Collection of oocytes and cumulus cells
All cell culture reagents were obtained from Sigma, unless otherwise specified. Bovine ovaries were obtained from a local abattoir and transported to the laboratory immersed in 0.85% saline supplemented with 100 mg/ml of Streptomycin and 80 mg/mL Sodium Penicillin G at a temperature of 35–38°C within 3 h of collection. Cumulus-oocyte complexes (COC) were obtained by aspirating follicles 3- to 8-mm in diameter with an 18 G needle connected to a vacuum pump at −50 mmHg. The follicular fluid was deposited in 60-ml tubes containing PBS-Dulbecco (8 mg/ml NaCl, 0.2 mg/ml KCl, 1.15 mg/ml KH2PO4, 0.10 mg/ml MgCl2 + 6H2O, 0.10 mg/ml CaCl2, 0.036 mg/ml sodium pyruvate, 1.00 mg/ml glucose) supplemented with BSA (3 mg/ml) and gentamicin (50 μg/ml).
Immunofluorescence for GM-CSF detection in bovine oocytes and granulosa cells
Granulosa cells and oocytes were washed in 0.1 M PBS (pH 7.4, Gibco BRL) and fixed in a mixture of Histochoice and ethanol (4:1). Cumulus cells were obtained by vortexing COC for 5 minutes in PBS-0.1% BSA. Cells were then permeabilized and blocked in a solution of 0.1 M PBS with 1% BSA, 5% skim milk and 0.3% Triton X-100 for 60 min at room temperature. Cells were incubated in blocking solution (without Triton X-100) containing polyclonal antibodies (1:200; N-20 and C-18 for the GM-CSF alpha and beta subunit receptors respectively, Santa Cruz Biotechnology, California, USA) raised against the carboxyl and amino terminals of the α- and β-GM-CSF receptor subunits, respectively. After three washes with PBS, cells were incubated with anti-rabbit, anti-goat and anti-mouse IgGs (1:300 in blocking buffer) conjugated to Alexa Fluor 488 and 594 nm (Molecular Probes, California, USA), respectively. Cells were again washed three times in PBS and mounted under coverslips in a solution containing 4’, 6-diamidino-2-phenylindole (DAKO Laboratories, Denmark). Samples were examined under confocal microscope and photos were obtained using photomicroscopy (Olympus Fluoview 1000, Tokyo, Japan).
In vitro maturation of cumulus oocyte complexes
After follicular aspiration, COC were classified into five groups based on the morphology of their surrounding cumulus cells . Group A: with many layers of compact cumulus cells; Group B: with partially removed cumulus cells; Group C: denuded oocytes; Group D: degeneration of oocyte cytoplasm; Group E: expanded cumulus cells. Only COC classified as Group A and B were used in this study. Cumulus-oocyte complexes were then washed twice in PBS-Dulbecco (Gibco BRL) and twice in maturation media according to each treatment. Maturation media consisted of SOF (107.7 mM NaCL, 7.16 mM KCl, 1.19 mM KH2PO4, 1.5 mM D-glucose, 5 mM Taurine, 1.71 mM CaCL2, 0.49 mM MgCl2, 3.3 mM Sodium lactate and 25.07 mM NaHCO3) at pH 7.4 supplemented with aminoacids BME 50× (20 μl/ml), MEM 100x (10 μl/ml), BSA FV (8 mg/ml) and gentamicin (50 μg/ml). Cumulus oocyte complexes were randomly assigned to SOF medium supplemented with Recombinant human GM-CSF (hGM-CSF 215-GM-010 (R&D System, Inc., Minneapolis, USA) at concentrations of 1 (n = 71), 10 (n = 59) and 100 ng/ml (n = 89) [0.07, 0.7 and 7 nM, respectively]. Two additional groups were incorporated in the experimental design: SOF alone (n = 75) and a positive control maturation media consisted of tissue culture medium (n = 95) [TCM; 15 mg/ml TCM 199, 2.2 mg/ml NaHCO3 at pH 7.4] supplemented with 10% FBS (Hyclone, Utah, USA), 0.2 μM Pyruvate, 5 μg/ml LH (Lutropin, Bioniche, Belleville, Canada), 40 mg/ml FSH (Folltropin, Bioniche, Bellevile, Canada) and 50 μg/ml gentamicin.
Groups of 10–15 COC were allocated for in vitro maturation (IVM) in 50-μl droplets of treatment media in Petri dishes under mineral oil for 22 h in humidified atmosphere consisting in 5% CO2 at 38.5°C.
