Sperm DNA methylation is predominantly stable in mice offspring born after transplantation of long-term cultured spermatogonial stem cells
Serrano JB, Tabeling NC, de Winter-Korver CM, van Daalen SKM, van Pelt AMM, Mulder CL, 07.04.2023
Abstract
Background: Spermatogonial stem cell transplantation (SSCT) is proposed as a fertility therapy for childhood cancer survivors. SSCT starts with cryopreserving a testicular biopsy prior to gonadotoxic treatments such as cancer treatments. When the childhood cancer survivor reaches adulthood and desires biological children, the biopsy is thawed and SSCs are propagated in vitro and subsequently auto-transplanted back into their testis. However, culturing stress during long-term propagation can result in epigenetic changes in the SSCs, such as DNA methylation alterations, and might be inherited by future generations born after SSCT. Therefore, SSCT requires a detailed preclinical epigenetic assessment of the derived offspring before this novel cell therapy is clinically implemented. With this aim, the DNA methylation status of sperm from SSCT-derived offspring, with in vitro propagated SSCs, was investigated in a multi-generational mouse model using reduced-representation bisulfite sequencing. Results: Although there were some methylation differences, they represent less than 0.5% of the total CpGs and methylated regions, in all generations. Unsupervised clustering of all samples showed no distinct grouping based on their pattern of methylation differences. After selecting the few single genes that are significantly altered in multiple generations of SSCT offspring compared to control, we validated the results with quantitative Bisulfite Sanger sequencing and RT-qPCRin various organs. Differential methylation was confirmed only for Tal2, being hypomethylated in sperm of SSCT offspring and presenting higher gene expression in ovaries of SSCT F1 offspring compared to control F1. Conclusions: We found no major differences in DNA methylation between SSCT-derived offspring and control, both in F1 and F2 sperm. The reassuring outcomes from our study are a prerequisite for promising translation of SSCT to the human situation. Keywords: DNA methylation; Multi-generational mouse model; Preclinical epigenetics; SSCT; Spermatogenesis; Spermatogonial stem cell transplantation; Spermatogonial stem cells.
Serrano JB, Tabeling NC, de Winter-Korver CM, van Daalen SKM, van Pelt AMM, Mulder CL. Sperm DNA methylation is predominantly stable in mice offspring born after transplantation of long-term cultured spermatogonial stem cells. Clin Epigenetics. 2023 Apr 7;15(1):58. doi: 10.1186/s13148-023-01469-x. PMID: 37029425; PMCID: PMC10080964.
Publication: https://doi.org/10.1186/s13148-023-01469-x Repository: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE213399
Disclaimer
The publication Sperm DNA methylation is predominantly stable in mice offspring born after transplantation of long-term cultured spermatogonial stem cells by Serrano JB, Tabeling NC, de Winter-Korver CM, van Daalen SKM, van Pelt AMM, Mulder CL is published under an open access license: https://creativecommons.org/licenses/by-nc/4.0/. Permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
Curation by the MFGA team Relevant data sets presented in the publication have been identified. If possible, annotations (title, general information, conditions, processed tissue types and processed cell types) have been added based on information from the publication. Data tables and images that provide a good overview on the publication's findings on the data set have been extracted from the publication and/or supplement. If not stated otherwise, images are depicted with title and description exactly as in the publication. Tables have been adjusted to the MFGA table format. Conducted adjustments are explained in the detailed view of the tables. However, titles and descriptions have been adopted from the publication.
Data set 1: Bisulfite conversion and RRBS
Methylome: Reduced-Representation Bisulfite Sequencing
Tissue Types
| BRENDA tissue ontology | Maturity | Description | Species | Replicates |
|---|---|---|---|---|
| BTO_0000408: epididymis | Adult | A system of ductules emerging posteriorly from the testis that holds sperm during maturation and that forms a tangled mass before uniting into a single coiled duct which is continuous with the vas deferens. | Mouse |
Cell Types
| Cell ontology | Maturity | Description | Species | Replicates | Cells per replicate |
|---|---|---|---|---|---|
| -: epididymal spermatozoa | adult, 16-18 months | SSCT F0 (transpanted mice) | Mouse | 3 | |
| -: epididymal spermatozoa | adult, 3 months | SSCT F1 | Mouse | 5 | |
| -: epididymal spermatozoa | adult, 3 months | SSCT F2/M (maternal line) | Mouse | 5 | |
| -: epididymal spermatozoa | adult, 3 months | SSCT F2/P (paternal line) | Mouse | 5 | |
| -: epididymal spermatozoa | adult, 16-18 months | control F0 | Mouse | 3 | |
| -: epididymal spermatozoa | adult, 16-18 months | control F1 | Mouse | 5 |
Images
Figure 1: Global unsupervised clustering heatmap of the average methylation of all the samples.
