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Characterization and Expression of Trypsinogen and Trypsin in Medaka Testis
Author(s): Sanath Rajapakse, Katsueki Ogiwara and Takayki Takahashi
Source: Zoological Science, 31(12):840-848. 2014.
Published By: Zoological Society of Japan
DOI: http://dx.doi.org/10.2108/zs140111
URL: http://www.bioone.org/doi/full/10.2108/zs140111
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¤ 2014 Zoological Society of JapanZOOLOGICAL SCIENCE 31: 840–848 (2014)
Characterization and Expression of Trypsinogen and Trypsin
in Medaka Testis
Sanath Rajapakse1, Katsueki Ogiwara2, and Takayki Takahashi2*
1Department of Molecular Biology and Biotechnology, Faculty of Science, University of Peradeniya,
Peradeniya 20400, Sri Lanka
2Laboratory of Reproductive and Developmental Biology, Faculty of Science, Hokkaido University,
Sapporo 060-0810, Japan
Previously, we reported that the medaka testis abundantly expresses the mRNA for trypsinogen,
which is a well-known pancreatic proenzyme that is secreted into and activated in the intestine.
Currently, we report our characterization of the medaka trypsin using a recombinant enzyme and
show that this protein is a serine protease that shares properties with trypsins from other species.
Two polypeptides (28- and 26-kDa) were detected in the testis extracts by Western blot analysis
using antibodies that are specific for medaka trypsinogen. The 28-kDa polypeptide was shown to
be trypsinogen (inactive precursor), and the 26-kDa polypeptide was shown to be trypsin (active
protease). We did not detect enteropeptidase, which is the specific activator of trypsinogen, in the
testis extract. Immunohistochemical analyses using the same trypsinogen-specific antibody pro-
duced a strong signal in the spermatogonia and spermatozoa of the mature medaka testis. Sub-
stantial staining was found with spermatocytes, whereas extremely weak signals were observed
with spermatids. In vitro incubation of testis fragments with the trypsinogen antibody strongly
inhibited the release of sperm from the testis into the medium. Trypsin activity was detected in
sperm extracts using gelatin zymographic analysis. Immunocytochemistry showed that trypsino-
gen and trypsin were localized to the cell membranes surrounding the sperm head. Collectively,
these results suggest that trypsin plays an important role in the testis function of the medaka.
Key words: teleost, medaka, testis, spermiation, protease, trypsin
INTRODUCTION
Trypsin, which is a member of the large and diverse ser-
ine protease family, is generated from the precursor protein
trypsinogen, which is synthesized in and secreted from the
pancreas. Trypsin plays a central role in pancreatic exocrine
physiology by triggering the activation of all other pancreatic
digestive zymogens, including its own inactive precursor
(Halfon et al., 2004). Trypsin and trypsinogen are among the
most extensively studied enzyme models of protein structure
and function as they are readily available in large quantities
from the lumen of the vertebrate gut (Walsh and Wilcox,
1970). In particular, trypsinogen and trypsin have become
prototypes for understanding the molecular mechanisms
underlying the auto-activation of zymogens and the auto-
degradation of the active proteases (Halfon et al., 2004;
Varallyay et al., 1998; Szilagyi et al., 2001; Chen and Ferec,
2004). Studies concerning mutants and variants of trypsin
have also provided a great deal of fundamental knowledge
concerning the substrate-specific action of serine proteases
(Hedstrom et al., 1992; Jelinek et al., 2004).
While mammalian trypsins are well characterized, there
are only a few reports of the molecular and biochemical
characterization of trypsin and trypsinogen from birds (Wang
et al., 1995; Szenthe et al., 2005). However, trypsins from a
variety of fish species have been isolated and characterized
(Simpson and Haard, 1984; Genicot et al., 1996; Cao et al.,
2000; Bezerra et al., 2001; Castillo-Yanez et al., 2005;
Kurtovic et al., 2006; Klomklao et al., 2006). Fish trypsins
are particularly interesting because these trypsins exhibit
higher catalytic activity than that of their mammalian coun-
terparts, making these proteins suitable for food processing
and biotechnology applications (Simpson and Haard 1987;
Simpson, 2000, Macouzet et al., 2005).
Recently, we reported the molecular and biochemical
characterization of the enteropeptidase serine protease of
the medaka Oryzias latipes (Ogiwara and Takahashi, 2007).
In that study, we cloned a cDNA for medaka trypsinogen,
which is the target zymogen of enteropeptidase, and
deduced its amino acid sequence. Using Northern hybridiza-
tion, we detected the trypsinogen mRNA in the medaka tes-
tis, spleen, and in the intestine (Ogiwara and Takahashi,
2007). More recently, Miura et al. (2009) provided evidence
that testicular trypsin is an important factor in the control of
meiosis, spermatogenesis, and of fertilization in the Japanese
eel. Their results suggested that active trypsin, but not the
zymogen trypsinogen, is involved in the control of meiosis,
spermiogenesis, and fertilization; however, the precise role
of this protease remains to be determined. These observa-
* Corresponding author. Tel. : +81-11-706-2748;
Fax : +81-11-706-4851;
E-mail: ttakaha@sci.hokudai.ac.jp
doi:10.2108/zs140111
Trypsin in the medaka testis 841
tions prompted us to further investigate the possible role of
trypsin in testicular physiology using the medaka, which is a
good non-mammalian vertebrate model (Nagahama et al.,
1994; Ozato and Wakamatsu, 1994; Ishikawa, 2000;
Wittbrodt et al., 2002; Kasahara et al., 2007). During prelim-
inary experiments using an in vitro medaka testis culture, we
observed that the release of sperm from the mature testis
into the medium was suppressed in the presence of soy-
bean trypsin inhibitor (SBTI). Therefore, we speculated that
trypsin might have a role in the spermiogenesis of the
medaka.
