Introduction: Intercellular communication governs the exchange of signalling molecules between cells. The different mechanisms employed in cell-to-cell communication include the secretion and receptor-mediated binding of diffusible messengers such as hormones and growth factors, the transport of small molecules through gap junctions, exosomes as well as tunnelling nanotubes. In this study, we report a new feature of intercellular transfer in cultured sheep bone marrow mesenchymal stem cells (BM-sMSCs). Methods: Bone marrow was harvested from iliac crest of healthy sheep. Mononuclear cells were isolated by gradient centrifugation, plated in culture flasks and incubated in the presence of Coon’s medium in a humidified atmosphere at 37 °C with 5% CO2. Non-adherent cells were discarded after 3 days and adherent cells were cultured until they reached near-confluence (10 days). The cells were trypsinized and the pellets were fixed with 3% glutaraldehyde and post-fixed in OsO4 1%. After dehydration and embedding in Epon 812, ultrathin sections were counterstained with lead citrate and uranyl acetate and observed at a transmission electron microscope. Results: The cell populations consisted of two undifferentiated cell types: electron-lucent and electron-dense cells. Both showed prominent RER, glycogen aggregates and filopodia. The electron-lucent cells were more numerous. The nucleus in electron-lucent cells was euchromatic and contained a prominent nucleolus, whereas in electron-dense cells it was irregular in shape and full of heterochromatin. No intercellular junctions were observed between either electron-lucent cells or electron-dense cells, whereas spot-like fusions of the plasma membrane occur between the electron-lucent cell surface and electron-dense cells. Across this distinct membranous connection, a flow of cytoplasm - but not of organules – occurs from electron-dense to electron-lucent cells. Conclusions: This finding shows a new cell-to-cell connection implicated in intercellular transfer and stimulates further studies to identify the cell types involved and the nature of the substances transferred.

Ultrastructural and biochemical features of efferent ducts (EDs) are indicative of an intense absorptive activity towards the luminal fluid. This function was investigated by 1) the immunohistochemical localization of different aquaporins, integral membrane water channels that facilitate rapid passive movement of water, and 2) the histochemical localization of lectins, known to have specific affinity for glycoconjugate residues. AQP1 was mostly revealed at the apical surface and adluminal cytoplasm of nonciliated cells and to a minor extent in their lateral plasma membrane, whereas it was absent in ciliated cells. Blood vessels showed AQP1-immunoreactivity, which was present in endothelial cells of venous vessels and capillaries and around the muscular sheath of arteries. AQP9 was expressed in the apical zone of ciliated and non-ciliated cells and in the lateral cell membrane. AQP2 and AQP5 were undetectable. Lectin histochemistry showed that non-ciliated cells contain glycans with terminal Neu5Aca2,3Galß1,3GalNAc, Neu5Aca2,3Galß1,4GlcNAc, Galß1,4GlcNAc, GalNAc (s-PNA, MAL II, RCA120, SBA reactivity) and with internal/terminal aMan (Con A affinity) at the luminal surface and the apical region. In addition, non-ciliated cells expressed oligosaccharides terminating with GalNAc and Neu5Aca2,6Gal/GalNAc (SNA reactivity) in the luminal surface and the apical zone, respectively. Ciliated cells revealed glycoconjugates only on cilia, which showed terminal Neu5Aca2,3Galß1,4GlcNAc (s- RCA120 staining) and GalNAc, as well as internal/ terminal aMan and GlcNAc (s-WGA, GSA II staining). Data provide evidence for the involvement of different pathways in the bulk reabsorption of water and low molecular weight solutes by the non-ciliated cell of the cat EDs. AQP-mediated trans-cellular route can be hypothesized, together with fluid phase endocytosis mediated by the glycocalix and a well-developed endocytotic apparatus. Epithelial ciliated cells, whose main function is the movement of luminal content, might also participate in absorptive processes to a lesser extent.

Recent investigations1 indicate that cat efferent ducts (EDs) play a role in reabsorption of the fluid and proteins leaving the testes, dependent also on androgen2. The males with Klinefelter syndrome (XXY) show a variable degree of androgen deficit responsible for testicular disfunction3. Since we demonstrated synthesis and secretion of glycoconjugates in normal cat EDs1, here we investigated the glycoprotein pattern of the EDs from a tricolor cat with Klinefelter syndrome (39,XXY), by means of the lectin histochemistry, utilizing a panel of 12 lectins in association with sialidase (s) treatment. Cilia of ciliated cells reacted with HPA, SBA, Con A, KOH-s-WGA, GSA II in normal cats and with MAL II, SNA, Con A and KOH-s-WGA in XXY cat. The luminal surface of non-ciliated cells bound MAL II, KOH-s-PNA, Con A in all samples, RCA120 and HPA only in normal subjects and PNA in XXY cat. The supra-nuclear cytoplasm of non-ciliated cells expressed SNA and Con A affinity in XXY cat and also MAL II, KOH-s-PNA, RCA120, SBA in normal cats. These results indicate that negative charges are mainly expressed on the cilia of XXY cat EDs, whereas a more complex glycoconjugate pattern, probably related to an more effective endocytotic apparatus, is expressed in the supra-nuclear cytoplasm of non-ciliated cells from normal EDs. References 1. Arrighi S et al. Histol. Histopathol. 2001, 25:433-44. 2. Jones RC. The epididymis: From molecule to clinical practice. Robaire and Hinton eds, Kluwer Academic/Plenum Publ. 2002,11-33. 3. Wikström AM and Dunkel L. Horm. Res. 2008, 69:317-26.

