Aydin Guzeloglu

Uterine-conceptus interactions and reproductive failure in cattle

The dialogue between trophectoderm cells of the conceptus and epithelial cells of the endometrium is critical to CL maintenance and embryo survival. The signal transduction mechanisms by which bovine interferon (IFN)-tau regulates cyclooxygenase (COX)-2 expression and secretion of prostaglandin F2alpha (PGF2alpha) in bovine endometrial (BEND) cells is examined. Stimulation of Protein Kinase C with a phorbol ester (phorbol 12, 13 dibutyrate [PDBu]) activates COX-2 gene expression and PGF2alpha secretion via the mitogen-activated protein kinase (MAPK) pathway. Interferon-tau attenuates PDBu activation of PGF2alpha secretion, but this inhibitory effect appears to be independent of the MAPK pathway. Embryonic IFN-tau, acting through a Type I IFN receptor, activates the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway resulting in activation or repression of interferon-stimulated genes. Experimental evidence is provided that IFN-tau regulation of STATs regulates gene expression of COX-2 in a manner that decreases secretion of PGF2alpha. Maternal regulation of the antiluteolytic pathway is discussed relative to the ability of the polyunsaturated fatty acid, eicosapentaenoic (EPA), to decrease endometrial secretion of PGF2alpha and progesterone to increase both conceptus development and IFN-tau secretion.

Polyunsaturated Fatty Acids and Bovine Interferon-τ Modify Phorbol Ester-Induced Secretion of Prostaglandin F2α and Expression of Prostaglandin Endoperoxide Synthase-2 and Phospholipase-A2 in Bovine Endometrial Cells

Abstract Embryonic mortality in cattle may occur because of inadequate inhibition of uterine secretion of prostaglandin (PG) F2α mediated by bovine interferon-τ (bIFN-τ). The objectives of the present study were to determine whether polyunsaturated fatty acids inhibit secretion of PGF2α from bovine endometrial cells induced by stimulating protein kinase C with phorbol 12,13 dibutyrate (PDBu) and to investigate possible mechanisms of action. Confluent cells were exposed for 24 h to 100 μM of linoleic, arachidonic (AA; C20:4, n-6), linolenic (LNA; C18:3, n-3), eicosapentaenoic (EPA; C20:5, n-3), or docosahexaenoic (DHA; C22:6, n-3) acid. After incubation, cells were washed and stimulated with PDBu. The EPA, DHA, and LNA attenuated secretion of PGF2α in response to PDBu. The EPA and DHA were more potent inhibitors than LNA. The EPA inhibited secretion of PGF2α at 6.25 μM. Secretion of PGF2α in response to PDBu decreased with increasing incubation time with EPA. Both bIFN-τ and EPA inhibited secretion of PGF2α, and their inhibitory effects were additive. The bIFN-τ, but not EPA, reduced the abundance of PG endoperoxide synthase-2 (PGHS-2) mRNA. Incubation with 100 μM EPA, DHA, or AA for 24 h followed by treatment with PDBu did not affect concentrations of PGHS-2 and phospholipase A2 proteins. The EPA and DHA inhibit secretion of PGF2α through a mechanism different from that of bIFN-τ. The effect of EPA on PGF2α secretion may be caused by competition with AA for PGHS-2 activity or reduction of PGHS-2 activity. The use of EPA and DHA to inhibit uterine secretion of PGF2α and to improve embryonic survival in cattle warrants further investigation.

Interferon-τ Modulates Phorbol Ester-Induced Production of Prostaglandin and Expression of Cyclooxygenase-2 and Phospholipase-A2 from Bovine Endometrial Cells

Abstract Antiluteolytic actions of bovine interferon-tau (bIFN-τ) require suppression of prostaglandin F2α (PGF2α) production. Our objective was to test whether bIFN-τ could block PGF2α production and synthesis of phospholipase A2 (PLA2) and cyclooxygenase-2 (COX-2) enzymes induced by a protein kinase C (PKC) stimulator (phorbol 12,13 dibutyrate; PDBu). Bovine endometrial epithelial (BEND) cells were treated with PDBu in the presence or absence of bIFN-τ. Medium samples were analyzed for concentrations of PGF2α, whole-cell extracts were analyzed for abundance of PLA2 and COX-2 by immunoblotting, and RNA extracts were examined for steady-state levels of COX-2 mRNA by Northern blotting. The PDBu stimulated production of PGF2α between 3 and 12 h, levels of COX-2 mRNA by 3 h and protein expression of COX-2 and PLA2 by 6 and 12 h, respectively. Added concomitantly with PDBu, bIFN-τ suppressed PGF2α production, steady-state levels of COX-2 mRNA, and expression of COX-2 and PLA2 proteins. Added after a 3-h stimulation with PDBu alone, bIFN-τ suppressed PGF2α production after 1 h. Bovine IFN-τ inhibited intracellular mechanisms responsible for PGF2α production in BEND cells, and this could be through both cytosolic and nuclear actions.

