Energy status and ovarian follicular development

Female reproduction is tightly linked to body energy status and it has become increasingly clear that disturbed energy metabolism can negatively affect reproductive performance. Nevertheless, the way how a disturbed energy status affects ovarian follicular reserve as well as follicular recruitment and growth is little investigated and not fully elucidated. Therefore, the overall goal of this thesis was to investigate the effects of an altered metabolism, and particularly an altered energy status, on ovarian follicular development. To achieve this goal, the first aim was to establish the role of autophagy in follicular degeneration under normal physiological conditions, with focus on preantral and antral follicles; The second aim was to elucidate the effects of a diet-induced reduction in thyroid hormone concentrations, affecting whole body metabolism, on ovarian follicular development; The third aim was to investigate the effect of an increased nutrient flux towards skeletal muscle on ovarian follicular development and the possible underlying mechanism. It is well known that granulosa cell death via apoptosis is the main cause of atresia of antral follicles, however, whether preantral follicular attrition makes use of the same cell death pathway is not clear. Therefore, in <strong>chapter 2</strong> I have investigated different cell death pathways in the adult rat ovary to examine whether they represent the reported histological differences between preantral and antral atretic follicles. Based on the results of studies in other organs, I used microtubule-associated light-chain protein 3 (LC3) and QSQTM1/p62 as markers of autophagy and cleaved caspase 3 (cCASP3) as marker of apoptosis, using immunohistochemistry, western blotting, and laser capture micro-dissection followed by qRT-PCR. The results showed that in the granulosa cells of atretic preantral follicles, p62 immunostaining was less intense compared to healthy preantral follicles, while no difference in LC3 immunostaining intensity was observed. In contrast, in antral follicles, no difference in both immunostaining and mRNA levels of LC3 and p62 were found between healthy and atretic follicles, indicating that autophagy was not responsible for attrition of antral follicles. cCASP3 immunostaining was scarce in the granulosa cells of atretic preantral follicles, whereas many cCASP3 positive apoptotic cells were present in atretic antral follicles, indicating that apoptosis is a major cell death pathway activated in antral follicle degeneration. Immunostaining for superoxide dismutase 2 (SOD2) was reduced in preantral and antral atretic follicles. This observation was confirmed by a concomitant down regulation of <em>Sod2</em> mRNA levels. These findings suggest that preantral follicular atresia mainly makes use of autophagy as cell death pathway, while antral follicles degenerate mainly via apoptosis. In <strong>chapter 3</strong>, the consequences of prolonged exposure to reduced thyroid hormone concentrations in adulthood on the size of the ovarian follicle pool are investigated. Besides having a direct effect on the functioning of many cells, changes in thyroid hormone levels also influence metabolism. In this study female rats at the age of 10 weeks were given a control diet or an iodide deficient diet in combination with perchlorate supplementation to inhibit iodide uptake by the thyroid, resulting in a relatively mild chronic hypothyroid condition. At the age of 26 weeks animals were sacrificed and ovaries histologically evaluated. Plasma concentrations of relevant hormones (thyroid-stimulating hormone (TSH), tri-iodothyronine (T3), thyroxine (T4), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and anti-Müllerian hormone (AMH) were determined. Primordial, primary and preantral follicle numbers were significantly lower in the hypothyroid ovaries compared to the euthyroid controls, while a downward trend in antral follicle numbers and corpora lutea was observed. The percentage of atretic follicles was not different between the two groups. Plasma AMH concentrations showed a significant correlation with the growing follicle population represented by the total number of primary, preantral and antral follicles per ovary. The data indicate that prolonged mild hypothyroidism negatively affects ovarian follicular reserve as well as the size of the growing follicle population, which may impact fertility. AMH can serve, also under mild hypothyroid conditions, as a surrogate marker to assess the size of the growing ovarian follicle population, offering a non-invasive way to evaluate the correlation