Mechanisms That Protect Mammalian Sperm from the Spontaneous Acrosome Reaction.

To acquire the capacity to fertilize the oocyte, mammalian spermatozoa must undergo a series of biochemical reactions in the female reproductive tract, which are collectively called capacitation. The capacitated spermatozoa subsequently interact with the oocyte zona-pellucida and undergo the acrosome reaction, which enables the penetration of the oocyte and subsequent fertilization. However, the spontaneous acrosome reaction (sAR) can occur prematurely in the sperm before reaching the oocyte cumulus oophorus, thereby jeopardizing fertilization. One of the main processes in capacitation involves actin polymerization, and the resulting F-actin is subsequently dispersed prior to the acrosome reaction. Several biochemical reactions that occur during sperm capacitation, including actin polymerization, protect sperm from sAR. In the present review, we describe the protective mechanisms that regulate sperm capacitation and prevent sAR.

Fer and FerT: A New Regulatory Link between Sperm and Cancer Cells.

Fer and its sperm and cancer specific variant, FerT, are non-receptor tyrosine kinases which play roles in cancer progression and metastasis. Recent studies have shed light on the regulatory role of these kinases in ensuring proper sperm function. Comparison of the regulatory cascades in which Fer and FerT are engaged in sperm and cancer cells presents an interesting picture, in which similar regulatory interactions of these enzymes are integrated in a similar or different regulatory context in the two cell types. These diverse compositions extend from the involvement of Fer in modulation of actin cytoskeleton integrity and function, to the unique regulatory interactions of Fer with PARP-1 and the PP1 phosphatase. Furthermore, recent findings link the metabolic regulatory roles of Fer and FerT in sperm and cancer cells. In the current review, we discuss the above detailed aspects, which portray Fer and FerT as new regulatory links between sperm and malignant cells. This perspective view can endow us with new analytical and research tools that will deepen our understanding of the regulatory trajectories and networks that govern these two multi-layered systems.

Sperm interaction with bacteria induces the spontaneous acrosome reaction.

Bacterial contamination in the semen deteriorates spermatozoa function. One mechanism through which this may occur is by inducing a premature form of the acrosome reaction (spontaneous acrosome reaction (sAR)) which has been shown to abrogate fertilization. To understand the mechanism by which bacteria affect sperm functions, we determined the effects of bacteria on sperm sAR and on other parameters involved in sperm capacitation. Sperm cells undergo biochemical changes in the female reproductive tract collectively called capacitation. Only capacitated sperm can undergo the physiological acrosomal exocytosis process near or on the oocyte, which allows the spermatozoon to penetrate and fertilize the egg. Bovine sperm incubated with the bacteria Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) or Pseudomonas aeruginosa (P. aeruginosa), revealed a sperm-bacteria interaction, however only E. coli and P. aeruginosa caused an increase in sperm sAR. This effect was seen only when the bacteria were present with the sperm during the full incubation under capacitation conditions but not when the bacteria were added to capacitated sperm. These results indicate that bacteria affect sperm during capacitation and not at the AR step. In addition, Ca2+ influx, protein kinase A, and protein tyrosine phosphorylation activities, three essential processes that promote capacitation, were inhibited by the bacteria. Moreover, increasing intracellular cAMP, which also occur during sperm capacitation, caused significant reverse of sAR induced by the bacteria.

Involvement of metabolic pathway in the sperm spontaneous acrosome reaction.

In order to fertilize the egg, spermatozoa must undergo a series of biochemical processes in the female reproductive tract collectively called capacitation. Only capacitated sperm can interact with the egg resulting in the acrosome reaction (AR), allowing egg penetration and fertilization. Sperm can undergo spontaneous AR (sAR) before reaching the egg, preventing successful fertilization. Here we investigated the metabolic pathways involved in sperm capacitation and sAR. Inhibition of glycolysis or oxidative phosphorylation did not affect capacitation or sAR levels; however, when both systems were inhibited, no capacitation occurred, and there was a significant increase in sAR. Under such ATP-starvation, the increase in sAR is triggered by Ca2+ influx into the sperm via the CatSper cation channel. Protein kinase A (PKA) is an essential key enzyme in sperm capacitation; there was no change in its activity when a single metabolic system was inhibited, while complete inhibition of was observed when the two systems were inhibited. Protein tyrosine phosphorylation (PTP), also known to occur in sperm capacitation, was partially reduced by inhibition of one metabolic system, and completely blocked when the two metabolic systems were inhibited. We conclude that ATP, PKA and PTP are involved in the mechanisms protecting sperm from sAR.

