Recognition Of Pregnancy In Ruminants Research Articles

Early transcriptomic changes in peripheral blood 7 days after embryo transfer in dairy cattle

A common goal of the dairy industry is to shorten the calving interval to reap several benefits associated with improved fertility. Early pregnancy detection is crucial to shorten this interval, allowing for prompt re-insemination of cows that failed to conceive after the first service. Currently, the industry lacks a method to accurately predict pregnancy within the first 3 wk. The polypeptide cytokine IFN-tau (IFNT) is the primary signal for maternal recognition of pregnancy in ruminants. As IFNT is released from the early conceptus, it initiates a cascade of effects, including upregulation of IFN-stimulated genes (ISG). Expression of ISG can be detected in the peripheral blood. The present study aimed to characterize peripheral transcriptomic changes, including the ISG, as early as d 7 after embryo transfer. A total of 170 Holstein heifers received in vitro-produced embryos. Whole blood was collected from these heifers within 24 h of the embryo transfer (d 0), d 7, and d 14 after embryo transfer. The heifers were divided into 2 groups, pregnant and nonpregnant, based on pregnancy diagnosis on d 28 via ultrasound. Total RNA was extracted from the peripheral blood of pregnant and nonpregnant heifers, pooled and sequenced. Expression analysis on d 7 heifers resulted in 13 significantly differentially expressed genes mostly related to innate immunity. Differential expression analysis comparing pregnant heifers on d 0 to the same heifers on d 14 showed 51 significantly differentially expressed genes. Eight genes were further quantified through reverse-transcription quantitative real-time PCR for biological validation. On d 7 after embryo transfer, mRNA transcriptions of EDN1, CXCL3, CCL4, and IL1A were significantly upregulated in pregnant heifers (n = 14) compared with nonpregnant heifers (n = 14), with respective fold changes of 8.10, 18.12, 29.60, and 29.97. Although on d 14 after embryo transfer, mRNA transcriptions of ISG15, MX2, OASY1, and IFI6 were significantly upregulated in the blood of pregnant heifers (n = 14) compared with the same heifers on d 0, with respective fold changes of 5.09, 2.59, 3.89, and 3.08. These findings demonstrate that several immune-related genes and ISG are activated during the first 2 wk after embryo transfer, which may explain how the maternal immune system accommodates the allogenic conceptus. To further investigate the diagnostic potentials of these genes, future studies are warranted to analyze the specificity and sensitivity of these biomarkers to predict early pregnancy.

Conceptus-modulated innate immune function during early pregnancy in ruminants: a review.

This review focuses on the innate immune events modulated by conceptus signaling during early pregnancy in ruminants. Interferon-tau (IFN-τ) plays a role in the recognition of pregnancy in ruminants, which involves more than the inhibition of luteolytic pulses of PGF2α to maintain corpus luteum function. For successful pregnancy establishment, the allogenic conceptus needs to prevent rejection by the female. Therefore, IFN-τ exerts paracrine and endocrine actions to regulate the innate immune system and prevent conceptus rejection. Additionally, other immune regulators work in parallel with IFN-τ, such as the pattern recognition receptors (PRR). These receptors are activated during viral and bacterial infections and in early pregnancy, but it remains unknown whether PPR expression and function are controlled by IFN-τ. Therefore, this review focuses on the main components of the innate immune response that are involved with early pregnancy and their importance to avoid conceptus rejection.

Arginine, Agmatine, and Polyamines: Key Regulators of Conceptus Development in Mammals.

