Platelet-activating Factor Treatment Research Articles

Role of superoxide and nitric oxide in platelet-activating factor-induced acute lung injury, hypotension, and mortality in rats.

To investigate the role of superoxide and nitric oxide in platelet-activating factor-induced acute lung injury, hypotension, and mortality. Prospective, randomized, controlled, experimental study. University research laboratory. Anesthetized male Wistar rats (180 to 220 g) were studied. In the first set of experiments, animals were divided into three groups. Group 1 received platelet-activating factor (2 microg/kg i.v.). Group 2 received recombinant human superoxide dismutase (50,000 U/kg i.v.) 30 mins before platelet-activating factor injection. Group 3 received vehicle agents. In the second set of experiments, animals were divided into six groups that received N(G)-nitro-L-arginine (L-NNA), a selective inhibitor of nitric oxide synthesis, or L-arginine, the physiologic precursor of nitric oxide synthesis: a) vehicles (i.v.); b) vehicle plus L-arginine (100 mg/kg i.v.); c) vehicle plus L-NNA (10 mg/kg i.v.); d) vehicle plus platelet-activating factor (2 microg/kg i.v.); e) L-arginine plus platelet-activating factor; and f) L-NNA plus platelet-activating factor. The first intravenous administration was given 5 mins before the second intravenous injection for each group. In the first set of experiments, vascular labeling with Monastral blue B demonstrated diffuse microvascular injury in the alveolar capillary beds 2 hrs after platelet-activating factor challenge. Thiobarbituric acid-reactive substances in the lung significantly increased at 2 hrs after platelet-activating factor injection. Platelet-activating factor treatment also resulted in an increased concentration of total protein, albumin, and Evans blue dye in bronchoalveolar lavage fluid at 2 hrs after administration, suggesting platelet-activating factor induction of increased alveolar permeability. The platelet-activating factor-induced alveolar microvascular injury, lipid peroxidation, and increased alveolar permeability were inhibited by pretreatment with recombinant human superoxide dismutase. Although L-NNA alone did not affect alveolar permeability in the second set of experiments, L-NNA treatment before platelet-activating factor challenge significantly aggravated platelet-activating factor-induced increased alveolar permeability 2 hrs after platelet-activating factor challenge. Platelet-activating factor also produced a rapid decrease in blood pressure that was not ameliorated by treatment with L-NNA. However, L-NNA pretreatment was associated with a significant increase in platelet-activating factor-caused mortality within 6 hrs. All rats survived with L-arginine treatment before platelet-activating factor challenge. L-NNA treatment decreased nitrate/nitrite concentration, an index of total nitric oxide production, in plasma. These results indicate that superoxide, the derived active oxygen species, and lipid peroxidation are implicated in the pathogenesis of platelet-activating factor-induced acute lung injury. Nitric oxide does not play a major role in platelet-activating factor-induced hypotension. Nitric oxide appears to play a protective role in the acute lung injury and mortality induced by platelet-activating factor.

Evidence for the Requirement of Autocrine Growth Factors for Development of Mouse Preimplantation Embryos in Vitro1

