Presence of platelet-activating factor in squirrel monkey (Saimiri boliviensis) spermatozoa: seasonal differences

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) (PAF) is a potent signaling phospholipid which has pleiotropic biological properties in addition to platelet activation. PAF has been detected in the spermatozoa in a number of species. The concentration of PAF is inversely related to human spermatozoal quality. There are no reports on the presence of PAF in nonhuman primate spermatozoa. Therefore, the primary objective of this study was to determine if PAF is present in the spermatozoa from the squirrel monkey (which is a seasonal breeder). A second objective was to determine if PAF levels change from the breeding to the nonbreeding season. Endogenous lipids were extracted from mature Bolivian squirrel monkeys (Saimiri boliviensis) spermatozoa and assayed for the presence of PAF by [¹²⁵I] radioimmunoassay. PAF was detected in all samples assayed. PAF levels were significantly higher (P< 0.01) during the breeding season (mean: 3.58 ng/10⁶ spermatozoa) than the nonbreeding season (mean: 0.76 ng/10⁶ spermatozoa). The data demonstrate that PAF is present in squirrel monkey spermatozoa, with higher levels found during the breeding season. Additional studies are warranted to elucidate the role of PAF in spermatozoa function. Am. J. Primatol. 45:301–305, 1998. © 1998 Wiley-Liss, Inc.     

Presence of platelet-activating factor in rhesus (Macaca mulatta) spermatozoa.

Platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-phosphocholine; PAF] is a unique signaling phospholipid which has been implicated in a number of biological activities (e.g., reproduction). PAF has been detected in the spermatozoa from a number of laboratory and domestic species, including, but not limited to, rabbit, bovine, and the mouse. The concentration of PAF is inversely related to human (Homo sapien) spermatozoal quality. Additionally, PAF levels are significantly higher in Bolivian squirrel monkey (Saimiri sciureus) spermatozoa obtained during the breeding season than spermatozoa obtained during the nonbreeding season. There are no reports on the presence of PAF in rhesus (Macaca mulatta) spermatozoa. Therefore, the primary objective of this study was to detect the presence of PAF in rhesus spermatozoa. A second objective was to determine if PAF spermatozoa levels differ between animals housed individually (single-caged) versus free-ranging (open corrals). Semen were collected from mature rhesus via electro-ejaculation. Spermatozoa were washed free of ejaculatory plug and quick frozen in PBS. Endogenous lipids were extracted from thawed spermatozoa and ejaculatory plugs then assayed for the presence of PAF by [125I]-radioimmunoassay. PAF was not detected in any ejaculatory plugs. PAF levels were significantly higher (P < 0.01) in spermatozoa obtained from free-ranging males (mean: 1.16 pmol/10(6) spermatozoa) than males housed individually in single cage units (mean: 0.53 pmol/10(6) spermatozoa). PAF was present in rhesus spermatozoa. Additionally, PAF levels were higher in spermatozoa obtained from corral-housed animals. Additional studies are warranted to elucidate the role of PAF in spermatozoa function.     

Characterization of3H-labelled platelet activating factor receptor complex solubilized from rabbit platelet membranes

Rabbit platelet membranes, preincubated with³H-labeled platelet activating factor ([³H]PAF), were solubilized with 2% digitonin. Sedimentation of the detergent extract in a sucrose density gradient revealed a major labeled component with a sedimentation coefficient (s_20,ω) of 10.5 S, which was substantially diminished when an excess of unlabeled PAF or L-652,731, (trans-2,5-bis(3,4,5-trimethoxyphenyl)tetrahydrofuran), (PAF antagonist) was present in the preincubation mixture, suggesting that the 10.5 S component is a specific receptor-bound [³H]PAF complex. Gel filtration of the [³H]PAF-receptor complex on Sephacryl S-300 revealed a single radiolabeled fraction with an apparent Stokes' radius of 4.9 nm. The apparent molecular weight and the frictional ratio of the agonist-receptor complex were computed to be 220 000 and 1.13, respectively. Dissociation of [³H]PAF from the radioligand-receptor complex was facilitated by Na⁺ and Li⁺, whereas K⁺ and Cs⁺ were ineffective. The guanine nucleotide, GTP, was also found to promote the dissociation in a manner that is additive with the effect of Na⁺, suggestive of the coupling of a guanine nucleotide binding protein to the solubilized PAF-receptor complex.     

