The oocyte spindle is preserved by 1,2-propanediol

during slow freezing

Ching-Chien Chang, Ph.D.,a Li-Ying Sung, Ph.D.,b,c Chih-Jen Lin, M.Sc.,b,c Hilton I. Kort, M.D.,a
Xiangzhong Yang, Ph.D.,b,c X. Cindy Tian, Ph.D.,b,c and Zsolt Peter Nagy, M.D., Ph.D.a
a
Reproductive Biology Associates, Atlanta, Georgia; b Center for Regenerative Biology, University of Connecticut, Storrs,
Connecticut; and c Department of Animal Science, University of Connecticut, Storrs, Connecticut

Objective: To investigate the specific changes in oocyte spindle subjected to severe challenges of low temperature,
as well as to examine the effect of cryoprotectants in preserving oocyte spindle during cryopreservation.
Design: In vitro experimental study.
Setting: Academic research laboratory.
Animal(s): B6D2F1 (C57BL/6 X DBA/2) mice.
Intervention(s): Mouse oocytes were cryopreserved using a slow freezing method in a sodium-depleted medium
with 1.5 mol/l 1,2-propanediol (PROH) and 0.3 M sucrose. To examine the spindle, oocytes were fixed before, dur-
ing, and after cryopreservation, and oocytes were analyzed by immunocytochemistry and confocal microscopy.
Result(s): The MII spindle was preserved during the slow freezing, because the cryoprotectant PROH was found
to support the organization of MII spindle in resisting the subzero temperature. In contrast, the MII spindle was
disassembled gradually during the thawing process with or without PROH. Most of the oocytes were able to re-
cover the MII spindle after thawing, but a portion of thawed oocytes could not sustain the meiotic spindle because
of parthenogenetic activation.
Conclusion(s): 1,2-Propanediol can support the organization of MII spindle to defy the subphysiologic tempera-
ture; however, the PROH cannot sustain oocyte spindle structure after the subsequent thawing process. (Fertil
Steril 2010;93:1430–9. 2010 by American Society for Reproductive Medicine.)
Key Words: Oocyte cryopreservation, meiotic spindle, 1, 2-propanediol

The meiotic spindle is composed of microtubules consisting
of a- and b-tubulin dimmers, which continuously change
their state in equilibrium between a free and polymerized
state. Temperature decrease can compel the equilibrium to-
ward depolymerization. Furthermore, the meiotic spindle is
extremely susceptible to freezing processes in oocyte cryo-
preservation because spindle integrity can be affected not
only by ice crystal formation, but also by the dramatic in-
crease of salt concentration that occurs during cellular dehy-
dration (‘‘solution effect’’) (1). Low temperature has been
reported to potentially result in significant meiotic spindle
disruption (2). Disassembly of the meiotic spindle is highly
associated with dispersion of metaphase chromosomes and
subsequent chromosomal anomalies (3–10). In contrast, sev-
eral studies also have indicated that the meiotic spindle can
endure freezing–thawing without chromosome dispersal in
the oocyte (11) or no increase of aneuploidies in the resulting
embryos (12, 13).
Received October 24, 2008; revised December 10, 2008; accepted Janu-
ary 9, 2009; published online March 26, 2009.
C.-C.C. had nothing to disclose. L.-Y.S. had nothing to disclose. C.-J.L.
has nothing to disclose. H.I.K. has nothing to disclose. X.Y. had nothing
to disclose. X.C.T. had nothing to disclose. Z.P. N. has nothing to dis-
close.
Presented in part at the annual meeting of American Society for Reproduc-
tive Medicine, Washington, DC, October13–17, 2007.
Reprint requests: Zsolt Peter Nagy, M.D., Ph.D., Reproductive Biology As-
sociates, 1150 Lake Hearn Drive, Suite 600, Atlanta, GA 30342 (FAX:
404-256-1912; E-mail: peter.nagy@rba-online.com).
Among the permeating cryoprotectants, propylene glycol
(1,2 propanediol; PROH) is the most commonly applied in
slow freezing cryopreservation recently. 1,2-Propanediol
can readily permeate cell membrane and establish hydrogen
bonds with water molecules to prevent ice crystallization.
During freezing, as the free water solidifies into ice, the re-
maining solution will contain progressively higher concen-
tration of electrolytes. The use of PROH can protect the
cell from stringent osmotic stress, in that the total concentra-
tion of both PROH and electrolytes are maintained to be con-
stant during the freezing process. In the freezing state, the
higher the concentration of PROH causes a lower concentra-
tion of electrolytes, thus confining the toxicity of osmotic
stress. Interestingly, it has been reported that low doses of
PROH (<1.0 M) can cause disassembly of the meiotic spin-
dle, whereas the structure of the meiotic spindle can be stabi-
lized by higher doses of PROH (1.5–2.0 M) (14). However, it
is reported that PROH can adversely affect oocyte physiology
by inducing intracellular calcium in metaphase II oocytes,
which may cause oocyte activation and render oocytes non-
fertilizable (15–17).
During oocyte slow freezing, intracellular ice formation,
cryoprotectant toxicity, osmotic stress, nonphysiologic tem-
peratures, and pH instability are factors believed to affect
the structure of the meiotic spindle. With more and more
human pregnancies derived from oocyte slow freezing
methods using PROH as a cryoprotectant (18–22), it is ex-
tremely important to know whether the meitotic spindle can

