Identificationof glycoconjugates in the zona pellucida of in vitro matured and tubal unfertilized bovine oocytes by lectin histochemestry

This study was undertaken to determine the pattern of saccharides of zona pellucida glycoconjugates in immature, in vitro matured and tubal unfertilized bovine oocytes using lectin horseradish peroxidase conjugates. Immature and in vitro matured oocytes were obtained from ovaries of recently slaughtered Chianina heifers. Oocytes present in follicles with a diameter < 5 mm were aspirated; 200 cumulus-intact oocytes were immediately fixed for histochemical analysis while 200 were placed in TCM 199 with 5% fetal calf serum and cultured at 39°C in a humidified incubator with 5% CO2 in air for 24 h. Tubal unfertilized oocytes were obtained from six Chianina heifers superovulated with human menopausal gonadotrophin. The heifers were slaughtered 48 h after standing estrous and the oviducts were flushed with D-PBS. Recovered oocytes (n = 66) were washed in D-PBS with 0.1% polyvinyl alcohol and then fixed for histochemical study. In order to identify neutral and acidic glycoconjugates, a pool of immature, in vitro matured and tubal unfertilized oocytes were fixed in Bouin`s fluid, embedded in paraffin and the sections processed by the periodic acid-Schiff (PAS), low iron diamine (LID) and high iron diamine (HID) methods. Characterization of terminal residues of glycoconjugates was carried out using nine lectin-horseradish peroxidase conjugates: Arachis hypogea (PNA), Griffonia simplicifolia (GSA- Il), Ulex europeus (UEA-I), Lotus tetragonolobus (LTA), Dolichos biflorus (DBA), Glycine maximus (SBA), Triticum vulgare (WGA), Canavalia ensiformis (Con-A) and Ricinus communis (RCA-I). Saponification and sialidase procedures, followed by lectin binding, were employed to visualize the distribution and to reveal the sequence of the sialoglycoconjugates in the zona pellucida. In the present study the zona pellucida of all the oocytes exhibited positive reactions to PAS, LID and HID. In particular, the positive Con-A, WGA, PNA, RCA-I and SBA stainings suggested the presence of glycoconjugates containing mannose, N acetylglucosamine, B-galactose and above all N acetylgalactosamine residues. Moreover, these results demonstrate no differences between carbohydrate compositions of the zona pellucida in immature and in vitro matured oocytes, whereas some variations in the content and distribution of terminal sugars of glycoconjugates in tubal unfertilized oocytes were observed.