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BOR - Papers in Press, published online ahead of print December 21, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.046821
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BIOLOGY OF REPRODUCTION 74, 666–673 (2006)
DOI: 10.1095/biolreprod.105.046821
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Sperm from Mice Genetically Deficient for the PCSK4 Proteinase Exhibit Accelerated Capacitation, Precocious Acrosome Reaction, Reduced Binding to Egg Zona Pellucida, and Impaired Fertilizing Ability1

Charles Gyamera-Acheampong 4, Julierut Tantibhedhyangkul 4, Wattana Weerachatyanukul 4, Haidy Tadros 4, Hongbin Xu 4, Jan-W. van de Loo 5, R.-Marc Pelletier 6, Nongnuj Tanphaichitr 4, and Majambu Mbikay 2 4

Ottawa Health Research Institute,4 Ottawa Hospital, University of Ottawa, Ottawa, Ontario, Canada K1Y 4E9 Cancer Research—Unit F2—Major Diseases DG Research,5 European Commission CDMA 2/47, B-1049 Brussels, Belgium Department of Pathology and Cell Biology,6 Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada H3T 1J4

ABSTRACT

The gene for proprotein convertase subtilisin/kexin-like 4 (PCSK4, previously known as PC4) is primarily transcribed in testicular spermatogenic cells. Its inactivation in mouse causes severe male subfertility. To better understand the reproductive function of PCSK4, we examined its subcellular localization in the testicular epithelium via immunohistochemistry, and on intact sperm via indirect immunofluorescence and immunoelectron microscopy. PCSK4 was detected in the acrosomal granules of round spermatids, in the acrosomal ridges of elongated spermatids, and on the sperm plasma membrane overlying the acrosome. We also investigated PCSK4 relevance for sperm acquisition of fertilizing ability by comparing wild-type and PCSK4-null sperm for their abilities in capacitation, acrosome reaction, and egg binding in vitro. PCSK4-null sperm underwent capacitation at a faster rate; they were induced to acrosome react by lower concentrations of zona pellucida; and their egg-binding ability was only half that of wild-type sperm. These sperm physiologic anomalies likely contribute to the severe subfertility of PCSK4-deficient male mice.

acrosome reaction, fertilization, sperm, sperm capacitation, sperm maturation

INTRODUCTION

Proprotein convertase subtilisin/kexin-like 4 (PCSK4, previously known as proprotein convertase 4 or PC4) belongs to a family of calcium-dependent serine proteinases that cleave secretory precursor proteins after selected pairs of basic residues. This family also includes PCSK1, PCSK2, PCSK3, PCSK5, PCSK6, and PCSK7 (previously known as PC1/3, PC2, furin, PC5/6, PACE4, and PC7/8, respectively). These enzymes are present in varying combinations in all cells. PCSK2, PCSK5, PCSK6, and PCSK7 are widely expressed. PCSK1 and PCSK2 are mostly found in endocrine and neuroendocrine cells [1, 2]. Pcsk4 mRNA is readily detectable only in testicular germ cells [3–5].

PCSK4 is specified by a 9-kb, 15-exon gene located on mouse chromosome 10 (locus symbol, Pcsk4) and human chromosome 19 (locus symbol, PCSK4) [6, 7]. This gene is transcribed into a major mRNA isoform and several minor mRNA isoforms generated by differential splicing [3, 6]. Pcsk4 transcripts are detectable by in situ hybridization near the lumen of rodent testicular tubules [3, 8], and by Northern blot analysis in testicular cell fractions enriched in spermatocytes and round spermatids [3]. Pcsk4 transcripts are absent in spermatogonia and in elongated spermatids, as well as in Sertoli and Leydig cells. The transcripts are first observed in mouse testis on postnatal Day 16, after pachytene spermatocytes have appeared [9]. In the ovary, they are found in very low amounts in macrophage-like cells [9]. The major Pcsk4 mRNA encodes a secretory precursor glycoprotein of 654 and 655 amino acids in rat and mouse, respectively [3, 4]. The encoded polypeptide is made up of an amino-terminal hydrophobic signal peptide, a prodomain, a subtilisin-like catalytic domain, a P domain, and a carboxyl-terminal domain. Similar to all PCSKs, PCSK4 cleaves its substrates at the carboxyl side of an Arg, when this P1 Arg is preceded by another basic amino acid at P2 (Lys-Arg{downarrow} or Arg-Arg{downarrow}) and/or at P4 (Lys-X-X-Arg{downarrow} or Arg-X-X-Arg{downarrow}, where X stands for any amino acid). Among PCSKs, PCSK4 is uniquely efficient at cleaving at Lys-X-X-Arg{downarrow} sites [10, 11].

