BACKGROUND
one of the most important events in the history of life was the eukaryotes evolution that started with the onset of intracellular compartmentalization, approximately  <dig> - <dig>  billion years ago  <cit> .
this fundamental step had enormous consequences on cell organization and function.
in fact, in one hand, the appearance of specialized districts within the cytoplasm increased the efficiency of metabolic and signaling pathways, in the other hand, cell complexity increased.
in fact, the development of organelles determined the existence of specific subcellular microenvironments characterized by different biochemical properties.
thus, the cell complexity arose either by the large number of molecular components involved in signal transduction and by the reciprocal connections and spatial relationship existing among them  <cit> .
in addition, the spatial segregation of molecules and chemical reactions allowed that the same molecule could be responsible of very different signals.
"we already have signaling "wires" distinguished by the identity of the molecules in the pathways.
compartmentalization duplicates these existing wires and separates them in space.
this multiplies the number of signals they can carry"  <cit> .
this increasing complexity imposes new biological problems as the emergence of new proprieties of cellular systems and the need of the presence of a/some conductor/s which harmonize the function of subcellular compartments.
recently the interpretation of the molecular mechanisms involved in cell signaling is made possible by new computational approaches that allow to modellize cellular functions as a network of integrated and coordinated signals operating within subcellular districts interconnected to each other  <cit> .
one clear example is represented by sperm cells, whose biochemical mechanisms leading to capacitation during the post-ejaculatory life, were recently described by using the biological networks formalism  <cit> .
the spermatozoa offer several advantages to adopt this kind of approach since:
- the main molecular events occurring during sperm capacitation are largely studied for biological and clinical reasons;
- their maturation can be experimentally reproduced under in vitro condition and assessed by measuring their ability to undergo acrosomal reaction  after zonae pellucidae stimulation  or to in vitro fertilize the oocytes ;
- their molecular composition is stable since the nucleus of the male germ cells is transcriptionally silent except for the mitochondrial protein translation of nuclear-encoded proteins  <cit> .
starting from these premises, the present work has been carried out, first, to study, using a computational approach, the mechanisms involved in the coordination of the molecular events that take place in the different subcellular districts during the post-ejaculatory spermatozoa maturation, the capacitation.
then, an experimental set-up was developed to confirm the model-based prediction.
to this aim the events potentially acting as coordinators were inhibited by specific drugs during the spermatozoa incubation under capacitating conditions.
the effects of this inhibition were assessed on functional spermatozoa status , and on the major cellular events involved in the acquisition of full fertilizing ability .
RESULTS
computational model of spermatozoa capacitation
the main topological parameters of the network representing the capacitation are shown in table  <dig>  the distribution of node linkages followed a power law, represented by the generic equation:
were
and
the number of nodes represent the total number of molecules involved, the number of edges represents the total number of interaction found, the clustering coefficient is calculated as ci = 2ni/k, where ni is the number of links connecting the ki neighbours of node i to each other, the network diameter is the largest distance between two nodes, the averaged n° neighbours represent the mean number of connection of each node, the char.
path length gives the expected distance between two connected nodes.
the r, r <dig> coefficients were, respectively,  <dig>  and  <dig> .
the clustering coefficient distribution does not follow a power law, thus, the results of power law fitting of clustering coefficient distribution were: r =  <dig> ; r <dig> =  <dig> .
the most connected nodes are showed in table  <dig>
the analysis of the network topology revealed that only tree nodes bound all the subcellular compartments involved in post-ejaculatory signalling: i, atp, and "actin polymerization".
i and atp are the most linked nodes and bound about one third of the nodes .
more interesting is the behaviour of the "actin polymerization" node, represented in figure  <dig>  that links a smaller number of nodes  located in all the subcellular compartments .
the removal of the "actin polymerization" node from the capacitation network had modest consequences on the network topological parameter , but caused the loss of  <dig> nodes: oam fusion, pm fusion, g-actin, f-actin, mitochondrial protein translation .
the number of nodes represent the total number of molecules involved, the number of edges represents the total number of interaction found, the clustering coefficient is calculated as ci = 2ni/k, where ni is the number of links connecting the ki neighbours of node i to each other, the network diameter is the largest distance between two nodes, the averaged n° neighbours represent the mean number of connection of each node, the char.
path length gives the expected distance between two connected nodes.
phalloidin staining
the distribution of the fluorescence within the spermatozoa was assessed, allowing the identification of two different patterns:
- pattern a) low actin polymeriziation: the phalloidin fluorescence emission is low and is mainly localized over the midpiece and the post acrosomial region ;
- pattern b) high actin polymerization: the phalloidin fluorescence emission is high and localized over the midpiece and the whole sperm head .
