New Approach to the Cryopreservation of GV Oocytes and Cumulus Cells through the Lens of Preserving the Intercellular Gap Junctions Based on the Bovine Model.

Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus-oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes (GJA1, GJA4; BCL2, BAX) and gene-specific epigenetic marks (DNMT3A) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs.

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.

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.

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.

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.

Collagens and DNA methyltransferases in mare endometrosis.

Inflammation and fibroproliferative diseases may be modulated by epigenetic changes. Therefore, we suggest that epigenetic mechanisms could be involved in equine endometrosis pathogenesis. DNA methylation is one of the methods to evaluate epigenetics, through the transcription of methyltransferases (DNMT1, DNMT3A, DNMT3B). The correlation between DNMTs and collagen (COL) transcripts was assessed for the different Kenney and Doig's (Current Therapy in Theriogenology. Philadelphia: WB Saunders; 1986) endometrium categories. Endometrial biopsies were randomly collected from cyclic mares. Histological classification (category I, n = 13; II A, n = 17; II B, n = 12; and III, n = 7) and evaluation of COL1A2, COL3A1 and DNMTs transcripts by qPCR, were performed. Data were analysed by one-way analysis of variance (ANOVA), Kruskal-Wallis test and Pearson correlation. As mares aged, there was an increase in endometrium fibrosis (p < .01), and in DNMT1 mRNA (p < .001). Considering DNMT3B transcripts for each category, there was an increase with fibrosis (p < .05). No changes were observed for DNMT1 and DNMT3A transcripts. However, DNMT3A mRNA levels were the highest in all categories (p < .01). In category I endometrium, a positive correlation was observed for transcripts of all DNMTs in both COLs (p < .01). In category IIA, this correlation was also maintained for all DNMTs transcripts in COL1A2 (p < .05), but only for DNMT3B in COL3A1 (p < .05). In category IIB, there was a positive correlation between DNMT3B and COL3A1 (p < .05). In category III, a positive correlation was only observed between DNMT3B and COL3A1 (p < .05). Our results suggest that there is a disturbance in COLs and DNMTs correlation during fibrosis.

Elastase inhibition affects collagen transcription and prostaglandin secretion in mare endometrium during the estrous cycle.

We have shown that bacteria induce neutrophil extracellular traps (NETs) in mare endometrium. Besides killing pathogens, NETs may contribute for endometrosis (chronic endometrium fibrosis). Since elastase (ELA) is a NETs component that regulates fibrosis and prostaglandin (PG) output, the aim was to evaluate if inhibition of ELA would affect collagen 1 (COL1) transcription and PGs secretion by endometrium explants, in different estrous cycle phases. Follicular-FP (n = 8) and mid luteal-MLP (n = 7) phases explants were cultured for 24-48 hr with medium alone (Control), ELA (0.5 µg/ml,1 µg/ml), sivelestat - ELA inhibitor (INH,10 µg/ml), or ELA (0.5 µg/ml,1 µg/ml) + INH (10 µg/ml). COL1 gene transcription was done by qRT-PCR and PGE2 and PGF2 α determination in culture medium by EIA. In FP, at 24 hr, ELA0.5 increased COL1 transcription (p < 0.001) but its inhibition (ELA0.5 + INH10) decreased COL1 transcription (p < 0.01) and PGF2 α production (p < 0.05). Also, ELA0.5 + INH10 or ELA1 + INH10 raised PGE2 production (p < 0.01). At 48 hr, ELA1 increased COL1 transcription (p < 0.01) and PGF2 α production (p < 0.001), but its inhibition (ELA1 + INH10) decreased these actions (p < 0.01; p < 0.05, respectively). Besides, ELA1 + INH10 incubation increased PGE2 (p < 0.05). PGF2 α also augmented with ELA0.5 (p < 0.001), but lowered with ELA0.5 + INH10 (p < 0.01). In MLP, ELA0.5 up-regulated COL1 transcription (24 hr, p < 0.01; 48 hr, p < 0.001), but ELA0.5 + INH10 decreased it (24 hr, p < 0.05; 48 hr, p < 0.001). At 48 hr, incubation with ELA1 also increased COL1 transcription and PGF2 α production (p < 0.05), but PGF2 α production decreased with ELA1 + INH10 incubation (p < 0.05). PGE2 production was higher in ELA1 + INH10 incubation (p < 0.05). Therefore, ELA inhibition may reduce the establishment of mare endometrial fibrosis by stimulating the production of anti-fibrotic PGE2 and inhibiting pro-fibrotic PGF2 α.

