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 5α-androstanedione (5α-A), a product of dareductive
metabolism of androstenedione. 5α-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 5α-A is a
metabolic product of 5α-reductase type 1 (5α-Rl), a steroidogenic enzyme present in
human GC and TC, the thesis also describes studies of the localisation and
gonadotrophic regulation of 5α-Rl in the rat ovary. Ovarian aromatase expression was
also investigated because 5α-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 5α-A was not. Additional investigations using oestradiol
radioimmunoassay (RIA) supported the findings. The lack of a method for
measurement of 5α-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 5α-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 5α-R1 in the rat ovary was determined to identify the ovarian
cell types responsible for 5α-A production. 5α-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 5α-R1 tissue expression pattern was similar,
levels were markedly reduced in adult ovaries. Northern analysis and in situ
hybridisation (ISH) clearly showed that 5α-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 5α-R1 expression.
ECG down-regulated 5α-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 5α-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 5α-R1
expression. However, expression is stimulated by LH, which rises around the time of
ovulation. Hence, 5α-R1 action and 5α-reduced androgen production are reduced
when follicle development begins, probably to prevent an inhibitory action on oestrogen
synthesis. The transient increase in 5α-R1 around ovulation suggests a regulatory role
of 5α-reduced steroids at this stage of follicle development.The location of 5α-R1 in rat TIC indicated that they were the cells of choice for
cell culture investigations of 5α-Rl activity. However, repeated attempts to
demonstrate 5α-R1 activity in TC in isolated cell cultures were unsuccessful, even
though the cited literature reported 5α-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 5α-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
