Abstract
Assisted reproductive technologies (ART) to generate developmentally competent oocytes necessitates light exposure due to the use of microscopes. Previous studies in several species have reported that the wavelength of light during the light exposure period is a critical factor in embryo development. However, the effects of different light wavelengths on embryo development in pigs remain unexplored. This study aimed to identify the optimal light conditions to enhance oocyte maturation, parthenogenetic activation of mature oocytes, and pre-implantation development of parthenogenetic embryos in pigs. Conducted irradiation experiments during in vitro maturation (IVM), parthenogenesis (PG), and in vitro culture (IVC) using visible (390–750 nm), blue (445–500 nm), green (500–575 nm), yellow (575–585 nm), and red (620–750 nm) light. Variation in light wavelengths during IVM, PG, or IVC did not significantly influence oocyte maturation of cumulus–oocyte complexes (COCs) derived from median antral follicles (MAFs), developmental competence of in vitro-culture COCs after PG, and the production of blastocysts. However, continuous irradiation with green light throughout the entire process (IVM, PG, and IVC) significantly enhanced in vitro blastocyst production, and the resulting blastocysts showed significantly elevated HSP70 expression and a numerical increase in PCNA expression. We recommend conducting all in vitro procedures (IVM, PG, and IVC) for producing blastocysts from MAFs in porcine models under green light. This study will contribute to yielding higher success rates of porcine ART and reduce artificial stress to oocytes and embryos caused by in vitro manipulation under strong light exposure.
Keywords: In vitro blastocyst production, In vitro culture, Light wavelength, Oocyte maturation, Pig
Processes such as folliculogenesis, fertilization, and preimplantation development in the reproductive systems of animals or humans occur in the absence of light within the body. Nevertheless, light is an essential element in microscopy applications during all stages of in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC). Therefore, determining the optimal light conditions for these stages in each animal or human is crucial to advancing various assisted reproductive technologies (ART) specific to different types of animals or humans.
Previous studies have shown that the exposure of oocytes or embryos to specific light wavelengths can lead to alterations in maturation or preimplantation development [1, 2]. For instance, in hamsters, red light exposure during invitro blastocyst production led to higher blastocyst yield compared to other wavelengths [1]. Similarly, in mice, yellow light exposure during invitro blastocyst production resulted in stronger blastocyst formation than other wavelengths [2]. These findings indicate that the ideal light wavelengths for maturation or preimplantation development under microscopy during in vitro embryo production stages can differ across species.
To date, no studies have explored the effects of light wavelength on porcine oocytes and embryos. Accordingly, in this study, we aimed to establish the optimal light conditions for the in vitro production of blastocysts from median antral follicles (MAFs) derived from the porcine ovarian cortex. For these, various light wavelengths were used in IVM to produce mature oocytes from MAFs, during parthenogenesis (PG) to generate parthenogenetic embryos from mature oocytes, during IVC to produce blastocysts from parthenogenetic embryos, and throughout the entire process involving IVM, PG, and IVC. Then, we investigated the nuclear and cytoplasmic maturation of immature oocytes and/or the preimplantation development of oocytes activated parthenogenetically from mature oocytes, along with conducting quantitative analyses of genes associated with environmental stress tolerance or embryonic developmental potential.
Materials and Methods
Experimental design
Light intensity was measured using an illuminometer (TEN01070; Tenmars, Taipei, Taiwan) positioned on the observation stage of a stereomicroscope (SMZ-1000; Nikon, Tokyo, Japan). As shown in Supplementary Fig. 1, all in vitro procedures for the maturation of immature cumulus–oocyte complexes (COCs) derived from MAFs, parthenogenetic embryo production, and the culture of parthenogenetic embryos were conducted under distinct light wavelength conditions, including 200 lux visible, blue, green, yellow, or red light wavelengths. Each light irradiation condition was implemented by covering the observation plane of a stereomicroscope (SMZ-1000) with the corresponding cellophane filter: no cellophane filter for 390–750 nm, blue cellophane filter for 445–500 nm, green cellophane filter for 500–575 nm, yellow cellophane filter for 575–585 nm, and red cellophane filter for 620–750 nm. These experiments were performed in a controlled, dark clean room environment maintained at a temperature of 25°C, with strict restrictions on additional light sources.
In Experiment 1, we exposed immature COCs to different light wavelengths at a constant light intensity during the IVM process. Subsequently, we explored their influence on the nuclear and cytoplasmic maturation of immature COCs. After the aspiration of MAFs from ovarian cortices, we collected immature COCs during a 20-min period under each light irradiation condition. The collected immature COCs were then cultured for 22 h in gonadotropin-supplemented IVM medium. Subsequently, immature COCs from each group were transferred into gonadotropin-free IVM medium within 3 min using the same light irradiation conditions employed during the collection of immature COCs, followed by an additional 22 h of culture. Subsequently, nuclear maturation (extrusion of the first polar body into the perivitelline space [PVS]), cytoplasmic maturation (PVS size and intra-oocyte glutathione [GSH] and reactive oxygen species [ROS] levels), and cumulus cell mucification (cumulus cell expansion score) were assessed in metaphase II (MII)-stage oocytes derived from each group. Additionally, the developmental competence of MII-stage oocytes in each group was evaluated by conducting PG and monitoring preimplantation development in the 2-cell and blastocyst stages, as well as the total cell number of blastocysts. This in vitro blastocyst production (PG and IVC) was performed under visible light irradiation at 200 lux.
