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Biomedical subjects

R M Roberts

Publications and source records attributed to R M Roberts.

At least 109 records · Page 6Linked to original sources
PubMed1992

Expression of bovine trophoblast interferon in conceptuses derived by in vitro techniques.

Expression of the trophoblast interferon, bovine trophoblast protein-1 (bTP-1), has been studied in embryos produced by in vitro maturation-in vitro fertilization (IVM-IVF). No bTP-1 production was noted until after embryos had reached the expanded blastocyst stage and had begun to hatch (Days 8-9 post-fertilization). Single blastocysts comprising 115 +/- 22 cells released 1.0 +/- 0.1 units of interferon activity/24 h. Amplification of conceptus mRNA by reverse transcription-polymerase chain reaction procedure with bTP-1-specific oligonucleotides confirmed that bTP-1 transcripts were present in blastocysts but were not detectable at earlier stages. Although cultured blastocysts produced by IVM-IVF procedures continued to secrete bTP-1 for a few days, they failed to attach to the substratum and form outgrowths, and soon lost structural integrity. However, when Day 8 blastocysts/morulae were transferred to the uteri of synchronized cows, recovered 4 days later, and placed into individual cultures, they attached and formed outgrowths that produced large amounts of bTP-1 (greater than 2000 units/culture/24 h after 14 days). Embryos thus first expressed bTP-1 when a functional trophectoderm was first formed, and induction did not require a period of in vivo development. However, continued viability of the blastocyst and bTP-1 production were not sustained in vitro and may require some exposure to the uterine environment.

Animals↗
PubMed1992

Steroid regulation of the synthesis and secretion of retinol-binding protein by the uterus of the pig.

The endometrium of the pig secretes retinol-binding protein (RBP) under the influence of progesterone (P4). The objective of this study was to determine how conceptus-derived estrogen might modulate this production of RBP around days 11-13 of pregnancy when conceptuses elongate from spheres to long thread-like forms. Concentrations of retinol and RBP were low (35 +/- 7 ng/ml) in uterine flushings obtained on days 10-12 of the estrous cycle or from pregnant gilts in which conceptuses had not elongated. Concentrations of retinol and RBP increased 7- to 8-fold (P less than 0.01) in flushings where filamentous conceptuses were present. Size exclusion and ion exchange chromatography demonstrated that virtually all retinol assayed in uterine flushings was associated with RBP. Northern blot analysis with a cDNA representing uterine RBP revealed a single endometrial mRNA 1.1 kilobases in length. Expression of RBP mRNA in uterine endometrium was measured in ovariectomized prepubertal gilts after the administration of steroids according to the following regimens: I, corn oil (days 0-16; n = 10); II, estradiol benzoate (EB; days 13-14; n = 11); III, EB (days 1-2; n = 12); IV, EB (days 1-2) plus P4 (days 3-16; n = 12); and V, EB (days 1-2) plus P4 (days 3-16) plus EB (days 13-14; n = 12). EB (200 micrograms) and P4 (100 mg) were administered twice daily. Treatment IV was designed to stimulate the estrous cycle, and treatment V simulated early pregnancy. All gilts were hysterectomized on day 16, and total uterine mRNA (3 micrograms) was analyzed by Northern blotting. No RBP mRNA was detected in groups I, II, or III. In group IV, 5 of 12 gilts had detectable RBP mRNA, as measured by densitometric scanning (OD = 0.35 +/- 0.14). RNA isolated from all gilts in group V (12 of 12) gave a strong hybridization signal (OD = 1.58 +/- 0.22) for RBP. Finally, RBP mRNA was examined in the uterine endometrium of mature gilts on day 13 of the estrous cycle (n = 4), day 13 of pseudopregnancy (2.5 mg EB given on days 11-12; n = 4), or day 13 of pregnancy after conceptuses had elongated (n = 4). RBP mRNA was present in all groups, but was enhanced approximately 12-fold (P less than 0.01) in pregnant and pseudopregnant gilts compared to that in control gilts.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Luteal function after intrauterine infusion of recombinant bovine interferon-alpha I1 into postpartum beef cows expected to have short or normal luteal phases.

