Tex19 genes are mammalian specific and duplicated to give Tex19.1 and Tex19.2 in some species, such as the mouse and rat. It has been demonstrated that mutant Tex19.1 males display a variable degree of infertility whereas they all upregulate MMERVK10C transposons in their germ line. In order to study the function of both paralogs in the mouse, we generated and studied Tex19 double knockout (Tex19DKO) mutant mice. Adult Tex19DKO males exhibited a fully penetrant phenotype, similar to the most severe phenotype observed in the single Tex19.1KO mice, with small testes and impaired spermatogenesis, defects in meiotic chromosome synapsis, persistence of DNA double-strand breaks during meiosis, lack of post-meiotic germ cells and upregulation of MMERVK10C expression. The phenotypic similarities to mice with knockouts in the Piwi family genes prompted us to check and then demonstrate, by immunoprecipitation and GST pulldown followed by mass spectrometry analyses, that TEX19 paralogs interact with PIWI proteins and the TEX19 VPTEL domain directly binds Piwi-interacting RNAs (piRNAs) in adult testes. We therefore identified two new members of the postnatal piRNA pathway.
In all sexually reproducing species, germ cells are the cell lineage that is specialized in transferring genetic information from one generation to the next (Hajkova, 2011). The genome of germ cells needs to be maintained stably and protected in all individuals throughout development. In particular, the genome must be protected against mobile transposable elements (TEs). These TEs can move, amplify themselves and transpose into new genomic loci prompting a serious threat to the genome. Although most TEs are inert, some elements have retained activity and become typically expressed in the germ line where they can transpose into new copies and pass onto the next generation. TEs account for almost half of the human and mouse genomes. They are classified into two categories, class I, which represents the majority of the TE families and class II, which accounts for ∼3% of the mammalian genome (Zamudio and Bourc'his, 2010; Goodier and Kazazian, 2008). Class I TEs are believed to be ancient traces of viral infections; they transpose themselves by using a ‘copy and paste’ mode involving an RNA intermediate, which is converted into cDNA before its integration. Class II TEs are DNA transposons that transpose using a ‘cut and paste’ mode without any transcription needed (Goodier and Kazazian, 2008; Lander et al., 2001; Zamudio and Bourc'his, 2010).
Despite an undeniable role in evolution (Furano et al., 2004; Ostertag and Kazazian, 2001; Zamudio and Bourc'his, 2010), TEs can be responsible for drastic mutagenic effects, as illustrated by the growing number of human diseases caused by their transposition and expression (Cowley and Oakey, 2013). Germ cells have developed sophisticated strategies to control TE activities, for example, at the transcriptional level through DNA methylation and the post-transcriptional level through RNA degradation or by blocking their integration (Gasior et al., 2006; Obbard et al., 2009; Rollins et al., 2006; Yoder et al., 1997; Zamudio and Bourc'his, 2010). For reasons that remain unclear, the control of TEs in the mammalian germ line is gender specific. Indeed, it has been shown that the protection of the male germline genome relies on the PIWI family of Argonaute proteins, which produce and bind small RNAs of ∼24–30 nucleotides long (named Piwi-interacting RNAs, piRNAs) (Ghildiyal and Zamore, 2009). In mice, the PIWI clade contains three members, namely MIWI (PIWIL1), MILI (PIWIL2) and MIWI2 (PIWIL4) (Kuramochi-Miyagawa et al., 2004), which interact with germline-specific members of the TUDOR family proteins, such as TDRD1, TDRD5, TDRD6, TDRD7 and TDRD9 (Chuma et al., 2003; Hosokawa et al., 2007; Vasileva et al., 2009; Zheng and Wang, 2012). They also interact with other factors such as MOV10L1, MVH (DDX4), MAEL or GASZ (ASZ1) (Costa et al., 2006; Ma et al., 2009; Soper et al., 2008; Zheng et al., 2010; Kotaja et al., 2006; Kuramochi-Miyagawa et al., 2004). Mutation of these piRNA pathway members give rise to a similar phenotype consisting of male infertility, due to meiosis defects that can take place at different stages of meiosis, and lead to an upregulation of most TEs (Zamudio and Bourc'his, 2010). Despite a large number of studies, particularly involving loss-of-function mice, the exact mechanism whereby piRNAs control TE expression and the link between the loss of TE control and meiosis defects are far from fully understood. Currently, only the process in males is beginning to be deciphered while that in females remains to be discovered.
