RESUMO
BACKGROUND: Paternal obesity has been associated with reduced live birth rates. It could lead to inheritance of metabolic disturbances to the offspring through epigenetic mechanisms. However, obesity is a multifactorial disorder with genetic or environmental causes. Earlier we had demonstrated differential effects of high-fat diet-induced obesity (DIO) and genetically inherited obesity (GIO) on metabolic, hormonal profile, male fertility, and spermatogenesis using two rat models. The present study aimed to understand the effect of DIO and GIO on DNA methylation in male germline, and its subsequent effects on the resorbed (post-implantation embryo loss) and normal embryos. First, we assessed the DNA methylation enzymatic machinery in the testis by Real-Time PCR, followed global DNA methylation levels in spermatozoa and testicular cells by ELISA and flow cytometry, respectively. Further, we performed Methylation Sequencing in spermatozoa for both the groups. Sequencing data in spermatozoa from both the groups were validated using Pyrosequencing. Expression of the differentially methylated genes was assessed in the resorbed and normal embryos sired by the DIO group using Real-Time PCR for functional validation. RESULTS: We noted a significant decrease in Dnmt transcript and global DNA methylation levels in the DIO group and an increase in the GIO group. Sequencing analysis showed 16,966 and 9113 differentially methylated regions in the spermatozoa of the DIO and GIO groups, respectively. Upon pathway analysis, we observed genes enriched in pathways involved in embryo growth and development namely Wnt, Hedgehog, TGF-beta, and Notch in spermatozoa for both the groups, the methylation status of which partially correlated with the gene expression pattern in resorbed and normal embryos sired by the DIO group. CONCLUSION: Our study reports the mechanism by which diet-induced and genetically inherited obesity causes differential effects on the DNA methylation in the male germline that could be due to a difference in the white adipose tissue accumulation. These differences could either lead to embryo loss or transmit obesity-related traits to the offspring in adult life.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Perda do Embrião/genética , Células Germinativas/metabolismo , Hereditariedade/genética , Obesidade/genética , Tecido Adiposo Branco/metabolismo , Animais , Estudos de Casos e Controles , Metilação de DNA , Perda do Embrião/metabolismo , Desenvolvimento Embrionário/genética , Epigênese Genética , Expressão Gênica , Masculino , Modelos Animais , Obesidade/metabolismo , Ratos , Ratos Wistar , Espermatogênese/genética , Espermatozoides/metabolismo , Testículo/enzimologiaRESUMO
doublesex regulates early embryonic sex differentiation in holometabolous insects, along with the development of species-, sex-, and morph-specific adaptations during pupal stages. How does a highly conserved gene with a critical developmental role also remain functionally dynamic enough to gain ecologically important adaptations that are divergent in sister species? We analyzed patterns of exon-level molecular evolution and protein structural homology of doublesex from 145 species of four insect orders representing 350 million years of divergence. This analysis revealed that evolution of doublesex was governed by a modular architecture: Functional domains and female-specific regions were highly conserved, whereas male-specific sequences and protein structures evolved up to thousand-fold faster, with sites under pervasive and/or episodic positive selection. This pattern of sex bias was reversed in Hymenoptera. Thus, highly conserved yet dynamic master regulators such as doublesex may partition specific conserved and novel functions in different genic modules at deep evolutionary time scales.
Assuntos
Proteínas de Ligação a DNA/genética , Evolução Molecular , Proteínas de Insetos/genética , Insetos/genética , Animais , Besouros/classificação , Besouros/genética , Proteínas de Ligação a DNA/química , Dípteros/classificação , Dípteros/genética , Éxons , Himenópteros/classificação , Himenópteros/genética , Proteínas de Insetos/química , Insetos/classificação , Lepidópteros/classificação , Lepidópteros/genética , Masculino , Filogenia , Domínios ProteicosRESUMO
Analysis of structural variation in domain superfamilies can reveal constraints in protein evolution which aids protein structure prediction and classification. Structure-based sequence alignment of distantly related proteins, organized in PASS2 database, provides clues about structurally conserved regions among different functional families. Some superfamily members show large structural differences which are functionally relevant. This paper analyses the impact of structural divergence on function for multi-member superfamilies, selected from the PASS2 superfamily alignment database. Functional annotations within superfamilies, with structural outliers or 'rebels', are discussed in the context of structural variations. Overall, these data reinforce the idea that functional similarities cannot be extrapolated from mere structural conservation. The implication for fold-function prediction is that the functional annotations can only be inherited with very careful consideration, especially at low sequence identities.