Comparison of Siglec-1 protein networks and expression patterns in sperm and male reproductive tracts of mice, rats, and humans.

Background: Sialic acid-binding immunoglobulin-like lectin 1 (Siglec-1) is a transmembrane glycoprotein involved in the sialic acid (Sia)-dependent regulation of the immune system. Siglec-1 expression has recently been identified in the male reproductive tract (MRT) of several species, including humans, cattle, horses, and sheep, and may play a role in modulating fertility in a Sia-dependent manner. Materials and Methods: In this study, protein-protein interaction (PPI) analysis of Siglec-1 was conducted to identify associated network protein conservation, and the expression of Siglec-1 in the MRT of mice and rats, including their accessory sex glands and spermatozoa was determined by immunostaining. Results: Network analysis of proteins with Siglec-1 in mice and rats demonstrated significant similarity to human Siglec-1 networks, suggesting a similar conservation of network proteins between these species and, hence, a potential conservation role in immune modulation and function. Specific immunostaining patterns of mouse and rat testes, epididymis, ductus deferens, accessory sex gland tissues, and sperm were detected using human Siglec-1. These results confirmed that the human Siglec-1 antibody could cross-react with mouse and rat Siglec-1, suggesting that the specific expression patterns of Siglec-1 in the MRT and sperm of both mice and rats are similar to those observed in other species. Conclusions: The conservation of Siglec-1 expression patterns in sperm and within the MRT and the similarity of protein networks for Siglec-1 across species suggest that Siglec-1 may function in a similar manner across species. These results also suggest that rodents may serve as a valuable model system for exploring the function of Siglecs in the reproductive system across species and their potential role in modulating fertility in a Sia-dependent manner.

The transcriptomic response of bovine uterine tissue is altered in response to sperm from high- and low-fertility bulls†.

Despite stringent quality control checks, some bulls with apparently normal semen quality yield lower than expected pregnancy rates. This study profiled the transcriptome and performed histological analysis of the bovine uterus in response to sperm from high-fertility (HF) and low-fertility (LF) bulls. Postmortem uterine biopsies and uterine explants were collected from heifers 12 h after a fixed time artificial insemination (AI) to a synchronized estrus with frozen-thawed semen from five HF (fertility rate 4.01% ± 0.25) and five LF (fertility rate - 11.29% ± 1.11; mean ± SEM) bulls. Uterine biopsies were also collected from control (CTRL) heifers, which were not inseminated. RNA-sequencing and histological analysis were performed for differential gene expression and neutrophil quantification. In the HF treatment relative to CTRL heifers, there were 376 genes significantly differentially expressed in the endometrium with just one gene differentially expressed in the LF treatment relative to CTRL heifers. Comparing the HF and LF treatments directly, there were 40 significantly differentially expressed genes (P < 0.05). Transcriptomic analysis shows a predominant role for the inflammatory marker Interleukin-1 alpha, which was further confirmed by immunohistochemistry. Quantification of neutrophils in the endometrium showed a significant effect of sperm; however, there was no difference in neutrophil numbers between HF and LF groups. In conclusion, this novel study clearly shows a distinct inflammatory response to sperm in the endometrium and a divergent transcriptomic response to semen from HF and LF bulls.

Biochemical and molecular characterization of sialylated cervical mucins in sheep†.

Sialic acid occupies terminal positions on O-glycans of cervical mucins, where they contribute to the increased viscosity of mucin thereby regulating sperm transport. This study characterized the sialylated cervical mucins from follicular phase mucus of six European ewe breeds with known differences in pregnancy rates following cervical artificial insemination (AI) using frozen-thawed semen at both synchronized and natural estrus cycles. These were Suffolk (low fertility) and Belclare (medium fertility) in Ireland, Ile de France and Romanov (both with medium fertility) in France, and Norwegian White Sheep (NWS) and Fur (both with high fertility) in Norway. Expression of mucin and sialic acid related genes was quantified using RNA-sequencing in cervical tissue from Suffolk, Belclare, Fur, and NWS only. Cervical tissue was also assessed for the percentage of cervical epithelial populated by mucin secreting goblet cells in the same four ewe breeds. Biochemical analysis showed that there was an effect of ewe breed on sialic acid species, which was represented by Suffolk having higher levels of Neu5,9Ac2 compared with NWS (P < 0.05). Suffolk ewes had a lower percentage of goblet cells than Fur and NWS (P < 0.05). Gene expression analysis identified higher expression of MUC5AC, MUC5B, ST6GAL1, and ST6GAL2 and lower expression of ST3GAL3, ST3GAL4, and SIGLEC10 in Suffolk compared with high fertility ewe breeds (P < 0.05). Our results indicate that specific alterations in sialylated mucin composition may be related to impaired cervical sperm transport.

