Membrane potential hyperpolarization: a critical factor in acrosomal exocytosis and fertilization in sperm within the female reproductive tract.

Hyperpolarization of the membrane potential (Em), a phenomenon regulated by SLO3 channels, stands as a central feature in sperm capacitation-a crucial process conferring upon sperm the ability to fertilize the oocyte. In vitro studies demonstrated that Em hyperpolarization plays a pivotal role in facilitating the mechanisms necessary for the development of hyperactivated motility (HA) and acrosomal exocytosis (AE) occurrence. Nevertheless, the physiological significance of sperm Em within the female reproductive tract remains unexplored. As an approach to this question, we studied sperm migration and AE incidence within the oviduct in the absence of Em hyperpolarization using a novel mouse model established by crossbreeding of SLO3 knock-out (KO) mice with EGFP/DsRed2 mice. Sperm from this model displays impaired HA and AE in vitro. Interestingly, examination of the female reproductive tract shows that SLO3 KO sperm can reach the ampulla, mirroring the quantity of sperm observed in wild-type (WT) counterparts, supporting that the HA needed to reach the fertilization site is not affected. However, a noteworthy distinction emerges-unlike WT sperm, the majority of SLO3 KO sperm arrive at the ampulla with their acrosomes still intact. Of the few SLO3 KO sperm that do manage to reach the oocytes within this location, fertilization does not occur, as indicated by the absence of sperm pronuclei in the MII-oocytes recovered post-mating. In vitro, SLO3 KO sperm fail to penetrate the ZP and fuse with the oocytes. Collectively, these results underscore the vital role of Em hyperpolarization in AE and fertilization within their physiological context, while also revealing that Em is not a prerequisite for the development of the HA motility, essential for sperm migration through the female tract to the ampulla.

Reorganization of the Flagellum Scaffolding Induces a Sperm Standstill During Fertilization.

Mammalian sperm delve into the female reproductive tract to fertilize the female gamete. The available information about how sperm regulate their motility during the final journey to the fertilization site is extremely limited. In this work, we investigated the structural and functional changes in the sperm flagellum after acrosomal exocytosis and during the interaction with the eggs. The evidence demonstrates that the double helix actin network surrounding the mitochondrial sheath of the midpiece undergoes structural changes prior to the motility cessation. This structural modification is accompanied by a decrease in diameter of the midpiece and is driven by intracellular calcium changes that occur concomitant with a reorganization of the actin helicoidal cortex. Although midpiece contraction may occur in a subset of cells that undergo acrosomal exocytosis, live-cell imaging during in vitro fertilization showed that the midpiece contraction is required for motility cessation after fusion is initiated. These findings provide the first evidence of the F-actin network's role in regulating sperm motility, adapting its function to meet specific cellular requirements during fertilization, and highlighting the broader significance of understanding sperm motility. Significant statement: In this work, we demonstrate that the helical structure of polymerized actin in the flagellum undergoes a rearrangement at the time of sperm-egg fusion. This process is driven by intracellular calcium and promotes a decrease in the sperm midpiece diameter as well as the arrest in motility, which is observed after the fusion process is initiated.

The Beta2-adrenergic agonist salbutamol synergizes with paclitaxel on cell proliferation and tumor growth in triple negative breast cancer models.

PURPOSE: Globally breast cancer accounts for 24.5% in incidence and 15.5% in cancer deaths in women. The triple-negative subtype lacks any specific therapy and is treated with chemotherapy, resulting in significant side-effects. We aimed to investigate if the dose of chemotherapeutic drugs could be diminished by co-administering it with the ß2-agonist salbutamol. METHODS: Cell proliferation was measured by thymidine incorporation; gene expression, by real-time PCR and protein phosphorylation by WB. Apoptosis was assessed by acridine orange / ethidium bromide and TUNEL tests. Public patient databases were consulted. Cells were inoculated to nude mice and their growth assessed. RESULTS: The ß2-agonist salbutamol synergizes in MDA-MB-231 cells in vitro with paclitaxel and doxorubicin on cell proliferation through ADRB2 receptors, while the ß-blocker propranolol does not. The expression of this receptor was assessed in patient databases and other cell lines. Triple negative samples had the lowest expression. Salbutamol and paclitaxel decreased MDA-MB-231 cell proliferation while their combination further inhibited it. The pathways involved were analyzed. When these cells were inoculated to nude mice, paclitaxel and salbutamol inhibited tumor growth. The combined effect was significantly greater. Paclitaxel increased the expression of MDR1 while salbutamol partially reversed this increase. CONCLUSION: While the effect of salbutamol was mainly on cell proliferation, suboptimal concentrations of paclitaxel provoked a very important enhancement of apoptosis. The latter enhanced transporter proteins as MDR1, whose expression were diminished by salbutamol. The expression of ADRB2 should be assessed in the biopsy or tumor to eventually select patients that could benefit from salbutamol repurposing.

Two-pore channel 1 and Ca2+ release-activated Ca2+ channels contribute to the acrosomal pH-dependent intracellular Ca2+ increase in mouse sperm.

