Vikas Dighe

Hypermethylation of estrogen receptor promoter region in adult testis of rats exposed neonatally to bisphenol A

BACKGROUND Bisphenol A (BPA) is an estrogenic endocrine disruptor commonly used in manufacture of polycarbonate plastics and epoxy resins. Due to its ubiquitous presence in the environment, health concerns are increasing. Earlier studies from our group have shown that neonatal exposure of male rats to BPA affected spermatogenesis leading to impairment in fertility during adulthood. Further we also observed an altered gene expression of ERα and ERβ in adult testis upon BPA exposure. Based on these results, we hypothesized that apart from endocrine action, BPA might mediate perturbations in expression of ERs via epigenetic mechanism. OBJECTIVES The present study was undertaken to determine the effect of exposure of neonatal male rats to BPA on DNA methylation profile of estrogen receptor promoter region and on DNA methylation machinery. METHODS In order to test this hypothesis, neonatal male rats were subcutaneously injected with 2.4μg of BPA/day for the first five days of life, i.e., on postnatal days (PND) 1-5, while control group received vehicle (sesame oil). Animals were sacrificed during adulthood (PND-125) and testes were dissected out for analysis. Methylation pattern of promoter region of ERα and ERβ was analyzed in the testis by bisulfite sequencing and expression levels of DNA methyltransferases by quantitative RT-PCR and Western blotting respectively. RESULTS Bisulfite sequencing revealed significant hypermethylation of ERα promoter to varying extents from 40% to 60%, and ERβ promoter region with varying extent from 20% to 65%. Approximately 2-fold increase in Dnmt3a and Dnmt3b expression at transcript and protein level was also observed. CONCLUSION The experimental evidence demonstrated that the neonatal exposure of rats to BPA led to aberrant DNA methylation in testis, indicating methylation mediated epigenetic changes as one of the possible mechanisms of BPA induced adverse effects on spermatogenesis and fertility.

Heterozygous Embryonic Stem Cell Lines Derived from Nonhuman Primate Parthenotes

Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC‐derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line‐dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients.

Epigenetic Reprogramming by Somatic Cell Nuclear Transfer in Primates

We recently demonstrated that somatic cells from adult primates could be reprogrammed into a pluripotent state by somatic cell nuclear transfer. However, the low efficiency with donor cells from one monkey necessitated the need for large oocyte numbers. Here, we demonstrate nearly threefold higher blastocyst development and embryonic stem (ES) cell derivation rates with different nuclear donor cells. Two ES cell lines were isolated using adult female rhesus macaque skin fibroblasts as nuclear donors and oocytes retrieved from one female, following a single controlled ovarian stimulation. In addition to routine pluripotency tests involving in vitro and in vivo differentiation into various somatic cell types, primate ES cells derived from reprogrammed somatic cells were also capable of contributing to cells expressing markers of germ cells. Moreover, imprinted gene expression, methylation, telomere length, and X‐inactivation analyses were consistent with accurate and extensive epigenetic reprogramming of somatic cells by oocyte‐specific factors. STEM CELLS 2009;27:1255–1264

Rapid label-free visual assay for the detection and quantification of viral RNA using peptide nucleic acid (PNA) and gold nanoparticles (AuNPs).

A rapid label-free visual assay for the detection of viral RNA using peptide nucleic acid (PNA) probes and gold nanoparticles (AuNPs) is presented in this study. Diagnosis is a crucial step for the molecular surveillance of diseases, and a rapid visual test with high specificity could play a vital role in the management of viral diseases. In this assay, the specific agglomerative behavior of PNA with gold nanoparticles was manipulated by its complementation with viral RNA. The assay was able to detect 5-10 ng of viral RNA from various biological samples, such as allantoic fluids, cell culture fluids and vaccines, in 100 μl of test solution. The developed assay was more sensitive than a hemagglutination (HA) test, a routine platform test for the detection of Newcastle disease virus (NDV), and the developed assay was able to visually detect NDV with as little as 0.25 HA units of virus. In terms of the specificity, the test could discriminate single nucleotide differences in the target RNA and hence could provide visual viral genotyping/pathotyping. This observation was confirmed by pathotyping different known isolates of NDV. Further, the PNA-induced colorimetric changes in the presence of the target RNA at different RNA to PNA ratios yielded a standard curve with a linear coefficient of R(2)=0.990, which was comparable to the value of R(2)=0.995 from real-time PCR experiments with the same viral RNA. Therefore, the viral RNA in a given samples could be quantified using a simple visual spectrophotometer available in any clinical laboratory. This assay may find application in diagnostic assays for other RNA viruses, which are well known to undergo mutations, thus presenting challenges for their molecular surveillance, genotyping and quantification.

