1240
BIOLOGY OF REPRODUCTION 59, 1240?1250 (1998)
Characterization of a Major Permeability Barrier in the Zebra?sh Embryo1
Mary Hagedorn,2,3 F.W. Kleinhans,4 Dmitri Artemov,5 and Ulrich Pilatus5
Reproductive Physiology Program,3 National Zoological Park, Smithsonian Institution, Washington,
District of Columbia 20008
Physics Department,4 IUPUI, Indianapolis, Indiana 46202
Radiology Department,5 Johns Hopkins School of Medicine, Baltimore, Maryland 21205
ABSTRACT
Fish embryos represent a class of multicompartmental bio-
logical systems that have not been successfully cryopreserved,
primarily because of the lack of understanding of how water
and cryoprotectants permeate the compartments. We are using
the zebra?sh embryo as a model to understand these kinetics.
Zebra?sh embryos have two major compartments, the blasto-
derm and the yolk, which is surrounded by the multinucleated
yolk syncytial layer (YSL). We determined the water and cryo-
protectant permeability in these compartments using two meth-
ods. First, we measured shrink/swell dynamics in optical volu-
metric experiments. Zebra?sh embryos shrank over time and did
not re-expand while immersed in dimethyl sulfoxide (DMSO) or
propylene glycol. Second, we measured DMSO uptake with dif-
fusion-weighted nuclear magnetic resonance spectroscopy.
DMSO uptake was rapid during the ?rst few minutes, then grad-
ual thereafter. We used one- and two-compartment models to
analyze the data and to determine the permeability parameters.
We found that the two-compartment model provided a better
?t to the data. On the basis of this model and in the presence
of DMSO, the yolk and blastoderm had very similar water per-
meabilities (i.e., 0.01 and 0.005 mm 3 min21atm21, respective-
ly), but they had different DMSO permeabilities separated by
three orders of magnitude (i.e., # 5 3 1026 and 1.5 3 1023 cm/
min, respectively). The low solute permeability of the yolk pre-
dicted that the yolk/YSL compartment should be more suscep-
tible to cryodamage. To test this, the yolk, blastoderm, and YSL
were examined at the ultrastructural level after vitri?cation.
Only the YSL incurred signi?cant damage after freezing and
thawing (p # 0.05).
INTRODUCTION
Yolk-laden embryos of fish, reptiles, birds, and amphib-
ians represent a class of multicompartmental biological sys-
tems that have not been successfully cryopreserved. Over
the past 50 years, the fish embryo has presented a challenge
to cryobiologists because of its interesting and complex
physiology. Successful cryopreservation of fish embryos
must address several important issues, including 1) the in-
trinsic biophysical properties of the cells (e.g., membrane
permeabilities and osmotic tolerance limits) and 2) deter-
mination of the procedural cryopreservation steps, based on
the cell?s biophysical properties, necessary to minimize cry-
odamage and maximize survival [1]. In this paper, we ex-
amine the kinetics of cryoprotectant permeability of the
Accepted July 8, 1998.
Received May 14, 1998.
1Supported by grants from the National Institutes of Health (R29
RR08769) (M.H.), and Maryland Sea Grant College (M.H.), and from IUP-
UI for a Sabbatical Year Award (F.W.K.), and the Smithsonian Institution
for a Smithsonian Short-Term Visitor Award (F.W.K.).
2Correspondence: M. Hagedorn, National Zoological Park, Smithson-
ian Institution, 3001 Conn. Ave., NW, Washington, DC 20008. FAX: 202
673 4733; e-mail: mhagedorn@nzp.si.edu
yolk and blastoderm compartments, and the effects of cry-
odamage on the fish embryo.
We are using the zebrafish embryo as a model to un-
derstand the cryobiological properties of a multicompart-
mental system. The zebrafish model is becoming one of the
most important vertebrate models for the study of devel-
opment and genetics. Transgenic and mutagenic studies of
zebrafish are expected to play an important role in human
health and disease. The human genome is being mapped,
but the functionality of many of the genes is not known.
On the other hand, the function of many of the genes in
the zebrafish genome is known because of the mutational
screens going on in a number of labs throughout the world.
When the zebrafish genome is fully mapped (hopefully
within the next few years), it will have great value, because
the organization of the fish and human genomes has been
relatively conserved throughout evolution [2]. Therefore,
cryopreservation of this embryo would be a great service
to the field. The zebrafish embryo is composed of two cel-
lular compartments: a large yolk and the developing blas-
toderm (Fig. 1). The major component of the yolk is vitel-
logenin, a large phospholipid (approximately 400 kDa; [3]),
stored in membrane-bound vesicles within the yolk. The
major developmental stages important for this work are
100% epiboly to the 3-somite stage. We did not consider
earlier developmental stages, because they are sensitive to
chilling [4, 5] and are mechanically fragile, thus making
them less attractive for cryopreservation. At the onset of
development, the blastoderm divides and forms a cap of
cells on the large yolk. At the 128-cell stage, epiboly begins
as the blastoderm envelopes the yolk and ceases once the
yolk is completely surrounded by blastoderm at 100% epib-
oly [6]. Underlying the blastoderm and covering the yolk
is the yolk syncytial layer (YSL). This is a multinucleated
layer (approximately 10 mm thick) of nonyolky cytoplasm
that begins to develop at approximately the 1000-cell stage
in the zebrafish embryo [7]. As the YSL develops, it re-
places the thin (approximately 2 mm thick), nonnucleated
yolk cytoplasmic layer [8]. The YSL begins to envelop the
yolk ahead of the blastoderm, and it surrounds the embryo
by approximately 50?75% epiboly [9]. The segmentation
period follows. The stages within this period are identified
by the number of muscle somites visible in the tail region
from the 1- to the 26-somite stage.
