Cervical mucus sialic acid content determines the ability of frozen-thawed ram sperm to migrate through the cervix.

The aim of this study was to investigate the properties and to functionally characterize the cervical mucus that modulates sperm transport through the cervix by using ewe breeds with a divergent pregnancy rate (Belclare and Suffolk; high and low, respectively) following cervical insemination using frozen-thawed semen. Sperm number, as well as sialic acid and fucose content in both the channels and in the lumen of different regions of the cervix were quantified in inseminated Belclare and Suffolk ewes. Expression of glycosyltransferase and MUC genes, glycosidase activity and sialic acid speciation in follicular phase cervical tissue and mucus were assessed. More spermatozoa were found in the cervical channels in the region closest to the cervical os in Belclare than Suffolk ewes (P < 0.05) and Suffolk ewes had a higher sialic acid content in the cervical channels than Belclare ewes (P < 0.05) in all regions of cervix. Suffolk ewes had significantly higher expression of FUT1, ST6GAL1 and MUC5AC than Belclare ewes. There was no difference between the breeds in glycosidase activity (P > 0.05). Levels of Neu5Ac were higher in Belclare than Suffolk ewes (P < 0.05) and levels of Neu5Gc was higher in Suffolk than Belclare ewes (P < 0.05). Competitive sperm penetration assays demonstrated that frozen-thawed sperm progression increased when cervical mucus was incubated with sialyllactose prior to a sperm penetration test (P < 0.05). These results suggest that the difference between Belclare and Suffolk ewes in sperm transport with frozen-thawed semen is due to the higher concentration of sialic acid within channels, which binds to spermatozoa and reduces their ability to traverse the cervix.

Siglec expression on the surface of human, bull and ram sperm.

Sialic acid (Sia) is a major constituent of both the sperm glycocalyx and female reproductive mucosal surface and is involved in regulating sperm migration, uterotubal reservoir formation and oocyte binding. Siglecs (sialic acid-binding immunoglobulin - like lectins) commonly found on immune cells, bind to Sia in a linkage- and sugar-specific manner and often mediate cell-to-cell interactions and signalling. Proteomic and transcriptomic analysis of human and bovine sperm have listed Siglecs, but to date, their presence and/or localisation on sperm has not been studied. Therefore, the aim of this study was to characterise the presence of Siglecs on the surface of bovine, human and ovine sperm using both immunostaining and Western blotting. Siglec 1, 2, 5, 6, 10 and 14 were identified and displayed both species- and regional-specific expression on sperm. Almost universal expression across Siglecs and species was evident in the sperm neck and midpiece region while variable expression among Siglecs, similar among species, was detected in the head and tail regions of the sperm. The possible role for these proteins on sperm is discussed.

Fertility and genomics: comparison of gene expression in contrasting reproductive tissues of female cattle.

To compare gene expression among bovine tissues, large bovine RNA-seq datasets were used, comprising 280 samples from 10 different bovine tissues (uterine endometrium, granulosa cells, theca cells, cervix, embryos, leucocytes, liver, hypothalamus, pituitary, muscle) and generating 260 Gbases of data. Twin approaches were used: an information-theoretic analysis of the existing annotated transcriptome to identify the most tissue-specific genes and a de-novo transcriptome annotation to evaluate general features of the transcription landscape. Expression was detected for 97% of the Ensembl transcriptome with at least one read in one sample and between 28% and 66% at a level of 10 tags per million (TPM) or greater in individual tissues. Over 95% of genes exhibited some level of tissue-specific gene expression. This was mostly due to different levels of expression in different tissues rather than exclusive expression in a single tissue. Less than 1% of annotated genes exhibited a highly restricted tissue-specific expression profile and approximately 2% exhibited classic housekeeping profiles. In conclusion, it is the combined effects of the variable expression of large numbers of genes (73%-93% of the genome) and the specific expression of a small number of genes (<1% of the transcriptome) that contribute to determining the outcome of the function of individual tissues.

N-glycan profiling of bovine follicular fluid at key dominant follicle developmental stages.

