Jan 27, 1992 - Cancer Society, Nordic .... by manual dissection. [24] and .... air-dried. Subsequently,. NTB2 nuclear track emulsion was applied and the ...
BIOLOGY
OF
(1992)
46, 1057-1068
REPRODUCTION
and Cell-Specific
Stage-
Expression
Protein PETER
LONNERBERG,23 Department
Kinases
MARTFI
of Medical
of Cyclic
TORE
Pediatric
Endocrinology
Unit,4
of Biomedicine, Institute
Karolinska
and
Neurobiology, S-10401,
University
University
Biochemistry,6
HANSSON,6
VIDAR
Hospital,
of Anatomy,5
Departnent
of Medical
JAHNSEN,6
Epithelium1
of Molecular Stockholm, Sweden
Laboratory
S-10401, Institute
3’,5’ -Monophosphate-Dependent
in Rat Seminiferous
PARVINEN,4’5
C/xmzslry,3
Adenosine
of Oslo,
Turku,
Sweden
SF-20520,
Blindern,
PERSSON3
Institute
Karolinska
Stockholm,
of
HAKAN
Finland
3, Norway
Oslo
ABSTRACT Expression
of mRNAs
crodissection
iniferous of the the
epithelium
same
levels
a maximum
reached
maximal
revealed late
of the
pachytene
levels
in the
levels
spermatids
spermatids
of later
stages
VIl-Vill
and
in some
in differentiating kinases
tubules male
at different
of stages
germ
cells
times
I-VI, with
mRNA
was
VII
and
of stages stages These
different
differences
were
further
Vll-XIIL
while data
PJI( show
patterns
increased
at later
Rilu
mRNA that
mENA was
mRNA5
of expression
was
seen
detected
detected
increase markedly
RIl
stages.
encoding for
In the
only PKA-R
each
at approximately at stages at stage
mRNA from
layers
are
expressed this
and
rat
detected
testIs over
spermatids
spermatid
and
increased
adult
was
of round
round
IV-V, VHa,b,
levels
the
mRNA
in the
subunit;
to be due
of
two
distinct
[5,7,8]. Subsequent ious tissues, together
region
of of
In a stage-specific
suggests
specific
roles
for
regulatory
studies
to the
FAX: 8-34-19-60.
1057
with
of proteins
cDNA
subunits. isolated
RI and
Rh
from
varat least
cloning, revealed for by separate genes, I, and Rut and RhI13 of type II [9-15]. Different isoforms have also been found for the C subunit [16-18]. Bovine, murine, and human Ca and C13 cDNAs have been isolated [16-19], and recently the cDNA for a third isoform, named C-y, has been cloned from human testis [19], Molecular clones for the various PKA subunits have been used as hybridization probes that have revealed expression of PKA mRNAs in many different tissues from mice and rats. Riot appears to be constitutively expressed in all tissues, whereas Rl(3 mRNA is predominantly expressed in the brain and developing male germ cells [9,20]. Like Riot mRNA, Rihot is widely expressed in many different tissues while expression of Ri113 mRNA is mostly confined to reproductive tissues and the brain [11, 13,21]. In situ hybridization has been used to demonstrate that in the adult mouse, RIot and RI(3 mRNA expression is high in many different brain regions [221. In contrast, Rilot and RiI(3 mRNA expression is more restricted in the adult mouse brain, with the highest levels of Rilot mRNA in medial habenula and the highest Ri113 mRNA levels in caudate-putamen, the supraoptic nucleus, and dentate gyms [22]. All four PKA-R subunits are expressed in rat testis, although the Riot and Rilot mRNAs are more abundant than mRNM for the two 3-subunits [20,23]. Distinct develop-
four distinct regulatory Riot and Rh13 of type
Borgen.
