Interferon-tau infusion into the ovine corpus luteum delays luteolysis.

Conceptus-derived interferon-tau (IFNT) initiates maternal recognition of pregnancy in ewes by paracrine actions on the endometrium and endocrine action on the corpus luteum (CL). To examine the effect of IFNT on the CL without inducing IFN stimulated genes (ISGs) in the endometrium, recombinant ovine IFNT (roIFNT) or bovine serum albumin (BSA) was delivered directly into CLs via osmotic pumps at a rate of 10, 50 or 100 ng/h from days 9 to 12 of the estrous cycle. Endometrial and CL samples were collected on day 12. Fifty ng/h of roIFNT induced ISG15 in the CL on day 12 without affecting endometrial ISG15 concentrations. In a second experiment, roIFNT (50 ng/h) was infused into the CL from days 10 to 17 of the estrous cycle and serum samples were collected daily. Serum progesterone concentrations were significantly higher on days 15 to 17 in roIFNT-infused ewes compared to controls. Levels of LHCGR, STAR, CYP11A1, HSL, OPA1 and PKA mRNA and proteins were higher in the roIFNT-infused CLs compared to the controls. Levels of ISG15 and MX1 mRNA increased in the CLs of roIFNT-infused ewes but not in the endometrium. Endometrial ESR1 mRNA and protein concentrations were higher in the controls compared to roIFNT-infused ewes. In conclusion, intra-luteal delivery of roIFNT induced ISGs, stabilized steroidogenesis in the CL and delayed luteolysis without inducing endometrial ISGs. Inhibition of ESR1 in the endometrium of roIFNT-infused ewes was observed suggesting that direct delivery of IFNT to the CL has an additional anti-luteolytic effect on the endometrium.

Induced endometrial inflammation compromises conceptus development in dairy cattle†.

Endometrial inflammation is associated with reduced pregnancy per artificial insemination (AI) and increased pregnancy loss in cows. It was hypothesized that induced endometritis alters histotroph composition and induces inflammatory signatures on conceptus that compromise development. In Experiment 1, lactating cows were assigned to control (CON; n = 23) or to an intrauterine infusion of Escherichia coli and Trueperella pyogenes (ENDO; n = 34) to induce endometritis. Cows received AI 26 days after treatment, and the uterine fluid and conceptuses were collected on day 16 after AI. In Experiment 2, Holstein heifers were assigned to CON (n = 14) or ENDO (n = 14). An embryo was transferred on day 7 of the estrous cycle, and uterine fluid and conceptuses were recovered on day 16. Composition of histotroph and trophoblast and embryonic disc gene expression were assessed. Bacterial-induced endometritis in lactating cows altered histotroph composition and pathways linked to phospholipid synthesis, cellular energy production, and the Warburg effect. Also, ENDO reduced conceptus length in cows and altered expression of genes involved in pathogen recognition, nutrient uptake, cell growth, choline metabolism, and conceptus signaling needed for maternal recognition of pregnancy. The impact of ENDO was lesser on conceptuses from heifers receiving embryo transfer; however, the affected genes and associated pathways involved restricted growth and increased immune response similar to the observed responses to ENDO in conceptuses from lactating cows. Bacterial-induced endometrial inflammation altered histotroph composition, reduced conceptus growth, and caused embryonic cells to activate survival rather than anabolic pathways that could compromise development.

Attenuated lymphocyte activation leads to the development of immunotolerance in bovine fetuses persistently infected with bovine viral diarrhea virus†.

Bovine viral diarrhea virus continues to cost the cattle industry millions of dollars each year despite control measures. The primary reservoirs for bovine viral diarrhea virus are persistently infected animals, which are infected in utero and shed the virus throughout their lifetime. The difficulty in controlling the virus stems from a limited understanding of transplacental transmission and fetal development of immunotolerance. In this study, pregnant bovine viral diarrhea virus naïve heifers were inoculated with bovine viral diarrhea virus on day 75 of gestation and fetal spleens were collected on gestational days 82, 97, 190, and 245. Microarray analysis on splenic RNA from days 82 and 97 revealed an increase in signaling for the innate immune system and antigen presentation to T cells in day 97 persistently infected fetuses compared to controls. Reverse transcription quantitative polymerase chain reaction on select targets validated the microarray revealing a downregulation of type I interferons and lymphocyte markers in day 190 persistently infected fetuses compared to controls. Protein was visualized using western blot and tissue sections were analyzed with hematoxylin and eosin staining and immunohistochemistry. Data collected indicate that fetal immunotolerance to bovine viral diarrhea virus developed between days 97 and 190, with mass attenuation of the immune system on day 190 of gestation. Furthermore, lymphocyte transcripts were initially unchanged then downregulated, suggesting that immunotolerance to the virus stems from a blockage in lymphocyte activation and hence an inability to clear the virus. The identification of lymphocyte derived immunotolerance will aid in the development of preventative and viral control measures to implement before or during pregnancy.

