Molecular contribution to embryonic aneuploidy and karyotypic complexity in initial cleavage divisions of mammalian development.

Embryonic aneuploidy is highly complex, often leading to developmental arrest, implantation failure or spontaneous miscarriage in both natural and assisted reproduction. Despite our knowledge of mitotic mis-segregation in somatic cells, the molecular pathways regulating chromosome fidelity during the error-prone cleavage-stage of mammalian embryogenesis remain largely undefined. Using bovine embryos and live-cell fluorescent imaging, we observed frequent micro-/multi-nucleation of mis-segregated chromosomes in initial mitotic divisions that underwent unilateral inheritance, re-fused with the primary nucleus or formed a chromatin bridge with neighboring cells. A correlation between a lack of syngamy, multipolar divisions and asymmetric genome partitioning was also revealed, and single-cell DNA-seq showed propagation of primarily non-reciprocal mitotic errors. Depletion of the mitotic checkpoint protein BUB1B (also known as BUBR1) resulted in similarly abnormal nuclear structures and cell divisions, as well as chaotic aneuploidy and dysregulation of the kinase-substrate network that mediates mitotic progression, all before zygotic genome activation. This demonstrates that embryonic micronuclei sustain multiple fates, provides an explanation for blastomeres with uniparental origins, and substantiates defective checkpoints and likely other maternally derived factors as major contributors to the karyotypic complexity afflicting mammalian preimplantation development.

A systematic review and meta-analysis on the transcriptomic signatures in alcohol use disorder.

Currently available clinical treatments on alcohol use disorder (AUD) exhibit limited efficacy and new druggable targets are required. One promising approach to discover new molecular treatment targets involves the transcriptomic profiling of brain regions within the addiction neurocircuitry, utilizing animal models and postmortem brain tissue from deceased patients with AUD. Unfortunately, such studies suffer from large heterogeneity and small sample sizes. To address these limitations, we conducted a cross-species meta-analysis on transcriptome-wide data obtained from brain tissue of patients with AUD and animal models. We integrated 36 cross-species transcriptome-wide RNA-expression datasets with an alcohol-dependent phenotype vs. controls, following the PRISMA guidelines. In total, we meta-analyzed 964 samples - 502 samples from the prefrontal cortex (PFC), 282 nucleus accumbens (NAc) samples, and 180 from amygdala (AMY). The PFC had the highest number of differentially expressed genes (DEGs) across rodents, monkeys, and humans. Commonly dysregulated DEGs suggest conserved cross-species mechanisms for chronic alcohol consumption/AUD comprising MAPKs as well as STAT, IRF7, and TNF. Furthermore, we identified numerous unique gene sets that might contribute individually to these conserved mechanisms and also suggest novel molecular aspects of AUD. Validation of the transcriptomic alterations on the protein level revealed interesting targets for further investigation. Finally, we identified a combination of DEGs that are commonly regulated across different brain tissues as potential biomarkers for AUD. In summary, we provide a compendium of genes that are assessable via a shiny app, and describe signaling pathways, and physiological and cellular processes that are altered in AUD that require future studies for functional validation.

Transcriptional profiling of mucus production in rhesus macaque endocervical cells under hormonal regulation.

OBJECTIVE: Endocervical mucus production is a key regulator of fertility throughout the menstrual cycle. With cycle-dependent variability in mucus quality and quantity, cervical mucus can either facilitate or block sperm ascension into the upper female reproductive tract. This study seeks to identify genes involved in the hormonal regulation of mucus production, modification, and regulation through profiling the transcriptome of endocervical cells from the non-human primate, the rhesus macaque (Macaca mulatta). INTERVENTION: We treated differentiated primary endocervical cultures with estradiol (E2) and progesterone (P4) to mimic peri-ovulatory and luteal-phase hormonal changes. Using RNA-sequencing, we identified differential expression of gene pathways and mucus producing and modifying genes in cells treated with E2 compared to hormone-free conditions and E2 compared to E2-primed cells treated with P4. MAIN OUTCOME MEASURES: We pursued differential gene expression analysis on RNA-sequenced cells. Sequence validation was done using qPCR. RESULTS: Our study identified 158 genes that show significant differential expression in E2-only conditions compared to hormone-free control, and 250 genes that show significant differential expression in P4-treated conditions compared to E2-only conditions. From this list, we found hormone-induced changes in transcriptional profiles for genes across several classes of mucus production, including ion channels and enzymes involved in post-translational mucin modification that have not previously been described as hormonally regulated. CONCLUSION: Our study is the first to use an in vitro culture system to create an epithelial-cell specific transcriptome of the endocervix. As a result, our study identifies new genes and pathways altered by sex-steroids in cervical mucus production.

