Exposure to nanoscale graphene oxide deteriorates the quality of porcine oocytes via induction of oxidative stress and the apoptosis

Abstract

Purpose

Nano graphene oxide (nGO), as a type of engineered carbon nanomaterial, has witnessed significant growth in biomedical applications. Given the likelihood of accumulation of these materials in human tissues or organs, it becomes imperative to comprehensively assess the toxicological profile of nGO, particularly concerning female reproductive health.

Methods

Germinal vesicle (GV) porcine oocytes were cultured at 38.5 °C to the specific developmental stage for subsequent analysis. The nGO was diluted with the maturation medium to the final concentrations of 10, 50, 100 and 200 μg/ml, respectively. Immunostaining and fluorescence intensity quantification were applied to assess the effects of nGO exposure on the key processes during the oocyte meiotic maturation.

Results

We observed that exposure to nGO led to compromised meiotic competency in porcine oocytes during in vitro culture. Specifically, nGO exposure resulted in reduced acetylation levels of α-tubulin and misattachment of kinetochore-microtubules, thereby disrupting spindle/chromosome organization and impeding meiotic progression. Furthermore, nGO exposure perturbed actin dynamics, potentially hindering spindle migration and cortical polarization during oocyte meiosis. Additionally, mislocalization and premature exocytosis of ovastacin were observed following nGO exposure. Notably, nGO exposure induced mitochondrial dysfunction, DNA damage, and oxidative stress, ultimately triggering apoptosis and impeding the maturation of porcine oocytes and the development of post-fertilized embryos.

Conclusion

Our findings underscore the potential deleterious effects of nGO on mammalian oocyte quality, while also contributing valuable insights into the impact of environmental nanoparticle release on female germ cell development.

Graphical Abstract

Introduction

Nano Graphene oxide (nGO) is a nanomaterial synthesized by chemically exfoliating graphite oxide, exhibiting unique physical, chemical, and mechanical properties. In recent years, nGO has garnered increasing attention in the fields of agriculture and food science. In agriculture, nGO serves as an effective pesticide carrier, enhancing formulation stability and enabling controlled release mechanisms that reduce dosage requirements while mitigating environmental contamination. Furthermore, nGO enhances soil physicochemical properties through improved water retention and nutrient retention capacity, thereby promoting plant growth and seed germination via microenvironment modulation. In food science, its inherent antibacterial activity and structural robustness render nGO suitable for advanced packaging systems to prolong shelf life. Additionally, the material's conductive and fluorescent properties facilitate the development of ultrasensitive biosensors for detecting contaminants such as heavy metals and microbial pathogens. Despite these advancements, rigorous safety evaluations remain imperative to assess the long-term impacts of nGO on human health and ecological systems.

Infertility represents a significant global public health challenge, approximately 17.5% of adults will experience infertility at some point in their lives. Environmental pollution is recognized as a key risk factor for infertility. Some studies have found that chemical substances in the environment, such as pesticides, chemicals in plastics (such as bisphenol A and phthalates), heavy metals, and air pollutants, may have adverse effects on the reproductive system. Despite these observations, mechanistic insights into pollutant-induced reproductive impairments remain insufficient, necessitating comprehensive investigations to develop targeted prevention strategies. This includes not only reducing the emission and exposure to these pollutants but also raising public awareness about the potential impacts of environmental pollution on reproductive health. In-depth investigation into the effects of environmental pollutants on reproductive health is of paramount importance for improving global fertility rates and enhancing public health outcomes.

Graphene-family nanomaterials (GFNs) have attracted significant interest owing to their versatile applications in biomedicine [26]. NGO, as a type of engineered carbon nanomaterial (CNM), has emerged as one of the most promising nanomaterials since its isolation in 2004 [15]. This type of nanomaterial has a particular surface area [18], excellent flexibility [21], high mechanical strength [17], and ease of surface functionalization (Y. [28]), making it a popular choice in electrocatalysis, electrode material, sensors, sorbents, and biological delivery [4]. The escalating global production of nGO, driven by its widespread adoption, has raised concerns regarding its potential environmental dispersion into hydrosphere, soil, atmosphere, biosphere, and human systems [1]. Unlike other carbon nanotubes and graphene, nGO possesses a wealth of hydrophilic surface oxygen functional groups. These functional groups significantly enhance nGO's affinity for polar solvents, enabling it to form stable suspensions in water and other solvents [7]. Additionally, these surface functional groups endow nGO with unique chemical reactivity, making it highly applicable in various fields such as biomedicine, environmental remediation, and composite materials [11]. Despite these advantages, the high dispersibility and stability of nGO heighten its potential risks to microbial and mammalian systems. While such nanomaterials deliver significant economic benefits, their proliferation necessitates rigorous evaluation of associated environmental and health hazards. Currently, the research on nGO is still in its nascent phase, necessitating further exploration and validation to understand its precise health implications.

