Glycohistochemical investigation on canine and feline zonae pellucidae of preantral and antral oocytes

Glycoconjugate modifications were analysed in the zona pellucida during development of oocytes in dog and cat using conventional histochemical staining methods with or without previous carbohydrate digestion. A series of lectins combined with desulphation and sialic acid degradation were applied. No differences were observed between dog and cat follicles using conventional histochemical staining methods. In both species, the zona pellucida and follicular fluid/intercellular matrix strongly reacted with PAS and high iron diamine stain (HID) and reacted moderately with low iron diamine stain (LID). Treatment with testicular hyaluronidase, chondroitinase ABC, chondroitinase AC and chondroitinase B treatment diminished HID and LID positivity of follicular fluid and intercellular matrix. Lectins that gave the most intense staining of the zona pellucida of both species were SBA, PNA, RCA-I, GSA-IB4 and WGA, indicating the presence of beta-D-GalNAc, D-Gal and GlcNAc residues. Sulpho- and asulpho-carbohydrates were identified in terminal and/or subterminal positions linked to sialic acid residues. In conclusion, the results indicate that glycosaminoglycans are not present in the zona pellucida of both species. Differences were observed in carbohydrate residues and in their spatial distribution, depending on species and developmental stage of the follicles. The similarity in lectin affinity between ooplasm and zona pellucida of oocytes present in follicles at different stages of development confirm the involvement of oocytes in zona pellucida production

Glycoconjugates in small antral ovarian follicles of the river buffalo (Bubalus bubalis L.)

Types and distribution patterns of glycoconjugates in antral ovarian follicles were investigated in the buffalo, using periodic-acid Schiff (PAS), high iron diamine (HID), low ion diamine (LID) and lectin histochemical staining methods. HID and LID staining procedures were preceded in some cases by digestion with testicular hyaluronidase, Streptomyces hyaluronidase, chondroitinase ABC and heparitinase (heparinase III). Lectin staining was performed with the use of 12 horseradish peroxidase (HRP) lectin conjugates. Some lectin staining procedures were preceded by neuraminidase digestion and saponification. Large amounts of isomeric chondroitin sulphates and a minor quantity of heparan sulphate and hyaluronic acid and/or chondroitin were found in follicular fluid. Lectin staining of buffalo follicular fluid revealed glycoconjugates with different glucidic determinants such as beta-N-acetylgalactosamine, beta-galactose-(1-3)-N-acetylgalactosamine, beta-galactose-(1-4)-N-acetylglucosamine, N-acetylglucosamine, alpha-fucose and alpha-glucose/alpha-mannose, and sialic acid residues. Glycosaminoglycans were absent in the zona pellucida of oocytes in small antral follicles. Acidic glycoconjugates in the zona pellucida were caused by sulphated groups and sialic acid residues. Our data show few internal glucidic residues, such as N-acetylglucosamine in the buffalo zona pellucida but many subterminal beta-N-acetylgalactosamine, alpha- and beta-galactose determinants masked by sialic acids. These findings demonstrate that buffalo follicular fluid has a very heterogeneous composition that is similar to that found in small and large bovine follicles. No differences in composition of the follicular fluid were observed in the follicles examined

Chromosomal analysis of embryos produced by artificially inseminated superovulated cattle

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Application of the cDNA-AFLP method for studying gene expression in Fibrobacter succinogenes S85 exposed to 134.2 kHz electromagnetic field

AbstractMany biological effects related to the exposure of cells and tissues to electromagnetic fields have been reported in the literature, including those influencing DNAs and RNAs structure and ..

Topographical localisation of glucidic residues and their variations in the canine zona pellucida during folliculogenesis.

In the present ultrastructural study, horseradish peroxidase-labelled lectins, in conjunction with antiperoxidase antibody and protein A-gold, were used to characterise and localise the oligosaccharide sequences of zona pellucida glycoproteins at different stages of follicular development in the canine ovary. Deacetylation and sialidase digestion were also performed before lectin cytochemistry. The zona pellucida of oocytes present in unilaminar primary follicles reacts with WGA- and RCA-I-lectins. The zona pellucida of oocytes present in bilaminar and trilaminar secondary follicles displays positivity to WGA, RCA-I, Con-A, UEA-I, and sialidase/SBA. This labelling pattern persists in the zona pellucida of oocytes present in antral tertiary follicles with the exception of WGA and RCA-I reactive sites which are differently distributed throughout the zona pellucida. The topographical distribution of these carbohydrates is not uniform throughout the zona pellucida, indicating the regionalization of oligosaccharide chains within three concentric bands of the zona matrix: an inner surface close to the oocyte plasma membrane, an intermediate portion and an outer layer in contact with the follicular cells. Our results demonstrated variations in the presence and distribution of the carbohydrate residues in the canine zona pellucida during different stages of follicular growth. We also observed the presence of vesicles in both the ooplasm and granulosa cells, showing a similar lectin binding pattern to that of the zona pellucida

GLYCOMOLECULE MODIFICATIONS IN THE SEMINIFEROUS EPITHELIAL CELLS AND IN THE ACROSOME OF POST-TESTICULAR SPERMATOZOA IN THE ALPACA.

