Melav2, an elav-like gene, is essential for spermatid differentiation in the flatworm Macrostomum lignano
© Sekii et al; licensee BioMed Central Ltd. 2009
Received: 11 June 2009
Accepted: 8 December 2009
Published: 8 December 2009
Failure of sperm differentiation is one of the major causes of male sterility. During spermiogenesis, spermatids undergo a complex metamorphosis, including chromatin condensation and cell elongation. Although the resulting sperm morphology and property can vary depending on the species, these processes are fundamental in many organisms. Studying genes involved in such processes can thus provide important information for a better understanding of spermatogenesis, which might be universally applied to many other organisms.
In a screen for genes that have gonad-specific expression we isolated an elav-like gene, melav2, from Macrostomum lignano, containing the three RNA recognition motifs characteristic of elav-like genes. We found that melav2 mRNA was expressed exclusively in the testis, as opposed to the known elav genes, which are expressed in the nervous system. The RNAi phenotype of melav2 was characterized by an aberrant spermatid morphology, where sperm elongation often failed, and an empty seminal vesicle. Melav2 RNAi treated worms were thus male-sterile. Further analysis revealed that in melav2 RNAi treated worms precocious chromatin condensation occurred during spermatid differentiation, resulting in an abnormally tightly condensed chromatin and large vacuoles in round spermatids. In addition, immunostaining using an early-spermatid specific antibody revealed that melav2 RNAi treated worms had a larger amount of signal positive cells, suggesting that many cells failed the transition from early spermatid stage.
We characterize a new function for elav-like genes, showing that melav2 plays a crucial role during spermatid differentiation, especially in the regulation of chromatin condensation and/or cell elongation.
Failure of spermatogenesis is one of the major causes of male sterility. Many cases of human infertility are associated with low sperm production (oligozoospermia), poor sperm motility (asthenozoospermia) and abnormal sperm morphology (teratozoospermia) . Also, it has been shown that abnormalities of sperm chromatin, which is important for properly transmitting genetic information to offspring, can often be observed in cases of infertility . Although spermatid cells undergo a complex metamorphosis in a species-specific manner, resulting in various types of sperm morphology and other traits depending on species, fundamental processes such as the reorganization of the nucleus, cell organelles, and cell shape are found in most organisms . Thus studying genes involved in such processes can provide important information for a better understanding of spermatogenesis, which may also be applied to many other organisms.
Macrostomum lignano (Macrostomorpha, Rhabditophora, Platyhelminthes) is a simultaneously hermaphroditic flatworm, namely with male and female gonads within one individual, and a suitable model organism for gametogenesis research. An EST database is available as a source for gene information http://flatworm.uibk.ac.at/macest/. In addition, basic experimental techniques for gene analysis such as in situ hybridization and gene specific RNA interference (RNAi) are already established [5, 6]. Monoclonal antibodies against various types of cells are also available for detailed analysis of tissues . The biggest advantage of M. lignano for spermatogenesis research is its transparency, which allows non-invasive observation of cells and tissues in live animals using light microscopy and thus allows very efficient screening of RNAi phenotypes. Finally, their frequent copulation and short generation time make it easy to examine mating behavior and the degree of reproductive contribution to the next generation . Also a lot of research has been done in terms of evolutionary biology, such as sex allocation adjustment, sexual conflict, and sperm competition [9–12]. Thus studying M. lignano allows the comprehensive understanding of spermatogenesis, not only at the developmental level but also its significance in evolutionary aspects.
In M. lignano, the testes mainly consist of male germ cells such as spermatogonia, spermatocytes, spermatids, and sperm. A thin layer of tunica cells encloses the testes. After meiosis, spermatids remain in four-cell clusters with cytoplasmic connections until just before the completion of sperm maturation . Mature sperm have a complex morphology, with distinct parts easily observed in the microscope. From anterior to posterior, sperm have a feeler, a body which ends in a pair of bristles, and a shaft which ends in a brush . Within the shaft the nucleus is condensed into connected packages of compact chromatin. The overall appearance of the sperm nucleus can be compared to connected railway carriages . This morphology will be referred to as "train-shape" in this manuscript. Sperm differentiation starts with the development of the anterior part which is followed by the elongation of spermatids and then the nucleus becomes enclosed in the shaft of the mature sperm. Here we reserve the term 'sperm' for cells that have completed spermatogenesis and call the aberrant cells of the RNAi phenotype 'aberrant spermatids'.
