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. 2026 May 31;21:139. doi: 10.1186/s13062-026-00845-w

Pyrroloquinoline quinone protects heat-stressed porcine oocytes and improves early embryonic development via maintaining mitochondrial integrity and redox homeostasis

Mianqun Zhang 1,#, Qimei Xu 1,#, Bo Jia 1,#, Xue Zhang 2,#, Khienduc Lay 1, Mengchan Li 1, Yan Zou 3, Ziyi Wang 3, Xu Peng 3, Yang Gao 3, Changyin Zhou 2,4,✉, Yanfeng Xue 1,✉, Yunhai Zhang 1,✉
PMCID: PMC13435474  PMID: 42226245

Abstract

Heat stress (HS) is a major environmental factor that impairs female fertility by disrupting oocyte quality and developmental competence. Pyrroloquinoline quinone (PQQ), a redox cofactor with strong antioxidant and mitochondrial-protective properties, has recently attracted attention for its cytoprotective roles in mammalian cells. Here, we investigated whether PQQ supplementation could alleviate HS-induced porcine oocyte damage and improve subsequent embryonic development. Our results showed that PQQ treatment significantly restored the rates of germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) in heat-stressed oocytes. PQQ markedly reduced intracellular reactive oxygen species (ROS) accumulation, enhanced mitochondrial membrane potential (ΔΨm), and maintained ATP levels, indicating improved mitochondrial function. Furthermore, PQQ attenuated DNA damage (γH2A.X foci) and apoptosis (Caspase-3 activation), preserved spindle morphology and actin distribution, and normalized cortical granule and ovastacin localization, suggesting improved cytoplasmic maturation. Importantly, embryos derived from PQQ-treated oocytes exhibited higher cleavage and blastocyst formation rates compared with the HS group. Collectively, these findings demonstrate that PQQ effectively mitigates HS-induced oocyte dysfunction by maintaining mitochondrial integrity, redox balance, and cytoskeletal organization, thereby improving oocyte competence and early embryonic development under heat stress.

Graphical Abstract

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Keywords: Pyrroloquinoline quinone, Heat stress, Porcine oocyte, Mitochondrial function, Oxidative stress, Embryonic development

Introduction

With the ongoing global rise in ambient temperature and the increased frequency of extreme heat events, livestock are increasingly exposed to environmental conditions exceeding their upper critical thermal limits. Heat stress (HS) disrupts the balance between heat production and heat dissipation, leading to systemic physiological compensation characterized by peripheral redistribution of blood flow [40], reduced feed intake, elevated oxidative stress, and inflammatory activation [23], ultimately impairing intestinal barrier integrity and metabolic homeostasis [12, 45]. At the production level, HS is closely associated with seasonal declines in reproductive performance, including reduced farrowing rates, compromised maintenance of early pregnancy, and impaired postnatal growth [42]. Gestational HS further disrupts placental function and skeletal muscle development, resulting in reduced fetal myofiber formation and greater adiposity later in life [18, 51].

Oocytes are particularly sensitive to changes in temperature and redox homeostasis during meiotic maturation and early embryonic development, processes that rely heavily on proper mitochondrial activity and controlled reactive oxygen species (ROS) levels [36]. Elevated oxidative stress or mitochondrial dysfunction reduces maturation rates, impairs spindle/chromosome organization, and diminishes developmental competence [5, 53]. Indeed, impaired oocyte maturation is considered a central factor linking HS to reduced conception and early embryonic failure [52]. In vitro, exposure of cumulus–oocyte complexes (COCs) to supra-physiological temperatures induces cumulus cell apoptosis, abnormal nuclear maturation, and delayed embryonic development [33]. Mitochondrial dysfunction and ROS accumulation are recognized as key mechanisms underlying this phenotype, affecting ATP production, cytoskeletal stability, and spindle integrity [10]. Additionally, HS has been shown to disrupt transzonal projections (TZPs) between oocytes and cumulus cells [26], indicating cytoskeletal dysregulation as another contributor to the decline in oocyte quality.

Pyrroloquinoline quinone (PQQ) was originally identified as a redox cofactor in bacterial dehydrogenases and exhibits a molecular structure distinct from classical vitamins and micronutrients [21, 22]. Although PQQ is not synthesized in mammals, it can be obtained from dietary sources such as fermented foods, vegetables, and milk [2]. PQQ deficiency has been associated with impaired growth, immune dysfunction, and reproductive abnormalities in mice, while supplementation alleviates these conditions [3]. Notably, PQQ displays strong antioxidant capacity—surpassing that of vitamin C—and regulates mitochondrial oxidative metabolism and ATP synthesis [39], thereby influencing mitochondrial biogenesis and cellular energy homeostasis [7]. These properties underlie its protective effects in oxidative stress–related conditions including neurodegeneration, metabolic disorders, and cardiovascular injury, as well as estrogen-deficiency– and aging-associated bone loss. However, whether PQQ can protect oocytes—cells highly dependent on mitochondrial integrity—from HS-induced damage remains unknown.

Based on this background, we hypothesized that PQQ may alleviate HS-induced impairment of oocyte quality by maintaining mitochondrial function and redox balance. To test this, we employed an in vitro HS model to evaluate the effects of PQQ on oocyte meiotic maturation and early embryonic developmental competence, and assessed mitochondrial membrane potential, ROS accumulation, and ultrastructural alterations. This study aims to clarify the protective role and physiological significance of PQQ in oocytes under heat stress conditions.

Materials and methods

Antibodies and chemicals

All reagents were of analytical grade unless otherwise specified. Porcine ovaries were collected from a local slaughterhouse, and no live animals were used in this study. Therefore, institutional ethical approval for animal experimentation was not required. All chemicals, including culture media components, were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise indicated.

Oocyte collection and in vitro maturation (IVM)

Porcine ovaries were collected from healthy, sexually mature sows at a local slaughterhouse and transported to the laboratory in pre-warmed (38.5 °C) sterile saline containing penicillin (100 IU/mL) and streptomycin (100 µg/mL) within 2 h. After rinsing, follicular fluid was aspirated from 3 to 6 mm follicles using an 18–20 G needle attached to a syringe to recover COCs. COCs with homogeneous cytoplasm and at least three layers of compact cumulus cells were selected under a stereomicroscope. Selected COCs were washed and cultured in IVM medium composed of TCM-199 supplemented with 10 ng/mL epidermal growth factor (EGF), 10 IU/mL pregnant mare serum gonadotropin (PMSG), 10 IU/mL human chorionic gonadotropin (hCG), 10% (v/v) porcine follicular fluid, 0.57 mM cysteine, and 25 µM β-mercaptoethanol. Approximately 50 COCs were placed in 500 µL maturation medium under mineral oil and incubated at 38.5 °C in 5% CO₂ and saturated humidity for 44 h. During the first 22 h, the medium contained PMSG and hCG; for the remaining 22 h, COCs were cultured in hormone-free maturation medium. At the end of in vitro maturation (IVM), cumulus cells were removed by brief exposure to 0.1% hyaluronidase and mechanical pipetting. Denuded oocytes were examined under an inverted microscope, and extrusion of the first polar body was used to identify oocytes that had reached the metaphase II (MII) stage for subsequent experiments.

