F1 Sperm Research Articles

PSIII-1 The effects of maternal diet on ram lamb reproductive characteristics

Abstract Maternal nutrition during gestation and resulting impacts on ram reproduction and epigenetics are poorly understood. We previously reported altered mRNA and epigenetic markers in F1 ram sperm due to poor maternal diet (restricted- and over-feeding) during gestation, yet the multigenerational effects on ram reproduction are unexplored. We hypothesized that F2 offspring born from restricted- and over-fed granddams would exhibit altered reproductive characteristics, and F1 and F2 offspring would display altered circulating concentrations of reproductive hormones. The objectives of this study were to determine the effects of poor maternal diet on 1) the reproductive function of F2 ram offspring, and 2) key reproductive hormones in F1 and F2 offspring. To evaluate these objectives, multiparous Dorset ewes (F0, n = 46) were fed 100% (CON), 60% (RES), or 140% (OVER) of National Research Council requirements from d 30 of gestation through parturition and a control diet during lactation. Offspring are identified as CON-F1 (n = 10 ewes; 12 rams), RES-F1 (n = 13 ewes; 21 rams), and OVER-F1 (n = 16 ewes; 15 rams). The F1 ewes (n = 37) were maintained on a control diet throughout and were bred between 16 and 19 mo of age to establish the F2 generation. Resulting male offspring are referred to as CON-F2 (n = 6 rams), RES-F2 (n = 13 rams), and OVER-F2 (n = 9 rams) in correspondence to the diets of the granddam. Semen samples of F2 rams were collected via electroejaculation at d 248.4 ± 0.36 (collection 1) and d 255.4 ± 0.36 (collection 2). Semen quality (volume, pH, sperm motility, and sperm concentration) was evaluated at both collections using mCASA; iSperm, Aidmics Biotechnology Co., LTD. Scrotal circumference was measured at collection 2. Sperm morphology was determined by contrast microscopy. Testosterone (T) and follicle stimulating hormone (FSH) were measured in F1 and F2 serum samples collected at d 256 ± 0.13 using commercially available kits. Data were analyzed using R Studio with P ≤ 0.05 considered significant and 0.05 < P ≤ 0.10 considered a tendency. An effect of granddam diet was not observed for F2 ram semen volume, pH, sperm motility, sperm concentration, scrotal circumference, nor histological sperm morphology (P ≥ 0.25). An effect of F0 diet was not observed for F1 or F2 offspring serum concentrations of T and FSH (P ≥ 0.28). In our model, F0 diet during gestation did not affect F2 male offspring sperm quality or circulating T and FSH in F1 and F2 generations. However, based on our previous reports that F0 diet altered F1 sperm epigenome, there may be an impact of maternal diet on epigenetic inheritance which warrants further exploration.

Sperm DNA methylation is predominantly stable in mice offspring born after transplantation of long-term cultured spermatogonial stem cells

BackgroundSpermatogonial stem cell transplantation (SSCT) is proposed as a fertility therapy for childhood cancer survivors. SSCT starts with cryopreserving a testicular biopsy prior to gonadotoxic treatments such as cancer treatments. When the childhood cancer survivor reaches adulthood and desires biological children, the biopsy is thawed and SSCs are propagated in vitro and subsequently auto-transplanted back into their testis. However, culturing stress during long-term propagation can result in epigenetic changes in the SSCs, such as DNA methylation alterations, and might be inherited by future generations born after SSCT. Therefore, SSCT requires a detailed preclinical epigenetic assessment of the derived offspring before this novel cell therapy is clinically implemented. With this aim, the DNA methylation status of sperm from SSCT-derived offspring, with in vitro propagated SSCs, was investigated in a multi-generational mouse model using reduced-representation bisulfite sequencing.ResultsAlthough there were some methylation differences, they represent less than 0.5% of the total CpGs and methylated regions, in all generations. Unsupervised clustering of all samples showed no distinct grouping based on their pattern of methylation differences. After selecting the few single genes that are significantly altered in multiple generations of SSCT offspring compared to control, we validated the results with quantitative Bisulfite Sanger sequencing and RT-qPCRin various organs. Differential methylation was confirmed only for Tal2, being hypomethylated in sperm of SSCT offspring and presenting higher gene expression in ovaries of SSCT F1 offspring compared to control F1.ConclusionsWe found no major differences in DNA methylation between SSCT-derived offspring and control, both in F1 and F2 sperm. The reassuring outcomes from our study are a prerequisite for promising translation of SSCT to the human situation.

