Depletion of SMARCB1 and BRD7, two SWI/SNF chromatin remodelling complex subunits, differentially impact porcine embryo development.

CONTEXT: SWI/SNF chromatin remodelling complexes are composed of multiple protein subunits and can be categorised into three sub-families, including the BAF, PBAF, and GBAF complexes. We hypothesised that depletion of SMARCB1 and BRD7, two subunits unique to different SWI/SNF sub-families, would differentially impact porcine embryo development. AIM: The aim of these experiments was to determine the developmental requirements of two SWI/SNF subunits, SMARCB1 and BRD7. METHODS: RNA interference assays were used to determine the developmental requirements of SMARCB1 and BRD7 in porcine embryos. KEY RESULTS: Our findings indicate that knockdown of SMARCB1 dramatically reduces embryo developmental potential, with few embryos developing beyond the pronuclear stage. The knockdown of BRD7 had a less severe impact on developmental potential. CONCLUSIONS: Our findings also demonstrate that knockdown of SMARCB1 alters the expression of NANOG and POU5F1 (also referred to as OCT4 ). IMPLICATIONS: These findings highlight the unique developmental requirements for sub-families of SWI/SNF chromatin remodelling complexes. This new knowledge will enable us to determine how discrete genomic loci are differentially remodelled during key points in embryo development.

Nuclear trafficking dynamics of Bromodomain-containing protein 7 (BRD7), a switch/sucrose non-fermentable (SWI/SNF) chromatin remodelling complex subunit, in porcine oocytes and cleavage-stage embryos.

In the work presented here, we investigated how bromodomain-containing protein 7 (BRD7), a subunit associated with switch/sucrose non-fermentable (SWI/SNF) chromatin remodelling complexes, is trafficked between cellular compartments during embryo development. SWI/SNF complexes are multi-subunit complexes that contain a core catalytic subunit (SWI/SNF related, Matrix associated, Actin dependent Regulator of Chromatin, subfamily A, member 4, or member 2; SMARCA4 or SMARCA2) and a collection of additional subunits that guide the complexes to their appropriate loci; BRD7 is one of these additional subunits. We hypothesised that BRD7 is exported from the nuclei of porcine oocytes and embryos in a Chromosome Region Maintenance 1 (CRM1)-dependent manner and imported into the nuclei using the karyopherin α/ß1 heterodimer. Porcine oocytes and embryos were treated with inhibitors of CRM1-mediated nuclear export and karyopherin α/ß1-mediated nuclear import to test this hypothesis. An RNA interference assay and a dominant negative overexpression assay were also performed to determine if karyopherin α7 serves a specific role in BRD7 trafficking. Our findings indicate that BRD7 shuttles between nuclear and cytoplasmic compartments during cleavage development. The shuttling of BRD7 indicates that it serves a unique role in remodelling chromatin during this developmental window.

Differential expression of key subunits of SWI/SNF chromatin remodeling complexes in porcine embryos derived in vitro or in vivo.

In vitro embryo production is an established method for both humans and animals, but is fraught with inferior development and health issues in offspring born after in vitro fertilization procedures. Analysis of epigenetic changes caused by exposure to in vitro conditions should shed light on potential sources of these phenotypes. Using immunocytochemistry, we investigated the localization and relative abundance of components associated with the SWI/SNF (Switch/Sucrose non-fermentable) chromatin-remodeling complex-including BAF155, BAF170, BAF180, BAF53A, BAF57, BAF60A, BAF45D, ARID1A, ARID1B, ARID2, SNF5, and BRD7-in oocytes and in in vitro-produced and in vivo-derived porcine embryos. Differences in the localization of BAF155, BAF170, BAF60A, and ARID1B among these sources indicate that improper timing of chromatin remodeling and cellular differentiation might occur in early preimplantation embryos produced and cultured in vitro.

CLOCK regulates mammary epithelial cell growth and differentiation.

