Assefa Workiyie Worie (MSc Thesis 2015)

In vitro culture of gonad explants in thicklip grey mullet (Chelon labrosus) in different gametogenic stages; a tool for the assessment of the effects of reproductive Endocrine Disrupting Chemicals (EDCs)
Supervisor: Ibon Cancio (UPV/EHU)
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 general and ultimate goal of this study was to develop a simple cost effective in vitro protocol to culture thicklip grey mullet (Chelon 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 intersex condition as a result of exposure to xenoestrogens. Fish were sampled in Plentzia port and in 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 analyzed measuring the amount of lactate dehydrogenase released into the culture medium. Cell proliferation in ovarian explants was checked by 5-bromo-2-deoxyuridine (BrdU) incorporation. Finally, 5S/18S rRNA ratio was determined in 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 cytotoxity were very high both in control and in 17β-estradiol incubated explants. BrdU incorporation only revealed very limited proliferation activity in a few somatic cells after hormone incubation, but not in ovarian explant germ cells. 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 maintenance of the time 0 transcription levels. piwil1 transcript levels were not affected by the culture conditions as a proof that there was not proliferation of mitotically dividing oogonia. These results demonstrate that the developed mullet ovary explant in vitro assay was effective 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 analyzed. 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 (EDCs). Keywords: Chelon labrosus, gene transcription, ex vivo tissue explant cultures, ovary explants, gametogenesis, cyp19a1a, tfiiia, piwil1, steroidogenesis, 17β-estradiol, testosterone, 5S/18S rRNA index, BrdU, reproductive endocrine disruption

In vitro culture of gonad explants in thicklip grey mullet (Chelon labrosus) in different gametogenic stages; a tool for the assessment of the effects of reproductive Endocrine Disrupting Chemicals (EDCs)

Supervisor: Ibon Cancio (UPV/EHU)

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 general and ultimate goal of this study was to develop a simple cost effective in vitro protocol to culture thicklip grey mullet (Chelon 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 intersex condition as a result of exposure to xenoestrogens. Fish were sampled in Plentzia port and in 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 analyzed measuring the amount of lactate dehydrogenase released into the culture medium. Cell proliferation in ovarian explants was checked by 5-bromo-2-deoxyuridine (BrdU) incorporation. Finally, 5S/18S rRNA ratio was determined in 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 cytotoxity were very high both in control and in 17β-estradiol incubated explants. BrdU incorporation only revealed very limited proliferation activity in a few somatic cells after hormone incubation, but not in ovarian explant germ cells. 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 maintenance of the time 0 transcription levels. piwil1 transcript levels were not affected by the culture conditions as a proof that there was not proliferation of mitotically dividing oogonia. These results demonstrate that the developed mullet ovary explant in vitro assay was effective 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 analyzed. 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 (EDCs).

Keywords: Chelon labrosus, gene transcription, ex vivo tissue explant cultures, ovary explants, gametogenesis, cyp19a1a, tfiiia, piwil1, steroidogenesis, 17β-estradiol, testosterone, 5S/18S rRNA index, BrdU, reproductive endocrine disruption

Assefa Workiyie Worie (MSc Thesis 2015)

In vitro culture of gonad explants in thicklip grey mullet (Chelon labrosus) in different gametogenic stages; a tool for the assessment of the effects of reproductive Endocrine Disrupting Chemicals (EDCs)

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