2.2.1 Experimental Animals
Experiments involving the use of Rhesus macaques and nonhuman primate tissues were approved by the Institutional Animal care and Use Committee of Magee-Womens Research Institute and the University of Pittsburgh, and they were performed according to the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. Seven adult Rhesus
macaques (Age: 72 to 108 months) and four prepubertal (Age: 35 to 40 months) housed in the nonhuman Primate vivarium of Magee-Womens Research Institute under a 12-hour cycle of light and darkness were used for this experiment. Monkeys were randomly divided into four groups:
one bolus group and three groups got BrdU in drinking water for 3 weeks followed by 0, 5 or 9 weeks of wash out (Figure 5).
2.2.2 BrdU bolus
One adult and one prepubertal Rhesus macaques were mildly sedated for venipuncture and intravenous BrdU administration. Then, 33 mg/kg of BrdU (Sigma-Aldrich, B5002, St. Louis, MO) was diluted in sterile normal saline and injected intravenously as previously described (Simorangkir et al., 2009a). Testis tissues were collected 3 hours after the injection.
2.2.3 BrdU in drinking water
All animals were conditioned to Kool-Aid® flavored drinking water, and a favorite flavor for each individual monkey was identified before commencing the experiment. BrdU (Sigma-Aldrich, B5002, St. Louis, MO) was added (1mg/ml) to the Kool-Aid flavored drinking water and offered to each monkey ad libitum (Walls et al., 2012). Consumption of Kool-Aid® flavored drinking water with BrdU was monitored and recorded each day to obtain the precise quantity of BrdU consumed by each animal. Testis was collected from each animal at the end of
experimental period.
2.2.4 Testicular tissue processing for anti-BrdU staining
Collected testis tissues were cut into small pieces and fixed in Bouin’s fluid in a 24-hour (hr) fixation. Fixed testis tissues were washed in 70% ethanol (3×1 hr), embed in paraffin wax block and sectioned (5μm per section). To avoid counting a cell twice, every fifth section was used for quantitative analysis.
2.2.5 Colorimetric BrdU staining in testis tissue sections
Sections were deparaffinized in xylene (2×15 minutes (mins)), rehydrated in graded ethanol series (2×100% for 10 mins, 1×95% for 5 mins, 1×80% for 5 mins, 1×70% for 5 mins, 1×50%
for 5 mins, 1×25% for 5 mins) and washed in 1X Gibco® Dulbecco’s phosphate-buffered saline (DPBS; Life Technologies, 14200-166, Grand Island, NY) for 3 mins. Sections were then incubated in peroxidase block (3% hydrogen peroxide in methanol) for 15 mins, washed in 1X DPBS (3×5 mins) and stained with mouse anti-BrdU, formalin grade (1:30, Roche Diagnostics, 11170376001, Indianapolis, IN), at 4°C overnight. The next day, sections were washed in 1X DPBS (3×5 mins) and then incubated with horseradish peroxidase-conjugated secondary antibody (1:200, Santa Cruz, SC-2055, Dallas, TX) for 45mins at room temperature. After washing in 1X DPBS (3×5 mins), metal enhanced 3, 3ι-diaminobenzidine (DAB) substrate solution (ThermoFisher, 34065, Waltham, MA) was then added to detect and develop positive signal for 5 mins. Sections were then washed in distilled water (dH2O) for 5 mins and stained with sufficient drops of periodic acid (Cancer Diagnostics, SS1011, Durham, NC) for 7 mins.
Sections were washed in tap H2O for 3 mins, dipped in dH2O and then stained with sufficient drops of Schiff’s reagent (Cancer Diagnostics, SS1011, Durham, NC) for 10 mins. Sections were
washed in tap H2O for 10 mins, followed by washing in dH2O for 5 mins and dipping in deionized, distilled water (ddH2O). Next, sections were counterstained with Gill’s number 3 hematoxylin (Sigma-Aldrich, GHS332, St Louis, MO) for 30 secs and washed in tap H2O for 10 mins, followed by dipping into acid rinse (2% glacial acetic acid in water) 10 times and dipping into H2O 10 times. Sections were briefly dipped in bluing solution (0.1% sodium bicarbonate) for 1 min and washed in tap H2O for 1 min. Tissue sections were then dehydrated in graded ethanol series (1×70% for 3 min, 1×95% for 3 min, 2×100% for 5 mins each, 2× xylene for 5 min each) and mounted with permount® (ThermoFisher, SP15, Grand Island, NY).
2.2.6 Colorimetric Ki67, cKIT or caspase 3 staining in testis tissue sections
Testis tissues used for these experiments were fixed in 4% paraformaldehyde (PFA) solution (at 4°C) for 24 hours. PFA-fixed testis tissues were then washed in DPBS (3×1 hour). Tissues were then embedded in paraffin wax block and sectioned (5μm). To avoid counting a cell twice, every fifth section was acquired and analyzed. Tissue sections were deparaffinized in xylene (2×15 mins). Deparaffinized sections were rehydrated in graded ethanol series (2×100% for 10 mins, 1×95% for 5 mins, 1×80% for 5 mins, 1×70% for 5 mins, 1×50% for 5 mins, 1×25% for 5 mins) and washed in 1× DPBS (1×3 mins). Antigen retrieval was performed by incubating slides in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6) for 30 mins at 97.5°C.
Slides in retrieval buffer were left on the bench to cool to room temperature. This was followed by washing twice in DBPS-T (0.1% Tween-20 in 1× DPBS) for 2 mins each. Goat blocking buffer (1X DPBS containing 3% bovine serum albumin, 0.1% Triton X-100 and 5% normal goat serum) was then used to block non-specific antigenic sites in tissue sections for 30 mins at room temperature. Mouse human Ki67 (BD Biosciences, 550609, San Jose, CA) or rabbit
anti-human cKIT (1:100, Agilent, A4502, Santa Clara, CA) or rabbit anti-anti-human caspase 3 (1:100, Abcam, AB13847, Cambridge, MA) primary antibodies were diluted in goat blocking buffer, and sufficient drops were added to tissue sections for 90 mins at room temperature. Sections were then washed in DPBS-T (3×5 mins) and incubated in peroxidase block for 15 mins. This was followed by another wash in DPBS-T (3×5 min). Sections were then washed in 1X DPBS (3×5 mins) and then incubated with goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (1:200, Santa Cruz, SC-2055, Dallas, TX) for 45 min at room temperature.
(Subsequent periodic acid, Schiff’s reagent and hematoxylin staining, as well as washing and mounting steps, were exactly as described in Colorimetric BrdU staining in testis tissue section, above).
2.2.7 Quantification of labeled germ cells and statistical analysis
Quantification of germ cells with or without BrdU, Ki67, cKIT and activated caspase 3 expressions involved counting events in round seminiferous tubule cross-sections in multiple (except otherwise stated, usually 7-8) tissue sections from each biological replicate. Number of biological replicates used for the experiments is indicated in each result section. Quantitative observations from biological replicates are presented as mean ± standard error of the mean (SEM). Two quantitative variables were compared using independent-sample student’s T-test analysis, while quantitative variables greater than 2 were compared with one-way analysis of variance (ANOVA) method. If significantly different F-value was obtained (*-P<0.05; **-P<0.01), multiple paired-wise comparison was then performed, by simultaneously using Scheffé multiple comparison test, Tukey’s honest significant difference (HSD) test and Bonferroni and Holm’s multiple comparison test, to identify variables with relative significant difference. All
statistical analyses were performed using freely available online (as at 03/30/2018) statistical tool at http://astatsa.com/OneWay_Anova_with_TukeyHSD/.