Drug

All the drugs were obtained from Sigma but without special instructions.

Collection and transport of test materials

The cattle ovaries used in this study were obtained from a slaughterhouse in China (Yanji). The ovarian specimens were placed in a thermos (temperature 23–30°C) and transported to the laboratory within 3 h. The surrounding tissues were excised in sterile saline (0.9% sodium chloride, 75 μg/ml of penicillin G, and 50 μg/ml of streptomycin). To address the formation of sulfate, the specimens were washed until the solution became clear, and the entire process was performed at 25°C.

Acid of oocytes and cultures in vitro

Fresh ovaries were cleaned once or twice with sterile saline (25°C), and the ovarian surface was disinfected with 75% alcohol. After the water absorbing paper was saturated, the specimen was washed once with physiological saline and then air dried. An 18-g needle was used to aspirate the cumulus–oocyte complexes (COCs) from follicles 2–8 mm in diameter, and the COCs were precipitated in another tube for 10 min. The supernatant was removed, and 2–3 ml of in vitro maturation (IVM) culture solution (90% TCM-199, 10% FBS, 25 μg/ml of gentamycin, 0.2 mM pyruvate, 1 μg/ml of FSH, 0.57 mM cysteine, 1 μg/ml of oestradiol) was added to the precipitate. The COCs were added to a disposable culture dish containing IVM solution and observed under a microscope. The COCs were washed three times using IVM culture solution. To evaluate whether glyphosate had dose-dependent effects on the in vitro culture, different concentrations (0, 10, 20, 50, 50 or 100 mM) of glyphosate were added, and the COCs were incubated at 38.5°C for 24 h. After determining the glyphosate culture concentration, glyphosate was added to the IVM solution and cultured oocytes according to the above method.

In vitro fertilization and in vitro development

Oocytes that were matured and cultured in vitro for 24 h were selected, and the outer cumulus cells of COCs were removed by gentle blowing with 1% hyaluronidase (HY). Next, the oocytes were cleaned 3–5 times with in vitro semen (IVF, Japan), and then the oocytes were placed into 100 IVF droplets and then in an incubator for fertilization. The semen were removed from the liquid nitrogen, left to stand at room temperature for 20 s, and then thawed in a water bath at 38°C for 20 s. The thawed sperm were placed in a 1.5-ml sperm product (BO) centrifuge. After the completion of sperm capacitation, the bottom fraction of the semen was injected into 100 IVF droplets, mixed with the oocytes, covered with mineral oil, cultured in a 38°C, 5% CO₂ incubator, and cultured in vitro to complete IVF. After oocyte fertilization in vitro, the oocytes were blown with 1% HY to remove all cumulus cells and obtain bare oocytes. Afterwards, the naked oocytes were washed five times with 0.4% bovine serum albumin (BSA)-CRI (IVC) culture media at 10 μl of IVC droplets per wash and covered with mineral oil. Next, the cells were cultured at 38.5°C in a 5% CO₂ incubator until the 8-cell stage (72 h), and then statistical analyses of the ovulation and 8-cell rates were performed. The cultivation in 10% FBS-CRI continued to the seventh experimental dose, and the test was repeated at least three times.

Determination of meiosis progression in oocytes

Fresh bovine oocytes were cultured in IVM for 22 h. Ruptured oocytes were eliminated with 0.1% HY, and cumulus cells were removed to obtain denuded oocytes. The cells were washed three or four times with 0.1% BSA-PBS, and denuded oocytes were incubated with Hoechst 33342 stain at 37°C for 20 min. Next, BSA-PBS was used to wash the residual stain, and a fluorescence microscope was used to photograph and count the number of discharged polar bodies. As shown in Figure 1, the nuclei of the stained oocytes were evaluated under a fluorescence microscope.

Figure 1.

Schematic diagram of meiosis of bovine oocyte. (A) Oocytes with diffusion or slight concentrated chromosomes are classified as the GV phase or GVBD phase. (B) Oocytes with clumped or strongly condensed chromatin form individual divalent (prometaphase) or metaphase plates, but irregular networks without polar bodies are classified as being in the MI phase. (C) Oocytes with polar bodies or two bright chromatin spots are classified as being in the MII stage. Blue, chromosome. Scale, 40 μm.