Transgenerational impact of grand-paternal lifetime exposures to both folic acid deficiency and supplementation on genome-wide DNA methylation in male germ cells.

DNA methylation (DNAme) erasure and reacquisition occur during prenatal male germ cell development; some further remodeling takes place after birth during spermatogenesis. Environmental insults during germline epigenetic reprogramming may affect DNAme, presenting a potential mechanism for transmission of environmental exposures across multiple generations. We investigated how germ cell DNAme is impacted by lifetime exposures to diets containing either low or high, clinically relevant, levels of the methyl donor folic acid and whether resulting DNAme alterations were inherited in germ cells of male offspring of subsequent generations. Female mice were placed on a control (FCD), 7-fold folic acid deficient (7FD) or10-to20-fold supplemented (10FS and 20FS) diet before and during pregnancy. Resulting F1 litters were weaned on the respective diets. F2 and F3 males received control diets. Genome-wide DNAme at cytosines (within CpG sites) was assessed in F1 spermatogonia, and in F1, F2 and F3 sperm. In F1 germ cells, a greater number of differentially methylated cytosines (DMCs) were observed in spermatogonia as compared with F1 sperm for all folic acid diets. DMCs were lower in number in F2 versus F1 sperm, while an unexpected increase was found in F3 sperm. DMCs were predominantly hypomethylated, with genes in neurodevelopmental pathways commonly affected in F1, F2 and F3 male germ cells. While no DMCs were found to be significantly inherited inter- or transgenerationally, we observed over-representation of repetitive elements, particularly young longinterspersednuclearelements(LINEs). These results suggest that the prenatal window is the time most susceptible to folate-induced alterations in sperm DNAme in male germ cells. Altered methylation of specific sites in F1 germ cells was not present in later generations. However, the presence of DNAme perturbations in the sperm of males of the F2 and F3 generations suggests that epigenetic inheritance mechanisms other than DNAme may have been impacted by the folate diet exposure of F1 germ cells.

Paternal genetic intergenerational and transgenerational effects of cadmium exposure on hormone synthesis disorders in progeny ovarian granulosa cells

To investigate the paternal genetic effects of cadmium (Cd) exposure on hormone synthesis disorders in the ovarian granulosa cells (GCs) of offspring. Here, male Sprague‒Dawley (SD) rats were gavaged with CdCl2 (0, 0.5, 2, 8 mg/kg) from postnatal day (PND) 28–56, followed by mating with newly purchased healthy adult females to produce F1, and F1 adult males (PND 56) were mated with newly purchased healthy adult females to produce F2. The serum levels of estradiol (E2) and progesterone (Pg) decreased in F1 but essentially returned to normal in F2. The levels of StAR, CYP11A1, CYP17A1, CYP19A1, and SF-1 showed different alterations in F1 and F2 ovarian GCs. The expression patterns of miRNAs and imprinted genes related to hormone synthesis in GCs of F1 and F2 differed, but methylation of hormone synthesis-related genes was not significantly altered (except for individual loci in F1). In addition, there were significant changes in the expression of imprinted genes and miRNAs in F0 and F1 sperm. We conclude that paternal Cd exposure causes intergenerational genetic effects (hormone synthesis disorders) and transgenerational effects (reparative changes in hormone synthesis function) in ovarian GCs. These genetic effects were related to the downregulation of StAR in F1 and the upregulation of CYP17A1, CYP19A1, StAR and SF-1 in F2. Important changes in miRNAs and imprinted genes were also observed, but not all alterations originated from paternal inheritance.

