All chemicals were purchased from Sigma-Aldrich (Milan, Italy), unless otherwise indicated.

Ovaries were collected at a local slaughterhouse (Surace Carne s.r.l., Noci, Bari) from prepubertal lambs (<3 months of age) subjected to routine veterinary inspection and transported to the laboratory at room temperature within 2–4 h from slaughter. The lamb’s prepubertal status was later confirmed at the ovarian level, in the laboratory, by evaluating the absence of growing follicles and corpora lutea. For the retrieval of COCs, the slicing technique was employed. The follicular contents were released into 60-mm sterile Petri dishes containing phosphate-buffered saline (PBS). COCs were selected using a Nikon SMZ 1500 optical stereomicroscope (Nikon Instruments, Firenze, Italy). Only those exhibiting at least three intact layers of CCs and homogeneous cytoplasm were selected for in vitro culture (24).

To evaluate the effects of PS-MPs on COC uptake and oocyte IVM, specific culture medium solutions were prepared by incorporating commercially available PS beads (Bangs Laboratories Inc., Fishers, IN, USA) at several concentrations. Fluorescent microspheres conjugated with Flash Red (200 nm diameter) were used for uptake assessment, while those not conjugated to the fluorophore (100 nm diameter) were employed to assess oocyte and CCs toxicity. IVM medium was prepared, as previously described (24), based on TCM-199 medium with Earle’s salts, buffered with 5.87 mmol/L HEPES and 33.09 mmol/L sodium bicarbonate and supplemented with 0.1 g/L L-glutamine, 2.27 mmol/L sodium pyruvate, calcium lactate pentahydrate (1.62 mmol/L Ca²⁺, 3.9 mmol/L lactate), 50 μg/mL gentamicin, 20% (v/v) fetal calf serum (FCS), 10 μg/mL of porcine follicle-stimulating hormone and luteinizing hormone (FSH/LH; Pluset^®, Calier, Barcelona, Spain) and 1 μg/mL 17-β-estradiol. On the day of experiments, PS-MPs stock solutions were diluted with IVM medium to cover a concentration range from 5 to 100 μg/mL, according to the experimental design. IVM medium without PS-MPs was used as a control. For each set of experiments, groups of 20–25 selected COCs were placed in four-well dishes (Nunc Intermed, Roskilde, Denmark) containing 400 μL of IVM, supplemented or not with PS-MPs, covered with an equal volume of pre-equilibrated lightweight mineral oil per well. IVM culture was performed for 22–24 h at 38.5 °C with 5% CO₂ in a humidified incubator (24).

Considering the functions of CCs in COCs, the effects of exposure to PS-MPs were verified over time. Specifically, the bioaccumulation of fluorescent PS-MPs in the CCs after 6 and 24 h of incubation was evaluated in the same experimental conditions previously described for COCs. Recovered CCs were centrifuged at 300 g for 5 min, the supernatant was aspirated, and the pellet was resuspended in 100 μL of PBS. Meanwhile, 10 slides were prepared, coverslipped inside 2 plates with 35-mm diameter wells. Subsequently, CCs were adhered onto the slides, and Dulbecco’s Modified Eagle Medium (DMEM) culture medium, implemented with 20% (v/v) FCS, with or without PS-MPs, was added.

After IVM, COCs were washed in PBS and fixed in 4% paraformaldehyde (PFA) solution in PBS. Subsequently, COCs were washed a second time in PBS, mounted on slides in a drop of glycerol–PBS solution with a ratio of 3:1 (v/v), covered with cover slips, and sealed with nail polish. Finally, the slides were stored at 4 °C in the dark. To evaluate the uptake of fluorescent PS-MPs, COCs and CCs monolayers were observed under a Nikon C1/TE2000-U confocal laser scanning microscope (Nikon Instruments, Firenze, Italy) at 600x magnification under oil immersion. To detect Flash Red fluorescence, a 633-nm helium/neon laser beam and an LP-650 filter were used. The fluorescence intensities were measured on the equatorial plane, with the aid of the EZ-CI GoldVersion 3.70 image analysis software platform for the Nikon CI confocal microscope (Nikon Instruments, Firenze, Italy). A circular area was traced to delineate the cytoplasmic area of the oocytes, whereas a polygonal area was traced by dividing CCs into sectors. Instead, for the fluorescence intensity of the CCs plated in a monolayer, a polygonal area was traced for each CC of the group present in the framed field. The fluorescence intensity within the programmed scanning area (512 × 512 pixels) was recorded and expressed as arbitrary densitometric units (ADU). The evaluations of all samples were carried out under fixed scanning conditions with regard to laser energy, signal detection (gain), and pinhole size.

