291 PRESENCE OF PLATELET-ACTIVATING FACTOR (PAF) RECEPTOR IN BULL SPERM AND POSITIVE CORRELATION OF SPERM PAF CONTENT WITH FERTILITY
B.G. Brackett A , P. Bosch A , R.A. McGraw A , J.M. DeJarnette B , C.E. Marshall C , J.B. Massey C and W.E. Roudebush CA University of Georgia, Athens, GA, USA;;
B Select Sires, Inc., Plain City, OH, USA;;
C Reproductive Biology Associates, Atlanta, GA, USA. email: roudebush@rba-online.com
Reproduction, Fertility and Development 16(2) 265-265 https://doi.org/10.1071/RDv16n1Ab291
Submitted: 1 August 2003 Accepted: 1 October 2003 Published: 2 January 2004
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, 60 min; followed by 65°C, 5 min. 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, 1 min; 55°C, 1 min; 72°C, 1 min for 35 cycles followed by 72°C, 7 min. 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 3 h, washed in PBS, then exposed to fluorescein isothiocyanate-conjugated anti-IgG for 90 min at 37°C, and again washed in PBS. Specimens were examined by epifluorescence microscopy at 400 ×. 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. 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.
