Introduction
In vitro production of porcine embryos is a challenging process. At present, the most popular integrated system used to produce porcine embryos in vitro include the processes of in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) (Sjunnesson, Reference Sjunnesson2020; Ozawa et al., Reference Ozawa, Nagai, Fahrudin, Karja, Kaneko, Noguchi, Ohnuma and Kikuchi2006). Despite the application of various adjustments to improve the quality of resultant embryos, the developmental competence of porcine in vitro produced (IVP) embryos is rather low compared with that of in vivo counterparts (Romek et al., Reference Romek, Gajda, Krzysztofowicz, Kucia, Uzarowska and Smorag2017; Martinez et al., Reference Martinez, Martinez and M.A.2018). Insufficient cytoplasmic maturation, slow zona reaction and improper embryo culture conditions are considered responsible for this low efficacy (Mohammadi-Sangcheshmeh et al., Reference Mohammadi-Sangcheshmeh, Held, Ghanem, Rings, Salilew-Wondim, Tesfaye, Sieme, Schellander and Hoelker2011; Nagai et al., Reference Nagai, Funahashi, Yoshioka and Kikuchi2006).
Previous studies suggested that supplements of fibroblast growth factor 2 (FGF2) (Yuan et al., Reference Yuan, Spate, Redel, Tian, Zhou, Prather and Roberts2017), leukaemia inhibitory factor (Yuan et al., Reference Yuan, Spate, Redel, Tian, Zhou, Prather and Roberts2017), insulin-like growth factor-I (IGF-1) (Yuan et al., Reference Yuan, Spate, Redel, Tian, Zhou, Prather and Roberts2017), nonessential amino acids (Gupta et al., Reference Gupta, Uhm, Lee and Lee2008), vitamins (Fang et al., Reference Fang, Tanga, Bang, Seong, Saadeldin, Qamar, Shim, Choi, Lee and Cho2022), insulin (Alvarez et al., Reference Alvarez, Barrios Expósito, Elia, Paz, Morado and P.D.2019) and sodium selenite (Uhm et al., Reference Uhm, Gupta, Yang, Lee and Lee2007; Tareq et al., Reference Tareq, Akter, Khandoker and Tsujii2012) in porcine IVM and IVC media could enhance the embryonic development potentials in vitro.
Insulin-transferrin-selenium (ITS) is a solution mainly composed of insulin, transferrin and selenium. Insulin promotes glucose and amino acid uptake, fat formation, intracellular transport and the biosynthesis of protein and nucleic acid (Houtkooper et al., Reference Houtkooper, Pirinen and Auwerx2012). In vitro maturation medium containing insulin or insulin-like growth factors could promote porcine oocyte maturation and embryonic development (Currin et al., Reference Currin, Glanzner, Gutierrez, de Macedo, Guay, Baldassarre and Bordignon2022; Serrano Albal et al., Reference Serrano Albal, Silvestri, Kiazim, Vining, Zak, Walling, Haigh, Harvey, Harvey and Griffin2022). Transferrin, a major iron-containing protein in plasma, acts as a chelator for oxygen radicals to limit oxidative stress (Kim et al., Reference Kim, Lee, Lee, Kim, Jeong, Kim, Kang, Lee and W.S.2005; Lee et al., Reference Lee, Kim, Hyun, Jeon, Nam, Jeong, Kim, Kim, Kang, Lee and Hwang2005). Selenium is a critical microelement in the organism, which promotes cell metabolism and prevents oxidative damage from reactive oxygen species (ROS) (Ren et al., Reference Ren, Wang, Zhang, Hu, Zhou, Li and Xu2020). ITS has been widely used to promote cell growth in serum-free culture media (Eppig et al., Reference Eppig, Wigglesworth and O’Brien1992; Liu et al., Reference Liu, Zhang, Wang, Shi, Pan and Pang2019). The addition of ITS into the IVM medium could improve porcine oocyte maturation and decrease polyspermy fertilization to promote subsequent embryonic development by increasing uptakes of glucose, amino acids and minerals and eliminating oxygen free radicals (Eppig et al., Reference Eppig, Wigglesworth and O’Brien1992; Jeong et al., Reference Jeong, Hossein, Bhandari, Kim, Kim, Park, Lee, Park, Jeong, Lee, Kim and WS2008; Cordova et al., Reference Cordova, Morato, Izquierdo, Paramio and Mogas2010; Hu et al., Reference Hu, Ma, Bao, Li, Cheng, Gao, Lei, Yang and Wang2011). Additionally, supplementing the IVC medium with ITS could significantly improve porcine parthenogenetic activation (PA) and IVF zygotes (Das et al., Reference Das, Gupta, Uhm and Lee2014).
