Embryos produced in vitro are lower in quality than those produced in vivo (Mahdavinezhad et al., 2019). Furthermore, the reduction of reactive oxygen species (ROS) by antioxidants in germ cells could improve their developmental potential until blastocyst (Aitken, 2020). The deficiency of antioxidant capacity within the follicle decreased the maturation competency of the oocyte and interfered with the process of fertilization and development rate (Kafi et al., 2019). The critical role of regulating mitochondrial ROS during in vitro culture (IVC) progression was demonstrated in our previous study using a mitochondrial-targeted antioxidant (Mito-TEMPO) in porcine embryos following their in vitro fertilization (IVF) (Yang et al., 2018). The mitochondria are important sources of ROS for the adenosine triphosphate (ATP) production process. Thus, we hypothesized that the protective effects of the Mito-TEMPO supplement in conjunction with the IVC medium could improve the bovine embryonic developmental competence during IVC.

Cryopreservation of oocytes and embryos is an assisted reproductive technique (ART), which is attracting attention as a means of solving female infertility (Rodriguez-Wallberg et al., 2019). The technique to cryopreserve mammalian embryos has become an especially integral part of assisted reproduction in humans and the resolution of female infertility using ART (Larman et al., 2014). Cryopreservation of IVP embryos can help the various utilization of genetically valuable animal embryos such as the transgenic of animal production and conservation of superior traits, along with increasing the opportunities for pregnancy (Ferré et al., 2020). However, vitrification-induced mitochondrial damage, oxidative stress by increasing ROS production, mitochondrial dysfunction, and low survival rates were also reported for vitrified-warmed bovine blastocysts following cryopreservation (Mesalam et al., 2020). Major problems in the current cryopreservation method are the survival rate and the compromised quality of frozen then thawed blastocysts. Therefore, to optimize the outcomes of IVP and vitrified-warmed blastocysts, the acquirement of the necessary factors is essential for bovine oocyte metabolism processes such as the response antioxidants to oxidative stress (Ali et al., 2003).

Previously, we reported that the supplementation of Mito-TEMPO in a vitrification medium has improved the survival rate and quality of vitrified-warmed bovine blastocysts (Jeong et al., 2021). Therefore, the cryopreservation-induced mitochondrial superoxide or ROS production may cause cryopreservation-associated deterioration of the embryonic cytoskeleton leading to low survival potential. Also, the commonly employed antioxidants have previously proven effective in embryo developmental capacity and protection response to cryo-injury. Therefore, using Mito-TEMPO to initiate these effects may improve the viability of bovine blastocysts after vitrification.

Many reports demonstrated the interaction between ROS production and embryo cytoskeleton structure for improving blastocyst developmental competence (Hardy et al., 2021). Additionally, ROS depletion in the embryo development process led to a significantly higher amount of F-actin (Munnamalai and Suter, 2009). F-actin structure and formation play essential roles in blastomeres division and cytokinesis in early embryonic development progression in pigs (Joe et al., 2022). Moreover, vitrification and the addition of antioxidant supplements to the frozen medium prior to vitrification can affect the embryonic cytoskeleton in pigs by improving their blastocyst developmental competence after cryopreservation (Somfai et al., 2007). Specifically, embryonic cytoskeleton stabilization after cryopreservation was closely connected to the re-expanded or survival response to disruptions in cortical F-actin or cytoskeleton formation at the adhesion-junction site after cryopreservation.

In the present study, we investigated whether bovine embryos undergoing 2 days of IVC in a 0.1 μM Mito-TEMPO supplied culture medium could improve the blastocyst developmental competence and support the recovery of the embryonic cytoskeleton from cryo-injury. Moreover, we investigated whether the survival capacity and quality of the embryos were enhanced through F-actin cytoskeleton stabilization following the freeze-thaw process with Mito-TEMPO treatment.

Chemicals

All the chemicals used in this study were purchased from Sigma Chemical Corporation (St. Louis, MO, USA) unless stated otherwise.