Previous research indicates that the extensive production of nGO inevitably increases human exposure to it through various routes such as skin contact, inhalation, and ingestion [6]. This exposure has been associated with cytotoxicity, genotoxicity, and organ toxicity, attributed to nGO's ability to damage cell membranes through extraction and cleavage processes (L. [2]). Research has demonstrated the potential for nGO to be transferred through the food chain, leading to its accumulation within cells of humans or animals. Subsequently, it distributes to various tissues and organs including the kidney, spleen, liver, lungs, among others. Additionally, recent investigations have identified nGO as a contaminant associated with reproductive toxicity [22]. The negative impacts have been demonstrated in zebrafish [1], Oryzias latipes [5], mice [25], humans (A. [23]), and other species [27]. Histological evidence shows nGO accumulation in Oryzias latipes gonads causes fertilized egg abnormalities and mortality [5]. In vitro experiments conducted by Wang revealed that treatment with nGO induced significant production of reactive oxygen species (ROS) in cells, exhibiting higher toxicity than other graphene derivatives (A. [23]). Singh confirmed that the exceptionally flat surface of nGO could intercalate into the DNA helix and disrupt physiological processes such as DNA damage repair and apoptosis [20]. Chen et al. demonstrated that exposure to nGO can induce cell differentiation and apoptosis through modulation of phosphorylation levels of proteins upstream and downstream of the ERK pathway (Y. [3]), which is an important protein kinase of the MAPK cascade [12]. Oral administration of nGO to maternal mice resulted in developmental delays in their offspring, including reduced weight and shortened body length [25]. Notably, studies revealed that graphene oxide (GO) disrupts cell cycle progression, while nGO impairs cell viability via apoptosis and DNA degradation (Hashemi et al., 2020), underscoring the necessity to investigate mitochondrial dysfunction, oxidative stress, and cytoskeletal dynamics, which are key parameters directly linked to oocyte developmental competence and genomic stability. Furthermore, exposure to graphene oxide nanoparticles (GO NPs) during critical windows of gametogenesis-namely, early germ cell differentiation and the gametogenesis stage in adulthood-induces inheritable reproductive toxicity in medaka (Oryzias latipes) [13], highlighting the urgency to elucidate its acute effects on oocyte quality and fertilization potential. While previous studies have reported on the reproductive toxicity induced by nGO, there remains a significant gap in our understanding regarding its effects and potential mechanisms on oocyte quality, particularly in terms of oocyte developmental competence.

In this study, we used porcine oocytes to explore the molecular mechanisms by which nGO impairs oocyte quality, aiming to reveal pathways associated with mitochondrial dysfunction, oxidative stress, and early apoptosis in oocytes. These findings contribute to our understanding of nGO-induced female gamete toxicity, particularly regarding oocyte quality.

Materials and method

Chemicals

Penicillin and streptomycin (Cat#: P1400) were purchased from Solarbio (Beijing, China). Follicle-stimulating hormone (FSH, Cat#: B2203151) and luteinizing hormone (LH, Cat#: B2301131) were purchased from Ningbo Second Hormone Factory (Ningbo, China). AF488-conjugated goat anti-mouse IgG (H + L) (Cat#: A11029) and AF555-conjugated goat anti-human IgG (H + L) (Cat#: A21433) were purchased from ThermoFisher Scientific (Waltham, MA, USA). Mito-Tracker Red (Cat#: MB6046) was provided by Meilunbio (Dalian, China). ROS assay kit (Cat#: S0033S) and Annexin V-FITC apoptosis detection kit (Cat#: C1062) were purchased from Beyotime (Shanghai, China). Unless otherwise specified, other chemicals utilized in this research were procured from Sigma Chemical Company (St Louis, MO, USA).

Collection and culture of porcine oocytes

Cumulus-oocyte complexes (COCs) were obtained from antral follicles (diameter 3–8 mm) of prepubertal gilt ovaries obtained from a nearby slaughterhouse, placed in 38.5 °C saline with 3% (v/v) penicillin and streptomycin and delivered to the laboratory within 2 h. The follicular contents were extracted using a 10 ml syringe to retrieve COCs from porcine ovaries, which were then rinsed thrice with 10 mM HEPES-buffered TCM199 supplemented with 0.1% polyvinyl alcohol (PVA). Subsequently, the COCs with 3–5 layers of cumulus cells and a homogeneous cytoplasm were placed in porcine oocyte maturation medium (TCM-199 supplemented with 10% porcine follicular fluid, 10 IU/ml FSH, 0.57 mM cysteine, 10 IU/ml LH and 0.91 mM sodium pyruvate) and cultured in a 5% CO₂ incubator at 38.5 °C for 26–28 h to the metaphase I (MI) stage and 44–46 h to the metaphase II (MII) stage. The calculation of cumulus cell expansion was based on the area using the formula: area = length × width × 0.7854 (X. [24]).

