William E. Roudebush

Immunofluorescent evidence of the platelet-activating factor receptor on human spermatozoa

OBJECTIVE To determine the presence of the platelet-activating factor (PAF) receptor on human spermatozoa. DESIGN Prospective analysis. SETTING University-based reproductive biology laboratory. PATIENT(S) Spermatozoa obtained from men undergoing routine semen analysis. INTERVENTION(S) Spermatozoa (n = 10) were exposed to PAF, sheep anti-PAF antibody, and fluorescein isothiocyanate-conjugated rabbit antisheep antibody, and then evaluated by fluorescent microscopy. MAIN OUTCOME MEASURE(S) Assessment of fluorescent intensity at four locations on the spermatozoa (tail, midpiece, proximal head, and acrosome region). RESULT(S) Immunofluorescence demonstrated the presence of the PAF receptor on human spermatozoa. The PAF receptor was most prevalent at two sites on the spermatozoa: the midpiece and the proximal head. CONCLUSION(S) The PAF receptor is present on human spermatozoa. Platelet-activating factor may affect the motility of spermatozoa through a receptor-mediated mechanism at the midpiece and/or proximal head.

Expression of the platelet-activating factor receptor in human spermatozoa: differences in messenger ribonucleic acid content and protein distribution between normal and abnormal spermatozoa

OBJECTIVE To determine the expression and distribution of the platelet-activating factor (PAF) receptor in normal and abnormal specimens of human spermatozoa. DESIGN Prospective analysis of membrane-bound PAF receptors by immunofluorescence and PAF receptor messenger RNA by quantitated reverse transcription-polymerase chain reaction in normal and abnormal spermatozoa. SETTING University-based reproductive genetics laboratory. PATIENT(S) Men undergoing routine semen analysis. INTERVENTION(S) Normal and abnormal spermatozoa were exposed to rabbit anti-PAF receptor antibody, fluorescein isothiocyanate-conjugated goat anti-rabbit antibody, and fluorescent microscopy or subjected to RNA isolation by acid-phenol extraction and quantitated (MIMIC Construction Kit [Clontech Laboratories, Inc., Palo Alto, CA]) reverse transcription-polymerase chain reaction. MAIN OUTCOME MEASURE(S) Fluorescent intensities at six locations along spermatozoa (end piece, principal tail, midpiece, neck, proximal head, and acrosomal region) and PAF receptor expression (messenger RNA) levels. RESULT(S) Immunofluorescence demonstrated a significant difference in PAF receptor distribution between normal and abnormal human spermatozoa, specifically at the neck region. Additionally, abnormal spermatozoa were found to have statistically significantly more PAF receptor messenger RNA than normal spermatozoa. CONCLUSION(S) Platelet-activating factor receptor expression and distribution are significantly altered in abnormal spermatozoa and this may be the result of some defect in gene transcription.

Platelet-activating factor content in human spermatozoa and pregnancy outcome

OBJECTIVE To determine whether platelet-activating factor (PAF) content in human spermatozoa from an isolated population is related to fertilization and pregnancy outcome. DESIGN Prospective analysis of PAF content in human spermatozoa after a Percoll gradient wash and its relation to fertilization and pregnancy outcome. SETTING University-based reproductive genetics laboratory. SUBJECT(S) Couples undergoing assisted reproduction. INTERVENTION(S) Lipids extracted from Percoll gradient spermatozoa were quantitated for PAF content by a specific radioimmunoassay. MAIN OUTCOME MEASURE(S) The relation between spermatozoa-derived PAF levels and motility, concentration, morphology, and fertilization and pregnancy rates were determined by using regression analysis and the Student t-test. RESULT(S) Radioimmunoassay and regression analysis showed a significant and positive relation between PAF content in human spermatozoa and concentration and motility indices and implantation rate. Patients who became pregnant had a significantly higher PAF content in the spermatozoa used (7.285 pmol/10(6) cells) than did patients who did not become pregnant (2.990 pmol/10(6) cells). CONCLUSION(S) The PAF content in human spermatozoa has a significant and positive relation with motility and concentration indices and implantation rate. Pregnancy rates but not fertilization rates may be predicted by measuring PAF levels in an isolated subpopulation of human spermatozoa.

