MOLECULAR REPRODUCTION AND DEVELOPMENT 60:517±534 (2001) Distribution of Lectin-Binding Glycosidic Residues in the Hamster Follicular Oocytes and Their Modifications in the Zona Pellucida After Ovulation MAJID EL-MESTRAH AND FREDERICK W. K. KAN* Department of Anatomy and Cell Biology, Faculty of Health Sciences, Queen's University, Kingston, Ontario, Canada ABSTRACT In the present study, we have employed a battery of colloidal gold-tagged lectins as probes in conjunction with quantitative analysis to demonstrate the distribution and changes of carbohy- drate residues in the hamster zona pellucida (ZP) during ovarian follicular development and during transit of the oocyte through the oviduct after ovulation. High-resolution lectin±gold cytochemistry performed on thin sections of LR White-embedded ovaries revealed a moderate to strong reactivity to WGA, PNA, DSA, AAA, and MAA over the entire thickness of the ZP of ovarian oocytes at different stages of follicular development. Labeling intensity over the ZP progres- sively increased as follicles matured in the ovary. In parallel, there was an association of labeling by gold particles with cortical granules, stacks of Golgi saccules, and complex structures called vesicular aggregates in the oocyte proper especially during the late stages of follicular growth. In contrary, labeling with each of HPA, DBA, and BSAIB 4 was absent in the ovary but was found to be localized over Golgi complexes and secretory granules in the non-ciliated secretory cells of the oviduct. When ovulated oocytes were labeled with each of HPA, WGA, RCA-I, PNA, DSA, BSAIB 4 , AAA, MAA, and DBA, the ZP and several organelles in the oocyte proper presented a differential distribution of lectin-binding sites. Quantitative analy- sis was also performed on labeling by lectin±gold complexes that bind specifically to the ZP of mature follicular and ovulated oocytes. Quantitative evaluation revealed heterogeneous labeling between the inner and the outer zone of the ZP. A significant increase in the labeling densities in both inner and outer ZP was noted when tissue sections of ovulated oocytes were labeled with RCA-I or AAA. Tissue sections of ovaries labeled with WGA demonstrated a significant increase in the density of labeling in the outer layer of the ZP. Labeling by PNA, DSA, and MAA, however, showed a significant decrease in both the inner and outer portions of the ZP. Together, these results suggest that in the hamster, glycoproteins carrying specific sugar residues are added to the ZP of ovarian follicles during the early stages of folliculogenesis and are processed through a common secretory machinery, and that there is a significant change in both the sugar moieties and distribution of glycoproteins in the ZP following ovulation. Our results also showed that the hamster oviduct plays an important role in contributing certain glycoproteins to the ZP suggesting that the sugar moieties of these oviductal glycoproteins may have functional significance in fertilization. Mol. Reprod. Dev. 60: 517±534, 2001. ß 2001 Wiley-Liss, Inc. Key Words: zona pellucida glycoproteins; carbohy- drate residues; folliculogenesis; oviductal secretions; lectin cytochemistry INTRODUCTION The zona pellucida (ZP), a thick extracellular matrix that surrounds mammalian oocytes, appears during follicular growth, increasing in width as oocytes in- crease in diameter (Bleil and Wassarman, 1980). This structure is composed of three major glycoproteins: ZP1, ZP2, and ZP3 in the hamster (Moller et al., 1990; Yanagimachi, 1994). Collectively, they account for the functions of the ZP that include: species-speci®city, blockage to polyspermy, induction of acrosome reaction, and protection of cleaving embryo (Wassarman, 1987; Yanagimachi, 1994). For some time, it was believed that the chemical composition of the ZP does not change throughout oocyte maturation or even after ovulation. According to Fowler and Grainge (1985), however, the zona matrix of mouse eggs loses glycosaminoglycans several hours before ovulation. Previous biochemical studies performed in several mammalian species in- cluding the human also demonstrated modi®cations in the zona properties during folliculogenesis. In the sheep, Crozet and Dumont (1984) reported that sperm only binds the zonae pellucidae of oocytes isolated from 2 mm antral follicles but not to those originating from 1 mm diameter ones. Human oocytes exposed to capacitated spermatozoa in vitro show a substantial increase in the penetrability of their ZP during the transition from metaphase I to metaphase II (Tesarik et al., 1988). A similar observation was reported by Shalgi et al. (1989) in rat oocytes. Other studies carried ß 2001 WILEY-LISS, INC. Grant sponsor: Canadian Institutes of Health Research (CIHR). *Correspondence to: Dr. Frederick W.K. Kan, Department of Anatomy and Cell Biology, Faculty of Health Sciences, Queen's University, Kingston, Ontario, Canada K7L 3N6. E-mail: kanfwk@post.queensu.ca Received 9 April 2001; Accepted 8 June 2001