Unexplained male infertility is a diagnosis reserved for men in whom routine semen analysis results are found within normal values and physical as well as endocrine abnormalities were ruled out. This assumption is based on the observation of low success rates of IVF and intrauterine insemination (IUI) in certain cases of unexplained infertility. The major cause of fertilization failure in conventional IVF is due to abnormalities of sperm-egg membrane interaction. Many molecular interactions in the form of protein-protein interactions, mediate the sperm-egg membrane interaction. Due to the various limitations of materials and difficulties in analyzing vivo membrane protein-protein interactions (PPI), many efforts have been failed to comprehensively elucidate the fusion mechanism and the molecular interactions that mediate sperm-egg membrane fusion. Understanding the molecular mechanism is crucial in solving problems with infertility and failed in vitro fertilization. The main purpose of this study was to identify possible protein interaction in human sperm-egg interaction using protein interaction network. Different databases have been applied to predict new interaction for constructing human sperm-egg interaction network. The protein interaction network represented new predicted interaction and confirmed the previous findings about the importance of the candidate genes, involved in this field. CD151 and CD9 in human oocyte have interaction with CD49 in sperm, and CD49 interacts to CD63 and CD81 in the oocyte. These results showed that the different tetraspanins in sperm may implicate in human sperm-egg interaction. It was also represented that ADAM2 in sperm implicates as a member of protein candidate that involved in sperm-egg membrane interaction, by having interaction with CD9 and ZP3 in the oocyte. Further experimental studies are required to investigate these new interactions. The application of this network approach may be an alternative tool to find novel important protein interactions in order to study new treatment methods for successful assisted reproductive technologies.
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Infertility is a common clinical problem affecting 13–15% of couples worldwide . The prevalence varies throughout developed and underdeveloped countries, being higher in the latter in which limited resources for diagnosis and treatment are available . A male factor is solely and partially implicated in 20-50% of the cases of infertility . However, despite advances in technologies and diagnostic methods in the field of Andrology, there remains a significant subset of these subfertile men who are classified as having unexplained male infertility (UMI).
The category ‘UMI’ is reserved for infertile men with infertility of unknown origin, possess normal semen and in which female infertility factors have been ruled out . Therefore, Normospermic infertile men may have defective sperm that are unable to fertilize. This assumption is based on the observation of low success rates of IVF and intrauterine insemination (IUI) in certain cases of unexplained infertility. The major cause of fertilization failure in conventional IVF is due to abnormalities of sperm-egg membrane interaction . Many molecular interactions in the form of protein-protein interactions mediate the sperm-egg membrane interaction . Janice P. Evans collected the list of candidate sperm proteins for participation in sperm-egg membrane interactions . A number of previous studies have attempted to find the molecules that are involved in the interaction process, but due to the various limitations of materials and difficulties in analyzing vivo membrane protein-protein interactions (PPI), many efforts have failed to comprehensively elucidate the fusion mechanism, leaving the molecular interactions that mediate sperm-egg membrane fusion still poorly understood. Thus the recognition of the candidate proteins that involved in the crucial step of fertilization can help to better understand for research into new treatment methods to successfully assist reproductive technologies. In this study, all the potential protein interactions that involve sperm-egg membrane interaction was investigated by constructing and analyzing a protein-protein interaction (PPI) network of all the membrane and surface proteins of the sperm and the oocyte proteins, identifying the essential PPI and their biological roles in the sperm-egg interaction process through various computational tools and online databases.
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Collection associated proteins with human sperm and egg
UniProt (http://www.uniprot.org/) was used to find human egg/oocyte and sperm related proteins with the keywords "human", "sperm", "spermatozoa", "spermatozoon", and "human", "oocyte", "egg".
Construction of the Protein-Protein Interaction (PPI) Network
All of the proteins identified by these methods were loaded into Cytoscape 2.8.3  using the MiMI plugin . MiMI Cytoscape plugin retrieves molecular interactions from Michigan Molecular Interactions (MiMI) database anddisplaythe interactionnetworkwith Cytoscape. MiMI gathers and merges data from well-knownprotein interaction databases including BIND, DIP, HPRD, RefSeq, SwissProt, IPI and CCSB-HI1 etc. All of the collated proteins have their own UniProt ID and these were also used as input for STRING database  and each protein-protein interaction network was retrieved; then all the network merge together using cytoscape to create the whole protein network. The STRING database consists of known and predicted protein interactions that include direct (physical) and indirect (functional) associations. STRING quantitatively integrates interaction data from four different sources: genomic context, high-throughput experiments, coexpression, and prior knowledge from research publications. As a last step, the protein network from MiMI plugin and STRING database were merged using Cytoscape. These methods were done for sperm profile and oocyte profile to map sperm and oocyte protein interaction network. This method enabled the researcher to consider all possible interactions between proteins of sperm and oocyte.