Although the individual effects of ITS have already been proven in a single porcine embryo IVP stage so far, the effect and potential event of applying ITS both in IVM and IVC media on porcine embryo production in vitro haven’t been elucidated. This study aimed to optimize the protocol based on dual-stage ITS supplements in the media for porcine embryo production in vitro.
Materials and Methods
Experimental design
ITS (100×) (41400045, Thermo Fisher Scientific Grand Island, NY) containing insulin 1 g/L, transferrin 0.55 g/L and selenium 0.00067 g/L was prepared in Earle’s Balanced Salt Solution without Phenol Red. To determine the effects of ITS on the efficiency of porcine embryo production in vitro, ITS was supplemented in IVM medium at four concentrations (0, 0.5, 1.0 or 2.0%) during the entire maturation period of 44 h, so was in the 7d-IVC medium. For the mitochondrial function test, oocytes at MII stage after 44 h-IVM were harvested, then ΔΦm was detected by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolyl-carbocyanineiodide (JC-1) staining, cellular ATP content was measured using an Enhanced ATP Assay Kit and ROS production was assayed by 2′,7′-dichlorofluorescin diacetate (DCFH-DA) staining. Once the optimal concentration of ITS was identified, the cumulus cell expansion grades were assessed at the end of 44 h-IVM.
Chemicals
All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specifically indicated.
Recovery and IVM of oocytes
Porcine ovaries were harvested from cross-bred pigs slaughtered at a local abattoir (the Shahe Fifth Meat Union Company, Beijing, China) and transported to the laboratory in saline solution at 33–37°C. The cumulus–oocyte complexes (COCs) were aspirated from the follicles (2–5 mm diameter) using a 10 ml syringe fitted with an 18 G needle.
IVM of oocytes was carried out as described previously (Jeong et al., Reference Jeong, Hossein, Bhandari, Kim, Kim, Park, Lee, Park, Jeong, Lee, Kim and WS2008; Kitaji et al., Reference Kitaji, Ookutsu, Sato and Miyoshi2015; Zhang et al., Reference Zhang, Ma, Hu, Ding and Xu2015). The COCs with uniform granulated cytoplasm and compact cumulus cells with no less than three layers were washed three times in HEPES-buffered Tyrode’s lactate (TL-HEPES) medium containing 1.0 mg/ml BSA (TL-HEPES-BSA) (Yang et al., Reference Yang, Liu, Miao, Mou, Liu, Wang, Huo and Du2021) and matured in groups of 80–100 in 500 μl of TCM-199 with Earle’s salts (Gibco BRL, Grand Island, NY) supplemented with 10% (v/v) porcine follicular fluid, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 10 ng/ml EGF, 10 IU/ml eCG (Ningbo Hormone Product Co., Ltd., Ningbo, China) and 10 IU/ml hCG (Ningbo Hormone Product Co., Ltd., Ningbo, China) under mineral oil at 38.5°C in a humidified atmosphere of 5% CO2 for 44 h.
In vitro fertilization and in vitro culture of embryos
After 44 h of maturation, the oocytes were stripped of cumulus cells by pipetting in TL-HEPES containing 0.1% hyaluronidase. After being washed twice in TL-HEPES and once in the modified Tris-buffered medium (mTBM), the oocytes were placed into groups of 10 oocytes per 45 μl droplets of the fertilization medium. Then, 5 μl of sperm suspension with a concentration of about 5 × 106 sperm/ml was added to the 45 μl oocyte-containing droplets. The sperm and oocytes were then co-incubated in a humidified atmosphere of 5% CO2 air at 38.5°C. After 6 h of fertilization, the zygotes were stripped of spermatozoa by pipetting in TL-HEPES supplemented with 0.4% (w/v) BSA and then washed three times with porcine zygote medium 3 (PZM-3) (Zhang et al., Reference Zhang, Zheng, Han, Kuang, Min, Wang, Zhao, Wang, Yang and Che2022) before being transferred to an IVC medium (PZM-3) for further culture. The day of IVF was defined as Day 0 (Nguyen et al., Reference Nguyen, Dang-Nguyen, Somfai, Men, Viet Linh, Xuan Nguyen, Noguchi, Kaneko and Kikuchi2020), and cleavage rates were assessed on Day 2. Blastocyst rates were recorded on Day 7 of cultivation and then fixed with 4% paraformaldehyde and subjected to 10 µg/ml Hoechst 33342 staining for 8–10 min in the dark.