In Experiment 2, we examined the influence of different light wavelengths at constant intensity during PG on the parthenogenetic activation (PA) of MII-stage oocytes and the preimplantation development of parthenogenetic embryos. After IVM under visible light irradiation at 200 lux, MII-stage oocytes were collected under 200 lux light irradiation at different wavelength within 15 min after completing IVM of immature COCs. Electrical PA was then conducted under same light irradiation conditions for 10 min. After completing chemical PA under irradiation-free conditions for 4 h, both parthenogenetic embryo transfer into culture medium and cleavage monitoring were performed under visible light irradiation at 200 lux.
Additionally, in Experiment 3, we explored the influence of different light wavelengths at constant intensity during IVC on the preimplantation development of parthenogenetic embryos. For these, IVM and PG were conducted under visible light irradiation at 200 lux. Subsequently, parthenogenetic embryo transfer into the culture medium was performed under 200 lux light irradiation at different wavelengths, with subsequent cleavage monitoring under the same light irradiation conditions. After subsequent chemical PA, the parthenogenetic embryos were transferred into PZM-3 medium for IVC within 5 min, and preimplantation development of the parthenogenetic embryos into the 2-cell stage was monitored for 2 min. Then, under visible light irradiation at 200 lux, preimplantation development into the blastocyst stage was monitored, and the total cell number of blastocysts were counted.
In Experiment 4, we investigated the influence of different light wavelengths at constant intensity during the entire in vitro process, from immature COC maturation to parthenogenetic embryo-derived blastocyst production, on the preimplantation development of parthenogenetic embryos. Light irradiation at all steps of IVM, PG, and IVC was conducted for equal time under 200 lux light irradiation at different wavelengths, and the monitoring and the total cell count of blastocysts in each group were performed under visible light irradiation at 200 lux.
Finally, in Experiment 5, we tried to identify how light of a specific wavelength enhances embryonic development during invitro manipulation. Among the conducted experiments (Experiment 1 to 4), heat shock protein 70 (HSP 70) and proliferating cell nuclear antigen (PCNA) genes associated with embryonic development were analyzed quantitatively in blastocysts derived from experiments demonstrating significant differences in blastocyst production using a real-time PCR.
Retrieval of immature COCs from MAFs
All animal experimental procedures were conducted and approved by the Institutional Animal Care and Use Committee of Kangwon National University (approval no. KW-230920-4). The ovaries were obtained from pre-pubertal domestic pigs slaughtered at a local abattoir, immersed in a 0.9% (w/v) sodium chloride solution (Daejung Chemicals & Metals Co., Ltd., Siheung, Korea) at 39°C, and promptly transported to the laboratory within 2 h. Only antral follicles within the ovarian cortex, measured as 3–8 mm in diameter using a digital caliper (Traceable Carbon Fiber Calipers; Control Company, Webster, TX, USA), were considered MAFs, and aspiration of COCs from these MAFs was performed using an 18-gauge syringe needle (Koreavaccine, Seoul, Korea). Immature COCs with homogeneous cytoplasm distribution and at least three uniform layers of unexpanded cumulus cells were collected and washed several times in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered Tyrode’s medium (TLH) supplemented with 0.05% (w/v) polyvinyl alcohol (PVA; Sigma-Aldrich, St. Louis, MO, USA).
IVM
Porcine follicular fluid (pFF) was collected from MAFs with a diameter of 3–8 mm using a syringe. The pFF was then centrifuges at 1,900 × g for 20 min, and the supernatant was filtered before being stored at –20°C for following experiments. The basic medium for IVM (herein as referred to the basic IVM medium) was prepared by supplementing 10% (v/v) pFF, 0.83 mM L-cysteine (Sigma-Aldrich), 0.4 mM sodium pyruvate (Sigma-Aldrich), 75 µg/ml kanamycin (Sigma-Aldrich), 15 ng/ml mouse epidermal growth factor (Sigma-Aldrich), and 1 µg/ml insulin (Sigma-Aldrich) in Medium 199 (Welgene, Daegu, Korea). Without an oil overlay, the collected immature COCs (25 per well) were cultured for 22 h in four-well culture dishes (Nunc, Roskilde, Denmark) containing 500 μl of gonadotropin-supplemented IVM medium, consisting of the basic IVM medium supplemented with 5 IU/ml human chorionic gonadotropin (hCG; Chorulon; Merck, Kenilworth, NJ, USA) and 10 IU/ml pregnant mare serum gonadotrophin (PMSG; Daesung Microbiological Labs, Uiwang, Korea), at 39°C under humidified air with 5% CO2. Subsequently, the cultured COCs were washed three times with the basic IVM medium without hCG and PMSG supplementation (herein as referred to the gonadotropin-free IVM medium) and incubated for 22 h in gonadotropin-free IVM medium at 39°C under humidified air with 5% CO2. Then, cumulus cells surrounding in vitro-matured COCs were denuded by gentle pipetting in gonadotropin-free IVM medium supplemented with 0.06% (w/v) hyaluronidase (Sigma-Aldrich).
