The Presence of Specific Binding Sites on Boar Spermatozoa for Porcine Relaxin and Its Action on Their Motility Characteristics.

Abstract

The purpose of this investigation was to examine whether boar spermatozoa possess specific binding sites for relaxin and to examine what effects relaxin actually exerts on their motility characteristics. Boar spermatozoa were collected from cauda epididymides. For the relaxin binding study, washed cauda epididymal spermatozoa were smeared on glass slides, and specific sites that bind relaxin were identified by sequential application of a biotinylated relaxin probe, antibiotine immunoglobulin G conjugated to 1 nm colloidal gold, and silver for signal amplification. For the motility characteristics study, cauda epididymal spermatozoa were diluted 300-fold in modified KRB + 2% cauda epididymal fluid (approximately 1 × 10 7 cell/ml). After preincubation for 30 min at 37 C in a CO2 incubator, the sperm aliquots were placed in culture dishes covered with mineral oil and mixed with porcine relaxin. The concentrations of relaxin and spermatozoa in the aliquot were 100 ng/ml and 5 × 106 cells/ml, respectively. The aliquots were incubated for a certain defined period under the same conditions. After 30, 60, 90 and 120 min of incubation, sperm motility was recorded using a videotape recorder. As an evaluation of motility characteristics, the percent motility, grade of forward progression and velocity were examined. The results of the relaxin binding study demonstrated that relaxin bound with specificity to the midpiece and tail. In addition, bindings were visible at the acrosome cap and neck of the head. The results of the motility study which examined the effects of relaxin on the motility characteristics of boar sperm showed that relaxin did not cause a significant alteration in percent motility, but it improved forward progression. Relaxin treatment for 90 min produced a significant 1.4-fold enhancement in sperm velocity compared to that of control. These results suggest that relaxin activates sperm motility through a specific receptor.