Analysis of changes in the proteomic profile of porcine corpus luteum during different stages of the oestrous cycle: effects of PPAR gamma ligands.

CONTEXT: The corpus luteum (CL) is an endocrine gland in the ovary of mature females during the oestrous cycle and pregnancy. There is evidence of a relationship between the secretory function of the CL and PPARs. AIMS: In this study, we investigated the changes in the proteome of the CL in relation to the phase of the oestrous cycle and the impact of PPARγ ligands on the proteomic profile of the CL during the mid- and late-luteal phase of the oestrous cycle. METHODS: The porcine CL explants were incubated in vitro for 6h in the presence of PPARγ ligands (agonist pioglitazone, antagonist T0070907) or without ligands. Global proteomic analysis was performed using the TMT-based LC-MS/MS method. KEY RESULTS: The obtained results showed the disparity in proteomic profile of the untreated CL - different abundance of 23 and 28 proteins for the mid- and late-luteal phase, respectively. Moreover, seven proteins were differentially regulated in the CL tissue treated with PPARγ ligands. In the mid-luteal phase, one protein, CAND1, was downregulated after treatment with T0070907. In the late-luteal phase, the proteins SPTAN1, GOLGB1, TP53BP1, MATR3, RRBP1 and SRRT were upregulated by pioglitazone. CONCLUSIONS: Comparative proteomic analysis revealed that certain proteins constitute a specific proteomic signature for each examined phase. Moreover, the study showed that the effect of PPARγ ligands on the CL proteome was rather limited. IMPLICATIONS: The results provide a broader insight into the processes that may be responsible for the structural luteolysis of the porcine CL, in addition to apoptosis and autophagy.

Peroxisome proliferator-activated receptor gamma ligands regulate the expression of inflammatory mediators in porcine endometrium during LPS-induced inflammation.

Inflammation in the female reproductive system is one of the most common causes of reproductive dysfunction such as infertility, delay of the reproductive cycle and a reduction in reproductive efficiency. In this study, we aimed to investigate the effect of peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the expression of selected inflammatory mediators: nuclear factor kappa (NF-κB), interleukin (IL)-1ß, IL-6, IL-4, IL-10, leukemia inhibitory factor (LIF) and toll-like receptor 4 (TLR4) in the porcine endometrium treated in vitro with lipopolysaccharide (LPS) on days 10-12 and 18-20 of the estrous cycle. In addition, two experimental protocols were applied to evaluate the role of PPARγ agonists in ongoing and developing inflammation. Endometrial slices were incubated in vitro in the presence of LPS (to induce inflammation) and PPARγ agonists, prostaglandin J2 or pioglitazone (natural or synthetic, respectively). The study showed that PPARγ agonists decreased the expression of pro-inflammatory (NF-κB, TLR4, IL-6) and increased the abundance of anti-inflammatory mediators (IL-10) in the inflamed endometrium of pigs. These findings indicate anti-inflammatory properties of the tested ligands.

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

Inflammation is a biological response of the immune system, which can be triggered by many factors, including pathogens. These factors may induce acute or chronic inflammation in various organs, including the reproductive system, leading to tissue damage or disease. In this study, the RNA-Seq technique was used to determine the in vitro effects of peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the expression of genes and long non-coding RNA, and alternative splicing events (ASEs) in LPS-induced inflammation of the porcine endometrium during the follicular phase of the estrous cycle. Endometrial slices were incubated in the presence of LPS and PPARγ agonists (PGJ2 or pioglitazone) and a PPARγ antagonist (T0070907). We identified 169, 200, 599 and 557 differentially expressed genes after LPS, PGJ2, pioglitazone or T0070907 treatment, respectively. Moreover, changes in differentially expressed long non-coding RNA and differential alternative splicing events were described after the treatments. The study revealed that PPARγ ligands influence the LPS-triggered expression of genes controlling the DNA damage response (GADD45ß, CDK1, CCNA1, CCNG1, ATM). Pioglitazone treatment exerted a considerable effect on the expression of genes regulating the DNA damage response.

PPARγ ligands modulate the immune response mediators in the pig myometrium - An in vitro study.

The current study was conducted with the aim to investigate effects of PPARγ ligands on synthesis of nuclear receptor κB (NF-κB) and selected cytokines (IL-1ß, IFNγ, TNFα, IL-4, IL-10, LIF) in the pig myometrium on days 14-15 of the estrous cycle (late-luteal phase) and days 14-15 of the gestational period (beginning of embryonic implantation). The myometrial slices were incubated in vitro for 6 h in medium containing PPARγ ligands, agonists: 15d-prostaglandin J2 or pioglitazone, and antagonist - T0070907. The mRNA transcript and protein abundances were evaluated in tissues and culture medium. During the estrous cycle, PPARγ ligands did not have an effect on the mRNA transcript abundance of the immune response mediators used for treatments. The IL-10 protein abundance in the tissue was less when there was inclusions of pioglitazone in the medium, while the treatment with T0070907 resulted in a larger abundance of NF-κB, IL-1ß (in the tissue) and IL-4 (in tissue and culture media). During the gestational period, pioglitazone or PGJ2 suppressed mRNA IFNγ and IL-10 transcript and protein abundances (in the tissue and culture media), whereas there was an enhanced NF-κB protein abundance (in the tissue). Treatment with T0070907 had diverse effects (e.g., for NFκB inhibited mRNA transcript abundance or enhanced protein abundance). The observed changes are related mainly in tissues from pregnant animals. Responses to PPARγ antagonist are indicative of the possible involvement of PPARγ-independent factors as well as ligand-independent activation of the receptor, ligand selectivity/functionality or tissue receptivity to the factors evaluated.