Experimental design

To evaluate the protective effects of PQQ on porcine oocytes exposed to heat stress, COCs were randomly assigned to five groups: (1) Control (38.5 °C, vehicle), (2) Heat stress (HS), (3) HS + PQQ 1 µg/mL, (4) HS + PQQ 5 µg/mL, and (5) HS + PQQ 10 µg/mL. PQQ stock solutions were prepared in dimethyl sulfoxide (DMSO) and diluted into the IVM medium to achieve the indicated working concentrations. The final DMSO concentration in all groups was kept below 0.1% (v/v), and an equal volume of vehicle was added to the Control and HS groups.

COCs in the Control group were cultured at 38.5 °C with 5% CO₂ for the entire 44 h IVM period. For the HS and HS + PQQ groups, COCs were exposed to 41.5 °C for the initial 24 h and then returned to 38.5 °C for an additional 20 h. PQQ was present throughout the 44 h maturation period in the respective treatment groups. For each replicate, approximately 40–50 COCs were cultured in 500 µL IVM medium under mineral oil. At the end of IVM, cumulus expansion, germinal vesicle breakdown (GVBD), and first polar body extrusion (PBE) were evaluated. MI- or MII-stage oocytes were subsequently collected for spindle/chromosome analysis, actin cytoskeleton visualization, assessment of cortical granule and ovastacin distribution, examination of mitochondrial distribution and membrane potential, measurement of intracellular ROS levels, and detection of γH2A.X and Annexin-V signals. A separate set of MII oocytes was subjected to parthenogenetic activation or in vitro fertilization (IVF) to assess cleavage and blastocyst formation rates. All experiments were independently repeated at least three times.

Immunofluorescence staining

Denuded oocytes were fixed in 4% (w/v) paraformaldehyde for 30 min and permeabilized in 0.5% Triton X-100 in PBS for 1 h at room temperature. Following permeabilization, oocytes were blocked in 1% BSA/PBS for 1 h and then incubated overnight at 4 °C with primary antibodies, including anti–α-tubulin, anti–γ-H2AX,. After three washes in PBST (0.1% Tween-20 in PBS), oocytes were incubated with the corresponding fluorophore-conjugated secondary antibodies for 1 h at room temperature. Nuclei were counterstained with Hoechst 33,342 or DAPI. Fluorescent images were acquired using a laser-scanning confocal microscope (Zeiss LSM800, Germany), and fluorescence intensity was quantified using ImageJ software (NIH, USA). Each experiment was repeated at least three times, and 15–20 oocytes were analyzed per group.

Detection of intracellular ROS

Intracellular ROS levels in MII oocytes were measured using H₂DCFDA (Beyotime, Shanghai, China). Oocytes were incubated in maturation medium containing 10 µM H₂DCFDA for 30 min at room temperature in the dark, washed three times in PBS, and placed in 4 µL PBS droplets in glass-bottom dishes. Fluorescent images were acquired using an inverted fluorescence microscope (Nikon Ti2eU, Japan), and fluorescence intensity was quantified with ImageJ software. For each group, 30–60 oocytes were analyzed, and all experiments were repeated at least three times.

Mitochondrial abundance

Mitochondrial content was assessed using MitoTracker Red CMXRos (Beyotime, Shanghai, China). MII oocytes were incubated in 200 nM MitoTracker solution for 30–60 min at 38.5 °C in the dark and rinsed in DPBS. Fluorescence images were captured using an inverted fluorescence microscope (Nikon Ti2eU, Japan), and signal intensity was quantified using ImageJ software. Each experiment was performed in triplicate, with 30–50 oocytes per group.

Sperm binding assay

Frozen–thawed boar semen was washed and resuspended in fertilization medium (mTBM supplemented with 2 mM caffeine and 3 mg/mL BSA) and incubated at 38.5 °C for 1 h for capacitation. MII oocytes were co-incubated with capacitated sperm at a final concentration of 0.25 × 10⁶ cells/mL for 1 h. After co-incubation, oocytes were washed gently to remove unbound sperm, fixed in 4% paraformaldehyde, and stained with Hoechst 33,342. The number of sperm attached to the zona pellucida (ZP) was counted under a fluorescence microscope. Each experiment was repeated at least three times.

In vitro fertilization (IVF)

MII oocytes were co-incubated with capacitated sperm at 0.25 × 10⁶ cells/mL for 6 h at 38.5 °C. Following fertilization, excess spermatozoa were removed by gentle washing, and presumptive zygotes were cultured in PZM-3 medium under standard incubation conditions. Fertilization success was assessed by the cleavage rate, defined as the percentage of embryos reaching the 2-cell stage after 24 h of culture. Each experiment was repeated independently at least three times.

Statistical analysis

All experiments were repeated at least three times. Data are presented as the mean ± SD. The number of oocytes or embryos analyzed per group is indicated in the figures. Statistical analyses were performed using GraphPad Prism (GraphPad Software, USA) or SPSS 18.0 (IBM, USA). Differences between two groups were evaluated using an unpaired two-tailed t-test, while comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test. A value of p < 0.05 was considered statistically significant. Fluorescence and signal intensities were quantified using ImageJ software.

Results

PQQ restores meiotic maturation of porcine oocytes under heat stress

To evaluate the impact of heat stress on meiotic progression, GV-stage porcine oocytes were cultured at 41.5 °C for 24 h and subsequently transferred to 38.5 °C for an additional 20 h of maturation. Cumulus expansion and the first PBE rates were assessed at the end of IVM. Cumulus expansion was categorized into three grades based on cumulus cell layer thickness: grade A (L ≥ 3D), grade B (2D ≤ L < 3D), and grade C (L < 2D), with grades A and B considered fully expanded. Compared with the control group, heat stress significantly suppressed cumulus expansion (Control: 91.8 ± 0.89%, n = 72, HS༚51.24 ± 0.51%, n = 70, p < 0.001, HS + PQQ༚69.9 ± 3.04%, n = 70, p < 0.01;Fig. 1A-B).

Fig. 1.

