Sara Zizza

Glycoconjugate histochemistry of the digestive tract of Triturus carnifex (Amphibia, Caudata)

SummaryIn this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4)n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4)n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4)noligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4)noligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H+K+-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4)noligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.

Morphometric and ultrastructural features of the mare oviduct epithelium during oestrus

Morphometric, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) investigations have displayed regional differences in the mare oviductal epithelium. The entire mucosa of the oviduct was lined with a pseudostratified epithelium, which consisted of two distinct cell types, ciliated and non-ciliated. Ciliated cells were predominant in the three different segments of the oviduct and their percentage increased from fimbriae to ampulla and significantly decreased in the isthmus. SEM revealed in the infundibulum finger-like mucosal folds, some of them interconnected, in the ampulla numerous and elaborated branched folds of the mucosa, whereas the isthmus displayed a narrow lumen, short and non-branched mucosal folds. In the ampulla and isthmus the majority of non-ciliated cells showed apical blebs provided or not of short microvilli. TEM displayed different ultrastructural features of ciliated and non-ciliated cells along the oviduct. Isthmus ciliated cells presented a more electron-dense cytoplasm than in infundibulum and ampulla cells and its cilia were enclosed in an amorphous matrix. The non-ciliated cells of infundibulum did not contain secretory granules but some apical endocytic vesicles and microvilli coated by a well developed glycocalyx. Non-ciliated cells of ampulla and isthmus contained secretory granules. Apical protrusions of ampulla displayed two types of secretory granules as well as occasional electron-lucent vesicles. Isthmus non-ciliated cells showed either electron-lucent or electron-dense cytoplasm and not all contained apical protrusions. The electron-dense non-ciliated cells displayed microvilli coated with a well developed glycocalyx. Three types of granules were observed in the isthmus non-ciliated cells. The regional differences observed along the epithelium lining the mare oviduct suggest that the epithelium of the each segment is involved in the production of a distinctive microenvironment with a unique biochemical milieu related to its functional role.

Localization of thyrotropin receptor and thyroglobulin in the bovine corpus luteum

The receptor of the Thyroid Stimulating Hormone (TSHr) and thyroglobin (TGB), are two proteic factors necessary for the synthesis of hormones, in the thyrocite. In mammals, many immuno-histochemical reports indicate the presence of the TSHr in extra-thyroidal tissues, but not in the ovary. Triiodothyronine (T(3)) and thyroxine (T(4)) have been widely shown to affect ovarian functions and the synthesis of progesterone (P(4)). The aim of this study was to determine if by immunohistochemistry techniques TSHr and TGB could be found in the bovine corpora haemorragica, lutea and albicantia. A primary rabbit polyclonal antibody against human TSHr and a primary rabbit polyclonal antibody against human TGB were employed. Furthermore, the accuracy of bovine thyroid to the antibodies used in this study was tested. A positivity reaction for the anti-TSHr serum in the large luteal cells and immunostaining of both small and large luteal cells with the anti-TGB serum occurred only in mature corpora lutea. No immunostaining was detected in stromal cells, blood and lymphatic vessels and in corpora haemorragica and albicantia. Bovine thyroid tissue showed immunostaining to both the antibodies employed. These data suggest that the luteal cells of mature corpora lutea may be involved in the synthesis of thyroid hormones, which may modulate P(4) synthesis, acting in an autocrine and paracrine way.

Lectin-binding sites on ejaculated stallion sperm during breeding and non-breeding periods.

Stallion sperm from semen collected in Southern Italy during the breeding (June-July) and non-breeding (December-January) periods were analyzed by means of twelve lectins to evaluate the glycoconjugate pattern and to verify whether there are any seasonal differences in the glycosylation pattern of the sperm glycocalyx. The acrosomal cap showed reactivity for Maackia amurensis (MAL II), Sambucus nigra (SNA), Arachis hypogaea (PNA), Glycine max (SBA), Helix pomatia (HPA), Canavalia ensiformis (Con A) Triticum vulgaris (WGA), and Griffonia simplicifolia isolectin II (GSA II) in breeding and non-breeding ejaculated sperm, suggesting the presence of oligosaccharides terminating with Neu5Ac alpha 2,3Gal beta 1,4GlcNAc, Neu5Ac alpha 2,6Gal/GalNAc, with Gal beta 1,3GalNAc, alpha/beta GalNAc and glycans with terminal/internal alpha Man and GlcNAc. During the non-breeding period, the acrosomal cap expressed oligosaccharides terminating with Gal beta 1,4GlcNAc (Ricinus communis(120) affinity) (RCA(120)) and L-Fuc alpha 1,2Gal beta 1,4GlcNAc beta (Ulex europaeus affinity) (UEA I). The equatorial segment placed between the acrosomal cap and post-acrosomal region did not display glycans terminating with GalNAc, GlcNAc, and alpha L-Fuc. The post-acrosomal region of sperm collected in the breeding and non-breeding periods bound Con A, MAL II, SNA, and SBA, thus showing the presence of N-linked oligosaccharides from high-Man content, terminating with Neu5Ac alpha 2,3Gal beta 1,4GlcNAc, Neu5Ac alpha 2,6Gal/GalNAc and GalNAc. In winter, the post-acrosomal region also expressed oligosaccharides terminating with alpha GalNAc, GlcNAc, and L-Fuc alpha 1,2Gal beta 1,4GlcNAc beta (HPA, GSA II, and UEA I staining). The tail of sperm from semen collected during the breeding and non-breeding periods showed a lectin binding pattern similar to the post-acrosomal region, except for the absence of HPA staining in sperm collected during the winter season. These results indicate that the surface of stallion sperm contains different glycocalyx domains and that the glycosylation pattern undergoes changes during the breeding and non-breeding periods.

