Showing papers by "Fuller W. Bazer published in 1996"

Spatial and temporal analyses of integrin and Muc-1 expression in porcine uterine epithelium and trophectoderm in vitro.

Abstract: The spatial and temporal expressions of Muc-1, selected integrin subunits, and extracellular matrix components in porcine uterine epithelium from estrous (Days 0, 4, 8, 10-15) and early pregnant (Days 10-15 of pregnancy) gilts and from steroid-treated ovariectomized gilts were analyzed using indirect immunofluorescence analyses on cryosectioned tissues to identify potential components of uterine receptivity. Integrin subunit and extracellular matrix expressions were also examined in Day 11-15 conceptuses. Intense Muc-1 staining was detected on apical uterine epithelium on Day 0 but was absent by Day 10 in both cyclic and pregnant gilts. The result of estrogen treatment (E2; 100 micrograms/day for 10 days) was similar to that of the corn oil vehicle control, while treatment with progesterone (P4; 200 mg/day for 10 days) or E2 + P4 decreased Muc-1 staining in ovariectomized gilts. Immunostaining performed with antibodies directed against integrin subunits (alpha 1, alpha 3, alpha 4, alpha 5, alpha v, beta 1, and beta 3) in uterine epithelium revealed low (integrin subunits alpha 1 alpha 3), high (integrin subunits alpha v and beta 3), or modulated (integrin subunits alpha 4, alpha 5, and beta 1) expression, with the lowest expression on Day 0 and maximum expression by Days 10-15. Additionally, no differences due to pregnancy status were detected in staining of uterine epithelium on Days 10-15. Uterine epithelium from steroid-treated ovariectomized gilts had low expression of alpha 4, alpha 5, and beta 1 subunits in the presence of E2 that increased in response to P4 and E2 + P4 treatments. The expression of integrin subunits alpha 3, alpha v and beta 3 was not affected by sex steroids. Trophectoderm also expressed alpha 1, alpha 4, alpha 5, alpha v, beta 1, and beta 3, integrin subunits. Extracellular matrix constituents (fibronectin, vitronectin, laminin, and collagen type IV) were also examined. Fibronectin and vitronectin were present on trophectoderm, but only vitronectin was detected on uterine epithelium. The alpha 4, alpha 5, alpha v, beta 1, and beta 3 integrin subunits, vitronectin, and fibronectin were detected at sites of attachment between uterine epithelial cells and trophectoderm on Days 12-15 of pregnancy. These studies indicate that down-regulation of Muc-1 coincides with the transition of the prereceptive uterus to the receptive uterus. Additionally, the expression of alpha 4, alpha 5, alpha v, beta 1, and beta 3 integrin subunits along with the extracellular matrix components of fibronectin and vitronectin correlates with the time of implantation in swine.

Ovine interferon tau suppresses transcription of the estrogen receptor and oxytocin receptor genes in the ovine endometrium

Abstract: Tau interferons (IFNtau) are a unique subclass of the omega interferons that are transiently produced by the trophectoderm of the conceptus (embryo and associated membranes) during early pregnancy in ruminants. IFNtau acts as an antiestrogen on the endometrium to suppress increases in estrogen receptor (ER) and oxytocin receptor (OTR) gene expression which prevents pulsatile production of prostaglandin F2alpha and regression of the corpus luteum or luteolysis. In this study, steady-state levels of ER mRNA and transcription rates of the ER and OTR genes were two-fold lower in the endometrium of pregnant as compared to cyclic ewes on day 15. Levels of ER mRNA and ER and OTR gene transcription were also two-fold lower in the endometrium of day 15 cyclic ewes receiving intrauterine injections of recombinant ovine IFNtau from day 11 to day 14 compared to control ewes. Results suggest that the antiluteolytic action of IFNtau is to suppress transcription of the ER gene by a negative-acting transcriptional mechan...

τ-Interferon: Pregnancy recognition signal in ruminants

Abstract: SummaryThe ovine uterus provides an excellent in vivo model system for the study of the cellular and molecular mechanisms of Type I IFN and steroid hormone regulation of hormone receptor gene expression in an epitheliomesenchymal organ. Future experiments will be directed toward determining the signal transduction pathway activated by IFN-τ and mechanisms involved in regulation of gene expression. These experiments include (i) cloning the Type I IFN receptor found on the endometrial luminal and superficial glandular epithelium; (ii) eluciding the signal transduction pathway activated by IFN-τ binding to the Type I IFN receptor; (iii) cloning and structural analysis of the ovine ER and OTR genes; (iv) determining mechanisms regulating transcription of the ovine ER and OTR genes, particularly those involving both IFN-τ and progesterone; (v) determining the role(s) of IFN-τ-induced proteins (e.g., Mx protein) in establishment of pregnancy; and (vi) investigating conceptus-endometrial and epithelial-stromal i...

Estrogen enhances endometrial estrogen receptor gene expression by a posttranscriptional mechanism in the ovariectomized ewe.

