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FSP95, A Testis-Specific 95-Kilodalton Fibrous Sheath Antigen That Undergoes Tyrosine Phosphorylation in Capacitated Human Spermatozoa¹

^a Center for Recombinant Gamete Contraceptive Vaccinogens, Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908 ^b Academic Computing Health Sciences, University of Virginia, Charlottesville, Virginia 22908 ^c W.M. Keck Biomedical Mass Spectrometry Laboratory, School of Medicine, University of Virginia, Charlottesville, Virginia 22908

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
Protein tyrosine phosphorylation has been associated with both capacitation and motility of mammalian sperm. During capacitation, human spermatozoa undergo tyrosine phosphorylation of a characteristic set of proteins, only one of which has thus far been cloned and localized. We report here the sequence of a fibrous sheath protein of 95 kDa (FSP95) that undergoes tyrosine phosphorylation during capacitation of human spermatozoa and has similarity to sperm A-kinase anchor proteins (AKAPs). FSP95 is both auto- and iso-antigenic in humans as it is recognized by sera containing antisperm antibodies from infertile men and women. The 853-residue protein has a calculated molecular weight of 94.6 kDa and an isoelectric point (pI) of 6.0, and it contains multiple potential phosphorylation sites for protein kinase C and casein kinase II as well as one potential tyrosine kinase phosphorylation site at amino acid 435. The sequence has amino acid homology to mouse sperm fibrous sheath AKAP82 (pro-mAKAP82, 34% identity) and to human sperm fibrous sheath AKAP82 (pro-hAKAP82, 32% identity). The gene encoding FSP95 has 5 exons separated by 4 introns and is located on chromosome 12 at locus p13.3. Northern analysis detected a single transcript of 3.0 kilobases, and Northern dot blot analysis of 50 human tissues revealed FSP95 mRNA expression only in testis. By employing sperm immobilization, indirect immunofluorescence, and immunoelectron microscopy with antisera to purified recombinant FSP95, the protein was localized to the ribs of the fibrous sheath in the principal piece of the sperm tail. FSP95 is the second fibrous sheath protein to be cloned, sequenced and localized in human spermatozoa.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
Once deposited in the female reproductive tract, ejaculated mammalian spermatozoa undergo an intricate series of membrane and metabolic changes, collectively termed capacitation, which prepare the sperm to undergo the acrosome reaction and to bind and penetrate the zona pellucida [1]. During capacitation of bull and human spermatozoa, an increase in intracellular Ca²⁺ [2, 3] and cAMP has been detected [4, 5]. Capacitated spermatozoa are also characterized by a rise in intracellular pH [6] and loss of cholesterol from the sperm plasma membrane [7]. Phosphorylation of tyrosine residues on a cohort of specific proteins in mouse [8], bovine [9], and human sperm [10–12] indicates that activation of sperm tyrosine kinases occurs during the capacitation process. Mouse caput epididymal sperm that lack the ability to undergo capacitation do not display tyrosine phosphorylation of this set of proteins [8].

Although distinct sets of high-molecular weight tyrosine-phosphorylated proteins have been recognized during capacitation in human (80–105 kDa) [10, 12], mouse (40–120 kDa) [8], and bovine spermatozoa (40–120 kDa) [9], little is known about the structure of these substrates of tyrosine phosphorylation, particularly in the case of human spermatozoa. Only one capacitation-induced tyrosine-phosphorylated protein of human spermatozoa has been reported so far, and it has been demonstrated to be a fibrous sheath-associated A-kinase anchor protein (AKAP) [10, 13]. It has been suggested that protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent protein kinase A (PKA) pathway [7]. Recently, PKA anchoring proteins have been demonstrated in the fibrous sheath of both mouse and human spermatozoa [13, 14]. AKAPs in other cell types have been implicated in signal transduction by forming complexes with protein kinases and phosphatases within specific cellular compartments [15, 16].

Sperm acquire the capacity for motility during epididymal maturation, become motile in the ejaculate, and express a distinct motility pattern called hyperactivation during capacitation [1]. The initiation and maintenance of motility of spermatozoa involves phosphorylation of sperm proteins [17, 18], and motility is highly regulated by a cascade of phosphorylation-dephosphorylation events affecting the activities of protein kinase substrates [19, 20].

Many sperm proteins are potent auto- and iso-antigens that evoke immune responses in both males and females, and antisperm antibodies (ASA) are capable of causing infertility in both human and animal models [21, 22]. For example, ASA can be measured in serum of up to 70% of men after vasectomy compared with 2–8% of men without vasectomy [23]. The incidence of ASA in infertile couples varies from 9% to 36%, whereas only 0.9–4% of fertile men and women have detectable antibodies [22]. The presence of ASA in the female genital tract can affect sperm-egg interaction by several mechanisms and consequently can result in reduced fertility [24]. The characterization of such sperm antigens may provide leads for improved diagnosis of antibody-mediated infertility and for identifying contraceptive vaccine candidates. In this regard, a two-dimensional (2D) human sperm proteome was developed recently [25] that has provided a means to select and microsequence proteins of interest.

The objective of this study was molecular characterization of a high-molecular weight tyrosine-phosphorylated auto- and iso-antigen of human spermatozoa, as well as initial evaluation of its role in antibody-mediated infertility and its potential as a candidate for a contraceptive vaccinogen.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
Preparation of Spermatozoa, Capacitation, and Extraction of Sperm Proteins

Human semen samples were obtained from healthy volunteers by masturbation following 3–4 days of sexual abstinence. Written consent was obtained, and all donors tested negative for HIV. Ejaculates with normal semen volume, sperm count, and motility were used in this study. After liquefaction of semen samples at room temperature, fresh sperm were separated from seminal plasma, germ cells, white blood cells, and epithelial cells by Percoll (Pharmacia Biotech, Bromma, Sweden) density gradient centrifugation and washed in Ham's F-10 medium (Gibco BRL, Life Technologies, Gaithersburg, MD) within 2–3 h of ejaculation as described earlier [25].

To obtain capacitated spermatozoa, motile sperm were prepared by the swim-up method for 60 min in Biggers, Whitten, and Whittingham (BWW) medium (Irvine Scientific, Santa Ana, CA) containing no human serum albumin. Motile spermatozoa were subsequently collected from the supernatant and incubated in BWW medium supplemented with 3.0% human serum albumin (HSA) (Sigma, St. Louis, MO) at 37°C in 5% CO₂ for 6 h to induce capacitation [11]. Capacitation was also performed in the presence of 200 µM genistein, a protein tyrosine kinase inhibitor [10].

Fresh and capacitated spermatozoa were solubilized in a lysis buffer containing 9.8 M urea, 2% octyl-ß-D-glucopyranoside (OBG) (ESA Inc., Chelmsford MA), 2% (v:v) ampholines, 100 mM dithiothreitol (Bio-Rad, Hercules, CA), 5 mM iodoacetamide (Sigma), 5 mM EDTA, and four protease inhibitors: 2 mM PMSF (Sigma), 3 mg/ml N-p-tosyl-L-lysine chloro-methyl ketone (TLCK; Boehringer Mannheim, Indianapolis, IN), 1.46 mM pepstatin A (Sigma), and 2.1 mM leupeptin (Sigma). Five hundred million sperm per milliliter were solubilized by shaking at 4°C for 45 min. Insoluble molecules were removed by centrifugation at 10 000 x g for 2 min, and the supernatant was used for 2D gel separation of sperm proteins according to our published protocols [25].