Nano graphene oxide treatment

Following the Nanogenotox protocol, nGO was dispersed in 1% bovine serum albumin (BSA) solution in MilliQ water to prepare a stock solution with a concentration of 200 mg/ml. Oocytes were randomly assigned to different experimental groups. The nanomaterial dispersion was diluted with porcine oocyte maturation medium and sonicated at 10% amplitude for 16 min to achieve final concentrations of 10, 50, 100, and 200 μg/ml, respectively.

Immunofluorescence staining and confocal microscopy

The oocytes were fixed in 4% paraformaldehyde (in phosphate-buffered saline (PBS)) for 15 min, after which there was permeabilization with 0.5% Triton X-100 in PBS for 30 min. After blocking with 2% BSA for 1 h, the oocytes were incubated with primary antibodies (1:200 for anti-α-tubulin-FITC, Cat#: F2168; 1:100 for anti-acetyl-α-tubulin, Cat#: T7451; 1:200 for Phalloidin-TRITC, Cat#: P1951) overnight, respectively. Subsequently, the specimens were treated for 1 h with secondary antibodies (1:200 for AF488-conjugated goat anti-mouse IgG (H + L); 1:200 for AF555-conjugated goat anti-human IgG (H + L)) after washing three times with DPBS containing 0.1% BSA. Following incubation, the specimens were stained with 1 μl/ml DAPI for 15 min to visualize DNA. Finally, the specimens were examined using a confocal laser-scanning microscope (FV3000, Olympus) after being put on glass slides. To detect and record the fluorescence signals, we used an excitation wavelength of 488 nm and detection wavelengths of 500–560 nm (green) and 590–617 nm (red). DIC images and DAPI images were also recorded using an excitation wavelength of 405 nm and a detection wavelength of 429–480 nm (blue). To accurately compare the fluorescence intensity between oocytes, all scanning parameters related to fluorescence intensity were fixed throughout the analysis.

Sperm binding assay

The sperm were incubated in in vitro fertilization (IVF) medium previously equilibrated with 5% CO₂ at 38.5 °C for 1 h under mineral oil to allow capacitation. Capacitated sperm were then co-incubated with matured oocytes or 2-cell embryos for 1 h to assess sperm binding. The 2-cell embryos served as a negative control for binding. Following incubation, samples were fixed in 4% paraformaldehyde for 30 min and stained with DAPI. The number of sperm bound to each oocyte or 2-cell embryo was quantified from Z-projection images obtained using the confocal microscope.

Determination of mitochondria and ROS

In accordance with the manufacturer's instructions, we utilized Mito-Tracker Red and ROS to investigate the distribution of mitochondria and ROS levels within oocytes. The oocytes underwent a brief exposure to Mito-Tracker Red (diluted at 1:10) and ROS (diluted at 1:10) for 30 min at 38.5 °C, 5% CO₂. Then, they were subjected to three rinses in DPBS supplemented with 0.1% BSA. Subsequently, the oocytes were subjected to confocal fluorescent microscopy under consistent parameters for analysis.

Annexin-V staining

Oocytes were stained in accordance with the manufacturer's instructions using an Annexin-V staining kit to measure the early apoptotic of oocytes. To clarify, oocytes were moved from the medium into a solution that contained 5 μl of Annexin-V-FITC and 45 μl of binding buffer for 30 min at 38.5 °C, 5% CO₂. A confocal microscope was used to evaluate the fluorescent signals after three PBS rinses.

Quantification of immunofluorescence imaging and statistical analysis

In order to assess the intensity of fluorescence, immunofluorescence-stained slides were placed on a laser confocal microscope, ensuring uniform parameters for observation across all samples remained consistent. The average fluorescence intensity within the region of interest (ROI) was calculated using Image J software. The resulting average values from all measurements were then compared between control and treatment groups. Statistical analysis was performed using t-tests with GraphPad Prism (La Jolla, USA) prior to graphical representation, with statistical significance set at P < 0.05. Data are presented as mean ± standard error of the mean (SEM) from three independent experiments, the number of oocytes was labelled in parentheses as (n).

