113 Specific Impacts of Mild Feed Restriction on Gene Expression of Endometrial Luminal, Glandular and Stromal Cells in Postpartum Dairy Cows

Abstract

Although numerous studies have examined global mRNA expression in the whole endometrium, very few studies aimed at characterising the global mRNA profile at specific cellular types within this tissue. In this study, we hypothesised that metabolic status during the postpartum period can affect specifically gene expression of endometrial luminal, glandular epithelial and stromal cells. To achieve this, endometrial biopsies were performed in 22 postpartum cows receiving either a control diet (n = 12, aiming for 35 kg/d of energy-corrected milk, ECM) or a mild restricted diet (n = 10, aiming for 25 kg/d of ECM). The diet affected the metabolic status of the cows as documented in Ntallaris et al. (2017 Theriogenology 90, 276-283). Three endometrial biopsies were collected for each cow between 12 and 15 days post-oestrus (oestrus was synchronized with CIDR and all cows had progesterone concentrations >5 ng mL⁻¹ at time of biopsy). Biopsies were frozen in Optimal Cutting Temperature compound and then 10-µm sections were obtained by using cryostat. Slides were fixed in ethanol and stained using cresol violet. Luminal and glandular epithelial cells as well as stromal cells were harvested by laser capture microdissection using the PixCell II LCM System. Following capture, mRNA was extracted from each cellular types using the Arcturus PicoPure RNA Isolation (Thermo Fisher Scientific, Waltham, MA, USA). A genome-wide transcriptomic profile of endometrial cells from biopsies was generated with RNAseq. Samples were sequenced in 50-length paired. The average number of reads across all samples was near 100 million. Reads were mapped on the UMD3.1 assembly of the Bos taurus genome using Tophat. Quantification of gene expression has been performed using HTSeq with annotations from Ensembl. The comparisons of interest have been performed using DESEqn 2 (R package) with the statistical method proposed by Love et al. (2014 Genome Biol. 15, 550). With an adjusted P-value of 0.05, 8, 571, and 2658 genes were found significantly differentially expressed between animals fed with low or control diets for glandular, stromal, and luminal cells respectively. These results provide novel insights into mechanisms regulating endometrium physiology in postpartum dairy cows. The number of differentially expressed genes according to diet was much higher in luminal epithelial and stromal cells than in glandular cells providing evidence that these cells express differential responses to the metabolic environment to which they are exposed. Therefore, transcriptomic analyses based only on full tissue, as possibly biased by differences in cell numbers of different types could be not fully relevant.

We acknowledge the financial support of ‘Prolific’ (EU grant KBBE; 31176) and RMUTSV (Thailand)