Tamara R. Cohen

Disruption of histone methylation in developing sperm impairs offspring health transgenerationally

Generations affected by histone changes Parent and even grandparent environmental exposure can transmit adverse health effects to offspring. The mechanism of transmission is unclear, but some studies have implicated variations in DNA methylation. In a mouse model, Siklenka et al. found that alterations in histone methylation during sperm formation in one generation leads to reduced survival and developmental abnormalities in three subsequent generations (see the Perspective by McCarrey). Although changes in DNA methylation were not observed, altered sperm RNA content and abnormal gene expression in offspring were measured. Thus, chromatin may act as a mediator of molecular memory in transgenerational inheritance. Science, this issue p. 10.1126/science.aab2006; see also p. 634 Overexpression of a histone demethylase in the mouse germ line reveals a mode of transgenerational epigenetic inheritance. [Also see Perspective by McCarrey] INTRODUCTION Despite the father transmitting half of the heritable information to the embryo, the focus of preconception health has been the mother. Paternal effects have been linked to complex diseases such as cancer, diabetes, and obesity. These diseases are increasing in prevalence at rates that cannot be explained by genetics alone and highlight the potential for disease transmission via nongenetic inheritance, through epigenetic mechanisms. Epigenetic mechanisms include DNA methylation, posttranslational modifications of histones, and noncoding RNA. Studies in humans and animals suggest that epigenetic mechanisms may serve in the transmission of environmentally induced phenotypic traits from the father to the offspring. Such traits have been associated with altered gene expression and tissue function in first and second offspring generations, a phenomenon known as intergenerational or transgenerational inheritance, respectively. The mechanisms underlying such paternal epigenetic transmission are unclear. RATIONALE Sperm formation involves rapid cell division and distinctive transcription programs, resulting in a motile cell with highly condensed chromatin. Within the highly compacted sperm nucleus, few histones are retained in a manner that suggests an influential role in development. Despite being the major focus of studies in epigenetic inheritance, the role of DNA methylation in paternal epigenetic inheritance is unresolved, as only minor changes in DNA methylation in sperm at CpG-enriched regions have been associated with transmission of environmentally induced traits. Instead, there may be a combination of molecular mechanisms underlying paternal transgenerational epigenetic inheritance involving changes in histone states and/or RNA in sperm. The function of sperm histones and their modifications in embryonic development, offspring health, and epigenetic inheritance is unknown. By overexpressing the human KDM1A histone lysine 4 demethylase during mouse spermatogenesis, we generated a mouse model producing spermatozoa with reduced H3K4me2 within the CpG islands of genes implicated in development, and we studied the development and fitness of the offspring. RESULTS Male transgenic offspring were bred with C57BL/6 females, generating the experimental heterozygous transgenic (TG) and nontransgenic (nonTG) brothers. Each generation from TG and nonTG animals (F1 to F3 in our transgenerational studies) was bred with C57BL/6 females, and the offspring (pups from generations F1 to F4) were analyzed for intergenerational and transgenerational effects. We found that KDM1A overexpression in one generation severely impaired development and survivability of offspring. These defects lasted for two subsequent generations in the absence of KDM1A germline expression. We characterized histone and DNA methylation states in the sperm of TG and nonTG sires. Overexpression of KDM1A was associated with a specific loss of H3K4me2 at more than 2300 genes, including many developmental regulatory genes. Unlike in other examples of paternal transgenerational inheritance, we observed no changes in sperm DNA methylation associated with primarily CpG-enriched regions. Instead, we measured robust and analogous changes in sperm RNA content of TG and nonTG descendants, as well as in their offspring, at the two-cell stage. These changes in expression and the phenotypic abnormalities observed in offspring correlated with altered histone methylation levels at genes in sperm. This study demonstrates that KDM1A activity during sperm development has major developmental consequences for offspring and implicates histone methylation and sperm RNA as potential mediators of transgenerational inheritance. Our data emphasize the complexity of transgenerational epigenetic inheritance likely involving multiple molecular factors, including the establishment of chromatin states in spermatogenesis and sperm-borne RNA. CONCLUSION Correct histone methylation during spermatogenesis is critical for offspring development and survival over multiple generations. These findings demonstrate the potential of histone methylation as a molecular mechanism underlying paternal epigenetic inheritance. Its modification by environmental influences may alter embryo development and complex disease transmission across generations. An essential next step is to establish functional links between environmental exposures, the composition of the sperm epigenome, and consequent altered gene expression and metabolic processes in offspring. Considering the mounting evidence, it may soon be reasonable to suggest that future fathers protect their sperm epigenome. Disruption of histone methylation in developing sperm by exposure to the KDM1A transgene in one generation severely impaired development and survivability of offspring. These defects were transgenerational and occurred in nonTG descendants in the absence of KDM1A germline expression. Developmental defects in offspring and embryos were associated with altered RNA expression in sperm and embryos. A father’s lifetime experiences can be transmitted to his offspring to affect health and development. However, the mechanisms underlying paternal epigenetic transmission are unclear. Unlike in somatic cells, there are few nucleosomes in sperm, and their function in epigenetic inheritance is unknown. We generated transgenic mice in which overexpression of the histone H3 lysine 4 (H3K4) demethylase KDM1A (also known as LSD1) during spermatogenesis reduced H3K4 dimethylation in sperm. KDM1A overexpression in one generation severely impaired development and survivability of offspring. These defects persisted transgenerationally in the absence of KDM1A germline expression and were associated with altered RNA profiles in sperm and offspring. We show that epigenetic inheritance of aberrant development can be initiated by histone demethylase activity in developing sperm, without changes to DNA methylation at CpG-rich regions.

