Hepatic Mitochondrial Dysfunction and Risk of Liver Disease in an Ovine Model of "PCOS Males".

First-degree male relatives of polycystic ovary syndrome (PCOS) sufferers can develop metabolic abnormalities evidenced by elevated circulating cholesterol and triglycerides, suggestive of a male PCOS equivalent. Similarly, male sheep overexposed to excess androgens in fetal life develop dyslipidaemia in adolescence. Dyslipidaemia, altered lipid metabolism, and dysfunctional hepatic mitochondria are associated with the development of non-alcoholic liver disease (NAFLD). We therefore dissected hepatic mitochondrial function and lipid metabolism in adolescent prenatally androgenized (PA) males from an ovine model of PCOS. Testosterone was directly administered to male ovine fetuses to create prenatal androgenic overexposure. Liver RNA sequencing and proteomics occurred at 6 months of age. Hepatic lipids, glycogen, ATP, reactive oxygen species (ROS), DNA damage, and collagen were assessed. Adolescent PA males had an increased accumulation of hepatic cholesterol and glycogen, together with perturbed glucose and fatty acid metabolism, mitochondrial dysfunction, with altered mitochondrial transport, decreased oxidative phosphorylation and ATP synthesis, and impaired mitophagy. Mitochondrial dysfunction in PA males was associated with increased hepatic ROS level and signs of early liver fibrosis, with clinical relevance to NAFLD progression. We conclude that excess in utero androgen exposure in male fetuses leads to a PCOS-like metabolic phenotype with dysregulated mitochondrial function and likely lifelong health sequelae.

Pubertal FGF21 deficit is central in the metabolic pathophysiology of an ovine model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS), affecting over 10% of women, is associated with insulin resistance, obesity, dyslipidaemia, fatty liver and adipose tissue dysfunction. Its pathogenesis is poorly understood and consequently treatment remains suboptimal. Prenatally androgenized (PA) sheep, a clinically realistic model of PCOS, recapitulate the metabolic problems associated with PCOS. Fibroblast Growth Factor 21 (FGF21) is a metabolic hormone regulating lipid homeostasis, insulin sensitivity, energy balance and adipose tissue function. We therefore investigated the role of FGF21 in the metabolic phenotype of PA sheep. In adolescence PA sheep had decreased hepatic expression and circulating concentrations of FGF21. Adolescent PA sheep show decreased FGF21 signalling in subcutaneous adipose tissue, increased hepatic triglyceride content, trend towards reduced fatty acid oxidation capacity and increased hepatic expression of inflammatory markers. These data parallel studies on FGF21 deficiency, suggesting that FGF21 therapy during adolescence may represent a treatment strategy to mitigate metabolic problems associated with PCOS.

Chlamydia inhibits progesterone receptor mRNA expression in SHT-290 cells.

Chlamydia trachomatis is the most commonly diagnosed sexually transmitted infection in the UK, with over 200,000 positive diagnoses annually. The infection is thought to cause reproductive complications including problems in conceiving a pregnancy through to miscarriage and early or stillbirth. One potential reason Chlamydia may impact upon pregnancy is through disrupting the embryo implantation at the earliest stages of pregnancy is by altering the ability of specific cells that line the uterus called stromal cells to respond to the hormone progesterone, the hormone responsible for preparing the uterus for pregnancy. The results of this study showed that Chlamydial infection of these uterus lining stromal cells decreased the levels of specific progesterone sensitive markers which are associated with early embryo implantation, suggesting a loss of responsiveness to progesterone treatment. These changes were accompanied by a decrease in the levels of RNA for the progesterone receptor which is responsible for progesterone activity, suggesting that this is a potential mechanism through which Chlamydia could directly inhibit the effects of progesterone on uterine cells.

Aberrant subcutaneous adipogenesis precedes adult metabolic dysfunction in an ovine model of polycystic ovary syndrome (PCOS).

