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Differential Expression of Genes: RESULTS(1)

RESULTS(1)

Histology and Semen Quality

No changes in the histology of testicular and epididymal tissues were observed in control rats at any time point (Fig. 1, A-C) or in the THP-exposed rats on Day 18 (data not shown; Supplemental Table is available online at www.biolreprod. org). Histological changes of seminiferous epithelia were noted in three of six exposed animals on Day 30 and in five of six rats on Day 42 (Supplemental Table 1). Sloughing of spermatids in the form of multinucleated bodies (Fig. 1, D-I) was the most frequent testicular anomaly and was consistent with the proposed mechanism of the THP action on Sertoli cells. This translated into the depletion of spermatogonia and spermatids from seminiferous epithelium and a reduction in the number of fully differentiated spermatozoa in the testis and epididymis.

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Differential Expression of Genes: MATERIALS AND METHODS(11)

Real-Time Quantitative PCR

Two-step RT-PCR, multiplex PCR, TaqMan probe-based chemistry, and the Comparative CT Method were used. TaqMan probes and primers (Table 2) were designed with Primer Express (Applied Biosystems). In all cases, the target amplicon spans an intron. A RealMasterMix Probe (Rox) was used as the real-time PCR reagent. On the basis of semiquantitative RT-PCR data from the present study and from a separate pilot study on the reproductive toxicity of DNB (unpublished results), two marker genes were selected: Ube2d3 and Psmbl.

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Differential Expression of Genes: MATERIALS AND METHODS(10)

METHODS(10)

The PCR products were resolved by electrophoresis on a 2% agarose gel stained poststain with ethidium bromide. Gels were photographed on top of an ultraviolet illuminator. The PCR products were confirmed as target sequences by sequencing with the Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, Foster City, CA) by means of Applied Biosystems Prism BigDye Terminator cycle sequencing chemistry at the DNA core facility of the University of Missouri at Columbia.

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Differential Expression of Genes: MATERIALS AND METHODS(9)

PCR reactions were performed with a Promega PCR kit, an Eppendorf PCR kit (Eppendorf, Westbury, NY), or a HotMaster Hot Start PCR kit from Eppendorf on an Eppendorf Mastercycler PCR machine. Gene-specific primers were designed to flank intron regions in order to eliminate false-positive PCR fragments generated from genomic DNA. Table 1 shows the primer sequences and the GenBank accession numbers of the genes (mRNA sequences). Actb (p-actin) was the endogenous control (reference gene) for the normalization of the quantification of the target mRNA to compensate for differences in the amount of total RNA added to each reaction.

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Differential Expression of Genes: MATERIALS AND METHODS(8)

METHODS(8)

Semiquantitative Relative RT-PCR and Multiplex and Singleplex PCR

A two-step RT-PCR was used. An oligo-dT primer (15n labeled; Promega, Madison, WI) was used in the first step of cDNA synthesis. A minimum mixture of total RNA (100 ng) and RNase-free distilled H2O was preheated at 65°C for 5 min and then chilled on ice rapidly for 10 min. The Qiagen Sensiscript RT-PCR kit was used for the RT-PCR reaction, and 20 il of RT mix was incubated at 37°C for 90 min. The cDNA was stored at —20°C. For singleplex PCR, the samples, along with a calibrator sample (liver), were processed with target and reference gene primers in the same PCR run.

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Differential Expression of Genes: MATERIALS AND METHODS(7)

Tissue sections were mounted on microscopy slides, deembedded, and incubated sequentially with the first and second antibodies listed above. Slides were examined under epifluorescence illumination as described above. For conventional histological analysis, deparaffinized tissue sections were stained with hematoxylin-eosin and observed under transmitted illumination.

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Differential Expression of Genes: MATERIALS AND METHODS(6)

METHODS(6)

Slides were photographed with a CoolSnap HQ CCD camera (Roper Scientific, Tucson, AZ) operated by MetaMorph software with appropriate epifluor-escence filters. Images were edited by Adobe Photoshop 5.5 (Adobe Systems, Inc., San Jose, CA).

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Differential Expression of Genes: MATERIALS AND METHODS(5)

For this purpose, the diagrams of the visible light scatter were generated by combining the forward and side scatter of the cells passing through the flow cytometer (see Fig. 3). The relative ubiquitin median values of low-fluorescent cells (median M2; x-axis) and high-fluorescent cells (median M3; y-axis) were arranged in scatter plots with regression lines color coded according to treatment (see Fig. 4C). Numeric data (ubiqitin medians and percent cells within each of the three markers) were entered into Microsoft Excel tables and analyzed by statistical analysis tools (e.g., MS Excel and the SAS 8.2 statistical package).

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Differential Expression of Genes: MATERIALS AND METHODS(4)

METHODS(4)

Flow cytometry was performed by a procedure developed previously for the measurement of sperm ubiquitin in human semen samples. Samples were analyzed with FACS Scan Analyzers (Becton Dickinson). Relative levels of ubiquitin-induced fluorescence (ubiquitin median) in 10 000 individual cells per sample were recorded.

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Differential Expression of Genes: MATERIALS AND METHODS(3)

Care was taken to eliminate pieces of tissue and to prevent contamination with epididymal epithelial cells, blood cells, and stromal tissue. Purity of sperm preparation was checked under a light microscope. Spermatozoa were fixed in 2% formaldehyde in PBS, washed in PBS, blocked by a 40-min incubation with 5% normal goat serum (Sigma), and incubated overnight with mouse monoclonal antibody KM691 (diluted 1:100; Kamiya Biomedical Company, Seattle, WA) and then incubated for 1 h with goat antimouse immunoglobulin G (IgG)-fluorescein isothiocyanate (FITC).

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