The efficacy of cryoprotectant permeation is crucial to
formulating successful cryopreservation procedures. Each
compartment may have different permeability to water and
cryoprotectants. To prevent cryodamage, the compartment
with the lowest permeability to water and cryoprotectant
must be considered when formulating cryopreservation pro-
cedures. Much of the work to date on cryoprotectant per-
meability of the teleost embryo has not addressed the em-
bryo?s multicompartmental nature. Harvey et al. [10] ex-
amined the permeability of zebrafish embryos at 50% epib-
1241DMSO PERMEABILITY OF ZEBRAFISH
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FIG. 1. A) Image of a zebra?sh embryo identifying the major compart-
ments (i.e., yolk and blastoderm). Although the YSL normally would not
be visible, its position is indicated on the embryo for clarity. The position
of the chorion is indicated even though this layer was removed in all of
our preparations. B and C) Method of sampling the zebra?sh embryo for
ultrastructural analysis. B) Locations from which thin sections were sam-
pled from the whole embryo. C) Location on the thin section where areas
of the blastoderm, YSL, and yolk were examined for ultrastructural integ-
rity.
oly to isotope-labeled glycerol and dimethyl sulfoxide
(DMSO). He found that DMSO permeated into dechorion-
ated embryos and that both solutes permeated into intact
embryos. Unfortunately, the amount and location (i.e., yolk
versus blastoderm) of these permeating cryoprotectants
were unclear. Subsequently, Harvey [11] reported that glyc-
erol protected only the blastoderm, and neither cryoprotec-
tant effectively preserved the entire embryo at 21968C. Re-
cently, Suzuki et al. ([12]; for carp, medaka, and rainbow
trout) and Lubzens et al. ([13]; for carp) reported an uptake
of DMSO solution into the perivitelline space and some
tissues, but the permeation level was insufficient for cry-
opreservation. Using optical measurements, Zhang and
Rawson [14, 15] described cryoprotectant permeation into
intact and dechorionated zebrafish embryos. Of the variety
of cryoprotectants that they tried, only methanol showed
clear evidence of permeation into the embryos.
We have examined the multicompartmental zebrafish
embryo using a variety of techniques that can reveal the
permeability response of the individual compartments [16,
17]. These experiments identified the YSL as a barrier to
the movement of cryoprotectants into the yolk. Specifically,
little or no DMSO or propylene glycol (PG) entered the
yolk, but methanol did. Unfortunately, methanol may not
be a suitable cryoprotectant for vitrification (a method
needed because of the embryo?s chill sensitivity), whereas
DMSO and PG may. Our previous studies [16] identified
the existence and location of the permeability barrier to
DMSO and PG, whereas the present study quantified the
water and solute permeability parameters for the yolk and
blastoderm. We used two methods to estimate the perme-
ability parameters. First we measured cryoprotectant-in-
duced volumetric changes by light microscopic analysis
and then cryoprotectant uptake with diffusion-weighted 1H
nuclear magnetic resonance (NMR) spectroscopy. Nonlo-
calized NMR spectroscopy was selected for this study in-
stead of single-embryo NMR microscopy, as used previ-
ously [16], because a larger sample size could be used. This
increased the signal-to-noise; therefore, smaller amounts of
cryoprotectants could be detected within the embryos.
When a single cell is exposed to a hyperosmotic solute
solution (e.g., 2 M DMSO or 2 M PG), water initially
leaves the cell and solute enters the cell in response to the
osmotic and solute gradients. Eventually, as the intracellu-
lar solute concentration rises, water re-enters the cell, pro-
ducing the typical shrink-swell volume curves seen in many
optical experiments. The rate at which the cell volume
changes in response to the water and solute flux reflects the
permeability of the cell membrane to water and solute.
These parameters are characterized by the hydraulic or wa-
ter permeability (Lp) and solute permeability (Ps), respec-
tively. In order to determine permeability parameters for
the multicompartmental zebrafish embryo from the NMR
and optical data, it is necessary to have a model of the
embryo. Initially we considered a one-compartment model
and then generalized to a more complex two-compartment
model. For modeling purposes, we did not distinguish the
YSL from the yolk and consider it part of the yolk com-
partment.
As stated earlier, all attempts to cryopreserve fish em-
bryos have produced lethal damage. This analysis of the
permeability parameters of the zebrafish embryo predicted
that a major site of the lethal cryodamage would occur
within the yolk compartment. Presumably, without suffi-
cient cryoprotectant entering the yolk, damaging ice-crys-
tals form. To test this hypothesis, we vitrified zebrafish em-
bryos and examined the cellular ultrastructure of the cry-
odamage in the blastoderm and the yolk compartments.
MATERIALS AND METHODS
Maintenance of Animals
Animals were maintained according to the maintenance
and culturing procedures described by Westerfield [6]. Brief-
ly, 12?16 fish were kept in 5-L aquaria (temp 5 28.58C;
pH 5 7.0) illuminated according to a 12L:12D cycle, and
fed dried copepods and krill (Argent Laboratories, Red-
mond, WA). Animals spawned in response to photoperiod,
producing 50?60 embryos per female. Embryos were col-
lected, dechorionated by enzymatic digestion, and main-
tained in embryo medium (a modified Hepes buffer at
0.040 Osm) as described in Westerfield [6]. All solutions
were made up in this embryo medium. Because the devel-
1242 HAGEDORN ET AL.
TC # 369
FIG. 2. NMR perfusion sample chamber. Diagram of the experimental
perfusion set-up and location of the NMR slices selected for the experi-
ment.
opmental temperature was 28.58C, all developmental stages
described here are similar to those in Westerfield [6].
Throughout the study, only embryos at 100% epiboly to
the 3-somite stage were used. At this time, there was no
epidermis covering the embryo that might act as a diffusion
barrier by hindering permeability to water and cryoprotec-
tants. Moreover, embryos still were relatively round, main-
tained a reproducible orientation when moved from solution
to solution, and were nonmotile, simplifying the optical
volumetric measurements.
Volumetric Measurements: Light Microscopy
After immersion in a hypertonic solute (2 M DMSO or
PG in embryo medium), changes in embryo size were mea-
sured using computer-assisted light microscopy. Embryos
were examined under a Zeiss compound microscope fitted
with a CCD camera (WV-BL200; Panasonic, Secaucus, NJ)
at room temperature (approximately 228C). Images were
digitized with a video frame-grabber card (LG3; Scion
Corp., Frederick, MD) and stored for later morphometric
analysis. Digitized images (3100) were displayed on a
high-resolution video monitor and analyzed using a com-
puter-aided morphometry package (Image 1.45; NIH, Be-
thesda, MD). The outline of each embryo was determined
by the computer, and linear, planar, and volumetric param-
eters of each embryo were calculated. The major and minor
axes were used to determine the volume of embryos at all
stages using a prolate spheroid formula (V 5 4/3p ab2),
where a and b were the major and minor semi-axes, re-
spectively. The data were modeled as described below.
