Adult female (6-8 weeks old) and male (8-12 weeks old) NMRI mice were cared for and used according to the guide for the care and use of laboratory animals of Tarbiat Modares university. Mice were housed under a 12 hr light: 12 hr dark regimen at 22-24°C and 40-50% humidity with food and water available ad libitum.

The purpose of this study was two fold. First, the effects of vitrification and warming procedure were studied on the developmental competence, mitochondrial distribution and ATP content of vitrified and non-vitrified mouse GV and MII oocytes.

Next, after in vitro culture of both vitrified and non-vitrified oocytes, their mitochondrial distribution and ATP content of collected oocytes from in vivo and in vitro condition were analyzed and compared.

Unless otherwise indicated, all chemicals were purchased from Sigma Aldrich (Germany).

Oocyte: Adult female mice (n = 30) were superovulated by Intraperitoneal injection (IP) of 10 IU pregnant mare serum gonad-otrophin (PMSG, Folligon; Intervet, Australia). To obtain GV oocytes, females were killed by cervical dislocation 48 hr after PMSG injection and dissected ovaries were placed in global total medium (Life Global, USA). Antral follicles were punctured using needles to release the GV oocytes. Cumulus cells were mechanically removed.

For MII oocyte harvesting, female mice (n = 50) were superovulated by IP injection of 10 IU PMSG followed with another injection of 10 IU human chorionic gonadotrophin (hCG, Sereno, Switzerland) 48 hr later. The mice were killed by cervical dislocation 12-16 hr after hCG injection and their oviducts were removed.

The Cumulus-Oocyte-Complex (COC) was released with a needle from the ampullary region of each oviduct and then they were exposed to 0.01% hyaluronidase for 1 min to allow cumulus cells to separate from the oocytes. Denuded MII oocytes were washed several times in global total medium. The collected GV and MII oocytes were considered as vitrified and non-vitrified ones.

Sperm was obtained from the adult male mice through several slits in the cauda epididymis in global total medium and capacitated for 1.5 hr at 37°C, 5% CO₂ in air.

Oocytes were vitrified by cryotop method ⁽¹⁴⁾. The Equilibration Solution (ES) contained 7.5% (v/v) Dimethyl Sulphoxide (DMSO) and 7.5% Ethylene Glycol (EG) in Ham's F10 containing 20% Human Serum Albumin (HSA). The Vitrification Solution (VS) contained 15% (v/v) DMSO and 15% EG and 0.5 M sucrose in Ham's F10 containing 20% HSA.

Three-five oocytes were equilibrated in ES for 10 min and then transferred to VS. Finally, the oocytes were loaded onto the strip end of cryotop with a small volume of VS (<0.1 µl) and then plunged into liquid nitrogen (LN) at least for 24 hr.

For warming, the cap of strip was removed and then the strip of cryotop was directly placed into 1 M sucrose at 37°C for 1 min. Next, it was sequentially transferred to 0.5 M sucrose for 3 min, 0.25 M sucrose for 3 min and was washed in Ham's F10. After these steps, GV oocytes were put into in vitro maturation media and MII in culture media, respectively. After 2 hr of incubation, the survival rate of thawed oocytes was assessed.

Oocytes were cultured in 20 µl droplet of global media supplemented daily with 75 mIU/ml rFSH (Sereno, Switzerland) and 10 IU/ml hCG under mineral oil at 37°C in 5% CO₂ in air for 24 hr. The extrusion of the first polar body was used as the criterion for maturation of GV-stage oocytes ⁽¹⁵⁾.

Both collected in vitro matured MII oocytes (IVM-MII) and in vivo MII oocytes (OV-MII) were inseminated with sperm in global medium containing 15 mg/ml BSA. After 4-6 hr, oocytes were washed and cultured in 20 µl droplet of global medium under mineral oil at 37°C in 5% CO₂ in air. The development of zygotes to hatching stage was recorded daily up to 5 days.

