Deletion of the Pluripotency-Associated Tex19.1 Gene Causes Activation of Endogenous Retroviruses and Defective Spermatogenesis in Mice

As genetic information is transmitted through successive generations, it passes between pluripotent cells in the early embryo and germ cells in the developing foetus and adult animal. Tex19.1 encodes a protein of unknown function, whose expression is restricted to germ cells and pluripotent cells. During male spermatogenesis, Tex19.1 expression is highest in mitotic spermatogonia and diminishes as these cells differentiate and progress through meiosis. In pluripotent stem cells, Tex19.1 expression is also downregulated upon differentiation. However, it is not clear whether Tex19.1 has an essential function in germ cells or pluripotent stem cells, or what that function might be. To analyse the potential role of Tex19.1 in pluripotency or germ cell function we have generated Tex19.1−/− knockout mice and analysed the Tex19.1−/− mutant phenotype. Adult Tex19.1−/− knockout males exhibit impaired spermatogenesis. Immunostaining and histological analysis revealed defects in meiotic chromosome synapsis, the persistence of DNA double-strand breaks during meiosis, and a loss of post-meiotic germ cells in the testis. Furthermore, expression of a class of endogenous retroviruses is upregulated during meiosis in the Tex19.1−/− testes. Increased transposition of endogenous retroviruses in the germline of Tex19.1−/− mutant mice, and the concomitant increase in DNA damage, may be sufficient to disrupt the normal processes of recombination and chromosome synapsis during meiosis and cause defects in spermatogenesis. Our results suggest that Tex19.1 is part of a specialised mechanism that operates in the germline to repress transposable genetic elements and maintain genomic stability through successive generations.


Introduction
The germ cells of sexually reproducing organisms have a unique role in generating genetic diversity and transmitting genetic information from one generation to the next. Establishment of the germline in mammals involves the induction of germ cells from pluripotent epiblast cells through the action of extra-embryonic ectoderm-derived bone morphogenetic proteins and occurs comparatively late in development, commencing around day 6.25 days post coitum (dpc) in mice [1][2][3][4]. At around 12.5 dpc -13.5 dpc the sexually dimorphic germ cells become committed to develop along either a male or a female pathway and start to initiate sex-specific differentiation [5]. Although there are numerous differences in the differentiation of the germline and in the timing and regulation of meiosis between the sexes, the fundamental events of meiosis that increase genetic diversity and reduce ploidy of the gametes are common to both.
The main group of genes that have been shown to be required for mammalian meiosis are those involved in the recombination and synapsis of homologous chromosomes. Mice carrying loss-offunction mutations in these genes, such as Atm, Dmc1, cH2AX, Mlh1, Msh5, Rec8, Rad51, Smc1b, Spo11, Sycp1, Sycp2, Sycp3, Syce2 and Tex14, typically exhibit defects in chromosome synapsis in both sexes, although male and female germ cells can exhibit different responses to these defects [6][7][8].
A second group of genes that are required for progression through meiosis are those involved in repression of transposable genetic elements. Retrotransposons, for example long interspersed repeats (LINEs), short interspersed repeats (SINEs), and endogenous retroviruses such as intracisternal A-particles (IAPs) are the major class of transposable genetic elements in mammals and comprise around 37.5% of the mouse genome [9]. To allow new transposition events to propagate through subsequent generations, retrotransposons have evolved to be active in the germline.
Accordingly, germ cells appear to have evolved mechanisms to reduce the mutational load of retrotransposon activity. Mutations in genes involved in mediating DNA methylation-dependent transcriptional repression of retrotransposons cause increased expression of retrotransposons and defects in chromosome synapsis during meiosis in either male or female germ cells [10,11]. For example Dnmt3L is a catalytically inactive member of the DNA methyltransferase family that is expressed in foetal germ cells but is absent by 6 days post partum (dpp) [11]. Male mice null for this gene do not methylate dispersed repeat DNA during foetal germ cell development, and express LINEs and a class of endogenous retroviruses known as intracisternal A-particles (IAPs) in the germline [11]. Dnmt3L mutant male mice also exhibit meiotic abnormalities that result in a loss of post-meiotic germ cells in the testis [11]. The Dnmt3L mutant phenotype suggests that epigenetic changes that occur in foetal germ cells can cause meiotic defects later in germ cell development. A second example is provided by the murine piwi-related genes, which encode germline-specific proteins that are associated with a class of small germline-specific piwi-interacting RNAs (piRNAs) and are also required to repress retrotransposons during spermatogenesis [12][13][14]. Mili and Miwi2 both appear to be involved in de novo methylation of LINE and IAP elements during germ cell development in male embryos, and both mutants exhibit reduced DNA methylation and increased expression of LINE and IAP element in the testis, and defects in chromosome synapsis during meiosis in male germ cells [12][13][14]. The mechanism by which increased expression of retrotransposons results in the defects in chromosome synapsis seen in these mutant mice is unknown, but the phenotype of these mutant mice suggests that repression of transposable genetic elements is required to allow germ cells to progress through meiosis.
A set of testis expressed (Tex) genes has been identified in a subtractive hybridisation screen for genes expressed in spermatogonia but not somatic tissue [15]. One of these genes, Tex19.1 (AAH53492.1), was found in a screen for potential RNA-targets of the germline-specific RNA binding protein Dazl by immunoprecipitation and microarray analysis [16]. Further unpublished work in this laboratory and work recently published by Kuntz et al. [17] confirmed an earlier report that Tex19.1 is a ''pluripotent cell expressed gene'' [18]. Humans and primates possess a single Tex19 gene in their genome, but in rodents a recent duplication has produced a two-gene family arranged as divergently transcribed genes separated by 29kb of DNA [17]. While expression of murine Tex19.1 is restricted to pluripotent stem cells and developing germ cells, Tex19.2 is expressed in the testis somatic tissues and does not appear to be restricted to germ cells or pluripotent stem cells in mice [17].
The expression pattern of Tex19.1 suggests that the protein could have an important role in pluripotency or germ cell function. Since the sequence of Tex19.1 gives no clue to the biochemical function of this protein we decided to take a genetic approach to determining the function of this gene in the germline. In this paper we report that targeted deletion of Tex19.1 in mice results in upregulation of endogenous retrovirus expression in testicular germ cells, perturbed chromosome synapsis during meiosis, and impaired spermatogenesis.

Generation of Tex19.1 Knockout Mice
Tex19.1 knockout mice were generated by replacing the Tex19.1 open reading frame with a neomycin selection cassette by homologous recombination in E14 embryonic stem cells [19]. Homologous regions were cloned by PCR from E14 embryonic stem cell genomic DNA using primers listed in Supplementary  Table S1. The Tex19.1 targeting vector was linearised, electroporated into E14 embryonic stem cells, and neomycin-resistant clones screened for the desired integration event by PCR and Southern blot. Tex19.1 +/2 ES cells were used to generate Tex19.1 2/2 knockout mice by blastocyst injection and breeding as described [19]. Mice were genotyped by multiplex PCR using primers listed in Supplementary Table S1. Phenotypic analysis was performed on mice with a 129/Ola x CD1 mixed genetic background. Generation and analysis of Tex19.1 2/2 knockout mice was performed under a UK Home Office project licence with approval from an institutional ethics committee.

Antibody Production
Anti-Tex19.1 antibodies were raised in rabbits using the synthetic peptide 78 ESEQEPGPEQDAWRG 92 (Eurogentec). This peptide was designed to be specific to Tex19.1 and is not present in the Tex19.2 protein sequence. Antibodies were affinity purified from sera using the immunising peptide immobilised on a Sulfolink column (Pierce) according to manufacturer's instructions.

