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Fuller W Bazer and William W Thatcher

It has been 38 years since a protein, now known as interferon tau (IFNT), was discovered in ovine conceptus-conditioned culture medium. After 1979, purification and testing of native IFNT revealed its unique antiluteolyic activity to prevent the regression of corpora lutea on ovaries of nonpregnant ewes. Antiviral, antiproliferative and immunomodulatory properties of native and recombinant IFNT were demonstrated later. In addition, progesterone and IFNT were found to act cooperatively to silence expression of classical interferon stimulated genes in a cell-specific manner in ovine uterine luminal and superficial glandular epithelia. But, IFNT signaling through a STAT1/STAT2-independent pathway stimulates expression of genes, such as those for transport of glucose and amino acids, which are required for growth and development of the conceptus. Further, undefined mechanisms of action of IFNT are key to a servomechanism that allows ovine placental lactogen and placental growth hormone to affect the development of uterine glands and their expression of genes throughout gestation. IFNT also acts systemically to induce the expression of interferon stimulated genes that influence secretion of progesterone by the corpus luteum. Finally, IFNT has great potential as a therapeutic agent due to its low cytotoxicity, anti-inflammatory properties and effects to mitigate diabetes, obesity-associated syndromes and various autoimmune diseases.

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Gwonhwa Song, Fuller W Bazer and Thomas E Spencer

Radical S-adenosyl methionine domain containing 2 (RSAD2) encodes a cytoplasmic antiviral protein induced by interferons (IFN). Interferon-induced with helicase C domain 1 (IFIH1) is a RNA helicase involved in innate immune defense against viruses, growth suppression, and apoptosis. Interferon tau (IFNT), a Type I IFN produced by the peri-implantation ruminant conceptus, acts on the uterine endometrium to signal pregnancy recognition and promote receptivity to implantation. Transcriptional profiling identified RSAD2 and IFIH1 as IFNT regulated genes in the ovine uterine endometrium. This study tested the hypothesis that RSAD2 and IFIH1 were induced in the endometrium in a cell type-specific manner by IFNT from the conceptus during early pregnancy. Endometrial RSAD2 and IFIH1 mRNA increased between days 12 and 16 of pregnancy, but not of the estrous cycle. In pregnant ewes, RSAD2 and IFIH1 mRNAs increased in endometrial glands, and stroma and immune cells, but not in the luminal epithelium. Neither gene was expressed in the trophectoderm of day 18 or 20 conceptuses. Progesterone (P4) treatment of ovariectomized ewes did not induce expression RSAD2 or IFIH1 mRNA in the endometrium; however, intrauterine injections of IFNT induced expression of RSAD2 and IFIH1 mRNA in endometria of ewes treated with P4, as well as in ewes treated with P4 and the progesterone receptor antagonist, ZK 136,317. These results indicate that conceptus IFNT induces both RSAD2 and IFIH1 in a P4-independent manner in the ovine uterine endometrium. These two IFNT-stimulated genes are proposed to have biological roles in the establishment of uterine receptivity to the conceptus during implantation through induction of an antiviral state and modulation of local immune cells in the endometrium.

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A review of the literature indicates that injection of gonadotrophic hormones or giving high-level-energy feeds will markedly increase the number of ovulations in polytocous animals. Higher ovulations rates are then followed by an increase in the precentage of embroys which die in utero. This increased embryonic death may be due either to inherent limitations within the individual embryo or to maternal limitations which affect the litter (Runner, 1951). In any event, the number of young at term reamins at a level characteristic for the species. Empbryonic death usually occurs before the time of attachment of the embryo to the endometrium (Boyd, 1965).