The present study was performed to gain insights into
the role of trypsin in the testis of adult medaka. We pro-
duced and characterized active recombinant medaka trypsin
and determined the localization of trypsinogen and of trypsin
in the testis by morphological analyses. We also examined
the effect of a trypsinogen-specific antibody on the release
of sperm from cultured testes. These results are reported in
this study.
MATERIALS AND METHODS
Animals
Individuals of the adult orange-red variant of medaka (Oryzias
latipes) were purchased from a local dealer and were kept in indoor
tanks under artificial reproductive conditions (photoperiod, 10-h
dark/14-h light; temperature, 27°C). The Committee of the Center
for Experimental Plants and Animals at Hokkaido University
approved all experiments conducted in this study.
Production and purification of recombinant medaka trypsino-
gen
Recombinant medaka trypsinogen was prepared as described
previously (Ogiwara and Takahashi, 2007). Briefly, a 684-bp cDNA
fragment (nucleotides 72–755, AB272106) containing the trypsino-
gen coding sequence, but without the putative signal sequence,
was amplified by PCR. The product was digested with EcoRI and
with HindIII, gel-purified and ligated into the pET30a expression
vector (Novagen, Madison, WI). The recombinant medaka trypsino-
gen was expressed in E. coli and initially purified using a Ni2+-
Sepharose column, as described previously (Ogiwara et al., 2005).
The purified recombinant protein was refolded by dialysis. The pro-
tein solution (20 ml) obtained from the above affinity chromatogra-
phy was placed in dialysis tubing (dialysis membrane, size 36,
Wako) and was extensively dialyzed against 50 mM Tris-HCl, pH
8.0 at 4°C. The dialyzed sample was then applied to a Resource Q
column previously equilibrated with the same buffer. The resulting
purified recombinant protein was a 279-residue fusion protein that
contained a vector-derived 52-residue peptide at the N-terminus of
the 227-residue medaka trypsinogen polypeptide. This recombinant
trypsinogen contained two enteropeptidase cleavage sites: one
from the vector sequence and the other from the trypsinogen
sequence.
Activation of medaka trypsinogen by immobilized enteropepti-
dase
The active 32-kDa C-terminal serine protease domain of
medaka enteropeptidase was prepared as described previously
(Ogiwara and Takahashi, 2007) and immobilized on CNBr-activated
Sepharose 4B (GE healthcare Biosciences, Piscataway, NJ)
according to the manufacturer’s protocol. Recombinant trypsinogen
was incubated with the immobilized enteropeptidase for 1 h at room
temperature. After removing the immobilized enteropeptidase by fil-
tration, the filtrate was concentrated for further experiments.
Antibody preparation
The recombinant fusion protein of medaka trypsinogen (279-
residues) prepared above was used as an antigen to raise anti-
medaka trypsinogen antibodies in rats.
Anti-medaka enteropeptidase antibodies were as prepared pre-
viously (Ogiwara et al., 2007).
The recombinant medaka kallikrein-like protease was produced
using an E. coli expression system. The coding region (nucleotides
70–828, AB242321) of the protein was amplified by PCR using
medaka ovary cDNA. The product was digested with EcoRI and
with XhoI, and the digested product was ligated into the pET30a
vector. The kallikrein-like protease expressed in E. coli was purified
using a Ni2+-Sepharose column as described above. Specific anti-
bodies were purified as previously described (Ogiwara et al., 2007).
Immunohistochemistry
Adult medaka testes were isolated and fixed with Bouin’s solu-
tion (5% acetic acid, 9% formaldehyde, and 0.9% picric acid) for 2 h
at room temperature. After fixing, specimens were dehydrated by
passing the specimens through increasing concentrations of etha-
nol. The specimens were incubated in benzene for 30 min twice and
then embedded into paraffin. Paraffin sections (5 μm thickness)
were deparaffinized in xylene for 10 min, hydrated by passing the
sections through decreasing concentrations of ethanol, and then
placed in distilled water. After the sections were incubated in PBS
including 3% H2O2 for 10 min at room temperature, the sections
were incubated in Block Ace (Dainippon-Sumitomo Seiyaku, Osaka,
Japan) for 60 min at room temperature. After blocking, the sections
were reacted with an anti-trypsinogen antibody diluted with Immuno
Shot immunostaining-fine (Cosmo Bio, Tokyo, Japan) for 60 min at
room temperature. As a control, an anti-trypsinogen antibody previ-
ously treated with the recombinant medaka trypsinogen protein was
used. After washing in PBS three times, the sections were reacted
with an anti-rat IgG peroxidase-linked antibody (GE healthcare
Biosciences) diluted with Immuno Shot immunostaining-fine for
60 min at room temperature. The sections were then washed with
PBS three times and stained using a TSA fluorescence kit (Perkin-
Elmer, Boston, MA) according to the manufacturer’s instructions.
