Intrauterine devices influence prostaglandin secretion by equine uterus: in vitro and in vivo studies.

BACKGROUND: Intrauterine devices (IUD) are used in the veterinary practice as the non-pharmacological method of oestrus suppression in mares. When placed in the uterus, IUD create a physical contact with the endometrium that mimics the presence of an equine embryo. However, the mechanism of their action has not been fully elucidated. The objective of the present study was to examine the effect of mechanical stimulation of IUD on mare`s endometrium in both in vitro and in vivo study. For this purpose, we demonstrated the effect of IUD on prostaglandin (PG) F2α and PGE2 secretion, and mRNA transcription of genes involved in PG synthesis pathway in equine endometrial cells in vitro. In the in vivo study, we aimed to compare short-term effect of IUD inserted on day 0 (oestrus) with day 5-6 post-ovulation (the specific time when embryo reaches uterus after fertilization) on PG secretion from equine endometrium. To determine the long-term effect on PG synthase mRNA transcription, a single endometrial biopsy was taken only once within each group of mares at certain time points of the estrous cycle from mares placement with IUD on days 0 or 5-6 post-ovualtion. RESULTS: We showed for the first time that the incubation of the endometrial cells with the presence of IUD altered the pattern of PG synthase mRNA transcription in equine epithelial and stromal endometrial cells. In vivo, in mares placement with IUD on day 0, PGE2 concentrations in blood plasma were upregulated between 1 and 6, and at 10 h after the IUD insertion, compared with the control mares (P < 0.05). Moreover, the decrease of PTGFS mRNA transcription on day 16- 18, associated with an elevation in PTGES mRNA transcription on day 20 -21 of the estrous cycle in endometrial biopsies collected from mares placement with IUD on days 5-6 suggest an antiluteolytic action of IUD during the estrous cycle. CONCLUSION: We conclude that the application of IUD may mimic the equine conceptus presence through the physical contact with the endometrium altering PG synthase transcription, and act as a potent modulator of endometrial PG secretion both in vitro and in vivo.

Bioinformatic analysis of endometrial miRNA expression profile at day 26-28 of pregnancy in the mare.

The establishment of the fetomaternal interface depends on precisely regulated communication between the conceptus and the uterine environment. Recent evidence suggests that microRNAs (miRNAs) may play an important role in embryo-maternal dialogue. This study aimed to determine the expression profile of endometrial miRNAs during days 26-28 of equine pregnancy. Additionally, the study aimed to predict target genes for differentially expressed miRNAs (DEmiRs) and their potential role in embryo attachment, adhesion, and implantation. Using next-generation sequencing, we identified 81 DEmiRs between equine endometrium during the pre-attachment period of pregnancy (day 26-28) and endometrium during the mid-luteal phase of the estrous cycle (day 10-12). The identified DEmiRs appear to have a significant role in regulating the expression of genes that influence cell fate and properties, as well as endometrial receptivity formation. These miRNAs include eca-miR-21, eca-miR-126-3p, eca-miR-145, eca-miR-451, eca-miR-491-5p, members of the miR-200 family, and the miRNA-17-92 cluster. The target genes predicted for the identified DEmiRs are associated with ion channel activity and sphingolipid metabolism. Furthermore, it was noted that the expression of mucin 1 and leukemia inhibitory factor, genes potentially regulated by the identified DEmiRs, was up-regulated at day 26-28 of pregnancy. This suggests that miRNAs may play a role in regulating specific genes to create a favorable uterine environment that is necessary for proper attachment, adhesion, and implantation of the embryo in mares.

Temperature Elevation during Semen Delivery Deteriorates Boar Sperm Quality by Promoting Apoptosis.

