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J Biol Chem, Vol. 274, Issue 41, 29426-29432, October 8, 1999

A Homologue of Pancreatic Trypsin Is Localized in the Acrosome of Mammalian Sperm and Is Released During Acrosome Reaction^*

Ko Ohmura, Nobuhisa Kohno, Yoshie Kobayashi, Kazuo Yamagata, Sayaka Sato, Shin-ichi Kashiwabara, and Tadashi Baba^§

From the Institute of Applied Biochemistry and Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, and the National Institute for Advanced Interdisciplinary Research (NAIR), Tsukuba Science City, Ibaraki 305-8572, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have identified cDNA and genomic clones encoding a homologue of pancreatic trypsin, termed TESP4, as a candidate protein involved in the sperm penetration of the egg zona pellucida in mouse. The deduced amino acid sequence indicates that TESP4 is 90% identical to pancreatic trypsin. Analysis of Northern blotting and reverse transcriptase-polymerase chain reaction reveals that the mouse TESP4 gene is ubiquitously expressed in all tissues tested, including the pancreas and testis, and the transcript is present in the haploid stages of male germ cells. Moreover, immunochemical analysis of mouse cauda epididymal sperm using an affinity-purified antibody against bovine pancreatic trypsinogen shows that TESP4 is localized only in the sperm acrosome and is released during the acrosome reaction induced by calcium ionophore A23187. These findings may open a new point of view regarding the molecular mechanisms of the sperm/egg interactions, including the sperm penetration of the egg zona pellucida.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mammalian fertilization requires the penetration of sperm through the zona pellucida (ZP),¹ an extracellular matrix of the egg (for reviews, see Refs. 1 and 2). This sperm/ZP interaction occurs following the acrosome reaction of sperm, a fusion (vesiculation) event between the overlying plasma and outer acrosomal membranes. Although there has long been a controversy concerning the importance of protease(s) present in the sperm acrosome (3), the creation of a penetration pathway for the motile sperm is thought to require limited hydrolysis of the ZP glycoprotein by the acrosomal enzyme(s). Indeed, various trypsin inhibitors have been reported to block effectively the sperm penetration of the ZP in vitro (4-7).

Acrosin, a serine protease with trypsin-like cleavage specificity, is one of the proteins present in the sperm acrosome (for reviews, see Refs. 8 and 9). Although this enzyme has long been believed to act by causing the limited hydrolysis of ZP, our previous study (10) using acrosin-deficient (Acr^/) mutant mice conclusively showed that sperm do not require acrosin to penetrate the ZP. Further experiments of the Acr^/ mouse sperm have provided evidence that the major role of acrosin may be to accelerate the dispersal of acrosomal components during the acrosome reaction of sperm (11, 12). Thus, acrosomal trypsin-like protease(s) other than acrosin are probably essential for the sperm penetration of ZP at least in mouse (10, 12).

We (13) have already identified two similar but different serine proteases, TESP1 and TESP2 (testicular serine proteases 1 and 2) in the acrosome of mouse sperm as candidates involved in the sperm penetration of egg ZP. However, the deduced amino acid sequences of TESP1 and TESP2 imply that these two proteases are unlikely capable of splitting the Arg/Lys-Xaa bond, in contrast to trypsin and acrosin (13). Because Acr^/ mouse sperm barely penetrate the ZP in the presence of p-aminobenzamidine, a competitive inhibitor toward trypsin and acrosin, a protease(s) sensitive to the inhibitor other than both acrosin and two TESPs must be present in the acrosome to enable the sperm to penetrate the ZP. On the basis of this possibility, we have re-attempted to identify acrosomal serine protease(s) with trypsin-like cleavage specificity in mouse. The cDNA clones encoding each of two serine proteases, termed TESP3 and TESP4, have been isolated from a mouse testis cDNA library. Interestingly, one of these two proteases is a homologue of pancreatic trypsin that is localized in the acrosome of mouse sperm, and is released by calcium ionophore-induced acrosome reaction. A possible function of TESP4 in the sperm/egg interaction is discussed.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Calcium ionophore A23187 and bovine pancreatic trypsin (Type III, T-8253) were purchased from Sigma. An IgG fraction of rabbit anti-bovine pancreatic trypsinogen antiserum was purchased from Biogenesis (Sandown, NH). Experimental animals, ddY and ICR mice, Wistar rats, Hartley guinea pigs, Golden hamsters, and New Zealand White rabbits, were obtained from Japan SLC. Acr^+/+ and Acr^/ male mice were obtained by mating Acr^+/ males and females, as described previously (10). Monkey testicles were provided by Dr. T. Sankai at the Tsukuba Primate Center for Medical Science, Tsukuba, Japan. Dog and cat testes were provided by Dr. M. Sakuma at the Sakuma Animal Hospital, Tsukuba, Japan. Porcine testis was obtained from a slaughterhouse.

