Steroid hormones, prostanoids, and angiogenic systems during rescue of the corpus luteum in pigs

Abstract

In order to characterize the transition of the corpora lutea (CL) from acquisition of luteolytic sensitivity to rescue of luteal function: i) the expression of 38 factors associated with steroids, prostanoids, and angiogenic systems and ii) concentrations of the main hormones responsible for maintenance of CL function in cyclic and pregnant pigs were examined. Additionally, the effect of prostaglandin (PG) E 2 and F 2a on luteal function during the estrous cycle and pregnancy was evaluated in vitro. Significantly up-regulated gene expression was revealed in CL collected on day 14 of the estrous cycle (CYP19A1, ESR2, PTGS2, HIF1A, and EDN1) and on days 12-14 of pregnancy (SCARB1, PGRMC1, STAR, HSD3B1, NR5A1, PTGFR, PTGER4, and VEGFA). Elevated concentrations of estradiol-17b and PGE 2 occurred in CL on days 12 and 14 of pregnancy respectively, while an increased intraluteal PGF 2a content was noted on day 14 of the estrous cycle. Both PGs increased the synthesis of progesterone by cultured luteal slices obtained on day 14 of pregnancy, in contrast to the action of PGF 2a on the corresponding day of the estrous cycle. PGE 2 stimulated cAMP production via PTGER2 and PTGER4, while PGF 2a elevated the content of CREB in cultured luteal slices from CL of pregnant pigs. In silico analysis showed that infiltration of lymphocytes and apoptosis of microvascular endothelium were activated in CL on day 12 of the estrous cycle vs pregnancy. Summarizing, an abundance of E 2 and PGE 2 during pregnancy regulates specific pathways responsible for steroidogenesis, the prostanoid signaling system and angiogenesis during rescue from luteolysis in porcine CL.

Clustered microRNAs: The molecular mechanism supporting the maintenance of luteal function during early pregnancy

“…Interestingly, miRNAs of the same cluster share thousands of common targets, including EDN1 , JUN , FOS, OXTR, PTGS2, HIF1A, CYP19A1 , and ESR2 (Figure 2B). The aforementioned genes were previously identified as less abundant in the porcine corpus luteum on Day 14 of pregnancy 13,14 . NR4A1, a gene encoding transcription factor found to be a negative regulator of steroidogenesis in the rodent corpus luteum and a marker of luteolysis in the bovine corpus luteum,36,37 was found among shared targets of miRNAs clustered in miR‐99b and miR‐532 clusters (Figure 2B).…”

“…Bioinformatics analysis using miRWalk 2.0 found thousands of targets for the studied miRNAs; thus, in our next approach, we aimed to identify among them genes known as important factors involved in the maintenance of luteal function. Therefore, lists of predicted targets for particular miRNAs were compared to a list of 50 genes analyzed in our previous studies,13,14 which were studied in the corpus luteum on the same days of the luteal phase and pregnancy. NR4A1 , which showed an elevated expression in the corpus luteum on Day 14 of the estrous cycle (Figure 3A,B), was included in this analysis.…”

“…In order to quantify qPCR results, real‐time PCR Miner Software was used to estimate the mean PCR amplification efficiency and Ct for each gene 25. The expression of analyzed genes was assessed relative to the most stable gene–– GAPDH as previously described 13,14…”

“…In order to examine the effect of luteotrophic hormones on the expression of miRNAs belonging to the ssc ‐miR‐99b cluster, the luteal slices were incubated in the culture medium M‐199 (negative control) in the presence of E 2 (1 ng/mL), PGE 2 (1 µM), or E 2 (1 ng/mL) + PGE 2 (1 µM) for 6, 12, and 24 hours. Doses for this treatment were selected based on our previous studies using porcine luteal tissue slices 14. Afterward, post‐incubation media were collected and stored for the further analysis of progesterone concentration, while tissue slices were washed in cold PBS and snap‐frozen in liquid nitrogen in order to isolate RNA and examine the expression of selected miRNAs as well as their potential targets.…”

“…We noticed a decreased expression of genes related to steroidogenesis and angiogenesis, while the elevated expression of some genes related to apoptosis in the corpus luteum on Day 14 of the estrous cycle. Simultaneously, plasma and intraluteal concentration of progesterone started to drop in cyclic vs pregnant animals 13,14. It indicates dynamic molecular and hormonal changes in both stages of the corpus luteum development.…”