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

Abstract

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 (Galβ1-3[SO3-GlcNAcβ1-6]GalNAc), fucosylated glycan (GlcNAcβ1-3(Fucα1-2Galβ1-3)GalNAc) and core 4 glycan (GlcNAcβ1-3[GlcNAcβ1-6]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.

Keywords: O-glycans; UPLC; cervical mucus; fertility.

Fig. 1

(A) Representative FLR-HILIC-UPLC chromatograms of O-glycans released by microwave-assisted nonreductive β-elimination in sheep follicular phase cervical mucin (six European ewe breeds ordered top-down from lowest to highest fertility). Blank in black. Retention times of all identified glycan peaks expressed in glucose units (GUs). Major components of each peak shown. (B) Structural symbols for the O-glycans and common core structures found in this study.

Fig. 2

Pie charts showing the relative abundance of (A) of the core glycan types and (B) the main types of glycan modifications in sheep cervical mucins at the follicular phase, including pictograms of representative structures of each type of core and modification, respectively.

Fig. 3

Nested plots of significantly different O-glycans in cervical mucus of six ewe European ewe breeds collected at the follicular phase of a natural cycle. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. NWS = Norwegian White Sheep.

Fig. 4

Nested plot of core 6 glycosylation of cervical mucin from six European ewe breeds at the follicular phase of a natural cycle. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. NWS = Norwegian White Sheep.

Fig. 5

Nested plots of significantly differentially expressed O-glycans in cervical mucus of six European ewe breed collected at the follicular phase of a synchronized cycle. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. NWS = Norwegian White Sheep.

Fig. 6

(A) Nested plots of significant feature changes (fucosylation, glucuronylation, Neu5Ac, core 1 and core 4) in cervical mucus of six European ewe breeds at the follicular phase of a synchronized cycle. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. (B) PCA plot shows the three core 4-associated peaks (12, 41 and 50), which are driving the significant difference between Suffolk and Belclare (samples within each breed are indicated by the same colors).

Fig. 7

Overview of the main findings of the characterization of O-glycans in cervical mucus across six ewe breeds with divergent fertility at the follicular phase (around the time of ovulation) of the estrous cycle.