The restricted expression of PCSK4 suggested that this enzyme might play a role in reproduction. We have confirmed this presumption by producing the Pcsk4tm1Mbi mouse lacking PCSK4 [12]. Male fertility is severely reduced in these mice, both in terms of fertile mating and of average litter size. This reduction, however, is not associated with any apparent spermatogenic defect. PCSK4-null sperm are motile, but their hyperactivated motility after capacitation is reduced. In vitro, they are less competent in fertilizing eggs, and fewer of the fertilized eggs develop into viable embryos [12]. Pcsk4tm1Mbi female mice are mildly subfertile: their rate of productive mating is normal, but the average size of their litter is reduced and their ovarian response to gonadotropin stimulation is diminished [9, 12].

The contribution of PCSK4 to sperm fertilization competence is currently unknown. Freshly ejaculated mammalian sperm are incapable of fertilizing eggs [13, 14]. However, as they travel in the female genital tract toward the ovulated egg, they attain fertilization competence by undergoing series of physiologic, biochemical, morphologic, and behavioral changes, collectively termed as capacitation [15]. In vitro capacitation studies have unraveled some molecular events that take place during capacitation, including increased sperm metabolism [16, 17], changes in plasma membrane fluidity [18], changes in lectin reactivity [19–21], hyperactivated motility of sperm [15, 22], elevated intracellular pH [23], membrane hyperpolarization [24], and increased protein tyrosine phosphorylation [25]. Fertilization is achieved as receptors on the sperm-head surface bind to ligands on the egg's zona pellucida (ZP). This binding triggers sperm intracellular signaling cascades that stimulate acrosomal exocytosis [26, 27]. Hydrolyzing enzymes within the acrosome perforate the ZP matrix layer, paving the way for the sperm to enter into the perivitelline space. Acrosome-reacted sperm bind to the egg plasma membrane and one is incorporated into the egg proper after fusion of its plasma membrane with the egg plasma membrane [28].

In this study, we have attempted to gain a better understanding of the physiologic role of sperm PCSK4 in fertilization. Specifically, we examined the cellular and subcellular localization of the PCSK4 protein in mouse spermatogenic and sperm cells; and, comparing wild-type and Pcsk4tm1Mbi mice, we also investigated how lack of this enzyme affects sperm acquisition of fertilizing ability.

MATERIALS AND METHODS

Animals and Antibodies

CF-1 female mice from Charles Rivers (Montreal, Quebec) and C57BL/6J (B6) male mice from our colony were used in this study. B6 mice were either wild type (+/+) or null congenics (–/–) for the Pcsk4tm1Mbi allele. They were housed in temperature-controlled rooms with 12L:12D cycles and were provided with food and drink ad libitum. Mice were handled according to the guidelines of the Canadian Council on Animal Care.

Two rabbit polyclonal antisera to PCSK4 were generated in our laboratories. The first antibody (Belg-{alpha}PCSK4) was produced using a purified recombinant mouse PCSK4 fragment produced in Escherichia coli as the antigen; it is suitable for immunoblotting [12]. The second antibody ({alpha}PCSK4–606) was produced by immunizing rabbits with a rat PCSK4-expression plasmid DNA; it is suitable for immunohistochemistry [29]. Horseradish-peroxidase (HRP)-conjugated goat antibody against rabbit IgG were purchased from Amersham (Piscataway, NJ); biotinylated antibody against rabbit IgG, fluorescein isothiocyanate (FITC)-conjugated goat antibody against rabbit IgG, and HRP-conjugated streptavidin were purchased from Molecular Probes (Eugene, OR); 15-nm gold-coupled goat antibody against rabbit IgG was purchased from EY Laboratory (San Monteo, CA).

Collection and Processing of Gonadal Tissues and Cells

For testis histology, male mice were anesthetized and perfused with Bouin fixative; testes were collected and processed as described elsewhere [30, 31]. For other studies, mice were killed under anesthesia by cervical dislocation. Sperm were collected from cauda epididymis and vas deferens (heretofore referred to as sperm) into Krebs Ringer bicarbonate (KRB)-HEPES medium (112.4 mM NaCl, 4.8 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM Mg2SO4, 4 mM NaHCO3, 21 mM HEPES, 25 mM sodium lactate, 1 mM sodium pyruvate, 5.6 mM glucose, and 28 µM phenol red) containing 0.3% bovine serum albumin (BSA).

