Ewe breed differences in the cervical transcriptome at the follicular phase of a synchronised oestrous cycle

Abstract

Background: Cervical artificial insemination (AI) with frozen-thawed semen results in unacceptably low pregnancy rates internationally. The exception is in Norway, where vaginal deposition of frozen-thawed semen to a natural oestrous routinely yields pregnancy rates in excess of 70%. Previous studies by our group has demonstrated that this is due to differences in cervical sperm transport. However, a potentially important contributory factor is that ewes are inseminated to a natural oestrous in Norway but to a synchronised oestrous across most of the rest of the world. In this study, we interrogated the gene expression of the sheep cervix of four ewe breeds with known differences in pregnancy rates following cervical AI using frozen-thawed semen under the effect of exogenous hormones to synchronise the oestrous cycle. These four ewe breeds (n = 8 to 11 ewes per breed) are from two countries: Ireland (Belclare and Suffolk; medium and low fertility, respectively) and Norway (Norwegian White Sheep (NWS) and Fur; both with high fertility compared to the Irish ewe breeds).

Results: RNA extracted from cervical biopsies collected from these breeds was analysed by RNA-sequencing and differential gene expression analysis. Using the low-fertility Suffolk breed as a reference level; 27, 1827 and 2641 genes were differentially expressed in Belclare, Fur and NWS ewes, respectively (P < 0.05 and FC > 1.5). Gene ontology (GO) analysis revealed that Fur and NWS had an up-regulation of enriched pathways involved in muscle contraction and development compared to Suffolk. However, there was a down-regulation of the immune response pathway in NWS compared to Suffolk. In addition, GO analysis showed similar expression patterns involved in muscle contraction, extracellular matrix (ECM) development and cell-cell junction in both Norwegian ewe breeds, which differed to the Irish ewe breeds.

Conclusions: This novel study has identified a number of conserved and breed-specific biological processes under the effect of oestrous synchronisation that may impact cervical sperm transport during the follicular phase of the reproductive cycle.

Keywords: Cervix; Oestrous synchronisation; Ovine; RNA-sequencing.

Fig. 1

Principal component analysis (PCA) plots show distribution of RNA-sequencing samples, where colours indicate the two ewe breeds in each comparison: Belclare (A), Fur (B) and Norwegian White Sheep (NWS) (C) compared to Suffolk at the follicular phase of a synchronised oestrous cycle

Fig. 2

Gene expression data presented as volcano plots at the follicular phase of a synchronised oestrous cycle for Belclare (A), Fur (B) and Norwegian White Sheep (NWS) (C) compared to the low fertility Suffolk breed using log values of the fold change and P-value. Each point represents a single gene, with those in black representing genes that survived the cut off thresholds of adjusted P <  0.05 and FC > 1.5 and red points represent genes with a P > 0.05

Fig. 3

Representative images of COX-1 expression (stained brown) in cervical tissue from Norwegian White Sheep (NWS), Fur and Suffolk at the follicular phase of a synchronised oestrous cycle using immunohistochemical COX-1 staining (magnification: 100x). A representative image of a negative control (without adding antibody) is also shown

Fig. 4

Gene co-expression analysis across the four ewe breeds (Suffolk, Belclare, Fur and NWS) at the follicular phase of a synchronised oestrous cycle. A Gene set enrichment analysis identified module 1, 2 and 3 to differ between Irish and Norwegian ewe breeds. B Over-representation analysis of genes showed the gene ontology terms in module 1, 2 and 3. The size of the circle is proportional to its normalized enriched score (NES) value. NWS = Norwegian White Sheep