Abstract: Prior influence of estrogen is required for many physiological effects of steroid hormones. This study addresses positive autoregulation of estrogen receptor gene expression in endometrium. Groups of ovariectomized ewes (n = 6) were treated with a single i.m. injection of 50 micrograms 17 beta-estradiol for 6, 12, 24, or 48 h or of vehicle for 24 h (control) prior to collection of endometrium. Three ewes received a regimen of estradiol-progesterone-estradiol (EPE) designed to mimic the estrous cycle. Northern analysis of endometrial RNA using an ovine estrogen receptor complementary RNA probe indicated that estradiol increased (p 0.25). These results suggest that a physiological dose of estradiol, similar to the preovulatory surge of estrogen, up-regulates endometrial estrogen receptor gene expression by a posttranscriptional mechanism.

High Yield Expression and Secretion of the Ovine Pregnancy Recognition Hormone Interferon-τ by Pichia pastoris

Abstract: The early conceptus (embryo and associated membranes) of domestic ruminants signals its presence to the maternal uterus through production of interferon-τ (IFN-τ). Production of IFN-τ ensures conti...

Unusual abundance of arginine and ornithine in porcine allantoic fluid.

Abstract: The concentrations of free arginine, ornithine, and glutamine in porcine allantoic and amniotic fluids were determined on Days 30,35, 40, and 45 of gestation. Arginine and ornithine were the most abundant amino acids in allantoic fluid on Days 35-40 and 45 of gestation, respectively. Arginine and ornithine nitrogen accounted for 40%, 50%, and 55% of the total free alpha-amino acid nitrogen in allantoic fluid on Days 35, 40, and 45 of gestation, respectively. Glutamine was the most abundant amino acid in amniotic fluid during early gestation and was also abundant in allantoic fluid. On Day 45 of gestation, glutamine nitrogen accounted for 41% of the total free alpha-amino acid nitrogen in amniotic fluid. The unusual abundance of arginine (2.5-4.1 mM) and ornithine (1.08-2.52 mM) in allantoic fluid on Days 35-40 of gestation has not been reported for any other biological fluid. These results are novel and interesting with respect to the role of these two basic amino acids in fetal-placental nutrition and metabolism.

Effects of interferon-tau and progesterone on oestrogen-stimulated expression of receptors for oestrogen, progesterone and oxytocin in the endometrium of ovariectomized ewes.

Abstract: The effects of recombinant ovine interferon-tau (IFN-tau) and progesterone on oestrogen-stimulated expression of endometrial receptors for oestrogen (ER), progesterone (PR) and oxytocin (OTR) were determined in ovariectomized ewes. Cyclic ewes (n = 16) were ovariectomized and fitted with uterine catheters on Day 4 of the oestrous cycle (Day O, oestrous) and assigned randomly in 2 x 2-factorial arrangement to receive daily intrauterine injections of either recombinant ovine IFN-tau (roIFN-tau; 2 x 10(7) anti-viral units) or control proteins from Day 11 to Day 15 and 50 mg progesterone from either Day 4 to Day 10 (E-P) or Day 4 to Day 15 (E+P). All ewes received 50 micrograms oestradiol-17 beta on Days 13, 14 and 15 and were hysterectomized on Day 16. In control ewes, endometrial ER mRNA, PR protein and OTR density were greater in E-P- than E+P- treated ewes. In E-P ewes, roIFN-tau decreased oestrogen-stimulated increases in ER and OTR, but not PR expression compared with control ewes. In E+P ewes, endometrial ER mRNA and protein, PR mRNA and protein, and OTR levels were lower in roIFN-tau-treated ewes than control ewes. Immunoreactive ER and PR were absent in the endometrial luminal and superficial glandular epithelium of roIFN-tau compared with control ewes, but were present in the deep glandular epithelium and stroma regardless of steroid or protein treatment. These results indicate that progesterone affects oestrogen-induced increases in endometrial ER, PR and OTR expression in the PR+ deep glandular epithelium and stroma, whereas IFN-tau suppresses oestrogen-induced increases ER, PR and OTR expression in the PR- luminal and superficial glandular epithelium. These combined actions of IFN-tau and progesterone to suppress oestrogen-induced increases in endometrial OTR formation would prevent pulsatile production of luteolytic prostaglandin F2 alpha by the endometrium during early pregnancy.

Differential expression of beta transforming growth factors (TGF beta 1, TGF beta 2, and TGF beta 3) and their receptors (type I and type II) in peri-implantation porcine conceptuses.