Polymerase Chain Reaction-- PCR was carried out using an Acr^/ mouse testis cDNA library (13) as a template. The following oligonucleotides were used as primers: SPP3, 5'-TCCATGGGTI(C/G)TI(A/T)(C/G)IGCIGCICA(C/T)TG-3'; SPP4, 5'-AGGATCCI(C/G)(A/T)(A/G)TCICC(C/T)TG(A/G)CAI(C/G)(A/T)(A/G)TC-3'. The reaction was performed in a 50-µl mixture containing 10 mM Tris/HCl, pH 8.8, 50 mM KCl, 1.5 mM MgCl₂, 0.1% Triton X-100, 0.2 mM each of dATP, dCTP, dGTP, and dTTP, 0.01 mM each of the primers, approximately 30 ng of the template DNA, and 5 units of Taq DNA polymerase (Wako, Osaka, Japan). The reaction program consisted of 35 cycles of 94 °C for 30 s, 50 °C for 120 s, and 72 °C for 30 s. The PCR products were digested by NcoI and BamHI, purified by polyacrylamide gel electrophoresis (PAGE), and then introduced into a pTV119N vector (Takara) at the NcoI/BamHI sites for sequence analysis.

Isolation of cDNA and Genomic Clones-- Approximately 4.5 × 10⁵ recombinant plaques from a ddY mouse testis cDNA library in gt11 (14) were screened by the plaque hybridization method (15), using a PCR-amplified DNA fragment encoding TESP4 as a probe, as described previously (16). The probe was labeled with [-³²P]dCTP (Bresatec, Adelaide, Australia) by the random-priming procedure (17). A positive clone was plaque-purified, and the cDNA insert was introduced into the EcoRI site of pUC19 for sequence analysis. A mouse 129/SvJ genomic DNA library in FIX II (Stratagene) was also screened using the above ³²P-labeled probe. The phage DNA was prepared from the positive clones, digested by various restriction enzymes, and subcloned into pUC19 for further characterization. Nucleotide sequence analysis was carried out using an ABI Prism 310 genetic analyzer.

Reverse Transcriptase-PCR-- RT-PCR was carried out using a full 3'-RACE kit (Takara) according to the protocol of the manufacturer. Briefly, first-strand cDNA was synthesized from total cellular RNA of various tissues and male germ cells by AMV RT XL using an oligo dT-3 sites adapter (Takara) as a primer. A portion of the synthesized cDNA was subjected to PCR, as described above, except that the reaction of the annealing and chain elongation was performed at 68 °C for 2 min. The following oligonucleotides were used as primers: TT13 (5'-GTCTGCAGCTCATTGCTACAAAA-3') and TT6 (5'-GGCATAATGACTTCAAAGGGATGCT-3') for TESP4; TT9 (5'-TCTGTCACCATGAGTGCACTTCTGA-3') and TT10 (5'-TCCATGAATTATAATTGGGGTGCCG-3') for pancreatic trypsin (Ta); TT14 (5'-TCTGCAGCTCACTGCTACAAGT-3') and TT6 for Td; TT1 (5'-CTGGCTACCACTTCTGTGGAGGTTCC-3') and TT2 (5'-TCCTGCTATTGAAGTTGGGATGCTT-3') for TESP4 and Td; G3PP1 (5'-AGTGGAGATTGTTGCCATCAACGAC-3') and G3PP2 (5'-GGGAGTTGCTGTTGAAGTCGCAGGA-3') for glyceraldehyde-3-phosphate dehydrogenase as a control. To amplify a cDNA fragment of TESP4 in various animal testes, RT-PCR was also carried out using TT16 (5'-AGGATCCTGTGGATGATGATGACAA-3') and TT17 (5'-TAAGCTTAGTTTGCGGCAATGGT-3') as primers. The PCR reaction consisted of 35 cycles of 94 °C for 30 s, 60 °C for 60 s, and 72 °C for 120 s. The amplified DNAs were separated by electrophoresis on agarose gels, stained with ethidium bromide, and transferred onto Hybond-N⁺ nylon membranes (Amersham Pharmacia Biotech). The blots were then subjected to Southern blot analysis, as described below.

