Gastrin-releasing peptide (GRP) in the ovine uterus: regulation by interferon tau and progesterone

Gastrin-releasing peptide (GRP) is abundantly expressed by endometrial glands of the ovine uterus and processed into different bioactive peptides, including GRP1-27, GRP18-27, and a C-terminus, that affect cell proliferation and migration. However, little information is available concerning the hormonal regulation of endometrial GRP and expression of GRP receptors in the ovine endometrium and conceptus. These studies determined the effects of pregnancy, progesterone (P4), interferon tau (IFNT), placental lactogen (CSH1), and growth hormone (GH) on expression of GRP in the endometrium and GRP receptors (GRPR, NMBR, BRS3) in the endometrium, conceptus, and placenta. In pregnant ewes, GRP mRNA and protein were first detected predominantly in endometrial glands after Day 10 and were abundant from Days 18 through 120 of gestation. Treatment with IFNT and progesterone but not CSH1 or GH stimulated GRP expression in the endometrial glands. Western blot analyses identified proGRP in uterine luminal fluid and allantoic fluid from Day 80 unilateral pregnant ewes but not in uterine luminal fluid of either cyclic or early pregnant ewes. GRPR mRNA was very low in the Day 18 conceptus and undetectable in the endometrium and placenta; NMBR and BRS3 mRNAs were undetectable in ovine uteroplacental tissues. Collectively, the present studies validate GRP as a novel IFNT-stimulated gene in the glands of the ovine uterus, revealed that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days induces GRP expression in endometrial glands.     

Developmental changes in polyamine levels and synthesis in the ovine conceptus

Polyamines (putrescine, spermidine, and spermine) are essential for placental growth and angiogenesis. However, little is known about changes in polyamine synthesis associated with development of the ovine conceptus (embryo/fetus and associated placental membranes). We hypothesized that rates of placental polyamine synthesis were maximal during the rapid placental growth that occurs in the first half of pregnancy. This hypothesis was tested using ewes between Days 30 and 140 of gestation. Columbia cross-bred ewes were hysterectomized on Days 30, 40, 60, 80, 100, 120, or 140 of gestation (Day 0 = mating; n = 4 ewes/day) to obtain placentomes, intercotyledonary placenta, intercaruncular endometrium, and allantoic as well as amniotic fluids. The tissues were analyzed for ornithine decarboxylase (ODC) and arginase activities; arginine, ornithine, and polyamine concentrations; and polyamine synthesis using radiochemical and chromatographic methods. Maximal ODC and arginase activities and the highest rates of polyamine synthesis were observed in all tissues on Day 40 of gestation. Concentrations of ornithine and polyamines in placentomes and intercaruncular endometrium also peaked on Day 40 of gestation. In ovine allantoic and amniotic fluids, polyamines were most abundant during early (Days 40-60) and late (Days 100-140) gestation, respectively. Amniotic fluid spermine increased progressively with advancing gestation. Results of the present study indicate metabolic coordination among the several integrated pathways that support high rates of polyamine synthesis in the placenta and endometrium during early pregnancy. Our findings may have important implications for both intrauterine growth retardation and fetal origins of diseases in adults.     

Molecular cloning and functional analysis of three type D endogenous retroviruses of sheep reveal a different cell tropism from that of the highly related exogenous jaagsiekte sheep retrovirus

Integrated into the sheep genome are 15 to 20 copies of type D endogenous loci that are highly related to two exogenous oncogenic viruses, jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV). The exogenous viruses cause infectious neoplasms of the respiratory tract in small ruminants. In this study, we molecularly cloned three intact type D endogenous retroviruses of sheep (enJS56A1, enJS5F16, and enJS59A1; collectively called enJRSVs) and analyzed their genomic structures, their phylogenies with respect to their exogenous counterparts, their capacity to form viral particles, and the expression specificities of their long terminal repeats (LTRs). In addition, the pattern of expression of enJSRVs in vivo was studied by in situ hybridization. All of the three enJSRV proviruses had open reading frames for at least one of the structural genes. In particular, enJS56A1 had open reading frames for all structural genes, but it could not assemble viral particles when highly expressed in human 293T cells. We localized the defect for viral assembly in the first two-thirds of the gag gene by making a series of chimeras between enJS56A1 and the exogenous infectious molecular clone JSRV(21). Phylogenetic analysis distinguished five ovine type D retroviruses: enJSRV groups A and B, ENTV, and two exogenous JSRV groups (African versus United Kingdom/North America isolates). Transient transfection assays indicated that the LTRs of the three enJSRVs were not preferentially active in differentiated lung epithelial cells. This suggests that the pulmonary tropic JSRV developed from a type D retrovirus that did not have lung specificity. Consistent with this, in situ hybridization of a panel of normal ovine tissues revealed high expression of enJSRV mRNA in the luminal epithelium and glandular epithelium of the uterus; lower expression was localized in the lamina propria of the gut and in the bronchiolar epithelium of the lungs.     

