RESUMEN
In order to penetrate the egg, spermatozoa must undergo the acrosome reaction in close proximity to the egg. This process can take place only after a series of biochemical changes in the sperm, collectively termed capacitation, occur in the female reproductive tract. Sperm cells can undergo spontaneous-acrosome reaction(sAR) before reaching the vicinity of the egg, preventing successful fertilization. Several mechanisms were shown to protect sperm from undergoing sAR, and all of them are involved in proper capacitation. Here, we describe the involvement of protein acetylation in the mechanism that protects bovine spermatozoa from sAR. Incubation of bovine sperm under non-capacitation conditions revealed a strong increase in sAR that was significantly reduced in the presence of deacetylase inhibitors. Protein kinase A (PKA) is an essential key enzyme in sperm capacitation, and its inhibition results in high sAR. The reduction in sAR by hyperacetylation was independent of PKA activity. We previously demonstrated that calmodulin-kinase II (CaMKII) activity protects sperm from sAR, and here we show that its activity is essential for reduction in sAR by hyperacetylation. We further show that the 'exchange protein directly activated by Camp' (EPAC) mediates both protein lysine acetylation and the reduced rate of sAR caused by hyperacetylation. In conclusion, these results suggest a PKA-independent and EPAC-CaMKII dependent hyperacetylation mechanism that protects sperm from sAR.
Asunto(s)
Reacción Acrosómica , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Acetilación , Acrosoma/metabolismo , Reacción Acrosómica/fisiología , Animales , Bovinos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Factores de Intercambio de Guanina Nucleótido/metabolismo , Masculino , Semen/metabolismo , Capacitación Espermática/fisiología , Espermatozoides/metabolismoRESUMEN
To interact and penetrate the egg, the spermatozoon must undergo a maturation step called the acrosome reaction (AR) in close proximity to the egg. This process can take place only after a series of biochemical changes to the sperm occur in the female reproductive tract, collectively called capacitation. Spermatozoa can undergo spontaneous-acrosome reaction (sAR) before reaching the vicinity of the egg, preventing successful fertilization. Several mechanisms were shown to protect spermatozoa from undergoing sAR. Here we describe the involvement of the actin cross-linker, Ezrin in the mechanism that protects spermatozoa from sAR. Inhibition of Ezrin stimulates sAR and inhibits actin polymerization. Ezrin is highly phosphorylated/activated during the first hour of the capacitation process, and its phosphorylation rate is subsequently decreased. Ezrin phosphorylation depends on protein kinase A (PKA) and calmodulin kinase II (CaMKII) activities, and to some extent on phosphatidyl-inositol-4-kinase (PI4K) activity. Inhibition of these three kinases stimulates sAR, in which the effect of PI4K inhibition, but not PKA or CaMKII inhibition, can be reversed by increasing p-Ezrin using a phosphatase inhibitor. All together, we showed that three kinases mediate Ezrin activation during spermatozoa capacitation, leading to actin polymerization in a mechanism that prevents sAR.
Asunto(s)
Reacción Acrosómica/efectos de los fármacos , Proteínas del Citoesqueleto/farmacología , Espermatozoides/efectos de los fármacos , Reacción Acrosómica/fisiología , Animales , Bencilaminas/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Bovinos , Regulación de la Expresión Génica , Masculino , Ácidos Fosfatidicos , Fosforilación , Capacitación Espermática/efectos de los fármacos , Sulfonamidas/farmacología , Wortmanina/farmacologíaRESUMEN
BACKGROUND: Sperm capacitation is essential for proper fertilization and is associated with increased sperm hyperactivity (HA) and acrosome reaction (AR). For successful fertilization, AR timing is critical; accordingly, early spontaneous AR may not facilitate fertilization. Paraoxonase 1 (PON1) possesses antioxidant properties which affect sperm capacitation. The association between PON1, semen parameters, and capacitation is not fully understood. OBJECTIVE: To study PON1 activity in relation to human sperm hyperactivity and AR. MATERIALS AND METHODS: Semen samples were collected, and parameters were determined (volume, concentration, total sperm count, percentage total motility, and percentage normal morphology) according to World Health Organization (WHO) guidelines. AR and hyperactivity were evaluated using FITC-PSA, staining, and computer-aided sperm analysis (CASA). PON1 activity was assessed using arylesterase activity assay. RESULTS: Purified PON1 inhibited both sperm hyperactivity and AR in a dose-dependent manner. Native semen PON1 activity was positively associated with higher sperm concentration and negatively associated with spontaneous acrosome reaction (sAR). DISCUSSION AND CONCLUSION: PON1 may have a positive effect on fertility via its ability to prevent early spontaneous sperm capacitation and AR before reaching the female genital tract.
