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.

Cervical immune activation during the luteal phase may compromise subsequent trans-cervical ram sperm transport

“…In contrast to our earlier findings where only minor differences in gene expression were detected between the Irish ewe breeds (Suffolk and Belclare) compared with both Norwegian ewe breeds during the follicular phase of a natural [ 7 ] and synchronized estrous cycle [ 8 ], larger numbers of DEGs were expressed at the luteal phase. Although the FC in gene expression was not of a high magnitude for the majority of these genes, there was remarkable consistency in terms of the functional classes and genes differentially expressed, thereby adding important biological insights into mechanisms that may inhibit frozen–thawed sperm transport in the cervix of the Suffolk at the follicular phase.…”

“…This could be indicative of a suboptimal microbial environment in the cervix of the low-fertility Suffolk breed. Levels of COX1 were also higher in Suffolk than in Fur (but not in the other ewe breeds) as we reported previously at the follicular phase of a natural [ 7 ] and a synchronized [ 8 ] cycle. Although its role in the cervix is unknown, high levels of COX1 contribute to gastric mucosal defense [ 30 ].…”

“…A more recent study by our group of six European ewe breeds has shown no relationship between gross cervical anatomy, mucus properties and previously reported pregnancy rates following cervical AI with frozen–thawed semen [ 6 ]. However, we have identified extensive differences in the cervical gene expression between high- and low-fertility ewe breeds at the follicular phase of both a natural [ 7 ] and a synchronized [ 8 ] estrous cycle. It has been reported that cervical gene expression fluctuates between the phases of the estrous cycle [ 9 , 10 ].…”

Metabolic signature of cervical mucus in ewe breeds with divergent cervical sperm transport: a focus on metabolites involved in amino acid metabolism

“…Therefore, the increased levels of these products from bacterial metabolism could lead to an active immune response against bacteria as well as sperm compromised by cryopreservation. Although more studies are required to investigate this, in our previous RNA sequencing analysis of cervical tissue collected from the same animals (Abril-Parreño, et al, 2022a ), we identified a heightened immune response in the cervix of the low fertility Suffolk breed. In addition, higher expression of a membrane transporter of polyamines ( SLC22A16 ) and a polyamine oxidase ( PAOX ) was identified in Suffolk compared to Fur ewes (high fertility) at the follicular phase of a synchronised estrous cycle.…”

“…Lanthionine and N -acetyl methionine sulfoxide, both which are involved in methionine metabolism, were significantly increased in the low fertility Suffolk breed at the synchronised cycle compared to the natural cycle. Some Gram-positive bacterial species produce lantibiotics, which are lanthionine containing antimicrobial peptides (Cotter, et al, 2005 ) that have been postulated to be induced by pathogens and cytokines as part of the innate host defense response (Diamond, et al, 2009 ), indicating an active immune response in the cervix of the Suffolk as we previously reported using RNA-sequencing analysis (Abril-Parreño, et al, 2022a ).…”