Semen delivery practice is crucial to the efficiency of artificial insemination using high-quality boar sperm. The present study aimed to evaluate the effect of a common semen delivery method, a Styrofoam box, under elevated temperatures on boar sperm quality and functionality and to investigate the underlying molecular responses of sperm to the temperature rise. Three pooled semen samples from 10 Duroc boars (3 ejaculates per boar) were used in this study. Each pooled semen sample was divided into two aliquots. One aliquot was stored at a constant 17 °C as the control group. Another one was packaged in a well-sealed Styrofoam box and placed in an incubator at 37 °C for 24 h to simulate semen delivery on hot summer days and subsequently transferred to a refrigerator at 17 °C for 3 days. The semen temperature was continuously monitored. The semen temperature was 17 °C at 0 h of storage and reached 20 °C at 5 h, 30 °C at 14 h, and 37 °C at 24 h. For each time point, sperm quality and functionality, apoptotic changes, expression levels of phosphorylated AMPK, and heat shock proteins HSP70 and HSP90 were determined by CASA, flow cytometry, and Western blotting. The results showed that elevated temperature during delivery significantly deteriorated boar sperm quality and functionality after 14 h of delivery. Storage back to 17 °C did not recover sperm motility. An increased temperature during delivery apparently promoted the conversion of sperm early apoptosis to late apoptosis, showing a significant increase in the expression levels of Bax and Caspase 3. The levels of phosphorylated AMPK were greatly induced by the temperature rise to 20 °C during delivery but reduced thereafter. With the temperature elevation, expression levels of HSP70 and HSP90 were notably increased. Our results indicate that a temperature increase during semen delivery greatly damages sperm quality and functionality by promoting sperm apoptosis. HSP70 and HSP90 could participate in boar sperm resistance to temperature changes by being associated with AMPK activation and anti-apoptotic processes.

Mare stromal endometrial cells differentially modulate inflammation depending on oestrus cycle status: an in vitro study.

The modulation of inflammation is pivotal for uterine homeostasis. Here we evaluated the effect of the oestrus cycle on the expression of pro-inflammatory and anti-inflammatory markers in a cellular model of induced fibrosis. Mare endometrial stromal cells isolated from follicular or mid-luteal phase were primed with 10 ng/mL of TGFß alone or in combination with either IL1ß, IL6, or TNFα (10 ng/mL each) or all together for 24 h. Control cells were not primed. Messenger and miRNA expression were analyzed using real-time quantitative PCR (RT-qPCR). Cells in the follicular phase primed with pro-inflammatory cytokines showed higher expression of collagen-related genes (CTGF, COL1A1, COL3A1, and TIMP1) and mesenchymal marker (SLUG, VIM, CDH2, and CDH11) genes; p < 0.05. Cells primed during the mid-luteal overexpressed genes associated with extracellular matrix, processing, and prostaglandin E synthase (MMP2, MMP9, PGR, TIMP2, and PTGES; p < 0.05). There was a notable upregulation of pro-fibrotic miRNAs (miR17, miR21, and miR433) in the follicular phase when the cells were exposed to TGFß + IL1ß, TGFß + IL6 or TGFß + IL1ß + IL6 + TNFα. Conversely, in cells from the mid-luteal phase, the treatments either did not or diminished the expression of the same miRNAs. On the contrary, the anti-fibrotic miRNAs (miR26a, miR29b, miR29c, miR145, miR378, and mir488) were not upregulated with treatments in the follicular phase. Rather, they were overexpressed in cells from the mid-luteal phase, with the highest regulation observed in TGFß + IL1ß + IL6 + TNFα treatment groups. These miRNAs were also analyzed in the extracellular vesicles secreted by the cells. A similar trend as seen with cellular miRNAs was noted, where anti-fibrotic miRNAs were downregulated in the follicular phase, while notably elevated pro-fibrotic miRNAs were observed in extracellular vesicles originating from the follicular phase. Pro-inflammatory cytokines may amplify the TGFß signal in the follicular phase resulting in significant upregulation of extracellular matrix-related genes, an imbalance in the metalloproteinases, downregulation of estrogen receptors, and upregulation of pro-fibrotic factors. Conversely, in the luteal phase, there is a protective role mediated primarily through an increase in anti-fibrotic miRNAs, a decrease in SMAD2 phosphorylation, and reduced expression of fibrosis-related genes.

The potential role of miRNAs and regulation of their expression in the development of mare endometrial fibrosis.

