Search

Paqr5b is a gene encoding membrane progesterone receptor γ (mPRγ), which is one of five mPR subtypes. Paqr5b belongs to the progestin and adipoQ receptor (PAQR) family, which consists of 11 genes. To elucidate the physiological functions of the mPR subtypes, we established gene knockout (KO) zebrafish strains by genetically editing seven paqr genes and analyzed their phenotypes. The null-mutant strain of paqr5b (paqr5b -/- ) that we established in this study showed low fecundity, reduced chorion elevation and a high percentage of abnormal embryos. Embryos showed curvature of the spine and an abnormal head morphology. Individuals with abnormal head morphology continued to develop a phenotype of markedly abnormal palatine bone. The length of the brain of paqr5b -/- zebrafish was short, and the position of the cerebellum moved to the front and overlapped with that of the midbrain. Micro-CT scans revealed that the olfactory rosettes (ORs) were so shrunken that they were difficult to identify and connected with the olfactory bulbs (OBs) by thread-like structures. Immunohistochemical staining of OR with an anti-Paqr5b antibody revealed that Paqr5b was extensively expressed in neurons in the OR in wild-type zebrafish, whereas signals were not detected in paqr5b -/- zebrafish. In histological sections, the neurons disappeared, and the lamellar layer of the OR became thinner. These results indicate that Paqr5b is required for the formation of neurons in the OR. This is the first report demonstrating a distinct role for the mPR gene., (© 2024. The Author(s).)

Successful fertilization in fish mating occurs when egg maturation in the ovary of the female, ovulation, sperm maturation in the testis of the male, and reproductive behaviors in both sexes are triggered in synchrony. The male sexual behavior of fish is induced by hormones and pheromones. In a previous study, we demonstrated that externally applied hormones added to the water can induce oocyte maturation and ovulation in female zebrafish. Here, we attempted to establish a similar method to induce the sexual behavior of male zebrafish. The male sex steroid testosterone (Tes) triggered sexual behavior within several hours in vivo when administered directly into the surrounding water. A selective agonist for membrane progesterone receptor (mPR), Org OD-02 (Org), also induced sexual behavior. Through trials of various combinations of compounds, we found that the most effective conditions were achieved by treatment with a mixture of testosterone (Tes) and Org. The effect of treatment was evaluated by the number of fertilized eggs obtained by pairing with females with induced ovulation in vivo. The period necessary for the induction of male sexual behavior was evaluated by time course experiments. The success rate of mating and the number of fertilized eggs reached the maximum level at 3-4 hours of treatment. The duration of hormonal treatment was confirmed by counting the number of hooking occurrences, which is the final cue to induce spawning by females. In summary, we have established a method to induce male sexual behavior in zebrafish in vivo. The method can be used to obtain fertilized eggs in zebrafish by simply adding agents into the water., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ahamed et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Post-myocardial infarction (MI) ventricular septal perforation (VSP) is a rare but life-threatening complication. Surgical repair is challenging and carries significant risks, particularly in the context of recurrent VSPs. This case study presents a patient with recurrent VSP after initial surgical repair following myocardial infarction., Case Presentation: A 65-year-old male were re-administered to our hospital due to recurrent VSP. He was during follow up after undergone emergency VSP closure surgery 2 months earlier, utilizing the bovine double patch technique via left ventriculostomy. The initial VSP was located in the apical part of the interventricular septum, while the recurrent VSP appeared in the upper middle portion of the interventricular septum (Fig. 1). As the previous patch remained intact, the second surgery employed the bovine double patch technique via right ventriculostomy. The patient's condition remained stable without the development of heart failure symptoms., Conclusion: Repairing recurrent VSPs remains a challenge, necessitating the mastery of appropriate approaches to achieve optimal outcomes. Further research and guidelines are required to refine management strategies for recurrent VSPs., (© 2024. The Author(s).)

Previously, we established a homogeneous assay for membrane progesterone receptor alpha (mPRα) ligands by conjugating semiconductor nanoparticles known as graphene quantum dots (GQDs) to mPRα. When mixed with a progesterone-BSA-fluorescein isothiocyanate conjugate (P4-BSA-FITC), fluorescence occurred by fluorescence resonance energy transfer (FRET) but was reduced by the ligand-receptor binding activity. The established way showed ligand specificity as mPRα protein. In this study, we tried to establish the same way for nuclear progesterone receptor (Pgr). The ligand-binding domain (LBD) of zebrafish Pgr (zPgrLBD) was expressed as a fusion protein with glutathione S-transferase (GST) (GST-zPgrLBD). The recombinant protein was then purified and coupled with GQDs to produce GQD-conjugated GST-zPgrLBD (GQD-GST-zPgrLBD). When mixed with a P4-BSA-FITC and activated by 370 nm light, fluorescence at 520 nm appeared by FRET mechanism. Fluorescence at 520 nm was reduced by adding free progesterone to the reaction mixture. Reduction of fluorescence was induced by zPgr ligands but not by steroids or chemicals that do not interact with zPgr. The results showed the formation of a complex of GQD-GST-zPgrLBD and P4-BSA-FITC with ligand-receptor binding. The binding of the compounds was further confirmed by a radiolabeled steroid binding assay. A homogenous ligand-binding assay for nuclear progesterone receptor has been established., Competing Interests: The authors declare that there no conflicts of interest., (© 2024 The Authors.)

Teratomas in mice, composed of different tissue types, are derived from primordial germ cells in the fetal gonads. Previously, we identified a locus responsible for experimental testicular teratoma (ETT) formation on chromosome 18, referred to as ett1. The strongest candidate sequence in the ett1 locus was found to be a missense mutation in the melanocortin 4 receptor (Mc4r), Mc4r G25S . We established a strain with a point mutation in the Mc4r gene in the ETT-nonsusceptible LT strain, called LT- Mc4r G25S , by genome editing. Surprisingly, highly developed ovarian teratomas (OTs), rather than testicular teratomas, appeared in the LT-Mc4r G25S strain. The results demonstrated that Mc4r is also one of the genes responsible for OT formation and suggested that missense mutations in Mc4r promote teratoma formation in both sexes. In this study, we performed ETT experiments in different host-graft combinations of the LT-Mc4r G25S and LT strains. Furthermore, the expression of MC4R in germ cells in the testis was demonstrated. Expression of Mc4r in testis was also confirmed by RT-PCR. The results demonstrated that MC4R is expressed in germ cells in the testis and that a point mutation in the Mc4r gene is responsible for ETT formation., (© 2023. The Author(s).)

