We have recently shown that many mediators of the JAK/STAT signaling pathway are present in ejaculated human spermatozoa. Among them, STAT3 is detected mainly in membranes and flagellar cytoskeletal fractions. In order to determine the importance of STAT3-mediated signaling, sperm were incubated with Stattic V, a specific inhibitor. Effects on motility were evaluated by CASA, sperm acrosomal integrity was evaluated by FITC conjugated lectin (PSA or PNA) staining, and protein phosphotyrosine content was assessed by Western blot using a monoclonal anti-phosphotyrosine antibody. INDO1-AM and JC-1 were used to measure sperm intracellular calcium and mitochondrial membrane potential, respectively, by flow cytometry, and reactive oxygen species (ROS) production was investigated by luminol-based assay. Percentages of motility and motility parameters were significantly affected by Stattic V. This later also significantly increased intracellular Ca(2+) levels, progesterone- and calcium ionophore (A23187)-induced acrosome reaction. On the other hand, a significant decrease in ATP content was measured when sperm were treated with Stattic V, associated with depolarization of mitochondrial membrane and elevated ROS production. These results suggest that STAT3 is involved in sperm functions, at least through regulation of mitochondrial activity. This further emphasizes that STAT3 mediates cellular activities in a manner different than strictly the activation of gene transcription.
Abstract We previously showed that Stattic V (Stat3 inhibitory compound V) reduces human sperm motility and cellular ATP levels, increases intracellular Ca 2+ concentration, and promotes mitochondrial membrane depolarization resulting in increased levels of extracellular reactive oxygen species ( ROS ). As these alterations in cellular function are highly similar to what is observed in a cell undergoing apoptosis, our goal was to determine if the immobilizing effect of Stattic V on spermatozoa results from apoptosis or was because of an oxidative stress. To address this question, spermatozoa were incubated with Stattic V in combination with a caspase inhibitor, a proteasome inhibitor or a cell permeant ROS scavenger. Following incubation in different conditions, sperm motility was evaluated by CASA , acrosomal integrity by FITC conjugated Pisum sativum agglutinin ( PSA ‐ FITC ) labeling, intracellular pH , and mitochondrial superoxide production by flow cytometry using BCECF and MitoSoxRed dye, respectively. Levels of reduced thiols were assessed by iodoacetamidofluorescein staining on total and on sperm surface proteins, and protein tyrosine phosphorylation was evaluated by western blot. The loss in sperm motility induced by Stattic V was associated with a slight intracellular acidification and an important increase in intracellular superoxide anion. Unlike caspase and proteasome inhibitors, low molecular weight thiols, such as N‐acetyl‐L‐cysteine ( NAC ), prevented Stattic V‐induced sperm immobilization and increase responsiveness to acrosome reaction inducers. NAC also efficiently prevented the production of superoxide anion, mitochondrial membrane depolarization, intracellular acidification and the oxidation of protein free thiols caused by Stattic V. These results show that the deleterious effects of Stattic V on sperm functions are caused directly or indirectly by excessive intracellular ROS production without causing sperm apoptosis or necrosis.
Medical fetal imaging is a crucial tool for improving medical care and postnatal prognosis. LUMIERE is a research platform with dedicated US/MRI machines for fetal imaging. The challenge is to create and organise an evolving database allowing listing, searching and visualisation of fetal imaging examinations for future educational purposes, diagnosis improvement or AI application. We acquired US/MRI data on volunteer women (NCT04142606) at LUMIERE and stocked their information in a dedicated Case Report Form (CRF). A thesaurus containing principal anatomies of interest in fetal imaging was added to the CRF for examination description. Regarding MRI/US DICOMs storage, we created local patient specific folders with a predefined hierarchy fitting CRF structure and thesaurus. We extracted the CRF into an Excel file which permit with a Python program to complete a SQL database designed around an easy-to-use processing platform coded in PHP and HTML, a questionnaire structured by fetal medicine referents, radiologists and obstetricians using a POST protocol for examination searching and an examination visualisation thanks to a DICOM plugin viewer. We secured all of this with a password encrypted in sha256. We obtained an evolving database with its research platform and exhaustive structured questionnaire secured by an authentication page. Regarding his need, the user search is specified according to fetal imaging exam characteristics such as the anatomy, the pathology, if applicable and the term of the pregnancy. Searching results shows the exams fitting the user request with a DICOM viewer on which we can apply some parameters changes. Creating a fetal MRI/US imaging database with patient specific results process, will help searchers to find multimodality images easily with specific chosen pregnancy parameters and allow with AI, the creation of a fundamental and powerful tool which can improve and ease searcher's network on obstetrics, gynecology and radiology.