Asunto(s)
Reacción Acrosómica/fisiología , Arildialquilfosfatasa/metabolismo , Capacitación Espermática/fisiología , Espermatozoides/fisiología , Antioxidantes/metabolismo , Humanos , Masculino , Análisis de Semen , Recuento de Espermatozoides , Motilidad Espermática/fisiologíaRESUMEN
Bromodomain testis-specific (BRDT) protein is essential for the normal process of spermatogenesis. Mutant mice that expressed truncated BRDT had impaired testicular histology with severely reduced sperm concentration and abnormal sperm morphology, while a model of knockout Brdt mice with no BRDT protein had complete meiotic arrest. A BRDT single nucleotide polymorphism (SNP) (rs3088232) was reported as being associated with infertility in men. We assessed testicular specimens of 276 azoospermic men who underwent testicular sperm extraction to search for specimens that showed spermatogenic impairments similar to those of mutant BRDT mice. Ten similar specimens were selected for BRDT gene sequencing and they revealed three NCBI-reported SNPs (rs10783071, rs3088232 and rs10747493) variously distributed among them. Bioinformatics analysis predicted that they would not affect protein activity. Further assessment of rs3088232 frequency in a large group of non-obstructive azoospermia men and fertile controls demonstrated no significant difference between them (27.2 and 21.7% respectively; p = 0.122, Fisher's exact test). We conclude that the testicular impairments observed in the 10 specimens were not a consequence of BRDT gene mutation. The association between BRDT rs3088232 and infertility that had been reported in other studies was not supported.
Asunto(s)
Azoospermia/genética , Proteínas Nucleares/genética , Polimorfismo de Nucleótido Simple , Espermatogénesis/genética , Testículo/patología , Secuencia de Aminoácidos , Frecuencia de los Genes , Humanos , Masculino , Datos de Secuencia MolecularRESUMEN
To enable fertilization, spermatozoa must undergo several biochemical processes in the female reproductive tract, collectively called capacitation. These processes involve protein kinase A (PKA)-dependent protein tyrosine phosphorylation including phosphatidylinositol-3-kinase (PI3K). It is not known how PKA, a serine/threonine (S/T) kinase, mediates tyrosine phosphorylation of proteins. We recently showed that inhibition of S/T phosphatase 1 (PP1) causes a significant increase in phospho-PI3K. In this study, we propose a mechanism by which PKA and PP1 mediate an increase in PI3K tyrosine phosphorylation and implicate calmodulin-dependent kinase II (CaMKII) in this process. Inhibition of sperm PP1 or PKC, stimulated CaMKII phosphorylation/activation, and inhibition of PKC enhanced PP1 phosphorylation/inactivation. Inhibition of CaMKII, using KN-93, caused significant reduction in phospho-PP1, indicating its activation. Moreover, KN-93 prevented the dephosphorylation/inactivation of PKC. We therefore suggest that CaMKII inhibits PKC, leading to PP1 inhibition and the reciprocal auto-activation of CaMKII. Thus, CaMKII can regulate its own activation by inhibiting the PKC/PP1 cascade. Inhibition of Src family kinases (SFK) caused significant inhibition of CaMKII and PP1 phosphorylation, suggesting that SFK activity results in PP1 inhibition and CaMKII activation. Activation of sperm PKA by 8Br-cAMP revealed an increase in phospho-CaMKII, which was inhibited by PKA inhibitor. Tyrosine phosphorylation of PI3K was stimulated by 8Br-cAMP and by PKC or PP1 inhibition and was abrogated by CaMKII inhibition. Furthermore, phosphorylation/activation of the tyrosine kinase Pyk2 was enhanced by PP1 inhibition, and this activation is blocked by CaMKII inhibition. Thus, PKA activates Src, which inhibits PP1, leading to CaMKII and Pyk2 activation, resulting in PI3K tyrosine phosphorylation/activation.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Fosfatidilinositol 3-Quinasa/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Fosfatasa 1/antagonistas & inhibidores , Espermatozoides/metabolismo , Animales , Bovinos , Activación Enzimática , Masculino , Fosforilación , Proteína Quinasa C/metabolismo , Proteína Fosfatasa 1/metabolismo , Transducción de Señal , Espermatozoides/enzimología , Familia-src Quinasas/metabolismoRESUMEN
Basic semen analysis is insufficient for determining the fertility potential. The aim of this study was to determine if hyperactivated motility (HAM) and acrosome reaction (AR) can be useful tests for evaluating semen quality during male infertility evaluations and to help the clinician decide whether regular insemination or intracytoplasmic sperm injection (ICSI) is preferable during in vitro fertilisation. A prospective study was conducted. Patients with normal sperm according to World Health Organization guidelines who underwent IVF treatment and planned regular insemination were asked to participate. A portion of sperm sample was evaluated for HAM and AR on day of ovum pick up. In HAM assessment, 93.3% of patients with increased HAM had a high fertilisation rate compared with 64% in the group without increased HAM (P = 0.059). For the AR evaluation, 91.7% of samples with a low rate of spontaneous AR had a high fertilisation rate compared with 39.3% in the group with a high rate of spontaneous AR (P = 0.004).