Attempts to increase the number of young born either by hormonal superovulation(Flower & Edwards, 1957; Adams, 1960; Hafez, 1964) or by the addition of embryos to the uterine horns (McLaren & Michine, 1959; Adams, 1960) have seldom been sucessful. In some

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Gwonhwa Song, Jo-Ann G W Fleming, Jinyoung Kim, Thomas E Spencer and Fuller W Bazer

Interferon τ (IFNT), the pregnancy recognition signal in ruminants, abrogates the luteolytic mechanism for maintenance of the corpus luteum for production of progesterone (P4). This study examined the expression of DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 (DDX58) and phospholipid scramblase 1 (PLSCR1) mRNAs in the ovine uterus as these genes were increased most in 2fTGH (STAT1 positive) cells by IFNT. The results of this study indicated that IFNT regulates expression of DDX58 and PLSCR1 mRNAs in the ovine uterus, which confirmed the results of the in vitro transcriptional profiling experiment with the 2fTGH (parental STAT1 positive) and U3A (STAT1 null) cell lines. Steady-state levels of DDX58 and PLSCR1 mRNAs increased in cells of the ovine uterus between days 12 and 20 of pregnancy, but not between days 10 and 16 of the estrous cycle. The expression of DDX58 and PLSCR1 mRNAs was greatest in endometrial stromal cells, but there was transient expression in uterine luminal and superficial glandular epithelial cells. P4 alone did not induce expression of DDX58 and PLSCR1 mRNAs; however, intrauterine injections of IFNT did induce expression of DDX58 and PLSCR1 mRNAs in the endometria of nonpregnant ewes independent of effects of P4. These results indicate that IFNT induces expression of DDX58 and PLSCR1 in ovine endometrial cells via the classical STAT1-mediated cell signaling pathway. Based on their known biological effects, DDX58 and PLSCR1 are IFN-stimulated genes, which may increase the antiviral status of cells of the pregnant uterus to protect against viral infection and/or enhance secretion of type I IFNs that inhibit viral replication.

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Thomas E Spencer, Greg A Johnson, Fuller W Bazer and Robert C Burghardt

Implantation in all mammals involves shedding of the zona pellucida, followed by orientation, apposition, attachment and adhesion of the blastocyst to the endometrium. Endometrial invasion does not occur in domestic ruminants; thus, definitive implantation is achieved by adhesion of the mononuclear trophoblast cells to the endometrial lumenal epithelium (LE) and formation of syncytia by the fusion of trophoblast binucleate cells with the LE. This review highlights new information on mechanisms regulating the implantation cascade in sheep. The embryo enters the uterus on day 4 at the morula stage of development and then develops into a blastocyst by day 6. The blastocyst sheds the zona pellucida (day 8), elongates to a filamentous form (days 11–16), and adheres to the endometrial LE (day 16). Between days 14 and 16, the binucleate cells begin to differentiate in the trophoblast and subsequently migrate and fuse with the endometrial LE to form syncytia. Continuous exposure of the endometrium to progesterone in early pregnancy downregulates the progesterone receptors in the epithelia, a process which is associated with loss of the cell-surface mucin MUC1 and induction of several secreted adhesion proteins. Recurrent early pregnancy loss in the uterine gland knockout ewe model indicates that secretions of the endometrial epithelia have a physiologic role in blastocyst elongation and implantation. A number of endometrial proteins have been identified as potential regulators of blastocyst development and implantation in sheep, including glycosylated cell adhesion molecule 1 (GlyCAM-1), galectin-15, integrins and osteopontin. The epithelial derived secreted adhesion proteins (GlyCAM-1, galectin-15 and osteopontin) are expressed in a dynamic temporal and spatial manner and regulated by progesterone and/or interferon tau, which is the pregnancy recognition signal produced by the trophoblast during blastocyst elongation. The noninvasive and protracted nature of implantation in domestic animals provides valuable opportunities to investigate fundamental processes of implantation that are shared among all mammals. Understanding of the cellular and molecular signals that regulate uterine receptivity and implantation can be used to diagnose and identify causes of recurrent pregnancy loss and to improve pregnancy outcome in domestic animals and humans.