Mare endometrial fibrosis (endometrosis), is one of the main causes of equine infertility. Despite the high prevalence, both ethology, pathogenesis and the nature of its progression remain poorly understood. Recent studies have shown that microRNAs (miRNAs) are important regulators in multiple cellular processes and functions under physiological and pathological circumstances. In this article, we reported changes in miRNA expression at different stages of endometrosis and the effect of transforming growth factor (TGF)-ß1 on the expression of the most dysregulated miRNAs. We identified 1, 26, and 5 differentially expressed miRNAs (DEmiRs), in categories IIA (mild fibrosis), IIB (moderate fibrosis), and III (severe fibrosis) groups compared to category I (no fibrosis) endometria group, respectively (Padjusted < 0.05, log2FC ≥ 1.0/log2FC ≤ - 1.0). This study indicated the potential involvement of miRNAs in the regulation of the process associated to the development and progression of endometrosis. The functional enrichment analysis revealed, that DEmiRs target genes involved in the mitogen-activated protein kinases, Hippo, and phosphoinositide-3-kinase (PI3K)-Akt signalling pathways, focal adhesion, and extracellular matrix-receptor interaction. Moreover, we demonstrated that the most potent profibrotic cytokine-TGF-ß1-downregulated novel-eca-miR-42 (P < 0.05) expression in fibroblasts derived from endometria at early-stage endometrosis (category IIA).

5-Aza-2'-Deoxycytidine (5-Aza-dC, Decitabine) Inhibits Collagen Type I and III Expression in TGF-ß1-Treated Equine Endometrial Fibroblasts.

Endometrosis negatively affects endometrial function and fertility in mares, due to excessive deposition of type I (COL1) and type III (COL3) collagens. The pro-fibrotic transforming growth factor (TGF-ß1) induces myofibroblast differentiation, characterized by α-smooth muscle actin (α-SMA) expression, and collagen synthesis. In humans, fibrosis has been linked to epigenetic mechanisms. To the best of our knowledge, this has not been described in mare endometrium. Therefore, this study aimed to investigate the in vitro epigenetic regulation in TGF-ß1-treated mare endometrial fibroblasts and the use of 5-aza-2'-deoxycytidine (5-aza-dC), an epigenetic modifier, as a putative treatment option for endometrial fibrosis. Methods and Results: The in vitro effects of TGF-ß1 and of 5-aza-dC on DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), COL1A1, COL3A1, and α-SMA transcripts were analyzed in endometrial fibroblasts, and COL1 and COL3 secretion in a co-culture medium. TGF-ß1 upregulated DNMT3A transcripts and collagen secretion. In TGF-ß1-treated endometrial fibroblasts, DNA methylation inhibitor 5-aza-dC decreased collagen transcripts and secretion, but not α-SMA transcripts. Conclusion: These findings suggest a possible role of epigenetic mechanisms during equine endometrial fibrogenesis. The in vitro effect of 5-aza-dC on collagen reduction in TGF-ß1-treated fibroblasts highlights this epigenetic involvement. This may pave the way to different therapeutic approaches for endometrosis.

Antioxidants and Oxidants in Boar Spermatozoa and Their Surrounding Environment Are Associated with AMPK Activation during Liquid Storage.

Activation of the AMP-activated protein kinase (AMPK) has been demonstrated to be beneficial for boar sperm quality and functionality, while the underlying mechanism of AMPK activation of boar spermatozoa remains obscure. This study aimed to explore the effect of antioxidants and oxidants in boar spermatozoa and their surrounding fluid (SF) on the activation of AMPK during the liquid storage. Ejaculates from Duroc boars, routinely used for semen production, were collected and diluted to a final concentration of 25 × 106/mL. In experiment 1, twenty-five semen samples from eighteen boars were stored at 17 °C for 7 days. In experiment 2, three pooled semen samples created from nine ejaculates of nine boars were used, and each sample was treated with 0, 0.1, 0.2, and 0.4 µM/L H2O2 and stored at 17 °C for 3 h. Sperm quality and functionality, antioxidants and oxidants in boar spermatozoa and SF, the intracellular AMP/ATP ratio, and the expression levels of the phosphorylated AMPK (Thr172) were determined. Sperm quality significantly decreased with storage time in terms of viability (p < 0.05). Antioxidant and oxidant levels were markedly affected with storage time, with a decline in the SF total antioxidant capacity (TAC) (p < 0.05), SF malondialdehyde (MDA) (p < 0.05), and the sperm's total oxidant status (TOS), as well as a fluctuation in sperm superoxidase dismutase-like (SOD-like) activity (p < 0.05). The intracellular AMP/ATP ratio increased (p < 0.05) on day 4 and subsequently decreased to its lowest value on days 6 and 7 (p < 0.05). The phosphorylated AMPK levels increased from day 2 to day 7 (p < 0.05). Correlation analyses indicate that sperm quality during liquid storage was correlated to antioxidants and oxidants in spermatozoa and SF (p < 0.05), which were correlated to the phosphorylation of sperm AMPK (p < 0.05). Treatment with H2O2 induced damages in sperm quality (p < 0.05), a decline in antioxidant levels (SF TAC, p < 0.05; sperm SOD-like activity, p < 0.01), an increase in oxidant levels (SF MDA, p < 0.05; intracellular ROS production, p < 0.05), a higher AMP/ATP ratio (p < 0.05), and phosphorylated AMPK levels (p < 0.05) in comparison with the control. The results suggest that antioxidants and oxidants in boar spermatozoa and SF are involved in AMPK activation during liquid storage.

