Biomedical subjects

Osman V Patel

Publications and source records attributed to Osman V Patel.

8 recordsLinked to original sources

PubMed2006

Novel algorithm for transcriptome analysis.

A growing body of evidence implicates the oocyte as a key regulator of ovarian folliculogenesis and early embryonic development. We have screened bovine cDNA microarrays (containing expressed sequence tags representing >15,000 unique genes) with Cy3- and Cy5-labeled cDNA derived from bovine oocyte samples collected at two different stages of meiotic maturation (germinal vesicle vs. metaphase II; n = 3 samples per group). Here, we present a novel data analysis approach that uses all available information from above experiments to obtain and index the transcriptome of bovine oocytes and changes in transcriptome composition in response to meiotic maturation. Signal intensities (Fg) for all housekeeping genes were omitted prior to analysis. A local threshold for gene expression was computed as background intensity (Bg) plus 2 times the standard deviation of background and foreground signals. Within each array, data were normalized by the LOWESS procedure. Subsequently, a two-stage mixed model was fitted to remove systematic variations. In the first stage, the response was the LOWESS normalized Fg with treatment as a fixed effect. In stage 2, the residuals from stage 1 were analyzed in a gene-specific model that included treatment group and spots nested within patch and array. A test for the difference between least squares means for the treatment effect was performed. A false discovery rate (FDR) adjustment on the p values for the difference was carried out. This novel algorithm was compared with approaches that ignore the FDR and the threshold described herein and stark differences obtained.

Algorithms↗

PubMed2006

Quantitative analysis of messenger RNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, beta-actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitro.

Real-time reverse transcription PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, measurement of gene expression generally requires selection of a valid reference (housekeeping gene) for data normalization to compensate for inherent variations. Given the dynamic nature of early embryonic development, application of this technology to studies of oocyte and early embryonic development is further complicated due to limited amounts of starting material and a paucity of information on constitutively expressed genes for data normalization. We have validated quantitative procedures for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA abundance during bovine meiotic maturation and early embryogenesis and utilized this technology to determine temporal changes in mRNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, and histone H2A. Quantification of amounts of specific exogenous RNAs added to samples revealed acceptable rates of RNA recovery and efficiency of reverse transcription with minimal variation. Progression of bovine oocytes to metaphase II resulted in reduced abundance of polyadenylated, but not total transcripts for majority of above genes; however phosphoglycerokinase exhibited a significant decline in both RNA populations. Abundance of mRNAs for above genes in early embryos generally remained low until the blastocyst stage, but abundance of ribosomal protein L-15 mRNA was increased at the morula stage and histone H2A mRNA showed dynamic changes prior to embryonic genome activation. Results demonstrate a valid approach for quantitative analysis of mRNA abundance in oocytes and embryos, but do not support constitutive expression of above genes during early embryonic development.

Actins↗

PubMed2005

Validation and application of a high fidelity mRNA linear amplification procedure for profiling gene expression.

The need for microgram quantities of RNA for microarray experiments has hindered application of this novel technology in cell types/tissue samples with limited abundance of RNA. In this study, potential application of T7-based linear RNA amplification was investigated for use in gene expression profiling experiments where starting material is limited. Yield and integrity of amplified antisense RNA (aaRNA), microarray hybridization intensities, and fidelity of differential gene expression detected were determined for arrays generated for unamplified versus amplified RNA from the same homogenous starting pools. Total RNA was extracted from bovine spleen and fetal ovary, serially diluted to concentrations ranging from 2 microg to 500 pg and amplified. Quality and quantity of total input RNA and aaRNA were assessed by spectrophotometry, gel electrophoresis and bioanalyzer. In experiment 1, we determined the optimal amounts of aaRNA generated from 20, 40, 200 ng and 2 microg input total RNA for use in cDNA synthesis, labeling and array hybridization that would yield robust and consistent hybridization signals on a bovine oocyte cDNA microarray. In experiment 2, comparison of microarray hybridization intensities and fidelity of differential gene expression between aaRNA generated from 2, 20 and 40 ng input total RNA versus unamplified RNA (uRNA) were conducted. The hybridization intensities for each of the 7000 spots per slide for microarrays conducted using aaRNA versus uRNA were highly correlated (2 ng = 0.84, 20 ng = 0.88, 40 ng = 0.90; P < 0.01). The false positive rate was low and similar (4.0% versus 4.4%) for arrays done with uRNA and aaRNA. Ninety-seven ESTs were detected as differentially expressed in the fetal ovary versus spleen at > 1.5- or < 0.5-fold using uRNA (P < 0.05). However, the number of genes detected in arrays using aaRNA was approximately 1.5-2.5 times greater than with uRNA. Approximately, 65-70% of differentially expressed genes were common between uRNA and aaRNA arrays. Relative fold-expression (Cy3/Cy5 ratios) for 25 overlapping abundant genes was comparable for uRNA versus aaRNA arrays with 2 and 20 ng total RNA as input. Results demonstrate that T7-based linear amplification of small amounts of input RNA and use of aaRNA in microarray experiments retains fidelity of detection of differential gene expression that is relatively comparable to experiments done with uRNA and provides a potentially viable approach to facilitate gene expression profiling using limited amounts of starting material.

