All experiments were performed at Jilin Agricultural University, China. Ethical approval for the present study was obtained from the Ethics Committee of Jilin Agricultural University.

The porcine ovaries were obtained from a local slaughterhouse and sent to the laboratory within 2 h at 25–30°C in 0.9% saline (w/v). Cumulus-oocyte complexes (COCs) were drawn from follicles with a diameter of 3–6 mm with an 18-gauge needle attached to a 20-ml disposable syringe. Before COCs were cultured in a four-well dish that contained 500 μl of IVM medium (NCSU-37 supplemented with 10 IU/ml pregnant mare serum gonadotropin (PMSG), 10 IU/ml human chorionic gonadotrophin (hCG) and 10 μg/ml epidermal growth factor (EGF)) in each well, they were washed 3 times with TL-HEPES-PVA (polyvinyl alcohol, 0.1%). They were incubated at 38.5°C under 5% CO₂ in 95% humidified air for IVM. After 22 h of culture, the previous medium was replaced with the same medium without PMSG/hCG, and then the culture was continued for 22 h.

First, the control group and the treatment group supplemented with 50 nM TG were cultured. After 22 h of culture, the media in the control group and one of the TG treatment groups were replaced with the new control medium to eliminate TG, and the media in the remaining TG treatment groups were replaced with 6 mM Gly medium (Control group, TG group and TG + 6 mM Gly group). PMSG/hCG were no longer added to the new medium.

The diameter expansion of cumulus cells was measured by microscopy (ix70, Olympus, Tokyo, Japan). COCs were measured using the two vertical diameters at maturity in all groups, and a total of 30 were measured in each group. In addition, the cumulus cells were denuded by 0.1% hyaluronidase. The marker of nuclear maturation is the discharge of the first polar body (PB1), and mature oocytes were collected for subsequent trials (Supplementary Figure S1).

The collected oocytes were activated with a direct current pulse (1.2 kV/cm, 30 μs) utilizing a BXT Electro-Cell Manipulator 2001 (BXT Inc., San Diego, CA) in 0.28 mol/L mannitol containing 0.1 mM MgSO₄ and 0.05 mM CaCl₂. After PA, oocytes were cultured in NCSU-37 medium [4 mg/ml bovine serum albumin (BSA) and cytochalasin B (CB)] for 3 h and then transferred into in vitro culture medium in the same culture environment as IVM for 2 days to record their morphology and cleavage rate. The blastocyst formation rate was recorded on day 7.

The intracellular GSH and ROS levels of oocytes at the metaphase II (MII) stage were measured by CellTracker Blue CMF2HC (4-chloromethy-6.8-difluoro-7-hydroxycoumarin; Invitrogen) and H₂DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) fluorescence assays. Fluorescence staining was performed as described in a previous report (Yu et al., 2021). The fluorescence was observed under a fluorescence microscope (Olympus Tokyo, Japan) and imaged with ImageJ software.

The mitochondrial ΔΨ m was analysed by a mitochondrial membrane potential assay kit with JC-1 according to the manufacturer’s protocol to determine the change in ΔΨ m in oocytes. After washing, mature-denuded oocytes were washed three times with PBS–PVA and stained for 15 min with fluorescent dyes at 38.5°C. Then, the oocytes were washed three times, examined with a Nikon fluorescence microscope (Tokyo, Japan) and analysed by ImageJ software.

The [Ca²⁺]_i levels were detected as described in a previous report (Yu et al., 2021). Briefly, oocytes were stained with 5 μM Fluo-3/AM (Beyotime) for 40 min at 38.5°C and then washed three times with PBS-PVA (without Ca²⁺). The [Ca²⁺]_ER levels were assessed by Mag-Fluo-4 AM, and the oocytes were incubated with 10 μM Mag-4 AM for 15 min at 37°C. The cells were washed three times with PBS-PVA and incubated without Mag-Fluo-4 AM. The [Ca²⁺]_m levels were stained with 10 μM Rhod-2 AM, and the dyeing operation was the same as that for Mag-Flou-4 AM. Finally, the fluorescence intensity was detected with an Olympus fluorescence microscope (Tokyo, Japan) and analysed by ImageJ software. The final average fluorescence was calculated from the fluorescence intensity of all oocytes.

