Decreased expression of genes associated with memory and x-linked mental retardation in boys with non-syndromic cryptorchidism and high infertility risk.

An elevated odds ratio for low IQ has been found for cryptorchid boys. Furthermore, poor school performance has been observed in cryptorchid boys with impaired mini-puberty. Gene expression analysis, qPCR and immunohistology were performed on testicular biopsies from 7 boys who underwent orchiopexy and had testicular histology typical of a high risk of infertility (HIR). The results were compared with 12 biopsies from cryptorchid boys with a low risk for developing infertility. The following genes associated with mental retardation were identically expressed: GDI1, OPHN1, PAK3, ARHGEF6, IL1RAPL, ACSL4, MECP2, RPS6KA3, ARX, and ATRX. However, boys in the HIR group had low or no expression of EGR4, FMR2 (AFF2) and VCX3A. In conclusion, impaired expression of genes known to encode proteins involved in signaling pathways that regulate cytoskeletal organization, synaptic vesicle transport and the establishment of connections between neuronal cells may contribute to reduced intellectual and cognitive functioning in infertile cryptorchid males.

Testicular gene expression in cryptorchid boys at risk of azoospermia.

Despite timely and successful surgery, 32% of patients with bilateral and 10% with unilateral cryptorchidism will develop azoospermia. Cryptorchid boys at risk of azoospermia display a typical testicular histology of impaired mini-puberty at the time of the orchidopexy. During mini-puberty increased gonadotropin and testosterone secretion stimulate transformation of gonocytes into Ad spermatogonia. In the azoospermia risk group this transformation is to a great extent impaired. This study aimed to analyze data on whole genome expression signatures of undescended testes at risk of developing azoospermia. Twenty-three testicular biopsies from 22 boys were analyzed (19 testes from 18 boys with cryptorchidism and 4 contralateral descended testes from patients with testicular agenesis). Expression profiling identified 483 genes not or under-expressed in the azoospermia risk group compared with the control and low risk for azoospermia (LAZR) groups. Annotated loci were associated with spermatogenesis. Other significant genes were cellular defense response genes and hormone-controlled loci involved in spermatogenesis. Some genes transcribed in normal adult meiotic and post-meiotic germ cells are activated in healthy juvenile Ad spermatogonia. Thus, molecular events initiating the testicular expression program at the onset of puberty and maintaining it during adulthood occur very early in prepubertal testes. This molecular event is to a great extent impaired in the high risk for azoospermia (HAZR) group lacking Ad spermatogonia (stem cells for spermatozoa) indicating impaired mini-puberty.

Deficient expression of genes involved in the endogenous defense system against transposons in cryptorchid boys with impaired mini-puberty.

Mini-puberty is the period between 30 and 80 days after birth when testosterone and gonadotropin surges occur in male infants to induce the transformation of gonocytes into adult/dark spermatogonia. Cryptorchid boys with impaired mini-puberty develop infertility despite timely and successful surgical treatment. The decreased germ cell count found in this group of boys could be the result of uncontrolled transposon activity inducing genomic instability and germ cell death. A genome-wide analysis of 18 cryptorchid and 4 control testes was performed with Affymetrix chips. We found that 5 of 8 genes that are important for transposon silencing were not expressed in the high azoospermia risk group of cryptorchid boys but were expressed in the low azoospermia risk and control groups. Two genes, CBX3 and DNMT1, were equally expressed in all 3 groups. Impaired expression of the DDX4, MAEL,MOV10L1, PIWIL2, PIWIL4, and TDRD9 genes in the group of cryptorchid boys at high risk of infertility indicates that gene instability induced by impaired expression of transposon silencing genes contribute to the development of azoospermia. Intact mini-puberty appears to be essential for the development of the endogenous defense system mediated by transposon silencing.

Decreased expression of FGFR1, SOS1, RAF1 genes in cryptorchidism.

