Characterization of the effects of heat stress on autophagy induction in the pig oocyte

Pig ovaries were obtained from a local abattoir for isolation of cumulus-oocyte-complexes (COCs) to be subjected to in vitro maturation (IVM) [44, 45]. Briefly, follicles (2–4 mm) were aspirated and COCs were collected and washed in TL-Hepes with 0.1% polyvinyl alcohol (PVA). Cumulus oocyte complexes were cultured in maturation media (Tissue Culture Media 199 (TCM-199)) containing 0.57 mM L-cysteine, follicle stimulating hormone (0.5 μg/mL), luteinizing hormone (0.5 μg/mL), and epidermal growth factor (10 ng/mL) for approximately 42 h at 38.5 °C in 5% CO₂. Prior to IVM, a representative sample of germinal vesicle-intact (GV) stage oocytes for each replication were randomly selected from the COC pool. Identification of polar body was performed with phase-contrast light microscopy. Oocytes used for analysis were stripped of cumulus cells via gentle vortex (6 to 8 min) in 1% hyaluronidase in TL-Hepes-PVA and washed in TL-Hepes-PVA. To observe the effect of HS on IVM of oocytes, oocytes underwent three different IVM temperature treatments: 1) TN conditions (38.5 °C) for the entirety of 42 h (TN/TN), 2) TN conditions for the first 21 h of IVM followed by HS (41 °C) conditions for the following 21 h (TN/HS), or 3) HS conditions for the first 21 h of IVM followed by TN conditions for the following 21 h (HS/TN). Following IVM oocytes were stripped of cumulus cells by vortexing 5–6 min in TL-Hepes-PVA supplemented with 1% hyaluronidase and washed in TL-Hepes-PVA.

To characterize the temporal change in abundance of LC3B-II, oocytes were matured under either the TN/TN or HS/TN treatments. A pool of oocytes was collected before introduction into maturation media (0-h). Oocytes were collected after 21 h of either TN (21-h TN) or HS conditions (21-h HS). The remainder of the oocytes that had experienced 21 h of TN conditions were allowed to continue maturation under TN conditions for a total of 42 h (42-h TN/TN), and the remainder of the oocytes that had experienced 21 h of HS conditions continued maturation under a subsequent 21 h of TN conditions, for a total of 42 h (42-h HS/TN).

Pools of 50 denuded oocytes per replicate were collected as described above after 21 or 42 h of IVM. Oocyte pools were lysed in 5 μL of Laemmli sodium dodecyl sulfate buffer at 95 °C for 4 min followed by 1 min on ice and then centrifugation at 1000 rpm for 1 min at room temperature. Lysates from fifty oocytes were loaded per lane of a 4–20% Tris glycine gel (Lonza PAGEr Gold Precast Gels). The BioRad Mini PROTEAN Tetra System was used to separate protein homogenates at 60 V for 30 min followed by 120 V for 90 min. The protein was transferred to a nitrocellulose membrane for 1 h at 100 V at 4 °C. Membrane blocking was conducted using 5% milk in phosphate buffered solution with 0.5% Tween 20 (PBST) for 1 h at room temperature. A rabbit anti-BECN1 (Cell Signaling Technology, 3495), rabbit anti-LC3B (Cell Signaling Technology, 3868), rabbit anti-ATG12 (Cell Signaling Technology, 4181), rabbit anti-BCL2L1 (Cell Signaling Technology, 2764), or normal rabbit IgG (Cell Signaling Technology, 2729) as a negative control were added (1:1000 dilution) to the membrane in 0.5% milk in PBS overnight at 4 °C. Following primary antibody incubation, the membranes were washed with PBST (PBS with 0.1% Tween) three times at room temperature for 10 min each. Donkey anti-Rabbit IgG (Amersham ECL NA934) was incubated (1:1000) with the membrane for 1 h at room temperature. The membrane was then washed three times for 10 min each at room temperature. Horseradish peroxidase substrate (Millipore, Billerica, MA) was added to the membrane for 1 min in the dark, and was exposed to x-ray film and developed for visualization. Average pixel intensity for the band corresponding to the primary antibody was compared for each blot using Image Studio Lite (Li-Core). Signal from detection of each protein of interest was normalized to α-tubulin.

