Differential extraction of sperm proteins involves various solubilization methods to preferentially enrich for peripheral membrane proteins, hydrophobic membrane associated proteins, or hydrophilic proteins. One well known and powerful technique to enrich the membrane associated hydrophobic proteins is temperature-induced phase partitioning in TX-114 [57], which allows the separation of hydrophobic integral membrane proteins from the hydrophilic proteins. The technique is based on the ability of the nonionic detergent TX-114 to partition into two distinct phases above 23 °C: a detergent-rich phase and a detergent-depleted or aqueous phase. Amphipathic membrane proteins, whether anchored to a lipid by a GPI moiety or a hydrophobic polypeptide, partition predominantly into the detergent-rich phase, whereas hydrophilic proteins partition predominantly into the aqueous phase [58]. Shetty et al. [20] exploited this technique to identify sperm membrane associated proteins. The sperm surface proteins were labeled before extraction using sulfo-NHS-LC-biotin and the proteins were resolved on large format 2-D gels capable of high resolution. Surface localized proteins were identified by avidin blotting of the biotinylated proteins transferred to nitrocellulose membranes. Figure 2.2 demonstrates the identification of several membrane associated sperm surface localized proteins by 2-D gel analysis and avidin blotting [20]. The method facilitated the identification of eight novel sperm proteins in addition to several known membrane proteins. For example, two acrosomal membrane proteins: SAMP14 [28], a GPI-anchored Ly6/uPAR superfamily protein, and SAMP32 [29], an autoantigenic protein, emerged from this approach. Antibodies against recombinant human SAMP14 and SAMP32 inhibited both the binding and the fusion of human sperm to zona free hamster eggs, suggesting that these molecules may have a role in sperm-egg interaction. Triton X-114 phase partitioning is routinely applied to characterize a protein or to check the presence or absence of a known hydrophobic protein under various experimental conditions [59–61].

Initial evidence for raft formation in male germ cells came from identification of the raft protein, caveolin-1, in the head and flagellum of mouse and guinea pig sperm, implicating these structures in the regulation of both motility and sperm–zona interaction [67]. In order to determine alteration of the protein composition in DRMs following capacitation, Sleight et al. [68] performed a proteomic analysis of mouse sperm proteins isolated in the light buoyant-density fraction. The immunoglobulin superfamily protein Izumo, a well-known sperm-egg fusion protein [69], was also one of the predominant protein enriched in the preparation. Thaler et al. [70] made a similar kind of study and reported that several individual proteins became enriched or depleted in DRM fractions following capacitation. Studies done on the pig [71] and boar [72] support the hypothesis that capacitation induced increased levels of sperm DRMs, with an enhanced zona pellucida affinity. Nixon et al. [73] have shown that DRMs isolated from spermatozoa possessed the ability to selectively bind to the zona pellucida of unfertilized, but not fertilized, mouse oocytes. Watanabe and Kondoh [74] recently demonstrated that mouse sperm undergo GPI-anchored protein release associated with lipid raft reorganization and acrosome reaction to acquire fertility. Collectively, these data provide compelling evidence that mouse spermatozoa possess membrane microdomains that provide a platform for the assembly of key recognition molecules on the sperm surface.

An alternative method for the enrichment and isolation of sperm membrane proteins involves preparation of membranes through hypoosmotic swelling, homogenization, and sonication [75]. Membranes are further isolated by differential centrifugation steps. Purified human sperm membrane proteins can be separated by 2-D gel electrophoresis and further analysis of the sperm antigens can be achieved. Oko’s group [76] has devised a methodology to obtain a sperm head fraction consisting solely of the inner acrosomal membrane (IAM) bound to the detergent-resistant perinuclear theca. On the exposed IAM surface of this fraction, they define an electron dense protein layer that was termed IAM extracellular coat (IAMC). Their approach has led to the identification of a novel inner acrosomal protein IAM38 from bovine sperm with a demonstrated role in sperm egg interaction along with the matrix metallo-proteinase2 (MMP2) and acrosin in the inner acrosomal membrane [76, 77]. Amaral et al. [12] focused on the sperm tail proteins, as the role of the sperm flagellum is specific and very distinct from the role of the sperm head. Isolation of the sperm tails was performed by sonication followed by ultracentrifugation in a sucrose gradient; the proteins were then solubilized in lysis buffer that was compatible with 2DE.

