Orange-red, outbred line of Japanese medaka (Oryzias latipes) was originated from the Duke University, Molecular Aquatic Toxicology Laboratory. In 2008, fish were transferred and maintained as a colony in the City University of Hong Kong. Fertilized eggs were collected daily and reared until hatch as described in . Fish that hatched in the same month were grouped as one cohort. Upon sexual maturation (approx. 3 month-old), males and females from the same cohort were randomly paired and transferred to aquarium tanks (39.5 L × 23.5 W × 27.5H cm) at a density of 15 pairs per tank. The fish were kept in the same set of tanks throughout their whole life.
All fish were kept under static condition of 26 ± 1°C, 7.2 ± 0.2 mg O2 L-1 and 14:10 hrs light-dark cycle. Half of the tank water is replaced with charcoal-filtered tap water every day. The fish were feed twice daily with Otohime β1 (Nisshin Co, Japan) and supplemented freshly hatched brine shrimp (Artemia nauplia) (Ocean Star International Lucky Brand, Utah, USA) 3 days per week. Fish health was closely monitored. Unhealthy fish, usually characterized by lack of appetite, inactivity, loss of golden-red color, hemorrhages, and/or external outgrowth, were promptly isolated into individual glass containers (diameter: 14.5 cm; height: 5 cm) containing 500 ml charcoal-filtered tap water. Every quarantined fish was reared under identical condition as described above and would not return to originated tank unless symptoms disappeared (‘recovered’) for at least 2 weeks. Records of stocking density and daily mortality of males and females in individual tank (hence individual cohort) were curated in SQLite 3 relational database management system (http://www.sqlite.org). These records were extracted for generating survival profiles and statistics.
Healthy fish at five different ages were sampled: 4- (‘young’), 8- (‘mature’), 12- (‘senior’), 15- (‘old’) and 22- (‘very old’) months. At each time point, 90 fishes (45 males and 45 females) were dissected. Fish were anesthetized in ice-cold aquarium water for 30 s, removed and measured for body length and weight. The fish was kept sedated by gently wrapping with paper towel fully soaked with ice-cold aquarium water. A cut was made to tail at 1–2 mm rostral to the caudal fin. Blood was drawn from the cut using P10 micropipette (Eppendorf, Hamburg, Germany) attached with heparinized pipette tips and instantly diluted with 8 μL double-distilled water to prevent clotting. The fish was then immediately decapitated. Gill and liver were isolated on ice bed and divided into two equal portions for parallel telomerase activity and telomeric length measurements. Blood and tissue samples from three fish of the same sex were pooled as one replicate (i.e. final sample number = 15 per sex per time-point). Pooled samples were snap-frozen in liquid nitrogen and stored at -80°C until further processing. Animal handling procedures as mentioned above were accepted by the Animal Ethics Committee, City University of Hong Kong.
Telomere length measurement by Southern blot analysis
Genomic DNA was extracted from the livers and gills of adult male and female medaka using the DNeasy® Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For each assay, 3 μg genomic DNA was digested to completion with RsaI and HinfI (New England Biolabs, Massachusetts, USA) at 37°C overnight. The digested DNA was resolved by electrophoresis on a 1% agarose gel, run in parallel with a λ HindIII/EcoRI molecular marker (Fermentas, Burlington, Canada) and subsequently transferred to Hybond-XL membrane (GE Healthcare, Little Chalfont, United Kingdom) for overnight through capillary transfer. The membrane was saturated with ExpressHyb™ Hybridization Solution (Clontech, California, USA) at 42°C for 30 mins, and hybridized with 100 pmol of DIG-labeled oligonucleotide probes (TTAGGG)5 (Invitrogen, California, USA) in ExpressHyb™ Hybridization Solution (Clontech Laboratories, California, USA). The DIG-labeling was achieved by the use of DIG Oligonucleotide Tailing Kit, 2nd generation (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions. After hybridization, the membrane was washed two times in 2X SSC buffer with 0.1% SDS at 25°C (5 mins each) and twice for 15 mins in 0.1X SSC with 0.1% SDS at 42°C. The washed membrane was blocked in 3% solution of non-fat milk powder (in phosphate buffered saline, PBS; pH 7.4) for 30 mins at room temperature, then incubated with 1:10000 anti-DIG, AP-conjugated antibodies (Roche). The membrane was then washed three times in PBS at RT and incubated with CDP-star (Roche) in darkness for 5 mins. Chemiluminescence was imaged by luminiscent image analyzer (Fujifilm LAS 4000, Tokyo, Japan) and saved lossless as TIFF. Telomere length was quantified in ImageJ as terminal restriction fragment (TRF) length following the procedure of .
