Calpain Regulates Reactive Oxygen Species Production during Capacitation through the Activation of NOX2 and NOX4.

Capacitation is a series of physiological, biochemical, and metabolic changes experienced by mammalian spermatozoa. These changes enable them to fertilize eggs. The capacitation prepares the spermatozoa to undergo the acrosomal reaction and hyperactivated motility. Several mechanisms that regulate capacitation are known, although they have not been fully disclosed; among them, reactive oxygen species (ROS) play an essential role in the normal development of capacitation. NADPH oxidases (NOXs) are a family of enzymes responsible for ROS production. Although their presence in mammalian sperm is known, little is known about their participation in sperm physiology. This work aimed to identify the NOXs related to the production of ROS in guinea pig and mouse spermatozoa and define their participation in capacitation, acrosomal reaction, and motility. Additionally, a mechanism for NOXs' activation during capacitation was established. The results show that guinea pig and mouse spermatozoa express NOX2 and NOX4, which initiate ROS production during capacitation. NOXs inhibition by VAS2870 led to an early increase in the capacitation and intracellular concentration of Ca2+ in such a way that the spermatozoa also presented an early acrosome reaction. In addition, the inhibition of NOX2 and NOX4 reduced progressive motility and hyperactive motility. NOX2 and NOX4 were found to interact with each other prior to capacitation. This interaction was interrupted during capacitation and correlated with the increase in ROS. Interestingly, the association between NOX2-NOX4 and their activation depends on calpain activation, since the inhibition of this Ca2+-dependent protease prevents NOX2-NOX4 from dissociating and ROS production. The results indicate that NOX2 and NOX4 could be the most important ROS producers during guinea pig and mouse sperm capacitation and that their activation depends on calpain.

FAK regulates actin polymerization during sperm capacitation via the ERK2/GEF-H1/RhoA signaling pathway.

Actin polymerization is a crucial process during sperm capacitation. We have recently described the participation of FAK during actin polymerization in guinea pig spermatozoa. However, the mechanism by which FAK mediates these processes is unknown. Our previous data have shown that MAPK1 (hereafter referred to as ERK2) is activated during the first minutes of capacitation, and inhibition of ERK2 blocked actin polymerization and the acrosome reaction. In this current study, we found that FAK is involved in ERK2 activation - as FAK was phosphorylated at tyrosine residue 925 and bound to Grb2 - and that inhibition of FAK results in a significant decrease of ERK2 activation. We also confirmed the presence of Rho guanine nucleotide exchange factor 2 (ARHGEF2, hereafter referred to as GEF-H1), which is able to associate with RhoA during capacitation. RhoA activation and its participation in actin polymerization were also analyzed. Inhibition of FAK or ERK1/2 impeded GEF-H1 phosphorylation, RhoA activation, and the association between GEF-H1 and RhoA. Finally, we observed the presence of fibronectin on the sperm surface, its role in sperm-sperm interaction as well as participation of ß-integrin in the activation of ERK2. Our results show that the signaling pathway downstream of fibronectin, via integrin, FAK, Grb2, MEK1/2, ERK2, GEF-H1 and RhoA regulates the actin polymerization associated with spermatozoa capacitation.

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa.

Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1-GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem-like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin-1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F-actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.

CDC42 drives RHOA activity and actin polymerization during capacitation.

Mammalian sperm cells acquire fertilizing capacity as a result of a process termed capacitation. Actin polymerization is important for capacitation; inhibiting actin polymerization prevents the adhesion and fusion of the sperm with the ovule. The main function of RHO proteins CDC42 and RHOA is to direct actin polymerization. Although these two RHO proteins are present in mammalian sperm, little is known about their role in capacitation, the acrosome reaction, and the way in which they direct actin polymerization. The purpose of this study was to determine the participation of CDC42 and RHOA in capacitation and the acrosome reaction and their relationship with actin polymerization using guinea pig sperm. Our results show that the inhibition of CDC42 and RHOA alters the kinetics of actin polymerization, capacitation, and the acrosome reaction in different ways. Our results also show that the initiation of actin polymerization and RHOA activation depend on the activation of CDC42 and that RHOA starts its activity and effect on actin polymerization when CDC42 reaches its maximum activity. Given that the inhibition of ROCK1 failed to prevent the acrosomal reaction, the participation of RHOA in capacitation and the acrosomal reaction is independent of its kinase 1 (ROCK1). In general, our results indicate that CDC42 and RHOA have different roles in capacitation and acrosomal reaction processes and that CDC42 plays a preeminent role.