Multigenerational effect of maternal bisphenol A exposure on DNA methylation in F1 sperm

Multigenerational effect of maternal bisphenol A exposure on DNA methylation in F1 sperm

Sperm DNA methylation alterations from cannabis extract exposure are evident in offspring

BackgroundCannabis legalization is expanding and men are the predominant users. We have limited knowledge about how cannabis impacts sperm and whether the effects are heritable.ResultsWhole genome bisulfite sequencing (WGBS) data were generated for sperm of rats exposed to: (1) cannabis extract (CE) for 28 days, then 56 days of vehicle only (~ one spermatogenic cycle); (2) vehicle for 56 days, then 28 days of CE; or (3) vehicle only. Males were then mated with drug-naïve females to produce F1 offspring from which heart, brain, and sperm tissues underwent analyses. There were 3321 nominally significant differentially methylated CpGs in F0 sperm identified via WGBS with select methylation changes validated via bisulfite pyrosequencing. Significant methylation changes validated in F0 sperm of the exposed males at the gene 2-Phosphoxylose Phosphatase 1 (Pxylp1) were also detectable in their F1 sperm but not in controls. Changes validated in exposed F0 sperm at Metastasis Suppressor 1-Like Protein (Mtss1l) were also present in F1 hippocampal and nucleus accumbens (NAc) of the exposed group compared to controls. For Mtss1l, a significant sex-specific relationship between DNA methylation and gene expression was demonstrated in the F1 NAc. Phenotypically, rats born to CSE-exposed fathers exhibited significant cardiomegaly relative to those born to control fathers.ConclusionsThis is the first characterization of the effect of cannabis exposure on the entirety of the rat sperm methylome. We identified CE-associated methylation changes across the sperm methylome, some of which persisted despite a “washout” period. Select methylation changes validated via bisulfite pyrosequencing, and genes associated with methylation changes were involved in early developmental processes. Preconception CE exposure is associated with detectable changes in offspring DNA methylation that are functionally related to changes in gene expression and cardiomegaly.These results support that paternal preconception exposure to cannabis can influence offspring outcomes.

Assessment of parental benzo[a]pyrene exposure-induced cross-generational neurotoxicity and changes in offspring sperm DNA methylome in medaka fish.

Previous studies have revealed that DNA methylation changes could serve as potential genomic markers for environmental benzo[a]pyrene (BaP) exposure and intergenerational inheritance of various physiological impairments (e.g. obesity and reproductive pathologies). As a typical aromatic hydrocarbon pollutant, direct BaP exposure has been shown to induce neurotoxicity. To unravel the inheritance mechanisms of the BaP-induced bone phenotype in freshwater medaka, we conducted whole-genome bisulfite sequencing of F1 sperm and identified 776 differentially methylated genes (DMGs). Ingenuity pathway analysis revealed that DMGs were significantly enriched in pathways associated with neuronal development and function. Therefore, it was hypothesized that parental BaP exposure (1 μg/l, 21 days) causes offspring neurotoxicity. Furthermore, the possibility for sperm methylation as an indicator for a neurotoxic phenotype was investigated. The F0 adult brains and F1 larvae were analyzed for BaP-induced direct and inherited toxicity. Acetylcholinesterase activity was significantly reduced in the larvae, together with decreased swimming velocity. Molecular analysis revealed that the marker genes associated with neuron development and growth (alpha1-tubulin, mbp, syn2a, shh, and gap43) as well as brain development (dlx2, otx2, and krox-20) were universally downregulated in the F1 larvae (3 days post-hatching). While parental BaP exposure at an environmentally relevant concentration could induce neurotoxicity in the developing larvae, the brain function of the exposed F0 adults was unaffected. This indicates that developmental neurotoxicity in larvae may result from impaired neuronal development and differentiation, causing delayed brain growth. The present study demonstrates that the possible adverse health effects of BaP in the environment are more extensive than currently understood. Thus, the possibility of multigenerational BaP toxicity should be included in environmental risk assessments.

