Human Reproduction Vol.20, No.12 pp. 3385–3389, 2005
doi:10.1093/humrep/dei236
Advance Access publication July 29, 2005.
The effect of chilling on membrane lipid phase transition
in human oocytes and zygotes
Yehudith Ghetler1,2*, Saar Yavin3*, Ruth Shalgi1 and Amir Arav3,4
1
Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, 2IVF Unit and
Obstetrics and Gynaecology Department, Meir Hospital, Sapir Medical Centre, Kfar Saba 44281 and 3Institute of Animal Science,
Volcani Centre, POB 6, Bet Dagan 50250, Israel
4
To whom correspondence should be addressed. E-mail: [email protected]
*These authors contributed equally to this work
BACKGROUND: Chilling injury occurs when the cell membrane undergoes a transition from the liquid state to the
gel state. Human oocytes and single-cell zygotes are of similar shape and size but the post-thawing survival rate of
oocytes is poorer. We set out to investigate the possible difference in membrane lipid phase transition (LPT) temperature between the two cell types. METHODS: The LPT temperature was measured with a Fourier Transform Infrared analyser, which detects the change in the vibration frequency of the CH2 bond stretches of the membrane lipid
molecules during temperature change. The LPT temperatures of unfertilized human oocytes, in vitro-matured
oocytes, and immature germinal vesicle (GV) stage oocytes were compared with that of abnormally fertilized human
zygotes. RESULTS: The LPT temperatures of zygotes and of mature and immature GV oocytes differ significantly
from each other (10.0 ± 1.2, 16.9 ± 0.9 and 24.4 ± 1.6°C respectively; P < 0.05). CONCLUSIONS: Zygotes show a
higher resistance to chilling injury compared to oocytes at different developmental stages; this might explain the relatively poor survival rates of cryopreserved human oocytes and indicates the necessity to adjust the cryopreservation
protocols in order to minimize cryoinjury.
Key words: chilling injury/cryopreservation/human oocyte/human zygote/lipid phase transition
Introduction
Cryopreservation of human sperm cells and embryos has been
successfully applied for several years and contributes to the
increasing success of assisted reproduction technologies.
Unfortunately, despite years of research, cryopreservation of
human oocytes still yields unsatisfactory results and is considered experimental. The ability to cryopreserve oocytes would
be beneficial in overcoming moral, ethical and legal issues that
arise from embryo cryopreservation. It might also offer the
prospect of preserving the fertility of women scheduled for
chemo or radiation therapy.
During the process of cryopreservation, cells are exposed to
mechanical, thermal and chemical stresses. Oocyte freezing is
accompanied by various types of injury, including damage to
the meiotic spindle and to the microfilaments (Pickering et al.,
1990; Baka et al., 1995; Park et al., 2000; Boiso et al., 2002;
Mullen et al., 2004; Bianchi et al., 2005) and zona pellucida
hardening. Due to injury inflicted during cryopreservation, survival rate is low as reviewed by Van der Elst (2003).
There are two main steps in the process of cryopreservation:
(i) chilling, i.e. lowering the temperature from the physiological temperature to the freezing point; and (ii) freezing,
further reducing the temperature to the storage temperature
(liquid nitrogen at −196°C). Chilling injury is defined as perman-
ent damage that occurs upon cooling to low but not freezing
temperatures. Chilling injury can modify the structure of membranes and, therefore, their integrity (Arav et al., 1996; Zeron
et al., 1999). Phospholipids are major components of cellular
membranes; the length of the fatty acid acyl chain, the number
and positions of the double bonds within the phospholipids
strongly influence the membrane properties (Stubbs and Smith,
1984; Arav et al., 2000a). High concentrations of cholesterol
and polyunsaturated fatty acids (PUFA) (Quinn, 1985; White,
1993) render the membrane more fluid at low temperatures and
decrease its sensitivity to chilling injury. The membrane lipid
composition is used as an indicator of changes in membrane
fluidity, which depends on the cholesterol level, the phospholipid
composition, the degree of unsaturation and the protein content
(Giraud et al., 2000). As cooling results in removal of thermal
energy, the molecular motion in the membrane lipid bilayer
decreases, allowing interaction between adjacent lipid molecules,
thus changing the fluidity and hence the functioning of the
membrane. The temperature at which the membrane phospholipids transform from a liquid crystalline phase (Lα ), characterized
by high rotational and lateral mobility of the lipids and therefore by a high degree of fluidity, to a crystal gel phase (Lβ ) in
which the mobility of the lipids is restricted, is called the ‘transition temperature’. The centre of the lipid phase transition (LPT)
© The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Y.Ghetler et al.
curve—the point of transition from Lα to Lβ —is designated Tm
and is determined by statistical analysis (Crowe et al., 1989).