the majority of freshly ejaculated spermatozoa localized a faint fluorescence emission in the midpiece and in the post acrosomal region .
after  <dig> h of incubation under control conditions, the majority of spermatozoa increased their content of f-actin that resulted to be localized over sperm head .
the cd treated spermatozoa, displayed a fluorescence signal similar to that recorded at t <dig> .
zp-induced ar
the percentage of acrosome reacted spermatozoa recorded during incubation in control condition ranged from about 5% at the beginning of incubation to about 10% after  <dig> h. the szp addition to the samples made possible to quantify the percentage of the spermatozoa responding to the physiological stimulus with the exocytosis of the acrosomal content , which after the  <dig> h incubation reached about the 30% .
the cd treated spermatozoa showed an incidence of spontaneous ar similar to that recorded in the control samples during all the  <dig> h of incubation , instead, the szp addition promoted the ar in a markedly reduced percentage of cells , see figure 4b.
chlortetracycline staining
the spermatozoa incubated under control conditions, at the beginning of incubation, showed three different patterns of chlortetracycline  stain in agreement with mattioli et al.
<cit> :
- about 80% of spermatozoa displayed a faint fluorescence uniformly distributed over the head ;
- about the 10% spermatozoa displayed the fluorescence concentrated in the post-acrosomal region ;
- about the 10% displayed the fluorescence concentrated in the acrosome : these spermatozoa, in keeping with mattioli et al.
<cit> , were considered to have completed the capacitation-related membrane reorganization.
after  <dig> h of incubation the percentage of the three patterns was markedly different: pattern a decreased to about 40%, pattern b remained at about 10%, while, pattern c reached 50%.
as evident in figure  <dig>  the treatment with cd did not affect significantly the percentage of spermatozoa showing the different ctc stain patterns.
tyrosine phosphorylation
the tyrosine phosphorilation pattern of freshly ejaculated male gametes , or of spermatozoa incubated under control condition , or in the presence of cd was assessed.
as summarized in figure  <dig>  after the incubation the tyrosine phosphorylation pattern changed regardless of the cultural condition adopted .
phospholipase c-γ <dig> relocalization
phospholipase c-γ <dig>  protein expression studied with western blotting revealed that this protein modified its localization during the interval of culture.
in fact, as shown in figure  <dig>  uncapacitated sperm cells  strongly expressed plc-γ <dig> within the cytoplasm, while, the incubation caused its translocation into the membrane compartment both in ctr and in cd samples.
ultrastructural analysis performed under pre-embedding condition, confirmed the membrane traslocation of plc-γ <dig> in incubated spermatozoa maintained either in the presence or absence of cd, confirming the western blot analysis.
in fact, uncapacitated spermatozoa displayed very few gold particles, indicative of plc-γ <dig>  on their membranes , while, in in vitro incubated cells, independently from the used treatment, the number of gold particles localizing the protein on the membrane notably grew .
zp-induced intracellular calcium concentration rise
the coincubation with the szp in spermatozoa capacitated under control conditions caused an evident i elevation in the 36% of sperm cells : the intracellular ca2+ peaked in 1- <dig> sec and lasted 20- <dig> sec before i returning at a basal level.
in the presence of cd the ability and the kinetic to respond with a i rise to the szp coincubation  was maintained in the 38%  of spermatozoa .
discussion
aim of this work was to study the molecular event/s involved in the coordination of the biochemistry of the subcellular compartments during spermatozoa acquisition of fertilizing ability  using a predictive computational modelisation and the in vitro experimental approach as a confirmation.
thanks the in silico experiment the node acting as a coordinator for the signaling cascades of different subcellular districts was identified.
then, the consequences on cellular function of its inhibition were experimentally evaluated in vitro.
this goal was targeted by representing the biochemical machinery that drives the capacitation as a biological network that, on the basis of its topological proprieties, was considered as a scale free network.
in fact, the node degree , k, which indicates how many links the node has to other nodes, followed a power-law distribution of the number of links per node and, in parallel, the tendency to develop clusters of nodes, the so called clustering coefficient ci  was independent of the number of links per node.
this architecture conferred to the capacitation network high signaling efficiency and robustness against random failure  <cit>  and, in keeping with the barabási-albert model  <cit> , made evident that a relatively small number of nodes  were highly connected and most of the nodes were scarcely linked.
thus, it was possible to identify the hubs, i.e.
