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Keywords:

  • intracellular calcium concentration;
  • Kazal-type protease inhibitor;
  • sperm activity modulation;
  • Spink–spermatozoa interaction;
  • trypsin-like activity

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The reproductive-derived serine protease inhibitor Kazal-type (Spink) has been identified in seminal plasma, and Spink–spermatozoa binding has been illustrated in many mammalian species including human. We used mice as experimental animal to study the mode of Spink action in the modulation of mammalian sperm activity. A Spink3-binding zone was cytochemically stained on the sperm head at apical hook separated from intact acrosome, whether the cells were capacitated or not. The Spink3–spermatozoa binding neither changed the population of cells in the uncapacitated, capacitated and acrosome-reacted status nor affected the capacitation-related protein phosphorylation and cell motility enhancement. Despite that, the Spink–spermatozoa interaction resulted in decreasing the intracellular calcium concentration ([Ca2+]i) of the cell head and suppressing both the acrosome reaction induced by Ca+2 ionophore A23187 and the cell fertility. Furthermore, Spink3 seen on the head of spermatozoa in the uterine cavity after coitus could be removed by the trypsin-like activity in the uterine fluid of oestrous females, and free Spink3 in the uterine cavity suppressed the protease activity. We integrated our data to shed light on the molecular mechanism of how Spink and its inhibiting protease are interplayed to modulate the activity of mammalian spermatozoa during their transit in the reproductive tract.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Low molecular-weight protease inhibitors are ubiquitous in the mammalian genital tracts (Fink & Fritz, 1976; Fritz et al., 1976; Meloun et al., 1983). It is believed that they are important in balancing protease activities for the protection of genital tract epithelium against damage by proteolysis (Tschesche et al., 1982). In addition, they provide a physiological function by regulating the fertilization process (Cechová & Jonáková, 1981; Huhtala, 1984).

There are 48 distinct families of protease inhibitors, one of which is the serine protease inhibitor Kazal (Spink) family (Rawlings et al., 2004). Spinks have been identified in the seminal plasma of different mammalian species: for example, human acrosin-trypsin inhibitor (Spink2) (Fink et al., 1990), bull inhibitor type IIA (Spink 6) (Meloun et al., 1983), porcine inhibitor (Tschesche et al., 1982), rabbit protease inhibitor (Zaneveld et al., 1971) and guinea pig seminal vesicle protease inhibitor (Fink & Fritz, 1976). Although a mouse seminal vesicle inhibitor (SVI) possessing many similarities to bovine pancreatic trypsin inhibitor has been identified (Haendle et al., 1965; Fritz et al., 1968; Poirier & Jackson, 1981), its molecular structure remains obscure. A Kazal-type protease inhibitor consisting of 57 amino acid residues with an inhibitory constant (Ki) of 0.15 nm to trypsin has been purified also from mouse seminal vesicle secretion (Lai et al., 1991). As this rather small protein was derived from the P12 cDNA cloned from the mouse ventral prostate by Mills et al. (1987), it was tentatively named P12. According to the Mouse Genome Informatics nomenclature committee, P12 is now renamed mouse Spink3 (NCBI reference sequences NP_033284). SVI may be identical to Spink3, as they are secreted from common origin and have similar molecular sizes. In the genital tracts of adult mice, Spink3 is exclusively expressed in the male accessory sexual glands, and the sperm cell possesses a single-type Spink3-binding site (1.49 × 106 sites/cell) with a Kd-value of 70 nm (Chen et al., 1998).

Under natural coitus, mammalian spermatozoa exhibit an intriguing sense of timing through a series of physiological changes at different sites in the reproductive tract to acquire fertilizing ability. Although the Spink–spermatozoa binding has been illustrated on different mammalian species such as mouse (Irwin et al., 1983; Chen et al., 1998), human (Dietl et al., 1976), boar (Schill et al., 1975) and bull (Veselsky & Cechová, 1980), the precise mechanism of how Spink is involved in modulating the activity of spermatozoa during their transit in the reproductive tract has not been convincingly demonstrated. In this study, we used mice as an animal model to study the mode of Spink action on spermatozoa. Here, we propose our findings to conclude that: (i) Spink3 binding on the apical hook of sperm head diminishes the acrosome reaction (AR) of capacitated spermatozoa via the decrease in the intracellular calcium concentration ([Ca2+]i) of the cell head to prevent spermatozoa from becoming infertile before encountering an egg; (ii) Spink3 on spermatozoa reduces in vitro fertility, and the Spink3-inhibiting trypsin-like activity (SITA) secreted from the uterus of oestrous females under natural coitus releases Spink3 from spermatozoa to restore their fertilization ability; (iii) the proteolytic damage to spermatozoa because of SITA is suppressed by free Spink3 in the uterine cavity.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Animals

Outbred ICR mice were purchased from Charles River Laboratories (Wilmington, MA, USA) and bred in the animal centre at the College of Medicine, National Taiwan University. All the animals were housed under conditions of controlled humidity, temperature and light regimen and fed ad libitum on standard mouse chow. Animal care was consistent with institutional guidelines for the care and use of experimental animals. Mice were killed humanely by cervical dislocation.

