Effect of Passage Number of Conditioned Medium Collected from Equine Amniotic Fluid Mesenchymal Stem Cells: Porcine Oocyte Maturation and Embryo Development

Oocyte in vitro maturation (IVM) is the most important first step in in vitro embryo production. One prerequisite for the success of IVM in oocytes is to provide a rich culture microenvironment that meets the nutritional needs of developing oocytes. We applied different equine amniotic fluid mesenchymal stem cell conditioned medium (eAFMSC-CM) from passages 7, 18, and 27 to porcine oocytes during IVM to determine its effects on oocyte development and subsequent embryo development, specifically. The eAFMSC-CM from passage 7 (eAFMSC-CMp7) has a considerable impact on 9 genes: BAX, BCL2, SOD2, NRF2, TNFAIP6, PTGS2, HAS2, Cx37, and Cx43, which are associated with cumulus cell mediated oocyte maturation. GSH levels and distribution of mitochondrial and cortical granules were significantly increased in oocytes incubated with eAFMSC-CMp7. In addition, catalase and superoxide dismutase activities were high after IVM 44 h with eAFMSC-CMp7. After in vitro fertilization, blastocyst quality was significantly increased in the eAFMSC-CMp7 group compared to control. Lastly, the antioxidant effect of eAFMSC-CMp7 substantially regulated the expression of apoptosis, pluripotency related genes and decreased autophagy activity in blastocysts. Taken together, this study demonstrated that the eAFMSC-CMp7 enhanced the cytoplasmic maturation of oocytes and subsequent embryonic development by generating high antioxidant activity.


Introduction
In vitro maturation (IVM) of oocytes is the most important first step in the production of embryos in vitro, and it is essential for biomedical studies and assisted reproduction techniques [1,2]. Despite the increasing number of studies on IVM application, only a few oocytes that mature in vitro develop into blastocysts, whereas the rate of embryonic development in several species such as pigs is much higher when oocytes are matured in vivo [3][4][5][6]. For the oocyte meiosis to be successful, chromosomal segregation during nuclear maturation and cytoplasmic maturation including organelle distribution are indispensable to enhance developmental competence [7,8]. Cytoplasmic oocyte maturation also affects the completion of oocyte nuclear maturation, fertilization, and early embryonic development [9,10]. However, the current porcine oocyte IVM system may be suboptimal for complete synchronization of nuclear and/or cytoplasmic maturation [11]. One prerequisite for the success of IVM in oocytes is the provision of a rich culture microenvironment in vitro that meets the nutritional needs of the developing oocyte. It has

Effects of eAFMSC-CM Passages during IVM on Oocyte Cytoplasmic Maturation (Experiment 2)
We evaluated the effects of eAFMSC-CM from passages 7, 18, and 27 (p7, p18, and p27, respectively) on oocyte cytoplasmic maturation after IVM for 44 h. Although nuclear maturation did not significantly differ among the four groups, the rate of maturation was significantly higher in the p7 than in the eAFMSC-CMp27 groups (81.7% ± 1.3% vs. 78.9% ± 1.7%, 76.3% ± 1.4%, and 72.0% ± 1.9%, respectively; p < 0.05; Figure 3A). The staining intensity of GSH was significantly increased (p < 0.05) in the eAFMSC-CMp7 group compared to the other groups, and was essentially the same among the control, p18, and p27 groups. The staining intensity of ROS was significantly decreased in the eAFMSC-CMp7 group compared to all other groups (p < 0.05; Figure 3B,C). intensity of GSH was significantly increased (p < 0.05) in the eAFMSC-CMp7 group compared to the other groups, and was essentially the same among the control, p18, and p27 groups. The staining intensity of ROS was significantly decreased in the eAFMSC-CMp7 group compared to all other groups (p < 0.05; Figure 3B,C). Mitochondrial distribution was heterogeneous throughout the cytoplasm in control oocytes, but not in homogeneous and non-aggregated in eAFMSC-CMp7 oocytes ( Figure  4A). The distribution rate of mitochondria was significantly higher in eAFMSC-CMp7 oocytes (p < 0.05) than in the other groups, whose distribution did not significantly differ from controls ( Figure 4B). Moreover, cortical granules (CGs) were distributed in cortical areas in the eAFMSC-CMp7 group, whereas they were distributed in the central cytoplasm in the other groups ( Figure 4C). Significantly more CGs were distributed in oocytes from eAFMSC-CMp7 groups than in those in the control and eAFMSC-CMp27 groups (p < 0.05; Figure 4D). Mitochondrial distribution was heterogeneous throughout the cytoplasm in control oocytes, but not in homogeneous and non-aggregated in eAFMSC-CMp7 oocytes ( Figure 4A). The distribution rate of mitochondria was significantly higher in eAFMSC-CMp7 oocytes (p < 0.05) than in the other groups, whose distribution did not significantly differ from controls ( Figure 4B). Moreover, cortical granules (CGs) were distributed in cortical areas in the eAFMSC-CMp7 group, whereas they were distributed in the central cytoplasm in the other groups ( Figure 4C). Significantly more CGs were distributed in oocytes from eAFMSC-CMp7 groups than in those in the control and eAFMSC-CMp27 groups (p < 0.05; Figure 4D).

