Biochemical Modifications Research Articles

Will climate changes enhance the impacts of e-waste in aquatic systems?

The increase of the worlds' population is being accompanied by the exponential growth in waste of electrical and electronic equipment (e-waste) generation as a result of the rapid technological implementations. The inappropriate processing and disposal of this e-waste, containing rare-earth elements (REEs) such as gadolinium (Gd), may enhance its occurrence in the environment. In particular, the presence of Gd in marine systems may lead to environmental risks which are still unknown, especially considering foreseen climate modifications such as water salinity shifts due to extreme weather events. Within this context, the present study intended to assess the combined effects of Gd at variable salinities. For that, biochemical modifications were assessed in mussels, Mytilus galloprovincialis, exposed to Gd (0 and 10 μg/L) and different salinity levels (20, 30 and 40), acting individually and in combination. A decrease in salinity, induced an array of biochemical effects associated to hypotonic stress in non-contaminated and contaminated mussels, including metabolism, antioxidant and biotransformation defenses activation. Moreover, in Gd-contaminated organisms, the increase in salinity was responsible for a significant reduction of metabolic and defense mechanisms, possibly associated with a mussels’ physiological response to the stress caused by the combination of both factors. In particular, Gd caused cellular damage at all salinities, but mussels adopted different strategies under each salinity to limit the extent of oxidative stress. That is, an increase in metabolism was associated to hypotonic stress and Gd exposure, an activation of defense enzymes was revealed at the control salinity (30) and a decrease in metabolism and non-activation of defenses, associated with a possible physiological defense trait, was evidenced at the highest salinity. The different strategies adopted highlight the need to investigate the risk of emerging contaminants such as REEs at present and forecasted climate change scenarios, thus providing a more realistic environmental risk assessment.

Mitochondrial Oxidation of the Cytoplasmic Reducing Equivalents at the Onset of Oxidant Stress in the Isoproterenol-Induced Rat Myocardial Infarction.

We have developed and characterized a model of isoproterenol (ISO)-induced myocardial necrosis, identifying three stages of cardiac damage: a pre-infarction (0–12 h), infarction (24 h), and post-infarction period (48–96 h). Using this model, we have previously found alterations in calcium homeostasis and their relationship with oxidant stress in mitochondria, which showed deficient oxygen consumption and coupled ATP synthesis. Therefore, the present study was aimed at assessing the mitochondrial ability to transport and oxidize cytoplasmic reducing equivalents (NADH), correlating the kinetic parameters of the malate-aspartate shuttle, oxidant stress, and mitochondrial functionality. Our results showed only discreet effects during the cardiotoxic ISO action on the endogenous malate-aspartate shuttle activity, suggesting that endogenous mitochondrial NADH oxidation capacity (Nohl dehydrogenase) was not affected by the cellular stress. On the contrary, the reconstituted system showed significant enhancement in maximal capacity of the malate-aspartate shuttle activity only at later times (post-infarction period), probably as a compensatory part of cardiomyocytes’ response to the metabolic and functional consequences of the infarcted tissue. Therefore, these findings support the notion that heart damage associated with myocardial infarction suffers a set of sequential biochemical and metabolic modifications within cardiomyocytes, where mitochondrial activity, controlling the redox state, could play a relevant role.

Effect of Static Compressive Force on Aldehyde Dehydrogenase Activity in Periodontal Ligament Fibroblasts

Background: The application of static compressive forces to periodontal ligament fibroblasts (PDLFs) in vivo or in vitro has been linked to the expression of biochemical agents and local tissue modifications that could be involved in maintaining homeostasis during orthodontic movement. An approach used for identifying mesenchymal cells, or a subpopulation of progenitor cells in both tumoral and normal tissues, involves determining the activity of aldehyde dehydrogenase (ALDH). However, the role of subpopulations of PDLF-derived undifferentiated cells in maintaining homeostasis during tooth movement remains unclear. Objective: This study aimed at analyzing the effect of applying a static compressive force to PDLFs on the activity of ALDH in these cells. Methods: PDLFs were distributed into two groups: control group (CG), where fibroblasts were not submitted to compression, and experimental group (EG), where fibroblasts were submitted to a static compressive force of 4 g/mm2 for 6 hours. The compressive force was applied directly to the cells using a custom-built device. ALDH activity in the PDLFs was evaluated by a flow cytometry assay. Results: ALDH activity was observed in both groups, but was significantly lower in EG than in CG after the application of a static compressive force in the former. Conclusion: Application of a static compressive force to PDLFs decreased ALDH activity.