Materials

The following materials were obtained from commercial sources: N-benzoyl-Phe-Val-Arg-p-nitroanilide HCl, fatty acid-free bovine serum albumin (BSA), polyvinyl alcohol, diethyl stibesterol (DES), Sephadex G-100, chlorotetracycline, tetramethylrhodamine isothiocyanate-conjugated peanut agglutinin (Arachis hypogea) (TRITC–PNA), Hoechst 33258 and trypsin (Sigma-Aldrich, St Louis, MO, USA); Percoll and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (GE Healthcare, Amersham Biosciences, Uppsala, Sweden); fluo-3 acetoxymethyl ester (Fluo-3 AM) (Molecular Probes, Eugene, OR, USA); Calcium ionophore A23187 (Fluka, Buchs, Switzerland); Anti-mouse IgG conjugated with horseradish peroxidase (HRP) (Cell signaling, Santa Clara, CA, USA); PY-99 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Enhanced chemiluminescence (ECL) plus (Amersham Pharmacia Biotech, Buckinghamshire, UK). All chemicals were of reagent grade.

Preparation of spermatozoa, Spink3 and R19L, and fractionation of uterine luminal fluid (ULF)

Adult male mice (8–12 weeks) were sacrificed by cervical dislocation. Spermatozoa from the caudal epididymes were harvested using a swim-up procedure with 30 and 60% Percoll density gradients (Miyake et al., 1989; Ruiz-Romero et al., 1995; Chen et al., 1998). Polyvinyl alcohol (1 mg/mL) was added as a sperm protectant (Bavister, 1981). The contents were individually collected from the uterine cavity and the oviductal lumen of female mice with a plugged vagina after coitus.

The sperm cells were extensively washed with PBS before use. The soluble portion of uterine cavity and ULF collected from oestrus females was stored at −70 °C for further use. Spink3 was purified from seminal vesicle secretion (Lai et al., 1991). The recombinant R19L and the rabbit antiserum against spink3 were prepared according to the previous procedures (Lai et al., 1994; Luo et al., 2004).

The immature female mice (21 days old) were injected subcutaneously with DES in corn oil at a daily dose of 100 ng/g of body weight for three consecutive days and sacrificed by cervical dislocation when 24-day old. Uterine luminal fluid was collected and the soluble portion of ULF was fractionated by gel chromatography on a Sephadex G-100 column as described earlier (Chu et al., 1996).

Cytological observations

The culture medium used in this study was modified 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) medium (HM) as described earlier (Huang et al., 2000). In brief, the modified HM contained 120.0 mm NaCl, 2.0 mm KCl, 1.20 mm MgSO4·7H2O, 0.36 mm NaH2PO4, 15 mm NaHCO3, 10 mm HEPES, 5.60 mm glucose, 1.1 mm sodium pyruvate and 1.7 mm CaCl2. The pH of the medium was adjusted to 7.3–7.4 with humidified air/CO2 (95 : 5) in an incubator at 37 °C for 48 h before use. Percoll-separated sperm suspension (106 cells/mL) was incubated in modified HM containing the specified chemicals at 37 °C for a total of 90 min under 5% CO2 and 100% humidity.

Using the method of Ward & Storey (1984), the sperm cells were stained with chlortetracycline. Their cell morphology was examined to distinguish the following three states: the F-form, the cells in the uncapacitated state; the B-form, cells that had undergone capacitation; and the AR-form, cells that had undergone AR.

The sperm preparations described above were further treated with 10 μm Ca2+-ionophore A23187 in 0.2% DMSO at 37 °C for 30 min to induce an AR. The cells were smeared on slides, fixed with methanol for 30 sec to permeate the plasma membrane and stained with 5.0 μg/mL of TRITC–PNA in the dark for 10 min to examine the outer membrane of the acrosome according to a previously described method (Mortimer et al., 1987).