Immunofluorescence staining

The intracellular ROS content in oocytes was detected using the fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime, China). The cumulus cells of COCs were removed, and the naked oocytes were incubated in 10 μM DCFH-DA in the dark at 37°C for 30 min. After washing with 0.1% BSA-TC M199 three times, quantitatively labelled oocytes were observed using a fluorescence microscope (Nikon, JP) with a 460-nm UV filter. Finally, Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA) was used to analyze the fluorescence intensity of the oocytes (green fluorescence, UV filter, 490 nm). Each experiment was repeated five times.

To assess the MMP, a JC-1 (Beyotime, China, AUM) assay kit was used. This process was performed according to the instructions given in the kit. The oocytes were incubated in a working solution containing 10 μM JC-1 in the dark at 38.5°C for 30 min. After washing with JC-1 buffer solution, the oocytes were observed in the same scan of each sample under the microscope. JC-1 aggregates (red fluorescence) were detected using the tetramethylrhodamine (TRITC) channel, indicating high membrane potential, while the JC-1 monomer (green fluorescence) was detected using the FITC channel, indicating a low membrane potential. The ratio of aggregates (red fluorescence) to monomers (green fluorescence) was calculated to quantify changes in MMP.

The colliculus cells of the COCs were removed to obtain denuded oocytes, and then the naked oocytes were washed with 1% BSA-PBS two or three times. The denuded oocytes were placed in 10 μL of the cell-tracking blue-fluorescent dye 4-chloromethyl-6,8-difluoro-7-hydroxy-coumarin at a concentration of 10 μmol/l. The oocytes were incubated at 37°C for 30 min in the dark and then washed with 1% BSA-PBS. They were observed and photographed under a fluorescence microscope using blue fluorescence.

RT-PCR

Each group of 30 oocytes cultured for 24 h was removed from the cumulus and washed with 1% BSA-PBS, and the samples were added to 50 μl of lysis buffer (5 mmol/ml of dithiothreitol, 20 U/mol of RNase inhibitor, 1% NP-40) according to the product’s instructions. The samples were fast frozen in liquid nitrogen and stored at −80°C for later use. mRNA was extracted using the Dynabeads mRNA Direct Kit according to the specifications. Next, a Dynabeads mRNA Direct Kit was used to synthesize cDNA by reverse transcription of the improved mRNA. Real-time fluorescence quantitative PCR was then performed using a Kapa SYBR^® FAST kit, and qPCR was performed using CFX Connect Optics Module PCR. The 20 μl PCR system comprised the following: 2 μl of cDNA, primer (comprising 1 μl each of the upstream primer and downstream primers), 10 μl of SYBR GREEN, and 7 μl of DDH₂O. The PCR procedure was as follows: predenaturation at 95°C for 3 min, 95°C for 3 s, 60°C for 30 s, and 72°C for 20 s. Forty target genes were identified, such as SOD1, SOD2, SIRT1, SIRT2, SIRT3, Caspase-4, Caspase-3, Survivin and BCL-XL. The reference gene was Actin. The primer sequences used to amplify each gene are shown in Table 1, and quantitative RNA data were statistically analyzed using the 2^−ΔΔCt method.

Table 1.

Primer sequences expressed in oocytes were analyzed by RT-PCR

Note: annealing temperature is 60°C. F: forward primer. R: reverse primer.

Data processing and analysis

SPSS 19.0 (Inc., Chicago, IL, USA) software was used for statistical analysis, and all the test results were expressed as means ± standard deviation (SD). A highly significant difference between the two groups was indicated by **P < 0.01, and a significant difference was indicated by *P < 0.05. An independent sample t-test was used for comparisons between the two groups. GraphPad Prism 6.01 software was used to generate the graphics. Fluorescence intensity analysis was performed using ImageJ software. The test was repeated at least three times.