Germ Cell Gene Research Articles

Mitf is a transcriptional activator of medaka germ genes in culture

Germ cells express a unique subset of genes called germ genes mostly encoding RNA-binding proteins such as Dazl, Dnd and Vasa. How germ gene expression is controlled remains illusive, because in no organism has a transcription factor been identified that regulate expression of these genes. Microphthalmia-associated transcription factor (Mitf) has been reported to show expression in male mouse germ cells of the adult testis. Here we report in the fish medaka (Oryzias latipes) that Mitf is a transcription activator of germ gene expression. Mitf is a master regulator of melanocyte development, which activates melanogenic genes through binding to the E-box containing consensus CANNTG. The E-box was found to be present in 23–26 copies in the promoters of medaka germ genes dazl, dnd and vasa. Importantly, forced Mitf expression enhanced the transcriptional activity of the three gene promoters by up to more than 10 fold and remarkably increased the level of endogenous dazl, dnd and vasa transcripts in cell culture. Transfection of Mitf expression vectors was sufficient to induce directed differentiation of medaka embryonic stem cells into melanocytes. Fluorescence in situ hybridization revealed the expression of both medaka mitf genes in adult germ cells of male and female gonads. Mitf is well-known as the melanocyte master regulator. Our results offer first evidence that Mitf may act as a transcriptional activator of germ gene expression in medaka.

Jarid1b targets genes regulating development and is involved in neural differentiation

H3K4 methylation is associated with active transcription and in combination with H3K27me3 thought to keep genes regulating development in a poised state. The contribution of enzymes regulating trimethylation of lysine 4 at histone 3 (H3K4me3) levels to embryonic stem cell (ESC) self-renewal and differentiation is just starting to emerge. Here, we show that the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) is dispensable for ESC self-renewal, but essential for ESC differentiation along the neural lineage. By genome-wide location analysis, we demonstrate that Jarid1b localizes predominantly to transcription start sites of genes encoding developmental regulators, of which more than half are also bound by Polycomb group proteins. Virtually all Jarid1b target genes are associated with H3K4me3 and depletion of Jarid1b in ESCs leads to a global increase of H3K4me3 levels. During neural differentiation, Jarid1b-depleted ESCs fail to efficiently silence lineage-inappropriate genes, specifically stem and germ cell genes. Our results delineate an essential role for Jarid1b-mediated transcriptional control during ESC differentiation.

Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes

To detect rare epigenetic effects associated with assisted reproduction, it is necessary to monitor methylation patterns of developmentally important genes in a few germ cells and individual embryos. Bisulfite treatment degrades DNA and reduces its complexity, rendering methylation analysis from small amounts of DNA extremely challenging. Here we describe a simple approach that allows determining the parent-specific methylation patterns of multiple genes in individual early embryos. Limiting dilution (LD) of bisulfite-treated DNA is combined with independent multiplex PCRs of single DNA target molecules to avoid amplification bias. Using this approach, we compared the methylation status of three imprinted (H19, Snrpn and Igf2r) and one pluripotency-related gene (Oct4) in three different groups of single mouse two-cell embryos. Standard in vitro fertilization of superovulated oocytes and the use of in vitro matured oocytes were not associated with significantly increased rates of stochastic single CpG methylation errors and epimutations (allele methylation errors), when compared with the in vivo produced controls. Similarly, we compared the methylation patterns of two imprinted genes (H19 and Snrpn) in individual mouse 16-cell embryos produced in vivo from superovulated and non-superovulated oocytes and did not observe major between-group differences. Using bovine oocytes and polar bodies as a model, we demonstrate that LD even allows the methylation analysis of multiple genes in single cells.