Developmental changes in nitric oxide synthesis in the ovine placenta

Nitric oxide (NO), synthesized from l-arginine by NO synthase (NOS), is a key regulator of placental angiogenesis and growth during pregnancy. However, little is known about placental NO synthesis associated with ovine conceptus development. This study was conducted to test the hypothesis that placental NO synthesis is greatest during early gestation. Columbia cross-bred ewes were hysterectomized on Days 30, 40, 60, 80, 100, 120, or 140 of gestation (n = 4 per day) to obtain placentomes, intercotyledonary placenta, and intercaruncular endometrium. Tissues were analyzed for constitutive NOS (cNOS) and inducible NOS (iNOS) activities, NO synthesis, tetrahydrobiopterin (BH4) and NADPH (essential cofactors for NOS), and GTP-cyclohydrolase I (GTP-CH, a rate-controlling enzyme in de novo synthesis of BH4) activity using radiochemical and chromatographic methods. Marked changes in NO synthesis, cNOS and iNOS activities, GTP-CH activity, and concentrations of BH4 and NADPH occurred in all placental and endometrial tissues between Days 30 and 140 of gestation. NO synthesis peaked on Day 60 of gestation in both intercotyledonary placenta and placentomes and on Days 40-60 in intercaruncular endometrium. NO synthesis in placentomes increased 100% between Days 80 and 100 of gestation, when placental and uterine blood flows increase continuously. In all placental and endometrial tissues, NO synthesis was positively correlated with total NOS activity, GTP-CH activity, and concentrations of BH4 and NADPH. Importantly, these results indicate a high degree of metabolic coordination among the several integrated pathways that support high rates of NO synthesis in the conceptus and uterus and establish a new base of information for future studies to define the roles of NO in fetal-placental growth and development.     

The transdominant endogenous retrovirus enJS56A1 associates with and blocks intracellular trafficking of Jaagsiekte sheep retrovirus Gag

The sheep genome harbors approximately 20 endogenous retroviruses (enJSRVs) highly related to the exogenous Jaagsiekte sheep retrovirus (JSRV). One of the enJSRV loci, enJS56A1, acts as a unique restriction factor by blocking JSRV in a transdominant fashion at a late stage of the retroviral cycle. To better understand the molecular basis of this restriction (termed JLR, for JSRV late restriction), we functionally characterized JSRV and enJS56A1 Gag proteins. We identified the putative JSRV Gag membrane binding and late domains and determined their lack of involvement in JLR. In addition, by using enJS56A1 truncation mutants, we established that the entire Gag protein is necessary to restrict JSRV exit. By using differentially tagged viruses, we observed, by confocal microscopy, colocalization between JSRV and enJS56A1 Gag proteins. By coimmunoprecipitation and molecular complementation analyses, we also revealed intracellular association and likely coassembly between JSRV and enJS56A1 Gag proteins. Interestingly, JSRV and enJS56A1 Gag proteins showed distinct intracellular targeting: JSRV exhibited pericentrosomal accumulation of Gag staining, while enJS56A1 Gag did not accumulate in this region. Furthermore, the number of cells displaying pericentrosomal JSRV Gag was drastically reduced in the presence of enJS56A1. We identified amino acid residue R21 in JSRV Gag as the primary determinant of centrosome targeting. We concluded that JLR is dependent on a Gag-Gag interaction between enJS56A1 and JSRV leading to altered cellular localization of the latter.     