Signals were viewed using a fluorescent microscope.
Immunocytochemistry of medaka spermatozoa
Adult medaka testes were isolated and gently torn with tweezers.
After the tissues were gently shaken in PBS, released spermatozoa
were collected by centrifugation and used for immunocytochemical
analysis. Spermatozoa were attached to the MAS-coated glass
slides (Matsunami Glass Industries, Osaka, Japan) and fixed with
4% paraformaldehyde (Wako, Osaka, Japan) in 0.1 M phosphate
buffer (pH 7.4) for 1 h. Then, slides were washed with PBS and
were permeabilized in 1% Triton X-100 in PBS for 30 min. Next, the
slides were incubated in Block Ace for 1 h at room temperature to
block the non-specific binding sites, followed by incubation with a
purified anti-medaka trypsinogen antibody for 1 h at room tempera-
ture and washed in PBS containing 0.1% Tween-20 (TPBS) three
times. After washing, the slides were reacted with anti-rat IgG per-
oxidase-linked antibody for 1 h at room temperature. After three
washes in TPBS, the sperm cells were stained using an AEC kit
(Vector Laboratories, Burlingame, CA) according to the manufac-
turer’s instructions. Purified Rat IgG (1:1000 diluted) was used as
the control.
Western blotting
Whole tissue samples of medaka testis, intestine, ovary, liver,
spleen, brain, heart, and kidney were separately homogenized in
PBS containing protease inhibitor cocktail IV (Wako) and 1 mM
EDTA and centrifuged at 15,000 rpm for 10 min to obtain superna-
tant fractions. Protein concentrations of the extracts were deter-
mined using a BCA assay kit (Thermo Scientific, Yokohama,
S. Rajapakse et al.842
Japan). Western blot analysis was performed with a trypsinogen
antibody raised in rats.
Enzyme activity assay
Unless otherwise stated, the activity of recombinant medaka
trypsin was determined at 37°C with a synthetic peptide substrate
containing 4-methylcoumaryl-7-amide (MCA), butyloxycarbonyl
(Boc)-Gln-Ala-Arg-MCA (Peptide Insitute, Osaka, Japan), according
to the method of Barrett (1980) with the slight modifications reported
in our previous study (Matsui and Takahashi, 2001). The release of
the fluorophore 7-amino-4-methyl coumarin was measured by spec-
trofluorometry using an excitation wavelength of 370 nm and an
emission wavelength of 460 nm.
The active trypsin concentration was determined using the
active site titrant p-nitrophenyl-p’-guanidinobenzoate HCl.
Active medaka trypsin was preincubated with various protease
inhibitors at 37°C in 500 μl of 0.1 M Tris-HCl buffer (pH 8.0). After
incubation for 15 min, enzyme reactions were started by adding the
substrate Boc-Gln-Ala-Arg-MCA.
Kinetic parameters
Kinetic parameters were determined for a variety of MCA-
containing peptide substrates. Initial velocities were extrapolated
from the plot of product versus time and transformed into double-
reciprocal plots (Lineweaver and Burk, 1934). Maximum velocity
(Vmax), Km, and kcat values were obtained from the intercepts of
these plots.
Digestion of protein substrates by recombinant medaka trypsin
Bovine plasma fibronectin (Sigma, St. Louis, MO), mouse laminin
(Sigma), and medaka collagen type I (5 μg each) were incubated
with recombinant medaka trypsin (0.1 μg for fibronectin and for
laminin and 0.5 μg for collagen type I) in 20 μl of 50 mM Tris-HCl
buffer (pH 8.0) for 16 h at 37°C. After incubation, 5 μl of SDS-PAGE
sample buffer was added to each reaction, and the mixtures were
boiled and subjected to SDS-PAGE under reducing conditions. After
electrophoresis, the gels were stained with 0.25% Coomassie bril-
liant blue. Medaka collagen type I was prepared as described pre-
viously (Horiguchi et al., 2008).
Medaka collagen type IV (1 μg) was incubated with 0.02 μg of
recombinant medaka trypsin in 20 μl of 50 mM Tris-HCl buffer (pH
8.0) for 16 h at 37°C. The reactions were terminated by adding 5 μl
of SDS-PAGE sample buffer. The mixtures were boiled and
subjected to SDS-PAGE under reducing conditions, followed by
Western blotting using the medaka collagen type IV antibody.
Medaka collagen type IV and anti-medaka collagen type IV α1
chain antibodies were prepared as described previously (Kato et al.,
2010).
Gelatin zymography
Gelatin zymography was performed as previously described
(Ogiwara et al., 2012).