1430 Fertility and Sterility Vol. 93, No. 5, March 15, 2010 0015-0282/10/$36.00
Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2009.01.106
be preserved or whether the capability of meiotic spindle re-
assembly can be restored following oocyte cryopreservation
by slow freezing. The aim of this study is to investigate the
meiotic spindle dynamics before and after slow freezing pro-
cedures using PROH as the cryoprotectant.
MATERIALS AND METHODS
Chemicals and Culture Media
Unless otherwise indicated, all chemicals purchased were
from Sigma Chemical Co. (St. Louis, MO). All media were
prepared fresh and filter sterilized through a 0.22-mm filter
(Acrodisc; Pall Gelman Laboratory, Ann Arbor, MI).
Animals and Recovery of Metaphase II Oocytes
The B6D2F1 (C57BL/6 X DBA/2) mice used were from
Charles River Laboratories (Wilmington, MA). All animal
treatments were approved by the Institutional Animal Care
and Use Committee of the University of Connecticut, Storrs,
CT.
We collected in vivo matured MII-stage oocytes from
B6D2F1 female mice subjected to the following hormone
priming protocol: superovulation was induced with 10 IU
of eCG followed 48 hours later with 10 IU of human cho-
rionic gonadotrophin (hCG). Oocytes at MII stage were har-
vested 14 hours after the hCG treatment and freed of cumulus
cells by brief exposure to 100 IU/mL of hyaluronidase at
37 C and gentle pipetting.
Cryopreservation Solutions
The sodium (Na)-depleted freezing media (18, 23, 24) were
employed for oocyte cryopreservation in this study. The basal
medium of cryopreservation solution was prepared using Na-
depleted system: choline chloride 137 mmol/L, KCl 2.6
mmol/L, NaH2PO4 8.0 mmol/L, and KH2PO4 1.4 mmol/L.
All salts were dissolved in the distilled water, and the pH
was adjusted to 7.4 by 5.0 mmol/L KOH. The freezing so-
lution consisted of Na-depleted basal medium supplemented
with 20% fetal calf serum (FCS; Hyclone, Logan, UT), 1.5
mol/L PROH, and 0.3 mol/L sucrose. The thawing procedure
was the step-wise dilution method. The first thawing solution
consisted of Na-depleted basal medium supplemented with
20% FCS (Hyclone) and 0.5 mol/L sucrose. The second
thawing solution was composed of Na-depleted basal me-
dium supplemented with 20% FCS (Hyclone) and 0.2 mol/
L sucrose. Finally, the washing solution was composed of
Na-depleted basal medium supplemented with 20% FCS
(Hyclone) without addition of sucrose.
Freezing Procedure
Before freezing, the cumulus cells were removed and the oo-
cytes were transferred into 1.0-mL freezing solution for 20
minutes equilibration at room temperature (RT) (22–25 C),
then loaded into 0.25-mL plastic straws (Paillette s Crystal
133mm; Cryo Bio System, France). The freezing process
Fertility and Sterility
was performed with a controlled rate freezer CL8000 (Cryo-
logic, Victoria, Australia). Temperature was lowered gradu-
ally from 18.1 C to 6.9 C at a rate of 2 C/min. Manual
seeding was induced during the 10 min holding ramp at
6.9 C, and the temperature was controlled to drop to
38 C at a rate of 0.3 C/min. The straws were then directly
plunged into liquid nitrogen and stored for later use.
Thawing Procedure
The frozen straws with cryopreserved oocytes were held in
air for 30 seconds and immersed into the 32 C to 35 C water
bath until all ice crystals had disappeared. The thawed con-
tents of straws were expelled into an empty dish. Oocytes
were then picked up and transferred into 1.0 mL of the first
thawing solution (0.5 M sucrose) for 10 minutes at RT. The
oocytes were subsequently transferred into the second thaw-
ing solution (0.2 M sucrose) for 10 minutes at RT. Then, oo-
cytes were washed in the washing solution (0 M sucrose) for
10 minutes at RT. Finally, the oocytes were incubated in
KSOM þ AA medium (Specialty Media, Phillipsburg, NJ)
before oocyte fixation.
Timing of Oocyte Fixation
To thoroughly investigate the meiotic spindle change during
freezing and thawing, oocytes were fixed at [1] 0 hours before
exposure to the freezing solution, [2] 20 minutes exposure to
the freezing solution, [3] 1 minutes after the straws were
immersed into the 32 C to 35 C water bath and expelled oo-
cytes into fixatives, [4] 10 minutes after the first thawing so-
lution (0.5 M sucrose), [5] 10 minutes after the second
thawing solution (0.2 M sucrose), [6] 10 minutes after wash-
ing solution (0 M sucrose), [7] 30 minutes incubation after
thawing procedures, [8] 1 hour incubation after thawing pro-
cedures, [9] 2 hours incubation after thawing procedures, and
[10] 4 hours incubation after thawing procedures.
Immunohistochemistry and Laser-Scanning Confocal
Microscopy
For examination of microtubules, oocytes were fixed in a mi-
crotubule stabilizing buffer containing 2% formaldehyde,
0.5% Triton X-100, 1 mM taxol, 10 units/mL aprotinin, and
50% deuterium oxide at 37 C for at least 30 minutes. They
were then washed in washing buffer (PBS containing 3 mM
NaN3, 0.01% Triton X-100, 0.2% nonfat dry milk, 2% normal
goat serum, 0.1 M glycine, and 2% bovine serum albumin)
three times and left in the washing buffer overnight at 4 C
for blocking and permeabilization (25). Oocytes were then
double stained to visualize microtubules and DNA. Briefly,
samples were incubated in mouse anti-a-tubulin antibody
(1:200) for 4 hours at 37 C or overnight at 4 C. After three
washes in washing buffer, the oocytes were incubated in fluo-
rescein isothiocyanate (FITC)-conjugated goat antimouse
immunoglobulin G (1:200) for 1 hour at 37 C. Finally, the
oocytes were washed, stained for DNA with 7.5 mM propi-
dium iodide, mounted in PBS containing 50% glycerol as
1431
an antifading reagent and 25 mg/mL NaN3, and examined maintained without detectable lessening or damage (Fig.
with a laser-scanning confocal microscope (Leica TCS 1D, Table 1; 31/31). We observed a high degree of resem-
SP2, Exton, PA). blance of MII spindle when comparing before and after freez-
ing (Fig. 1).