Limiting Excess Weight Gain in Healthy Pregnant Women: Importance of Energy Intakes, Physical Activity, and Adherence to Gestational Weight Gain Guidelines

Few studies have investigated if compliance with energy intakes, physical activity, and weight gain guidelines attenuate postpartum weight retention (PPWR) in mothers attending prenatal classes. We investigated whether (a) daily energy intakes within 300 kcal of estimated energy requirements (EERs), (b) walking more than 5000 steps/day, (c) targeting the recommended weight gain goals for prepregnancy BMI, and/or (d) achieving weekly or total gestational weight gain (GWG) recommendations minimized PPWR in 54 women attending prenatal classes in Montreal/Ottawa, Canada. Participants completed a validated pregnancy physical activity questionnaire (PPAQ), 3 telephone-validated 24-hr dietary recalls, and wore a pedometer for one week. PPWR was measured 6 weeks after delivery. Results showed that 72% had healthy prepregnancy BMIs. However, 52% consumed >300 kcal/day in excess of their EER, 54% exceeded recommended GWG, and more overweight (93%) than normal weight women (38%) cited nonrecommended GWG targets. Following delivery, 33% were classified as overweight, and 17% were obese. Multiple logistic regressions revealed that women targeting “recommended weight gain advice” were 3 times more likely to meet total GWG recommendations (OR: 3.2, P < 0.05); women who complied with weekly GWG goals minimized PPWR (OR: 4.2, P < 0.02). In conclusion, appropriate GWG targets, lower energy intakes, and physical activity should be emphasized in prenatal education programs.

Foot-to-foot bioelectrical impedance accurately tracks direction of adiposity change in overweight and obese 7- to 13-year-old children.

Body composition measurements are valuable when evaluating pediatric obesity interventions. We hypothesized that foot-to-foot bioelectrical impedance analysis (BIA) will accurately track the direction of adiposity change, but not magnitude, in part due to differences in fat patterning. The purposes of this study were to examine the accuracy of body composition measurements of overweight and obese children over time using dual-energy x-ray absorptiometry (DXA) and BIA and to determine if BIA accuracy was affected by fat patterning. Eighty-nine overweight or obese children (48 girls, 41 boys, age 7-13 years) participating in a randomized controlled trial providing a family-centered, lifestyle intervention, underwent DXA and BIA measurements every 3 months. Bland-Altman plots showed a poor level of agreement between devices for baseline percent body fat (%BF; mean, 0.398%; +2SD, 8.685%; -2SD, -7.889%). There was overall agreement between DXA and BIA in the direction of change over time for %BF (difference between visits 3 and 1: DXA -0.8 ± 0.5%, BIA -0.7 ± 0.5%; P = 1.000) and fat mass (FM; difference between visits 3 and 1: DXA 0.7 ± 0.5 kg, BIA 0.6 ± 0.5 kg; P = 1.000). Bioelectrical impedance analysis measurements of %BF and FM at baseline were significantly different in those with android and gynoid fat (%BF: 35.9% ± 1.4%, 32.2% ± 1.4%, P < .003; FM: 20.1 ± 0.8 kg, 18.4 ± 0.8, P < .013). Bioelectrical impedance analysis accurately reports the direction of change in FM and FFM in overweight and obese children; inaccuracy in the magnitude of BIA measurements may be a result of fat patterning differences.