Southern and Northern Blot Analyses-- DNA and RNA were separated by agarose gel electrophoresis and blotted onto Hybond-N⁺ nylon membranes. The blots were probed by ³²P-labeled DNA fragments, as described previously (18). After washing, the blots were analyzed by a BAS2000 Bio-Image Analyzer (Fuji Photo Film, Tokyo).

Western Blot Analysis-- An IgG fraction of rabbit anti-bovine pancreatic trypsinogen antiserum was purified by affinity chromatography on a column of Sepharose 4B that had been substituted by bovine trypsin previously treated with diisopropyl fluorophosphate, as described before (18). Proteins were separated by SDS-PAGE (19) and transferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore). After blocking with 1% skim milk, the blots were probed by affinity-purified antibody against bovine trypsinogen and then incubated with goat anti-rabbit IgG horseradish peroxidase conjugate (Jackson Immunoresearch Laboratories). The immunoreactive proteins were detected by an ECL Western blotting detection kit (Amersham Pharmacia Biotech).

Immunoprecipitation-- Cauda epididymal sperm from eight ICR mice were extracted in 5 ml of 50 mM Tris/HCl, pH 7.5, containing 0.15 M NaCl and 1% Nonidet P-40 at 4 °C for 3 h. The mixture was centrifuged, and the affinity-purified anti-pancreatic trypsinogen was added to a portion (2 ml) of the supernatant solution. After incubation at 4 °C overnight, 50 µl of protein A immobilized on agarose beads (5 ml of gel/20 ml of phosphate-buffered saline (PBS), Pierce) was mixed with the above solution, and the mixture was incubated at 4 °C for 3 h. The agarose beads were washed five times with 10 mM Tris/HCl, pH 7.5, containing 0.6 M KCl and 0.05% Triton X-100, and twice with PBS by centrifugation. The pellet was then dissolved in 8 M urea, and subjected to SDS-PAGE in the presence of 0.1% gelatin to detect proteins exhibiting gelatin-hydrolyzing activity (12). The antibody solution, which had been pre-treated with the inactive trypsinogen-coupled agarose beads described above, was used as a control.

Immunocytochemical Analysis-- Sperm suspensions were placed onto glass slides that had been coated with Vectabond (Vector Laboratories, Burlingame, CA), treated with PBS containing 4% paraformaldehyde on ice for 30 min, and washed three times with PBS. After the fixation, the sperm samples on the slides were treated with 0.3% hydrogen peroxide, washed with PBS containing 0.1% Tween 20, and blocked with 1.5% normal goat serum in PBS and with an avidin/biotin blocking kit (Vector Laboratories). The slides were incubated with affinity-purified anti-trypsinogen antibody, washed three times with the above blocking solution, and then treated with biotin-conjugated goat anti-rabbit IgG (Vector Laboratories) followed by an ABC solution containing horseradish peroxidase-conjugated avidin (Vector Laboratories). After washing with PBS, the sperm samples were stained using 3,3'-diaminobenzidine as a chromogen, mounted, and viewed under an Olympus BX50 microscope.

