Genome-wide comparative methylation analysis reveals the fate of germ stem cells after surrogate production in teleost

Nayak, Rigolin; Franěk, Roman; Laurent, Audrey; Pšenička, Martin

doi:10.1186/s12915-024-01842-z

Genome-wide comparative methylation analysis reveals the fate of germ stem cells after surrogate production in teleost

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Published: 16 February 2024

Volume 22, article number 39, (2024)
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Genome-wide comparative methylation analysis reveals the fate of germ stem cells after surrogate production in teleost

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Rigolin Nayak ORCID: orcid.org/0000-0002-5327-801X¹,
Roman Franěk^1,2,
Audrey Laurent³ &
…
Martin Pšenička¹

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Abstract

Background

Surrogate production by germline stem cell transplantation is a powerful method to produce donor-derived gametes via a host, a practice known as surrogacy. The gametes produced by surrogates are often analysed on the basis of their morphology and species-specific genoty**, which enables conclusion to be drawn about the donor’s characteristics. However, in-depth information, such as data on epigenetic changes, is rarely acquired. Germ cells develop in close contact with supporting somatic cells during gametogenesis in vertebrates, and we hypothesize that the recipient’s gonadal environment may cause epigenetic changes in produced gametes and progeny. Here, we extensively characterize the DNA methylome of donor-derived sperm and their intergenerational effects in both inter- and intraspecific surrogates.

Results

We found more than 3000 differentially methylated regions in both the sperm and progeny derived from inter- and intraspecific surrogates. Hypermethylation in the promoter regions of the protocadherin gamma gene in the intraspecific surrogates was found to be associated with germline transmission. On the contrary, gene expression level and the embryonic development of the offspring remained unaffected. We also discovered MAPK/p53 pathway disruption in interspecific surrogates due to promoter hypermethylation and identified that the inefficient removal of meiotic-arrested endogenous germ cells in hybrid gonads led to the production of infertile spermatozoa.

Conclusions

Donor-derived sperm and progeny from inter- and intraspecific surrogates were more globally hypermethylated than those of the donors. The observed changes in DNA methylation marks in the surrogates had no significant phenotypic effects in the offspring.

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Background

Epigenetics plays a critical role in regulating the expression of genes during embryonic development and tissue differentiation in living organisms. The epigenome is defined as the structural adaptation of chromosomal regions [1], which maintains stable cellular processes in living individuals unless disrupted by external factors. The epigenetic modifications involve complex mechanisms governed by many structural and molecular regulators. The common mechanisms include (a) DNA methylation, (b) histone protein modification, and (c) non-coding RNAs [2]. Among all epigenetic marks, DNA methylation has been the most extensively studied [3]. The most common structural modification is the addition of a methyl group to the 5th carbon atom of a cytosine base, forming 5-methylcytosine (5-mC). The 5-mC modification in a DNA segment changes gene activity without changing the gene sequence [2, 4]. Hypermethylation in regulatory regions, such as promoters in the genome, is known to suppress transcription [5, 6]. In contrast, the loss of methylation marks, leading to hypomethylation, stimulates the expression of certain genes [7]. A methyl group-bound cytosine base is typically followed by guanine base in the sequence; this short segment is known as a CpG dinucleotide. In vertebrates, methylation most commonly occurs in the CpG context [8]. The percentage of methylated CpG sites varies by cell type [9] and individual organism. Any minor change to the epigenome can exert an impact on cellular function [10].

Surrogate production involves advanced reproductive biology-based biotechnology, which enables the production of donor-derived gametes via germline chimeras. Isolated germ stem cells (GSCs) from a donor species are implanted into the suitable sterile recipient (endogenous germ-cell-free individuals) to produce donor-derived gametes [11]. Surrogate production technologies have offered the possibility of preserving the germplasm of large-bodied commercially valuable species in smaller species with a shorter sexual maturation cycle [12, 13]. This technique provides a unique opportunity to cryopreserve maternal genetic information in fish, as neither fish eggs nor embryos can be cryopreserved. Therefore, there are efforts to develop techniques to conserve endangered species such as sturgeon [14,15,16]. Despite the recent advancements in this field, the practical application of this technology in aquaculture production is still questionable. Our understanding is limited to the morphological characteristics of the gametes and progeny produced via surrogacy. There are several questions pertaining to the reliability of this approach must be answered to maximize the possibility of applying surrogate production technologies in aquaculture. Epigenetic change is a major question associated with germ cell transplantation (GCT) that has never been reported. This mechanism is sensitive to internal and external environmental factors and can induce heritable phenotypical alterations [17]. Our knowledge of how and to what degree GCT can modify the epigenome (DNA methylation) of the donor germline is still scarce. Elucidating the epigenetic perturbations and their functional consequences will provide us insight into the reliability of the surrogate production approach.