In 2001, Wang et al. described a set of testis-expressed (Tex) genes, including Tex19 (Wang et al., 2001). In a previous study, we showed that Tex19 is a mammal-specific gene, which is duplicated in the mouse genome into two paralogs, Tex19.1 and Tex19.2. By multiple sequence alignment of mammalian TEX19 proteins, two highly conserved domains, named MCP and VPTEL, respectively, were identified. However, neither of them has any homology with known proteins, preventing functional prediction (Kuntz et al., 2008). Both genes are co-expressed in the ectoderm, then in primordial germ cells (PGCs). Later on, they are co-expressed from embryonic day (E)13.5 until adulthood in testes. However, only Tex19.1 transcripts are present in developing and adult ovaries as well as in the placenta (Celebi et al., 2012). Tex19.1-knockout (Tex19.1KO) mice show a variable degree of male infertility due to impaired meiosis associated with an upregulation of MMERVK10C retrotransposon expression (Öllinger et al., 2008; Tarabay et al., 2013). More recently, it was shown that Tex19.1 plays an important role in placenta function, as Tex19.1KO mouse embryos exhibit intra-uterine growth retardation and have small placentas due to reduced number of spongiotrophoblast, glycogen trophoblast and sinusoidal trophoblast giant cells (Reichmann et al., 2013; Tarabay et al., 2013). Tex19.2KO mice are fertile presenting only a subtle phenotype with discrete seminiferous tubule degeneration in adult male testes (Y.T., M.T. and S.V. unpublished observations). The co-expression of Tex19.1 and Tex19.2 in PGCs, gonocytes and spermatocytes suggests that these two genes could play redundant functions during spermatogenesis.
By generating a double knockout of both Tex19.1 and Tex19.2 genes (Tex19DKO), we demonstrate here that TEX19 paralogs exhibit redundant functions, which are essential for male fertility. We show that the Tex19DKO phenotype is fully penetrant, mimicking the most severe phenotype observed in single Tex19.1KO mice. All adult DKO males are infertile and display impaired spermatogenesis: meiosis is blocked at the pachytene stage – during which chromosome synapsis is not correctly formed or incomplete – and this leads to testis degeneration. Furthermore, MMERVK10C retrotransposon expression is upregulated during meiosis in Tex19DKO testes. Through GST pulldown and immunoprecipitation (IP) experiments, we found that TEX19 paralogs interact with PIWI proteins and their accessory partners; moreover, they have the ability to bind RNAs of ∼30 nucleotides, a diagnostic size for piRNAs, but are not required for their biogenesis. Detailed analysis further showed that TEX19 directly interacts with piRNAs via its VPTEL domain. Taken together, our study provides novel evidence that TEX19 proteins are new members of the PIWI/piRNA pathway acting during post-natal spermatogenesis.
Tex19 paralogs play an essential role in male fertility
Tex19.1KO mice show a variable level of male infertility: one third of the KO male mice are almost indistinguishable from wild-type (WT) mice, one third display a mild phenotype and the last third a severe phenotype with a complete meiosis arrest at pachytene (Öllinger et al., 2008; Tarabay et al., 2013). Independently of the severity of the phenotype, a twofold upregulation of MMERVK10C elements was consistently observed among Tex19.1KO animals (Öllinger et al., 2008; Tarabay et al., 2013). In contrast, Tex19.2KO mice are fertile and their testes are undistinguishable from controls. However, some seminiferous tubules show epithelium vacuolization (Y.T., M.T. and S.V. unpublished observations). Considering the overlapping profile of Tex19.1 and Tex19.2 expression (Celebi et al., 2012), we wondered whether there was any functional redundancy between the two genes in testes. We therefore generated conditional double KO mice for Tex19.1 and Tex19.2 genes, which exist as tandem duplication (Fig. 1A).
The KO of both alleles (called Tex19DKO) was confirmed by PCR (Fig. 1B) and verifying the absence of the encoded proteins by western blotting of testes extracts from double wild-type (DWT), Tex19.1KO, Tex19.2KO or Tex19DKO samples (Fig. 1C and data not shown).
Interbreeding of Tex19 double heterozygote (DHZ) animals resulted in a statistically significant deviation of the frequency of double homozygous animals from the expected Mendelian 1:2:1 ratio [207 (29.3%) Tex19DWT mice, 440 (62.3%) Tex19 double heterozygote (DHZ) mice and 59 (8.3%) Tex19DKO mice, P<0.05, Student's t-test]. Yang et al., 2010 previously described higher level of lethality in females among Tex19.1KO animals, an observation which was not shared by other independent studies including ours (Öllinger et al., 2008; Tarabay et al., 2013; Yang et al., 2010). Nonetheless, we found that the number of Tex19DKO females surviving for 2 weeks or more was significantly reduced when compared to Tex19DKO males (32.2% of animals were females and 67.8% males, P<0.05), suggesting a gender-specific lethality. Surviving Tex19DKO mice did not present gross somatic abnormalities. Females displayed normal fertility, but males were sterile.
