Cryobiology 73 (2016) 7e14

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Cryobiology
journal homepage: www.elsevier.com/locate/ycryo

The contribution of apoptosis and necrosis in freezing injury of sea

urchin embryonic cells
Andrey V. Boroda, Yulia O. Kipryushina, Konstantin V. Yakovlev, Nelly A. Odintsova*
Laboratory of Cytotechnology, A.V. Zhirmunsky Institute of Marine Biology, The Far Eastern Branch of the Russian Academy of Sciences, Vladivostok, 690041,
Russia

a r t i c l e i n f o a b s t r a c t

Article history: Sea urchins have recently been reported to be a promising tool for investigations of oxidative stress, UV
Received 30 March 2016 light perturbations and senescence. However, few available data describe the pathway of cell death that
Received in revised form occurs in sea urchin embryonic cells after cryopreservation. Our study is focused on the morphological
20 June 2016
and functional alterations that occur in cells of these animals during the induction of different cell death
Accepted 25 June 2016
Available online 27 June 2016
pathways in response to cold injury. To estimate the effect of cryopreservation on sea urchin cell cultures
and identify the involved cell death pathways, we analyzed cell viability (via trypan blue exclusion test,
MTT assay and DAPI staining), caspase activity (via ow cytometry and spectrophotometry), the level of
Keywords:
Apoptosis
apoptosis (via annexin V-FITC staining), and cell ultrastructure alterations (via transmission electron
Caspases microscopy). Using general caspase detection, we found that the level of caspase activity was low in
Cryoinjuries unfrozen control cells, whereas the number of apoptotic cells with activated caspases rose after freezing-
Cryopreservation thawing depending on cryoprotectants used, also as the number of dead cells and cells in a late
Freezing-thawing apoptosis. The data using annexin V-binding assay revealed a very high apoptosis level in all tested
Necrosis samples, even in unfrozen cells (about 66%). Thus, annexin V assay appears to be unsuitable for sea
Cell death pathway urchin embryonic cells. Typical necrotic cells with damaged mitochondria were not detected after
Sea urchin
freezing in sea urchin cell cultures. Our results assume that physical cell disruption but not freezing-
induced apoptosis or necrosis is the predominant reason of cell death in sea urchin cultures after
freezing-thawing with any cryoprotectant combination.
2016 Elsevier Inc. All rights reserved.

1. Introduction
Our understanding of the mechanisms involved in the process of
cell death in mammalian cells may become clearer from the study
of different cellular responses to external stresses in simpler
Deuterostome organisms, such as Echinoderms. A variety of
extracellular signals, including heat shock, osmotic stress, pro-
inammatory cytokines and UV exposure, results in activation of
stress proteins in different organisms [14,21,44]. Particularly, sea
urchin embryos and larvae are strongly inuenced by their envi-
ronment and can be used as bioindicators in ecotoxicological
studies [3,31,32,34] or as a promising tool for investigations of
Abbreviations: CMFSS, CaCl2 and MgCl2-free salt solution; EG, ethylene glycol;
hsp, heat shock proteins; LN, liquid nitrogen; Me2SO, dymethyl sulfoxide; MTT, 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RT, room tempera-
ture; STS, staurosporine; SW, seawater; Tr, trehalose; UV, ultraviolet.
* Corresponding author.
E-mail address: nelodin54@gmail.ru (N.A. Odintsova).
oxidative cell damage and senescence [10]. One of the reason for
their sensitivity is that the largest group of genes in the sea urchin
genome is represented by genes that encode sensory proteins
[9,25].
The development of cryopreservation methods for sea urchin
cells is important for these studies, but freezing-thawing results in
different cell injuries, including a higher degree of aberrations and a
delay in development in cryopreserved embryos, abnormalities in
mitosis and cytoskeleton disassembly of sea urchin embryonic cells
[30,33]. To improve the cryopreservation outcomes of cells, we
focused on cryoinjuries and identifying the predominant pathway
of sea urchin embryonic cell death after freezing-thawing.
Apoptosis and necrosis can occur independently, sequentially,
or simultaneously [13,50]. The induction of these two processes is
regulated by many of the same biochemical factors [28], and the
degree of stimulation often determines whether the cells die via
apoptosis or necrosis. Necrosis, which is characterized by cell
swelling and a loss of cell membrane integrity, is triggered by
multiple stresses, such as osmotic shock, hypothermia, hypoxia,