Polycystic ovary syndrome (PCOS) affects over 10% of women. Insulin resistance, elevated free fatty acids (FFAs) and increased adiposity are key factors contributing to metabolic dysfunction in PCOS. We hypothesised that aberrant adipogenesis during adolescence, and downstream metabolic perturbations, contributes to the metabolic phenotype of adult PCOS. We used prenatally androgenised (PA) sheep as a clinically realistic model of PCOS. During adolescence, but not during fetal or early life of PA sheep, adipogenesis was decreased in subcutaneous adipose tissue (SAT) accompanied by decreased leptin, adiponectin, and increased FFAs. In adulthood, PA sheep developed adipocyte hypertrophy in SAT paralleled by increased expression of inflammatory markers, elevated FFAs and increased expression of genes linked to fat accumulation in visceral adipose tissue. This study provides better understanding into the pathophysiology of PCOS from puberty to adulthood and identifies opportunity for early clinical intervention to normalise adipogenesis and ameliorate the metabolic phenotype.

Early pregnancy maternal progesterone administration alters pituitary and testis function and steroid profile in male fetuses.

Maternal exposure to increased steroid hormones, including estrogens, androgens or glucocorticoids during pregnancy results in chronic conditions in offspring that manifest in adulthood. Little is known about effects of progesterone administration in early pregnancy on fetal development. We hypothesised that maternal early pregnancy progesterone supplementation would increase fetal progesterone, affect progesterone target tissues in the developing fetal reproductive system and be metabolised to other bioactive steroids in the fetus. We investigated the effects of progesterone treatment during early pregnancy on maternal and fetal plasma progesterone concentrations, transcript abundance in the fetal pituitary and testes and circulating steroids, at day 75 gestation, using a clinically realistic ovine model. Endogenous progesterone concentrations were lower in male than female fetuses. Maternal progesterone administration increased male, but not female, fetal progesterone concentrations, also increasing circulating 11-dehydrocorticosterone in male fetuses. Maternal progesterone administration altered fetal pituitary and testicular function in ovine male fetuses. This suggests that there may be fetal sex specific effects of the use of progesterone in early pregnancy, and highlights that progesterone supplementation should be used only when there is clear evidence of efficacy and for as limited time as necessary.

Fetal androgen exposure is a determinant of adult male metabolic health.

Androgen signalling is a critical driver of male development. Fetal steroid signalling can be dysregulated by a range of environmental insults and clinical conditions. We hypothesised that poor adult male health was partially attributable to aberrant androgen exposure during development. Testosterone was directly administered to developing male ovine fetuses to model excess prenatal androgenic overexposure associated with conditions such as polycystic ovary syndrome (PCOS). Such in utero androgen excess recreated the dyslipidaemia and hormonal profile observed in sons of PCOS patients. 1,084 of 15,134 and 408 of 2,766 quantifiable genes and proteins respectively, were altered in the liver during adolescence, attributable to fetal androgen excess. Furthermore, prenatal androgen excess predisposed to adolescent development of an intrahepatic cholestasis-like condition with attendant hypercholesterolaemia and an emergent pro-fibrotic, pro-oxidative stress gene and protein expression profile evident in both liver and circulation. We conclude that prenatal androgen excess is a previously unrecognised determinant of lifelong male metabolic health.

Urinary estrogens as a non-invasive biomarker of viable pregnancy in the giant panda (Ailuropoda melanoleuca).

Female giant pandas show complex reproductive traits, being seasonally monoestrus, displaying a variable length embryonic diapause and exhibiting pseudopregnancy. Currently, there is no confirmatory non-invasive biomarker of blastocyst implantation or pregnancy. This study aimed to monitor urinary estrogens across gestation in pregnancy (n = 4), pseudopregnancy (n = 4) and non-birth cycles (n = 5) in the giant panda. A pregnancy-specific profile of estrogens corrected for urinary specific gravity was identified during the gestation period. Pregnant females showed increasing concentrations of estrogens for 29 days until birth, no increase was observed during pseudopregnancy and the two profiles were distinguishable from each other for the final 2 weeks of the cycle suggesting the estrogens are of placental origin. This allowed a nomogram, starting at a known fixed point during the cycle, to be created and tested with cycles of known outcome, and cycles which were inseminated but did not result in a birth. Non-birth profiles showed deviations from that of pregnancy. We believe these deviations indicate the point of failure of the placenta to support a developing cub. Non-invasive longitudinal monitoring of estrogen concentrations therefore has the potential to be developed as a panda pregnancy test to predict viable cub development.

Urinary specific gravity as an alternative for the normalisation of endocrine metabolite concentrations in giant panda (Ailuropoda melanoleuca) reproductive monitoring.