Evaluation of cumulus cell expansion
After completing the IVM of immature COCs, cumulus cell expansion scores were determined as 0 (no response), 1 (minimum observable response, with the outermost cumulus cells becoming round and glistening), 2 (expansion of outer cumulus cell layers), 3 (expansion of all cumulus cell layers except the corona radiata), or 4 (expansion of all cumulus cell layers, including the corona radiata).
PVS size measurement
Photographs of cumulus cell-free MII-stage oocytes were taken using a digital camera (DS-Ri1; Nikon) equipped with an inverted microscope (TE300; Nikon) and analyzed using ImageJ v1.52 software (National Institutes of Health, Bethesda, MD, USA). Oocyte diameter and zona pellucida inner diameter were determined based on the mean of the maximum vertical or horizontal diameters (Supplementary Fig. 2). PVS size was calculated by subtracting the oocyte diameter from the zona pellucida inner diameter.
Measurement of GSH and ROS levels
Following previously described methods [3, 4] GSH and ROS levels were measured in cumulus cell-free MII-stage oocytes. To measure intra-oocyte GSH levels, MII-stage oocytes were stained with 10 µM Cell-Tracker Blue CMF2HC (4-chloromethyl-6,8-difluoro-7-hydroxycoumarin; Invitrogen, Carlsbad, CA, USA) diluted in TLH supplemented with 0.05% (w/v) PVA (referred to as TLH-PVA) for 30 min on a warm plate (MATS-USP; Tokai Hit Co., Ltd., Shizuoka, Japan) set to 39°C. The stained oocytes were then cultured for 30 min in PZM-3 medium [4] at 39°C under humidified 95% air and 5% CO2. To measure intra-oocyte ROS levels, MII-stage oocytes were stained with 10 µM H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate; Invitrogen) diluted in TLH-PVA for 30 min on a warm plate set to 39°C. The stained MII-stage oocytes were subsequently rinsed several times with Dulbecco’s phosphate-buffered saline (DPBS; Welgene) supplemented with 0.1% (w/v) PVA. Fluorescence of the stained MII-stage oocytes was detected using an epifluorescence microscope (TE300; Nikon). Unstained MII-stage oocytes were used as a negative control to normalize fluorescence signal intensity. Fluorescence images were acquired at a single focal plane for intensity measurement, with exposure settings (30 msec and gain 100) and image resolution (1280 × 1024 pixels) maintained consistently across all experiments to ensure comparability. The cytoplasmic region (inner area of the oolemma) of each oocyte was defined as the region of interest (ROI), and fluorescence intensity was quantified using ImageJ v1.52 software. The measured fluorescence intensity was normalized to that of unstained MII-stage oocytes.
PA
Cumulus cell-free MII-stage oocytes, exhibiting an extruded first polar body, were introduced into a fusion chamber containing 1.5 ml of activation medium consisting of a 280 mM D-mannitol solution (Sigma-Aldrich) supplemented with 0.05 mM magnesium chloride hexahydrate (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) and 0.1 mM calcium chloride dihydrate (FUJIFILM Wako Pure Chemical Corp.). Electrical activation was performed by administering two pulses of 120 V/mm direct current for 60 μsec using a cell fusion generator (LF101; NepaGene, Chiba, Japan). The electrically activated MII oocytes were then exposed to PZM-3 medium supplemented with 5 μg/ml cytochalasin B (Sigma‐Aldrich) for 4 h. PA embryos were rinsed several times with PZM-3 medium for subsequent processing.
IVC of parthenogenetic embryos and measurement of blastocyst total cell numbers
Parthenogenetic embryos were cultured for 7 days in PZM-3 medium at 39°C under a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. The termination of PA was considered as 0 h, and subsequent cleavage or blastocyst formation was evaluated under a stereomicroscope (SZX16; Olympus, Tokyo, Japan) at 48 or 168 h following PA. Following preimplantation development into the blastocysts, the blastocysts were stained with 5 µg/ml bisbenzimide H33342 (Sigma-Aldrich) for 15 min, and the stained blastocysts were placed on a glass slide. After covering a coverslip, the blue-stained cell nuclei were counted using an epifluorescence microscope (TE300; Nikon).