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Gwonhwa Song, M Carey Satterfield, Jinyoung Kim, Fuller W Bazer and Thomas E Spencer

The actions of leukemia inhibitory factor (LIF) via LIF receptor (LIFR) and its co-receptor, IL6 signal transducer (IL6ST), are implicated in uterine receptivity to conceptus implantation in a number of species including sheep. The present study determined the effects of the estrous cycle, pregnancy, progesterone (P4), and interferon tau (IFNT) on the expression of LIFR and IL6ST in the ovine uterus. LIFR mRNA and protein were localized to the endometrial luminal (LE) and superficial glandular epithelia (sGE), whereas IL6ST mRNA and protein were localized primarily in the middle to deep GE. Both LIFR and IL6ST mRNAs and protein were more abundant in pregnant than cyclic ewes and increased from days 10 to 20 of pregnancy. Treatment of ovariectomized ewes with P4 and/or infusion of ovine IFNT increased LIFR and IL6ST in endometrial LE/sGE and GE respectively. Co-expression of LIFR and IL6ST as well as phosphorylated STAT3 was observed only in the upper GE of the endometrium as well as in the conceptus trophectoderm on days 18 and 20. In mononuclear trophectoderm and GE cells, LIF elicited an increase in phosphorylated STAT3 and MAPK3/1 MAPK proteins. Collectively, these results suggest that LIFR and IL6ST are both stimulated by IFNT and regulated by P4 in a complex stage- and cell-specific manner, and support the hypothesis that LIF exerts effects on the endometrial GE as well as conceptus trophectoderm during early pregnancy in sheep. Thus, LIF and STAT3 may have biological roles in endometrial function and trophectoderm growth and differentiation.

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Hyo Won Ahn, Jennifer L Farmer, Fuller W Bazer and Thomas E Spencer

During early pregnancy in ruminants, progesterone (P4) and interferon tau (IFNT) act on the endometrium to regulate genes hypothesized to be important for conceptus development and implantation. The present study was conducted to verify several candidate genes (actin α-2, smooth muscle, aorta (ACTA2), collagen, type III, α-1 (COL3A1), periostin (POSTN), secreted protein acidic cysteine-rich (SPARC), S100 calcium-binding protein A2 (S100A2), STAT5A and transgelin (TAGLN)) regulated by pregnancy, P4, and/or IFNT in the endometrium determined using a custom ovine cDNA array. S100A2 mRNA was detected primarily in endometrial epithelia and conceptuses. S100A2 mRNA increased in endometrial epithelia from days 10 to 16 in cyclic ewes and from days 10 to 14 in pregnant ewes and declined thereafter. The abundance of S100A2 mRNA was less in endometrial luminal epithelium of IFNT-infused ewes receiving P4. Expression of COL3A1, SPARC, ACTA2, and TAGLN was independent of pregnancy, P4, or IFNT. POSTN mRNA was detected primarily in compact stroma of intercaruncular and caruncular endometria, but not in the conceptus. Endometrial POSTN mRNA increased between days 12 and 14 in pregnant but not cyclic ewes, and POSTN mRNA was more abundant in uterine stroma of ewes treated with P4. POSTN protein was detected in uterine flushings of pregnant ewes and found to mediate attachment and stimulate migration of ovine trophectoderm cells in vitro. These results support the ideas that POSTN and S100A2 are regulated by P4 and IFNT respectively, and that POSTN is involved in conceptus elongation during early pregnancy.

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Irene Ruiz-González, Megan Minten, Xiaoqiu Wang, Kathrin A Dunlap and Fuller W Bazer

Toll-like receptors (TLRs) belong to the innate immune system and regulate inflammatory events that affect mammalian reproduction. In Study 1, we demonstrated that abundance of ovine TLR1TLR9 mRNAs in the uterus differs due to reproductive status (TLR2, TLR3, TLR7, and TLR8) and the day of the estrous cycle and pregnancy (TLR1TLR3, TLR5TLR7, and TLR9). Expression of TLR7 and TLR8 proteins was localized primarily to uterine epithelia and stroma and regulated in a temporal manner. In Study 2, we determined that ovine conceptuses express TLR7 and TLR8 on all days studied and that expression of the envelope protein of ovine endogenous retrovirus (enJSRV-Env) declined in conceptus trophectoderm from Day 13 to Day 16 of pregnancy. In Study 3, loss-of-function experiments were conducted in vivo using morpholino antisense oligonucleotides (MAOs) injected into the uterine lumen to block synthesis of TLR7 and TLR8 proteins, individually and jointly. Conceptuses were recovered on Day 16 to assess their morphology. MAO-treated conceptuses were developmentally retarded, produced less interferon tau (IFNT), and had fewer binucleate cells (BNCs) compared with MAO-Controls. Moreover, expression of enJSRV-Env mRNA in MAO-TLR7 conceptuses was greater than that for MAO-Control and MAO-TLR8 conceptuses, but similar to MAO-TLR7/TLR8 conceptuses. Results of this study indicated differences in TLR1TLR9 expression due to reproductive status and the day of the estrous cycle and pregnancy. TLR7 and TLR8 also influence development, enJSRV-Env abundance, secretion of IFNT, and formation of BNCs by conceptuses. These findings corroborate our hypothesis that TLR7 and TLR8 mediate pathways whereby enJSRV-Env regulates key peri-implantation events in conceptus development and differentiated functions of trophectoderm cells.

