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Immunology |
Complement Biology Group,3 Department of Medical Biochemistry and Immunology, University of Wales College of Medicine, Cardiff CF14 4XN, United Kingdom
Department of Immunology,4 University of Liverpool, Liverpool L69 3BX, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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acrosome reaction, immunology, sperm, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A role for MCP in reproduction was first suggested by the demonstration that the human spermatozoal protein (trophoblast-leukocyte common antigen; TLX) was identical to MCP [13–15]. TLX/MCP was detectable on sperm only after the acrosome reaction, indicating that it was restricted to the inner acrosomal membrane and effectively excluding a role in complement regulation on spermatozoa, as it would be only transiently expressed in vivo. MCP on human spermatozoa differs from MCP in other tissues in that it has a smaller molecular mass (43–50 kDa versus 51–58 kDa and 59–68 kDa for the major ubiquitous MCP isoforms) due to trimming of N-linked carbohydrate to less complex structures [16, 17]. Spermatozoal MCP may contain exclusively cytoplasmic tail 2, and the three N-linked sugar groups are trimmed to small, simple structures [18]. Surprisingly, incubation with monoclonal antibodies (mAbs) against the first short consensus repeat (SCR) domain of MCP inhibited the interaction of human sperm with oocytes in both heterologous (hamster egg) and homologous (human egg) systems [19–21]. These observations provoked several investigators to propose specific roles for MCP in sperm-egg interaction, although no common model has yet emerged. The association of abnormalities of expression of MCP in testis with infertility in a small percentage of infertile men supports a role for MCP in sperm function but leaves many questions unanswered [22, 23].
MCP expression has also been characterized in nonhuman primates, pigs, and rodents. In primates and pigs, MCP is widely distributed, being expressed on most cell types including erythrocytes [24, 25]. In contrast, MCP in mice, rats, and guinea pigs is predominantly or exclusively expressed in the testis when assessed by Northern blot analyses of mRNA expression [26–28]. In mice and rats, the role of MCP as a ubiquitous cell surface complement regulator is subsumed by the rodent-specific regulator Crry [29]. Despite the presence of Crry, expression of MCP has been retained in male reproductive tissues, adding support to the conjecture that MCP may play an important noncomplement role at this site. Lacking is any detailed analysis of rodent MCP expression at the protein level, and, without this information, it is impossible to predict whether rodents are appropriate models for examining the functional role of spermatozoal MCP.
In order to address this deficit, we have generated a specific mAb against rat MCP using a recombinant fusion protein comprising the second and third SCR domains fused to human IgG-Fc. We have used these reagents to characterize the expression of the protein in immature and adult rats as well as during embryonic development and have assessed the cofactor function of the expressed protein.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All tissue culture reagents were purchased from Gibco-BRL (Paisley, UK). The Chinese hamster ovary (CHO) cell line, SP2/0-A14 cells (the myeloma fusion partner), and Jurkat human T cell line were obtained from the European Collection of Animal Cell Cultures (ECACC, Salisbury, UK). The CHO cell line was grown in Ham F12 medium with 5% fetal calf serum (FCS), 50 U/ml penicillin/streptomycin, 1 µg/ml amphotericin B, 2 mM glutamine, and 1 mM sodium pyruvate. SP2/0-A14 cells and Jurkat cells were grown in RPMI 1640 medium supplemented with 10% FCS and the same additives.
Proteins
Human and rat C3 and fI were prepared in-house according to published methods [30]. Soluble complement receptor 1 (sCR1) was a gift from T Cell Sciences Inc. (Needham, MA). Human and rat C3 were methylamine-activated (hC3ma and rC3ma, respectively) by incubating C3 (0.5 mg/ml in borate-buffered saline, pH 8.0) with 0.1 M methylamine for 2 h at 37°C. The protein was dialyzed into PBS. The C3/30 mAb against human C3b has been described previously [31].
Preparation of Recombinant Ig Fusion Proteins
Ig fusion proteins comprising either rat MCP SCR 2 and 3 (MCP23-Ig) or rat CD59 (CD59-Ig) attached to IgG-Fc were prepared as described [32, 33]. Briefly, cDNA encoding SCR2 and 3 of rat MCP or the extracellular domain of rat CD59 [28, 34] was cloned into the expression vector pDR2
EF1
(gift from Dr I. Anegon, INSERM U437, Nantes, France) upstream of and in-frame with DNA encoding the hinge and Fc domains of human IgG. For rat MCP, the signal sequence of CD33 was included as described previously [32]. Vent DNA proofreading polymerase was used in the polymerase chain reaction (PCR) reactions, and sequencing the products confirmed that no errors had been introduced by PCR. CHO cells were transfected with these plasmids using lipofectamine (Gibco BRL) according to the manufacturer's instructions. Stable lines were selected with 400 µg/ml hygromycin B (Gibco BRL) in Ham F12 medium with 10% FCS and maintained in hygromycin B (100µ/ml) in Ham F12 medium with 5% FCS.
To isolate the fusion protein, spent medium was collected and passed over a Prosep A column (Bioprocessing Ltd, Consett, UK); the column was washed with PBS and with 0.1 M citrate buffer, pH 5.0, to remove contaminating bovine Ig; and then the fusion protein was eluted with 0.1 M glycine/HCl, pH 2.5. The eluted protein was neutralized, dialyzed into PBS, and concentrated by ultrafiltration.
Expression of Rat MCP in CHO Cells
CHO cells were transfected with the full-length rat MCP cDNA [28] in the expression vector pDR2EF1, as described previously (CHO/ MCP). Control cells were transfected with the vector alone (vector control; CHO/VC). Transfectants were selected and maintained in Ham F12 medium with hygromycin as described previously.
Cell lysates of CHO/MCP, CHO/VC, spermatozoa, or Jurkat cells were prepared by solubilization in 1% NP40 in PBS for 30 min on ice. Lysates were centrifuged at 5000 x g for 15 min at 4°C and the supernatants stored at –80°C.
MAb Production and Purification
BALB/c mice were immunized with MCP23-Ig; mAb were generated using published methods [32]. Fusion wells were screened in ELISA for reactivity against MCP23-Ig and the control CD59-Ig proteins, and those wells specifically positive for the former were cloned by limiting dilution. Monoclonal populations were rescreened, isotyped using the IsoStrip mouse monoclonal antibody isotype kit (Boehringer Mannheim, Indianapolis, IN), and expanded in culture.
The mAb were purified from culture supernatant on Prosep A as described earlier [32]. For purification of IgG1 mAb, the hybridoma culture supernatant was mixed 1:1 (v:v) with 1.5 M glycine and 3 M NaCl and then adjusted to pH 8.9 with NaOH prior to application to the column. The column was then washed with the same buffer at pH 8.9 and bound IgG1 eluted with 0.1 M citrate phosphate buffer, pH 5.18. The eluted protein was dialyzed into PBS and concentrated by ultrafiltration.
The mAb were fluorescein isothiocyanate (FITC)-labeled by incubation with NHS-FITC according to the manufacturer's instructions (Pierce, Rockford, IL).
Spermatozoa Preparation and Analysis
Motile epididymal spermatozoa were obtained by a modification of the swim-up technique for human spermatozoa as described previously [35]. Briefly, epididymis from an adult male Wistar rat humanely killed using UK Home Office approved methods was minced in 3 ml of Ham F12 medium and incubated at room temperature for 15 min to allow sedimentation of large cellular aggregates. The supernatant was removed to a fresh tube and carefully overlaid with 3 ml of Ham F12 medium at room temperature. The tube was incubated for 90 min at 37°C, and the top 2.5 ml of Ham F12 medium, which contained motile spermatozoa, were then carefully removed. Swim-up isolated spermatozoa were used for immunofluorescence (IF) analyses, for preparing sperm lysates, and for the acrosome reaction.
For IF, 20 µl of the sample were spotted onto glass slides and air-dried overnight. The slides were fixed in acetone at room temperature for 5 min and stored at –20°C until use. To prepare sperm lysates, swim-up spermatozoa from the cauda epididymis of a single rat were pelleted by centrifugation and incubated with mixing in 1 µl of lysate buffer (PBS containing 2% NP40, 1 mM phenylmethylsulfonyl fluoride, 10 mM EDTA, 1 µg/ml leupeptin, and 1 µg/ml pepstatin) for 30 min on ice. Insoluble debris was removed by centrifugation (5000 x g, 15 min at 4°C) and the supernatant lysate stored in aliquots at –80°C.
The acrosome reaction was performed using minor modifications of a published method [35]. Briefly, the swim-up spermatozoa (1 x 106 in 1 ml of Ham F12 medium) were incubated for 1 h at 37°C with the calcium ionophore A23187 (Sigma-Aldrich, Dorset, UK) at 1 µM to induce the acrosome reaction. Control cells were incubated without ionophore. Acrosome-reacted and control spermatozoa were gently smeared on glass slides and immediately air-dried. Viability of spermatozoa following acrosome reaction was >80% as assessed by exclusion of propidium iodide. To confirm the subcellular localization of rat MCP, we used an acrosome-specific mAb, 18-6 (kind gift of Dr. H. Moore; University of Sheffield, UK) [36]. This mAb was generated by immunization of mice with hamster spermatozoa and is recognized as a marker of the acrosome in hamster and human [36]. The antigen recognized by 18-6 is undefined. Here we first confirmed that mAb 18-6 specifically stained the acrosome in rat spermatozoa. For double staining, mAb 18-6 (neat tissue culture supernatant, 50 µl) was first incubated on the glass slides smeared with the spermatozoa, followed by rhodamine-labeled donkey anti-mouse IgG (1: 200 in PBS; Jackson ImmunoResearch Laboratories, Philadelphia, PA). After this procedure, specimens were blocked with nonimmune mouse IgG and finally incubated with 50 µl of FITC-MM.1 anti-MCP mAb (diluted 1:50 in PBS).
Fresh human semen was obtained by masturbation after 3 days of abstinence from healthy volunteer donors of proven fertility. Informed consent was obtained from all donors in accordance with local ethical guidelines. After liquefaction at room temperature, samples were either fractionated by centrifugation at 1000 x g to separate spermatozoa from seminal plasma or used unfractionated. Whole semen was mixed 1:1 with lysis buffer and incubated as described previously prior to separation on SDS-PAGE. Separated spermatozoa were washed in PBS prior to suspension in lysis buffer and analyses. Human spermatozoa were also isolated by swim-up as described previously.
Preparation of Tissue Lysates
Epididymis and testis were obtained fresh from killed rats. The tissue was finely minced, suspended in 3 ml lysate buffer (for a pair of epididymi or one testis), and incubated for 60 min on ice. After incubation, insoluble debris was removed by centrifugation (15 000 x g, 15 min at 4°C), and supernatants were stored in aliquots at –80°C until use.
SDS-PAGE and Western Blot Analysis
Lysates were mixed 1:1 with sample buffer for SDS-PAGE and separated under nonreducing conditions on 10% gels. Separated proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, London, UK), and the membrane was blocked with 5% (w:v) nonfat milk in PBS (PBS-M). Membranes were then probed with the primary mAb diluted in PBS-M, washed in PBS containing 0.1% Tween-20 (PBS-T), then probed with horseradish peroxidase (HRPO)-conjugated goat anti-mouse Ig (BioRad) in PBS-M. To detect Ig-fusion proteins in Western blots, the membrane was incubated with HRPO-conjugated goat anti-human Fc antibody (1:1000 in PBS-M; Sigma Aldrich). To detect polyclonal antibody in Western blots, the membrane was incubated with HRPO-conjugated donkey anti-rabbit IgG antibody (1:1000 in PBS-M; Jackson). After further washing in PBS-T, bands were developed using ECL (Perbio Science; Helsingborg, Sweden) and captured on autoradiographic film (Kodak).