The heatmap clustering of all the samples is based on the significantly differential methylation between each sample (n = 3 in the F0, n = 5 per offspring subgroup in the F1 and F2) and the average methylation of all the samples (in duplo), included in the heatmap as the last 2 rows in medium light gray for the top-500 most altered DMRs (both hypermethylated and hypomethylated)
Licensed under: https://creativecommons.org/licenses/by-nc/4.0/
Table 1:
All DMCs and DMRs that are significantly different between control F0 or control F1 and various SSCT groups and subgroups (n = 3 in the F0, n = 5 per offspring subgroup in the F1 and F2)
Licensed under: https://creativecommons.org/licenses/by-nc/4.0/
Data set 2: Quantitative Bisulfite Sanger sequencing PCR
Transcriptome: High-resolution bisulfite Sanger PCR
Cell Types
| Cell ontology | Maturity | Description | Species | Replicates | Cells per replicate |
|---|---|---|---|---|---|
| -: epididymal spermatozoa | adult, 16-18 months | SSCT F0 (transpanted mice) | Mouse | 3 | |
| -: epididymal spermatozoa | adult, 3 months | SSCT F1 | Mouse | 5 | |
| -: epididymal spermatozoa | adult, 3 months | SSCT F2/M (maternal line) | Mouse | 5 | |
| -: epididymal spermatozoa | adult, 3 months | SSCT F2/P (paternal line) | Mouse | 5 | |
| -: epididymal spermatozoa | adult, 16-18 months | control F0 | Mouse | 3 | |
| -: epididymal spermatozoa | adult, 16-18 months | control F1 | Mouse | 5 |
Images
Figure 4: Validation of RRBS results with BSP of Tal2 from individual sperm samples.
Overall CpGs presented the following averages per group: control F1 24.7% ± 4.4%, SSCT F1 17.4% ± 4.5%, SSCT F2/M 22.7% ± 9.3%, SSCT F2/P 14.5% ± 3.2% presenting the highest difference of 10% methylation difference between SSCT and control (n = 5 per offspring subgroup in the F1 and F2). F2/M—F2 generated from the maternal line, F2/P—F2 generated from the paternal line
Licensed under: https://creativecommons.org/licenses/by-nc/4.0/
Data set 3: Gene expression analysis
Transcriptome: RT-qPCR
Tissue Types
| BRENDA tissue ontology | Maturity | Description | Species | Replicates |
|---|---|---|---|---|
| BTO_0001363: testis | adult, 3 months | SSCT F1 | Mouse | 5 |
| BTO_0001363: testis | adult, 3 months | SSCT F2/M (maternal line) | Mouse | 5 |
| BTO_0001363: testis | adult, 3 months | SSCT F2/P (paternal line) | Mouse | 5 |
| BTO_0001363: testis | adult, 16-18 months | control F1 | Mouse | 5 |
| BTO_0000975: ovary | adult, 3 months | SSCT F1 | Mouse | 5 |
| BTO_0000975: ovary | adult, 3 months | SSCT F2/M (maternal line) | Mouse | 5 |
| BTO_0000975: ovary | adult, 3 months | SSCT F2/P (paternal line) | Mouse | 5 |
| BTO_0000975: ovary | adult, 16-18 months | control F1 | Mouse | 5 |
| BTO_0000671: kidney | adult, 3 months | SSCT F1 | Mouse | 5 |
| BTO_0000671: kidney | adult, 3 months | SSCT F2/M (maternal line) | Mouse | 5 |
| BTO_0000671: kidney | adult, 3 months | SSCT F2/P (paternal line) | Mouse | 5 |
| BTO_0000671: kidney | adult, 16-18 months | control F1 | Mouse | 5 |
Images
Figure 5: Comparative Tal2 gene expression analysis with RT-qPCR.
A testis, B ovary, and C kidney between control F1 and SSCT offspring in F1 and F2/M and F2/P. The bar chart and statistical analyses with ANOVA followed by Tukey’s post hoc testing represent the RNA expression between groups of all generations, (n = 5 per organ, per offspring subgroup in the F1 and F2, RT-PCR performed in technical triplos). *Statistically significant differences observed between groups (p < 0.05). F2/M—F2 generated from the maternal line, F2/P—F2 generated from the paternal line
Licensed under: https://creativecommons.org/licenses/by-nc/4.0/