This study was conducted to determine whether intrauterine infusion of recombinant bovine interferon-alpha I1 (rboIFN-alpha I1), which has 70% sequence identity to bovine trophoblast protein-1, will prevent regression of corpora lutea anticipated to have a short lifespan. Twenty-six beef cows in good body condition were allotted to four treatment groups at parturition in a 2 x 2 factorial design. Treatments were: group 1, saline; group 2, rboIFN-alpha I1; group 3, norgestomet-saline; and group 4, norgestomet-rboIFN-alpha I1. Norgestomet implants were inserted on days 21-24 postpartum and removed 9 days later (before injection of human chorionic gonadotrophin (hCG)). Ovulation was induced 30 to 33 days postpartum with 5000 or 10,000 iu hCG. Groups 1 (n = 7) and 3 (n = 5) were given intrauterine infusions (rectocervical approach) twice daily with saline on days 1-12 or 13-24 after hCG injection, respectively. Cows allotted to groups 2 (n = 8) and 4 (n = 6) were given intrauterine infusions (rectocervical approach) of 2 mg rboIFN-alpha I1 twice daily on days 1-12 or 13-24 after hCG injection, respectively. Treatment with both norgestomet and rboIFN-alpha I1 delayed (P less than 0.01) luteolysis. Lengths of luteal phases (days; mean +/- SEM) were 8.4 +/- 0.7 (group 1, saline), 14.1 +/- 1.0 (group 2, rboIFN-alpha I1), 18.6 +/- 1.3 (group 3, norgestomet-saline) and 20.8 +/- 1.2 (group 4, norgestomet-rboIFN-alpha I1). Concentration of progesterone in serum was similar among all groups the first 6 days following hCG-induced ovulation, but differed (P less than 0.01) thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Role of interferons in maternal recognition of pregnancy in ruminants.

It has recently become evident that a type I interferon (IFN) subtype signals the presence of a viable conceptus to the mother during early pregnancy in cattle, sheep, and related mammalian species. This IFN, which is a product of the epithelium (trophectoderm) of the expanding trophoblast, is expressed in extremely large quantities for a few days just prior to implantation. It appears to be involved in modulating the release of the luteolytic hormone, prostaglandin F2 alpha, from the uterine endometrium and, hence, preventing the destruction of the corpus luteum that normally occurs at the end of an estrous cycle if an egg has not been fertilized. These trophoblast IFN have antiviral, antiproliferative, and immunomodulatory properties quite similar to other type I IFN, such as IFN-alpha, -beta, and -omega. However, they constitute a structurally and serologically distinct subtype. In addition, they are poorly inducible by virus, and the promoter regions of their genes are organized differently than other type I IFN. The genes for these trophoblast IFN are confined to ruminant species in the Artiodactyla order and probably evolved from IFN-omega less than 55 million years ago. There is no evidence for comparable production of type I IFN by trophoblast and placental tissues of mammals outside this ruminant group. Recent experiments have indicated that IFN treatment may have value in improving reproductive performance of sheep when provided during the period of maternal recognition of pregnancy, when much embryonic loss is believed to occur.

Animals↗
PubMed1991

Identification of the major pregnancy-specific antigens of cattle and sheep as inactive members of the aspartic proteinase family.

Pregnancy in cattle and sheep can be diagnosed by the presence of a conceptus-derived antigen in maternal serum that is secreted by trophoblast and placental tissue primarily as an acidic component of Mr 67,000. Molecular cloning of its cDNA reveals that the antigen belongs to the aspartic proteinase family and has greater than 50% amino acid sequence identity to pepsin, cathepsin D, and cathepsin E. The inferred sequences of the ovine and bovine polypeptides show approximately 73% identity to each other. Critical amino acid substitutions at the active site regions suggest that both proteins are enzymatically inactive. The antigen is a product of trophoblast binucleate cells that invade maternal endometrium at implantation sites.

Amino Acid Sequence↗
PubMed1991

Uteroferrin contains complex and high mannose-type oligosaccharides when synthesized in vitro.