JC-1 staining
Mitochondrial membrane potential (ΔΦ) was determined as described by the mitochondrial probe, JC-1, with the monomers accumulating preferentially within mitochondria and subsequently aggregating as a function of the ΔΦ (Smiley et al., Reference Smiley, Reers, Mottola-Hartshorn, Lin, Chen, Smith, Steele and Chen1991). Oocytes after IVM were incubated in TL-HEPES containing 10 μg/ml JC-1 (BV-1130, Biovision, USA) at 37°C for 15 min. After incubation, the oocytes were washed with PVA/PBS solution for three times before observing under a Nikon A1HD25 confocal microscope (Minato, Tokyo, Japan). ΔΦm was calculated as a ratio of JC-1 monomers (green fluorescence) and J-aggregate fluorescence (red fluorescence) at 590 nm in response to 488 nm excitation. The obtained images were processed using Image-Pro Plus software (Media Cybernetics Inc., Silver Spring, MD, USA) under fixed thresholds across all the slides.
Assessment of ATP
Cellular ATP content was measured using an Enhanced ATP Assay Kit (S0027; Beyotime Biotechnology) according to the manufacturer’s instructions. Briefly, serial dilutions of ATP were prepared (from 0 to 32 pmol). Five denuded oocytes after IVM were collected in 50 μl lysis buffer on ice for direct detection or storage at –80°C. Before measurement, ATP assay buffer was added to 96-well black plates and equilibrated for 3–5 min at room temperature. Then, standard solutions or cell samples were added to each well, and luminescence activity was measured using an automatic chemiluminescent analyser (Infinite® F200, Tecan Group Ltd., Mannedorf, Switzerland). ATP content was calculated using a standard curve and the total amount of ATP measured was divided by the number of oocytes in each sample to obtain the mean ATP content (pmol) per oocyte. Three separate experiments were performed with three replicates in each.
ROS staining
Intracellular ROS levels were determined with a ROS Assay Kit (S0033, Beyotime Institute of Biotechnology, China) (Jiang et al., Reference Jiang, He, Li, Ni, Li, Peng, Luo, Rui and Ju2021). Briefly, the matured oocytes were incubated in PVA/PBS containing 10 μM DCFH-DA at 38.5°C for 30 min. After three times of wash, oocytes were placed in a confocal dish and imaged under the confocal microscope. Image-Pro Plus software (Media Cybernetics Inc., Silver Spring, MD, USA) was used to analyse fluorescence intensities of the oocytes and the average relative fluorescence intensity of the oocytes from the control group was set to 1, while those from other treatment groups were expressed relative to this value.
Assessment of cumulus cell expansion
Cumulus cell expansion of COCs was assessed at the end of the IVM using a subjective scoring method (Tao et al., Reference Tao, Xia, Bo, Zhou, Zhang and Wang2004; Choi et al., Reference Choi, Lee, Yoon, Hwang, Cai, Kim, Kim, Oh, Kim and Hyun2021): grade 0, no expansion; grade 1, minimum observable expansion; grade 2, expansion of outer COCs layers; grade 3, expansion of all COCs layers except the corona radiate; grade 4, expansion of all COCs layers.
Statistical analysis
There were at least three replicates for each treatment unless otherwise stated. Statistical analysis of experiments with two groups, such as assessment of cumulus expansion, was carried out using the unpaired t-test; data including the cleavage rate, the blastocyst rate and the cell number per blastocyst were arc sine transformed and analysed with analysis of variance. A Duncan, multiple comparison test, was used to locate differences. The software used was the Statistics Package for Social Sciences (SPSS 11.5, SPSS Inc. Chicago, IL). Data were expressed as mean ± standard deviation, and P < 0.05 was considered significant.
