Involvement of Bicarbonate-Induced Radical Signaling in Oxysterol Formation and Sterol Depletion of Capacitating Mammalian Sperm During In Vitro Fertilization1

Abstract

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 ORP-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: hyperactivated motility, tyrosin phosphorylation, and 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 ORP-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.

Capacitation in the presence of methyl-β-cyclodextrin results in enhanced zona pellucida-binding ability of stallion spermatozoa

“…Although media containing mbcd promoted high levels of heterologous ZP interaction, a recent study of murine and porcine sperm capacitation has suggested that the artificial depletion of sterols mediated by this agent may result in low IVF rayes (Boerke et al 2013). Such results purportedly stem from the dual action of this reagent in circumventing the bicarbonate-induced reactive oxygen species (ROS) oxysterol signalling pathway that leads to the removal of sperm surface sterols, in addition to its ability to induce oocyte degeneration in these species (Boerke et al 2013).…”

“…Such results purportedly stem from the dual action of this reagent in circumventing the bicarbonate-induced reactive oxygen species (ROS) oxysterol signalling pathway that leads to the removal of sperm surface sterols, in addition to its ability to induce oocyte degeneration in these species (Boerke et al 2013). While the effect of mbcd on equine IVF rates and equine oocyte quality is yet to be examined, the very low concentration encountered by oocytes in our study is likely to limit any detrimental influence of this reagent.…”

“…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). Understandably, BSA is a reliable macromolecule to facilitate cholesterol loss in stallion sperm (Rathi et al 2003, McPartlin et al 2008; nevertheless, if a capacitation medium is to be used regularly in equine assisted reproduction, an alternative macromolecule of nonanimal origin would be required due to the legitimate concern of zoonotic disease transmission and to ensure the reproducibility of the media.…”

Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm

“…It has recently been shown that ROS generation during capacitation may be necessary for cholesterol efflux from the sperm plasma membrane. ROS oxidizes cholesterol to form oxysterols; this appears to be associated with depletion of both oxysterols and unoxidized cholesterol from the plasma membrane (Brouwers et al 2011, Boerke et al 2013. It is postulated that formation of oxysterols may activate the sterol transporter proteins, which make sterols available to cholesterol acceptors such as albumin (Boerke et al 2013).…”

“…Interestingly, the cholesterol complexing agent, methyl-b-cyclodextrin (MbCD), does not require cholesterol oxidation in order to effectively remove cholesterol from the sperm plasma membrane (Boerke et al 2013).…”