Mature uteroferrin (Uf; Mr = 35,500) is a progesterone-induced acid phosphatase secreted by the pig uterus. It contains a single, unphosphorylated, high mannose-type oligosaccharide. Endometrial explants cultured in vitro secrete Uf with a Mr of 37,000 (37k Uf) having phosphorylated high mannose oligosaccharides. In this report we demonstrate that 37k Uf contains two N-linked oligosaccharides which are a mixture of complex and high mannose-type oligosaccharides. The complex-type glycopeptides are biantennary and a portion may be fucosylated on the GlcNac of the chitobiose core proximal to the peptide. Only a portion of the high mannose-type oligosaccharides are phosphorylated. The remainder appear to be typical Man6-4GlcNac2 oligosaccharides found on mature Uf.

Acid Phosphatase↗
PubMed1991

Constitutive and trophoblast-specific expression of a class of bovine interferon genes.

The early conceptus in sheep and cattle secretes a low molecular weight protein called ovine and bovine trophoblast protein 1 (TP-1) that is critical for establishment of pregnancy. TP-1 is a type I interferon (IFN) and is most related to IFN-omega. Here we have determined if TP-1 genes are regulated similarly to other type I IFNs. Single day 18 bovine conceptuses secrete approximately 10(5) units of IFN antiviral activity per hour in culture, amounts approximately 300 times higher than those produced by Sendai virus-induced leukocytes. Although conceptuses express mRNA for IFN-alpha, IFN-omega, and TP-1, TP-1 constitutes greater than 99% of the IFN produced. In contrast, leukocytes produced predominantly IFN-alpha, although TP-1 mRNA is inducible by Sendai virus to very low levels. TP-1 mRNA is detectable by Northern analysis in conceptuses from early pregnancy but is absent in late gestation placenta and several adult tissues. Transfected bovine TP-1 genes are expressed in human choriocarcinoma (JAR) cells in the absence of any specific stimulus, whereas these cells do not secrete antiviral activity constitutively or after transfection with a bovine IFN-omega gene. The transfected TP-1 gene is not expressed in nontrophoblast cells (mouse L929 and hamster Chinese hamster ovary), however. The 5' promoter region of the TP-1 gene is sufficient to direct trophoblast-specific expression onto a human growth hormone reporter gene in JAR cells. Deletion of the promoter from -450 to -126 results in a 4- to 5-fold decrease in expression. Together these data demonstrate that the genes for TP-1 are inducible by virus but are expressed preferentially in trophoblast cells and are functionally distinct from IFN-omega genes.

Animals↗
PubMed1991

The genes for the trophoblast interferons and the related interferon-alpha II possess distinct 5'-promoter and 3'-flanking sequences.

The genes for trophoblast interferons (IFN) bovine trophoblast protein-1 (bTP-1) and ovine trophoblast protein-1 (oTP-1) are expressed massively in the trophectoderm of preimplantation bovine and ovine concepti during the period of maternal recognition of pregnancy. These 172-amino acid IFN are closely related to the IFN-alpha II, a family of "long" IFN expressed in virus-induced leukocytes. Genomic Southern blotting with a full-length bTP-1 cDNA revealed about 15 genes that bind the probe with varying intensities. By using more specific probes for the 3'-ends of the cDNA, we have shown that only four to five of these represent bTP-1 genes, whereas no more than another four are IFN-alpha II. Genes for bTP-1 and IFN-alpha II, all of which are intronless, have been isolated from bovine genomic libraries, and their nucleotide sequences were compared. Additional bTP-1 genes and two distinct oTP-1 genes have been isolated from bovine and ovine genomic DNA by using the polymerase chain reaction procedure in conjunction with specific 3'- and 5'- primers (derived from the bTP-1 gene sequences determined above). The promoter region up to 100 bases upstream from the transcription start sites of the trophoblast IFN are almost completely conserved across different genes and across species, yet show only limited sequence identity with IFN-alpha II. Two putative binding regions for interferon regulatory factor-1 as well as several GAAANN motifs (where N is any nucleotide) that have been implicated in viral responsiveness of alpha 1- and beta-IFN genes are retained at identical positions in all of the trophoblast genes. Putative interferon regulatory factor-1-binding nucleotide hexamers and GAAANN motifs are also present in the IFN-alpha II genes, but these are organized very differently than in the trophoblast IFN genes. A GAAATG motif is present in IFN-alpha II promoters but is absent in trophoblast IFN genes. Based on the evidence presented, it is proposed that the trophoblast interferons constitute a separate subclass of IFN-alpha distinct from IFN-alpha II and IFN-alpha I.

