Transcriptomic profiling of mare endometrium at different stages of endometrosis.

In the current study, transcriptome profiles of mare endometrium, classified into categories I, IIA, and IIB according to Kenney and Doig, were compared using RNA sequencing, analyzed, and functionally annotated using in silico analysis. In the mild stage (IIA) of endometrosis compared to category I endometrium, differentially expressed genes (DEGs) were annotated to inflammation, abnormal metabolism, wound healing, and quantity of connective tissue. In the moderate stage (IIB) of endometrosis compared to category I endometrium, DEGs were annotated to inflammation, fibrosis, cellular homeostasis, mitochondrial dysfunction, and pregnancy disorders. Ingenuity pathway analysis (IPA) identified cytokines such as transforming growth factor (TGF)-ß1, interleukin (IL)-4, IL-13, and IL-17 as upstream regulators of DEGs associated with cellular homeostasis, metabolism, and fibrosis signaling pathways. In vitro studies showed the effect of these cytokines on DEGs such as ADAMTS1, -4, -5, -9, and HK2 in endometrial fibroblasts at different stages of endometrosis. The effect of cytokines on ADAMTS members' gene transcription in fibroblasts differs according to the severity of endometrosis. The identified transcriptomic changes associated with endometrosis suggest that inflammation and metabolic changes are features of mild and moderate stages of endometrosis. The changes of ADAMTS-1, -4, -5, -9, in fibrotic endometrium as well as in endometrial fibroblast in response to TGF-ß1, IL-4, IL-13, and IL-17 suggest the important role of these factors in the development of endometrosis.

The effect of basic fibroblast growth factor 2 on the bovine corpus luteum depends on the stage of the estrous cycle and modulates prostaglandin F2α action.

The roles of fibroblast growth factor 2 (FGF2) in the corpus luteum (CL) function and its modulatory effect on prostaglandin (PG) F2α during the bovine estrous cycle were studied using the following design of in vivo and in vitro experiments: (1) effects of FGF2 and FGF receptor 1 inhibitor (PD173074) on bovine CL function in the early (PGF2α-resistant) and mid (PGF2α-responsive) luteal stage in vivo, (2) the modulatory effect of FGF2 on PGF2α action during the luteal phase in vivo and (3) effects of FGF2 and PD173074 on bovine CL secretory function in vitro. Cows were treated by injection into the CL with: (1) saline (control), (2) FGF2, (3) PD173074, (4) FGF2 followed by intramuscular (i.m.) PGF2α, (5) PD173074 followed by i.m. PGF2α and (6) i.m. PGF2α as a positive control. For in vitro experiments, CL explants were treated with the aforementioned factors. Progesterone (P4) concentrations of blood samples or culture media were determined by radioimmunoassay. Relative mRNA expressions of the genes involved in angiogenesis and steroidogenesis were determined by quantitative real-time PCR. Although FGF2 treatment on day 4 of the estrous cycle did not change the cycle length, FGF2 with PGF2α decreased the P4 concentrations observed during the estrous cycle compared to the control group (P < 0.001). Moreover, FGF2 treatment on day 10 prolonged CL function as indicated by a significantly greater concentration of P4 on day 21 compared to the control group. In the in vitro study, FGF2 decreased cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase (HSD3B1) mRNA expression (P < 0.01) and decreased P4 production in the early-stage CL (P < 0.001). However, FGF2 + PGF2α or PGF2α alone resulted in an elevation of steroidogenic acute regulatory protein and CYP11A1 mRNA expression and P4 secretion in the early-stage CL (P < 0.01). In the mid-luteal phase, FGF2 upregulated CYP11A1 and HSD3B1 mRNA expression (P < 0.01), while FGF2 + PGF2α increased only HSD3B1 mRNA expression (P < 0.001). In conclusion, FGF2 seems to play a modulatory role in CL development or luteolysis, differentially regulating steroidogenesis and angiogenic factors as well as PGF2α actions.

Luteinizing hormone and ovarian steroids affect in vitro prostaglandin production in the equine myometrium and endometrium.