Arginine is a key amino acid in pregnant females as it is the precursor for nitric oxide (NO) via nitric oxide synthase and forpolyamines (putrescine, spermidine, and spermine) by either arginase II and ornithine decarboxylase to putrescine or via arginine decarboxylase to agmatine and agmatine to putrescine via agmatinase. Polyamines are critical for placental growth and vascularization. Polyamines stabilize DNA and mRNA for gene transcription and mRNA translation, stimulate proliferation of trophectoderm, and formation of multinucleated trophectoderm cells that give rise to giant cells in the placentae of species such as mice. Polyamines activate MTOR cell signaling to stimulate protein synthesis and they are important for motility through modification of beta-catenin phosphorylation, integrin signaling via focal adhesion kinases, cytoskeletal organization, and invasiveness or superficial implantation of blastocysts. Physiological levels of arginine, agmatine, and polyamines are critical to the secretion of interferon tau for pregnancy recognition in ruminants. Arginine, polyamines, and agmatine are very abundant in fetal fluids, fetal blood, and tissues of the conceptus during gestation. The polyamines are thus available to influence a multitude of events including activation of development of blastocysts, implantation, placentation, fetal growth, and development required for the successful establishment and maintenance of pregnancy in mammals.

Heat stress on oocyte or zygote compromises embryo development, impairs interferon tau production and increases reactive oxygen species and oxidative stress in bovine embryos produced in vitro.

Interferon tau (IFNT) is the cytokine responsible for the maternal recognition of pregnancy in ruminants and plays a role modulating embryo-maternal communication in the oviduct inducing a local response from immune cells. We aimed to investigate IFNT production, reactive oxygen species, and oxidative stress under the influence of heat stress (HS) during different stages of bovine in vitro embryo production. HS was established when the temperature was gradually raised from 38.5°C to 40.5°C in laboratory incubator, sustained for 6 hr, and decreased back to 38.5°C. To address the HS effects on IFNT production, reactive oxygen species, and oxidative stress, ovaries from a slaughterhouse were used according to treatments: control group (38.5°C); oocytes matured under HS; oocytes fertilized under HS; zygotes cultured in the first day under HS; and cells submitted to HS at oocyte maturation, fertilization, and the first day of zygote culture. The HS negatively affected cleavage and blastocyst rates, in all HS groups. On Day 7, all HS-treated embryos showed decrease IFNT gene and protein expressions, whereas reactive oxygen species were increased in comparison to the control. In conclusion, the compromised early embryo development due to higher temperatures during in vitro oocyte maturation, fertilization, and/or zygote stage have diminished IFNT expression and increased reactive oxygen species in bovine.

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.

Physiological mechanisms of pregnancy recognition in ruminants

Maternal recognition of pregnancy in sheep, cattle and goats involves physiological mechanisms that result in protection of corpora lutea from luteolysis by modification or inhibition of uterine production of luteolytic pulses of prostaglandin (PG) F-2 alpha. Ovine, bovine and caprine luteal cells release oxytocin in a pulsatile manner during late dioestrus. Oxytocin then binds to its endometrial receptors and initiates luteolytic pulses of PGF-2 alpha. Ovine, bovine and caprine trophoblast protein-1 (oTP-1, bTP-1 and cTP-1) are secreted by the trophectoderm of conceptuses between Days 10 and 21-24 of pregnancy. These antiluteolytic proteins (oTP-1 and bTP-1) are primarily responsible for inhibiting uterine production of luteolytic amounts of PGF-2 alpha. During early pregnancy, the numbers of endometrial receptors for oxytocin are significantly lower in ewes and cows, and stimulatory effects of exogenous oxytocin on uterine production of PGF-2 alpha are correspondingly reduced or absent for ewes, cows and goats. Exogenous oestrogens can, through a uterine-dependent mechanism, stimulate synthesis of endometrial receptors for oxytocin and uterine production of PGF-2 alpha; an effect which is significantly attenuated during early pregnancy. These results suggest that oTP-1, bTP-1 and possibly cTP-1 exert their antiluteolytic effect(s) by: (1) inhibiting effects of oestrogen and/or progesterone necessary for synthesis of endometrial receptors for oxytocin; (2) inhibiting endometrial synthesis and/or recycling of oxytocin receptors directly; or (3) inducing the endometrium to synthesize an inhibitor of an enzyme(s) necessary for synthesis of PGF-2 alpha.

