Pulmonary vascular reactivity: effect of PAF and PAF antagonists.

We investigated the effects of two different platelet-activating factor (PAF) antagonists, SRI 63-441 and WEB 2086, on PAF-, angiotensin II-, and hypoxia-induced vasoconstrictions in isolated rat lungs perfused with a physiological salt solution. Bolus injection of PAF (0.5 micrograms) increased pulmonary arterial and microvascular pressures and caused lung edema. Both SRI 63-441, a PAF-analogue antagonist, and WEB 2086, a thienotriazolodiazepine structurally unrelated to PAF, completely blocked PAF-induced vasoconstriction and lung edema at 10(-5) M. At a lower concentration (10(-6) M), WEB 2086 was more effective than SRI 63-441. WEB 2086 also blocked the pulmonary vasodilation induced by low-dose PAF (15 ng) in blood-perfused lungs preconstricted with hypoxia. SRI 63-441 and CV 3988 (another PAF analogue antagonist), but not WEB 2086, caused acute pulmonary vasoconstriction at 10(-5) M and severe lung edema at a higher concentration (10(-4) M). PAF-induced but not SRI- or CV-induced pulmonary vasoconstriction and edema were inhibited by WEB 2086. In addition, SRI 63-441 potentiated angiotensin II- and hypoxia-induced vasoconstrictions. This effect of SRI 63-441 is not due to PAF receptor blockade because 1) addition of PAF (1.6 nM) to the perfusate likewise potentiated angiotensin II-induced vasoconstriction and 2) WEB 2086 did not cause a similar response. We conclude that both SRI 63-441 and WEB 2086 are effective inhibitors of PAF actions in the rat pulmonary circulation. However, antagonists with structures analogous to PAF (SRI 63-441 and CV 3988) can have significant pulmonary vasoactive side effects.     

Pharmacological actions of Y-24180, a new specific antagonist of Platelet Activating Factor (PAF): II. Interactions with PAF and benzodiazepine receptors

The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of ³H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC₅₀ 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC₅₀ values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC₅₀ 0.84 nM) was more potent than WEB 2086 (IC₅₀ 4.21 nM) and etizolam (IC₅₀ 998 nM). Y-24180, WEB 2086 and etizolam displaced ³H-PAF binding from the washed-platelets of rabbits with an IC₅₀ value of 3.50, 9.35 and 29.5 nM, respective- ly. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180(1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced ³H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 μ M. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets.     

Pharmacological actions of Y-24180, a new specific antagonist of platelet activating factor (PAF): II. Interactions with PAF and benzodiazepine receptors

The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-t hieno [3,2-f][1,2,4]triazolo [4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of 3H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC50 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC50 values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC50 0.84 nM) was more potent than WEB 2086 (IC50 4.21 nM) and etizolam (IC50 998 nM). Y-24180, WEB 2086 and etizolam displaced 3H-PAF binding from the washed-platelets of rabbits with an IC50 value of 3.50, 9.35 and 29.5 nM, respectively. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180 (1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced 3H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 microM. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets.     

Sperm DNA adducts impair fertilization during ICSI but not during IVF

Many studies emphasize the influence of the status of spermatozoal nucleus on fertilization, mainly with regard to DNA fragmentation. This study was undertaken to analyze the influence of DNA adducts content in spermatozoa on fertilization during assisted reproduction. Ovarian hyperstimulation, oocyte retrieval and laboratory work-up in 61 IVF (in vitro fertilization) and 118 ICSI (intracytoplasmic sperm injection) first cycles were performed according to the same protocol. Semen analysis was made according to WHO Manual (1999). DNA adducts assay in spermatozoa was performed by 32Ppostlabeling method. In total 331 fertilizable oocytes were obtained during IVF and 659 during ICSI. Both groups differed significantly by sperm count, motility and morphology but not by the concentration of DNA adducts in spermatozoa (0.0306 +/- 0.0217 in IVF versus 0.0373 +/- 0.0321 in ICSI). The fertilization rate during IVF was significantly influenced by sperm count (p=0.0002) and motility (p=0.0037) but not by DNA adducts concentration (p=0.30528), whereas during ICSI was positively influenced by sperm motility (p=0.04669) and negatively by DNA adducts concentration (p=0.00796). DNA adducts concentration in spermatozoa significantly negatively influences fertilization rate during ICSI, but not during IVF.     