RESULTS
Influence of the Freezing Process on the MII Spindle
We investigated the organization of the MII spindle during
the course of oocyte freezing process. To do this, we fixed oo-
cytes before undergoing the freezing program when oocytes
had been treated with cryopreservation solution for 20 min-
utes at RT. We also fixed the cryopreserved oocytes immedi-
ately after the ice crystal was melted. The organization of the
MII spindle structure was analyzed with respect to microtu-
bule apparatus and chromosome configuration before and af-
ter freezing. As shown in Figure 1, of 35 treated oocytes, all
of the MII spindles were well maintained in a barrel-shaped
structure before freezing (Fig. 1A, Table 1; 35/35). In the oo-
cytes immediately after thawing, the MII spindle was still
Effect of Cryoprotectants on Preserving MII Spindle
Because the nature of microtubules is temperature sensitive,
we next sought to investigate the effect of cryoprotectants
during subphysiologic temperature. Two kinds of cryoprotec-
tants were supplemented in the cryopreservation solution.
One is 1.5 M PROH as a permeating cryoprotectant, and
the other is 0.3 M sucrose as a nonpermeating cryoprotectant.
Therefore, four different combinations of cryoprotectants
were used to treat oocytes at 4 C for 30 minutes, and the
MII spindle configuration was compared (Fig. 2). In the con-
trol group (without PROH and sucrose), none of the oocytes
(0%; 0/27) had bipolar spindles (Fig. 2A), but only a scarce
residual microtubule structure was found adjacent to the

FIGURE 1
Influence of the freezing process on the MII spindle. Confocal images of a-tubulin (A,D), chromatin (B,E), and
merge of chromatin and a-tubulin (C,F) of representative oocytes treated with freezing solution for 20 minutes at
RT (n ¼ 35; A–C) and oocytes that have been fixed immediately upon thawing (without removal of
cryoprotectants) (n ¼ 31; D–F). Oocytes have been frozen and thawed display stabilized MII spindle with
chromosomes aligned on the metaphase plate (D–F) resembling oocytes before the freezing process (A–C).