Exogenous platelet-activating factor stimulates cell proliferation in mouse pre-implantation embryos prior to the fourth cell cycle and shows isoform-specific stimulatory effects.

Mouse embryos secrete molecules homologous to platelet-activating factor (PAF), termed embryo-derived PAF (EPAF), which act in an autocrine/paracrine fashion to stimulate embryonic development in vitro. Mouse EPAF is thought to consist predominantly of hexadecyl (C16) and octadecyl (C18) PAF-like components. Mouse pre-implantation embryos cultured with exogenous PAF from the early cleavage stages exhibit increased blastocyst cell numbers and rates of mitosis around the 8-cell stage. We investigated whether exogenous PAF could specifically stimulate embryonic cell proliferation prior to the blastocyst stage in the mouse and also compared the biological activities of the C16 and C18 PAF isoforms as follows. Embryos were cultured for either 24 h or 120 h from the 2-cell stage and their total cell numbers were determined or their development assessed in terms of their incidence of successful zona-hatching respectively. In each case, embryos were cultured in unsupplemented medium or in medium supplemented with either C16 or C18 PAF (0.5 microM). Compared with controls, culture with C16 PAF produced a significant stimulation of mean total per number per embryo and a significant increase in the incidence of successful zona-hatching, whilst culture with C18 PAF had no significant effect. We then cultured 1-cell zygotes for 48 h in unsupplemented medium or medium supplemented with either an equimolar mixture of C16 and C18 PF or with either C16 or C18 PF alone (each at 0.2 microM). Embryos were also scored for cell number at 4 h and 30 h of culture. Although no significant effect on mean cell number per embryo was seen following 4 h or 30 h of culture with a mixed C16/C18 PAF preparation, culture for 48 h with a mixed C16/C18 PAF preparation or with C16 PAF alone produced a significant increase in mean cell number per embryo compared with controls - an effect that is likely to be receptor-mediated, since culture with an equivalent concentration of C18 PAF had no significant effect compared with controls. We have demonstrated that mouse zygotes/embryos can respond in a specific manner to exogenous hexadecyl PAF in terms of increased rates of cell proliferation prior to cavitation, and must be capable of doing so at some time between the first and third, and also between the second and fourth, cell cycles. Such embryos presumably express one or more classes of functional PAF-receptor molecule during this period (i.e. as early as during the 1-, 2- or 4-cell stages). We have also demonstrated that embryonic response to exogenous PAF is significantly isoform-specific, which may reflect differences between the two isoforms either in affinity for binding to putative embryonic PAF-receptor molecules or in their ability to elicit a stimulatory response following binding. This observation calls into question the use of preparations containing a mixture of hexadecyl and octadecyl PAF isoforms, particularly in dose-response studies, in the mouse.

Effect of platelet-activating factor (PAF) on preimplantation mouse B6D2F1/J embryo formation.

Platelet‐activating factor (1‐O‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine; PAF) is a potent signaling phospholipid that has been implicated in a variety of reproductive processes. Human, rabbit, and mouse preimplantation embryos produce and secrete PAR Anti‐PAF antibodies interfere with mouse preimplantation development. A controversy exists on whether exogenous PAF is beneficial to preimplantation embryo development. The study objective was to determine the effect of exogenous PAF on embryo formation. One‐cell mouse B6D2F1/J embryos were collected from PMSG/hCG primed females mated with fertile males. Embryos were exposed to PAF (0–10 μM) in MEM (0.3% BSA) for 15 min, then cultured in MEM (0.3% BSA) in a 5% CO2 in air, 95% relative humidity at 37°C atmosphere, for 120 hr to the hatched blastocyst stage. PAF (0.1 or 0.01 μM) significantly (P < 0.05) improved preimplantation embryo development and formation in vitro. PAF at higher doses had no significant effect. Supplementation of culture medium with exogenous PAF was beneficial to preimplantation embryo development in B6D2F1/J mice.

PRESENCE OF PLATELET-ACTIVATING FACTOR IN RHESUS (MACACA MULATTA) SPERMATOZOA

Abstract: 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/106 spermatozoa) than males housed individually in single cage units (mean: 0.53 pmol/106 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.