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.

Experimentally Induced Endometritis Impairs the Developmental Capacity of Bovine Oocytes†.

Uterine infection is associated with infertility in women and dairy cows, even after the resolution of infection. However, the mechanisms causing this persistent infertility are unclear. Here, we hypothesized that induced endometritis in non-lactating dairy cows would reduce the developmental competence of oocytes. Non-lactating Holstein cows received an intrauterine infusion of endometrial pathogenic bacteria (Escherichia coli and Trueperella pyogenes; n = 12) or vehicle control (n = 11) on day 2 of the estrous cycle. Bacterial infusion increased expression of endometrial inflammatory mediators, and a mucopurulent discharge in the vagina confirmed the establishment of endometritis. Oocytes were collected by transvaginal ultrasound-guided ovum pickup on days 2, 24, 45, and 66 following infusion and subjected to in vitro fertilization and embryo culture. Bacterial infusion resulted in fewer cleaved oocytes developing to morulae compared to vehicle-infused controls (30.7 versus 45.0%), with the greatest effect observed in oocytes collected on day 24. Development to morula was inversely correlated with endometrial expression of IL6 on day 6. The expression of genes associated with embryo quality did not differ significantly between morulae from bacteria-infused and control cows. Artificial insemination 130 days after intrauterine infusion resulted in normal, filamentous embryos that produced interferon tau 16 days after conception in both infusion groups. This model of experimentally induced uterine infection successfully resulted in endometritis and a reduction in the proportion of oocytes that developed to morulae following in vitro fertilization. In conclusion, endometritis reduced the capacity of oocytes to develop to morulae.

Pregnancy-induced changes in metabolome and proteome in ovine uterine flushings.

Mass spectrometry (MS) approaches were used herein to identify metabolites and proteins in uterine flushings (UF) that may contribute to nourishing the conceptus. Ovine uteri collected on Day 12 of the estrous cycle (n = 5 ewes exposed to vasectomized ram) or Days 12 (n = 4), 14 (n = 5), or 16 (n = 5) of pregnancy (bred with fertile ram) were flushed using buffered saline. Metabolites were extracted using 80% methanol and profiled using ultraperformance liquid chromatography (LC) tandem mass spectrometry. The proteome was examined by digestion with trypsin, followed by the analysis of peptides with LC-MS/MS. Metabolite profiling detected 8510 molecular features of which 9 were detected only in UF from Day 14-16 pregnant ewes that function in fatty acid transport (carnitines), hormone synthesis (androstenedione like), and availability of nutrients (valine). Proteome analysis detected 783 proteins present by Days 14-16 of pregnancy in UF, 7 of which are as follows: annexin (ANX) A1, A2, and A5; calcium-binding protein (S100A11); profilin 1; trophoblast kunitz domain protein 1 (TKDP); and interferon tau (IFNT). These proteins function in endocytosis, exocytosis, calcium signaling, and inhibition of prostaglandins (annexins and S100A11); protecting against maternal proteases (TKDP); remodeling cytoskeleton (profilin 1); and altering uterine release of prostaglandin F2 alpha as well as inducing IFNT-stimulated genes in the endometrium and the corpus luteum (IFNT). Identifying metabolites and proteins produced by the uterus and conceptus advances our understanding of embryo/maternal signaling and provides insights into possible the causes of reproductive failure.

Paracrine and endocrine actions of interferon tau (IFNT).

This review focuses on the paracrine and endocrine actions of interferon tau (IFNT) during pregnancy recognition and establishment in ruminants. Pregnancy recognition involves the suppression of the endometrial luteolytic mechanism by the conceptus to maintain progesterone production by the corpus luteum (CL). The paracrine antiluteolytic effects of conceptus-derived IFNT inhibit upregulation of oxytocin receptors in the endometrial epithelia of the uterus, thereby preventing the production of luteolytic prostaglandin F2 alpha (PGF2α) pulses. In the endometrium, IFNT induces or upregulates a large number of classical IFN-stimulated genes (ISGs) and regulates expression of many other genes in a cell-specific manner that are likely important for conceptus elongation, implantation and establishment of pregnancy. Further, IFNT has endocrine effects on extrauterine cells and tissues. In sheep, IFNT induces luteal resistance to PGF2α, thereby ensuring survival of the CL for maintenance of pregnancy. The ISGs induced in circulating peripheral blood mononuclear cells by IFNT may also be useful as an indicator of pregnancy status in cattle. An increased knowledge of IFNT and ISGs is important to improve the reproductive efficiency in ruminants.