Current views on the role of fatty acids in the diagnosis of cardiovascular diseases (review)

An additional informative tool in the diagnosis of cardiovascular diseases in the early stages can be the analysis of changes in the fatty acid profile, which can be considered as a marker of various pathological conditions. The study of the effects of fatty acids and the mechanisms of changes in the fatty acid profile in connection with cardiovascular diseases remains relevant. We have analyzed modern data from foreign and domestic literature on issues related to the importance of fatty acids as possible markers in the diagnosis of cardiovascular diseases. Basic information about the structure of fatty acids, their functions in the human body, the relationship between the level of free fatty acids and indicators of the development of pathological processes of the cardiovascular system is presented. The factors influencing the dynamics of fatty acid concentrations both in normal conditions and during the development of pathological processes are reflected. The processes of biochemical modification of the fatty acid composition of the lipid matrix of the cell membrane are considered. The expediency of using fatty acids, together with some protein markers in the diagnosis of diseases of the cardiovascular system, has been shown. The collection and analysis of the accumulated information on the role of fatty acids helps to optimize the use of laboratory markers to determine the stages of pathogenesis of circulatory organ damage, to develop a system for evaluating the effectiveness of therapy for cardiovascular diseases, and to create a set of laboratory and instrumental tests for monitoring the condition of patients.

Unravelling the molecular mechanism of mutagenic factors impacting human health.

Environmental mutagens are chemical and physical substances in the environment that has a potential to induce a wide range of mutations and generate multiple physiological, biochemical, and genetic modifications in humans. Most mutagens are having genotoxic effects on the following generation through germ cells. The influence of germinal mutations on health will be determined by their frequency, nature, and the mechanisms that keep a specific mutation in the population. Early prenatal lethal mutations have less public health consequences than genetic illnesses linked with long-term medical and social difficulties. Physical and chemical mutagens are common mutagens found in the environment. These two environmental mutagens have been associated with multiple neurological disorders and carcinogenesis in humans. Thus in this study, we aim to unravel the molecular mechanism of physical mutagens (UV rays, X-rays, gamma rays), chemical mutagens (dimethyl sulfate (DMS), bisphenol A (BPA), polycyclic aromatic hydrocarbons (PAHs), 5-chlorocytosine (5ClC)), and several heavy metals (Ar, Pb, Al, Hg, Cd, Cr) implicated in DNA damage, carcinogenesis, chromosomal abnormalities, and oxidative stress which leads to multiple disorders and impacting human health. Biological tests for mutagen detection are crucial; therefore, we also discuss several approaches (Ames test and Mutatox test) to estimate mutagenic factors in the environment. The potential risks of environmental mutagens impacting humans require a deeper basic knowledge of human genetics as well as ongoing research on humans, animals, and their tissues and fluids.

What if the cold days return? Epigenetic mechanisms in plants to cold tolerance.

The epigenetic could be an important, but seldom assessed, mechanisms in plants inhabiting cold ecosystems. Thus, this review could help to fill a gap in the current literature. Low temperatures are one of the most critical environmental conditions that negatively affect the growth, development, and geographic distribution of plants. Exposure to low temperatures results in a suit of physiological, biochemical and molecular modifications through the reprogramming of the expression of genes and transcription factors. Scientific evidence shows that the average annual temperature has increased in recent years worldwide, with cold ecosystems (polar and high mountain) being among the most sensitive to these changes. However, scientific evidence also indicates that there would be specific events of low temperatures, due it is highly relevant to know the capacity for adaptation, regulation and epigenetic memory in the face of these events, by plants. Epigenetic regulation has been described to play an important role in the face of environmental stimuli, especially in response to abiotic stress. Several studies on epigenetic mechanisms have focused on responses to stress as drought and/or salinity; however, there is a gap in the current literature considering those related to low temperatures. In this review, we focus on systematizing the information published to date, related to the regulation of epigenetic mechanisms such as DNA methylation, histone modification, and non-coding RNA-dependent silencing mechanisms, in the face of plant´s stress due to low temperatures. Finally, we present a schematic model about the potential responses by plants taking in count their epigenetic memory; considering a global warming scenario and with the presence or absence of extreme specific events of low temperatures.

