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Gamete Biology |
Center for Research in Contraceptive and Reproductive Health, School of Medicine, University of Virginia, Charlottesville, Virginia 22908
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, fertilization, gametogenesis, sperm maturation, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Acrosomal biogenesis is divided into four distinct steps, Golgi, cap, elongation (acrosome), and maturation phases, based initially on light microscopic analysis of periodic acid-Schiff (PAS)-stained testicular sections of several species, including humans [4], and subsequently on ultrastructural observations (see [5] and references therein). The initial Golgi phase is marked by synthesis, translocation, and clustering of Golgi-derived PAS-positive proacrosomal granules into a single acrosomal granule that attaches to the nuclear envelope or perinuclear theca. During the cap phase, fusion of additional Golgi-derived vesicles occurs around the granule, resulting in the formation of the acrosomal vesicle, which becomes segregated into discrete electron-dense and electron-lucent areas. This initially rounded vesicle flattens and spreads over the nucleus as the cap phase progresses while the Golgi apparatus migrates to the opposite pole of the cell. As the spermatid begins to elongate during the acrosomal phase, manchette microtubules start to grow from the nuclear ring, the acrosomal contents condense into a uniformly electron-dense matrix, and the acrosomal cap undergoes further elongation. The acrosome reaches its definitive shape covering the anterior two thirds of the nucleus during the maturation phase, and most of the spermatid cytoplasm and organelles are discarded in the cytoplasmic droplet and residual body.
The equatorial segment of the acrosome is of considerable functional importance to fertilization because it 1) remains intact following the acrosomal reaction, 2) underlies the domain of the plasma membrane involved in fusion with the egg membrane, and 3) is the site where breakdown of the sperm nuclear envelope is initiated after fertilization [6]. In developing a comprehensive human sperm proteome [7], two-dimensional Western blots were employed to assess the concanavalin A (Con-A)-binding abilities of human sperm proteins. Here, we describe the coring and microsequencing of two protein spots that bound Con-A, leading to the cloning and characterization of a novel protein, equatorial segment protein (ESP; GenBank accession AF275321), that is localized to the equatorial segment of the human sperm early in development and defines a distinct equatorial segment subcompartment in the acrosomal vesicles of Golgi, cap, elongation, and maturation phase spermatids. Although other researchers have described acrosomal biogenesis in various species [8–13], the specification of a discrete equatorial segment domain segregating and persisting in the acrosomal vesicle during subsequent phases of acrosomal biogenesis has not been reported. We also propose a modified model for acrosomal morphogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human sperm proteins for two-dimensional (2-D) gel electrophoresis were solubilized and separated on 2-D polyacrylamide gels as previously described by Naaby-Hansen et al. [7]. Standard one-dimensional SDS-PAGE [14] for recombinant proteins and prokaryotic cell lysates was performed on a 16- x 18-cm gel electrophoresis apparatus (Bio-Rad, Hercules, CA) with 0.75-mm spacers and 12% polyacrylamide separating gels. Protein or cellular samples were either suspended in standard Laemmli buffer [15] and boiled for 10 min before addition to the gel or were treated with iodoacetic acid using the procedure of Crestfield et al. [16]. Escherichia coli lysates consisted of 1 OD-ml of bacterial culture, which was pelleted before extraction in Laemmli loading buffer.
Electrophoretic transfer of separated proteins to nitrocellulose membranes was performed in transblot buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine, and 20% methanol) for 1 amp-h. The membranes were either utilized intact for 2-D gels or cut into strips for standard protein gels, blocked with 5% fat-free dry milk in PBS-Tween 20 (1x PBS, pH 7.4, 0.05% Tween 20), and then incubated in rat antiserum to ESP for 1 h at 22°C. Immunodetection was performed with horseradish peroxidase-conjugated goat anti-rat IgG (Jackson ImmunoResearch, West Grove, PA) at a 1:5000 dilution in blocking buffer and visualized with diaminobenzidine (Sigma, St. Louis, MO) in H2O2 or with TMB (Kierkegard and Perry Laboratories, Gaithersburg, MD). Western blots of 2-D gel-separated human proteins were also screened with a 1:2000 dilution of serum from infertile patients containing anti-sperm antibodies [17] as determined by the IBT test [18] and with a 1:2000 dilution of fertile control serm. Bound anti-sperm antibodies were detected by incubation of thrice-washed blots for 1 h at 22°C with secondary enzyme-conjugated goat 
-human IgG + IgM (H+L) F(ab')2 antibodies (Jackson) diluted 1:5000 in PBS-Tween 20. Horseradish peroxidase conjugates were visualized by enhanced chemiluminescence (ECL) using the manufacturer's protocol (Amersham, Buckinghamshire, U.K.).
