I, Walter. U, 1984. High-affinity binding of the reg- ulatory subunit. (Rh) of cAMP- .... trout testis cells. Biochemistry. 21:1869-77. Slaughter. GR, Means. AR, 1989.
BIOLOGY
OF
43, 46-54
REPRODUCTION
(1990)
Subunits of Cyclic Adenosine 3’, 5’-Monophosphate-Dependent Protein Kinase Differential and Distinct Expression Patterns during Germ Cell Differentiation: Alternative Polyadenylation in Germ Cells Gives Rise to Unique Smaller-Sized mRNA Species1 OLE
0YEN,2’3’4
FRODE
MYKLEBUST,3
JOHN
VIDAR
Institute
of Pathology3
D.
Rikshospitalet, University
Department
of Physiology
Department
of Pharmacology6
and
Oslo of
and
University
SCOTF,5
HANSSON,4
Oslo,
GARY TORE
Institute
of Ca4fornia
Irvine,
University
G.
STANLEY
McKNIGHT,6
of Medical
Biochemistry4
3, Norway
Oslo
and
CADD,6
JAHNSEN3’4
1, Norway, Blindrn,
Biophysics,
G.
Show
Department Irvine,
of Biological
California
of Washington,
Chemistry5
92717
Seattle,
Washington
98195
ABSTRACT Cyclic
AMP
functions,
spermatozoal
ument
the
presence
demonstrate
to be The
The this
may late
be
as motility, mRNA
expression
patterns may
furthermore, truncated use
of alternative
the
delayed
after
for cell
subunits stages,
convey
presence and
RIIB,
polyadenylation
translation
cessation
site
observed
of
is well
established
that
cyclic
for
unique
C,. mRNAs signals.
Recent
studies
on
and
sequencing,
AMP
(cAMP)
ing
is involved
Tash
subunits
these
the gene/mRNA et al., 1983), RI
and
Means,
1983).
Numerous
diverse and complex the role played by dependent germ cell
of significant
umented dependent
1980;
to elucidate
in sperm its effector
(PKA). Also (spermatocytes,
levels
Kopf,
have focused on enzyme, cAMPat earlier stages spermatids),
of PKA has
been
firmly
(Conti et al., 1983). However, the role regulatory mechanisms during earlier
spermatogenesis tial evidence positive
and
investigations
functions cAMP and
protein kinase differentiation
presence
(Garbers
has exists
effector
remained suggesting
of growth
obscure, a general
and
although role for
differentiation
R1I
(C)
February
Received
August
‘This work wegian Research (Tj.,
V.H.),
16, 28,
of the doc-
AndersJahres
Promotion
and
at haploid
Our
with
of mRNA
of Science
(Tj.,
PKA have level. (Clegg
with
data
suggest
higher for
stability,
at
translation
0027
Norgrant Nordic Oslo
cDNA
unexpected
clonmulti-
different gene products. Four (R) and two different catalytic now
been
identified,
at least
at
et al., 1986; Levy et al., 1988), C,, (Uhler et (Showers and Maurer, 1986; Uhler et al., give rise to type I PKA, whereas JUl
give
isolated
cDNA
rise
to type
II. In addition,
a third
catalytic
for
human
testis,
and
(and PICAS in general), less been considered
1978).
V.H.),
involving an
These have been designated RI,, (Lee et a!., 1988), RIl,, (Scott et al., 1987),
expression for this subunit In previous investigations
Society (Tj., V.H.), (TJ., V.H), Torsteds
from the NIH (G.S.M.). of Pathology, Rikshospitalet,
primarily
representing subunits
for
(Jahnsen
subunits
and
for the
and
RiB)
compared
cells.
revealed
substanPKA as a
(Russell,
by the Norwegian Cancer Science and the Humanities
Foundation
cells
germ
have
from
1990.
Insulin Foundation (T.J., V.H.) and grants 2Reprint requests: Ole Oyen. Institute
expressed
PKAs,
of cAMPstages of
1989.
was supported Council for
and
sperm.
species,
levels
doc-
al., 1986a), and C 1986b). RI subunits
and the only and type II. demonstrated Accepted
for RI (Ri,, first
we cells
of transcription.
and
reaction
mRNA
certain
of essential study,
in germ
C,,)
in germ
in the
ensure
plicity in isoforms different regulatory
acrosome
are
mRNAs
of shorter
may
and
mature
and
to be selected
regulation In this
RNA5
R1IB)
cells
in the regulation of highly specialized spermatozoal functions such as motility, epididymal maturation, capacitation, the
and
germ
selection
IUIB,
Messenger
smaller-sized
This
in the reaction.
RIB, R.IL,
RI! (RU,,
appear
The
in spermatids.
