Genome-scale requirements for dynein-based transport revealed by a high-content arrayed CRISPR screen.

The microtubule motor dynein plays a key role in cellular organization. However, little is known about how dynein's biosynthesis, assembly, and functional diversity are orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a genome-wide gRNA library, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints revealed co-functional proteins involved in many cellular processes, including several candidate novel regulators of core dynein functions. Further analysis of one of these factors, the RNA-binding protein SUGP1, indicates that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our data represent a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organization captured by our high-content imaging.

Understanding Intracellular Biology to Improve mRNA Delivery by Lipid Nanoparticles.

Poor understanding of intracellular delivery and targeting hinders development of nucleic acid-based therapeutics transported by nanoparticles. Utilizing a siRNA-targeting and small molecule profiling approach with advanced imaging and machine learning biological insights is generated into the mechanism of lipid nanoparticle (MC3-LNP) delivery of mRNA. This workflow is termed Advanced Cellular and Endocytic profiling for Intracellular Delivery (ACE-ID). A cell-based imaging assay and perturbation of 178 targets relevant to intracellular trafficking is used to identify corresponding effects on functional mRNA delivery. Targets improving delivery are analyzed by extracting data-rich phenotypic fingerprints from images using advanced image analysis algorithms. Machine learning is used to determine key features correlating with enhanced delivery, identifying fluid-phase endocytosis as a productive cellular entry route. With this new knowledge, MC3-LNP is re-engineered to target macropinocytosis, and this significantly improves mRNA delivery in vitro and in vivo. The ACE-ID approach can be broadly applicable for optimizing nanomedicine-based intracellular delivery systems and has the potential to accelerate the development of delivery systems for nucleic acid-based therapeutics.

Lipid-peptide nanocomplexes for mRNA delivery in vitro and in vivo.

Despite recent advances in the field of mRNA therapy, the lack of safe and efficacious delivery vehicles with pharmaceutically developable properties remains a major limitation. Here, we describe the systematic optimisation of lipid-peptide nanocomplexes for the delivery of mRNA in two murine cancer cell types, B16-F10 melanoma and CT26 colon carcinoma as well as NCI-H358 human lung bronchoalveolar cells. Different combinations of lipids and peptides were screened from an original lipid-peptide nanocomplex formulation for improved luciferase mRNA transfection in vitro by a multi-factorial screening approach. This led to the identification of key structural elements within the nanocomplex associated with substantial improvements in mRNA transfection efficiency included alkyl tail length of the cationic lipid, the fusogenic phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol. The peptide component (K16GACYGLPHKFCG) was further improved by the inclusion of a linker, RVRR, that is cleavable by the endosomal enzymes cathepsin B and furin, and a hydrophobic motif (X-S-X) between the mRNA packaging (K16) and receptor targeting domains (CYGLPHKFCG). Nanocomplex transfections of a murine B16-F10 melanoma tumour supported the inclusion of cholesterol for optimal transfection in vivo as well as in vitro. In vitro transfections were also performed with mRNA encoding interleukin-15 as a potential immunotherapy agent and again, the optimised formulation with the key structural elements demonstrated significantly higher expression than the original formulation. Physicochemical characterisation of the nanocomplexes over time indicated that the optimal formulation retained biophysical properties such as size, charge and mRNA complexation efficiency for 14 days upon storage at 4 °C without the need for additional stabilising agents. In summary, we have developed an efficacious lipid-peptide nanocomplex with promising pharmaceutical development properties for the delivery of therapeutic mRNA.

Mechanistic Studies of an Automated Lipid Nanoparticle Reveal Critical Pharmaceutical Properties Associated with Enhanced mRNA Functional Delivery In Vitro and In Vivo.

