Equine endometrial gene expression changes during and after maternal recognition of pregnancy.

The mechanism for maternal recognition of pregnancy (MRP) in horses is unknown. To maintain a pregnancy, a mobile conceptus must be recognized by the uterus before d 14 postovulation (PO). This recognition prevents endometrial secretion of PGF2α on d14 through 16, which would otherwise initiate luteolysis. The objective of this study was to evaluate gene expression in the endometrium of pregnant and nonpregnant mares during and after MRP to identify possible genes involved during this time. Twelve normally cycling mares were used in a crossover design and randomly assigned to a specific collection day. Endometrial samples were collected from a pregnant and nonpregnant (nonmated) mare on cycle d 12, 14, 16, and 18 (n = 3/d) PO. Microarray analysis comparing the endometrial gene expression in pregnant and nonpregnant mares revealed no differences at d 12. Ten genes were identified to have consistently higher or lower expression levels in the endometrium from pregnant versus nonpregnant mares on d 14, 16, and 18 (P < 0.001). The expression of these 10 genes was further analyzed with real-time PCR. d 14, 16, and 18 gene expression patterns were consistent with the microarray analysis, but on d 12, 4 of the 10 were identified as differentially expressed. Endometrial samples were then collected on d 13 PO (n = 3) and processed for western blot and immunohistochemical analysis of 2 proteins due to their reproductive significance. SPLA2 and DKK1 antibody specificity were confirmed via western blot analysis but were not different in samples from pregnant and nonpregnant mares (P = 0.114 and P = 0.514, respectively) and cellular localization was examined by immunohistochemical analysis. This is the first study to describe gene expression and cellular localization in the endometrium at the time of MRP for these genes and suggests that the uterus does not prepare to support a pregnancy until d 14. The function of these genes may be critical in the process of MRP.

Impact of preovulatory estradiol concentrations on conceptus development and uterine gene expression.

This experiment was conducted to determine the effect of altering preovulatory estradiol concentrations, through manipulation of length of proestrus, on peripheral progesterone concentrations, conceptus development, interferon tau (IFNT) production and uterine gene expression in cattle. Approximately 6 days after a time-synchronized ovulation, all antral follicles (≥5 mm) were ablated from the ovaries in beef heifers. To manipulate preovulatory estradiol concentrations, the length of proestrus prior to the GnRH-induced LH surge was altered between treatments. Heifers were administered PGF(2α) either -2.5 days (2.5 days of proestrus; HiE2; n=5) or -1.5 days (1.5 days of proestrus; LoE2; n=5) prior to GnRH (Day 0 of the experiment; 6.75 days after follicle ablation). Follicular dynamics and estradiol concentrations were evaluated during proestrus and progesterone concentrations were analyzed in the subsequent estrous cycle. On Day 7, embryos were transferred into all heifers using standard procedures. On Day 15.5 heifers were slaughtered, the reproductive tract was flushed to collect the conceptus and uterine flush media, and the uterine tissue was processed for subsequent analyses. Peripheral progesterone concentrations, conceptus development and IFNT production were similar between treatments. However, amount of nuclear progesterone receptor in the deep glandular epithelium and mRNA concentrations for estradiol receptor alpha (ESR1) in the uterine endometrium were less in the LoE2 than HiE2 treatment. These changes in uterine characteristics in heifers with lower preovulatory estradiol concentrations were not related to aspects of conceptus development monitored, however, it is speculated that the alterations in mRNA and receptor protein detected may contribute to pregnancy failure subsequent to day 15.5 of gestation.

Endocrine actions of interferon-tau in ruminants.

