Yoichi Hanaoka

Primary action of steroid hormone at the surface of amphibian oocyte in the induction of germinal vesicle breakdown

An insoluble steroid derivative was prepared by the coupling of desoxycorticosterone with aminoethylated agarose beads. When the naked full-grown Xenopus oocytes were incubated in contact with the steroid-bound agarose beads, the dissociation of oocyte nuclei or the initial step in the maturation of amphibian oocyte was induced in 30-100% of total oocytes. The induction did not occur in the oocytes covered with follicle cells and when the naked oocytes were placed apart from the steroid-bound agarose beads in the medium. The above findings confirmed that the oocyte surface was a primary site for this particular steroid action, eliminating the possibility of participation of free steroid artifactually released from the agarose. The 105,000g supernatant fraction of oocytes showed no sign of the presence of steroid receptor. This was not inconsistent with the assumption mentioned above.

Morphological and functional maturation of the thyroid during early development of anuran larvae

In Bufo bufo japonicus, synthesis of thyroid hormones, as measured by 131I-incorporation, developed very early, and small but significant amounts of iodothyronines were formed as early as stage 26, at which time the thyroid was recognized only as a simple thickening of the pharyngeal epithelium. The thyroid appeared in its typical form, and 131I-labeled thyroglobulin was formed in an appreciable amount at stage 33. Before this stage, soluble 131I-proteins were formed only in small amounts, and the molecular sizes of these proteins were smaller than that of thyroglobulin. The changes in iodoamino acid formation were also quite critical between stages 32 and 33. The main iodotyrosine was monoiodotyrosine and the main iodothyronine was triiodothyronine before stage 32. After stage 33, diiodotyrosine was the main iodotyrosine and thyroxine the main iodothyronine. Similar sequential maturation of the thyroid function was observed also in Xenopus laevis.

Production and characterization of a monoclonal antibody against the β-subunit of bullfrog lutropin

We generated a high-affinity and highly specific monoclonal antibody (BL4B11)-producing hybridoma against bullfrog lutropin (LH) beta by fusing mouse myeloma, X63.Ag8.653, with spleen lymphocytes obtained from BALB/c mice immunized with bullfrog LH-IV (pI 9.3) beta-subunit. The resultant antibody-secreting hybridoma was injected into intraperitoneally pristane-primed BALB/c mice to obtain a large amount of antibody. Noncompetitive binding tests revealed that the ascitic fluid (BL4B11) could be diluted up to 1:12,000 for 50% binding to 125I-labeled bullfrog LH beta and also bound strongly to bullfrog intact LH, but not to LH alpha, follitropin (FSH), FSH alpha, FSH beta, and rat glycoprotein hormones (LH, FSH, and thyrotropin (TSH) significantly. The immunoblotting results also showed a similar immunological specificity of BL4B11. Cross-reactivities of bullfrog LH, FSH beta, FSH, LH alpha, and FSH alpha against BL4B11 were 9.69, 3.76, 2.40, 1, and 1%, respectively, when compared with bullfrog LH beta in the competitive inhibition assay system. The affinity constant (Ka) of the BL4B11 was 1.09 X 10(9) M-1. In the sexually mature bullfrog pituitary, immunoreactive LH cells which were revealed by this BL4B11 were distributed throughout the pars distalis except the rostral region. They were especially large, numerous, and polygonal, with well-developed cytoplasm. In the rostral region, immunoreactive LH cells were larger and more intense than those in the central region. In the case of young bullfrog, several immunoreactive LH cells were found only in the dorsocaudal region of the pars distalis. The distribution and histological characteristics of immunoreactive LH cells were different from those of immunoreactive TSH cells revealed by anti-human TSH beta serum.