Anti-platelet activating factor (PAF) antibody inhibits CFW mouse preimplantation embryo development

ObjectiveOur purpose was to investigate the effect of anti-PAF antibodies on CFW mouse embryo development in vitro.DesignWe studied the in vitro development of CFW mouse one-cell-stage embryos cultured in MEM supplemented with anti-PAF, anti-IgG, or MEM alone to the hatched blastocyst stage.ResultsMouse embryos cultured with anti-PAF (1∶5 dilution; 61%) significantly decreased embryo development compared to controls (MEM alone; 93%), whereas embryos cultured in anti-mouse IgG-supplemented MEM (1∶ 10 dilution; 93%) had no effect.ConclusionsThe results provide additional evidence that PAF is produced and secreted by cleavage-stage embryos and is required during the preimplantation period.

Alpha-minimum essential medium (MEM) enhances in vitro hatched blastocyst development and cell number per embryo over Ham's F-10

ProblemThe development of mouse embryos in vitro is affected primarily by mouse strain-genotype and culture conditions. Embryo culture studies evaluate the effectiveness of culture conditions in supporting one- or two-cell mouse embryo development to the blastocyst stage by reporting the percentage blastocyst formation rate.MethodDetermining the cell number per cultured blastocyst may also help in determining embryo culture medium quality. The objective of this study was to determine the effect of MEM and Hams F-10 on overall CFW mouse embryo development and hatched blastocyst cell number in vitro. CFW embryos cultured in MEM had significantly higher (87%; P<0.001 hatched blastocyst rates than embryos cultured in F-10 (56%).ResultsA significant difference in nuclei per hatched blastocyst was found between MEM and F-10 (P<0.001). The results demonstrate that inbred mouse embryos have significantly higher blastocyst hatching rates and higher cell numbers per blastocyst when cultured in MEM.

Presence of Platelet-Activating Factor and Its Receptor in Baboon (Papio spp) Spermatozoa

Platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF] is a novel potent signaling phospholipid which has unique pleiotropic biological properties in addition to platelet activation. PAF has been detected in the spermatozoa in several species. The concentration of PAF is inversely related to human spermatozoal quality. PAF is present in squirrel monkey (a seasonal breeder) spermatozoa and is significantly higher during the breeding season. PAFs mechanism of action is a receptor-mediated event. There is no report on the presence of PAF or the PAF-receptor in nonhuman Old World primate spermatozoa. Therefore, the primary objective of this study was to determine if PAF is present in the spermatozoa from baboons, which are year-round breeders. A secondary objective was to determine the presence and localization of the PAF-receptor in spermatozoa. We extracted endogenous lipids from mature hybrid baboon (Papio spp) epididymal spermatozoa and assayed them for the presence of PAF by [125I]-radioimmunoassay. We also exposed baboon spermatozoa to PAF-receptor antibody followed by FITC-conjugated antibody. PAF was in all samples assayed (mean: 2.29 (±0.63) pmol/106spermatozoa). Baboon spermatozoa possess PAF-receptors most prevalently along the neck and midpiece regions. The data demonstrates that PAF and its receptor are present in baboon spermatozoa. Additional studies will elucidate the role of PAF in spermatozoal function.

Human follicular fluid and mouse cumulus cells act synergistically to enhance preimplantation mouse Balb/cJ embryo development

ProblemThe development of preimplantation mammalian embryos in vitro is less than optimal. Follicular fluid and cumulus cells have both been used, independently, to improve preimplantation embryo quality in culture.MethodTo determine the ability of mouse cumulus cell coculture in the presence of human follicular fluid to support preimplantation mouse Balb/cJ embryo development in vitro.ResultsCulture of preimplantation mouse Balb/cJ embryos independently in human follicular fluid or on mouse cumulus cells had no significant affect on blastocyst development or total cell number per blastocyst. The coculture of mouse Balb/cJ preimplantation-stage embryos on mouse cumulus cells in the presence of human follicular fluid significantly (P<0.07) improved blastocyst development and the total number of cells per blastocyst.ConclusionsCumulus cells and follicular fluid have a positive synergistic affect on preimplantation mouse Balb/cJ embryo development and formation in vitro.