The ovine conceptus releases interferon-tau (IFNT), which prevents upregulation of the endometrial estrogen receptor (ESR1) and, consequently, oxytocin receptor (OXTR), thereby disrupting pulsatile release of prostaglandin F2alpha (PGF) in response to oxytocin. IFNT, through paracrine action on the endometrium, protects the corpus luteum (CL) during maternal recognition of pregnancy. Pregnancy also induces IFN stimulated genes (ISGs) in peripheral blood mononuclear cells (PBMCs), which is interpreted to reflect a "prompted" antiviral and immune cell response peripherally in ruminants. IFNT was recently demonstrated to be released from the uterus in amounts of 200 microg (2 x 10(7) U)/24 h via the uterine vein and to induce ISGs in the CL during maternal recognition of pregnancy. Delivery of recombinant ovine (ro) IFNT into the uterine vein in a location that is upstream of the utero-ovarian plexus from Day 10 to 17 maintained serum progesterone concentrations and extended normal 16-17 d estrous cycles to beyond 32 d. It is concluded from these studies that IFNT is released into the uterine vein and initiates a peripheral antiviral response to protect pregnancy from maternal viral infection. It also may have endocrine action through inducing luteal resistance to PGF and longer-term survival of the CL and maintenance of pregnancy.

Regulation of interferon-stimulated genes in peripheral blood leukocytes in pregnant and bred, nonpregnant dairy cows.

In ruminants, pregnancy results in up-regulation of a large number of IFN-stimulated genes (ISG) in the uterus. Recently, one of these genes was also shown to increase in peripheral blood leukocytes (PBL) during early pregnancy in sheep. Our working hypothesis is that conceptus signaling activates maternal gene expression in PBL in dairy cattle. The objectives of this study were to characterize ISG expression in PBL from pregnant (n = 20) and bred, nonpregnant (n = 30) dairy cows. Steady-state levels of mRNA for Mx1, Mx2, beta2-microglobulin, ISG-15, IFN regulatory factor-1, and IFN regulatory factor-2 were quantified. Holstein cows were synchronized to estrus and artificially inseminated (d 0). Blood samples were collected (coccygeal venipuncture) on d 0 and 16, 18, and 20 d after insemination for progesterone analysis and PBL isolation. Pregnancy was confirmed by transrectal ultrasonography at approximately 40 d after breeding. A status x day interaction was detected for Mx1, Mx2, and ISG-15 gene expression. When analyzed within day, levels of mRNA for ISG-15 and Mx1 were greater in pregnant compared with bred, nonpregnant cows on d 18 and 20, respectively. Expression of the Mx2 gene increased in the pregnant group compared with bred, nonpregnant cows on d 16, 18, and 20 after insemination. beta2-Microglobulin, IFN regulatory factor-1, and IFN regulatory factor-2 were not different between groups. The results clearly indicated that components of the innate immune response are activated in PBL during the period of pregnancy recognition and early embryo signaling. The physiological implications of these changes on maternal immune function are as yet unknown; however, they do provide a unique opportunity to identify bred, nonpregnant, cows 18 d after insemination in dairy cattle.

Inhibition of phorbol ester-induced PGF2alpha secretion by IFN-tau is not through regulation of protein kinase C.

Inhibitory effect of IFN-tau on phorbol ester (PdBu)-induced PGF2alpha secretion was hypothesized to be manifested by the regulation of protein kinase C (PKC) in bovine endometrial (BEND) cells. Following 12 h stimulation with PdBu, cells were unresponsive to freshly added PdBu. Pretreatment of cells with a PKC inhibitor abolished PGF2alpha secretion in response to PdBu. Therefore, PdBu induction of PGF2alpha secretion is through activation of PKC. The alpha, epsilon, iota and lambda isotypes of the PKC family were identified by Western blotting. Cells were then treated with medium alone (control), PdBu or PdBu + IFN-tau for 3 or 6 h. The PdBu-induced secretion of PGF2alpha was suppressed by IFN-tau. At 3 and 6 h, PKCalpha and PKCepsilon were detected both in the cytosolic and membrane fractions of unstimulated cells. There was a clear reduction of PKCalpha in the cytoplasm induced by PdBu and PdBu + IFN-tau at 3 and 6 h. The total abundance (cytoplasm and membrane fractions) of PKCalpha was lower in the PdBu + IFN-tau than PdBu alone. These temporal responses indicate a PKCalpha responsiveness of BEND cells to PdBu and PDBu + INF-tau with some evidence that IFN-tau causes a slight but detectable reduction in PKCalpha when added with PdBu. However, IFN-tau-induced decrease in the total abundance of PKCalpha was not enough to affect negatively the translocation of the PKCalpha to the membrane. Therefore, IFN-tau's ability to suppress secretion of PGF2alpha is unlikely due to an interference with the PdBu-induced activity of PKC.