Fig. 1

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PQQ supplementation improves the meiotic maturation of heat-stressed porcine oocytes. (A) Representative images showing cumulus–oocyte complexes (COCs) and denuded oocytes (DOs) from control, heat-stressed (HS), and PQQ-treated (HS + PQQ, 5 µM) groups after in vitro maturation. Cumulus expansion and first polar body extrusion (PBE) were visualized under a confocal microscope. Scale bars: 25 μm. (B) Quantification of cumulus expansion. Cumulus expansion was measured as the area of the expanded cumulus layers relative to oocyte diameter. (C) Representative images showing first polar body extrusion (PBE) from control, heat-stressed (HS), and PQQ-treated (HS + PQQ, 5 µM) groups after in vitro maturation. Scale bars: 25 μm. (D) Quantification of PBE rates in control, HS, and PQQ-treated oocytes (1, 5 and 10 µmol/L) following 44 h of culture. Data are presented as mean percentages (mean ± SD) from at least three independent replicates. **p < 0.01, ***p < 0.001

To determine whether PQQ supplementation could attenuate the heat stress-induced reduction in first polar body extrusion (PBE) in porcine oocytes, different concentrations of PQQ (1, 5, and 10 µg/mL) were included during IVM under heat stress. Cumulus expansion was analyzed using 5 µM PQQ, which showed the most pronounced protective effect, restoring expansion toward control levels. For PBE analysis, all three concentrations were tested, and 5 µM PQQ exerted the strongest rescuing effect (Control: 72.69 ± 1.0%, n = 81;HS༚39.54 ± 0.49%, n = 81, p < 0.001༛1 µg/mL༚56.5 ± 0.64%, n = 82, p < 0.001༛5 µg/mL༚59.6 ± 0.57%, n = 80, p < 0.001༛10 µg/mL༚53.59 ± 0.69%, n = 85, p < 0.001༛Fig. 1C-D). Collectively, these results indicate that PQQ at 5 µM can partially reverse heat stress–induced defects in cumulus expansion and meiotic maturation of porcine oocytes, while PBE is improved in a dose-dependent manner.

PQQ preserves spindle organization and chromosome alignment in porcine oocytes under heat stress

To determine whether meiotic failure under heat stress is associated with defects in spindle assembly and chromosome alignment, MI-stage oocytes were subjected to immunofluorescence staining. Control oocytes exhibited a typical barrel-shaped spindle with chromosomes aligned along the metaphase plate (Fig. 2A). In contrast, heat-stressed oocytes showed a significantly higher incidence of abnormal spindle structures (including nonpolar, multipolar, elongated, or dispersed forms) and chromosome misalignment (spindle defects: Control: 15.27 ± 2.1%, n = 88;HS: 60.42 ± 4.07%, n = 88, p < 0.001; chromosome misalignment: Control: 23.3 ± 3.75%, n = 89༛HS༚57.89 ± 4.0%, n = 88, p < 0.001; Fig. 2B-C). Supplementation with PQQ during IVM markedly reduced these abnormalities in heat-stressed oocytes (spindle defects: 42.58 ± 2.23%, n = 90, p < 0.01༛chromosome misalignment: 40.41 ± 3.98%, n = 86, p < 0.01; Fig. 2B-C). These findings indicate that PQQ helps maintain spindle architecture and proper chromosome alignment, thereby supporting meiotic maturation under heat stress conditions.

Fig. 2.

Fig. 2

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PQQ preserves spindle organization and chromosome alignment in heat-stressed porcine oocytes. (A) Confocal images of spindle morphology and chromosome alignment in control, HS, and PQQ-treated oocytes at the metaphase I stage. Scale bar: 5 μm. (B) Proportion of oocytes with abnormal spindle structures. (C) Proportion of oocytes with misaligned chromosomes. Data in (B) and (C) represent mean ± SD from at least three independent experiments. **p < 0.01, ***p < 0.001

PQQ stabilizes microtubules in porcine oocytes under heat stress

As microtubule dynamics play a critical role in spindle assembly regulation, we next examined whether the spindle assembly defects observed in heat-stressed oocytes resulted from impaired microtubule stability. To address this, we evaluated the acetylation level of α-tubulin, a well-established marker of microtubule stabilization. Immunofluorescence analysis revealed that heat stress markedly reduced the acetylated α-tubulin signals compared with the control group (Control: 37.84 ± 7.34, n = 72; HS: 20.61 ± 6.01, n = 72, p < 0.001; Fig. 3A-B). Remarkably, supplementation with PQQ effectively restored the acetylation level of α-tubulin in heat-stressed oocytes (HS + PQQ: 27.35 ± 8.1, n = 70, p < 0.01; Fig. 3A-B), indicating that PQQ protects spindle assembly integrity by maintaining microtubule stability under heat stress conditions.

Fig. 3.

Fig. 3

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PQQ stabilizes microtubule structures in heat-stressed porcine oocytes. (A) Representative immunofluorescence images of acetylated α-tubulin in control, HS, and PQQ-treated oocytes at metaphase I. Scale bar: 5 μm. (B) Quantification of fluorescence intensity of acetylated α-tubulin. Data are expressed as mean ± SD from three independent experiments. **p < 0.01, ***p < 0.001. (C) Immunostaining images of microtubule fibers after nocodazole challenge. Oocytes were incubated at 4 °C for 5 min before α-tubulin staining. Scale bar: 5 μm

To further verify the compromised microtubule stability in heat-stressed oocytes, nocodazole, a microtubule depolymerizing agent, was applied to disrupt the spindle structure. As shown in Fig. 3C, following treatment with 20 µg/mL nocodazole for 5 min, microtubule fibers remained partially visible in control oocytes despite spindle disorganization. In contrast, the same treatment completely depolymerized microtubules in heat-stressed oocytes, confirming reduced microtubule resilience. Importantly, PQQ supplementation markedly preserved the microtubule network against nocodazole-induced depolymerization during heat stress (Fig. 3C).

PQQ preserves actin cytoskeletal integrity in porcine oocytes under heat stress

The actin cytoskeleton is crucial for maintaining oocyte morphology and spindle migration during meiosis. F-actin staining with phalloidin-TRITC revealed a uniform cortical distribution with strong fluorescence in control oocytes, whereas heat stress markedly reduced and disrupted actin signals, indicating cytoskeletal damage (Control: 16.11 ± 1.25, n = 74;HS: 8.09 ± 1.41, n = 72, p < 0.001༛Fig. 4A). PQQ supplementation effectively restored the cortical actin network and fluorescence intensity. Quantitative analysis confirmed that PQQ significantly mitigated the heat stress–induced decline in actin fluorescence (HS + PQQ: 13.33 ± 1.44, n = 72, p < 0.001༛Fig. 4B), demonstrating its protective role in maintaining cytoskeletal stability under heat stress.

Fig. 4.

