BIOLOGY
OF
REPRODUCTION
Nonhistone
17,
769-779
(1977)
Chromosomal NATHANIEL
Proteins
C. MILLS2 Dept. Baylor
of the Developing
and
ANTHONY
of Cell College
R. MEANS
Biology, of
Houston,
Rat Testis’
Medicine,
Texas
ABSTRACT Chromatins were isolated from rat testes at 5 day intervals during development and solubilized in sodium dodecyl sulfate. Nonhistone chromosomal proteins were then separated by electrophoresis on SDS-polyacrylamide gels. This technique resolved the proteins into 40-50 distinct bands separated primarily on the basis of molecular weight. The chromosomal proteins derived from testes containing large portions of supporting (immature Sertoli) cells, spermatogonia and primary spermatocytes (5-20 days of age) contain many high molecular weight proteins. Chromatin preparations enriched in round spermatid chromatin (65 days of age) showed diminished amounts of high molecular weight proteins with the exception of two bands at 81,000 and 87,000 molecular weight which appeared to be specific for spermatids. Analysis of chromatin from Sertoli cell-enriched (SCE) testes, which are devoid of germinal cells, revealed that most of the nonhistones present are proteins of less than 80,000 molecular weight. Between 22 and 40 days of age, a large decrease in proteins at 123,000 and corresponding increase in proteins at 48,000 molecular weight was noted in SCE testis preparations. Thus, the metabolically active cells (supporting cells or immature Sertoli cells, spermatogonia and primary spermatocytes) have many high molecular weight nonhistone proteins, whereas less active spermatids contain predominantly smaller species. These data suggest that the high molecular weight proteins may represent enzymes or structural proteins needed for mitosis, meiosis and cell growth.
INTRODUCTION
The onset of spermatogenesis represents an opportunity development setting. postnatal
and The rat
populations, progenitors
the all
the reproductive supporting cells The hum
orderly from
to
lifetime of the which give rise
progression stem cells
of the through
1975),
entiated non et Histones
allows
assignment
1975;
a unique
of
the early of two cell
which produced
are the during
arginine-rich into the regression shige,
germinal epithespermatogenesis
of specific
functions to particular al., 1971; Mills and of the early germ cells
precisely cells to Means et
histones in the primary spermaet al., 1975; Grimes et al., al., 1977b) and this process is
by
replacement
of
histones
protein for packaging sperm head which results of the genome (Marushige
1975).
In
only somatic Nevertheless,
animal, and the to Sertoli cells.
(Clermont and Percy, 1957) and the timed conversion of immature Sertoli mature ones (Steinberger et al., 1970; a!.,
meiotic (Branson Mills et
in
tubules primarily
gonocytes, germ cells
specific tocyte
mammal cellular
culminated
differentiation
seminiferous testis consist of
in the study
contrast,
Sertoli
histones differentiation
(Mills
cells et
and
differ-
results in marked metabolic et al., 1977; Means et al.,
1976;
et al.,
bly
Fakunding
involve
1976).
remodeling
nonhistone much evidence in specific
gene
regulatory
Kodohama tempted
769
regulation
(Teng
little
in these onset of and
to
the
genome
and
by
Indeed, proteins Hamilton,
and Allfrey, 1970; O’Malley et et al., 1976b). In spite of the on nonhistone proteins as gene
substances,
ing changes during the
of
in the protein and somatic most proba-
chromosomal proteins. exists implicating these
1969; Shelton al., 1972; Tsai emphasis placed
Accepted July 18, 1977. Received May 18, 1977. ‘Submitted in partial fulfillment of the requirement for the Doctor of Philosophy degree (Nathanial C. Mills) Vanderbilt University, Nashville, Tennessee. 2 address: Div. of Endocrinology, Dept. of Medicine, M. S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033.
contain
a!., 1977b). maturation of
the Sertoli cell changes (Griswold
The large changes that occur composition of testicular germ cells during postnatal development
cell types (VerMeans, 1972). are replaced by
with
the DNA in complete and Maru-
study
is known
chromosomal spermatogenesis.