Reverse transcription and polymerase chain reaction (RT-PCR)
of testicular enteropeptidase cDNA fragment
Total RNA was isolated from mature medaka testis using Iso-
gen (Nippon Gene, Tokyo, Japan) following the manufacturer’s
instructions. The amount and purity of the fraction were determined
by spectrophotometry. RT-PCR was performed with the RNA using
a SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA).
The PCR amplification mixture consisted of the RT-PCR reaction,
1 × PCR buffer, 0.2 mM of each dNTP, 2.5 mM MgCl2, 0.2 μM of
each primer, and 0.5 U Takara Ex Taq Hot start Version (Takara
Bio, Ohtsu, Japan) (10 μl total volume). The following primers were
used: EP-SS1, 5′-AGAACATCACAGGTGAACCGGTGA-3′ (sense,
nucleotide no. 1-24, AB272104); EP-AS1, 5′-TAAGACATTAGAAT-
GGACAGAGTC-3′ (antisense, no. 3497–3520). The amplification
Fig. 1. Preparation of recombinant medaka trypsin. (A) Strategy
for the preparation of active medaka trypsin. Our E. coli expression
system using the pET30a expression vector was designed to pro-
duce inclusion bodies containing a 279-residue fusion protein with
two enteropeptidase cleavage sites: one derived from the vector
(EP1) and the other from the intrinsic medaka trypsinogen activation
site (EP2). Enteropeptidase digestion thus generates two forms
(236 and 222 residues) of the protein. The vector-derived 52-
residue peptide (white), 5-residue medaka trypsinogen activation
peptide (grey), and active trypsin (black) are indicated. The appar-
ent molecular masses (38-, 31-, and 24-kDa) observed by SDS-
PAGE analysis are shown for each polypeptide. (B) Proteolytic con-
version of the 38-kDa trypsinogen fusion protein to smaller polypep-
tides (31- and 24-kDa) by treatment with enteropeptidase. T, a
recombinant trypsinogen was incubated alone for 20 min; T+E, a
recombinant trypsinogen was incubated with the medaka entero-
peptidase serine protease domain at room temperature for 5 to
20 min; E, medaka enteropeptidase was incubated alone for 20 min.
The incubated samples were subjected to SDS-PAGE under reduc-
ing conditions (upper panel). The same samples were electrophore-
sed under nonreducing conditions and analyzed by gelatin
zymography (lower panel). The molecular masses of the polypep-
tides are shown on the right. (C) Recombinant trypsinogen fusion
proteins treated with or without enteropeptidase (E) for 1 h at room
temperature were analyzed by SDS-PAGE under reducing condi-
tions. Untreated (–) and treated (+) samples are shown.
Table 1. Kinetic parameters of recombinant medaka trypsin as
measured on MCA-containing substrates.
Substrates Vmax Kmkcat kcat/Km
μmol/min/mg mM min–1 mM–1/min–1
Boc-Gln-Ala-Arg MCA 1.80 0.10 42.9 429
Boc-Val-Pro-Arg-MCA 1.27 0.13 30.2 233
Boc-Leu-Lys-Arg-MCA 1.31 0.19 31.2 164
Boc-Glu-Lys-Lys-MCA 2.20 0.61 52.4 85.9
Z-Val-Val-Arg-MCA 0.57 0.16 13.6 84.8
Z-Leu-Arg-MCA 0.22 0.16 5.24 32.8
Suc-Leu-Leu-Val-Tyr-MCA ND
Ac-Ile-Glu-Thr-Asp-MCA ND
Suc-Ala-Ala-Pro-Phe-MCA ND
Boc, butyloxycarbonyl; Z, benzyloxycarbonyl; Suc, succinyl, Ac,
acetyl; ND, not detected.
Trypsin in the medaka testis 843
products were cloned into the pBluescript II
KS(–) vector (Stratagene, La Jolla, CA) for
sequencing.
Medaka testis culture
Adult medaka testes were removed, gently
washed with L15 medium supplemented with 10%
fetal bovine serum, 1 × penicillin-streptomycin-
glutamine (Invitrogen) and with 10 mM HEPES,
and incubated in the same medium. After incu-
bating for 1.5 h at 26°C, the testes were
removed carefully and washed gently with the
medium. Then, the testes were individually
placed in sterilized culture plate inserts (milli-
cells, 0.4 μm pore size, Millipore Corporation,
MA) with the same medium containing soybean
trypsin inhibitor (SBTI, 0.4 mg/ml), purified rat
anti-medaka trypsinogen antibodies (100 μg/ml),
or rat normal IgG (100 μg/ml) for further incuba-
tion for 4.5 h. Twenty microliters of medium from
each testis and from the control sample were
removed, and the number of released sperma-
tozoa were counted using a hemocytometer.
The weight of each testis was measured, and
the amount of released spermatozoa per milli-
gram of testis was calculated.
Statistical analysis
All experiments were conducted at least
three times to confirm the reproducibility of the
results. The data are presented at the mean ±
S.E.M.
RESULTS
Characterization of recombinant medaka trypsin
Recombinant medaka trypsinogen was produced as a
279-amino acid (38-kDa) fusion protein in an E. coli expres-
sion system. The serine protease domain of recombinant
medaka enteropeptidase was used to convert the fusion
protein into a 24-kDa form via a 31-kDa intermediate (Fig.
