Biology subjects

Russell, D. L.

Publications and source records attributed to Russell, D. L..

7 recordsLinked to original sources

bioRxiv2026

Proprotein convertase activity regulates cumulus-oocyte-complex matrix integrity and cumulus cell migration during ovulation via a GDF9-dependent mechanism

Cumulus cells have well-established roles early in ovulation but the key molecules that drive their behavior in later stages, leading to follicle rupture, remain underexplored. Here, we observed that inhibition of proprotein convertases (PCSKs) via a pan-inhibitor (PCI) impaired follicular rupture and disrupted the cumulus matrix integrity within intact follicles. Reduced cumulus cell adherence to the cumulus-oocyte-complex (COC) matrix was also observed in isolated COCs and notably occurred late during the maturation window without affecting oocyte maturation. Visualization of PCSK transcript and protein expression, as well as selective inhibition of specific PCSKs, determined that the observed phenotype in COCs is likely attributed to PCSK5A inhibition. We conducted bulk RNA-sequencing and proteomics of PCI-treated COCs which revealed that PCSK inhibition caused dysregulation of extracellular matrix organization, cell migration/adhesion, and TGF-{beta} signaling pathways. Subsequent validation showed that this inhibition translated to disrupted matrix organization and altered migratory and adhesive behaviors in cumulus cells. The TGF-{beta} ligand GDF9 has a predicted PCSK cleavage site, and supplementation with GDF9 rescued matrix integrity suggesting its role as a downstream substrate of PCSKs to regulate matrix organization. Altogether, this study identified PCSK5A and GDF9 as key regulators of COC matrix integrity and cumulus cell migration during late ovulation. These findings highlight novel factors required for follicle rupture which can be leveraged for the development of fertility therapeutics and contraceptives.

cell biology↗

bioRxiv2025

A network of steroid receptor transcription factors regulates ovarian chromatin remodelling in the transition to ovulation

Steroid receptors are transcription factors activated by progesterone (PGR), androgen (AR) and glucocorticoid (GR), with shared canonical DNA binding sequence. In the ovary, PGR is the key determinant of ovulation while AR and GR play important roles in growing follicles. However, the mechanism that defines the unique physiological roles of these conserved receptors, and whether their functions overlap remains elusive. We investigated the relationship between AR, GR and PGR during folliculogenesis and ovulation. In response to ovulatory hormones, PGR and GR jointly gained binding to novel chromatin sites and had a substantial effect on periovulatory gene regulation, whereas AR-chromatin interactions were repressed. Two modes of PGR action to drive gene activation were identified. Induction of PGR leads to cooperative PGR/GR recruitment to novel promoters, increased histone acetylation and chromatin accessibility, culminating in transcription activation, with PGR being the key component in the unique ovulatory transcriptional complex. Alternatively, PGR tethered to enhancers interacts with pre-accessible, AR/GR-bound promoters to promote gene activation. Our findings illustrate the multi-faceted ovarian steroid receptor interactions which explain how the progressive change in steroid environments throughout folliculogenesis programs granulosa cells during the transition to ovulation.

genomics↗

bioRxiv2024

Cortico-amygdala synaptic structural abnormalities produced by templated aggregation of α-synuclein

Parkinsons disease (PD) and Dementia with Lewy bodies (DLB) are characterized by neuronal -synuclein (-syn) inclusions termed Lewy Pathology, which are abundant in the amygdala. The basolateral amygdala (BLA), in particular, receives projections from the thalamus and cortex. These projections play a role in cognition and emotional processing, behaviors which are impaired in -synucleinopathies. To understand if and how pathologic -syn impacts the BLA requires animal models of -syn aggregation. Injection of -synuclein pre-formed fibrils (PFFs) into the striatum induces robust -synuclein aggregation in excitatory neurons in the BLA that corresponds with reduced contextual fear conditioning. At early time points after aggregate formation, cortico-amygdala excitatory transmission is abolished. The goal of this project was to determine if -syn inclusions in the BLA induce synaptic degeneration and/or morphological changes. In this study, we used C57BL/6J mice injected bilaterally with PFFs in the dorsal striatum to induce -syn aggregate formation in the BLA. A method was developed using immunofluorescence and three-dimensional reconstruction to analyze excitatory cortico-amygdala and thalamo-amygdala presynaptic terminals closely juxtaposed to postsynaptic densities. The abundance and morphology of synapses were analyzed at 6- or 12-weeks post-injection of PFFs. -Syn aggregate formation in the BLA did not cause a significant loss of synapses, but cortico-amygdala and thalamo-amygdala presynaptic terminals and postsynaptic densities with aggregates of -synuclein show increased volumes, similar to previous findings in human DLB cortex, and in non-human primate models of PD. Transmission electron microscopy showed that PFF-injected mice showed reduced intervesicular distances similar to a recent study showing phospho-serine-129 -synuclein increases synaptic vesicle clustering. Thus, pathologic -synuclein causes major alterations to synaptic architecture in the BLA, potentially contributing to behavioral impairment and amygdala dysfunction observed in synucleinopathies.

neuroscience↗

bioRxiv2023

dFLASH; dual FLuorescent transcription factor Activity Sensor for Histone integrated live-cell reporting and high-content screening