Amino acids in ram testicular fluid and semen and their metabolism by spermatozoa

1. The testis of the ram secretes considerable amounts of amino acids (200μmoles/day) into the fluid collected from the efferent ducts. The principal amino acid in this testicular fluid is glutamate, which is present in concentrations about eight times those in testicular lymph or in blood from the internal spermatic vein. 2. The concentration of glutamate in seminal plasma from the tail of the epididymis is about ten times that in testicular fluid, and, though glutamate is the major amino acid in ejaculated seminal plasma, its concentration is less than in epididymal plasma. 3. After the intravenous infusion of [U-¹⁴C]glucose, labelled glutamate was found in the testicular fluid. Radioactivity was also detected in alanine, glycine, serine plus glutamine and aspartate. Alanine had the highest specific activity, about 50% of the specific activity of blood glucose. 4. When [U-¹⁴C]glutamate was infused, the specific activity of glutamate in testicular fluid was only about 2% that in the blood plasma. 5. Testicular and ejaculated ram spermatozoa oxidized both [U-¹⁴C]glutamate and [U-¹⁴C]leucine to a small extent, but neither substrate altered the respiration from endogenous levels. 6. No radioactivity was detected in testicular spermatozoal protein after incubation with [U-¹⁴C]glutamate or [U-¹⁴C]leucine. Small amounts of radioactivity were detected in protein from ejaculated ram spermatozoa after incubation with [U-¹⁴C]glutamate. 7. The carbon of [U-¹⁴C]glucose was incorporated into amino acids by testicular spermatozoa; most of the radioactivity occurred in glutamate.     

Platelet-activating factor content in boar spermatozoa correlates with fertility

The objective of this study was to evaluate the level of platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF] content in spermatozoa between two groups of boars that differ in farrow rate percentages. The boar farrow rate was defined as High if it was > or = 70% and Low if it was < 70%. Fresh, extended semen was collected from sexually mature boars and used in the PAF extractions. Platelet-activating factor was detected in all semen samples assayed. The amount of PAF detected in spermatozoa obtained from the High group ranged from 1.90 to 11.30 pM/10(6) cells. The level of PAF in the Low group ranged from 0.92 to 4.96 pM/10(6) cells. Regression analysis revealed a positive (R2 = 0.369) and significant (P = 0.021) relationship between PAF content in boar spermatozoa and farrow rate. Spermatozoa-derived PAF levels (mean +/- SEM) were significantly higher (P = 0.015) in the High-farrow group (6.75 +/- 1.25 pM/10(6) cells) than in the Low-farrow group (2.45 +/- 0.51 pM/10(6) cells). The PAF content in spermatozoa was significantly higher (P = 0.035) in the High-average (> or = 10.5/litter) number of piglets born group (5.78 +/- 1.24 pM/10(6) cells) than in the Low-average (< 10.5/litter) number of piglets born group (3.34 +/- 1.19 pM/10(6) cells). Additionally, PAF content in spermatozoa was significantly higher (P = 0.034) in the High-average (> or = 9/litter) number of piglets born alive group (6.82 +/- 1.35 pM/10(6) cells) than the Low-average (< 9/litter) number of piglets born alive group (3.00 +/- 0.87 pM/10(6) cells). The data demonstrate that PAF is present in boar spermatozoa and that levels are significantly higher in individuals with a high-farrow rate status and high-number of piglets born and born-alive.     

Characterization of 3H-labelled platelet activating factor receptor complex solubilized from rabbit platelet membranes

Rabbit platelet membranes, preincubated with 3H-labeled platelet activating factor ([3H]PAF), were solubilized with 2% digitonin. Sedimentation of the detergent extract in a sucrose density gradient revealed a major labeled component with a sedimentation coefficient (s20,omega) of 10.5 S, which was substantially diminished when an excess of unlabeled PAF or L-652,731, (trans-2,5-bis(3,4,5-trimethoxyphenyl)tetrahydrofuran), (PAF antagonist) was present in the preincubation mixture, suggesting that the 10.5 S component is a specific receptor-bound [3H]PAF complex. Gel filtration of the [3H]PAF-receptor complex on Sephacryl S-300 revealed a single radiolabeled fraction with an apparent Stokes' radius of 4.9 nm. The apparent molecular weight and the frictional ratio of the agonist-receptor complex were computed to be 220,000 and 1.13, respectively. Dissociation of [3H]PAF from the radioligand-receptor complex was facilitated by Na+ and Li+, whereas K+ and Cs+ were ineffective. The guanine nucleotide, GTP, was also found to promote the dissociation in a manner that is additive with the effect of Na+, suggestive of the coupling of a guanine nucleotide binding protein to the solubilized PAF-receptor complex.     

