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

in Reproduction
Correspondence should be addressed to S Fair; Email: sean.fair@ul.ie

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.

Downloadable materials

  • Supplementary Table 1
  • Supplementary Figure 1: Glycosidase activity (log scale) in cervical mucus (mean &#x00B1; s.e.m). Ewes were slaughtered at 42 (Belclare n=7; Suffolk n=6) or 57 (Belclare n=8; Suffolk n=4) h post pessary removal and swabs were taken from the mid-cervical region for glycosidase analysis.

 

    Society for Reproduction and Fertility

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    Isolated cervical regions, comprising of regions from just after the cervical os (Region 1) to just before the uterus (Region 4).

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    Mean (with 95% C.I.) number of spermatozoa in a selection of cervical sections of Belclare (n = 18) and Suffolk (n = 14) ewes after cervical insemination with 200 × 106 frozen-thawed sperm. Ewes were slaughtered 4 h after cervical insemination with frozen-thawed semen and the cervix was dissected into eight regions along the length of the cervix. Four alternate cervical regions from the vaginal end (Region 1) to the uterine end (Region 4) were sectioned and stained with haematoxylin and eosin. The number of spermatozoa in four sections from each region was categorized according to location (cervical channels in the top panel, and cervical lumen in the bottom panel). Representative images of spermatozoa in cervical channels and lumen. Bar = 50 μm.

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    Representative images of sections of cervical tissue from Belclare and Suffolk ewes stained for sialic acid and fucose in the cervical channels and lumen, showing similar levels of fucose for both breeds and higher sialic acid levels in the cervical channels of Suffolk ewes compared with Belclare ewes. Sialic acid and fucose were visualized using the lectins, Sambucus nigra and Ulex europaeus, respectively. Bar = 200 μm.

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    Mean (with 95% C.I.) percentage staining for sialic acid (A) and fucose (B) in the cervical epithelial cells of channels vs the lumen (location). Belclare (n = 18) and Suffolk (n = 14) ewes were slaughtered 4 h post cervical insemination with frozen-thawed semen and the cervix was dissected into regions along the length of the cervix. The proportion of sialic acid and fucose in the cervical epithelium was measured, using Sambucus nigra and Ulex europaeus lectins, respectively, in regions 1, 2, 3 and 4 (see ‘Materials and methods’ section) and these data are combined here.

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    Mean (± s.e.m.) levels of Neu5Gc and Neu5Ac in cervical mucus from Belclare and Suffolk ewes as analysed using high performance liquid chromatography (HPLC). Mucus was collected from Belclare and Suffolk ewes at 42 h (n = 13) or 57 h (n = 12) post pessary removal. Mucus was purified from six ewes (three per breed) and the remainder pooled according to breed and time post pessary removal. As time post pessary removal was not significant, data from both time points were pooled within the ewe breed.

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    Mean log number of spermatozoa (with 95% C.I.) penetrating cervical mucus following the addition of sialyllactose to (A) cervical mucus or (B) spermatozoa. Mucus was collected from ewes of various breeds (n = 11) at 57 h post pessary removal and pooled. In experiment (A) mucus was incubated with 6′-sialyllactose at various concentrations for 1 h at 4°C prior to a mucus penetration test. In (B) spermatozoa were incubated for 20 min with 6′-sialyllactose at various concentrations prior to a mucus penetration test.

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