(Cameo and. Blaquier,. 1976;. Brooks and Higgins,. 1980;. Jones et al., 1980,. 1981;. Brooks,. 1983a; .... glass vials containing. 1 ml of medium, essentially.
BIOLOGY
OF
REPRODUCTION
38,
487-496
(1988)
Characterization
and Hormonal
Regulation
by the Murine MICHAEL
of
for
Reproductive
Vanderbilt
ORGEBIN-CRIST
& Gynecology
Obstetrics
Center
and
Biology
University Nashville,
Synthesis
Epididymis’
and MARIE-CLAIRE
K. HOLLAND2
Departments
of Protein
School
Cell
Biology
Research
of Medicine
Tennessee
37232
ABSTRACT Protein The able
synthesis
and
incorporation of label incorporated
secretion
were
examined
in vitro
label into tissue plus medium into protein increased with
was
the
by
linear
incubating for
weight
minced
tissue
with
the 5 h of incubation. The of tissue used. Approximately
[35S]methionine. percentage 13% of
of avail the label
incorporated appeared in the medium after 5 h of incubation. Release of radioactive protein into the medium was characterized by an initial slow release (1-2 h) followed by a more rapid linear release between 3 and 5 h. Polyacrylamide gel electropboresis revealed that the pattern of radioactive proteins present in the medium was different from and less complex than the tissue proteins. Substantial differences in protein patterns from different epididymal regions could be detected. The caput epididymidis was particularly active in secreting proteins characteristic of this region, whereas the corpus and cauda synthesized and secreted similar proteins. At least one of these proteins characteristic of the caput is stabilized by disulpbide bonds. Short-term (9 day) castration resulted in reduced synthesis and secretion of several of these epididymal proteins. Testosterone administered after 9 days of castration reinitiated synthesis of some but not all of these epididymal proteins. INTRODUCTION
nochemical
The mammalian epididymis intraluminal environment in
techniques
Fournier-Delpech
provides a specific which immature sper-
Garberi
(Fournier-Delpech,
et
et al.,
al.,
1979;
1973;
Kohane
1968;
Lea
et al.,
et
al.,
1979,
1978;
1980a,b,
matozoa from the testis undergo a series of changes, collectively termed maturation, that result in the spermatozoa becoming fully functional. This specific intraluminal environment is maintained by an active
1983; Faye et al., 1980; Moore, 1980, 1981b; Jones and Brown, 1982; Wong and Tsang, 1982; Brooks and Tiver, 1983; Thomas et al., 1984; Rifkin and Olson,
process didymal
the epididymis (Brooks, 1981; 1984; Klinefelter
of
absorption epithelium.
and secretion Electrophoretic
by the analyses
epididymal fluid obtained by micropuncture have demonstrated the presence of proteins specific to the epididymis (Koskimies and Kormano, 1975; Turner et al., 1979; Wong and Tsang, 1982; Olson and Hinton,
1985;
Flickinger
proteins
have
been
or fluid, and, and localization
isolated
et al., 1986). from
Some
epididymal
in some studies, their has been determined
of
these et
shown
has
that
(Cameo and Blaquier, 1980; Jones et al., 1980,
Kohane proteins al.,
et
al.,
interact
1982;
1983)
1976; 1981;
and
that
with
spermatozoa
and
Tiver,
Brooks
Klinefelter and Hamilton, 1985). Protein and secretion have also been demonstrated
1983;
synthesis by radio-
autography (Neutra and Leblond, 1966; Vendrely and Durliat, 1968; Kopecny, 1971; Bennett et a!., 1974; Kanka and Kopecny, 1977; Kopecny and Pech, 1977; Flickinger, 1979, 1981, 1983, 1985; Flickinger
School
University
1983a;
(Voglmayr
site of synthesis by using immu-
Gynecology,
Vanderbilt
some
incorporation
synthesizes and secretes proteins Sylvester et al., 1986; Thomas et al., and Hamilton, 1985) under the
of androgens and Higgins,
Brooks,
cytosol
Grant HD03820 from the National Development and by a grant from to M.C.O.C. The Center for Reproby NIH Grant HD05797. K. Holland, Dept. of Obstetrics &
Nashville, TN
control Brooks
of these
Accepted September 21, 1987. Received November 28, 1986. ‘This study was supported by Institute of Child Health & Human the Andrew W. Mellon Foundation ductive Biology Research is supported Reprint requests: Dr. Michael
MCN,
Radiolabel
1985).
epiof
of Medicine,
Room
et al., 1984; Orgebin-Crist and Menezo, 1980; FainMaurel et al., 1981, 1983; Orgebin-Crist et al., 1986). A few studies have reported that interaction with epididymal
D-2318
(Brandt
37232.
