Comparing the viability and in vitro maturation of cumulus germinal vesicle break down (GVBD) oocyte complexes using two vitrification techniques in mice

Abstract

AbstractBackground: Vitrification is assumed to be a promising method to cryopreserve human oocytes but still needs optimizing. Objective: The aim of this study was to improve the single step and step-wise vitrification effects on maturing mouse GVBD oocytes by ethylene glycol (EG) in conventional straws. Materials and Methods: Oocytes with compact cumulus cells were cultured for 3hr in TCM199 supplemented with 10% fetal bovine serum (FBS) in 5% CO2 in air. GVBD oocytes were randomly allocated into three groups. (1) Control (non-vitrified group), (2) exposed to single-step vitrification (contained of EG 20%+0.5M sucrose), (3) exposed to step-wise vitrification (2%, 5%, 10%, 20%EG +0.5M sucrose). In vitrification groups,oocytes were thawed and underwent additional 21 hr maturation. Viability of oocytes and maturation to MII stage were analyzed using inverted microscope and additionally by staining of propidium iodide and Hoechst 33342.Results: All non-vitrified oocytes were viable after 24 hr; however, viability of vitrified samples in single-step group was significantly lower than that of the step-wise and control Groups. Also, the maturation rate in the step-wise group was significantly higher (p < 0.05) compared to single-step.Conclusion: These results suggest that step-wise vitrification of GVBD oocytes as compared to single step vitrification was better in the rate of survival and in vitro maturation of oocytes.

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Cryopreservation of oocytes has important roles
in the preservation and management of genetic
resources, genetic engineering and nuclear transfer
procedure. It has the potential to become an

Correspondence Author :
Dr. Aligholi Sobhan, Department of Anatomy. and Vali-
e-Asr Reproductive Health Research Center, Faculty of
Medicine, Tehran University of Medical Sciences,
Tehran, Iran.
E-mail: Sobhania@tums.ac.ir
Iranian Journal of Reproductive Medicine Vol.5. No.4. pp: 165-170, Autumn 2007

Comparing the viability and in vitro maturation of
cumulus germinal vesicle break down (GVBD) oocyte
complexes using two vitrification techniques in mice

Somaye Khosravi Farsani¹ M.Sc., Reza Mahmoudi² Ph.D., Mir Abbas Abdolvahhabi¹ Ph.D.,
Mehdi Abbasi¹ Ph.D., Fateme Malek1 Ph.D., Aligholi Sobhani1 Ph.D.

1 Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences,
Tehran, Iran.
2 Department of Anatomy, Faculty of Medicine, Yasuj University of Medical Sciences, Yasuj,
Iran.

Received: 7 October 2007; accepted: 27 December 2007

Abstract
Background: Vitrification is assumed to be a promising method to cryopreserve human
oocytes but still needs optimizing.
Objective: The aim of this study was to improve the single step and step-wise
vitrification effects on maturing mouse GVBD oocytes by ethylene glycol (EG) in
conventional straws.
Materials and Methods: Oocytes with compact cumulus cells were cultured for 3hr in
TCM199 supplemented with 10% fetal bovine serum (FBS) in 5% CO2 in air. GVBD
oocytes were randomly allocated into three groups. (1) Control (non-vitrified group), (2)
exposed to single-step vitrification (contained of EG 20%+0.5M sucrose), (3) exposed
to step-wise vitrification (2%, 5%, 10%, 20%EG +0.5M sucrose). In vitrification
groups,oocytes were thawed and underwent additional 21 hr maturation. Viability of
oocytes and maturation to MII stage were analyzed using inverted microscope and
additionally by staining of propidium iodide and Hoechst 33342.
Results: All non-vitrified oocytes were viable after 24 hr; however, viability of vitrified
samples in single-step group was significantly lower than that of the step-wise and
control Groups. Also, the maturation rate in the step-wise group was significantly higher
(p < 0.05) compared to single-step.
Conclusion: These results suggest that step-wise vitrification of GVBD oocytes as
compared to single step vitrification was better in the rate of survival and in vitro
maturation of oocytes.

Key words: Vitrification, Viability, In vitro maturation, GVBD, Oocyte, Mouse.

