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Opioid use disorder (OUD) is a neuropsychological disease that has a devastating impact on public health. Substantial individual differences in vulnerability exist, the neurobiological substrates of which remain unclear. To address this question, we investigated genome-wide gene transcription (RNA-seq) and chromatin accessibility (ATAC-seq) in the medial prefrontal cortex (mPFC) of male and female rats exhibiting differential vulnerability in behavioral paradigms modeling different phases of OUD: Withdrawal-Induced Anhedonia (WIA), Demand, and Reinstatement. Ingenuity Pathway Analysis (IPA) of RNA-seq revealed greater changes in canonical pathways in Resilient (vs. Saline) rats in comparison to Vulnerable (vs. Saline) rats across 3 paradigms, suggesting brain adaptations that might contribute to resilience to OUD across its trajectory. Analyses of gene networks and upstream regulators implicated processes involved in oligodendrocyte maturation and myelination in WIA, neuroinflammation in Demand, and metabolism in Reinstatement. Motif analysis of ATAC-seq showed changes in chromatin accessibility to a small set of transcription factor (TF) binding sites as a function either of opioid exposure (i.e., morphine versus saline) generally or of individual vulnerability specifically. Some of these were shared across the 3 paradigms and others were unique to each. In conclusion, we have identified changes in biological pathways, TFs, and their binding motifs that vary with paradigm and OUD vulnerability. These findings point to the involvement of distinct transcriptional and epigenetic mechanisms in response to opioid exposure, vulnerability to OUD, and different stages of the disorder.

The complex nature of the transcriptional networks underlying addictive behaviors suggests intricate cooperation between diverse gene regulation mechanisms that go beyond canonical activity-dependent pathways. Here, we implicate in this process a nuclear receptor transcription factor, retinoid X receptor alpha (RXRα), which we initially identified bioinformatically as associated with addiction-like behaviors. In the nucleus accumbens (NAc) of male and female mice, we show that although its own expression remains unaltered after cocaine exposure, RXRα controls plasticity- and addiction-relevant transcriptional programs in both dopamine receptor D1- and D2-expressing medium spiny neurons, which in turn modulate intrinsic excitability and synaptic activity of these NAc cell types. Behaviorally, bidirectional viral and pharmacological manipulation of RXRα regulates drug reward sensitivity in both non-operant and operant paradigms. Together, this study demonstrates a key role for NAc RXRα in promoting drug addiction and paves the way for future studies of rexinoid signaling in psychiatric disease states., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Standardizing RNA quality is key to interpreting RNA-seq data as a compromised sample can mask the underlying biology. The challenge remains when evaluating RNA quality in samples with high RNA fragmentation. For example, programmed fragmentation and cytoplasmic expulsion, integral to sperm maturation, is a prime example of the complexities of interpreting RNA-seq data, given that fragmentation can be random and\or targeted. To meet this challenge, we developed an algorithm that accurately measures RNA quality in samples with high fragmentation, such as spermatozoa. The integrity of 1,000 previously identified abundant sperm transcripts were independently visualized and evaluated using the Transcript Integrity Index (TII) algorithm to identify intact transcripts. Full-length transcripts from visual and the TII algorithm were evaluated for testis preference in humans using the GTEx tissues database. Samples were then filtered by the Interquartile Range (IQR), identifying those in which the greatest number of transcripts failed to pass the visual or TII thresholds. Transcript lists were overlapped, forming the set of intact transcripts used as TII standards. Each sample was re-evaluated as a function of this TII set of intact transcripts, with poor quality samples identified as those failing in the largest number of transcripts. While ontologically enriched in roles related to spermatogenesis and/or fertilization, samples did not segregate based on birth outcome. The TII algorithm proved an effective means to identify samples of similar quality from sperm, a cell type enriched in biologically fragmented RNAs. The algorithm should facilitate other studies using samples compromised by high levels of RNA fragmentation, such as Formalin-Fixed Paraffin-Embedded samples. Requisite to assessing male health, TII provides a solution to the long-sought-after standard that identifies samples of similar quality.