In the process of spermiogenesis, post-transcriptional control of mRNA is very important [15, 16]. In an elongating spermatid, the nucleus has to be condensed into a compact shape, which causes cessation of transcription. Therefore, transcription of genes that are necessary for later stages has to be completed before chromatin condensation, but their translation needs to be controlled until they are needed. For this post-transcriptional control, such as transport, translational repression and storage of mRNAs, the involvement of various types of RNA binding proteins has been reported [15–19]. For example, during spermiogenesis DNA-binding histones are gradually replaced by transition proteins (Tnp1 and Tnp2) and then protamines (Prm1 and Prm2), and it is suggested that protamine-1 mRNA binding protein (PRBP) has a role for proper translational activation of prm-1 mRNA .
Elav genes are RNA binding proteins that are characterized by three RNA recognition motifs (RRMs) and a hinge region between the second and the third RRM [20, 21]. RRMs are the most common protein domains found in all kingdoms and each RRM consists of 80-90 amino acids containing two conserved sequences called RNP-1 and RNP-2 [22–25]. Structurally, a RRM has two α helices and four anti-parallel β strands, forming two β-α-β structures. It is suggested that proteins containing one or several RRMs are capable of interacting with RNA molecules [23–25]. Molecular functions of the elav gene family are quite diverse, including mRNA stability, splicing, translatability and transport [20, 26].
In this paper, we study a Macrostomum elav-like gene, melav2 (Macrostomum elav-like gene 2), using differential interference contrast microscopy, in situ hybridization, monoclonal antibodies, histology, and electron microscopy. We found that melav2 is expressed exclusively in the testes. Moreover, we show that melav2 RNA interference causes aberrant spermatid morphology, abnormal chromatin condensation, and an empty seminal vesicle leading to male-sterility. We thus prove that melav2 plays a crucial role for proper sperm differentiation and male fertility in M. lignano.
Melav2 is an elav-like gene encoding three RNA recognition motifs
Melav2 was identified and isolated during the process of screening for gonad-specific genes in M. lignano. We found this gene in our EST database with the keyword 'sex-lethal (sxl)'. Sxl is a well-known gene involved in sex determination in Drosophila melanogaster, although its function is not conserved among all insects [27–31]. However, we found that, as described in detail below, melav2 had a higher similarity with elav genes than with sxl. We therefore named it melav2 (M acrostomum elav-like gene 2) to distinguish it from melav1, another elav-like gene present in our EST database. Given the scope of our paper we focused on melav2. The entire open reading frame of this gene was obtained from the EST clone ANGU919. This gene was predicted to encode 422 amino acids.
The structure of the RRM is important for the interaction with RNA. We therefore examined the structural similarity of melav2 and other elav genes. An RRM contains two α helices and four anti-parallel β strands, forming two β-α-β structures [22–25]. Structural analysis using the SWISS-MODEL program revealed that melav2 has two such β-α-β structures in each RRM region and that they correspond to the β-α-β region of other elav genes (Figure 1A). In the third RRM of melav2, however, the β-α-β pattern was less distinct. We further analyzed melav2 using the SMART program for identification and annotation of protein domains (Figure 1B). According to this program, melav2 was considered to have three RRMs, corroborating the similar overall gene structure to other elav genes. The e-value of the first and second RRM was 4.89e-18 and 2.16e-10, respectively, and the e-value of the third RRM was 4.61e-05. All these e-values were for the RRM in the SMART database (SMART accession number SM00360).
BLASTX analysis also showed a certain similarity between melav2 and another elav related gene, sxl (data not shown). Structurally, sxl has two RRMs. The third RRM of melav2 was not well conserved compared to other elav-like genes, but despite the similarity with sxl, the phylogenetic analysis revealed that melav2 seemed to belong to the elav-like gene family (Figure 1C).