Results
The effect of IVM/IVC media containing ITS on porcine embryo production in vitro
We first compared the rate of first polar body (PB1) extrusion after 44 h-IVM among groups with different concentrations of ITS (0, 0.5, 1.0 and 2.0% v/v). As shown in Table 1, with the concentration of ITS increasing, the rate of PB1 extrusion notably increased, and 1.0% of ITS yielded the highest rate of oocyte maturation, which was significantly different from the control group (P < 0.05) and followed by 0.5% group (P < 0.05) and 2.0% group (P < 0.05). Next, we examined the dose-dependent effect of ITS on IVF embryos and found that the addition of different concentrations of ITS into IVM and IVC media had no effect on cleavage rates (P >0.05; Table 1). Still, the blastocyst rate was higher in the 1.0% group than in the other three groups (P < 0.05). Additionally, the cell number per blastocyst was higher in the 1.0 and 2.0% groups compared with the control group (P < 0.05) (Table 1); yet, there was no statistical difference between the 0.5% group and the 2% group (P > 0.05).
Table 1. The effect of IVM/IVC media containing ITS on the porcine embryo production in vitro
ITS = insulin-transferrin-selenium; IVM = in vitro maturation; IVC = in vitro culture; IVF = in vitro fertilization. The cleavage rate was based on the number of incubated oocytes, while the blastocyst rate was based on the number of cleaved embryos in each group. Values with different superscripts (a, b, and c) within the same column statistically differ (P < 0.05). Each group was repeated three times with one replicate of 90–110 oocytes.
ANumber of oocytes for IVM experiments.
BNumber of oocytes for IVM-IVF-IVC experiments.
The effect of IVM medium containing ITS on mitochondrial function of MII stage porcine oocytes
Effect of ITS supplement on mitochondrial function of MII stage porcine oocytes were exhibited by ΔΦm, ATP production and ROS production.
As shown in Figure 1A and 1B, the results of JC-1 staining, specific for the detection of cellular ΔΦm levels, indicated that adding 0.5, 1.0 and 2.0% of ITS could significantly elevate the mitochondrial ΔΦm in the porcine MII oocytes than that in the control group (P < 0.05), among those three ITS groups, 1.0% group yielding the highest ΔΦm, which was significantly higher than 0.5% group (P < 0.05) but not different from that in 2.0% group (P > 0.05).
Figure 1. The effect of IVM medium containing ITS on mitochondrial function of MII porcine oocytes. (A) Determination of ΔΦm. High ΔΦm in red fluorescence and low ΔΦm in green fluorescence were observed after staining with JC-1. Bar = 100 μm. (B) Quantitative analysis of ΔΦm values after ITS supplementation. Values with different superscript letters (a, b, c) between different columns statistically differ (P < 0.05). Each group was repeated three times with one replicate of 30–40 oocytes. (C) Intracellular ATP content in oocytes. Data are presented as mean ± SEM of six independent experiments. Values with different superscript letters (a, b) between different columns statistically differ (P < 0.05). (D) Staining of porcine oocytes with ROS (DCFDA, green). Bar = 100 μm. (E) Quantitative analysis of ROS levels. Data are presented as mean ± SEM of three independent experiments with one replicate of 30–40 oocytes. Values with different superscript letters (a, b) between different columns statistically differ (P < 0.05). IVM, in vitro maturation; ITS, insulin-transferrin-selenium; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolyl-carbocyanineiodide; ROS, reactive oxygen species.
The results of the ITS supplement on ATP content are shown in Figure 1C. Oocytes cultured in medium containing 1.0% ITS had higher ATP contents compared with those cultured without ITS (3.72 ± 0.35 pmol/oocyte vs. 3.19 ± 0.23 pmol/oocyte, P < 0.05), while in groups of 0.5 and 2.0%, ATP contents in single oocyte did not differ from the control group (3.47 ± 0.27 pmol (0.5%) and 3.49 ± 0.23 pmol (2.0%) vs. 3.19 ± 0.23 pmol (0%), P > 0.05).
