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

Introduction

Planarians are free-living freshwater flatworms belonging to the phylum Platyhelminthes. They have a primitive morphology with simple triploblastic bodies (based on body-plan categories). They are acoelomate: the body cavities between the digestive tract and the outer body wall are filled with parenchymal cells. They have no vascular system, but they possess a dorso-ventral polarity and a central nervous system (CNS). Some planarians, those belonging to the genus Dugesia, can be classified into at least three races according to their mode of reproduction: exclusively asexual worms, exclusively sexual worms, and worms that switch between the two reproductive modes depending on the season (Jenkins, 1967). Asexual planarians lack sexual organs and reproduce by fission. Sexual planarians are hermaphroditic and are not likely to undergo fission under natural conditions, but they can regenerate after artificial ablation (Teshirogi and Suto, 1968). Although their existing sexual organs degenerate after ablation, new sexual organs are soon formed (Teshirogi and Fugiwara, 1970). Such worms gradually develop a pair of ovaries, testes, copulatory apparatus, yolk glands, and a genital pore.

The hormone system and CNS are important for regulating internal communication and signal transduction in metazoans. In vertebrates, steroid hormones are necessary for normal development and function of the female reproductive system. In invertebrates, the hormones for molting or metamorphosis have been well studied (Butenandt and Karlson, 1954; Roller et al., 1967), but the hormones for the reproductive system remain unknown. In hydra, a particular hormone stimulates growth and budding but prevents sexual reproduction (Pascoe et al., 2002; Fukuhori et al., 2005). Thus it is expected that some hormone in planarian could play a role in regeneration and formation of sexual organs. Vigorous molecular studies have examined the CNS, which is composed of the cephalic ganglion and a pair of ventral nervous cords (Agata et al., 1998; Nakazawa et al., 2003), but none have investigated the steroidal hormone system.

17[beta]-estradiol (E2) is a steroid compound, named for its importance in the estrous cycle, that functions as the primary female sex hormone. Bisphenol A (BPA), a monomer of epoxy resins and polycarbonates, is used as a stabilizer or antioxidant in industrial chemicals (Richter et al., 2007). Originally introduced as a synthetic estrogen in the 1930s, it has been shown to have uterotropic activity in the rodent uterus and binds to the estrogen receptors (Dodds and Lawson, 1936, 1938; Kuiper et al., 1998). In Xenopus leavis, BPA induces the feminization of tadpoles, which reveals its estrogenic potency to influence sexual development in amphibians (Levy et al., 2004). BPA is well-known as an endocrine disrupter--that is, an exogenous substance that causes adverse health effects in an intact organism or its progeny, secondary to changes in endocrine function (Diamanti-Kandarakis et al., 2009). However, the actual estrogenic potency of BPA, especially at low doses, is still a matter of controversy (Ben-Jonathan and Steinmetz, 1998; Sheehan, 2000).

Although sex steroids have not been found in planarians, high-pressure liquid chromatography has been used to detect testosterone, and the level of testosterone increased during spermatogenesis (Fukushima et al., 2008). However, the results could not prove that testosterone was actually active in spermatogenesis. The objective of our study was to examine the endocrine system of the free-living flatworm by investigating the effects of E2 (a steroid) and BPA (an estrogenic endocrine disrupter) on the formation of sexual organs in the sexual (hermaphroditic) planarian Dugesia ryukyuensis Kawakatsu. We show evidence that E2 and BPA are actually involved in the development of yolk glands. These data indicate that these hormone molecules may have various effects in planarians.

Materials and Methods

Chemicals

17[beta]-estradiol (E2) and bisphenol A (BPA) were purchased from Sigma Chemical Company (St. Louis, Missouri). All additional chemicals used during the experiments were of analytical grade.

Animals

The Dugesia ryukyuensis OH strain, an exclusively fissiparous (i.e., asexual) strain, was provided by Dr. S. Ishida of Hirosaki University. The worms were maintained at 20 [degrees]C in dechlorinated tap water and fed chicken liver. The sexual worms were prepared by using the Kobayashi feeding procedure in which they were fed frozen worms of Bdellocephala brunnea every day for 5 weeks (Kobayashi et al., 1999).