http://dx.doi.org/10.1016/j.cryobiol.2016.06.007
0011-2240/ 2016 Elsevier Inc. All rights reserved.
8 A.V. Boroda et al. / Cryobiology 73 (2016) 7e14
and mechanical or chemical injuries [11]. In contrast, apoptosis is a
highly organized and evolutionarily conserved cellular process that
occurs under normal physiological conditions and is accompanied
by de novo gene expression of caspases or other enzymes respon-
sible for controlled cellular dissolution [11,18,19,39,43,46,51]. Many
stress factors are well-known initiators of necrosis and apoptotic
cell death. Necrosis is frequently observed during the cryopreser-
vation of mammalian cells [8]. Apoptosis may also be initiated by a
variety of extracellular and intracellular stresses, including expo-
sure to extreme temperatures [5].
Abundant experimental evidences indicate that apoptosis in sea
urchin embryos occurs in response to oxidative stress or specic
molecular perturbations, such as UV light treatment and environ-
mental pollutants [22,26,27,36,38,47,48]. As in vertebrate cells,
apoptosis in sea urchin cells appears to involve a cascade of com-
plex genetically encoded reactions, but this process also has unique
features. At least two novel pro-apoptotic proteins that are asso-
ciated with a death domain and an unusually large number of
caspase genes have been detected in sea urchins [35]. The authors
suggest that the increased number of caspases may be connected to
the involvement of caspases in developmental remodeling during
sea urchin metamorphosis.
Although some data concerning the molecular mechanisms that
respond to environmental stress have been reported for mammals
[16,23,45,49], there is no published attempt to describe the
pathway of cell death that occurs during freezing-thawing of ma-
rine invertebrate cells. New approaches have recently been devel-
oped for mammalian cells that focus on the inhibition of the cell
death-associated apoptosis and/or necrosis responses after
freezing-thawing and improve the cellular state after cryopreser-
vation [8]. These approaches may be useful for marine invertebrate
cells, but further studies are required to increase our understanding
of the mechanisms that underlie cell death pathways after
cryopreservation.
An effective cryopreservation protocol for sea urchin cells (up to
65e80% of viable cells) including three-step freezing with a low
cooling rate (1e2
C/min) and a combination of non-penetrating
and penetrating cryoprotectants was developed previously [7,30].
The current study was undertaken to determine the predominant
reason of sea urchin cell death caused by freezing-thawing. We
conducted a detailed morphological and functional analysis of sea
urchin embryonic cells after freezing-thawing with the aim of
lling this knowledge gap and improving post-thaw cell outcomes.
Cell viability, the level of apoptosis, caspase activity and cell ul-
trastructure alterations were analyzed to characterize the damage
that occurs after cold injury in sea urchin embryonic cell cultures.
Data describing the cell death process in sea urchin cell cultures
after cryopreservation are presented for the rst time, and the
possible impact of apoptosis and necrosis in death induced by
freezing-thawing is discussed.
2. Materials and methods
2.1. Materials
Adult sea urchins (Strongylocentrotus intermedius) were
collected from Peter the Great Bay of the Sea of Japan and kept in
tubs lled with aerated running seawater (SW) for one-two months
at 10e14
C until needed. All the experiments on animals were
reviewed and approved by the Ethics Committee of A.V. Zhir-
munsky Institute of Marine Biology of the Far Eastern Branch of the
Russian Academy of Sciences. Spawning was induced via an intra-
coelomic injection of 0.5 M KCl (0.2e0.5 ml per animal). Developing
embryos were cultivated in 5 L tanks at 18
C and harvested until
the mesenchymal blastula stage (17e18 h post fertilization) for
obtaining embryonic cell culture, as described previously [1].
2.2. Cell freezing and thawing
The cryoprotective solutions (cooled to 4
C) were prepared
with sterile 32 SW and contained different combinations of
cryoprotectants:the non-penetrating cryoprotectants, disaccharide
trehalose (Tr) at a nal concentration of 10e15 mg/ml, and the
penetrating cryoprotectants, dymethyl sulfoxide (Me2SO) and
ethylene glycol (EG) at nal concentrations of 5e15% each. The
resulting cell suspension was cooled to 196
C via three-step
freezing, as described previously [30].
After storage in liquid nitrogen for 1e60 days, the cryotubes
were placed into a 30
C circulating water bath until the ice was
completely thawed. The samples were gradually diluted ten-fold
with sterile cold SW (5e10
C) over 3e5 min with gentle shaking,
centrifuged (at 600 g for 5 min), and re-suspended in SW supple-
