Presence of Platelet-Activating Factor (PAF) Receptor in Bull Sperm and Positive Correlation of Sperm PAF Content with Fertility

BRACKETT, B. G.; BOSCH, P.; MCGRAW, R. A.; DEJARNETTE, J. M.; MARSHALL, C. E.; MASSEY, J. B.; ROUDEBUSH, W. E.

Portland, OR USA

Congreso; 30 Annual Congress of the International Embryo Transfer Society; 2004

International Embryo Transfer Society

Male fertility involves the capacity to obtain a 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 PAFs action is a receptor-mediated event reported to affect intracellular calcium levels. Bull sperm contain PAF and its contents has a positive relationship with motility. While the PAF-rector 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. 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 37oC, 60 minutes followed 65oC, 5 minutes. Reverse transcription (cDNA) products were amplified with Taq polymerase, dNTP, and PAF-receptor specific primer pair (upper, 5’-AATCCAGTCACCCTGGTTGTAG-3’; lower, 5’-TGGACTCAGAGTTCCGATACAC-3’) at 94oC, 1 minute; 55oC, 1 minute; 72oC, 1 minute for 35 cycles followed by 72oC, 7 minutes. 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 4oC for 3 h, washed in PBS, then exposed fluorescein isothiocyanate-conjugated anti-IgG for 90 min at 37oC, and again washed in PBS. Specimens were examined epifluoresence microscopy at 400X. PAF content in bull sperm ranged from 1.39 ng/106 sperm cells to 13.68 ng/106 sperm cells. There was a positive correlation (P<0.05) between PAF content and ERCR. The presence of PAF-receptors on bull sperm was confirmed by immunofluorescene. However, distribution of the PAF-receptor 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.