Calcium Ionophore-induced Acrosome Reaction-- Capacitated cauda epididymal sperm (4 × 10⁶ sperm/ml) in 0.2 ml of a modified Krebs-Ringer bicarbonate solution (TYH medium, see Ref. 20) were induced to undergo acrosome reaction by addition of calcium ionophore A23187 at a final concentration of 5 µg/ml followed by incubation at 37 °C under 5% CO₂ in air for 60 min (12). The sperm suspension was transferred into a 1.5-ml microcentrifuge tube, centrifuged at 3,000 rpm for 10 min, and then subjected to immunocytochemical analysis, as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To isolate cDNA clones coding for a novel serine protease(s) present in mouse sperm, we designed two oligonucleotide primers, SPP3 and SPP4, corresponding to the consensus sequences of trypsin-like proteases around the active-site residues, His and Ser. The antisense primer SPP4 corresponded to the Asp-Ser-Cys-Gln-Gly-Asp-Ser-Gly sequence, where first Asp and seventh Ser function as the substrate recognition and active-site residues in trypsin (21), respectively. When PCR was carried out using an Acr^/ mouse testis cDNA library (13) as a template, two DNA fragments encoding different serine proteases, termed TESP3 and TESP4, were obtained (the details of TESP3 will be reported elsewhere²). Screening of a ddY mouse testis cDNA library and a mouse genomic DNA library, using the PCR-amplified DNA fragment for TESP4 as a probe, yielded a single cDNA clone and 23 genomic clones, respectively.

The cDNA sequence of TESP4 (data not shown, see AB009661 in GenBank^TM/EMBL Data Bank) shared a high degree of identity (87%) with that of mouse pancreatic trypsin (22), suggesting that TESP4 is a member of the gene family of pancreatic trypsin. Indeed, it has been reported that 5-10 homologues of the trypsin gene are located on the mouse genome (22). To identify the genomic clones encoding the mouse TESP4 gene among the above 23 clones isolated, two sets of oligonucleotides, TT1/TT2 and TT9/TT10, were prepared according to the cDNA sequences of TESP4 and pancreatic trypsin, respectively, and used as PCR primers. A genomic clone, mTRYG11, gave a positive DNA band for the pancreatic trypsin gene, termed the Ta gene (22), whereas the positive signal for the TESP4 gene was apparently found in four clones, mTRYG3, mTRYG5, mTRYG7, and mTRYG9 (data not shown). Further analysis of restriction mapping and partial sequencing indicated that these four clones were divided into three groups; the TESP4 gene was encoded by mTRYG3, whereas mTRYG7 and mTRYG9 coded for another gene (Td gene) that also belongs to the gene family of mouse trypsin (22), as described below. mTRYG5 was distinguished from these three clones by restriction mapping (data not shown). Thus, mTRYG3, mTRYG7, and mTRYG11 were selected for further characterization, because only partial sequences of the trypsin family genes in mouse have been reported (22).

The mouse TESP4 gene as well as the Ta (pancreatic trypsin) and Td genes consisted of five exons interrupted by four introns, although the size of the TESP4 gene was approximately 200 nucleotides longer than those of the two other genes mainly because of the length of first intron (Fig. 1 and Table I). When mouse genomic DNA was digested by several restriction enzymes and subjected to Southern blot analysis using two DNA fragments as probes, the hybridization patterns were consistent with the restriction map of the TESP4 gene (Fig. 1). Thus, the TESP4 gene is a single copy gene on the mouse genome.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Exon/intron organization of the mouse TESP4 (Tc) and Td genes and Southern blot analysis of mouse genomic DNA. A genomic clone, mTRYG3, encoding the TESP4 (Tc) gene (mTRYG7 and mTRYG9 for the Td gene) has been identified from a mouse genomic library. The TESP4 and Td genes carrying five exons (closed boxes) interrupted by four introns are closely located on the mouse genome in a tail-to-tail manner (22). The sites of restriction enzymes are shown as follows: E, EcoRI; H, HindIII; K, KpnI. The mouse genomic DNA was digested by these three enzymes and subjected to Southern blot analysis using 32P-labeled PstI/EcoRI (Probe 1) and EcoRI/KpnI (Probe 2) fragments as probes.