Asunto(s)
Reacción Acrosómica , Fertilización In Vitro/métodos , Inyecciones de Esperma Intracitoplasmáticas/métodos , Motilidad Espermática , Femenino , Humanos , Masculino , Estudios Prospectivos , Capacitación EspermáticaRESUMEN
BACKGROUND: Hyper-activated motility (HAM) is part of the sperm capacitation process, which is necessary for fertilization. In this study, we investigated the effect of visible light on sperm motility and hyperactivation and evaluated pathways mediating these effects. METHODS: Human sperm (1 × 107 cells/ml) in capacitation media were irradiated for 3 min with 40 mW/cm² visible light (400-800 nm with maximum energy at 600 nm). Sperm motility was assessed and analyzed by computer-assisted sperm analysis. The involvement of sperm capacitation factors was investigated as follows. The generation of reactive oxygen species (ROS) was measured using 20,70-dichlorofluorescein diacetate. Protein kinase A (PKA) and sarcoma protein kinase (Src) activity were measured using western blot analysis and inhibited using 50 µM H89 and 10 µM PP2, respectively. Soluble adenlyl cyclase was inhibited using 20 µM 2-OH-Estradiol. The intracellular concentration of free Ca(2+) was assessed using the fluorescent calcium indicator, Fluo-4/AM. Sperm DNA fragmentation was determined using the sperm chromatin dispersion test. RESULTS: Light irradiation of human sperm caused a significant increase in hyper-HAM but not total motility. The production of ROS and activation of soluble adenylyl cyclase and PKA mediated the effect of light on HAM. Light irradiation also activated Src, and inhibition of Src significantly reduced the effect of light on HAM. Light irradiation caused a rapid increase in intracellular Ca²âº concentration and the increase in HAM was significantly reduced when voltage-dependent-Ca²âº-channel activity was blocked or when Ca²âº-deficient medium was used. CONCLUSIONS: Light irradiation of human sperm for a short time causes a significant increase in HAM in a mechanism mediated by ROS production, activation of PKA, Src and Ca²âº influx.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Luz , Capacitación Espermática/efectos de la radiación , Espermatozoides/efectos de la radiación , Familia-src Quinasas/fisiología , Señalización del Calcio , AMP Cíclico/metabolismo , Fragmentación del ADN , Humanos , Masculino , Especies Reactivas de Oxígeno/metabolismo , Motilidad Espermática/fisiología , Motilidad Espermática/efectos de la radiación , Espermatozoides/fisiologíaRESUMEN
In order to acquire fertilization competence, spermatozoa have to undergo biochemical changes in the female reproductive tract, known as capacitation. Signaling pathways that take place during the capacitation process are much investigated issue. However, the role and regulation of phosphatidylinositol 3-kinase (PI3K) in this process are still not clear. Previously, we reported that short-time activation of protein kinase A (PRKA, PKA) leads to PI3K activation and protein kinase C(alpha)(PRKCA, PKC(alpha)) inhibition. In the present study, we found that during the capacitation PI3K phosphorylation/activation increases. PI3K activation was PRKA dependent, and down-regulated by PRKCA. PRKCA is found to be highly active at the beginning of the capacitation, conditions in which PI3K is not active. Moreover, inhibition of PRKCA causes significant activation of PI3K. Similar activation of PI3K is seen when the phosphatase PPP1 is blocked suggesting that PPP1 regulates PI3K activity. We found that during the capacitation PRKCA and PPP1CC2 (PP1gamma2) form a complex, and the two enzymes were degraded during the capacitation, suggesting that this degradation enables the activation of PI3K. This degradation is mediated by PRKA, indicating that in addition to the direct activation of PI3K by PRKA, this kinase can enhance PI3K phosphorylation indirectly by enhancing the degradation and inactivation of PRKCA and PPP1CC2.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteína Quinasa C-alfa/fisiología , Espermatozoides/enzimología , Espermatozoides/fisiología , Animales , Bovinos , Activación Enzimática/fisiología , Técnicas In Vitro , Masculino , Fosfoproteínas Fosfatasas/fisiología , Fosforilación , Transducción de Señal/fisiología , Capacitación Espermática/fisiologíaRESUMEN
