Annual rhythmicity in the switching of reproductive mode in planarians.

Species of planarians include both asexually reproducing individuals (reproduce through fission and regeneration) and sexually reproducing individuals (hermaphrodites that mate to produce cocoons). While some individuals can switch between the asexual and sexual modes of reproduction. In this study, we examined the reproductive modes and ploidy of Dugesia japonica and Dugesia ryukyuensis from three spring wells in Okinawa (Japan) during two consecutive years. D. japonica are mostly asexual and triploid. In contrast, only 40 % of D. ryukyuensis are asexual and triploid; the remaining are sexual, and diploid or triploid. The sexually reproductive season of D. ryukyuensis is winter. In July, the reproductive organs disappear, and the individuals start asexual reproduction through fission and regeneration. In January of the following year, the individuals develop ovaries and necessary reproductive organs and start sexual reproduction. When these species were lab-reared for a longer period, the reproductive cycles in three strains were repeated for three years. These results confirm that D. ryukyuensis population in Okinawa switches between reproductive modes on an annual cycle, even when kept under constant temperature and no light/dark cycle.

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.

Selective Intracellular Delivery of Ganglioside GM3-Binding Peptide through Caveolae/Raft-Mediated Endocytosis.

Glycosphingolipids are major components of the membrane raft, and several kinds of viruses and bacterial toxins are known to bind to glycosphingolipids in the membrane raft. Since the viral genes and pathogenic proteins that are taken into cells are directly delivered to their target organelles, caveolae/raft-mediated endocytosis represents a promising pathway for specific delivery. In the present study, we demonstrated the ability of an artificial pentadecapeptide, which binds to ganglioside GM3, to deliver protein into cells by caveolae/raft-mediated endocytosis. The cellular uptake of a biotinylated GM3-binding peptide (GM3BP)-avidin complex into HeLa cells was observed, and the cellular uptake of this complex was inhibited by an incubation with sialic acid or endocytic inhibitors such as methyl-ß-cyclodextrin, and also by an incubation at 4 °C. These results indicate that the GM3BP-avidin complex bind to GM3 in membrane raft, and are taken into cell through caveolae/raft-mediated endocytosis. The GM3BP-avidin complex was transported into cells and localized around the nucleus more slowly than a human immunodeficiency virus type 1 TAT peptide. Furthermore, the uptake of a green fluorescent protein (GFP) linked with GM3BP into HeLa cells was similar to that of the GM3BP-avidin complex, and the localization of the GM3BP-GFP fusion protein was markedly different with that of the TAT-GFP fusion protein. The uptake and trafficking of GM3BP were distinguished from conventional cell-penetrating peptides. GM3BP has potential as a novel peptide for the selective delivery of therapeutic proteins and materials into cells in addition to being a cell-penetrating peptide.

Triploid planarian reproduces truly bisexually with euploid gametes produced through a different meiotic system between sex.

Although polyploids are common among plants and some animals, polyploidization often causes reproductive failure. Triploids, in particular, are characterized by the problems of chromosomal pairing and segregation during meiosis, which may cause aneuploid gametes and results in sterility. Thus, they are generally considered to reproduce only asexually. In the case of the Platyhelminthes Dugesia ryukyuensis, populations with triploid karyotypes are normally found in nature as both fissiparous and oviparous triploids. Fissiparous triploids can also be experimentally sexualized if they are fed sexual planarians, developing both gonads and other reproductive organs. Fully sexualized worms begin reproducing by copulation rather than fission. In this study, we examined the genotypes of the offspring obtained by breeding sexualized triploids and found that the offspring inherited genes from both parents, i.e., they reproduced truly bisexually. Furthermore, meiotic chromosome behavior in triploid sexualized planarians differed significantly between male and female germ lines, in that female germ line cells remained triploid until prophase I, whereas male germ line cells appeared to become diploid before entry into meiosis. Oocytes at the late diplotene stage contained not only paired bivalents but also unpaired univalents that were suggested to produce diploid eggs if they remained in subsequent processes. Triploid planarians may therefore form euploid gametes by different meiotic systems in female and male germ lines and thus are be able to reproduce sexually in contrast to many other triploid organisms.

Innate sexuality determines the mechanisms of telomere maintenance.

Recently, telomere length has been shown to be differentially regulated in asexually and sexually reproducing planarians. In addition, it was found that asexual worms maintain telomere length somatically during reproduction by fission or when regeneration is induced by amputation, whereas sexual worms only achieve telomere elongation through sexual reproduction. We have established an experimental bioassay system to induce switching from asexual to sexual reproduction in planarians, that is, sexualization. In this study, the relationship between the reproductive mode and telomere maintenance was investigated using innate asexually reproducing worms, innate sexually reproducing worms, and experimentally sexualized worms. Here, we show that innate asexual planarians maintain telomere length during cell division and that innate sexual planarians exhibit telomere shortening. However, experimental sexualized worms maintain telomere length during cell division. These results indicate that innate sexuality is linked to the mechanism of telomere maintenance.

Stem cells from innate sexual but not acquired sexual planarians have the capability to form a sexual individual.

Planarian species may harbor as many as three populations with different reproductive strategies. Animals from innate asexual (AS) and innate sexual (InS) populations reproduce only by fission and cross-fertilization, respectively, whereas the third population switches seasonally between the two reproductive modes. AS worms can be experimentally sexualized by feeding them with minced InS worms; we termed the resulting animals "acquired sexual" (AqS) worms. Both AqS and InS worms exhibit sexualizing activity when used as feed, suggesting that they maintain their sexual state via endogenous sexualizing substances, although the mechanisms underlying determination of reproductive strategy and sexual switching in these metazoans remain enigmatic. Therefore, we compared the endogenous sexualizing activity of InS worms and AqS worms. First, we amputated mature worms and assessed if they could re-enter a sexual state. Regenerants of InS worms, but not AqS worms, were only sexual, indicating that sexual state regulation comprises two steps: (1) autonomous initiation of sexualizing substance production and (2) maintenance of the sexual state by continuous production of sexualizing substances. Next, InS neoblasts were characterized by transplantation, finding that they successfully engrafted, proliferated, and replaced all recipient cells. Under such conditions, the AS recipients of InS worm neoblasts, but not those of AqS worms, became sexual. These results clearly show that there is a neoblast-autonomous determination of reproductive strategy in planarians.

Reproductive mode and ovarian morphology regulation in chimeric planarians composed of asexual and sexual neoblasts.

Planarians are comprised of populations with different reproductive strategies: exclusively innately asexual (AS), exclusively innately sexual (InS), and seasonally switching. AS worms can be sexualized experimentally by feeding them with minced InS worms, and the resultant worms are characterized as acquired sexual (AqS). Differences between InS and AqS worms are expected to provide important clues to the poorly understood mechanism underlying the regulation of their reproductive mode. Morphological differences were found between InS and AqS worm ovaries, and we showed that the pluripotent stem cells (neoblasts) from InS worms, but not those of AqS worms, have the capacity to initiate the sexual state autonomously via neoblast fraction transplantation. To compare their reproductive mode and ovarian morphology regulation, InS donor neoblast fractions were transplanted into non-lethally X-ray-irradiated AS recipients. All transplants showed stable chimerism and reproduced sexually, suggesting that InS worm neoblasts can initiate sexual state autonomously, even when coexisting with AS worm neoblasts. The chimeras formed extraordinarily large and supernumerary ovaries equivalent to AqS worms, which were not seen in InS worms, suggesting that regulation of ovarian morphology in AS worm-derived cells in response to endogenous sexualizing stimulation distinctly differs from that of InS worms.

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.