Explore the vast range of titles available.

Ancestral environmental exposures have previously been shown to promote epigenetic transgenerational inheritance and influence all aspects of an individual's life history. In addition, proximate life events such as chronic stress have documented effects on the development of physiological, neural, and behavioral phenotypes in adulthood. We used a systems biology approach to investigate in male rats the interaction of the ancestral modifications carried transgenerationally in the germ line and the proximate modifications involving chronic restraint stress during adolescence. We find that a single exposure to a common-use fungicide (vinclozolin) three generations removed alters the physiology, behavior, metabolic activity, and transcriptome in discrete brain nuclei in descendant males, causing them to respond differently to chronic restraint stress. This alteration of baseline brain development promotes a change in neural genomic activity that correlates with changes in physiology and behavior, revealing the interaction of genetics, environment, and epigenetic transgenerational inheritance in the shaping of the adult phenotype. This is an important demonstration in an animal that ancestral exposure to an environmental compound modifies how descendants of these progenitor individuals perceive and respond to a stress challenge experienced during their own life history.

All individuals are directly exposed to extant environmental endocrine-disrupting chemicals (EDCs), and indirectly exposed through transgenerational inheritance from our ancestors. Although direct and ancestral exposures can each lead to deficits in behaviors, their interactions are not known. Here we focused on social behaviors based on evidence of their vulnerability to direct or ancestral exposures, together with their importance in reproduction and survival of a species. Using a novel “two hits, three generations apart” experimental rat model, we investigated interactions of two classes of EDCs across six generations. PCBs (a weakly estrogenic mixture Aroclor 1221, 1 mg/kg), Vinclozolin (antiandrogenic, 1 mg/kg) or vehicle (6% DMSO in sesame oil) were administered to pregnant rat dams (F0) to directly expose the F1 generation, with subsequent breeding through paternal or maternal lines. A second EDC hit was given to F3 dams, thereby exposing the F4 generation, with breeding through the F6 generation. Approximately 1200 male and female rats from F1, F3, F4 and F6 generations were run through tests of sociability and social novelty as indices of social preference. We leveraged machine learning using DeepLabCut to analyze nuanced social behaviors such as nose touching with accuracy similar to a human scorer. Surprisingly, social behaviors were affected in ancestrally exposed but not directly exposed individuals, particularly females from a paternally exposed breeding lineage. Effects varied by EDC: Vinclozolin affected aspects of behavior in the F3 generation while PCBs affected both the F3 and F6 generations. Taken together, our data suggest that specific aspects of behavior are particularly vulnerable to heritable ancestral exposure of EDC contamination, that there are sex differences, and that lineage is a key factor in transgenerational outcomes.

Endocrine-disrupting chemicals (EDCs) are increasingly associated with disease prevalence in exposed individuals. A classic example is the insecticide dichlorodiphenyltrichloroethane (DDT), brought to attention by Rachel Carson in her seminal 1962 book Silent Spring, DDT use in the United States was widespread from the 1940s until its ban in the 1970s. Nevertheless, DDT is still used for malaria control in some countries. In this issue of Environmental Health Perspectives, Lismer et al. provide compelling evidence on the lasting imprint of DDT--that it causes widespread disruption of the sperm epigenome and that these changes are likely to be heritable. Men from the VhaVenda tribe in South Africa are directly exposed to DDT through the spraying of their homes to eliminate insects carrying malaria and have high body burdens of the pesticide. Greenlandic Inuit men are exposed via the bioaccumulation of DDT in the food chain through consumption of marine mammals.