Introduction

important technique for preserving gamets for
female whose fertility may become compromised
by medical treatment (such as chemotherapy and
Radiotherapy ) . Oocyte preservation has been
successfully applied to livestock including Cattle,
Goats, Sheep, and other animals, but it is still an
open challenge in most mammalian species, due to
the extreme sensitivity of gamets to chilling
injuries (1, 2).
Oocyte cryopreservation accompanied with in
vitro maturation is important in women either are
at risk of ovarian hyper stimulation, or fail to
hormonal stimulation response and finally in those
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All chemicals were obtained from sigma
(St.Louis, MO) unless indicated otherwise.

Experimental design
Two experimental and one control groups were
used in the present study. Experimental groups
were vitrified immediately after getting to GVBD
in step-wise or single step methods. While non-
vitrified GV oocytes in control group were
opposed to maturation protocols only (for 24 hr).

Khosravi Farsani et al
Iranian Journal of Reproductive Medicine Vol.5. No.4. Autumn 2007
166
patients with polycystic ovarian syndrome (3).
Over one decade after the first pregnancy was
achieved using frozen and thawed oocyte (4),
Fabbri et al (2001) reported a modified slow
freezing and rapid thawing method to freeze
human oocytes that gave higher rates of oocyte
survival and embryo development (5) than before
(6,7). The results obtained using this method
revealed that frozen- thawed oocytes had
significantly lower cleavage rate than non- frozen
controls (8-10). Vitrification can be used to freeze
mammalian oocytes and embryos (11-13). It is an
alternative to traditional freezing methods (slow
freezing) to avoid chilling injury and ice crystal
formation (14, 15). This method is simple and
rapid compared with slow freezing; however, it
still needs more studies, including animal research
(16). Since the major concerns about spindle
damage in oocyte freezing are all linked to the use
of mature oocytes (MII) (17,18), several attempts
were made to freeze immature oocytes (GV), in
which the meiotic spindle is not yet formed.
Unfortunately not the nucleus, but the cytoplasm
appears to be the main problem in immature
oocytes in cryopreservation procedure (19-21).
Therefore, choosing an intermediate stage, such as
germinal vesicle breakdown (GVBD), may
circumvent some of the problems associated with
the cryopreservation of GV and MII oocytes. With
regard to this fact that the oocytes have distinct
properties in each species, our research has been
done on GVBD oocytes of mouse. Since 1998,
there have done limited appropriate researches on
GVBD oocyte stage; on the other hand some
researches have only been done on bovine GVBD
oocyte (20-23). Therefor, the aim of the present
study was to evaluate the effects of step-wise and
single step vitrification on survival and maturation
rates on GVBD oocytes.

Materials and methods

Animals and collection of GV oocytes
Female NMRI virgin mice aged 3 to 4 weeks
old were prepared from pasture institute (Iran) and
maintained for adaptation in anatomical laboratory
of Tehran Medical Sciences University for 2
weeks. The animals were injected intra-peritoneal
(IP) with 10 IU of pregnant mare serum
gonadotropin (PMSG) for ovulation induction. The
animals were killed by cervical dislocation 48 hr
after PMSG administration, and the ovaries were
removed and transferred into a holding medium,
which consisted of TCM199 (Gibco, UK)
supplemented with 10% fetal bovine serum. GV
oocytes were obtained
complexes (COCs) by puncturing antral follicles in
the ovaries with a 29-G needle in the holding
medium. In all experiments, only full-grown
COCs, surrounded by at least two layers of
cumulus cells, were selected, whereas partly or
completely naked oocytes were discarded.

In vitro maturation of GV oocytes
Fresh GV oocytes were cultured in 100-µl
droplets of TCM-199 supplemented with 10% fetal
bovine serum (FBS), 0.23 mM sodium pyruvate ,
10 ng/mL epidermal growth factor , 100 mIU FSH
(GONAL- F serono), 75 µg/ml of penicillin G-K
salts , and 50µg/ml of streptomycin sulfate (Sigma)
under liquid paraffin oil at 37°C in an atmosphere
of 5% CO2 in humidified air, as described by
Albarracin et al. (23), with some modifications. In
experimental groups the maturation duration for
getting to GVBD oocytes before vitrification was 3
to 4 hr.