Epitranscriptome engineering in crop improvement

Biochemical modifications of RNAs, collectively designated as the epitranscriptome, have emerged as fundamental mechanisms underlying gene regulation. Recent studies have shown that N6-methyladenosine (m6A) and 5-methycytosine (m5C) modifications govern multifaceted developmental processes and environmental responses in plants. The potential of engineering the epitranscriptome in crop improvement was nicely demonstrated in a recent study (Yu et al., 2021), where the authors took advantage of epitranscriptome engineering to vigorously increase crop yield.

Effects of early noise exposure on hippocampal-dependent behaviors during adolescence in male rats: influence of different housing conditions.

Central nervous system (CNS) development is a very complex process that can be altered by environmental stimuli such as noise, which can generate long-term auditory and/or extra-auditory impairments. We have previously reported that early noise exposure can induce hippocampus-related behavioral alterations in postnatal day (PND) 28 adolescent rats. Furthermore, we recently found biochemical modifications in the hippocampus (HC) of these animals that seemed to endure even in more mature animals (i.e. PND35) and that have not been studied along with behavioral correlates. Thus, the aim of this work was to reveal novel data about the effects of early noise exposure on hippocampal-dependent behaviors in more mature animals. Additionally, extended enriched environment (EE) housing was evaluated to determine its capacity to induce behavioral modifications, either by its neuroprotective ability or the greater stimulation that it generates. Male Wistar rats were exposed to different noise schemes at PND7 or PND15. Upon weaning, some animals were transferred to EE whereas others were kept in standard cages. At PND35, different hippocampal-dependent behavioral assessments were performed. Results showed noise-induced behavioral changes that differed according to the scheme and age of exposure used. In addition, housing in an EE was effective either in preventing some of these changes or in inducing the appearance of new behavioral modifications. These findings suggest that CNS development would be sensitive to the effects of different type of environmental stimuli such as noise or enriched housing, leading to maladaptive behavioral changes that last even until adolescence.

Investigation of single and combined effects of repeated oral cadmium and lead administration in ewes

Cadmium is considered one of the most toxic elements in the environment, with its ubiquity, a multi-organs damage and long elimination half-life. This heavy metal can pose serious health problems and lead to various biological disorders. Our research investigated the adverse health effects related to subchronic oral exposure at regular low- dose treatment in Ouled Djellal non- lactating ewes. The study aimed to ascertain whether the subchronic co-exposure to Cd and Pb in Ouled Djellal non- lactating ewes exacerbates the toxic effects and what are the most sensitive indicator parameters related to the blood Cd concentration. Haematological and biochemical modifications as well as the interaction with Zn and Cu were investigated. The study was conducted during two periods: in the first one, which lasted four weeks, the ewes were not contaminated: animals during this period acted as a control group and all values obtained during this period are considered as control values. Two groups of five animals were used: the Cd group received cadmium chloride orally at a dose of 2 mg/kg/day and the Cd+Pb group received cadmium chloride orally at a dose of 2mg/kg/day in addition to lead nitrate at a dose of 2.5 mg/kg/day during 9 weeks. Mean blood Cd has increased after the cadmium administration in both Cd group and Cd+Pb group. The evolution of Cd levels in the blood over time is slightly different for the Cd+Pb group. ANOVA showed a time effect (P˂0.001). The effects of treatment and animals effect were not significant (P>0.05). Disturbance in haematological and biochemical parameters was observed. The balance of the essential elements was altered; resulting in a significant decrease mainly in Cu plasma concentrations. Student's t-test indicates that toxic effects were similar for the two groups. This multi-indicator approach based on changes in different sensitive parameters can is be pertinent for risk assessment.

Towards a druggable epitranscriptome: Compounds that target RNA modifications in cancer.

Epitranscriptomics is an exciting emerging area that studies biochemical modifications of RNA. The field has been opened up by the technical efforts of the last decade to characterize and quantify RNA modifications, and this has led to a map of post-transcriptional RNA marks in normal cell fate and development. However, the scientific interest has been fuelled by the discovery of aberrant epitranscriptomes associated with human diseases, mainly cancer. The challenge is now to see whether epitrancriptomics offers mechanisms that can be effectively targeted by low MW compounds and are thus druggable. In this review, we will describe the principal RNA modifications (with a focus on mRNA), summarize the latest scientific evidence of their dysregulation in cancer and provide an overview of the state-of-the-art drug discovery to target the epitranscriptome. Finally, we will discuss the principal challenges in the field of chemical biology and drug development to increase the potential of targeted-RNA for clinical benefit. LINKED ARTICLES: This article is part of a themed issue on New avenues in cancer prevention and treatment (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.12/issuetoc.

