Developing an in Vitro Thicklip Grey Mullet (Chelon labrosus) Gonad Explant Culture for Assessing Effects of Reproductive Endocrine Disrupting Chemicals

Workiye Worie Assefa, Ibon Cancio

Abstract


Sex steroid hormones necessary for regulation of gametogensis are produced through the coordinate actions occurring along the hypothalamic-pituitary-gonadal (HPG) axis. The process is complex with various potential steps liable to disruption due to exposure to external chemical stimuli such as the presence of reproductive endocrine disrupting chemicals (EDCs), eventually leading to reproductive failure. The toxic effects of reproductive EDCs in aquatic organisms can be elucidated effectively using in vitro culture techniques. The goal of this study was to develop a simple cost effective in vitro protocol to culture C. labrosus gonad explants to investigate the effects of EDCs. Mullets are useful sentinel organisms of environmental pollution in the Southern Bay of Biscay, where they have been shown to suffer an intersex condition as a result of exposure to xenoestrogens. Fish were sampled in Plentzia port and in the Bilbao river during the spring months (April–June 2015). The ovaries were sliced and explants cultured in L-15 supplemented media for 5 days, to study the effects of incubation with different concentrations of 17β-estradiol and testosterone at 18oC. The morphology of explants was evaluated histologically and the cell viability was analysed measuring the amount of lactate dehydrogenase released into the culture medium. Finally, 5S/18S rRNA ratio was determined in the ovaries and the transcriptional levels of steroidogenesis (cyp19a1a), and germ cell differentiation genes (tfiiia and piwil1) were quantified by qPCR. Histologically, explant culture conditions did not alter the morphology of oocytes, either previtellogenic or vitellogenic, but disruption of the stromal connective tissue and follicular cells around the oocytes was observed in some ovary explants after 5 days of culture. Cell viability analysis revealed no significant differences after 2 and 5 days in culture, but the levels of cyto-toxicity were very high both in control and in 17β-estradiol incubated explants. Culture conditions did not affect the production of 5S rRNA which was high both in previtellogenic (PV) and vitellogenic (V) ovarian explants at time 0, 5S rRNA transcript levels being higher in ovaries with PV oocytes than in those with V oocytes. A significant down-regulation of cyp19a1a was observed in the cultures, both under control and hormone incubation conditions. The same happened with tfiiia after 5 days of in vitro culture, but incubation with E2 this time resulted in the maintenance of the time 0 transcription levels. piwil1 transcript levels were not affected by the culture conditions. These results demonstrate that the developed mullet ovary explant in vitro assay was effective in preserving oocyte morphology and function, but not in somatic cell maintenance. In the present culturing circumstances, no effective analysis of the steroidogenic process can be undertaken, but the analysis of exogenous supply of steroid hormones on oocytes could be effectively analysed. Improving gonad explants culturing conditions will be a prerequisite in order to apply such explants as cost-effective, in vivo test substituting and high-throughput technology for the analysis of basic thicklip grey mullet reproductive endocrinology and of the effects of reproductive endocrine disrupting chemicals.

Keywords


Gene Transcription; Gametogenesis; cyp19a1a; tfiiia; piwil1; 17β-estradiol; 5S/18S rRNA Index

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