Journal of Animal Reproduction and Biotechnology 2024; 39(3): 169-178
Published online September 30, 2024
https://doi.org/10.12750/JARB.39.3.169
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Min-Jee Oh1,3,# , Baasanjav Batmunkh1,# , Ji-Yeon Mo2 and Sang-Hwan Kim1,2,3,*
1General Graduate School of Animal Life Convergence Science, Hankyong National University, Anseong 17579, Korea
2School of Animal Life Convergence Science, Hankyong National University, Anseong 17579, Korea
3Institute of Applied Humanimal Science, Hankyong National University, Anseong 17579, Korea
Correspondence to: Sang-Hwan Kim
E-mail: immunoking@hknu.ac.kr
#These authors contributed equally to this work.
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Typical difficulties encountered during in vitro fertilization (IVF) to produce embryos in pigs include poor pronucleus formation and poor-quality fertilized embryos because of high polysperm invasion. In this study, we evaluated the effects of supplementation with apple seed extract (ASE) and coculture systems on porcine in vitro-fertilized embryo culture.
Methods: Slaughterhouse-derived ovaries were used to obtain cumulus-oocyte complexes (COCs). COCs were conventionally used to perform IVF. We examined the differences in apoptosis and metabolism during development following addition of ASE to normal culture and coculture systems. Matrix metalloproteinases (MMPs), cell development-related factors, and apoptotic proteins were compared in porcine embryos produced under different conditions.
Results: The expression of genes related to insulin-like growth factor (IGF) signaling was increased in the coculture system. In the ASE group, early apoptosis and necrosis were reduced in fertilized embryos and the late survival rate increased. Supplementation of the coculture system with ASE led to increased expression of BCL-2 and decreased expression of Casp-3 in the cytoplasm, thereby lowering the apoptosis rate and inducing MMP expression. In addition, compared with the extract-supplemented group in normal culture, the activity of MMP-2 decreased in the coculture system supplemented with ASE, activity of MMP-9 increased, and the expression of dynactin p62 and BrdU in the cytoplasm was higher than that in the other groups.
Conclusions: The coculture system increased the activity of the embryonic cytoplasm compared with the non-coculture system. Supplementation with ASE may induce cell activity and inhibit the expression of apoptotic factors.
Keywords: apple seed, coculture, embryo, microtubule, porcine
Ovarian follicles grow oocytes by release mature oocytes at the time of ovulation, after which final maturation is completed in the microenvironment of the ovary during oocytes development (Loutradis et al., 1999; Fair, 2010; Scaramuzzi et al., 2011). Oocyte development results from the close interaction between hormones and follicular cells (Milewski et al., 2018; Liu et al., 2020). Several methods have been attempted to induce stable development of embryos after maturity; however, inducing this development remains challenging. Particularly, the current in vitro fertilization and culture technology used for pigs reduces the rates of oocyte development because of incomplete cytoplasm, polyspermy invasion, low maturation rates, and blastocysts compared with that in other livestock. Therefore, an efficient method for producing pig embryos is needed (Dang-Nguyen et al., 2010; Isom et al., 2012). In IVP technology in pigs, several hormones are used during the in vitro maturation process or co-culture with oocytes to induce nuclear maturation of pig embryos (Oktay et al., 2000; Qian et al., 2005). Addition of porcine ovarian epithelial cells during in vitro fertilization (IVF) and pig embryo development not only increases the number of blastocysts formed but also improves blastocyst quality (Teplitz et al., 2020). Therefore, coculture with homologous cells induces embryonic development and efficiently improves the quality of pig embryos. However, embryonic cells sometimes degrade or cell development is stopped at the 2- or 4-cell phase after IVF in pigs (Jin et al., 2017; Kim and Yoon, 2018a). This phenomenon results in cell cycle arrest during cell division, leading to chromosomal abnormalities and DNA damage (Vandaele et al., 2007; Song et al., 2022). Particularly, high expression of Casp-3 and inhibition of BCl-2 during embryo development lead to pig embryo mortality (Kim and Yoon, 2018a; 2018b). Methods need to be established to inhibit cell death while maintaining the microenvironment to promote the development of porcine embryonic cells. Several studies have investigated the reduction of oxidative stress in in vitro embryos to achieve IVF in pigs while inhibiting apoptosis that can occur during embryonic cell differentiation (Chen et al., 2022). Several studies have suggested that successful cell growth is mediated by activating the Nrf2/ARE signaling pathway, thereby preventing the production of reactive oxygen species and inducing cell differentiation (Combelles et al., 2009; Kim et al., 2019; Kim, 2022). Portion natural extracts can maintain mitochondrial function in embryonic cells by activating the expression of endogenous antioxidant molecules, such as glutathione and various antioxidant enzymes (Beker et al., 2002; Brad et al., 2003; Svoradová et al., 2022). In addition, based on our previous findings that apple seed extract (ASE) can control the apoptosis of endometrial cells and influence oxidative stress and apoptosis (Kim, 2022), in this study, we analyzed whether apple seed extract (ASE) can also participate in maintaining embryo cell metabolism during in vitro culture.