Immunohistochemistry
Testes were recovered from mice, fixed at 4uC overnight in 4% paraformaldehyde in phosphate buffered saline (PBS) and embedded in paraffin wax. 6 mm-thick sections were dewaxed in xylene, rehydrated, and antigen retrieval performed by boiling slides for 15 minutes in 0.01 M sodium citrate, pH 6.0. Sections were blocked, incubated with rabbit anti-Tex19.1 primary antibody at 1:50, and bound antibody detected using the DAKOvision ABC diaminobenzidine (DAB) kit as described by the manufacturer (DakoCytomation). For peptide competition, anti-Tex19.1 antibodies were pre-incubated with 5 nM immunising peptide. For immunostaining of cultured cells, cells were fixed for 30 minutes at room temperature with 3.7% formaldehyde in PBS, then blocked with PBS containing 5% serum and 0.01% Tween-20. Cells were incubated with rabbit anti-Tex19.1 primary antibody at 1:100, then fluorescently labelled secondary antibodies

Author Summary
The germ cells-eggs in females and sperm in males-are responsible for passing genetic information from one generation to the next. As any genetic changes that arise in the germ cells can be transmitted to the next generation, germ cells are a prime target for the activity of mobile genetic elements. Mobile genetic elements make up around 40% of a mammalian genome, and many of these elements are derived from retroviruses that have infected germ cells, or early embryonic precursors to germ cells, and have integrated into the genome. Here, we characterise the function of Tex19.1, a gene whose expression is restricted to germ cells and the pluripotent cells that are early embryonic precursors to germ cells. We show that when Tex19.1 is deleted from mice, germ cells have problems progressing through meiosis, and sperm production is impaired. Furthermore, we show that, in the absence of Tex19.1, endogenous retroviruses are activated in male germ cells attempting to go through meiosis. Our results suggest that Tex19.1 is part of a specialised mechanism that guards against mutagenic endogenous retrovirus activity in germ cells and pluripotent cells and thus helps to maintain the integrity and stability of the genome through successive generations. at 1 mg/mL (Invitrogen). DNA was counterstained with 2 mg/mL DAPI.
Nuclear/Cytoplasmic Fractionation E14 embryonic stem cells or 13 dpp postnatal testes were lysed in cytoplasmic lysis buffer (10 mM Hepes pH 7.6, 3 mM MgCl 2 , 40 mM KCl, 50 mM b-glycerophosphate, 5% glycerol, 0.5% Igepal CA-630, 2 mM NaF, 1 mM Na 3 VO 4 , 2 mM DTT and protease inhibitors) for 5 minutes on ice and the whole cell lysate centrifuged for 5 minutes at 1000g at 4uC. The nuclear pellet was resuspended in Laemmli buffer, boiled for 5 minutes and sonicated to disrupt genomic DNA. The cytosolic supernatant was mixed with Laemmli buffer and boiled for 5 minutes. Equivalent proportions of each fraction were separated by SDS-PAGE then Western blotted.

Southern Blotting
Non-radioactive Southern blots were performed using a digoxigenin-labeled DNA probe generated using primers listed in Supplementary Table S1, and alkaline phosphatase-conjugated anti-digoxigenin antibodies, essentially as described by the manufacturer (Roche).

RT-PCR and Quantitative PCR
Testis RNA was isolated with Trizol (Invitrogen) according to the manufacturer's protocol and reverse transcription performed with Superscript III (Invitrogen) on 1 mg RNA per reaction using oligo dT primer. Primers for RT-PCR are listed in Supplementary  Table S1.
For quantitative PCR (qPCR), random-primed cDNA was generated from total RNA using Superscript III (Invitrogen). qPCR was performed using SYBR Green PCR System (Applied Biosystems) and a PTC-200 thermal cycler equipped with a Chromo4 continuous fluorescence detector and Opticon Monitor software (MJ Research). Primers for qPCR are listed in Supplementary Table S1. Five technical replicates were performed for each biological sample, and the relative changes in gene expression determined using the DD 22Ct method as described [21]. As Tex19.1 is expressed in the germ cells in the testis the Sertoli cell marker Sdmg1 [22] was used to normalise cDNAs prepared from different animals to reduce the probability that the cDNAs were being normalised to a transcript whose level could be influenced by loss of Tex19.1.

Testis Weight and Sperm Count
Both testes from each adult animal (6-36 weeks old) were weighed, and the mean testis weight was used for statistical comparison. For sperm count one epididymis from each animal was homogenised in 1 mL 1% sodium citrate and incubated for 5 minutes at room temperature to allow the debris to settle. Sperm in the supernatant was then counted with a hemocytometer.

Histology
Testes were fixed for 4-6 hours in Bouin's solution (Sigma-Aldrich) at room temperature, then embedded in wax. For histological analysis 6 mm sections were dewaxed with xylene, rehydrated, then stained with hematoxylin and eosin.

Immunostaining of Meiotic Chromosome Spreads
Immunostaining of chromosome spreads from meiotic spermatocytes was performed essentially as described [23]. Briefly, testes were homogenized in PBS and 0.1 mL of cells were incubated in 0.5 mL 5% sucrose on a microscope slide for 1 hour. Cells were lysed with 0.1 mL 0.05% Triton-X-100 for 10 minutes, and fixed with 0.8 mL of fixing solution (2% paraformaldehyde, 0.02% SDS in PBS) for 1 hour. The slides were then washed, blocked with 5% serum, 0.1% Tween in PBS and incubated with primary antibodies for 1 hour. Mouse anti-Sycp3 antibodies (Abcam) were used at a 1:2000 dilution, rabbit anti-Sycp1 antibodies (Abcam) at 1:250, rabbit anti-cH2AX antibodies (Upstate Biotechnology) at 1:200, and mouse anti-Rad51 antibodies (Upstate Biotechnology) at 1:125. Fluorescently labelled secondary antibodies were used at 1 mg/mL (Invitrogen), and DNA was stained with 2 mg/mL DAPI.

Metaphase I Analysis of Spermatocytes
Chromosome spreads for metaphase I analysis were prepared as described in [24]. Briefly, testes were incubated for 20 minutes in 1% sodium citrate, minced with scissors, and the cells harvested by centrifugation. Cells were then washed and resuspended in fixing solution (3:1 methanol:glacial acetic acid), dropped onto slides, and the resulting chromosome spreads were stained with Giemsa solution. 100 metaphase I spreads were scored per animal, and two animals scored for each genotype.

In Situ Hybridisation and Northern Blotting
A 460 bp fragment of the MMERVK10C endogenous retrovirus was amplified by RT-PCR from Tex19.1 2/2 mutant testes using primers listed in Supplementary Table S1 and cloned into pBluescript II SK+ (Stratagene). Sense and anti-sense digoxigenin-labelled riboprobes were generated using T3 and T7 RNA polymerase according to the supplier's instructions (Roche). In situ hybridisation on 6 mm wax sections of Bouin's-fixed testis tissue was performed essentially as described [25] using 100 ng/mL digoxigenin-labelled probe and a hybridisation temperature of 50uC. Bound probe was detected with alkaline phosphatase-conjugated anti-digoxigenin antibodies (Roche) and BCIP/NBT precipitating stain (Vector Labs), then sections counterstained with nuclear fast red according to manufacturer's instructions. The antisense MMERVK10C digoxigenin-labelled riboprobe was also used for non-radioactive Northern blotting of 1 mg testis RNA as described [22].

