Oxidative stress has been implicated in an extensive range of age-related pathologies including neurodegenerative disease, and is a well-known cause of infertility. The negative impacts of oxidative stress in the male germline are primarily underpinned by the peroxidation of fatty acids, resulting in the production of highly reactive lipid aldehydes, such as 4-hydroxynonenal (4HNE). In many cell types, the portion of the cell proteome that is targeted for 4HNE-modification often experiences severe protein misfolding that, in turn, leads to a disruption of protein homeostasis (proteostasis). This study was designed to explore a relationship between oxidative stress and protein aggregation in the male germline with a key focus on determining the protective mechanisms employed by germ cells to prevent protein damage.
Through the development of several robust strategies for the detection of protein aggregates, this study has revealed a causative role for oxidative stress in the induction of protein aggregation in both pachytene spermatocytes and round spermatids. Specifically, the exogenous application of 4HNE to these cells resulted in a significant increase in aggregation (P<0.005) detectable with the amyloid-specific fluorophores Proteostat and Thioflavin T, and the conformer specific antibodies anti-A11 and anti-OC. In this study, nucleocytoplasmic transport machinery was examined as a potential mechanism for the subcellular compartmentalisation of aggregating proteins. The inhibition of transport proteins karyopherin beta 1/alpha 2 (KPNB1/A2) and exportin 1 (XPO1), resulted in a significant increase in cellular protein aggregates (P < 0.005). Similarly, the inhibition of the molecular chaperone Heat Shock Protein 90 (HSP90) also resulted in a significant increase in protein aggregation (P<0.005). These results shed light on two mechanisms that may assist in the management of misfolded proteins in the male germline. These important leads provide new targets to enhance the innate defences of germ cells against oxidative stress and better maintain germline proteostasis.