BACKGROUND: In recent years, several genes were found to be involved in the process of epididymo-testicular descent, the most frequently cited ones include INSL3, HOXA10, GNRHR, and KAL1. In this study, we analyzed the differences in gene expression profiles between cryptorchid and descended testes. In particular, we analyzed expression of all recently published genes known to be associated with undescended testis. PATIENTS AND METHODS: Twenty-two testicular biopsies from 18 boys were analyzed. We analyzed gene expression in 16 cryptorchid and 6 descended testes using Affymetrix Human Genome U133 Plus 2.0 GeneChips, and validated the results with qPCR. RESULTS: 3,688 transcripts were differentially expressed with an adjusted p value of <0.05 and a change of at least 1.5-fold. The list contained 1,866 downregulated and 1,822 upregulated transcripts in the cryptorchid testes. A novel observation in our study was that the fibroblast growth factor receptor 1 gene (FGFR1) and its mediators SOS1 and RAF1 were expressed less in undescended testes. CONCLUSION: Based on our results, it is possible that a subtle dysfunction (expression) of the FGFR1, SOS1 and RAF1 genes is involved in the development of the most common male reproductive tract disorder - unilateral or bilateral cryptorchidism.

EGR4 is a master gene responsible for fertility in cryptorchidism.

The purpose of early medical or surgical treatment of boys with undescended testes is to prevent the development of infertility. However, early and successful surgery cannot prevent infertility in cryptorchid boys who lack type A dark (Ad) spermatogonia. The aim of this study was to compare the gene expression pattern of patients with completed transformation of gonocytes into Ad spermatogonia, associated with low infertility risk, with patients that had failed to undergo this process and had a high infertility risk. Genes expressed in the 16 cryptorchid testes were estimated using Affymetrix whole-genome microarray and compared to the expression profiles from four contralateral gonads of boys with unilateral testicular agenesis. Whole-genome expression profiling showed that boys in the high infertility risk group according to testicular histology, showed decreased or lack of expression of most of the genes essential for hypothalamo-pituitary-testicular axis function relative to low or intermediate risk group as well as controls. In particular, EGR4, which is involved in regulating the secretion of luteinizing hormone, was virtually not expressed. Thus, we found multiple differences in gene expression between the high and low infertility risk groups, confirming the importance of an intact hypothalamo-pituitary testicular axis and EGR4 in fertility development.

Transcriptional profiling of myotubes from patients with type 2 diabetes: no evidence for a primary defect in oxidative phosphorylation genes.

AIMS/HYPOTHESIS: Microarray-based studies of skeletal muscle from patients with type 2 diabetes and high-risk individuals have demonstrated that insulin resistance and reduced mitochondrial biogenesis co-exist early in the pathogenesis of type 2 diabetes independently of hyperglycaemia and obesity. It is unknown whether reduced mitochondrial biogenesis or other transcriptional alterations co-exist with impaired insulin responsiveness in primary human muscle cells from patients with type 2 diabetes. METHODS: Using cDNA microarray technology and global pathway analysis with the Gene Map Annotator and Pathway Profiler (GenMapp 2.1) and Gene Set Enrichment Analysis (GSEA 2.0.1), we examined transcript levels in myotubes established from obese patients with type 2 diabetes and matched obese healthy participants, who had been extensively metabolically characterised both in vivo and in vitro. We have previously reported reduced basal lipid oxidation and impaired insulin-stimulated glycogen synthesis and glucose oxidation in these diabetic myotubes. RESULTS: No single gene was differently expressed after correction for multiple testing, and no biological pathway was differently expressed using either method of global pathway analysis. In particular, we found no evidence for differential expression of genes involved in mitochondrial oxidative metabolism. Consistently, there was no difference in mRNA levels of genes known to mediate the transcriptional control of mitochondrial biogenesis (PPARGC1A and NRF1) or in mitochondrial mass between diabetic and control myotubes. CONCLUSIONS/INTERPRETATION: These results support the hypothesis that impaired mitochondrial biogenesis is not a primary defect in the sequence of events leading to insulin resistance and type 2 diabetes.

5-Methylcytosine DNA glycosylase participates in the genome-wide loss of DNA methylation occurring during mouse myoblast differentiation.

Changes in gene expression during mouse myoblast differentiation were monitored by DNA microarray hybridisation. Four days after the onset of differentiation 2.37% of the genes increased in activity from a value of zero, whereas during the same time 1.68% of total genes had decreased expression. During the first 24 h of differentiation an average of 700 000 CpG sites per haploid genome were demethylated. Maximal loss of DNA methylation is attained after 2 days of differentiation, followed by a gradual remethylation. The highest demethylation is observed in highly repeated DNA sequences, followed by single copy sequences. When DNA replication is inhibited by aphidicolin or L-mimosine this genome-wide demethylation is still observed. During the first 3 h of differentiation there is an increase in the number of hemimethylated CpG sites, which disappear rapidly during the course of genome-wide hypomethylation. Transfection of cells with an antisense morpholino oligonucleotide to 5-methylcytosine DNA glycosylase (G/T mismatch DNA glycosylase) decreases both the activity of the enzyme and genome-wide demethylation. It is concluded that the genome-wide loss of DNA methylation in differentiating mouse myoblasts occurs in part by formation of hemimethylated CpG sites, which can serve as the substrate for 5-methylcytosine-DNA glycosylase.

