Abstract
During the period of maternal recognition of pregnancy (MRP) in the mare, the embryo needs to signal its presence to the endometrium to prevent regression of the corpus luteum and prepare for establishment of pregnancy. This is achieved by mechanical stimuli and release of various signaling molecules by the equine embryo while migrating through the uterus. We hypothesized that embryo’s signals induce changes in the endometrial gene expression in a highly cell type-specific manner. A spatiotemporal transcriptomics approach was applied combining laser capture microdissection and low-input-RNA sequencing of luminal and glandular epithelium (LE, GE), and stroma of biopsy samples collected from days 10–13 of pregnancy and the estrous cycle. Two comparisons were performed, samples derived from pregnancies with conceptuses ≥ 8 mm in diameter (comparison 1) and conceptuses ≤ 8 mm (comparison 2) versus samples from cyclic controls. The majority of gene expression changes was identified in LE and much lower numbers of differentially expressed genes (DEGs) in GE and stroma. While 1253 DEGs were found for LE in comparison 1, only 248 were found in comparison 2. Data mining mainly focused on DEGs in LE and revealed regulation of genes related to prostaglandin transport, metabolism, and signaling, as well as transcription factor families that could be involved in MRP. In comparison to other mammalian species, differences in regulation of genes involved in epithelial barrier formation and conceptus attachment and implantation reflected the unique features of equine reproduction at the time of MRP at the molecular level.
Highlights
The approach used in our study to unravel unknown MRP signaling in the mare was based mainly on three points: (i) up to date, it is not completely clear when MRP is occurring in the mare; (ii) it is expected that the endometrial response to conceptus signaling depends on the developmental stage of the conceptus; and (iii) previous studies on cyclic control samples collected from day 10 to day 13 showed quite similar gene expression on these days of the estrous cycle[26]
The average sizes of the recovered conceptuses gradually increased from day 10 to day 13 after ovulation, but with expected variability for the same day of P as it has been described for this period of rapid growth[27]
The further approach was based on performing two different comparative analyses by grouping samples with larger conceptuses (≥ 8 mm, comparison 1) and samples with smaller conceptuses (≤ 8 mm, comparison 2) and comparing both to the same controls (C10 and C13). This approach agrees with the assumption that larger conceptuses will induce a stronger endometrial response and revealing a higher number of DEGs in comparison 1 and a much lower number in comparison 2. This contrasts with Klohonatz et al.[12], where they found the same number of DEGs on days 9 and 11 and less on day 13
Summary
The aim of the present study was to perform a more extensive cell type-specific gene expression analysis by: (1) using an improved approach of the LCM and low-input RNAsequencing that provide high quality RNA samples and RNA-seq libraries, and (2) analyzing different time points of pregnancy from day 10 to 13 to reveal new insights into MRP in the mare
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