Pharmacological treatments

The E2 and BPA stock solutions were prepared at a concentration of 1000 mg/ml in methanol and 100 mg/ml in methanol, respectively. The methanol concentration in all test solutions was less than 0.001% (v/v). The culture water was prepared by using a 1:300 dilution of artificial seawater (containing 430 mmol [l.sup.-1] NaCl, 9 mmol [l.sup.-1] KCl, 9 mmol [l.sup.-1] [CaCl.sub.2], 23 mmol [l.sup.-1] [MgCl.sub.2], 25 mmol [l.sup.-1] [MgSO.sub.4], and 10 mmol [l.sup.-1] N-2-hydroxy-ethylpiperazine-N'-3-sulfonic acid [EPPS], pH 8.2). The running solutions of E2 and BPA were prepared by diluting the stock solution with the culture water and kept in the glass bottles. After surgical ablation (see next section), 15 planarians were observed for regeneration and were given pharmacological treatment for 7 weeks.

Regeneration experiments with sexual planarians

For the regeneration experiments, 15-mm-long sexual planarians were cut transversally at different anterior-posterior levels into three segments (Salo and Baguna, 1984), and the trunk sections were used for further experiments. The worms were fed weekly with raw chicken liver, starting from week 2 after surgical ablation.

Histological analysis

Specimens were fixed in 8% (v/v) formalin, dehydrated through an ethanol series, and embedded in Paraplast Plus (Sherwood Medical, St. Louis, MO). Transverse and sagittal sections of 4 [micro]m were cut, stained with hematoxylin and eosin (HE), and used for in situ hybridization.

In situ hybridization

The procedure for whole-mount in situ hybridization and in situ hybridization of sections, including the procedures for fixation, bleaching, hybridization, and washing, was carried out as described previously (Kobayashi et al., 1998). The digoxigenin (DIG)-labeled antisense and sense ribonucleic acid (RNA) probes were synthesized with T7 RNA polymerase and T3 RNA polymerase, respectively. The region used in the northern blot analysis was also used for the probe.

Reverse transcriptase-polymerase chain reaction

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed using total RNA from worms and the following specific primers:

Dryg-F, 5'-TTGGACGGATTTCTATTCTATGCC-3'

Dryg-R, 5'-CACTGGGTTTCTGATCTCTTTCG-3'

Dractl-F, 5'-CAGAAATGAGAACAGCATCAGC-3'

Dractl-R, 5'-CAATTCCAGGTCCAGATTGTC-3'

The annealing temperature was 55 [degrees]C. In all, 36 cycles were performed. Control reactions were performed in the absence of reverse transcriptase.

Results

Minimum lethal concentration of 17[beta]-estradiol and bisphenol A

As a preliminary investigation, we determined the appropriate concentration of E2 or BPA. To examine the lethal dose of E2 or BPA for planaria, five planarians were treated with each concentration of E2 or BPA, ranging from 100 ppb to 10 ppm (0.00001%-0.001% (w/v)). We observed lethality in blastemas regenerated within 5 days when exposed to E2 or BPA at concentrations greater than 1 ppm (data not shown). Thus, in this study, the worms were treated with E2 or BPA at a concentration not exceeding 100 ppb.

Appearance of regenerated sexual organs in sexual planarians after exposure to 17[beta]-estradiol and bisphenol A after ablation

During planarian regeneration, the existing organs first degenerate and then regenerate by a process similar to normal organogenesis. The existing pair of ovaries is located in the head part and not in the trunk part. After ablation, the ovaries regenerate and are now located in the ventral portion of the trunk. The regenerated ovaries, copulatory apparatus, genital pore, and seminal vesicle can be judged by their appearance. To investigate the effects of E2 and BPA on the morphogenesis of sexual organs in planarians, we observed the appearance of the ovaries, copulatory apparatus, and genital pore in 30 regenerated trunk parts of sexual planarians every week until 7 weeks after ablation (Fig. 1). Comparison of the E2-treated (1-100 ppb) and BPA-treated (1-100 ppb) planarians with normal regenerated planarians revealed no clear difference in the external structures of the ovaries, copulatory apparatus, and genital pore of each of the 30 worms. The regeneration of genital pores in the control planarians started 2 weeks after ablation. Although the regeneration of the genital pores of the worms treated with 1 ppb and 10 ppb of E2 and 100 ppb of BPA started later than that of the controls, 30 planarians finally regenerated them in the 7 weeks after ablation. Further, the 30 planarians treated with 10 ppb of BPA required 4 weeks to start regenerating their genital pores, and some planarians could not regenerate genital pores within 7 weeks.