Detection of Tyrosine-Phosphorylated Proteins

After a 6.0-h incubation period in capacitation medium, sperm were solubilized in the lysis buffer [25]. The solubilized proteins were separated by 2D SDS-PAGE and either stained with silver [26] or the proteins were electrotransferred to nitrocellulose membranes, and the nonspecific binding sites on the membrane were blocked with 1% BSA in 10 mM Tris (pH 7.5), 0.1 M NaCl, and 0.05% Tween 20 for 20 min at 37°C. The blocked membrane was then incubated with horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antibody RC-20 (Transduction Laboratories, Lexington, KY) at a 1:2500 dilution in the above buffer for 20 min at 37°C. The membrane was washed thoroughly, and positive immunoreactive spots were detected by enhanced chemiluminescence assay (Amersham Corp., Buckinghamshire, UK) according to the manufacturer's instructions. RC-20 is a well-characterized monoclonal antibody with a specificity for phosphotyrosine [27, 28].

Immunoblotting of Sperm Proteins with Infertile Sera Prescreened by Immunobead Binding Test

Sera were obtained from infertile men and women with unexplained infertility, having no diagnosed hormonal, infective, or physical causes of their infertility. ASA in the infertile patient sera were detected by the indirect immunobead binding test [29]. Sera chosen for immunoblotting analysis had a high immunobead binding test score; i.e., more than 60% of spermatozoa revealed bead binding, indicating IgG-, IgA-, and/or IgM-specific ASA. The selected sera all had antibodies directed against the sperm head or the entire cell.

For immunoblotting, sperm proteins were extracted and separated by a 2D SDS-PAGE system as described earlier [25]. After electrophoretic transfer of the proteins, the membranes were rinsed in PBS (pH 7.4) and blocked with 5% dry milk in PBS-Tween (10 mM PBS with 0.05% Tween 20). The blots were then incubated with test serum diluted 1:1000 at 4°C overnight. A horseradish peroxidase-conjugated goat anti-human IgG/IgM secondary antibody (Jackson ImmunoResearch Lab, West Grove, PA) was then incubated with the blots for 1 h at a 1:5000 dilution in PBS-Tween, and the immunoreactive spots were visualized by enhanced chemiluminescence using the manufacturer's protocol (Amersham Corp.).

Microsequencing of a 95-kDa Tyrosine-Phosphorylated Auto- and Iso-Antigen

After identification of a 95-kDa phosphotyrosine containing auto- and iso-antigen on 2D immunoblots, a Coomassie blue-stained protein spot was cored from a 1.5-mm-thick 2D SDS-PAGE gel. The gel core was diced into small fragments. The protein in these fragments was destained in methanol, reduced in 10 mM dithiothreitol, and alkylated in 50 mM iodoacetamide in 0.1 M ammonium bicarbonate. After the reagents were removed, the gel pieces were incubated with 12.5 ng/µl trypsin in 50 mM ammonium bicarbonate overnight at 37°C. Peptides were extracted from the gel pieces in 50% acetonitrile in 5% formic acid and evaporated to 25 µl for microsequencing by tandem mass spectrometry (W.M. Keck Biomedical Mass Spectrometry Laboratory at the University of Virginia) using a Finnigan-MAT TSQ7000 system equipped with an electrospray ion source interfaced to a 8-cm x 75-µm internal diameter POROS 10 R2 reverse-phase capillary column. Approximately 50 fmol (4% of the digest) was injected for each experiment, and peptides were eluted with an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.6 µl/min. Molecular weights of the peptides were determined by capillary liquid chromatography-electrospray mass spectrometry, and then peptide sequences were determined by collision-activated dissociation (CAD) using capillary liquid chromatography-electrospray tandem mass spectometry with argon as the collision gas. The peptides were interpreted manually. The data were analyzed by searching nonredundant and expressed sequence tag (EST) databases using the Sequest algorithm (available through GenBank).

Cloning, Sequencing, and Analysis of the cDNA

A completely degenerate deoxyinosine-containing sense primer (5'-A/T-C/G-I GTI TT-C/T TT-C/T AA-C/T TT-C/T A/T/C-TI A/C-GI-3') was designed from peptide number 6 (SVFFNFI/LR) obtained by mass spectrometry, and the oligonucleotide was synthesized by GIBCO BRL (Life Technologies, CA). Using this forward primer and an adapter primer, a 3' rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR) was performed using 0.25 ng of human testicular Marathon ready cDNA (Clontech, Palo Alto, CA) in a 25-µl assay system for 40 cycles. Thermal cycling was done in an MJ Research (Watertown, MA) thermal cycler (PTC-200 DNA engine) using a program of one cycle at 94°C for 1.5 min and 40 cycles of 94°C for 30 sec, 46°C for 1 min and 68°C for 2 min. PCR products were separated on a 1.7% NuSieve (FMC, Rockland, ME) agarose gel. A 1.0-kilobase (kb) DNA fragment was isolated, reamplified, cloned into the pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA), and sequenced on a Perkin-Elmer Applied Biosystems DNA sequencer using Big Dye terminator chemistry with Taq DNA polymerase. The 3' clone contained a 786-base pair (bp) open reading frame (ORF) and a 218-bp untranslated region. The 5' end of the cDNA was also amplified by 5' RACE PCR from the same template using an adapter primer and an antisense 3' gene specific primer (5'-AGC CTG GGG GGA GAA GAC GCC AAC GGT C-3'), which was 118 bp downstream from the 5' end of the 1.0-kb 3' clone. A product of 2081 bp was obtained and cloned into the pCR 2.1-TOPO vector. The 5' clone revealed a 162-bp untranslated region and an ORF of 1919 bp. Complementary DNA clones were sequenced in both directions using vector-derived and insert specific primers. The nucleotide and amino acid sequence data were analyzed using the Genetics Computer Group (Madison, WI) program package.

Northern and Dot Blot Analysis

A Northern blot containing 2 µg of poly(A)⁺ RNA from eight selected human tissues and a normalized RNA dot blot containing 89–514 ng of mRNA from 50 different human tissues were obtained from Clontech. The Northern blot was probed with a ³²P-labeled 2337-bp DNA fragment generated from the 5' end of the coding region and a 1007-bp DNA fragment from the 3' end of the cDNA. Probes were prepared by random oligonucleotide primer labeling. Hybridization was performed in ExpressHyb solution (Clontech) at 68°C for 1 h and was followed by three washes in double-strength SSC (single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), 0.05% SDS at room temperature, and two washes in 0.1-strength SSC, 0.1% SDS for 20 min at 50°C. The blot was exposed to x-ray film (Kodak Blue XB-1; Eastman-Kodak, Rochester, NY) at -70°C for 60 h with two intensifying screens. A control Northern analysis was also performed on the same blot using human ß-actin as a probe.