Increasing evidence supports an association of endocrine-disrupting chemical (EDC) exposures with adverse biological effects in humans and wildlife. Recent studies reveal that health consequences of environmental exposures may persist or emerge across generations. This creates a dual conundrum: that we are exposed to contemporary environmental chemicals overlaid upon the inheritance of our ancestors' exposure profiles. Even when legacy EDCs are phased out, they may remain relevant due to persistence in the environment together with intergenerational inheritance of their adverse biological effects. Thus, we all possess a body burden of legacy contaminants, and we are also increasingly exposed to new generations of EDCs. We assessed the effects of direct and ancestral exposures to EDCs across six generations on anxiety-like behaviors in male rats using our \"two hits, three generations apart\" multigenerational EDC exposure experimental model. We investigated two classes of EDCs with distinct hormonal actions and historical use-the weakly estrogenic polychlorinated biphenyl (PCB) mixture Aroclor 1221 (A1221) and the anti-androgenic fungicide vinclozolin (VIN)-in both the maternal and paternal line. We also determined if a hormonal mechanism drives these effects across generations. Rats were gestationally exposed to A1221, VIN, or vehicle [dimethyl sulfoxide (DMSO)] in the F1 generation. Three generations later, the F4 generation was given the same or a different exposure. Anxiety-like behavior was measured in the open field test, light:dark box, and elevated plus maze across generations. Serum was collected at the end of the experiment, and concentrations of estradiol and corticosterone were analyzed. Although direct exposure did not affect behavior in F1 males, ancestral exposure to VIN decreased anxiety-like behavior in the F3 paternal line compared to vehicle. In the F4 paternal line, ancestral A1221 followed by direct exposure to VIN increased anxiety-like behavior compared to controls. In the F6 maternal line, relative to vehicle, the double ancestral hits of A1221/VIN decreased anxiety-like behavior. Serum hormones weakly predicted behavioral changes in the F4 paternal line and were modestly affected in the F4 and F6 maternal lines. Our data suggest that anxiety-like behavioral phenotypes emerge transgenerationally in male rats in response to EDC exposure and that multiple hits of either the same or a different EDC can increase the impact in a lineage-specific manner. https://doi.org/10.1289/EHP15684.

Endocrine-disrupting chemicals (EDCs) are exogenous chemical compounds that interfere with the normal function of the endocrine system and are linked to direct and inherited adverse effects in both humans and wildlife. Legacy EDCs such as polychlorinated biphenyls (PCBs) are no longer used yet remain detectable in biological specimens around the world; concurrently, we are exposed to newer EDCs like the fungicide vinclozolin (VIN). This combination of individuals' direct environmental chemical exposures and any heritable changes caused by their ancestors' chemical exposures leads to a layered pattern of both direct and ancestrally inherited exposures that might have cumulative effects over generations. We assessed consequences of both direct and ancestral exposure to EDCs over six generations, examining anxiety-like behaviors in maternal and paternal lines of female rats. We used the \"two hits, three generations apart\" multigenerational exposure model to explore how two distinct EDCs-the weakly estrogenic PCB mixture Aroclor 1221 (A1221) and the antiandrogenic VIN-interact on behavior across generations. We also explored serum hormones as a potential mechanism. Rats were prenatally exposed to A1221, VIN, or vehicle (DMSO) in the F1 generation, and a second exposure (same or different) was administered to the F4 generation. Anxiety-like behavior was measured in the Open Field test, Light:Dark box, and Elevated Plus Maze in the F1, F3, F4, and F6 generations. Serum concentrations of estradiol and corticosterone were analyzed. Behavioral effects were not detectable in the F1 generation but emerged and became more robust across generations. Rats with ancestral VIN exposure demonstrated less anxiety-like behavior in the F3 paternal line in comparison with controls. Rats exposed to ancestral then prenatal A1221/VIN and VIN/A1221 had more anxiety-like behavior in the F4 maternal line, and those with two ancestral hits of VIN/VIN had more anxiety in the F6 paternal line, in comparison with controls. Our findings suggest that anxiety-like behavioral phenotypes can manifest in rats following germline exposure to EDCs and that subsequent exposures across generations can intensify these effects in a lineage-dependent manner. https://doi.org/10.1289/EHP15621.

