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Title: CHARACTERIZING THE GLYCOCALYX OF POULTRY SPERMATOZOA: I. IDENTIFICATION AND DISTRIBUTION OF CARBOHYDRATE RESIDUES USING FLOW CYTOMETRY AND EPIFLUORESCENCE MICROSCOPY

Author
item PELAEZ, JESUS - USDA,ARS
item Long, Julie

Submitted to: Journal of Andrology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/9/2006
Publication Date: 3/1/2007
Citation: Pelaez, J., Long, J.A. 2007. Characterizing the glycocalyx of poultry spermatozoa: i. identification and distribution of carbohydrate residues using flow cytometry and epifluorescence microscopy. Journal of Andrology. 28(2):342-352.

Interpretive Summary: The sperm glycocalyx represents the primary interface between the male gamete and its environment. Most of the data for sperm glycocalyx pertaining to composition or functionality has been derived from mammalian species. Here we provide the first comprehensive characterization of the sugar residues comprising the glycocalyx of poultry sperm. Our approach was to first delineate the types of sugar residues present using flow cytometry and, secondly, to localize specific sugar residues with respect to the morphological regions of the sperm cell. For this second objective, two methods of lectin labelling (unfixed sperm in suspension versus fixed sperm air-dried on slides) were compared. The glycocalyx of turkey and chicken spermatozoa is extensively sialylated and contains residues of '-mannose/'-glucose, '- and '-galactose, '-fucose, '- and '-galactosamine, N-acetyllactosamine, as well as monomers and dimers of N-acetylglucosamine in variable amounts. Sugar residues specific for STA and UEA-I do not appear to exist in either of the two species; whereas those for PSA and Jacaline appear to only exist in turkey spermatozoa. Chicken spermatozoa are considerably richer in dimers of N-acetylglucosamine and residues of N-acetyllactosamine than turkey sperm; whereas turkey spermatozoa contain a higher amount of WFA-recognized N-acetylgalactosamine residues. Monomers of N-acetylglucosamine, and glycoconjugates containing GNA-recognized mannose residues appear to be restricted to the acrosomal region.

Technical Abstract: The aim of the present work was (1) to study the carbohydrate content of sperm glycocalyx in the turkey and chicken species using flow cytometry analysis of lectin binding, and (2) to evaluate the distribution of existing sugars over the sperm plasma membrane surface with epifluorescent microscopy. Sugar residues (lectins) investigated included galactose (GS-I, Jacalin, RCA-I, PNA), mannose (ConA, PSA, GNA), N-acetylglucosamine (GS-II, succinyl-WGA, STA), N-acetylgalactosamine (SBA, WFA), fucose (Lotus, UEA-I), sialic acid (LFA, LPA) and N-acetyllactosamine (ECA). Fresh, ejaculated spermatozoa were assessed before and after treatment with neuraminidase to remove sialic acid. Mean Fluorescence Intensity was used as indicator of lectin binding in the flow cytometry analysis. Non-treated spermatozoa showed high fluorescence intensity values when incubated with RCA-I, ConA, LFA and LPA in the two species, and also when incubated with succinyl-WGA in the chicken. Fluorescence intensity was low for the rest of lectins. Treatment with neuraminidase reduced the fluorescence intensity of samples incubated with LFA and LPA, and increased significantly that of the rest of lectins in virtually all cases. Major differences in fluorescence intensity between species included significantly higher values for succinyl-WGA and ECA in chicken, and significantly higher values for WFA in turkey. Evaluation of the distribution of carbohydrates revealed segregation of some sugar residues into membrane domains. These data suggest that (1) turkey and chicken sperm glycocalyx contains a diversity of sugar residues, (2) these residues are extensively masked by sialic acid, (3) the composition is species-specific, and (4) some sugar residues appear to be segregated into membrane domains as reported also for mammals.