Recombinant human IL-7 was purchased from PeproTech (London, UK). Unless otherwise indicated, all the chemicals and reagents used in this study were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Porcine ovaries were collected from a local abattoir and transported to the laboratory within 1 h in 0.9% (v/v) NaCl solution at 37°C. At the laboratory, the ovaries were washed twice with 0.9% (v/v) NaCl solution, and thereafter, cumulus-oocyte complexes (COCs) were retrieved from medium sized (3–7 mm in diameter) follicles (24) using an 18-gauge needle and a 10 mL disposable syringe and collected into 15 mL conical tubes. The COCs were washed with HEPES-buffered Tyrode's medium containing 0.05% (w/v) polyvinyl alcohol (TLH-PVA). Then, 50–60 COCs with compact cumulus cell layers and evenly granulated cytoplasm were cultured in a four-well dish (Nunc, Roskilde, and Denmark) containing 500 μL IVM medium, consisting of TCM-199 (Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 0.6 mM cysteine, 0.91 mM sodium pyruvate, 10 ng/mL epidermal growth factor, 75 μg/mL kanamycin, 1 μg/mL insulin, and 10% (v/v) porcine FF. The IVM process took place over a period of 42 h. This was followed by the incubation of the COCs with 10 IU/mL equine chorionic gonadotropin and 10 IU/mL human chorionic gonadotropin at 39°C in a humidified 5% CO₂ atmosphere first for 22 h, after which the COCs were then transferred to a hormone-free IVM medium and further incubated for the remaining 20 h.

PA was performed according to a previously reported protocol (25). Briefly, after IVM, metaphase II—stage (MII) oocytes were obtained by mechanically pipetting enclosing cumulus cells in the presence of 0.1% hyaluronidase for 1 min. After washing twice with the activation solution (280 mM mannitol solution containing 0.01 mM CaCl₂ and 0.05 mM MgCl₂), the MII oocytes were activated in 2 mL of the same solution with two direct electrical pulses of 120 V/mm for 60 μs, using a cell fusion generator (LF101; NepaGene, Chiba, Japan). Thereafter, the oocytes were cultured in the IVC medium (porcine zygote medium 5; PZM5) (26) containing 5 μg/mL of cytochalasin B for 4 h in a humidified atmosphere of 5% CO₂, 5% O₂, and 95% N₂. The electrically activated embryos then were washed three times in droplets of IVC medium (30 μL) and incubated in the same droplets (10 embryos per droplet) covered with mineral oil. PA was carried out on day 0. The embryos were moved to fresh droplets 48 (day 2) and 96 h (day 4) after PA. On day 2, the embryo cleavage rates were analyzed in five categories (1-, 2–3-, 4–5-, 6–8-cell, and fragmented embryos). On day 7, embryonic development was quantitatively evaluated by determining the cleavage and blastocyst formation rates in three groups, according to blastocyst morphology (early, expanded, and hatched). During the entire IVC period, IL-7 was added to the IVC media at concentrations of 0 (control), 0.1, 1, 10, and 100 ng/mL.

Intracellular GSH and ROS levels were measured using a previously reported method (27). The 4 to 5-cell stage embryos in each group were sampled on day 2 to make these measurements. CellTracker Blue 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (CMF₂HC; Invitrogen) and 2'7'-dichlorodihydrofluorescein diacetate (H₂DCFDA; Invitrogen) were used to measure intracellular GSH (blue fluorescence) and ROS (green fluorescence) levels in the embryo cytoplasm. In brief, the embryos were transferred to TLH-PVA medium containing 10 μM CMF₂HC or H₂DCFDA and stained in the dark for 30 and 10 min, respectively. Thereafter, the embryos were washed three times with TLH-PVA and transferred to 8 μL droplets of TLH-PVA. GSH and ROS levels were then imaged using a fluorescence microscope (TE300; Nikon, Tokyo, Japan) with UV filters (370 nm for GSH and 460 nm for ROS). Finally, Adobe Photoshop 2021 was used to analyze the fluorescence intensity of each embryo, which was normalized to that of the control group.