Effect of preincubation of cryopreserved porcine epididymal sperm

Preincubation of spermatozoa is important for capacitation and successful fertilization in vitro. The effects of preincubation time on frozen-thawed boar epididymal spermatozoa as measured by sperm motility, acrosomal integrity and fertilization ability in vitro were examined. Epididymal spermatozoa were collected from three Large White boars and frozen. The thawed spermatozoa were preincubated for 0, 15, 30, 60 and 120 min. Their motility was evaluated by a sperm motility analyzer and then the sperm motility indexes (SMIs) were calculated. The status of their acrosomal integrity was evaluated by triple-staining. Then, their fertilization ability was examined by in vitro fertilization (IVF) using porcine oocytes matured in vitro. SMIs of spermatozoa and the incidences of acrosome-intact live spermatozoa from the three boars were high (21-39 for SMI and 50-61% for acrosome-intact live spermatozoa) just after thawing, but both decreased as the duration of preincubation was prolonged (2-10 and 23-40%, respectively). The incidences of sperm penetration were high (61-89% of inseminated oocytes) when the sperm were preincubated for 0-60 min. However, sperm penetration decreased as the preincubation period was prolonged to 120 min. The degree of this decrease differed depending upon the boar from which the spermatozoa were obtained (10-72%). When the two parameters, sperm motility and acrosomal integrity, were analyzed statistically, the latter parameter rather than the former one showed a significant effect on penetration ability in vitro after each duration of preincubation. These results suggest that preincubation of frozen-thawed boar epididymal spermatozoa is not required for IVF and also that the maintenance of acrosomal integrity in unreacted status, rather than the maintenance of sperm motility, is important for fertilization ability after thawing and during preincubation of boar epididymal spermatozoa.     

Live mice from cryopreserved embryos derived in vitro with cryopreserved ejaculated spermatozoa

This study was undertaken to try to reduce the number of animals required to maintain mouse strains by banking of embryos or spermatozoa. The principal objective was to cryopreserve ejaculated mouse spermatozoa, using a method recently developed for epididymal spermatozoa. Within 30 min after mating, ejaculated spermatozoa were flushed from the uterus of mated females; shortly afterwards, epididymal spermatozoa were also collected from the same males that had mated with the females. The average values for spermatozoal motility and viability of ejaculated specimens of nine males were 43 and 46%, respectively, and for epididymal specimens, the corresponding values were 60 and 52%. In experiment 1, ejaculated or epididymal spermatozoa were incubated with oocytes for 0.5 to 4 h. As evidenced by development into two-cell embryos within 24 h, kinetics of fertilization of the two spermatozoa types were similar. In experiment 2, ejaculated and epididymal spermatozoa of three males were separately cryopreserved in medium containing raffinose, glycerol, and egg yolk. Samples were cooled and seeded at -4 degrees C, cooled to -70 degrees C at 20 degrees C/min, and then were placed into liquid nitrogen for storage. When cryopreserved epididymal or ejaculated spermatozoa were thawed at > 1,000 degrees C/min and used for in vitro fertilization, > 60% of oocytes cleaved, and approximately 95% of cleaved embryos developed into morulae or blastocysts. When embryos produced with cryopreserved spermatozoa were transferred into recipients, 18 and 22 live pups were obtained from 62 and 54 embryos resulting from ejaculated or epididymal spermatozoa, respectively. This study documented the feasibility of cryopreserving ejaculated spermatozoa as an effective alternative to preserving germ plasm from genetically valuable mice.     

Brief coincubation of gametes in porcine in vitro fertilization: role of sperm:oocyte ratio and post-coincubation medium