Circadian clocks influence virtually all physiological processes, including lactation. Here, we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late-pregnant wild-type and ClockΔ19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand whether these effects were due, in part, to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with short hairpin RNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein (P < 0.05). HC11 lines carrying shClock had four-fold higher growth rates (P < 0.05), and the percentage of cells in G1 phase was significantly higher (90.1 ± 1.1% of shClock vs. 71.3 ± 3.6% of WT-HC11) following serum starvation. Quantitative-PCR (qPCR) analysis showed shClock had significant effects (P < 0.0001) on relative expression levels of Ccnd1, Wee1, and Tp63 qPCR analysis of the effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures and cultures treated 96 h with dexamethasone, insulin, and prolactin (differentiated) found levels were reduced by twofold and threefold, respectively (P < 0.05), in shClock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with shClock These data support how CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland.

Tissue-specific changes in molecular clocks during the transition from pregnancy to lactation in mice.

Circadian clocks regulate homeostasis and mediate responses to stressors. Lactation is one of the most energetically demanding periods of an adult female's life. Peripartum changes occur in almost every organ so the dam can support neonatal growth through milk production while homeostasis is maintained. How circadian clocks are involved in adaptation to lactation is currently unknown. The abundance and temporal pattern of core clock genes' expression were measured in suprachiasmatic nucleus, liver, and mammary from late pregnant and early lactation mice. Tissue-specific changes in molecular clocks occurred between physiological states. Amplitude and robustness of rhythms increased in suprachiasmatic nucleus and liver. Mammary rhythms of core molecular clock genes were suppressed. Attenuated rhythms appeared to be a physiological adaptation of mammary to lactation, because manipulation of timing of suckling resulting in significant differences in plasma prolactin and corticosterone had no effect on amplitude. Analysis of core clock proteins revealed that the stoichiometric relationship between positive (CLOCK) and negative (PER2) components remained 1:1 in liver but was increased to 4:1 in mammary during physiological transition. Induction of differentiation of mammary epithelial cell line HC11 with dexamethasone, insulin, and prolactin resulted in similar stoichiometric changes among positive and negative clock regulators, and prolactin induced phase shifts in HC11 Arntl expression rhythm. Data support that distinct mechanisms drive periparturient changes in mammary clock. Stoichiometric change in clock regulators occurs with gland differentiation. Suppression of mammary clock gene expression rhythms represents a physiological adaptation to suckling cues. Adaptations in mammary clock are likely needed in part to support suckling demands of neonates.

Identification of SWI/SNF Subcomplex GBAF Presence, Intra-Complex Interactions, and Transcriptional Dynamics during Early Porcine Development.

Understanding the complex interplay between genetics and environmental factors is vital for enhancing livestock production efficiency while safeguarding animal health. Despite extensive studies on production-specific genes in livestock, exploring how epigenetic mechanisms and heritable modifications govern animal growth and development remains an under-explored frontier with potential implications across all life stages. This study focuses on the GBAF chromatin remodeling complex and evaluates its presence during embryonic and fetal development in swine. Immunocytochemistry and co-immunoprecipitation techniques were employed to investigate the presence and interactions of GBAF subunits BRD9 and GLTSCR1 in porcine oocytes, preimplantation embryos, and cell lines, and transcriptional dynamics of GBAF subunits across these key developmental stages were analyzed using existing RNA-seq datasets. BRD9 and GLTSCR1 were identified across all represented stages, and an interaction between GLTSCR1 and BAF170 was shown in PTr2 and PFF cells. Our findings highlight the ubiquitous presence of GBAF in porcine early development and the potentially novel association between GLTSCR1 and BAF170 in swine. The transcriptional dynamics findings may suggest GBAF-specific contributions during key developmental events. This study contributes to the growing understanding of epigenetic regulators in both swine and mammalian development, emphasizing the implications of GBAF as a modulator of key developmental events.

The contribution of initial concussive forces and resulting acrolein surge to ß-amyloid accumulation and functional alterations in neuronal networks using a TBI-on-a-chip model.