Superovulation was induced in CF-1 female mice by intraperitoneal injection of 5 IU pregnant mare's serum gonadotropin in the middle of the light cycle, followed by 5 IU of human chorionic gonadotropin 48–50 h later. Cumulus-free eggs were prepared as described by Hogan et al. [32]. Ovarian ZP were isolated and solubilized as described by Tanphaichitr et al. [33].

Western Blot Analysis

Testis extracts were prepared by homogenization and sonication in RIPA buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.25% Na-deoxycholate; 150 mM NaCl; and 1 mM EDTA). Protein concentrations were determined by the Bradford dye-binding method [34], using reagents and a protocol from Bio-Rad Laboratories (Mississauga, ON). The extracts were analyzed by Western blotting using the Belg-{alpha}PCSK4 antibody (1:1000 dilution) and a HRP-conjugated goat anti-rabbit IgG antibody (1:2000 dilution) as the primary and secondary antibodies, respectively. The antigen-antibody complex was detected using a Western Lightning Chemiluminescence Reagent Plus Kit (Perkin-Elmer, Boston, MA), following the manufacturer's instructions.

Immunohistochemistry

Endogenous peroxidase activity in testicular sections was inhibited with 0.6% H2O2, and immunolabeling was conducted following a previously described protocol [35]. Briefly, after blocking nonspecific binding sites with 0.5% milk, the sections were successively incubated with the {alpha}PCSK4–606 antiserum (1:200 dilution), biotinylated anti-rabbit IgG (1:1000 dilution), HRP-conjugated streptavidin (1:200 dilution), and, finally, with 0.01% H2O2 and 0.05% diaminobenzidine tetrachloride (pH 7.7) for revelation. Control sections were incubated with the secondary antibody alone or with nonimmune rabbit serum IgG. The stained sections were viewed under an Axiophot 2 Carl Zeiss microscope (Carl Zeiss Canada, Mississauga, ON) at 1100x magnification. Photographs were captured on Technical Pan films from Eastman Kodak (Rochester, NY).

Indirect Immunofluorescence

Live sperm from wild-type and PCSK4-null mice were washed with phosphate-buffered saline (PBS) by centrifugation at 350 x g. They were then sequentially incubated for 30 min at room temperature, at ~107 cell/ml in PBS containing, first, 2% BSA to saturate nonspecific antibody-binding sites; then {alpha}PCSK4–606 antibody (1:200 dilution); and finally, 25 µg/ml FITC-conjugated goat anti-rabbit IgG. The sperm were washed with PBS after each incubation. Negative controls were sperm that were not treated with the primary antibody or were incubated with normal rabbit serum IgG instead of the primary antibody, but were otherwise processed the same as sperm treated with the {alpha}PCSK4–606 antibody. An aliquot of the final sperm suspension was plated on a microscope slide and examined under a Zeiss Axioplan epifluorescence microscope (Carl Zeiss Canada) at x500 magnification.

Immunogold Transmission Electron Microscopy

Caudal epididymal sperm were collected into PBS. They were washed twice (350 g, 10 min) in the same medium. This medium was used throughout the whole procedure. Non-specific binding of antibody was blocked with 5% goat serum. Thereafter, the sperm were incubated with 4 µg/ml rabbit anti-PCSK4 IgG ({alpha}PCSK4–606) for 60 min at room temperature, washed, and then exposed to goat anti-rabbit IgG (1:100) coupled with 15-nm gold particles. After successive washes with PBS, sperm were fixed with 4% glutaraldehyde in PBS and routinely processed for embedding in LR-white resin (London Resin, Berkshire, UK). Negative control sperm were prepared in a similar manner to those described in the Indirect Immunofluorescence section. Ultra-thin sections mounted on nickel grids were counterstained with uranyl acetate and lead citrate before viewing under a Hitachi H-7100 transmission electron microscope at 75 kV.