Yeasts of the genus Malassezia are commensals of the normal skin microbial flora of humans and animals. These yeasts may become pathogenic under certain circumstances and their pathogenic role may be related to host immune system as well to yeast virulence factors (e.g., phospholipase production and biofilm formation). This study aims to evaluate the in vitro ability of M. pachydermatis strains to produce biofilm, and its relationship with phospholipase activity and the genetic make-up of isolates from lesioned (n=32) and healthy (n=30) dog skin. The production of biofilm was determined by crystal violet staining and scanning electronic microscopy (SEM). Biofilm was produced by almost all M. pachydermatis isolates (95.2%) from dogs with and without skin lesions at variable level and different structure. At the SEM, biofilm matrix presented adhering blastoconidia clustered in multi- or monolayer structures with variable quantity of extracellular production. Of the three genotypes detected, genotype B showed the lowest ability to produce biofilm. Of the 59 isolates producing biofilm, 33 (55.9%) produced phospholipase, with a higher biofilm formation (p<0.05) in strains collected from animals with skin lesions. It is here suggested that phospholipase production might act in synergism with the biofilm formation by inducing or exacerbating skin lesions in dogs. The results provide evidences for a better understanding of the interactions between yeasts and host immune system, toward revealing the pathogenicity of M. pachydermatis in animals.

The presence of the μ-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 mare's physiological condition.

The Octopus vulgaris farming is impaired by the high mortality of the paralarvae during the first month of life. Several factors have been investigated in this regard, but no data exist on the body surface mucus, which represents the interface with the outside environment. This study included morphometric analysis and glycoconjugates characterization of skin mucus in reared Octopus vulgaris paralarvae during the first month of life. Four types of mucous cells were distinguished: mucous 1 (m1) and mucous 2 (m2) cells were scattered in the mantle epidermis, mucous 3 (m3) and mucous 4 (m4) in the epithelium surrounding the sucker. Except for the presence of fucosylated and neutral glycoconjugates in all mucous cells, each cell type expressed a characteristic glycopattern. m2 and m4 contained also suphate and acid non-sulphate glycans, m3 lacked suphate glycoproteins. Lectin histochemistry showed that mantle mucous cells (m1,m2) expressed GlcNAc and lactosamine terminating glycans. m2 also contained GalNAc terminal or penultimate to sialic acid. m3 was distinguished by mannosylated glycans terminating with lactosamine and m4 by α2,6 sialoglycans. Glycoproteins terminating with lactosamine, Galβ1,3GalNAc, and α1,6-linked fucose were a common feature of paralarvae surface layer. Morphometry revealed a significant decrease of m1 and m2 abundance during the first month of life, afterwards the reared paralarvae died. Since the glycopattern did not change during the investigated period, the mantle mucous cells abundance could be related to the Octopus vulgaris paralarvae survival.

Oviductal environment affects preparation of gametes for fertilization, fertilization itself, and subsequent embryonic development. The aim of this study was to evaluate the effect of oviductal fluid and the possible involvement of deleted in malignant brain tumor 1 (DMBT1) on IVF in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase in the monospermic fertilization rate and that this effect is canceled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase in the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the estrus cycle was shown by western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus–oocyte complexes was shown by western blot analysis, and the localization of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatic and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization.

Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern differed between animals and COC source (IVM versus preovulatory). Among the 12 carbohydrate residues investigated, the IVM COCs from these two species shared: (a) sialo- and bN-acetylgalactosamine (GalNAc)-termi- nating glycans in the ZP; (b) sialylated and fucosylated glycans in cumulus cells; and (c) GalNAc and N-acetylglucosamine (GlcNAc) glycans in the ooplasm. Differences in the preovulatory COCs of the two species included: (a) sialoglycans and GlcNAc terminating glycans in the equine ZP versus terminal GalNAc and internal GlcNAc in the porcine ZP; (b) terminal galactosides in equine cumulus cells versus terminal GlcNAc and fucose in porcine cohorts; and (c) fucose in the mare ooplasm versus lactosamine and internal GlcNAc in porcine oocyte cytoplasm. Furthermore, equine and porcine cumulus cells and oocytes contributed differently to the synthesis of ZP glycoproteins. These results could be attributed to the different in vitro fertilization efficiencies between these two divergent, large-animal models.

The buffalo has a seasonal reproduction activity with mating and non-mating periods occurring from late autumn to winter and from late spring to beginning of autumn, respectively. Sperm glycocalyx plays an important role in reproduction as it is the first interface between sperm and environment. Semen quality is poorer during non-mating periods, so we aimed to evaluate if there were also seasonal differences in the surface glycosylation pattern of mating period spermatozoa (MPS) compared with non-mating period spermatozoa (NMPS). The complexity of carbohydrate structures makes their analysis challenging, and recently the high-throughput microarray approach is now providing a new tool into the evaluation of cell glycosylation status. We adopted a novel procedure in which spermatozoa was spotted on microarray slides, incubated with a panel of 12 biotinylated lectins and Cy3-conjugated streptavidin, and then signal intensity was detected using a microarray scanner. Both MPS and NMPS microarrays reacted with all the lectins and revealed that the expression of (i) O-glycans with NeuNAcα2-3Galβ1,3(±NeuNAcα2-6)GalNAc, Galβ1,3GalNAc and GalNAcα1,3(l-Fucα1,2)Galβ1,3/4GlcNAcβ1 was not season dependent; (ii) O-linked glycans terminating with GalNAc, asialo N-linked glycans terminating with Galβ1,4GlcNAc, GlcNAc, as well as α1,6 and α1,2-linked fucosylated oligosaccharides was predominant in MPS; (iii) high mannose- and biantennary complex types N-glycans terminating with α2,6 sialic acids and terminal galactose were lower in MPS. Overall, this innovative cell microarray method was able to identify specific glycosylation changes that occur on buffalo bull sperm surface during the mating and non-mating periods.

