17 FLOW CYTOMETRY ANALYSIS OF THE TURKEY SPERM GLYCOCALYX: EFFECTS OF NEURAMINIDASE TREATMENT

Abstract

The surface of all eucaryotic cells consists of a carbohydrate-rich zone known as the glycocalyx which mediates a variety of specific cell-recognition processes, including sperm-egg interactions. Several carbohydrate residues are known to be necessary for poultry sperm to traverse the female reproductive tract and for sperm-egg recognition. Alteration of the sperm glycocalyx during semen storage could adversely impact fertility. Accordingly, the aim of this work was to elucidate the sugar residues existing in the surface of turkey spermatozoa as a first step in understanding the deleterious effects of semen storage on sperm physiology and function. Nine turkey toms were randomly assigned to 1 of 3 groups (3 males/group) and semen was pooled for each group. Seminal plasma was removed and isolated sperm were resuspended in Tris-NaCl buffer at either (1) pH 7.4 for immediate staining with 1 of 16 FITC-conjugated lectins (100 µg/mL; 30 min; 25^°C; 100 × 10⁶ cells/mL), or (2) pH 6.0 for neuraminidase treatment (30 min; 37^°C; 1 IU/10⁹ cells), followed by washing (700 g, 5 min; 4 times) and lectin staining. The control consisted of sperm cells incubated only in Tris-NaCl buffer. Stained and control samples were washed twice (700 g, 5 min) and counterstained with 12 µM propidium iodide (PI); the green fluorescence intensity of PI-negative cells was assessed using a Coulter EPICS XL-MCL" Flow Cytometer (Beckman Coulter, Inc., Fullerton, CA, USA) and single-parameter histogram analysis (6 replicates). Median fluorescence intensity (MdFI) was used to assess differences in the presence of sugar residues among the control, non-neuraminidase-treated, and neuraminidase-treated samples (Kruskal-Wallis test; Tukey's HSD test in post-hoc comparisons). Lectin staining occurred in the PI-positive subpopulation, or membrane-disrupted cells, of all samples. By contrast, only intact cells were labeled by LPA, ConA, RCA-I, and s-WGA lectins in the non-neuraminidase-treated samples, indicating that only residues of sialic acid, ±-mannose/±-glucose, ²-galactose, and N-acetylglucosamine oligomers were on the surface. As expected, removal of sialic acid significantly (P < 0.05) decreased the fluorescence intensity of LPA-labeled cells. This treatment significantly (P < 0.05) increased the affinity of ConA, RCA-I, and s-WGA lectins, and exposed sugar residues previously undetected using ECA (N-acetyllactosamine), GNA (±-mannose), GS-I (±-galactose), GS-II (N-acetylglucosamine monomers), LOTUS (±-l-fucose), PNA (²-galactose), SBA (±-N-acetylgalactosamine), and WFA (²-N-acetylgalactosamine) lectins. Low levels of MdFI were observed for cells labeled by JAC (±-galactose), PSA (±-mannose/±-glucose), STA (N-acetylglucosamine polymers) and UEA-I (±-l-fucose) irrespective of neuraminidase treatment (P > 0.05). These observations suggest that (1) the carbohydrate content of turkey sperm glycocalyx is extensively masked by sialic acid molecules, (2) it contains a diversity of sugar residues, and (3) discrimination between intact and damaged cells needs to be done in order to properly identify surface binding.