Expressions of lipoprotein receptors and cholesterol efflux regulatory proteins during luteolysis in bovine corpus luteum.

The corpus luteum (CL) synthesises and secretes progesterone (P4), which is essential for the establishment and maintenance of pregnancy in mammals. P4 is synthesised from cholesterol. Cholesterol is internalised by low-density lipoprotein receptor (LDLR) and/or scavenger receptor B1 (SR-BI), and is effluxed by ATP-binding cassette (ABC) transporter A1 (ABCA1) and G1 (ABCG1). To test the hypothesis that lipoprotein receptors and ABC transporters are involved in functional luteolysis, we examined the expression of LDLR, SR-BI, ABCA1 and ABCG1 in bovine CL during the luteal stages and after injection of prostaglandin (PG) F2α on Day 10 after ovulation. Expression of LDLR and SR-BI mRNA and protein was lower in the regressed luteal than late luteal stage. Injection of cows with a PGF2α did not affect LDLR mRNA and protein levels in the CL. Although expression of SR-BI mRNA did not change, SR-BI protein expression decreased 12 and 24h after PGF2α injection. The overall findings of the present study suggest that the decreased expression of SR-BI induced by PGF2α is one of the factors responsible for the continuous decrease in P4 production during functional luteolysis.

The Effect of Coumestrol on Progesterone and Prostaglandin Production in the Mare: In Vitro and In Vivo Studies.

Coumestrol (Cou) is a plant-derived phytoestrogen that induces various pathologies in the female reproductive tract. Although effects of phytoestrogens on reproductive function in other species are well documented, their influence on progesterone (P4) and prostaglandin (PG) secretion in the mare is unknown. The aim of this study was to determine if Cou directly affects P4 and PG concentrations (in vivo) and endometrial PG secretion (in vitro) in the mare. In experiment 1, the mares (n = 4) were fed for 14 days on a diet containing increasing proportions of alfalfa pellets (250 g-1 kg/day). An additional 4 mares were fed a standard diet (control group). Sequential blood samples were obtained for 8 h after feeding on Days 13 and 14 (1 kg/day alfalfa pellets). Feeding the mares alfalfa pellets up-regulated PGE2 and 13,14-dihydro-15-ketoprostaglandin F2alpha (PGFM) and down-regulated P4 in the blood plasma compared to those in the control group (P < 0.05). In experiment 2, epithelial and stromal cells were exposed to E2 (10-9 M) or Cou (10-8 M) for 24 h. In the in vitro study, Cou increased PG secretion in epithelial and stromal cells (P < 0.05). In both types of endometrial cells, Cou up-regulated PTGS-2 protein expression (P < 0.05). Moreover, PGES and PGFS proteins were up-regulated by Cou in epithelial cells (P < 0.01). These results indicate that Cou can disturb reproductive function by affecting reproductive hormone secretion and altering the endometrial milieu through PG stimulation. Coumestrol therefore may impair physiologic regulation of the estrous cycle and early pregnancy.

Evidence for a PGF2α auto-amplification system in the endometrium in mares.

In mares, prostaglandin F2α (PGF2α) secreted from the endometrium is a major luteolysin. Some domestic animals have an auto-amplification system in which PGF2α can stimulate its own production. Here, we investigated whether this is also the case in mares. In an in vivo study, mares at the mid-luteal phase (days 6-8 of estrous cycle) were injected i.m. with cloprostenol (250 µg) and blood samples were collected at fixed intervals until 72 h after treatment. Progesterone (P4) concentrations started decreasing 45 min after the injection and continued to decrease up to 24 h (P < 0.05). In turn, 13,14-dihydro-15-keto-PGF2α (PGFM) metabolite started to increase 4h after an injection and continued to increase up to 72 h (P < 0.05). PGF receptor (PTGFR) mRNA expression in the endometrium was significantly higher in the late luteal phase than in the early and regressed luteal phases (P < 0.05). In vitro, PGF2α significantly stimulated (P < 0.05) PGF2α production by endometrial tissues and endometrial epithelial and stromal cells and significantly increased (P < 0.05) the mRNA expression of prostaglandin-endoperoxide synthase-2 (PTGS2), an enzyme involved in PGF2α synthesis in endometrial cell. These findings strongly suggest the existence of an endometrial PGF2α auto-amplification system in mares.