Trauma-induced Alzheimer's disease (AD) is rapidly emerging as a major consequence of traumatic brain injuries (TBI), with devastating social and economic impacts. Unfortunately, few treatment options are currently available due to a limited understanding of the underlying mechanisms. A clinically-relevant, in vitro experimental model that emulates in vivo scenarios with high levels of spatial and temporal resolution is critical for demystifying the pathways of post-TBI AD. Using a unique, recently established "TBI-on-a-chip" system with murine cortical networks, we demonstrate the correlative elevation of oxidative stress (acrolein), inflammation (TNF-α), and Aß42 aggregation, with concomitant reduction of neuronal network electrical activity post-concussive impact. These findings confirm that TBI-on-a-chip could provide a novel paradigm to supplement in vivo studies of trauma, while simultaneously validating the interaction of these alleged, key-pathological factors in post-TBI AD development. Specifically, we have shown that acrolein, acting as a diffusive factor of secondary injury, is both critical and sufficient in promoting inflammation (TNF-α) and Aß42 aggregation, two known contributors of AD pathogenesis. Furthermore, using a cell-free preparation with TBI-on-a-chip, we have confirmed that both force and acrolein can independently and directly stimulate the aggregation of purified Aß42, highlighting the key capabilities of primary and secondary injury mechanisms towards inducing Aß42 aggregation, independently and synergistically. In addition to morphological and biochemical assessment, we also demonstrate parallel monitoring of neuronal network activity, further validating the chief pathological role of acrolein in not only inflicting biochemical abnormalities, but also functional deficits in neuronal networks. In conclusion, through this line of investigations, we have shown that by recapitulating clinically-relevant events, the TBI-on-a-chip device is capable of quantitatively characterizing parallel force-dependent increases in oxidative stress, inflammation, protein aggregation, and network activity, offering a unique platform for mechanistic investigations of post-TBI AD, and trauma-induced neuronal injury in general. It is expected that this model could provide crucial insights into pathological mechanisms which will be critical in developing novel, effective diagnostics and treatment strategies that significantly benefit TBI victims.

Zebrafish germline chimeras produced by transplantation of ovarian germ cells into sterile host larvae.

High frequency production of zebrafish germline chimeras was achieved by transplanting ovarian germ cells into sterile Danio hybrid recipients. Ovarian germ cells were obtained from 3-mo-old adult Tg(vasa:DsRed2-vasa);Tg(bactin:EGFP) double transgenic zebrafish by discontinuous Percoll gradient centrifugation. An average of 755 ± 108 DsRed-positive germ cells was recovered from each female. For transplantations, a total of approximately 620 ± 242 EGFP-positive cells of which 12 ± 4.7 were DsRed-positive germ cells were introduced into the abdominal cavity under the swim bladder of 2-wk-old sterile hybrid larvae. Six weeks after transplantation, a total of 10 recipients, obtained from 2 different transplantations, were examined, and 2 individuals (20%) were identified that possessed a large number of DsRed- and EGFP-positive cells in the gonadal region. The transplanted ovarian germ cells successfully colonized the gonads and differentiated into sperm in the male hybrid recipients. Of 67 adult recipients, 12 (18%) male chimeric fish reproduced and generated normal offspring when paired with wild-type zebrafish females. The fertilization efficiency ranged from 23% to 56%. Although the fertile male chimeras were generated by transplantation of ovarian germ cells, the F1 generation produced by the male chimeras contained both male and female progeny, indicating that male sex determination in zebrafish is not controlled by sex chromosome heterogamy. Our findings indicate that a population of ovarian germ cells that are present in the ovary of adult zebrafish can function as germline stem cells, able to proliferate and differentiate into testicular germ cells and functional sperm in male recipients. The high frequency of germline chimera formation achieved with the ovarian germ cells and the convenience of identifying the chimeras in the sterile host background should make this transplantation system useful for performing genetic manipulations in zebrafish.

Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis.

The role the mammary epithelial circadian clock plays in gland development and lactation is unknown. We hypothesized that mammary epithelial clocks function to regulate mammogenesis and lactogenesis, and propose the core clock transcription factor BMAL1:CLOCK regulates genes that control mammary epithelial development and milk synthesis. Our objective was to identify transcriptional targets of BMAL1 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) mammary epithelial cells (HC11) using ChIP-seq. Ensembl gene IDs with the nearest transcriptional start site to ChIP-seq peaks were explored as potential targets, and represented 846 protein coding genes common to UNDIFF and DIFF cells and 2773 unique to DIFF samples. Genes with overlapping peaks between samples (1343) enriched cell-cell adhesion, membrane transporters and lipid metabolism categories. To functionally verify targets, an HC11 line with Bmal1 gene knocked out (BMAL1-KO) using CRISPR-CAS was created. BMAL1-KO cultures had lower cell densities over an eight-day growth curve, which was associated with increased (p<0.05) levels of reactive oxygen species and lower expression of superoxide dismutase 3 (Sod3). RT-qPCR analysis also found lower expression of the putative targets, prolactin receptor (Prlr), Ppara, and beta-casein (Csn2). Findings support our hypothesis and highlight potential importance of clock in mammary development and substrate transport.