Inhibitory effect of azelastine hydrochloride on synthesis and release of platelet activating factor from human alveolar macrophages

The effect of azelastine hydrochloride (azelastine) on synthesis and release of platelet activating factor (PAF) in alveolar macrophages obtained from asthmatic and non-asthmatic subjects was examined. Alveolar macrophages (AMs) were preincubated with or without azelastine and stimulated with f-Met-Leu-Phe (fMLP, 10 μM) for 15 min. PAF activity was detected by aggregation of washed guinea pig platelets. PAF activity released from alveolar macrophages (AMs) from asthmatics without preincubation of azelastine was 15.97 [2.17] (mean [SD], ng/10⁷ cells) in supernatants and 42.52 [10.16] in cell pellets. After preincubation with 10⁻⁸, 10⁻⁶, and 10⁻⁴ M of azelastine, PAF activity reduced to 10.71 [2.73] (mean [SD], ng/10⁷ cells), 7.86 [0.94], and 3.52 [0.31] in the supernatants, and 35.58 [7.37], 21.57 [4.36], and 14.77 [0.99] (n = 15) in the cell pellets, respectively.PAF activity in non-asthmatic subjects without preincubation of azelastine was 8.55 [1.16] (mean [SD], ng/10⁷ cells) in supernatants and 32.64 [3.37] in cell pellets. After preincubation with 10⁻⁸, 10⁻⁶, and 10⁻⁴ M of azelastine, PAF activity reduced to 6.68 [0.78] (mean [SD], ng/10⁷ cells), 4.47 [0.51], and 2.97 [0.36] in the supernatants, and 29.53 [3.75], 14.78 [1.95], and 6.16 [0.55] (n = 20) in the cell pellets, respectively.Our results showed that preincubation with azelastine caused a dose-dependent inhibition of intra- and extracellular PAF activity from asthmatic and non-asthmatic macrophages in the same manner.     

Phospholipid metabolism in boar spermatozoa and role of diacylglycerol species in the De Novo formation of phosphatidylcholine

We have investigated pathways of lipid metabolism in boar spermatozoa sperm cells incubated for up to 3 days with [¹⁴C]palmitic acid, [¹⁴C]glycerol, [¹⁴C]choline, or [¹⁴C]arachidonic acid or incorporated these precursors into diglycerides and/or phospholipids. When spermatozoa were incubated with [¹⁴C]palmitic acid or [¹⁴C]glycerol, there was first an incorporation into phosphatidic acid, followed by labelling of 1,2-diacylglycerol (DAG) and then phosphatidyl-choline (PC). This indicates that the de novo pathway of phospholipid synthesis is active in these cells. However, not all DAG was converted to PC. A pool of di-saturated DAG, which represented a considerable proportion of the high basal levels of DAG, accumulated the majority of label. Another DAG pool, containing saturated fatty acids in position 1 and unsaturated fatty acids in position 2 and representing the remaining basal DAG, was in equilibrium with PC. When spermatozoa were incubated with [¹⁴C]arachidonic acid, there was a considerable incorporation of label into PC, which indicates the presence of an active deacylation/reacylation cycle. The behaviour of certain lipid pools varied depending on the temperature at which spermatozoa were incubated. For example, in the presence of [¹⁴C]palmitic acid or [¹⁴C]arachidonic acid, there was more incorporation of label into PC when spermatozoa were incubated at 25°C than when incubated at 17°C. Taken together, these results indicate that spermatozoa have an active lipid synthetic capacity. It may therefore be possible to design methods to evaluate the metabolic activity of boar spermatozoa based on the incorporation of lipid precursors under standardized conditions. Mol. Reprod. Dev. 47:105–112, 1997. © 1997 Wiley-Liss, Inc.     

