The endometrial transcriptomic response to pregnancy is altered in cows after uterine infection.

Pregnancy induces changes in the transcriptome of the bovine endometrium from 15 days after insemination. However, pregnancy is less likely to occur if cows had a postpartum bacterial infection of the uterus, even after the resolution of disease. We hypothesized that uterine bacterial infection alters the endometrial transcriptomic signature of pregnancy after the resolution of disease. To examine the endometrial transcriptomic signature of pregnancy, cows were inseminated 130 days after intrauterine infusion of pathogenic Escherichia coli and Trueperella pyogenes, subsequently endometrium was collected 16 days after insemination for RNA sequencing. We found 171 pregnancy regulated genes in cows 146 days after bacterial infection. When comparing our findings with previous studies that described the endometrial transcriptomic signature of pregnancy in healthy cows, 24 genes were consistently differentially expressed in pregnancy, including MX1, MX2 and STAT1. However, 12 pregnancy regulated genes were found only in the endometrium of healthy cows, including ISG15 and TRANK1. Furthermore, 28 pregnancy regulated genes were found only in the endometrium of cows following bacterial infection and these were associated with altered iNOS, TLR, and IL-7 signaling pathways. Although 94 predicted upstream regulators were conserved amongst the studies, 14 were found only in the endometrium of pregnant healthy cows, and 5 were found only in cows following bacterial infection, including AIRE, NFKBIA, and DUSP1. In conclusion, there were both consistent and discordant features of the endometrial transcriptomic signature of pregnancy 146 days after intrauterine bacterial infusion. These findings imply that there is an essential transcriptomic signature of pregnancy, but that infection induces long-term changes in the endometrium that affect the transcriptomic response to pregnancy.

Maternal Influenza A Virus Infection Restricts Fetal and Placental Growth and Adversely Affects the Fetal Thymic Transcriptome.

Maternal influenza A viral infections in humans are associated with low birth weight, increased risk of pre-term birth, stillbirth and congenital defects. To examine the effect of maternal influenza virus infection on placental and fetal growth, pregnant C57BL/6 mice were inoculated intranasally with influenza A virus A/CA/07/2009 pandemic H1N1 or phosphate-buffered saline (PBS) at E3.5, E7.5 or E12.5, and the placentae and fetuses collected and weighed at E18.5. Fetal thymuses were pooled from each litter. Placentae were examined histologically, stained by immunohistochemistry (IHC) for CD34 (hematopoietic progenitor cell antigen) and vascular channels quantified. RNA from E7.5 and E12.5 placentae and E7.5 fetal thymuses was subjected to RNA sequencing and pathway analysis. Placental weights were decreased in litters inoculated with influenza at E3.5 and E7.5. Placentae from E7.5 and E12.5 inoculated litters exhibited decreased labyrinth development and the transmembrane protein 150A gene was upregulated in E7.5 placentae. Fetal weights were decreased in litters inoculated at E7.5 and E12.5 compared to controls. RNA sequencing of E7.5 thymuses indicated that 957 genes were downregulated ≥2-fold including Mal, which is associated with Toll-like receptor signaling and T cell differentiation. There were 28 upregulated genes. It is concluded that maternal influenza A virus infection impairs fetal thymic gene expression as well as restricting placental and fetal growth.

Analysis of the uterine lumen in fertility-classified heifers: II. Proteins and metabolites†.

Survival and growth of the bovine conceptus is dependent on endometrial secretions or histotroph. Previously, serial blastocyst transfer was used to classify heifers as high fertile (HF), subfertile (SF), or infertile (IF). Here, we investigated specific histotroph components (proteins and metabolites) in the uterine lumen of day 17 fertility-classified heifers. Interferon tau (IFNT) was more abundant in uterine lumenal fluid (ULF) of pregnant HF than SF animals as the conceptus was longer in HF heifers. However, no differences in endometrial expression of selected classical and nonclassical interferon-stimulated genes (ISGs) were observed, suggesting that IFNT signaling in the endometrium of pregnant HF and SF heifers was similar. Pregnancy significantly increased the abundance of several proteins in ULF. Based on functional annotation, the abundance of a number of proteins involved in energy metabolism, oxidative stress, amino acid metabolism, and cell proliferation and differentiation were greater in the ULF of pregnant HF than SF heifers. Metabolomics analysis found that pregnancy only changed the metabolome composition of ULF from HF heifers. The majority of the metabolites that increased in the ULF of pregnant HF as compared to SF heifers were associated with energy and amino acid metabolism. The observed differences in ULF proteome and metabolome are hypothesized to influence uterine receptivity with consequences on conceptus development and survival in fertility-classified heifers.