INTRODUCTION It
(RI,.,
in developing
involved
acrosome
spermatogenesis. mRNAs
functions
the
of RI,,, RI!,,,
during
to be the
and
subunits
PKA
whereas
specific
believed
are
capacitation,
5 different
for these
demonstrates forms
for stages,
maturation,
levels germ
subunits
(PKAs)
kinases
epididymal
significant
to the
essential
spermatid
such
protein
at premeiotic
Specific,
to be due
cAMP-dependent
PKA
study,
cells.
and
induced
individual
present
somatic
of
differential
C,. appear stages.
(cAMP)
1.
type
46
found
recently C.5,
(Beebe et al., 1990). regarding PKAs in germ
possible decrease progress to be the
complex pool
has been in type of spermatid major
form
spermatids (Conti et al., 1983). The probes for the different subunits
abled arately
us to investigate at the mRNA expression
have designated
a testis-specific
gating cDNA
cell-specific
Norway.
II was
demonstrated
the enzyme one homogenous
distinction selective with the
A
we
subunit,
the expression level. We have of mRNAs
or
between type I I PKA has been development, present
in elon-
newly developed of PKA have en-
of each previously for
cells
has more of molecules,
subunit reported
Pd,, (previously
septhe de-
UNIQUE
noted
RI),
RII
(previously
ferent testicular cell al., 1987). Recently,
et al.,
R1I51),
OF
PKA
and
C,, in dif-
the
(#{248}yenet high-level
cDNA
mRNA species for Ril,, at late stages during spermatid elongation (#{248}yen
1988b).
We
have
now
been
able
to investigate
the
all known subunits of PKA at the mRNA highly specific cDNA (and oligonucleotide) present
study,
we
report
sion during demonstrate
spermatogenesis the presence
adenylation
mechanism
mRNA
SUBUNITS
types (somatic and germinal) we have demonstrated the
expression of a unique of spermatogenesis-e.g.
denoted
EXPRESSION
expression
level
characteristic
patterns
for several of a unique in germ
of expres-
subunits, alternative
cells
that
of
by means of probes. In the and
favors
we poly-
smaller
species.
ORE,
Preparation
of various
cells.
Tissue
ovary
(hypophysectomized
samples
[Jahnsen et al., (nonpathologic) patient. and
METHODS
tissues and isolated taken from rat liver,
were
and
samples
were
at -75#{176}Cfor
Cultured Sertoli dibutyryl-cAMP),
later
frozen
rats
stimulated cells,
whole testis tissue from as previously described
kb)
of rat germinal
isolated
old
rats.
in liquid
N2
with Leydig
0.1 cell
rats of different by #{248}yenet al.
from
A cell
cell fractions.
seminiferous
tubules
preparation
was
(RST),
in 44-day-old gravity
and
elongating
rats)
were
sedimentation
of 32- and by
quence
then
(ES;
isolated
by the
using
44-day(#{248}yen sper-
present StaPut
a BSA gradient,
tially as described by Grootegoed et al. (1977). The cells were examined both by phase-contrast microscopy by regular light microscopy after fixation and staining. purities of these germ cell fractions were evaluated
Preparation
on
1988)
kb
RNA
of total
RNA
extraction
from
RI
probe
containing kb SAII-BglII
was
a 1.5 kb EcoRI
the
entire
fragment
ORE. (Scott
fragment
The et a!.,
rat
(Clegg
RII,, cDNA 1987)
from
gel
phate grams
running buffer total RNA was and
and
with
transferred
translated subunits
was
kb)
a 1.5 kb rat
and
1986).
noncoding
Furthermore,
we
sequence
samples
(Showers
were
and
electrophoresed
containing
formaldehyde
recirculating
20 mM
as denasodium-phos-
(#{248}yen et al., 1987). Twenty used in each lane, separated to a nylon
SSC
filter
at 50#{176}C, and
the
filter
was
(Biotrans;
finally
ing Hyperfilm MP (Amersham, were estimated by comparison Research Laboratories, Bethesda, (40
microon the
ICN Biomed-
conserved was
among
species
as described
[y-32P]-ATP
(Amersham,
kinase.
Northern
usRNA sizes (Bethesda
probe. An 3’ nontrans-
(Maldonado and Hanks, from Genetic Designs 3’ region of C,, is very
(Uhler
performed
using
autoradiographed
oligonucleotide to the terminal
lated region of human C,, cDNA 1988), nts 2462-2501, was purchased Inc. (Houston, TX). The terminal well
for the different with 0. 1X-0.5X
UK). Messenger to RNA standards MD).
with C,,-eczfic corresponding
mer)
et a!.,
1986a).
by Maxam PB10218)
nylon
filters
End-
and
Gilbert
and
were
T4 poly-
probed
un-
der the following prehybridization/hybridization tions: 40% formamide, 5X SCC, 5X Denhardt’s mM sodium phosphate (pH 6.5), 0.1% SDS,
solution, 50 250 g/ml
denatured
salmon
at 42#{176}C. Fil-
ters
then
were
sperm
DNA,
washed
with
50 pg/ml
0.5X-1X
condi-
tRNA, SSC
at 50#{176}C.
