Perfluorooctanoic acid (PFOA) inhibits steroidogenesis and mitochondrial function in bovine granulosa cells in vitro

Abstract

Perfluorooctanoic acid (PFOA) is a persistent environmental contaminant. Due to the ubiquitous presence of PFOA in the environment, the impacts of PFOA exposure not only affect human reproductive health but may also affect livestock reproductive health. The focus of this study was to determine the effects of PFOA on the physiological functions of bovine granulosa cells in vitro. Primary bovine granulosa cells were exposed to 0, 4, and 40 μM PFOA for 48 and 96 h followed by analysis of granulosa cell function including cell viability, steroidogenesis, and mitochondrial activity. Results revealed that PFOA inhibited steroid hormone secretion and altered the expression of key enzymes required for steroidogenesis. Gene expression analysis revealed decreases in mRNA transcripts for CYP11A1, HSD3B, and CYP19A1 and an increase in STAR expression after PFOA exposure. Similarly, PFOA decreased levels of CYP11A1 and CYP19A1 protein. PFOA did not impact live cell number, alter the cell cycle, or induce apoptosis, although it reduced metabolic activity, indicative of mitochondrial dysfunction. We observed that PFOA treatment caused a loss of mitochondrial membrane potential and increases in PINK protein expression, suggestive of mitophagy and mitochondrial damage. Further analysis revealed that these changes were associated with increased levels of reactive oxygen species. Expression of autophagy related proteins phosphoULK1 and LAMP2 were increased after PFOA exposure, in addition to an increased abundance of lysosomes, characteristic of increased autophagy. Taken together, these findings suggest that PFOA can negatively impact granulosa cell steroidogenesis via mitochondrial dysfunction.

Keywords: Perfluorooctanoic acid; autophagy; bovine; granulosa cells; mitochondria; steroidogenesis.

Figure 1.. PFOA exposure inhibits steroid secretion by bovine granulosa cells.

Primary cultures of bovine granulosa cells were incubated for 48 h or 96 h with increasing concentrations of PFOA (0, 4 and 40 μM). Progesterone (P4) secretion (Panel A) in conditioned media and estradiol (E2) secretion (Panel B) in conditioned media following 48 h incubation. P4 secretion (Panel C) in conditioned media and E2 secretion (Panel D) in conditioned media following 96 h incubation. Results are percent of control means ± SEM, n = 4. **P < 0.01; ***P <0.001; ****P <0.0001 compared to control.

Figure 2.. PFOA inhibits the expression of genes required for steroid production by granulosa cells.

Primary cultures of bovine granulosa cells were incubated for 48 h or 96 h with increasing concentration of PFOA (0, 4 and 40 μM). mRNA was collected and prepared for RT-PCR. Quantification of (Panel A) STAR, (Panel B) CYP11A1, (Panel C) HSD3B, and (Panel D) CYP19A1 mRNA expression following 48 h exposure to PFOA. Quantification of (Panel E) STAR, (Panel F) CYP11A1, (Panel G) HSD3B, and (Panel H) CYP19A1 mRNA expression following 96 h exposure to PFOA. The results are average relative fold-changes compared to controls and presented means ± SEM, n = 4. *P < 0.05; **P < 0.01.

Figure 3.. PFOA inhibits the expression of proteins required for steroid production by granulosa cells.

Primary cultures of bovine granulosa cells were incubated for 48 h or 96 h with increasing concentration of PFOA (0, 4 and 40 μM). Protein lysates were collected and subject to Western blotting. (Panel A) Representative western blot of enzymes associated with steroid synthesis following 48 and 96 h exposure to PFOA. Quantification of STAR (Panel B), CYP11A1 (Panel C), HSD3B (Panel D), and CYP19A1 (Panel E) protein expression following 48 h exposure to PFOA. Quantification of STAR (Panel F), CYP11A1 (Panel G), HSD3B (Panel H), and CYP19A1 (Panel I) protein expression following 96 h exposure to PFOA. Results are averages of the fold-changes compared to control in each experiment. Data are shown as means ± SEM, n = 4. **P < 0.01; ***P <0.001; ****P <0.0001.

Figure 4.. PFOA does not stimulate proliferation of bovine granulosa cells.

Primary cultures of bovine granulosa cells were incubated for 48 h or 96 h with increasing concentrations of PFOA (0, 4 and 40 μM). Cell counts were performed by Trypan blue staining to determine effects of PFOA on proliferation. Cell counts following 48 h of PFOA exposure (Panel A) and following 96 h of PFOA exposure (Panel B). (Panel C) Representative micrographs showing nuclear DNA staining using DAPI (blue) and mitochondria staining (white) as described in the Methods. Concentrations of PFOA (top to bottom); incubation time (left to right). Quantification of labelled DNA following 48 h PFOA exposure (Panel D) and following 96 h PFOA exposure (Panel E). Micron bar represents 20 μm. Results are expressed as means ± SEM, n = 3

Figure 5.. PFOA exposure inhibits metabolic activity of bovine granulosa cells.

Primary cultures of bovine granulosa cells were incubated for 48 h or 96 h with increasing concentration of PFOA (0, 4 and 40 μM). MTT assays were performed to determine cellular metabolic activity. Results are expressed as average percent of control in each experiment. Data are represented as means ± SEM, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6.. Treatment with PFOA decreases mitochondrial membrane potential in bovine granulosa cells.

