Effect of addition of FSH, LH and proteasome inhibitor MG132 to in vitro maturation medium on the developmental competence of yak (Bos grunniens) oocytes

Dulbecco’s phosphate-buffered saline (DPBS) was purchased from Hyclone Laboratories Inc. (Logan, UT), FSH from Bioniche Inc. (Belleville, Ontario, Canada) and fetal calf serum (FCS) from Gibco (Grand Island, NY). All other chemicals and reagents were cell-culture tested and were obtained from Sigma-Aldrich (St. Louis, MO). Synthetic oviductal fluid (SOF) medium was prepared according to the formula of Tervit et al.[15] minus glucose. All media were filtered through 0.2 μm Millipore filter (Carrigtwohill, Co. Cork, Ireland) and placed in an incubator (Forma Serial II, USA) to equilibrate for four to six hours in an atmosphere of 5% CO₂ in air.

Collection and IVM of yak oocytes were performed in accordance with the method of Zi et al. [13, 16]. Briefly, ovaries were collected from yaks at local abattoir from October to December, and transported to the laboratory in DPBS maintained at 29–33°C. Cumulus-oocyte complexes (COCs) were aspirated from follicles (2–8 mm diameter) using a hand-held 10-ml syringe connected to an 18 ga needle. COCs were collected in DPBS supplemented with 6 mg/ml BSA under a low-power (20×) stereomicroscope. Unselected COCs were rinsed three times in DPBS containing 5% (v/v) FCS and twice in TCM 199 supplemented with 20% (v/v) FCS, 1 μg/ml estradiol-17β, 100 U/ml penicillin and 100 μg/ml streptomycin, different concentrations of FSH and LH according to the experimental design (IVM medium). Only COCs having one or more layers of cumulus cells and evenly granulated ooplasm were selected. Up to 30 COCs were placed in each culture well (Nunc Inc., Naperville IL, USA) containing 600 μl of maturation medium covered with 300 μl mineral oil. COCs were allowed to mature for approximately 24 h at 38.6°C in an atmosphere of 5% CO₂ in humidified air.

Sperm preparation and IVF were conducted according to previously described procedures [13, 16] with some modifications. Frozen semen was thawed and washed by centrifugation through a Percoll gradient (30%/45%) containing Hepes-buffered SOF medium supplemented with 5 mg/ml BSA, 50 μg/ml caffeine, 30 μg/ml glutathione and 20 μg/ml heparin (sperm medium) and centrifuged at 500 × g for 10 min to separate motile sperm. After centrifugation, 120 μl of the concentrated sperm fraction was removed and placed into 200 μl of sperm medium and incubated at 38.6°C for 15 min.

After oocyte maturation, excess cummulus cells were removed by gently swirling the COCs in a 36 mm Petri dish containing SOF with 6 mg/ml BSA (IVF medium). The COCs were further washed twice in IVF medium before being transferred into four-well plates (up to 30 per well) containing 500 μl IVF medium covered with 300 μl mineral oil per well. Each well received 50 μl of the sperm suspension (for a final concentration of 1 × 10⁶ motile sperm/ml). Oocytes and sperm were allowed to coincubate for 24–26 h at 38.6°C in an atmosphere of 5% CO₂ in humidified air. Remnant cumulus cells were removed from the putative zygotes by gentle pipetting after approximately 26 h of coincubation. The putative zygotes were then washed three times in SOF medium. Groups of 25 embryos were cultured in 600 μl of SOF medium supplemented with 6 mg/ml BSA, 0.5 mg/ml myoinositol, 3% (v/v) essential amino acids, 1% (v/v) nonessential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin and 100 μg/ml L-glutamine (culture medium) under 300 μl mineral oil, cultured in a humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂ at 38.5°C. The number of zygotes that cleaved was recorded 48 h post insemination (hpi). The culture medium was changed at 96 hpi and blastocyst development was determined on Days 7 to 9 post insemination (Day 0 = insemination). This experiment was replicated three to five times for each group.

GAPDH gene was chosen as reference gene for normalizing expression levels of target genes. Primers specific for target goat genes were designed with Beacon Designer 7.0 software (Premier Biosoft International, Palo Alto, CA USA) according to manufacturers guidelines (FSHR: 5'-TTCAATGGGACAACGCTGATTTC-3'/5'-TGTGGCAATTAGCGTCTGAATG GA-3'; LHR: 5'-AGTGACACCAAGATAGCCAAGC-3/5-GGTAGAACAGGACCAGGAGG AT-3'; GAPDH: 5'-AGTTCCACGGCACAGTCAAG-3'/5'-ACTCAGCACCAGCATCACC- 3'). Total RNA of cumulus cells after in vitro maturation (different concentrations of FSH and LH in IVM medium) was extracted, and reverse transcribed as described previously [17]. Real-time PCR was performed using on an iCycler iQ5 Real-time Detection System (Bio-Rad, CA, USA) with the SsoFast™ EvaGreen Supermix (Bio-Rad, CA, USA) in a volume of 10 μl. The cycle parameters were 3 min at 95°C, followed by 45 cycles of denaturation at 95°C for 10 s and annealing at 60.7°C (FSHR), 58.9°C (LHR) or 57.7°C (GAPDH) for 10 s, and finally, melt curve analysis. The real-time PCR amplification efficiency (E) of each primer pair and mean Ct (threshold cycles) values were calculated and used for determination of target gene RNA transcript levels [18], which includes a correction for differences in E between the target and housekeeping gene. Results were expressed, however, as relative expression ratios (RE) rather than as fold changes from a calibrator sample. The formula, therefore, was as RE = (1 + E ref) ^{Ct ref} /(1 + E target) ^{Ct target} . Each sample was tested in triplicate, and each experiment was replicated four times with cumulus cells from 30–50 COCs for each replicate.