RESULTS
whole as
Cell-spec(fic ious
mRJVA Expression
Figure 1 shows PKA subunits
testis.
RIB has been
specific manner, of PKA regulatory
et a!., was
RII
(#{248}yenet al., 1987) cDNA probes of PKA. Washing was performed
nucleotide
germ and The by
inside
et al.,
The
agarose
agent
(1977)
from isolated testicular cells was performed described (#{248}yen et al., 1987) by homogeniza-
for (1.2
3’ noncoding
turing
unitessen-
Complementay DNA pmbes. The mouse RI,, probe was a 0.6 kb PstI cDNA fragment (McKnight GS; unpublished data) from inside the open reading frame (ORF), and the
0.7
(Jahnsen
0.5
a 1.5%
labeling
tion in guanidinium isothiocyanate. Total RNA was isolated by centrifugation through a cesium chloride gradient and purified by phenol/chloroform extractions.
mouse
and
only
counting cells: PS, 32 days, 90-95%; RST, 32 days, 85-90%; PS, 44 days, 75-80%; RST, 44 days, 65-70% (contamination mostly ES); ES, 45-55% (contamination mostly RST). testes and previously
used
3’ coding
Maurer, 1986). Northern analysis.
oligo
cells
consecutive
treatment (PS), round
spermatids
method
Germ
obtained
collagenase, trypsin, DNAse, and mechanical et a!., 1987). Pachytene spermatocytes matids
regions
Hybridization
Preparation
probe
used a 0.6 kb EcoRI fragment of mouse C,, containing 3’ coding region and about 160 bp of 3’ untranslated sequence (Uhler et al., 1986a). The bovine C probe was a 1.5 kb DraI fragment, representing I kb of coding se-
(1987). were
the both
(SDS), and 250 pg/ml denatured salmon sperm DNA, at 42#{176}C, and hybridized under the same conditions using nick-
RNA extraction.
cells (control and cultured peritubular
tumor tissue, and ages were prepared
lung,
testicular tissue from a 14-yr-old
at once
whereas
47
icals, Costa Mesa, CA) by capillary blotting technique. The resulting filter was prehybridized in 50% formamide, 5X SSC (Maniatis et a!., 1982), 5X Denhardt’s solution, 50 mM sodium phosphate (pH 6.5), 0.1% sodium dodecyl sulfate
testicular heart,
estradiol/FSH-treated
1986]), and brain. Human was obtained at surgery
All tissue stored
mM
AND
CELLS
GERM
containing
(0.3
gel,
MATERIALS
IN
a
the mRNA in different found
RS, Lane
4), giving
of postpubertal
expression rat testicular
to be expressed
pattern of the varcelLs and in whole in a highly
tissue-
and has been denoted a brain-specific subunits (Clegg et al., 1988). This
ern blot demonstrated be present at significant testis
in Rat Testis
rise
the
single 2.6 kb levels in germ
to a corresponding rats
(Lane
9).
The
form North-
RIB mRNA cells (PS,
also Lane
signal
in whole
RIB cDNA
probe
to 3;
48
#{216}YEN ET AL.
RIp
RII *
6.Okb
2.6kb’1.7
4.
kb
RI
12
34
67
5
89
12
34
67
5
kb
#{149}4.7
FIG. 1. Northern blot showing levels of mRNA for RIB, Rh,,, and CB in different representing the different cell types in rat testis. Lanes I and 2: Cultured Sertoli 48 h (Lane 2). Lanes 3 and 4: StaPut germinal cell fractions: pachytene spermatocytes peritubular cells. Lanes 6 and 7 (similar): Rat Leydig cell tumor tissue. Lanes 8 Twenty micrograms of total RNA was loaded in each lane and the resulting filter autoradiographed.
also
detected
tion
with
a lower
and very
3), which faint but
the
band
RI,, 1.7 kb is also distinct
representing
mRNA
cross-hybridiza-
(#{248}yenet al.,
induced in germ RIB mRNA band
1987)
2
cells. In addition, a was detected in the
peritubular cells (Lane 5). These cells have been cultured for 13 days, which excludes the possibility that the RIB signal was due to germ cell contamination. The
RI!,,
6.0
sues (Scott all testicular strongest mediate mM
kb
mRNA,
et a!., 1987; cells, but
RII,, levels
single were
which
is expressed
#{248}yenet al., 1987), in variable amounts
dibutyryl-cAMP;
Lane
2) and
Leydig
Lanes 6 and 7), whereas very in germ cells (may represent
matic
cells
in the the
in most
tis-
detected (Fig. 1).