Identification of 67 Histone Marks and Histone Lysine Crotonylation as a New Type of Histone Modification

We report the identification of 67 previously undescribed histone modifications, increasing the current number of known histone marks by about 70%. We further investigated one of the marks, lysine crotonylation (Kcr), confirming that it represents an evolutionarily-conserved histone posttranslational modification. The unique structure and genomic localization of histone Kcr suggest that it is mechanistically and functionally different from histone lysine acetylation (Kac). Specifically, in both human somatic and mouse male germ cell genomes, histone Kcr marks either active promoters or potential enhancers. In male germinal cells immediately following meiosis, Kcr is enriched on sex chromosomes and specifically marks testis-specific genes, including a significant proportion of X-linked genes that escape sex chromosome inactivation in haploid cells. These results therefore dramatically extend the repertoire of histone PTM sites and designate Kcr as a specific mark of active sex chromosome-linked genes in postmeiotic male germ cells.

Regulation of gene expression in mouse germ cells upon exposure to tetrabutyltin

Tetrabutyltin is the starting material of the tributyltin and dibutyltin compounds. These tin compounds are starting materials for a wide range of organotin compounds used as stabilizers for PVC, biocides, fungicides, and anti-biofouling agents. It is commonly known that some of the environmental chemicals affect the human endocrine system. Since the 1970s, the harmful effects of endocrine disrupting chemical (EDC), Tetrabutyltin (TTBT) have been extensively studied. In the present study, we analyzed the effect of TTBT on gene regulation and expression of genes in mouse germ cells. Mouse germ cells were treated with TTBT for 3 and 24 hrs, and global gene expression was analyzed by using customized Agilent mouse arrays. We identified genes that were >2-fold differentially expressed in TTBT-treated cells than control cells (P<0.05) and analyzed their functions through Gene Ontology analysis. In a total number of 687 genes, there was differential expression between TTBT-treated and control cells. About 471 and 350 genes exhibited 2-fold (P<0.05) increased or decreased expression at 3 and 24 hrs, respectively. Additionally, in 134 genes, common and significant changes were observed in two-time series. Functional analysis of these genes showed that there were significant enrichments in some of the processes related to the reproduction such as, female gamete generation, homeostatic process, cell differentiation, response to chemical stress and reproductive process. In conclusion, the present study provides an insight on the effects of TTBT on mouse germ cells. Furthermore, it also increases our knowledge on the underlying molecular mechanisms on the adverse effects of organotin on the reproductive system.

Different doses of bone morphogenetic protein 4 promote the expression of early germ cell-specific gene in bone marrow mesenchymal stem cells

Bone morphogenetic protein (BMP)-4 has a crucial role on primordial germ cells (PGCs) development in vivo which can promote stem cell differentiation to PG-like cells. In this study, we investigated the expression of Mvh as one of the specific genes in primordial germ cells after treatment with different doses of BMP4 on bone mesenchymal stem cells (BMSCs)-derived PGCs. Following isolation of BMSCs from male mouse femur and tibia, cells were cultured in medium for 72 h. Passage 4 murine BMSCs were characterized by CD90, CD105, CD34, and CD45 markers and osteo-adipogenic differentiation. Different doses of BMP4 (0, 0.01, 0.1, 1, 5, 25, 50, and 100 ng/ml) were added to BMSCs for PGCs differentiation during 4-days culture. Viability percent, proliferation rates, and expression of Mvh gene were analyzed by RT-qPCR. Data analysis was done with ANOVA test. CD90(+), CD105(+), CD34(-), and CD45(-) BMSCs were able to differentiate to osteo-adipogenic lineages. The results revealed that proliferation rate and viability percent were raised significantly (p ≤ 0.05) by adding 1, 5, 25 ng/ml of BMP4 and there were decreased to the lowest rate after adding 100 ng/ml BMP4 (p ≤ 0.05). There were significant up regulation (p ≤ 0.05) in Mvh expression between 25, 50, and 100 ng/ml BMP4 with other doses. So the selective dose of BMP-4 for treatment during 4-day culture was 25 ng/ml. The results suggest that addition of 25 ng/ml BMP4 had the best effects based on gene-specific marker expression.