The dot blot was probed with a ³²P-labeled 2337-bp 5' cDNA. The blot was hybridized in ExpressHyb solution containing salmon sperm DNA and human placental Cot-1 DNA overnight at 65°C. The blot was then washed three times in double-strength SSC, 1% SDS at 65°C and twice in 0.1-strength SSC, 0.5% SDS at 55°C. Autoradiography was performed by exposing the filter to x-ray film for 18 h at -70°C with two intensifying screens. The distribution of mRNAs from 50 human tissues in the dot blot were as follows: A1, whole brain; A2, amygdala; A3, caudate nucleus; A4, cerebellum; A5, cerebral cortex; A6, frontal lobe; A7, hippocampus; A8, medulla oblongata; B1, occipital lobe; B2, putamen; B3, substantia nigra; B4, temporal lobe; B5, thalamus; B6, subthalamic nucleus; B7, spinal cord; C1, heart; C2, aorta; C3, skeletal muscle; C4, colon; C5, bladder; C6, uterus; C7, prostate; C8, stomach; D1, testis; D2, ovary; D3, pancreas; D4, pituitary gland; D5, adrenal gland; D6, thyroid gland; D7, salivary gland; D8, mammary gland; E1, kidney; E2, liver; E3, small intestine; E4, spleen; E5, thymus; E6, peripheral leukocyte; E7, lymph node; E8, bone marrow; F1, appendix; F2, lung; F3, trachea; F4, placenta; G1, fetal brain; G2, fetal heart; G3, fetal kidney; G4, fetal liver; G5, fetal spleen; G6, fetal thymus; G7, fetal lung.

Expression of FSP95 in Escherichia coli and Purification of the Recombinant Protein

To express the molecule, 91% of the FSP95 cDNA (ORF) encoding amino acids (aa) 1–779 was amplified by PCR from human testicular Marathon ready cDNA (Clontech). Primers were designed to create an NdeI site at the 5' end and an XhoI site at the 3' end of the PCR product. The amplified DNA was ligated into the NdeI-XhoI sites of the pET28b expression vector (Novagen, Madison, WI). The resulting construct appended 28 amino acids from the vector including six residues of histidine tag on either side of the protein. The FSP95 coding sequence was preceded by the promotor for phage T7 RNA polymerase, an initiator ATG, and six consecutive His codons. The recombinant vector was introduced into the E. coli strain NovaBlue(DE3) cells, which contain a chromosomal copy of T7 RNA polymerase under the control of the lac promotor. The expression plasmid was sequenced at the 5' and 3' ends to verify the reading frame of the construct.

A single positive colony was used to inoculate 10 liters of Luria-Bertani broth with 30 µg/ml kanamycin in New Brunswick Scientific fermenter (Edison, NJ), and the culture was grown at 37°C until the A₆₀₀ was 0.6. Then recombinant protein expression was induced by addition of 1.0 mM isopropyl-1-thio-ß-D-galactopyranoside (IPTG), and growth was continued for another 3.0 h. The cells were pelleted, resuspended in single-strength binding buffer (20 mM Tris-HCl pH 7.9, 0.5 M NaCl, 5 mM imidazole) containing 0.1% NP-40 (Sigma) and 0.1 mg/ml lysozyme on ice for 30 min, and sonicated briefly. Upon centrifugation at 15 000 x g for 15 min, the resulting insoluble pellet was dissolved in 6 M urea in single-strength binding buffer for 1 h on ice. The urea-dissolved supernatant obtained at 15 000 x g for 15 min was loaded onto an Ni²⁺ activated His-binding resin column (Novagen) following the manufacturer's protocol, and the recombinant protein was eluted with 300 mM imidazole in single-strength binding buffer containing 6 M urea. The affinity-purified recombinant protein was further purified by preparative SDS-PAGE to remove some lower-molecular-weight breakdown products of the full-length form. The affinity-purified rFSP95 was dissolved in the sample loading buffer, after urea and imidazole were removed, and separated by preparative electrophoresis on 1.5-mm-thick curtain gels. The 95-kDa band was visualized by incubation in cold 0.1 M KCl and excised. The protein was then isolated in Centricon 10 concentrators using micro-electroelutor (Amicon, Bedford, MA) at 170 volts for 4.5 h.

Production of Antisera to Recombinant FSP95 (rFSP95) and Immunoblot Analysis of Specificity

Female Lewis rats were immunized with 200 µg of gel-purified recombinant protein in Freund's Complete adjuvant, boosted twice at intervals of 2 wk with 200 µg of protein in incomplete Freund's adjuvant, and bled 1 wk after each boost. Specificity of the rat antisera for FSP95 was tested by Western blotting against rFSP95 as well as human sperm protein extracts.

For immunoblotting, 3.5 µg of the recombinant protein was subjected to 10% SDS-PAGE, and the protein was electrophoretically transferred to nitrocellulose membrane. The membrane was cut into strips (each containing 150 ng), blocked with 5% dry fat-free milk in PBS-Tween (10 mM PBS pH 7.4, 0.05% Tween 20) for 1 h, and incubated with rat antisera at a 1:5000 dilution in the blocking buffer for 1 h. Immunodetection was performed with horseradish peroxidase-conjugated goat anti-rat IgG (Jackson ImmunoResearch) at a 1:5000 dilution in blocking buffer and visualized with a chromogenic substrate, diaminobenzidine (Sigma), in H₂O₂. For immunoblotting of sperm proteins, Percoll-washed sperm were solubilized in lysis buffer containing urea and OBG (see sperm preparation) and subjected to 2D SDS-PAGE analysis. After electrophoretic transfer of the proteins to nitrocellulose, the membrane was blocked and probed with the primary and secondary antibodies as above.

Micro Sperm Immobilization Assay

Fresh ejaculates were diluted 1:2 with BWW medium and centrifuged at 450 x g for 5 min. The resulting pellet was washed twice in BWW containing HSA (10 mg/ml). The pellet was layered with BWW containing HSA (30 mg/ml) for 1 h at 37°C in 5% CO₂. An aliquot of swim-up cells, containing about 98% motile sperm, was diluted in the swim-up medium to 20 million/ml as a working stock. For testing in the immobilization assay, rat antisera against the rFSP95 were diluted 1:1 with swim-up medium and decomplemented at 56°C for 30 min before use. Guinea pig serum, which was used as the source of complement, was absorbed twice with sperm for 30 min at 4°C. The micro sperm immobilization assay [30] was performed after modification by incubating 10 µl of 1:1 diluted and decomplemented antiserum, 1 µl of the 20 million/ml swim-up sperm, and 2 µl of the 1:1 diluted guinea-pig complement (containing 10–15 CH50 U/reaction) in a micro PCR tube for 60 min at 37°C. The percentage of motile sperm was determined microscopically in a counting chamber (Humagen Fertility Diagnostics, Charlottesville, VA) at x200. Heat-inactivated complement in each test served as a control. The experiment was performed along with CD59 (BioSource International, Camarillo, CA) and SAGA-1 monoclonal antibodies (which are known to recognize surface antigens) as positive controls [31, 32] and with a monoclonal antibody (MHS-10) that recognizes an acrosomal sperm antigen, SP10, as a negative control [33]. The sperm immobilization value (SIV) was calculated by dividing the percentage of motile sperm in the control (inactivated complement) by that in the test sera with active complement. When the SIV is 2 or more, the test serum is judged as positive for sperm-immobilizing antibodies.