The Role of NF-κB in Endometrial Diseases in Humans and Animals: A Review.

The expression of genes of various proinflammatory chemokines and cytokines is controlled, among others, by the signaling pathway of the nuclear factor kappaB (NF-κB) superfamily of proteins, providing an impact on immune system functioning. The present review addresses the influence and role of the NF-κB pathway in the development and progression of most vital endometrial diseases in human and animal species. Immune modulation by NF-κB in endometritis, endometrosis, endometriosis, and carcinoma results in changes in cell migration, proliferation, and inflammation intensity in both the stroma and epithelium. In endometrial cells, the NF-κB signaling pathway may be activated by multiple stimuli, such as bacterial parts, cytokines, or hormones binding to specific receptors. The dysregulation of the immune system in response to NF-κB involves aberrant production of chemokines and cytokines, which plays a role in endometritis, endometriosis, endometrosis, and endometrial carcinoma. However, estrogen and progesterone influence on the reproductive tract always plays a major role in its regulation. Thus, sex hormones cannot be overlooked in endometrial disease physiopathology. While immune system dysregulation seems to be NF-κB-dependent, the hormone-independent and hormone-dependent regulation of NF-κB signaling in the endometrium should be considered in future studies. Future goals in this research should be a step up into clinical trials with compounds affecting NF-κB as treatment for endometrial diseases.

Inhibition of Myeloperoxidase Pro-Fibrotic Effect by Noscapine in Equine Endometrium.

Myeloperoxidase is an enzyme released by neutrophils when neutrophil extracellular traps (NETs) are formed. Besides myeloperoxidase activity against pathogens, it was also linked to many diseases, including inflammatory and fibrotic ones. Endometrosis is a fibrotic disease of the mare endometrium, with a large impact on their fertility, where myeloperoxidase was shown to induce fibrosis. Noscapine is an alkaloid with a low toxicity, that has been studied as an anti-cancer drug, and more recently as an anti-fibrotic molecule. This work aims to evaluate noscapine inhibition of collagen type 1 (COL1) induced by myeloperoxidase in equine endometrial explants from follicular and mid-luteal phases, at 24 and 48 h of treatment. The transcription of collagen type 1 alpha 2 chain (COL1A2), and COL1 protein relative abundance were evaluated by qPCR and Western blot, respectively. The treatment with myeloperoxidase increased COL1A2 mRNA transcription and COL1 protein, whereas noscapine was able to reduce this effect with respect to COL1A2 mRNA transcription, in a time/estrous cycle phase-dependent manner (in explants from the follicular phase, at 24 h of treatment). Our study indicates that noscapine is a promising drug to be considered as an anti-fibrotic molecule to prevent endometrosis development, making noscapine a strong candidate to be applied in future endometrosis therapies.

Post-Thaw Storage Temperature Influenced Boar Sperm Quality and Lifespan through Apoptosis and Lipid Peroxidation.