Expression of Tex19.1 Protein during Spermatogenesis
Spermatogenesis in the adult testis involves the differentiation of a small pool of spermatogonial stem cells into large numbers of mature sperm. Within the testis spermatogenesis takes place in the seminiferous tubules where the mitotic spermatogonia reside at the outermost edge of the tubule, and progressive stages of differentiation are found as layers of meiotic spermatocytes then haploid spermatids located more and more centrally towards the lumen of the tubule [26]. Published RT-PCR expression data for Tex19.1 in purified spermatogenic cell populations suggests that Tex19.1 expression is highest in mitotic spermatogonia, decreases as the spermatocytes progress through meiosis, and is present at low levels in round spermatids [27]. To establish the expression pattern of Tex19.1 protein in male testis during spermatogenesis we raised anti-peptide antibodies to Tex19.1. Immunohistochemistry on mouse testis shows strong cytoplasmic expression of Tex19.1 in spermatogonia that is downregulated as these cells differentiate and progress through meiosis ( Figure 1A-C). We were able to detect cytoplasmic Tex19.1 protein in some meiotic spermatocytes ( Figure 1C), but not in others ( Figure 1B) suggesting that Tex19.1 protein expression is switched off as the germ cells proceed through meiosis. The expression of Tex19.1 protein in spermatogonia and spermatocytes is consistent with that observed for Tex19.1 mRNA by in situ hybridisation ( Figure S1A-C).
Our finding that Tex19.1 is present in the cytoplasm of spermatogonia and early spermatocytes in the adult testis is not consistent with the published nuclear localisation of Tex19.1 protein in embryonic stem cells [17]. We confirmed that our anti-Tex19.1 antibody detects Tex19.1 and not a cross-reacting antigen by blocking the anti-Tex19.1 immunohistochemistry signal by competition with the immunising peptide, and by immunohistochemistry on Tex19.1 2/2 knockout testes ( Figure S1D-F). We were also able to detect a predominantly cytoplasmic subcellular localisation of Tex19.1 by immunostaining germ cells isolated from 14.5 dpc embryonic testes ( Figure S1G). Again the cytoplasmic anti-Tex19.1 immunostaining could be competed with the immunising peptide, and was absent in germ cells from Tex19.1 2/2 knockout embryos ( Figure S1H, I). These data suggest that the anti-Tex19.1 antibody used in this present study specifically recognises endogenous Tex19.1 in germ cells, and that at least some Tex19.1 is present in the cytoplasm of spermatogonia and early spermatocytes in adult mouse testes. To investigate whether the discrepancy between the cytoplasmic localisation of Tex19.1 in germ cells presented in this study and the nuclear localisation of Tex19.1 in embryonic stem cells described previously [17] is caused by the difference between the cell types studied we performed immunostaining for Tex19.1 on embryonic stem cells. In contrast to the previous report [17], we found that Tex19.1 is predominantly cytoplasmic in embryonic stem cells ( Figure 1D-F).
To exclude the possibility that our anti-Tex19.1 antibody is unable to detect a nuclear population of Tex19.1 due to loss or masking of the epitope during the immunohistochemical procedures we biochemically fractionated 13 dpp prepubertal testes and embryonic stem cells into nuclear and cytoplasmic fractions. On Western blots the 42 kDa Tex19.1 band was barely detectable in the nuclear fraction, but was easily detectable in equivalent loadings of the whole cell lysate and cytoplasmic fractions ( Figure 1G,H). The observed size of the anti-Tex19.1 band in the Western blots (42 kDa) correlates well with the predicted molecular weight of Tex19.1 (40.4 kDa). This 42 kDa band appears to be endogenous Tex19.1 as it is not present in testes from Tex19.1 2/2 knockout animals ( Figure 2E). The biochemical fractionation of embryonic stem cells and testes therefore confirms the predominantly cytoplasmic subcellular localisation of Tex19.1 that we have observed by immunohistochemistry and immunostaining. Taken together the data presented here strongly suggests that Tex19.1 is a predominantly cytoplasmic protein in embryonic stem cells and germ cells.
Germ cells in many species possess specialised cytoplasmic structures termed nuage that are implicated in RNA metabolism. Although Tex19.1 appears to be a predominantly cytoplasmic germ cell protein, the subcellular localisation and cell-type distribution of Tex19.1 appears to be distinct from the nuage component Tdrd1 [28] ( Figure S2). Thus Tex19.1 does not appear to be a novel component of nuage. As the subcellular localisation of Tex19.1 does not provide any major insight into what the cellular function of this protein might be, and the Tex19.1 protein sequence does not contain any functional domains to illuminate the potential biochemical function of this protein, we decided to take a genetic approach to analyse the function of Tex19.1 in the germline.
The surviving Tex19.1 2/2 knockout mice are apparently healthy, with overtly normal morphology and behaviour. However both male and female Tex19.1 2/2 knockout mice have reduced fertility. Tex19.1 2/2 knockout females have a mean litter size of 5.062.2 SD, n = 9 compared to a litter size of 10.662.9, n = 23 for Tex19.1 +/2 heterozygous females (Student's t-test p,0.01). The reduced fertility in Tex19.1 2/2 homozygous females is consistent with expression of Tex19.1 in embryonic ovaries [17], and a detailed analysis of the cause of the subfertility in the Tex19.1 2/2 knockout female mice will be published elsewhere. Similarly, Tex19.1 2/2 knockout male mice are also severely subfertile when test-mated with wild-type female mice. Although one out of the eleven Tex19.1 2/2 knockout males tested for fertility was able to sire offspring, the remaining Tex19.1 2/2 knockout males were infertile. The sterile Tex19.1 2/2 knockout male mice were apparently able to mate with the wild-type females to produce a copulation plug, but these females did not give birth to any pups. Tex19.1 2/2 knockout male mice have smaller testes ( Figure 3A, B), with the median testis weights of adult animals reduced from 111 mg in Tex19.1 +/+ wild type and Tex19.1 +/2 heterozygous mice to 42.5 mg in Tex19.1 2/2 knockout littermates (Mann Whitney U-test, p,0.01). Furthermore, the median epididymal sperm count is reduced to 1.3610 5 in Tex19.1 2/2 knockout mice from 1.3610 7 in wild type and heterozygous littermates (Mann Whitney U-test, p,0.01, Figure 3C) suggesting that spermatogenesis is defective in the Tex19.1 2/2 knockout testes. We have not been able to detect any difference in testis weight or sperm count between Tex19.1 +/+ wildtype and Tex19.1 +/2 heterozygous animals.
The extent of the spermatogenesis defect in the Tex19.1 2/2 knockout males varied between individual animals. When sperm was counted, a strong reduction was observed for most of the animals, but for the single fertile animal the sperm count was close to normal levels ( Figure 3C). A similar variation in testis weight was also evident amongst Tex19.1 2/2 knockout animals ( Figure 3B). This phenotypic variation was not influenced by the age of the mice at the time of analysis. Age-matched adult mice were analysed at 6 weeks, 3 months, 6 months and 9 months during the course of this study, and there appeared to be no correlation between the severity of the phenotype and the age at which the adult mice were examined. Rather phenotypic variation was observed in adult mice at all ages (R.O. and I.R.A., data not shown). The outbred component of the genetic background of these mice may contribute to this variation.