Using an in vivo assay, we selected 11 genes that were highly upregulated during the induction of ovulation in zebrafish using microarray analysis and RNA sequencing. The starmaker gene ( stm ) was one of these genes. Although stm has been previously reported to be involved in otolith formation during the early development of zebrafish, we detected its expression in eggs and showed that stm was related to fertilization by establishing an stm gene knockout strain using the CRISPR/Cas9 system. Further phenotypic analysis of stm knockout fish was conducted in this study. With a higher nonfertilization rate, the stm mutant strain showed an extremely low survival rate. Otoliths of stm homozygous mutant zebrafish showed abnormal morphology in embryos and adult fish. However, fish did not show any abnormalities in swimming behaviour in either embryos or adults. Stm proteins were detected on the chorion of ovulated eggs before spawning. Fibre-supported knob-like structures on the fertilization envelope (FE) also showed abnormal structures in stm mutants. The Stm protein is necessary for otolith formation, and a lack of Stm causes abnormal otolith formation. The partial defect of otolith formation does not cause defects in swimming behaviour. The Stm protein is expressed in the chorion and is responsible for the formation of fibre-supported knob-like structures on the FE. It was suggested that a lack of Stm caused a lower fertilization rate due to inadequate formation of the FE., Lay Summary: In zebrafish, the protein Starmaker (Stm) was identified as having a role in ovulation. Stm is also known to be required for the formation of ear stones (otoliths) which are needed to keep the body in balance. Zebrafish lacking Stm were produced by genome editing. As expected, Stm-deficient fish formed abnormal otoliths. To investigate the role of Stm in ovulation, fertilization and early development, we tried mating of Stm mutants and observed their juveniles. Although no problem found in ovulation, we found low fertilization rate and abnormal structure of knob-like structure (small pit) on the egg membrane. Survival rate of embryos with abnormal egg membrane was extremely low. It was demonstrated that Stm protein is necessary to form the functional egg membrane to protect embryos from the outside environment., Competing Interests: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this study., (© 2021 The authors.)

Teratomas in mice, composed of different tissue types, are derived from primordial germ cells (PGCs) in the foetal gonads. The strongest candidate gene in the testicular teratoma locus (Ter) responsible for testicular teratoma formation was identified as mutation in Dnd1, Dnd1R178*. However, the phenotype of mice with a mutated Dnd1 gene was germ cell loss. This suggests that other genes are involved in teratoma formation. Testicular teratomas can also be induced experimentally (experimentally testicular teratomas: ETTs) in 129/Sv mice by transplanting E12.5 foetal testes into adult testes. Previously, we mapped the ett1 locus, which is the locus responsible for ETT formation on chromosome 18. By exome sequence analysis of the 129 and LTXBJ (LT) strains, we identified a missense mutation in the melanocortin 4 receptor (MC4R) gene among 8 genes in the ett1 region. The missense mutation causes a substitution of glycine 25 by serine. Thus, this gene is a candidate for ETT formation. We established the LT-ett1 congenic strain, which introduced the locus responsible for ETT formation genetically into the genomes of a testicular teratoma non-susceptible strain. In this study, we crossed LT-ett1 and a previously established LT-Ter strain to establish the double congenic strain LT-Ter-ett1. Also, we established a strain with a point mutation in the MC4R gene of the LT strain by genome editing, LT-MC4R G25S . Furthermore, double genetically modified strain LT-Ter-MC4R G25S was established to address the relation between Ter and MC4R. Surprisingly, highly developed ovarian teratomas (OTs), instead of testicular teratomas, appeared not only in the LT-Ter-MC4R G25S and LT-MC4R G25S strains but also in the LT-ett1 and LT-Ter-ett1 strains. The incidence of OT formation was high in double genetically modified strains. The results demonstrated that MC4R is one of the genes responsible for OT formation. It was suggested that the effect of the missense mutation in MC4R on teratoma formation was promoted by abnormal germ cell formation by the mutation in DND1.

DNA methylation is a prevalent epigenetic modification in vertebrates, and it has been shown to be involved the regulation of gene expression and embryo development. However, it remains unclear how DNA methylation regulates sexual development, especially in species without sex chromosomes. To determine this, we utilized zebrafish to investigate DNA methylation reprogramming during juvenile germ cell development and adult female-to-male sex transition. We reveal that primordial germ cells (PGCs) undergo significant DNA methylation reprogramming during germ cell development, and the methylome of PGCs is reset to an oocyte/ovary-like pattern at 9 days post fertilization (9 dpf). When DNA methyltransferase (DNMT) activity in juveniles was blocked after 9 dpf, the zebrafish developed into females. We also show that Tet3 is involved in PGC development. Notably, we find that DNA methylome reprogramming during adult zebrafish sex transition is similar to the reprogramming during the sex differentiation from 9 dpf PGCs to sperm. Furthermore, inhibiting DNMT activity can prevent the female-to-male sex transition, suggesting that methylation reprogramming is required for zebrafish sex transition. In summary, DNA methylation plays important roles in zebrafish germ cell development and sexual plasticity., (Copyright © 2021.)

