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Title: Role of 5α-reduced androgens in the ovary
Author: Mubiru, Josephine
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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Ovarian androgens are products of steroidogenic processes that are integral to follicular development, which culminates in ovulation. Follicle development involves growth and differentiation of the different follicular cell types. These developments enable maturing follicles to become steroidogenically competent and eventually release mature oocytes capable of fertilisation. The follicular cells involved in steroidogenesis are granulosa (GC) and theca cells (TC). Androgens are synthesised in TC. The main ovarian androgens are androstenedione and testosterone, which are predominandy substrates for aromatisation by GC into oestrogens, the most physiologically important steroids in the female. Follicle stimulating hormone (FSH) and luteinising hormone (LH) regulation of steroid synthesis is well documented. It is now also known that locally produced regulators within the ovary modulate this endocrine control. Autocrine/paracrine control modulates gonadotrophin-induced ovarian proliferation, differentiation and steroidogenesis. This thesis researches steroid 50C-androstanedione (50C-A), a product of dareductive metabolism of androstenedione. 5a-A is an aromatase inhibitor which inhibits oestradiol production, and therefore has a potential intraovarian paracrine role in modulating oestrogen biosynthesis. I investigated whether 5a-A was a product of androstenedione metabolism in the rat ovary, as an animal model. Since 5a-A is a metabolic product of 5a-reductase type 1 (5a-Rl), a steroidogenic enzyme present in human GC and TC, the thesis also describes studies of the localisation and gonadotrophic regulation of 5a-Rl in the rat ovary. Ovarian aromatase expression was also investigated because 5a-Rl activity is associated with inhibition of oestrogen production, which depends on aromatase activity. The metabolism of androstenedione was investigated in in vitro cultures of isolated ovarian GC and TCs. The steroid metabolites were investigated using radiochromatography. In GC cultures, oestradiol was detected among the steroid metabolites, but 5a-A was not. Additional investigations using oestradiol radioimmunoassay (RIA) supported the findings. The lack of a method for measurement of 5a-A led to the development and validation of a new RIA for this purpose. However, the assay crossreacted significantly with androsterone and androstenedione as well as 5CC-A. Although thin layer chromatography (TLC) could resolve the steroids, incorporation of the separation technique in the assay of biological samples was problematic due to inconsistent steroid recoveries. Therefore, the assay could only be used to measure total 17keto-androgens (17KAs) in unpurified biological samples. The location of 50C-R1 in the rat ovary was determined to identify the ovarian cell types responsible for 5a-A production. 5CC-R1 messenger RNA (mRNA) and protein were detected in the theca/interstitial cells (TIC) of the rat ovary, and strongly expressed in immature ovaries. Although 50C-R1 tissue expression pattern was similar, levels were markedly reduced in adult ovaries. Northern analysis and in situ hybridisation (ISH) clearly showed that 50C-R1 mRNA was abundantly expressed in the TIC. Additional evidence by immunocytochemistry (ICC) depicted the same location of 5a-Rl protein. 5a-Rl was not localised in rat GC. The expression of 50C-R1 in the immature rat ovary was developmentally regulated by gonadotrophins because eCG decreased its expression while it was transiently up-regulated by hCG. Because each hormone acts on either granulosa or theca cells in immature ovaries, hCG stimulation of 5CC-R1 expression suggested that LH acts on theca cells to stimulate 5CC-R1 expression. ECG down-regulated 50C-R1 expression, suggesting that FSH induced this action by local agents produced in the granulosa cells. These factors are yet to be identified, but this action of FSH indicates a paracrine product of GC that inhibits 5cc-R1 expression. ECG and hCG induced developmental changes reflecting the follicular changes that occur before ovulation. The findings, therefore, suggest that FSH, (the secretion of which is known to increase early in follicle development) decreases ovarian 50C-R1 expression. However, expression is stimulated by LH, which rises around the time of ovulation. Hence, 50C-R1 action and 5CC-reduced androgen production are reduced when follicle development begins, probably to prevent an inhibitory action on oestrogen synthesis. The transient increase in 5CC-R1 around ovulation suggests a regulatory role of 5a-reduced steroids at this stage of follicle development. The location of 5CC-R1 in rat TIC indicated that they were the cells of choice for cell culture investigations of 5a-Rl activity. However, repeated attempts to demonstrate 50C-R1 activity in TC in isolated cell cultures were unsuccessful, even though the cited literature reported 5a-Rl activity in rat GC and TC. The results of aromatase investigations were similar to reported findings. Aromatase mRNA was expressed abundantly in the GC of eCG-treated ovaries, but absent in GC of immature or hCG-treated ovaries as well as the TIC. The findings also matched the well-known stimulatory role of FSH on aromatase action. Finally, other investigators had implicated 5CC-A in dysfunctional follicle growth in women presenting with polycystic ovary syndrome (PCOS). Therefore, the concluding clinical section of the thesis is devoted to the measurement of 50C-A levels in follicular fluids (FF) obtained from the ovaries of women with normal ovarian function and those with PCOS. Measurements of androstenedione and 50C-A in normal and PCOS FF showed that both androgens were elevated in PCOS subjects, whether ovulatory or anovulatory, compared to normal FF. The findings illustrated abnormal steroid production in PCOS ovaries and are reflective of the characteristic hyperandrogenaemia of the condition. In summary, this is the first definitive description of the spatiotemporal expression of 50C-R1 in the mammalian ovary. 5α-R1 gene expression was located to rat TIC and shown to be regulated developmentally by gonadotrophins. A new RIA was developed and used to measure the 5α-reduced androgens and androstenedione in normal and PCOS FF. These androgens were shown to be elevated in PCOS, where they probably contribute to the local intraovarian paracrine control that impairs oestradiol production in anovulatory PCOS.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available