The Fer tyrosine kinase protects sperm from spontaneous acrosome reaction.

The physiological acrosome reaction occurs after mammalian spermatozoa undergo a process called capacitation in the female reproductive tract. Only acrosome reacted spermatozoon can penetrate the egg zona-pellucida and fertilize the egg. Sperm also contain several mechanisms that protect it from undergoing spontaneous acrosome reaction (sAR), a process that can occur in sperm before reaching proximity to the egg and that abrogates fertilization. We previously showed that calmodulin-kinase II (CaMKII) and phospholipase D (PLD) are involved in preventing sAR through two distinct pathways that enhance F-actin formation during capacitation. Here, we describe a novel additional pathway involving the tyrosine kinase Fer in a mechanism that also prevents sAR by enhancing actin polymerization during sperm capacitation. We further show that protein-kinase A (PKA) and the tyrosine-kinase Src, as well as PLD, direct Fer phosphorylation/activation. Activated Fer inhibits the Ser/Thr phosphatase PP1, thereby leading to CaMKII activation, actin polymerization, and sAR inhibition.

The Role of Zinc in Male Fertility.

Several studies proposed the importance of zinc ion in male fertility. Here, we describe the properties, roles and cellular mechanisms of action of Zn2+ in spermatozoa, focusing on its involvement in sperm motility, capacitation and acrosomal exocytosis, three functions that are crucial for successful fertilization. The impact of zinc supplementation on assisted fertilization techniques is also described. The impact of zinc on sperm motility has been investigated in many vertebrate and invertebrate species. It has been reported that Zn2+ in human seminal plasma decreases sperm motility and that Zn2+ removal enhances motility. Reduction in the intracellular concentration of Zn2+ during epididymal transit allows the development of progressive motility and the subsequent hyper activated motility during sperm capacitation. Extracellular Zn2+ affects intracellular signaling pathways through its interaction with the Zn2+ sensing receptor (ZnR), also named GPR39. This receptor was found in the sperm tail and the acrosome, suggesting the possible involvement of Zn2+ in sperm motility and acrosomal exocytosis. Our studies showed that Zn2+ stimulates bovine sperm acrosomal exocytosis, as well as human sperm hyper-activated motility, were both mediated by GPR39. Zn2+ binds and activates GPR39, which activates the trans-membrane-adenylyl-cyclase (tmAC) to catalyze cAMP production. The NHE (Na+/H+-exchanger) is activated by cAMP, leading in increased pHi and activation of the sperm-specific Ca2+ channel CatSper, resulting in an increase in [Ca2+]i, which, together with HCO3-, activates the soluble adenylyl-cyclase (sAC). The increase in [cAMP]i activates protein kinase A (PKA), followed by activation of the Src-epidermal growth factor receptor-Pphospholipase C (Src-EGFR-PLC) cascade, resulting in inositol-triphosphate (IP3) production, which mobilizes Ca2+ from the acrosome, causing a further increase in [Ca2+]i and the development of hyper-activated motility. PKA also activates phospholipase D1 (PLD1), leading to F-actin formation during capacitation. Prior to the acrosomal exocytosis, PLC induces phosphadidylinositol-4,5-bisphosphate (PIP2) hydrolysis, leading to the release of the actin-severing protein gelsolin to the cytosol, which is activated by Ca2+, resulting in F-actin breakdown and the occurrence of acrosomal exocytosis.

Ca2+ signaling in mammalian spermatozoa.