ER richness were evaluated using the ER-Tracker Red (Molecular Probes). And fluorescently labelled ER-Tracker Red was used to quantify the ER mass. Mature-denuded oocytes were washed in PBS-PVA three times and stained with ER-Tracker Red (1:200) for 20 min at 37°C. Images of oocytes were captured using an Olympus fluorescence microscope (Tokyo, Japan) after washing three times. ImageJ software was used to quantify ER mass. The fluorescence signal was calculated as the average intensity after background subtraction.

The Caspase 3 Activity and Apoptosis Detection Kit for Live Cells (Beyotime) was used to determine Caspase 3 activity and Annexin-V signals in oocytes following the instructions. The oocytes were directly added to the solution prepared using the kit and incubated for 20 min for fluorescence microscopy detection (Olympus, Tokyo, Japan). Then, the oocytes were viewed with a fluorescence microscope, and the fluorescence intensities of oocytes were assayed by the ImageJ program.

MII oocytes subjected to different treatments were immobilized with 4% paraformaldehyde for 30 min at room temperature (RT) and then washed three times in PBS-PVA for 5 min each time. Next, MII oocytes were permeabilized with 1% Triton X-100 at RT for at least 10 min and transferred to blocking solution (PBS +3% BSA) for 1 h. For IP₃R1 or VDAC1 staining, oocytes were incubated overnight at 4°C with primary antibodies [rabbit polyclonal anti-IP₃R1 (Invitrogen, PA1901), 1:500; VDAC1 rabbit monoclonal antibody (Beyotime, AF1027), 1:200]. After three washes in PBS-PVA for 10 min each time, oocytes were incubated with goat antirabbit IgG [(Invitrogen A27034), 1:250] for 1 h at RT in the dark. Then, oocytes were stained with 10 μg/ml Hoechst 33,342 at 37°C for 10 min. Finally, samples were mounted on glass slides. Distribution patterns of IP₃R1 and VDAC1 were identified by laser-scanning confocal microscopy (Leica TCS SP5). Twenty oocytes were assayed in each group.

For analysis of gene expression, 50 mature denuded oocytes in each group were placed in 1.5-ml centrifuge tubes and stored at −80°C until RNA was extracted utilizing the Arcturus^® PicoPure^® RNA Isolation Kit (Thermo Fisher Scientific, KIT0204, United States) according to the manufacturer’s instructions. The concentration of extracted RNA was measured employing a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific). The PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) was utilized to synthesize complementary DNA (cDNA). The CFX96 Real-Time PCR Detection System was utilized for real-time PCR (Yu et al., 2021). Specific primer sequences (Table 1) were devised utilizing the NCBI database. Relative gene expression levels were calculated utilizing the 2^−∆∆CT strategy, and 18S ribosomal RNA (18S) served as the loading control. For helpful comparison, the average expression level of each gene was normalized to the control group.

All experiments were repeated at least three times. The data are expressed as the mean ± standard error of the mean (SEM). Statistical analysis utilized univariate analysis of variance (ANOVA) followed by Duncan’s multiple range test conducted with the IBM-SPSS 23.0 statistical program. To ensure the homogeneity of variance, the percentage data were arcsine-transformed before analysis. Differences in gene expression were compared by Student’s t-test, and p < 0.05 was considered statistically significant.

We first detected the effects of Gly on cumulus expansion and the oocyte maturation rate. The results showed that Gly is not only conducive to cumulus diffusion but also improves the IVM rate of porcine oocytes (Supplementary Figure S2).

Then we detected the effects of Gly on cumulus expansion and the oocyte maturation rate after exposure to TG. The detection indexes were the expansion diameter of cumulus cells and the PB1 extrusion rate. The results showed that both cumulus expansion and the oocyte maturation rate were decreased by TG. The diameter of COCs was measured and recorded after IVM (Figures 1A,B). As expected, the cumulus expansion diameter was significantly reduced in the TG-treated group (p < 0.05). Importantly, the cumulus expansion of oocytes exposed to TG returned to the control level after Gly treatment.

The oocyte maturation rate and death rate showed similar results. Compared with the rates of the control group and TG + 6 mM Gly group (73.05 ± 3.01, 67.30 ± 1.98), the maturation rate was decreased significantly in the TG treatment group (54.63 ± 2.2, p < 0.05). There were no significant differences between the control group and the TG + 6 mM Gly group. The death rate of the TG group was significantly higher than the death rates of the control group and the TG + 6 mM Gly group, but the rescue group was not significantly different from the control group (Table 2).

