Characterization of candidate genes related to estrogenic activity in Oreochromis mossambicus
Thesis (PhD (Botany and Zoology))--Stellenbosch University, 2008.
Endocrine disruption is an alteration of the chemical messaging processes in the body. The value of studies‐ and monitoring of endocrine disruption using techniques included in the field of toxicogenomics is undoubtedly supported by scientific literature over the past four decades, as is demonstrated in Chapter 1 where I review relevant literature on the topic. Clearly, well sustained bio‐monitoring will include studies both in vitro and in vivo, and very well on transcriptional and translational levels. Animals are providing good models for in vivo studies to report or monitor endocrine disruption. It is imperative though to first understand such an animal’s biology, especially its endocrine system, and characterize what is considered “normal” for a species before engaging in endocrine disrupting exposures. A multitude of studies report endocrine disruption in relation to reproductive systems, with more recent work illustrating alteration of metabolism related to thyroidogenic disruption within the last decade. It is therefore essential to consider sex determination and ‐differentiation when studying sentinel species. Apart from the obvious academic interest in the matter of sex differentiation, altered patterns of sex differentiation in certain appropriate species provide for a very convincing endpoint in monitoring estrogenic endocrine disruption. As I approach to study a potential sentinel species for the southern African subcontinent, I set forward to study aspects of endocrine disruption influencing the reproductive system in a piece‐meal manner, starting with estrogenic endocrine disruption as this is the best studied facet of the endocrine disruption hypothesis to date. Yet, one learn from vast amounts of literature that in cases where sex is not exclusively determined by the genetic fraction of an individual, a number other characteristics may very well be used to determine estrogenic disruption in ecosystems. Quantitative production of the egg yolk precursor protein (vitellogenin) resides under these characteristics, and in the proposed sentinel, South African tilapiine, Oreochromis mossambicus phenotypic sex can be altered by environmental sex determination. The present study therefore targeted firstly the product most often used in tier I screening processes, vitellogenin (VTG). Specimens of O. mossambicus were cultured for this purpose from wild breeding stock, sampled at 5 day intervals and the transcription levels of vitellogenin gene (vtg) studied in those. Hereby, Chapter 2 describes the cloning of partial vtg gene and subsequent temporal expression of vtg quantitatively in O. mossambicus. To shed light on the state of gonadal differentiation sub‐samples were subjected to histology, illustrated in Chapter 3. In addition the quantitative vtg responses has been described in this study at a transcriptional level, both of adult males and juveniles subjected to low and very high levels of natural estrogens. In addition, a 3 kb 5’ flanking region of vtg was cloned and sequenced, and several putative binding sites identified for transcription factors of vtg, including several estrogen responsive elements (EREs). These indicate the expected regulational process of vtg by estrogens. Subsequently I measured the transcription levels of the only enzyme capable of aromatizing androgens into estrogens, Cytochrome P450 19 (cyp19) as has been characterized in Chapter 3. For stable binding of an estrogen to an ERE, binding of the ligand to its specific nuclear receptor (Estrogen receptor, ESR) is required. Since E2 is known to have different mechanisms of action in vertebrates, the expression levels of the ESRs were evaluated in our sample set after cloning 3 different homologues of ESR in O. mossambicus. The results on this matter is discussed in Chapter 4 and provides in addition to data on vtg and cyp19 a platform of “normal” transcription levels of these candidate genes involved in estrogenic endocrine disruption of O. mossambicus. Ultimately, characterization of those candidate genes involved extensively in phenotypic sex, contribute to our understanding of sex determination and differentiation in this species in a small way.