Levels of Antioxidant Biomarkers in Fresh CM and IVM Medium with eAFMSC-CM (Experiment 3)
We analyzed SOD and CAT activities in fresh CM and IVM culture medium in the four groups after IVM for 22 and 44 h. The SOD and CAT activities were significantly higher in eAFMSC-CMp7 with the pre and first than in the TCM199. The activities of SOD and CAT were significantly higher in the second IVM medium with eAFMSC-CMp7 than

Levels of Antioxidant Biomarkers in Fresh CM and IVM Medium with eAFMSC-CM (Experiment 3)
We analyzed SOD and CAT activities in fresh CM and IVM culture medium in the four groups after IVM for 22 and 44 h. The SOD and CAT activities were significantly higher in eAFMSC-CMp7 with the pre and first than in the TCM199. The activities of SOD and CAT were significantly higher in the second IVM medium with eAFMSC-CMp7 than those in the control, p18 and p27 groups (55.63 ± 0.93 vs.12.57 ± 1.98, 28.71 ± 2.07, and 36.02 ± 2.21, respectively; p < 0.05; Figure 5A; 1.92 ± 0.01 vs. 1.82 ± 0.01, 1.86 ± 0.02, and 1.79 ± 0.02, respectively; p < 0.05, Figure 5B).
The expression of mRNAs associated with apoptosis, pluripotency, and autophagy in IVF blastocysts determined by qRT-PCR revealed a significantly decreased BAX/BCL2 ratio in the eAFMSC-CMp7 group compared to control group (p < 0.05; Figure 7), whereas Beclin1 expression did not differ between these groups. The expression of SOX2 and NANOG that are associated with pluripotency significantly differed between the control and eAFMSC-CMp7 groups (p < 0.05). The subcellular localization of LC3 of blastocysts was investigated by immunofluorescent staining ( Figure 8A). The fluorescence intensity of LC3 is significantly lower in the eAFMSC-CMp7 group than in the control group (11.5% ± 0.61 vs. 13.0% ± 0.58, p < 0.05; Figure 8B). The subcellular localization of LC3 of blastocysts was investigated by immunofluorescent staining ( Figure 8A). The fluorescence intensity of LC3 is significantly lower in the eAFMSC-CMp7 group than in the control group (11.5% ± 0.61 vs. 13.0% ± 0.58, p < 0.05; Figure 8B).