In vitro production (IVP) of bovine embryos

Bovine ovaries were collected from a local slaughterhouse (Gyeongsan, Gyeongbuk, Korea) and transported to the laboratory in 0.9% saline supplemented with 75 μg/mL potassium penicillin G while maintained at 30-35℃. Cumulus-oocyte complexes (COCs) were aspirated from 3- to 6-millimeter (mm) follicles with a disposable 10 mL syringe and an 18-gauge needle. Afterward, the COCs were selected along with the surrounding cumulus cells and homogeneous cytoplasm. The COCs were washed three times in Tyrode’s lactate-N-2-hydroxyethyl piperazine-N’-2-ethane sulfonic acid (TL-HEPES) and twice in the in vitro maturation (IVM) medium. The IVM medium used for oocyte maturation was TCM-199 (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco Life Technologies, Inc.), 10 IU/mL pregnant mare serum gonadotropin (PMSG), 10 IU/mL human chorionic gonadotropin (hCG), 0.6 mM cysteine, 0.2 mM sodium pyruvate, 10 ng/mL epidermal growth factor (EGF), 25 μM β-mercaptoethanol, 25 μg/mL gentamycin and 1 μg/mL 17 β-estradiol. Approximately 15 COCs were matured under paraffin oil in a 60 mm dish with 50 μL of IVM medium for 22 hours at 38.5℃ under 5% CO₂. After culturing for IVM, 15 oocytes were fertilized at 2 × 10⁶ cells/mL in 50 μL fertilization medium with previously frozen and thawed sperm (Fert-TALP). The addition of the sperm to the fertilization drops was performed in parallel with the addition of 2 μg/mL heparin, 20 μM penicillamine, 10 μM hypotaurine, and 1 μM epinephrine (PHE). The cumulus-enclosed oocytes were detached 22 hours after insemination using gentle vortexing and transferred to Charles Rosenkrans amino acid (CR1aa) medium for in vitro culture (IVC), which contained: 0.3% BSA, 1× Basal Medium Eagle (BME) amino acids, and 1× minimum essential medium (MEM) nonessential amino acids. Following 2 days of initial culturing, the cleaved embryos were further cultured for 5 days in a medium consisting of 50 μL CR1aa (with 10% FBS) at 38.5℃ under 5% CO₂. During the culture periods, presumptive embryos were treated with 0.1 μM Mito-TEMPO (Jeong et al., 2021) through its addition to the culture medium. The rates of blastocyst-stage embryos were determined on day 7. Mito-TEMPO (0.1 μM) was added in IVC medium for 2 days after IVC (at 22 hours after IVF).

Vitrification and warming procedure

Cryopreservation was performed using a Cryotop (Kitazato Supply Co, Fujinomiya, Japan) for vitrification via a slightly modified procedure previously described by Kim et al. 2020 (Kim et al., 2020). Briefly, two or three blastocysts were transferred into an equilibration solution (ES) consisting of 7.5% ethylene glycol (EG) and 7.5% dimethylsulfoxide (DMSO) in PBS supplemented with 20% FBS for 5 minutes at room temperature. Next, blastocysts were transferred into a vitrification solution (VS) consisting of 15% EG, 15% DMSO, and 0.5 M sucrose dissolved in PBS containing 20% FBS. After 40-45 seconds, the blastocysts were loaded into a Cryotop and snap-frozen in liquid nitrogen. The process from exposure in VS to cryopreservation in liquid nitrogen was completed within 1 minute at room temperature. Vitrified blastocysts were warmed by immersing the Cryotop directly into a warming solution (1.0 M sucrose dissolved in PBS containing 20% FBS) for 1 minute, before being transferred to a dilution solution (0.5 M sucrose dissolved in PBS containing 20% FBS) for 3 minutes, and then transferred into dilution solution (0.25 M sucrose dissolved in PBS containing 20% FBS) for 5 minutes at room temperature. Finally, the blastocysts were incubated for 5 minutes in a washing solution (PBS containing 20% FBS). The survival of the vitrified-warmed blastocysts was determined according to their re-expansion rates following a 24-hour recovery period in a culture medium.

