Dicalcin inhibits fertilization through its binding to a glycoprotein in the egg envelope in Xenopus laevis.

Fertilization comprises oligosaccharide-mediated sperm-egg interactions, including sperm binding to an extracellular egg envelope, sperm penetration through the envelope, and fusion with an egg plasma membrane. We show that Xenopus dicalcin, an S100-like Ca(2+)-binding protein, present in the extracellular egg envelope (vitelline envelope (VE)), is a suppressive mediator of sperm-egg interaction. Preincubation with specific antibody greatly increased the efficiency of in vitro fertilization, whereas prior application of exogenous dicalcin substantially inhibited fertilization as well as sperm binding to an egg and in vitro sperm penetration through the VE protein layer. Dicalcin showed binding to protein cores of gp41 and gp37, constituents of VE, in a Ca(2+)-dependent manner and increased in vivo reactivity of VE with a lectin, Ricinus communis agglutinin I, which was accounted for by increased binding ability of gp41 to the lectin and greater exposure of gp41 to an external environment. Our findings strongly suggest that dicalcin regulates the distribution of oligosaccharides within the VE through its binding to the protein core of gp41, probably by modulating configuration of oligosaccharides on gp41 and the three-dimensional structure of VE framework, and thereby plays a pivotal role in sperm-egg interactions during fertilization.

Estradiol and corticosterone stimulate the proliferation of a GH cell line, MtT/S: Proliferation of growth hormone cells.

OBJECTIVES: Estrogens are known as a potent growth-stimulator of the anterior pituitary cells such as prolactin cells and somatomammotroph cell lines, while glucocorticoids often inhibit cellular proliferation in the pituitary gland as well as in the extra-pituitary tissues. In this study, the involvement of these steroid hormones in the regulation of proliferation was examined in the MtT/S cells, secreting growth hormone (GH). DESIGN: Effects of estrogens and glucocorticoids were examined in MtT/S cells grown in the medium containing dextran-coated charcoal treated serum. The relative cell density after culture was estimated by the Cell Titer-Glo Luminescent Cell Viability Assay System, and the proliferation rate was determined by the BrdU incorporation method. The mRNA levels were determined by real-time PCR. RESULTS: Estradiol and the specific agonist for both estrogen receptor (ER) α and ERß stimulated MtT/S growth at a dose dependent manner. The membrane impermeable estrogen, 17ß-estradiol-bovine serum albumin conjugate also stimulated the MtT/S proliferation. The effects of all estrogens were inhibited by an estrogen receptor antagonist, ICI182780. Corticosterone stimulated the proliferation of MtT/S cells at doses lower than 10nM without stimulating GH gene transcription, whereas it did not change the proliferation rate at 1µM. The effects of corticosterone were inhibited by glucocorticoid receptor inhibitor, RU486, but not by the mineralocorticoid receptor antagonist, spironolactone. Both estrogens and glucocorticoids were found to stimulate the proliferation of MtT/S, increasing the mRNA expression of cyclins D1, D3, and E. CONCLUSIONS: The results suggest that estrogens and glucocorticoids may be involved in the mechanisms responsible for the proliferation of GH cells in the course of pituitary development, to maintain the population of GH cells in the adult pituitary gland, and also in the promotion of GH cell tumors.

A novel Xenopus laevis SRY-related gene, xSox33.

The sex-determining region Y (SRY) gene and its related Sox genes encode transcriptional regulatory factors. In this study, we isolated and sequenced a novel Sox cDNA from African clawed frog (Xenopus laevis). The Sox gene was named xSox33. xSox33 was revealed to encode 244 amino acids. Reverse transcription-polymerase chain reaction (RT-PCR) showed that xSox33 was expressed at very low levels in some frog tissues including lung, ovary, skeletal muscle, testis, brain and heart. Its embryonic expression was also studied by RT-PCR. After the mid-blastula transition, the zygotic expression was initiated during gastrulation and the level was elevated as the embryogenesis proceeded. Electrophoretic mobility shift assay (EMSA) indicated that a recombinant xSox33 polypeptide was capable of binding to the nucleotide sequence AACAAT.