Discussion
Oocyte developmental competence is affected by direct contact with CCs and by the microenvironment created by the COC during maturation. We found that eAFMSC-CM in porcine IVM medium facilitated oocyte cytoplasmic maturation and embryo development through cumulus cells.
We initially assessed the effects of eAFMSC-CM passaged 7, 18, and 27 times on IVM to establish the optimal number of passages required for the successful development of in vitro fertilized porcine embryos. Oocyte cytoplasmic maturation was significantly improved in eAFMSC-CMp7 via the expression of genes associated with competent cumulus expansion and cumulus cell apoptosis and improvement in intracellular GSH and ROS, mitochondrial distribution, and CG distribution. We found that eAFMSC-CMp7 significantly improved blastocyst formation, total cell number, and pluripotency-related genes, which are indicators of embryo developmental potential that consequently determine implantation success. In addition, eAFMSC-CMp7 significantly reduced autophagy activity and the expression of associated genes. These results indicated that supplementation of eAFMSC-CMp7 during IVM provides an optimal cytoplasmic maturation microenvironment and preimplantation milieu to develop quality porcine embryos in vitro.
Communication between oocytes and CCs is important for the acquisition of developmental competence [34,35]. The degree of cumulus cell expansion is routinely used as a gross indicator of oocyte maturation and fertilization in vitro [36]. A decrease in cumulus cell apoptosis increases oocyte maturation capacity and developmental potential [37]. The present study found a significant decrease in the BAX/BCL2 ratio in the eAFMSC-CMp7 group compared to the control group. SOD2 and NRF2 neutralize ROS and protect oocytes and embryos from damage [38]. NRF2 regulates the basal expression of SOD2 and the transcription of genes encoding a variety of antioxidant enzymes [39]. Our results suggested that eAFMSC-CMp7 promotes the expression of antioxidant enzymes in cumulus cells by upregulating NRF2 and SOD2 expression. The mRNA levels of hyaluronan synthase 2 (HAS2), TNFAIP6, and PTGS2 genes affiliated with cumulus cell expansion and Cx37 and Cx43 associated with cumulus cell gap junctions were upregulated in porcine cumulus cells in the eAFMSC-CMp7 group. We thus concluded that eAFMSC-CMp7 could provide sufficient bioactive materials through cumulus cells to enhance oocyte competence.

