Involvement of Bicarbonate-Induced Radical Signaling in Oxysterol Formation and Sterol Depletion of Capacitating Mammalian Sperm During In Vitro Fertilization

Biology of Reproduction (Impact Factor: 3.32). 10/2012; 88(1). DOI: 10.1095/biolreprod.112.101253
Source: PubMed


This study demonstrates for the first time that porcine and mouse sperm incubated in capacitation media supplemented with bicarbonate produce oxysterols. The production is dependent on a reactive oxygen species (ROS) signaling pathway that is activated by bicarbonate and can be inhibited or blocked by addition of vitamin E or vitamin A or induced in absence of bicarbonate with pro-oxidants. The oxysterol formation was required to initiate albumin dependent depletion of 30% of the total free sterol and >50% of the formed oxysterols. Incubation of bicarbonate treated sperm with oxysterol binding proteins (ORP-1 or -2) caused a reduction of >70% of the formed oxysterols in the sperm pellet but no free sterol depletion. Interestingly, both ORP and albumin treatments led to similar signs of sperm capacitation: hyper-activated motility, tyrosin phosphorylation, aggregation of flotillin in the apical ridge area of the sperm head. However, only albumin incubations led to high in vitro fertilization rates of the oocytes whereas the ORP-1 and -2 incubations did not. A pretreatment of sperm with vitamin E or A caused reduced in vitro fertilization rates with 47% and 100%, respectively. Artificial depletion of sterols mediated by methyl-beta cyclodextrin bypasses the bicarbonate ROS oxysterol signaling pathway but resulted only in low in vitro fertilization rates and oocyte degeneration. Thus bicarbonate induced ROS formation causes at the sperm surface oxysterol formation and a simultaneous activation of reverse sterol transport from the sperm surface which appears to be required for efficient oocyte fertilization.