Methylglyoxal-derived hemoglobin advanced glycation end products induce apoptosis and oxidative stress in human umbilical vein endothelial cells

The presence of excess glucose promotes hemoglobin glycation via the biochemical modification of hemoglobin by dicarbonyl products. However, the precise effects of Hb-AGEs in human umbilical vein endothelial cells (HUVECs) are not known to date. Therefore, we investigated the tentative effects of Hb-AGEs in HUVECs. Initially, we used the AGE formation assay to examine the selectivity of MGO toward various proteins. Among all proteins, MGO-Hb-AGEs formation was higher compared to the formation of other dicarbonyl-mediated AGEs. Our next data demonstrated that treatment with 0.5 mg/mL of Hb-AGEs-4w significantly reduced cell viability in HUVECs. Further, we evaluated the role of MGO in conformational and structural changes in Hb. The results showed that Hb demonstrated a highly altered conformation upon incubation with MGO. Moreover, Hb-AGEs-4w treatment strongly increased ROS production, and decreased mitochondrial membrane potential in HUVECs, and moderately reduced the expression of phosphorylated forms of p-38 and JNK. We observed that Hb-AGEs-4w treatment increased the number of apoptotic cells and the Bax/Bcl-2 ratio and cleaved the nuclear enzyme PARP in HUVECs. Finally, Hb-AGEs also inhibited migration and proliferation of HUVECs, thus be physiologically significant in endothelial dysfunction. Taken together, our data suggest that Hb-AGEs may play a critical role in inducing vascular endothelial cell damage. Therefore, this study may provide a plausible explanation for the potential Hb-AGEs in human endothelial cell dysfunction of diabetic patients.

Exogenous Applications of Bio-fabricated Silver Nanoparticles to Improve Biochemical, Antioxidant, Fatty Acid and Secondary Metabolite Contents of Sunflower.

The present study involved the bio-fabrication of silver nanoparticles (AgNPs) by using the Euphorbia helioscopia L. leaves aqueous extract to improve the production of secondary metabolites in industrially important sunflower (Helianthus annuus L.) plants. Phyto-fabrication of AgNPs was confirmed by using spectrophotometry, SEM imaging and X-ray diffraction analysis. The morphological and optical characterization manifested that the AgNPs are crystalline and exist in the size range of 30–100 nm. Various concentrations (10, 20, 40, 60, 80 and 100 mg/L) of AgNPs were applied in combinations on sunflower seeds and crop plants. The effects of biosynthesized AgNPs were evaluated for agro-morphological parameters (plant height, flowering initiation and seed weight), biochemical metabolites (chlorophyll, proline, soluble sugar, amino acid and protein contents) and enzymatic activities (superoxide dismutase and ascorbate peroxidase) in sunflower and 60 mg/L concentration of AgNPs on sunflower seeds and foliar sprays on plants in combination were found to be effective to elicit biochemical modifications to improve secondary metabolites. It was also observed experimentally that 60 mg/L concentration of AgNPs improved the biochemical, fatty acid and enzymatic attributes of sunflower plants, which in turn improved the plant agro-morphological parameters. Near-infrared spectroscopic analysis results confirmed the improvement in the seed quality, oil contents and fatty acid composition (palmitic acid, oleic acid and linoleic acid) after the applications of AgNPs. The findings of the present investigation confirm the exogenous applications of bio-fabricated AgNPs in combinations on seeds and plants to improve the plant yield, seed quality and secondary metabolite contents of the sunflower plants.

A novel mechanosensitive channel controls osmoregulation, differentiation, and infectivity in Trypanosoma cruzi.

