290 ENZYMATIC ACTIVITY LEVEL OF SOME GLYCOSIDASES IN BOVINE OVIDUCTAL FLUID AT DIFFERENT STAGES OF THE ESTROUS CYCLE

Abstract

Carbohydrates play a key role in different reproductive events such as the sperm–oviductal cell interaction and sperm–oocyte recognition. In this way, α-d-mannosyl (Amari et al. 2006 Mol. Reprod. Dev. 59, 221–226) and α-2,3-sialic acid (Velasquez et al. 2006 Mol. Reprod. Dev. in press) residues contained in the zona pellucida have been identified as sperm receptors in bovine oocytes. The glycosidases, enzymes that remove carbohydrates, could play an important role in the reproductive tract, modulating decisive physiological events mediated by carbohydrates. However, the enzymatic activity level of these enzymes or its fluctuations throughout the estrous cycle in the bovine oviductal fluid (BOF) has not been studied. The objective of this work was to compare the enzymatic activity level of 7 different glycosidases in the oviductal fluid of cows at different stages of the estrous cycle. Oviducts were collected from the abattoir and classified according to the macroscopic aspect of the genital tract (Grippo et al. 1995 J. Reprod. Fertil. 105, 57–64) as early follicular (presence of growing follicles), late follicular (presence of a dominant follicle), early luteal phase (ovaries showing a corpus hemorrhagicum or a recent corpus luteum), and late luteal phase (old corpus luteum or corpus albicans). Oviductal fluid samples were collected by aspiration with an automatic pipette making simultaneous manual pressure from the isthmus toward the ampulla. Samples (9 per group) were centrifuged (7000g, 10 min) and supernatant was stored at −20°C until assay. Total activity levels were fluorimetrically measured at 450 nm, with the corresponding substrate conjugated to 4-methylumbelliferil for each enzyme (Abascal et al. 1998 Biochem. J. 333, 201–207) using a fluorometer Fluostar Galaxy (BMG LABTECH GmbH, Offenberg, Germany). Enzymatic assays were done in duplicate for 4 h at 37°C, and the reactions were stopped by adding glycine-calcium carbonate buffer. Fluorescences were corrected for tissue and substrate blanks. Fluorescence results of each enzyme and oviduct phase were analyzed using a 1-way ANOVA, with estrous cycle phase being the main factor. Results (mean counts of fluorescence) showed that the level of activity changes during the estrous cycle and the activity of some enzymes increases close to or after ovulation, suggesting a role of some glycosidases in the fertilization process. Preliminary assays for neuraminidase were negative for all samples. Future studies are necessary to identify the biological role played by the glycosidases present in the bovine oviductal fluid.

Table 1.  Enzymatic activity level of some glycosidases in bovine oviductal fluid at different stages of the estrous cycle

This work was supported by Fundación Séneca (03018/PI/05) and MEC (Project code 3495).