The binding of capacitated sperm to the egg's zona pellucida stimulates it to undergo the acrosome reaction, a process which enables the sperm to penetrate the egg. Mammalian sperm capacitation and the acrosome reaction require remodeling of actin filaments. An increase in phospholipase D (PLD)-dependent actin polymerization occurs during capacitation whereas the increase in sperm intracellular calcium after its binding to the egg causes very fast actin depolymerization prior to the acrosome reaction. Protein kinase A (PKA) and C (PKC) can both activate sperm PLD and actin polymerization under in vitro incubation, however under physiological conditions, actin polymerization depends primarily on PKA activity. We suggest that PKA indirectly activates phosphatidylinositol 4-kinase to produce phosphatidylinositol 4,5-bisphosphate which is a cofactor for PLD activation. In addition, activation of PKA during capacitation inactivates phospholipase C resulting in preventing PKC activation. It appears that PKA activation promotes sperm capacitation whereas too early activation of PKC during capacitation would jeopardize this process. Thus, a refined balance between the two pathways is required for optimal and sustained activation during sperm capacitation.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteína Quinasa C/metabolismo , Capacitación Espermática/fisiología , Animales , Bovinos , Femenino , Masculino , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
Mammalian spermatozoa should reside in the female reproductive tract for a certain time before gaining the ability to fertilize. During this time, the spermatozoa undergo a series of biochemical processes collectively called capacitation. We recently demonstrated that actin polymerization is a necessary step in the cascade leading to capacitation. We demonstrate here for the first time a role for phospholipase D (PLD) in the induction of actin polymerization and capacitation in spermatozoa. The involvement of PLD is supported by specific inhibition of F-actin formation during sperm capacitation by PLD inhibitors and the stimulation of fast F-actin formation by exogenous PLD or phosphatidic acid (PA). Moreover, PLD activity is enhanced during capacitation before actin polymerization. Protein kinase A (PKA), known to be active in sperm capacitation, and protein kinase C (PKC), involved in the acrosome reaction, can both activate PLD and actin polymerization. We suggest that PKA- and PKC-dependent signal transduction pathways can potentially lead to PLD activation; however, under physiological conditions, actin polymerization depends primarily on PKA activity. Activation of PKA during capacitation causes inactivation of phospholipase C, and as a result, PKC activation is prevented. It appears that PKA activation promotes sperm capacitation whereas early activation of PKC during capacitation would jeopardize this process.
Asunto(s)
Actinas/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfolipasa D/metabolismo , Proteína Quinasa C/metabolismo , Capacitación Espermática , Animales , Bovinos , MasculinoRESUMEN
The generation of reactive oxygen species (ROS) has been implicated in the regulation of sperm capacitation and acrosome reaction; however, the mechanisms underlying this regulation remain unclear. To examine the cellular processes involved, we studied the effect of different concentrations of hydrogen peroxide (H(2)O(2)) on protein tyrosine phosphorylation under various conditions. Treatment of spermatozoa with H(2)O(2) in medium without heparin caused a time- and dose-dependent increase in protein tyrosine phosphorylation of at least six proteins in which maximal effect was seen after 2 h of incubation with 50 microM H(2)O(2). At much higher concentrations of H(2)O(2) (0.5 mM), there is significant reduction in the phosphorylation level, and no protein tyrosine phosphorylation is observed at 5 mM H(2)O(2) after 4 h of incubation. Exogenous NADPH enhanced protein tyrosine phosphorylation similarly to H(2)O(2). These two agents, but not heparin, induced Ca(2+)-dependent tyrosine phosphorylation of an 80-kDa protein. Treatment with H(2)O(2) (50 microM) caused approximately a twofold increase in cAMP, which is comparable to the effect of bicarbonate, a known activator of soluble adenylyl cyclase in sperm. This report suggests that relatively low concentrations of H(2)O(2) are beneficial for sperm capacitation, but that too high a concentration inhibits this process. We also conclude that H(2)O(2) activates adenylyl cyclase to produce cAMP, leading to protein kinase A-dependent protein tyrosine phosphorylation.