Detection of Con-A-binding sperm proteins was performed by washing Western blots of 2-D gels of human sperm proteins first with Tris-buffered saline (TBS; 10 mM Tris-HCl, pH 8.0, 150 mM NaCl) then blocking with TBS containing 1% gelatin (TBSG) for 1 h [19]. After rinsing in TBS, the membrane was incubated with peroxidase-conjugated Con-A (5 µg/ml; Sigma) in TBSG containing 1 mM MnCl2 and 1 mM CaCl2 for 1 h at 22°C before rinsing briefly twice with TBSG. After washing the membrane four times for 15 min each with TBSG + cations, the reaction product was developed with 0.06% diaminobenzidine in 0.1% H2O2. Sequencing by liquid chromatography-mass spectrometry was performed by the W.M. Keck Foundation Center for Biomedical Mass Spectrometry (University of Virginia, Charlottesville, VA) on an LC-Q apparatus (Finnigan, Austin, TX) according to the manufacturer's instructions and as described previously [20].
Reverse Transcription Polymerase Chain Reaction
Oligonucleotides designed from the expressed sequence tag (EST) and cDNA library clones were manufactured by GibcoBRL (Gaithersburg, MD). Oligonucleotides (EST forward primer: 5'-CTTGCTCTAGCAGCAGCAGAAC-3'; EST reverse primer: 5'-TCATAACACATGACACATAAAGATGTTGGC-3') corresponding to the EST (GenBank accession AA913806) were utilized to generate a 430-base pair (bp) probe by reverse transcription of 1 µg poly(A)+ human testicular RNA (Clontech, Palo Alto, CA) in a 40-µl reaction as previously described [21] utilizing avian myeloblastosis virus reverse transcriptase. After the addition of 60 µl of water treated with diethyl pyrocarbonate, 1-µl aliquots of the cDNA solution were amplified for 40 cycles with denaturation at 94°C, annealing at 60°C for 30 sec, and polymerization at 68°C for 3 min as specified by the manufacturer of the recombinant Thermophilus thermophilus (rTth) polymerase (Perkin Elmer Life Sciences, Boston, MA) on a Dual Block PCR Engine (MJ Research, Watertown, MA). Separation and isolation of the PCR products were achieved by electrophoresis of reaction aliquots in 1.5% agarose gels made to 1x concentration in TAE (40 mM Tris-acetate, 1 mM EDTA) buffer followed by ethidium bromide staining, ultraviolet visualization, and photography. Collection of specific reverse transcription polymerase chain reaction (RT-PCR) fragments was performed by electroelution from the agarose gel followed by precipitation, quantitation on agarose gels versus standards, and ligation into a pCR2.1-TOPO cloning vector according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Sequencing was performed by the University of Virginia Biomolecular Research Facility.
Screening of cDNA Library and Northern Blotting
The sequenced 430-bp insert obtained by RT-PCR was purified by EcoRI digestion of the pCR2.1-TOPO cloning vector and collecting the released cDNA fragments by band excision after agarose gel separation. Fifty nanograms of the purified fragment was denatured by boiling and then radiolabeled with [-32P]-dCTP by including the two degenerate oligonucleotides described above in the random priming procedure [22]. The radiolabeled fragment was purified on an Elutip-D column (Schleicher and Schuell, Keene, NH) and hybridized to six 137-mm plaque lifts (Magna Nylon Transfer Membranes; MSI, Westboro, MA) containing an estimated 240 000 phage from a human testicular 
DR2 5'-stretch cDNA library (Clontech) in a solution containing 50% formamide, 5x saline sodium citrate (SSC), 5x Denhardt solution, 0.25 µg/ml yeast RNA, 0.5% SDS, and 0.05 M sodium phosphate (pH 7.0) at 42°C. After overnight hybridization, the filters were washed in a final solution of 0.2x SSC/0.2% SDS at 52°C before mounting, exposure to XAR-5 film (Kodak, Rochester, NY), and development. Twenty primary isolates were rescreened twice, and the remaining eight positive isolates were converted from DR2 to pDR2 according to the manufacturer's instructions in AM1 cells. Sequencing was performed (University of Virginia Biomolecular Research Facility) in both directions, and the nucleotide and amino acid sequences were analyzed using the Genetics Computer Group and SEQWeb (Madison, WI) program packages.
To generate a full-length probe for tissue specificity analysis, 50 ng of the purified 1337-bp ESP cDNA was radiolabeled as described above omitting the EST oligonucleotides. The radiolabeled cDNA was isolated and hybridized in ExpressHyb (Clontech) to either a Human Multiple Tissue Northern Blot (Clontech) or a Human RNA Master Blot (Clontech). All prehybridizations, hybridizations, and washings were performed according to the manufacturer's instructions. The Human Multiple Tissue Northern blot contained 2 µg of poly(A)+ RNA from spleen, thymus, prostate, testis, ovary, small intestine, mucosal lining of the colon, and peripheral blood leukocytes per lane, and the Human RNA Master Blot contained various amounts (100–500 ng) of poly(A)+ RNAs from 76 different tissues normalized to various housekeeping genes.