Live-cell reporting of regulated transcription factor (TF) activity has a wide variety of applications in synthetic biology, drug discovery, and functional genomics. As a result, there is high value in the generation of versatile, sensitive, robust systems that can function across a range of cell types and be adapted toward diverse TF classes. Here we present the dual FLuorescent transcription factor Activity Sensor for Histone integrated live-cell reporting (dFLASH), a modular sensor for TF activity that can be readily integrated into cellular genomes. We demonstrate readily modified dFLASH platforms that homogenously, robustly, and specifically sense regulation of endogenous Hypoxia Inducible Factor (HIF) and Progesterone receptor (PGR) activities, as well as regulated coactivator recruitment to a synthetic DNA-Binding Domain-Activator Domain fusion proteins. The dual-colour nuclear fluorescence produced normalised dynamic live-cell TF activity sensing with facile generation of high-content screening lines, strong signal:noise ratios and reproducible screening capabilities (Z = 0.68-0.74). Finally, we demonstrate the utility of this platform for functional genomics applications by using CRISPRoff to modulate the HIF regulatory pathway, and for drug screening by using high content imaging in a bimodal design to isolate activators and inhibitors of the HIF pathway from a [~]1600 natural product library.

synthetic biology↗

bioRxiv2023

N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

The cell adhesion molecule N-cadherin (CDH2) is a membrane component of adherens junctions which regulates tissue morphogenesis and architecture. In the follicles of mammalian ovaries, N-cadherin adherens junctions are present between granulosa cells, cumulus cells and at the interface of cumulus cell transzonal projections and the oocyte. We demonstrate a mechanosensory role of N-cadherin integrating tissue structure and hormonal regulation of follicular morphogenic events including expansion of the cumulus oocyte complex (COC) matrix, oocyte maturation and ovulation. Two small molecule N-cadherin antagonists inhibited COC maturation in vitro. Transcriptome profiling revealed that targets of {beta}-catenin and YAP1 pathways were dysregulated by N-cadherin antagonists. In vivo, N-cadherin antagonist significantly reduced ovulation in mice compared to controls (11 vs 26 oocytes/ovary; p=5.8x10-6). Ovarian follicles exhibited structural dysgenesis with granulosa and cumulus cell layers becoming disorganised and the connection between cumulus cells and the oocyte disrupted and the transcriptome again indicated altered mechanical sensing causing dysregulation of the Hippo/YAP and {beta}-catenin pathways and ECM reorganization. Granulosa specific N-cadherin depletion in Cdh2Fl/FL;Amhr2-Cre also showed significantly altered mechanosensitive gene expression and reduced ovulation. Our findings demonstrate a critical role for N-cadherin in ovarian follicular development and ovulation, and the potential to inhibit ovulation through targeting this signalling mechanism.

cell biology↗

bioRxiv2022

Core and Flanking bHLH-PAS:DNA interactions mediate specificity and drive obesity

The basic-Helix-Loop-Helix Per-Arnt-Sim (PAS) homology domain (bHLH-PAS) transcription factor (TF) family comprises critical biological sensors of physiological (hypoxia, tryptophan metabolites, neuronal activity, and appetite) and environmental (diet derived metabolites and environmental pollutants) stimuli to regulate genes involved in signal adaptation and homeostasis1. bHLH TFs bind DNA as homo or heterodimers via E-box (CANNTG) response elements, however the DNA binding specificity of the PAS domain-containing bHLH subfamily remains unresolved1. We systematically analysed cognate DNA binding hierarchies of prototypical bHLH-PAS family members (ARNT, ARNT2, HIF1, HIF2, AhR, NPAS4, SIM1) and demonstrate distinct core (NNCGTG) specificities for different heterodimer classes. The results also show that bHLH-PAS TFs bind over a large footprint 12-15bp and recognise preferential DNA sequences flanking the core. For example, specificity beyond otherwise identical core binding by SIM1 and the HIFs is mediated through N-terminal HIF-DNA interactions. We also reveal an intimate relationship between DNA shape and both core and flanking TF binding allowing motif sequence flexibility and underpinning TF binding specificity. Furthermore, DNA-shape affinity relationships revealed that novel downstream PAS-A-loop DNA interactions are associated with AT-rich sequences that lead to high-affinity binding, and that loss of this function underpins a monogenic cause of human hyperphagic obesity in a recapitulated SIM1.R171H knock-in mouse model. Importantly, models of protein-DNA binding accurately predict in vivo occupancy, while response element methylation blocks DNA binding and predicts cell type specific chromatin occupancy. These data provide a definitive and accurate map of bHLH-PAS TF specificity and target selectivity through novel flanking protein-DNA interactions that are crucial for in vivo biological function.

molecular biology↗

bioRxiv2021

Progesterone receptor-A isoform interaction with RUNX transcription factors controls chromatin remodelling at promoters during ovulation

Progesterone receptor (PGR) plays diverse roles in reproductive tissues and thus coordinates mammalian fertility. In the ovary, acutely induced PGR is the key determinant of ovulation through transcriptional control of a unique set of genes that culminates in follicle rupture. However, the molecular mechanisms for PGRs specialised function in ovulation is poorly understood. To address this, we assembled a detailed genomic profile of PGR action through combined ATAC-seq, RNA-seq and ChIP-seq analysis in wildtype and isoform-specific PGR null mice. We demonstrated the unique action of PGR-A isoform in the ovary through a transcriptional complex involving physical interaction with RUNX and JUN/FOS transcription factors. The assembly of this unique complex directs targeting of PGR binding to proximal promoter regions and enables chromatin accessibility, leading to ovulatory gene induction. This PGR signalling mechanism is specific to ovulation and provides potential targets for infertility treatments as well as new contraceptives that block ovulation.

molecular biology↗