mented with 2% fetal calf serum and penicillin-streptomycin (to
nal concentrations of 100 U/mL and 100 mg/mL, respectively) for
cultivation at 18
C in 24-well plates (Nunc, Nunclon Surface, Ros-
kilde, Denmark) at a concentration of 1.5e2.5  106 cells/well. Cell
viability was assessed in control cultures and immediately after
thawing by a trypan blue exclusion test and the colorimetric MTT
assay, as described previously [30]. We used staurosporine (LC
Laboratories, Woburn, MA, USA) to induce apoptosis in unfrozen
sea urchin embryonic cells. Staurosporine was dissolved in Me2SO
at 12.5 mM, added to the cells at nal concentrations of 1 mM; then
cells were incubated for 4 h at 18
C.
2.3. Flow cytometry analysis
Intact cells, cells undergoing induced apoptosis and cells
immediately after a freeze-thaw cycle (1e2  106) were re-
suspended in 1 mL of annexin-binding buffer (CMFSS with
2.5 mM CaCl2) for subsequent caspase activity detection and
annexin V labeling.
2.3.1. General caspase detection
We used the Vybrant FAM Poly Caspases Assay Kit (Molecular
Probes, Eugene, OR, USA) which provided the FAM-VAD-FMK
FLICA reagent (a generic probe for the general evaluation of
caspase activation, including caspase-1, -3, -4, -5, -6, -7, -8, and -9)
in combination with DAPI staining for dead cell detection. The FAM-
VAD-FMK FLICA reagent was added to 90 ml of a cell suspension,
according to the manufacturers instructions, and left in the dark at
room temperature (RT) for 45 min. Then, the cell suspension was
diluted with 1.5 mL of SW, centrifuged (600 g for 5 min) and re-
suspended in 95 ml of fresh SW. DAPI was added to generate a
nal concentration of 1 mg/ml (Biolegend, San Diego, CA, USA). The
samples were left in the dark at RT for an additional 5 min, diluted
with 400 ml of SW and analyzed in a CytoFlex ow cytometer,
equipped with three lasers (405, 488 and 638 nm) and connected to
a computer running the CytExpert software (version 1.2.10.0,
Beckman-Coulter, Inc., Indianapolis, IN, USA). At least 50 000 events
were evaluated for each sample.
2.3.2. Apoptosis detection via annexin V-FITC/DAPI staining
Annexin V is a phosphatidylserine-binding protein, and its
complex with FITC can be used for the sensitive detection of
phosphatidylserine translocation to the outer membrane surfaces
of apoptotic cells [11]. In intact cells, the membrane lipid phos-
phatidylserine is restricted to the inner leaet of the membrane [4].
Five microliters of annexin V-FITC staining solution was added to
90 ml of a cell suspension in previously prepared annexin-binding
buffer. After 10-min incubation at RT in the dark, DAPI (to a nal
 A.V. Boroda et al. / Cryobiology 73 (2016) 7e14
concentration of 1 mg/ml) was added to the cells and incubated for
an additional 5 min. Then, the cell solutions were diluted 1:4 in
annexin-binding buffer. Apoptosis-associated uorescence (FITC)
and necrosis-associated uorescence (DAPI) were measured within
10 min using a CytoFlex ow cytometer (Beckman-Coulter, Inc.,
Indianapolis, IN, USA).
2.4. Colorimetric assay of caspase activity
The quantitative determination of caspase proteolytic activity in
lysates of intact cells, cells undergoing induced apoptosis and
experimental (frozen) cells was performed via incubation with
labeled synthetic substrates for the relevant caspase (ApoTarget
Caspase Colorimetric Protease Assay Sampler Kit, Invitrogen,
Frederick, MD, USA), according to the manufacturers instructions.
Upon cleavage of the substrates by caspases, the absorption of light
by the cleavage products was quantied with a xMark microplate
spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA) at
400 nm.
2.5. Transmission electron microscopy
For transmission electron microscopy, samples of thawed sea
urchin embryonic cells (1 h and 4 h after freezing) were centrifuged
at 3000  g for 5 min. The pellets were incorporated into 1.5%
agarose pieces (2  3 mm) that were previously soaked with SW.
The samples were xed with a solution comprised of 2% glutaral-
dehyde, 100 mM HEPES-KOH, pH 7.4, 350 mM NaCl, and 140 mM
mannitol for 2 h at 16
C, and then rinsed several times in washing
solution (100 mM HEPES-KOH, pH 7.4, 350 mM NaCl, and 340 mM
mannitol). The osmolarity of the xative and washing solutions was
equal to 36 SW. The samples were then post-xed for 1 h at 4
C
using 1% OsO4 in distilled water, washed in distilled water and
treated with 1% uranyl acetate (water solution) for 30 min at 4
C.
After washing, the samples were processed through ethanol series,
absolute ethanol, a propylene oxide mixture and pure propylene
oxide. The material was embedded in Spurr resin (Sigma-Aldrich,
St. Louis, MO, USA). Ultrathin sections were cut using a Leica EM
UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany) and
stained with uranyl acetate followed by lead citrate. The grids were