Cumulus-oocyte-complexes were collected and subjected to IVM under the HS/TN treatment as mentioned above, with the addition of either DMSO vehicle control, 1.0 nM rapamycin, 10 nM rapamycin, or 100 nM rapamycin. In addition to rapamycin, IVM media for this study contained 10 ng/mL leukemia inhibitory factor (LIF; Sigma-Aldrich L5283), 40 ng/mL basic fibroblast growth factor (Sigma-Aldrich F0291), and 20 ng/mL insulin-like growth factor 1 (Sigma-Aldrich I36769). After 21 h of HS IVM followed by a subsequent 21 h of TN IVM, oocytes were denuded and healthy metaphase II oocytes containing extruded polar bodies were counted as a percent of total oocytes per treatment. Pools of 50 metaphase II arrested oocytes were then flash frozen in liquid nitrogen to be used for downstream analysis.

Oocytes were collected and denuded, as described above, from different time points during IVM and then fixed and mounted to slides as previously described [46, 47]. Briefly, oocytes were fixed in 4% paraformaldehyde in PBS overnight at 4 °C, and then transferred to 70% ethanol in PBS at 4 °C. Oocytes were permeabilized in 0.5% Triton X-100™ (Sigma-Aldrich, St. Louis, MO) for 30 min at room temperature. Next, oocytes were blocked in 5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO) for 45 min at room temperature, then incubated with primary antibody overnight at 4 °C. After approximately 24 h, oocytes were washed twice in PBS for 30 min, and incubated with secondary antibody for 1 h at room temperature. The oocytes were then washed twice in PBS for 30 min, and mounted to microscope slides using SlowFade Gold Mountant containing DAPI (S36939, ThermoFisher Scientific, Pittsburgh, Pennsylvania).

Primary antibodies derived from different species were used so that secondary antibodies with different fluorophores would recognize and bind to the separate primary antibodies within the same fixed oocyte. This made it possible to view two different fluorophores conjugated to secondary antibodies at different excitation wavelengths. 30 oocytes per temperature treatment per time point were visually assessed using a confocal microscope. Three oocytes per temperature treatment per time point of IVM were used in colocalization analysis on sequential focal planes (step size of 1 μm) measuring spatial fluorescence over approximately 50 μm of each oocyte. Fluorescence of DAPI stained chromatin, labeled rabbit anti-BCL2L1 and mouse anti-BAX, or labeled rabbit anti-BECN1 and mouse anti-BCL2L1 was measured.

Statistical analysis of maturation rate, western blot data, and co-fluorescence data was conducted using PROC MIXED in SAS Enterprise Miner Workstation version 14.1 (Carry, NC), where a standard student t-test was used to compare statistical differences. Statistical significance was determined when P values were less than or equal to 0.05. The PDC colocalization plugin [48] in the ImageJ processing program [49] was used to calculate Pearson’s and Spearman’s correlation coefficient and scatter plots representing colocalization of signal intensity collected from confocal microscopy of individual oocytes. Corresponding z-stacks with blocks of pixels randomly scrambled were used to determine that the probability of the measured value for Pearson’s or Spearman’s correlation between two color channels was significantly greater than would be calculated if there was only random overlap of the same information.

To characterize the effect of HS on maturation, oocytes underwent IVM during one of three temperature treatments: TN conditions throughout the entire 42-h maturation period (TN/TN), HS conditions during the second half of IVM (TN/HS), or HS conditions during the first half of IVM (HS/TN). Oocytes were subject to 21-h HS intervals because we have previously observed that 42 h of HS greatly decreases oocyte maturation and subsequent embryo development [50]. This also allowed us to compare maturation rate of different temperature treatments with downstream protein abundance and localization. Both HS treatments decreased maturation compared to TN/TN control, where the maturation rate of TN/TN oocytes was 66.9 ± 5.0%, the maturation rate of TN/HS oocytes was decreased to 44.9% ± 5.0% (P < 0.01), and the maturation rate of HS/TN oocytes was decreased to 33.2 ± 1.6% (P < 0.01; Table 1). The maturation rate of HS/TN oocytes decreased compared to the maturation rate of TN/HS (P ≤ 0.05; Table 1). These are comparable to previous results from our group [50].

The abundance of BECN1, fomation of the ATG12-ATG5 complex, and the cleavage of LC3B was measured via western blotting to characterize the effect of HS on autophagy induction in the oocyte. The abundance of BECN1 and BCL2 like 1 (BCL2L1) were unaffected by temperature treatment (Fig. 1A and B). The abundance of ATG12 in complex with ATG5 was increased in oocytes collected after 42 h of IVM of each temperature treatment (TN/TN, TN/HS, or HS/TN) compared to GV-intact oocytes collected before IVM. There was increased abundance (~ 1.5 fold; P < 0.01) of ATG12 in complex with ATG5 after 42 h of IVM under the HS/TN temperature treatment compared to the TN/TN (P < 0.01) and TN/HS (P < 0.01; Fig. 1A and B).