1A). The 24-kDa product was capable of hydrolyzing gelatin
(Fig. 1B), indicating that this product was active medaka
trypsin. For further enzyme characterization, the pure 24-
kDa enzyme sample generated by enteropeptidase diges-
tion was used (Fig. 1C).
The activity of the recombinant medaka trypsin was
tested against various synthetic MCA-containing peptide
substrates (Table 1). The enzyme hydrolyzed substrates
that contained arginine or lysine at the P1 position; however,
the enzyme did not hydrolyze substrates that contained
tyrosine, phenylalanine, and asparagine at that location. The
best substrate (highest kcat/Km value) was Boc-Gln-Ala-
Arg-MCA, followed by Boc-Val-Pro-Arg-MCA. Both of these
substrates had a small, hydrophobic residue at the P2
position. Substrates with a bulky or basic residue at the P2
position (Boc-Leu-Lys-Arg-MCA, Boc-Glu-Lys-Lys-MCA, Z-
Val-Val-Arg-MCA, and Z-Leu-Arg-MCA) displayed smaller
kcat/Km values.
The medaka trypsin also successfully degraded four
extracellular matrix proteins, collagen type I, collagen type
IV, fibronectin, and laminin (Fig. 2).
The effects of protease inhibitors on the activity of
medaka trypsin were examined using the best peptide sub-
strate, Boc-Gln-Ala-Arg-MCA (Table 2). The activity of the
enzyme was strongly suppressed by well-known serine pro-
tease inhibitors. Interestingly, the enzyme was also substan-
tially inhibited by chymostatin, which is a chymotrypsin-like
protease inhibitor. Overall, these results strongly suggest
that medaka trypsin is a serine protease that cleaves specif-
ically at the carboxyl side of basic amino acid residues.
Presence of active trypsin in medaka testis
A Western blot analysis of medaka tissue extracts was
conducted using antibodies specific for medaka trypsinogen.
Clear signals were only detected in testis, intestine, and
spleen tissues (Fig. 3A). The protein detected by the anti-
body appeared in two forms, a 28-kDa polypeptide that
predominated in the testis and in the spleen and a 26-kDa
polypeptide that was primarily visible in the intestine. The
presence of both polypeptide species in the testis was con-
firmed using an excessive amount of the tissue extract (Fig.
3B). The signal intensity of the 26-kDa polypeptide was
approximately 1/5 that of the 28-kDa species. The relation
between the two polypeptides was examined by treating the
testis extract with recombinant medaka enteropeptidase ser-
ine protease. The 28-kDa polypeptide was proteolytically
converted to the 26-kDa protein by the treatment (Fig. 3C).
A similar proteolytic conversion was observed when the
intestinal extract was treated with the enteropeptidase; the
28-kDa band disappeared, and only the 26-kDa band was
detected. These results indicate that the 28- and 26-kDa
polypeptides are the precursor form (trypsinogen) and active
trypsin, respectively.
The medaka testis extract exhibited enzymatic activity
Fig. 2. Activity of recombinant medaka trypsin against protein substrates. (A) Digestion
of medaka collagen type I by medaka trypsin. Medaka collagen type I was incubated in
the absence (–) or presence (+) of medaka trypsin and analyzed by SDS-PAGE under
reducing conditions. Col(I), collagen type I. (B) Digestion of medaka collagen type IV by
medaka trypsin. Medaka collagen type IV was incubated in the absence (–) or presence
(+) of medaka trypsin and analyzed by SDS-PAGE (under reducing conditions)/Western
blotting using specific antibodies for medaka collagen type IV α1 chain. Col(IV), collagen
type IV. (C) Digestion of fibronectin (FN) and laminin (LM) by medaka trypsin. Bovine
plasma fibronectin and mouse laminin were separately incubated in the absence (–) or
presence (+) of medaka trypsin and analyzed by SDS-PAGE under reducing conditions.
Note that collagen type I, collagen type IV, fibronectin and laminin were all degraded by
the medaka protease. Each experiment was performed in triplicate, with similar results,
and one representative image is shown.
S. Rajapakse et al.844
toward Boc-Gln-Ala-Arg-MCA. This activity was drastically
reduced by pretreatment with SBTI and with the trypsinogen
antibody (Fig. 4). Antibodies against the medaka kallikrein-
like protein had no effect on this activity. These results indi-
cate that active trypsin is present in the medaka testis.
Analyses of the expression of enteropeptidase in
medaka testis
Medaka enteropeptidase cDNA was produced by RT-
PCR using total RNA isolated from mature medaka testis.
As expected, RT-PCR using the primer set EP-SS1/EP-AS1
produced a 3.5 kb cDNA (Data not shown). Parallel experi-
ments using the intestinal total RNA as the template pro-
duced the same 3.5 kb cDNA. However, no immunoreactive
material was detected in a Western blot analysis of the testis
extract using antibodies specific for medaka enteropepti-
dase. These results indicate that although the medaka testis
contains enteropeptidase mRNA that encodes the functional
protein, it does not contain detectable levels of the trans-
lated product.