Expression of CA-125 by progestational bovine endometrium: prospective regulation and function.

Cancer antigen 125 (CA-125) is expressed by malignant human ovarian surface epithelial cells and derivatives of the Müllerian duct system. This study explored the expression, regulation, and function of CA-125 in the bovine uterus. CA-125 was localized by immunohistochemistry to the apical surfaces of epithelial cells lining the endometrium and proximal glands of the late luteal phase and early pregnancy; antigen was not detected during oestrus or the postpartum period. Production of CA-125 by bovine endometrial cells in vitro was upregulated by progesterone and interferon-tau. Immunopurified CA-125 from uterine flushes of dioestrous or pregnant cows was similar in biochemical composition (as determined by gel electrophoresis and amino acid content) to the human antigen isolated from incubation medium conditioned by the ovarian cancer cell line OVCAR-3. Bovine CA-125 inhibited complement-induced lysis of antibody-sensitized sheep erythrocytes. It is suggested that endometrial CA-125 exerts a progestational role in part by protecting maternal and embryonic cells from immune targeting and lysis.

[Spontaneous abortion. Drug treatment versus surgery]. / Spontan abort. Medicinsk versus kirurgisk behandling.

INTRODUCTION: Studies of conservative management of early miscarriage have questioned the need for post abortem curettage. METHODS: A prospective, randomised study was carried out to clarify the effect of vaginal administration of a prostaglandin E1 analogue (gemeprost) versus surgical management (curettage) of miscarriages at up to twelve weeks of gestation. A questionnaire revealed discomfort as bleeding and pain. RESULTS: The study comprised 61 patients: group 1 (n: 27) with an endometrial thickness less than 10 mm managed by expectancy, and group 2 with an endometrial thickness greater than 10 mm; group 2 was randomised to group 2A (n: 17), given gemeprost, and group 2B (n: 17), underwent curettage. On entry the mean gestational ages were 51 and 67.5 days for groups 1 and 2, respectively; transvaginal ultrasonography revealed a mean endometrial thickness of 8 mm in group 1 and 19 mm in group 2. One week later this was reduced to 4 mm in group 1 and 5.7 mm in group 2. The duration of vaginal bleeding was similar in all groups, with a mean of 1 week (2-3 days of moderate/heavy bleeding and 6-10 of no bleeding or spotting). The discomfort experienced was similar in all groups (a mean of 36-48 hours of moderate/strong pain and 7-10 days of no or insignificant pain). DISCUSSIONS: Conservative treatment can substitute general anesthesia and curettage in the management of complete spontaneous abortions with fresh vaginal bleeding and an endometrial thickness of up to 10 mm. Vaginal administration of 1 mg gemeprost can substitute general anesthesia and curettage in the management of incomplete spontaneous abortions of up to 12 weeks of gestation and absence of a gestation sac.

Interferon-tau suppresses prostaglandin F2alpha secretion independently of the mitogen-activated protein kinase and nuclear factor kappa B pathways.

Pregnancy is established in ruminants through inhibitory actions of interferon (IFN)-tau on the release of prostaglandin F2alpha (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-tau attenuated phorbol ester (PDBu)-induced secretion of PGF, and 3) establish the site at which rbIFN-tau 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-tau (50 or 500 ng/ml), PDBu + rbIFN-tau, or PDBu + PD98059 (MEK-1 inhibitor; 50 microM). Secretion of PGF was induced (P < 0.0001) by PDBu within 180 min, but induction was inhibited 74% by the addition of rbIFN-tau (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-tau. 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-tau. Treatment of BEND cells with rbIFN-tau also did not attenuate PDBu-induced degradation of IkappaBalpha, suggesting that the IkappaBalpha/NFkappaB pathway is not a site of IFN-tau inhibition of PGF. However, rbIFN-tau 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-tau. Because rbIFN-tau inhibits COX-2 mRNA within 30 min, we hypothesized that transcription factors activated by rbIFN-tau rapidly and directly attenuate COX-2 gene expression, thereby suppressing secretion of PGF.

