
temperature agarose in modified Ringer’s). Oocytes were
cultured for 15–18 h after isolation before use; this culture
R.R. Kalinowski et al. / Developmental Biology 267 (2004) 1–13
2

Page 3

period allows the oocytes to recover their protein synthesis
capability after collagenase treatment (Smith et al., 1991).
Oocytes were injected with 50 nl of RNA, using a
Picospritzer (General Valve Corporation, Fairfield, NJ),
or 50 nl of an antibody solution, using a syringe-controlled
injection system and pipets backfilled with mercury (see
Runft et al., 1999). The volume of a Xenopus oocyte is
approximately 1000 nl.
DHP was used as the maturation-inducing steroid for
Xenopus oocytes because we found that it usually caused
GVBD at a somewhat lower concentration than was seen
with progesterone. GVBD was scored by observation of a
white spot at the animal pole using a stereoscope, and
confirmed by fixing for 5 min in 4% trichloroacetic acid
and halving the oocytes with a scalpel, to determine whether
the GV was present (Gallo et al., 1995). Oocytes were
photographed using a stereoscope (Wild M3C, Leica Inc.,
Rockleigh, NJ) and a DC4800 digital camera (Eastman
Kodak, Rochester, NY). Chromosomes and polar bodies
were fluorescently labeled by incubating live defolliculated
oocytes in 10 Ag/ml H33258 Hoechst stain (Gallo et al.,
1995). Chromosomes were photographed using a Zeiss
Axioskop with a 10?, 0.3 NA neofluar objective (Carl
Zeiss, Inc., Thornwood, NY) and a Kodak DC4800 digital
camera. These and other data figures were assembled using
Adobe Photoshop 6.0.
Culture and microinjection of follicle-enclosed and isolated
mouse oocytes
NSA (CF1) mice were purchased from Harlan Sprague–
Dawley (Indianapolis, IN). For experiments with follicle-
enclosed oocytes, antral follicles were dissected from the
ovaries of 22- to 25-day-old, unprimed mice using fine
forceps and 30-gauge needles (Mehlmann et al., 2002).
Approximately 10–20 follicles were obtained per mouse,
ranging in size from approximately 260 to 470 Am in
diameter, and were cultured in 200-Al drops of medium
under light mineral oil (Fisher Scientific, Pittsburgh, PA) on
a tray maintained at 37jC. The medium used was MEM
with Earle’s salts, L-glutamine, nonessential amino acids,
120 U/ml penicillin G (potassium salt), 50 Ag/ml strepto-
mycin sulfate, 0.24 mM sodium pyruvate, 0.1% polyvinyl
alcohol, and 20 mM HEPES, pH 7.2, and was supplemented
with 1 mg/ml BSA (Fraction V, Calbiochem; other reagents
from Sigma).
Follicle-enclosed mouse oocytes were microinjected in a
chamber constructed of two pieces of coverglass that were
held apart by a 300-Am spacer composed of three layers of
double-stick tape (see Mehlmann et al., 2002). The tape held
a small piece of coverslip hanging from the top coverslip,
forming a 1-mm wide ledge in which the follicles were
placed using a mouth-controlled pipet. This assembly was
mounted on a U-shaped plastic slide over a reservoir of
medium. The chamber was observed using an upright
microscope (Zeiss Axioskop) with a 20? lens (0.5 or 0.75
N.A.). A micropipet was used to roll the follicle in order to
position the oocyte near the upper surface for optimized
viewing of the oocyte. Only oocytes in which a nucleolus
could be seen were injected and used for these experiments.
Two follicles were put in the injection chamber at a time and
were kept in the chamber at 18–22jC for 10–30 min; after
injection, they were transferred back to a culture dish at
37jC (1–10 follicles per 200-Al drop).
Oocytes were injected using a syringe-controlled injec-
tion system and pipets backfilled with mercury (see Mehl-
mann et al., 2002; Jaffe and Terasaki, in press). Injection
volumes (14 pl) were calibrated by drawing up a compara-
ble length of oil into the injection pipet, then expelling the
oil and measuring the diameter of the drop. (The volume of
a mouse oocyte is approximately 200 pl.) The success of the
injection was confirmed by including calcium green 10-kDa
dextran (Molecular Probes) in the RNA solution (final
concentration in the oocyte = 10 AM) and checking the
oocytes for fluorescence (see Mehlmann et al., 2002).
For these experiments, we divided the follicles obtained
from a single mouse into two dishes, choosing follicles of
equivalent size and appearance for each dish. One dish was
used for injection and the other was set aside as a control.
Injections were performed within approximately 1–2 h after
the dissection. Six hours after injection, follicles were
opened using 30-gauge needles to determine if the oocyte
had undergone GVBD. After opening the injected follicles,
the follicles in the control dish were also opened. If GVBD
in the control dish exceeded 25%, the results from injections
into follicles from that mouse were disregarded; 10% of the
mice we tested were unacceptable by this criterion. Dis-
counting these, the rate of spontaneous GVBD in control,
uninjected oocytes was 7%.
For experiments with isolated oocytes, 4- to 12-week-old
mice were used. Fully grown (approximately 70–75 Am
diameter) immature oocytes were collected, pipetted to
removed cumulus cells, and injected as previously described
(Mehlmann and Kline, 1994). The medium used was MEM
as described above, supplemented with 250 AM dbcAMP
during dissection and injection. Oocytes were subsequently
transferred to MEM with 4 mM hypoxanthine.
Culture and microinjection of follicle-enclosed zebrafish
oocytes
Zebrafish (Danio rerio, wild type) were kindly provided
by Dr. Stephen DeVoto (Wesleyan University, Middletown,
CT) or purchased from Carolina Biological (Burlington,
NC). Males and females were maintained together in a
filtered, aerated 30-gal aquarium containing deionized water
and 1.5 g/gal Tropic Marin sea salts (Marinus Inc., Long
Beach, CA). The temperature was 25–28jC, and the light
cycle was 14 h light and 10 h dark. Fish were fed twice a
day with dry fish food (TetraMin) supplemented two times
per week with live brine shrimp or frozen Drosophila. Fish
were killed between 1 and 3 h after the light turned on by
R.R. Kalinowski et al. / Developmental Biology 267 (2004) 1–13
3