Unexpectedly, the nucleotide sequence of the TESP4 gene (Fig. 2) was identical to the 482-nucleotide sequence of the Tc gene, one of the trypsin gene family (22), carrying the promoter region, first exon, and first intron. Moreover, the TESP4 gene had the same restriction map as the Tc gene. We therefore concluded that the mouse TESP4 gene corresponds to the Tc gene. Indeed, overlapping among three genomic clones encoding each of the Tc (TESP4) and Td genes (Fig. 1) is consistent with the fact that these two genes are closely located on the mouse genome in a tail-to-tail manner (22).

View larger version (99K): [in this window] [in a new window]   Fig. 2.   DNA sequence of the mouse TESP4 (Tc) gene. Upper and lower case letters indicate the exon and intron sequences of the mouse TESP4 (Tc) gene, respectively. The amino acid sequence is shown below the nucleotide sequence numbered in the 5' to 3' direction. Three active- site residues of TESP4 are indicated by arrowheads. A putative polyadenylation signal, AATAAA, is underlined.

Alignment of the DNA sequences among the Ta, Tc, and Td genes indicated that the exon and intron sequences of the Tc gene were highly homologous to those of the Td gene, except for first and second introns (Table I). Despite relatively low degrees of identity in the intron sequences between the Ta and Tc (or Td) genes, the exon sequences were still 77-100% homologous among these three genes.

The amino acid sequence deduced from the DNA sequence indicated that mouse Tc (TESP4) is initially synthesized as a 246-residue preproprotein with a calculated molecular mass of 26,276 Da (Figs. 2 and 3). Tc shared high degrees of sequence identity (90 and 98%) with Ta (22) and Td, respectively (Fig. 3). The locations of ten Cys residues, three active-site residues, and a substrate recognition residue (21) are all conserved among the three proteins. Thus, these data demonstrate that both Tc and Td are homologues of pancreatic trypsin (Ta), as described above. The sequence homology also suggests that the N-terminal 15-residue and subsequent 8-residue sequences of the Tc preproprotein correspond to a signal peptide for a nascent protein and a proenzyme segment (activation peptide), respectively.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Comparison of the amino acid sequence of mouse TESP4 (Tc) with those of mouse pancreatic trypsin (Ta) and a trypsin homologue Td. The sequences of the TESP4 (Tc), Ta, and Td preproproteins are aligned. Dashes represent identical residues among the sequences of these three proteins. Closed and open arrows indicate the sites of removals of the amino-terminal 15-residue signal peptide and 8-residue proenzyme segment (activation peptide), respectively. The locations of three active-site residues as a serine protease and a substrate recognition residue for cleavage of the Arg/Lys-Xaa bond are shown by asterisks and a closed triangle, respectively.