2Correspondence.
il-VI,
was to
of spermatogenesis,
Accepted January 27, 1992. Received November 7, 1991. 1This work was supported by grants from Petrus and Augusta Hedlunds Stiftelse, The Swedish Natural Science Research Council, Funds from the Karolinska Institute, The Academy of Finland, Norwegian Research Council for Science and the Humanities (NAVF), The Norwegian Cancer Society, Nordic Insulin Foundation, and Astri and
expression
presence
shown
kinases (PKAs) was studied. A mivarious stages of the cycle of the semresults showed a differential expression
VIII, In situ hybridization of sections and round spermatids of all stages. RI
Protein phosphorylation, mediated by protein kinases, regulates a large number of cellular processes. Among the best characterized protein kinases is the family of cAMPdependent protein kinases (PKA) (for review, see [1]). PKAs appear to mediate most, if not all, of the effects of cAMP in regulating cell growth, metabolism, and differentiation [2]. The PKAs thus far isolated exist as an inactive tetrameric holoenzyme complex containing two catalytic (C) and two regulatory (R) subunits. On binding of two molecules of cAMP to each of the R subunits, the holoenzyme dissociates into the free, active C complex (which phosphorylates target proteins on serine and threonine residues) and a complex of cAMP and the R subunits [3,4]. Fractionation on DEAE-cellulose has shown that PKA consists of two different holoenzymes, designated type I and type II [5,6]. The two holoenzymes differ in several functional aspects: type I has a high-affinity binding site for MgATP and type II is characterized by an autophosphorylation These
at stages
low
by a reduced
INTRODUCTiON
site.
protein
representhg
tubules
and in situ hybridization, The stages of the cycle. Mu mRNA mRNA was low at stages XlIl-1II, started
spermatocytes
and round
elongating
R13
at stages
of pachytene
(cAMP)-dependent
at various
of Rilu
spermatocytes and
protein
level
AMP
and VIII, followed
VIIc,d
layers
of
The
cyclic
(PKA-R)
expression
maximal
VI with
mRNA
VIl-VIlI
these
blots
subunits
V1II-Xl.
at stages
stages manner
Northern
with
while
at stages
at stage RIa
of seminiferous
regulatory
stages
at all
reached
significantly
encoding 10 pools
rat
used
in combination
isoforms
four
testis
to isolate
in the
was
method
subunits,
coded
1058
LONNERBERG
mental
changes
observed
in mRNA expression PKA-R subunits
in the [20].
for all four
Moreover,
several
different
sizes
testis have been described; they regulated during ontogeny in the germ cells followed by Northern
testis
have
been
El
idine 0.1
of transcripts
unique
AL.
to
appear to be differentially testis. Fractionation of male blot analysis has indicated
isothiocvanate,
0.025
M sodium
M 13-mercaptoethanol
using
citrate
7.0),
(pH
a Pasteur
pipette.
and
The
in-
dividual samples were then loaded on top of a 2-mi cushion of 5.7 M CsC1 in 0.025 M sodium acetate (pH 5.5) and centrifuged for 16 h at 42 000 rpm in a Beckman SW55 ro-
tor (Beckman
Instr.,
Palo Alto,
CA). The
pellet
was
dissolved
that the smaller size PKA-R transcripts are all associated with male germ cells 120]. These findings indicate that PKA-R mRNA expression in the testis is strictly regulated during
in 0.5 ml of 0.01 M Tris-HC1 (pH 7.5). 1 mM EDTA, and 1% SDS, then extracted with an equal mixture of phenol chloroform, and ethanol-precipitated. The recovery of RNA was
postnatal
quantified
development
and
possibly
also
during
sperma-
togenesis in the adult animal. They further suggest different PKA-R subunits exert specific functions oping germ cells and in mature spermatozoa. In the
present
report
we
that the in devel-
taining
and
was electrophoresed
0.7%
mide.
After
formaldehyde
and
electrophoresis
the
40
g
in a 1%
0.1
tg
gel was
of RNA
agarose
from
gel con-
of ethidium examined
bro-
under
DV
explored this possibility further by analyzing the expression of PKA-R mRNAs in the testis at various stages of the cycle of the seminiferous ep-
light to ensure equal amounts of RNA in each lowed by blotting onto a nitrocellulose filter Hybond-C-Extra; Amersham Corp., Arlington
ithelium. tis were
The filter was then hybridized as previously described [28] to the indicated cDNA fragments labeled with ot-32P-dCTP by nick-translation to a specific activity of approximately i09
Cells expressing also identified
have
spectrophotometrically
each sample
PKA-R mRNAs in the adult by in situ hybridization.