Several factors may have a significant impact on the donor’s methylome during GCT. Donor GSC is subjected to systematic procedures and is exposed to a variety of chemical compounds during surrogate production. The complex gonadal tissue is then reduced to single cells and transplanted into recipients. Cell proliferation and differentiation of donor GSCs occur in the recipient’s gonad after transplantation, supported by the gonadal somatic cells of the recipient. Very few of the hundreds of transplanted cells survive to be incorporated into the recipient gonad. In a previous study, Saito et al. [18] reported the size difference of the larvae produced by surrogate in zebrafish. Franek et al. [13] observed different morphological characteristics in male and female donor-derived gametes. Therefore, we hypothesize that the methylome of donor GSCs may undergo alterations in the recipient gonad and may thus lose or gain some crucial methylation marks. Considering these reports, it is now crucial to think beyond the morphological characteristics of the gametes produced by the surrogate parent. The number of methylation studies on donor-derived gametes produced via GCT is considerably lower than any other epigenetic study. A study of spermatogonia transplantation in mice has been reported, and it described the methylation of donor-derived spermatozoa and their offspring, but the results were limited to only a few imprinting genes [19]. In vitro culturing and transplantation of foetal germ cells have been conducted in mice, and the derived offspring were reported to exhibit growth abnormalities due to differential methylation [20], whereas fresh testicular tissue grafting into the recipients led to no epigenetic abnormalities in the germ cells or the offspring [21]. However, no information on methylation alterations in donor-derived gametes in fish has been reported.

It was previously reported that in zebrafish (Danio rerio), DNA methylation pattern was preserved in the germline [22], and the paternal methylome was inherited by the embryos [23, 24], which is why it is crucial to decode the methylome of sperm produced by the surrogate. It has been shown that after endogenous germ cell depletion, all sterile zebrafish transdifferentiated into males [25, 26] and there was no female germline chimera after transplantation. Therefore, we used donor-derived sperm samples and progeny for this study. Sterilization to deplete the endogenous germ cells of the host species is a crucial step in surrogate production to prevent the production of recipient-derived gametes. Sterilization of a host can be achieved by chromosomal manipulation [27], gene knockdown, gene knockout [28], UV exposure [29], and chemical treatment [30]. In this study, we used vas::EGFP transgenic zebrafish as donors and two types of hosts: (1) dead end (dnd) gene knockdown (dnd gene is exclusively expressed in primordial germ cells (PGCs) and plays a crucial role in PGC migration to the gonadal ridge during embryonic development, dnd-morpholino (dnd-MO) prevents PGC migration by blocking the translation of dead end protein [31]; hence the recipient becomes sterile with no endogenous germ cells) and (2) zebrafish and pearl danio (Danio albolineatus) hybrids (cross-breeding between two closely related species interferes with homologous chromosomal pairing during meiosis or induces mitotic arrest [32]).

Zebrafish have been used in many studies to produce germline chimeras [18, 3 for map** results). We calculated the average methylation level for all the genome cytosines in three different genomic contexts. We found that 87% of CpGs were methylated in the sperm samples for all three groups, and the average methylation level for the CHG and CHH contexts ranged between 0.35 and 0.4% (see Additional file 4, cytosine coverage in different contexts). In contrast, the progeny samples exhibited low CpG methylation levels, ranging between 79 and 81%, in which the CHH and CHG contexts accounted for 0.37 to 0.46%. When analysing the genome-wide methylation level, we did not observe a considerable difference between sperm and progeny samples in all three groups (Fig. 2A). Then, we examined methylation density at the whole-genome level. Methylation density showed high conservation among all groups and a trend similar to the methylation level, indicating a close relationship between methylation level and methylation density (Violin plots, Figure S2, Additional file 1). Since CHG and CHH methylation was negligible in all the samples, we focused only on CpG methylation in subsequent analyses.

