Kazufumi Takamune

Aromatase inhibitor and 17α-methyltestosterone cause sex-reversal from genetical females to phenotypic males and suppression of P450 aromatase gene expression in Japanese flounder (Paralichthys olivaceus)

The sex of Japanese flounder (Paralichthys olivaceus) is easily altered by water temperature or sex steroid hormone treatment during the period of sex determination. We have previously shown that rearing the genetically female larvae at high water temperature caused the suppression of P450 aromatase (P450arom) gene expression in the gonad and phenotypic sex‐reversal of the individuals to males (Kitano et al. 1999. J Mol Endocrinol 23:167–176). In the present study, we show that treatment of genetically female larvae with fadrozole (aromatase inhibitor) or 17α‐methyltestosterone induces sex‐reversal as well as suppression of P450arom gene expression. The effect of fadrozole was counteracted by co‐administration of estradiol‐17β. Effective periods for fadrozole treatment to induce sex‐reversal were similar to those for high water temperature treatment. RT‐PCR did not detect P450arom mRNA in gonad of the sex‐reversed, phenotypic males. These results indicate that sex‐reversal of the genetically female larvae by aromatase inhibitor (or 17α‐methyltestosterone) may be due to the suppression of P450arom gene expression and the resultant decrease in the amount of estrogen. Mol. Reprod. Dev. 56:1–5, 2000.

Tdrd1/Mtr-1, a tudor-related gene, is essential for male germ-cell differentiation and nuage/germinal granule formation in mice

Embryonic patterning and germ-cell specification in mice are regulative and depend on zygotic gene activities. However, there are mouse homologues of Drosophila maternal effect genes, including vasa and tudor, that function in posterior and germ-cell determination. We report here that a targeted mutation in Tudor domain containing 1/mouse tudor repeat 1 (Tdrd1/Mtr-1), a tudor-related gene in mice, leads to male sterility because of postnatal spermatogenic defects. TDRD1/MTR-1 predominantly localizes to nuage/germinal granules, an evolutionarily conserved structure in the germ line, and its intracellular localization is downstream of mouse vasa homologue/DEAD box polypeptide 4 (Mvh/Ddx4), similar to Drosophila vasa-tudor. Tdrd1/Mtr-1 mutants lack, and Mvh/Ddx4 mutants show, strong reduction of intermitochondrial cement, a form of nuage in both male and female germ cells, whereas chromatoid bodies, another specialized form of nuage in spermatogenic cells, are observed in Tdrd1/Mtr-1 mutants. Hence, intermitochondrial cement is not a direct prerequisite for oocyte development and fertility in mice, indicating differing requirements for nuage and/or its components between male and female germ cells. The result also proposes that chromatoid bodies likely have an origin independent of or additional to intermitochondrial cement. The analogy between Mvh-Tdrd1 in mouse spermatogenic cells and vasa-tudor in Drosophila oocytes suggests that this molecular pathway retains an essential role(s) that functions in divergent species and in different stages/sexes of the germ line.

Mouse Tudor Repeat-1 (MTR-1) is a novel component of chromatoid bodies/nuages in male germ cells and forms a complex with snRNPs.

Characteristic ribonucleoprotein-rich granules, called nuages, are present in the cytoplasm of germ-line cells in many species. In mice, nuages are prominent in postnatal meiotic spermatocytes and postmeiotic round spermatids, and are often called chromatoid bodies at the stages. We have isolated Mouse tudor repeat-1 (Mtr-1) which encodes a MYND domain and four copies of the tudor domain. Multiple tudor domains are a characteristic of the TUDOR protein, a component of Drosophila nuages. Mtr-1 is expressed in germ-line cells and is most abundant in fetal prospermatogonia and postnatal primary spermatocytes. The MTR-1 protein is present in the cytoplasm of prospermatogonia, spermatocytes, and round spermatids, and predominantly localizes to chromatoid bodies. We show that (1) an assembled form of small nuclear ribonucleoproteins (snRNPs), which usually function as spliceosomal complexes in the nucleus, accumulate in chromatoid bodies, and form a complex with MTR-1, (2) when expressed in cultured cells, MTR-1 forms discernible granules that co-localize with snRNPs in the cell plasm during cell division, and (3) the deletion of multiple tudor domains in MTR-1 abolishes the formation of such granules. These results suggest that MTR-1, which would provide novel insights into evolutionary comparison of nuages, functions in assembling snRNPs into cytoplasmic granules in germ cells.