Northern blot analysis of total cellular RNAs from various mouse tissues, using a cDNA fragment of Tc as a probe, demonstrated the presence of a weak but significant mRNA signal with a size of 1.2 kilobases in the testis (Fig. 4). To ascertain that the hybridized signal corresponded to the Tc mRNA, we designed three sets of oligonucleotides, TT9/TT10, TT6/TT13, and TT6/TT14, specific for Ta, Tc, and Td, respectively, and used them as primers for RT-PCR (Fig. 4). In Ta and Td, the amplified DNA fragments were found in the pancreas, lung, and kidney among the mouse tissues tested and were completely missing in the testis. The positive signals for Tc were detected in all tissues, including the pancreas and testis, thus indicating that the mouse Tc gene is ubiquitously expressed. RT-PCR analysis also revealed that the Tc gene is initially transcribed in the testis at 20th day after birth, and the transcript is present only in the round and elongating spermatids (Fig. 4). These data demonstrate that expression of the mouse Tc gene is restricted in the haploid stages of male germ cells in the testis.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Expression of the mouse Tc (TESP4) gene in various tissues and male germ cells. A, Northern blot analysis of total cellular RNAs (10 µg each) from testis (T), heart (H), lung (L), brain (B), kidney (K), liver (I), epididymis (E), ovary (O), and uterus (U). The blot was first probed by a 32P-labeled cDNA fragment encoding mouse TESP4 and then re-probed by the DNA fragment coding for mouse glyceraldehyde-3-phosphate dehydrogenase (GDH). Note that the 1.2-kb signal for TESP4 was also found in the pancreatic tissue. However, the data is not shown because of a very strong intensity. B, RT-PCR analysis of Tc, Ta, Td, and glyceraldehyde-3-phosphate dehydrogenase (GDH). First-strand cDNA was synthesized from total cellular RNAs of various mouse tissues, including pancreas (P), and a portion of the synthesized cDNA was subjected to PCR followed by Southern blot analysis using specific internal 32P-labeled probes, as described under "Experimental Procedures." C, RT-PCR analysis of Tc and glyceraldehyde-3-phosphate dehydrogenase (GDH) in mouse testis. Total cellular RNAs from the testicular tissues of mice at 12-60 days after birth were used as templates. D, RT-PCR analysis of Tc and glyceraldehyde-3-phosphate dehydrogenase (GDH) in male germ cells of mouse. Total cellular RNAs from purified populations of pachytene spermatocytes (P), round spermatids (R), and elongating spermatids (E) were used as templates.

To examine whether a gene corresponding to the mouse Tc gene is expressed in testes of other mammalian species, the testicular RNAs from nine mammals, including mouse, were subjected to RT-PCR using TT16 and TT17 as primers. A DNA fragment of approximately 700 nucleotides was amplified in these nine mammals (Fig. 5). The DNA sequences of the amplified fragments were all identical, except that only mouse Tc sequence contained cytosine (Cyt) at nucleotide 2,288 (Fig. 2) instead of thymine (Thy). However, the amino acid sequences derived from the amplified fragments were the same between mouse and other mammals because the TTG codon still coded for Leu. No DNA fragment encoding a trypsin homologue other than Tc was amplified from the mouse testicular RNA. Thus, the presence of the trypsin homologue corresponding to mouse Tc is widely conserved at least in the testis, regardless of the mammalian species.

View larger version (43K): [in this window] [in a new window]   Fig. 5.   Expression of a trypsin homologue gene corresponding to the mouse Tc gene in mammalian testes. A, RT-PCR analysis of Tc. First-strand cDNA was synthesized from total cellular RNAs of testicular tissues from monkey (1), pig (2), dog (3), cat (4), rabbit (5), guinea pig (6), hamster (7), rat (8), and mouse (9). A portion of the synthesized cDNA was subjected to PCR using TT16 and TT17 as primers, followed by Southern blot analysis using a specific internal 32P-labeled probe for mouse Tc, as described in Fig. 4. B, conservation of the cDNA sequence encoding Tc in the testes among nine mammalian species. The DNA sequences of the amplified fragments were all identical, except that only mouse Tc sequence contained cytosine (underline) at nucleotide 233 in the cDNA sequence (nucleotide 2,288 in the genomic sequence, see Fig. 2), instead of thymine. The DNA sequence in monkey was identical to those in pig, dog, cat, rabbit, guinea pig, hamster, and rat (not shown).

An IgG fraction of rabbit antiserum against bovine trypsinogen was purified by affinity column chromatography, and the immunoreactivity of the affinity-purified antibody with a recombinant Tc protein produced in Escherichia coli cells was confirmed by Western blot analysis (data not shown). When Nonidet P-40 extracts of mouse cauda epididymal sperm were separated by SDS-PAGE under reducing conditions and subjected to Western blot analysis, a sperm protein with a size of 25 kDa immunoreacted with the affinity-purified anti-trypsinogen antibody (Fig. 6). Following immunoprecipitation of the sperm protein extracts using the same anti-trypsinogen antibody, SDS-PAGE in the presence of gelatin under nonreducing conditions revealed the presence of a 21.5-kDa gelatin-hydrolyzing protein. The sizes of the reduced and nonreduced sperm proteins immunoreacted were consistent with those of bovine pancreatic trypsin. These data strongly suggest that a trypsin homologue present in mouse sperm corresponds to Tc.