The susceptibility of oocytes to chilling injury had been
reported elsewhere (Didion et al., 1990; Parks and Ruffing,
1992; Arav et al., 1993; Otoi et al., 1995), and it had been
suggested that chilling injury also affects the oocyte microtubules (Albertini and Eppig, 1995), the cytoskeletal organization (Overstrom et al., 1990) and the zona pellucida (Vajta
et al., 1998). LPT was associated with direct chilling injury in
pig sperm membranes (Drobnis et al., 1993) and in bovine
(Arav et al., 1996), sheep (Zeron et al., 2002a) and zebra fish
(Pearl and Arav, 2000) oocytes.
It was demonstrated that a low LPT was associated with
chilling resistance (Arav et al., 1993), therefore we decided to
evaluate the LPT of relatively highly resistant single-cell
human zygotes that had been successfully cryopreserved in
IVF programmes. The relatively poor survival rate of frozen–
thawed oocytes compared with that of single-cell 2-pronuclei
(PN) zygotes that are similar in shape and size, led us to investigate whether there is a difference in LPT temperature between
human oocytes and zygotes.
Materials and methods
Biological material
Oocytes included in this study were donated by consenting patients
undergoing IVF/ICSI procedures and represent material that is otherwise discarded. The study was approved by the Local Review
Board.
The ovulation induction protocol (Shulman et al., 1996) and the
ICSI procedure (Van Steirteghem et al., 1993) were as described previously. Sperm-injected oocytes that had failed to be fertilized, or
immature oocytes not suitable for ICSI were used (Ghetler et al.,
1998). Metaphase I oocytes were matured in vitro for 24 h in Cook
Culture System (Cook, Brisbane, Australia) until they reached the
metaphase II (MII) stage. They were analysed, together with the
unfertilized oocytes, 24 h after retrieval, whereas the germinal vesicle
(GV) oocytes were analysed on the retrieval day. The zygotes used in
the present study were abnormally fertilized oocytes that exhibited
one or three pronuclei 16–20 h after ICSI. According to the ICSI procedure, all oocytes were free of cumulus cells after treatment with
hyaluronidase (Cook).
Evaluation of membrane phase transition
Each analysed oocyte or zygote was individually placed between
two quartz slides separated by a thin layer of silicone grease and
compressed until the specimen was flattened to a thin layer of
cytoplasm. Care was taken to keep the membrane intact. The
quartz slides containing the sample were loaded into a temperature
controller and placed on the microscope stage. The temperature
was regulated by a microprocessor feedback system that measured
the sample temperature with a thermocouple attached to the glass.
The temperature was adjusted to the desired level and allowed to
equilibrate before the specimen was scanned. Scanning was performed at the range of 28 to 2, at 2°C intervals. A total of 66 scans
at a resolution of 4 cm−1 were collected for each sample at each
temperature. The evaluation was conducted with a Bruker Equinox
55 Fourier Transform Infrared (FTIR) analyser connected to a
Bruker A590 FTIR microscope equipped with a liquid nitrogencooled mercury cadmium telluride detector (Arav et al., 1996,
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2000a; Zeron et al., 2002a). The Tm values were calculated from
the frequency–temperature plots by statistical analysis as described
by Crowe et al. (1989).
Statistics
The significance of differences between experimental groups was
determined by one-way analysis of variance; P < 0.05 was considered
significant.
Results
FTIR spectroscopy measures the vibrational frequency of
methylene (CH2) groups, most of which are in the hydrocarbon
chain of lipid molecules. The membrane phase transition was
detected by monitoring the change in the vibration frequency
of the CH2 bond stretches as the temperature changed. The
peak showed a shift in wave number when the lipid underwent
a phase transition. Figure 1 shows a schematic illustration of
the wave shift following the change in CH2 frequency of the
membrane lipids (change from liquid phase Lα to gel phase
Lβ), with change in temperature.
The evaluated value of Tm differed among the groups examined. As demonstrated by Crowe et al. (1999), the changes in
FTIR frequency in the isolated membranes were in excellent
agreement with the data they obtained with differential scanning calorimetry, indicating that our FTIR results are reliable.
Furthermore, when the plasma membrane and the internal
membranes were analysed separately, Crowe et al. (1999)
recorded similar transitions.