Discussion
Oocyte developmental competence is affected by direct contact with CCs and by the microenvironment created by the COC during maturation. We found that eAFMSC-CM in porcine IVM medium facilitated oocyte cytoplasmic maturation and embryo development through cumulus cells.
We initially assessed the effects of eAFMSC-CM passaged 7, 18, and 27 times on IVM to establish the optimal number of passages required for the successful development of in vitro fertilized porcine embryos. Oocyte cytoplasmic maturation was significantly improved in eAFMSC-CMp7 via the expression of genes associated with competent cumulus expansion and cumulus cell apoptosis and improvement in intracellular GSH and ROS, mitochondrial distribution, and CG distribution. We found that eAFMSC-CMp7 significantly improved blastocyst formation, total cell number, and pluripotency-related genes, which are indicators of embryo developmental potential that consequently determine implantation success. In addition, eAFMSC-CMp7 significantly reduced autophagy activity and the expression of associated genes. These results indicated that supplementation of eAFMSC-CMp7 during IVM provides an optimal cytoplasmic maturation microenvironment and preimplantation milieu to develop quality porcine embryos in vitro.
Communication between oocytes and CCs is important for the acquisition of developmental competence [34,35]. The degree of cumulus cell expansion is routinely used as a gross indicator of oocyte maturation and fertilization in vitro [36]. A decrease in cumulus cell apoptosis increases oocyte maturation capacity and developmental potential [37]. The present study found a significant decrease in the BAX/BCL2 ratio in the eAFMSC-CMp7 group compared to the control group. SOD2 and NRF2 neutralize ROS and protect oocytes and embryos from damage [38]. NRF2 regulates the basal expression of SOD2 and the transcription of genes encoding a variety of antioxidant enzymes [39]. Our results suggested that eAFMSC-CMp7 promotes the expression of antioxidant enzymes in cumulus cells by upregulating NRF2 and SOD2 expression. The mRNA levels of hyaluronan synthase 2 (HAS2), TNFAIP6, and PTGS2 genes affiliated with cumulus cell expansion and Cx37 and Cx43 associated with cumulus cell gap junctions were upregulated in porcine cumulus cells in the eAFMSC-CMp7 group. We thus concluded that eAFMSC-CMp7 could provide sufficient bioactive materials through cumulus cells to enhance oocyte competence.
The endogenous antioxidant, GSH, can protect cells from free radicals and ROS, thus playing an important role in the antioxidant defense system of oocytes [40,41]. Excessive ROS generation induced by environmental stress in vitro damages cell structures and activates apoptotic pathways in many types of cells [42]. CCs surrounding oocytes are structurally and metabolically linked to oocytes via gap junctions and they regulate the synthesis and accumulation of GSH in ooplasm [34]. Direct supplementation of antioxidative reagents into the maturation medium improves the GSH content of oocytes [43]. We found that supplementing IVM medium with eAFMSC-CMp7 consistently resulted in significantly generating more GSH and less ROS than the other groups, which indicates powerful antioxidant properties.
Upon maturation, mitochondria migrate towards the central region, and homogeneous mitochondrial localization is considered a sign of cytoplasmic maturity that is associated with oocyte developmental capacity [44][45][46]. Mammalian CGs are oocyte-specific vesicles located in the subcortical region of fully grown oocytes and function as post-fertilization blocks to polyspermy [47]. Any dysfunction or dislocation of the CGs reduces oocyte competence and affects embryo quality [48]. The ratios of oocytes with mitochondria were higher and the CG distribution was greater in the eAFMSC-CMp7 group, suggesting enhanced cytoplasmic maturation. Mitochondria are critical for energy production via glycolysis and TCA cycle pathways in CCs and as an energy supply for oocyte maturation [49]. Moreover, persistent interaction between CCs and oocytes stabilizes CG distribution, whereas interrupting it induces CG exocytosis that might be responsible for the reduced penetrability of oocytes [50].
Cells use enzymatic antioxidants to resist ROS, with SOD and CAT being the major antioxidants. Intracellular ROS levels in human umbilical cord MSCs are higher at p11 and p17 than at p4, suggesting that senescence occurred according to the cumulative number of passages [51]. Even in human and equine MSCs, successive passages lead to alterations in immune phenotype, differentiation capacity, and cellular senescence [52]. Therefore, we investigated antioxidant biomarker activity in fresh eAFMSC-CM from passages 7, 18, and 27 in the 1st and 2nd IVM media. Our results were consistent with the findings that factors secreted from MSCs into CM contain antioxidants and are predominant elements, and that they exert anti-oxidative effects via paracrine mechanisms [53,54]. Moreover, the findings revealed that oocytes in the second IVM medium supplemented with eAFMSC-CMp7 had significantly more SOD and CAT activities than the other groups. Favorable IVM conditions generated by the release of active antioxidants during early eAFMSC-CM passages might explain the improved oocyte maturation and antioxidant expression in CCs. Next, we used seven passages in subsequent experiments.
We evaluated embryo development and gene expression in oocytes cultured in eAFMSC-CMp7 to further explore this notion. Supplementation with eAFMSC-CMp7 increased anti-apoptotic activities (BCL2) and pluripotency (SOX2 and Nanog) of IVF blastocysts. Moreover, the formation rate and total number of blastocysts significantly improved with eAFMSC-CMp7 through the regulation of apoptotic genes. These results supported our notion that eAFMSC-CM supplementation during the IVM period improves the quality of mature porcine oocytes.
Various stimuli induce both ROS and autophagy, and the induction of autophagy is antagonized by antioxidants [55][56][57]. The autophagy pathway might be induced under various stress conditions to restore homeostasis as a pro-survival adaptive response to stress. Microtubule-associated light chain protein 3 is a marker of autophagosomes, and changes in its expression reflect levels of autophagy [58,59]. Therefore, autophagy probably protects oocyte maturation under stress. The present findings showed that eAFMSC-CMp7 decreased the intensity of LC3 immunofluorescence emission in porcine blastocysts, indicating reduced autophagy.
To the best of our knowledge, this is the first study to evaluate the effects of the different passage numbers of eAFMSC-CM on porcine oocytes. We demonstrated that supplementation of eAFMSC-CM during IVM clearly improved oocyte quality and subse-quent embryo development. The antioxidant competence of CM from the lower passage of eAFMSC was significantly effective for oocyte cytoplasmic maturation and blastocyst formation. Therefore, eAFMSC-CM would provide an abundant antioxidant-environment influencing the oocyte and embryo developmental competence, and we recommend the usage of eAFMSC-CM from passage 7 for porcine IVM. In a further study, we plan to analyze various factors (many growth factors including cytokines and bioactive factors) that could affect the oocytes in addition to the SOD and CAT investigated in the current study.