Oocytes were stained for mitochondria by Mito Tracker Green (Molecular Probes, Eugene, OR) as described previously by Stojkovic et al. Briefly, the oocytes were incubated in pre-warmed and equilibrated global medium (at 37°C in 5% CO₂ in air) containing 0.2 mM Mito Tracker Green for 10 min at 37°C. Then the oocytes were washed several times in global medium and mounted on glass slides and observed under fluorescent microscope at 490 wavelengths ⁽¹⁰⁾. Then the photos of individual oocytes in each group were captured and imported into Image J software (National Institutes of Health, Bethesda). The fluorescence intensity of each oocyte was quantified and analyzed by this software.

The ATP content of completely denuded single oocytes was measured using methods described previously ⁽¹⁶⁾. Briefly, oocytes of all groups (n = 20 per each group) were transferred individually in 20 µl of ultrapure water into micro tubes and stored at -80°C until assay. ATP levels were quantified by measuring the luminescence (Berthold LB 9501 lumino-meter) generated in an ATP-dependent luciferin-luciferase bioluminescence assay (Bioluminescence Somatic Cell Assay System; Sigma, USA). A six-point standard curve (0-5 pmol) was considered in each series of assay. The ATP content was defined according to the standard curve.

All experiments were repeated at least four times. The oocyte maturation, fertilization and developmental rates were analyzed by one-way ANOVA and Tukey's HSD was used as the post hoc test. ATP levels and fluorescence intensity of oocytes were analyzed by independent t-test. Statistical significance was described as p < 0.05.

Data relating to this section is shown in Table 1. The survival rate of vitrified-warmed oocytes at GV and MII stages were 87.39 and 89.50%, respectively and there was no significant difference in this regard.

The maturation rates of GV oocytes in vitrified and non-vitrified groups (which reached to MII stage) were 69.48 and 77.45%, respectively and there was no significant difference between the groups.

The fertilization rates in vitrified and non-vitrified IVM-MII oocytes were 64.36 and 72.72% and these rates in OV-MII oocytes were 66.14 and 71.79%, respectively. The blastocyst hatching rates in vitrified IVM-MII and OV-MII groups were 9.89 and 12.14% and these rates in non-vitrified samples were 9.93 and 13.21%, respectively. There was no significant difference in maturation, fertilization and hatching rates between vitrified and non-vitrified oocytes in both in vitro and in vivo samples.

The comparison of ATP levels in different groups is shown in Figure 1. The ATP contents of GV oocyte in vitrified and non-vitrified groups were 0.2692±0.06216 and 0.2974±0.02588 pmol, respectively. The ATP levels in IVM-MII and OV-MII oocytes in control (non-vitrified) group were 0.2970± 0.02542 and 0.2850±0.01454 pmol and concentrations for IVM-MII and OV-MII in vitrified group were 0.3165±0.01020 and 0.3165± 0.03804 pmol. This content value was not sig-nificantly different between groups.

The mitochondrial pattern of vitrified and non-vitrified GV oocytes was similar and mitochondria were diffused throughout the oo-plasm of GV oocytes in vitrified (Figure 2A) and non-vitrified (Figure 2B) groups.

The mitochondria distribution in MII oocytes obtained from in vitro matured GV oocytes in both non-vitrified (Figure 3A) and vitrified (Figure 3B) groups was similar and they showed homogenous aggregation pattern throughout the ooplasm (spot distribution).

However, in MII oocytes obtained from in vivo condition in both non-vitrified (Figure 3C) and vitrified (Figure 3D) groups, this pattern was not seen. Their mitochondria had diffused pattern. The large aggregate of mitochondria was prominent in in vitro matured oocytes than in vivo MII oocytes.

The intensities of florescent (Figure 4) in GV oocyte in non-vitrified (fresh) and vitrified groups were 48.47±1.8 and 38.69±1.1 and in IVM-MII and OV-MII oocytes in non-vitrified group were 47.21±1.4 and 44.43± and in IVM-MII and OV-MII of vitrified group were 41.60±1 and 34.05±3.9, respectively. These rates were significantly different between vitrified and non-vitrified groups and between in vitro matured group and in vivo groups (p < 0.05).