Platelet-activating factor-receptor sequence analysis between motile and nonmotile spermatozoa

Abstract

Objective: Platelet-activating factor (PAF) plays a significant role in fertility. Human spermatozoa synthesize PAF (sPAF) and this sPAF is required for motility and fertilization. PAF’s mechanism of action is a receptor-mediated event and its expression (mRNA content) and presence (distribution along the cell’s membrane) have been reported in the spermatozoa. Whereas, PAF-receptor content and distribution are significantly different between normal and abnormal specimens, limited information is available on differences in expression (i.e. mRNA) sequences between motile and non-motile spermatozoa. Therefore, the study objective was to determine if a difference exists between motile and non-motile spermatozoa with regard to PAF-receptor mRNA sequences. Design: Motile and nonmotile human sperm subjected to RNA extraction, reverse transcription-polymerase chain reaction, and sequence analysis. Materials/Methods: Motile and non-motile spermatozoa were separated by silane-coated silica particle suspension wash (Promotor™, CERES Fertility, San Diego, CA) and total RNA purified by acid phenol extraction and ethanol percipitation. Complementary DNA were synthesized by reverse transcriptase with oligo dNTP and random primers at 80°C, 30 minutes; 42°C, 60 minutes; 95°C, 5 minutes. The RT products were amplified with Taq polymerase, dNTP, and PAF receptor specific primer pair at 94°C, 30 seconds; 60°C, 1 minute; 72°C, 1 minute for 30 cycles followed by 72°C, 1 minute. The RT-PCR products were analyzed by agarose gel electrophoresis. The purified RT-PCR products were sequenced by an automated thermal cycle sequencer with Taq DNA polymerase and fluoresecnt dye-labeled terminators. The PAF-receptor sequence data generated was queried for homology via BLAST (http://www.ncbi.nlm.nih.gov/blast/). Results: Analysis of cDNA sequences for the PAF-receptor generated from motile and non-motile spermatozoa showed homology with known PAF-receptor sequences. There were significant differences (p <0.05) between the motile (∼92% homology) and non-motile (∼83% homology) populations with respect to control PAF-receptor cDNA sequences. Conclusions: The data confirms the presence of PAF-receptor mRNA in human spermatozoa. Whereas, significant differences between motile and nonmotile sperm PAF-receptor sequences were found, no single point mutations were observed. The mechanism of PAF’s action in spermatozoa may be due to a receptor sequence defect resulting in depressed receptor content and, or distribution along the cell as well as in event(s) following ligand-receptor binding. Additional studies are warranted to elucidate the mechanism of PAF’s action in spermatozoa. Supported by: Organon, Inc, West Orange, NJ.