Identification and characterization of O-linked glycans in cervical mucus as biomarkers of sperm transport: A novel sheep model.

Cervical mucus plays an important role in female fertility, since it allows the entry of motile and morphological normal sperm while preventing the ascent of pathogens from the vagina. The function of cervical mucus is critically linked to its rheological properties that are in turn dictated by O-glycosylated proteins, called mucins. We aimed to characterize the O-glycan composition in the cervical mucus of six European ewe breeds with known differences in pregnancy rates following cervical/vaginal artificial insemination with frozen-thawed semen, which are due to reported differences in cervical sperm transport. These were Suffolk (low fertility) and Belclare (medium fertility) in Ireland, Ile de France and Romanov (both with medium fertility) in France, and Norwegian White Sheep (NWS) and Fur (both with high fertility) in Norway (n = 28-30 ewes/breed). We identified 124 O-glycans, from which 51 were the major glycans with core 2 and fucosylated glycans as the most common structures. The use of exogenous hormones for synchronization did not affect the O-glycan composition in both high-fertility ewe breeds, but it did in the other four ewe breeds. There was a higher abundance of the sulfated glycan (Galß1-3[SO3-GlcNAcß1-6]GalNAc), fucosylated glycan (GlcNAcß1-3(Fucα1-2Galß1-3)GalNAc) and core 4 glycan (GlcNAcß1-3[GlcNAcß1-6]GalNAc) in the low-fertility Suffolk breed compared with NWS (high fertility). In addition, core 4 glycans were negatively correlated with mucus viscosity. This novel study has identified O-glycans that are important for cervical sperm transport and could have applications across a range of species including human.

Exploiting the MUC5AC Antigen for Noninvasive Identification of Pancreatic Cancer.

Pancreatic cancer (PC) remains the fourth leading cause of cancer death; therefore, there is a clinically unmet need for novel therapeutics and diagnostic markers to treat this devastating disease. Physicians often rely on biopsy or CT for diagnosis, but more specific protein biomarkers are highly desired to assess the stage and severity of PC in a noninvasive manner. Serum biomarkers such as carbohydrate antigen 19-9 are of particular interest as they are commonly elevated in PC but have exhibited suboptimal performance in the clinic. MUC5AC has emerged as a useful serum biomarker that is specific for PC versus inflammation. We developed RA96, an anti-MUC5AC antibody, to gauge its utility in PC diagnosis through immunohistochemical analysis and whole-body PET in PC. Methods: In this study, extensive biochemical characterization determined MUC5AC as the antigen for RA96. We then determined the utility of RA96 for MUC5AC immunohistochemistry on clinical PC and preclinical PC. Finally, we radiolabeled RA96 with 89Zr to assess its application as a whole-body PET radiotracer for MUC5AC quantification in PC. Results: Immunohistochemical staining with RA96 distinguished chronic pancreatitis, pancreatic intraepithelial neoplasia, and varying grades of pancreatic ductal adenocarcinoma in clinical samples. 89Zr-desferrioxamine-RA96 was able to detect MUC5AC with high specificity in mice bearing capan-2 xenografts. Conclusion: Our study demonstrated that RA96 can differentiate between inflammation and PC, improving the fidelity of PC diagnosis. Our immuno-PET tracer 89Zr-desferrioxamine-RA96 shows specific detection of MUC5AC-positive tumors in vivo, highlighting the utility of MUC5AC targeting for diagnosis of PC.

Sialylated Cervical Mucins Inhibit the Activation of Neutrophils to Form Neutrophil Extracellular Traps in Bovine in vitro Model.