observed under a JEM 100S transmission electron microscope (JEOL
Ltd., Tokyo, Japan).
2.6. Data analysis
Each experiment was performed independently at least three
times, and all assays were performed in triplicate. The results were
subjected to a one-way analysis of variance (ANOVA) followed by
Tukeys multiple comparison test with the use of Ofce Excel 2013
software (Microsoft Corporation, CA, USA) to test whether the
values of the means among each experiment group were signi-
cantly different. A p-value < 0.05 was used to assess statistical
signicance of treatment differences in all data analyses.
3. Results
3.1. Cell survival of sea urchin embryonic cells
As shown in Fig. 1, the results of cell survival after freezing-
thawing obtained by the trypan blue exclusion test (Section 1)
and the MTT assay (Section 2) were similar. Both tests revealed a
high level of cell viability in unfrozen cells and signicant differ-
ences in cell viability after freezing-thawing depending on the
cryoprotectants used. The viability of cells frozen in Me2SO alone
reached approximately 75e78% of that of the unfrozen control cells
9
(estimated both by the trypan blue exclusion test and the MTT
assay), whereas the viability of cells frozen in Me2SO with trehalose
was signicantly lower and reached only 40% (by the MTT assay) of
that of the unfrozen control cells. The lowest cell survival after
cryopreservation was observed in a complex mixture of penetrating
cryoprotectants (Me2SO and EG). Cell viability in that mixture was
almost three times lower than that of the control unfrozen cells.
3.2. Flow cytometry analysis
A ow cytometry analysis of caspase activation in cell culture via
the FLICA staining is presented in Fig. 2. The cell viability (via
DAPI staining) of intact unfrozen cells (K) was approximately 85%
versus 16e43% after cryopreservation. Staurosporine slightly
induced caspase activity in unfrozen cells, increasing the total
apoptotic cells from 3.94% (in K) to 7.94% (in K STS). The number
of apoptotic cells increased signicantly to 55e83% after freezing-
thawing. Late apoptotic cells prevailed over early apoptotic cells
(39.70% versus 20.36%) after freezing with Me2SO alone (A). Most of
the cells frozen with additional cryoprotectants (trehalose (B) or EG
(C)) were in the late apoptotic stage (49.53% and 77.22%,
respectively).
Annexin-V-FITC/DAPI staining was used to distinguish apoptotic
cells from viable non-apoptotic cells and dead cells, as presented in
Fig. 3. The quantity of live cells decreased from 30.65% (unfrozen
cells) to 28.26% (frozen with Me2SO alone) after cryopreservation,
with some samples decreasing to 4.96% (frozen with Me2SO and
EG). Staurosporine did not signicantly increase the proportion of
apoptotic cells (66.01% early apoptotic cells and 2.87% late apoptotic
cells in intact samples versus 72.07% and 4.13%, respectively, in
samples treated with staurosporine). It is worth noting that the
early apoptotic fraction in unfrozen cells was greater (66.01%) than
that observed in cryopreserved cells (approximately 20e40%). The
general tendency of cryopreservation-associated apoptosis is
consistent with caspase activation, as described previously. How-
ever, the percentage of late apoptotic cells increased signicantly
from 2.87% in intact cells to 30.72% in cells frozen in Me2SO alone,
even reaching 67.76% in some samples (in cells frozen in Me2SO
with EG).
To clearly show the differences in the proportions of live, early
apoptotic, late apoptotic and dead cells before and after cryopres-
ervation, we combined the ow cytometry data of both staining
methods in Fig. 4 (Quantication of the tests).
3.3. Colorimetric analysis of caspase activity
The activity of caspases-2, -3, -6, -8, -9 in the total protein ex-
tracts of cells before and after cryopreservation is presented in
Fig. 5. Interestingly, intact cells rather than cells with
staurosporine-induced apoptosis had the highest levels of indi-
vidual caspase activity. Freezing-thawing decreased the activity
levels of all tested caspases by approximately 2e3 times regardless
of the cryoprotectants used in comparison to intact unfrozen cells.
3.4. Electron microscopy analysis
Transmission electron microscopy was used to detect the
morphological characteristics of the cells after freezing-thawing in
dissociated sea urchin embryonic cell cultures. We chose the
cryoprotectant combination (Me2SO and EG) that showed the
worst outcome of live cells to have an opportunity to see all con-
sequences of freezing-thawing. In 1-h and 4-h cultures after
thawing, we did not nd any morphological differences between
live cells (Fig. 6A, C). In both cases, the cells were round or looked
similar to mesenchyme-like cells. The cytoplasm of the cells
10 A.V. Boroda et al. / Cryobiology 73 (2016) 7e14