Reproductive monitoring for captive breeding in giant pandas is based on behavioural observation and non-invasive hormone analysis. In urine, interpretation of results requires normalisation due to an animal's changing hydration. Correction of urinary concentrations based on creatinine is the gold standard. In this study, a largely unexplored, easy-to-perform normalisation technique, based on urinary specific gravity (USpG), was examined and compared to creatinine. To this extent, six cycles from two female pandas (SB741(1) and SB569(5)) were monitored through urine analysis for oestrogen, progesterone, ceruloplasmin and 13,14-dihydro-15-keto-PGF2a (PGFM). The Pearson's correlation between creatinine and USpG was high (r = 0.805-0.894; p < 0.01), indicative for a similar performance of both normalisation methods. However, generally lower values were observed during pro-oestrus and primary (progesterone) rise. This could be associated with huge shifts in appetite, monitored by faecal output (kg) with an averaged > 50% decrease during oestrus and >50% increase during primary progesterone rise. In parallel, respectively highest and lowest creatinine and USpG levels, were measured, with creatinine obviously more affected as a result of linkage with muscle tissue metabolism affected by reproductive hormones. As a consequence, metabolite levels were significantly different between both corrected datasets with significantly higher oestrogen peak levels during oestrus ranging from 2.13-86.93 and 31.61-306.45 ng/mL (USpG correction) versus 2.33-31.20 and 36.36-249.05 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively, and significant lower progesterone levels during primary progesterone rise ranging from 0.35-3.21 and 0.85-6.80 ng/mL (USpG correction) versus 0.52-10.31 and 2.10-272.74 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively. Consequently, USpG correction rendered unbiased profiles, less subject to variation and metabolic artefacts and therefore allowed a more straightforward identification of peak oestrogen and onset of secondary progesterone rise, being potentially advantageous for future studies unravelling key giant panda reproductive events, including (delayed) implantation. The alternative application of USpG as a normalisation factor was further supported by its easy application and environmental and technical robustness.

Preferential activation of HIF-2α adaptive signalling in neuronal-like cells in response to acute hypoxia.

Stroke causes severe neuronal damage as disrupted cerebral blood flow starves neurons of oxygen and glucose. The hypoxia inducible factors (HIF-1α and HIF-2α) orchestrate oxygen homeostasis and regulate specific aspects of hypoxic adaptation. Here we show the importance of HIF-2α dependant signalling in neuronal adaptation to hypoxic insult. PC12 and NT2 cells were differentiated into neuronal-like cells using NGF and retinoic acid, and exposed to acute hypoxia (1% O2). Gene and protein expression was analysed by qPCR and immunoblotting and the neuronal-like phenotype was examined. PC12 and NT2 differentiation promoted neurite extension and expression of neuronal markers, NSE and KCC2. Induction of HIF-1α mRNA or protein was not detected in hypoxic neuronal-like cells, however marked induction of HIF-2α mRNA and protein expression was observed. Induction of HIF-1α target genes was also not detected in response to acute hypoxia, however significant induction of HIF-2α transcriptional targets was clearly evident. Furthermore, hypoxic insult dramatically reduced both neurite number and length, and attenuated expression of neuronal markers, NSE and KCC2. This correlated with an increase in expression of the neural progenitor and stem cell-like markers, CD44 and vimentin, suggesting HIF-2α molecular mechanisms could potentially promote regression of neuronal-like cells to a stem-like state and trigger neuronal recovery following ischaemic insult. Our findings suggest the HIF-2α pathway predominates over HIF-1α signalling in neuronal-like cells following acute hypoxia.

Waddlia chondrophila infects and multiplies in ovine trophoblast cells stimulating an inflammatory immune response.

