Direct observation of cortactin protecting Arp2/3-actin filament branch junctions from GMF-mediated destabilization.

How cells tightly control the formation and turnover of branched actin filament arrays to drive cell motility, endocytosis, and other cellular processes is still not well understood. Here, we investigated the mechanistic relationship between two binding partners of the Arp2/3 complex, glia maturation factor (GMF) and cortactin. Individually, GMF and cortactin have opposite effects on the stability of actin filament branches, but it is unknown how they work in concert with each other to govern branch turnover. Using TIRF microscopy, we observe that GMF's branch destabilizing activities are potently blocked by cortactin (IC50 = 1.3 nM) and that this inhibition requires direct interactions of cortactin with Arp2/3 complex. The simplest model that would explain these results is competition for binding Arp2/3 complex. However, we find that cortactin and GMF do not compete for free Arp2/3 complex in solution. Further, we use single molecule analysis to show that cortactin's on-rate (3 ×107 s-1 M-1) and off-rate (0.03 s-1) at branch junctions are minimally affected by excess GMF. Together, these results show that cortactin binds with high affinity to branch junctions, where it blocks the destabilizing effects of GMF, possibly by a mechanism that is allosteric in nature. In addition, the affinities we measure for cortactin at actin filament branch junctions (Kd = 0.9 nM) and filament sides (Kd = 206 nM) are approximately 20-fold stronger than previously reported. These observations contribute to an emerging view of molecular complexity in how Arp2/3 complex is regulated through the integration of multiple inputs.

Examining the key determinants of the jordanian customer's adoption of genetically modified food.

Genetically modified food (GMF) is one of the most debated issues in the food market. There has been considerable interest from both academic researchers and policy makers regarding the antecedents and consequences of the commercial adoption of GMF applications. Conceptually, GMF can be defined as "Genetically modified (hereafter GM) foods are produced from genetically modified seeds or ingredients derived from plants or animals whose DNA has been manipulated using genetic engineering methods" [1, p. 2861]. However, only a limited number of studies have tested the related issues of GMF products from a customer perspective. Thus, this project intends to discover and examine the main drivers and hindrances in predicting customers' intention and buying decision behaviour in developing Arabian countries (i.e., Jordan). A diffusion of innovations (DOIs) model was selected as the theoretical basis for the current study project. A field survey study was conducted to collect the requested quantitative data from a convenience sample of Jordanian customers. Statistical results largely supported the role of relative advantage, compatibility, trialability, social approval, awareness, perceived risk and price value on the behavioural intention to adopt GMF products, which in turn significantly predicted actual adoption behaviour. The results of the current project will hopefully expand the current academic understanding of the main factors that predict Jordanian customers' perception and adoption of GMF products.

Identification and Characterization of Glycine- and Arginine-Rich Motifs in Proteins by a Novel GAR Motif Finder Program.

Glycine- and arginine-rich (GAR) motifs with different combinations of RG/RGG repeats are present in many proteins. The nucleolar rRNA 2'-O-methyltransferase fibrillarin (FBL) contains a conserved long N-terminal GAR domain with more than 10 RGG plus RG repeats separated by specific amino acids, mostly phenylanalines. We developed a GAR motif finder (GMF) program based on the features of the GAR domain of FBL. The G(0,3)-X(0,1)-R-G(1,2)-X(0,5)-G(0,2)-X(0,1)-R-G(1,2) pattern allows the accommodation of extra-long GAR motifs with continuous RG/RGG interrupted by polyglycine or other amino acids. The program has a graphic interface and can easily output the results as .csv and .txt files. We used GMF to show the characteristics of the long GAR domains in FBL and two other nucleolar proteins, nucleolin and GAR1. GMF analyses can illustrate the similarities and also differences between the long GAR domains in the three nucleolar proteins and motifs in other typical RG/RGG-repeat-containing proteins, specifically the FET family members FUS, EWS, and TAF15 in position, motif length, RG/RGG number, and amino acid composition. We also used GMF to analyze the human proteome and focused on the ones with at least 10 RGG plus RG repeats. We showed the classification of the long GAR motifs and their putative correlation with protein/RNA interactions and liquid-liquid phase separation. The GMF algorithm can facilitate further systematic analyses of the GAR motifs in proteins and proteomes.