Calcium is an essential ion which regulates sperm motility, capacitation and the acrosome reaction (AR), three processes necessary for successful fertilization. The AR enables the spermatozoon to penetrate into the egg. In order to undergo the AR, the spermatozoon must reside in the female reproductive tract for several hours, during which a series of biochemical transformations takes place, collectively called capacitation. An early event in capacitation is relatively small elevation of intracellular Ca2+ (in the nM range) and bicarbonate, which collectively activate the soluble adenylyl cyclase to produce cyclic-AMP; c-AMP activates protein kinase A (PKA), leading to indirect tyrosine phosphorylation of proteins. During capacitation, there is an increase in the membrane-bound phospholipase C (PLC) which is activated prior to the AR by relatively high increase in intracellular Ca2+ (in the µM range). PLC catalyzes the hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (PIP2) to diacylglycerol and inositol-trisphosphate (IP3), leading to activation of protein kinase C (PKC) and the IP3-receptor. PKC activates a Ca2+- channel in the plasma membrane, and IP3 activates the Ca2+- channel in the outer acrosomal membrane, leading to Ca2+ depletion from the acrosome. As a result, the plasma-membrane store-operated Ca2+ channel (SOCC) is activated to increase cytosolic Ca2+ concentration, enabling completion of the acrosome reaction. The hydrolysis of PIP2 by PLC results in the release and activation of PIP2-bound gelsolin, leading to F-actin dispersion, an essential step prior to the AR. Ca2+ is also involved in the regulation of sperm motility. During capacitation, the sperm develops a unique motility pattern called hyper-activated motility (HAM) which is essential for successful fertilization. The main Ca2+-channel that mediates HAM is the sperm-specific CatSper located in the sperm tail.

Signaling pathways involved in human sperm hyperactivated motility stimulated by Zn2.

To fertilize the egg, sperm cells must reside in the female reproductive tract for several hours during which they undergo chemical and motility changes collectively called capacitation. During capacitation, the sperm develop a unique type of motility known as hyperactivated motility (HAM). The semen contains Zn2+ in millimolar concentrations, whereas in the female reproductive tract the concentration is around 1 µM. In this study, we characterize the role of Zn 2+ in human sperm capacitation focusing on its effect on HAM. Western blot analysis revealed the presence of G protein-coupled receptor 39 (GPR39) type Zn-receptor localized mainly in the sperm tail. Zn 2+ at micromolar concentration stimulates HAM, which is mediated by a cascade involving GPR39-AC-cAMP-PKA-Src-EGFR and phospholipase C. Both the transmembrane adenylyl cyclase (AC) and the soluble-AC are involved in the stimulation of HAM by Zn 2+ . The development of HAM is precisely regulated by cyclic adenosine monophosphate, in which relatively low concentration (5-10 µM) stimulated HAM, whereas at 30 µM no stimulation occurred. A similar response was seen when different concentrations of Zn 2+ were added to the cells; low Zn 2+ stimulated HAM, whereas at relatively high Zn 2+ , no effect was seen. We further demonstrate that the Ca 2+ -channel CatSper involved in Zn 2+ -stimulated HAM. These data support a role for extracellular Zn 2+ acting via GPR39 to regulate signaling pathways in sperm capacitation, leading to HAM induction.

PKA and PI3K activities during capacitation protect sperm from undergoing spontaneous acrosome reaction.

In order to fertilize the egg, the spermatozoon should undergo a process called acrosomal exocytosis or acrosome reaction (AR), a process which can take place only after a series of biochemical changes collectively called capacitation occur in the female reproductive tract. We present here for the first time the involvement of protein-kinase A (PKA) and phosphatidyl-inositol-3-kinase (PI3K) in protecting sperm from undergoing spontaneous AR (sAR) which decreases fertilization rate. Previously we showed that Calmodulin-kinase II (CaMKII) and phospholipase-D (PLD) prevent the occurrence of sAR in two distinct pathways by enhancing actin polymerization. Here we show that PKA mediates PLD activation, and inhibition of PKA resulting in an increase of sAR and a decrease of F-actin levels, two functions which can be recovered by adding phosphatidic acid (PA), the product of PLD activity. PKA is known to induce CaMKII activation. Inhibition of CaMKII, also enhanced sAR and reduced F-actin levels which can be recovered by adding PA. Inhibition of the PLD pathway resulted in an increase in sAR and a decrease in F-actin levels which can be artificially reversed by inhibition of protein - phosphatase 1 (PP1), and this effect is mediated by PI3K. All together, we showed that PKA via PLD and PI3K activation protects the sperm from undergoing sAR by enhancing actin polymerization.