Animals↗

PubMed2004

Quantitative analysis throughout pregnancy of placentomal and interplacentomal expression of pregnancy-associated glycoproteins-1 and -9 in the cow.

The multigenic pregnancy-associated glycoproteins (PAG) exhibit spatially and temporally distinct pattern across gestation in the bovine. The majority of the bovine bPAG are localized to the binucleate cells (BNC) while some are expressed throughout the trophectoderm. Bovine (b)PAG-1 and -9 are both localized to the BNC but are differentially transcribed. In addition, the anatomical location of BNC does influence protein expression in the ungulates. Therefore, the objective of the present study was to compare and contrast bPAG-1 and -9 transcriptions in the placentomal (cotyledonary, caruncular) and interplacentomal (intercotyledonary, intercaruncular) tissues throughout pregnancy in the bovine using real-time reverse transcription PCR (RT-PCR) and by in-situ hybridization. The levels of bPAG-9 transcription in the fetal membrane at peri-implantation were significantly (P<0.01) higher than bPAG-1. The expression of bPAG-9 in the placentomal and interplacentomal tissues were significantly (P<0.01) higher than bPAG-1 during the first trimester of gestation. The transcription of bPAG-1 in the placentomal and interplacentomal tissues were significantly (P<0.01) higher than bPAG-9 from mid-gestation to peripartum. The expression of bPAG-1 and -9 throughout gestation were significantly (P<0.01) affected by the anatomical location of BNC. In situ analysis paralleled the expression patterns of bPAG-1 and -9 across gestation. These findings indicate that bPAG-9 expression in the placentomal and interplacentomal tissues predominates in the first trimester of gestation while bPAG-1 transcription was primarily higher in the last two trimesters of gestation. The cellular location had significant effect on bPAG-1 and -9 transcription.

Animals↗

PubMed2003

Expression of trophoblast cell-specific pregnancy-related genes in somatic cell-cloned bovine pregnancies.

We compared the expression of bovine prolactin-related protein-1 (bPRP-1), placental lactogen (bPL), and pregnancy-associated glycoproteins-1 (bPAG-1) and -9 (bPAG-9) genes in artificially inseminated (AI) and nuclear transferred (NT) cows during the first trimester of gestation using real-time reverse transcription-polymerase chain reaction and in situ hybridization. Placentomal (cotyledonary, caruncular) and interplacentomal (intercotyledonary, intercaruncular) tissues of AI and NT cows carrying either motile (M) or immotile (IM) fetuses were examined. Transcripts for bPL and bPAG-9 were lower (P < 0.01) in the fetal membranes of NT (n = 4) cows at Day 30 of gestation, compared with AI (n = 4) cows. There was no difference in the mean (+/- SEM) levels of expressions of bPRP-1, bPL, and PAG-1 in the placentomal and interplacentomal tissues of AI (n = 5) and NT (M, n = 4) cows at Day 60 of gestation. The mRNAs for bPRP-1, bPL, bPAG-1, and bPAG-9 genes were higher (P < 0.01) in the caruncular tissue of AI cows, compared with NT (IM, n = 4) cows at Day 60 of gestation. Expression of bPRP-1, bPL, bPAG-1, and bPAG-9 in the placentomal and interplacentomal tissues of the NT (n = 3) group varied considerably more, compared with the AI (n = 4) group at Day 100 of gestation. These findings suggest defective binucleate cell-specific gene transcriptional commands in NT cows.