Background: The onset and persistence of addiction phenotypes are, in part, mediated by transcriptional mechanisms in the brain that affect gene expression and, subsequently, neural circuitry. ΔFosB is a transcription factor that accumulates in the nucleus accumbens (NAc)-a brain region responsible for coordinating reward and motivation-after exposure to virtually every known rewarding substance, including cocaine and opioids. ΔFosB has also been shown to directly control gene transcription and behavior downstream of both cocaine and opioid exposure, but with potentially different roles in D1 and D2 medium spiny neurons (MSNs) in NAc., Methods: To clarify MSN subtype-specific roles for ΔFosB and investigate how these coordinate the actions of distinct classes of addictive drugs in NAc, we developed a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-based epigenome editing tool to induce endogenous ΔFosB expression in vivo in the absence of drug exposure. After inducing ΔFosB in D1- or D2-MSNs or both, we performed RNA sequencing on bulk male and female NAc tissue (n = 6-8/group)., Results: We found that ΔFosB induction elicits distinct transcriptional profiles in NAc by MSN subtype and by sex, establishing for the first time that ΔFosB mediates different transcriptional effects in males versus females. We also demonstrated that changes in D1-MSNs, but not those in D2-MSNs or both, significantly recapitulate changes in gene expression induced by cocaine self-administration., Conclusions: Together, these findings demonstrate the efficacy of a novel molecular tool for studying cell type-specific transcriptional mechanisms and shed new light on the activity of ΔFosB, a critical transcriptional regulator of drug addiction., (Copyright © 2021 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Depression is a common disorder that affects women at twice the rate of men. Here, we report that long non-coding RNAs (lncRNAs), a recently discovered class of regulatory transcripts, represent about one-third of the differentially expressed genes in the brains of depressed humans and display complex region- and sex-specific patterns of regulation. We identified the primate-specific, neuronal-enriched gene LINC00473 as downregulated in prefrontal cortex (PFC) of depressed females but not males. Using viral-mediated gene transfer to express LINC00473 in adult mouse PFC neurons, we mirrored the human sex-specific phenotype by inducing stress resilience solely in female mice. This sex-specific phenotype was accompanied by changes in synaptic function and gene expression selectively in female mice and, along with studies of human neuron-like cells in culture, implicates LINC00473 as a CREB effector. Together, our studies identify LINC00473 as a female-specific driver of stress resilience that is aberrant in female depression., Competing Interests: Declaration of Interests The authors declare no competing interests, (Copyright © 2020 Elsevier Inc. All rights reserved.)

Recent studies have shown that tissue-specific transcriptomes contain multiple types of RNAs that are transcribed from intronic and intergenic sequences. The current study presents a tool for the discovery of transcribed, unannotated sequence elements from RNA-seq libraries. This RNA Element (RE) discovery algorithm (REDa) was applied to a spectrum of tissues and cells representing germline, embryonic, and somatic tissues and examined as a function of differentiation through the first set of cell divisions of human development. This highlighted extensive transcription throughout the genome, yielding previously unidentified human spermatogenic RNAs. Both exonic and novel X-chromosome REs were subject to robust meiotic sex chromosome inactivation, although an extensive de-repression occurred in the post-meiotic stages of spermatogenesis. Surprisingly, 2.4% of the 10,395 X chromosome exonic REs were present in mature sperm. Transcribed genomic repetitive sequences, including simple centromeric repeats, HERVE and HSAT1, were also shown to be associated with RE expression during spermatogenesis. These results suggest that pervasive intergenic repetitive sequence expression during human spermatogenesis may play a role in regulating chromatin dynamics. Repetitive REs switching repeat classes during differentiation upon fertilization and embryonic genome activation was evident., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Male fertility and spermatogenesis are directly linked to the Sertoli cell's ability to produce factors associated with germ cell development. Sertoli cells express receptors for FSH and testosterone, and are the major regulators of spermatogenesis. Recent studies report that regulatory RNA molecules, such as microRNAs (miRNAs), are able to modulate testicular function during spermatogenesis and that their altered expression may be involved in male infertility. miRNAs may play a role in the response to xenobiotics that have an adverse consequences to health. An important group of xenobiotic organic compounds with toxic potential are dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Experimental models of TCDD exposure in mice demonstrated that TCDD exposure causes low sperm count and delayed puberty. This study below examines the mechanism of TCDD's action in human Sertoli cells, through interrogating the expression profile of miRNAs and mRNAs, that enabled us to identify dysregulated molecular pathawys in Sertoli cell. 78 miRNAs presented altered expression, with positive regulation of 73 and negative regulation of 5 miRNAs when compared to the control group. Regarding gene expression profile, 51 genes were deregulated, of which 46 had positive regulation and 5 genes with negative regulation. Important pathways have been altered by the action of TCDD as AhR pathway, GPR68, FGF2 and LIF. This study has opened the door to new perspectives on the TCDD toxicity pathway as it affects Sertoli cells physiology that can ultimately