Melav2is expressed exclusively in the testes
Melav2RNAi causes an aberrant spermatid morphology
Melav2is necessary for spermiogenesis
Melav2RNAi interrupts the transition from spermatid to sperm
Melav2 encodes for an elav-like gene
Our results clearly show a new biological function of an elav-like gene, namely the involvement of melav2 in spermatid differentiation in M. lignano. Elav family genes have been mainly described to be involved in neural processes, such as the transition between proliferation and differentiation, maturation, maintenance of neurons, and learning [20, 26, 32–34] (. Recently, however, also other biological functions have been reported. For example, RBP9, a Drosophila paralog of elav, is expressed during oogenesis and is required for female fertility . EXC-7, a C. elegans ELAV homologue, is involved in the developent of excretory canals . And some elav family members are expressed in other tissues or ubiquitously , suggesting non-neuronal functions. In our study, we did not find remarkable effects of melav2 RNAi on the nervous system, although we cannot exclude the possibility of melav2 functions in neural cells in pre-hatching stages. However, we found another elav-like gene in M. lignano, melav1 (Figure 1). As previously outlined, the focus of the current study was on the testis-specific expression of melav2, and we therefore did not explore the function of melav1. We speculate that it may have a role in nervous system similar to other organism's elav-like genes (particularly given that it appears to more closely resemble these genes, Figure 1C).
Elav genes are characterized by three RRMs with a hinge region between the second and the third RRM. RRM is a quite common protein domain in eukaryotes [22, 23, 25]. For example, a recent analysis of ESTs in the planarian Schmidtea mediterranea revealed that RRM was the second most common domain . It is considered that slight changes in RRM can lead to different interactions with other proteins, leading to various types of functions . In fact, recent findings revealed diverse molecular functions for members of the elav gene family. For example, elav has been shown to regulate alternative splicing of neuroglian, erect wing and armadillo transcripts in Drosophila [40–42]. The human elav gene Hel-N1 (HuB) has been suggested to stabilize GLUT1 mRNA and to increase its translational efficiency . In melav2, the similarity of the third RRM was not well conserved compared to the first and the second RRM. Recently, it has been indicated that the Elav protein can form multimeric complexes [44, 45], and in vitro experiments suggested that the third RRM has a role for the multimerization, although this is not its only function . It might be possible that the difference in the third RRM of melav2 contributes to a new interaction with the target mRNAs involved in spermatogenesis.
Melav2is necessary for spermatid differentiation
The melav2 RNAi phenotype showed severe defects in spermatids and an abnormally condensed chromatin. We do not consider these features as signs of an apoptotic process, but instead we found several indications for necrosis. First, cell components such as mitochondria were observed outside of cells (Figure 7D), suggesting that cells were dying in a necrotic way. Second, we observed neither nuclear fragmentation nor an apoptotic body, which are typical characteristics of apoptosis also in M. lignano . Third, cells often showed cytoplasmic vacuolization (Figure 7D), which is unusual for apoptosis. Fourth, we observed an accumulation of cells that failed to proceed properly into the later spermatid stages (Figure 8B), suggesting that the elimination of such failed cells by programmed cell death did not occur.
We propose two possible explanations for the abnormal chromatin condensation and the failure of spermatid elongation. One possibility is that melav2 regulates the genes that mediate chromatin condensation, and chromatin condensation occurred too early as a result of melav2 RNAi, leading to cessation of further sperm maturation, possibly due to an early stop in the transcription of essential genes. A similar phenotype was shown in spermiogenesis of transgenic mice where pre-mature translation of prm-1 occurred . Prm-1 should be repressed until a later spermatid stage for a proper transition from nucleohistones to nucleo-protamines. Premature translation of prm-1 caused early chromatin condensation in round spermatids, a subsequent arrest in spermiogenesis and an aberrant spermatid morphology. These features correspond well with the melav2 RNAi phenotype shown here (Figure 7, 8 and 5, respectively) and therefore it appears possible that melav2 has a function in regulating prm-1 or other comparable genes.
