Biomedical subjects

Yoshitaka Nagahama

Publications and source records attributed to Yoshitaka Nagahama.

At least 19 recordsLinked to original sources

PubMed2006

Foxl2 up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with ad4 binding protein/steroidogenic factor 1.

Increasing evidence suggests the crucial role of estrogen in ovarian differentiation of nonmammalian vertebrates including fish. The present study has investigated the plausible role of Foxl2 in ovarian differentiation through transcriptional regulation of aromatase gene, using monosex fry of tilapia. Foxl2 expression is sexually dimorphic, like Cyp19a1, colocalizing with Cyp19a1 and Ad4BP/SF-1 in the stromal cells and interstitial cells in gonads of normal XX and sex-reversed XY fish, before the occurrence of morphological sex differentiation. Under in vitro conditions, Foxl2 binds to the sequence ACAAATA in the promoter region of the Cyp19a1 gene directly through its forkhead domain and activates the transcription of Cyp19a1 with its C terminus. Foxl2 can also interact through the forkhead domain with the ligand-binding domain of Ad4BP/SF-1 to form a heterodimer and enhance the Ad4BP/SF-1 mediated Cyp19a1 transcription. Disruption of endogenous Foxl2 in XX tilapia by overexpression of its dominant negative mutant (M3) induces varying degrees of testicular development with occasional sex reversal from ovary to testis. Such fish display reduced expression of Cyp19a1 as well as a drop in the serum levels of 17beta-estradiol and 11-ketotestosterone. Although the XY fish with wild-type tilapia Foxl2 (tFoxl2) overexpression never exhibited a complete sex reversal, there were significant structural changes, such as tissue degeneration, somatic cell proliferation, and induction of aromatase, with increased serum levels of 17beta-estradiol and 11-ketotestosterone. Altogether, these results suggest that Foxl2 plays a decisive role in the ovarian differentiation of the Nile tilapia by regulating aromatase expression and possibly the entire steroidogenic pathway.

Animals↗

PubMed2006

Knock-down of DMY initiates female pathway in the genetic male medaka, Oryzias latipes.

DMY is the second vertebrate sex-determining gene identified from the fish, Oryzias latipes. In this study, we used two different ways of sex reversal, DMY knock-down and estradiol-17beta (E2) treatment, to determine the possible function of DMY during early gonadal sex differentiation in XY medaka. Our findings revealed that the mitotic and meiotic activities of the germ cells in the 0 day after hatching (dah) DMY knock-down XY larvae were identical to those of the normal XX larvae, suggesting the microenvironment of these XY gonads to be similar to that of the normal XX gonad, where DMY is naturally absent. Conversely, E2 treatment failed to initiate mitosis in the XY gonad, possibly due to an active DMY, even though it could initiate meiosis. Present study is the first to prove that the germ cells in the XY gonad can resume the mitotic activity, if DMY was knocked down.

Animals↗

PubMed2006

Molecular cloning and analysis of gonadal expression of Foxl2 in the medaka, Oryzias latipes.

Foxl2 is a member of the winged helix/forkhead family of transcription factors and is known to be involved in ovarian development in some vertebrates. To address the role of Foxl2 in ovarian differentiation in medaka, we isolated Foxl2 cDNA and analyzed its expression patterns during sex differentiation. Expression of Foxl2 started in somatic cells surrounding germ cells in XX gonads, just after initiation of ovarian differentiation, and was maintained in granulosa cells throughout ovarian development. In the adult ovary, Foxl2 was expressed in previtellogenic and vitellogenic follicles, but expression ceased in postvitellogenic follicles. In contrast, Foxl2 mRNA could not be detected in testes. In addition, Foxl2 and aromatase mRNAs were co-localized in some somatic cells located on the ventral side of developing XX gonads. Our results suggested that Foxl2 was not involved in ovarian determination, but was involved in differentiation of granulosa cells in medaka.

Amino Acid Sequence↗

PubMed2006

Phosphorylation of the p34(cdc2) target site on goldfish germinal vesicle lamin B3 before oocyte maturation.

