CONSIDERACIONES FINALES

1.4.1 Hypothalamic-pituitary-gonadal axis in fish

Regulation of sexual differentiation, gonad development, function and re- production in fish is under control of the hypothalamic-pituitary-gonadal (HPG) axis and the synthesis and release of endocrine hormones, which in turn are regulated by feedback mechanisms. The mechanisms of these steroid feedback regulations are described hereafter and illustrated in Fig- ure 1.1. In response to sex steroids, the hypothalamus releases the go- nadotropin releasing hormone, which binds to gonadotropin releasing hor- mone receptors (GnRHR) in the pituitary resulting in the release of two gonadotropins, i.e. follicle stimulating hormone (FSH) and luteinizing hor- mone (LH) into the blood stream. In female gonads, FSH and LH control the oocyte development in the ovary. They bind to their receptors in the follicular theca- and granulosa cells and induce the production of testos- terone in the theca cells as well as the upregulation of aromatase, which converts testosterone to estradiol in the granulosa cells. In the testis, go- nadotropins regulate the spermatogenesis and the testosterone synthesis.

1.4 Fundamental background of endocrine disruption 9

FSH binds to the receptor in Sertoli-cells and activates a cAMP pathway which induces the transformation of germ cells to spermatoids. LH acts on the Leydig cell membrane and causes, also via a cAMP dependent path- way, the de novo testosterone synthesis from cholesterol. In contrast to mammals, downstream enzymes, i.e. 11β -hydroxylase (11βH, encoded by cyp11b) and 11β-hydroxysteroid dehydrogenase (11βHSD) further convert testosterone to 11-ketotestosterone in fish, which is the major androgen in fish [Kime, 1998]. The steroids produced in the gonads are then released into the blood and act via negative or positive feedback on GnRH neurons, regulating the gonadotropin production. Because GnRH neurons do not express steroid receptors, this regulation occurs indirectly via kiss1 neurons and neuropeptide Y (NPY) neurons, which do express steroid receptors. Kiss1 neurons are located mainly in two hypothalamic areas, the arcu- ate nucleus (ARC) and the anteroventral periventricular nucleus (AVPV), whereas NPY neurons are present in the ARC, but not in the AVPV. Steroid binding to those neurons leads to the secretion of the neurotrans- mitter kisspeptin 1 and NPY.

Steroids can either stimulate (positive feedback) or inhibit (negative feedback) the gonadotropin release. The negative feedback, required for maintaining the gonadotropin and steroid balance, is triggered by both, estrogen and androgen, and involves mainly the kiss1 and npy neurons in the ARC nucleus. Estrogen and androgen receptors mediate the steroid signalling on kiss1 neurons, which in turn secrete kisspeptin1 [Smith et al., 2005; Irwig et al., 2004; Rometo et al., 2007; Smith et al., 2007; Garc´ıa- Galiano et al., 2012]. Kisspeptin 1 can directly excite GnRH neurons and it has also been shown to bind to the kiss1 receptor, expressed by npy neu- rons resulting in NPY secretion which also contributes to GnRH neuron stimulation [Kim et al., 2010]. The positive feedback is mainly required for the induction of the preovulatory LH surge, taking place in the AVPV and is mainly induced by estradiol in females. Although the kiss1 neurons in the AVPV nucleus also express the androgen receptors, the estrogen re- ceptors alpha and beta have been identified to mediate the feedback effects

[Smith et al., 2005]. Testosterone is also able to activate kiss1 neurons in the AVPV nucleus. However, since the AVPV is sexually dimorphic and less kiss1 neurons are present in male AVPV nuclei than in females, the androgen-driven positive feedback has been proposed to be rather irrele- vant [Smith et al., 2005]. Apart from the spatial divergences, little is known about the exact mechanism leading to either positive or negative feedback. However, there is some evidence that molecular differences in steroid re- ceptor signalling might contribute to the stimulating or inhibiting effect. The negative feedback in the ARC involves steroid receptors, but does not necessarily require classical gene signalling. In contrast, inhibition of the classical signalling in the AVRV kiss1 neurons blocks the positive feedback [Gottsch et al., 2009; Glidewell-Kenney et al., 2007].

