Co-localization of DrPiwi-1 and DrPiwi-2 in the oogonial cytoplasm is essential for oocyte differentiation in sexualized planarians.

P-Element-induced wimpy testis (Piwi) subfamily proteins form complexes that bind to Piwi-interacting RNA. This interaction is crucial for stem cell regulation and formation, maintenance of germline stem cells, and gametogenesis in several metazoans. Planarians are effective models for studying stem cells. In the planarian Dugesia ryukyuensis, DrPiwi-1 is essential for the development of germ cells, but not somatic cells and sexual organs. DrPiwi-2 is indispensable for regeneration. In this study, we aimed to investigate the effects of Piwi on the differentiation of germ cells using monoclonal antibodies against DrPiwi-1 and DrPiwi-2. DrPiwi-1 and DrPiwi-2 co-localized more in immature germ cells than in mature germ cells in the ovary. DrPiwi-1 was found in the cytoplasm of early oogonia as undifferentiated germ cells, whereas DrPiwi-2 was found to localize not only in the nuclei but also in the cytoplasm of early oogonia. In descendant germ cells (oocytes), DrPiwi-2 was not present in the cytoplasm, but was strongly detected in the nucleolus. Moreover, we found that DrPiwi-1 forms a complex with DrPiwi-2. The cause of DrPiwi-1 depletion may be the severe reduction in the DrPiwi-2 level in the cytoplasm of oogonia. These results suggest that the formation of the DrPiwi-1 and DrPiwi-2 complex in the cytoplasm of oogonia is essential for oocyte differentiation. Our findings support the conclusion that DrPiwi-1 forms a complex with DrPiwi-2 in the cytoplasm of undifferentiated germ cells, and it signifies the start of gametogenesis. In contrast, in the testes, Drpiwi-1 was found in undifferentiated germ cells (spermatogonia), whereas DrPiwi-2 was found in descendant germ cells (spermatocytes). The process of germ cell differentiation from adult stem cells in planarians may be regulated in different ways in female and male germ lines by the Piwi family.

Correction to: A comprehensive comparison of sex-inducing activity in asexual worms of the planarian Dugesia ryukyuensis: the crucial sex-inducing substance appears to be present in yolk glands in Tricladida.

[This corrects the article DOI: 10.1186/s40851-018-0096-9.].

A comprehensive comparison of sex-inducing activity in asexual worms of the planarian Dugesia ryukyuensis: the crucial sex-inducing substance appears to be present in yolk glands in Tricladida.

BACKGROUND: Turbellarian species can post-embryonically produce germ line cells from pluripotent stem cells called neoblasts, which enables some of them to switch between an asexual and a sexual state in response to environmental changes. Certain low-molecular-weight compounds contained in sexually mature animals act as sex-inducing substances that trigger post-embryonic germ cell development in asexual worms of the freshwater planarian Dugesia ryukyuensis (Tricladida). These sex-inducing substances may provide clues to the molecular mechanism of this reproductive switch. However, limited information about these sex-inducing substances is available. RESULTS: Our assay system based on feeding sex-inducing substances to asexual worms of D. ryukyuensis is useful for evaluating sex-inducing activity. We used the freshwater planarians D. ryukyuensis and Bdellocephala brunnea (Tricladida), land planarian Bipalium nobile (Tricladida), and marine flatworm Thysanozoon brocchii (Polycladida) as sources of the sex-inducing substances. Using an assay system, we showed that the three Tricladida species had sufficient sex-inducing activity to fully induce hermaphroditic reproductive organs in asexual worms of D. ryukyuensis. However, the sex-inducing activity of T. brocchii was sufficient only to induce a pair of ovaries. We found that yolk glands, which are found in Tricladida but not Polycladida, may contain the sex-inducing substance that can fully sexualize asexual worms of D. ryukyuensis. CONCLUSIONS: Our results suggest that within Tricladida, there are one or more common compounds or functional analogs capable of fully sexualizing asexual worms of D. ryukyuensis; namely, the crucial sex-inducing substance (hydrophilic and heat-stable, but not a peptide) produced in yolk glands.

Global identification and analysis of isozyme-specific possible substrates crosslinked by transglutaminases using substrate peptides in mouse liver fibrosis.

The transglutaminase (TG) family comprises eight isozymes that form the isopeptide bonds between glutamine and lysine residues and contribute to the fibrotic diseases via crosslinking-mediated stabilization of ECM and the activation of TGF-ß in several tissues. However, despite a growing body of evidence implicating TG2 as a key enzyme in fibrosis, the causative role of TG2 and the involvement of the other isozymes have not yet been fully elucidated. Therefore, here we clarified the distributions of TG isozymes and their in situ activities and identified the isozyme-specific possible substrates for both TG1 and TG2 using their substrate peptides in mouse fibrotic liver. We found that TG1 activity was markedly enhanced intracellularly over a widespread area, whereas TG2 activity increased in the extracellular space. In total, 43 and 42 possible substrates were identified for TG1 and TG2, respectively, as involved in chromatin organization and cellular component morphogenesis. These included keratin 18, a biomarker for hepatic injury, which was accumulated in the fibrotic liver and showed the partly similar distribution with TG1 activity. These findings suggest that TG1 activity may be involved in the functional modification of intracellular proteins, whereas TG2 activity contributes to the stabilization of extracellular proteins during liver fibrosis.

