Aromatase inhibitor and 17alpha-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 17alpha-methyltestosterone induces sex-reversal as well as suppression of P450arom gene expression. The effect of fadrozole was counteracted by co-administration of estradiol-17beta. 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 17alpha-methyltestosterone) may be due to the suppression of P450arom gene expression and the resultant decrease in the amount of estrogen.

Suppression of P450 aromatase gene expression in sex-reversed males produced by rearing genetically female larvae at a high water temperature during a period of sex differentiation in the Japanese flounder (Paralichthys olivaceus).

The phenotypic sex of many teleost fishes including flounders can be experimentally altered by treating embryos or larvae with varied temperatures or sex-steroid hormones. To analyse the sex determination mechanism, especially the role of cytochrome P450 aromatase (P450arom), an enzyme that catalyses the conversion of androgens to estrogens, in temperature-dependent gonadal sex differentiation in the Japanese flounder, we generated two populations of larvae, both having XX (genetic females) but each growing up to display all phenotypic females or males, by rearing the larvae at normal (18 degrees C) or high (27 degrees C) water temperatures from days 30 to 100 after hatching respectively. The larvae (XX) were produced artificially by mating normal females (XX) with gynogenetic diploid males (XX) which had been sex-reversed to phenotypic males by 17alpha-methyltestosterone. To study the role of P450arom in sex determination in the flounder, we first isolated a P450arom cDNA containing the complete open reading frame from the ovary. RT-PCR showed that P450arom mRNA was highly expressed in the ovary and spleen but weakly in the testis and brain. Semi-quantitative analyses of P450arom mRNA in gonads during sex differentiation showed that there was no difference in the levels of P450arom mRNA between the female and male groups when the gonad was sexually indifferent (day 50 after hatching). However, after the initiation of sex differentiation (day 60), the mRNA levels increased rapidly in the female group, whereas they decreased slightly in the male group. Similarly, estradiol-17beta levels rose remarkably in the female group, yet remained constant in the male group. These results suggest that induction of sex reversal of genetically female larvae to phenotypic males by rearing them at a high water temperature caused a suppression of P450arom gene expression. Furthermore, we suggest that the maintenance of P450arom mRNA at very low levels is a prerequisite for testicular differentiation, while the increased levels are indispensable for ovarian differentiation.

A family with Liddle's syndrome caused by a new missense mutation in the beta subunit of the epithelial sodium channel.

Liddle's syndrome is an autosomal dominant form of salt sensitive hypertension caused by mutations in the beta or gamma subunit of the epithelial sodium channel. Systematic mutagenesis studies revealed that a conserved PPPXY sequence (PY motif) of the C-terminus of the alpha, beta, or gamma subunits might be involved in the regulation of the channel activity. However, only two missense mutations in the PY motif of the beta subunit have been reported to cause Liddle's syndrome. We sequenced the C-termini of the beta and gamma subunits of the epithelial sodium channel in a Japanese family clinically diagnosed as having Liddle's syndrome and found a new missense mutation in the PY motif of the beta subunit, P615S. Expression studies with P615S mutant in Xenopus oocytes resulted in an about 3-fold increase in the amiloride-sensitive sodium current compared to the wild type (p = 0.001). These findings provide further clinical evidence for the hypothesis that a conserved PY motif may be critically important for the regulation of the epithelial sodium channel.

Primary structures of sperm-specific basic nuclear proteins and gene expression in Japanese newt, Cynops pyrrhogaster.

Electrophoretic analyses of acid extracts from mature sperm of newt, Cynops pyrrhogaster, on acid/urea/Triton X-100 polyacrylamide gel showed the exclusive occurrence of sperm-specific nuclear basic proteins (SBPs), which moved faster than somatic histones on the gel. These SBPs were eluted separately by reversed phase-high-performance liquid chromatography as two large peaks and a few small peaks. Of these, only the small peaks disappeared with treatment of the acid extracts with alkaline phosphatase before they were injected into the column, so that there were only two distinct components: NP1 and NP2. Determination of amino acid sequences by the Edman method as well as by sequencing of cDNA for both components indicated that each protein consisted of 43 (NP1) or 48 (NP2) amino acid residues, rich in arginine residues (53.5% in NP1; 47.9% in NP2), forming the clusters. They had molecular masses of 5,386 Da (NP1) and 5,748 Da (NP2), respectively. Northern blot analysis using cDNAs as probes indicated that mRNAs for both NP1 and NP2 occurred not in primary spermatocytes but in round spermatids. In situ hybridization analyses using antisense RNA for NP1 as a probe clearly showed the first appearance of NP1 mRNA at the late stage of round spermatid.