The causative agent of Chagas disease undergoes drastic morphological and biochemical modifications as it passes between hosts and transitions from extracellular to intracellular stages. The osmotic and mechanical aspects of these cellular transformations are not understood. Here we identify and characterize a novel mechanosensitive channel in Trypanosoma cruzi (TcMscS) belonging to the superfamily of small-conductance mechanosensitive channels (MscS). TcMscS is activated by membrane tension and forms a large pore permeable to anions, cations, and small osmolytes. The channel changes its location from the contractile vacuole complex in epimastigotes to the plasma membrane as the parasites develop into intracellular amastigotes. TcMscS knockout parasites show significant fitness defects, including increased cell volume, calcium dysregulation, impaired differentiation, and a dramatic decrease in infectivity. Our work provides mechanistic insights into components supporting pathogen adaptation inside the host, thus opening the exploration of mechanosensation as a prerequisite for protozoan infectivity.

Polydopamine-assisted immobilization of silk fibroin and its derived peptide on chemically oxidized titanium to enhance biological activity in vitro

Biochemical modification can endow the surface of implants with superior biological activity. Herein, silk fibroin (SF) protein and its anionic derivative peptides (Cs) were covalently immobilized onto a titanium implant surface via a polydopamine layer. The successful conjugation of SF and Cs was revealed by X-ray photoelectron spectroscopy (XPS), field-emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), and contact angle measurements. The addition of Cs prevented the conformational transition of silk fibroin to silk II. The deposition of apatite on its surface was significantly accelerated, and the bioactive composite coating was observed to enhance protein adsorption and cell proliferation. More importantly, it also promoted the osteogenic differentiation of bone marrow stem cells (BMSCs) for the quantitative and qualitative detection of alkaline phosphatase (ALP) and alizarin red (ARS). Overall, the stable performance and enhanced osteogenic property of the composite coating promote an extensive application for clinical titanium-based implants.

Mapping the residue specificities of epigenome enzymes by yeast surface display

Histone proteins are decorated with a combinatorially and numerically diverse set of biochemical modifications. Here, we describe a versatile and scalable approach which enables efficient characterization of histone modifications without the need for recombinant protein production. As proof-of-concept, we first use this system to rapidly profile the histone H3 and H4 residue writing specificities of the human histone acetyltransferase, p300. Subsequently, a large panel of commercially available anti-acetylation antibodies are screened for their specificities, identifying many suitable and unsuitable reagents. Furthermore, this approach enables efficient mapping of the large binary crosstalk space between acetylated residues on histones H3 and H4 and uncovers residue interdependencies affecting p300 activity. These results show that using yeast surface display to study histone modifications is a useful tool that can advance our understanding of chromatin biology by enabling efficient interrogation of the complexity of epigenome modifications.

Mummified embalmed head skin: SR-FTIR microspectroscopic exploration

This case report details the examination of the skin of an Egyptian mummified head with a possible skin disorder. The head, thought to be dated in the first half of the 18th Dynasty, New Kingdom (1570–1400 BCE) belongs to the Museum of Forensic Anthropology, University of Madrid. Initial histological examination demonstrated evidence of chronic inflammation, which was confirmed by immunohistochemistry and Transmission Electron Microscopy (TEM). However, confirmation of pathology could be confounded by both the age of the specimen and the process of preservation by mummification. In this case report, Synchrotron Radiation Fourier Transform Microspectroscopy (SR-µFTIR) was used to add novel insights into embalmed mummified tissue. More precisely, FTIR is used for the first time on the specific specimens, while no other similar studies have been performed on these samples priorly. Additionally, modern skin tissue was examined too, in order to compare the amount of degradation to the mummified one. Whilst the FTIR results confirmed the results from the initial histological study, they also showed a biochemical modification of the mummified skin that could be indicative of tissue degradation. The latter was supported by comparing it to FTIR results of the modern tissue used.

Silver nanoparticles elicited physiological, biochemical, and antioxidant modifications in rice plants to control Aspergillus flavus