the molecules playing a key role in post-ejaculatory maturation of spermatozoa and to localize them in different subcellular districts.
in addition, the functional meaning and the localization of all the molecules involved in capacitation were studied and the nodes that interconnect the intracellular compartments were identified.
it is really impressive that these nodes were only three: i, atp and "actin polymerization".
more in detail i and atp are linked with a high number of nodes  and exerted the function of ubiquitous second messengers  or of metabolic sustain .
it is important to note that it has been recently demonstrated that their ablation from the network causes the collapse of network structures  <cit> .
on the contrary, the "actin polymerization" bounds in a specific way, having only  <dig> links, all the subcellular compartments involved in capacitation .
it is suggestive that it does not link the nucleus, the only compartment that remains stable until fertilization.
in particular it links:
- phosphatidic acid : it is known that actin polymerization depends on pld activation, which occurs via the hco <dig> 2/camp/pka pathway or via the g-protein coupled receptor  /pkc pathway  <cit> ;
- atp: the actin is an atpase and its polymerization depends on atp cellular concentration, in addition, the atp is the most important molecule of spermatozoa energy metabolism.
in fact, the metabolic energy production in spermatozoa depends on glycolysis and/or on mitochondrial oxidative  <cit> ;
- f-actin and g-actin: for recent reviews on actin polymerization see  <cit> ;
- i: the intracellular calcium is one of the most important second messengers that drives the intracellular signaling during capacitation;
- oam and pm fusion: see below for explanation;
- protein synthesis: the incubation with the mitochondrial translation inhibitor d-chloramphenicol almost completely blocked spermatozoa actin polymerization  <cit> .
in this context it is important to note that the links of actin polymerization involve three of the most linked nodes found in capacitation  and that this node was per se one of the most linked node.
thus, it is possible to hypothesize that a message involving the actin polymerization node could reach all the cell districts, perturbing the whole signal transduction system of spermatozoa.
in addition, it has been found that the removal of "actin polymerization" node caused an important effect: the main topological parameters of the capacitation network were virtually unaffected but some nodes remained out from the network: oam fusion, pm fusion, g-actin, f-actin, mitochondrial protein translation.
this finding allows to predict that the functional ablation of actin polymerization during the in vitro capacitation could cause the spermatozoa inability to undergo ar  while, the different signalling pathways remained unaffected.
to obtain an experimental confirmation of the model-based prediction, the actin polymerization was inhibited, as clearly confirmed by confocal analyses, by the incubation of spermatozoa under capacitating condition in the presence of cd.
the effect of this drug on the whole cell function was immediately evident: the treated spermatozoa lost the ability to undergo ar when exposed to the physiological agonist, the szp.
in parallel, the biochemical analyses showed that the main signaling pathways involved in capacitation were unaffected by the treatment.
particularly the capacitation implies a marked calcium-dependent rearrangement of membrane structure that, in turn, determines the increase of the plasma membrane  and of the outer acrosome membrane  fusogenicity.
this event resulted to be unaffected by cd administration as demonstrated by ctc staining.
another important signaling pathway involves the protein tyrosine phosphorylation, via sac/camp/pka.
it is known that the protein tyrosine phosphorylation pattern of spermatozoa changes during the capacitation in several species such as human, mice, cattle, pig, hamster and cat  <cit> .
in boar spermatozoa a typical protein phosphorylation pattern has been identified  <cit> , and the cd treatment did not exert any detectable effect on this parameter.
the plc-γ <dig> plays a key role in coupling actin cytoskeleton and membrane dynamics to the calcium metabolism.
when activated this enzyme migrates from the cytosol to the membrane  <cit>  where it hydrolyses the phosphatidylinositol  <dig> -bisphosphate  generating inositol  <dig> ,5-trisphosphate, a universal calcium-mobilizing second messenger, and diacylglycerol, an activator of protein kinase c. the present results showed that the plc-γ <dig> translocation was unaffected by the presence of cd in the capacitation medium.
ultimately, the effect of spermatozoa incubation under capacitating condition in presence of cd on calcium metabolism was tested.
in fact, the spermatozoa signaling machinery strictly depends on intracellular calcium concentration  <cit> .
during the capacitation the ca2+ behaves as a second messenger converting extracelluar stimuli in chemical response involving a myriad of molecular system, such as, protein kinase c , protein kynase c , actin, and many others.
the completion of capacitation enables the spermatozoa to respond to the interaction with oocyte zona pellucida with a very fast surge of i, during ar.