Ovaries of pigs slaughtered at slaughterhouses were placed in physiological saline (0.85% w/v of NaCl, 300 mOsm/L or 9.0 g per liter, Penicillin G 100 IU/mL, Streptomycin 100 mg/mL) at 37℃, transported to our laboratory within 2 h, and washed three to several times with saline. An 18-gauge needle attached to a 10-mL syringe was used to collect the ovum and follicular fluid from antral follicles 2-6 mm in diameter. Cumulus-oocyte complexes (COCs) were observed under a stereomicroscope. Only COCs with three or more uniform layers of compact cumulus cells and a homogeneous cytoplasm were selected for further study. The sorted oocytes were washed three times with washing medium (liquid TCM, 10% antibiotic-antimycotic) supplemented with bovine serum albumin (Sigma, St. Louis, MO, USA). The oocytes were matured in two steps in maturation culture medium (TCM-199; Gibco, Grand Island, NY, USA) supplemented with 10% porcine follicular fluid. The cells were incubated in IVM 1 medium containing 2.5 µg/mL gonadotrophic hormone (Sigma) and 15 ng/mL epidermal growth factor (Sigma), and then incubated in IVM 2 medium, which did not contain the hormones. COCs (50-70 per well) were incubated at 39℃ in a 5% CO2 humid atmosphere for 22 h during each step.
Semen (1-2 mL) were transferred into a 15 mL conical tube and centrifuged at 1,500 rpm for 3 min. The supernatant was removed and washed twice with 3 mL modified Tris buffer medium. Twenty mature oocytes were suspended in 30 µL mM Tris buffer medium in a 60-mm culture dish and covered with mineral oil. Sperm (1 × 106) were added to the oocytes and incubated for 6 h in a 5% CO2 humid atmosphere incubator at 39℃ for IVF.
The in vitro culture was divided into four groups defined below. The NC group was used as a negative control, and the culture medium did not contain ASE. In the NA group, the culture medium was supplemented with ASE. The CC group was a co-culture system group (Using Cumulus cell) without ASE supplementation. The CA co-culture system group was supplemented with ASE. All coculture systems used Cumulus cells.
In the in vitro culture method, the in vitro culture (IVC) for all groups used porcine zygote medium (PZM-5) supplemented with 10% fetal bovine serum. After 6 h of in vitro fertilization, cumulus, and corona radiata cells were removed by pipetting, and the cells were washed in a porcine tissue culture medium (pTCM-199). Zygotes were identified, and each was placed in the prepared media group and cultured at 39℃ in a 5% CO2 humidified atmosphere for 6 days. The zygotes were transferred to freshly prepared culture media every 2 days. The cells used in the coculture group were Granulosa cells collected from follicles, initially cultured in PZM-5, and confirmed to have pore size inserts of approximately 0.4 µm. Afterwards, according to the treatment group, IVC media were used for coculture (Zeyneloglu and Kahraman 2009; Chung et al., 2018). For the ASE treatment group, 1 µg/500 µL ASE was treated in PZM-5 media. Embryos were checked under a microscope on the 4 and 6 days after in vitro culture. On the 4 day, embryos at approximately the 4cell stage were selected, and on the 6 day, embryos that had not developed beyond approximately the 8cell stage were used for the experiment.
For zymography and enzyme-linked immunosorbent assay (ELISA), total protein was extracted from cultured embryos using PRO-PREP (Intron Biotechnology, Seongnam, Korea), according to the manufacturer’s protocol. Protein samples were mixed with loading buffer (0.06% bromophenol blue, 10% sodium dodecyl sulfate, and 2% glycerol) and electrophoresed for 90 min at 100 V; sodium dodecyl sulfate-polyacrylamide gels (12%) containing 100 mg/mL gelatin A/B were used. The gels were washed twice with renaturation buffer (2.5% Triton X-100) and distilled water for 20 min and then incubated at 37℃ for 18 h in zymography reaction buffer. After the reaction, the gels were stained with 1% Coomassie blue for zymography (40% methanol, 10% acetic acid, 0.5 g/500 mL CBBR-250) staining solution for 1 h and destained with destain solution (Bio-Rad, Hercules, CA, USA). Bands were measured to evaluate MMP-2 and MMP-9 activity.
The MMP-9 (ab58803, Abcam, Cambridge, UK), MT1-MMP (sc-30074, Santa Cruz Biotechnology, Dallas, TX, USA), TIMP-2 (sc-9905, Santa Cruz Biotechnology), TIMP-3 (sc-6836, Santa Cruz Biotechnology), IGF-R1 (sc-712, Santa Cruz Biotechnology), PI3K (sc-365290, Santa Cruz Biotechnology), Akt (sc-5298, Santa Cruz Biotechnology), and mTOR (sc-517464, Santa Cruz Biotechnology) primary antibodies were added to 96-well ELISA plates to analyze the expression levels these proteins; incubation was performed at 4℃ overnight. After washing twice with washing buffer (1x PBS containing 2.5% Triton X-100), the wells were blocked for 2 h at RT with 1% skim milk solution. After washing, anti-rabbit (AB6721-1; Abcam), anti-mouse (AB205719; Abcam), and anti-goat (AB6741; Abcam) secondary antibodies were added to each well and incubated for 2 h at RT. The cells were washed with washing buffer and incubated with substrate solution (LIFE TECH, USA). The reaction was stopped by adding 1 M NH2SO4, and absorbance was measured at 450 nm.