Stanniocalcin (STC) in the endometrial glands of the ovine uterus: regulation by progesterone and placental hormones

Stanniocalcin (STC) is a hormone in fish that regulates calcium levels. Mammals have two orthologs of STC with roles in calcium and phosphate metabolism and perhaps cell differentiation. In the kidney and gut, STC regulates calcium and phosphate homeostasis. In the mouse uterus, Stc1 increases in the mesometrial decidua during implantation. These studies determined the effects of pregnancy and related hormones on STC expression in the ovine uterus. In Days 10-16 cyclic and pregnant ewes, STC1 mRNA was not detected in the uterus. Intriguingly, STC1 mRNA appeared on Day 18 of pregnancy, specifically in the endometrial glands, increased from Day 18 to Day 80, and remained abundant to Day 120 of gestation. STC1 mRNA was not detected in the placenta, whereas STC2 mRNA was detected at low abundance in conceptus trophectoderm and endometrial glands during later pregnancy. Immunoreactive STC1 protein was detected predominantly in the endometrial glands after Day 16 of pregnancy and in areolae that transport uterine gland secretions across the placenta. In ovariectomized ewes, long-term progesterone therapy induced STC1 mRNA. Although interferon tau had no effect on endometrial STC1, intrauterine infusions of ovine placental lactogen (PL) increased endometrial gland STC1 mRNA abundance in progestinized ewes. These studies demonstrate that STC1 is induced by progesterone and increased by a placental hormone (PL) in endometrial glands of the ovine uterus during conceptus (embryo/fetus and extraembryonic membranes) implantation and placentation. Western blot analyses revealed the presence of a 25-kDa STC1 protein in the endometrium, uterine luminal fluid, and allantoic fluid. The data suggest that STC1 secreted by the endometrial glands is transported into the fetal circulation and allantoic fluid, where it is hypothesized to regulate growth and differentiation of the fetus and placenta, by placental areolae.     

Osteopontin is synthesized by uterine glands and a 45-kDa cleavage fragment is localized at the uterine-placental interface throughout ovine pregnancy

Osteopontin (OPN) is a phosphorylated and glycosylated, secreted protein that is present in various epithelial cells and biological fluids. On freezing and thawing or treatment with proteases, the native 70-kDa protein gives rise to 45- and 24-kDa fragments. Secreted OPN functions as an extracellular matrix (ECM) protein that binds cell surface receptors to mediate cell-cell adhesion, cell-ECM communication, and cell migration. In sheep and humans, OPN is proposed to be a secretory product of uterine glandular epithelium (GE) that binds to uterine luminal epithelium (LE) and conceptus trophectoderm to mediate conceptus attachment, which is essential to maintain pregnancy through the peri-implantation period. Cell-cell adhesion, communication, and migration likely are important at the interface between uterus and placenta throughout pregnancy, but to our knowledge, endometrial and/or placental expression of OPN beyond the peri-implantation period has not been documented in sheep. Therefore, the present study determined temporal and spatial alterations in OPN mRNA and protein expression in the ovine uterus between Days 25 and 120 of pregnancy. The OPN mRNA in total ovine endometrium increased 30-fold between Days 40 and 80 of gestation. In situ hybridization and immunofluorescence analyses revealed that the predominant source of OPN mRNA and protein throughout pregnancy was the uterine GE. Interestingly, the 45-kDa form of OPN was detected exclusively, continuously, and abundantly along the apical surface of LE, on conceptus trophectoderm, and along the uterine-placental interface of both interplacentomal and placentomal regions through Day 120 of pregnancy. The 45-kDa OPN is a proteolytic cleavage fragment of the native 70-kDa OPN, and it is the most abundant form in uterine flushes during early pregnancy. The 45-kDa OPN is more stimulatory to cell attachment and cell migration than the native 70-kDa protein. Collectively, the present results support the hypothesis that ovine OPN is a component of histotroph secreted by the uterine GE that accumulates at the uterine-placental interface to influence maternal-fetal interactions throughout gestation in sheep.     