Low concentrations of lead decrease the sperm fertilization ability by altering the acrosome reaction in mice.

Lead (Pb) exposure at high concentrations is associated with poor sperm quality, acrosome alterations, and low fertilization rate. Sperm capacitation and the acrosome reaction (AR) are required for successful fertilization. Actin polymerization is crucial for correct capacitation, and small GTPases, such as RhoA, Rac1, and Cdc42, are involved. This study aimed to evaluate the effects of Pb on sperm fertilization ability, capacitation, AR, and the mechanisms involved in mice exposed to low Pb concentrations. CD1 mice were exposed to 0.01% Pb2+ for 45 days through their drinking water and their spermatozoa were collected from the cauda epididymis-vas deferens to evaluate the following: AR (oAR: initial, sAR: spontaneous, and iAR: induced) using the PNA-FITC assay, sperm capacitation (P-Tyr levels), actin polymerization (phalloidin-TRITC), MDA production (stress oxidative marker), the RhoA, Rac1, and Cdc42 protein levels, and the in vitro fertilization (IVF). After the treatment, the blood Pb (PbB) concentration was 9.4 ±â€¯1.6 µg/dL. Abnormal sperm morphology and the oAR increased (8 and 19%, respectively), whereas the iAR decreased (15%) after a calcium ionophore challenge, and the actin polymerization decreased in the sperm heads (59%) and tails (42%). Rac1 was the only Rho protein to significantly decrease (33%). Spermatozoa from the Pb-treated mice showed a significant reduction in the fertilization rate (19%). Our data suggest that Pb exposure at environmental concentrations (PbB < 10 µg/dL) decreases the acrosome function and affects the sperm fertilization ability; this is probably a consequence of the low Rac1 levels, which did not allow adequate actin polymerization to occur.

TMEM16A inhibition impedes capacitation and acquisition of hyperactivated motility in guinea pig sperm.

Ca2+ -activated Cl- channels (CaCCs) are anionic channels that regulate many important physiological functions associated with chloride and calcium flux in some somatic cells. The molecular identity of CaCCs was revealed to be TMEM16A and TMEM16B (also known as Anoctamin or ANO1 and ANO2, respectively) in all eukaryotes. A recent study suggests the presence of TMEM16A in human sperm and a relationship with the rhZP-induced acrosome reaction. However, to the best of our knowledge, little is known about the role of TMEM16A in other spermatic processes such as capacitation or motility. In this study, we evaluated the effects of two TMEM16A antagonists on capacitation, acrosome reaction, and motility in guinea pig sperm; these antagonists were T16Ainh-A01, belonging to a second generation of potent antagonists of TMEM16A, and niflumic acid (NFA), a well-known antagonist of TMEM16A (CaCCs). First of all, we confirmed that the absence of Cl- in the capacitation medium changes motility parameters, capacitation, and the progesterone-induced acrosome reaction. Using a specific antibody, TMEM16A was found as a protein band of ∼120 kDa, which localization was in the apical crest of the acrosome and the middle piece of the flagellum. Inhibition of TMEM16A by T16Ainh-A01 affected sperm physiology by reducing capacitation, blocking the progesterone-induced acrosome reaction under optimal capacitation conditions, inhibiting progressive motility, and the acquisition of hyperactivated motility, diminishing [Ca2+ ]i, and increasing [Cl- ]i. These changes in sperm kinematic parameters provide new evidence of the important role played by TMEM16A in the production of sperm capable of fertilizing oocytes.