Immunohistochemical Analysis of Rat MCP Distribution
Male and female Wistar rats (12–16 wk) were killed, and the major organs and tissues, including heart, lung, liver, spleen, kidney, intestine, adrenal gland, bladder, ovary, oviduct, uterus, skin, eye, adipose tissue, skeletal muscle, femoral artery, femoral vein, cerebrum, cerebellum, spinal cord, sciatic nerve, testis, and epidermis, were removed, cut into blocks, and snap-frozen in isopentane at –40°C. Organs and tissues were also collected from 2-wk-old male Wistar rats, and intact rat embryos (15-day gestation) were additionally obtained for analysis. Frozen tissues were sectioned in a cryostat at 7 µm for brain, epididymis, and adipose tissue; 15 µm for ovary; and 5 µm for all other tissues. All sections were fixed in acetone at room temperature for 5 min. To observe the binding of mAbs, fixed sections were incubated with the optimal dilution (5 µg/ml) of mAb (determined by prior titration experiments), followed by FITC-labeled goat anti-mouse IgG antibody (1:200; BioRad) preabsorbed with normal Wistar rat serum. Counterstaining of nuclei was obtained by addition of DAPI (4'-6-diamino-2-phenylindole-2 HCl; 100 ng/ml final concentration; Sigma) with the secondary antibody.
Purification of Native Rat MCP
An affinity matrix was made comprising 5 mg of the mAb immobilized on CNBr-activated Sepharose 4B according to the manufacturer's instructions (Amersham Biosciences; Uppsala, Sweden). Tissue or cell lysates were diluted 1:1 in PBS containing 1 M NaCl and 0.2% NP40 and applied to the matrix packed in a column. After washing in the same buffer, bound MCP was eluted with 0.1 M glycine (pH 2.5) in 0.2% NP40, dialyzed into PBS/0.2% NP40, and concentrated by ultrafiltration. The purified protein was characterized by SDS-PAGE and Western blot analysis.
Cofactor Assay
All procedures were performed essentially as previously described [37]. For assessment of cofactor activity, hC3ma (50 µg/ml final concentration) was incubated in a final volume of 50 µl with rat fI (10 µg/ml) and either fractions containing rat MCP from the affinity column or sCR1 as a positive control. Samples were incubated for 4 h at 37°C, and the reaction was stopped by adding an equal volume of reducing SDS-PAGE loading buffer. The samples were briefly boiled, then loaded onto a 10% SDS-PAGE. Gels were Western blotted and blots probed with polyclonal sheep anti-human C3d (1:1000 in PBS-M; The Binding Site, Birmingham, UK) followed by HRPO-labeled donkey anti-sheep IgG (1:1000 in PBS-M; Sigma) as the secondary antibody and developed as described previously.
Deglycosylation of Rat Testis MCP
O-glycosidase and N-glycosidase (Roche, Mannheim, Germany) were used for the removal of O-linked and N-linked sugars, respectively, from rat MCP according to the manufacturer's instructions. Briefly, rat testis lysate prepared as described previously or a similar lysate prepared from the unfractionated cell pellet from human semen as a positive control was diluted (2:9 v:v) in 0.1 M sodium phosphate buffer, pH 7.4, containing 20 mM EDTA, 0.1% sodium dodecyl sulfate, and 1% NP40. N-glycosidase (0.5 U) and/or O-glycosidase (0.25 mU) was added to 60-µl aliquots of diluted lysate and incubated at 37°C for 24 h. Neuraminidase (Roche; 25 mU) was included in O-deglycosylation tubes to remove terminal disaccharide. Samples were then separated on SDS-PAGE and Western blotted as described previously. Blots were probed using the specific mAb or a polyclonal rabbit anti-human MCP (kind gift of Dr. T. Seya, Osaka, Japan).
Binding of C3ma by Rat Spermatozoa and Competition with MCP Fusion Protein
Rat spermatozoa, freshly isolated by swim-up and either acrosome-reacted or unreacted, were incubated at 106/ml in Ham F12 medium with hC3ma (50 µg/ml) at 37°C for 30 min and washed in PBS. The cells were then smeared onto glass slides, air-dried, fixed by brief immersion in acetone, and again air-dried. The fixed cells were washed in cold PBS, then stained by incubation with the anti-human C3b mAb C3/30 (10 µg/ml in PBS containing 1% BSA, 30 min on ice) followed by FITC-labeled donkey anti-mouse IgG (1:200 in PBS containing 1% BSA, 30 min on ice; Jackson) prior to mounting and imaging. The hC3ma was used in these preliminary experiments because of the availability of a mAb to detect its binding.