In order to combat invading pathogens neutrophils can release neutrophil extracellular traps (NETs). However, since NETs can also damage endogenous cells, several control mechanisms for the formation of NETs must work effectively. For instance, neutrophil activation is silenced within blood circulation by the binding of sialylated glycoconjugates to sialic acid binding immunoglobulin-like lectins (Siglecs) on neutrophils. As neutrophils are recruited within the female reproductive tract, after mating, a comparable mechanism may also take place within the bovine cervix to prevent an exaggerated NET formation and thus, infertility. We examined, if the highly glycosylated mucins, which are the major functional fraction of biomolecules in mucus, represent a potential regulator of NET formation. The qPCR data revealed that in polymorphonuclear neutrophils (PMNs) inhibitory Siglecs are the most frequently expressed Siglecs and might be a potential target of sialylated glycans to modulate the activation of PMNs. Remarkably, the addition of bovine cervical mucins significantly inhibited the formation of NET, which had been induced in response to lipopolysaccharides (LPS) or a combination of phorbol myristate acetate (PMA) and ionomycin. The inhibitory effects were independent of the stage of estrous cycle (estrus, luteal, and follicular mucins). PMNs retained their segmented nuclei and membrane perforation was prevented. However, the inhibitory effects were diminished, when sialic acids were released under acidic conditions. Comparable results were achieved, when sialic acids were targeted by neuraminidase digestion, indicating a sialic acid dependent inhibition of NET release. Thus, bovine cervical mucins have an anti-inflammatory capability to modulate NET formation and might be further immunomodulatory biomolecules that support fertility.

Use of Recombinant Mucin Glycoprotein to Assess the Interaction of the Gastric Pathogen Helicobacter pylori with the Secreted Human Mucin MUC5AC.

There is intense interest in how bacteria interact with mucin glycoproteins in order to colonise mucosal surfaces. In this study, we have assessed the feasibility of using recombinant mucin glycoproteins to study the interaction of the gastric pathogen Helicobacter pylori with MUC5AC, a mucin which the organism exhibits a distinct tropism for. Stable clonal populations of cells expressing a construct encoding for a truncated version of MUC5AC containing N- and C-termini interspersed with two native tandem repeat sequences (N + 2TR + C) were generated. Binding of H. pylori to protein immunoprecipitated from cell lysates and supernatants was assessed. High molecular weight mucin could be detected in both cell lysates and supernatants of transfected cells. Recombinant protein formed high molecular weight oligomers, was both N and O glycosylated, underwent cleavage similar to native MUC5AC and was secreted from the cell. H. pylori bound better to secreted mucin than intracellular mucin suggesting that modifications on extracellular MUC5AC promoted binding. Lectin analysis demonstrated that secreted mucin was differentially glycosylated compared to intracellular mucin. H. pylori also bound to a recombinant C-terminus MUC5AC protein, but binding to this protein did not inhibit binding to the N + 2TR + C protein. This study demonstrates the feasibility of using recombinant mucins containing tandem repeat sequences to assess microbial mucin interactions.

In vitro generation of polysialylated cervical mucins by bacterial polysialyltransferases to counteract cytotoxicity of extracellular histones.

Neutrophil extracellular traps (NET) are formed against pathogens. However, various diseases are directly linked to this meshwork of DNA. The cytotoxic properties of extracellular histones especially seem to be an important trigger during these diseases. Furthermore, NET accumulation on implants is discussed to result in an impaired efficiency or failure, depending on the category of implant. Interestingly, mucins have been investigated as surface coatings potentially capable of reducing neutrophil adhesion. Similarly, polysialic acid was shown to inactivate the cytotoxic properties of extracellular histones. We wanted to combine the probability to decrease the adhesion of neutrophils using mucins with the capability of sialic acid polymers to counteract histone-mediated cytotoxicity. To this end, we elongate cervical mucins using bacterial polysialyltransferases. Subsequent cell-based experiments demonstrated the activity of elongated mucins against histone-mediated cytotoxicity. Thus, polysialylated mucins may represent a novel component to coat implants or to combat diseases with exaggerated NET formation.