The glycoconjugates of the mucosal surface of urinary bladder act as a barrier against invasion by pathogenic microorganisms and injury from toxic substances. Furthermore, they serve as a source of soluble urinary glycoproteins, which actively modify the urine composition. Glycoconjugate pattern characterizes distinct cellular populations, cell differentiation and maturation, as well as cell morpho-functional changes. We investigated the glycan pattern in the bladder epithelium by lectin histochemistry, comparatively in the horse and donkey. Tissue fragments were fixed in 4% (w/v) neutral formalin and then embedded in paraffin wax. Sections were stained with a panel of twelve lectins, in combination with sialidase (s) digestion. The entire urothelium reacted with SNA, s-PNA, s-SBA, Con A, GSA II in horse bladder, and also with MAL II and GSA I-B4 in the donkey one. The urothelium luminal surface bound MAL II, DBA, LTA in the horse and DBA and LTA in the donkey. In addition, the horse bladder stained with PNA, SBA and GSA I-B4 in the basal and luminal regions and with UEA I in the adluminal zone. These results demonstrate remarkable inter-specific difference of the glycoprotein pattern in the bladder urothelium of two Equine species, despite their close taxonomic vicinity. It is noteworthy that glycans binding PNA and UEA I lack in the epithelium lining the donkey urinary bladder.

The oviduct plays an essential role in the mammalian reproduction and it undergoes significant endocrine-induced morphological, biochemical and physiological changes during the oestrous cycle. The functions of the oviduct epithelium are controlled by the ovarian steroids, oestrogen and progesterone1. In this study the glycoconjugate pattern of oviduct obtained from hormonally (FSH-P and eCG) superovulated ewes for oocytes recoverywas analyzed. Oviducts from treated and control sheep were collected by laparatomy, fixed in 4% (w/v) neutral formalin, embedded in paraffin wax and sections processed for lectin histochemistry. In the ampulla, the luminal surface of all specimens showed strong reactivity with MAL II,SNA, PNA after KOH-sialidase (s) treatment, RCA120, HPA, SBA, KOH-s-WGA, and GSA I-B4, whereas it stained strongly with GSA II, UEA I, and LTA in treated sheep which showed reactivity with KOH-s-PNA, SBA, GSA I-B4, GSA II also in the apical cytoplasm of non-ciliated cells. In the isthmus, the luminal surface showed same staining reactivity with RCA120, SBA, and GSA I-B4 in all specimens, and a stronger affinity for MAL II, UEA I and LTA in treated ones. A distinctive feature of hormonized isthmus was the binding of the entire cytoplasm of ciliated cells and non-ciliated cells with MAL II, SNA, RCA120, SBA, GSA II, UEA I, and LTA. These results indicate that ampulla and isthmus of ovine oviduct express a different glycoconjugate pattern and that the hormone administration for superovulation produces different effect along the oviduct. These differences could be related to the different functions of each segment that constitutes the ovine oviduct. 1. Buhi WC Reproduction 2002, 123:355-62.

Introduction: The umbilical cord is the lifeline between the fetus and placenta. The glycoproteins are involved in many biological activities including cell proliferation and cell differentiation. Because of their negative charge and terminal location, sialic acids present a high potential in modulating epithelial permeability barrier and cytoprotective function in the urinary system. The aim of the present study was to examine and characterize sialoglycoproteins in the epithelium lining the allantoic duct by means of the lectin histochemistry. Methods: Fragments of the umbilical cord from fetuses were fixed in 4% (w/v) neutral formalin and embedded in paraffin wax. Sections (5 µm thick) were stained with SNA, MAL II, PNA, DBA, Con A, WGA before or after KOH-sialidase (Ks) treatment. Results: The luminal surface bound MAL II, SNA, Ks-PNA, SBA and Ks-WGA; MAL II did not reacted with the luminal surface of the proximal tract. The urothelium apical region bound MAL II, SBA,SNA, PNA and Ks-WGA in the proximal tract, whereas in the other tracts did not react with MAL II and SBA. Except for vacuolized cells, the entire cytoplasm of the urothelium throughout the ductus bound Con A, whereas some elongated cells reacted with Ks-PNA; the elongated cells of the distal region bound also SBA. Basal region of urothelium linked Ks-WGA. Conclusions: These results demonstrated the presence of sialoglycoconjugates mostly in the lumnal surface of the epithelium lining the horse allantoic duct as well as their regional difference. These glycoconjugates belong to N-linked type (Con A and Ks-WGA reactivity). Althought similar investigations on the allantoic duct lack, our findings agree with studies on the implication of N-linked sialoglycans in the protection and activity of urothelium. It is noteworthy that asialo- or sialylGalNAcα1,3(LFucα1,2)Galβ1,3/4GlcNAcβ1 terminating glycans (DBA and Ks-DBA reactivity) lack in the horse umbilical cord.

