Early life programming of adult leydig cell function
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Kilcoyne2014.doc (15.66Mb)
Date
05/07/2014Author
Kilcoyne, Karen
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Abstract
There is increasing evidence to suggest that fetal events can predetermine
reproductive health and general wellbeing in adulthood, a process termed 'fetal
programming'. This refers to the association between altered fetal
growth/development and health disorders in adulthood e.g. the metabolic
syndrome, which is linked to low male testosterone levels. Studies from both
Europe and the USA have shown that adult male testosterone levels have been
declining, independent of age. As low testosterone levels in aging men are
associated with increased morbidity and mortality, this highlights the
importance of investigating how testosterone levels are determined or
potentially ‘programmed’ during fetal development.
Evidence from human and rodent studies have shown that reduced fetal
androgen exposure results in lower adult testosterone levels, although the
mechanism(s) is unknown, to date. One way to explain how a fetal insult (e.g.
androgen deficiency) could affect (testosterone producing) adult Leydig cells in
adulthood, is if their progenitor cells were present during fetal life and were
thus affected by such an insult. This hypothesis has been unexplored to date,
due to the lack of a unifying/defining marker for adult Leydig progenitor cells.
An earlier study promoted the hypothesis for the studies in this thesis, namely
that chicken ovalbumin upstream promoter transcription factor-II (COUP-TFII)
might constitute such a marker, as inducible knockout of COUP-TFII in pre-pubertal
male mice results in failure of adult Leydig cells to develop. Therefore,
the hypothesis which was explored in this thesis was that 'fetal programming' of
COUP-TFII+ adult Leydig progenitor cells prior to their differentiation into adult
Leydig cells, would explain how fetal events could predetermine adult
testosterone levels.
To investigate whether adult Leydig cells (ALC) develop from COUP-TFII+
interstitial cells, firstly an adult Leydig cell ablation/regeneration model was
used, which involved a single injection of ethane dimethane sulphonate (EDS).
This identified that in rats, ALC derive from COUP-TFII+ interstitial cells which
do not express any other phenotypical adult Leydig or interstitial cell markers
prior to differentiation. Secondly, COUP-TFII+ adult Leydig progenitor cells are
abundant in the fetal testis and conserved across species, including man.
Thirdly, fetal interstitial cells which differentiated into ALC, as evident from an
ALC lineage tracer model, also expressed COUP-TFII. Overall, these findings
suggest that the COUP-TFII+ interstitial cells which differentiate into ALC are
'adult Leydig progenitor cells'.
The findings from this thesis also show that the identified adult Leydig
progenitor cells express the androgen receptor (AR) in fetal life. Furthermore,
experimental reduction of androgen action in fetal life in transgenic mice (AR
knockout) or chemical manipulations to reduce fetal testosterone levels (di(n-butyl)
phthalate; DBP exposure) resulted in a similar reduction (~40%) in
progenitor cell numbers from birth through to adulthood. A parallel reduction
of adult Leydig cell numbers across postnatal development was found in mice,
but not rats, but as a result of altered fetal androgen action, both models showed
evidence for compensated adult Leydig cell failure. This is defined as
normal/low testosterone and elevated luteinising hormone (LH) levels. Cell-selective
knockout of AR in peritubular myoid (PTM) cells (PTM-ARKO) or
Sertoli cells (SC-ARKO) did not affect the numerical development of adult Leydig
progenitor cells. To manipulate testicular testosterone action in postnatal life,
rats were exposed to a potent AR antagonist, flutamide, which reduced the
number of adult Leydig progenitor cells but did not affect ALC number/function.
However, the combination of fetal DBP+postnatal flutamide exposure reduced
adult Leydig progenitor cells and resulted in compensated ALC failure. Overall,
these studies highlight the importance of fetal androgens for the normal
development of adult Leydig progenitor cells and for the subsequent
development of normally functioning adult Leydig cells.
As fetal deficits in androgen exposure resulted in adult Leydig cell dysfunction,
this thesis also investigated three separate models to determine whether
increased fetal androgen exposure could increase/enhance adult Leydig
progenitor cell development, resulting in a 'gain of adult Leydig cell function'. In
the first model to increase fetal androgen exposure, pregnant dams injected
with testosterone propionate (TP; 20mg/kg/day e14-21.5) were discarded, due
to confounding factors including fetal growth restriction and aromatisation of
TP. The second model utilised dihydrotestosterone (DHT; 10mg/kg/day),
administered to pregnant dams, but there were no effects found in adulthood to
male offspring. It was concluded that the administered dose was not sufficient
to increase intratesticular testosterone levels in the fetus. The third model
utilised an inducible nitric oxide synthase knockout (iNOS-/-) mouse model, for
which previous evidence showed increased testis weight, Leydig and Sertoli cell
number (~50%), and normal testosterone but low LH levels in adulthood.
Stereological quantification showed an increase in the number of adult Leydig
progenitor cells in postnatal, but not fetal life, which resulted in the conclusion
that the observed changes were a consequence of postnatal effects.
Finally, a potential mechanism to explain how DBP-induced androgen deficiency
in fetal life, could result in adult Leydig cell dysfunction in adulthood was
investigated. Analysis of testicular genes in adulthood, involved in the
steroidogenic pathway, showed a reduction in 3b-hsd and StAR. The reduced
StAR expression was associated with increased repressive histone methylation
(H3K27me3) in its proximal promoter region, as demonstrated by a chromatin
immunoprecipitation (ChIP) assay, qPCR, and densitometrical analysis.
Accordingly, adult Leydig cells were shown to express increased H3K27me3 by
immunohistochemistry, a change also evident in adult Leydig progenitor cells in
the fetal testis. This would provide a potential mechanism to explain how fetal
events can 'programme' adult Leydig cell testosterone production, namely via
an epigenetic change to adult Leydig progenitor cells. In summary, the results in
this thesis show how fetal events, including androgen action on progenitor cells,
can potentially programme adult Leydig cell function and thus determine
testosterone levels. As testosterone is crucial to man, the findings reported in
this thesis may have important implications for the general health and longevity
of man.