Immunofluorescence Localization of FSP95 in Live or Permeabilized Sperm

Swim-up spermatozoa were prepared by layering 0.5 ml of semen below 2 ml of BWW medium (Irvine Scientific) containing 3 mg/ml HSA for 1.5 h at 37°C in 5% CO₂. The swim-up cells were then washed and allowed to capacitate in BWW medium containing 30 mg/ml HSA at 37°C in 5% CO₂ for 6 h. The motile capacitated sperm were air-dried onto poly-L-lysine-coated slides (Polysciences, Warrington, PA), permeabilized in methanol at -20°C for 30 min, air-dried, and blocked in 10% normal goat serum in PBS-Tween (10 mM PBS with 0.01% Tween 20) for 30 min. The sperm were then incubated with a 1:100 dilution of the primary antibody (rat anti-rFSP95) in the blocking buffer for 2 h at 37°C and then incubated with the fluorescence (Cyanine, Cy3)-conjugated anti-rat (IgG) secondary antibody (Zymed Labs., South San Francisco, CA) at a 1:100 dilution in the blocking buffer for 1 h at 37°C. The slides were washed in PBS, coated with slow fade (Molecular Probes, Eugene, OR), and mounted with coverslips. Images were obtained with in an Axioplan fluorescence microscope (Zeiss, Oberkochen, Germany) at x1000. For indirect immunofluorescence of live sperm, capacitated cells were incubated in capacitation medium containing a 1:100 dilution of primary antibody for 2 h at 37°C in 5% CO₂. Sperm were then washed twice at 600 x g for 5 min in capacitation medium lacking HSA and air-dried onto poly-L-lysine-coated slides. The sperm preparations were then incubated with blocking buffer for 30 min and treated with secondary antibody as described above.

Electron Microscopic Immunolocalization of FSP95

Pooled sperm were washed twice in Ham's F-10 (GIBCO BRL) containing 3% sucrose (wash buffer), and were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde in wash buffer for 15 min at 22°C. After the fixative was removed by three changes of wash buffer, cells were dehydrated through a series of graded ethanols from 40% to 100% and embedded in Lowicryl K4M (Electron Microscopy Sciences, Fort Washington, PA). The blocks were polymerized with UV light for 72 h at -20°C, and ultrathin sections (100-nm thick) were made.

Immunostaining of thin sections was modified from Berryman and Rodewald [34]. The sections were blocked in undiluted normal goat serum for 15 min at 22°C and then incubated for 16 h at 4°C with either rat anti-rFSP95 or the preimmune sera from the same animal at a 1:50 dilution in wash buffer containing 1% normal goat serum, 1% BSA, and 0.1% Tween 20. After being rinsed in wash buffer four times, the sections were incubated with a 1:100 dilution of 5 nm gold-conjugated goat anti-rat IgG (Goldmark Biologicals, Phillipsburg, NJ) for 1.5 h at 22°C. The specimens were washed in distilled water and stained with uranyl acetate before examination with a JEOL (Peabody, MA) 100CX electron microscope.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
Identification of a High-Molecular Weight Major Acidic Tyrosine-Phosphorylated Protein in Capacitated Human Spermatozoa

In order to identify human sperm proteins that undergo tyrosine phosphorylation during in vitro capacitation, actively motile sperm obtained after swim-up were incubated for 6 h under conditions known to allow capacitation to proceed [1]. Extracts containing sperm proteins were separated by 2D SDS-PAGE and visualized by silver staining (Fig. 1A). Tyrosine-phosphorylated proteins were localized on 2D immunoblots by reaction with the anti-phosphotyrosine monoclonal antibody RC20. Fresh, uncapacitated sperm demonstrated a subset of proteins with a low level of phosphorylation on tyrosine residues (Fig. 1B) compared with sperm that were capacitated for 6 h (Fig. 1C), and showed increased immunoreactivity of several protein groups ranging in size from 95 to 55 kDa and pI from 4.0 to 5.7. A prominent 95-kDa protein with a pI of 5.3 was seen to be one of the major acidic high-molecular weight tyrosine-phosphorylated proteins of human sperm (Fig. 1, A and C). The reaction of anti-phosphotyrosine antibody was considered to be specific because preincubation of the RC20 monoclonal antibody with 20 mM phosphotyrosine abolished the immunoreactivity (data not shown). Furthermore, tyrosine phosphorylation was almost completely abolished when capacitated cells were incubated with a tyrosine kinase inhibitor, genistein (Fig. 1D).

View larger version (112K): [in this window] [in a new window]   FIG. 1. Identification of FSP95 as a high-molecular weight major acidic tyrosine-phosphorylated protein in capacitated human spermatozoa by 2D immunoblot. Fresh swim-up sperm were capacitated in the absence and presence of genistein, a protein tyrosine kinase inhibitor. The location of the FSP95 spot in the 2D sperm proteome is indicated with a white half-rectangular box (A) in a silver-stained gel. The high-molecular weight acidic proteins (box area of A) that showed phosphorylation of tyrosine residues are shown in blots probed with anti-phosphotyrosine antibody before (B) and after capacitation (C). Prominent high-molecular weight acidic tyrosine-phosphorylated proteins (95 kDa, pI 5.1–5.5) showed increased phosphorylation with capacitation (C). A lack of phosphorylation of the 95-kDa proteins was observed in capacitated cells in the presence of genistein (D). The spot cored for microsequencing FSP95 is indicated with a white circle along with an arrow (A). IEF, isoelectic focusing. Molecular weight (x 10-3) markers are on the left

FSP95 as an Auto- and Iso-Antigen

Immunoblotting of human sperm proteins with sera from infertile male (Fig. 2A) and female (Fig. 2B) subjects previously screened for antisperm antibodies (ASA) revealed strong immunoreactivity to a group of sperm proteins with a molecular size of 95 kDa. Six serum samples out of 15 from infertile males (40%) and 2 of 6 from infertile females bound the 95-kDa protein group. Among 5 fertile subjects of each sex, 2 male and 1 female sera showed only weak immunoreactivity to the 95-kDa proteins (Fig. 2, C and D). These findings demonstrated that the 95-kDa proteins were isoantigenic in some women and autoantigenic in some men.