Chang. Oocyte spindle in slow freezing process. Fertil Steril 2010.

1432 Chang et al. Oocyte spindle in slow freezing process Vol. 93, No. 5, March 15, 2010
 TABLE 1
The dynamics of the meiotic spindle during oocyte cryopreservation.
Completely Anaphase/
Bipolar Disorganized disappeared telophase
spindle spindle spindle spindle
Fresh control MII oocytes 100% (33/33) 0 (0/33) 0 (0/33) 0 (0/33)
Freezing Sol.-Equilibration 20 100% (35/35) 0 (0/35) 0 (0/35) 0 (0/35)
minutes, room temperature
Fixed immediately 100% (31/31) 0 (0/31) 0 (0/31) 0 (0/31)
upon thawinga
Thawing (0.5 M Sucrose) 10 100% (22/22) 0 (0/22) 0 (0/22) 0 (0/22)
minutes, room temperature
Thawing (0.2 M Sucrose) 10 0 (0/19) 100% (19/19) 0 (0/19) 0 (0/19)
minutes, room temperature
Washing (0 M Sucrose) 10 0 (0/23) 100% (23/23) 0 (0/23) 0 (0/23)
minutes, room temperture
30 minutes (37 C) postthawing 0 (0/29) 69.0% (20/29) 31% (9/29) 0 (0/29)
1 hour (37 C) postthawing 76.7% (23/30) 0 (0/30) 16.6% (5/30) 6.7% (2/30)
2 hours (37 C) postthawing 81.2% (26/32) 0 (0/32) 6.3% (2/32) 12.5% (4/32)
4 hours (37 C) postthawing 85.0% (34/40) 0 (0/40) 10.0% (4/40) 5.0 % (2/40)
a
The frozen straw with cryopreserved oocytes was held in air for 30 seconds and immersed into water bath at 32 C until ice
has fully melted (approximately 15 seconds), and all the liquid contents with oocytes was directly transferred into the
fixative upon the ice crystal has been melted.
Chang. Oocyte spindle in slow freezing process. Fertil Steril 2010.

metaphase chromosomes (Fig. 2C). In the group with only
sucrose added, we observed largely diminished microtubule
apparatus with two distinctive spindle poles (100%; 33/33),
and the MII spindle could not be fully stabilized by the non-
permeating cryoprotectant (Fig. 2F). In contrast, if the per-
meating cryoprotectant PROH was present in the solution,
the organization of bipolar spindles was not affected by the
subphysiologic temperature with (Fig. 2J–L; 35/35) or with-
out (Fig. 2G–I; 37/37) sucrose presence.
MII Spindle Organization during the Thawing Process
We examined the MII spindle at 10 minutes in 0.5 M sucrose
thawing solution, and another 10 minutes in 0.2 M sucrose
thawing solution, and, finally, 10 minutes for washing in
HEPES buffered solution without any sucrose (Fig. 3, Table
1). We found that the MII spindle started to disorganize
when thawed oocytes were transferred into 0.2 M sucrose
thawing solution (Fig. 3D–F, Table 1; 19/19). We also found
that the microtubule was detached from the metaphase plate
after serial dilutions of the thawing process (Fig. 3G–I, Table
1; 23/23).
Because the cryoprotectants in cryopreservation solution
can preserve the MII spindle during the freezing process,
we performed control experiments to look into whether
PROH, which has been used in the freezing process, can
also preserve the MII spindle during the thawing process.
Hence, in place of the serial dilution thawing protocol, we
directly subjected the frozen oocytes to the complete
cryopreservation solution with 1.5 M PROH and 0.3 M su-
crose for 30 minutes at RT. We found that the MII spindle still
disorganized when the oocyte was thawed in the solution with
cryoprotectants (Fig. 3J–L; 34/34).
After thawing, the thawed oocytes were further examined
at 30 minutes incubation of 37 C. We observed the MII spin-
dle was totally disassembled in a portion of oocytes (9/29;
Fig. 4A–C, Table 1), and the others had scarce microtubule
apparatus attached to the metaphase chromosomes (20/29;
Fig. 4D–F, Table 1).
Recovery of the MII Spindle after the Thawing Process
Frozen and thawed oocytes were fixed for examining the be-
havior of the MII spindle 1 hour, 2 hours, and 4 hours post-
thawing to more closely follow the recovery of the MII
spindle. After 1 hour incubation, we observed three situa-
tions: [1] a recovered bipolar spindle was found with com-
plete chromosome alignment (Fig. 5A, Table 1; 23/30). [2]
No spindle was reassembled with condensed chromosomes
(Fig. 5B; 5/30). [3] A spindle was undergoing anaphase/telo-
phase with two sets of divided chromosomes (Fig. 5C; 2/30).
By 2 hours, 81.2% of oocytes had recovered their MII spindle
(Fig. 5A, Table 1; 26/32), 6.3% of oocytes had no spindle re-
assembled with condensed chromosomes (Fig. 5B; 2/32), and

