The treatment of porcine ovaries used in this study followed the guidelines of the International University–Vietnam National University, Ho Chi Minh City, Viet Nam.

Porcine ovaries were collected from local slaughterhouses and transported to the laboratory within two hours in phosphate-buffered saline (PBS; Dulbecco), supplemented with 100 IU penicillin and 100 mg/ml streptomycin. The SAFs (2–3 mm) and the LAFs (4–6 mm) were dissected from the ovaries. To collect EAFs, the ovarian cortex was sectioned into slices 1–2 mm thick using a surgical blade (No. 21). These slices were then placed under a microscope to dissect using a surgical blade (No. 11). After dissection, the follicles were washed in 25 mM HEPES-buffered TCM-199 containing 0.1% polyvinyl alcohol. The follicles were then placed in HEPES and opened using an 18-gauge needle under a microscope. Healthy OCGCs derived from these follicles, characterized by multilayered cumulus and granulosa cells with uniform ooplasm, were selected for the experiment.

Agarose gel was used to mimic the in vivo microenvironment of the porcine ovarian matrix. To prepare a 1.5% agarose solution, mix low-gelling-temperature agarose (A9539) powder with PBS at a concentration of 1.5% (w/v), heat until fully dissolved, then transfer the hot solution to 1.5 mL centrifuge tubes and store at 4°C. For coating a 96-well dish, reheat the stored agarose gel in boiling water for 5 minutes to remelt it, then apply 30 µL of the molten solution to the bottom of each well. The agarose hardens at room temperature for 10 minutes, and before use, the equilibrated medium is replaced with fresh medium8.

The OCGCs from EAFs were transferred to an IVG medium and cultured under different IVG conditions for 4 days in an incubator with 5% CO₂ in humidified air at 38.5°C. Every well was filled with 120 µL IVG medium and covered with 50 µL mineral oil. Each well could culture 3–5 OCGCs.

The basic IVG medium contained α-MEM supplemented with 3% polyvinylpyrrolidone, 5% follicle fluid, 10% fetal bovine serum, 0.91 mM NaPy, 3 mM hypoxanthine, and 1 µg/mL estradiol. This study was divided into two main experiments, with each IVG culture medium supplemented with different concentrations of antioxidants. In the first experiment, groups of Mela were divided into three different concentrations (1 µM/mL, 10 µM/mL, and 100 µM/mL) to determine the optimal concentration for the next experiment. In the next experiment, Asta was combined at three different concentrations (0.05 µM/mL, 0.1 µM/mL, and 0.25 µM/mL) with the optimal concentration of Mela.

The OCGCs were cultured in a dish coated with agar. Half of the culture medium was replaced after 2 days. After IVG, the survival rate of OCGCs (n = 54) was evaluated by calculating the percentage of healthy cultured OCGCs. Oocyte diameter (n = 25) was measured after removing the surrounding cells, and both horizontal and vertical dimensions were recorded using a digital microscope.

The mRNA extraction and RT-PCR were performed on the same day to prevent mRNA degradation. According to the manufacturer's instructions, total mRNA was extracted from samples (10–15 oocytes) using the Dynabeads™ mRNA DIRECT™ Kit (Invitrogen, Thermo Fisher Scientific). RT-PCR was performed using the Applied Biosystems™ Power SYBR™ Green RNA-to-CT™ 1-Step Kit (Invitrogen, Thermo Fisher Scientific), and the relative abundance of target mRNAs was examined with specific primers. Act- β was used as the reference gene. The primer sequences were listed in Table 1. Each RT-PCR reaction had a total volume of 10.0 µL, consisting of 3.0 pmol/µL of each primer, 5 µL Master Mix, 0.08 µL Enzyme Mix, and 1 µL mRNA template. RT-PCR was processed using a thermocycler (Applied Biosystems™ QuantStudio™ 5 Real-Time PCR System, 96-well, 0.2 mL) over 45 cycles. The RT-PCR cycle conditions were performed as follows:

Table 1

Genes	Primer sequence (5'-3')	Product size
Act- β	Forward: ATCGTGCGGGACATCAAGGA	179bp
	Reverse: AGGAAGGAGGGCTGGAAGAG
BMP15	Forward: ACCATGGTGAGGCTGGTGAG	178bp
	Reverse: CATGGCAGGAGAGGTGGAAG
CAT	Forward: ACGTTGGAAAGAGGACACCC	137bp
	Reverse: TCCAACGAGATCCCAATTACCA
SOD-1	Forward: CACTGTGTACATCGAAGATTCTGTGA	97bp
	Reverse: GCCCAAGTCATCTGGTTTTTCA
GPX4	Forward: ACGGGCACATGGTGAACCT	85bp
	Reverse: ACCTCCGTCTTGCCTCATTG

Each group of cultured oocytes was washed twice in an electro-permeabilization (EP) medium containing 0.25 M D-glucose, 100 µM CaCl₂, 100 µM MgSO₂ and 0.1% PVP. They were then transferred into a drop of EP medium supplemented with 10 mM BrUTP for 2 minutes. The oocytes were placed in 20 µL of EP medium containing 10 mM BrUTP between two parallel stainless steel electrodes in a chamber. Electro-permeabilization was conducted with one direct current pulse at 30 V and 80 µs. Two minutes post-permeabilization, the oocytes were cultured in IVG medium for 1 hour at 38.5°C with 5% CO₂. Oocytes were fixed and immunostained, as described in a previous study36. Indirect immunofluorescence detected newly synthesized transcripts incorporating BrUTP and histone methylation H3K4 (Me-H3-K4). The primary antibodies used include mouse monoclonal anti-bromodeoxyuridine (6 µg/mL, Roche-1117037600) and rabbit anti-methyl-histone H3K4. The secondary antibodies, Alexa-Fluor-568-labeled goat anti-mouse IgG and Alexa-Fluor-488-labeled goat anti-rabbit IgG, were used. DNA was counterstained with 2 µg/mL 4,6-diamidino-2-phenylindole (DAPI) for 30 minutes. The levels of Me-H3-K4 expression and BrUTP were analyzed based on fluorescence intensity using the Nikon analysis program. Fluorescence intensity within each nucleus was measured across five different regions, and the cytoplasmic background value was subtracted. The resulting value was then multiplied by the nuclear volume to estimate the total fluorescence for the nucleus. Due to the limited sample size, no statistical analysis could be performed.

Each treatment was replicated at least 3 times. Descriptive statistics were performed to assess normality and homogeneity of variance prior to parametric analyses, using skewness values as part of the evaluation. One-way ANOVA was performed with the Statistical Package for Social Sciences (SPSS) version 20, with a significance level of p < 0.05. Data were reported as the mean ± standard error of the mean (SEM). RT-PCR data were analyzed using the Log2FC method, with statistically significant differences at p < 0.05. Based on the Log2FC method, the non-treated group was used as the reference. Therefore, the expression on the graph shows the increase or decrease relative to the non-treated group.

Figure 1

After IVG culture, antrum formation in OCGCs did not show significant differences between the groups supplemented with Mela (Figure 1B). Nevertheless, Mela positively affected oocytes and granulosa cells, improving survival after 4 days of IVG. The OCGCs supplemented with Mela 10 μM had the highest survival rate, yet no significant differences were observed among the treatment groups (Figure 1C). Based on gene expression studies, Mela slightly promoted BMP15 expression in growing oocytes (Figure 1D). In addition, Mela enhanced the expression of the CAT and GPX4 genes, thereby increasing antioxidant enzyme activity and reducing the effects of ROS. However, the differences between each treatment group were negligible, resulting in nearly identical oocyte qualities among the treatment groups.