Role of spermatozoal platelet-activating factor in fertilization

Platelet-activating factor (PAF), a potent lipid mediator of inflammation, has been shown to play a role in both the implantation and viability of mammalian embryos. We examined whether human and mouse spermatozoa release PAF during in vitro incubation and assessed the effect of exogenous PAF and the PAF receptor antagonist WEB 2086, a thieno-triazolodiazepine, on mouse in vitro fertilization (IVF) rate. PAF biological activity was detected in 11 samples of leukocyte-free, purified human spermatozoa (28 pg PAF/10(6) cells/24 hr) and 5 samples of epididymal mouse spermatozoa (7.8 pg PAF/10(6) cells/3 hr). Exogenous PAF (10(-8) and 10(-6) M) increased (p less than 0.01) the fertilization rate 2- and 3-fold, respectively of mouse oocytes by mouse epididymal spermatozoa. 10(-4) M PAF, however, reduced sperm motility and decreased (p less than 0.05) the fertilization rate. 10(-6) M WEB 2086, decreased IVF to approximately 50% of the control fertilization rate (42% vs. 89%). WEB 2086 treatment also promoted the attachment of supernumerary spermatozoa to both fertilized and unfertilized oocytes. The fertilization rate in the presence of WEB 2086 returned to control levels when zona-pellucida-free oocytes were employed, indicating that WEB 2086 did not interfere with the spermatozoal acrosome reaction. These data suggest that PAF, of spermatozoal origin, may be important in mammalian fertilization.     

Morphometric characterization of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa using computer-assisted spermatozoa analysis (ASMA)

As part of a larger study on sperm quality and cryopreservation methods, the present study characterized the head morphometry of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa, using both scanning electron microscopy (SEM) and computer‐assisted morphology analysis (ASMA). The latter method has been used rarely in fish and this is its first application on sharpsnout sea bream and gilthead sea bream spermatozoa. Results obtained using SEM are expensive and time‐consuming, while ASMA provides a faster and automated evaluation of morphometric parameters of spermatozoa head. For sharpsnout sea bream spermatozoa, similar head measurement values were obtained using both ASMA and SEM, having a mean ± standard error length of 2.57 ± 0.01 μm vs 2.54 ± 0.02 μm, width of 2.22 ± 0.02 μm vs 2.26 ± 0.04 μm, surface area of 4.44 ± 0.02 μm² vs 4.50 ± 0.04 μm² and perimeter of 7.70 ± 0.02 μm vs 7.73 ± 0.04 μm using ASMA and SEM, respectively. Although gilthead sea bream spermatozoa were found to be smaller than those of sharpsnout sea bream, spermatozoal head morphometry parameters were also found to be similar regardless of evaluation method, having a mean head length of 1.97 ± 0.01 μm vs 1.94 ± 0.02 μm, head width of 1.80 ± 0.01 μm vs 1.78 ± 0.02 μm, surface area of 3.16 ± 0.03 μm² vs 3.18 ± 0.06 μm² and perimeter of 6.52 ± 0.04 μm vs 6.56 ± 0.08 μm using ASMA and SEM, respectively. The results demonstrate that ASMA can be considered as a reliable technique for spermatozoal morphology analysis, and can be a useful tool for studies on fish spermatozoa, providing quick and objective results.     

Production and Metabolism of Platelet-Activating Factor in the Normal and Ischemic Fetal Rat Brain