BACKGROUND: Waddlia chondrophila (W. chondrophila) is an emerging abortifacient organism which has been identified in the placentae of humans and cattle. The organism is a member of the order Chlamydiales, and shares many similarities at the genome level and in growth studies with other well-characterised zoonotic chlamydial abortifacients, such as Chlamydia abortus (C. abortus). This study investigates the growth of the organism and its effects upon pro-inflammatory cytokine expression in a ruminant placental cell line which we have previously utilised in a model of C. abortus pathogenicity. METHODOLOGY/PRINCIPAL FINDINGS: Using qPCR, fluorescent immunocytochemistry and electron microscopy, we characterised the infection and growth of W. chondrophila within the ovine trophoblast AH-1 cell line. Inclusions were visible from 6 h post-infection (p.i.) and exponential growth of the organism could be observed over a 60 h time-course, with significant levels of host cell lysis being observed only after 36 h p.i. Expression of CXCL8, TNF-α, IL-1α and IL-1ß were determined 24 h p.i. A statistically significant response in the expression of CXCL8, TNF-α and IL-1ß could be observed following active infection with W. chondrophila. However a significant increase in IL-1ß expression was also observed following the exposure of cells to UV-killed organisms, indicating the stimulation of multiple innate recognition pathways. CONCLUSIONS/SIGNIFICANCE: W. chondrophila infects and grows in the ruminant trophoblast AH-1 cell line exhibiting a complete chlamydial replicative cycle. Infection of the trophoblasts resulted in the expression of pro-inflammatory cytokines in a dose-dependent manner similar to that observed with C. abortus in previous studies, suggesting similarities in the pathogenesis of infection between the two organisms.

The pancreas is altered by in utero androgen exposure: implications for clinical conditions such as polycystic ovary syndrome (PCOS).

Using an ovine model of polycystic ovary syndrome (PCOS), (pregnant ewes injected with testosterone propionate (TP) (100 mg twice weekly) from day (d)62 to d102 of d147 gestation (maternal injection - MI-TP)), we previously reported female offspring with normal glucose tolerance but hyperinsulinemia. We therefore examined insulin signalling and pancreatic morphology in these offspring using quantitative (Q) RT-PCR and western blotting. In addition the fetal pancreatic responses to MI-TP, and androgenic and estrogenic contributions to such responses (direct fetal injection (FI) of TP (20 mg) or diethylstilbestrol (DES) (20 mg) at d62 and d82 gestation) were assessed at d90 gestation. Fetal plasma was assayed for insulin, testosterone and estradiol, pancreatic tissue was cultured, and expression of key ß-cell developmental genes was assessed by QRT-PCR. In female d62MI-TP offspring insulin signalling was unaltered but there was a pancreatic phenotype with increased numbers of ß-cells (P<0.05). The fetal pancreas expressed androgen receptors in islets and genes involved in ß-cell development and function (PDX1, IGF1R, INSR and INS) were up-regulated in female fetuses after d62MI-TP treatment (P<0.05-0.01). In addition the d62MI-TP pancreas showed increased insulin secretion under euglycaemic conditions (P<0.05) in vitro. The same effects were not seen in the male fetal pancreas or when MI-TP was started at d30, before the male programming window. As d62MI-TP increased both fetal plasma testosterone (P<0.05) and estradiol concentrations (P<0.05) we assessed the relative contribution of androgens and estrogens. FI-TP (commencing d62) (not FI-DES treatment) caused elevated basal insulin secretion in vitro and the genes altered by d62MI-TP treatment were similarly altered by FI-TP but not FI-DES. In conclusion, androgen over-exposure alters fetal pancreatic development and ß-cell numbers in offspring. These data suggest that that there may be a primary pancreatic phenotype in models of PCOS, and that there may be a distinct male and female pancreas.

Cortisol inactivation by 11beta-hydroxysteroid dehydrogenase-2 may enhance endometrial angiogenesis via reduced thrombospondin-1 in heavy menstruation.