To complete meiosis II, cyclin B is degraded in a short period by the inactivation of M-phase promoting factor (MPF). Previously, we showed that the destruction of cyclin B was initiated by the ubiquitin-independent proteolytic activity of the 26 S proteasome through an initial cut in the N-terminus of cyclin (at K57 in the case of goldfish cyclin B). We hypothesized that this cut allows cyclin to be ubiquitinated for further destruction by the ubiquitin-dependent proteolytic pathway, which leads to MPF inactivation. In this study, we aimed to identify the ubiquitination site for further degradation. The destruction of cyclin B point mutants in which lysine residues in a lysine-rich stretch following the cut site of cyclin B had been mutated was analyzed. All the lysine point mutants except K57R (a point mutant in which K57 was substituted with arginine) were susceptible to proteolytic cleavage by the 26 S proteasome. However, the degradation of the K77R and K7677R mutants in Xenopus egg extracts was significantly slower than the degradation of other mutants, and a 42 kDa truncated form of cyclin B was detected during the onset of the degradation of these mutants. The truncated form of recombinant cyclin B, an N-terminal truncated cyclin BΔ57 produced as cut by the 26 S proteasome, was not further cleaved by the 26 S proteasome but rather degraded in Xenopus egg extracts. The injection of the K57R, K77R and K7677R cyclin B proteins stopped cleavage in Xenopus embryos. From the results of a series of experiments, we concluded that cyclin B degradation involves a two-step mechanism initiated by initial ubiquitin-independent cleavage by the 26 S proteasome at lysine 57 followed by its ubiquitin-dependent destruction by the 26 S proteasome following ubiquitination at lysine 77.

Spontaneous testicular teratomas (STTs) derived from primordial germ cells (PGCs) in the mouse embryonic testes predominantly develop in the 129 family inbred strain. Ter (spontaneous mutation) is a single nucleotide polymorphism that generates a premature stop codon of Dead end1 (Dnd1) and increases the incidence of STTs in the 129 genetic background. We previously found that DND1 interacts with NANOS2 or NANOS3 and that these complexes play a vital role in male embryonic germ cells and adult spermatogonia. However, the following are unclear: (a) whether DND1 works with NANOS2 or NANOS3 to regulate teratoma incidence, and (b) whether Ter simply causes Dnd1 loss or produces a short mutant DND1 protein. In the current study, we newly established a conventional Dnd1-knockout mouse line and found that these mice showed phenotypes similar to those of Ter mutant mice in spermatogenesis, oogenesis, and teratoma incidence, with a slight difference in spermiogenesis. In addition, we found that the amount of DND1 in Dnd1+/Ter embryos decreased to half of that in wild-type embryos, while the expression of the short mutant DND1 was not detected. We also found that double mutants for Dnd1 and Nanos2 or Nanos3 showed synergistic increase in the incidence of STTs. These data support the idea that Ter causes Dnd1 loss, leading to an increase in STT incidence, and that DND1 acts with NANOS2 and NANOS3 to regulate the development of teratoma from PGCs in the 129 genetic background. Thus, our results clarify the role of Dnd1 in the development of STTs and provide a novel insight into its pathogenic mechanism., Competing Interests: The authors have declared that no competing interests exist.

The molecular mechanisms by which environmental light conditions affect cerebellar development are incompletely understood. We showed that circadian disruption by light-at-night induced Purkinje cell death through pineal allopregnanolone (ALLO) activity during early life in chicks. Light-at-night caused the loss of diurnal variation of pineal ALLO synthesis during early life and led to cerebellar Purkinje cell death, which was suppressed by a daily injection of ALLO. The loss of diurnal variation of pineal ALLO synthesis induced not only reduction in pituitary adenylate cyclase-activating polypeptide (PACAP), a neuroprotective hormone, but also transcriptional repression of the cerebellar Adcyap1 gene that produces PACAP, with subsequent Purkinje cell death. Taken together, pineal ALLO mediated the effect of light on early cerebellar development in chicks., Competing Interests: SH, MK, TT, KT, MS, MN, MO, IS, GH, VC, XL, TM, JK, TN, KM, HO, TT, TT, AM, KT No competing interests declared, (© 2019, Haraguchi et al.)

Next-generation effects and further transgenerational effects of an endocrine disruptor, bisphenol A (BPA), were investigated in zebrafish. The effects of BPA treatment through dietary administration in male and female zebrafish on reproductive factors, such as gonadal activity, fertility, hatching rate and malformation in subsequent generations, were examined through the third filial (F3) generation. BPA treatment of initial generation (F0) not only caused retraction of the ovaries and testes but also lowered the survival rate and increased the rate of malformation of the offspring. Although the overall phenotypes of the surviving first filial (F1) generation offspring of treated fish initially appeared to be normal, we found abnormalities in their reproductive tissues after they matured to adulthood. Although the juveniles were fed a normal diet, the ovaries of 40% of the F1 generation fish remained small and did not develop vitellogenic oocytes. Moreover, sterile male fish appeared at a higher percentage (48%) than control (10%). Adverse transgenerational effects on the fecundity of the second filial (F2) and F3 generation fish were also observed. In each generation, survival rate of embryos were significantly low and abnormal embryos were appeared in offspring from BPA treated ancestral. These results demonstrate that the effects of BPA are transferred to subsequent generations not only through oocytes but also through sperm.

Pyoderma gangrenosum (PG) is a skin disease characterized by an unknown neutrophilic infiltration in dermis and a nonbacterial destructive ulcer. Post-operative PG is an extremely rare type that occurs around surgical sites during the immediate post-operative period. It is usually diagnosed as surgical site infection at the time of presentation. The condition rapidly worsens despite antibiotic treatment and debridement. We report on a case of post-operative PG in a 64-year-old man after radical prostatectomy. Following the operation, redness and pus from surgical site rapidly progress although repeated antibiotic therapy and debridement were performed. Although the patient received appropriate debridement and broad-spectrum antibiotic treatment, the ulcerative lesion spread surrounding drain region and the condition of the skin region deteriorated. The diagnosis of PG was made by a skin biopsy that presented only neutrophilic invasion in the dermis without vasculitis, tumor, or malignancy. Finally, the patient died of lesion progression in whole body and multiple organ dysfunction. Considering PG along with ulcers, wounds, and post-operative complications is critical for prompt diagnosis and proper treatment., Competing Interests: All authors declare that they have no conflict of interest.The case report approval was obtained from the Hospital Research Ethics Board (Approval Number: 1763).