Abstract: Production and metabolism of platelet-activating factor (PAF) in the fetal rat brain under normal and under ischemic stress conditions were examined. Endogenous PAF levels, determined by a bioassay using PAF-stimulated platelet release of [³H]serotonin, averaged 2.32 ± 2.14 pg/mg in control brains and was reduced to 1.10 ± 1.06 pg/mg after 20 min of maternal-fetal blood flow occlusion. [³H]PAF administered intracranially into the fetuses in utero was removed in a biphasic, time-dependent manner: a rapid component with an estimated elimination rate constant of 0.067 min^?1 and t_1/2 of 10 min and a slower component with an elimination rate of 0.017 min^?1 and t_1/2 of 41 min. In fetal brains subjected to ischemia a delayed elimination of [³H]PAF was noticed in the slow component (t_1/2 = 59 min), indicating a possible difference between the clearance of exogenous and endogenous PAF. The disappearance of [³H]PAF was accompanied by an increase in the radioactivity associated with lyso-PAF that reached a plateau after 2.5 min, possibly indicating the degradation of the fast component. A steady increase in the alkyl-acyl-glycerophosphorylcholine radioactivity commenced after 5 min and continued up to 30 min. The endogenous production of PAF and the rapid degradation due to maternal-fetal blood flow occlusion indicate an additional target for therapeutic intervention in the pathology of intrauterine ischemia. Addition of the calcium ionophore A23187 stimulated in vitro formation of PAF and lyso-PAF from [³H]-choline-labeled fetal brain phospholipids, suggesting that intracellular calcium may play a major stimulatory role in PAF production. Degradation of polyphosphoinositides by a phospholipase C may constitute a major target for PAF generated either by decapitation or after blood flow occlusion, as evident from the protective effect of the in vivo administered BN52021 PAF antagonist.     

Enzyme leakage during cryopreservation of ram spermatozoa

Spermatozoal motility and enzyme leakage during cryopreservation of ram spermatozoa was evaluated in the presence and absence of antifreeze proteins (AFP). Loss of spermatozoal motility due to the cryopreservation process was reduced by 10% in the presence of AFP. Samples of the diluted semen at various stages of the cryopreservation process were assayed for aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), acid phosphatase (ACP) and lactate dehydrogenase (LDH). The levels of AST, ALT and ACP were low in ram spermatozoa and were not considered suitable for the objective measurement of spermatozoal damage. ALP leaked during the cooling and freezing process, whereas LDH leakage was prevalent during cooling, freezing and post-thaw incubation of spermatozoa. Changes in ALP and LDH could be used as marker enzymes in the development of semen processing protocols and of semen diluents.     

Lysozyme activities and immunoglobulin concentrations in seminal plasma and spermatozoa of different teleost species and indications on its significance for sperm function

The occurrence of lysozyme and immunoglobulin (Ig) in semen of different teleost species (brown trout—Salmo trutta, perch—Perca fluviatilis, burbot—Lota lota) was studied. In all investigated species lysozyme activities (1.13-1.45 U ml⁻¹) and Ig concentrations (T-Ig: 1.11-1.61 μg ml⁻¹, IgG [measured only in brown trout]: 1.49 μg ml⁻¹) were detected in seminal plasma. Ig was also found in spermatozoa (T-Ig: 0.234-0.357 μg/g protein, IgG: 0.198 μg ml⁻¹) while spermatozoal lysozyme activities were low and fluctuating (0.093-0.164 U/g protein). In Salmo trutta lysozyme activities and immunoglobulin levels were compared between semen samples with high and low sperm motility as motility is an indicator for sperm fertility. Lysozyme activities were higher in seminal plasma of samples with high motility than in those with low motility while seminal plasma and spermatozoal immunoglobulin concentrations (T-Ig, IgG) were increased in samples with low motility in comparison to samples with high motility. Seminal plasma and spermatozoal IgG concentrations and seminal plasma lysozyme activities showed significant correlations with the sperm motility rate and swimming velocity. Moreover, lysozyme improved the viability of spermatozoa in in vitro experiments. Possible physiological meanings of these results are discussed.     

A specific, photolabile and irreversible antagonist (L662,025) of the PAF-receptor

PAF (0.2 microM) induced maximal platelet aggregation in human PRP and [3H]-PAF (1-5 nM) binding to platelet membrane preparations had Kd value of 3.8 nM and Bmax of 200 fmoles/mg of protein. Without UV irradiation, a synthetic azido tetrahydrofuran derivative L662,025 was a reversible and competitive PAF-receptor antagonist with IC50 values of 5.6 +/- 0.3 microM (platelet aggregation) and 1.0 +/- 0.25 microM (receptor binding). Photolysis of L662,025 in the presence of PRP produced an irreversible inhibition of platelet aggregation and specific binding of [3H]-PAF (1 nM). L662,025 did not affect collagen- or ADP-induced human platelet aggregation before or after photolysis. It is a new probe that can be used to identify and characterize the PAF-receptor.     