The combined impact of testosterone and Western-style diet on endometriosis severity and progression in rhesus macaques†.

Polycystic ovary syndrome (PCOS) is associated with irregular menstrual cycles, hyperandrogenemia, and obesity. It is currently accepted that women with PCOS are also at risk for endometriosis, but the effect of androgen and obesity on endometriosis has been underexplored. The goal of this study was to determine how testosterone (T) and an obesogenic diet impact the progression of endometriosis in a nonhuman primate (NHP) model. Female rhesus macaques were treated with T (serum levels approximately 1.35 ng/ml), Western-style diet (WSD; 36% of calories from fat compared to 16% in standard monkey chow) or the combination (T + WSD) at the time of menarche as part of a longitudinal study for ~7 years. Severity of endometriosis was determined based on American Society for Reproductive Medicine (ASRM) revised criteria, and staged 1-4. Stages 1 and 2 were associated with extent of abdominal adhesions, while stages 3 and 4 were associated with presence of chocolate cysts. The combined treatment of T + WSD resulted in earlier onset of endometriosis and more severe types associated with large chocolate cysts compared to all other treatments. There was a strong correlation between glucose clearance, homeostatic model assessment for insulin resistance (HOMA-IR), and total percentage of body fat with presence of cysts, indicating possible indirect contribution of hyperandrogenemia via metabolic dysfunction. An RNA-seq analysis of omental adipose tissue revealed significant impacts on a number of inflammatory signaling pathways. The interactions between obesity, hyperandrogenemia, and abdominal inflammation deserve additional investigation in NHP model species.

Single-cell sequencing of primate preimplantation embryos reveals chromosome elimination via cellular fragmentation and blastomere exclusion.

Aneuploidy that arises during meiosis and/or mitosis is a major contributor to early embryo loss. We previously showed that human preimplantation embryos encapsulate missegregated chromosomes into micronuclei while undergoing cellular fragmentation and that fragments can contain chromosomal material, but the source of this DNA was unknown. Here, we leveraged the use of a nonhuman primate model and single-cell DNA-sequencing (scDNA-seq) to examine the chromosomal content of 471 individual samples comprising 254 blastomeres, 42 polar bodies, and 175 cellular fragments from a large number (N = 50) of disassembled rhesus cleavage-stage embryos. Our analysis revealed that the aneuploidy and micronucleation frequency is conserved between humans and macaques, and that fragments encapsulate whole and/or partial chromosomes lost from blastomeres. Single-cell/fragment genotyping showed that these chromosome-containing cellular fragments (CCFs) can be maternally or paternally derived and display double-stranded DNA breaks. DNA breakage was further indicated by reciprocal subchromosomal losses/gains between blastomeres and large segmental errors primarily detected at the terminal ends of chromosomes. By combining time-lapse imaging with scDNA-seq, we determined that multipolar divisions at the zygote or two-cell stage were associated with CCFs and generated a random mixture of chromosomally normal and abnormal blastomeres with uniparental or biparental origins. Despite frequent chromosome missegregation at the cleavage-stage, we show that CCFs and nondividing aneuploid blastomeres showing extensive DNA damage are prevented from incorporation into blastocysts. These findings suggest that embryos respond to chromosomal errors by encapsulation into micronuclei, elimination via cellular fragmentation, and selection against highly aneuploid blastomeres to overcome chromosome instability during preimplantation development.

Effects of early daily alcohol exposure on placental function and fetal growth in a rhesus macaque model.