Depleted iron stores and iron deficiency anemia associated with reduced ferritin and hepcidin and elevated soluble transferrin receptors in a multiethnic group of preschool-age children

Iron deficiency anemia is prevalent in subgroups of the Canadian population. The objective of this study was to examine iron status and anemia in preschool-age children. Healthy children (n = 430, 2-5 years old, Montreal, Quebec, Canada) were sampled from randomly selected daycares. Anthropometry, demographics, and diet were assessed. Biochemistry included hemoglobin, ferritin, soluble transferrin receptors (sTfR), ferritin index, markers of inflammation (C-reactive protein, interleukin 6 (IL-6), and tumour necrosis factor alpha (TNFα)), and hepcidin. Iron deficiency and anemia cutoffs conformed to the World Health Organization criteria. Differences among categories were tested using mixed-model ANOVA or χ(2) tests. Children were 3.8 ± 1.0 years of age, with a body mass index z score of 0.48 ± 0.97, and 51% were white. Adjusted intakes of iron indicated <1% were at risk for deficiency. Hemoglobin was higher in white children, whereas ferritin was higher with greater age and female sex. Inflammatory markers and hepcidin did not vary with any demographic variable. The prevalence of iron deficiency was 16.5% (95% confidence interval (CI), 13.0-20.0). Three percent (95% CI, 1.4-4.6) of children had iron deficiency anemia and 12.8% (95% CI, 9.6-16.0) had unexplained anemia. Children with iron deficiency, with and without anemia, had lower plasma ferritin and hepcidin but higher sTfR, ferritin index, and IL-6, whereas those with unexplained anemia had elevated TNFα. We conclude that iron deficiency anemia is not very common in young children in Montreal. While iron deficiency without anemia is more common than iron deficiency with anemia, the correspondingly reduced circulating hepcidin would have enabled heightened absorption of dietary iron in support of erythropoiesis.

Changes in eating behavior and plasma leptin in children with obesity participating in a family-centered lifestyle intervention

The goal of childhood obesity lifestyle interventions are to positively change body composition, however it is unknown if interventions also modulate factors that are related to energy intake. This study aimed to examine changes in eating behaviors and plasma leptin concentrations in overweight and obese children participating in a 1-year family-centered lifestyle intervention. Interventions were based on Canadian diet and physical activity (PA) guidelines. Children were randomized to 1 of 3 groups: Control (Ctrl; no intervention), Standard treatment (StnTx: 2 servings milk and alternatives/day (d), 3x/wk weight-bearing PA), or Modified treatment (ModTx: 4 servings milk and alternatives/day; daily weight-bearing PA). Study visits occurred every 3-months for 1-y; interventions were held once a month for 6-months with one follow-up visit at 8-months. Ctrl received counselling after 1-y. Caregivers completed the Childrens Eating Behavior Questionnaire (CEBQ) and reported on diet and activity. Plasma leptin were measured from morning fasted blood samples. Seventy-eight children (mean age 7.8 ± 0.8 y; mean BMI 24.4 ± 3.3 kg/m2) participated; 94% completed the study. Compared to baseline, at 6-months StnTx reduced Emotional Overeating and Desire to Drink scores (p < 0.05) while Food Responsiveness scores were reduced in both StnTx and ModTx (p < 0.05). At 1-year, scores for Desire to Drink in StnTx remained reduced compared to baseline (p < 0.05). Plasma leptin concentrations were significantly lower in ModTx at 6-months compared to baseline (p < 0.05). This study resulted in intervention groups favorably changing eating behaviors, supporting the use family-centered lifestyle interventions using Canadian diet and PA recommendations for children with obesity.

Assessment of pedometer accuracy in capturing habitual types of physical activities in overweight and obese children

Background:Currently, there is a limited amount of research exploring physical activity measurement tools in overweight and obese (OW/OB) children using pedometers. Thus, our objective was to determine the accuracy of one spring-levered (SC-T2) and two piezoelectric pedometers (NL-1000 and Piezo) in OW/OB children.Methods:A total of 26 boys and 34 girls (n = 60) participated. Pedometer step-counts were compared to observed step counts for walking (walking, stair ascent and decent) and hopping tests. Pedometer accuracies were compared with Friedman tests while Bland-Altman plots were used to establish the accuracy of each pedometer against direct observations.Results:Boys (n = 26) and females (n = 34) were 96 and 91% OB, respectively. The two piezoelectric pedometers (NL-1000 and Piezo) were accurate for walking and stair climbing tasks, however all pedometers were inaccurate for hopping tests. Averaged over all three walking activities, the NL-1000 was the most accurate with 6.7% median error (interquartile range (IQR): 0.0–13.3); followed by the Piezo with 10.0% median error (IQR: 3.3–18.1); SC-T2 was the least accurate with −14.7% median error (IQR: −54.8–3.5).Conclusion:These results support the use of the piezoelectric pedometers for walking and stair climbing types of activities, which are typical for OW/OB children in a nonlaboratory setting.