Advanced paternal age increased metabolic risks in mice offspring

ObjectivesThe Developmental Origins of Health and Disease Science indicate that chronic diseases in adulthood are associated with prenatal and early-life traits. Our study aimed to explore the metabolic phenotype of offspring from advanced paternal age (APA) and the inherited alterations in sperm. Methods3-month-old (Young father, YF-F0) and 21-month-old male (Old Father, OF-F0) C57BL/6J mice were used to study paternal aging's effect on offspring. Blood glucose testing, lipid analysis, indirect calorimetry and RNA sequencing were performed. ResultsThe characterized metabolic changes in OF-F1 male mice offspring were glucose intolerance, hepatic lipid accumulation, increased adipocytes and impaired energy balance that lasted until they were elderly. Gene expression in both 8-week-old and 52-week-old offspring livers significantly altered in lipid metabolism- and thermogenesis-related pathways. PPAR signaling pathway was activated in both young and elderly offspring livers as indicated by significant upregulation of Cyp7a1, Cyp8b1, Cyp4a10, Cyp4a31, Fabp2, and Scd1. These targeted genes were also confirmed to be increased in offspring adipocytes. Furthermore, when examined the differentially expressed genes in F0 and F1 sperm, upregulated pathways including cholesterol metabolism, type II diabetes mellitus and endocrine resistance were strongly related to the APA offspring phenotype. Importantly, approximately 46.7% of enriched pathways in the sperm of APA offspring were consistent with those of APA fathers. ConclusionsThese findings added evidence of the connection between paternal gametes and alterations in progeny genome and raised the possibility that inherited alterations in sperm contribute to the intergenerational effects of paternal aging offspring's chronic metabolic risks.

Prenatal inflammation exposure-programmed hypertension exhibits multi-generational inheritance via disrupting DNA methylome.

The multi-generation heredity trait of hypertension in human has been reported, but the molecular mechanisms underlying multi-generational inheritance of hypertension remain obscure. Recent evidence shows that prenatal inflammatory exposure (PIE) results in increased incidence of cardiovascular diseases, including hypertension. In this study we investigated whether and how PIE contributed to multi-generational inheritance of hypertension in rats. PIE was induced in pregnant rats by intraperitoneal injection of LPS or Poly (I:C) either once on gestational day 10.5 (transient stimulation, T) or three times on gestational day 8.5, 10.5, and 12.5 (persistent stimulation, P). Male offspring was chosen to study the paternal inheritance. We showed that PIE, irrespectively induced by LPS or Poly (I:C) stimulation during pregnancy, resulted in multi-generational inheritance of significantly increased blood pressure in rat descendants, and that prenatal LPS exposure led to vascular remodeling and vasoconstrictor dysfunction in both thoracic aorta and superior mesenteric artery of adult F2 offspring. Furthermore, we revealed that PIE resulted in global alteration of DNA methylome in thoracic aorta of F2 offspring. Specifically, PIE led to the DNA hypomethylation of G beta gamma (Gβγ) signaling genes in both the F1 sperm and the F2 thoracic aorta, and activation of PI3K/Akt signaling was implicated in the pathologic changes and dysregulated vascular tone of aortic tissue in F2 LPS-P offspring. Our data demonstrate that PIE reprogrammed DNA methylome of cells from the germline/mature gametes contributes to the development of hypertension in F2 PIE offspring. This study broadens the current knowledge regarding the multi-generation effect of the cumulative early life environmental factors on the development of hypertension.

Intrauterine hyperglycemia impairs memory across two generations

Studies on humans and animals suggest associations between gestational diabetes mellitus (GDM) with increased susceptibility to develop neurological disorders in offspring. However, the molecular mechanisms underpinning the intergenerational effects remain unclear. Using a mouse model of diabetes during pregnancy, we found that intrauterine hyperglycemia exposure resulted in memory impairment in both the first filial (F1) males and the second filial (F2) males from the F1 male offspring. Transcriptome profiling of F1 and F2 hippocampi revealed that differentially expressed genes (DEGs) were enriched in neurodevelopment and synaptic plasticity. The reduced representation bisulfite sequencing (RRBS) of sperm in F1 adult males showed that the intrauterine hyperglycemia exposure caused altered methylated modification of F1 sperm, which is a potential epigenetic mechanism for the intergenerational neurocognitive effects of GDM.

Paternal MTHFR deficiency leads to hypomethylation of young retrotransposons and reproductive decline across two successive generations.