Fig. 4

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PQQ maintains actin filament polymerization in heat-stressed porcine oocytes. (A) Representative confocal images of F-actin distribution in control, HS, and PQQ-treated oocytes at metaphase I. Scale bar: 20 μm. (B) Quantification of actin fluorescence intensity. Data represent mean ± SD from at least three independent experiments. ***p < 0.001

PQQ restores cortical granule and ovastacin distribution in porcine oocytes exposed to heat stress

Cortical granules (CGs) are specialized vesicular organelles in oocytes that play an essential role in preventing polyspermy. Their proper localization beneath the oolemma is a hallmark of cytoplasmic maturation. As shown in Fig. 5A, heat stress markedly weakened CG fluorescence signals in porcine oocytes, whereas supplementation with PQQ partially restored their intensity and peripheral distribution. Quantitative analysis confirmed a significant decrease in CG fluorescence in heat-stressed oocytes compared with controls (Control: 26.03 ± 3.56, n = 78; HS: 9.34 ± 2.97, n = 78, p < 0.001;Fig. 5B), which was substantially recovered by PQQ treatment during in vitro maturation (HS + PQQ: 19.43 ± 3.35, n = 80, p < 0.01༛Fig. 5B). Consistently, ovastacin—a major component of CGs—showed a comparable localization pattern across groups (Control: 7.6 ± 2.67, n = 80༛HS: 3.33 ± 1.53, n = 80, p < 0.001༛Fig. 5D). Measurement of ovastacin fluorescence further demonstrated that its signal intensity was markedly reduced following heat stress but significantly elevated after PQQ supplementation (HS + PQQ: 6.9 ± 2.43, n = 78, p < 0.001༛Fig. 5D). Collectively, these findings indicate that PQQ mitigates heat stress–induced cytoplasmic maturation defects in porcine oocytes by restoring the proper distribution of CGs and ovastacin.

Fig. 5.

Fig. 5

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PQQ restores the distribution of cortical granules (CGs) and ovastacin in heat-stressed porcine oocytes. (A) Representative images of CG localization in control, HS, and PQQ-treated oocytes at metaphase II. Zona pellucida was removed prior to LCA–FITC staining to avoid non-specific signals. Scale bar: 20 μm. (B) Quantification of CG fluorescence intensity. (C) Representative images showing ovastacin localization. Scale bar: 20 μm. (D) Quantification of ovastacin fluorescence intensity. Data in (B) and (D) are expressed as mean ± SD from at least three independent experiments. **p < 0.01, ***p < 0.001

PQQ enhances mitochondrial distribution in porcine oocytes under heat stress

We further examined the mitochondrial distribution in porcine oocytes using MitoTracker staining as previously described. In control oocytes, mitochondria were predominantly localized in the subcortical region, whereas heat stress disrupted this pattern and caused an irregular distribution (Fig. 6A). Notably, PQQ supplementation partially restored the normal subcortical localization of mitochondria (Fig. 6A). Quantitative analysis of fluorescence intensity confirmed that the mitochondrial signal was markedly reduced after heat stress but significantly increased following PQQ treatment (Control: 9.05 ± 1.66, n = 78;HS: 3.25 ± 0.96, n = 83, p < 0.001༛HS + PQQ: 5.65 ± 1.41, n = 75༛ p < 0.01; Fig. 6B). These findings indicate that PQQ effectively preserves mitochondrial organization under heat stress conditions.

Fig. 6.

Fig. 6

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PQQ enhances mitochondrial distribution and function in heat-stressed porcine oocytes. (A) Representative confocal images of mitochondrial distribution in control, HS, and PQQ-treated oocytes at metaphase II. Scale bar: 25 μm. (B) Quantification of mitochondrial fluorescence intensity. Data are expressed as mean ± SD from three independent experiments. **p < 0.01, ***p < 0.001

PQQ attenuates excessive ROS accumulation, DNA damage, and apoptosis in porcine oocytes under heat stress

Heat exposure is known to trigger oxidative stress, leading to cellular dysfunction and apoptosis in multiple cell types. We hypothesized that similar mechanisms contribute to oocyte impairment under heat stress. To test this, intracellular ROS levels were evaluated using DCFH-DA staining as previously described [24]. Control oocytes displayed faint cytoplasmic fluorescence, whereas heat-stressed oocytes showed markedly elevated ROS signals (Fig. 7A). Remarkably, PQQ supplementation effectively reduced ROS accumulation (Control: 6.26 ± 1.75, n = 64; HS: 19.39 ± 2.71, n = 67, p < 0.001; HS + PQQ: 9.02 ± 2.41, n = 65, p < 0.001; Fig. 7B).

Fig. 7.

Fig. 7

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PQQ reduces excessive ROS accumulation in heat-stressed porcine oocytes. (A) Representative images showing ROS levels detected by DCFH-DA staining in control, HS, and PQQ-treated oocytes at metaphase II. Scale bar: 30 μm. (B) Quantification of DCF fluorescence intensity. Data are presented as mean ± SD from at least three independent experiments. ***p < 0.001

Because oxidative stress is often accompanied by DNA damage and apoptosis, we next examined γH2A.X and Annexin-V staining in each group. Heat-stressed oocytes exhibited a pronounced increase in γH2A.X fluorescence compared with controls, while PQQ supplementation markedly reduced this signal (Control: 7.44 ± 3.02, n = 56; HS: 36.85 ± 3.88, n = 60, p < 0.001;HS + PQQ: 12.95 ± 4.30, n = 62, p < 0.001; Fig. 8A-B). Similarly, Annexin-V staining revealed distinct apoptotic labeling on the plasma membrane of heat-stressed oocytes, which was notably decreased after PQQ treatment (Control: 4.26 ± 1.39, n = 71; HS: 9.59 ± 3.06, n = 71, p < 0.001; HS + PQQ: 5.58 ± 1.73, n = 70, p < 0.001; Fig. 9A-B). Together, these findings indicate that PQQ mitigates heat stress–induced oxidative injury in porcine oocytes by reducing ROS accumulation, limiting DNA damage, and suppressing apoptosis.

Fig. 8.

Fig. 8

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PQQ alleviates DNA damage in heat-stressed porcine oocytes. (A) Representative images showing γH2A.X signals in control, HS, and PQQ-treated oocytes at metaphase II. Scale bar: 20 μm. (B) Quantification of γH2A.X fluorescence intensity. Data represent mean ± SD from at least three independent experiments. ***p < 0.001

Fig. 9.

Fig. 9

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PQQ suppresses early apoptosis in heat-stressed porcine oocytes. (A) Representative confocal images of Annexin-V staining in control, HS, and PQQ-treated oocytes at metaphase II. Scale bar: 30 μm. (B) Quantification of Annexin-V fluorescence intensity. Data are expressed as mean ± SD from at least three independent experiments. ***p < 0.001

PQQ promotes early embryonic development from heat-stressed porcine oocytes

Given that PQQ improved the quality of porcine oocytes exposed to heat stress, we next evaluated their developmental competence after parthenogenetic activation. As shown in Fig. 10, most embryos derived from control oocytes developed to the blastocyst stage, whereas only a small proportion reached this stage under heat stress (Control: 50.97 ± 2.78%, n = 91; HS: 21.93 ± 3.58%, n = 88, p < 0.001; Fig. 10A-B). Remarkably, supplementation with PQQ significantly increased the blastocyst formation rate in heat-stressed oocytes (HS + PQQ: 39.5 ± 3.3%, n = 91, p < 0.01; Fig. 10A-B), indicating that PQQ effectively restores the developmental potential of oocytes impaired by heat stress.

