Do arachidonic acid metabolites affect apoptosis in bovine endometrial cells with silenced PPAR genes?

Peroxisome proliferator-activated receptors (PPARs) are expressed in bovine uterus, and their agonists are arachidonic acid (AA) metabolites. We hypothesised that silencing of PPAR genes in bovine endometrial stromal cells (ESC) would change the intracellular signalling through PPAR and affect apoptosis after cell treatment with different AA metabolites. The study's aims are detection of apoptosis and examining the influence of prostaglandins and leukotrienes on apoptosis occurring in physiological ESC and cells with silenced PPAR (α, δ, and γ) genes. Silencing the PPARα and PPARδ genes in cells resulted in increased DNA fragmentation and mRNA and protein expression of caspase (CASP) -3 and -8 (P < 0.05). Neither DNA fragmentation nor the mRNA and protein expression of CASP3 and -8 in cells with silenced PPARγ gene were changed compared to physiological cells (P > 0.05). Among PPARs, PPARα and PPARδ appear to inhibit apoptosis, and AA metabolites, as PPAR agonists, modify this process in bovine ESC.

The influence of experimentally induced endometritis on the PPAR expression profile in the bovine endometrium.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear receptor superfamily. The PPARs activity can be modified e.g. by arachidonic acid metabolites. Escherichia coli (E. coli) is one of the main infectious agent of endometritis in dairy cows. We hypothesized that PPAR expression profile change in the bovine endometrium under the influence of LPS or E. coli. In experiment 1, endometrial explants were obtained post mortem from heifers and incubated without (control) or with LPS for 12, 24, 48, 72 and 96 h. In experiment 2, heifers were intrauterine infused with 0.9% NaCl (control) or with E. coli suspension in 0.9% NaCl. Endometrial biopsies were performed before (0 h) and 12, 24, 48, 72, 96 h after the infusions. In experiment 1, the increase in protein expression was observed for PPARα 48 h, for PPARß/δ 24, 72 and 96 h, whereas for PPARγ 12, 24 and 96 h after LPS treatment relative to the control groups. In experiment 2, the up-regulation in protein expression was observed for PPARα 48 and 72 h, for PPARß/δ 72 and 96 h, for PPARγ1 and PPARγ2 12 and 96 h after the intrauterine infusion with E. coli suspension compared to the control group. Changes in mRNA and protein PPAR expression profile in endometrial explants under the exposure of LPS indicate participation of these nuclear receptors in signal transduction during stimulation with LPS. The patterns of mRNA and protein PPAR expression in endometrial bioptates suggest that during experimentally induced endometritis in vivo, PPARs role may be connected both with enhancement of inflammation as well restoring physiological conditions in uterus.

The influence of arachidonic acid metabolites on PPAR and RXR expression in bovine uterine cells.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the superfamily of nuclear receptors. Three isoforms have been described: alpha (PPARα), delta (PPARδ), and gamma (PPARγ). PPARs heterodimerize with retinoid X receptors (RXRs: RXRα, RXRß and RXRγ). PPAR activity can be modulated by several ligands, including arachidonic acid (AA) metabolites. The aims of the study were to determine the effect of AA metabolites (prostaglandin [PG]E2, PGF2α, leukotriene [LT]B4, and LTC4) on mRNA (real-time PCR) and protein expression (Western blotting) of PPARα, PPARδ, and PPARγ, and on mRNA expression of RXRα, RXRß, and RXRγ, in bovine epithelial, stromal, and myometrial primary uterine cells and in bovine stromal cells with silenced PPAR genes (N = 10). All PPAR and RXR isoforms were expressed. Prostaglandins affected expression of PPARs only in stromal cells, whereas LTs modulated PPARγ mRNA expression in epithelial and myometrial primary cells. Blockade of signal transduction through PPARs prevented interactions between AA metabolites and PPARs and changed RXR expression comparing with primary stromal cells. In primary stromal uterine cells, mRNA expression of RXRs was higher than that of PPARs. In uterine stromal cells in which intracellular signaling through PPARs was blocked, RXRs seem to take over the role of PPARs and are pivotal for cell functions. This study revealed the reaction of PPARs and RXRs to agonists which naturally occur in the bovine uterus.

PPAR expression throughout the oestrous cycle in the bovine endometrium.

Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors that is composed of three isoforms: PPARα, PPARß/δ and PPARγ. The ratio of two isoforms of PPARγ (1 and 2) varies among both species and tissues. The activity of PPARs can be modified by a number of endogenous compounds, including arachidonic acid (AA), its eicosanoid metabolites and synthetic ligands. Many studies have revealed that PPARs are important in reproduction. We hypothesized that the profiles of PPARs expression vary in the bovine endometrium during certain days of the oestrous cycle. The aim of this study was to determine the immunolocalization, mRNA expression and protein expression of PPARα, PPARß/δ and PPARγ in the bovine endometrium throughout the oestrous cycle. Endometrial tissues were obtained post mortem from heifers on days 0 (oestrus phase, n = 6), 2-5 (early luteal phase, n = 6), 8-12 (mid-luteal phase, n = 6), 15-17 (late luteal phase, n = 6) and 19-21 (follicular phase, n = 6) of the oestrous cycle. PPARs immunolocalization was determined in the endometrium using immunohistochemistry. The mRNA and protein expression were evaluated by real-time PCR and Western blotting, respectively. The results were statistically analysed by one-way ANOVA followed by a Bonferroni test. Immunolocalization revealed the protein expression of PPARα, PPARß/δ and PPARγ in bovine endometrial structures throughout the oestrous cycle. PPARγ1 mRNA and protein expression fluctuated in the tissue depending on the studied days of the oestrous cycle, whereas the transcript and protein levels of PPARα and PPARß/δ did not display significant differences during the oestrous cycle. We observed the highest PPARγ1 mRNA expression at the oestrus phase and the lowest expression at the mid-luteal phase. During the late luteal and follicular phases, the mRNA and protein expression of PPARγ1 were detectable at similar levels compared to the early luteal and mid-luteal phases of the oestrous cycle. The overall results indicate the presence of PPARα, PPARß/δ and PPARγ in endometrial tissues, but the mRNA and protein expression of only PPARγ1 changed throughout the oestrous cycle, especially during the oestrus and mid-luteal phases. Our findings suggest an association between the expression of PPARs in the bovine endometrium and stage of the oestrous cycle that may be a consequence of changes in ovarian steroids.