Functional roles of arginine during the peri-implantation period of pregnancy. III. Arginine stimulates proliferation and interferon tau production by ovine trophectoderm cells via nitric oxide and polyamine-TSC2-MTOR signaling pathways.

In mammal species, arginine is a multifunctional amino acid required for survival, growth, and development of conceptuses (embryo/fetus and associated extraembryonic membranes) during the peri-implantation period of pregnancy. However, functional roles of arginine with respect to it being a substrate for production of nitric oxide (NO) and polyamines on trophectoderm cell proliferation and function remain largely unknown. To systematically assess roles of arginine in conceptus development and its effect on interferon tau (IFNT) production for pregnancy recognition signaling in ruminants, an established ovine trophectoderm (oTr1) cell line isolated from Day-15 ovine conceptuses were used to determine their response to arginine, putrescine, and NO donors, as well as their associated inhibitors. Arginine at physiological concentration (0.2 mM) stimulated maximum oTr cell proliferation (increased 2.0-fold at 48 h and 2.6-fold at 96 h; P < 0.05), stimulated IFNT production (IFNT/cell increased 3.1-fold; P < 0.05), and increased total protein per cell by more than 1.5-fold (P < 0.05). It also increased phosphorylated tuberous sclerosis protein (p-TSC2) and phosphorylated mechanistic target of rapamycin (MTOR) abundance by more than 2.7- and 4.3-fold (P < 0.0001) after long-term incubation, respectively. When Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME; NO synthase inhibitor), DL-α-difluoromethylornithine hydrochloride hydrate (DFMO; ornithine decarboxylase inhibitor), and the combination (L-NAME + DFMO) were added, the effects of arginine on cell proliferation was reduced by 10.7%, 16.1%, and 22.3% (P < 0.05) at 48 h, and 15.3%, 27.2%, and 39.1% (P < 0.05) at 96 h of incubation, respectively, but values remained 1.5-fold higher (P < 0.05) than for the arginine-free control, which suggests that arginine, per se, serves as a growth factor. Both putrescine and NO stimulate cell proliferation via activation of the TSC2-MTOR signaling cascade, whereas only putrescine increased IFNT production. Collectively, our results indicate that arginine is essential for oTr1 cell proliferation and IFNT production via the NO/polyamine-TSC2-MTOR signaling pathways, particularly the pathway involving polyamine biosynthesis.

Embryo mortality in Isg15-/- mice is exacerbated by environmental stress.

The interferon-stimulated gene 15 (Isg15) encodes a ubiquitin-like protein that is induced in the endometrium by pregnancy in mice, humans, and ruminants. Because ISG15 is a component of the innate immune system, we hypothesized that development of the embryo, fetus, and postnatal pup may be impaired in mice lacking Isg15 (Isg15(-/-)) and that this development would be further impaired in response to environmental insults such as hypoxia. The number of implantation sites, resorption sites, dead embryos, and the changes in overall gross morphology of the uterus were evaluated in Isg15(-/-) mice on Days 7.5 and 12.5 postcoitum (dpc). Postnatal development also was monitored from birth to 12 wk of age. On 7.5 dpc, the number of implantation sites and serum progesterone concentrations were similar. However, embryo mortality increased (P < 0.05) in Isg15(-/-) dams by 12.5 dpc, resulting in smaller litter sizes (4.26 ± 0.21 embryos; n = 83 litters) compared to Isg15(+/+) females (7.78 ± 0.29 pups; n = 47 litters). Embryo mortality in Isg15(-/-) mice was further exacerbated to 70% when dams were stressed through housing under hypoxic conditions (PB = 445 mmHg; 6.5-12.5 dpc). Transmission electron microscopy revealed lesions in antimesometrial decidua as well as trophoblast cells adjacent to decidual cells on 7.5 dpc. ISG15 was localized to mesometrial decidua on 7.5 dpc. By 12.5 dpc, ISG15 was intensely localized to the labyrinth of the placenta. By 7.5 dpc, uterine natural killer cell migration into the mesometrial pole was diminished by 65% and was less prevalent in Isg15(-/-) compared to Isg15(+/+) deciduum. Postnatal growth rate of offspring that survived to birth from Isg15(-/-) and Isg15(+/+) dams was not different. Embryo mortality occurs in pregnant Isg15(-/-) mice, is exacerbated by environmental insults like maternal hypoxia, and might result from impaired early decidualization, vascular development, and formation of the labyrinth.

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.

Functional role of arginine during the peri-implantation period of pregnancy. II. Consequences of loss of function of nitric oxide synthase NOS3 mRNA in ovine conceptus trophectoderm.