As shown in Figure 1D and 1E, the fluorescence intensity of ROS in the MII oocytes stained with DCFDA was much lower in the 1.0% ITS group than in the control group (P < 0.05). The fluorescence intensity tended to increase in groups of 0.5 and 2.0% compared with that in the control group, but the difference was not significant (P > 0.05).
The effect of IVM medium containing ITS on porcine cumulus cell expansion
Furthermore, the cumulus cell expansion grades were assessed at the end of IVM. According to the scoring criterion in Figure 2A, the percentage of COCs with grade 0 or 1 was significantly lower in the 1.0% ITS group than the control group (P < 0.05), while the proportion of COCs with grade 2 was significantly higher than the control group (P < 0.05) (Figure 2B).
Figure 2. The effect of 1.0% ITS supplementation in IVM medium on porcine cumulus cell expansion. (A) Criteria for ranking porcine COC expansion. The expansion of COCs was assessed from grade 0 to grade 4 after 44 h culture. Grade 0, no expansion; grade 1, minimum observable expansion; grade 2, expansion of outer COC layers; grade 3, expansion of all COC layers except the corona radiate; grade 4, expansion of all COC layers. Bar = 90 μm. (B) Quantitative analysis of ITS effect on cumulus expansion. Values with different superscript letters (a, b) between different columns statistically differ (P < 0.05). Each group was repeated three times with one replicate of 40–60 oocytes. ITS, insulin-transferrin-selenium; IVM, in vitro maturation; COCs, cumulus–oocyte complexes.
Based on the above results, IVM/IVC media containing 1.0% ITS demonstrated positive effects on the efficiency of porcine embryo production.
Discussion
IVM or IVC medium supplemented with arginine (Redel et al., Reference Redel, Tessanne, Spate, Murphy and Prather2015), leptin (Craig et al., Reference Craig, Zhu, Dyce, Wen and Li2005), melatonin (Niu et al., Reference Niu, Zhou, Nie, Shin and Cui2020; Wang et al., Reference Wang, Gao, Chen, Nie, Cheng, Liu, Schatten, Zhang and Miao2017), sonic hedgehog (Nguyen et al., Reference Nguyen, Lin, Yen, Tseng, Chuang, Chen, Lin, Chang and Ju2009; Nguyen et al., Reference Nguyen, Lo, Chuang, Jian and Ju2011), IGF-1 (Kim et al., Reference Kim, Lee, Lee, Kim, Jeong, Kim, Kang, Lee and W.S.2005; Oberlender et al., Reference Oberlender, Murgas, Zangeronimo, da Silva, Menezes, Tde, Pontelo and Vieira2013), insulin (Song et al., Reference Song, Xu, Hao, Liang, Lu and Lu2010) and ITS (Das et al., Reference Das, Gupta, Uhm and Lee2014; Luchetti et al., Reference Luchetti, Lorenzo, Elia, Teplitz, Cruzans, Carou and Lombardo2023; Cordova et al., Reference Cordova, Morato, Izquierdo, Paramio and Mogas2010; Hu et al., Reference Hu, Ma, Bao, Li, Cheng, Gao, Lei, Yang and Wang2011; Jeong et al., Reference Jeong, Hossein, Bhandari, Kim, Kim, Park, Lee, Park, Jeong, Lee, Kim and WS2008) have shown to promote porcine embryos production in vitro. In the present study, we further optimize the protocol based on dual-stage ITS supplements in the media for porcine embryo production in vitro. Our results demonstrated that 1.0% ITS supplementation in IVM/IVC media promotes the expansion of the cumulus, yields raised ΔΦm and ATP content yet reduced ROS level in matured oocytes, while increases the blastocyst rate and the number of blastocyst cells, simultaneously. Thus, this new system has advantages for the generation of cloned and transgenic pigs.