Proteomic and network analysis of pregnancy-induced changes in the porcine endometrium on Day 12 of gestation.

Conceptus attachment is a time-sensitive process that requires a synchronized uterine environment created by molecular changes in the endometrium in response to ovarian hormones and conceptus signals. Porcine conceptuses undergo rapid elongation and differentiation, and secrete estrogens that serve as maternal-recognition-of-pregnancy signals during the peri-implantation period (Days 11-12). Pregnancy-induced proteomic changes in the porcine endometrium were measured during this period using two-dimensional differential gel electrophoresis of endometrial protein lysates from Day-12 pregnant versus non-pregnant animals (n = 4 each). Forty-four differentially abundant proteins in the pregnant endometrium were identified by mass spectrometry. The pregnant endometrium was associated with a unique protein profile, revealed by principal component analysis. A pregnancy-dependent increase in the abundance of serpins, cofilin, annexin A2, aldose reductase, cyclophilin, protein disulphide isomerase A3, and peroxiredoxin 1 was observed. Western blotting for some of the selected proteins confirmed their enrichment during pregnancy. Ingenuity pathway analysis identified several functions specifically over-represented among the differentially abundant proteins in the pregnant endometrium, including calcium signaling, angiogenesis, leukocyte migration, and cell movement. Interleukin-1 beta and beta-estradiol were identified as upstream regulators of several high-abundance proteins from pregnancy. Therefore, signals from porcine conceptuses, such as estrogens, interleukin 1B, and epidermal growth factor, either alone or in coordination with other factors, prepare the uterus for implantation. Mol. Reprod. Dev. 83: 827-841, 2016 © 2016 Wiley Periodicals, Inc.

Leukotriene production profiles and actions in the bovine endometrium during the oestrous cycle.

We have previously shown the influence of leukotrienes (LTs) on reproductive functions in vivo: LTB4 is luteotrophic and supports corpus luteum function inducing PGE2 and progesterone (P4) secretion, whereas LTC4 is luteolytic and stimulates PGF2α secretion in cattle. The aim of this study was to examine expression and production profiles of LTs and their actions in the endometrium. LT receptors (LTB4R for LTB4 and CysLTR2 for LTC4), 5-lipoxygenase (LO), 12-LO synthase (LTCS) and LTA4 hydrolase (LTAH) mRNA and protein expression, as well as LT production were measured in bovine endometrial tissue during the luteal phases of the oestrous cycle. The action of LTs on uterine function was studied by measuring the level of PGs after stimulating uterine slices with LTs on Days 8-10 of the cycle. Expression of 5-LO and LTB4R mRNA and protein were highest on Days 2-4 of the cycle, while CysLTR2 and LTCS were highest on Days 16-18 (P<0.05). LTB4 concentration was highest on Days 2-4 of the cycle, whereas the greatest LTC4 level was on Days 16-18 (P<0.05). Both LTB4 and C4 increased the content of PGE2 and F2α in endometrial slices at a dose of 10(-7)M (P<0.05). In summary, mRNA expression and activation of receptors for LTB4 and production occur in the first part of the cycle, whereas LTC4 and its receptors predominate at the end of the cycle. The 12-LO and 5-LO pathways are complementary routes of LT production in the bovine uterus.

Galectin-3 contributes to luteolysis by binding to Beta 1 integrin in the bovine corpus luteum.

Luteolysis is characterized by a reduction in progesterone (P4) production and tissue degeneration in the corpus luteum (CL). One of major events during luteolysis is luteal cell death. Galectin-3, a ubiquitously expressed protein involved in many cellular processes, serves as an antiapoptotic and/or proapoptotic factor in various cell types. Although galectin-3 is detected in the bovine CL, its role remains unclear. The expression of galectin-3 in the bovine CL was higher at the regressed stage than at the other luteal stages. Galectin-3 was localized on luteal steroidogenic cells (LSCs). When cultured LSCs were exposed to prostaglandin F2alpha (PGF) for 48 h, the expression and secretion of galectin-3 increased. When the cultured LSCs were treated with galectin-3 for 24 h, cleaved caspase-3 expression was increased, and the cell viability was decreased, whereas P4 production did not change. Beta 1 integrin, a target protein of galectin-3, was expressed in bovine CL and possessed glycans, which galectin-3 binds. Furthermore, galectin-3 bound to glycans of luteal beta 1 integrin. The decreased cell viability of cultured LSCs by galectin-3 was suppressed by beta 1 integrin antibody. The overall findings suggest that the secreted galectin-3 stimulated by PGF plays a role in structural luteolysis by binding to beta 1 integrin.