Prostaglandins (PGs) play crucial roles in the regulation of the oestrus cycle and establishment of pregnancy in animals. Luteinizing hormone (LH) and ovarian steroids are involved in regulating endometrial PG production in many species. Their effects on PG production and associated pathways in the mare myometrium and endometrium are the subjects of our interest. This study aimed to evaluate the specific effects of LH and ovarian steroids on equine myometrial and endometrial tissues on (i) PGE2 and PGF2α secretion and (ii) transcription of genes encoding specific enzymes responsible for PG synthesis, such as prostaglandin-endoperoxide synthase (PTGS2), PGE2 synthases (PGES), PGF2α synthases (PGFS), and PGI2 synthases (PGIS), using equine myometrial and endometrial explants. Equine myometrial and endometrial tissues were collected at the mid-luteal (n = 6) and follicular (n = 6) phases of the oestrus cycle and were exposed to: (1) vehicle (control), (2) arachidonic acid (AA, 50 ng/mL, positive control), (3) LH (10 ng/mL), (4) progesterone (P4, 10-7M) and (5) 17-ß oestradiol (E2, 10-9M) for 24 h. After exposure, PGF2α and PGE2 concentrations were determined using direct enzyme immunoassays. Alterations in PG synthase mRNA expression were determined using RT-qPCR. After 24 h, LH and P4 increased PGE2 and PGF2α secretion by myometrial tissues at the mid-luteal phase (P < 0.05), whereas PG secretion was augmented by LH and E2 during the follicular phase (P < 0.01). In contrast, LH and E2 increased PGE2 and PGF2α secretion by endometrial tissues during the mid-luteal phase (P < 0.05), while E2 enhanced PGE2 secretion during the follicular phase of the oestrus cycle (P < 0.01). These results indicate that LH and ovarian steroids modulate PG production in equine myometrial and endometrial tissues and affect PG synthase expression at the mRNA level. We conclude that the equine myometrium is an alternative source of PG production and participates in the regulation of uterus function during the oestrus cycle.

Effects of follicular ablation and induced luteolysis on LH and follicular fluid factors during the periovulatory period in mares.

Haemorrhagic anovulatory follicles (HAFs) are the most common pathological anovulatory condition in the mare. To enhance understanding of the physiopathology of HAFs, the aim of the present study was to determine the effects of an induced-follicular wave on LH concentrations and follicular fluid factors relevant to the ovulatory process. Mares were allocated to treatment or control groups (n = 7/group) in a crossed over design during 14 oestrous cycles with a period of one cycle occurring when there were no treatments between the times when treatments were administered. In the treatment group, all antral follicles ≥8 mm were ablated on Day 10 after ovulation followed by administration of a luteolytic dose of PGF2α. All mares of both groups were treated with 1500 IU of hCG when a follicle ≥32 mm was detected (Hour 0), and follicular fluid was aspirated 35 h later. Blood samples were collected every 48 h from Day 10 until Hour 0 from all mares. Follicular fluid was assayed for PGE2, estradiol and progesterone. Plasma was assayed for LH concentrations. A follicular wave followed follicle ablation in the treated mares. Concentrations of LH were greater (P = 0.05) in mares ot the treatment compared with control group. Concentrations of PGE2, estradiol and progesterone in follicular fluid did not differ between groups (P > 0.05). Treatment resulted in an earlier increase in circulating LH, however, there was no effect on concentrations of intra-follicular PGE2, estradiol or progesterone in hCG-stimulated preovulatory follicles.

Effects of prostaglandin F2α on angiogenic and steroidogenic pathways in the bovine corpus luteum may depend on its route of administration.

Prostaglandin (PG) F2α and its analogs (aPGF2α) are used to induce regression of the corpus luteum (CL); their administration during the middle stage of the estrous cycle causes luteolysis in cattle. However, the bovine CL is resistant to the luteolytic actions of aPGF2α in the early stage of the estrous cycle. The mechanisms underlying this differential luteal sensitivity, as well as acquisition of luteolytic sensitivity by the CL, are still not fully understood. Therefore, to characterize possible differences in response to aPGF2α administration, we aimed to determine changes in expression of genes related to (1) angiogenesis-fibroblast growth factor 2 (FGF2), fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2); and (2) steroidogenesis-steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (P450scc), and hydroxy-delta-5-steroid dehydrogenase, 3 ß- and steroid delta-isomerase 1 (HSD3B) in early- and middle-stage CL that accompany local (intra-CL) versus systemic (i.m.) aPGF2α injection. Cows at d 4 (early stage) or d 10 (middle stage) of the estrous cycle were treated as follows: (1) systemic saline injection, (2) systemic aPGF2α injection (25 mg), (3) local saline injection, and (4) local aPGF2α injection (2.5 mg). Progesterone (P4) concentration was measured in jugular vein blood samples during the entire set of experiments. After 4 h of treatment, CL were collected by ovariectomy, and mRNA and protein expression levels were determined by reverse transcription quantitative-PCR and Western blotting, respectively. Local and systemic aPGF2α injections upregulated FGF2 expression but decreased expression of VEGFA in both CL stages. Both aPGF2α injections increased the expression of STAR in early-stage CL, but downregulated it in middle-stage CL. In the early-stage CL, local administration of aPGF2α upregulated HSD3B, whereas systemic injection decreased its mRNA expression in early- and middle-stage CL. Moreover, we observed a decrease in the P4 level earlier after local aPGF2α injection than after systemic administration. These results indicate that aPGF2α acting locally may play a luteotrophic role in early-stage CL. The systemic effect of aPGF2α on the mRNA expression of genes participating in steroidogenesis seems to be more substantial than its local effect in middle-stage CL.

