Reproductive and metabolic programming by exogenous steroids

Abstract

Polycystic ovary syndrome (PCOS) is a heterogeneous disorder encompassing reproductive and metabolic phenotypes. Genetic analysis, targeting candidate genes has to date proven unsuccessful in the search for a truly dominant genetic link. Another hypothesis to explain the etiology of PCOS is that of fetal programming in the context of developmental origins of health and disease. Extensive animal studies, validated by human data, support the fetal origins hypothesis of PCOS and highlight that PCOS may arise due to excess androgen exposure in fetal life. Previous reports from our laboratory found metabolic dysfunction in 11 month old prenatally androgenised females (d62-102 of fetal life), which included pancreatic and hepatic alterations. The pancreatic alterations seemed to result from gene expression changes induced in fetal life. Therefore, chapter 3 focuses on the gluconeogenic response in the day 90 fetus following maternal androgenisation from day 62 of gestation. Interestingly hepatic gluconeogenic enzymes, specifically phosphoenolpyruvate caboxykinase (PEPCK) and glucose 6 phosphatase (G6PC), were not altered. However they were decreased in the kidney, in a sex specific manner with PEPCK significantly decreased (P<0.01) and G6PC showing a strong trend toward reduction (P=0.056) in females only. This chapter progresses to explore regulatory pathways involved in gluconeogenic regulation. It seems probable that the female specific increase in circulating testosterone (P<0.001), with increased renal androgen reception (P<0.01), may be accountable for the altered expression of gluconeogenic enzymes in the kidney. Chapter 4 investigates why testosterone concentrations were not increased in the male fetus, after maternal androgenisation, by focusing on the site of testosterone production, the fetal testis. Results demonstrate that the day 90 fetus is capable of responding to prenatal androgenisation by decreasing luteinising hormone (P<0.01) and thus testicular testosterone production, such that there was a global down regulation in steroidogenic enzyme expression, in vivo testosterone production (P<0.001) and Leydig cell morphology was altered (P<0.001). As prenatal androgenisation is administered through the maternal route and placental aromatisation may occur, a novel method whereby the fetus was directly injected was utilised to assess the effects of control oil (C), testosterone (TP) or diethylstilboestrol (DES) on the fetal testis. Unlike DES, direct fetal injection with TP mimics the results found from maternal androgenisation. When the testis are examined at a later date, day 112, ten days after androgen treatment ceases, Leydig cell morphology and steroidogenic gene expression return to control values, although fascinatingly, an overshoot of in vivo testosterone production (P<0.01) was observed. When the maternal androgenisation window is extended to begin at day 30 of fetal life, further changes are noted including increased circulating testosterone (P<0.01), a strong trend toward decreased testis weight (P=0.0519) and altered expression of Sertoli and germ cell specific markers. These studies are followed up by assessing the legacy effect of testosterone on the peripubertal male testis in Chapter 5. At ten weeks of postnatal life, males, exposed to androgens from day 62-102 of fetal life had reduced testis weight (P<0.05). However, functional or cellular alterations were not observed and by 12 weeks of age, when LH had normalised, testicular weight and stimulated testosterone secretion of prenatally TP-treated males was comparable to controls. This highlights the remarkable plasticity of the testis and the unremarkable legacy of altered prenatal androgen exposure. The legacy effect of testosterone on the fetal ovary is examined in Chapter 6. Previous studies from our laboratory found minor functional alterations but no structural alterations in the fetal ovary at day 90 following androgenisation from day 62. However, as this was at a time of a highly androgenic environment we assessed the function and morphology of the ovary ten days after the removal of testosterone at day 112. In marked contrast to the normalisation of the male gonad, we observe structural changes with an increase in recruited follicles from the primordial to primary stage in the testosterone treated group (P<0.01). The chapter continues with an investigation of pathways involved in the altered follicular dynamics that may account for the change in follicular recruitment. Furthermore, the functional changes which were previously noted in the day 90 ovary were also examined in response to direct exogenous steroid treatment including, C, TP, DES and dexamethasone (DEX) and also when the window of maternal androgenisation was extended to begin at day 30. Interesting changes are observed such that the direct fetal injection treatments induce similar changes to each other, regardless of the steroid, whilst maternal androgenisation induces a different response. This highlights the complexity of the pathways involved in female gonadal development.