Jared J. Romero

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

ABSTRACT 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.

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.

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

ABSTRACT 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.

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

ABSTRACT 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.

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

ABSTRACT 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.

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

Abstract 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. Summary Sentence Discovery of metabolites and proteins in uterine flushings contributes to understanding how the conceptus communicates with the endometrium during early pregnancy in sheep.