Figure 2

The addition of combined Mela 1μM + Asta did not significantly affect antrum formation after 4 days of IVG (Figure 2A). However, supplementation with combined Mela 10 μM + Asta reduced the ability to form an antrum after the IVG stage (Figure 2D). The combined supplementation of Mela 1 μM + Asta significantly increased the oocyte survival rate after the IVG stage. Nevertheless, no significant differences were detected among the treatment groups (Figure 2 B). Similarly, the combined Mela 10 μM + Asta supplementation increased survival after 4 days of the IVG stage (Figure 2E). However, the quality of oocytes after 4 days of culture showed significant differences. In this case, supplementation with combined Mela 1 μM + Asta may enhance the expression of specific genes. In contrast, the combined Mela 10 μM + Asta groups generally decreased the expression of most of the genes studied (Figure 2C, F). OCGCs supplemented with combined Mela 1 μM + Asta 0.05 μM showed the highest quality after 4 days of IVG, yielding results comparable to fully grown oocytes (LAF). In contrast, groups supplemented with higher concentrations of Mela and Asta exhibited a significant reduction in quality. Both combined Mela 1 μM + Asta and Mela 10 μM + Asta groups demonstrated an increase in CAT expression; however, it was still lower than LAF. OCGCs supplemented with combined Mela 1 μM + Asta showed increased expression of SOD1 and GPX4, while a decrease was observed in the combined Mela 10 μM + Asta group. Among the treatment groups, the combined Mela 1 μM + Asta 0.05 μM group exhibited the highest expression for all genes studied. Therefore, supplementation with combined Mela 1 μM + Asta 0.05 μM significantly increased the oocyte survival rate and quality after IVG.

Figure 3

SAF is the preceding stage of LAF; therefore, growing oocytes isolated from SAF must undergo a pre-IVM phase to attain competence equivalent to that of growing oocytes isolated from LAF5. Since the growing oocytes isolated from EAF are of a size comparable to those isolated from SAF, SAF is used for comparative evaluation after 2 days of IVG (Figure 3C). After 2 days of IVG, oocytes supplemented with Mela increased Me-H3-K4 expression compared to the non-treated group (Figure 3B). After 4 days of IVG, Me-H3-K4 levels decreased in all treatment groups as well as in the non-treated group. In Figure 3D, Me-H3-K4 is observed to be concentrated only in the heterochromatin ring, indicating the repression of transcriptional activities. This suggests that the addition of antioxidants does not lead to abnormalities in chromosome configuration26. Therefore, the evaluation of Me-H3-K4 was only performed at IVG day 2. Due to the increase in gene activity, a significant rise in oocyte size was also observed after 4 days of IVG (Figure 3E). Although oocytes exhibited a considerable increase in size following IVG, their diameter remained smaller than that of LAF. This suggests that Mela supplementation during IVG can enhance oocyte quality; however, it has not yet reached the level of LAF.

Figure 4

The groups supplemented with the combined Mela 1 μM + Asta 0.05 μM and the combined Mela 10 μM + Asta 0.05 μM showed an increase in Me-H3-K4 expression compared to the other groups (Figure 4B, C). However, both the combined Mela 1 μM + Asta 0.05 μM and combined Mela 10 μM + Asta 0.05 μM groups showed lower intensity than SAF. Therefore, while the oocytes cultured for 2 days had a size similar to SAF, their quality was still lower than that of SAF.

Figure 5

The transcriptional activity of the oocytes supplemented with the combined Mela 1 μM + Asta 0.05 μM was higher, as indicated by the greater BrUTP intensity compared to the other two groups, although the combined Mela 10 μM + Asta 0.05 μM group showed a trend towards higher intensity than the non-treated group (Figure 5A, B). Therefore, supplementation with combined Mela 1 μM + Asta 0.05 μM enhances global transcription, improving oocyte quality through increased mRNA storage. Because global transcription only occurs during the FC/SC stage, by day 4 the oocytes have reached the GV stage, where transcription activity has ceased. Therefore, the assessment of global transcription cannot be performed on day 4. Corresponding to the increase in global transcription, the groups supplemented with appropriate antioxidants also showed a significant increase in size compared to the non-treated group. Among these, the combined Mela 1 μM + Asta 0.05 μM group resulted in a notable size increase compared to the other groups, although it was still smaller than LAF (Figure 5C). Therefore, antioxidant supplementation improved both oocyte quality and size, but it has not yet reached the level of LAF.