We previously reported the microarray-based selection of three ovulation-related genes in zebrafish. We used a different selection method in this study, RNA sequencing analysis. An additional eight up-regulated candidates were found as specifically up-regulated genes in ovulation-induced samples. Changes in gene expression were confirmed by qPCR analysis. Furthermore, up-regulation prior to ovulation during natural spawning was verified in samples from natural pairing. Gene knock-out zebrafish strains of one of the candidates, the starmaker gene (stm), were established by CRISPR genome editing techniques. Unexpectedly, homozygous mutants were fertile and could spawn eggs. However, a high percentage of unfertilized eggs and abnormal embryos were produced from these homozygous females. The results suggest that the stm gene is necessary for fertilization. In this study, we selected additional ovulation-inducing candidate genes, and a novel function of the stm gene was investigated.

Background: Two essential processes, oocyte maturation and ovulation, are independently induced, but proceed cooperatively as the final step in oogenesis before oocytes become fertilizable. Although these two processes are induced by the same maturation-inducing steroid, 17α, 20β-dihydroxy-4-pregnen-3-one (17, 20β-DHP), in zebrafish, it has been suggested that the receptor, and thus the signal transduction pathway is different for each process. Although much progress has been made in understanding the molecular mechanisms underlying the induction of oocyte maturation, the mechanisms for inducing ovulation remain under investigation. In the present study, in vivo induction techniques that permit the induction of oocyte maturation and ovulation in living zebrafish ( in vivo assays) were used to select highly up-regulated genes (genes associated with ovulation). Using an in vivo assay, ovarian tissues that induced only oocyte maturation could be obtained. This made it possible for the first time to distinguish maturation-inducing genes from ovulation-inducing genes. Using a genome-wide microarray of zebrafish sequences, the gene expression levels were compared among an ethanol (EtOH)-treated group (non-activated group), a diethylstilbestrol (DES)- or testosterone (Tes)-treated group (maturation-induced group), and a 17, 20β-DHP-treated group (maturation- and ovulation-induced group). Ovulation-specific up-regulated genes were selected. The mRNA expression levels of the selected genes were measured by quantitative polymerase chain reaction (qPCR)., Results: Among 34 genes identified, three that showed ovulation-specific increases were selected as candidates potentially associated with ovulation. The ovulation-specific up-regulation of three candidates, slc37a4a, zgc:65811 and zgc:92184 was confirmed by qPCR., Conclusion: Our in vivo assay provides a new approach to precisely select genes associated with ovulation.

Developing methods to efficiently convert lignocellulosic polymers, i.e. cellulose, hemicellulose, and lignin into user-friendly carbon resources, such as fermentable sugars, is critical for improving plant biomass utilization. Here, we report the identification of genes that increase enzymatic saccharification efficiency in cultured Arabidopsis wood cells. We overexpressed a set of genes that were upregulated during the early stages of in vitro xylem vessel cell differentiation, including transcription factor and CAZYme genes, in Arabidopsis and tested their effects on enzymatic saccharification efficiency. Of the 96 transgenic seedlings sampled, 37 and 17 lines showed significant increases and decreases in glucose yields, respectively. Further analysis of 20 overexpression lines with high glucose yields in seedling samples indicated that compared to wild type, the glucose and xylose yields from inflorescence stem samples were higher in lines overexpressing genes encoding BETA-XYLOSIDASE 2, UDP-GLUCOSYL TRANSFERASE 88A1, AT3G15350 (a class GT14 glycosyltransferase protein), and the Dof-type transcription factor Dof4.6, whose detailed molecular functions have not yet been characterized. No apparent defect in growth or inflorescence stem structure was detected in these overexpression lines. Therefore, these four genes might represent novel factors that can be used to increase saccharification efficiency in wood tissues without negatively affecting total biomass production. Furthermore, our results confirm the validity of our screening strategy for isolating factors related to the saccharification efficiency of lignocellulosic biomass.

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 17,20β-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.

The origins of spin exchange in crystalline thin films of Copper Octabutoxy Phthalocyanine (Cu-OBPc) are investigated using Magnetic Circular Dichroism (MCD) spectroscopy. These studies are made possible by a solution deposition technique which produces highly ordered films with macroscopic grain sizes suitable for optical studies. For temperatures lower than 2 K, the contribution of a specific state in the valence band manifold originating from the hybridized lone pair in nitrogen orbitals of the Phthalocyanine ring, bears the Brillouin-like signature of an exchange interaction with the localized d-shell Cu spins. A comprehensive MCD spectral analysis coupled with a molecular field model of a σπ - d exchange analogous to sp-d interactions in Diluted Magnetic Semiconductors (DMS) renders an enhanced Zeeman splitting and a modified g-factor of -4 for the electrons that mediate the interaction. These studies define an experimental tool for identifying electronic states involved in spin-dependent exchange interactions in organic materials.

Membrane progestin receptors (mPRs) are responsible for mediating the rapid, nongenomic activity of progestins and belong to the G protein-coupled receptor (GPCR) family. mPRs are also considered as attractive proteins to draw a new medicinal approach. In this study, we optimized a procedure for the expression and purification of recombinant human mPRα protein (hmPRα) by a methylotropic yeast, Pichia pastoris, expression system. The protein expressed in crude membrane fractions exhibited a binding affinity of Kd = 3.8 nM and Bmax = 288.8 fmol/mg for progesterone. These results indicated that the hmPRα expressed in yeast was active. Solubilized hmPRα was purified through three column chromatography steps. A nickel-nitrilotriacetic acid (Ni-NTA) column was first used, and the mPRα proteins were then bound to cellulose resin with free amino groups (Cellufine Amino) and finally passed through an SP-Sepharose column. The optimization of expression and purification conditions resulted in a high yield of purified hmPRα (1.3-1.5 mg from 1 L culture). The purified hmPRα protein demonstrated progesterone binding (Kd = 5.2 nM and Bmax = 111.6 fmol/mg). The results indicated that we succeeded in solubilizing and purifying hmPRα in an active form. Sufficient amount of active hmPRα protein will support the establishment of applications for the screening of ligands for mPRα.