Another possible explanation is that cells retained their round shape because of the disruption of spermatid elongation. It has been reported that a human neuronal Elav protein, Hel-N1 (HuB) gene, upregulates the translation of neurofilament M (NF-M) mRNA by using human embryonic teratocarcinoma cells (hNT2) transfected with Hel-N1 . Overexpressed Hel-N1 did not affect the level of NF-M mRNA expression but instead recruited it into heavy polysomes more efficiently, resulting in the formation of neurites. If a similar molecular function is performed by melav2, it is possible that melav2 is involved in promoting translation of some cytoskeletal elements such as sperm-specific intermediate filaments. The shortage of these elements caused by melav2 RNAi could result in cessation of elongation, as is often seen in the posterior part at later spermatid stage (Figure 5F-I) and the failed chromatin reorganization, in which the chromatin was condensed but never built in a train shape (Figure 7D).
Although melav2 has three RRMs, we have no direct evidence whether melav2 really functions as RNA-binding protein and, if so, what kinds of mRNA are targeted. Thus, identifying the target genes of melav2 and their interaction might be important follow-up experiments. Recently, a transcriptome and a genome sequencing project have been initiated in M. lignano. Therefore it will soon be possible to identify melav2 downstream genes by microarrays or in silico comparison of transcriptomes of control animals with melav2 RNAi treated worms.
Melav2RNAi treated worms are male-sterile
The seminal vesicles of melav2 RNAi treated worms were empty, and in consequence they cannot transfer sperm to a mating partner, suggesting they are male-sterile. However, M. lignano is hermaphroditic and the female reproductive function of melav2 RNAi treated worms was normal (Figure 3). So a melav2 RNAi treated animal (or a loss of function mutant) would still be able to contribute offspring to next generation as a female after copulation with normal worms. Thus, future experiments aimed at following their reproductive success of such 'females' and determining if such functional females could spread within a hermaphroditic population, would provide a better understanding of the evolution of different reproductive modes. In addition, the formation of the stylet and the male accessory glands in melav2 RNAi treated worms was normal (Figure 3), which offers interesting possibilities for studies on sexual conflicts. Recent experiments have suggested that male accessory glands and their secretions have important roles for increasing male reproductive success by manipulating the female reproductive physiology and behavior, such as increasing egg production, decreasing female's motivation toward further copulations with other partners, and decreasing female's longevity [50–52]. Although we still need to analyze if melav2 RNAi treated worms can in fact transfer ejaculates without sperm, such worms have a great potential to study the effects of seminal fluids in hermaphroditic organisms, by removing the effect of the transferred sperm itself.
We found that melav2 has a similarity with the elav gene family at the protein sequence level. Elav genes are mainly known to be involved in the nervous system in other organisms, but our study clearly shows that melav2 plays a crucial role during spermatid differentiation in M. lignano. Melav2 RNAi disturbed the proper regulation of chromatin condensation and/or the cell elongation, resulting in aberrant spermatid morphology and male sterility.
The free-living flatworm M. lignano  was cultured in glass Petri dishes with nutrient-enriched artificial seawater (Guillard's f/2 medium ) and fed with the diatom Nitzschia curvilineata. The worms were kept in a climate chamber with 60% humidity at 20°C in a 14:10 h day-night cycle . Animal experimentation was carried out in accordance to Austrian legal and ethical standards.
Gene isolation and analyses
The full length open reading frame of the clone ANGU919 http://flatworm.uibk.ac.at/macest/ was sequenced by GATC (Konstanz, Germany) using M13 standard primers. Sequences were analyzed by BLASTX searches at the EMBL-EBI database http://www.ebi.ac.uk/Tools/blast2/. Conserved protein domains were identified using the SMART databases http://smart.embl-heidelberg.de/. Protein structural analysis was performed using the SWISS-MODEL program http://swissmodel.expasy.org/[56, 57].