Identification of 5-methylcytosine in complex genomes.

Cytosine methylation is attracting new attention for regulatory roles in gene expression and there is an increasing interest in detecting, at a single-base resolution, any 5-methylcytosine in genes from complex genomes. Differential base modification by chemicals followed by PCR-based genomic sequencing procedures can provide the resolution, sensitivity, and specificity required for such a goal. The various methods available are not devoid of artifacts but if used carefully and in combination, very reliable information can be obtained. We compare the methods using bisulfite and conventional PCR with those using either hydrazine or potassium permanganate and ligation-mediated PCR and provide a step-by-step description of the corresponding procedures.

Quantification of 5-methylcytosine in DNA by the chloroacetaldehyde reaction.

The study of changes in genome-wide levels of DNA methylation has become a key focus for understanding the epigenetic regulation of gene expression. Many procedures exist to study DNA methylation, falling into two categories: gene-specific and genome-wide. Genome-wide methylation analysis is best performed by DNA hydrolysis followed by HPLC; however, it requires access to an HPLC machine, which is not always available. Alternative procedures, such as the radioactive labeling of CpG sites using SssI DNA methyltransferase, have been developed to address this problem, but it can only monitor CpG methylation changes, and CpNpG methylation is not detected. Here, we present a method for the analysis of DNA methylation in any sequence context by fluorescent labeling. We present control analyses using synthetic oligonucleotides of known methylation levels and a comparison of genomic DNA from two transgenic tobacco lines known to differ in their methylation levels. The results indicate that hygromycin-induced hypermethylation acts equally on all classes of methylatable cytosine, perhaps indicating a common mechanism.

DNA methylation analysis: a review of current methodologies.

The relationship between levels of DNA methylation and gene activity has been known for some time. Many of the early procedures developed gave only somewhat limited information about methylation patterns, for example, the total level of 5-methyl cytosine in the genome or the frequency of methylation of cytosines within certain restriction sites. However, in the last few years, there has been an explosion of interest in DNA methylation, and with it, many new and powerful techniques have been developed to facilitate its study. In this paper, the key techniques currently available are reviewed and the advantages, disadvantages, and potential artifacts of each are discussed.

Molecular analysis of animal and plant cells is facilitated by their attachment to collodion (cellulose nitrate) films.

A procedure for the efficient transfer of cell monolayers, cultured on glass coverslips, to microscopy slides has been developed. This technique involves the coating of the upper surface of an ethanol-fixed cultured cell layer with a film of collodion dissolved in n-amyl acetate. The dry collodion-coated cell layer can then be detached by rehydrating it for 1 h under water or phosphate-buffered saline and then carefully peeling it away from the coverslip using a pair of tweezers. Once a cell layer has been so mounted, it can be subjected to rough treatment such as proteolytic degradation (which greatly improves the signal-to-noise ratio in procedures like in situ hybridization [ISH] or primed in situ synthesis [PRINS]) without running the risk of cell detachment because of the partial or total degradation of the extracellular matrix. As an example of its application, we show a PRINS of telomeres from mouse fibroblasts. The high mechanical strength of collodion ensures that the structural integrity and morphology of the cell layer is maintained under experimental conditions where the collodion itself is insoluble. In addition to its use on cell layers, collodion can be used for the production of support films for (i) attaching suspension cell cultures, (ii) immobilizing cells normally cultured in suspension (such as tobacco BY2 cells or germinating tobacco pollen grains) or (iii) planting cryostat sections to microscopy slides. The value of this technique lies in its ease of use and the large number of different applications, in both the plant and animal fields of research, to which it may be applied.

Multiple domains are involved in the targeting of the mouse DNA methyltransferase to the DNA replication foci.