Solute Uptake: NMR Spectroscopy
NMR data were acquired on an Omega 400 NMR (Gen-
eral Electric, Fremont, CA) spectrometer (equipped with
GE Accustar actively shielded gradients up to 130 G/cm in
three directions) using a specially designed probe for per-
fusion experiments with 5-mm NMR tubes. Embryos (each
sample consisted of 20 embryos at 100% epiboly) rested
as a monolayer on a porous filter (Fritware; Bel-Art Prod-
ucts, Pequannock, NJ, polyethylene sheets, 70-micron pore
size, 3.2 mm thickness) placed in a bottom-cut 5-mm NMR
tube (Fig. 2). Embryo medium was pumped through the
NMR tube, perfusing the embryos from below at 0.3 ml/
min with a peristaltic pump (Masterflex; Cole-Palmer,
Vernon Hills, IL) for 20 min and was then changed to 2.0
M DMSO for 60 min. To follow any rapid changes in the
embryos, localized spectra (two scans/spectrum, 1 sec rep-
etition time) were acquired every 44 sec at two different
cross-sectional locations in the NMR tube, one slice con-
taining the embryos and the other in the solution just above
the embryos (Fig. 2). 1H-NMR spectra were obtained with
stimulated echo pulse sequences using high and low dif-
fusion-weighting with the spatial localization in the z di-
rection (i.e., parallel to the NMR tube) obtained by slice
selective radio frequency pulses following the methods of
Frahm et al. [18].
NMR spectra of the slice containing the embryos were
obtained with high diffusion-weighting, which was suffi-
cient to suppress all extracellular water and DMSO, allow-
ing only intracellular water and DMSO to be detected [16].
Low diffusion-weighted spectra were obtained in the upper
buffer slice to show the time course of the appearance of
DMSO in the buffer. The rate at which DMSO signal ap-
peared in the buffer slice placed a lower limit on the mea-
surable embryo response time. Changes in intracellular
DMSO were obtained from the high diffusion-weighted
spectra from the lower slice (embryo slice) and were quan-
tified by measuring the change in area under the spectral
peaks of the DMSO. Since the intensity of diffusion-
weighted signals depends on many factors, such as relax-
ation processes that may interfere with quantification of in-
tracellular DMSO, we were not able to measure the abso-
lute intracellular quantity of DMSO. However, the signal
intensity (area under the signal) is proportional to the quan-
tity of intracellular DMSO. These intensities were arbitrar-
ily normalized, and then three separate NMR runs were
averaged to yield the data presented here. In all experi-
ments, the embryos were counted before introduction and
after removal from the magnet to insure that changes ob-
served were due to physiological changes and not due to
the embryo number diminishing during the experiment. Ini-
tially, NMR experiments were performed using PG. Un-
fortunately, a signal of an endogenous (unidentified) com-
pound within the embryos appeared at approximately the
same position as PG. This caused large errors in quantify-
ing intracellular PG, especially when low concentrations
had to be determined, so the PG NMR experiments were
terminated. The DMSO data were modeled as described
below.
Permeability Parameters
The objective of the modeling was to determine the per-
meability parameters of the zebrafish embryo from the op-
tical and NMR data. In the optical experiments, we mea-
sured the changing embryo volume (Vc), which depends on
the flux of water and solute into or out of the embryo. In
1243DMSO PERMEABILITY OF ZEBRAFISH
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TABLE 1. Parameters used for the one- and two-compartment model
analyses of the NMR solute uptake and optical volume measurements;
embryos were at approximately 100% epiboly for these experiments.
Parameter Value Comments
General parameters
Mino 0.300 Osm Assumed cytoplasmic and yolk osmolali-
ty.a
Men 0.040 Osm Osmolality of external, nonpermeating
embryo medium.
Mes 2.39 Osm Osmolality of external 2 M DMSO or 2
M PG.
V? s (DMSO) 0.069 l/mol Partial molar volume of DMSO.
V? s (PG) 0.070 l/mol Partial molar volume of PG.
T 294?296 K Temperature of NMR and optical experi-
ments.
One compartment model parameters
Vo 0.2 mm3 Volume of ``standard'' embryo.
A 1.65 mm2 Area of ``standard'' embryo, assumed to
be spherical.
vb 0.36 Osmotically inactive fraction of em-
bryo.b
Lsp variable Hydraulic conductivity in presence of
solute.
Ps variable Solute permeability.
Two compartment parameters
Vo 0.2 mm3 Volume of ``standard'' embryo.
fy 0.77 Fraction of embryo volume that is yolk.c
fb 0.23 Fraction of embryo volume that is blas-
toderm.c
Ay 1.39 mm2 Assumed surface area of yolk compart-
ment.d
Ab 1.65 mm2 Surface area of blastoderm compart-
ment.d
vby 0.41 Osmotically inactive fraction of yolk.e
vbb 0.20 Osmotically inactive fraction of blasto-
derm.e
Lspy variable Hydraulic conductivity of yolk compart-
ment in presence of solute.
Lspb variable Hydraulic conductivity of blastoderm
compartment in presence of solute.
Psy variable Solute permeability of yolk compart-
ment.
