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.

Bacteria causing pyometra in bitch and queen induce neutrophil extracellular traps.

Neutrophils are capable of releasing their DNA in response to infectious agents to form neutrophil extracellular traps (NETs) to destroy pathogens. Even though pyometra in queens and bitches is a common disease, its pathogenesis is not fully understood. The aim of this study was to assess the presence of NETs in the endometrium of queens and bitches suffering from pyometra. Pyometra and normal uteri were obtained after ovariohysterectomy from adult queens and bitches in diestrus. Uterine contents were evaluated for bacterial isolation and identification and for NETs presence. Escherichia coli were isolated in 5/5 queens and 4/5 bitches, and Streptococcus spp in one bitch. Sterile glass coverslips were placed on the endometrium surface to obtain material for NETs that were evaluated by immunocytochemistry (histone, neutrophil elastase or myeloperoxidase), fluorescence microscopy or scanning electron microscopy. NETs in endometrium content were positively stained by DNA histone DAPI, myeloperoxidase and by neutrophil elastase. NETs were spread in all observed queen and bitch endometria of pyometra cases. Ultrastructure images of NETs depicted clusters of globular material with fine filaments deposited on or around thick filaments and trapped bacteria. To the best of our knowledge these are the first findings confirming NETs endometrial presence in queen and bitch pyometra. Nevertheless, the precise role of NETs in pyometra in the bitch and queen, either to contribute to the defeat of infection or to its persistence remains to be unraveled.

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.

Oestrous cycle-dependent expression of Fas and Bcl2 family gene products in normal canine endometrium.

During the oestrous cycle canine endometrium undergoes cyclical cellular proliferation, apoptosis and differentiation. To study the regulation of endometrial apoptosis and proliferation events the expression of apoptosis-related genes was analysed by real-time polymerase chain reaction and cellular expression of their proteins was identified through immunohistochemistry. Cellular apoptosis and proliferation events were detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL) and proliferation marker Ki67 immunostaining, respectively. The highest proliferative index was observed in the follicular phase (all endometrial cellular components) and at early dioestrus (basal glands). This was associated with a low apoptotic index and a strong expression of anti- (Bcl2) and pro-apoptotic proteins (Fas, FasL, Bax). Subsequently (Days 11-45 of dioestrus), basal glandular epithelium experienced the highest apoptotic index, coincidental with a decrease of Bcl2 expression and a low ratio of Bcl2/Bax transcription. An increase in the apoptotic index of crypts, stromal and endothelial cells was observed at late dioestrus and the beginning of anoestrus. These results indicate that pro- and anti-apoptotic proteins regulate the balance between cell proliferation and death in the canine endometrium during the oestrous cycle. High Bcl2 expression in both the follicular and early dioestrous phases stimulate glandular proliferation and prevent apoptosis but, in the non-pregnant uterus, a decrease in Bcl2 expression together with an increase in pro-apoptotic proteins induces apoptosis of basal glandular epithelium cells.

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.

Neutrophil extracellular traps formation by bacteria causing endometritis in the mare.

Besides the classical functions, neutrophils (PMNs) are able to release DNA in response to infectious stimuli, forming neutrophil extracellular traps (NETs) and killing pathogens. The pathogenesis of endometritis in the mare is not completely understood. The aim was to evaluate the in vitro capacity of equine PMNs to secrete NETs by chemical activation, or stimulated with Streptococcus equi subspecies zooepidemicus (Szoo), Escherichia coli (Ecoli) or Staphylococcus capitis (Scap) strains obtained from mares with endometritis. Ex vivo endometrial mucus from mares with bacterial endometritis were evaluated for the presence of NETs. Equine blood PMNs were used either without or with stimulation by phorbol-myristate-acetate (PMA), a strong inducer of NETs, for 1-3h. To evaluate PMN ability to produce NETs when phagocytosis was impaired, the phagocytosis inhibitor cytochalasin (Cyt) was added after PMA. After the addition of bacteria, a subsequent 1-h incubation was carried out in seven groups. NETs were visualized by 4',6-diamidino-2-phenylindole (DAPI) and anti-histone. Ex vivo samples were immunostained for myeloperoxidase and neutrophil elastase. A 3-h incubation period of PMN + PMA increased NETs (p < 0.05). Bacteria + 25 nM PMA and bacteria + PMA + Cyt increased NETs (p<0.05). Szoo induced fewer NETs than Ecoli or Scap (p < 0.05). Ex vivo NETs were present in mares with endometritis. Scanning electron microscopy showed the spread of NETs formed by smooth fibers and globules that can be aggregated in thick bundles. Formation of NETs and the subsequent entanglement of bacteria suggest that equine NETs might be a complementary mechanism in fighting some of the bacteria causing endometritis in the mare.