Cultured embryos were fixed in 4% paraformaldehyde overnight at 4℃, washed for 30 min in PBS, and permeabilized with 0.2% Triton X-100 for 30 min at RT. After blocking with 3% bovine serum albumin in PBS, the samples were incubated with an antibody against the active forms of BCl-2 (sc-492, Santa Cruz Biotechnology), Casp-3 (sc-373730, Santa Cruz Biotechnology), dynactin p62 (sc-25730, Santa Cruz Biotechnology), BrdU (sc-32323, Santa Cruz Biotechnology), TNF-R (sc-31349, Santa Cruz Biotechnology), and Bax (Cell Signaling, Danvers, MA, USA) at a 1:300 dilution. The samples were washed and incubated with anti-rabbit IgG conjugated to Alexa Fluor 488 (A11094, Invitrogen, Carlsbad, CA, USA), anti-mouse IgG (AB150116, Abcam), or anti-goat IgG (AB150132, Abcam) conjugated to Alexa Fluor 594. Nuclei were counterstained with 1 g/mL Hoechst 33258 and mounted using fluorescence mounting medium. Images were acquired using a confocal microscope (TIRFF-C1; Nikon, Tokyo, Japan). ImageJ software (NIH Image, Bethesda, MD, USA) was used to measure the blastomere diameter and examine the shape and size of the embryos. The brightness of fluorescence was compared relatively using the Image J program (Image J 1.54f; NIH, WI, USA).
ELISA data were analyzed using the Statistical Package for Social Sciences (IBM SPSS Statistics for Windows, version 23.0; IBM Corp, Armonk, NY). Duncan’s multiple range test was used to determine the differences in mean values for each treatment group. Statistical significance was set at p < 0.05.
Fig. 1 shows the expression patterns of apoptotic factors in embryos with cell cycle arrest for in vitro culture methods and with addition of ASE after IVF.
In all treatment groups, pro-Casp-3 was expressed in the cytoplasm of embryonic cells in which the cell cycle had stopped, and cytoplasmic collapse was confirmed. On day 4 of embryo culture in non-coculture medium, Casp-3 was highly expressed in the cytoplasm, whereas on day 6, a protein similar to BCL-2 accumulated. However, the NA group treated with ASE showed high accumulation of BCL-2 on day 4, but the expression of pro-Casp-3 was increased on day 6, showing opposite results from the NC group. The coculture group showed different results from those of the non-coculture group. In the group without ASE, pro-Casp-3 expression was increased on day 6 compared to that on day 4. However, in the group treated with ASE, BCL-2 was highly expressed on both days 4 and 6, and the expression of Casp-3 was relatively low compared to that in all other groups (Fig. 1A and 1B). TNF-R and BAX were detected in all groups, with high expression of TNF-R (Fig. 2A and 2B). In both the regular culture and coculture groups, TNF-R expression tended to be higher than that in the ASE-treated group from day 4 to 6. The non-coculture group treated with ASE showed high expression of BAX, and the coculture group treated with ASE showed high expression of TNF-R (Fig. 2).
We detected the expression levels of the cytoskeleton-forming factor dynactin p62 and proteins related to BrdU and IGF signals, which are active in S phase of the cell cycle. The level of BrdU was similar in regular medium and coculture without ASE. In all groups, the levels of dynactin p62 were very high on day 6, except for on day 4 of non-coculture and on days 4 and 6 of co-culture. The group treated with ASE showed higher levels of dynactin p62 than did the group without ASE. Particularly, in the coculture group with ASE, dynactin p62 expression rather than BrdU expression was detected throughout the cytoplasm (Fig. 3A and 3B).
3D stereoscopic comparison of the cytoplasmic expression of dynactin p62 and BrdU in day 6 embryos of each group confirmed that the expression of these proteins in the cytoplasm was drastically increased in both the normal culture and coculture groups treated with ASE (Fig. 3C and 3D).
The expression of IGF-R showed a similar pattern in both the groups with and without ASE, and the coculture group showed increased expression from day 4 to 6 compared to that in the non-coculture group (Fig. 4). PI3K expression decreased from day 4 to 6, regardless of ASE treatment. However, the reaction of AKT1 showed conflicting results in the regular culture and coculture groups, and the ASE-supplemented group showed a significantly higher level of AKT1 than did the non-supplemented group. The mTOR response significantly decreased from day 4 to 6 in all groups that did not contain ASE but significantly increased from day 4 to 6 in the regular medium containing ASE group. However, unlike the expression of genes linked to IGF signaling, the expression in the ASE-treated coculture group significantly decreased from day 4 to 6. The coculture group with ASE generally showed higher compared to relative protein expression.