Turkington this
important
concernproteins Only
(1974)
have class
of
atpro-
MILLS
770
teins in techniques ation tion
rat
testis selected
of the
nonhistone
of only
In changes
the
histone
and even in for separation proteins
20-25
protein
present that occur
proteins
at
report the fraction-
and allowed
resolu-
bands.
study in the 5-day
testicular development. in nonhistone proteins
their
AND
we have population intervals
examined of nonduring
In addition, of the Sertohi
rat
alterations cell were
investigated using the (SCE) testis (Tindall et isolation and fractionation
Sertohi cell-enriched al., 1975). Chromatin procedures were
adapted
protein
to
techniques distinct molecular
minimize
weights MATERIALS
Chemicals
losses.
resulted in resolution bands on SDS gels which
and
using
protein AND
of were
These 40-50 assigned
standards.
METHODS
Supplies
The sodium dodecyl sulfate (sequenal grade) was obtained from the Pierce Chemical Co. Glycine was purchased from J. T. Baker Co. Coomassie brilliant blue, R 250, was a product of Colab and bromophenol blue was purchased from Mallinckrodt. The protein molecular weight standards, myoglobin, chymotrypsinogen A, ovalbumin, and albumin were purchased from Schwarz/Mann. The E. coli RNA polymerase used as a molecular weight standard was isolated from late log E. coli K-12 by a modification of the procedures by Burgess (1969) and Bautz and Dunn (1969) as presented by Tsai et al. (1976a). Triton X-100, phosphorylase A and thyroglobulin Type I were purchased from Sigma Chemical Co. Collagenase was obtained from Worthington. Myosin was purified from rabbit muscle by the method of Perry (1955). Animals Male rats were ordered from the Holtzmann Co. at specific ages with a designated body weight and weight range (Mills et al., 1977a). To obtain animals with testes free of germinal epithelium, rat fetuses were X-irradiated in utero on Day 20 of gestation as described by Tindall et al. (1975). The biochemical characterization of these Sertoli cell-enriched (SCE) testes has been previously documented (Fakunding et a!., 1976).
Nuclei
and Chromatin
Preparation
Male rats were killed by cervical dislocation and the testes were placed on ice. The tunica albuginea was removed and the testicular tissue was processed for nuclei isolation by homogenization in 0.32 M sucrose containing 50 mM Tris, pH 7.5, and 3 mM MgCI2. After filtering the homogenate through 3 layers of organza the nuclei were pelleted at 1000 g using the Sorval HB-4 rotor in a refrigerated centrifuge. The nuclear pellet was resuspended in 10 volumes of 2.2 M sucrose containing 50 mM Tris, pH 7.5 and 3 mM Mga . The nuclei were pelleted from the dense sucrose by centrifugation at 15,000 g for 45 mm in a swinging bucket rotor. Chromatin was prepared from
MEANS
the purified
by the procedure
nuclei
of Spelsberg
et al.
(1971).
The yield of chromatin per gram of tissue at 5 or 10 days of age was 4 times greater than that obtained from rat testes at 35 days of age. The decrease in chromatin yield is partially accounted for by the decrease of the DNA content of the testes during development which is approximately 8 mg DNA/gm tissue at 5 days and 2 mg DNA/gm tissue at 35 days of age (Mills et al., 1977a). The protein to DNA ratio from the isolated chromatins was slightly less than 2:1 throughout testicular development. The total histone to DNA content ranged from 1.00 to 1.22 mg histone/mg DNA (average 1.13). The nonhistone chromosomal proteins averaged 0.62 mg protein/mg DNA and ranged from 0.5-0.7. SDS
Gel Electropboresis
Total chromosomal proteins were separated according to molecular weight using the Tris, glycine, SDS polyacrylamide gel system of Laemmli (1970). Samples of 80 ag of DNA as whole chromatin (approximately 160 ig total protein) were dissolved in 200 MI of SDS sample buffer (5 percent glycerol, 5 percent 2-mercaptoethanol, 3.3 percent SDS, 0.75 g percent Tris-HC1, pH 6.8, v/v) by heating to 90#{176}C for 5 mm and separated on a 6 mm diameter, 8 percent acrylamide gel (pH 8.8) which had a 3 percent acrylamide stacking gel (pH 6.8). Ten Ml of 0.1 percent bromophenol blue was added as a dye marker. The samples were stacked at 0.5 mAmps/gel and dcctrophoresed at 1.25 mAmps per tube for separation. Electrophoresis was stopped when the dye was less than 1 cm from the bottom of the 10 cm separating gel. The polyacrylamide gels were immediately removed from the tubes and fixed in 40 percent methanol, 10 percent acetic acid and 50 percent water. The fixation process required 12 to 24 h during which time 3 changes of fixative were added. After fixation the gels were stained with 0.1 percent Coomassie brilliant blue (w/v) dissolved in the fixative. After staining for 4 h the dye solution was removed and the gels were destained by diffusion. The destained gels were scanned with a GCA/McPherson recording spectrophotometer at 600 nm with a 0.2 mm slit width. RESULTS
Protein
standards
SDS 8 percent value (distance the dye front of Rf
were
electrophoresed
the molecular weight values for each protein
standards
On 8 percent less than 23,000 at the dye front.