Fig. 1. Cell viability before and after cryopreservation assessed by the trypan blue exclusion test (I) and the MTT assay (II) in a blastula-derived cell culture. Treatment key: intact
unfrozen cells (K); cells frozen without cryoprotectants (K-); cells frozen with Me2SO (A); cells frozen with Me2SO and trehalose (B); and cells frozen with Me2SO and EG (C). The
signicance levels are *P < 0.05; and **P < 0.01.

contained multiple yolk granules. Cells with a necrotic appearance intracellular organelles, a large number of transparent vacuoles and
were also found in both time points tested (Fig. 6AeC). These cells membrane remnants; in addition, many of these cells were without
had a compact nucleus with coarsely aggregated chromatin, few a cytosol. The nuclear membrane retained its integrity in most dead

Fig. 2. Flow cytometry analysis of caspase activation before and after cryopreservation. Dot plots of FAM-VAD-FMK FLICA staining intensity (a marker of early apoptosis) versus
DAPI staining intensity (dead cells with damaged membranes) in cell culture. Intact unfrozen cells (K); unfrozen cells undergoing staurosporine-induced apoptosis (K STS); cells
frozen with Me2SO (A); cells frozen with Me2SO and trehalose (B); and cells frozen with Me2SO and EG (C).
 A.V. Boroda et al. / Cryobiology 73 (2016) 7e14 11

Fig. 3. Flow cytometry analysis of sea urchin embryonic cells before and after cryopreservation. Dot plots of annexin V detection (a marker of late apoptosis) versus DAPI staining
intensity (dead cells with damaged membranes) in cell culture. Intact unfrozen cells (K); unfrozen cells undergoing staurosporine-induced apoptosis (K STS); cells frozen with
Me2SO (A); cells frozen with Me2SO and trehalose (B); and cells frozen with Me2SO and EG (C).

cells (Fig. 6B). Interestingly, few mitochondria and yolk granules in cellular disruption that leads to the destruction of the cytoplasm
dead cells appeared to be externally unchanged. It is possible that while maintaining the integrity of mitochondria.
the major cause of cell death after a freezing-thawing cycle is Apoptotic cells were rare in 1-h cultures after thawing (Fig. 6B).

Fig. 4. The number of live, early apoptotic, late apoptotic and dead cells before and after cryopreservation, as indicated by ow cytometry (combined data for general caspase
activation and annexin V detection) in cell cultures. I e total caspase activation; II e annexin V detection. Treatment key: unfrozen intact cells (K); unfrozen cells undergoing
staurosporine-induced apoptosis (K STS); cells frozen with Me2SO (A); cells frozen with Me2SO and trehalose (B); and cells frozen with Me2SO and EG (C).
12 A.V. Boroda et al. / Cryobiology 73 (2016) 7e14

dead cells in 4-h cultures (Fig. 6D) was similar to that of dead cells
in 1-h cultures (Fig. 6A). The mechanism by which these dead cells
are removed remains unclear. We investigated the uptake of cells
that have died by different mechanisms. Typical necrotic cells with
damaged mitochondria were not detected after either 1-h cultiva-
tion or 4-h cultivation. Using cytomorphological analysis, we could
detect only the later events of apoptosis. We have already found no
apoptotic cells after 4-h cultivation, indicating that apoptosis is rare
in cell cultures after a freezing-thawing cycle. Our ndings suggest
the hypothesis that the major cause of cell death is not freezing-
induced apoptosis or necrosis but actually freezing itself.