Results

nGO inhibites the in vitro porcine oocyte developmental maturation

To examine the effects of nGO on porcine oocyte development, different concentrations of nGO (10, 50, 100 or 200 μg/ml) were added to the maturation medium. As illustrated in Fig. 1A, the majority of the cumulus cells encircling the control group showed notable growth after 44 h of in vitro maturation (IVM). Conversely, cumulus cells surrounding oocytes exposed to nGO displayed only partial expansion. Furthermore, exposure to nGO led to a dose-dependent decrease in the proportion of first polar body (PB1) extrusion, with a notable effect observed at 200 μg/ml nGO (86.9 ± 2.3%, n = 103, control vs 59.7 ± 3.8%, n = 101, 200 μg/ml nGO, P < 0.001; Fig. 1B). Hence, a concentration of 200 μg/ml nGO was selected for further examinations. Subsequently, we quantified the cumulus expansion in oocytes exposed to nGO and observed a significant inhibition of this process compared to controls (350032 ± 22651 μm², n = 35, control vs 151700 ± 12,939 μm², n = 27, 200 μg/ml nGO, P < 0.01; Fig. 1C). Taken together, these findings imply that exposure to nGO has detrimental effects on the developmental competence of porcine oocytes.

Fig. 1.

Effects of nGO exposure on the porcine oocyte maturation in vitro. (A) Representative images of oocyte meiotic progression in control and nGO-exposed oocytes. Scale bar, 360 μm (a, d); 120 μm (b, e); 40 μm (c, f). (B) The percentage of PB1 extrusion was quantified in control (n = 103) and nGO-exposed (10 μg/ml: n = 97, 50 μg/ml: n = 102, 100 μg/ml: n = 98, 200 μg/ml: n = 101) oocytes after culture for 44 h in vitro. (C) The cumulus expansion area for COCs was recorded in control (n = 35) and nGO-exposed (n = 27) oocytes. Data were presented as mean ± SEM from at least three independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

nGO exposure disrupts spindle morphology and chromosome alignment in porcine oocytes

To explore the determinants implicated in aberrant embryonic development, we analyzed spindle organization and chromosome arrangement during the maturation process of porcine oocytes. Anti-α-tubulin-FITC antibody staining was used to examine spindle morphology, and propidium iodide (PI) staining was used to evaluate chromosome alignment. As depicted in Fig. 2A, the control group exhibited a characteristic barrel-shaped spindle with chromosomes neatly aligned on the metaphase plate; however, the nGO-exposed oocytes displayed various abnormal spindles and misaligned chromosomes. Furthermore, our statistical analysis revealed a markedly higher frequency of abnormal spindle shapes and misaligned chromosomes in nGO-exposed oocytes compared to controls (spindle: 17.9 ± 2.9%, n = 95, control vs 54.7 ± 2.4%, n = 95, nGO, P < 0.001; chromosome: 18.9 ± 1.3%, n = 97, control vs 55.8 ± 2.8%, n = 97, nGO, P < 0.001; Fig. 2B-C). These findings suggest that nGO exposure induces abnormalities in the spindle and chromosome structure of porcine oocytes.

Fig. 2.

nGO exposure disturbs MI spindle assembly and chromosome alignment in porcine oocytes. (A) Representative images of spindle/chromosome structure in control and nGO-exposed oocytes. Scale bar, 10 μm. (B) The rate of aberrant spindle were recorded in both control (n = 95) and nGO-exposed (n = 97) oocytes. (C) The rate of misaligned chromosomes were recorded in both control (n = 95) and nGO-exposed (n = 97) oocytes. Data were presented as mean ± SEM from at least three independent experiments. ^***P < 0.001

nGO exposure decreases acetylation level of α-tubulin disrupts and kinetochore-microtubule attachment in porcine oocytes

The observed impairment in spindle assembly suggests a potential compromise in microtubule stability and dynamics within nGO-exposed oocytes. To explore this proposition, we evaluated the α-tubulin acetylation status, a recognized marker of microtubule stability. As depicted in Fig. 3A-B, the fluorescence intensity of acetylated α-tubulin was notably decreased in nGO-exposed oocytes compared to controls (23.2 ± 0.4, n = 32, control vs 15.0 ± 0.3, n = 31, nGO, P < 0.001). Thus, the findings indicate that exposure to nGO during porcine oocyte meiotic maturation may perturb spindle structure and chromosome arrangement through downregulation of α-tubulin acetylation.

Fig. 3.