Translational Significance of GMF-ß Inhibition by Indazole-4-yl-methanol in Enteric Glial Cells for Treating Multiple Sclerosis.

In the emerging context of gut-brain control of multiple sclerosis (MS), developing therapeutics targeting proinflammatory proteins controlling the gut-brain immunomodulation is welcoming. One such immunomodulator is glia maturation factor-ß (GMF-ß). GMF-ß activation following GMF-ß-ser-83 phosphorylation upregulates proinflammatory responses and exacerbates experimental autoimmune encephalomyelitis (EAE). Notably, GMF-ß-/- mice exhibited no EAE symptoms. Thus, we identified 1H-indazole-4-yl-methanol (GMFBI.1) inhibitor which blocked GMF-ß-ser-83 phosphorylation critical in EAE suppression. To establish gut GMF-ß's role in EAE in the context of gut-brain involvement in neurodegenerative diseases, we altered gut GMFBI.1 bioavailability as an index of EAE suppression. At first, we identified Miglyol 812N as a suitable biocompatible GMFBI.1 carrier compared to other FDA-approved carriers using in silico molecular docking analysis. GMFBI.1 administration in Miglyol 812N enhanced its retention/brain permeability. Subsequently, we administered GMFBI.1-Miglyol 812N by subcutaneous/oral routes at different doses with differential GMFBI.1 bioavailability in gut and brain to assess the role of local GMFBI.1 bioavailability in EAE reversal by a pharmacokinetic approach. Deprival of gut GMFBI.1 bioavailability led to partial EAE suppression despite having sufficient GMFBI.1 in circulation to inhibit brain GMF-ß activity. Restoration of gut GMFBI.1 bioavailability led to complete EAE reversal. Molecular pathology behind partial/full EAE reversal was associated with differential GMF-ß-Ser-83 phosphorylation/GM-CSF expression levels in enteric glial cells owing to GMFBI.1 bioavailability. In addition, we observed leaky gut reversal, tight junction protein ZO-1 restoration, beneficial gut microbiome repopulation, recovery from gut dysbiosis, and upregulation of Treg cells. GMFBI.1's dual gut/brain targeting of GMF-ß has therapeutical/translational potential in controlling autoimmunity in MS.

Innovative Application of SERS in Food Quality and Safety: A Brief Review of Recent Trends.

Innovative application of surface-enhanced Raman scattering (SERS) for rapid and nondestructive analyses has been gaining increasing attention for food safety and quality. SERS is based on inelastic scattering enhancement from molecules located near nanostructured metallic surfaces and has many advantages, including ultrasensitive detection and simple protocols. Current SERS-based quality analysis contains composition and structural information that can be used to establish an electronic file of the food samples for subsequent reference and traceability. SERS is a promising technique for the detection of chemical, biological, and harmful metal contaminants, as well as for food poisoning, and allergen identification using label-free or label-based methods, based on metals and semiconductors as substrates. Recognition elements, including immunosensors, aptasensors, or molecularly imprinted polymers, can be linked to SERS tags to specifically identify targeted contaminants and perform authenticity analysis. Herein, we highlight recent studies on SERS-based quality and safety analysis for different foods categories spanning the whole food chain, 'from farm to table' and processing, genetically modified food, and novel foods. Moreover, SERS detection is a potential tool that ensures food safety in an easy, rapid, reliable, and nondestructive manner during the COVID-19 pandemic.

Ovarian toxicity of plant-derived edible oils: a 28 days hormonal and histopathological study in Wistar rat.

Initial evidence on the endocrine-disrupting effects of genetically modified (GM) food motivated us to evaluate the reproductive toxicity of GM and non-GM plant-derived edible oils in female Wistar rats. Sunflower (non-GM), maize (GM), and canola (GM) oils as popular resource dietary oils were purchased from the local market. After tracking the target sequence of CaMV 35S and Nos terminator in all selected batch numbers of edible oils by real-time PCR, oil samples were daily gavaged to 10 weeks Wistar rats for 28 days. Clinical factors, serum lipid levels, sex hormones, and gonadotropins as well as the histopathological changes were compared among groups by statistical analysis. Besides normal lipid profile, gonadotropin levels, and LH/FSH ratio at day 28, serum estradiol levels were raised in both GM (canola oil (p=0.04)) and non-GM (sunflower oil (p=0.008)) groups. In necropsy studies, ovarian atrophies were detected in canola (p<0.001) and sunflower groups (p<0.043) although uterine remained unchanged in all groups. In histopathological evaluations, all sections showed severe congestion and multiple follicular cysts in the sunflower oil group. Simple and secondary cysts in the maize group were the other type of ovarian toxicity in this short period of time. Remarkable estrogenic properties of GM and non-GM plant-derived edible oils with signs of ovarian atrophy, congestion, and cysts may contribute to phthalate or other xenoestrogenic contaminations; therefore, analytical studies of samples and further human populations studies are highly recommended.