Recently, lipid nanoparticles (LNPs) have attracted attention due to their emergent use for COVID-19 mRNA vaccines. The success of LNPs can be attributed to ionizable lipids, which enable functional intracellular delivery. Previously, the authors established an automated high-throughput platform to screen ionizable lipids and identified that the LNPs generated using this automated technique show comparable or increased mRNA functional delivery in vitro as compared to LNPs prepared using traditional microfluidics techniques. In this study, the authors choose one benchmark lipid, DLin-MC3-DMA (MC3), and investigate whether the automated formulation technique can enhance mRNA functional delivery in vivo. Interestingly, a 4.5-fold improvement in mRNA functional delivery in vivo by automated LNPs as compared to LNPs formulated by conventional microfluidics techniques, is observed. Mechanistic studies reveal that particles with large size accommodate more mRNA per LNP, possess more hydrophobic surface, are more hemolytic, bind a larger protein corona, and tend to accumulate more in macropinocytosomes, which may quantitatively benefit mRNA cytosolic delivery. These data suggest that mRNA loading per particle is a critical factor that accounts for the enhanced mRNA functional delivery of automated LNPs. These mechanistic findings provide valuable insight underlying the enhanced mRNA functional delivery to accelerate future mRNA LNP product development.

Arrayed CRISPR Screening Identifies Novel Targets That Enhance the Productive Delivery of mRNA by MC3-Based Lipid Nanoparticles.

Modified messenger RNAs (mRNAs) hold great potential as therapeutics by using the body's own processes for protein production. However, a key challenge is efficient delivery of therapeutic mRNA to the cell cytosol and productive protein translation. Lipid nanoparticles (LNPs) are the most clinically advanced system for nucleic acid delivery; however, a relatively narrow therapeutic index makes them unsuitable for many therapeutic applications. A key obstacle to the development of more potent LNPs is a limited mechanistic understanding of the interaction of LNPs with cells. To address this gap, we performed an arrayed CRISPR screen to identify novel pathways important for the functional delivery of MC3 lipid-based LNP encapsulated mRNA (LNP-mRNA). Here, we have developed and validated a robust, high-throughput screening-friendly phenotypic assay to identify novel targets that modulate productive LNP-mRNA delivery. We screened the druggable genome (7795 genes) and validated 44 genes that either increased (37 genes) or inhibited (14 genes) the productive delivery of LNP-mRNA. Many of these genes clustered into families involved with host cell transcription, protein ubiquitination, and intracellular trafficking. We show that both UDP-glucose ceramide glucosyltransferase and V-type proton ATPase can significantly modulate the productive delivery of LNP-mRNA, increasing and decreasing, respectively, with both genetic perturbation and by small-molecule inhibition. Taken together, these findings shed new light into the molecular machinery regulating the delivery of LNPs into cells and improve our mechanistic understanding of the cellular processes modulating the interaction of LNPs with cells.

Divalent cannabinoid-1 receptor ligands: A linker attachment point survey of SR141716A for development of high-affinity CB1R molecular probes.

The cannabinoid-1 receptor (CB1R) inverse agonist SR141716A has proven useful for study of the endocannabinoid system, including development of divalent CB1R ligands possessing a second functional motif attached via a linker unit. These have predominantly employed the C3 position of the central pyrazole ring for linker attachment. Despite this precedent, a novel series of C3-linked CB1R-D2R divalent ligands exhibited extremely high affinity at the D2R, but only poor affinity for the CB1R. A systematic linker attachment point survey of the SR141716A pharmacophore was therefore undertaken, establishing the C5 position as the optimal site for linker conjugation. This linker attachment survey enabled the identification of a novel divalent ligand as a lead compound to inform ongoing development of high-affinity CB1R molecular probes.

Using macropinocytosis for intracellular delivery of therapeutic nucleic acids to tumour cells.

Nucleic acids are a rapidly emerging therapeutic modality with the potential to become the third major drug modality alongside antibodies and small molecules. Owing to the unfavourable physico-chemical characteristics of nucleic acids, such as large size and negative charge, intracellular delivery remains a fundamental challenge to realizing this potential. Delivery technologies such as lipids, polymers and peptides have been used to facilitate delivery, with many of the most successful technologies using macropinocytosis to gain cellular entry; mostly by default rather than design. Fundamental knowledge of macropinocytosis is rapidly growing, presenting opportunities to better tailor design strategies to target this pathway. Furthermore, certain types of tumour cells have been observed to have high levels of macropinocytic activity and traffic cargo to favourable destinations within the cell for endosomal release, providing unique opportunities to further use this entry route for drug delivery. In this article, we review the delivery systems reported to be taken up by macropinocytosis and what is known about the mechanisms for regulating macropinocytosis in tumour cells. From this analysis, we identify new opportunities for exploiting this pathway for the intracellular delivery of nucleic acids to tumour cells. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.