Prolactin role in the bovine uterus during adenomyosis.

Adenomyosis is uterine dysfunction defined as the presence of endometrial glands within the myometrium. It is suggested that adenomyosis is estrogen-dependent pathology, and prolactin (PRL) also affects its development. In the uterus of ruminants, PRL stimulates gland proliferation and function. We hypothesized that in the bovine uterus, the expression of PRL and its receptors (PRLRs) during adenomyosis is disturbed and modulated by estradiol (E2). Uterine tissues were collected postmortem from cows; epithelial, stromal, and myometrial cells were isolated; and cultured and treated with E2. Material was divided into 2 groups: control (nonadenomyotic) and uteri with adenomyosis. In adenomyotic uterine tissue, PRL and its long-form receptor protein were increased, as determined by Western blotting. Immunohistostaining showed that during adenomyosis, PRL and its receptors are highly expressed in adenomyotic lesions. In cultured myometrial cells, protein expression of PRL and its receptors was increased during adenomyosis. Estradiol decreased PRLRs protein expression in nonadenomyotic stromal cells and in adenomyotic myometrial cells, and increased PRL secretion by adenomyotic myometrial cells. Moreover, PRL secretion was increased in untreated epithelial and stromal cells during adenomyosis. On the other hand, in stromal cells, PRLRs messenger RNA and protein expression was decreased, as determined by real-time PCR and Western blotting, respectively. Obtained results show that significant changes in PRL and PRLRs expression are observed in uterine tissue and cells during adenomyosis, which were also affected by E2. These data suggest involvement of PRL in adenomyosis development and the link between PRL and E2 actions during the dysfunction in cows.

In vitro cow uterine response to Escherichia coli, leukotrienes and cytokines.

The aim was to determine the dynamic profile of interactions between Escherichia coli (E. coli) and the actions of leukotrienes (LTs) and TNF and INFγ (cytokines) in the uterus in vitro. Uterine explants (N=6) were incubated for 2, 12 and 24h either as E. coli-treated (106CFU) or non-treated and/or with: LTB4 and C4 (10-6M, for both LTs), LTs receptors antagonists (aLTR; 10-6M) and/or cytokines (each 10ng/ml). Toll Like Receptor 4 (TLR4) mRNA expression increased in explants incubated with E. coli, cytokines and LTs after 2 and 12h and aLTR inhibited the effect of LTs in explants incubated with E. coli (P<0.05). IL-6 mRNA expression was up-regulated in E.coli-treated explants with cytokines after 2h and cytokines with LTs after 12h (P<0.05). E. coli increased prostaglandin (PG)E2 output after all examined time points, and PGF2α and IL-6 levels in E.coli-treated explants after 12 and 24h with cytokines, with LTs (P<0.05). aLTR inhibited LT stimulating action on PGs and IL-6 output in explants incubated with E. coli after 12 and 24h (P<0.05). LTs modify and enhance experimentally induced infection: TLR4 and IL-6 mRNA expression, IL-6 and PGs secretion, and cytokines participate in this process.

Estradiol Reduces Connexin43 Gap Junctions in the Uterus during Adenomyosis in Cows.

Adenomyosis is defined as the presence of glandular foci external to the endometrium of the uterus, either in the myometrium or/and perimetrium, depending on the progress of this dysfunction. To date, we showed that steroids secretion and prolactin expression and proliferative processes are disturbed during uterine adenomyosis in cows. During endometriosis in eutopic endometrium in women, gap junctions are down regulated. The transmembrane gap junction protein, connexin (Cx43) is necessary for endometrial morphological, biochemical and angiogenic functions. The aim of this study is recognition of adenomyosis etiology by determination of the role of Cx43 in this process. Immunolocalization and comparison of Cx43 mRNA and protein expression in healthy (N=9) and adenomyotic uterine tissue (N=9), and Cx43 mRNA expression (real time PCR) in uterine stromal - myometrium co-culture under 24-hour stimulation with 17-beta estradiol (10-7M) isolated from healthy (N=5) and adenomyotic (N=5) cows were determined. Cx43 was localized in healthy and adenomyotic uteri. mRNA and protein expression was down-regulated in uterine tissue in adenomyotic compared with healthy cows (p<0.05). Estradiol stimulated Cx43 mRNA expression in myometrial cell culture and co-culture of stromal and myometrial cells in adenomyotic compared with healthy cows (p<0.05). In summary, down-regulation of Cx43 expression in the junction zone might play an important role in pathogenesis of adenomyosis. Estradiol modulates gap junctions during adenomyosis.

Is adenomyosis a problem in reproduction and fertility?

Adenomyosis is defined as the presence of glandular foci beside the endometrium of uterus: in the myometrium and/or perimetrium depending on the progress of the disorder. So far, adenomyosis has been diagnosed in women and rodents, and studies conducted on cows have been rare. In this review we: (1) summarize the knowledge regarding adenomyosis, (2) compare the symptoms and aetiopathology between women and cows, (3) describe angiogenic uterine processes related to adenomyosis development and (4) outline the influence of adenomyosis on proper fertility processes in cattle (conception and fertility rates).