Our study reported that supplementation with the combination of Mela and Asta at appropriate concentrations improved oocyte quality by increasing Me-H3-K4 and global transcriptional activity of growing oocytes after 2 days of IVG. Finally, there was a significant increase in the expression of BMP15, an important folliculogenesis regulator gene, as well as antioxidant genes (CAT, SOD1, GPX4), thereby reducing the negative effects of ROS on oocytes after IVG. Our culture system supported the growth of oocytes isolated from EAFs to achieve a size and quality equivalent to fully grown oocytes isolated from LAF.

Mela has been shown to mediate its effects on oocytes through MT1 and MT2 membrane receptors, binding to melatonin receptors in the plasma membrane and binding to intracellular proteins such as calmodulin and calreticulin, which helps increase the follicle survival rate13. It has proven effective in IVM culture by stimulating the resumption of meiosis37. Additionally, Mela has been demonstrated to improve granulosa cell quality and mitochondrial activity and to reduce cathepsin B activities31, 38, 39, thereby enhancing oocyte quality. This is consistent with the significant increase in oocyte diameter and marker gene activity (Figure 1D, Figure 3E). Although the differences in marker gene activity were not substantial compared to the non-treated group, a significant improvement in oocyte size was observed in the Mela-supplemented groups. However, OCGCs supplemented with 100 µM Mela exhibited a tendency toward reduced oocyte size compared to those treated with 1 µM and 10 µM Mela. This suggests that while 100 µM Mela still has some positive effects, its adverse impacts begin to emerge, leading to a reduced overall efficacy compared to lower concentrations.

Asta neutralizes reactive oxygen, nitrogen, sulfur, and carbon species by donating electrons or forming stable bonds with free radicals to inactivate them. Asta also induces antioxidant genes and suppresses apoptotic genes in bovine embryos40. Asta may prevent glutamate-induced apoptosis by preserving redox balance and suppressing glutamate-triggered calcium influx and endoplasmic reticulum stress41. It is reported that Me-H3-K4 is highly enriched at transcription start sites, and its presence in enhancers has also been linked to increased transcriptional activity42. Thus, increased Me-H3-K4 was associated with increased gene expression. Moreover, combining both Mela and Asta in IVG is promising due to their different mechanisms of action. We found that combining Asta and Mela at optimal concentrations increased Me-H3-K4 levels and global transcription activity, resulting in improved oocyte quality after IVG. This also significantly improved the survival rate compared to the non-treated group.

Following a 4-day IVG culture, growing oocytes derived from EAFs exhibit chromatin morphology and oocyte diameter similar to those of fully grown oocytes derived from LAFs. Gene expression studies on oocytes supplemented with Mela alone showed a minimal increase compared to the non-treated group. OCGCs cultured in IVG medium supplemented with Mela and Asta also exhibited a significant increase in CAT activity, similar to supplementation with both ascorbic acid and resveratrol43 or coenzyme Q10, which increased antioxidant gene activities44. In mammals, BMP15 is known to play a role in oocyte maturation and cholesterol biosynthesis, as well as enhancing oocyte competence and promoting early embryo development45. Interestingly, combinatory supplementation of astaxanthin and melatonin at lower concentrations demonstrated enhanced BMP15 activity levels in growing oocytes. Similarly, supplementation with Eugenol resulted in significantly higher BMP15 levels46. Therefore, the significant increase in BMP15 and antioxidant gene activities contributes to a notable improvement in oocyte quality after IVG. Consequently, the supplementation of astaxanthin at a concentration of 0.05 and melatonin at a concentration of 1 during the IVG culture emerges as the most optimal culture condition.

However, supplementation with high concentrations of Mela led to a significant increase in ROS, consequently reducing oocyte quality47, 48. This is consistent with the significant reduction in the activity of antioxidant genes and BMP15 gene expression (Figure 2F). Moreover, a notable decrease in oocyte size was observed compared to the group supplemented with antioxidants at appropriate concentrations. While the supplementation of 10 µM Mela and Asta did not directly induce oocyte death, it still exhibited negative effects on oocyte quality. This has been demonstrated, as supplementation with high concentrations of Kaempferol and ascorbic acid resulted in lower oocyte quality compared to supplementation with either compound alone49. However, due to certain limitations of the study, it was not possible to clearly distinguish between healthy oocytes and those initiating the apoptosis pathway. As a result, it remains unclear whether the combination of Mela and Asta at inappropriate concentrations truly triggers apoptosis at IVG day 4.