The mitotic stimuli in the early mammalian embryo have not been unequivocally identified. One hypothesis is that the embryo releases autocrine growth factors (GFs) that have a role in such growth. To determine whether such putative GFs were limited by dilution, and hence secreted, development was observed at various embryo concentrations in culture. Embryos were collected at the zygote or 2-cell stage. Zygotes were produced by fertilization in situ (ISF) or in vitro (IVF). Two-cell-stage embryos had a high rate of development to the blastocyst stage across an embryo concentration range of 1/microl-0.001/microl. By contrast, zygotes produced by either ISF or IVF were adversely affected by reducing the embryo concentration over this range (p < 0.001), with approximately 80% of ISF zygotes developing to blastocysts at the highest concentration but only 26% at the lowest. For IVF zygotes the corresponding results were 64% and 6%. For all three embryo types, the number of cells in each blastocyst was significantly lower with reduced embryo concentration. The major determinant of zygote development was the concentration of embryos in culture rather than the absolute volume of culture medium or the actual number of embryos present. A concentration of 1 embryo/microl (in the form of 10 embryos/10microl) gave the best development rates and highest cell numbers per blastocyst. Varying the albumin concentration influenced development rates; a 10-fold reduction in BSA concentration (to 0.3 mg/ml) resulted in significantly more IVF zygotes developing to the blastocyst stage. Platelet-activating factor (PAF) is released by embryos, and albumin can act as a competitive inhibitor of PAF's action on cells. ISF embryos released more PAF (p < 0.05) into media than did similarly treated IVF embryos. There was no difference in the amount of PAF remaining associated with the resulting 2-cell embryos. The amount of PAF released by both these groups was markedly less (p < 0.001) than the amount released by 2-cell embryos collected fresh from the reproductive tract and cultured for 24 h. PAF supplementation of media caused a significant increase in the rate of blastocyst development of IVF zygotes at embryo concentrations of 0.1/microl (1 ng/ml) and 0.01/microl (100 ng/ml). Insulin-like growth factor (IGF)-I (30 ng/ml) and IGF-II (1 ng/ml) also stimulated development of IVF zygotes when cultured at an embryo concentration of 1/10 microl. Epidermal growth factor was without effect over the range 0.2-2000 ng/ml. Supplementation of media with both PAF and IGF-II gave no additional benefit over that caused by IGF-II alone, but this treatment was marginally better (p < 0.05) than PAF treatment alone. The results show that factors necessary for normal embryo development are diluted to suboptimal levels during culture at low embryo concentration. The ability of PAF, IGF-I, and IGF-II to partially compensate for the adverse effects of low embryo concentration during culture is consistent with their having roles as autocrine embryotrophic factors. The use of IVF and low embryo concentrations in culture may provide a functional multiple ablation model that will help to define the range of GFs required for normal embryo development.

Regulation of platelet-activating factor receptor expression in human B cells and B cell lines.

We extended our previous data regarding the modulation of human platelet-activating factor receptor (hPAF-R) expression on human B cell lines as well as normal B cells. First, we showed that hPAF-R mRNA was present in B cell lines expressing membrane hPAF-R, but was absent from cell lines devoid of hPAF-R. Second, enhanced hPAF-R membrane expression induced in IM9 line by IL4 was preceeded by hPAF-R mRNA accumulation that was detectable by 8 h and which peaked at 24 h. Similar results were observed for 10 nM platelet-activating factor treatment, which increased hPAF-R mRNA content up to 120% at 48 h, whereas hPAF-R membrane expression was up-regulated by 130%. Third, our data indicate that functional hPAF-R are expressed on resting, as well as on activated, B cells and that B cell activation is required for maintaining hPAF-R membrane and mRNA expression. Thus, in normal B cells, as well as in B cell lines, transcriptional regulation and/or messenger stability control hPAF-R expression.

Involvement of protein kinases in platelet activating factor-induced acrosome reaction of human spermatozoa.

The involvement of protein kinases in platelet activating factor (PAF)-induced acrosome reaction of human spermatozoa was investigated using specific inhibitors of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK). PAF (10(-9)-10(-11) M) treatment of spermatozoa enhanced the acrosome reaction in a dose-dependent manner (32 +/- 4% at 10(-9) M, 28 +/- 4% at 10(-10) M and 24 +/- 3% at 10(-11) M respectively). When spermatozoa were preincubated with PKA, PKC and PTK inhibitor (KT5720, calphostin C and genistein) for 15 min prior to addition of PAF, there was a significantly reduced acrosome reaction induced by PAF, but complete inhibition was not observed. On the other hand, combined use of three inhibitors completely inhibited PAF-induced acrosome reaction to levels of non-treated samples. These results suggest that the induction of the acrosome reaction by PAF treatment may involve the activation of PKA, PKC and PTK signalling pathways, and that interaction between these pathways may regulate complex mechanisms of PAF-induced acrosome reaction.