Effects of nGO exposure on the acetylation level of α-tubulin and the stability of kinetochore-microtubule attachment in porcine oocytes. (A) Representative images of acetylated α-tubulin in control and nGO-exposed oocytes. Scale bar, 10 μm. (B) The fluorescence intensity of acetylated α-tubulin was measured in both control (n = 32) and nGO-exposed (n = 31) oocytes. (C) Representative images of Kinetochores-microtubules attachments in control and nGO-exposed oocytes. Scale bar, 10 μm. (D) The rate of defective kinetochore-microtubule attachments were recorded in both control (n = 95) and nGO-exposed (n = 97) oocytes. Data were presented as mean ± SEM from at least three independent experiments. ^***P < 0.001

Given that abnormal spindle assembly and inaccurate chromosome positioning consistently correlate with impaired kinetochore-microtubule interactions, we evaluated whether the misalignment of chromosomes resulting from nGO exposure stemmed from impaired interaction between kinetochores and microtubules, we examined the stability of kinetochore-microtubule attachment. This involved using anti-tubulin-FITC antibody to visualize spindles, Crest immunostaining to detect kinetochores, and DAPI counterstaining to identify chromosomes. As shown in Fig. 3C-D, nGO exposure significantly increased the frequency of kinetochore-microtubule misattachments (18.0 ± 1.4%, n = 95, control vs 55.0 ± 2.6%, n = 97, nGO, P < 0.001). Overall, the attachment between kinetochores and microtubules appears less stable following nGO exposure, potentially contributing to the inability to align chromosomes.

nGO exposure perturbs the actin cytoskeleton in porcine oocytes

Actin, an essential component of the cytoskeleton, plays a pivotal role in porcine oocyte nuclear maturation. To investigate whether actin dynamics contribute to the disruption of oocyte meiosis by exposure to nGO, we stained F-actin with Phalloidin-TRITC. In Fig. 4A, F-actin filaments showed a steady distribution with significant signals along the plasma membrane in control oocytes, whereas the signals were diminished in nGO-exposed oocytes. This observation was corroborated by quantitative analysis, revealing a notable disparity in F-actin fluorescence intensity between control and nGO-exposed oocytes (22.4 ± 0.3, n = 32, control vs 13.5 ± 0.3, n = 32, nGO, P < 0.001; Fig. 4B-C). Thus, these data imply that nGO exposure disrupts the actin dynamics, which might contribute to poor oocyte quality and the breakdown of meiotic maturation in oocytes.

Fig. 4.

Effects of nGO exposure on the actin cytoskeleton in porcine oocytes. (A) Representative images of F-actin filaments on the plasma membrane in control and nGO-exposed oocytes. Scale bar, 25 μm. (B) The fluorescence intensity profiling of phalloidin-TRITC was shown in control and nGO-exposed oocytes. Lines were drawn across the oocytes, and pixel intensities were quantified along the lines. (C) The fluorescence intensity of F-actin signals was quantified in control (n = 32) and nGO-exposed (n = 32) oocytes. Data were presented as mean ± SEM from at least three independent experiments. ^***P < 0.001

nGO exposure disrupts the distribution of mitochondria and ovastacin in porcine oocytes

Mitochondria play a vital role in supplying ATP for cellular processes, making their distribution a key indicator of oocyte cytoplasmic maturation. To assess mitochondrial distribution after nGO exposure, we stained oocytes with MitoTracker™ Red CMXRos. In Fig. 5A, two main patterns of mitochondrial distribution were evident in the control group: polarized distribution surrounding the spindle and uniform distribution throughout the cytoplasm. Conversely, in nGO-exposed oocytes, there was a marked rise in the percentage of mitochondria that display abnormal clustering within the cytoplasm, departing from their usual arrangement around the chromosomes. Quantitative analysis further revealed a notable decrease in the fluorescent intensity of mitochondria in oocytes exposed to nGO when compared to the control group (39.9 ± 0.5, n = 35, control vs 17.8 ± 0.6, n = 34, P < 0.001, nGO; Fig. 5A-B). The findings suggest that mitochondrial function may be compromised following exposure to nGO.

Fig. 5.

Effects of nGO exposure on the mitochondrial distribution and dynamics of ovastacin in porcine oocytes. (A) Representative images of mitochondria in control and nGO-exposed oocytes. Scale bar, 25 μm. (B) The fluorescence intensity of mitochondria was measured in control (n = 35) and nGO-exposed (n = 34) oocytes. Data were presented as mean ± SEM from at least three independent experiments. ^***P < 0.001. (C) Representative images of ovastacin distribution in control and nGO-exposed oocytes. Scale bar, 25 μm. (D) The fluorescence intensity of ovastacin was measured in control (n = 31) and nGO-exposed (n = 30) oocytes. Data were presented as mean ± SEM feast three independent experiments. ^***P < 0.001