Polyclonal or monoclonal antibodies may also be used for the identification, isolation, and characterization of sperm antigens that are relevant to fertility. A number of sperm antigens have been identified by means of monoclonal antibodies [59, 69, 78–86]. Among the most significant findings was the discovery of the immunoglobulin superfamily protein Izumo1, by using monoclonal antibody OBF13, which interfered with sperm-egg interaction [69]. Izumo1 was shown to localize within the acrosome and to have a definitive role in sperm-egg fusion during fertilization. Izumo1 was identified by separation of the crude extracts from mouse sperm by 2-D gel electrophoresis and subsequent immunoblotting with the monoclonal antibody. Recent studies by Clark and Naz [87] have shown that significant percentage of immunoinfertile female sera have circulating isoantibodies against this protein with none of the fertile women’s sera showing any reactivity, suggesting a possible application of Izumo1 in diagnosis and treatment of infertility, and human contraceptive vaccine development.

In mammals, more than 200 cell surface proteins with various functions, such as hydroxylation, cellular adhesion, and receptor activity, are anchored to the membrane by a covalently attached GPI moiety [88–90]. GPI deficiency causes developmental abnormalities, failure of skin barrier formation, and female infertility in mice indicating that GPI anchor is essential for cell integrity [91, 92].

A few GPI anchored proteins discovered from sperm are CD59, CD52, TESP5, TEX101, Ly6K, PH20 [hyaluronidase], and SAMP14. PH-20 is proposed to have multiple functions involved in cell signaling and serve as a receptor for the zona pellucida, in addition to its hyaluronidase activity [93]. The discovery that a testicular isoform of angiotensin converting enzyme (ACE) is a GPI-anchored protein-releasing factor which is crucial for fertilization [94] has shown the importance of GPI-anchored proteins in the fertilization process. It is known that TEX101 has to be shed and to disappear from testicular germ cells by the GPI-anchored protein-releasing activity of ACE for the correct localization of ADAM3 on the mature sperm surface. LY6K a recently discovered GPI-anchored protein interacted with TEX101 and ADAM3 in the testicular germ cells but disappeared from mature spermatozoa and is believed to be a new factor involved in sperm fertilizing ability [95].

A standard method to isolate GPI-anchored molecule from the cell surface is treatment of the cells with GPI-specific phospholipase C (PIPLC) that cleaves GPI anchors specifically, leaving the lipid moiety in the membrane and releasing the protein with a terminal cyclic phosphoinositol [96]. Even though there is no single report on the identification of GPI anchored molecules from the sperm surface on a global scale, an experiment carried out on oocytes by Coonrod et al. [97] investigated human sperm-hamster oocyte interactions and determined that PI-PLC cleavable glycosylphosphatidylinositol (GPI)-anchored proteins are involved in sperm-egg binding and fusion. Two-dimensional electrophoresis was then utilized to visualize proteins released from hamster oocytes following PI-PLC treatment. The authors demonstrated that treatment of hamster oocytes with PI-PLC inhibits sperm-egg interaction and releases a 25–40 kDa protein cluster (pI 5–6) from the oolemma suggesting that this released protein cluster represents an oolemmal GPI-linked surface protein(s) which is involved in human sperm-hamster egg interaction. A comprehensive search for all the GPI-anchored molecules in spermatozoa and oocyte by proteomic analysis may be needed to identify the full repertoire of molecules that are directly involved in fertilization.

The recent development of 2D-DIGE [98] is beginning to have an impact on the field of reproductive immunology assisting in the identification and characterization of potential fertility related proteins. This technology is based on the creation of a family of size and charge-matched spectrally resolvable dyes that are used to label different protein preparations prior to 2-D gel electrophoresis, allowing up to three distinct protein mixtures to be separated within a single 2-D PAGE gel. By running such differentially labeled protein mixtures on the same gel, between-gel differences in electrophoretic migration patterns can be entirely eliminated. Baker et al. [99] used this technique to isolate and characterize those proteins that undergo processing in rat spermatozoa as they transit the epididymal tract. The technique can be effectively applied to determine the post-translational modifications of the sperm at various functional states (non-capacitated, capacitated and acrosome reacted) and also to identify fertility related proteins by using sperm proteins from healthy fertile specimens versus infertile specimens. Hamada et al. [100] utilized the technique to identify the relative abundance of proteins in pooled reactive oxygen species (ROS)–positive (ROS+) and ROS-negative (ROS−) semen samples and found significantly different expression of protective proteins against oxidative stress in ROS-compared with ROS+ samples. Since so few genes related to human infertility are now known, this method may open up the field of male infertility genetics by “back-tracking” from the proteomes of affected individuals compared to fertile controls.

