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a Institute of Biochemical Sciences, College of Science, National Taiwan University, and
b Institute of Biological Chemistry, Academia Sinica, Taipei 10746, Taiwan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have purified a PI of Mr 6126 from mouse seminal vesicle secretions (SVS) and identified it as a Kazal-type trypsin inhibitor with very strong affinity to trypsin [10]. It is a basic polypeptide of 57 amino acid residues with no glycoconjugate. Its primary structure is identical to P12 deduced from P12 cDNA that has been cloned from mouse ventral prostate by Mills et al. [11]. Recently, a recombinant P12 with full activity of the naturally occurring P12 was recovered from a chimerical polypeptide of glutathione-S-transferase and P12 (GST-P12) expressed in Escherichia coli [12].
The P12 RNA message is detectable in the male accessory sexual glands of adult mice while its expression is constitutive in pancreas [13]. Substantial progress has been made to establish its genomic structure. As a result, the DNA-binding sites for some transcription factors such as GC2 and SP1 have been identified in this gene [14, 15]. On the other hand, less progress has been made in studying the role of P12 in reproductive biology despite its presence in SVS. Accordingly, we conducted this work to better understand the characteristics of P12-sperm binding as well as the developmental profiles of P12 in the seminal vesicle and its binding sites during sperm generation in the testis. In addition, we assessed the ability of P12 to suppress Ca2+ uptake by spermatozoa.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The seminal vesicles of mature male mice (ICR, 8–12 wk; Charles River Laboratories, Wilmington, MA) killed by cervical dislocation were carefully dissected free of the adjacent coagulating glands. The secretions collected from 100 mice were expressed directly into 100 ml of ice-cold 5% acetic acid. P12 was purified from SVS according to our previous procedure [10]. A fusion protein of GST-P12 was prepared from the expression of a recombinant DNA in E. coli [12]. A recombinant P12 with the full activity of the naturally occurring P12, with regard to its inhibitory effect on trypsin activity, was recovered from the chimerical polypeptide.
Detection of P12 and Its mRNA in Reproductive Tracts
The protein in normal saline (1.0 mg/ml) was mixed with an equal volume of complete adjuvant. Rabbits received intrasplenic injection of 0.25 ml of the mixture. After 12 wk, the animals were treated in the same way with 100 µg of antigen in a mixture of normal saline and incomplete adjuvant (1:1 by volume). The antiserum was collected 2 wk later. The P12 antibody was partially purified by affinity chromatography on a protein A-Sepharose (Pharmacia, Uppsala, Sweden) column.
The protein extract of tissue homogenate was resolved on a 15% polyacrylamide gel slab (6.5 cm x 10.5 cm x 0.075 cm) by the method of Laemmli [16]. The proteins on the gel were stained with Coomassie brilliant blue dye or transferred to a nitrocellulose membrane. After transfer, protein blots were immunodetected by Western blot procedures, using the P12-induced antisera or the partially purified antibody as the primary antibody and goat anti-rabbit IgG conjugated with horseradish peroxidase as the secondary antibody.
The total RNA of fresh tissue was prepared by the acid guanidium thiocyanate/phenol/chloroform method [17]. RNA samples were analyzed by separation on a 1.0% agarose/formaldehyde-containing gel [18], followed by capillary transfer to a nylon membrane, and were hybridized to 32P-labeled nucleic acid probes [19]. 32P-Labeled random-primed probe was prepared with a Promega random-priming kit (Promega, Madison, WI) using a template of P12 cDNA (369 base pairs [bp]) [12] or a cDNA segment of mouse ß-actin gene inserted into a pGEM4Z vector.
Preparation of Spermatozoa
Cells were extruded from the distal portion of the caudal epididymis of adult male mice in a modified Tyrode's solution containing 125 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 0.36 mM NaH2PO4, 4.5 mM glucose, 0.09 mM pyruvate, 8.9 mM lactate, 0.5 mM taurine, 100 IU/ml penicillin, and 100 µg/ml streptomycin at pH 7.4 [20]. The cell suspension was gently filtered through two layers of nylon gauze, layered on top of a linear gradient of 10–90% Percoll (Pharmacia), and centrifuged at 150 x g for 10 min. Two distinct cell layers were formed. The lower layer, which contained more than 95% of viable cells with progressive motility [21, 22], was diluted with three volumes of modified Tyrode's solution and centrifuged at 150 x g to remove Percoll solution. The cells were resuspended in modified Tyrode's solution (1.0 x 107 cells/ml) and used for the study. Throughout this study we used only the preparation in which more than 70% of cells were motile.
