2D Electrophoretic pattern of bovine placental proteins during early-mid pregnancy.

The Placenta, like every tissue, possesses its own characteristic protein profile, which may change within the course of pregnancy. These changes can be used for the elucidation of the mechanisms related to both physiology of pregnancy and pathological events. The aim of the study was to describe proteinergic profiles of maternal and fetal parts of bovine placenta during early-mid pregnancy by the use of 2D electrophoresis and MALDI TOF/TOF MS identification to evaluate dynamics of the possible changes necessary for placentation. Placental samples were collected from six pregnant cows (3-5 months) in the local abattoir. Placentomes were separated, and proteins were extracted and subjected to 2D electrophoresis and MALDI TOF/TOF identification. Out of 907 spots identified by the statistical analysis of gels, 54 were identified. Out of this number, 36 spots were significantly different between examined samples. Moreover, the obtained patterns differed between maternal and fetal parts of the placenta with regard to the intensity of staining, suggesting quantitative differences in protein content. These preliminary results are unique for this period of pregnancy. Such data are important for further experiments to obtain full protein profiles necessary to understand biochemical mechanisms underlying the attachment between fetal and maternal parts of the placenta during placentation. Moreover, the outcomes may help in elucidating pregnancy biomarkers in the future.

Regulation of conceptus interferon-tau gene subtypes expressed in the uterus during the peri-implantation period of cattle.

Conceptus interferon tau (IFNT), produced by the embryonic trophectoderm, is known as a major signaling protein essential for the process of maternal recognition of pregnancy in ruminants. Similar to other IFN gene families such as IFNA and IFNB, multiple IFNT genes exist. The number of IFNT genes actively transcribed and regulated in conceptuses of cattle has, however, not been well characterized. In this study, IFNT transcripts in utero were studied through the use of next generation sequencer. Among 38 IFN genes registered and eight annotated as IFNT, only two transcripts, IFNT1 and IFNTc1, were found in conceptuses in utero. Relative abundance of transcription factor(s) involved in the regulation of IFNT genes were investigated by real-time PCR. Transcriptional activity of IFNT1 and IFNTc1 were investigated using bovine non-trophoblast ear fibroblast (EF) cells, which were co-transfected with luciferase reporter constructs with upstream (-631 to -51) promoter regions of IFNT1 or IFNTc1 and various transcription factor expression plasmids, CDX2, AP1 (JUN), ETS2 and/or CREBBP. CDX2 with AP1 and ETS2 was found to increase luciferase activity of IFNT1 and IFNTc1 approximately 14- and 11-fold, respectively, in EF cells, which do not express the CDX2 gene. These results indicated that two isoforms of conceptus IFNT genes of cattle could be regulated differently in utero. Furthermore, IFNT1 and IFNTc1 were found to have similar antiviral activity, suggesting that both IFNT genes could function to increase conceptus signaling to the uterine endometrium for the process of maternal recognition of pregnancy during the pre-implantation period.

Transcriptional regulation of two conceptus interferon tau genes expressed in Japanese black cattle during peri-implantation period.

Interferon tau (IFNT), produced by the mononuclear trophectoderm, signals the process of maternal recognition of pregnancy in ruminants. However, its expression in vivo and its transcriptional regulation are not yet well characterized. Objectives of this study were to determine conceptus IFNT gene isoforms expressed in the bovine uterus and to identify differences in promoter sequences of IFNT genes that differ in their expression. RNA-seq data analysis of bovine conceptuses on days 17, 20, and 22 (day 0  =  day of estrus) detected the expression of two IFNT transcripts, IFNT1 and IFNTc1, which were indeed classified into the IFNT gene clade. RNA-seq and quantitative RT-PCR analyses also revealed that the expression levels of both IFNT mRNAs were highest on day 17, and then decreased on days 20 and 22. Bovine ear-derived fibroblast (EF) cells, a model system commonly used for bovine IFNT gene transcription study in this laboratory, were cotransfected with luciferase reporter constructs carrying upstream (positions -637 to +51) regions of IFNT1 or IFNTc1 gene and various transcription factor expression plasmids including CDX2, AP-1 (Jun) and ETS2. CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. The upstream regions of IFNT1 and IFNTc1 loci were then serially deleted or point-mutated at potential CDX-, AP-1-, and ETS-binding sites. Compared to the wild-type constructs, deletion or mutation at CDX2 or ETS2 binding sites similarly reduced the luciferase activities of IFNT1- or IFNTc1-promoter constructs. However, with the AP-1 site mutated construct, IFNT1- and IFNTc1-reporters behaved differently. These results suggest that two forms of bovine conceptus IFNT genes are expressed in utero and their transcriptional regulations differ.