Analysis of Bovine Interferon-tau Gene subtypes Expression in the Trophoblast and Non-trophoblast cells

Interferon-tau (IFNT) is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants. Also, multiple interferon (IFNT) genes exist in cattle, However, molecular mechanisms of these bIFNT genes whose expressions are limited have not been characterized. We and others have observed that expression levels of bovine subtype IFNT genes in the tissues of ruminants; thus, bIFNT1 and other new type I (bIFNTc1/c2/c3) gene co-exist during the early stages of conceptus development and non-trophoblast cells. Its genes transcription could be regulated through caudal-relatedhomeobox-2 (CDX2) and ETS2 and AP1(JUN) and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Bovine earderived fibroblast (EF) cells, were co-transfected with luciferase reporter constructs carrying upstream (positions −1000 to +51) regions of bIFNT1 and other new type I (bIFNT c1/c2/c3) gene and various transcription factor expression plasmids. Compared to each -1kb-bIFNT1/c1/c2/c3-Luc increased when this constructs were co-transfected with CDX2, ETS2, AP1 and/or CREBBP. Also, Its genes was had very effect on activity by CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. However, the degree of transcriptional activation of the bIFNTc1 gene was not similar to that bIFNT1/c2/c3 gene by expression plasmid. Furthermore, RNA-seq analyses also revealed that the expression levels of bIFNT1/ c2/c3 gene mRNAs expression were highest on day 17, 20 and 22 trophoblast and Madin-Darby bovine kidney (MDBK), Bovine ear fibroblast (EF) and (Endo) non-trophoblast cells. But, bIFNTc1 mRNA had not same expression level, bIFNTc1 lowest levels than those of IFNT1/c2/c3 gene in both trophoblast and non-trophoblast cells. These results demonstrate that bovine subtype bIFNT genes display differential, in the trophoblast and non-trophoblast cells.

Changes in expression of ISG15, progesterone receptor and progesterone-induced blocking factor in ovine thymus during early pregnancy

Interferon-tau (IFNT) is the main signal for the maternal recognition of pregnancy in ruminants, and exerts its effects by stimulating the expression of interferon-stimulated genes, including the expression of interferon-stimulated gene15 kDa protein (ISG15). Progesterone (P4) exerts significant immune effects on the uterus during early pregnancy in ruminants that are partly mediated by progesterone-induced blocking factor (PIBF). The thymus is necessary for the normal development of immunologic function. In this study, thymuses were obtained on day 16 of the estrous cycle and on days 13, 16 and 25 of pregnancy (n = 6 for each group) from ewes. Our results showed that the expression of ISG15, P4 receptor (PGR) and PIBF mRNA and the expression of ISG15 and ISG15-conjugated proteins were upregulated in the thymuses during early pregnancy, and the 89-kDa PGR isoform and the 80-kDa PIBF variant were expressed constantly in the thymuses. However, there was no expression of the 60-kDa PGR isoform and the 62-kDa PIBF variant on day 16 of the estrous cycle. ISG15 and ISG15-conjugated proteins were limited to the epithelial reticular cells, capillaries and thymic corpuscles. This paper reports for the first time that early pregnancy exerts its effects on the thymus through IFNT and P4 in sheep.

Interferon-τ regulates prostaglandin release in goat endometrial stromal cells via JAB1 - unfolded protein response pathway

Prostaglandins (PGs) are major products of the uterine endometrium, and they are critical for recognition of pregnancy in ruminants. During the peri-implantation period of pregnancy, interferon tau (IFN-τ) plays an important role in the regulation of endometrial PGs synthesis, but the underlying mechanisms remain poorly understood. In this work, the results demonstrated that IFN-τ increased the PGE2/PGF2α ratio, up-regulated the expression of JAB1 and activated the unfolded protein response (UPR). Knockdown of JAB1 reduced the PGE2/PGF2α ratio and inhibited the expression of UPR markers in endometrial stromal cells (ESCs) under IFN-τ treatment. Pre-treatment with endoplasmic reticulum (ER) stress activator thapsigargin (Tg) activated UPR and restored the PGE2/PGF2α ratio in shJAB1 groups under IFN-τ treatment. In conclusion, our results indicated that IFN-τ regulated the PGE2/PGF2α ratio via cooperation between JAB1 and UPR, and the reduction of JAB1 led to the down-regulation of the PGE2/PGF2α ratio, which inhibits UPR, and thus is harmful to early pregnancy. Activation of UPR could restore JAB1 reduction, resulting in a reduced PGE2/PGF2α ratio. These findings extend our understanding and may provide new insights into the mechanism of IFN-τ regulation of PG secretion in ESCs and the biological functions of JAB1 and UPR.

