The interactive effects of cytoskeleton disruption and mitochondria dysfunction lead to reproductive toxicity induced by microcystin-LR

Abstract

The worldwide occurrence of cyanobacterial blooms evokes profound concerns. The presence of microcystins (MCs) in waters and aquatic food increases the risk to human health. Some recent studies have suggested that the gonad is the second most important target organ of MCs, however, the potential toxicity mechanisms are still unclear. For a better understanding of reproductive toxicity of MCs on animals, we conducted the present experimental investigation. Male rats were intraperitoneally injected with MC-LR for 50 d with the doses of 1 and 10 µg/kg body weight per day. After prolonged exposure to MC-LR, the testes index significantly decreased in 10 µg/kg group. Light microscope observation indicated that the space between the seminiferous tubules was increased. Ultrastructural observation showed some histopathological characteristics, including cytoplasmic shrinkage, cell membrane blebbing, swollen mitochondria and deformed nucleus. Using Q-PCR methods, the transcriptional levels of some cytoskeletal and mitochondrial genes were determined. MC-LR exposure affected the homeostasis of the expression of cytoskeletal genes, causing possible dysfunction of cytoskeleton assembly. In MC-LR treatments, all the 8 mitochondrial genes related with oxidative phosphorylation (OXPHOS) significantly increased. The reactive oxygen species (ROS) level significantly increased in 10 µg/kg group. The mitochondria swelling and DNA damage were also determined in 10 µg/kg group. Hormone levels of testis significantly changed. The present study verified that both cytoskeleton disruption possibly due to cytoskeletal reorganization or depolymerization and mitochondria dysfunction interact with each other through inducing of reactive oxygen species and oxidative phosphorylation, and jointly result in testis impairment after exposure to MC-LR.

Figure 1. Effect of MC-LR on testis index rats in comparison to control rats.

Figure 2. Testis microstructures of rats exposed to MC-LR by intraperitoneal injection at a dose of 1 or 10 µg/kg/day for 50 d (stained with H&E).

(A) Control group (100×). (B) Control group (400×). (C) The 1 µg/kg/day group (100×), showing the enlarged spaces between the seminiferous tubules (gray arrow), enlargement of the lumen of the seminiferous tubules (white arrow), and blockage in seminiferous tubules (black arrow). (D) The 1µg/kg/day group (400×), showing the enlargement of the lumen of the seminiferous tubules (white arrow) and blockage in seminiferous tubules (black arrow). (E) The 10 µg/kg/day group, showing the enlarged spaces between the seminiferous tubules (gray arrow), enlargement of the lumen of the seminiferous tubules (white arrow), and blockage in seminiferous tubules (black arrow) (100×). (F) The 10 µg/kg/day group (400×), showing the blockage in seminiferous tubules (black arrow). Bar = 1000 µm (A, C, E) or 100 µm (B, D, F).

Figure 3. Toxic effect on testis ultrastructures of male rats treated with MC-LR.

(A) showing spermatogonia of control rat, 2500×. (B) showing the normal mitochondria, 10000×. (C) showing the normal nucleus, 10000×. (D) showing the shrank cell (asterisk), margination of chromatin (arrow), 2500×. (E) showing the condensation of chromatin (arrow), 2500×. (F) showing the swollen mitochondria (black arrow), margination of chromatin (white arrow), 2500×. (G) showing the swollen mitochondria, 10000×. (H) showing the dissolved nucleus membrane (black arrow) and margination of chromatin (white arrow), 10000×. (I) showing the blebbing of spermatogonia (white arrow), swollen mitochondria (black arrow), 2500×. (J) showing the shrank cell (asterisk), margination of chromatin (arrow), 2500×. (K) showing the condensation of chromatin (white arrow), swollen mitochondria (black arrow), 2500×. (L) showing the nuclear shape alteration (white arrow), condensation of chromatin (asterisk), swollen mitochondria (black arrow), 2500×. (M) showing the dissolved nucleus membrane, 2500×. (N) showing the swollen mitochondria (black arrow), condensation of chromatin (white arrow), 2500×, (O) showing the swollen mitochondria, 10000×. (P) showing the swollen mitochondria (black arrow), margination of chromatin (white arrow), 10000×.

Figure 4. MC-Expose induced swelling of male rat testis mitochondria.

(A) MPT was measured as a decrease in absorbance at a 520 nm wavelength (A₅₂₀) in isolated rat testis mitochondria. (B) A total change in A₅₂₀ over 30 min of mitochondrial swelling, **, p<0.01 versus control.

Figure 5. MC-induced mitochondrial DNA damage of rat testis.

Electrophoresis was carried out for 0.5 h in 1% agarose gel. DL 2000 DNA marker was served as molecular size standard. MtDNA were stained with GelGreen.

Figure 6. Transcriptional changes of cytoskeletal genes in the testis of male rats exposure to MC-LR compared with controls.

Quantitative real-time PCR was used to test the expression levels of cytoskeletal genes. GAPDH was used as an internal control. *, p<0.05 versus control, **, p<0.01 versus control.

Figure 7. Transcriptional changes of mitochondrial genes in the testis of male rats exposure to MC-LR compared with controls.

Quantitative real-time PCR was used to test the expression levels of mitochondrial genes. GAPDH was used as an internal control. *, p<0.05 versus control, **, p<0.01 versus control.