Dickkopf-related protein 1 is a progestomedin acting on the bovine embryo during the morula-to-blastocyst transition to program trophoblast elongation.

Progesterone regulates the endometrium to support pregnancy establishment and maintenance. In the ruminant, one action of progesterone early in pregnancy is to alter embryonic development and hasten the process of trophoblast elongation around day 14-15 of pregnancy, which is required for maternal recognition of pregnancy. Here we demonstrate that the WNT antagonist DKK1, whose expression is increased by progesterone treatment, can act on the bovine embryo during day 5 to 7.5 of development (the morula to blastocyst stage) to promote embryonic elongation on day 15 of pregnancy. Embryos were produced in vitro and exposed to 0 or 100 ng/ml recombinant human DKK1 from day 5 to 7.5 of culture. Blastocysts were transferred into synchronized recipient cows on day 7.5 (n = 23 for control and 17 for DKK1). On day 15, cows were slaughtered and embryos recovered by flushing the uterus. Embryo recovery was n = 11 for controls (48% recovery) and n = 11 for DKK1 (65% recovery). Except for two DKK1 embryos, all embryos were filamentous. Treatment with DKK1 increased (P = 0.007) the length of filamentous embryos from 43.9 mm to 117.4 mm and the intrauterine content of the maternal recognition of pregnancy signal IFNT (P = 0.01) from 4.9 µg to 16.6 µg. Determination of differentially expressed genes (DEG), using the R environment, revealed 473 DEG at p < 0.05 but none at FDR < 0.05, suggesting that DKK1 did not strongly modify the embryo transcriptome at the time it was measured. However, samples clustered apart in a multidimensional scaling analyisis. Weighted gene co-expression analysis of the transcriptome of filamentous embryos revealed a subset of genes that were related to embryo length, with identification of a significant module of genes in the DKK1 group only. Thus, several of the differences between DKK1 and control groups in gene expression were due to differences in embryo length. In conclusion, DKK1 can act on the morula-to-blastocyst stage embryo to modify subsequent trophoblast elongation. Higher pregnancy rates associated with transfer of DKK1-treated embryos may be due in part to enhancements of trophoblast growth and antiluteolytic signaling through IFNT secretion. Given that progesterone can regulate both timing of trophoblast elongation and DKK1 expression, DKK1 may be a mediator of progesterone effects on embryonic development.

Temporal Release, Paracrine and Endocrine Actions of Ovine Conceptus-Derived Interferon-Tau During Early Pregnancy.

The antiviral activity of interferon (IFN) increases in uterine vein serum (UVS) during early pregnancy in sheep. This antiviral activity in UVS collected on Day 15 of pregnancy is blocked by anti-IFN-tau (anti-IFNT) antibodies. Conceptus-derived IFNT was hypothesized to induce IFN-stimulated gene (ISG) expression in endometrium and extrauterine tissues during pregnancy. To test this hypothesis, blood was collected from ewes on Days 12-16 of the estrous cycle or pregnancy. Serum progesterone was >1.7 ng/ml in pregnant (P) and nonpregnant (NP) ewes until Day 13, then declined to <0.6 ng/ml by Day 15 in NP ewes. A validated IFNT radioimmunoassay detected IFNT in uterine flushings (UFs) on Days 13-16 and in UVS on Days 15-16 of pregnancy. IFNT detection in UF correlated with paracrine induction of ISGs in the endometrium and occurred prior to the inhibition of estrogen receptor 1 and oxytocin receptor expression in uterine epithelia on Day 14 of pregnancy. Induction of ISG mRNAs in corpus luteum (CL) and liver tissue occurred by Day 14 and in peripheral blood mononuclear cells by Day 15 in P ewes. Expression of mRNAs for IFN signal transducers and ISGs were greater in the CL of P than that of NP ewes on Day 14. It is concluded that: 1) paracrine actions of IFNT coincide with detection of IFNT in UF; 2) endocrine action of IFNT ensues through induction of ISGs in peripheral tissues; and 3) IFNT can be detected in UVS, but not until Days 15-16 of pregnancy, which may be limited by the sensitivity of the IFNT radioimmunoassay.

