Germline Defects Caused by Smed-boule RNA-Interference Reveal That Egg Capsule Deposition Occurs Independently of Fertilization, Ovulation, Mating, or the Presence of Gametes in Planarian Flatworms

Few animals are known to lay eggs in the absence of ovulation or copulation, as it is presumably energetically wasteful and subjected to negative selection. Characterization of Smed-boule, a member of the DAZ family of germline RNA-binding proteins, revealed that egg capsule (or capsule) production and deposition occurs independently of the presence of gametes in the planarian flatworm Schmidtea mediterranea. Reduction of Smed-boule expression by RNA-interference (RNAi) causes ablation of spermatogonial stem cells and the inability of ovarian germline stem cells to undergo oogenesis. Although animals subjected to Smed-boule RNAi lose their gametes and become sterile, they continue to lay egg capsules. Production of sterile capsules is even observed in virgin Smed-boule(RNAi) and control planarians maintained in complete isolation, demonstrating that egg production in S. mediterranea occurs independently of ovulation, fertilization, or mating. Evidence suggests that this is a conserved feature amongst Platyhelminthes, and therefore relevant to the pathology and dissemination of parasitic flatworms. These findings demonstrate that Smed-boule functions at different stages during male and female germline stem cell development, and also demonstrate that egg capsule production by planarian flatworms occurs independently of signals produced by mating or ova.


Introduction
The characterization of developmental processes involved in sexual reproduction has important implications towards reproductive medicine, stockbreeding, farming, and for controlling the dissemination of infectious disease. Evolutionarily conserved molecular processes involved in metazoan germline development have been identified through decades of research using model organisms. For example, post-transcriptional regulation of gene expression by conserved germline-specific RNA-binding proteins is one of the conserved molecular processes that ensure development of gametes [1][2][3]. On the other hand, there is great diversity in the processes that occur during and after fertilization, many of which are the outcome of speciation events [4,5].
Planarian flatworms belong to the phylum Platyhelminthes, and are well known for their extraordinary regenerative abilities, which are founded in the availability of a pluripotent stem cell population throughout their life [6][7][8][9]. The evolutionary history of these organisms has yielded extreme divergence of reproductive strategies, both between and within populations of different planarian species. For example, there are planarians that rely exclusively or temporally on asexual reproduction, which involves transverse fission and stem cell driven regeneration [7,10]. There are also populations of planarians that reproduce predominantly through parthenogenesis (Pongratz et al., 2003). However, the default reproductive strategy of turbellarians is believed to be hermaphroditic sexual reproduction [11], more specifically for planarians, through cross-fertilization and oviparity [12]. By contrast, some parasitic flatworms (i.e. schistosomes or blood flukes) have complex life cycles that involve dioecious and asexual reproductive phases during transitions between vertebrate and invertebrate hosts, respectively [13]. Since the complex life cycle of schistosomes complicates husbandry and experimentation in laboratory settings, researchers have begun to use planarian flatworms as a model to dissect the molecular mechanisms behind the extensive lifespan and reproduction of their parasitic cousins [14]. One aspect of particular interest is the continuous production of thousands of eggs that both facilitate dissemination and sustain the pathology of schistosomes by populating organs of their host [13,14].
Planarian flatworms have become useful models for the study of metazoan germline development [12,15]. In general, the specification of germline stem cells can occur through mechanisms that involve: 1) inherited material deposited in the cytoplasm of the maturing oocyte (preformation); or 2) embryonic stem cell differentiation in response to inductive cell-to-cell interactions (epigenesis) [16,17]. Inductive determination occurs in mice and is also observed in planarians, both initially and during regeneration of fragments that lack germ cells, and it occurs through differentiation of pluripotent somatic stem cells called neoblasts [18]. In the planarian species Schmidtea mediterranea, germline stem cells are first detected as dorsolateral clusters in the area where testes develop [18]. In other planarian species, such as Dugesia ryukyuensis, germline stem cells are first detected in the area of the ovaries [19,20]. Upon feeding and growth, planarians that reproduce sexually develop a hermaphroditic reproductive system and their gonads begin continuous production of gametes [12,19]. Germline stem cells in the ovary enter oogenesis and produce oocytes of approximately 40 μm in diameter that exit the ovary and are fertilized by sperm deposited in the tuba [12,15]. Even-though oocytes are large in comparison to other planarian cells (e.g. neoblasts are~8 μm diameter) these do not hold the nutrients necessary for embryonic development, as is normally observed in eggs of insects, amphibians or fish (to name a few). Instead, planarian yolk glands (vitellaria in other flatworms) produce separate cells that provide material required for egg capsule shell formation and nurturing embryonic development [21,22].
The development of planarian germline stem cells depends on conserved post-transcriptional regulators such as Nanos and Bic-C [15,18]. Boule is an RNA-binding protein encoded by the basal member of the Deleted in AZoospermia (DAZ) gene family, which is required for germ cell development in species ranging from sea anemone to humans [23,24]. How DAZ family homologs contribute to germline development in planarians remains unknown. In this study, we characterize a Boule homolog in the planarian Schmidtea mediterranea and demonstrate that it functions at different stages during male and female germline development. Functional analyses by RNA-interference (RNAi) revealed that Smed-boule is required for development and maintenance of spermatogonial stem cells, but disposable for the existence of their oogonial counterparts, uncovering the presence of sex-specific germline stem cells in planarian hermaphrodites. Long-term analysis of Smed-boule knockdowns revealed that egg capsule deposition in planarians is not triggered by gametogenesis, ovulation, oocyte activation, fertilization, or mating. These results demonstrate that egg capsule formation occurs regardless of signals from sexual activity or germ cell activity in S. mediterranea. These findings also provide a unique opportunity to identify internal mechanisms that influence capsule production in Platyhelminthes, which is central in the dissemination and pathology of parasitic members of this phylum.