Follicular fluid (FF), an important microenvironment for the development of oocytes, contains many proteins that are glycosylated with N-linked glycans. This study aimed i) to present an initial analysis of the N-linked glycan profile of bovine FF using hydrophilic interaction liquid chromatography, anion exchange chromatography, high performance liquid chromatography (HPLC)-based separations and subsequent liquid chromatography-mass spectrometry/mass spectrometry analysis; ii) to determine differences in the N-glycan profile between FF from dominant and subordinate follicles from dairy heifers and lactating dairy cows and iii) to identify alterations in the N-glycan profile of FF during preovulatory follicle development using newly selected, differentiated (preovulatory) and luteinised dominant follicles from dairy heifers and lactating cows. We found that the majority of glycans on bovine FF are based on biantennary hypersialylated structures, where the glycans are sialylated on both the galactose and N-acetylglucosamine terminal sugars. A comparison of FF N-glycans from cows and heifers indicated higher levels of nonsialylated glycans with a lower proportion of sialylated glycans in cows than in heifers. Overall, as the follicle develops from Selection, Differentiation and Luteinisation in both cows and heifers, there is an overall decrease in sialylated structures on FF N-glycans.

Interaction of Eimeria tenella with intestinal mucin in vitro.

The mucus gel layer overlying the gastrointestinal epithelium plays an important role in host-pathogen interactions. The initial interaction between the coccidian parasite Eimeria tenella and host cells of the intestinal epithelium must occur across this mucus interface. In this study, we examined the relationship between E. tenella and avian mucin, in particular the effect of purified intestinal regional mucin on parasite adherence and invasion in vitro. Secreted mucin from the chicken duodenum and cecum was purified by density gradient centrifugation and gel chromatography. Parasite invasion studies were performed in the Madin-Darby bovine kidney cell model. Eimeria tenella adherence to chicken duodenal mucin was detected, whereas adherence to cecal or bovine mucin was not shown. Parasite invasion into epithelial cells was not influenced by bovine mucin, whereas chicken mucin purified from the duodenum and cecum significantly inhibited invasion. Inhibition of E. tenella invasion into cells by mucin from the duodenum was marginally greater than that of the cecum, but this was not significant. This study demonstrated E. tenella interaction with native chicken intestinal mucin, which in turn inhibited parasite invasion into epithelial cells in vitro.

The role of mucins in host-parasite interactions: Part II - helminth parasites.

Some parasites express mucin-like molecules. These have possible roles in attachment and invasion of host cells and in the avoidance of host immune processes. Enzymes of parasite origin might also facilitate infection, either by degrading host mucus barriers or by generating binding sites on host cells. Host mucins have roles in preventing parasite establishment or in parasite expulsion. They, in turn, might be exploited by parasites, either as sources of fuel or binding sites, or as host-finding targets. Here, we describe the biochemical properties of mucins and mucin-like molecules in relation to interactions (established and putative) between helminth parasites and their hosts.

Distribution of MUC1 in the normal human oral cavity is localized to the ducts of minor salivary glands.

The change in expression of MUC1 from health to disease forms the basis of its use as a potential disease marker. Previous attempts at isolating MUC1 from normal, healthy human oral mucosa have, however, drawn conflicting conclusions as to its presence. Furthermore, when MUC1 was detected in the oral glycocalyx, it was not clear which cells were synthesising it. We examined human oral glycocalyx using pooled buccal smears from 50 normal individuals. Following isopycnic density centrifugation and membrane extraction with octyl glucoside and saponin, MUC1 was detected with the polyclonal antibody CT1. Immunohistochemistry using antibodies CT1 and BC2 was performed on sections from eight labial, seven palatal, four buccal, three retromolar pad, three dorsum of tongue and two ventral surface of tongue biopsies. In-situ hybridisation using MUC1 and cytoplasmic tail oligoprobes on sections from four palatal, seven labial and two retromolar pad biopsies was also carried out. MUC1 mRNA could only be detected in the minor salivary mucous glands. MUC1 has already been identified in the ducts of normal parotid and submandibular gland, and our findings demonstrate a similar distribution in minor salivary glands. We conclude that when present in the normal oral glycocalyx, the only oral source of MUC1 is from cell membranes of the minor salivary glands.

The topography of the anterior surface of the cat's cornea.

The topography of the cornea in nine unanaesthetised domestic cats was studied by the computer analysis of photokeratographs obtained using a Wessley Jessen P.E.K. 2000 photokeratoscope . Mean figures were obtained for: Central corneal radius in the steeper (9.06 mm) and flatter (9.20 mm) corneal meridians. Corneal astigmatism (0. 61D ). Corneal shape factor in the steeper (0.33) and flatter (0.31) corneal meridians and the diameter of the corneal cap in the vertical (6.23 mm) and horizontal (7.08 mm) meridians. The results are discussed and compared with previous such estimates for corneal topography. Dimensions for an hydrophilic contact lens suitable for general therapeutic or research use in the cat are suggested.

