Interferons and the maternal–conceptus dialog in mammals

Two-way communication between the conceptus and the mother during early pregnancy is essential if the pregnancy is to survive. In this review, our primary focus is on biochemical communication between the conceptus and mother in the ruminant ungulate species. We emphasize, in particular, the role played by interferon-tau (IFNT) in triggering maternal responses in cattle and sheep and how maternal factors intervene to up-regulate IFNT gene (IFNT) expression in trophoblast. However, we also consider the possibility that different signaling cytokines or the physical presence of trophoblast may induce a partial IFN response in endometrium of those species where there is no evidence for large scale trophoblast IFN production. Conceivably, disparate signaling mechanisms trigger common downstream events necessary to secure a successful pregnancy.     

Interferons and the maternal-conceptus dialog in mammals

Two-way communication between the conceptus and the mother during early pregnancy is essential if the pregnancy is to survive. In this review, our primary focus is on biochemical communication between the conceptus and mother in the ruminant ungulate species. We emphasize, in particular, the role played by interferon-tau (IFNT) in triggering maternal responses in cattle and sheep and how maternal factors intervene to up-regulate IFNT gene (IFNT) expression in trophoblast. However, we also consider the possibility that different signaling cytokines or the physical presence of trophoblast may induce a partial IFN response in endometrium of those species where there is no evidence for large scale trophoblast IFN production. Conceivably, disparate signaling mechanisms trigger common downstream events necessary to secure a successful pregnancy.     

Porcine conceptuses secrete an interferon during the preattachment period of early pregnancy

Maternal recognition of pregnancy in sheep and cattle is thought to be initiated by the conceptus secretory proteins ovine trophoblast protein-1 (oTP-1) and bovine trophoblast protein-1 (bTP-1), respectively. Recently, these proteins have been shown to be members of the interferon-alpha (IFN-alpha) family. In this study, we have examined whether pig conceptuses also produce IFN during early pregnancy. conceptuses were collected at Days 8, 10, 11, 12, 13, 15, and 17 of pregnancy and cultured in serum-free medium for 24 h. Day 11 conceptuses secreted a dominant 22,000-24,000 Mr cluster of acidic proteins (pI 5.2-5.4), that appeared to cross-react on immunoblots with antiserum against human IFN-alpha but not against oTP-1. Antiviral activity characteristic of an IFN was present in conceptus culture medium and uterine flushings from Day 11 through Day 17 of pregnancy, but was absent in flushings prior to Day 11 of pregnancy and in flushings from Day 12 nonpregnant gilts. The antiviral activity coeluted with a 22,000-24,000 Mr protein during partial purification through a gel filtration column. The activity was extremely labile, but could be restored by sequential protein denaturation, reduction, and renaturation. We conclude that production of IFN by early conceptuses is not restricted to ruminant species, and may therefore represent a more general phenomenon.     

Amino acid differences in interferon-tau (IFN-τ) of Bos taurus Coreanae and Holstein

Interferons (IFNs) are commonly grouped into type I and type II IFN. Type I IFNs are known as antiviral IFNs including IFN-α, IFN-β, and IFN-ω whereas type II IFN is referred to immune IFN and IFN-γ is only member of the type II IFN. Type I IFNs are induced by virus invading however type II IFN is produced by mitogenic or antigenic stimuli. IFN-τ was first identified in ruminant ungulates as a pregnancy recognition hormone, trophoblastin. IFN-τ constitutes a new class of type I IFN, which possesses the common features of type I IFN, such as the ability to prevent viral infection and to limit cell proliferation. In addition, IFN-τ is unique in that it is induced by pregnancy unlike other type I IFNs. We cloned Bos taurus (B. T.) Coreanae IFN-τ from peripheral blood mononuclear cells. The amino acid sequence of B. T. Coreanae IFN-τ shares only 90.3% identity with that of Holstein dairy cow. Recombinant B. T. Coreanae and Holstein IFN-τ proteins were expressed in Escherichia coli and the antiviral activity of IFN-τ proteins were examined. Both recombinant proteins were active and protected human WISH and bovine MDBK cells from the cytopathic effect of vesicular stomatitis virus. The recombinant IFN-τ protein of B. T. Coreanae and Holstein properly induced the expression of antiviral genes including 2',5'-oligoadenylate synthetase (OAS) and Mx GTPase 1 (Mx-1).     