Smed-boule is required for male and female germline development
We identified a boule homolog in the planarian flatworm S. mediterranea with a region of amino acid sequence 55% identical with that of the RNA recognition motif of human BOLL (E-value = 1e-23; Fig 1A). The protein encoded by this gene shared highest homology with members of the Boule-like subfamily, as compared with other members of the DAZ family of proteins ( Fig 1B). Expression of this gene (from here on referred to as Smed-boule or boule) was detected by whole-mount in situ hybridization (ISH) in testes and ovaries of sexually mature planarians that are actively laying egg capsules (Fig 2A-2D). Smed-boule expression was also detected in testis primordia of hatchlings and sexually immature animals ( Fig 2D and  2D'). To better understand the distribution of Smed-boule expression in testes and ovaries, we performed detailed analysis by double fluorescent in situ hybridization (FISH) with the germline stem cell marker Smed-nanos [18] (Fig 3). Detection of Smed-boule mRNA overlapped with that of Smed-nanos in testes ( Fig 3A) and partially in ovaries ( Fig 3B). The presence of Smed-boule mRNA was also robustly detected in the spermatogonial layer of the testes ( Fig  3A). Detection of Smed-boule expression was not apparent in the soma, and thus we conclude that expression of this gene is restricted to the germline in S. mediterranea.
To test the function of Smed-boule in planarian germline development and sexual reproduction we subjected planarians to three months of RNAi. Planarians continuously turn over all cells in their body from a continuous population of pluripotent stem cells, which allowed us to assess whether Smed-boule is required for normal germline development in sexually mature adults using germ cell markers (Fig 4A-4F; S1 and S2 Figs). Groups of seven sexually mature planarians were fed liver supplemented with 100 ng/μl of double-stranded RNA (dsRNA)    Capsule production (J) and hatching (K) from groups of sexually mature planarians subjected to continuous twice per week. Smed-boule knockdowns (Smed-boule(RNAi)) were compared to control (RNAi) planarians. DsRNA corresponding to a planarian Cytoplasmic Polyadenylation Element Binding Protein 1 homolog (CPEB1), which is required for yolk gland development and egg capsule production (below), was administered to an additional group (CPEB1(RNAi)) as readout of RNAi effectiveness overtime. At the end of three months of RNAi, we observed that both oocytes ( (Fig 4F), but their ovaries displayed abnormal morphology and distribution of oocyte marker expression ( Fig 4C). From these results we concluded that Smed-boule is required for development of sperm and ova in S. mediterranea.
Neoophoran flatworms rely on a particular approach to oviparity in which nutrients for the developing embryo (yolk) are not accumulated in the developing ova. Instead, nutritional support is contributed by yolk cells (vitellocytes), which are transferred from yolk glands to the planarian uterus and encapsulated with embryos during egg capsule deposition. We checked for the presence of yolk glands using the yolk cell marker Smed-surfactant b (S1 Fig), which proved to be of comparable abundance and distribution in control(RNAi) and Smed-boule (RNAi) animals ( Fig 4G and 4H). However, the presence of yolk glands in CPEB1(RNAi) animals was severely reduced (Fig 4I). We looked for other possible defects in the development of somatic reproductive structures but were unable to find any abnormalities other than the absence of accumulated sperm in the seminal vesicles of Smed-boule(RNAi) (Fig 5), which is due to their inability to produce sperm ( Fig 4E). There was also a noticeable difference in size of CPEB1(RNAi) planarians, which were on average 30.8% larger than control animals maintained under the same conditions (n = 14; unpaired two-tailed t-test, p < 0.05; S3 Fig). The normality and functionality of the accessory reproductive system in Smed-boule(RNAi) was further supported by quantitative analyses of egg capsule production (below).