Animals↗

PubMed2003

Characterization of gene expression profiles in early bovine pregnancy using a custom cDNA microarray.

Gene expression analysis comparing nonpregnant with pregnant bovine uteri, including placenta, was performed with a custom cDNA microarray containing 1,933 independent genes. These genes were classified into six categories according to biological function, as follows: cell and tissue structural dynamics (108 genes), intercellular communication (221), intracellular metabolism (265), cell cycle and apoptosis (26), regulation of gene expression (113), expressed sequence tag (EST) and function unknown (617), and uncomplemented genes (583 clones). This array possessed bovine placental/endometrial specificity, as it included many pregnancy-specific molecules, such as pregnancy-associated glycoprotein-1 (PAGs), placental lactogen (PLs), and prolactin-related protein-1 (PRPs). A total of 77 genes were induced and 12 repressed in the placenta/endometrium. Our results point to a fundamental role for bovine placental-specific genes such as PAGs, PLs, and PRPs, in implantation and placentogenesis, and document that cDNA microarray analysis from bovine placenta/endometrium is possible and is a specific tool for monitoring genome-wide gene expression during the establishment and maintenance of pregnancy.

Animals↗

PubMed2002

Implantation and placental development in somatic cell clone recipient cows.

Successful somatic cloned animal production has been reported in various domesticated species, including cattle; however, it is associated with a high rate of pregnancy failure. The low cloning yield could possibly arise from either an abnormal and/or poorly developed placenta. In comparison to control cows, fewer placentomes were found in somatic cell nuclear recipient (NT) cows at day 60 of gestation, suggesting a retardation of fetal/placental growth in these animals. NT cows not only had fewer numbers of chorionic villi but also had poorly developed caruncles. Macroscopic examination revealed atypical development of the placentome in terms of shape and size. Histological disruption of chorionic villi and caruncular septum was found in NT cows. Of particular interest was that the expression of genes, as well as proteins in the placentome, was disparate between NT and artificially inseminated cows, especially placental lactogen (PL) and pregnancy-associated glycoprotein (PAG). In contrast, prolactin-related protein-1 (PRP-1) signals were comparable across cows, including NT cows carrying immotile fetuses. The expression of extracellular matrix degrading molecule, heparanase (HPA), in NT cows was divergent from that of control cows. Microarray data suggest that gene expression was disorientated in early stages of implantation in NT cows, but this was eliminated with progression of gestation. These findings strongly support a delay in trophoblast development during early stages of placentation in NT cows, and suggest that placental specific proteins, including PLs, PAGs, and HPA, are key indicators for the aberration of gestation and placental function in cows.

Animals↗

PubMed2002

Plasma steroid profiles following follicle-stimulating hormone or equine chorionic gonadotropin injection in cows chronically treated with gonadotropin-releasing hormone agonist.

Plasma steroid profiles following follicle-stimulating hormone (FSH) or equine chorionic gonadotropin (eCG) injection were studied in chronically gonadotropin releasing hormone agonist (GnRH-A)-treated cows. Follicular development and irINH secretion were stimulated by FSH or eCG injection. The plasma concentrations of estradiol-17 beta (E(2)) and testosterone (T) were markedly increased following eCG injection. However, significant increases of the plasma E(2) and T concentrations were not detected in FSH-treated cows. Ovulation of developed follicles were depended on the hCG injection in both groups. These results show: 1) Follicular response to an exogenous gonadotropin is still remained, 2) Ovulation of developed follicles is induced by hCG injection and 3) FSH and eCG cause disparate plasma steroid profiles, under the influence of repeated GnRH-A treatment.

Animals↗