In addition to the proteomic methods described above, several other strategies being employed to identify interesting sperm proteins include the identification of phosphoproteins and glycoproteins. It is well established that capacitation, a prerequisite event for fertilization requires a cyclic AMP-dependent increase in tyrosine phosphorylation. One of the approaches employed to target phosphoproteins involved in the capacitation event is two-dimensional gel analysis coupled to anti-phosphotyrosine immunoblots and tandem mass spectrometry [101]. The molecular probe, Pro-Q^® diamond phosphoprotein gel staining, is an advanced method that provides a precise strategy for selectively staining phosphoproteins in polyacrylamide gels [102]. The glycoproteins, on the other hand, may be identified by lectin blotting coupled to 2-D gel electrophoresis [41]. A commercially available fluorescent dye Lissamine rhodamine B sulfonyl hydrazine (LRSH) has been introduced recently to specifically stain the glycoproteins [103]. Suryavathi et al. [104] utilized this technique successfully to analyze the glycosylation sites on the mouse Equatorial Segment Protein 1 (SPESP1) using a glycoprotein specific fluorescent stain with the trade name Glycoprofile (Sigma). Mouse testis SPESP1 was immunoprecipitated with the SPESP1 antibody and analyzed by 2D-SDS-PAGE Western blotting and glycoprofile stain (Fig. 2.3).

An important challenge in most of the proteomic methods described above is the identification and mining of antibody reactive, low abundant proteins. Pre-fractionation of the proteins by continuous elution preparative electrophoresis (using PrepCell from Bio-Rad) and preparative isoelectric focusing (using Rotofor from Bio-rad) are useful methods of choice for enriching these low abundant proteins.

In excising proteins from a 2-D gel, it is essential to precisely define the boundaries of individual protein spots to obtain pure proteins for microsequencing. Figure 2.4 demonstrates exceptionally useful tools for coring spots of interest from a silver-stained 2-D gel. These custom-made coring tools consists of hollow cylindrical steel tubes with specialized tips milled to a razor sharp edge. Different bore sizes range from a diameter of 0.5 to 5 mm and each coring tool holds a solid piston within the tube for expulsing gel plugs. A tube of appropriate diameter is chosen to just encompass an individual protein spot and the acrylamide plug is precisely drawn into the tube by placing the tube vertically on the center of the spot with the coring tip down and applying gentle pressure. The piston is used to extrude the gel piece into a sample tube. Use of these precision coring tools has resulted in a high incidence of unique and novel peptide sequence resulting from mass spectrometry. Such sequences may match an Expressed Sequence Tag (EST) sequence which may then be used to clone the corresponding gene by PCR. In the absence of any known sequence in the data base, the gene can be cloned by using a completely degenerate deoxyinosine-containing sense primer and adaptor primer, and performing a 5′ and 3′ rapid amplification of cDNA ends (RACE) PCR [42].

Mass spectrometry proteomic-based technologies have proven to be powerful tools in the determination of sperm antigens and their associated post-translational modifications (PTMs). In a typical experiment, proteins are digested with enzymes and the resulting peptides separated by high performance liquid chromatography (HPLC) into a mass spectrometer over a period of 0.5–4 h depending on the complexity of the sample. This type of experiment is often referred to as LC-MS(MS). Modern mass spectrometers take high resolution spectra (R = 15–250 K) in both MS (molecular weight determination – MW) and MS/MS (fragmentation for identification) modes. In an experiment to identify as many proteins as possible (shotgun proteomics), the mass spectrometer is programmed to take 1 MS scan followed by 5–20 MS/MS scans and repeat this loop for the length of the HPLC gradient. These types of runs produce MW/fragment information for 5000–100,000 species in a given sample. The resulting fragment spectra (MS/MS) are searched by algorithms such as Sequest, Mascot, etc. against databases comprised of proteins predicted from the organism’s genome/transcriptome. The end result is a list of peptides identified and mapped to parent proteins. Often shotgun experiments yield identifications for thousands of proteins in a sample [105]. The large amounts of data collected during a mass spectrometry run can be searched looking for differential mass addition to determine the presence and locations of PTMs. In some cases, a particular protein involved in a cellular event may be isolated to study its PTMs [104] while in others a shotgun type experiment can be performed to look at thousands of PTMs in a specific cell type [106]. Identification of proteins and PTMs gives a catalog listing of species present (qualitative analysis) but is often just the first step in determining biological activity. In most conditions or diseases, the amount of a certain protein and/or PTM and how that amount changes under different treatments (quantative analysis) is equally important. Quantitative changes can be determined relative to a standard condition using label free (ion current peak areas, spectral counts) or labeled (Stable Isotopes – SILAC, iTRAQ) approaches [107, 108]. In these cases, the actual absolute amount is not known, only how it changes between conditions. In some cases, the absolute amount of the protein or PTMs needs to be determined between conditions. This type of quantitation is most often done by MRM (multiple reaction monitoring) with either an internal isotopically labeled standard or external standard curve [109, 110]. Using these techniques, proteins, peptides, and PTMs can be identified and quantified for any given set of samples providing fundamental data necessary for further experiments.

