RNA-sequencing Based Analysis Of Bovine Endometrium During The Maternal Recognition Of Pregnancy

Abstract

Background: Reproductive efficiency is crucial to the production of food animals and overall profitability of the farm. The majority of pregnancy losses occur in the first month, especially around Day-19 of gestation, mainly due to the inability of the uterus to support conceptus growth or abnormal development of conceptus. Since the incidence of pregnancy failure does not occur in a single day of Day-19, then days 15-17 is a critical period for the maternal recognition and establishment of pregnancy. We hypothesize that RNA-Sequencing based analysis of bovine endometrial tissues during the critical period of maternal recognition of pregnancy will reveal important genes and biological pathways required for the conceptus growth and development. Objectives: Hence, the objectives of the current study are: 1) To identify the important differentially expressed genes (DEGs) and biological pathways in the bovine caruncular endometrium among the groups (Pregnant vs. Cyclic) and (Pregnant vs. Non-Pregnant), and 2) To validate the most highly up-regulated DEGs using quantitative polymerase chain reaction (qPCR). Methods: Grass-fed Angus heifers (2-3 years old) were used for sampling. The estrous cycles of heifers (n=21) were synchronized using the intramuscular injection of a Prostaglandin F2 alpha (on Day-0 and -11). Fifteen heifers were bred by natural mating at estrus. Endometrial samples were collected at Day 15-17 of gestation (pregnant), of estrous cycle (cyclic), and absence of conceptus (nonpregnant) heifers. Total RNAs were isolated and were subjected to high throughput RNA-sequencing (n=4/group). The genes with at least two-fold change, and Benjamini and Hochberg q-value <= 0.05 were considered as differentially expressed. The mRNA expression of selected candidate genes in the bovine endometrium was also validated using qPCR. Results: A total of 107 genes (pregnant vs. cyclic), and 98 genes (pregnant vs. Nonpregnant) were differentially expressed (FDR <0.05) in the pregnant endometrium. The most highly up-regulated genes in the pregnant endometrium were MRS2, CST6, FOS, VLDLR, ISG15, IFI6, MX2, C15H11ORF34, EIF3M, PENK, PRSS22, MS4A8, CLDN4, TINAGL1, and R3HDM1. Gene ontology analysis revealed that the biological process related to Type-1 interferon signaling (MX1, MX2, IFI6, IRF1, and ISG15), immune response (IL23A, and RSAD2), extracellular matrix organization (COL1A1, COL1A2, COL3A1, and TIMP2) and ion transporters (SLC34A2, SLC2A1, SLC16A11, SLC16A4 and ATP1B1) were significantly enriched in the pregnant endometrium. The qPCR results confirmed the significantly higher (P <0.05) mRNA expression of MRS2, CST6, FOS, VLDLR, ISG15, IFI6, MX2, C15H11ORF34, PRSS22, TINAGL1, and MS4A8 in the presence of conceptus in the bovine endometrium. Conclusions: Both the RNA-Seq and qPCR confirmed the differential expression of several pre-discovered and novel genes, and their biological pathways during the maternal recognition of pregnancy (Day 15-17 of gestation) compared to cyclic and non-pregnant endometrium. Interferon signaling, immune response, nutrient transporter, synthesis, and secretion of proteins are crucial pathways during the maternal recognition of pregnancy. Overall, this study identified the differentially expressed genes and their pathways in the pregnant caruncular endometrium compared to cyclic and non-pregnant. In this study, using RNA-sequencing, we found some novel genes (MRS2, C15H11ORF34, and PRSS22). The study demonstrated that the presence of conceptus on day 15-17 of gestation could actively affect the endometrial gene expression during the maternal recognition of pregnancy. In summary, this study provides a comprehensive dataset of transcripts associated with maternal recognition of pregnancy.