Integration of molecules to construct the processes of conceptus implantation to the maternal endometrium.

During the peri-implantation period, ruminant conceptuses go through rapid elongation, followed by their attachment to the uterine endometrial epithelial cells, during which interferon-tau (IFNT), a trophectodermal cytokine required for the process of maternal recognition of pregnancy, is expressed in a temporal and spatial manner. On day 22 (day 0 = day of estrus), 2 to 3 d after the initiation of bovine conceptus attachment to the uterine epithelium, when IFNT production begins to subside, the expression of molecules related to epithelial-mesenchymal transition, zinc finger E-box binding homeobox 1, snail family transcriptional repressor 2, N-cadherin, and vimentin was found in the trophectoderm. Through the use of in vitro coculture system with bovine trophoblast CT-1 and endometrial epithelial cells, a series of experiments have been conducted to elucidate mechanisms associated with the regulation of IFNT gene transcription and conceptus implantation, including epithelial-mesenchymal transition processes. Expression of IFNT, both up- and downregulation, during the peri-implantation period is tightly controlled. Cytokines and cell adhesion molecules such as epidermal growth factor, basic fibroblast growth factor, transforming growth factor beta, activin A, L-selectin-podocalyxin, and vascular cell adhesion molecule 1-integrin α4 expressed in utero all contribute to the initiation of epithelial-mesenchymal transition in the trophectoderm. These results indicate that conceptus implantation to the uterine endometrium proceeds while elongated conceptuses and endometria express cell adhesion molecules and their receptors, and the trophectoderm experiences epithelial-mesenchymal transition. Data accumulated suggest that while the conceptus and the endometrial epithelium adhere, trophectodermal cells must gain more flexibility for binucleate and possibly trinucleate cell formation during the peri-implantation period, and that understanding and constructing the conditions throughout implantation processes is key to improving ruminants' fertility.

The Phylogeny of Placental Evolution Through Dynamic Integrations of Retrotransposons.

Trophoblasts, a major constituent of the placenta, are known to express genes derived from various endogenous retroviruses (ERVs) as well as LTR retrotransposons. However, the evolutionary significance of ERV-derived genes involved in placental development has not been well characterized. In this review, we catalog the diverse morphology of placental structure among mammalian species with note of counterintuitive developments. We then detail the history of ancient placenta development with paternally expressed gene 10 (Peg10/Sirh1), Peg11/Sirh2, and Sirh7/Ldoc1 as LTR retrotransposons, followed by independent captures of ERV-env-related genes such as Syncytin-1, -2, -A, -B, -Rum1, and Fematrin-1 responsible for trophoblast cell fusion, resulting in multinucleate syncytiotrophoblast formation, and possibly morphological diversification of placentas. Because the endogenization of retroviral infections has occurred multiple times independently in different mammalian lineages, and some use the same molecules in their transcriptional activation, we speculate that ERV gene variants integrated into mammalian genomes in a locus-specific manner have replaced the genes previously responsible for cell fusion. Moreover, ERVs also work as transcriptional regulators of various genes such as interferon (IFN)-stimulated genes. The "baton pass" hypothesis suggests that evolutionary events caused by multiple successive retrotransposon integrations, possibly resulting in effective fusogenic activity, downstream gene transcription in a temporal and spatial manner, and/or increased diversity of placental structures.

A transcriptional cofactor YAP regulates IFNT expression via transcription factor TEAD in bovine conceptuses.