In this study, a short coincubation time of 10 min was used to determine the effect of different sperm:oocyte ratios during in vitro fertilization (IVF), and different periods of post-coincubation in a medium that is not appropriate for IVF, on fertilization parameters. In the first experiment, a total of 1624 in vitro matured oocytes, from 4 replicates, were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000, 1500, 1000 and 500 sperm:oocyte) and coincubated for 10 min or 6 h. The oocytes from 10 min of coincubation were washed in IVF medium to remove spermatozoa not bound to the zona pellucida and transferred to another droplet of the same medium (containing no spermatozoa) for 6h. The oocytes from the other group remained with the spermatozoa for 6h. Oocytes from both groups were then cultured in embryo culture medium (IVC) for 12h to assess fertilization parameters. In the second experiment, 1872 in vitro matured oocytes, in 3 replicates were inseminated with frozen-thawed spermatozoa using the same sperm:oocyte ratios as in the first experiment. The oocytes were coincubated for 10 min and transferred directly to IVC medium for 18 h (group A), to IVF medium (containing no sperm) only for 2h and then to IVC medium for 16 h (group B), or to IVF medium (containing no sperm) for 6h and then to IVC medium for 12 h (group C or control). There was an effect of sperm:oocyte ratio on all fertilization parameters in experiment 1. The efficiency of IVF (number of monospermic oocytes/total number inseminated) was higher (P<0.05) for oocytes coincubated with spermatozoa for 10 min and inseminated with 1500 and 1000 sperm:oocyte (35.8+/-3 and 37.6+/-2.7%, respectively) and for those coincubated for 6h with 500 spermatozoa per oocyte (37.2+/-3.1%). In experiment 2, the penetration and efficiency rates obtained in group A were poor (between 3 and 15%) irrespective of the sperm:oocyte ratio. However, in group B the fertilization parameters were similar to the controls and were also affected by the sperm:oocyte ratio. These results demonstrate that coincubation time may be reduced to 10 min to increase the efficiency of fertilization depending on the sperm:oocyte ratio, and that the spermatozoa bound to the zona pellucida require a maximum of 2h in an appropriate medium to penetrate the oocytes.     

In vitro deveropment of porcine oocytes fertilized in vitro with spermatozoa preincubated in two different media

This study was conducted to clarify the effects of sperm concentration and media during preincubation on fertilization and development of porcine oocytes fertilized in vitro (IVF). The effect of porcine oviduct epithelial cell aggregates (POECA) on in vitro development of IVF embryos was also examined. Oocytes matured in vitro for 48 to 50 h were inseminated with epididymal spermatozoa preincubated at 2 sperm concentrations (1 - 2 x 10(8)/ ml vs 4 - 5 x 10(8)/ ml) for 3 h in either Dulbecco's phosphate buffered saline (PBS) or Brackett and Oliphant medium (BO). For capacitation, spermatozoa were treated with heparin (100microg / ml) for 15 min at 38.5 degrees C under 5% CO(2) in air. Cleavage and development to the blastocyst stage were evaluated on Day 3 and Day 8 after culture with or without POECA. The effect of sperm concentration on preincubation did not affect the fertilization rate, but preincubation in PBS medium did result in a higher fertilization rate (P < 0.05) than did the BO medium. The proportion of embryos undergoing cleavage and development to the blastocyst stage was significantly higher (P < 0.05) in the POECA co-culture group than in the group without POECA co-culture. The present results indicate that PBS medium can be utilized as a simple preincubation medium for porcine spermatozoa and that the presence of POECA during in vitro culture improved the development of IVF oocytes to the blastocyst stage.     

Production of virus-free seronegative pups from murine embryos arising from in vitro fertilization with mouse minute virus-exposed spermatozoa

In the present study, the risk of transmission of mouse minute virus (MMV) to recipients of murine embryos arising from in vitro fertilization (IVF) of oocytes with MMV-exposed spermatozoa and to resulting pups was evaluated. Also, the time of seroconversion of recipients and pups was investigated. To achieve this goal, IVF of oocytes with cryopreserved spermatozoa from the inbred C3HeB/FeJ mouse strain was performed, and the resulting embryos were transferred to suitable Swiss recipients. Three groups were investigated: 1) oocytes or the developing embryos were continuously exposed to 10(4) TCID(50) MMVp per milliliter in the fertilization (human tubal fluid [HTF]), culture (KSOM), and embryo transfer (M2) media (positive control); 2) oocytes and spermatozoa were exposed to MMVp in the HTF medium only and transferred after a standard washing procedure with 10 washing steps in virus-free KSOM and M2; and 3) oocytes and spermatozoa were exposed to virus-free HTF, KSOM, and M2 (negative control). To detect antibodies to MMV in recipients and progeny, serological analyses were performed by ELISA on Days 14, 21, 28, and 42, and on Days 42 and 63, respectively, after embryo transfer. The presence of MMV in the washing drops was analyzed by PCR and an in vitro infectivity assay, while organs of some recipients and pups were analyzed by PCR. Using 10(4) of the tissue culture infective dose of MMVp per millilitre in the fertilization medium only, the present results demonstrate that 10 washing steps in the IVF-ET procedure are sufficient to remove the virus to a noninfectious dose, producing MMV-free seronegative recipients and pups. As such, there is minimal risk of transmission of MMV to recipients and pups if spermatozoa become contaminated with such viral loads.     