Osteopontin expression in uterine stroma indicates a decidualization-like differentiation during ovine pregnancy

Osteopontin (OPN) is a component of the extracellular matrix that interacts with cell surface receptors, including integrins, to mediate cell adhesion, migration, differentiation, survival, and immune function. In pregnant mice and primates, OPN has been detected in decidualized stroma and is considered to be a gene marker for decidualization. Decidualization involves transformation of spindle-like fibroblasts into polygonal epithelial-like cells that are hypothesized to limit conceptus trophoblast invasion through the uterine wall during invasive implantation. Decidualization is not considered characteristic of species with noninvasive implantation, such as domestic animals. However, the extent of trophoblast invasion between sheep and pigs differs, with sheep exhibiting erosion of the uterine luminal epithelium (LE) and fusion of trophectoderm with LE to form syncytia, and pigs maintaining an intact LE throughout pregnancy. Therefore, the present study measured changes in the decidualization marker genes OPN, desmin, and alpha smooth muscle actin (alphaSMA) in ovine and porcine uterine stroma throughout pregnancy. The morphology of endometrial stromal cells in pregnant ewes changes following conceptus attachment, with cells increasing in size and becoming polyhedral in shape by Day 35 of pregnancy. Expression of OPN mRNA and protein, as well as desmin and alphaSMA proteins, was observed in this same uterine stromal compartment. In contrast, no morphological changes in uterine stroma nor induction of OPN mRNA and protein, or desmin protein, were detected during porcine pregnancy. Interestingly, alphaSMA protein was absent on Day 20, but prominent in uterine stroma of pregnant pigs on Day 45. Collectively, these results indicate that the uterine stroma of sheep undergoes a program of differentiation similar to decidualization in invasive implanting species, whereas porcine stroma exhibits differentiation that is more limited than that in sheep, rodents, or primates. Results suggest that uterine stromal decidualization is common to species with different types of placentation, but the extent is variable and correlates with the depth of trophoblast invasion during implantation.     

Interferon activity is not detected in blastocyst secretions and does not induce decidualization in mice.

Mouse trophoblast cells synthesized and secreted proteins during the peri-implantation period, some in the molecular size range of alpha interferons (IFN-alpha), known mediators of the maternal recognition of pregnancy in sheep and cows. However, conditioned media samples containing secreted proteins from Day-5 mouse blastocysts or from trophoblast outgrowths did not contain detectable levels of antiviral activity indicative of IFN. In addition, it was not possible to induce a decidual reaction in suitably sensitized uteri with intraluminal instillation of IFN-alpha. The results indicate that IFNs are probably not involved in the maternal recognition of pregnancy at the time of implantation in the mouse.     

A deer (subfamily Cervinae) genetic linkage map and the evolution of ruminant genomes

Comparative maps between ruminant species and humans are increasingly important tools for the discovery of genes underlying economically important traits. In this article we present a primary linkage map of the deer genome derived from an interspecies hybrid between red deer (Cervus elaphus) and Père David's deer (Elaphurus davidianus). The map is approximately 2500 cM long and contains >600 markers including both evolutionary conserved type I markers and highly polymorphic type II markers (microsatellites). Comparative mapping by annotation and sequence similarity (COMPASS) was demonstrated to be a useful tool for mapping bovine and ovine ESTs in deer. Using marker order as a phylogenetic character and comparative map information from human, mouse, deer, cattle, and sheep, we reconstructed the karyotype of the ancestral Pecoran mammal and identified the chromosome rearrangements that have occurred in the sheep, cattle, and deer lineages. The deer map and interspecies hybrid pedigrees described here are a valuable resource for (1) predicting the location of orthologs to human genes in ruminants, (2) mapping QTL in farmed and wild deer populations, and (3) ruminant phylogenetic studies.     

干扰素-tau的原核表达、纯化和活性测定

干扰素-tau (IFN-tau)是一种新发现的I型干扰素,为了更清楚的研究它的生物学功能,在已克隆IFN-tau cDNA的基础上,PCR扩增出IFN-tau ORF,与原核表达载体pBV220重组后,成功的构建了IFN-tau的原核表达质粒pBV220/IFN-tau。重组质粒转化大肠杆菌BL21,该菌经过诱导,用凝胶过滤色谱层析的方法获得了纯化的目的蛋白,该蛋白经过氨基酸序列分析证实是IFN-tau ;用细胞病变抑制法测定IFN-tau经过透析复性后的活性为2.09×106IU/ml,比活性为2.35×106IU/mg。     