Tex19DKO males displayed a constant and severe reduction in testis size (Fig. 2A). The weight of Tex19DKO adult testes (from 19 mg up to 35 mg, mean 24.4 mg±10.5 mg per testis) was threefold less than that of WT testes (from 109 mg up to 135 mg, mean 113.5 mg±5.52 mg per testis) (Fig. 2B). In contrast to WT seminiferous tubules (Fig. 2C), Tex19DKO testes histology showed degenerate seminiferous epithelium with tubules containing only early spermatogenic cells, but a complete lack of post-meiotic germ cells, suggesting a meiotic arrest (Fig. 2D); consequently, the epididymis was free of spermatozoa (Fig. S1A,B). Adult Tex19DKO seminiferous tubules exhibited a drastically increased level of apoptosis when compared to WT (Fig. S1C). This phenotype is comparable to the most severe phenotype observed in the single Tex19.1KO (Tarabay et al., 2013). Thus, Tex19 paralogs are required for male meiosis and are essential for male fertility.
To further investigate the meiotic defect of Tex19DKO mutants, we tested the assembly of the synaptonemal complex by co-immunostaining of the central and lateral elements with antibodies against SYCP1 and SYCP3, respectively. In contrast to WT spermatocytes where all autosomes were fully synapsed at pachytene (Fig. 2E), synapsis failed to occur properly in Tex19DKO, as evidenced by the complete or partial absence of the SYCP1 signal (Fig. 2F). Furthermore, as progression of homologous recombination and chromosome synapsis are interdependent, we checked whether homologous recombination was also affected by studying DNA double-strand break (DSB) distribution, by immunostaining against phosphorylated histone γH2AX. γH2AX staining is normally present throughout chromatin during the zygotene stage. As synapsis proceeds, the DSBs are resolved, resulting in γH2AX disappearance from autosomes, but not from the sex chromosomes. In WT pachytene cells, synapsis was complete and only the sex chromosomes were stained for γH2AX (Fig. 2G). However, the incompletely synapsed pachytene chromosomes in Tex19DKO nuclear spreads exhibited strong diffuse γH2AX staining, suggesting that the disruption of Tex19 paralogs caused meiotic arrest at the pachytene stage because of the persistence of DSBs (Fig. 2H). Alternatively, γH2AX retention in spermatocytes from Tex19DKO may be a consequence of asynapsis.
In order to date the developmental onset of the Tex19DKO phenotype, we analyzed testis sections at post-natal day (P)10, P16 and P20 and at 16 weeks of age (Fig. 3). At P10, Tex19DKO sections were histologically indistinguishable from WT and Tex19DHZ testes (Fig. 3A–C). Tex19DKO showed vacuolization and seminiferous epithelium degeneration as soon as P16 (Fig. 3D–F). Thus, the Tex19DKO testes phenotype became histologically visible between P10 and P16, corresponding to the timing when the first spermatocytes reach the pachytene stage during the first post-natal wave of spermatogenesis. This confirmed a spermatogenesis arrest occurring between the zygotene and the early pachytene stages of meiosis prophase I.
Finally, increased TE expression has been proposed to be responsible for impaired chromosome synapsis and meiotic defects during spermatogenesis in Dnmt3L, Miwi2 and Mili mutant mice (Aravin et al., 2007; Bestor and Bourc'his, 2004; Carmell et al., 2007; Kuramochi-Miyagawa et al., 2004; Zamudio et al., 2015). Overexpression of MMERVK10C retrotransposons could similarly be responsible for the meiotic defects seen in Tex19.1KO mice (Öllinger et al., 2008; Tarabay et al., 2013). In Tex19.DKO mice, we observed a twofold and fourfold upregulation of MMERVK10C expression at P10 and P16, respectively; no upregulation was observed at other TEs (LINEs and IAP) (Fig. S2), confirming the specific impact of Tex19 deficiency in the control of MMERVK10C.
TEX19 associate with PIWI proteins and pachytene piRNAs
To gain insight into TEX19 function, we sought to further identify its protein partners through GST pulldown and IP experiments. For the GST pulldown experiment, GST alone, GST–TEX19.1 or GST–Tex19.2 was incubated with total testicular protein extract of adult mice (Fig. S3); complexes were then analyzed by mass spectrometry (MS). In GST–TEX19.1 and GST–TEX19.2 samples, we detected bands corresponding to MAEL, MIWI, MILI, TDRD6, RANBP9 and MVH, while these bands were absent in the GST-only sample (Fig. 4B). These interactions were further confirmed by western blotting after co-immunoprecipitation experiments. Using a specific anti-TEX19.1 monoclonal antibody, we further immunopurified TEX19.1 complexes in adult testes and identified individual polypeptides by MS. Among the identified proteins, we found MAEL, MIWI, MILI and MVH, which were already identified by GST pulldown, but also TDRD8, TDRD6, EDC4, PABPC2, PSMC3, RANBP9, ANGEL1, SRPK1, DDX20 and DDX46. We confirmed the presence of MAEL, MIWI, MILI, MVH, EDC4, TDRD6 and RANBP9 in TEX19.1 immunopurified complexes isolated from cytoplasmic adult testes extracts by western blotting (Fig. 4C,D). These interactions were further validated by the reciprocal detection of TEX19.1 in immunopurified MAEL, MIWI, MILI, MVH, EDC4 and RANBP9 complexes (Fig. 4D). Taken together these results suggest that TEX19.1 and TEX19.2 interact with proteins of the post-natal PIWI/piRNA pathway, namely core components such as MIWI and MILI, and accessory proteins such as EDC4, TDRD6, RANBP9 and MVH.