Quantitative real-time PCR
Total mRNA was extracted from blastocysts (10 blastocysts per independent experiment) using the RNeasy® Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, and then cDNA was synthesized using the ReverTra AceTM qPCR RT Master Mix with gDNA remover kit (Toyobo, Osaka, Japan). Real-time quantitative of the specific gene expression was performed using Prime Q-Mastermix (GeNet Bio, Daejeon, Korea) with qTOWER3 Real-time PCR Thermal Cycler (Analytik Jena, Jena, Germany), and identification of PCR specificity was verified by analyzing melting curve data. The relative mRNA levels were calculated as 2-ΔΔCt, where Ct = threshold cycle for target amplification, ΔCt = Cttarget gene (specific genes for each sample) - Ctinternal reference (β-actin for each sample), and ΔΔCt = ΔCtsample (treatment sample in each experiment) - ΔCtcalibrator (control sample in each experiment). Primer3Plus software (Whitehead Institute/MIT Center for Genome Research) with porcine cDNA sequence data obtained from GenBank was used for designing primer sequences. Supplementary Table 1 shows general information and sequences of primers.
Statistical analysis
All statistical analyses were performed using SAS v9.4 software (SAS Institute, Cary, NC, USA). The effect of each treatment was assessed using a generalized linear model (PROC-GLM) within the SAS program. Additionally, Duncan’s method was employed to identify significant main effects following analysis of variance using SAS. Significant differences were evaluated at a level of P < 0.05.
Results
Experiment 1: Effects of different light wavelengths during IVM on nuclear and cytoplasmic maturation of immature COCs and preimplantation development post-parthenogenesis
The variation in light wavelengths during IVM did not show significant differences in the nuclear maturation of immature COCs derived from MAFs (Table 1). Additionally, the cumulus cell expansion score, which indicates the degree of cumulus cell mucification in mature COCs, did not differ significantly among the various light wavelengths (visible, blue, green, yellow, and red) (Fig. 1A). Similarly, no significant alterations were observed in the PVS size (Fig. 1B), GSH level (Fig. 1C), and ROS level (Fig. 1D) among cytoplasmic maturation parameters due to different light wavelengths. Furthermore, no significant differences in preimplantation development into the 2-cell and blastocyst stages, nor in the total cell number of blastocysts, were detected among parthenogenetic embryos derived from mature oocytes produced under different light wavelengths (Table 1). These results suggested that the acquisition of high-quality oocytes from immature COCs derived from MAFs was not dependent on the specific light wavelength employed during IVM; thus, light wavelength conditions do not significantly enhance oocyte maturity during IVM.
Table 1. Effects of exposure of different light wavelength irradiation to immature COCs derived from MAFs during IVM on nuclear maturation of immature COCs, preimplantation development of mature oocytes activated parthenogenetically and total cell number of blastocysts developed from parthenogenetic embryos.
| Light irradiation wavelength a (nm) selected by color filter | No. of in-vitro-matured immature COCs | No. (%) b of MII stage oocytes | No. of MII stage oocytes activated parthenogenetically | No. (%) c of parthenogenetic embryos developed to |
No. (Mean ± SD) of total cells in blastocysts | |
|---|---|---|---|---|---|---|
| ≥ 2-cell [48] d |
Blastocyst [168] d |
|||||
| 390–750 (visible light, no filtering) | 399 | 335 (84.0) | 335 | 306 (91.3) | 139 (41.5) | 36.4 ± 14.6 |
| 445–500 (blue light) | 200 | 166 (83.0) | 166 | 153 (92.2) | 76 (45.8) | 39.5 ± 13.4 |
| 500–575 (green light) | 199 | 166 (83.4) | 166 | 155 (93.4) | 78 (47.0) | 36.2 ± 13.4 |
| 575–585 (yellow light) | 195 | 170 (87.2) | 170 | 159 (93.5) | 83 (48.8) | 37.1 ± 13.7 |
| 620–750 (red light) | 200 | 171 (85.5) | 171 | 158 (92.4) | 71 (41.5) | 35.7 ± 14.9 |
Model effects in the number of MII stage oocytes, the development to the ≥ 2-cell and blastocyst stages and the number of total cells in blastocysts, which is indicated as P value, were 0.7676 and 0.8994, 0.4569, and 0.0551, respectively. IVM of immature COCs derived from porcine MAFs was conducted by culturing immature COCs derived from MAFs of porcine ovary in gonadotropin-supplemented IVM medium for 22 hours and subsequent in gonadotropin-free IVM medium for 22 hours. All procedures in IVM (collection of immature COCs and transfer of COCs cultured in gonadotropin-supplemented IVM medium to gonadotropin-free IVM medium) were conducted for an equal time under the light wavelength of each ray, and all subsequent experimental procedures were performed for an equal time under visible light with the light intensity of 200 lux. a Light irradiation wavelength was selected by color filter and the light strength of 200 lux was used during immature COCs collection and COC transfer into the gonadotropin-free IVM medium. b Percentage of the number of in vitro-matured immature COCs. c Percentage of the number of MII stage oocytes activated parthenogenetically. d Each number in parenthesis indicates hours post-parthenogenetic activation. Eight independent experiments. SD, standard deviation.