CONTEXT: Heavy menstrual bleeding (HMB; menorrhagia) impairs quality of life for women and requires medication or surgery. Because glucocorticoids inhibit angiogenesis in other organs, we hypothesized that endometrium of women with HMB is subject to decreased local glucocorticoid exposure and enhanced angiogenesis, thereby increasing menstrual bleeding. DESIGN: Endometrium was collected from 29 women with menstrual complaints. Menstrual blood loss was measured by alkaline-hematin assay (n = 12, > 80 ml (HMB); n = 17, < 80 ml). Quantitative RT-PCR for thrombospondin-1 (TSP-1) and glucocorticoid-metabolizing enzymes, 11beta-hydroxysteroid dehydrogenases-1 and -2 (11betaHSD1,2) was performed. Glucocorticoid effects on endometrial stromal cells and uterine endothelial cells (UECs) were determined. RNA interference studies in UECs examined the effect of TSP-1 ablation on cortisol action. RESULTS: Secretory phase endometrium mRNA levels for the cortisol inactivating enzyme 11betaHSD2 were higher [3.78 +/- 1.29 vs. 1.40 +/- 0.6 (arbitrary units), P < 0.05], whereas TSP-1 mRNA was lower [0.40 +/- 0.13 vs. 1.66 +/- 1.02 (arbitrary units), P < 0.05] in women with HMB. In cultured endometrial stromal cells and UECs, cortisol increased TSP-1 expression. Both cortisol and TSP-1 inhibited new vessel formation in endometrial explants embedded in Matrigel. In UECs cortisol inhibition of tube-like structure formation was blocked by small interfering RNA (siRNA) against TSP-1 (25 +/- 2.5% cortisol inhibition with scrambled siRNA vs. 0% cortisol inhibition with TSP-1 siRNA inactivation, P<0.01). CONCLUSIONS: Enhanced inactivation of cortisol by 11betaHSD2 in endometrium from women with HMB may explain reduced TSP-1 levels and hence endothelial cell dysfunction and abnormal angiogenesis. Inhibition of 11betaHSD2 may be a rational novel therapy for heavy menstrual bleeding.

Effects of prenatal undernutrition on emotional reactivity and cognitive flexibility in adult sheep.

The experiment reported was designed to test two hypotheses: that prenatal undernutrition (a) increases emotional reactivity and (b) impairs cognitive flexibility in sheep. The mothers of one group were fed live weight maintenance requirements throughout pregnancy (control, C) while those of another group were fed 50% of that amount from days 1 to 95 of pregnancy and 100% from then onwards (prenatal undernutrition, PU). At 18 months of age, PU sheep were more active during restraint (P < 0.05) and approached a novel stimulus more slowly (P = 0.02). In response to a sudden stimulus, PU males and C females showed a higher initial level of locomotion compared to C males, which only gradually declined, while PU females started at a high initial rate, changing rapidly to immobility. In a T-maze, PU resulted in a shift of side preferences (laterality) from a general right-bias to neutrality in males and to a left-bias in females (P < 0.05). In the two reversal tasks, C males and PU females had a preference for one side over the other, while PU males showed no preference. In contrast to C males, PU males failed to improve their learning speed from the first to the second reversal (P < 0.05). It is concluded that PU can lead to increased emotional reactivity and changes in side preference in both sexes and impaired cognitive flexibility in males. Undernutrition during pregnancy, therefore, not only affects the welfare of the dam, but also the personality of her offspring.

Lysyl oxidase gene expression and enzyme activity in the rat ovary: regulation by follicle-stimulating hormone, androgen, and transforming growth factor-beta superfamily members in vitro.

Lysyl oxidase (LOX) catalyzes the final enzymatic reaction required for cross-linking of collagen and elastin fibers and therefore has a crucial role in regulating the formation and maintenance of extracellular matrix in the ovary. LOX mRNA is abundantly expressed in rat granulosa cells. To examine how regulation of LOX in the ovary might influence follicular development, we studied LOX mRNA expression and enzyme activity in rat granulosa cells from late preantral/early antral follicles in vitro. FSH dose dependently inhibited LOX mRNA and enzyme activity (50% reduction at 10 ng/ml) in vitro, and FSH action was mimicked by 8-bromo-cAMP, suggesting FSH action via elevation of cAMP. Dihydrotestosterone alone enhanced LOX mRNA and enzyme activity, but potentiated the effect of FSH, causing a further reduction. TGFbeta1 alone dose dependently enhanced LOX mRNA (5-fold increase at 10 ng/ml) and activity (1.5-fold increase). FSH dose dependently inhibited the increase in LOX mRNA and activity caused by TGFbeta1 (by up to 84% and 80%, respectively). Growth differentiation factor-9 (GDF-9) and activin A, at the same concentration as TGFbeta1 (10 ng/ml), stimulated LOX mRNA and activity within 6 h, although overall expression was higher at 48 h. All three factors when combined with FSH further reduced both mRNA and enzyme activity (by up to 60%) compared with FSH alone. These findings indicate control of LOX at endocrine, paracrine, and autocrine levels within the ovary and suggest coordinated regulation of ovarian extracellular matrix during follicular development, with FSH determining whether local factors act as stimulators or inhibitors of LOX.