Performance, gut morphology and carcass characteristics of fattening rabbits as affected by particle size of pelleted diets.

A review of past literature revealed inconsistencies in recommended feed particle size for optimal growth and productive performance of rabbits. Changing diet formulation and subsequent processing conditions may improve pellet texture and potentially affect rabbit performance. In the current study, two isoenergetic and isonitrogenous pelleted diets were formulated, which varied in the particle size of the concentrates (2 and 8 mm, respectively). The objective was to evaluate the effect of different particle sizes of compound diets on performance, nutrient utilisation, gut morphology, and carcass characteristics of fattening Italian White breed rabbits. The finely ground diet led to a significant improvement in feed efficiency and apparent digestibility of crude protein, ether extract, crude fibre and NDF, without any negative effect on gut morphology. Furthermore, a smaller particle size of concentrates in pelleted diets improved carcass traits. Meat colour parameters showed significant differences in longissimus lumborum and biceps femoris due to dietary treatments, but in both muscles pH values 1 h and 24 h after slaughter remained unchanged. It is concluded that a finely ground pelleted diet can be used to improve growth performance of rabbits without affecting carcass parameters.

Changes in the expression of the μ-opioid receptor in the mare oviduct during oestrus and anoestrus

The presence of the mu-opioid receptor (MOR) was investigated in the mare oviduct during oestrus and anoestrus, by means of immunoblotting and immunohistochemistry. Immunoblotting analysis showed that the MOR protein is expressed as 65, 50 and 30 kDa forms in the infundibulum and ampulla both in oestrus and anoestrus, while the 30 kDa form is absent in the isthmus. Moreover, different levels of expression were observed along the ampulla in the two periods examined. Immunohistochemistry revealed MOR in the mucosal epithelium, stromal cells, myocytes and blood vessels. Ciliated cells expressed MOR in the apical cytoplasm and, except for the isthmus of oestrous mares, also in the nucleus. Non-ciliated cells showed MOR only in the isthmus segment during oestrus. Stromal cells showed different immunoreactivity along the oviduct segments and during the oestrous and anoestrous phases. The myosalpinx displayed immunostained myocytes in the intrinsic musculature of the ampulla and in the extrinsic and intrinsic musculature of the isthmus without significant differences between anoestrus and oestrus. Blood vessels expressed MOR in endothelial cells and smooth muscle cells in the isthmus myosalpinx of oestrous mares only. In conclusion, these findings show diverse MOR expression in the three segments constituting the oviduct, as well as changes in MOR expression linked to the mares physiological condition.

Histochemical and immunohistochemical evidence for a gradient in gastric juice production in the greater horseshoe bat, Rhinolophus ferrumequinum (Schreber, 1774)

ABSTRACT Histochemical and immunohistochemical investigations were performed on the gastric mucosa of the greater horseshoe bat, Rhinolophus ferrumequinum (Schreber, 1774) to estimate the presence of a gradient of pepsinogen and hydrochloric acid along an oro-aboral axis of the stomach, similar to that found in some non-mammals. Paraffin sections were stained with DBA-lectin binding, Bowie and fluorescent anti-H+/K+-ATPase &agr;-subunit immunostaining to detect the chief and parietal cells in the gastric mucosa. The stomach of the bat presents a short cardias, a wide fundus and a small pylorus. Chief and parietal cells were found in the fundic glands and their number varied from the oral to the aboral region of fundus. In the oral region several chief cells with Bowie-positive pepsinogen granules were observed in the basal part of glands, whereas parietal cells positive to DBA-lectin binding and immuroreactive with anti-H+/K+-ATPase &agr;-subunit were concentrated in the the upper part of the glands. In the aboral fundus chief cells were lacking, whereas the number of parietal cells increased and they were distributed along the glands. A gradient of pepsinogen and hydrochloric acid secretion similar to that found in some non-mammals can be hypothesised. The possibility that this gradient is the ancestral condition in Chiroptera and Eutheria and its functional meaning are discussed.