View larger version (70K): [in this window] [in a new window]   FIG. 2. Antigenicity of FSP95 in men and women with antisperm antibodies. Human sperm proteins were separated by 2D SDS-PAGE, transferred to nitrocellulose membranes, and probed with sera from an infertile male (A) and from an infertile female (B) previously screened for antisperm antibodies by the immunobead binding test. The immunoreactive proteins were compared to Western blots probed with sera from clinically fertile male (C) and female (D) subjects. Note the strong immunoreactivity exhibited by proteins at 95 kDa, including FSP95 (arrow, the protein spot selected for microsequencing), with sera from infertile subjects of both sexes compared to only faint immunoreactivity observed in the 95-kDa group with sera from fertile subjects. IEF, isoelectric focusing. Molecular weight (x 10-3) markers are on the left

Microsequencing of the 95-kDa Antigen by Mass Spectrometry

To obtain structural information on the identity of one of the 95-kDa tyrosine-phosphorylated antigens, microsequencing of a Coomassie blue-stained 2D SDS-PAGE protein spot was undertaken. The location of the spot cored from the 2D gel is indicated by a white circle in Figure 1A. Because of the low amount of the protein available in a well-resolved 2D gel, amino acid sequencing was performed by tandem mass spectrometry on peptides generated by overnight trypsin digestion at 37°C of the protein spot within pieces of the gel. The extracted peptides were concentrated and analyzed by capillary column liquid chromatography electrospray-tandem mass spectrometry. A total of 18 peptide sequences were obtained (Table 1). Database searches using both the molecular size information (mass mapping by MS-Fit, available through GenBank) and sequence information by using Sequest, allowing a 15% gel-derived protein mass tolerance and a 1-dalton peptide mass tolerance, did not identify any known protein at that point.

Cloning and Sequence Analysis of FSP95

To isolate the cDNA encoding the 95-kDa tyrosine-phosphorylated protein, a completely degenerate inosine containing forward primer designed from peptide number 6 (Table 1) was used to amplify a 1.0-kb piece of cDNA by 3' RACE PCR from human testicular Marathon ready cDNA (Clontech). The 5' cDNA including an untranslated region was also cloned by PCR, producing a 2.1-kb cDNA with a 118-bp overlap at the 3' end. The nucleotide sequence of the full-length cDNA (Fig. 3) consisted of 2942 bp with an in-frame start codon at nucleotides 163–165 conforming to a Kozak [35] consensus for the translation initiation site. The translation start site was further authenticated by the presence of two in-frame stop codons at 45 bp and 72 bp upstream of the first ATG sequence. The cDNA contained a 2559-bp ORF with untranslated regions of 162 bp at the 5' end, 218 bp at the 3' end, and a polyadenylation signal (ATTAAA) [36] 11 bp upstream from the poly(A) tail. The ORF encoded a protein of 853 amino acids with a predicted molecular weight of 94.6 kDa and a pI of 6.0. All of the 18 tryptic peptides obtained by microsequencing the 95-kDa protein spot were recovered in the predicted amino acid sequence of the molecule (Fig. 3, underlines), validating that the protein originally identified and cored from the gel had been cloned.

View larger version (68K): [in this window] [in a new window]   FIG. 3. Nucleotide and deduced amino acid sequences of the human sperm protein FSP95. The deduced amino acid sequence of human sperm FSP95 is shown below the cDNA sequence. The numbers on the left refer to the nucleotide sequence; numbers on the right refer to the amino acid sequence. The consensus ATG of the ORF and the polyadenylation signal (ATTAAA) are indicated in bold letters. The termination codon (TAA) is marked with an asterisk. The 5' and 3' untranslated regions are 162 bp and 218 bp, respectively, and are shown in italics. The calculated molecular mass and pI of the predicted protein were 94.6 kDa and 6.0, respectively. The 18 underlined sequences indicate the tryptic peptides obtained by microsequencing. The putative tyrosine kinase phosphorylation site is indicated in bold within a box (residue number 435). The nucleic acid sequence was submitted to the GenBank (accession number AF087003).

Analysis of the predicted amino acid sequence of FSP95 revealed no N-terminal eukaryotic secretory signal peptide cleavage site [37]. Comparison of the protein sequence with the Prosite database [38] demonstrated the presence of five potential N-linked glycosylation sites (aa 87, 117, 180, 502, 763), fifteen potential casein kinase II phosphorylation sites (aa 21, 34, 52, 102, 109, 120, 223, 280, 440, 448, 549, 659, 691, 713, 816), eleven possible protein kinase C phosphorylation sites (aa 2, 89, 102, 217, 223, 236, 303, 367, 408, 484, 597), eight myristoylation sites (aa 68, 116, 346, 366, 648, 722, 724, 814), and one tyrosine kinase phosphorylation site at residue 435. Four potential O-linked glycosylation sites were also found at residue numbers 168, 504, 557, and 745 [39]. The deduced sequence of FSP95 revealed no apparent transmembrane regions.

Comparison of the deduced FSP95 sequence to the GenBank data base using BLAST [40] and FASTA [41] revealed that the human sperm FSP95 had closest amino acid similarity to a mouse sperm fibrous sheath AKAP, precursor of mouse AKAP82 (pro-mAKAP82; identity 33.6%, similarity 42.5%), and to a human sperm fibrous sheath AKAP, precursor of human AKAP82 (pro-hAKAP82; identity 32.4%, similarity 39.4%) [13, 42]. The amino acid sequence alignment of these proteins revealed that the middle and part of the N-terminal and C-terminal regions of these molecules contained conserved domains (Fig. 4). The 32–33% identities between FSP95 and these AKAPs suggest that the human sperm FSP95 is encoded by a previously unreported gene. Interestingly, the two potential intracellular anchoring domains of mouse sperm pro-mAKAP82 [42] were conserved in both the pro-hAKAP82 [13] and the human sperm FSP95 cDNAs. Both these intracellular targeting regions were located in the highly basic N-terminal region while the C-termini of these proteins were more acidic, containing residues with long aliphatic side chains. However, the RII-binding domains of human and mouse AKAP82 were not conserved in FSP95.

View larger version (121K): [in this window] [in a new window]   FIG. 4. Homology comparison of the deduced amino acid sequences of human sperm FSP95 with those of mouse and human sperm fibrous sheath AKAPs (mouse: pro-mAKAP82, accession #I48968; human: pro-hAKAP82, accession #AF072756). The sequences are listed in descending order of homology from the FSP95. The alignment was constructed by use of the GCG-PILEUP (Genetics Computer Group, Madison, WI) program and formatted with ALSCRIPT version 2.0 [67]. The shaded areas indicate the amino acid identities and similarities among the molecules (cut off 8 in ALSCRIPT). The conserved AKAP-like intracellular targeting domains are shown in boxes. The N-terminal RII-binding domain of the mouse and human AKAP82 is highlighted with an underline

Genomic Structure and Chromosomal Localization of FSP95

The complete genomic sequence of human sperm FSP95 was obtained from a BAC genomic clone (no. RPCI11-500M8) from GenBank (accession no. AC005832). The genomic organization of the FSP95 gene is presented in Figure 5. The FSP95 gene spans 29.6 kb. It contains five exons and four introns. The sizes of the individual exons and introns and the sequences immediately flanking the exon-intron junctions are presented in Table 2. All intron sequences at these junctions follow the gt-ag rule. The first two introns occur in the 5'-untranslated region of the mRNA. Introns 3 and 4 interrupt the coding region of the gene at codon 258 and 2568, respectively. The BAC genomic clone RPCI11-500M8 is mapped to the chromosomal locus 12p13.3, which is the distal-most band to the centromere on the short arm.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Schematic structure of human sperm FSP95 gene and mRNA. A) Genomic organization as obtained from genomic clone, RPCI11-500M8 (GenBank accession no. AC005832). The horizontal line denotes the locations and relative sizes of the introns (I-1 to I-4). Vertical solid lines and boxes denote the locations and relative sizes of the exons (E-1 to E-5). B) The FSP95 mRNA as deduced from the genomic sequence. The shaded part represents the coding region, also indicated by the initiation (ATG) and stop (TAA) codons

FSP95 mRNA Expression in Human Tissues

Northern blot (Fig. 6A) and dot blot (Fig. 6B) hybridization were used to analyze the expression of FSP95 mRNA in different human tissues. Northern blots were hybridized with either of two FSP95 probes, a 2337-bp 5' cDNA and a 1.0-kb 3' cDNA. Both probes hybridized to an mRNA of 3.0 kb present only in testis. A broader screening was performed using the 2337-bp 5' cDNA as a probe to hybridize a RNA dot blot containing poly(A)⁺ RNAs from 50 different human tissues. Among the tissues examined, a strong hybridization signal was observed only from testis mRNA, indicating a testis-specific expression pattern of human sperm FSP95 gene.