Psb variable Solute permeability of blastoderm com-
partment.
a The internal osmolality of the zebra?sh is unknown. The embryos are
held in embryo medium at 0.040 Osm. However, a number of arguments
point to an internal embryo osmolality closer to 0.300 Osm. It is not
understood how zebra?sh embryos can regulate their internal osmotic
environment while living in such a hypo-osmotic environment. Vapor
pressure osmometer measurements suggest an osmolality of approximate-
ly 0.240 Osm (Hagedorn, unpublished data). We choose 0.300 Osm for
these models to match the osmolality of a typical vertebrate cell.
b This is determined by adding together the components of the two-com-
partment model (vb 5 fy 3 vby 1 fb 3 vbb).
c These fractional compartmental volumes are taken from measurements
of the yolk and blastoderm as a function of developmental stage [5].
d At 100% epiboly, the embryo has an approximately spherical yolk com-
partment surrounded by a thin layer of blastoderm cells. The area of the
blastoderm compartment is taken to be just the outer surface area of the
embryo, assuming a spherical volume. The yolk compartment is a sphere
of somewhat smaller size (containing 77% of the volume), with a surface
area of 1.39 mm2.
e Hagedorn et al. [17], see text.
the NMR experiments, we measured the (relative) quantity
of solute taken up by the embryo as the number of moles
of solute (Ns) in the embryo over time. The flux of water
into a cell is determined by its water permeability or hy-
draulic conductivity (Lp). Experimentally, it is often found
that Lp depends on whether the osmotic driving gradient is
produced by a permeating (e.g., DMSO) or nonpermeating
(e.g., NaCl) solute. To distinguish between these two cases,
we referred to the water permeability in the presence of a
permeating solute as . The flux of solute into the cell issLp
determined by the solute permeability (Ps). The task was
to relate the permeability parameters and Ps to Vc andsLp
Ns.
The embryo volume (Vc) is the sum of the water (Vw),
solute (Vs), and solids (Vsolids) volumes:
i
?V 5 V 1 V 1 V 5 V 1 M V V 1 v V ,c w s solids w s w s b co (1)
where vb is the fractional volume of the osmotically inac-
tive cell contents, is the solute partial molar volume, and?Vs
Vco is the isotonic volume of the cell. Thus the problem
was reduced to one of finding the water volume (Vw) and
the solute content (Ns) of the embryo. These two quantities
were given by a pair of coupled, differential equations that
must be numerically integrated along with subsidiary de-
fining equations. These equations are outlined below.
The water flux into a cell is given by:
s i edV /dt 5 L ART(M 2 M ),w p (2)
where A is the cell surface area, R and T the gas constant
and absolute temperature, respectively, and Mi and Me are the
intracellular and extracellular medium osmolality. The sol-
ute flux is given by:
e idN /dt 5 P A(M 2 M ),s s s s (3)
where Ns is the number of osmoles of solute in the cell,
and and are the extracellular and intracellular solutee iM Ms s
osmolalities, respectively. Sometimes it is more convenient
to express the solute flux in terms of the intracellular solute
osmolality . Substituting for Ns ini i(M 5 N /V ) V Ms s w w s
equation 3 and utilizing equation 2 yielded:
i e idM /dt 5 P (A/V )(M 2 M )s s w s s
s i e i
1 L (A/V )M RT(M 2 M ).p w s (4)
The external medium osmolality (Me) was set by the ex-
perimental conditions:
e e eM 5 M 1 M ,n s (5)
where is the external nonpermeating solute concentra-eMn
tion and is the external permeating solute concentrationeMs(DMSO or PG). The intracellular osmolality Mi was com-
puted assuming a linear Boyle van?t Hoff cell response:
i i iM 5 M (V /V ) 1 M ,no wo w s (6)
where the subscripts n, o, and w refer to nonpermeating
solute, initial (t 5 0) conditions, and water, respectively.
The cell volume (Vc) and intracellular solute (Ns) were
modeled by simultaneously integrating equations 2 and 3
or equations 2 and 4. In the NMR experiments, only the
relative solute concentration (Nrel) was measured; thus in
equation 3, a scaling factor (Sf) must be introduced so that
Ns 5 Sf 3 Nrel. Integration was done using the Stiff Episode
integrator in the program Scientist (MicroMath, Salt Lake
City, UT). The permeability parameters, and Ps (and SfsLp
in the NMR experiment) were obtained by iteratively vary-
ing them until a good fit was obtained between the model
calculations and the experimental observables, specifically
Vc, Nrel, or Vc and Nrel. Least-squares fitting was performed
using the Powell variant of the Levenberg-Marquardt meth-
od as implemented in the program Scientist. In some cases,
a wide range of Ps values gave a satisfactory fit to the
experimental data. In these cases, Ps was manually varied
to find the acceptable range of values as judged by visual
evaluation of the fits. A number of fixed parameters appear
1244 HAGEDORN ET AL.
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in the fitting equations, and their values are listed in Table
1. Some justifications for the fixed parameter values appear
in the next section.
Typical initial volumes of 100% epiboly embryos (or
slightly larger) in the optical experiments were observed to
scatter approximately at 0.20 mm3. Therefore, all initial
volumes were normalized to this value. Further, all data for
a given experimental series were averaged before fitting to
reduce noise and reduce the computational burden. In the
NMR experiments, the data were first (arbitrarily) normal-
ized, then averaged.
Modeling the Zebra?sh Embryo
Two different structural models were used in fitting the
data. The simpler was a one-compartment model assuming
a spherical embryo of initial volume 0.20 mm3 with a fixed
area of 1.65 mm2 and an osmotically inactive fraction (vb)
of 0.36 (see below). A two-compartment model was also
utilized in which the yolk and blastoderm were considered
as separate, distinct compartments, of volume and water
content appropriate to the 100% epiboly stage. For two
compartments, the permeability equations must essentially
be solved in duplicate. The fixed and variable parameters
of this model are listed in Tables 1 and 2.
Some justification is required for our choice of fixed
parameters in the two-compartment model (Table 1). Prior
NMR studies [16] show that the yolk and blastoderm were
responding differently to cryoprotectants, and this formed
the basis for our decision to divide the embryo into a yolk
and blastoderm compartment. The volumes and areas of the
two compartments were determined from the morphometric
data in previous papers [5, 17]. The most difficult parameter
to determine was the osmotically inactive fraction of the
yolk (vby) and of the blastoderm (vbb). For the blastoderm,
we used our estimate of vbb 5 0.2 from Hagedorn et al.
[17]. However, depending on the method of measurement,
we obtained vby?s for the yolk ranging from 0.41 to 0.78
[17]. To help chose an appropriate value, we turned to the
PG optical shrink data. In some experiments with exposure
times up to 225 min in 2 M PG, the embryos were observed
to shrink in a linear fashion by 40?50% (data not shown).
