| In this study, we set BaP exposure concentration referred to the real pollution situation in china offshore. We selected the scallop Chlamys farreri as the research species to study the influences of BaP on steroid levels in gonad and haemolymph, its effects on DNA single-strand breaks, protein carbonyl content and MDA contents, screened differentially expressed genes under BaP stress. Finally, we verify scallop gonads toxicity by using microscopic tissue slicing technology. The experimental results were as follows:1. Screening and analysis of differentially expressed genes in scallop gonad under BaP stressThis research used digital gene expression (DGE) technology to screen the differentially expressed genes of scallop gonads between control and 0.5 ug/L BaP exposure group after exposure for 10 days. A total of 12,485,055 and 14,454,127 clean reads were generated from control and BaP exposure DGE libraries in testis of scallop, respectively. The matching rates were74.24% and 77.27% compared with the unigene of scallop transcriptome (SRP018044). After comparing two libraries,1,051 differentially expressed genes were detected, with 223 up-regulated and 828 down-regulated genes. GO function cluster analysis showed that the differentially expressed genes were mainly related to aromatic compound catabolic processes, ATP metabolism, immune defense, apoptosis and spermatid development. K.EGG pathway analysis showed a significant enrichment of metabolic pathways, including the krebs cycle pathways, oxidative phosphorylation pathway, NOD-like receptor signaling pathways and steroid synthesis pathway.9 differentially expressed genes (HSP90n CYP3A、HGD、CDK2、IAP、TSSK2、PDE5、17HSD、E2SULT) were performed by Quantitative real-time PCR to verify DGE. A total of 13,666,882 and 14,276,579 clean reads were generated from control and BaP exposure DGE libraries in ovary of scallop, respectively. The matching rates were 74.55% and 78.46%. After comparing two libraries,124 differentially expressed genes were detected, with 42 up-regulated and 82 down-regulated genes. Combing the overall analysis of the whole data in this study, the reproductive response changes of C. farreri exposed to BaP stress can be summarized as "detoxification process" "damage effects to extracellular matrix and cell interactions", "endocrine disrupting effects" as well as "tissue damage responses".From the above analysis, we can see that the detoxification metabolism, energy metabolism and immune defense function were changed in scallop gonads under BaP stress. BaP stress also induced apoptosis, interfered steroids (hormone precursors) synthesis and the formation of sperm. BaP stress changed the relative genes expression of detoxification metabolism, cytoskeleton and gonad development and resulted in cytoplasmic matrix loose, affected ovarian development and so on.2. Study of reproductive endocrine disrupting effects in scallop gonad under BaP stressIn this study, scallop was exposed to 0,0.1,0.5,2.5μg/L BaP, and sampled at 0, 1d, 3d,6d, 10d,15d and 21d. The contents of sex hormone in ovary, testis and hemolymph were detected by the Elisa kit. We researched the expression levels of differentially expressed genes related with cell adhesion, apoptosis, sex hormone synthesis and gonadal development using qRT-PCR technology and explored the expression pattern of key reproductive toxicity responsed genes under.more BaP concentration gradient and time points stress. Three hormones contents of scallop gonad and hemolymph were all affected under BaP stress. The three hormones contents were significantly decreased following exposure concentrations increase in the ovary tissue. The estradiol and testosterone contents increased at day 0-3, then decreased significantly, and finally recovered to the control level. While progesterone content was lower than the control group with all the experiment period. In the testis, BaP significantly inhibited the content of estradiol, but increased the contents of testosterone, which showed an androgenic effect, and different concentrations of BaP had different effects on progesterone. The progesterone could be increased by the low BaP concentrations, and suppressed by the high concentrations. The three hormones contents of male scallop hemolymph increased in each exposure group under BaP stress, and then recovered to significantly lower levels than the control group. The experimental results suggested that the changes of sex hormones level between hemolymph and gonad were inconsistent. In addition, the effects of BaP on the three hormones contents were also different in testis and ovary. Overall, BaP can significantly affect the estradiol, progesterone and testosterone contents in the male and female gonadal and scallop hemolymph, which showed strong endocrine disruptors effects of BaP. BaP also significantly influenced the expression level of detoxification damage response, gonadal hormone synthesis related and developmental related genes in scallop gonads, and different BaP exposure concentrations had the different effects. For the same gene, BaP showed different patterns of induction between the male and female scallop emerging its toxic effects and endocrine disrupting effects.3. Study of damage effects in scallop gonad under BaP stressIn this study, scallop was exposed to 0,0.1,0.5,2.5μg/L BaP, and sampled at 0, Id, 3d,6d, 10d,15d and 21d. We identified the effect of BaP to three nutrients by detecting alkaline unwinding F value, lipid and protein carbonyl levels LPO content. Studies have shown that the DNA single-strand breaks, protein carbonyl content, MDA contents of scallop ovary and testis were significantly influenced under BaP stress. In each exposure group, F value of scallop gonads decreased during the experiment, especially the 0.5 and 2.5μg/L BaP exposure group. During the exposure time, protein carbonyl content increased first and then decreased, and significantly higher than the control level at the end of the experiment. MDA contents of each groups in gonad increased during exposure peoriod, and the 2.5μg/L BaP exposure group MDA contents significantly higher than the control level at day 15. Our results suggested that different contents of BaP have significantly effect on DNA damage, lipid LPO and protein carbonyl. Varying of protein carbonyl contents showed an emergency and repair process and a significant increase of MDA at the end of experiment showed the detoxification response in scallop under BaP stress. BaP could result toxic effects in scallop gonad by oxidative damage. These toxic effects can be repaired following the extent of exposure time, and there are also differences between the different exposure groups.4. Effects of BaP on microstructure of C. farreri gonadIn this study, scallop was exposed to 0,0.1,0.5 and 2.5μg/L BaP, the testis and ovary tissue sections were made at day 15 and 20 to investigate the effects of BaP on scallop gonad microstructure. Compared with the control group, the interspace occurred and increased gradually in ovarian follicles of C. farreri with BaP exposure concentration increased. The oocytes were more loosely arranged and deformed. Until day 21, the interspaces between follicles and within oocytes were larger and some degenerating oocytes appeared to be severe deformation and surrounding follicular tissues were damaged as well as the nuclear membranes were obscure compared with the control. Similar responses occurred in the testis of C. farreri. The spaces between follicles became larger and the numbers of sperm decreased and arrangement without rules after BaP exposure. The above results demonstrate that BaP could influence the development of C. farreri gonad, delay the growth of germ cells in the follicular and poison the germ cells to deformation and disorderly arrangement. In addition, BaP could damage the follicular structure. Thus BaP showed the endocrine disrupting effects and tissue damage effects. |
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