Protein kinase A inhibition induces EPAC-dependent acrosomal exocytosis in human sperm.

To interact with the egg, the spermatozoon must undergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the involvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent; however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l-1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02-0.1 µmol l-1), whereas higher concentrations (>5 µmol l-1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing-protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca2+-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization and/or activation of effectors downstream to F-actin breakdown that lead to acrosomal exocytosis.

Signaling pathways involved in human sperm hyperactivated motility stimulated by Zn2.

To fertilize the egg, sperm cells must reside in the female reproductive tract for several hours during which they undergo chemical and motility changes collectively called capacitation. During capacitation, the sperm develop a unique type of motility known as hyperactivated motility (HAM). The semen contains Zn2+ in millimolar concentrations, whereas in the female reproductive tract, the concentration is around 1 µM. In this study, we characterize the role of Zn2+ in human sperm capacitation focusing on its effect on HAM. Western blot analysis revealed the presence of GPR39-type Zn-receptor localized mainly in the sperm tail. Zn2+ at micromolar concentration stimulates HAM, which is mediated by a cascade involving GPR39-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A-tyrosine kinase Src (Src)-epidermal growth factor receptor and phospholipase C. Both the transmembrane AC and the soluble-AC are involved in the stimulation of HAM by Zn2+ . The development of HAM is precisely regulated by cAMP, in which relatively low concentration (5-10 µM) stimulated HAM, whereas at 30 µM no stimulation occurred. A similar response was seen when different concentrations of Zn2+ were added to the cells; low Zn2+ stimulated HAM, whereas at relatively high Zn2+ , no effect was seen. We further demonstrate that the Ca2+ -channel CatSper involved in Zn2+ -stimulated HAM. These data support a role for extracellular Zn2+ acting via GPR39 to regulate signaling pathways in sperm capacitation, leading to HAM induction.

Actin cytoskeleton and sperm function.

For the acquisition of the ability to fertilize the egg, mammalian spermatozoa should undergo a series of biochemical transformations in the female reproductive tract, collectively called capacitation. The capacitated sperm can undergo the acrosomal exocytosis process near or on the oocyte, which allows the spermatozoon to penetrate and fertilize it. One of the main processes in capacitation involves dynamic cytoskeletal remodeling particularly of actin. Actin polymerization occurs during sperm capacitation and the produced F-actin should be depolymerized prior to the acrosomal exocytosis. In the present review, we describe the mechanisms that regulate F-actin formation during sperm capacitation and the F-actin dispersion prior to the acrosomal exocytosis. During sperm capacitation, the actin severing proteins gelsolin and cofilin are inactive and they undergo activation prior to the acrosomal exocytosis.

Role and regulation of Glycogen synthase kinase-3 beta in bovine spermatozoa.