Next, the correlation between replicates in each group was calculated using the Pearson correlation coefficient to determine closeness. The whole genome was divided into 2-kb bins, and the number of methylated cytosines (mCpG) was calculated for each bin. Replicates in each group showed a strong correlation, with r² value > 0.92 (Figure S3, Additional file 1). Next, we checked the correlation between groups of similar sample types. We compared the sperm samples, which showed high homogeneity with r² value > 0.91, and the result was similar to the progeny samples, in which r² > 0.9.

Differential methylation analysis suggests no massive epigenetic remodelling in the surrogates

Data from the replicates were combined for the differential methylation analysis between comparison groups at the whole-genome level (Fig. 2B, Circos plot). We observed that the hypermethylation rate in the HybH and MOH groups was slightly higher than that of the DOH group, and the difference in the methylation level was high among all three compared groups. Similarly, the sperm samples from all three groups showed higher differences with a different methylation pattern in all 25 chromosomes. Surprisingly, the landscape of CpG methylation in all genomic functional regions followed a very similar pattern with methylation depletion at the transcription start site (TSS) (Fig. 2C,D). Compared to those in other regions, introns and the repeat elements were hypermethylated and retained a similar shape in the plotted data for all the groups with no substantial difference. Compared to other genomic features, CpG islands (CGIs), which have been reported to predominate the promoter regions in vertebrates and to be involved in regulating gene expression [36, 37], were hypomethylated in all the samples and retained a similar methylation pattern in all studied groups. These results indicate that the sperm and progeny produced by the surrogates did not undergo a high degree of epigenetic remodelling.

DMRs in the compared groups reveal hypermethylation in both surrogates

Since we did not see any differences in the functional regions of the genome, we next investigated the differentially methylated regions (DMRs) in all groups. First, we calculated the number of DMRs in progeny samples across the genome. We found 3788 DMRs between the HybH and DOH, 3814 between the MOH and DOH, and 3766 between the MOH and HybH (Figure S4, Additional file 1). Then, we looked at hypomethylated (hypoDMR) and hypermethylated DMR (hyperDMR) in the compared groups. We annotated the DMRs according to their position in the genome (see Additional file 5 for the number of DMR distributions in all the genomic regions). More DMRs were found in introns and repeat elements than in other functional regions (Fig. 3A). When comparing HybH with DOH, we found more hyperDMRs in all the regions compared to the number of hypoDMRs. Then, we compared MOH and DOH and found almost the same number of both types of DMRs in all regions. However, the scenario was entirely different when we compared MOH with HybH, as we observed a higher number of hypoDMRs in all the regions, indicating higher methylation in HybH. Next, we calculated the methylation level of all the DMRs in the individual groups (Fig. 3B). The median value of the methylation level of the DMRs associated with the HybH was higher than that in the MOH and DOH. When we evaluated the DMR distribution with a violin plot, most DMRs were found to have accumulated with a higher methylation level in MOH and HybH.

Next, we asked whether the DMRs in sperm samples were similar to those in the progeny groups. To this end, we found 3639 DMRs between the HybS and DOS, 3527 between the MOS and DOS, and 3708 between the MOS and HybS. We annotated the DMRs, assessed their distribution (Fig. 3C), and calculated the methylation levels (Fig. 3D). The DMR distribution in all the sperm groups was similar to that in the progeny groups. The violin plots show that most of the DMRs in HybS and MOS groups exhibited higher methylation levels compared to those in the DOS group (also see the cluster heatmap of the DMRs in Figure S5, Additional file 1). The median value was higher, suggesting that sperm samples from both surrogates carried more hyperDMRs than the donors. These results indicate that both sperm and progeny derived from the surrogates were hypermethylated compared to the methylation level of the donors.