The Spink3-binding zone on the sperm head was examined using an indirect fluorescence technique as described previously (Chen et al., 1998). The cells being treated at conditions previously specified were extensively washed with PBS, smeared on slides and washed twice with PBS. After incubation in blocking solution (5% nonfat milk in PBS) for 60 min, the slides were incubated with anti-Spink3 antiserum and diluted at 1 : 200 in a blocking solution for 60 min. The slides were washed three times with PBS to remove excess antibodies and further incubated with fluorescein-conjugated goat anti-rabbit IgG diluted at 1 : 400 in the blocking solution for 60 min. The same slide was then washed with PBS and further treated with methanol to permeate the plasma membrane, and stained with TRITC–PNA to examine the outer membrane of the acrosome.

Images of the cells after specific treatments were immediately captured at room temperature on a fluorescent microscope (BX61; Olympus, Tokyo, Japan) equipped with ×40 NA 0.90 objective using a digital camera (DP-71; Olympus). The acquiring software was DP Controller and DP Manager (DP-71; Olympus). Tagged image file format images were processed using Photoimpact software (version 10.0; Corel, Ottawa, Canada).

Detection of protein tyrosine phosphorylation

After incubation of spermatozoa under the specified conditions, the soluble fraction of cell lysate was prepared according to a method described earlier by Visconti et al. (1995). Resolution of the protein components in the soluble extract was performed on an 8% polyacrylamide gel slab (10 × 8 × 0.075 cm) according to the method described by Laemmli (1970). The proteins on the gel were transferred to a filter of nitrocellulose membrane by electrophoresis conducted at 50 V for 2 h at 4 °C, a method described by Towbin et al. (1979). The protein blots on the filter were immunodetected by Western blot analysis using a monoclonal antibody PY-99 against phosphotyrosine as the primary antibody and anti-mouse IgG conjugated with HRP as the secondary antibody. The enzyme-stained bands were enhanced by chemiluminescence detection using an ECL kit according to the manufacturer’s instructions.

Measurement of [Ca2+]i

The Percoll-separated sperm suspension (106 cells/mL) was incubated with Fluo-3 AM (5 μm) in modified HM at 37 °C in the dark for 15 min under 5% CO2 and 100% humidity. After 15 min incubation, the sperm suspension was washed twice with modified HM to remove any free Fluo-3 AM. The Fluo-3 AM loaded spermatozoa were treated at specified conditions and fluorescence images of the spermatozoa were captured. [Ca2+]i was measured using a flow cytometer (FACScan; Becton Dickinson Biosciences, Mountain View, CA, USA).

Assay of in vitro fertility

Insemination proceeded in modified potassium simplex optimized medium (mKSOM) consisting of 95 mm NaCl, 2.5 mm KCl, 0.35 mm KH2PO4, 0.2 mm MgSO4, 10 mm sodium lactate, 5.56 mm glucose, 0.2 mm sodium pyruvate, 25 mm NaHCO3, 1.71 mm CaCl2, 1 mm glutamine, 0.01 mm EDTA and 0.1 mm non-essential amino acid (Summers et al., 2000). Epididymal spermatozoa in 150 μL mKSOM (105 cells/mL) under mineral oil were capacitated with 0.3% BSA in the presence of 0–60 μm Spink3 or R19L at 37 °C for 90 min in 5% (v/v) CO2 in humidified air. Oocyte–cumulus complexes collected by superovulation treatment (Gates & Bozarth, 1978) in the same medium were added to the sperm preparation for insemination. After 30 min, eggs were removed and transferred serially through several drops of mKSOM to remove spermatozoa associated loosely with the egg surface. Oocytes were gently washed and placed on slides. Coverslips were applied, and the number of sperm bound per egg was scored under a phase-contrast microscope. The oocytes obtained after an 8-h insemination were washed with mKSOM, fixed on slides with 4% paraformaldehyde, stained with Hoechst 33258 (5 μg/mL) for 1 min and observed under a fluorescence microscope. The unfertilized and the fertilized eggs were scored. Embryos with two pronuclei were considered fertilized.