Isolation and analysis of α-expansin genes in the tree Anthocephalus chinensis (Rubiaceae).

Expansins are cell wall-associated proteins that induce wall extension and relax stress by disrupting noncovalent bonds between cellulose microfibrils and cross-linking glycan chains, thereby promoting wall creep. Anthocephalus chinensis is a very fast-growing economically important tree found mainly in South Asia. Sixteen cDNAs, designated AcEXPA1 to AcEXPA16 (GenBank accession Nos. FJ417847, JF922686-JF922700) with corresponding genomic DNA sequences (GenBank accession Nos. GQ228823, JF922701-JF922715), were isolated by amplifying conserved domain binding with genomic walking and RACE techniques from four differential growth tissues in A. chinensis. These α-expansin homologues were highly conserved in size and sequence; they had the same sequence structures as an N-terminal signal peptide, three exons and two introns. Their amino acid alignment showed that A. chinensis expansin genes are divided into three subgroups: A, B and C. This study is the first report on expansin genes from A. chinensis. It will be used for a tissue-specific expression model and for studying the relationship between expansin genes, growth rate and wood quality of the xylem in this fast-growing tree.

Quantitative proteomic analysis of pregnancy-related proteins from peripheral blood mononuclear cells during pregnancy in pigs.

Information obtained from peripheral blood could help us understand the underlying mechanisms in autoimmune diseases, cancer, pregnancy, and other conditions. In this paper, we present the protein map of porcine peripheral blood mononuclear cells (PBMC) to better understand the molecular expression changes that occur during pregnancy using proteomic analysis. We detected 94 differentially expressed proteins in pregnant vs. non-pregnant (NP) pigs, and a representative set of the proteins was subjected to LC-MS/MS analysis. Furthermore, the identified proteins were categorized according to their biological process and molecular function. By classifying the proteins according to their functions, a large number of differentially regulated proteins involved in anti-oxidant, detoxification and stress response pathways were found, including peroxiredoxin (PRX) 1, 2, and 6, glutathione-S-transferase (GST), annexin A2, and A6, and heat shock protein 27 (HSP 27) during pregnancy (pregnancy d of E40, embryonic day 40; E70, embryonic day 70; and E93, embryonic day 93) compared with non-pregnancy. In this study, a proteomic approach utilizing 2-DE and LC-MS/MS was applied to evaluate specific molecular expression changes during pregnancy compared with non-pregnancy. Together, these data offer new information about the proteome map and factors that are differentially regulated during maintenance of normal pregnancy.

Identification of interferon-tau isoforms expressed by the peri-implantation goat (Capra hircus) conceptus.

Interferon-tau (IFN-tau) is the maternal recognition of pregnancy factor in pecoran ruminants. The aims of this study were to identify the various IFN-tau transcripts in the peri-implantation caprine (ca) conceptus and to compare these nucleotide sequences phylogenetically with established mRNA sequences from the goat. Conceptuses (n = 5) were collected from Boer crossbred and Angora female goats by laparotomy at days 17 and 18 of pregnancy. Total cellular RNA was extracted and RT-PCR was performed by standard procedures using a DNA polymerase with proofreading activity and gene-specific primers complimentary to non-coding regions of the published caIFN-tau sequence. Nine distinct nucleotide sequences were isolated that encode five distinct caIFN-tau proteins. These caIFN-tau have greater sequence homology with ovine IFN-tau (94-96% nucleotide identity; 90-93% amino acid identity) than with bovine IFN-tau (<92% nucleotide identity; <85% amino acid identity). The novel caIFN-tau isoforms contained pronounced nucleotide and amino acid sequence identity with one another (97-99% nucleotide identity; 94-99% amino acid identity) but only moderate sequence identity with the previously identified caIFN-tau (94-96% nucleotide identity; 87-90% amino acid identity). In conclusion, multiple caIFN-tau mRNA species are expressed during peri-implantation conceptus development and distinct clusters of caIFN-tau genes appear to have evolved in this species.

TEPP, a new gene specifically expressed in testis, prostate, and placenta and well conserved in chordates.

We have combined computer-based screening and experimental expression analysis to identify genes that are expressed in normal prostate and/or prostate cancer but not in essential human tissues. Using this approach we identified a new gene that is specifically expressed in testis, prostate, and placenta. The gene has one major transcript of 1.0kb in size and encodes for a protein of 30.7kDa molecular weight. We named this gene TEPP (expressed in testis, prostate, and placenta). The amino acid sequence analysis of TEPP using SignalP program shows that it has a signal peptide with a predicted cleavage site between amino acids 19 and 20, indicating that it might be a secreted protein. Analysis of the predicted TEPP orthologs from different species shows that these proteins are highly conserved in chordates. In addition we have identified a splice variant of TEPP, which encodes a 37kDa protein. In conclusion, a combination of bioinformatic and molecular approaches is useful in the identification of genes expressed in specific tissues. Selective expression of TEPP in testis, prostate, and in placenta and its high conservation among different species indicate that TEPP might have a role in reproductive biology.