Evaluation of genes involved in prostaglandin action in equine endometrium during estrous cycle and early pregnancy

The aim was to evaluate expression of genes involved in the biosynthesis of prostaglandins (PTG), Prostaglandin H Synthase-1 (PTGS1) and PTGS2, PGF synthase (PTGFS), and PGE synthase (PTGES), PGF receptor (PTGFR), PGE receptors (PTGER2 and PTGER4), prostaglandin transporter (SLCO2A1) and hydroxyprostaglandin dehydrogenase-15 (HPGD). Endometrial biopsies were obtained from mares on day of ovulation (d0, n=4), late diestrus (LD, n=4), early luteolysis (EL, n=4) and after luteolysis (AL, n=4) during the cycle. Stages of the cycle were confirmed by plasma progesterone concentrations measured daily and ultrasound examinations. Biopsies were also taken on days 14 (P14; n=4), 15 (P15, n=4), 18 (P18, n=4) and 22 (P22; n=4) of pregnancy. Relative mRNA expressions were quantified using real-time RT-PCR. A mixed model was fitted on the normalized data and least significant difference test (α=0.05) was employed. Expression of PTGS1 mRNA was low throughout the estrous cycle and early days of pregnancy, but upregulated on P18 and P22. PTGS2 expression was increased on EL, but it was suppressed by pregnancy on P15, P18, and P22. PTGFS expression was upregulated in both cyclic and pregnant mares compared to d0 and its level was the highest on LD. PTGFR expression was transiently increased on LD and EL and was suppressed during early pregnancy. Both PTGES and PTGER2 expressions were increased on LD, EL, and early pregnancy, but were decreased after the luteolysis in cyclic mares as they remained high on P18 and P22. PTGER4 expression did not change throughout the cycle and early pregnancy. Levels of HPGD and SLCO2A1 were significantly increased only on P22. In conclusion, PTGS2 expression increases around the time of luteolysis and concurrent upregulation of PTGFS and PTGES indicates that equine endometrium has increased capability of PTG production around the time of luteolysis. However, pregnancy reduces PTGS2 expression, but maintains the high levels of PTGES during early pregnancy along with PTGER2 while PTGFR expression was suppressed. These findings suggest that possible luteotrophic action of PGE₂ is required in early equine pregnancy. PTGS1 is only upregulated later in the early pregnancy suggesting that it is not involved in luteolysis, but could be the main PTGS enzyme at this time during early pregnancy. An increase in HPGD and SLCO2A1 levels on P22 indicates a tight regulation of PTG action by pregnancy.

Interferon-Tau Suppresses Prostaglandin F2α Secretion Independently of the Mitogen-Activated Protein Kinase and Nuclear Factor κ B Pathways

Abstract Pregnancy is established in ruminants through inhibitory actions of interferon (IFN)-τ on the release of prostaglandin F2α (PGF), which allows the corpus luteum to survive and continue to produce progesterone. Experiments were designed to 1) delineate the signal transduction pathway coordinating the synthesis of PGF, 2) determine how rapidly recombinant bovine (rb) IFN-τ attenuated phorbol ester (PDBu)-induced secretion of PGF, and 3) establish the site at which rbIFN-τ attenuates the secretion of PGF in cultured bovine endometrial (BEND) cells. BEND cells were untreated (control) or treated for 5, 10, 60, 180, or 300 min with PDBu (100 ng/ml), rbIFN-τ (50 or 500 ng/ml), PDBu + rbIFN-τ, or PDBu + PD98059 (MEK-1 inhibitor; 50 μM). Secretion of PGF was induced (P < 0.0001) by PDBu within 180 min, but induction was inhibited 74% by the addition of rbIFN-τ (P < 0.0001) and was ablated completely by PD98059. Parallel results were obtained for cyclooxygenase (COX)-2 protein expression. PDBu induced (P < 0.05) activation of the Raf-1/MEK-1/ERK-1/2 pathway, which was obligatory for the expression of COX-2 and secretion of PGF but was not altered by cotreatment with rbIFN-τ. PDBu induced (P < 0.05) transcription of c-jun and c-fos mRNAs within 30 min; induction was inhibited (P < 0.05) by cotreatment with PD98059 but not by cotreatment with rbIFN-τ. Treatment of BEND cells with rbIFN-τ also did not attenuate PDBu-induced degradation of IκBα, suggesting that the IκBα/NFκB pathway is not a site of IFN-τ inhibition of PGF. However, rbIFN-τ did block transcription of the COX-2 gene induced by PDBu within 30 min. In conclusion, COX-2 expression and PGF secretion induced by PDBu is mediated through the Raf-1/MEK-1/ERK-1/2 pathway, but this pathway is not disrupted by rbIFN-τ. Because rbIFN-τ inhibits COX-2 mRNA within 30 min, we hypothesized that transcription factors activated by rbIFN-τ rapidly and directly attenuate COX-2 gene expression, thereby suppressing secretion of PGF.

