Platelet activating factor (PAF) is a unique signaling phospholipid that has many biologic properties in addition to platelet activation. PAF roles in reproduction involve ovulation, fertilization, embryo development, implantation and parturition. It may also serve as a biomarker for normal sperm function. The presence of PAF receptor on the spermatozoa of 10 stallions was investigated by immunofluorescence microscopy. Statistical analysis revealed that the fluorescence intensity, FI (Mean+/-SEM), in the post- acrosomal region (FI= 2.60+/-0.15) was significantly higher (P< 0.01) than that in any other region of stallion spermatozoa. The effect of synthetic PAF on stallion spermatozoal motility, capacitation, and the acrosome reaction (AR) were evaluated. Treatment of 10 stallion semen samples with 10 â 4 to 10 â 13 M PAF resulted in statistically significant differences in motility and capacitation (r2 = 0.81 and 0.83 respectively). The concentration of PAF, incubation time and their interaction were highly significant (P< 0.01) for their effect on motility. Concentrations of PAF ranging from 10-9 to10-11 M were able to induce capacitation. Following capacitation in vitro with PAF, and induction of the acrosomal reaction by progesterone, transmission electron microscopy (TEM) was conducted on the spermatozoa of 3 stallions, to detect the true AR. Differences in PAF concentrations were highly significant as indicated by R-square (for intact: 97.2, reacted: 89.8, and for vesiculated: 98.1). Treating spermatozoa from 3 stallions with the PAF antagonist FR-49175 inhibited calcium release and fluorescence intensity with a median inhibitory concentration (IC50) of 10-7.5 M (r2=0.82, P<0.01) and 10-8 M (r2=0.92, P<0.01) respectively, suggesting a receptor mediated process for the mechanism of action of PAF. Although the exact mechanisms of PAF action on equine spermatozoa remain unclear, it is widely reported that PAF acts by a receptor-mediated mechanism and that the PAF receptor is a member of the family of G-protein coupled receptors with phospholipase C as the effector. Since the limited success in equine ART (e.g. IVF) is in part due to lack of efficient treatment of stallion spermatozoa for capacitation, PAF may be useful to help capacitate stallion spermatozoa. Without proper capacitation, spermatozoa are unable to initiate the acrosome reaction which is a prerequisite for fertilization. / Ph. D.

Ataxia telangiectasia and Rad3-related (ATR) is well known for its regulatory role in DNA damage responses (DDR) as a checkpoint kinase that phosphorylates hundreds of protein substrates. However, its role in cellular non-DNA damage stress responses (NDDR) is unknown. Necrosis is one form of cell death and traditionally has been regarded as a passive and uncontrolled cell death. Recently, evidence has emerged to support the concept that necrosis also may occur in a programmed manner and that PARP1 can be a mediator. Active poly (ADP-ribose) polymerase 1 (PARP1) hydrolyzes nicotinamide adenine dinucleotide (NAD+) to produce poly (ADP-ribose) (PAR) polymers on target proteins or itself. As a result, hyper-activity of PARP1 may lead to necrosis by excessively depleting ATP pool which results in mitochondrial energetic collapse. On the other hand, it is known that Ca2+ overload induces necrosis, but much still remains unknown about how Ca2+ overload-induced necrosis is regulated in cells. In this study, we show that ATR, besides its hallmark regulatory role in DDR, also plays a role in NDDR by suppressing ionomycin-induced necrosis. Ionomycin as a Ca2+ ionophore can dramatically raise the intracellular level of Ca2+, leading to necrosis. We found that this Ca2+ overload-induced necrosis occurs without inducing DDR in cells. Instead, the hyper-poly(ADP-ribosyl)ation (PARylation) activity of activated PARP1 could be a reason leading to necrosis, as NAD+ supplied to media can rescue ionomycin-induced necrosis. In vitro PARylation assay also demonstrates that PARP1 hyper-activation is Ca2+ dependent. In cells, ATR-PARP1 interaction happened after ionomycin treatment. Furthermore, ionomycin treatment induces more full-length PAR polymers formed in ATR-deficient cells than in ATR-proficient cells. The interaction of kinase-dead ATR and PARP1 dramatically decreased as compared to wild-type ATR. Therefore, ATR plays a novel role in NDDR wherein it is able to suppress Ca2+ overload-induced PARP1-mediated necrosis. Ca2+ overload-induced cell death is a major cause of many human medical conditions and diseases, such as brain injury, stroke and ischemia et al. Our ongoing studies will help to define the molecular mechanisms of the anti-necrosis activities of ATR, which may support ATR as a new clinical target for therapeutic treatment of those diseases.

The mechanism of microparticle shedding from the plasma membrane of calcium-loaded cells has been investigated in erythrocytes and platelets. Recent studies have revealed the physiological and clinical importance of microparticle release from nucleated cells such as lymphocytes and endothelium. The experiments of this study were designed to address whether simple mechanisms discovered in platelets and erythrocytes also apply to the more complex nucleated cells. Four such mechanisms were addressed: potassium efflux, transbilayer phosphatidylserine migration, cytoskeleton degradation, and membrane lipid order. The rate and amount of microparticle release in the presence of a calcium ionophore, ionomycin, was assayed by light scatter at 500 nm. To inhibit the calcium-activated potassium current, cells were exposed to 1 mM quinine or a high-potassium buffer. Both interventions substantially attenuated microparticle shedding induced by ionomycin. Microparticle release was also greatly reduced in a lymphocyte cell line deficient in the expression of scramblase, the enzyme responsible for calcium-stimulated phosphatidylserine migration to the cell surface. This result indicated that such phosphatidylserine exposure is also required for microparticle shedding. The importance of cytoskeletal rearrangement was evaluated through the use of E64-d, a calpain inhibitor, which appeared to have no affect on release. Thus, if cytoskeleton degradation is important for microparticle release, a different enzyme or protein must be involved. Finally, the effect of membrane physical properties was addressed by varying the experimental temperature (32–42 °C). A significant positive trend in the rate of microparticle release as a function of temperature was observed. Fluorescence experiments with trimethylammoniumdiphenylhexatriene and patman revealed significant differences in the level of apparent membrane order along that temperature range. Ionomycin treatment appeared to cause further disordering of the membrane, although the magnitude of this change was minimally temperature-sensitive. Thus, it was concluded that microparticle release depends more on the initial level of membrane order than on the change imposed by calcium uptake. In general, mechanisms involved in particle release from platelets and erythrocytes appeared relevant tolymphocytes with the exception of the hydrolytic enzyme involved in cytoskeletal degradation.