Identification of Functional Platelet-Activating Factor Receptors on Human Keratinocytes

Platelet-activating factor (PAF) is a potent inflammatory mediator that has been shown to be produced by human keratinocytes and is thought to play a role in cutaneous inflammation. Immunofluorescence and radioligand binding studies were used to characterize PAF receptors (PAF-R) on human keratinocytes and the human epidermoid cell lines A-431 and HaCaT. Indirect immunofluorescence studies demonstrated anti-PAF-R staining of primary cultures of human keratinocytes, A-431 cells, and HaCaT cells. Primary cultures of human fibroblasts and the melanoma cell line SK-30 failed to show immunostaining above that seen with control antiserum. With indirect immunofluorescence studies of sections of normal human skin, a granular anti-PAF-R staining pattern was noted on the keratinocyte cell membranes. A-431 cells readily metabolized PAF by deacetylation-reacylation at 37 degrees C, but not at 4 degrees C. Binding studies on crude membrane preparations of A-431 cells conducted at 4 degrees C demonstrated specific binding that reached saturation by 120 min. Scatchard analysis of PAF binding data revealed a single class of high-affinity (KD = 6.3 +/- 0.3 nM) PAF binding sites. The immunofluorescence and radioligand binding sites were shown to be functional PAF-Rs, as 10 pM to 1 microM PAF increased intracellular calcium in primary cultures of human keratinocytes, A-431 cells, and HaCaT cells, whereas PAF treatment of primary cultures of human fibroblasts or the melanoma cell line SK-30 did not result in changes in the intracellular calcium concentration. The structurally dissimilar PAF-R antagonists CV-6209, Ro19-3704, and alprazolam all inhibited the PAF-induced calcium changes in A-431 cells. The CV-6209 inhibition was seen at doses that competed with the PAF binding to these cells. These studies provide the first evidence for the presence of a functional PAF-R expressed on human keratinocytes, suggesting that this lipid mediator may play an important role in normal keratinocytes or in inflammatory dermatology.

Differences in oxidative response of subpopulations of neutrophils from healthy subjects and patients with rheumatoid arthritis.

To determine whether blood neutrophils from healthy individuals and blood and synovial fluid neutrophils from patients with rheumatoid arthritis (RA) responded differently to priming agonists and stimuli of the oxidative burst and, if so, whether this was a property of a subpopulation of neutrophils. Continuous flow electrophoresis was used to separate neutrophils into subpopulations based upon quantitative differences in net negative surface charge. The generation of superoxide anion (O2-) was used as a measure of oxidative activity using 10(-7) mol/l N-formyl-methionylleucyl-phenylalanine (FMLP) as the stimulating agonist and 10(-8) mol/l platelet activating factor (PAF) as the priming agent. The production of O2- by blood and synovial fluid neutrophils from RA patients in response to FMLP was greater than that observed with control blood neutrophils (p < 0.001). Priming of normal blood neutrophils with PAF increased their FMLP induced oxidative burst (p < 0.001), but PAF treatment had no effect on rheumatoid neutrophils. Neutrophils from synovial fluid of RA patients were less electronegative than paired blood samples and exposure of blood neutrophils to FMLP but not PAF reduced their surface charge. Continuous flow electrophoresis isolated three neutrophil subpopulations: cells of least surface electronegativity were ascribed to pool P1 and cells of greatest surface electro-negativity to P3. Normal blood neutrophils from P3, but not P1, showed increased oxidative activity after PAF priming (twofold increase; p < 0.01), whereas the responsiveness of rheumatoid blood and synovial fluid neutrophils from P1 and P3 was not modified by PAF treatment under the same conditions. It is suggested that most of the circulating neutrophils in RA are already in a state of readiness to generate O2- upon activation by an inflammatory stimulus. This is in contrast to normal blood neutrophils, which have both responsive and non-responsive subpopulations with respect to priming agonists.