Effects of body condition and leptin on the reproductive performance of Lusitano mares on extensive systems.

The aim of this study was to investigate the effects of body condition (BC), BC changes, and plasma leptin concentrations on the reproductive performance of Lusitano broodmares on extensive systems. Data from 119 mares (ranging from 4 to 22 years of age) were collected over a period of four consecutive breeding seasons. Each case was considered as one foaled mare bred in 1 year. Body condition changes at conception (ΔBCScon) showed a strong effect on fertility at the first two postpartum estrous cycles and a significant interaction with body condition score at conception (BCScon) was observed (P < 0.01). The best fertility results were obtained with positive and greater ΔBCScon. The best predictive value of fertility (91%) was achieved when the BCScon was 3.0 and ΔBCScon was 0.375. Global foaling rate for the 4-year period was 74.5%. Mean foaling interval and gestation length were, respectively, 368.0 ± 2.8 and 340.3 ± 1.0 days. Gestation length was influenced by the month of foaling (P < 0.05) and decreased as mares got older (P < 0.01). The number of estimated estrous cycles per live foal was 1.78 ± 0.12, including foal heat ovulation. Leptin was correlated with BCS (0.41; P < 0.001), but in the present study, plasma leptin concentrations on late gestation and early lactation did not influence fertility results. At 90 days of age, lower growth performances were obtained in foals which dams presented negative BCS changes on the first 3 months of lactation (P < 0.05). In conclusion, BC changes at early postpartum period influence the reproductive efficiency of broodmares and the growth of their suckling foals. These findings are important to help breeders on management options concerning feeding strategies in the Lusitano production systems and others under similar conditions.

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.

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.

Oestrous cycle-related changes in production of Toll-like receptors and prostaglandins in the canine endometrium.

The objectives of this study were to evaluate the following events in the canine endometrium over the course of the oestrous cycle: (i) the transcriptional profiles of genes encoding the Toll-like receptors (TLR1-TLR7 and TLR9); (ii) the transcription and protein expression levels of TLR2 and TLR4; (iii) the gene transcription profile of prostaglandin synthesis enzymes (PTGS2, PGES and PGFS); (iv) the response pattern of PGF(2α) and PGE(2) following exposure of endometrial explants to LPS and LTA. TLR1-TLR7 and TLR9 genes were transcribed in the endometrium of bitches throughout the oestrous cycle, which indicates that TLR-mediated immune surveillance is an important component of the defence mechanisms within the uterus. Canine endometrial mRNA and protein expression of TLR2 and TLR4 was up-regulated at the late dioestrus and anoestrus and was the lowest in the follicular phase and early dioestrus. The decreased mRNA and protein levels observed at early dioestrus may favour implantation, but may also be linked to the high prevalence of pyometra at this stage of the oestrous cycle. After LPS and LTA stimulation, endometrial explants produced more PGF(2α) than PGE(2), which may be related to the early demise of the corpus luteum observed in vivo in canine pyometra cases. Overall, these results indicate that TLRs are involved in the activation of the inflammatory response associated with pyometra in the bitch. TLRs may therefore be therapeutic targets for the control of uterine bacterial infections in the bitch and potentially in other species.

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.

Effects of cell storage and passage on basal and oxytocin-regulated prostaglandin secretion by equine endometrial epithelial and stromal cells.