Filamentous actin (F-actin) staining

Bovine blastocysts were washed with 0.1% PVA in PBS, then fixed in 4% PBS-paraformaldehyde (PFA) at room temperature for 1 hour. Afterward, they were incubated with 0.2% Triton X-100 at room temperature to promote permeability and then incubated in 0.1% PVA in PBS containing 1% BSA overnight at 4℃. Samples were then incubated in PVA in PBS with 1% BSA for 1 hour at room temperature. The sample was subsequently incubated with the secondary antibodies, rhodamine-conjugated goat anti-rabbit IgG (cat. no. 31463; Thermo Scientific, Rockford, IL), and then diluted 1:50 for 2 hours at room temperature. The samples were then washed and incubated in Hoechst 33342 (w/v) for 20 minutes at room temperature. Finally, the samples were mounted on glass slides and examined with a confocal laser-scanning microscope (Zeiss LSM 700 META; Carl Zeiss, Jena, Germany).

Statistical analysis

All percentage data obtained in this study were presented as the means ± standard deviation (SD). Moreover, the results of the DCFDA experiments were presented as the means ± standard error of the mean (SEM). All experiments were replicated three times, after which the results were analyzed by one-way ANOVA followed by Bonferroni’s Multiple Comparison Test using t-tests. All data analyses were performed using the GraphPad Prism 5.0 software package (San Diego, CA, USA). Differences were considered significant at *p < 0.05, **< 0.01, and ***< 0.001.

Effect of Mito-TEMPO on the developmental rate of bovine embryos in vitro

To observe the effects of 0.1 μM Mito-TEMPO supplement in an IVC medium, the cleaved and blastocyst developmental rate was investigated in bovine embryos (Table 1). The bovine blastocyst developmental rate of Mito-TEMPO exposed embryos increased slightly compared with the non-treated group (p < 0.05, non-treated group: 21.2 ± 4.2% vs 0.1 μM Mito-TEMPO treated group: 25.6 ± 5.6%). Additionally, the bovine expanded blastocyst formation in embryos significantly elevated in the 0.1 μM Mito-TEMPO treated group (Fig. 1A and 1B). Simultaneously, the green fluorescence intensity and aggregation diameter of F-actin significantly reduced in 0.1 μM Mito-TEMPO treated bovine embryos compared to the non-treated group (1.68-fold, Mito-TEMPO treated embryos: 3.94 μm vs non-treated embryos: 6.64 μm; Fig. 1C and 1D). These results showed that the blastocyst developmental competence improves through the stabilization of F-actin by Mito-TEMPO treatment in bovine embryos during IVC.

Table 1. Percentages of bovine blastocysts formation with Mito-TEMPO in the culture medium.

Mito-TEMPO (μM)	No. of embryos cultured	No. of cleaved embryos (%)	No. of blastocysts (%)
Non-treated	348	271 (77.6 ± 3.4)	75 (21.2 ± 4.2)
0.1	350	276 (78.8 ± 4.4)	92 (25.6 ± 5.6)

Data are expressed as the mean ± SD, and the non-normally distributed data are expressed as the median (interquartile range). No, number..

Figure 1.Effects of Mito-TEMPO on the blastocyst developmental rate and F-actin structure in bovine embryos. (A) Representative images of blastocysts according to the 0.1 μM Mito-TEMPO treatment in bovine embryos. Scale bar = 500 μm. (B) The percentage of expanded blastocysts derived from Mito-TEMPO treated bovine embryos. Scale bar = 100 μm. Data are expressed as mean ± SD and were analyzed using a t-test. Differences were considered significant at p < 0.05. (C) Accumulation of adhesive junction F-actin and aggregation F-actin in Mito-TEMPO treated bovine embryos. (D) Fluorescence expressions were presented with F-actin (green) and nuclei (blue) by immunofluorescent (IF) and DAPI staining in bovine blastocyst derived from Mito-TEMPO treated embryos. Scale bar = 50 μm. Data are presented in the bar graph as the mean ± SD from three independent experiments. Differences were considered significant at *p < 0.05 and **p < 0.01 in comparison to the non-treated group.