Expression Patterns and miRNA Regulation of DNA Methyltransferases in Chicken Primordial Germ Cells

DNA methylation is widespread in most species, from bacteria to mammals, and is crucial for genomic imprinting, gene expression, and embryogenesis. DNA methylation occurs via two major classes of enzymatic reactions: maintenance-type methylation catalyzed by DNA (cytosine-5-)-methyltransferase (DNMT) 1, and de novo methylation catalyzed by DNMT 3 alpha (DNMT3A) and -beta (DNMT3B). The expression pattern and regulation of DNMT genes in primordial germ cells (PGCs) and germ line cells has not been sufficiently established in birds. Therefore, we employed bioinformatics, RT-PCR, real-time PCR, and in situ hybridization analyses to examine the structural conservation and conserved expression patterns of chicken DNMT family genes. We further examined the regulation of a candidate de novo DNA methyltransferase gene, cDNMT3B by cotransfection of cDNMT3B 3′UTR- and cDNMT3B 3′UTR-specific miRNAs through a dual fluorescence reporter assay. All cDNMT family members were differentially detected during early embryonic development. Of interest, cDNMT3B expression was highly detected in early embryos and in PGCs. During germ line development and sexual maturation, cDNMT3B expression was reestablished in a female germ cell-specific manner. In the dual fluorescence reporter assay, cDNMT3B expression was significantly downregulated by four miRNAs: gga-miR-15c (25.82%), gga-miR-29b (30.01%), gga-miR-383 (30.0%), and gga-miR-222 (31.28%). Our data highlight the structural conservation and conserved expression patterns of chicken DNMTs. The miRNAs investigated in this study may induce downregulation of gene expression in chicken PGCs and germ cells.

Expression of BCL-2 family genes in germ cells undergoing apoptosis during the first wave of spermatogenesis in the rat

Apoptosis is a key event controlling sperm output both in normal and pathological conditions. However, the mechanisms involving germ cell apoptosis is far from being understood. In this work, we have immunoisolated germ cells undergoing apoptosis by taking advantage of the up-regulation of Fas, a dead receptor involved in apoptosis induction in these cells. Analysis of specific markers showed that this cell population is composed of spermatogonia and meiotic spermatocytes. We measured the mRNA levels of several apoptosis-inducing proteins belonging to the BCL-2 family (BAX, BAD, PUMA, BOK and BAK) as well as those that prevent apoptosis (BCL-2, BCL-W and BCL-XL). Results showed that apoptotic germ cells have elevated mRNA levels of all studied genes (both pro and anti-apoptotic) compared with non-apoptotic cells. Our data would help to define the molecular mechanisms involving germ cell apoptosis under physiological conditions.

Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development.

BackgroundWe present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development.ResultsThe genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements.ConclusionsAnalyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution.

Germ Cell Genes and Cancer

In fruit flies, genes that help to program germ cells also play a role in a brain cancer.

Biallele Expression of PEG10 Gene in Primordial Germ Cells Derived from Day 27 Porcine Fetuses

Primordial germ cells (PGCs) from day 27 porcine fetuses have often been isolated to establish pluripotent embryonic germ (EG) cell lines, but little is known regarding their imprinted gene status. In our study, we attempted to detect the imprinted gene expression of cloned embryos and EG cells derived from individual PGC of day 27 and day 35, using single nucleotide polymorphism (SNP) analysis of the paternally expression gene 10 (PEG10) as a sign of parental-origin-specific expression. The results showed biallelic gene expression of the SNP that occurred in EG cell colonies and almost all of the cloned blastocysts, demonstrating that aberrant imprinted gene expression of PEG10 occurs in the day 27 porcine PGCs, whereas monoallelic expression of the PEG10 gene occurs in all the PGC clones derived from day 35 PGCs. In addition, the same imprinted gene status was observed for blastocysts derived from both male and female PGCs, indicating that the parental genomic imprinting is erased in male and female germlines.

Regulation of Translational Suppression in Post-Meiotic Germ Cells: Crosstalk Between MicroRNAs and Actin-Associated Proteins.