This strategy combines recombinant DNA technology with the experimental approach as described by Hjort and Griffin [111]. Typically a testis lambda expression library is used in this strategy and is plated with a chosen strain of E.coli as host bacterium. After growth at 42 °C and induction with isopropyl-b-thiogalactoside (IPTG), the nitrocellulose filters are screened with the sperm antibody of interest. Bound recombinant protein is detected by use of an isotype specific secondary antibody coupled to horse-radish peroxidase. The cDNA insert of the positive clone is utilized again to re-probe the testis lambda cDNA library to confirm the sequence of the identified clone and also to identify any additional clones.

Wright et al. [112] identified the SP-10 cDNA (ARCV1 gene) using the MHS-10 monoclonal antibody with this method. Diekman and Goldberg [113] used sera from ASA-positive infertile patients and vasectomized men to identify an antigen, designated AgX, expressed by recombinant bacteriophage in a human testis library. Later studies used similar methods to identify additional sperm/testis antigens [114, 115]. In another study, the FA-1 mAb was used for a screening of murine testis lambda gt11 cDNA expression library [116] and the novel sperm protein discovered was named FA-1 for its potential role in fertilization. A similar strategy was employed by the same group to identify two more sperm antigens NZ-1 [117] and NZ-2 [118] from mouse and human origin, respectively. Two monoclonal antibodies, designated S71 and S72 (by World Health Organization workshop on antisperm antibodies), were used to isolate their corresponding cDNA clones from a human testis λZAP cDNA library by Westbrook et al. [85]. The cloned gene was named SPAN-X for sperm protein associated with the nucleus on the X chromosome.

cDNA library screening techniques can also be used as a strategy to isolate the complete cDNA for an unknown candidate molecule when a small region of the cDNA corresponding to the gene is known (e.g. through EST data base). Shetty et al. [28] applied the technique to clone the full-lenth cDNA for SAMP14 using a PCR amplified partial cDNA (derived from EST) clone from human testis as a probe to screen a λDR2 cDNA library. A similar strategy was used earlier by Wolkowicz et al. [119] to clone the full-length cDNA for a human sperm flagellar protein tectin-B1. PCR-based cloning strategy and cDNA library screening utilizing the known sequences of potential drug targets and primary mediators of signaling network has also led to the discovery of novel testis specific targets. Testis specific serine threonine kinases (TSSK 1–6) are one such example. TSSK1 and 2, the first two members of the family, were initially discovered using degenerate oligos corresponding to highly conserved motifs within the protein kinase catalytic domain and by cDNA library screening [120, 121]. Recent studies have validated these testis specific, post-meiotically expressed kinases as candidate male contraceptive targets [122–124].

In an attempt to identify peptide sequences that might be involved in zona pellucida (ZP) binding, Naz et al. [55] screened FliTrx random phage display library with solubilized human ZP. A novel dodecamer sequence, designated as YLP₁₂, was identified that is involved in sperm-ZP recognition/binding. A subtractive cDNA hybridization technology was employed by Naz et al. [125] to obtain sperm specific antigens that could be targeted as potential contraceptive vaccines.

Chen et al. [126] constructed phage-display peptide libraries to select epitope peptides derived from human posterior head 20 (hPH20) and homo sapiens sperm acrosome associated 1 (hSPACA1) using sera collected from infertile women harboring antisperm antibodies.

The studies led to the identification of four epitope peptides. The BSA-coupled synthetic peptides generated a sperm-specific antibody response with a contraceptive effect in both male and female mice. Samoylova et al. [127] used a modified phage display by using intact oocytes surrounded by ZP proteins in native conformation. The procedure led to the identification of an antigneic 12 aa peptide that produced antisperm antibody recognizing acrosomal region of the sperm surface.