Proteomic and Biochemical Comparison of the Cellular Interaction Partners of Human VPS33A and VPS33B.

Multi-subunit tethering complexes control membrane fusion events in eukaryotic cells. Class C core vacuole/endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS) are two such complexes, both containing the Sec1/Munc18 protein subunit VPS33A. Metazoans additionally possess VPS33B, which has considerable sequence similarity to VPS33A but does not integrate into CORVET or HOPS complexes and instead stably interacts with VIPAR. It has been recently suggested that VPS33B and VIPAR comprise two subunits of a novel multi-subunit tethering complex (named "CHEVI"), perhaps analogous in configuration to CORVET and HOPS. We utilized the BioID proximity biotinylation assay to compare and contrast the interactomes of VPS33A and VPS33B. Overall, few proteins were identified as associating with both VPS33A and VPS33B, suggesting that these proteins have distinct sub-cellular localizations. Consistent with previous reports, we observed that VPS33A was co-localized with many components of class III phosphatidylinositol 3-kinase (PI3KC3) complexes: PIK3C3, PIK3R4, NRBF2, UVRAG and RUBICON. Although VPS33A clearly co-localized with several subunits of CORVET and HOPS in this assay, no proteins with the canonical CORVET/HOPS domain architecture were found to co-localize with VPS33B. Instead, we identified that VPS33B interacts directly with CCDC22, a member of the CCC complex. CCDC22 does not co-fractionate with VPS33B and VIPAR in gel filtration of human cell lysates, suggesting that CCDC22 interacts transiently with VPS33B/VIPAR rather than forming a stable complex with these proteins in cells. We also observed that the protein complex containing VPS33B and VIPAR is considerably smaller than CORVET/HOPS, suggesting that the CHEVI complex comprises just VPS33B and VIPAR.

VPS18 recruits VPS41 to the human HOPS complex via a RING-RING interaction.

Eukaryotic cells use conserved multisubunit membrane tethering complexes, including CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting), to control the fusion of endomembranes. These complexes have been extensively studied in yeast, but to date there have been far fewer studies of metazoan CORVET and HOPS. Both of these complexes comprise six subunits: a common four-subunit core and two unique subunits. Once assembled, these complexes function to recognise specific endosomal membrane markers and facilitate SNARE-mediated membrane fusion. CORVET promotes the homotypic fusion of early endosomes, while HOPS promotes the fusion of lysosomes to late endosomes and autophagosomes. Many of the subunits of both CORVET and HOPS contain putative C-terminal zinc-finger domains. Here, the contribution of these domains to the assembly of the human CORVET and HOPS complexes has been examined. Using biochemical techniques, we demonstrate that the zinc-containing RING (really interesting new gene) domains of human VPS18 and VPS41 interact directly to form a stable heterodimer. In cells, these RING domains are able to integrate into endogenous HOPS, showing that the VPS18 RING domain is required to recruit VPS41 to the core complex subunits. Importantly, this mechanism is not conserved throughout eukaryotes, as yeast Vps41 does not contain a C-terminal zinc-finger motif. The subunit analogous to VPS41 in human CORVET is VPS8, in which the RING domain has an additional C-terminal segment that is predicted to be disordered. Both the RING and disordered C-terminal domains are required for integration of VPS8 into endogenous CORVET complexes, suggesting that HOPS and CORVET recruit VPS41 and VPS8 via distinct molecular interactions.

Real-Time Measurement of Cannabinoid Receptor-Mediated cAMP Signaling.