Cryopreservation deteriorates boar sperm quality and lifespan, which restricts the use of artificial insemination with frozen-thawed boar semen in field conditions. The objective of this study was to test the effects of post-thaw storage time and temperature on boar sperm survival. Semen ejaculates from five Landrace boars (one ejaculate per boar) were collected and frozen following a 0.5 mL-straw protocol. Straws from the five boars were thawed and diluted 1:1 (v:v) in BTS. The frozen-thawed semen samples were aliquoted into three parts and respectively stored at 5 °C, 17 °C, and 37 °C for up to 6 h. At 0.5, 2, and 6 h of storage, sperm motility, viability, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels and apoptotic changes were measured. Antioxidant and oxidant levels were tested in boar sperm (SPZ) and their surrounding environment (SN) at each timepoint. The results showed significant effects of post-thaw storage time and temperature and an impact on boar sperm quality (total and progressive motility, VCL, viability, acrosome integrity), early and late sperm apoptotic changes, and changes in MDA levels in SPZ and SN. Compared to storage at 5 °C and 37 °C, frozen-thawed semen samples stored at 17 °C displayed better sperm quality, less apoptotic levels, and lower levels of SPZ MDA and SN MDA. Notably, post-thaw storage at 17 °C extended boar sperm lifespan up to 6 h without obvious reduction in sperm quality. In conclusion, storage of frozen-thawed boar semen at 17 °C preserves sperm quality for up to 6 h, which facilitates the use of cryopreserved boar semen for field artificial insemination.

Metallopeptidades 2 and 9 genes epigenetically modulate equine endometrial fibrosis.

Endometrium type I (COL1) and III (COL3) collagen accumulation, periglandular fibrosis and mare infertility characterize endometrosis. Metalloproteinase-2 (MMP-2), MMP-9 and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) are involved in collagen turnover. Since epigenetic changes may control fibroproliferative diseases, we hypothesized that epigenetic mechanisms could modulate equine endometrosis. Epigenetic changes can be reversed and therefore extremely promising for therapeutic use. Methylation pattern analysis of a particular gene zone is used to detect epigenetic changes. DNA methylation commonly mediates gene repression. Thus, this study aimed to evaluate if the transcription of some genes involved in equine endometrosis was altered with endometrial fibrosis, and if the observed changes were epigenetically modulated, through DNA methylation analysis. Endometrial biopsies collected from cyclic mares were histologically classified (Kenney and Doig category I, n = 6; category IIA, n = 6; category IIB, n = 6 and category III, n = 6). Transcription of COL1A1, COL1A2, COL3A1, MMP2, MMP9, TIMP1, and TIMP2 genes and DNA methylation pattern by pyrosequencing of COL1A1, MMP2, MMP9, TIMP1 genes were evaluated. Both MMP2 and MMP9 transcripts decreased with fibrosis, when compared with healthy endometrium (category I) (P < 0.05). TIMP1 transcripts were higher in category III, when compared to category I endometrium (P < 0.05). No differences were found for COL1A1, COL1A2, COL3A1 and TIMP2 transcripts between endometrial categories. There were higher methylation levels of (i) COL1A1 in category IIB (P < 0.05) and III (P < 0.01), when compared to category I; (ii) MMP2 in category III, when compared to category I (P < 0.001) and IIA (P < 0.05); and (iii) MMP9 in category III, when compared to category I and IIA (P < 0.05). No differences in TIMP1 methylation levels were observed between endometrial categories. The hypermethylation of MMP2 and MMP9, but not of COL1A1 genes, occurred simultaneously with a decrease in their mRNA levels, with endometrial fibrosis, suggesting that this hypermethylation is responsible for repressing their transcription. Our results show that endometrosis is epigenetically modulated by anti-fibrotic genes (MMP2 and MMP9) inhibition, rather than fibrotic genes activation and therefore, might be promising targets for therapeutic use.

Molecular Mechanism of Equine Endometrosis: The NF-κB-Dependent Pathway Underlies the Ovarian Steroid Receptors' Dysfunction.