Implantation and Establishment of Pregnancy in Ruminants.

The establishment of pregnancy in ruminants occurs during the peri-implantation period and involves the suppression of the endometrial luteolytic mechanism to maintain progesterone production by the corpus luteum (CL). Reciprocal interactions between the elongating conceptus (embryo/fetus and associated extraembryonic membranes) and endometrium culminate in implantation. Antiluteolytic effects of the conceptus are due to the production of interferon tau (IFNT) by the trophoblast that has a paracrine effect to inhibit the upregulation of oxytocin receptors in the endometrial epithelia, thereby disrupting uterine release of luteolytic prostaglandin F2 alpha (PGF) pulses. Additionally, IFNT is released into the uterine vein and has endocrine actions to induce ISGs in peripheral tissues. For example, IFNT may induce luteal resistance to PGF, thereby ensuring survival of the CL and maintenance of pregnancy. Survival of the blastocyst and elongation of the conceptus requires embryotrophic factors from the epithelia of the uterus, and those embryotrophic factors are regulated by ovarian progesterone as well as conceptus-derived factors including IFNT and prostaglandins. This review provides new concepts on mechanisms of the establishment of pregnancy and implantation in ruminants with emphasis on conceptus-maternal signaling associated with elongation of the blastocyst and endometrial responses to the presence of a conceptus.

Functional role of arginine during the peri-implantation period of pregnancy. I. Consequences of loss of function of arginine transporter SLC7A1 mRNA in ovine conceptus trophectoderm.

Arginine, the common substrate for production of nitric oxide (NO) and polyamines in mammals, increases in the uterine lumen during the peri-implantation period of pregnancy. However, functional roles of arginine within the uterine lumen for conceptus (embryo and extraembryonic membranes) development have not been elucidated in vivo. To assess roles of arginine in reproductive tissue for survival and development of the conceptus, we conducted an in vivo morpholino antisense oligonucleotide (MAO)-mediated knockdown of SLC7A1 mRNA, the arginine transporter in ovine conceptus trophectoderm (Tr). Translational knockdown of SLC7A1 mRNA resulted in retarded conceptus development and abnormal function compared to MAO control. Use of MAO-SLC7A1 knockdown in conceptuses decreased arginine transport (73%, P<0.01), the abundance of ornithine decarboxylase, and nitric oxide synthase (NOS3) proteins, arginine-related amino acids [citrulline (76%, P<0.05) and ornithine (40%, P<0.05)], and polyamines, which likely accounts for their retarded development. Also, no alternative arginine precursors (glutamine and glutamate), isoforms of nitric oxide synthase (NOS1 and NOS2), or alternative pathways for polyamine biosynthesis via arginine decarboxylase and agmatinase were activated to rescue conceptus development. Collectively, SLC7A1 is the key transporter of arginine by conceptus Tr, and arginine is essential for conceptus survival and development.-Wang, X., Frank, J. W., Little, D. R., Dunlap, K. A., Satterfield, M. C., Burghardt, R. C., Hansen, T. R., Wu, G., and Bazer, F. W. Functional role of arginine during the peri-implantation period of pregnancy. I. Consequences of loss of function of arginine transporter SLC7A1 mRNA in ovine conceptus trophectoderm.

Pregnancy-associated genes contribute to antiluteolytic mechanisms in ovine corpus luteum.