Blastocysts were stained with TUNEL to determine the number of apoptotic cells using an in situ cell death detection kit (Roche, Mannheim, Germany), according to a previously reported method (28). On day 7, the blastocysts derived from the IVC medium were treated with 0, 0.1, 1, 10, and 100 ng/mL of IL-7, washed three times in 0.1% PBS-PVA (PVS), and fixed in 4% paraformaldehyde in PBS for 30 min at 25°C (room temperature; RT). In the next step, the blastocysts were washed twice in 0.1% PVS with 0.1% Tween 20 and 0.01% Triton X-100 (v/v) and permeabilized with 0.3% TritonX-100 in PBS for 1 h at 37°C. Finally, TUNEL assay was performed using fluorescein-conjugated deoxyuridine triphosphate (dUTP) and terminal deoxynucleotidyl transferase (Roche, Mannheim, Germany) for 1 h 30 min at 37°C. After washing twice in 0.1% PVS, the blastocysts were counterstained with 5 μg/mL Hoechst-33342 for 10 min at RT to visualize nuclei.

The mRNA expression was analyzed using qRT-PCR for 19 specific genes associated with different functions, such as apoptosis-related genes: BCL2 associated X (BAX), BCL2 like 1 (BCL2L1), caspase-3 (CASP3), and myeloid cell leukemia-1 (MCL1); IL-7 signaling-related genes: phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), AKT serine/threonine kinase 1 (AKT1), and extracellular-regulated kinase 1/2 (ERK1/2; also known as MAPK3/1); embryonic development-related genes: proliferating cell nuclear antigen (PCNA), POU class 5 homeobox 1 (OCT4), NANOG, caudal type homeobox 2 (CDX2), and GATA binding protein 6 (GATA6); maternal effect genes (MEGs): KH domain containing 3 like (KHDC3L; Filia), nucleophosmin/nucleoplasmin 2 (NPM2), and zygote arrest 1 (ZAR1); zygotic genome activation (ZGA)–related genes: solute carrier family 34 member 2 (SLC34A2), developmental pluripotency associated 2 (DPPA2), and eukaryotic translation initiation factor 1A (EIF1A). All primer sequences are listed in Supplementary Table 1.

On day 2, embryos and blastocysts were washed thrice with PVS and sampled at −80°C before the experiment. RNA extraction was performed using TRIzol reagent (TaKaRa Bio, Inc., Otsu, Shiga, Japan), according to the manufacturer's protocol. Then, the extracted RNA (1 μg of total RNA) was converted to complementary DNA (cDNA) using SuperScript IV VILO Master Mix (Thermo Fisher Scientific, MA, USA). Finally, the synthesized cDNA, 2× SYBR Premix Ex Taq (TaKaRa Bio, Inc.), and 5 pmol of specific primers (Macrogen, Inc., Seoul, Republic of Korea) were used for qRT-PCR. The mRNA expression was analyzed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The cycling parameters were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 56°C for 15 s, and 72°C for 30 s. Relative quantification was performed by comparing the threshold cycle (Ct) at constant fluorescence intensity. Relative mRNA expression (R) was calculated using the equation R = 2^{−[ΔCtsample−ΔCtcontrol]} (29). The R values were normalized to those of RN18S in the blastocysts of each group.