The oviduct isthmus is considered to be a sperm reservoir prior to ovulation in the reproductive tract of mammals. Ovarian steroids regulate the synthesis and secretion of specific molecules such as glycoproteins that are involved in the interactions between germ cells or embryos and oviductal epithelial cells. The objective of the present study was to investigate the effects on histmus glycoprotein pattern from hormonally stimulated ewes by means of lectin histochemistry. Isthmus fragments were separated from oviducts immediately after laparatomy, and, after fixed in 4% (w/v) neutral formalin, they were embedded in paraffin wax. Then, the sections were stained with a panel of lectins which revealed: 1) an increase of reactivity with MAL II, SNA, RCA120, SBA, GSA I-B4, GSA II, UEA I and LTA in the whole cytoplasm of ciliated and non-ciliated cells of hormonally treated females, 2) a reduction of DBA affinity in the luminal surface, 3) no staining pattern modification with PNA, KOH-sialidase (s)- PNA, HPA, Con A and KOH-s-WGA. These results indicate that exogenous gonadotropin administration for superovulation may alter the oligosaccharidic composition of glycoproteins produced in the ovine isthmus.

The characterization of mucus O-linked glycans in the proximal and distal mouse colon was performed by conventional histochemical methods and by lectin histochemistry in combination with enzymatic treatment (PNGase, α1,2 fucosidase, sialidase digestion), with and without prior desulfation. We demonstrated the presence of sialo- and sulfomucins in both the proximal and distal colon of the mouse. In the distal colon the sulfomucins were clearly prevalent, although there were always sialomucins with sialyl residues linked α2,6 to the subterminal galactose. Sialic acid was poorly O-acetylated, especially in the distal colon. The lectin binding pattern indicates a massive presence of fucose α1,2 linked to galactose in O-glycans and smaller quantities of fucose linked α1,6 to N-acetylglucosamine in the core of N-linked glycans. Lectin histochemistry also demonstrated the presence of glycosidic residues of N-acetylglucosamine, N-acetylgalactosamine, and galactose in oligosaccharide chains of highly sulfated mucins.

In vitro matured oocytes (IVM) suffer some inadequacies when compared with in vivo matured ones1. Inadequate IVM can yield under- or overmature oocytes, which will not undergo normal fertilization and embryo development2. Glycoconjugates play a key role in oocyte maturation, and in oocyte-sperm interactions leading to fertilization 3,4, thus the knowledge of oligosaccharide pattern of equine COCs could provide useful information about the comparison between immature and matured COCs. Cumulus enclosed oocytes from abbattoir ovaries were fixed in Bouin’s solution and embedded in paraffin wax either before or after IVM. Sections were stained with 13 lectin (MAL II, SNA, PNA, DBA, RCA120, SBA, HPA, Con A, WGA, GSA I-B4, GSA II, UEA I, LTA). The radiata zone of immature COCs reacted with all used lectins, whereas matured COCs stained with MAL II, SNA, HPA, SBA, and Con A. The zona pellucida of both COCs types bound MAL II, SNA, SBA, and Con A, whereas immature COCs reacted also with RCA120, WGA, and matured ones stained with UEA I. The ooplasm of both types of COCs reacted with HPA, Con A, GSA II, UEA I and LTA, whereas immature oocytes bound also SNA, SBA, WGA, GSA I-B4. These results indicate that IVM modifies glycoprotein pattern of equine COCs and prompted us to undergo further studies to investigate the role of the modified oligosaccharides in oocyte viability, capacity to undergo fertilization and normal embryonic development. References 1. Deleuze S et al Theriogenology. 2009, 72:203-9. 2. Hinrichs K & Di Giorgio LM J Reprod Fertil Suppl 1991, 44:369–74. 3. Dell A et al Biochim Biophys Acta 1999, 1473:196-205. 4. Clark GF & Dell A. J Biol Chem 2006, 281:13853-6.

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.

