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

Abstract

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.

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

“…2007 , 2019 ). In a recent study, we found fewer sperm in the cervical crypts of Suffolk ewes (low fertility) compared with Belclare ewes (high fertility), which was associated with higher sialic acid content in the cervix of Suffolk ewes compared with Belclare ewes ( Richardson et al. 2019 ).…”

“…In the current study, sialyl-Tn antigen (Neu5Gcα2–6GalNAc, peak 18) was related to fertility, having lower abundance in Belclare ewes (medium fertility) than in Fur ewes (high fertility). However, Richardson et al. (2019 ) demonstrated increased amounts of Neu5Ac/Neu5Gc α2–6Gal- GalNAc in Suffolk ewes (low fertility) using a lectin assay.…”

The biological mechanisms regulating sperm selection by the ovine cervix

“…An investigation into the rheology of the mucus between the breeds found that Suffolk ewes tended to have higher elastic and complex moduli than that from Belclare ewes leading to greater mucus penetration, as assessed in vitro (Richardson et al 2011). Glycosylation of cervical mucins also varied between breeds, with low fertility breeds (Suffolk) containing a significantly higher sialic acid content in the cervical channels than high fertility breeds (Belclare), while in vitro, the addition of sialic acid to spermatozoa increased mucus penetration (Richardson et al 2019). The immunoprotection of spermatozoa against immune recognition in the female uterus has been shown to be mediated by sialic acid (Alkhodair et al 2018), and therefore, differential glycosylation levels may mediate higher immunoreactivity and lower fertility in vivo.…”

“…It is not known if spermatozoa interact with these cilia in a similar manner to the ciliated epithelial cells in the oviduct. The presence of folds along the FRT suggests that they may have evolved to accommodate sperm transit as evidenced by the presence of spermatozoa deep within the cervical channels in cervices from ewes recovered shortly after insemination with F/T semen (Richardson et al 2019). The flow of mucus within these crypts and folds is thought to differ in composition and be slower than the stronger flow in the lumen of the cervix, which is critical for the protection of the upper FRT against the infiltration of pathogens (Cone 2009).…”

Biochemical and molecular characterization of sialylated cervical mucins in sheep

“…Richardson et al. [ 17 ] reported that glycosylation of cervical mucins differs between Suffolk (low fertility) and Belclare (medium fertility), with higher sialic acid content in the cervical channels (where sperm progression occurs) of Suffolk compared with Belclare. In addition, there was higher expression of the sialyltransferase ST6GAL1 in the cervical channels of Suffolk compared with Belclare ewes, which supports the evidence of the role of the sialic acid in the inhibition of sperm transit through the cervix.…”