Statistical analysis

Data are presented as mean ± SD. Difference was analyzed by one-way anova followed by the Turkey–Kramer multiple comparison test using Instat software (Graph Pad, San Diego, CA, USA). A p-value <0.05 was considered to be significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The Spink3–spermatozoa binding reduces the head [Ca2+]i and suppresses AR

Percoll-separated spermatozoa from the caudal epididymides were used in this study unless otherwise stated. Cells with intact acrosomal outer membrane could be stained with TRITC-conjugated peanut agglutinin (TRITC–PNA). No fluorescence appeared on the spermatozoa after they had been immunoreacted successively with the Spink3 antiserum and fluorescein-conjugated anti-rabbit IgG [Fig. 1, column (1)]. When the cells without permeation with methanol were pre-incubated with Spink3 before cytochemical staining, a crescent fluorescence zone was examined on the apical head hook of cells with intact acrosome [Fig. 1, column (2)]. Both the stainable intact acrosome and the Spink3-binding zone were observed even when the sperm cells were exposed to 0.3% BSA at 37 °C for 90 min [Fig. 1, column (3)]. Apparently, the Spink3-binding sites reside on the plasma membrane overlaying the apical hook whether the sperm cells are capacitated or not.

image

Figure 1.  Cytochemical observations of the Spink3-binding zone on the sperm head. Mouse epididymal spermatozoa (106 cells/mL) were incubated alone as control [column (1)] or with 10 μm Spink3 for 15 min at 37 °C, followed by an additional 90 min incubation in the absence or presence of 0.3% BSA [columns (2) and (3)]. The FITC-stained Spink3-binding zone on the apical hook (B) and the tetramethylrhodamine isothiocyanate-conjugated peanut agglutinin (TRITC–PNA)-stained outer membrane of the intact acrosome (C) were examined on the same slide (Materials and Methods). The cell morphology was observed using a differential interference contrast microscope (A), and the fluorescence because of FITC or TRITC–PNA was seen using a fluorescence microscope. Scale bar, 20 μm. Cells without the signals arising from both FITC and TRITC–PNA are denoted by arrows. The FITC signal only appears on the TRITC–PNA-stainable cells.

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We assessed whether the Spink3 action caused the change in spermatozoal status. Figure 2(A) displays the population of F-form (uncapacitated state), B-form (capacitated state) and AR-form (acrosome-reacted state) in sperm incubation in modified HM under various treatments. Around 70, 17 and 13% of the controls were counted as F-form, B-form and AR-form, respectively [(a), Fig. 2(A)], whereas cell incubation in the presence of 0.3% BSA remarkably reduced the number of F-form, greatly increased the number of B-form and slightly increased number of AR-form. Around 63% of the spermatozoa were B-form and 14% were AR-form [cf. (a) and (c) in Fig. 2(A)]. The incubation of either the control cells or the BSA-treated cells in the presence of 60 μm Spink3 resulted in little change in the spermatozoal status [cf. (a) and (b); (c) and (d) of Fig. 2(A)]. Meanwhile, we performed Western blot analysis of the tyrosine-phosphorylated proteins in the cell extract. The protein tyrosine phosphorylation in the control cells was limited mainly to a 105-kDa band of hexokinase (Kalab et al., 1994) [Fig. 2(B), lane 1], whereas several proteins in the range Mr 55 000–160 000 were tyrosine-phosphorylated in the BSA-treated cells [Fig. 2(B), lane 2]. The protein phosphorylation pattern of either cell preparation was persistent even when as much as 120 μm Spink3 was added to the cell incubation [cf. lanes 1 and 3, and lanes 2 and 6 of Fig. 2(B)]. Moreover, neither the cell motility enhancement associated with the capacitation induced by BSA was not affected by Spink3. When sperm incubated for 90 min without Spink3 were stimulated with 10 μm Ca2+ ionophore A23187 for another 30 min, the population of AR-form increased greatly [Fig. 2(C)]. Around 40% of the control cells became AR-form, and a remarkable decrease in the B-form was accompanied by a large increase in the AR-form in the BSA-treated cells; 67% of the BSA-treated cells were AR-form. The increase in the AR-form seemed to stem predominantly from transition of the capacitated cell population of either the control cells or the BSA-treated cells. However, addition of Spink3 to the cell incubation greatly suppressed the ionophore-induced AR in a dose-dependent manner [Fig. 2(C)]. Taken together, the Spink3–spermatozoa binding greatly inhibited the A23187-induced AR, but did not interfere with the BSA capacity in the induction of sperm capacitation by which the membrane modification did not release Spink3 on spermatozoa. On the Spink3 molecule, R19 is the reactive site for protease inhibition and D22, and/or Y21 are mainly responsible for the binding to spermatozoa (Luo et al., 2004). R19L, a recombinant polypeptide in which R19 of Spink3 is replaced by L, gives no inhibitory effect on the protease activity. We found that R19L retained the capacity to suppress the A23187-induced AR; despite that, this was not as strong as with the parent protein [cf. • and bsl00001 of Fig. 2(C)].