Cannabinoid receptors, like other GPCRs, signal via a spectrum of related signaling pathways. Recently, monitoring GPCR-mediated cAMP signaling has become significantly easier with the development of genetically encoded, transfectable cAMP biosensors. Cell lines transfected with these biosensors can be monitored continuously, allowing the analysis of receptor-mediated signaling in unprecedented detail. Here, we describe a protocol for transfectable biosensors which report cellular cAMP concentrations by bioluminescence resonance energy transfer (BRET). This assay system has been utilized to elucidate the temporal nature of agonists and allosteric modulators of the cannabinoid receptor CB1. In particular, the CB1 allosteric modulator ORG27569 has been shown to modify receptor agonism in a time-dependent fashion; a characteristic which would not have been observed via traditional endpoint methods of detecting cAMP signaling. BRET cAMP biosensors are suitable for miniaturization and automation, and as such are valuable and cost-effective tools for moderate- to high-throughput experimental protocols.

Gαs signalling of the CB1 receptor and the influence of receptor number.

BACKGROUND AND PURPOSE: CB1 receptor signalling is canonically mediated through inhibitory Gαi proteins, but occurs through other G proteins under some circumstances, Gαs being the most characterized secondary pathway. Determinants of this signalling switch identified to date include Gαi blockade, CB1 /D2 receptor co-stimulation, CB1 agonist class and cell background. Hence, we examined the effects of receptor number and different ligands on CB1 receptor signalling. EXPERIMENTAL APPROACH: CB1 receptors were expressed in HEK cells at different levels, and signalling characterized for cAMP by real-time BRET biosensor -CAMYEL - and for phospho-ERK by AlphaScreen. Homogenate and whole cell radioligand binding assays were performed to characterize AM6544, a novel irreversible CB1 receptor antagonist. KEY RESULTS: In HEK cells expressing high levels of CB1 receptors, agonist treatment stimulated cAMP, a response not known to be mediated by receptor number. Δ9 -THC and BAY59-3074 increased cAMP only in high-expressing cells pretreated with pertussis toxin, and agonists demonstrated more diverse signalling profiles in the stimulatory pathway than the canonical inhibitory pathway. Pharmacological CB1 receptor knockdown and Gαi 1 supplementation restored canonical Gαi signalling to high-expressing cells. Constitutive signalling in both low- and high-expressing cells was Gαi -mediated. CONCLUSION AND IMPLICATIONS: CB1 receptor coupling to opposing G proteins is determined by both receptor and G protein expression levels, which underpins a mechanism for non-canonical signalling in a fashion consistent with Gαs signalling. CB1 receptors mediate opposite consequences in endpoints such as tumour viability depending on expression levels; our results may help to explain such effects at the level of G protein coupling.

Circumventing the stability-function trade-off in an engineered FN3 domain.

The favorable biophysical attributes of non-antibody scaffolds make them attractive alternatives to monoclonal antibodies. However, due to the well-known stability-function trade-off, these gains tend to be marginal after functional selection. A notable example is the fibronectin Type III (FN3) domain, FNfn10, which has been previously evolved to bind lysozyme with 1 pM affinity (FNfn10-α-lys), but suffers from poor thermodynamic and kinetic stability. To explore this stability-function compromise further, we grafted the lysozyme-binding loops from FNfn10-α-lys onto our previously engineered, ultra-stable FN3 scaffold, FN3con. The resulting variant (FN3con-α-lys) bound lysozyme with a markedly reduced affinity, but retained high levels of thermal stability. The crystal structure of FNfn10-α-lys in complex with lysozyme revealed unanticipated interactions at the protein-protein interface involving framework residues of FNfn10-α-lys, thus explaining the failure to transfer binding via loop grafting. Utilizing this structural information, we redesigned FN3con-α-lys and restored picomolar binding affinity to lysozyme, while maintaining thermodynamic stability (with a thermal melting temperature 2-fold higher than that of FNfn10-α-lys). FN3con therefore provides an exceptional window of stability to tolerate deleterious mutations, resulting in a substantial advantage for functional design. This study emphasizes the utility of consensus design for the generation of highly stable scaffolds for downstream protein engineering studies.

Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model.

