Article

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: 3.45). 10/2012; DOI: 10.1095/biolreprod.112.101253
Source: PubMed

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 -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.

Download full-text

Full-text

Available from: Chris Van de Lest, Jun 06, 2014
2 Followers
 · 
111 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Binder of SPerm (BSP) proteins are ubiquitous amongst mammals and are exclusively expressed in male genital tract. The main function associated with BSP proteins is their ability to promote sperm capacitation. In mice, two proteins (BSPH1 and BSPH2) have been studied. Using recombinant strategies, BSPH1 was found to bind to epididymal sperm membranes and promote sperm capacitation in vitro. The goal of the present study was to evaluate the role of native murine BSPH1 protein in sperm capacitation induced by bovine serum albumin (BSA) and High-Density Lipoproteins (HDL). The effect of antibodies, antigen-binding fragments (Fabs) and F(ab')2 specific against murine BSPH1 on BSA- and HDL-induced capacitation was tested. Results show that BSPH1 has no direct role in BSA-induced capacitation. However, antibodies, Fabs and F(ab')2 could block capacitation induced by HDL and could inhibit the HDL-induced increase in tyrosine phosphorylation, suggesting a specific interaction between HDL and BSPH1. Results suggest that murine BSPH1 proteins in mice could be a new important piece of the puzzle in sperm capacitation induced by HDL. Since murine BSPH1 is orthologous to human BSPH1, this study could also lead to new insights on the functions and the importance of the human protein in male fertility.
    Reproduction (Cambridge, England) 01/2015; 149(4). DOI:10.1530/REP-14-0559 · 3.26 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Repeatable methods for in vitro fertilization have not been established in the horse, reflecting the failure of standard capacitating media to induce changes required for fertilization capacity in equine sperm. One important step in capacitation is membrane cholesterol efflux, which in other species is triggered by cholesterol oxidation and is typically enhanced using albumin as a sterol acceptor. We incubated equine sperm in the presence of calcium, bovine serum albumin (BSA) and bicarbonate, independently or in combination. Bicarbonate induced an increase in reactive oxygen species (ROS) that was abolished by the addition of calcium or BSA. Bicarbonate induced protein tyrosine phosphorylation (PY), even in the presence of calcium or BSA. Incubation at high pH enhanced PY but did not increase ROS production. Notably, no combination of these factors was associated with significant cholesterol efflux, as assessed by fluorescent quantitative cholesterol assay and confirmed by filipin staining. In contrast, sperm treated with methyl-β-cyclodextrin (MβCD) showed significant cholesterol reduction, but no significant increase in PY or ROS. Presence of BSA increased sperm binding to bovine zonae pellucidae in three of three stallions. These results show that presence of serum albumin is not associated with reduction of membrane cholesterol in equine sperm, highlighting the failure of equine sperm to exhibit core capacitation-related changes in standard capacitating medium. These data suggest an atypical relationship among cholesterol efflux, ROS production and PY in equine sperm. Our findings may help to elucidate factors affecting failure of equine in vitro fertilization under standard conditions.
    Reproduction (Cambridge, England) 01/2015; 149(1):87-99. DOI:10.1530/REP-14-0457 · 3.26 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Spermatozoa are vulnerable to oxidative attack because they contain an abundance of polyunsaturated fatty acids that are susceptible to lipid peroxidation. In addition, functionally important proteins and DNA are also subject to oxidative modification and adduction by aldehydes, such as 4-hydroxynonenal (4HNE), generated as a consequence of the peroxidative process. The proteins adducted by 4HNE include elements of the mitochondrial electron transport chain, such as succinic acid dehydrogenase. The net result of such electrophilic attack is to stimulate generation of mitochondrial reactive oxygen species (ROS) in a self-perpetuating lipid peroxidation–ROS generation cycle that ultimately triggers the intrinsic apoptotic pathway, leading to a rapid loss of motility and cell death. A major point of difference between apoptosis in spermatozoa and somatic cells is that in the former, nuclear DNA is located in a compartment (the head) separate from the mitochondria and most of the cytoplasm (the midpiece). As a result, nucleases activated and released in the midpiece during apoptosis cannot gain access to the DNA in the sperm head in order to cleave the DNA. However, the ROS generated during apoptosis can readily gain access to the sperm nucleus and generate oxidative base adducts, typically 8-hydroxy, 2′-deoxyguanosine (8OHdG), which are converted into abasic sites by 8-oxoguanine glycosylase (OGG1), the only enzyme of the base excision repair pathway possessed by spermatozoa. These abasic sites subsequently become the foci of DNA fragmentation. Because defective sperm function and DNA damage are frequently associated with oxidative stress, there is a great deal of interest in the use of antioxidants in a therapeutic context. This presentation examines the fundamental relationships between oxidative stress and sperm function and considers the implications of recent findings for the management of sperm function and fertility in stallions.
    Journal of Equine Veterinary Science 01/2014; 34(1):17–27. DOI:10.1016/j.jevs.2013.10.120 · 0.89 Impact Factor