During male germ cell development, the vast majority of mRNAs is translationally repressed at the post-meiotic stage. While individual components implicated in the transcriptional and posttranscriptional control of specific germ cell gene expression have been identified, the mechanism that coordinates widespread translational suppression during germ cell differentiation remains unclear. In this study, we established that the cross-talk between miRNAs and actin-associated protein sets the tone for the mRNA-specific as well as general uncoupling of transcription and translation during post-meiotic germ cell maturation. Our study reveals that loss of the miRNA-processing enzyme Dicer function in haploid spermatids causes altered translational activation of several post-meiotic germ cell-specific transcripts, resulting in abnormal chromatin compaction and impaired fertility. We identified a specific miRNA-targeted actin-associated protein 2/3 complex (Arpc) subunit that plays a crucial role in this process as it sequesters germ cell-specific transcripts through their 3' UTR into translationally inert RNP complexes, and making them inaccessible to translational machinery. Interestingly, this actin-associated protein mediates translational suppression by interacting with GW182, a component of processing and chromatoid bodies, discrete cytoplasmic foci in the somatic and germ cells, respectively, believed to play an instrumental role in the storage and degradation of translationally suppressed mRNA. Collectively, our findings suggest that miRNAs are important checkpoint regulators that control timely expression of genes critical for post-meiotic germ cell differentiation and add a new dimension to the increasing role of actin and actin-associated proteins in post-transcriptional gene regulation. In light of recent studies showing an association between altered expression of actin-binding proteins and poor semen quality in human males, we propose that miRNA(s) targeting this Arpc subunit may serve as novel biomarkers for defective sperm and impaired male fertility. This research is funded by the NIH (HD053774-03). (platform)

KIT ligand and bone morphogenetic protein signaling enhances human embryonic stem cell to germ-like cell differentiation

Signaling mechanisms involved in early human germ cell development are largely unknown and believed to be similar to mouse germ cell development; however, there may be species specific differences. KIT ligand (KITL) and Bone morphogenetic protein 4 (BMP4) are necessary in mouse germ cell development and may play an important role in human germ cell development. KITL signaling studies were conducted by differentiating human embryonic stem cells (hESCs) on KITL wild-type, hetero- or homozygous knockout feeders for 10 days, and the effects of BMP signaling was determined by differentiation in the presence of BMP4 or its antagonist, Noggin. The formation of germ-like cells was ascertained by immunocytochemistry, flow cytometry and quantitative RT-PCR for germ cell markers. The loss of KITL in enrichment and differentiation cultures resulted in significant down-regulation of germ cell genes and a 70.5% decrease in germ-like (DDX4+ POU5F1+) cells, indicating that KITL is involved in human germ cell development. Moreover, endogenous BMP signaling caused germ-like (DDX4+ POU5F1+) cell differentiation, and the inhibition of this pathway caused a significant decrease in germ cell gene expression and in the number of DDX4+ POU5F1+ cells. Further, we demonstrated that eliminating feeders but maintaining their secreted extracellular matrix is sufficient to sustain the increased numbers of DDX4+ POU5F1+ cells in culture. However, this resulted in decreased germ cell gene expression. From these studies, we establish that KITL and BMP4 germ cell signaling affects in vitro formation of hESC derived germ-like cells and we suggest that they may play an important role in normal human germ cell development.

Characterization of Maternal Antigen That Embryos Require (MATER/NLRP5) Gene and Protein in Pig Somatic Tissues and Germ Cells

Maternal effect genes produce mRNA or proteins that accumulate in the egg during oogenesis and control the developmental program until embryonic genome activation takes place. NLRP5 (NLR family, Pyrin domain containing 5), also called MATER (Maternal Antigen That Embryos Require) is one of the genes required for normal early embryonic development, although its precise function remains to be elucidated. The aim of the present study was to analyze the NLRP5 gene expression pattern and protein distribution in somatic tissues and germ cells in the pig. Reverse transcription was performed on mRNA from germinal vescicle (GV) oocytes and total RNA from spermatozoa and tissues from different organs. The transcript for NLRP5 gene was identified only in ovaries and oocytes. The presence of NLRP5 protein was detected only in ovaries by western blot analysis and immunohistochemistry.