View larger version (65K): [in this window] [in a new window]   Fig. 6.   Identification of a trypsin homologue in mouse cauda epididymal sperm by analysis of Western blotting and immunoprecipitation. Nonidet P-40 extracts of mouse cauda epididymal sperm (SE, 150 µg of proteins) and bovine pancreatic trypsin (BT, 0.25 µg) were separated by SDS-PAGE under reducing conditions and subjected to Western blot analysis using affinity-purified anti-bovine pancreatic trypsinogen antibody. A sperm protein and bovine trypsin with the sizes of 25 and 26 kDa, respectively, immunoreacted with the affinity-purified antibody (left panel). When the Nonidet P-40 extracts (SE, approximately 5.4 mg of proteins) were immunoprecipitated with the affinity-purified anti-trypsinogen antibody, and subjected to SDS-PAGE in the presence of gelatin under nonreducing conditions, a 21.5-kDa gelatin-hydrolyzing protein was found (right panel). The antibody solution, which had been pre-treated with the inactive trypsinogen-coupled agarose beads, was used as a negative control (Con). Bovine pancreatic trypsin (BT) migrated as a 21.0-kDa gelatin-hydrolyzing protein.

To assess the localization of Tc in cauda epididymal sperm, immunocytochemical analysis was carried out (Fig. 7). Affinity-purified anti-bovine trypsinogen antibody gave the signals solely in the acrosome of mouse sperm. Porcine epididymal sperm also exhibited weak but significant signals in the acrosome. In the mouse sperm, the positive signals disappeared from the acrosome after the calcium ionophore A23187-induced acrosome reaction. Thus, Tc is localized in the sperm acrosome and is released during the acrosome reaction.

View larger version (50K): [in this window] [in a new window]   Fig. 7.   Location of a trypsin homologue in mouse and pig cauda epididymal sperm. Cauda epididymal sperm from mouse (a, b, and c) and pig (d and e) were stained by the avidin/biotin peroxidase complex method using affinity-purified anti-bovine pancreatic trypsinogen antibody. The signals stained only in the sperm acrosome are indicated by arrowheads (b and e). The antibody solution, which had been pre-treated with the inactive trypsinogen-coupled agarose beads, was used as a negative control (a and d). The positive signals disappeared from the acrosome of mouse sperm after calcium ionophore A23187-induced acrosome reaction (c).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It is widely known that trypsin is selectively synthesized as an inactive precursor at a high level in the exocrine pancreas of mammals. The role of trypsin is implicated in the digestion of foods by itself and by activation of other zymogens. Trypsin has been also reported to form a multigene family on mammalian genomes (22, 23). However, little is known of the expression and function of trypsin and its homologues in tissues except the pancreas (24, 25). In mouse, although five different genes encoding each of pancreatic trypsin (Ta) and its homologues, Tb, Tc (TESP4), Td, and Te, have been already identified (22), these genes are still partially characterized.

Despite high degrees of identities in the gene sequences among Ta, Tc, and Td (Table I), the Tc gene is distinguished from the others by the expression pattern; the Tc gene is ubiquitously expressed in all tissues tested, including the testis, whereas expression of the Ta and Td genes in the testis is completely missing (Fig. 4). Because the DNA fragments encoding trypsin homologues other than Tc were not amplified by RT-PCR using the testicular RNA as a template, as described above, it is most likely that only the Tc gene among the trypsin genes is expressed in the testis. A possible implication of Tc in the acrosome reaction of sperm and/or in sperm/egg interaction at the early stages of fertilization appears to be supported by the following observations: haploid cell-specific expression of the Tc gene in the testis (Fig. 4), conservation of the cDNA sequence encoding Tc in the testes among nine mammalian species (Fig. 5), and the presence of Tc in the acrosome of mouse and pig sperm (Figs. 6 and 7). However, ubiquitous expression of the Tc gene (Fig. 4) also suggests that this trypsin homologue may play an important role(s) in various cellular events of other tissues.