After IVM, COCs underwent a denuding procedure through the enzymatic action of hyaluronidase and mechanical pipetting of a Gilson pipette. Collected CCs were centrifuged at 400 g for 5 min at room temperature, and the resulting pellet was immediately stored at −80 °C. Total RNA extraction was performed using the Rneasy^® Plus Micro Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions (25). Single-stranded complementary DNA (cDNA) was synthesized by using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) in a thermal cycler (Eppendorf, Hamburg, Germany) according to the following thermal program: (1) primers annealing at 25 °C for 10 min, (2) DNA polymerization at 37 °C for 120 min, and (3) enzyme deactivation at 85 °C for 5 min. Real-time PCR was used to perform gene expression analysis. For each sample, the reaction mix (20 μL) contained: 11 μL SYBR Green PCR Master Mix powerup (Applied Biosystems, 2x), 1 μL forward primer (Table 1), 1 μL reverse primer (Table 1), 1 μL cDNA, and RNase-free water up to the final volume. Each reaction was performed using a StepOne thermal cycler (Applied Biosystems, Foster City, CA, USA) with the following thermal cycling parameters: (1) 40 cycles of denaturation at 95 °C for 15 s each, (2) annealing at 60 °C for 1 min, (3) extension at 60 °C for 1 min. The specificity of each primer was confirmed through melting curve analysis. Each amplification was performed in duplicate, and the relative quantification of gene expression was conducted using the 2^-ΔΔCt method (Livak method) using β-actin as the housekeeping gene.

For the TUNEL assay, CCs were centrifuged at 300 g for 5 min, and the resulting pellet was used to assess DNA fragmentation (26) using the Click-iT^® Plus TUNEL Assay (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Briefly, CCs were fixed in 4% PFA for 15 min at room temperature and then permeabilized with 0.5% Triton X-100 for 20 min. After washing with deionized water, CCs were incubated in 50 μL drops of the TUNEL reagent in the dark for 1 h at 37 °C in a humidified atmosphere. The total nuclei were stained with 2.5 μg/mL Hoechst 33258 in a 3:1 (v/v) glycerol/PBS solution, mounted onto microscope slides, covered with a coverslip, sealed with nail polish, and stored at 4 °C in the dark. CCs were observed under a Nikon E-600 fluorescence microscope equipped with excitation filters of 365 nm for Hoechst 33258 and 495 nm for TUNEL staining. For each condition, approximately 15 different fields were examined to evaluate at least 100 cells in total. The apoptotic index was determined as the percentage of TUNEL-positive cells relative to the total number of cells stained with Hoechst 33258. Fluorescence quantification was performed by manually selecting the area of each TUNEL-positive cell (27).

After IVM culture and COC denuding, oocytes were washed in PBS with 3% BSA and incubated for 30 min in the same medium containing 280 nmoL/L of MitoTracker Orange CMTM Ros (Thermo Fisher Scientific, Waltham, MA, USA) at 38.5 °C under 5% CO₂ in air (28). Then, the cells were washed in PBS with 0.3% BSA and incubated in the same medium with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) for 15 min at 38.5 °C under 5% CO₂ in air for intracellular reactive oxygen species (ROS) detection (28, 29). After incubation, oocytes were washed in pre-warmed PBS without BSA and fixed overnight in 4% PFA at 4 °C.