Regulation of ALF Promoter Activity in Xenopus Oocytes

BackgroundIn this report we evaluate the use of Xenopus laevis oocytes as a matched germ cell system for characterizing the organization and transcriptional activity of a germ cell-specific X. laevis promoter.Principal FindingsThe promoter from the ALF transcription factor gene was cloned from X. laevis genomic DNA using a PCR-based genomic walking approach. The endogenous ALF gene was characterized by RACE and RT-PCR for transcription start site usage, and by sodium bisulfite sequencing to determine its methylation status in somatic and oocyte tissues. Homology between the X. laevis ALF promoter sequence and those from human, chimpanzee, macaque, mouse, rat, cow, pig, horse, dog, chicken and X. tropicalis was relatively low, making it difficult to use such comparisons to identify putative regulatory elements. However, microinjected promoter constructs were very active in oocytes and the minimal promoter could be narrowed by PCR-mediated deletion to a region as short as 63 base pairs. Additional experiments using a series of site-specific promoter mutants identified two cis-elements within the 63 base pair minimal promoter that were critical for activity. Both elements (A and B) were specifically recognized by proteins present in crude oocyte extracts based on oligonucleotide competition assays. The activity of promoter constructs in oocytes and in transfected somatic Xenopus XLK-WG kidney epithelial cells was quite different, indicating that the two cell types are not functionally equivalent and are not interchangeable as assay systems.ConclusionsOverall the results provide the first detailed characterization of the organization of a germ cell-specific Xenopus promoter and demonstrate the feasibility of using immature frog oocytes as an assay system for dissecting the biochemistry of germ cell gene regulation.

Regulation of Germ Cell Promoter Activity in Xenopus Oocytes

Here we evaluate the feasibility of using Xenopus laevis oocytes as a model system for assaying the activity and architecture of a germ cell promoter. To test this approach the promoter from the X. laevis ALF transcription factor gene was cloned and evaluated for transcription start site usage and DNA methylation status. Comparison with human, chimpanzee, macaque, mouse, rat, cow, pig, horse, dog, chicken and X. tropicalis revealed low homology and demonstrated the difficulty in the computer prediction of regulatory elements. However, the activity of ALF promoter‐luciferase reporter constructs assayed after microinjection into oocytes was very high and could be localized to a 63 base pair segment. Interestingly, promoter activity in oocytes and somatic Xenopus XR epithelial cells were very different, indicating that these cell types are not interchangeable as assay systems. The use of site‐specific promoter mutants and deletion constructs identified two distinct functional elements (A and B) in the promoter, both of which were required for activity and recognized by proteins present in oocyte extracts. Rearrangement of the two elements displayed effects on activity ranging from minimal to severe. Overall, the results show that microinjection of a germ cell‐specific promoter into Xenopus oocytes provides a viable model system for dissecting the biochemistry of germ cell gene regulation.

Gonadoblastoma locus and the TSPY gene on the human Y chromosome

The gonadoblastoma (GBY) locus is the only oncogenic locus on the human Y chromosome. It is postulated to serve a normal function in the testis, but could exert oncogenic effects in dysgenetic gonads of individuals with intersex and/or dysfunctional testicular phenotypes. Recent studies establish the testis-specific protein Y-encoded (TSPY) gene to be the putative gene for GBY. TSPY serves normal functions in male stem germ cell proliferation and differentiation, but is ectopically expressed in early and late stages of gonadoblastomas, testicular carcinoma in situ (the premalignant precursor for all testicular germ cell tumors), seminomas, and selected nonseminomas. Aberrant TSPY expression stimulates protein synthetic activities, accelerates cell proliferation, and promotes tumorigenicity in athymic mice. TSPY binds to type B cyclins, enhances an activated cyclin B-CDK1 kinase activity, and propels a rapid G(2)/M transition in the cell cycle. TSPY also counteracts the normal functions of its X-homologue, TSPX, which also binds to cyclin B and modulates the cyclin B-CDK1 activity to insure a proper G(2)/M transition in the cell cycle. Hence, ectopic expression and actions of the Y-located TSPY gene in incompatible germ cells, such as those in dysgenetic or ovarian environments and dysfunctional testis, disrupt the normal cell cycle regulation and predispose the host cells to tumorigenesis. The contrasting properties of TSPY and TSPX suggest that somatic cancers, such as intracranial germ cell tumors, melanoma, and hepatocellular carcinoma, with detectable TSPY expression could exhibit sexual dimorphisms in the initiation and/or progression of the respective oncogenesis.