ABSTRACT: An increase in protein tyrosine phosphorylation occurs during sperm capacitation in numerous species. The involvement of Src-related tyrosine kinases in this phenomenon has been demonstrated using different inhibitors specifically targeting this family of enzymes. In mammals, this group of nonreceptor tyrosine kinases is made up of 8 members with similar SRC homology domain 3 (SH3) and SH2 domains. Although some members of this group of enzymes can compensate for one another, showing some redundancy, each is unique and may perform specific functions during male germ cell development. To further characterize the importance of Src-related tyrosine kinases in the events leading to proper sperm formation, and because no inhibitor affecting a single gene product exists, expression of Src, Yes1, Fyn, Lyn, Lck, Hck, Blk, and Fgr was assessed by real-time polymerase chain reaction in developing mouse testes and in enriched populations of mouse spermatogenic cells, revealing distinct expression profiles for each kinase during testis development and in isolated male germ cells. Immunolocalization of SRC, LYN, and HCK in adult mouse testes as well as in mature spermatozoa further confirmed differential localization of these kinases during spermatogenesis. Although mRNA levels of these latter kinases were higher in spermatogonia and spermatocytes than in spermatids, protein levels were highest in spermatids, suggesting delayed transcript translation. Taken together, these results clearly show an uneven expression of each kinase in different spermatogenic cells, indicating that each member may play a different role during spermatogenesis, in addition to highlighting the complexity of Src-related kinase expression regulation in male germ cells. Furthermore, differential localization of these tyrosine kinases in mature spermatozoa also suggests a specific role for each member in sperm function and integrity.
Previous studies from our laboratory have shown that a decrease in the calmodulin binding properties of a few sperm proteins occurs during the capacitation process, an effect associated with a decrease in intracellular calmodulin concentrations. Using biotinylated-calmodulin nitrocellulose overlay assay on protein extracts of subcellular fractions of bull spermatozoa, one of these proteins (p32) is detected in the flagellar-enriched fractions, whereas p30 is found in the fraction enriched with sperm heads. This latter calmodulin binding protein, p30, appears to be associated with the perinuclear theca. None of these binding proteins was solubilized by nonionic detergents. Sodium dodecyl sulfate was effective solubilizing p32, whereas p30 was extracted only in conditions reported to isolate the perinuclear theca. Cellular localization of calmodulin binding proteins was also achieved by incubating spermatozoa fixed on slides with biotinylated calmodulin and revealed in a further step by fluorescein-conjugated streptavidin. Using this procedure, it was found that calmodulin binds to the sub- and postacrosomal areas of the sperm head along with the midpiece in the presence of Ca2+. Only a sharp band of fluorescence at the subacrosomal area was observed when this procedure was performed in the absence of Ca2+ in the presence of EGTA. The pattern of cellular calmodulin binding was highly decreased when spermatozoa were incubated under capacitating conditions, in the presence of heparin, in agreement with the published effect of capacitation on calmodulin binding proteins.