PCA-4248, a PAF receptor antagonist, inhibits PAF-induced phosphoinositide turnover

The effect of a new PAF(platelet activating factor; 1- O-alkyl-2-acetyl-sn-glycero-3-phosphoryl-choline) receptor antagonist, PCA-4248 (2-phenylthio)ethyl-5-metoxycarbonyl-2,4,6-trimethyl-1, 4-dihydropyridine-3-carboxylate), on phosphoinositide turnover evoked by PAF was investigated. PAF treatment resulted in an increased 32P incorporation into phosphoinositides and phosphatidic acid in rabbit platelets. Treatment with PCA-4248 abolished both effects in a dose-dependent manner, 10 μM being the most effective dose. In thrombin stimulated platelets, phosphoinositide turnover was not influenced by PCA-4248. In addition, PAF caused a rapid and significant increase in protein phosphorylation. Thus, PAF treatment resulted in a marked phosphorylation of two proteins of 47 kDa and 20 kDa. Treatment with PCA-4248 resulted in an inhibition of these actions. Serotonin secretion evoked by PAF was also inhibited by PCA-4248. It is concluded that PCA-4248 antagonizes the PAF effects by acting as a competitive antagonist at the PAF receptor level as evidenced from binding studies.

Platelet activating factor treatment of spermatozoa enhances rabbit oocyte fertilization following subzonal sperm insertion.

The objective of this study was to evaluate the effects of spermatozoal platelet activating factor (PAF) treatment on fertilization rates employing subzonal sperm insertion in the rabbit. Oocytes (n = 130) with distinct first polar bodies were injected with three to seven motile spermatozoa. Half (n = 65) of the oocytes were injected with PAF-treated sperm, whereas the other half (n = 65) served as controls and were injected with nontreated sperm. Fertilization rates were assessed by the formation of two pronuclei followed by cleavage to the two-cell stage. In the group of oocytes injected with PAF-treated sperm, we noted a significant (P < 0.001) improvement in fertilization rates, 61.5% (40/65), vs. control nontreated sperm, 20.0% (13/65). Subsequent cleavage to the two-cell stage was noted for 95% (38/40) in the treated vs 92.3% (12/13) in the nontreated fertilized zygotes. We conclude that PAF treatment of rabbit spermatozoa prior to subzonal insertion improves fertilization rates and may be of clinical significance in assisted reproductive programs.

Activation of NF- KB and Immunoglobulin Expression in Response to Platelet-Activating Factor in a Human B Cell Line

Nuclear factor kappa B (NF- K B) has been shown to be an important transcriptional regulatory protein in multiple cell types in response to a number of physiological signals. In lymphocytes it has been implicated in transcriptional regulation of the kappa light chain, MHC, IL-6, and IL-2 receptor genes, depending on the differentiation state of the cell. In the present study we demonstrate that platelet-activating factor (PAF), a phospholipid molecule, activates NF- K B and increases kappa light chain mRNA in a human B cell line. Treatment of Ramos cells with PAF (10 -9 to 10 6 M) increased RNA levels of the NF- K B p50 precursor, known as p105, in a dose-dependent manner, p105 RNA levels increased to a maximum observed at 8-10 hr and then diminished by 24 hr; this induction was not blocked by cycloheximide (CHX). PAF induced nuclear kappa B binding at similar concentrations, but more rapidly, attaining maximal levels within 15 min. CHX did not block this activity either. PAF treatment of Ramos cells also resulted in increased levels of RNA for kappa light chain. These results suggest that PAF activates NF- K B by at least two mechanisms in these cells, one at the level of post-translational protein activation and the other by increasing the level of RNA for NF- K B p105.

Platelet activating factor enhances the acrosome reaction, fertilization in vitro by subzonal sperm injection and resulting embryonic development in the rabbit.