Effect of proinflammatory cytokines on endometrial collagen and metallopeptidase expression during the course of equine endometrosis.

Equine endometrosis (endometrial fibrosis) is a degenerative chronic process that occurs in the uterus of the mare and disturbs proper endometrial function. Fibrosis is attributed to excessive deposition of extracellular matrix (ECM) components. The turnover of ECM is mediated by matrix metallopeptidases (MMP). Previously, it was shown that cytokines modulate MMP expression in other tissues and may regulate fibrosis indirectly by attracting inflammatory cells to the site of inflammation and directly on various tissues. However, the regulation of MMP expression in equine endometrosis is still relatively unknown. Thus, our aim was to determine if interleukin (IL)-1ß and IL-6 regulate ECM, MMPs, or their inhibitors (TIMPs) and whether this regulation differs during endometrosis in the mare. Endometrial fibrosis was divided into four categories according to severity: I (no degenerative changes), IIA (mild degenerative changes), IIB (moderate degenerative changes) and III (severe degenerative changes) according to Kenney and Doig classification. Endometrial explants (n = 5 for category I, IIA, IIB and III according to Kenney and Doig) were incubated with IL-1ß (10 ng/ml) or IL-6 (10 ng/ml) for 24 h. Secretion and mRNA transcription of collagen type 1 (Col1a1) and type 3 (Col3a1), fibronectin (Fn1), Mmp-1, -2, -3, -9, -13, Timp-1, -2 were analyzed by real-time PCR and ELISA, respectively. IL-1ß treatment up-regulated secretion of COL1, MMP-2, TIMP1, and TIMP2 in category I endometrial fibrosis tissues (P < 0.05). IL-6 treatment up-regulated secretion of ECM, MMP-2, and MMP-3 and down-regulated secretion of MMP-9 in category I tissues (P < 0.05). In category IIA tissues, IL-1ß and IL-6 treatment up-regulated secretion of COL3 (P < 0.05; P < 0.05), and IL-6 treatment also down-regulated secretion of MMP-9 (P < 0.05). In category IIB tissues, IL-1ß treatment down-regulated secretion of COL3 (P < 0.05) and up-regulated secretion of MMP-3 (P < 0.01), while IL-6 treatment up-regulated secretion of MMP-3, MMP-9, and MMP-13 (P < 0.05). In category III tissues, IL-1ß treatment up-regulated secretion of COL1, MMP-1, MMP-9 and TIMP-2 (P < 0.05), and IL-6 up-regulated secretion of all investigated ECM components, MMPs and TIMPs. These results reveal that the effect of IL-1ß and IL-6 on equine endometrium differs depending on the severity of endometrial fibrosis. Our findings indicate an association between inflammation and development of endometrosis through the effect of IL-1ß and IL-6 on expression of ECM components, MMPs, and TIMPs in the mare.

Effect of transforming growth factor -ß1 on α-smooth muscle actin and collagen expression in equine endometrial fibroblasts.

Transforming growth factor (TGF)-ß1 not only regulates cell growth, development, and tissue remodeling, but it also participates in the pathogenesis of tissue fibrosis. In the equine endometrium, the concentration of TGF-ß1 is correlated with endometrosis (equine endometrial fibrosis). In other tissues, TGF-ß1 induces differentiation of many cell types into myofibroblasts. These cells are characterized by α-smooth muscle actin (α-SMA) expression and an ability to deposit excessive amounts of extracellular matrix (ECM) components. The aim of the study was to determine whether TGF-ß1 plays a role in the development of equine endometrosis. In Exp. 1, endometrial expression of α-SMA in different stages of endometrosis was determined. In endometrial tissues from the mid luteal phase (n = 6 for each stages of endometrosis) and the follicular phase of the estrous cycle (n = 5 for each stages of endometrosis), mRNA transcription and protein expression of α-Sma were evaluated by Real-time PCR and Western-blot, respectively. The α-Sma mRNA transcription and protein expression levels were correlated with the severity of endometrosis (P < 0.05). In both phases of the estrous cycle, α-SMA protein expression was up-regulated in final stage of endometrosis compared to initial stage (P < 0.05). In Exp. 2, the dose- and time-dependent effects of TGF-ß1 on expression of α-SMA and ECM components were determined, as well as cell proliferation of equine fibroblasts. Equine endometrial fibroblasts (n = 6, Kenney and Doig category I) were stimulated with vehicle or TGF-ß1 (1, 5, 10 ng/ml) for 24, 48 or 72 h. Then, mRNA transcription of α-Sma, collagen type I (Col1a1), collagen type III (Col3a1) and fibronectin 1 (Fn1) were determined by Real-time PCR. The production of ECM components was determined by ELISA. Transforming growth factor-ß1 increased the mRNA transcription of α-Sma and ECM components in a dose- and time-dependent manner in cultured endometrial fibroblasts (P < 0.05). Additionally, TGF-ß1 at a dose of 10 ng/ml increased α-SMA protein expression and COL1, COL3, FN production after 72 h of stimulation (P < 0.05). The data showed a positive linkage between the presence of myofibroblasts and severity of endometrosis. We conclude that TGF-ß1 may participate in pathological fibrotic changes in equine endometrial tissue by induction of myofibroblast differentiation, increased production of ECM components and fibroblast proliferation.