Molecular characterization of novel variants of interferon-tau (IFNT) gene in Garole breed of sheep (Ovis aries)

The survivability of embryo, especially during the early embryonic life is dependent on the effective maternal recognition of pregnancy. Interferon-tau (IFNT), secreted from the elongating blastocyst, acts as the primary signal for maternal recognition of pregnancy in ruminant ungulates. IFNT has been studied extensively in many domesticated and wild ruminant species. In the present study, we have cloned and characterized the IFNT gene of Garole sheep, a popular Indian micro-sheep breed, which is known across the world for its high prolificacy and fecundity. The 588 bp sequences of two variants of IFNT gene described in this study are novel variants, compared to the variants reported previously in sheep. It exhibited more than 96% identity with other ovine IFNT variants and phylogenetically placed in a single clad containing the ovine, caprine and musk ox IFNT variants. The IFNT of Garole sheep demonstrated the highest identity with the genomic derived and highly expressed ovine IFNT variants.     

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.     

Mosaic evolution of ruminant stomach lysozyme genes

The genomes of ruminant artiodactyls, such as cow and sheep, have approximately 10 lysozyme genes, 4 of which are expressed in the stomach. Most of the duplications of the lysozyme genes occurred 40-50 million years ago, before the divergence of cow and sheep. Despite this, the coding regions of stomach lysozyme genes within a species (e.g., cow, sheep, or deer) are more similar to each other than to lysozyme genes in other ruminants. This observation suggests that the coding regions of the stomach lysozyme genes have evolved in a concerted fashion. Our previous characterization of 3 cow stomach lysozyme genes suggested that it was only the coding exons that had participated in concerted evolution. To determine whether the introns and flanking regions of ruminant stomach lysozyme genes are evolving in a concerted or a divergent fashion, we have isolated and characterized 2 sheep stomach lysozyme genes. Comparison of the sequences of the sheep and cow stomach lysozyme genes clearly shows that the introns and flanking regions have evolved, like the 3' untranslated region of the mRNAs, in a divergent manner. Thus, if the four coding exons are evolving by concerted evolution, then a mosaic pattern of concerted and divergent evolution is occurring in these genes. The independent concerted evolution of coding exons of the ruminant stomach lysozyme gene may have assisted in the accelerated adaptive evolution of the lysozyme to new function in the early ruminant.     

The interferon system of teleost fish

Interferons (IFNs) are secreted proteins, which induce vertebrate cells into an antiviral state. In mammals, three families of IFNs (type I IFN, type II IFN and IFN-lambda) can be distinguished on the basis of gene structure, protein structure and functional properties. Type I IFNs, which include IFN-alpha and IFN-beta, are encoded by intron lacking genes and have a major role in the first line of defense against viruses. The human IFN-lambdas have similar biological properties as type I IFNs, but are encoded by intron containing genes. Type II IFN is identical to IFN-gamma, which is produced by T helper 1 cells in response to mitogens and antigens and has a key role in adaptive cell mediated immunity. IFNs, which show structural and functional properties similar to mammalian type I IFNs, have recently been cloned from Atlantic salmon, channel catfish, pufferfish, and zebrafish. Teleost fish appear to have at least two type I IFN genes. Phylogenetic sequence analysis shows that the fish type I IFNs form a group separated from the avian type I IFNs and the mammalian IFN-alpha, -beta and -lambda groups. Interestingly, the fish IFNs possess the same exon/intron structure as the IFN-lambdas, but show most sequence similarity to IFN-alpha. Recently, IFN-gamma genes have also been cloned from several fish species and shown to have the same exon/intron structure as mammalian IFN-gamma genes. The antiviral effect of mammalian type I IFN is exerted through binding to the IFN-alpha/beta-receptor, which triggers signal transduction through the JAK-STAT signal transduction pathway resulting in expression of Mx and other antiviral proteins. Putative IFN receptor genes have been identified in pufferfish. Several interferon regulatory factors and members of the JAK-STAT pathway have also been identified in various fish species. Moreover, Mx and several other interferon stimulated genes have been cloned and studied in fish. Furthermore, antiviral activity of Mx protein from Atlantic salmon and Japanese flounder has recently been demonstrated.     

