Sattar, Saeeda (2003) The study of cellular and molecular aspects of the human fallopian tube epithelium. (PhD thesis), Kingston University, .
Abstract
The Fallopian tube provides physiological support to both gametes and the embryo. Experimental approaches to improve the outcome of infertility treatment have included co-culture systems, whereby sperm, ova and/or embryos are cultured in a monolayer of Fallopian tube epithelial cells. This approach has been shown to be superior to other methods based on co-culture with other cell types. In addition, the role of gap junction intercellular communication (GJIC) in the regulation of cell proliferation and differentiation is becoming increasingly recognised as one of the major cellular functions. GJIC plays an important role in fertilisation as well as in the normal development of both the embryo and foetus. It is also involved in the sexual maturation of the adult and in the maintenance of health throughout life. As such, GJIC may be the key element in the understanding of cellular functions especially during the reproductive process. Therefore, the aims of this thesis were to examine some of the cellular and molecular aspects of the human Fallopian tube epithelium in relation to infertility. In the first part of this study, the gap junction proteins, connexins were examined by immunohistochemical and Western blotting techniques. Fallopian tubes were removed from women at different stages of the ovarian cycle. The results demonstrated the presence of gap junction proteins, connexins (cx) in the human Fallopian tube throughout the ovarian cycle. Fimbrial and ampullary regions were separated and subsequently processed for Western blotting analysis using a range of monoclonal or polyclonal antibodies directed against cx26, cx32 and cx43.The intensity of staining varied depending upon the hormonal status of the patients examined and appeared to be upregulated during the secretory stage as opposed to the proliferative phase of the ovarian cycle. Human Fallopian tube did not express cx32, regardless of the anatomical site examined. In all cases though, the expression of both cx26 and 43 appeared to be more prevalent in the ampullary region. The second part of this investigation focussed on transforming Fallopian tube epithelial cells into an immortal cell line. Normal cells cannot be sustained indefinitely, as cell degeneration occurs with continuous passaging. In most IVF treatments, sperm and ova are allowed to fertilise in a conventional medium but there is the potential that the Fallopian tube epithelial cells may be employed in IVF treatments in the future. Therefore, current techniques used to isolate and culture epithelial cells from the Fallopian tube were evaluated, in order to assess the best method that provides an optimal yield of Fallopian tube epithelial cells, both qualitatively and quantitatively. The cells were isolated using (i) a mechanical technique, whereby the mucosal layer of the tube were minced finely using a pair of scissors and (ii) an enzymatic method which consisted of the enzymes, trypsin and pancreatin. The results showed that enzymatic isolation provided a large number of cells, but there was a significant detrimental effect on cell survival and their secretory status. In contrast, cells isolated by the mechanical method were fewer in number than those obtained by the enzymatic method but cell survival and secretory status were relatively better. Regardless, of the isolation technique employed, the Fallopian tube epithelial cells had a limited lifespan in culture. Bearing this in mind and coupled with the realisation that the interactions between the tubal epithelial cells and gametes/embryos cannot adequately be studied in vivo, the next part of the study investigated ways of establishing an immortalised human Fallopian tube cell line. Such a cell line would be able to survive in culture indefinitely and therefore could be a potential model, which could mimic the in vivo milieu more closely. In order to obtain a 'bank' of Fallopian tube epithelial cells, cells isolated by either the mechanical or enzymatic method were cultured in growth medium for 24-48 hours prior to being transfected with a transforming gene, the SV40 Large T-antigen. Various techniques were used to transfect the epithelial cells in culture; these included well-established methods such as DNA co-precipitation as well as more advanced methods using cationic liposomes. The results from this study demonstrated that human Fallopian tube epithelial cells can be transiently transfected and could survive for over 102 days in culture, whilst still retaining many of the morphological characteristics of the original epithelial cell type. By examining both the cellular and molecular aspects of the tube as described above, it was anticipated that further insight into the complex nature of this structure would be achieved. Furthermore, it was hoped to improve the current understanding and knowledge of the Fallopian tube's functions, thereby facilitating a greater awareness of the issues important in establishing and maintaining an in vitro model of the Fallopian tube.
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