The hypothesis that ovine luteal gene expression differs due to pregnancy status and day of estrous cycle was tested. RNA was isolated from corpora lutea (CL) on days 12 and 14 of the estrous cycle (NP) or pregnancy (P) and analyzed with the Affymetrix bovine microarray. RNA also was isolated from luteal cells on day 10 of estrous cycle that were cultured for 24 h with luteolytic hormones (OXT and PGF) and secretory products of the conceptus (IFNT and PGE2). Differential gene expression (>1.5-fold, P < 0.05) was confirmed using semiquantitative real-time PCR. Serum progesterone concentrations decreased from day 12 to day 15 in NP ewes (P < 0.05) reflecting luteolysis and remained >1.7 ng/ml in P ewes reflecting rescue of the CL. Early luteolysis (days 12-14) was associated with differential expression of 683 genes in the CL, including upregulation of SERPINE1 and THBS1. Pregnancy on day 12 (55 genes) and 14 (734 genes) also was associated with differential expression of genes in the CL, many of which were ISGs (i.e., ISG15, MX1) that were induced when culturing luteal cells with IFNT, but not PGE2. Finally, many genes, such as PTX3, IL6, VEGF, and LHR, were stabilized during pregnancy and downregulated during the estrous cycle and in response to culture of luteal cells with luteolytic hormones. In conclusion, pregnancy circumvents luteolytic pathways and activates or stabilizes genes associated with interferon, chemokine, cell adhesion, cytoskeletal, and angiogenic pathways in the CL.

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.

Endocrine delivery of interferon tau protects the corpus luteum from prostaglandin F2 alpha-induced luteolysis in ewes.

Paracrine release of ovine interferon tau (oIFNT) from the conceptus alters release of endometrial prostaglandin F2 alpha (PGF) and prevents luteolysis. Endocrine release of oIFNT into the uterine vein occurs by Day 15 of pregnancy and may impart resistance of the corpus luteum (CL) to PGF. It was hypothesized that infusion of recombinant oIFNT (roIFNT) into the uterine or jugular veins on Day 10 of the estrous cycle would protect the CL against exogenous PGF-induced luteolysis. Osmotic pumps were surgically installed in 24 ewes to deliver bovine serum albumin (BSA; n = 12) or roIFNT (200 µg/day; n = 12) for 24 h into the uterine vein. Six ewes in each treatment group received a single injection of PGF (4 mg/58 kg body weight) 12 h after pump installation. In a second experiment, BSA or roIFNT was delivered at 20 or 200 µg/day into the uterine vein or 200 µg/day into the jugular vein for 72 h in 30 ewes. One half of these ewes received an injection of PGF 24 h after pump installation. Concentrations of progesterone in serum declined in BSA-treated ewes injected with PGF, but were sustained in all ewes infused with 20 µg/day of roIFNT into the uterine vein and 200 µg of roIFNT into the jugular vein followed 24 h later with injection of PGF. All concentrations of roIFNT and modes of delivery (uterine or jugular vein) increased luteal concentrations of IFN-stimulated gene (i.e., ISG15) mRNA. Infusion of 200 µg of IFNT over 24 h induced greater mRNA concentrations for cell survival genes, such as BCL2-like 1 (BCL2L1 or Bcl-xL), serine/threonine kinase (AKT), and X-linked inhibitor of apoptosis (XIAP) and decreased prostaglandin F receptor (PTGFR) mRNA concentrations, when compared to controls. It is concluded that endocrine delivery of roIFNT, regardless of route (uterine or jugular vein), effectively protects CL from the luteolytic actions of PGF by mechanisms that involve ISGs and stabilization of cell survival genes.

Gene profiling of inflammatory genes in day 18 endometria from pregnant and non-pregnant mares.

Maternal recognition of pregnancy is a physiological process that primarily describes endometrial responses to a conceptus. Recognition of a conceptus prevents the release of prostaglandin F(2α) , thereby ensuring survival of the corpus luteum and continued progesterone production. Exactly how this occurs in the mare is poorly understood. Because prostaglandin F(2α) is a pro-inflammatory hormone, we hypothesized that differential gene expression in the endometrium at the time of maternal recognition reflects an anti-inflammatory event leading to decreased prostaglandin F(2α) secretion. Mares were inseminated, and endometrial biopsies were recovered from pregnant mares on Day 18 post-ovulation. In subsequent estrous cycles, mares were not inseminated and Day 18 post-ovulation endometrial biopsies were collected (non-pregnant control, matched per individual). Endometrial gene expression profiles were examined by screening an Affymetrix equine GeneChip containing probes specific for genes related to inflammatory processes. Microarray analysis revealed 118 genes that were up-regulated and 93 genes that were down-regulated (P < 0.001) at least 1.5-fold in the endometrium of pregnant versus non-pregnant mares. Quantitative, real-time RT-PCR confirmed the microarray results for three up-regulated genes homologous to TSC22D3, PPAPDC2, and KLF6, and three down-regulated genes homologous to ESR1, MARCKSL1, and EPSTI1 (P < 0.05). It is concluded that the presence of the equine embryo induces differential gene expression in the endometrium of Day 18 pregnant mares, and that these genes are associated with inflammatory processes and pathways involving cellular growth and proliferation. The results from this study provide important new insights into endometrial gene expression in response to early equine pregnancy.