Ovarian steroids, oxytocin, and tumor necrosis factor modulate equine oviduct function.

The oviduct plays important roles in the early reproductive process. The aim of this study was to evaluate gene transcription and protein expression of progesterone receptor (PGR), estrogen receptors 1 (ESR1) and 2 (ESR2); oxytocin receptor (OXTR); prostaglandin F2α synthase (AKR1C3), and prostaglandin E2 synthase (Ptges) in mare oviduct in different estrous cycle stages. Estradiol (E2), progesterone (P4), oxytocin (OXT), and tumor necrosis factor α (TNF) effect on in vitro PGE2 and prostaglandin F2α (PGF2α) secretion by equine oviduct explants or by oviductal epithelial cells (OECs) were also assessed. During the breeding season, oviduct tissue was obtained post mortem from cyclic mares. Protein of ESR1, ESR2, PGR, AKR1C3, and Ptges was present in OECs, whereas OXTR was shown in oviduct stroma. In follicular phase, protein expression of ESR1, ESR2, PGR, and OXTR increased in oviduct explants (P < 0.05), whereas no estrous cycle effect was noted for AKR1C3 or Ptges. In follicular phase, mRNA transcription was upregulated for Pgr but downregulated for Oxtr, Ptges, and Akr1c3 (P < 0.05). Nevertheless, Esr1 and Esr2 mRNA levels did not change with the estrous cycle. In the ampulla, Esr1, Esr2, and Oxtr mRNA transcription increased, but not for Pgr or Ptges. In contrast, Akr1c3 mRNA level was upregulated in the infundibulum (P < 0.05). In follicular phase, E2, P4, and OXT downregulated PGE2 production by OEC (P < 0.05), but no difference was observed in mid-luteal phase. Explants production of PGE2 rose when treated with OXT in follicular phase; with TNF or OXT in early luteal phase; or with TNF, OXT, or P4 in mid-luteal phase. PGF2α production by OEC was downregulated by all treatments in follicular phase but upregulated in mid-luteal phase (P < 0.05). Oviduct explants PGF2α production was stimulated by TNF or OXT in all estrous cycle phases. In conclusion, this work has shown that ESR1, ESR2, OXTR, Ptges, and AKRLC3 gene transcription and/or translation is estrous cycle dependent and varies with oviduct portion (infundibulum vs ampulla) and cell type. Ovarian steroid hormones, OXT and TNF stimulation of PGF2α and/or PGE2 production is also estrous cycle dependent and varies in the different portions of mare oviduct. Differential transcription level and protein localization in various portions of the oviduct throughout the estrous cycle, as well as PG production, suggest coordinated physiologic actions and mechanisms of steroid hormones, OXT, and TNF in the equine oviduct.

Lipopolysaccharides, cytokines, and nitric oxide affect secretion of prostaglandins and leukotrienes by bovine mammary gland during experimentally induced mastitis in vivo and in vitro.

The aim of the study was to determine the effects of lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF), interleukin-1-alpha (IL-1α), and nitric oxide donor (NONOate) on both in vivo and in vitro secretion of prostaglandin (PG)E2, PGF2α, leukotriene (LT)B4, and LTC4 by the bovine mammary gland. In the first experiment, tissues isolated from the teat cavity and lactiferous sinus were treated in vitro with LPS (10 ng/mL), TNF (10 ng/mL), IL-1α (10 ng/mL), NONOate (10(-4) M), and the combination of TNF + IL-1α + NONOate for 4 or 8 h. PGE2 or PGF2α secretion was stimulated by all treatments (P < 0.05) excepting NONOate alone, which did not stimulate PGF2α secretion. Moreover, all factors increased LTB4 and LTC4 secretion (P < 0.05). In the second experiment, mastitis was experimentally mimicked in vivo by repeated (12 h apart) intramammary infusions (5 mL) of (1) sterile saline; (2) 250-µg LPS; (3) 1-µg/mL TNF; (4) 1-µg/mL IL-1α; (5) 12.8-µg/mL NONOate; and (6) TNF + IL-1α + NONOate into 2 udder quarters. All infused factors changed PGE2, 13,14-dihydro,15-keto-PGF2α, and LT concentrations in blood plasma collected from the caudal vena cava, the caudal superficial epigastric (milk) vein, the jugular vein, and the abdominal aorta (P < 0.05). In summary, LPS and other inflammatory mastitis mediators modulate PG and LT secretion by bovine mammary gland in both in vivo and in vitro studies.