Assessment of cumulus expansion and oocyte nuclear maturation
After 22 h of IVM, oocytes were collected and evaluated according to the cumulus expansion and then nuclear maturation. Cumulus expansion was determined using three different methods: 1) higher and a lower diameter for each COC were measured using a micrometric rule previously calibrated using a 0.1 mm objective (Nikon); 2) oocytes were microphotographed and higher and lower diameters were measured using a Fluoview software (FV 1000-ASW 1.4.3; Olympus, Corporation, Japan); and 3) a subjective scale was used  to estimate the degree of cumulus expansion. The degree of cumulus expansion was measured as follows: 0, no expansion; +1, separation of only the outermost layer of cumulus cells; + 2, further expansion involving the outer half of the cumulus oophorus; +3, further expansion up to, but not including, the corona radiate; +4, complete expansion, including the innermost corona radiate cells. A cumulus expansion index (CEI)  was calculated according to the subjective scale previously described using the following formula: CEI = (+1xn) + (+2xn) + (+3xn) + (+4xn) / N. Where CEI is the index for a given treatment, n is the total number of COC observed for each scale value in each treatment and N is the total number of COC in each treatment.
After cumulus expansion evaluation, cumulus cells were removed mechanically by vortex in PBS-0.1% BSA and washed twice in the same solution. Oocytes were placed between glass and cover slides with silicone and fixed with a mixture of acetic acid and ethanol (1:3) overnight at room temperature. Oocytes were then stained using 1% aceto-orcein for 1 h and destained using a mixture of acetic acid, glycerol and distilled water (1:1:3). Stained oocytes were examined under a phase contrast microscope for intact nucleus with germinal vesicle (GV), germinal vesicle breakdown (GVBD) or metaphase II (MII)-arrested.
Determination of cumulus cell number and viability
Cumulus oocyte complexes (n = 52-60/per group) were randomly assigned to the following in vitro maturation media: SOF alone, SOF supplemented with GM-CSF at a concentration of 1, 10 or 100 ng/ml of GM-CSF or TCM 199 as described above. Groups of 10–15 COC were allocated for in vitro maturation (IVM) in 50-μl droplets of treatment media in Petri dishes under mineral oil for 22 h in humidified atmosphere consisting of 5% CO2 at 38.5°C. An additional sample of COC (n = 40-45/per group) was in vitro matured in SOF medium alone or supplemented with 10 and 100 μM of LY294002 a PI 3-kinase inhibitor or DMSO (DMSO was used as a diluent control). Cumulus cells were removed mechanically by vortex in PBS-0.1% BSA at 22 h. A 50 μl aliquot of cell suspension was mixed with 5 μl of Trypan Blue for cell viability using a Neubauer chamber.
Assessment of oocyte cytoplasmic maturation
Cumulus oocyte complexes were randomly assigned to the following in vitro maturation media: 1- SOF without GM-CSF supplementation (n = 123), 2- SOF supplemented with 100 ng/ml of GM-CSF or 3- TCM 199 as described above (n = 159).
Immunohistochemical staining for cortical granules was also performed for evaluation of oocyte cytoplasm maturation. The type of cortical granules (type I, aggregates; type II, aggregates with some dispersion and type III, dispersion of granules) was evaluated as previously described . Briefly, the zona pellucida was removed using 0.5% (w/v) pronase and oocytes were fixed in 4% (w/v) paraformaldehyde for 30 minutes. Oocytes were permeabilized with 0.25% Triton X-100 and washed with blocking solution (PBS containing 2% (w/v) BSA, 2% non-fat milk and 0.15 M glycine). Staining was performed using 10 mg/ml lens culinaris conjugated to fluorescein isothiocyanate (FITC, Sigma L9267, St Louis, USA).
Oocytes were examined and evaluated under epifluorescence inverted microscope (Nikon Corporation, Tokyo, Japan).