The chemokine receptor CXCR4 and its ligand CXCL12 are activated during implantation and placentation in sheep.

BACKGROUND: The progression of implantation and placentation in ruminants is complex and is regulated by interplay between sex steroids and local signaling molecules, many of which have immune function. Chemokines and their receptors are pivotal factors in implantation and vascularization of the placenta. Based on known critical roles for chemokine receptor 4 (CXCR4) during early pregnancy in other species, we hypothesized that CXCR4 and its ligand CXCL12 would increase in the endometrium and conceptus in response to implantation in ewes. The objectives of the current study were to determine if CXCL12 and CXCR4 were upregulated in: endometrium from pregnant compared to non-pregnant ewes and in, conceptuses, cotyledons, caruncles and intercaruncular tissue. METHODS: Tissues were collected from sheep on Days 12, 13, 14, and 15 of either the estrous cycle or pregnancy and from pregnant ewes on Days 35 and 50. Blood samples from jugular and uterine vein were also collected on all days. Conceptuses were collected from mature ewes on Days 13, 15, 16, 17, 21 and 30 of gestation. Real time PCR was used to determine relative mRNA concentrations for CXCL12 and CXCR4 and Western blot analysis was employed to confirm protein concentration. RESULTS: Differences described are P < 0.05. In the endometrium, CXCR4 mRNA and protein was greater on Day 15 of pregnancy compared to the estrous cycle. CXCL12 and CXCR4 mRNA in conceptuses was greater on Days 21 and 30 compared to earlier days. CXCL12 mRNA was greater in cotyledons on Day 35 compared to Day 50. On Day 35 of gestation, CXCR4 was greater compared to Day 50 in caruncle and intercaruncular tissue. White blood cells obtained from jugular and uterine vein collection had the greatest mRNA concentration of CXCL12 on Day 35 of pregnancy. CONCLUSIONS: A comprehensive analysis of CXCL12 and CXCR4 expression in fetal and maternal tissues during early pregnancy is reported with noteworthy differences occurring during implantation and placentation in sheep. We interpreted these data to mean that the CXCL12/CXCR4 pathway is activated during implantation and placentation in sheep and is likely playing a role in the communication between trophoblast cells and the maternal endometrium.

Prostaglandin F2alpha represses IGF-I-stimulated IRS1/phosphatidylinositol-3-kinase/AKT signaling in the corpus luteum: role of ERK and P70 ribosomal S6 kinase.

Little is known about the early intracellular events that contribute to corpus luteum regression. Experiments were designed to determine the effects of prostaglandin F2alpha (PGF2alpha) on phosphatidylinositol-3-kinase (PI3K)/Akt signaling in the corpus luteum in vivo and in vitro. Treatment of midluteal-phase cows with a luteolytic dose of PGF2alpha resulted in a rapid increase in ERK and mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (p70S6K1) signaling and a rapid suppression of Akt phosphorylation in luteal tissue. In vitro treatment of primary cultures of luteal cells with PGF2alpha also resulted in an increase in ERK and mTOR/p70S6K1 signaling and a diminished capacity of IGF-I to stimulate PI3K, Akt, and protein kinase C zeta activation. Accounting for the reductions in PI3K and Akt activation observed in response to PGF2alpha treatment, we found that PGF2alpha promoted the phosphorylation of serine residues (307, 612, 636) in the insulin receptor substrate 1 (IRS1) peptide sequence in vivo and in vitro. Serine phosphorylation of IRS1 was associated with reduced formation of IGF-I-stimulated IRS1/PI3Kp85 complexes. Furthermore, treatment with inhibitors of the MAPK kinase 1/ERK or mTOR/p70S6K1 signaling pathways prevented PGF2alpha-induced serine phosphorylation of IRS1 and abrogated the inhibitory actions of PGF2alpha on Akt activation. Taken together, these experiments provide compelling evidence that PGF2alpha treatment stimulates IRS1 serine phosphorylation, which may contribute to a diminished capacity to respond to IGF-I. It seems likely that the rapid changes in phosphorylation events are among the early events that mediate PGF2alpha-induced corpus luteum regression.