MATERIALS
AND
Transillumination-Assisted Seminiferous
METHODS
cpm/p.g. clones
Microdissection
of the
Tubules
Twelve
young
Stockholm,
adult
Sweden)
decapsulated iniferous
(3-5 were
mo)
Sprague-Dawlev
used
as donors
rats
of testes
(Alab,
that
were
and placed in cooled PBS solution. The semtubules were separated from the interstitial tissue
by manual
dissection
[24] and
subjected
tion under the stereomicroscope, [25]. On the basis of appearance the following weak spot,
four dark
principal spot, and
erous tubules were The pale zone was fIrst 2 mm flanking
resent
rat tes-
stages
zones were dark [26]. The
recognized: isolated
pale, seminif-
cut into 2-mm segments as follows [27). subdivided into two sections, with the the dark zone being selected to rep-
IX-XI and
assigned to the also subdivided
to transillumina-
as described previously under transillumination,
pool into
the
remainder
of the
pale
zone
being
of stage XII. The weak spot zone was two segments, the first representing
The following fragments purified from human were used as probes: for Riot, a 630-bp EcoRI
rived
from
the
3’ end
of the
the stringency used. washed at reduced
Following stringency
SSC:
g NaCI
and
pH
adjusted
800
175.3 ml H2O;
middle,
stages
VlIc-d;
ning of the pale segments assigned using weight
and
homogenous the first
long dark representing
and
the
last
darkness
was
zone
subdivided VIIa-b; the
2 mm
was stages before
ing
microdissection, dry
ice
Preparation Frozen iniferous
for
begin-
zone, stage VIII. The total length of the to each of these pools was measured
a 2-mm scale placed beneath the Petri of 1 cm of tubule was approximately
on
the
consid-
the mRNA
of RNA
samples
were
dish. The wet 1 mg. Follow-
immediately
brought
to 1 L] 0.1%
SDS
films
the
times.
for
In Situ
frozen
and
Northern samples disrupted
Blot
a rat cDNA 1324 were
the filters were SSC, [20-strength citrate
1 M HCI
at 50#{176}C) and
dissolved and
exposed
from adult (200 g body weight) dissected out and immediately tissue
was
probes
mRNA were used: sequence of Kuno sequence
The
in
volume to x-ray
Hybridization
Testes rats were The
indicated
clones.
then
in a cryostate
(14-i.m-thick
on glass slides pretreated The sections were then [33,34]. The following
complementary
for Riot, nucleotides et al. [351; for R13,
[36]; for Rut, of Scott
cut
Sprague-Dawley frozen at -70#{176}C.
nucleotides
et al. [13]; and
in a rat cDNA clone [37]. labeled at their 3’ end
to their 280-327 nucleotides 1131-1178
with hybrid48-mer
respective in the cDNA 32-79 of in the
cDNA
for R1113, nucleotides
1267-
The oligonucleotide to a specific activity
probes of ap-
frozen
analysis.
microdissected epithelium were
g sodium
to 7.0 with
oligonucleotide
spots
VI. Finally, zones: the
cDNA
to one another, but rat mRNAs under
hybridization, (0.2-strength
88.2
tween
strong
respective
four fragments did not cross-hybridize readily hybridized to their corresponding
sections) and thaw-mounted poly-L-lysine (50 i.g/ml). ized as previously described
stage three
cDNA frag-
ment [29]; for R1t3, a 2 400-bp EcoRl fragment [30]; for Rut, a 1 300-bp Ec0PJ fragment [31], and for RII3, a 3259-bp EcoRJ fragment [32]. The Riot and RIlu fragments were de-
stages XIII-XIV and the second, stage I. Similarly, the dark spot zone was divided into two parts, the first comprising stages Il-Ill and the second, stages IV-V. The transition beered into
sample, fol(Amersham Heights, IL).
