Developing follicles of the spotted ray Torpedo marmorata express different glycoside residues in relation to granulosa differentiation and vitelline envelope formation.

Lectins constitute a class of proteins/glycoproteins that specifically bind to terminal glycoside residues. The present investigation aimed to identify lectin-binding sites in developing follicles of Torpedo marmorata. Using eleven lectins (WGA, GSI-A4, GSI-B4, PSA, UEA-I, PNA, MPA, Con-A, DBA, LCA, BPA, SBA), we demonstrated that the biochemical nature and the distribution of carbohydrate residues significantly change during oogenesis in the granulosa cells and the vitelline envelope. In fact, a progressive appearance of surface glycoproteins bearing terminated ss-GlcNAc O-linked side chains was observed in the granulosa during the differentiation of pyriform-like cells from the small ones via intermediate cells simultaneously with a significant reduction of the D-Gal chains present in their nucleus. Glycoproteins bearing ss-GlcNAc O-linked side chains were first evident on the surface of small cells in contact with the oocyte, then on the intermediate ones, and finally on pyriform-like cells. The distribution pattern of such glycoproteins over the differentiated granulosa cells remained unchanged during the subsequent stages of the oocyte growth so granulosa cells preserved the same sugar distribution pattern. Furthermore, a progressive loss of D-Gal residues was evident in the nucleus of granulosa cells. In fact, staining for D-Gal was intense in the nucleus of small follicle cells and progressively reduced till disappearing in differentiated pyriform-like cells. Conversely, the small follicle cells located under the basal lamina were devoid of ss-GlcNAc residues, and the nuclear content in D-Gal remained unchanged. This finding strongly suggests that surface glycoproteins containing ss-GlcNAc residues, and the nuclear content in D-Gal might be related to the differentiation of pyriform-like cells. The present investigation also demonstrates that the content of the sugar residues of the vitelline envelope (VE) changes during oocyte growth, suggesting that pyriform-like cells may contribute to its formation.

Type I and II metabotropic glutamate receptors modulate periaqueductal grey glycine release: interaction between mGlu2/3 and A1 adenosine receptors.

In this study we investigated the effects of type I and II mGlu receptors ligands in glycine extracellular concentrations at the periaqueductal gray (PAG) level by using in vivo microdialysis, in conscious rats. An agonist of type I mGlu receptors, (S)-3,5-DHPG (1 and 5 mM), but not a selective agonist for mGlu5 receptors, CHPG (3 and 5 mM), was noticed to increase the dialysate glycine levels in a concentration-dependent manner (60+/-15% and 136+/-13%, respectively). CPCCOEt (1mM), a selective mGlu1 receptor antagonist, perfused in combination with (S)-3,5-DHPG, counteracted the effect induced by (S)-3,5-DHPG, but did not change per se the extracellular PAG glycine values, even at the highest dosage used (2 mM). MPEP (1 and 2 mM), a selective antagonist of mGlu5 receptor, did not modify extracellular glycine level. An agonist of type II mGlu receptors, 2R,4R-APDC (25 and 50 microM), decreased the dialysate glycine in a concentration-dependent manner (-26+/-4% and -54+/-6%, respectively). The 2R,4R-APDC-induced decrease in extracellular glycine was prevented by EGlu (0.5 mM), a selective type II mGlu receptors antagonist. EGlu (0.5 and 1 mM), per se, led to a significant decrease (-56+/-7% and -57+/-2%, respectively) in extracellular PAG glycine too. This effect was prevented by DPCPX (100 microM), a selective antagonist for A1 adenosine receptors, but was not affected by CPA (1 mM), a selective A1 adenosine receptors agonist. Intra-PAG perfusion of CPA (0.1-1 mM) decreased the extracellular PAG glycine values (-47+/-13%) with 1 mM concentration. The CPA-induced effect was prevented by DPCPX (100 microM), and resulted to be additive with the 2R,4R-APDC-induced decrease in glycine values. DPCPX (1 mM) increased per se extracellular glycine (48+/-7%) at the highest dose used. Dipyridamole (100 microM), an inhibitor of both adenosine reuptake and phosphodiesterases, decreased extracellular glycine (-28+/-7%). Extracellular concentrations of glutamine never changed throughout this study. These data show opposing effects of type I and II mGlu receptors in the regulation of PAG glycine values. Moreover, functional interaction between type II mGlu and adenosine A1 receptors, which possibly operate through a common transductional pathway, may be relevant in the physiological control of glycine release in awake, freely moving rats at the periaqueductal gray matter.

Surface glycoproteins bearing alpha-GalNAc terminated chains accompany pyriform cell differentiation in lizards.

The present investigation demonstrates that in squamate reptiles, as already reported for Podarcis sicula (Andreuccetti et al., 2001), the differentiation of pyriform cells from small, stem follicle cells is characterized by the progressive appearance on the cell surface of glycoproteins bearing alpha-GalNAc terminated O-linked side chains. Using a lectin panel (WGA, GSI-A4, GSI-B4, PSA UEA-I, PNA, Con-A, DBA, LCA, BPA, SBA), we demonstrated that, during previtellogenesis, the pattern of distribution of DBA binding sites over the follicular epithelium dramatically changes. In fact, binding sites first appear in follicular epithelium at the time that small cells begin to differentiate; in such follicles, labeling is evident on the cell surfaces of small and intermediate cells. Later on, as the differentiation progresses, the binding sites also become evident on the cell surface of pyriform cells. Once differentiated, the pattern of the distribution of DBA binding sites over the follicular epithelium does not change. By contrast, during the phase of intermediate and pyriform cell regression, DBA binding sites gradually decrease, so that the monolayered follicular epithelium of vitellogenic follicles, constituted only by small cells, shows no binding sites for DBA. It is noteworthy that binding sites for DBA are present on small cells located in contact with the oocyte membrane, but not on those located under the basal lamina or among pyriform cells, and therefore not engaged in the differentiation into pyriform cells. This finding demonstrates that, in squamates, the pattern of distribution of alpha-N-GalNAc containing glycoproteins significantly changes during previtellogenesis, and that these modifications are probably related to the differentiation of small stem cells into highly specialized pyriforms.