Endometrosis is a frequently occurring disease decreasing mares' fertility. Thus, it is an important disease of the endometrium associated with epithelial and stromal cell alterations, endometrium gland degeneration and periglandular fibrosis. Multiple degenerative changes are found in uterine mucosa, the endometrium. However, their pathogenesis is not well known. It is thought that nuclear factor-κB (NF-κB), a cell metabolism regulator, and its activation pathways take part in it. The transcription of the profibrotic pathway genes of the NF-κB in fibrotic endometria differed between the follicular (FLP) and mid-luteal (MLP) phases of the estrous cycle, as well as with fibrosis progression. This study aimed to investigate the transcription of genes of estrogen (ESR1, ESR2) and progesterone receptors (PGR) in equine endometria to find relationships between the endocrine environment, NF-κB-pathway, and fibrosis. Endometrial samples (n = 100), collected in FLP or MLP, were classified histologically, and examined using quantitative PCR. The phase of the cycle was determined through the evaluation of ovarian structures and hormone levels (estradiol, progesterone) in serum. The transcription of ESR1, ESR2, and PGR decreased with the severity of endometrial fibrosis and degeneration of the endometrium. Moreover, differences in the transcription of ESR1, ESR2, and PGR were noted between FLP and MLP in the specific categories and histopathological type of equine endometrosis. In FLP and MLP, specific moderate and strong correlations between ESR1, ESR2, PGR and genes of the NF-κB pathway were evidenced. The transcription of endometrial steroid receptors can be subjected to dysregulation with the degree of equine endometrosis, especially in both destructive types of endometrosis, and mediated by the canonical NF-κB pathway depending on the estrous cycle phase.

Collagen Type III as a Possible Blood Biomarker of Fibrosis in Equine Endometrium.

Collagen pathological deposition in equine endometrium (endometrosis) is responsible for infertility. Kenney and Doig's endometrial biopsy histopathological classification is the gold standard method for endometrosis evaluation, whereby blood biomarkers identification would be less invasive and could provide additional information regarding endometrosis diagnosis and fertility prognosis. This study aimed to identify blood biomarkers for endometrosis diagnosis (42 mares were used in experiment 1), and fertility assessment (50 mares were used in experiment 2). Reproductive examination, endometrial biopsy histopathological classification (Kenney and Doig) and blood collection were performed. Endometrium and serum collagen type I (COL1) and type III (COL3), and hydroxyproline concentrations were measured (ELISA). Serum COL3 cut-off value of 60.9 ng/mL allowed healthy endometria (category I) differentiation from endometria with degenerative/fibrotic lesions (categories IIA, IIB or III) with 100% specificity and 75.9% sensitivity. This cut-off value enabled category I + IIA differentiation from IIB + III (76% specificity, 81% sensitivity), and category III differentiation from others (65% specificity, 92.3% sensitivity). COL1 and hydroxyproline were not valid as blood biomarkers. Serum COL3 cut-off value of 146 ng/mL differentiated fertile from infertile mares (82.4% specificity, 55.6% sensitivity), and was not correlated with mares' age. Only COL3 may prove useful as a diagnostic aid in mares with endometrial fibrosis and as a fertility indicator.

Evolution of the Concepts of Endometrosis, Post Breeding Endometritis, and Susceptibility of Mares.

In this paper, the evolution of our understanding about post breeding endometritis (PBE), the susceptibility of mares, and events leading to endometrosis are reviewed. When sperm arrive in the uterus, pro-inflammatory cytokines and chemokines are released. They attract neutrophils and induce modulatory cytokines which control inflammation. In susceptible mares, this physiological defense can be prolonged since the pattern of cytokine release differs from that of resistant mares being delayed and weaker for anti-inflammatory cytokines. Delayed uterine clearance due to conformational defects, deficient myometrial contractions, and failure of the cervix to relax is detected by intrauterine fluid accumulation and is an important reason for susceptibility to endometritis. Multiparous aged mares are more likely to be susceptible. Untreated prolonged PBE can lead to bacterial or fungal endometritis called persistent or chronic endometritis. Exuberant or prolonged neutrophilia and cytokine release can have deleterious and permanent effects in inducing endometrosis. Interactions of neutrophils, cytokines, and prostaglandins in the formation of collagen and extracellular matrix in the pathogenesis of fibrosis are discussed. Endometritis and endometrosis are interconnected, influencing each other. It is suggested that they represent epigenetic changes induced by age and hostile uterine environment.

The NF-κB-signalling pathway in mare's endometrium infiltrated with the inflammatory cells.