The acrosome is a lysosome-related vesicular organelle located in the sperm head. The acrosomal reaction (AR) is an exocytic process mediated by Ca2+ and essential for mammalian fertilization. Recent findings support the importance of acrosomal alkalinization for the AR. Mibefradil (Mib) and NNC 55-0396 (NNC) are two amphipathic weak bases that block the sperm-specific Ca2+ channel (CatSper) and induce acrosomal pH (pHa ) increase by accumulating in the acrosomal lumen of mammalian sperm. This accumulation and pHa elevation increase the intracellular Ca2+ concentration ([Ca2+ ]i ) and trigger the AR by unknown mechanisms of Ca2+ transport. Here, we investigated the pathways associated with the pHa increase-induced Ca2+ signals using mouse sperm as a model. To address these questions, we used single-cell Ca2+ imaging, the lysosomotropic agent Gly-Phe-ß-naphthylamide (GPN) and pharmacological tools. Our findings show that Mib and NNC increase pHa and release acrosomal Ca2+ without compromising acrosomal membrane integrity. Our GPN results indicate that the osmotic component does not significantly contribute to acrosomal Ca2+ release caused by pHa rise. Inhibition of two-pore channel 1 (TPC1) channels reduced the [Ca2+ ]i increase stimulated by acrosomal alkalinization. In addition, blockage of Ca2+ release-activated Ca2+ (CRAC) channels diminished Ca2+ uptake triggered by pHa alkalinization. Finally, our findings contribute to understanding how pHa controls acrosomal Ca2+ efflux and extracellular Ca2+ entry during AR in mouse sperm. KEY POINTS: The acrosomal vesicle is a lysosome-related organelle located in the sperm head. The acrosome reaction (AR) is a highly regulated exocytic process mediated by Ca2+ , which is essential for fertilization. However, the molecular identity of Ca2+ transporters involved in the AR and their mechanisms to regulate Ca2+ fluxes are not fully understood. In mammalian sperm, acrosomal alkalinization induces intracellular Ca2+ concentration ([Ca2+ ]i ) increase and triggers the AR by unknown molecular mechanisms of Ca2+ transport. In this study, we explored the molecular mechanisms underlying Ca2+ signals caused by acrosomal alkalinization using mouse sperm as a model. TPC1 and CRAC channels contribute to [Ca2+ ]i elevation during acrosomal alkalinization. Our findings expand our understanding of how the acrosomal pH participates in the physiological induction of the AR.

High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding.

The exclusive expression of CatSper in sperm and its critical role in sperm function makes this channel an attractive target for contraception. The strategy of blocking CatSper as a male, non-hormonal contraceptive has not been fully explored due to the lack of robust screening methods to discover novel and specific inhibitors. The reason for this lack of appropriate methodology is the structural and functional complexity of this channel. We have developed a high-throughput method to screen drugs with the capacity to block CatSper in mammalian sperm. The assay is based on removing external free divalent cations by chelation, inducing CatSper to efficiently conduct monovalent cations. Since Na+ is highly concentrated in the extracellular milieu, a sudden influx depolarizes the cell. Using CatSper1 KO sperm we demonstrated that this depolarization depends on CatSper function. A membrane potential (Em) assay was combined with fluorescent cell barcoding (FCB), enabling higher throughput flow cytometry based on unique fluorescent signatures of different sperm samples. These differentially labeled samples incubated in distinct experimental conditions can be combined into one tube for simultaneous acquisition. In this way, acquisition times are highly reduced, which is essential to perform larger screening experiments for drug discovery using live cells. Altogether, a simple strategy for assessing CatSper was validated, and this assay was used to develop a high-throughput drug screening for new CatSper blockers.

Extending resolution within a single imaging frame.

The resolution of fluorescence microscopy images is limited by the physical properties of light. In the last decade, numerous super-resolution microscopy (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on the Mean Shift theory, which extends spatial resolution of single fluorescence images beyond the diffraction limit of light. MSSR works on low and high fluorophore densities, is not limited by the architecture of the optical setup and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image stacks, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other SRM approaches. Along with its wide accessibility, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.

The early molecular events leading to COFILIN phosphorylation during mouse sperm capacitation are essential for acrosomal exocytosis.

The actin cytoskeleton reorganization during sperm capacitation is essential for the occurrence of acrosomal exocytosis (AR) in several mammalian species. Here, we demonstrate that in mouse sperm, within the first minutes of exposure upon capacitating conditions, the activity of RHOA/C and RAC1 is essential for LIMK1 and COFILIN phosphorylation. However, we observed that the signaling pathway involving RAC1 and PAK4 is the main player in controlling actin polymerization in the sperm head necessary for the occurrence of AR. Moreover, we show that the transient phosphorylation of COFILIN is also influenced by the Slingshot family of protein phosphatases (SSH1). The activity of SSH1 is regulated by the dual action of two pathways. On one hand, RHOA/C and RAC1 activity promotes SSH1 phosphorylation (inactivation). On the other hand, the activating dephosphorylation is driven by okadaic acid-sensitive phosphatases. This regulatory mechanism is independent of the commonly observed activating mechanisms involving PP2B and emerges as a new finely tuned modulation that is, so far, exclusively observed in mouse sperm. However, persistent phosphorylation of COFILIN by SSH1 inhibition or okadaic acid did not altered actin polymerization and the AR. Altogether, our results highlight the role of small GTPases in modulating actin dynamics required for AR.

Seeing is believing: Current methods to observe sperm acrosomal exocytosis in real time.

Acrosomal exocytosis (AR) is a critical process that sperm need to undergo to fertilize an egg. The evaluation of the presence or absence of the acrosome is usually performed by using lectins or dyes in fixed cells. With this approach, it is neither possible to monitor the dynamic process of exocytosis and related molecular events while discriminating between live and dead cells, nor to evaluate the acrosomal status while sperm reside in the female reproductive tract. However, over the last two decades, several new methodologies have been used to assess the occurrence of AR in living cells allowing different groups to obtain information that was not possible in the past. These techniques have revolutionized the whole study of this process. This review summarizes current methods available to analyze AR in living cells as well as the important information that emerged from studies using these approaches.