Effect of the administration of flunixin meglumine on pregnancy rates in holstein heifers

Fifty-two 15-month-old Holstein heifers were synchronised with single or double injections of prostaglandin F2α, followed by an injection of gonadotrophin-releasing hormone (gnrh) 48 hours later, and inseminated 12 to 14 hours after the injection of gnrh (day 0). Half of them were then injected twice intramuscularly with 1·1 mg/kg flunixin meglumine 12 hours apart, on the evening of day 15 and the morning of day 16, and the other 26 were not treated. Pregnancy was diagnosed by ultrasound 29 and 65 days after they were inseminated. On day 29, 20 of the treated heifers were pregnant compared with 13 of the control heifers (P<0·05); on day 65, 18 of the treated heifers were still pregnant compared with 12 of the control heifers (P<0·10).

Rating of putative housekeeping genes for quantitative gene expression analysis in cyclic and early pregnant equine endometrium

The aim was an evaluation of a set of housekeeping genes (HKGs) to be used in the normalization of gene expression in the equine endometrium. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine ribosyl transferase 1 (HPRT1), ubiquitin B (UBB), tubulin alpha 1 (TUBA1), ribosomal protein L32 (RPL32), beta-2-microglobulin (B2M), 18S rRNA (18S), and 28S rRNA (28S) HKGs were evaluated using real-time PCR and were compared in different physiological stages of the endometrium. Endometrial biopsies were obtained from mares on day of ovulation (d0, n=4), at late diestrus (LD, n=4), after luteolyis (AL, n=4) of the cycle and on days 14 (P14; n=3), 18 (P18, n=3) and 22 (P22; n=3) of pregnancy. A model based on REML with support of descriptive statistics was proposed in accordance with experimental design and was further confirmed with principal component analysis (PCA). Results were compared with widely used software including geNorm, BestKeeper, and NormFinder. Results indicated that GAPDH was the most stable HKG and RPL32 was ranked as the second best. 18S and 28S were found to be the least stable. The proposed model, PCA, geNorm, and BestKeeper were in agreement in detecting the most stable and the least stable HKGs in the equine endometrium during the estrous cycle and early pregnancy.

Expression of wingless type (WNT) genes and their antagonists at mRNA levels in equine endometrium during the estrous cycle and early pregnancy

WNT signaling pathway plays important roles in reproductive events. Aims were to (1) determine presence of WNT genes and their antagonists in equine endometrium; and (2) to evaluate their expression profiles during early pregnancy. Endometrial biopsies were obtained from mares on day of ovulation (d0, n=4) and on days of 14 (P14, n=4), 18 (P18, n=4), 22 (P22, n=4) of early pregnancy. Biopsies were also collected from cyclic mares during late diestrus (LD, on day of 13.5-14, n=4) and after luteolysis in estrus phase (AL, on day of 17.5-18, n=4) of the cycle. PCR was used to detect expression of genes studied and then relative expression levels were quantified using real-time PCR analysis. A mixed model was fitted on the normalized data and least significant difference test (α=0.05) was employed. Eleven WNT genes (WNT2, WNT2B, WNT4, WNT5A, WNT5B, WNT7A, WNT8A, WNT9B, WNT10B, WNT11 and WNT16) and their antagonists (SFRP1, SFRP2, SFRP5, DKK1, DKK2 and WIF-1) were detected in equine endometrium. Compared to d0, WNT2, WNT5B, WNT7A and SFRP1 expressions were downregulated by the pregnancy while DKK1 was upregulated. WNT5A, WNT11 and WIF-1 were upregulated on P14 and P18, but WNT2B increased only on P14. When LD and P14 were compared, level of WNT8A decreased on P14 while increase in WNT4 level on P14 was slightly significant (P<0.06). Levels of WNT7A and SFRP1 decreased while DKK1 and WIF-1 increased by the pregnancy on P18 compared to AL. Moreover, WNT2B, WNT5A, WNT9B, WNT10B, WNT11, WNT16 DKK1 and WIF-1 were upregulated on LD compared to AL whereas WNT4, WNT7A, SFRP1 were downregulated. In conclusion, the results demonstrate that WNT genes and their antagonists appear to be regulated during early pregnancy in equine endometrium possibly due to embryonic factors and/or maternal progesterone.