Relative expression of IGF-2 gene transcript in bovine cumulus cells and oocytes were determined in COC (n = 30 per group) in vitro matured in TCM, SOF alone or supplemented with 100 ng/ml of GM-CSF. Total RNA was extracted from lysed cells using the RNAeasy extraction mini kit (Qiagen Inc., Valencia, CA, USA). All subsequent RNA purification steps were carried out according to the manufacturer’s instructions. cDNA was synthesized using the oligo-dT method (Promega Corp., Madison, WI, USA) with 1 μg of total RNA as a template in a reaction volume of 20 μl. Sequences of forward and reverse bovine IGF-2 were: ATCCAGCCGCATAAACCG and GGACGGTACAGGGATTTCAG. A reaction mixture containing a volume of 50 μl was prepared (5 μl 10× PCR buffer, 2 μl dNTPs mix 10 mM, 2.5 μl forward and reverse primers 10 μM, molecular biology grade water and 0.5 μl Taq DNA polymerase). All the reagents were acquired from Promega. The reaction was heated on a Stratagene Thermo Cycler (GRI Systems, UK) to 95°C for 7 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, and a final extension step of 72°C for 10 min. As a normalization control for RNA loading, parallel reactions in the same multiwell plate were performed using GAPDH as a target. Quantification of gene amplification was made following quantitative PCR by determining the threshold cycle (CT) number for SYBR fluorescence within the geometric region of the semilog plot generated during PCR. Within this region of the amplification curve, each difference of one cycle is equivalent to a doubling of the amplified product of the PCR. The relative quantification of the target gene expression across treatment was evaluated using the comparative ∆∆CT method. The CT value was determined by subtracting the GAPDH CT value from the target CT value of the sample. Calculation of ∆∆ CT involved using target gene expression on immature control (sample with the highest CT value or lowest target expression) as an arbitrary constant to subtract from all other CT sample values. Relative target mRNA expression was calculated as fold changes in relation to immature control sample and expressed as 2-∆∆CT value.
In vitro fertilization and embryo development
A sample of COC was randomly assigned to in vitro maturation media consisting of: 1) SOF alone (SOF, n = 212); 2) SOF supplemented with 100 ng/ml of GM-CSF (SOF + GM-CSF, n = 224) or 3) Tissue Culture Medium (TCM 199, n = 216) and then subsequently in vitro fertilized and cultured for 9 days.
Embryos were produced using standard protocols for in vitro maturation, fertilization and culture [36–39]. Frozen-thawed semen from bulls of proven fertility (ABS, American Breeders Service, DeForest, WI, USA) was used for in vitro fertilization (IVF). The content of one 0.25-ml straw of frozen Holstein Friesian semen was thawed in water at 35–37°C. Thawed sperm were washed in a discontinuous gradient of 45/90% Percoll using centrifugation at 700 g for 20 min. The pellet was resuspended with washing medium TALP (Tyrode’s albumin lactate pyruvate) containing 6 mg/mL BSA (Fraction V), 1.0 mM Sodium Pyruvate and 5 μg/mL of gentamicin and centrifuged once again at 250 g for 5 minutes. After being centrifuged, the spermatozoa in pellets were counted and the volume adjusted to give a concentration of approximately 1.5-2 × 106 sperm/ml of heparin-containing (1 μg/ml) TALP-IVF medium (TALP without glucose supplemented with 4 mg/ml BSA, 100 IU/ml penicillin and streptomycin, 0.1 mM pyruvate, and 2 μg/ml heparin). The sperm suspension was pippeted into 35 mm-petri dishes in 50 μl microdrops and covered with mineral oil. Thereafter, 10–12 matured COC per drop were added and incubated in 5% CO2 and 5% O2 in humidified air at 38.5°C. After 18–20 h, the presumptive zygotes were vortexed in PBS-0.1% BSA medium to remove the cumulus cells. Denuded zygotes were cultures in 30 μl of bicarbonate-buffered SOF medium for 7 days in a humid chamber under an atmosphere containing 5% CO2, 5% O2 and 90% N2.
Embryo development and total cell number of blastocysts
Early cleavage was evaluated on Day 2 after in vitro fertilization (Day 0 = in vitro fertilization) and blastocyst formation were recorded on Days 7 and 9 of in vitro culture. Blastocysts from days 9 of in vitro culture (n = 30/per group) were used for cell number determination. Embryos were placed on a slide with dye bisbenzimide (Bis, Hoechst 33342, 10 μg/ml) for 5 min at 39°C. Hoechst dye was removed, and cover lips were mounted with wax and then firmly pushed onto the slide to spread the embryo. Staining nuclei was visualized with an epifluorescence microscope (Olympus, Tokyo, Japan).
Single point measurements such as the difference among treatments for cumulus expansion, nuclear and cytoplasmic maturation, and IGF-2 mRNA levels were estimated using one-way analysis of variance (ANOVA). Tukey’s multiple comparison was used as a post-hoc test when a significant difference was detected. Data from cumulus diameter was normalized to a logarithmic scale in order to accomplish homocedasticity. CEI and number of cells values were compared among treatments using non-parametric Kruskal-Wallis and multicomparison tests. Data from cellular viability were arcsin-transformed and analyzed using one-way ANOVA and Tukey’s test. All statistical analyses were performed using the Statistica 7.0 (StatSoft, Inc., Oklahoma, USA) software package.