Androgen Receptor Roles in Spermatogenesis and Fertility: Lessons from Testicular Cell-Specific Androgen Receptor Knockout Mice

Androgens are critical steroid hormones that determine the expression of the male phenotype, including the outward development of secondary sex characteristics as well as the initiation and maintenance of spermatogenesis. Their actions are mediated by the androgen receptor (AR), a member of the nuclear receptor superfamily. AR functions as a ligand-dependent transcription factor, regulating expression of an array of androgen-responsive genes. Androgen and the AR play important roles in male spermatogenesis and fertility. The recent generation and characterization of male total and conditional AR knockout mice from different laboratories demonstrated the necessity of AR signaling for both external and internal male phenotype development. As expected, the male total AR knockout mice exhibited female-typical external appearance (including a vagina with a blind end and a clitoris-like phallus), the testis was located abdominally, and germ cell development was severely disrupted, which was similar to a human complete androgen insensitivity syndrome or testicular feminization mouse. However, the process of spermatogenesis is highly dependent on autocrine and paracrine communication among testicular cell types, and the disruption of AR throughout an experimental animal cannot answer the question about how AR in each type of testicular cell can play roles in the process of spermatogenesis. In this review, we provide new insights by comparing the results of cell-specific AR knockout in germ cells, peritubular myoid cells, Leydig cells, and Sertoli cells mouse models that were generated by different laboratories to see the consequent defects in spermatogenesis due to AR loss in different testicular cell types in spermatogenesis. Briefly, this review summarizes these results as follows: 1) the impact of lacking AR in Sertoli cells mainly affects Sertoli cell functions to support and nurture germ cells, leading to spermatogenesis arrest at the diplotene primary spermatocyte stage prior to the accomplishment of first meiotic division; 2) the impact of lacking AR in Leydig cells mainly affects steroidogenic functions leading to arrest of spermatogenesis at the round spermatid stage; 3) the impact of lacking AR in the smooth muscle cells and peritubular myoid cells in mice results in similar fertility despite decreased sperm output as compared to wild-type controls; and 4) the deletion of AR gene in mouse germ cells does not affect spermatogenesis and male fertility. This review tries to clarify the useful information regarding how androgen/AR functions in individual cells of the testis. The future studies of detailed molecular mechanisms in these in vivo animals with cell-specific AR knockout could possibly lead to useful insights for improvements in the treatment of male infertility, hypogonadism, and testicular dysgenesis syndrome, and in attempts to create safe as well as effective male contraceptive methods.

Expression of mx gene in chicken male germ cells in vivo

Expression of mx gene in chicken male germ cells in vivo

DAZL binds to the transcripts of several Tssk genes in germ cells

The Dazl gene encodes a germ-cell-specific RNA-binding protein which is essential for spermatogenesis. It has been proposed that this protein (DAZL) binds to RNA in the cytoplasm of germ cells and controls spermatogenesis. Using the specific nucleic acids associated with proteins (SNAAP) technique, we identified 17 target mRNAs bound by mDAZL. Among these transcripts, we focused on TSSK2, which encodes a testis-specific serine/threonine kinase. To date, five TSSK family members have been cloned, and all are exclusively expressed in the testis. We demonstrated that in addition to the TSSK1 3'UTR , the 3'UTRs of TSSKs 2 and 4 were bound by human and mouse DAZL, and that human DAZL (hDAZL) bound to the 3'UTR of human TSSK5 (hTSSK5). Our results suggest that the Dazl gene may play different roles in human and mouse spermatogenesis by regulating different members of the downstream gene family.