Page 4

incubation in an ice bath for 30 s followed by decapitation.
Ovaries were placed into 3 ml of the same medium
described above for Xenopus oocytes, but at pH 7.2, on
2% agarose-coated dishes. Follicle-enclosed oocytes were
isolated using fine forceps under a dissecting microscope;
the follicle was pinched from the ovary at the stalk, resulting
in an oocyte surrounded by a three-layer follicular epithe-
lium approximately 10 Am in thickness (Selman et al., 1993,
1994), which we were unable to remove without damaging
the oocytes. Before use, the follicle-enclosed oocytes were
cultured for 2–3 h to identify and remove any that had been
damaged by the isolation procedure. Approximately 15–
130 follicles were obtained per fish; follicles from 1 to 4 fish
were pooled for each experiment. No more than 20 follicles
were cultured in a 35-mm dish.
Follicles of 650–700 Am in diameter were used for the
experiments. All oocytes in this size range underwent
GVBD in response to 30 nM DHP and none underwent
spontaneous GVBD in the absence of DHP. For microin-
jection, follicles were placed in a chamber constructed from
a plastic slide supporting two parallel coverslips, with the
follicles resting on the bottom coverslip. The slide had a
rectangular cut out 15 mm long, 5 mm deep, and 1.5 mm
thick, with the coverslips attached above and below with
silicon grease. The slide was held on the stage of an upright
microscope, and viewed with a 10? objective, 0.3 N.A.
Injections were made using pipets backfilled with mercury
(see Jaffe and Terasaki, in press). Injection volumes were
calibrated by measuring the decrease in the length of the
column of solution in a loading capillary (0.5 mm inner
diameter, Drummond Scientific Company, Broomall, PA)
that was held on the injection slide. Injection volumes were
5–10 nl, corresponding to approximately 3–5% of the
oocyte volume (180 nl). After injection, the oocytes were
removed from the chamber and incubated in 4 ml of
medium in a 35-mm culture dish, at 18–20jC, or at
25jC. Follicles were photographed using a stereoscope
(Wild M3C) and a Kodak DC4800 digital camera. Indirect
fiber optic lighting was used to visualize the GVand oocyte
clearing (see Fig. 4B).
Immunoblotting
For immunoblotting, Xenopus oocyte membranes (Gallo
et al., 1996; ‘‘type 2’’ membranes) and a homogenate of
whole mouse brain (Mehlmann et al., 2001) were prepared
as previously described. Mouse oocytes were prepared by
freezing oocytes with liquid N2, then just before use,
solubilizing them in SDS sample buffer (Mehlmann et al.,
1998). Zebrafish samples were prepared by lysing approx-
imately 20 oocytes in a glass homogenizer in 20 mM
HEPES, pH 7.0, 1 mM EDTA, 2 Ag/ml aprotinin, 0.1 mM
Pefabloc, and 10 Ag/ml leupeptin, followed by centrifuga-
tion at 1000 ? g for 5 min at 4jC. The supernatants were
used for gel samples. The protein content of mouse oocytes
was estimated as 25 ng per oocyte (Schultz and Wassarman,
1977). A BCA protein assay (Pierce Chemical Co., Rock-
ford, IL) using BSA as a standard was performed to
determine the protein concentration for samples from frog
and fish oocytes. Proteins were separated by SDS-PAGE
and blots were incubated with the Gasantibody (1.7 Ag/ml)
or the Gaqantibody (1.0 Ag/ml), and developed with ECL
Plus reagents (Amersham Life Science, Inc., Arlington
Heights, IL). Immunodensities were compared using
scanned images of the films and NIH Image (available at
http://rsb.info.nih.gov/nih-image/).
Online supplemental material
Video1.mov. Microinjection of a follicle-enclosed mouse
oocyte. The micropipet was advanced towards the follicle
from the left, pushed through the mural granulosa cell
layers, pulled back, then pushed forward again into the
oocyte. A drop of silicon oil was introduced into the oocyte
cytoplasm as a consequence of the injection. After the pipet
was withdrawn from the follicle, the success of the injection
was confirmed by the presence of a fluorescent marker
(calcium green dextran) in the oocyte.
The movie was made by imaging the follicle using a
Zeiss Axioskop with a 20?/0.75 N.A. fluar lens, and
recording the video signal from a Kodak DC 4800 camera
using a Sony PC5 camcorder. The movie was downloaded
by firewire into a Macintosh computer using iMovie and
then cropped using Adobe Premiere 6.0. The images were
collected at 30 frames per second; every other frame was
subsequently deleted, to reduce the file size, so the Quick-
time movie is 15 frames per second.
Results
Injection of a dominant negative form of Gs causes
resumption of meiosis in Xenopus oocytes
To examine whether inhibiting Gas with a dominant
negative form of the Gas protein (GsDN) would cause
Xenopus oocytes to resume meiosis, we injected oocytes
with GsDN RNA. Oocytes injected with z1 ng of this RNA
underwent GVBD, as indicated by the formation of a white
spot at the animal pole (Fig. 1A) and confirmed by fixing
oocytes in 4% trichloroacetic acid, halving them with a
scalpel and observing the absence of the germinal vesicle
using a stereoscope.
The time course of GVBD in response to injection of
GsDN RNA was similar to that seen in uninjected oocytes
incubated with 10 AM of a maturation-inducing progester-
one derivative, 4-pregnen-17a20h-diol-3-one (DHP) (Fig.
2A); this indicated that the translation of the injected RNA
to make protein occurs rapidly. Other studies have shown
that protein production in Xenopus oocytes can be detected
as early as 1 h after RNA injection (e.g., Martı ´nez-Torres
and Miledi, 2001), so the lack of an obvious delay in GVBD
R.R. Kalinowski et al. / Developmental Biology 267 (2004) 1–13
4