The identification of TEX19 paralogs as partners of the PIWI proteins along with the phenotypic similarities observed between Tex19KO mice and piRNA mutant mice prompted us to check whether TEX19s could bind small RNAs, and in particular piRNAs. For this purpose, we immunoprecipitated TEX19.1, TEX19.2 and MILI (as a positive control) in adult mouse (aged 12 weeks) testes extracts and assessed for the presence of small RNAs by 32P 5′ end-labeling and gel electrophoresis (Fig. 5). As expected, MILI associated with ∼26–30 nucleotide (nt) small RNAs. TEX19.1 purification also revealed an interaction with small RNAs ranging in size between 26 and 30 nt, migrating similarly to MILI-associated piRNAs. Using the 6Tex:4D2 antibody, which recognizes both TEX19 paralogs, on an adult Tex19.1KO testicular extract from a normal size testis from a 12-week-old male, we showed that TEX19.2 also interacted with small RNAs of the same size range (Fig. 5). All bands disappeared upon RNase treatment, confirming that these entities were RNA. Furthermore, no such band was detected in the ascitic fluid control sample or in the sample precipitated with a specific anti-TEX19.1 monoclonal antibody in Tex19.1KO testicular extract, confirming the specificity of the interaction between TEX19.1 and small RNAs. Thus, both TEX19 paralogs interact with small RNAs in a size range compatible with piRNAs.
To further identify the small RNA species associated with TEX19 proteins, we generated small RNA-seq libraries by Illumina sequencing after TEX19 and TEX19.1 IP from adult WT testes. To infer the TEX19.2-bound fraction, we analyzed TEX19 complexes in Tex19.1KO testes. Size distribution and genomic content were then compared to available datasets from MILI and MIWI-bound piRNAs from adult testes (Robine et al., 2009). MIWI was formerly shown to preferentially associate with 29–30 nt piRNA species, while the main piRNA size in MILI complexes is shorter, at ∼26–27 nt. Interestingly, TEX19-, TEX19.1- and TEX19.2-associated small RNAs were found to segregate in size with MIWI-bound piRNAs, with a diagnostic 30 nt size (Fig. 6A). TEX19.2 showed a stronger association to 22–23 nt long RNAs, which is a diagnostic size for microRNAs; however, as small RNA libraries are expressed as relative rather than absolute values, this may reflect a weaker affinity of TEX19.2 towards piRNAs but a genuine preferential association with miRNAs cannot be excluded.
Because the three libraries were very similar in size and content, but the TEX19.1 sample had a better quality, we focused the rest of our analysis on the latter. Out of 1.6 million reads, we noticed a strong preference (94.42%) for a uridine at position one (U1 bias), in agreement with the biogenesis mode of piRNAs (Aravin et al., 2006; Frank et al., 2010; Kawaoka et al., 2011) (Table S1, Fig. S4). The genomic distribution appeared identical to both MILI and MIWI IPs (Fig. 6B): the majority (70%) of the TEX19.1-derived reads originated from intergenic regions, while less than 20% of the reads mapped to repeated elements. This is in line with the known genomic distribution of pachytene piRNA species, which are produced in adult testes from a discrete number of unique and intergenic piRNA clusters. Accordingly, 90% TEX19.1-associated reads uniquely mapped onto the genome (Fig. 6C), and, moreover, we found the large majority of TEX19.1-associated small RNAs to be derived from the same piRNA loci clusters previously defined as regulated by MILI and MIWI (Fig. 6D) (Beyret et al., 2012). We nonetheless examined the content of the repeated fraction of the TEX19.1 library and found that ∼50% were derived from long terminal repeat (LTR) retrotransposons, among which ∼1% belonged to the MMERVK10C class (Fig. 6B). This means that among all 25–30 nt-bound RNAs, only 0.1% derived from MMERVK10C elements. Finally, to address the role of TEX19.1 in the biogenesis of piRNAs, we sequenced whole testes small RNA libraries in three adult (12 weeks of age) Tex19.1KO animals showing normal size testis: the size distribution appeared similar to WT testes, and, in particular, there was no relative redistribution towards 22–23 nt microRNA species, as an indication of impaired piRNA production (Fig. 6E). Enrichment in 30 nt small RNAs was even higher in the Tex19.1KO background compared to WT: this likely reflects different cellular composition, and in particular an overrepresentation of pachytene cells in Tex19.1KO testes, due to the meiotic arrest. Taken together, these results suggest that TEX19 proteins sample the same set of primary, unique piRNA species as MILI and MIWI, at the time of their production at the pachytene stage in the adult testis. However, they are not required for their synthesis, and likely act as simple cargo.