Our results indicated that Gly could effectively restore the cumulus expansion and maturation of porcine oocytes that were exposed to TG.

ER stress influences mitochondrial function. ROS are an index of the oxidative stress in mitochondria. To determine whether TG could induce oxidative stress in oocytes and whether Gly could alleviate this situation, we detected the GSH and ROS levels in porcine oocytes after TG treatment and Gly supplementation (Figure 2A). The GSH levels of the 50 nM TG group were significantly lower than those of the control group (p < 0.05). After adding Gly, the level of GSH returned to that of the control group level in porcine oocytes subjected to IVM (Figure 2B). In contrast, the ROS levels were significantly higher in the TG group. However, the addition of Gly can downregulate the level of ROS and the increase in ROS caused by ER stress induced by TG (Figure 2C) (p < 0.05).

We measured the mitochondrial ΔΨ m, which is an indicator of mitochondrial function in porcine oocytes. Because ER stress may provoke mitochondrial dysfunction. Plot (Figures 3A,B) shows that ΔΨ m was significantly affected in the culture group with only TG added, and the addition of Gly rescued the significant decrease in mitochondrial ΔΨ m caused by ER stress (p < 0.05). These results indicated that the addition of Gly could alleviate mitochondrial dysfunction, which was triggered by TG-induced ER stress.

In fact, the addition of Gly alone significantly increased the level of ER in oocytes compared with the blank control group. In ER stress related genes, the results showed that CHOP, Xbp1, and ATF4 were significantly decreased in mature oocytes after Gly treatment (Supplementary Figure S3). After determining the role of Gly, to detected the effects of Gly on ER function in oocytes in exposure to TG, we tested the level of ER in cells by ER-Tracker Red. Compared with the control group and Gly group, the ER levels showed an obvious downward trend in the TG group (p < 0.05). However, the addition of Gly successfully improved the TG-induced decrease in ER levels (Figures 4A,B). To further confirm the effect of Gly on ER function, we detected the mRNA expression of typical genes associated with ER stress by qPCR. As shown in the results, Bip/Grp78, ATF4, ATF6, Xbp1, and CHOP were significantly increased in oocytes that received TG relative to the control oocytes. We then examined those putative central genes in the Gly treatment group, and the mRNA expression levels of UPR signalling pathway genes were normal (Figure 4C) (p < 0.05). Real-time PCR analysis revealed the induction of ER stress by TG, which increased the expression of most UPR marker genes. We hypothesized that there was a protective role of Gly on TG-induced ER stress. Based on these results, we determined that porcine COCs at 44 h would be used for subsequent experiments.

As shown in Figure 5A, we tested the Ca²⁺ levels in porcine oocytes, including [Ca²⁺]_i levels, [Ca²⁺]_ER levels and [Ca²⁺]_m levels. Our Fluo-3AM (5 μM) fluorescent staining evidenced that the level of [Ca²⁺]_i in the TG group was visibly higher than those of other groups (p < 0.05), reaching almost twice as high as the control group. In the TG + 6 mM Gly group, although the level of [Ca²⁺]_i was still higher than that in the control group, it was not significantly different from that in the control group (Figure 5B).

Importantly, to detect the whole dynamic balance of calcium ions in cells, we also stained the calcium ions in the ER and mitochondria with specific probes. The results showed that the trend for the mitochondrial calcium changes in the three groups was the same as that of [Ca²⁺]_i levels, while the calcium in the ER was opposite to the first two. The [Ca²⁺]_ER in the TG group decreased significantly, while the calcium ions in the control group and Gly group were significantly higher than those in the TG group (Figure 5C). In addition, we found that the levels of [Ca²⁺]_i and [Ca²⁺]_m were opposite to the mitochondrial ΔΨ m levels. In the TG group, the levels of [Ca²⁺]_i and [Ca²⁺]_m were increased significantly (Figure 5D), while the mitochondrial ΔΨ m of mitochondria decreased significantly, indicating that a high level of [Ca²⁺]_i was not conducive to the normal function of mitochondria in oocytes. In addition, to further determine the changes in calcium ion levels, we detected calcium ion-related genes by qPCR. The mRNA expression of calreticulin (CALR) was also significantly increased in the TG group (Figure 5E). To maintain the calcium ion balance in cells, [Ca²⁺]_ER may flow out violently after ER stress, leading to an increase in the calcium concentration in the cytoplasm and mitochondria. The results showed that in TG-induced ER stress, the addition of Gly could effectively regulate the dynamic balance of calcium, ameliorate the excessive [Ca²⁺]_i and [Ca²⁺]_m caused by ER stress, and inhibit [Ca²⁺]_ER outflow. Adding Gly could improve mitochondrial dysfunction and promote oocyte maturation. Therefore, the addition of 6 mM Gly could effectively change oocyte Ca²⁺ levels, which were increased by TG.