The positive effect of Mito-TEMPO on survival and cytoskeletal structure of vitrified-warmed bovine blastocysts

The survival rate after the vitrification-warming process was confirmed in developed blastocysts following 0.1 μM Mito-TEMPO treated bovine embryos. The viability rate of vitrified-warmed blastocysts in the Mito-TEMPO treated group was significantly higher than the non-treated group (p < 0.05, non-treated group: 66.7 ± 3.2% vs 0.1 μM Mito-TEMPO treated group: 79.2 ± 5.9%) (Table 2 and Fig. 2A). Moreover, Mito-TEMPO supplementation during the IVC procedure significantly reduced (p < 0.01) the length of F-actin aggregation in vitrified-warmed bovine blastocysts (2.04-fold, Mito-TEMPO treated embryos: 4.05 μm vs non-treated embryos: 8.25 μm; Fig. 2C and 2D). Furthermore, the F-actin fluorescence intensity significantly decreased in the vitrified-warmed blastocysts treated with 0.1 μM Mito-TEMPO compared to the non-treated group.

Table 2. TUNEL-positive cell rates for freeze-thawed bovine blastocysts following Mito-TEMPO treatment.

Mito-TEMPO (μM)	No. of blastocysts vitrified	No. of blastocysts thawed	No. of survived blastocysts (%)
Non-treated	46	45	29 (66.7 ± 3.2)a
0.1	42	29	23 (79.2 ± 5.9)b

Data are expressed as the mean ± SD, and the non-normally distributed data are expressed as the median (interquartile range). Different superscript letters a and b denote significant differences (p < 0.05). No, number..

Figure 2.Changes of viability and F-actin aggregation in vitrified-warmed blastocysts from Mito-TEMPO treated bovine embryos. (A) Representative images of the survival morphologies in vitrified-warmed blastocysts from Mito-TEMPO treated bovine embryos. Scale bar = 500 μm. (B) The percentage of survival in vitrified-warmed blastocysts derived from Mito-TEMPO treated bovine embryos. Data are expressed as mean ± SD and were analyzed using a t-test. Differences were considered significant at p < 0.05. (C) Fluorescence expressions were presented with F-actin (green) and nuclei (blue) by IF and DAPI staining in Mito-TEMPO treated bovine vitrified-warmed blastocysts. Scale bar = 50 μm. (D) F-actin aggregation and fluorescence intensity in Mito-TEMPO treated bovine vitrified-warmed blastocysts. Data are presented in the bar graph as the mean ± SD from three independent experiments. Differences were considered significant at *p < 0.05 and **p < 0.01 as compared to the non-treated group.

Previous studies mainly discussed the antioxidative effects on the morphological variations regarding the in vitro embryo development and vitrification embryos, such as cleavage rate, blastocyst rate, expanded blastocyst rate, the total blastocyst cell numbers, and the survival rate following the freeze-thawing process (Jeong et al., 2021). The vitrification method represents a technique that can potentially provide the successful fertilization and indefinite storage of female germ cells. However, the low quality and survival rate of freeze-thawed embryos currently remain a problem (Hwang and Hochi, 2014). This current study, therefore, aimed to elucidate the effects of a 0.1 μM Mito-TEMPO supplementation during IVC on the viability of vitrified-warmed bovine blastocysts and analyze their subsequent F-actin mediated cytoskeleton structures.

Mito-TEMPO predominantly serves as a potent free radical scavenger and mitochondrial specific-superoxide antioxidant (Zhan et al., 2018). Our previous studies demonstrated the positive effects Mito-TEMPO supplementation could provide towards improving embryonic development potential including promoting the activity of mitochondrial fission, ATP production, meiotic maturation, and dividing blastomeres during in vitro production (IVP) progression (Park et al., 2018; Yang et al., 2018; Yang et al., 2019; Park et al., 2020). Few studies have previously investigated the effects of Mito-TEMPO as a mitochondrial superoxide scavenger in regard to developmental competence until such analyses on blastocysts in bovine embryos. Moreover, Mito-TEMPO was rarely used as a supplement in the freeze-thawing medium of mammalian embryos.