Recently, we reported the conversion of bisphenol A (BPA) to 4-(2-(4-hydroxyphenyl)propan-2-yl)benzene-1,2-diol (hydroxy-BPA) by hyper lignin-degrading fungus Phanerochaete sordida YK-624 under non-ligninolytic condition. In the present study, the metabolism of hydroxy-BPA by P. sordida YK-624 was demonstrated under non-ligninolytic condition. Under these conditions, approximately 66% of hydroxy-BPA was degraded after 7 d of incubation. High-resolution electrospray ionization mass spectra and nuclear magnetic resonance analyses of the metabolites isolated from the culture broth indicated that hydroxy-BPA was metabolized to 4-(2-(4-hydroxyphenyl)propan-2-yl)-2-methoxyphenol (methoxy-BPA) and to 4-(2-(3,4-dimethoxyphenyl)propan-2-yl)phenol (dimethoxy-BPA) by sequential methylation events. These metabolites showed reduced estrogenic activity compared to BPA. These results suggested that the hydroxy BPA is methylated to two low toxic-methylation metabolites., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Membrane progestin receptors (mPRs) are key mediators of rapid, nongenomic actions of progestins on plasma membranes. We established a procedure for the expression and purification of recombinant goldfish mPRα using the methylotropic yeast Pichia pastoris. In P. pastoris, the recombinant protein, which carried C-terminal histidine and c-Myc tags, was expressed in an active form as the receptor for maturation-inducing steroids of fish. Expressed proteins were bound reversibly with a high affinity (Kd = 9.4 nM) at a single binding site that could be saturated. After solubilization of mPRα with n-dodecyl-β-D-maltoside (DDM) from yeast membranes, the recombinant protein was purified using three different columns: first it was affinity-purified over nickel-nitrilotriaceticacid (Ni-NTA), then bound to a cellulose resin with free amino groups and finally to a column with affinity for the c-Myc epitope. The identity of the purified protein was verified by MALDI-TOF/MS analysis and its capacity to bind progestin remained. Expression and purification of mPRα protein in its functional form will enable the screening of ligands and the determination of its three dimensional structure.

This study investigated whether undifferentiated germ and/or somatic stem cells remain in the differentiated ovary of a species that does not undergo sex changes under natural conditions and retain their sexual plasticity. The effect of aromatase inhibitor (AI)-treatment on sexually mature female zebrafish was examined. A 5-month AI treatment caused retraction of the ovaries after which testes-like organs appeared, and cyst structures filled with spermatozoa-like cells were observed in sections of these tissues. Electron microscopic observations revealed that these cells appeared as large sperm heads without tails. Sperm formation was re-examined after changing the diet to an AI-free food. A large number of normal sperm were obtained after eight weeks, and no formation of ovarian tissue was observed. Artificial fertilization using sperm from the sex-changed females was successful. These results demonstrated that sex plasticity remains in the mature ovaries of this species.

Bisphenol A (BPA) is one of the representative compounds of the endocrine disrupting compounds group and the highest volume chemicals produced worldwide. As a result, BPA is often detected in many soil and water environments. In this study, we demonstrated the transformation of BPA from liquid cultures inoculated with hyper lignin-degrading fungus Phanerochaete sordida YK-624. Under non-ligninolytic condition, approximately 80% of BPA was eliminated after 7d of incubation. High-resolution electrospray ionization mass spectra and nuclear magnetic resonance analyses of a metabolite isolated from the culture supernatant suggested that BPA was metabolized to hydroxy-BPA, 4-(2-(4-hydroxyphenyl)propan-2-yl)benzene-1,2-diol, which has a much lower estrogenic activity than BPA. In addition, we investigated the effect of the cytochrome P450 inhibitor piperonyl butoxide (PB) on the hydroxylation of BPA, markedly lower transformation activity of BPA was observed in cultures containing PB. These results suggest that cytochrome P450 plays an important role in the hydroxylation of BPA by P. sordida YK-624 under non-ligninolytic condition., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Because of the presence of sperm-storage tubules (SST) in the utero-vaginal junction (UVJ) in the oviduct, once ejaculated sperm have entered the female reproductive tract, they can survive for a prolonged time in domestic birds, although the specific mechanisms involved in the sperm uptake into, maintenance within, and controlled release from the SST remain to be elucidated. In this report, we provide evidence that progesterone triggers the release of the resident sperm from the SST in the UVJ. The ultrastructural observation of the SST indicated that the resident sperm are released from the SST around 20 h after oviposition. When laying birds were injected with progesterone, most of the sperm were released from the SST within 1 h of injection. In situ hybridization analyses demonstrated the presence of the transcripts of membrane progestin receptor α in the UVJ, and the translated proteins were detected in the UVJ extracts by Western blotting. Moreover, the number of secretory granules in the SST epithelial cells fluctuates during the ovulatory cycle, and the progesterone administration mimics this phenomena. A binding assay using [(3)H]-progesterone indicated the presence of a high affinity, limited capacity, saturable and single binding site for [(3)H]-progesterone in the membrane fraction of the UVJ, and this receptor did not interact with the synthetic antiprogestin RU486. These results demonstrated for the first time that the progesterone stimulates the release of the resident sperm from the SST and that the release of the sperm might occur via membrane progestin receptor α-mediating signal transduction.

Background: The African clawed frog, Xenopus laevis, is widely used in studies of oogenesis, meiotic cell cycle and early embryonic development. However, in order to perform such studies, eggs are normally collected after the injection of hCG into the dorsal lymph sac of fully-grown female frogs following pre-injection of PMSF. Although this protocol is established and used as standard laboratory approach, there are some concerns over whether the injections could cause the transmission of deleterious microorganisms. Moreover, these injection protocols require a competent skilled worker to carry out the procedure efficiently., Methods: Recently, we established a novel method to induce fish ovulation by simply adding the natural maturation-inducing hormone of teleosts, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-DHP), into the surrounding water. In the present study, we demonstrate how we can induce ovulation in frogs using the same methodology., Results: In frogs, progesterone was effective in the induction of oocyte maturation in vitro. We then examined the ability of progesterone to induce ovulation in frogs. However treatment of frogs with progesterone alone only occasionally induced ovulation in vivo. The number of oocytes and the frequency of ovulation were significantly lower than that induced by hCG-injection. Thus, conditions were improved by using a combination of progesterone with estradiol and by pre-treating frogs with low concentrations of progesterone or estradiol. Finally, we established an efficient means of inducing ovulation in frogs which involved pre-treatment of frogs with salt solution followed by a mixture of estradiol and progesterone at high concentration. The frequency and numbers of oocytes obtained were identical to those resulting from PMSG-hCG induction. Fertilization rate of eggs ovulated by the new treatment method was comparable to eggs obtained by hCG-injection and juveniles developed normally., Conclusions: To conclude, we have successfully developed a novel method to induce ovulation in frogs but without the need for a potentially harmful injection strategy.