Cloning and sequencing of an Escherichia coli K12 gene which encodes a polypeptide having similarity to the human ferritin H subunit.

SummaryUsing lambda phage clones containing segments of the Escherichia coli K12 chromosome as hybridization probes, we found one gene at 42 min on the E. coli chromosome map, the expression of which was affected by RNase III. The sequence of the DNA fragment containing this gene (gen-165) revealed the presence of an open reading frame encoding a polypeptide of 165 amino acid residues. The amino acid sequence deduced from the nucleotide sequence exhibited a remarkable similarity to that of the human ferritin H chain.

Nuclear Basic Proteins of Xenopus laevis Sperm: Their Characterization and Synthesis during Spermatogenesis

Nuclear basic proteins from morphologically and functionally mature sperm of Xenopus laevis were analyzed by acid/urea/Triton X‐100 polyacrylamide gel electrophoresis (AUT‐PAGE). Six sperm‐specific proteins (SP1‐6) were identified in addition to somatic histones H3, H4 and smaller amount of H2A and H2B, but not H1. Of these, SP3–6 were unique in containing 33–41% arginine and having very low lysine/arginine ratios, while SP2 was more similar to H3 and H4 in having a lower arginine and higher lysine content. Fractionations of testicular cells at different spermatogenic stages by unit gravity sedimentation showed that primary spermatocytes and acrosomal vesicle spermatids possess typical somatic type histones but no SPs. Injection of [14C]‐arginine into the testis and its tracing by fluorography on AUT‐PAGE gels indicated that all somatic histones are synthesized during the stages between spermatogonia and primary spermatocytes, whereas SPs are synthesized at differentially regulated rates during the stages after acrosomal vesicle formation. In indirect immunofluorescence studies with anti‐SP3‐5 rabbit antiserum, a positive reaction was observed in the last step of spermiogenesis after the commencement of nuclear coiling.

The Properties of the Oviducal Pars Recta Protease which Mediates Gamete Interaction by Affecting the Vitelline Coat of a Toad Egg

SDS‐PAGE analyses of the vitelline coats (VCs) of coelomic eggs (CEVC) and uterine eggs (UEVC) of Bufo japonicus revealed that the UEVC lacks the 40K–52K molecular weight components present in the CEVC; this is concomitant with the increased stainability of a 39K component and the appearance of a 36K component. These macromolecular alterations, accompanied by the acquisition of egg fertilizability, were induced when coelomic eggs were treated with the contents of secretory granules obtained from the oviducal pars recta (PRG). Gel‐filtration of PRG in combination with hydrolytic assays employing either fluorescamine‐labeled CEVC or a variety of synthetic substrates showed that the CEVC to UEVC alterations are ascribable to the action of a protease hydrolyzing specifically peptidyl‐Arg‐MCAs in a highly Ca2+‐dependent way. This enzyme, which has an optimal pH of 8.0–8.2, is inhibited by soybean trypsin inhibitor and leupeptin, as well as by such serine protease inhibitors as DFP and p‐APMSF. On the basis of a SDS‐PAGE analysis, its molecular weight is estimated to be 66K. Treatment of coelomic eggs with the partially purified PR protease did not render the eggs fertilizable, although CEVC to UEVC macromolecular alterations were effected. We conclude that the action of this oviducal protease in partially hydrolyzing the VC is a prerequisite but insufficient in itself to render the coelomic eggs fully accessible to a fertilizing sperm.