4. Discussion

In this study, we analyzed the morphological and functional

Fig. 5. Colorimetric assay of individual caspase activation in cells before and after
cryopreservation. Treatment key: unfrozen intact cells (K); unfrozen cells undergoing
staurosporine-induced apoptosis (K STS); cells frozen with Me2SO (A); cells frozen
with Me2SO and trehalose (B); and cells frozen with Me2SO and EG (C).The data are
shown as the mean SEM.
In contrast to the cells with a necrotic appearance, the cell mem-
branes retained their integrity. The apoptotic cells were shrunken
and had a condensed cytoplasm with externally unchanged mito-
chondria. The nuclei of apoptotic cells contained large star-like
inclusions, which may reect chromosomal condensation. In 4-h
cultures after thawing, live cells formed aggregates. A portion of
the yolk granules in the cytoplasm was lysed, indicating active
consumption of stored yolk (Fig. 6C, D). A small proportion of the
dying cells appeared to be phagocytozed subsequently by adjacent
live cells; the phagosomes contained dead cells and cellular debris,
which was degraded within phagosomes (Fig. 6C, D). Thus, we
found that the engulfment of dying sea urchin cells by surviving
cells resulted in the utilization of dead cells. The morphology of
variations that occur in sea urchin embryonic cells after freezing-
thawing. In addition, cell viability was determined via the trypan
blue exclusion test, MTT assay and DAPI staining. All these data are
consistent with our previously published results on the effective-
ness of different cryoprotectants [30]. The single parameter never
denes an apoptosis in all systems, hence we have used a combi-
nation of four methods for reliable detection of apoptosis in sea
urchin blastula-derived culture after freezing-thawing: FAM-VAD-
FMK FLICA reagent, annexin V staining, ApoTarget Caspase
Colorimetric Protease Assay Sampler Kit (for individual caspases),
and transmission electron microscopy. We suggest that the ob-
tained results of two methods (annexin V staining and ApoTarget
kit, Figs. 3 and 5, consequently) are not reliable and, probably, these
methods are not suitable for detection of apoptosis in sea urchin
blastula cells. Two other methods (the FLICA reagent staining,
detecting general caspase activity, and electron microscopy)
showed a low level of apoptosis (1e3%) in intact unfrozen cells and
could be used for detection of apoptosis in sea urchin cells.
The data obtained using the annexin V assay showed a very high
apoptosis level in unfrozen cells (approximately 66%). This fact may