Immunohistochemical localization of trypsinogen and of
trypsin in medaka testis
The mature medaka testis is a type of testis represent-
ing restricted distribution of spermatogonia in the germinal
compartment (Grier, 1981; Schulz et al., 2010). The testis is
composed of many cysts (Gresik et al., 1973; Grier et al.,
1980; Shibata and Hamaguchi, 1986; Iwai et al., 2006).
Spermatogonia are present in the cysts in the peripheral
region of the testis, and the cysts migrate toward the center
as the germ cells differentiate. Generally, cysts housing
spermatogonia, spermatocytes, spermatids, and spermato-
zoa are distributed in that order from the periphery to the
center of the testis (Fig. 5A). Strong signals detected using
a trypsinogen-specific antibody were associated with sper-
matogonia and with spermatozoa (Fig. 5B and 5C). Signals
were also observed, although to a lesser extent, with sper-
matocytes. Weak signals were detected with spermatids.
Effect of trypsinogen antibodies on sperm release from
medaka testes
Knowing that trypsinogen was detected in association
with the spermatogonia and with the spermatozoa of the
mature medaka testes, next, we conducted experiments
examining the role of trypsin, specifically its possible
involvement in the process of spermiation. In the in vitro cul-
ture of testis fragments, an inclusion of trypsinogen-specific
antibodies dramatically reduced the release of sperm into
the medium (Fig. 6A). The antibody caused a reduction in
sperm release to an extent comparable to that caused by
SBTI (Fig. 6B). Western blot analysis using the antibody
with the sperm extracts showed the 28-KDa trypsinogen
Table 2. Effects of protease inhibitors on recombinant medaka
trypsin activity.
Inhibitor Concentration inhibition (%)
Antipain 1 mM 100
Aprotinin 0.1 mg/ml 100
Leupeptin 1 mM 100
SBTI 0.2 mg/ml 100
Benzamidine 2 mM 90
DFP 1 mM 88
E-64 1 mM 25
Pepstatin 2 mM 0
TPCK 2 mM 0
TLCK 2 mM 100
Chymostatin 0.2 mM 50
EDTA 1 mM 25
Enzyme activities of the medaka recombinant trypsin were deter-
mined using Boc-Gln-Ala-Arg-MCA in the presence of inhibitors.
Activities are expressed as the percentages of the respective con-
trols. DFP, diisopropyl fluorophosphate; SBTI, soybean trypsin
inhibitor; TPCK, N
α
-p-tosyl-L-phenylalanine chloromethyl ketone;
TLCK, N
α
-p-tosyl-L-lysine chloromethyl ketone; EDTA, ethylenedi-
amene-tetraacetic acid.
Fig. 3. Detection of active trypsin in the medaka testis. (A) West-
ern blot analyses of medaka tissue extracts using specific anti-
medaka trypsinogen antibodies. Various tissue extracts (0.5 μg pro-
tein) were electrophoresed in the presence of SDS under reducing
conditions. (B) Occurrence of both the 26- and 28-kDa polypeptides
in the medaka testis. Varying amounts (0.3, 0.5, 2.0, and 5.0 μg per
lane) of the extract were analyzed by SDS-PAGE/Western blotting
using specific anti-medaka trypsinogen antibodies. (C) Conversion
of the 28-kDa polypeptide to the 26-kDa polypeptide by treatment
with enteropeptidase (E). Extracts of medaka intestine (0.25 μg) and
testis (2.5 μg) were separately incubated with active recombinant
medaka enteropeptidase serine protease (0.05 μg) at 37°C for 1 h in
50 mM Tris-HCl (pH 8.0), and then analyzed by SDS-PAGE (reduc-
ing conditions)/Western blotting using specific anti-medaka trypsino-
gen antibodies. Note that enteropeptidase-treated intestine
(intestine, +E) and testis extract (testis, +E) gave only a polypeptide
band of 26-kDa. All experiments were performed in triplicate, and a
representative image is shown for each.
Trypsin in the medaka testis 845
apparently with no 26-kDa active trypsin
(Fig. 7A). However, the presence of the
26-kDa active enzyme in the sperm
extracts was confirmed by gelatin
zymography (Fig. 7B). An immunocy-
tochemical analysis using anti-trypsino-
gen antibodies revealed that signals
were associated with the cell mem-
branes surrounding the sperm head
(Fig. 7C). No significant staining was
observed with the middle piece or with
the tail of the sperm. These results indi-
cate that active trypsin in the testes
may have a role in the process of
sperm release.
DISCUSSION
Trypsin is a pancreatic enzyme that is secreted into the
intestine as an inactive precursor, trypsinogen. Previously,
we found that mature medaka testes also abundantly
express trypsinogen mRNA (Ogiwara and Takahashi, 2007).
Intrigued, we pursued the biochemical and immunological
analyses reported in this study. We found that medaka
testes contain not only the inactive precursor protein
trypsinogen but also the active enzyme trypsin. This strongly
suggests a role for trypsin in testis function.
