In the spermatozoa of infertile patients that have failed in vitro fertilisation, we have previously identified reduced levels of the molecular chaperone Heat Shock Protein A2 (HSPA2) as a major feature of their underlying pathophysiology. Moreover, we have established that conditions of oxidative stress induce modification of HSPA2 by the lipid peroxidation product 4-hydroxynonenal (4HNE) resulting in a dramatic reduction in sperm-egg interaction. While these studies have revealed the susceptibility of HSPA2 to oxidative insult in mature spermatozoa, they also raise questions as to the damage that may befall HSPA2 during testicular germ-cell development. While oxidised proteins have long been considered targets for proteolysis in somatic cells, the fate of protein substrates modified by 4HNE in the male germ line has not been examined. Given this, the current study sought to explore the effects of 4HNE on the stability of HSPA2 in developing germ cells.
Round spermatids (RS), pachytene spermatocytes (PS) and a germ-cell derived cell line (GC-2 cells) were isolated and treated with 50-200 µM 4HNE. Under such conditions, HSPA2 protein expression was significantly reduced in RS and GC-2 cells but not in PS. Using the GC-2 cell line as a model, immunoprecipitation revealed the modification of HSPA2 by 4HNE and its subsequent ubiquitination. Additionally, HSPA2 expression was able to be stabilised through the use of a proteasome inhibitor, MG132. Coupled with the finding that proteasome activity increases significantly in GC-2 cells exposed to 4HNE, this study provides multiple lines of evidence in support of a proteasome-dependent degradation pathway occurring in response to oxidative stress in the male germ-line. Moreover, using a proximity ligation assay, 4HNE exposure was shown to induce the dissociation of HSPA2 from its protective co-chaperone BCL2-Associated Athanogene 6 (BAG6), an event that may greatly enhance the susceptibility of HSPA2 to degradation in testicular germ cells.