Surface mucus in the non-glandular region of the equine stomach.

In horses, ulceration of the non-glandular region of the stomach is common and has been attributed to the lack of a protective mucus covering. This study aimed to determine whether the non-glandular region is covered by a mucus layer. A mixture of antibodies raised against human gastric mucin (MUC 5 AC) showed a tissue distribution in the glandular region of the equine stomach similar to that seen in humans. Dot blots of mucus from the glandular and non-glandular regions showed cross-reactivity with these antibodies. Various histological fixation and processing techniques were compared for their ability to preserve mucus in the non-glandular region. Fixing frozen sections on-slide for 20 seconds in 20 per cent formalin/1 per cent cetylpyridinium chloride was considered the best method. In conclusion, the equine stomach expresses a gene homologous to human MUC 5 AC. Its product is expressed as a neutral mucin, which is present in the mucus that covers both the glandular and non-glandular regions. Future comparison of mucus composition in the healthy and ulcerated stomach will improve our understanding of gastric ulceration in the horse.

Biomicroscopy of the tear film: the tear film of the pekingese dog.

Polarised light biomicroscopy was used to examine the normal pre-corneal tear film in 21 eyes of 12 pekingese dogs. The purpose of the study was to examine the influence of excessive exophthalmos on the pre-corneal tear film in the dog. The majority of the animals were found to have high levels of ocular surface contamination by particulate material and plaques of viscous mucus. Other abnormalities included surface lipid with an abnormal granular (three dogs) or 'curdled' (two dogs) appearance; excessive thinning of the lipid layer of the tear film; and the presence of dark globular structures in two dogs, which were presumed to be abnormal meibomian lipid. Break up of the tear film was observed in one dog. Grossly, a thread of viscous mucus was frequently observed along the margin of the lower eyelid. It is postulated that this thread forms because of the excessively exophthalmic conformation of the breed, which prevents the normal access of effete mucus and entrapped debris to the lower conjunctival fornix. The combination of the above factors in the pekingese is suggested as the mechanism whereby the tear film has a reduced stability, thus enhancing the risk from factors more usually considered to initiate corneal ulceration in the breed. The possible adverse effects of lid splitting for the mass removal of distichiae in exophthalmic dogs is discussed.

Improving bovine semen diluents: insights from the male and female reproductive tracts, and the potential relevance of cervical mucins.

The commercial applicability of bovine artificial insemination (AI) depends on the effectiveness of diluents for maintaining sperm fertility. Challenges faced by the AI industry due to recent advances in assisted reproduction, and the limitations inherent in using fresh and frozen-thawed sperm for AI, could be overcome with the development of better semen diluents. Research into the different microenvironments of bovine sperm as they progress towards maturity, capacitation and fertilisation is revealing various mechanisms that could be exploited to improve the formulation of semen diluents. These are reviewed here. A rationale for a more detailed investigation of bovine cervical mucus for factors that may allow further progress towards this goal are also discussed.

Glycoproteins and glycosidases of the cervix during the periestrous period in cattle.

The cervix and its secretions undergo biochemical and physical changes under the differential influences of estrogen and progesterone. These include changes in the glycoprotein profile of the endocervix and its secretions. A comprehensive survey of such changes in cervical epithelium and cervical secretions was performed on bovine samples throughout the periestrous period. Cervical tissue samples and swabs were collected from synchronized beef heifers that were slaughtered 1) 12 h after controlled intravaginal progesterone-releasing device (CIDR) removal, 2) 24 h after CIDR removal, 3) at the onset of estrus, 4) 12 h after the onset of estrus, 5) 48 h after the onset of estrus, and 6) 7 d after the onset of estrus. Histological staining with hematoxylin and eosin, periodic acid Schiff, Alcian blue, and high-iron diamine was carried out to map overall patterns of stored glycoproteins and tissue structure. Biotinylated lectins were also used to detect the presence and distribution of a range of saccharide structures. The activities of ß-galactosidase, α-L-fucosidase, ß-N-acetyl-hexosaminidase, and sialidase were measured in cervical swabs using specific substrates. The epithelial layer of the cervix exhibited dynamic changes in cellular hypertrophy and amounts of stored glycoprotein. The greatest content of neutral and acidic mucins was observed 48 h after onset of estrus (P < 0.05). Sialylated mucins predominated at the bases of cervical folds, whereas sulfated mucins were more abundant (P < 0.05) at their apices. The stained area of core mucin glycans changed (P < 0.05) in association with follicular versus luteal phases, whereas terminal glycans changed (P < 0.05) mainly at the time of estrus and shortly thereafter. The greatest activity of ß-galactosidase and sialidase was observed 12 h after onset of estrus, whereas ß-hexosaminidase and α-fucosidase peaked at the luteal time point (P < 0.05). Taken together, we suggest that the well-known changes in the endocervix and its secretions that are associated with the physiological modulation of sperm transport and function of the cervical barrier are, in part, driven by glycosylation changes.