TEX19 paralogs interact directly with piRNAs
In order to assess a direct interaction between TEX19.1, TEX19.2 and piRNAs, we performed electrophoretic mobility shift assays (EMSA) using GST–TEX19.1 and GST–TEX19.2 proteins as well as different domains of TEX19.1 fused to GST (Figs S5 and S6) against the piR-117061 piRNA. We choose this piRNA because it has a very significant absolute count (2361 and 2028) in two biological replicates prepared using anti-TEX19.1 antibody (Table S2) and it has a U at position one (denoted U1). We found that the piR-117061 was ‘shifted’ up upon electrophoresis in the presence of GST–TEX19.1, GST–TEX19.2, GST–VPTEL-Cter (amino acids 125–351) or GST–VPTEL of TEX19.1, but not in the presence of GST alone or GST–MCP (amino acids 1–124) or GST–Cter (amino acids 163–351) of TEX19.1. This result stresses the specificity of the interaction between piR-117061 and TEX19.2 and TEX19.1 via the VPTEL domain (Fig. 7A). The specificity of this interaction was further confirmed by showing that a modified form of piR-117061, in which we replaced U1 and A10 by a C nucleotide was unable to bind to TEX19.1, its VPTEL domain or TEX19.2 (Fig. 7B) and was not able to replace piR-117061 in a competition experiment (Fig. 7C). By adding an increased concentration of the non-radiolabeled piR-117061 to a fixed concentration of GST–VPTEL (5 µM pmole) and radiolabeled piR-117061, we showed that the non-radiolabeled piR-117061 completely displaced the radiolabeled piR-117061 when the concentration was 100-fold higher (Fig. 7D). Finally, we showed that the quantity of piR-117061 ‘shifted’ up was proportional to the quantity of the TEX19.1 VPTEL domain (Fig. 7D).
Tex19 genes are specific to mammals and can be found as a tandem duplication in the mouse and rat genomes. In the mouse, Tex19.1 and Tex19.2 expression is restricted to the germ line and the placenta (Celebi et al., 2012). Analyses of Tex19.1KO mice have previously highlighted a variable phenotype. In the severest form, Tex19.1-deficient males were infertile and presented an arrest at the pachytene stage of meiosis in association with a failure to silence MMERVK10C elements (Öllinger et al., 2008; Tarabay et al., 2013). Tex19.2KO males are fertile; however, a subtle phenotype of testes degeneration could be observed in adult mice (Y.T., M.T. and S.V. unpublished observations). The present study describes the functional consequences of deleting both TEX19 paralogs and the involvement of these proteins in the piRNA pathway. Surviving Tex19DKO male and female mice were perfectly healthy, but males exhibited a fully penetrant phenotype similar to the severest Tex19.1KO phenotype (Öllinger et al., 2008; Tarabay et al., 2013). Indeed, Tex19DKO males have highly degenerated testes, with a lack of germ cells beyond the pachytene stage, which is linked to defects in chromosome synapsis during meiosis. Compared to Tex19.1KO testes (Öllinger et al., 2008; Tarabay et al., 2013), we showed, here, a stronger upregulation of MMERVK10C retrotransposon in the double knockout (Tex19DKO) testes of twofold at P10 and fourfold at P16 compared to WT counterparts. This fully penetrant phenotype confirms the hypothesis of a functional redundancy between Tex19.1 and Tex19.2. Moreover, this phenotype bears striking resemblance to the Dnmt3L, Miwi2 and Mili mutant phenotypes (Aravin et al., 2007; Bestor and Bourc'his, 2004; Carmell et al., 2007; Kuramochi-Miyagawa et al., 2004). One explanation was proposed by Zamudio et al., who suggested that improper TE silencing could alter the meiotic process through local remodeling of the chromatin landscape (Zamudio et al., 2015).
The essential role of TEX19 may extend beyond the context of spermatogenesis as we report here a severe lethality of Tex19DKO animals, with only 8.3%, instead of the expected 25%, mutant animals surviving at over 2 weeks of age. Moreover, this lethality was sexually dysmorphic as among the survivors, only one third were females. We did not observe such lethality in our single Tex19.1KO mouse models (Tarabay et al., 2013), but Yang et al. (2010) had reported such female-specific lethality in their Tex19.1KO mice and suggested that the variable severity among different studies could be linked to strain specificities (Yang et al., 2010; Kwon et al., 2003). However, our studies on Tex19.1KO or Tex19DKO mice were conducted in the same genetic background (C57Bl/6). A strong bias in sexual lethality was also observed in C57Bl/6 mice by Reichmann et al., but only when females were lactating and nursing a pre-existing litter during pregnancy (Reichmann et al., 2013). However, none of our studies were conducted under such lactating conditions. Consequently, this difference between Tex19.1KO and Tex19DKO phenotypes is quite surprising since Tex19.2 is not expressed in the placenta, neither in WT nor in Tex19.1KO animals. This early postnatal lethality could be indicative of defects in placenta (Reichmann et al., 2013; Tarabay et al., 2013), but also suggests a functional redundancy between Tex19.1 and Tex19.2 during embryo development. The exact reason for the preferential female lethality of the Tex19DKO mutation remains unclear, but may be linked to X chromosome-specific effects.