Genesis of newt sperm axial fiber: cDNA cloning and expression of a 29 kDa protein, a major component of the axial fiber, during spermatogenesis.

Newt sperm has a unique structure: the tail consists of axial fiber, undulating membrane and flagellum. The genesis and chemical composition of the axial fiber remain unknown. The axial fiber consists of about 10 major components, as evidenced by SDS-polyacrylamide gel electrophoresis. In order to clarify the biochemical properties of the components of the axial fiber and study the mechanism of axial fiber formation, we focused our attention on a 29 kDa protein, the major constituent of the axial fiber. Immunofluorescent antibody technique showed that the 29 kDa protein was first expressed in the cytoplasm of early round spermatids but was expressed on fibers in the periphery of the cyst in late round spermatids. Double staining with tubulin antibody and 29 kDa antibody showed that the fibers around the cysts in early round spermatids were flagella alone but those in late round spermatids consisted of flagella and 29 kDa protein. These results indicated that 29 kDa proteins are synthesizsed in the cytoplasm of round spermatids and enter the preformed flagella in late round and elongated spermatids. A cDNA clone for 29 kDA protein was isolated. A database search could not find any homologous clones, indicating that the 29 kDa protein is a new one. Northern blot with the cDNA showed that mRNA for 29 kDa protein was highly expressed in round spermatids but barely in primary spermatocytes, indicating that the mRNA for 29 kDa protein is haploid-expressed.

Structure of genes for sperm-specific nuclear basic protein (SP4) in Xenopus laevis.

Nuclear basic proteins in sperm of Xenopus laevis consist of 6 sperm-specific proteins (SPs1-6) in addition to somatic core histones. Using a cDNA for SP4 as a probe, we cloned genomic DNA containing SP4 genes from a genomic library constructed from recombinant lambda bacteriophage containing 12.0 kbp-EcoRI digests of J-strain X. laevis liver DNA. Construction of restriction maps based on Southern blot analysis revealed the existence of a total of five SP4 genes which are arranged in a tandemly repeated array forming a cluster of simple multigenes per haploid genome, over a range of 18 kbp. Among these genes, the one located at the most upstream position differed from others in possessing a single base substitution which gave rise to a replacement of one out of 78 amino acid residues. The DNA containing the second to the fourth SP4 genes, arranged at about 3 kbp intervals each, was totally sequenced for 10,165 bp. Each gene was found to contain one intron, typical TATA and CCAAT boxes in the 5'-flanking region, and a polyadenylation signal in the 3'-flanking region. Comparative sequence analyses revealed three regions of extensive homology within the upstream non-coding region among three genes, suggesting a possible relevance to their expression at a particular phase of spermatogenesis and/or in testis.

Characteristic features of preleptotene spermatocytes in Xenopus laevis: increase in the nuclear volume and first appearance of flattened vesicles in these cells.

In Xenopus laevis, the spermatogenic cells derived from a primary spermatogonium divide synchronously and form a cyst surrounded by somatic Sertoli cells. To clarify how many times the spermatogonia divide mitotically before differentiating to primary spermatocytes, the number of spermatogenic cells in the cysts was counted. Each cyst which consisted of cells morphologically identical to secondary spermatogonia contained discrete numbers of cells such as 2(6), 2(7), or 2(8) cells. On the other hand, the number of the germ cells in a cyst of zygotene/pachytene primary spermatocytes was approximately 2(8). These results indicate that spermatogenic cells which had finished eight mitotic divisions and showed a similar morphology to secondary spermatogonia were preleptotene spermatocytes. The nuclei of the preleptotene spermatocytes increased in volume concomitantly with premeiotic DNA replication and differentiation into zygotene/pachytene primary spermatocytes. Therefore the cells morphologically indistinguishable from secondary spermatogonia but with larger nuclei than typical secondary spermatogonia were identified as preleptotene spermatocytes. Electron microscopic observations of the preleptotene spermatocytes identified as above showed that flattened vesicles, 0.1-0.5 micron in length, about 0.05 micron in width, and with more electron-dense membrane than typical endoplasmic reticulum, first appeared in the cytoplasmic area of preleptotene spermatocytes.

The occurrence of a gene-encoded variant of nuclear basic protein (SP4) in sperm of Xenopus laevis.

