Oocyte aspiration and in vitro maturation
Bovine (Bos taurus) ovaries from a local abattoir (Cargill Meat Solutions, Guelph, ON, Canada) were aspirated to retrieve cumulus oocyte complexes (COCs) into collection medium, consisting of 1 M HEPES 52 (Sigma Aldrich, Oakville, Canada; H3375)-buffered Ham’s F-10 (Sigma Aldrich N6635) supplemented with 2% (v/v) steer serum (Cansera International Inc., Etobicoke, Canada), heparin (2 IU/mL) (Sigma Aldrich H3149), sodium bicarbonate, and penicillin/streptomycin (1%) (Gibco, 15,140–122). Oocyte maturation was carried out using the protocol previously established in our lab . In brief, COCs were collected and washed in serum in vitro maturation medium (S-IVM) consisting of HEPES buffered TCM199 (Sigma Aldrich M4530) supplemented with 2% steer serum and sodium pyruvate (Sigma Aldrich P4562). Oocytes with a dark cytoplasm and tightly packed cumulus cells were selected, washed in S-IVM, and randomly divided between the four treatments, in groups of 60 COCs.
Each group of COCs was washed twice in S-IVM supplemented with hormones (S-IVM + H) prepared as follows: 10 μL of LH (1 μg/mL – NIH, California, United States; AP117438), 12.6 μL of FSH (0.5 μg/mL - Folltropin V; Vetoquinol, Quebec, Canada; 00867357), 10 μL of Estradiol (1 μg/mL – Sigma Aldrich E2785), and 800 μL of Fetal Bovine Serum (FBS) (10% total serum - Gibco, Whitby, Canada 12,483–020) into 10 mLs of S-IVM. The groups were then washed twice in S-IVM + H with the treatments before being placed into 80 μL micro-drops of S-IVM + H. Treatment groups were prepared using 2.5 ml of S-IVM + H for each group: no further additives (control), 2.5 μl of 0.1% ethanol (vehicle), 2.5 μl of BPA (Sigma Aldrich 239,658) and 2.5 μl of BPS (Sigma Aldrich 43,034) dissolved in 0.1% ethanol for a final concentration of 0.05 mg/mL.
Sixty COCs were placed into the micro-drops of the S-IVM + H + treatment with 15–20 COCs per micro-drop. They were then covered with mineral oil (Sigma Aldrich M5310) and placed in an incubator at 38.5 °C in 5% CO2 to mature for 24 h. After maturation, 30 COCs were removed and washed 3X in sterile phosphate buffered saline (PBS) (Multicell, Wisent Bioproducts, Quebec, Canada; 311–010) with 0.01% polyvinyl alcohol (PVA) (Sigma Aldrich P8136) and snap-frozen in liquid nitrogen. The remaining 30 COCs were transferred into 500 μL of hyaluronidase (2 mg/mL) from Bovine Testes (Sigma; H3506-1G) for enzymatic and mechanical removal (via micropipette) of the cumulus cells. Oocytes were then washed 3X in PBS + 0.01% PVA and snap-frozen in liquid nitrogen. The remaining cumulus cells were immediately removed from the hyaluronidase and pelleted with PBS + 0.01% PVA, washed in PBS + 0.01% PVA to ensure removal of hyaluronidase and snap-frozen in liquid nitrogen.
In vitro embryo production
After maturation, COCs were washed twice in HEPES with Bovine Serum Albumin (BSA) (Sigma Aldrich A8806) and twice in BSA-supplemented IVF TALP (Tyrode albumin lactate pyruvate). COCs were then placed in 80 μL droplets of IVF-TALP + BSA covered with mineral oil in an incubator at 38.5 °C and 5% CO2. Bos taurus semen (Semex, Guelph, ON) was thawed and assessed for motility. The swim-up protocol was performed in 1.5 mL of HEPES Sperm TALP supplemented with 15% BSA and incubated at 38.5 °C and 5% CO2 for 45 min. A concentration of 1 × 106 sperm cells/mL/drop was added into each IVF droplet and incubated at 38.5 °C and 5% CO2 in the separate treatment groups for 18 h.
Presumptive zygotes (PZs) were mechanically stripped of any remaining cumulus cells and sperm, then washed 3X in HEPES Sperm TALP. They were then washed in Synthetic Oviductal Fluid (SOF) medium supplemented with freshly prepared sodium pyruvate, essential and non-essential amino acids (Sigma Aldrich M5550; Sigma Aldrich M7145), Gentamicin (Sigma Aldrich G1272), 15% BSA and 2% FBS. PZs were then placed in 30 μL droplets of the in vitro culture medium (IVC medium) covered with mineral oil in a low oxygen (5% O2) incubator until day-8 blastocysts. Cleavage and blastocyst rates were recorded ~ 40 h and 8 days, respectively, post-fertilization.