Alignment and phylogenetic analyses
Amino acid alignments were performed using the Multiple Sequence Alignment Program CLUSTALW of the EMBL-EBI database http://www.ebi.ac.uk/Tools/clustalw2/ with default alignment parameters. The phylogenetic tree was calculated using the MrBayes 3.2.2 [58, 59]. Each run was performed using default parameters and comprised 5,000,000 generations.
Elav sequences were from Mus musculus ELAV-like protein1 [UniProtKB:P70372], Xenopus laevis ELAV-like2 [GenBank:NP_001081035], Danio rerio ELAV-like 1 [GenBank:NP_571527], Drosophila melanogaster Protein elav [UniProtKB:P16914], Aplysia californica ELAV 2-like protein [GenBank:AAY42042], Platynereis dumerilii Elav [GenBank:ABO93208]. Elav sequences of Schmidtea mediterranea and Hydra magnipapillata were generated by computational work using available EST database, SmedGD v1.3.14 http://khan.neuro.utah.edu/index.html and dbEST_HMAG070214 http://www.compagen.org, respectively. The Schmidtea sequence was generated from contig ec1.03596.005, and the Hydra sequence was generated by assembling three clones, tai96e09.y2 [CX056199], tai87h08.y1 [CV182751] and tai87h08.x1 [CV182482]. Melav1, another elav-like gene from M. lignano was obtained from EST clone Ml_aw_006_D03 in a same way as the melav2 gene.
Sex lethal sequences were from Drosophila melanogaster Protein sex-lethal [UniProtKB:P19339], Drosophila subobscura Protein sex-lethal [UniProtKB:Q24668], Musca domestica Sex-lethal homolog [UniProtKB:O17310], Ceratitis capitata Sex-lethal homolog [UniProtKB:O61374], Chrysomya rufifacies Sex-lethal homolog [UniProtKB:O97018], Megaselia scalaris Sex-lethal homolog [UniProtKB:O01671], Anopheles gambiae TPA:sex-lethal [GenBank:CAJ55784], Acyrthosiphon pisum sex-lethal [GenBank:NP_001119609], Bombyx mori sex-lethal [GenBank:NP_001036780]. As an outgroup for the calculation of phylogeny, Drosophila melanogaster CG4787-PA [UniProtKB:Q9VAX1] was used. Square brackets indicate accession number.
Whole mount in situhybridization
The sequence region for the in situ RNA probe was amplified with the melav2 specific primers 5'-GGC CTT CTC AGA TGA CGA GT-3' and 5'-GGA CAG ATG TTG ATG GAC CTG-3'. The PCR condition was 2 min at 94°C, 35 cycles (30 sec at 94°C, 30 sec 55°C, 90 sec at 72°C), 7 min at 72°C. Obtained PCR products were sub-cloned into pGEM®-T (Promega). Then PCR with M13 standard primers was performed to generate the template for RNA probe synthesis, including SP6 and T7 RNA polymerase promoter sequences. Digoxygenin-labeled RNA probe was generated using DIG RNA Labeling KIT SP6/T7 (Roche), following the manufactor's protocol. Whole mount in situ hybridization for M. lignano was performed as previously described . The signal was developed at 37°C using the NBT/BCIP system (Roche). Specimens were examined with a Leica DM5000 microscope. Image acquisition and analysis were performed using a Leica DFC490 digital camera, the Leica Application Suite 2.8.1 software, and the Adobe® Photoshop® 7.0 software.
By adding T7 RNA polymerase promoter sequence to the primers that were used to generate the templates for in situ RNA probe, two different templates for dsRNA probe were generated: one had a T7 promoter at the 5' end for producing sense RNA, and the other had it at the 3' end for producing anti-sense RNA, respectively. The PCR condition was 2 min at 94°C, 35 cycles (30 sec at 94°C, 30 sec 55°C, 90 sec at 72°C), 7 min at 72°C. The in vitro synthesis of dsRNA was performed using T7 RiboMax TM Express RNAi System (Promega). As a negative control, firefly luciferase dsRNA was produced from the pGEM®-luc Vector (Promega). RNAi treatment for M. lignano was performed by soaking as previously described . We used 31 animals for control and 55 animals for melav2 RNAi treatment. One-day old hatchlings were maintained in 24-well plates (20 worms per well) and incubated with 250 μl dsRNA solution (3.0 ng/μl) in f/2 medium containing the antibiotics Kanamycin and Ampicillin (50 μg/ml, respectively) and diatoms. dsRNA solution was changed twice a day. After 3-4 weeks of RNAi treatment, the specimens were examined with a Leica DM5000 microscope using DIC optics. Image acquisition and analysis were performed using the same imaging set-up as described in the "Whole mount in situ hybridization" section. For the observation of testis components and sperm morphology, live worms were squeezed under a cover slip until the testis ruptured and the contents of the testis were directly observed.