View larger version (42K): [in this window] [in a new window]   FIG. 6. Analysis of human sperm FSP95 expression. A) Northern blot (Clontech) containing 2 µg of poly(A)+ mRNA from human tissues per lane was hybridized with radiolabeled FSP95 cDNA and exposed for 60 h. The migration of RNA markers (kb) are indicated on the left. A single transcript of 3.0 kb was apparent only in testis. The blot was subsequently stripped and rehybridized with human ß-actin as a probe to assess the levels of RNA in each lane (lower panel). B) Dot blot containing poly(A)+ RNA from 50 human tissues (obtained from Clontech) was hybridized with a radiolabeled FSP95 cDNA, and signals were visualized by autoradiography. Hybridization signal was found only in testis (box-D1) after 18 h of exposure. The dot blot contained normalized amounts (89–514 ng) of poly(A)+ RNA from 50 tissues (for details, see Materials and Methods)

Expression of rFSP95 and Western Blot Analysis

The cDNA sequence encoding the FSP95 from residues 1 to 779 was cloned into the bacterial expression vector pET28b. When the bacteria were induced with IPTG for 1.5 or 3 h, rFSP95 with a molecular size of 97 kDa was produced (Fig. 7A). The recombinant protein was purified by using the high affinity of the six-His domain for Ni²⁺ ions immobilized on Sepharose [43]. The affinity-purified rFSP95 was further purified by preparative gel electrophoresis (Fig. 7B) and used as an immunogen to inoculate rats. The rat antibody to rFSP95 recognized both the rFSP95 and the "native" FSP95 present in sperm extracts, and stained the 95-kDa spot at a pI 5.3, which was originally microsequenced (Fig. 7, C and D). Preimmune rat sera did not react with either rFSP95 or with sperm proteins.

View larger version (42K): [in this window] [in a new window]   FIG. 7. Isolation of rFSP95 from E. coli and immunoblotting of recombinant and sperm FSP95. A portion of the human sperm FSP95 cDNA (encoding residues 1–779 of the protein) was expressed in E. coli using the pET28b plasmid, induced by addition of 1.0 mM IPTG, and purified by nickel ion affinity column chromatography followed by preparative PAGE. A) Bacterial extracts stained with Coomassie blue; lane 1, uninduced; lanes 2 and 3, 1.5 h and 3.0 h after induction; expressed FSP95 is indicated by the arrow. B) Coomassie blue-stained purified rFSP95 (2.3 µg), used for immunization). C) Immunoblot of gel-purified rFSP95 probed with rat antisera against rFSP95 at a 1:5000 dilution; lane 1, preimmune serum; lane 2, immune serum. D) 2D Blot of human sperm proteins probed with rat serum against gel-purified rFSP95. The 95-kDa region at pI 5.3 originally microsequenced immunoreacted with the antibody (arrow). The pH gradient is indicated at the top. Molecular weight (x 10-3) markers are on the left

Localization of FSP95 in Human Spermatozoa

The possibility that FSP95 is exposed on the plasmalemma of fresh noncapacitated human spermatozoa was evaluated first by a modified micro sperm immobilization assay [30]. Immobilization of fresh sperm was not observed in presence of rat antiserum against the rFSP95 and guinea pig complement (SIV = 0.91 ± 0.12; mean ± SD; n = 6). Furthermore, live capacitated sperm did not show immunofluorescent staining over any domain (data not shown). Together, these results indicate a lack of FSP95 at the cell surface.

Indirect immunofluorescence analysis of capacitated and methanol-permeabilized ejaculated human spermatozoa using rat serum against rFSP95 localized FSP95 to the entire length of the principal piece of the flagellum (Fig. 8B) of 100% sperm. The head, midpiece, and end piece remained unreactive (Fig. 8, A and B). Preimmune antisera showed no immunofluorescence (Fig. 8, C and D).

View larger version (90K): [in this window] [in a new window]   FIG. 8. Phase contrast (A, C) and indirect immunofluorescence staining (B, D) of FSP95 in capacitated permeabilized human spermatozoa. Immunofluorescence was noted throughout the principal piece (PP) of the flagellum (bar) with immune sera (B) while no fluorescence was observed in the head, midpiece (MP), or end piece (EP). No immunostaining was observed with preimmune sera (D) or by using immune sera on live capacitated unpermeabilized sperm (data not shown)

The intracellular distribution of FSP95 was examined at the ultrastructural level by post-embedding immunolabeled ultrathin sections of washed ejaculated human spermatozoa. Gold particles indicating the localization of FSP95 were associated with the entire thickness of the fibrous sheath in both longitudinal and cross sections. The gold particles were observed over the circumferential ribs whereas the central zone of the longitudinal columns remained unstained (Fig. 9, A and B, indicated by arrows). No label was associated with the outer dense fibers or the axoneme. Only a rare gold particle was observed in sections exposed to preimmune control rat sera (Fig. 9, C and D).

View larger version (180K): [in this window] [in a new window]   FIG. 9. Electron microscopic immunogold localization of FSP95 in a longitudinal (A) and a cross section (B) of the principal piece of ejaculated human spermatozoa. Gold particles (arrows) were detected in the ribs of fibrous sheath (FS) but not within the central portion of the longitudinal columns (LC). No immunoreactivity was detected on the outer dense fibers (ODF) or on the axoneme (AXO). Only a rare gold particle was detected on control sections (C and D) treated with preimmune rat serum

FSP95 as a Capacitation-Induced Tyrosine-Phosphorylated Protein

To provide further evidence that the protein which had been selected, microsequenced, and cloned did indeed undergo tyrosine phosphorylation during in vitro capacitation, the following experiment was performed. Protein extracts from both capacitated and noncapacitated sperm were resolved by high-resolution (23 x 23-cm) 2D SDS-PAGE, electroblotted, and analyzed with rat antisera against rFSP95 and anti-phosphotyrosine antibody RC20. Immunoreactive forms of FSP95 with a pI of 5.3, evident in extracts of noncapacitated sperm (Fig. 10A), became less abundant in capacitated sperm (Fig. 10B). Concomitantly, new immunoreactive forms of FSP95 with more acidic pIs appeared. This shift in charge towards acidic FSP95 isoforms was accompanied by increased tyrosine phosphorylation after capacitation (compare Fig. 10, C and D) and the appearance of more acidic FSP95 isoforms that were phosphorylated. This demonstration using antibody to rFSP95 provided formal proof that the FSP95 which was cloned and sequenced undergoes tyrosine phosphorylation during capacitation and has isoforms that become more acidic in the process.