The maturation of fish oocytes is a well-characterized system induced by progestins via non-genomic actions. In a previous study, we demonstrated that diethylstilbestrol (DES), a non-steroidal estrogen, induces fish oocyte maturation via the membrane progestin receptor (mPR). Here, we attempted to evaluate the effect of DES as an environmental endocrine disrupting chemical (EDC) upon fish oocyte maturation using live zebrafish. DES triggered oocyte maturation within several hours in vivo when administrated directly into the surrounding water. The natural teleost maturation-inducing hormone, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17,20beta-DHP) also induced oocyte maturation in vivo. Steroids such as testosterone, progesterone or 17alpha-hydroxyprogesterone were also effective in vivo. Further studies indicated that externally applied 17,20beta-DHP even induced ovulation. In contrast to 17,20beta -DHP, DES induced maturation but not ovulation. Theoretically this assay system provides a means to distinguish pathways involved in the induction of ovulation, which are known to be induced by genomic actions from the pathway normally involved in the induction of oocyte maturation, a typical non-genomic action-dependent pathway. In summary, we have demonstrated the effect of EDCs on fish oocyte maturation in vivo. To address the effects, we have explored a conceptually new approach to distinguish between the genomic and non-genomic actions induced by steroids. The assay can be applied to screens of progestin-like effects upon oocyte maturation and ovulation for small molecules of pharmacological agents or EDCs.

Although there have been some reports describing the serostatus of cytomegalovirus, strain-specific antibody responses and their distribution remain unknown. In this study, ELISA using fusion proteins encompassing epitope of glycoprotein H from both AD169 and Towne strains was used to test 352 blood donors. Of these 352 donors, 207 were analyzed for strain-specific glycoprotein H antibodies. Of the 44 donors whose serum contained antibodies against both AD169 and Towne, 27 (60%) were aged 50 years or over (p = 0.0003). This may indicate serological evidence of reinfection with cytomegalovirus in the elder population. The nucleotide sequence analysis of cytomegalovirus glycoprotein H from the peripheral blood of the cytomegalovirus-positive renal transplant recipients showed that our strain-specific ELISA can reveal cytomegalovirus reinfection after transplantation.

Background: Rpt6-1 is a thermosensitive yeast mutant with a deletion of a gene encoding a regulatory subunit of the 26S proteasome, RPT6, which is able to grow at 25 degrees C but not at 37 degrees C. In this study, peptidase activities, activation profiles, and the subunit composition of the 20S proteasome purified from the rpt6-1 mutant was characterized., Results: The 20S proteasome purified from rpt6-1 exhibited low levels of peptidase activities in the absence of activators, but nearly same activated activities in the presence of activators, suggesting a gating defect in the proteasome channel. Detailed analyses of the composition of the 20S proteasome through separation of all subunits by two-dimensional gel electrophoresis followed by identification of each subunit using MALDI-TOF-MS revealed that two subunits, alpha1 and alpha7, differed from those of wild-type cells in both electrophoretic mobility and pI values. The changes in these two alpha-subunits were apparent at the permissive temperature, but disappeared during stress response at the restrictive temperature. Interestingly, upon disappearance of these changes, the levels of peptidase activity of the 20S proteasome in the rpt6-1 mutant were restored as the wild-type. These results suggest that two different forms of the alpha-subunits, alpha1 and alpha7, block the proteasome channel in the rpt6-1 mutant., Conclusion: Two alpha-subunits (alpha1 and alpha7) of the 20S proteasome in the rpt6-1 mutant differed from their wild-type counterparts and peptidase activities were found to be lower in the mutant than in the wild-type strain.

Numerous studies have shown that protocol biopsies have predictive power. We retrospectively examined the histologic findings and C4d staining in 89 protocol biopsies from 48 ABO-incompatible (ABO-I) transplant recipients, and compared the results with those of 250 controls from 133 ABO-compatible (ABO-C) transplant recipients given equivalent maintenance immunosuppression. Others have shown that subclinical rejection (borderline and grade I) in ABO-C grafts decreased gradually after transplantation. In our study, however, subclinical rejection in the ABO-I grafts was detected in 10%, 14% and 28% at 1, 3 and 6-12 months, respectively. At 6-12 months, mild tubular atrophy was more common in the ABO-C grafts whereas the incidence of transplant glomerulopathy did not differ between the two groups (ABO-C: 7%; ABO-I: 15%; p = 0.57). In the ABO-I transplants, risk factors for transplant glomerulopathy in univariate analysis were positive panel reactivity (relative risk, 45.0; p < 0.01) and a prior history of antibody-mediated rejection (relative risk, 17.9; p = 0.01). Furthermore, C4d deposition in the peritubular capillaries was detected in 94%, with diffuse staining in 66%. This deposition, however, was not linked to antibody-mediated rejection. We conclude that, in the ABO-I kidney transplantation setting, detection of C4d alone in protocol biopsies might not have any diagnostic or therapeutic relevance.

Background: Cytomegalovirus (CMV) is the most important pathogen affecting the outcome of renal transplantation. The combination of CMV-seronegative transplant recipients with CMV-seropositive transplant donors places recipients at the highest risk of CMV disease. In cases of congenital CMV infection, existing immunity only partially protected mothers from reinfection with a different genotypic strain. The effect of differences in infected CMV strains between CMV-seropositive transplant donors and CMV seropositive transplant recipients on the outcome of transplantation remains unclear., Methods: In this prospective multicenter study, the presence of antibodies against strain-specific glycoprotein H epitopes in 84 CMV-seropositive transplant donor/CMV-seropositive transplant recipient renal transplantation cases were determined, and their relationships to acute transplant rejection, CMV infection, degree of antigenemia, and CMV disease were evaluated., Results: Among the 84 donor/recipient pairs, 45 and 32 had matched and mismatched strain-specific glycoprotein H antibodies, respectively. Acute transplant rejection in the mismatched group was more frequent than it was in the matched group (63% vs. 22%; P=.005). CMV disease was also more frequently observed in the mismatched group (28% vs. 9%; P=.026). The mismatched group had a higher level of antigenemia (P=.019)., Conclusions: Our results illustrate more adverse events in the cases with a CMV-seropositive transplant donor and a CMV-seropositive transplant recipient in which the glycoprotein H antibodies are mismatched, suggesting that reinfection with a different CMV strain results in more complications.