Caprine pregnancy-associated glycoproteins (PAG): their cloning, expression, and evolutionary relationship to other PAG.

Pregnancy-associated glycoproteins (PAG) are structurally related to aspartic proteinases and belong to an extensive, rapidly evolving family of recently duplicated genes expressed in the placentas of artiodactyl species. The aim of the present study was to clone PAG from the goat, study their temporal and cell-specific expression, and determine their phylogenetic relationship to PAG from other species. RT-PCR was used to generate PAG cDNA from pooled placental RNA obtained between days 45 and 115 of pregnancy. A total of 11 cDNA, which differed by > 5% from each other, were selected for complete bidirectional sequencing from 60 clones analyzed. A group of nine (caPAG1, caPAG3-7(var), caPAG9-11), which displayed > 80% sequence identity with each other, were expressed after day 45 of pregnancy and were localized to trophoblast binucleate cells. These PAG demonstrated an unusually high ratio of nonsynonymous (amino acid changing) to synonymous nucleotide differences. CaPAG2, by contrast, was detectable only in early pregnancy (days 18 and 19) and expressed throughout trophectoderm. It was of more ancient origin than the PAG1 group, but more recent than caPAG8. The latter was expressed at all stages examined (days 18 to 115). The data confirm that many PAG genes, with different patterns of temporal and spatial expression, are transcribed in the placenta of the goat. The data also suggest that the recently duplicated PAG genes are being selected for rapid diversification of function.

A classification for the interferon-tau.

An attempt has been made to provide a rational organization for the many interferon-tau (IFN-tau) sequences entered in GenBank based on phylogenetic analysis and common amino acid substitutions, which might form the basis for a universal nomenclature scheme. Over the 13 years since these genes were first discovered, large numbers of cDNA and gene sequences have been reported, and there is reason to suspect that representatives of all the major ovine and bovine forms have now been described. The data are consistent with the presence of many genes and also allelic variants in sheep and cattle analogous to what has been observed for the IFN-alpha in the human. Future variants should be easily accommodated into the scheme outlined here. A flexible system of nomenclature, based on that used for HuIFN, is needed to provide a common base for comparison between research done in different laboratories and to assign relative biologic potencies to these molecules.

The cross-species antiviral activities of different IFN-tau subtypes on bovine, murine, and human cells: contradictory evidence for therapeutic potential.

It is claimed that interferon-tau (IFN-tau) has broad cross-species reactivity and less cytotoxicity than other type I IFN when used at high concentration either in vitro or in living animals. It can also amelioriate the development of experimental allergic encephalomyelitis (EAE) without the usual side effects of IFN therapy in mice autoimmunized with myelin basic protein. For these reasons, IFN-tau may have therapeutic potential in humans. Here, the antiviral (AV) activities of eight different recombinant IFN-tau were compared with those of several bovine, human, and murine type I IFN on bovine MDBK cells, murine L929 cells, and human WISH cells. The data show that only one of the IFN-tau, OvIFN-tau4, has broad cross-species reactivity. It was comparable in this respect to HuIFN-omega1 and HuIFN-alpha1. The other IFN-tau, including the variant form (OvIFN-tau1mod) tested by others in cytotoxicity experiments and for its ability to protect mice against EAE, had relatively weak AV activity on mouse and human cells. It is possibly because this particular bioengineered form of IFN-tau binds the common type I receptor of these two species with such low affinity that it lacks cytotoxic effects. The basis for its potent anti-EAE activity is unclear, but it seems possible that it does not involve the type I IFN receptor.

Glycodelin from seminal plasma is a differentially glycosylated form of contraceptive glycodelin-A.

Glycodelin-A is a human amniotic fluid-derived glycoprotein with contraceptive and immunosuppressive activities. An immunoreactive form of glycodelin was detected in seminal plasma over a decade ago, but definitive characterization of this glycoprotein was not pursued. We considered it unlikely that the seminal plasma of fertile men would contain an appreciable amount of contraceptive glycodelin-A. To address this issue we purified seminal plasma glycodelin (glycodelin-S) and performed comparative studies with glycodelin-A. Glycodelin-S behaved differently when compared with glycodelin-A during sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing but identically after enzymatic deglycosylation. N-terminal sequencing of glycodelin-A and glycodelin-S gave identical results, and digestion with trypsin gave identical peptide fragments. The glycoproteins were also found to be indistinguishable from each other based upon immunological analyses. These results indicate that glycodelin-S and glycodelin-A have similar overall protein structure, suggesting the likelihood that these glycoproteins are differentially glycosylated forms of very similar proteins. This latter possibility is supported by lectin binding studies indicating that, unlike glycodelin-A, glycodelin-S does not manifest any affinity for lectins from Wisteria floribunda or Sambucus nigra. The results of sugar analysis and neuraminidase digestion also lead us to conclude that glycodelin-S and glycodelin-A are differentially glycosylated forms of similar proteins. Our evidence indicates that glycodelin-A mediated its biological activities via its unusual oligosaccharide sequences that are not associated with glycodelin-S. In lectin-immunoassay no appreciable amount of contraceptive glycodelin-A was found in the 22 seminal plasma samples studied.

