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.
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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.
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    • "While bicarbonate and calcium fluxes are required for rapid cAMP production and increases in membrane fluidity (Harrison et al. 1993), albumin-mediated sterol depletion is critical to achieve increases in protein tyrosine phosphorylation, often considered a hallmark of sperm functional competence in model species such as the mouse (Visconti et al. 1995b, Galantino-Homer et al. 1997). Sterol loss occurs either through the contribution of an active cholesterol transporter that provides free cholesterol to the hydrophobic pocket of albumin (Flesch et al. 2001) or possibly through the oxidation of membrane sterols and the subsequent scavenging of the hydrophilic oxidation products by albumin (Boerke et al. 2013). "
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    ABSTRACT: While in vitro fertilisation has been widely successful in other domesticated species, the development of a robust IVF system for the horse remains an elusive and highly valued goal. A major impediment to the development of equine IVF, is the fact that optimized conditions for the capacitation of equine spermatozoa have not yet been developed. Conversely, it is known that stallion spermatozoa are particularly susceptible to damage arising as a consequence of capacitation-like changes induced prematurely in response to semen handling and transport conditions. To address these limitations, this study sought to develop an effective system to both suppress and promote the in vitro capacitation of stallion spermatozoa. Our data demonstrated that the latter could be achieved in a bicarbonate rich media supplemented with a phosphodiesterase inhibitor, a cAMP analogue, and methyl-β-cyclodextrin, an efficient cholesterol-withdrawing agent. The populations of spermatozoa generated under these conditions displayed a number of hallmarks of capacitation, including: elevated levels of tyrosine phosphorylation, a reorganisation of the plasma membrane leading to lipid raft coalescence in the peri-acrosomal region of the sperm head and a dramatic increase in their ability to interact with heterologous bovine ZP and undergo agonist induced acrosomal exocytosis. Furthermore, this functional transformation was effectively suppressed in media devoid of bicarbonate. Collectively, these results have highlighted the importance of efficient cholesterol removal in priming stallion spermatozoa for ZP binding in vitro.
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