Llama Sperm Binding to Oviductal Epithelium Involves N-Acetyl Galactosamine Recognition

APICHELA, SILVANA A.; JIMÉNEZ-DÍAZ, MARÍA. A.; VALZ-GIANINET, JORGE; ROLDAN-OLARTE, EUGENIA M.; MICELI, DORA C.

Budapest, Hungría

Congreso; 16th INTERNATIONAL CONGRESS ON ANIMAL REPRODUCTION; 2008

SOCIEDAD INTERNACIONAL DE REPRODUCCIÓN ANIMAL

INTRODUCTION: Sperm binding to oviductal epithelium would be involved in the sperm reservoirs formation. In other animals, a species-specific carbohydrate recognition take part in the sperm –oviduct interaction but in llama it remains unknown. The type of carbohydrate residues that could participate in the adhesion of spermatozoa to the oviductal epithelium in the llama was one of the objectives of this study. MATERIAL AND METHODS:, The distribution of glycoconjugates in llama oviducts was examined by lectin- histochemistry. Uterotubal Junction (UTJ), Isthmus and Ampulla where separated, fixed, and embedded in paraplast. Cross-sections where labelled with WGA, WGAs, UEA 1, DBA, RCA 120, Con A, PSA, LCA, PHA E, PHA L, GSL and SJA lectins-FITC/rhodamine conjugated and observed by Confocal Laser Scanning Microscopy. Competition binding assays with carbohydrates and lectins were used for  assessing the sperm binding to oviductal epithelium in vitro. Epithelial cell explants (CEC) were obtained from UTJ epithelium oviducts of non mated females. CECs were incubated with 10 ug/ml of DBA, WGA, UEA1 or PNA for 20 min (39°C, 5% CO) and then aliquots of washed fresh motile sperm (obtained by artificial vagina) were added. In parallel, fresh sperm were incubated with 10 ug/ml of glucose, manose, galactose or N-acetyl galactosamine and then co-incubated with CECs. After one hour, the explants were fixed, stained, rinsed, and the sperm binding index (BI=sperm number / 0.1 mm2) were calculated. RESULTS AND DISCUSSION: No differences where found between oviductal segments regarding the carbohydrate residues distribution on the apical cell surface. Abundant µ-mannopyranosyl, µ-glucopyranosyl, N-Acetyl glucosamine, and N-acetyl-neuraminic acid residues and scarce µ-linked N-acetyl-galactosamine and b-galactosyl residues were detected. Neither µ-L- N-acety- galactosamine fucopyranosyl nor b-N-acetyl-galactosamine residues were distinguished. N-Acetyl galactosamine inhibited sperm-CECs binding while other carbohydrates had no effect. In addition DBA lectin was the sole lectin that inhibited sperm-oviduct binding. Taken into account these results, the binding sites for Nacetyl galactosamine on sperm were corroborated by using N-acetyl galactosamine-PAA-FITC conjugate. In agreement, sperm were strongly labelled suggesting that N-Acetyl galactosamine could be implicated in a specific interaction between llama sperm and UTJ oviductal epithelium.