Tex19.1 2/2 Knockout Spermatocytes Exhibit Defects in Progression through Meiosis
We investigated the spermatogenesis defect in Tex19.1 2/2 knockout mice further by examining the testis histology in these animals. We did not detect any overt differences in testis histology between Tex19.1 +/+ wild-type and Tex19.1 +/2 heterozygous animals. However, Tex19.1 2/2 knockout testes have considerably narrower seminiferous tubules than their wild-type or heterozygous littermates due to a reduction in the number of post-meiotic germ cells ( Figure 4A, B). This phenotype was also subject to some heterogeneity. In animals with a more severe phenotype all postmeiotic cell-types were missing and the most advanced meiotic cells were in pachytene stage ( Figure 4C, D). In animals with a less severe phenotype a proportion of cells were able to complete meiosis and haploid cells could be detected, although often in comparatively low numbers ( Figure 4C, E). Like the testis weight and sperm count phenotypes described in the previous section, there appeared to be no correlation between the severity of the testis histology phenotype and the age at which the adult mice were analysed (from 6 weeks to 9 months).
To test whether the reduction in the number of post-meiotic germ cells in Tex19.1 2/2 knockout testes arises from a decrease in the spermatogonial mitotic divisions or apoptosis of differentiating germ cells, we counted the number of B-type spermatogonia, early meiotic cells and apoptotic cells in testis sections from Tex19.1 +/2 heterozygous and Tex19.1 2/2 knockout animals. B-spermatogonia and early meiotic cells were identified by their location and histological appearance in the seminiferous tubules [26], and apoptotic cells were identified using the TUNEL assay to label fragmented chromatin. Whereas the number of B-type spermato- gonia and early meiotic cells did not differ between Tex19.1 +/2 heterozygous and Tex19.1 2/2 knockout testes (R.O., data not shown), TUNEL staining showed an increase in the number of dying cells in adult Tex19.1 2/2 knockout testis ( Figure S3A, B, N). In more severe Tex19.1 2/2 knockout seminiferous tubules, TUNEL-positive cells were found within or next to layers of meiotic germ cells ( Figure S3C), but even at high magnification the nuclear morphology of these TUNEL-positive cells was not distinct enough to allow their developmental stage to be unambiguously identified ( Figure S3H-J). In less severe Tex19.1 2/2 knockout seminiferous tubules, TUNEL-positive cells could also be found between the layers of meiotic germ cells and post-meiotic round spermatids ( Figure S3D). At higher magnification, some of these TUNEL-positive cells could be identified as metaphase I spermatocytes ( Figure S3E-G).
In order to further define the point during spermatogenesis when the Tex19.1 2/2 knockout cells are dying we examined the synchronous first wave of spermatogenesis that occurs in prepubertal mice. The first wave of spermatogenic germ cells initiates meiosis at around 10 dpp in the prepubertal testis, and progresses through the pachytene stage of meiosis from around 14 to 20 dpp to produce the first post-meiotic round spermatids around 21 dpp, and mature sperm at around 31 dpp [29,30]. Analysis of apoptosis ( Figure S3N) and testis histology ( Figure S4) at various stages of prepubertal testis development revealed no overt differences in testis histology and no statistically significant increase in apoptosis at 16 dpp in Tex19.1 2/2 knockout testes. However by 19-22 dpp, a reduction in the number of meiotic and post-meiotic germ cells and an increase in the frequency of cell death are both evident in Tex19.1 2/2 knockout testes ( Figures  S3N, S4). In 22 dpp testes, clusters of TUNEL-positive cells can be seen within the layer of pachytene germ cells that line the lumen of the seminiferous tubule suggesting that at least some apoptosis is occurring at the pachytene stage of meiosis ( Figure S3K-M). The high level of apoptosis in the Tex19.1 2/2 knockout testes increases by 29-31 dpp to the level seen in adult testes ( Figure S3N). This data suggests that the reduction in the number of post-meiotic germ cells and increased levels of apoptosis seen in the adult Tex19.1 2/2 knockout testes is at least partly due to some Tex19.1 2/2 knockout germ cells initiating apoptosis during the pachytene stage of meiosis, and some Tex19.1 2/2 knockout germ cells initiating apoptosis during metaphase I.
Although the vast majority of the Tex19.1 2/2 null testes examined contained differentiating germ cells, two of thirty analysed knockout animals had an extremely severe phenotype with one testis that completely lacked germ cells. One of these agametic testes was isolated from a 31 dpp prepubertal mouse ( Figure 4F) suggesting that this extreme phenotype is indicative of defects occuring during embryonic or early post-natal germ cell development rather than a progressive loss of spermatogonial stem cells in an ageing adult testis. However, as only a small number of testes exhibited this phenotype, we were not able to study this extreme phenotype further and instead focused on the meiotic phenotype evident in the vast majority of the Tex19.1 2/2 mutant testes.
Chromosome Synapsis during Male Meiosis Is Impaired in the Absence of Tex19.1 We next attempted to determine the cause of the increased apoptosis in Tex19.1 2/2 null testes. Defects in homologous chromosome synapsis or homologous recombination during meiotic prophase can cause apoptosis in late pachytene spermato- cytes [31,32]. Therefore we used immunocytochemistry on meiotic chromosome spreads to analyse chromosome synapsis and homologous recombination in Tex19.1 2/2 knockout testes. In order to analyse chromosome synapsis, meiotic chromosome spreads were stained using Sycp3 as a marker for lateral elements of meiotic chromosomes and Sycp1 as a marker for synapsed homologous chromosomes [6]. In wild-type pachytene cells the autosomal chromosome axes stain completely for both markers, whereas the X and Y sex chromosomes remain largely asynapsed with only a small area of Sycp1 staining in the pseudo-autosomal region ( Figure 5A). In contrast, about half the pachytene cells in Tex19.1 2/2 homozygotes have Sycp3-stained autosomal chromosomal axes that lack Sycp1 staining ( Figure 5B-D, I). The asynapsed chromosomes in Tex19.1 2/2 knockout cells did not appear to be arranged in homologous pairs ( Figure 5D). However it is not clear whether the asynapsed chromosomes have never paired in Tex19.1 2/2 knockout spermatocytes, or have paired but have subsequently fallen apart. In some of the incompletely synapsed Tex19.1 2/2 knockout cells, some chromosomes appeared to form chains linked by regions of apparent non- homologous synapsis ( Figure 5C, asterisk). Incompletely synapsed pachytene cells comprise less than 1% of spreads from Tex19.1 +/+ wild-type or Tex19.1 +/2 heterozygotous testes ( Figure 5I). Thus Tex19.1 2/2 knockout animals exhibit defects in homologous chromosome synapsis during male meiosis.
During meiotic prophase, homologous recombination starts prior to homologous chromosome pairing and synapsis [33]. As progression of homologous recombination and chromosome synapsis are interdependent on each other [6,7], we investigated whether the chromosome synapsis defect in Tex19.1 2/2 knockout spermatocytes was a consequence of an earlier defect in the initiation of homologous recombination. The appearance of DNA double strand breaks and the formation of early recombination foci during meiotic prophase can be detected by immunostaining for the phosphorylated histone cH2AX and the recombinase enzyme Rad51 respectively [33,34]. cH2AX staining is normally present on chromatin during the leptotene and zygotene stages of early meiotic prophase. As synapsis proceeds during zygotene, the DNA double strand breaks are resolved, resulting in cH2AX staining disappearing from the autosomal chromosomes, but not the sex chromosomes. In normal Tex19.1 +/+ wild-type pachytene cells, chromosome synapsis is complete and only the sex chromosomes stain for cH2AX ( Figure 5E). However, the incompletely synapsed pachytene cells in Tex19.1 2/2 knockout testes, exhibit strong diffuse cH2AX staining ( Figure 5F). This cH2AX staining is localised to the regions of the chromosome spreads that contain the unsynapsed chromosomes ( Figure 5F). Similarly, immunostaining for the early recombination foci marker Rad51, which largely disappears from autosomal chromosomes as synapsis proceeds, suggests that Rad51 foci are formed in Tex19.1 2/2 knockout spermatocytes, but are not resolved or matured on the unsynapsed chromosomes ( Figure 5G, H). Thus the formation of DNA double strand breaks and the assembly of early recombination foci both appear to be occurring in Tex19.1 2/2 knockout spermatocytes. This suggests that the defect in meiotic chromosome synapsis that we have observed in Tex19.1 2/2 spermatocytes does not appear to be a secondary consequence of impaired initiation of homologous recombination. Rather, the presence of DNA double strand breaks and early recombination foci in the unsynapsed regions of the incompletely synapsed Tex19.1 2/2 pachytene spermatocytes is consistent with impaired chromosome synapsis. Furthermore, the presence of DNA double strand breaks and early recombination foci in unsynapsed regions of incompletely synapsed Tex19.1 2/2 pachytene spermatocytes indicates that the unsynapsed chromosomes arise from a failure to initiate synapsis rather than premature desynapsis. The unsynapsed chromosomes in the incompletely synapsed pachytene Tex19.1 2/2 knockout cells are presumably sufficient to trigger apoptosis at the pachytene checkpoint [31,32], and would account for the increased levels of cell death seen in pachytene stage meiotic germ cells in Tex19.1 2/2 knockout testes ( Figure S3).
Although incompletely synapsed pachytene cells could explain the increased levels of cell death in the pachytene meiotic germ cells in Tex19.1 2/2 knockout testes, the presence of apoptotic metaphase I spermatocytes in these animals suggests that there may be an additional defect later in spermatogenesis to account for cell death at the metaphase I stage. Around half of the Tex19.1 2/2 knockout pachytene cells did not appear to have any overt defects in chromosome synapsis ( Figure 5I), and would therefore presumably be able to progress to metaphase I and continue through spermatogenesis. To investigate whether there might be additional defects in chromosome behaviour at later stages of meiosis in Tex19.1 2/2 knockout spermatocytes, we prepared and analysed meiotic metaphase I chromosome spreads. During metaphase I of meiosis, homologous chromosomes are held together as bivalents by chiasmata ( Figure 5J). 94% of the metaphase I spreads from Tex19.1 +/2 heterozygous testes contained only bivalent metaphase I chromosomes, 5% contained univalent sex chromosomes, and 1% contained univalent autosomes. However, only 34% of the metaphase I spreads from Tex19.1 2/2 knockout testes contained only bivalent metaphase I chromosomes, while 56% of the spreads contained univalent sex chromosomes, and 33% contained univalent autosomes ( Figure 5K). 23% of the Tex19.1 2/2 knockout metaphase I spreads feature univalent autosomes and univalent sex chromosomes. Thus Tex19.1 2/2 knockout testes contain increased numbers of univalent chromosomes at meiotic metaphase I that could potentially trigger apoptosis at the metaphase I checkpoint [35,36] and account for the apoptotic metaphase I cells seen in Tex19.1 2/2 knockout testes. Furthermore, the presence of univalent chromosomes in metaphase I spreads from Tex19.1 2/2 knockout testes is indicative of a defect in the formation or maintenance of chiasmata in post-pachytene spermatocytes.