This study was conducted to investigate the effect of platelet activating factor (PAF) on the acrosome reaction and fertilizing capacity of spermatozoa, and development of the resulting embryos in the rabbit. Rabbit spermatozoa were exposed to PAF, lyso-PAF, or high ionic strength medium (HIS) prior to subzonal sperm injection (SUZI) into 326 mature oocytes, or morphological assessment of the acrosome reaction. The rates of fertilization and blastocyst formation were compared among the three treatment groups. Acrosome reaction was assessed by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) staining and electron microscopy. PAF-treated spermatozoa fertilized the oocytes at a significantly higher rate (56.1%) than did lyso-PAF-(36.8%, P < 0.01) or HIS- (38.2%, P < 0.05) treated spermatozoa. The embryos produced by PAF-treated spermatozoa showed significantly higher blastocyst formation rates (34.0%) than lyso-PAF- (8.6%, P < 0.05) or HIS-(8.8%, P < 0.05) treated spermatozoa. FITC-PSA staining demonstrated a significantly higher incidence of acrosome reaction in PAF-treated spermatozoa (45.8%) than in lyso-PAF- (28.0%, P < 0.01) or HIS- (34.9%, P < 0.01) treated spermatozoa. Acrosome reaction of PAF-treated spermatozoa was also confirmed by electron microscopy. PAF treatment of spermatozoa enhances fertilizing capacity for SUZI possibly by augmenting the acrosome reaction. Enhanced embryonic development was also found in the oocytes fertilized by SUZI of PAF-treated spermatozoa.

Platelet-activating factor treatment of human spermatozoa enhances fertilization potential

International Journal of Gynecology & ObstetricsVolume 44, Issue 1 p. 93-93 Citations from the literature fertility and sterility Platelet-activating factor treatment of human spermatozoa enhances fertilization potential B.S. Minhas, B.S. MinhasSearch for more papers by this author B.S. Minhas, B.S. MinhasSearch for more papers by this author First published: January 1994 https://doi.org/10.1016/0020-7292(94)90036-1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume44, Issue1January 1994Pages 93-93 RelatedInformation

The effect of platelet‐activating factor on the responsiveness of the human nasal airway

1. The effects of inhaled platelet-activating factor (PAF) on responsiveness of the human nasal airway were examined in normal subjects by measuring nasal airway resistance in response to histamine and bradykinin at 2, 6, 24, 48 h and 7 d after PAF administration. Eosinophil cationic protein (ECP) in nasal secretions was also measured. 2. Intranasal aerosol administration of PAF, 30 or 60 micrograms per nostril to normal human subjects induced an increased responsiveness to inhaled histamine, 50 to 400 micrograms and bradykinin, 100 micrograms per nostril at 2, 6 and 24 h following PAF treatment. However the effect was not apparent at 48 h or 7 days after PAF administration. 3. Intranasal administration of lyso-PAF, 60 micrograms by aerosol did not increase the reactivity of the nasal airway in response to histamine, 200 micrograms. 4. There was no difference in the time course of the PAF-induced hyperresponsiveness to histamine or bradykinin. 5. PAF-induced nasal hyperresponsiveness at 2 and 6 h was associated with increases in the ECP concentration of the nasal lavage fluid. 6. Vitamin E pretreatment of subjects resulted in the attenuation of the PAF-induced hyperresponsiveness to histamine, and a decrease in ECP levels of the nasal lavage fluid. 7. The results suggest that in the human nasal airway, PAF induces a non-specific hyperresponsiveness which is accompanied by eosinophil activation in the nasal cavity. Free radical production induced by PAF may contribute to the hyperresponsiveness and the activation of eosinophils.

Andrology: Effects of platelet activating factor on human sperm function in vitro

The direct effects of platelet activating factor (PAF) and the specific PAF receptor antagonist, CV-3988, on the fertilizing ability of human spermatozoa were investigated. PAF (10(-7)-10(-11) M) increased the human sperm penetration rates in a sperm penetration assay at all doses > 10(-11) M. In contrast, treatment of the spermatozoa with 10(-5) CV-3988 caused a significant decrease in human sperm penetration of zona-free hamster oocytes and adversely affected sperm motility after 24 h of incubation. This suppression was reversed by the addition of PAF. The acrosome reaction was also enhanced by PAF treatment of spermatozoa but this effect was not observed in calcium-free medium. While 10(-5) M CV-3988 decreased the acrosome reaction, the inhibition was also reversed by the addition of PAF. These results suggest that PAF may have a direct role in the fertilizing capacity of human spermatozoa. These findings also suggest that PAF may have a clinical application in an in-vitro fertilization programme.