The main findings provided by our work relate to the identification of the protein partners of TEX19 in adult testes. Through GST pulldown and IP experiments, we indeed showed for the first time that TEX19.1 exists in a complex with members of the PIWI and/or piRNA pathway: MAEL, MIWI, MILI and MVH. We also confirmed a significant level of interaction between TEX19.1 and UBR2 by IP, as has been previously reported (Yang et al., 2010). Most importantly, we found that both TEX19 paralogs bind small RNAs that have all the characteristics of piRNAs as they are ∼30 nt in size and there is a strong preference for a uridine as the first nucleotide (Frank et al., 2010; Kawaoka et al., 2011; Brennecke et al., 2007). Finally, this interaction seems to be direct and to occur through the well-conserved VPTEL domain of TEX19 proteins. We can therefore conclude that TEX19 paralogs are new members of the family of proteins that bind piRNAs and that the VPTEL domain could be a new small RNA-binding motif. While this study reveals the piRNA-binding function of the VPTEL domain, we cannot speculate about the function of the MCP domain, which lacks homology with any known proteins. PIWI proteins have, in addition to their ability to bind piRNAs, an RNA-guided nuclease function linked to the catalytic triad DDH localized in the PIWI domain and which is required for piRNA biogenesis (Martinez and Tuschl, 2004). No such triad can be found in TEX19 proteins. Accordingly, the production of small RNAs of 25–30 nt is not altered in Tex19-deficient testes. This suggests that while TEX19 proteins bind to piRNAs, they are not involved in the catalysis of piRNA synthesis.
Finally, it is now well established that TEX19 paralogs are required for the control of MMERVK10C transposons in testes (Öllinger et al., 2008; Tarabay et al., 2013). Despite a consistent effect of Tex19 deficiency on these elements, we found that MMERVK10C-derived reads only represent 0.1% of all TEX19-bound small RNAs of 25–30 nt. In contrast, TEX19 mostly binds unique, non-transposon-derived small RNAs in adult testes, which are similar to the MILI and MIWI-associated piRNAs. This is not surprising, as the post-natal PIWI machinery is responsible for producing piRNAs derived from discrete intergenic regions as male germ cells enter the pachytene stage (Aravin et al., 2006; Girard et al., 2006), although the function of these mammalian post-natal piRNAs has not been fully determined (Goh et al., 2015). This poor enrichment of TEX19-bound small RNAs in MMERVK10C fragments raises the question as to whether the reactivation of MMERVK10C in Tex19.1KO and Tex19DKO mice reflects a direct role of TEX19 proteins in silencing these elements – likely via post-transcriptional mechanisms – or an indirect effect of the Tex19 mutation. Further work should be dedicated to resolving this question.
In conclusion, our analysis of the double-mutant mice for the two TEX19 paralogs undoubtedly reveals the crucial role of these proteins for male fertility in mammals, likely via their association with the PIWI/piRNA pathway.
MATERIALS AND METHODS
Generation of Tex19 double knockout mice
The Tex19 double mutant mouse line was established at the MCI (Mouse Clinical Institute, Institut Clinique de la Souris, Illkirch, France; http://www-mci.u-strasbg.fr). For Tex19.1, the targeting vector (Fig. 1A) was constructed by sequentially subcloning three PCR fragments (from 129S2/SvPas ES cell genomic DNA) into an MCI proprietary vector, namely, one 2.2 kb fragment corresponding to inter-loxP fragment encompassing exons 2 and 3, and a 4.5 kb and a 3.1 kb fragment corresponding to the 5′ and 3′ arms, respectively. The MCI vector contains a LoxP site as well as a floxed and flipped neomycin resistance cassette. A unique PacI restriction site was inserted downstream to the 3′ loxP site. The linearized construct was electroporated in 129S2/SvPas mouse embryonic stem (ES) cells. After G418 selection, targeted clones were identified by PCR using external primers and further confirmed by Southern blotting with 5′ and 3′ external probes.
