Acquisition Of Motility Research Articles

Episodic ozone exposure in Long-Evans rats has limited effects on cauda sperm motility and non-coding RNA populations

Epidemiological evidence suggests the potential for air pollutants to induce male reproductive toxicity. In experimental studies, exposure to ozone during sensitive windows in the sperm lifecycle has been associated with impaired sperm motility. Subsequently, we sought to investigate the effects of episodic exposure to ozone during sperm maturation in the rat. Long-Evans rats were exposed to either filtered air or ozone (0.4 or 0.8 ppm) for five non-consecutive days over two weeks. Ozone exposure did not impact male reproductive organ weights or sperm motility ∼24 hours following the final exposure. Furthermore, circulating sex hormones remained unchanged despite increased T3 and T4 in the 0.8 ppm group. While there was indication of altered adrenergic signaling attributable to ozone exposure in the testis, there were minimal impacts on small non-coding RNAs detected in cauda sperm. Only two piwi-interacting RNAs (piRNAs) were altered in the mature sperm of ozone-exposed rats (piR-rno-346434 and piR-rno-227431). Data across all rats were next analyzed to identify any non-coding RNAs that may be correlated with reduced sperm motility. A total of 7 microRNAs (miRNAs), 8 RNA fragments, and 1682 piRNAs correlated well with sperm motility. Utilizing our exposure paradigm herein, we were unable to substantiate the relationship between ozone exposure during maturation with sperm motility. However, these approaches served to identify a suite of non-coding RNAs that were associated with sperm motility in rats. With additional investigation, these RNAs may prove to have functional roles in the acquisition of motility or be unique biomarkers for male reproductive toxicity.

SMPD3 expression is spatially regulated in the developing embryo by SOXE factors

During epithelial-to-mesenchymal transition (EMT), significant rearrangements occur in plasma membrane protein and lipid content that are important for membrane function and acquisition of cell motility. To gain insight into how neural crest cells regulate their lipid content at the transcriptional level during EMT, here we identify critical enhancer sequences that regulate the expression of SMPD3, a gene responsible for sphingomyelin hydrolysis to produce ceramide and necessary for neural crest EMT. We uncovered three enhancer regions within the first intron of the SMPD3 locus that drive reporter expression in distinct spatial and temporal domains, together collectively recapitulating the expression domains of endogenous SMPD3 within the ectodermal lineages. We further dissected one enhancer that is specifically active in the migrating neural crest. By mutating putative transcriptional input sites or knocking down upstream regulators, we find that the SOXE-family transcription factors SOX9 and SOX10 regulate the expression of SMPD3 in migrating neural crest cells. Further, ChIP-seq and nascent transcription analysis reveal that SOX10 directly regulates expression of an SMPD3 enhancer specific to migratory neural crest cells. Together these results shed light on how core components of developmental gene regulatory networks interact with metabolic effector genes to control changes in membrane lipid content.

Comparative genomic analysis of Periweissella and the characterization of novel motile species.

The genus Periweissella was proposed as a novel genus in the Lactobacillaceae in 2022. However, the phylogenetic relationship between Periweissella and other heterofermentative lactobacilli, and the genetic and physiological properties of this genus remain unclear. This study aimed to determine the phylogenetic relationship between Periweissella and the two closest genera, Weissella and Furfurilactobacillus, by the phylogenetic analysis and calculation of (core gene) pairwise average amino acid identity. Targeted genomic analysis showed that fructose bisphosphate aldolase was only present in the genome of Pw. cryptocerci. Mannitol dehydrogenase was found in genomes of Pw. beninensis, Pw. fabaria, and Pw. fabalis. Untargeted genomic analysis identified the presence of flagellar genes in Periweissella but not in other closely related genera. Phenotypes related to carbohydrate fermentation and motility matched the genotypes. Motility genes were organized in a single operon and the proteins shared a high amino acid similarity in the genus Periweissella. The relatively low similarity of motility operons between Periweissella and other motile lactobacilli indicated the acquisition of motility by the ancestral species. Our findings facilitate the phylogenetic, genetic, and phenotypic understanding of the genus Periweissella.ImportanceThe genus Periweissella is a heterofermentative genus in the Lactobacillaceae which includes predominantly isolates from cocoa fermentations in tropical climates. Despite the relevance of the genus in food fermentations, genetic and physiological properties of the genus are poorly characterized and genome sequences became available only after 2020. This study characterized strains of the genus by functional genomic analysis, and by determination of metabolic and physiological traits. Phylogenetic analysis revealed that Periweissella is the evolutionary link between rod-shaped heterofermentative lactobacilli and the coccoid Leuconostoc clade with the genera Weissella and Furfurilactobacillus as closest relatives. Periweissella is the only heterofermentative genus in the Lactobacillaceae which comprises predominantly motile strains. The genomic, physiological, and metabolic characterization of Periweissella may facilitate the potential use of strains of the genus as starter culture in traditional or novel food fermentations.