The role of toll-like receptors 2 and 4 in the pathogenesis of feline pyometra.

Pyometra is the most common uterine disease in queens. To protect itself from infection, the female reproductive tract possesses several immune mechanisms that are based on germline-encoded pattern recognition receptors (toll-like receptors [TLRs]). The aim of our study was to examine endometrial immunolocalization of TLR2/4, study the influence of lipopolysaccharide (LPS) and tumor necrosis factor (TNF) α on messenger RNA expression of both receptors in pyometric queens, and compare these patterns between estrous cycling queens and those hormonally treated with medroxyprogesterone acetate (MPA). Thirty-six queens, ranging in age from 7 months to 11 years, were allocated into seven groups (anestrus, estrus, mid-diestrus and late diestrus, short-term and long-term hormonally treated queens, and pyometric queens). At the messenger RNA level, the real-time polymerase chain reaction was applied, whereas at the TLR2/4 protein level, the expression was tested by immunohistochemistry. In queens at estrus, gene expression of TLR2 was upregulated after stimulation of endometrial explants by TNF (P < 0.001) and by TNF together with the LPS (P < 0.01). Moreover, gene expression of TLR2 was significantly upregulated after stimulation by TNF (P < 0.001) and LPS (P < 0.01) explants derived from queens that had been long-term hormonally treated with MPA. Endometrial gene expression of TLR4 was significantly upregulated after incubation of explants with TNF (P < 0.001) in queens at estrus and with LPS (P < 0.05) in queens short-term hormonally treated with MPA. Immunolocalization reported that TLR2/4 receptors are mainly localized in the surface and glandular epithelia. These data show that short-term and especially long-term administration of progesterone derivatives impairs TLRs in the endometrial epithelium, presumably enabling pathogens to break through this first natural barrier and thereby increase the risk of pyometra development.

Physiopathologic mechanisms involved in mare endometrosis.

Endometrosis is a degenerative chronic process, characterized by paramount fibrosis development in mare endometrium. This condition is one of the major causes of subfertility/infertility in mares. As in other organs, fibrosis might be a pathologic sequel of many chronic inflammatory diseases. However, aetiology and physiopathologic mechanisms involved in endometrial fibrosis are still controversial. This review presents new hypotheses based on our newest data. As the first line of innate immune defence, systemic neutrophils arrive in the uterus at mating or in the presence of pathogens. A novel paradigm is that neutrophils cast out their DNA in response to infectious stimuli and form neutrophil extracellular traps (NETs). We have shown that bacterial strains of Streptococcus zooepidemicus, Escherichia coli or Staphylococcus capitis, known to cause endometritis in mares were able to induce NETs release in vitro by equine PMN to different extents. An intriguing dilemma is the dual action of NETs. While NETs play a desirable role fighting micro-organisms in mare uterus, they may also contribute to endometrial fibrosis. A long-term in vitro exposure of mare endometrium explants to NETs components (myeloperoxidase, elastase and cathepsin G) up-regulated fibrosis markers TGFß and Tissue inhibitor of metalloproteinase (TIMP-1). Also, pro-fibrotic cytokines regulated collagen deposition and fibrosis. Changes in expression of connective tissue growth factor (CTGF), interleukins (IL)1-α, IL-1ß, IL-6 and receptors in endometrium with different degrees of fibrosis and/or inflammation were observed. A putative role of CTGF, IL and NETs components in endometrosis development should be considered. Additionally, we speculate that in sustained endometritis in mares, prostaglandins may not only cause early luteolysis or early pregnancy loss, but may also be related to endometrial fibrosis pathogenesis by stimulating collagen deposition.

Bisphenol A alters ß-hCG and MIF release by human placenta: an in vitro study to understand the role of endometrial cells.

A proper fetomaternal immune-endocrine cross-talk in pregnancy is fundamental for reproductive success. This might be unbalanced by exposure to environmental chemicals, such as bisphenol A (BPA). As fetoplacental contamination with BPA originates from the maternal compartment, this study investigated the role of the endometrium in BPA effects on the placenta. To this end, in vitro decidualized stromal cells were exposed to BPA 1 nM, and their conditioned medium (diluted 1 : 2) was used on chorionic villous explants from human placenta. Parallel cultures of placental explants were directly exposed to 0.5 nM BPA while, control cultures were exposed to the vehicle (EtOH 0.1%). After 24-48 h, culture medium from BPA-treated and control cultures was assayed for concentration of hormone human Chorionic Gonadotropin ( ß -hCG) and cytokine Macrophage Migration Inhibitory Factor (MIF). The results showed that direct exposure to BPA stimulated the release of both MIF and ß -hCG. These effects were abolished/diminished in placental cultures exposed to endometrial cell-conditioned medium. GM-MS analysis revealed that endometrial cells retain BPA, thus reducing the availability of this chemical for the placenta. The data obtained highlight the importance of in vitro models including the maternal component in reproducing the effects of environmental chemicals on human fetus/placenta.