The serine/threonine kinase Glycogen synthase kinase 3 (GSK-3) is a master switch that regulates a multitude of cellular pathways, including the acrosome reaction in sperm. In epididymal sperm cells, for example, GSK-3 activity correlates with inhibition of motility-yet no direct pathways connecting GSK-3 activation with loss of motility have been described. Indeed, the details of how GSK-3 is regulated during sperm capacitation and the acrosome reaction remains obscure. To this end, we addressed the involvement of the GSK-3 beta isoform in several known pathways that contribute to motility and the acrosome reaction. We established that Protein kinase A (PKA) is the main regulator of GSK-3ß in sperm, as pre-treatment of cells with a GSK-3 inhibitor prior to addition of H89, an inhibitor of PKA, attenuated the motility loss induced by blocking PKA activity. Both induced and spontaneous acrosome reactions also occurred less frequently in sperm treated with GSK-3 inhibitors. Finally, we observed a slow decline in phosphorylation of GSK-3ß on Ser 9, which represents an inhibited state, during sperm capacitation; this phenotype is reversed during the induced acrosome reaction, in parallel to activation of Protein phosphatase 1. These results suggest that maintenance of sperm motility and acrosome reaction timing are mediated by PKA through the regulation of GSK-3 beta activity. Mol. Reprod. Dev. 84: 8-18, 2017. © 2016 Wiley Periodicals, Inc.

CaMKII prevents spontaneous acrosomal exocytosis in sperm through induction of actin polymerization.

In order to interact with the egg and undergo acrosomal exocytosis or the acrosome reaction (AR), mammalian spermatozoa must undergo a series of biochemical changes in the female reproductive tract, collectively called capacitation. We showed that F-actin is formed during sperm capacitation and fast depolymerization occurs prior to the AR. We hypothesized that F-actin protects the sperm from undergoing spontaneous-AR (sAR) which decreases fertilization rate. We show that activation of the actin-severing protein gelsolin induces a significant increase in sAR. Moreover, inhibition of CaMKII or PLD during sperm capacitation, caused an increase in sAR and inhibition of F-actin formation. Spermine, which leads to PLD activation, was able to reverse the effects of CaMKII inhibition on sAR-increase and F-actin-decrease. Furthermore, the increase in sAR and the decrease in F-actin caused by the inactivation of the PLD-pathway, were reversed by activation of CaMKII using H2O2 or by inhibiting protein phosphatase 1 which enhance the phosphorylation and oxidation states of CaMKII. These results indicate that two distinct pathways lead to F-actin formation in the sperm capacitation process which prevents the occurrence of sAR.

New insights into the role of the Brdt protein in the regulation of development and spermatogenesis in the mouse.

The bromodomain testis-specific (BRDT) protein belongs to the bromodomain extra-terminal (BET) family of proteins. It serves as a transcriptional regulator of gene expression during spermatogenesis, and is an essential factor for the normal spermatogenesis process. In this study, we characterized mice of several age groups who lacked the Brdt gene. The testes of Brdt mutant mice aged 8 weeks exhibited complete spermatocyte maturation arrest with a significantly increased number of apoptotic cells. The weights of the testes and accessory glands as well as the testosterone levels of the mutant mice were significantly lower compared to the normal mice. The mutant mice had delayed puberty, with normal levels of testosterone and accessory gland weights at the age of 14 and 28 weeks. The testes of the mutant mice at older ages also exhibited round spermatids. The presence of the BRDT protein was identified in the mice pituitary gland. Microarray analysis of mice pituitaries showed that 28 genes were down-regulated while 26 genes were up-regulated in the absence of the Brdt gene. Our results suggest that in addition to its critical role in the spermatogenesis process, the BRDT protein is also responsible for scheduling male puberty by regulation of the pituitary-gonad axis.

The role and importance of cofilin in human sperm capacitation and the acrosome reaction.

The spermatozoon is capable of fertilizing an oocyte only after undergoing several biochemical changes in the female reproductive tract, referred to as capacitation. The capacitated spermatozoon interacts with the egg zona pellucida and undergoes the acrosome reaction, which enables its penetration into the egg and fertilization. Actin dynamics play a major role throughout all these processes. Actin polymerization occurs during capacitation, whereas prior to the acrosome reaction, F-actin must undergo depolymerization. In the present study, we describe the presence of the actin-severing protein, cofilin, in human sperm. We examined the function and regulation of cofilin during human sperm capacitation and compared it to gelsolin, an actin-severing protein that was previously investigated by our group. In contrast to gelsolin, we found that cofilin is mainly phosphorylated/inhibited at the beginning of capacitation, and dephosphorylation occurs towards the end of the process. In addition, unlike gelsolin, cofilin phosphorylation is not affected by changing the cellular levels of PIP2. Despite the different regulation of the two proteins, the role of cofilin appears similar to that of gelsolin, and its activation leads to actin depolymerization, inhibition of sperm motility and induction of the acrosome reaction. Moreover, like gelsolin, cofilin translocates from the tail to the head during capacitation. In summary, gelsolin and cofilin play a similar role in F-actin depolymerization prior to the acrosome reaction but their pattern of phosphorylation/inactivation during the capacitation process is different. Thus, for the sperm to achieve high levels of F-actin along the capacitation process, both proteins must be inactivated at different times and, in order to depolymerize F-actin, both must be activated prior to the acrosome reaction.

