310 DETERMINATION OF THE PORCINE OVIDUCTAL GLYCOSIDASES DURING THE ESTROUS CYCLE

Abstract

Carbohydrates play a key role in different reproductive events, such as the sperm–oviductal cell interaction and sperm–oocyte recognition. For example, β-d-galactose and α-d-mannose residues contained in the zona pellucida have been identified as sperm receptors in porcine oocytes (Song et al. 1999 J. Mamm. Ova Res. 16). 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 their fluctuations throughout the estrous cycle in the porcine oviductal fluid (POF) has not been studied. The objective of this work was to compare the enzymatic activity level of 7 glycosidases in the POF 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 please (presence of growing follicles), late follicular phase (presence of several grown follicles), early luteal phase (ovaries showing corpora hemorrhagica or recent corpora lutea), and late luteal phase (old corpora lutea or corpora albicans). After classification, oviducts were dissected and oviductal fluid samples were collected by aspiration with an automatic pipette while applying manual pressure from the isthmus toward the ampulla. Samples (6 per group) were centrifuged (7000g, 10 min) and the supernatant was stored at −20°C until assay. Total activity levels were measured fluorimetrically at 450 nm with the corresponding substrate conjugated to 4-methylumbelliferyl for each enzyme (Abascal et al. 1998 Biochem. J. 333, 201–207) using a FLUOstar Galaxy fluorometer (BMG Lab Technologies, Offenburg, Germany). Enzymatic assays were done in duplicate for 4 h at 37°C, and the reactions were stopped by adding glycine–calcium carbonate buffer. Fluorescence was corrected for tissue and substrate blanks. Fluorescence results of each enzyme and oviduct phase were analyzed using a one-way ANOVA with estrous cycle phase being the main factor. Results (mean counts of fluorescence, see Table 1) showed that changes of enzymatic activity during the estrous cycle and activity of some enzymes were modified during or after ovulation, suggesting a role of some glycosidases in the fertilization process. Preliminary assays for neuraminidase were negative in all samples. Future studies are necessary to identify the biological role played by the glycosidases present in the POF.

Table 1.  Changes in porcine oviductal glycosidases during the estrous cycle

Supported by Fundacion Seneca (03018/PI/05) and Ministerio de Educacío y Ciencia (Project code 3495).