Characterization of the bovine pregnancy-associated glycoprotein gene family--analysis of gene sequences, regulatory regions within the promoter and expression of selected genes.

BACKGROUND: The Pregnancy-associated glycoproteins (PAGs) belong to a large family of aspartic peptidases expressed exclusively in the placenta of species in the Artiodactyla order. In cattle, the PAG gene family is comprised of at least 22 transcribed genes, as well as some variants. Phylogenetic analyses have shown that the PAG family segregates into 'ancient' and 'modern' groupings. Along with sequence differences between family members, there are clear distinctions in their spatio-temporal distribution and in their relative level of expression. In this report, 1) we performed an in silico analysis of the bovine genome to further characterize the PAG gene family, 2) we scrutinized proximal promoter sequences of the PAG genes to evaluate the evolution pressures operating on them and to identify putative regulatory regions, 3) we determined relative transcript abundance of selected PAGs during pregnancy and, 4) we performed preliminary characterization of the putative regulatory elements for one of the candidate PAGs, bovine (bo) PAG-2. RESULTS: From our analysis of the bovine genome, we identified 18 distinct PAG genes and 14 pseudogenes. We observed that the first 500 base pairs upstream of the translational start site contained multiple regions that are conserved among all boPAGs. However, a preponderance of conserved regions, that harbor recognition sites for putative transcriptional factors (TFs), were found to be unique to the modern boPAG grouping, but not the ancient boPAGs. We gathered evidence by means of Q-PCR and screening of EST databases to show that boPAG-2 is the most abundant of all boPAG transcripts. Finally, we provided preliminary evidence for the role of ETS- and DDVL-related TFs in the regulation of the boPAG-2 gene. CONCLUSION: PAGs represent a relatively large gene family in the bovine genome. The proximal promoter regions of these genes display differences in putative TF binding sites, likely contributing to observed differences in spatial and temporal expression. We also discovered that boPAG-2 is the most abundant of all boPAG transcripts and provided evidence for the role of ETS and DDVL TFs in its regulation. These experiments mark the crucial first step in discerning the complex transcriptional regulation operating within the boPAG gene family.

Characterization of the bovine type I IFN locus: rearrangements, expansions, and novel subfamilies.

BACKGROUND: The Type I interferons (IFN) have major roles in the innate immune response to viruses, a function that is believed to have led to expansion in the number and complexity of their genes, although these genes have remained confined to single chromosomal region in all mammals so far examined. IFNB and IFNE define the limits of the locus, with all other Type I IFN genes except IFNK distributed between these boundaries, strongly suggesting that the locus has broadened as IFN genes duplicated and then evolved into a series of distinct families. RESULTS: The Type I IFN locus in Bos taurus has undergone significant rearrangement and expansion compared to mouse and human, however, with the constituent genes separated into two sub-loci separated by >700 kb. The IFNW family is greatly expanded, comprising 24 potentially functional genes and at least 8 pseudogenes. The IFNB (n = 6), represented in human and mouse by one copy, are also present as multiple copies in Bos taurus. The IFNT, which encode a non-virally inducible, ruminant-specific IFN secreted by the pre-implantation conceptus, are represented by three genes and two pseudogenes. The latter have sequences intermediate between IFNT and IFNW. A new Type I IFN family (IFNX) of four members, one of which is a pseudogene, appears to have diverged from the IFNA lineage at least 83 million years ago, but is absent in all other sequenced genomes with the possible exception of the horse, a non-ruminant herbivore. CONCLUSION: In summary, we have provided the first comprehensive annotation of the Type I IFN locus in Bos taurus, thereby providing an insight into the functional evolution of the Type I IFN in ruminants. The diversity and global spread of the ruminant species may have required an expansion of the Type I IFN locus and its constituent genes to provide broad anti-viral protection required for foraging and foregut fermentation.

Expression of bovine interferon-tau variants according to sex and age of conceptuses.

Interferon-tau (IFNT), which plays a major role in maternal recognition of pregnancy in cattle, is transcribed from multiple genes. Moreover, there are at least 12 cDNA variants, many presumably allelic. Although the IFNT locus is autosomal, Day 8 female blastocysts produced approximately twice as much antiviral activity as males. The questions addressed here are whether male and female blastocysts differed in the kind and number of IFNT they expressed, and whether this pattern changed over development. Day 8, in vitro-produced blastocysts were bisected, and one half of each was sexed by PCR. Demi-embryos (n=64) were grouped according to whether they were male or female, to provide two pools of each sex. Individual cDNA were sequenced after RT-PCR amplification and shot-gun cloning to provide comparisons between male and female blastocysts, elongating conceptuses of various developmental ages (Days 14-19), and a female trophoblast cell line (CT-1). A total of 376 cDNA clones were sequenced. Six additional cDNA were identified, in addition to the forms described earlier. There were no differences between male and female blastocysts (P=0.54), and between blastocysts and a trophoblast model system (CT-1 cells; P=0.24) in the IFNT transcripts expressed, indicating that sexual dimorphism was not correlated with particular IFNT variants. There were differences in variant frequencies (P<0.001) among conceptuses of different age, although two, representing boIFN1a and boIFN3c, predominated throughout development. Notably, no alteration in overall IFNT variant diversity was detected in CT-1 cells over time (P=0.124).

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.

Effect of interferon-tau administration on endometrium of nonpregnant ewes: a comparison with pregnant ewes.

In ruminants, conceptus interferon-tau (IFNT) alters maternal physiology to accommodate a pregnancy. We hypothesized that the effectiveness of IFNT on extending corpus luteum (CL) life span in nonpregnant ewes would depend upon the dose and manner of administration and would be correlated with the response in gene expression in endometrium. We anticipated that IFNT, whether administered im or by uterine infusion, would mimic changes observed in pregnancy. Ewes were assigned to five treatments: 1) uterine infusion of saline; 2) uterine infusion of ovine IFNT4 (200 microg/d); 3) saline im injection; 4) im injection of IFNT4 at low dose (200 microg/d); and 5) high dose (2 mg/d). CL life span was increased in groups 2 and 5, but not in 1, 3, and 4. Endometrial RNA extracted from groups 1-5 on d 14 and from d 14 pregnant and nonbred (cyclic) ewes was used to assess expression of 70 genes on microarrays. When pregnant and cyclic ewes were compared, 30 genes were up-regulated and nine down-regulated during pregnancy. Responses were slightly less in groups 2 and 5 but were much lower in group 4. The majority of the highly up-regulated genes were associated with antiviral responses. Those down-regulated included ones for IGF-II, hypoxia-inducible factor 1alpha, oxytocin receptor, prostaglandin F synthase, and cyclooxygenase-2. Quantitative PCR for selected genes confirmed these data and revealed that similar gene expression changes occurred in the CL of pregnant and group 2 ewes. IFNT treatment mimics pregnancy, but relatively high doses of im-injected IFNT are required to elicit a full endometrial response.