Mouse models of preterm birth: Suggested assessment and reporting guidelines

Preterm birth affects approximately 1 out of every 10 births in the United States, leading to high rates of mortality and long-term negative health consequences. To investigate the mechanisms leading to preterm birth so as to develop prevention strategies, researchers have developed numerous mouse models of preterm birth. However, the lack of standard definitions for preterm birth in mice limits our field\u27s ability to compare models and make inferences about preterm birth in humans. In this review, we discuss numerous mouse preterm birth models, propose guidelines for experiments and reporting, and suggest markers that can be used to assess whether pups are premature or mature. We argue that adoption of these recommendations will enhance the utility of mice as models for preterm birth

Inhibition of de novo ceramide biosynthesis affects aging phenotype in an in vitro model of neuronal senescence.

Although aging is considered to be an unavoidable event, recent experimental evidence suggests that the process can be counteracted. Intracellular calcium (Ca2+i) dyshomeostasis, mitochondrial dysfunction, oxidative stress, and lipid dysregulation are critical factors that contribute to senescence-related processes. Ceramides, a pleiotropic class of sphingolipids, are important mediators of cellular senescence, but their role in neuronal aging is still largely unexplored. In this study, we investigated the effects of L-cycloserine (L-CS), an inhibitor of thede novoceramide biosynthesis, on the aging phenotype of cortical neurons cultured for 22 days, a setting employed as anin vitromodel of senescence. Our findings indicate that, compared to control cultures, 'aged' neurons display dysregulation of [Ca2+]ilevels, mitochondrial dysfunction, increased generation of reactive oxygen species (ROS), altered synaptic activity as well as the activation of neuronal death-related molecules. Treatment with L-CS positively affected the senescent phenotype, a result associated with recovery of neuronal [Ca2+]isignaling and reduction of mitochondrial dysfunction and ROS generation. The results suggest that thede novoceramide biosynthesis represents a critical intermediate in the molecular and functional cascade leading to neuronal senescence and identify ceramide biosynthesis inhibitors as promising pharmacological tools to decrease age-related neuronal dysfunctions

Genome Maintenance by Selenoprotein H in the Nucleolus

Selenoprotein H (SELENOH) is a nucleolar oxidoreductase with DNA binding properties whose function is not well understood. To determine the functional and physiological roles of SELENOH, a knockout of SELENOH was generated in cell lines using CRISPR/Cas9-mediated genomic deletion and in mice by targeted disruption. Based on the sequenced genome, the results of deduced protein sequences indicated various forms of mutants in the CRISPR/Cas9-mediated knockout, including a frame-shift by aberrant splicing and truncated SELENOH by early termination of the translation process. Loss of SELENOH in HeLa cells induced slow cell proliferation, the formation of giant multinucleated cells, accumulation of unrepaired DNA damage and oxidative stress, and cellular senescence. SELENOH cells were enlarged and possessed a single large nucleolus. Atomic force microscope showed increased stiffness in the nucleoli of SELENOH knockout cells, which suggests that SELENOH maintains the flexible structure of the nucleolus. Furthermore, the knockout of SELENOH led to a large-scale reorganization of the nucleolar architecture with the movement of nucleolar protein into nucleolar cap regions in response to oxidative stress. The nucleolar reorganization is dependent on ATM signaling. Altogether, results suggest that SELENOH appears to be a sensor of oxidative stress that plays critical roles in redox regulation and genome maintenance within the nucleolus. To determine the physiological role of SELENOH in vivo, Selenoh knockout mice were generated by targeted deletion through homologous recombination. Selenoh+/− mice were fertile and phenotypically indistinguishable from wild-type littermates. Results from matings of Selenoh+/− mice showed a significantly reduced fraction of Selenoh−/− offspring on the basis of Mendelian segregation. Since some Selenoh−/− were born, it is likely that Selenoh is a partially essential gene in mice. Live-born Selenoh−/− mice were viable and born without apparent phenotypes. Selenoh−/− mice at 2-month of age showed increased GPX activity in the lung but not in the brain and liver. Furthermore, loss of Selenoh resulted in the aggravated formation of aberrant crypt foci in the colon of Selenoh+/− mice that were injected with azoxymethane. Altogether, SELENOH has critical roles in embryogenesis and colorectal carcinogenesis

Healing and repair mechanisms in fetal membrane defects after trauma, strain and fetal surgery