The localization and characterization of oligosaccharide sequences in the testis of Senegalese sole Solea senegalensis was investigated using 12 lectins in combination with KOH saponification and sialidase digestion (K-s). The interstitial compartment contained all the sugar residues investigated, those bearing oligosaccharides terminating with sialic acid (Neu5Ac) alpha 2,3Gal beta 1,4GlcNAc, Neu5AcGalNAc alpha 1,3(LFuc beta 1,2) Gal beta 1,3/4GlcNAc beta 1 and GalNAc alpha 1,3(LFuc1,2) Gal beta 1,3/4GlcNAc beta 1 being more abundant in the medullar region than in the cortex. The melanomacrophage centres found in the interstitial compartment displayed glycans terminating with Gal beta 1,3GalNAc. The basal lamina separating the germinal and interstitial compartments exhibited glycans with terminal/ internal mannose, internal beta GlcNAc, and terminal Neu5Ac alpha 2,6Gal/GalNAc, and Neu5AcGal beta 1,3GalNAc, Gal beta 1,3GalNAc (PNA), Gal beta 1,4GlcNAc, GalNAc, alpha Gal, and alpha L-Fuc. In the germinal compartment, the Sertoli cells expressed only glycans terminating with Neu5Ac alpha 2,3Gal beta 1,4GlcNAc in the apical and supranuclear lateral surface of the spermatonial cysts located in the distal part of the seminiferous lobules. Primary spermatocytes exhibited oligosaccharides terminating with Gal beta 1,3GalNAc and alpha GalNAc in the cytoplasm and nucleus, respectively. The spermatids contained highly mannosylated glycans terminating with GalNac, alpha Gal, and alpha L-Fuc. The head of spermatozoa expressed a more complex glycosylation pattern characterized by the additional presence of oligosaccharides terminating with Neu5Ac alpha 2,3Gal beta 1,4GlcNAc, Neu5AcGal beta 1,3GalNAc, Neu5AcGalNAc alpha 1,3(LFuc alpha 1,2)Gal beta 1,3/4GlcNAc beta 1, GalNAc alpha 1,3(LFuc alpha 1,2)Gal beta 1,3/4GlcNAc beta 1. The comparison with previous lectin histochemical studies carried out in other fish species reveals a specific glycosylation pattern of Senegalese sole testicular structures and spermatozoa head.

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 Neu5Aca2,3Galb1,4GlcNAc, Neu5Aca2,6Gal/GalNAc, with Galb1,3GalNAc, a/bGalNAc and glycans with terminal/internal aMan and GlcNAc. During the non-breeding period, the acrosomal cap expressed oligosaccharides terminating with Galb1,4GlcNAc (Ricinus communis120 affinity) (RCA120) and LFuca1,2Galb1,4GlcNAcb (Ulex europaeus affinity) (UEA I). The equatorial segment placed between the acrosomal cap and postacrosomal region did not display glycans terminating with GalNAc, GlcNAc, and aL-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 Neu5Aca2,3Galb1,4GlcNAc, Neu5Aca2,6Gal/GalNAc and GalNAc. In winter, the post-acrosomal region also expressed oligosaccharides terminating with aGalNAc, GlcNAc, and L-Fuca1,2- Galb1,4GlcNAcb (HPA, GSA II, and UEA I staining). The tail of sperm from semen collected during the breeding and nonbreeding 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.

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.

The effect of culture-rearing conditions on glycoprotein pattern of Senegalese sole Solea senegalensis developing ovarian follicles was investigated by means of the lectin histochemistry. Ovaries from cultured specimens stopped the oocyte development in the late vitellogenic stage, whereas they reached the hydration stage in wild specimens. Compared to ovaries from wild specimens, in cultured S. senegalensis1 the follicular layer showed the appearance of sialic acid (Neu5Ac) linked to Galβ1,3GalNAc in perinucleolus stage and the lack of Neu5Acα2,6Gal/GalNAc residues2, the zona radiata expressed mannosylated glycans in cortical alveoli stage3, the cortical alveoli, during the late vitellogenic phase, revealed the presence of Neu5AcGalβ1,3GalNAc, Neu5Acα2,6Gal/GalNAc as well as asialogalactosides4, yolkglobules expressed mannosilated oligosaccharides only in the late vitelogenic phase5, the oocyte nucleoplasm did not show sialilated and fucosilated glycans. These findings demonstrate that the glycan pattern of fish ovarian follicles is affected by culture-rearing conditions.

This study describes regional differences in the oviduct of the one-humped camel (Camelus dromedarius) during the growth phase (GP) and the mature phase (MP) of the follicular wave by means of morphometry, scanning electron microscopy (SEM) and glycohistochemistry investigations. Epithelium height significantly increased in the ampulla and decreased in the isthmus passing from the GP to the MP. Under SEM, non-ciliated cells displayed apical blebs (secretory) or short microvilli. Cilia glycocalyx expressed glycans terminating with sialic acid linked 2,6 to Gal/GalNAc (SNA affinity) throughout the oviducts of GP and MP and sialic acid linked 2,3 to Gal1,3GalNAc (MAL II and KOH-sialidase (K-s)-PNA staining) throughout the MP oviducts. Non-ciliated cells displayed lectin-binding sites from the supra-nuclear cytoplasm to the luminal surface. Ampulla non-ciliated cells showed O-linked (mucin-type) sialoglycans (MAL II and K-s-PNA) during GP and MP and N-linked sialoglycans (SNA) during the MP. Isthmus non-ciliated cells expressed SNA reactivity in GP and MP, also K-s-PNA binders in MP, and MAL II and PNA affinity (Gal1,3GalNAc) during GP. Gal1,3GalNAc was sialilated in the non-ciliated cells of GP UTJ. Luminal surface lacked of Gal1,3GalNAc in GP and MP, whereas it expressed 2,6- and 2,3-linked sialic acids. In GP intraluminal substance reacted with SNA, MAL II, K-s-PNA in ampulla and only with MAL II in the isthmus and UTJ. These results demonstrate that the morphology and the glycan pattern of the camel oviductal epithelium vary during the follicular wave and that could relate to the region-specific functions.