Prostaglandin F2alpha stimulates the expression and secretion of transforming growth factor B1 via induction of the early growth response 1 gene (EGR1) in the bovine corpus luteum.

In most mammals, prostaglandin F2alpha (PGF2alpha) is believed to be a trigger that induces the regression of the corpus luteum (CL), whereby progesterone synthesis is inhibited, the luteal structure involutes, and the reproductive cycle resumes. Studies have shown that the early growth response 1 (EGR1) protein can induce the expression of proapoptotic proteins, suggesting that EGR1 may play a role in luteal regression. Our hypothesis is that EGR1 mediates the actions of PGF2alpha by inducing the expression of TGF beta1 (TGFB1), a key tissue remodeling protein. The levels of EGR1 mRNA and protein were up-regulated in the bovine CL during PGF2alpha-induced luteolysis in vivo and in PGF2alpha-treated luteal cells in vitro. Using chemical and genetic approaches, the RAF/MAPK kinase (MEK) 1/ERK pathway was identified as a proximal signaling event required for the induction of EGR1 in PGF2alpha-treated cells. Treatment with PGF2alpha increased the expression of TGFB1 mRNA and protein as well as the binding of EGR1 protein to TGFB1 promoter in bovine luteal cells. The effect of PGF2alpha on TGFB1 expression was mimicked by a protein kinase C (PKC)/RAF/MEK1/ERK activator or adenoviral-mediated expression of EGR1. The stimulatory effect of PGF2alpha on TGFB1 mRNA and TGFB1 protein secretion was inhibited by blockade of MEK1/ERK signaling and by adenoviral-mediated expression of NAB2, an EGR1 binding protein that inhibits EGR1 transcriptional activity. Treatment of luteal cells with TGFB1 reduced progesterone secretion, implicating TGFB1 in luteal regression. These studies demonstrate that PGF2alpha stimulates the expression of EGR1 and TGFB1 in the CL. We suggest that EGR1 plays a role in the expression of genes whose cognate proteins coordinate luteal regression.

Expression of interferon (IFN)-stimulated genes in extrauterine tissues during early pregnancy in sheep is the consequence of endocrine IFN-tau release from the uterine vein.

The ruminant conceptus synthesizes and secretes interferon (IFN)-tau, which presumably acts via an intrauterine paracrine mechanism to signal maternal recognition of pregnancy. The aims of this study were to determine whether IFN-stimulated genes (ISG) such as ISG15 and OAS-1 are differentially expressed in blood cells circulating in the uterus of ewes; whether extrauterine components of the reproductive tract such as the corpus luteum (CL) also express mRNA for these ISG, and whether antiviral activity is greater in uterine vein than in uterine artery during early pregnancy. The concentrations of mRNA for both ISG were significantly greater (P < 0.0001) in endometrium and jugular blood of 15-d pregnant ewes than in nonpregnant ewes. ISG15 and OAS-1 mRNA concentrations were also greater (P < 0.05) in CL from 15-d pregnant ewes than in nonpregnant ewes. Immunohistochemistry revealed intense staining for ISG15 in large luteal cells on d 15 of pregnancy. Blood cells from uterine artery and vein of 15-d pregnant ewes had similar ISG15 and OAS-1 mRNA concentrations, suggesting that these cells were not conditioned by IFN-tau within the uterus. By using an antiviral assay, uterine venous blood was found to contain 500- to 1000-fold higher concentrations of bioactive IFN-tau than in uterine arterial blood on d 15 of pregnancy. It is concluded that uterine vein releases IFN-tau, which induces ISG in extrauterine tissues such as the CL during the time of maternal recognition of pregnancy.

The postimplantation embryo differentially regulates endometrial gene expression and decidualization.