AKAP3 degradation in sperm capacitation is regulated by its tyrosine phosphorylation.

BACKGROUND: The A-kinase anchoring protein (AKAP) family is essential for sperm motility, capacitation and the acrosome reaction. PKA-dependent protein tyrosine phosphorylation occurs in mammalian sperm capacitation including AKAP3. In a recent study, we showed that AKAP3 undergoes degradation under capacitation conditions. Thus, we tested here whether AKAP3 degradation might be regulated by its tyrosine phosphorylation. METHODS: The intracellular levels of AKAP3 were determined by western blot (WB) analysis using specific anti-AKAP3 antibodies. Tyrosine phosphorylation of AKAP3 was tested by immunoprecipitation and WB analysis. Acrosome reaction was examined using FITC-pisum sativum agglutinin. RESULTS: AKAP3 is degraded and undergoes tyrosine-dephosphorylation during sperm capacitation and the degradation was reduced by inhibition of tyrosine phosphatase and enhanced by inhibition of tyrosine kinase. Sperm starvation or inhibition of mitochondrial respiration, which reduce cellular ATP levels, significantly accelerated AKAP3 degradation. Treatment with vanadate, or Na(+) or bicarbonate depletion, reduced AKAP3-degradation and the AR rate, while antimycin A or NH4Cl elevated both AKAP3-degradation and the AR degree. Treatment of sperm with NH4Cl enhanced PKA-dependent phosphorylation of four proteins, further supporting the involvement of AKAP3-degradation in capacitation. To demonstrate more specifically that sperm capacitation requires AKAP3-degradation, we inhibited AKAP3-degradation using anti-AKAP3 antibody in permeabilized cells. The anti-AKAP3-antibody induced significant inhibition of AKAP3-degradation and of the AR rate. CONCLUSION: Sperm capacitation process requires AKAP3-degradation, and the degradation degree is regulated by the level of AKAP3 tyrosine phosphorylation. GENERAL SIGNIFICANCE: Better understanding of the molecular mechanisms that mediate sperm capacitation can be used for infertility diagnosis, treatment and the developing of male contraceptives.

Regulation of Sperm Capacitation and the Acrosome Reaction by PIP 2 and Actin Modulation.

Actin polymerization and development of hyperactivated (HA) motility are two processes that take place during sperm capacitation. Actin polymerization occurs during capacitation and prior to the acrosome reaction, fast F-actin breakdown takes place. The increase in F-actin during capacitation depends upon inactivation of the actin severing protein, gelsolin, by its binding to phosphatydilinositol-4, 5-bisphosphate (PIP 2 ) and its phosphorylation on tyrosine-438 by Src. Activation of gelsolin following its release from PIP 2 is known to cause F-actin breakdown and inhibition of sperm motility, which can be restored by adding PIP 2 to the cells. Reduction of PIP 2 synthesis inhibits actin polymerization and motility, while increasing PIP 2 synthesis enhances these activities. Furthermore, sperm demonstrating low motility contained low levels of PIP 2 and F-actin. During capacitation there was an increase in PIP 2 and F-actin levels in the sperm head and a decrease in the tail. In spermatozoa with high motility, gelsolin was mainly localized to the sperm head before capacitation, whereas in low motility sperm, most of the gelsolin was localized to the tail before capacitation and translocated to the head during capacitation. We also showed that phosphorylation of gelsolin on tyrosine-438 depends upon its binding to PIP 2 . Stimulation of phospholipase C, by Ca 2 + -ionophore or by activating the epidermal-growth-factor-receptor, inhibits tyrosine phosphorylation of gelsolin and enhances enzyme activity. In conclusion, these data indicate that the increase of PIP 2 and/or F-actin in the head during capacitation enhances gelsolin translocation to the head. As a result, the decrease of gelsolin in the tail allows the maintenance of high levels of F-actin in this structure, which is essential for the development of HA motility.