ABSTRACT Mucosal epithelium of pyloric caeca was studied in normal and in GnRH-treated Atlantic bluefin tuna Thunnus thynnus L., using morphological analysis, conventional and lectin glycohistochemistry. The lining epithelium consisted of columnar (absorptive) cells, goblet cells and intraepithelial leucocytes. The epithelium from normal animals was significantly taller than GnRH-treated samples. Conventional histochemistry displayed the same staining pattern in normal and hormone-treated specimens which showed a mixture of neutral and sulphated acidic glycoconjugates in the luminal surface and goblet cells, and neutral glycans in apical granules of enterocytes. Lectin histochemistry revealed a different glycoconjugate pattern in normal and GnRH-treated tunas. In normal specimens the luminal surface expressed sialoglycoconjugates which bound MAL II, SNA, KOH-sialidase-PNA, KOH-sialidase-SBA as well as asialoglycans stained with HPA, SBA, GSA I-B4, LTA. N-linked glycans were highlighted by Con A and KOH-sialidase- WGA. In GnRH-treated tunas the luminal surface did not react with SNA, SBA and LTA. The columnar cells of normal tunas bound KOH-sialisase-PNA in the apical region, KOH-sialidase- PNA, KOH-sialidase-DBA, HPA, SBA, KOH-sialidase-SBA and KOH-sialidase-WGA in apical granules, GSA I-B4 and LTA in the supranuclear region. GnRH-treated specimens showed some columnar cells that stained with KOH-sialidase-WGA in the apical granules and with GSA I-B4 in the supranuclear region. The goblet cells of normal animals produced mucins positive to PNA, HPA, KOH-sialidase-DBA, SBA, GSA II. The latter three binding sites lacked in GnRH-treated tunas. The results suggest that the mucosal epithelium of Thunnus thynnus L. pyloric caeca expresses a complex glycan pattern that is affected by GnRH-treatment.

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

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. Brunner’s 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.

Fusarium spp. are fungi worldwide distributed in soil, plants, water and other organic substrates which sometimes act as pathogens causing superficial and or disseminated infections in humans and animals. Several skin and shell diseases have been described to occur in sea turtles but the role of Fusarium spp. as a primary cause of infections remains anecdotic. The present study was designed to investigate the occurrence of Fusarium spp. on sea turtles with or without shell and skin lesions. A total of 90 animals (i.e., 38 without and 52 with lesions) were enrolled in the study. Shell and skin scraps were collected from each turtle on three anatomical sites (i.e., carapace, flipper and plastron) examined microscopically (i.e., cytological examination with Calcofluor and scanning electron microscope -SEM analysis) and cultured. Seven (7.8%) out of 90 samples gave positive results at the microscopic examination with calcoflour (i.e., 3 animals without lesions and 4 animals with lesions). SEM analysis revealed the presence of fungal hyphae on and in the internal structures of the carapace in all samples collected from animals with lesions whereas no fungal structures were observed in animals without lesions. Fusarium spp. was isolated from 23 (44.2%) isolates collected from animals with shell and skin lesions and from 3 (7.9%) without lesions. Fusarium spp. were also isolated and identified from the sand of the filter of the rescue centres. Occurrence of lesions and recovering time spent in the rescue centre were identified as the most significant risk factors (p<0.05). Fusarium solani (7.8%), Fusarium oxysporum (5.6%), Fusarium acuminatum (0.74%) and Fusarium brachygibbosum (0.37%) were morphologically and molecularly identified. The results of this study suggest that fusariosis should be included in the differential diagnosis of shell and skin infections in sea turtles and that the diagnosis of infections should be performed using SEM analysis associated with fungal culture. Finally, in the rescue centre, management procedures (e.g., protocols of disinfection) are suggested in order to control the infections.

OBJECTIVE: To analyze within-/between-subject, in vivo versus in vitro maturation (IVM), and age-related variations of mitochondrial (mt) bioenergy potential and oxidative status of metaphase II (MII) oocytes recovered from hormonally stimulated sheep. DESIGN: Prospective study. SETTING: Academic basic research laboratory. SUBJECT(S): Ten adult ewes. INTERVENTION(S):Estrus synchronization, controlled ovarian hyperstimulation (COH), ovariohysterectomy; follicular and oviductal oocyte retrieval; IVM of follicular oocytes. MAIN OUTCOME MEASURE(S): Mean ± SD, within-subject (CV(w)) and between-subject (CV(b)) variation coefficients of mt activity, intracellular reactive oxygen species (ROS) levels, and mt/ROS colocalization in sheep oocytes from young and aged donors and matured in vivo (in vivo MIIs) or in vitro (IVM MIIs). RESULT(S): Within- and between-subject, in vivo versus IVM, and age-related variations of mt activity were observed in MII oocytes from hormonally stimulated donor sheep. ROS levels increased significantly in oocytes from aged donors. Mt-ROS colocalization was consistently higher in in vivo MIIs compared with IVM MIIs. Oviductal energy/antioxidant ability is influenced by COH. CONCLUSION(S): Oocyte energy/oxidative status is affected by within-/between-subject, in vivo versus IVM, and age-related variations. Mt/ROS colocalization is a reliable marker of in vivo MII oocytes.