in the present work the incubation of spermatozoa in presence of cd did not exert any detectable effect on this event.
from these findings it is evident that the spermatozoa inability to fulfill the physiological goal, the zp-induced ar, is paralleled by the maintained efficiency of the main signaling pathways, in agreement with the computational model-based prediction.
the data until now available attribute to the actin dynamics a structural role during capacitation and ar.
in particular, it is known that gradually the pm acquires the ability to fuse with the oam thanks to the remodelling of its lipid composition and architecture  <cit> .
the developing network of actin acts as a diaphragm between the two membranes avoiding their fusion.
once the capacitation was completely achieved and the physiological stimulus  was detected, the ar takes place and the zp-induced calcium peak causes the fast depolymerisation of actin structure.
if this model was correct it was reasonably to expect that the block of actin polymerization  in early phases of capacitation leads the increase in the percentage of spermatozoa showing the loss of acrosome integrity due to the inefficiency of separation of pm and oam.
in our experiments, on the contrary, it was found that, even in the absence of the actin network, the pm and oam did not fuse.
thus, the results of the present work could complete and revamp the current concept, leading us to hypothesize that in swine spermatozoa, as demonstrated in other cells, the role of actin cytoskeleton overcomes this merely structural function.
it is possible to conceive that, linking all the cellular districts involved in capacitation, the actin polymerization could have a role of general coordination of this pivotal biological event.
in particular, it is noteworthy that this node links three hubs of the system, thus, strengthening the hypothesis that actin polymerization acts as an important node of coordination trough the information flow.
this supposition is in keeping with the newly emerging evidence that in different cellular systems the cytoskeleton is not only a mechanical support for the cell, but exerts a key role in signaling.
in fact, it was proposed that "independent of its mechanical strength, the filaments of the cytoskeleton form a continuous, dynamic connection between nearly all cellular structures, and they present an enormous surface area on which proteins and other cytoplasmic components can dock"  <cit> .
this is strengthened by the finding that the plasma membrane surface area of a 20-μm-diameter generic cell is on the order of  <dig> μm <dig>  in contrast, the total surface area of a typical concentration of  <dig> mg/ml f-actin is  <dig>  μm <dig>  <cit>  and that the diffusion along cytoskeletal tracks could be a reliable alternative to other established ways of intracellular trafficking and signaling, and could therefore provide an additional level of cell function regulation  <cit> .
one implication of this role is that, together with other well known molecules involved in intracellular signaling, such as ca2+ or atp, the actin cytoskeleton might provide a signal transduction route and macromolecular scaffold, which, during eukaryotic evolution, contributes to the spatial organization of signaling pathways components  <cit> .
CONCLUSIONS
from these data it is possible to take some considerations:
<dig>  it is possible to speculate that the male germ cell representation as a biological network could be an important tool, because it would allow to study not only the molecular components of these cells, but also their intracellular localization and intermolecular interactions in signaling pathways.
in this context it is important to note that in complex systems the whole system is more than the sum of its single components;
<dig>  the present findings could be discussed as the evidence that spermatozoa behave as complex systems.
in this optic the acquisition of the fertilizing ability is a system propriety that emerges when these cells are considered in their whole signaling network.
if the single subcellular element is unperturbed by an external factor, but the coordination among all of them is broken, the male gamete becomes unable to express the ability to physiologically respond to the stimulus .
in addition, the molecule/s acting as controller of cross-talking among the different intracellular compartments could become a sort of "achilles' heel" of cellular signaling machinery, when compromised.
<dig>  it was already known that the actin cytoskeleton is a highly dynamic structure involved in capacitation.
from the present data it emerges that actin polymerization could be involved in coordination of signaling during the capacitation.
in our opinion these findings strength the usefulness of the combined use of a computational and an experimental approach, could have important implications in diagnostics and therapeutics of male infertility, as well in contraceptive strategies, and could contribute to the knowledge of the role played by cytoskeleton in cell signal transduction.

