Breeding and housing conditions
All animal procedures were conducted in accordance with the Home Office Animals (Scientific Procedures) Act 1986 (Project License number: 70/6693). Female and male C57BL/6 J mice were obtained from Charles Rivers UK, aged 6–8 weeks and 10 weeks, respectively. Mice were maintained on a 12 h light/dark cycle (07:00–19:00) and housed at a constant temperature and humidity. After 1 week of acclimatisation in the mouse facility on standard chow (control diet, 20% protein), matings were set up by transferring one, or sometimes two, females into a male’s cage in the late afternoon. On the discovery of a vaginal plug the next morning (designated 0.5 days post coitum), pregnant females were singly housed and given ad libitum access to either a PR diet (8% protein) or maintained on the CT diet. Breeding males were housed individually for the duration of the breeding period. Females were maintained on the respective diet until offspring were weaned. Whole litters were weighed at 7 and 14 days. Upon weaning at 21 days, male offspring from each litter were put on either a CT or PR diet until death. Only litters with 5–10 pups were included. Litter sizes had no impact on the conclusions reported here (Additional file 1: Figures S8 and S9). Male offspring were housed in cages containing 3–5 mice from weaning and weighed individually every week from weaning until they were killed at 11–13 weeks of age.
Diets
The CT diet was PicoLab® Mouse Diet 20 Extruded (5R58*), consisting of a standard chow containing roughly 20% of calories from protein. The PR diet was a custom diet obtained from Special Diet Services and was isocaloric with the control diet but contained only 8% of calories from protein (code: 829277, name: RB 8% CP ISO E (P)) (Diet compositions outlined in Additional file 1: Table S1).
Adult male dissection and phenotyping
Mice were fasted for 16 h before being killed by CO2 asphyxiation. After weighing the whole animal and measuring its length from nose to base of the tail, cardiac puncture was performed using a 23 G needle and 1 mL syringe. Between 100 and 500 μL of blood was collected. A drop of blood from the syringe (0.6 μL) was placed on a glucose measurement strip and blood glucose concentration was measured using a Bayer NEXT Contour Glucose meter. The remaining blood was decanted into a 1.5 mL Eppendorf tube and allowed to clot at room temperature before being placed on ice. In male mice, the epididymis was next dissected from the base of the testes and transferred to a 2 mL Eppendorf tube containing pre-made sperm motility medium warmed to 37 °C in a water bath (sperm motility medium: 1 M NaCl, 100 mM KCl, 25 mM KH2PO4, 20 mM MgSO4, 0.6% sodium lactate, 500 mM NaHCO3, 25 mM sodium pyruvate, 25 mM CaCl, 500 mM HEPES, 34.5 mg/mL of BSA). Epididymides were homogenised using a fine pair of scissors in the tube for 5 min and placed in a water bath for 30 min at 37 °C, with regular inversion, to allow the sperm to swim out. After incubation, the tube was briefly spun down using a nanofuge to collect debris, then the supernatant containing free-swimming sperm was removed and placed in a new 1.5 mL Eppendorf tube and stored on ice for the rest of the dissection.
Liver, kidneys and visceral gonadal white adipose tissue deposits were dissected out, weighed and flash frozen in liquid nitrogen. A small amount of pancreas and small intestine were also removed and flash frozen. Next, the subcutaneous inguinal white adipose tissue deposits on each side of the mouse and the interscapular brown adipose tissue deposits on the back of the mouse were removed, weighed and flash frozen. Finally, a small section of ear was flash frozen.
Phenol:chloroform DNA extraction
For DNA extraction, one-quarter of the extracted sperm was incubated overnight in 600 μL of PK buffer (10 mM Tris-HCl, 100 mM NaCl, 25 mM EDTA, 1% SDS) with 2 μL of Proteinase K enzyme (19 mg/mL) and 0.1 M DTT at 55 °C with slow rotation. Phenol (750 μL) was added to the samples and agitated for several minutes before spinning at 17,000x g for 5 min at 4 °C. The upper aqueous phase was transferred to a new tube and the process repeated with phenol:chloroform, then chloroform alone. After the final spin, 5 μL of Rnase was added and samples were incubated at 37 °C for 60 min. Then, 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2.5 volumes of 100% EtOH were added and samples incubated at −20 °C for 1 h. Samples were spun at 17,000x g for 10 min at 4 °C and the pellet was washed with 75% EtOH. Finally, the pellet was air-dried at 37 °C, resuspended in 200 μL of TE buffer and incubated at 50 °C for 3 h before storage at 4 °C. DNA was quantified using the High Sensitivity Qubit® kit (Thermo Fish Scientific, Cat. Q32851) as per the protocol. Sperm purity was confirmed by Bis-PCR-Seq of imprinting control regions associated with MEST, MCTS2, NESP and IGF2/H19.