Expression and Isolation of Recombinant Human ESP
The ESP open reading frame (ORF) minus the leader peptide was adapted for ligation into pET-28b+ (Novagen, Madison, WI) by designing adaptor primers (ESP/pET-28b+/Forward, 5'-CATGCATGCCATGGATCCGAGCATAACTGTGACACCTGATGAA-3'; ESP/pET-28b+/Reverse, 5'-GAGTCGCTCGAGATAAACTTTTAATAAGGCTGT-GACTCTCCTTG-3') containing in-frame NcoI and XhoI sites in the 5' and 3' primers, respectively. The complete 1337-bp cDNA was subjected to PCR (see above) with rTth DNA polymerase according to the manufacturer's instructions (Perkin-Elmer) using primers. After pET-adaption, the resulting 990-bp product was separated and isolated as described above, digested, reisolated on an agarose gel, and ligated into the restricted pET-28b+ vector. After transformation into Novablue host cells, a clone was chosen for sequencing (University of Virginia Biomolecular Research Facility) to ascertain the proper insertion of the insert into the pET-28b+ expression vector and the retention of the leaderless ESP reading frame.
For expression, purified BL21 (DE3, pLysS) host cells containing the pET-28b+/ESP construct were grown in four 1-L shake-flasks. Cultures were grown to an OD600 between 0.5 and 0.7 before the addition of isopropyl ß-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM to induce protein expression. After 3 h of fermentation, recombinant human ESP (rec-h-ESP) containing a C-terminal (His)6-Tag was isolated by binding prepared cell-pellet fractions to a His-Bind Resin (Novagen) column, all according to the manufacturer's instructions and as reported previously [20]. Final purification of the rec-h-ESP was accomplished by elution from the column with Elute Buffer (300 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and dialysis into 1x PBS prior to loading onto a Prep-Cell (Bio-Rad), where the protein sample was electrophoresed and fractionated.
Generation of Rat Monospecific Polyclonal Serum
Three virgin female young adult Lewis rats were immunized i.m. and in selected fat pads with 100 µg of purified rec-h-ESP in squalene monooleate. After 1 mo, the 3 rats were given two booster injections of 50 µg rec-h-ESP in squalene monooleate 2 wk apart, and blood was collected 1 wk after each injection. Preimmune and immune serum samples were collected from tail veins, and titers were determined by both ELISA (data not shown) and Western blot analysis. Specificity of each rat's antiserum for rec-h-ESP (-rec-h-ESP) was tested by Western blot analysis against rec-h-ESP and an SDS lysate of human sperm proteins separated on polyacrylamide gels and Western blotted as described above.
Immunofluorescent Microscopy of Ejaculated Human Sperm
Swim-up sperm were prepared as described [7] in Ham F10 medium. Preparations of noncapacitated sperm were derived by incubating swim-up sperm at 37°C for 2 h in 5% CO2, after which they were washed with Ham F10 medium and air dried on slides. Acrosome-reacted sperm were prepared by incubating swim-up sperm at 37°C for 2 h in 5% CO2 and then in Biggers, Whitten, and Whittingham medium supplemented with 0.3% BSA at 37°C for 18 h in 5% CO2 followed by a 30-min incubation in 5% CO2 at 37°C in 500 µM progesterone before air drying on slides. Immunofluorescent staining was performed by permeabilizing mounted sperm with methanol for 30 sec, treatment with 10% normal goat serum (NGS) for 15 min at 22°C, and incubation with either -rec-ESP rat serum (1:50) or preimmune rat serum in 10% NGS/PBS for 2 h at 22°C. After washing in PBS, the mounted sperm were incubated with goat -rat IgG + IgM (H+L) F(ab')2-tetramethyl rhodamine isothiocyanate (TRITC) (Jackson Immunoresearch Labs, West Grove, PA) (1:50) in 10% NGS/PBS for 1 h at 22°C before visualization on a Axioplan microscope (Carl Zeiss, Thornwood, NY) fitted with a digital camera (Hamamatsu, Hamamatu-City, Japan). The PSA-FITC staining pattern was used to assess acrosomal status [23].
Electron Microscopy
Electron microscopy on ejaculated human sperm was performed by preparing noncapacitated or capacitated sperm as described above prior to fixation in 4% paraformaldehyde in Ham F10 medium for 30 min. The fixed sperm were then washed with PBS, dehydrated through a graded series of ethanols, and embedded in Lowicryl K4M resin (Electron Microscopy Sciences, Fort Washington PA) according to the manufacturer's recommendations. Ultrathin 0.1-nm sections on nickel grids were blocked with 10% NGS for 30 min at 22°C. Immune serum from one of the squalene monooleate/rec-h-ESP-immunized rats was diluted 1:25 in PBS with 10% NGS, pH 8.6, and incubated with the sections overnight at 4°C. After being washed with PBS, the sections from noncapacitated and capacitated sperm were incubated with 10-nm gold-conjugated goat anti-rat IgG diluted 1:50 in PBS with 10% NGS, pH 8.6, for 2 h at 22°C. The sections were then washed and stained with 5% uranyl acetate in 50% ethanol for 20 min before viewing in a JEOL 100CX electron microscope.