A lectin histochemical investigation of the seminiferous epithelium and acrosomes of spermatozoa present in the efferent ductules and epididymal regions was carried out in the alpaca. The histochemical characterization was performed using a battery of different lectins: Con-A, UEA-I, LTA, WGA, GSA-IB4, SBA, PNA, ECA, DBA, MAL-II and SNA. Sialidase digestion and deglycosilation pre-treatments were also employed. The cytoplasm of the Sertoli cells contained N-linked oligosaccharides with alpha-D-Man/alpha-D-Glc and GlcNAc and O-linked glycans with alpha-L-Fuc, beta-GalNAc, beta-D-Gal-(1-4)-D-GlcNAc, alpha-Gal and Neu5Acalpha2,6alpha-GalNAc moieties whereas beta-D-Gal-(1-3)-D-GalNAc residues were included in both O- and N-glycoproteins. Spermatogonia expressed alpha-D-Man/alpha-D-Glc residues included in N-glycoproteins and alpha-Fuc in O-glycoproteins. Spermatocytes contained the N-glycoproteins residues alpha-D-Man/alpha-D-Glc and GlcNAc and the O-glycoproteins residues alpha-L-Fuc, beta-D-Gal-(1-4)-D-GlcNAc, alpha-Gal, beta-GalNAc, Neu5Acalpha2,6alpha-GalNAc and Neu5Acalpha2,6beta-D-Gal-(1-3)-D-GalNAc. The results of the present study show differences in the presence and distribution of lectin reactive sites throughout the acrosomal development in the alpaca. In particular, Fuc moieties were found only during the Golgi-phase of spermatids, alpha-Gal were found in the acrosome of Golgi- and cap-phase spermatids, sialic-acid/alpha-GalNAc sequence was revealed during the cap-phase and elongated spermatids, and alpha-D-Man/alpha-D-Glc and GlcNAc were detected only in the acrosomes of elongated spermatids. Finally, beta-GalNAc, beta-D-Gal-(1-3)-D-GalNAc and beta-D-Gal-(1-4)-D-GlcNAc were added to acrosomal glycoproteins in the early stages of spermatogenesis and remained unchanged during the later phases. Differences in the carbohydrate expression were also demonstrated on the sperm acrosomes during passage through the post-testicular ducts

Exercise-induced up-regulation of MMP-1 and IL-8 genes in endurance horses

<p>Abstract</p> <p>Background</p> <p>The stress response is a critical factor in the training of equine athletes; it is important for performance and for protection of the animal against physio-pathological disorders.</p> <p>In this study, the molecular mechanisms involved in the response to acute and strenuous exercise were investigated using peripheral blood mononuclear cells (PBMCs).</p> <p>Results</p> <p>Quantitative real-time PCR (qRT-PCR) was used to detect modifications in transcription levels of the genes for matrix metalloproteinase-1 (<it>MMP-1</it>) and interleukin 8 (<it>IL-8</it>), which were derived from previous genome-wide expression analysis. Significant up-regulation of these two genes was found in 10 horses that had completed a race of 90–120 km in a time-course experimental design.</p> <p>Conclusion</p> <p>These results suggest that <it>MMP-1 </it>and <it>IL-8 </it>are both involved in the exercise-induced stress response, and this represents a starting point from which to understand the adaptive responses to this phenomenon.</p

Exercise induced stress in horses: Selection of the most stable reference genes for quantitative RT-PCR normalization

<p>Abstract</p> <p>Background</p> <p>Adequate stress response is a critical factor during athlete horses' training and is central to our capacity to obtain better performances while safeguarding animal welfare.</p> <p>In order to investigate the molecular mechanisms underlying this process, several studies have been conducted that take advantage of microarray and quantitative real-time PCR (qRT-PCR) technologies to analyse the expression of candidate genes involved in the cellular stress response.</p> <p>Appropriate application of qRT-PCR, however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability.</p> <p>Results</p> <p>The expression of nine potential reference genes was evaluated in lymphocytes of ten endurance horses during strenuous exercise. These genes were tested by qRT-PCR and ranked according to the stability of their expression using three different methods (implemented in <it>geNorm</it>, <it>NormFinder </it>and <it>BestKeeper</it>). Succinate dehydrogenase complex subunit A (<it>SDHA</it>) and hypoxanthine phosphoribosyltransferase (<it>HPRT</it>) always ranked as the two most stably expressed genes. On the other hand, glyceraldehyde-3-phosphate dehydrogenase (<it>GAPDH</it>), transferrin receptor (<it>TFRC</it>) and ribosomal protein L32 (<it>RPL32</it>) were constantly classified as the less reliable controls.</p> <p>Conclusion</p> <p>This study underlines the importance of a careful selection of reference genes for qRT-PCR studies of exercise induced stress in horses. Our results, based on different algorithms and analytical procedures, clearly indicate <it>SDHA </it>and <it>HPRT </it>as the most stable reference genes of our pool.</p

Mitochondrial DNA diversity of five Italian autochtonous donkey breeds

AbstractTo investigate the mitochondrial DNA diversity of five Italian donkey breeds (Amiata, Martinafranca, Romagnolo, Asinara, and Ragusano), we sequenced the HVR I region (D-loop, 288 bp) and cytochrome b gene (274 bp) in 121 individuals. In the D-loop we found nineteen mutations corresponding to fourteen different haplotypes, while in cyt b coding gene only six mutations were found, originating five different haplotypes. In particular, three mutations out of six were non-synonymous, causing an aminoacidic substitution. About the D-loop region, the value of nucleotide diversity (π) observed within breeds was relatively low, but not far from values detected in other European breeds. Phylogenetic and network analyses disclosed the presence of two divergent maternal lineages within Italian donkeys. These haplogroups correspond to the well known lineages of ancestors (Equus asinus somaliensis and E. a. africanus), as donkeys were domesticated from distinct wild subspecies living in Eastern Africa regions. ..