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Irene Ruiz-González, Jing Xu, Xiaoqiu Wang, Robert C Burghardt, Kathrin A Dunlap and Fuller W Bazer

Conceptus–endometrial communication during the peri-implantation period of pregnancy ensures establishment of pregnancy. We hypothesized that this dialog involves exosomes, ovine endogenous jaagsiekte retroviruses (enJSRV) and toll-like receptors (TLR) which regulate the secretion of interferon tau (IFNT), the pregnancy recognition signal in ruminants. First, exosomes isolated from uterine flushings from cyclic and pregnant ewes were analyzed for exosomal content and uterine expression of heat shock protein 70 (HSC70). Then, conceptus trophectoderm cells (oTr1) treated with different doses of exosomes were analyzed for the expression of genes involved in TLR-mediated cell signaling. The results revealed that exosomes contain mRNAs for enJSRV-ENV, HSC70, interleukins, and interferon (IFN)-regulatory factors. Exosomal content of enJSRV-ENV mRNA and protein decreased from days 10 and 12 to day 16 of gestation, and uterine expression of HSC70 increased in pregnant ewes compared with cyclic ewes. The oTr1 cells proliferated and secreted IFNT in a dose-dependent manner in response to exosomes from cyclic ewes. The expression of CD14, CD68, IRAK1, TRAF6, IRF6, and IRF7 mRNAs that are key to TLR-mediated expression of type 1 IFNs was significantly influenced by day of pregnancy. This study demonstrated that exosomes are liberated into the uterine lumen during the estrous cycle and early pregnancy; however, in pregnant ewes, exosomes stimulate trophectoderm cells to proliferate and secrete IFNT coordinately with regulation of TLR-mediated cell signaling. These results support our hypothesis that free and/or exosomal enJSRV act on the trophectoderm via TLR to induce the secretion of IFNT in a manner similar to that for innate immune responses of macrophages and plasmacytoid dendritic cells to viral pathogens.

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Fuller W Bazer, Thomas E Spencer, Greg A Johnson, Robert C Burghardt and Guoyao Wu

Uterine receptivity to implantation of blastocysts in mammals includes hatching from zona pellucida, precontact with uterine luminal (LE) and superficial glandular (sGE) epithelia and orientation of blastocyst, apposition between trophectoderm and uterine LE and sGE, adhesion of trophectoderm to uterine LE/sGE, and, in some species, limited or extensive invasion into the endometrial stroma and induction of decidualization of stromal cells. These peri-implantation events are prerequisites for pregnancy recognition signaling, implantation, and placentation required for fetal–placental growth and development through the remainder of pregnancy. Although there is a range of strategies for implantation in mammals, a common feature is the requirement for progesterone (P4) to downregulate expression of its receptors in uterine epithelia and P4 prior to implantation events. P4 then mediates its effects via growth factors expressed by stromal cells in most species; however, uterine luminal epithelium may express a growth factor in response to P4 and/or estrogens in species with a true epitheliochorial placenta. There is also compelling evidence that uterine receptivity to implantation involves temporal and cell-specific expression of interferon (IFN)-stimulated genes that may be induced directly by an IFN or induced by P4 and stimulated by an IFN. These genes have many roles including nutrient transport, cellular remodeling, angiogenesis and relaxation of vascular tissues, cell proliferation and migration, establishment of an antiviral state, and protection of conceptus tissues from challenges by the maternal immune cells.