Calpain inhibition prevents flotillin re-ordering and Src family activation during capacitation.

Prior to fertilization, mammalian sperm undergo several molecular, biochemical and physiological changes in a process termed capacitation. However, the mechanisms explaining the involvement of cytoskeletal remodeling and membrane re-ordering in each process prior to fertilization remain poorly understood. We found that the migration of both flotillin microdomains and Src family kinases towards the apical ridge of guinea pig sperm occurs under capacitating conditions. This re-ordering is associated with spectrin cleavage by calpain. Moreover, Src, Fyn, Lyn and Hck interact with flotillin-1; this interaction increases in a capacitation-dependent manner and the increased autophosphorylation of these kinases is linked to flotillin-1 association. The aforementioned results are prevented by the inhibition of calpain by calpeptin. Thus, spectrin cytoskeleton cleavage during capacitation seems to precede the reorganization of flotillin microdomains and Src family kinases towards the apical ridge of the sperm head in order to initiate the signaling cascade required for proper capacitation and further acrosome reaction. The significance of the Src family kinase reorganization for capacitation is demonstrated by the inhibition of calpain during capacitation also preventing the Src-family-kinase-dependent phosphorylation of FAK at Tyr576/577. Our work further highlights the scaffolding properties of flotillin microdomains and reveals the importance of their large-scale segregation during capacitation.

Absence of aryl hydrocarbon receptor alters CDC42 expression and prevents actin polymerization during capacitation.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates the toxicity of a variety of environmental chemicals. The absence of this receptor causes serious reproductive complications. Ahr-knockout (Ahr-KO) male mice, for example, are considerably less fertile: Half of the few spermatozoa they produce exhibit morphological alterations, and those with typical morphology may have pathologic modifications. We therefore investigated the consequences of AHR loss on capacitation and the acrosome reaction, and asked if these effects are a consequence of changes to actin polymerization and the expression of Cdc42, which encodes Cell division control protein 42 (CDC42), a RHO protein that controls assembly of the actin cytoskeleton in somatic cells as well as during spermatogenesis. Nearly 50% of spermatozoa produced by Ahr-KO mice had alterations in the flagellum. Ahr-KO spermatozoa were frequently capacitated, but showed reduced spontaneous and progesterone-induced acrosome reaction-which is related to low CDC42 abundance and very limited actin polymerization during capacitation. Thus, the expression of CDC42 might be regulated by AHR, and both proteins are fundamental to the development of normal spermatozoa and the acrosome reaction. Mol. Reprod. Dev. 83: 1015-1026, 2016 © 2016 Wiley Periodicals, Inc.

Association of membrane/lipid rafts with the platelet cytoskeleton and the caveolin PY14: participation in the adhesion process.

Platelets are the most prominent elements of blood tissue involved in hemostasis at sites of blood vessel injury. Platelet cytoskeleton is responsible for their shape modifications observed during activation and adhesion to the substratum; therefore the interactions between cytoskeleton and plasma membrane are critical to modulate blood platelet functions. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to membrane/lipid rafts (MLR) and regulate lateral diffusion of membrane proteins and lipids. Resting, thrombin-activated, and adherent human platelets were processed for biochemical studies including western-blot and immunprecipitation assays and confocal analysis were performed to characterize the interaction of MLR with the main cytoskeleton elements and ß-dystroglycan as well as with the association of caveolin-1 PY14 with focal adhesion proteins. We transfected a megakaryoblast cell line (Meg-01) to deplete ß-dystroglycan, subsequent to their differentiation to the platelet progenitors. Our data showed a direct interaction of the MLR with cytoskeleton to regulate platelet shape, while an association of caveolin-1 PY14 with vinculin is needed to establish focal adhesions, which are modulated for ß-dystroglycan. In conclusion, caveolin-1 PY14 in association with platelet cytoskeleton participate in focal adhesions dynamics.

ADAM15 participates in fertilization through a physical interaction with acrogranin.