RNA extraction and small RNA library preparation
For RNA extraction, three-quarters of the extracted sperm were incubated at 60 °C for 15 min with slow rotation in 33.3 μL of sperm lysis buffer (6.4 M Guanidine HCl, 5% Tween 20, 5% Triton, 120 mM EDTA, 120 mM Tris; pH 8.0) with 3.3 μL of Proteinase K (19 mg/mL) and 3.3 μL of 0.1 M DTT. After the incubation, one volume (100 μL) of ultra-pure water was added, followed by 700 μL of Qiazol Lysis reagent (QIAGEN, Cat. 79,306) and samples were vortexed for 5 min. Chloroform (140 μL) was added and samples were shaken vigorously for 30 s before 3 min incubation at room temperature. Samples were centrifuged at 12,000x g at 4 °C for 15 min then the upper aqueous phase was transferred to a new reaction tube. One volume of 70% EtOH was added and mixed thoroughly. Samples were transferred to a RNeasy mini spin column and the protocol from the miRNeasy Mini Kit (Qiagen, Cat. 217,004) was then followed, including the separation of the small RNA and large RNA fractions using an RNA MinElute spin column (Qiagen, Cat. 74,204). The small RNA fraction was eluted in 14 μL of RNase-free water and quantified using the microRNA kit from Qubit® (Cat. Q32880). Them, 6 μL of the small RNA fraction was used for small RNA library preparation using the NEBNext® Small RNA library prep set for Illumina (Cat. E7330S) as per the protocol. Each sample was uniquely barcoded using one of the NEBNext® Index Primers for Illumina (Cat. E7300S, E7580S, E7710S, E7730S). For PCR amplification, 15 cycles were used. Libraries were purified using the QIAQuick PCR purification kit (Qiagen, Cat. 28,104) and DNA was eluted into 32 μL of nuclease-free water. An aliquot of each library was diluted and 1 μL was run on an Agilent 2100 Bioanalyzer using the Agilent High Sensitivity DNA kit (Cat. 5067-4626) to assess the size distribution of the library. The libraries were pooled using equal volumes and size selected for between 140 and 200 bp (corresponding to an insert size of 13–73 bp) using a BluePippin machine (Sage Science) with 3% agarose cassettes (Sage Science, Cat. BDF3010).
Sequencing and data analysis
DNA from sperm was diluted to a concentration of 11 ng/μL and 45 μL of each sample was sent for sequencing to the Genome Centre Facility at Charterhouse Square, QMUL. Bis-PCR-Seq was performed using the 48.48 layout on the Fluidigm® C1 system (Fluidigm®, USA), coupled with Illumina MiSeq sequencing using version2 chemistry (150 bp, paired-end). See Additional file 1: Table S3 for the primer sequences. Small RNA libraries were initially sequenced using Illumina MiSeq Nano sequencing (75 bp, single-end) and read counts for each samples were used to re-balance the library pool. The final pool was then sequenced using Illumina NextSeq sequencing (75 bp, single-end).
Bismark (v0.7.12) was used to align Bis-PCR-Seq data to the mm10 reference genome (imprinting control region data; Additional file 1: Figure S4) or to the adjusted consensus rDNA reference, using Bowtie2 (v2.1.0). Only reads that mapped to the correct starting position and perfectly matched the consensus were used for further analysis. For rDNA analysis, the R package RSamtools was used to identify each read as either having an A or a C at position −104 bp and to determine the methylation status at position −133 bp of each read. Reads could therefore be assigned to either Am, Au, Cm, or Cu. The total number of reads in each group was summed and %A ((Am + Au)/(Cm + Cu)) and CpG –133 A meth % (Am/(Am + Au)) calculated for each sample. Methylation of imprinting control regions were assessed using a custom program (https://bitbucket.org/lowelabqmul/methylation-extractor).
Small RNA sequencing data was mapped to the whole genome (UCSC, mm10), piRNA, tRNA, miRNA and rRNA databases using the SPORTS 1.0 pipeline (https://github.com/junchaoshi/sports1.0.git). Total read counts for each small RNA class were expressed as percentages of number of reads mapped to the genome for each sample in the composition analysis (Fig. 4a). Differential expression analysis of tRNA fragments was performed using edgeR (glmQLFTest) using the number of reads mapping to the genome as the library sizes for normalisation.
Statistics
All statistical analysis and plotting were performed using R (v3.2.3). For all phenotype plots, a linear model was run on individuals and P values were derived by using robust standard errors to account for the relatedness between siblings in each diet group (R packages plm [34] and lmtest [35]) and corrected for n = 3 tests using Bonferroni’s correction. A Pearson’s product moment correlation coefficient was calculated to describe the relationship between %A and CpG –133 A methylation percentage in each diet group (cor) and a linear model with robust standard errors was then run to obtain P values, which were then corrected for n = 4 comparisons using Bonferroni’s correction (P
lin
). All mice were used in the analyses instead of litter averages and linear models with robust standard errors were used to correct for any biases due to sibling relatedness (a full justification is provided in Additional file 1: Supplementary methods). Fisher’s exact test was used to assess the differences in distribution of the small RNA compositions compared to CTCT, using percentages of mapped reads corresponding to each species in each sample and these were rounded to the nearest integer (corrected for n = 3 tests using Bonferroni’s correction). ANOVA was used to assess whether the %A or %C and CpG –133 A meth % or CpG –133 C meth % were different between the diet groups.