| Due to the rapid development of the mining, power generation and metal smelting and other modern industry, pollution incidents especially heavy metals cadmium often occur in recent years. Cadmium pollution often caused severe damage in the local biological populations and ecosystems. Studies found that cadmium stress can affect material, energy and other metabolic processes, or even seriously affect the reproduction and oogenesis of organism. Accumulation of yolk proteins is one of the major changes of crustacean ovarian development, and the formation and accumulation of vitellin (Vn) is main features during oocyte maturation. Vitellogenin (Vg) can be further modified by the formation and processing of lipid and carbohydrate to Vn, and accumulate in the yolk body. Under the stress from external environment, lipid and other substances accumulated in the organism would be consumed to produce sufficient energy for the synthesis of metallothionein (MT) to relieve external toxicity. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) as an important reducing power in organism, not only participating in the maintaining the balance of glutathione system to alleviate the external stress but also involving in the process of lipid biosynthesis.Basis on our previous studies, effects of cadmium on lipid metabolism and reproductive development mechanism of Sinopotamon henanense was studied. Meanwhile, the biochemical and reproductive parameters was explored as the important indicators for the studies of the impact of pollutants on biological populations and scientific basis. The experimental methods included the subchronic exposure, colorimetry, transmission electron microscopy, immunology and gel filtration chromatograph. These biochemical indicators include lipid content, hemolymph triglyceride (TG), ATP/ADP, NADH/NAD, mitochondrial membrane potential (Δψm) levels, NADPH/NADP+; lipid digestion metabolism related pancreatic lipase (PL), lipid transfer associated lipoprotein lipase (LPL), lipid synthesis and metabolism involved in acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS); mitochondrial energy metabolism associated Glucokinase (GK), NAD+ -isocitrate dehydrogenase (NAD+-IDH), cytochrome C oxidase (COX) activities, and IDH and COX mRNA expression; NADPH-dependent enzyme synthesis:Glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphate dehydrogenase:(6PGDH), malic enzyme (ME), NADP+dependent isocitrate dehydrogenase (NADP+-IDH); glutathione system:reduced glutathione (GSH) and oxidized glutathione (GSSG) content, GSH/GSSG, glutathione reductase (GR) activity, and levels of MT and lipid peroxidation index (malondialdehyde, MDA). Using transmission electron microscopy (TEM), changes of cadmium on mitochondrial, endoplasmic reticulum and other microscopic structures of Sinopotamon henanense were observed. By transcriptomics, the sequence of Vg was obtained, and also was analyzed by the bioinformatics methods. Based on the sequence of Vg, phylogenetic tree of S. henanense was constructed. Moreover, the method of vitellin (Vn) and Vn-ELISA was studied and established. Vn was purified from mature ovaries by gel filtration chromatography. Using SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight and the quantity of Vn subunit were determined. Based on the purified Vn and Vn anti-serum, the reaction parameters for an enzyme-linked immunosorbent assay (ELISA) was developed. Finally, Vn-ELISA and real-time quantitative PCR (RT-PCR) were used to detect possible effects of cadmium on Vn content, Vg mRNA expression, carbohydrate and protein metabolism in hepatopancreas and ovary.The Results had shown that:First, with significantly increased Cd accumulation in exposed crabs, lipid content in hepatopancreas and ovary showed a time-dependent and concentration-dependent reduction, being at least one of the reasons for a lower ovarian index (OI) and hepatopancreatic index (HI).10-day exposure increased TG level in hemolymph and PL activity of the hepatopancreas, suggesting an increased nutritional lipid uptake. With increasing exposure time, the crabs at 15 and 20-day exposure contained less lipid and TG, suggesting that more energy was consumed during the exposure time. Meanwhile, the level of NADPH, ATP and the activity of PL, LPL, FAS, ACC was down-regulated suggesting an impairment of the crab metabolism by Cd in addition to causing a lower lipid level.Second, an experimental 10-day exposure increased ATP, ADP and provided a higher ATP/ADP ratio in cells of Sinopotamon henanense. Corresponding significantly increased levels of NADH, NAD+, NADH/NAD+ ratios and Δψm in