Immune Suppression of Glia Maturation Factor Reverses Behavioral Impairment, Attenuates Amyloid Plaque Pathology and Neuroinflammation in an Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is an irreversible progressive neurodegenerative disorder recognized by accumulation of amyloid-plaques (APs) and neurofibrillary tangles (NFTs) and eventually loss of memory. Glia maturation factor (GMF), a neuroinflammatory protein first time isolated and cloned in our laboratory plays an important role in the pathogenesis of AD. However, no studies have been reported on whether anti-GMF antibody administration could downregulate neuroinflammation and attenuate amyloid pathology in AD brain. We investigated the potential effect of single dose of (2 mg/kg b.wt/mouse) intravenously (iv) injected with anti-GMF antibodyon cognitive function, neuroprotection, neuroinflammation and Aß load in the brain of 9-month-old 5XFAD mice. Following 4 weeks of anti-GMF antibody delivery in mice, we found reduced expression of GMF, astrocytic glial fibrillary acidic protein (GFAP) and microglial ionizing calcium binding adaptor molecule 1 (Iba1) as well as improvement inneuroinflammatory response via inhibition of pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) production and amyloid pathology in the cerebral cortex and hippocampal CA1 region of 5XFAD mice. Correspondingly, blockade of GMF function with anti-GMF antibody improved spatial learning, memory, and long-term recognition memory in 5XFAD mice. The present study demonstrates that the immune checkpoint blockade of GMF function with anti-GMF antibody coordinates anti-inflammatory effects to attenuate neurodegeneration in the cortex and hippocampal CA1 region of 5XFAD mouse brain. Further, our data suggest, that pharmacological immune neutralization of GMF is a promising neuroprotective strategy totherapeutically target neuroinflammation and neurodegeneration in AD. Graphical Abstract 5XFAD mice Polyclonal anti-GMF antibody.

NLRP3 inflammasome and glia maturation factor coordinately regulate neuroinflammation and neuronal loss in MPTP mouse model of Parkinson's disease.

Neuroinflammation plays an active role in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease (PD). Earlier studies from this laboratory showed that glia maturation factor (GMF), a proinflammatory mediator; is up-regulated in the brain in neurodegenerative diseases and that deficiency of GMF showed decreased production of IL-1ß and improved behavioral abnormalities in mouse model of PD. However, the mechanisms linking GMF and dopaminergic neuronal death have not been completely explored. In the present study, we have investigated the expression of NLRP3 inflammasome and caspase-1 in the substantia nigra (SN) of human PD and non-PD brains by immunohistochemistry. Wild-type (WT) and GMF-/- (GMF knock-out) mice were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine (MPTP) and the brains were isolated for neurochemical and morphological examinations. NLRP3 and caspase-1 positive cells were found significantly increased in PD when compared to non-PD control brains. Moreover, GMF co-localized with α-Synuclein within reactive astrocytes in the midbrain of PD. Mice treated with MPTP exhibit glial activation-induced inflammation, and nigrostriatal dopaminergic neurodegeneration. Interestingly, increased expression of the inflammasome components in astrocytes and microglia observed in the SN of MPTP-treated WT mice were significantly reduced in GMF-/- mice. Additionally, we show that NLRP3 activation in microglia leads to translocation of GMF and NLRP3 to the mitochondria. We conclude that downregulation of GMF may have beneficial effects in prevention of PD by modulating the cytotoxic functions of microglia and astrocytes through reduced activation of the NLRP3 inflammasome; a major contributor of neuroinflammation in the CNS.