This large shrinkage requires a large percentage of water
(or a small percentage of inactive volume) in the zebrafish
embryo. For this reason, we used the lowest yolk value, vby
5 0.41, suggested by our earlier experiments. Combining
the compartment volumes and vb data yields an overall os-
motically inactive volume (vb) for the embryo of 0.36 (see
Table 1).
In either case, it must be understood that the permeabil-
ity parameters determined are phenomenological parame-
ters characterizing the average response of a very complex
structure, and caution should be used in attributing them to
any specific membrane. Because of the complexity of this
system and the required approximations, none of the deter-
mined permeability parameters are considered to be accu-
rate to better than a factor of two.
EM Analysis
We examined the type of cryodamage evident at the lev-
el of the electron microscope in various compartments of
the zebrafish embryo after vitrification. We chose vitrifi-
cation as a method because zebrafish embryos are chill-
sensitive [4, 5], and the rapid rate of cooling used to vitrify
may ultimately increase embryo survival. Embryos at
100% epiboly were immersed into 2 M PG for 2.5 h, then
6 M PG for 2?4 min, drawn into 0.25-ml straws, cooled in
liquid nitrogen vapor for 3 min, then plunged into liquid
nitrogen for 1?2 min. To thaw, straws were held horizon-
tally at room temperature (22?238C) for 10 sec, then
plunged into room temperature water until thawed (approx-
imately 30 sec). Afterwards, the embryos were diluted by
steps into 3 M, 2 M, then 1 M PG for 20 min each. Control
embryos were exposed to all chemicals but were not sub-
jected to freezing. To prepare for EM, both experimental
and control embryos (n 5 3 each) were placed in a fixative
(2.5% glutaraldehyde, 1% paraformaldehyde in cacodylate
buffer) 10 min after thawing for 2?3 h at 228C, and then
for 1?3 h at 48C. Embryos were stored for 24 h in caco-
dylate buffer at 48C, fixed in 1% osmium tetroxide in cac-
odylate buffer for 1 h, en bloc stained with 2% uranyl ac-
etate for 1 h, then dehydrated in a series of alcohols (70,
70, 80, 90 and 100, 100, 100% ethyl alcohol) for 5?6 min
each, followed by propylene oxide (1:1, 1:2, and 1:3 for 5?
6 min each) and 100% propylene oxide for 40 min. Em-
bryos were infiltrated with Spurr?s plastic resin (for 18 h),
embedded in fresh plastic, and placed in a 708C oven for
polymerization. Sections were cut at 600?800 A? and
mounted onto copper grids that were stained for 1.5 min in
lead citrate, and then examined using a Zeiss EM10 CA
transmission electron microscope.
To evaluate whether the cellular morphology of the fro-
zen and nonfrozen embryos was similar, thin sections were
made at three positions (i.e., through the center of the em-
bryo and 200 mm to each side, see Fig. 1B). Then, on each
thin section, three areas including the blastoderm and YSL
were randomly chosen and scored for intact cells, nuclear
membranes, the presence and normal appearance of various
organelles (e.g., mitochondria, Golgi bodies, endoplasmic
reticulum, etc.), and the presence of yolk droplets (Fig. 1C).
In the yolk, the presence of intact membranes and smooth
yolk material was assessed. Each intact class of organelle
or membrane was given one point for its presence in the
tissue. A maximum of 10 points was possible in the blas-
toderm and the YSL, and two points in the yolk. Mean
scores from the yolk, YSL, and blastoderm of each treat-
ment were analyzed with a two-tailed t-test and then
graphed. Representative areas of the specimens were pho-
tographed and printed.
RESULTS
Biophysical Data: Volumetric and NMR
When zebrafish embryos were exposed for long periods
of time (i.e., 60 min) to 2 M DMSO or PG, their light
microscopic volumes exhibited an approximately linear de-
crease over time without any re-expansion (Fig. 3). The
steady volume decrease observed over the long duration of
the experiments suggested that the embryo experienced
mostly dehydration with little to no solute permeation. At
some point during the dehydrating events, the embryonic
structure began to lose integrity. For example, after embry-
os were immersed in 2 M DMSO for 90?120 min, the
blastoderm began to shed cells, and a rupture of the yolk
in that area followed shortly thereafter.
NMR spectroscopy allows the solute uptake to be mea-
sured directly. When embryos were perfused with a 2 M
DMSO solution, DMSO uptake was rapid during the first
few minutes, then gradual thereafter (Fig. 4). The response
time of the DMSO perfusion system was determined by
measuring the NMR signal rise-time in the buffer slice (po-
sitioned above the embryos, data not shown). The rise-time
1245DMSO PERMEABILITY OF ZEBRAFISH
TC # 369
FIG. 3. Volume changes in zebra?sh embryo as a function of time in 2
M DMSO (n 5 7) and 2 M PG (n 5 9) at 22?238C measured with light
microscopy. A decrease of approximately 20% in the averaged volume
was observed. The data from each group were averaged and normalized
to a standard volume of 0.2 mm3 for each graph.
FIG. 4. NMR measured DMSO uptake by embryos at 100% epiboly
(218C). Three measurements were run on samples containing 20 embryos
each. The measurements were individually normalized, then averaged.
The embryos were perfused with 2 M DMSO beginning at time zero. The
observed initial rise time is partially limited by the time required for the
perfusate to reach 2 M concentration in the sample chamber.
of the DMSO signal from 10% to 90% was 2.3 min. This
imposed a lower limit on the attainable time resolution of
the embryo response to DMSO exposure.
The NMR data suggested some embryo permeability to
DMSO, whereas the light microscopic data suggested little
embryo permeability to DMSO. At first glance, this pre-
sented a somewhat contradictory picture of the permeability
within the embryo. In order to estimate the permeability
parameters, we analyzed these data with a one- and two-
compartment model.