BACKGROUND: Prenatal alcohol exposure is the most common cause of birth defects and intellectual disabilities and can increase the risk of stillbirth and negatively impact fetal growth. OBJECTIVE: To determine the effect of early prenatal alcohol exposure on nonhuman primate placental function and fetal growth. We hypothesized that early chronic prenatal alcohol would alter placental perfusion and oxygen availability that adversely affects fetal growth. STUDY DESIGN: Rhesus macaques self-administered 1.5 g/kg/d of ethanol (n=12) or isocaloric maltose-dextrin (n=12) daily before conception through the first 60 days of gestation (term is approximately 168 days). All animals were serially imaged with Doppler ultrasound to measure fetal biometry, uterine artery volume blood flow, and placental volume blood flow. Following Doppler ultrasound, all animals underwent both blood oxygenation level-dependent magnetic resonance imaging to characterize placental blood oxygenation and dynamic contrast-enhanced magnetic resonance imaging to quantify maternal placental perfusion. Animals were delivered by cesarean delivery for placental collection and fetal necropsy at gestational days 85 (n=8), 110 (n=8), or 135 (n=8). Histologic and RNA-sequencing analyses were performed on collected placental tissue. RESULTS: Placental volume blood flow was decreased at all gestational time points in ethanol-exposed vs control animals, but most significantly at gestational day 110 by Doppler ultrasound (P<.05). A significant decrease in total volumetric blood flow occurred in ethanol-exposed vs control animals on dynamic contrast-enhanced magnetic resonance imaging at both gestation days 110 and 135 (P<.05); moreover, a global reduction in T2∗, high blood deoxyhemoglobin concentration, occurred throughout gestation (P<.05). Similarly, evidence of placental ischemic injury was notable by histologic analysis, which revealed a significant increase in microscopic infarctions in ethanol-exposed, not control, animals, largely present at middle to late gestation. Fetal biometry and weight were decreased in ethanol-exposed vs control animals, but the decrease was not significant. Analysis with RNA sequencing suggested the involvement of the inflammatory and extracellular matrix response pathways. CONCLUSION: Early chronic prenatal alcohol exposure significantly diminished placental perfusion at mid to late gestation and also significantly decreased the oxygen supply to the fetal vasculature throughout pregnancy, these findings were associated with the presence of microscopic placental infarctions in the nonhuman primate. Although placental adaptations may compensate for early environmental perturbations to fetal growth, placental blood flow and oxygenation were reduced, consistent with the evidence of placental ischemic injury.

Blocking the IL-1 receptor reduces cardiac transplant ischemia and reperfusion injury and mitigates CMV-accelerated chronic rejection.

Ischemia-reperfusion injury (IRI) is an important risk factor for accelerated cardiac allograft rejection and graft dysfunction . Utilizing a rat heart isogeneic transplant model, we identified inflammatory pathways involved in IRI in order to identify therapeutic targets involved in disease. Pathway analyses identified several relevant targets, including cytokine signaling by the IL-1 receptor (IL-1R) pathway and inflammasome activation. To investigate the role of IL-1R signaling pathways during IRI, we treated syngeneic cardiac transplant recipients at 1-hour posttransplant with Anakinra, a US Food and Drug Administration (FDA)-approved IL-1R antagonist; or parthenolide, a caspase-1 and nuclear factor kappa-light-chain-enhancer of activated B cells inhibitor that blocks IL-1ß maturation. Both Anakinra and parthenolide significantly reduced graft inflammation and cellular recruitment in the treated recipients relative to nontreated controls. Anakinra treatment administered at 1-hour posttransplant to recipients of cardiac allografts from CMV-infected donors significantly increased the time to rejection and reduced viral loads at rejection. Our results indicate that reducing IRI by blocking IL-1Rsignaling pathways with Anakinra or inflammasome activity with parthenolide provides a promising approach for extending survival of cardiac allografts from CMV-infected donors.

Transcriptome analysis during photostimulated recrudescence reveals distinct patterns of gene regulation in Siberian hamster ovaries†.