Functional significance and cortisol dependence of the gross morphology of ovine placentomes during late gestation

The gross morphological appearance of ovine placentomes is known to alter in response to adverse intrauterine conditions that increase fetal cortisol exposure. The direct effects of fetal cortisol on the placentome morphology, however, remain unknown, nor is the functional significance of the different placentome types clear. The present study investigated the gross morphology of ovine placentomes in relation to placental nutrient delivery to sheep fetuses during late gestation and after experimental manipulation of the fetal cortisol concentration. As fetal cortisol levels rose naturally toward term, a significant decrease was observed in the proportion of the D-type placentomes that had the hemophagous zone everted over the bulk of the placentomal tissue. When the prepartum cortisol surge was prevented by fetal adrenalectomy, there were proportionately more everted C- and D-type placentomes and fewer A-type placentomes with the hemophagous zone inverted into the placentome compared with those of intact fetuses at term. Raising cortisol concentrations by infusion before term reduced the incidence of D-type placentomes and lowered the proportion of individually tagged placentomes that became more everted during the 10- to 15-day period between tagging and delivery. Cortisol, therefore, appears to prevent hemophagous zone eversion in ovine placentomes during late gestation. The distribution of placentome types appeared to have no effect on the net rates of placental delivery of glucose and oxygen to the fetus under normal conditions. When fetal cortisol levels were raised by exogenous infusion, however, placental delivery of glucose, but not oxygen, to the fetus, measured as umbilical uptake, was reduced to a greater extent in fetuses with a higher proportion of C- and D-type placentomes. The gross morphology of the ovine placentomes is, therefore, determined, at least in part, by the fetal cortisol concentration and may influence placental nutrient transfer when fetal cortisol concentrations are high during late gestation. These findings have important implications for the placental control of fetal growth and development, particularly during adverse intrauterine conditions.     

Cloning of the bovine antiapoptotic regulator, BCL2-related protein A1, and its expression in trophoblastic binucleate cells of bovine placenta

This report studied the identification and sequence of a full-length cDNA for the bovine BCL2 antiapoptotic family member, BCL2-related protein A1 (BCL2A1), and its localized and quantitative expression in the placenta to clarify the regulatory mechanism of trophoblast cell proliferation and differentiation during implantation and placental development. We cloned a full-length bovine BCL2A1 cDNA with 725 nucleotides and an open-reading frame corresponding to a protein of 175 amino acids. The predicted amino acid sequence shared 78% homology with human BCL2A1. All BCL2 homology domains (BH1, BH2, BH3, and BH4) in bovine BCL2A1 were conserved as well as in other mammalian BCL2A1. In the placentomes, in situ hybridization demonstrated that the BCL2A1 was limited in binucleate cells expressing various pregnancy-specific molecules like placental lactogen. BCL2-associated X protein (BAX) was also expressed in binucleate cells. Quantitative real-time RT-PCR detection exhibited a high-level expression of BCL2A1 in the conceptus at Day 21 of gestation, and it was expressed and increased in the extraembryonic membrane, cotyledon, and intercotyledon from implantation to term. BAX expression intensity increased with progression of gestation and remained elevated in postpartum. Caspase-3 protein (CASP3) and mRNA (CASP3) were detected from late gestation to postpartum in placenta as well as in the results of TUNEL detection. We believe that the apoptosis of binucleate cells may be regulated by the balance of the BCL2A1 and BAX. BCL2A1 genes produced a BCL2A1 protein in the mammalian cell-expression system. This molecule is a new candidate for antiapoptotic maintenance of the binucleate cells that support placental functions throughout gestation in bovine.     

Effect of morphology on placentome size, vascularity, and vasoreactivity in late pregnant sheep

