Mastitis, inflammation of the mammary gland, is the most prevalent disease in dairy cattle that has a potential impact on profitability and animal welfare. Specifically designed multi-omics studies can be used to prioritize candidate genes and identify biomarkers and the molecular mechanisms underlying mastitis in dairy cattle. Hence, the present study aimed to explore the genetic basis of bovine mastitis by integrating microarray and RNA-Seq data containing healthy and mastitic samples in comparative transcriptome analysis with the results of published genome-wide association studies (GWAS) using a literature mining approach. The integration of different information sources resulted in the identification of 33 common and relevant genes associated with bovine mastitis. Among these, seven genes—CXCR1, HCK, IL1RN, MMP9, S100A9, GRO1, and SOCS3—were identified as the hub genes (highly connected genes) for mastitis susceptibility and resistance, and were subjected to protein-protein interaction (PPI) network and gene regulatory network construction. Gene ontology annotation and enrichment analysis revealed 23, 7, and 4 GO terms related to mastitis in the biological process, molecular function, and cellular component categories, respectively. Moreover, the main metabolic-signalling pathways responsible for the regulation of immune or inflammatory responses were significantly enriched in cytokine–cytokine-receptor interaction, the IL-17 signaling pathway, viral protein interaction with cytokines and cytokine receptors, and the chemokine signaling pathway. Consequently, the identification of these genes, pathways, and their respective functions could contribute to a better understanding of the genetics and mechanisms regulating mastitis and can be considered a starting point for future studies on bovine mastitis.

Various endometrial genes in ruminant ungulates are regulated by conceptus interferon tau (IFNT). However, the effect of each IFNT isoform has not been carefully evaluated. In this study, the effects of 2 IFNT isoforms, paralogs found in utero, and interferon alpha (IFNA) on uterine epithelial and Mardin-Darby bovine kidney (MDBK) cells were evaluated. Expression vectors of the bovine interferon (bIFNT) genes bIFNT1, bIFNTc1, and bIFNA were constructed, and recombinant bIFNs (rbIFNs) were produced by 293 cells. Bovine uterine epithelial or MDBK cells were cultured in the presence or absence of increasing concentrations of each rbIFN for 24, 48, or 72 h. Transcript levels of the IFN-stimulated genes (ISGs) ISG12, ISG15, MX1, and MX2 were analyzed using quantitative reverse transcription-polymerase chain reaction. These messenger RNAs were up-regulated by rbIFN in a time- and concentration-dependent manner. In the epithelial cells, the ISG12 transcript level increased at 48 h after rbIFN treatment but slightly decreased at 72 h, whereas the transcript level of ISG15 increased at 24 h and was maintained through 72 h. Expressions of MX1 and MX2 increased at 72 h after rbIFN treatment. MX1 expression increased in all treatment groups, but MX2 increased only by bIFNTc1. In MDBK cells, the expression of ISG12 was increased by bIFNT1 and bIFNTc1 after 24 and 72 h; however, it was unchanged by rbIFNA. ISG15 increased following the same pattern as that seen in uterine epithelial cells, and MX1 showed a similar expression pattern. MX2 expression was increased by bIFNTc1 treatment in uterine epithelial cells, and its expression was increased by both bIFNT1 and bIFNTc1 in MDBK cells. These results show that epithelial and MDBK cell responses to IFNs differ, suggesting that IFNs possess common functions, but may have acquired different functions following gene duplication.

To directly assess the biological role of oligosaccharides in recombinant equine chorionic gonadotropin (rec-eCG) functioning, cDNA encoding the full-length eCGβ-subunit was fused with the mature protein part of the α-subunit, and we examined the expression levels of deglycosylated eCG mutants, the ovulation rate for deglycosylated mutants in C57BL/6 mice. The characterizations of heterodimeric and tethered mutants were studied following their respective secretions in culture medium, molecular weight and ovulation in vivo. Rec-eCG variants containing mutations at glycosylation sites at Asn82 of the α-subunit (eCGβ/αΔ82) and Asn13 of the β-subunit (eCGβΔ13/α) were not efficiently secreted into the culture medium from transfected cells. Western blot analysis revealed that the rec-eCGβ/α proteins have an approximate broad range of molecular weights of 40–46 kDa. Three rec-eCG mutants—a deglycosylated site at Asn56 of the α-subunit (eCGβ/αΔ56), a deletion of the C-terminal region of the β-subunit (eCGβ-D/α), and the double mutant (eCGβ-D/αΔ56)—turned out to have clearly lower (approximately 4–23 kDa) molecular weights. Protein N-glycosydase F (PNGase F) treatment markedly decreased the molecular weight to approximately 2–10 kDa. Normal oocytes were significantly more abundant in the natural eCG–treated group than in mutant rec-eCG–treated groups. In particular, numbers of nonfuntional oocytes were remarkably lower in all rec-eCG groups. Our results indicate that the ovulation rates of oocytes are not affected by the deglycosylated rec-eCGβ/α mutant proteins. There are around 20% non-functional oocytes with natural eCG and only 2% with the rec-eCGs tested. These results provide insight into the molecular mechanisms underlying the production of rec-eCG hormones with excellent bioactivity in vivo.