Ovarian steroid-dependent tumor necrosis factor-α production and its action on the equine endometrium in vitro.

Tumor necrosis factor-α (TNF) is a cytokine that plays important roles in functions of the endometrium. The aims of this study were to determine whether (i) ovarian steroids modulate TNF production by endometrial cells (Experiment 1); (ii) TNF effects on prostaglandin (PG) production in cultured equine endometrial cells and tissue (Experiment 2). Epithelial and stromal cells were isolated from equine endometrium (Days 2-5 of the estrous cycle; n=20) and treated after passage 1. In Experiment 1, epithelial and stromal cells were exposed to progesterone (P4; 10(-7)M), 17-ß estradiol (E2; 10(-9)M) or P4+E2 (10(-7)/10(-9)M) for 24h. Then, TNF mRNA transcription was determined using Real-time PCR. Additionally, TNF protein production was investigated in response to ovarian steroids for 24h using Enzyme-Linked Immunosorbent Spot (EliSpot). In Experiment 2, epithelial and stromal cells and endometrial explants (mid-luteal phase of the estrous cycle; n=5) were exposed in vitro to TNF (10 ng/ml) and to oxytocin (OT; positive control; 10(-7)M) for 24h. The concentrations of PGE2 and PGF2α were determined using a direct enzyme immunoassay (EIA) method. The transcription of prostaglandin-endoperoxide synthase-2 (PTGS-2), prostaglandin E2 synthase (PGES) and PGF2α synthase (PGFS) mRNAs in the endometrial explants was determined using Real-time PCR. Results showed that TNF is produced by two types of equine endometrial cells and its production is up-regulated by ovarian steroids (P<0.05) in stromal cells and by P4 (P<0.05) and E2 (P<0.01) in epithelial cells. Epithelial and stromal cells can also produce PG in response to TNF. In endometrial explants, TNF stimulated PGE2 production to a large extent and PGF2α secretion to a lesser extent. These actions are mediated by up-regulation of PG synthases mRNA transcription. The study indicates that TNF production is closely related to ovarian steroid actions and that the interaction between TNF and PG regulates physiologic processes in the equine endometrium.

Expression of aldo-keto reductase 1C23 in the equine corpus luteum in different luteal phases.

Regression of the corpus luteum (CL) is characterized by a decay in progesterone (P4) production (functional luteolysis) and disappearance of luteal tissues (structural luteolysis). In mares, structural luteolysis is thought to be caused by apoptosis of luteal cells, but functional luteolysis is poorly understood. 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes P4 into its biologically inactive form, 20α-hydroxyprogesterone (20α-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20α-HSD activity. To clarify whether AKR1C23 is associated with functional luteolysis in mares, we investigated the expression of AKR1C23 in the CL in different luteal phases. The luteal P4 concentration and levels of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA were higher in the mid luteal phase than in the late and regressed luteal phases (P<0.05), but the level of 3ß-HSD protein was higher in the late luteal phase than in the regressed luteal phase (P<0.05). The luteal 20α-OHP concentration and the level of AKR1C23 mRNA were higher in the late luteal phase than in the early and mid luteal phases (P<0.05), and the level of AKR1C23 protein was also highest in the late luteal phase. Taken together, these findings suggest that metabolism of P4 by AKR1C23 is one of the processes contributing to functional luteolysis in mares.

Interleukins affect equine endometrial cell function: modulatory action of ovarian steroids.