Asunto(s)
Reacción Acrosómica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Capacitación Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Animales , Calcio/metabolismo , Bovinos , Técnicas In Vitro , Masculino , Fosforilación/efectos de los fármacos , Espermatozoides/fisiología , Tirosina/metabolismoRESUMEN
The binding to the egg's zona pellucida stimulates the spermatozoon to undergo acrosome reaction, a process which enables the sperm to penetrate the egg. Prior to this binding, the spermatozoa underago in the female reproductive tract a series of biochemical transformations, collectively called capacitation. The first event in capacitation is cholesterol efflux leading to the elevation of intracellular calcium and bicarbonate to activate adenylyl cyclase (AC) to produce cyclic-AMP, which activates protein kinase A (PKA) to indirectly phosphorylate certain proteins on tyrosine. During capacitation, there is also an increase in protein tyrosine phosphorylation dependent actin polymerization and in the membrane-bound phospholipase C (PLC). Sperm binding to zona-pellucida causes further activation of cAMP/PKA and protein kinase C (PKC), respectively. PKC opens a calcium channel in the plasma membrane. PKA together with inositol-trisphosphate activate calcium channels in the outer acrosomal membrane, which leads to an increase in cytosolic calcium. The depletion of calcium in the acrosome will activate a store-operated calcium entry mechanism in the plasma membrane, leading to a higher increase in cytosolic calcium, resulting in F-actin dispersion which enable the outer acrosomal and the plasma membrane to come into contact and fuse completing the acrosomal reaction.
Asunto(s)
Reacción Acrosómica , Calcio/metabolismo , Capacitación Espermática , Actinas/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Calcio/fisiología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Humanos , Masculino , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Motilidad Espermática , Zona Pelúcida/fisiologíaRESUMEN
Mitogen-activated protein (MAP) kinase has been reported to be activated during oocyte meiotic maturation in a variety of mammalian species. However, the mechanism(s) responsible for MAP kinase activation and the consequence of its premature activation during gonadotropin-induced oocyte meiotic resumption have not been examined. The present experiments were conducted to investigate the possible role of MAP kinase in FSH-induced and spontaneous oocyte meiotic resumption in the mouse. MAP kinase kinase (MAPKK, MEK) inhibitor, PD98059 or U0126, produced a dose-dependent inhibitory effect on both FSH-induced oocyte meiotic resumption and MAP kinase activation in the oocytes. However, the same inhibitor did not block spontaneous meiotic resumption of either denuded or cumulus cell-enclosed mouse oocytes, despite the activity of MAP kinase being totally inhibited. Immunoblotting the oocytes and the cumulus cells with the anti-active MAP kinase antibody showed that MAP kinase activity in the oocytes was detected at 8 h of FSH treatment, prior to germinal vesicle breakdown and increased as maturation progressed in the following culture period. In the cumulus cells, MAP kinase was activated even faster, its activity was detected at 1 h of FSH stimulation and increased gradually until 8 h of FSH treatment, then decreased and diminished after 12 h of FSH action. These data demonstrated that the MEK-MAP kinase pathway is implicated in FSH-induced but not spontaneous oocyte meiotic resumption.