Animals↗
PubMed1991

A role for interferons in early pregnancy.

In order to survive, the developing conceptus must interrupt the normal ovarian cycle of the mother and extend the production of progesterone by the corpus luteum. An unusual Type 1 interferon (IFN), related structurally to the IFN-alpha molecule and produced in massive amounts for only a few days by the first epithelium (trophectoderm) of the preimplantation conceptus, has been implicated as the antiluteolytic agent in sheep and cattle. IFN-alpha therapy during this critical period can also improve pregnancy success in sheep. It remains unclear, however, whether the trophoblast IFN have specialized biological properties or whether they are unique merely in the timing, magnitude and site of their expression.

Animals↗
PubMed1991

Quantitative immunogold ultracryomicrotome studies of the distribution of periimplantation proteins in the sheep.

Several mammalian uterine and conceptus proteins are produced at specific stages of implantation. Ovine trophoblast protein-1 (OTP-1) is only synthesised in vitro by conceptus tissue from between 13 and 21 days of pregnancy (dpc). This immunogold ultracryosection study shows that, during this period, OTP-1 immunoreactivity is only found in the Golgi body of the trophectodermal cells. A second protein, of 14 kD molecular weight (14 K protein), has a more varied distribution being found in membrane-bounded crystals in uterine epithelium and trophectodermal cells, and distributed throughout the cytosol and nucleoplasm of the uterine epithelium. There are only trace amounts of the 14 K protein in the fetomaternal syncytium which replaces the uterine epithelium during implantation, and no crystals are found in the trophectoderm after cotyledonary villus formation is initiated at 24-25 dpc. The crystals containing 14 K protein persist throughout pregnancy in the intercotyledonary areas. The narrow time window of OTP-1 occurrence reinforces the suggestion that this represents an important developmental signal, whereas the distribution of the 14 K protein indicates a more general nutritive function.

Animals↗
PubMed1991

Unique features of the trophoblast interferons.

The trophoblast interferons (IFN) are Type I IFN with about 50% amino acid sequence identity to the leukocyte IFN (IFN-alpha). They are the major secretory products of the trophoblast of ruminant ungulate species during pregnancy in the period immediately preceding attachment and implantation when they have been implicated in the phenomenon known as maternal recognition of pregnancy. The trophoblast IFN have antiviral and antiproliferative activities typical of other Type I IFN, but unlike IFN-alpha, -beta and -omega are poorly responsive to viral induction and have a highly restricted pattern of expression. Nevertheless, a recombinant bovine IFN-alpha can mimic many of the properties of the trophoblast IFN and has been used pharmacologically to improve pregnancy success in sheep. It still remains unclear, however, whether the trophoblast IFN have unique biological properties or whether they are unusual merely by virtue of the location, magnitude and temporal nature of their expression at a critical time during pregnancy.

Animals↗
PubMed1991

Induction of trophoblastic interferon expression in ovine blastocysts after treatment with double-stranded RNA.