Developmental and arrested rates
Developmental rates were measured at key timepoints : cleavage rate at 40–45 h post-fertilization, 2–4 cells between 45 and 50 h post-fertilization, 8–16 cells between 75 and 80 h post-fertilization, and blastocyst rates at Day-8 post-fertilization. All embryos were produced from oocytes matured in the four treatment groups mentioned above: Control, Vehicle, BPA (0.05 mg/mL) and BPS (0.05 mg/mL). The number of embryos arrested at the 2–4 cell stage, the 8–16 cell stage, and the morula stage, were counted on day-8 post-fertilization in order to determine the rates of embryonic arrest at significant developmental stages . All the rates were calculated as a percentage over the number of cleaved zygotes. Zygotes were considered cleaved if 2 distinct blastomeres were present.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed using the In Situ Cell Death Detection Kit, Fluorescein (Roche; 11,684,795,910) following the Manufacturer’s instructions in order to detect apoptosis. For each embryo, ImageJ software was used to count the number of cells by detection of the number of stained nuclei (blue). The number of cells with apoptosis was counted manually based on observation of the TUNEL label (green) and apoptosis rates were taken as a ratio of the number of apoptotic cells over the total number of cells. Positive controls were not quantified, they were observed exclusively to assure the success of the technique in each run.
After 8 days of culture in a low oxygen incubator (5% O2), blastocysts and arrested embryos were washed 3X in 0.5 mL of PBS with 1% PVA (PBS/PVA) and then fixed in 4% PFA for 20 min at RT and stored in 1% PFA in PBS until used. Embryos were washed 3X in 0.5 mL of PBS/PVA and permeabilized in 0.5 mL of Triton X solution (0.5% Triton X in 1X PBS) for 1 h at RT. After 40 min, the embryos used for the positive control were removed, washed twice in 0.5 mL of PBS/PVA and incubated in a solution of DNase I (Thermo Fisher Scientific, EN0521) for the remaining 20 min in a dark, humidified chamber at 37–38 °C.
After 1 h, all embryos were washed twice in 0.5 mL of PBS/PVA, except for the positive controls which remained in the DNase I. Then all embryos, including the positive controls, were transferred to a micro-well dish, with each well containing 10 μL of the TUNEL reaction mixture (1-part enzyme + 9-parts FITC label). The negative control embryos were placed in a well containing 10 μL of the FITC label only, without the TUNEL enzyme. Embryos were incubated in the TUNEL solution for 1 h in a dark, humidified chamber at 37–38 °C, washed 2X 5 min in 10 μL of Triton X solution, 1X 5 min in 10 μL of PBS/PVA, 1X in 10 μL of RNase buffer at RT and incubated into 10 μL of RNase A (Thermo Fisher Scientific; EN0531) solution for 1 h in a dark, humidified chamber at 37–38 °C in order to digest nuclear RNA. Embryos were then washed twice in 10 μL of RNase buffer, incubated in 10 μL of Hoechst stain solution for 30 min in a dark, humidified chamber at 37–38 °C and mounted onto slides using the same protocol mentioned above and imaged using an Olympus FV1200 Confocal Microscope at 20X or 40X objectives with laser wavelengths of 405 nm for Hoechst (blue) and 488 nm for the TUNEL FITC label (green) using the Fluoview software.
RNA extraction and reverse transcription
Total RNA was extracted from the frozen groups of either 30 COCs, 30 denuded oocytes, or cumulus cells corresponding to 30 oocytes using the Qiagen RNeasy Plus Micro Kit (Qiagen, Toronto, ON) with a modified protocol. To each sample, 350 μL of Buffer RLT Plus was added, samples were then transferred to a gDNA Eliminator spin column placed in a 2 mL collection tube, centrifuged for 30 s at 10,000 RPM and then 350 μL of 70% ethanol was added to the flow-through. Samples were transferred to a RNeasy MinElute spin column and centrifuged for 15 s at 10,000 RPM. After washes with buffers and 80% ethanol, each column was centrifuged at 13,000 RPM for 5 min to dry the membrane, transferred to a new 1.5 mL collection tube and 17 μL of RNase-free water was added directly to the center of the spin column membrane, followed by 1 min centrifugation at 13,000 RPM to elute the RNA.