Effect of neonatal exposure on male rats to bisphenol A on the expression of DNA methylation machinery in the postimplantation embryo.

Bisphenol A (BPA) is used in the production of polycarbonate plastics and epoxy resins. Our previous studies have demonstrated that neonatal exposure of male rats to BPA causes decrease in sperm count and motility, increase in postimplantation loss (POL), ultimately leading to subfertility during adulthood. Epigenetic mechanisms such as DNA methylation play an important role in embryo development. DNA methyltransferases (Dnmts) are the key players involved in regulating DNA methylation marks. The objective of the present study was to determine the mechanism involved in resorption of embryo as a result of BPA exposure. The results of the present study demonstrate that neonatal exposure of male rats to BPA down regulates the gene expression of Dnmts and related transcription factors in resorbed embryos as compared with the viable embryo. Thereby, suggesting that BPA may have altered the sperm epigenome, which might have affected the embryo development and leading to an increase in the POL.

DNA methylation programming and reprogramming in primate embryonic stem cells

DNA methylation is an important epigenetic mechanism, affecting normal development and playing a key role in reprogramming epigenomes during stem cell derivation. Here we report on DNA methylation patterns in native monkey embryonic stem cells (ESCs), fibroblasts, and ESCs generated through somatic cell nuclear transfer (SCNT), identifying and comparing epigenome programming and reprogramming. We characterize hundreds of regions that are hyper- or hypomethylated in fibroblasts compared to native ESCs and show that these are conserved in human cells and tissues. Remarkably, the vast majority of these regions are reprogrammed in SCNT ESCs, leading to almost perfect correlation between the epigenomic profiles of the native and reprogrammed lines. At least 58% of these changes are correlated in cis to transcription changes, Polycomb Repressive Complex-2 occupancy, or binding by the CTCF insulator. We also show that while epigenomic reprogramming is extensive and globally accurate, the efficiency of adding and stripping DNA methylation during reprogramming is regionally variable. In several cases, this variability results in regions that remain methylated in a fibroblast-like pattern even after reprogramming.

Multiple antigenic peptide (MAP): a synthetic peptide dendrimer for diagnostic, antiviral and vaccine strategies for emerging and re-emerging viral diseases

The peptide dendrimer provides novel strategies for various biological applications. Assembling of peptide in macromolecular structure is expected to give rational models as drugs, their delivery and diagnostic reagents. Improved understanding of virus structure and their molecular interactions with ligands have paved the way for treatment and control of emerging and re-emerging viral diseases. This review presents a brief account of a synthetic peptide dendrimer used for diagnostic, therapeutic and prophylactic applications. The designs comprise of multiple antigenic peptides which are being used as alternate synthetic antigens for different viruses.

Efficacy and safety of a herbo-mineral ayurvedic formulation 'Afrodet Plus(®)' in male rats.

Background: Reverse pharmacology for drug development has been highly productive and cost-effective in recent past as it is based on the documented therapeutic effects of plants in ancient texts. Afrodet Plus® is formulated for the treatment of male infertility, which contains ancient herbo-minerals. Its efficacy and safety are validated through this animal study in reverse pharmacology mode. Objectives: This study was undertaken to evaluate efficacy and safety of an Ayurvedic formulation Afrodet Plus® in adult male rats. Materials and Methods: Twelve male rats (Holtzman) between 8 and 10 weeks of age were randomly selected and animals were assigned to a control and two treatment groups. Dosing was performed daily. Various parameters such as weekly body weight, hematology, serum testosterone levels, epididymal sperm count, and efficiency of Daily Sperm Production (DSP) were evaluated. Results: It was found that epididymal sperm count had significantly increased in both low-dose (+27.39%) and high-dose (+40.5%) groups as compared to control group. The DSP also showed an increase of 43.7% at high dose of 180 mg/kg body weight as compared to the control group. An increase in sperm motility and especially progressive motility was observed when evaluated by Computer Assisted Semen Analyzer. Histological evaluation of testicular tissue for spermatogenic index revealed that the index had increased in treatment group as compared to control group. Conclusion: This study revealed that oral administration of Afrodet Plus® resulted in significant increase in DSP in the testis along with increase in epididymal sperm count and progressive motility as compared to control group without producing any treatment-related adverse effects. These findings provide the documentary evidence that the use of Afrodet Plus® at 90 and 180 mg/kg body weight is effective and safe for the treatment of male infertility especially to improve sperm count and progressive motility.