Endometritis is an important issue decreasing mares' fertility. In the case of endometritis, both inflammatory cells infiltration and proinflammatory molecules production are regulated by various cellular and gene regulatory mechanisms, including the nuclear factor-κB (NF-κB)-dependent pathway. NF-κB-signalling pathway has been recently studied in the equine endometrium in the context of endometrosis. Thus, this study aimed to determine gene transcription of NF-κB subunits (RelA; NF-κB1; NF-κB2), proinflammatory molecules (MCP-1; IL-6) and hyaluronan synthases (HAS 1; HAS 2; HAS 3) in endometritis and compare them with the intensity and type of inflammatory cell infiltration. Endometrial samples, collected post-mortem from cyclic mares in oestrus or dioestrus, were classified histologically and examined using quantitative PCR. Transcription NF-κB subunits genes did not differ with either inflammatory intensity or type of inflammatory cell infiltration. Transcription of MCP-1 and IL-6 genes increased with the severity of inflammation, with the involvement of HAS 3 and HAS 2 genes, as opposed to HAS 1 genes. These proinflammatory molecules and hyaluronan synthases in the equine inflamed endometrium do not seem to be regulated by the NF-κB pathway. Hence, separate signalling pathways for the development and progression of equine endometritis and endometrosis may be suggested.

Equine Endometrosis Pathological Features: Are They Dependent on NF-κB Signaling Pathway?

Endometrosis is an important mares' disease which considerably decreases their fertility. As classic endometrial classification methods might be insufficient for tissue pathological evaluation, further categorization into active/inactive and destructive/non-destructive types was developed by Hoffmann and others. This study aimed to compare NF-κB pathway genes transcription among histopathological types of endometrosis, following Hoffmann and co-authors' classification. Endometrial samples, collected postmortem from cyclic mares (n = 100) in estrus or diestrus, were classified histologically and used for gene transcription assessment. Gene transcription of NF-κB subunits (RelA, NF-κB1, NF-κB2), pro-inflammatory molecules (MCP-1, IL-6), and hyaluronan synthases (HAS 1, HAS 2, HAS 3) was compared among endometrosis types (active, non-active, destructive, non-destructive). Most individual mRNA samples showed high expression of RelA, NF-κB1, and MCP-1 gene transcripts and the destructive type of endometrosis, simultaneously. The expression of RelA and NF-κB1 genes was higher in active destructive group than in the other groups only in the follicular phase, as well as being higher in the inactive destructive group than in the others, only in the mid-luteal phase. The increase in gene transcription of the NF-κB canonical activation pathway in destructive endometrosis may suggest the highest changes in extracellular matrix deposition. Moreover, the estrous cycle phase might influence fibrosis pathogenesis.

The Inhibitory Effect of Noscapine on the In Vitro Cathepsin G-Induced Collagen Expression in Equine Endometrium.

Cathepsin G (CAT) is a protease released by neutrophils when forming neutrophil extracellular traps that was already associated with inducing type I collagen (COL1) in equine endometrium in vitro. Endometrosis is a fibrotic condition mainly characterized by COL1 deposition in the equine endometrium. The objective was to evaluate if noscapine (an alkaloid for cough treatment with anti-neoplastic and anti-fibrotic properties) would reduce COL1A2 transcription (evaluated by qPCR) and COL1 protein relative abundance (evaluated by western blot) induced by CAT in equine endometrial explants from follicular and mid-luteal phases treated for 24 or 48 h. The explants treated with CAT increased COL1 expression. Noscapine decreased COL1A2 transcription at both estrous cycle phases, but COL1 relative protein only at the follicular phase, both induced by CAT. Additionally, the noscapine anti-fibrotic action was found to be more effective in the follicular phase. The CAT treatment caused more fibrosis at the longest period of treatment, while noscapine acted better at the shortest time of treatment. Our results showed that noscapine could act as an anti-fibrotic drug in equine endometrosis by inhibiting CAT in vitro. Noscapine offers a new promising therapeutic tool for treating fibrosis as a single non-selective agent to be considered in the future.

Enzymes Present in Neutrophil Extracellular Traps May Stimulate the Fibrogenic PGF2α Pathway in the Mare Endometrium.