Analysis
from 50 cm of rat semin 3 ml of 4 M guan-
FIG. 1. Northern blot analysis of PKA-R mRNA expression at various stages of the rat seminiferous epithelium. Total cellular RNA (40 p.g/slot) from the indicated stages of adult rat seminiferous epithelium was fractionated on a formaldehyde-containing agarose gel, transferred to a nitrocellulose filter, and hybridized to eDNA probes for the indicated PKA-R subunits. All filters were exposed to x-ray films for 12 h.
EXPRESSION
OF
PKA
rnRNA
IN THE
1059
TESTIS
RIa 3.2kb
-
2.9kb
-
1.7kb
-
R1f3 2.6kb
-
RIkx 2.2kb
-
RII1 3.2kb-
1.6kb-
I
Il-Ill
IV-V
VI
Vilab
VIIcd
VIII
IX-XI
XII
XIII-XIV
1060
LONNERBERG
ET
AL
A
B
I.
Ix-xI
S
xII-xIv 1’
‘.
-
EXPRESSION
proximately
i09
cpm/p.g
cleotidvltransferase. bridized
to a 50-mer
brain-derived
a3iSdATP
using
As a control,
adjacent
sections
oligonucleotide
neurotrophic
sponding Following 20-mm
washes air-dried.
was
applied
distilled
Subsequently, and
the
sections
counterstained
in Entellan
lightly
(Merck,
were
hto rat
mRNA
SSC;
the’
corre-
60%
NTB2
nuclear
track
were
exposed
for
with
cresvl
Darmstadt,
and
were
water,
95%
violet
Germany)
then
ethanol, emulsion
for
mounted
Expression
of PKA-R
of the Rat
Northern
inRNAc
at D[ferent
Serninferous
blot
mRNA
for
of RNA
all four
Stages
(Fig.
of the
from
subunits.
cline lowed
stage
RIot, a pre-
with
expression VI-VIIc,d
abundant at stages
to a main mRNAs low
at stages (Fig.
than
two 1.7-
amount transcripts
at the
a single
other
2.6 kh mRNA.
RUt
VIIc,d-VIII
and
the
at stages Il-Ill
2.2-kb
decreased
levels at stages at stage VIIc,d.
1.6-kb
showed levels
two
IV-VIII
of R113 mRNA
thereafter to very low by a marked increase
1). Both sion,
An
same
other
detected
with The
was ‘en’ low at stages XIII-III with IV, and maximal expression at stages
Expression
XII.
the
the
at stages
of this mRNA levels at stage I).
while
RI[3 probe
expression
hybridized
of the
adult rat testis were hybridized to specific for the different PKA-R
localize
same
mRNA.
cells
length
This
in the
and
probe
RIot probe
G/C
readily
expressing cells in the brain beling over cells in the testis The
same IX-I
reaching
in the
Hybridization
testis
As a hybridization specificity hybridized in parallel with
spermatocvtes
individual
For
in approximately
level
1). The
(Fig.
maximal
probe
to
mRNA
expressing
control, adjacent an oligonucleo-
content
specific
detects
BDNF
[38] hut did not (see Fig. 7).
labeled
cells
show
in the
for
mRNAan’
la-
seminiferous
intensity of labeling of the semimiferous
at stages
XIII-XIV
(Fig.
2B).
Rio.
prepared
PKA-R
analyzed,
a higher
The level increasing after
present
was
10 stages
VIII-XI
in order
BDNF
Epitheliuin
analysis
RNA transcripts
stages
the probes
PKA-R
epithelium (Fig. 2A). Higher magnification revealed labeling over the layer where round spermatids are located at stages I-VIII and also over the layers of pachvtenc spermatocvtes at stages VII-XII and of diakinetic and dividing
dominating mRNA of 1.7 kh was seen together minor mRNAs of 2.9 kh and 3.2 kh, respectively.
showed
through
Riot.
pools seminiferous tubules representing 10 different stages of the rat seminiferous epithelium showed expression of
at all
mRNAs
b) in Situ
Testis
these mRNAs. sections were
Jipressing
tubules at all stages, with the highest over cells located in the middle part RESULTS
kh
of Cells
Rat
tide
1061
TESTIS
Sections oligonucleotide
microscopic
examinations.
cycle
Adult
rat
2 to 3 wk,
and
IN TIlE
identification
746-795 of pig BDNF mRNA [38]. the sections were subjected to four
at 40#{176}C in 0.1-strength
through
and
(BDNF)
PKA mRNA
deoxvnu-
complementan’
factor
to nucleotides hybridization,
transferred
then
with
OF
a minor
mRNA (Fig.
followed maximal
h
At the
layer of pachvtene
of round
level
Rut. the middle VII-XI
(Fig.
trated
to the
parts
stages. layer
center also
at stages
1).
labeled
(Fig.