Oocytes
Immature oocytes
The Tm of GV oocytes (n = 6) was 24.4 ± 1.6°C (mean ± SE)
(Figure 2a).
Liquid phase
Lα
2853cm-1
Gel phase
Lβ
2851cm-1
WaveNumber
Chilling
37˚C
0˚C
Figure 1. Schematic illustration of the wave shift following the
change in CH2 frequency of the membrane lipids (change from liquid
phase Lα to gel phase Lβ) with change in temperature.
Lipid phase transition in human oocytes and zygotes
significant difference (P > 0.05) between their Tm values (15.7 ±
3.2 and 18.9 ± 1.3°C respectively), therefore they were pooled
into one group of mature oocytes.
The phase transition temperature of this group of mature
oocytes (n = 20) was 16.86 ± 0.86°C (Figure 2b).
Zygotes
The LPT of single-cell zygotes (n = 10) occurred at a significantly
lower temperature than that of oocytes, i.e. at 10.04 ± 1.22°C
(Figure 2c).
Statistical evaluation of the measured data showed significant differences between all the study groups (Table I).
Discussion
During cryopreservation, cells are exposed to low, nonphysiological temperatures and thus become vulnerable to
chilling injury because of the thermotropic LPT. The lipid
composition of the membrane strongly influences its properties, including its resistance to thermal stress (Arav et al.,
2000a; Zeron et al., 2001, 2002b).
The chilling injury is temperature dependent; it is caused by
changes in the membrane properties and integrity and is
responsible for the extensive cell damage that occurs during the
process of cryopreservation. We examined, for the first time,
the LPT of human mature oocytes and human zygotes, and our
findings matched the preliminary results reported by ReuvenofPass et al. (2003) regarding the Tm of GV oocytes. Since the
availability of MII human oocytes donated for research is scarce
and sporadic, the ‘failed to fertilize’ and ‘in vitro-matured’
oocytes were chosen as the closest alternative for mature
oocytes. These oocytes were incubated at the same culture conditions and culture medium as the tested human zygotes.
Human zygotes, used in the current research, presented an
abnormal number of PN after ICSI treatment. These fertilized
oocytes failed to either form the male PN (1PN), or to extrude
the 2nd polar body (3PN). These zygotes can, however, represent a normal zygote (2PN) since the developmental abnormality
is not due to inadequate cortical granule exocytosis (Ghetler
et al., 1998) and their membranes, similar to normally fertilized
oocytes, were exposed to cortical granule exudates.
Since the oocytes and zygotes were free of surrounding
cumulus cells and were tightly compressed between the two
quartz slides, the analysed section contained mostly the analysed cell membrane and a minimal amount of cytoplasm
(Arav et al., 1996; Crowe et al., 1999).
Figure 2. The lipid phase transition (LPT) as measured by Fourier
Transform Infrared analyser. (a) Immature germinal vesicle (GV)
oocyte; (b) unfertilized mature oocyte (UF); (c) 3-pronuclei (3PN)
zygote. The centre of the LPT curve, from liquid crystalline to gel
phase, is designated Tm, which indicates the temperature at which the
LPT occurred, and its values were calculated from the frequency–
temperature plots according to Crowe et al. (1989).
Mature oocytes
When the unfertilized oocytes (n = 13) and the in vitro-matured
MII oocytes (n = 7) were analysed separately we found no
Table I. Lipid phase transition temperatures (Tm) of human immature
germinal vesicle (GV) oocytes, mature oocytes and zygotes of the study
groups
Study group
n
Tm (°C)*
Immature (GV) oocytes
Mature oocytes
Zygotes
6
20
10
24.4 ± 1.6a
16.9 ± 0.9b
10.0 ± 1.2c
*Values represent (mean ± SE).
a,b,c
Different superscripts within the column denote significant differences
(P < 0.05).
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Y.Ghetler et al.
Our results clearly demonstrate a significant difference
between the phase transition temperatures in zygotes and oocytes
(at different developmental stages). Cryopreservation of
zygotes is widely used as a routine procedure in IVF treatment,
and it yields satisfactory results (Al-Hasani et al., 1996). The
Tm of human zygotes is low, indicating a relatively good resistance of the membrane to chilling injury, nevertheless the Tm of
human oocytes is significantly higher than that of zygotes,
which suggests a greater sensitivity to chilling. In the present
study the most sensitive group was the GV oocytes. Arav et al.