The nuclear membranes surrounding fish and frog oocyte germinal vesicles (GVs) are supported by the lamina, an internal, mesh-like structure that consists of the protein lamin B3. The mechanisms by which lamin B3 is transported into GVs and is assembled to form the nuclear lamina are not well understood. In this study, we developed a heterogeneous microinjection system in which wild-type or mutated goldfish GV lamin B3 (GFLB3) was expressed in Escherichia coli, biotinylated, and microinjected into Xenopus oocytes. The localization of the biotinylated GFLB3 was visualized by fluorescence confocal microscopy. The results of these experiments indicated that the N-terminal domain plays important roles in both nuclear transport and assembly of lamin B3 to form the nuclear lamina. The N-terminal domain includes a major consensus phosphoacceptor site for the p34(cdc2) kinase at amino acid residue Ser-28. To investigate nuclear lamin phosphorylation, we generated a monoclonal antibody (C7B8D) against Ser-28-phosphorylated GFLB3. Two-dimensional (2-D) electrophoresis of GV protein revealed two major spots of lamin B3 with different isoelectric points (5.9 and 6.1). The C7B8D antibody recognized the pI-5.9 spot but not the pI-6.1 spot. The former spot disappeared when the native lamina was incubated with lambda phage protein phosphatase (lambda-PP), indicating that a portion of the lamin protein was already phosphorylated in the goldfish GV-stage oocytes. GFLB3 that had been microinjected into Xenopus oocytes was also phosphorylated in Xenopus GV lamina, as judged by Western blotting with C7B8D. Thus, lamin phosphorylation appears to occur prior to oocyte maturation in vivo in both these species. Taken together, our results suggest that the balance between phosphorylation by interphase lamin kinases and dephosphorylation by phosphatases regulates the conformational changes in the lamin B3 N-terminal head domain that in turn regulates the continual in vivo rearrangement and remodeling of the oocyte lamina.

Active Transport, Cell Nucleus↗

PubMed2006

Promoter activity and chromosomal location of the Rana rugosa P450 aromatase (CYP19) gene.

Sex is determined genetically in amphibians, but is reversed occasionally by steroid hormones. The phenotypic sex of some amphibian species can be reversed from male to female by estrogens. Estrogens, which are synthesized from testosterone irreversibly by the enzyme P450 aromatase (CYP19), are essential for ovarian development in vertebrates. CYP19 expression is reportedly regulated by steroidogenic factor-1 (SF-1), also designated as Ad4BP, in fish and mammals. However, it is unknown if this is also the case in amphibians. Thus, to elucidate the role of SF-1 in CYP19 gene expression in the gonad of amphibians, it is necessary to isolate and characterize the promoter region of the CYP19 gene of amphibians. For this purpose, we first cloned the promoter region of CYP19 from genomic DNA fragments of the frog Rana rugosa. As a result, a potential binding site of SF-1 was found in the region. When a luciferase promoter assay in HEK 293 cells was carried out to examine the ability of SF-1 as a transcriptional regulator, we found that R. rugosa SF-1 stimulated the expression of the CYP19 gene of the tilapia Oreochromis niloticus, but not that of the frogs R. rugosa and Xenopus laevis. RT-PCR analysis revealed that CYP19 mRNA was expressed at a higher level in the indifferent gonads of females than in those of males. This was also true to SF-1 mRNA In addition, FISH analysis showed that the CYP19 gene was located on chromosome 3 of R. rugosa. Taken together, our data suggest that CYP19, an autosomal gene, is expressed in the undifferentiated gonads to an extent greater in females than in males, but its expression probably is not regulated by SF-1 alone. Another factor(s) may be required if SF-1 promotes the expression of the CYP19 gene in R. rugosa as it does in fish and mammals.

Animals↗

PubMed2005

Induction and inhibition of oocyte maturation by EDCs in zebrafish.

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.

Animals↗

PubMed2005

Forebrain gonadotropin-releasing hormone neuronal development: insights from transgenic medaka and the relevance to X-linked Kallmann syndrome.

