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. 2018 Mar 1;19(3):699.
doi: 10.3390/ijms19030699.

Effects of Fullerenol Nanoparticles on Rat Oocyte Meiosis Resumption

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Free PMC article

Effects of Fullerenol Nanoparticles on Rat Oocyte Meiosis Resumption

Runhong Lei et al. Int J Mol Sci. .
Free PMC article

Abstract

The excellent biocompatibility and biological effects of fullerenol and its derivatives make their biomedical application promising. The potential effects of fullerenol in mammals have been extensively studied, but little is known about its effects on female reproduction. Using canonical oocyte-granulosa cell complexes (OGCs) in vitro maturation culture model, we investigated the effect of fullerenol on the first oocyte meiotic resumption. In the surrounding granulosa cells, fullerenol nanoparticles occluded the extracellular domain of the epidermal growth factor receptor (EGFR) to reduce EGFR-ligand binding and subsequent extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation, which involved the regulation of connexin 43 (CX43) expression and internalization. Downregulation of CX43 expression and the retraction of transzonal projections (TZPs) interrupted the gap junction channel and TZPs based mass transportation. This effect decreased cyclic adenosine monophosphate (cAMP) levels in the oocyte and thereby accelerated rat oocyte meiosis resumption. Moreover, perinuclear distribution of CX43 and EGFR was observed in granulosa cells, which could further exacerbate the effects. Fullerenol nanoparticles interfered with the strict process of oocyte meiosis resumption, which likely reduced the oocyte quality.