Asunto(s)
Hormona Folículo Estimulante/farmacología , Meiosis/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oocitos/citología , Oocitos/enzimología , Animales , Butadienos/farmacología , Activación Enzimática/efectos de los fármacos , Femenino , Flavonoides/farmacología , Cinética , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Nitrilos/farmacología , Oocitos/fisiologíaRESUMEN
Protein kinase C (PKC) has been implicated in the sperm acrosome reaction. In the present study, we demonstrate induction of the acrosome reaction and activation of sperm PKCalpha by lysophosphatidic acid (LPA), which is known to induce signal transduction cascades in many cell types via binding to specific cell-surface receptors. Under conditions by which LPA activates PKCalpha, there is significant stimulation of the acrosome reaction, which is inhibited by PKC inhibitors. Protein kinase Calpha belongs to the Ca(2+)-dependent classical PKC family of isoforms, and indeed we show that its activation depends upon the presence of Ca(2+) in the incubation medium. Protein kinase Calpha is a known regulator of phospholipase D (PLD). We investigated the possible regulatory relationships between PKCalpha and PLD1. Using specific antibodies against PLD1, we demonstrate for the first time its presence in bovine sperm. Furthermore, PLD1 coimmunoprecipitates with PKCalpha and the PKCalpha-PLD1 complex decomposes after treatment of the cells with LPA or 12-O:-tetradecanoyl phorbol-13-acetate, resulting in the translocation of PKCalpha to the plasma membrane and translocation of PLD1 to the particulate fraction. A possible bilateral regulation of PKCalpha and PLD1 activation during the sperm acrosome reaction is suggested.
Asunto(s)
Reacción Acrosómica/efectos de los fármacos , Isoenzimas/metabolismo , Lisofosfolípidos/farmacología , Fosfolipasa D/fisiología , Proteína Quinasa C/metabolismo , Espermatozoides/efectos de los fármacos , Animales , Western Blotting , Bovinos , Activación Enzimática/efectos de los fármacos , Exocitosis/efectos de los fármacos , Inmunohistoquímica , Técnicas In Vitro , Masculino , Pruebas de Precipitina , Proteína Quinasa C-alfa , Transducción de Señal , Espermatozoides/enzimología , Fracciones Subcelulares/enzimología , Fracciones Subcelulares/metabolismoRESUMEN
Protein kinase C (PKC), the major cell target for tumor-promoting phorbol esters, plays a central role in signal transduction pathways. In many biological systems where Ca(2+) serves as a second messenger, regulatory control is mediated by PKC. The activation of PKC depends on its binding to RACK1 receptor, which is an intracellular protein anchor for activated PKC. We demonstrate that the conventional PKC (cPKC) isoforms, PKC-alpha, PKC-betaI, and PKC-betaII, as well as RACK1, are expressed in mouse oocytes (germinal vesicle [GV]) and mature eggs (metaphase II [MII]). In GV oocytes, PKC-alpha, PKC-betaII, and RACK1 were uniformly distributed in the cytoplasm, while PKC-betaI was localized in the cytoplasm and in the plasma membrane as well. Treatment of GV oocytes with the biologically active phorbol ester, 12-o-tetradecanoyl phorbol-13-acetate (TPA), resulted in a rapid translocation of the cytosolic PKC-alpha, but not PKC-betaI, PKC-betaII, or RACK1, to the plasma membrane. This was associated with inhibition of GV breakdown. In MII eggs (17 h post-hCG), PKC-alpha was uniformly distributed in the cytoplasm while PKC-betaI and -betaII were distributed in the cytoplasm and in the plasma membrane as well. Treatment with TPA resulted in a rapid translocation of PKC-alpha from the cytoplasm to the plasma membrane and a significant decrease of PKC-betaI throughout the cytoplasm, while it also remained in the cell periphery. No change in the distribution of PKC-betaII or RACK1 was observed. TPA also induced pronucleus formation. Physiological activation of MII eggs by sperm induced cortical granule exocytosis associated with significant translocation of PKC-alpha and -betaI, but not -betaII, to the plasma membrane. Overall, these results suggest a possible involvement of cPKC isoforms in the mechanism of mouse oocyte maturation and egg activation.