ABSTRACT5,10-Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme in the folate metabolic pathway with a key role in generating methyl groups. As MTHFR deficiency impacts male fertility and sperm DNA methylation, there is the potential for epimutations to be passed to the next generation. Here, we assessed whether the impact of MTHFR deficiency on testis morphology and sperm DNA methylation is exacerbated across generations in mouse. Although MTHFR deficiency in F1 fathers has only minor effects on sperm counts and testis weights and histology, F2 generation sons show further deterioration in reproductive parameters. Extensive loss of DNA methylation is observed in both F1 and F2 sperm, with >80% of sites shared between generations, suggestive of regions consistently susceptible to MTHFR deficiency. These regions are generally methylated during late embryonic germ cell development and are enriched in young retrotransposons. As retrotransposons are resistant to reprogramming of DNA methylation in embryonic germ cells, their hypomethylated state in the sperm of F1 males could contribute to the worsening reproductive phenotype observed in F2 MTHFR-deficient males, compatible with the intergenerational passage of epimutations.

Gestational arsenic exposure induces site-specific DNA hypomethylation in active retrotransposon subfamilies in offspring sperm in mice

BackgroundEnvironmental impacts on a fetus can disrupt germ cell development leading to epimutations in mature germ cells. Paternal inheritance of adverse health effects through sperm epigenomes, including DNA methylomes, has been recognized in human and animal studies. However, the impacts of gestational exposure to a variety of environmental factors on the germ cell epigenomes are not fully investigated. Arsenic, a naturally occurring contaminant, is one of the most concerning environmental chemicals, that is causing serious health problems, including an increase in cancer, in highly contaminated areas worldwide. We previously showed that gestational arsenic exposure of pregnant C3H mice paternally induces hepatic tumor increase in the second generation (F2). In the present study, we have investigated the F1 sperm DNA methylomes genome-widely by one-base resolution analysis using a reduced representation bisulfite sequencing (RRBS) method.ResultsWe have clarified that gestational arsenic exposure increases hypomethylated cytosines in all the chromosomes and they are significantly overrepresented in the retrotransposon LINEs and LTRs, predominantly in the intergenic regions. Closer analyses of detailed annotated DNA sequences showed that hypomethylated cytosines are especially accumulated in the promoter regions of the active full-length L1MdA subfamily in LINEs, and 5′LTRs of the active IAPE subfamily in LTRs. This is the first report that has identified the specific positions of methylomes altered in the retrotransposon elements by environmental exposure, by genome-wide methylome analysis.ConclusionLowered DNA methylation potentially enhances L1MdA retrotransposition and cryptic promoter activity of 5′LTR for coding genes and non-coding RNAs. The present study has illuminated the environmental impacts on sperm DNA methylome establishment that can lead to augmented retrotransposon activities in germ cells and can cause harmful effects in the following generation.

Gestational arsenic exposure and paternal intergenerational epigenetic inheritance

A growing body of evidence has shown that gestational exposure to environmental factors such as imbalanced diet, environmental chemicals, and stress can lead to late-onset health effects in offspring and that some of these effects are heritable by the next generation and subsequent generations. Furthermore, altered epigenetic modifications in DNA methylation, histone modifications and small RNAs in a single sperm genome have been shown to transmit disease phenotypes acquired from the environment to later generations. Recently, our group found that gestational exposure of F0 pregnant dams to an inorganic arsenic, sodium arsenite, increases the incidence of hepatic tumors in male F2 mice, and the effects are paternally transmitted to the F2. Here, we first overview the epigenetic changes involved in paternal intergenerational and transgenerational inheritance caused by exposure to environmental factors. Then, we discuss our recent studies regarding paternal inheritance of the tumor-augmenting effects in F2 mice by gestational arsenite exposure, in which we investigated alterations of DNA methylation status in F2 tumors and causative F1 sperm. We also discuss the possible targets of the F2 effects. Finally, we discuss future perspectives on the studies that are needed to fully understand the health effects of arsenic exposure.

Folic acid supplementation reduces multigenerational sperm miRNA perturbation induced by in utero environmental contaminant exposure.