Disclosure: R. Gillette: None. M. Aguirre: None. K. Bell: None. Y. Nadeem: None. L. Thompson: None. A.C. Gore: None. Extracellular vesicles (EVs) transfer a complex repertoire of small non-coding RNAs (sncRNA) to sperm as they transit the caput of the epididymis. These molecules are essential for the functional competence of sperm and are directly influenced by environmental factors such as stress, diet, and drug use. We hypothesized that prenatal exposure to EDCs would alter the sncRNA contents of caput extracellular vesicles, and that an exposure to a previously established contextual fear cue would exacerbate this effect. Pregnant dams were fed Nilla Wafers with either 3% DMSO (vehicle control) or Aroclor 1221 (1 mg/kg; a weakly estrogenic EDC) from embryonic day (E)8 until E18 and post-natal day (PND)1 to P21. In adulthood male rat offspring were subjected to a contextual fear conditioning paradigm in which each rat was exposed to an odorant paired with a shock (four 500 ms shocks at 0.7 mA) for three consecutive days. Two weeks later, all rats were returned to the conditioning chambers and exposed to the odorant for 5 minutes (no shocks). The next day, male rats were euthanized and the epididymis was dissected. EVs and total RNA isolated from the caput portion were sequenced on a NovaSeq 6000 SP in a single-end 75 bp format. Reads were checked for quality, trimmed for adapter sequences, aligned to the rat genome, and assigned an annotation designation across 10 different categories of sncRNA. We found that the majority of caput EV sncRNA were either tRNA fragments or piRNA and that unlike mice, miRNA accounted for a small portion of the total reads. The third largest category of aligned reads was in intergenic space and not associated with canonical small non-coding (snc) RNA loci. In-depth investigation determined these reads (∼19 nt) aligned strictly within the boundaries of known CpG islands (CpGi), which have not previously been reported to express any form of sncRNA. Re-exposure to a fear-associated scent 24 hours prior to sample collection substantially reduced the proportion tRNAs in EVs, which were supplanted by an increase in piRNA, CpGi sRNA, and lncRNA. This effect was exacerbated by prenatal EDC exposure and was particularly prevalent in the lncRNA and CpGi sRNA categories. Taken together our data show that the small RNA contents of caput epididymosomes of male rats is substantially different from previous observations reported in mice. We demonstrate that exposure to a contextual fear cue is a powerful influence on sncRNA composition of caput extracellular vesicles and that it is uniquely altered by prenatal EDC exposure. Finally, we provide evidence for what we believe may be a novel category of sncRNA. Presentation: Saturday, June 17, 2023

This study investigates the differential gene expression in an immortalized cell line of mouse skeletal myoblasts (C2C12)-derived myotube cells subjected to hyperthermia (40°C) with and without insulin treatment to elucidate the impact of thermal stress on skeletal muscle physiology. Hyperthermia, which occurs during intense physical activity or environmental heat exposure, is known to challenge muscle homeostasis and influence metabolic function. mRNA sequencing revealed that hyperthermia robustly altered gene expression-upregulating key genes involved in glycolysis, oxidative phosphorylation, heat shock response, and apoptosis. These changes are suggestive of an elevated metabolic state and enhanced cellular stress; however, these results remain preliminary without complementary protein or metabolic assays. Notably, insulin treatment moderated many of the hyperthermia-induced transcriptional alterations, particularly affecting genes linked to glucose uptake and metabolism. Together, these findings provide hypothesis-generating insights into the interplay between thermal stress and insulin signaling in C2C12 myotubes, and they underscore potential targets for future mechanistic studies.