To confirm binding in a homologous system, rC3ma (50 µg/ml in 0.15 M bicarbonate buffer, pH 9.6) was used to coat alternate rows of wells in a 12-well plastic plate (Nunclon delta surface; NUNC, Roskilde, Denmark) by incubation at 37°C for 2 h. All wells were then blocked by incubation with 2% BSA in PBS followed by washing in PBS. Swim-up acrosome-reacted or unreacted spermatozoa (viability >80%), suspended at 106/ml in Ham F12 medium were applied to pairs of rC3ma-coated and control (block only) wells in triplicate and incubated for 1 h at 37°C. Wells were gently washed five times with cold Ham F12 medium. Bound spermatozoa were counted under 200x magnification in each well. The binding ratio of acrosome-reacted or unreacted spermatozoa to C3ma-coated and control wells was calculated for each pair of wells following the formula:
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In some experiments, the fusion protein MCP23-Ig at either 0.5, 0.1, or 0.01 mg/ml in PBS was preincubated for 15 min at 37°C with the C3ma-coated and BSA-coated wells prior to the addition of acrosome-reacted or unreacted spermatozoa as described previously. Control wells were pre incubated with PBS alone. The binding ratios were calculated as done previously.
Data are shown as the mean ± SEM of the ratios for triplicate sets of wells. The statistical analysis was performed by Student paired t-test, and a P-value less than 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although numerous hybridoma supernatants were posi tive for antibody reactive with MCP23-Ig in early screen ing, only a single strongly positive clone was retained through subsequent limiting dilution cloning. This IgG1 mAb, termed MM.1, was strongly reactive against MCP23-Ig but not CD59-Ig in ELISA and Western blotting (data not shown). The MM.1 mAb specifically detected rat MCP as a single band of approximate molecular mass (Mr) 38 kDa in Western blots of lysates from transfected CHO cells but not vector control cells (Fig. 1A).
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The Anti-Rat MCP mAb MM.1 Detects Native Rat MCP in Cell and Tissue Lysates
In Western blots of lysates from swim-up spermatozoa, epididymis, and testis, the MM.1 mAb specifically detected a single band of 
38 kDa (Fig. 1A shows results from a testis lysate; identical results were obtained with spermatozoa and epididymis). Lysate from unfractionated human semen, purified human spermatozoa, and the Jurkat human T cell line were also run and stained with rabbit polyclonal anti-human MCP as a comparator (Fig. 1A). This revealed the expected spermatozoa-specific MCP band in unfractionated seminal plasma and purified spermatozoa at an approximate size of 45 kDa, compatible with the published size [16]. A MCP band at approximately 60 kDa was present in Jurkat cells and also in unfractionated seminal plasma, presumably contributed by MCP from leukocytes in this preparation. Rat spermatozoal MCP was consistently smaller than human sperm MCP by 5 kDa.
Enzymatic Deglycosylation Reveals the Presence of N-Linked Sugars in Rat MCP
Treatment of immunoaffinity purified rat MCP with N-glycosidase reduced the molecular mass by approximately 3–5 kDa, demonstrating the presence of N-linked sugar on the protein (Fig. 1B). Treatment with O-glycosidase did not detectably alter the molecular mass, suggesting that O-linked sugars were resistant to enzymatic removal (Fig. 1B). As controls for the activities of the deglycosidases, human MCP from sperm-free seminal plasma (high-molecular mass form) was treated in an identical manner. Treatment with either N-glycosidase or O-glycosidase reduced the molecular mass of human MCP by 8–10 kDa, and the combination of the two enzymes reduced the molecular mass of human MCP by approximately 18 kDa (Fig. 1B). The doublets observed in human MCP likely represent the commonly expressed BC and C isoforms.