Progestin induction of oocyte maturation (OM) in fish is a useful model for investigating endocrine disruption of nongenomic steroid actions. Although diethylstilbestrol (DES) analogs have been shown to mimic the actions of progestins to induce meiotic maturation of goldfish and zebrafish oocytes, their molecular mechanisms of action remain unclear. The ability of these endocrine-disrupting chemicals (EDCs) to interact with the progestin receptor mediating OM was investigated in receptor binding assays using plasma membranes from goldfish ovaries and breast cancer cells transfected with goldfish membrane progestin receptor (mPR)-alpha. Membranes prepared from both ovaries and mPRalpha-transfected cells showed high-affinity, saturable, displaceable, single binding sites specific for the goldfish maturation-inducing steroid, 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-DHP). DES and DES analogs (dipropionate-DES and hexestrol), which induce OM in goldfish, bound to the receptor and caused concentration-dependent displacement of [3H]-17,20beta-DHP, whereas dimethyl ether-DES had no affinity for the receptor. Scatchard plot analysis of specific 17,20beta-DHP binding in the presence of different amounts of DES showed that DES binding is of the noncompetitive type. The activities of DES and DES analogs to induce meiotic maturation of goldfish oocytes were examined in an in vitro bioassay. Whereas a concentration-dependent induction of OM was observed in response to DES, dipropionate-DES, and hexestrol, dimethyl ether-DES did not show any OM-inducing activity. The close correspondence between binding of DES and its analogs to the mPRalpha protein and their OM-inducing activities suggests a mechanism of endocrine disruption mediated by binding to mPRalpha resulting in its activation, thereby mimicking the nongenomic action of the progestin 17,20beta-DHP.

Many temporarily functioning proteins are generated during the replacement of nucleoproteins in the nuclei of late spermatids and seem to be degraded in the nucleus. This study was designed to clarify the involvement of the ubiquitin-proteasome degradation system in the nucleus of rat developing spermatids. Thus, we studied the nuclear distribution of polyubiquitinated proteins (pUP) and proteasome in spermiogenic cells and sperm using postembedding immunoelectron microscopy. We divided the nuclear area of late spermatids into two regions: (1) a dense area composed of condensed chromatin and (2) a nuclear pocket in the neck region. The latter was located in the caudal nuclear region and was surrounded by redundant nuclear envelope. We demonstrated the presence of pUP in the dense area and nuclear pocket, proteasome in the nuclear pocket, and clear spots in the dense area of rat spermatids. Using quantitative analysis of immunogold labeling, we found that fluctuation of pUP and proteasome levels in late spermatogenesis was mostly synchronized with disappearance of histones and transitional proteins reported previously. In the nuclei of human sperm, pUP was detected in the dense area, whereas proteasome was in the nuclear vacuoles and clear spots. These results strongly suggest that pUP occur in the dense nuclear area of developing spermatids and that the ubiquitin-proteasome system is more actively operational in the nuclear pocket than dense area. Thus, the nuclear pocket might be the degradation site for temporarily functioning proteins generating during condensation of chromatin in late spermatids.

Several protocols allow the successful ABO incompatible living-related kidney transplantation (ABO-ILKT), yet no single method has emerged as the best. We have made several substantial changes to our ABO-ILKT protocol over the past decade and a half and have attempted to determine whether the changes in immunosuppressive agents have resulted in a better outcome. We used methylprednisolone (MP), cyclosporine (CsA), azathioprine (AZ), antilymphocyte globulin (ALG) and deoxyspergualine (DSG) in the 105 cases of ABO-ILKT (group 1) between 1989 and 1999, and MP, tacrolimus (FK506), mycophenolate mofetil (MMF) in the 117 cases of ABO-ILKT (group 2) between 2000 and 2004. We compared the patient and graft survival rates as well as the incidence rate of acute rejection in these two eras, when different regimens were used. There were significant differences in the 1- and 5-year graft survival rates between groups 1 and 2 (1-year: 78% in group 1 vs. 94% in group 2; 5-year: 73% in group 1 vs. 90% in group 2, p = 0.008). Also, a higher incidence rate of acute rejection was significantly observed in group 1 (50/105, 48%) than in group 2 (18/117, 15%) (p < 0.001). We conclude that the FK/MMF combination regimen provides excellent graft survival results in ABO-ILKT.

Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make "ApoB-crescents." ApoB-crescents were seen in <10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50-100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12-24 h of proteasomal inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways.

Background: Oocyte maturation in lower vertebrates is triggered by maturation-inducing hormone (MIH), which acts on unidentified receptors on the oocyte surface and induces the activation of maturation-promoting factor (MPF) in the oocyte cytoplasm. We previously described the induction of oocyte maturation in fish by an endocrine-disrupting chemical (EDC), diethylstilbestrol (DES), a nonsteroidal estrogen., Methods: In this study, stimulatory and inhibitory effects of EDCs and natural steroids on oocyte maturation were examined in zebrafish. For effective agents, some details about the mechanism in induction or inhibition of maturation were examined. Possible groups of DES interacting with the MIH receptor are discussed based on relative potency of steroids to induce maturation., Results: Among agents tested, tamoxifen (TAM) and its metabolite 4-hydroxytamoxifen (4-OHT) showed stimulatory activity similar to DES. The time courses of the change in germinal vesicle breakdown and an intracellular molecular event (the synthesis of cyclin B) induced by TAM were indistinguishable from those induced by MIH. In contrast, pentachlorophenol (PCP) had a potent inhibitory effect on MIH-induced oocyte maturation. PCP inhibited not only MIH-induced maturation but also DES- and TAM-induced maturation. Methoxychlor also inhibited maturation when oocytes were pre-treated with this agent., Conclusion: These results suggest that EDCs act as agonists or antagonists in the induction of oocyte maturation in fish.