Autoradiographic localization of platelet-activating factor (PAF) binding sites in the rabbit endometrium during the peri-implantation period

Summary This communication describes the use of in-vivo and in-vitro autoradiography to map specific platelet-activating factor (PAF) receptors in the rabbit uterus. Specific [³H]PAF uptake was predominantly localized on epithelial, but not on stromal or myometrial cells. Very few silver grains were associated with the luminal epithelial cells in the uterus of the estrous rabbit, primarily because of the non-differentiated state of the epithelium. In the differentiated pregnant uterus, significantly more [³H]PAF was bound to the glandular epithelial cells, with the stromal cells binding consistently significantly less. The highest density of silver grains was observed at the implantation sites on day 7 of pregnancy. There was no apparent difference in [³H]PAF C18:0 uptake between the epithelial cells at the inter-implantation zone on day 7 and on day 6. Bound [³H]PAF was displaceable by lyso-PAF, U66985, CV3988, but not U66982, L652,731, SRI 63,441 or the inactive PAF isomer, oleoyl PAF. Bovine serum albumin (BSA) significantly inhibited tissue uptake of [³H]PAF C18:0. Intraluminally administered [³H]PAF C18:0 and intravenously injected [³H]methylcarbamyl-PAF, a non-metabolizable PAF analog, penetrated the implanted blastocyst and bound to the embryoblast. This event was reproducible in vitro with pre-implantation blastocysts from day-6 pregnant rabbits, which suggests that uterine-derived PAF may translocate into the blastocyst after attachment.     

Development of a chemically defined ram semen diluent (RSD-1)

A chemically defined ram semen diluent (RSD-1) has been developed. RSD-1 maintained spermatozoal motility of diluted semen containing approximately 800 million spermatozoa ml⁻¹ during cooling to 15°C and its storage for 1 h. Motility was further maintained when the cooled semen was diluted to 100 million spermatozoa ml⁻¹ and incubated at 38°C for about 24 h. In contrast, a conventional milk-based diluent supported motility for less than 6 h at 38°C. Spermatozoal motility was influenced by the buffering capacity, osmolarity and the presence or absence of macromolecules and calcium in the chemically defined diluent. Among the organic buffers tested, MOPS (3-(N-morpholino)propanesulphonic acid) had a marked influence on the maintenance of spermatozoal motility. The presence of MOPS also overcame the detrimental effects of 2 mM calcium in Krebs Ringer improved (KR-I) buffer.     

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.     

Insulin-like Growth Factor I Regulation of Swarm Rat Chondrosarcoma Chondrocytes in Culture

Insulin-like growth factor I (IGF-I) is anabolic for chondrocytes and is thought to be important in regulating such normal cartilaginous tissues as the epiphyseal growth plate. In the present studies, we have investigated the role of IGF-I in the regulation of neoplastic cartilage. Chondrocytes cultured from a transplantable rat chondrosarcoma were analyzed for responsiveness to IGF-I with respect to DNA and glycosaminoglycan synthesis as determined by labeling with radioactive thymidine and sulfate, respectively. Stimulation of [³H]thymidine and [³⁵S]sulfate incorporation by IGF-I was two to four times that in serum-free controls, with half-maximal stimulation at 1 × 10^-9M. The efficacy of IGF-I was approximately one-half of that of serum in stimulating [³H]thymidine incorporation and was comparable to that of serum for [³⁵S]sulfate incorporation. When Swarm rat chondrosarcoma chondrocytes were cultured in the presence of IGF-I and exposed to graded concentrations of anti-IGF-I antibody, [³H]thymidine incorporation and [³⁵S]sulfate incorporation were attenuated in a dose-dependent fashion to 29 and 25% of antibody-free controls, respectively. Nonspecific antibody not raised against IGF-I was not inhibitory. These observations suggest that the majority of IGF-I action on these cells is susceptible to immunoinhibition. To estimate the contribution of IGF-I to the regulation of these cells by serum, Swarm rat chondrosarcoma chondrocytes were cultured with graded concentrations of either calf serum or fetal calf serum in the presence of anti-IGF-I antibody, nonspecific antibody, or no other additives. Specific antibody attenuated the effect of calf serum on both [³H]thymidine and [³⁵S]sulfate incorporation with overall inhibition of 52% (P < 0.01) and 48% (P < 0.001), respectively. Nonspecific antibody superimposed small, variably stimulatory or inhibitory effects on those of calf serum. When chondrosarcoma chondrocytes were incubated with fetal calf serum, anti-IGF-I antibody exerted a minimal inhibitory effect, reducing both [³H]thymidine and [³⁵S]sulfate incorporation by less than 25%. The immunoinhibition of both pre- and postnatal serum could be overcome in a dose-dependent fashion by increasing serum concentrations. These results suggest that the factors influencing Swarm rat chondrosarcoma chondrocytes may be developmentally regulated and that the contribution of IGF-I to the action of serum increases between fetal and postnatal life. These data support the hypothesis that chondrosarcoma is a somatomedin-responsive neoplasm and suggest that this tumor may be susceptible to interventions directed toward mechanisms that block insulin-like growth factor action.     