Pregnancy rest-activity patterns are related to salivary cortisol rhythms and maternal-fetal health indicators in women from a disadvantaged population.

Irregular rest-activity patterns can disrupt metabolic and hormonal physiology and potentially lead to disease. Little is known regarding rest-activity patterns during gestation and their association with hormonal rhythms and health in pregnant women. We conducted a pilot study to determine if 24 h rest-activity was related to saliva cortisol rhythms and maternal-fetal health in an economically disadvantaged population. Primiparous women wore a wrist actigraphy device for a week to record activity during gestational weeks 22 (G22; n = 50) and 32 (G32; n = 46) and postpartum week one (PPW1; n = 39). Participants collected saliva samples every 4 hr over a 24 hr period during G22 (n = 22), G32 (n = 20) and 24-48 hr postnatal (n = 20), and cortisol concentrations were measured with ELISA. Circadian rhythmicity was assessed using autocorrelation coefficient (r24) and cosinor analysis. Blood glucose levels, body mass index (BMI), gestational disease data, and gestational age of infant at birth were abstracted from medical charts. Time of cortisol peak (acrophase) during G22 was related with acrophase of activity (r = 0.66; p = 0.001) and blood glucose levels (r = 0.58; p = 0.006). During G22, minutes of wake after sleep onset was positively related to cortisol mesor and AUC (p <0.05). Rest-activity r24, R2, and mesor during G32 were positively (p<0.05) associated with gestational age of infant at birth. Across all three time points r24 of activity was related with cortisol amplitude (r = 0.33; p = 0.01). Findings support a relationship between rest-activity patterns and saliva cortisol rhythms during pregnancy. The association of less robust activity rhythms with earlier gestational age of infant at birth indicates a potential link between circadian system disruption and maternal-fetal health outcomes.

Relationship Between Sleep Quality, Depression Symptoms, and Blood Glucose in Pregnant Women.

Sleep quality during pregnancy affects maternal/child health. We aimed to assess changes in sleep quality during pregnancy and determine its relationship to maternal mood, blood glucose, and work schedule among primiparous women. We conducted a prospective/longitudinal/observational study. Ninety-two pregnant women were recruited from Midwestern hospital. Mood and sleep quality data were collected using Edinburgh Postnatal Depression Scale/Pittsburgh Sleep Quality Index at Gestational Weeks 22 and 32. Forty-three women completed the study. Twenty-six women (63%) were African American and the mean age was 23.64 (SD = 3.82) years. Rate of poor sleep quality increased during pregnancy with 25% of women had scores indicative of depression symptoms. Poor sleep quality score was related to mood scores (p < .05) and work schedule. Blood glucose was not significantly related to sleep duration. In conclusion, poor sleep quality during pregnancy was associated with poor mood and work schedule, suggesting that interventions targeting mental health and lifestyles are needed.

Delayed Lactogenesis II is Associated With Lower Sleep Efficiency and Greater Variation in Nightly Sleep Duration in the Third Trimester.

BACKGROUND: Metabolic and hormonal disturbances are associated with sleep disturbances and delayed onset of lactogenesis II. RESEARCH AIMS: The aim of this study was to measure sleep using wrist actigraphy during gestation weeks 22 and 32 to determine if sleep characteristics were associated with blood glucose, body mass index, gestational related disease, delayed onset of lactogenesis II, or work schedule. METHODS: Demographic data were collected at study intake from primiparous women who wore a wrist actigraph during gestation weeks 22 (n = 50) and 32 (n = 44). Start and end sleep time, total nighttime sleep, sleep efficiency, wake after sleep onset, and sleep fragmentation were measured. Night to night variability was assessed with the root mean square of successive difference. Blood glucose levels, body mass index, and gestational disease data were abstracted from medical charts. Timing of lactogenesis II was determined by survey. RESULTS: Between gestation week 22 and 32, sleep efficiency decreased and fragmentation increased (p < .05). During gestation week 32, blood glucose was negatively correlated with sleep duration, and positively related to fragmentation (p < .05). Women who experienced delayed lactogenesis II had lower sleep efficiency and greater fragmentation (p < .05), and greater night-to-night variability in sleep start and end time, efficiency, and duration during gestation week 32 (p < .05). CONCLUSION: Women with better sleep efficiency and more stable nightly sleep time are less likely to experience delayed onset of lactogenesis II. Interventions to improve sleep may improve maternal health and breastfeeding adequacy.