Tex19.1 2/2 Homozygous Testes
Meiotic defects similar to those present in the Tex19.1 2/2 knockout testes have been observed in various different mouse mutants that carry defects in genes encoding components of meiotic chromosomes, the meiotic recombination machinery, or the synaptonemal complex [6,7]. However, we have been unable to detect any Tex19.1 protein physically associated with meiotic chromosomes by immunostaining (R.O., data not shown), and our finding that Tex19.1 is predominantly localised to the cytoplasm rather than the nucleus suggests that Tex19.1 is unlikely to be a component of meiotic chromosomes or the synaptonemal complex. We therefore reasoned that the meiotic defects present in the Tex19.1 2/2 knockout testes are unlikely to be a direct effect of Tex19.1 on meiotic chromosome structure or function but rather may be an indirect consequence of changes in meiotic gene expression.
In order to detect changes in gene expression in the testis of Tex19.1 2/2 knockout mice, we performed microarray analysis using an Illumina MouseWG-6 v1.1 Whole Genome Gene Expression Beadchip containing 48,318 different probes. To exclude potential differences in transcript levels due to the loss of post-meiotic germ cells in the Tex19.1 2/2 testes we performed this analysis on testes from 16 dpp prepubertal mice during the first synchronous wave of spermatogenesis. At this stage of testis development, some germ cells are already in the pachytene stage of meiosis, but no obvious changes in cell composition were apparent between Tex19.1 +/+ wild type and Tex19.1 2/2 knockout testes ( Figure S4). RNAs from two different 16 dpp Tex19.1 2/2 knockout testes were compared with Tex19.1 +/+ wild type or Tex19.1 +/2 heterozygous littermates, and transcripts that had consistent and greater than three fold changes in relative gene expression between the two groups of animals were identified. The Mouse Genome Database (http://www.informatics.jax.org) currently lists 97 mutations that are known to give rise to meiotic arrest during spermatogenesis [8]. These male meiotic arrest genes include genes that encode components of meiotic chromosomes, the meiotic recombination machinery and the synaptonemal complex such as Atm, Dmc1, cH2AX, Mlh1, Msh5, Rec8, Rad51, Smc1b, Spo11, Sycp1, Sycp2, Sycp3, Syce2 and Tex14. None of the male meiotic arrest genes listed in the Mouse Genome Database showed a consistent change in expression level in Tex19.1 2/2 knockout testes compared to littermate controls (I.R.A., data not shown). However, analysis of the microarray data suggested that the class II LTR-retrotransposon MMERVK10C [37] is upregulated by around four-fold in the testis RNA from each of the 16 dpp Tex19.1 2/2 knockout animals relative to their littermate controls (I.R.A., data not shown). The mouse genome contains around 16 approximately full-length copies of the MMERVK10C sequence in the genome, and a further 1200 fragments of the MMERVK10C endogenous retrovirus. Increased retrotransposon expression has been proposed to be responsible for impaired chromosome synapsis and meiotic defects during spermatogenesis in Dnmt3L, Miwi2 and Mili mutant mice [11][12][13][14]. As overexpression of the MMERVK10C retrotransposons could similarly be responsible for the meiotic defects seen in Tex19.1 2/2 mutant mice we sought to determine whether MMERVK10C expression is indeed upregulated in the testis in the absence of Tex19.1.
The levels of MMERVK10C expression in testis cDNA from two Tex19.1 2/2 knockout animals relative to their Tex19.1 +/+ wild-type littermates were each tested by quantitative PCR ( Figure 6A). The Sertoli cell marker Sdmg1 [22] was used to normalise cDNAs from different animals. Although there was no significant change in the expression of the ubiquitously expressed b-actin gene, or the germ cell marker Dazl [38], expression of the MMERVK10C endogenous retrovirus was increased by a factor of approximately four-fold in both Tex19.1 2/2 knockout animals (Student's t-test, p,0.01) ( Figure 6A). Expression of LINE, SINE or IAP retrotransposons showed no significant change in the absence of Tex19.1 ( Figure 6A).
To further validate the potential upregulation of MMERVK10C transcripts in the Tex19.1 2/2 knockout mice we performed Northern blots on testis RNA from the same two 16 dpp Tex19.1 2/2 knockout animals and their Tex19.1 +/+ wild type littermates. Using a probe derived from the env gene of the MMERVK10C endogenous retrovirus we were able to detect a predominant 3.2 kb MMERVK10C env transcript in mouse testes, and some weaker MMERVK10C env transcripts at around 4.5 kb and 7.5 kb ( Figure 6B). The Northern blot profile for MMERVK10C env transcripts is comparable to that of envcontaining transcripts from HERV-K endogenous retroviruses in human teratocarcinoma cell lines [39]. Northern blotting confirmed that the predominant 3.2 kb MMERVK10C env transcript is consistently more abundant in testes from Tex19.1 2/2 knockout animals than in testes from their wild-type littermates at 16 dpp ( Figure 6B).
In order to determine which cell types are accumulating MMERVK10C transcripts in the Tex19.1 2/2 knockout testes we performed in situ hybridisation on testis sections using a MMERVK10C env probe ( Figure 6C-N). In Tex19.1 +/+ wild-type and Tex19.1 +/2 heterozygous testes at 16 dpp, low levels of MMERVK10C env transcripts were present in some meiotic spermatocytes ( Figure 6C,G). However, MMERVK10C transcripts were generally more abundant in Tex19.1 2/2 knockout testes than in testes from Tex19.1 +/+ wild-type or Tex19.1 +/2 heterozygous littermates at 16 dpp ( Figure 6D, H). The increased levels of MMERVK10C env transcript in 16 dpp Tex19.1 2/2 testes appeared to be largely due to the presence of strongly expressing cells located towards the centre of the tubules where meiotic spermatocytes are present ( Figure 6K, L). Similarly in adult animals MMERVK10C env transcripts were upregulated in meiotic germ cells in the testes from adult Tex19.1 2/2 knockout animals relative to their heterozygous littermates ( Figure 6E, F, I, J). A total of nine different Tex19.1 2/2 knockout animals at various ages were assayed for MMERVK10C expression in the testes by in situ hybridisation, and MMERVK10C expression in Tex19.1 2/2 knockout testes was consistently higher that in Tex19.1 +/+ or Tex19.1 +/2 littermate controls. No in situ hybrid-isation signals were detected on testis sections using a sense MMERVK10C control probe ( Figure 6M, N).
Taken together, the quantitative PCR, Northern blotting and in situ hybridisation data all suggest that transcripts from the MMERVK10C endogenous retrovirus are upregulated in the meiotic spermatocytes of Tex19.1 2/2 knockout testes.
The upregulation of retrotransposons in Dnmt3L, Mili and Miwi2 mutant mice is associated with defects in de novo DNA methylation of IAP and LINE elements in the male germline, which presumably allows increased transcription of these elements during spermatogenesis [11][12][13][14]. In order to investigate whether the upregulation of MMERVK10C retrotransposons in Tex19.1 2/2 knockout testis was caused by a similar mechanism, we investigated the DNA methylation status of CpG dinucleotides in MMERVK10C elements by bisulphite sequencing MMERVK10C elements from 16 dpp prepubertal Tex19.1 2/2 knockout testes. The MMERVK10C element includes a weak CpG island overlapping the LTR and 59untranslated region ( Figure S5). As promoters with weak CpG islands are good candidates for regulation by DNA methylation [40], we examined DNA methylation at CpG dinucleotides within this region. Sequence analysis of 30 independent clones from each of Tex19.1 +/+ wild-type, Tex19.1 +/2 heterozygous and Tex19.1 2/2 homozygous 16 dpp testes showed that CpG dinucleotides in this region of the MMERVK10C element are predominantly methylated in the testis at this age ( Figure S5). The MMERVK10C element was also methylated to a similarly high level in liver taken from the same animals as a somatic tissue control ( Figure S5). The prepubertal testis is composed of approximately equal numbers of germ cells and somatic cells at 16 dpp [29,30], therefore around half the clones analysed by bisulphite sequencing are likely to be derived from testicular germ cells and around half from testicular somatic cells. As all of the 16 dpp testis clones represented highly methylated DNA sequences ( Figure S5), the MMERVK10C element appears to be highly methylated in both the germ cell and somatic cell compartments of Tex19.1 +/+ wild-type, Tex19.1 +/2 heterozygous and Tex19.1 2/2 homozygous 16 dpp testes.
Although we have been unable to find any evidence that the methylation status of MMERVK10C elements in the testis changes in the absence of Tex19.1 ( Figure S5), we cannot exclude the possibility that the absence of Tex19.1 causes reduced DNA methylation in a subset of MMERVK10C elements in the genome, or in a subset of germ cells in 16 dpp testes. If only a subset of germ cells have altered DNA methylation at MMERVK10C elements in Tex19.1 2/2 mutant testes then we estimate that this subset would need to represent less than 25% of the germ cell population to be below our detection limit in this assay (x 2 -test, p,0.05). Nevertheless, our observations that loss of Tex19.1 causes the upregulation of MMERVK10C retrotransposon elements in the testis, but not IAP or LINE elements, combined with the absence of a detectable change in DNA methylation levels in MMERVK10C elements in Tex19.1 2/2 knockout testes, suggests that Tex19.1-mediated repression of retrotransposons may involve a mechanism that is distinct from Dnmt3L/Miwi2/Mili-mediated repression of retrotransposons. Thus we conclude that Tex19.1 is part of a novel genetic pathway that represses retrotransposons in the male germline.