Regulation of conceptus interferon-tau gene subtypes expressed in the uterus during the peri-implantation period of cattle

Conceptus interferon tau (IFNT), produced by the embryonic trophectoderm, is known as a major signaling protein essential for the process of maternal recognition of pregnancy in ruminants. Similar to other IFN gene families such as IFNA and IFNB, multiple IFNT genes exist. The number of IFNT genes actively transcribed and regulated in conceptuses of cattle has, however, not been well characterized. In this study, IFNT transcripts in utero were studied through the use of next generation sequencer. Among 38 IFN genes registered and eight annotated as IFNT, only two transcripts, IFNT1 and IFNTc1, were found in conceptuses in utero. Relative abundance of transcription factor(s) involved in the regulation of IFNT genes were investigated by real-time PCR. Transcriptional activity of IFNT1 and IFNTc1 were investigated using bovine non-trophoblast ear fibroblast (EF) cells, which were co-transfected with luciferase reporter constructs with upstream (−631 to −51) promoter regions of IFNT1 or IFNTc1 and various transcription factor expression plasmids, CDX2, AP1 (JUN), ETS2 and/or CREBBP. CDX2 with AP1 and ETS2 was found to increase luciferase activity of IFNT1 and IFNTc1 approximately 14- and 11-fold, respectively, in EF cells, which do not express the CDX2 gene. These results indicated that two isoforms of conceptus IFNT genes of cattle could be regulated differently in utero. Furthermore, IFNT1 and IFNTc1 were found to have similar antiviral activity, suggesting that both IFNT genes could function to increase conceptus signaling to the uterine endometrium for the process of maternal recognition of pregnancy during the pre-implantation period.

Interferon-tau and fertility in ruminants.

Establishment of pregnancy in domestic ruminants includes pregnancy recognition signalling by the conceptus, implantation and placentation. Despite the high fertilisation success rate in ruminants, a significant amount of embryo loss occurs, primarily during early gestation. Interferon-tau (IFNT), a type I interferon that is exclusively secreted by the cells of the trophectoderm of the ruminant conceptus, has been recognised as the primary agent for maternal recognition of pregnancy in ruminants. It produces its antiluteolytic effect on the corpus luteum by inhibiting the expression of oxytocin receptors in the uterine epithelial cells, which prevents pulsatile, luteolytic secretion of prostaglandin F2α by the uterine endometrium. While the importance of IFNT in maternal recognition of pregnancy and prevention of luteolysis in ruminants is unequivocal, important questions, for example, relating to the threshold level of IFNT required for pregnancy maintenance, remain unanswered. This paper reviews data linking IFNT with measures of fertility in ruminants.

Interferon-τ increases BoLA-I for implantation during early pregnancy in dairy cows.

Interferon-τ (IFN-τ) signals pregnancy recognition in ruminants. We investigated the effects of IFN-τ produced by embryo trophoblastic cells (ETCs) on expression of bovine leukocyte antigen-I (BoLA-I), a bovine analogue of human MHC-I, in endometrial luminal epithelial cells (EECs) during early pregnancy in dairy cows. Expression of IFN-τ and BoLA-I was increased in endometrial tissues during early pregnancy. Expression of the anti-inflammatory cytokine IL-10 was increased in endometrial tissues, while expression of the pro-inflammatory cytokine IL-6 was decreased, indicating immunosuppression. Progesterone increased IFN-τ expression in EECs. IFN-τ increased p-STAT1 and p-STAT3 levels in EECs, but reduced TRAF3 levels. In addition, IFN-τ increased expression of BoLA-I and IL-10, but decreased expression of IL-6 in EECs. These results indicate that IFN-τ enables stable implantation in dairy cows by increasing expression of BoLA-I, and by immunosuppression mediated by increased IL-10 and decreased IL-6 expression.