of molecular
weight
a linear greater
migrate
but
shown
in
a consistent by
the
graph
log
1.
23,000 and 100,000 form with molecular weights as
Rf
versus the calculated plotted on a linear
scale, is shown in Figure polyacrylamide gels, proteins molecular weight migrate Protein
on
polyacrylamide gels to allow the protein migrated/distance moved) determinations. The
of
between
plot. than
nonlinear the
standards.
Proteins 100,000 fashion As
TESTICULAR
judged
by
the
variations
migration on the molecular range
more
portion weights
of the greater
more
for
standard
to
4,000
curve. Proteins than 100,000 determinations
molecular (Fig.
on
of not
the
linear
with molecular vary considerably due
of the migration proteins from
assigned
to
weights
using
this
standard
the of
the
on the molecular
the
some peak. gel
scans.
idea The
scans
distance weight
data
provide
migrated of each
by each molecule,
of the relative proportions large bands shown at the
are
stacking
These
caused gel
and
by refractive the
top
properties
of the
separating
340
K
COLI
RNA
POLYMERASE
58
a
x
4
-Jz Ui -J
0
n
A 94
K POLYMERSSE
rat
401
20 MYOGLOBIN
I’Ko
at 5 and
0.5 Rf
0.6
F3
as described
histones or near
proteins
50 protein shoulders,
of
(F2b, dye
gel. patterns
of age
several smaller
(Fig.
2A
prominent bands. The
these
reveal
the
nonhistone
between
40
and
bands, recognized as either peaks to be present. Particularly prominent bands
are
of 34, 39, 51, and 220 thousand As 2C-21),
10 days
show many scans
chromosomal
55,
those
with
60, (K).
69,
molecular 73,
testicular development the nonhistone protein
or
weights
123,
135,
proceeds bands
K molecular weight those at 69 K and
0.7
of the decreased
220 after
prominent, 34 K and
testicular Microscopic
tatively
148
(Figure of 115 K
increase 73 K
in intensidiminish in
K molecular 10 days
0.8
weight of age.
most of the protein 60 K remain through-
development. examination
of
nuclei
prepara-
0.9
when
nuclei
through
2.2
1.0
FIG. 1. Standard curve of known molecular weight proteins as analyzed from SDS polyacrylamide gels. Molecular weight standards were electrophoresed on Tris-glycine, SDS, 8 percent polyacrylamide gels and their Rf values were determined. The curve shown is a composite from several different gels. This curve was used to assign tentative molecular weights to the nonhistone chromosomal protein bands shown in subsequent figures.
were
nuclei of round nuclei of the pelleting quanti-
purified
M sucrose.
by Late
the
pellet
the
majority
did
contain of
pellicle. of the
nuclei
for
and
X-100
(w/v)
Sertoli
cell
To obtain a mature semichromatin
using a buffer mM Tris, pH
containing
0.25
to
cytoplasmic
remove
were the Although spermatids,
and
the
were purified M sucrose, 50
MgCl2
round
spermatid
found in representative
epithelium,
paration of 0.32
a few
early
centrifu-
spermatids
primary spermatocyte nuclei cell components in the pellet.
niferous 04
testes
nuclei were preparation
In
0.3
and
The smaller migrate in
spectrophotometer
and major
02
histones
respectively) bands and
gation
30
0.1
be
(1972). F2A1)
tions revealed that the haploid spermatids and the voluminous mature Sertoli cells were not
50 RNA
of
front.