To assure reproducibility, all samples were reversed transcribed consistently in groups of 30. RNA was reverse transcribed into cDNA by adding 4 μL of QuantaBio qScript cDNA SuperMix (VWR, Mississauga, Canada; 95,048) to the 16 μL of RNA eluted for each sample (T100 Thermal Cycler, BioRad, Mississauga, ON) and incubated as previously described . Samples were stored at − 20 °C until processed. Additionally, a group of 300 oocytes was reverse transcribed and diluted to a concentration of 3 oocytes/μL and used as a calibrator and for standard curves.
Quantitative polymerase chain reaction (qPCR)
qPCR was used to measure AMH and AMHRII mRNA expression profiles using a CFX96 Touch Real-Time PCR Detection System (BioRad). Four biological replicates, consisting each of either 30 COCs, 30 denuded oocytes or equivalent stripped cumulus cells, were analyzed for each target and reference gene listed in Table 1. Glyceradehyde-3-phosphate dehydrogenase (GAPDH) and peptidylprolyl isomerase A (PPIA) were used as reference genes based on previous studies performed in our laboratory showing that these reference genes are not affected by the treatments . Relative quantification analysis was performed with a use of Standard curve for each set of primers, by the ΔΔCt method and a calibrator was run in each plate to account for interrun variations.
Each mRNA target was amplified using SsoFast EvaGreen supermix (Biorad 1,725,201) with 5 μL of EvaGreen, 2 μL of RNase-free water and 1 μL of a working dilution of the forward and reverse primers (25 μM) to a final volume of 8 μL. 2 μL of each cDNA sample was added and reactions performed at: 95 °C for 5 min, followed by 44 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s and elongation at 72 °C for 10 s and acquisition of fluorescence at 95 °C for 10 s, ending with the melt curve acquisition from 72 to 95 °C by 0.5 °C increments.
Quantification of AMH and AMHRII protein in 30 COCs, denuded oocytes and corresponding stripped cumulus cells in each of the four treatment groups was performed by western blotting. Results were analyzed using a minimum of 3 biological replicates. β-Actin (Cell Signalling Technology, Whitby, Canada; 4967) was used as a loading control and densitometry performed using the Bio-Rad Image Lab software and analyzed as a ratio to β-actin expression.
Samples were lysed in 20 μL radioimmunoprecipitation assay (RIPA) buffer and 1% (v/v) protease inhibitors (Biotool, Florida, United States; B14001 and B15001), followed by a freeze-thaw cycle in liquid nitrogen, repeated 4 times. Samples were then placed in a water bath sonicator for 30 min followed by centrifugation at 12,000 RPM at 4 °C for 10 min. Equal volumes of 3X reducing buffer with β-mecaptoethanol (Sigma Aldrich M6250) were added to each sample. Polyacrylamide gels (12%) were prepared using Bio-Rad standard gel recipes.
Proteins were heated at 90 °C for 6 min to denature disulfide bonds and reduce tertiary and quaternary proteins prior to use. Samples were run on the 12% gel in an Invitrogen wet transfer western blot apparatus (Invitrogen, Burlington, ON) at 125 V for 2 h. The gel was then placed in a transfer chamber full of cold 1X transfer buffer of Tris, Glycine and water and was run at 25 V for 3 h in order to transfer the protein onto nitrocellulose paper (Biorad 1,620,115). Nitrocellulose blots were washed 2X 10 min in Tris buffered saline pH 7.6 with 0.1% Tween 20 (Thermo Fisher Scientific, Whitby, Canada; BP337) (TBST), ponceau stained to ensure protein transfer, blocked for 1 h in 5% skim milk in TBST and incubated with each primary antibody of interest at 4 °C overnight: AMH at 1:500 (Abcam, Cambridge, United States; ab229212) and AMHRII 1:2000 (Abcam; ab197148) dilutions, respectively.
After 3X washes in TBST and blots were incubated with the secondary antibody, antirabbit IgG HRP-linked antibody (Cell signaling Technology, Whitby, Canada; 70,745) at 1:5000 dilution for 1 hour, then washed 3X in TBST and incubated with Clarity Western ECL Blotting Substrate (Bio-Rad 170–5060) for 5 min. Blots were imaged on a ChemiDoc XRS + Imaging System (Bio-Rad) and densitometric analysis was used to quantify relative protein expression of AMH and AMHR against the loading control, ß-actin .