Neurons that synthesize and release GnRH are essential for the central regulation of reproduction. Evidence suggests that forebrain GnRH neurons originate in the olfactory placode and migrate to their final destinations, although this is still a matter of controversy. X-linked Kallmann syndrome (X-KS), characterized by failed gonadal function secondary to deficient gonadotropin secretion, is caused by a mutation in KAL1, which is suggested to regulate the migration of forebrain GnRH neurons. Because rodents lack Kal1 in their genome and have GnRH neurons scattered throughout their forebrain, the development of forebrain GnRH neurons and the pathogenesis of X-KS have been difficult to study. In the present study, we generated transgenic medaka that expressed green fluorescent protein under the control of the gnrh1 and gnrh3 promoters for analyzing forebrain GnRH neuronal development. Our data revealed the presence of the following four gnrh1 neuronal populations: an olfactory region-derived ventral preoptic population, a dorsal preoptic population that migrates from the dorsal telencephalon, a medial ventral telencephalic population that migrates from the anterior telencephalon, and a nonmigratory ventral hypothalamic population. We found that all forebrain gnrh3 neurons, extending from the terminal nerve ganglion to the anterior mesencephalon, arise from the olfactory region and that trigeminal ganglion neurons express gnrh3. Maternal gnrh3 expression was also observed in oocytes and early embryos. We subsequently identified a KAL1 ortholog and its paralogous form in the medaka. Consistent with the X-KS phenotype, antisense knockdown of the medaka KAL1 ortholog resulted in the disruption of forebrain GnRH neuronal migration. Thus, these transgenic medaka provide a useful model system for studying GnRH neuronal development and disorders of GnRH deficiency.

Animals↗

PubMed2005

Cloning and identification of a membrane progestin receptor in goldfish ovaries and evidence it is an intermediary in oocyte meiotic maturation.

Previously, a cDNA clone encoding a protein that satisfies the criteria for its designation as a membrane progestin receptor, mPRalpha, was discovered in spotted seatrout ovaries. Moreover, preliminary evidence was obtained for a role for mPRalpha in maturation-inducing hormone (MIH) induction of oocyte maturation in this species. Here, we describe the cloning of the mPRalpha cDNA from a goldfish ovarian cDNA library. Northern blot analysis indicates the presence of a major 2.6kb transcript in ovaries that encodes a 354 amino acid protein which shows high sequence identity with seatrout (81%), zebrafish (93%), and human (55%) mPRalphas. Western blot analysis using a polyclonal goldfish mPRalpha antibody shows a major immunoreactive band of the predicted molecular weight (40kDa) in goldfish ovarian membranes. Computer modeling predicts that the deduced protein has seven transmembrane domains, typical of G protein-coupled receptors. Treatment of full grown, late vitellogenic stage follicle-enclosed oocytes in vitro with gonadotropin increased mPRalpha protein levels. A correlation between mPRalpha protein levels and the ability of oocytes to undergo GVBD in response to the MIH (maturational competence) was observed after treatment with gonadotropin. Microinjection of goldfish oocytes with a morpholino antisense oligonucleotide to mPRalpha blocked both the induction of oocyte maturational competence and mPRalpha protein upregulation by gonadotropin. These results with the goldfish mPRalpha protein are similar to those obtained previously with spotted seatrout, further supporting the hypothesis that the mPRalpha acts as an intermediary in MIH induction of oocyte maturation in teleosts.

Amino Acid Sequence↗

PubMed2005

Suppression of steroidogenic enzyme expression during androgen-induced sex reversal in Nile tilapia (Oreochromis niloticus).

Exogenous sex steroids markedly alter sex differentiation in fish. The endocrine and molecular mechanisms involved in these changes remain unclear. To further clarify the mechanism of androgen-induced testicular differentiation, we treated female tilapia Oreochromis niloticus with methyltestosterone (MT at a dose of 50 microg/g diet) and examined the expression of P450 cholesterol-side-chain-cleavage, 3beta-hydroxysteroid dehydrogenase, and cytochrome P450 aromatase (P450arom) in the gonads. MT treatment resulted in 100% masculinization. Untreated fish showed normal ovarian differentiation with strong expression of all three steroidogenic enzymes. In gonads of MT-treated fish, expression of all three steroidogenic enzymes was attenuated within 15 days and completely disappeared within 30 days of treatment. Our results indicate that exogenous androgen treatment suppresses the expression of key steroidogenic enzymes, including P450arom throughout sex differentiation in tilapia, thus masculinizing the animal. Whether the absence of aromatase or the presence of androgens is responsible for testicular differentiation remains to be determined.

3-Hydroxysteroid Dehydrogenases↗

PubMed2005

Whole-cell electrophysiology of gonadotropin-releasing hormone neurons that express green fluorescent protein in the terminal nerve of transgenic medaka (Oryzias latipes).

Gonadotropin-releasing hormone (GnRH) controls reproduction in vertebrates. Most studies have focused on the population of GnRH neurons in the hypothalamus that ultimately controls gonadal function. However, all vertebrates studied to date have two to three anatomically distinct populations of GnRH neurons that express different forms of this hormone. The purpose of the present study was to develop a new model for studying the population of GnRH neurons in the terminal nerve (TN) associated with the olfactory bulb and then to characterize their pattern of action potential firing to provide a foundation for understanding the role of these neurons in regulating reproduction. A stable line of transgenic medaka (Oryzias latipes) was generated in which a DNA construct containing the salmon GnRH (Gnrh3) promoter linked to green fluorescent protein (GFP) was expressed in TN-GnRH3 neurons. This population of GnRH neurons is located at or near the ventral surface of the brain, making them ideally situated for electrophysiological analysis. Whole-cell and loose-patch recordings in current-clamp mode were performed on these neurons from excised, intact brains of adult males in which afferent and efferent neural connections remained intact. All TN-GnRH3-GFP neurons that we recorded showed a beating pattern of spontaneous action potential firing. Action potentials were blocked by tetrodotoxin, indicating they are generated by a voltage-sensitive Na+ current; however, an oscillation in subthreshold membrane potential persisted. The present results indicate that this transgenic fish will provide an excellent model for studying the cell physiology of an extrahypothalamic population of GnRH neurons.

Action Potentials↗

PubMed2005

Changes in steroidogenic enzyme and steroidogenic acute regulatory protein messenger RNAs in ovarian follicles during ovarian development of rainbow trout (Oncorhynchus mykiss).

In salmonid fishes, estradiol-17beta (E2) and 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) are the major steroid hormones controlling oocyte growth (vitellogenesis) and final maturation (resumption of meiosis). The aim of this study was to determine changes in mRNAs encoding ovarian steroidogenic enzymes and steroidogenic acute regulatory protein (StAR) during ovarian development in female rainbow trout. We analyzed the levels of mRNAs encoding the enzymes P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17alpha-hydroxylase/C17-C20 lyase (P450c17), aromatase (P450arom), and carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase (20beta-HSD), and StAR in developing ovarian follicles of rainbow trout by Northern blot, in relation to the pattern of serum E2 and 17,20beta-P levels. Serum E2 levels were elevated during vitellogenesis and decreased prior to an ovulatory increase in 17,20beta-P. Transcripts for P450scc and P450c17 increased in late vitellogenic follicles, then decreased in post-ovulatory follicles. In contrast, P450arom transcripts were abundant during vitellogenesis and then declined as vitellogenesis was completed and were barely detectable in post-ovulatory follicles. 3beta-HSD mRNA levels increased in late vitellogenic follicles and were maintained at high levels in post-ovulatory follicles. 20beta-HSD and StAR mRNA levels were very low during vitellogenesis, and then strongly increased during late vitellogenesis to a peak in post-ovulatory follicles. These results indicate that the expression of genes encoding steroidogenic enzymes and StAR change dynamically, dependent on the developmental stages of rainbow trout follicles. The acquisition of the ability of later stage follicles to rapidly produce large quantities of 17,20beta-P appears to be supported by a preparatory increase in mRNAs encoding StAR and other steroidogenic enzymes.

3-Hydroxysteroid Dehydrogenases↗

PubMed2005

Testicular type Sox9 is not involved in sex determination but might be in the development of testicular structures in the medaka, Oryzias latipes.

Testicular type Sox9 is the most upstream conserved gene in the sex determining cascade among vertebrate. However, in medaka, only one Sox9 gene was identified as expressed in the ovary; no other Sox9 gene was reported expressed in the testis. We explored the medaka genome and cloned a novel testicular type Sox9 cDNA. Phylogenetic analysis revealed that both our isolated Sox9 and the already reportedly cloned medaka Sox9 belongs zebrafish Sox9a branch. Therefore, we named our gene Sox9a2. Unexpectedly, Sox9a2 mRNA was expressed in somatic cells surrounding germ cells at similar high levels in both sexes during early gonadal sex differentiation. However, at the initial stage of testicular tubules development, the expression of Sox9a2 was maintained only in XY gonads, and was remarkably reduced in XX gonads. These results suggest that Sox9a2 is not involved in early sex determination and differentiation, but is involved in the later development of testicular tubules in medaka.