487
secretory et al.,
1980;
proteins Thomas
changes et al.,
sperm
function
1984;
Orgebin-
488
HOLLAND
Crist 1984;
and Fournier-Delpech, Gonzalez Echeverria
the epididymal extensively in of
rat
spermatozoa
rat eggs in vitro The model
secretory the rat, and
their
to
Cuasnicu et al., 1984). Although has been studied in vitro viability
poor
ability
functional
provides
in which
1982; al.,
function the short
has limited
mouse
spermatozoa media and
et
study
these
remain motile for can fertilize eggs in
appropriate as mouse
hours synthetic
in
artificial media in
vitro. Furthermore, a number of strains of mice bearing gene mutations that affect motility or fertility have been described (Green, 1981). For some of these mutants, the exact site affected that results in infertility is not known. One potential site is the epididymis. Clearly, any such invaluable tools to assess the mal function for fertility. In the
present
study,
mutants importance
we have
used
would provide of epididytissue
minces
mutant
mice
(see
Holland
and
to
AND
albumin (Fraction methylsulphonyl
V), fluoride
METHODS
of analytical blue R250, bovine amino (PMSF)
Fig.
6).
Tissue
from
each
small pieces (800 Ci/mmol) was purchased from New England Nuclear (Boston, MA). The reagents used for polyacrylamide gel electrophoresis came from Bio-Rad
centrifuged supernatants Small removed,
(Richmond,
as carrier protein, added to a final precipitate was
CA).
Animals
Mature C57BL6/J male mice were obtained as retired breeding animals from the Jackson Labora-
(100 pCi) of the tissue and
was added. incubation,
pieces were homogenized
the
supple1 mg of glucose. medium
homogenized, tissue were
at 100,000 X g for 30 mm at 2#{176}C.The were retained at -70#{176}C. aliquots of each supernatant (10 pl) were 50 pg of bovine serum albumin were added and trichloroacetic acid concentration of 10% collected on Millipore
(TCA) (w/v). filters
was The and
tories (Bar Harbor, ME) and were housed at 25#{176}C on a lighting regimen of 16L:8D, with free access to food and water. Animals were killed by an overdose
washed with 5% (w/v) TCA containing 10 mM methionine; the dried filters were immersed in 5 ml of toluene containing 4 g/l of 2,5-diphenyloxa.zole and 0.1 g/l of 1,4,bis(5-phenyloxazolyl) benzene, and counted in a liquid scintillation spectrometer.
of ether, and blood samples were drawn directly from the heart. The animals were then perfused, via the
The residual supernatant was made with TCA and left at 4#{176}C overnight.
heart, with saline to clear as much blood as possible.
proteins
was
removed
and cleared
the testis and epididymis Each testis and epididymis of fat.
The
testicular
of
capsule
were
TCA, then v/v) before
pelleted,
washed
twice
to with
10% (w/v) Precipitated 10%
extracted twice with ethanol:ether being dried under vacuum. The pellet
(w/v) (1:1 was
PROTEIN
dissolved sodium 30 mM
in
dodecyl Tris-HC1,
material the
50
pl
of
centrifuged
supernatant
was
tubes. Small radioactivity
(2
proteins
measured
containing prepared
for
buffer
sulphate [SDS], 20% pH 6.8) and boiled
was
was
dispersion
SYNTHESIS
(10,000
transferred
p1) aliquots incorporated
were into of
THE
[w/v} glycerol, for 5 mm. The 4 mm),
carefully
to
removed, the
counts (Brooks
489
EPIDIDYMIS
phosphorylase
13-galactosidase-1 with
and
0.1%
fresh
omitted raphy (1983).
Samples
Coomassie
X-omat
AR5
1980).
,400,
was
performed
blue
(1:3:6), same solution
and
R250
in
and destaining with the dye
x-ray
film.
RESULTS
Polyacrylamide
Gel
Electrophoresis
Protein
Electrophoresis was
performed
in
slab
gels
(15
X
13 X 1.5 cm). Denaturing gels consisted of linear 10% to 15% gradients of acrylamide using the SDS system of
brilliant
B-97
before gels were impregnated for fluorogby the method of Skinner and Griswold The impregnated gels were exposed at -70#{176}C
to Kodak
were then and Higgins,
Staining
16,000.
(w/v)
acetic acid:methanol:water was performed in the
and the solubilized
above.