1
OF
REPRODUCTION
Nonhistone
17,
769-779
(1977)
Chromosomal NATHANIEL
Proteins
C. MILLS2 Dept. Baylor
of the Developing
and
ANTHONY
of Cell College
R. MEANS
Biology, of
Houston,
Rat Testis’
Medicine,
Texas
ABSTRACT Chromatins were isolated from rat testes at 5 day intervals during development and solubilized in sodium dodecyl sulfate. Nonhistone chromosomal proteins were then separated by electrophoresis on SDS-polyacrylamide gels. This technique resolved the proteins into 40-50 distinct bands separated primarily on the basis of molecular weight. The chromosomal proteins derived from testes containing large portions of supporting (immature Sertoli) cells, spermatogonia and primary spermatocytes (5-20 days of age) contain many high molecular weight proteins. Chromatin preparations enriched in round spermatid chromatin (65 days of age) showed diminished amounts of high molecular weight proteins with the exception of two bands at 81,000 and 87,000 molecular weight which appeared to be specific for spermatids. Analysis of chromatin from Sertoli cell-enriched (SCE) testes, which are devoid of germinal cells, revealed that most of the nonhistones present are proteins of less than 80,000 molecular weight. Between 22 and 40 days of age, a large decrease in proteins at 123,000 and corresponding increase in proteins at 48,000 molecular weight was noted in SCE testis preparations. Thus, the metabolically active cells (supporting cells or immature Sertoli cells, spermatogonia and primary spermatocytes) have many high molecular weight nonhistone proteins, whereas less active spermatids contain predominantly smaller species. These data suggest that the high molecular weight proteins may represent enzymes or structural proteins needed for mitosis, meiosis and cell growth.
INTRODUCTION
The onset of spermatogenesis represents an opportunity development setting. postnatal
and The rat
populations, progenitors
the all
the reproductive supporting cells The hum
orderly from
to
lifetime of the which give rise
progression stem cells
of the through
1975),
entiated non et Histones
allows
assignment
1975;
a unique
of
the early of two cell
which produced
are the during
arginine-rich into the regression shige,
germinal epithespermatogenesis
of specific
functions to particular al., 1971; Mills and of the early germ cells
precisely cells to Means et
histones in the primary spermaet al., 1975; Grimes et al., al., 1977b) and this process is
by
replacement
of
histones
protein for packaging sperm head which results of the genome (Marushige
1975).
In
only somatic Nevertheless,
animal, and the to Sertoli cells.
(Clermont and Percy, 1957) and the timed conversion of immature Sertoli mature ones (Steinberger et al., 1970; a!.,
meiotic (Branson Mills et
in
tubules primarily
gonocytes, germ cells
specific tocyte
mammal cellular
culminated
differentiation
seminiferous testis consist of
in the study
contrast,
Sertoli
histones differentiation
(Mills
cells et
and
differ-
results in marked metabolic et al., 1977; Means et al.,
1976;
et al.,
bly
Fakunding
involve
1976).
remodeling
nonhistone much evidence in specific
gene
regulatory
Kodohama tempted
769
regulation
(Teng
little
in these onset of and
to
the
genome
and
by
Indeed, proteins Hamilton,
and Allfrey, 1970; O’Malley et et al., 1976b). In spite of the on nonhistone proteins as gene
substances,
ing changes during the
of
in the protein and somatic most proba-
chromosomal proteins. exists implicating these
1969; Shelton al., 1972; Tsai emphasis placed
Accepted July 18, 1977. Received May 18, 1977. ‘Submitted in partial fulfillment of the requirement for the Doctor of Philosophy degree (Nathanial C. Mills) Vanderbilt University, Nashville, Tennessee. 2 address: Div. of Endocrinology, Dept. of Medicine, M. S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033.
contain
a!., 1977b). maturation of
the Sertoli cell changes (Griswold
The large changes that occur composition of testicular germ cells during postnatal development
cell types (VerMeans, 1972). are replaced by
with
the DNA in complete and Maru-
study
is known
chromosomal spermatogenesis.