In embryonic cells, the activity of active-MMP-9 and MMP-2 was increased in all groups supplemented with ASE. MMP expression was generally low in the co-culture group and significantly high in the ASE-treated coculture group compared to that in the ASE-treated non-coculture group (Fig. 5).
The detection of MT-MMP, which induces MMP activity, significantly increased from day 4 to 6 in the non-coculture group; the coculture group showed the same results on day 4 and 6. In the ASE-supplemented groups, MT-MMP significantly increased from days 4 to 6 in the coculture group, with the highest expression level observed on day 6.
Expression of the MMP inhibitors TIMP-2 and TIMP-3 was increased from day 4 to 6 in non-coculture; in coculture, only TIMP-2 increased from day 4 to 6. However, the expression of TIMP-3 significantly increased. In all ASE-treated groups, the expression of TIMPs decreased from day 4 to 6.
Embryonic cell development in pig IVF embryos acts on the follicles and microenvironment provided by the uterus, during which the follicles grow and gradually gain more developmental ability until they go through backpack destruction, phase I, and phase II (Day, 2000; Cushman et al., 2002). Depending on the environmental conditions of the in vitro maturation medium, nuclear condensation may not occur during embryo maturation, which can result in termination of the dictyotene stage of the first meiosis (Day, 2000; Abeydeera, 2002). IVF embryos in pigs are sensitive to environmental stress during cell division and may undergo apoptosis because of mitochondrial damage caused by oxidative stress (Combelles et al., 2009; Chen et al., 2022).
In this study, in vitro-fertilized embryos were cultured by adding ASE to the non-coculture and coculture, and the effects on cell development were analyzed by assessing cell death and the levels of cell survival factors in embryos showing cell cycle arrest on day 4 and 6. Our results confirm that intracellular proteins are maintained after cell cycle arrest, and some embryonic cytoplasmic forms are maintained (Kidson et al., 2004; Gajda, 2009). Although there are many causes of embryonic cell death, the main cause is Casp-3, which induces excessive metabolic stress in the mitochondria (Jeong and Seol, 2008; Sinha et al., 2013). Apoptosis in the embryo is aggravated by excessive expression of Casp-3 and Bax during the embryonic cell development in pigs (Sinha et al., 2013; Kim and Yoon, 2018a). However, compared with the non-ASE-treated group, the ASE-treated group maintained some BCL-2 activity (Prenek et al., 2017; Abate et al., 2020), which inhibited apoptosis. The expression of dynactin p62 and BrdU proteins, including IGF signal-related factors, was maintained, particularly in the ASE-treated coculture group. Reducing death of the embryonic cytoplasm through the antioxidant system may reduce oxidative stress caused by hydrogen peroxide (Brown et al., 2021; Wang et al., 2021). In practice, ASE-regulated cell death in the co-culture group began on early day 4, when embryonic cell development increased and continued until late day 6. Addition of ASE can affect the coculture system, and the activity of dynactin p62, a cytoplasmic skeleton-forming microtubule-forming factor, can ensure morphological maintenance and intracellular cell cycle activity (Skop and White, 1998; Torisawa and Kimura, 2022). The activity of MMPs associated with cytoplasmic activity was significantly high in ASE-treated groups, and MMP-9 was not controlled by TIMP-3. To date, many studies have been conducted to control antioxidant effects with the aim of developing IVF embryos in pigs. Recent studies have focused on reducing reactive oxygen species-induced cell death through the Nrf2/ARE signaling pathway (Combelles et al., 2009; Chen et al., 2022). Thus, it is important to minimize oxidative stress in mitochondria. However, it remains unclear whether this method can maintain substrate levels in the cell and increase nuclear reactions apart than these stress-reducing effects (Milewski et al., 2018; Kim et al., 2019). ASE may positively affect the development of pig embryos by inducing stable growth of the matrix and cytoskeleton in embryonic cells and controlling initial cell death.
During the development of pig IVF embryos, apoptosis action resulting in early cell death and cytoplasm collapse are challenges that must be addressed to efficiently produce embryos in pigs using IVF. Particularly, it is important to reduce mitochondria-induced oxidative stress and control apoptosis. In this study, addition of ASE to the coculture system suppressed early cell death. Increasing cytoskeleton formation in the cytoplasm may maintain cytoplasmic stability. It remains unclear which substances act directly or indirectly on the coculture, but addition of ASE influenced activation of the matrix of embryonic cells and stably developing embryos. These results may be useful in developing methods for embryo production in pigs using IVF, provide insights into the applications of ASE, and improve the understanding of the molecular mechanisms of pig embryo development.
None.
Conceptualization, S-H.K.; data curation, S-H.K.; formal analysis, B.B., M-J.O.; funding acquisition, S-H.K.; investigation, S-H.K., M-J.O.; methodology, M-J.O., J-Y.M.; project administration, S-H.K.; resources, S-H.K.; supervision, S-H.K.; roles/writing - original draft, M-J.O., B.B., J-Y.M.; writing - review & editing, B.B., S-H.K.
None.
Not applicable.
Not applicable.
Not applicable.
Not applicable.
