Nicotinic acid protects germinal vesicle oocyte meiosis against toxicity of benzo(a)pyrene in mice and humans

Abstract

In brief: Low concentrations of benzo(a)pyrene in the follicular fluid of smokers disrupt oocyte maturation, leading to meiotic defects. Nicotinic acid (NA) partially rescues these defects, offering insights into potential strategies for protecting fertility.

Abstract: Benzo(a)pyrene (BaP), a carcinogen present in cigarette smoke, was detected in human follicular fluid at concentrations of approximately 5 nM in smokers and 7 nM in cases of assisted reproductive failure. However, whether a low concentration of BaP affects germinal vesicle (GV) oocyte maturation remains unclear. Here, we investigated the effects of 5 nM BaP on GV oocyte maturation in both mice and humans. In mice, GV oocytes were treated with 5 or 50 nM BaP, while human oocytes were exposed to 5 nM BaP. Our results demonstrated that 5 or 50 nM BaP exposure significantly inhibited first polar body extrusion during oocyte maturation. Mechanistic investigations revealed that BaP treatment downregulated Sirt1 protein expression in both GV and metaphase II (MII) stage mouse oocytes. Moreover, BaP exposure induced multiple cellular abnormalities, including spindle disorganization, cortical actin cap disruption, mitochondrial dysfunction and DNA damage in MII oocytes. Importantly, 15 μM NA supplementation increased Sirt1 expression and significantly rescued most of the abnormal effects. Subsequently, 5 nM BaP exposure impaired meiotic progression by reducing mitochondrial membrane potential and causing significant reactive oxygen species accumulation in human GV oocytes. Importantly, 15 μM NA supplementation partially rescued human GV oocytes from the toxicity of BaP during in vitro maturation (IVM). The present study indicated that a low BaP concentration in follicular fluid can significantly disrupt GV oocyte IVM, inducing meiotic defects in both mice and humans. NA has been shown to provide partial protection to GV oocyte meiosis against the toxicity of BaP during IVM.

Keywords: in vitro maturation; benzo(a)pyrene; germinal vesicle oocyte; nicotinic acid; oxidative stress.

Figure 1

Effects of BaP exposure on mouse oocyte maturation in vitro. (A) A Schematic presentation of the experimental protocol in mouse oocytes. (B) Representative oocyte morphologies in different groups. Red arrowheads point to oocytes with symmetrical division or a large polar body. Yellow arrows point to oocytes with no polar body. Scale bar: 80 μm. (C) Oocyte maturation rate in the control (n = 199), BaP-5 nM (n = 176) and BaP-50 nM (n = 196). BaP, benzo(a)pyrene. (D) Representative MII oocytes of CM-H₂DCFDA fluorescence. Scale bar: 80 μm. (E) Quantification of relative ROS levels in the control (n = 12), BaP-5 nM (n = 73) and BaP-50 nM (n = 81). Each data point represents an oocyte. (F) Sirt1 protein was verified by western blot analysis in different groups. The relative amount of SIRT1 was estimated based on the level of actin (n = 100 per group). (G) MII oocytes were stained with α-tubulin antibody to visualize the spindle (green) and counterstained with Hoechst 33342 (red) for chromosomes. Representative images of spindle morphologies and chromosome alignment in the control, BaP-5 nM and BaP-50 nM treated oocytes. Scale bar: 25 μm. (H) Quantification of control (n = 131), BaP-5 nM (n = 246) and BaP-50 nM (n = 262) with spindle disorganization. (I) Quantification of control (n = 137), BaP-5 nM (n = 192) and BaP-50 nM (n = 180) with chromosome defects. Data were presented as the mean percentage ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

NA protected against meiotic maturation in BaP exposure mouse oocytes. (A) Representative oocyte morphologies in different groups. Red arrowheads point to oocytes with symmetrical division or a large polar body. Yellow arrows point to oocytes with no polar body. Scale bar: 80 μm. (B) NA protected against meiotic maturation in 5 nM BaP exposure mouse oocytes. The oocyte maturation rate in control (n = 112), BaP-5 nM (n = 120), BaP-5 nM + NA-7.5 μM (n = 191), BaP-5 nM + NA-15 μM (n = 173) and BaP-5 nM + NA-60 μM (n = 154). (C) NA protected against meiotic maturation in 50 nM BaP exposure mouse oocytes. The oocyte maturation rate in the control (n = 89), BaP-50 nM (n = 162), BaP-50 nM + NA-7.5 μM (n = 140), BaP-50 nM + NA-15 μM (n = 175) and BaP-50 nM + NA-60 μM (n = 160). NA, nicotinic acid. (D) Sirt1 protein was verified by western blot analysis in different groups. The relative amount of Sirt1 was estimated based on the level of actin (n = 100 per group). Data were presented as the mean percentage ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Effects of NA on the subcellular structure in BaP-exposed mouse oocytes. (A) MII oocytes were labeled with MitoTracker Red to visualize mitochondrial distribution and counterstained with Hoechst 33342 (blue) for chromosomes. Representative images of mitochondrial distribution patterns in MII oocytes. Scale bar: 25 μm. (B) Quantification of control (n = 180), BaP-5 nM (n = 125), BaP-50 nM (n = 101), BaP-5 nM + NA (n = 81) and BaP-50 nM + NA (n = 84) with each mitochondrial distribution pattern. (C) MII oocytes were labeled with phalloidin to visualize actin (green) and were counterstained with propidium iodide (PI) (red) for chromosomes. White arrowheads point to MII oocytes with actin filaments on the membrane. Scale bar: 25 μm. (D) Quantitative analysis of control (n = 115), BaP-5 nM (n = 120), BaP-50 nM (n = 126), BaP-5 nM + NA (n = 124) and BaP-50 nM + NA (n = 141) with normal actin cap formation. (E) MII oocytes were stained with anti-γ-H2A.X antibody to visualize DNA damage (green) and counterstained with Hoechst 33342 (blue) for chromosomes. Scale bar: 50 μm. (F) Quantification of the fluorescence intensity of DNA damage in control (n = 17), BaP-5 nM (n = 18), BaP-50 nM (n = 17), BaP-5 nM + NA (n = 17) and BaP-50 nM + NA (n = 17). Data were presented as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

NA protected against meiotic maturation in BaP exposure human oocytes. (A) Schematic presentation of the experimental protocol in human oocytes. (B) Representative oocyte morphologies in different groups. Scale bar: 100 μm. (C) The oocyte maturation rate in control (n = 21), BaP-5 nM (n = 22) and BaP-5 nM + NA (n = 16). (D) Representative images of CM-H₂DCFDA fluorescence. The samples were GV oocytes cultured in vitro for 24–30 h. Scale bar: 100 μm. (E) The fluorescence intensity of ROS in control (n = 15), BaP-5 nM (n = 7) and BaP-5 nM + NA (n = 10). Each data point represents an oocyte. Each data point represents an oocyte. Data were presented as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Diagram of the experiment in mouse and human oocytes.