Ovine placentomes vary in shape, with type A placentomes being concave, type D convex, and types B and C intermediate in morphology. It has been speculated that as placentomes advance in type they differ in vascularity and nutrient transport capacity. Our objective was to determine cellularity and vascularity measurements, angiogenic factor expression, and arterial vasoactivity within different morphologic types of placentomes. On Day 130 of gestation, placentomes were collected from multiparous ewes (n = 38) and were evaluated for size, cellularity estimates, angiogenic factor mRNA expression, capillary vascularity (capillary size, capillary surface density [CSD], capillary number density [CND], and capillary area density [CAD]), and vasoreactivity to potassium chloride and angiotensin II. The average weight and size of type A and B placentomes were less (P < 0.01) than those of type C and D placentomes. Placentome morphology did not affect (P > or = 0.24) cotyledonary or caruncular cellularity estimates or percentage of cellular proliferation. Placentome morphology affected (P > or = 0.41) neither caruncular CAD, CND, CSD, or capillary size nor cotyledonary CND, CSD, or capillary size. Cotyledonary CAD was increased (P < 0.01) in type B and D placentomes compared with type A placentomes. Furthermore, placentome type did not affect (P > or = 0.06) angiogenic factor gene expression in the cotyledon or the caruncle. Size, but not morphologic type of placentome, was associated with greater caruncular artery contractility to potassium chloride and angiotensin II (P < 0.01 for both). Placentome size, but not morphologic type, may be important for vascularity and nutrient transfer in the placenta of the pregnant ewe.     

Insulin-like growth factor-I and binding proteins 1, 2, and 3 in bovine nuclear transfer pregnancies

In cloned pregnancies, placental deficiencies, including increased placentome size, reduced placentome number, and increased accumulation of allantoic fluid, have been associated with low cloning efficiency. To assess differences in paracrine and endocrine growth regulation in cloned versus normal bovine placentomes and pregnancies, we have examined the expression of insulin-like growth factor (IGF)-I and -II and their binding proteins (IGFBP)-1 through -3 in placentomes of artificially inseminated (AI), in vitro-produced (IVP), and nuclear transfer (NT) pregnancies at Days 50, 100, and 150 of gestation. Fetal, maternal, and binucleate cell counts in representative placentomes were performed on Days 50-150 of gestation in all three groups. Increased numbers of fetal, maternal, and binucleate cells were present in NT placentomes at all stages of gestation examined. Immunolocalization studies showed that spatial and temporal patterns of expression of IGFBP-2 and -3 were markedly altered in the placentomes of NT pregnancies compared to AI/IVP controls. Concentrations of IGF-I in fetal plasma, as determined by RIA, were significantly higher (P = 0.001) in NT pregnancies (mean +/- SEM, 30.3 +/- 2.3 ng/ml) compared with AI (19.1 +/- 5.5 ng/ml) or IVP (24.2 +/- 2.5 ng/ml) pregnancies on Day 150 of gestation. Allantoic fluid levels of IGFBP-1 were also increased in NT pregnancies. These findings suggest that endocrine and paracrine perturbations of the IGF axis may modulate placental dysfunction in NT pregnancies. Furthermore, increased cell numbers in NT placentomes likely have significant implications for fetomaternal communication and may contribute to the placental overgrowth observed in the NT placentomes.     

Cloning and expression of a new member of prolactin-related protein in bovine placenta: bovine prolactin-related protein-VII

This study reports the identification and sequence of a full-length cDNA for a new member of bovine prolactin-related protein (bPRP-VII) and its quantitative and localized expression in the placenta. A full-length bPRP-VII cDNA was cloned with a 929-nucleotide open-reading-frame corresponding to a protein of 238 amino acids. The predicted amino acid sequence shares 63% homology with bPRP-I and 70% with bPRP-VI. bPRP-VII has eight cysteine residues with four disulfide bonds, which is more abundant than that of other bPRPs. RT-PCR detected bPRP-VII only in the placenta. In the placenta, mRNA was expressed in the cotyledon and intercotyledonary tissues throughout gestation. Quantitative real-time RT-PCR analysis exhibited a high expression of bPRP-VII mRNA in the fetal membrane at Day 27 of gestation. In the placentome on Day 60 of gestation, in situ hybridization analysis evidenced bPRP-VII mRNA in binucleate cells. bPRP-VII gene produced a mature protein in mammalian cell expression system. Approximately 29kDa protein was confirmed in this by the Western blot analysis with FLAG epitope tag. Expression profiles and localization were similar to those of bPRP-I. Although the functional data remain to be examined, a new member of the bPRP-VII gene was cloned. In addition to bPRP-I, bPRP-VII may take on an important functional role in implantation.     