Dual roles of cellular communication network factor 6 (CCN6) in the invasion and metastasis of oral cancer cells to bone via binding to BMP2 and RANKL.

The acquisition of motility via epithelial-mesenchymal transition (EMT) and osteoclast induction are essential for the invasion and metastasis of oral squamous cell carcinoma (OSCC) to bone. However, the molecule suppressing both EMT and osteoclastogenesis is still unknown. In this study, we found that cellular communication network factor 6 (CCN6) was less produced in a human OSCC cell line, HSC-3 with mesenchymal phenotype, than in HSC-2 cells without it. Notably, CCN6 interacted with bone morphogenetic protein 2 (BMP2) and suppressed the cell migration of HSC-3 cells stimulated by BMP2. Moreover, knockdown of CCN6 in HSC-2 cells led to the promotion of EMT and enhanced the effect of transforming growth factor-β (TGF-β) on the promotion of EMT. Furthermore, CCN6 combined with BMP2 suppressed EMT. These results suggest that CCN6 strongly suppresses EMT in cooperation with BMP2 and TGF-β. Interestingly, CCN6 combined with BMP2 increased the gene expression of receptor activator of nuclear factor-κB ligand (RANKL) in HSC-2 and HSC-3 cells. Additionally, CCN6 interacted with RANKL, and CCN6 combined with RANKL suppressed RANKL-induced osteoclast formation. In metastatic lesions, increasing BMP2 due to the bone destruction led to interference with binding of CCN6 to RANKL, which results in the promotion of bone metastasis of OSCC cells due to continuous osteoclastogenesis. These findings suggest that CCN6 plays dual roles in the suppression of EMT and in the promotion of bone destruction of OSCC in primary and metastatic lesions, respectively, through cooperation with BMP2 and interference with RANKL.

Dengue Virus Infection Alters Inter-Endothelial Junctions and Promotes Endothelial-Mesenchymal-Transition-Like Changes in Human Microvascular Endothelial Cells.

Dengue virus (DENV) is a pathogenic arbovirus that causes human disease. The most severe stage of the disease (severe dengue) is characterized by vascular leakage, hypovolemic shock, and organ failure. Endothelial dysfunction underlies these phenomena, but the causal mechanisms of endothelial dysfunction are poorly characterized. This study investigated the role of c-ABL kinase in DENV-induced endothelial dysfunction. Silencing c-ABL with artificial miRNA or targeting its catalytic activity with imatinib revealed that c-ABL is required for the early steps of DENV infection. DENV-2 infection and conditioned media from DENV-infected cells increased endothelial expression of c-ABL and CRKII phosphorylation, promoted expression of mesenchymal markers, e.g., vimentin and N-cadherin, and decreased the levels of endothelial-specific proteins, e.g., VE-cadherin and ZO-1. These effects were reverted by silencing or inhibiting c-ABL. As part of the acquisition of a mesenchymal phenotype, DENV infection and treatment with conditioned media from DENV-infected cells increased endothelial cell motility in a c-ABL-dependent manner. In conclusion, DENV infection promotes a c-ABL-dependent endothelial phenotypic change that leads to the loss of intercellular junctions and acquisition of motility.