There was no effect of temperature treatment on the abundance of LC3B II in oocytes after IVM with any of the three temperature treatments (P > 0.05). Although, there was decreased LC3B-II in TN/TN (P < 0.01), TN/HS (P < 0.01) and HS/TN oocytes (P < 0.01) compared to oocytes collected at 0 h of IVM (Fig. 1A and B). Compared to oocytes collected at 0-h, there was an approximately 24-fold lower abundance of LC3B-II in oocytes collected after TN/TN IVM, approximately 17-fold lower abundance in oocytes collected after TN/HS IVM, and approximately 73-fold lower abundance of LC3B-II in oocytes collected after HS/TN IVM (Fig. 1A and B).

The observed sharp decrease in LC3B-II due to temperature treatment provided rationale for an IVM experiment in which oocytes were collected after 21 h to determine the temporal effects of HS on LC3B-II. Although the abundance of LC3B-II did not differ between TN/TN oocytes and HS/TN oocytes at 42 h of IVM (P = 0.92), there was decreased abundance of LC3B-II after 21 h of HS (0.21 ± 0.6 relative band intensity) compared to oocytes collected after 21 h of TN (0.9 ± 0.26; P < 0.01; Fig. 2A and B). These data suggest that HS affects the utilization of autophagy-related proteins in the oocyte.

Because HS in the first half of IVM sharply decreased oocyte LC3B-II, the interaction of BCL2L1 with either BAX or BECN1 was determined to assess a potential HS-induced regulatory mechanism. The TN/HS treatment was not used in further experiments because it appeared to have a lesser affect on LC3B-II turnover. Oocytes that underwent IVM in either TN/TN or HS/TN conditions were fixed for immunohistochemistry (IHC) to determine BCL2L1, BECN1, or BAX co-localization. Based on the Pearson’s (r_P) or Spearman’s (r_S) correlation coefficients, where a r_P or r_S closer to 1 is indicative of co-localization (Table 2) and overlap of fluorescent signal (Fig. 3A and B), neither the time point nor the temperature treatment affected colocalization of BCL2L1 and BAX. Based on level of fluorescence, BAX was numerically increased, but not significantly increased, at 21 h of HS compared to TN conditions (Fig. S2B). There were no effects of time or temperature treatment on the abundance of BCL2L1 based on level of fluorescence (Fig. S2C).

Based on the r_P and r_S correlation coefficients (Table 2) and overlap of fluorescent signal (Fig. 4A and B), there was little colocalization of anti-BECN1 and anti-BCL2L1 antibodies regardless of time point during IVM or temperature treatment (Table 2). BECN1 staining appeared punctate at all time points and temperature treatment with no overlap with BCL2L1 staining (Fig. 4A and B). There appeared to be more intense punctate BECN1 focal staining in oocytes (Fig. 4A and B). This data suggests that BCL2L1 is participating in the regulation of apoptosis in IVM oocytes under TN or HS conditions, while not appearing to inhibit autophagy.

To determine if inducing autophagy in oocytes during HS increases the oocyte’s ability to mature to MII arrest, oocytes underwent IVM while experiencing HS during the first 21 h and TN conditions the following 21 h in the presence of vehicle control, 100 nM, 10 nM, or 1 nM rapamycin. Oocytes treated with all four concentrations of rapamycin were subjected to the HS/TN IVM treatment, based upon the effect of this temperature treatment on ATG12-ATG5 complex and LC3B-II abundance. The maturation rate of oocytes in the presence of DMSO vehicle control was 50.0 ± 4.1, 43.6 ± 5.6% for 100 nM rapamycin, 47.8 ± 5.5% for 10 nM rapamycin, and 65.8 ± 5.0% for 1 nM rapamycin (Fig. 5). Including 100 nM or 10 nM rapamycin in the IVM media had no effect on maturation rate after HS/TN compared to vehicle control (P = 0.31 and P = 0.72, respectively), though oocytes matured in the presence of 1 nM rapamycin had an approximately 1.3-fold increase in maturation rate compared to vehicle control (P = 0.03; Fig. 5). This data suggests low concentrations of rapamycin may provide the oocyte some resistance to HS and improve their ability to reach MII arrest, potentially through induction of autophagy.