MCP Is Expressed Only in the Male Reproductive Tract in Adult Rats
Testis and spermatozoa in the lumen of the epididymis stained strongly with the MM.1 mAb (Table 1). An exhaustive search failed to reveal any specific staining with the MM.1 mAb in any other cells or tissues. Of particular relevance, no staining was observed in the female reproductive tissues, ovary, oviduct, and uterus; oocytes were clearly visible in the ovary and were negative.
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In the testis, differential staining with the MM.1 mAb was noted on cells in various stages of spermiogenesis (Fig. 2, A–F). The earliest precursors, spermatogonia, in the outer regions of the seminiferous tubules and round spermatocytes were not stained. Elongated spermatids, the immediate precursors of spermatozoa, stained strongly in the vicinity of the developing acrosome. Sertoli cells and phagocytic and secretory cells situated close to the lumen of the seminiferous tubule were not stained. Interstitial areas between the seminiferous tubules including the hormone-secreting Leydig cells and vascular elements, were also negative. Immature spermatozoa in the lumen of the seminal vesicles were strongly stained but only in the acrosomal region (Fig. 2, A–D).
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In the epididymis, staining was noted only on the spermatozoa in the lumen of the epididymal duct and was again restricted to the acrosomal region of spermatozoa (Fig. 2, G and H); epididymal epithelium and interstitium were not stained.
In immature male rats (2 wk), which lack late spermatozoal precursors and mature sperm and have seminiferous tubules containing predominantly Sertoli cells, the testis and epididymis were completely negative for MM.1 mAb staining (Table 1). Sections from embryos (15-day gestation) were also stained with the MM.1 mAb and were negative.
Rat MCP Is Expressed Only on the Inner Acrosomal Membrane
Whereas more than 95% of unfixed freshly isolated spermatozoa were not stained with the MM.1 mAb, following acetone fixation, 100% of sperm cells were stained with MM.1, and staining was restricted to the acrosomal region (Fig. 2, I and J). There was no positive staining with an isotype-matched control IgG (Fig. 2, K and L). To confirm the apparent restriction of expression to the inner acrosomal membrane, the acrosome reaction was induced by incubation of fresh sperm with the calcium ionophore A23187. After ionophore treatment, more than 50% of unfixed sperm were strongly stained by MM.1 with a typical acrosome-restricted pattern, confirming that MCP on rat spermatozoa was sequestered on the inner acrosomal membranes (Fig. 3, A and E). MM.1 staining on acrosome reacted spermatozoa completely coincided with that of mAb 18-6 (Fig. 3E), a specific marker of the acrosome (Fig. 3C) [35]. In contrast, unfixed swim-up sperm were not stained with either MM.1 or mAb 18-6 (Fig. 3, B, D, and F).
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Acrosome-Reacted Rat Spermatozoa Bind Methylamine-Activated C3
When unfixed, acrosome-reacted rat spermatozoa were incubated with hC3ma and then stained with the C3/30 mAb to detect bound hC3ma, 20%–30% of the cells were stained (Fig. 3, G and H). The staining was not solely acrosomal (evident from comparison with plates 3A and 3C) but was restricted to the head region of spermatozoa and was patchy/granular in appearance. Non-acrosome-reacted unfixed spermatozoa did not bind hC3ma (Fig. 3, I and J). In the absence of added hC3ma, the C3/30 mAb did not stain spermatozoa (data not shown).
In order to address further this C3-binding activity, unfixed acrosome-reacted spermatozoa were incubated in wells coated with rC3ma. Many cells became firmly adherent, through the head region, to the plastic (Fig. 4A). In contrast, very few nonreacted spermatozoa bound to rC3ma-coated wells (Fig. 4B), and neither acrosome-reacted nor nonreacted spermatozoa bound BSA-coated control wells (Fig. 4, C and D). Bound spermatozoa were counted in triplicate wells and results expressed as a ratio of binding to rC3ma-coated and control wells such that no increased binding would give a ratio of 0 (Fig. 4E). The mean binding ratio was 4.88 for acrosome-reacted spermatozoa versus 0.61 for nonreacted cells (P < 0.05). Binding to control wells was consistently low and was not different for acrosome-reacted and nonreacted cells.