Fig. 1.
Comparison of cumulus cell expansion score, perivitelline space size, and GSH and ROS levels among MII-stage oocytes invitro-matured under different light wavelengths (visible, blue, green, yellow, or red). The irradiation experiments during IVM procedure of immature COCs derived from MAFs involved culturing immature COCs from porcine ovarian MAFs in gonadotropin-supplemented IVM medium for 22 h, followed by gonadotropin-free IVM medium for another 22 h. During the IVM procedure, the collection of immature COCs and transfer of COCs cultured in gonadotropin-supplemented IVM medium to gonadotropin-free IVM medium were conducted for an equal time under each light wavelength at a light intensity of 200 lux. After nuclear maturation, the cumulus cell expansion score, perivitelline space size, and GSH and ROS levels in cumulus cell-free MII-stage oocytes were analyzed. Cumulus cell-free MII-stage oocytes were stained with Cell-Tracker Blue (to analyze GSH levels) or H2DCFDA (to analyze ROS level) (Supplementary Fig. 3), and the fluorescence intensity of the stained MII-stage oocytes was determined through fluorescence microscopy. Relative levels of GSH and ROS were calculated by dividing the fluorescence intensity of MII-stage oocytes matured under different light wavelengths (treatment groups) by those matured under visible light (control group). Differences in light wavelengths did not significantly affect cumulus cell expansion score (A), perivitelline space size (B), or GSH (C) or ROS (D) levels. Data are means ± standard deviation (SD) of three independent experiments. n = the number of MII-stage oocytes tested.
Experiment 2: Effects of different light wavelengths during PG on parthenogenetic embryo production and preimplantation development post-parthenogenesis
PG (MII-stage oocyte selection and PA) under each light wavelength condition did not show any significant differences in the preimplantation development of PA oocytes into the 2-cell stage, indicating no effect on parthenogenetic embryo production (Table 2). Additionally, parthenogenetic embryos generated through PG under each light wavelength did not exhibit significant differences in preimplantation development into the blastocyst stage and the total cell number of blastocysts. These findings suggested that the acquisition of parthenogenetic embryos with good developmental competence was not dependent on specific light wavelengths during PG; thus, light wavelength conditions do not significantly enhance the developmental competence of parthenogenetic embryos.
Table 2. Effects of exposure of different light wavelength irradiation to MII stage oocytes during PG on preimplantation development of mature oocytes activated parthenogenetically and total cell number of blastocysts developed from parthenogenetic embryos.
| Light irradiation wavelength a (nm) selected by color filter | No. of MII stage oocytes activated parthenogenetically | No. (%) b of parthenogenetic embryos developed to |
No. (Mean ± SD) of total cells in blastocysts | |
|---|---|---|---|---|
| ≥ 2-cell [48] c |
Blastocyst [168] c |
|||
| 390–750 (visible light, no filtering) | 326 | 281 (86.2) | 173 (53.1) | 32.1 ± 11.8 |
| 445–500 (blue light) | 150 | 126 (84.0) | 70 (46.7) | 30.3 ± 11.5 |
| 500–575 (green light) | 183 | 165 (90.2) | 94 (51.4) | 30.7 ± 9.6 |
| 575–585 (yellow light) | 137 | 116 (84.7) | 68 (49.6) | 33.8 ± 10.8 |
| 620–750 (red light) | 131 | 111 (84.7) | 72 (55.0) | 33.8 ± 10.6 |
The model effects in the development to the ≥ 2-cell and blastocyst stages, and the number of total cells in blastocysts, which is indicated as P value, were 0.4975, 0.6104, and 0.1595 respectively. All IVM procedure of immature COCs derived from porcine MAFs was conducted for an equal time under visible light with the light intensity of 200 lux. Subsequently, PG (MII stage oocyte selection and PA) were conducted for an equal time under the light wavelength of each ray, and all procedures in IVC (parthenogenetic embryo transfer into the PZM-3 medium and embryo monitoring 48 hours post-culture of parthenogenetic embryos) were conducted for an equal time under visible light with the light intensity of 200 lux. a Light irradiation wavelength was selected by color filter and the light strength of 200 lux was used during MII stage oocyte selection and PA. b Percentage of the number of MII stage oocytes activated parthenogenetically. c Each number in parenthesis indicates hours post-parthenogenetic activation. Seven independent experiments. SD, standard deviation.
