Epigenetic Inheritance of 5-AZA-2'-Deoxycytidine (5-AZA-CdR) Induced Alterations

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Title: Epigenetic Inheritance of 5-AZA-2'-Deoxycytidine (5-AZA-CdR) Induced Alterations
Author: Cisneros, Francisco Javier
Advisors: Dr. Ross Leidy, Committee Member
Dr. Ida Smoak, Committee Member
Dr. Robert Smart, Committee Member
Dr. Stacy Branch, Committee Chair
Abstract: Methylation of DNA regulates in part genomic imprinting which plays a critical role in fetal development. Changes in methylation patterns can induce alterations of gene expression leading to abnormal growth and/or morphological defects. 5-AZA-CdR inhibits DNA methylation, thus altering gene expression. It was believed that in mammals, epigenetic defects could not be inherited because they are cleared and reset during gametogenesis and/or embryogenesis. However, recent publications sustain the existence of epigenetic inheritance. The aim of this study were: 1) to investigate if 5-AZA-CdR would induce heritable alterations in development; 2) to determine 5- AZA-CdR effects on post-natal development and reproductive capacity; 3) to confirm if these effects were due to alterations in energy metabolism or in circulating IGF-I levels; and 4) to analyze the nature of the effect on male reproductive capacity. To determine if 5-AZA-CdR would induce heritable alterations in development, pregnant mice were administered 1 mg/kg of 5-AZA-CdR, and three subsequent generations were examined. Male and female treated offspring were mated avoiding consanguinity. In each generation, 50% of pregnant mice were killed and fetuses used for necropsy and skeletal analysis while the other 50% were allowed to give birth to produce the next generation. Leg and tail abnormalities, abnormal male mating behavior, retarded post-natal body development, cleft palate and global DNA hypermethylation were observed in exposed F1 mice. In the F2, cleft palate and increased levels of global DNA methylation were observed, while in the F3, cleft palate was evident. Previous work determined genes whose expression is altered in developing limb buds exposed in utero by 5-AZA-CdR. Methylation status in abnormal palate tissue was determined to clarify if methylation of the promoter regions of developmentally related genes were affected. Statistically significant differences were not observed. To elucidate the effects of 5-AZA-CdR on post-natal development and reproductive capacity, pregnant mice were administered 1 mg/kg of 5-AZA-CdR. Mothers gave birth and pups were weaned at 21 days of age. Number of pups per litter and weight were recorded. Body weight of males and females were collected at different ages. Lower weights in treated F1 males and females were observed every recorded time. Effects were more pronounced with age. 5-AZA-CdR F1 males and females and control mice were killed and levels of serum IGF-1, corticosterone and glucose were determined to investigate if growth retardation was a consequence of altered energy metabolism or serum IGF-1 levels induced by the treatment. No treatment effect was observed in serum corticosterone or glucose levels. However, statistically reduced levels of IGF-1 were observed in 5-AZA-CdR F1 males only. To understand the effects on reproductive capacity, 5-AZA-CdR F1 males and females were mated with control females and males respectively. Reproductive capacity of in utero exposed male mice was adversely affected. However, testis histology, daily sperm production and testosterone levels were not. To clarify if the altered reproductive capacity was a behavioral phenomenon, a male sexual behavior test was conducted. A significantly decreased number of mounts and a significant increase of mount latency were observed in exposed mice. Additionally, although the treatment affected the male:female offspring ratio, presence of Sry gene (an indicator of Y chromosome presence) in exposed male mice was not affected by the treatment. Our data suggest that altered methylation patterns (induced initially by 5-AZA-CdR embryonic exposure) affect normal fetal and post-natal development and male sexual behavior. Induced growth retardation could be associated with lower serum IGF-1 levels. Furthermore, 5-AZA-CdR-induced alteration may be inherited but the offspring.
Date: 2002-12-18
Degree: PhD
Discipline: Comparative Biomedical Sciences
URI: http://www.lib.ncsu.edu/resolver/1840.16/4762

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