In Siberian hamsters, exposure to short days (SDs, 8 h light:16 h dark) reduces reproductive function centrally by decreasing gonadotropin secretion, whereas subsequent transfer of photoinhibited hamsters to stimulatory long days (LDs, 16 L:8 D) promotes follicle stimulating hormone (FSH) release inducing ovarian recrudescence. Although differences between SD and LD ovaries have been investigated, a systematic investigation of the ovarian transcriptome across photoperiod groups to identify potentially novel factors that contribute to photostimulated restoration of ovarian function had not been conducted. Hamsters were assigned to one of four photoperiod groups: LD to maintain ovarian cyclicity, SD to induce ovarian regression, or post transfer (PT), where females housed in SD for 14-weeks were transferred to LD for 2-days or 1-week to reflect photostimulated ovaries prior to (PTd2) and following (PTw1) the return of systemic FSH. Ovarian RNA was extracted to create RNA-sequencing libraries and short-read sequencing Illumina assays that mapped and quantified the ovarian transcriptomes (n = 4/group). Ovarian and uterine masses, plasma FSH, and numbers of antral follicles and corpora lutea decreased in SD as compared to LD ovaries (P < 0.05). When reads were aligned to the mouse genome, 18 548 genes were sufficiently quantified. Most of the differentially expressed genes noted between functional LD ovaries and regressed SD ovaries; however, five main expression patterns were identified across photoperiod groups. These results, generally corroborated by select protein immunostaining, provide a map of photoregulated ovary function and identify novel genes that may contribute to the photostimulated resumption of ovarian activity.

Comprehensive Molecular Characterization of Papillary Renal-Cell Carcinoma.

BACKGROUND: Papillary renal-cell carcinoma, which accounts for 15 to 20% of renal-cell carcinomas, is a heterogeneous disease that consists of various types of renal cancer, including tumors with indolent, multifocal presentation and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal-cell carcinoma, and no effective forms of therapy for advanced disease exist. METHODS: We performed comprehensive molecular characterization of 161 primary papillary renal-cell carcinomas, using whole-exome sequencing, copy-number analysis, messenger RNA and microRNA sequencing, DNA-methylation analysis, and proteomic analysis. RESULTS: Type 1 and type 2 papillary renal-cell carcinomas were shown to be different types of renal cancer characterized by specific genetic alterations, with type 2 further classified into three individual subgroups on the basis of molecular differences associated with patient survival. Type 1 tumors were associated with MET alterations, whereas type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2-antioxidant response element (ARE) pathway. A CpG island methylator phenotype (CIMP) was observed in a distinct subgroup of type 2 papillary renal-cell carcinomas that was characterized by poor survival and mutation of the gene encoding fumarate hydratase (FH). CONCLUSIONS: Type 1 and type 2 papillary renal-cell carcinomas were shown to be clinically and biologically distinct. Alterations in the MET pathway were associated with type 1, and activation of the NRF2-ARE pathway was associated with type 2; CDKN2A loss and CIMP in type 2 conveyed a poor prognosis. Furthermore, type 2 papillary renal-cell carcinoma consisted of at least three subtypes based on molecular and phenotypic features. (Funded by the National Institutes of Health.).

Long-lasting effect of obesity on skeletal muscle transcriptome.

BACKGROUND: Reduced physical activity and increased intake of calorically-dense diets are the main risk factors for obesity, glucose intolerance, and type 2 diabetes. Chronic overnutrition and hyperglycemia can alter gene expression, contributing to long-term obesity complications. While caloric restriction can reduce obesity and glucose intolerance, it is currently unknown whether it can effectively reprogram transcriptome to a pre-obesity level. The present study addressed this question by the preliminary examination of the transcriptional dynamics in skeletal muscle after exposure to overnutrition and following caloric restriction. RESULTS: Six male rhesus macaques of 12-13 years of age consumed a high-fat western-style diet for 6 months and then were calorically restricted for 4 months without exercise. Skeletal muscle biopsies were subjected to longitudinal gene expression analysis using next-generation whole-genome RNA sequencing. In spite of significant weight loss and normalized insulin sensitivity, the majority of WSD-induced (n = 457) and WSD-suppressed (n = 47) genes remained significantly dysregulated after caloric restriction (FDR ≤0.05). The MetacoreTM pathway analysis reveals that western-style diet induced the sustained activation of the transforming growth factor-ß gene network, associated with extracellular matrix remodeling, and the downregulation of genes involved in muscle structure development and nutritional processes. CONCLUSIONS: Western-style diet, in the absence of exercise, induced skeletal muscle transcriptional programing, which persisted even after insulin resistance and glucose intolerance were completely reversed with caloric restriction.