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.
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.
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.
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).
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).
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.
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).
Food consumption was measured weekly during the study period. The start date of each group was coordinated to accommodate a single necropsy and tissue collection procedure. The left and right testis and epididymis were collected, weighed, and processed for molecular and microscopic end points.
Animals, Treatments, and Sample Isolation
All procedures and experimental protocols were reviewed and approved by the Animal Care and Use Committee at Groton Laboratories (Pfizer, Inc., New York, NY), and animals were housed in facilities accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. Groups of 10-wk-old male Crl:CD (SD)BR VAF/Plus rats (—175 g) were housed singly and given THP daily at 0 ppm (five animals per group) or 8000 ppm (five animals per group) in feed for 18, 30, or 42 consecutive days (T1633; Sigma, St. Louis, MO).
At high concentrations, THP is a documented male reprotoxic agent thought to induce infertility by incapacitating the nurturing Sertoli cells, thus resulting in the premature release of late differentiating spermatogenic cells, round spermatids. This leads to the depletion of spermatids and mature spermatozoa from the adluminal compartment of the seminiferous epithelium, ultimately causing testicular atrophy. The effect of THP on the epididymal epithelium is poorly studied, although we have recently shown that an increased accumulation of ubiquitin in the secretory sites along epididymal epithelia of THP-exposed rats coincides with exposure.