Fig. 10.

Fig. 10

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PQQ improves early embryonic development derived from heat-stressed porcine oocytes. (A) Representative bright-field images of embryos at the blastocyst stage from control, HS, and PQQ-treated groups. Scale bar: 200 μm. (B) Blastocyst formation rate in each group. Data are expressed as mean ± SD from at least three independent replicates. ***p < 0.001

Discussion

Elevated ambient temperatures exert multiple detrimental effects on living organisms, including heat stress, endotoxemia, and systemic inflammatory responses [28, 38]. Increasing evidence indicates that mitochondria are the primary subcellular targets of heat stress. Mitochondrial dysfunction leads to insufficient energy supply, excessive accumulation of ROS, and ultimately triggers apoptotic cell death [6, 9, 44]. However, effective strategies to mitigate heat stress-induced cellular damage remain lacking. Previous studies have demonstrated that supplementation with PQQ improves the quality of mouse and porcine oocytes by restoring mitochondrial function, thereby protecting them from aging- or environmental pollutant–induced damage, such as that caused by diisobutyl phthalate (DiBP) exposure [43, 50]. Based on these findings, we hypothesized that PQQ may alleviate heat stress–induced oocyte damage by preserving mitochondrial function.

To verify the potential role of PQQ in regulating porcine oocyte maturation under heat stress, we first examined the first PBE rate and cumulus expansion, two morphological indicators of meiotic progression. Consistent with previous reports, heat stress markedly impaired the meiotic maturation of porcine oocytes in vitro [10, 30]. PQQ supplementation significantly increased the PBE rate and promoted cumulus expansion, suggesting a protective effect on oocyte maturation under heat stress. During oocyte maturation, the dynamics of microtubules and actin filaments are essential for asymmetric division and accurate chromosome alignment [16, 17]. Proper spindle assembly and chromosome congression are critical for the successful completion of meiosis; disruption of these processes may result in oocyte aneuploidy, a leading cause of fertilization failure and embryonic loss in livestock exposed to heat stress [19, 31, 35]. Further analysis revealed that PQQ restored cytoskeletal integrity by stabilizing microtubules and maintaining actin dynamics, thereby ameliorating the cytoskeletal disruption induced by heat stress. These findings suggest that heat stress hinders nuclear maturation by impairing spindle assembly and actin polymerization, whereas PQQ supplementation effectively reverses these meiotic defects and enhances oocyte maturation quality.

Another key finding of this study is that PQQ promotes cytoplasmic maturation of porcine oocytes under heat stress. CGs form a uniform layer beneath the oolemma and release their contents, such as ovastacin, to cleave ZP2 at the N-terminus of the ZP, thereby preventing polyspermy after fertilization. Premature exocytosis of CGs before fertilization can cause precocious ZP2 cleavage and fertilization failure [37, 47]. Thus, proper CG distribution is regarded as a critical indicator of cytoplasmic maturation [4, 41]. Our results showed that heat stress markedly reduced both the number of subcortical CGs and ovastacin levels, indicating that heat stress compromises cytoplasmic maturation and fertilization competence. In contrast, PQQ supplementation significantly restored CG and ovastacin localization patterns, suggesting that PQQ supports cytoplasmic maturation of heat-stressed oocytes.

Mitochondria are the principal sites of ATP production and ROS generation in oocytes, and they also participate in maintaining intracellular Ca²⁺ homeostasis [14, 54]. Abnormal cytosolic Ca²⁺ levels are often associated with disrupted oxidative phosphorylation and redox imbalance, which can lead to mitochondrial dysfunction and opening of the mitochondrial permeability transition pore (mPTP) [11, 13, 32]. Therefore, mitochondrial abundance and activity are critical determinants of oocyte quality. During maturation, mitochondria not only provide energy but also maintain redox homeostasis. Mitochondrial impairment often results in excessive ROS production, which triggers apoptosis [29, 34]. Although ROS are normal by-products of oxidative phosphorylation, damaged mitochondria exacerbate ROS overproduction, damaging mitochondrial membranes and mitochondrial DNA, thereby establishing a vicious cycle of mitochondrial dysfunction and oxidative stress [8, 48].

As expected, heat-stressed oocytes exhibited excessive ROS accumulation and elevated cytosolic Ca²⁺ concentrations. PQQ supplementation effectively mitigated these changes. As an antioxidant compound, PQQ modulates the activity of redox enzymes, reduces intracellular calcium overload, and preserves mitochondrial oxidative phosphorylation to ensure sufficient ATP supply [20, 55]. Previous studies have shown that melatonin can improve the survival, maturation, and cytoskeletal organization of oocytes under heat stress by reducing ROS levels and regulating related genes [46]. Resveratrol has also been reported to alleviate heat-induced damage by increasing GSH levels and decreasing ROS, although in some models its effect alone may be weaker than that of melatonin or combined treatments [27]. Meanwhile, supplementation with nicotinamide mononucleotide (NMN) has been shown to restore multiple meiotic processes disrupted by heat stress and improve oocyte quality [25]. Due to differences in species, experimental conditions, and dosages across studies, direct head-to-head comparisons are limited, but the protective effects of PQQ observed in our study are at least comparable to those of these antioxidants.

Consistently, our results demonstrated that PQQ restored mitochondrial function and reduced ROS accumulation by maintaining calcium homeostasis in heat-stressed oocytes. Excessive oxidative stress and mitochondrial dysfunction are closely linked to DNA damage and apoptosis. Indeed, we observed increased γ-H2AX expression in metaphase I chromosomes, indicating DNA damage in heat-stressed oocytes. Moreover, excessive ROS and DNA damage were accompanied by early apoptotic features, whereas PQQ supplementation markedly attenuated both DNA damage and apoptosis under heat stress conditions [49].

In summary, our findings demonstrate that PQQ protects porcine oocytes from heat stress–induced autophagy and apoptosis by suppressing ROS overproduction and preserving mitochondrial function. More importantly, this study provides new evidence that PQQ significantly enhances oocyte quality under thermal stress, offering a promising strategy to improve oocyte developmental competence and reproductive performance in livestock exposed to high-temperature environments [1, 15].

Author contributions

M.Z., Y.Z., and Y.X. conceived and designed the study. Y.Z., Q.X., and B.J. performed the experiments and collected the data. D.Z., K.L., M.L., and Z.W. contributed to data analysis, software development, and visualization. M.Z., Q.X., and Y.G. wrote the original manuscript draft and supervised manuscript revision and editing. We sincerely thank Z. Cui, C. Zhou, and Y. Miao for their valuable support and insightful suggestions, and for kindly providing the antibodies used in this study.