Interferon-t Induces Degradation of Prostaglandin H Synthase-2 Messenger RNA in Bovine Endometrial Cells Through a Transcription-Dependent Mechanism 1

Abstract A series of experiments were undertaken to examine the effects of interferon (IFN)-τ on regulation of prostaglandin H synthase (PGHS)-2 mRNA in bovine endometrial (BEND) cells as a means to elucidate the actions of IFN-τ to maintain pregnancy. The objective was to determine if IFN-τ mediates posttranscriptional regulation of PGHS-2 mRNA. Cells were treated with phorbol 12,13-dibutyrate (PdBu) for 3 h to induce PGHS-2 mRNA expression. Actinomycin D (0 or 1 μg/ml) or the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580 (1 μM), were added at 3 h, followed by addition of IFN-τ (0 or 50 ng/ml) at 3.5 h and extraction of RNA at 4.5 h. The concentrations of PGHS-2 mRNA were stable between 3 and 4.5 h regardless of actinomycin D. Simultaneous treatment of PdBu-treated cells with actinomycin D and SB203580 (1 μM) decreased PGHS-2 mRNA. Addition of IFN-τ (50 ng/ml) reduced PGHS-2 mRNA, which was not observed when actinomycin D was present. Concurrent treatments of cells with SB203580 and IFN-τ (5 ng/ml) decreased concentrations of PGHS-2 mRNA in an additive manner. Although IFN-τ reduced PGHS-2 mRNA concentrations, phosphorylation of p38 MAPK was induced by IFN-τ, PdBu, and PdBu combined with IFN-τ after 10 min of treatment. Both the p38 MAPK inhibitor and IFN-τ decreased prostaglandin F2α secretion, and decreases were additive when the two were given together. In summary, activation of p38 MAPK by PdBu is required for continued presence of PGHS-2 mRNA and secretion of prostaglandin F2α in BEND cells. Interferon-τ mediates a transcription-dependent mechanism, which induces degradation of PGHS-2 mRNA. However, the consequences of an IFN-τ-induced activation of p38 MAPK warrant further investigation, because inhibition of p38 MAPK caused a degradation of PGHS-2 mRNA.

Expression profile of interferon tau–stimulated genes in ovine peripheral blood leukocytes during embryonic death

Early and efficient detection of embryonic death (ED) has a valuable impact as important as early pregnancy diagnosis in ruminants. Among early pregnancy diagnosis methods, detection of the expression of interferon tau-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) is well documented in cows and ewes. Therefore, we hypothesized that the expression profile of ISGs in PBLs might also be useful for detecting ED in these animals. For this purpose, pregnant ewes were used as an experimental model. Pregnancy was detected on Day 18 after mating by transrectal ultrasonography. Pregnant ewes were divided into a control group (sham injection on Day 18, n = 10) and ED group (treated with 75 μg synthetic PGF2α on Day 18, n = 12). PBLs and plasma were collected on Days 0 (mating day), 15, 18, 19, 20, 21, 23, and 25 by jugular venipuncture. Total RNA was isolated from PBLs. ISGs expression levels were determined by real-time polymerase chain reaction in triplicate. Electrochemiluminescence immunoassay was used to measure progesterone (P4) levels in plasma. In the ED group, the P4 level declined to less than 1 ng/mL on Day 19 and remained at a low level until the end of the study. Compared with that on Day 0, receptor transporter protein 4 (RTP4) and ISG15 expression was upregulated on Day 15 and remained high until Day 21 in both groups, and RTP4 and ISG15 mRNA levels were attenuated on Days 23 and 25 only in the ED group (P < 0.001). Myxovirus resistance 1 expression was upregulated on Day 15 and remained high until Day 23 in both groups, but was attenuated on Day 25 in the ED group (P < 0.05). The B2-microglobulin mRNA level did not change significantly during the study in either group. These results indicate that the decline in P4 concentration was an immediate response to PGF2α and that the embryo may have survived longer than the CL on the basis of the extended period of ISGs expression. This suggests that the absence of P4 could be the reason for ED rather than a direct effect of PGF2α. In conclusion, the expression of ISGs, including ISG15, RTP4, and myxovirus resistance 1, but not B2-microglobulin, in PBLs may serve as a marker of ED.