Page 5

to account for the time required for protein synthesis is not
surprising. Analyses of cellular levels of Gsand another Gs-
linked receptor (h-adrenergic) have shown that Gs is in
approximately 200? molar excess (Post et al., 1995),
making it reasonable that even a small amount of a mutant
Gsprotein with high receptor affinity (Berlot, 2002) could
inhibit receptor–Gscoupling.
To examine whether GsDN-injected oocytes proceeded
through meiosis and arrested normally at metaphase II, we
stained the injected oocytes with a DNA specific dye
(H33258), 24 h after injection, and examined them by
fluorescence microscopy. Observation of the animal pole
of the live oocytes showed condensed chromosomes and a
polar body (Fig. 1B). This pattern of condensed chromo-
somes and a polar body was similar to that seen after
injection of a Gsinhibitory antibody or exposure to steroid
(Gallo et al., 1995) and indicated that the GsDN-injected
oocytes had progressed to metaphase II.
Specificity controls
As controls, we injected oocytes with two different
RNAs, each of which encode an assubunit with a single
amino acid substitution (R280K or G226A). These two
substitutions are also present in GsDN. When transfected
Fig. 2. Kinetics and concentration dependence of GVBD in response to
injection of Gsdominant negative (GsDN) RNA into Xenopus oocytes. (A)
Oocytes were injected with 1–50 ng of GsDN RNA or exposed to 10 AM
DHP, and scored for GVBD at various times after injection or DHP
application. The numbers in parentheses indicate the number of oocytes and
number of animals tested. (B) Oocytes were injected with various amounts
of GsDN RNA or a control RNA (R280K or G226A) and scored for GVBD
24 h later. (C) Immunoblot of Xenopus oocyte membranes showing
expression of GsDN, R280K, and G226A proteins after injection of 1 ng of
GsDN RNA, or 50 ng of R280K or G226A RNA. Membranes were
prepared 24 h after RNA injection and 10 Ag of protein was loaded in each
lane of the gel. The blot was probed with an antibody against the a subunit
of Gs(RM). The lower bands show endogenous Gsand the upper bands
show the exogenously expressed proteins. GsDN migrates slightly more
slowly than R280K and G226A (see Berlot, 2002). The densities of the
R280K and G226A bands were 2.5? the density of the GsDN band.
Fig. 1. Injection of Gsdominant negative RNA causes meiotic maturation
of Xenopus oocytes. (A) White spot formation indicative of GVBD.
Oocytes were injected with 10 ng of RNA and photographed 18 h later.
Scale bar = 2.0 mm. (B) DNA staining showing the first polar body (arrow)
(5/6 oocytes) and second metaphase chromosomes (arrowhead) (6/6
oocytes). Oocytes were injected with 3 ng of RNA; 24 h later, they were
stained with 10 Ag/ml Hoechst 33258 and visualized by fluorescence
microscopy. Scale bar = 50 Am.
R.R. Kalinowski et al. / Developmental Biology 267 (2004) 1–13
5