Electron microscopy of ESP in testicular human sperm was performed by obtaining human testis from an orchiectomized patient with prostate cancer. The testis was cut into 4-mm cubes and fixed for 1.5 h in 4% paraformaldehyde, 0.5% glutaraldehyde in isotonic PBS, pH 7.6. After washing and dehydration through a graded ethanol series, the tissue cubes were trimmed again and embedded in Lowicryl K4M resin according to the manufacturer's recommendations. Ultrathin 100-nm sections were blocked with 10% NGS in PBS, pH 8.6, for 30 min at 22°C followed by incubation with rat polyclonal serum raised against rec-h-ESP at a 1:30 dilution in 10% NGS in PBS, pH 8.6, overnight at 4°C. After washing four times in PBS, sections were incubated for 1.5 h at 22°C with 5-nm gold-conjugated secondary antibody, goat anti-rat IgG (Goldmark Biologicals, Phillipsburg, NJ) diluted 1:50 in 10% NGS in PBS, pH 8.6. The sections were then washed with distilled water and stained with uranyl acetate before examination with a JEOL 100CX electron microscope.
Immunofluorescence of ESP in the Seminiferous Epithelium of Human Testis
Testes were obtained from three patients undergoing elective orchiectomies for prostate cancer. Testes were sliced once with a razor blade and immersed in neutral buffered formalin (4%) solution (Sigma) for 1 h. Tissue was minced and placed into fresh fixative overnight and then dehydrated in a graded series of ethanols, cleared in xylene, and embedded in paraffin. Sections (2.5 mm thick) were cut, mounted onto slides, deparaffinized, rehydrated, and permeabilized with 100% methanol. Sections were then incubated in blocking solution containing 10% NGS in PBS, incubated with anti-rec-h-ESP antiserum or preimmune serum (1:200) in PBS containing 1% NGS (PBS-NGS), washed, incubated with TRITC-labeled goat anti-rat IgG (1:400; Jackson) in PBS-NGS, washed, and mounted with Slow Fade (Molecular Probes, Eugene, OR) containing 4',6'-diamidino-2-phenylindole (DAPI) II counterstain (Vysis, Downers Grove, IL). Sections were observed by epifluorescence microscopy using a Zeiss microscope. Individual blue and red fluorescent images were obtained using a digital camera (Hamamatsu) and compiled using Openlab software (Im-provision, Boston, MA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Comparison of silver-stained 2-D gels (Fig. 1A) with complementary Western blots reacted with Con-A lectin (Fig. 1C) allowed cross-referencing of silver-stained human sperm proteins with those that were glycosylated and therefore likely to be of secretory or surface origin. Peroxidase-conjugated Con-A bound to spots at 30, 40, 45, 75, and 90 kDa (data not shown) and to a region between 32 and 36 kDa with an isoelectric point (pI) of 5.0–5.1 that reacted strongly (Fig. 1C). A train of silver-stained proteins differing slightly in charge and mass resided in this region (Fig. 1, A and B), and the corresponding positive Con-A staining of several of these spots (Fig. 1C) encouraged us to microsequence several proteins in this region.
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Cloning and Analysis of the ESP (C7/C8) Transcript
Two protein spots having relative molecular masses of 34 and 36 kDa with estimated pIs of 5.0 and 5.1 (designated C7 and C8, respectively, in the Sperm Proteome) were excised from a Coomassie blue-stained 2-D gel (Fig. 1B, arrows). Microsequencing of peptides derived from tryptic digestion of the protein spots performed by both Edman degradation and tandem mass spectrometry yielded the sets of peptide sequences listed in Table 1. Several peptides obtained from each spot were similar (peptides 4, 6, and 7 of C7, corresponding to peptides 7, 9, and 10 of C8, respectively). Comparison of these peptide sequences to the nonredundant and EST NCBI databases revealed several ESTs of testicular origin with significant matches to all three peptides found in both protein spots (assession numbers AA913806, AI33709, AI123225). AA913806 was derived from a mixed pool of tissues, including human testis, lung, and B cells; AI33709 was derived from a human germ cell tumor; and AA782995 was derived from human testis. These three peptides were present in the same ORF in these ESTs, suggesting that all are part of a single protein. Further database analysis with these ESTs did not reveal any significant match to any known protein.
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Oligonucleotides manufactured to the 5' and 3' ends of the EST (AA913806) allowed a 432-bp PCR product to be transcribed from human testis RNA. This product was collected, sequenced, labeled with 32P, and used to probe a human testicular DR2 cDNA library, leading to the sequencing (Fig. 2) of a 1337-bp cDNA. Subsequently, a start codon (bp 59) with a consensus start site containing purines at the -6 and +4 positions [24] was found.
