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.
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L Richardson, J P Hanrahan, T Tharmalingam, S D Carrington, P Lonergan, A C O Evans, and S Fair
T Tharmalingam-Jaikaran, S W Walsh, P A McGettigan, O Potter, W B Struwe, A C O Evans, P M Rudd, and S D Carrington
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.
K Alkhodair, H Almhanna, J McGetrick, S Gedair, M E Gallagher, B Fernandez-Fuertes, T Tharmalingam, P B Larsen, E Fitzpatrick, P Lonergan, A C O Evans, S D Carrington, and C J Reid
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.