Experiment 3: Effects of different light wavelengths during IVC on preimplantation development of parthenogenetic embryos
No significant differences in preimplantation development into the 2-cell and blastocyst stages, nor in the total cell number of blastocysts, were induced by irradiating different light wavelengths during IVC of parthenogenetic embryos (Table 3). These results suggested that the acquisition of high-quality blastocysts was not dependent on specific light wavelengths during the IVC process; thus, light wavelength conditions do not significantly enhance the preimplantation development of parthenogenetic embryos.
Table 3. Effects of exposure of different light wavelength irradiation to parthenogenetic embryos during IVC on preimplantation development of parthenogenetic embryos and total cell number of blastocysts developed from parthenogenetic embryos.
| Light irradiation wavelength a (nm) selected by color filter | No. of MII stage oocytes activated parthenogenetically | No. (%) b of parthenogenetic embryos developed to |
No. (Mean ± SD) of total cells in blastocysts | |
|---|---|---|---|---|
| ≥ 2-cell [48] c |
Blastocyst [168] c |
|||
| 390–750 (visible light, no filtering) | 208 | 188 (90.4) | 87 (41.8) | 35.2 ± 14.6 |
| 445–500 (blue light) | 107 | 99 (92.5) | 46 (43.0) | 36.5 ± 16.1 |
| 500–575 (green light) | 101 | 90 (89.1) | 47 (46.5) | 40.3 ± 16.4 |
| 575–585 (yellow light) | 111 | 100 (90.1) | 52 (46.8) | 38.7 ± 15.2 |
| 620–750 (red light) | 106 | 96 (90.6) | 45 (42.5) | 36.2 ± 13.5 |
The model effects in the development to the ≥ 2-cell and blastocyst stages, and the number of total cells in blastocysts, which is indicated as P value, were 0.9438, 0.8855, and 0.3664, respectively. All IVM procedure of immature COCs derived from porcine MAFs and PG of mature oocytes (MII stage oocyte selection and PA) was conducted for an equal time under visible light with the light intensity of 200 lux. Subsequently, all procedures in IVC (parthenogenetic embryo transfer into the PZM-3 medium and embryo monitoring 48 hours post-culture of parthenogenetic embryos) were conducted for an equal time under the light wavelength of each ray. a Light irradiation wavelength was selected by color filter and the light intensity of 200 lux was used during IVC procedure. b Percentage of the number of MII stage oocytes activated parthenogenetically. c Each number in parenthesis indicates hours post-parthenogenetic activation. Five independent experiments. SD, standard deviation.
Experiment 4: Effects of different light wavelengths during the entire process of in vitro production of blastocysts from MAFs on preimplantation development post-parthenogenesis
In contrast to our findings regarding light wavelength irradiation at specific stages, sustained green light irradiation during the entire process (IVM, PG, and IVC) of in vitro production of blastocysts from MAFs significantly improved the preimplantation development of parthenogenetic embryos into the blastocyst stage compared to other light wavelengths (Table 4). However, no significant differences in preimplantation development into the 2-cell stage or total cell number of blastocysts were observed among the various light wavelengths (visible, blue, yellow, and red) (Table 4). These results demonstrated that a large number of blastocysts from MAFs can be acquired using green light during the entire process encompassing IVM, PG, and IVC, whereas other light wavelengths (visible, blue, yellow, or red) do not yield similar results.
Table 4. Effects of exposure of different light wavelength irradiation during in vitro MAF-derived blastocyst production (IVM, PG, and IVC) on preimplantation development of mature oocytes activated parthenogenetically and total cell number of blastocysts developed from parthenogenetic embryos.
| Light irradiation wavelength a (nm) selected by color filter | No. of MII stage oocytes activated parthenogenetically | No. (%) b of parthenogenetic embryos developed to |
No. (Mean ± SD) of total cells in blastocysts | |
|---|---|---|---|---|
| ≥ 2-cell [48] c |
Blastocyst [168] c |
|||
| 390–750 (visible light, no filtering) | 460 | 394 (85.7) | 249 (54.1) d | 43.2 ± 15.7 |
| 445–500 (blue light) | 109 | 96 (88.1) | 54 (49.5) d | 41.9 ± 16.0 |
| 500–575 (green light) | 112 | 101 (90.2) | 75 (67.0) e | 43.3 ± 18.8 |
| 575–585 (yellow light) | 107 | 97 (90.1) | 50 (46.7) d | 44.5 ± 13.6 |
| 620–750 (red light) | 105 | 95 (90.5) | 52 (50.0) d | 42.5 ± 15.8 |
The model effects in the development to the ≥ 2-cell and blastocyst stages, and the number of total cells in blastocysts, which is indicated as P value, were 0.4004, 0.0209, and 0.9382, respectively. All procedures in IVM of immature COCs derived from porcine MAFs (collection of immature COCs and transfer of COCs cultured in gonadotropin-supplemented IVM medium to gonadotropin-free IVM medium), PG of mature oocytes (MII stage oocyte selection and PA), and IVC of parthenogenetic embryos (parthenogenetic embryo transfer into the PZM-3 medium, and embryo monitoring 48 hours post-culture of parthenogenetic embryos) were conducted for an equal time under the light wavelength of each ray. a Light irradiation wavelength was selected by color filter and the light strength of 200 lux was used during IVM procedure and invitro blastocyst production. b Percentage of the number of MII stage oocytes activated parthenogenetically. c Each number in parenthesis indicates hours post-parthenogenetic activation. de Different superscripts within the same column are significantly different, P < 0.05. Five independent experiments. SD, standard deviation.
