The antagonistic activity of Padina arborescens extracts on mPRα.

The current study attempted to evaluate the antagonistic activity of compounds isolated and purified from the marine algae Padina arborescens during cultivation. The compounds were collected on a filter, concentrated on ODS columns and separated by HPLC. Two peaks that showed competitive progesterone binding activity with membrane progesterone receptor α (mPRα) were purified. Their physiological activity was further uncovered by in vitro and in vivo oocyte maturation and ovulation-inducing assays using zebrafish. The compounds inhibited the induction of oocyte maturation and ovulation. Moreover, the results showed that the compounds have antagonistic activity against mPRα. The purified compounds with antagonistic activity against mPRα would be considered as new pharmaceutical candidate.

Purification and Identification of the 20S Proteasome Complex from Zebrafish.

The proteasome is a large polymeric protease complex responsible for degradation of intracellular proteins and generation of peptides. In this study, we purified a native 20S proteasome protein complex from zebrafish (Danio rerio) from the whole body. The cytosolic fraction of zebrafish hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA (Suc-LLVY-MCA), a well-known substrate for the proteasome, in the presence of sodium dodecyl sulfate. From the cytosolic fraction, the 20S proteasome was purified using five column chromatography steps: DEAE cellulose, Q-Sepharose, Sephacryl S-300 gel, hydroxylapatite, and phenyl Sepharose. Electrophoresis and Western blot analyses showed that zebrafish 20S proteasome subunits have molecular masses ranging from 22 to 33 kDa. The subunit composition of the purified 20S proteasome was identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation. Fourteen kinds of 20S subunits were found. As a special characteristic of zebrafish, two proteins of the α1 subunit were identified. In addition, the results suggested that the α8 subunit is in the 20S complex instead of the α4 subunit. In this study, we demonstrated the subunit composition of the 20S proteasome complex present in zebrafish cells.

Establishment of a steroid binding assay for membrane progesterone receptor alpha (PAQR7) by using graphene quantum dots (GQDs).

Currently, semiconductor nanoparticles known as quantum dots (QDs) have attracted interest in various application fields such as those requiring sensing properties, binding assays, and cellular imaging and are the very important in the acceleration of drug discovery due to their unique photophysical properties. Here, we applied graphene quantum dots (GQDs) for the binding assay of membrane progesterone receptor alpha (mPRα), one of the probable membrane receptors that have potential in drug discovery applications. By coupling the amino groups of mPRα with GQDs, we prepared fluorogenic GQD-conjugated mPRα (GQD-mPRα). When mixed with a progesterone-BSA-fluorescein isothiocyanate conjugate (P4-BSA-FITC) to check the ligand receptor binding activity of GQD-mPRα, fluorescence at 520 nm appeared. The fluorescence at 520 nm was reduced by the addition of free progesterone into the reaction mixture. GQD-coupled BSA (GQD-BSA) did not show a reduction in fluorescence at 520 nm. The results demonstrated the formation of a complex of GQD-mPRα and P4-BSA-FITC with ligand receptor binding. We established a ligand binding assay for membrane steroid receptors that is applicable for high-throughput assays.

Pax2a is expressed in oocytes and is responsible for early development and oogenesis in zebrafish.

Eleven genes, including pax2a, were selected as candidate ovulation-inducing genes on the basis of microarray analysis and RNA sequencing in our previous study. The purpose of this study was to investigate the role of the pax2a gene in the ovulation-inducing process. F2 pax2a homozygous mutant zebrafish possessing a deletion of 6 nucleotides were established in this study. However, the deletion included the start codon (ATG) of the pax2a gene, and the Pax2a protein was still detected, which indicated that the deletion caused a shift in the start codon to the next ATG, resulting in a 12-amino acid deletion. F2 pax2a homozygous mutant zebrafish showed ovulation. However, the embryos showed an abnormal oval shape at the epiboly stage that resulted in yolk and tail formation abnormalities and heart edema. The surviving F3 homozygous mutants did not develop ovaries. Pax2a was detected in oocytes and eggs but not after the Prim-22 stage. It is suggested that pax2a is expressed as a maternal gene in oocytes and is necessary for oogenesis and early development.

Characterization of membrane progestin receptor α (mPRα) of the medaka and role in the induction of oocyte maturation.

Oocyte maturation in medaka is induced by the maturation-inducing hormone (MIH) via its membrane receptor. The most likely candidates for the membrane receptor are membrane progestin receptors (mPRs). In order to characterize the mPRα subtype of medaka, a human cell line expressing the mPRα gene of medaka was established and its steroid binding property was assessed. The α subtype exhibited high binding affinity for 17,20ß-DHP, the MIH in medaka. Treatment with a morpholino antisense oligonucleotide to mPRα blocked oocyte maturation in vivo. These results suggest that the medaka mPRα protein acts as an intermediary during MIH-induced oocyte maturation in medaka in a manner similar to that described previously for fish species.