It has been shown that, during the S-phase of the cell cycle, the mouse DNA methyltransferase (DNA MTase) is targeted to sites of DNA replication by an amino acid sequence (aa 207-455) lying in the N-terminal domain of the enzyme [Leonhardt, H., Page, A. W., Weier, H. U. and Bestor, T. H. (1992) Cell , 71, 865-873]. In this paper it is shown, by using enhanced green fluorescent protein (EGFP) fusions, that other peptide sequences of DNA MTase are also involved in this targeting. The work focuses on a sequence, downstream of the reported targeting sequence (TS), which is homologous to the Polybromo-1 protein. This motif (designated as PBHD) is separated from the reported targeting sequence by a zinc-binding motif [Bestor , T. H. (1992) EMBO J , 11, 2611-2617]. Primed in situ extension using centromeric-specific primers was used to show that both the host DNA MTase and EGFP fusion proteins containing the targeting sequences were localized to centromeric, but not telomeric, regions during late S-phase and mitosis. Also found was that, in approximately 10% of the S-phase cells, the EGFP fusions did not co-localize with the centromeric regions. Mutants containing either, or both, of these targeting sequences could act as dominant negative mutants against the host DNA MTase. EGFP fusion proteins, containing the reported TS (aa 207-455), were targeted to centromeric regions throughout the mitotic stage which lead to the discovery of a similar behavior of the endogenous DNA MTase although the host MTase showed much less intense staining than in S-phase cells. The biological role of the centromeric localization of DNA MTase during mitosis is currently unknown.

Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation.

Changes in DNA methylation during tobacco pollen development have been studied by confocal fluorescence microscopy using a monoclonal anti-5-methylcytosine (anti-m5C) antibody and a polyclonal anti-histone H1 (anti-histone) antibody as an internal standard. The specificity of the anti-m5C antibody was demonstrated by a titration series against both single-stranded DNA and double-stranded DNA substrates in either the methylated or unmethylated forms. The antibody was found to show similar kinetics against both double- and single-stranded DNA, and the fluorescence was proportional to the amount of DNA used. No signal was observed with unmethylated substrates. The extent of methylation of the two pollen nuclei remained approximately constant after the mitotic division that gave rise to the vegetative and generative nuclei. However, during the subsequent development of the pollen, the staining of the generative nucleus decreased until it reached a normalized value of (1)/(5) of that of the vegetative nucleus. The use of a confocal microscope makes these data independent of possible focusing artefacts. The anti-histone antibody was used as a control to show that, while the antibody staining directed against 5-methylcytosine changed dramatically during pollen maturation, the histone signal did not. We observed the existence of structural dimorphism amongst tobacco pollen grains, the majority having three pollen apertures and the rest with four. However, the methylation changes observed occurred to the same extent in both subclasses.

Antibiotics induce genome-wide hypermethylation in cultured Nicotiana tabacum plants.

Plant genomic DNA methylation was analyzed by an improved SssI methyltransferase assay and by genomic sequencing with sodium bisulfite. Kanamycin, hygromycin, and cefotaxime (also called Claforan) are commonly used as selective agents for the production of transgenic plants. These antibiotics caused DNA hypermethylation in tobacco plants grown in vitro, which was both time- and dose-dependent. An exposure of the plantlets to 500 mg/liter cefotaxime for 1 month caused the de novo methylation of 3 x 10(7) CpG sites/haploid genome of 3.5 x 10(9) base pairs. It occurred in high, moderate, and low repetitive DNA and was not reversible upon the removal of the antibiotics. Reversion was only observed in progeny grown in the absence of drugs. Analysis of the promoter regions of two single-copy genes, an auxin-binding protein gene and the class I chitinase gene, showed the hypermethylation to be heterogeneous but biased toward CpGs. The hypermethylation of the class I chitinase and the auxin-binding protein promoters was not a consequence of a drug-induced gene amplification.

Non-symmetrical cytosine methylation in tobacco pollen DNA.

We have detected sequence-specific non-symmetrical cytosine methylation within a 140 bp region of the promoter for the tobacco auxin-binding protein gene T85 in pollen DNA. Direct sequencing of the population of bisulphite reaction products showed that, in this region. 10 out of a possible 49 cytosine residues were methylated at a high frequency in pollen whereas the corresponding region from somatic cells (leaf DNA) did not show a detectable level of methylation. The context of these sites was 1 x m5CpTpC, 1 x m5CpGpT, 1 x m5CpCpT, 2 x m5CpTpT, 2 x m5CpGpG, and 3 x m5CpApT of which only m5CpGpG and m5CpGpT fitted the consensus sequence for symmetrical methylation in plants.