HOXBES2: A Novel Epididymal HOXB2 Homeoprotein and Its Domain-Specific Association with Spermatozoa

Abstract The sperm from the testis acquires complete fertilizing ability and forward progressive motility following its transit through the epididymis. Acquisition of these characteristics results from the modification of the sperm proteome following interactions with epididymal secretions. In our attempts to identify epididymis-specific sperm plasma membrane proteins, a partial 2.83-kb clone was identified by immunoscreening a monkey epididymal cDNA library with an agglutinating monoclonal antibody raised against washed human spermatozoa. The sequence of the 2.83-kb clone exhibited homology to the region between 1 and 1097 bp of the homeobox gene, Hoxb2. This sequence was found to be species conserved, as revealed by RT-PCR analysis. To obtain a full-length clone of the sequence, 5′ RACE-PCR (rapid amplification of cDNA ends PCR) was carried out using rat epididymal RNA as the template. It resulted in a full-length 1.657-kb cDNA encoding a 32.9-kDa putative protein. The protein designated HOXBES2 exhibited homology to the conserved 61-amino acid homeodomain region of the HOXB2 homeoprotein. However, characteristic differences were noted in its amino and carboxyl termini compared with HOXB2. A putative 30-kDa protein was detected in the tissue extracts from adult rat epididymis and caudal spermatozoa, and a 37-kDa protein was detected in the rat embryo when probed with a polyclonal antibody against HOXB2 protein. Multiple tissue Western blot and immunohistochemical analysis further indicated its expression in the cytoplasm of the principal and basal epithelial cells, with maximal expression in the distal epididymal segments. Northern blot analysis detected a single ~2.5-kb transcript from the adult epididymis. Indirect immunofluorescence localized the protein to the acrosome, midpiece, and equatorial segments of rat caudal and ejaculated human and monkey spermatozoa, respectively. In conclusion, we have identified and characterized a novel epididymal homeoprotein different from HOXB2 protein and hereafter referred to as HOXBES2, (HOXB2 homeodomain containing epididymis-specific sperm protein) with a probable role in fertilization.

Fipronil-induced genotoxicity and DNA damage in vivo Protective effect of vitamin E

Fipronil, an insecticide of the phenylpyrazole class has been classified as a carcinogen by United States Environmental Protection Agency, yet very limited information is available about its genotoxic effects. Adult male and female animals were gavaged with various doses of fipronil (2.5, 12.5, and 25 mg/kg body weight (bw)) to evaluate micronucleus test (mice), chromosome aberration (CA), and comet assay (rats), respectively. Cyclophosphamide (40 mg/kg bw; intraperitoneal) was used as positive control. Another group of animals were pretreated with vitamin E orally (400 mg/kg bw) for 5 days prior to administration of fipronil (12.5 mg/kg). Fipronil exposure in both male and female mice caused significant increase in the frequency of micronuclei (MN) in polychromatic erythrocytes. Similarly, structural CAs in bone marrow cells and DNA damage in the lymphocytes was found to be significantly higher in the male and female rats exposed to fipronil as compared to their respective controls. The average degree of protection (male and female animals combined together) shown by pretreatment of vitamin E against fipronil-induced genotoxicity was 63.28%: CAs; 47.91%: MN formation; and 74.70%: DNA damage. Findings of this study demonstrate genotoxic nature of fipronil regardless of gender effect and documents protective role of vitamin E.