Previous studies reported that porcine IVM media containing ITS improved nuclear maturation and cortical granules distributions in the cortex (Hu et al., Reference Hu, Ma, Bao, Li, Cheng, Gao, Lei, Yang and Wang2011), increased the concentration of intracellular glutathione, decreased polyspermy fertilization and promoted male pronucleus formation, blastocyst rate and the cell number in blastocyst (Jeong et al., Reference Jeong, Hossein, Bhandari, Kim, Kim, Park, Lee, Park, Jeong, Lee, Kim and WS2008). According to our results, porcine IVM media containing ITS stimulates cumulus cell expansion of COCs, and in turn, promotes porcine oocyte maturation in vitro. Das et al. (Reference Das, Gupta, Uhm and Lee2014) previously reported that the IVC medium containing ITS could stimulate the development of the embryos produced by PA, IVF or somatic cell nuclear transfer and decrease the level of ROS in PA embryos (Das et al., Reference Das, Gupta, Uhm and Lee2014). In the present study, we found that IVM and IVC media supplemented with ITS can improve the rate and the cell number per blastocyst. Yet, Das et al. did not show the beneficial effect of ITS in IVC medium on IVF embryo development in vitro (Das et al., Reference Das, Gupta, Uhm and Lee2014). Data from this study imply that ITS may be more critical for oocyte maturation than early embryo development.
ITS was widely used in the process of IVM to facilitate oocyte development in vitro of mouse (Eppig et al., Reference Eppig, Wigglesworth and O’Brien1992), bovine (Dos Santos Mendonça-Soares et al., Reference Dos Santos Mendonça-Soares, Guimarães, Fidelis, Franco and Dode2022), goat (Fengjun et al., Reference Fengjun, Yuling, Zijun, Guohua and Yong2008) and porcine (Jeong et al., Reference Jeong, Hossein, Bhandari, Kim, Kim, Park, Lee, Park, Jeong, Lee, Kim and WS2008; Hu et al., Reference Hu, Ma, Bao, Li, Cheng, Gao, Lei, Yang and Wang2011; Das et al., Reference Das, Gupta, Uhm and Lee2014). Yet, the mechanism by which ITS promotes oocyte maturation in vitro is complex. Normally, insulin stimulates amino acid transport, protein synthesis and accumulation of glucose carbon (Arnqvist, Reference Arnqvist1974; Yuli et al., Reference Yuli, Incerpi, Luly and Shinitzky1982), transferrin can act as a chelator for oxygen radicals to limit oxidative stress (Lee et al., Reference Lee, Kim, Hyun, Jeon, Nam, Jeong, Kim, Kim, Kang, Lee and Hwang2005; Kim et al., Reference Kim, Lee, Lee, Kim, Jeong, Kim, Kang, Lee and W.S.2005). Selenium has been proven to promote cell metabolisms such as glycolysis, gluconeogenesis, fatty acid synthesis and the pentose phosphate pathway (Stapleton, Reference Stapleton2000; Chen et al., Reference Chen, Wang, Zhang, Li and Zhang2022) and prevent oxidative damage from ROS (Uhm et al., Reference Uhm, Gupta, Yang, Lee and Lee2007; Luo et al., Reference Luo, Wang, Lin, Liu, Gu, Liu and Xiao2020). According to our results, the positive effects of ITS on oocytes during IVM contribute to the development of preimplantation embryos produced in vitro but do not affect fertilization and cleavage. Still, the exact mechanism needs to be investigated further.
The processes of mammalian oocyte maturation and embryo development consume lots of energy with mitochondria being the main source (Li et al., Reference Li, Wang, Chen, Tian, Gao and Lei2022) and producing ATP via oxidative phosphorylation and the citric acid cycle (Mitchell & Moyle, Reference Mitchell and Moyle1967). Relying on the electron transport chain, mitochondria gain a highly negative mitochondrial membrane potential (ΔΦm) via pumping hydrogen ions (protons) across the inner mitochondrial membrane (Al-Zubaidi et al., Reference Al-Zubaidi, Liu, Cinar, Robker, Adhikari and Carroll2019), which is the driving force for ATP production and can usually be assayed by a potentiometric cationic fluorescent indicator, JC-1. There is a close temporal correlation between ΔΦm and ATP content during oocyte maturation (Al-Zubaidi et al., Reference Al-Zubaidi, Liu, Cinar, Robker, Adhikari and Carroll2019; Liu et al., Reference Liu, Li, Yan, Du, Peng, Li, Cui, Zhang, Yang, Lu and Liang2023; Dalton et al., Reference Dalton, Szabadkai and Carroll2014), ΔΦm and ATP levels are commonly used as an indicator of mitochondrial function in and viability of oocytes (Zhan et al., Reference Zhan, Cao, Zhang, Guo, Xu, Wang, Yang, Yang, Che, Lu and Ma2022; Liang et al., Reference Liang, Yuan, Kwon, Ahn, Cui, Bang and Kim2016). In the present study, both ΔΦm and ATP content were increased after 1.0% ITS supplementation, which indicated that ITS supplementation in IVM medium enhanced porcine oocyte maturation and embryo development potential might come from the enhanced mitochondrial function exhibited as up-regulated ΔΦm and ATP content.
