Proteolytic degradation of heat shock protein A2 occurs in response to oxidative stress in male germ cells of the mouse.

STUDY QUESTION:Does oxidative stress compromise the protein expression of heat shock protein A2 in the developing germ cells of the mouse testis? SUMMARY ANSWER:Oxidative stress leads to the modification of by the lipid aldehyde 4-hydroxynonenal (4HNE) and initiates its degradation via the ubiquitin-proteasome system. WHAT IS KNOWN ALREADY:Previous work has revealed a deficiency in Hduanyu18422 protein expression within the spermatozoa of infertile men that have failed fertilization in a clinical setting. While the biological basis of this reduction in Hduanyu18422 remains to be established, we have recently shown that the Hduanyu18422 expressed in the spermatozoa of normozoospermic individuals is highly susceptible to adduction, a form of post-translational modification, by the lipid aldehyde 4HNE that has been causally linked to the degradation of its substrates. This modification of Hduanyu18422 by 4HNE adduction dramatically reduced human sperm-egg interaction in vitro. Moreover, studies in a mouse model offer compelling evidence that the co-chaperone BCL2-associated athanogene 6 (BAG6) plays a key role in regulating the stability of Hduanyu18422 in the testis, by preventing its ubiquitination and subsequent proteolytic degradation. STUDY DESIGN, SIZE, DURATION:Dose-dependent studies were used to establish a 4HNE-treatment regime for primary culture(s) of male mouse germ cells. The influence of 4HNE on Hduanyu18422 protein stability was subsequently assessed in treated germ cells. Additionally, sperm lysates from infertile patients with established zona pellucida recognition defects were examined for the presence of 4HNE and ubiquitin adducts. A minimum of three biological replicates were performed to test statistical significance. PARTICIPANTS/MATERIALS, SETTING, METHODS:Oxidative stress was induced in pachytene spermatocytes and round spermatids isolated from the mouse testis, as well as a GC-2 cell line, using 50-200 µM 4HNE or hydrogen peroxide (H2O2), and the expression of Hduanyu18422 was monitored via immunocytochemistry and immunoblotting approaches. Using the GC-2 cell line as a model, the ubiquitination and degradation of Hduanyu18422 was assessed using immunoprecipitation techniques and pharmacological inhibition of proteasomal and lysosomal degradation pathways. Finally, the interaction between BAG6 and Hduanyu18422 was examined in response to 4HNE exposure via proximity ligation assays. MAIN RESULTS AND THE ROLE OF protein levels were significantly reduced compared with controls after 4HNE treatment of round spermatids (P < 0.01) and GC-2 cells (P < 0.001) but not pachytene spermatocytes. Using GC-2 cells as a model, Hduanyu18422 was shown to be both adducted by 4HNE and targeted for ubiquitination in response to cellular oxidative stress. Inhibition of the proteasome with MG132 prevented Hduanyu18422 degradation after 4HNE treatment indicating that the degradation of Hduanyu18422 is likely to occur via a proteasomal pathway. Moreover, our assessment of proteasome activity provided evidence that 4HNE treatment can significantly increase the proteasome activity of GC-2 cells (P < 0.05 versus control). Finally, 4HNE exposure to GC-2 cells resulted in the dissociation of Hduanyu18422 from its regulatory co-chaperone BAG6, a key mediator of Hduanyu18422 stability in male germ cells. LIMITATIONS, REASONS FOR CAUTION:While these experiments were performed using a mouse germ cell-model system, our analyses of patient sperm lysate imply that these mechanisms are conserved between mouse and human germ cells. WIDER IMPLICATIONS OF THE FINDINGS:This study suggests a causative link between non-enzymatic post-translational modifications and the relative levels of Hduanyu18422 in the spermatozoa of a specific sub-class of infertile males. In doing so, this work enhances our understanding of failed sperm-egg recognition and may assist in the development of targeted antioxidant-based approaches for ameliorating the production of cytotoxic lipid aldehydes in the testis in an attempt to prevent this form of infertility. LARGE SCALE DATA:Not applicable. STUDY FUNDING/COMPETING INTEREST(S):This work was supported by the National Health and Medical Research Council of Australia (APP1101953). The authors have no competing interests to declare.

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