Interferon tau (IFNT) is the pregnancy recognition protein in all ruminants, and its expression is restricted to trophoblast cells. Interferon tau production increases as the conceptus elongates; however, its expression is downregulated soon after the initiation of conceptus attachment to the uterine epithelium. Our previous study identified that among 8 bovine IFNT genes, only 2 forms of IFNTs, IFNT2 and IFN-tau-c1, were expressed by the conceptuses during the periattachment period. To characterize whether Hippo signaling including a transcription cofactor yes-associated protein (YAP) was involved in the IFNT regulation, we examined the expression and effects of YAP and/or TEAD in human choriocarcinoma JEG3 and bovine trophoblast CT-1 cells, and in bovine conceptuses obtained from day 17, 20 or 22 pregnant animals (pregnant day 19.5 = day of conceptus attachment to the endometrium). YAP was expressed in bovine conceptuses and transfection of YAP or TEAD4, a transcription factor partner of YAP, expression plasmid increased the luciferase activity of IFNT2 and IFN-tau-c1 reporter plasmids in JEG3 cells. In the presence of YAP expression plasmid, TEAD2 or TEAD4 expression plasmid further upregulated transcriptional activity of IFNT2 or IFN-tau-c1 constructs, which were substantially reduced in the absence of the TEAD-binding site on IFNT2 or IFN-tau-c1 promoter region in JEG3 cells. In CT-1 cells, treatment with TEAD2, TEAD4, or YAP small-interfering RNA downregulated endogenous IFNT expression. It should be noted that TEAD2 and TEAD4 were predominantly localized in the nuclei of trophectoderm of Day 17 conceptuses, but nuclear localization appeared to be lower in those cells of conceptuses on days 20 and 22 of pregnancy. Moreover, the binding of TEAD4 to the TEAD-binding site of the IFN-tau-c1 promoter region in day 17 conceptuses was less in day 20 and 22 conceptuses. Furthermore, the level of YAP phosphorylation increased in day 20 and 22 conceptuses. These results indicated that although YAP/TEAD had the ability to up-regulate IFNT gene transcription on day 17, IFNT2 or IFN-tau-c1 was down-regulated following changes in the localization of TEAD2 and TEAD4 from the nucleus to the cytoplasm and increases in phosphorylation and degradation of YAP. These data suggest that TEAD relocation and/or YAP degradation following its phosphorylation down-regulates IFNT gene transcription after conceptus attachment to the uterine endometrium.

Regulation of decidualization in human endometrial stromal cells through exchange protein directly activated by cyclic AMP (Epac).

INTRODUCTION: Human endometrial stromal cells (ESCs) undergo differentiation during the decidualization process. Decidualization is characterized by their enhanced production of IGF binding protein-1 (IGFBP-1), prolactin (PRL), and the forkhead transcriptional factor FOXO1, and transformation into more rounded cells, during the secretory phase of the menstrual cycle and subsequent pregnancy. Protein kinase A (PKA)-mediated cAMP signaling is crucial for this process. The present study was undertaken to examine the involvement of a mediator of cAMP signaling, exchange protein directly activated by cAMP (Epac), in decidualization of cultured ESCs. RESULTS: Treatment of ESCs with the Epac-selective cAMP analog 8-CPT-2-OMe-cAMP (CPT) had no effect on IGFBP-1, PRL, and FOXO1 mRNA expression. However, CPT potentiated IGFBP-1 and PRL expression stimulated by the PKA-selective cAMP analog N(6)-Phe-cAMP (Phe) and activated Rap1, a downstream regulator of Epac signaling. Knock-down of Epac1, Epac2, or Rap1 significantly inhibited the Phe- or Phe/CPT-induced increase in IGFBP-1 and PRL expression, as well as Rap1 activation. Furthermore, CPT enhanced IGFBP-1 and PRL expression and the morphological differentiation induced by ovarian steroids, whereas Epac1, Epac2, or Rap1 knock-down suppressed these events. CONCLUSION: These data provide evidence for the involvement of the Epac/Rap1 signaling pathway in cAMP-mediated decidualization of human ESCs.

Expression of inhibin/activin subunits in the equine uteri during the early pregnancy.

The establishment of equine pregnancy is a unique and long process during which a series of physical and possibly biochemical interactions are required between the conceptus and uterus. In this study, we investigated the expression pattern of inhibin/activin subunits in the uterus during early pregnancy. The uteri from four adult mares on cyclic day 13 or pregnancy day 25 were obtained. Immunohistochemical experiments suggested that inhibin/activin subunits were immunolocalized in the luminal and glandular epithelium on pregnancy day 25. In addition, the inhibin α and inhibin/activin ßB subunits were not detected, and inhibin/activin ßA subunit was detected, in the luminal and glandular epithelium on cyclic day 13. Real-time polymerase chain reaction and Western blotting results for the inhibin/activin subunits suggested a significant increase in the expression of inhibin/activin subunit ßB and a significant decrease in the expression of inhibin/activin subunit ßA on pregnancy day 25 compared with those on cyclic day 13. Enzyme-linked immunosorbent assays suggested a significant decrease in the concentration of activin A in endometrium extracts from cyclic day 13 to pregnancy day 25. These results suggest that inhibins or activins synthesized in the uterus, as endocrine factors and necessary nutriments, have different expression patterns and may play different, important roles during early embryonic development of the equine.

