Submission Number: EDS-4-35-9

Abstract Number: 396

TERMINAL CARBOHYDRATE CHARACTERIZATION OF MULTIPLE FORMS OF ESTRUS-ASSOCIATED PROTEIN IN BOVINE OVIDUCTAL FLUID USING LECTIN PROBES.

Edson G Vieira* ^1,2, David A Chapman ¹ and Gary J Killian* ¹

The J. O. Almquist Research Center, The Pennsylvania State University, University Park, PA, USA ¹
Departamento de Zootecnia, UNESP, Campus de Ilha Solteira, SP, Brazil ²

Abstract:
Estrus-associated protein (EAP) is a highly glycosilated protein found in oviductal fluid (ODF) which likely plays multiple roles in sperm function, fertilization, and early embryonic development. Previous studies have determined that multiple forms of EAP exist in ODF, and their presence differ with oviduct region and estrous cycle stage. The purpose of this study was to determine if different forms of EAP have different compliments of terminal carbohydrates. Daily ODF was obtained from indwelling catheters in ampullary (A) and isthmic (I) oviducts during the non-luteal phase (NL; serum P₄ = 1.5 ng/ml) or luteal phase (L; serum P₄ > 1.5 ng/ml). Samples were subjected to two-dimensional SDS-PAGE, Western blotted and probed with anti-97kDa EAP, and a battery of lectins: Concanavalin A (ConA; -linked mannose), Dolichos biflorus agglutinin (DBA; -linked acetylgalactosamine), Peanut agglutinin (PNA; galatosyl (-1,3)-N-acetylgalactosamine), Ricinus communis agglutinin (RCA120; terminal galactose and sometimes N-actylgalactosamine), Soybean agglutinin (SBA; terminal - or -linked N-acetylgalactosamine or sometimes galactose), Ulex europaeus agglutinin I (UEA I; -linked fucose), and Wheat germ agglutinin (WGA; terminal N-acetylglucosamine or chitobiose). The detected forms of EAP, region of the oviduct and stage of the cycle of occurrence, and lectins with corresponding spots in companion gels were 99-104 kDa, pI 6.4-9.3 (ANL; RCA); 74-90 kDa, pI 5.7-9.8 (AL, ANL, IL, INL; DBA, WGA); 74-80 kDa, pI 7.3-8.3 (IL, INL; DBA, SBA, UEA); 72-73 kDa, pI 6.7-8.5 (ANL, IL; DBA, RCA, UEA, WGA); 64-70 kDa, pI 6.8-9.8 (AL, ANL, IL; ConA, DBA, SBA, UEA, WGA); 47-57 kDa, pI 7.3-9.8 (AL, ANL, IL, INL; ConA, DBA, PNA, RCA, SBA, UEA, WGA); 31-41 kDa, pI 7.9-9.8 (AL, ANL, IL, INL; ConA, DBA, PNA, RCA); 26-28 kDa, pI 7.9-8.8 (AL, IL, INL; DBA,SBA, UEA). These results indicate that the compliment of terminal carbohydrates vary with EAP form, stage of the cycle and region of the oviduct. Because specific carbohydrates associated with glycoproteins may mediate signalling, it is possible that responses of sperm or ova to types of ODF or EAP treatment are initiated by differences in specific terminal carbohydrates of ODF glycoproteins. (Supported by USDA grant#96-3523-3428 and FAPESP grant#98/5345-4).

Keywords: EAP; oviductal fluid; estrous cycle; glycoprotein; lectins

This abstract is being presented at: 8:00 AM in session:
Uterus/Oviduct I