Chlortetracycline Assay for Sperm Capacitation

Sperm were collected from individual wild-type and PCSK4-null mice into KRB-HEPES medium containing no BSA. For standard capacitation, sperm pellets were carefully resuspended in KRB containing 0.3% BSA to a density of 107 sperm/ml. Aliquots were immediately taken for zero-time point chlortetracycline (CTC) staining. The rest of the sperm were incubated for 90 min at 37°C under 5% CO2, and aliquots were taken every 15 min for the CTC staining assay. The assay was a slight modification [36] of the procedure described by Ward and Storey [37]. Briefly, 49.5 µl of sperm was added to 49.5 µl CTC working solution [500–750 µM CTC-HCl (Sigma), 130 mM NaCl, 5 mM cysteine, and 20 mM Tris-Cl, pH 7.8] and incubated in the dark at room temperature for 1 min. The sperm were fixed by adding 1 µl of 25% glutaraldehyde in 2.5 M Tris base (1:1); 8 µl were spotted onto a microscope slide, covered with coverslips, and placed in the dark at 4°C. A minimum of 200 sperm per slide were microscopically analyzed within 24 h using the FITC filter on a Zeiss Axioplan 2 Imaging Microscope (Carl Zeiss Canada), and three slides were prepared for each sperm sample. Sperm were scored according to A, B, or AR patterns, as described by Lee and Storey [38]. Pattern A (also referred to as pattern F) consisted of fluorescence over the entire head and equatorial region: it identified noncapacitated sperm; pattern B consisted of a fluorescence-free band in the postacrosomal region, it identified capacitated acrosome-intact sperm; pattern AR consisted of very low fluorescence over the entire head, except for bright fluorescence along the equatorial region, it identified capacitated acrosome-reacted sperm. Percentages of sperm of the three CTC staining patterns were averaged from the three slides analyzed on each experimental day. The experiment was conducted with four separate mice of each genotype on different days and the final percentages of sperm with various CTC staining patterns were calculated as the mean ± SD of the mean values from the four experiments.

ZP-Induced Acrosome Reaction

On each experimental day, sperm collected from a wild-type male and a PCSK4-null male were precapacitated in KRB-BSA, as described above. They were then washed by centrifugation at 600 x g and resuspended in KRB supplemented with 0.01% BSA at 106 sperm/ml. Solubilized mouse ZP were added to this sperm suspension to the final concentration of 2–8 ZP/µl. The sperm suspension was incubated at 37°C under 5% CO2 for 60 min to induce the acrosome reaction. The sperm were washed, fixed in 4% paraformaldehyde, treated with 0.04% Coomassie brilliant blue G-250 in 3.5% perchloric acid, applied to a slide, and then viewed under a Zeiss Axioskop light microscope at 400x magnification. Acrosome-intact and -reacted sperm were differentiated by the presence and the absence of an intense blue stain of the acrosomal ridge of the acrosome, respectively. Two hundred sperm in various fields were assessed in each slide for their acrosomal status. Three replicate slides were made for each sperm sample. The average percentage of acrosome-reacted sperm from the three slides in each experiment was recorded. The experiment was repeated four times, and the final percentage of acrosome-reacted sperm for each sample was expressed as a mean ± SD of the mean values from the four experiments.

In Vitro Sperm-ZP Binding

The assay for in vitro sperm-ZP binding was performed as described previously [33]. Approximately 20–25 cumulus-free eggs were incubated (37°C, 30 min, 5% CO2) with 6 x 104 sperm of wild-type or PCSK4-null mice in a 60-µl droplet of KRB-BSA. One to two droplets of gamete co-incubates were set up for each sperm type. Subsequently, sperm-egg complexes were washed gently in three fresh KRB-BSA droplets through a drawn Pasteur pipet with a bore size of 200-µm diameter to remove loosely attached sperm. The complexes were then placed into a well of a sera culture slide, overlaid with mineral oil, and examined under an inverted phase-contrast microscope. Because numerous sperm bound to the egg ZP, only those observed in the same focal plane of the egg diameter were counted. To determine the percentage of sperm that bound nonspecifically to ZP, two-cell embryos were co-incubated with sperm and washed under the same conditions. Based on the number of bound sperm per two-cell embryo, only 5% of sperm bound to the egg ZP could be considered nonspecific. The sperm-ZP binding assay was performed three times on different days and the results were expressed as mean ± SD of the average numbers of sperm bound per egg on the three different days.