Persistent organic pollutants (POPs) can induce epigenetic changes in the paternal germline. Here, we report that folic acid (FA) supplementation mitigates sperm miRNA profiles transgenerationally following in utero paternal exposure to POPs in a rat model. Pregnant founder dams were exposed to an environmentally relevant POPs mixture (or corn oil) ± FA supplementation and subsequent F1–F4 male descendants were not exposed to POPs and were fed the FA control diet. Sperm miRNA profiles of intergenerational (F1, F2) and transgenerational (F3, F4) lineages were investigated using miRNA deep sequencing. Across the F1–F4 generations, sperm miRNA profiles were less perturbed with POPs+FA compared to sperm from descendants of dams treated with POPs alone. POPs exposure consistently led to alteration of three sperm miRNAs across two generations, and similarly one sperm miRNA due to POPs+FA; which was in common with one POPs intergenerationally altered sperm miRNA. The sperm miRNAs that were affected by POPs alone are known to target genes involved in mammary gland and embryonic organ development in F1, sex differentiation and reproductive system development in F2 and cognition and brain development in F3. When the POPs treatment was combined with FA supplementation, however, these same miRNA-targeted gene pathways were perturbed to a lesser extend and only in F1 sperm. These findings suggest that FA partially mitigates the effect of POPs on paternally derived miRNA in a intergenerational manner.

Assessment of Glyphosate Induced Epigenetic Transgenerational Inheritance of Pathologies and Sperm Epimutations: Generational Toxicology

Ancestral environmental exposures to a variety of factors and toxicants have been shown to promote the epigenetic transgenerational inheritance of adult onset disease. One of the most widely used agricultural pesticides worldwide is the herbicide glyphosate (N-(phosphonomethyl)glycine), commonly known as Roundup. There are an increasing number of conflicting reports regarding the direct exposure toxicity (risk) of glyphosate, but no rigorous investigations on the generational actions. The current study using a transient exposure of gestating F0 generation female rats found negligible impacts of glyphosate on the directly exposed F0 generation, or F1 generation offspring pathology. In contrast, dramatic increases in pathologies in the F2 generation grand-offspring, and F3 transgenerational great-grand-offspring were observed. The transgenerational pathologies observed include prostate disease, obesity, kidney disease, ovarian disease, and parturition (birth) abnormalities. Epigenetic analysis of the F1, F2 and F3 generation sperm identified differential DNA methylation regions (DMRs). A number of DMR associated genes were identified and previously shown to be involved in pathologies. Therefore, we propose glyphosate can induce the transgenerational inheritance of disease and germline (e.g. sperm) epimutations. Observations suggest the generational toxicology of glyphosate needs to be considered in the disease etiology of future generations.

Passing experiences on to future generations: endocrine disruptors and transgenerational inheritance of epimutations in brain and sperm

ABSTRACTAll animals have body burdens of polychlorinated biphenyls (PCBs) despite their ban decades ago. These and modern endocrine-disrupting chemicals (EDCs) such as the fungicide vinclozolin (VIN) perturb hormone signaling and lead to dysfunctions following prenatal exposures. Beyond direct exposures, transgenerational disease phenotypes can persist for multiple generations without subsequent exposure. The mechanisms of action of these EDCs differ: VIN is anti-androgenic while the PCB mixture Aroclor 1221 (A1221) is weakly estrogenic. Based on limited evidence for the inheritance of epimutations in germline, we measured DNA methylation in brain and sperm of rats. Pregnant dams were exposed from day 8–18 of gestation to low dosages of VIN, A1221, or the vehicle. To produce paternal lineages, exposed F1 males were bred with untreated females, creating the F2 and subsequently F3 generations. In adult F1 and F3 males, mature sperm was collected, and brain nuclei involved in anxiety and social behaviors (CA3 of the hippocampus; central amygdala) were selected for assays of epimutations in CpG islands using reduced representation bisulfite sequencing. In F1 sperm, VIN and PCBs induced differential methylation in 215 and 284 CpG islands, respectively, compared to vehicle. The majority of effects were associated with hypermethylation. Fewer epimutations were detected in the brain. A subset of differentially methylated regions were retained from the F1 to the F3 generation, suggesting a common mechanism of EDC and germline epigenome interaction. Thus, EDCs can cause heritable epimutations in the sperm that may embody the future phenotype of brain-behavior disorders caused by direct or transgenerational exposures.