Preserved correlation matrices pinpoint extracellular matrix organization as a critical factor in pancreatic ductal adenocarcinoma

Background Gene co-expression correlations frequently signal shared biological functions with coordinated regulation. We hypothesized that maintained correlations might be essential for cellular survival, representing potential vulnerabilities of cancer cells. We aimed to reveal correlations preserved in pancreatic ductal adenocarcinomas (PDAC) across normal and tumor tissues. Methods: We searched the NCBI GEO for raw microarray data and the TCGA project for RNA-seq data. The microarray dataset consisted of 248 tumors and 108 normal samples, allowing the analysis of 12,210 genes. The RNA-seq dataset incorporated 177 tumors, four normal samples from TCGA, and 248 normal samples from GTEx, enabling the analysis of 21,479 genes. Genes with an altered expression were identified with a Mann-Whitney U test at p<0.01, and a Pearson correlation was performed to identify preserved correlations. Results: Altogether 371 significant correlations involving 262 genes were preserved across normal samples and tumors in both RNA-seq and gene chip platforms. The identified close-knit gene network is mainly responsible for extracellular matrix organization. Seven genes (SPARC, COL6A3, MMP2, HTRA1, FN1, PALLD, and COL3A1) were heavily overrepresented in maintained correlations, some of them participating in as many as 58 interactions. High expression of 28 genes was linked to poor disease outcome at FDR ≤ 10%, out of which FN1, an extracellular matrix component, was both overrepresented in maintained correlations and associated with worse overall survival (p = 0.00097, FDR ≤ 5%). The growing expression of two genes, MYL12A and MYL12B, across normal tissues, primary, and metastatic tumors may drive the acquisition of motility by cancer cells. Conclusions: Our results propose novel prognostic biomarkers of PDAC and pinpoint fundamental cellular interactions as potential targets for combination therapies. Furthermore, the presence of significant correlations across different data platforms substantiates the validity of our findings.

Live imaging of delamination in Drosophila shows epithelial cell motility and invasiveness are independently regulated

Delaminating cells undergo complex, precisely regulated changes in cell–cell adhesion, motility, polarity, invasiveness, and other cellular properties. Delamination occurs during development and in pathogenic conditions such as cancer metastasis. We analyzed the requirements for epithelial delamination in Drosophila ovary border cells, which detach from the structured epithelial layer and begin to migrate collectively. We used live imaging to examine cellular dynamics, particularly epithelial cells’ acquisition of motility and invasiveness, in delamination-defective mutants during the time period in which delamination occurs in the wild-type ovary. We found that border cells in slow border cells (slbo), a delamination-defective mutant, lacked invasive cellular protrusions but acquired basic cellular motility, while JAK/STAT-inhibited border cells lost both invasiveness and motility. Our results indicate that invasiveness and motility, which are cooperatively required for delamination, are regulated independently. Our reconstruction experiments also showed that motility is not a prerequisite for acquiring invasiveness.

The Massive Impact of Ram's Sperm Starvation on the Fertilization and Blastocyst Rates in Terms of Sperm Quality and Capacitation

To obtain a high in vitro fertilization rate while ensuring the ideal development of the
 embryos, thus eliminating the most critical problems of assisted reproductive
 technology (ART) (implantation and low pregnancy rates). Therefore, sperm taken
 from the caudal epididymis must undergo the acquisition of progressive motility and
 fertilization. This process takes place through a series of physiological and biochemical
 changes, known as capacitation, a prerequisite for fertilization. In this work, we have
 developed a new way to incubate sperm using four different treatments in terms of
 energy substrates and different incubation methods. In this sperm energy restriction and
 recovery (RSE) treatment, after starving sperm for 50 min until they lost their capacity
 for motility, they were given glucose and incubated for 60 min. As for the other
 treatments, the sperm were not exposed to starvation and were incubated by the
 standard methods of maturation and capacitation of sperm, but with different energy
 substrates. According to the results obtained, the sperm was hyperactive and showed a
 significant increase in the ability to fertilize oocytes in the treatment that involved
 starvation and then saving the sperm by adding glucose, compared to other treatments.
 In conclusion, the effects of this feature persisted until after fertilization, which led to
 the production of a high percentage of blastocysts, as well as it has found that starvation
 and rescue sperm are adequate for fertilizing and developing embryos in sheep.