Experiment 1 was conducted to investigate the optimal concentration of FSH and LH in IVM medium to improve yak oocyte competence for development after fertilization. COCs were matured in IVM medium that was supplemented with different combinations of LH (50 or 100 IU/ml) and FSH (0, 1, 5, 10 μg/ml).

Experiment 2 was conducted to investigate the effect of addition of FSH and LH in IVM media on FSHR and LHR mRNA expression in cumulus cells after 24 h IVM. COCs were matured in IVM medium that was supplemented with different combinations of LH (0, 50 or 100 IU/ml) and FSH (0, 1, 5, 10 μg/ml). Cumulus cells were collected at the end of IVM for each combination to determine FSHR and LHR mRNA expression.

Experiment 3 was conducted to investigate the concentration-dependent effects of MG132 added at the end of oocyte maturation on embryonic development. The MG132 was dissolved in dimethyl sulfoxide (DMSO) and was added to maturation drops so that the final concentration of DMSO was not greater than 0.5% (v/v). The control oocytes were cultured with medium supplemented with a similar amount of DMSO during IVM as for oocytes treated with MG132. COCs were matured in IVM medium (containing 5 μg/ml FSH and 50 IU/ml LH) that was supplemented with 0, 10, 20 or 30 μM MG132 from 18 h to 24 h after initiation of maturation. Treatment was achieved by washing COCs after 18 h of maturation and placing them in fresh medium containing MG132.

Experiment 4 was conducted to determine whether timing of MG132 treatment altered effects of the inhibitor on embryonic development. COCs were untreated or treated with 10 μM MG132 at two times [0–6 h of maturation (during the initiation of maturation) or 18–24 h of maturation (at the end of maturation)] using a 2 × 2 factorial arrangement of treatments. The COCs were placed in appropriate treatments at 0 h (MG132), washed at 6 h, placed in fresh IVM medium (containing 5 μg/ml FSH and 50 IU/ml LH) without MG132, washed at 18 h of maturation, and placed in fresh medium with appropriate treatment. Thus, some cultures received MG132 at 0–6 h and 18–24 h, some received MG132 from 0–6 h only, some received MG132 from 18–24 h only, and some did not receive MG132 treatment during in vitro maturation.

Data were analyzed statistically as follows. For each replicate, percentage of oocytes that cleaved and percentage of cleaved embryos that became blastocysts were calculated for all oocytes or embryos within the same treatment. Thus, the group of oocytes treated alike within each replicate was the experimental unit. Statistical analyses were performed using the Statistical Analysis System (version 9.2, SAS Institute Inc., Cary, NC, USA). Data were analyzed using the General Linear Models procedure. Percentage data were arcsine- transformed prior to analysis to maintain homogeneity of variance. Results are expressed as least-squares means ± standard error (SE) of the untransformed data.

Effects of FSH and LH concentration in IVM medium on subsequent embryonic development of the yak were shown in Table 1. The optimal concentration of FSH and LH in IVM medium was 5 μg/ml FSH and 50 IU/ml LH, i.e. both the cleavage rate and the blastocyst rate were the highest at this condition. There was a tendency for high LH concentration (100 IU/ml) in IVM medium to decrease the subsequent cleavage rate.

FSHR and LHR mRNA expressions of cumulus cells in media supplemented with different concentrations of FSH and LH were shown in Figure 1. Both FSHR and LHR mRNA were detected in yak cumulus cells after in vitro maturation, and the greatest expression was observed when the concentration of FSH and LH in IVM medium was 5 μg/ml FSH and 50 IU/ml LH. The cumulus cells had higher numbers of FSH receptors than LH receptors. Addition of FSH in IVM media had a greater effect on increase of these receptors than that of LH.

Results for COCs treated from 18 to 24 h of maturation with 0, 10, 20 or 30 μM MG132 are shown in Table 2. Treatment of COCs with 10 μM MG132 in IVM medium increased (P < 0.05) the percentage of cleaved embryos (i.e., that were ≥2 cells) becoming blastocysts. There was, however, no effect of 10 μM MG132 on the cleavage rate. Treatment with 20 μM MG132 had no effect on the cleavage rate and the percentage of cleaved embryos becoming blastocysts. However, treatment with 30 μM MG132 had negative effect on subsequent development.

Results are in Table 3. Addition of MG132 from 0–6 h of maturation reduced fertilization rate regardless of whether MG132 was also added at 18–24 h of maturation (P < 0.05), but treatment of COCs with 10 μM MG132 increased (P < 0.05) the percentage of cleaved embryos becoming blastocysts, as also shown in Experiment 2.