Kaoru Ohno

A blue-light-activated adenylyl cyclase mediates photoavoidance in Euglena gracilis.

Blue light regulates processes such as the development of plants and fungi and the behaviour of microbes. Two types of blue-light receptor flavoprotein have been identified: cryptochromes, which have partial similarity to photolyases, and phototropins, which are photoregulated protein kinases. The former have also been found in animals with evidence of essential roles in circadian rhythms. Euglena gracilis, a unicellular flagellate, abruptly changes its swimming direction after a sudden increase or decrease in incident blue light intensity, that is, step-up or step-down photophobic responses, resulting in photoavoidance or photoaccumulation, respectively. Although these photobehaviours of Euglena have been studied for a century, the photoreceptor molecules mediating them have remained unknown. Here we report the discovery and biochemical characterization of a new type of blue-light receptor flavoprotein, photoactivated adenylyl cyclase, in the photoreceptor organelle of Euglena gracilis, with molecular genetic evidence that it mediates the step-up photophobic response.

A relaxin-like peptide purified from radial nerves induces oocyte maturation and ovulation in the starfish, Asterina pectinifera

Gonad-stimulating substance (GSS) of starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to the vertebrate luteinizing hormone (LH). Here, we purified GSS of starfish, Asterina pectinifera, from radial nerves and determined its amino acid sequence. The purified GSS was a heterodimer composed of 2 different peptides, A and B chains, with disulfide cross-linkages. Based on its cysteine motif, starfish GSS was classified as a member of the insulin/insulin-like growth factor (IGF)/relaxin superfamily. The cDNA of GSS encodes a preprohormone sequence with a C peptide between the A and B chains. Phylogenetic analyses revealed that starfish GSS was a relaxin-like peptide. Chemically synthesized GSS induced not only oocyte maturation and ovulation in isolated ovarian fragments, but also unique spawning behavior, followed by release of gametes shortly after the injection. Importantly, the action of the synthetic GSS on oocyte maturation and ovulation was mediated through the production of cAMP by isolated ovarian follicle cells, thereby producing the maturation-inducing hormone of this species, 1-methyladenine. In situ hybridization showed the transcription of GSS to occur in the periphery of radial nerves at the side of tube feet. Together, the structure, sequence, and mode of signal transduction strongly suggest that GSS is closely related to the vertebrate relaxin.

Neuronal peptides induce oocyte maturation and gamete spawning of sea cucumber, Apostichopus japonicus.

Extracts prepared from tissues containing buccal ring nerve or longitudinal radial nerve of sea cucumber induce oocyte maturation and ovulation from ovarian tissues. We purified two small peptides, a pentapeptide and a heptapeptide, from the buccal tissues of Japanese common sea cucumber, Apostichopus japonicas. Both peptides induced oocyte maturation and gamete spawning. The pentapeptide was identified as NGIWYamide. This peptide induced in vitro germinal vesicle breakdown and ovulation of fully-grown oocytes at less than 1 pM and in vivo spawning at 10 nM. A synthetic derivative of the pentapeptide, NGLWYamide, was 10-100 times more potent compared to the natural NGIWYamide. The heptapeptide was less potent, inducing ovulation at 1 muM. NGIWYamide and NGLWYamide induced a characteristic spawning behavior when injected into sexually matured individuals. Mature eggs artificially spawned were fertilized, and developed normally and metamorphosed into young sea cucumbers. The details of the production and the mechanism of action of NGIWYamide are still unclear, but the high biopotency of the peptide will aid understanding of the neuronal and hormonal control of reproduction of sea cucumber.

Molecular characterization of the major yolk protein of the Japanese common sea cucumber (Apostichopus japonicus) and its expression profile during ovarian development

The most abundant protein in the coelomic fluid of the Japanese common sea cucumber (Apostichopus japonicus) was purified through two steps of liquid chromatography. Subsequent peptide sequencing and cDNA cloning demonstrated that the purified fraction contained two similar but distinct proteins. Deduced amino acid sequences of these proteins revealed about 30% identity with those of sea urchin major yolk protein (MYP) and therefore they were designated AjMYP1 and AjMYP2. The full-length cDNAs for AjMYP1 and AjMYP2 consisted of 4600 and 4420bp, with predicted protein lengths of 1365 and 1345 amino acid residues, respectively. RT-PCR detected transcripts for both types of AjMYPs in all the tissues and organs examined. The transcript levels of both AjMYPs in the ovary were apparently elevated at late stages of ovarian development whereas the MYP content of the ovary examined by SDS-PAGE remained stable throughout ovarian development.

Silk Gland Factor-2, Involved in Fibroin Gene Transcription, Consists of LIM Homeodomain, LIM-interacting, and Single-stranded DNA-binding Proteins

Background: Silk gland factor-2 (SGF-2) is a key factor regulating tissue-specific expression of the fibroin gene. Results: SGF-2 is a 1.1-MDa heteromeric complex containing Awh, Ldb, Lcaf, and fibrohexamerin proteins. Conclusion: Awh, Ldb, and Lcaf interact functionally in SGF-2 to control fibroin gene expression. Significance: This study provides new insight into the functional role of single-stranded DNA-binding proteins in protein-protein interaction and transcriptional regulation. SGF-2 binds to promoter elements governing posterior silk gland-specific expression of the fibroin gene in Bombyx mori. We purified SGF-2 and showed that SGF-2 contains at least four gene products: the silkworm orthologues of LIM homeodomain protein Awh, LIM domain-binding protein (Ldb), a sequence-specific single-stranded DNA-binding protein (Lcaf), and the silk protein P25/fibrohexamerin (fhx). Using co-expression of these factors in Sf9 cells, Awh, Ldb, and Lcaf proteins were co-purified as a ternary complex that bound to the enhancer sequence in vitro. Lcaf interacts with Ldb as well as Awh through the conserved regions to mediate transcriptional activation in yeast. Misexpression of Awh in transgenic silkworms induces ectopic expression of the fibroin gene in the middle silk glands, where Ldb and Lcaf are expressed. Taken together, this study demonstrates that SGF-2 is a multisubunit activator complex containing Awh. Moreover, our results suggest that the Ldb·Lcaf protein complex serves as a scaffold to facilitate communication between transcriptional control elements.

Ovarian aromatase loss-of-function mutant medaka undergo ovary degeneration and partial female-to-male sex reversal after puberty

Although estrogens have been generally considered to play a critical role in ovarian differentiation in non-mammalian vertebrates, the specific functions of estrogens during ovarian differentiation remain unclear. We isolated two mutants with premature stops in the ovarian aromatase (cyp19a1) gene from an N-ethyl-N-nitrosourea-based gene-driven mutagenesis library of the medaka, Oryzias latipes. In XX mutants, gonads first differentiated into normal ovaries containing many ovarian follicles that failed to accumulate yolk. Subsequently, ovarian tissues underwent extensive degeneration, followed by the appearance of testicular tissues on the dorsal side of ovaries. In the newly formed testicular tissue, strong expression of gsdf was detected in sox9a2-positive somatic cells surrounding germline stem cells suggesting that gsdf plays an important role in testicular differentiation during estrogen-depleted female-to-male sex reversal. We conclude that endogenous estrogens synthesized after fertilization are not essential for early ovarian differentiation but are critical for the maintenance of adult ovaries.