Histological Studies
Tissues were fixed in freshly prepared Bouin's solution (0.2% picric acid and 2% formaldehyde in PBS) overnight, dehydrated in ethanol, infiltrated, and embedded in paraffin (Paraplast m.p. 56–57; Curtin Matheson Scientific Co., Houston, TX). Each tissue section (7 µm) was mounted on a slide that had been precoated with a solution containing 0.25% gelatin (Gibco, Grand Island, NY) and 0.025% chromium potassium sulfate. Sections were dried at 45°C, deparaffinized in xylene, and rehydrated through a gradient from alcohol to distilled water. The rehydrated sections were placed in a saturated lead thiocyanate solution and heated according to the method of von Wasielewski et al. [23]. After the slides had been cooled at room temperature for 15 min, they were rinsed in distilled water and PBS each for 5 min. The slides were immersed in a blocking solution (5% nonfat skim milk in PBS) in a moisture chamber at 25°C for 1 h and washed with PBST (0.05% Tween in PBS) four times, each for 15 min. The testis sections on slides were preincubated with 3.0 µM P12 in PBS at 25°C for 1 h, and the slides were washed with PBST as above. The slides prepared from each tissue were incubated for 2 h in the presence of the P12-induced antiserum diluted 1:250 in blocking solution. After the slides had been gently agitated in four changes of PBST for 15 min each, the antibody to P12 were immunodetected with alkaline phosphatase-conjugated anti-rabbit IgG (Sigma, St. Louis, MO) diluted 1:1000 in the blocking solution. The sections were covered with 50% glycerol in PBS and photographed with a microscope (AH3-RFCA; Olympus, Tokyo, Japan) after they had been washed with three changes of PBS for 15 min each.
Spermatozoa were air-dried on a glass slide and washed twice with PBS before cytochemical staining. The slides were immersed in 3.5% perchloric acid containing 0.04% Coomassie G-250 to manifest the intact acrosomes of spermatozoa according to previously described methods [24, 25]. To examine P12-binding region on spermatozoa, the slides were incubated with 3.0 µM P12 for 40 min, washed with PBS, and incubated with the P12-induced antiserum diluted 1:250 in the blocking solution for 30 min. The slides were washed three times with PBS to remove excess antibody before they were incubated with fluorescein-conjugated goat anti-rabbit IgG (Sigma) diluted 1:100 in the blocking solution for 50 min. All of the slides were rinsed with PBS and covered with PBS:glycerol (1:1 by volume) before observation under a microscope equipped with epifluorescence (AH3-RFCA).
45Ca2+ Uptake by Spermatozoa
Ca2+ uptake by epididymal spermatozoa was measured according to the method of Coronel and Lardy [26]. Before this measurement, the spermatozoa in the modified Tyrode's solution (1.0 x 107 cells/ml) were preincubated with 1.5 µM P12 or recombinant P12 at 37°C for 10 or 60 min. Then they were incubated in the presence of 200 µM CaCl2 labeled with 45Ca2+ (10–40 µCi/mg calcium; Amersham, Arlington Heights, IL) at 37°C for 10 min, and the cells were collected by vacuum filtration under 0.16 atm through a cellulose acetate filter. The cells on the filters were washed with six volumes of modified Tyrode's solution. The filters were dried and transferred into scintillation vials containing 5.0 ml of scintillation counting cocktail (Merck, Darmstadt, Germany). Radioactivity was counted by a liquid scintillation counter (Parker; LKB, Stockholm, Sweden).