It is important to highlight that the localization of ovastacin, a zinc metalloendoprotease responsible for cleaving the zona pellucida to inhibit sperm binding, is considered a key marker of oocyte cytoplasmic maturation. To investigate the factors underlying nGO-induced oocyte quality compromise, we examined the localization of ovastacin. Our findings demonstrated that in control specimens, ovastacin was primarily concentrated in the subcortical area of the oocyte and was not present in regions devoid of cortical granules. In contrast, nGO-exposed oocytes showed various aberrant patterns of ovastacin localization. The incidence of abnormal ovastacin localization was notably elevated in nGO-exposed oocytes compared to controls (16.4 ± 0.4, n = 31, control vs 9.8 ± 0.4, n = 30, nGO, P < 0.001; Fig. 5D). These findings imply that exposure to nGO may lead to the mislocalization and premature exocytosis of ovastacin, potentially contributing to the compromised quality of porcine oocytes.

nGO exposure disrupts the sperm binding ability and the fertilization ability of porcine oocytes

To assess the potential impact of nGO exposure on the sperm binding ability of nGO-exposed oocytes and its consequent effect on fertilization rate, we conducted a sperm-oocyte binding assay. Subsequently, the amount of sperm attached to the zona pellucida was measured by labeling sperm heads with DAPI. In control oocytes, the zona pellucida demonstrated strong affinity for sperm attachment. However, it was no longer able to promote further sperm adhesion in 2-cell embryos due to the depletion of sperm binding sites after fertilization. Notably, in nGO-exposed oocytes, there was a significant reduction in the number of sperm binding to the zona pellucida compared to controls (108.4 ± 1.5, n = 33, control vs 48.3 ± 1.4, n = 32, nGO, P < 0.001; Fig. 6A-B).

Fig. 6.

Effect of nGO exposure on the sperm binding ability and fertilization ability in porcine oocytes. (A) Representative images of sperm binding to the zona pellucida of control and nGO-exposed oocytes. Oocytes and 2-cell embryos from control and nGO-exposed oocytes were incubated with capacitated sperm for 1 h. After washing with a wide-bore pipette to remove all but 2–6 sperm on normal 2-cell embryos (negative control), oocytes and embryos with sperm were fixed and stained with DAPI. Scale bar, 25 μm. (B) The sperm binding to the surface of the zona pellucida surrounding oocytes from control (n = 33) and nGO-exposed (n = 32) oocytes were counted. (C) Representative images of early embryos developed from control and nGO-exposed oocytes. Scale bar, 120 μm. (D) The fertilization rate was recorded in the control (n = 108) and nGO-exposed (n = 105) oocytes. Data were presented as mean ± SEM feast three independent experiments. ^***P < 0.001

After that, we investigated the possibility that nGO exposure would reduce the ability of exposed oocytes to fertilize. We discovered through IVF experiments that the majority of control oocytes underwent fertilization, leading to the generation of 2-cell embryos, whereas the fertilization rate of nGO-exposed oocytes was notably lower than that of the control group (62.0 ± 1.1%, n = 108, control vs 26.7 ± 1.4%, n = 105, nGO, P < 0.001; Fig. 6C-D). These findings highlight that nGO exposure has the potential to disrupt the fertilization ability of porcine oocytes and impair their subsequent embryonic development.

nGO exposure causes DNA damage and early apoptosis by increasing ROS levels

Elevated oxidative stress often coincides with aberrant mitochondrial function. To investigate this correlation, we evaluated the levels of ROS using DCFHDA staining after exposure to nGO. As depicted in Fig. 7A-B, the ROS levels, as indicated by DCFHDA staining, were notably elevated in nGO-exposed oocytes compared to the control group (5.5 ± 0.3, n = 33, control vs 22.3 ± 0.4, n = 34, nGO, P < 0.001). Elevated levels of ROS often overwhelm the cellular antioxidant defense mechanisms, leading to DNA damage and apoptosis [19]. Hence, we proceeded to assess the potential induction of DNA damage during oocyte maturation following nGO exposure by staining γH2A.X, a marker indicative of DNA damage. The results depicted in Fig. 7C-D revealed a notable elevation in the fluorescence intensity of γH2A.X signals in nGO-exposed oocytes compared to controls, as supported by quantitative analysis (9.4 ± 0.3, n = 32, control vs 15.9 ± 0.3, n = 31, nGO, P < 0.001). Moreover, we conducted an analysis of the apoptotic rate of oocytes using Annexin-V staining. The findings revealed minimal green fluorescent signals in control oocytes, in contrast to the observable signals localized on the membrane of nGO-exposed oocytes. Statistical analysis confirmed a significant enhancement in the fluorescence signals of Annexin-V in nGO-exposed oocytes compared to controls (5.0 ± 0.2, n = 35, control vs 19.7 ± 0.4, n = 34, nGO, P < 0.001; Fig. 7E-F). In summary, our results indicate a possible link between the identified apoptotic oocytes, which result from increased levels of superoxide and DNA damage during aging, and the diminished quality of oocytes after exposure to nGO.