Fertility and Sterility 1433

 FIGURE 2
The effect of cryoprotectants on preserving the MII spindle during low-temperature treatment (4 C for 30 minutes). Confocal
images of a-tubulin (A,D,G,J), chromatin (B,E,H,K), and merge of chromatin and a-tubulin (C,F,I,L) of representative oocytes
treated with choline-based freezing solution without PROH and sucrose (A–C), choline-based freezing solution with 0.3 M
sucrose but without PROH (D–F), choline-based freezing solution with 1.5 M PROH but without sucrose (G–I), and complete
freezing solution with both 1.5 M PROH and 0.3 M sucrose (J–L). In the absence of PROH and sucrose, oocytes display
disorganized MII spindle after low-temperature treatment (A–C). The 1.5 M PROH stabilized MII spindle during low-
temperature treatment (G–I and J–L). However, sucrose alone was not able to prevent the spindle disassembly caused by low-
temperature treatment, and those oocytes revealed diminished microtubule apparatus with two distinctive spindle poles (D–F).

Chang. Oocyte spindle in slow freezing process. Fertil Steril 2010.

1434 Chang et al. Oocyte spindle in slow freezing process Vol. 93, No. 5, March 15, 2010
 FIGURE 3
MII spindle organization during the thawing processes. Confocal images of a-tubulin (A,D,G,J), chromatin
(B,E,H,K), and merge of chromatin and a-tubulin (C,F,I,L) of cryopreserved oocytes first thawing in 0.5 M sucrose
solution at RT for 10 minutes (A–C), secondly transferred into 0.2 M sucrose solution at RT for another 10 minutes
(D–F), and finally washed by the HEPES-buffered solution for 10 minutes at RT (G–I). The cryopreserved oocytes
were directly thawed into freezing solution with 1.5 M PROH and 0.3 M sucrose for 30 minutes at RT (without
removal of any cryoprotectants) (J–L). The MII spindle started to disassemble following thawing, and it is
noteworthy that the microtubule apparatus was detached from the metaphase plate when the cryoprotectants
have been completely removed (I). On the other hand, the disorganized MII spindle was not avoidable when the
cryopreserved oocytes were directly thawed into freezing solution with complete cryoprotectants (J–L).

Chang. Oocyte spindle in slow freezing process. Fertil Steril 2010.

Fertility and Sterility 1435

 FIGURE 4
MII spindle underwent disassembling and reassembling during the first hour after thawing. Confocal images of a-
tubulin (A,D), chromatin (B,E), and merge of chromatin and a-tubulin (C,F) of cryopreserved oocytes fixed after
the complete thawing processes (after removal of cryoprotectants) and subsequent incubation for 30 minutes at
37 C. Approximately, one-third of the thawed oocytes (9/29) did not have detectable microtubule structure
surrounding chromosomes (A–C), and the rest of oocytes (20/29) revealed that metaphase chromosomes were
attached with scarce microtubule structure (D–F).

Chang. Oocyte spindle in slow freezing process. Fertil Steril 2010.

12.5% of oocytes had been activated with an anaphase/telo-
phase spindle (Fig. 5C; 4/32). At 4 hours of postthawing in-
cubation, 85% of oocytes had a recovered MII spindle
(Fig. 5A, Table 1; 34/40), 2.5% of oocytes had no spindle
reassembled with condensed chromosomes (Fig. 5B; 1/40),
and 12.5% of oocytes had been activated either with an ana-
phase/telophase spindle (Fig. 5C; 2/40) or with an early stage
of pronucleus (Fig. 5D; 3/40).
DISCUSSION
These results describe for the first time the systematic kinet-
ics of MII spindle during the complete course of a slow freez-
ing and thawing process. The results indicate that the MII
spindle was preserved during the slow freezing process and
that the permeating cryoprotectant PROH can support the or-
ganization of the MII spindle to resist the subphysiologic
temperature. Interestingly, the MII spindle was disassembled
gradually during the thawing process whether the cryoprotec-
tant PROH was present or not. Most of the oocytes were able
to recover their MII spindles after thawing; however, a portion
of oocytes could not sustain metaphase arrest because of this
slow freezing and thawing processes.
Previous studies involving the structure of oocyte spindle
during cryopreservation have been complicated by contradic-
tive observations among different labs and protocols that
have been used. Thus far, the whole picture of the change
of oocyte spindles during the freezing and thawing protocols
remains unclear. Unlike what we report here in mouse oocyte
cryopreservation, the oocytes in previous studies displayed
profound spindle alterations immediately upon thawing
(26), and only slight recovery was observed upon removal
of the cryoprotectants, indicating that the cryoprotectants (di-
methyl sulfoxide) and the freezing process caused a huge im-
pact on spindle structure detected by immunocytochemistry.