Oocytes vitrification and thawing

Vitrification: The GVBD oocytes were
vitrified by solid surface vitrification, as described
by Aono et al. (10), with some modifications. The
holding medium used for handling oocytes during
vitrification was TCM199 containing 10% FBS.
All vitrification solutions were prepared using this
holding medium. Manipulation of oocytes and
vitrification process were performed at room
temperature (25◦C). In the single step group,
oocytes were exposed to %20 ethylene glycol (EG)
+0.5 M sucrose in a holding medium for 40-60 sec.
Oocyte in the stepwise group was exposed to
vitrification solution of %2 EG for 5 min (step
one), %5 EG for 3 min (step two), %10 EG for 2
min (step three) and %20 EG +0.5 M sucrose for
40-60 sec (step four). Oocytes were loaded into
straws by aspiration and the straws were plunged
as cumulus–oocyte
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Step-wise 160 (100%) 148 (92.50%)
Single step 142 (100%)
GV= Germinal vesicle, GVBD = GV breakdown. *=p<0.05

Discussion

In this study, we showed that
cryopreservation of mouse GVBD oocytes by
step-wise and single step vitrification enabled
oocytes to survive and mature .
Successful cryopreservation of GVBD oocytes
has been reported only in bovine and calf (20,22
Viability and in vitro maturation of GVBD oocyte
Iranian Journal of Reproductive Medicine Vol.5. No.4. Autumn 2007
167
directly into liquid nitrogen. The straws were
stored for 7 days in liquid nitrogen.

Thawing: The straws were thawed in air for 10
sec, and immediately plunged into a water bath at
37◦C for 10 sec.
Thawing was carried out in four steps using
sucrose solution in a holding medium containing
%10 FBS at 37◦C, after which the oocytes were
exposed to decreasing concentrations of sucrose
(0.5, 0.2, 0.1 and 0.05 M) for 1 minute. GVBD
oocytes were washed three times at 37◦C in
maturation medium before being transferred for
maturation protocol. After thawing, GVBD
oocytes were matured for additional 21 hr to fulfill
the 24 hr maturation requirement.

Assessment of oocyte viability
Oocyte survival was evaluated morphologically
based on the integrity of the oolema and zona
pellucida; Oocytes were also assessed for viability
based on oolema integrity by propidium iodide (PI)
and Hoechst.
For this purpose oocytes were stained with PI
(10μg/ml) and Hoechst 33342 (10μg/ml) for 10
minutes, washed, and then observed under a
fluorescence microscope.The dead cells showed
red fluorescence (PI-positive) for disruption of
cellular membrane. The viable cells showed blue
fluorescence without red
negative) for the intact cell membrane (24).

Statistical analysis
Collected data were analyzed by chi-square test.
The differences in the values of survival and
maturation rates, were considered significant when
p<0.05.


Results
Table I . Viability and in vitro maturation of vitrified mouse GVBD oocyte
Treatment groups

GV oocytes
Control (non vitrified) 120 (100%)
fluorescence (PI-

A total of 422 GV oocytes with cumulus cells were
obtained from 20 ovaries that were used for 2
vitrified and 1 non-vitrified (control) groups. The
number of oocytes collected averaged 21.1 per
mouse ovary.

Morphology of GV oocytes
Morphological figures of vitrified GVBD
oocytes using non-vitrified (control) and either
single step or step-wise method is shown in figure
1 (1A, 1B and 1C). Cumulus-oocyte complexes in
the single step method show dispersed cumulus
cells (Figure 1C). In step-wise method (Figure 1B)
cumulus-oocyte complexes were almost similar to
those in the control group (Fig. 1A). Also, viability
assessment of oocytes based on oolema integrity
by Hoechst and PI showed red fluorescence
(Figure 2A) for dead cells for disruption of cellular
membrane (PI-positive) and blue fluorescence
(Figure 2B) for viable cells for the intact cell
membrane (PI-negative).

Viability and in vitro maturation of vitrified GV
oocytes
The survival and maturation rates of GV
oocytes after different treatments including
exposure to 2 type of vitrification and non-vitrified
oocytes shown in Table I. The survival and
maturation rate in the step-wise group (81.97% and
68.43% respectively) were significantly (p<0.05)
higher than single step group (58.04% and 57.12%
respectively). But maturation rate in control group
was significantly higher than single step and
stepwise vitrified groups
vitrifying/warming process in two experimental
groups were greatly reduced the developmental
capacity of GVBD oocytes compared to control as
assessed by in vitro maturation (p<0.05).