Continuous production of sterile egg capsules by Smed-boule(RNAi)
As expected, from planarians with underdeveloped yolk glands (Fig 4I), CPEB1(RNAi) ceased laying eggs within a month of RNAi (Fig 4J). The rate of egg production in Smed-boule(RNAi) was unaffected during the three months of RNAi treatment (Fig 4J). Both the control(RNAi) and Smed-boule(RNAi) groups continuously laid eggs for the entirety of the experiment ( Fig  4J). In fact, an increase of 33% and 30% in egg capsule production was observed in Smed-boule (RNAi) when compared to control(RNAi) planarians during the second and third months of RNAi treatment, respectively (unpaired two-tailed t-test, p < 0.05; Fig 4J). Given the surprising result that planarians devoid of gametes continued to deposit egg capsules, we monitored and quantified the number of fertile capsules (yielding progeny) produced by the different knockdown groups for two months after capsule deposition. From this, we discovered that egg capsules produced by animals subjected to two months of Smed-boule RNAi completely ceased to hatch (Fig 4K). Egg capsules produced by control(RNAi) groups hatched 22% to 48% of the time (Fig 4K). From these results, we concluded that Smed-boule function is required for germline development and sexual reproduction in S. mediterranea, but dispensable for production control, Smed-boule, or CPEB1 RNAi treatments for three months (first, second, and third month represented by column from left to right in each group). Quantification of the number of capsules laid (J) and the number of fertile capsules (K) show that capsules deposited by Smed-boule(RNAi) animals ceased being fertile and that CPEB1(RNAi) ceased capsule production as a result of RNAi. Asterisks (*) represent statistically significant results compared to controls of same month by unpaired two-tailed t-test (p > 0.05). Scale bars = 1 mm.
doi:10.1371/journal.pgen.1006030.g004 of egg capsules. Furthermore, the continuous production of egg capsules by groups of Smedboule(RNAi) planarians (Fig 4J) suggested that production and deposition of egg capsules do not require fertilization, contributions from sperm, ovulation, or the presence of oocytes.

Continuous egg production in planarians regardless of isolation and absence of gametes
Given the fact that Smed-boule(RNAi) planarians were capable of producing sterile egg capsules in the absence of germ cells (and therefore fertilization events), we hypothesized that control animals would also produce sterile egg capsules in the absence of fertilization events. To test this hypothesis, we obtained 1 week-old hatchlings (which lack ovaries, testes, yolk glands, and accessory reproductive organs) and maintained them in isolation for four months under continuous RNAi regimens. Planarians were maintained in isolation throughout the experiment, which allowed us to test whether egg capsule production is independent of signals produced during mating or the presence of potential mates altogether. Since planarians in this experiment were subjected to Smed-boule RNAi within a week of being born, which is a point when no sperm has developed, this approach also allowed us to verify that lingering sperm in adult knockdowns used in the previous experiment was not contributing to egg capsule production. Two categories of isolated virgins were maintained on either liver containing Smedboule dsRNA or control dsRNA and were fed twice per week. These animals were expected to grow and eventually reach sexual maturity under these husbandry conditions. The production of egg capsules would only occur if independent from stimuli produced during mating, fertilization, embryonic development and, in the case of Smed-boule(RNAi), the absence of gametes.
Indeed, both control and Smed-boule(RNAi) isolated animals produced egg capsules during the third and fourth months of the experiment (Fig 6A). The number of capsules produced during the length of the experiment by isolated individuals from each category ranged from none to six (Fig 6B). The average number of capsules deposited by individuals in the control category was slightly, but not significantly higher than those of Smed-boule(RNAi) (unpaired two-tailed t-test, p = 0.25) (Fig 6B). As expected from results observed in animals subjected to RNAi in the presence of potential mates (Fig 4J and 4K), none of the egg capsules produced by Smed-boule(RNAi) individuals were fertile (n = 0/28 capsules). Capsules produced by control RNAi animals were also completely sterile (n = 0/43 capsules), suggesting that the production of egg capsules in these animals were not due to self-fertilization or parthenogenesis. We verified that normal gamete development was present in control animals at the end of the isolation experiment (Fig 6C and 6E) and absent in Smed-boule(RNAi) flatworms (Fig 6D and 6F), which was expected from analyses of knockdowns not maintained in isolation (Fig 4A, 4B, 4D and 4E). We also validated successful development of yolk glands in control and Smed-boule (RNAi) planarians raised in isolation (Fig 6G and 6H). Collectively, these results demonstrate that production of egg capsules in S. mediterranea occurs in response to internal triggers that do not require the presence of a mate, mating, or fertilization events. Furthermore, the production of egg capsules by Smed-boule(RNAi) planarians suggests that this trigger is detached from signals originating from sperm and oocyte development or ovulation.