For Tex19.2, the targeting vector was constructed by sequentially subcloning three PCR fragments (from 129S2/SvPas ES cell genomic DNA) into an MCI proprietary vector, namely, a 2.0 kb fragment corresponding to inter-lox P fragment encompassing exons 2 and 3, and a 4.4 kb and a 3.5 kb fragment corresponding to the 5′ and the 3′ arms, respectively. The MCI vector contains a Lox5171 site as well as a F3-surrounded hygromycin resistance cassette. A unique PacI restriction site was inserted upstream of the 5′ loxP site. The linearized construct was electroporated in one fully valid Tex19.1 KO clone. After hygromycin selection, targeted clones were identified by PCR using external primers and further confirmed by Southern blotting with 5′ and 3′ external probes. Restriction digestion with PacI followed by Southern blotting allowed selection of a clone showing targeting of both constructs on the same allele. This clone was injected into C57BL/6J blastocysts, and male chimeras tested for germline transmission. The NeoR and HygroR cassettes were deleted by breeding the F1 mice with Flp mice (Rodríguez et al., 2000). Excision of both Tex19.1 and Tex19.2 inter-Lox fragments was performed by breeding the mice with a Cre deleter line (Dupe et al., 1997). The absence of Tex19.1 and Tex19.2 mRNA and protein was tested by RT-PCR, using oligonucleotides described previously (Tarabay et al., 2013), and by western blotting, respectively.
All animal experimental procedures were performed according to the European authority guidelines.
Antibody production, western blotting, and preparation of cytoplasmic and nuclear fractions
Injecting the entire protein or the peptide DLGPEDAEWTQALPWRC into mice allowed the generation of anti-TEX19 monoclonal antibodies. The specificity of both antibodies was tested as described elsewhere (Tarabay et al., 2013). The 7Tex:1F11 antibody is TEX19.1 specific, whereas the 6Tex:4D2 antibody detects both TEX19.1 and TEX19.2. Cytoplasmic and nuclear fractions were prepared as described at http://openwetware.org/wiki/Cytoplasm_and_nuclear_protein_extraction
RT-PCR and real-time quantitative RT-PCR
RNA was prepared using the RNeasy mini or micro kit (Qiagen) following the manufacturer's instructions. After DNase I digestion (Roche, Mississauga, ON, Canada), 1 µg RNA was reverse-transcribed by random priming using Superscript II (Invitrogen/Life Technologies, Burlington, ON, Canada). The quantitative PCR was performed by using SYBR® green JumpStartTM Taq ReadyMixTM (Sigma-Aldrich, Oakville, ON, Canada) and LightCycler 480 (Roche, Mississauga, ON, Canada). The efficiency and specificity of each primer pair was checked using a cDNA standard curve. All samples were normalized to β-actin (Actb) and Rrm2 expression (Tarabay et al., 2013).
Testes were collected and fixed in Bouin's fluid for 48 h and then embedded in paraffin. Adult male testis were dissected at 16 weeks of age. For histological analyses, 5-µm-thick sections were stained with hematoxylin and eosin. All slides were examined using a DMLA microscope (Leica) with 10×, 20×, 40× and 100× objectives with apertures of 0.3, 0.5, 0.7 and 1.3, respectively. Images were taken with a digital camera (CoolSnap; Photometrix) using CoolSnap v.1.2 software and then processed with Photoshop CS2 v.9.0.2 (Adobe).
Immunostaining of meiotic chromosome spreads
Meiotic chromosome spreads were prepared as described previously (Mark et al., 2008).
TEX19 protein production and purification
Tex19.1 cDNA or its domains were cloned, through RT-PCR starting from a testis cDNA library, into pCR®4-TOPO (Invitrogen, Life Technologies, Burlington, ON, Canada), then into pENTR 1A (Life Technologies) then by LR recombinase into pHGGWA or pGGWA. The GST-tagged recombinant proteins TEX19, MCP, VPTEL-Cter, VPTEL, Cter and protein GST alone were individually expressed in E. coli BL21 cells. The induction by 0.1 mM of IPTG was performed at a cell density of A600=0.8 at 20°C overnight and a portion of the proteins examined by SDS-PAGE (10% gels). Then, sonication was performed for 3 s ON and 3 s OFF for 5 min at amplitude 33%. Cleared lysates were prepared in lysis buffer (50 mM Tris-HCl pH 9, 400 mM NaCl, 2 mM DTT, Triton 1% and protease inhibiting cocktail) and centrifuged for 1 h at 20,000 rpm. The soluble fusion proteins were purified by a batch procedure, incubating the lysate with 500 µl of glutathione–Sepharose (Amersham Biosciences) for 1 h at 4°C with gentle shaking. Then the glutathione–Sepharose was collected by centrifugation at 1000 rpm for 5 min at 4°C and washed five times with lysis buffer. After boiling, the bound proteins were analyzed by SDS-PAGE (10% gels) followed by western blotting using anti-TEX19 monoclonal antibodies 7Tex:1F11 (1:1000) or 6Tex:4D2 (1:1000).
For GST pulldown assays, total testicular extracts of adult mice (aged 12 weeks) were added to recombinant protein fixed to glutathione–Sepharose beads for 2 h at 4°C. After five washes, proteins were separated on 10% SDS-PAGE gels and subsequently stained with Bio-Safe Coomassie (Bio-Rad, Hercules, CA). The gel was then sent to Taplin Biological Mass Spectrometry Facility to be analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS; Taplin, Boston, MA). Purified recombinant proteins were used in EMSA assays as described below.