[FIGURE 1 OMITTED]

We performed histological analysis of the effects of BPA and E2 on the morphogenesis of sexual organs (Figs. 2, 3). After ablation, the ovaries and yolk glands regenerated in the ventral portion of the trunk parts, and testes regenerated in the dorsal side of the trunk parts. In the control planarians, the ovaries, testes, and yolk gland appeared in the regenerated trunk parts of sexual planarians 6 or 7 weeks after ablation. However, the yolk glands were not developed enough to be visible in 6 weeks after the planarians were treated with 1 ppb, 10 ppb, or 100 ppb of E2 (Fig. 2Cb-d); or 10 ppb, 100 ppb of BPA (Fig. 3Cb, c), and 7 weeks after they were treated with 1 ppb, 10 ppb, or 100 ppb of E2 (Fig. 2Cf-h); or 100 ppb of BPA (Fig. 3Cf). The worms treated with 1 ppb, 10 ppb, or 100 ppb of E2 for 6 and 7 weeks, or with 10 ppb or 100 ppb of BPA for 6 weeks, or 100 ppb of BPA for 7 weeks showed only the primordial yolk glands. Some individuals did not develop mature oocytes 7 weeks after treatment with 100 ppb of BPA (Fig. 3Af). There was no clear difference in their testes.

Dryg expression in regenerated sexual planarians following exposure to BPA or E2 after ablation

To confirm the effects of E2 and BPA on yolk glands, in situ hybridization of the sections was performed with Dryg, a molecular marker of the yolk gland (Hase et al., 2003) (Figs. 4, 5). During the control experiment, the planarians developed yolk glands all over their bodies at 6 or 7 weeks after ablation (Figs. 4Aa, 5Aa), and the Dryg signal disappeared in particular in the trunk and the tail parts. In addition, on comparing the Dryg mRNA signal in the E2-treated (1-100 ppb) head fragments with that in the normal regenerated head region, we observed that the Dryg mRNA signal was weaker in the E2-treated planarians. The signals in those treated for 7 weeks almost disappeared (Fig. 4B, C). Similarly, comparison of the Dryg mRNA signal in the BPA-treated (10 or 100 ppb) head region with that in the normal head region revealed that the Dryg mRNA signal almost disappeared after treatment with 100 ppb of BPA (Fig. 5B, C).

Figure 6 shows the RT-PCR products of Dryg and D80 for whole E2- or BPA-treated planarians and corresponding normal planarians. D80 codes for actin, a housekeeping gene in planarians, which is useful as an internal control. The RNA levels in the 1 ppb E2-treated worms were almost one-fifth of those in the control planarians, and the RNA levels in the 100 ppb BPA-treated worms were lower than that. Therefore, we concluded that E2 and BPA treatment decreased the expression of Dryg.

[FIGURE 6 OMITTED]

Discussion

In this study, by investigating the effects of 17[beta]-estradiol (E2) and bisphenol A (BPA) on the formation of sexual organs in sexual planarians, we have developed an attractive model for studying the primitive endocrine-like system, which the reproduction system regulates through the steroid substances, among metazoans. After ablation, the yolk glands of worms treated with E2 or BPS showed a clear difference from those of the controls, but no difference was seen in the other sexual organs.

The yolk gland is a special organ in planarians: it produces a source of nutrition for offspring in a cocoon containing the composite egg. The components of the yolk gland in planarians are still unknown, but we cloned Dryg as a molecular marker of the yolk gland (Hase et al., 2003). Dryg is expressed in the first stage of yolk gland development; using Dryg, we showed that the regenerated planarians had only primordial yolk glands in the head parts. A major component of the yolk gland might presumably be a substance similar to vitellogenin, which is the precursor of yolk components, or to vitellogenin-related proteins in vertebrates and in many aquatic invertebrates (Byrne et al., 1989). Their production of vitellogenin is controlled by estrogen; estrogenic compounds can also promote its production (Ryffel, 1978; Sumpter and Jobling, 1995; Mat-tozzo et al., 2008).