The effect of a dietary probiotic blend on the carbohydrate composition of mucins secreted by the Brunner's glands in the duodenum of growing-finishing pigs was investigated by means of conventional (periodic acid-Schiff, Alcian Blue pH 2.5, high iron diamine staining) and lectin (15 lectins) histochemistry. Pigs were assigned to two dietary treatments: a control basal diet without the probiotic blend (No-Pro) and a test diet that included the probiotic blend (Pro). Duodenal tissue fragments were fixed in 4% phosphate-buffered-saline-buffered paraformaldehyde, dehydrated through a graded alcohol series, and embedded in paraffin wax. The secretory cells of the Brunner's glands from No-Pro pigs primarily produced neutral glycoproteins and a small amount of acidic non-sulphated mucins. This glycan pattern was opposite that of the Brunner's glands from Pro animals. A comparison of lectin-binding profiles of the secretory cells of Brunner's glands in these two groups showed that in Pro pigs, there was (i) a decrease in N-linked glycans containing α1,2-linked fucose (Con A, UEA I); (ii) a loss of complex types of N-glycans (PHA-L, PHA-E) terminating with lactosamine (RCA120), α1,6- and α1,3-linked fucose (LTA), and α-galactose (GSA I-B4), as well as of O-glycans with terminal Galβ1,3GalNAc (PNA); and (iii) an increase in O-glycans containing GalNAc HPA. No-Pro and Pro samples showed no change in the expression of α2,6 sialoglycans and terminal GlcNAc residues and no affinity for MAL II, DBA, and SBA. These results indicate that probiotic supplementation affects the glycan composition of mucins produced in the Brunner's glands of growing-finishing pigs. These changes could effectively act on the gastrointestinal function and health status of these animals because the probiotic blend induced higher growth performance and meat quality in the test probiotic group than it did in the control basal diet group (Tufarelli et al., 2017).

Blackbelly rosefish Helicolenus dactylopterus is a zygoparous fish whose males are equipped with the copulating organ named urogenital papilla (UP). This study deals with the morphology and the glycoconjugate pattern of the UP epidermis, which is the male tissue interacting with the female internal body during copulation. The carbohydrate content was studied by means of conventional and lectin histochemistry. The epidermis was shown to be a stratified cuboidal epithelium and to exhibit characteristic intraepithelial pits in the apical zone. The mucous cells are scattered in the epidermis. The epidermal cell layers and their thickness as well as the size of mucous cells varied along the UP. Conventional histochemistry showed that the mucous cells contained i) only neutral glycoproteins in the basal zone; ii) both neutral and acidic nonsulphated glycans as well as only acidic non-sulphated or sulphated glycoconjugates in the intermediate zone; iii) neutral and sulphated glycoconjugates in the apical zone. The mucous cells in the basal region expressed O-linked (mucin type) glycans terminating with αGalNAc, Galβ1,3GalNAc which could be α2,3-linked to sialic acid, and high mannose type N-linked glycans terminating with fucose, lactosamine, and sialic α2,6-linked to galactose/N-acetylgalactosamine; terminal Gal and terminal/internal GlcNAc were also found. The mucous cells in the intermediate zone lacked Galβ1,3GalNAc and showed less terminal α2,3-linked sialic acid, lactosamine, fucose, galactose, and internal N-acetylglucosamine residues. In the apical region, mucous cells only exhibited O-glycans terminating with GalNAc and N-acetylglucosamine. The demonstrated region-specific differences in the UP skin provide new insights into the reproductive biology of fishes with internal fertilization.

The presence of Aquaporins 1 (AQP1) and 9 (AQP9), integral membrane water channels that facilitate rapid passive movement of water and solutes, was immunohistochemically detected in the excurrent ducts collected from sexually mature buffalo bulls of proven fertility during the mating (late autumn-winter) and non-mating (late spring to the beginning of autumn) seasons. Furthermore, the research was performed also on the epididymal cauda of a senile buffalo bull with inactive testis. Aquaporins 1 and 9 were immunolocalized at distinct levels. In the efferent ducts, AQP1 immunoreactivity was strongly evidenced at the apical surface of the non-ciliated cells and weakly along the basal membrane of the epithelial cells. The latter reactivity disappeared during the non-mating season. No AQP1 immunoreactivity was detected in the epithelium of epididymis and vas deferens, whereas AQP1 was expressed in the smooth muscle layer of the vas deferens. Aquaporin 1 was present in the blood vessels and in small nerve bundles all along the genital tract. The supranuclear zone of the epididymal principal cells was AQP9 immunoreactive, limited to the corpus and cauda regions, and vas deferens. The samples collected in the two reproductive seasons showed a weaker AQP9 immunoreactivity during the non-mating season. A typical AQP9 immunoreactivity was noticed in the old buffalo examined. The tested AQP molecules showed a different expression pattern in comparison with laboratory mammals, primates, equine, dog and cat. In addition, seasonal differences were noticed which are possibly useful in regard to the comprehension of the morphophysiology of reproduction in the bubaline species, which are still a matter of debate