Interleukin-1 receptor antagonist expression in the equine endometrium during the peri-implantation period.

To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0=day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was found in the extracts of day 25 gravid endometrium and was immunochemically localized to the glandular epithelium/luminal cavity of the pregnant uterus. High concentrations of estradiol-17beta (E(2)) were detected in day 25 conceptuses. Concentrations of E(2) were higher in the gravid endometrial portion than in other endometrial regions. On the other hand, progesterone concentrations did not differ among endometrial samples analyzed. Furthermore, the expression of IL-1RN mRNA was up-regulated in endometrium culture samples treated with 10 ng/mL E(2) and 10 ng/mL progesterone. In the analysis of related gene expression, increased amounts of IL-1alpha and IL-6 mRNA were also found in the day 25 gravid endometrium; however, these expressions in endometrial culture samples were not up-regulated by the steroid treatment. These results indicate that expression of IL-1RN in the endometrium is likely regulated by E(2) and progesterone and suggest that IL-1RN regulates the degree of IL-1 signal transduction and thereby plays an important role in the establishment of equine pregnancy.

Analysis of transcriptional control elements in the 5'-upstream region of ovine interferon-tau gene using feeder-independent caprine trophoblast cell line, HTS-1.

Interferon-tau (IFNtau) is a protein secreted from the embryonic trophectoderm of ruminant ungulates during peri-implantation period. This protein acts on the uterine endometrium, which indirectly maintains corpus luteum function, and is therefore considered essential for the process of maternal recognition of pregnancy. Transcriptional regulation of IFNtau genes had been examined using human choriocarcinoma cell lines, JEG-3 or JAR, however, molecular mechanisms by which cell and term specific IFNtau expression are regulated have not been elucidated. Recently, a feeder cell free-trophoblast cell line derived from Shiba-goat placenta, termed HTS-1, was established. In the present investigation, the 5'-upstream region of ovine IFNtau (oIFNtau) gene was analysed using this cell line, which would provide a more suitable system for studies of the ovine trophoblast specific gene than human choriocarcinoma cells. Variously modified 5'-upstream sequences of the oIFNtau gene fused to a luciferase reporter gene were transiently transfected into HTS-1 cells, and human JEG-3 cells were used as a control. These results and co-transfection with expression vectors revealed that Ets-2 binding site in the promoter region was important in HTS-1, whereas AP-1 that binds to the enhancer region was a major activator in JEG-3. By electrophoretic mobility shift assay, a nuclear protein from HTS-1 cells was confirmed to bind specifically to the Ets-2 site of oIFNtau promoter region. Differences in amounts of AP-1 and Ets-2 protein were demonstrated in nuclear extracts from HTS-1, JEG-3 and ovine conceptuses. Substantial differences on oIFNtau gene transcriptions found between caprine HTS-1 and human JEG-3 cells suggest that this cell line could be valuable in the elucidation of a molecular mechanism(s) by which oIFNtau gene expression is regulated in a cell specific manner.

Establishment of feeder-independent cloned caprine trophoblast cell line which expresses placental lactogen and interferon tau.

