Ovary apoptosis in zebrafish (Danio rerio): pathways characterization, role in sexual differentiation and as endocrine disruptors

Some chemicals present in the environment are known for altering the normal endocrine function of animals, including humans(1); these were named endocrine disruptor compounds (EDCs). Among EDCs are pharmaceuticals used in human and veterinarian medicine(2). Developmental and reproductive effects of EDCs depend on the precise timing the endocrine signal is being released. The critical window, sex differentiation, is of particular relevance to understand EDCs effects on fish reproductive function(3). When gonads are still sexually undifferentiated, fish gonad differentiation remains susceptible to the action of endogenous and exogenous factors, mainly factors that act as sex steroids. Sex steroids action is mediated by receptors that act on specific DNA sequences controlling the expression of target genes.

During development, D. rerio develops as undifferentiated gonochoristic(4), all individuals develop an immature ovary and afterwards these non‑functional ovaries develop into mature ovaries or undergo transition into testis(3‑6). The massive disappearance of oocytes, within gonad transition from ovary‑like tissue to testis, is caused by apoptosis, where oocytes disappear between 23-27 dph with simultaneous appearance of spermatogonia(5). The latter study suggested that a proapoptotic signalling pathway specific to males may be present in the oocytes that it may be stimulated by sex-determining genes or by changes in oestrogen level, and finally that it is possible that oocyte apoptosis followed by spermatogenesis during sex differentiation may be induced by the depletion of aromatase and oestrogen. In a posterior study(7), it was showed that an aromatase inhibitor (fadrozole), leading to depletion of estrogens, induced oocyte apoptosis.

Exposure to estrogens during the sex differentiation period is known to alter D. rerio populations’ sex ratio. Studies show that at concentrations of 0.5 ng/L there is already a reduction on males proportion(8) and that populations exposed to 2‑4 ng/L(3,9‑11) are composed only by females and fish with undifferentiated gonads. However, after estrogens removal and a recovery period the sex ratio is equal to unexposed populations(3;12).

In D. rerio, apoptosis is widely studied during embryogenesis and several genes related with apoptosis pathways are described(13‑14), but studies focusing in the role of estrogens on apoptosis pathways during gonad differentiation are absent, though this is a key factor to understand D. rerio sexual development and a may be even a helpful tool to estrogen‑related cancer research.

Two key questions underlie this project proposal. Are estrogens directly regulating D. rerio ovarian apoptosis pathways during sexual differentiation and how EDCs interfere with this? In order to tackle both questions, D. rerio will be exposed to two estrogens and one aromatase inhibitor used as pharmaceuticals, 17a-ethynilestradiol (EE2), genistein (G) and fadrozole (F) and several targets will be evaluated. The main aims are: study the expression of genes involved in cell apoptosis and their regulation by estrogens using the D. rerio model; determine apoptosis pathways involved in ovary apoptosis; identify possible female-male differences in gene expression and contribute for the knowledge of D. rerio sex differentiation and estrogens apoptosis regulation.

For answering this, in D. rerio model, we intent to: identify ovarian apoptosis genes responsive to estrogens; understand how sex hormones and EDCs determine sexual differentiation by control of ovarian apoptosis and evaluate how EE2, G and F concentrations present in environment may interfere with this mechanisms.

In summary, effects of EDCs, including pharmaceuticals, in fish have recently deserved increased attention. D. rerio is a species that does not have sex chromosomes and where all individuals develop initially as females and only after some differentiate into males. Ovary transformation into testis engages oocytes apoptosis. It is known that exposure to (xeno)estrogens can result on D. rerio all female populations and that this is reversible, thus these must interfere with ovarian apoptosis. Although, a large number of studies focus on D. rerio reproduction, there are no studies relating estrogenic effects or EDCs with ovarian apoptosis and testicular development. This project aims to fill this gap of knowledge, evaluating apoptosis gene expression regulation by three EDCs and its importance to sexual differentiation.

(Xeno)estrogens effects in fish reproduction are well studied and D. rerio is one of the most used models; but, its mechanisms of sexual development and differentiation are still poorly known. Knowledge of the estrogenic inhibition process/pathways of ovarian apoptosis and of the reversible character of this process is essential to fulfil this hole. Also, this can be helpful to oncologic research on estrogen-dependent cancers, since this study can reveal mechanistic insights in important pathways of estrogen action on apoptosis and action of G and F as anti-cancer medicines.