Antibodies were chemically cross-linked to protein-G–Sepharose beads by using dimethyl pimelimidate (DMP; Sigma-Aldrich, D-8388) and used to purify TEX19.1, TEX19.2, MAEL, MIWI, MILI and MVH complexes from cytoplasmic fractions of adult mouse testes (aged 12 weeks). 30 mg of clarified DWT testes cytoplasmic extracts were incubated with 300 µl protein-G–Sepharose beads coupled to antibodies as follows: anti-TEX19.1 (7Tex:1F11, 100 µl), anti-TEX19.1/TEX19.2 (6Tex:4D2, 10 ml of hybridoma supernatant), anti-MAEL (18 µg, Abcam, Toronto, ON, Canada), anti-MIWI (20 µg, Aviva System Biology, San Diego, CA), anti-MILI (40 µg, Abcam), anti-MVH (12 µg, Abcam), anti-TDRD66 (50 µg, Millipore/Upstate, Etobicode, ON, Canada), anti-EDC4 (12 µg, Abcam), anti-RANBP9 (8 µg, Proteintech, Rosemonte, IL, USA) or an ascitic fluid control (negative control, 100 µl) in co-IP buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, complete protease inhibitor) overnight at 4°C. After five washes with co-IP buffer, the retained proteins were denatured by using Laemmli buffer with 5% β-mercaptoethanol heated to 100°C, then separated by SDS-PAGE as described above. If required, gels were stained with Coomassie Blue and sent to Taplin Biological Mass Spectrometry Facility to be analyzed by LC/MS/MS (Taplin, Boston, MA).
Small RNA IP and purification
TEX19 libraries were prepared as described elsewhere (Reuter et al., 2009).
For 32[P] labeling, piR-117061 (5′-UAGGACCUGAGAACUUAACCUUGUUAUGGG-3′) and piR-nonspecific (5′-CAGGACCUGCGAACUUAACCUUGUUAUGGG-3′) were incubated for 60 min at 37°C in 20 µl kinase buffer containing 5μl RNA (0.5 nmol), 2 μl buffer B, 4 µl polyethylene glycol (PEG), 10 U T4 PNK kinase (10 U/µl, Fermentas), 40 U RNasin (40 U/µl), 2 μl [γ-32P]ATP (6000 Ci/mmole) (Perkin Elmer) and 5 μl double-distilled H2O.
EMSA assays were performed with 5 µM GST alone, or GST-tagged TEX19.1, MCP, VPTEL-Cter, VPTEL or Cter and 125 femtomoles (8000 cpm) of denatured radiolabeled piRNA in the binding buffer (250 µg herpain, 10 mM MOPS pH 7, 50 mM MgCl2, 1 mM DTT, 10% glycerol, 40 U RNasin, 10 ng/µl tRNA from E.coli) as described previously (Bendak et al., 2012).
Small RNA sequencing and analysis
Small RNA-seq of whole adult testes and TEX19 IP were performed by using Illumina HiSeq 2000 on libraries (18–45 nt RNAs size-fractionated on an acrylamide gel) prepared with the Illumina TruSeq small RNA protocol. For the analysis, adapters were removed using Cutadapt (Martin, 2011). The trimmed sequencing reads of each library were then mapped and annotated with ncPRO-seq (Chen et al., 2012) onto the mouse reference genome (mm9). Briefly, read alignment was performed by using Bowtie v0.12.8, allowing a sum of qualities of mismatching bases lower than 50 (denoted –e 50). A number of 5000 hits per aligned read were allowed. Aligned reads were then annotated by using RepeatMasker and refGene databases, and the positional read coverage was weighted by mapping site numbers.
The piRNA cluster analysis was performed as described previously (Beyret et al., 2012). A cluster was defined as a group of piRNAs where the reads were less than 1500 bp away from each other. Clusters with at least 1000 normalized read counts were reported. The UCSC genome tracks were generated by using the HOMER software (v4.3) using unique mapped reads.
We would like to thank Robert Drillien for his critical reading of the manuscript. We are grateful to the Institute of Genetics and Molecular and Cellular Biology (IGBMC) platforms.
The authors declare no competing or financial interests.
Y.T., M.A., M.T., T.Y., A.T., M.M., D.B., S.V.: Data analysis and interpretation, manuscript writing, final approval of manuscript.
The study was funded by Agence Nationale de la Recherche (ANR-11-BSV2-002 – TranspoFertil) and Agence de la Biomédecine – AMP, diagnostic prénatal et diagnostic génétique. This work was supported by the French Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), the Ministère de l'Enseignement Supérieur, de la Recherche Scientifique et des Technologies de l'Information et de la Communication, and the Université de Strasbourg (University Hospital of Strasbourg).
Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.188763.supplemental
- Received March 17, 2016.
- Accepted February 27, 2017.
- © 2017. Published by The Company of Biologists Ltd