image

Figure 2.  Impact of the Spink3 action on the sperm activity. (A) Epididymal spermatozoa (106 cells/mL) were incubated at 37 °C for 90 min in the presence of Spink3 or BSA or both. The cell stages were quantified by the chlortetracycline fluorescence technique: F-forms, open column; B-forms, grey column; AR-forms, black column. Two hundred cells were randomly selected and scored for each of the three cell stages. The population of each stage was estimated from the average of three determinants. The standard deviation is represented by a vertical line. (B) Spermatozoa (2 × 107cells/mL) were incubated in the presence of 0–0.3% BSA and 0–120 μm Spink3 at 37 °C for 90 min. Detection of protein tyrosine phosphorylation was described in Materials and Methods. (C) Cell incubation in the presence of 0–60 μm Spink3 proceeded as described in (A) before another 30 min incubation in the absence or presence of 10 μm A23187. The extent of acrosome reaction (AR) was reciprocal to the percentage of spermatozoa with an intact acrosome which could be stained with tetramethylrhodamine isothiocyanate (TRITC)-conjugated peanut agglutinin (PNA): Δ, control cells without exposure to the ionophore; bsl00066, control cells exposed to the ionophore; ○, BSA-treated cells without exposure to the ionophore; •, BSA-treated cells exposed to the ionophore; bsl00001, replacement of Spink3 with its mutant R19L in BSA-treated cells exposed to the ionophore.

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We intended to examine the subcellular [Ca2+]i using the Fluo-3 AM-loaded spermatozoa. The cells maintained an intact acrosome even when they were incubated in the presence of 0.3% BSA at 37 °C for 90 min. Relative to the Ca2+-Fluo-3 signal, auto-fluorescence of spermatozoa was not prominent. The level of [Ca2+]i was monitored as the intensity of fluorescent Ca2+-Fluo-3 using flow cytometry. As shown in Fig. 3(A), BSA elevated [Ca2+]i to 204 ± 4% of the control level. Addition of Spink3 to a final concentration of 0–60 μm in the cell incubation reduced [Ca2+]i in both the BSA-treated cells and control cells in a dose-dependent manner. The fluorescent Ca2+-Fluo-3 in both the BSA-treated cells and control cells was intense from the head to the mid-piece, and became very faint in the tail portion [(a) and (c) of Fig. 3(B)]. The complex Ca2+-Fluo-3 in the sperm head was predominantly distributed in the space between the plasma membrane and the outer membrane of the acrosome, as the fluorescence was no longer observed once they were permeated with methanol. The presence of Spink3 at a final concentration of 60 μm in the cell incubation completely abolished Ca2+-Fluo-3 fluorescence in the head of both the control and the BSA-treated cells, but had no impact on the Ca2+-Fluo-3 fluorescence in the mid-piece [cf. (a)/(b) and (c)/(d) in Fig. 3(B)].

image

Figure 3.  Reduction of [Ca2+]i in the mouse sperm head by Spink3. (A) The fluo-3 acetoxymethyl ester (Fluo-3 AM) loaded spermatozoa were incubated with 0–60 μm Spink3 for 15 min at 37 °C, followed by an additional 90 min incubation in the absence (open circles) or presence (solid circles) of 0.3% BSA before they were subjected to flow cytometry to measure the [Ca2+]i. The relative [Ca2+]i of spermatozoa expressed as a percentage was compared with that of the control cells. Each data point is the mean ± SD of three independent determinations. Data obtained from spermatozoa treated with BSA or Spink3 alone were compared with that of control spermatozoa using the Turkey–Kramer multiple comparison test (†< 0.01, and ††< 0.001). The same test was applied to compare the data obtained from spermatozoa treated with BSA and Spink3 together with that of spermatozoa treated with BSA, but without Spink3 (*< 0.01, and **< 0.001). (B) Sperm cells were incubated the same way as described in (A) under conditions listed to the right side of the figure. The fluorescent images of Ca2+-Fluo-3 were captured under a microscope. Scale bar, 20 μm.