Effects of oviductal and cumulus cells on in vitro fertilization and embryo development of porcine oocytes fertilized with epididymal spermatozoa

This study was designed to evaluate the effects of adding porcine oviductal epithelial cell (POEC) monolayers before or during the fertilization of denuded or cumulus-enclosed oocytes, in terms of fertilization results and subsequent embryo development. The variables determined were: penetration rate, mean number of spermatozoa per oocyte, male pronucleus formation rate, monospermy rate, cleavage rate after 48 h of fertilization, blastocyst rate, and mean number of nuclei per blastocyst. We used cumulus-free and cumulus-enclosed oocytes preincubated or fertilized in the presence of POEC, once the purity in epithelial cells of these cultures had been assessed. All the experiments involved the use of frozen-thawed epididymal spermatozoa to avoid replicate variability. The POEC cultures prepared showed a high proportion of epithelial cells (over 95%). Preincubation of oocytes with POEC before fertilization showed no effects on the fertilization variables determined. In contrast, during IVF under our experimental conditions, these cells attached to the cumulus cells and their interaction had a significant effect on some of the fertilization variables analyzed. The presence of POEC and cumulus cells during IVF increased oocyte penetrability. Moreover, in the absence of POEC, cumulus cells resulted in a reduced monospermy rate. On subsequent embryo culture, a lower cleavage and blastocyst formation rate were recorded when the oocytes had been preincubated with POEC before IVF.     

Effect of partial incision of the zona pellucida by piezo-micromanipulator for in vitro fertilization using frozen-thawed mouse spermatozoa on the developmental rate of embryos transferred at the 2-cell stage.

Cryopreservation of mouse spermatozoa is widely used, although considerable strain differences in fertilization rates using frozen-thawed mouse spermatozoa have been described. The C57BL/6 mouse strain is a very widely used for establishment of transgenic mice, but the fertilization rate associated with the use of cryopreserved C57BL/6 spermatozoa is very low compared with rates for other inbred strains. We have recently solved this difficulty by in vitro fertilization (IVF) in combination with partial zona pellucida dissection (PZD). However, this technique requires culture of fertilized eggs with PZD in vitro up to morula or blastocyst stage before transfer into the uterus because blastomeres are lost after transfer into the oviduct because of the relatively large artificial slit in the zona pellucida. To overcome this problem, we performed a partial zona pellucida incision by using a piezo-micromanipulator (ZIP) for IVF with frozen-thawed mouse spermatozoa. The blunt end of the micropipette touched the surface of the zona pellucida of the oocytes, and piezo pulses were used to incise the zona pellucida while the pipette was moved along by the surface of zona pellucida. The length of the incision was pir/6 microm. When cumulus-free ZIP and PZD oocytes were inseminated with frozen-thawed genetically modified C57BL/6J spermatozoa, the fertilization rates of ZIP and PZD oocytes were 52% and 48%, respectively. After embryo transfer at the 2-cell stage, 18% and 2% of the transferred embryos with ZIP and PZD developed to term, respectively. This difference was significant (P < 0.05). When ZIP and PZD zygotes were cultured to blastocyst stage and subsequently transferred to uterine horns of recipient animals, the difference between ZIP and PZD zygotes for development rate to full term was not significant. Our results indicate that ZIP is an effective alternative technique for IVF using cryopreserved mouse spermatozoa and subsequent embryo transfer.     

Pharmacological actions of Y-24180: I. A potent and specific antagonist of platelet-activating factor