To evaluate nuclear chromatin status, fixed oocytes in PFA were stained with 2.5 μg/mL Hoechst 33258 in a 3:1 (v/v) glycerol/PBS mixture, mounted on microscope slides, and stored at 4 °C in the dark. Slides were analyzed using an epifluorescence microscope (Nikon Eclipse 600; ×400 magnification) equipped with a B-2A filter (excitation 346 nm/emission 460 nm). Oocytes were classified according to their meiotic stage as germinal vesicles (GV), metaphase to telophase I (MI to TI), or metaphase II (MII) with the first polar body (PB) extruded. Oocytes displaying one pronucleus with extruded PB, irregular chromatin clumps, absence of chromatin, or a multipolar meiotic spindle were categorized as abnormal (28).

To detect and localize mitochondria and ROS, mature oocytes were observed using a Nikon C1/TE2000-U laser scanning confocal microscope (Nikon Instruments, Firenze, Italy) equipped with the Apo 60×/NA 1.40 Nikon Plan objective in oil immersion. A 543 nm helium/neon laser and a G-2A filter were used to detect the MitoTracker Orange CMTM Ros (551 nm excitation and 576 nm emission). A 488-nm argon ion laser and a B-2A filter were used to detect dichlorofluorescein (DCF) (495 nm excitation and 519 nm emission). Oocytes were observed in 25 optical sections with a step size of 0.45 μm, thus allowing 3D distribution analysis. The mitochondrial distribution pattern was evaluated as “perinuclear and subcortical (P/S)”, index of cytoplasmic maturity, “finely granular”, typical of immature oocytes and “abnormal”, with irregular mitochondria distribution (24). Concerning intracellular ROS localization, oocytes with intracellular ROS diffused throughout the cytoplasm, together with areas/sites of mitochondria/ROS overlapping, were considered viable.

In each individual oocyte, MitoTracker and DCF fluorescence intensities and the Manders’ overlap coefficient (30), indicating the extent of mitochondria/ROS colocalization, were measured at the equatorial plane using the EZ-C1 Gold Version 3.70 image analysis software platform for a Nikon C1 confocal microscope. A circular area was drawn around the ooplasm for the quantification analysis. The fluorescence intensity within the scanned area was recorded, and 16-bit images were obtained. Mitochondrial membrane potential and intracellular ROS concentrations were recorded as the fluorescence intensity emitted by each probe and expressed as arbitrary densitometric units (ADUs). Variables related to fluorescence intensity, such as laser energy, signal detection (gain), and pinhole size, were maintained at constant values for all measurements. In the mitochondria/ROS colocalization analysis, threshold levels were kept constant at 10% of the maximum pixel intensity for all measurements.

Metaphase II (MII) oocytes were fixed for 1 h at 37 °C in a microtubule-stabilizing buffer and subsequently stored at 4 °C in blocking solution (31). Samples were incubated overnight at 4 °C with a mixture of mouse monoclonal antibodies directed against α-tubulin (1:1000 dilution) and β-tubulin (1:100 dilution). Immunolabeling was then performed using donkey anti-mouse secondary antibodies conjugated with fluorescein isothiocyanate (FITC-Alexa Fluor 488; 1:100 dilution; Life Technologies, Invitrogen, Carlsbad, CA, USA), in combination with rhodamine–phalloidin (1:150 dilution; Invitrogen, Carlsbad, CA, USA), for 1 h at room temperature. Nuclear chromatin was counterstained with Hoechst 33258 (10 μg/mL). Confocal imaging of meiotic spindle microtubules, chromatin organization, and cortical F-actin architecture was carried out using a laser scanning confocal microscope (Leica TCS SP5, Leica, Wetzlar, Germany) equipped with Ar/He/Ne lasers and a 40 × oil-immersion objective. Hoechst 33258, FITC, and rhodamine–phalloidin were excited at 358 nm, 488 nm, and 551 nm, respectively, with fluorescence emission signals collected at 461 nm, 550 nm, and 595 nm. Oocytes were optically sectioned along the Z-axis, and cortical F-actin images were acquired at the equatorial plane. Oocytes were classified based on meiotic spindle morphology (normal symmetrical barrel-shaped spindles versus abnormal disorganized, clumped, or dispersed structures), chromosomal alignment (proper alignment versus misalignment or dispersion at the metaphase plate), and cortical F-actin distribution (continuous, uniformly distributed subplasmalemmal F-actin layer versus irregular or discontinuous organization) (31). The proportion of oocytes displaying normal or aberrant spindle, chromatin, and cortical F-actin configurations was quantified for each experimental group.