in The
was seen in peritubular cells. Interdetected in stimulated Sertoli cells (0.1
(LCT; served reported
was
germ
cell
differential
cell
fractions).
regulation
tumor
faint bands contamination We
have
of PKA subunits
were
found
to
be
below
our labeling/hybridization shown the mRNA expression in rat testicular
Developmental testis. At early
cells
the
level
conditions). patterns
(#{248}yenet a!.,
changes puberty
(15-25
days
of age
mental mRNA
there
can
be
predicted
from
the
age
variation
of mRNAs pattern.
in the
the
both
6.0
found
kb
as
testis
According
declining
of 35 days.
(2.9
of this
Eli,,
levels
the
mRNA
dominant with
25spe-
cell localiby develop-
appearance and
forms age.
from mRNA
The
the in
of this 3.2
kb
RIIB
somatic
cells,
2.4 kb C,, mRNA,
in
an enrichment from 30 to 35 days RI!,, mRNA species (2.2 kb) appeared 40 days of age (#{248}yenet al., 1988b). In mRNAs for RIIB and C,, apA 1.6 kb RIIB signal (below
A C,, mRNA
band,
described intensities
located
closely
(Jahnsen beyond below
the
et al., age
the
usual
2.4 kb mRNA, appeared from 30 to 35 days of age (we will refer to this unique band as the 2.3 kb mRNA). The CB 4.7 kb mRNA was detected very faintly in whole testis preparations and showed a declining tendency with age (not shown).
of these
cells)
RI,, mRNAs
1.7 kb RI,, mRNA
an induction
the faint 1.9 kb mRNA previously 1986) was detected at variable
ization
vs. germ
fainter
unique smaller-sized in postpubertal testis.
in whole
larger
studies, which demonstrated from 30 to 35 days of age.
Accordingly, mRNA,
tions.
cells
usually indicating
addition, peared
(under
two
cies in meiotic germ cells. The specific germ zation of the RIB 2.6 kb mRNA was supported
is an exponential increase in germ cells, which at later ages dominate the testis and dilute the signals from somatic cells in whole testis preparations. In this way, the cellular local(somatic
of the of age,
demonstrated and a unique levels from
by cAMP
in rats)
expression 30 days
of the
kb), which have been found to be the most forms in somatic cells (#{248}yen et al., 1987), dewith age, whereas there was a marked increase in
contrast, of age, at high
1987).
expression
2), expression
3.2
showed
We have previously for RI,,, RIIB, and C,,
in mRWA
(Fig.
obof so-
previously
of detection
kb and abundant creased
tissue
analogues in Sertoli cells (#{248}yenet a!., 1988a). For CB, low levels of the 4.7 kb mRNA were detected in peritubular cells and LCT. In germ cells and Sertoli cells, CB was
testicular cell types. Total RNA was extracted from various cells/tissues cells; control (Lane I) and stimulated with 0.1 mM dibutyryl-cAMP for (PS, Lane 3) and round spermatids (RST, Lane 4). Lane 5: Cultured and 9: Whole rat testis: 20 days (Lane 8) and 30 days (Lane 9) of age. was probed succesively with 32P-labeled cDNA5 for RIB, Rll, and CB and
to this
(Figs.
89
RWA
Messenger The
3) should
expression
Northern be
fractions
kb RI,, mRNA,
blots
interpreted (listed as suggested
on
in
isolated
the
germ
in light
of the
in Materials from
the
and
germ cell fraccell fractions (Fig. estimated
purities
Methods).
The
developmental
studies,
1.7
UNIQUE
EXPRESSION
PKA
OF
SUBUNITS
IN
GERM
49
CELLS
RIp
RI 3.2 kb 2.9 kb
-.2.6
1.7 kba-
-
i.
-
,
kb
1.7 kb
.
RI 5 10152025303540506080100
RIIp
RII 6.0kb-
**#{243} -.3.2kb
2.2kb..-
-.1.9kb -‘1.6kb
-
5
5 10152025303540506080100
20 25 30 35 40 50 60 80 100
1015
Ca
24kb
5
10152025303540506080100
Age
(days)
FIG. 2. Northern blot showing levels of mRNA for RI,, RIB, RII,,, Rh5, and C, in testis from rats of different ages. testis of animals of various ages. Twenty micrograms total RNA was loaded in each lane on the gel and the resulting nick-translated cDNAs for RI,,, RI5, RIl,,, RUB, and C,. The abscissa shows increasing rat ages. Approximate mRNA sizes
was
found
and
RST
to be present (postmeiotic
day-old rats, while stage (ES present and
germ
at high cells)
levels
isolated
declining levels only in 44-day-old
cell-specific
in PS (meiotic from
both
were found rats). For
RIB subunit,
a similar
RST
and
ES fractions
of 44-thy-old
of this band in the PS fraction accounted for by ES contamination kb RI!,, signal was not present
rats.