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Available from: Chris Van de Lest, Jun 06, 2014
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    • "Importantly, in vitro capacitation of sperm in the presence of albumin did not result in cholesterol depletion in the DRM fraction (in contrast to MBCD treatment; van Gestel et al. 2005b). Nevertheless, MBCD treatment allows cholesterol depletion in sperm and this does result in increased zona binding of stallion sperm (Bromfield and Nixon 2013a, b) and to some extent in vitro fertilization of porcine oocytes (Boerke et al. 2013). Note that sperm capacitation is a process essential for sperm to become competent to fertilize the oocyte (Gadella et al. 2008; Aitken and Nixon 2013). "
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    ABSTRACT: Lipid rafts are micro-domains of ordered lipids (L o phase) in biological membranes. The L o phase of cellular membranes can be isolated from disordered lipids (L d phase) after treatment with 1 % Triton X-100 at 4 °C in which the L o phase forms the detergent-resistant membrane (DRM) fraction. The lipid composition of DRM derived from Madin-Darby canine kidney (MDCK) cells, McArdle cells and por-cine sperm is compared with that of the whole cell. Remarkably, the unsaturation and chain length degree of aliphatic chains attached to phospholipids is virtually the same between DRM and whole cells. Cholesterol and sphingomyelin were enriched in DRMs but to a cell-specific molar ratio. Sulfatides (sphingolipids from MDCK cells) were enriched in the DRM while a seminolipid (an alkylacylglycerolipid from sperm) was depleted from the DRM. Treatment with<5 mM methyl-ß-cy-clodextrin (MBCD) caused cholesterol removal from the DRM without affecting the composition and amount of the phospholipid while higher levels disrupted the DRM. The substantial amount of (poly)unsaturated phospholipids in DRMs as well as a low stoichiometric amount of cholesterol suggest that lipid rafts in biological membranes are more fluid and dynamic than previously anticipated. Using negative staining, ultrastructural features of DRM were monitored and in all three cell types the DRMs appeared as multi-lamellar vesicular structures with a similar morphology. The detergent resistance is a result of protein–cholesterol and sphingolipid interactions allowing a relatively passive attraction of phospholipids to maintain the L o phase. For this special issue, the relevance of our findings is discussed in a sperm physiological context.
    Cell and Tissue Research 09/2015; DOI:10.1007/s00441-015-2272-y · 3.57 Impact Factor
    • "ROS are well recognized as key regulators of mammalian sperm capacitation , yet a fine balance exists between a 'beneficial' presence of ROS to promote cholesterol efflux (Boerke et al., 2013), cAMP production (Zhang and Zheng, 1996; Ickowicz et al., 2012), protein tyrosine phosphorylation (Aitken et al., 1998; Leclerc et al., 1997) and the acrosome reaction (de Lamirande et al., 1998) and the 'detrimental' presence of excess ROS that can lead otherwise viable cells down an intrinsic apoptotic-like pathway (Aitken et al., 2012b). "
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    ABSTRACT: Study question: How does oxidative stress impact upon human sperm-egg interaction and in particular the formation of zona pellucida-receptor complexes on the sperm surface? Summary answer: 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. What is known already: 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. Study design, size, duration: 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. Participants/materials, setting, 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. Main results and the role of chance: 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. Limitations, reasons for caution: 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. Wider implications of the findings: 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. Study funding/competing interests: This work was supported by the National Health and Medical Research Council. Grant # APP1046346. The authors have no competing interests to declare. Trial registration number: N/A.
    Human Reproduction 09/2015; DOI:10.1093/humrep/dev214 · 4.57 Impact Factor
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    • "Nevertheless, cyclodextrins have been shown also to induce some capacitation responses in sperm [13]. Both cyclodextrins at a very narrow concentration range in absence of albumin result in IVF rates [13] under conditions in which the cyclodextrins already induce oocyte deterioration [13,14,63]. Recently, it has been shown that cholesterol incorporation into mammalian sperm by cholesterol-complexed cyclodextrins improves cryopreservation of sperm and subsequent IVF results of thawed treated sperm [64,65]. "
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    ABSTRACT: The fusion of a sperm with an oocyte to form new life is a highly regulated event. The activation-also termed capacitation-of the sperm cell is one of the key preparative steps required for this process. Ejaculated sperm has to make a journey through the female uterus and oviduct before it can approach the oocyte. The oocyte at that moment also has become prepared to facilitate monospermic fertilization and block immediately thereafter the chance for polyspermic fertilization. Interestingly, ejaculated sperm is not properly capacitated and consequently is not yet able to fertilize the oocyte. During the capacitation process, the formation of competent lipid-protein domains on the sperm head enables sperm-cumulus and zona pellucida interactions. This sperm binding allows the onset for a cascade reaction ultimately resulting in oocyte-sperm fusion. Many different lipids and proteins from the sperm surface are involved in this process. Sperm surface processing already starts when sperm are liberated from the seminiferous tubules and is followed by epididymal maturation where the sperm cell surface is modified and loaded with proteins to ensure it is prepared for its fertilization task. Although cauda epididymal sperm can fertilize the oocyte IVF, they are coated with so-called decapacitation factors during ejaculation. The seminal plasma-induced stabilization of the sperm surface permits the sperm transit through the cervix and uterus but prevents sperm capacitation and thus inhibits fertilization. For IVF purposes, sperm are washed out of seminal plasma and activated to get rid of decapacitation factors. Only after capacitation, the sperm can fertilize the oocyte. In recent years, IVF has become a widely used tool to achieve successful fertilization in both the veterinary field and human medicine. Although IVF procedures are very successful, scientific knowledge is still far from complete when identifying all the molecular players and processes during the first stages the fusion of two gametes into a new life. A concise overview in the current understanding of the process of capacitation and the sperm surface changes is provided. The gaps in knowledge of these prefertilization processes are critically discussed. Copyright © 2015 Elsevier Inc. All rights reserved.
    Theriogenology 08/2015; DOI:10.1016/j.theriogenology.2015.07.018 · 1.80 Impact Factor
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