No abstract

Cadmium exposure is ubiquitous and has been linked to diseases including cancers and reproductive defects. Since cadmium is nonmutagenic, it is thought to exert its gene dysregulatory effects through epigenetic reprogramming. Several studies have implicated germline exposure to cadmium in developmental reprogramming. However, most of these studies have focused on maternal exposure, while the impact on sperm fertility and disease susceptibility has received less attention. In this study, we used reduced representation bisulfite sequencing to comprehensively investigate the impact of chronic cadmium exposure on mouse spermatozoa DNA methylation. Adult male C57BL/J6 mice were provided water with or without cadmium chloride for 9 weeks. Sperm, testes, liver, and kidney tissues were collected at the end of the treatment period. Cadmium exposure was confirmed through gene expression analysis of metallothionein-1 and 2, 2 well-known cadmium-induced genes. Analysis of sperm DNA methylation changes revealed 1788 differentially methylated sites present at regulatory regions in sperm of mice exposed to cadmium compared with vehicle (control) mice. Furthermore, most of these differential methylation changes positively correlated with changes in gene expression at both the transcription initiation stage as well as the splicing levels. Interestingly, the genes targeted by cadmium exposure are involved in several critical developmental processes. Our results present a comprehensive analysis of the sperm methylome in response to chronic cadmium exposure. These data, therefore, highlight a foundational framework to study gene expression patterns that may affect fertility in the exposed individual as well as their offspring, through paternal inheritance.

Objective: Turner syndrome women (monosomy X) have high risk of aortopathies consistent with a role for sex chromosomes in disease development. We demonstrated that sex chromosomes influence regional development of Ang II (angiotensin II)–induced aortopathies in mice. In this study, we determined if the number of X chromosomes regulates regional development of Ang II–induced aortopathies. Approach and Results: We used females with varying numbers of X chromosomes (XX female mice [XXF] or XO female mice [XOF]) on an C57BL/6J (ascending aortopathies) or low-density lipoprotein receptor deficient ( Ldlr −/− ) background (descending and abdominal aortopathies) compared with XY males (XYM). To induce aortopathies, mice were infused with Ang II. XOF (C57BL/6J) exhibited larger percent increases in ascending aortic lumen diameters than Ang II–infused XXF or XYM. Ang II–infused XOF ( Ldlr −/− ) exhibited similar incidences of thoracic (XOF, 50%; XYM, 71%) and abdominal aortopathies (XOF, 83%; XYM, 71%) as XYM, which were greater than XXF (XXF, 0%). Abdominal aortic lumen diameters and maximal external diameters were similar between XOF and XYM but greater than XXF, and these effects persisted with extended Ang II infusions. Larger aortic lumen diameters, abdominal aortopathy incidence (XXF, 20%; XOF, 75%), and maximal aneurysm diameters (XXF, 1.02±0.17; XOF, 1.96±0.32 mm; P =0.027) persisted in ovariectomized Ang II–infused XOF mice. Data from RNA-seq demonstrated that X chromosome genes that escape X-inactivation (histone lysine demethylases Kdm5c and Kdm6a ) exhibited lower mRNA abundance in aortas of XOF than XXF ( P =0.033 and 0.024, respectively). Conversely, DNA methylation was higher in aortas of XOF than XXF ( P =0.038). Conclusions: The absence of a second X chromosome promotes diffuse Ang II–induced aortopathies in females.

Huanglongbing (HLB) is considered the most destructive disease in Citrus production and threatens the future of the industry. Microbial-derived defense elicitors have gained recognition for their role in plant defense priming. This work assessed a 5% (V/V) microbial fermentation application (MFA) and its role in the elicitation of defense responses in HLB-infected Citrus sinensis trees following a foliar application with a pump sprayer. Using a PCR detection method, HLB infection levels were monitored in healthy and infected trees for 20months. Nutrient analysis assessed N, P, K, Ca, Mg, Mn, Zn, Fe, B, and Cu concentrations in the trees. MFA significantly increased Cu concentrations in treated trees and resulted in the stabilization of disease index (DI) in infected trees. Initial real-time qPCR analysis of defense-associated genes showed a significant increase in pathogenesis-related protein 2 (PR2) and phenylalanine ammonia lyase (PAL) gene expression in healthy and HLB-infected trees in response to MFA. Gene expression of PR2 and PAL peaked 6h post-microbial fermentation application during an 8-h sampling period. A transcriptomic assessment using GeneChip microarray of the hour 6 samples revealed differential expression of 565 genes when MFA was applied to healthy trees and 909 genes when applied infected citrus trees when compared to their respective controls. There were 403 uniquely differentially expressed genes in response to MFA following an intersectional analysis of both healthy and infected citrus trees. The transcriptomic analysis revealed that several genes associated with plant development, growth, and defense were upregulated in response to MFA, including multiple PR genes, lignin formation genes, ROS-related genes, hormone synthases, and hormone regulators. This study provides further evidence that MFA may play an important role as a plant elicitor in an integrated pest management strategy in citrus and other agronomically important crops.

Millions of people are exposed to cadmium and chronic‐low dose exposure has been linked to increased risks of breast and endometrial cancer, osteoporosis, renal dysfunction, cardiovascular disease and reproductive defects. One possible mechanism is dysregulation of gene expression through oxidative stress. Since cadmium is not a mutagen, we hypothesize that it could also exert its effects through epigenetic reprogramming, modulating regulatory targets with consequences in gene expression. Studies have reported that these dysregulations in gene expression is transgenerational; however these studies have focused heavily on maternal exposure while the impact on sperm and fertility has received very little attention. This discrepancy is due to the long‐held belief that, DNA methylation, one of the most stable epigenetic marks, is erased during fertilization. Recent evidence now suggests that at least some methylation marks are retained and can be passed on to the next generation. Here, we investigate the impact of chronic‐low dose cadmium exposure on DNA methylation patterns in sperm using a mouse model and an environmentally relevant cadmium dose. We hypothesize that chronic‐low dose exposure to cadmium modifies DNA methylation patterns in sperm leading to developmental effects. Our preliminary data shows that exposure to cadmium did not cause any histological changes to the testes of cadmium‐treated compared to control animals. However, using the 5‐mC DNA ELISA method we demonstrated a global decrease in DNA methylation in our treatment group. Additionally, Reduced Representation Bisulfite Sequencing (RRBS) which measures DNA methylation changes at specific genomic loci, further validated the impact of cadmium exposure on DNA methylation. A stringent statistical cut‐off (q<0.01 with at least 25% difference between the groups and at least 10X reads), showed that chronic‐low dose cadmium exposure leads to 1,788 differentially methylated sites. Importantly, some of these sites, which we are now validating, were at promoters and are likely to have functional consequences in gene expression. Our results show for the first time that mice sperm methylome is altered in response to cadmium exposure while the testes integrity remain intact. Our data highlight a foundational framework to study gene expression patterns that may affect offspring in a paternal inheritance context. Support or Funding Information UK‐CARES Grant P30 ES026529