We prepared recombinant medaka trypsin and showed
that this protein is enzymatically active and exhibited many
features common to trypsins from other species. The
medaka trypsin is a serine protease that hydrolyzes internal
peptide bonds at the carboxyl terminal side of basic amino
acids, such as arginine and lysine. Trypsins detected in the
intestine and testis of the medaka were not distinguishable
at the molecular level. The spleen also expressed trypsino-
gen to a considerable extent. A mechanism for the regula-
tion of this tissue-specific expression of the trypsinogen
Fig. 4. Effect of soybean trypsin inhibi-
tor (SBTI) and antibodies specific for
medaka trypsinogen on ability of medaka
testis extract to hydrolyze Boc-Gln-Ala-
Arg-MCA. Testis extracts (25 μg) were
preincubated with SBTI (0.2 mg/ml) or
with purified anti-medaka trypsinogen
antibodies (120 μg/ml) in 500 μl of 0.1 M
Tris-HCl (pH 8.0) for 18 h at 4°C. The
extract was also preincubated with puri-
fied anti-medaka kallikrein antibodies
(120 μg/ml) as a control. After incubation,
the amydolytic activity of each sample
was determined using Boc-Gln-Ala-Arg-
MCA as a substrate. Enzyme activities
are expressed as the percentage of
activity relative to the control (no
addi-
tion). The data are presented as the mean
values
±
S.E.M. (n = 3).
100
80
60
40
20
0
Relative activity (%)
No addition
SBTI
Trypsinogen Ab
Kallikrein-like Ab
Fig. 5. Immunohistochemical detection of trypsinogen and trypsin in medaka testis. A tes-
tis section was stained with hematoxylin-eosin to identify individual cysts that contained
spermatogonia (SG), spermatocytes (SC), spermatids (ST), or spermatozoa (SZ). Neighbor-
ing sections of the testis were incubated with a purified anti-medaka trypsinogen antibody (B
and D) or with absorbed antibody (C and E). Boxed areas in (B) and (C) are shown at higher
magnification in (D) and in (E), respectively. Positive signals were stained green. The scale
bars are indicated in each panel. The reproducibility of the results was confirmed by repeat-
ing the experiments three times, and the results of a representative experiment are shown.
S. Rajapakse et al.846
gene in the medaka remains to be clarified. A recent obser-
vation by Miura et al. (2007) that trypsinogen is upregulated
in the eel testis by 17,20-dihydroxy-4-pregnen-3-one (DHP),
which is the steroid hormone that induces oocyte maturation
in teleosts, is interesting in this context. DHP may also be a
critical factor for testicular expression of the trypsinogen
gene in the medaka.
As established by several studies using mammalian
species, trypsinogen is activated by the specific processing
enzyme enteropeptidase. Previously, we demonstrated that
this activation is also true for medaka trypsinogen in the
intestine (Ogiwara and Takahashi, 2007). Because the testis
extract contained active trypsin in addition to the inactive
precursor trypsinogen and because the precursor protein
present in the extract could be readily converted in vitro to
its active trypsin by recombinant medaka enteropeptidase,
we suggest that the same activation mechanism involving
enteropeptidase may operate in mature medaka testes.
However, we did not detect enteropeptidase protein,
although an mRNA transcript encoding the complete
sequence of enteropeptidase was present in the testis. At
present, we have no data concerning the mechanism of
trypsinogen activation in the medaka testis; however, the fol-
lowing explanations may be possible: (a) the testis may pro-
duce active enteropeptidase in amounts that are sufficient
for trypsinogen activation but that are too small to detect
with our Western blot analysis, (b) an unknown protease
may be involved in the activation process, and (c) trypsino-
gen may be autocatalytically activated by trypsin (Halfon
and Craik, 2004). However, in the last case, the question of
how trypsinogen is initially activated remains. Further stud-
ies are necessary to solve this problem.
Spermatogenesis in medaka is categorized as a cystic
type (Gresik et al., 1973; Grier et al., 1980; Shibata and
Hamaguchi, 1986; Iwai et al., 2006; Schulz et al., 2010).
Spermatogenesis proceeds within a cyst that is delimited by
somatic Sertoli cells. Each cyst is initially formed by a single
primary spermatogonium and by several Sertoli cells in the
peripheral regions of the testis near the tunica albuginea
(Schulz et al., 2010). Mitotic divisions of the primary sper-
matogonium produce a cohort of secondary spermatogonia.
As the cells enter meiosis, the cysts migrate toward the cen-
ter of the testis. During spermatogenic progression, a clonal
group of isogenic spermatocytes, spermatids, and sperma-
tozoa is produced. Mature sperm are eventually released
into the spermatic duct; this process is called spermiation
(Pudney, 1995; Schulz et al., 2010). Our present observa-
tion that strong signals for trypsinogen and for trypsin were
detected with spermatogonia and with spermatozoa most
likely indicates roles of the proteins in the processes rele-
vant to these cells. In this connection, the inhibition of sperm
release from testis fragments by SBTI and by a trypsinogen-
specific antibody in our in vitro experiment could be
explained in several ways, given the involvement of active
trypsin. During spermiation, spermatozoa should be freed
from Sertoli cells, which play an indispensable role through-
Fig. 7. Detection of trypsinogen and trypsin associated with
medaka spermatozoa. (A) Adult medaka spermatozoa were col-
lected, and the extract was prepared. The sperm extract (2.0 μg)
was analyzed by Western blotting using an anti-trypsinogen anti-
body. Intestine (0.25 μg) and testis (1 μg) extracts were also ana-
lyzed for comparison. (B) The sperm extract (2.0 μg) was analyzed
using gelatin zymography. The reproducibility of the result was con-
firmed by three independent experiments. An arrowhead indicates
the position corresponding to 26-kDa. (C) Spermatozoa were col-
lected from the testis of adult medaka and immune-stained with an
anti-trypsinogen antibody. Sperm heads positively stained are indi-
cated by an arrow. The scale bar (10 μm) is indicated. Experiments
were conducted in triplicate, and the result of a representative
experiment is shown.