In Vitro Fertilization

Cumulus masses enclosing approximately 15–40 ZP-intact eggs, retrieved from oviducts of superovulated mice, were incubated (37°C, 5% CO2) with 106 million motile PGC sperm of wild-type or PCSK4-null mice in 1 ml of KSOM (95 mM NaCl, 2.5 mM KCl, 1.7 mM CaCl2, 0.35 mM KH2PO4, 0.2 mM Mg2SO4, 25 mM NaHCO3, 10 mM sodium lactate, 0.2 mM sodium pyruvate, 0.2 mM glucose, 1 mM glutamine, and 0.01 mM EDTA) supplemented with 0.1% BSA. One-to-two dishes of sperm-cumulus mass co-incubates were set up for each sperm type. Dispersion of cumulus cells was monitored at x100 magnification under an inverted Nikon Diaphot microscope, as a function of time after insemination (i.e., 0, 1.5, and 9 h). At 9 h, the number of eggs containing two pronuclei (evidence of fertilization) was determined, and the in vitro fertilization (IVF) rate was expressed as percent eggs fertilized, with the total number of eggs inseminated defined as 100%. Experiments were repeated on three different days.

Statistical Analysis

Differences between wild-type and PC-null sperm in ZP-binding and in IVF rate were analyzed for significance by Student t-test; differences in the ZP-induced acrosome reaction were analyzed by ANOVA.

RESULTS

Temporal Expression and Subcellular Localization of PCSK4

Pcsk4 transcripts are first detected in the testis on postnatal Day 16 [9]. To determine when translation of these transcripts begins, we conducted an immunoblotting analysis of testicular extracts from mice of different postnatal ages. An immunoreactive band of 54 kDa, presumably corresponding to the mature enzyme, was first detected on Day 16 and became more abundant with age (Fig. 1A). A smaller form, of 45-kDa, was also consistently observed.


Figure 1
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  		FIG. 1. PCSK4 expression in the testis. A) Immunoblot analysis of PCSK4 expression during ontogeny. The analysis was conducted using 20 µg of protein extracts and the Belg-{alpha}PCSK4 at a 1:1000 dilution. B) Immunohistochemical analysis of testicular sections for PCSK4 expression. Thin arrows: round and elongated spermatids; wide arrows: spermatocytes; arrowheads: residual bodies. Original magnification x1100.

To determine which testicular cell types express PCSK4, immunohistochemistry was performed on testis sections of wild-type mice. Strong PCSK4-specific immunoreactivity was detected in the acrosomal granules of round spermatids and the acrosomal ridges of elongated spermatids (Fig. 1B, a–d; thin arrows). Spermatocytes contained minute PCSK4-positive granules (Fig. 1B, a, b, and d; wide arrows). Surprisingly, PCSK4-specific immunoreactivity was also observed in residual bodies engulfed by Sertoli cells at spermatogenic stages VIII and IX (Fig. 1B, b and c; arrowheads).

Indirect immunofluorescence analysis of live sperm from wild-type and PCSK4-null mice showed an intense PCSK4-specific fluorescent signal over the sperm-head convex ridge of wild-type sperm (Fig. 2A, a), but not on that of PCSK4-null sperm (Fig. 2A, b). Finally, when immunogold transmission electron microscopy was performed on intact wild-type mouse sperm, gold particles were observed on the sperm plasma membrane overlying the acrosome (Fig. 2B, a and b). Control sperm revealed minimal gold deposition (Fig. 2B, c).


Figure 2
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  		FIG. 2. Localization of PCSK4 on intact sperm. A) Indirect immunofluorescence analysis of intact mouse sperm: a and b, sperm from +/+ and –/– mice, respectively. B) Immunogold transmission electron microscopy localization of live cauda epididymal sperm with anti-PCSK4 IgG (a and b). a) Horizontal section of the sperm head through the dorsal aspect of the acrosomal region. b) Oblique section through the postacrosomal region and the caudal end of the acrosome. c) For control, sperm was processed as in a and b, except that the primary antibody was omitted. N, Nucleus; Ac, acrosome.

Increased Rate of Capacitation of PCSK4-Null Sperm

In an effort to understand the reduced ability of PCSK4-null sperm to fertilize eggs in vitro [12], we examined the physiologic steps that lead to sperm acquisition of fertilizing ability. We first compared the rate of capacitation of wild-type and PCSK4-null sperm using the CTC staining technique. The results are shown in Figure 3. During the initial 30 min, the rate of capacitation of PCSK4-null sperm was significantly faster than that of wild-type sperm. The time required for 50% of the sperm to be capacitated, but still remain acrosome intact, was 19 and 34 min for PCSK4-null and wild-type sperm, respectively. Furthermore, although ~70% of PCSK4-null sperm were of pattern B at 30 min, only ~ 28% of the wild-type sperm had gained that pattern. After 45 min, however, the level of capacitation reached a similar plateau, of 85%–90%, for both sperm types. The rate of spontaneous acrosome reaction after 90 min was 10–20% for either sperm type.