Turkington this
important
concernproteins Only
(1974)
have class
of
atpro-
MILLS
770
teins in techniques ation tion
rat
testis selected
of the
nonhistone
of only
In changes
the
histone
and even in for separation proteins
20-25
protein
present that occur
proteins
at
report the fraction-
and allowed
resolu-
bands.
study in the 5-day
testicular development. in nonhistone proteins
their
AND
we have population intervals
examined of nonduring
In addition, of the Sertohi
rat
alterations cell were
investigated using the (SCE) testis (Tindall et isolation and fractionation
Sertohi cell-enriched al., 1975). Chromatin procedures were
adapted
protein
to
techniques distinct molecular
minimize
weights MATERIALS
Chemicals
losses.
resulted in resolution bands on SDS gels which
and
using
protein AND
of were
These 40-50 assigned
standards.
METHODS
Supplies
The sodium dodecyl sulfate (sequenal grade) was obtained from the Pierce Chemical Co. Glycine was purchased from J. T. Baker Co. Coomassie brilliant blue, R 250, was a product of Colab and bromophenol blue was purchased from Mallinckrodt. The protein molecular weight standards, myoglobin, chymotrypsinogen A, ovalbumin, and albumin were purchased from Schwarz/Mann. The E. coli RNA polymerase used as a molecular weight standard was isolated from late log E. coli K-12 by a modification of the procedures by Burgess (1969) and Bautz and Dunn (1969) as presented by Tsai et al. (1976a). Triton X-100, phosphorylase A and thyroglobulin Type I were purchased from Sigma Chemical Co. Collagenase was obtained from Worthington. Myosin was purified from rabbit muscle by the method of Perry (1955). Animals Male rats were ordered from the Holtzmann Co. at specific ages with a designated body weight and weight range (Mills et al., 1977a). To obtain animals with testes free of germinal epithelium, rat fetuses were X-irradiated in utero on Day 20 of gestation as described by Tindall et al. (1975). The biochemical characterization of these Sertoli cell-enriched (SCE) testes has been previously documented (Fakunding et a!., 1976).
Nuclei
and Chromatin
Preparation
Male rats were killed by cervical dislocation and the testes were placed on ice. The tunica albuginea was removed and the testicular tissue was processed for nuclei isolation by homogenization in 0.32 M sucrose containing 50 mM Tris, pH 7.5, and 3 mM MgCI2. After filtering the homogenate through 3 layers of organza the nuclei were pelleted at 1000 g using the Sorval HB-4 rotor in a refrigerated centrifuge. The nuclear pellet was resuspended in 10 volumes of 2.2 M sucrose containing 50 mM Tris, pH 7.5 and 3 mM Mga . The nuclei were pelleted from the dense sucrose by centrifugation at 15,000 g for 45 mm in a swinging bucket rotor. Chromatin was prepared from
MEANS
the purified
by the procedure
nuclei
of Spelsberg
et al.
(1971).
The yield of chromatin per gram of tissue at 5 or 10 days of age was 4 times greater than that obtained from rat testes at 35 days of age. The decrease in chromatin yield is partially accounted for by the decrease of the DNA content of the testes during development which is approximately 8 mg DNA/gm tissue at 5 days and 2 mg DNA/gm tissue at 35 days of age (Mills et al., 1977a). The protein to DNA ratio from the isolated chromatins was slightly less than 2:1 throughout testicular development. The total histone to DNA content ranged from 1.00 to 1.22 mg histone/mg DNA (average 1.13). The nonhistone chromosomal proteins averaged 0.62 mg protein/mg DNA and ranged from 0.5-0.7. SDS
Gel Electropboresis
Total chromosomal proteins were separated according to molecular weight using the Tris, glycine, SDS polyacrylamide gel system of Laemmli (1970). Samples of 80 ag of DNA as whole chromatin (approximately 160 ig total protein) were dissolved in 200 MI of SDS sample buffer (5 percent glycerol, 5 percent 2-mercaptoethanol, 3.3 percent SDS, 0.75 g percent Tris-HC1, pH 6.8, v/v) by heating to 90#{176}C for 5 mm and separated on a 6 mm diameter, 8 percent acrylamide gel (pH 8.8) which had a 3 percent acrylamide stacking gel (pH 6.8). Ten Ml of 0.1 percent bromophenol blue was added as a dye marker. The samples were stacked at 0.5 mAmps/gel and dcctrophoresed at 1.25 mAmps per tube for separation. Electrophoresis was stopped when the dye was less than 1 cm from the bottom of the 10 cm separating gel. The polyacrylamide gels were immediately removed from the tubes and fixed in 40 percent methanol, 10 percent acetic acid and 50 percent water. The fixation process required 12 to 24 h during which time 3 changes of fixative were added. After fixation the gels were stained with 0.1 percent Coomassie brilliant blue (w/v) dissolved in the fixative. After staining for 4 h the dye solution was removed and the gels were destained by diffusion. The destained gels were scanned with a GCA/McPherson recording spectrophotometer at 600 nm with a 0.2 mm slit width. RESULTS
Protein
standards
SDS 8 percent value (distance the dye front of Rf
were
electrophoresed
the molecular weight values for each protein
standards
On 8 percent less than 23,000 at the dye front.