Fig. 7.

Effects of nGO exposure on the ROS levels, DNA damage accumulation and occurrence of apoptosis in porcine oocytes. (A) Representative images of ROS levels in control and nGO-exposed oocytes. Scale bar, 25 μm. (B) The fluorescence intensity of ROS was measured in control (n = 33) and nGO-exposed (n = 34) oocytes. (C) Representative images of DNA damage in control and nGO-exposed oocytes. Scale bar, 10 μm. (D) The fluorescence intensity of γH₂A.X signals were measured in control (n = 32) and nGO-exposed (n = 31) oocytes. (E) Representative images of apoptotic oocytes in control and nGO-exposed oocytes. Scale bar, 25 μm. (F) The fluorescence intensity of Annexin-V signals was measured in control (n = 35) and nGO-exposed (n = 34) oocytes. Data were presented as mean ± SEM from at least three independent experiments. ^***P < 0.001

Discussion

With the widespread application of nGO in various fields, there is a growing concern about its impact on health. Although the unique properties of nGO make it an ideal material for many applications, its microscopic scale and special structure have also raised concerns about health safety. Previous studies have shown that exposure to nGO may lead to cytotoxicity, causing adverse effects on human cells. This toxicity may involve a series of cellular damage mechanisms, including cell membrane disruption, oxidative stress, and mitochondrial dysfunction. Additionally, increasing evidence suggests that exposure to nGO may have negative effects on the reproductive system. The potential adverse effects of in vitro nGO exposure on oocyte development and its underlying molecular mechanisms remain unclear. To close this gap, we employed porcine oocytes as a model system due to their physiological and developmental resemblance to humans. Our aim was to investigate how nGO exposure influences oocyte quality and maturation capacity by exploring the associated molecular mechanisms.

The significance of cumulus cells in facilitating oocyte development is widely acknowledged, with research indicating decreased rates of IVM and later development of the embryo in their absence. Conversely, the substantial expansion of cumulus cells is a crucial event during oocyte maturation, affecting both the nucleus and cytoplasm. Therefore, the degree of cumulus cell growth is a consistent indicator of oocyte developmental potential. In the meantime, maintaining fertility in females relies heavily on the production of high-quality mature oocytes, a process regulated by precise control of oocyte meiotic arrest and resumption progression. Any disruption to this delicate process can lead to oogenesis failure, significantly impacting both fertility and female health [9]. Embryonic exposure to graphene oxide nanoparticles (GO NPs) leads to an abnormal increase in germ cell numbers with potential lethality, and continuous exposure for 21 days results in reduced fertility, an effect that can be transmitted to the F1 and F2 generations [13]. Hence, our initial investigation focused on evaluating the impact of nGO on the maturation potential of porcine oocytes and the expansion of cumulus cells in vitro. Subsequent to germinal vesicle breakdown (GVBD), oocytes advance to the MI stage. During this phase, the PB1 is expelled, carrying a fraction of cytoplasm, thereby concluding the MI stage for an oocyte containing a single set of chromosomes [14]. As the concentration of nGO increased, a greater number of embryos exhibited arrest at the MI stage. The spindle, a crucial organelle in oocyte meiosis, exhibited abnormal assembly and chromosome misalignment under nGO exposure during the MI stage. Corresponding to these spindle-chromosome anomalies, a notable increase in unstable microtubules and a consequent rise in misattachments between kinetochores and microtubules were observed. Accurate chromosome segregation during meiosis, vital for genome integrity, depends on duplicated chromosomes adhering correctly in a bi-oriented manner to spindle microtubules using kinetochores. Therefore, regulating the rate of attachment and detachment of spindle microtubules to kinetochores is crucial for successful meiosis. Based on these findings, we infer that the misattachments of kinetochores and microtubules, alongside the instability of microtubules, may underlie the meiotic defects induced by nGO. Exposure to nGO leads to a significant increase in intracellular ROS and activates apoptotic pathways, thereby disrupting the redox homeostasis of the cell. Studies have shown that excessive ROS accumulation can damage spindle structure, destabilize microtubules, and impair the accurate alignment and segregation of chromosomes, ultimately interfering with the meiotic progression of oocytes. Moreover, ROS can activate the p53 signaling pathway, increase mitochondrial membrane permeability, induce the release of cytochrome c, and subsequently trigger the caspase cascade, leading to programmed cell death. These alterations not only compromise oocyte viability and maturation capacity but may also result in failed oocyte development. The completion of meiosis requires accurate spatial and temporal synchronization between cytokinesis and karyokinesis. During meiotic maturation, several processes such as spindle assembly, alignment, and rotation, along with chromosomal movements, polar body expulsion, and pronuclear migration, rely on the control of the cytoskeletal system (Z.-Y. [29]). We noted that exposure to nGO led to a disruption in actin polymerization during oocyte maturation, thereby compromising cytoskeleton organization.