Mammalian fertilization is completed by direct interaction between sperm and egg. This process is primarily mediated by both adhesion and membrane-fusion proteins found on the gamete surface. ADAM1, 2, and 3 are members of the ADAMs protein family, and have been involved in sperm-egg binding. In this study, we demonstrate the proteolytic processing of ADAM15 during epididymal maturation of guinea pig spermatozoa to produce a mature form a size of 45 kDa. We find that the size of the mature ADAM15, 45 kDa, in cauda epididymal spermatozoa indicates that the pro-domain and metalloprotease domain are absent. In addition, using indirect immunofluorescence, ADAM15 was found throughout the acrosome, at the equatorial region and along the flagellum of guinea pig spermatozoa. After acrosome reaction, ADAM15 is lost from the acrosomal region and retained in the equatorial region and flagellum. In this study, we also report the first evidence of a complex between ADAM15 and acrogranin. By immunoprecipitation, we detected a protein band of 65 kDa which co-immunoprecipated together ADAM15. Analysis of the N-terminal sequence of this 65 kDa protein has revealed its identity as acrogranin. In addition, using cell-surface labeling, ADAM15 was found to be present on the cell surface. Assays of heterologous fertilization showed that the antibody against acrogranin inhibited the sperm-egg adhesion. Interestingly, ADAM15 and acrogranin were also found associated in two breast cancer cell lines. In conclusion, our results demonstrated that ADAM15 and acrogranin are present on and associated with the surface of guinea pig spermatozoa; besides both proteins may play a role during sperm-egg binding.

Mouse sperm membrane potential hyperpolarization is necessary and sufficient to prepare sperm for the acrosome reaction.

Mammalian sperm are unable to fertilize the egg immediately after ejaculation; they acquire this capacity during migration in the female reproductive tract. This maturational process is called capacitation and in mouse sperm it involves a plasma membrane reorganization, extensive changes in the state of protein phosphorylation, increases in intracellular pH (pH(i)) and Ca(2+) ([Ca(2+)](i)), and the appearance of hyperactivated motility. In addition, mouse sperm capacitation is associated with the hyperpolarization of the cell membrane potential. However, the functional role of this process is not known. In this work, to dissect the role of this membrane potential change, hyperpolarization was induced in noncapacitated sperm using either the ENaC inhibitor amiloride, the CFTR agonist genistein or the K(+) ionophore valinomycin. In this experimental setting, other capacitation-associated processes such as activation of a cAMP-dependent pathway and the consequent increase in protein tyrosine phosphorylation were not observed. However, hyperpolarization was sufficient to prepare sperm for the acrosome reaction induced either by depolarization with high K(+) or by addition of solubilized zona pellucida (sZP). Moreover, K(+) and sZP were also able to increase [Ca(2+)](i) in non-capacitated sperm treated with these hyperpolarizing agents but not in untreated cells. On the other hand, in conditions that support capacitation-associated processes blocking hyperpolarization by adding valinomycin and increasing K(+) concentrations inhibited the agonist-induced acrosome reaction as well as the increase in [Ca(2+)](i). Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the acrosome reaction.

Participation of the Cl-/HCO(3)- exchangers SLC26A3 and SLC26A6, the Cl- channel CFTR, and the regulatory factor SLC9A3R1 in mouse sperm capacitation.