We tried to establish the technique of retroperitoneoscopic live donor nephrectomy (RPLDN). Between July 2001 and March 2004, 135 renal transplant donors underwent RPLDN. Low (average: 7 mmHg) CO2 gas pressure was employed during the procedure. All procedures were performed through a three-port retroperitoneal approach without opening the peritoneal cavity. The hand-assisted technique was not used. One hundred and twenty-seven cases were of left and eight cases were of right nephrectomy. Donor nephrectomy was carried out successfully in all patients. In one donor, the procedure was changed to open donor nephrectomy because of severe adhesion around the renal vein due to previous surgery. No serious complications, such as massive bleeding or bowel injury were encountered. Return of bowel function took 0.7 days on average. Post-operative hospital stay was 4.9 days on average, and return to work was 12 days on average. Ureteral complications occurred in 2 patients and were treated with temporally retrograde ureteral stenting. Average serum creatinine levels were 1.5 mg/dL, 1.3 mg/dL and 1.3 mg/dL at 3, 7 and 14 days after transplantation, respectively. No patients required hemodialysis after transplantation due to acute tubular necrosis. RPLDN could be an option for laparoscopic live donor nephrectomy.

In Japan, the complement-dependent cytotoxicity (CDC-crossmatch) test and the anti-donor antibody flow cytometric assay (FCXM) are used to evaluate presensitization among transplantation candidates. We introduced the flow cytometric panel reactive antibody method (FlowPRA) at our institution, and in this paper, we compared the results of FCXM and FlowPRA. Sera of a total of 238 patients receiving the first graft were analyzed by FlowPRA retrospectively. Specimens from 125 of these patients were also analyzed by FCXM, and the results obtained using the two methods were compared. In addition, postoperative pathological findings by graft biopsy were examined in patients with PRA class 1(+) or PRA class 2(+). (i) Class 1 antibodies were detected in 36 of the 238 patients (15%), class 2 antibodies in six patients (3%), and both class 1 and class 2 antibodies in five patients (2%). (ii) Totally 125 patients analyzed by both FCXM and FlowPRA, 28 patients (22%) who tested negative by FCXM were, however, found to be positive by FlowPRA, and 16 of these 28 patients (57%) had shown evidence of humoral rejection suspected of antibody-mediated in the early postoperative stage. A large proportion of patients who tested negative by FCXM but positive by FlowPRA experienced rejection. Thus, for detecting 'high responders' in patients receiving the first graft, use of FlowPRA to detect antibodies may be superior to that of FCXM.

Background: The 26S proteasome is the proteolytic machinery of the ubiquitin-dependent proteolytic system responsible for most of the regulated intracellular protein degradation in eukaryotic cells. Previously, we demonstrated meiotic cell cycle dependent phosphorylation of alpha4 subunit of the 26S proteasome. In this study, we analyzed the changes in the spotting pattern separated by 2-D gel electrophoresis of alpha subunits during Xenopus oocyte maturation., Results: We identified cDNA for three alpha-type subunits (alpha1, alpha5 and alpha6) of Xenopus, then prepared antibodies specific for five subunits (alpha1, alpha3, alpha5, alpha6, and alpha7). With these antibodies and previously described monoclonal antibodies for subunits alpha2 and alpha4, modifications to all alpha-type subunits of the 26S proteasome during Xenopus meiotic maturation were examined by 2D-PAGE. More than one spot for all subunits except alpha7 was identified. Immunoblot analysis of 26S proteasomes purified from immature and mature oocytes showed a difference in the blots of alpha2 and alpha4, with an additional spot detected in the 26S proteasome from immature oocytes (in G2-phase)., Conclusions: Six of alpha-type subunits of the Xenopus 26S proteasome are modified in Xenopus immature oocytes and two subunits (alpha2 and alpha4) are modified meiotic cell cycle-dependently.

BK polyomavirus (BKV) is ubiquitous in the human population, infecting children without obvious symptoms, and persisting in the kidney in a latent state. In immunosuppressed patients, BKV is reactivated and excreted in urine. BKV isolates have been classified into four subtypes (I-IV) using either serological or genotyping methods. To elucidate the subtypes of BKV prevalent in Japan, the 287 bp typing region in the viral genome was PCR-amplified from urine samples of 45 renal transplant (RT) and 31 bone-marrow transplant (BMT) recipients. The amplified fragments were subjected to a phylogenetic or RFLP analysis to determine the subtypes of BKV isolates in urine samples. Subtypes I, II, III and IV were detected, respectively, in 70-80, 0, 2-3 and 10-20 % of the BKV-positive patients in both patient groups. This pattern of distribution was virtually identical to patterns previously demonstrated in England, Tanzania and the United States, suggesting that BKV subtypes are distributed similarly in various human populations. Furthermore, transcriptional control regions (TCRs) were PCR-amplified from the urine samples of 25 RT and 20 BMT recipients, and their nucleotide sequences were determined. The basic TCR structure (the so-called archetype configuration) was observed in most isolates belonging to subtypes I, III and IV (subtype II isolates were not available), albeit with several nucleotide substitutions and a few single-nucleotide deletions (or insertions). Only three TCRs carried extensive sequence rearrangements. Thus, it was concluded that the archetypal configuration of the BKV TCR has been conserved during the evolution of BKV.

An endocrine-disrupting chemical, diethylstilbestrol (DES), a nonsteroidal estrogen, triggers oocyte maturation in fish. The morphology (the time course of the change in germinal vesicle breakdown) and an intracellular molecular event (the de novo synthesis of cyclin B) induced by DES were indistinguishable from those induced by a natural maturation-inducing hormone, 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-DHP). A synergistic action of DES on 17,20beta-DHP-induced oocyte maturation was observed. Both 17,20beta-DHP- and DES-induced oocyte maturation was inhibited by an antibody against the maturation-inducing hormone receptor. The structural requirement for the action of DES is discussed based on results obtained with DES analogs.