Bovine granulosa cells were treated with increasing concentrations of PFOA (0, 4, or 40 μM) for 48 h and 96 h and subject to confocal microscopy. (Panel A) Representative micrographs showing the effects of PFOA on mitochondrial membrane potential as measured by JC-1. From left to right; 48 h exposure to PFOA (merge of red and green; panels a, c, and e) and 96 h exposure to PFOA (merge of red and green; panels b, d, and f). Quantitative analyses of the mean fluorescence intensity of JC-1 red/green following 48 h exposure to PFOA (Panel B) and following 96 h exposure to PFOA (Panel C). Micron bar represents 20 μm. Results are means ± SEM, n = 3. ****P <0.0001.

Figure 7.. PFOA exposure stimulates the expression of mitophagy associated protein PINK1 granulosa cells.

Primary cultures of bovine granulosa cells were incubated for 48 h or 96 h with increasing concentration of PFOA (0, 4 and 40 μM). Protein lysates were collected and subject to Western blotting. (Panel A) Representative western blot of PINK1 expression following 48 h exposure to PFOA. (Panel B) Quantification of PINK1 protein expression following 48 h treatment with PFOA. (Panel C) Representative western blot of PINK1 expression following 96 h exposure to PFOA. (Panel D) Quantification of PINK1 protein expression following 96 h treatment with PFOA. Results are average relative fold-change means ± SEM, n = 4. *P < 0.05.

Figure 8.. Treatment with PFOA stimulates the production of reactive oxygen species (ROS) in bovine granulosa cells.

Bovine granulosa cells were treated with increasing concentrations of PFOA (0, 4, or 40 μM) for 48 h and 96 h and subject to confocal microscopy. (Panel A) Representative micrographs showing the effects of PFOA on ROS production as measured by CellROX. From left to right; Merge of Mitochondria, DNA, and CellROX (48 h; panels a, e, and i), CellROX (48 h; panels b, f, and j), Merge of Mitochondria, DNA, and CellROX (96 h; panels c, g, and k), and CellROX (96 h; panels d, h, and l). (Panels B, C) Quantitative analyses of the mean fluorescence intensity of CellROX following 48 h exposure to PFOA (Panel B) and following 96 h exposure to PFOA (Panel C). Micron bar represents 20 μm. Results are means ± SEM, n = 3. ****P <0.0001.

Figure 9.. Treatment with PFOA stimulates the activation of autophagy and accumulation of lysosomes in bovine granulosa cells.

Bovine granulosa cells were treated with increasing concentrations of PFOA (0, 4, or 40 μM) for 48 h and 96 h and subject to live-cell imaging using a confocal microscope. (Panel A) Representative micrographs showing the effects of PFOA on activation of autophagy (CytoID, green) and lysosomal accumulation (Lysotracker, red) following 48 h exposure. From left to right; Lysotracker (panels a, d, and g), CytoID (panels b, e, and h), Merge of Lysotracker with CytoID (panels c, f, and i). (Panel B) Quantitative analyses of the mean fluorescence intensity of CytoID following 48 h exposure to PFOA. (Panel C) Quantitative analyses of the mean fluorescence intensity of Lysotracker following 48 h exposure to PFOA. (Panel D) Representative micrographs showing the effects of PFOA on activation of autophagy (CytoID green reagent) and lysosomal (Lysotracker) accumulation following 96 h exposure. From left to right; Lysotracker (panels a, d, and g), CytoID (panels b, e, and h), Merge of Lysotracker with CytoID (panels c, f, and i). (Panel E) Quantitative analyses of the mean fluorescence intensity of CytoID following 96 h exposure to PFOA. (Panel F) Quantitative analyses of the mean fluorescence intensity of Lysotracker following 96 h exposure to PFOA. Micron bar represents 20 μm. Results are means ± SEM, n = 3. *P < 0.05, ****P <0.0001.

Figure 10.. PFOA triggers the expression of proteins involved in autophagy initiation.

Primary cultures of bovine granulosa cells were incubated for 48 h or 96 h with increasing concentration of PFOA (0, 4 and 40 μM). Protein lysates were collected and subject to Western blotting. (Panel A) Representative western blot of proteins associated with autophagy initiation following 48 h exposure to PFOA. Quantification of phospho-ULK1 (Ser555) and total ULK1 ratio (Panel B) and LAMP2 (Panel C) protein expression. (Panel D) Representative western blot of proteins associated with autophagy initiation following 96 h exposure to PFOA. Quantification of phospho-ULK1 (Ser555) and total ULK1 ratio (Panel E) and LAMP2 (Panel F) protein expression. Results are average relative fold-change means ± SEM, n = 3. ^#P<0.1; *P <0.05; **P < 0.01.

Figure 11.. Schematic model of how PFOA may inhibit steroid hormone synthesis in bovine granulosa cells.

PFOA increases cellular levels of reactive oxygen species (ROS) and decreases mitochondrial membrane potential (ΔΨM), potentially impacting the enzymatic activity of the cholesterol side chain cleavage enzyme CYP11A1 and the aromatase enzyme CYP19A1. These changes result in reductions in progesterone and estradiol synthesis, respectively.