Secretion of angiogenic activity by placental tissues of cows at several stages of gestation

Samples of maternal and fetal placental tissues were obtained from cows on Days 100 (N = 4), 150 (N = 5), 200 (N = 6) and 250 (N = 6) of gestation and incubated for 24 h. Conditioned media from caruncular explants were mitogenic for bovine aortic endothelial cells (BAEC) on all days of gestation. Media from intercaruncular endometrium were stimulatory for proliferation of BAEC on Day 100 but inhibitory on Days 150, 200 and 250. Media from cotyledonary and intercotyledonary tissues inhibited proliferation of BAEC on all days. Caruncular-conditioned media stimulated migration of BAEC on Days 150, 200 and 250. Cotyledonary-conditioned media inhibited migration of BAEC on all days. Effects of media from intercaruncular and intercotyledonary tissues on migration of BAEC varied with stage of gestation. Angiogenic activity of media from caruncular (all stages) and intercaruncular (Day 100) tissues appeared to have an Mr greater than 100,000. In cows, therefore, the maternal placentome (caruncle) appears to be the primary source of placental angiogenic activity throughout gestation. The fetal placentome (cotyledon) secretes activity which inhibits two major components of angiogenesis (proliferation and migration of endothelial cells) throughout gestation. Intercaruncular and intercotyledonary tissues may modulate placental angiogenesis throughout gestation. Placental vascular development in the cow is therefore probably controlled by an interaction between stimulatory and inhibitory factors produced by the placenta itself.     

Receptor usage and fetal expression of ovine endogenous betaretroviruses: implications for coevolution of endogenous and exogenous retroviruses

Betaretroviruses of sheep include two exogenous viruses, Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV), and a group of endogenous viruses known as enJSRVs. The exogenous JSRV and ENTV are the etiological agents of ovine pulmonary adenocarcinoma (OPA) and enzootic nasal tumor (ENT), respectively. Sheep affected by OPA or ENT do not show an appreciable antibody response to JSRV or ENTV. Consequently, it is conceivable that enJSRV expression in the fetal lamb tolerizes sheep to the related exogenous viruses. In this study, possible mechanisms of interference between the sheep exogenous and endogenous betaretroviruses were investigated. In situ hybridization detected enJSRV RNAs in lymphoid cells associated with the lamina propria of the small intestine and in the thymus of sheep fetuses. Low-level expression of enJSRVs was also detected in the lungs. In addition, expression of enJSRVs was found to block entry of the exogenous JSRV, presumably via mechanisms of receptor interference. Indeed, enJSRVs, like JSRV and ENTV, were found to utilize hyaluronidase-2 as a cellular receptor.     

Regulation of conceptus adhesion by endometrial CXC chemokines during the implantation period in sheep

To gain a better understanding of biochemical mechanisms of conceptus adhesion to the maternal endometrium in ruminant ungulates, the present study was performed to clarify roles of chemokines and extracellular matrix (ECM) components in the regulation of ovine blastocyst attachment to the endometrium. In addition to the chemokine, interferon-gamma inducible protein 10 kDa (IP-10, CXCL10), the chemokine receptor, CXCR3, also recognizes two other chemokines; monokine induced by IFN-gamma (MIG, CXCL9) and IFN-inducible T cell alpha chemoattractant (I-TAC, CXCL11). Similar to CXCL10, CXCL9, and CXCL11 were expressed in the uterus during the peri-implantation period, and CXCL9 mRNA expression was stimulated in endometrial explants from day 14 cyclic ewes by the addition of IFN-tau or IFN-gamma. Without ECM components, conceptus cell adhesion was low on day 14 of gestation and exhibited a 2.5-fold increase on day 17; adhesiveness on day 20 was 1/10 of that on day 14. Among various ECM components examined, trophoblast adhesion was greatest when fibronectin was used. Although day 14 conceptuses did not show much adhesive activity to fibronectin, day 17 trophoblast, and day 20 chorionic membrane exhibited 2.3-fold and 50-fold increase, respectively, which was enhanced by treatment with CXCL9 or CXCL10. These results indicate that through endometrial fibronectin and chemokines, ovine conceptus cells gain the ability to attach to the endometrium during pre-implantation period; however, elucidation of molecular mechanisms by which the conceptus acquires the adhesive ability during this time period awaits further investigation.