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Suppression of fertility by Spink3 on spermatozoa and release of Spink3 by SITA in uterine luminal fluid

We performed in vitro fertilization of cumulus-intact oocytes and epididymal spermatozoa (see Materials and Methods for details). The spermatozoa–egg association was represented by the spermatozoa/egg ratio (S/E) and the fertility rate by the percentage of eggs that were fertilized. Figure 4 gives the fertility indices obtained from several inseminations. An S/E value of 1.93 and a fertility rate of 17.5% were determined for the uncapacitated sperm preparation. The low fertility might be attributed to the small portion of B-form in the cell preparation. The spermatozoa capacitated with 0.3% BSA without Spink3 as the control yielded an S/E value of 10.59 and a fertilization rate of 53% [Fig. 4(A)]. These two fertility indices greatly reduced as the sperm capacitation proceeded in the presence of Spink3 in a dose-dependent manner [cf. (B) and (C) of Fig. 4]. The S/E was 0.57 and almost no eggs were fertilized at a dose of 60 μm Spink3. Apparently, Spink3 on spermatozoa prohibited fertilization. Although not as strong as its parent protein, the mutant R19L retained the ability to suppress fertility. Spermatozoa capacitated in the presence of 60 μm R19L had an S/E value of 5.04 and a fertilization rate of 21% [Fig. 4(D)].

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Figure 4.  Suppression of fertility by Spink3 in mouse spermatozoa. Epididymal spermatozoa in modified potassium simplex optimized medium (mKSOM; 105 cells/mL) were capacitated with 0.3% BSA for 90 min at 37 °C in the presence of the chemicals listed in the bottom of the figure. The spermatozoa prepared without further treatment were assayed for their in vitro fertility (Materials and Methods). The spermatozoa/egg ratio (S/E) was determined after insemination for 30 min, and the fertilized eggs were scored after insemination for 8 h based on the observation that an unfertilized egg had one pronucleus and a fertilized egg had two pronuclei (inlet). Data are the mean ± SD from four independent experiments. *< 0.001 in comparison relative to the fertilization rate with BSA only.

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Spink3 was traced in the female reproductive tract. It was undetectable in ULF of oestrous female, but appeared in the content of uterine cavity after coitus [Fig. 5(A)]. Meanwhile, Spink3 was mapped on the apical head hook of a considerable portion of spermatozoa in the uterine cavity, but it disappeared on the spermatozoa in the oviduct lumen [Fig. 5(B)], suggesting that release of Spink3 on the ejaculated spermatozoa was likely to be carried out by some component as yet uncharacterized in the uterine cavity before their traverse to the oviduct. As it was formidable to completely separate the sperm cells from the endometrial cellular debris, Spink3 detected in the endometrial cellular debris may arise from the non-specific adsorption of free Spink3.

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Figure 5.  Examination of Spink3 in the female reproductive tracts after coitus. (A) Immunodetection of Spink3. The seminal vesicle fluid (lane 1, 10 μg protein), the uterine fluid of oestrous female (lane 2, 10 μg protein), the soluble portion collected from the content of uterine cavity of mating females (lane 3, 10 μg protein), and the purified Spink3 (lane 4, 2 μg protein) were resolved by SDS-PAGE. The proteins on the gel were stained with Coomassie blue G250 (left side) or transferred to a nitrocellulose filter and immunoblotted with the anti-Spink3 serum (right side). (B) Cytochemical staining for Spink3. Spermatozoa separately collected from the lumens of uterus and oviduct were immunoreacted with Spink3 antiserum and FITC-conjugated anti-rabbit IgG. The cell morphology was examined by differential interference contrast microscope. The Spink3-binding zone was mapped on the spermatozoa head and detected by a fluorescence microscope. Spermatozoa with the Spink3-binding zone are denoted by arrows. Non-specific adsorption of free Spink3 was examined on the endometrial cellular debris. Scale bar, 20 μm.

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As shown in Fig. 6(A)), the trypsin-like activity was detected in the ULF of both the oestrous females and the diethyl stibesterol (DES)-stimulated immature females, but it became weak in the soluble portion collected from uterine cavity of mating females. By gel chromatography on a Sephadex G-100 column, ULF from DES-stimulated baby mice was resolved into three peaks denoted as 1, 2 and 3 on Fig. 6(B), and the trypsin-like activity was assayed for the three peak samples [Fig. 6(C)]. There emerged two features. First, the relative protease activity was in the order of peak-3 > peak-2 > peak-1. Second, both peak-1 and peak-2 activity were inhibited slightly, in contrast to peak-3 activity, which was substantially suppressed by Spink3. These data manifested the presence of SITA in the peak-3 sample. Furthermore, we assayed SITA in the peak-3 sample (300 μg/mL) containing different concentrations of Spink3 or a serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) in the reaction buffer [Fig. 6(D)]. The enzyme activity could be inhibited by PMSF, indicative of SITA because of serine protease. According to the inhibitory effect of Spink3 in a dose-dependent manner, the enzyme activity was completely inhibited by 18.75 nm Spink3, suggesting a meagre amount of serine protease responsible for SITA in the ULF.