Progress in the effect of human epididymis protein 4 on sperm maturation

Human epididymis protein 4(HE4) is a secretory glycoprotein found in human distal epididymis epithelial cells. It is often used in the early diagnosis, efficacy evaluation and monitoring of ovarian cancer, and also has been considered as an effective serum marker for many other types of cancer. However, its function in the process of sperm maturation is not fully unknown. The maturation of sperm in epididymis is characterized by the acquisition of motility and fertilization. As a member of the whey acid protein (WAP) family, several studies proposed the importance of HE4 in the maturity of sperm in epididymis. This article reviews the effect of HE4 on spermatozoa maturation in epididymis, which provides basis for the evaluation of male reproductive ability, early detection, early diagnosis and pathogenesis of male infertility.

P-089 Characterization of Epididymosomes and Prostasomes in Seminal Plasma of Infertile Males

Abstract Study question Could Extracellular Vesicles such as Epididymosomes and Prostasomes be employed as a new tool to characterize the male fertility potential? Summary answer Extracellular vesicles (EVs) content represents a new approach to the discovery of novel molecular mechanisms involved in sperm cell maturation and acquisition of fertilizing ability. What is known already Cells throughout the reproductive tract release EVs that are sorted by the endosomal compartment. These vesicles attract great attention because they contain lipids, proteins, nucleic acids that have been shown to be transferred to maturing sperm cells. EVs in seminal plasma, such as epididymosomes and prostasomes, contribute about 3% of the total associated seminal plasma proteins and are recognized as being responsible for sperm maturation, positively influencing actual fertilization. Several studies have documented the proteomic profile of these vesicles, but little is known about their specific contribution to sperm maturation, motility and acquisition of fertilizing capacity. Study design, size, duration 48 Caucasian males underwent semen evaluation at the IVF Unit, Siena University Hospital, from October 2020 to November 2021. According to seminal parameters (WHO,2010), they were divided into Normozoospermic (N; n = 18); OligoAsthenoTeratozoospermic (OAT; n = 15); Azoospermic (A; n = 15). We excluded patients with an intake of spermiotoxic drugs, smoking, alcohol, or drugs abuse. The mean age was 33 years; the BMI ranged between 18-25. All participants signed a written informed consent, approved by the ethics committee. Participants/materials, setting, methods EVs from seminal plasma were isolated by an in-house modified EvGAG®-polymer precipitation-based protocol and characterized for their size and morphology by Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), respectively. Full proteomic profiling of these vesicles was obtained by 2-D electrophoresis and mass spectrometry, and further investigations by functional Omics analysis by bioinformatic tools (as. Metacore, STRING). Gene expression was evaluated by digital PCR (ddPCR). Main results and the role of chance The present work highlights the impact of EVs on male fertility due to their ability to transport selected molecules to maturing sperm cells, thus providing a specific proteomic and transcriptomic profile based on their fertilizing capacity. Proteomic profiling of OAT and A vs N samples provided crucial information regarding differential protein expression, thus obtaining information on the correlation between the encapsulated content of EVs and sperm parameters. The ddPCR analysis of specific genes encoding for proteins expressed differently among patients confirmed that EVs may not only be potential biomarkers for male infertility, but at the same time contribute as an important regulator of specific pathways concerning sperm cell modification through the male reproductive tract. Genes involved in sperm-egg binding and zona pellucida interactions were overexpressed in N compared to OAT and A. Congruent results were obtained for genes involved in sperm cell metabolism. Meanwhile, regarding the acquisition of motility, some genes were found overexpressed in the OAT groups compared to N, indicating that some proteins delivered by EVs have a negative impact on sperm cell motility and function. Limitations, reasons for caution This study represents a preliminary experiment. A confirmatory study on an enlarged number of samples is needed to provide more statistical strength by reducing the effect of inter-sample variability. Wider implications of the findings Seminal extracellular vesicles secreted along the male reproductive tract have been demonstrated to be involved in the process of sperm maturation and could represent a new appealing therapeutic and diagnostic tool in the field of human reproduction for male fertility/infertility. Trial registration number Not applicable

Male SIRT1 insufficiency leads to sperm with decreased ability to hyperactivate and fertilize.