 one of the most important events in the history of life was the eukaryotes evolution that started with the onset of intracellular compartmentalization, approximately - billion years ago. this fundamental step had enormous consequences on cell organization and function. in fact, in one hand, the appearance of specialized districts within the cytoplasm increased the efficiency of metabolic and signaling pathways, in the other hand, cell complexity increased. in fact, the development of organelles determined the existence of specific subcellular microenvironments characterized by different biochemical properties. thus, the cell complexity arose either by the large number of molecular components involved in signal transduction and by the reciprocal connections and spatial relationship existing among them. in addition, the spatial segregation of molecules and chemical reactions allowed that the same molecule could be responsible of very different signals. "we already have signaling "wires" distinguished by the identity of the molecules in the pathways. compartmentalization duplicates these existing wires and separates them in space. this multiplies the number of signals they can carry". this increasing complexity imposes new biological problems as the emergence of new proprieties of cellular systems and the need of the presence of a/some conductor/s which harmonize the function of subcellular compartments. recently the interpretation of the molecular mechanisms involved in cell signaling is made possible by new computational approaches that allow to modellize cellular functions as a network of integrated and coordinated signals operating within subcellular districts interconnected to each other. one clear example is represented by sperm cells, whose biochemical mechanisms leading to capacitation during the post-ejaculatory life, were recently described by using the biological networks formalism. the spermatozoa offer several advantages to adopt this kind of approach since: - the main molecular events occurring during sperm capacitation are largely studied for biological and clinical reasons; - their maturation can be experimentally reproduced under in vitro condition and assessed by measuring their ability to undergo acrosomal reaction after zonae pellucidae stimulation or to in vitro fertilize the oocytes; - their molecular composition is stable since the nucleus of the male germ cells is transcriptionally silent except for the mitochondrial protein translation of nuclear-encoded proteins. starting from these premises, the present work has been carried out, first, to study, using a computational approach, the mechanisms involved in the coordination of the molecular events that take place in the different subcellular districts during the post-ejaculatory spermatozoa maturation, the capacitation. then, an experimental set-up was developed to confirm the model-based prediction. to this aim the events potentially acting as coordinators were inhibited by specific drugs during the spermatozoa incubation under capacitating conditions. the effects of this inhibition were assessed on functional spermatozoa status, and on the major cellular events involved in the acquisition of full fertilizing ability.  computational model of spermatozoa capacitation the main topological parameters of the network representing the capacitation are shown in table the distribution of node linkages followed a power law, represented by the generic equation: were and the number of nodes represent the total number of molecules involved, the number of edges represents the total number of interaction found, the clustering coefficient is calculated as ci = 2ni/k, where ni is the number of links connecting the ki neighbours of node i to each other, the network diameter is the largest distance between two nodes, the averaged n° neighbours represent the mean number of connection of each node, the char. path length gives the expected distance between two connected nodes. the r, r coefficients were, respectively, and. the clustering coefficient distribution does not follow a power law, thus, the results of power law fitting of clustering coefficient distribution were: r =; r =. the most connected nodes are showed in table the analysis of the network topology revealed that only tree nodes bound all the subcellular compartments involved in post-ejaculatory signalling: i, atp, and "actin polymerization". i and atp are the most linked nodes and bound about one third of the nodes. more interesting is the behaviour of the "actin polymerization" node, represented in figure that links a smaller number of nodes located in all the subcellular compartments. the removal of the "actin polymerization" node from the capacitation network had modest consequences on the network topological parameter, but caused the loss of nodes: oam fusion, pm fusion, g-actin, f-actin, mitochondrial protein translation. the number of nodes represent the total number of molecules involved, the number of edges represents the total number of interaction found, the clustering coefficient is calculated as ci = 2ni/k, where ni is the number of links connecting the ki neighbours of node i to each other, the network diameter is the largest distance between two nodes, the averaged n° neighbours represent the mean number of connection of each node, the char. path length gives the expected distance between two connected nodes. phalloidin staining the distribution of the fluorescence within the spermatozoa was assessed, allowing the identification of two different patterns: - pattern a) low actin polymeriziation: the phalloidin fluorescence emission is low and is mainly localized over the midpiece and the post acrosomial region; - pattern b) high actin polymerization: the phalloidin fluorescence emission is high and localized over the midpiece and the whole sperm head. the majority of freshly ejaculated spermatozoa localized a faint fluorescence emission in the midpiece and in the post acrosomal region. after h of incubation under control conditions, the majority of spermatozoa increased their content of f-actin that