1436 Chang et al. Oocyte spindle in slow freezing process Vol. 93, No. 5, March 15, 2010
 FIGURE 5
Nuclear and microtubule dynamics of frozen and thawed oocytes after incubation at 37 C. Confocal images
depict microtubules (green) and chromatin (red). (A) A representative oocyte reveals a well-organized bipolar
spindle. (B) A representative oocyte lacks detectable microtubule structure. (C) A representative oocyte exhibits
telophase of meiotic spindle with two sets of separated chromosomes. (D) A representative oocyte reveals an
early stage pronucleus in the cytoplasm. Arrow indicates the pronucleus.

Chang. Oocyte spindle in slow freezing process. Fertil Steril 2010.

In contrast, in human oocyte cryopreservation, the oocyte
spindle can survive through the freezing process (with
PROH as a cryoprotectant), in that the spindle was detectable
in approximately 24% to 35% of oocytes immediately after
thawing (27, 28) with a computer-assisted polarization mi-
croscopy system (Polscope). Different consequences for oo-
cyte spindles were found under variations in factors such as
species (mouse vs. human), detection systems (immunocyto-
chemistry vs. Polscope), and cryoprotectants (dimethyl sulf-
oxide vs. PROH) still exist. Therefore, in our present study, to
elucidate the potential effects of cryopreservation on oocyte
spindles, we employ mouse oocytes treated with PROH in
a slow freezing protocol and analyze oocyte spindles by im-
munocytochemistry. Surprisingly, the oocyte spindle was not
only maintained before the freezing process, but also 100% of
oocyte spindles (Fig. 1D; 29/29) remained intact immediately
upon thawing. It suggests that the structure of the MII spindle
was not significantly altered during the freezing process and
might be sustained by cryoprotectants such as PROH and su-
crose in the freezing solution.
In fact, the MII spindle is highly susceptible to low temper-
atures, and even transient exposure to RT for only 10 minutes
causes irreversible disruption of the meiotic spindle in human
oocytes (29–31), whereas the MII spindle can be completely
disorganized at 4 C (5, 31, 32). However, in combination
with PROH and sucrose, our results demonstrated that the
MII spindle can survive through the freezing process. Our ex-
planations of the mechanisms involved in stabilizing the spin-
dle during subphysiologic temperatures are because of the
effects of sucrose and PROH. Therefore, to investigate the ef-
fect of cryoprotectant on oocyte spindles during low temper-
ature, we treated the oocytes at 4 C for 30 minutes and