Deficient sperm motility is a frequent cause of the age-related male sub-/infertility. Since the protein sirtuin 1 (SIRT1) develops anti-aging action and participates in sperm motility and ATP synthesis in mitochondria, we investigated its role in the acquisition of hyperactivated motility during capacitation. For this, the dynamics of sperm subpopulations were studied, using males of Sirt1+/- heterozygous mutant mice. After 2hr of capacitation, we observed reduced percentage of hyperactivated spermatozoa in Sirt1+/- males. Interestingly, prior to capacitation, Sirt1+/- spermatozoa showed higher mitochondrial superoxide levels, which could render mitochondrial injury and thereby motility defects. Accordingly, the fertilization rate of Sirt1+/- males after mating was decreased. We elucidated that SIRT1 male insufficiency underlies posterior sperm defects to hyperactivate during capacitation and propose Sirt1+/- males as a model for the study of the age-related infertility.

Increased ATG5 Expression Predicts Poor Prognosis and Promotes EMT in Cervical Carcinoma.

Cervical cancer has the second-highest incidence and mortality of female malignancy. The major causes of mortality in patients with cervical cancer are invasion and metastasis. The epithelial–mesenchymal transition (EMT) process plays a major role in the acquisition of metastatic potential and motility. Autophagy-related genes (ARGs) are implicated in the EMT process, and autophagy exerts a dual function in EMT management at different phases of tumor progression. However, the role of specific ARGs during the EMT process has not yet been reported in cervical cancer. Based on the data from the Cancer Genome Atlas (TCGA) cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) sequencing database, we performed the prognosis analysis for those ARGs obtained from the Human Autophagy database. ATG5 was identified as the only important harmful marker influencing survival of cervical cancer patients by univariate Cox regression (HR 1.7; 95% CI: 1.0–2.8, p = 0.047), and the 5-years survival rate for the high- and low-ATG5 expression groups was 0.486 (0.375–0.631) and 0.782 (0.708–0.863), respectively. TCGA CESC methylation data showed that eight methylation sites of ATG5 could also be significantly associated with the overall survival (OS) of cervical cancer patients. Single-sample gene-set enrichment and gene functional enrichment results showed that ATG5 was correlated with some cancer-related pathways, such as phagocytosis-related genes, endocytosis-related genes, immune-related genes, EMT score, and some EMT signature-related genes. Next, cell migration and invasion assay and Western blot were applied to detect the function of ATG5 in EMT of cervical cancer. In cervical cancer cells, ATG5 knockdown resulted in attenuation of migration and invasion. The functional study showed that knockdown of ATG5 could reverse EMT process by P-ERK, P-NFκBp65, P-mTOR pathways, and so on. In conclusion, the present study implies that ATG5 was a major contributor to EMT regulation and poor prognosis in cervical cancer.

Caput Ligation Renders Immature Mouse Sperm Motile and Capable to Undergo cAMP-Dependent Phosphorylation.

Mammalian sperm must undergo two post-testicular processes to become fertilization-competent: maturation in the male epididymis and capacitation in the female reproductive tract. While caput epididymal sperm are unable to move and have not yet acquired fertilization potential, sperm in the cauda epididymis have completed their maturation, can move actively, and have gained the ability to undergo capacitation in the female tract or in vitro. Due to the impossibility of mimicking sperm maturation in vitro, the molecular pathways underlying this process remain largely unknown. We aimed to investigate the use of caput epididymal ligation as a tool for the study of sperm maturation in mice. Our results indicate that after seven days of ligation, caput sperm gained motility and underwent molecular changes comparable with those observed for cauda mature sperm. Moreover, ligated caput sperm were able to activate pathways related to sperm capacitation. Despite these changes, ligated caput sperm were unable to fertilize in vitro. Our results suggest that transit through the epididymis is not required for the acquisition of motility and some capacitation-associated signaling but is essential for full epididymal maturation. Caput epididymal ligation is a useful tool for the study of the molecular pathways involved in the acquisition of sperm motility during maturation.