Fig. 6. Ultrastructure of sea urchin embryonic cells cultivated for 1 h (A, B) and 4 h (C, D) after freezing-thawing in the presence of the penetrating cryoprotectants (Me2SO and EG).
(A) Live and dead cells; (B) Dead and apoptotic cells; (C) Live cells; (D) Live and dead cells. Some live cells have phagosomes that contain cellular debris. Dead cells are marked by
arrows. Arrowheads indicate intact mitochondria. Black asterisks show yolk granules. White asterisks detect lysed yolk granules. ph are phagosomes. n e nucleus.
 A.V. Boroda et al. / Cryobiology 73 (2016) 7e14
be explained by the lower density of the cytoplasmic membranes of
marine invertebrate cells [41] in comparison to mammalian cells. A
lower membrane density can facilitate easier ip-op trans-
location of membrane lipids, including phosphatidyl serine mole-
cules. This process may increase the false detection of non-
apoptotic but annexin V-positive cells. Moreover, a long-term
cultivation (up to 42 days) of sea urchin embryonic cells,
described previously [2], would be impossible due to such a high
number of apoptotic cells detected by annexin V. Thus, annexin V
assay appears to be unsuitable for sea urchin cells. Staurosporine
insignicantly induced apoptosis in unfrozen cells, as indicated by
both the low activation of caspases (Fig. 2, K STS), and the slight
increase in the number of annexin V-positive cells (Fig. 3, K STS).
Cryopreservation reliably increased the number of early and late
apoptotic cells with activated caspases (Figs. 2 and 4). In contrast to
the ow cytometry data, the aolorimetric assay results showed
decreasing caspase activity after cryopreservation (Fig. 5). The
lower level of caspases appears to be connected to the presence of
some inhibiting proteins in the total protein extracts of cells after
freezing (colorimetric assay). This inhibition probably occurred
only in protein extracts but not in whole cells (ow cytometry
analysis).
Live cells have intact membranes that exclude a variety of dyes
that easily penetrate the damaged membranes of non-viable cells.
Changes in phosphatidyl serine asymmetry, which is analyzed by
measuring annexin V-binding to the cell membrane, can be
detected before morphological changes associated with apoptosis
and before a loss of membrane integrity [11]. Additionally, the
morphological phenotype of apoptotic cells depends on the activity
of caspases [17,40]. Nematostella, Hydra, Amphioxus, and sea ur-
chin have representatives of more caspase subtypes than nema-
todes and insects [51]. To date, research has indicated that there are
two main apoptotic pathways: the death receptor-mediated
pathway and the mitochondrial pathway. These two pathways are
linked, and molecules in one pathway can inuence the other
pathway [15].
General caspase inhibitors (for example, the FLICA reagent)
have been used to analyze the activity of caspases in the molluscan
apoptotic pathway with contradictory results [39], suggesting that
different genotoxic stresses can trigger diverse apoptotic molecular
mechanisms. The data on general level of caspase activity are
consistent with our electronic microscopic data and with results on
sea urchin coelomocyte cultures after UV exposure [27]; the
number of apoptotic cells rose with increasing intensity of stress.
An activation of the caspase cascade and pronounced alterations in
membrane asymmetry appeared to be characteristics specic to
cryopreserved sea urchin cells.
Using transmission electron microscopy, which is considered to
be the gold standard for conrming apoptosis, we found three main
cell populations after freezing-thawing. The cells can be identied
as live or dead (probably late apoptotic and necrotic) cells based on
morphological characteristics. The early phase of both modes of cell
death may involve a similar change in mitochondrial membrane
permeability. The mitochondrial morphology in sea urchin cells
after cold stress was not similar to that of typical necrotic cells in
vertebrate animals [20]. Necrosis usually occurs when cells are
exposed to extreme physiological conditions (e.g., hypothermia,
hypoxia, etc.), which may cause damage to the plasma membrane
and mitochondria.
To assess the cell death pathways in sea urchin cultures after
freezing-thawing, the cells should be analyzed within a few hours
or almost immediately after a freeze-thaw cycle, because the cells
and the remaining cell fragments ultimately swell and lyse. As
shown for mammalian tissues, apoptotic cells detached from their
adjacent cells and were often found in tissue vacuoles (apoptotic
13
bodies) [6]. The apoptotic cells were rapidly recognized and
phagocytized by either macrophages or adjacent cells [42]. Our
observations of phagocytic cells after freezing-thawing in 4-h cul-
tures of sea urchin cells conrm the evolutionary conservation of
the phagosomal pathway from worms to humans [29].
No information on the apoptotic pathways and caspase activa-
tion in different species of sea urchins exposed to a cryopreserva-
tion has been published yet. However, there is the data on the
synthesis of the sea urchin heat shock proteins (hsp) under a heat
stress for some sea urchin species. The ability to synthesize hsp in
response to stress has been previously reported to present during
oogenesis and after hatching, at least in sea urchins Paracentrotus
lividus and Sphaerechinus granularis [37]. The rise in temperature
needed to elicit hsp synthesis for Mediterranean species such as
P. lividus or Arbacia lixula [12] may differ for species from other
regions.
Some vertebrate cells (particularly murine endothelial cells)
have been shown to undergo necrosis after repeated freeze-thaw
cycles [24]. Taken together, our data suggest that a balance exists
between the possible mechanisms of cell death (necrosis and
apoptosis), but the predominant mode of cell death after a freezing-
thawing cycle in sea urchin cultures is cellular disruption caused by
cryoinjuries.
Acknowledgments
This study was supported by the Russian Science Foundation
(grant no. 14-14-00035). The work was partially performed at the
Far Eastern electron microscopy center (A.V. Zhirmunsky Insti-
tute of Marine Biology, FEB RAS, Vladivostok, Russia). We thank Dr.
I.V. Kudryavtsev (Institute of Experimental Medicine, RAMS, Saint-
Petersburg) for his help with ow cytometry.
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