ESR study of molecular orientation and dynamics of TEMPO derivatives in CLPOT 1D nanochannels.

The molecular orientations and dynamics of 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO) radical derivatives with large substituent groups at the 4-position (4-X-TEMPO) in the organic one-dimensional nanochannels within the nanosized molecular template 2,4,6-tris(4-chlorophenoxy)-1,3,5-triazine (CLPOT) were examined using ESR. The concentrations of guest radicals, including 4-methoxy-TEMPO (MeO-TEMPO) or 4-oxo-TEMPO (TEMPONE), in the CLPOT nanochannels in each inclusion compound (IC) were reduced by co-including 4-substituted-2,2,6,6-tetramethylpiperidine (4-R-TEMP) compounds at a ratio of 1 : 30-1 : 600. At higher temperatures, the guest radicals in each IC underwent anisotropic rotational diffusion in the CLPOT nanochannels. The rotational diffusion activation energy, Ea , associated with MeO-TEMPO or TEMPONE in the CLPOT nanochannels (6-7 kJ mol(-1) ), was independent of the size and type of substituent group and was similar to the Ea values obtained for TEMPO and 4- hydroxy-TEMPO (TEMPOL) in our previous study. However, in the case in which TEMP was used as a guest compound for dilution (spacer), the tilt of the rotational axis to the principal axis system of the g-tensor, and the rotational diffusion correlation time, τR , of each guest radical in the CLPOT nanochannels were different from the case with other 4-R-TEMP. These results indicate the possibility of controlling molecular orientation and dynamics of guest radicals in CLPOT ICs through the appropriate choice of spacer. Copyright © 2016 John Wiley & Sons, Ltd.

Planarian D-amino acid oxidase is involved in ovarian development during sexual induction.

To elucidate the molecular mechanisms underlying switching from asexual to sexual reproduction, namely sexual induction, we developed an assay system for sexual induction in the hermaphroditic planarian species Dugesia ryukyuensis. Ovarian development is the initial and essential step in sexual induction, and it is followed by the formation of other reproductive organs, including the testes. Here, we report a function of a planarian D-amino acid oxidase, Dr-DAO, in the control of ovarian development in planarians. Asexual worms showed significantly more widespread expression of Dr-DAO in the parenchymal space than did sexual worms. Inhibition of Dr-DAO by RNAi caused the formation of immature ovaries. In addition, we found that feeding asexual worms 5 specific D-amino acids could induce the formation of immature ovaries that are similar to those observed in Dr-DAO knockdown worms, suggesting that Dr-DAO inhibits the formation of immature ovaries by degrading these D-amino acids. Following sexual induction, Dr-DAO expression was observed in the ovaries. The knockdown of Dr-DAO during sexual induction delayed the maturation of the other reproductive organs, as well as ovary. These findings suggest that Dr-DAO acts to promote ovarian maturation and that complete sexual induction depends on the production of mature ovaries. We propose that Dr-DAO produced in somatic cells prevents the onset of sexual induction in the asexual state, and then after sexual induction, the female germ cells specifically produce Dr-DAO to induce full maturation. Therefore, Dr-DAO produced in somatic and female germline cells may play different roles in sexual induction.

Existence of two sexual races in the planarian species switching between asexual and sexual reproduction.

In certain planarian species that are able to switch between asexual and sexual reproduction, determining whether a sexual has the ability to switch to the asexual state is problematic, which renders the definition of sexuals controversial. We experimentally show the existence of two sexual races, acquired and innate, in the planarian Dugesia ryukyuensis. Acquired sexuals used in this study were experimentally switched from asexuals. Inbreeding of acquired sexuals produced both innate sexuals and asexuals, but inbreeding of innate sexuals produced innate sexuals only and no asexuals. Acquired sexuals, but not innate sexuals, were forced to become asexuals by ablation and regeneration (asexual induction). This suggests that acquired sexuals somehow retain asexual potential, while innate sexuals do not. We also found that acquired sexuals have the potential to develop hyperplastic and supernumerary ovaries, while innate sexuals do not. In this regard, acquired sexuals were more prolific than innate sexuals. The differences between acquired and innate sexuals will provide a structure for examining the mechanism underlying asexual and sexual reproduction in planarians.

The Dr-nanos gene is essential for germ cell specification in the planarian Dugesia ryukyuensis.