Asunto(s)
Isoenzimas/análisis , Oocitos/enzimología , Oocitos/fisiología , Proteína Quinasa C/análisis , Animales , Transporte Biológico/efectos de los fármacos , Membrana Celular/enzimología , Citoplasma/enzimología , Exocitosis , Femenino , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Oocitos/ultraestructura , Péptidos/análisis , Péptidos/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Cinasa C Activada , Interacciones Espermatozoide-Óvulo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Calcium influx is required for the mammalian sperm acrosome reaction (AR), an exocytotic event occurring in the sperm head prior to fertilization. We show here that thapsigargin, a highly specific inhibitor of the microsomal Ca(2+)-Mg(2+)-ATPase (Ca(2+) pump), can initiate acrosomal exocytosis in capacitated bovine and ram spermatozoa. Initiation of acrosomal exocytosis by thapsigargin requires an influx of Ca(2+), since incubation of cells in the absence of added Ca(2+) or in the presence of the calcium channel blocker, La(3+), completely inhibited thapsigargin-induced acrosomal exocytosis. ATP-Dependent calcium accumulation into nonmitochondrial stores was detected in permeabilized sperm in the presence of ATP and mitochondrial uncoupler. This activity was inhibited by thapsigargin. Thapsigargin elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)), and this increase was inhibited when extracellular Ca(2+) was chelated by EGTA, indicating that this rise in Ca(2+) is derived from the external medium. This rise of [Ca(2+)](i) took place first in the head and later in the midpiece of the spermatozoon. However, immunostaining using a polyclonal antibody directed against the purified inositol 1,4,5-tris-phosphate receptor (IP(3)-R) identified specific staining in the acrosome region, in the postacrosome, and along the tail, but not in the midpiece region. No staining in the acrosome region was observed in sperm without acrosome, indicating that the acrosome cap was stained in intact sperm. The presence of IP(3)-R in the anterior acrosomal region as well as the induction, by thapsigargin, of intracellular Ca(2+) elevation in the acrosomal region and acrosomal exocytosis, implicates the acrosome as a potential cellular Ca(2+) store. We suggest here that the cytosolic Ca(2+) is actively transported into the acrosome by an ATP-dependent, thapsigargin-sensitive Ca(2+) pump and that the accumulated Ca(2+) is released from the acrosome via an IP(3)-gated calcium channel. The ability of thapsigargin to increase [Ca(2+)](i) could be due to depletion of Ca(2+) in the acrosome, resulting in the opening of a capacitative calcium entry channel in the plasma membrane. The effect of thapsigargin on elevated [Ca(2+)](i) in capacitated cells was 2-fold higher than that in noncapacitated sperm, suggesting that the intracellular Ca pump is active during capacitation and that this pump may have a role in regulating [Ca(2+)](i) during capacitation and the AR.
Asunto(s)
Acrosoma/metabolismo , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Calcio/metabolismo , Endocitosis/fisiología , Espermatozoides/metabolismo , Adenosina Trifosfato/fisiología , Animales , ATPasa de Ca(2+) y Mg(2+)/antagonistas & inhibidores , Radioisótopos de Calcio , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/metabolismo , Bovinos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Endocitosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Ovinos , Capacitación Espermática , Espermatozoides/efectos de los fármacos , Espermatozoides/enzimología , Tapsigargina/farmacología , Desacopladores/farmacologíaRESUMEN
Binding to the zona pellucida of an egg stimulates the spermatozoon to undergo the acrosome reaction, a process that enables it to penetrate the egg. Before this binding, the spermatozoon undergoes a series of biochemical transformations in the female reproductive tract, collectively called capacitation. Only capacitated spermatozoa can bind to the zona pellucida and undergo the acrosome reaction. Protein kinases may be involved in the regulation of intracellular Ca2+ during capacitation and the acrosome reaction. The first event in capacitation is the increase in intracellular calcium, bicarbonate and hydrogen peroxide, which collectively activate adenylyl cyclase to produce cyclic AMP, which activates protein kinase A to phosphorylate certain proteins. During capacitation, there is an increase in membrane-bound phospholipase C, and this binding is highly stimulated by the addition of epidermal growth factor to the cells. The capacitated spermatozoon binds to the zona pellucida of the egg via specific receptors and it is suggested that the zona pellucida binds to at least two different receptors in the sperm head plasma membrane. One is a Gi-coupled receptor that can activate phospholipase Cbeta1 and may regulate adenylyl cyclase to further increase cyclic AMP concentrations. The cyclic AMP activates protein kinase A to open a calcium channel in the outer acrosomal membrane, resulting in a relatively small increase in cytosolic calcium. This increase in Ca2+ leads to activation of phospholipase Cgamma, which is coupled to the second tyrosine kinase receptor. The products of phosphatidyl-inositol bisphosphate hydrolysis by phospholipase C, diacylglycerol and inositol-trisphosphate, induce the activation of protein kinase C and a calcium channel in the outer acrosomal membrane, respectively. Protein kinase C opens a calcium channel in the plasma membrane and, together with the inositol-trisphosphate-activated calcium channel, leads to a second and higher increase in cytosolic calcium. In addition, the depletion of calcium in the acrosome activates a capacitative calcium entry mechanism in the plasma membrane, leading to a rapid increase in cytosolic calcium (300-500 nmol l(-1)). This increase in intracellular calcium concentration (and pH) leads to membrane fusion and the acrosome reaction.