G protein-coupled receptors (GPCRs) are important therapeutic targets and therefore extensively studied. Like most transmembrane proteins, there has been considerable difficulty in developing reliable specific antibodies for them. To overcome this, epitope tags are often used to facilitate antibody recognition in studies on fundamental receptor signalling and trafficking. In our study of cannabinoid CB1/dopamine D2 interactions we sought to generate HEK293 cells expressing FLAG-tagged D2 for use in antibody-based assays of GPCR localisation and trafficking activity, however observed that stable FLAG-hD2 expression was particularly challenging to maintain. In contrast, when expressed in cell lines expressing hCB1 robust and stable FLAG-hD2 expression was observed. We hypothesised that co-expression of CB1 might stabilise surface FLAG-hD2 expression, and therefore investigated this further. Here, we describe the observation that co-expression of either cannabinoid CB1 or CB2 receptors in HEK293 decreases the sulfation of a FLAG epitope appended at the N-terminus of the dopamine D2 receptor. Sulfation alters epitope recognition by some anti-FLAG antibodies, leading to the detection of fewer receptors, even though expression is maintained. This demonstrates that cannabinoid receptor expression modifies posttranslational processing of the FLAG-hD2 receptor, and importantly, has wider implications for the utilisation and interpretation of receptor studies involving epitope tags.

Structural and dynamic properties that govern the stability of an engineered fibronectin type III domain.

Consensus protein design is a rapid and reliable technique for the improvement of protein stability, which relies on the use of homologous protein sequences. To enhance the stability of a fibronectin type III (FN3) domain, consensus design was employed using an alignment of 2123 sequences. The resulting FN3 domain, FN3con, has unprecedented stability, with a melting temperature >100°C, a ΔG(D-N) of 15.5 kcal mol(-1) and a greatly reduced unfolding rate compared with wild-type. To determine the underlying molecular basis for stability, an X-ray crystal structure of FN3con was determined to 2.0 Å and compared with other FN3 domains of varying stabilities. The structure of FN3con reveals significantly increased salt bridge interactions that are cooperatively networked, and a highly optimized hydrophobic core. Molecular dynamics simulations of FN3con and comparison structures show the cooperative power of electrostatic and hydrophobic networks in improving FN3con stability. Taken together, our data reveal that FN3con stability does not result from a single mechanism, but rather the combination of several features and the removal of non-conserved, unfavorable interactions. The large number of sequences employed in this study has most likely enhanced the robustness of the consensus design, which is now possible due to the increased sequence availability in the post-genomic era. These studies increase our knowledge of the molecular mechanisms that govern stability and demonstrate the rising potential for enhancing stability via the consensus method.

Increasing the flexibility of the LANCE cAMP detection kit.

INTRODUCTION: The detection of cAMP signalling is a common endpoint in the study of G-protein coupled receptors. A number of commercially available kits enable easy detection of cAMP. These kits are based on competition for a cAMP binding site on an antibody or cAMP binding protein and as such have a limited dynamic range. Here, we describe the optimisation of the commercially-available LANCE cAMP detection kit (PerkinElmer) to enable detection in cell lysates. This kit has been designed for use with live cells, with detection reagents applied to cells without wash steps. The standard protocol therefore requires that all assay reagents are compatible with the antibody and the final fluorescent detection stage, limiting the range of assay media and test compounds that can be utilised. The entire experiment must be repeated if cAMP levels fall outside the limited dynamic range. Here we describe a modified protocol that enables the assay to be performed on cell lysates, thereby overcoming these limitations. METHODS: In this modified protocol, cells are stimulated for a cAMP response in standard media/buffers, washed and then lysed. The cell lysate is then assayed using a modified protocol for the LANCE cAMP detection kit. Samples were tested for stability following a freeze-thaw cycle. RESULTS: The modified LANCE cAMP detection protocol gives a reproducible measurement of cAMP in cell lysate. Lysate samples remain stable when stored at -80°C. DISCUSSION: Separating the stimulation and detection phases of this cAMP assay allows a vast array of cell stimulation conditions to be tested. The lysate-modified protocol for the LANCE cAMP detection kit therefore increases the flexibility, versatility and convenience of the assay. As samples are insensitive to freeze-thaw, it enables retesting of samples under different dilution conditions to ensure that all samples remain within the dynamic range of the standard curve.