Fertility and Sterility 1437

compared the spindle structure in the cryopreservation solu-
tions with the complete (with 0.3 M sucrose and 1.5 M
PROH) and incomplete (without either 0.3 M sucrose or
1.5 M PROH, or both) formula. Devoid of any cryoprotectant,
it is not surprising to see that the microtubule apparatus can
not endure the 4 C harsh conditions (Fig. 2A–C). Similarly,
unable to permeate through the plasma membrane, sucrose
alone only partially maintained the spindle structure with di-
minished microtubule apparatus and two distinctive spindle
poles (Fig. 2D–F) because sucrose might only induce water
molecules to move out of the oocyte and raise the concentra-
tion of tubulin molecules in the oocyte. However, the PROH
plays a critical role in resisting the damage of the low temper-
ature condition by preventing microtubule disassembly. It is
worth noting that the MII spindle can be stabilized by
PROH only when its concentration is above 1.5 M (14).
In contrast to the freezing process, the oocyte spindle dis-
integrated during the thawing process when the intracellular
PROH was replaced and the sucrose concentration was seri-
ally diluted by water molecules. To clarify whether the cryo-
protectants can also preserve the meiotic spindle during
thawing, we thawed the oocytes directly into the cryopreser-
vation solution with 1.5 M PROH and 0.3 M sucrose for 30
minutes (Fig. 3J–L). Surprisingly, the meiotic spindle could
not be preserved during the thawing process even when the
PROH remained in the solution. These observations dis-
closed a very essential finding that PROH can protect oocyte
spindle when the temperature declines; however, even with
the aid of PROH, the oocyte spindles disassembled during
the process of thawing. This situation may have huge impli-
cations for oocyte cryopreservation, especially the slow
freezing method. Because one is aware that the spindle disas-
sembly is not avoidable after the freezing and thawing pro-
cess, frozen and thawed oocytes must have sufficient time
and adequate conditions to recover their spindles before ad-
ministering further fertilization. Hence, during oocyte cryo-
preservation, efforts should be focused on how to restore
the ability to reassemble the meiotic spindle.
Rienzi et al. (27) reported that the meiotic spindle re-
mained intact in human oocytes after 10 minutes of RT equil-
ibration with the cryoprotectants PROH and sucrose. In fact,
the oocyte spindle depolymerization was observed during the
thawing process (the removal of the cryoprotectant) (27).
However, it was still not clear whether this phenomenon
was a direct effect of PROH on the microtubule themselves
or as a result of cellular dehydration. As well, it has been
speculated that the disintegration of the meiotic spindle dur-
ing the thawing procedure might be because of the rehydra-
tion steps (when water enters into the oocyte in place of the
cryoprotectant) (27).
To our knowledge, the current study is the first one to doc-
ument that the preservation of oocyte spindle during the
freezing procedure was mainly because of the effect of
PROH rather than cellular dehydration by sucrose. The loss
of spindle organization was always found during the final
phase of rehydration after recovery from low temperature
1438 Chang et al. Oocyte spindle in slow freezing process
storage; however, we clarified that the disintegration of the
meiotic spindle was caused by the thawing process per se in-
stead of the rehydration steps. Our study has clearly proven
for that PROH can support the organization of the MII spindle
to resist the subphysiologic temperature; however, the PROH
cannot sustain the oocyte spindle structure after the subse-
quent thawing process. Therefore, future efforts to optimize
oocyte cryopreservation should consider how to preserve or
recover the oocyte spindle efficiently after the freezing and
thawing processes. The information provided here is valuable
for further improvement of oocyte cryopreservation by the
slow freezing method.
REFERENCES
1. Ambrosini G, Andrisani A, Porcu E, Rebellato E, Revelli A, Caserta D,
et al. Oocytes cryopreservation: state of art. Reprod Toxicol 2006;22:
250–62.
2. Magistrini M, Szollosi D. Effects of cold and of isopropyl-N-phenylcar-
bamate on the second meiotic spindle of mouse oocytes. Eur J Cell Biol
1980;22:699–707.
3. Nasmyth K. Separating sister chromatids. Trends Biochem Sci 1999;24:
98–104.
4. Glenister PH, Wood MJ, Kirby C, Whittingham DG. Incidence of chro-
mosome anomalies in first-cleavage mouse embryos obtained from fro-
zen–thawed oocytes fertilized in vitro. Gamete Res 1987;16:205–16.
5. Sathananthan AH, Ng SC, Trounson AO, Bongso A, Ratnam SS, Ho J,
et al. The effects of ultrarapid freezing on meiotic and mitotic spindles
of mouse oocytes and embryos. Gamete Res 1988;21:385–401.
6. Kola I, Kirby C, Shaw J, Davey A, Trounson A. Vitrification of mouse