Cell cultures are useful for determining the responses of specific cell types to various factors under controlled conditions and for obtaining a better understanding of in vivo physiologic processes. The aims of the present study were (i) to establish methodologies for isolation, culture and cryopreservation of equine endometrial epithelial and stromal cells; and (ii) to determine the effect of passage and cryopreservation on endometrial cell physiology, based on their basal and oxytocin (OT)-stimulated prostaglandin (PG) release. Epithelial and stromal cells were obtained by enzymatic digestion of equine endometrium collected from Days 2-5 of the estrous cycle (n = 16). Primary epithelial and stromal cells, as well as cryopreserved cells were stimulated with OT (10(-7)m) for 24 h. The concentrations of PGE(2) and PGF(2α) in the culture medium were measured by enzyme-linked immunosorbent assay (EIA). Oxytocin increased PGE(2) and PGF(2α) release by primary cultures of unfrozen epithelial cells until passage I (P < 0.01) and by the primary culture of unfrozen and cryopreserved/thawed stromal cells until passage IV (P < 0.01). Cryopreserved/thawed stromal cells cultured up to passage IV and unfrozen epithelial cells derived from passage I have physiological properties similar to those observed in primary culture and may be successfully used for in vitro studies of PG secretion.

Cytokines tumor necrosis factor-α and interferon-γ participate in modulation of the equine corpus luteum as autocrine and paracrine factors.

Knowledge on the regulation of corpus luteum (CL) function in the mare is scarce. In this study, the presence of cytokines tumor necrosis factor alpha (TNF) and interferon gamma (IFNG), and their receptors (TNFRI, TNFRII and IFNRI), was investigated in equine CL throughout the luteal phase. The effects of TNF and IFNG on secretory function and viability of luteal cells were defined in vitro. Cytokine ligands and receptors were present in steroidogenic and endothelial cells. Protein expression for TNF was greater in mid-phase and regressing CL, while TNFRI was increased in regressing CL and TNFRII did not change. IFNG and IFNRI showed the highest expression in regressing CL. Transcription of mRNA for TNF increased from mid-phase to regressing CL and both TNFRI and TNFRII decreased from early to regressing CL. Transcription of mRNA for IFNG was lower in CL from early phase than in mid or regressing luteal phase, while IFNRI expression was not changed. In the early CL, TNF acted to increase P(4) and PGE(2) but decrease PGF(2α) secretion. In the mid luteal phase, TNF increased PGF(2α) secretion and TNF+IFNG decreased PGE(2) secretion. In the regressing luteal phase, TNF, IFNG and TNF+IFNG decreased P(4) and PGE(2) secretion, but TNF and TNF+IFNG increased PGF(2α) secretion by luteal cells. Cell viability was reduced by TNF+IFNG in regressing CL. These data show the presence of cytokines TNF and IFNG, and their receptors, in the equine CL and indicate their potential involvement in regulation of luteal function.

Nitric oxide stimulates progesterone and prostaglandin E2 secretion as well as angiogenic activity in the equine corpus luteum.

Cytokines and nitric oxide (NO) are potential mediators of luteal development and maintenance, angiogenesis, and blood flow. The aim of this study was to evaluate (i) the localization and protein expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) in equine corpora lutea (CL) throughout the luteal phase and (ii) the effect of a nitric oxide donor (spermine NONOate, NONOate) on the production of progesterone (P4) and prostaglandin (PG) E(2) and factor(s) that stimulate endothelial cell proliferation using equine luteal explants. Luteal tissue was classified as corpora hemorrhagica (CH; n = 5), midluteal phase CL (mid-CL; n = 5) or late luteal phase CL (late CL; n = 5). Both eNOS and iNOS were localized in large luteal cells and endothelial cells throughout the luteal phase. The expression of eNOS was the lowest in mid-CL (P < 0.05) and the highest in late CL (P < 0.05). However, no change was found for iNOS expression. Luteal explants were cultured with no hormone added or with NONOate (10(-5) M), tumor necrosis factor-α (TNFα; 10 ng/mL; positive control), or equine LH (100 ng/mL; positive control). Conditioned media by luteal tissues were assayed for P4 and PGE(2) and for their ability to stimulate proliferation of bovine aortic endothelial cells (BAEC). All treatments stimulated release of P4 in CH, but not in mid-CL. TNFα and NONOate treatments also increased PGE(2) levels and BAEC proliferation in CH (P < 0.05). However, in mid-CL, no changes were observed, regardless of the treatments used. These data suggest that NO and TNFα stimulate equine CH secretory functions and the production of angiogenic factor(s). Furthermore, in mares, NO may play a role in CL growth during early luteal development, when vascular development is more intense.