Control and melav2 RNAi treated animals were relaxed in 7.14% MgCl2 and fixed for 1 hour at 4°C in 2.5% glutardaldehyde in 0.1 M cacodylate buffer (pH 7.4) containing 9% sucrose. After several washes with buffer, specimens were postfixed in 1% osmium tetroxide in 0.05 M cacodylate puffer (pH 7.4) for 1 hour. After washing in buffer and subsequent dehydration in an ethanol series, animals were embedded in SPURR"s low viscosity resin . Complete series of 1.5 μm thick semi-thin sections were cut with a prototype of a Butler diamond knife (Diatome) and mounted on glass slides. After drying the sections were stained for 2 minutes in a methylen blue Azur II mixture after Richardson  and mounted in cedar wood oil. One control animal and one melav2 RNAi treated animal were examined with a Leica 5000B microscope. Image acquisition and analysis were performed using the same imaging set-up as described in the "Whole mount in situ hybridization" section.
Histology after whole mount in situhybridization
After performing melav2 whole mount in situ hybridizations as described above, specimens were fixed with BOUIN's fluid overnight. After subsequent dehydration in an ethanol series, animals were embedded in SPURR's low viscosity resin. Complete series of 2 μm thick semi-thin sections were cut with the same sectioning set-up as described in the "Histology" section. Two animals were examined with a Leica 5000B microscope. Image acquisition and analysis were performed using the same imaging set-up as described in the "Whole mount in situ hybridization" section.
Transmission electron microscopy
Control and melav2 RNAi treated animals were fixed and embedded as described above. Semi-thin and ultra-thin sections were cut with a diamond knife on an Ultracut S (Leica) ultramicrotome, double stained with uranyl acetate and lead citrate. One control animal and one melav2 RNAi treated animal were examined with a ZEISS Libra 120 energy filter electron microscope. Image acquisition and analysis were performed using a 2 k Vario Speed SSCCD camera (Droendle) and the iTEM software (TEM imaging platform, Olympus).
Immunocytochemistry for M. lignano was performed as previously described [6, 7]. As the primary antibody, the spermatid-specific mouse monoclonal MSp-1 antibody  was used (1:200). As the secondary antibody, a FITC-conjugated goat-anti-mouse antibody (1:250, DAKO) was used. Two control animals and three melav2 RNAi treated animals were examined with a Zeiss LSM 510 confocal laser scanning microscope system. Image acquisition and analysis were performed using the LSM510 ver.3.2 software, the LSM image browser ver.188.8.131.52 software and the Adobe® Photoshop® 7.0 software.
RNA recognition motif.
We are very grateful to the people at the Institute of Zoology, University of Innsbruck. Especially, we thank Daniela Pfister and Georg Kuales for the discussion about molecular biological work, Helene Heiss for the information about early spermatogenesis, Bernhard Egger for the discussion about flatworms, Thomas Ostermann for the support of computational work, and Anna-Maria Mitterstiller for the management of experimental materials. Also, we are very grateful to Dita Vizoso at the University of Basel for the critical and helpful comments on earlier versions of the manuscript. We are also grateful to three anonymous reviewers for improving the quality of our manuscript. KS was supported by a Swiss Government Scholarship for Foreign Students. This work was further supported by a personal FWO grant to KDM (Belgium), a SNF grant (3100A0-113708) to LS (Switzerland), and a FWF grant (18099) to PL (Austria).
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