detected
a faint
3.1
kb
The
signal
suggested
from
the
developmental
studies,
was
This smaller RIIB mRNA was found predominantly RST fraction. The presence of a smaller 2.3 kb mRNA was also clearly demonstrated in the germ
hibited a pattern RI,, 1.7 kb mRNA: of detection
1988b),
a marked
C,,-Related responding (Maldonado
same was
expression less
of expression significant
decline
pattern
from whole rat succesively with
as the
usual
Both
similar expression
to that found for the both in PS and RST
in ES. CB mRNA
was
C,, mRNAs
2.4
abundant.
below
the
in the isolated germ cells. mRNAs. A C,,-specific oligonucleotide, to the far 3’ region of the and Hanks, 1988), detected
ex-
level cor-
human C,, cDNA (Fig. 4) both the
usual “somatic” 2.4 kb mRNA and the postpubertal 2.3 kb band (as well as the corresponding human C,, 2.8 kb mRNA). In addition, a 1.8 kb mRNA was detected faintly by the C,, cDNA
probe
testis, human
as opposed testis-specific
been
shown
al., cells,
the but
and
studto be inthe RI!,,
(#{248}yenet al.,
which may represent a nuclear RNA precursor. The presence of a 1.6 kb RIIB mRNA in germ
showed
C,, mRNA,
levThe in
presence
developmental RI!,, mRNA In addition,
kb
was
pattern
of 44-thy-old rats could be of this fraction. The 2.2 in the more purified RST
fraction from 32-day-old rats, and the ies (Fig. 2) suggested the small-sized duced at the ES (or late RST) stage. cDNA
44-
at the ES the brain-
demonstrated; the 2.6 kb mRNA was found at significant els in both PS and RST, and declined at the ES stage. very abundant 2.2 kb RI!,, mRNA was located primarily the
mRNA
cells)
32- and
RNA was extracted filter was probed are indicated.
in some
blots
(Fig.
5),
but
to the rather ubiquitous mRNA of the same
to represent
a third
C subunit
was
only
seen
(C.5; Beebe
1990).
as
confirmed. in the C,,-related cells. This
in
2.4 kb mRNA. A size (1.8 kb) has
DISCUSSION This study documents the presence of significant mRNA levels for 5 different PKA subunits in germ cells, and dem-
et
50
#{216}YEN ET AL.
3.2 kb
RI 2.9
RIp
kb
2.6 kb
-
1.7 kb PS
RST
______
PS
RST 44
_________
32
ES
PS
RST
PS
RST 44
32
RIIQ
ES
RIIp
6.0 kb
3.1kb’-
-.3.2kb
2.2 kb’-
-.
4,;
PS
RST
______
PS
_________
32
RST 44
PS
ES
RST
PS
RST 44
32
1.9 kb
-
1.6kb
ES
Ca
-2.4kb
Germ cell fr.: Age (days):
PS
RST
PS
32
RST 44
ES
FIG. 3. Northern blot showing mRNA levels for subunits of PKA in different germ cell fractions (PS: pachytene ES: elongating spermatids). RNA was extracted from germinal cell fractions obtained by the StaPut unit-gravity day-old rats and 44-day-old rats (ES present only in 44-day-old rats). Twenty micrograms total RNA was loaded filter was probed with nick-translated cDNAs for RI,,, RI5, RIl,,, Rll5, and C,,.
onstrates differential expression patterns for these subunits during spermatogenesis. Some of these subunits (RIB and RuB) have been found to be expressed in a very limited number of cell types. When taking into account both the developmental studies
and
the
studies
performed
on
isolated
germ
cell
frac-
tions, rather conclusive statements can be made regarding the stage-specific expression of the different PKA subunits during germ cell differentiation. and C,, 2.4/2.3 kb mRNAs meiotic
stages,
to 30 in the in
PS germ
crease at the signals
as they fractions.
