Characterization of Sterility and Germline Defects Caused by \u3cem\u3esmed-boule\u3c/em\u3e RNA-Interference

Evolutionarily conserved molecular processes involved in construction of the germline and embryonic development are essential for the procreation of many species. Infertility affects 15% of couples in the world and can be caused by dysfunctions during egg and sperm development, anatomic defects, as well as faulty embryonic development. Although there are some infertility disorders that are genetically defined, such as Turner and Klinefelter syndromes, many clinical infertility cases are diagnosed as idiopathic due to the lack of understanding of basic fertility mechanisms. Schmidtea mediterranea is a freshwater planarian species that has the ability to regenerate complete organisms, including germ cells and reproductive structures, from small tissue fragments containing pluripotent somatic stem cells. The developmental plasticity of planarians provides a wonderful opportunity to investigate the molecular mechanisms behind the differentiation and development of specialized cells, including gametes. Smed-boule encodes for an RNA-binding protein and is the most ancestral member of the Deleted in AZoospermia (DAZ) gene family. DAZ family genes function in different aspects of germ cell development and fertility in species ranging from sea anemone to humans. Whole-mount in situ hybridization experiments revealed Smed-boule expression is enriched in the testes and ovaries of planarian flatworms. Interestingly, Smed-boule RNA-interference (RNAi) planarians lost the ability to produce gametes, yet still were able to deposit sterile egg capsules. Virgin Smed-boule(RNAi) and control planarians maintained in isolation also continuously produced sterile egg capsules. Altogether these results demonstrate that egg capsule production in S. mediterranea occurs independently of ovulation, fertilization, and mating events. In addition, detailed analysis of gametogenesis defects revealed that Smed-boule functions at different stages during male and female germline development. These findings provide novel information about the evolution of boule and DAZ-family gene expression and function in sexual reproduction

Germline Defects Caused by \u3cem\u3eSmed-boule\u3c/em\u3e 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

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

<div><p>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 <i>Smed-boule</i>, 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 <i>Schmidtea mediterranea</i>. Reduction of <i>Smed-boule</i> 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 <i>Smed-boule</i> RNAi lose their gametes and become sterile, they continue to lay egg capsules. Production of sterile capsules is even observed in virgin <i>Smed-boule(RNAi)</i> and control planarians maintained in complete isolation, demonstrating that egg production in <i>S</i>. <i>mediterranea</i> 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 <i>Smed-boule</i> 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.</p></div

Egg capsules are produced in the absence of gametes or mating events.

<p><b>(A-B)</b> Egg capsules produced per month by planarian hatchlings (≤ 1 week old) raised in isolation on a diet of liver containing control (left) and <i>Smed-boule</i> (right) dsRNA for four months. The average number of egg capsules deposited per month per isolated individual <b>(A)</b>, as well as the total number of capsules deposited per each isolated individual throughout the experiment (single dot in <b>(B)</b>) show no significant difference (unpaired two-tailed <i>t</i>-test, <i>p > 0</i>.<i>05</i>) in egg capsule production between control and <i>Smed-boule(RNAi)</i> isolated virgins. Dashed lines in <b>(B)</b> represent the mean of total number of capsules produced per animal. Vertical lines represent standard deviation <b>(C-H)</b> The reproductive anatomy of isolated planarian virgins monitored in (A and B) was analyzed using <i>synaptotagmin XV</i> as an oocyte marker <b>(C-D)</b>, DAPI staining for testes <b>(E-F)</b> and <i>surfactant b</i> to assess yolk gland development <b>(G-H)</b>. <i>Smed-boule(RNAi)</i> lacked oocytes <b>(D)</b> and sperm <b>(F)</b> seen in control animals <b>(C and E)</b>, but developed yolk glands comparably <b>(G-H)</b>. Scale bars = 1 mm.</p

<i>Smed-boule</i> is required for maintenance of testicular germ cells, but not for all ovarian germ cells.

<p><b>(A-B)</b> Whole-mount <i>in situ</i> hybridization (ISH) analysis of neoblast and germ cell (GC) distribution through detection of <i>germinal histone H4</i> (<i>gH4</i>) mRNA in planarians subjected to three months of control <b>(A)</b> or <i>Smed-boule</i> <b>(B)</b> RNAi. GCs were detected in the ovary region (pink arrowheads) of both controls <b>(A’)</b> and <i>Smed-boule(RNAi)</i> <b>(B’)</b>. Testicular GCs (blue arrowheads) detected in control samples <b>(A”)</b> were absent in <i>Smed-boule(RNAi)</i> samples <b>(B”)</b>. The fraction of animals displaying the phenotype represented by the image is shown at the bottom-left corner of each frame. Scale bars = 1 mm.</p

<i>Smed-boule</i> expression is restricted to the planarian germline.