View larger version (55K): [in this window] [in a new window]   FIG. 10. Tyrosine phosphorylation of FSP95 during in vitro capacitation of human spermatozoa. Proteins of uncapacitated sperm (A, C) were compared to 6-h capacitated sperm (B, D) after 2D SDS-PAGE, electroblotting, and probing with rat antisera against rFSP95 (A, B) and anti-phosphotyrosine monoclonal antibody (C, D). Immunoreactive forms of FSP95 with pI of 5.3 (A) were less abundant after capacitation (B), while more acidic forms of FSP95 were immunoreactive after capacitation (B, arrows). These acidic charge shifts in immunoreactive forms of FSP95 after capacitation were accompanied by increased tyrosine phosphorylation and the appearance of more acidic phosphotyrosine containing isoforms (C, D, arrows). Molecular weight (x 10-3) markers are on the left

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
In this report, we describe the cloning and characterization of a human sperm antigen, designated FSP95 in consideration of its fibrous sheath localization and calculated molecular weight of 95 kDa. In order to characterize FSP95 from a 2D SDS-PAGE protein spot-derived microsequence, the 3' cDNA of the molecule was amplified using a single gene-specific inosine containing primer and an adapter primer from human testicular Marathon cDNA. The obtained cDNA revealed the presence of 4 FSP95 microsequenced peptides embedded in its ORF. The 5' end of the cDNA was then similarly cloned by 5' RACE, revealing two in-frame stop codons upstream of the most 5' methionine identified. This indicates that the reported cDNA represents the complete coding sequence of FSP95. The translation start site also conforms to the essential Kozak [35] consensus sequence. Embedded within the deduced amino acid sequence are all 18 microsequenced peptides obtained from the FSP95 protein spot (Table 1; Fig. 3), validating that the protein spot originally cored was cloned. The success of this cloning strategy using a single microsequence-derived primer is important because of its efficiency in time and reagents. Therefore, this approach may be an important avenue in converting microsequence data into complete sequence information and highlights the increasing importance of proteome-based cloning.

From a methodological view, it is important to note that the peptide sequence used to initiate the cloning experiment (peptide 6, Table 1) was confirmed by combining interpretation of the CAD spectrum of the peptide in the digest, interpretation of the CAD spectrum of the N-terminal-derivatized peptide [44], and comparison of CAD spectra of the digest peptide with a synthetic peptide. Minor discrepancies were observed in comparing the amino acid sequences derived from the mass spectrometry data and the cDNA data for peptides 6, 12, and 18 because of changes in a single base. The differences produce an F-to-S substitution in peptide 6, either an E-to-V or a T-to-A substitution in peptide 12, and an E-to-G substitution in peptide 18. These substitutions could be due to an inter-individual DNA polymorphism between the donors from which FSP95 was microsequenced and the donor from whom the Clontech Marathon testicular cDNA was constructed.

Characterization of proteins whose patterns of tyrosine phosphorylation change with capacitation in human spermatozoa is an important step in dissecting the involvement of various signal transduction pathways in capacitation-related changes that are preparatory for mammalian fertilization. By using 1D gel electrophoresis, human spermatozoa were previously shown to undergo increased tyrosine phosphorylation of two major high-molecular weight proteins of 105 and 80 kDa during the process of capacitation [10, 12, 45]. In the present study, 2D Western analysis comparing capacitated, uncapacitated, and genistein-treated sperm revealed multiple high-molecular weight proteins that showed increased tyrosine phosphorylation during capacitation, including the antigen FSP95 (Fig. 2C). On the basis of its abundance on phosphotyrosine-containing blots, FSP95 emerges as one of the major acidic high-molecular weight tyrosine-phosphorylated proteins of capacitated human spermatozoa thus far described. Indeed, a potential tyrosine kinase phosphorylation site was identified in FSP95 at residue number 435. The change in pI of FSP95 toward acidic isoforms during in vitro capacitation as examined with antibodies to phosphotyrosine residues and to recombinant FSP95 (Fig. 10) provides formal proof that FSP95 is a tyrosine kinase substrate. The anti-phosphotyrosine immunostain at pI 5.3 after capacitation (Fig. 10D) is due to another major capacitation-induced tyrosine-phosphorylated fibrous sheath protein, pro-hAKAP82, as determined by microsequencing (data not shown).

The inhibition of tyrosine phosphorylation in capacitated sperm by genistein (Fig. 2D) suggests that a tyrosine kinase is active and present in human sperm. The presence of a tyrosine kinase has previously been demonstrated in ejaculated human spermatozoa and in the midpiece tail region of boar spermatozoa using antibodies against tyrosine kinase purified from boar male germ cells [46]. The decrease in protein phosphorylation of tyrosine residues observed in capacitated sperm in the presence of genistein below that of the uncapacitated cells (Fig. 1) is probably due to the presence of phosphatases, perhaps activity of the calmodulin-dependent protein phosphatase calcineurin [47]. Such an interpretation is supported by the observation that Ca²⁺-induced dephosphorylation is calmodulin-calcineurin-dependent [10]. As tyrosine phosphorylation of mammalian spermatozoa is associated with sperm hyperactivation [7], the capacitation-induced tyrosine phosphorylation of FSP95 could play a role in sperm motility. Furthermore, availability of a major tyrosine kinase substrate may provide a means of characterizing the participating tyrosine kinase(s) involved in these sperm functions.

Although two sequences identical (99.5%) to FSP95 are now available in the database (Genbank accession nos. AF093408 and U85715), comparison of the deduced amino acid sequence of the FSP95 cDNA with other known sequences reveals the highest homology with mouse and human sperm pro-AKAP82 (34% and 32% amino acid identity, respectively), which are protein kinase A anchor proteins that sequester PKA to subcellular locations. This suggests that FSP95 is a protein with structural homology to sperm AKAPs. Alignment analysis of FSP95 with these AKAPs reveals that FSP95 possesses two potential intracellular targeting domains (Fig. 4). Both these domains lie within an N-terminal basic region similar to the AKAPs 75 and 79 [48, 49]. Interestingly, however, the predicted RII-binding domain of these sperm AKAPs was found not to be conserved in FSP95 as judged by low similarity and lack of the putative amphipathic helix binding motif in this region [50]. In a recent study, the presence of an AKAP of approximately 110 kDa and of the regulatory subunits of PKA (RII, RIIß, and RIß) have been demonstrated in human, bovine, and monkey spermatozoa [51]. Moreover, it has been suggested that the anchoring of the regulatory subunit of PKA to bovine sperm AKAP, independent of PKA catalytic activity, is essential for the regulation of sperm motility [51]. Therefore, considering the unique testis-specific expression pattern of FSP95 (Fig. 6), and its similarity to sperm AKAPs, FSP95 could be explored for the possible development of a cell-permeable anchoring inhibitor peptide for the formulation of a topical spermiostatic agent for humans. Furthermore, the identification of FSP95 as a tyrosine kinase substrate and its similarity to sperm AKAP82 may suggest a possible interrelationship between PKA and tyrosine kinase signaling pathways, because tyrosine phosphorylation and capacitation in other mammals has been shown to be up-regulated by a cAMP/PKA-dependent pathway [7, 9, 52].