Endogenous Nitric Oxide Protects against Platelet-Activating Factor-Induced Bowel Injury in the Rat

Platelet-activating factor (PAF) causes bowel necrosis in animal models that is histologically identical to that seen in neonatal necrotizing enterocolitis, but little is known about endogenous mechanisms that might protect against PAF-induced bowel injury. We hypothesized that endogenous nitric oxide might represent such a protective mechanism. Adult male Sprague-Dawley rats were pretreated with 2.5 mg/kg NG-nitro-L-arginine methyl ester (L-NAME), a potent nitric oxide synthase inhibitor, and given injections of 1.5 micrograms/kg PAF 15 min later. Animals treated with normal saline placebo, L-NAME alone, and PAF alone were also studied. Superior mesenteric artery blood flow and blood pressure were continuously recorded. At the end of 2 h or upon death of the animal, hematocrit was measured and intestinal samples were taken for histologic examination and determination of myeloperoxidase activity, a measure of intestinal neutrophil content. Compared with animals given PAF alone, animals pretreated with L-NAME followed by PAF developed significantly worse bowel injury (median injury scores: 2.5 versus 0.5, p = 0.005), hemoconcentration (final hematocrit 65.2 +/- 2.0% versus 53.9 +/- 1.0%, p < 0.001), and intestinal myeloperoxidase activity (12.45 +/- 1.94 U/g versus 6.51 +/- 0.57 U/g, p < 0.01). The last two effects were further accentuated when 10 mg/kg L-NAME was given before PAF. Treatment with sodium nitroprusside, a nitric oxide donor, for 10 min before and after PAF administration reversed the effects of L-NAME. Animals pretreated with phenylephrine rather than L-NAME did not develop worse injury than animals treated with PAF alone despite comparable reductions in superior mesenteric blood flow before PAF treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet-activating factor treatment of human spermatozoa enhances fertilization potential

The purpose of this study was to evaluate the effects of platelet-activating factor treatment on human spermatozoa fertilizing capability, as assessed by the zona-free hamster penetration assay. Twenty-three semen specimens were divided into two groups on the basis of initial motile sperm count greater or less than 20 x 10(6)/ml. Each sample served as its own treatment control. Twice-washed spermatozoa were treated with 1 x 10(-7) mol/L of platelet-activating factor before performing the sperm penetration assay. For the two groups the percent penetrations and penetration index were compared with the paired t test, and an independent t test was used to compare parameters between groups. Significant enhancement of both percent penetrations (p < 0.0001) and penetration index (p < 0.005) were noted on platelet-activating factor treatment in both groups. Enhancement of both indices were noted to be independent of initial motile sperm counts. Platelet-activating factor treatment of human spermatozoa may be of potential therapeutic value in certain cases of asthenospermia or fertilization failure in human in vitro fertilization.

Platelet-Activating Factor Stimulates Protein Kinase C Translocation in Cerebral Microvessels

The effects of platelet-activating factor (PAF) on distribution of protein kinase C activity in brain microvessels have been examined. Our results indicate that PAF caused an increase of protein kinase C activity in membrane, accompanied by a loss of activity in the cytosol. This effect resulted to be dose-dependent and the translocation was evident after 30 min of PAF treatment. These results suggest that PAF could play a role in transport processes in the blood-brain barrier, involving protein phosphorylation by activation of protein kinase C.

Enhanced embryo development of rabbit oocytes fertilized in vitro with platelet activating factor (PAF)-treated spermatozoa

The purpose of this study was to determine the effects of PAF treatment of rabbit spermatozoa on in vitro fertilization and subsequent blastocyst formation. Rabbit spermatozoa were exposed to PAF (10(-7) M), lyso-PAF (10(-7) M), or HIS (385 mOsm/kg) for 15 min prior to insemination of ovulated oocytes. Fertilized oocytes were cultured to the hatched blastocyst stage. Fertilization rates with PAF were significantly higher than those of fresh (P < 0.001), lyso-PAF-treated (P < 0.01), HIS-treated (P < 0.05) spermatozoa. Two-cell embryos produced from oocytes inseminated with PAF-treated spermatozoa had significantly higher hatched blastocysts than oocytes inseminated with fresh (P < 0.01), lyso-PAF-treated (P < 0.05), or HIS-treated (P < 0.05) spermatozoa. We conclude that PAF treatment of spermatozoa increases fertilization rates and subsequent embryonic development.