Molecular Evolution of the Porcine Type I Interferon Family: Subtype-Specific Expression and Antiviral Activity

Type I interferons (IFNs), key antiviral cytokines, evolve to adapt with ever-changing viral threats during vertebrate speciation. Due to novel pathogenic pressure associated with Suidae speciation and domestication, porcine IFNs evolutionarily engender both molecular and functional diversification, which have not been well addressed in pigs, an important livestock species and animal model for biomedical sciences. Annotation of current swine genome assembly Sscrofa10.2 reveals 57 functional genes and 16 pseudogenes of type I IFNs. Subfamilies of multiple IFNA, IFNW and porcine-specific IFND genes are separated into four clusters with ∼60 kb intervals within the IFNB/IFNE bordered region in SSC1, and each cluster contains mingled subtypes of IFNA, IFNW and IFND. Further curation of the 57 functional IFN genes indicates that they include 18 potential artifactual duplicates. We performed phylogenetic construction as well as analyses of gene duplication/conversion and natural selection and showed that porcine type I IFN genes have been undergoing active diversification through both gene duplication and conversion. Extensive analyses of the non-coding sequences proximal to all IFN coding regions identified several genomic repetitive elements significantly associated with different IFN subtypes. Family-wide studies further revealed their molecular diversity with respect to differential expression and restrictive activity on the resurgence of a porcine endogenous retrovirus. Based on predicted 3-D structures of representative animal IFNs and inferred activity, we categorized the general functional propensity underlying the structure-activity relationship. Evidence indicates gene expansion of porcine type I IFNs. Genomic repetitive elements that associated with IFN subtypes may serve as molecular signatures of respective IFN subtypes and genomic mechanisms to mediate IFN gene evolution and expression. In summary, the porcine type I IFN profile has been phylogenetically defined family-wide and linked to diverse expression and antiviral activity, which is important information for further biological studies across the porcine type I IFN family.     

Identification of a second group of type I IFNs in fish sheds light on IFN evolution in vertebrates

In this report, three type I IFN genes were identified in rainbow trout (rt) Oncorhynchus mykiss and are classified into two groups based on their primary protein sequences: group I containing two cysteine residues; and group II containing four cysteines residues. The group I rtIFNs were induced in fibroblasts (RTG-2 cells), macrophages (RTS-11 cells), and head kidney leukocytes when stimulated with polyinosinic:polycytidylic acid, whereas group II IFN was up-regulated in head kidney leukocytes but not in RTG-2 and RTS-11 cells. Recombinant group I rtIFNs were potent at inducing Mx expression and eliciting antiviral responses, whereas recombinant group II rtIFN was poor in these activities. That two subgroups of type I IFN exist in trout prompted a survey of the genomes of several fish species, including zebrafish, medaka, threespine stickleback and fugu, the amphibian Xenopus tropicalis, the monotreme platypus and the marsupial opossum, to gain further insight into possible IFN evolution. Analysis of the sequences confirmed that the new IFN subgroup found in trout (group II IFN) exists in other fish species but was not universally present in fish. The IFN genes in amphibians were shown for the first time to contain introns and to conserve the four cysteine structure found in all type I IFNs except IFN-betaepsilon and fish group I IFN. The data overall support the concept that different vertebrate groups have independently expanded their IFN types, with deletion of different pairs of cysteines apparent in fish group I IFN and IFN-betaepsilon of mammals.     

Type I interferon genes from the egg-laying mammal, Tachyglossus aculeatus (short-beaked echidna)

The type I IFN are an important group of multifunctional cytokines that have, for whatever reason, evolved to a high level of complexity in eutherian mammals such as humans and mice. However, until recently, little was known about the type I IFN systems of the other two groups of extant mammals, the marsupials and the egg-laying monotremes. Preliminary partial type I IFN sequences from the short-beaked echidna were previously found to cluster only with the IFN-beta subtype in phylogenetic analyses, but a lack of sequence information made interpretation of these results tenuous. Here, we report cloning of the full-length genes of representatives from the two previously defined groups of echidna type I IFN by genomic walking PCR. Along with analysis of conserved cysteine placement and promoter elements, phylogenetic analysis incorporating these sequences strongly suggest that the two groups of echidna type I IFN genes are in fact homologous to IFN-alpha and IFN-beta, confirming that the duplication leading to these two major classes of type I IFN occurred prior to the divergence of eutherians and monotremes some 180 million years ago. Thus, even though there are major differences in gene copy number and heterogeneity, separate IFN-alpha and IFN-beta gene families are a feature of the cytokine networks of all three groups of living mammals.