Constitutive expression of GmF6'H1 from soybean improves salt tolerance in transgenic Arabidopsis.

Coumarin plays a pivotal role in plant response to biotic stress, as well as in the mediation of nutrient acquisition. However, its functions in response to abiotic stresses are largely unknown. In this work, a homologous gene, GmF6'H1, of AtF6'H1, which encodes the enzyme catalyzing the final rate-limiting step in the biosynthesis pathway of coumarin, was isolated from soybean. GmF6'H1 protein shares very high amino acid identity with AtF6'H1, and expression of GmF6'H1 in atf6'h1 can successfully restore the decreased coumarin production in the T-DNA insertion mutant. Further study revealed that the expression of GmF6'H1 in soybean was remarkably induced by salt stress. Constitutive expression of GmF6'H1 in Arabidopsis, driven by 35S promoter, significantly enhanced the resistance to salt of transgenic Arabidopsis. All these results suggest that GmF6'H1 can be used as a potential candidate gene for the engineering of plants with improved resistance to both biotic and abiotic stresses.

First description of enhanced expression of transforming growth factor-alpha (TGF-α) and glia maturation factor-beta (GMF-ß) correlate with severity of neuropathology in border disease virus-infected small ruminants.

Border disease (BD) is caused by Pestivirus and characterized by severe neuropathology, and histopathologically observed severe hypomyelination. We have previously shown that small ruminants infected with border disease virus (BDV) play an important role for neuropathology and pathogenesis of severe oxidative damage in brain tissue, neuronal mtDNA; in the production of high pathologic levels of nitric oxide; in glial cell activation and stimulation of intrinsic apoptosis pathway. This study aimed to investigate the relationship between glia maturation factor beta (GMF-ß) and transforming growth factor alpha (TGF-α) expressions and the causes of BDV-induced neuropathology and to investigate their role in neuropathogenesis in a way that was not presented before. Expression levels of GMF-ß and TGF-α were investigated. Results of the study revealed that the levels of GMF-ß (P < 0.005) and TGF-α (P < 0.005) expression in the brain tissue markedly increased in the BDV-infected animals compared to the non-infected healthy control group. While TGF-α expressions were predominantly observed in neurons, GMF-ß expressions were found in astrocytes, glial cells and neurons. These results were reasonable to suggest that BDV-mediated increased GMF-ß might play a pivotal role neuropathogenesis and a different type of role in the mechanism of neurodegeneration/neuropathology in the process of BD. The results also indicated that increased levels of GMF up-regulation in glial cells and neurons causes neuronal destruction, suggesting pathological pathway involving GMF-mediated brain cell cytotoxicity. It is clearly indicated that the cause of astrogliosis is due to severe TGF-a expression. This is the first study to demonstrate the expression of GMF-ß and TGF-α in neurons and reactive glial cells and its association with neuropathology in BD.

Genetically Modified Labeling Policies: Moving Forward or Backward?

One of the priorities to address food security is to increase the access of farmers to biotechnology, through the application of scientific advances, such as genetically modified organisms and food (GMF). However, the spread of (mis)information about their safety strengthens the clamor for mandatory GMF labeling. This paper provides an overview of food labeling policies, considering the principles suggested by the Codex Alimentarius Commission, and analyzes the consequences for the world food security of the Brazilian labeling policies compared to developed countries. We discuss the discriminatory application of GMF mandatory labeling in the absence of any scientific evidence as it has the potential of causing social harm and jeopardizes research, production, and distribution of food and consumers' right to information.

Analysis of the interactions between GMF and Arp2/3 complex in two binding sites by molecular dynamics simulation.

The Arp2/3 complex plays a key role in nucleating actin filaments branching. The glia maturation factor (GMF) competes with activators for interacting with the Arp2/3 complex and initiates the debranching of actin filaments. In this study, we performed a comparative analysis of interactions between GMF and the Arp2/3 complex and identified new amino acid residues involved in GMF binding to the Arp2/3 complex at two separate sites, revealed by X-ray and single particle EM techniques. Using molecular dynamics simulations we demonstrated the quantitative and qualitative changes in hydrogen bonds upon binding with GMF. We identified the specific amino acid residues in GMF and Arp2/3 complex that stabilize the interactions and estimated the mean force profile for the GMF using umbrella sampling. Phylogenetic and structural analyses of the recently defined GMF binding site on the Arp3 subunit indicate a new mechanism for Arp2/3 complex inactivation that involves interactions between the Arp2/3 complex and GMF at two binding sites.