3D US is increasingly used to explore fetal brain anomalies. The limitations encountered are the shadowing of the fetal skull ossification depending on the acquisition angles and the term of the pregnancy; and the poor quality of the reconstructed C-plane in 3D US. The objective of this offline US-US fusion technique is to minimise the skull shadowing, the attenuation of the US and the poor multiplanar reconstruction of each 3D volume by fusing two 3D volumes acquired in two perpendicular planes. This is a prospective study at Necker Hospital (NCT04142606) conducted from January to June 2021. Normal singleton pregnancies were scanned between 20 and 36 WP by transabdominal and/or transvaginal imaging. Each axial volume was fused with a sagittal or coronal volume using 4D View Fusion (GE Healthcare) in a semiautomatic manner by 4 landmarks. Fused volumes were compared to the two reconstructed axial C-plane on both skull shadowing and the qualitative aspect. 85 patients were included. We obtained 33 volumes of high quality that could be merged. We obtained a workable reconstructed C-plane and reduced the shadowing effect (figure 1). Depending on the native acquisition angle, both peripheric brain parenchyma and median brain structures were clearly visible and assessed on the C-plane with fusion imaging. Eventually, starting from two native volumes (e.g., coronal and axial), we finally obtained a single merged volume on which it is possible to carry out a morphological evaluation in the 3 erected planes. FI Software may improve the capacities for anatomical brain assessment by increasing the quantity of information provided on the same volume. It is a very promising tool, easy to handle using volumic ultrasound acquisitions. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Abstract Calmodulin is a small, highly conserved acidic protein present at high levels in spermatozoa that mediates numerous intracellular Ca 2+ ‐dependent events. Sperm motility and fertilizing ability results from an array of biochemical pathways under Ca 2+ control, in which the importance of calmodulin is not fully understood. The role of calmodulin in sperm function has been mostly assessed using antagonists. Nevertheless, few known calmodulin‐regulated enzymes have been described in spermatozoa regarding their involvement in sperm function. To further understand the role of this important Ca 2+ mediator in spermatozoa, different studies were also undertaken to investigate and to identify sperm calmodulin‐binding proteins and determine their localization and subcellular distribution as an attempt to elucidate the role of this important Ca 2+ mediator. In the present study, sperm calmodulin‐binding proteins were identified by mass spectrometry after Ca 2+ ‐dependent biotinylated‐calmodulin binding on sperm head proteins subjected to 2D electrophoresis and transferred on a polyvinylidene difluoride membrane. Calmodulin binding protein identification was also done on detergent extracted whole sperm proteins pulled down in a Ca 2+ ‐dependent manner by calmodulin‐conjugated sepharose beads. In this latter group, 300 proteins were identified in at least two experiments out of three, and those identified in the three independent experiments were analyzed for overrepresented biological processes using the Bos taurus Gene Ontology database. Proteins with known function in reproductive processes, fertilization, sperm‐egg recognition, sperm binding to the zona pellucida, regulation of sperm capacitation, and sperm motility were identified and further emphasize the importance of calmodulin in sperm function.
Haptoglobin (Hp), TNF-alpha, and neutrophils are parts of a highly interactive ensemble participating in inflammatory processes. Hp is taken up by neutrophils, stored within a cytoplasmic granular compartment, and is secreted during phagocytosis by those cells. In the present study, the effects of TNF-alpha on the release of Hp from human neutrophils were investigated. Incubation of neutrophils with TNF-alpha induced the release of Hp from cells in a time- and concentration-dependent manner as revealed by Western blot analysis and immunofluorescence. The release of Hp induced by TNF-alpha was not due to nonspecific lysis of the cells. TNF-alpha is a highly pleiotropic cytokine that mediates its effects by binding to two distinct receptors (p55 and p75). Administration of TNF-alpha mutants binding specifically either to the p55 or to the p75 TNF receptors showed that there is a preference of TNF-alpha for the p55 receptor in the mediation of Hp release by neutrophils. A stimulated release of Hp was also induced by the chemotactic tripeptide fMLP. The TNF-alpha-induced release of Hp from neutrophils was inhibited by erbstatin, a tyrosine kinase inhibitor. These findings suggest that TNF-alpha may promptly increase the level of Hp at sites of infection or injury, leading to the modulation of the acute inflammatory response.