Transgenic frogs expressing the highly fluorescent protein Venus under the control of a strong mammalian promoter suitable for monitoring living cells

To easily monitor living cells and organisms, we have created a transgenic Xenopus line expressing Venus, a brighter variant of yellow fluorescent protein, under the control of the CMV enhancer/chicken β‐actin (CAG) promoter. The established line exhibited high fluorescent intensity not only in most tissues of tadpoles to adult frogs but also in germ cells of both sexes, which enabled three‐dimensional imaging of fluorescing organs from images of the serial slices of the transgenic animals. Furthermore, by using this transgenic line, we generated chimeric animals by brain implantation and importantly, we found that the brain grafts survived and expressed Venus in recipients after development, highlighting the boundary between fluorescent and nonfluorescent areas in live animals. Thus, Venus‐expressing transgenic frogs, tadpoles, and embryos would facilitate their use in many applications, including the tracing of the fluorescent cells after tissue/organ transplantation. Developmental Dynamics 233:562–569, 2005.

Involvement of Xtr (Xenopus tudor repeat) in microtubule assembly around nucleus and karyokinesis during cleavage in Xenopus laevis

We have previously shown that the transcriptional product of the novel gene, Xenopus tudor repeat (Xtr), occurred exclusively in germline cells and early embryonic cells and that the putative Xtr contained plural tudor domains which are thought to play a role in the protein–protein interactions. To understand the role of Xtr, we produced an antibody against a polypeptide containing Xtr tudor domains as an antigen and investigated the distribution and the function of the Xtr. Immunoprecipitation/Western blot and immunohistochemical analyses indicated a similar occurrence of the Xtr to the mRNA except for a slightly different profile of its amount during spermatogenesis. In spite of a large amount of Xtr mRNA at late‐secondary spermatogonial stage, the amount of Xtr was kept at a low level until this stage and increased after entering into the meiotic phase. Depletion of the Xtr function in the activated eggs by injection of the anti‐Xtr antibody caused the inhibition both of microtubule assembly around nucleus and of karyokinesis progression after prophase, but not of the oscillation of H1 kinase activity. These results suggest that the karyokinesis of at least early embryonic cells are regulated by unique mechanisms in which the Xtr is involved.

Functional demonstration of the ability of a primary spermatogonium as a stem cell by tracing a single cell destiny in Xenopus laevis

In Xenopus, although primary spermatogonium (PG), the largest cell in the testis, is believed to be spermatogonial stem cell by histological observations, functional evidence has never been obtained. In the present study, we first indicated that culture of juvenile testis in a medium supplemented with follicle stimulating hormone resulted in no proliferation of PG. In this culture system, early secondary spermatogonia could undergo mitotic divisions with a concomitant decrease in their size, so that they became distinguishable in size from PG. Because the subcutaneous environment of juveniles permitted aggregates of the dissociated testicular cells to reconstruct the normal testis structure, we next inserted a genetically marked PG isolated from cultured testes into the aggregate and transplanted it subcutaneously. In this system, 73.9% of the aggregates contained a marked PG. When we observed the aggregates 12 weeks after transplantation, most aggregates (70.0%) contained marked PG that had self‐renewed. Among these, fully growing aggregates contained many spermatogenic cells at the later developmental stage. These results suggested that isolated PG from the cultured testes had the ability as stem cells, and that purification of the spermatogenic stem cells became reliable in Xenopus.

Genes for Sperm‐specific Basic Nuclear Proteins in Bufo and Xenopus Are Expressed at Different Stages in Spermatogenesis

The expression of the genes for sperm‐specific basic nuclear proteins was examined, using the cDNA clones encoding protamine (P2) of Bufo japonicus and SP4 of Xenopus laevis as probes. Northern analyses showed that the mRNAs for these proteins were present only in the testes. Analyses with total RNA extracted from testicular cells at various spermatogenic stages revealed that in Bufo the transcripts of protamine genes are present in the spermatids, while in Xenopus the mRNAs for SP4 are present in both primary spermatocytes and spermatids. In situ hybridization studies with radiolabeled antisense RNA probes generated from cDNAs indicated that the Bufo protamine mRNAs accumulated first in round spermatids, while the Xenopus SP4 mRNAs did so in the pachytene stage of primary spermatocytes and thereafter.