of molecular
weight
a linear greater
migrate
but
shown
in
a consistent by
the
graph
log
1.
23,000 and 100,000 form with molecular weights as
Rf
versus the calculated plotted on a linear
scale, is shown in Figure polyacrylamide gels, proteins molecular weight migrate Protein
on
polyacrylamide gels to allow the protein migrated/distance moved) determinations. The
of
between
plot. than
nonlinear the
standards.
Proteins 100,000 fashion As
TESTICULAR
judged
by
the
variations
migration on the molecular range
more
portion weights
of the greater
more
for
standard
to
4,000
curve. Proteins than 100,000 determinations
molecular (Fig.
on
of not
the
linear
with molecular vary considerably due
of the migration proteins from
assigned
to
weights
using
this
standard
the of
the
on the molecular
the
some peak. gel
scans.
idea The
scans
distance weight
data
provide
migrated of each
by each molecule,
of the relative proportions large bands shown at the
are
stacking
These
caused gel
and
by refractive the
top
properties
of the
separating
340
K
COLI
RNA
POLYMERASE
58
a
x
4
-Jz Ui -J
0
n
A 94
K POLYMERSSE
rat
401
20 MYOGLOBIN
I’Ko
at 5 and
0.5 Rf
0.6
F3
as described
histones or near
proteins
50 protein shoulders,
of
(F2b, dye
gel. patterns
of age
several smaller
(Fig.
2A
prominent bands. The
these
reveal
the
nonhistone
between
40
and
bands, recognized as either peaks to be present. Particularly prominent bands
are
of 34, 39, 51, and 220 thousand As 2C-21),
10 days
show many scans
chromosomal
55,
those
with
60, (K).
69,
molecular 73,
testicular development the nonhistone protein
or
weights
123,
135,
proceeds bands
K molecular weight those at 69 K and
0.7
of the decreased
220 after
prominent, 34 K and
testicular Microscopic
tatively
148
(Figure of 115 K
increase 73 K
in intensidiminish in
K molecular 10 days
0.8
weight of age.
most of the protein 60 K remain through-
development. examination
of
nuclei
prepara-
0.9
when
nuclei
through
2.2
1.0
FIG. 1. Standard curve of known molecular weight proteins as analyzed from SDS polyacrylamide gels. Molecular weight standards were electrophoresed on Tris-glycine, SDS, 8 percent polyacrylamide gels and their Rf values were determined. The curve shown is a composite from several different gels. This curve was used to assign tentative molecular weights to the nonhistone chromosomal protein bands shown in subsequent figures.
were
nuclei of round nuclei of the pelleting quanti-
purified
M sucrose.
by Late
the
pellet
the
majority
did
contain of
pellicle. of the
nuclei
for
and
X-100
(w/v)
Sertoli
cell
To obtain a mature semichromatin
using a buffer mM Tris, pH
containing
0.25
to
cytoplasmic
remove
were the Although spermatids,
and
the
were purified M sucrose, 50
MgCl2
round
spermatid
found in representative
epithelium,
paration of 0.32
a few
early
centrifu-
spermatids
primary spermatocyte nuclei cell components in the pellet.
niferous 04
testes
nuclei were preparation
In
0.3
and
The smaller migrate in
spectrophotometer
and major
02
histones
respectively) bands and
gation
30
0.1
be
(1972). F2A1)
tions revealed that the haploid spermatids and the voluminous mature Sertoli cells were not
50 RNA
of
front.
1
