| With the advancement and development of science,a large number of chemicals are manufactured and continue to enter human life.People call this substance "environmental endocrine disruptors"(EDCs).EDCs,as a research hotspot in recent years,are one of the leading topics discussed by people.Because they are insoluble in water and not easily degraded,they can continuously migrate in various environments,and most EDCs will eventually enter the water environment,causing certain pollution to the water body.Fish,as a type of animal that exists in a large amount of water,live in the water environment for life,so its various organ systems can be interfered by EDCs.If they enter the human body through the food chain,the consequences will be immeasurable.At the same time,fish are very sensitive to the toxicity of EDCs.Therefore,studying the interference of EDCs on fish will provide some theoretical support for human health and safety from a long-term perspective.In this study,17α-methyltestosterone,which was mostly used in medicine in real life,was used as a toxic agent.It was used to change the body color of fish or to obtain a large number of economically valuable male fish.It is a very representative synthetic androgen.Pseudorasbora parva(P.parva)is used as the test object because it is cheap,easy to obtain,and more sensitive to EDCs.The experiment was divided into four parts.First,the obtained P.parva is reared in the laboratory for one month.This process can not only familiarize the P.parva with the laboratory environment,but also screen some unhealthy P.parva.In this step,the female and male fish can be separated according to the body characteristics of P.parva,and the sex ratio should be kept at 1: 1 as much as possible.Then we set up a control group,three exposure groups.The concentration of the exposure group is 50 ng/L,100 ng/L and 200 ng/L in order,and the domesticated P.parva will be randomly divided into these four groups of fish tanks,then respectively exposed for 7,14,and 21 days.The exposed P.parva were sampled,and their body length,body weight,and gonad weight were measured respectively.Part of the collected gonads were made by paraffin section,stained with H-E staining method,and histopathological analysis was performed under an electron microscope observed.The gonads collected in the other part were first extracted with Trizol RNA,then reverse-transcribed into c DNA,and then q RT-PCR was used to detect the expression of four genes that play a key role in t he synthesis of sex hormones.Each test group had 3 replicates to ensure the accuracy of the data.The sampled P.parva was then ground for whole fish,and the ground supernatant was subjected to protein extraction.First,the BCA protein concentration was measured as a calibration after measuring the hormone level,and then the ELISA method was used to determine and the level of VTG five sex hormones.At the same time,the control group and the 200 ng/L concentration group of P.parva gonads were selected for high-throughput sequencing to deeply understand the damage caused by MT.Combined with the results of previous sections,we can analyze the disturbing effects on the hypothalamus-pituitary-gonadal-liver axis of P.parva by MT.In this study,the effects of MT on the development of gonads,steroid hormones,and transcriptome of P.parva were experimentally investigated.It also provided a certain theoretical basis for P.parva as a sensitive material for acute toxicity tests.The main results obtained in this study are as follows:1 Analysis of gonad weight and GSI of w P.parva showed that the GSI and ovarian weight of females were significantly reduced(P <0.05)after 7 days of MT exposure.After MT exposure for 21 days at 100 ng/L and 200 ng / L concentrations,their body weight were significantly lower(P < 0.05)than the control group,indicating that MT inhibited normal ovarian growth.2 The results of H-E stained paraffin sections showed that after 7 and 14 days of exposure to 50 ng/L and100 ng/L MT,the number of Poc in immature cells in the ovary increased significantly,while the number of Voc in mature oocytes was significant reduction.With 14 and 21 days of 200 ng/L MT exposure,there was almost no Voc cells in the ovary,which showed that after MT exposure,the development of mature oocytes was inhibited,and the number of immature oocytes in the ovary increased.The results of male section showed that with increasing MT concentration and prolonged exposure time,the number of mature sperm SZ decreased gradually,and the proportion of immature spermatocyte S increased gradually.And after MT was exposed,the testis showed obvious vacuolization,especially at the high concentrations,which showed that MT not only inhibited the development of mature sperm,but also caused abnormalities in the testis.3 The overall performance of 11β-HSD2 showed an upward trend after MT exposed female P.parva.The m RNA expression of cyp11 b,cyp17a1,and cyp19a1 a genes changed significantly(P <0.05),and the trend gradually changed with the increase of exposure concentration and exposure time.In the P.parva testis,The m RNA expression of cyp11 b genes in the treatment group with MT was significantly down-regulated(P < 0.05),and the m RNA expression trends of cyp17a1 and cyp19a1 a genes were similar.This indicateed that females and males respond differently to MT interference,and different steroid synthesis-related gene expressions respond differently.This may be due to the differences in female and male endogenous estrogen androgen levels.4 For females,the levels of 6 hormones decreased after 7 days with the exposure to MT at 50 ng/L.With the increase of exposure time,the hormone content increased.When 100 ng/L MT exposed P.parva,the hormone content decreased significantly(P < 0.05),and the trend of 200 ng/L was similar.For males,the levels of the VTG and hormones decreased after 7 days of exposure to 50 ng/L MT,and the decline disappeared with the prolonged exposure time.After the exposure with 100 ng/L MT for 7 and 21 days,P.parva also showed a trend of first decline and then increase,indicating that MT significantly affects the synthesis of VTG and sex hormones and may be related to interference with the H-P-G-L axis.5 Four DGE libraries were constructed in this study,including two female fish groups(Ova-C and Ova-MT)and two male fish testis groups(Tes-C and Tes-MT).A total of 7,578 DEGs screened in the ovaries,of which 3,592 DEGs are up-regulated and 3,986 DEGs are down-regulated.These differential genes are involved in 695 GO enrichment projects and 165 KEGG pathways in females.A total of 11,543 DEGs were selected from the testis,of which 6,799 DEGs were up-regulated and 4,744 DEGs were down-regulated.Differential genes were involved in 774 GO enrichment projects and 192 KEGG enrichment pathways in males.In summary,the study has analyzed the effects of MT on the gonads of P.parva from different perspectives.These results fully showed that the interference of MT on the gonads of P.parva include the inhibition of the normal development of gonads,the key enzymes that disrupt the synthesis of hormones,and the H-P-G-L axis,which affects the synthesis of VTG and sex hormones.High-throughput sequencing results show that in addition to harming the gonads,MT also affects the normal regulation of more tissue systems,which provides more scientific basis for the study of MT. |
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