Is adenomyosis a problem in reproduction and fertility?

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

Influence of prostaglandin F2α analogues on the secretory function of bovine luteal cells and ovarian arterial contractility in vitro.

Although prostaglandin (PG) F2α analogues are routinely used for oestrus synchronisation in cattle, their effects on the function of the bovine corpus luteum (CL), and on ovarian arterial contractility, may not reflect the physiological effects of endogenous PGF2α. In the first of two related experiments, the effects of different analogues of PGF2α (aPGF2α) on the secretory function and apoptosis of cultured bovine cells of the CL were assessed. Enzymatically-isolated bovine luteal cells (from between days 8 and 12 of the oestrous cycle), were stimulated for 24h with naturally-occurring PGF2α or aPGF2α (dinoprost, cloprostenol or luprostiol). Secretion of progesterone (P4) was determined and cellular [Ca(2+)]i mobilisation, as well as cell viability and apoptosis were measured. Naturally-occurring PGF2α and dinoprost stimulated P4 secretion (P<0.05), whereas cloprostenol and luprostiol did not influence P4 synthesis. The greatest cytotoxic and pro-apoptotic effects were observed in the luprostiol-treated cells, at 37.3% and 202%, respectively (P<0.001). The greatest effect on [Ca(2+)]i mobilisation in luteal cells was observed post-luprostiol treatment (200%; P<0.001). In a second experiment, the influence of naturally-occurring PGF2α and aPGF2α on ovarian arterial contraction in vitro, were examined. No differences in the effects of dinoprost or naturally-occurring PGF2α were found across the studied parameters. The effects of cloprostenol and luprostiol on luteal cell death, in addition to their effects on ovarian arterial contractility, were much greater than those produced by treatment with naturally-occurring PGF2α.

Growth and regression in bovine corpora lutea: regulation by local survival and death pathways.

The bovine corpus luteum (CL) is a transient gland with a life span of only 18 days in the cyclic cow. Mechanisms controlling CL development and secretory function may involve factors produced both within and outside this gland. Although luteinizing hormone (LH) surge is the main trigger of ovulation and granulosa cells luteinization, many locally produced agents such as arachidonic acid (AA) metabolites, growth factors and cytokines were shown to complement gonadotropins action in the process of CL development. Bovine CL is a highly vascular gland, where the very rapid angiogenesis rate (until Day 5 of the cycle) results in the development of a capillary network, endowing this gland with one of the highest blood flow rate per unit mass in the body. Angiogenesis in the developing CL is later followed by either controlled regression of the microvascular tree in the non-fertile cycle or maintenance and stabilization of the blood vessels, as seen during pregnancy. Different luteal cell types (both steroidogenic and accessory luteal cells: immune cells, endothelial cells, pericytes and fibroblasts) are involved in the pro- and/or anti-angiogenic responses. The balance between pro- and anti-angiogenic responses to the main luteolysin - prostaglandin F2α (PGF2α) could be decisive in whether or not PGF2α induces CL regression. Fibroblast growth factor-2 (FGF2) may be one of the factors that modulate the angiogenic response to PGF2α. Manipulation of local production and action of FGF2 will provide new tools for reproductive management of dairy cattle. Luteolysis is characterized by a rapid decrease in progesterone production, followed by structural regression. Factors like endothelin-1, cytokines (tumour necrosis factorα, interferons) and nitric oxide were all shown to play critical roles in functional and structural regression of the CL by inhibiting steroidogenesis and inducting apoptosis.

Coumestrol and its metabolite in mares' plasma after ingestion of phytoestrogen-rich plants: potent endocrine disruptors inducing infertility.