Abstract This study was carried out to analyze the effects of biogenic silver nanoparticles (AgNPs) on physiological, biochemical, and enzymatic attributes of rice plants against Aspergillus flavus. The plant-based AgNPs were synthesized by using the aqueous extract of Moringa oleifera leaves. The characterization of AgNPs was accomplished through UV-visible spectrophotometry, SEM, and energy-dispersive X-ray (EDX) analysis, which confirmed that the nanoparticles are crystalline and are less than 100 nm in size. The exogenous applications of different concentrations of AgNPs (25, 50, 75, and 100 mg/L) on rice plants in field experiments were used to control the proliferation of A. flavus. The effects of biosynthesized AgNPs were evaluated for physiological (relative water content, membrane stability index, and chlorophyll content), nonenzymatic metabolites (total phenolic, total flavonoid, proline, soluble sugar, and protein contents), and enzymatic metabolites (superoxide dismutase, peroxidase, and catalase) in rice plants under biotic stress, and 50 mg/L concentration of AgNPs was found to be effective to elicit biochemical modifications to reduce biotic stress. The 50 mg/L concentration of AgNPs was also effective in controlling the proliferation of fungal pathogen. The applications of AgNPs reduced the biotic stress by decreasing the production level of osmolytes, enzymatic, and nonenzymatic compounds but significantly increased the protein content.

Biochemical Modification of Titanium Oral Implants: Evidence from In Vivo Studies.

The discovery of osseointegration of titanium implants revolutionized the dental prosthesis field. Traditionally, implants have a surface that is processed by additive or subtractive techniques, which have positive effects on the osseointegration process by altering the topography. In the last decade, innovative implant surfaces have been developed, on which biologically active molecules have been immobilized with the aim of increasing stimulation at the implant–biological tissue interface, thus favoring the quality of osseointegration. Among these molecules, some are normally present in the human body, and the techniques for the immobilization of these molecules on the implant surface have been called Biochemical Modification of Titanium Surfaces (BMTiS). Different techniques have been described in order to immobilize those biomolecules on titanium implant surfaces. The aim of the present paper is to present evidence, available from in vivo studies, about the effects of biochemical modification of titanium oral implants on osseointegration.

Exposure to Iron Oxide Nanoparticles Coated with Phospholipid-Based Polymeric Micelles Induces Renal Transitory Biochemical and Histopathological Changes in Mice.

The renal toxicity induced by the intravenously injected iron oxide nanoparticles (IONPs) encapsulated in phospholipid-based polymeric micelles was studied in CD1 mice for 2 weeks. Two doses of 5 and 15 mg of Fe/kg bodyweight of NPs or saline solution (control) were tested, and the levels of antioxidant enzyme activities, oxidative stress parameters, and the expressions of kidney fibrosis biomarkers were analyzed. The enzymatic activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, glutathione reductase, and glucose-6-phosphate dehydrogenase in the kidney were significantly decreased compared to the control in the first 3 days followed by a recovery up to 14 days. Concomitantly, a significant increase in lipid peroxidation (malondialdehyde) levels and a decrease in protein thiol groups were recorded. Moreover, increases in the expressions of T cell immunoglobulin and mucin domain 1 (TIM-1) and transforming growth factor-β (TGF-β) were observed in mouse tissue samples in the first week, which were more pronounced for the higher dose. The results suggested the role of oxidative stress as a mechanism for induced toxicity in mice kidneys after the IV administration of IONPs encapsulated in phospholipid-based polymeric micelles but also the capacity of the kidneys’ defense systems to revert efficiently the biochemical modifications that were moderate and for short duration.

Membrane hyperpolarization abolishes calcium oscillations that prevent induced acrosomal exocytosis in human sperm.

Sperm capacitation is essential to gain fertilizing capacity. During this process, a series of biochemical and physiological modifications occur that allow sperm to undergo acrosomal exocytosis (AE). At the molecular level, hyperpolarization of the sperm membrane potential (Em) takes place during capacitation. This study shows that human sperm incubated under conditions that do not support capacitation (NC) can become ready for an agonist stimulated AE by pharmacologically inducing Em hyperpolarization with Valinomycin or Amiloride. To investigate how Em hyperpolarization promotes human sperm's ability to undergo AE, live single-cell imaging experiments were performed to simultaneously monitor changes in [Ca2+ ]i and the occurrence of AE. Em hyperpolarization turned [Ca2+ ]i dynamics in NC sperm from spontaneously oscillating into a sustained slow [Ca2+ ]i increase. The addition of progesterone (P4) or K+ to Valinomycin-treated sperm promoted that a significant number of cells displayed a transitory rise in [Ca2+ ]i which then underwent AE. Altogether, our results demonstrate that Em hyperpolarization is necessary and sufficient to prepare human sperm for the AE. Furthermore, this Em change decreased Ca2+ oscillations that block the occurrence of AE, providing strong experimental evidence of the molecular mechanism that drives the acquisition of acrosomal responsiveness.