Amino Acid Sequence↗

PubMed2005

Molecular cloning and expression of Ad4BP/SF-1 in the serial sex changing gobiid fish, Trimma okinawae.

Involvement of Ad4BP/SF-1 was investigated using the serial sex changing goby Trimma okinawae. First, a cDNA encoding Ad4BP/SF-1 was cloned from ovarian follicles. The open reading frame of goby Ad4BP/SF-1 encodes a protein of 489 amino acids. Ad4BP/SF-1 was expressed in gonadal tissues, brain, and kidney. Second, transcript levels of Ad4BP/SF-1 were measured in the ovary and testis of the same individuals. Expressions were related to sexual phases. Moreover, ovarian expression of Ad4BP/SF-1 increased during vitellogenesis and declined sharply during the post-vitellogenic period. Finally, the expression profile of Ad4BP/SF-1 was measured during serial sex change in the ovary. Ad4BP/SF-1 increased in parallel with the onset of the female-phase and decreased as female became male. Therefore, Ad4BP/SF-1 probably acts as an important indirect regulator of oocyte growth and maturation at female-phase of serial sex changing gobiid fish T. okinawae.

Amino Acid Sequence↗

PubMed2005

Two types of aromatase with different encoding genes, tissue distribution and developmental expression in Nile tilapia (Oreochromis niloticus).

We isolated a novel type of aromatase cDNA from a Nile tilapia (Oreochromis niloticus) ovary cDNA library. Because this aromatase is phylogenetically related to brain aromatase (CYP19b) of goldfish, zebrafish and sea bass, we named it tilapia CYP19b (tCYP19b). tCYP19b encodes a protein that is predicted to consist of 495 residues and have 63.8% homology with the aromatase (tCYP19a) we previously isolated from the same source. In vitro transient transfection of cultured COS7 cells demonstrated that tCYP19b codes a functional protein to catalyze estrogen production from an androgen substrate. RT-PCR and Northern hybridization analysis showed that tCYP19b was expressed at a high level in the brain and at a low level in a wide variety of other tissues, whereas tCYP19a was mainly present in the ovary and its level significantly increased during the vitellogenic stage. RT-PCR also detected tCYP19b expression in brain and gonad tissues of both female and male tilapia during sex differentiation, but tCYP19a was only found in the ovary of the fry at that period. These results suggest that tCYP19a plays a key role in sex differentiation and ovarian development. We also isolated genes of two tilapia aromatases. Based on the location of the transcription initiation site, we predicted that there is one promoter for tCYP19a and three promoters for tCYP19b. Although the two aromatase isoforms have similar gene structures in the coding region, we found that the binding regions of SF-1/Ad4 BP region, WT1-KTS and SRY, which are sex-determining factors in mammals, are present in the 5' flank region of tCYP19a but not tCYP19b. A similar situation is present in promoters of zebrafish and goldfish aromatase isoforms. This data indicates that CYP19a plays a decisive role in sex differentiation of those species. The unique presence of the ERE motif in the tCYP19b promoter and the high expression of tCYP19b in the brain support that CYP19b is mainly involved in estrogen-mediated neural estrogen synthesis.

Amino Acid Sequence↗

PubMed2005

Cloning of cDNAs and the differential expression of A-type cyclins and Dmc1 during spermatogenesis in the Japanese eel, a teleost fish.

We cloned A-type cyclins (cyclins A1 and A2) and Dmc1 cDNAs from the eel testis. Cyclin A1 mRNA was predominantly expressed in the livers, ovaries, and testes of the eels. In contrast to cyclin A1 mRNA, a very high expression of cyclin A2 mRNA was observed in the brains, livers, kidneys, spleens, ovaries, and testes of the eels. Dmc1 mRNA was predominantly expressed in the testes and ovaries; expression in the brain was also detected. In the eel testis, a few type-A spermatogonia incorporating 5-bromo-2'-deoxyuridine (BrdU) were seen before the initiation of spermatogenesis by hormonal induction. On day 1 after hormonal induction, the number of BrdU-labeled spermatogonia increased remarkably, and after 3 and 6 days, many labeled type-B spermatogonia were also observed. The expression of cyclin A2 increased 1 day after the induction of spermatogenesis and reached a plateau after 6 days, when many type-B spermatogonia with high proliferative activity were found. In contrast, the expression of cyclin A1 mRNA was detected after 9 days, coincident with the first appearance of spermatocytes. Cyclin A1 mRNA was localized in germ cells of all stages, from primary spermatocytes to round spermatids, whereas cyclin A2 mRNA was specifically localized in spermatogonia, secondary spermatocytes, round spermatids, and testicular somatic cells, including Sertoli cells. Dmc1 was localized only in the earlier stages of primary spermatocytes; before this stage, cyclin A1 mRNA was not detectable. Overall, cyclin A2, Dmc1, and cyclin A1 are expressed in spermatogenic cells sequentially before and during meiosis in the eel testis.