Hypermethylation in promoters regions of protocadherin gene (pcdh) in MOH and HybS

Because most of the DMRs found were accumulated in intronic regions and repeat elements, we focused only on the promoter DMRs to see if any biological function was disrupted in the chimera groups (The list of significant DMPs between the compared group is provided as Additional file 6). Promoter regions are located within 1 kb from the TSS (− 1 to 0). Methylation in the immediate vicinity of a TSS hinders transcription initiation, whereas methylation in the gene body does not and may even stimulate transcription elongation [37]. Hence, we extracted all the differentially methylated promoters (DMPs) and analysed their GO terms; terms with p-value less than 0.05 were considered to be significantly enriched (Figure S6, Additional file 1). To our surprise, in the comparison groups for the progeny samples, more DMPs associated with biological processes were significantly enriched in the MOH compared to HybH and DOH. Biological functions such as homophilic cell adhesion, regulation of odontogenesis, and tooth mineralization were highly enriched in the MOH compared to HybH and DOH. We individually checked all the gene IDs for the promoters associated with this term in the Integrative Genome Viewer (IGV). The identified promoter regions encoded pcdh1gc6, pcdh1g22, pcdh1g30, pcdh1g33, pcdh1g2, and pcdh1g31 (IGV Snapshots for pcdh1g DMPs, Fig. 4A). When visualizing individual replicates from each group of the progeny samples in IGV, the identified DMPs were found to be hypermethylated only in the MOH samples, and the pattern was consistent among the replicates. Next, we wondered whether these marks were present in the sperm samples. We compared the MOS and DOS groups and did not find significantly enriched DMPs related to these genes (Fig. 4B). When checking the methylation pattern in IGV for these DMPs, we see that HybS and DOS have similar methylation level, and the difference only arise in the progeny, where HybH and DOH show low methylation and MOH remains higher, suggesting that these loci in the HybH and DOH were demethylated while those in the MOH remained unchanged. These results indicated that hyperDMPs within the pcdh gene in MOH were transmitted through the germline.

We also found very few functions like cell–cell adhesion and homophilic cell adhesion enrichment for hyperDMPs in HybS, and the gene promoters targeted for these functions are pcdh1g, pcdh2g, and pcdh1a (Fig. 4C). However, the hybrid progenies were unaffected by this hypermethylation since we did not see any function enrichment in the GO analysis. We measured the mRNA expression levels for selected genes, such as pcdh1g2, pcdh1g31, pcdh1g22, and pcdh1g33 by RT‒qPCR (see Fig. 4D, individual data values for qPCR are provided in Additional file 7). However, We did not observe any significant difference in the gene expression level, which suggested that the hyperDMPs in pcdh did not affect the expression of these genes. Next, we analysed the hypoDMPs between MOH and DOH and found that functions such as regulation of angiogenesis, positive regulation of the developmental process, and positive regulation of vasculature development were significantly enriched. Angiogenesis is the process of blood and lymphatic vessel formation during embryonic development [38]. DMPs in genes itga5 and itga6l were found to be hypomethylated. Integrin alpha 5 (itga5) is essential for heart development [39], but RT-qPCR showed no significant difference in the mRNA expression levels for itga5. Next, we examined the hypoDMPs in the comparison groups HybH and DOH for enriched GO terms. We found very few basic enriched molecular functions like nickel cation binding, adenylate kinase activity, nucleotide kinase activity, and phosphotransferase activity. The promoters associated with these terms in the progeny samples were predicted to have transcription factor activity. The genes are si:dkey-14o6.4, si:dkey-8o9.5, and si:dkey-54j5.2 (complete list is provided as Additional file 8). These HypoDMPs were not significant in the sperm samples of any surrogate groups.

Promoter hypermethylation in HybS reveals that MAPK/p53 pathway disruption leads to low-quality sperm production

Next, we investigated the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment for DMPs to determine whether any signalling pathway was disrupted. Pathways with a p-value lower than 0.05 in Fisher’s exact test were regarded as significant. When analysing the hypoDMPs between the MOH and DOH samples, we found that metabolic pathways, oxidative phosphorylation, and cardiac muscle contraction were highly enriched functional pathways (Figure S7, Additional file 1). The finding of cardiac muscle contraction enrichment was congruent with GO angiogenesis term enrichment in the MOH group. However, we did not find any significant pathway enrichment in the MOS group. Metabolic pathways are critical mechanisms, and oxidative phosphorylation is an essential function; enriched DMPs were found mainly in the mitochondrial genome (the complete list is provided as Additional file 9). Moreover, mitochondria are maternally inherited by progeny [7). Data are shown as mean ± SD; unpaired t-test for data with Gaussian distribution (mapk12a, mapk8a, map2k2a, and bcl2l1 dataset) and the Mann–Whitney U test for data with non-Gaussian distribution (for Tp53, apaf1, and map4k2 dataset) with adjusted p < 0.05, n = 4 biological replicates, asterisks represent significant difference between the groups, ns = not significant

Full size image

In a previous comparison study by Franek et al. [65].

