Conserved and breed-specific differences in the cervical transcriptome of sheep with divergent fertility at the follicular phase of a natural oestrus cycle

Abstract

Background The outcome of cervical artificial insemination (AI) with frozen-thawed semen in sheep is limited by the inability of sperm to traverse the cervix of some ewe breeds. Previous research has demonstrated that cervical sperm transport is dependent on ewe breed, as sperm can traverse the cervix in greater numbers in some higher fertility ewe breeds. However, the molecular mechanisms underlying ewe breed differences in sperm transport through the cervix remain unknown. In this study, we aimed to characterise the cervical transcriptome of four European ewe breeds with known differences in pregnancy rates following cervical AI using frozen-thawed semen at the follicular phase of a natural oestrous cycle. Cervical post mortem tissue samples were collected from two Irish ewe breeds (Belclare and Suffolk; medium and low fertility, respectively) and from two Norwegian ewe breeds (Norwegian White Sheep (NWS) and Fur; high fertility compared to both Irish breeds) at the follicular phase of a natural oestrous cycle (n = 8 to 10 ewes per breed). Results High-quality RNA extracted from biopsies of the mid-region of the cervix was analysed by RNA-sequencing and Gene Ontology (GO). After stringent filtering (P < 0.05 and FC > 1.5), a total of 11, 1539 and 748 differentially expressed genes (DEGs) were identified in Belclare, Fur and NWS compared to the low fertility Suffolk breed, respectively. Gene ontology analysis identified significantly enriched biological processes involved in muscle contraction, extracellular matrix (ECM) development and the immune response. Gene co-expression analysis revealed similar patterns in muscle contraction and ECM development modules in both Norwegian ewe breeds, which differed to the Irish ewe breeds. Conclusions These breed-specific biological processes may account for impaired cervical sperm transport through the cervix in sheep during the follicular phase of the reproductive cycle. This novel and comprehensive dataset provides a rich foundation for future targeted initiatives to improve cervical AI in sheep.

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

“…The differential expression of multiple informative immune genes in this dataset supports the case for a divergent immune profile between ewe breeds – particularly between the NWS and Suffolk breeds. The response between ewe breeds previously reported during the follicular phase of a natural oestrous cycle [ 20 ], is again evident in the profiles detected in this study after the use of hormones for oestrous synchronisation, leading to the conclusion that oestrous synchronisation does not seem to be the principal factor of the known ewe breed differences in fertility.…”

“…All libraries were sequenced on an Illumina NovaSeq sequencer by Macrogen, Inc. (Seoul, Republic of Korea) where they were sequenced using an Illumina NovaSeq. Sequencing was performed for each sample at 2 × 150 bp paired end reads (50 M reads) as previously described [ 20 ].…”

“…Given that the aforementioned ewe breed differences in sperm transport across the cervix are likely due to subtle molecular differences in the cervix or its secretions and the fact that cervical AI is performed to a natural oestrous in Norway but to a synchronised oestrous across the rest of the world. We undertook an RNA-sequencing analysis to interrogate cervical gene expression in these ewe breeds at the synchronised cycle following our previous gene expression analysis of the same ewe breeds at the natural oestrous cycle [ 20 ] . Therefore, the aim of this study was to profile the transcriptome of the cervix in two Norwegian and two Irish ewe breeds, with known differences in pregnancy rates following cervical AI with frozen-thawed semen, at the follicular phase of a synchronised oestrous cycle.…”

Biochemical and molecular characterization of sialylated cervical mucins in sheep

“…All libraries were sequenced on an Illumina NovaSeq sequencer. Sequencing was performed for each sample at 2 × 150 bp paired end reads (50 M reads) as previously described [ 22 ].…”

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

“…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 ].…”