Discussion
The Tex19.1 2/2 Knockout Phenotype This study describes the functional consequences of deleting the pluripotency-associated Tex19.1 gene in mice. Our data shows that loss of Tex19.1 causes impaired spermatogenesis and defects in Figure 6. Expression of the endogenous retrovirus MMERVK10C is upregulated in Tex19.1 2/2 null testes. (A) Quantitative PCR showing relative expression of retrotransposons and marker genes in testes from 16 dpp Tex19.1 2/2 knockout animals relative to their Tex19.1 +/+ wild-type littermates. Expression levels in cDNAs prepared from different animals were normalised to Sdmg1, animals represented in the first and third columns chromosome synapsis during meiosis. Mutations in genes that are involved in various aspects of meiotic chromosome behaviour such as the initiation of recombination between homologous chromosomes, or the assembly of the synaptonemal complex, all typically cause defective chromosome synapsis during meiosis, and apoptosis in the male germline [6,7]. However, although there is some similarity between these phenotypes and the Tex19.1 mutant phenotype, we have been unable to detect any localisation of Tex19.1 to meiotic chromosomes by immunostaining testis sections or testis chromosome spreads (R.O., data not shown). Indeed our data suggest that Tex19.1 is a predominantly cytoplasmic protein and is therefore unlikely to play a direct role in meiotic chromosome behaviour. Thus, although Tex19.1 mutant mice exhibit defects in chromosome pairing during meiosis, we do not believe that Tex19.1 is a component of meiotic chromosomes and favour the interpretation Tex19.1 is influencing meiotic chromosome behaviour indirectly.
Our finding that Tex19.1 is a predominantly cytoplasmic protein in germ cells and embryonic stem cells contradicts a previous study suggesting that Tex19.1 is a nuclear protein in embryonic stem cells [17]. The reason for the discrepancy between these studies is not yet clear. Kuntz et al. [17] raised monoclonal antibodies to Tex19.1 and observed nuclear staining with those antibodies in embryonic stem cells and pre-implantation embryos. The Tex19.1 peptide used by Kuntz et al. [17] to raise the monoclonal anti-Tex19.1 antibody is located C-terminally to the peptide that we have used to raise the anti-Tex19.1 antibodies in this study. Both peptides, and indeed the entire Tex19.1 open reading frame, lie within a single exon. In our study we have shown that Tex19.1 is predominantly cytoplasmic in embryonic stem cells by immunostaining and by Western blotting of subcellular fractions. We have also shown that Tex19.1 has a predominantly cytoplasmic localisation in germ cells by immunostaining germ cells isolated from embryonic testes, by immunohistochemistry on wax sections of adult testis and by Western blotting of subcellular fractions from prepubertal testes. Furthermore we have demonstrated the specificity of our antibody in the assays that we use by immunostaining and Western blotting on material from Tex19.1 2/2 knockout animals. As the cytoplasmic anti-Tex19.1 staining patterns that we present in this paper are lost in Tex19.1 2/2 knockout animals, at least some of the Tex19.1 protein that is present in germ cells and embryonic stem cells is cytoplasmic. However we cannot exclude the possibility that the two different antibodies raised in these two studies recognise mutually exclusive isoforms of Tex19.1 that have different subcellular localisations. Alternatively, the discrepancy between our study and the study by Kuntz et al. [17] could be caused by procedural differences, or by cross-reaction of anti-Tex19.1 antibodies with an unrelated antigen.
Tex19.1 2/2 null male mice showed some phenotypic variation between individuals ranging from completely agametic testes to fertility. This phenotypic variability may be partly due to the genetic heterogeneity in the outbred component of the genetic background used for this study. However, as some germ cells are more severely affected by the loss of Tex19.1 than other germ cells in the same animal, there is also some phenotypic variability in the absence of genetic variation. Furthermore, our finding that loss of Tex19.1 can impair spermatogenesis even in this heterogeneous genetic background suggests that mutations in the single human homologue, TEX19, could contribute to fertility problems in human populations. The human TEX19 gene contains two premature stop codons in the open reading frame that truncates the Tex19 protein from 351 residues in mouse to 164 residues in human [17]. The first premature stop codon in the human TEX19 gene is conserved in other primates suggesting that the C-terminal region of Tex19 is dispensable for function in primates [17]. The significance of this major difference in structure between human and mouse is at present unclear given our current level of understanding of the mechanisms underlying the phenotype in mouse.
The Tex19 genomic locus has undergone a duplication event in rodents to generate two closely related divergently transcribed genes [17]. The mutation that we have engineered removes the entire Tex19.1 open reading frame, but leaves Tex19.2 intact. Therefore Tex19.2 could potentially provide some functional redundancy with Tex19.1. Although Tex19.1 and Tex19.2 are reported to be expressed in testicular germ cells and testicular somatic cells respectively [17], there appears to be a moderate upregulation of Tex19.2 in Tex19.1 2/2 knockout testes as judged by quantitative RT-PCR (I.R.A., data not shown). It is not clear at present whether this upregulation of Tex19.2 occurs in the germ cells or somatic cells of the testis, but any upregulation of Tex19.2 that is occurring does not seem to be able to fully compensate for loss of Tex19.1. Nevertheless, deletion of the entire Tex19 locus may be required to rule out the possibility of some functional redundancy between these genes and may reveal additional functions for Tex19.1 in the germline.
This study demonstrates that Tex19.1 has a function in progression through meiosis in the male germline. Characterisation of the meiotic defect in Tex19.1 2/2 knockout spermatocytes indicates that homologous recombination is being initiated in the Tex19.1 2/2 knockout spermatocytes but that, for some chromosomes, synapsis does not occur. As homologous recombination and chromosome synapsis progress interdependently during meiosis, it is possible that the chromosome synapsis defect that we describe in Tex19.1 2/2 knockout spermatocytes is a secondary consequence of a defect in the progression of homologous recombination, or a secondary consequence of defects in the pairing between homologous chromosomes that normally precedes chromosome synapsis [7]. Further work is needed to dissect the molecular basis of the Tex19.1 chromosome synapsis defect in more detail, and to understand if and how the upregulation of MMERVK10C retrotransposons that we detect in Tex19.1 2/2 spermatocytes causes these defects in meiotic chromosome synapsis. Tex19. 1