As reported by Franek et al. [65], and that explains the germ stem cells are more protected against epigenetic alterations. We presume that there is a strong selection bias after their transplantation into the host, which explains the reason that only a few cells survive to colonize the host gonad. For interspecific transplantation, this scenario for methylome pattern may differ if the host is phylogenetically farther species, which needs to be investigated in the future. Our data cannot exclude the possibility that this scenario may vary from species to species and between different sterilization treatments. Zebrafish are highly polymorphic [67], and the methylation pattern can also differ between the individuals derived from the same parents. The advantage of using zebrafish as a model in this study is their lack of epigenetic reprogramming postfertilization. This makes them a suitable model to study epimutations caused by surrogate production and their intergenerational inheritance compared to other species like Japanese medaka, which undergo genome-wide reprogramming during embryonic development, and the majority of inherited marks are erased. However, whether the environmental stressor-induced DNA methylation marks escape the global epigenetic reprogramming in medaka is unclear. Our study is designed based on the most commonly used germ cell transplantation method. Gonial cell transplantation into larvae is easier than other methods, such as PGC transplantation. PGC isolation and transplantation are skill-sensitive and more complicated than gonial cell transplantation. Although PGC transplantation is well established in zebrafish, gonial cells are most commonly used for germ cell transplantation. There are several advantages of gonial cells over PGCs, including their abundance and transplantation into the hatchlings, which is considerably easier than PGC transplantation.

Moreover, we used freshly isolated testicular cells for transplantation in our study, and it will be interesting to understand the changes when transplanting the cryopreserved germ cells. Dimethyl sulfoxide (DMSO) is widely used as a cryoprotectant for the long-term storage of cell lines. In surrogate production techniques, cryopreserved germ stem cells are often used for transplantation. As per the previous study, DMSO significantly impacts genome-wide methylation in mouse embryonic cells and changes cell fate [68]. Future studies will explore the potential changes in the gamete produced by the transplantation of cryopreserved germ cells. The surrogate production technique is exploited to reduce the maturation period of some bigger species into fast-growing small fish [69, 70] and preserve the germplasm of endangered species [71].

Conclusions

In summary, our study addresses the question of DNA methylation alteration in surrogate production and their persistence in the offspring by using teleost model species zebrafish. We report that (1) the genome of donor-derived sperm and progeny from both chimeras showed higher levels of methylation than the donor, and the observed hypermethylation in the surrogates did not cause any functional or phenotypical abnormalities in the produced offsprings, (2) protocadherin gamma was the most affected gene in the MO chimera by promoter hypermethylation and transmitted to the F1 progeny, (3) hypermethylated pcdh-gamma promoters did not affect gene expression or larval development in zebrafish and (4) low fertility in the interspecific hybrid was a result of apoptosis resistance in the meiotic-arrested germ cell. Our data sets the stage for further research on surrogates produced by evolutionary, more divergent species. Future studies will focus on the donor-derived gametes produced by the transplantation of cryopreserved germ cells.