Asunto(s)
Reacción Acrosómica , Mamíferos/metabolismo , Proteínas Quinasas/metabolismo , Capacitación Espermática , Animales , ATPasa de Ca(2+) y Mg(2+) , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Humanos , Masculino , Proteínas Tirosina Quinasas/metabolismo , Interacciones Espermatozoide-ÓvuloRESUMEN
The expression and phosphorylation/dephosphorylation of mitogen-activated protein (MAP) kinases during mouse spermatogenesis and epididymal sperm maturation have been investigated by immunoblotting and immunohistochemical staining with commercially available anti-ERK2 and anti-Active MAPK antibodies. Two forms of MAP kinases, p42ERK2 and p44ERK1, were expressed in a similar amount in spermatogenic cells at different stages. ERK1 and ERK2 were phosphorylated (activated) in early spermatogenic cells from primitive spermatogonia to zygotene primary spermatocytes, while only a small quantity of phosphorylated MAP kinases could be detected in pachytene primary spermatocytes and spermatids. MAP kinase activity in primative spermatogonia and preleptotene primary spermatocytes was the highest among spermatogenic cells. ERK1 and ERK2 were also present in epididymal spermatozoa, and their phosphorylation was increased while spermatozoa pass through epididymis and vas deferens for maturation. It would appear that MAP kinase activation may contribute to the mitotic proliferation of primative spermatogonia, an early phase of spermatogenic meiosis, and, later, sperm motility acquirement.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Epidídimo/fisiología , Regulación Enzimológica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos , Maduración del Esperma , Espermatogénesis , Espermatozoides/fisiología , Animales , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Fosforilación , Espermatogonias/citología , Espermatogonias/enzimología , Espermatogonias/fisiología , Espermatozoides/citología , Espermatozoides/enzimologíaRESUMEN
Mitogen-activated protein (MAP) kinase in human eggs has been investigated by using immunoblotting with both anti-Active MAPK and anti-ERK2 antibodies. The results showed that the main form of MAP kinase was p42ERK2. It was in a dephosphorylated form in oocytes at the germinal vesicle stage, but fully phosphorylated in unfertilised mature eggs. MAP kinase phosphorylation was significantly decreased when pronuclei were formed after intracytoplasmic sperm injection. Neither MAP kinase expression nor activity was detected in morphologically degenerated eggs. Although MAP kinase still existed in early embryos arrested at the 8-cell or morula stages, little, if any, activity could be detected. These data suggest that MAP kinase may play an important role in the cell cycle regulation of human eggs, as in other mammalian species.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Embrión de Mamíferos/enzimología , Proteínas Quinasas Activadas por Mitógenos , Óvulo/enzimología , Adulto , Proteínas Quinasas Dependientes de Calcio-Calmodulina/inmunología , Femenino , Humanos , Masculino , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Oocitos/enzimologíaRESUMEN
A very sensitive method was established for detecting the activity of mitogen-activated protein (MAP) kinase in mouse eggs, and used to follow temporal changes of this kinase during fertilization and spontaneous or chemically-induced parthenogenic activation. MAP kinase activity increased between 1 and 2.5 h post-insemination, at which time the second polar body was emitted and sperm chromatin was dispersed; its activity decreased sharply at 8 h. when pronuclei were formed. Both calcium ionophore A23187 and ethanol simultaneously induced pronuclear formation and MAP kinase inactivation in aged eggs 8 h after incubation but less effectively in fresh eggs. The protein kinase inhibitor staurosporine induced pronuclear formation and MAP kinase inactivation more quickly than other treatments, with MAP kinase inactivation occurring slightly proceeding pronuclear formation. Okadaic acid, a specific inhibitor of protein phosphatase 1 and 2A, induced increase in MAP kinase activity, and overcame pronuclear formation induced by various stimuli. MAP kinase inactivation preceded pronuclear formation in eggs spontaneously activated by aging in vitro, perhaps due to cytoplasmic degeneration and thus delayed response of nuclear envelope precursors to MAP kinase inactivation. These data suggest that MAP kinase is a key protein kinase regulating the events of mouse egg activation. Increased MAP kinase activity is temporally correlated with the second polar body emission and sperm chromatin decondensation. Although different stimuli (including sperm) may initially act through different mechanisms, they finally inactivate MAP kinase, probably by allowing the action of protein phosphatase, and thus induces the transition to interphase.