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Figure 6.  Demonstration of Spink3-inhibiting trypsin-like activity (SITA) in the mouse uterine luminal fluid (ULF). (A) Assay of trypsin-like activity. N-benzoyl-Phe-Val-Arg-p-nitroanilide was used as a trypsin substrate. The soluble protein (50 μg) from the uterine cavity of mating females (bsl00066), the ULF of estrous female (♦) or diethyl stibesterol (DES)-stimulated baby mice (bsl00001) and 60 μm substrate in 0.1 m Tris-20 mm CaCl2 (pH 8.0) was incubated at 25 °C. The time course of substrate hydrolysis was recorded by absorbance at 405 nm. (B) Fraction of ULF from DES-stimulated baby mice. Around 4.0 mL of ULF was applied to a Sephadex G-100 column (2.5 × 100 cm) pre-equilibrated with 0.1 m ammonium acetate. A 2.0 mL sample was collected for each fraction and the absorbance was monitored at 280 nm. Three peaks denoted as 1, 2 and 3 appear in the chromatographic profile. (C) Confirmation of SITA in the peak-3 sample. Similar to the assay of (A), each peak sample from (B) in the buffer at a final concentration of 300 μg/mL was incubated at 25 °C for 3 min alone (peak-1, bsl00001; peak-2, bsl00066; peak-3, •) or in the presence of 6.25 nm Spink3 (peak-1, □; peak-2, Δ; peak -3, ○) before addition of the trypsin substrate. (D) The inhibitory effects of PMSF and Spink3 on the SITA of peak-3. The enzyme activity of peak-3 was performed in the presence of 0–18.75 nm Spink3 (bsl00001, solid line) or 0–4 mm PMSF (•, dashed line). The protease activity was reflected by the substrate hydrolysis, which was monitored after 3 min incubation.

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To assess the capacity of SITA to release Spink3 on the sperm head, we treated the sperm cells being incubated in the presence of 10 μm Spink3 at specified conditions described in Table 1 and counted the population of cytochemically stainable Spink3 on the sperm head after each cell treatment. None of the cell treatments changed the cell morphology. The Spink3-binding spermatozoa were examined on 80 ± 1% of the control cells. The population was slightly diminished by peak-1, peak-2 and PMSF, but was greatly reduced by both trypsin and peak-3 in a dose-dependent manner. Only around 40% of the trypsin-treated cells and 26% of the cells incubated with 1.5 μg/μL peak-3 retained the stainable Spink3-binding zone. Moreover, removal of Spink3 from spermatozoa by peak-3 could be suppressed by PMSF. These data together support that SITA arising from a serine protease secreted from the uterus of oestrous females is capable of releasing Spink3 on spermatozoa.

Table 1.   The capacity of Spink3-inhibiting trypsin-like activity (SITA) from uterine luminal fluid (ULF) to release Spink3 from spermatozoa
Cell treatmentaSpink3-binding cells (%)
  1. aSpermatozoa from caudal epididymis were smeared and fixed on slides. The slides were incubated with 10 μm Spink3 in PBS at 37 °C for 15 min. After washing with PBS, the slides were further incubated in PBS containing the chemicals listed in the table for 30 min. Two hundred cells were randomly selected to determine the population of Spink3-binding cells, which were examined by the indirect fluorescence technique described in the text. Data are the mean ± SD from three independent experiments.

Control80 ± 1.0
PMSF (4 mm)73 ± 1.8
Trypsin (10 nm)40 ± 3.0
Trypsin (10 nm) + PMSF (4 mm)70 ± 2.5
Peak-1 (0.5 μg/μL)78 ± 2.0
Peak-2 (0.5 μg/μL)72 ± 1.2
Peak-3 (0.5 μg/μL)49 ± 1.8
Peak-3 (0.5 μg/μL) + PMSF (4 mm)67 ± 3.1
Peak-3 (1.0 μg/μL)38 ± 2.5
Peak-3 (1.5 μg/μL)26 ± 1.1