Iatrogenic preterm premature rupture of the fetal membranes (iPPROM) is a major complication after diagnostic or invasive fetal surgical interventions, often associated with adverse perinatal outcomes. Strategies to seal human FM defects with glues, collagen or fibrin plugs have failed to restore structural function of the FM and promote tissue regeneration and defect repair, and none are in routine clinical use. In this thesis, the mechanisms of human FM healing after iatrogenic rupture were investigated, to translate this to pregnancies affected by spontaneous PPROM. The concentration of the gap-junction protein Connexin 43 (Cx43) has been associated with poor wound healing mechanisms in skin models and its expression was increased in FM defect sites taken from fetoscopic interventions. The present study examined the healing mechanisms in amniotic membrane (AM) defects after trauma, cyclic tensile strain (CTS) and fetal surgery. There was evidence that Cx43 increased in expression in term AM following in vitro trauma concurrent with αSMA-expressing myofibroblast differentiation and collagen polarisation examined by SHG imaging. Both the wound healing marker TGFβ1 and Cx43 gene expression were significantly increased after trauma. To investigate mechanotransduction mechanisms, preterm AM was subjected to 2% CTS for 24 hours. Collagen fibres were polarised in the direction of strain concurrent with differential effects on markers for ECM (collagen, elastin, GAGs), inflammation (PGE2) and healing (Cx43 and TGFβ1) in a donor and tissue-dependent manner. Morphological changes in AM cell populations in iPPROM defects and spontaneous PPROM showed myofibroblast differentiation, changes in collagen alignment and lack of wound closure. The results obtained in this study contribute to our knowledge on AM wound healing and repair capabilities, mechanotransduction mechanisms in preterm AM and the influence of fetal surgical interventions on AM cell types and collagen integrity. Establishing how Cx43 regulates AM cell function and healing after trauma and CTS could be an approach to repair defects in FM

Myometrial cyclic AMP function

Background Uncovering the processes that drive labour onset is essential to reduce the adverse consequences of dysfunctional labour. Myometrial cAMP signalling is upregulated during pregnancy promoting uterine quiescence. Changes in its components and effectors have been identified at the onset of term labour. Preterm labour (PTL) treatments targeting this pathway have limited effectiveness and serious maternal effects. In this study, real-time FRET imaging was used to investigate compartmentalised cAMP signals at distinct cellular sites. Methods Myometrial biopsies were obtained from women at term or in distinct causes of PTL. Tissues were processed for mRNA and protein extraction or cell isolation. Primary myometrial cells (HPMCs) and an hTERT-HM cell line expressed either a cytosolic (EPAC-SH187) or plasmalemma (AKAP79-CUTie) genetically encoded FRET sensor. The florescence emission changes were monitored following isoproterenol and PGE2 treatment to determine intracellular cAMP concentrations. Results Differences in cAMP signalling components were detected in PTL compared to term with variations in effector predominance and an associated increase in OTR expression in twin-PTL. Stimulus-specific subcellular compartmentalisation of cAMP was identified in both cell types with differential regulation by phosphodiesterases (PDEs). Significant disparities were detected in the amplitude, kinetics, and regulation of cAMP signals between the two cell types. For the HPMCs, a prolonged time in culture was associated with a reduction in PDE activity and altered cell phenotype. Conclusion The cAMP signalling system is influential in the final pathway of labour, primarily regulating OTR expression. This study established the technique of FRET imaging in human myometrial cells, determining the cell model of choice and culture conditions to explore localised cAMP signalling. The findings provide new insights into the spatial and temporal dynamics of cAMP in the human myometrium and pave the way for unravelling the details of how this fundamental pathway operates and its role in pregnancy and labour.Open Acces

Apoptosis in spermatozoa of infertile men, clinical correlations

The methods for evaluation of male infertility include not only routine investigations, standardized by the WHO, but also complementary techniques, developed over the last years, in order to improve the predictive value of seminal analysis for natural conception and assisted reproduction. With reference to these new methods, studies suggest that sperm with certain levels of DNA fragmentation serve as a strong predictor of reduced male fertility. We studied subjects who underwent seminal fluid evaluation, because of an infertility condition, at the Department of Biomedical Sciences of the University of Sassari.The samples collected by masturbation were evaluated according to the World Health Organisation (1999).The samples was washed twice in PBS and cytocentrifuged for 10 min at 1800 rpm on polylysine-coated slides that were fixed in methanol at room temperature. The apoptosis was evaluated using the TUNEL (In Situ Cell Death Detection Kit, Fluorescein, Roche, Cat.No. 1 684 795). At fluorescent miscroscopy are counted at least 300 cells. Quantitative evaluation of apoptosis by the TUNEL method confirmed that apoptosis did not seem to be correlated with sperm concentration or morphology; however, we found a higher apoptotic rate in semen from patients affected by andrologic diseases, such as varicocele, than from those with alteration of semen characteristics. Apoptosis analysis might be used in infertile patients in order to understand the etiology of unexplained infertility and to improve therapeutic effectiveness