Besides ensuring genomic stability through nuclear maturation, oocytes need to go through cytoplasmic maturation in order to become fertile and enable the creation of subsequent embryos. Assessing cytoplasmic maturation often involves evaluating the distribution patterns of mitochondria and the status of key cytoplasmic components like ovastacin. Mitochondria, as the powerhouse of the cell, are vital for ATP production and various cellular processes. Our findings revealed that exposure to nGO led to aberrant mitochondrial distribution in the MI oocytes, suggesting potential disruptions in energy metabolism and cellular function. Additionally, ovastacin, a critical component of cortical granules that is involved in the cortical reaction during fertilization, plays a pivotal role in preventing polyspermy. Our study demonstrated that nGO exposure also perturbed the localization or function of ovastacin, indicating impaired fertilization competence and early embryonic development. These observations highlight the multifaceted impact of nGO exposure on oocyte cytoplasmic maturation, encompassing alterations in mitochondrial dynamics and disruption of essential cytoplasmic factors. Furthermore, nGO exposure adversely affects IVF by disrupting essential steps such as sperm binding and fusion, which are critical for successful fertilization and subsequent embryonic development. Upon exposure to nGO, we observed significant impairments in the ability of sperm to bind to oocytes, suggesting interference with the initial recognition and interaction between gametes. These findings underscore the intricate interplay between nanomaterial exposure and reproductive processes, highlighting the need for further investigation into the underlying mechanisms. Understanding these effects is crucial for elucidating the mechanisms underlying nanomaterial-induced reproductive toxicity and informing strategies to mitigate adverse outcomes. Further investigation into the specific molecular pathways involved in these disruptions could provide valuable insights into potential therapeutic interventions or preventive measures.

Assessing nGO-induced cytotoxicity typically includes measuring oxidative stress levels and plasma membrane integrity [10]. The interaction between cells and nGO leads to cell membrane obstruction, hindering the transport of essential nutrients and oxygen, ultimately leading to cell death. Moreover, due to its amphiphilic nature, nGO can induce membrane puncturing and lipid extraction. Upon cellular uptake, nGO tends to accumulate in the cytoplasm, exerting toxic effects [16]. Additionally, within cells nGO triggers ROS generation, leading to oxidative stress, apoptosis, and DNA damage. Thus, the compromised oocyte quality caused by nGO exposure results from ROS accumulation, a known trigger for cell apoptosis. We substantiated this hypothesis by detecting significantly elevated ROS levels and DNA damage in nGO-exposed oocytes. Furthermore, Annexin-V staining revealed early signs of apoptosis in these oocytes. Our study sheds important light on the complex processes that underlie nGO toxicity, especially with regard to its deleterious effects on the reproductive potential and mitochondrial function of oocytes. These findings have the potential to shape public health strategies regulating exposure to nGO and other emerging nanomaterials.

This research presents novel evidence indicating that nGO diminishes the maturation and fertilization capacity of oocytes by disturbing redox homeostasis and mitochondrial function. The implications of these findings are significant, suggesting new therapeutic strategies in the realms of female health and reproductive medicine, particularly regarding oocyte quality and mitochondrial function. The overarching recommendation is that populations exposed to high concentrations of nanoscale graphene oxide should recognize the associated risks of graphene contamination and take necessary measures to safeguard their health. These strategies could involve avoiding settings containing graphene or selecting items with minimal or no graphene content. Moreover, it is recommended to periodically assess graphene levels and apply appropriate corrective actions as needed.

Authors contributions

M.Z., F.B., D.Z. and Y.G. designed the research; M.Z., Y.G., K.T., M.L. and M.W. performed the experiments; D.Z., Y.Z., Z.L., M.L. and K.T. analyzed the data; M.Z., F.B. and Y.G. wrote the manuscript.

Funding

This work was funded by the Startup Foundation for Advanced Talents of Anhui Agricultural University (RC392004), National Natural Science Foundation (32272881), the Anhui Provincial Natural Science Foundation (2308085QC82) and the Natural Science Research Project of Anhui Province (KJ2020A0123).

Data availability

Data and materials will be made available on request.

Declarations

Ethical approval

Material used for this project was abattoir-derived only and no review was required by an Animal Welfare and Ethical Review Body.

Conflict of interest

The authors declare no conflicts of interest with regard to the study.