Transgenerational transmission of hedonic behaviors and metabolic phenotypes induced by maternal overnutrition

Maternal overnutrition has been associated with increased susceptibility to develop obesity and neurological disorders later in life. Most epidemiological as well as experimental studies have focused on the metabolic consequences across generations following an early developmental nutritional insult. Recently, it has been shown that maternal high-fat diet (HFD) affects third-generation female body mass via the paternal lineage. We showed here that the offspring born to HFD ancestors displayed addictive-like behaviors as well as obesity and insulin resistance up to the third generation in the absence of any further exposure to HFD. These findings, implicate that the male germ line is a major player in transferring phenotypic traits. These behavioral and physiological alterations were paralleled by reduced striatal dopamine levels and increased dopamine 2 receptor density. Interestingly, by the third generation a clear gender segregation emerged, where females showed addictive-like behaviors while male HFD offspring showed an obesogenic phenotype. However, methylome profiling of F1 and F2 sperm revealed no significant difference between the offspring groups, suggesting that the sperm methylome might not be the major carrier for the transmission of the phenotypes observed in our mouse model. Together, our study for the first time demonstrates that maternal HFD insult causes sustained alterations of the mesolimbic dopaminergic system suggestive of a predisposition to develop obesity and addictive-like behaviors across multiple generations.

Intergenerational impact of paternal lifetime exposures to both folic acid deficiency and supplementation on reproductive outcomes and imprinted gene methylation.

Do paternal exposures to folic acid deficient (FD), and/or folic acid supplemented (FS) diets, throughout germ cell development adversely affect male germ cells and consequently offspring health outcomes? Male mice exposed over their lifetimes to both FD and FS diets showed decreased sperm counts and altered imprinted gene methylation with evidence of transmission of adverse effects to the offspring, including increased postnatal-preweaning mortality and variability in imprinted gene methylation. There is increasing evidence that disruptions in male germ cell epigenetic reprogramming are associated with offspring abnormalities and intergenerational disease. The fetal period is the critical time of DNA methylation pattern acquisition for developing male germ cells and an adequate supply of methyl donors is required. In addition, DNA methylation patterns continue to be remodeled during postnatal spermatogenesis. Previous studies have shown that lifetime (prenatal and postnatal) folic acid deficiency can alter the sperm epigenome and increase the incidence of fetal morphological abnormalities. Female BALB/c mice (F0) were placed on one of four amino-acid defined diets for 4 weeks before pregnancy and throughout pregnancy and lactation: folic acid control (Ctrl; 2 mg/kg), 7-fold folic acid deficient (7FD; 0.3 mg/kg), 10-fold high FS (10FS, 20 mg/kg) or 20-fold high FS (20FS, 40 mg/kg) diets. F1 males were weaned to their respective prenatal diets to allow for diet exposure during all windows of germline epigenetic reprogramming: the erasure, re-establishment and maintenance phases. F0 females were mated with chow-fed males to produce F1 litters whose germ cells were exposed to the diets throughout embryonic development. F1 males were subsequently mated with chow-fed female mice. Two F2 litters, unexposed to the experimental diets, were generated from each F1 male; one litter was collected at embryonic day (E)18.5 and one delivered and followed postnatally. DNA methylation at a global level and at the differentially methylated regions of imprinted genes (H19, Imprinted Maternally Expressed Transcript (Non-Protein Coding)-H19, Small Nuclear Ribonucleoprotein Polypeptide N-Snrpn, KCNQ1 Opposite Strand/Antisense Transcript 1 (Non-Protein Coding)-Kcnq1ot1, Paternally Expressed Gene 1-Peg1 and Paternally Expressed Gene 3-Peg3) was assessed by luminometric methylation analysis and bisulfite pyrosequencing, respectively, in F1 sperm, F2 E18.5 placenta and F2 E18.5 brain cortex. F1 males exhibited lower sperm counts following lifetime exposure to both folic acid deficiency and the highest dose of folic acid supplementation (20FS), (both P < 0.05). Post-implantation losses were increased amongst F2 E18.5 day litters from 20FS exposed F1 males (P < 0.05). F2 litters derived from both 7FD and 20FS exposed F1 males had significantly higher postnatal-preweaning pup death (both P < 0.05). Sperm from 10FS exposed males had increased variance in methylation across imprinted gene H19, P < 0.05; increased variance at a few sites within H19 was also found for the 7FD and 20FS groups (P < 0.05). While the 20FS diet resulted in inter-individual alterations in methylation across the imprinted genes Snrpn and Peg3 in F2 E18.5 placenta, ≥50% of individual sites tested in Peg1 and/or Peg3 were affected in the 7FD and 10FS groups. Inter-individual alterations in Peg1 methylation were found in F2 E18.5 day 10FS group brain cortex (P < 0.05). Not applicable. The cause of the increase in postnatal-preweaning mortality was not investigated post-mortem. Further studies are required to understand the mechanisms underlying the adverse effects of folic acid deficiency and supplementation on developing male germ cells. Genome-wide DNA and histone methylome studies as well as gene expression studies are required to better understand the links between folic acid exposures, an altered germ cell epigenome and offspring outcomes. The findings of this study provide further support for paternally transmitted environmental effects. The results indicate that both folic acid deficiency and high dose supplementation can be detrimental to germ cell development and reproductive fitness, in part by altering DNA methylation in sperm. This study was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR #89944). The authors declare they have no conflicts of interest.