The aim of the present study was to investigate the interaction between ovarian steroids, interleukins and prostaglandins (PG) in equine epithelial and stromal cells in vitro. In Experiment 1, cells were exposed to IL-1α (10 ng/mL), IL-1ß (10 ng/mL) or IL-6 (10 ng/mL) for 24 h and cell proliferation was determined using MTT. In Experiment 2, cells were exposed to progesterone (P4; 10(-7) M); 17-ß estradiol (E2; 10(-9) M) or P4+E2 for 24 h and later medium was replaced with a fresh one treated with IL-1α, IL-1ß or IL-6 (10 ng/mL, each) for 24 h. The oxytocin (OT; 10(-7) M) was used as a positive control. In Experiment 3, cells were exposed to P4 (10(-7) M), E2 (10(-9) M) or P4+E2 for 24 h and the IL receptor mRNAs transcription was determined using Real-time PCR. Prostaglandins concentration was determined using the direct enzyme immunoassay (EIA) method. Our findings reveal a functional linking between ovarian steroids and IL-stimulated PG secretion by equine endometrial cells. This interaction could be one of the mechanisms responsible for endometrial local orchestrating events during the estrous cycle and early pregnancy.

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.

Impairment of the interleukin system in equine endometrium during the course of endometrosis.

The aim of the study was to characterize endometrial mRNA transcription, immunolocalization, and protein expression of interleukin (IL) 1alpha, IL1beta, IL6, and IL1RI, IL1RII, and IL6Ralpha/beta in the course of endometrosis during the estrous cycle. Additionally, the influence of IL1alpha, IL1beta, and IL6 on prostaglandin (PG) secretion and PG synthase mRNA transcription in endometrial tissue during endometrosis was investigated. The endometrial samples were obtained at the early (n = 12), mid- (n = 12), and late (n = 12) luteal phases and at the follicular (n = 12) phase of the estrous cycle. Within each of these phases, there were four samples within each category I, II, and III of endometrium, according to the Kenney classification. In experiment 1, transcription of IL1alpha, IL1beta, IL6, and their receptor's (IL1RI, IL1RII, and IL6Ralpha/beta) mRNAs and their immunolocalization and protein expression were determined using real-time PCR and immunohistochemistry, respectively. In Experiment 2, endometrial samples (n = 5 samples within categories I, II, and III) were obtained for tissue culture in the midluteal phase of the estrous cycle. The endometrial tissues were stimulated with IL1alpha (10 ng/ml), IL1beta (10 ng/ml), IL6 (10 ng/ml), and oxytocin (positive control; 10⁻7 M) for 24 h. The PG concentration was determined using ELISA. In addition, transcription of PTGS-2, PGES, and PGFS mRNAs was determined using real-time PCR. ILs were found to regulate PG secretion via modulation of PG synthases in equine endometrium. The alterations in IL and the expression of their receptors, and in endometrial secretory functions, were observed during the course of endometrosis, and suggest serious changes in the endometrial microenvironment. The described disturbances may be closely related to impaired endometrial processes responsible for the subfertility or the infertility in endometrosis.

Role of tumor necrosis factor-α, interferon-γ and Fas-ligand on in vitro nitric oxide activity in the corpus luteum.

Normal reproductive function involves the expression of inflammatory mediators. Regarding the corpus luteum (CL), cytokines promote the cross-talk between immune, vascular and steroidogenic cells, among others. Moreover, TNF, IFNG and FASL were shown to regulate equine CL establishment and regression. We hypothesized that cytokines action on equine CL may be mediated by nitric oxide (NO), through the regulation of endothelial NO synthase (eNOS) expression. TNF increased eNOS mRNA level and NO metabolite (nitrite) production during CL growth. Cytokines combined action (TNF+IFNG+FASL) promoted eNOS protein upregulation in mid-CL and nitrite production in mid and late-CL. However, in late-CL, TNF alone decreased nitrite secretion. These results indicate that in equine CL, cytokines TNF, IFNG and FASL regulate NO activity, via eNOS expression modulation.

Cytokines and angiogenesis in the corpus luteum.

In adults, physiological angiogenesis is a rare event, with few exceptions as the vasculogenesis needed for tissue growth and function in female reproductive organs. Particularly in the corpus luteum (CL), regulation of angiogenic process seems to be tightly controlled by opposite actions resultant from the balance between pro- and antiangiogenic factors. It is the extremely rapid sequence of events that determines the dramatic changes on vascular and nonvascular structures, qualifying the CL as a great model for angiogenesis studies. Using the mare CL as a model, reports on locally produced cytokines, such as tumor necrosis factor α (TNF), interferon gamma (IFNG), or Fas ligand (FASL), pointed out their role on angiogenic activity modulation throughout the luteal phase. Thus, the main purpose of this review is to highlight the interaction between immune, endothelial, and luteal steroidogenic cells, regarding vascular dynamics/changes during establishment and regression of the equine CL.