Study On Reproductive Toxicity Effects Of Benzo[α] Pyrene On Scallop Chlamys Farreri

Polycyclic Aromatic Hydrocarbons (PAHs) are the typical persistent organic pollutions and have the ability of endocrine disruption. Lately, the PAHs pollution condition has been becoming worse because of exploitation of petroleum, development of steamship transportation and outlet of anthropogenic contaminants. The studies on reproductive toxicity effects of PAHs on bivalves, which are widely employed as sentinel organisms to monitor aquatic pollution, are limited. In this study, B[α]P was used as the representative of PAHs to investigate its influences on steroid contents, reproduction related gene expression and histological damage of Chlamys farreri gonad and the reproductive toxicity mechanism was discussed. The following results were obtained:1 Effects of B[α]P on testosterone and 17β- estradiol contents in C. farreri gonad Steroids play an important role in organism's growth, development, reproduction and other physiological processes. In this study, scallops were exposed to different concentrations of B[α]P (0.1μg/L, 0.5μg/L, 2.5μg/L) for 15 days, and sampled on day 6 and day 15. Radio immunoassay (RIA) was used to detect the contents of testosterone and 17β- estradiol in scallop gonads. The results show that B[α]P can reduce the contents of testosterone and 17β- estradiol in gonads. The influences of all the B[α]P treatment groups on the steroids contents in spermary and ovary are not significant on both 6d and 15d (P>0.05), except the 2.5μg/L B[α]P treatment group on 15d which reduce the 17β- estradiol content in ovary significantly (P<0.05). It can be drawn from the experiment that B[α]P can exert its reproductive toxicity effects by reducing the contents of testosterone and 17β- estradiol.2 Cloning and sequence analysis of estrogen receptor (ER) in C. farreri ovary The degenerate primers were designed from ER protein multiple sequence alignments from related species. The complete ER cDNA sequence was obtained through the technology of RT-PCR and RACE. The results show that ER is highly conserved when compared among species, especially the DNA binding domain (DBD). The identity of C. farreri ER DBD compared with Crassostrea gigas ER DBD is 98%, and 87% when compared with homo ERαDBD. The ligand binding domain (LBD) of ER is not as conserved as DBD among species. And it can be drawn from the experiment that ER mRNA expresses in all tissues tested and is highly expressed in ovary.3 Cloning and sequence analysis of vitellogenin (VTG) in C. farreri ovaryThe codehop web site is used to design the degenerate primers depending on the VTG amino acid in related species. Partial cDNA sequence was obtained through the technology of RT-PCR and RACE. The VTG identity of C. farreri is higher when compared with species in the same family, which is 78%-89%. When compared with species in the same order, the VTG identity is 36%-43%, but is much lower when compared with other invertebrate and oviparous vertebrates, which is only 21%-33%. VTG mRNA expresses only in C. farreri ovary, which shows that VTG expression in C. farreri is female specific.4 Effects of B[α]P on ER and VTG mRNA expression in C. farreri ovaryScallops were exposed to different concentrations of B[α]P (0.1μg/L, 0.5μg/L, 2.5μg/L) for 15 days, and sampled on 1d, 3d, 6d, 10d and 15d. The technology of RT-PCR was used to detect ER and VTG mRNA expression. The results show that both gene expressions in treatment groups are a little higher than the control level on 6d, which are not significant, afterwards decrease and are significantly lower than the control level in 2.5μg/L B[α]P on 15d (P<0.05). During the experiment, VTG expression is not detected in spermary and ER expression of spermary in treatment groups have no significant difference with the control level. It can be concluded that B[α]P exerts its antiestrogenic activity by reduce the expression of estrogen responsed gene.5 Effects of B[α]P on microstructure of C. farreri gonadThe quality of gonad is the key factor for reproduction. In this experiment, C. farreri were exposed to to 2.5μg/L B[α]P for 15 days and the microstructure of C. farreri gonad was observed. The results show that B[α]P can delay the maturation of spermary and ovary of C. farreri. And ovaries in B[α]P treatment groups display numerous degenerating ovarian follicles and the connective tissues contain numerous haemocytes in the periphery of the follicles indicative of dramatic toxic effects of B[α]P.