No potential conflict of interest relevant to this article was reported.
Journal of Animal Reproduction and Biotechnology 2024; 39(3): 169-178
Published online September 30, 2024 https://doi.org/10.12750/JARB.39.3.169
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Min-Jee Oh1,3,# , Baasanjav Batmunkh1,# , Ji-Yeon Mo2 and Sang-Hwan Kim1,2,3,*
1General Graduate School of Animal Life Convergence Science, Hankyong National University, Anseong 17579, Korea
2School of Animal Life Convergence Science, Hankyong National University, Anseong 17579, Korea
3Institute of Applied Humanimal Science, Hankyong National University, Anseong 17579, Korea
Correspondence to:Sang-Hwan Kim
E-mail: immunoking@hknu.ac.kr
#These authors contributed equally to this work.
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Typical difficulties encountered during in vitro fertilization (IVF) to produce embryos in pigs include poor pronucleus formation and poor-quality fertilized embryos because of high polysperm invasion. In this study, we evaluated the effects of supplementation with apple seed extract (ASE) and coculture systems on porcine in vitro-fertilized embryo culture.
Methods: Slaughterhouse-derived ovaries were used to obtain cumulus-oocyte complexes (COCs). COCs were conventionally used to perform IVF. We examined the differences in apoptosis and metabolism during development following addition of ASE to normal culture and coculture systems. Matrix metalloproteinases (MMPs), cell development-related factors, and apoptotic proteins were compared in porcine embryos produced under different conditions.
Results: The expression of genes related to insulin-like growth factor (IGF) signaling was increased in the coculture system. In the ASE group, early apoptosis and necrosis were reduced in fertilized embryos and the late survival rate increased. Supplementation of the coculture system with ASE led to increased expression of BCL-2 and decreased expression of Casp-3 in the cytoplasm, thereby lowering the apoptosis rate and inducing MMP expression. In addition, compared with the extract-supplemented group in normal culture, the activity of MMP-2 decreased in the coculture system supplemented with ASE, activity of MMP-9 increased, and the expression of dynactin p62 and BrdU in the cytoplasm was higher than that in the other groups.
Conclusions: The coculture system increased the activity of the embryonic cytoplasm compared with the non-coculture system. Supplementation with ASE may induce cell activity and inhibit the expression of apoptotic factors.
Keywords: apple seed, coculture, embryo, microtubule, porcine
Ovarian follicles grow oocytes by release mature oocytes at the time of ovulation, after which final maturation is completed in the microenvironment of the ovary during oocytes development (Loutradis et al., 1999; Fair, 2010; Scaramuzzi et al., 2011). Oocyte development results from the close interaction between hormones and follicular cells (Milewski et al., 2018; Liu et al., 2020). Several methods have been attempted to induce stable development of embryos after maturity; however, inducing this development remains challenging. Particularly, the current in vitro fertilization and culture technology used for pigs reduces the rates of oocyte development because of incomplete cytoplasm, polyspermy invasion, low maturation rates, and blastocysts compared with that in other livestock. Therefore, an efficient method for producing pig embryos is needed (Dang-Nguyen et al., 2010; Isom et al., 2012). In IVP technology in pigs, several hormones are used during the in vitro maturation process or co-culture with oocytes to induce nuclear maturation of pig embryos (Oktay et al., 2000; Qian et al., 2005). Addition of porcine ovarian epithelial cells during in vitro fertilization (IVF) and pig embryo development not only increases the number of blastocysts formed but also improves blastocyst quality (Teplitz et al., 2020). Therefore, coculture with homologous cells induces embryonic development and efficiently improves the quality of pig embryos. However, embryonic cells sometimes degrade or cell development is stopped at the 2- or 4-cell phase after IVF in pigs (Jin et al., 2017; Kim and Yoon, 2018a). This phenomenon results in cell cycle arrest during cell division, leading to chromosomal abnormalities and DNA damage (Vandaele et al., 2007; Song et al., 2022). Particularly, high expression of Casp-3 and inhibition of BCl-2 during embryo development lead to pig embryo mortality (Kim and Yoon, 2018a; 2018b). Methods need to be established to inhibit cell death while maintaining the microenvironment to promote the development of porcine embryonic cells. Several studies have investigated the reduction of oxidative stress in in vitro embryos to achieve IVF in pigs while inhibiting apoptosis that can occur during embryonic cell differentiation (Chen et al., 2022). Several studies have suggested that successful cell growth is mediated by activating the Nrf2/ARE signaling pathway, thereby preventing the production of reactive oxygen species and inducing cell differentiation (Combelles et al., 2009; Kim et al., 2019; Kim, 2022). Portion natural extracts can maintain mitochondrial function in embryonic cells by activating the expression of endogenous antioxidant molecules, such as glutathione and various antioxidant enzymes (Beker et al., 2002; Brad et al., 2003; Svoradová et al., 2022). In addition, based on our previous findings that apple seed extract (ASE) can control the apoptosis of endometrial cells and influence oxidative stress and apoptosis (Kim, 2022), in this study, we analyzed whether apple seed extract (ASE) can also participate in maintaining embryo cell metabolism during in vitro culture.
