Ibrahim Mariam, Grochowska Ewa, Bednarczyk Marek, Stadnicka Katarzyna
Faculty of Health Sciences, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland.
PBS Doctoral School, Bydgoszcz University of Science and Technology, Bydgoszcz, Poland.
Sci Rep. 2025 Aug 22;15(1):30940. doi: 10.1038/s41598-025-16387-6.
Epigenetic mechanisms play a pivotal role in hereditary processes, shaping phenotypic outcomes across generations. This study investigates the transgenerational impacts of in ovo injection of bioactive substances on gene expression and DNA methylation in the male gonads using the Green-legged Partridgelike chickens as a model organism. Synbiotic PoultryStar (Biomin; PS) and choline were injected in ovo on the 12th day of egg incubation. In the F1 generation, three groups were established: (1) control (C, 0.9% physiological saline); (2) PS synbiotic (SYN, 2 mg/embryo); and (3) PS synbiotic combined with choline (SYNCH, 2 mg/embryo of synbiotic and 0.25 mg/embryo of choline). In subsequent F2 and F3 generations, groups SYN and SYNCH were further divided into two subgroups each: (A) only injected in F1 embryos (SYNs and SYNCHs); and (B) repeatedly injected in every generation (SYNr and SYNCHr). At 21 weeks post-hatching, gonadal tissues were sampled from F2 and F3 male chickens for transcriptomic and reduced representation bisulfite sequencing (RRBS). Synbiotic alone produced minimal and diminishing changes in gene expression across generations. In contrast, the single co-administration of synbiotic and choline in F1 embryos (SYNCHs) led to 1,897 differentially expressed genes (DEGs) and 786 differentially methylated regions (DMRs) in F3. Repeated administration across generations (SYNCHr) resulted in an even greater number of DEGs (2,804) and DMRs (2,880) in F3, reflecting a cumulative exposure effect. DEGs in SYNCH groups were mainly enriched in pathways related to cytoskeletal organization and extracellular matrix. In SYNCHs, methylation changes were associated with TGF-beta signaling, whereas SYNCHr showed additional enrichment in Wnt signaling, focal adhesion, and adipocytokine signaling pathways. Integrative analysis revealed coordinated changes in gene expression and DNA methylation, particularly in the F3 generation, identifying 37 genes (47 DMRs) in SYNCHs and 194 genes (306 DMRs) in SYNCHr. This study highlights the potential of prenatal epigenetic interventions to induce gene expression and DNA methylation changes across generations in reproductive tissues.
表观遗传机制在遗传过程中起着关键作用，塑造了跨代的表型结果。本研究以绿脚鹧鸪鸡为模式生物，研究了卵内注射生物活性物质对雄性性腺基因表达和DNA甲基化的跨代影响。在卵孵化的第12天，向卵内注射合生元家禽之星（百奥明；PS）和胆碱。在F1代中，建立了三组：（1）对照组（C，0.9%生理盐水）；（2）PS合生元组（SYN，2mg/胚胎）；（3）PS合生元与胆碱联合组（SYNCH，2mg/胚胎的合生元和0.25mg/胚胎的胆碱）。在随后的F2和F3代中，SYN组和SYNCH组进一步各分为两个亚组：（A）仅在F1胚胎中注射（SYNs和SYNCHs）；（B）在每一代中重复注射（SYNr和SYNCHr）。在孵化后21周，从F2和F3雄性鸡中采集性腺组织进行转录组学和简化代表性亚硫酸氢盐测序（RRBS）。单独使用合生元在各代中引起的基因表达变化最小且逐渐减少。相比之下，在F1胚胎中单独联合使用合生元和胆碱（SYNCHs）在F3代导致1897个差异表达基因（DEG）和786个差异甲基化区域（DMR）。跨代重复给药（SYNCHr）在F3代导致更多的DEG（2804个）和DMR（2880个），反映了累积暴露效应。SYNCH组中的DEG主要富集在与细胞骨架组织和细胞外基质相关的途径中。在SYNCHs中，甲基化变化与TGF-β信号传导相关，而SYNCHr在Wnt信号传导、粘着斑和脂肪细胞因子信号传导途径中显示出额外的富集。综合分析揭示了基因表达和DNA甲基化的协同变化，特别是在F3代，在SYNCHs中鉴定出37个基因（47个DMR），在SYNCHr中鉴定出194个基因（306个DMR）。本研究强调了产前表观遗传干预在生殖组织中诱导跨代表达和DNA甲基化变化的潜力。