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Like human Spink2, mouse SVI/Spink3 can bind to the human sperm head (Boettger-Tong et al., 1993), showing the similarity of the Spink3-binding site on mouse spermatozoa to the Spink2-binding site on human spermatozoa. Thus, this work adds knowledge of the way in which Spink from males and SITA from females are interplayed to modulate the activity of mammalian spermatozoa during their transit through the reproductive tract. Mouse sperm head has the binding site of trypsin-directed inhibitors such leupeptin, aprotinin, benzamidine and N-ρ-tosyl-L-Lys chloromethyl ketone (Saling, 1981). We found that the Spink3–spermatozoa binding was not inhibited by previous exposure of the cells for 30 min at 37 °C to over excess of any one of these trypsin inhibitors. Apparently, the sensitive sites of trypsin-directed inhibitors are not identical to the Spink3 binding sites on the plasma membrane overlaying the apical head hook of mouse spermatozoa. Sperm capacitation results in neither the concealment of the Spink3-binding site nor the removal of Spink3 from the cells (Fig. 1). Our previous study does not support the acrosin/proacrosin and the ZP3-binding site as the Spink3-binding site on the spermatozoa (Chen et al., 1998). Recently, Ou et al. (2010) have identified a membrane-bound testis-specific serine protease-like protein (TESPL) devoid of proteolytic activity as a potential Spink3-binding site on the mouse sperm head. They found TESPL was not identical to mouse TESP 1–5 (testicular serine protease 1–5) (Kohno et al., 1998; Ohmura et al., 1999; Honda et al., 2002), although these proteins are likely to have a common evolutionary origin.

The capacitation processes elevate the spermatozoal [Ca2+]i. The increase of [Ca2+]i in the middle piece/tail initiates an intensified flagellar motion to propel the capacitated spermatozoa towards the fertilization site. Thereupon, subsequent events raise the level of [Ca2+]i in the head region of capacitated spermatozoa to a critical threshold for acrosomal exocytosis, a necessary preliminary event for sperm–oocyte interaction before the penetration of spermatozoa during fertilization. These two Ca+2-dependent modulations happen simultaneously, but independently. Previously, we showed the ability of Spink3 to suppress 45Ca+2-uptake by the capacitated spermatozoa (Chen et al., 1998). This study further demonstrated that the binding of Spink3 to either the un-capacitated or capacitated spermatozoa does not suppress [Ca2+]i in the spermatozoal middle piece/tail, but results in a pronounced reduction of [Ca2+]i in the space between the plasma membrane and the acrosomal outer membrane [Fig. 3(B)]. This may partially account for why the decrease of [Ca2+]i in the sperm head by Spink3 affects neither the cell motility nor the protein tyrosine phosphorylation associated with the BSA-induced capacitation, but suppresses the A23187-induced AR. Integration of our data may strengthen the possibility that the Spink–spermatozoa binding prohibits the acrosomal exocytosis of capacitated cells to prevent them from becoming infertile before they encounter eggs. Spink3 and R19L have similar gross conformation according to their CD spectra (Luo et al., 2004). Replacement of R19 with L may modify the configuration of D22/Y21 to lessen their affinity to spermatozoa. This may explain why R19L is not as strong as Spink3 to inhibit AR and the in vitro fertility [Fig. 2(C) and Fig. 4].

Zaneveld et al. (1971) illustrated the inhibition of rabbit fertilization in vivo by pancreatic and seminal plasma trypsin inhibitors. The results of our in vitro fertility assay demonstrate that Spink3 on the capacitated mouse spermatozoa prohibits sperm–egg association and thereby reduces the fertilization rate (Fig. 4), suggesting the need for the release of Spink3 on spermatozoa before they fertilize eggs under natural coitus. According to the tertiary structure, the binding of D22/Y21 on the Spink3 molecule to the sperm head from one direction would turn R19 towards the other side. Such a structural feature allows the trypsin-like protease binding (Lin et al., 2006). This together with the high local concentration of Spink3 on the sperm head may facilitate the attack of SITA, and even its amount in the uterine cavity of mating females is limited (Fig. 6). As the free Spink3 in the uterine cavity is not completely hydrolyzed by SITA as suggested by the results shown in Fig. 5(A), it may suppress SITA to reduce its proteolytic damage to ejaculated spermatozoa and endometrial epithelium. Thus, our results support the theory that the interplay of Spink3 and SITA in the uterine cavity is important to bring about successful fertilization. Our preliminary study suggests that SITA arises from an airway trypsin-like protease, which has been identified in the adrenal gland (Hansen et al., 2004). More studies are needed to understand its role in reproduction.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported in part by the grants 98R 0066-030 from National Taiwan University (NTU) and NSC 98-2311-0-001-005-MY2 from the National Science Council, Taipei, Taiwan. Some of the work described in this article forms part of a dissertation submitted by CMO in partial fulfilment of the requirement for a PhD at the NTU. We thank Mr. Chung-Hao Lu for his technical assistance.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References