Immunofluorescence was conducted according to the methods of Yoon et al. (8), with a few modifications. Briefly, the MII oocytes and embryos were fixed with 4% paraformaldehyde in PBS for 30 min at RT, permeabilized in 0.5% Triton X-100 for 1 h at RT and washed twice with 0.1% PVS. The MII oocytes or embryos were treated with Image-iT™ FX Signal Enhancer (Invitrogen, Carlsbad, CA, USA) for 30 min at RT and blocked in PBS with 3% BSA and 0.05% Tween 20, for 1 h 30 min at RT. The samples were then incubated overnight at 4°C with the following primary antibodies: rabbit anti-IL-7 (ab 175380, 1:50 dilution in blocking solution; Abcam, Cambridge, UK), rabbit anti-IL-7R (ab 180521, 1:100; Abcam), mouse anti-SOX2 (sc-365823, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and mouse anti-phospho-STAT5 (ab 106095, 1:25; Abcam). On the following day, the samples were washed three times for 5 min with 0.1% Tween 20 and 0.01% Triton X-100 in 0.1% PVS (TTVS) at RT and then incubated with the appropriate secondary antibodies: goat anti-mouse IgG (H + L) Alexa Fluor^TM 488 (A11029, 1:200; Invitrogen Corporation, Carlsbad, CA, USA), donkey anti-rabbit IgG (H + L) Alexa Fluor^TM 594 (A21207, 1:400; Invitrogen) for 2 h at RT. After washing three times in TTVS, the samples were counterstained with Hoechst-33342 for 10 min and mounted on glass slides in an anti-fade mounting medium (Molecular Probes, Inc., Eugene, OR, USA). The stained samples were analyzed using an epifluorescence microscope (TE300; Nikon) with UV filters. SOX2-positive cells were specifically examined in blastocysts with more than 30 cells on day 7.

Statistical analysis was performed using SPSS 21.0 (SPSS Inc., Chicago, IL, USA). The experiments were performed at least in triplicates unless stated otherwise. Further, the results were presented as the mean ± SEM. Percentage data (cleavage and blastocyst formation rates) and average data (intracellular GSH and ROS levels in cleavage, TUNEL assay in blastocysts, relative gene expression levels, and quantitative analysis in immunofluorescence) were analyzed using one-way analysis of variance or Student's t-test. Statistical significance was set at p < 0.05.

To identify the locations of IL-7 and IL-7R in porcine oocytes and embryos at each stage, we collected MII oocytes after IVM, and sampled 2-, 4-, and 8-cell embryos and blastocysts at 24, 48, 72, and 168 h after PA, respectively. The localization of IL-7 and IL-7R proteins during preimplantation development was investigated by immunofluorescence. Both IL-7 and IL-7R were expressed in MII oocytes (Figure 1A). Furthermore, these proteins were observed in the 2-, 4-, and 8-cell stage embryos (Figures 1B–D). IL-7 and its receptor were also found to be localized to the cytoplasm until the blastocyst stage (Figure 1E).

We added IL-7 at various concentrations during IVC to determine its optimal concentration for porcine embryonic development after PA. The cleavage rates were significantly increased (p < 0.05) in the 10 and 100 ng/mL IL-7-treated groups compared to the control group (Table 1 and Figure 2). Furthermore, blastocyst formation rates were significantly higher (p < 0.05) in the group treated with 10 ng/mL IL-7 than in the control group. The cleavage patterns on day 2 also revealed that the rates of fragmented embryos were significantly lower (p < 0.05) in the 1 and 10 ng/mL IL-7-treated groups than in the control group (Figure 2B).

To examine the effect of IL-7 on oxidative stress, we analyzed intracellular GSH and ROS levels in 4-cell embryos on day 2 after PA (Figure 3). The 1 and 10 ng/mL IL-7 treatment groups showed significantly higher (p < 0.05) intracellular GSH levels than the control group. Further, embryos in the 10 ng/mL IL-7 treatment group showed significantly lower (p < 0.05) intracellular ROS levels than those in the control group.

The total number of cells and apoptotic nuclei were counted to investigate the quality of porcine blastocysts treated with IL-7. Supplementing the IVC media with IL-7 did not influence the total number of nuclei compared with that in the control group (Figures 4A,B). Nevertheless, the number of apoptotic nuclei and the apoptotic index were significantly reduced (p < 0.05) in blastocysts treated with 1 and 10 ng/mL IL-7 compared to those in the control (Figures 4A,C,D).