Sperm capacitation is required for fertilization and involves several ion permeability changes. Although Cl(-) and HCO(3)(-) are essential for capacitation, the molecular entities responsible for their transport are not fully known. During mouse sperm capacitation, the intracellular concentration of Cl(-) ([Cl(-)](i)) increases and membrane potential (Em) hyperpolarizes. As in noncapacitated sperm, the Cl(-) equilibrium potential appears to be close to the cell resting Em, opening of Cl(-) channels could not support the [Cl(-)](i) increase observed during capacitation. Alternatively, the [Cl(-)](i) increase might be mediated by anion exchangers. Among them, SLC26A3 and SLC26A6 are good candidates, since, in several cell types, they increase [Cl(-)](i) and interact with cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) channel present in mouse and human sperm. This interaction is known to be mediated and probably regulated by the Na(+)/H(+) regulatory factor-1 (official symbol, SLC9A3R1). Our RT-PCR, immunocytochemistry, Western blot, and immunoprecipitation data indicate that SLC26A3, SLC26A6, and SLC9A3R1 are expressed in mouse sperm, localize to the midpiece, and interact between each other and with CFTR. Moreover, we present evidence indicating that CFTR and SLC26A3 are involved in the [Cl(-)](i) increase induced by db-cAMP in noncapacitated sperm. Furthermore, we found that inhibitors of SLC26A3 (Tenidap and 5099) interfere with the Em changes that accompany capacitation. Together, these findings indicate that a CFTR/SLC26A3 functional interaction is important for mouse sperm capacitation.

Cytoskeletal proteins F-actin and ß-dystrobrevin are altered by the cryopreservation process in bull sperm.

The cryopreservation process has an important impact on sperm structure and physiology. The negative effects have been mainly observed on the plasma membrane, which is directly stabilized by the cytoskeleton. Since cytoskeleton proteins are osmosensitive and thermosensitive, the aim of this study was to evaluate the damage caused to the bull sperm cytoskeleton by cryopreservation (freezing-thawing). Fresh and frozen-thawed bull semen samples were exposed to a treatment with the neutral detergent Brij 36-T. Electron microscopy evidenced important damages at the sperm perinuclear theca after the protein extraction protocol; the perinuclear theca was partially solubilized, the perinuclear theca substructure disappeared in the cryopreserved samples. Furthermore, the sperm head's shape was significantly altered on the cryopreserved samples. Fluorescence analysis showed a decrease of the intensity of actin and dystrobrevin on the frozen-thawed samples. Western blot assays revealed a stronger signal for actin and ß-dystrobrevin in the frozen-thawed sperm samples than in the fresh ones. Our results suggest that the cryopreservation process highly alters the sperm cytoskeleton stability, causing its proteins to become more fragile and therefore more susceptible to be extracted.

Participation of signaling proteins in sperm hyperactivation.

Sperm hyperactivation is described as a fast whip movement of the flagellum, an irregular trajectory, and an asymmetrically flagellum bend. This motility pattern is achieved during the passage of the sperm along the female genital tract. It helps the spermatozoa to cross through different viscous ambient fluids to finally reach the oocyte. Important signaling proteins are located in the sperm head and flagellum, and they all play an important role in the cascade that controls the sperm hyperactivation. The presence of HCO3- modulates the activity of the soluble adenylyl cyclase (sAC), leading to the production of cAMP. In turn, cAMP modulates the sperm-specific Na+/H+ exchanger (sNHE) and the t-complex protein 11 (TCP11) which play an essential role on the signaling pathway (cAMP/PKA and tyrosine phosphorylation) and sperm hypermotility. sNHE, cystic fibrosis transmembrane conductance regulator (CFTR), and voltage-gated proton channel (Hv) mainly contribute to the regulation of the intracellular pH (pHi) during capacitation. HCO3- entrance and the removal of H+ from the cytoplasm induces the alkalization of pHi, and this change will contribute to the activation of the cation channel of sperm (CatSper). Recently, it was described the participation on sperm motility and the regulation of calcium channels of an autophagy-related protein, the microtubule-associated protein light chain 3 (LC3). This review gathers important literature about the essential roles of sAC, sNHE, CFTR, Hv, and CatSper in the acquisition of sperm hyperactivation, and provides an integrated overview of recently described roles of TCP11 and LC3 on the sperm signaling pathway. Additionally, we provide insight into the infertility induced by the dysfunction of these critical proteins.

Perfluorooctane sulfonate and perfluorooctanoic acid induce plasma membrane dysfunction in boar spermatozoa during in vitro capacitation.