Ovine trophoblast protein-1 (oTP-1) is an interferon (IFN) related to the IFN-omega. The objectives of this research were: (i) to attempt to induce oTP-1 mRNA in day-11 ovine conceptuses with polyinosinic-polycytidylic acid (poly(I).poly(C], and (ii) to determine if IFN-omega mRNA is also produced on day 11 of gestation. In experiment I, conceptuses were cultured in presence of 100 micrograms/ml poly(I).poly(C) (n = 5) or medium alone (control, n = 3) for up to 8 h. In situ hybridization was used to assess effects of treatment on mRNA concentrations for oTP-1 and actin (positive hybridization control). Poly(I).poly(C) increased oTP-1 mRNA concentrations approximately 2.5-fold (p less than 0.01), but had no effect on actin mRNA. In experiment II, the presence of mRNA for oTP-1 and ovine IFN-omega was determined by using reverse transcription-polymerase chain reaction (RT-PCR) analysis of conceptus total RNA coupled with Southern blot hybridization of the PCR reaction products with specific cDNA probes. oTP-1 mRNA was detectable in all poly(I).poly(C)-treated (n = 7) and control (n = 6) conceptuses, whereas IFN-omega mRNA was detected in only three of seven poly(I).poly(C)-treated conceptuses and not in any controls. Together these results demonstrate that expression of oTP-1 mRNA can be enhanced by treatment with poly(I).poly(C) and that oTP-1 is the primary but not the only type I-IFN inducible in conceptuses on day 11 of gestation.

Animals↗
PubMed1991

Slowed transcription and rapid messenger RNA turnover contribute to a decline in synthesis of ovine trophoblast protein-1 during in vitro culture.

Ovine trophoblast protein-1 (oTP-1) is produced in massive amounts by conceptuses during the Day-12-20-period of early pregnancy. The rate of production of oTP-1 declines during culture of conceptuses, however, suggesting a role for critical intrauterine factors for continued production. The present study was conducted to define the mechanism responsible for the in vitro decline in oTP-1 synthesis. Over 24 h culture, synthesis of oTP-1 was initially rapid but then declined such that there was little increase in either the protein or its antiviral activity after 12 h. By contrast, the release of total protein into the medium continued at an approximately linear rate for the entire 24-h period of culture. By using in situ hybridization to tissue sections and dot-blot analysis of tissue extracts with labeled cDNA probes, it was shown that the quantity of oTP-1 mRNA fell 3- to 5-fold during culture, whereas the amount of actin mRNA remained relatively constant. To determine whether this selective fall in levels of oTP-1 mRNA resulted from decreased transcription rate and/or high turnover rate of existing mRNA, conceptuses were either provided continuously with [3H]uridine for 24 h or labeled for 9 h and then exposed to medium enriched in unlabeled uridine and cytidine for a further 15 h. Specific incorporation of 3H into oTP-1 mRNA was assessed by hybridization to excess oTP-1 cDNA immobilized on nitrocellulose membranes. In the continuous labeling study, 3H in total RNA increased at an approximately linear rate for at least 18 h, whereas the content of 3H in oTP-1 mRNA peaked at 9 h and declined about 10-fold by 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The retinol-binding protein of the expanding pig blastocyst: molecular cloning and expression in trophectoderm and embryonic disc.

Retinol-binding protein (RBP) is a major secretory product of the porcine conceptus. Using an oligonucleotide probe corresponding to a highly conserved region of all known mammalian RBP, we have isolated an apparently full-length cDNA clone for porcine conceptus RBP from a cDNA library constructed from pig conceptuses collected between days 13-17 of pregnancy. The cDNA was 937 base-pairs in length and coded for a protein whose inferred amino-terminal sequence was identical to that reported for both porcine conceptus RBP and porcine serum RBP. Its length was consistent with the size (approximately 1 kilobase) of the RBP message in porcine conceptuses. Porcine conceptus RBP and human serum RBP share 91% amino acid sequence identity. The inferred differences in sequence were evenly distributed throughout the length of the polypeptide. RBP mRNA was detectable within the trophoblast of day 11 porcine conceptuses by in situ hybridization with a 618-basepair 35S-labeled probe corresponding to the 3' end of porcine RBP. Silver grain density was distributed relatively uniformly over the trophoblast and the inner cell mass. Western blot analysis of conceptus culture medium demonstrated that the conceptuses of cattle (on day 19) and sheep (on day 15) as well as pigs secrete RBP during early pregnancy. Secretion of large quantities of RBP by the trophoblast of preimplantation pig conceptuses suggests important roles for vitamin A and RBP near the time of conceptus elongation.