To evaluate the effects of PS-MPs exposure on developmental competence, oocytes were parthenogenetically activated (PA) after IVM with 5 μM ionomycin in TCM-199 for 5 min, followed by 4 h of culture in TCM-199 with 2 mM 6-dimethylaminopurine (6-DMAP) in a humidified atmosphere with 5% CO₂ at 38.5 °C (32). PA oocytes underwent in vitro embryo culture (IVEC) for 7 days in four-well dishes containing 500 μL/well of synthetic oviductal fluid medium (SOFM) with essential and non-essential amino acids at oviductal concentrations and 0.4% bovine serum albumin (BSA) under mineral oil in humidified atmosphere with 5% CO₂, 5% O₂, and 90% N₂ at 38.5 °C (3). Embryo development was followed by conventional morphology assessment under phase contrast microscopy. The cleavage was evaluated morphologically after 24 and 48 h of culture, while development to the blastocyst stage was assessed on day 7 (32). Blastocysts were classified according to the expansion and hatching status as early (initiating blastocoelic cavity formation), cavitated (full blastocoelic cavity formation), expanded (increased size with blastocelic cavity greater than half of the embryo volume and thinner zona pellucida), hatching (beginning of the exit from the zona pellucida), or hatched (full exit from the zona pellucida). Moreover, blastocyst diameter was evaluated by Oosight™ Research Instruments as the distance between the outside borders of the trophectoderm. Finally, blastocyst formation was confirmed on the last day of the culture by observing blastomere nuclear chromatin under epifluorescence microscopy after staining with Hoechst 33258, as indicated before.

Oocyte chromatin configurations, mitochondria distribution patterns, cytoskeletal configurations, cleavage rates, blastocyst rates, and apoptotic index were compared between conditions using the Chi-square test. PS-MPs uptake, bioenergetic oxidative status parameters, and gene expression were compared using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Data were analyzed with GraphPad Prism (Software version 8.0.2, San Diego, CA, USA). Differences with p < 0.05 were considered statistically significant.

The uptake of fluorescent 200 nm PS-MPs was assessed on a total of 30 ovine COCs at the end of IVM culture. Only the lowest and highest PS-MPs concentrations were used for this test (5 μg/mL and 100 μg/mL, respectively). For oocytes, no significant differences in intracellular fluorescence intensity were observed in comparison to the control condition, regardless of PS-MPs concentration. In contrast, for CCs, increased PS-MPs uptake was observed after exposure to the highest tested concentration (p < 0.05; Figure 2A). Bioaccumulation of fluorescent PS-MPs over time in CCs was analyzed in monolayer-cultured CCs. Increased PS-MPs bioaccumulation was observed already after 6 h of in vitro exposure, both at 5 μg/mL (p < 0.05) and 100 μg/mL (p < 0.0001) concentrations compared to the control condition. After 24 h of exposure, the intracellular uptake of PS-MP was confirmed at the highest concentration (p < 0.0001; Figure 2B).

The real-time PCR analysis of CCs isolated from 356 COCs exposed to PS-MPs during IVM revealed significant alterations in the expression of genes related to oxidative stress and apoptosis across the different treatment groups (Figure 3). The expression levels of GPX and SOD, key enzymes involved in antioxidant defense, were reduced at the concentration of 100 μg/mL (p < 0.05). CAT expression did not vary at any tested concentration. Regarding apoptosis-related gene expression, BAX, a proapoptotic gene, was significantly upregulated only at 5 μg/mL (p < 0.05), whereas BCL2 expression did not reach statistical significance.