Homologs of nanos are required for the formation and maintenance of germline stem cell (GSC) systems and for gametogenesis in many metazoans. Planarians can change their reproductive mode seasonally, alternating between asexual and sexual reproduction; they develop and maintain their somatic stem cells (SSCs) and GCSs from pluripotent stem cells known as neoblasts. We isolated a nanos homolog, Dr-nanos, from the expressed sequence tags (ESTs) of the sexualized form of Dugesia ryukyuensis. We examined the expression of Dr-nanos in asexual and sexualized planarians by in situ hybridization and analyzed its function using RNA interference (RNAi) together with a planarian sexualization assay. A nanos homolog, Dr-nanos, was identified in the planarian D. ryukyuensis. Dr-nanos expression was observed in the ovarian primordial cells of the asexual worms. This expression increased in proportion to sexualization and was localized in the early germline cells of the ovaries and testes. In X-ray-irradiated worms, the expression of Dr-nanos decreased to a large extent, indicating that Dr-nanos is expressed in some subpopulations of stem cells, especially in GSCs. During the sexualization process, worms in which Dr-nanos was knocked down by RNAi exhibited decreased numbers of oogonia in the ovaries and failed to develop testes, whereas the somatic sexual organs were not affected. We conclude that Dr-nanos is essential for the development of germ cells in the ovaries and testes and may have a function in the early stages of germ cell specification, but not in the development of somatic sexual organs.

Drpiwi-1 is essential for germline cell formation during sexualization of the planarian Dugesia ryukyuensis.

A piwi homolog is required for the regulation of stem cells, formation and maintenance of germline stem cells, and gametogenesis in many metazoans. Planarians can change their reproductive mode seasonally, both asexually and sexually, and develop and maintain germ cells and sexual organs. They have many pluripotent stem cells (neoblasts) that can differentiate into both somatic and germline stem cells. Thus, we searched for a piwi subfamily in the planarian Dugesia ryukyuensis. Four piwi homologs, identified as Drpiwi-1, -2, -3, and -4, were expressed in sexually reproductive worms. We then selectively destroyed the neoblasts by irradiating the worms with X-rays. In such worms, Drpiwi-1, -2, and -3 were not expressed at all, whereas Drpiwi-4 was expressed to the same degree as that in non-irradiated controls, indicating that Drpiwi-1, -2, and -3, but not Drpiwi-4, are expressed in neoblasts. During the regeneration process, Drpiwi-2(RNAi) and -3(RNAi) worms failed to regenerate after ablation, but Drpiwi-1 and -4(RNAi) worms regenerated. During the sexualizing process, Drpiwi-1(RNAi) worms failed to develop ovaries and testes, but somatic sexual organs were unaffected. Germ cell development was normal in Drpiwi-4(RNAi) worms. Therefore, Drpiwi-2 and -3 may be related to the regulation of neoblasts important for maintaining homeostasis, and Drpiwi-1 is essential for the development of germ cells but not somatic sexual organs. DrPiwi-1 is localized in the cytoplasm of stem cells and germline cells and may be involved in regulating some gene expression. We suggest that planarian Piwi controls germline formation via RNA silencing mechanisms.

Effects of 17ß-estradiol and bisphenol A on the formation of reproductive organs in planarians.

Planarians have a remarkable capacity for regeneration after ablation, and they reproduce asexually by fission. However, some planarians can also reproduce and maintain their sexual organs. During the regenerative process, their existing sexual organs degenerate and new ones develop. However, little is known about hormonal regulation during the development of reproductive organs in planarians. In this study, we investigated the effects of 17ß-estradiol (a steroid) and bisphenol A (an endocrine disrupter) on the formation of sexual organs in the hermaphroditic planarian Dugesia ryukyuensis. Under control conditions, all worm tissues regenerated into sexual planarians with sexual organs within 4 weeks after ablation. However, in the presence of bisphenol A or 17ß-estradiol, although they apparently regenerated into sexual planarians, the yolk glands, which are one of the female sexual organs, failed to regenerate even 7 weeks after ablation. These data suggest that planarians have a steroid hormone system, which plays a key role in the formation and maturation of sexual organs.

The Dugesia ryukyuensis database as a molecular resource for studying switching of the reproductive system.

The planarian Dugesia ryukyuensis reproduces both asexually and sexually, and can switch from one mode of reproduction to the other. We recently developed a method for experimentally switching reproduction of the planarian from the asexual to the sexual mode. We constructed a cDNA library from sexualized D. ryukyuensis and sequenced and analyzed 8,988 expressed sequence tags (ESTs). The ESTs were analyzed and grouped into 3,077 non-redundant sequences, leaving 1,929 singletons that formed the basis of unigene sets. Fifty-six percent of the cDNAs analyzed shared similarity (E-value<1E -20) with sequences deposited in NCBI. Highly redundant sequences encoded granulin and actin, which are expressed in the whole body, and other redundant sequences encoded a Vasa-like protein, which is known to be a component of germ-line cells and is expressed in the ovary, and Y-protein, which is expressed in the testis. The sexualized planarian expressed sequence tag database (http://planaria.bio.keio.ac.jp/planaria/) is an open-access, online resource providing access to sequence, classification, clustering, and annotation data. This database should constitute a powerful tool for analyzing sexualization in planarians.