Expression and characterization of a novel recombinant version of the secreted human mucin MUC5AC in airway cell lines.

Molecular manipulation and expression of mucins, large glycoproteins that provide the structural framework of mucus, are challenging due to mucins' size and numerous domains, including variable number tandem repeat (VNTRs) regions that are sites of O-glycosylation. Only individual human mucin domains have been expressed in mammalian cells. We produced recombinant versions of MUC5AC, a major secreted mucin in the respiratory tract, encoding the N-terminus, C-terminus, N- and C-termini together, and N- and C-termini interspersed with two native tandem repeat sequences (N+2TR+C) in both tracheal and bronchial cell lines. The latter protein contains all of the functional domains required for the biosynthesis and secretion of glycosylated mucin. The N-terminus protein was found in monomeric and higher molecular mass forms suggesting that secreted MUC5AC may form a branched netlike structure analogous to that described for MUC2. At the C-terminus, proteins underwent cleavage, polymerization, and glycosylation. Thus, they appear to undergo pivotal processing steps as predicted for native MUC5AC, which is analogous to that for other individual recombinant mucin domains. Secretion occurred when cells were grown on transwell filter inserts but not on plastic, indicating that the extracellular environment likely plays a role in mucin processing. The secreted N+2TR+C protein differed in molecular mass from the intracellular form, indicating that additional processing occurred. These recombinant proteins, expressed in different backgrounds, can potentially address the role of different mucin domains on MUC5AC processing and function as well as the role of MUC5AC in health and disease.

Modulation of expression in BEAS-2B airway epithelial cells of α-L-fucosidase A1 and A2 by Th1 and Th2 cytokines, and overexpression of α-L-fucosidase 2.

Chronic Th2-driven airway inflammation with excessive mucus production occurs in asthma. The regulation of FUCA1 and FUCA2 gene expression and enzyme activity in response to asthma-associated Th2 cytokines and, for contrast, Th1 cytokine IFN-γ, were investigated in a human airway cell line. BEAS-2B cells were supplemented with Th2-derived cytokines (IL-13, IL-4, IL-5) or/and IFN-γ. RNA and cell supernatants from stimulated and unstimulated cells were collected over a period of 3 h. Alpha-L-fucosidase A1 and A2 gene expression were assessed using real time RT-PCR, while enzymatic activities were measured using a fluorescent assay. To characterise α-L-fucosidase A2, CHO-K1 and BEAS-2B cell lines were transiently transfected, the FUCA2 gene was overexpressed, and the protein was immunoprecipitated. The transcription of FUCA1 was upregulated (p < 0.01) in response to IFN-γ, suggesting that FUCA1 transcription and fucosidase activity are regulated in a Th1-dependent manner. The gene expression was the highest for 30 min after IFN-γ stimulation (>twofold induction), whereas secreted enzyme activity in BEAS-2B cells was significantly increased 1 h after IFN-γ addition. IL-4, IL-5 and IL-13 had no effect on FUCA1 and FUCA2 expression and activity. The IFN-γ-induced increase in expression and activity was repressed by the presence of the Th2 cytokine IL-5. Enzymatically active α-L-fucosidase 2 was immunoprecipitated from BEAS-2B cells, with highest activity at pH 4.9. IL-13, IL-4 and IL-5 have no effect on the expression of FUCA1 and FUCA2, but its expression is upregulated by IFN-γ, a Th1 cytokine. Active α-L-fucosidase 2 was overexpressed in BEAS-2B cells.

Divergent mechanisms of interaction of Helicobacter pylori and Campylobacter jejuni with mucus and mucins.