In order to confirm the regulatory effect of Gly on calcium ion, Gly was added separately and compared with the blank control group. The results showed that added Gly could decrease the [Ca²⁺]_i and [Ca²⁺]_m but could increase the [Ca²⁺]_ER, so Gly could affect calcium levels (Supplementary Figure S4).

Since apoptosis will be induced by some stress reactions, we then examined the activity of the apoptosis-associated protein Caspase 3 and Annexin-V signals to prove whether ER stress induced by TG could cause apoptosis in oocytes in vitro. As shown in the results, the almost invisible Caspase 3 expression and Annexin-V signals were very weak in the control group. TG treatment greatly increased Caspase 3 expression and Annexin-V signals in comparison with controls levels of fluorescence intensity (Figure 6A). TG-treated oocytes showed clear positive Caspase 3 and Annexin-V signals, indicating the occurrence of apoptosis. Remarkably, we found that adding Gly altered this phenomenon, and the Caspase 3 activity and Annexin-V signals were normalized in oocytes from the TG + 6 mM Gly group (Figures 6B,C).

On the other hand, to improve the accuracy of apoptosis detection caused by ER stress induced by TG, we evaluated the mRNA expression of genes related to apoptosis in mature oocytes. As shown in the results, Bax, Cyto C, and Caspase 3 were significantly upregulated, but the Bcl2 gene was downregulated in the TG group compared to the control group (p < 0.05). Conversely, the expression of proapoptotic genes was obviously decreased after Gly treatment (Figure 6D). These data indicate the protective role of Gly against TG-induced apoptosis.

In maintaining intracellular calcium homeostasis, the IP₃R1 and VDAC1 proteins are important proteins. Our results showed that Gly inhibited the expression of IP₃R protein and the mRNA levels of IP₃R1 (Supplementary Figure S5) and promoted the aggregation of cells in the cortex. In order to deepen the study of the effects of Gly on calcium signalling pathway in oocytes in exposure to TG, we tested the localization of IP₃R1 and VDAC1. Normally, IP₃R1 and VDAC1 were distributed in the cortical portion in the control group. Nevertheless, the regular distribution of IP₃R1 and VDAC1 was disturbed, and they were observed in the cytoplasm in oocytes in the TG group. Here, we showed that TG-induced ER stress led to the cortical redistribution of IP₃R1 and VDAC1, as oocytes that matured in the presence of TG lacked IP₃R1 and VDAC1 cortical clusters. These proteins were evenly distributed in the cytoplasm rather than accumulated in the cortex. Furthermore, the distribution patterns of IP₃R1 and VDAC1 in the Gly group redistributed into the cortical portion, similar to those of the controls (Figures 7A–D). The qPCR analysis further verified that the mRNA expression of IP₃R1, VDAC1 and Grp75 was significantly increased in the TG group (Figure 7E) (p < 0.05). Their change trends showed consistent results.

To detect the early embryonic development of pigs treated with TG or Gly, MII oocytes were parthenogenetically activated. From the development morphology of blastocysts, the blastocysts in the TG group were significantly smaller than those in the other two groups (Figure 8A). The statistical results showed that there was an apparent decline in the cleavage rate in the TG group, but Gly reduced this damage, and the cleavage rate even reached the level of the control group (89.61 vs. 71.03% and 84.43%, p < 0.05) (Figure 8B). The blastocyst rate also showed the same result in which adding 6 mM Gly increased the rate to a level that was higher than that in the TG group but not the control group (67.27 vs. 53.59% and 55.4%, p < 0.05) (Figure 8C). And the total cell numbers shown the results consistent with the above two indicators (Figures 8D,E). Based on these results, we hypothesized that adding 6 mM Gly could ameliorate ER stress and was beneficial to oocyte IVM and the subsequent development of PA embryos.