Hormones generally need an optimal dose to function (Nishikawa et al., 1999). The optimal dose is very important for their physical activity. In the planarians, E2 could not influence the regeneration of yolk glands in a dose-dependent manner: 1 ppb of E2 inhibited yolk gland regeneration to a higher extent than did 10 or 100 ppb of E2. The steroid could not act with dose dependence. Especially in fish or mammalian estrogen receptor systems, BPA is about 1/10,000 weaker than E2 (Sheeler et al., 2000; Pawlowski et al., 2000); however, we used a narrow concentration range of chemicals. If an even greater range of concentrations is used in future experiments, the effects may be more definitive. As the optimal dose of E2 is important for the generation of yolk glands, E2 could be essential for the development of sexual organ in planarians. These substances promote the production of yolk components in amphibians (Wallace, 1985), but they had an opposite effect on the planarians. This result shows that the effect of estrogen or BPA on the reproduction system of planarians is different from their effects on other oviparous animals that have a common endocrine system. Thus, it is expected that the estrogen-like hormone in planaria may differ from E2. In fact, the ortholog of the estrogen receptor was not identified from the genome of the planarian Schmidtea mediterranea. However, the basis of the endocrine system seems to be evolutionary conserved, because the effect of E2 or BPA was specific to the yolk gland.

Molting in invertebrates is controlled by steroid hormones, such as ecdysteroids in arthropods (Jegla, 1990). In coelenterates (Sturaro et al., 1982) and even sponges (Costantino et al., 2000), ecdysteroids have also been isolated and chemically identified, but steroid compounds with sex hormone activity have not been found in these primitive invertebrates. However, many examples of reciprocal endocrine interactions between parasites and hosts have been found in insects, arthropods, and mammals, and also in parasitic platyhelminths. For example, the platyhelminth tapeworm Taenia crassiceps affects the serum concentration of steroid hormones and the reproductive behavior of its male mouse host (Romano et al., 2003). Invertebrates probably have the capability for sex steroid regulation, the basic system of which might be common to vertebrates. In this study, we found that steroids have an effect on planarian development, and that their endocrine system is involved in morphogenesis and sexual maturation. E2 or BPA clearly inhibited the formation of yolk glands. Judging by this effect, it could be expected that those compounds play roles as sexual hormones. Moreover, a search of the sexualized planarian EST database (in preparation) revealed the presence of cytochrome P450 genes, which are the enzymes for the steroidogenic pathway, suggesting that the planaria might have a system for the synthesis of sex steroids and produce an E2-like hormone.

Recently, extensive research has been performed on the toxicity of steroid compounds by using concentrations in the parts-per-million range. In fish or amphibian tadpoles, these substances can be added to the rearing water; however, they may accumulate in the gills, making it difficult to clearly detect their effect (Kloas et al., 1999; Luts and Kloas, 1999; Kashiwada et al., 2002). Because planarians have no gills, the substances can be absorbed directly and will act very effectively on the worms.

Our data suggest that planarians have a primitive steroid hormone system that plays a key role in the formation or maturation of the yolk glands. Because planarians are acoelomate and do not have vascular systems, the steroid hormones might act with the autocrine or paracrine systems but not with the endocrine system. Additional studies will provide new insights into the mechanisms of planarian reproduction.

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research (No. 13027284) from the Ministry of Science, Culture, Sports and Education, Japan.

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HITOSHI MIYASHITA (1), *, HARUKA NAKAGAWA (1), *, KAZUYA KOBAYASHI (2), MOTONORI HOSHI (3), AND MIDORI MATSUMOTO (1), [dagger]

(1) Department of Biological Sciences and Informatics, Keio University, 3-14-1, Hiyoshi, Kouhoku-ku, Yokohama 223-8522, Japan; Center for Integrated Medical Research, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; and Open University of Japan, 2-11, Wakaba, Mihama-ku, Chiba 261-8586, Japan

Received 13 October 2010; accepted 29 December 2010.

* These authors have contributed equally to this work.

[dagger] To whom correspondence should be addressed. E-mail: mmatsumo@bio.keio.ac.jp

Abbreviations: BPA, bisphenol A; E2, 17[beta]-estradiol.
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Author:Miyashita, Hitoshi; Nakagawa, Haruka; Kobayashi, Kazuya; Hoshi, Motonori; Matsumoto, Midori
Publication:The Biological Bulletin
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Date:Feb 1, 2011
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