Helicobacter pylori and Campylobacter jejuni colonize the stomach and intestinal mucus, respectively. Using a combination of mucus-secreting cells, purified mucins, and a novel mucin microarray platform, we examined the interactions of these two organisms with mucus and mucins. H. pylori and C. jejuni bound to distinctly different mucins. C. jejuni displayed a striking tropism for chicken gastrointestinal mucins compared to mucins from other animals and preferentially bound mucins from specific avian intestinal sites (in order of descending preference: the large intestine, proximal small intestine, and cecum). H. pylori bound to a number of animal mucins, including porcine stomach mucin, but with less avidity than that of C. jejuni for chicken mucin. The strengths of interaction of various wild-type strains of H. pylori with different animal mucins were comparable, even though they did not all express the same adhesins. The production of mucus by HT29-MTX-E12 cells promoted higher levels of infection by C. jejuni and H. pylori than those for the non-mucus-producing parental cell lines. Both C. jejuni and H. pylori bound to HT29-MTX-E12 mucus, and while both organisms bound to glycosylated epitopes in the glycolipid fraction of the mucus, only C. jejuni bound to purified mucin. This study highlights the role of mucus in promoting bacterial infection and emphasizes the potential for even closely related bacteria to interact with mucus in different ways to establish successful infections.

The expression of mucin genes and the presence of mucin gene products in the equine endometrium.

In the equine reproductive tract, little is known about mucin gene expression and the role of mucins in barrier function and host-cell interaction. The aims of the study were to identify equine orthologs of mammalian mucin genes using available equine sequence data, to profile expression of equine orthologous mucin genes in the endometrium using reverse transcriptase polymerase chain reaction (RT-PCR), to determine spatial expression patterns of mucin genes using in situ hybridisation, and to confirm the presence of mucin gene products using Western blotting and equine-specific mucin antibodies during oestrus and dioestrus. While the mucin gene expression pattern in equine endometrium is similar to that of other mammals, several mucins appear to be uniquely expressed in this tissue (eqMUC3B, 7, 18, and 20) and one is hormonally regulated (eqMUC3B).

Molecular aspects of mucin biosynthesis and mucus formation in the bovine cervix during the periestrous period.

Mucus within the cervical canal represents a hormonally regulated barrier that reconciles the need to exclude the vaginal microflora from the uterus during progesterone dominance, while permitting sperm transport at estrus. Its characteristics change during the estrous cycle to facilitate these competing functional requirements. Hydrated mucin glycoproteins synthesized by the endocervical epithelium form the molecular scaffold of this mucus. This study uses the bovine cervix as a model to examine functional groups of genes related to mucin biosynthesis and mucus production over the periestrous period when functional changes in cervical barrier function are most prominent. Cervical tissue samples were collected from 30 estrus synchronized beef heifers. Animals were slaughtered in groups starting 12 h after the withdrawal of intravaginal progesterone releasing devices (controlled internal drug releases) until 7 days postonset of estrus (luteal phase). Subsequent groupings represented proestrus, early estrus, late estrus, metestrus, and finally the early luteal phase. Tissues were submitted to next generation RNA-seq transcriptome analysis. We identified 114 genes associated with biosynthesis and intracellular transport of mucins, and postsecretory modifications of cervical; 53 of these genes showed at least a twofold change in one or more experimental group in relation to onset of estrus, and the differences between groups were significant (P < 0.05). The majority of these genes showed the greatest alteration in their expression in the 48 h postestrus and luteal phase groups.

The activity and expression of chitinase in the equine lung and its activity in normal horses and animals with recurrent airway obstruction.

Recurrent airway obstruction (RAO) is a chronic inflammatory condition in equine lung, which may share a common immunological basis with human asthma, in which dysregulated Th2 responses occur. Mammals express chitinases and chitinase-like proteins, two of which are active enzymes, chitotriosidase and acidic mammalian chitinase (AMCase). Both enzymes are upregulated in a range of inflammatory conditions, including asthma. We investigated the activity of chitinase in bronchoalveolar lavage fluid from horses with and without RAO in response to organic dust challenges. No significant differences were found in activity, although in one study RAO animals had elevated chitinase activity that fell short of statistical significance. The pH optimum and pH lability of the activity was consistent with the presence of chitotriosidase. RT-PCR amplification of the mRNA encoding chitotriosidase and AMCase in normal equine lung showed that chitotriosidase, but not AMCase, is expressed in trachea, bronchi, and peripheral lung tissue. The gene for chitotriosidase was identified from the Equus caballus (horse) genome 1.1 database and its similarity to the same genes from other species was determined. The results of this study indicate that the involvement of chitotriosidase in RAO is uncertain.