Zebrafish primordial germ cell cultures derived from vasa::RFP transgenic embryos.

Although embryonic germ (EG) cell-mediated gene transfer has been successful in the mouse for more than a decade, this approach is limited in other species due to the difficulty of isolating the small numbers of progenitors of germ cell lineage (PGCs) from early-stage embryos and the lack of information on the in vitro culture requirements of the cells. In this study, methods were established for the culture of PGCs obtained from zebrafish embryos. Transgenic embryos that express the red fluorescent protein (RFP) under the control of the PGC-specific vasa promoter were used, making it possible to isolate pure populations of PGCs by fluorescence-activated cell sorting (FACS) and to optimize the culture conditions by counting the number of fluorescent PGC colonies produced in different media. Cultures initiated from 26-somite-stage embryos contained the highest percentage of PGCs that proliferated in vitro to generate colonies. The effect of growth factors, including Kit ligand a and b (Kitlga and Kitlgb) and stromal cell-derived factor 1a and 1b (Sdf-1a and Sdf-1b), on PGC proliferation was studied. Optimal in vitro growth and survival of the zebrafish PGCs was achieved when recombinant Kitlga and Sdf-1b were added to the culture medium through transfected feeder cells, resulting in a doubling of the number of PGC colonies. Results from RT-PCR and in situ hybridization analysis demonstrated that PGCs maintained in culture expressed the kita receptor, even though receptor expression was not detected in PGCs isolated by FACS directly from dissociated embryos. In optimal growth conditions, the PGCs continued to proliferate for at least 4 months in culture. The capacity to establish long-term PGC cultures from zebrafish will make it possible to conduct in vitro studies of germ cell differentiation and EG cell pluripotency in this model species and may be valuable for the development of a cell-mediated gene transfer approach.

Homologous recombination in zebrafish ES cells.

Targeted insertion of a plasmid by homologous recombination was demonstrated in zebrafish ES cell cultures. Two selection strategies were used to isolate ES cell colonies that contained targeted plasmid insertions in either the no tail or myostatin I gene. One selection strategy involved the manual isolation of targeted cell colonies that were identified by the loss of fluorescent protein gene expression. A second strategy used the diphtheria toxin A-chain gene in a positive-negative selection approach. Homologous recombination was confirmed by PCR, sequence and Southern blot analysis and colonies isolated using both selection methods were expanded and maintained for multiple passages. The results demonstrate that zebrafish ES cells have potential for use in a cell-mediated gene targeting approach.

Zebrafish embryo cells remain pluripotent and germ-line competent for multiple passages in culture.

Mouse embryonic stem (ES) cell lines are routinely used to introduce targeted mutations into the genome, providing an efficient method to study gene function. Application of similar gene knockout techniques to other organisms has been unsuccessful due to the lack of germ-line competent ES cell lines from non-murine species. Previously, we reported the production of zebrafish germ-line chimeras using short-term primary embryo cell cultures. Here we demonstrate that zebrafish embryo cells, maintained for several weeks and multiple passages in culture, remain pluripotent and germ-line competent. Zebrafish germ-line chimeras were generated from passage 5 and 6 cultures initiated from blastula- and gastrula-stage embryos. In addition to the germ line, the cultured cells contributed to multiple tissues of the host embryo, including muscle, liver, gut, and fin. To facilitate the identification of germ-line chimeras, ES cells expressing the green fluorescent protein (GFP) were introduced into host embryos, and germ-line contribution was detected by the presence of GFP+ cells in the region of the gonad. The germ-line competent embryo cell cultures will be useful for the development of a gene targeting strategy that will increase the utility of the zebrafish model for studies of gene function.