Methods

Recipient production and transplantation

Adult AB line (wild type) zebrafish for recipient production were obtained from the European Zebrafish Resource Center (EZRC, Germany). Transgenic Tg(ddx4:egfp) (ZDB-TGCONSTRCT-210809–1, referred to as vas::EGFP) strain expressing enhanced green fluorescence protein in the germ cells driven by the promoter of the germ-cell-specific vasa gene [72], from the University of Liege, Belgium, and maintained in our facility for several generations at 28 ℃, 14/10-h light/dark photoperiod. All chemicals were procured from Sigma-Aldrich unless stated otherwise. The recipients were derived from the same parent (AB fish) and divided into two groups for dnd-MO injection (MO sequence 5′- GCTGGGCATCCATGTCTCCGACCAT-3′ [73], GeneTools, LLC) and hybrid production. dnd-MO (0.1 mM with 0.2 mM KCl) was injected into 1- to 2-cell-stage embryos. Hybrids were produced from AB females and pearl danio males as per the protocol described by Wong et al. [74]. Individual fish were first anaesthetized with 0.05% tricaine methanesulfonate (MS222). Then, the oocytes were collected from the ovulated AB females by applying gentle abdominal pressure, and milt was collected from the pearl danio (3-month-old) males; in vitro fertilization was then performed.

Testicular cell preparation from vas::EGFP donors and transplantation were performed according to the methods described by Franěk et al. [Histology of gonads

Histology was performed to evaluate the sterility of non-transplanted recipients. Three-month-old sterile recipients and intact controls (n = 3 for each group) were anaesthetized with an overdose of MS222 and carefully degutted. The whole torso was fixed with Bouin fixative for 24 h, followed by dehydration with a series of ethanol dilutions, embedded in JB-4 resin (JB4 embedding kit), using a plastic mold, and cut into 5-µm sections with a rotary microtome (Leica Biosystems). The sections were stained with hematoxylin and eosin according to the method described by Sullivan-Brown et al. [77]. Stained sections were then imaged under the Olympus microscope (Olympus BX51) and analysed to identify germ cells.

RT‒qPCR for hyper- and hypoDMP genes

Total RNA was extracted from the larvae (5 dpf, n = 5 biological replicates) and the testis (n = 4 biological replicates) using a PureLink RNA Mini Kit (Thermo Fisher Scientific), followed by cDNA synthesis using WizardScript™ RT FDmix (Wizbiosolutions). Synthesized cDNA samples were used as templates for RT-qPCR. PCR was performed with PowerTrack SYBR Green Master Mix on a QuantStudio™ 5 System (Applied Biosystems), and the PCR cycling conditions were as follows: 95 ℃ for 2 min, 40 cycles (95 ℃ for 15 s, and 60 ℃ for 60 s) followed by a dissociation curve. The housekee** gene eukaryotic translation elongation factor 1 alpha 1 (eef1a1l1) was used as the reference gene to normalize the expression of target genes. The primer sequences used for RT-qPCR are presented in Additional file 10. The level of gene expression was calculated using the 2^−ΔΔCT method. Four to five biological replicates were established for each group, and two technical replicates for each sample were established for RT-qPCR.

Raw data processing and map** to the reference genome

Before analysis, the reference data for zebrafish, which included the reference sequence fasta file, the annotation file in gtf format, the GO annotation file, the description file, and the gene region file in bed format, were prepared. As for the bed files, we predict repeats through RepeatMasker (version 4.1.2), followed by getting CGI track from the genome using cpgIslandExt. Bismark software (version 0.16.3, [78]) was used to perform alignments of bisulfite-treated reads to the zebrafish reference genome GRCz11. The reference genome was first transformed into a bisulfite-converted version (C-to-T and G-to-A) and then indexed using bowtie2 [79]. Sequence reads were also transformed into fully bisulfite-converted versions (C-to-T and G-to-A conversions) before they were aligned to similarly converted versions of the genome in a directional manner. Sequence reads that produced the best unique alignment from two alignment processes (the original top and bottom strand) were then compared to those from the normal genomic sequence, and the methylation state of all cytosine positions in each read was inferred. The reads that aligned to the same regions of the genome were regarded as duplicated reads. The sequencing depth and coverage were summarized using deduplicate reads. The results of methylation extraction (bismark_methylation_extractor) were transformed into bigWig format for visualization using the IGV browser. The sodium bisulfite nonconversion rate was calculated as the percentage of cytosine residues sequenced at cytosine reference positions in the lambda genome.