further in RST, whereas ES stage. In fact, most seen in the ES fraction
studies Their
and
from clearly
expression
were tended
Days
found
sedimentation in each
lane
of this fraction. to be induced
on
the
(or late RST) stage, since it first appeared The data on the less abundant 1.6 kb dicated postmeiotic induction, as it first of age
and
was
undetectable
25
present to in-
a marked decline was found or all of the RI,, 1.7 kb and C,, can be accounted for by RST
mRNA
did
germ cells. Overall, induced the the
not it
seem appears
at premeiotic
in the
to be expressed that
the
germ
RIls are first expressed major part of the RH,,
place at later spermatid kb mRNA. These findings
gel
and
The high-level postmeiotical!y,
stages,
resulting
RI!,, 2.2 at the
kb ES
at 40 days of age. mRNA also inappeared at 35 days
PS fraction
of 32-thy-
to be expressed declined in ES. CB
at detectable
mRNAs cell
the
RIIB
old rats. However, this RIIB mRNA seemed predominantly in RST and its expression
The RI,, 1.7 kb, RIB 2.6 kb, induced at premeiotic/
appeared/increased
developmental cell
were
contamination mRNA was
RST: round spermatids; method on testes of 32-
spermatocytes;
for the whereas
levels
RIs and
in
C,, are
mRNAs
for
at haploid stages. Furthermore, 2.2 kb mRNA expression takes
stages are
compared in agreement
with
the RI! 1.6 with previous
UNIQUE
C
EXPRESSION
OF
PKA
oligo
SUBUNITS
IN
the
substrate flexibility
specificities. This view of the cAMP signalling
observed
ulated separate not
various
51
whereas
fects
2.4 kb
CELLS
GERM
in cells,
by cAMP. functions
been
catalytic
subunits
lular
6O,
as in the
spermatozoa,
rat Testis
human Testis
provided.
studies
on
kinase
activity
demonstrating
that
type!
PKA is
the dominating form in PS and RST, whereas type I! PKA is the major form in ES (Conti et al., 1983). What are the functional implications of these characteristic
and
differential
during spermatogenesis? in isoforms of PKA transduction.
Various
expression
patterns
for
PKA
subunits
However,
isoforms
may
differentiate
cAMP
specific
compartments/structures a!., are
1984; targets
heterogeneity regulatory
De Camilli et al., for phosphorylation;
(Va!lee 1986)
et al., where this could
1981;
Lohmann
been
et al., 1985).
kDa)
(Horowitz
patible
with
when
stim-
interactions
has
between
structures have been Lohmann et al., 1984). a characteristic subcel-
demonstrated
for
RI and
RI! in
The
et a!., RI!,,.
interaction
of Rul with
documented (Horowitz mass of this flagellar 1988;
It seems
Paupard
very
et a!.,
likely,
then,
the
sperm
et al., 1984, ElI (56-57 1988)
that
is com-
the
unique,
abundant RI!,, 2.2 kb mRNA induced at late spermatogenic stages encodes the RI! form interacting with flagellar structures. Thus, RI!,, may be responsible for anchoring kinase activity to the sperm flagella, where cAMP-dependent phosphorylation of certain substrates is believed to be essential for sperm motility (Tash et a!., 1986; Brokaw, 1987). RI has been shown to be predominant in the membrane fraction of spermatozoa (Horowitz et al., 1984), and immunogold
electron
ticularly
re-
has
flagellum has been well 1988) and the molecular
The recently discovered diversity offers a new model for cAMP signal
sponses at the level of the kinase, Different regulatory subunits may localize the kinase activity to specific subcellular
the great diverse ef-
by immunogold electron microscopy (Pariset et al., and by cell fractionation studies (Horowitz et al., 1984;
Atherton
FIG. 4. Northern blot showing mRNAs detected by a C,-specific oIlgonucleotide probe. Total RNA was extracted from rat (15 and 60 days old) and human testes (14-year-old patient) and subjected to Northern analysis. The resulting filter was hybridized with the end-labeled C,,-specific oligonucleotide (40 mer; see Materials and Methods). The smaller arrow on the left indicates the fainter 2.3 kb band seen in postpubertah rats.
in their
So far, conclusive evidence demonstrating attached to a specific PKA subunit,
localization
sperm 1989)
differ
may help explain system and the
type !i PKA and various subcellular documented (Vallee et a!., 1981; Concerning germ cells particularly, A(days):15
may
microscopy
confined
to
the
has
suggested
acrosomal
(head)
that
RI is par-
membranes
(Pariset et al., 1989), However, the data on RI are still too scarce to suggest any specific functions in sperm or to differentiate RI,,/RIB at the protein and functional levels.
et
certain substrates be related to the
As concerns the catalytic subunits, expressed in significant amounts in ever,
cDNAs
for
a third
form
only C,, seems rat germ cells.
of C, designated
C,
to be Howhave
re-
cently been isolated from human testis (Beebe et al., 1990), and detectable levels of the 1.8 kb C mRNA was found only
observed in the aminoterminal part of the subunits (Levy et a!., 1988; #{248}yenet a!., 1989),
cDNA
..#{149}
$IWt24kb .1.8kb
Cy? C 0.1
1.0
mM db cAMP
Sertoli cells
5 15
25
Age
(days)
50
Testis
FIG. 5. Northern blot showing C,-related mRNAs in various rat cells and tissues using C,, cDNA as probe. Total RNA was extracted from the following rat cells and tissues: liver, heart, brain, lung, and ovary (hypophysectomized and estradiol/FSH-treated rats), Sertohi cells (control (Cl and stimulated with 0.1 or 1.0 mM dibutyryl-cAMP for 48 h) and testis from rats of various ages. Twenty micrograms of total RNA was loaded in each lane and the resulting filter was probed with the nick-translated C,, cDNA (mouse). A possible (cross-hybridizing) C mRNA (1.8 kb) seen in rat testis is indicated.