Keywords: EGFR/ERK1/2; fullerenol; gap junction; granulosa cells; oocyte meiosis resumption.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Fullerenol accelerates transzonal projection (TZP) retraction. Freshly isolated oocyte-granulosa cell complexes (OGCs) were cultured in medium with different concentrations of fullerenol (0, 1, 10, and 100 μg/mL) for different times (0.5, 1, and 2 h). TZP number and integrity were evaluated using fluorescent labeling. (a) Representative images of the F-actin-rich TZPs between oocyte and surrounding granulosa cells are shown (red, scale bar: 20 μm). Blue color indicates granulosa cell nuclei; (b) Intact TZPs were counted and expressed as mean ± SEM (n = 5–6). Experiment was performed three times, * p < 0.05.
Figure 2
Figure 2
Fullerenol downregulates connexin 43 (CX43) expression. Oocyte-granulosa cell complexes (OGCs) were cultured in medium with different concentrations of fullerenol (0, 10, and 100 μg/mL) for 2 h, and then fixed for immunofluorescent staining or harvested after removal of oocytes for immunoblotting and polymerase chain reaction (PCR) analysis. (a) Reduced CX43 protein levels in granulosa cells. Green, CX43, Blue, nuclei; scale bar: 20 μm; (b) Quantitative analysis of CX43 fluorescence intensity normalized to nuclear fluorescence intensity. Data are mean ± standard error of the mean (SEM, n = 6–9 OGCs); (c) Immunoblotting detection of CX43 protein and (d) mRNA levels in granulosa cells after 2 h fullerenol treatment. Data are mean ± standard deviation (SD). At least three individual experiments were performed. The mean value was presented in the quantitative image. GF, green fluorescence intensity (CX43). BF, blue fluorescence intensity (nuclei). Each experiment was performed at least three times with similar results; * p < 0.05 compared with the indicated group.
Figure 3
Figure 3
Fullerenol induces connexin 43 (CX43) perinuclear distribution. Freshly isolated granulosa cells were routinely cultured for 4 days for attachment and were then treated with 100 μg/mL fullerenol for 2 h. Cellular distribution of CX43 was evaluated. (a) Immunofluorescent analysis of CX43 (green, scale bar: 20 μm). Fullerenol treatment caused CX43 perinuclear distribution compared with the control group (indicated by white arrow). The white squares indicate the distinctive cells and are zoomed. Blue and red color indicate nuclear staining and F-actin skeleton, respectively; (b) Percentage of granulosa cells with CX43 perinuclear distribution; * indicates the test statistic was greater than the critical value when the two percentages were compared.
Figure 4
Figure 4
Immunofluorescent analysis of cyclic adenosine monophosphate (cAMP) in oocytes (red, scale bar: 20 μm). Oocyte-granulosa cell complexes (OGCs) were cultured in M2 medium with different concentrations of fullerenol (0, 10, and 100 μg/mL) for 0.5 and 2 h and then fixed. (a) Representative images of cAMP in OGCs; (b) Quantitative analysis of intraoocyte cAMP fluorescence intensity (n = 7–14 oocytes). Blue and red color indicate nuclei staining and cAMP, respectively. Experiment was performed three times with similar results; * p < 0.05 compared with the indicated groups.
Figure 5
Figure 5
Fullerenol reduces extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation by blocking epidermal growth factor receptors (EGFRs). Freshly isolated oocyte-granulosa cell complexes (OGCs) were cultured in M2 medium with 100 μg/mL fullerenol (presented as C60) and 2 μg/mL EGFR antibody (presented as Ab) for 2 h, and then granulosa cells were harvested after removing oocytes following continuous stimulation with or without EGF (20 pg/mL, 10 min). (a) Fullerenol treatment (100 μg/mL) significantly reduced ERK1/2 activation in granulosa cells; (b) Fullerenol or extracellular domain targeted anti-EGFR antibody pretreatment (100 μg/mL for 2 h) reduced EGF-induced-ERK1/2 activation. Mean values are presented in the quantitative image. Ab, extracellular domain targeted anti-EGFR antibody. At least three individual experiments were performed. Data are shown as mean ± standard deviation (SD); * p < 0.05 compared with the control.
Figure 6
Figure 6
Fullerenol reduces extracellular domain targeted anti-epidermal growth factor receptor (EGFR) antibody binding. Freshly isolated oocyte-granulosa cell complexes (OGCs) were quickly fixed for immunofluorescent staining. During the standard procedure, OGCs were exposed to 100 μg/mL fullerenol for 10 min, and then gently washed before incubation with primary antibody. (a) Representative images of EGFR immunofluorescently labeled with an extracellular domain-targeted antibody; (b) Relative quantitative analysis of EGFR fluorescence intensity (n = 9–10 views) normalized to fluorescence intensity of nuclei. Green and blue indicate EGRF and nuclei, respectively. GF, green fluorescence intensity (EGFR). BF, blue fluorescence intensity (nuclei). Scale bar: 40 μm. Mean values are presented in quantitative image. Data are mean ± standard error of the mean (SEM); * p < 0.05 compared with the control.
Figure 7
Figure 7
Fullerenol changes distribution pattern of epidermal growth factor receptor (EGFR). Adherent granulosa cells or oocyte-granulosa cell complexes (OGCs) were treated with or without 100 μg/mL fullerenol for 2 h and then harvested or fixed. (a) Fullerenol treatment reduced EGFR membrane distribution in adherent granulosa cells (green, scale bar: 20 μm). White squares indicate distinctive cells and are zoomed. White arrows indicate membrane-distributed EGFR; (b) Portion of adherent granulosa cells with EGFR membrane distribution; (c) Expression of EGFR in granulosa cells was not significantly altered after 100 μg/mL fullerenol treatment for 2 h. Green, blue and red color indicate EGFR, nuclei staining and F-actin, respectively. At least three individual experiments were performed to detect EGFR levels.
Figure 8
Figure 8
Schematic representation of effects of fullerenol nanoparticles on oocyte meiosis resumption. Fullerenol nanoparticles occluded extracellular domain of epidermal growth factor receptor (EGFR) to inhibit ligand-EGFR binding-mediated extracellular regulated kinase 1 and 2 (ERK1/2) activation. Then, connexin 43 (CX43) expression was downregulated, resulting in TZP retraction (dashed arrows) and reduction of gap junction-based material transport, which decreased cAMP in oocytes. Moreover, fullerenol nanoparticles caused perinuclear distribution of CX43 and EGFR, which further exacerbated the effects. Black arrows mean the signal transduction of EGFR mediated ERK1/2 activation and black cross (X) means fullerenol’s inhibitory effect. Orange arrows indicate material flow from granulosa cells to oocyte via TZPs.

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