Assay of P12 Binding to Spermatozoa
P12 was labeled with 125I-Na in the presence of Iodobead (Pierce, Rockford, IL) according to previously described method [27]. The radiolabeled P12 was used to characterize the P12-binding site on sperm. Epididymal spermatozoa were incubated with 125I-P12 at 25°C. The cells were collected on a Whatman (Clifton, NJ) GF/C glass microfiber filter by rapid filtration at a pressure of 0.5 atm. The filters had been blocked with 1% nonfat skim milk in 50 mM Tris buffer at pH 7.4 for 30 min and washed with ice-cold buffer before they were used. The filter was washed with six changes of 0.2 ml of ice-cold PBS and air-dried on a filter paper. Radioactivity of the filter was counted by a 
-counter. The nonspecific binding of 125I-P12 to cells was measured by addition of a 100-fold excess of nonradioactive ligand. Each assay was performed in triplicate, and specific binding was the difference between the averages of the total binding and the nonspecific binding.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hypodermic injection of P12 to rabbits poorly induced the production of P12 antibody. Instead, we immunized the animals by intrasplenic injection of P12. The antiserum prepared from this method showed strong immunoaffinity to P12 as well as to GST-P12 but did not cross-react with GST (cf. lanes 1–3 of Fig. 1). Among the protein components of SVS, which contained a trace amount of P12 (Fig. 1A, lane 4), the antiserum immunoreacted only with P12 (Fig. 1B, lane 4), showing the high specificity of the P12 antibody in the antiserum. Therefore, we used the antiserum for the immunodetection of P12 throughout this study.
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We examined the distribution of P12 in the genital tracts of adult mice by Western blot analysis. In the male reproductive tracts, P12 was demonstrated in the homogenates of seminal vesicle, coagulating gland, and prostate, but it was not detectable in the homogenates of other sexual organs such as testis, epididymis, and vas deferens (Fig. 2). This is in agreement with the previous examination of the distribution of P12 mRNA in the male sexual organs [13]. The protein was not found in the female reproductive tracts including ovary, oviduct, uterus, and vagina. The existence of this protein in the three accessory sexual glands of adult male mice was also confirmed in the immunohistochemical staining patterns of P12 in the tissue sections shown in Figure 3. Abundant P12 appeared in the concretion of the glandular alveolar in the prostate and in the fluid in the lumen of both the seminal vesicle and coagulating gland. In the seminal vesicle, P12 was immunolocalized on the luminal epithelium of primary and secondary mucosa folds, but it was absent in smooth muscle cells, indicative of the secretion of P12 from the epithelium of mucosa folds to lumen. The secretion of P12 from the epithelium of mucosa folds was also noted in the coagulating gland. In the prostate, the secretion of P12 from the fold projecting into the lumen of glandular alveolus was obvious.
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Developmental Profile of P12 Expression in the Seminal Vesicle
It is well known that accumulation of SVS, which contains a group of proteins that constitute the major protein components of seminal plasma, becomes prominent in the postpubertal period. We compared the levels of P12 and its message in the seminal vesicle at different ages of male mice in order to assess whether the P12 expression coincided with the development of this accessory sexual gland. The P12 RNA message was undetectable in the mice younger than 3 wk old, increased remarkably when they were 4 wk old, and remained at that level thereafter (Fig. 4A). The protein level corresponding to this change was examined by Western blot analysis, shown in Figure 4B. The protein did not appear in mice younger than 4 wk old, but it increased progressively with age after 5 wk. Apparently, P12 is synthesized by the translation at a constant level of P12 mRNA in the epithelium of mucosa folds, and the successive secretion from the epithelial cell results in its accumulation in the lumen. The positive correlation of P12 and its RNA message with the development of the seminal vesicle prompted us to assess the effect of androgen on P12 expression in the accessory sexual gland. Figure 5 displays the Western blot analyses of P12 in the tissue homogenates of seminal vesicles from mice (14 wk) treated in several ways. The protein disappeared in the tissue of mice that had been castrated 3 wk earlier (Fig. 5, lane 2). The protein was not detectable in the tissues of castrated mice that had received a daily injection of corn oil only (Fig. 5, lane 3). The protein reappeared in the tissues of castrated mice after they had received daily injections of testosterone propionate in corn oil for 8 consecutive days (s.c., 5 mg/kg of body weight per day; Fig. 5, lane 4). As compared with the P12 level in seminal vesicles of normal adults (Fig. 5, lane 1), the absence of P12 in the control castrated mice and its recovery soon after the hormonal administration revealed androgenic stimulation of P12 expression in the seminal vesicle.