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The ORF (Fig. 2, uppercase letters) terminated at bp 1108 with a stop codon at bp 1109–1111 and was followed by a 226-bp 3' untranslated region (UTR) that contained a polyadenylation signal [25] beginning at bp 1316. Examination of the predicted protein revealed that the first 19 amino acids of the protein are hydrophobic residues and therefore likely represent a signal peptide. Additionally, the residues PAYP at amino acids 18–21 constitute a signal peptide cleavage site [26]. These observations suggest that the ORF of the predicted protein begins at bp 59. The resulting ORF predicts a 349-amino acid protein with a molecular mass of 37 kDa after cleavage of the signal peptide and 39.6 kDa prior to this posttranslational modification, with predicted pIs of 5.4 and 5.44, respectively. These predictions agree with the estimated migration of the spots at 34–36 kDa and pI 5.0–5.1 on 2-D gels.
Further examination of the predicted protein sequence revealed a consensus N-glycosylation site, NVSI [27], starting at amino acid 134, which is in accord with the lectin-binding property of the protein (Fig. 1C). Numerous cAMP-dependent kinase [28], protein kinase C [29], and casein kinase 2 [30] consensus phosphorylation sites were also found. The three common peptide sequences detected by mass spectrometry were present in the ORF (amino acids 264–274, 310–317, and 328–325; see bold, underlined amino acids in Fig. 2). Identification of an additional peptide microsequence, SPVTTLDK (C8 peptide 5, Table 1) at amino acids 179–186 reinforced the conclusion that the authentic cDNA corresponding to the microsequenced 2-D protein spots had been cloned.
Analysis of the complete ESP ORF with the Blast search engine identified a 68-amino acid (amino acids 272–337) C-terminal region that contained a 29% identity and 49% similarity (conservative amino acid substitutions) to a portion (amino acids 67–134) of murine osteoglycin [31] (Fig. 2, boxed and shaded amino acids), which is known to bind extracellular matrix proteins [32]. Also identified within this region by the ProDomo algorithim [33] was an additional overlapping subdomain with 58% homology (amino acids 274–304 of ESP) to the bacterial type II membrane-binding proteins, which anchor the bacterial DNA to the cell wall (Fig. 2). Further examination of smaller regions of ESP by the Blocks algorithm revealed an N-terminal domain (amino acids 58–99 of ESP) that has a bacterial thiol-activated cytolysin protein signature (Fig. 2). Cytolysins are known to bind cholesterol [34].
The Human Genome Nomenclature Committee has assigned the gene symbol SP-ESP to the human ESP locus on human chromosome 15q22 (assession AC027088). The ESP gene consists of two exons, the first of which contains the initial 123 bp of the ORF, followed by a 14 879-bp intron and a second 1212-bp exon. The 5' UTR of the NOX5 gene (assession AF325190) utilizes the same genomic segment as the first exon of the SP-ESP gene locus, but the NOX5 gene product is generated by a different ORF. Homologous gene transcripts for ESP were discovered in Macaca fasicularis (assession AB072796; 94% identical) and Mus musculus (assessions AK015620 and AK14843; 81% identical). The Macaca protein translated from AB072796 is 93% identical overall to the human protein and is highly conserved (97.6%, 90%, and 94%) in the cytolysin [34], type II membrane-binding protein, and osteoglycin domains [32], respectively, suggesting that these motifs may be functionally important.
Tissue Distribution of ESP Expression
To ascertain the expression pattern of the gene transcript, a Northern blot containing mRNA from various human tissues was probed with the radiolabeled ESP cDNA (Fig. 3A). A single prominent band of approximately 1.4 kilobases (kb) was observed only in the lane containing testicular mRNA. This size transcript also verified that a near full-length cDNA had been isolated. An array of 76 different human tissue mRNAs was hybridized to a radiolabeled ESP probe on a dot-blot (Fig. 3B) to examine a larger repertoire of tissues. The results confirmed that ESP is indeed transcribed predominantly in adult testis (Fig. 3B, sample F8), although a faint signal was also observed in placenta (sample B8) and in fetal lung (sample G11). The EST database was next analyzed with the cDNA and revealed high-similarity matches to sequences derived from normal kidney, breast, medulla, hypothalamus, hippocampus, and placenta (assessions AI985457, H15004, BI826643, BG715794, BG700740, and BG623286, respectively) and samples of tumorigenic origin such as parathyroid and germ cell tumors (assessions AI027115 and AI652853, respectively). It is not known at the present time whether these nontesticular transcripts are translated, but the existence of ESTs suggests that ESP may be expressed at low levels in other tissues even though the appropriate mRNA samples were not detected in the experiment shown in Figure 3B (i.e., hippocampus, 2F; medula, 2G; kidney, 7A; mammary, 9F).