Two gelatin-hydrolyzing serine proteases with sizes of 42 and 41 kDa, which do not correspond to acrosin, are present in the protein extracts of Acr^+/+ mouse sperm, whereas Acr^/ mouse sperm contain the 42-kDa protease and apparently lack the 41-kDa protease (10, 12, 26). The gelatin-hydrolyzing activity of the 41-kDa protease is found when the protein extracts of the Acr^/ mouse sperm are treated with bovine pancreatic trypsin (26), indicating that exogenous trypsin is capable of compensating for the absence of the acrosin activity in vitro. In addition, the activities of the 42- and 41-kDa proteases in the Acr^+/+ mouse sperm are increased by the trypsin treatment (26). These data suggest that Tc alone or in cooperation with acrosin may serve to activate latent forms of acrosomal proteins by the proteolytic activity during the acrosome reaction of mouse sperm. However, the 41-kDa gelatin-hydrolyzing protease is not detectable in the protein extracts of the Acr^/ mouse sperm untreated with pancreatic trypsin (10, 12, 26) despite the presence of Tc in the acrosome of the mutant mouse sperm (data not shown). The reason for this discrepancy may be explained by the probability that mouse sperm contain only a very small amount of Tc in the acrosome. In fact, we were previously unable to detect gelatin-hydrolyzing proteases, including Tc, in the mouse sperm extracts except for the 42- and 41-kDa proteases, and acrosin (10, 12, 26). Moreover, the possible function of Tc in mouse sperm seems inapplicable to other mammalian sperm because the quantity and quality of serine proteases, including acrosin, are highly divergent at least between mouse and other rodents (26).

Trypsin-like protease(s) other than acrosin have not yet been identified in the acrosome of mammalian sperm. Although the importance of the acrosin activity in the sperm penetration of the ZP has long been believed, Acr^/ mouse sperm are still capable of penetrating the ZP, and the sperm penetration is effectively inhibited by p-aminobenzamidine (10, 12). Therefore, it is also possible that Tc itself is a key enzyme involved in the penetration event of mouse sperm. Even if so, the involvement of Tc in the sperm penetration seems most unlikely in other mammals because the amount of the acrosin activity in mouse sperm is estimated to be much smaller than those in other mammalian sperm (26). Perhaps the mechanism of the sperm penetration through the egg ZP in mouse is basically similar to, but may be different from those in other mammals, including rat and hamster. At any rate, further characterization of Tc in mammalian sperm, including mouse sperm, is necessary to elucidate the function of Tc in the sperm/egg interaction. Moreover, the role of this trypsin homologue in the other tissues and cells remains to be clarified.

	FOOTNOTES

^* This study was supported in part by grants from the Ministry of Education, Science, Sports, and Culture in Japan (to K. Y., and T. B.), the Sasakawa Scientific Research Grant of the Japan Science Society (to K. Y.), and by TARA Sakabe/Shoun-project, NAIR "Molecular Mechanism and Design" Taira-project, and CREST/JST Tohyama-project (to T. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequences reported in this paper have been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession numbers AB009661 (TESP4 (Tc) cDNA), AB017030 (Ta gene, AB017031 (Tc gene), and AB017032 (Td gene).

These authors have contributed equally to this paper.

^§ To whom correspondence should be addressed: Institute of Applied Biochemistry, University of Tsukuba, Tsukuba Science City, Ibaraki, 305-8572, Japan. Fax: +81-298-53-6632; E-mail: acroman@sakura. cc.tsukuba.ac.jp.

² S. Sato, N. Kohno, K. Yamagata, and T. Baba, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: ZP, zona pellucida; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RT, reverse transcriptase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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