Identification of 9-O-acetyl-N-acetylneuraminic acid in normal canine pre-ocular tear film secreted mucins and its depletion in Keratoconjunctivitis sicca.

O-Acetylated sialic acids have been reported in many sialoglycoproteins where they mediate a variety of immune and other biological events. We have previously demonstrated that the protective mucus barrier on the surface of the canine eye contains sialoglycoproteins. We have also investigated the occurrence of O-acetylated sialic acids in these ocular mucins. Mucus aspirated from the surface of normal dog eyes and those with keratoconjunctivitis sicca (KCS) was fractionated into three pools by density gradient centrifugation. Sialic acids comprised 0.6-0.9% of the dry weight of the mucins isolated. The sialic acid profile in these pools was examined using HPLC. O-Acetylated sialic acids, mainly Neu5,9Ac2, were detected in normal animals and made up 10-30% of the total sialic acids detected. A doubling of the sialic acid content was found in KCS mucins, but the level of 9-O-acetylated sialic acid was reduced below 4% of total. Histological analysis of conjunctival tissue from normal and KCS dogs showed the presence of sialic acids, detected with the alpha(2-6) sialic acid-specific lectin Sambucus nigra, in the goblet cells and corresponding to the staining pattern for MUC5AC, the major ocular-secreted mucin gene product. In KCS animals a disruption of the normal pattern of conjunctival goblet cells was seen with preservation of the pattern of lectin binding observed in normal animals. Thus the data demonstrate the presence of mono-O-Acetylated sialic acids in normal canine ocular mucins and a loss of this population of sialic acids in dry eye disease in spite of a significant increase in total sialic acids in KCS mucin.

Trichinella spiralis: enteric mucin-related response to experimental infection in conventional and SPF pigs.

Duodenal and jejunal responses to infection with Trichinella spiralis were compared in weaned piglets with a "normal dirty" vs. a "clean SPF" gut flora. Histochemical staining of neutral, acidic, sialylated, and sulphated residues was used to assess biosynthetic responses in mucin-secreting goblet cells. Peanut and Ulex lectins were also used to assess responses within the intestinal glycocalyx. Histomorphometric analysis was undertaken to evaluate the distribution and staining patterns of goblet cells in villi and crypts. Our analysis showed that stored mucin within goblet cells increased more in the infected conventional animals than in the infected SPF group. This was accompanied by changes in the pattern of sulphation and sialylation in the duodenum and jejunum. The thickness of the glycocalyx was increased in both duodenum and jejunum in both infected groups. However, this effect was greater for the infected SPF animals than the infected conventional animals. No significant differences were observed between uninfected conventional and uninfected SPF pigs.

Corneal thickness and diameter in the domestic cat.

Two sample groups of domestic cats were assessed for (a) central corneal thickness (17 eyes), using optical pachymetry, and (b) horizontal and vertical corneal diameter, using a photographic method (20 eyes). The average corneal thickness was 0.755 +/- 0.33 mm. The mean horizontal diameter was 16.5 +/- 0.60 mm and the mean vertical diameter was 16.2 +/- 0.61 mm. The horizontal corneal diameter was significantly greater than the vertical corneal diameter (p less than 0.001, paired t test).

Regional differences in phagocytosis by the corneal endothelium in the domestic cat.

The phagocytosis of IgG-coated latex microspheres by the central and peripheral regions of the corneal endothelium in the domestic cat was investigated by organ culture. The endothelium was exposed to 1.357 X 10(7) microspheres/mm2, and counts of ingested microspheres were made by scanning electron microscopy. Initial assessments of phagocytosis over 8-h and 24-h periods of culture gave extremely low levels of incorporation. Therefore, subsequently, a 48-h period of culture was adopted. Microsphere uptake levels per square millimeter over 48 h were as follows: peripheral endothelium 6,021 +/- 3,162; central endothelium 2,000 +/- 1,529; overall 4,011 +/- 3,294. The greater uptake of microspheres in the peripheral cornea was statistically significant (P less than 0.002, paired t-test). The involvement of microvilli in phagocytosis by corneal endothelial cells was proposed.