Funding

This work was supported by the National Key Research and Development Program of China (2023YFD13000502), the National Natural Science Foundation of China (32272881), the Natural Science Foundation of Anhui Province (2308085QC82) and Suzhou City Health and Wellness Scientific Research Project (SZWJ2023a013). We gratefully acknowledge Professor Bo Xiong from Zhejiang University for his valuable support and insightful advice.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

The authors confirm that all necessary institutional approvals related to ethical use of experimental animals were obtained and are clearly stated in the manuscript. We also confirm compliance with institutional and national regulations regarding intellectual property and publication policies.

Consent for publication

All authors have reviewed and approved the final version of the manuscript and consent to its submission for publication.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Mianqun Zhang, Qimei Xu, Bo Jia and Xue Zhang contributed equally to this work.

Contributor Information

Changyin Zhou, Email: zhoucy@gd2h.org.cn.

Yanfeng Xue, Email: xueyanfeng1990@163.com.

Yunhai Zhang, Email: dkzyh@ahau.edu.cn.

References

  • 1.Bian D-D, Xing Z-Y, Dai S-G, Lu X, Zhang D-Z, Tang B-P, Zhu B-J, Liu Q-N. Toxicological Insights Into Multiple Pesticide Exposures in Red Swamp Crayfish (Procambarus clarkii): Molecular Mechanisms, Risk Assessments, and Future Perspectives for Aquaculture. Reviews Aquaculture. 2026;18(1):e70125. 10.1111/raq.70125. [Google Scholar]
  • 2.Canovai A, Williams PA. Pyrroloquinoline quinone: A potential neuroprotective compound for neurodegenerative diseases targeting metabolism. Neural Regeneration Res. 2025;20(1):41. 10.4103/NRR.NRR-D-23-01921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Charrier D, Cerullo G, Carpenito R, Vindigni V, Bassetto F, Simoni L, Moro T, Paoli A, Charrier D, Cerullo G, Carpenito R, Vindigni V, Bassetto F, Simoni L, Moro T, Paoli A. Metabolic and biochemical effects of pyrroloquinoline quinone (PQQ) on inflammation and mitochondrial dysfunction: potential health benefits in obesity and future perspectives. Antioxidants (Basel). 2024;13(9). 10.3390/antiox13091027. [DOI] [PMC free article] [PubMed]
  • 4.Chen J, Miao Y, Gao Q, Cui Z, Xiong B. Exposure to perfluorooctane sulfonate in vitro perturbs the quality of porcine oocytes via induction of apoptosis. Environ Pollut. 2021;284:117508. 10.1016/j.envpol.2021.117508. [DOI] [PubMed] [Google Scholar]
  • 5.Chen T, Li S, Mu X, Yang T, Wang L, Tian M, Yu M, Luo L, Xie Y, Xuan W, Noctor G, Han Y. A Coupled GSH/GSNOR System Denitrosylates TRXh5 to Allow Activation of SA Signalling by Oxidative Stress. Plant Cell Environ. 2026;49(5):2691–705. 10.1111/pce.70423. [DOI] [PubMed] [Google Scholar]
  • 6.Chen X, Ji Y, Liu R, Zhu X, Wang K, Yang X, Liu B, Gao Z, Huang Y, Shen Y, Liu H, Sun H. Mitochondrial dysfunction: Roles in skeletal muscle atrophy. J Translational Med. 2023;21(1):503. 10.1186/s12967-023-04369-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Cheng Q, Chen J, Guo H, Lu J, Zhou J, Guo X, Shi Y, Zhang Y, Yu S, Zhang Q, Ding F. Pyrroloquinoline quinone promotes mitochondrial biogenesis in rotenone-induced Parkinson’s disease model via AMPK activation. Acta Pharmacol Sin. 2021;42(5):665–78. 10.1038/s41401-020-0487-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Chianese R, Pierantoni R. Mitochondrial reactive oxygen species (ROS) production alters sperm quality. Antioxidants (Basel). 2021;10(1). 10.3390/antiox10010092. [DOI] [PMC free article] [PubMed]
  • 9.Christen F, Desrosiers V, Dupont-Cyr BA, Vandenberg GW, François L, Tardif NR, Dufresne J-C, Lamarre F, S. G., Blier PU. Thermal tolerance and thermal sensitivity of heart mitochondria: Mitochondrial integrity and ROS production. Free Radic Biol Med. 2018;116:11–8. 10.1016/j.freeradbiomed.2017.12.037. [DOI] [PubMed] [Google Scholar]
  • 10.Clare CE, Pestinger V, Kwong WY, Tutt DAR, Xu J, Byrne HM, Barrett DA, Emes RD, Sinclair KD, Clare CE, Pestinger V, Kwong WY, Tutt DAR, Xu J, Byrne HM, Barrett DA, Emes RD, Sinclair KD. Interspecific variation in one-carbon metabolism within the ovarian follicle, oocyte, and preimplantation embryo: consequences for epigenetic programming of DNA methylation. Int J Mol Sci. 2021;22(4). 10.3390/ijms22041838. [DOI] [PMC free article] [PubMed]
  • 11.Cui T, Li Z, Gao J, Miao Z, Li F, Shao Y, Wang Z, Sun J, Song X, Qi K, Tu J. Outer membrane vesicles secreted by avian pathogenic Escherichia coli promote its survival within macrophages and systemic infection by inducing endoplasmic reticulum stress-mediated autophagy flux blockade. Vet Res. 2026;57(1):34. 10.1186/s13567-025-01679-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Gao Q, Wu Y, Huang J, Xu S, Chen R, Jiang H, Huang Y, He Q, Wang Y, Xiao J, Cao H. Chronic neonicotinoid exposure disrupts survival, development, digestive enzymes, and gut microbiome in honeybee queen larvae (Apis mellifera L). Pestic Biochem Physiol. 2026;218:106959. 10.1016/j.pestbp.2026.106959. [DOI] [PubMed] [Google Scholar]
  • 13.Garbincius JF, Elrod JW. Mitochondrial calcium exchange in physiology and disease. Physiol Rev. 2022;102(2):893–992. 10.1152/physrev.00041.2020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Gualtieri R, Kalthur G, Barbato V, Nardo MD, Adiga SK, Talevi R, Gualtieri R, Kalthur G, Barbato V, Nardo MD, Adiga SK, Talevi R. Mitochondrial dysfunction and oxidative stress caused by cryopreservation in reproductive cells. Antioxidants (Basel). 2021;10(3). 10.3390/antiox10030337. [DOI] [PMC free article] [PubMed]