Animals↗

PubMed2005

Aromatase immunoreactivity and the role of enzymes in steroid pathways for inducing sex change in the hermaphrodite gobiid fish Trimma okinawae.

The role of aromatase (Arom) in the process of bi-directional sex change in the gobiid fish Trimma okinawae was investigated by immunohistochemical methods. Irrespective of sexual phase, gonads comprised both ovarian and testicular tissues. In each sexual phase of females, the 2nd (2DF-M) and 4th (4DF-M) days after initiation of sex change to male, males, and the 2nd (2DM-F), 4th (4DM-F) and 6th (6DM-F) days after the initiation of reversion from male to female, ovarian and testicular histological observations were made. During the female, 2DF-M, 4DF-M and 6DM-F phases, the ovary contained vitellogenic and previtellogenic oocytes, compared with previtellogenic oocytes in the other phases. Although sperm was found in the testis in every phase, sperm ducts were apparent in the male phase, but not the female phase. Arom immunoreactivity was detected in the interstitial cells between the oocytes in all phases. On the other hand, it was localized in the thecal and granulosa cells of the follicular layer enclosing the oocytes in the female, 2DF-M, 4DF-M and 6DM-F phases. Activity of Arom in the thecal and granulosa cells is thought to be important for the development of oocytes and subsequent sex change.

Animals↗

PubMed2005

Identification of a protein kinase which phosphorylates a subunit of the 26S proteasome and changes in its activity during meiotic cell cycle in goldfish oocytes.

The proteasome is involved in the progression of the meiotic cell cycle in fish oocytes. We reported that the alpha4 subunit of the 26S proteasome, which is a component of the outer rings of the 20S proteasome, is phosphorylated in immature oocytes and dephosphorylated in mature oocytes. To investigate the role of the phosphorylation, we purified the protein kinase from immature oocytes using a recombinant alpha4 subunit as substrate. A protein band which well corresponded to the kinase activity was identified as casein kinase Ialpha (CKIalpha). Two-dimensional (2D) PAGE analysis showed that part of the alpha4 subunit was phosphorylated by CKIalpha in vitro. This spot was detected in purified immature 26S proteasome but not in mature 26S proteasome, demonstrate that the alpha4 subunit is phosphorylated by CKIalpha meiotic cell cycle dependently.

Amino Acid Sequence↗

PubMed2005

Gonadal structure of the serial-sex changing gobiid fish Trimma okinawae.

In order to obtain basic information about the role played by endogenous sex hormones in bringing about sex changes in the serial-sex changing gobiid fish Trimma okinawae, the gonadal structure of male and female phases were observed histologically. Steroid-producing cells (SPC; Leydig cells in a testis) were observed ultrastructurally in the ovaries and testes of both female-phase and male-phase fish. In addition, gonadal expression of P450 cholesterol side-chain-cleavage (scc) was examined immunohistochemically. Gonads of fish in female and male phases were observed to have both ovaries and testes simultaneously. Female-phase fish had matured with many developed vitellogenic oocytes, while male-phase individuals had immature ovaries with many numbers of previtellogenic oocytes at the perinucleolus stage. Testes of fish in different sexual phases had active spermatogenic germ cells. Organellae of SPC in the ovaries of female-phase fish had active structures of steroid production. In contrast, SPC in the ovaries of male-phase fish did not show active structures of steroid production. Immunopositive reactions against the scc antibody in the ovaries of female-phase fish were very strong, but immunoreactions in the ovaries of male-phase fish were very weak. In the testis, moderate immunopositive signals were obtained from dual-phase male/females.

Animals↗