Spermatozoa require the capacitation, a series of biochemical events, to perform fertilization. Many toxic compounds can interfere in this process, including perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), which belong to the perfluoroalkyl substances (PFAS). Since both substances are found in many everyday materials and are highly persistent, they accumulate in organisms where they have been associated with fertility problems. This study analyzes the effects of PFOS and PFOA on the functionality of boar spermatozoa, and changes in the plasma membrane (PM) during capacitation. The median lethal concentrations (LC50) of PFOS and PFOA were 460 and 1894 µM, respectively, while the mean inhibitory concentrations of capacitation (ICC50) were 274 µM and 1458 µM, respectively. The ICC50 of PFOA was insufficient to reduce the capacitation, but 950 µM (½ LC50) of PFOA and the ICC50 of PFOS significantly reduced the number of capacitated spermatozoa. PFOS and PFOA also impeded the progesterone (P4)-induced acrosomal reaction (iAR). These effects occur despite the accumulation of [Ca2+]i under capacitating conditions. The accumulation of [Ca2+]i produces saturation, which prevents its entry through ionophore A23187 and P4 in the presence of PFOS. Membrane potential (Emv) was deregulated. Both PFAS affected lipid membrane conductance mediated by valinomycin. The spermatozoa presented 49% and 47% of membrane dysfunction with PFOS and PFOA, respectively. By causing membrane damage, both substances prevented the release of cholesterol and altered the organization of membrane microdomains (MMDs). Data indicate that both PFAS caused alterations in PM functionality.

Calpain modulates capacitation and acrosome reaction through cleavage of the spectrin cytoskeleton.

Research on fertilization in mammalian species has revealed that Ca(2+) is an important player in biochemical and physiological events enabling the sperm to penetrate the oocyte. Ca(2+) is a signal transducer that particularly mediates capacitation and acrosome reaction (AR). Before becoming fertilization competent, sperm must experience several molecular, biochemical, and physiological changes where Ca(2+) plays a pivotal role. Calpain-1 and calpain-2 are Ca(2+)-dependent proteases widely studied in mammalian sperm; they have been involved in capacitation and AR but little is known about their mechanism. In this work, we establish the association of calpastatin with calpain-1 and the changes undergone by this complex during capacitation in guinea pig sperm. We found that calpain-1 is relocated and translocated from cytoplasm to plasma membrane (PM) during capacitation, where it could cleave spectrin, one of the proteins of the PM-associated cytoskeleton, and facilitates AR. The aforementioned results were dependent on the calpastatin phosphorylation and the presence of extracellular Ca(2+). Our findings underline the contribution of the sperm cytoskeleton in the regulation of both capacitation and AR. In addition, our findings also reveal one of the mechanisms by which calpain and calcium exert its function in sperm.

Dietary Biotin Supplementation Impairs Testis Morphology and Sperm Quality.

Supplements containing pharmacological concentrations of biotin are commercially available over the counter. Classical toxicity studies have considered biotin administration as harmless; however, recent investigations have shown that biotin supplementation modifies tissue morphology without changes in toxicity markers, raising concerns about the consequences of morphological changes on tissues' functions and the safety of pharmacological concentrations of the vitamin. Testes are very sensitive to toxicants, and testicular histology is a reliable method to study its function. In this work, we investigated the effects of dietary biotin supplementation on testis morphology and spermatogenesis function using an experimental model, in which we have not observed unfavorable effects on other tissue functions or toxicity markers. Male BALB/cAnNHsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for 8 weeks. Compared to the control group, the biotin-supplemented mice presented remarkable testis morphology changes, including increased spermatogonia layers; the cellular mechanism involved is related to increased proliferation. Sperm count and serum testosterone levels were not affected, but spermatozoa motility and morphology were significantly impaired in the biotin-supplemented mice. These results caution against the use of supplements with high concentrations of biotin and indicate that biotin's pharmacological effects on morphology need to be considered in toxicological studies.

beta-Dystroglycan modulates the interplay between actin and microtubules in human-adhered platelets.