Methylation level estimation

The sequence was divided into multiple bins of size 10 kb for the methylation level calculation. The sum of the methylated and unmethylated read counts in each window was calculated. The methylation level (ML) for each window or C (cytosine) site shows the fraction of methylated cytosines (mC) and is defined as Eq. 1. The calculated ML was further corrected on the basis of the bisulfite nonconversion rate as described in a previous study [80]. Given the bisulfite nonconversion rate “r”, the corrected ML was estimated as Eq. 2.

$$ML\left(C\right)=reads \left(mC\right)\div reads \left(mC\right)+reads (C)$$

(1)

$$ML\left(corrected\right) =ML-r\div 1 -r$$

(2)

DMR analysis

Differentially methylated regions (DMRs) were identified using DSS software [81,82,83]. The core of DSS is a new dispersion shrinkage method for estimating the dispersion parameter from Gamma-Poisson or Beta-Binomial distributions. According to the distribution of the DMRs throughout the genome, the genes related to DMRs were identified as genes with a gene body region (from TSS to TES) or promoter region (upstream 2 kb from the TSS) that overlaps with a DMR. The DMR filtering command and parameters are as follows: smoothing = TRUE, smoothing.span = 200, delta = 0, p.threshold = 1e-05, minlen = 50, minCG = 3, dis.merge = 100, pct.sig = 0.5. Gene Ontology (GO) enrichment analysis of genes related to DMRs was implemented with the GOseq R package [39], in which gene length bias was corrected. GO terms with corrected P values less than 0.05 were considered to be significantly enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg) is a database resource for understanding high-level functions and utilities of the biological system [84]. We used KOBAS software [39] to test the statistical enrichment (P < 0.05) of DMR-related genes in KEGG pathways.

Statistical analysis

Data for relative gene expression was first checked for normal (Gaussian) distribution with the Shapiro‒Wilk test. Differences among the three groups were calculated using one-way ANOVA followed by Tukey’s multiple comparisons for normally distributed samples and the Kruskal–Wallis test for data with non-Gaussian distribution. Unpaired t-test was used to calculate the significance between the two groups for normally distributed data and the Mann–Whitney U test for data with non-Gaussian distribution with the adjusted p-value < 0.05. The statistical analysis for RT-qPCR data was performed using GraphPad Prism 9.

Availability of data and materials

The raw data files and the processed methylation data are publicly available at the Gene Expression Omnibus (GEO) under accession number GSE212876 [85]. Supplementary figures are provided in Additional file 1, and other data supporting the conclusions of this article are provided in Additional files 2, 3, 4, 5, 6, 7, 8, 9 and 10. All data generated or analysed during this study are included in this published article and its supplementary information files.

Abbreviations

HybS:: Sperm derived from the hybrid chimera
HybH:: Progeny derived from the hybrid chimera
DoS:: Sperm derived from the donor
DOH:: Progeny derived from the donor
MOS:: Sperm derived from the MO chimera
MOH:: Progeny derived from the MO chimera
5-mC:: 5- Methylcytosine
GSC:: Germ stem cell
MO:: Morpholino antisense nucleotide
PGC:: Primordial gem cell
WGBS:: Whole-genome bisulfite sequencing
GO:: Gene Ontology
KEGG:: Kyoto Encyclopedia of Genes and Genomes
TSS:: Transcription start site
TES:: Transcription end site
TE:: Transposable element
DMP:: Differentially methylated promoter
DMR:: Differentially methylated region
GCT:: Germ cell transplantation
IGV:: Integrative Genomics Viewer
ML:: Methylation level
mC:: Methylated cytosine
MAPK:: Mitogen-activated protein kinases

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Acknowledgements

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Funding

The work was supported by the Ministry of Education, Youth and Sports of the Czech Republic—project Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370), and the Czech Science Foundation (grant number 22-31141J, and grant to RF 22-01781O). This project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 871108 (AQUAEXCEL3.0). This output reflects only the author’s view, and the European Union cannot be held responsible for any use that may be made of the information contained therein.

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Authors and Affiliations

The University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zatisi 728/II, 389 25, Vodnany, Czech Republic
Rigolin Nayak, Roman Franěk & Martin Pšenička
Department of Genetics, The Silberman Institute, The Hebrew University of Jerusalem, Jerusalem, Israel
Roman Franěk
Fish Physiology and Genomics Laboratory, INRAE, Campus de Beaulieu, 35000, Rennes, France
Audrey Laurent

Authors

Rigolin Nayak
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Roman Franěk
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Audrey Laurent
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Martin Pšenička
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Contributions

MP conceptualized and supervised the study. RF performed the transplantation and provided the biological samples, RN performed the experiments, data analysis, and wrote the manuscript. AL helped with the manuscript review. All authors read and approved the final manuscript.