Study on possible protein interaction involved in sperm-egg interactions
In this study, the focus was on the proteins of the spermatozoa and oocyte identified by MS proteomics technology. As a methodological approach, all the articles that were retrieved from PubMed search were considered for inclusion, with the keywords "human", "sperm", "spermatozoa", "spermatozoon", and "human", "oocyte", "egg" combined with the key word "proteome", "proteomics" or "mass spectrometry". The only far-reaching human sperm proteome and oocyte proteome analysis are available to date on which the work is done by Baker et al  and Assou et al , respectively. The proteins involved in these studies were collected with own UniProt ID using UniProt ID mapping. Since the aim was to identify protein interaction involved in sperm-egg interactions, only those proteins are addressed that contain a signal sequence and/or transmembrane domain by selecting signal peptide and transmembrane features from sequence annotation (features) in UniProt (www.uniprot.org).
Sperm profile network and oocyte profile network were merged and then the above collected proteins nodes were extracted as two groups (sperm proteins nodes and oocyte protein nodes) with the aim to study direct interactions between the two groups.
Construction of the PPI Network
The collected proteins from UniProt were loaded into Cytoscape 2.8.3  using the MiMI plugin to construct PPI networks. The networks for sperm and egg/oocyte associated proteins contain 409 and 2076 protein nodes, 2746 and 8565 interactions between those proteins, respectively (figure 1).
Extraction of protein-protein interaction from sperm-egg interaction network
The two networks above were merged and the identified sperm and oocyte proteins by MS proteomics technology that contain a signal sequence and/or transmembrane domain were selected from the merged network as two groups: sperm protein group and oocyte protein group. The direct interaction between two groups has finally been studied (Table 1).
Table 1 includes the direct protein-protein interaction between sperm and oocyte proteins that were identified by protein network approach. Some of these interactions have already been identified and confirmed the role of them in human sperm-egg interaction processes and successful fertility. Monoclonal antibodies approach identified notable sperm proteins, including IZUMO1  and ADAM1 and ADAM2 . The ADAM family has been of interest, building on the identification of sperm ADAM2 (fertilinβ) in studies, with a fertilization-blocking antibody and characterization as one of the founding members of the ADAM family [14, 15]. Sperm ADAMs are binding partners for several members of the integrin family (table 4 in Reference ); a number of these integrins are expressed in eggs and can participate in sperm-egg interactions. ADAM3 has been proposed to be a sperm protein that mediates sperm-ZP interaction on the basis of findings that Adam3-null sperm bind poorly to the ZP (61, 62) and that incubation of solubilized ZP proteins with sperm lysates pulls down ADAM3 (73). Although, these data may indicate that ADAM3 binds a ZP component(s) directly, it is also possible that Adam3-null sperm lack critical proteins for ZP interaction [as Adam3-null sperm have an altered surface proteome, with reduced amounts of several ADAMs (62, 65, 66), and thus may lack other proteins as well]. In this model, ADAM3 would be associated with this molecule(s) on wild-type sperm and thus would pull down in a complex with ZP proteins. IZUMO1 on the sperm is an immunoglobulin superfamily member [with an immunoglobulin-like domain (Ig)] that is essential for sperm-oocyte fusion . The function of IZUMO1 is not entirely clear; IZUMO1 may function by interacting with a molecule on the egg (in trans) and/or may act through IZUMO1-associated proteins (in cis). The tetraspanin CD9 is the major player identified so far in the mouse egg [18-20] and is likely to function in conjunction with another tetraspanin, CD81, as Cd9−/−/Cd81−/− female mice are completely infertile . CD9 may work by interacting with a sperm protein in trans. A Little is known of tetraspanin involvement in human fertilization, although there are data from antibody inhibition studies. Human sperm-egg fusion is partially inhibited by treating eggs with an antibody to a different tetraspanin, CD151 . These data preliminarily raise the possibility that sperm-egg interaction in different mammalian species may rely on different members of the tetraspanin family. In this study, the protein interaction network has been applied using different databases that consist of known and predicted protein interactions.
