While microcystins (MCs) have been reported to exert reproductive toxicity on fish with a sexdependent effect, the underlying mechanism has been rarely investigated. In the present study, zebrafish were exposed to 1, 5 and 20 μg/L MC-LR for 30 d. The gonad-somatic index declined in all treated males. 17β-estradiol (E 2 ), testosterone (T), 11-keto testosterone (11-KT) and follicle-stimulating hormone (FSH) levels increased in serum from all treated females, while T, FSH and luteinizing hormone (LH) levels changed in all treated males. Histomorphological observation showed that MC-LR exposure evidently retarded oogenesis and spermatogenesis. Transcriptional changes of 22 genes of the hypothalamic-pituitary-gonad (HPG) axis exhibited sex-specific responses, and the relationship between gene transcriptions and gametogenesis was evaluated by principle component analysis (PCA). Major contributors to PC1 (gnrh2, gnrhr3, ar, lhr, hmgra, hmgrb and cyp19a) were positively correlated with the number of post-vitellogenic oocytes, while PC1 (gnrh2, lhβ, erβ, fshr, cyp11a and 17βhsd) were positively correlated with the number of spermatozoa. The protein levels of 17βHSD and CYP19a were affected in both females and males. In conclusion, this study first investigated the sex-dependent effects of microcystins on fish reproduction and revealed some important molecular biomarkers related to gametogenesis in zebrafish suffered from MC-LR.Cyanobacteria are photosynthetic, oxygen producing, gram-negative microorganisms found ubiquitously in nature even at extreme climatic conditions 1,2 . Increasing global temperature, nutrient and pollutant enrichment via anthropogenic runoff, drought and flooding lead to eutrophication and outbreak of cyanobacterial blooms 3,4 . The toxic cyanobacterial blooms can produce and release cyanotoxins, the secondary metabolites of cyanobacteria, into water 5 . Among all the cyanobacterial toxins, microcystins (MCs) represent a family of potent hepatotoxins and are considered as the most resistant of cyanotoxins to degradation because of their stable cyclic peptide structure 6 . To date, over 100 variants of MCs have been isolated, and microcystin-LR (MC-LR), a hydrophobic variant, is considered to be the most commonly occurring and lethal toxin 7 . In fish, MCs accumulate mainly in liver and therefore liver is considered as the first target organ of MCs 8,9 . A provisional safety guideline of 1.0 μg/L MC-LR in drinking water was recommended by the World Health Organization (WHO) 10 . MCs are released from the cyanobacterial cells into the water bodies where fish spends their whole life stage, including growth, reproduction and embryonic development 11 . Gonad has been recognized as the second important target organ of MCs 12 and MCs exert negative effects on the reproductive system of fish [13][14][15] . Lysis of the gonadosomatic tissue in ovary and disruption of spermatogenesis in testis were observed in medaka fish exposed to MC-LR 13. Hou et al. also suggested MC-LR had adverse effects on histologic...

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Obtaining oocytes from the adult female zebrafish (Danio rerio) ovary has enormous importance in the studies of developmental biology, toxicology, and genetics. It is vital to establish a simple and effective approach to ensure the quantity and quality of oocytes, which will enable the success of follow‐up experimental investigation finally. Usually, oocytes are separated with mechanical or enzymatic methods, however, little studies have been done with concerns about the comparative effects. The present study separated zebrafish oocytes of Stage III with five frequently used methods, including stripping, pipetting, hyaluronidase (1.6 mg/ml), collagenase (0.4 mg/ml), and trypsin (0.1%). The cell viability, oxidative stress, mitogen‐activated protein kinase (MAPK) protein phosphorylation, and apoptosis levels were selected as main biomarkers to evaluate the oocytes health status. The results showed that both trypsin and hyaluronidase isolation significantly upregulated germinal vesicle breakdown (GVBD) rates and downregulated p38 MAPK activity simultaneously. GVBD rates and survival rates were decreased notably in oocytes separated by the collagenase method. Above results indicate that zebrafish oocytes in vitro are sensitive to enzymatic treatments and the enzymatic isolation is not the suitable mean for collecting zebrafish oocytes although it is time‐saving. The mechanical strategy of pipetting remarkably increased the reactive oxygen species and malondialdehyde level in isolated oocytes. Interestingly, oocytes separated with stripping show less physiological and biochemical damages. Therefore, stripping isolation is comparatively recommended as the optimum method for separating and collecting numerous intact and healthy zebrafish oocytes in vitro for the subsequent developmental research.