lead to male infertility., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Study Question: Are preconception phthalate and phthalate replacements associated with sperm differentially methylated regions (DMRs) among men undergoing IVF?, Summary Answer: Ten phthalate metabolites were associated with 131 sperm DMRs that were enriched in genes related to growth and development, cell movement and cytoskeleton structure., What Is Known Already: Several phthalate compounds and their metabolites are known endocrine disrupting compounds and are pervasive environmental contaminants. Rodent studies report that prenatal phthalate exposures induce sperm DMRs, but the influence of preconception phthalate exposure on sperm DNA methylation in humans is unknown., Study Design, Size, Duration: An exploratory cross-sectional study with 48 male participants from the Sperm Environmental Epigenetics and Development Study (SEEDS)., Participants/materials, Setting, Methods: The first 48 couples provided a spot urine sample on the same day as semen sample procurement. Sperm DNA methylation was assessed with the HumanMethylation 450 K array. Seventeen urinary phthalate and 1,2-Cyclohexane dicarboxylic acid diisononyl ester (DINCH) metabolite concentrations were measured from spot urine samples. The A-clust algorithm was employed to identify co-regulated regions. DMRs associated with urinary metabolite concentrations were identified via linear models, corrected for false discovery rate (FDR)., Main Results and Role of Chance: Adjusting for age, BMI, and current smoking, 131 DMRs were associated with at least one urinary metabolite. Most sperm DMRs were associated with anti-androgenic metabolites, including mono(2-ethylhexyl) phthalate (MEHP, n = 83), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP, n = 16), mono-n-butyl phthalate (MBP, n = 22) and cyclohexane-1,2-dicarboxylic acid-monocarboxy isooctyl (MCOCH, n = 7). The DMRs were enriched in lincRNAs as well as in regions near coding regions. Functional analyses of DMRs revealed enrichment of genes related to growth and development as well as cellular function and maintenance. Finally, 13% of sperm DMRs were inversely associated with high quality blastocyst-stage embryos after IVF., Limitations, Reasons for Caution: Our modest sample size only included 48 males and additional larger studies are necessary to confirm our observed results. Non-differential misclassification of exposure is also a concern given the single spot urine collection., Wider Implications of the Findings: To our knowledge, this is the first study to report that preconception urinary phthalate metabolite concentrations are associated with sperm DNA methylation in humans. These results suggest that paternal adult environmental conditions may influence epigenetic reprogramming during spermatogenesis, and in turn, influence early-life development., Study Funding/competing Interest(s): This work was supported by grant K22-ES023085 from the National Institute of Environmental Health Sciences. The authors declare no competing interests., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Human populations are exposed to a wide spectrum of environmental contaminants, some of which are considered reproductive toxins. The influence of such toxins on the male reproductive system has been investigated extensively in animal models, while epidemiological studies seek to understand the effect of human exposures. The basic tenant of epidemiological studies in male human reproduction is to infer how one or more substances alter the hormonal profile, seminal characteristics, or both. Determining if a substance alters semen quality may not always provide the underlying mechanism. The mechanisms by which toxins may alter human sperm and semen quality are typically examined as a function of hormonal changes and cellular damage. The possibility that more subtle epigenetic alterations underlie some of the reproductive changes has, until recently, received little attention. In this review, we discuss the roles of epigenetics in human spermatogenesis, while considering the impact of reproductive toxicants on the epigenome.

Objective: To evaluate the effect of infertility and intracytoplasmic sperm injection (ICSI) on DNA methylation of offspring., Design: Microarray analysis of DNA methylation in archived neonatal bloodspots of in vitro fertilization (IVF)/ICSI-conceived children compared with controls born to fertile and infertile parents., Setting: Academic research laboratory., Patient(s): Neonatal blood spots of 137 newborns conceived spontaneously, through intrauterine insemination (IUI), or through ICSI using fresh or cryopreserved (frozen) embryo transfer., Intervention(s): None., Main Outcome Measure(s): The Illumina Infinium HumanMethylation450k BeadChip assay determined genome-wide DNA methylation. Methylation differences between conception groups were detected using a Bioconductor package, ChAMP, in conjunction with Adjacent Site Clustering (A-clustering)., Result(s): The methylation profiles of assisted reproductive technology and IUI newborns were dramatically different from those of naturally (in vivo) conceived newborns. Interestingly, the profiles of ICSI-frozen (FET) and IUI infants were strikingly similar, suggesting that cryopreservation may temper some of the epigenetic aberrations induced by IVF or ICSI. The DNA methylation changes associated with IVF/ICSI culture conditions and/or parental infertility were detected at metastable epialleles, suggesting a lasting impact on a child's epigenome., Conclusion(s): Both infertility and ICSI alter DNA methylation at specific genomic loci, an effect that is mitigated to some extent by FET. The impact of assisted reproductive technology and/or fertility status on metastable epialleles in humans was uncovered. This study provides an expanded set of loci for future investigations on IVF populations., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)