Predicting of molecules mediating an interaction between bovine embryos and uterine epithelial cells

Abstract

Embryo-maternal reproductive tract interactions are pivotal for successful pregnancy. The present study predicted the molecules modulating embryo-uterine communication by comparing two sets of differentially expressed genes (DEGs): DEGs in uterine epithelial cells (UECs) collected from the uterus with and without blastocysts and DEGs between blastocysts developed in vivo and in vitro. Cows were subjected to super ovulation (SOV), followed by insemination or non-insemination at estrus (SOV + AI and SOV cows). Seven days after estrus, the uterus was flushed to collect UECs, and the presence of blastocysts in the uterus was confirmed. UECs were subjected to RNA-Sequencing (RNA-Seq) to identify DEGs. Publicly available RNA-Seq data of in vivo and in vitro developed bovine blastocysts were used to determine DEGs. Then, using ingenuity pathway analysis, activated- and inhibited-upstream regulators (USRs) for UECs in blastocysts were compared with those for blastocysts developed in vivo. RNA-Seq of UECs revealed that the DEGs were associated with immune response and cell adhesion pathways. The activated and inhibited USRs of UECs derived from SOV+ AI cows overlapped with the activated and inhibited USRs of blastocysts developed in vivo. Overlapping activated USRs include leukemia inhibitory factor, interleukin 6, fibroblast growth factor-2, transforming growth factor beta-1, and epidermal growth factor. In conclusion, the present study predicted the molecules that potentially mediate communication between the developing embryo and the uterus in vivo and prepare the uterus for pregnancy.

Keywords: Blastocyst; Gene expression; Ingenuity pathway analysis; Upstream regulators; Uterine epithelial cells.

Fig. 1.

The cows were administered a controlled internal drug-releasing (CIDR) device containing progesterone and estradiol benzoate (EB). Five days after treatment, the cows received follicular stimulating hormone (FSH, total 30 IU; six decreasing doses at 12 h intervals). Forty-eight hours after the first dose of FSH, prostaglandin F2a (1 ml, PGF2α) was administered intramuscularly, and 12 h after the PGF2α treatment, the CIDR was removed. One days after the removal of the CIDR, the cows were treated with GnRH. One day after the GnRH treatment, cows in estrus were artificially inseminated. Uterine flushing was performed seven days after insemination to collect the specimens for analysis.

Fig. 2.

PCA of the RNA-seq data of UECs from SOV with or without AI. Black circles represent SOV without AI and while circles represent SOV with AI.

Fig. 3.

Design of experiments. Cows were subjected to super ovulation followed by artificial insemination (SOV + AI) or not (SOV). The two groups of uterine epithelial cells (UECs) were subjected to RNA-Seq. Differentially expressed genes (DEGs) and associated pathways were determined. In addition, upstream regulators of UECs of SOV + AI were predicted using Ingenuity Pathway Analysis (IPA). Upstream regulators of in vivo-developed blastocysts were predicted from DEGs obtained from publicly available RNA-Seq dataset of in vivo- and in vitro developed blastocysts. Comparison of the two groups of upstream regulators predicted the molecules potentially involved in embryo-uterine interactions. For example, 27 molecules including EGF, FGF2, Insulin, IL6, Insulin, and TGFB1 were predicted as overlapped activated upstream regulators. Number of overlapped activated- and inhibited upstream regulators was described as a ven diagram.