The ability of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a] [1,4]diazepine to inhibit platelet-activating factor (PAF)-induced reactions was investigated. Y-24180 (0.0003–0.003 mg/kg, i.v.) dose-dependently inhibited PAF-induced bronchoconstriction in guinea pigs, but even at a high dose of 10 mg/kg, i.v., it was either inactive or weakly active against the bronchoconstriction induced by histamine, serotonin, acetylcholine, arachidonic acid, bradykinin, or leukotriene D₄. Oral doses (0.003–0.1 mg/kg) of Y-24180 also prevented hemoconcentration due to PAF in a dosedependent manner and produced a parallel shift of the PAF dose-response curve. Y-24180 (0.0003–0.1 mg/kg, i.v.) and WEB 2086 (0.03–1 mg/kg, i.v.) dose-dependently reversed PAF-induced hypotension in anesthetized rats. In mice, PAF-induced lethality was inhibited by Y-24180 and WEB 2086 with ED₅₀ values of 0.022 and 1.42 mg/kg, p.o., and 0.023 and 0.12 mg/kg, i.v., respectively. This protective effect of Y-24180 given p.o. persisted for at least 6 hr. In actively sensitized mice lethal anaphylactic shock was prevented by oral doses of Y-24180 and WEB 2086 with ED₅₀ values of 0.095 and 0.69 mg/kg, respectively. These results suggested that Y-24180 is an extremely potent and specific PAF antagonist with a good duration of action.     

Separation of motile populations of spermatozoa prior to freezing is beneficial for subsequent fertilization in vitro: a study with various mouse strains

Success with in vitro fertilization (IVF) using inbred strains of mice varies considerably and appears to be related to the proportion of motile spermatozoa present in epididymal sperm samples of different strains. In this study, motile spermatozoa were separated from the original samples using a column of Sephadex G25. IVF rates were compared between separated and nonseparated samples of epididymal spermatozoa before and after cryopreservation. Oocytes and spermatozoa were obtained from FVB, DBA/2, C57BL/6J, and BALB/c inbred mice; and from F1 (C57BL/6J;ts DBA/2) hybrid mice, and isogenic gametes were used for IVF. These strains of mice were chosen because of their common use in transgenesis and mutagenesis studies. Dulbecco PBS was used for sperm separation on Sephadex, 18% raffinose, and 3% skim milk for cryopreservation; T6 medium for IVF; and mKSOM(AA) for embryo culture. There was a marked improvement in the rate of fertilization using fresh spermatozoa after motile spermatozoa were separated in C57BL/6J and BALB/c strains (92% vs. 58%, 79% vs. 44%) but no differences were found in fertilization rates between separated and nonseparated spermatozoa in F1, FVB, and DBA/2 strains (99% vs. 83%, 95% vs. 93%, 86% vs. 87%, respectively). After cryopreservation, higher rates of fertilization were obtained with separated motile samples in all strains; the greatest improvements were obtained with spermatozoa from C57BL/6J and BALB/c strains (40% vs. 16% and 51% vs. 14% for separated and nonseparated spermatozoa, respectively). No differences were found between the proportions of 14.5-day fetuses developing from embryos derived from separated and nonseparated spermatozoa with or without cryopreservation (33% to 46%). In conclusion, the fertility of frozen-thawed mouse epididymal spermatozoa improves significantly when highly motile populations of spermatozoa are separated for freezing.     

Positive effect of partial zona pellucida digestion on in vitro fertilization of mouse oocytes with cryopreserved spermatozoa

Cryopreservation of mouse spermatozoa has been widely used; however, fertility of frozen spermatozoa in some strains, especially when inseminating cryopreserved oocytes, is low and may be improved by assisted fertilization techniques. The present study was performed to investigate the effect of partial zona pellucida (ZP) digestion on the in vitro fertilization (IVF) capacity of frozen mouse spermatozoa. Mouse oocytes were subjected to partial ZP digestion using acidic Tyrode's solution (pH 3.1). Fertilization rates in digestion groups (30 or 45 s) were higher (P < 0.05) than that of zona-intact control (78.3% or 86.3% vs. 52.5%). The recovery rate at 45 s was lower (P < 0.05) than that at 30 s (84.2% vs. 97.3%). Among vitrified oocytes, the fertilization rate in treatment group (digested for 30 s) was higher (P < 0.05) than that of zona-intact group (50.8% vs. 22.1%). After embryo transfer at the two-cell stage, 17.7% and 11.8% of transferred embryos derived from fresh and vitrified digested oocytes developed to term and showed no significant difference as compared with that from zona-intact oocytes (24.1%, P > 0.05). These results indicate that partial ZP digestion improves IVF efficiency of fresh and vitrified oocytes with frozen mouse spermatozoa, which can provide valuable information for in vitro assisted fertilization using cryopreserved gametes in the re-establishment of mouse colonies.     