A feeder-independent cloned trophoblast cell line, HTS-1, was established from a mature placenta of Shiba goat (Capra hircus). During the growth phase, single HTS-1 cells exhibited ruffled membranes or lamellipodia often accompanied by elongated cell shape, indicating highly motile nature of the cells. At or near confluence, HTS-1 cells formed monolayers with few sign of cellular overlapping. Binucleate cells were found at a high frequency especially in the peripheral regions of monolayers. In small colonies and the monolayers, majority of HTS-1 cells assumed polygonally shaped cobble-stone like morphology characteristic to epithelial cells, although considerable variations in cellular morphology were observed despite of repeated cloning. Time-lapse video recordings of HTS-1 cells during culture revealed that not only the small colonies but also the monolayers near or at confluence were remarkably motile, often causing extreme elongation of the cells within them. The extremely plastic nature of HTS-1 cells in vitro is likely to be the reflection of the extraordinary capacity of caprine trophoblast cells to be stretched to extreme thinness in vivo as shown by electron microscopy. HTS-1 cells cultured on matrigel are highly invasive, and express MT1-MMP which, in the mouse, has been known to be expressed at the invasive edge of trophoblast both in vitro and in vivo. HTS-1 cells express placental lactogen (PL) and interferon-tau (IFNtau), as confirmed by immunocytochemistry, Western blotting and RT-PCR analysis. Both PL and IFNtau expression in the cells appeared to be down-regulated by cell-cell contact. In the medium conditioned by HTS-1 cells, the presence of secretory form of PL and IFNtau was confirmed by Western blotting. The HTS-1 cell line will serve as a useful in vitro model for the analysis of the molecular and/or cellular mechanisms underlying synepitheliochorial placentation in bovidae animals.

Regulation of endometrial granulocyte macrophage-colony stimulating factor (GM-CSF) in the ewe.

Granulocyte macrophage-colony stimulating factor (GM-CSF) increases ovine interferon-tau (oIFNtau) secretion by ovine conceptuses, but endometrial production of GM-CSF has not been characterized. Endometrial GM-CSF expression was evaluated in ovariectomized ewes implanted with estradiol-17beta (E(2)) and/or progesterone (P(4)) for 14 days, in day 14 cyclic and day 14 pregnant ewes. Relative levels of endometrial GM-CSF mRNA were 3-fold higher in E(2)- and E(2)/P(4)-treated ewes than that of control or P(4)-treated ovariectomized ewes. Levels of endometrial GM-CSF mRNA for cyclic ewes were similar to E(2)- and E(2)/P(4)-treated ewes, but amounts of GM-CSF mRNA in pregnant ewes were 2-fold higher. GM-CSF concentrations in endometrial culture media, determined by GM-CSF bioassay, for cyclic and E(2)/P(4)-treated ovariectomized ewes were 3-fold higher than those of control, E(2)- and P(4)-treated ovariectomized ewes; however, amounts of GM-CSF in pregnant ewes were 2-fold higher. Immunoreactive GM-CSF, examined by western blot, was detected in the culture medium from E(2)/P(4)-treated ovariectomized, cyclic and pregnant ewes. Luminal and glandular epithelia and stromal regions were determined to be sites of GM-CSF expression by immunohistochemistry and in situ hybridization techniques. Data indicate that combined E(2) and P(4) treatment of ovariectomized ewes is sufficient to restore GM-CSF expression to the level found in cyclic ewes; however, GM-CSF mRNA and protein in pregnant ewes is 2-fold greater than in ovariectomized or cyclic ewes. These data suggest that the conceptus, in addition to steroids, may play a role in the regulation of endometrial production of GM-CSF.

The casein mRNA decay changes in parallel with the poly(A) tail length in the mouse mammary gland.

Using beta- and gamma-casein mRNAs, the relationship between poly(A) tail length and half-life of mRNA is determined in the mouse mammary gland during pregnancy and lactation. beta- and gamma-Casein mRNAs increase before and after parturition, respectively. The poly(A) tail as well as the half-life of casein mRNA becomes longer upon the active casein mRNA synthesis. The poly(A) tail is shortened gradually as lactation progresses. The half-life of mRNA decreases approximately from 20 h at early to 4 h at late lactation. Northern blot analysis reveals that nuclear RNA has the same poly(A) tail length as casein mRNA in the cytoplasm does. Thus, the mammary gland changes the poly(A) tail length of casein mRNA. The poly(A) tail length changes in parallel with the level of poly(A) polymerase (PAP) mRNA during pregnancy and lactation, suggesting that the mammary gland determines the poly(A) tail length of casein mRNA through the change in the PAP gene expression. As the half-life of casein mRNA is related with the degree of polyadenylation, we conclude that the poly(A) tail elongation and shortening is a mechanism in regulating the mRNA decay.

Detection of transforming growth factor-alpha and epidermal growth factor receptor mRNA and immunohistochemical localization of their proteins in the ovine uterus during the early implantation period.