Effects of the Parkinsonian toxin MPP+ on electrophysiological properties of nigral dopaminergic neurons.

Although MPP(+) (1-methyl-4-phenylpyridinium) has been widely used to damage dopaminergic neurons of the Substantia Nigra pars compacta (SNc) and produce animal and cellular models of Parkinson's disease, the action of this toxin on ion channels and electrophysiological properties of these neurons remains controversial. Previous work has attributed the early effects of MPP(+) on the membrane potential and firing frequency of SNc neurons either to block of hyperpolarisation-activated (Ih) current, or to activation of ATP-sensitive K(+) (KATP) channels. Using a combination of electrophysiological and pharmacological techniques, we investigated the acute effects of MPP(+) (20 µM) on SNc neurons in rat midbrain slices. Our results show that MPP(+) inhibits the activity of these neurons in distinct stages involving different mechanisms. The early phase of inhibition was dependent on D2 autoreceptors, but [(3)H]raclopride membrane binding and cAMP production assays demonstrated that the toxin (0.001-100 µM) did not directly bind to these receptors nor activated the Gi-linked signalling pathway. Depletion of vesicular dopamine with Ro4-1284 attenuated the early inhibitory effect, indicating that D2 autoreceptors were activated by dopamine released from the somato-dendritic region. After longer exposure (>10-20 min), MPP(+) produced a late phase of inhibition which mainly involved activation of KATP channels, and required uptake of the toxin via dopamine transporter. Although Ih current mediated by hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels was reduced by MPP(+), neither inhibition of firing nor membrane potential hyperpolarisation was significantly attenuated by blocking HCN channels with ZD7288. Our results indicate that the initial cellular events that lead to activation of cell death pathways by MPP(+) are complex and include KATP, and dopamine-dependent components, and show that the inhibitory effect of the toxin is independent of Ih block.

Fibroblast growth factor 2 induces the precocious development of endothelial cell networks in bovine luteinising follicular cells.

The transition from follicle to corpus luteum represents a period of intense angiogenesis; however, the exact roles of angiogenic factors during this time remain to be elucidated. Thus, the roles of vascular endothelial growth factor (VEGF) A, fibroblast growth factor (FGF) 2 and LH in controlling angiogenesis were examined in the present study. A novel serum-free luteinising follicular angiogenesis culture system was developed in which progesterone production increased during the first 5 days and was increased by LH (P<0.01). Blockade of signalling from FGF receptors (SU5402; P<0.001) and, to a lesser extent, VEGF receptors (SU1498; P<0.001) decreased the development of endothelial cell (EC) networks. Conversely, FGF2 dose-dependently (P<0.001) induced the precocious transition of undeveloped EC islands into branched networks associated with a twofold increase in the number of branch points (P<0.001). In contrast, VEGFA had no effect on the area of EC networks or the number of branch points. LH had no effect on the area of EC networks, but it marginally increased the number of branch points (P<0.05) and FGF2 production (P<0.001). Surprisingly, progesterone production was decreased by FGF2 (P<0.01) but only on Day 5 of culture. Progesterone production was increased by SU5402 (P<0.001) and decreased by SU1498 (P<0.001). These results demonstrate that FGF and VEGF receptors play a fundamental role in the formation of luteal EC networks in vitro, which includes a novel role for FGF2 in induction of EC sprouting.

The expression, regulation and function of secreted protein, acidic, cysteine-rich in the follicle-luteal transition.