Even though the gonads are not yet developed in embryos of zebrafish and only gonadal precursors, the gonadal primordium, exist in medaka [Nakamura et al., 2006], steroids are present in both and have a crucial role for sexual differentiation. The steroids are mostly maternally derived and are taken up from the yolk. At a later stage, some steroids are pro- duced in the brain of fish embryos [Kishida et al., 2001; Iwamatsu et al., 2006; Brooks et al., 1997; Hsu et al., 2002; Vosges et al., 2012; Pikulkaew et al., 2010]. The presence of steroid receptors in embryos (section 1.5.1.3) further underscore the relevance of steroids in early life stages [Pikulkaew et al., 2010]. Thus, steroids elicit effects in fish embryos and endocrine pro- cesses in the hypothalamus and pituitary regions are active and susceptible to disruption at these developmental stages.

1.4.2 Mechanisms of endocrine disruption in brief

Endocrine active agents can disrupt the endogenous hormone balance in many different ways. They can act as agonists or antagonists of steroid receptors and thereby mimic or inhibit endogenous steroid hormone sig- nalling or they can interfere with other sites of hormone synthesis or metabolism. The main mechanisms are described in this chapter.

1.4 Fundamental background of endocrine disruption 11 ERAR Kisspeptin Neuropeptide Y ER AR Kisspeptin kiss1r npyr ER ER Gonadotropin relea- sing hormone Pituitary Ovary Testis Gonads Kiss1 neuron Kiss1 neuron kiss1r Kisspeptin NPY neuron Gnrh neuron FSH LH +

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feedback negative feedback

Figure 1.1: Steroid feedback regulation The hypothalamic gonadotropin releasing hormone induces the release of the gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary, which are transported to the gonads and stimulate the production of estrogen (E2) and testosterone (T). E2 and T autoregulate their production by either positive or negative feedback for enhancing or lowering the steroid levels, respectively. The negative feedback is triggered by both, E2 and T, and involves the kiss and npy neurons in the arcuate nucleus. In the absence of steroid signals, kiss1 and NPY neurons secrete the neurotransmitter kisspeptin1 and neuropeptide X respectively, which both excite gnrh neurons. Also, kisspeptin1 can indirectly enhance the gnrh production by stimulating NPY neurons. Binding of E2 and T to cytosolic and membrane estrogen receptors as well as androgen receptors in the kiss1 neurons inhibits this signalling cascade and leads to impaired steroid production. The positive feedback is mainly induced by estrogen in females and involves kiss neurons located in the AVPV. The estrogen receptors mediate the positive feedback effects and lead to an enhanced kisspeptin production and therefore to gonadotropin release. Abbreviations: AR (androgen receptor); ER (estrogen receptor); kiss1r (kiss 1 receptor); npyr (neuropeptide Y receptor); gnrhr (gonadotropin releasing hormone receptor).

1.4.2.1 Receptor mediated signalling

Most endocrine disruptors are ligands of steroid receptors and activate or suppress receptor signalling. In the classical, receptor-mediated sig- nal transduction, steroids diffuse across membranes and bind to nuclear steroid receptors, inducing the expression of steroid responsive genes. Prin- cipally, ligand binding induces a conformational change of the receptor leading to the dimerization of the receptors, which thereby become a nu- clear transcription factor. The activated receptor then regulates gene ex- pression by binding with its DNA binding domain to steroid responsive elements in the promoter or enhancer regions of target genes or by recruit- ing other transcription factors. This process is relatively slow and takes at least several hours. Yet, steroids can also induce rapid actions, relying on nongenomic pathways. Though the exact signalling mechanisms are not fully understood yet, it is known that membrane bound steroid recep- tors are mediating this process. One category of these plasma membrane receptors are receptors identical to nuclear receptors, which translocate from the nucleus to the cell membrane through posttranslational modifi- cations [Razandi et al., 2003] or interaction with cytoplasmatic adaptor molecules [Song et al., 2004, 2002]. Binding of ligands to such a mem- brane bound steroid receptor initiates interaction with several signalling molecules and induces the activation of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K)/Akt signalling cascades. Another class of non-genomic signalling involves G-protein coupled recep- tors, i.e. the GPR 30, which mediate the rapid actions of estradiol. This signalling induces rapid cellular responses such as MAP kinase activation, phosphoinositide 3-kinase (PI3K) activation, calcium flux elevations, el- evated cyclic adenosine monophosphate (cAMP) concentrations and in- creased c-fos oncogene transcription of oncogenes (e.g., c-fos) [Andersen et al., 2003; Str¨ahle et al., 2012; Thomas et al., 2005; Albanito et al., 2007; Filardo et al., 2000; Vivacqua et al., 2006; Li et al., 2010]. Notably, apart from inducing cellular responses, some of these signalling cascades can ultimately alter gene expression, despite being termed “non-genomic”