Hirohito Yamaguchi

Implantation-Dependent Expression of Trophinin by Maternal Fallopian Tube Epithelia during Tubal Pregnancies: Possible Role of Human Chorionic Gonadotrophin on Ectopic Pregnancy

Trophinin, tastin, and bystin have been identified as molecules potentially involved in human embryo implantation. Both trophoblasts and endometrial epithelial cells express trophinin, which mediates apical cell adhesion through homophilic trophinin-trophinin binding. We hypothesized that trophinins function in embryo implantation is unique to humans and investigated the expression of trophinin, tastin, and bystin in ectopic pregnancy, a condition unique to humans. In tubal pregnancies, high levels of all three were found in both trophoblasts and fallopian tubal epithelia. Trophinin expression in maternal cells was particularly high in the area adjacent to the trophoblasts, whereas trophinin was barely detectable in intact fallopian tubes from women with in utero pregnancies or without pregnancies. When explants of intact fallopian tube were incubated with the human chorionic gonadotrophin (hCG), trophinin expression was enhanced in epithelial cells. Since the trophectoderm of the human blastocyst secretes hCG before and after implantation, these results suggest that hCG from the human embryo induces trophinin expression by maternal cells. As both beta-subunit of hCG and trophinin genes have diverged in mammals, the present study suggests a unique role of hCG and trophinin in human embryo implantation, including the pathogenesis of ectopic pregnancy.

Corticosterone is required for the prolactin receptor gene expression in the late pregnant mouse mammary gland.

In order to clarify the prolactin receptor (PRL-R) gene expression at lactogenesis, the levels of the long and short forms of PRL-R mRNA were determined by the competitive RT-PCR in the pregnant, lactating and ovariectomized midpregnant mouse mammary gland. Plasma concentrations of corticosterone and progesterone were determined by RIA. The long form of PRL-R mRNA level was low until 10:00 on day 18, increased 3.3-fold at 22:00 on day 18 of pregnancy and further increased to 4.6-fold at 10:00 on day 0 of lactation. The short form of PRL-R mRNA level remained unchanged during this time period. The corticosterone:progesterone ratio increased 15.5-fold during the last 1.5 days of pregnancy. Corticosterone increased the long form of PRL-R mRNA level when the tissues on day 17 were cultured. On day 12 of pregnancy and following ovariectomy, corticosterone was exceedingly high from 2 h to 8 h and the corticosterone:progesterone ratio changed prior to the increase in the long form of PRL-R mRNA level. We conclude that corticosterone increases the PRL-R gene expression in the mammary gland before the onset of parturition.

Identification of a functional transcriptional factor AP-1 site in the sheep interferon τ gene that mediates a response to PMA in JEG3 cells

To examine regulatory mechanisms of sheep interferon tau (oIFNtau) gene expression, potential enhancer/silencer elements of the oIFNtau gene were examined using a transient transfection system with oIFNtau gene-chloramphenicol acetyltransferase (oIFNtau-CAT) reporter constructs in human choriocarcinoma cells, JEG3. Experiments with 5-deletion constructs revealed that the upstream regions from bases -654 to -607 and from bases -606 to -555 were essential for oIFNtau gene expression. In a heterologous transcriptional system in which the upstream regions of oIFNtau were inserted in front of simian virus 40 (SV40) promoter, the regions between bases -654 and -555 were determined as being the enhancer region required for oIFNtau-SV40-CAT transactivation. A subsequent study with the oIFNtau-CAT constructs lacking the upstream region between bases -542 and -124 revealed that, in addition to the further upstream region between bases -1000 and -654, the sequences from bases -543 to -452 seemed to act as silencer regions. The oIFNtau-CAT constructs with site-specific mutagenesis revealed that multiple enhancer elements existed between bases -654 and -555 of the oIFNtau gene. On the basis of nucleotide sequence analysis, there are numerous sites between bases -654 and -555 to which potential transcriptional factors, AP-1, GATA and GATA-related proteins, could bind. Furthermore, gel mobility-shift assays revealed that AP-1 or other nuclear factors could bind to these elements. In co-transfection studies, the expression of c-Jun plus c-Fos enhanced the transactivation of oIFNtau-CAT but the expression of GATA-1, GATA-2 or GATA-3 did not. Taken together, these results suggest that the upstream region between bases -654 and -555 could be considered as the enhancer region for oIFNtau gene transactivation.

REMOVAL OF MILK BY SUCKLING ACUTELY INCREASES THE PROLACTIN RECEPTOR GENE EXPRESSION IN THE LACTATING MOUSE MAMMARY GLAND

To determine the effect of suckling on the prolactin receptor (PRL-R) gene expression, we measured the quantity of PRL-R mRNA in the lactating mouse mammary gland. When the pups were separated from their mother on day 5 of lactation, the long form of PRL-R (PRL-R[L]) mRNA disappeared with a half-life of 12.5 h for the first 9 h and 3.0 h for the following 9-15 h. By supplying pups to mice which had been weaned for 24 h, PRL-R(L) mRNA increased 2.5-fold during the next 6 h-period. The increase in PRL-R(L) mRNA was found in the mammary glands from which the pups removed milk. The number of mammary PRL-R protein decreased or increased following weaning or following the removal of milk by suckling, respectively. From these observations. it was concluded that the removal of milk acutely increases the level of PRL-R(L) mRNA during lactation.

Enhancer regions of ovine interferon-τ 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.