  • 15.Guo Y, Ma C, Meng T, Tu T, Cui S, Ling Y, Zhang Y, Liu H, Song N. ELF5 modulates casein synthesis in goat mammary epithelial cells via JAK2/STAT5 signaling pathway. Anim Bioscience. 2026;39(2):250181. 10.5713/ab.25.0181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Harasimov K, Uraji J, Mönnich EU, Holubcová Z, Elder K, Blayney M, Schuh M. Actin-driven chromosome clustering facilitates fast and complete chromosome capture in mammalian oocytes. Nat Cell Biol. 2023;25(3):439–52. 10.1038/s41556-022-01082-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Hernandez B, Tetlak P, Domingo-Muelas A, Akizawa H, Skory RM, Ardestani G, Biro M, Liu X, Bissiere S, Sakkas D, Plachta N. Actin organizes chromosomes and microtubules to ensure mitotic fidelity in the preimplantation embryo. Science. 2025;388(6749):eads1234. 10.1126/science.ads1234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Huang L, Li M, Deng C, Qiu J, Wang K, Chang M, Zhou S, Gu Y, Shen Y, Wang W, Huang Z, Sun H, Huang L, Li M, Deng C, Qiu J, Wang K, Chang M, Zhou S, Sun H. Potential therapeutic strategies for skeletal muscle atrophy. Antioxidants (Basel). 2022;12(1). 10.3390/antiox12010044. [DOI] [PMC free article] [PubMed]
  • 19.Huang Z-H, Yang J-Y, Hou C-P, Liu S-H, Li B, Wang J, Huang S, Xu J-P. Diapause termination alters the m6A methylation profiles and regulates gene expression associated with early embryonic development in the silkworm (Bombyx mori). J Insect Physiol. 2026;169:104944. 10.1016/j.jinsphys.2026.104944. [DOI] [PubMed] [Google Scholar]
  • 20.Ji W, Tang X, Du W, Lu Y, Wang N, Wu Q, Wei W, Liu J, Yu H, Ma B, Li L, Huang W. Optical/electrochemical methods for detecting mitochondrial energy metabolism. Chem Soc Rev. 2022;51(1):71–127. 10.1039/D0CS01610A. [DOI] [PubMed] [Google Scholar]
  • 21.Jiang B, Zhang H, Xu Q, Jiang Z, He R, Fu Q, Sun Y. Pyrroloquinoline Quinone Is an Effective Senomorphic Agent to Target the Pro-Inflammatory Phenotype of Senescent Cells. Aging Cell. 2025;24(9):e70138. 10.1111/acel.70138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Kirschning A. On the evolution of coenzyme biosynthesis. Nat Prod Rep. 2022;39(11):2175–99. 10.1039/D2NP00037G. [DOI] [PubMed] [Google Scholar]
  • 23.Lan R, Luo H, Wu F, Wang Y, Zhao Z, Lan R, Luo H, Wu F, Wang Y, Zhao Z. Chitosan oligosaccharides alleviate heat-stress-induced lipid metabolism disorders by suppressing the oxidative stress and inflammatory response in the liver of broilers. Antioxidants (Basel). 2023;12(8). 10.3390/antiox12081497. [DOI] [PMC free article] [PubMed]
  • 24.Lang LI, Wang ZZ, Liu B, Chang-Qing SHEN, Jing-Yi TU, Shi-Cheng WANG, Rui-Ling LEI, Si-Qi PENG, Xiong XIAO, Yong-Ju ZHAO, Qiu XY. The effects and mechanisms of heat stress on mammalian oocyte and embryo development. J Therm Biol. 2024;124:103927. https://pubmed.ncbi.nlm.nih.gov/39153259. [DOI] [PubMed] [Google Scholar]
  • 25.Li L, Han Q, Chen Y, Zhang M, Wang L, An X, Zhang S, Zhai Y, Dai X, Tang B, Li Z, Xie G. β-nicotinamide mononucleotide rescues the quality of aged oocyte and improves subsequent embryo development in pigs. PLoS ONE. 2023;18(10):e0291640. [DOI] [PMC free article] [PubMed]
  • 26.Li X, Wu Q, Gao H, Cheng J, Chen H, Duan J, Sun W, Zhang Z, Yang L, Hua R, Li L, Li Q, Jiang Z. Isorhamnetin protects porcine oocytes from heat stress by maintaining cumulus cells-oocyte communications. Free Radic Biol Med. 2025;237:239–50. 10.1016/j.freeradbiomed.2025.05.418. [DOI] [PubMed] [Google Scholar]
  • 27.Li Y, Wang J, Zhang Z, Yi J, He C, Wang F, Tian X, Yang M, Song Y, He P, Liu G. Resveratrol compares with melatonin in improving in vitro porcine oocyte maturation under heat stress. J Anim Sci Biotechnol. 2016;7:33. 10.1186/s40104-016-0093-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Lim CL. Heat sepsis precedes heat toxicity in the pathophysiology of heat stroke—a new paradigm on an ancient disease. Antioxidants (Basel). 2018;7(11). 10.3390/antiox7110149. [DOI] [PMC free article] [PubMed]
  • 29.Liu M, Lu J, Hu J, Chen Y, Deng X, Wang J, Zhang S, Guo J, Li W, Guan S. Sodium sulfite triggered hepatic apoptosis, necroptosis, and pyroptosis by inducing mitochondrial damage in mice and AML-12 cells. J Hazard Mater. 2024;467:133719. 10.1016/j.jhazmat.2024.133719. [DOI] [PubMed] [Google Scholar]
  • 30.Liu Y, He Y, Chen M, Sun Q, Zhang B, Zhang G, Cao A, Li Q, Wang W, Ju S, Liu Y, He Y, Chen M, Sun Q, Zhang B, Zhang G, Cao A, Li Q, Wang W, Ju S. Alpha-tocopherol protects porcine oocytes from acetamiprid-induced meiotic defects by alleviating oxidative stress-mediated ferroptosis. Antioxidants (Basel). 2025;14(11). 10.3390/antiox14111304. [DOI] [PMC free article] [PubMed]
  • 31.Mihajlović AI, Byers C, Reinholdt L, FitzHarris G. Spindle assembly checkpoint insensitivity allows meiosis-II despite chromosomal defects in aged eggs. EMBO Rep. 2023;24(11):EMBR202357227. 10.15252/embr.202357227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Morciano G, Naumova N, Koprowski P, Valente S, Sardão VA, Potes Y, Rimessi A, Wieckowski MR, Oliveira PJ. The mitochondrial permeability transition pore: An evolving concept critical for cell life and death. Biol Rev. 2021;96(6):2489–521. 10.1111/brv.12764. [DOI] [PubMed] [Google Scholar]
  • 33.Olexiková L, Dujíčková L, Makarevich AV, Bezdíček J, Sekaninová J, Nesvadbová A, Chrenek P, Olexiková L, Dujíčková L, Makarevich AV, Bezdíček J, Sekaninová J, Nesvadbová A, Chrenek P. Glutathione during post-thaw recovery culture can mitigate deleterious impact of vitrification on bovine oocytes. Antioxidants (Basel). 2022;12(1). 10.3390/antiox12010035. [DOI] [PMC free article] [PubMed]