Reconhecimento materno da gestação em animais de produção

O reconhecimento materno da gestação é o período em que o concepto sinaliza a sua presença para a mãe. Esse é um importante momento da gestação, que pode ser decisivo para o progresso da prenhez. Durante a gestação, ocorre uma série de eventos hormonais, envolvendo progesterona e prostaglandinas, sendo fundamental a função do corpo lúteo, uma glândula temporária formada a partir da ovulação de um folículo. O corpo lúteo produz a progesterona, esteroide envolvido tanto na ciclicidade ovariana quanto no estabelecimento e manutenção da gestação, na maioria dos mamíferos. Algumas particularidades dos eventos envolvidos na relação materno-fetal durante o reconhecimento da gestação em espécies domésticas foram abordadas nesta revisão; entre estas, foi descrito o papel fundamental da proteína interferon-tau (INF-T) com ação antiluteolítica comprovada e que está associada ao reconhecimento materno da gestação em ruminantes; a movimentação do embrião equino, mantendo a sua sinalização; os estrogênios produzidos pelos conceptos suínos com função luteotrófica para a espécie, mas que também possui função luteolítica em outras espécies.

Transcriptional Regulation of Two Conceptus Interferon Tau Genes Expressed in Japanese Black Cattle during Peri-Implantation Period

Interferon tau (IFNT), produced by the mononuclear trophectoderm, signals the process of maternal recognition of pregnancy in ruminants. However, its expression in vivo and its transcriptional regulation are not yet well characterized. Objectives of this study were to determine conceptus IFNT gene isoforms expressed in the bovine uterus and to identify differences in promoter sequences of IFNT genes that differ in their expression. RNA-seq data analysis of bovine conceptuses on days 17, 20, and 22 (day 0 = day of estrus) detected the expression of two IFNT transcripts, IFNT1 and IFNTc1, which were indeed classified into the IFNT gene clade. RNA-seq and quantitative RT-PCR analyses also revealed that the expression levels of both IFNT mRNAs were highest on day 17, and then decreased on days 20 and 22. Bovine ear-derived fibroblast (EF) cells, a model system commonly used for bovine IFNT gene transcription study in this laboratory, were cotransfected with luciferase reporter constructs carrying upstream (positions −637 to +51) regions of IFNT1 or IFNTc1 gene and various transcription factor expression plasmids including CDX2, AP-1 (Jun) and ETS2. CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. The upstream regions of IFNT1 and IFNTc1 loci were then serially deleted or point-mutated at potential CDX-, AP-1-, and ETS-binding sites. Compared to the wild-type constructs, deletion or mutation at CDX2 or ETS2 binding sites similarly reduced the luciferase activities of IFNT1- or IFNTc1-promoter constructs. However, with the AP-1 site mutated construct, IFNT1- and IFNTc1-reporters behaved differently. These results suggest that two forms of bovine conceptus IFNT genes are expressed in utero and their transcriptional regulations differ.

Conceptus-derived prostaglandins regulate gene expression in the endometrium prior to pregnancy recognition in ruminants

In cattle, the blastocyst hatches from the zona pellucida on days 8-9 and then forms a conceptus that grows and elongates into an ovoid and then filamentous shape between days 9 and 16. The growing conceptus synthesizes and secretes prostaglandins (PGs) and interferon τ (IFNT). Our hypothesis was that the ovoid conceptus exerts a local effect on the endometrium prior to maternal recognition of pregnancy on day 16 in cattle. In study one, synchronized cyclic heifers received no blastocysts or 20 in vitro-produced blastocysts on day 7 and their uteri were collected on day 13. IFNT was not detected by RIA in the uterine flushing samples of pregnant heifers containing multiple ovoid conceptuses; however, total PG levels were higher in the uterine lumen of pregnant heifers than in that of cyclic heifers. Microarray analysis revealed that the expression of 44 genes was increased in the endometria of day 13 pregnant heifers when compared with that in the endometria of cyclic heifers, and many of these genes were classical Type I IFN-stimulated genes (ISGs). In studies two and three, the effects of infusing PGs at the levels produced by the elongating day 14 conceptus into the uterine lumen of cyclic ewes on ISG expression in the endometrium were determined. Results indicated that the infusion of PGs increased the abundance of several ISGs in the endometrium. These studies support the hypothesis that the day 13 conceptus secretes PGs that act locally in a paracrine manner to alter gene expression in the endometrium prior to pregnancy recognition in cattle.