The specific binding of the platelet-activating factor (PAF) receptor antagonist WEB 2086 and the benzodiazepine flunitrazepam to rat hepatocytes

Thieno-triazolodiazepines WEB 2086 and BN 50739 have been described as the potent PAF receptor antagonists. Binding of radiolabeled [³H]WEB 2086 has been widely employed to characterize PAF receptors in different cells. In a search for a PAF receptor in isolated rat hepatocytes, we discovered that the binding of [³H]WEB to rat hepatocytes was highly specific but had a relatively low affinity with a K_d of 113 nM and B_max of 0.65 pmol/10⁶ cells in freshly isolated cell suspension and K_d of 1.65 μM and B_max of 2.0 pmol/plate in cultured hepatocytes. No consistent specific binding of [³H]PAF itself was found in the same cell preparations. The binding of [³H]flunitrazepam in the presence of the peripheral type of benzodiazepine receptor antagonist Ro 5-4864 was saturated and exhibited a K_i of 3.8 nM and B_max of 3.5 pmol/plate. The central type of benzodiazepine receptor antagonist clonazepam also competed for the [³H]flunitrazepam binding, however with a much lower affinity. Various antagonists inhibited the binding of [³H]WEB 2086 with a rank order BN 50739⪢Ro 5-4864≥clonazepam. Interestingly, bicuculline, a specific antagonist of GABA(A) recognition sites, also significantly reduced the binding of [³H]WEB 2086. The binding of [³H]flunitrazepam was inhibited with a rank potency BN 50739⪢WEB 2086. Taken together, these findings suggest that the specific binding of PAF receptor antagonists WEB 2086 and BN 50739 in rat hepatocytes does not involve PAF receptors and occurs via peripheral benzodiazepine and, possibly GABA(A) receptor sites.     

The benzodiazepine receptor ligands RO 5-4864 and RO 15-1788 do not block the inhibition of PAF-induced platelet aggregation seen with the hetrazepine WEB2086

The hypothesis was tested that the hetrazepine WEB 2086 acts as an inhibitor of PAF-induced platelet aggregation via interaction with the platelet benzodiazepine receptor(BDZR). WEB 2086 is a potent inhibitor of rabbit platelet aggregation and ATP secretion induced by 370 nM PAF. The two BDZR ligands RO 5-4864 and RO 15-1788 (7-96 microM) are inactive as PAF antagonists. When platelets were pretreated with either BDZR ligand, and then exposed to various concentrations of WEB 2086, there was no alteration of the dose-response relationship of the hetrazepine on PAF-induced aggregation, as reflected by threshold concentration, ED50, or maximum inhibition seen with WEB 2086. Pretreatment of platelets with the BDZR ligands also failed to block the inhibitory action of WEB 2086 on PAF-induced ATP release. The data are consistent with the notion that WEB 2086 acts as a PAF antagonist through its action at a specific PAF receptor, and is dissociated from, and independent of, interaction with the benzodiazepine receptor.     

Effect of Platelet-Activating Factor on the Process of Cellular Differentiation of Herpetomonas muscarum muscarum

ABSTRACT. The effects of platelet-activating factor (PAF), at doses ranging from 10⁻⁶ M to 10⁻¹⁰ M, on cell growth and on cell differentiation of Herpetomonas muscarum muscarum were investigated. Cell differentiation was evaluated by both light and electron microscopy. At the concentrations used, PAF did not interfere with the protozoan growth. However, parasites grown in the presence of PAF (10⁻⁶ M) were significantly more differentiated than those grown in the absence of PAF, since the first day of culture. On the first two days of culture, PAF doses ranging from 10⁻¹⁰ M to 10⁻⁷ M, did not significantly interfere with the differentiation of these parasites, although after the third day of culture, all PAF doses used significantly increased the protozoan differentiation. Specific PAF receptor antagonists totally abrogated (WEB 2086 and WEB 2170)or significantly decreased (BN 52021) PAF effect on cell differentiation. These findings indicate PAF triggers the process of cell differentiation in Herpetomonas muscarum muscarum and suggest these parasites have receptors for PAF.     