Ovaries of pigs slaughtered at slaughterhouses were placed in physiological saline (0.85% w/v of NaCl, 300 mOsm/L or 9.0 g per liter, Penicillin G 100 IU/mL, Streptomycin 100 mg/mL) at 37℃, transported to our laboratory within 2 h, and washed three to several times with saline. An 18-gauge needle attached to a 10-mL syringe was used to collect the ovum and follicular fluid from antral follicles 2-6 mm in diameter. Cumulus-oocyte complexes (COCs) were observed under a stereomicroscope. Only COCs with three or more uniform layers of compact cumulus cells and a homogeneous cytoplasm were selected for further study. The sorted oocytes were washed three times with washing medium (liquid TCM, 10% antibiotic-antimycotic) supplemented with bovine serum albumin (Sigma, St. Louis, MO, USA). The oocytes were matured in two steps in maturation culture medium (TCM-199; Gibco, Grand Island, NY, USA) supplemented with 10% porcine follicular fluid. The cells were incubated in IVM 1 medium containing 2.5 µg/mL gonadotrophic hormone (Sigma) and 15 ng/mL epidermal growth factor (Sigma), and then incubated in IVM 2 medium, which did not contain the hormones. COCs (50-70 per well) were incubated at 39℃ in a 5% CO2 humid atmosphere for 22 h during each step.
Semen (1-2 mL) were transferred into a 15 mL conical tube and centrifuged at 1,500 rpm for 3 min. The supernatant was removed and washed twice with 3 mL modified Tris buffer medium. Twenty mature oocytes were suspended in 30 µL mM Tris buffer medium in a 60-mm culture dish and covered with mineral oil. Sperm (1 × 106) were added to the oocytes and incubated for 6 h in a 5% CO2 humid atmosphere incubator at 39℃ for IVF.
The in vitro culture was divided into four groups defined below. The NC group was used as a negative control, and the culture medium did not contain ASE. In the NA group, the culture medium was supplemented with ASE. The CC group was a co-culture system group (Using Cumulus cell) without ASE supplementation. The CA co-culture system group was supplemented with ASE. All coculture systems used Cumulus cells.
In the in vitro culture method, the in vitro culture (IVC) for all groups used porcine zygote medium (PZM-5) supplemented with 10% fetal bovine serum. After 6 h of in vitro fertilization, cumulus, and corona radiata cells were removed by pipetting, and the cells were washed in a porcine tissue culture medium (pTCM-199). Zygotes were identified, and each was placed in the prepared media group and cultured at 39℃ in a 5% CO2 humidified atmosphere for 6 days. The zygotes were transferred to freshly prepared culture media every 2 days. The cells used in the coculture group were Granulosa cells collected from follicles, initially cultured in PZM-5, and confirmed to have pore size inserts of approximately 0.4 µm. Afterwards, according to the treatment group, IVC media were used for coculture (Zeyneloglu and Kahraman 2009; Chung et al., 2018). For the ASE treatment group, 1 µg/500 µL ASE was treated in PZM-5 media. Embryos were checked under a microscope on the 4 and 6 days after in vitro culture. On the 4 day, embryos at approximately the 4cell stage were selected, and on the 6 day, embryos that had not developed beyond approximately the 8cell stage were used for the experiment.
For zymography and enzyme-linked immunosorbent assay (ELISA), total protein was extracted from cultured embryos using PRO-PREP (Intron Biotechnology, Seongnam, Korea), according to the manufacturer’s protocol. Protein samples were mixed with loading buffer (0.06% bromophenol blue, 10% sodium dodecyl sulfate, and 2% glycerol) and electrophoresed for 90 min at 100 V; sodium dodecyl sulfate-polyacrylamide gels (12%) containing 100 mg/mL gelatin A/B were used. The gels were washed twice with renaturation buffer (2.5% Triton X-100) and distilled water for 20 min and then incubated at 37℃ for 18 h in zymography reaction buffer. After the reaction, the gels were stained with 1% Coomassie blue for zymography (40% methanol, 10% acetic acid, 0.5 g/500 mL CBBR-250) staining solution for 1 h and destained with destain solution (Bio-Rad, Hercules, CA, USA). Bands were measured to evaluate MMP-2 and MMP-9 activity.
The MMP-9 (ab58803, Abcam, Cambridge, UK), MT1-MMP (sc-30074, Santa Cruz Biotechnology, Dallas, TX, USA), TIMP-2 (sc-9905, Santa Cruz Biotechnology), TIMP-3 (sc-6836, Santa Cruz Biotechnology), IGF-R1 (sc-712, Santa Cruz Biotechnology), PI3K (sc-365290, Santa Cruz Biotechnology), Akt (sc-5298, Santa Cruz Biotechnology), and mTOR (sc-517464, Santa Cruz Biotechnology) primary antibodies were added to 96-well ELISA plates to analyze the expression levels these proteins; incubation was performed at 4℃ overnight. After washing twice with washing buffer (1x PBS containing 2.5% Triton X-100), the wells were blocked for 2 h at RT with 1% skim milk solution. After washing, anti-rabbit (AB6721-1; Abcam), anti-mouse (AB205719; Abcam), and anti-goat (AB6741; Abcam) secondary antibodies were added to each well and incubated for 2 h at RT. The cells were washed with washing buffer and incubated with substrate solution (LIFE TECH, USA). The reaction was stopped by adding 1 M NH2SO4, and absorbance was measured at 450 nm.