(p<0.05). The
No. of
No. of
GVBD oocytes
109 (90.83%)
No. of survived
oocytes (after vitrification)
-
121 (81.97%)*
78 (58.04%)*
No. MII oocytes
92 (84.40%)*
83 (68.43%)
44 (57.12%)* 133(93.66%)
the


,23), but there were low rates of survival capacity
after cryopreservation. As some of researcher
reported, oocytes at different maturation stages
respond to cryopreservation differently (19, 25).
In fact, several authors have reported
that GV oocytes are just as sensitive to chilling
injury (26) or more sensitive to chilling or
cryopreservation than MII oocytes (27,28).Bovine
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Khosravi Farsani et al
Iranian Journal of Reproductive Medicine Vol.5. No.4. Autumn 2007
168
oocytes at the GVBD stage have been described
as more resistant to cooling than GV or MII
oocytes. However, when cryopreservation was
attempted, Men et al (2002) observed that a
significantly higher proportion of cleaved bovine
embryos from vitrified MII oocytes developed
into blastocysts than those derived from vitrified
GVBD oocytes (20).
In addition to GVBD oocytes, attempts have
also been made on vitrifying oocytes at other
maturation stages. Thus when bovine oocytes
were vitrified 0, 6, 12 or 24 hr after the onset of
maturation, Hochi et al (1998) found the best
stage for vitrification was that of oocytes matured
for 12 hr (22). The reason for the high sensitive
nature of bovine oocytes at GVBD oocytes to
cryopreservation is unknown. In bovine and other
mammalian oocytes, as Hunter and Moor (1987)
reported, active transcription and translation occur
at GVBD and later stages of meiotic maturation
(28,30).Therefore, in addition to the detrimental
effect on cytology, the biochemical process with
the oocytes may also
cryopreservation. Consequently, the impaired
biochemical process will negatively influence the
cytoplasmic maturation of oocytes. In addition to
meiotic stages, the protocol of vitrification is
important in survival, in vitro maturation and
cleavage rate of oocytes after thawing. In the
present study, we used step-wise and single-step
vitrification methods to cryopreserve GVBD
oocyte of mice. We found high rates of survival
and IVM rates when the oocytes were vitrified
after step-wise method. The survival and in vitro
maturation in single step was lower in comparison
to step-wise. Our results in step-wise group in
survival rate are consistent with Men et al (20)
that reported vitrification of bovine GVBD


A B C
be affected by
oocytes after a two-step vitrification
(81.97% versus 79.59%). Four-step vitrification in
our study might be responsible for higher survival
rate than the study of Men et al, in where they
used two-step vitrification. Other reasons could
explain such differences between their results
and ours: the different size of oocyte
in bovine and mouse and the different
cryoprotectant agents (CPAs) in it.
Mahmmoudi et al (2005) reported that intact
mouse oocytes had a higher developmental
competence than denuded oocytes (31) and
damage to connection between oocyte and
cumulus cells after exposure to cryopreservation
has adverse effect on in vitro maturation after
thawing. Hurt et al (2000) reported that
vitrification with EG would lead to expansion of
cumulus cells probably due to damage of the gap
junction between cumulus and oocytes (32). In
this study, as Cetin and Bastan stated (33),
contrary to Hurt et al. (32) in step-wise
vitrification with EG, distribution of cumulus cells
did not occur after dissolution.
Finally, as reported by other investigators, the
cryodamage of the oocytes is the major
determinant to the significant low maturation rate
in cryopreserved oocytes. Therefor, we do not
regard present vitrification protocol as a definitive
protocol. Anyway, further research will help
clarify the cellular and molecular mechanisms of
cryopreservation-indused injury.

Conclusion

Our results suggest that step-wise vitrification
of GVBD oocytes as compared to single step
vitrification was better in rate of survival and in
vitro maturation of oocytes.

Figure 1. GVBD A, before vitrification. B, after step wise vitrification C, after single step vitrification

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Viability and in vitro maturation of GVBD oocyte
Iranian Journal of Reproductive Medicine Vol.5. No.4. Autumn 2007
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A B

Figure 2. GVBD stained by propidium iodide and Hoechst. A, Dead B, Viable



Acknowledgements

This project financially supported by research
deputy of Tehran University of Medical Sciences
and Health services grant No. 85-04-30-4669. So,
we would like to thank for their helps.

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