Transcriptome analysis of porcine endometrium after LPS-induced inflammation: effects of the PPAR-gamma ligands in vitro†

Abstract

Female fertility depends greatly on the capacity of the uterus to recognize and eliminate microbial infections, a major reason of inflammation in the endometrium in many species. This study aimed to determine the in vitro effect of PPARγ ligands on the transcriptome genes expression and alternative splicing in the porcine endometrium in the mid-luteal phase during LPS-stimulated inflammation using RNA-seq technology. The endometrial slices were incubated in vitro in the presence of LPS and PPARγ agonists—PGJ2 or pioglitazone and antagonist—T0070907. We identified 222, 3, 4 and 62 differentially expressed genes after LPS, PGJ2, pioglitazone or T0070907 treatment, respectively. In addition, we detected differentially alternative spliced events: after treatment with LPS—78, PGJ2–60, pioglitazone—52 or T0070907–134. These results should become a basis for further studies explaining the mechanism of PPARγ action in the reproductive system in pigs.

PPARγ regulates the expression of genes involved in the DNA damage response in an inflamed endometrium

“…Surprisingly, in the current study, both PPARγ ligands (agonist and antagonist) exerted similar inhibitory effects on the abundance of cyclin G1 mRNA. Such phenomena were frequently noted by our team and other researchers 19 , 22 . Several explanations are possible, but the most likely one is that intracellular metabolic pathways are activated independently of PPARγ.…”

“…Interestingly, the gene expression profile in LPS-treated endometrium was different in the follicular phase (current study) and the mid-luteal phase of the estrous cycle 22 . In our previous study, 222, 3, 4, and 62 DEGs were identified after treatment with LPS, PGJ 2 , pioglitazone or T0070907, respectively.…”

“…Our recent studies have shown that PPARγ is engaged in the synthesis of inflammatory mediators in the porcine endometrium under physiological conditions (during the luteal phase of the estrous cycle and early pregnancy) 19 . We have also demonstrated that PPARγ is involved in LPS-stimulated inflammation of the porcine endometrium during the mid-luteal phase of the estrous cycle 22 . PPARγ blocking by a specific antagonist exerted pro-inflammatory effects, and this mechanism involved various intracellular pathways, including the cellular response to IL-1, cell migration and granulocyte chemotaxis.…”

“…Twenty RNA samples with an RNA Integrity Number (RIN) > 7 were selected for future analysis. The library preparation and sequencing procedure was described previously 22 . In brief, the library was prepared with the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA).…”

“…Differentially expressed genes were validated by real-time PCR with the use of the AriaMx real-time PCR System (Agilent Technology, USA), as described previously 22 . Primer sequences (Supplemental Table S6 ) for reference and target genes ( CCL5, CCL19, CDK1, CCNA1, CCNG1, TNFAIP3 ) were designed using Primer Express Software 3 (Applied Biosystems, USA).…”