291PRESENCE OF PLATELET-ACTIVATING FACTOR (PAF) RECEPTOR IN BULL SPERM AND POSITIVE CORRELATION OF SPERM PAF CONTENT WITH FERTILITY

Abstract

Male fertility involves the capacity to obtain viable pregnancy and offspring after insemination. Currently, the most common way to measure bull fertility is through non-return rates (NRR) calculated after insemination of many females. However, this method is time-consuming and expensive. A number of biochemical molecules in sperm have been proposed as potential predictors of male fertility, e.g. platelet-activating factor (PAF). Platelet-activating factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphorylcholine) is a ubiquitous phospholipid that is implicated in the mediation of a wide variety of reproductive processes. The mechanism of PAF’s action is a receptor-mediated event reported to affect intracellular calcium levels. Bull sperm contain PAF and its content has a positive relationship with motility. While the PAF-receptor has been reported in other species, it has not been demonstrated in bull sperm. Therefore, our objectives were to determine: 1. the relationship between PAF content in bull sperm and Estimated Relative Conception Rates (ERCR, a 3-year rolling average of NRR); and 2. the presence of the PAF-receptor in bull sperm. Sperm PAF content for bulls (n=8) with different ERCR was determined by radioimmunoassay. PAF-receptor expression was determined as follows: total RNA was purified by acid phenol extraction and ethanol precipitation. Complementary DNAs were synthesized by reverse transcriptase with dNTPs and random primers at 37°C, 60min; followed by 65°C, 5min. Reverse transcription (cDNA) products were amplified with Taq polymerase, dNTP, and PAF receptor primer pair (upper, 5′-AATCCAGTCACCCTGGTTGTAG-3′; lower, 5′-TGGACTCAGAGTTCCGATACAC-3′) at 94°C, 1min; 55°C, 1min; 72°C, 1min for 35 cycles followed by 72°C, 7min. RT-PCR products were analyzed by 2% agarose gel electrophoresis. PAF-receptor protein was determined as follows: PBS-washed bull sperm was exposed to human PAF-receptor antibody at 4°C for 3h, washed in PBS, then exposed to fluorescein isothiocyanate-conjugated anti-IgG for 90min at 37°C, and again washed in PBS. Specimens were examined by epifluorescence microscopy at 400×. PAF content in bull sperm ranged from 1.39ng/106 sperm cells to 13.68ng/106 sperm cells. There was a positive correlation (P<0.05) between PAF content and ERCR. Presence of PAF-receptors in bull sperm was confirmed by immunofluorescence. However, distribution of PAF-receptors in bull sperm was not uniform within or between specimens. A cDNA clone containing the coding region for PAF-receptor was isolated from bull sperm using a reverse transcription-polymerase chain reaction protocol. There is a positive correlation (R=0.40; P<0.05) between PAF content in sperm and in vivo fertility of individual bulls as determined by NRR. Molecular and immunofluorescence data confirm the presence of PAF-receptor (mRNA and protein) in bull sperm. Additional studies are warranted to elucidate the mechanism of PAF’s action in sperm. Early selection for fertility in bulls represents a potentially valuable application to enhance efficiency in cattle breeding.