Role of peroxisomes in granulosa cells, follicular development and steroidogenesis in the mouse ovary

Abstract

Human disorders with peroxisomal deficiency show a range of pathologies in the reproductive system leading to human infertility. In recent years, the relationship between peroxisomes and male infertility has been examined in different groups. However, until now nobody has focused on the pathological consequences happening in the ovary in case of peroxisomal deficiency. Peroxisomes are cell organelles involved in fatty acid ß-oxidation, plasmalogen synthesis and cholesterol synthesis on which steroidogenesis depends. Besides this, peroxisomes are able to maintain the equilibrium between production and scavenging of ROS, which are involved in the regulation of follicular development. The aim of this thesis was therefore to study the role of peroxisomes in granulosa cells, follicular development and steroid synthesis in the ovary.Paraformaldehyde-fixed paraffin-embedded (FFPE) sections of adult mouse ovaries were stained to analyze peroxisome distribution and regulation during follicular development. A highly differentiated mouse granulosa tumor cell line (KK-1 cells) was established as cell culture model to study peroxisomal function in granulosa cells. Peroxisome related gene expression and protein abundance were compared before and after hCG treatment in KK-1 cells. Moreover, the Pex13 gene, encodes a peroxisomal biogenesis protein, was knocked down in KK-1 cells by Pex13 RNAi. Moreover, the effects of the peroxisomal deficiency on mitochondrial cholesterol transport (StAR protein) as well as steroidogenesis were analyzed. As trial to stimulate StAR cholesterol transportation, the cloned A kinase anchoring protein Akap1 was overexpressed in KK-1 cells. Thereafter, H2O2 treatment and a-Tocopherol treatment were used to figure out the influence of ROS and antioxidants on steroidogenesis respectively. The results of this thesis provide clear evidence that peroxisomal proteins are highly abundant in the mouse ovary and exhibit a distinct heterogeneous location pattern during folliculogenesis. Generally, the abundance of peroxisomal proteins is increasing in oocytes during follicular development, suggesting that peroxisomes are protecting mature oocytes against ROS and lipotoxicity. The in vitro studies showed with KK-1 cells that hCG induced the upregulation of peroxisomal proteins and their corresponding mRNAs, suggesting a strong regulatory effect of gonadotropic hormones on the peroxisomal compartment in granulosa cells. Under peroxisome deficiency conditions, induced by the Pex13 KD, the steroidogenic pathway in granulosa cells was disturbed and the peroxisome deficiency-exerted reduction of the steroid hormone secretion is at least partially mediated via StAR inhibition. The underlying mechanisms for this may at least partly result from increased oxidative stress after the Pex13 KD. In this thesis it was indeed shown that ROS were increased after the Pex13 KD and it is well known that excessive ROS leads to the inhibition of steroidogenesis. This hypothesis is further confirmed by the protective effect of tocopherol on StAR protein and progesterone synthesis under oxidative stress induced by the peroxisomal deficiency. Moreover, the results of this thesis provide also evidence that an increase of AKAP1 can also partly compensate the peroxisomal deficiency- induced mitochondrial alteration. In addition, it was shown in the literature that the lipid structure and the lipid composition of cell membrane are related to the activity of adenylyl cyclase (AC) and G protein-coupled receptor signaling, but the direct relationship between peroxisomal deficiency with adenylyl cyclase needs to be clarified in the future.