International Journal of

     Introduction
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 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 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
 
     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).
 
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 as cumulus–oocyte 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 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 fluorescence (PI-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
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 (p<0.05). The 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).



Discussion
 
     In this study, we showed that the 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 (20,22,23), 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 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 be affected by 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 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.



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.