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P12 Binding Site on Sperm Surface
No fluorescence appeared on the epididymal spermatozoa after they had been immunoreacted successively with the P12 antiserum and the fluorescein-conjugated anti-rabbit IgG, manifesting the lack of P12 on the cell surface. When the cells were preincubated with P12 before cytochemical staining, a crescent fluorescence zone on the anterior region of the sperm acrosome was visible (Fig. 6), indicating P12-binding sites on the acrosomal region. The proportion of epididymal spermatozoa having intact acrosomes was compared to the proportion of epididymal sperm with the P12-binding zone after they had been incubated in a modified Tyrode's solution according to the previously mentioned study [26] (Table 1). Around 86% and 85% of the fresh cells from caudal epididymis had an intact acrosome and P12-binding zone, respectively. The population of cells with an intact acrosome decreased progressively as long as the incubation proceeded. This was parallel to the decrease in the cell population with the P12-binding zone. At any incubation time, the population of cells with an intact acrosome was nearly equal to that with the P12-binding zone, and the cell morphology showed no visible change in the presence of P12 in the cell incubation, indicating P12-binding sites on intact acrosome.
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125I-P12 was used for the quantitative characterization of sperm-P12 binding. To minimize the nonspecific binding of P12 to the glass microfiber filters used in the binding assay, the filters had been blocked with 1.0% skim milk for 30 min. This resulted in low background binding of the radiolabeled P12 to the filters. Consequently, the nonspecific binding of ligand to the cells was sufficiently low to allow the measurement of specific binding. Figure 7 displays the data from one representative determination from the cell incubation (1.0 x 107 cells/ml) in 50 mM Tris-1.0% skim milk (pH 7.4) in the presence of 100 nM 125I-P12. The equilibrium binding of 125I-P12 to sperm reached a plateau after a 45-min incubation. Therefore, a fixed number of sperm cells (1.0 x 107 cells/ml) were incubated with an increasing ligand concentration for 60 min to determine the steady-state ligand saturation (Fig. 8). The nonspecific binding of 125I-P12 to the cells was determined by addition of a 100-molar excess of unlabeled P12. The amount of 125I-P12 bound to the sperm surface increased with increasing ligand concentration until the available binding sites were saturated. The isotherm saturation suggested 1.2 x 106 P12-binding sites per sperm cell. A Scatchard plot of the data on equilibrium binding due to specific binding yielded a linear plot (Fig. 8 inset) indicating that a single type of binding site on sperm is most likely for P12. The apparent dissociation constant (Kd) was determined to be 70.15 ± 5.25 nM, and 1.49 ± 0.06 x 106 binding sites/cell was estimated from the maximum binding capacity (Bmax). The latter is comparable to that estimated from the isotherm saturation.
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We examined the development of P-12 binding sites during sperm generation in the testis. In contrast to the cell morphology in the tissue (Fig. 9A), no P12 appeared in the seminiferous tubules or Leydig cells (Fig. 9C). P12 could be immunolocalized (Fig. 9B) in spermatids and spermatozoa but remained undetectable in Leydig cells, Sertoli cells, and precursor cells such as spermatogonia and spermatocytes in the tissue slices preincubated with P12 (Fig. 9B).
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Suppression of Calcium Transport into Spermatozoa by P12
We measured 45Ca2+ uptake by epididymal mouse spermatozoa incubated in modified Tyrode's solution (1.0 x 107 cells/ml) in the absence or presence of 1.5 µM P12 or recombinant P12 at 37°C. On the basis of the study by Coronel and Lardy [26], cell surface binding was represented by the uptake of 45Ca2+ by fresh spermatozoa without cell capacitation. Thus, the induced 45Ca2+ uptake, which reflected the calcium transport into cells, was estimated by subtraction of the cell surface binding from the total 45Ca2+ uptake. At any incubation time, the induced 45Ca2+ uptake by cells incubated in the presence of P12 was less than by those incubated in the absence of P12 (Table 2). About 50% of Ca2+ transport to the control cells during a 60-min incubation could be suppressed by P12. Recombinant P12 was as strong as the naturally occurring P12 in suppressing the incorporation of Ca2+ by spermatozoa. These data support the identification of P12 as a caltrin.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The synthesis of some secretory proteins from male sexual glands and several aspects associated with the growth of genital tracks are androgen-dependent as are some differentiation processes. As shown, the castration of male adults results in the disappearance of P12 mRNA in the seminal vesicle, and the gene expression is restored in the sexual gland after administration of testosterone to the castrated males. This is consistent with the lack of P12 as well as its RNA message in the seminal vesicle before puberty, when serum androgen is at a low level (Fig. 5), representing a positive correlation of the androgen-dependent P12 expression in the accessory sexual gland with the animal's maturation.