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Recognition of Recombinant ESP by Polyclonal Serum
To localize and characterize the immune response to human ESP, the ORF minus the putative leader peptide was cloned into pET28 and expressed in E. coli (Fig. 4A, lanes 1 and 2), and a monospecific polyclonal serum was generated for immunological studies against the purified recombinant material (Fig. 4A, lane 3). Antiserum to the purified rec-h-ESP raised in virgin female Lewis rats was reactive with the recombinant immunogen by Western blotting (Fig. 4A, lane 5) and with a series of bands around 
36 kDa in an extract of sperm proteins (lane 7). Smaller bands at 24 and 18 kDa most likely represent proteolytic products. The preimmune rat serum failed to recognize rec-h-ESP (lane 4) or any bands of the appropriate size in the Celis-extracted sperm proteins (lane 6). When a 2-D blot of human sperm proteins (Fig. 4B, B1) was exposed to rat--rec-h-ESP (Fig.4B, B3), numerous human ESP-positive isoforms were identified in the area originally cored for microsequencing. Although a majority of the ESP isoforms migrated between 34 and 38 kDa, areas of higher molecular mass were also stained, and the isoforms varied in pI from 5.1 to 5.4. These observations indicate considerable heterogeneity of posttranslationally modified forms of the ESP present in human sperm.
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Immunohistochemical Localization of ESP to the Equatorial Segment
The rat -rec-h-ESP serum was utilized to examine the immunohistochemical localization of the ESP within human sperm (Fig. 5). Live human sperm were not stained (data not shown), indicating that the ESP was not located in a surface-accessible region in sperm that had not undergone the acrosome reaction. However, when human sperm were mounted, permeabilized, and probed with the rat -rec-h-ESP serum, an oval banded region at the posterior of the acrosome was fluorescent (Fig. 5D). Preimmune serum (Fig. 5B), used as a control, demonstrated no fluorescence. In addition, sperm samples in which ESP was colocalized with Con-A fluorescence to assess acrosomal status demonstrated that ESP was present in 98% and 96% of noncapacitated and capacitated Con-A-positive sperm, respectively (data not shown). These results indicate that 1) human ESP most likely resides in the equatorial segment of the acrosome and 2) in sperm with functioning acrosomes, a high proportion contain ESP.
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To study further details of the equatorial segment localization of the ESP, human sperm were studied by electron microscopy following immunogold staining (Fig. 6). The preimmune control serum demonstrated a random background of a few particles (Fig. 6, A1 and A2). Gold particles were predominantly present within the equatorial segment of the acrosome in both noncapacitated (panels B1 and B2) and capacitated (panels C1 and C2) sperm stained with immune serum. On the basis of these immunofluorescent and electron microscopic localizations within the equatorial segment, the protein was designated ESP and the gene was designated SP-ESP (sperm equatorial segment protein).
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Immunohistochemical Localization of ESP During Spermatogenesis
Immunolocalization of the ESP was studied in paraffin-embedded sections of human testis (Fig. 7). Red immunofluorescent staining demonstrated the ESP in the postmeiotic stages of spermiogenesis in round (Fig. 7, a–f) and elongating (g and h) spermatids and in testicular spermatozoa (i). In Golgi phase early round spermatids, immunofluorescent staining was observed within small proacrosomal granules close to the nucleus (a). In subsequent stages, the ESP localized to the acrosomal vesicle as the Golgi-derived proacrosomal granules coalesced (b). Even at this early stage of acrosomal development, the ESP occupied a toroid-shaped region, which suggests that it was relegated to the periphery of the acrosomal vesicle (b). In cap phase spermatids with enlarged acrosomal vesicles and in acrosomal vesicles that had begun to flatten over the anterior nucleus (c–e), the ESP remained associated with the margin of the acrosomal vesicle. In acrosomal (elongation) phase spermatids in which the acrosome had extended posteriorly over the condensing nucleus (f), the ESP migrated posteriorly to the leading edge of the developing acrosome (f and g). In late phase spermatids and testicular spermatozoa, the ESP was localized to the equatorial segment of the acrosome. Testicular cells stained with preimmune serum showed no immunofluorescent staining (data not shown).