Given the highest amount of lipid droplets and also longer maturation periods, porcine oocytes are more prone to oxidative stress such as accumulation of ROS due to lipid peroxidation with potential damage to the cell membrane (Dalbies-Tran et al., Reference Dalbies-Tran, Cadoret, Desmarchais, Elis, Maillard, Monget, Monniaux, Reynaud, Saint-Dizier and Uzbekova2020). Oxidative phosphorylation in the mitochondria is the largest source of ROS in the oocyte, and ROS can disrupt the ΔΦm and reduce ATP production in porcine oocytes and embryos (Zhan et al., Reference Zhan, Cao, Zhang, Guo, Xu, Wang, Yang, Yang, Che, Lu and Ma2022; Zhao et al., Reference Zhao, Liang, Kim, Cui and Kim2015), ultimately activating the mitochondria-mediated apoptosis pathways in porcine oocytes (Li et al., Reference Li, Miao, Chen and Xiong2021). In the present study, the ROS level in porcine MII oocytes was decreased in the 1.0% ITS group. It implied that ITS supplementation could protect oocytes from oxidative damage by scavenging ROS during IVM.
The degree of cumulus cell expansion is a well-known predictor of oocyte maturation and embryo development after IVF (Kang et al., Reference Kang, Jeong, Kim, Joo, Gwon, Jeon, Song, Kim, Lee and Sim2022). Cumulus cell expansion regulates oocyte maturation via the synthesis of hyaluronic acid-rich glycosaminoglycan in the extracellular space, which functions as a structural component of expanded cumuli (Nagyová et al., Reference Nagyová, Němcová and Camaioni2021). Optimum cumulus cell expansion is vital for oocyte maturation and fertilization for the signals transduction and nutritional exchange between oocytes and surrounding cumulus cells(Yokoo et al., Reference Yokoo, Kimura and Sato2010; Jin et al., Reference Jin, Sun, Jiang, Cui, Bian, Lee, Zhang, Lee and Liu2022), while insufficient cumulus cell expansion always results in low average oocyte retrieval rate, low No. of flushing per follicle, high ROS level, low cleavage and blastocyst formation rates (Yin et al., Reference Yin, Mao, Liu, Shu, Yuan, Cui, Hou and Liu2021; Saini et al., Reference Saini, Sharma, Ansari, Kumar, Thakur, Malik, Kumar and Malakar2022). Furthermore, the expanded cumulus cell mass induces an acrosome reaction in boar sperm and promotes penetration into the oocyte(Mattioli et al., Reference Mattioli, Lucidi and Barboni1998). In the present study, we obtained an increase in cumulus cell expansion after 1.0% ITS supplementation, which might contribute to the improved porcine oocyte maturation and embryo development potential.
In conclusion, porcine IVM/IVC media supplemented with 1.0% ITS demonstrated positive effects on the efficiency of embryo production in vitro by increasing ΔΦm and ATP content, scavenging ROS and promoting cumulus cell expansion.
Author contributions
Juan Lin and Zhuqing Ji contributed equally. Juan Lin, Zhuqing Ji and Shenming Zeng designed the experiments. The manuscript was mainly written by Juan Lin, and the draft was revised by Shenming Zeng and Juan Lin. All the authors contributed to, read and approved the final manuscript.
Funding
This work was supported by grants from the National Key Research and Development Program of China (2022YFD1300400).
Competing interests
None.
Ethical standards
All experiments were performed in accordance with the institutional guidelines established by the Animal Care and Use Committee of China Agricultural University (ID: SKLAB-B-2010-003).