Immunocytochemical Localization of Secretory Phospholipase A2-like Protein in the Pituitary Gland and Surrounding Tissue of the Bullfrog, Rana catesbeiana

Previously, we obtained a protein that has considerable amino acid sequence homology with secretory phospholipase A2 (PLA2) from a bullfrog pituitary fraction obtained during the purification of thyrotropin (TSH). Subsequently, partial amino acid sequence (N-terminal 45 amino acid residues) analysis revealed this protein to be identical to the N-terminal amino acid sequence of otoconin-22, the major protein of aragonitic otoconia in the Xenopus saccule. In this study we developed an antibody against the N-terminal peptide of the bullfrog protein and applied it for immunocytochemical study of the pituitary and its surrounding tissue. Western blotting analysis showed that this antibody recognizes a 20.4-kD protein that has a molecular mass close to that of otoconin-22. Immunohistochemical reaction with the antibody was not found in any anterior pituitary cells but was intense in the monolayer epithelial cells of the endolymphatic sac surrounding the pituitary gland, which is a major storage site of calcium carbonate in amphibians. An electron microscopic study revealed that the cuboidal cells in the endolymphatic sac contained large, polymorphic secretory granules in their apical cytoplasm. Immunogold particles indicating the presence of a PLA2-like protein were observed predominately in these secretory granules. These findings support the view that this PLA2-like protein obtained during purification of TSH was derived from the endolymphatic sac adhering to the pituitary and that this protein is a bullfrog otoconin. (J Histochem Cytochem 49:631–637, 2001)

Xenopus laevis Pancreatic DNase I: Purification and Immunological Characterization

Deoxyribonuclease I (DNase I) was purified from Xenopus laevis pancreas to apparent electrophoretic homogeneity using a series of column chromatographies. The purified enzyme showed a molecular mass of about 36 kDa and maximum activity at pH 7.0–8.0, required divalent cations, Ca2+ and Mg2+, for its activity, and was inhibited by EDTA, EGTA and an antibody specific to the enzyme, but not by G-actin. The N-terminal amino acid sequence of the enzyme up to the 37th residue shared 38–44% homology with that of mammalian DNases I derived from bovine, ovine, porcine, rat, mouse, rabbit and human. A systematic survey of DNase I activity distribution in 20 different kinds of frog tissues showed that the pancreas and rectum produced higher amounts than other tissues. This is the first report concerning the purification and chemical and immunological characterization of frog pancreatic DNase I.

DIF-1, an anti-tumor substance found in Dictyostelium discoideum, inhibits progesterone-induced oocyte maturation in Xenopus laevis

Differentiation-inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one) is a putative morphogen that induces stalk-cell formation in the cellular slime mold Dictyostelium discoideum. DIF-1 has previously been shown to suppress cell growth in mammalian cells. In this study, we examined the effects of DIF-1 on the progesterone-induced germinal vesicle breakdown in Xenopus laevis, which is thought to be mediated by a decrease in intracellular cAMP and the subsequent activation of mitogen-activated protein kinase (MAPK) and maturation-promoting factor, a complex of cdc2 and cyclin B, which regulates germinal vesicle breakdown. DIF-1 at 10-40 microM inhibited progesterone-induced germinal vesicle breakdown in de-folliculated oocytes in a dose-dependent manner. Progesterone-induced cdc2 activation, MAPK activation, and c-Mos accumulation were inhibited by DIF-1. Furthermore, DIF-1 was found to inhibit the progesterone-induced cAMP decrease in the oocytes. These results indicate that DIF-1 inhibits progesterone-induced germinal vesicle breakdown possibly by blocking the progesterone-induced decrease in [cAMP](i) and the subsequent events in Xenopus oocytes.

Hyperpolarization-activated Cl- current elicited by pituitary adenylate cyclase activating polypeptide in Xenopus oocytes

We examined the electrophysiological effect of pituitary adenylate cyclase activating polypeptide (PACAP) in isolated Xenopus laevis oocytes in vitro. In conventional two-electrode voltage clamp experiments, PACAP (1-10 microM) activated an inward rectifier current at membrane potentials more negative than -60 mV without causing any significant change in currents at potentials more positive than -60 mV both in the follicle-enclosed oocyte and in the defolliculated oocyte. This current reversed at -22.5 mV, close to the theoretical value of Cl- equilibrium potential and the reversal potential of this current was shifted positively by reducing [Cl-]o. This current was blocked by Cl- channel blocker SITS and Ba2+. Furthermore, VIP and adenylate cyclase activator forskolin did not elicit the currents. In conclusion, PACAP elicited the hyperpolarization-activated Cl- current in Xenopus laevis oocytes. This current may modulate the membrane potential of the oocyte, thereby affecting the oocyte physiology.