Transcriptomal changes in the uterine endometrium induced in response to the implanting embryo remain largely unknown. In this study, using Affymetrix mRNA expression microarray analysis, we identified genes differentially expressed in the murine endometrium in the presence or absence of the embryo. Compared with the pseudopregnant deciduoma induced by a mechanical stimulus in the absence of an embryo, approximately 1500 genes (753 up-regulated, 686 down-regulated; P < 0.05) were differentially expressed by at least 1.2-fold in the uterine decidua of pregnancy. Most of these genes fall into five major biological categories that include binding (45%), catalysis (24%), signal transduction (10%), transcriptional regulators (5%), and transporters (5%). This strong, embryo-induced transcriptomal impact represented approximately 10% of the total number of genes expressed in the decidualizing endometrium. Validation studies with mRNA and protein confirmed existence of the phylogenetically conserved, embryo-regulated genes involved in the following: 1) hemostasis and inflammation; 2) interferon signaling; 3) tissue growth and remodeling; and 4) natural killer cell function. Interestingly, whereas expression of many growth factors and their cognate receptors were not different between the decidual and deciduomal endometria, a number of proteases that degrade growth factors were selectively up-regulated in the decidual tissue. Increased expression of IGF and activin A neutralizing factors (i.e. HtrA1 and Fstl3) correlated with reduced stromal cell mitosis, tissue growth, and mitogenic signaling in the decidual endometrium. These results support the hypothesis that the implanting murine embryo takes a proactive role in modulating endometrial gene expression and development during early gestation.

The hinge region between two ubiquitin-like domains destabilizes recombinant ISG15 in solution.

Interferon-stimulated gene (ISG) 15 mediates antiviral responses and also is upregulated within the endometrium in response to the developing embryo during early pregnancy. Structurally, ISG15 resembles two ubiquitin domains (30% identical) that are separated by a hinge region. Recombinant (r) bovISG15 is not stable in solution. It was hypothesized that the hinge region contributed to the instability of rbovISG15. Within 24 h of dialysis, rbovISG15 formed complexes as detected by reducing and denaturing SDS-PAGE. However, chemical perturbations of cysteine prevented formation of rbovISG15 complexes over time. Furthermore, a site-directed mutant of rbovISG15 (Cys80Ser) was isomeric and more stable than rbovISG15. Neither wild-type nor mutant rbovISG15 was able to interact with the ISG15 E1 initiating enzyme, UBE1L, in an in vitro pull-down assay. Ovine (ov) ISG15 has three additional amino acids within the hinge region that were hypothesized to increase stability and the degree of interaction with UBE1L because of increased separation of the ubiquitin-like domains. Over time in solution, rovISG15 the level of rovISG15 secondary structure was diminished, whereas the Cys80Ser rovISG15 structure did not change. A GST-Cys80Ser rovISG15 fusion protein had increased structural stability and enhanced protein-protein interaction with UBE1L after dialysis for 48 h, when compared to the GST-rovISG15 fusion protein or rbovISG15. Models of bovISG15, Cys80Ser bovISG15, and ovISG15 were constructed, which confirmed that the hinge region between the two ubiquitin domains destabilizes rbovISG15 in solution.

Low blood ISG15 mRNA and progesterone levels are predictive of non-pregnant dairy cows.

ISG15 is induced by conceptus-derived interferon-tau in the endometrium on days 15-45 of pregnancy. It was hypothesized that pregnancy induces blood cell ISG15 gene expression and that low blood ISG15 mRNA levels provide an indication of non-pregnant cows on day 18. Blood was collected either on day 18 (n = 78) or on days 15-21, 25, and 32 (n = 21; serial collection) from dairy cows following artificial insemination (AI). Plasma progesterone concentration was determined using RIA. ISG15 mRNA levels were determined using real-time PCR. Pregnancy was diagnosed on day 32 using transrectal ultrasound. ISG15 mRNA levels increased after day 16, peaked at day 20 and then declined to day 16 levels by 32 days following AI. The average pregnancy rate was 43% based on blood cell ISG15 mRNA. The average pregnancy rate was 33% based on the transrectal ultrasound. Lower levels of ISG15 mRNA or progesterone during serial collections were 100% accurate in predicting non-pregnant cows based on day 32 transrectal ultrasound. However, detection of ISG15 mRNA yielded 78% accuracy in predicting pregnant cows, while progesterone yielded 58% accuracy. Average plasma progesterone based on pregnancy status according to ultrasound was consistently higher in pregnant (> 4 ng/ml) when compared with non-pregnant cows from days 15 to 32, except on day 16. It is concluded that detection of low blood ISG15 mRNA levels during serial collection from days 17 to 25 serves as an accurate indicator of cows that are not pregnant, thus allowing re-synchronization and insemination.