Experiment 5: Quantitative comparison of HSP70 and PCNA transcription in blastocysts produced under different light wavelengths during the entire in vitro production process from MAFs
Subsequently, we identified how continuous irradiation with green light throughout the processes of IVM, PG, and IVC enhances embryonic development by analyzing transcription of HSP70, which indicate enhanced tolerance to environmental stress [5,6,7,8,9] and PCNA, which serve an indicator of embryonic developmental potential [10,11,12]. The blastocysts produced under green light showed significantly stronger transcription of HSP70 compared to those produced under other light wavelengths (visible, blue, yellow, or red) (Fig. 2A), and the transcription of PCNA was numerically the strongest in blastocysts produced under green light compared to those produced under other light wavelengths (visible, blue, yellow, or red) (Fig. 2B). These findings suggest that cumulative irradiation with green light throughout the in vitro production stages (IVM, PG, and IVC) of blastocysts from MAFs in pigs may enhance blastocyst production by increasing tolerance to in vitro culture stress and improving embryonic developmental competence. Therefore, green light can contribute to advancing ART through the production of blastocysts from MAFs derived from the ovarian cortex in pigs.
Fig. 2.
Quantitative comparison of HSP70 and PCNA expression in blastocysts produced under different light wavelengths during the entire in vitro production process from MAFs. All procedures in IVM of immature COCs derived from porcine MAFs (collection of immature COCs and transfer of COCs cultured in gonadotropin-supplemented IVM medium to gonadotropin-free IVM medium), PG of mature oocytes (MII stage oocyte selection and PA), and IVC of parthenogenetic embryos (parthenogenetic embryo transfer into the PZM-3 medium, and embryo monitoring 48 hours post-culture of parthenogenetic embryos) were conducted for an equal time under the light wavelength of each ray with the light strength of 200 lux. Subsequently, mRNA levels of HSP70 (A) or PCNA (B) in blastocysts were quantified by real-time PCR. As the results, the blastocysts produced under green light showed significantly stronger transcription of HSP70 compared to those produced under visible, blue, yellow, or red light (A), and the transcription of PCNA was numerically the strongest in blastocysts produced under green light compared to those produced under visible, blue, yellow, or red light (B). All data are represented as the means ± standard deviation (SD) of three independent experiments. * P < 0.05.
Discussion
In this study, we investigated an irradiation protocol using specific light wavelengths to maximize the production of blastocysts from parthenogenetic embryos derived from oocytes obtained after the maturation of MAFs through sequential progression of IVM, PG, and IVC in pigs. Throughout each stage, variation in light wavelength did not significantly enhance the maturation and preimplantation development of parthenogenetic embryos, or the numerical or qualitative superiority of blastocysts. Green light irradiation throughout the entire process promoted blastocyst acquisition, but did not affect the quality or cell number of blastocysts. These results suggested that green light may have a beneficial role in in vitro blastocyst production from MAFs in pigs, indicating the importance of light wavelength exposure during the in vitro production process.
Invivo, oocyte maturation, fertilization and embryo development take place without any exposure to light. However, in invitro conditions, oocytes and embryos are frequently exposed to artificial light sources, which may increase their vulnerability to light-induced (phototoxic) stress. Visible light can penetrate cells and induce the generation of ROS, resulting in oxidative stress that can damage DNA, disrupt mitochondrial function and denature intracellular proteins [13,14,15,16,17,18,19,20,21]. These cellular disturbances may ultimately compromise the developmental potential of embryos. Previous studies have demonstrated that the longer the embryo was irradiated to light, the more detrimental effects occurred on embryonic development [22, 23], meiosis resumption [24] and DNA stability [25, 26]. In addition, specific wavelengths of light exerted protective or beneficial effects depending on the species. For instance, red light has been reported to support hamster embryo development and alleviate the negative effects caused by other wavelengths [1], while yellow light has been shown to promote blastocyst development in mouse embryos [2]. In bovine embryos, although green light did not directly enhance blastocyst development rates, it was found to reduce the expression of HSPs, indicating a potential stress-relieving effect [27]. However, in this study, variations in the wavelength of light utilized during the steps of IVM, PG, or IVC did not prompt any alterations in oocyte maturation, embryogenesis, and developmental competency. The maximum cumulative light irradiation time (25 min) was shorter than the minimum cumulative time (60 min) reported in previous studies [28]. Therefore, the lack of significant effects may be due to the insufficient irradiation time. Additionally, any potential damage to preovulatory follicles or embryos caused by light irradiation might have been mitigated by completing microscopic manipulations within 25 min.