Transgenerational inheritance of heart disorders caused by paternal bisphenol A exposure

Bisphenol A (BPA) is an endocrine disruptor used in manufacturing of plastic devices, resulting in an ubiquitous presence in the environment linked to human infertility, obesity or cardiovascular diseases. Both transcriptome and epigenome modifications lie behind these disorders that might be inherited transgenerationally when affecting germline. To assess potential effects of paternal exposure on offspring development, adult zebrafish males were exposed to BPA during spermatogenesis and mated with non-treated females. Results showed an increase in the rate of heart failures of progeny up to the F2, as well as downregulation of 5 genes involved in cardiac development in F1 embryos. Moreover, BPA causes a decrease in F0 and F1 sperm remnant mRNAs related to early development. Results reveal a paternal inheritance of changes in the insulin signaling pathway due to downregulation of insulin receptor β mRNAs, suggesting a link between BPA male exposure and disruption of cardiogenesis in forthcoming generations.

F0 maternal BPA exposure induced glucose intolerance of F2 generation through DNA methylation change in Gck

BPA, a common environmental endocrine disruptor, has been reported to induce epigenetic changes and disrupt glucose homeostasis in F1 offspring through maternal exposure. However, no studies have examined whether maternal BPA exposure can exert multigenerational effects of glucose metabolic disorder on F2 generation through the altered epigenetic information. The aim of the current study was to investigate whether BPA exposure can disrupt glucose homeostasis in F2 offspring and the underlying epigenetic mechanism. In the present study, F0 pregnant dams were orally administered at a daily dose of 40μg/kg body weight during gestation and lactation. The F1 and F2 generations were obtained and not exposed to BPA anymore. The glucose and insulin tolerance tests were carried out to evaluate the glucose homeostasis level. The relative hormone level and the relative gene expression were also examined. F2 generation was found to exhibited glucose intolerance and insulin resistance in ipGTT and ipITT, as well as the downregulation of glucokinase (Gck) gene in liver. DNA methylation pattern of Gck promoter in the F2 generation of hepatic tissue and F1 generation of sperm was then performed. The Gck promoter in F2 hepatic tissue became completely methylated in the all CpG sites compared with five unmethylated sites in controls. In the F1 sperm, the global DNA methylation was decreased. However, there is only CpG site −314 was differently methylated between BPA and controls in sperm. In conclusion, F0 maternal BPA exposure during gestation and lactation can induce impaired glucose homeostasis in the F2 offspring through the transmission of sperm. The underlying epigenetic modifications in the sperm of F1 generation remain to be further elucidated