Ethics
The Institutional Animal Care and Use Committee (IACUC) of Chungnam National University approved the use of ovaries (Approval no: 202103A-CNU-002).

Reagents and Chemicals
We purchased all chemicals and reagents from Sigma Chemical Co. (St. Louis, MO, USA), unless otherwise stated.

Preparation of Equine Amniotic Fluid Stem Cell-Conditioned Medium (eAFMSC-CM)
We cultured eAFMSC for 7, 18, and 27 passages until they reached 70% confluence in 100-mm dishes with Dulbecco's modified Eagle's medium (DMEM) (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37 • C under 5% CO 2 . The CM was replaced with StemPro ® MSC serum-free medium (Gibco; Thermo Fisher Scientific Inc.). After 48 h, media were collected and centrifuged at 300× g for 10 min at 4 • C. The supernatant was passed through a 0.22 µM filter and the filtrate was regarded as eAFMSC-CM.

Maturation In Vitro
Porcine ovaries obtained locally within 3 h of slaughter were placed in sterilized 0.9% normal saline at 30 • C. Cumulus oocyte complexes (COCs) were aspirated from antral follicles (3-6 mm) using a 10 mL syringe. Then, COCs with three layers of cumulus cells were selected and cultured at 38.5 • C in a humidified 5% CO 2 atmosphere in tissue culture medium (TCM)-199 (500 µL) containing 2.5 mM fructose, 0.4 mM L-cysteine, 1 mM sodium pyruvate, 0.13 mM kanamycin, 10% (v/v) porcine follicular fluid, 10 ng/mL epidermal growth factor (EGF), 10 IU/mL pregnant mare serum gonadotropin (PMSG), and human chorionic gonadotropin (hCG). After 22 h of maturation, the COCs were washed in fresh IVM medium without hormones and cultured for 20-22 h. The maturity of the oocytes was assessed as extrusion of the first polar body in the perivitelline space after denuding. Cumulus cells that separated from the COCs were stored at −80 • C.

Evaluation of Cumulus Cell Expansion
We quantified degrees of cumulus cell expansion in COCs on a scale of 0-2 after 44 h of IVM as described [60,61]. Degrees of 0, 1, and 2, respectively, indicated no expansion or detachment of cumulus cells from the oocyte, moderate expansion between degrees 0 and 2, and full expansion including the corona radiata.

Assessment of Mitochondrial Distribution
We assessed the mitochondrial distribution in oocytes as follows. Oocytes were washed three times in TCM-199 and incubated for 30 min at 38.5 • C with 250 nM Mi-toTracker™ Red CMXRos (Thermo Fisher Scientific Inc.). After incubation, they were again washed in TCM-199 and fixed in PBS with 4% paraformaldehyde in phosphate buffered saline (PFA-PBS; w/v) for 30 min at room temperature (RT). Finally, the oocytes were mounted on glass slides, covered with coverslips, and visualized using an LSM 880 confocal laser scanning microscope (Zeiss, Jena) with filters (between 579 nm and 599 nm). Digital images of oocytes were analyzed with ImageJ software in order to measure the average fluorescence intensity (normalized to the background average intensity).

Assessment of Cortical Granule Distribution
The distribution of cortical granules in oocytes was measured using fluorescein isothiocyanate (FITC)-labeled peanut agglutinin. Oocytes were fixed in 4% PVA-PBS (w/v) for 30 min at RT and washed three times with PBS. The oocytes were then permeabilized with 0.5% Triton X-100 for 30 min and rinsed three times with 0.05% Tween-20 (5 min each). Non-specific antigen binding was blocked with 2% BSA in PBS for 1 h, and then the oocytes were incubated with FITC for 2 h at RT. After three washes with 0.05% Tween-20 (5 min each), the sections were mounted on glass slides and examined using the LSM 880 confocal laser-scanning microscope with a filter (488 nm). Digital images of oocytes were analyzed with ImageJ software in order to measure the average fluorescence intensity (normalized to the background average intensity).