Platelet activating factor treatment of spermatozoa enhances fertilization rates of rabbit oocytes utilizing subzonal insertion of sperm

We examined the effects of co-culture with oviductal epithelial cells, cumulus cells, trophoblastic vesicles or amniotic sac cells on the development of bovine eight-cell embryos derived from in vitro maturation and fertilization into blastocysts. Frozen-thawed spermatozoa were treated with caffeine plus Ca-ionophore A23187 for capacitation and were then co-incubated for 4 h with oocytes matured in vitro. Ova resulting from this in vitro fertilization were cultured in HEPES-buffered TCM-199 + 10% fetal calf serum(FCS) for 68 h and then removed from the cumulus cell mass. The eight-cell embryos were cultured using four co-culture systems either without cells(controls) or within rabbit oviducts. The co-culture of oviductal epithelial cells, trophoblastic vesicles or amniotic sac cells significantly (P<0.05) increased development into blastocysts (39.0 to 50.7%) when compared with co-culture with cumulus cells, control or rabbit oviducts(1.9 to 29.3%). Six of 16 recipients became pregnant with frozen embryos derived from co-culture with oviductal epithelial cells(1/2), trophoblastic vesicles(2/7) or amniotic sac cells(3/7). Eight calves, including two sets of twins, were obtained.

Effects of platelet activating factor on mouse sperm function.

Platelet activating factor (PAF) has been implicated in a variety of reproductive processes. This study was designed to investigate the effect of PAF and the specific PAF receptor antagonist, CV-3988, on capacitation and the acrosome reaction in mouse spermatozoa using an in vitro fertilization (IVF) system. When spermatozoa were preincubated for 30 min in medium containing PAF (10(-7) to 10(-11) M), a significant increase in the fertilization rate with both cumulus-free and zona-free oocytes was observed. In contrast, treatment of the spermatozoa with 10(-5) M CV-3988 caused a significant decrease in both sperm motility and fertilization rates with zona-intact and zona-free oocytes. This suppression was reversed by the addition of PAF. Furthermore, the acrosome reaction was enhanced by PAF treatment of spermatozoa in a dose-dependent manner. This stimulation of the acrosome reaction by PAF required the presence of calcium ions in the medium. While 10(-5) M CV-3988 inhibited the acrosome reaction, the inhibition was also reversed by the addition of PAF. These results suggest that PAF can stimulate not only the capacitation process but also the acrosome reaction, both of which are dependent on extracellular calcium.

The effect of platelet activating factor on different phases of murine in vitro fertilization.

Our purpose was to examine the effect of platelet activating factor (PAF) in different phases of mouse in vitro fertilization and optimal parameters that would enhance the fertilization rate. Various PAF concentrations (10(-7) to 10(-5) M) were selected to investigate its effect on three phases of mouse in vitro fertilization (i.e., sperm capacitation, sperm/oocyte coincubation, and preimplantation embryo growth) in three experimental groups: (I) with PAF treatment in the first phase, (II) with PAF treatment adopted in the first and second phases, and (III) with PAF treatment implemented in all three phases. The improvement of the fertilization rate in PAF treatment groups over the control group ranges from 6.5 to 19.0% (P < 0.05-P < 0.001). The highest enhancement of fertilization rate was achieved under the condition of PAF treatment (10(-6) M) through sperm capacitation and sperm/oocyte coincubation phases. The PAF concentration of 10(-6) M in sperm capacitation and sperm/oocyte coincubation yielded the greatest improvement in fertilization. However, continuing PAF treatment after sperm/oocyte coincubation had no beneficial effect on fertilization and preimplantation development.