hepatopancreas and ovaries supported high energy production. With increasing exposure time, however, energy production was in decline. Excessive energy consumption was explained by mitochondrial impairment. Ultrastructurally, there were changes in mitochondria and endoplasmic reticulum with swelling, membrane disruption, shortening of cristae or the rupture and disappearance of entire mitochondria, leading the decreased NADH, Δψm and ATP levels.Third, the results show that G6PDH,6PGDH, ME and NADP-IDH activity were induced by cadmium in early treatment of cadmium. Enhanced pentose phosphate pathway (PPP) led to higher NADPH synthesis metabolism, and activity of GR was up-regulated compared to control group, suggesting that the glutathione redox recycling pathway was enhanced and the GSH synthesis metabolism was accelerated. With Cd exposure, NADPH and GSH synthesis metabolism was weakening. After 20-exposure, the activity of NADPH-dependent enzymes were inhibited, leading to the GSH synthesis caused the reduction and depletion of GSH. Lower MT level showed reduced capacity of Cd chelation in S. henanense, and higher MDA level suggested that the crabs was under a serious state of oxidative stress during Cd exposure. The results showed that short-term Cd treatment induced the NADPH synthesis, and enhanced generation of intracellular reducing power. Moreover, GSH synthesis rate increased, and intracellular antioxidant capacity and anti-cadmium stress was increased. However, long-term Cd treatment could inhabit generation of reducing power, and caused reduced glutathione level, weakened antioxidant capacity and affected lipid biosynthesis of crabs.Fourth. the results showed that the length of Vg is about 7764 bp, and the open reading frame is 7701 bp which could encode about 2566 the amino acid. Moreover, Vn was composed of three subunits (102、84 and 70KD). According to these optimal parameters, the standard linear equation was established for the determination of Vn concentration with a valid range of 125-2000 ng/mL, and qualitative detection can be 15.62 ng/mL. In verification experiments, Vn content in the ovaries showed no significant differences, and the mean coefficients of variation of inter-assay were 7.81%, suggesting that the developed ELISA of this study was precise, stable and repeatable. Moreover, Cd decreased the diameter of the oocyte, and caused a time-dependend and dose-dependend down-regulation of Vg mRNA and Vn level. The lower levels of carbohydrates and proteins in exposed crabs were also observed compared to the controls, and the activities of alanine aminotransferase and aspartate aminotransferase increased, indicating increased demand for energy to reduce cadmium stress.Conclusions:One, cadmium could affect lipid storage and metabolism in hepatopancreas and ovary of Sinopotamon henanense. Short-term Cd exposure can increase related enzymes activities causing strengthening of lipid digestion and lipid synthesis metabolism. However, with prolonged exposure time, cadmium can significantly inhibit lipid digestion, lipid transport and lipid synthesis process, leading to the lipid damage and negatively affecting hepatopancreas and ovary development.Two, short-term Cd exposure had a significant impact on the S. henanens energy metabolism in the hepatopancreas and ovary cells, showing the stress response. It benefited with fatty acids synthesis to alleviate damage of Cd-induced lipid peroxidation in cell membrane. With prolonged exposure time, the structure of mitochondria and rough endoplasmic reticulum of S. henanens was damaged seriously. The activity and mRNA expression of IDH, COX was inhibited, and then affecting the respiratory chain and synthesis of ATP.Three, NADPH synthesis of S. henanens was enhanced after short-term Cd exposure, which induce the generation of reducing power and faster GSH synthesis, suggesting the increased intracellular antioxidant capacity and anti-Cd stress. However, the long-term Cd exposure will inhibit tissue reducing power generation, and also cause lower glutathione content, weaken antioxidant levels, affecting the lipid biosynthesis of S. henanens.Four, the results suggested that inhabitation of Cd on the Vg mRNA express and cause decreased carbohydrates, proteins, leading the lower level of Vn in S. henanense, suggesting that Cd slows down oocyte maturation and vitellogenesis in S. henanense.Five, these biological effects can be used as an important basis for the further study of crabs reproductive regulation and long-term evaluation of environmental cadmium pollution. |
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