The role of pit-1 in the regulation of the rat growth hormone-releasing hormone receptor gene transcription by glucocorticoids.

Glucocorticoids are involved in the regulation of the rat growth hormone-releasing hormone (GHRH) receptor gene expression, but they act only in the presence of the pituitary specific transcription factor, pit-1. In this study, the role of pit-1 in the glucocorticoid stimulation of the GHRH-receptor gene transcription was examined. The results suggest the presence of a silencer element in the promoter and it is postulated that pit-1 permits glucocorticoid action through suppressing the inhibitory effect of an as yet unknown factor that binds to this element. The present results also suggest that the synergistic activation of the rat GHRH-receptor gene transcription depends on the proper distance between the proximal glucocorticoid response element and the pit-1 binding site.

A composite hormone response element regulates transcription of the rat GHRH receptor gene.

To further elucidate the molecular mechanisms underlying the transcriptional regulation of the GHRH receptor (GHRH-R) gene, hormonal regulation of the promoter activity of this gene was examined. An approximately 3-kb genomic fragment spanning the promoter region of the gene was sequenced and the transcription start site was determined by RT-PCR and RNase protection assay. A major start site was localized at -105 (relative to the translation initiation codon, ATG), and a pit-1 binding sequence characteristic of pituitary specific genes was found at -155 to -146. Deletion and mutation studies demonstrated this site to be functional. In the presence of dexamethasone, the GHRH-R promoter (from -2935 to -11) directed luciferase expression in MtT-S cells, a somatotropic cell line, but not in the PC12 cells that normally do not express GHRH-R. While T(3), all trans-RA, and 9cis-RA alone weakly enhanced the reporter gene expression, each of these substances was found to act as a synergistic enhancer in the presence of dexamethasone. Additional deletion and mutation analyses demonstrated a functional RA response element at -1090 to -1074. Two functional glucocorticoid response elements and a T(3) response element were found in an 80-bp 5'-flanking sequence of the pit-1 site. Interestingly, it is suggested that the 6-bp half-site AGGACA (from -209 to -204) functions as a 3'-half-site of T(3) response element as well as a 5'-half-site of one of the glucocorticoid response elements.

Expression and characterization of Xenopus laevis SRY-related cDNAs, xSox17alpha1, xSox17alpha2, xSox18alpha and xSox18beta.

Sox is a large family of genes related to the sex-determining region Y gene (designated as the SRY gene). Sox genes encoding DNA-binding transcriptional factors are found in many animals and are involved in developmental events. In this study, we newly isolated and sequenced novel Sox cDNAs from African clawed frog (Xenopus laevis). Five clones isolated here were classified into four distinct Sox genes designated as xSox17alpha1, xSox17alpha2, xSox18alpha and xSox18beta. All four belong to a subtype of SOX family, type F. The cDNA xSox17alpha1 contains essentially the same nucleotide sequence as that identified as Sox17alpha in a previous work (Cell 91 (1997) 397), whereas xSox17alpha2 is a distinct gene with high homology to xSox17alpha1. The clones, xSox18alpha and xSox18beta, are highly homologous to each other over the entire nucleotide sequences. The xSox18alpha and xSox18beta genes encode 363 and 361 amino acids, respectively. Genomic Southern hybridization analysis showed the existence of two copies of the xSox18. Northern analysis indicated that the xSox18 gene was expressed in the spleen and kidney and the size of the transcript was estimated to be 2.4 knt. Electrophoretic mobility shift assays indicated that recombinant xSox18 polypeptide was capable of binding to the HMG consensus nucleotide sequence, AACAAT.

The Polycomb-group protein ENX-2 interacts with ZAP-70.

