Cytochemical characterization of oligosaccharide side chains of the glycoproteins of rat zona pellucida: An ultrastructural study

Abstract

Background: The zona pellucida (ZP), an extracellular matrix which surrounds mammalian oocytes, is formed by different glycoproteins. Several studies have revealed that carbohydrate residues present in glycoproteins of ZP play a key role in the sperm-egg recognition. However, the origin and the biochemical composition of ZP remain to be completely resolved.Methods: ZP glycoproteins from rat ovarian follicles were investigated at light and electron microscopic level by the application of lectins conjugated to peroxidase, digoxigenin, and colloidal gold in combination with enzyme and chemical treatment. A quantitative analysis was also performed.Results: ZP shows reactivity to WGA, DSA, LFA, AAA, RCA I, and MAA. SBA and PNA showed a variable reactivity ranging from negative to strongly positive. A uniform pattern of binding throughout ZP was observed with DSA, Con A, AAA, MAA, and LFA. However, labeling by RCA I and SBA was higher in the outer Z P while PNA and WGA showed a higher binding in the inner ZP. Lectin reactivity was detected in cortical granules, endoplasmic reticulum, Golgi apparatus, vesicles, and multivesicular bodies of oocytes.Conclusions: ZP contained the terminal disaccharides Galpl,4GlcNAc, Galpl,3GalNAc, and GalNAcpl,3Gal and the trisaccharides Neu5Aca2, 3Galpl,4GlcNAc, Neu5Ac-Galpl,3GalNAc, and Neu5Ac-GalNAcp1,3Gal sequences. The occurrence of Fucose residues (Y 1,6 linked to the inner core region of N-linked glycoproteins of Z P was demonstrated by the use of several fucose-specific lectins. Methylation-saponification treatment in combination with lectin cytochemistry reveals that Gal, GalNAc, and polyllactosamine residues of rat Z P glycoproteins contain sulphated groups. The reactivity observed in ooplasmic vesicles was similar to that of ZP, thus suggesting that the oocyte is the site of synthesis of ZP glycoproteins.

Histochemical detection of the lectin-binding carbohydrates in the zona pellucida during oocyte growth in the wild boar (Sus scrofa scrofa)

“…Non uniform staining in antral oocytes for SO 4 β-D-Gal (1-3)-D-GalNAc (desulphation/PNA) and GlcNAc was similar to that previously reported in various domestic mammals using lectins at light microscopy [14][15][16]25] and confirmed in the rat [3] and hamster [19] at ultrastructural level; the ZP appears to be composed of a number of concentric zones, each characterised by different types and distribution patterns of the oligosaccharide chains.…”

“…Previous biochemical studies have demonstrated that the glycan portion of the glycoproteins ZPB of the pig [29] and of ZP3 [6,24,28] of the mouse may be the complementary sperm receptors mediating the primary binding between the spermatozoon and the ZP. These findings encouraged the use of plant lectins for the histochemical characterisation of the ZP glycoproteins in rats [3], humans [4] and various domestic mammals [12][13][14][15][16][17]25]. In those studies, lectin-staining was combined with treatments such as neuraminidase, desulphation and saponification to explore the carbohydrate composition and linkage types of the oligosaccharide side chains which constitute the mammalian ZP glycoproteins.…”

Pyriform cell differentiation in Podarcis sicula is accompanied by the appearance of surface glycoproteins bearing α‐galNAc terminated chains

“…Such changes represent for the first time in a nonmammalian species evidence of a gradual modification of lectin binding sites over the follicular epithelium in the follicles of nonmammalian vertebrates so far studied. Heterogeneity for lectin binding within the follicular epithelium has already been reported in mammals and has been related to the differentiation program, which induces the granulosa cells to respond differently to the gonadotropin hormones (Erickson et al, 1985;Aviles et al, 1994). Unlike the follicular epithelium, the ZP did not show any changes in carbohydrate composition and spatial distribution during oocyte development.…”

“…Indeed, the Podarcis ZP, which was found to be rich in GalNAc, GluNAc, Man, and Gal residues, exhibited the same lectin-binding pattern throughout oocyte growth. In mammals, differences in the carbohydrate composition and/or spatial distribution within the ZP of preantral, antral, and ovulated oo- cytes have been extensively documented in several species (Kaufman et al, 1989;Roux and Kan, 1991;Shalgi et al, 1991;Araki et al, 1992;Aviles et al, 1994;Parillo et al, 1998;Parillo and Verini Supplizi, 1999). Such differences have generally been related to the unequal distribution and composition of sperm receptors that are localized in the mammalian ZP (Bleil and Wassarman, 1980;Yamagata, 1985;Wassarman, 1995).…”

Identification of Apoptotic Cells by Means of Lectin Histochemistry: State of the Art Review

“…Their molecular structure, carbohydrate-binding properties and biological activities have been covered in detail in several reviews [18][19][20][21][22][23][24][25]. These proteins have been widely used for the detection of carbohydrate in various tissues and organs, e.g., the respiratory system [26][27][28][29], digestive system [30][31][32], and male [33][34][35][36][37] and female [38][39][40] reproductive systems, and in diverse pathological situations [41]. The use of lectins for the histochemical detection of glycans in tissue sections (lectin histochemistry) is an area of important applications for these molecules.…”