Early life programming of adult leydig cell function

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

Experimentally-induced ‘Testicular dysgenesis syndrome’ originates in the masculinization programming window

The testicular dysgenesis syndrome (TDS) hypothesis, which proposes that common reproductive disorders of newborn and adult human males may have a common fetal origin, is largely untested. We tested this hypothesis using a rat model involving gestational exposure to dibutyl phthalate (DBP), which suppresses testosterone production by the fetal testis. We evaluated if induction of TDS via testosterone suppression is restricted to the “masculinization programming window” (MPW), as indicated by reduction in anogenital distance (AGD). We show that DBP suppresses fetal testosterone equally during and after the MPW, but only DBP exposure in the MPW causes reduced AGD, focal testicular dysgenesis, and TDS disorders (cryptorchidism, hypospadias, reduced adult testis size, and compensated adult Leydig cell failure). Focal testicular dysgenesis, reduced size of adult male reproductive organs, and TDS disorders and their severity were all strongly associated with reduced AGD. We related our findings to human TDS cases by demonstrating similar focal dysgenetic changes in testes of men with preinvasive germ cell neoplasia (GCNIS) and in testes of DBP-MPW animals. If our results are translatable to humans, they suggest that identification of potential causes of human TDS disorders should focus on exposures during a human MPW equivalent, especially if negatively associated with offspring AGD

Effects of environmental Bisphenol A exposures on germ cell development and Leydig cell function in the human fetal testis

<div><p>Background</p><p>Using an organotypic culture system termed human Fetal Testis Assay (hFeTA) we previously showed that 0.01 μM BPA decreases basal, but not LH-stimulated, testosterone secreted by the first trimester human fetal testis. The present study was conducted to determine the potential for a long-term antiandrogenic effect of BPA using a xenograft model, and also to study the effect of BPA on germ cell development using both the hFETA and xenograft models.</p><p>Methods</p><p>Using the hFeTA system, first trimester testes were cultured for 3 days with 0.01 to 10 μM BPA. For xenografts, adult castrate male nude mice were injected with hCG and grafted with first trimester testes. Host mice received 10 μM BPA (~ 500 μg/kg/day) in their drinking water for 5 weeks. Plasma levels of total and unconjugated BPA were 0.10 μM and 0.038 μM respectively. Mice grafted with second trimester testes received 0.5 and 50 μg/kg/day BPA by oral gavage for 5 weeks.</p><p>Results</p><p>With first trimester human testes, using the hFeTA model, 10 μM BPA increased germ cell apoptosis. In xenografts, germ cell density was also reduced by BPA exposure. Importantly, BPA exposure significantly decreased the percentage of germ cells expressing the pluripotency marker AP-2γ, whilst the percentage of those expressing the pre-spermatogonial marker MAGE-A4 significantly increased. BPA exposure did not affect hCG-stimulated androgen production in first and second trimester xenografts as evaluated by both plasma testosterone level and seminal vesicle weight in host mice.</p><p>Conclusions</p><p>Exposure to BPA at environmentally relevant concentrations impairs germ cell development in first trimester human fetal testis, whilst gonadotrophin-stimulated testosterone production was unaffected in both first and second trimester testis. Studies using first trimester human fetal testis demonstrate the complementarity of the FeTA and xenograft models for determining the respective short-term and long term effects of environmental exposures.</p></div

Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells

The insulin family of growth factors (insulin, IGF1, and IGF2) are critical in sex determination, adrenal differentiation, and testicular function. Notably, the IGF system has been reported to mediate the proliferation of steroidogenic cells. However, the precise role and contribution of the membrane receptors mediating those effects, namely, insulin receptor (INSR) and type-I insulin-like growth factor receptor (IGF1R), have not, to our knowledge, been investigated. We show here that specific deletion of both Insr and Igf1r in steroidogenic cells in mice leads to severe alterations of adrenocortical and testicular development. Double-mutant mice display drastic size reduction of both adrenocortex and testes, with impaired corticosterone, testosterone, and sperm production. Detailed developmental analysis of the testes revealed that fetal Leydig cell (LC) function is normal, but there is a failure of adult LC maturation and steroidogenic function associated with accumulation of progenitor LCs (PLCs). Cell-lineage tracing revealed PLC enrichment is secondary to Insr and Igf1r deletion in differentiated adult LCs, suggesting a feedback mechanism between cells at different steps of differentiation. Taken together, these data reveal the cell-autonomous and nonautonomous roles of the IGF system for proper development and maintenance of steroidogenic lineages.-Neirijnck, Y., Calvel, P., Kilcoyne, K. R., Kühne, F., Stévant, I., Griffeth, R. J., Pitetti, J.-L., Andric, S. A., Hu, M.-C., Pralong, F., Smith, L. B., Nef, S. Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells