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.

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.

Apoptosis, autophagic cell death, and necroptosis: different types of programmed cell death in bovine corpus luteum regression.

In mammals, the corpus luteum (CL) is a transient organ that secretes progesterone (P4). In the absence of pregnancy, the CL undergoes regression (luteolysis), which is a crucial preparation step for the next estrous cycle. Luteolysis, initiated by uterine prostaglandin F2α (PGF) in cattle, is usually divided into two phases, namely functional luteolysis characterized by a decline in P4 concentration and structural luteolysis characterized by the elimination of luteal tissues from the ovary. Programmed cell death (PCD) of luteal cells, including luteal steroidogenic cells (LSCs) and luteal endothelial cells (LECs), plays a crucial role in structural luteolysis. The main types of PCD are caspase-dependent apoptosis (type 1), autophagic cell death (ACD) via the autophagy-related gene (ATG) family (type 2), and receptor-interacting protein kinase (RIPK)-dependent programmed necrosis (necroptosis, type 3). However, these PCD signaling pathways are not completely independent and interact with each other. Over the past several decades, most studies on luteolysis have focused on apoptosis as the principal mode of bovine luteal cell death. Recently, ATG family members were reported to be expressed in bovine CL, and their levels increased during luteolysis. Furthermore, the expression of RIPKs, which are crucial mediators of necroptosis, is reported to increase in bovine CL during luteolysis and is upregulated by pro-inflammatory cytokines in bovine LSCs and LECs. Therefore, apoptosis, ACD, and necroptosis may contribute to bovine CL regression. In this article, we present the recent findings regarding the mechanisms of the three main types of PCD and the contribution of these mechanisms to luteolysis.

Assessment of stem cell factor expression and its c-KIT receptor in patients with vitiligo.

Introduction: Melanocytes show antigen expressions characteristic for the immune response effector cells, and the immune reactions in the skin, especially those with inflammation background, significantly affect the function of melanocytes. Among the cytokines produced by keratinocytes, the stem cell factor (SCF) plays a leading role in stimulating melanogenesis. Aim: To compare the expression level of stem cell factor (mSCF, pSCF) and the c-Kit receptor in the centre of the vitiligo patch and in the area of healthy skin adjacent to the vitiligo patch. Material and methods: The research material consisted of skin samples from a vitiligo lesion and from non-lesional skin adjacent to the vitiligo patch. Real Time PCR analysis (Applied Biosystems 7900HT) was performed to determine the expression level of the studied genes. Results: The studies showed a statistically significant increase in the amount of mSCF within the vitiligo patch compared to both healthy skin of patients with vitiligo and controls. In patients with vitiligo, c-Kit receptor expression was significantly decreased in the area of the lesional skin compared to the healthy skin of the same patient and the skin of the control group. Conclusions: The membrane-bound form of the SCF is overexpressed within the vitiligo skin, which may indicate the participation of mSCF in the stimulation of melanogenesis in response to melanocyte damage. Decreased expression of C-Kit receptor by melanocytes in the vitiligo patch disrupts the ligand-receptor interaction and may therefore be related to melanocytes dysfunction and/or loss.

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.

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.

Peroxisome proliferator-activated receptor expression in the canine endometrium with cystic endometrial hyperplasia-pyometra complex.

The most common uterine diseases in bitches occurring during diestrus are cystic endometrial hyperplasia (CEH) and pyometra. These diseases can coexist as CEH-pyometra complex (CEH-P). Their pathogenesis has not been fully explained. Peroxisome proliferator-activated receptors (PPARs) are important factors regulating mammalian reproductive function and inflammatory processes. Although there is a lack of data concerning the expression of PPARs in the canine endometrium during CEH and CEH-P, we hypothesized that they might be involved in the development of pathological disorders of the canine endometrium. Therefore, the current study was conducted to evaluate and compare PPARα, PPARδ and PPARγ mRNA expression using quantitative real-time PCR (qPCR) and their immunolocalization using immunohistochemistry (IHC) staining in the endometrium of clinically healthy bitches (control group; n = 8) and those with CEH (n = 8) or CEH-P (n = 8). For quantification, the arithmetic means of all intensities of immunostaining from the cells were measured with the optical density. PPARα, PPARδ and PPARγ were detected in the luminal epithelium, glandular epithelium and stromal cells. The mRNA transcription of PPARα was higher in the CEH group than in the control group (p < .05). Additionally, the mRNA expression and immunostaining intensities of PPARδ and PPARγ in the endometrium in the CEH-P group were downregulated relative to those in the control group (p < .05). Moreover, the serum progesterone concentration measured by direct radioimmunoassay was decreased in the CEH-P group compared to the control group (p < .001) and CEH group (p < .05). The obtained results indicate that PPARs are present in the canine endometrium and that their mRNA profile and intensity levels change under pathological conditions such as CEH and CEH-P. This finding may suggest a correlation between changes in the PPAR expression profile and hormonal disturbances, as well as the potential involvement of PPARs in signal transduction during inflammatory processes occurring in the endometrium during CEH-P. These results pave the way to further research into the role of PPARs in the pathogenesis of CEH and CEH-P in female dogs.

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.

Effects of cortisol on prostaglandin F2α secretion and expression of genes involved in the arachidonic acid metabolic pathway in equine endometrium - In vitro study.

Glucocorticoids (GCs) are known to play an important role in maintaining basal and stress-related homeostasis by interacting with endocrine mediators and prostaglandins (PGs). Although a growing body of evidence shows that GCs exert their regulatory action at a multitude of sites in the reproductive axis through corticosteroid receptors, little is known about the direct role of cortisol, an active form of GCs, in the equine endometrium. Thus, the study aimed to determine the effect of cortisol on PGF2α synthesis in the endometrial tissue and cells in vitro. In Exp.1, the immunolocalization and the expression of the glucocorticoid receptor (GCR) in the endometrium throughout the estrous cycle were established. In Exp. 2 and 3, the effects of cortisol on PGF2α secretion and transcripts associated with the arachidonic acid (AA) cascade in endometrial tissues, and cells were defined. Endometrial tissues obtained from the early, mid, and late luteal phases and the follicular phase of the estrous cycle were exposed to cortisol (100, 200, and 400 nM) for 24 h. Endometrial epithelial and stromal cells (early phase of estrous cycle) were exposed to cortisol (100 nM) for 24 h. Then, PGF2α secretion and transcripts associated with the AA cascade (PLA2G2A, PLA2G4A, PTGS2, and PGFS) were assessed. GCR was expressed in the cytoplasm and the nucleus in the luminal and glandular epithelium as well as in the stroma. Endometrial GCR protein abundance was up-regulated at the late luteal phase compared to the mid-luteal phase of the estrous cycle. Cortisol dose-dependently decreased PGF2α secretion, PLA2G2A and PLA2G4A transcripts in endometrial tissues. Additionally, cortisol treatment decreased PGF2α secretion from endometrial epithelial and stromal cells. Moreover, it affected PLA2G2A, PLA2G4A, and PTGS2 transcripts in endometrial stromal cells. These findings suggest that cortisol suppresses the synthesis of PGF2α by affecting the AA cascade in the equine endometrium during the estrous cycle.

Death Processes in Bovine Theca and Granulosa Cells Modelled and Analysed Using a Systems Biology Approach.

In this paper, newly discovered mechanisms of atresia and cell death processes in bovine ovarian follicles are investigated. For this purpose the mRNA expression of receptor interacting protein kinases 1 and 3 (RIPK1 and RIPK3) of the granulosa and theca cells derived from healthy and atretic follicles are studied. The follicles were assigned as either healthy or atretic based on the estradiol to progesterone ratio. A statistically significant difference was recorded for the mRNA expression of a RIPK1 and RIPK3 between granulosa cells from healthy and atretic follicles. To further investigate this result a systems biology approach was used. The genes playing roles in necroptosis, apoptosis and atresia were chosen and a network was created based on human genes annotated by the IMEx database in Cytoscape to identify hubs and bottle-necks. Moreover, correlation networks were built in the Cluepedia plug-in. The networks were created separately for terms describing apoptosis and programmed cell death. We demonstrate that necroptosis (RIPK-dependent cell death pathway) is an alternative mechanism responsible for death of bovine granulosa and theca cells. We conclude that both apoptosis and necroptosis occur in the granulosa cells of dominant follicles undergoing luteinisation and in the theca cells from newly selected follicles.

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.

The Effects of Prostaglandin E2 Treatment on the Secretory Function of Mare Corpus Luteum Depends on the Site of Application: An in vivo Study.

We examined the effect of prostaglandin (PG) E2 on the secretory function of equine corpus luteum (CL), according to the application site: intra-CL injection vs. an intrauterine (intra-U) administration. Moreover, the effect of intra-CL injection vs. intra-U administration of both luteotropic factors: PGE2 and human chorionic gonadotropin (hCG) as a positive control, on CL function was additionally compared. Mares were assigned to the groups (n = 6 per group): (1) an intra-CL saline injection (control); (2) an intra-CL injection of PGE2 (5 mg/ml); (3) an intra-CL injection of hCG (1,500 IU/ml); (4) an intra-U saline administration (control); (5) an intra-U administration of PGE2 (5 mg/5 ml); (6) an intra-U administration of hCG (1,500 IU/5 ml). Progesterone (P4) and PGE2 concentrations were measured in blood plasma samples collected at -2, -1, and 0 (pre-treatment), and at 1, 2, 3, 4, 6, 8, 10, 12, and 24 h after treatments. Moreover, effects of different doses of PGE2 application on the concentration of total PGF2α (PGF2α and its main metabolite 13,14-dihydro-15-keto-prostaglandin F2α- PGFM) was determined. The time point of PGE2, hCG, or saline administration was defined as hour "0" of the experiment. An intra-CL injection of PGE2 increased P4 and PGE2 concentrations between 3 and 4 h or at 3 and 12 h, respectively (p < 0.05). While intra-U administration of PGE2 elevated P4 concentrations between 8 and 24 h, PGE2 was upregulated at 1 h and between 3 and 4 h (p < 0.05). An intra-CL injection of hCG increased P4 concentrations at 1, 6, and 12 h (p < 0.05), while its intra-U administration enhanced P4 and PGE2 concentrations between 1 and 12 h or at 3 h and between 6 and 10 h, respectively (p < 0.05). An application of PGE2, dependently on the dose, supports equine CL function, regardless of the application site, consequently leading to differences in both P4 and PGE2 concentrations in blood plasma.

Comparison of Intra-CL Injection and Peripheral Application of Prostaglandin F2α Analog on Luteal Blood Flow and Secretory Function of the Bovine Corpus Luteum.

We investigated the effects of different doses of dinoprost injected directly into the bovine corpus luteum (CL) on (i) concentrations of progesterone (P4) and oxytocin (OT) in peripheral blood and (ii) mRNA levels of steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (P450scc), hydroxy-delta-5-steroid dehydrogenase, 3 ß- and steroid delta-isomerase 1 (HSD3B), and receptor-interacting protein kinases 1 and 3 (RIPK1, RIPK3) in CL tissue. Moreover, we examined the effects of dinoprost, injected intra-CL or administered intramuscularly (IM), on CL secretory function and on indicators of CL vascular network status: luteal tissue area (LTA), CL blood flow (CLBF), and the CLBF:LTA ratio (Adj. CLBF), in cows at the early and mid-luteal phases. In the Experiment 1, cows (day 10 of the cycle) were allocated to (i) an intra-CL injection of saline (control; n = 3); (ii) an intra-CL injection of dinoprost (1.25 mg; 2.5 mg, or 5 mg; n = 3 for each dose); (iii) an IM administration of saline (control; n = 3); or (iv) an IM administration of dinoprost (25 mg; positive control; n = 3). Concentrations of OT and P4 were measured in plasma samples. The mRNA expression of steroidogenesis- or necroptosis-related factors was determined in CL tissue 4 h after treatments. In Experiment 2, cows on day 4 (n = 12) or day 10 (n = 12) were allocated to (i) an intra-CL injection of dinoprost (2.5 mg/0.5 ml; n = 6), or (ii) IM administration of dinoprost (25 mg/5 ml; n = 6). Concentrations of P4 were measured in plasma samples. Luteal tissue area, CLBF, and Adj. CLBF were assessed based on color Doppler ultrasonography. An intra-CL injection of dinoprost increased OT and decreased P4 levels in the jugular vein (JV) in a dose-dependent manner in cows at the mid-luteal phase. Increased CLBF and Adj. CLBF, accompanied by reduced P4 levels, were observed 2 h after intra-CL dinoprost injection in middle-stage CL. Decreased STAR and increased RIPK1 and RIPK3 mRNA levels confirmed that 2.5 mg dinoprost injected directly into CL is the minimum dose that induces luteolytic cascade. Injection of dinoprost directly into the CL (at a dosage lower than recommended for peripheral application) results in a pattern similar to IM dinoprost administration.

The In Vitro Inhibitory Effect of Sivelestat on Elastase Induced Collagen and Metallopeptidase Expression in Equine Endometrium.

Neutrophil extracellular traps (NETs) fight endometritis, and elastase (ELA), a protease found in NETs, might induce collagen type I (COL1) accumulation in equine endometrium. Metallopeptidases (MMPs) are involved in extracellular matrix balance. The aim was to evaluate the effects of ELA and sivelestat (selective elastase inhibitor) on MMP-2 and MMP-9 expression and gelatinolytic activity, as well as the potential inhibitory effect of sivelestat on ELA-induced COL1 in equine endometrium. Endometrial explants from follicular (FP) and mid-luteal (MLP) phases were treated for 24 or 48 h with ELA, sivelestat, and their combination. Transcripts of COL1A2, MMP2, and MMP9 were evaluated by qPCR; COL1 protein relative abundance by Western blot, and MMP-2 and MMP-9 gelatinolytic activity by zymography. In response to ELA treatment, there was an increase in MMP2 mRNA transcription (24 h) in active MMP-2 (48 h), both in FP, and in MMP9 transcripts in FP (48 h) and MLP (24 h) (p < 0.05). Sivelestat inhibited ELA-induced COL1A2 transcripts in FP (24 h) and MLP (24 h, 48 h) (p < 0.05). The sivelestat inhibitory effect was detected in MMP9 transcripts in FP at 48 h (p < 0.05), but proteases activity was unchanged. Thus, MMP-2 and MMP-9 might be implicated in endometrium fibrotic response to ELA. In mare endometrium, sivelestat may decrease ELA-induced COL1 deposition and hinder endometrosis development.

Neutrophils, monocytes and other immune components in the equine endometrium: Friends or foes?

The innate and adaptive immune mechanisms are key components of regulation of reproductive physiological function and uterine disorders in equine uterus. The predominant immunological response in equine endometrium, characterized by an innate immune response, occurs under estrogens influence, in the follicular phase. Although, the increase in immune-related genes in equine endometrium during estrus has been suggested to play a role in uterine clearance after mating, immune cells and their product, i.e. cytokines play also mandatory role in the luteal development and maintenance, regression of equine corpus luteum, as well as in early pregnancy. Innate immune response is nonspecific and acts as the first line of defense against pathogens, foreign stimuli that include constituents of seminal fluid and local infections (endometritis). It has been recently established that a phagocytosis-independent mechanism to restrain bacteria, by means of neutrophil extracellular traps (NETs) formation, is involved in pathogenesis of in mare endometrial fibrosis (endometrosis). Moreover, persistent macrophages and mast cell activation could also have pro-fibrotic roles by secreting great amounts of pro-fibrotic factors and lead to fibrosis. This review will highlight the involvement of immune key components of the innate and adaptive immune system and their products in equine uterus and their contribution to reproductive physiological function and uterine disorders.

Matrix metallopeptidase expression and modulation by transforming growth factor-ß1 in equine endometrosis.

Equine endometrial fibrosis (endometrosis) is described as a degenerative chronic condition in the uterus. Its characteristic feature is excessive deposition of extracellular matrix (ECM) components around the endometrial glands and stroma. Although matrix metallopeptidases (MMPs) that mediate ECM turnover are important factors in the process of fibrosis, knowledge of their expression and regulation in endometrosis is limited. In other species, one of the important regulators of MMPs and tissue inhibitors of MMPs (TIMPs) is transforming growth factor (TGF)-ß1. The goal of this study was to determine (i) endometrial expression of MMPs and TIMPs during endometrosis and (ii) the effect of TGF-ß1 on expression of MMPs and TIMPs in equine endometrial fibroblasts and epithelial cells. In the follicular phase of the estrous cycle, MMP-1, -2, -9, and TIMP concentrations were higher during endometrosis than in healthy endometrium (P < 0.05). In the midluteal phase, MMP-3 concentration was lower in severe endometrosis compared to healthy endometrium (P < 0.05). In fibroblasts, TGF-ß1 upregulated MMP-1, -9, -13, and TIMP1, but downregulated MMP-3 secretion (P < 0.05). In epithelial cells, TGF-ß1 upregulated MMP-1, -9, -13, and TIMP secretion (P < 0.05). Endometrial expression of MMPs and TIMPs is altered during endometrosis. TGF-ß1 is a regulator of endometrial ECM remodeling via its effect on MMPs and TIMPs in equine endometrial fibroblasts and epithelial cells.

Changes in the expression and distribution of junction and polarity proteins in the porcine endometrium during early pregnancy period.

The maternal endometrium undergoes transformations during early pregnancy period to regulate the paracellular permeability across the epithelium and to enable adhesion between the trophoblast and endometrial epithelial cells. These transformations, under the influence of ovarian hormones, are associated with a partial loss in polarity of epithelial cell that is regulated by tight junctions (TJ), adherens junctions (AJ) and associated polarity protein complexes. This study examined the change in expression and distribution of proteins associated with TJs, AJs and apical partition defective (PAR) complex in porcine endometrium on Days 10, 13 and 16 of estrous cycle and pregnancy. Moreover, effect of hormones, progesterone (P4) and 17-ß estradiol (E2) on polar phenotype of endometrial epithelial cells was also investigated in vitro. There was pregnancy induced increase in gene and protein expression of TJ associated claudin-1 (CLDN1) on Day 13 of pregnancy as compared to corresponding day of estrous cycle and a decrease in TJ protein, zona occludens-1 (ZO-1) and PAR complex associated PAR6 expression levels on Day 16 of pregnancy (P < 0.05). Immunofluorescence studies revealed that on Days 10 and 13, TJ proteins occludin (OCLN) and ZO-1were primarily present in the apical region of lateral epithelial membrane. On Day 16 of pregnancy, whereas, OCLN redistributed into cytoplasm, ZO-1 decreased apically but was found to localize in the basal epithelium. The AJ proteins cadherin and ß-catenin were located at the apical epithelium on Day 10 of estrous cycle and pregnancy and Day 13 of estrous cycle. On Days 13 and 16 of pregnancy both proteins were expressed in the lateral membrane and co-localization between these proteins was observed on Day 16. On Day 10, PAR complex proteins PAR3, cell division control protein 42 (CDC42) and atypical protein kinase C (aPKC) ζ were observed in apical epithelium and in lateral membrane and CDC42 was also present in the cytoplasm of epithelium. Pregnancy induced redistribution of aPKCζ to cytoplasm and CDC42 to apical surface of luminal epithelium was observed on Days 13 and 16. The in vitro P4 and E2 treatment of epithelial cells mimicked in vivo results. These results indicate that P4 and E2 regulate alterations in epithelium that may facilitate embryo implantation and given the role of cadherin, catenin and CDC42 in embryo invasion, change in distribution of these proteins may limit the invasiveness of porcine conceptuses into the stroma.

Proteomic Analysis of Porcine Pre-ovulatory Follicle Differentiation Into Corpus Luteum.

The luteinization of the follicular cells, following a LH surge, causes extensive molecular and structural changes in preovulatory follicles (POF) that lead to ovulation and ultimate formation of the corpus luteum (CL). The objective of this study was to identify proteins expressed in porcine POF before the LH surge and a new CL formed, 2-3 days after ovulation, and evaluate proteome changes associated with formation of the CL from a follicle. We used 2D-gel electrophoresis-based proteomics and tandem mass spectrometry followed by a functional analysis using Ingenuity Pathway analysis (IPA) to evaluate functional pathways associated with the luteinization process. Protein lysates were prepared from isolated POFs and from the newly formed CL. A total of 422 protein spots were identified in both structures. A total of 15 and 48 proteins or their proteoforms were detected only in the POFs and CL, respectively. An IPA analysis of a POF proteome showed that most of the follicular proteins were involved in cellular infiltration, endoplasmic stress responses, and the protein ubiquitination pathway. Most of the early luteal proteins were associated with steroid metabolism, cell death and survival, free radical scavenging, and the protein ubiquitination pathway. A comparison of a follicular proteome with that of an early luteal proteome revealed that 167 identified proteins or their proteoforms were differentially regulated between POFs and the newly formed CL (p < 0.05 and a fold change of >1.8). Proteins that were significantly more abundant in follicles included cAMP-dependent protein kinase, histone binding protein RBBP4, reticulocalbin, vimentin, and calumenin; more abundant luteal proteins included albumin, farnesyl diphosphate synthase, serine protease inhibitors, elongation factor-1, glutaredoxin, and selenium-binding protein. Proteins that were significantly altered with luteal formation were found to be associated with cholesterol biosynthesis, cell death and survival, and acute phase response. Moreover, upstream regulators of differentially abundant proteins in CL were identified that included insulin growth factor-1, sterol regulatory element-binding transcription factor-1, and nuclear factor erythroid-derived 2. We have identified novel proteins that advance our understanding of (1) processes associated with differentiation of POFs into the CL, (2) possible mechanisms of luteal cell survival, and (3) pathways regulating steroidogenesis in the newly formed CL.