52
#{216}YEN El
in human with
testicular
C,, in the
tissue.
human
same size as the was demonstrated rat C..
Future is also
Apart mature
from sperm,
showed
system.
will
expressed
cross-hybridization
A C,,-related
low-abundant in rat testis
studies
subunit
C
mRNA
sized The
reveal
if this
recently
mRNAs
in the
germ
cells.
the proposed functions PKA may play a role
in germ ElI,,,
to be expressed RI,, mRNA, shown also been detected matic have
cells, been
the the
these
observations,
PKA isoforms in as a positive effector at earlier stages has yet been
that
1989)
with
2.3
kb
cells. smaller-
somatic
we
postulate
the
presence
unique
mRNAs
to the
and testis
rat
2.2
polyadenylation 18 nucleotides resembling
UAUAAA
in RI!,,,
has
been
did the
not contain resembling
variant polyadeny!ation also been reported et a!., 1987)
site signal a calmodulin
in
corresponding
(UACAAA)
was
found
12 nucleotides
6). It should sequence
mRNA species expressed in spermatids 1989; here called CaM II! 1.0 kb). The induction of unique mRNA ferent
PKA subunits,
of atypical
mRNA
and
the
polyadenylation
presence
of the poly(A) residue) motif
addition (upstream
site), and a CAYUG or downstream)
to play a role in cleavage/polyadenylation 1985). Such sequences were found
c-abl
upstream
in the
nuclear
data
site
of alternative
=
by partial
pyrimidine are believed
(Birnstiel et a!., germ cell-specific
FIG.
6.
Putative
polyadenylation
site
kb signals
in four
putative polyadenylation et al., 1986), mouse c-abl cDNA (Meijer et al., 1987), and are uniquely expressed in germ cells and all are smaller canonical AATAAA is found in these sequences. the
poly(A)-tail,
with
the
species
(0.75
and
for
referring
signals,
Evidence
has
been
ribonucleoprotein
Means,
several
to the
strongly
dif-
presence
suggests
mechanisms
the
in germ
instances, smaller-sized likely that the polyade-
presented particles
(Birnstiel
complementarity
between
RNP5 and sequences (Berget, 1984). This
surrounding leads to the
snRNPs
define
signals. What
in germ
cells
functional
reasons
suggesting are
et
1985),
the
al.,
involved
in
perhaps
RNA moiety
the po!y(A) speculation
alternative would
that small
(RNPs)
of the
addition site that specific
polyadenylation there
be
to favor alternative mRNA species? Sequences nontranslated region of mRNAs are involved
mRNA.
RIIa 2.0 kb rat Rh8 1.6 kb mouse c-abl 4.0 kb rat calmodulin 0.75
(Slaughter
po!yadenylation
cleavage/polyadenylation
of the poly(A)-
humafl
before
to con(Meijer
point that less(downstream
(Y also
(Fig. 6) (prCM79;
nylation machinery in germ cells is able to accept alternative polyadenylation site signals, which are preferred when they are located 5’ to the usual (somatic) polyadenylation
hexanucleotide
be emphasized at this elements, G/T-clusters
(GAUAAA) cDNA
to a smaller-sized
site signal.
tail (Fig. conserved
site was
kb) mRNA in rat brain and found to be significantly less abundant than the predominant 2.2 kb mRNA, Interestingly, this shorter transcript was found to be the predominant
than the usual somatic transcripts) was tain the usual polyadeny!ation site signal et al., 1987). However, a resembling
not
for
site
germ
cells
within the 3’ in the regu-
TGATACAAAGTCCAAAGTATAAACATGCTCCTTTCCTCTC
(A)
CATGTTTAAGAAGATAATTAAAAGATGTACTCATAGGCCG
(A)
ACTGTACCTGCACCTTTGATGCTTACAAACTGTCCCCGAG ATTGACTGAGAATCTGATAAAGCAACAAAAGATTTGTCCC
(A) ( A)
different
mRNA5
in
previously
the usual po!yadenylation hexanucleotide AUUAAA
cells. Furthermore, in each of these transcripts are generated. It seems
(AAUAAA)
polyadenylUACAAA
not been found at detectable levels other tissue other than testis. This
has been shown to contain the same coding region as the larger somatic transcript. Furthermore, the smaller 2.3 kb C,, mRNA was also detected (Fig. 4) by an oligonucleotide from the 3’ region of the C,, cDNA. Recently, a germ cell-specific c-abl mRNA (also smaller shown
hexanucleotide
reported
and
rat
site signal upstream of
c-abl
Sherbany
smaller the pos-
RI!,,
closely
(#{248}yen et al., cell-specific
possible mRNA5,
A
proteins
kb
kb), the usual (Fig. 6); and the
cDNA germ
UAUAAA was found. Neither of these ation site signals in germ cell-specific
has
represent different po!yRII,, cDNA (#{248}yen et al.,
unique
poly(A)-tail,
RI!,,
to the
present.
