| As a group of steroid hormones,progestins are widely used in oral contraceptives and hormone replacement therapy in humans to prevent pregnancy and treat many endocrine diseases.Moreover,progestins are also used to promote growth and fattening of animals in order to increase the animal production and economic benefits.Because of incomplete removal in wastewater treatment plants or direct excretion of animals or direct discharge of wastes,progestins have been found in different aquatic environments.The reported concentrations for various progestins ranged from a few of ng L-1 to tens of thousands of ng L-1 in aquatic environments.Because of their high bioactivity,there progestins may affect aquatic organisms such as fish.Some previous have studies showed toxicological effects of several progestins,for instance,the inhibition of egg production in fish,histopathological and alterations in gene expression.However,due to their different chemical structures,it is necessary to study the effects of different progestins on aquatic organisms.In addition,though increasing number of studies showed various adverse effects of progestins on the physiology,biochemistry and transcription,the exact mechanisms underlying these effects remain elusive.The thesis focused on the toxicological effect of three progestins?norgestrel,dydrogesterone and medroxyprogesterone acetate?in zebrafish at transcriptome,transcription and histology levels.The main findings in present study are described as follows:?1?The first experiment aimed to investigate the effect of norgestrel?NGT?on the toxicological mechanism of gonadal development in adult zebrafish.The effects of NGT on transcriptome and gonadal development in adult zebrafish were analyzed.Microarray analysis was used to enrich the Gene ontology?GO?and pathway after 14 days of exposure to 871 ng L-1.The microarray data revealed 11545 transcripts were identified.GO analysis showed that genes related to organ development,system development,multicellular organismal development,single-organism developmental process and developmental process were significantly enriched.Venn diagram displayed that 434 target genes involved in organ development were common in these five development related processes.Kyoto Encyclopedia of Genes and Genomes?KEGG?analysis showed that Notch signaling pathway was the most affected pathway and selected as the target pathway for further q PCR study at environmentally relevant concentrations(6.7 ng L-1,83 ng L-1 and 912 ng L-1).The q PCR analysis revealed significant and dose-dependent alterations of most target genes involved in the Notch signaling pathway in the gonads,even at an environmentally relevant concentration of 6.7 ng L-1.The transcriptional patterns were consistent with the Notch signaling cascade.In addition,NGT significantly increased the frequency of mature sperms and decreased the frequency of immature sperms at all concentrations.Meanwhile,NGT treatment at 912 ng L-1 increased the percentage of mature vitellogenic oocyte and atretic follicles,but decreased the percentage of immature vitellogenic oocyte.Thus this experiment demonstrated significant developmental toxicity in the gonad of adult zebrafish even at environmentally relevant NGT concentrations.?2?The second experiment aimed to investigate the toxicological effect of dydrogesterone?DDG?in adult zebrafish after a short term exposure.The effects of DDG on transcriptional expression of target genes involved in hypothalamic-pituitary-gonadal?HPG?and Notch pathway and gonadal development in adult zebrafish were analyzed.The results showed that DDG strongly increased the transcriptional expression of target genes involved in the gonadotropin–releasing hormone?Gn RH?pathway in females even at environmentally relevant concentrations,while significantly influenced the transcriptional expression of steroidogenesis pathway related genes in males.The transcriptional levels of these cascade genes?gnrh2,gnrhr4,atf4 b,lhb and fshb?were consistent with the corresponding cascade response in Gn RH pathway after DDG exposure.Furthermore,the transcriptional alterations of Gn RH pathway and steroidogenesis pathway related genes induced by DDG were different between females and males.Meanwhile,a significant increased frequency of atretic follicles was observed in the ovary at 2486 ng L-1.In addition,DDG short exposure had no significant effect on the transcriptional expression of notch pathway,which was consistent with the histological changes.The above VI findings showed that DDG exposure could cause transcriptional alterations at environmentally relevant concentrations and histrionic damage at higher concentration.?3?The third experiment aimed to investigate the toxicological effect of dydrogesterone in zebrafish after a long term exposure?20 dpf to 144 dpf?at low concentrations(4 ng L-1,39 ng L-1 and 379 ng L-1).The effects of DDG on sex differentiation,gonadogenesis and transcriptome were analyzed.The results showed DDG affected the sex ratio,and caused the male bias at 379 ng L-1 compared with the solvent control.Exposure to 379 ng L-1 DDG increased the transcriptional expression of dmrt1,casp1 b,casp3a and casp9,but decreased the transcriptional expression of cyp19a1 a.The transcriptional alteration of circadian rhythm network was observed in brain of both male and female at all concentrations,and might influence the metabolism of steroid hormone and DNA repair after DDG exposure.In the testis,microarray analysis displayed spermatogenesis that related GOs?endothelial barrier and immune response?was significantly predicted at all doses.Importantly,phagosome,lysosome and sphingolipid metabolism pathways involved in sperm maturation were predicted to be upregulated in the testis at high dose,which explained the increased frequency of mature spermatids.In the ovary,the upregulation of casp3 a gene suggested that DDG caused the oocyte apoptosis,which was consistent with the increased frequency of atretic follicles.The transcriptional expression of target genes involved in major histocompatibility complex class I?mhc1ufa and mhc1uea?was increased at 39 ng L-1.Mhc1 ufa and mhc1 uea always express on the surface of regression organization,which induced phagocytosis.The enhanced expression of mhc1 ufa and mhc1 uea suggested that the ovarian follicle were clearance and engulfment by phagocytic cell.Taking together,DDG displayed strong androgen effect,which may lead to ecological risk of reproductive development and even population population dynamics.?4?The fourth experiment aimed to investigate toxicological effect of medroxyprogesterone acetate?MPA?in zebrafish after a long term exposure?20 dpf to 144 dpf?at low concentrations(4.3 ng L-1,43 ng L-1 and 408 ng L-1).The effects of MPA on sex differentiation,gonadogenesis and transcription were analyzed.The results showed that MPA had a weak androgenic effect,causing sex differentiation at 43 ng L-1.Meanwhile,MPA exposure increased transcriptional expression of hsd17b3 and dmrt1 at 35 dpf.Furthermore,MPA also affected transcriptional expression of HPG axis related genes,resulting in the dysregulation of HPG axis.However,slight effect was observed in gonad.Weak effect on transcriptional expression of notch pathway related genes was also observed in gonad,which was consistent with the histological changes after a long term exposure.However,exposure to 43 ng L-1 increased the frequency of immature spermid,which was different compared with norgestrel and dydrogesterone.MPA exposure increased the transcriptional expression of dmrt1 at 43 ng L-1.The dmrt1 is important for testis differentiation.Therefore,MPA might enhance the sperm division by affecting the transcriptional expression of dmrt1.In addition,MPA exposure could cause dysregulation of circadian rhythm.The transcriptional change of circadian rhythm related genes was also not consistent to the circadian rhythm cascade.This study showed toxicity of MPA in zebrafish,causing a wide range of responses.In summary,NGT,DDG and MPA exposure could significantly affect the zebrafish at transcription and histology levels,and had potential risk for the aquatic organisms. |
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