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ESP was also localized during acrosome biogenesis in developing spermatids, as revealed by electron microscopy of sectioned human testis (Fig. 8). In early round spermatids (Fig. 8A), the nascent acrosomal vesicle contained a central electron-dense acrosomal granule and lateral electron-lucent regions. Most of the gold particles localizing ESP were restricted to the lateral electron-lucent regions surrounding the acrosomal granule. Similarly, in early cap phase spermatids containing acrosomal vesicles that were flattened over the anterior nucleus, three regions of electron density were discerned: the acrosomal granule, regions of intermediate electron density, and lateral electron-lucent regions (Fig. 8B). Gold particles were concentrated in the peripheral electron-lucent regions of the acrosomal vesicle and not in the electron-dense regions of the acrosomal granule or adjacent areas of intermediate electron density. Examination of elongating spermatids (Fig. 8C) revealed gold particles associated with the matrix and membranes of the equatorial segment. Gold particles were particularly concentrated in dilated regions of the equatorial bulb that had not yet narrowed to form the definitive equatorial segment. These results verify the progressive localization of ESP observed by immunofluorescence of staged developing spermatids (Fig. 7) and in mature ejaculated sperm (Figs. 5 and 6). The ESP appeared at an early stage in the biogenesis of the developing acrosome and also served as a marker of a relatively electron-lucent peripheral subcompartment in the acrosomal vesicle of Golgi and cap phase spermatids. These observations indicate that a subcompartment that subsequently becomes the equatorial segment is specified in early acrosomal vesicles and persists as a definable domain as cap, elongation, and maturation phases proceed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Both immunofluorescent and immunoelectron microscopic evidence support the concept that ESP, a novel 349-amino acid protein, resides predominantly within the equatorial segment of ejaculated acrosome-intact sperm. Antiserum to recombinant ESP cross-reacted on both one-dimensional and 2-D gel Western blots with "native" ESP in sperm extracts at the appropriate molecular masses. Additionally, this immunoreagent localized ESP to the equatorial region on the heads of virtually all acrosome-intact cells present in samples of fixed, permeabilized sperm. This localization was confirmed with immunoelectron microscopy, which revealed gold particles concentrated in the equatorial segment matrix.
Several research groups have previously identified immunoreagents that react with equatorial segment proteins similar in size to ESP [8, 10, 11]. Noor and Moore [10] purified and injected hamster sperm heads into mice and raised monoclonal antibodies with strong affinity for spermatozoa. One of their monoclonal antibodies (M1) recognized the equatorial segment of hamster sperm, as revealed by immunofluorescence microscopy, and bound protein bands of 37.5 and 34 kDa on a Western blot. The M1 monoclonal antibody inhibited sperm-egg fusion but not sperm-oolemma binding or sperm motility. ESP may be a human ortholog of this hamster protein on the basis of its cellular localization and protein size. Auer et al. [11] utilized antibodies eluted from human sperm from infertile individuals to identify a 36-kDa protein with a pI of 
5.5 that localized to the equatorial segment. Although antibodies raised to the native protein reduced sperm-egg binding, no sequence data have been published for this protein. Toshimori et al. [8] generated the MN9 monoclonal antibody against mouse sperm proteins. This immunoreagent reacted positively to the equatorial segment and to 38- and 48-kDa bands on one-dimensional Western gels in addition to blocking sperm oocyte fusion. The protein recognized by MN9 has been named equatorin, and in contrast to ESP, it is not recognized by MN9 before the acrosome reaction [35], indicating that the MN9 epitope is acquired by a change in the conformation of the protein(s). Although these researchers detected antigens of size and location similar to those of ESP and provided data implicating these antigens in the equatorial segment-mediated fusion event, none of these studies involved cloning or characterization of the specific molecule(s) responsible for the equatorial segment immunoreactivity.
Computer modeling of the ESP sequence has predicted alternating -helical coils and ß-sheets and the absence of any hydrophobic transmembrane domain, suggesting a globular soluble protein (data not shown). The purified recombinant ESP is extremely soluble in nondenaturing buffer systems such as PBS (data not shown), and this property may be mirrored by the matrix location of the major portion of the ESP within the equatorial segment. Olson et al. [9] presented both immunofluorescent and electron microscopic evidence that acrosomal matrix proteins such as AM22 and AM29 are excluded from the equatorial segment of the acrosome and that there appear to be structurally distinct matrix domains in the mature acrosome. Our electron microscopic analysis confirmed the presence of different central (acrosomal granule) and lateral electron-lucent subdomains, respectively, in the developing acrosome during the Golgi and cap phases. Although the mechanism by which ESP is sequestered to the equatorial segment subdomain is unknown at present, ESP may have an affinity for or may be complexed with other molecules within this defined compartment of the human sperm.
ESP Polymorphism
On 2-D Western blots, the antiserum to recombinant ESP recognized several proteins of differing charges and masses in addition to the two immunoreactive spots that correspond to the proteins originally cored to obtain ESP microsequences. One explanation for the additional immunoreactive spots is that they represent posttranslational modifications of ESP, and the lower mass forms represent proteolytic products. Sequence analysis of ESP revealed consensus glycosylation and phosphorylation sites, which may explain in part the polymorphism. Although the additional immunoreactive spots have not been cored and microsequenced to determine whether they contain only ESP peptide sequences, the two that were cored and sequenced did yield identical sequences, confirming that polymorphic charge and mass variants of ESP are present in human sperm. Many of the proteins immunoreactive with ESP antiserum on the 2-D Western blots are found clustered in the same molecular mass and pI range as the original spots used for cloning, which suggests that posttranslational modification of a single protein has occurred. Although a polyclonal immunoreagent to recombinant ESP was employed in this analysis, the preimmune serum did not recognize any proteins in the Celis extract, supporting the conclusion that the immunoreactive proteins observed are likely authentic ESP isoforms. In further support of ESP polymorphism, we recently generated monoclonal antibodies to recombinant ESP and observed the same immunoreactive cluster of ESP spots with one of the monoclonal supernatants. The possibility that there are other ESP-like proteins in human sperm is not supported by either cDNA or genomic analyses, which show at present a single ESP gene and transcript in the human and mouse databases. The observation of a single tight band on tissue Northern blots further supports a single gene transcript (Fig. 3) and gives no evidence of alternative splicing.