The role of the tissue remodelling protein, secreted protein, acidic, cysteine-rich (SPARC), in key processes (e.g. cell reorganisation and angiogenesis) that occur during the follicle-luteal transition is unknown. Hence, we investigated the regulation of SPARC in luteinsing follicular cells and potential roles of SPARC peptide 2.3 in a physiologically relevant luteal angiogenesis culture system. SPARC protein was detected mainly in the theca layer of bovine pre-ovulatory follicles, but its expression was considerably greater in the corpus haemorrhagicum. Similarly, SPARC protein (western blotting) was up-regulated in luteinising granulosa but not in theca cells during a 6-day culture period. Potential regulatory candidates were investigated in luteinising granulosa cells: LH did not affect SPARC (P>0.05); transforming growth factor (TGF) B1 (P<0.001) dose dependently induced the precocious expression of SPARC and increased final levels: this effect was blocked (P<0.001) by SB505124 (TGFB receptor 1 inhibitor). Additionally, fibronectin, which is deposited during luteal development, increased SPARC (P<0.01). In luteal cells, fibroblast growth factor 2 decreased SPARC (P<0.001) during the first 5 days of culture, while vascular endothelial growth factor A increased its expression (P<0.001). Functionally, KGHK peptide, a SPARC proteolytic fragment, stimulated the formation of endothelial cell networks in a luteal cell culture system (P<0.05) and increased progesterone production (P<0.05). Collectively, these findings indicate that SPARC is intricately regulated by pro-angiogenic and other growth factors together with components of the extracellular matrix during the follicle-luteal transition. Thus, it is possible that SPARC plays an important modulatory role in regulating angiogenesis and progesterone production during luteal development.

Fibroblast growth factor 2 is a key determinant of vascular sprouting during bovine luteal angiogenesis.

Fibroblast growth factor (FGF) 2 and vascular endothelial growth factor (VEGF) A are thought to be key controllers of luteal angiogenesis; however, their precise roles in the regulation and coordination of this complex process remain unknown. Thus, the temporal and spatial patterns of endothelial network formation were determined by culturing mixed cell types from early bovine corpora lutea on fibronectin in the presence of FGF2 and VEGFA (6 h to 9 days). Endothelial cells, as determined by von Willebrand factor immunohistochemistry, initially grew in cell islands (days 0-3), before undergoing a period of vascular sprouting to display a more tubule-like appearance (days 3-6), and after 9 days in culture had formed extensive intricate networks. Mixed populations of luteal cells were treated with SU1498 (VEGF receptor 2 inhibitor) or SU5402 (FGF receptor 1 inhibitor) or control on days 0-3, 3-6 or 6-9 to determine the role of FGF2 and VEGFA during these specific windows. The total area of endothelial cells was unaffected by SU1498 treatment during any window. In contrast, SU5402 treatment caused maximal reduction in the total area of endothelial cell networks on days 3-6 vs controls (mean reduction 81%; P<0.001) during the period of tubule initiation. Moreover, SU5402 treatment on days 3-6 dramatically reduced the total number of branch points (P<0.001) and degree of branching per endothelial cell island (P<0.05) in the absence of changes in mean island area. This suggests that FGF2 is a key determinant of vascular sprouting and hence critical to luteal development.

Expression of renin-angiotensin system components in the early bovine embryo.

The renin-angiotensin system (RAS), mainly associated with the regulation of blood pressure, has been recently investigated in female reproductive organs and the developing foetus. Angiotensin II (Ang II) influences oviductal gamete movements and foetal development, but there is no information about RAS in the early embryo. The aim of this study was to determine whether RAS components are present in the pre-implantation embryo, to determine how early they are expressed and to investigate their putative role at this stage of development. Bovine embryos produced in vitro were used for analysis of RAS transcripts (RT-PCR) and localisation of the receptors AGTR1 and AGTR2 (immunofluorescent labelling). We also investigated the effects of Ang II, Olmesartan (AGTR1 antagonist) and PD123319 (AGTR2 antagonist) on oocyte cleavage, embryo expansion and hatching. Pre-implanted embryos possessed AGTR1 and AGTR2 but not the other RAS components. Both receptors were present in the trophectoderm and in the inner cell mass of the blastocyst. AGTR1 was mainly localised in granular-like structures in the cytoplasm, suggesting its internalisation into clathrin-coated vesicles, and AGTR2 was found mainly in the nuclear membrane and in the mitotic spindle of dividing trophoblastic cells. Treating embryos with PD123319 increased the proportion of hatched embryos compared with the control. These results, the first on RAS in the early embryo, suggest that the pre-implanted embryo responds to Ang II from the mother rather than from the embryo itself. This may be a route by which the maternal RAS influences blastocyst hatching and early embryonic development.