Phytoestrogens exist in plants that are present in forages fed to horses. They may compete with 17-ß estradiol and influence the estrous cycle. Therefore, the objective was to determine whether coumestrol from clover-mixed pastures is present in mare's plasma after their ingestion (experiment I), and when this phytoestrogen was present in mare's plasma after ingestion (experiment II). The effect of a long-term ingestion of phytoestrogens on estrous cycle disruption was assessed (experiment III; clinical case). Experiment I was carried out in nonpregnant anestrous and cyclic Lusitano mares (n = 14) kept on clover and grass-mixed pastures, and supplemented with concentrate and hay or cereal straw. Blood and feedstuff were obtained from November to March. In experiment II, stabled cyclic Lusitano mares (n = 6) were fed for 14 days with increasing amounts of alfalfa pellets (250 g to 1 kg/day). Sequential blood samples were obtained for 8 hours after feed intake on Day 0 (control) and on Days 13 and 14 (1 kg/day alfalfa pellets). Experiment III mares were fed with a mixture of alfalfa and clover haylage for 5 months (group 1; n = 4) or for 9 months (group 2; n = 12). Estrous cycle was determined on the basis of plasma estradiol (E2), progesterone (P4), and ultrasound (experiment III). Concentrations of phytoestrogen coumestrol and its metabolite methoxycoumestrol were determined by high-performance liquid chromatography coupled with mass spectrometry. Phytoestrogens decreased in pasture from November until March (P < 0.01) (experiment I), but were always detected in mares' plasma. In experiment II, plasma-conjugated forms of coumestrol and methoxycoumestrol were higher on Days 13 and 14 than in control (P < 0.05). The highest concentrations of conjugated form of coumestrol were at 1.5 and 4 hours (P < 0.001), whereas its free forms peaked at 1 and at 3.5 hours after ingestion (P < 0.05). Methoxycoumestrol-conjugated form concentration was the highest at 1.5 and 5 hours (P < 0.001), whereas its free form peaked at 1 hour (P < 0.05) and at 1.5 hours (P < 0.001). Long-term intake of coumestrol caused lack of ovulation, uterine edema, and uterine fluid accumulation (experiment III). Coumestrol and methoxycoumestrol in both forms were higher in group 2 (while still ingesting haylage) than in group 1, after haylage withdrawal (P < 0.001). These data show that in the mare, coumestrol and its metabolite increase in blood after ingestion of estrogenic plants and can influence reproduction in mares as potent endocrine disruptors.

Prostaglandin endoperoxide synthase 2 (PTGS2) and prostaglandins F2α and E2 synthases (PGFS and PGES) expression and prostaglandin F2α and E2 secretion following oestrogen and/or progesterone stimulation of the feline endometrium.

Sex steroids in synergy with prostaglandins (PG) are involved in the regulation of cyclic ovarian function. In this study, we investigated the mRNA expression of three genes involved in arachidonic acid (AA) metabolism and hence PG production in domestic cats: PG-endoperoxide synthase (PTGS2), PGF(2α) synthase (PGFS) and PGE(2) synthase (PGES). Feline endometria (n = 16) were collected at oestrus and mid and late phases of pseudopregnancy. In addition, the effects of E(2) and/or P(4) on PG secretion and gene expression on endometrial explants were studied in an in vitro culture system. Expression levels of all examined genes were up-regulated at the mid phase of pseudopregnancy. The effects of E(2) and/or P(4) treatment on both PG secretion and expression of the genes were observed after 12 h of culture. Expression of PGES was significantly up-regulated by E(2) plus P(4) at oestrus and the mid phase of pseudopregnancy and was also up-regulated by a single treatment with P(4) at late pseudopregnancy (p < 0.05). Simultaneous incubation with E(2) and P(4) up-regulated PTGS2 gene expression at oestrus and mid-luteal phase (p < 0.05). Progesterone plus E(2) significantly increased PGE(2) secretion at oestrus and the mid phase of pseudopregnancy. However, treatment with E(2) and/or P(4) affected neither PGF(2α) secretion nor PGFS expression at any phase after 12 h of culture. The overall findings indicate that genes involved in PG synthesis are up-regulated at the mid phase of pseudopregnancy. An increase in PGE(2) secretion and up-regulation of PGES and PTGS2 are the main responses of the endometrium to treatment with E(2) and P(4) at oestrus and the mid phase of pseudopregnancy in the cat. These data support the hypothesis that ovarian sex steroids via endometrial PGE(2) are involved in endocrine homoeostasis, especially at oestrus and the mid, but not the late, phase of pseudopregnancy in cats.

Effect of cytokines and ovarian steroids on equine endometrial function: an in vitro study.

Regulation of immune-endocrine interactions in the equine endometrium is not fully understood. The aims of the present study were to: (1) investigate the presence of tumour necrosis factor alpha (TNF), interferon gamma (IFNG), Fas ligand (FASLG) and their receptors in the mare endometrium throughout the oestrous cycle; and (2) assess endometrial secretory function (prostaglandins), angiogenic activity and cell viability in response to TNF, oestradiol (E2), progesterone (P4) and oxytocin (OXT). Transcription of TNF and FASLG mRNA increased during the early and late luteal phase (LP), whereas IFNG mRNA increased in late LP. Transcription of the mRNA of both TNF receptors was highest in the mid-LP. All cytokines and receptors were expressed in surface and glandular epithelium, as well as in the stroma. Expression of TNF and its receptor TNFRSF1A increased during the follicular phase (FP) and mid-LP. IFNG was expressed in the mid-LP, whereas its receptor IFNR1 was expressed in the in mid- and late LP. The highest expression of FASLG and FAS occurred during the late LP. OXT increased the secretion of prostaglandin (PG) E2 and PGF2α in the FP and mid-LP. In the mid-LP, E2 and P4+E2 stimulated PGF2α secretion, whereas TNF and P4 increased cell viability. All treatments, with the exception of P4, increased nitric oxide and angiogenic activity in both phases. The coordinated action of cytokines and ovarian hormones may regulate secretory, angiogenic and proliferative functions in the equine endometrium.

