Comparative Stepwise Pattern of Reactive Oxygen Species Production during In Vitro Development of Fertilized and Nuclear Transferred Goat Embryos

Abattoir-derived ovaries were used for oocyte in vitro maturation (IVM) as described previously (4). In brief, cumulus oocyte complexes (COCs) were aspirated from antral follicles and cultured in maturation medium comprised of tissue culture medium199 (TCM199) supplemented with 10% fetal calf serum (FCS), Na-pyruvate (2.5 mM), L-glutamine (1 mM), penicillin (100 IU/mL), streptomycin (100 mg/mL), follicle-stimulating hormone (FSH, 10 mg/mL), luteinizing hormone (LH, 10 mg/mL), estradiol-17β (1 mg/mL), cysteamine (0.1 mM), epidermal growth factor (EGF, 100 ng/mL) plus insulin-like growth factor (IGF, 100 ng/mL) for 20-22 hours under mineral oil at 38.5°C, 5% CO₂, and maximum humidity.

Ear biopsy of a healthy pre-pubertal female goat was taken, cut into 2-3 mm² fragments and cultured as explants in Dulbecco’s Modified Eagle Medium F-12 (DMEM/F-12) containing 10% FCS and antibiotic (1% penicillin-streptomycin) at 37°C, 5% CO₂ in air. Cell started to shed out of the explants. Eventually, these cells proliferate to forms a confluent monolayer within 2-3 weeks. Obtained cells were used for investigation of fibroblast phenotype using differential immunostaining with anti-vimentin (for fibroblasts) and pan-cytokeratin (for epithelial cells) antibodies (7). Confirmed fibroblasts at passages 3-5 were used for SCNT experiments. In order to provide a synchronized population of G0/G1, cells were first cultured at 2.5×104 cells/cm², and at the next day, the cells were washed thrice with phosphate buffer saline (PBS) before being cultured in medium that contained 0.5% FCS for 4-5 days. Serum starved cells were subsequently trypsinized and used for SCNT procedure.

In vitro matured oocytes were denuded by vortexing in presence of 300 IU/mL hyalorunidase. Only good quality oocytes with homogenous cytoplasm and extruded first polar body were used for the experiments. The process of zona free enucleation was carried out as described previously by Nasr-Esfahani et al. (4). In brief, zona was removed by brief enzymatic digestion [5 mg pronase in 1 mL of Hepes-TCM199 (HTCM) for 1 minute] followed by incubation in TCM199 free of pronase and containing 20% FCS to neutralize the remaining enzyme. It has been demonstrated that goat matured oocytes revealed a cytoplasmic extrusion cone which is clearly visible upon zona removal (14). This extrusion is considered as a hallmark of MII spindle during enucleation. The cytoplasmic extrusion was gently aspirated into a 2-3 μm pipette and with a gentle touch against the blind needle (5-10 μm), the MII extrusion was separated from the oocyte. Successful enucleation was confirmed by staining the separated MII extrusion with H33342 (5 μg/ml, 5 minutes). During this procedure enucleated oocyte are not exposed to UV.

Nuclear transfer (NT) was carried out according to Hosseini et al. (15). In brief, individual fibroblasts were adhered to oocytes in medium containing 10 mg/ml phytohemagglutinin. Subsequently, the couplets were electrofused in 290 mOsm fusion buffer [0.3 M Mannitol, 100 μM MgSO₄, 50 μM CaCl2, 500 μM hepes, 0.05% bovine serum albumin (BSA)]. The reconstructed oocytes were rested for 0.5 hours before being activated using ionomycin (5 μM, 1 minut) followed by incubation with 2 mM 6-DMAP for 2 hours. Reconstituted-activated oocytes were then cultured in groups of five to seven in modified synthetic oviductal fluid (mSOF) under mineral oil at 38.5°C, 5% CO₂, 5% O₂ and humidified air for 7 days in 20 μl wells.

According to Forouzanfar et al. (16), matured COCs were washed in fertilization medium and groups of 20-25 COCs were transferred into 100 μl droplets of fertilization medium under mineral oil. Five straws of frozen spermatozoa were thawed at 37ºC for 1 minute, pooled and washed through Pure Sperm (Nidacon, Gothenburg, Sweden) gradient (40 and 80%) to separate the motile spermatozoa from the immotile by centrifugation [700 g for 15 minutes at room temperature (RT)]. Matured COCs were inseminated with a final concentration of two million sperm per ml. The inseminated COCs were incubated for 22 hours in 5% CO₂ in humidified air at 38.5°C. Twenty-two hours after insemination, cumulus cells attached to oocytes were mechanically removed via pipetting. Then, the zona was removed by brief enzymatic digestion as described above. The presumptive zygotes were then cultured in groups of five to seven as described for SCNT embryos.