Bovine interferon-tau stimulates the Janus kinase-signal transducer and activator of transcription pathway in bovine endometrial epithelial cells.

Trophoblastic bovine interferon-tau (bIFN-tau) suppresses luteolytic pulses of endometrial prostaglandin F(2alpha) (PGF(2alpha)) at the time of maternal recognition of pregnancy. This results in maintenance of the corpus luteum in cattle. The hypothesis that effects of bIFN-tau in the endometrium were through activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway of signal transduction was tested. Whole cell, cytosolic, and nuclear extracts from bovine endometrial cells treated with bIFN-tau were analyzed by immunoprecipitation, immunoblotting, and electrophoretic mobility shift assays in a series of dose- and time-dependency experiments. Bovine IFN-tau stimulated tyrosine phosphorylation, homo- and heterodimer formation, nuclear translocation, and DNA binding of STAT proteins 1, 2, and 3. Moreover, bIFN-tau induced synthesis of interferon-regulatory factor. In conclusion, bIFN-tau stimulates the JAK-STAT pathway in the bovine endometrium. It is proposed that activation of the JAK-STAT pathway is involved in regulating the antiluteolytic effects of bIFN-tau.

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.

Interferon-tau modulates phorbol ester-induced production of prostaglandin and expression of cyclooxygenase-2 and phospholipase-A(2) from bovine endometrial cells.

Antiluteolytic actions of bovine interferon-tau (bIFN-tau) require suppression of prostaglandin F(2 alpha) (PGF(2 alpha)) production. Our objective was to test whether bIFN-tau could block PGF(2 alpha) production and synthesis of phospholipase A(2) (PLA(2)) 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-tau. Medium samples were analyzed for concentrations of PGF(2 alpha), whole-cell extracts were analyzed for abundance of PLA(2) 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 PGF(2 alpha) between 3 and 12 h, levels of COX-2 mRNA by 3 h and protein expression of COX-2 and PLA(2) by 6 and 12 h, respectively. Added concomitantly with PDBu, bIFN-tau suppressed PGF(2 alpha) production, steady-state levels of COX-2 mRNA, and expression of COX-2 and PLA(2) proteins. Added after a 3-h stimulation with PDBu alone, bIFN-tau suppressed PGF(2 alpha) production after 1 h. Bovine IFN-tau inhibited intracellular mechanisms responsible for PGF(2 alpha) production in BEND cells, and this could be through both cytosolic and nuclear actions.

Production, purification, and carboxy-terminal sequencing of bioactive recombinant bovine interferon-stimulated gene product 17.

An interferon (IFN)-stimulated gene (ISG) encodes a bovine 17-kDa protein (bISG17) that is released from endometrial cells but also conjugates to intracellular proteins through a ubiquitinlike mechanism. During early pregnancy in ruminants, conceptus-derived IFN-tau induces endometrial ISG17. The present experiments were designed to generate bioactive recombinant (r) bISG17. The Pichia pastoris yeast expression system was used because previous experiments expressing the human ISG15 ortholog in bacteria were confounded by inherent carboxypeptidase activity that cleaved C-terminal residues resulting in an inactive protein. In a series of extensive yeast culture experiments using shaker-bath and fermentation approaches, optimal conditions were determined for a transformant containing a multi-ISG17 gene insertion. Recombinant bISG17 was purified. Carboxy-terminal sequencing revealed that rbISG17 retained the C-terminal Gly that is potentially critical for the first step in covalent attachment to targeted intracellular proteins. The rISG17 induced (P < 0.0001) IFN-gamma mRNA (reverse transcription-polymerase chain reaction) and release of IFN-gamma protein (ELISA) by bovine peripheral blood mononuclear cells. The IFN-gamma mRNA also was upregulated (P < 0.0001) in endometrium from pregnant (Day 18) when compared with nonpregnant (Days 14 and 18) cows. It is concluded that rbISG17 generated in a yeast expression system retains cytokine/hormonal activity. This is the first description coupling the biology of two distinct IFNs (gamma and tau) through the intermediary ubiquitin homolog ISG17.