RAC1B: A Guardian of the Epithelial Phenotype and Protector Against Epithelial-Mesenchymal Transition.

The small GTPase Ras-related C3 botulinum toxin substrate 1B (RAC1B) has been shown to potently inhibit transforming growth factor (TGF)-β1-induced cell migration and epithelial-mesenchymal transition (EMT) in pancreatic and breast epithelial cells, but the underlying mechanism has remained obscure. Using a panel of pancreatic ductal adenocarcinoma (PDAC)-derived cell lines of different differentiation stages, we show that RAC1B is more abundantly expressed in well differentiated as opposed to poorly differentiated cells. Interestingly, RNA interference-mediated knockdown of RAC1B decreased expression of the epithelial marker protein E-cadherin, encoded by CDH1, and enhanced its TGF-β1-induced downregulation, whereas ectopic overexpression of RAC1B upregulated CDH1 expression and largely prevented its TGF-β1-induced silencing of CDH1. Conversely, knockdown of RAC1B, or deletion of the RAC1B-specific exon 3b by CRISPR/Cas-mediated genomic editing, enhanced basal and TGF-β1-induced upregulation of mesenchymal markers like Vimentin, and EMT-associated transcription factors such as SNAIL and SLUG. Moreover, we demonstrate that knockout of RAC1B enhanced the cells’ migratory activity and derepressed TGF-β1-induced activation of the mitogen-activated protein kinase ERK2. Pharmacological inhibition of ERK1/2 activation in RAC1B-depleted cells rescued cells from the RAC1B knockdown-induced enhancement of cell migration, TGF-β1-induced downregulation of CDH1, and upregulation of SNAI1. We conclude that RAC1B promotes epithelial gene expression and suppresses mesenchymal gene expression by interfering with TGF-β1-induced MEK-ERK signaling, thereby protecting cells from undergoing EMT and EMT-associated responses like acquisition of cell motility.

Transition from mesenchymal to bleb-based motility is predominantly exhibited by CD133-positive subpopulation of fibrosarcoma cells.

Metastatic disease is caused by the ability of cancer cells to reach distant organs and form secondary lesions at new locations. Dissemination of cancer cells depends on their migration plasticity - an ability to switch between motility modes driven by distinct molecular machineries. One of such switches is mesenchymal-to-amoeboid transition. Although mesenchymal migration of individual cells requires Arp2/3-dependent actin polymerisation, amoeboid migration is characterised by a high level of actomyosin contractility and often involves the formation of membrane blebs. The acquisition of amoeboid motility by mesenchymal cells is often associated with enhanced metastasis. We studied the ability of mesenchymal HT1080 fibrosarcoma cells to switch to amoeboid motility. We induced the transition from lamellipodium-rich to blebbing phenotype either by down-regulating the Arp2/3 complex, pharmacologically or by RNAi, or by decreasing substrate adhesiveness. Each of these treatments induced blebbing in a subset of fibrosarcoma cells, but not in normal subcutaneous fibroblasts. A significant fraction of HT1080 cells that switched to blebbing behaviour exhibited stem cell-like features, such as expression of the stem cell marker CD133, an increased efflux of Hoechst-33342 and positive staining for Oct4, Sox2 and Nanog. Furthermore, the isolated CD133+ cells demonstrated an increased ability to switch to bleb-rich amoeboid phenotype both under inhibitor's treatment and in 3D collagen gels. Together, our data show a significant correlation between the increased ability of cells to switch between migration modes and their stem-like features, suggesting that migration plasticity is an additional property of stem-like population of fibrosarcoma cells. This combination of features could facilitate both dissemination of these cells to distant locations, and their establishment self-renewal in a new microenvironment, as required for metastasis formation. These data suggest that migration plasticity is a new feature of cancer stem-like cells that can significantly facilitate their dissemination to a secondary location by allowing them to adapt quickly to challenging microenvironments. Moreover, it complements their resistance to apoptosis and self-renewal potential, thus enabling them not only to disseminate efficiently, but also to survive and colonise new niches.

