| The steroid hormones present in the aquatic environment have drawn a global attention as they have the high biological activities. As an important group of steroid hormones, progestins are widely used in oral contraceptives and hormone replacement therapy in humans, in order to prevent pregnancy and treat all kinds of endocrine diseases. In addtion, progestins are often used for growth promoting and fattening of animals, as well as controlling synchronization of ovulation and prevention of female abortion in the livestock industry, in order to improve the animal production and economic benefits. Progestins occur in the aquatic environment(e.g., wastewater treatment plant effluents, animal farm wastewaters and surface water) due to their incomplete removal in wastewater treatment plants and direct excretion of animals and direct discharge of wasters. And their concentrations ranged from a few of ng L-1 to tens of thousands of ng L-1. Environmental residues of progestins may pose potential risks to aquatic organisms in the aquatic environment. However, so far, very little attention has been paid to ecotoxicological effects and potential environmental health risk on fish caused by progestins, and in combination with estrogens. Therefore, the present study, with its focus on the endocrine disrupting effects of selected two progestins, i.e. progesterone(P4) and norgestrel(NGT), on the embryonic, juvenile and adult zebrafish(Danio rerio), and then investigated the effects of binary mixtures of P4 with estradiol(E2), and NGT with ethinylestradiol(EE2) on the embryonic zebrafish. The main findings from this study are given as follows:1. The aim of this study was to investigate the effects of P4(6 ng L-1, 45 ng L-1 and 90 ng L-1) and NGT(6 ng L-1, 45 ng L-1 and 87 ng L-1) on the transcriptional expression levels of genes related to hypothalamic-pituitary-gonadal(HPG), hypothalamic-pituitary-adrenal(HPA) and hypothalamic-pituitary-thyroid(HPT) axes in the early stage of zebrafish. For the receptor signaling pathways, P4 and NGT significantly inhibited the expression of Pgr messenger(m)RNA mainly at 96 h post fertilization(hpf) for all treatment groups. However, a significant up-regulation of Vtg1 m RNA was observed at 6 ng L-1 P4 and NGT or higher at different time points. For the steroidogenic pathways, the transcriptional expression of Cyp11a1 and Hsd17b3 m RNAs was mediated by 6 ng L-1 P4 and NGT or higher according to different exposure time points. In addition, P4 and NGT mainly resulted in a significant induction of Cyp19a1 a and Cyp11 b m RNA expression while it caused a significant inhibition of Hsd11b2 m RNA expression above 6 ng L-1. For the hypothalamic and pituitary hormones, P4 and NGT mainly modulated the transcripts of Gnrh2, Fshb and Lhb genes at 6 ng L-1 or higher. Furthermore, a strong induction of Nis m RNA by P4 and NGT was observed in zebrafish embryos-larvae, but the effects of P4 and NGT on other genes related to HPT axis were generally low. The overall results from the present study indicate that P4 and NGT at environmentally relevant concentrations could cause the potential effects on zebrafish reproductive, adrenal and thyroid endocrine systems by interfering with the HPG, HPA and HPT axes.2. The aim of this study was to investigate the effects of P4(4 ng L-1, 33 ng L-1 and 63 ng L-1) and NGT(4 ng L-1, 34 ng L-1 and 77 ng L-1) on the sex differentiation of zebrafish by measuring the sex ratio and transcriptions of genes related to sex differentiation(Amh, Dmrt1, Figa, Sox9 a and Sox9 b genes) as well as sex hormone levels and transcriptional expression profiles along the HPG, HPA and HPT axes in juvenile zebrafish. The results showed that exposure to P4 caused a significant increase in proportion of females as well as significant down-regulation of Amh gene and up-regulation of Figa at a concentration of 63 ng L-1. However, the shift in the sex ratio toward males was observed following exposure to 34 ng L-1 and 77 ng L-1 NGT, which came along with the significant induction of Dmrt1 gene and inhibition of Figa gene. The sex hormones in exposed fish were measured with estrone being detected only in the fish exposed to the highest P4 concentration; whereas estradiol and androstenedione were detected only in the fish of the control and lowest NGT concentration. Furthermore, the increase in females was associated with the significant up-regulation of several key genes controlling the synthesis of sex hormones(i.e., Cyp17, Cyp19a1 a and Hsd3b) following exposure to 63 ng L-1 P4 whereas the significant down-regulation of Cyp11a1, Cyp17, Cyp19a1 a and Hsd3 b genes was observed in the male-biased populations caused by 34 ng L-1 and 77 ng L-1 NGT. The overall results imply that both P4 and NGT could significantly affect sex differentiation in zebrafish, and that changes may be reflected by altered sex hormone levels and transcriptional expression profiles of genes related to synthesis of sex hormones.3. The aim of this study was to investigate the effects of P4(2 ng L-1, 11 ng L-1 and 16 ng L-1) and NGT(6 ng L-1, 29 ng L-1 and 69 ng L-1) on reproductive endocrine system in adult zebrafish by measuring egg production, histologcal alterations and transcriptional expression levels of genes associated with HPG axis. The results showed that NGT caused no significant effects on egg production and histological changes of gonads at all treatment groups. However, NGT significantly induced transcriptional expression levels of Fshb, Lhb, Pgr, Esr1 and Ar genes in the brain, and Hsd20 b and Hsd11b2 genes in the ovaries of females at 6 ng L-1 or higher. A significant inhibition of Star, Cyp11a1, Cyp11 b, Hsd20 b, Hsd17b3, Hsd11b2 and Ar m RNAs expression were observed in the testes of males at 6 ng L-1 NGT or higher. P4 at 11 ng L-1 or higher resulted in a significant inhibition of Gnrh2, Gnrh3, Fshb and Esr1 genes in the brain of females while it caused a significant induction of Fshb, Lhb and Ar genes in the brain of males. The overall results indicate that P4 and NGT could cause the potential effects on adult zebrafish reproductive endocrine system by interfering with transcriptional expression of genes related to HPG axis.4. The aim of this study was to investigate the effects of binary mixtures of P4 with E2(42-5076 ng L-1) and NGT with EE2(36-5513 ng L-1) on the transcriptional expression levels of genes related to HPG axis and circadian rhythm signaling during the early development of zebrafish. The results showed that exposure to single P4 and E2 and their binary mixtures have only minor alterations in m RNA expression of a series of genes realted to HPG axis and circadian rhythm signaling, except for Cyp19a1 b and Lhb genes. The highest concentration of E2 and its combination with P4 caused a strong induction of Cyp19a1 b and Lhb genes. For the effects of binary mixtures of NGT and EE2, most of gene transcriptions of HPG axis and circadian rhythm signaling displayed most pronounced alterations following exposure to the binary mixtures, even at the low NGT+EE2 concentrations, but not for single chemical exposures, even at high concentrations of NGT or EE2. This finding suggests exposure to the binary mixtures of NGT and EE2 produced significantly enhanced effects in fish as compared to single chemical exposures, and their coexistence could have significant environmental implications.The results of the present study imply that P4 and NGT could cause the potential effects on zebrafish reproductive endocrine system by interfering with transcriptional expression of genes related to HPG axis as well as sex hormone levels and sex differentiation in embryonic, juvenile and adult zebrafish. The binary mixtures of NGT and EE2 caused a strong induction of most of gene transcriptions of HPG axis and circadian rhythm signaling as compared to signile chemical exposures, and consequently enhanced the disruptive effects on zebrafish reproductive endocrine system. |
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