Lipopolysaccharides, cytokines, and nitric oxide affect secretion of prostaglandins and leukotrienes by bovine mammary gland epithelial cells.

The aims of this study were to determine the effects of lipopolysaccharides (LPS), tumor necrosis factor (TNF), interleukin 1 alpha (IL-1α), nitric oxide donor (NONOate), or the combination of TNF + IL-1α + NONOate on the following: (i) secretion of prostaglandin (PG)-F(2α), PGE(2), leukotriene (LT)-B(4), and LTC(4) by epithelial cells of the teat cavity and lactiferous sinus of bovine mammary gland; (ii) messenger RNA (mRNA) transcription of enzymes responsible for arachidonic acid (AA) metabolism (prostaglandin-endoperoxide synthase 2 [PTGS2], prostaglandin E synthase [PTGES], prostaglandin F synthase [PGFS], and arachidonate 5-lipooxygenase [ALOX5]); and (iii) proliferation of the cells. The cells were stimulated for 24 h. Prostaglandins and LT were measured by enzyme immunoassay, mRNA transcription of enzymes was determined by real-time reverse transcription polymerase chain reaction, and the cell viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. All factors increased PG secretion, but the highest stimulation was observed after TNF and IL-1α (P < 0.001). Tumor necrosis factor, NONOate, and TNF + IL-1α + NONOate increased LTB(4) production (P < 0.01), whereas LTC(4) was increased by LPS, TNF, and IL-1α (P < 0.01). Lipopolysaccharides, TNF, IL-1α, and the reagents combination increased PTGS2, PTGES, and PGFS mRNA transcription (P < 0.01), whereas ALOX5 mRNA transcription was increased only by TNF (P < 0.001). Lipopolysaccharides, TNF, IL-1α, NONOate, and the combination of reagents increased the cell number (P < 0.001). Mediators of acute-clinical Escherichia coli mastitis locally modulate PG and LT secretion by the epithelial cells of the teat cavity and lactiferous sinus, which might be a useful first line of defense for the bovine mammary gland. Moreover, the modulation of PG and LT secretion and the changing ratio of luteotropic (PGE(2), LTB(4)) to luteolytic (PGF(2α), LTC(4)) metabolites may contribute to disorders in reproductive functions.

mRNA transcription of prostaglandin synthases and their products in the equine endometrium in the course of fibrosis.

Accurate regulation of the reproductive cycle and successful implantation depend on proper functioning of the endometrium. The aim of this study was to determine whether mRNA transcription of specific enzymes responsible for prostaglandin (PG) synthesis (prostaglandin-endoperoxide synthase, PTGS-2; prostaglandin F(2α) synthase, PGFS; and prostaglandin E(2) synthases, PGES) and PG concentrations in endometrial extracts would change in moderate (Kenney's Category II) and severe phases of fibrosis (Kenney's Category III; endometrosis), compared with healthy endometrium (Kenney's Category I), during the estrous cycle. Endometrial tissues samples were obtained from mares at the early (n = 12), mid (n = 12) and late (n = 12) luteal phases and the follicular phase (n = 12) of the estrous cycle. Additionally, all endometria were classified microscopically as belonging to Categories I and II or III according to the Kenney classification, resulting in allocation of 4 samples for each subcategory, e.g., mid luteal I, II and III. Relative mRNA transcription was quantified using Real-time PCR. Concentrations of PGE(2) and PGF(2α) in the endometrial extracts were determined using enzyme-linked immunosorbent assay (EIA). In Category I, PTGS-2 mRNA transcription was upregulated at the mid (P < 0.05) and late luteal phases (P < 0.001) and at the follicular phase (P < 0.05) compared to the early luteal phase. PGFS mRNA transcription as well as PGF(2α) concentrations increased at the mid (P < 0.01) and late (P < 0.05) luteal phases compared to the early luteal phase in Category I. PGES mRNA transcription was higher at the mid (P < 0.01) and late luteal phases (P < 0.05) compared to the early luteal and follicular phases in Category I. Prostaglandin E(2) concentration in Category I was higher at the mid luteal phase (P < 0.01) compared to all other phases of the estrous cycle. During incipient endometrosis (Category II) and under full endometrosis (Category III), PTGS-2, PGFS and PGES mRNA transcription and PG concentration were altered compared to the respective estrous phases in healthy endometria (P < 0.05). It may be concluded that serious changes in mRNA transcription of PG synthases and PG production that occur in the equine endometrium during the course of fibrosis in the estrous cycle could be responsible for disturbances leading to disorders of the estrous cycle and early embryo losses.