Gene transcription of TLR2, TLR4, LPS ligands and prostaglandin synthesis enzymes are up-regulated in canine uteri with cystic endometrial hyperplasia-pyometra complex.

Escherichia coli (E. coli) is the most frequent bacterium isolated in cases of cystic endometrial hyperplasia-pyometra complex, the most frequent endometrial disorder in the bitch. Toll-like receptors (TLRs) play an essential role in the innate immune system. The aim of this study was to compare transcription of genes encoding TLR2, TLR4 and LPS ligands (CD14, MD-2, LBP), prostaglandin synthesis enzymes (COX1, COX2, PGES1 and PGFS), and to compare COX1 and COX2 protein expression and PGE(2) and PGF(2alpha) endometrial content in the endometrium of canine diestrous uteri with (n=7) or without (n=7) pyometra. All cases of pyometra were hyperplastic and E. coli was the only isolated bacteria, while diestrous normal uteri did not present signs of cystic endometrial hyperplasia and were negative for bacteriology. Except for COX1, transcription of all genes was significantly higher in pyometra than in normal endometria. COX1 protein was observed in both normal and pyometra uteri, but COX2 protein was only present in pyometra cases. Endometrial PGE(2) and PGF(2alpha) content were significantly higher in pyometra than in normal diestrous endometria. In conclusion, data obtained in this study provides evidence that pyometra-isolated E. coli induces the up-regulation of TLR2 and TLR4 genes in the canine diestrous endometrium. This up-regulation, which is probably the result of the stimulation by LPS and lipoprotein E. coli constituents, leads to the endometrial up-regulation of PG synthesis genes. This, in turn, results in a higher endometrial concentration of PGE(2) and PGF(2alpha), which may further regulate the local inflammatory response.

Prostaglandin synthesis genes are differentially transcripted in normal and pyometra endometria of bitches.

Pro-inflammatory stimuli, such as endotoxins released by Gram-negative bacteria, are potent stimulators of prostaglandin (PG) synthesis. The aim of this study was to evaluate the gene transcription pattern of PG synthesis enzymes in normal (anestrous, n = 6 and diestrous, n = 8) and pyometra (n = 7) endometria of bitches. Uteri were collected during routine ovariohysterectomy, processed for histopathological evaluation and uterine contents cultured. Gene transcription of COX-1, COX-2, mPGES-1 and PGF-synthase (PGFS) were evaluated by relative real-time PCR and normalized with the ribosomal protein L27 (RPL27) housekeeping gene. Normal uteri had no histological abnormalities and were negative for bacteriology. All pyometra uteri were hyperplasic and Escherichia coli was the only isolated bacterium. Except for COX-1, gene transcription was significantly higher in pyometra than in normal endometria. No significant differences in gene transcription were observed between normal diestrous and anestrous endometria. COX-2 gene transcription was 19 and 69 times higher in pyometra than in diestrous and anestrous endometria (p < 0.001), while PGFS gene transcription had a 3- and 600-fold increase in pyometra endometria compared to normal diestrous and anestrous endometria (p < 0.001). Gene transcription of mPGES-1 was 9 times higher in pyometra than in normal uteri (p < 0.01). Based on these results, we suggest that pyometra-associated E. coli endotoxin release stimulates the up-regulation of COX-2 PGFS and mPGES-1 gene transcription in the endometrium.

Testicular angiogenic activity in response to food restriction in rabbits.