<p><b>(A)</b> Simplified depiction of the reproductive anatomy of planarian hermaphrodites. Reproductive organs located dorsally (top) and ventrally (bottom) are illustrated separately. <b>(B-D)</b> <i>Smed-boule</i> expression is detected in testes and ovaries by whole mount <i>in situ</i> hybridization in dorsal <b>(B and D)</b> and ventral <b>(C)</b> views of sexually mature <i>S</i>. <i>mediterranea</i>, respectively. Arrows in <b>(B)</b> indicate individual testis lobes within testes regions found dorsolaterally in the animal. Magnified view of signal from ovaries (open arrowheads) is shown in <b>(C’)</b> inset. <i>Smed-boule</i> expression is also detected in germline stem cells (black arrowheads) of 1 week-old hatchlings in the dorso-lateral region where testes develop <b>(D’)</b>, as well as continuously in maturing animals <b>(D).</b> Scale bars = 1 mm.</p

<i>Smed-boule</i> is expressed in germline stem cells of testes and ovaries.

<p>Detailed analysis by FISH and confocal microscopy of testes <b>(A)</b> and ovaries <b>(B)</b> of <i>S</i>. <i>mediterranea</i> reveals <i>Smed-boule</i> mRNA (magenta) is detected in germline stem cells, which are recognized through expression of <i>nanos</i> (green). <i>Smed-boule</i> mRNA was detected in all <i>nanos(+)</i> cells of the testes (yellow arrows) <b>(A”-A”’)</b>. <i>Smed-boule</i> mRNA was also detected in some (yellow arrows) but not all (orange arrows) <i>nanos(+)</i> cells of the ovary <b>(B’-B”’).</b> DAPI staining <b>(A and B)</b> and merged images <b>(A”’ and B”’)</b> show the nuclei of all cells present in the imaged frames. Scale bars = 25 μm.</p

<i>Smed-boule</i> encodes for a member of the germline-specific Boule-Like subfamily of proteins.

<p><b>(A)</b> Illustration of human Boule-Like (BOLL) protein architecture, which includes a RNA-recognition motif (RRM; yellow) and a DAZ domain (blue). The amino acid sequence conservation within the RRM of <i>Schmidtea mediterranea</i> and <i>Homo sapiens</i> homologs is shown. <b>(B)</b> Neighbor-joining phylogenetic tree depicting the closer association of Smed-Boule predicted amino acid sequence with members of the Boule-Like (green) subfamily of DAZ proteins, than with members of the DAZ (blue) and DAZ-Like (magenta) subfamilies. Phylogenetic analysis was performed using Clustal Omega with default parameters [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006030#pgen.1006030.ref044" target="_blank">44</a>] and sequences obtained from NCBI accession NP_932074.1, XP_001169371.2, XP_001086915.2, XP_005640556.1, NP_001095585.1, NP_083543.2, XP_006245003.1, XP_004942650.1, NP_001261614.1, XP_315505.3, NP_001005785.1, XP_001138045.3, XP_002803072.1, NP_034151.3, NP_001102884.1, NP_989549.1, NP_571599.1, NP_989079.1, NP_004072.3, XP_003319020.2, CCD81039, NP_011092, NP_001177740.1. Abbreviations used for species names included <i>Anopheles gambiae</i> (Ag), <i>Bos taurus</i> (Bt), <i>Canis lupus familiaris</i> (Cl), <i>Danio rerio</i> (Dr), <i>Drosophila melanogaster</i> (Dm), <i>Gallus gallus</i> (Gg), <i>Homo sapiens</i> (Hs), <i>Macaca mulatta</i> (Mmul), <i>Mus musculus</i> (Mmus), <i>Pan troglodytes</i> (Pt), <i>Rattus norvegicus</i> (Rn), <i>Saccharomyces cerevisiae</i> (Sc), <i>Schmidtea mediterranea</i> (Smed), <i>Schistosoma mansoni</i> (Sm), and <i>Xenopus tropicalis</i> (Xt). Scale bar represents 0.05 substitutions per amino acid position.</p

<i>Smed-boule</i> is required for germline maintenance and production of fertile capsules.

<p><b>(A-I)</b> The reproductive anatomy of RNAi animals monitored in (J-K) was analyzed by whole mount <i>in situ</i> hybridization using <i>synaptotagmin XV</i> <b>(A-C)</b> or <i>surfactant b</i> <b>(G-I)</b> as oocyte and yolk gland markers, respectively (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006030#pgen.1006030.s001" target="_blank">S1 Fig</a>). DAPI staining was used to visualize sperm development in the testes <b>(D-F)</b>. The fraction of animals displaying the phenotype represented by the image is shown at the bottom-left corner of each frame. <b>(J-K)</b> Capsule production <b>(J)</b> and hatching <b>(K)</b> from groups of sexually mature planarians subjected to continuous control, <i>Smed-boule</i>, or <i>CPEB1</i> 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 <b>(J)</b> and the number of fertile capsules <b>(K)</b> show that capsules deposited by <i>Smed-boule(RNAi)</i> animals ceased being fertile and that <i>CPEB1(RNAi)</i> ceased capsule production as a result of RNAi. Asterisks (*) represent statistically significant results compared to controls of same month by unpaired two-tailed <i>t</i>-test (<i>p > 0</i>.<i>05</i>). Scale bars = 1 mm.</p