Endothelial to Mesenchymal Transition: Role in Physiology and in the Pathogenesis of Human Diseases.

Numerous studies have demonstrated that endothelial cells are capable of undergoing endothelial to mesenchymal transition (EndMT), a newly recognized type of cellular transdifferentiation. EndMT is a complex biological process in which endothelial cells adopt a mesenchymal phenotype displaying typical mesenchymal cell morphology and functions, including the acquisition of cellular motility and contractile properties. Endothelial cells undergoing EndMT lose the expression of endothelial cell-specific proteins such as CD31/platelet-endothelial cell adhesion molecule, von Willebrand factor, and vascular-endothelial cadherin and initiate the expression of mesenchymal cell-specific genes and the production of their encoded proteins including α-smooth muscle actin, extra domain A fibronectin, N-cadherin, vimentin, fibroblast specific protein-1, also known as S100A4 protein, and fibrillar type I and type III collagens. Transforming growth factor-β1 is considered the main EndMT inducer. However, EndMT involves numerous molecular and signaling pathways that are triggered and modulated by multiple and often redundant mechanisms depending on the specific cellular context and on the physiological or pathological status of the cells. EndMT participates in highly important embryonic development processes, as well as in the pathogenesis of numerous genetically determined and acquired human diseases including malignant, vascular, inflammatory, and fibrotic disorders. Despite intensive investigation, many aspects of EndMT remain to be elucidated. The identification of molecules and regulatory pathways involved in EndMT and the discovery of specific EndMT inhibitors should provide novel therapeutic approaches for various human disorders mediated by EndMT.

Potassium channel activity controls breast cancer metastasis by affecting \u03b2-catenin signaling

Potassium ion channels are critical in the regulation of cell motility. The acquisition of cell motility is an essential parameter of cancer metastasis. However, the role of K+ channels in cancer metastasis has been poorly studied. High expression of the hG1 gene, which encodes for Kv11.1 channel associates with good prognosis in estrogen receptor-negative breast cancer (BC). We evaluated the efficacy of the Kv11.1 activator NS1643 in arresting metastasis in a triple negative breast cancer (TNBC) mouse model. NS1643 significantly reduces the metastatic spread of breast tumors in vivo by inhibiting cell motility, reprogramming epithelial–mesenchymal transition via attenuation of Wnt/β-catenin signaling and suppressing cancer cell stemness. Our findings provide important information regarding the clinical relevance of potassium ion channel expression in breast tumors and the mechanisms by which potassium channel activity can modulate tumor biology. Findings suggest that Kv11.1 activators may represent a novel therapeutic approach for the treatment of metastatic estrogen receptor-negative BC. Ion channels are critical factor for cell motility but little is known about their role in metastasis. Stimulation of the Kv11.1 channel suppress the metastatic phenotype in TNBC. This work could represent a paradigm-shifting approach to reducing mortality by targeting a pathway that is central to the development of metastases.

Expressional and functional analyses of epididymal SPINKs in mice

Epididymal maturation is critical for acquisition of motility and fertilizing capacity by sperm. During epididymal transit, the surface of sperm undergoes prominent sequential changes through interactions with secreted proteins, including protease inhibitors. In the present study, we characterized three epididymis-specific SPINKs (serine protease inhibitors, Kazal-type): SPINK8, SPINK11, and SPINK12. We found that these epididymal SPINKs are expressed in an epididymal region-specific manner and their expression is developmentally regulated. Remarkably, cellular analyses revealed that SPINK8 and SPINK12 are transferred to the sperm. To investigate the in vivo properties of SPINK12, we analyzed knockout mice generated by CRISPR/Cas9-mediated genome editing. Loss of SPINK12 did not alter epididymal tubule structure or sperm phenotypes. Spink12 mutant mice exhibited normal fertility, suggesting that SPINK12 is functionally redundant in the epididymis.