Endometrial ISG17 mRNA and a related mRNA are induced by interferon-tau and localized to glandular epithelial and stromal cells from pregnant cows.

The interferon stimulated gene product, ISG17, conjugates to bovine uterine proteins in response to conceptus-derived interferon (IFN)-tau. The objectives of the present experiments were to examine induction of ISG17 (0.65 kb) and a related 2.5 kb mRNA in response to IFN-tau and pregnancy using Northern blotting procedures, and to determine cell types in the endometrium that expressed ISG17 mRNA using in situ hybridization. RNA was isolated from endometrial explants or from bovine endometrial (BEND) cells cultured in the absence (control) or presence of 25 nM recombinant (r) bolFN-tau for 0, 3, 6, 12, 24, or 48 h. The major ISG17 0.65 kb mRNA and a minor 2.5 kb mRNA were induced (p<0.05) after 6 h (explants) or 3 h (BEND cells) treatment with rboIFN-tau. Both mRNAs were present in endometrium from day 18 pregnant cows, but were absent in endometrium from nonpregnant cows. The ISG17 mRNA was localized to stromal and glandular epithelial cells on d 18 of pregnancy. The 2.5 kb mRNA may encode a novel ISG17 homolog, or a unique polyISG17 repeat that is similar in structure to the polyubiquitin genes. Because ISG17 mRNA is induced in stromal and glandular epithelial cells, it could be assumed that ISG17 has a role in regulating intracellular proteins in both cell types.

Cloning of interferon-stimulated gene 17: the promoter and nuclear proteins that regulate transcription.

A member of the interferon-stimulated gene (ISG) family encodes a 17-kDa ubiquitin homolog called ISG17 that is induced in the bovine uterine endometrium by interferon-tau (IFN-tau) during early pregnancy. The bovine (b) ISG17 cDNA shares 30% identity with a tandem ubiquitin repeat and 70% identity with human (h) ISG15. The present experiments were designed to sequence the bISG17 gene, compare general structure with the hISG15 gene, and to identify transcription factors that were induced by IFN-tau in bovine endometrial (BEND) cells. The promoter of the bISG17 gene was similar to the hISG15 gene in placement of a tandem IFN-stimulatory response element (ISRE) at position -90, but unique in the presence of three additional ISREs at positions -123, -332, and -525. IFN-tau (25 nM) induced nuclear proteins in BEND cells that interacted with a tandem bISG17 ISRE in electrophoretic mobility shift assay (EMSA). IFN-regulatory factor-1 (IRF-1) bound to this ISRE based upon supershift EMSA using antiserum against IRF-1. IFN-tau activated STAT-1 (signal transducer and activator of transcription-1) and -2 by 0.5 h, and IRF-1 by 2 h in BEND cells. It is concluded that the bISG17 gene is similar to the hISG15 gene, retains an ISRE that interacts with IRF-1, and is possibly induced initially by the STATs and later by IRF-1 in response to IFN-tau during early pregnancy.

Expression of the interferon tau inducible ubiquitin cross-reactive protein in the ovine uterus.