resulted to be localized over sperm head. the cd treated spermatozoa, displayed a fluorescence signal similar to that recorded at t. zp-induced ar the percentage of acrosome reacted spermatozoa recorded during incubation in control condition ranged from about 5% at the beginning of incubation to about 10% after h. the szp addition to the samples made possible to quantify the percentage of the spermatozoa responding to the physiological stimulus with the exocytosis of the acrosomal content, which after the h incubation reached about the 30%. the cd treated spermatozoa showed an incidence of spontaneous ar similar to that recorded in the control samples during all the h of incubation, instead, the szp addition promoted the ar in a markedly reduced percentage of cells, see figure 4b. chlortetracycline staining the spermatozoa incubated under control conditions, at the beginning of incubation, showed three different patterns of chlortetracycline stain in agreement with mattioli et al.: - about 80% of spermatozoa displayed a faint fluorescence uniformly distributed over the head; - about the 10% spermatozoa displayed the fluorescence concentrated in the post-acrosomal region; - about the 10% displayed the fluorescence concentrated in the acrosome: these spermatozoa, in keeping with mattioli et al., were considered to have completed the capacitation-related membrane reorganization. after h of incubation the percentage of the three patterns was markedly different: pattern a decreased to about 40%, pattern b remained at about 10%, while, pattern c reached 50%. as evident in figure the treatment with cd did not affect significantly the percentage of spermatozoa showing the different ctc stain patterns. tyrosine phosphorylation the tyrosine phosphorilation pattern of freshly ejaculated male gametes, or of spermatozoa incubated under control condition, or in the presence of cd was assessed. as summarized in figure after the incubation the tyrosine phosphorylation pattern changed regardless of the cultural condition adopted. phospholipase c-γ relocalization phospholipase c-γ protein expression studied with western blotting revealed that this protein modified its localization during the interval of culture. in fact, as shown in figure uncapacitated sperm cells strongly expressed plc-γ within the cytoplasm, while, the incubation caused its translocation into the membrane compartment both in ctr and in cd samples. ultrastructural analysis performed under pre-embedding condition, confirmed the membrane traslocation of plc-γ in incubated spermatozoa maintained either in the presence or absence of cd, confirming the western blot analysis. in fact, uncapacitated spermatozoa displayed very few gold particles, indicative of plc-γ on their membranes, while, in in vitro incubated cells, independently from the used treatment, the number of gold particles localizing the protein on the membrane notably grew. zp-induced intracellular calcium concentration rise the coincubation with the szp in spermatozoa capacitated under control conditions caused an evident i elevation in the 36% of sperm cells: the intracellular ca2+ peaked in 1- sec and lasted 20- sec before i returning at a basal level. in the presence of cd the ability and the kinetic to respond with a i rise to the szp coincubation was maintained in the 38% of spermatozoa. discussion aim of this work was to study the molecular event/s involved in the coordination of the biochemistry of the subcellular compartments during spermatozoa acquisition of fertilizing ability using a predictive computational modelisation and the in vitro experimental approach as a confirmation. thanks the in silico experiment the node acting as a coordinator for the signaling cascades of different subcellular districts was identified. then, the consequences on cellular function of its inhibition were experimentally evaluated in vitro. this goal was targeted by representing the biochemical machinery that drives the capacitation as a biological network that, on the basis of its topological proprieties, was considered as a scale free network. in fact, the node degree, k, which indicates how many links the node has to other nodes, followed a power-law distribution of the number of links per node and, in parallel, the tendency to develop clusters of nodes, the so called clustering coefficient ci was independent of the number of links per node. this architecture conferred to the capacitation network high signaling efficiency and robustness against random failure and, in keeping with the barabási-albert model, made evident that a relatively small number of nodes were highly connected and most of the nodes were scarcely linked. thus, it was possible to identify the hubs, i.e. the molecules playing a key role in post-ejaculatory maturation of spermatozoa and to localize them in different subcellular districts. in addition, the functional meaning and the localization of all the molecules involved in capacitation were studied and the nodes that interconnect the intracellular compartments were identified. it is really impressive that these nodes were only three: i, atp and "actin polymerization". more in detail i and atp are linked with a high number of nodes and exerted the function of ubiquitous second messengers or of metabolic sustain. it is important to note that it has been recently demonstrated that their ablation from the network causes the collapse of network structures. on the contrary, the "actin polymerization" bounds in a specific way, having only links, all the subcellular compartments involved in capacitation. it is suggestive that it does not link the nucleus, the only compartment that remains stable until fertilization. in particular it links: - phosphatidic acid: it is known that actin polymerization depends on pld activation, which occurs via the hco 2/camp/pka pathway or via the g-protein coupled receptor /pkc pathway; - atp: the actin is an atpase and its polymerization depends on atp cellular concentration, in addition, the atp is the most important molecule of spermatozoa energy metabolism. in fact, the metabolic energy