MURINE
albumin-66,000,
[w/v]
X g for
as described
equal numbers electrophoresis
(1%
BY
(1970).
Laemmli
Gels
were
calibrated
with
mixture of proteins with lar weight (a-lactalbumin-
subunits 14,200,
20,100, 29,000,
carbonic anhydrase-phosphate dehydrogenase
trypsinogen-24,000, glyceraldehyde-3
subunit-36,000,
bovine
Tissue
Mouse
epididymal
Secretion
tissue
release them of radioactive
incorporated tissue added
also (Fig.
maintained
methionine
tissue increased linearly incubation (Fig. la).
serum
by
in Vitro
L-[35S]
selectively incorporation
a
and
Epididymal
incorporate
of known molecutrypsin inhibitor-
egg albumin-45,000,
Synthesis
la,
with The
increased inset).
in vitro
into the
into label time total
proteins
medium. into medium over the quantity
with Generally,
the
could and Total plus
5 h of the of label
weight of 1-2% of the
(0)
(b)
z
>
o S.-.
LJU
0
U)
aU)
z 0 C-,
I
2
3 Time (h)
4
3
5 TIME(h)
FIG. 1. (a) Time course of incorporation of [35 SI methionine into proteins by epididymal tissue in vitro as a function of tissue weight and (b) the course of release of these proteins into the medium. The epididymis was minced and washed as described in Materials and Methods, and 5 mg (0), 10 mg (.), 15 mg (o), 20 mg (.), or 25 mg () of tissue were incubated in 1 ml of medium containing 100 zCi 13’SI methionine for the times indicated. Samples were homogenized and acid-insoluble radioactivity was measured as described in Materials and Methods. Each point represents the means of two incubations. time
HOLLAND
490
available label was
incorporated
not 1.1)
shown). Approximately per flask was used
this
yielded
without this
adequate
utilizing quantity
quantities
excessive of
tissue
into protein
15 mg of tissue each incubation,
in
100
pCi
was
a slow
inversely
phase,
dependent
the
upon
ployed,
followed
radioactive released
protein. The into the medium
of tissue into the
(Fig. lb, medium
comparing
the
by
inset). was
pattern
the
a more
methio-
of
weight rapid
which
of
tissue
linear
total quantity increased with That the release selective can be
revealed
The
by
the
the
of tissue gel
(see Fig. Pattern
proteins,
as
electrophoresis
4b, c).
2-6,
Fig.
caput
secreted
em-
proteins
release
of
3b).
Secretion
Indeed,
secreted
epididymis.
These
of proteins verified by
into
the
of
medium
regions. epididymis
A
caput
6).
and
distal
was
Of
initial
segment
twice
the
from
all
other
other
segments
the
segments
of
the
secreted
of
compara-
protein. There was substantial in the pattern of proteins secreted by epididymal
active
most
tissue
from
secreting
in
corpus and cauda of secreted proteins
interest caput
of
quantity
different
number of proteins characteristic were detectable (Fig. 4a, Lanes
and the profiles
and
the
approximately
ble quantities regional variation
present
of Protein
Epididymis
was
6
is a protein (Segments
weight of 79,000 (Fig. 4a, Lanes
unique
generally (Fig. 4a,
secreted 3 and
of the 2-6). showed Lanes 5
from
4) that
in the absence 3 and 4), but
pro-
the mid
ran
with
a
of mercapthat disap-
5
molecular toethanol
3
peared in the presence of mercaptoethanol. When this protein disappeared, two others, with molecular weights of 40,500 and 33,500 appeared (Fig. 4b). None of the other major characteristic epididymal proteins changed location in the presence or absence of mercaptoethanol and, thus, were not stabilized by
0
inter- or intra-chain disuiphide Epididymal tissue proteins
I’
-Jw
pattern
The epididymis was divided into 5 segments, as described in Figure 3a. Secretory activity, based on equal wet weights of tissue, was different from that of the testis (Line 1) and decreased moving from proximal to distal segments of the epididymis (Lines
teins, similar
wlI
the
polyacrylamide
Regional
The
>w
with
of proteins the weight
proteins
of radioactive
medium
from
incorporation
Release of proteins (Fig. ib). Initially, duration
in the
and fluorography
Using
total radioactivity released into
after 5 h of incubation. in a biphasic manner
there
L-[35S]
limit
±
ORGEBIN-CRIST
protein
of animals.
nine, availability of label did not into medium plus tissue (Fig. 2). Approximately 13% of the incorporated into protein was medium occurred
(data
(15.2 because
of radioactive
numbers and
AND
bonds. showed
a very
different
Ol-.
u,