One-Compartment Model
The NMR and optical data were first analyzed with a
one-compartment model (Tables 1 and 2) to determine
whether this would yield a useful phenomenological model
of the embryo. The optical volumetric data gave a water
permeability ( ) for 2 M DMSO of 0.007 mm 3sLp
min21atm21 and an upper limit to the solute permeability
(Ps) of 4 3 1025 cm/min (Fig. 5A; Table 3). In contrast,
the NMR DMSO-uptake data gave a water permeability
( ) of 0.03 mm 3 min21atm21 and a Ps of 0.003 cm/minsLp(Fig. 5B). Although good fits were obtained in each case
(Fig. 5), the optical and NMR permeability parameters did
not agree. Comparing the resultant permeability values (Ta-
ble 3) reveals a 4-fold difference in Lp values and at least
a 75-fold difference in Ps values between the optical and
NMR one-compartment fits. Another way to illustrate this
discrepancy was to simultaneously fit the optical and NMR
data with a single set of parameters (Fig. 5C). This resulted
in an intermediate pair of permeability parameters that
yielded a poor fit to both the optical and NMR data.
Analysis of the optical volumetric data for embryos im-
mersed in 2 M PG (data from Fig. 3) yielded similar water
and solute permeabilities similar to those of DMSO (fit not
shown; Table 3).
Two-Compartment Model
The optical and NMR data were also analyzed using a
two-compartment model, the blastoderm being one com-
partment, and the yolk, the second. By fitting these data
simultaneously, sufficient constraints were obtained to fit
values for of the yolk and blastoderm and Ps of thesLp
blastoderm, and to find an upper limit for Ps for the yolk.
In the presence of 2 M DMSO, the yolk and blastoderm
had very similar water permeabilities (i.e., 0.01 and 0.005
mm 3 min21atm21, respectively) but different solute per-
meabilities separated by three orders of magnitude (i.e., #
5 3 1026 and 1.5 3 1023 cm/min, respectively; Fig. 6,
Table 3). Empirically, it was found that the permeability
parameters for the yolk were largely fixed by the volumetric
data, whereas those for the blastoderm were fixed by the
solute uptake data.
Although NMR PG-uptake data could not be obtained,
it was still possible to do a two-compartment analysis of
the volumetric PG data. As noted above, the volumetric
data primarily reflect the response of the yolk. Thus, the
yolk permeability parameters are only weakly constrained
by the NMR solute uptake data. We made the assumption
that the blastoderm water and solute permeabilities in the
presence of PG are less than or equal to twice those found
for DMSO. With these assumptions, a two-compartment fit
to the volumetric PG data for the yolk compartment yielded
slightly smaller water and solute permeabilities for PG than
for DMSO (Table 3).
Cryodamage
If there is a permeability barrier in the yolk compart-
ment, how might cryopreservation effect the blastoderm,
YSL, and yolk? To test this, the embryos were subjected
to a vitrification procedure. During freezing, the external
solution (6 M PG) surrounding the embryos remained clear,
suggesting that the external solution vitrified. All of the
frozen embryos (n 5 26) died after thawing, whereas 32%
of the solution-control embryos (which passed through all
solutions, but were not frozen; n 5 25) survived and con-
tinued to develop. The normal laminar structure of the ze-
brafish embryo is shown in Figure 7. Control and frozen
1246 HAGEDORN ET AL.
TC # 369
FIG. 5. One-compartment model ?t to the zebra?sh embryo. Tables 1
and 2 show the ?xed parameters used. A) The optical volumetric data
from Figure 3 (2 M DMSO) were ?t with a one-compartment model,
which yielded a well-de?ned value of 5 0.007 mm 3 min21atm21, butsLp
only an upper limit of Ps # 4 3 1025 cm/min. The solid curve illustrates
an acceptable ?t with Ps 5 1 3 1025 cm/min. B) The NMR DMSO-uptake
data from Figure 4 were ?t with a one-compartment model. The solid
curve shows a model ?t assuming the zebra?sh embryo is a single com-
partment, yielding an 5 0.03 mm 3 min21atm21 and Ps 5 0.003 cm/sLp
min. C) The optical (open circles) and NMR (solid circles) data were si-
multaneously ?t with a one-compartment model yielding 5 0.01 mmsLp
3 min21atm21, Ps 5 3 3 1024 cm/min, and a very poor ?t. Note that the
scaling of the NMR data is arbitrary and different from that in Figure 5B.
FIG. 6. Two-compartment model ?t to the zebra?sh embryo in 2 M
DMSO at 22?238C. The experimental optical volume data (in mm3) are
plotted as open circles, and the NMR-measured DMSO uptake (in arbi-
trary units) are plotted as solid circles. The solid curves are a two-com-
partment model ?t to the combined data using a single set of water and
solute permeabilities for the yolk and blastoderm compartments (i.e., sLpy
5 0.01 mm 3 min21atm21, 5 0.005 mm 3 min21atm21, Psy 5 1 3sLpb
1026 cm/min, and Psb 5 0.0015 cm/min). The data only constrain Psy to
Psy # 5 3 1026 cm/min.
TABLE 2. Summary of two-compartment model of zebra?sh embryo at
100% epiboly.
Yolk Blastoderm Embryo
Volume (mm3)
Volume fraction of embryo
Water fraction of compartment
Solids fraction of compartment
0.15
0.77
0.59
0.41
0.05
0.23
0.80
0.20
0.20
1.00
0.64
0.36
embryos were examined and scored at the ultrastructural
level to identify areas of the embryo that were damaged.
Control embryos showed little or no damage to the blas-
toderm, yolk, or YSL as a result of the chemical treatment
(i.e., cell structures were intact and showed normal mor-
phology; p $ 0.05; Fig. 8). In contrast, experimental em-
bryos exhibited altered morphology only in the YSL (p #
0.05). Specifically, after freezing, many of the organelles in
the YSL were destroyed, and the cellular membrane was
destroyed (usually in the area of blastopore closure); some
YSL nuclei were present, but the chromatin was congested,
and the nuclear membrane was not always intact (Fig. 9).
Morphologically, the yolk was not affected by freezing, as
the yolk membranes were intact and the yolk contents ap-
peared to be normal. We concluded that cryodamage within
the yolk may be tolerated. However, damage to the YSL
(the only other major organizing structure related to the
yolk) may destroy the integrity of the yolk/YSL compart-
ment, thus leading to the lethal effects observed after thaw-
ing.