After determining the optimal IL-7 supplementation concentration during the IVC period to be 10 ng/mL, we conducted subsequent IVC experiments using 10 ng/mL IL-7. To determine the mechanism of the observed increase in embryonic developmental efficiency upon IL-7 supplementation, transcript levels in day 2 embryos and blastocysts were analyzed via qRT-PCR. Thus, we observed that in the IL-7-treated group relative to the control group, the mRNA level of the pro-apoptotic gene, BAX was significantly decreased (p < 0.001), while that of the anti-apoptotic gene, MCL1 was significantly increased (p < 0.05) (Figure 5A). The expression levels of genes associated with IL-7 signaling, including PIK3R1, AKT1, and ERK1, were not significantly different between the IL-7 supplementation and control groups. However, ERK2 levels were significantly higher (p < 0.05) for the IL-7-supplemented group relative to the control group (Figure 5B). Furthermore, the IL-7-treated embryos showed significantly higher (p < 0.05) NANOG transcript levels than the control embryos (Figure 5C).

A previous study revealed that porcine ZGA occurs at the 4-cell stage (30). It has also been reported that during the maternal-to-zygotic transition (MZT), perturbation in the degradation of maternal RNAs and ZGA can lead to embryonic arrest, indicating the importance of these processes (31). Therefore, we investigated whether IL-7 is involved in maternal mRNA clearance and ZGA by detecting the mRNA levels of MEGs (Filia, NPM2, and ZAR1) and ZGA-related genes (SLC34A2, DPPA2, and EIF1A). Compared with the control group, Filia and NPM2 transcript levels were significantly lower (p < 0.01) following IL-7 treatment (Figure 5D). Conversely, the mRNA expression levels of DPPA2 and EIF1A were significantly increased (p < 0.05) on day 2 after the embryos were treated with IL-7 (Figure 5E).

Additionally, blastocysts treated with IL-7 showed significantly (p < 0.05) higher MCL1 and lower BAX levels than the the control embryos (Figure 6A). Further, among the IL-7 signaling-related genes, the expression levels of PI3KR1, AKT1, and ERK2 were significantly increased in blastocysts treated with IL-7 compared with those in the control group (Figure 6B). We also observed that the expression levels of genes related to embryonic development, OCT4 and NANOG, were significantly increased (p < 0.01 and p < 0.05, respectively), while those of CDX2, GATA6, and PCNA did not show any significant difference between the control and the IL-7 treatment group (Figure 6C).

IL-7 activates the JAK/STAT pathway, which when stimulated leads to STAT5 phosphorylation in thymocytes and granulosa cells (32, 33). Therefore, to determine whether IL-7 supplementation during IVC stimulated the JAK/STAT pathway at the porcine blastocyst stage, we examined the expression of pSTAT5 protein via immunofluorescence. The pSTAT5 protein level was significantly (p < 0.01) increased in blastocysts treated with IL-7 compared to that in the control (Figure 7).

Based on qRT-PCR, IL-7-treated blastocysts showed significantly increased levels for the epiblast marker, NANOG, but not the trophectoderm marker, CDX2. Further, we hypothesized that IL-7 supplementation affects ICM development during lineage segregation in porcine blastocysts. Thus, blastocysts were stained for SOX2, a faithful ICM marker (34), to determine the effect of IL-7 on the number of ICM cells (Figure 8). Supplementation with IL-7 during the IVC period did not affect the total cell number (Figure 8B). However, embryos treated with IL-7 developed into blastocysts with significantly increased (p < 0.001) numbers of SOX2+ cells compared to the control (4 cells in IL-7 vs. 2.4 cells in controls) (Figure 8C). The ICM ratio was also significantly enhanced (p < 0.001) by ~1.7-fold in porcine blastocysts treated with IL-7 (10.3% in IL-7 vs. 5.9% in control) (Figure 8D).