Figure 3
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  		FIG. 3. Chlortetracycline assay of the kinetics of in vitro capacitation of sperm from +/+ and –/– mice (n = 4). Rate of capacitation is presented as a line plot of the mean ± SD percentage of sperm fluorescing in the A, B, and AR CTC staining patterns with capacitation time. Symbols corresponding to each pattern for either genotype are identified in the inset.

Increased Sensitivity of PCSK4-Null Sperm to Zona-Induced Acrosomal Exocytosis

We also examined whether the ZP-induced acrosomal exocytosis differed between capacitated wild-type and PCSK4-null sperm. We first studied the time course of the acrosome reaction in the presence of eight solubilized ZP/µl, a concentration commonly used to induce maximum acrosome reaction in mouse sperm, in 60 min at 37°C [39]. The percentage of reacted sperm (unstained by Coomassie brilliant blue at the head anterior ridge) was similar between wild-type and PCSK4-null sperm at all time points (Fig. 4A), indicating that the two types of sperm underwent the acrosome reaction with similar kinetics at this most effective ZP concentration. In a separate experiment, we studied the dependence of the acrosome reaction on ZP concentrations. Capacitated sperm from wild-type mice were incubated in medium containing ZP at 2, 4, 6, and 8 ZP/µl for 60 min. The rate of the acrosome reaction in the absence of ZP (spontaneous) was 10–20% for both sperm types. Capacitated wild-type sperm showed gradual increases in the percentages of acrosome reacted sperm after treatment with 2, 4, and 6 ZP/µl (18.0% ± 4.2%, 18.8% ± 4.5%, and 26.6% ± 5.1%, respectively); this percentage then rose to 63.8% ± 6.5% in the presence of 8 ZP/µl. In contrast, when capacitated PCSK4-null sperm were treated with the lowest experimental concentration of 2 ZP/µl, nearly half (48.8% ± 6.8%) underwent the acrosome reaction. This percentage rose to 54% ± 9.0% when the ZP concentration was increased to 4 and 6 ZP/µl. Finally, it reached a level comparable to that of wild-type sperm (62.7% ± 10.0%) at 8 ZP/µl (Fig. 4B). These results suggested that PCSK4-null sperm were more sensitive to ZP-induced exocytosis.


Figure 4
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  		FIG. 4. Ability of wild-type and PCSK4-null sperm to undergo acrosome reaction. These experiments were conducted on four separate occasions using one +/+ and one –/– mouse per experiment. In each sample, 200 sperm were assessed for acrosomal status, and the percentage of acrosome-reacted cells was determined. The results are expressed as mean ± SD of percent acrosome reaction for the four mice of each genotype. Black and white bars represent sperm from +/+ and –/– mice, respectively. A) Time-dependence of the acrosome reaction. B) Zona concentration-dependence of percentage of acrosome-reacted sperm (AR) after a 60-min incubation.

Decreased Ability of PCSK4-Null Sperm to Bind to the ZP and to Fertilize Eggs In Vitro

The localization of PCSK4 at the sperm surface overlying the acrosome, the site of ZP interaction, suggested that this proteinase might contribute to proteolytic events that facilitate fertilization. To determine whether PCSK4 is necessary for penetration of sperm through the egg vestment, wild-type or PCSK4-null sperm were incubated with cumulus masses, and the dispersion of these masses were monitored after 0, 1.5, and 9 h of incubation. No difference in cumulus cell dissociation was observed between the two sperm genotypes (Fig. 5A; a, c, and e vs. b, d, and f). However, after 9 h of incubation, no fertilized two-pronucleus egg was observed among eggs exposed to PCSK4-null sperm, whereas 40–51% of the eggs incubated with wild-type sperm were fertilized (Fig. 5A, g; and Table 1).


Figure 5
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  		FIG. 5. Ability of wild-type and PCSK4-null sperm to initiate and complete fertilization. A) Cumulus dispersion assay and fertilization. Sperm from +/+ (a, c, and e) and –/– (b, d, and f) mice (n = 3) were incubated with cumulus-intact cells; cumulus dispersion was microscopically assessed after 0 h (a and b), 1.5 h (c and d), and 9 h (e and f) of incubation. After 9 h, successful fertilization was assessed by determining the percentage of eggs with two pronuclei (magnified in g vs. h). Original magnification x100. Bars: black = 500 µm; white = 100 µm; gray = 25 µm. B) Egg binding assay. Sperm were incubated with unfertilized, zona-intact eggs, and the sperm-egg complexes were microscopically examined. Black and white bars represent sperm from +/+ and –/– mice, respectively. Data were expressed as mean ± SD of the average number of bound sperm/egg from three experiments. *P < 0.001; n = 4.