Accumulated evidence suggests that growth factors of the epidermal growth factor (EGF) family play an important role in the murine implantation process. In the sheep, however, the uterine distribution of these factors and their receptor, EGF receptor (EGF-R), during implantation is not known. This study examined the presence of mRNA transcripts and immunohistochemical localization for transforming growth factor-alpha (TGF-alpha), the potent EGF-family member, and EGF-R in the ovine uterus during the early implantation period. By reverse transcriptase-polymerase chain reaction and sequencing of the products, the presence of TGF-alpha and EGF-R mRNA transcripts were detected in the endometrium on Days 14, 16 and 20 (Day 0 = day of mating). Immunohistochemical analysis revealed that the luminal and glandular epithelial cells and some stromal cells of the endometrium and the trophectoderm were positive for TGF-alpha and EGF-R on Days 14 and 15. Distinct staining for TGF-alpha was observed in the glandular epithelium of deep endometrial areas and strong immunoreactivity for EGF-R was found in the trophectoderm. On Days 16, 18 and 20, although the staining pattern for TGF-alpha was similar to that on the previous days, the immunoreactivity for EGF-R in the stromal cells increased and that in the gland decreased. A distinct immunoreactivity for EGF-R was found in the trophectoderm throughout the days examined. These results suggest that TGF-alpha expressed in the endometrium and trophectoderm may exert effects locally on these tissues during implantation in sheep. Furthermore, it is speculated that the temporal changes in the uterine EGF-R distribution may be related to the endometrial microvascular development.

Evidence of H beta 58, a gene involved in mammalian placental development, in the three-toed skink, Chalcides chalcides (Squamata: Scincidae), a viviparous placentotrophic reptile.

The H beta 58 gene, whose disruption in mice causes reabsorption of the embryo at 9.5 days post-conception, is believed to be essential for development of the placenta. Although the H beta 58 gene is well conserved in some Amniota, nothing is known about its presence in reptiles, some species of which have developed a chorioallantoic placenta. In this work, we investigated the expression of H beta 58 mRNA and protein in the three-toed skink, Chalcides chalcides. H beta 58 protein expression was found in the uterine epithelium beginning from the peri-ovulatory stage. However, it increased strongly at the moment of placental formation, when a high level of expression of mRNA and protein was also observed in the extra-embryonic membranes. The expression of H beta 58 mRNA and protein was maintained, although to a lesser degree, in the placenta during late pregnancy. It was also present in the early embryo. Finally, cloning and sequencing of a gene fragment revealed strong homology of the reptile gene with that of mammals. The high degree of conservation of the gene in amniote vertebrates and its presence in a viviparous squamate reptile (as in mammals) indicates an important role of this gene in the chorioallantoic placenta formation and development.

Gastric proteinase digestion of caseins in newborn pups of the mouse.

Casein micelles of mouse milk consist of alpha-, beta-, gamma-, and kappa-caseins. By digestion with alkaline phosphatase, they were separated as an independent band by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The compositions of alpha-, beta-, gamma-, and kappa-caseins were 24.3, 25.1, 9.4, and 41.2% in colostrum, and 36.8, 15.6, 11.9, and 35.7% in mature milk, respectively. Zero-day-old pups were allowed to access either colostrum or mature milk, and the aggregated milk in the stomach was analyzed by SDS-PAGE. Caseins in colostrum were digested more rapidly and efficiently than those in mature milk. Among the seven peptides present in the aggregated caseins, four peptides were colostrum-specific and derived from alpha- and gamma-caseins. It was expected that colostrum-specific and soluble peptides were generated from alpha- and gamma-caseins through gastric proteinase digestion. Amino acid sequence analysis and the pH of the aggregated milk suggested that caseins in the stomach were digested by a chymotrypsin-like proteinase. Caseins in colostrum were different from those in mature milk, with respects to the casein composition as well as the gastric proteinase sensitivity. It is concluded that the lactating mice on the day of parturition supply particular caseins to their young.

Reactivation of feline foamy virus from a chronically infected feline renal cell line by trichostatin A.