The initial objective of the study was to determine the effects of a 2-day Mito-TEMPO treatment on further embryonic developmental competence following IVC and until the blastocyst stage in bovine embryos. The blastocyst developmental rate in the Mito-TEMPO (0.1 μM) treatment group was slightly higher than the non-treated group. Moreover, the survival of the freeze-thawed blastocysts was significantly increased in the Mito-TEMPO treated bovine embryos compared with the non-treated group (p < 0.05) (Table 2 and Fig. 2). These results are in concordance with those reported by Mehaisen et al. (2015), where melatonin supplements promoted improved development of both the fresh and vitrified rabbit embryos through antioxidant capacity. Moreover, our previous study similarly indicated that supplementation of Mito-TEMPO during cryopreservation could protect the apoptosis-related ROS and mitochondrial superoxide, which occurred through mitochondrial activation and improved the developmental competence of the freeze-thawed blastocysts (Jeong et al., 2021).

The embryos that survived following their freezing exhibited defects such as a disruption in the cytoskeleton structure and stabilization, high levels of ROS production, and oxidative stress following vitrification (García-Martínez et al., 2020). Therefore, the F-actin fluorescence expression was evaluated in all experimental groups, as it is representative of the cytoskeletal components for the F-actin length alongside the aggregation at the adhesion junction sites in blastomeres. A higher blastocyst development rate alongside high viability was observed in IVP and vitrified-warmed blastocysts from the Mito-TEMPO treated embryos (Table 1 and 2). Moreover, in the Mito-TEMPO treated group, a high level of F-actin stabilization was observed in both the IVP and vitrified-warmed blastocysts, indicating that these embryos were extremely sensitive to damage from ROS and mitochondrial superoxide induced by vitrification (Gaviria et al., 2019). Vitrified-warmed blastocysts and the developed blastocysts significantly reduced the F-actin aggregation and intensity following treatment with Mito-TEMPO when compared to control embryos (Fig. 1 and 2). Indeed, an increase in F-actin aggregation was previously reported to cause disruption in embryonic developmental competence and viability following the freeze-thawing process (López et al., 2021). Our study highlights the capability of Mito-TEMPO to reduce the adhesive junction F-actin aggregation in both the bovine IVP and vitrified-warmed blastocysts.

Overall, the present data illustrate that exogenous Mito-TEMPO enhances the developmental competence of IVP and vitrified-warmed blastocysts including the F-actin-mediated cytoskeleton stabilization, which improves embryonic development (Fig. 3, graphical summary). Importantly, Mito-TEMPO was found to reduce mitochondrial superoxide production, which improved the embryonic development and survival rate of IVP and vitrified-warmed bovine embryos. Thus, Mito-TEMPO could be used as a supportive tool to facilitate F-actin stabilization and improve the overall survival of cryo-injured bovine blastocysts.

Figure 3.Graphical summary. Here, the antioxidant capabilities of Mito-TEMPO induced blastocyst developmental competence through F-actin structure stabilization in bovine IVC embryos. Moreover, the Mito-TEMPO provided protection from cryo-injury in vitrified-warmed blastocysts, increasing the survival rate of blastocysts through direct mitochondria superoxide scavenger and F-actin stabilization in bovine embryos. Therefore, these findings contribute to the improvement of vitrification and warming procedures to overcome infertility.

None.

Conceptualization: J-H.J., H-J.P., and D-B.K.; methodology: J-H.J. and S-G.Y.; investigation: J-H.J.; data curation: J-H.J., S-G.Y., and H-J.P.; writing - original draft: J-H.J.; writing - review & editing: S-G.Y., H-J.P., and D-B.K.; supervision: H-J.P. and D-B.K.; project administration: J-H.J. and H-J.P.; funding acquisition: H-J.P., S-G.Y., and D-B.K.

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF-2021R1C1C2009469, NRF-2022R1A2C1002800, and NRF-2021R1A6A3A01087623) funded by and the Ministry of Science and ICT, Republic of Korea.

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No potential conflict of interest relevant to this article was reported.