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Novel protein interaction in human sperm-egg interaction
In table 1, the novel interactions are shown that were involved in human sperm-egg interaction using protein network approach. The results represent SERPINE1 (plasminogen activator inhibitor) and PPP1R3A (Protein phosphatase 1 regulatory subunit 3A) in sperm play a positive role in sperm-egg interaction by interacting to SERPING1 (Plasma protease C1 inhibitor) in the oocyte.
The plasminogen activation system is involved in a diversity of physiological and pathological processes such as thrombolysis, morphogenesis, wound healing and regeneration, as well as in tumor invasion . Plasmin, a proteinase capable of degrading several extracellular matrix proteins and also capable of activating other proteases, is generated from the zymogen plasminogen by proteolytic cleavage [24, 25]. SERPING1 was found on the surface, in the acrosome and in the tail of mature spermatozoa . Sperm-egg interaction has been described as sensitive to serine proteinase inhibitors . Together, these results suggest a role of the plasminogen activation system in sperm-egg interaction . Hypoxia plays a crucial role in many pathophysiological conditions, including cancer biology, whereas hypoxia-inducible factor (HIF) regulates transcriptional responses under hypoxia . It has been reported that hypobaric hypoxia is responsible for the altered male reproductive function. Themechanism of action concerning fertility has not been clearly established . Several PPP family members have been shown to be expressed in sperm, suggesting an important function in this cell . Another type of protein phosphatase 1 (PP1γ2), is localized to the posterior region of the sperm head, the equatorial region, implicated in sperm-egg binding .
Our protein network represented that tetraspanin, CD151 and CD9 in human oocyte has interaction with CD49 in sperm, and CD49 interacts to CD63 and CD81 in the oocyte. Several tetraspanins, including CD9 and CD81, have apparently indirect (and still poorly defined) roles in membrane fusion processes . Cd81-null females have a moderate loss of reproductive function . CD81 is a related tetraspanin that is 45% identical to CD9. The Cd81−/− mouse also showed defects in female fertility and sperm-egg interaction with in vivo–fertilized and in vitro–fertilized eggs . Ziyyat and his colleagues found that Human sperm-egg fusion is partially inhibited by treating eggs with an antibody to a different tetraspanin, CD151 . CD9 is a member of the tetraspanin family (named so, because members have four transmembrane domains) [33, 34]. Cd9−/− females, are severely subfertile .
It has been demonstrated that the different tetraspanin in sperm may implicate in human sperm-egg interaction. This approach showed that CD9 in oocyte plays some role in sperm-egg interaction process by interacting with CD49, IZUMO1 and ADAM2 in sperm. IZUMO1 is essential for the sperm to bind with eggs and that CD9 is essential for eggs to bind with sperm; therefore, it is tempting to speculate that they interact with each other to form a fusogenic complex. If these proteins do indeed interact, it is likely that they both require associating proteins on the sperm and egg cell surfaces, and the identity of these putative factors should be investigated . Several sperm ADAMs (where ADAM denotes “a disintegrin and a metalloprotease”) have been implicated in sperm-egg interaction. Although, no single ADAM is essential, there appears to be a correlation between the presence/levels of certain ADAM proteins and the ability of sperm to interact with the egg membrane. According to the results, ADAM2 in sperm implicates as a member of protein candidate that is involved in sperm-egg membrane interaction, by having interaction with CD9 and ZP3 in the oocyte. In a number of these Adam knockouts, the sperm shows reduced migration into the oviduct through the uterotubal junction, reduced binding to the ZP and/or reduced binding and fusion to the egg plasma membrane, as well as the abnormalities in the sperm surface proteome with the loss of multiple ADAMs [36, 37]. The Adam2−/− knockout has the most serious defects in gamete membrane interactions and has the lowest overall levels of several ADAM proteins on the sperm surface [38-40], but other Adam knockouts have little or no apparent effect on male reproduction [41, 42].
The first protein interaction network of human membrane and surface sperm-egg interaction proteins have been created using computational approaches. This protein network enabled the researcher to identify a set of candidate protein interactions between sperm and egg in human. New predicted protein-protein interactions were reported like CD49-CD151, CD49-CD63, CD49-CD9, ADAM-ZP3, IZUMO1-CD9, ADAM2-IZUMO1, in addition to some new interactions in the plasminogen activation system that may play important roles in sperm-egg interaction. These predicted interactions showed that the different tetraspanins in sperm may implicate in human sperm-egg interaction. The results also revealed that some necessary proteins for successful fertility like CD151 in human might play an important role in building interaction between sperm and egg. Further experimental studies will be required to confirm the importance of these new predicted interactions in human sperm-egg interaction field.