  • 34.Pang B, Dong G, Pang T, Sun X, Liu X, Nie Y, Chang X. Emerging insights into the pathogenesis and therapeutic strategies for vascular endothelial injury-associated diseases: Focus on mitochondrial dysfunction. Angiogenesis. 2024;27(4):623–39. 10.1007/s10456-024-09938-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Qi X, Liu Y, Peng Y, Fu Y, Fu Y, Yin L, Li X. UHRF1 promotes spindle assembly and chromosome congression by catalyzing EG5 polyubiquitination. J Cell Biol. 2023;222(11):e202210093. 10.1083/jcb.202210093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Richani D, Dunning KR, Thompson JG, Gilchrist RB. Metabolic co-dependence of the oocyte and cumulus cells: Essential role in determining oocyte developmental competence. Hum Reprod Update. 2021a;27(1):27–47. 10.1093/humupd/dmaa043. [DOI] [PubMed] [Google Scholar]
  • 37.Richani D, Dunning KR, Thompson JG, Gilchrist RB. Metabolic co-dependence of the oocyte and cumulus cells: Essential role in determining oocyte developmental competence. Hum Reprod Update. 2021b;27(1):27–47. 10.1093/humupd/dmaa043. [DOI] [PubMed] [Google Scholar]
  • 38.Sampath V, Shalakhti O, Veidis E, Efobi JAI, Shamji MH, Agache I, Skevaki C, Renz H, Nadeau KC. Acute and chronic impacts of heat stress on planetary health. Allergy. 2023;78(8):2109–20. 10.1111/all.15702. [DOI] [PubMed] [Google Scholar]
  • 39.Sangha V, Aboulhassane S, Qu QR, Bendayan R. Protective effects of pyrroloquinoline quinone in brain folate deficiency. Fluids Barriers CNS. 2023;20(1):84. 10.1186/s12987-023-00488-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Schmidt CJ, Lamont SJ. Integrated genomic approaches to characterize and mitigate heat stress in poultry. Ann Rev Anim Biosci. 2025;13:121–141. 10.1146/annurev-animal-111523-102021. [DOI] [PubMed]
  • 41.Song M, Li Y, Zhou Y, Yan J, Zhou X, Gao Q, Miao Y, Xiong B. Nicotinamide mononucleotide supplementation improves the quality of porcine oocytes under heat stress. J Anim Sci Biotechnol. 2022;13(1):68. 10.1186/s40104-022-00716-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.van Wettere WHEJ, Kind KL, Gatford KL, Swinbourne AM, Leu ST, Hayman PT, Kelly JM, Weaver AC, Kleemann DO, Walker SK. Review of the impact of heat stress on reproductive performance of sheep. J Anim Sci Biotechnol. 2021;12(1):26. 10.1186/s40104-020-00537-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Wang S, Wang Q, Min L, Cao H, Adetunji AO, Zhou K, Zhu Z, Wang S, Wang Q, Min L, Cao H, Adetunji AO, Zhou K, Zhu Z. Pyrroloquinoline quinone improved boar sperm quality via maintaining mitochondrial function during cryopreservation. Antioxidants (Basel). 2025;14(1). 10.3390/antiox14010102. [DOI] [PMC free article] [PubMed]
  • 44.Wu H, Tong Y, Wang C, Luan Y, Huang B. MrBZIP, a MrWETA-interacting transcription factor, mediates oxidative and heat stress tolerance in conidia of Metarhizium robertsii. J Invertebr Pathol. 2026;216:108558. 10.1016/j.jip.2026.108558. [DOI] [PubMed] [Google Scholar]
  • 45.Xia B, Wu W, Fang W, Wen X, Xie J, Zhang H. Heat stress-induced mucosal barrier dysfunction is potentially associated with gut microbiota dysbiosis in pigs. Anim Nutr. 2022;8:289–99. 10.1016/j.aninu.2021.05.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Yang L, Zhao Z, Cui M, Zhang L, Li Q. Melatonin restores the developmental competence of heat stressed porcine oocytes, and alters the expression of genes related to oocyte maturation. Animals (Basel). 2021;11(4):1086. 10.3390/ani11041086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Yang P, Chen T, Liu Y, Hou Z, Wu K, Cao Y, Zhang J, Wang Z, Zhao H. The critical role of ZP genes in female infertility characterized by empty follicle syndrome and oocyte degeneration. Fertil Steril. 2021;115(5):1259–69. 10.1016/j.fertnstert.2020.11.003. [DOI] [PubMed] [Google Scholar]
  • 48.Yang S, Li Y, Zong C, Pan X, Wang H, Fang Q, Shi L, Dong F, Cao H. LDH-based avermectin nanopesticide modulates bioaccumulation, antioxidant responses, and microbiota stability in earthworms’ gut. Pestic Biochem Physiol. 2026;219:107010. 10.1016/j.pestbp.2026.107010. [DOI] [PubMed] [Google Scholar]
  • 49.Yu D, Wang Y, Xie R, Zhou R, Liu Z, Shi N, Xie X, Yang Y, Qu J, Yang G, Huang B. Acetylation dynamics of MrATG4 governing autophagy-mediated conidiation in an entomopathogenic fungus. PLoS Pathog. 2026;22(1):e1013883. 10.1371/journal.ppat.1013883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Yuan X, Xu H, Liu X, Zhang J, Li J, Liang Q, An B, Paternò GM, Zhang M, Tang Y, Zhang C, Xu D, Zhong C, Li K, Wang X. Engineered Living Energy Materials. Interdisciplinary Mater. 2025;4(3):412–55. 10.1002/idm2.12245. [Google Scholar]
  • 51.Zhang L, Hui H, Faheem M, Xue Y, Chen N, Zhou X. Dietary defective jujube as a corn substitute: impacts on growth performance, meat traits, and alternaria toxin exposure in lambs. Animals (Basel). 2026;16(2). 10.3390/ani16020255. [DOI] [PMC free article] [PubMed]
  • 52.Zhang L, Wu K, Li H, Zhan M, Wang S, Song E, Zhang Q, He J, He X, Xu M, Deng H, Su Y, Liu Z, Yan Z, Tan P, He M, Weng S, Ge W, He J, Wang M. Vitellogenin receptor mediates heat adaptability of oocyte development in mud crabs and zebrafish. Nat Commun. 2025;16(1):3722. 10.1038/s41467-025-59035-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Zhang M, Wang W, Zhang D, Zhang Y, Li Y, Fang F, Zhang Z, Zhang Y. Prothioconazole exposure disrupts oocyte maturation and fertilization by inducing mitochondrial dysfunction and apoptosis in mice. Free Radic Biol Med. 2024;213:274–84. 10.1016/j.freeradbiomed.2024.01.027. [DOI] [PubMed] [Google Scholar]
  • 54.Zhang X, Zhang L, Xiang W. The impact of mitochondrial dysfunction on ovarian aging. J Translational Med. 2025;23(1):211. 10.1186/s12967-025-06223-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Zhou J, Jiang C. Targeting Multilayered Metabolic Networks in Brain Diseases: Emerging Perspectives on Nanodelivery Strategies. Adv Sci. 2025;12(41):e03645. 10.1002/advs.202503645. [DOI] [PMC free article] [PubMed] [Google Scholar]

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