Presence of Transcription Factor OCT4 Limits Interferon-tau Expression during the Pre-attachment Period in Sheep.

Interferon-tau (IFNT) is thought to be the conceptus protein that signals maternal recognition of pregnancy in ruminants. We and others have observed that OCT4 expression persists in the trophectoderm of ruminants; thus, both CDX2 and OCT4 coexist during the early stages of conceptus development. The aim of this study was to examine the effect of CDX2 and OCT4 on IFNT gene transcription when evaluated with other transcription factors. Human choriocarcinoma JEG-3 cells were cotransfected with an ovine IFNT (-654-bp)-luciferase reporter (-654-IFNT-Luc) construct and several transcription factor expression plasmids. Cotransfection of the reporter construct with Cdx2, Ets2 and Jun increased transcription of -654-IFNT-Luc by about 12-fold compared with transfection of the construct alone. When cells were initially transfected with Oct4 (0 h) followed by transfection with Cdx2, Ets2 and/or Jun 24 h later, the expression of -654-IFNT-Luc was reduced to control levels. OCT4 also inhibited the stimulatory activity of CDX2 alone, but not when CDX2 was combined with JUN and/or ETS2. Thus, when combined with the other transcription factors, OCT4 exhibited little inhibitory activity towards CDX2. An inhibitor of the transcriptional coactivator CREB binding protein (CREBBP), 12S E1A, reduced CDX2/ETS2/JUN stimulated -654-IFNT-Luc expression by about 40%, indicating that the formation of an appropriate transcription factor complex is required for maximum expression. In conclusion, the presence of OCT4 may initially minimize IFNT expression; however, as elongation proceeds, the increasing expression of CDX2 and formation of the transcription complex leads to greatly increased IFNT expression, resulting in pregnancy establishment in ruminants.

Non-classical mechanisms of steroid sensing in the ovary: Lessons from the bovine oxytocin model

Steroidogenic tissues such as the ovary, testes or adrenal glands are paradoxical in that they often indicate actions of steroid hormones within a dynamic range of ligand concentration in a high nanomolar or even micromolar level, i.e. at the natural concentrations existing within those organs. Yet ligand-activated nuclear steroid receptors act classically by direct interaction with DNA in the picomolar or low nanomolar range. Moreover, global genomic studies suggest that less than 40% of steroid-regulated genes involve classical responsive elements in gene promoter regions. The bovine oxytocin gene is a key element in the maternal recognition of pregnancy in ruminants and is regulated via an SF1 site in its proximal promoter. This gene is also regulated by steroids acting in a non-classical manner, involving nuclear receptors which do not interact directly with DNA. Dose-response relationships for these actions are in the high nanomolar range. Similar ‘steroid sensing’ mechanisms may prevail for other SF1-regulated genes and predict alternative pathways by which environmental endocrine disruptors might influence the functioning of steroid-producing organs and hence indirectly the steroid-dependent control of physiology and development.