A comparison of the brain uptake of N-(cyclopropyl[11C]methyl)norbuprenorphine ([11C]buprenorphine) and N-(cyclopropyl[11C]methyl)nordiprenorphme ([11C]diprenorphine) in baboon using PET

Buprenorphine and diprenorphine were radiolabeled with ¹¹C and their distributions in the baboon brain were studied using positron emission tomography (PET). Specific binding was demonstrated in the striatum (but not in the cerebellum) by pretreating the baboon with (−)naloxone. The absolute striatal uptakes and time courses were similar for these two radioligands but the ratio of radioactivity in the striatum to cerebellum in the baboon was higher for [¹¹C]diprenorphine than for [¹¹C]buprenorphine. Analysis of baboon plasma indicated that both [¹¹C]diprenorphine and [¹¹CJbuprenorphine are rapidly metabolized. Analysis of radioactivity in mouse brain indicated that these two radioligands are stable to metabolic transformation. At 30 min after injection, 86–90% of extracted radioactivity was due to unchanged ¹¹C-labeled radioligands. These results suggest that both [¹¹C]diprenorphine and [¹¹C]buprenorphine may be useful radioligands for studying opioid receptors in humans, although [¹¹C]diprenorphine may be a better radioligand than [¹¹C]buprenorphine for this purpose because of its more rapid clearance from the cerebellum.     

Relationship between platelet-activating factor concentration in rhesus monkey (Macaca mulatta) spermatozoa and sperm motility.

Platelet-activating factor (PAF) is a potent signaling phospholipid that has been implicated in a number of biological activities. PAF concentration in primate spermatozoa has a positive correlation with fertility. While PAF is present in rhesus spermatozoa, there are no relational reports on its concentration and the cell's motility. The study objective was to determine if PAF concentration in rhesus spermatozoa was correlated with motility indices (percent motility and forward progression). Semen was collected from sexually mature males and cell counts, and percent motilities and forward progressions were recorded prior to PAF measurement by radioimmunoassay. Spermatozoa-derived PAF concentration ranged from a low of 0.9 picomoles/10(6) cells to a high of 13.0 picomoles/10(6) cells. The overall mean (+/-SEM) PAF concentration was 4.6 (+/-1.6) picomoles/10(6) spermatozoa. Regression analysis revealed a positive and significant relationship between PAF concentration in the spermatozoa and percent motility (R2 = 0.914; P < 0.01) as well as forward progression (R2 = 0.849; P < 0.05). A receiver-operator characteristic curve and the calculation of the probability that a positive forward progression will be predicted indicated a cutoff limit of 1.5 picomoles/10(6) cells for PAF concentration in rhesus sperm. Rhesus monkey spermatozoa motility was significantly greater (P < 0.01) in the high-PAF (> or =2 picomoles/10(6) cells) group (31.0 +/- 7.6) than in the low-PAF (<2 picomoles/10(6) cells) group (6.8 +/- 2.1). Rhesus monkey spermatozoa forward progression was significantly greater (P < 0.05) in the high-PAF (> or =2 picomoles/10(6) cells) group (3.0 +/- 1.0) than in the low-PAF (<2 picomoles/10(6) cells) group (0.7 +/- 0.3). The data demonstrate that PAF concentration in rhesus spermatozoa has a significant relationship with percent motility and the cell's forward progression. Determining PAF concentration in spermatozoa may be a significant predictor of fertility in the primate. Additional studies will elucidate the role of PAF in spermatozoa function and the significance PAF plays in primate fertility.