Abstract 1090: The role of connexin43 in prostate cancer motility

Abstract Prostate cancer (PC) is one of the most common male cancers in United States. PC lethality is due to metastatic growth, when cancer cells move from the primary tumor site to a secondary site. A key step in metastsis is the acquisition of motility. Connexins (Cx) play a major role in cell-cell communication and growing evidence demonstrates their contribution to cell motility. Cx43 is up-regulated in cultured PC3 metastatic prostate cancer cells. To understand the role of Cx43 in migration we asked whether silencing Cx43 would decrease PC3 cell migration. Using cells in which short interfering RNA (sh-RNA) silenced Cx43 mRNA, we showed that silencing Cx43 decreased motility of sh-Cx43 PC3 cells in Boyden chamber assays. To confirm these results in another biological system, we performed cellular wound healing assays. Silencing Cx43 decreased motility of PC3 cells reducing movement to wounded area, leaving more area open in the Cx43 inhibited cell lines. The wound healing assay showed that knockdown Cx43 altered directional movement, velocity and distance. These results supported those of our Boyden assays showing that loss of Cx43 decreases the migratory ability of PC3. Conversely, the overexpression of Cx43 in LNCaP cells, non-migratory prostate cancer cells that lack Cx43 expression, increased their motility. A comparison of cell lines engineered to overexpress or to silence Cx43 confirms the importance of Cx43 in the motility of prostate cancer cells in vitro. Although the mechanism whereby Cx43 regulates motility is still unclear, our preliminary data revealed that Cx43 co-immunoprecipitated with N-cadherin, suggesting that they form a complex in PC3 cells. Cell fractionation studies of PC3 control cells showed that N-cadherin was located in the cell membrane and not detected free in the cytoplasm, but in sh-Cx43 PC3 cells that lack Cx43, the distribution of N-cadherin protein was altered and significant levels of N-cadherin were also detected in cytoplasmic fractions. Whether changes in N-cadherin distribution is a direct or indirect result of loss of Cx43 in sh-Cx43 PC3 cells, is unclear. However, modulation of Cx43 levels in prostate cancer cells does alter motility and N-cadherin distribution. These results indicate that Cx43 and N-cadherin are in close association in PC3 cells and their interaction may play an important role in prostate cancer cell motility. A better understanding of the requirements of migratory PC cells may lead to more effective targeting of key steps in PC progression to lethal metastatic disease. Citation Format: Abdulaziz A. Aloliqi. The role of connexin43 in prostate cancer motility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1090.

Changes in Protein O-GlcNAcylation During Mouse Epididymal Sperm Maturation.

After leaving the testis, sperm undergo two sequential maturational processes before acquiring fertilizing capacity: sperm maturation in the male epididymis, and sperm capacitation in the female reproductive tract. During their transit through the epididymis, sperm experience several maturational changes; the acquisition of motility is one of them. The molecular basis of the regulation of this process is still not fully understood. Sperm are both transcriptionally and translationally silent, therefore post-translational modifications are essential to regulate their function. The post-translational modification by the addition of O-linked β-N-acetylglucosamine (O-GlcNAc) can act as a counterpart of phosphorylation in different cellular processes. Therefore, our work was aimed to characterize the O-GlcNAcylation system in the male reproductive tract and the occurrence of this phenomenon during sperm maturation. Our results indicate that O-GlcNAc transferase (OGT), the enzyme responsible for O-GlcNAcylation, is present in the testis, epididymis and immature caput sperm. Its presence is significantly reduced in mature cauda sperm. Consistently, caput sperm display high levels of O-GlcNAcylation when compared to mature cauda sperm, where it is mostly absent. Our results indicate that the modulation of O-GlcNAcylation takes place during sperm maturation and suggest a role for this post-translational modification in this process.