Isolation and sequence of an interferon-tau-inducible, pregnancy- and bovine interferon-stimulated gene product 15 (ISG15)-specific, bovine ubiquitin-activating E1-like (UBE1L) enzyme.

Bovine (bov) interferon-stimulated gene product 15 (ISG15) is produced in the endometrium in response to conceptus-secreted interferon (IFN)-tau. ISG15 conjugates to endometrial proteins through an enzymatic pathway that is similar to ubiquitinylation. Ubiquitin-activating enzyme 1-like protein (UBE1L) initiates enzymatic conjugation by forming a thioester bond with ISG15, thus preparing it for transfer to the next series of enzymes. The bovUBE1L has not been described. We hypothesized that bovUBE1L was induced by pregnancy and IFN-tau in the endometrium. A 110-kDa protein was purified from bovine endometrial (BEND) cells based on affinity with recombinant (r) glutathione S-transferase (GST)-ISG15. This protein was digested in gel with trypsin. Seven peptides were purified using HPLC, sequenced using liquid chromatography-mass spectroscopy-mass spectroscopy and found to share 43-100% identity with human UBE1L. The full-length bovUBE1L cDNA was isolated from a BEND cell cDNA library, sequenced, and found to share 83% identity with human UBE1L cDNA. Northern blot revealed two mRNAs that were detected in greater (P<0.05) concentrations in endometrium from Day 17-21 pregnant versus nonpregnant cows. Western blots using antihuman UBE1L antibody revealed a similar pattern of pregnancy-associated expression of UBE1L protein in the uterus. The bovUBE1L mRNA was localized, using in situ hybridization, primarily to glandular and luminal epithelium, with more diffuse localization to stroma of the endometrium from pregnant cows. Because bovUBE1L was purified through its interaction with rGST-ISG15 and shares significant amino acid and cDNA sequence identity with human UBE1L, it is concluded that it mediates conjugation of ISG15 to uterine proteins in response to the developing and attaching conceptus.

Localization of ISG15 and conjugated proteins in bovine endometrium using immunohistochemistry and electron microscopy.

The interferon-stimulated gene ISG15, a ubiquitin homolog, becomes conjugated to and regulates uterine proteins in response to conceptus-derived interferon-tau on d 18 of pregnancy. It was hypothesized here that cellular localization of ISG15 within endometrial cells might provide insight regarding function. Uteri were collected from cows (approximately 21-d estrous cycle) on d 17-21/0 of the estrous cycle and pregnancy and d 23, 45, and 50 of pregnancy. Intracellular ISG15 and its conjugates were present on d 17 of pregnancy, peaked to highest levels from d 18 to 23 and then declined to low but detectable levels by d 45 (P < 0.05) based on Western blotting. ISG15 and its conjugates were not detected on d 50 of pregnancy or during the estrous cycle. Immunohistochemistry revealed that ISG15 was localized throughout the endometrium on d 18-23, with heaviest staining in the sublumenal stratum compactum and the glandular epithelium throughout the stratum spongiosum. By d 45 and 50, ISG15 was lightly stained only in the stratum compactum immediately beneath the lumenal epithelium. Using transmission electron microscopy and immunogold labeling, ISG15 was specifically localized to organelles and compartments of endometrial epithelial cells and stromal cells: nucleus, perinuclear space, cytosol, mitochondria, rough endoplasmic reticulum, and cell membrane. This specific localization in epithelial and stromal cells led to the conclusion that ISG15 has diverse intracellular functions. The sustained presence of conjugated ISG15 through d 50 of pregnancy might reflect stabilization of conjugated proteins in response to implantation and the development of the placenta.