Spatial memory impairment by TRPC1 depletion is ameliorated by environmental enrichment.

Canonical transient receptor potential (TRPC) channels are widely expressed throughout the nervous system whereas their functions remain largely unclear. Here we investigated the effects of TRPC1 deletion on spatial memory ability of mice and the potential intervention by environmental enrichment (EE). Significant spatial memory impairment assessed by conditional fearing test, Y maze test and step-down test in TRPC1 knockout mice was revealed. The behavioral abnormality were attenuated by the treatment of EE. Proteomic analysis by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with a matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) and tandem mass spectrometry (MS) revealed that TRPC1 deletion caused differential expression of a total of 10 proteins (8 up-regulated and 2 down-regulated) in hippocampus. EE treatment resulted in differential expression of a total of 22 proteins (2 up-regulated and 20 down-regulated) in hippocampus of TRPC1 knockout mice. Among these differentially expressed proteins, the expression of α-internexin and glia maturation factor ß (GMF-ß), two proteins shown to impair memory, were significantly down-regulated in hippocampus of TRPC1 knockout mice by EE treatment. Taken together, these data suggested that TRPC1 regulated directly or indirectly the expression of multiple proteins, which may be crucial for the maintenance of memory ability, and that EE treatment modulated spatial memory impairment caused by TRPC1 depletion and the mechanisms may involve the modulation of EE on the expression of those dys-regulated proteins such as α-internexin and GMF-ß in hippocampus.

Combinatorial genetic analysis of a network of actin disassembly-promoting factors.

The patterning of actin cytoskeleton structures in vivo is a product of spatially and temporally regulated polymer assembly balanced by polymer disassembly. While in recent years our understanding of actin assembly mechanisms has grown immensely, our knowledge of actin disassembly machinery and mechanisms has remained comparatively sparse. Saccharomyces cerevisiae is an ideal system to tackle this problem, both because of its amenabilities to genetic manipulation and live-cell imaging and because only a single gene encodes each of the core disassembly factors: cofilin (COF1), Srv2/CAP (SRV2), Aip1 (AIP1), GMF (GMF1/AIM7), coronin (CRN1), and twinfilin (TWF1). Among these six factors, only the functions of cofilin are essential and have been well defined. Here, we investigated the functions of the nonessential actin disassembly factors by performing genetic and live-cell imaging analyses on a combinatorial set of isogenic single, double, triple, and quadruple mutants in S. cerevisiae. Our results show that each disassembly factor makes an important contribution to cell viability, actin organization, and endocytosis. Further, our data reveal new relationships among these factors, providing insights into how they work together to orchestrate actin turnover. Finally, we observe specific combinations of mutations that are lethal, e.g., srv2Δ aip1Δ and srv2Δ crn1Δ twf1Δ, demonstrating that while cofilin is essential, it is not sufficient in vivo, and that combinations of the other disassembly factors perform vital functions.

Primary cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome): part I. Diagnosis: clinical and histopathologic features and new molecular and biologic markers.

Mycosis fungoides (MF) and Sézary syndrome (SS) comprise approximately 53% of cutaneous lymphomas. Both MF and SS may clinically and histologically mimic benign skin conditions, posing a diagnostic challenge to the dermatologist. Precise clinicopathologic correlation is necessary to support a diagnosis, especially in the early stages of disease. In addition to the identification of histopathologic criteria, ancillary studies, including the identification of CD4(+) T cells with aberrant immunophenotypes and T-cell receptor gene rearrangements within skin lesions and peripheral blood are used to support the diagnosis. Recent studies evaluating the pathogenesis of MF have found that the skin microenvironment, including immune cells, such as dendritic cells and reactive cytotoxic and regulatory T cells, plays a crucial supporting role in MF. The skin-homing ability of malignant T cells is the result of chemokines, cytokines, adhesion molecules, and defective apoptosis, and is believed to play a role in disease pathogenesis and progression. In addition, recent studies have also suggested that MF and SS arise from distinct memory T cell subsets and advanced/erythrodermic MF and SS may be distinguished by identification of certain molecules, including Programmed-Death-1.