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  		Table 1. Fertilization rate: Number of two-pronucleus eggs per total number of eggs scored.

The fertilization incompetence of PCSK4-null sperm could be partly caused by their inability to bind to ZP. Indeed, when egg-sperm-ZP binding assays were conducted using capacitated wild-type or mutant sperm, the average number of bound sperm per egg was 13.4 ± 2.4 and 6.2 ± 4.0 for wild-type and PCSK4-null sperm, respectively (n = 3; P < 0.001; Fig. 5C), an indication that lack of PCSK4 made sperm less efficient in egg binding.

DISCUSSION

In a previous study of PCSK4 expression in neonatal mice, we reported that transcripts for this protein were first detected in the testis on postnatal Day 16 [9]. Pachytene spermatocytes are known to appear in mouse testicular seminiferous tubules at this age [40]. In this study, we show by immunoblotting that PCSK4 immunoreactivity becomes detectable on the same day, indicating that transcription of the Pcsk4 gene and translation of its mRNA are coordinated. This coordinated expression probably occurs in spermatocytes and round spermatids only, because the Pcsk4 mRNA virtually disappears in elongated spermatids [3, 5].

Based on their primary sequence and the presence of a single N-glycosylation site, proPCSK4 and PCSK4 are predicted to have molecular masses of ~70 and ~60 kDa, respectively. Two immunoreactive bands of apparent molecular masses of 54 and 45 kDa were observed in testicular extracts at the same postnatal ages. In a previous study using the same antibody, the 54-kDa form was shown to be absent in PCSK4-null sperm extracts [12], indicating that it is indeed PCSK4. The lower than expected molecular mass suggests that, similar to proPC1 [41, 42], proPCSK4 may be activated through multiple endoproteolytic events occurring at the amino (removal of the prodomain) and the carboxyl termini (truncation). Transduction in insect and mammalian cells of rat PCSK4 tagged with a V5 epitope at its C-terminus invariably leads to the loss of this epitope on the membrane-bound enzyme (unpublished data), supporting the likelihood of a C-terminal processing. The identity of the 45-kDa immunoreactive band is currently unknown. It may represent a PCSK4 degradation product or a variant protein encoded by one of the alternate Pcsk4 mRNA isoforms found in the testis [3, 6].

Our immunohistochemical analysis confirmed that PCSK4 expression in the testis is restricted to the spermatogenic cells of the seminiferous tubules. PCSK4 immunoreactivity is most evident in the acrosomal granule of round spermatids and the acrosomal ridges of elongated spermatids. At some spermatogenic stages, it is also observed in residual bodies engulfed by Sertoli cells. Whether this immunoreactivity originates from active forms of PCSK4 or from their degradation products is still unclear.

Similar to all proprotein convertases [1, 2], PCSK4 is most likely produced in the endoplasmic reticulum as a secretory proprotein that is transported and modified through the Golgi cisternae before reaching its final destinations, including the acrosomal granule and the plasma membrane of the sperm head convex ridge. The association of mouse PCSK4 with the sperm plasma membrane was unexpected. Indeed, although the hydropathy plot of PCSK4 shows the presence of a conserved 18-amino acid hydrophobic domain (GYYYNTGTLYYCTLLLYGTA559–578) in the carboxyl terminal region, this domain does not meet the standard criteria of membrane-spanning domains when tested using a variety of algorithms (http://us.expasy.org/). In contrast, a bona fide transmembrane domain has been identified in human and Xenopus PCSK4 [43]. We presume that rodent PCSK4 associates with the plasma membrane through its C-terminal 127-residue domain because a truncated form of rat PCSK4 lacking this domain is secreted into the culture medium when stably expressed in Drosophila Hi5 cells [11].