and Repression of Retrotransposons in the Germline
The Tex19.1 mutant phenotype bears some resemblance to the Dnmt3L, Miwi2 and Mili mutant phenotypes in that they all exhibit defects in meiotic chromosome synapsis and increased expression of retrotransposons in the germline [11][12][13][14]. However it is not yet clear whether there is a direct causal relationship between these two events. The increase in retrotransposon expression does not appear to be caused by defects in meiotic chromosome synapsis [11,13], but it is not clear whether or how the increase in retrotransposon expression causes the defects in meiotic chromosome synapsis in any of these mutant mice. Increased transposition of mobile genetic elements could introduce quantitative, qualitative, or temporal changes in the DNA double strand breaks normally present during early meiotic prophase that could interfere with the homologous recombination events that normally precede and initiate chromosome pairing. Support for this model comes from the observation that mutating genes involved in piRNA function in flies activates the DNA damage signalling pathway [41,42]. Alternatively, it is possible that repression of retrotransposons is important for the fidelity of homolog pairing and synapsis during meiosis, and that increased expression of these repetitive elements either interferes with homolog recognition and synapsis, or promotes pairing between non-homologous chromosomes. A third possibility is that proteins encoded by the MMERVK10C endogenous retrovirus mediate the defects in meiotic chromosome synapsis by interfering with host cell proteins involved in meiotic chromosome behaviour or regulation of the meiotic cell cycle. In this regard it is important to note that transgenic mice expressing the rec protein derived from the HERVK human endogenous retrovirus exhibit defects in spermatogenesis [43]. Lastly, there may not be a direct causal relationship between retrotransposon de-repression and chromosome asynapsis. Rather the Tex19.1, Dnmt3L, Miwi2 and Mili mutants may all cause defects in meiotic chromosome structure that lead to both retrotransposon de-repression and defective chromosome synapsis. Clearly further work is needed to clarify the molecular mechanism underlying the chromosome synapsis defect in the Tex19.1 mutant mice presented here, and in the Dnmt3L, Miwi2 and Mili mutant mice [11][12][13]. However, this study provides further evidence demonstrating a correlation between de-repression of retrotransposons and impaired chromosome synapsis during mouse meiosis.
Although there are gross similarities between the Tex19.1 mutant phenotype and the Dnmt3L, Miwi2 or Mili mutant phenotypes, there are also important differences. Dnmt3L, Miwi2 and Mili are all required to repress LINE and IAP retrotransposons in the germline, and these three genes appear to converge on DNA methylation and transcriptional repression of these sequences in the genome [11][12][13][14]. However, repression of LINE and IAP retrotransposons is not perturbed in Tex19.1 2/2 knockout testes suggesting that Tex19.1 is not involved in the transcriptional repression of LINE or IAP elements. Rather our data shows that transcripts from the MMERVK10C class of endogenous retroviruses accumulate in the germ cells in the absence of Tex19.1. These differences between the Tex19.1 mutant phenotype and the Dnmt3L, Miwi2 and Mili mutant phenotypes may reflect the existence of multiple mechanisms with different specificities to repress retrotransposons in the germline.
The Tex19.1 mutant phenotype is characterised by the accumulation of MMERVK10C retrotransposon transcripts, but the molecular basis for this phenotype is not yet clear. The upregulation of MMERVK10C transcripts could be caused by changes acting at any level of gene expression from the initiation of transcription to mRNA turnover. We have not been able to find any difference in the level of DNA methylation at MMERVK10C elements in Tex19.1 mutant testes. This provides further evidence that Tex19.1 belongs to a different genetic pathway than Miwi2, Mili and Dnmt3L for repression of retrotransposons in the germline. However, we cannot exclude the possibility that DNA methylation may be altered in a subset of MMERVK10C elements in a subset of germ cells in the Tex19.1 mutant testes, and that this subset of elements is responsible for the upregulation of MMERVK10C transcripts that we describe in the Tex19.1 mutant testes. An alternative model to explain the upregulation of MMERVK10C elements in Tex19.1 mutant testes is that Tex19.1 could be a transcriptional repressor of MMERVK10C elements. The nuclear localisation of Tex19.1 reported by Kuntz et al. [17] would be consistent with this type of mechanism operating. However, although we cannot exclude the possibility that some Tex19.1 acts in the nucleus in the germ cells in the adult testes, our finding that Tex19.1 is predominantly cytoplasmic in these cells would be more consistent with Tex19.1 acting to regulate gene expression at a post-transcriptional level. We are able to detect MMERVK10C transcripts in wild-type testes ( Figure 6B,G) suggesting that some MMERVK10C transcripts must escape DNA methylation or transcriptional repression, and that posttranscriptional regulation of MMERVK10C mRNA may play a role in repressing the activity of this retrotransposon. The upregulation of MMERVK10C transcripts in Tex19.1 mutant testes does not appear to be the result of changes in RNA splicing as the MMERVK10C isoforms present in Tex19.1 mutant testes do not appear to be qualitatively different from those present in wild-type testes. However, the accumulation of MMERVK10C transcripts in Tex19.1 knockout testes would be consistent with Tex19.1 promoting degradation of MMERVK10C mRNA. Investigation into the biochemical function of Tex19.1 should provide a ready test of these models and generate some insight into the molecular mechanism of Tex19.1-dependent repression of MMERVK10C endogenous retroviruses.
Repression of retrotranposons in the mammalian germline requires mechanisms to distinguish retrotransposons from endogenous genes to allow repression to be targeted to the correct loci. piRNAs, a group of small RNAs that are physically associated with the piwi class of proteins, are abundant in male germ cells and some piRNAs have sequence homology to various classes of retrotransposon [14,[44][45][46]. The sequence homology between some piRNA molecules and retrotransposons is presumably used to target DNA methylation to retrotransposons rather than endogenous genes. Although there is good genetic evidence that the piwi class of proteins is involved in transcriptional repression of retrotransposons [12][13][14], there is also good biochemical evidence that piwi proteins and piRNAs are physically associated with the translational machinery in male germ cells [46,47], suggesting a role in translation or mRNA turnover. Thus piRNA-mediated repression of retrotransposons may be working at multiple levels of gene expression in male germ cells. It will be informative to investigate whether the Tex19.1 pathway for repression of retrotransposons that we describe here also utilises piRNAs to target repression to MMERVK10C elements.