Although acute infection of feline foamy virus (FeFV) is normally highly cytopathogenic in Crandell feline kidney (CRFK) cells, a noncytopathic persistent infection was established in the cells after cocultivation of the initially infected cells with uninfected cells four times. To investigate reactivation of persistent infection, CRFK cells chronically infected with FeFV were treated with trichostatin A (TA), a histone deacetylase inhibitor. TA induced higher FeFV production from the Coleman strain carrier culture and also induced marked syncytium formation. In contrast, human foamy virus, which contains less homologous long terminal repeat (LTR) and putative internal promoter (IP) sequences, persistently infecting baby hamster kidney cells was not reactivated by TA. The Sammy-1 strain of FeFV, from which a part of the U3 region in the LTR is naturally deleted, showed less reactivation. The Coleman LTR promoter-based beta-Gal-expressing plasmid was activated in the persistently Coleman-infected cells in the presence of TA, whereas the Sammy-1 LTR was not activated. Furthermore, the amounts of Gag protein expressed did not change in the presence or absence of TA. Because the putative IP region was very similar between the two strains, the initiation by TA is relatively specific for LTR sequences, and, therefore, histone deacetylation is at least in part responsible for reactivation of FeFV from carrier cell culture.

Enhancer regions of ovine interferon-tau gene that confer PMA response or cell type specific transcription.

Interferon-tau (IFNtau), produced by the trophectoderm of peri-implantation conceptuses in ruminant ungulates, attenuates the uterine production of a luteolytic factor, prostaglandin F(2alpha), resulting in the maintenance of corpus luteum function. However, molecular mechanisms regulating the temporal/spatial expression of IFNtau gene are not clearly understood. The 5'-upstream region of the sheep IFNtau (oIFNtau) gene was examined for its transcriptional regulation in two different cell types; JEG3 cells supported the transactivation of oIFNtau-reporter construct, but HeLa cells did not. In a heterologous SV40 enhancer-oIFNtau promoter or oIFNtau enhancer-SV40 promoter systems, elements required for such cell specific transactivation were localized between -654 and -555 bases, the enhancer, but not the basal promoter region of the oIFNtau gene. In these combinations, high degrees of transactivation were observed in JEG3 cells and the activity was further enhanced by the addition of phorbol 12-myristate 13-acetate (PMA), while those responses were absent in HeLa cells. To identify nucleotide sequences responsible for cell specific expression, transient transfection studies with sequential point mutations in the enhancer elements were executed. Transactivation of oIFNtau enhancer-reporter constructs was primarily regulated by three regions containing AP-1 site, GATA like sequence and site(s) unidentified. In gel mobility shift assays (GMSAs), the AP-1 site located in the enhancer region was recognized by nuclear extracts from both cell types. However, one of the GMSA probes containing GATA-like sequence exhibited different DNA-protein complex patterns in JEG3 and HeLa cells. Observations, in which the same upstream sequence behaved differently due possibly to kinds of nuclear factors available in these cell lines, suggest that such a sequence may be involved in cell specific transactivation of the oIFNtau gene. Furthermore, the same enhancer sequences were also recognized by nuclear extracts from sheep trophoblasts, suggesting that the enhancer sequences between -654 and -555 bases of oIFNtau gene may be functioning in vivo.

Changes in the temporal and spatial expression of H beta 58 during formation and maturation of the chorioallantoic placenta in the Rat.

Cloning and sequencing of a cDNA amplified by RNA fingerprinting at the implantation site of pregnant rats revealed 80% similarity with H beta 58, previously shown to be essential for formation of the chorioallantoic placenta in the mouse. H beta 58 mRNA was detected in the endometrium of hormonally sensitized rats stimulated to undergo decidualization and in the contralateral uterine horns lacking a decidual stimulus, indicating that uterine expression of H beta 58 mRNA did not require decidualization or the presence of a blastocyst. Immunodetection in the early postimplantation uterus (Days 6-8 of pregnancy) showed H beta 58 localized in the luminal and glandular epithelia and some stromal cells. Decidual cells at Day 6 of pregnancy expressed H beta 58, and by Day 9 of pregnancy, the protein localized throughout the maternal decidua. The temporal and spatial distribution of H beta 58 in the developing chorioallantoic placenta was assessed at Days 10, 12, and 14 of pregnancy. Immunoreactive H beta 58 localized to erythroid cells within the developing fetal vasculature of the chorioallantoic primordia at Day 10 of pregnancy. By Day 12, the fetal vasculature extended into the placental labyrinth, and the erythroid stem cells continued to strongly express H beta 58. At Day 14 of pregnancy, immunoreactivity became evident in the trophoblast giant cells and syncytiotrophoblast of the fetal placenta. As the chorioallantoic placenta matured (Day 18), H beta 58 mRNA was 3.6-fold higher in the labyrinth compared with the junctional region. Stable cell lines (HRP/LRP) isolated from the rat labyrinthine placenta expressed H beta 58 mRNA and protein. The expression pattern of H beta maternal and fetal placental tissues and its early expression in fetal erythroid stem cells during formation and maturation of the chorioallantoic placenta suggest that H beta 58 plays key roles in the regulatory networks that control hematopoietic development and placentation.