Extracellular vesicles (EVs) released from the epididymal epithelium, referred to as epididymosomes, impart functional competence on sperm as they transit the epididymis by merging with sperm and releasing a complex repertoire of molecules. The cargo of epididymal EVs includes small noncoding RNAs (sncRNAs) that are modified by external factors such as stress, nutrition, and drug use, which are delivered to sperm and affect offspring development and outcomes. The rat is an important translational model system for many fields including reproduction and transgenerational epigenetics, but there is little work on rat epididymosomes and their sncRNA cargo. To fill this gap in knowledge, in the current study we performed a comprehensive characterization of an epididymal EV preparation that we refer to as extracellular particles (EPs) because of the potential presence of other non-vesicular extracellular particles along with the EVs. EPs were collected from the caput epididymis, isolated, subjected to verification of the presence of EVs, and sncRNAs were sequenced. We considered nearly all known categories of small RNA and their fragment sub-types and in the process identified a subset that did not strictly fit the definition of any known category. These unique small RNAs are expressed strictly from within the boundaries of CpG islands and have a distinct 19-nt length. Their functional significance remains unknown, but they have characteristics of RNA fragments that can associate with the Argonaute/PIWI family of proteins and therefore could have regulatory function via RNA induced silencing or de novo DNA methylation.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Added new data shown in Figure 2 for electron microscopy and cryo-EM of extracellular particles. This work was done by Dana L Sheinhaus who was now added as a co-author.

Peripubertal exposure to the phthalate metabolite mono-(2-ethylhexyl) phthalate (MEHP) in rodents causes testicular inflammation, spermatocyte apoptosis, and disruption of the blood-testis barrier. The MEHP-induced inflammation response includes an infiltration of macrophages and neutrophils to the testes, although the cause and purpose of this response is unknown. Recently, a population of testicular macrophages phenotypically distinct from those resident in the interstitium was described in mice. Testicular peritubular macrophages aggregate near the spermatogonial stem cell niche and are believed to stimulate their differentiation. We hypothesized that if testicular peritubular macrophages do indeed stimulate spermatogonial differentiation, MEHP exposure would result in an increase of peritubular macrophages to stimulate the replacement of lost spermatocytes. Male rats were exposed to 700 mg/kg MEHP or corn oil (vehicle control) via oral gavage at PND 28 and euthanized at 48 hours, 1 week, or 2 weeks later. Tubules were stained with immunofluorescent markers for macrophages and undifferentiated spermatogonia. Peritubular macrophages were observed in rat testis similar to those previously described in mice: MHC-II+ cells on the surface of seminiferous tubules with heterogeneous morphology. Quantification of MHC-II+ cells revealed that, unlike in the mouse, their numbers did not increase through puberty. MEHP increased macrophage presence by six-fold 48-hours after exposure and remained elevated by two-fold two weeks after exposure. An increase of differentiating spermatogonia occurred two weeks after MEHP exposure. Taken together, our results suggest that peritubular macrophages play a crucial role in the testis response to acute injury and the subsequent recovery of spermatogenesis.

Hormonal contraceptives (HCs) are commonly used among reproductive aged women and alter the physiological state of the user by interfering with endogenous hormone concentrations and their actions on the reproductive tract. As hormones such as estradiol and progesterone modulate the incidence of substance abuse disorders in women, it is important to consider the influence HCs have on the female brain and behavior. This experiment explores how female sex steroid hormonal states associated with the rat estrous cycle, and modulating those states with HCs, influences measures of drug preference and responsivity. First, rats underwent food-light Pavlovian conditioning to measure conditioned orienting, a known predictor of amphetamine (AMP) place preference. Then, rats were conditioned and tested for AMP place preference with either an HC-implant or during estrous cycle stages associated with different ovarian hormone levels (i.e., proestrus (P) or metestrus/diestrus (M/D)) while recording ultrasonic vocalizations (USVs) as an index of hedonic responsivity. Because of dopamine's (DA) role in modulation of AMP actions, DA cell activity and availability were examined using tyrosine hydroxylase and FOS immunohistochemistry after final AMP challenge. Conditioned orienting did not differ between cycling and HC-implanted. P rats emitted more USVs during conditioning, showed higher AMP place preference throughout testing, and had higher DA cell activity in the substantia nigra compared to M/D and HC-implanted rats. Sex steroid hormone serum concentration and uterine horn thickness predicted some but not all of these measures. This experiment suggests ovarian hormones affect drug preference and responsivity, while providing novel insight into how hormone-altering contraceptives may reduce these measures. Competing Interest Statement The authors have declared no competing interest.