The use of bone marrow-derived mesenchymal stem cells (MSCs) for clinical and experimental studies is increasing, but full characterization of MSCs in veterinary species is hindered by the variability in species-specific cell surface marker expression and antibody cross reactivity. Recent studies demonstrated that the glycans in the glycocalyx of MSCs are promising candidates as cell biomarkers. In the present study, we analysed the glycocalyx of canine MSCs (cMSCs), ovine MSCs (oMSCs), and equine MSCs (eMSCs) by using a cell microarray procedure in which MSCs were spotted on microarray slides and incubated with a panel of 14 biotinylated lectins and Cy3-conjugated streptavidin. The signal intensity was then detected by using a microarray scanner. The lectin-binding signals indicated that the MSC surface of the investigated species contained both N- and O-linked glycan types, with N-glycosylation predominating over O-glycosylation and fucosylation being more abundant than sialylation. Relative quantification revealed an interspecific difference between these glycans. In addition, cMSCs expressed more α2,3-linked sialic acid (MAL II), terminal lactosamine (RCA120), and α1,6 and α1,3 fucosylated oligosaccharides (PSA, LTA); oMSCs exhibited more T antigen (Jacalin), GalNAcα1,3(LFucα1,2)Galβ1,3/4GlcNAcβ1 (DBA), chitotriose (succinylated WGA), and α1,2-linked fucose (UEA I); and eMSCs showed a higher density of α2,6 sialic acids (SNA) and high mannose N-glycans (Con A). By using cell microarray methodology, we have for the first time demonstrated differences in the glycosylation profiles of cMSC, oMSC, and eMSC surfaces. These results could be valuable as resources and references for MSC differentiation and molecular remodeling in clinical cell-based therapy and tissue engineering studies.

Bone marrow mesenchymal stem cells (BM-MSCs) in combination with bioceramics have been used to repair bone lesions but the effects of these biomaterials on BM-MSCs are little known. This study reports the changes observed in the ultrastructure of sheep BM-MSCs incubated with silicon-stabilized tricalcium phosphate (TSP). MSCs were isolated from bone marrow of iliac crest and cultured according to Crovace et al. (VCOT, 21, 2008), TSP was added to some cultures and cells were incubated for 10 days. Control and TPS-incubated cells were fixed with GTA, post-fixed in OsO4 and embedded in Epon 812. Control and TSP cultures consisted of differently electron-dense polygonal cells containìng euchromatic nuclei with prominent nucleoli, In control coltures, the electron-lucent cells were characterized by dilated rER cisternae whereas the moderately electron-dense cells showed dark bodies as prominent features, Small blebs and filopodia were present on the surface of light and dark cells, respectively. In TSP cultures, thè light cells showed large surface blebs, peripheral cytoplasm poor in organelles which were packed with contractile filaments around the nucleus, The dark elongated cells displayed pseudopodia and filopodia, peripheral vacuoles, dense bodies, round or elongated mitochondria, These findings demonstrate that TSP modifies differently the ultrastructure of celi types constituting the BM-MSCs of sheep.

Bioceramics are being used in experimental bone engineering application in association with bone marrow derived mesenchymal stem cells (BM-MSCs) as a new therapeutic tool, but their effects on the ultrastructure of BM-MSCs are yet unknown. In this study we report the morphological features of ovine (o)BM-MSCs cultured with Skelite, a resorbable bioceramic based on silicon stabilized tricalcium phosphate (SiTCP), able to promote the repair of induced bone defect in sheep model. oBM-MSCs were isolated from the iliac crest, cultured until they reached near-confluence and incubated with SiTCP. After 48 hours the monolayers were highly damaged and only few cells adhered to the plastic. Thus, SiTCP was removed, and after washing the cells were cultured until they became confluent. Then, they were trypsinizated and processed for transmission electron microscopy (TEM) and RT-PCR analysis. RT-PCR displayed that oBM-MSCs express typical surface marker for mesenchymal stem cells. TEM revealed the presence of electron-lucent cells and electron-dense cells, both expressing the CD90 surface antigen. The prominent feature of electron-lucent cells was the concentration of cytoplasmic organelles around the nucleus as well as large surface blebs containing glycogen or profiles of endoplasmic reticulum. The dark cells had a multilocular appearance by the presence of peripheral vacuoles. Some dark cells contained endocytic vesicles, lysosomes, and glycogen aggregates. oBM-MSCs showed different types of specialized interconnections. The comparison with ultrastructural features of untreated oBM-MSCs suggests the light and dark cells are two distinct cell types which were differently affected by SiTCP bioceramic.

Ovine bone marrow-derived mesenchymal stromal cells (oBM-MSCs) represent a good animal model for cell-based therapy and tissue engineering. Despite their use as a new therapeutic tool for several clinical applications, the morphological features of oBM-MSCs are yet unknown. Therefore, in this study the ultrastructural phenotype of these cells was analysed by transmission electron microscopy (TEM). The oBM-MSCs were isolated from the iliac crest and cultured until they reached near-confluence. After trypsinization, they were processed to investigate their ultrastructural features as well as specific surface marker proteins by flow cytometry and immunogold electron microscopy. Flow cytometry displayed that all oBM-MSCs lacked expression of CD31, CD34, CD45, HLA-DR whereas they expressed CD44, CD58, HLAI and a minor subset of the cell population (12%) exhibited CD90. TEM revealed the presence of two morphologically distinct cell types: cuboidal electron-lucent cells and spindle-shaped electron-dense cells, both expressing the CD90 antigen. Most of the electron-lucent cells showed glycogen aggregates, dilated cisternae of RER, moderately developed Golgi complex, and secretory activity. The electron-dense cell type was constituted by two different cell-populations: type A cells with numerous endosomes, dense bodies, rod-shaped mitochondria and filopodia; type B cells with elongated mitochondria, thin pseudopodia and cytoplasmic connectivity with electron-lucent cells. These morphological findings could provide a useful support to identify “in situ” the cellular components involved in the cell-therapy when cultured oBM-MSCs are injected.