The TUNEL assay revealed a dose-dependent increase in positive CCs (green-stained cells with fragmented DNA) isolated from a total of 68 COCs exposed to PS-MPs during IVM (46.6% at 5 μg/mL, p < 0.05; 64% at 50 μg/mL, p < 0.0001; and 81% at 100 μg/mL, p < 0.0001) compared to the control condition, which showed only 34% of TUNEL-positive cells (Figure 4).

The evaluation of the effects of non-functionalized 100 nm PS-MPs was performed on a total of 426 cultured COCs after IVM (5 replicates). As shown in Table 2, exposure to PS-MPs impaired meiosis resumption, particularly at higher tested concentrations. The percentage of oocytes reaching the MII + PB stage was significantly lower at 50 and 100 μg/mL PS-MPs (p < 0.01), whereas exposure to 5 μg/mL did not affect the maturation rate compared to untreated oocytes. These data were associated with a significant increase in the GV rates, particularly at 50 μg/mL and 100 μg/mL (p < 0.05). Notably, no significant differences were observed in the rates of abnormal chromatin configurations at all tested exposure concentrations.

Qualitative and quantitative parameters of bioenergetic/oxidative status were assessed in oocytes reaching nuclear maturation after IVM as a measure of cytoplasmic quality and developmental competence. Regarding the qualitative parameter, the rates of MII + PB oocytes displaying heterogeneous P/S distribution patterns (43% for control [9/21], 43% for 5 μg/mL [10/23], 24% for 50 μg/mL [4/17], and 31% for 100 μg/mL [5/16]) were not significantly affected by PS-MPs exposure.

The quantification of bioenergetic/oxidative status parameters revealed increased intracellular ROS levels at all PS-MPs exposure concentration compared to the control group (p < 0.05 for 5 μg/mL, p < 0.0001 for 50 μg/mL and 100 μg/mL; Figure 5A) whereas no differences were observed for mitochondrial membrane potential (Figure 5B). Finally, mitochondria/ROS colocalization showed a significant increase in oocytes exposed to 5 μg/mL (p < 0.05) and 50 μg/mL (p < 0.0001) (Figure 5C).

Oocyte exposure to PS-MPs resulted in perturbations in meiotic spindle assembly, chromosome alignment, and actin filament distribution. Indeed, the rate of oocytes exhibiting a classical barrel-shaped meiotic spindle structure was lower compared to untreated oocytes, regardless of PS-MPs concentration (p < 0.0001 for 5 μg/mL, p < 0.05 for 50 μg/mL, and p < 0.05 for 100 μg/mL) (Figure 6A). The rate of oocytes with correctly aligned chromosomes was also found to be reduced, particularly after exposure to 5 and 50 μg/mL (p < 0.0001 and p < 0.05, respectively) (Figure 6B). Finally, the percentage of oocytes with normally distributed actin filaments was reduced at all PS-MPs concentrations (p < 0.05 for 5 μg/mL, p < 0.001 for 50 μg/mL and p < 0.05 for 100 μg/mL), with oocytes displaying irregular staining in the area beneath the oolemma or spots staining within the ooplasm (Figure 6C).

The evaluation of the effects of PS-MPs exposure on developmental competence was performed on a total of 151 oocytes (three replicates). Only the lowest and intermediate PS-MPs concentrations were used for this test (5 μg/mL and 50 μg/mL, respectively). After 24 h of PA, the total cleavage rate was significantly reduced in oocytes exposed to 50 μg/mL (p < 0.01; Table 3), whereas no differences were found at 5 μg/mL compared to the control. After 48 h, no differences were observed in the cleavage rate, regardless of PS-MPs concentration (Table 3). After 7 days of IVEC, the blastocyst formation rate was not statistically different among conditions (Table 3). Regarding blastocyst quality, no differences were observed in either the mean diameter or the total number of nuclei, among experimental groups (Table 3). Nevertheless, hatched blastocysts were only obtained in the control group.