Successful in vitro production of embryos in the red deer (Cervus elaphus) and the sika deer (Cervus nippon)

The aim of our study was to define the conditions for IVM and IVF of oocytes in 2 common deer species as models for endangered related subspecies. Immature oocytes were recovered during the breeding season from postmortem ovaries (red deer) or by repeated laparoscopic follicular aspiration (sika deer). Oocytes were cultured for 24 h in IVM medium supplemented with EGF or FSH and follicular fluid. Stag semen was collected by electroejaculation (both species) or by epididymal flushing (red deer) and cryopreserved. For IVF, oocytes were exposed to different concentrations of thawed spermatozoa in a modified Tyrode albumin lactate pyruvate medium supplemented with 20% (v/v) estrus sheep serum for 18 h. After IVF, presumptive zygotes were allowed to develop in vitro for 7 days in synthetic oviduct fluid (SOF) supplemented with fetal calf serum (10%, v/v). In both species, the presence of ovine FSH and follicular fluid improved the in vitro maturation rate. In the sika deer, the optimal sperm concentration for IVF was 10(6)/mL and some fertilized oocytes reached the early morula stage (20 to 25 cells). In the red deer, after IVF with ejaculated or epididymal spermatozoa (2.0 x 10(6)/mL), 20% of zygotes developed to the blastocyst stage (50 to 80 cells).     

In vitro fertilization and development of llama (Lama glama ) oocytes using epididymal spermatozoa and oviductal cell co-culture

A study was designed to determine the feasibility of developing in vitro maturation, fertilization and culture systems utilizing follicular oocytes and epididymal spermatozoa collected from llamas at slaughter. From a total of 1324 cumulus oocyte complexes (COCs) recovered, 972 were cultured in 50-ul drops of TCM-199 medium with 10% heat inactivated steer serum (DBS) and hormones for 30 h. After maturation, the oocytes were randomly allocated into 4 groups in a 2x2 factorial design: cumulus-enclosed oocytes, 2 ug/ml heparin (Group 1); cumulus-enclosed oocytes, 5 ug/ml heparin (Group 2); denuded oocytes, 2 ug/ml heparin (Group 3); and denuded oocytes, 5 ug/ml heparin (Group 4). Denuded oocytes were obtained for groups 3 and 4 by vortexing. Epididymides were also collected at slaugther and fresh spermatozoa (for each replicate) were obtained by mincing the cauda epididymis with a scalpel blade. A total of 721 oocytes were inseminated with 2-3 x 10(6) epididymal spermatozoa/ml in a 50-ul drop of FERT-TALP medium. After 18 h of in vitro insemination, 234 oocytes were placed in a llama oviductal epithelial cell (LLOEC) co-culture in TCM-199 for 9 d. All cultures were done at 38.5 degrees C under 5% CO(2) in air with high humidity. The rate of fertilization, initial cleavage and development in co-culture were evaluated and compared. Of 192 oocytes examined for signs of fertilization, 56 (29.2%) were penetrated by spermatozoa with 57.1% (32 56 ) of the penetrated oocytes having a male and female pronucleus. There were no differences among treatment groups in total fertilization. However, the frequency of oocytes fertilized normally tended to be higher in the denuded oocytes 67.7% (21 31 ) than the oocytes inseminated with cumulus cells 44.0% (11 25 ) independent of heparin concentration (P<0.06). The total embryo development rate to the 2 cells to blastocyst stage was 32.1% (75 234 ). There was no difference in development rate between groups. From the 234 oocytes co-cultured in LLOEC for 9 d, 15.8% developed into 2 to 16 cells, 5.6% into morulae, 6.0% into early/expanded blastocysts and 4.7% into hatching/hatched blastocysts. The results indicate that an in vitro fertilization system is possible in the llama utilizing slaughterhouse material and that llama oocytes can be fertilized in the presence of heparin and epididymal spermatozoa.     

Effect of sperm concentration during preincubation in a defined medium on fertilization in vitro of pig follicular oocytes

Pig follicular oocytes cultured in a defined medium for 28-29 h were inseminated in vitro by epididymal or ejaculated boar spermatozoa that were preincubated in a modified KRB solution at various sperm concentrations for 4 h at 37 degrees C. Sperm concentration at insemination was 2 X 10(6) cells/ml. When epididymal spermatozoa were preincubated at concentrations of 4-16 X 10(8) cells/ml, 71-75% of oocytes were penetrated. In contrast, preincubation at a low concentration (0.8 X 10(8) cells/ml) resulted in a low penetration rate (11%). Epididymal spermatozoa preincubated at a concentration of 4 X 10(8) cells/ml could also penetrate denuded oocytes. None of the oocytes were penetrated by epididymal spermatozoa that were exposed to seminal plasma before preincubation or by ejaculated spermatozoa. After preincubation, whiplash motility was observed in the epididymal spermatozoa, but not in the ejaculated spermatozoa.