Defective germline reprogramming rewires the spermatogonial transcriptome

Abstract

Defective germline reprogramming in Piwil4 (Miwi2)- and Dnmt3l-deficient mice results in the failure to reestablish transposon silencing, meiotic arrest and progressive loss of spermatogonia. Here we sought to understand the molecular basis for this spermatogonial dysfunction. Through a combination of imaging, conditional genetics and transcriptome analysis, we demonstrate that germ cell elimination in the respective mutants arises as a result of defective de novo genome methylation during reprogramming rather than because of a function for the respective factors within spermatogonia. In both Miwi2−/− and Dnmt3l−/− spermatogonia, the intracisternal-A particle (IAP) family of endogenous retroviruses is derepressed, but, in contrast to meiotic cells, DNA damage is not observed. Instead, we find that unmethylated IAP promoters rewire the spermatogonial transcriptome by driving expression of neighboring genes. Finally, spermatogonial numbers, proliferation and differentiation are altered in Miwi2−/− and Dnmt3l−/− mice. In summary, defective reprogramming deregulates the spermatogonial transcriptome and may underlie spermatogonial dysfunction.

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Fig. 1: MIWI2 but not DNMT3L is expressed in adult mouse undifferentiated spermatogonia.
Fig. 2: MIWI2 is not required for homeostatic or regenerative spermatogenesis.
Fig. 3: Defective reprogramming affects undifferentiated spermatogonia numbers, proliferation and differentiation.
Fig. 4: Deregulation of IAP, but no DNA damage, is found in Miwi2/ and Dnmt3l/ undifferentiated spermatogonia.
Fig. 5: IAPs deregulate gene expression in Miwi2/ and Dnmt3l/ undifferentiated spermatogonia.
Fig. 6: Increase in de novo–identified weakly expressed transcripts in Miwi2- or Dnmt3l-deficient undifferentiated spermatogonia.
Fig. 7: Defective de novo DNA methylation is associated with deregulated gene expression in Miwi2/ and Dnmt3l−/− undifferentiated spermatogonia.

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Acknowledgements

The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)–ERC grant agreement GA 310206. This study was technically supported by the EMBL Genomic Core facility, as well as EMBL Monterotondo’s genome engineering, flow cytometry and microscopy core facilities.

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Contributions

L.V. contributed to the design, execution and analysis of the majority of experiments. R.V.B. generated the whole-genome bisulfite libraries and performed the bioinformatic analysis of genomic methylation under the supervision of W.R. L.V. and I.I. performed the in vivo characterization of spermatogonia. L.V. and C.C. designed, generated and validated the Dnmt3lV5 allele. A.J.E. performed all gene expression and transposon bioinformatic analyses. D.O’C. conceived and supervised this study. L.V. and D.O’C. wrote the final version of the manuscript.

Corresponding author

Correspondence to Dónal O’Carroll.

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Integrated supplementary information

Supplementary Figure 1 Generation and validation of the Dnmt3lV5 allele.

a, Targeting strategy for generation of the Dnmt3lV5 allele by CRISPR–Cas9 gene-editing technology. b, Validation of the Dnmt3lV5 allele by PCR using primers flanking the initial ATG codon. In the presence of the wild-type allele, a 411-bp fragment is amplified; in the presence of the Dnmt3lV5 allele, a 459-bp fragment is amplified. c, Normal testicular weight of adult Dnmt3lV5/+ and Dnmt3lV5/V5 mice. Error bars represent s.d. of the mean (n = 6 animals for Dnmt3lV5/+ and 5 animals for Dnmt3lV5/V5). Individual data points are shown. d, Representative images of hematoxylin- and eosin-stained testis cross-sections of wild-type and Dnmt3lV5/V5 mice. e, Representative images of wild-type and Dnmt3lV5/+ E16.5 fetal testis cross-sections stained with antibody against V5. f, Miwi2 and Dnmt3l mRNA expression levels in juvenile P14 undifferentiated spermatogonia as determined by RNA-seq. Source data for c are provided online. Source data

Supplementary Figure 2 MIWI2 is not required for homeostatic or regenerative spermatogenesis.

a, Conversion of the Miwi2FL allele to the Miwi2 (Miwi2null) allele in Miwi2iKO mice after tamoxifen administration. b, Expression of Miwi2 transcript in Miwi2CTL and Miwi2iKO mice 2 weeks after the last tamoxifen injection. After tamoxifen administration, Miwi2iKO mice express Miwi2 transcript without exon 17, which was flanked by two loxP sites. Here primers spanning exons 16 and 17 were used for RT–qPCR analysis. Error bars represent s.d. of the mean (n = 3 animals). c, Scheme representing a timeline for inducible Miwi2 deletion (tamoxifen (Tmx) administration is depicted as black arrows) and the analysis time point (red bar) 2 weeks after the last tamoxifen injection. d, Representative images of hematoxylin- and eosin-stained testis cross-sections of tamoxifen-treated Miwi2CTL and Miwi2iKO mice. Respective time points after tamoxifen administration are shown. e, Representative images of hematoxylin- and eosin-stained testis cross-sections of tamoxifen- and busulfan-treated Miwi2CTL and Miwi2iKO mice. Respective time points after busulfan administration are shown.