Analysis of rat placental plasma membrane proteins by two-dimensional gel electrophoresis.

The placenta plays an essential role in fetal growth and the maintenance of pregnancy and its functions are strictly controlled in a stage-specific manner. To gain an insight into placental functions and their regulation, we analyzed the plasma membrane proteins of rat placenta by two-dimensional polyacrylamide gel electrophoresis (2D/E). Plasma membrane fractions of the placenta obtained on days 12, 14, 16, 18 and 20 of pregnancy were purified by Percoll gradient centrifugation, and subjected to 2D/E analysis. After the proteins on the 2D/E gels had been visualized by silver staining, the patterns on the gels at different stages of pregnancy were compared using image analysis software. Proteins within an isoelectric point (pI) range of 4.0 to 7.0 and a molecular weight (Mw) range of 20-100 kDa were analyzed in detail, and about 800 proteins on average were recognized on each gel. Of these, the expression of 150 proteins was found to change dramatically according to the stage of pregnancy. According to their expression patterns, these proteins were categorized into two groups, Group I and Group II. The proteins belonging to Group I showed a higher intensity of expression on day 12 and disappeared on day 20. They included 119 plasma membrane proteins and were divided into five subgroups. Group II, which consisted of three subgroups, included 31 proteins showing a low or negligible expression on day 12 and higher expression on day 20. Most of the other membrane proteins (about 600) were expressed constantly during pregnancy. On the basis of our data, we constructed a database for plasma membrane proteins of the rat placenta.

Characterization of murine carcinoembryonic antigen gene family members.

The carcinoembryonic antigen (CEA) is a human tumor marker whose gene belongs to a family with more than 20 members. This gene family codes for a group of proteins with in vitro cell adhesion properties and for a group of abundantly expressed pregnancy-specific glycoproteins (PSG) with unknown functions. As a basis for in vivo functional studies, we have started to analyze the murine CEA gene family and have identified five new members (Cea-2 to Cea-6). cDNA clones were isolated for Cea-2, Cea-3, and Cea-6. The deduced amino acid sequences of Cea-2 and Cea-6 indicate three IgV-like (N), followed by one IgC-like (A) domain (N1-N2-N3-A). We have also partially characterized the Cea-2 gene and two additional ones, Cea-4 and Cea-5. Cea-2 and Cea-4 are separated by only 16 kb, suggesting a close linkage of murine CEA-related genes, as found for the human CEA gene family. Cea-5 was located to the proximal region of mouse Chromosome (Chr) 7, which is syntenic to part of human Chr 19, containing the human CEA gene family cluster. Cea-2, Cea-3, and a Cea-4-like gene are differentially transcribed in the placenta during pregnancy, but not in other organs tested. This expression pattern strongly suggests that they represent counterparts of the human PSG subgroup members, despite the presence of multiple IgV-like domains, a feature not found for human PSGs. The more distantly related Cea-5 seems to be ubiquitously expressed. The putative promoter region of Cea-2 lacks typical TATA- or CAAT-boxes, but contains other conserved motifs that could play a role in the initiation of transcription.

Tissue anticoagulants in the human placenta: preliminary study with a heparin-like anticoagulant and review of the literature.

In this study a new heparin-like anticoagulant in the human placenta is described and an extensive review of the literature is given. An anticoagulant with a heparin-like activity was isolated from an extract of the human placenta. A two-stage purification procedure was applied: affinity chromatography on heparin-Sepharose and ion-exchange chromatography on DEAE-trisacryl. The isolated anticoagulant fraction was found to prolong both thrombin clotting time and activated partial thromboplastin time and to increase the activity of antithrombin III and heparin cofactor II. The placental-tissue anticoagulants described in the literature are divided by the author into five groups: (1) acidic peptides of low molecular weight; (2) thrombomodulin; (3) phospholipid-binding protein anticoagulants; (4) heparin-like anticoagulants, and (5) others. The anticoagulant described in the present study has been classified to the 4th group.