Functional significance of aromatase in zebrafish during development
Estrogens, and their local synthesis by aromatase, are responsible for brain and gonadal differentiation during development, while in the adult, they play a major role in reproduction by regulating germ cell maturation in the gonads, in response to gonadotropin secretions from the pituitary and sexual behavior. In addition, at least in mammals, estrogens are known to play a neurotrophic role in development and have wider functions in the adult nervous system such as neuroprotection and regulating neuronal plasticity. In lower vertebrates, including fish, phenotypic sexual differentiation is regulated by sexual steroid hormones, such as estrogens, although the processes which trigger sexually dimorphic steroid synthesis and secretion in the developing fish are not yet understood. Even less is known on the effects of estrogen synthesis in the brain during early development (embryo) and gonadal differentiation (juvenile) of fish. This thesis aims to fill in the gaps of knowledge on the role of estrogens in sexual and nervous system differentiation of zebrafish.
Using the zebrafish as a model organism, we set out to investigate the role of aromatase during development. Zebrafish have two aromatase genes, cyp19a1 and cyp19a2, primarily expressed in the adult ovary and brain respectively, with some overlap. As a first step in studying the zebrafish aromatases, we developed new and adjusted existing tools. Six polyclonal and monoclonal antibodies were characterized. One of the polyclonal antibodies was found to be specific for the CYP19A2 protein, in western blots and immunohistochemistry. Using the anti-CYP19A2 antibody, we were able to map the distribution of aromatase positive cells in the brain. In particular, immunoreactive cells and processes were detected mostly in the telencephalon and diencephalon, in line with previous findings that have demonstrated the presence of aromatase mostly in reproductive nuclei of the preoptic area-hypothalamus. In addition, we have been able to adjust and improve a quantitative Real Time RT-PCR method for the detection of cyp19 transcripts. Our analyses showed that the two aromatase genes responded as expected on the basis of their regulatory elements after treatment with (xeno)estrogens.
It has been recently shown that inhibition of aromatase enzyme activity during the critical stage of gonadal development leads to irreversible changes in phenotypic sex. Just before the onset of sexual differentiation, the brain specific isozyme, cyp19a2, is expressed in a dimorphic manner. It has been hypothesized that, similar to mammals, aromatase can masculinize the developing fish brain and at the same time could also be the triggering signal for gonadal differentiation. To test this hypothesis, developing zebrafish undergoing gonadal differentiation were collected, using the heads partly for quantification of cyp19a2 transcript abundance and partly for CYP19A2 immunohistochemistry, while the bodies were used for histological evaluation of gonad differentiation status. Our data do not demonstrate a sexually dimorphic expression of cyp19a2 levels in the brain but rather show a pronounced variation within one and the same sex. The observation of large intrasexual variation is confirmed by immunohistochemical studies on CYP19A2 expression in the brains of sexually differentiating zebrafish. These studies further showed that the distribution pattern of CYP19A2 in the brain of zebrafish is similar between sexes. Finally, commitment to either sex, as evidenced from gonadal morphological differentiation, preceded upregulation of brain aromatase expression.
The fourth chapter of the thesis addressed the question on the functional significance of aromatase expression during embryonic and larval stages of zebrafish. We know that both zebrafish aromatase enzymes are expressed early in ontogeny, long before the initiation of gonadal differentiation, suggesting a role in development beyond phenotypic sex differentiation. In search of the aromatase gene functions during embryonic development, we found that cyp19a1 expression is localized in zebrafish lateral line neuromasts, the organs of the mechanosensory system of fish and amphibians. We demonstrated that cyp19a1 aromatase gene inactivation by morpholino oligonucleotide knockdown leads to decreased numbers of lateral line neuromasts. Coinjection of embryos with morpholino oligonucleotides and capped cyp19a1 RNA could rescue the phenotype, pointing to the specificity of the effect. The reduction in neuromast numbers did not associate with increased apoptotic cell death. Whole mount immunohistochemistry with a neuronal marker revealed that although neuromasts are properly innervated by nerves, hair cells fail to develop. This is the first study to provide evidence that lack of estrogen synthesis during zebrafish embryonic development is associated with decreased sensory hair cell differentiation.
In conclusion, this thesis advanced understanding of the functional significance of aromatase in zebrafish during development on two key aspects. The first aspect is the role of the brain-specific aromatase expression during sexual differentiation. The results of this thesis provide strong evidence against the hypothesis, as put forward in the literature that cyp19a2 is expressed in a sexually dimorphic manner during ontogeny and thereby guides sexual differentiation of the gonads. Furthermore, should sexual dimorphisms exist in the brain of developing or adult zebrafish, a role of cyp19a2 in this process during sexual differentiation is unlikely on the basis of the present findings. The second aspect is the role of aromatase in the differentiation of sensory neurons during embryonic development. This thesis provides strong evidence that aromatase expression is crucial for the differentiation of sensory neurons. This novel finding is suggestive on the principal role of aromatase and estrogens in the development of the nervous system in fish. Since this is the first time a direct effect of local estrogen synthesis by aromatase on the differentiation of sensory neurons is demonstrated, our findings can have a great impact in the current understanding of how the vertebrate senses are developed.