Cultured embryos were fixed in 4% paraformaldehyde overnight at 4℃, washed for 30 min in PBS, and permeabilized with 0.2% Triton X-100 for 30 min at RT. After blocking with 3% bovine serum albumin in PBS, the samples were incubated with an antibody against the active forms of BCl-2 (sc-492, Santa Cruz Biotechnology), Casp-3 (sc-373730, Santa Cruz Biotechnology), dynactin p62 (sc-25730, Santa Cruz Biotechnology), BrdU (sc-32323, Santa Cruz Biotechnology), TNF-R (sc-31349, Santa Cruz Biotechnology), and Bax (Cell Signaling, Danvers, MA, USA) at a 1:300 dilution. The samples were washed and incubated with anti-rabbit IgG conjugated to Alexa Fluor 488 (A11094, Invitrogen, Carlsbad, CA, USA), anti-mouse IgG (AB150116, Abcam), or anti-goat IgG (AB150132, Abcam) conjugated to Alexa Fluor 594. Nuclei were counterstained with 1 g/mL Hoechst 33258 and mounted using fluorescence mounting medium. Images were acquired using a confocal microscope (TIRFF-C1; Nikon, Tokyo, Japan). ImageJ software (NIH Image, Bethesda, MD, USA) was used to measure the blastomere diameter and examine the shape and size of the embryos. The brightness of fluorescence was compared relatively using the Image J program (Image J 1.54f; NIH, WI, USA).
ELISA data were analyzed using the Statistical Package for Social Sciences (IBM SPSS Statistics for Windows, version 23.0; IBM Corp, Armonk, NY). Duncan’s multiple range test was used to determine the differences in mean values for each treatment group. Statistical significance was set at p < 0.05.
Fig. 1 shows the expression patterns of apoptotic factors in embryos with cell cycle arrest for in vitro culture methods and with addition of ASE after IVF.
In all treatment groups, pro-Casp-3 was expressed in the cytoplasm of embryonic cells in which the cell cycle had stopped, and cytoplasmic collapse was confirmed. On day 4 of embryo culture in non-coculture medium, Casp-3 was highly expressed in the cytoplasm, whereas on day 6, a protein similar to BCL-2 accumulated. However, the NA group treated with ASE showed high accumulation of BCL-2 on day 4, but the expression of pro-Casp-3 was increased on day 6, showing opposite results from the NC group. The coculture group showed different results from those of the non-coculture group. In the group without ASE, pro-Casp-3 expression was increased on day 6 compared to that on day 4. However, in the group treated with ASE, BCL-2 was highly expressed on both days 4 and 6, and the expression of Casp-3 was relatively low compared to that in all other groups (Fig. 1A and 1B). TNF-R and BAX were detected in all groups, with high expression of TNF-R (Fig. 2A and 2B). In both the regular culture and coculture groups, TNF-R expression tended to be higher than that in the ASE-treated group from day 4 to 6. The non-coculture group treated with ASE showed high expression of BAX, and the coculture group treated with ASE showed high expression of TNF-R (Fig. 2).
We detected the expression levels of the cytoskeleton-forming factor dynactin p62 and proteins related to BrdU and IGF signals, which are active in S phase of the cell cycle. The level of BrdU was similar in regular medium and coculture without ASE. In all groups, the levels of dynactin p62 were very high on day 6, except for on day 4 of non-coculture and on days 4 and 6 of co-culture. The group treated with ASE showed higher levels of dynactin p62 than did the group without ASE. Particularly, in the coculture group with ASE, dynactin p62 expression rather than BrdU expression was detected throughout the cytoplasm (Fig. 3A and 3B).
3D stereoscopic comparison of the cytoplasmic expression of dynactin p62 and BrdU in day 6 embryos of each group confirmed that the expression of these proteins in the cytoplasm was drastically increased in both the normal culture and coculture groups treated with ASE (Fig. 3C and 3D).
The expression of IGF-R showed a similar pattern in both the groups with and without ASE, and the coculture group showed increased expression from day 4 to 6 compared to that in the non-coculture group (Fig. 4). PI3K expression decreased from day 4 to 6, regardless of ASE treatment. However, the reaction of AKT1 showed conflicting results in the regular culture and coculture groups, and the ASE-supplemented group showed a significantly higher level of AKT1 than did the non-supplemented group. The mTOR response significantly decreased from day 4 to 6 in all groups that did not contain ASE but significantly increased from day 4 to 6 in the regular medium containing ASE group. However, unlike the expression of genes linked to IGF signaling, the expression in the ASE-treated coculture group significantly decreased from day 4 to 6. The coculture group with ASE generally showed higher compared to relative protein expression.