Since it is recognized by sera with antisperm antibodies from both men and women, FSP95 is both iso- and auto-antigenic. This finding is in concert with the remarkable testis specificity observed for expression of the FSP95 transcript. Many sperm proteins are antigenic in nature because they are tissue-specific and do not appear until puberty, when meiosis is initiated and sperm-specific genes begin to be transcribed [53]. During the induction of self-tolerance during the neonatal period, such neo-antigens are probably not recognized by the immune system [53], while after meiosis the later stages of male germ cells are sequestered from the immune system by the blood-testis barrier and the blood-epididymis barrier [54, 55]. Although several human sperm-specific auto- and iso-antigens are recognized by infertile patient sera (e.g., SAGA-1, SPAG2, SP17, FA1) [32, 56–58], only a very few have been found on the spermatozoal surface (e.g., SAGA-1) [32]—a critical criterion for the selection of a potential contraceptive immunogen [59]. Evaluation of FSP95 localization using sperm immobilization assay, immunofluorescence on live cells, and immunoelectron microscopic localization, along with lack of a putative eukaryotic secretory signal sequence and no apparent transmembrane domains in the FSP95 deduced amino acid sequence, all suggest that the molecule is not present on the sperm plasma membrane. Thus FSP95 is probably not involved in antibody-mediated infertility and consequently is not a contraceptive vaccinogen candidate.

Instead, immunocytochemistry using antisera against rFSP95 localized FSP95 to the cytoplasm of the principal piece of the tail. Immunoelectron microscopy identified the antigen in association with the ribs of the fibrous sheath (Fig. 9), which are believed to be involved in defining the shape of the flagellar beat [60]. In demembranated mouse spermatozoa, it has been demonstrated that sliding of the fibrous sheath towards the head is accompanied by extrusion of microtubules towards the distal end [61]. The sliding of the fibrous sheath was cAMP-dependent, and the sliding velocity depended on the ATP concentration. Although several proteins have been identified on gels of isolated human sperm fibrous sheath (molecular sizes: 97, 76, 62, 55, 33, 28, 25 kDa) [62], to our knowledge only the human sperm fibrous sheath protein, hAKAP82, other than FSP95, has been characterized at the molecular level [13]. The recently reported human testis-specific gene termed "hi" encodes a predicted protein with 92% identity to the human sperm fibrous sheath protein pro-hAKAP82 and 27% identity with FSP95, but its localization in spermatozoa has not been demonstrated [63]. Although a similar-size protein of M_r 95 000 was identified in the fibrous sheath of human spermatozoa by monoclonal antibody 4F7 [64], it is not clear at present if FSP95 is identical. The cognate 95-kDa antigen was reported not to be present in bull and mouse spermatozoa, whereas FSP95 homologues are now known to be present in bull (accession #AF093407; identity: 80%) and mouse (accession #AF093406; identity: 78%) (see Note Added in Proof below). Further, the cognate 95-kDa antigen of Escalier et al. [64] was reported to be distributed on both the ribs and columns of the fibrous sheath, whereas FSP95 was localized to only the ribs of the fibrous sheath in the present study.

Anomalies in fibrous sheath structure, particularly disorganization of components and asymmetrical location of the longitudinal columns, have been shown to be associated with severe sperm immobility, related to sterility and flagella dyskinesia, respectively [65, 66]. Although the molecular basis of these defects is presently unknown, mutations of the genes encoding fibrous sheath proteins may be related to the disorganization of fibrous sheath structure, leading to flagellar dyskinesia. Further studies on disruption of the FSP95 gene would clarify its role in fibrous sheath organization as well as aid in evaluating the importance of tyrosine phosphorylation of FSP95 in hyperactivation and capacitation.

	NOTE ADDED IN PROOF

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
The human FSP95 sequence reported in this paper was deposited in GenBank on August 25, 1998, as accession AF087003. This sequence became available to the public on September 16, 1998. At the time of our deposit, our GenBank search revealed no homologous sequences to FSP95 other than the AKAP 82 proteins. On March 23, 1999, a month after this paper was submitted, a nearly identical sequence to FSP95 was released by GenBank. This sequence had been deposited by A. Lefevre and collegues on January 17, 1997, as "SOB1, a human gene coding for a specific sperm protein likely involved in oocyte binding." A paper by A. Lefevre et al. entitled "Cloning and Characterization of SOB1, a New Testis-Specific cDNA Encoding a Human Sperm Protein Probably Involved in Oocyte Recognition" appeared in the May 1999 issue of BBRC (BBRC 1999; 259:60–66). The FSP95 and SOB1 sequences are 99% identical, and our work agrees with a human chromosomal locus of 12p13.3. for this gene. However, as we demonstrate in this paper, the immunolocalization of FSP95 in human sperm to the principal piece of the tail, specifically to the ribs of the fibrous sheath, coupled to a lack of cell surface immunofluorescence, a lack of cytotoxicity of antibodies to FSP95 in the sperm immobilization test, and no cell surface immunolocalization at the electron microscopic level mitigates against a "direct" role for FSP95 at the sperm surface as a receptor for zona pellucida or oolemmal proteins. Given its role as a structural component of the fibrous sheath and its function as a protein kinase A anchoring protein, FSP95/SOB1 may have only an "indirect" role in oocyte binding. The term "sperm-oocyte binding protein-1" may not accurately depict the true function of this new protein.

On Sept. 21, 1998, Vijayaraghavan et al. deposited in GenBank a nearly identical human sequence to FSP95, accession AF093408, as well as homologous bovine (accession AF093407) and mouse (accession AF093406) sequences. These latter three deposits were named AKAP 110, and a report by Vijayaraghavan et al. on AKAP 110 appeared in Mol Endocrinol 1999; 13:705–717, identifying a protein kinase A RII binding domain to amino acids 124 through 141 of FSP95/SOB1/AKAP 110. It should be noted that there is a discrepancy between the apparent mass for the human FSP95 protein reported in our paper and the mass of the human protein reported as AKAP 110. The open reading frame for human FSP95 predicts a protein of 94.6 kDa. This is in close accord with our calculations of apparent mass from 2D SDS-gels. We suggest AKAP 95 may be a more accurate name for the human protein. One additional point deserves comment: whereas the present paper localized human FSP95 specifically to the principal piece in human sperm, the Vijayaraghavan report found intense AKAP 110 staining on the acrosomal cap and less intense staining of the flagellum of bovine sperm. These potential species differences in the localization of this new protein deserve further exploration.

	FOOTNOTES

 
¹ This work was supported in part by grants from Fogarty International Center D43 TW/HD 00654, NIH HD U54 29099, P30 28934, the Andrew W. Mellon Foundation, and Schering AG.

² Correspondence: John C. Herr, Department of Cell Biology, University of Virginia Health Sciences Center, Box 439, Charlottesville, VA 22908. FAX: 804 982 3912; jch7k{at}virginia.edu

Accepted: June 17, 1999.

Received: February 22, 1999.

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