Nitric oxide (NO) is a gaseous molecule that regulates angiogenesis and vasodilation via activation of the cGMP pathway. However, functional roles of NO during embryonic development from spherical blastocysts to elongated filamentous conceptuses (embryo and extraembryonic membrane) during the peri-implantation period of pregnancy have not been elucidated in vivo. In order to assess roles of NO production in survival and development of the ovine conceptus, we conducted an in vivo morpholino antisense oligonucleotide (MAO)-mediated knockdown trial of nitric oxide synthase-3 (NOS3) mRNA, the major isoform of NO synthase, in ovine conceptus trophectoderm (Tr). Translational knockdown of NOS3 mRNA results in small, thin, and underdeveloped conceptuses, but normal production of interferon-tau, the pregnancy recognition signal in sheep. MAO-NOS3 knockdown in conceptuses decreased the abundance of NOS3 (72%, P < 0.05) and the arginine transporter SLC7A1 proteins in conceptus Tr. Furthermore, the amounts of ornithine and polyamines were less (P < 0.01) in uterine fluid, whereas the amounts of arginine (58%, P < 0.01), citrulline (68%, P < 0.05), ornithine (68%, P < 0.001), glutamine (78%, P < 0.001), glutamate (68%, P < 0.05), and polyamines (P < 0.01) were less in conceptuses, which likely accounts for the failure of MAO-NOS3 conceptuses to develop normally. For MAO-NOS3 conceptuses, there were no compensatory increases in the expression levels of either nitric oxide synthase-1 (NOS1) or nitric oxide synthase-2 (NOS2) or in expression of enzymes for synthesis of polyamines (ornithine decarboxylase, arginine decarboxylase, agmatinase) from arginine or ornithine with which to rescue development of MAO-NOS3 conceptuses. Thus, the adverse effect of MAO-NOS3 to reduce NO generation and the transport of arginine and ornithine into conceptuses is central to an explanation for failure of normal development of MAO-NOS3, compared to control conceptuses. The study, for the first time, created an NO-deficient mammalian conceptus model in vivo and provided new insights into the orchestrated events of conceptus development during the peri-implantation period of pregnancy. Our data suggest that NOS3 is the key enzyme for NO production by conceptus Tr and that this protein also regulates the availability of arginine in conceptus tissues for synthesis of polyamines that are essential for conceptus survival and development.

ISGylation: a conserved pathway in mammalian pregnancy.

Successful pregnancy includes remodeling and differentiation of the endometrium in response to sex steroid hormones, development of maternal immunotolerance to the implanting embryo, and modification of the local uterine environment by the embryo to suit its own needs. The major signal released by the ruminant conceptus during establishment of pregnancy is interferon-tau (IFNT) that stimulates the expression of many genes in the endometrium and ovary. One of these genes is called interferon stimulated gene 15 (ISG15), which encodes a ubiquitin homolog with a C-terminal Gly that becomes covalently attached to Lys residues on targeted proteins through an ATP-dependent multi-step enzymatic reaction called ISGylation. The conceptus-derived induction of endometrial ISGs also occurs in mouse and human deciduas and placenta, in response to pregnancy presumably through action of cytokines such as interleukins and type I IFN. Described herein is evidence to support the concept that ISGylation is a maternal response to the developing conceptus, implantation and placentation that is conserved across mammalian pregnancy. Although the precise role for ISG15 remains elusive during pregnancy, it is clear that up-regulation in response to pregnancy may impart a pre-emptive defense to infection or other environmental insults, and protection of the conceptus against inflammatory insults across species.

Epigenetic Modifications of White Blood Cell DNA Caused by Transient Fetal Infection with Bovine Viral Diarrhea Virus.

Bovine viral diarrhea virus (BVDV) infections cause USD 1.5-2 billion in losses annually. Maternal BVDV after 150 days of gestation causes transient fetal infection (TI) in which the fetal immune response clears the virus. The impact of fetal TI BVDV infections on postnatal growth and white blood cell (WBC) methylome as an index of epigenetic modifications was examined by inoculating pregnant heifers with noncytopathic type 2 BVDV or media (sham-inoculated controls) on Day 175 of gestation to generate TI (n = 11) and control heifer calves (n = 12). Fetal infection in TI calves was confirmed by virus-neutralizing antibody titers at birth and control calves were seronegative. Both control and TI calves were negative for BVDV RNA in WBCs by RT-PCR. The mean weight of the TI calves was less than that of the controls (p < 0.05). DNA methyl seq analysis of WBC DNA demonstrated 2349 differentially methylated cytosines (p ≤ 0.05) including 1277 hypomethylated cytosines, 1072 hypermethylated cytosines, 84 differentially methylated regions based on CpGs in promoters, and 89 DMRs in islands of TI WBC DNA compared to controls. Fetal BVDV infection during late gestation resulted in epigenomic modifications predicted to affect fetal development and immune pathways, suggesting potential consequences for postnatal growth and health of TI cattle.

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.

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.

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.