Human ENX-2 is a homologue of Drosophila Enhancer of zeste, which is a member of Polycomb-group proteins regulating the expression of homeotic genes as chromatin-associated proteins. In this study, we demonstrate that ENX-2 plays an important role as a signaling molecule involved in T cell receptor-mediated signaling pathway. In immunoprecipitation experiments, ENX-2 and zeta associated protein-70 (ZAP-70) were co-precipitated from T cell lysate. When probed with an anti-phospho-tyrosine antibody, ENX-2 was found to be phosphorylated on tyrosine. On the other hand, ENX-2 was not phosphorylated on tyrosine in the mutant Jurkat cell, J.Cam1.6 lacking the activity of lymphocyte protein tyrosine kinase p56(lck). The interaction between ENX-2 and ZAP-70 was abolished in the mutant cell. Furthermore, in-vitro kinase assay using purified p56(lck) demonstrated that ENX-2 became tyrosine phosphorylated by this kinase. These findings show that the phosphorylation of ENX-2 is responsible for the interaction between ENX-2 and ZAP-70.

Regulation of gammadelta versus alphabeta T lymphocyte differentiation by the transcription factor SOX13.

alphabeta and gammadelta T cells originate from a common, multipotential precursor population in the thymus, but the molecular mechanisms regulating this lineage-fate decision are unknown. We have identified Sox13 as a gammadelta-specific gene in the immune system. Using Sox13 transgenic mice, we showed that this transcription factor promotes gammadelta T cell development while opposing alphabeta T cell differentiation. Conversely, mice deficient in Sox13 expression exhibited impaired development of gammadelta T cells but not alphabeta T cells. One mechanism of SOX13 function is the inhibition of signaling by the developmentally important Wnt/T cell factor (TCF) pathway. Our data thus reveal a dominant pathway regulating the developmental fate of these two lineages of T lymphocytes.

Regulation of 5'-promoter activity of the rat growth hormone and growth hormone-releasing hormone receptor genes in the MtT/S and MtT/E cells.

The MtT/E and MtT/S cells have been established from a mammotrophic pituitary tumor, and postulated to be progenitor and premature growth hormone (GH) cells, respectively. The difference in the regulation of GH and GH-releasing hormone (GHRH) receptor gene transcription in relation to the developmental stage of GH cells were examined in these two cell lines. In MtT/S cells, triiodothyronine (T3), all-trans retinoic acid (RA) and 9-cis retinoic acid (9cRA) stimulated GH promoter activity but dexamethasone (DEX) did not. On the other hand, DEX stimulated GHRH-receptor promoter alone. T3, RA and 9cRA showed little effect alone but each of them augmented the effect of DEX when used together with DEX. In MtT/E cells, DEX, RA and 9cRA showed similar effect as observed in MtT/S cells on both GH and GHRH-receptor promoter activity. However, T3 neither stimulated GH promoter activity nor augmented the DEX-induced GHRH-receptor gene transcription in MtT/E cells. RT-PCR analyses revealed that both cell types expressed TRalpha1, TRbeta1 and TRalpha2, but expression of TRbeta2, a pituitary specific isoform of TR, was only detected in MtT/S cells. However, the deficiency of TRbeta2 for its own sake does not appear to be a reason why T3 action was not observed in MtT/E cells, because co-transfection of expression plasmids for TRbeta2 and RXRalpha failed in conferring on the cells an ability to respond to T3 by increased GH or GHRH-receptor promoter activity. These results suggest that the acquisition of mechanisms responsible for the regulation of GH or GHRH-receptor transcription by T3 may be involved in the process of functional development of GH cells.

Nuclear translocation of Xenopus laevis paxillin.

Paxillin has been recognized as a focal adhesion adapter protein that participates in the integrin-mediated signaling. An earlier study [Ogawa et al. Biochim. Biophys. Acta 1519 (2001) 235] found that frog paxillin was expressed in the kidney epithelial cell line A6 and localized in the nucleus. Here, in this study, we have found that the expression of frog paxillin is up-regulated in the S phase of cell cycle. The protein became phosphorylated on tyrosine when the cells were grown on vitronectin; the tyrosine phosphorylation was not detectable when the cells were cultured on fibronectin, laminin or poly-D-lysine. On the other hand, MAP kinase was revealed to phosphorylate frog paxillin on serine. Both phosphorylation events, namely on tyrosine and serine, were essential for the nuclear translocation of this protein. Our results suggest that the integrin-mediated signaling pathway and the MAP kinase pathway meet at paxillin.