DISCUSSION
Using a one-compartment model (Fig. 5), we found that
no single set of parameters simultaneously fit the optical
and NMR data. However, a two-compartment model pro-
vided a better fit to the data (Fig. 6). In the presence of
DMSO, the yolk and blastoderm compartments had similar
water permeabilities but substantially different solute per-
meabilities. The yolk compartment was essentially imper-
meable to DMSO (Ps # 5 3 1026 cm/min), whereas the
blastoderm compartment had a ??typical?? membrane solute
permeability (Ps 5 1.5 3 1023 cm/min). This readily ex-
plains the different behavior seen in the DMSO uptake and
volumetric experiments. In the volumetric experiments,
when the blastoderm cells were exposed to 2 M DMSO,
they experienced a rapid shrink/swell response. This is not
1247DMSO PERMEABILITY OF ZEBRAFISH
TC # 369
FIG. 7. Control image of the zebra?sh
embryo at the 3-somite stage showing the
normal laminar structure of the blastoderm
(B), YSL, and yolk (Y). In all areas, mem-
branes are intact and nuclei (N) are visible
in the blastoderm and YSL. The dense,
granular appearance of the YSL is due to
the abundance of ribosomes. The yolk has
a smooth appearance and resides within
membrane-bound yolk-vesicles (bar, lower
right 5 1 mm).
TABLE 3. Permeability parameters of zebra?sh embryo exposed to 2 M DMSO at 218C (NMR experiments), 22?238C (optical experiments), and 2 M
PG at 22?238C (optical experiments): one- and two-compartment permeability results ( and Ps) of the optical volume versus time and the NMR uptakesLp
versus time data.a
Experiment Solute Lsp Ps Lspy Lspb Psy Psb
One-compartmentb
Optical volume
NMR uptake
Optical volume
DMSO
DMSO
PG
0.007
0.03
0.004
#4 3 1025
0.003
#5 3 1026
?
?
?
?
?
?
?
?
?
?
?
?
Two-compartmentc
NMR & optical
Optical volumed
DMSO
PG
?
?
?
?
0.01
0.006
0.005
#0.01
#5 3 1026
#2 3 1026
0.0015
#0.003
a The dimensions of L are micrometers 3 min21atm21 and of Ps are cm/min.sp
b Model: one-compartment, two-parameter model.
c Model: two-compartment (yolk and blastoderm), two parameter model.
d The values of the blastoderm permeability (L and Psb) could not be calculated and were assumed to be less than twice the values found in thespb
DMSO experiments.
evident in the data, because the shrink/swell response was
most likely small and occurred quickly (i.e., between the
first and second experimental data points). Since the yolk
compartment is essentially impermeable to DMSO, it
showed a slow, steady loss of water to the hyperosmotic
DMSO environment, yielding a steady decrease in volume,
which dominated the volumetric data. In the NMR uptake
experiments, primarily the blastoderm was permeable to the
DMSO, and the data reflect this response. Initially there
was a rapid influx of DMSO due to the large solute gra-
1248 HAGEDORN ET AL.
TC # 369
FIG. 8. Histogram quantifying the number of ultrastructural elements in 3-
somite embryos after exposing embryos to a solution-control or cryoprotec-
tant, then freezing, and thawing. Only the YSL in the frozen sample showed
a loss of structural elements (*p , 0.05), whereas the ultrastructure of the
blastoderm and the yolk appeared the same in both treatments (p $ 0.05).
FIG. 9. After freezing and thawing, the
ultrastructure of the YSL was destroyed.
No clear YSL layer remained as the riboso-
mal material from the YSL was scattered
throughout the blastoderm (B) and yolk
(Y). However, the areas not in direct con-
tact with the YSL appeared normal. Most
membranes and nuclei (N) of the blasto-
derm (in the upper part of the ?gure) and
yolk (area not shown) remained intact (bar,
lower right 5 1 mm).
dient; however, as equilibrium was approached, the influx
of DMSO slowed. Figure 10 shows the individual responses
of the two-compartment model and how they combine to
yield the observed DMSO uptake and volumetric response.
Of course, one-compartment models are used routinely
to characterize single cells. Even in multicellular systems,
a one-compartment model may yield useful phenomenolog-
ical information (e.g., in mouse embryos [19], pancreas
[20], and Drosophila [21]). A two- or multicompartmental
model is only useful if there are sufficient experimental data
to characterize the two compartments, and if the responses
of the two compartments are so different that a single set
of phenomenological parameters do not adequately char-
acterize the system. This is the case with the zebrafish data
presented here. The volumetric and DMSO uptake experi-
ments provide a comprehensive data set, and the two pu-
tative compartments of the zebrafish embryo (i.e., blasto-
derm and yolk/YSL) differ by three or more orders of mag-
nitude in their cryoprotectant (DMSO) permeability, 1.5 3
1023 and # 5 3 1026 cm/min, respectively. In comparison,
mouse oocytes in DMSO or PG have Ps 5 0.96 to 1.4 3
1023 cm/min [22], and goat oocytes in PG have Ps 5 0.87
1249DMSO PERMEABILITY OF ZEBRAFISH
TC # 369
FIG. 10. Two-compartment model simulations showing the response of
a zebra?sh embryo exposed to 2 M DMSO at room temperature. This
illustrates how the response of the each compartment contributes to yield
the experimentally observed response of the entire zebra?sh embryo. The
simulations were generated using the two-compartment parameters
shown in Tables 1 and 2 and their permeability values in Table 3. A) The
two different dashed lines indicate the changing volume of the blastoderm
and yolk compartments. Combining these yields the volume of the entire
embryo (solid line). This simulation is directly comparable with the opti-
cally determined volume versus time data shown in Figure 3. B) The two
different dashed lines indicate the changing DMSO solute in the blasto-
derm and yolk compartments. Combining these yields the total DMSO
uptake by the embryo (solid line), which is directly comparable with the
NMR DMSO uptake data shown in Figure 4.
to 1.2 3 1023 cm/min [23]. Because the yolk is completely
surrounded by the blastoderm at 100% epiboly, it might be
argued that the low apparent solute permeability of the yolk
compartment simply results from the surrounding layer of
blastoderm cells, which block ready access of the extracel-
lular medium to the yolk. However, if this were the case,
we would expect the yolk compartment to have a very low
water permeability as well. This suggests that the blasto-
derm does not create the solute permeability barrier around
the yolk compartment, but the YSL does.