ERK1/2 mediates sperm acrosome reaction through elevation of intracellular calcium concentration.

Mammalian sperm acquire fertilization capacity after residing in the female reproductive tract for a few hours in a process called capacitation. Only capacitated sperm can bind the zona pellucida (ZP) of the egg and undergo the acrosome reaction, a process that allows penetration and fertilization. Extracellular signal regulated kinase (ERK1/2) mediates signalling in many cell types, however its role in sperm function is largely unknown. Here we show that ERK1/2 is highly phosphorylated/activated after a short incubation of mouse sperm under capacitation conditions and that this phosphorylation is reduced after longer incubation. Further phosphorylation was observed upon addition of crude extract of egg ZP or epidermal growth factor (EGF). The mitogen-activated ERK-kinase (MEK) inhibitor U0126 abolished ERK1/2 phosphorylation, in vitro fertilization rate and the acrosome reaction induced by ZP or EGF but not by the Ca2+-ionophore A23187. Moreover, inhibition of ERK1/2 along the capacitation process diminished almost completely the sperm's ability to go through the acrosome reaction, while inhibition at the end of capacitation attenuated the acrosome reaction rate by only 45%. The fact that the acrosome reaction, induced by the Ca2+ -ionophore A23187, was not inhibited by U0126 suggests that ERK1/2 mediates the acrosome reaction by activating Ca2+ transport into the cell. Direct determination of intracellular [Ca2+] revealed that Ca2+ influx induced by EGF or ZP was completely blocked by U0126. Thus, it has been established that the increase in ERK1/2 phosphorylation/activation in response to ZP or by activation of the EGF receptor (EGFR) by EGF, is a key event for intracellular Ca2+ elevation and the subsequent occurrence of the acrosome reaction.

Zn2+-stimulation of sperm capacitation and of the acrosome reaction is mediated by EGFR activation.

Extracellular zinc regulates cell proliferation via the MAP1 kinase pathway in several cell types, and has been shown to act as a signaling molecule. The testis contains a relatively high concentration of Zn(2+), required in both the early and late stages of spermatogenesis. Despite the clinical significance of this ion, its role in mature sperm cells is poorly understood. In this study, we characterized the role of Zn(2+) in sperm capacitation and in the acrosome reaction. Western blot analysis revealed the presence of ZnR of the GPR39 type in sperm cells. We previously demonstrated the presence of active epidermal growth factor receptor (EGFR) in sperm, its possible transactivation by direct activation of G-protein coupled receptor (GPCR), and its involvement in sperm capacitation and in the acrosome reaction (AR). We show here that Zn(2+) activates the EGFR during sperm capacitation, which is mediated by activation of trans-membrane adenylyl cyclase (tmAC), protein kinase A (PKA), and the tyrosine kinase, Src. Moreover, the addition of Zn(2+) to capacitated sperm caused further stimulation of EGFR and phosphatydil-inositol-3-kinase (PI3K) phosphorylation, leading to the AR. The stimulation of the AR by Zn(2+) also occurred in the absence of Ca(2+) in the incubation medium, and required the tmAC, indicating that Zn(2+) activates a GPCR. The AR stimulated by Zn(2+) is mediated by GPR39 receptor, PKA, Src and the EGFR, as well as the EGFR down-stream effectors PI3K, phospholipase C (PLC) and protein kinase C (PKC). These data support a role for extracellular zinc, acting through the ZnR, in regulating multiple signaling pathways in sperm capacitation and the acrosome reaction.