between female reproductive health and thyroid status. Subsequently, in <strong>chapter 4, </strong>the long-term effects of chronic hypothyroidism initiated already in the foetal/neonatal period on ovarian follicular development were investigated. In contrast to the experiments described in <strong>chapter 3</strong>, the rats in this experiment were exposed to reduced thyroid hormone levels from the moment of conception until necropsy. Effects on the ovarian follicular reserve and ovulation rate in prepubertal (12-day-old) and adult (64-day-old and 120-day-old) rats were studied. Besides, antioxidant gene expression, mitochondrial density and the occurrence of oxidative stress were analyzed. The results of this investigation showed that continuous fetal/postnatal hypothyroidism resulted in lower preantral and antral follicle numbers in adulthood, accompanied by a higher percentage of atretic follicles, when compared to euthyroid age-matched controls. Not surprisingly, ovulation rate was lower in the hypothyroid rats. At the age of 120 days, the mRNA and protein content of superoxide dismutase 1 (SOD1) was significantly increased, while catalase (CAT) mRNA and protein content was significantly decreased, suggesting a disturbed antioxidant defense capacity of ovarian cells in the hypothyroid animals. This was supported by a significant reduction in peroxiredoxin 3 (<em>Prdx3</em>), thioredoxin reductase 1 (<em>Txnrd1</em>), and uncoupling protein 2 (<em>Ucp2</em>) mRNA content and a downward trend in glutathione peroxidase 3 (<em>Gpx3</em>) and glutathione S-transferase mu 2 (<em>Gstm2</em>) mRNA content. These changes in gene expression were likely responsible for the increased immunostaining of the oxidative stress marker 4-hydroxynonenal. Together these results suggest that chronic hypothyroidism initiated in the foetal/neonatal period resulted in a decreased ovulation rate associated with a disturbance of the antioxidant defense system in the ovary. In contrast to hypothyroidism induced in adulthood (<strong>chapter 3</strong>), no reduction in primordial or primary follicle numbers was observed, suggesting that the ovarian reserve was not affected. <strong>C</strong><strong>hapter 5</strong> addressed the question what the consequences were of a change in nutrient flux on ovarian follicular development. In this chapter mice were employed that ectopically express uncoupling protein 1 (UCP1) in skeletal muscle (UCP1-TG). This did not affect adiposity, but led to a redistribution of energy sources away from the ovaries towards skeletal muscle tissue,; a model of skeletal muscle pseudo-starvation. The results showed that UCP1-TG female mice had increased energy expenditure, reduced body size, unchanged adiposity, increased plasma fibroblast growth factor 21 (FGF21) concentrations and reduced insulin-like growth factor 1 (IGF1) levels. UCP1-TG mice had a 30% lower number of healthy follicle compared to WT mice. Primary and preantral follicle numbers were decreased by 40%, while the number of atretic follicles was significantly increased and corpora lutea (CL) were absent in 40% of the ovaries of UCP1-TG mice. The latter suggested that these mice did not ovulate and thus were infertile. The elevated circulating FGF21 concentrations were not responsible for the ovarian phenotype, since UCP1-TG and UCP1-TG/FG21-/- mice show the same ovarian follicular phenotype. Significant correlation of circulating IGF1 levels with antral follicle, CL numbers and differentially activated AKT in healthy antral follicles and activated IRS2 in atretic follicles between WT and UCP1-TG mice shows, that IGF1 is, at least partly, responsible for the ovarian phenotype of these mice. Together, our data show that an energy drain towards skeletal muscle tissue negatively impacts growing pool of ovarian follicles and ovulation rate in female mice, which is, at least in part, mediated by IGF1, and not by FGF21. In conclusion, the results of my thesis research shows that preantral atresia occurs mainly through autophagy. Dietary induced chronic hypothyroidism, an intervention that reduces basal metabolic rate, initiated either during foetal/neonatal or adulthood impairs ovarian follicle development. The age at onset of hypothyroidism modified the effects of this condition on ovarian follicular development. A change in nutrient flux away from the ovaries towards skeletal muscle tissue negatively affects ovarian follicle development. Overall, the results of my thesis have provided new insights in the mechanisms of follicular attrition and shows that conditions that alter metabolic fuel use impact on ovarian follicular development.