High-resolution 2-D gel techniques are increasingly demonstrating the polymorphism and microheterogeneity of many types of proteins. For example, we recently reported a highly polymorphic calcium-binding tyrosine phosphorylation-regulated protein, CABYR, which is localized in the sperm fibrous sheath [36].
ESP: A Postmeiotic Gene Product that Marks an Equatorial Segment Subcompartment in Early Acrosomal Biogenesis
Examination of human testis by both immunofluorescence and electron microscopy revealed that ESP is a postmeiotically expressed protein confined to the developing acrosome of germ cells. A model depicting the locations of the ESP subcompartment at each phase of acrosome biogenesis in humans is presented in Figure 9. The protein first appeared in the proacrosomal granules of postmeiotic, Golgi-phase round spermatids. In spermatids with spherical acrosomal vesicles, ESP occupied electron-lucent regions surrounding the acrosomal granule. In spermatids with flattened acrosomal vesicles, immunofluorescence revealed that ESP was concentrated at the vesicle periphery, and electron microscopy confirmed a concentration of ESP in an electron-lucent subcompartment. As acrosome development proceeded through cap and elongation phases, the peripheral localization of ESP in the acrosomal vesicle persisted as a defined collar that migrated posteriorly along the nuclear envelope. ESP was particularly prominent in the expanded equatorial bulbs of elongated spermatids where narrowing of the equatorial segment was not complete.
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These findings indicate that ESP can serve as a marker for the acrosome and specifically for the first appearance of the presumptive equatorial segment. Moreover, these results suggest the following hypotheses: 1) an equatorial segment subcompartment is specified as early as the acrosomal vesicle stage of acrosome biogenesis, and 2) this acrosomal subcompartment persists as a segregated domain in subsequent phases of acrosomal cap formation, elongation, and maturation. ESP may be the first equatorial segment protein to demonstrate segregation and concentration within a subcompartment of the developing acrosomal vesicle and to persist as a defined peripheral equatorial segment subdomain as acrosomal flattening, capping, and prolongation proceeds.
These observations of changes in ESP localization suggest that the equatorial segment subcompartment is mobile and is capable of being positioned by the stage-specific morphogenic forces acting within the developing sperm during acrosome biogenesis. It is not known at the present time whether the mechanisms exerting these pulling and pushing forces are passive physical ones or whether active biochemical processes are at work. Nonetheless, these observations are corroborated by the staged immunofluorescence data directly showing the accretion of the ESP into a peripheral collar in the acrosomal vesicle and the movement of an ESP-defined equatorial segment subcompartment posteriorly. Whether the equatorial segment compartment plays a direct role in the morphogenic movements during the cap and elongation phases of acrosome development remains to be determined, but its position at the leading edge of the flowing acrosomal vesicle suggests that it might have an active role, and ESP, which is present during these processes, may be essential for acrosome biogenesis. An alternative hypothesis is that ESP is segregated as a result of the condensation of other matrix proteins, excluding ESP (for a review of storage during secretory granule biogenesis, see [2]). Membrane-associative domains such as the region ESP shares with the type II membrane-binding protein or the putative leader peptide in unprocessed ESP may favor association with acrosomal vesicle membranes rather than with the other matrix proteins, which condense to form the electron-dense secretory granule.
The model presented in Figure 9 for equatorial segment biogenesis during human spermiogenesis provides a basis for comparative studies. The ESP orthologues in mice and monkeys that we have reported here may be helpful in determining whether this new model of equatorial segment development can be applied to other primates and to rodents. The identification of a molecule such as ESP, which is unique to the equatorial segment and can serve as a marker for early specification of the equatorial segment, will be useful in understanding the molecular mechanisms underlying key aspects of equatorial segment biology, including 1) membrane trafficking during acrosome development, 2) the basis for stability and retention of the equatorial segment during the acrosome reaction, 3) molecular mediators of sperm-egg binding and fusion, and 4) the fate of the equatorial segment after fertilization, including its role as the initiation site for breakdown of the sperm nuclear envelope [37].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: John C. Herr, Department of Cell Biology, University of Virginia Health System, School of Medicine, P.O. Box 800732, University of Virginia, Charlottesville, VA 22908. Fax: 434 982 3912; jch7k{at}virginia.edu
Received: 26 February 2003.
First decision: 26 March 2003.
Accepted: 8 May 2003.
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