The impact of oxidative stress on chaperone-mediated human sperm-egg interaction.
OBJECTIVE
How does oxidative stress impact upon human sperm-egg interaction and in particular the formation of zona pellucida-receptor complexes on the sperm surface?


CONCLUSIONS
Oxidative stress during human sperm capacitation resulted in the chemical alkylation of the molecular chaperone heat shock protein A2 (HSPA2), a concomitant reduction in surface expression of the zona pellucida-receptor arylsulphatase A (ARSA) and a severe loss of zona pellucida binding ability.


BACKGROUND
An inability to bind to the zona pellucida is commonly encountered in the defective spermatozoa generated by male infertility patients; however, the underlying mechanisms remain unresolved. Recent studies have revealed that zona pellucida binding is mediated by molecular chaperones, particularly HSPA2, that facilitate the formation of multimeric zona pellucida-receptor complexes on the surface of mammalian spermatozoa during capacitation.


METHODS
Spermatozoa were collected from healthy normozoospermic donors (n = 15). Low levels of oxidative stress were induced in populations of non-capacitated spermatozoa by a 1 h treatment with 4-hydroxynonenal (4HNE) or hydrogen peroxide (H2O2) and then these insults were removed and cells were capacitated for 3 h.


METHODS
Motility, membrane fluidity, protein tyrosine phosphorylation and lipid raft distribution were evaluated after sperm capacitation to determine the impact of oxidative stress on this process. The surface expression of ARSA and sperm adhesion molecule 1 (SPAM1) was observed using fluorescence microscopy, and the ability of treated cells to interact with homologous human zonae pellucidae was assessed through gamete co-incubation. Proximity ligation was used to evaluate the state of the HSPA2-laden zona pellucida-receptor complex and an immunoprecipitation approach was taken to establish the chemical alkylation of HSPA2 by the cytotoxic lipid aldehyde 4HNE. The validity of these findings was then tested through treatment of oxidatively stressed cells with the nucleophile penicillamine in order to scavenge lipid aldehydes and limit their ability to interact with HSPA2. All experiments were performed on samples pooled from two or more donors per replicate, with a minimum of three replicates.


RESULTS
The oxidative treatments employed in this study did not influence sperm motility or capacitation-associated changes in membrane fluidity, tyrosine phosphorylation and lipid raft redistribution. However, they did significantly impair zona pellucida binding compared with the capacitated control (P < 0.01). The reduction in zona pellucida binding was associated with the impaired surface expression (P < 0.02) of a zona pellucida-receptor complex comprising HSPA2, SPAM1 and ARSA. Proximity ligation and immunoprecipitation assays demonstrated that impaired zona pellucida binding was, in turn, associated with the chemical alkylation of HSPA2 with 4HNE and the concomitant disruption of this zona pellucida-receptor complex. The use of penicillamine enabled a partial recovery of ARSA surface expression and zona pellucida adherence in H2O2-treated cells. These data suggest that the ability of low levels of oxidative stress to disrupt sperm function is mediated by the production of lipid aldehydes as a consequence of lipid peroxidation and their adduction to the molecular chaperone HSPA2 that is responsible for co-ordinating the assembly of functional zona pellucida-receptor complexes during sperm capacitation.


CONCLUSIONS
While these results extend only to one particular zona pellucida-receptor complex, we postulate that oxidative stress may more broadly impact upon sperm surface architecture. In this light, further study is required to assess the impact of oxidative stress on additional HSPA2-laden protein complexes.


CONCLUSIONS
These findings link low levels of oxidative stress to a severe loss of sperm function. In doing so, this work suggests a potential cause of male infertility pertaining to a loss of zona pellucida recognition ability and will contribute to the more accurate diagnosis and treatment of such conditions.
Priority Research Centre for Reproductive Biology, School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia elizabeth.bromfield@uon.edu.au.
Elizabeth G
EG
Bromfield
Priority Research Centre for Reproductive Biology, School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
R John
RJ
Aitken
Priority Research Centre for Reproductive Biology, School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
Amanda L
AL
Anderson
Priority Research Centre for Reproductive Biology, School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
Eileen A
EA
McLaughlin
Priority Research Centre for Reproductive Biology, School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
Brett
B
Nixon
D000328:Adult; D001192:Arylsulfatases / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008297:Male; D018832:Molecular Chaperones / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*; D013075:Sperm Capacitation / Q000502:physiology*; D013084:Sperm-Ovum Interactions / Q000502:physiology*; D015044:Zona Pellucida / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; C091879:HSPA2 protein, human; D018832:Molecular Chaperones; D001192:Arylsulfatases
4-hydroxynonenal; HSPA2; capacitation; molecular chaperone; oxidative stress; protein; receptor; spermatozoa; sperm–egg recognition; zona pellucida