oocytes results in aneuploid zygotes and malformed fetuses. Teratology
1988;38:467–74.
7. Bouquet M, Selva J, Auroux M. The incidence of chromosomal abnor-
malities in frozen–thawed mouse oocytes after in-vitro fertilization.
Hum Reprod 1992;7:76–80.
8. Bouquet M, Selva J, Auroux M. Effects of cooling and equilibration in
DMSO, and cryopreservation of mouse oocytes, on the rates of in vitro
fertilization, development, and chromosomal abnormalities. Mol Reprod
Dev 1995;40:110–5.
9. Boiso I, Marti M, Santalo J, Ponsa M, Barri PN, Veiga A. A confocal
microscopy analysis of the spindle and chromosome configurations of
human oocytes cryopreserved at the germinal vesicle and metaphase
II stage. Hum Reprod 2002;17:1885–91.
10. Stachecki JJ, Cohen J, Garrisi J, Munne S, Burgess C, Willadsen SM.
Cryopreservation of unfertilized human oocytes. Reprod Biomed Online
2006;13:222–7.
11. Gook DA, Osborn SM, Johnston WI. Cryopreservation of mouse and hu-
man oocytes using 1,2-propanediol and the configuration of the meiotic
spindle. Hum Reprod 1993;8:1101–9.
12. Cobo A, Rubio C, Gerli S, Ruiz A, Pellicer A, Remohi J. Use of
fluorescence in situ hybridization to assess the chromosomal status of
embryos obtained from cryopreserved oocytes. Fertil Steril 2001;75:
354–60.
13. Huang JY, Chen HY, Tan SL, Chian RC. Effect of choline-supplemented
sodium-depleted slow freezing versus vitrification on mouse oocyte mei-
otic spindles and chromosome abnormalities. Fertil Steril 2007;88:
1093–100.
14. Joly C, Bchini O, Boulekbache H, Testart J, Maro B. Effects of 1,2-pro-
panediol on the cytoskeletal organization of the mouse oocyte. Hum
Reprod 1992;7:374–8.
15. Shaw JM, Trounson AO. Parthenogenetic activation of unfertilized
mouse oocytes by exposure to 1,2-propanediol is influenced by
temperature, oocyte age, and cumulus removal. Gamete Res 1989;24:
269–79.
16. Van der Elst J, Nerinckx S, Van Steirteghem AC. In vitro maturation of
mouse germinal vesicle-stage oocytes following cooling, exposure to
Vol. 93, No. 5, March 15, 2010
 cryoprotectants and ultrarapid freezing: limited effect on the morphology
of the second meiotic spindle. Hum Reprod 1992;7:1440–6.
17. Larman MG, Katz-Jaffe MG, Sheehan CB, Gardner DK. 1,2-propanediol
and the type of cryopreservation procedure adversely affect mouse oo-
cyte physiology. Hum Reprod 2007;22:250–9.
18. Boldt J, Cline D, McLaughlin D. Human oocyte cryopreservation as an
adjunct to IVF-embryo transfer cycles. Hum Reprod 2003;18:
1250–5.
19. Borini A, Bonu MA, Coticchio G, Bianchi V, Cattoli M, Flamigni C.
Pregnancies and births after oocyte cryopreservation. Fertil Steril
2004;82:601–5.
20. Chen SU, Lien YR, Chen HF, Chang LJ, Tsai YY, Yang YS. Observa-
tional clinical follow-up of oocyte cryopreservation using a slow-freez-
ing method with 1,2-propanediol plus sucrose followed by ICSI. Hum
Reprod 2005;20:1975–80.
21. Levi Setti PE, Albani E, Novara PV, Cesana A, Morreale G. Cryopreser-
vation of supernumerary oocytes in IVF/ICSI cycles. Hum Reprod
2006;21:370–5.
22. Gook DA, Edgar DH. Human oocyte cryopreservation. Hum Reprod
Update 2007;13:591–605.
23. Stachecki JJ, Cohen J, Willadsen SM. Cryopreservation of unfertilized
mouse oocytes: the effect of replacing sodium with choline in the freez-
ing medium. Cryobiology 1998;37:346–54.
24. Stachecki JJ, Cohen J, Willadsen S. Detrimental effects of sodium during
mouse oocyte cryopreservation. Biol Reprod 1998;59:395–400.
Fertility and Sterility
25. Carabatsos MJ, Combelles CM, Messinger SM, Albertini DF. Sorting
and reorganization of centrosomes during oocyte maturation in the
mouse. Microsc Res Tech 2000;49:435–44.
26. Eroglu A, Toth TL, Toner M. Alterations of the cytoskeleton and poly-
ploidy induced by cryopreservation of metaphase II mouse oocytes. Fer-
til Steril 1998;69:944–57.
27. Rienzi L, Martinez F, Ubaldi F, Minasi MG, Iacobelli M, Tesarik J, et al.
Polscope analysis of meiotic spindle changes in living metaphase II hu-
man oocytes during the freezing and thawing procedures. Hum Reprod
2004;19:655–9.
28. Bianchi V, Coticchio G, Fava L, Flamigni C, Borini A. Meiotic spindle
imaging in human oocytes frozen with a slow freezing procedure involv-
ing high sucrose concentration. Hum Reprod 2005;20:1078–83.
29. Pickering SJ, Braude PR, Johnson MH, Cant A, Currie J. Transient cool-
ing to RT can cause irreversible disruption of the meiotic spindle in the
human oocyte. Fertil Steril 1990;54:102–8.
30. Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. Limited
recovery of meiotic spindles in living human oocytes after cooling-re-
warming observed using polarized light microscopy. Hum Reprod
2001;16:2374–8.
31. Zenzes MT, Bielecki R, Casper RF, Leibo SP. Effects of chilling to 0 de-
grees C on the morphology of meiotic spindles in human metaphase II
oocytes. Fertil Steril 2001;75:769–77.
32. Pickering SJ, Johnson MH. The influence of cooling on the organization
of the meiotic spindle of the mouse oocyte. Hum Reprod 1987;2:207–16.
1439