Determination of sex ratio via qPCR
qPCR was used to measure DDX3Y and USP9Y mRNA expression profiles using a CFX96 Touch Real-Time PCR Detection System (BioRad). Thirty biological replicates (consisting of individual blastocysts) were processed in each treatment group and GAPDH and PPIA were used as reference genes (Table 1). The same qPCR protocol as for AMH and AMHRII was used.
Previous experiments from our laboratory assessing RNA-based blastocyst sexing, proved that three transcripts are consistently expressed in all male embryos tested and not in females . We selected two of these transcripts, DDX3Y and USP9Y, in order to determine the sex ratio of blastocysts via qPCR. Blastocysts that expressed both of the two exclusively male transcripts selected were classified as male, blastocysts that did not express these transcripts were classified as female and blastocysts that expressed one of the two transcripts were deemed inconclusive and excluded from the results. All samples were run in technical triplicates. RNA was extracted, reverse transcribed and analyzed from a single blastocyst as described previously.
AMH and AMHRII immunofluorescence detection
Blastocysts were stained with the specific antibody for AMH and AMHRII used for Western Blotting and visualized under confocal microscopy in order to localize and quantify AMH and AMHRII. COCs were used as positive controls as the presence of AMH and AMHRII has been previously confirmed by immunofluorescence in COCs . Semiquantitative analyses was performed using the ImageJ software. On this software each Hoeschst-stained nuclei (blue) were separated and counted, and the mean expression of Alexa Fluor-488 (green) surrounding or within each nucleus were quantified for every blastocyst. An average of the fluorescent levels surrounding or within the nuclei was calculated as the total relative quantification for each blastocyst. Ten biological replicates, consisting each of a single blastocyst in each treatment group, were analyzed for both AMH and AMHRII.
After 8 days of culture, blastocysts were fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature (RT) and then stored in 1% PFA in PBS at 4 °C. Fixed embryos were blocked in 1X PBS supplemented with 0.1% Triton X-100 (Sigma-Aldrich; 9002-93-1) and 5% normal donkey serum (NDS) (Sigma-Aldrich; D9663) for 1 h at RT. Embryos were then washed in 1X PBS for 20 min in a dark, humidified chamber between 37 and 38 °C. Next, embryos were incubated with the primary antibody, either AMH (Abcam ab229212) or AMHRII (Abcam ab197148) at a 1:50 dilution, overnight at 4 °C. Primary antibodies were diluted in antibody dilution buffer containing 1X PBS, 0.005% Triton X and 0.5% NDS. Embryos were washed 3X 30 min in the antibody dilution buffer in a dark, humidified chamber at 37–38 °C and incubated in Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa-Fluor 488 (Invitrogen, Thermo Fisher Scientific; A21206) at a 1:200 dilution in antibody dilution buffer. Embryos were incubated in the secondary antibody for 1 h in a dark, humidified chamber at 37–38 °C. After 1 h, blastocysts were incubated for 45 min in bisbenzimide H 33258 (Hoechst) nuclear stain (Sigma-Aldrich; B2883-25MG) in a dark, humidified chamber at 37–38 °C. Blastocysts were washed again 3X 30 min in antibody dilution buffer in a dark, humidified chamber at 37–38 °C to decrease background fluorescence.
Blastocysts were then mounted onto slides with Vectashield antifade mounting medium (MJS BioLynx Inc., Brockville, Canada; VECTH1000). Slides were sealed and stored at 4 °C until imaged. Embryos were imaged using an Olympus FV1200 Confocal Microscope at a 40X objective with laser wavelengths of 405 nm for Hoechst (blue) and 488 nm for Alexa-Fluor 488 (green) using the Fluoview software.
Statistical analyses were performed on all data sets using GraphPad Prism 6 and SPSS statistics software. qPCR expression levels were measured and normalized relative to the reference genes as well as to a calibrator using the delta delta Ct method. Western blot protein expression levels were normalized as a ratio to the loading control expression levels. Before assessing statistical differences, each set of data was analyzed for normality using Kolomogorov-Smirnov and Shapiro Wilk tests. If the data were normally distributed, one-way analysis of variance (ANOVA) was used. If the data were not normally distributed, a Kruskal-Wallis test was used. There was a minimum of three biological replicates analyzed per data set. Statistical significance was defined as a p-value less than 0.05. Tukey’s post-hoc test was used to compare differences between each treatment group. For embryo sexing data, qPCR expression levels were measured and normalized relative to the reference genes. As blastocyst sex was determined based on the presence or absence of mRNA expression, statistical analyses were performed using a Chi-Squared Post Hoc test for nominal/categorical variables to compare the average number of male and female blastocysts per group.