Analysis of possible silencer elements of ovine interferon-tau gene.

During the peri-implantation period significant production of ovine interferon-tau (olFNtau) by the trophectoderm is detected in day 13-16 conceptuses, but its level rapidly declines thereafter. To understand molecular mechanisms by which oIFNtau gene expression is down-regulated, a variety of deletion constructs were prepared from upstream sequences of the oIFNtau gene and examined for possible silencer regions by using transient transfection into human choriocarcinoma, JEG3, cells. Two regions between -700 to -654 bases (distal region) and from -503 to -453 bases (proximal region) were found to be the possible negative regulatory regions. With probes prepared from these regions, gel mobility shift assay (GMSA) was then conducted. DNA-protein complexes were observed, but the gel shift pattern was different between nuclear extracts from days 14 (active oIFNtau production) and 20 (minute oIFNtau production) ovine trophoblasts. Day 20 nuclear extracts exhibited more band patterns than those of day 14; most notably the distal region between -692 and -668 bases exhibited the distinct band with nuclear extracts from day 20, but not from day 14 trophoblasts. In addition, the band patterns from day 20 trophoblast nuclear proteins were similar to those detected with JEG3 and HeLa cell nuclear extracts. Taken together, these observations suggest that the upstream sequences identified could serve as negative regulatory regions to which various nuclear factors bind, resulting in reduction of oIFNtau gene transcription.

Determination of genes involved in the process of implantation: application of GeneChip to scan 6500 genes.

Using the high-density arrays of oligonucleotides (GeneChip) technology, the expression of uterine genes was examined before and after conceptus implantation in mice. Of the 6500 genes analyzed, levels of 399 gene expressions changed; 192 genes increased levels of expression while the remaining 207 genes declined. The findings suggest that both gene activation and deactivation (suppression) are required for successful implantation.

Genetic mapping of a locus associated with bovine chronic interstitial nephritis to chromosome 1.

Chronic interstitial nephritis with diffuse zonal fibrosis (CINF) occurs in Japanese Black cattle (Wagyu) as an autosomal recessive disorder leading to death prior to puberty, first six months or a year of life. We performed a genome-wide scan using microsatellite markers in a Wagyu pedigree segregating for CINF and mapped the CINF locus to bovine chromosome 1. CINF was closest to microsatellites BM9019 and INRA49 (Z score = 12.0; P < 3.4 x 10(-10)).

The poly(A) tail length of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of milk.

The length of casein mRNA from the lactating mouse mammary gland, as assessed on Northern blots, is shorter after weaning, but is elongated following the removal of milk. In order to investigate this phenomenon, the molecular structures of beta- and gamma-casein mRNAs were analysed. The coding and non-coding regions of the two forms were the same length, but the long form of casein mRNA had a longer poly(A) tail than the short form (P<0.05). In order to examine the stability of casein mRNA under identical conditions, casein mRNAs with the long and short poly(A) tails were incubated in the rabbit reticulocyte lysate (RRL) cell-free translation system. Casein mRNA with the long poly(A) tail had a longer half-life than that with the short tail (P<0.05). The beta- and gamma-casein mRNAs were first degraded into 0.92 and 0.81 kb fragments respectively. With undegraded mRNA, the poly(A) tail shortening by exoribonuclease was not observed until the end of the incubation. Northern blot analysis showed that casein mRNA with the long poly(A) tail was protected efficiently from endoribonucleases. We conclude that the length of the poly(A) tail of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of the gland's milk, and we show that the longer poly(A) tail potentially protects the mRNA from degradation by endoribonucleases.