Antioxidant Enzyme Activity Assays
SOD and catalase (CAT) activities were quantified in non-conditioned medium (TCM199) and in eAFMSC-CM before and after IVM using colorimetric OxiSelect™ assay kits (Cell Biolabs Inc., San Diego, CA, USA), as described by the manufacturer. The activities of SOD and CAT were assessed by measuring absorbance at 490 and 520 nm, respectively.

Embryo Development and Total Blastocyst Counts
Cleavage and blastocyst rates were evaluated at 2 (48 h) and 7 (168 h) days after IVF (Day 0). Depending on the experiment, total blastocysts were counted, RNA was extracted, and the blastocysts were visualized by immunofluorescence staining. The total numbers of blastocysts were determined on day 6. Then, the blastocysts were washed in NCSU-W and immediately stained for 15 min with 5 µg/mL of Hoechst-33342. Blastocysts from each group were mounted on glass slides with a drop of glycerol, covered with coverslip, and evaluated using fluorescence microscopy.

Immunofluorescence Staining
Blastocysts were washed three times with PBS, fixed in 4% PFA-PBS (w/v) at RT, permeabilized with 0.5% Triton X-100 for 30 min at RT, and rinsed three times with 0.05% Tween-20 (5 min each). Non-specific antigen binding was blocked with 1% BSA in PBS for 1 h, and then the blastocysts were incubated overnight at 4 • C with 1:500-diluted rabbit anti-microtubule-associated protein 1A/1B-light chain 3 (LC3) (ab51520; Abcam, Cambridge, UK). They were then washed three times with 0.05% Tween-20 (5 min each) and labeled with 1:200-diluted Alexa Fluor 488 goat anti-rabbit IGF (ab150077; Abcam, Cambridge, UK) for 1 h at RT. After five washes with 0.05% Tween-20 for 5 min each, blastocysts were counterstained with 10 µg/mL PI for DNA labeling and visualized using the LSM 880 confocal laser-scanning microscope with filter (488 nm). Digital images of oocytes were analyzed with ImageJ software in order to measure the average fluorescence intensity (normalized to the background average intensity).

Analysis of Gene Expression by Quantitative Real-Time PCR
We extracted RNA from cumulus cells and blastocysts using RNAqueousTM Micro Total RNA Isolation Kits (Invitrogen, USA) as described by the manufacturer. The total RNA concentration was determined using a Biospec-nano spectrophotometer (Shimadzu Corp., Kyoto, Japan). Complementary DNA (cDNA) was synthesized using the Maxime RT premix kit (iN-tRON Biotechnology Inc., Seongnam, Korea) and amplified by Quantitative Real-Time PCR (qRT-PCR) using a CFX96 Touch Real-Time PCR system (Bio-Rad Laboratories Inc., Hercules, CA, USA) as described by the manufacturer, with minor modifications. Each qRT-PCR mixture contained 10 µL SYBR ® Green Supermix (Bio-Rad Laboratories Inc.), 0.4 µL (10 pmol/µL) each of forward and reverse primers, 8.2 µL nuclease-free water, and 1 µL cDNA in a PCR plate. The temperature program comprised 40 cycles of 30 s at 95 • C, 30 s at 55 • C, and 30 s at 72 • C. The expression of each target gene in the CCs and blastocysts was quantified relative to that of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), using the equation: R = 2 −[∆Ct sample−∆Ct control . Table 1 shows the primers included in the reactions.

Statistical Analysis
All data generated from at least triplicate experiments were statistically analyzed using GraphPad Prism 9.0.0 (GraphPad, San Diego, CA, USA) and are presented as means ± SEM. All data from experiments 1-3 were evaluated using a univariate analysis of variance (ANOVA) with Tukey tests. All data from experiment 4 were evaluated using Student t-tests and the results are presented as means ± standard deviation. Values were considered statistically significant at * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001).

Conclusions
This study demonstrated that eAFMSC-CM treatment acts as an antioxidant in the optimal passage of eAFMSC during IVM of porcine oocyte. Moreover, eAFMSC-CM improves oocyte quality and embryonic development after in vitro fertilization by reducing ROS generation and increasing GSH levels during meiosis. These findings provide evidence that eAFMSC-CM would provide an abundant antioxidant-environment influencing the oocyte and embryo developmental competence, and we recommend the usage of eAFMSC-CM from passage 7 for porcine IVM.