In embryonic cells, the activity of active-MMP-9 and MMP-2 was increased in all groups supplemented with ASE. MMP expression was generally low in the co-culture group and significantly high in the ASE-treated coculture group compared to that in the ASE-treated non-coculture group (Fig. 5).
The detection of MT-MMP, which induces MMP activity, significantly increased from day 4 to 6 in the non-coculture group; the coculture group showed the same results on day 4 and 6. In the ASE-supplemented groups, MT-MMP significantly increased from days 4 to 6 in the coculture group, with the highest expression level observed on day 6.
Expression of the MMP inhibitors TIMP-2 and TIMP-3 was increased from day 4 to 6 in non-coculture; in coculture, only TIMP-2 increased from day 4 to 6. However, the expression of TIMP-3 significantly increased. In all ASE-treated groups, the expression of TIMPs decreased from day 4 to 6.
Embryonic cell development in pig IVF embryos acts on the follicles and microenvironment provided by the uterus, during which the follicles grow and gradually gain more developmental ability until they go through backpack destruction, phase I, and phase II (Day, 2000; Cushman et al., 2002). Depending on the environmental conditions of the in vitro maturation medium, nuclear condensation may not occur during embryo maturation, which can result in termination of the dictyotene stage of the first meiosis (Day, 2000; Abeydeera, 2002). IVF embryos in pigs are sensitive to environmental stress during cell division and may undergo apoptosis because of mitochondrial damage caused by oxidative stress (Combelles et al., 2009; Chen et al., 2022).
In this study, in vitro-fertilized embryos were cultured by adding ASE to the non-coculture and coculture, and the effects on cell development were analyzed by assessing cell death and the levels of cell survival factors in embryos showing cell cycle arrest on day 4 and 6. Our results confirm that intracellular proteins are maintained after cell cycle arrest, and some embryonic cytoplasmic forms are maintained (Kidson et al., 2004; Gajda, 2009). Although there are many causes of embryonic cell death, the main cause is Casp-3, which induces excessive metabolic stress in the mitochondria (Jeong and Seol, 2008; Sinha et al., 2013). Apoptosis in the embryo is aggravated by excessive expression of Casp-3 and Bax during the embryonic cell development in pigs (Sinha et al., 2013; Kim and Yoon, 2018a). However, compared with the non-ASE-treated group, the ASE-treated group maintained some BCL-2 activity (Prenek et al., 2017; Abate et al., 2020), which inhibited apoptosis. The expression of dynactin p62 and BrdU proteins, including IGF signal-related factors, was maintained, particularly in the ASE-treated coculture group. Reducing death of the embryonic cytoplasm through the antioxidant system may reduce oxidative stress caused by hydrogen peroxide (Brown et al., 2021; Wang et al., 2021). In practice, ASE-regulated cell death in the co-culture group began on early day 4, when embryonic cell development increased and continued until late day 6. Addition of ASE can affect the coculture system, and the activity of dynactin p62, a cytoplasmic skeleton-forming microtubule-forming factor, can ensure morphological maintenance and intracellular cell cycle activity (Skop and White, 1998; Torisawa and Kimura, 2022). The activity of MMPs associated with cytoplasmic activity was significantly high in ASE-treated groups, and MMP-9 was not controlled by TIMP-3. To date, many studies have been conducted to control antioxidant effects with the aim of developing IVF embryos in pigs. Recent studies have focused on reducing reactive oxygen species-induced cell death through the Nrf2/ARE signaling pathway (Combelles et al., 2009; Chen et al., 2022). Thus, it is important to minimize oxidative stress in mitochondria. However, it remains unclear whether this method can maintain substrate levels in the cell and increase nuclear reactions apart than these stress-reducing effects (Milewski et al., 2018; Kim et al., 2019). ASE may positively affect the development of pig embryos by inducing stable growth of the matrix and cytoskeleton in embryonic cells and controlling initial cell death.
During the development of pig IVF embryos, apoptosis action resulting in early cell death and cytoplasm collapse are challenges that must be addressed to efficiently produce embryos in pigs using IVF. Particularly, it is important to reduce mitochondria-induced oxidative stress and control apoptosis. In this study, addition of ASE to the coculture system suppressed early cell death. Increasing cytoskeleton formation in the cytoplasm may maintain cytoplasmic stability. It remains unclear which substances act directly or indirectly on the coculture, but addition of ASE influenced activation of the matrix of embryonic cells and stably developing embryos. These results may be useful in developing methods for embryo production in pigs using IVF, provide insights into the applications of ASE, and improve the understanding of the molecular mechanisms of pig embryo development.
None.
Conceptualization, S-H.K.; data curation, S-H.K.; formal analysis, B.B., M-J.O.; funding acquisition, S-H.K.; investigation, S-H.K., M-J.O.; methodology, M-J.O., J-Y.M.; project administration, S-H.K.; resources, S-H.K.; supervision, S-H.K.; roles/writing - original draft, M-J.O., B.B., J-Y.M.; writing - review & editing, B.B., S-H.K.
None.
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No potential conflict of interest relevant to this article was reported.
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