This suggests that excessive supplementation of antioxidants may lead to the disruption of cellular metabolism. When Asta was supplemented with 1 µM Mela, enhanced transcription and Me-H3-K4 levels were observed during 2 days of IVG. It can be concluded that the beneficial effects of Asta are obtained only when combined with lower concentrations of Mela, resulting in improved oocyte quality and overall outcomes in culture. Although OCGCs have been shown to improve quality through evaluations conducted after IVG, further in-depth assessment is necessary at the pre-implantation stage. This would provide a better understanding of mRNA accumulation throughout the IVG process, which could contribute to improving embryo quality. Additionally, this study has not yet evaluated the concentrations of ROS-related compounds such as superoxide, hydrogen peroxide, hydroxyl ions, peroxyl radicals, and hypochlorite ions. Moreover, since the method for assessing nascent mRNA has not been fully optimized, the oocyte survival rate after BrUTP incorporation remains low, resulting in a limited sample size for evaluation. Therefore, additional methods should be implemented to further assess nascent mRNA synthesis capacity.

Growing oocytes isolated from EAFs can attain competence nearly equivalent to fully grown oocytes isolated from LAFs when cultured in our optimal IVG culture systems. Supplementation of antioxidants at appropriate concentrations supports increases in oocyte size, survival rate, and quality by significantly enhancing Me-H3-K4 intensity, global transcription (nascent mRNA), BMP15 expression, as well as antioxidant gene expression (CAT, SOD1, GPX4).

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4′,6-diamidino-2-phenylindole (DAPI), alpha minimum essential medium (α-MEM), analysis of variance (ANOVA), astaxanthin (Asta), beta-actin (ActB), bone morphogenetic protein 15 (BMP15), bromouridine triphosphate (BrUTP), carbon dioxide (CO₂), catalase (CAT), early antral follicles (EAFs), electro-permeabilization (EP), fetal bovine serum (FBS), filamentous/stringy chromatin (FC/SC), germinal vesicle (GV), glutathione (GSH), glutathione peroxidase 4 (GPX4), histone H3 lysine 4 methylation (Me-H3-K4), in vitro growth (IVG), in vitro maturation (IVM), large antral follicles (LAFs), log2 fold change (Log2FC), melatonin (Mela), melatonin receptor types 1 and 2 (MT1/MT2), nuclear factor erythroid 2-related factor 2 (NRF2), oocyte cumulus granulosa cell complexes (OCGCs), ovarian tissue cryopreservation (OTC), oxidative stress (OS), phosphate-buffered saline (PBS), polyvinylpyrrolidone (PVP), pre-antral follicles (PAFs), quantitative polymerase chain reaction (qPCR), reactive oxygen species (ROS), Real-time polymerase chain reaction (RT-PCR), small antral follicles (SAFs), sodium pyruvate (NaPy), standard error of the mean (SEM), Statistical Package for the Social Sciences (SPSS), superoxide dismutase 1 (SOD1), and tissue culture medium-199 (TCM-199).

We would like to express our heartfelt gratitude to the International University – National University HCMC for providing an excellent research environment in the Cell Reprogramming Laboratory. We extend our thanks to the slaughterhouse Vissan, Tan Binh, and Ut Hao for supplying pig ovaries for research.

L.C.T and L.D.T.P designed the experiments, analyzed, investigate, writing – original draft. P.T.D and N.L.B.L and L.B.A.M and C.D.S.P and N.N.T.V and N-T.N: investigated, performed, and analyzed the experiments. N.V.T and H-T.B: project administration, methodology, discussed the results, writing – review & editing. All authors read and approved the final manuscript.

This research is funded by Vietnam National University HoChiMinh City (VNU-HCM) under grant number B2022-28-01.

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Not applicable.

Not applicable.

The authors declare that they have no competing interests.