Molecular mechanisms underlying equine endometrial cup regression: a transcriptomic analysis

Abstract

The gonadotropin-secreting equine endometrial cups (EC) have a major role in pregnancy maintenance, but an intriguingly short lifespan. First seen in the maternal endometrium at days (d) 38-40 post-ovulation, the EC undergo degeneration via poorly understood mechanisms starting around d 60-70, with complete dehiscence at approximately 120-150 d of gestation. Pregnancy losses during the EC physiological lifespan and EC pathological persistence after abortion or uncomplicated parturition usually lead to irregular estrous cyclicity and subfertility, with only anecdotal and inconsistent therapeutic options currently available. A holistic understanding of the cellular signaling leading to EC regression may provide insight for development of effective therapies for retained or persistent EC. We therefore hypothesized that comparing the transcriptome of EC during development and regression could elucidate key regulators and molecular pathways leading to EC trophoblast cell demise. Samples of EC were dissected from the adjacent endometrium of pregnant pony mares during early development (DEC, 42-47 d post-ovulation, n = 5), and regression (REC, 96 – 120 d, n = 5). Next generation RNA sequencing was performed with Illumina NovaSeq6000 and reads mapped to EquCab3.0 (STAR-2.5.2b). Differentially expressed genes (DEGs) were identified with Cuffdiff-2.2.1 (FDR < 0.05). A total of 2,504 DEG were identified, being 1,648 upregulated and 856 downregulated in REC when compared to DEC. Pathway Analysis of DEGs was also performed (IPA, Qiagen). Interestingly, the main pathways affected in REC included oxidative phosphorylation (P = 1.08e-38);mitochondrial dysfunction (P = 2e-25); estrogen receptor signaling (P = 2.2e-10); Sirtuin signaling pathway (P = 9.64e-16), which has central regulatory roles in cell senescence and apoptosis; and Granzyme A Signaling (3.29e-15), associated with Cytotoxic T Lymphocyte and Natural Killer-mediated apoptosis via caspase-independent pathways. Upstream regulator analysis highlighted the roles of mitochondrial protein ubiquinol-cytochrome c reductase complex-3; rapamycin-insensitive companion of mTOR, an essential molecule in cellular homeostasis; Torin-1, a potent mTOR inhibitor; dead box protein 5, which may act as a co-activator of estrogen receptor signaling; and transducing-like enhancer protein-3, a member of a family of proteins that regulate cell fate through the Notch signaling pathway. Importantly, the main upstream regulators and downstream molecules are also involved in cancer cell biology (P = 1.32e-07 – 1.71e-122), including the downstream effectors of neoplastic and epithelial cell death secreted phosphoprotein 1 (Spp1), and secreted protein acidic and cysteine rich (Sparc). Along with aromatase, Spp1 and Sparc were upregulated in REC in comparison to DEC. In summary, we have highlighted key molecular pathways and regulators of EC regression, which merit further molecular and functional investigations. Since multiple identified molecules have also been targeted in human oncological treatments, further investigation of their effectiveness in hastening EC demise is warranted.