Supplementary Figure 3 Generation and validation of the Dnmt3l allele.

a, Targeting strategy for generation of the Dnmt3l allele by homologous recombination. b, Southern blotting of the fragments generated by the BamHI restriction enzyme from wild-type (WT), Dnmt3lNeo/+ and Dnmt3l+/– genomic DNA hybridized with the external 3′ probe. c, Testicular weight of adult Dnmt3l+/– and Dnmt3l–/– mice. Error bars represent s.d. of the mean (n = 5 animals for Dnmt3l+/– and 7 animals for Dnmt3l–/–). Individual data points are shown. d, Representative images of hematoxylin- and eosin-stained testis cross-sections of WT and Dnmt3l–/– mice. e, Representative images of adult WT and Dnmt3l–/– testis cross-sections stained with antibody against LINE1 ORF1p. Source data for c are provided online. Source data

Supplementary Figure 4 CD45CD51c-KitCD9+ surface expression identifies undifferentiated spermatogonia.

FACS analysis of testicular cell suspensions from adult Miwi2tdTom/+; Gfrα1GFP/+ mice. Representative example of a gating strategy for analyzing undifferentiated spermatogonia in adult Miwi2tdTom/+; Gfrα1GFP/+ mice, where a single-cell suspension of testicular cells was stained with CD45-biotin, CD51-biotin, streptavidin-qDot605, c-Kit-PE-Cy7 and CD9-APC antibodies. Only CD45CD51c-KitCD9+ cells express Miwi2-tdTom, Gfrα1-GFP or both Miwi2-tdTom and Gfrα1-GFP; thus, this surface stain identifies undifferentiated spermatogonia.

Supplementary Figure 5 Representative example of a gating strategy for sorting and/or analyzing Miwi2-tdTomato-expressing undifferentiated spermatogonia.

a, A single-cell suspension of testicular cells was stained with CD45-biotin, CD51-biotin, streptavidin-qDot605, c-Kit-PE-Cy7 and CD9-FITC antibodies to isolate and analyze CD45CD51c-KitMiwi2-tdTomato+CD9+ or analyze CD45CD51c-KitCD9+ cell populations. b, A single-cell suspension of testicular cells was stained with CD45-biotin, CD51-biotin, streptavidin-APC and c-Kit-PE-Cy7 antibodies and Hoechst to determine the DNA content of CD45CD51c-KitMiwi2-tdTomato+ cell populations.

Supplementary Figure 6 Impact of Miwi2 and Dnmt3l deficiency on juvenile P14 spermatogonia.

a, Total testicular cell number from Miwi2tdTom/+ (WT), Miwi2tdTom/tdTom (Miwi2–/–) and Dnmt3l–/–; Miwi2tdTom/+ (Dnmt3l–/–) juvenile P14 mice. Error bars represent s.d. of the mean (n = 8 animals for WT and 7 animals for Miwi2–/– and Dnmt3l–/–). Individual data points are shown. b, FACS analysis of live CD45CD51 gated testicular cells from juvenile P14 mice of the indicated genotypes. Numbers indicate the percentage of live CD45CD51c-KitCD9+ cells. c, Enumeration of live CD45CD51c-KitCD9+ cells per testis from juvenile P14 mice of the indicated genotypes (as defined in a). Error bars represent s.d. of the mean. Individual data points are shown. d, FACS analysis of live CD45CD51 gated testicular cells from juvenile P14 mice of the indicated genotypes. Numbers indicate the percentage of live CD45CD51c-KitMiwi2-tdTomato+ and CD45CD51c-Kit+Miwi2-tdTomato+ cells. e,f, Enumeration of live CD45CD51c-KitMiwi2-tdTomato+ (e) and live CD45CD51c-Kit+Miwi2-tdTomato+ (f) cells per testis from juvenile P14 mice of the indicated genotypes (as defined in a). Error bars represent s.d. of the mean. Individual data points are shown. The number of animals (n) in c, e and f is 8 for WT and Dnmt3l–/– and 7 for Miwi2–/–, except that 6 animals were used for Dnmt3l–/– in f. Significance in a, c, e and f was assessed using two-sided t test. n.s., not significant; *P < 0.01, **P < 0.001, ***P < 0.0001. Source data for a, c, e and f are provided online. Source data

Supplementary Figure 7 Methylation changes in Miwi2–/– and Dnmt3l–/– juvenile P14 undifferentiated spermatogonia.

a, Comparison of the percentage of CpG methylation in WT and Miwi2–/– as well as WT and Dnmt3l–/– undifferentiated spermatogonia. Blue dots represent significant DMRs. Analysis was performed in biological triplicate (n = 3 animals). b,c, Percentages of CpG methylation levels measured by WGBS of WT (gray), Miwi2–/– (blue) and Dnmt3l–/– (green) undifferentiated spermatogonia for the indicated genomic features or TEs. An averaged value from biological triplicates (n = 3 animals) for each genotype is shown. The middle line represents the median of the data points with whiskers indicating the data range.

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Vasiliauskaitė, L., Berrens, R.V., Ivanova, I. et al. Defective germline reprogramming rewires the spermatogonial transcriptome. Nat Struct Mol Biol 25, 394–404 (2018). https://doi.org/10.1038/s41594-018-0058-0

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