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The specificity of the binding reaction for MCP was addressed by examining competition by the MCP23-Ig fusion protein. Preincubation of the rC3ma-coated wells with MCP23-Ig completely blocked binding of acrosome-reacted spermatozoa at all concentrations tested; the data shown are with MCP23-Ig at the lowest concentration, 0.01 mg/ml (Fig. 4E). The CD59-Ig fusion protein used as a control caused minimal inhibition of binding of spermatozoa at this concentration (not shown).
Cofactor Function of Sperm-Derived Rat MCP
We have shown previously that a recombinant soluble form of rat MCP has cofactor activity for the fI-mediated cleavage of methylamine-activated human C3 [28]. Here, we have sought to extend these observations to the native protein.
Used as a positive control, sCR1 catalyzed the specific production of a 68-kDa -chain cleavage product and also a subsequent fI cleavage event, unique to CR1, to produce a 41-kDa fragment (Fig. 5A, lane 1; Fig. 5B). Rat MCP, partially purified from testis lysate by immunoaffinity chromatography on MM.1, catalyzed the first of these cleavage events in the presence of fI, yielding the 68-kDa -chain product with no further cleavage. Omission of fI prevented cleavage catalyzed by either sCR1 or rat MCP, confirming the specificity of the reaction (Fig. 5A, lanes 3 and 5). In the absence of any cofactor, fI did not cleave C3ma (Fig. 5A, lane 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In rats, mice, and guinea pigs, MCP mRNA is significantly expressed only in testis [26, 28, 42], implying that the molecule has been retained at this site because it plays an essential and unique role in this organ. In New World primates, only MCP expressed in testis retains SCR1, again implying a special role [43]. In man and primates, MCP is strongly expressed on the inner acrosomal membrane of spermatozoa, a site that is exposed only to the external milieu immediately prior to interaction with the egg, and transgenic expression of human MCP in mice yields an identical acrosome-restricted expression [18].
In order to utilize rodents as models for the study of the role of MCP in sperm-egg interaction, it is first necessary to discover whether MCP is expressed at the relevant sites. Our analyses by Northern blotting of mRNA expression in rat tissues demonstrated that MCP was strongly expressed only in testis; however, Reverse transcription-polymerase chain reaction analyses did indicate low levels of mRNA in other tissues [28]. In order to further analyze the distribution of rat MCP and to confirm the cellular and subcellular localization of the protein in testis, we undertook to generate mAb against rat MCP. Here, we report a specific mAb reactive against rat MCP, MM.1, that has been obtained by immunizing mice with a Fc fusion protein containing the second and third SCR domains of the protein. The mAb detected a protein band of 38 kDa in Western blots of lysates from epididymis, testis, and sperm purified by swim-up. MCP on human sperm has a reported approximate size of 45 kDa [16], and we found a similar size in our analyses (Fig. 1). In humans, sperm express only the C2 isoform of MCP, containing the short STP-C and the 23 amino acid cytoplasmic tail 2; the low molecular mass of sperm MCP compared with that of the C2 isoform in other tissues is caused by the postsynthetic trimming of the three large N-linked carbohydrate groups to small, simple structures [18]. The predominant mRNA species obtained from rat testis comprised four SCR domains: a short STP region resembling STP C in humans, undefined and transmembrane regions, and a very short cytoplasmic region of just seven amino acids and related to the intracytoplasmic anchor of human MCP [28, 42]. Absence of the tail region in rat MCP likely explains the lower apparent molecular mass when compared with human sperm MCP. Rat MCP also differs from human in that the N-glycosylation site in SCR2 is absent; enzymatic N-deglycosylation of MCP in rat testis lysate caused a decrease in molecular mass of 3–5 kDa, suggesting that the two remaining N-glycans were simple structures as described for human sperm MCP in humans and in the human MCP transgenic mouse [18]. O-deglycosylation did not detectably alter the molecular mass, indicating that the O-linked sugars were resistant to digestion, as reported for MCP on human sperm [18]. Testis-derived rat MCP expressed cofactor activity for fI-mediated cleavage of C3b, confirming that it was the functional analogue of human MCP.