One of the interesting aspects of the Tex19.1 phenotype is that although the MMERVK10C subclass of retrotransposons is upregulated in Tex19.1 mutant testes, LINE, SINE and IAP retrotransposons are not. It is not clear how Tex19.1 determines specificity for the MMERVK10C element. Notably, IAP elements belong to the same subclass of endogenous retroviruses as MMERVK10C elements (class II LTR retrotransposons) but are not upregulated in Tex19.1 mutant testes. Sequences within the MMERVK10C promoter or transcript could be involved in targeting Tex19.1 activity to this element. Alternatively Tex19.1 may have the potential to regulate a wider range of retrotransposons than we have been able to identify here, but alternative mechanisms to repress retrotransposon expression during spermatogenesis, such as DNA methylation, may limit the phenotypic effects of losing Tex19.1 to a subset of its potential targets. Furthermore, as Tex19.1 expression is not restricted to spermatogenesis but also occurs in primordial germ cells, oocytes and pluripotent stem cells, it will be of interest to determine if Tex19.1 is involved in repressing MMERVK10C elements and other classes of retrotransposons in these cell types.
In addition to its role in the germline, Tex19.1 is also expressed in pluripotent cells. Like germ cells, pluripotent cells are viable targets for retrotransposon activity as any new transposition events could be propagated through successive generations. Therefore pluripotent cells presumably also need to modulate retrotransposon activity to ensure that the mutational load on the genome is not too high. Our finding that Tex19.1 2/2 homozygotes are born at a sub-Mendelian frequency is consistent with a role for Tex19.1 in pluripotent cells in early embryonic development. Further work is required to determine whether the loss of Tex19.1 2/2 homozygotes during embryogenesis is caused by defects in pluripotent cells, and whether pluripotent cells upregulate retrotransposon expression in Tex19.1 2/2 knockout embryos.
The ongoing battle between retrotransposons and the host genome has important consequences for evolution, and for genetic disease. Retrotransposons that can successfully evade genome defences in germ cells and pluripotent cells will be selected for during evolution, whereas germ cells and pluripotent cells are under selective pressure to keep the mutational load on the genome at sustainable levels. The striking differences in the relative abundance of different classes of retrotransposable elements between the mouse and human genomes suggest that this conflict is ongoing during mammalian evolution [9]. Although low levels of mutation and retrotransposition in the germline are required to generate the genetic variation essential for evolution, high levels of mutation or retrotransposition are deleterious to the survival of a species. In humans, endogenous retroviruses with intact coding sequences comprise a very small proportion of the genome [48], yet intact endogenous retroviral particles are found in human pluripotent stem cells, and in testicular germ cell tumours where the expression of endogenous retroviral proteins has been suggested to contribute to tumourigenesis [39,43,49]. Furthermore, a number of human genetic diseases are associated with de novo mutagenic retrotransposition events that disrupt the function of endogenous human genes [50,51]. Our data suggests that Tex19.1 is part of a mechanism that protects the genome from the deleterious effects of retrotransposon activity in the germline, and thereby helps to maintain genomic stability through successive generations. immunising peptide (+pep) the signal is not present. (F) Immunohistochemistry with anti-Tex19.1 antibodies on Tex19.1 2/2 knockout testes gives no signal. (G-I) Immunofluorescence on single cell suspensions from 14.5 dpc male embryonic gonads. Isolated gonads were trypsinised to single cell suspensions, then attached to poly lysine-coated slides, fixed with 4% paraformaldehyde in PBS and immunofluorescence was performed as described for cell spreads. Anti-Tex19.1 primary antibody is shown in green, nuclei counterstained with DAPI are shown in red. (G) Anti-Tex19.1 staining on a suspension of 14.5 dpc embryonic male gonadal cells gives strong signal in the germ cells. This signal is predominantly localized to the cytoplasm (inset in G). (H) This signal is not present when the antibody is blocked with immunising peptide (+pep). (I) Anti-Tex19.1 antibodies give no signal on gonadal cell suspensions from a 14.5 dpc male Tex19.1 2/2 knockout embryo. spermatocytes are present and there is no obvious difference between the cell types present in the testes of knockout and wildtype littermates. (C, G) By 20 dpp, the germ cells appear to be greatly reduced in number in Tex19.1 2/2 knockout testes (D, H) At 29 dpp, round spermatids and some elongating spermatids are present in heterozygous testes, but these cell types are reduced in number in testes from Tex19.1 2/2 knockout littermates. Found at: doi:10.1371/journal.pgen.1000199.s004 (6.2 MB TIF) Figure S5 MMERVK10C retrotransposons show no detectable change in DNA methylation status in Tex19.1 2/2 knockout testes. A schematic diagram showing the genomic organisation of the 5end of the MMERVK10C retrotransposon is shown at the top of the figure. The long terminal repeat (LTR), 59untranslated region (59utr) and the start of the gag open reading frame are indicated, and the region analysed by bisulphite sequencing shown below with CpG dinucleotides indicated by grey circles. The DNA methylation status of CpG dinucleotides in 30 independent clones isolated from the liver or testes from 16 dpp Tex19.1 +/+ wild-type, Tex19.1 +/2 heterozygous and Tex19.1 2/2 homozygous animals is also shown. Black circles indicate methylated CpGs protected from bisulphite conversion, white circles indictate unmethylated CpGs. 500 ng genomic DNA from these tissues was bisulphite treated using the EZ DNA Methylation Gold kit (Zymo Research) then used as a template for nested PCR using the primers 59-AGGTTTATAAAAGTAGTATTAG-39 and 59-ATAACAAT-TAAAACAATAACATA-39, then 59-TAAAAGTAGTAT-TAGTTTTGGG-39 and 59-AAACAAACAACACAATCCCA-39. The resulting 480 bp PCR product was then blunt-end cloned into pBluescript II SK+ (Stratagene) and independent plasmid clones were isolated and sequenced. Around 95% of the non-CpG cytosine residues were converted to thymine in the analysed sequences indicating succesful bisulphite conversion. Found at: doi:10.1371/journal.pgen.1000199.s005 (8.2 MB TIF)