Ubiquitin cross-reactive protein (UCRP) is a 17-kDa protein that shows cross-reactivity with ubiquitin antisera and retains the carboxyl-terminal Leu-Arg-Gly-Gly amino acid sequence of ubiquitin that ligates to, and directs degradation of, cytosolic proteins. It has been reported that bovine endometrial UCRP is synthesized and secreted in response to conceptus-derived interferon-tau (IFNtau). In the present studies, UCRP mRNA and protein were detected in ovine endometrium. Ovine UCRP mRNA was detectable on Day 13, peaked at Day 15, and remained high through Day 19 of pregnancy. The UCRP mRNA was localized to the luminal epithelium (LE), stromal cells (ST) immediately beneath the LE, and shallow glandular epithelium (GE) on Day 13, but it extended to the deep GE, deep ST, and myometrium of uterine tissues by Day 15 of pregnancy. Western blotting revealed induction of UCRP in the endometrial extracts from pregnant, but not cyclic, ewes. Ovine UCRP was also detected in uterine flushings from Days 15 and 17 of pregnancy and immunoprecipitated from Day 17 pregnant endometrial explant-conditioned medium. Treatment of immortalized ovine LE cells with recombinant ovine (ro) IFNtau induced cytosolic expression of UCRP, and intrauterine injection of roIFNtau into ovariectomized cyclic ewes induced endometrial expression of UCRP mRNA. These results are the first to describe temporal and spatial alterations in the cellular localization of UCRP in the ruminant uterus. Collectively, UCRP is synthesized and secreted by the ovine endometrium in response to IFNtau during early pregnancy. Because UCRP is present in the uterus and uterine flushings, it may regulate endometrial proteins associated with establishment and maintenance of early pregnancy in ruminants.

Pregnancy-specific protein B induces release of an alpha chemokine in bovine endometrium.

Pregnancy-specific protein B (PSPB), is secreted from binucleate trophoblast of the bovine conceptus as early as day 15 of pregnancy. The objective of this experiment was to determine if PSPB induced uterine proteins. PSPB was purified from day 120 cotyledons using antibody-based affinity chromatography. Endometrium from day 14 nonpregnant cows (n = 3) was prepared for explant (3H-Leu added) culture. Radiolabeled proteins released into medium were dialyzed, separated using 1D-PAGE, and detected using fluorography and densitometry. PSPB (0, 0.5, 5, 25 & 50 nM) caused a concentration-dependent increase in the release of a radiolabeled 8-kDa uterine protein. Western blots revealed that the 8-kDa protein cross-reacted with antibody against granulocyte chemotactic protein-2 (GCP-2). PSPB also induced release of GCP-2 by bovine endometrial (BEND) cells in primary culture. The induction of GCP-2 by PSPB was blocked by addition of antiserum against PSPB (1:4 molar ratio). This is the first indication that PSPB has a hormonal role in inducing GCP-2, an alpha chemokine that also is induced by interferon-tau during early pregnancy. This chemotactic cytokine may be integral to mediating adhesion, inflammation and angiogenesis associated with early implantation.

Mechanism of action of interferon-tau in the uterus during early pregnancy.

Early pregnancy is maintained in ruminants through the actions of conceptus-derived interferon (IFN)-tau on the endometrium. IFN-tau alters uterine release of PGF2 alpha' which results in rescue of the corpus luteum and continued release of progesterone. The mechanism of action of IFN-tau includes inhibition of oestradiol receptors, consequent reduction in oxytocin receptors, activation of a cyclooxygenase inhibitor, and a shift in the PGs to favour PGE2 over PGF2 alpha' IFN-tau also induces several endometrial proteins that may be critical for survival of the developing embryo. One endometrial protein induced by pregnancy and IFN-tau has been identified as bovine granulocyte chemotactic protein-2 (bGCP-2). This chemotactic cytokine (chemokine) has been used as a marker to delineate IFN-tau from IFN-alpha responses in the endometrium. A second protein, called ubiquitin cross-reactive protein (UCRP), resembles a tandem ubiquitin repeat. UCRP becomes conjugated to cytosolic endometrial proteins in response to IFN-tau and pregnancy. Proteins conjugated to UCRP are either modulated or targeted for processing through the proteasome. The action of IFN-tau is mediated by induction of signal transducer and activator of transcription 1 (STAT-1), STAT-2 and interferon regulatory factor 1 (IRF-1) transcription factors. Induction of these transcription factors, the alpha chemokines and UCRP is the prelude to maternal recognition of pregnancy in ruminants.