production in spermatozoa depends on glycolysis and/or on mitochondrial oxidative; - f-actin and g-actin: for recent reviews on actin polymerization see; - i: the intracellular calcium is one of the most important second messengers that drives the intracellular signaling during capacitation; - oam and pm fusion: see below for explanation; - protein synthesis: the incubation with the mitochondrial translation inhibitor d-chloramphenicol almost completely blocked spermatozoa actin polymerization. in this context it is important to note that the links of actin polymerization involve three of the most linked nodes found in capacitation and that this node was per se one of the most linked node. thus, it is possible to hypothesize that a message involving the actin polymerization node could reach all the cell districts, perturbing the whole signal transduction system of spermatozoa. in addition, it has been found that the removal of "actin polymerization" node caused an important effect: the main topological parameters of the capacitation network were virtually unaffected but some nodes remained out from the network: oam fusion, pm fusion, g-actin, f-actin, mitochondrial protein translation. this finding allows to predict that the functional ablation of actin polymerization during the in vitro capacitation could cause the spermatozoa inability to undergo ar while, the different signalling pathways remained unaffected. to obtain an experimental confirmation of the model-based prediction, the actin polymerization was inhibited, as clearly confirmed by confocal analyses, by the incubation of spermatozoa under capacitating condition in the presence of cd. the effect of this drug on the whole cell function was immediately evident: the treated spermatozoa lost the ability to undergo ar when exposed to the physiological agonist, the szp. in parallel, the biochemical analyses showed that the main signaling pathways involved in capacitation were unaffected by the treatment. particularly the capacitation implies a marked calcium-dependent rearrangement of membrane structure that, in turn, determines the increase of the plasma membrane and of the outer acrosome membrane fusogenicity. this event resulted to be unaffected by cd administration as demonstrated by ctc staining. another important signaling pathway involves the protein tyrosine phosphorylation, via sac/camp/pka. it is known that the protein tyrosine phosphorylation pattern of spermatozoa changes during the capacitation in several species such as human, mice, cattle, pig, hamster and cat. in boar spermatozoa a typical protein phosphorylation pattern has been identified, and the cd treatment did not exert any detectable effect on this parameter. the plc-γ plays a key role in coupling actin cytoskeleton and membrane dynamics to the calcium metabolism. when activated this enzyme migrates from the cytosol to the membrane where it hydrolyses the phosphatidylinositol -bisphosphate generating inositol,5-trisphosphate, a universal calcium-mobilizing second messenger, and diacylglycerol, an activator of protein kinase c. the present results showed that the plc-γ translocation was unaffected by the presence of cd in the capacitation medium. ultimately, the effect of spermatozoa incubation under capacitating condition in presence of cd on calcium metabolism was tested. in fact, the spermatozoa signaling machinery strictly depends on intracellular calcium concentration. during the capacitation the ca2+ behaves as a second messenger converting extracelluar stimuli in chemical response involving a myriad of molecular system, such as, protein kinase c, protein kynase c, actin, and many others. the completion of capacitation enables the spermatozoa to respond to the interaction with oocyte zona pellucida with a very fast surge of i, during ar. in the present work the incubation of spermatozoa in presence of cd did not exert any detectable effect on this event. from these findings it is evident that the spermatozoa inability to fulfill the physiological goal, the zp-induced ar, is paralleled by the maintained efficiency of the main signaling pathways, in agreement with the computational model-based prediction. the data until now available attribute to the actin dynamics a structural role during capacitation and ar. in particular, it is known that gradually the pm acquires the ability to fuse with the oam thanks to the remodelling of its lipid composition and architecture. the developing network of actin acts as a diaphragm between the two membranes avoiding their fusion. once the capacitation was completely achieved and the physiological stimulus was detected, the ar takes place and the zp-induced calcium peak causes the fast depolymerisation of actin structure. if this model was correct it was reasonably to expect that the block of actin polymerization in early phases of capacitation leads the increase in the percentage of spermatozoa showing the loss of acrosome integrity due to the inefficiency of separation of pm and oam. in our experiments, on the contrary, it was found that, even in the absence of the actin network, the pm and oam did not fuse. thus, the results of the present work could complete and revamp the current concept, leading us to hypothesize that in swine spermatozoa, as demonstrated in other cells, the role of actin cytoskeleton overcomes this merely structural function. it is possible to conceive that, linking all the cellular districts involved in capacitation, the actin polymerization could have a role of general coordination of this pivotal biological event. in particular, it is noteworthy that this node links three hubs of the system, thus, strengthening the hypothesis that actin polymerization acts as an important node of coordination trough the information flow. this supposition is in keeping with the newly emerging evidence that in different cellular systems the cytoskeleton is not only a mechanical support for the cell, but exerts a key role in signaling. in fact, it was proposed that "independent of its mechanical strength, the filaments of the cytoskeleton form a continuous, dynamic