Effect of Sorting Boar Spermatozoa by Sex Chromosomes on Oviduct Cell Binding

Abstract Text:  The following study examined how flow cytometrically sorted sperm bind to oviduct cells and purified oviduct glycans. Previous data has shown that there are two oviduct glycan motifs, bi-sialylated lactosamine (bi-SiaLN) and Lewis X trisaccharide (Le^X) that bind noncapacitated boar spermatozoa with high affinity and specificity. The sperm rich fraction from boars (n=5) was collected, sperm were stained with Hoechst 33342 and sorted in Wisconsin. Sperm were separated into either X or Y chromosome-bearing cells and placed into the following treatments: 1) sperm sorted for the X chromosome, 2) sorted for the Y, 3) an equal mixture of sorted X and Y, and 4) a control of non-sorted sperm from the same collection. Samples were then transported to Illinois and tested for oviduct cell binding within 12 h of sorting. Additionally we observed motility characteristics, acrosome status and glycan binding to three soluble fluoresceinated glycans bi-SiaLN, sulfated Le^X (suLe^X) and the control lactosamine disaccharide (LacNAc). Results showed that the number of sperm binding to oviduct cells was reduced by more than half in the three sorted samples compared to the control. When binding of fluoresceinated soluble glycans was investigated, the proportion of sperm that bound bi-SiaLN or suLe^X averaged 81% whereas 42% of sperm bound LacNAc. The glycans bound to sperm in three patterns (Pattern A: glycan binding to the apical ridge and post-acrosomal area, Pattern B: post-acrosomal binding only and Pattern C: apical ridge binding only). For suLe^X and bi-SiaLN glycans, pattern A was present on 38% of the sperm, pattern B on 29%, pattern C on 20% and no fluorescence was observed on 12% of sperm from each of the four samples. The percentage of sperm that were motile in the sorted samples was reduced on average by 15% from the unsorted control. However, Computer Assisted Semen Analysis did not detect other differences in motility parameters between the sorted and control samples. All samples maintained >97% acrosome integrity after the sorting process. In conclusion, sperm binding to the complex matrix around oviductal cell aggregates was reduced after sorting but binding to purified soluble fluoresceinated glycans was not different among sperm preparations, probably due to a requirement for higher affinity binding and motility to contact and bind intact oviduct cells. The reduction in sperm fertility observed following sorting may be due to reduced ability to bind the oviduct epithelium. 

Keywords: sperm, sex sorting, oviduct