Mouse strains and mutants
Mice (wild-type C57BL/6J, IMSR, JAX: 000664, RRID: IMSR_JAX:000664; Miwi−/− mutation, MGI: 2182488; and pachytene piRNA mutations listed in Supplementary Table 1) were housed and euthanized according to the guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Chan Medical School in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited barrier facility at controlled temperature (22 ± 2 °C), relative humidity (40 ± 15%) and a 12-h day–light cycle. All experimental animals were 2–6 months old6,15,48. sgRNAs (Supplementary Table 1) were designed using a CRISPR design tool (https://www.idtdna.com/site/order/designtool/index/CRISPR_SEQUENCE). sgRNAs were transcribed with T7 RNA polymerase and then purified by electrophoresis on 10% denaturing polyacrylamide gels. gRNA (20 ng µl–1) and Cas9 mRNA (50 ng µl–1, TriLink Biotechnologies, L-7206) were injected together into the pronucleus of one-cell C57BL/6 zygotes in M2 medium (Sigma, M7167). After injection, the zygotes were cultured in EmbryoMax Advanced KSOM medium (Sigma, MR-106-D) at 37 °C under 5% CO 2 until the blastocyst stage (3.5 days), then transferred into the uterus of pseudopregnant ICR females 2.5 days post coitum. To screen for founders with the mutation, gDNA extracted from tail tissues was analysed by PCR using the primers listed in Supplementary Table 1. All mutant strains were maintained in a C57BL/6 background; all experimental animals were the progeny of at least two backcrosses.
Mouse fertility
Fertility was measured as previously described6,15,48. In brief, each 2–6-month-old male mouse was continuously housed with one 2–4-month-old C57BL/6 female. For male mice that did not produce pups after 3 months (around 3 cycles), the original female was replaced with a new female and the fertility test continued.
To generate E8.5 or E14.5 embryos, one male mouse was housed with two C57BL/6 females. When a copulatory plug was observed, the female was housed separately until the experiment was completed.
Epididymal sperm count
Sperm counts were obtained as previously described6,15,48. In brief, to quantify sperm abundance, cauda epididymides were collected from mice and placed in PBS. A few incisions were made in the epididymides with scissors to release the sperm, followed by incubation at 37 °C and 5% CO 2 for 20 min. A 20 µl aliquot of sperm suspension was diluted in 480 µl of 1% (w/v) paraformaldehyde (PFA) and sperm cells were counted using a Leica DMi8 bright-field microscope equipped with a ×10, NA 0.4 objective.
TUNEL immunohistochemistry
Mouse testes were fixed in Bouin’s solution overnight, washed with 70% ethanol, embedded in paraffin and sectioned at 5 µm thickness. A Click-iT TUNEL Colorimetric IHC Detection kit (Thermo Fisher, C10625) was used to detect DNA breaks according to the manufacturer’s protocol. In brief, testes were fixed and embedded as described above, then were de-paraffinized in three changes of xylene for 5 min each, gradually re-hydrated in 100% (v/v), 95% (v/v) and 70% (v/v) ethanol for 5 min each, and then washed in 1× PBS for 5 min. After pretreating the slides with 20 µg ml–1 proteinase K at room temperature for 15 min, slides were washed with water twice (2 min each). Positive-control slides were treated with 1.0 U Turbo DNase (Thermo Fisher, AM2238) at room temperature for 30 min. Slides were then incubated with TdT reaction buffer containing terminal deoxynucleotidyl transferase in a humidified chamber at 37 °C for 1 h. The reaction was quenched with 2× SSC for 15 min, then washed twice in PBS. Peroxidase activity was quenched in 3% (v/v) H 2 O 2 at room temperature for 5 min. Slides were incubated with biotin azide and copper sulfate in a humidified chamber at 37 °C for 30 min, then stained with peroxidase substrate at room temperature for 10 min. Nuclei were counterstained with haematoxylin I, and the slides were sealed with EcoMount (Biocare Medical, EM897L). Images were captured using a Leica DMi8 bright-field microscope equipped with a ×20 objective with 0.4 NA (HC PL FL L ×20/0.40 CORR PH1, Leica Microbiosystems).
In vitro fertilization and embryo transfer
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