The P12-binding sites are on the anterior region of the acrosome of mouse sperm. Corresponding to the histological localization of the cells developed during spermatogenesis and spermiogenesis in the testis, they are present in the cell layers closely restricted to the lumen of seminiferous tubules. Their absence in the spermatogenic cells such as spermatogonia and spermatocytes indicates that the P12-binding sites are synthesized predominantly in the postmeiotic cells such as spermatids and spermatozoa. Thus, the biosynthesis of P12-binding sites is likely to start in the early haploid spermatids during sperm generation.
The study of Boettger-Tong et al. [30] shows the inhibitory effect of a PI (P12) on the binding of ZP3 to sperm. The recent study of Thaler and Cardullo [31] indicates the presence of 3.0 x 104 ZP3-binding sites/sperm cell and the complexity of ZP3-sperm binding, which is attributed to the cooperative interaction between a low-affinity ZP3-binding site with a Kd value of 50 nM and a high-affinity ZP3-binding site with a Kd value of 0.72 nM. They suggest the involvement of multiple receptors on the sperm surface and/or multiple ligand moieties. This work illustrates a single type of P12-binding site ([1.49 ± 0.06] x 106 sites/cell) on the sperm surface with a Kd value of 70.1 ± 5.3 nM. Apparently, the ZP3-binding sites cannot account for the capacity and characteristics of the P12-binding sites, even though the binding strength of P12-sperm binding sites is as strong as that of the low-affinity ZP3-binding sites.
Trypsin shows a strong affinity to P12, with a Kd value of 0.15 nM [10]. Since acrosin is a serine protease of the trypsin superfamily, this raises the question of whether acrosin or/and its zymogen form (proacrosin) is involved in the P12-binding sites in the acrosomal region. No active acrosin is present in intact spermatozoa [32], and it is released from the cell once proacrosin is activated during acrosomal exocytosis at the fertilization sites in the oviduct. Thus, no acrosin but proacrosin exists in spermatozoa before their passage to the oviduct. Since proacrosin is not exposed on the sperm surface but is located in the acrosomal matrix of intact sperm [33], P12 is unlikely to interact with the zymogen unless P12 penetrates into the acrosomal region. In fact, there is one protein component, which is distinct from acrosin/proacrosin but is capable of binding a PI of seminal vesicle origin, in the supernatant of frozen-thawed murine epididymal sperm suspension [34].
As stated by Barros [35], the fertile condition of spermatozoa is not a terminal condition but rather a transient one. It is well recognized that mammalian sperm from epididymis should undergo some Ca2+-dependent modifications before fertilization. In the reproductive tract, the Ca2+ concentration is sufficient to elevate intracellular Ca2+ in the induction of these cell modifications at any time earlier than the sperm-egg encounter. However, these modifications far from the oviduct would cause spermatozoa to become infertile. Thus, the calcium movement across the membrane of spermatozoa should be prohibited at ejaculation until the cells reach the oviduct. In this regard, caltrin is believed to play a most important role in the inhibition of Ca2+ uptake by sperm. This kind of protein was identified and purified originally from bovine seminal plasma and subsequently from seminal vesicle fluid of the guinea pig [36]. Our results support the idea that P12 is a caltrin-like protease inhibitor. The P12-sperm binding leading to the suppression of Ca2+ movement across the cell membrane sheds some light into the function of the P12-binding sites. The primary structure of P12 shows a high degree of similarity to that of a caltrin containing 56 amino acid residues in rat seminal vesicle fluid [9]. A caltrin consisting of 75 amino acid residues in mouse seminal vesicle fluid has been characterized by Coronel et al. [9]. This caltrin and P12, though they coexist in seminal vesicle fluid, show no appreciable sequence similarity. It is possible that they exhibit different mechanisms in the regulation of calcium movement across the membranes of sperm. Future study is needed to elucidate how the dual roles of P12 as a protease inhibitor and as a caltrin interplay in the regulation of sperm activity.
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FOOTNOTES |
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2 Correspondence: Yee-Hsiung Chen, Institute of Biochemical Sciences, College of Science, National Taiwan University, P.O. Box 23–106, Taipei 10746, Taiwan. FAX: 886 22363 5038; bc304{at}gate.sinica.edu.tw
Accepted: July 14, 1998.
Received: May 13, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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