Anxiety behavior is reduced, and physical growth is improved in the progeny of rat dams that consumed lipids from goat milk: an elevated plus maze analysis.

The goat milk contains conjugated linoleic acid (CLA), which can influence physical growth and brain development. This study investigated the impact of a diet containing goat milk fat (GMF) on physical parameters of gestating (G) and/or lactating (L) rat dams, and their progeny's physical growth, and anxiety behavior. In the dams, body weight was evaluated during gestation and lactation. Maternal physical parameters, thoracic and abdominal circumference and liver weight were measured at weaning. In the progeny, indicators of somatic development, and consolidation of reflex responses (palm grasp, righting, free-fall righting, vibrissa placing, auditory startle response, negative geotaxis and cliff avoidance) were determined. Anxiety behavior was tested on the elevated plus maze (EPM). Compared to the controls, GMF-pups presented higher body weight and tail length at days 18 and 21 (groups G+L and L). In the L-group, cliff avoidance and free-fall righting responses were respectively delayed, and accelerated. Fur appearance was anticipated in G+L pups. On postnatal day 35, the EPM responses of the G group indicated less anxiety than in the controls. Data show developmental and behavioral modifications in the progeny of dams fed the GMF-rich diet consumed during gestation and lactation, suggesting the involvement of CLA in such effects.

Glia maturation factor (GMF) interacts with Arp2/3 complex in a nucleotide state-dependent manner.

Glia maturation factor (GMF) is a member of the actin-depolymerizing factor (ADF)/cofilin family. ADF/cofilin promotes disassembly of aged actin filaments, whereas GMF interacts specifically with Arp2/3 complex at branch junctions and promotes debranching. A distinguishing feature of ADF/cofilin is that it binds tighter to ADP-bound than to ATP-bound monomeric or filamentous actin. The interaction is also regulated by phosphorylation at Ser-3 of mammalian cofilin, which inhibits binding to actin. However, it is unknown whether these two factors play a role in the interaction of GMF with Arp2/3 complex. Here we show using isothermal titration calorimetry that mammalian GMF has very low affinity for ATP-bound Arp2/3 complex but binds ADP-bound Arp2/3 complex with 0.7 µM affinity. The phosphomimetic mutation S2E in GMF inhibits this interaction. GMF does not bind monomeric ATP- or ADP-actin, confirming its specificity for Arp2/3 complex. We further show that mammalian Arp2/3 complex nucleation activated by the WCA region of the nucleation-promoting factor N-WASP is not affected by GMF, whereas nucleation activated by the WCA region of WAVE2 is slightly inhibited at high GMF concentrations. Together, the results suggest that GMF functions by a mechanism similar to that of other ADF/cofilin family members, displaying a preference for ADP-Arp2/3 complex and undergoing inhibition by phosphorylation of a serine residue near the N terminus. Arp2/3 complex nucleation occurs in the ATP state, and nucleotide hydrolysis promotes debranching, suggesting that the higher affinity of GMF for ADP-Arp2/3 complex plays a physiological role by promoting debranching of aged branch junctions without interfering with Arp2/3 complex nucleation.

Modes of acrosin functioning during fertilization.

Mammalian fertilization is a complex process that involves gamete recognition, penetration, and fusion. Biochemical studies that identified the role of acrosome components during sperm-ova interaction especially the zona pellucida (ZP) provided major advances in sperm cell biology. Acrosin (a typical serine protease) functions during fertilization in several significant ways which include: a) activation of acrosome components, b) secondary binding with the ZP, and c) hydrolysis of the ZP. However, studies using knockout (KO) acrosin-deficient mice cast doubt on the traditional role of acrosin in fertilization. The KO acrosin-deficient mice exhibit normal fecundity except for delayed fertilization. Despite the doubt cast on the traditional role of acrosin by the KO acrosin-deficient mouse studies, acrosin still remains a major protease involved in multiple processes of fertilization. In this review, we assess the functional profile of acrosin and briefly summarize recent findings on proteases involved in fertilization. We propose a refined scheme for the functional role of acrosin in fertilization. We particularly emphasize the role of acrosin in acrosome exocytosis and activation of other acrosome components based on advanced technology like structural X-ray analysis.