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.

Keywords: Mitochondria; Nano graphene oxide; Oocyte maturation; Oocyte quality; Oxidative stress Apoptosis.

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

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

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

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

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

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

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