Conversely, cumulative light irradiation throughout IVM, PG, and IVC produced different results compared to short-term irradiation during individual stages. Previous researches have shown that the effects of light wavelengths on cellular or embryonic physiology vary by specie [1, 2, 22, 24, 29,30,31]. Furthermore, no beneficial or detrimental effects of green light on cell or embryo physiology have been documented. However, this study demonstrated that the highest production of blastocysts from porcine MAFs was achieved by conducting the entire process under green light, compared to visible, blue, yellow, and red light (Table 4). Simultaneously, as illustrated in Fig. 2, the increase of tolerance to in vitro culture stress and the improvement of embryonic developmental competence were induced by irradiating cumulatively green light throughout the in vitro production stages (IVM, PG, and IVC) of blastocysts from MAFs. Therefore, green light may be the ideal wavelength for the in vitro manipulation of porcine follicles or embryos.
In this study, the increased expression of HSP70 under green light conditions suggests a cellular protective response to oxidative stress and other environmental factors. HSP70 is a molecular chaperone that prevents protein misfolding and aggregation while maintaining cellular homeostasis, and its appropriate expression level can support embryo development and enhance cellular resilience [5,6,7,8,9]. However, excessive HSP70 expression may indicate exposure to suboptimal culture conditions, potentially leading to negative effects on embryo quality and developmental potential over time [32, 33]. Therefore, while our findings suggest that green light may offer protective benefits for embryo development, further optimization of IVP conditions is required to minimize potential stress-related risks. Additionally, the increased expression of PCNA in blastocysts exposed to green light indicates a potential enhancement in cell proliferation and embryonic developmental competence. PCNA is a key regulator of DNA replication and cell cycle progression [10,11,12], suggesting that green light exposure may promote cell division and blastocyst development. However, whether this increased PCNA expression translates into improved implantation and fetal development post-transfer remains to be confirmed through further invivo studies. Furthermore, this study is limited by the analysis of only two genes, HSP70 and PCNA, which does not fully reflect the range of molecular responses triggered by light exposure. To better understand the biological effects of green light, future studies should examine a broader selection of genes involved in DNA damage, apoptosis, stress response, and key signaling pathways. In addition, transcriptomic analyses such as RNA-sequencing (RNA-seq) would be valuable for providing a more comprehensive and unbiased insights into the underlying cellular and molecular mechanisms.
While green light exposure was associated with an increased blastocyst production, its long-term impact on embryo viability is still uncertain. Unlike previous studies in bovine embryos, where green light did not enhance developmental rates but reduced stress marker expression, our study observed both an increase in blastocyst formation and an elevation in stress-related gene expression. This suggests that while green light may offer short-term benefits during the IVP process, careful adjustments to light intensity, exposure duration, and culture conditions are essential for optimizing long-term embryo development.
Further research is needed to better understand the role of green light in porcine embryo production. First, this study used colored cellophane filters to expose porcine embryos to specific wavelengths of light. However, these filters transmit a relatively broad range of wavelengths centered on a specific color, rather than isolating a single, precise wavelength. In addition, the halogen light source emits a continuous spectrum across the visible range. As the results, the light that reached the embryos was likely a mixture of wavelengths, not a single, defined one. This may limit the accuracy of interpreting the specific effects on individual wavelengths and represents a methodological limitation of this study. Future research should use more advanced optical systems capable of isolating specific wavelengths to better clarify how each component of light affects embryonic development. Second, we used parthenogenetic embryos as a model to investigate the effects of light wavelength on oocyte maturation, embryo activation, and preimplantation development. While PG is a useful tool for assessing cytoplasmic maturation and early embryogenesis, it does not fully recapitulate the physiological processes of fertilization. Therefore, additional studies using IVF are necessary to determine whether the beneficial effects of green light observed in PG-derived embryos can also be applied to embryos produced via IVF. Future studies should also focus on evaluating implantation and pregnancy rates through embryo transfer experiments, as well as conducting in-depth investigations into the cellular and molecular effects of green light.
In conclusion, we recommend conducting the entire in vitro procedure (IVM, PG, and IVC) for blastocyst production from MAFs in porcine models under green light. Furthermore, our study provides new insights into the potential role of green light in enhancing porcine embryo development. Adopting these tailored light conditions is expected to significantly enhance the in vitro production of blastocysts from porcine MAFs and reduce experimental errors due to light wavelength variability. Ultimately, this optimization strategy may contribute to higher success rates of porcine ART and improve the accuracy of data from porcine embryo experiments.
Conflict of interests
The authors declare no conflicts of interest.
Supplementary
Acknowledgments
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No.2023R1A2C1005625).
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