Endometrosis, a fibrotic disease of mare endometrium, impairs uterine function. Prostaglandins (PG), despite modulating reproductive physiological functions, may also cause local pathological collagen deposition (fibrogenesis). We have previously shown that neutrophil extracellular traps (NETs) may also favor mare endometrosis. The aim of this study was to investigate the effect of enzymes present in NETs on PGF2α-pathway activation. Kenney and Doig's type I/IIA and IIB/III mare endometria, from follicular phase (FLP) and mid-luteal (MLP) phase, were cultured in vitro in the presence of NETs enzymes (elastase, cathepsin-G or myeloperoxidase). Production of PGF2α (EIA) and transcription (qPCR) of its synthases (PTGS2, AKR1C3) and receptor (PTGFR) genes were evaluated. PGF2α and PTGFR were influenced by endometrial category and estrous cycle phase. In FLP endometrium, NETs enzymes induced both high PGF2α production and/or PTGFR transcription. In MLP type I/IIA tissues, down-regulation of PTGFR transcripts occurred. However, in MLP type IIB/III endometrium, high levels of PTGFR transcripts were induced by NETs enzymes. As PGF2α-pathway activation facilitates fibrogenesis in other tissues, PGF2α may be involved in endometrosis pathogenesis. In the mare, the endocrine microenvironment of healthy and pathological endometrium might modulate the PGF2α pathway, as well as fibrosis outcome on endometrium challenged by NETs enzymes.

Noscapine Acts as a Protease Inhibitor of In Vitro Elastase-Induced Collagen Deposition in Equine Endometrium.

Endometrosis is a reproductive pathology that is responsible for mare infertility. Our recent studies have focused on the involvement of neutrophil extracellular traps enzymes, such as elastase (ELA), in the development of equine endometrosis. Noscapine (NOSC) is an alkaloid derived from poppy opium with anticough, antistroke, anticancer, and antifibrotic properties. The present work investigates the putative inhibitory in vitro effect of NOSC on collagen type I alpha 2 chain (COL1A2) mRNA and COL1 protein relative abundance induced by ELA in endometrial explants of mares in the follicular or mid-luteal phases at 24 or 48 h of treatment. The COL1A2 mRNA was evaluated by qPCR and COL1 protein relative abundance by Western blot. In equine endometrial explants, ELA increased COL 1 expression, while NOSC inhibited it at both estrous cycle phases and treatment times. These findings contribute to the future development of new endometrosis treatment approaches. Noscapine could be a drug capable of preventing collagen synthesis in mare's endometrium and facilitate the therapeutic approach.

Microvascularization and Expression of Fibroblast Growth Factor and Vascular Endothelial Growth Factor and Their Receptors in the Mare Oviduct.

The oviduct presents the ideal conditions for fertilization and early embryonic development. In this study, (i) vascularization pattern; (ii) microvascular density; (iii) transcripts of angiogenic factors (FGF1, FGF2, VEGF) and their receptors-FGFR1, FGFR2, KDR, respectively, and (iv) the relative protein abundance of those receptors were assessed in cyclic mares' oviducts. The oviductal artery, arterioles and their ramifications, viewed by means of vascular injection-corrosion, differed in the infundibulum, ampulla and isthmus. The isthmus, immunostained with CD31, presented the largest vascular area and the highest number of vascular structures in the follicular phase. Transcripts (qPCR) and relative protein abundance (Western blot) of angiogenic factors fibroblast growth factor 1 (FGF1) and 2 (FGF2) and vascular endothelial growth factor (VEGF), and their respective receptors (FGFR1, FGFR2, VEGFR2 = KDR), were present in all oviduct portions throughout the estrous cycle. Upregulation of the transcripts of angiogenic receptors FGF1 and FGFR1 in the ampulla and isthmus and of FGF2 and KDR in the isthmus were noted. Furthermore, in the isthmus, the relative protein abundance of FGFR1 and KDR was the highest. This study shows that the equine oviduct presents differences in microvascular density in its three portions. The angiogenic factors VEGF, FGF1, FGF2 and their respective receptors are expressed in all studied regions of the mare oviduct, in agreement with microvascular patterns.