Fig. 6. Effects of specific anti-medaka trypsinogen antibodies and
SBTI on sperm release from the testis in vitro. (A) Mature medaka
testes were cultured at 26°C for 4.5 h in L15 medium with SBTI
(0.4 mg/ml) or with purified rat anti-medaka trypsinogen antibodies
(100 μg/ml), as described in the Materials and Methods. Controls
were incubated without SBTI or with normal rat IgG (100 μg/ml).
After incubation, the numbers of released sperm were counted. The
data are presented as the mean values ± S.E.M. (n = 4). (B) Micro-
photographs of media from testes cultured in the presence of normal
IgG (upper panel) or of anti-medaka trypsinogen antibodies (lower
panel). The experiments were performed in triplicate, and the result
of a representative experiment is shown. The scale bars (20 μm) are
indicated in each panel.
Trypsin in the medaka testis 847
out the course of spermatogenesis. The termination of Sertoli
cell-germ cell contact may require proteolytic enzymes. We
speculate that trypsin may be one such enzyme. The local-
ization of trypsinogen and trypsin on the surface of the
sperm head seems to favor this possibility. The reduction in
the number of sperm released from the tissue into the cul-
ture medium by SBTI and by the antibody may reflect the
deterioration in the process of Sertoli cell-germ cell detach-
ment due to trypsin inhibition by the protease inhibitor or by
the antibody. However, further studies demonstrating that
trypsin would facilitate the release of spermatozoa from
Sertoli cells in vivo are required in the future. Evidence for
the critical role of proteolytic enzymes in the regulation of
the Sertoli cell-germ cell interaction has been reported in
mammals (Mruk et al., 1997; Longin et al., 2001; Siu and
Chang, 2004). Notably, the inhibition of sperm release from
the testis fragments by SBTI or by a trypsinogen antibody
may have occurred through a trypsin-involving mecha-
nism(s) different from the above-speculated mechanisms.
For example, the protease might be involved in the process
of passage from the spermatic duct to outside. Regardless,
at present, the precise mechanism behind the release of
sperm from the tissue remains unclear. However, our results
strongly suggest the implication of trypsin in the process.
A previous study by Miura et al. (2007) reported that
trypsin was detectable in the membranes of spermatozoa
and found to be associated with fertilization in Japanese eel.
Considering that this medaka counterpart is also localized to
the membrane of spermatozoa, the medaka trypsin may
play a role in fertilization, similar to the case of the eel. Inter-
estingly, the expression of trypsin associated with sper-
matogonia is another common feature between the medaka
and the Japanese eel (Miura et al., 2007). Such similarities
in the expression and in the distribution of trypsin in the two
fish tempt us to speculate concerning the biological impor-
tance of the protease in the male reproduction of teleost
species.
Another interesting finding of the present study is that
trypsinogen and trypsin were expressed in high abundance
in the spermatogonia and in the spermatozoa compared
with spermatocytes or with spermatids. This finding sug-
gests a mechanism(s) regulating the differential expression
of the trypsinogen gene during the development of germ
cells. These cells express the trypsinogen gene during the
early stage of spermatogenesis; however, its expression
declines when the germ cells enter meiosis. However, germ
cells resume trypsinogen gene expression in the extremely
late stage of spermatogenesis. Considering that trypsinogen
and trypsin proteins were detected in the lowest abundance
in spermatids but in large abundance in spermatozoa, we
speculate that the resumption of transcription and translation
of the gene may occur in late spermatids, such that active
trypsin expressed in the germ cells may be synchronized for
their detachment from Sertoli cells. The validity of this
assumption would be further strengthened by providing evi-
dence that levels of trypsinogen mRNA expression is
enhanced in late spermatids. Recent studies using mamma-
lian species reveal that after meiosis, haploid germ cells
initiate a gene expression program necessary for the differ-
entiation of spermatids and of spermatozoa (Sassone-Corsi,
2002; Rousseaux et al., 2008; Gaucher et al., 2010, 2012;
Montellier et al., 2013; Boussouar and Benahmed, 2004;
Alves et al., 2013; Boussouar et al., 2014).
In summary, we have demonstrated the presence of
trypsinogen and trypsin in the medaka testis. Our present
results suggest that active trypsin may play a role in testis
function in medaka.
ACKNOWLEDGMENTS
This work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science
and Technology of Japan (24247010 to T. T.).
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(Received May 15, 2014 / Accepted August 12, 2014)
