title: Proteolytic degradation of heat shock protein A2 occurs in response to oxidative stress in male germ cells of the mouse.
abstract: Does oxidative stress compromise the protein expression of heat shock protein A2 (HSPA2) in the developing germ cells of the mouse testis?
  
  
  
  Oxidative stress leads to the modification of HSPA2 by the lipid aldehyde 4-hydroxynonenal (4HNE) and initiates its degradation via the ubiquitin-proteasome system.
  
  
  
  Previous work has revealed a deficiency in HSPA2 protein expression within the spermatozoa of infertile men that have failed fertilization in a clinical setting. While the biological basis of this reduction in HSPA2 remains to be established, we have recently shown that the HSPA2 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 HSPA2 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 HSPA2 in the testis, by preventing its ubiquitination and subsequent proteolytic degradation.
  
  
  
  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 HSPA2 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.
  
  
  
  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 HSPA2 was monitored via immunocytochemistry and immunoblotting approaches. Using the GC-2 cell line as a model, the ubiquitination and degradation of HSPA2 was assessed using immunoprecipitation techniques and pharmacological inhibition of proteasomal and lysosomal degradation pathways. Finally, the interaction between BAG6 and HSPA2 was examined in response to 4HNE exposure via proximity ligation assays.
  
  
  
  HSPA2 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, HSPA2 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 HSPA2 degradation after 4HNE treatment indicating that the degradation of HSPA2 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 HSPA2 from its regulatory co-chaperone BAG6, a key mediator of HSPA2 stability in male germ cells.
  
  
  
  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.
  
  
  
  This study suggests a causative link between non-enzymatic post-translational modifications and the relative levels of HSPA2 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.
  
  
  
  Not applicable.
  
  
  
  This work was supported by the National Health and Medical Research Council of Australia (APP1101953). The authors have no competing interests to declare.
authors.affiliation: Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: Elizabeth G
authors.initials: EG
authors.lastname: Bromfield
authors.affiliation: Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: R John
authors.initials: RJ
authors.lastname: Aitken
authors.affiliation: Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.|School of Biological Sciences, University of Auckland, Auckland, New Zealand.
authors.forename: Eileen A
authors.initials: EA
authors.lastname: McLaughlin
authors.affiliation: Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: Brett
authors.initials: B
authors.lastname: Nixon
mesh_terms: D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D051379:Mice; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress; D061251:Primary Cell Culture; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D055550:Protein Stability / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D013087:Spermatids / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D013090:Spermatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D013091:Spermatogenesis / Q000235:genetics; D013737:Testis / Q000166:cytology / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D000447:Aldehydes; C484266:Bag6 protein, mouse; D018840:HSP70 Heat-Shock Proteins; C490434:Hspa2 protein, mouse; D007976:Leupeptins; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D006861:Hydrogen Peroxide; D046988:Proteasome Endopeptidase Complex; C027576:4-hydroxy-2-nonenal; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
keywords: chaperone; degradation; germ cell; infertility; oxidative stress; proteasome; sperm; ubiquitin; zona pellucida