(1996) also found that the membrane of GV-stage bovine
oocytes was much more susceptible to cryoinjury than that of
mature oocytes. It became more tolerant to chilling after electrofusion with large fragments from membranes of mature
oocytes, indicating that alteration of the membrane composition could change its thermal behaviour and consequently its
chilling resistance. The changes in the thermal behaviour of
oocyte membranes at different developmental stages might
suggest that there are changes in the phospholipid composition
and the cholesterol concentration.
The slow-freezing, rapid-thawing protocol, with 1,2-propanediol cryoprotectant, that is routinely used for cryopreservation
of human zygotes, involves exposure to cryoprotectants at
room temperature, prior to the freezing process, which starts at
20°C. This protocol is suitable for zygote cryopreservation,
since the phase transition temperature of zygotes is far below
20°C, and the protocol is indeed successfully employed in IVF
treatments. In contrast, the Tm of mature oocytes is very close
to room temperature, at which a part of the procedure takes
place, and that of GV oocytes is above this temperature, which
renders their membranes prone to chilling injury. The high
LPT temperature may explain, at least in part, the poor survival
rate of human female gametes under cryopreservation. Lim
et al. (1991) demonstrated that bovine single-cell zygotes have
higher post-thaw survival rates and better developmental
capacities than those of MII oocytes. Furthermore, the survival,
fertilization and developmental rates of MII oocytes after slow
freezing were better than those of GV oocytes. Also for mouse
oocytes, higher cryosensitivity was found in immature than in
mature oocytes (Schroeder et al., 1990; Candy et al., 1994).
We have demonstrated in our present study that the Tm of
human zygotes is low. Since biological processes are very slow
at low temperatures the kinetics of injury are slow as well,
which enables the critical Tm to be passed with relatively minor
damage. The high Tm values of MII and GV oocytes might be
the underlying cause of the extensive cryoinjury to membranes.
Alternative methods for cryopreservation of human oocytes
rely on passing through the transition phase temperature rapidly,
and so partially avoiding the induction of membrane cryoinjury
by the LPT. Among these methods are the vitrification method
(Hong et al., 1999; Arav et al., 2000b; Cha et al., 2000; Yoon
et al., 2000; Jelinkova et al., 2002), and the rapid vitrification
method with accelerated cooling. Accelerated cooling is
achieved by using open pooled straws (Vajta et al., 1998),
electron microscope grids (Martino et al., 1996; Park et al.,
2000), minimal drop size (Arav and Zeron, 1997; Yavin and
Arav, 2001) or supercooled liquid nitrogen (Arav et al., 2002).
Changing the handling temperature to 37°C was reported to
3388
improve the outcome of cryopreservation in mouse GV
oocytes (Isachenko and Nayudu, 1999).
Artificial alteration of the lipid membrane composition, e.g. by
incorporation of liposomes into the membrane (Zeron et al.,
2002b), can create chilling-resistant oocytes. This method can be
used to improve the outcome of oocyte cryopreservation and also
to contribute to the development of better cryobanking programmes (Ledda et al., 2001). Enriching ewes’ diets with polyunsaturated fatty acid (PUFA) (Zeron et al., 2002a) demonstrated
changes in the fatty acid profile of the oocytes. These oocytes
exhibited changes in the physical properties of their membranes
and a decrease in the lipid transition temperature from 15 to 4°C,
which indicates enhanced resistance to chilling injury.
Membranes contain a variety of phospholipids, all of which
affect the Tm. The fact that membranes of zygotes and of
oocytes at different developmental stages have different Tm
values, suggests a meaningful difference between their compositions, the Tm being strongly related to the PUFA content
(Zeron et al., 2002a). Matorras et al. (1998) demonstrated that
mature human oocytes contain mainly (79.22%) saturated fatty
acids and only 6.5% PUFA. Certain changes in membrane
composition might occur during oocyte maturation from the
GV to the MII stage, and still greater changes after fertilization. We can speculate that one of the reasons for this change
in zygotes might be the contribution of exocytosis of cortical
granules that occurs at fertilization. More information about
the composition of the membranes of human zygotes and of
human oocytes at different developmental stages is needed.
Acknowledgements
We thank Tal Rom and Ayelet Itzkovitz, IVF Unit, Sapir Medical
Centre, Kfar Saba, for their technical assistance. This work is in
partial fulfilment of the requirements for the PhD degree of Y.Ghetler,
Sackler Faculty of Medicine, Tel Aviv University.
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Submitted on April 14, 2005; resubmitted on June 28, 2005; accepted on July
4, 2005
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