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Correspondence to Rigolin Nayak.

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Ethics approval and consent to participate

The study was conducted at the Faculty of Fisheries and Protection of Waters (FFPW), the University of South Bohemia in Ceske Budejovice, Vodnany, Czech Republic. The facility has the competence to perform experiments on animals (Act no. 246/1992 Coll., ref. number 16OZ19179/2016–17214). The Institutional Animal Care and Use Committee of the FFPW approved the methodological protocol of the current study according to the law on the protection of animals against cruelty (reference number: MSMT-6406/2019–2). The study did not involve endangered or protected species. The co-authors of the study (RF and MP) own the Certificate of Professional Competence for designing experiments and experimental projects under Sect. 15d (3) of Act no. 246/1992 Coll. on the Protection of Animals Against Cruelty.

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The authors declare that they have no competing interests.

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Supplementary Information

Additional file 1: Figure S1.

vas::EGFP testicular cell transplantation into the sterile recipients. Table S1. Data from transplantation. Figure S2. Violin plots for methylation density in all three contexts. Figure S3. Correlation analysis for CG context. Figure S4. Venn plot for the number of DMRs in all the compared groups. Figure S5. Cluster heatmap for DMR methylation level. Figure S6. GO term enrichment between the compared groups. Figure S7. KEGG enrichment scatter plot for DMP-related pathway. Table S2. In vitro fertilization success of the germline chimeras and the donor. Figure S8. Images of the testis histological sections. Figure S9. EGFP expression in the sperm samples. Figure S10. Progenies derived from donor-derived sperm show no phenotypical alterations.

Additional file 2.

The statistics of the genome coverage. File containing the genome coverage statistics for WGBS data.

Additional file 3.

Summary of map** results. File containing map** results of the sequencing reads to the reference genome.

Additional file 4.

The coverage statistics of cytosine in three different contexts. File containing the summary of the total percentage of cytosine bases covered in three different contexts.

Additional file 5.

Summary of the DMR distribution in different genomic regions. File containing the distribution summary of the DMRs between the compared groups in different genomic regions.

Additional file 6.

The number of significant DMPs in all the compared groups. File containing the number of significant DMPs in all the compared groups.

Additional file 7.

Individual data values for qPCR. File containing relative gene expression data values for the individual samples.

Additional file 8.

Gene ontology analysis of DMPs. File containing GO functional enrichment analysis of observed DMPs between all the compared groups.

Additional file 9.

KEGG signaling pathway analysis of DMPs. File containing KEGG signaling pathway enrichment analysis of observed DMPs between all the compared groups.

Additional file 10.

Primer sequences used in RT-qPCR. File containing the primer sequences used in RT-qPCR.

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Nayak, R., Franěk, R., Laurent, A. et al. Genome-wide comparative methylation analysis reveals the fate of germ stem cells after surrogate production in teleost. BMC Biol 22, 39 (2024). https://doi.org/10.1186/s12915-024-01842-z

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Received: 27 March 2023
Accepted: 09 February 2024
Published: 16 February 2024
DOI: https://doi.org/10.1186/s12915-024-01842-z

Genome-wide comparative methylation analysis reveals the fate of germ stem cells after surrogate production in teleost

Abstract

Background

Results

Conclusions

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Background

Differential methylation analysis suggests no massive epigenetic remodelling in the surrogates

DMRs in the compared groups reveal hypermethylation in both surrogates

Hypermethylation in promoters regions of protocadherin gene (pcdh) in MOH and HybS

Promoter hypermethylation in HybS reveals that MAPK/p53 pathway disruption leads to low-quality sperm production

Conclusions

Methods

Recipient production and transplantation

RT‒qPCR for hyper- and hypoDMP genes

Raw data processing and map** to the reference genome

Methylation level estimation

DMR analysis

Statistical analysis

Availability of data and materials

Abbreviations

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Acknowledgements

Funding

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Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Additional information

Publisher’s Note

Supplementary Information

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