There is a major difference in the predictions that the
one- and two-compartment models make regarding cry-
opreservation protocols. We modeled the time required for
DMSO to reach two thirds of its final concentration (ap-
proximately 2 M) within the embryo and yolk for the one-
and two-compartment models, respectively, using the upper
limits of the solute permeability given in Table 3. The one-
compartment model predicted a minimum time of 2.2 h for
the embryo to reach the projected DMSO concentration.
This is a reasonable treatment time given the constraints of
the rapidly developing embryo. However, the two-com-
partment model predicted a minimum of 9 h for the yolk
to reach the projected concentration. This is an unreason-
able treatment time, because of toxicity and the large
change in development that the embryo would experience
by the end of the test period. Clearly, the predictions of the
two-compartment model will impose some important con-
straints on future cryopreservation protocols.
Previously, we measured the water permeability of the
zebrafish embryo in the absence of cryoprotectants using
optical volumetric methods and a one-compartment model
[17]. The value of Lp we obtained at the 100% epiboly stage
was 0.05 mm 3 min21atm21, and may be compared with
our one-compartment analyses of the DMSO and PG op-
tical volumetric data (Table 3). This revealed that Lp
dropped by an order of magnitude in the presence of either
DMSO or PG and is consistent with a growing body of
evidence suggesting that Lp is modified in the presence of
permeating cryoprotectants for a variety of membrane types
[5, 24, 25]. Clearly, for most cryobiological purposes, it is
important to measure membrane water permeability in the
presence of proposed cryoprotective solutes. Other inves-
tigators have also measured the water permeability of fish
embryos in the presence of cryoprotectants. Zhang and
Rawson [15] report zebrafish Lps of 0.10 and 0.17 mm 3
min21atm21 in the presence of PG and methanol, respec-
tively. Their permeabilities are more than an order of mag-
nitude larger than our similarly obtained, optical volumetric
results. Some of this may be due to a difference in mod-
eling parameters or in the osmolality of the buffer used to
make the solutions. Nevertheless, in the presence of solutes,
their Lps appear to be substantially larger than those ob-
tained here.
Previously, we identified the YSL, and not the yolk it-
self, as the barrier to the movement of cryoprotectant into
the yolk [16]. In this study, we have characterized a per-
meability barrier in the yolk compartment of the zebrafish
embryo, but what are the consequences of this barrier to
cryopreservation? At the ultrastructural level, the most ob-
vious consequence of our vitrification protocol was a de-
struction of the YSL surrounding the yolk. This is not to
suggest that other, more subtle changes did not occur in the
blastoderm and the yolk after the tissue was frozen and
thawed. For example, the mere presence of ultrastructural
elements within a tissue does not mean they are present in
sufficient quantity for normal cell function. A study of se-
rial sections quantifying the number of ultrastructural ele-
ments per area of tissue may have shown a clear difference
in the blastoderm tissue of the controls and frozen embryos.
Additionally, the high concentration of cryoprotectants
used in vitrification (approximately 6 M or greater) could
have caused a disruption of cytoskeletal elements. Using
fluorescent antibodies, Hotamisligil et al. [26] observed a
disruption in the actin filaments of the mouse embryo after
exposure to 4 M ethylene glycol for more than 10 min.
Hypothetically, if little or no cryoprotectant reached the
yolk because of the impermeable YSL, ice-crystals may
have formed, leading to some type of damage in both the
yolk and YSL regions. However, these more subtle issues
are moot, because the test vitrification protocol caused the
complete destruction of the YSL.
Our ultrastructural experiments suggested that cryoda-
mage led to the death of the embryos, in part because of
insufficient cryoprotectant entering the yolk/YSL region.
Isotope-uptake experiments have quantified how little cryo-
protectant enters the blastoderm, and the yolk in particular.
Harvey et al. [10] measured a DMSO concentration in the
whole, dechorionated zebrafish embryo of 100 mM after 60
min in 1 M DMSO. After incubating carp embryos in 1.5
M isotope-labeled DMSO for 2 h, Magnus et al. [27] ex-
tracted yolk fractions with a micropipette and found ap-
proximately 100 mM DMSO. All of these values are well
in line with the predictions that the two-compartment model
made about the permeability of the zebrafish yolk com-
partment to DMSO after 2 h (approximately 150 mM) and
lends support for the two-compartment model when con-
sidering the yolk specifically.
Now that we have characterized and quantified the per-
meability parameters in the zebrafish embryo, we must ex-
amine how we can overcome this permeability barrier to
successfully cryopreserve fish embryos. The YSL is a com-
mon morphological feature in all teleost fish and will be an
important consideration for cryopreservation in most threat-
ened species and for important cultured species, such as
catfish, salmonids, tilapia, sturgeon, and striped bass. It is
clear that exposure alone may not allow enough cryopro-
1250 HAGEDORN ET AL.
TC # 369
tectant to enter the yolk for successful cryopreservation. A
means for getting cryoprotectant into and past the YSL bar-
rier is required. The vast arsenal of molecular biological
methods created for transfecting cells, such as the intro-
duction of pores in the YSL through chemical (e.g., anti-
biotics) or physical (e.g., electroporation) means, may help
overcome this permeability barrier, thus opening the door
for successful cryopreservation protocols for fish embryos.
ACKNOWLEDGMENTS
We would like to thank Dr. P. Mazur at the Oak Ridge National Lab-
oratory (Oak Ridge, TN), Dr. S. Blackband at the University of Florida
(Gainsville, FL), and Dr. V. Chacko at Johns Hopkins School of Medicine
(Baltimore, MD) for their discussion on many of the ideas in this manu-
script; Dr. E. Hsu at Duke University Medical Center (Durham, NC) for
his insightful comments on the manuscript; and Dr. Lee-Ann Hayek of the
Smithsonian Institution (Washington, DC) for statistical assistance.
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