of alternative
alternate
the
ElI6 cDNA signal, but
genes or by alternate splicing. 3’ fragments (McKnight GS; unthat the three RI,, mRNAs are
of a single gene sites. A human
corresponding
encode
was
mRNA (2.2 also absent
testis
analogous
ever, a 1.6 kb mRNA has in rat ovary, nor in any
appear
RI,, mRNAs (2.9 kb and 3.2 kb) ones (#{248}yen et al., 1987). From
mechanisms in germ cells, favoring The data available so far disprove these
cells.
C,, mRNAs
to be
(Birnstiel et al, 1985). An RuB cDNA (Fig. 6), which corresponds well with the size of the 1.6 kb RI!B mRNA seen in spermatids, has been cloned from rat ovary tissue (Jahnsen et al., 1986). How-
exclusively in the germ cells. The 1.7 kb to be highly enriched in germ cells, has in various somatic cells; however, in so-
by representing different Hybridization data with published data) indicate transcripts adenylation
and
in human
shown
ElI,,
for
as compared
RI!B,
two larger dominant
polyadenylation mRNA species. sibility
cells
1.6 kb
1989),
discovered
concerning PKA function in early germ study documents the induction of unique
2.2 kb
Furthermore, of the
human C mRNA (1.8 kb) (Fig. 5) and may represent
of growth and differentiation (Russell, 1978) of spermatogenesis. However, no evidence provided This
AL.
(cDNAs)
site signals underlined, rat calmodulin (prCM79) than the mRNA species
found
to be uniquely
expressed
in germ
cells.
The
last 40 nucleotides
are shown for human RIl, cDNA (#{248}yenet al., 1989), rat Rl15 cDNA cDNA (Sherbany et al., 1987). The corresponding mRNAs (sizes predominating in somatic cells. No hexanucleotide corresponding
(Jahnsen indicated) to the
UNIQUE
lation
of transcript
Strickland rich)
in the
expressed ble
stability
et al., 1988). have
destabilization
1986).
half-life
Concerning
sequence
region demonstrated
of the
transcripts
c-abl
the
motifs
of various
been
PKA
(Shaw
Brokaw Clegg
CH,
Conti
indicated
Garbers
(Geremia
et a!., 1977).
mediating the differentiation stages, must depend on Several
investigators
translational Heidaran
Therefore,
protein
processes mRNA made
taking
have
demonstrated
a characteristic
stages, before
beyond
nuclear
the
stage
of
but remain the protein condensation
(Hecht,
1986;
Kleene et a!., 1984). In light of these cell features, the selection of particularly cies may be essential to ensure certain translation at late spermatid stages-after
characteristic germ stable mRNA spelevels of mRNA for the cessation of
transcription. sary for late
of proteins, and mature
functions,
Thereby, spermatid be
may
adequate levels differentiation
neces-
and
transgenic
animal
date specific functions. alternative polyadenylation
To
studies
may
help
to await
elucidation
mechanisms
until
involved
is known
about
the
Opsahi,
Anne
acknowledge
Keiserud,
the
and
luger
technical
5:343-55
Differ symhesis
NB,
Gro
Knutsen,
1986.
pendent
of a tissue
kinase. Berget
SM, ation.
Mol 1984. Nature
specific
isoform
Endocrinol
Are U4 small 309:179-82
(In
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catalytic
(C,) subunit
from
human
of cAMP-dependent
J Biol Jahnsen
involved
in rat caudal
W, Leiser
Chem
Molen
HJ, 1977.
isolated
and
in the
from
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Ribonucleic
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259:832-
Kleene
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the
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P, Einig
(Rh)
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F, Hanks
Chem
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Dev
Isolation
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of a cDNA
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clone
protein
kinase,
AM, Gilbert
sequence
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A cDNA
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U, 1984.
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and
pre-
regulatory
protein
Proc
kinase
J, 1982.
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M, van Della
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