A Potential Autocrine Role for Interferon Tau in Ovine Trophectoderm

Interferon tau (IFNT), a type I IFN produced by the conceptus trophectoderm, is the signal for maternal pregnancy recognition in ruminants. The purpose of this study was to investigate whether IFNT effected on the proliferation of ovine trophectoderm cells in an autocrine manner. Elongated ovine conceptuses (Days 15, Day 0=day of mating) were collected for isolation of mononuclear ovine trophectoderm (oTr-1) cells, and conceptuses (Days 15 and 20, n=4 and 3, respectively) were collected for RNA extraction. We demonstrated that the IFNT receptor, IFNAR1, was expressed in trophectoderm of day 15 and 20 conceptuses. Interestingly, the ovine trophectoderm cell line oTr-1 cultured in the presence of recombinant bovine IFNT (rbIFNT) displayed increased expressions of IFN-stimulated genes (ISGs), such as IFN-stimulated gene 15 (ISG15), 2-5-oligoadenylate synthetase 1 (OAS1) and bone marrow stromal cell antigen 2 (BST2). Meanwhile, the presence of rbIFNT in the culture media could promote the cell proliferation in a dose-dependent manner. Furthermore, the connective tissue growth factor, which has diverse functions in cell proliferation and is involved in conceptus elongation, was upregulated in oTr-1 cell by rbIFNT treatment in vitro. These data indicated that IFNT could act as an autocrine factor to regulate trophectoderm cell proliferation.

HOMOLOGY MODELING OF BUFFALO (<i>BUBALUS BUBALIS</i>) INTERFERON-TAU PROTEIN

Interferon-Tau (IFNt) contributes towards maternal recognition of pregnancy in ruminants (like, cattle, buffalo, goat, giraffe). IFNt has been extensively studied in most of the ruminants except for buffalo Bubalus bubalis). The present study has been undertaken to predict the secondary structure of Interferon-tau in buffalo. The available amino acid sequence of bubaline IFNt (sequence database of SwissProt) was subjected to protein-BLAST to find similar sequences with high scores and low e-values. The ovine IFNt sequence (PDB code: 1B5L) was selected for further computational analysis of the bubaline IFNt sequence to predict the secondary and tertiary structure. The secondary structure of the modeled bubaline IFNt was predicted using STRIDE. The 3D structure was generated using academic version of MODELER9v6. The probability density functions (pdf) were used to restrain Cα-Cα distances, main chain N-O distances as well as main-chain and side-chain dihedral angles. The energy minimization and van der waal contacts were taken care of using ACCELRYS DS Modeling 2.0. The residue profiles of the obtained three-dimensional models were checked by VERIFY3D. The energetic architecture and the correctness of the generated model revealed that the predicted secondary model was correct and acceptable. The predicted structure of the bubaline IFNt constructed through homology modeling from ovine IFNt, could be used for further profiling the species specific difference in IFNt activity.

Oxytocin Receptor Down-Regulation Is Not Necessary for Reducing Oxytocin-Induced Prostaglandin F2α Accumulation by Interferon-τ in a Bovine Endometrial Epithelial Cell Line

Interferon-tau (IFNtau) is the embryonic signal responsible for pregnancy recognition in ruminants. The primary action of IFNtau is believed to be mediated through inhibition of prostaglandin F(2alpha) (PGF(2alpha)) released from the endometrial epithelial cells in response to oxytocin (OT). Our working hypothesis was that the antiluteolytic effect of IFNtau also involved modulation of PG production downstream of OT receptor (OTR) and/or cyclooxygenase 2 (COX2). There is currently no OT-sensitive endometrial cell line to study the molecular mechanisms underlying our hypotheses. Therefore, we established an immortalized bovine endometrial epithelial cell line (bEEL) exhibiting OT response. These cells were cytokeratin positive, expressed steroid receptors, and exhibited preferential accumulation of PGF(2alpha) over PGE(2). The bEEL cells were highly sensitive to OT, showing time- and concentration-dependent increase in COX2 transcript and protein and PGF(2alpha) accumulation. Interestingly, IFNtau (20 ng/ml) significantly reduced OT-induced PGF(2alpha) accumulation, but surprisingly, the effect was not mediated through down-regulation of either OTR or COX2. Rather, IFNtau up-regulated COX2 in a time- and concentration-dependent manner while decreasing OT-induced PG accumulation. This suggests that COX2 is not a primary target for the antiluteolytic effect of IFNtau. Because IFNtau reduced OT-stimulated PGF(2alpha) accumulation within 3 h, the mechanism likely involves a direct interference at the level of the OT signaling or transcription in addition to the down-regulation of OTR observed in vivo. In summary, bEEL cells offer a unique in vitro model for investigating the cellular and molecular mechanisms underlying OT and IFNtau response in relation with luteolysis and recognition of pregnancy in the bovine.