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

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 peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the transcriptome genes expression and alternative splicing in the porcine endometrium in the mid-luteal phase of the estrous cycle 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.

Peroxisome proliferator-activated receptor alpha regulates the expression of the immune response mediators in the porcine endometrium during the estrous cycle and early pregnancy.

PROBLEM: Cytokines are immune response mediators that play an important role in the regulation of reproductive functions. An association between cytokines and peroxisome proliferator receptors (PPARs) has been reported in various tissues, including the endometrium. The present study aimed to evaluate the impact of PPARα ligands on the expression of nuclear factor kappa B (NF-κB) and cytokines (interleukin [IL]-1ß, IL-4, IL-6, IL-8, IL-10, and LIF) in the porcine endometrium in different reproductive stages. METHODS OF STUDY: Endometrial slices were collected from gilts on days 10-12 or 14-16 of the estrous cycle and pregnancy. Endometrial tissue explants were incubated in vitro in the presence or absence of PPARα agonist WY-14643 and antagonist MK886. Expression of mRNA and protein for NF-ĸB and selected cytokines was evaluated by real-time PCR and immunoblot. RESULTS: PPARα agonist WY-14643 decreased the mRNA expression of NF-κB in most of the analyzed stages (excluding days 10-12 of the estrous cycle), but increased the expression of NF-κB protein (excluding days 14-16 of pregnancy). The WY-14643 increased expression of IL-1ß and IL-6 proteins, and the mRNA expression of IL-8 and LIF, decreased IL-4 expression, and did not affect the mRNA and protein expression of IL-10. CONCLUSION: The obtained results demonstrate that PPARα is involved in the regulation of NF-κB and cytokine expression in the porcine endometrium. PPARα ligands exert a varied influence on immune system components, which could be attributed to differences in the receptivity of porcine endometrial tissue during the reproductive cycle.

PPARß/δ ligands regulate the expression of immune response mediators in the porcine endometrium - An in vitro study.

The peroxisome proliferator-activated receptor (PPAR) ß/δ belongs to a group of nuclear receptors that act as transcription factors. PPAR ß/δ plays a significant role in the regulation of female reproductive processes. It has been demonstrated that PPARß/δ is expressed in mouse, rat and porcine endometrium during the estrous cycle and pregnancy. The current study aimed to investigate the effect of selected PPARß/δ ligands on the expression of nuclear factor kappa (NF-κB) and selected cytokines - interleukin (IL)-1ß, IL-6, IL-8, IL-4, IL-10, leukemia inhibitory factor (LIF), in the porcine endometrium on days 10-12 and 14-16 of the estrous cycle (mid- and late-luteal phases corresponding to the full activity and luteolysis of the corpus luteum, respectively) and pregnancy (maternal recognition of pregnancy and beginning of implantation, respectively). Endometrial slices were incubated in vitro in the presence of PPARß/δ agonist L-165,041 (1 or 10 µM) or antagonist GW9662 (10 µM). The expression of mRNA and protein of the immune response mediator in the tissues was determined by real-time PCR and Western Blot. In general, the PPARß/δ agonist inhibited endometrial NF-κB mRNA expression during all analyzed reproductive stages, but it did not change protein expression. In turn, the PPARß/δ antagonist increased NF-κB protein levels on days 10-12 of the estrous cycle or pregnancy. The presence of the PPARß/δ agonist stimulated mRNA expression of LIF, IL-1ß and IL-8 and decreased the expression of IL-6. The presence of PPARß/δ ligands had a varied effect on protein expression in different stages on the analyzed period. The obtained results indicate that PPARß/δ regulates the expression of endometrial NF-κB and selected cytokines in pigs. The effects of PPARß/δ ligands on immune response mediators varied subject to the reproductive status of females and could be associated with differences in endometrial receptivity.

Peroxisome proliferator-activated receptor gamma ligands affect NF-kB and cytokine synthesis in the porcine endometrium-An in vitro study.

PROBLEM: Cytokines, mediators of the immune response, are involved in the regulation of female reproductive processes during the estrous cycle and pregnancy. The present study aimed to investigate the effect of selected peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the expression of nuclear factor kappa B (NF-κB) and selected cytokines, such as interleukin (IL)-1ß, -4, -6, -8, -10, and the leukemia inhibitory factor, in the porcine endometrium on days 10-12 and 14-16 of the estrous cycle (mid- and late luteal phase, respectively) or pregnancy (maternal recognition of pregnancy and beginning of implantation, respectively). METHOD OF STUDY: Endometrial slices were incubated in vitro in the presence of PPARγ agonists, 15-deoxy-Δ12, 14-prostaglandin J2 or rosiglitazone, and PPARγ antagonist T0070907. mRNA and protein levels in tissues were determined by real-time PCR and Western blot. RESULTS: On days 10-12 of the estrous cycle and days 14-16 of pregnancy, PPARγ ligands enhanced the expression of NF-κB, mRNA cytokines, and/or proteins. During the late luteal phase of the estrous cycle (days 14-16) and maternal recognition of pregnancy (days 10-12), PPARγ ligands inhibited the expression of NF-κB, and they differentially affected the expression of mRNA and proteins of cytokines. CONCLUSION: Our results indicate that PPARγ is engaged in the endometrial synthesis of NF-κB and selected cytokines in pigs. The influence of PPARγ ligands on the tested components of the immune system varied subject to the physiological status of females, and it could be associated with differences in endometrial receptivity.