Amino Acid Sequence↗
PubMed1991

Effect of injected bovine interferon-alpha I1 on estrous cycle length and pregnancy success in sheep.

In Exp. 1 twice daily i.m. injections of 2 mg recombinant bovine IFN-alpha I1 (rboIFN-alpha I1) (N = 24) or placebo (N = 25) were administered to ewes from Day 12 to Day 16 during a normal oestrous cycle. Treatment did not increase (P greater than 0.10) oestrous cycle length (20.7 +/- 1.2 versus 18.5 +/- 1.4 days). In Exp. 2, ewes were injected twice daily with 2 mg IFN (N = 34) or placebo (N = 36) from Days 11 to 18 after natural mating. The rboIFN-alpha I1 significantly (P = 0.05) improved pregnancy rate (79% versus 58%) as determined by a failure of ewes to return to oestrus within 50 days. The number of ewes that lambed was greatest in the rboIFN-alpha I1-treatment group (71% versus 50%; P = 0.07), and no teratogenic effects were observed in the young born to IFN-treated ewes. The study was repeated a second year with a more fecund group of ewes (Exp. 3). More (P = 0.08) ewes injected with rboIFN-alpha I1 (58/65) than placebo-treated ewes (48/61) were judged pregnant by ultrasound. Again more ewes lambed (55 versus 45) and more lambs were born (98 versus 80) from the rboIFN-alpha I1-treated group. Combining the data from both studies revealed a significant (P = 0.01) effect of treatment. The amount of antiviral activity in jugular vein blood of ewes injected with rboIFN-alpha I1 (2 mg) was determined over time in Exp. 4. Activity rose to a maximum (approximately 450 IRU/ml) within 1-2 h and declined by over 75% in 24 h. Single injections of 1, 2 and 5 mg in buffer or 2 mg emulsified in sesame oil all gave similar profiles of antiviral activity in jugular blood over a 48-h period. In Exp. 5, antiviral activity was measured in uterine vein, ovarian artery and jugular vein serum of untreated pregnant (N = 7) and non-pregnant (N = 11) ewes at Day 15 after mating. Activity was detected in the uterine vein (58 +/- 19 IRU/ml) of all pregnant ewes. The observations in Exps 1-5 are consistent with a role for conceptus-derived IFN-alpha in maternal recognition of pregnancy and suggest that supplemental IFN-alpha might be useful in improving pregnancy success in sheep.

Animals↗
PubMed1991

Induction of the synthesis of the pregnancy-specific protein p70 in the endometrium by intramuscular injection of recombinant bovine interferon-alpha I1 in nonpregnant ewes.

We examined the effect of recombinant bovine interferon-alpha I1 (rboIFN-alpha I1) or recombinant bovine trophoblast protein-1 (rbTP-1) on protein synthesis by endometrial explants from Day-13 cyclic ewes and studied the ability of rboIFN-alpha I1 injected i.m. to influence subsequent protein secretion by endometrial tissue explants. In Expt 1, ewes were injected with either 2 mg rboIFN-alpha I1 or vehicle alone at 12 h intervals beginning on Day 11 of the oestrous cycle and ending on the morning of Day 13; 8 h after the last injection, ewes were hysterectomized and endometrial explant cultures were prepared. Explants were cultured for 24 h in leucine-deficient medium supplemented with 250 microCi L-[3H]leucine per culture. For Expt 2, additional explants were prepared from Expt 1 controls. Explants were cultured in the presence of 0, 20 or 200 ng/ml of either rboIFN-alpha I1 or rbTP-1 for 24 h in leucine-deficient medium supplemented with 250 microCi L-[3H]leucine per culture. Secreted proteins were analysed by two-dimensional electrophoresis and fluorography. There was a marked enhancement of a 70 kDa acidic protein, p70, in explants cultured in the presence of rboIFN-alpha I1 or rbTP-1. This polypeptide is a product of the gravid uterine horn from Day 14 to Day 20 of pregnancy and is a useful marker of the action of interferon-alpha (IFN-alpha) on endometrium. Enhanced production of p70 also occurred in ewes injected i.m. with rboIFN-alpha I1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