An immunohistological survey of rat tissues using the MM.1 mAb demonstrated that MCP is expressed only in the testis and epididymis from adult rats, more specifically, only by spermatozoa and late spermatozoal precursors. Early precursors in the process of spermiogenesis, spermatogonia, and spermatocytes were negative, as were the interdigitating Sertoli cells in the seminal vesicles. The staining pattern on fixed spermatozoa and precursors mirrored the course of acrosome formation [44]. MCP was not detected in female reproductive tissues, such as ovary, oviduct, and uterus, and was absent in the testis prior to puberty. Acrosome-restricted expression was confirmed in isolated spermatozoa where staining was detected only after fixation or induction of the acrosome reaction. This observation was further supported by demonstration of colocalization with mAb 18-6, an acrosome-specific mAb [36]. These results resemble the findings in humans, where sperm MCP expression is restricted to the inner acrosomal membrane; indeed, staining with anti-MCP is used as an indicator of the acrosome reaction in clinical analyses of sperm function [45, 46].
Expression of MCP correlated precisely with the appearance of the acrosome in late precursors and mature spermatozoa. The acrosome reaction, involving exocytic exposure of the inner acrosomal membrane and release of acrosomal contents, occurs after the sperm has made contact with the cells of the cumulus and zona pellucida complex that surrounds the oocyte and is essential to enable penetration of the zona [44, 47]. Enzymes released from the acrosome aid penetration, and receptors on the inner acrosomal membrane mediate oocyte binding and fusion events. The nature of the interacting molecules is incompletely understood, and the part that MCP plays in this complex process remains unknown. A decade ago, Anderson and colleagues [48] demonstrated that MCP on acrosome-reacted human spermatozoa specifically bound dimeric C3b and implicated this association in the adhesion of sperm and oocyte. These authors suggested that C3b or other C3 fragments bind MCP on sperm as well as complement receptors on the oocyte, providing a bridging interaction essential for fertilization. However, mAbs that block the C3b-binding site on MCP did not efficiently block sperm-oocyte interaction, whereas mAb binding SCR1 in MCP did block this interaction, indicating that sites other than the C3b-binding site are involved in the association with spermatozoa [19–21]. Here, we have shown that acrosome-reacted rat spermatozoa also express an MCP-dependent capacity to bind immobilized activated C3 and that sperm MCP has fI cofactor activity. Studies are ongoing to address the roles of activated C3 and MCP in sperm-oocyte interactions in rats.
The observation that some infertile males may have an unexplained sperm-specific MCP deletion, while others have low levels of soluble MCP in seminal plasma, lends further weight to suggested involvement of MCP in fertilization [17, 23, 49]. However, C3 deficiency in humans and mouse is not associated with infertility, weakening the case for an essential role of C3 in the process. While this work was in preparation, others reported MCP-gene deleted mice whose phenotype was surprising [50]. Far from being infertile, the male mice were apparently hyperfertile and produced larger litter sizes than MCP-positive controls. Analyses of sperm in vitro demonstrated instability of the acrosome and an accelerated acrosome reaction, suggesting a role for MCP in the maintenance of acrosome integrity. No clear explanation for these confounding results has yet emerged.
The demonstration that MCP in the rat is expressed exclusively on the inner acrosomal membrane and the availability of a specific mAb against rat MCP, as well as recombinant protein, open new possibilities for experimentation. It will now be possible to examine the effects of both MCP blockade with mAb and addition of soluble rat MCP on in vitro sperm-oocyte interaction, on sperm survival in the face of complement attack, and on in vivo fertility. These studies will enable the roles and mechanisms of action of MCP in sperm survival and sperm-egg interaction to be elucidated, providing new avenues for investigation of these processes in humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: B. Paul Morgan, Complement Biology Group, Department of Medical Biochemistry and Immunology, University of Wales College of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, United Kingdom. FAX: 44 2920744905; morganbp{at}cardiff.ac.uk
Received: 24 March 2004.
First decision: 13 April 2004.
Accepted: 8 June 2004.
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