Lectin-binding pattern of the senegalese sole Solea senegalensis oogenesis

The glycoconjugate pattern of developing ovarian follicles in wild and cultured Senegalese sole Solea senegalensis was investigated by means of lectin histochemistry. Ovaries from cultured fish contained oocytes up to the late vitellogenic stage, whereas they reached the hydration stage in wild specimens. The follicular cells bound MAL II, SBA, HPA, DBA, Con A, KOH‐sialidase (K‐s)‐WGA, GSA I‐B4 in the late vitellogenic stage, and in wild fish also SNA and K‐s‐PNA, whereas in the hydration stage SBA, HPA, DBA, and GSA I‐B4 only. The zona radiata reacted with SBA, HPA, DBA, Con A, and GSA I B4 in the late vitellogenic stage and in cultured fish also with UEA I, whereas in the hydration stage it stained with SBA only. The cortical alveoli bound SBA, HPA, RCA120 during the late vitellogenic stage, also SNA, PNA, K‐s‐PNA, GSA I‐B4 in cultured fish, DBA, and K‐s‐WGA in wild ones which stained with SBA, HPA, and GSA I‐B4 in the hydrated stage. The yolk reacted with Con A in the late vitellogenic oocytes, and also with MAL II, SNA, K‐s‐PNA, SBA, HPA, K‐s‐WGA, GSA I‐B4, UEA I in the hydrated ones. From perinucleolus to late vitellogenic stages, the oocyte nucleoplasm bound Con A, GSA I‐B4, GSA II, UEA I, and in wild fish also MAL II, SNA, LTA but only GSA I‐B4 reactivity in the early maturation stage. These findings demonstrate that the glycan pattern of fish ovarian follicles changes during the maturative stages and that it is affected by culture‐rearing conditions. Microsc. Res. Tech. 75:1124–1135, 2012.

Morphometric features and glycoconjugate pattern of rabbit intestine are affected by particle size of pelleted diets.

Feed particle size effects on morphology and glycoconjugate pattern was investigated in the rabbit intestine. Rabbits fed with fine particles (2 mm) displayed more irregularly shaped, higher duodenal villi and deeper crypts in distal colon as well as higher number of goblet cells than coarse (8 mm) fed ones. Brush border expressed: (i) in duodenum, neutral/sulfated glycoconjugates and glycans binding MAL II, SNA, Con A than KOH‐sialidase‐PNA and DBA reactivity in fine and coarse fed rabbits, respectively, (ii) in cecum, mainly sulfoglycans in coarse fed rabbits, MAL II and PNA staining in all samples, and (iii) in distal colon few sulfoglycans and MAL II reactivity. Enterocytes bound MAL II in duodenum, Con A in cecum, DBA, and Con A in distal colon of all rabbits, SNA in distal colon of coarse fed ones. Brunners glands displayed high presence of acidic/sulfated mucins in fine fed rabbits, neutral glycoconjugates and reactivity with MAL II, SNA, PNA, KOH‐sialidase‐PNA, and Con A in all rabbits. Goblet cells exhibited: (i) in duodenum neutral and sulfomucins as well as MAL II and KOH‐sialidase‐PNA staining, than SNA and DBA in fine and coarse fed rabbits, respectively, (ii) in cecum sulfated glycans, MAL II, SNA, KOH‐sialidase‐PNA, DBA reactivity, and (iii) in distal colon acidic/sulfomucins, MAL II and SNA staining, and DBA reactivity in fine fed specimens. Crypt cells exhibited neutral and PNA reactive glycoconjugates in the cecum. In the distal colon also acidic/sulfated glycans, and MAL II, KOH‐sialidase‐PNA, DBA; SNA staining showed weaker reactivity in fine fed rabbits, which bound Con A. Anat Rec, 2011.

In situ characterization of glycans in the urothelium of donkey bladder: Evidence of secretion of sialomucins

The glycoprotein pattern was investigated by lectin histochemistry in the urothelium lining the urinary bladder of the donkey Equus asinus. Tissue sections were stained with a panel of twelve lectins, in combination with saponification and sialidase digestion (K-s). The urinary bladder urothelium has three distinct layers from the basal zone to the lumen consisting of basal, intermediate and superficial cells (umbrella cells). Cytoplasm of basal cells reacted with SNA, PNA, K-s-PNA, GSA I-B4 and Con A showing glycans ending with Neu5Acα2,6Gal/GalNAc, Neu5AcGalβ1,3GalNAc, αGal and with terminal/internal αMan. The cytoplasm of umbrella cells displayed an increase of Neu5AcGalβ1,3GalNAc and the appearance of Neu5AcGalβ1,3GalNAc, Neu5acα2,3Galβ1,4GlcNAc and Neu5AcGalNAc residues (MAL II, K-s-SBA and K-s-HPA staining). Scattered umbrella cells were characterized by glycans terminating with GalNAc binding DBA, SBA and HPA. The mucosa forms folds with a crypt-like appearance where the urothelium shows a different pattern of glycans. The bladder luminal surface stained with K-s-PNA, K-s-DBA, KOH-s-SBA, and K-s-HPA displaying a coating of sialoglycoproteins belonging to O-linked glycans (typical secretory moieties). These findings show that different glycosylation patterns exist along the donkey bladder urothelium, and different sub-populations of umbrella cells are present secreting the sialoglycans which constitute the protective gel layer lining the bladder.