Equine chorionic gonadotropin (eCG), which comprises highly glycosylated α-subunit and β-subunit, is a unique member of the glycoprotein hormone family as it elicits both follicle-stimulating hormone (FSH)-like and luteinizing hormone (LH)-like responses in non-equid species. To examine the biological function of glycosylated sites in eCG, the following glycosylation site mutants were constructed: eCGβ/αΔ56, substitution of Asn⁵⁶ of α-subunit with Gln; eCGβ-D/α, deletion of the O-linked glycosylation site at the carboxyl-terminal peptide (CTP) region of the β-subunit; eCGβ-D/αΔ56, double mutant. The recombinant eCG (rec-eCG) mutants were expressed in Chinese hamster ovary suspension (CHO-S) cells. The FSH-like and LH-like activities of the mutants were examined using CHO-K1 cells expressing rat lutropin/CG receptor (rLH/CGR) and rat FSH receptor (rFSHR). Both rec-eCGβ/α and rec-eCGβ/αΔ56 were efficiently secreted into the CHO-S cell culture medium on day 1 post-transfection. However, the secretion of eCGβ-D/α and eCGβ-D/αΔ56, which lack approximately 12 O-linked glycosylation sites, was slightly delayed. The expression levels of all mutants were similar (200–250 mIU/mL) from days 3 to 7 post-transfection. The molecular weight of rec-eCGβ/α, rec-eCGβ/αΔ56 and rec-eCG β-D/α were in the ranges of 40–45, 37–42, and 34–36 kDa, respectively. Treatment with peptide-N-glycanase F markedly decreased the molecular weight to approximately 5–10 kDa. Rec-eCGβ/αΔ56 exhibited markedly downregulated LH-like activity. The signal transduction activity of both double mutants was completely impaired. This indicated that the glycosylation site at Asn⁵⁶ of the α-subunit plays a pivotal role in the LH-like activity of eCG. Similarly, the FSH-like activity of the mutants was markedly downregulated. eCGβ-D/α exhibited markedly downregulated LH-like and FSH-like activities. Rec-eCGβ/α exhibits potent biological activity in cells expressing rLH/CGR and rFSHR. The findings of this study suggest that the LH-like and FSH-like activities of eCG are regulated by the N-linked glycosylation site at Asn⁵⁶ of the eCG α-subunit and/or by the O-linked glycosylation sites of the eCG β-subunit. These findings improved our understanding of the mechanisms underlying both LH-like and FSH-like activities of eCG.

Eel luteinizing hormone (eelLH) is composed of a common a-subunit and hormone specific beta-subunit, both of which contain asparagine-linked carbohydrate residues, located at positions 56 and 79 on the a-subunit and position 10 on the beta-subunit. The specific roles of the individual carbohydrate chains are poorly defined in eel. Thus, we characterized the biologically active single chains by fusing the a-subunit to the carboxyl terminal region of the eelLH beta-subunit. Site-directed mutagenesis of the three N-linked glycosylation sites was performed to examine the function of individual glycosylation sites in secretion and signal transduction. The absence of the Asn(79) N-linked sugar chain slightly reduced secretion in Chinese hamster ovary (CHO) cells. The expression of eelLH beta/alpha (wild-type) in CHO suspension cells was increased by approximately 2-fold higher than that of attached CHO cells. By western blotting analysis, the molecular weight of wild-type was found to be 32 kDa. Mutants (beta/alpha Delta 56, beta/alpha Delta 79, and beta Delta 10/alpha) of the oligosaccharide chain at a single site showed molecular weights that were reduced by approximately 10%. However, the double mutant (beta/alpha Delta 56.79) molecular weight was decreased by more than 20% compared to the wild-type. Enzymatic digestion of oligosaccharides using PNGaseF treatment showed that the molecular weights of all mutants, including wild-type, were reduced to 25 kDa. The results obtained in the absence of carbohydrates at Asn(56) of the a-subunit and at Asn(10) of the beta-subunit revealed their roles in signal transduction through the eelLH receptor. The EC50 value of the cAMP response at Asn(79) of the a-subunit was increased by 5-fold, whereas the maximum response was dramatically reduced to 17.8% of wild-type levels. Specifically, removal of the carbohydrates at double mutant (beta/alpha Delta 56.79) is approximately 85% to wild-type levels in biopotency. These results revealed the site specific roles of eelLH carbohydrate residues. Our data suggest that N-linked oligosaccharide chains play a pivotal role in biological activity through the eelLH receptor.