Complex induction of bovine uterine proteins by interferon-tau.

Interferon-tau (IFN-tau) is released by the conceptus and induces two uterine proteins during early pregnancy: ubiquitin cross-reactive protein (UCRP) and granulocyte chemotactic protein-2 (GCP-2). The present experiments were designed to determine whether detection (Western blot) of cytosolic UCRP and release of GCP-2 could be used to examine IFN-tau signal transduction in cultured endometrial explants and primary epithelial cells. Recombinant (r) type 1 IFNs (rboIFN-tau and rboIFN-alpha; 5, 25, 100 nM) induced UCRP, but only rboIFN-tau induced GCP-2 in explant culture. Recombinant boIFN-tau and conceptus secretory proteins containing native IFN-tau induced UCRP and GCP-2 in cultured primary epithelial cells. All concentrations of rboIFN-alpha (25, 50, 100 nM) induced UCRP, but only the highest concentration induced GCP-2 in cultured primary epithelial cells. Interestingly, phorbol ester (100, 500, 1000 ng/ml) induced GCP-2, but it had no effect on UCRP. Because type 1 IFNs induce UCRP, IFN-tau probably interacts with the janus kinase (Jak)-associated IFN-alpha receptor to phosphorylate signal transducers and activators of transcription (STAT) and/or interferon regulatory factor-1 (IRF-1). However, IFN-tau-specific induction of GCP-2 may involve a variant type 1 receptor subunit or activators of transcription that are associated with protein kinase C and the Jak/STAT/IRF-1 pathway.

Inhibition by tocopherol of prostaglandin-induced apoptosis in ovine corpora lutea.

Accumulation of toxic oxidants within corpora lutea is a prelude of apoptotic cell death. Vitamin E (alpha-tocopherol) is a biological antioxidant that protects cells from the inductive effects of reactive oxygen on DNA damage and nuclear/cytoplasmic condensation that dictate apoptosis. Ewes were challenged with a luteolytic dose of PGF2 alpha on d 10 of the estrous cycle. The acute decline in circulatory progesterone indicative of the onset of functional luteolysis was not affected by systemic administration of alpha-tocopherol; however, corpora lutea consequently (beyond 24 h) rebounded from the steroidogenic insult. Luteal tissues obtained at 24 h after PGF2 alpha revealed that internucleosomal DNA fragmentation and cellular collapse were inhibited by alpha-tocopherol. These observations indicate that regressive corpora lutea can be spared from terminal involution by diminishing the apoptotic influence of luteolytic hormone with an antioxidant.

Transient ubiquitin cross-reactive protein gene expression in the bovine endometrium.

Bovine ubiquitin cross-reactive protein (boUCRP) is secreted by the endometrium from days 15 to 26 of pregnancy in response to conceptus-derived interferon-tau (IFN-tau). We hypothesized that the gene encoding boUCRP was under transcriptional control by the conceptus and IFN-tau. Northern blots using radiolabeled UCRP cDNA revealed a single UCRP transcript of approximately 700 b that was present (P < 0.05) in endometrial cells cultured with 25 nM rboIFN-tau. The UCRP mRNA was not detected in endometrium on days 15, 17, 18 or 19 of the estrous cycle (n = 4 cows on each day) or in spleen, kidney, liver, corpus luteum or muscle. Bovine UCRP mRNA was detectable (P < 0.05) in endometrium from pregnant cows by day 15, reached highest levels by day 17, remained elevated on days 18, 19 and 21, and then declined to amounts on day 26 that were not detectable. Northern blot using radiolabeled ubiquitin cDNA revealed presence of the two major ubiquitin transcripts UbB (1.2 Kb) and UbC (2.6 Kb) in all tissues examined. The bovine UCRP cDNA did not cross-hybridize with these ubiquitin transcripts. We conclude that transcription of the UCRP gene is transient during early pregnancy and regulated by IFN-tau.