In our recent analyses [1] of five tandemly arranged genes encoding a major sperm-specific basic nuclear protein (SP4) of Xenopus laevis, we found a gene containing a single base substitution which will give rise to the replacement of the 69th residue among the 78 amino acids of SP4. In this study, the polypeptide from sperm nuclei which were separated by reversed-phase HPLC as a distinct entity from SP4 were collected for their peptide mapping with V8 protease and partial amino acid sequence analyses. It resulted that a polypeptide exhibiting an amino acid replacement at exactly the same position as predicted from a single base substitution of SP4 occurs in approximately one-fourth of the amount of SP4. This finding suggests that the relative amount of SP4 and its variant directly depends on the relative number of genes of SP4 and its variant.

Primary structure of toad sperm protamines and nucleotide sequence of their cDNAs.

Acid extract of mature sperm of the toad, Bufo japonicus, exclusively comprised sperm-specific basic proteins which moved faster than somatic histones on acid/urea/Triton X-100 polyacrylamide gel electrophoresis. When these proteins were purified by reversed-phase high-performance liquid chromatography they were found to consist of three components; one of these was a phosphorylated form of another, so that there were only two distinct components (P1 and P2). Amino acid sequence analyses indicated that the components both contained 39 amino acid residues, with 43.6% Arg, and differed only in the 28th amino acid residue (P1, Asp; P2, Glu). They had molecular masses of 5092 Da (P1) and 5106 Da (P2). The nucleotide sequences of cDNA clones encoding P1 (245 bases) and P2 (305 bases) showed that the difference in the amino acid residue between P1 and P2 was due to the difference of a nucleotide at position +87. Both cDNAs possessed a canonical signal (AATAAA) for polyadenylation and/or cleavage of transcript at the 3' untranslational region. Statistical analyses of amino acid sequence similarities suggested that the Bufo protamines are homologous with the protamines of fishes rather than with those of avian/mammalians.

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

Using 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.

Classification, inhibition, and specificity studies of the vitelline coat lysin from toad sperm.

The sonicated supernatant of the sperm of the toad, Bufo japonicus, can digest easily the vitelline coat (VC) of uterine eggs, and to a lesser extent the VC of coelomic eggs, but not that of activated eggs. The VC lysis and fertilization were competitively inhibited in the presence of t-butyloxycarbonyl-L-Gln-L-Arg-L-Arg-4-methylcoumaryl-7-amide (Boc-Gln-Arg-Arg-MCA), suggesting the involvement of proteases in the fertilization process. Starting from a sonicated supernatant, a potent VC lysin, possessing hydrolytic activity on Boc-Gln-Arg-Arg-MCA, was obtained by anion-exchange chromatography and gel filtration. The activity of the partially purified lysin was inhibited by diisopropyl fluorophosphate (DFP) and by such trypsin inhibitors as soybean trypsin inhibitor, leupeptin, and (p-amidinophenyl) methanesulfonyl fluoride hydrochloride, but not by chymostatin, E-64, and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The molecular weight of the lysin was estimated to be 32K, based on the fluorographic image of 3H-DFP binding to the lysin on sodium dodecyl sulfate gel electrophoresis. The VC lysin was most active at pH 7.0-7.6 and under low ionic strength equivalent to fresh water. The release of the VC lysin was induced upon incubation of sperm with the contents of oviducal pars recta granules (PRG), which are known to induce the acrosome reaction. We conclude that the protease studied here represents the VC lysin of toad sperm that is involved in fertilization by digesting the VC of uterine eggs, probably released as a result of the acrosome reaction induced by PRG.

Detection of acrosome-reacted toad sperm based on specific lectin binding to the inner acrosomal membrane.

When the sperm of the toad Bufo japonicus were treated with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA), soybean agglutinin (SBA), or Dolichos biflorus agglutinin (DBA), a few sperm fluoresced at the acrosomal region. The number of sperm showing this lectin binding to the acrosome increased significantly upon mild sonication of the sperm suspension. Electron microscopy revealed that ferritin-conjugated PNA bind not to the outer acrosomal and overlying plasma membranes, but specifically to the surface of the inner acrosomal membrane exposed by sonication. Both the percentage of FITC-PNA-labeled sperm and the activity of vitelline coat lysin released by sperm increased in good correlation with increasing sonication time, although the PNA-labeled sperm decreased in number upon longer sonication. These results indicate that the binding of FITC-PNA to the sperm provides a reliable measure of the acrosome reaction of Bufo sperm.