The process of ROS measurement was as described previously (13). In brief, stock solutions of 2, 7-dichloro dihydroflourescein diacetate (DCHFDA, Sigma D6883, 5 mM) were prepared in dimethyl sulfoxide (DMSO) and stored at -20°C in dark. For each experiment, 5 μM working solution were prepared by dilution in TCM199 containing 1 mg/ mL poly vinyl alcohol (PVA). To measure ROS levels, 15-30 embryos per replicate derived from zona free IVF or SCNT were pooled from different stages of embryo development (2-cells, 4-cells, 5-8-cells, and greater than 8-cells, morula and expanded blastocysts). Zona free metaphase-II (MII) oocytes were also simultaneously assessed. Samples were incubated with 5 μM of DCFHDA in TCM199 for 30 minutes in incubator. Samples were then washed in TCM199, placed in 5 μl droplets covered by mineral oil and then immediately exposed to UV light of a fluorescent microscope (Olympus, IX71, Japan) and observed using filter sets (excitation wavelength: 450-490 nm, emission wavelength: 515-565 nm). Digital images of individual oocyte or embryo were taken with a high sensitive camera (DP-72, Olympus, Japan). Background, positive and negative controls were taken to account for fluorescence or inter-experimental variations. Fluorescent intensity of each taken image was assessed by Image J (National Institute of Mental Health, Bethesda, MD, USA). To reduce variations and possible errors, when comparison between different groups was required, experiments were designed so that oocytes and embryos from each group were available for assessment at the same period. To further minimize inter-experimental variation, the relative fluorescence intensity of each embryonic stage to the mean intensity of MII-oocytes in the same experiment was calculated according to the below formula:

The relative intensity is defined as the difference in the intensity of embryos from the mean intensity of MII-oocytes/mean intensity of MII-oocytes. It is important to note that for assessment of ROS at each embryonic stage at least three replicates were carried out. For each replicate at least 30 to 80 embryos and 55 to 95 oocytes were assessed.

Percentages data were transformed by ArcSin and analyzed by one way ANOVA model of SPSS version 17 (SPSS, Science, Chicago, IL, USA). Differences were compared by the Tukey multiple comparison post hoc test. All data are expressed as mean ± SEM and differences were considered as significant at P<0.05.

Figure 1 shows fluorescence images of goat MII-oocyte, zona free SCNT and IVF embryos at different stages of pre-implantation embryo development following staining with DCFHDA for ROS measurement. As depicted, irrespective of embryo production method, fluorescence intensity increased as the embryo progressed toward blastocyst stage.

Figure 2 shows the mean relative intensities of embryos relative to MII-oocytes at different stages of development in zona free IVF and SCNT derived embryos. As shown, in zona free IVF embryos, the mean relative ROS levels significantly decreased at 4 and 8- cell stages relative to the mean intensity of MII-oocytes and then began to rise by 16 cell stage which resulted in a significant increases by compact and blastocyst stages relative to all the earlier stages. Moreover, the relative increase at the blastocyst stage was significantly higher than compact embryos.

The trend of ROS production in SCNT embryos appear to follow the same trend as those of zona free IVF embryos. In zona free IVF embryos the decrement in ROS occur after 2-cell stage while in zona free SCNT reconstructs, the decrements being at earlier stage (post reconstruction). Subsequently, the increment in ROS production in zona free IVF embryos begin at around 8-cell stage while the increment in zona free SCNT embryo begin at around 4-cell stage. Therefore, despite similar trend of ROS production, a significant difference between the two groups were observed at 2- (lower in SCNT group), 4- (higher in SCNT group) and 8- (higher in SCNT group) cell stages. A significant difference was also observed at blastocyst stage. The degree of ROS production was significantly higher in blastocyst derived from zona free IVF embryos in comparison to zona free SCNT reconstructs.

During this study, unlike the zona free IVF embryos, in some of the SCNT embryos one or more blastomeres showed higher fluorescence intensity compared to other blastomeres (Fig .3).

Also, it has been reported that some blastomeres, due to asymmetrical division, are non-nucleated. In order to understand if this phenomenon has any relation to intensive ROS levels within blastomeres, the SCNT embryos with non-uniformed ROS staining were also stained with viable chromatin dye (H33342, 5 μg/ml for 5 minutes) and no relation was observed between ROS intensity with presence or absence of chromatin in each blastomeres. As shown in Figure 3, except for one blastomere without nucleus (arrow), all the other blastomeres with high ROS intensity had nuclei.