connection between nearly all cellular structures, and they present an enormous surface area on which proteins and other cytoplasmic components can dock". this is strengthened by the finding that the plasma membrane surface area of a 20-μm-diameter generic cell is on the order of μm in contrast, the total surface area of a typical concentration of mg/ml f-actin is μm and that the diffusion along cytoskeletal tracks could be a reliable alternative to other established ways of intracellular trafficking and signaling, and could therefore provide an additional level of cell function regulation. one implication of this role is that, together with other well known molecules involved in intracellular signaling, such as ca2+ or atp, the actin cytoskeleton might provide a signal transduction route and macromolecular scaffold, which, during eukaryotic evolution, contributes to the spatial organization of signaling pathways components.  from these data it is possible to take some considerations: it is possible to speculate that the male germ cell representation as a biological network could be an important tool, because it would allow to study not only the molecular components of these cells, but also their intracellular localization and intermolecular interactions in signaling pathways. in this context it is important to note that in complex systems the whole system is more than the sum of its single components; the present findings could be discussed as the evidence that spermatozoa behave as complex systems. in this optic the acquisition of the fertilizing ability is a system propriety that emerges when these cells are considered in their whole signaling network. if the single subcellular element is unperturbed by an external factor, but the coordination among all of them is broken, the male gamete becomes unable to express the ability to physiologically respond to the stimulus. in addition, the molecule/s acting as controller of cross-talking among the different intracellular compartments could become a sort of "achilles' heel" of cellular signaling machinery, when compromised. it was already known that the actin cytoskeleton is a highly dynamic structure involved in capacitation. from the present data it emerges that actin polymerization could be involved in coordination of signaling during the capacitation. in our opinion these findings strength the usefulness of the combined use of a computational and an experimental approach, could have important implications in diagnostics and therapeutics of male infertility, as well in contraceptive strategies, and could contribute to the knowledge of the role played by cytoskeleton in cell signal transduction. 
thus, it was suggested that actin polymerization could be involved in the signaling coordination of different subcellular districts, and that its functional ablation could compromise spermatozoa ability to complete the capacitation. in the present work the coordination of subcellular signalling, during the boar sperm capacitation, was studied by a computational and experimental approach. the model based-hypothesis was confirmed by the reported data obtained with the in vitro experiments, which strengthen the idea that the actin cytoskeleton is not only a mechanical support for the cell, but that it exerts a key role in signaling during the sperm capacitation. the experiments, carried out inhibiting the actin polymerization in capacitating boar spermatozoa by the administration of cytocalasin d, demonstrated that the cd treatment inhibited spermatozoa ability to reach the full fertilizing ability, while, the examined signaling pathways remained effective, thus, confirming the model-based hypothesis. it is the case of the pathways involved in spermatozoa acquisition of fertilizing ability. the analysis of computational model pointed out that the "actin polymerization" node had some important and unique features: - it links in a specific manner all the intracellular compartments, the signalling cascades involved in many biological processes require the coordination of different subcellular districts. as first the biological network representing all the molecular interactions involved in capacitation was build and analyzed, then, an experimental set up was carried out to confirm the computational model-based prediction.
 the signalling cascades involved in many biological processes require the coordination of different subcellular districts. it is the case of the pathways involved in spermatozoa acquisition of fertilizing ability. in the present work the coordination of subcellular signalling, during the boar sperm capacitation, was studied by a computational and experimental approach. as first the biological network representing all the molecular interactions involved in capacitation was build and analyzed, then, an experimental set up was carried out to confirm the computational model-based prediction.  the analysis of computational model pointed out that the "actin polymerization" node had some important and unique features: - it is one of the most connected nodes, - it links in a specific manner all the intracellular compartments, - its removal from the network did not affect the global network topology but caused the loss of five important nodes. thus, it was suggested that actin polymerization could be involved in the signaling coordination of different subcellular districts, and that its functional ablation could compromise spermatozoa ability to complete the capacitation. the experiments, carried out inhibiting the actin polymerization in capacitating boar spermatozoa by the administration of cytocalasin d, demonstrated that the cd treatment inhibited spermatozoa ability to reach the full fertilizing ability, while, the examined signaling pathways remained effective, thus, confirming the model-based hypothesis.  the model based-hypothesis was confirmed by the reported data obtained with the in vitro experiments, which strengthen the idea that the actin cytoskeleton is not only a mechanical support for the cell, but that it exerts a key role in signaling during the sperm capacitation. 