The localization of PCSK4 at the sperm surface overlying the acrosome suggested that this protease might be implicated in proteolytic events associated with sperm maturation and capacitation, as well as fertilization. The ability to undergo the acrosome reaction is one of the consequences of capacitation. Here, we show that, relative to wild-type sperm, PCSK4-null sperm are more reactive to capacitating conditions and zona-induced exocytosis. The molecular mechanism of this accentuated response is unclear. One can speculate that lack of PCSK4 and impaired processing of its physiologic substrates may cause qualitative, quantitative, and functional alterations in the signal transduction machinery of sperm. Spermatogenic cells produce a number of precursors to signaling molecules that depend on PCSKs for their processing. Among these proteins are precursors to growth hormone-releasing hormone, secretin, gonadotropin-releasing hormone, pituitary adenylate cyclase-activating peptides (PACAP), and insulin-like growth factors (IGFs) I and II [44], as well as IGF-I receptor [45] and hepatocyte growth factor receptor [46]. Of these precursors, only pro-PACAP has been shown to depend exclusively on PCSK4 for proteolytic activation, because its products, PACAP-38 and PACAP-27, are undetectable in the testis of PCSK4-null mice [47]. More recently, proIGF-II processing in placental cells was also found to be partly mediated by PCSK4 [29], suggesting that this enzyme plays the same role in the testis. PACAPs strongly activate mitogen-activated protein kinases (MAPK) of the extracellular signal-regulated kinase (ERK) types 1 and 2 in rat spermatids, through a mechanism involving cytosolic receptors [48]. PCSK4-null epididymal sperm contain reduced amounts of these kinases [48]. ERK-type MAPKs have been implicated in capacitation of human sperm [49–51]. How reduced expression of these kinases in PCSK4-null sperm correlates with the apparently accelerated capacitation displayed by these cells is unclear. Some insights may be gained by comparing the kinetics of activation of the MAPKs during in vitro capacitation of wild-type and mutant sperm. Pathways involving cAMP/protein kinase A pathway, membrane-bound phospholipase C and protein kinase C all contribute to sperm capacitation [52]. Their functionality in PCSK4-deficient sperm remains to be investigated.

It is highly probable that PCSK4 is present on the sperm surface as well as within the acrosome. Sperm surface PCSK4 is likely to be involved in a few steps of an early part of the fertilization process. Although PCSK4-null sperm could induce cumulus cell dispersion to the same extent as wild-type sperm, they bound less efficiently to zona-intact eggs. The reduced egg-binding ability of PCSK4-null sperm may be caused by impaired proteolytic processing of sperm surface proteins that serve as ZP binding ligands. However, this reduced binding capacity may be offset by a higher ZP affinity of the receptors on the PCSK4-null sperm surface or by increased responsiveness of the downstream signaling pathway, resulting in precocious acrosome reaction, as described in this report. On the other hand, PCSK4 in the acrosome, which is likely released during the acrosome reaction, may be involved in ZP matrix digestion because all ZP glycoproteins contain several potential cleavage sites of PCSK4 in their amino acid sequences (NCBI accession nos. Q62005, NP_035905.1, and NP_035906.1, for mouse ZP-1, 2, and 3, respectively). This postulation is supported by our observation herein that PCSK4-null sperm had a minimal ability to fertilize ZP intact eggs. In addition, it is also possible that PCSK4 deficiency leads to a relative decrease of several other sperm proteins involved in fertilization-related steps. This phenomenon has been observed in calmegin, fertilin ß, and cyritestin knockout mice [53–55]. Proteomics studies are under way to identify alterations of protein structure and protein-protein interactions in PCSK4-null sperm.

The total lack of fertilization competence exhibited by the mutant sperm in this study is a more serious defect than the IVF inefficiency described in a previous report [12]. This discrepancy may be caused by differences of genetic background: in the previous report, the mice were of a mixed B6-129Sv background; in this study, they were B6 congenics.

In conclusion, we have shown that PCSK4 is found on the acrosomal plasma membrane and that lack of this enzyme makes sperm more sensitive to capacitating conditions, more susceptible to the ZP-induced acrosome reaction, inefficient at binding to ZP, and incompetent at fertilization.

ACKNOWLEDGMENTS

The authors thank Ms. Adrianna Gambarotta and Mr. Andrew Chen for their help in animal maintenance and surgery.

FOOTNOTES

1 Supported by a grant from the Natural Sciences and Engineering Research Council of Canada. Back

2 Correspondence: M. Mbikay Ottawa Health Research Institute, 725 Parkdale Avenue, Ottawa, Ontario, Canada K1Y 4E9. FAX: 613 761 4355; mmbikay{at}ohri.ca Back

Received: 24 August 2005.

First decision: 18 September 2005.

Accepted: 12 December 2005.

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