Abstract: Beta transforming growth factors (TGF beta 1, TGF beta 2, and TGF beta 3) and type I and II TGF beta receptors were immunohistochemically localized in peri-implantation porcine conceptuses (embryos and associated membranes) collected on Day 10 through Day 14 of gestation. Our results indicate specific immunolocalization of TGF beta isoforms and their receptors in conceptuses during these gestational days. In parietal endoderm, TGF beta 1 immunoreactions were weak to undetectable, TGF beta 2 immunoreactions were intense, and TGF beta 3 immunoreactions were intermediate in intensity to TGF beta 2 and TGF beta 1. In contrast to immunoreactions in endoderm, TGF beta 1 and TGF beta 3 immunostaining in trophectoderm (Tr) was intense. Differences in TGF beta 2 immunostaining of Tr were observed from Days 10 to 14 of gestation. A drastic decrease in cytoplasmic immunostaining of ectoderm and mesoderm was detected from Days 12 to 14 for all TGF beta isoforms and type II receptor; however, type I receptor immunoreactions were consistently detected between Days 10 and 14. Concurrent expression of both type I and type II receptors in the peri-implantation conceptuses suggests that porcine conceptuses are capable of binding and responding to TGF beta s during this period. Differential expression of the three TGF beta isoforms suggests different roles for TGF beta s 1, 2, and 3 in conceptus development. Our results suggest possible roles for TGF beta s in early growth and differentiation of the embryo, differentiation of the Tr, and implantation.

Characterization of a polarized porcine uterine epithelial model system.

Abstract: A porcine uterine epithelial cell (pUE) culture system that retains structural and functional properties of the surface epithelium in vivo was developed. Uterine luminal epithelial cells were isolated after pancreatin-dispase enzymatic release of epithelium from hysterectomized gilts. Cells were seeded on Millicell filters precoated with Matrigel in 24-well plates and subsequently allowed to proliferate to confluence. Purity of the isolation was confirmed by the presence of > 99% cytokeratin-positive cells. Epithelial cells became polarized in vitro and compared favorably in morphology to uterine epithelial cells in situ once a transepithelial resistance of > 600 omega cm2 was established. Microscopic analysis confirmed the presence of a simple columnar epithelium with prominent microvilli on the apical cell surface and a well-developed junctional complex containing tight junctions, belt and spot desmosomes, and interdigitating lateral cell processes. Indirect immunofluorescence of the tight junction-associated protein, ZO-1, indicated the formation of tight junctional complexes in the subapical region of the polarized cells. Functional polarity of epithelial cultures was also verified by 1) electrical resistance measurements, 2) basal preference for the secretion of prostaglandins F2 alpha and E2, 3) apical preference for the release of 35S-methionine-incorporated secretory proteins, and 4) apically and basally distinct secretory protein profiles. Steroid treatment (estrogen, progesterone, or estrogen plus progesterone) of the polarized pUE cells affected the release of radiolabeled methionine-incorporated secretory proteins. In addition, the protein profiles as compared to samples treated with fetal bovine serum or charcoal/dextran-stripped fetal bovine serum were altered. Steroid treatments did not alter the electrical resistance or the basal preference for prostaglandin secretion. This culture system may be useful for in vitro analysis of maternal recognition of pregnancy paradigms as well as the study of the direct actions of hormones, prostaglandin secretion, and epithelial-stromal interactions.

Effects of recombinant interferon-tau on ovine lentivirus replication

Abstract: As a pregnancy recognition signal, sheep trophoblast cells secrete a type I interferon, ovine interferon-tau (OvIFN-tau), which has potent antiviral activity. We studied the effects of a recombinant protein (rOv-IFN-tau) on the replication of ovine lentivirus (OvLV) in goat synovial membrane cells. The amount of provirus DNA, as measured by polymerase chain reaction (PCR), the virus titers, and the number of OvLV-induced syncytia were 76.5%, 82%, and 95%, respectively, lower in cultures treated with rOv-IFN-tau than in placebo-treated controls (p < 0.01). rOv-IFN-tau also reduced OvLV reverse transcriptase activity and protected cells from OvLV-induced cell lysis, but the effect was less dramatic. The antiviral activity increased with the concentration up to a maximum with 256 antiviral units of rOv-IFN-tau per ml.

Enzyme-Linked Immunosorbent Assay for Ovine Interferon-τ

Abstract: Ovine interferon-tau (OvIFN-tau) is a trophectoderm secretory protein responsible for maintenance of corpus luteum function during early pregnancy. Methods for its quantitation include an antiviral assay and radioimmunoassay (RIA), both of which have disadvantages. We therefore developed an ELISA for OvIFN-tau that is specific, rapid, and 40-fold more sensitive than the current RIA. It uses a monoclonal antibody (Ab) HL129, which binds both native and recombinant (r) OvIFN-tau. The ELISA accurately detected known amounts of rOvIFN-tau added to yeast and tissue culture medium and to sheep serum at concentrations between 0.005 and 50,000 ng/ml. Inter- and intraassay coefficients of variation were 9.3 +/- 0.8 and 6.1 +/- 1.4%, respectively. Data are given for the reliability and reproducibility of the ELISA for measuring native OvIFN-tau and rOvIFN-tau.

Human interferon tau compositions and methods of use

Abstract: The present invention describes the isolation and characterization of multiple forms of human interferon-τ. Protein and nucleic acid coding sequences for the multiple forms are disclosed. In further aspects, the invention relates to methods of producing and using human interferon-τ molecules.