Telomerase activity assays by the real-time quantitative telomeric repeat amplification protocol (RTQ-TRAP)
Frozen tissues samples were thawed on ice bed and lysed in ice-cold CHAPS-containing lysis buffer (10 mM Tris–HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA pH 8.0, 0.5% CHAPS, 10% glycerol, 0.1 mM PMSF, 5 mM β-mercaptoethanol) (Sigma-Aldrich, Missouri, USA). The lysates were centrifuged at 16000 × g, 4°C for 30 mins. The supernatants were carefully transferred to new sterilized microfuge tubes. Protein concentration was determined using the Bradford protein assay (Bio-Rad, California, USA), with reference to standard curve constructed from serial dilutions of protein standard (bovine serum albumin, BSA; Sigma-Aldrich).
Telomerase activities in liver and gill samples of medaka were assessed by the RTQ-TRAP assay following the optimized protocol by . Briefly, 40 ng of protein extract was added to a 25 μL reaction mixture of TRAP buffer [20 mM Tris-HCl (pH 8.3), 63 mM KCl, 3.5 mM MgCl2, 1 mM EGTA (pH 8.0), 0.1 mg/mL BSA, 0.005% Tween 20], 100 μM dNTPs, 1:25000 SYBR Green I dye, 10 nM ROX reference dye, 1.25 U HotStar Taq polymerase (Qiagen), 0.1 μg telomerase substrate primer (a.k.a. ‘TS’; 5′-AATCCGTCGAGCAGAGTT-3′; HPLC grade; Invitrogen), and 0.065 μg anchored return primer (a.k.a. ‘ACX’; 5′-GCGCGG(CTTACC)3CTAACC-3′; HPLC grade; Invitrogen). The reaction mixture was first incubated at 25°C for 30 mins to allow the telomerase in the protein extract to elongate the TS primer by adding TTAGGG-repeats. Next, the TRAP reaction was halted and the Taq polymerase was activated by heating at 95°C for 15 min. The activation was followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 60 s. Melting curve analysis was automatically carried out after completion of the 40th cycle to verify the amplification specificity. Thermal cycling was conducted using the ABI 7500 Fast Real Time PCR System (Applied Biosystems, California, USA). Samples were run in triplicate. The Cq values were determined from semi-log amplification plots (log increase in fluorescence signal against cycle number). The relative telomerase activity was calculated by the 2-ΔCq method .
Sex hormone analysis
Blood samples were diluted with 300 μL double-distilled water and mixed with 2 mL of diethyl ether (Sigma-Aldrich). The mixture was vortexed for 10 seconds and centrifuged at 2000 × g for 10 mins. The upper, organic phase was transferred to glass centrifuge tube carefully. The extraction procedure was repeated 3 times to ensure complete recovery of the sex hormones. Diethyl ether was then evaporated under gentle stream of nitrogen gas (Hong Kong Oxygen Ltd, Hong Kong). Concentrations of estradiol, testosterones and 11-ketotestosterone were quantified using commercially available enzyme immunoassay kit (Cat.582251, Cat.582701, Cat.582751, respectively; Cayman Chemical, Michigan, USA) following the manufacturer’s recommended procedures. Plasma protein concentration of the whole blood was measured by the use of Bradford protein assay (Bio-Rad) and was used to normalize the concentration of sex hormones.
All statistical analyses were performed in the R environment 2.15.1. Survival probability of male and female medaka across age was estimated using the Kaplan-Meier method adjusted for left-truncation (< 3 month-old) and right-censoring (up-to-date survivorship data at the time of this writing). Sex difference in longevity was indicated by contrasting the survival curves of the two sexes using logrank test. The age-dependent TRF length was modeled as exponential decay: TRF
– Plateau = (TRF0 – Plateau) · e–λt , where TRF
is the modeled mean TRF length at age t, TRF0 is the extrapolated initial mean TRF length, Plateau is the horizontal asymptote, and λ is the rate constant. Model parameters were fitted using self-starting nonlinear least squares asymptotic regression model (R/nls in conjunction with SSasymp function).
Sex-dependency of the telomerase activity and telomeric length were tested using Student’s t-test. Age-dependent variation in plasma concentration of the three sex hormones’ level was illustrated by the use of one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) test. The relationship between telomere length and telomerase activity and the circulatory levels of sex hormones were illustrated by Spearman correlation analyses. Whenever applicable, the normality and homoscedascity assumptions for all parametric inferences were verified by the Shapiro-Wilk test and Levene’s test on median center, respectively. p-values from multiple comparisons were adjusted to control for false discovery rate (FDR) by use of the Benjamini and Hochberg procedure .