To maintain the continuity of an injured blood vessel, platelets change shape, secrete granule contents, adhere, aggregate, and retract in a haemostatic plug. Ordered arrays of microtubules, microfilaments, and associated proteins are responsible for these platelet responses. In full-spread platelets, microfilament bundles in association with other cytoskeleton proteins are anchored in focal contacts. Recent studies in migrating cells suggest that co-ordination and direct physical interaction of microtubules and actin network modulate adhesion development. In platelets, we have proposed a feasible association between these two cytoskeletal systems, as well as the participation of the dystrophin-associated protein complex, as part of the focal adhesion complex. The present study analysed the participation of microtubules and actin during the platelet adhesion process. Confocal microscopy, fluorescence resonance transfer energy and immunoprecipitation assays were used to provide evidence of a cross-talk between these two cytoskeletal systems. Interestingly, beta-dystroglycan was found to act as an interplay protein between actin and microtubules and an additional communication between these two cytoskeleton networks was maintained through proteins of focal adhesion complex. Altogether our data are indicative of a dynamic co-participation of actin filaments and microtubules in modulating focal contacts to achieve platelet function.

Presence, processing, and localization of mouse ADAM15 during sperm maturation and the role of its disintegrin domain during sperm-egg binding.

Successful fertilization requires gametes to complete several stages, beginning with maturation and transport along the male and female reproductive tracts and ending with the interaction between the sperm and the egg. This last step involves sperm-egg adhesion and membrane fusion. ADAMs (disintegrin and metalloprotease domain proteins) are a family of membrane-anchored glycoproteins that are thought to play diverse roles in cell-cell adhesion through their interaction with integrins. This study analyzes the presence, location, processing, and possible role of ADAM15 in mouse sperm. The presence of ADAM15 in mouse spermatozoa was detected by Western blotting, which revealed that ADAM15 is post-translationally processed, during epididymal sperm maturation and the acrosome reaction. The 35 kDa antigen present in the acrosome-reacted sperm is the last proteolytic product of the 110/75 kDa ADAM15 found in non-capacitated sperm. This 35 kDa protein contains the disintegrin domain. By indirect immunofluorescence, ADAM15 was identified in the acrosomal region and along the flagellum of mouse spermatozoa. In acrosome-reacted sperm, ADAM15 was lost from the acrosomal region, but remained diffusely distributed throughout the head and flagellum. Furthermore, the ADAM15 disintegrin domain (RPPTDDCDLPEF) partially inhibited fusion and almost completely inhibited sperm-oolemma adhesion. In conclusion, our data indicate that ADAM15 is present in the testis and in spermatozoa from the caput, corpus, and cauda epididymis, as well as in non-capacitated and acrosome-reacted gametes. Results also indicate that ADAM15 is processed during epididymal maturation and acrosome reaction and that it may play a role during sperm-egg binding.

Focal adhesion kinase is required for actin polymerization and remodeling of the cytoskeleton during sperm capacitation.

Several focal adhesion proteins are known to cooperate with integrins to link the extracellular matrix to the actin cytoskeleton; as a result, many intracellular signaling pathways are activated and several focal adhesion complexes are formed. However, how these proteins function in mammalian spermatozoa remains unknown. We confirm the presence of focal adhesion proteins in guinea pig spermatozoa, and we explore their role during capacitation and the acrosome reaction, and their relationship with the actin cytoskeleton. Our results suggest the presence of a focal adhesion complex formed by ß1-integrin, focal adhesion kinase (FAK), paxillin, vinculin, talin, and α-actinin in the acrosomal region. Inhibition of FAK during capacitation affected the protein tyrosine phosphorylation associated with capacitation that occurs within the first few minutes of capacitation, which caused the acrosome reaction to become increasingly Ca(2+) dependent and inhibited the polymerization of actin. The integration of vinculin and talin into the complex, and the activation of FAK and paxillin during capacitation, suggests that the complex assembles at this time. We identify that vinculin and α-actinin increase their interaction with F-actin while it remodels during capacitation, and that during capacitation focal adhesion complexes are structured. FAK contributes to acrosome integrity, likely by regulating the polymerization and the remodeling of the actin cytoskeleton.