Spermatogonial stem cells are lost and oogonial stem cells display early oogenesis defects after Smed-boule RNAi
We decided to evaluate the severity of germline development defects caused by Smed-boule RNAi. The most severe phenotype would be the loss of germline stem cells, which are specified and maintained post-embryonically through neoblast differentiation [18]. Germline stem cells in the planarian ovaries and testes can be identified by the characteristic expression of germinal Oviparity in Sterile Smed-boule(RNAi) Flatworms histone H4 and nanos [18,20,25,26]. We tested for the presence of germline stem cells in control(RNAi) and Smed-boule(RNAi) by nanos ISH after 3-4 months of RNAi (at the end of experiments in Figs 4J-4K and 6A-6B). Whole-mount ISH analysis of germinal histone H4 and nanos expression revealed the presence of germline stem cells in the testes region of control(RNAi) animals (Figs 7A and 8A). However, germline stem cells were completely absent from the testes region of Smed-boule(RNAi) planarians (Figs 7B and 8B). Surprisingly, germline stem cells in the ovary region of both control(RNAi) and Smed-boule(RNAi) planarians were readily detectable (Figs 7A', 7B', 8C and 8D). Identical results were observed from hatchlings raised subjected to Smed-boule RNAi while maintained in isolation (S4 Fig). Furthermore, analysis of germline stem cells in presumptive testis primordia present in asexual strains of S. mediterranea (Wang et al., 2007) were also lost after Smed-boule RNAi (S5 Fig). From these results, we conclude that spermatogenesis defects in Smed-boule(RNAi) are due to the absence of male germline stem cells, whereas defects in oogenesis occur further downstream in the differentiation pathway.
To better evaluate the progression of oogenesis in Smed-boule(RNAi) ovaries, we analyzed control and Smed-boule knockdowns stained with DAPI by confocal microscopy (Fig 8E and  8F; S1 and S2 Movies). DAPI is retained by DNA and allowed for the visualization of numerous large oocytes with condensed chromosomes in the ovaries of control samples (Fig 8E; S1 Movie). In contrast, neither oocytes, cells with condensed chromosomes, or otherwise recognizable mid-to late-oogenic intermediates, were detectable in ovaries of Smed-boule(RNAi) flatworms (Fig 8F; S2 Movie). From these results we conclude that Smed-boule function is required during the initial stages of oogenesis, sometime before development of primary oocytes, but after specification of ovarian germline stem cells.
The different outcomes observed on germline stem cells of testes and ovaries following Smed-boule(RNAi) reveal that these are two fundamentally distinct germline stem cell populations that require Smed-boule function at different developmental stages. Smed-boule function is necessary for neoblast differentiation into male germline stem cells and/or maintenance of male germline stem cells, whereas ovarian germline stem cells only require Smed-boule for progression through early stages of oogenesis ( Fig 8G). Furthermore, the severe defects in germline development observed after Smed-boule RNAi further support the hypothesis that egg capsule production and deposition occur independently of gametes, ovulation, parthenogenesis, fertilization, mating, or embryonic development in S. mediterranea.