The objective of this study was to evaluate the effects of two different levels of food restriction on testicular angiogenic activity, microvascularization, tissue growth, and regression, using the rabbit as a study model. The rabbits (Oryctolagus cuniculus cuniculus) were randomly assigned to a control group (A, n=5), fed ad libitum, and to groups B (n=5) and C (n=5), with two different levels of food restriction. Food restriction was responsible for a 21.2% decrease in body weight in group B and 34.7% in group C. Testis explants were cultured for 24 h and conditioned media were tested for their ability to stimulate mitogenesis of bovine aortic endothelial cells (BAEC). There was an increase in testicular microvascular area and mitogenesis of BAEC in group C rabbits. Despite no change in testicular DNA concentration among groups, food restriction decreased both RNA and protein compared with control. No treatment differences in the percentage of seminiferous tubules filled with all stages of spermatogenesis (spermatogonia, spermatocytes, and spermatids) and spermatozoa, as well as the area occupied by seminiferous tubules, were observed. Nevertheless, serum testosterone was markedly less in group C compared with groups A and B. These results suggest that angiogenesis may play a role in overcoming testicular nutritional impairment in rabbits subjected to food restriction.

Regulation of luteal function and corpus luteum regression in cows: hormonal control, immune mechanisms and intercellular communication.

The main function of the corpus luteum (CL) is production of progesterone (P4). Adequate luteal function to secrete P4 is crucial for determining the physiological duration of the oestrous cycle and for achieving a successful pregnancy. The bovine CL grows very fast and regresses within a few days at luteolysis. Mechanisms controlling development and secretory function of the bovine CL may involve many factors that are produced both within and outside the CL. Some of these regulators seem to be prostaglandins (PGs), oxytocin, growth and adrenergic factors. Moreover, there is evidence that P4 acts within the CL as an autocrine or paracrine regulator. Each of these factors may act on the CL independently or may modify the actions of others. Although uterine PGF(2 alpha) is known to be a principal luteolytic factor, its direct action on the CL is mediated by local factors: cytokines, endothelin-1, nitric oxide. The changes in ovarian blood flow have also been suggested to have some role in regulation of CL development, maintenance and regression.

Blood lymphocyte subpopulations, neutrophil phagocytosis and proteinogram during late pregnancy and postpartum in mares.

The aim of this study was to evaluate peripheral blood lymphocyte subpopulations, neutrophil phagocytic capacity and proteinogram characteristics in mares, during the last trimester of pregnancy and in postpartum. Measurement of phagocytosis and quantification of T-lymphocyte subsets were done by flow cytometry. Quantification of T-lymphocyte subsets was performed with monoclonal antibodies specific for CD2, CD3, CD4 and CD8 cell markers. Natural killer and B-cell counts were estimated mathematically. Serum proteinogram was obtained by electrophoresis. No significant differences were observed between gestation and postpartum on CD4(+), CD8(+) and NK(+) lymphocyte subsets, CD4 : CD8 ratio and phagocytosis. The percentage of cells expressing CD3 (64.2 +/- 1.8) and CD2 (68.4 +/- 1.7) (Mean +/- SEM) was reduced during gestation vs postpartum (69.7 +/- 1.5 and 73.8 +/- 1.4 respectively) (p < 0.05). During pregnancy, CD19(+) (31.6 +/- 1.7) was higher than in postpartum (26.2 +/- 1.4) (p < 0.05). Total T cells (2911 +/- 227 cells/mul), T helper cells (2144 +/- 169 cells/mul) and T-cytotoxic cells (767 +/- 68 cells/mul) were depressed in pregnancy, when compared with postpartum (4093 +/- 337 cells/mul; 3004 +/- 276 cells/mul; 1089 +/- 94 cells/mul respectively) (p < 0.01). Total white blood cell count was reduced during pregnancy (8815 +/- 427 cells/mul) with respect to postpartum (10742 +/- 446 cells/mul) (p < 0.01), while neutrophil count did not change. Total proteins, albumin, alpha(1),alpha(2),beta(1), beta(2), gamma globulins and albumin : globulin did not differ. Our results suggest that the physiological immune depression occurring in mares, during gestation might be due to T-helper and T-cytotoxic lymphocytes reduction.