Discussion
Collectively, the data presented demonstrate that production and deposition of the egg capsules that ensure development of planarian embryos occur independently of fertilization events. Rather, it seems that egg capsule deposition, at least in S. mediterranea, is driven by intrinsic signals that are activated once these flatworms grow past a certain size and develop their yolk glands and other accessory reproductive organs. Given these findings, conclusions regarding planarian "fecundity" previously calculated from the rate of capsule production [27,28], may need to be re-evaluated. Additionally, knowledge of the separation between capsule deposition and fertility should assist in the study of planarian germline and embryonic development, as well as in generation of methodologies for transgenesis, which have proven elusive to this point.
Planarian reproduction can occur asexually through transverse fission, or sexually through post-embryonic development of a hermaphroditic reproductive system [7]. In planarians committed to sexualization, the development of gonads and gametes precedes formation of the oviducts, sperm ducts, and copulatory organs [19]. Yolk gland development in S. mediterranea, which is essential for production of egg capsules, is initiated towards the end of sexual development depending on sufficient nutritional intake and growth. The rate of egg capsule production observed in our experiments (1 to 5 egg capsules per animal per month; Fig 4J) is comparable to those observed in different planarian species both in their natural habitat and raised under laboratory conditions following a similar liver-only diet [29,30]. Therefore, we believe that the conditions used for husbandry of S. mediterranea in the laboratory are conducive to normal egg capsule production rates, and that this is not the limiting factor in reproductive output. However, the low yield of fertile egg capsules observed from control animals in our experiments (22% to 48%; Fig 4J and 4K) suggests that optimal laboratory husbandry conditions need to established to promote oocyte production, ovulation, or mating (either of which may be rate limiting in actual reproductive output).
How can triggering egg production independently of fertilization be an efficient approach to survival of planarian populations? First, we must consider that in terms of sexual reproduction, S. mediterranea performs rather poorly under laboratory conditions. This is supported by the studies of Jenkins and Brown [29] who observed D. dorotocephala yield an average of 16.5 hatchlings per egg (approximately 10-fold higher from what is observed in our laboratory for fertile egg capsules of S. mediterranea). Studies in S. polychroa have shown that siblings emerging from a single egg result from different fertilization events, which is possible because sperm  The fraction of animals displaying the phenotype represented by the image is shown at the from one or more partners can be stored for at least a month after insemination [31]. The ability to store sperm for an extended period of time after insemination, combined with the delay in development of yolk glands in comparison to the rest of the reproductive system, presents a scenario that would benefit from a mechanism that triggers capsule formation independently of copulation. In fact, it would be optimal if the activation of capsule formation also triggers ovulation of the many fully-grown oocytes present in ovaries of sexually mature planarians (Consequential Model; . It is possible that the release of early oocyte precursors from the ovary triggers capsule formation. However, this hypothesis is challenged by the fact that dozens of hatchlings often emerge from single capsules of different planarian species, and the observation that the rate of capsule production was not compromised in Smed-boule(RNAi) when compared to control planarians (which contained both oocytes and precursors). Nevertheless, current and previous observations do support a model by which a sustainable approach to oviparity could rely on a trigger for capsule formation that is independent of mating, fertilization, or ovulation.
Inside the phylum Platyhelminthes, free-living species (such as S. mediterranea) are evolutionarily distant from members of parasitic groups (Trematoda, Monogenea, and Cestoda). However, the non-causative relationship between ovulation/fertilization and capsule production appears to be conserved in some cestodes and trematodes, whose dissemination and pathology depend on the continuous production of egg capsules. Parasitic flatworms of the genus Schistosoma have been reported to produce egg capsules from females after single-sex infections of mammalian hosts [32,33]. Although female schistosomes depend on interactions with a mate to fully grow and develop their gonads, they are also able to develop some vitelline cells and immature ovaries on their own. Shaw [33] observed the production of infertile capsules from females without male stimuli, probably through mechanisms conserved with those reported here for planarian flatworms. Similarly, parasitic flatworms belonging to the class Cestoda (tapeworms) have been reported to produce unviable egg capsules in the absence of fertilization events when cultured in vitro [34,35]. Thus, given that continuous production and deposition of egg capsules is central to dissemination and pathology of different types of parasitic flatworms, the molecular machinery involved in egg capsule production (and not germline development alone) becomes a desirable target for therapeutic developments.

Planarian culture
A laboratory sexual strain of Schmidtea mediterranea [36] was used all experiments, except for those presented with asexual planarians [37] [37]. Planarians were exposed to room temperature and light during weekly feedings of pureed organic beef liver (Vantage USA, Oak Park, Illinois). Experimental animals were starved at least seven days before experimentation.

RNAi
Double-stranded RNAi feedings were performed twice every seven days and the protocol was followed as previously described [43]. DsRNA corresponding Escherichia coli ccdB sequence, which does not affect planarian development or behavior was used for unaffected control groups. For isolated RNAi samples, each planarian was fed individually and in isolation. For other experiments, planarians were maintained in groups of seven animals.

Analysis of egg capsule production and hatching
Groups of seven sexual planarians of 0.5 to 0.7 cm size and with a visible gonopore were maintained in glass Petri dishes and subjected to dsRNA feedings as described above. For isolated experiments, single 1 week-old hatchlings were maintained in isolation in glass Petri dishes throughout the experiment, under the husbandry conditions described above. Isolated planarians were fed liver containing control or Smed-boule dsRNA twice per week, at which point any capsules present were collected and isolated. DsRNA corresponding to E. coli ccdB sequence was used for control samples. Egg capsules were monitored for hatchling events weekly for a period of three months after deposition.