5A).
tubules FIG. 2. In situ hybridization of sections through the adult rat testis hybridized to a probe specific for RIo mRNA. (A) Dark-field photomicrographs of sections hybridized to the Rio mRNA-specific probe. Four different stage groups of the seminiferous epithelium cycle, indicated in the figure, were identified by microscopic examination of the sections based on the morphology and relative position of spermatids in the tubules. The position of the basement membrane is indicated by arrowheads in the upper left photograph. The Star indicates the center of the tubule. Bar = 40 m. (B) Higher magnification bright-field photomicrographs of cresyl violet-stained sections hybridized to the Rio probe. Arrows point to some of the labeled cells. Abbreviations: ps, pachytene spermatocytes; Ips, late pachytene spermatocytes; rs, round spermatides. Bar = 20 m.
found
were
sections
an
oligonucleotide
probes is not
stages
was
number in all
found
stages
at the
of other
over
the the
(IX-XIV)
level
of elongating
from is of the
expressed
intensity
stages..
was
located
layer
was
(Fig. 5B). specifici adult
rat
specific
for
same
length
PKA-R labeling
of the
in situ
were
BDNF and
seen
hvhridiza-
hybridized This
content
However,
rat an
to oligoas
BI)NF
in the over
of round in all tu-
mRNA. G/C
dismost
h’hridization layer
labeled
levels was
tubules while
The to the
testis
VIII
labeling
of
also
mRNA.s.
at detectable no
VII and
At stages I-VI, some in their middle parts
This
the
epithelium
at stages the
at these
VlI-VIII. for the
seminiferous
probes
(D(-XIV)
stages
SB).
for the various
in accordance,
the than
at later
of the
Ril3
unlabeled
tion,
nucleotide
parts the
at these
bules at stages A.s a control
middle 1-VI, al-
4B).
declined. labeling
(Fig.
and
labeling
concen-
The
at stages
lower
and
middle
At later
spermatids
the
seen in at stages
was
4A).
tubules
predominantly
with
tubules
labeled cells labeling at
labeling
of labeling
VII-VIlI
over the tubules played intense
to the
and to late vast fliajority
any showed
(Fig.
significantly
spermatids,
The
were
over
intensity
(Fig.
RI! (3.
restricted
probe were epithelium
the
tubules
in a few
were
was
spermatids
expres-
the
At stages
labeling
XII-XIV,
of the
labeled
of round
contain stages
with the Rut seminiferous
4A). At stages
were
was
spermatids.
Cells labeled parts of the
showed
an increased
labeling
of stages VIl-VIlI of stages lX-XII. The
the
tubules
(Fig.
the
at stages 1-VI did not A few tubules at these
both
a de-
level,
of tubules (Fig. 3B).
labeled
mRNA
levels
cellular
round spermatids spermatocvtes
though
II to VIIa,b, folThe RII3 probe
stage-dependent
3A).
abruptly 1) and
3.2-kb
over
The RI probe showed a low hut specific laheling cells in the seminiferous tubules of stages VII-Xlll (Fig.
testis part
the
mRNA [38]; of the
LONNERBERG
1062
ET AL.
A
Ix-xI
B
1-VI
ux-xI
xII-xIv
I
vu-Vu’
xi i-xiv
OF PKA mRNA
EXPRESSION
sections after the labeling
hybridization patterns seen
with with
specific probes do not appear hybridization of these probes
this probe (Fig. 6). Hence, the various PKA-R mRNA to be due to the testis
to a nonspecific sections.
dependent protein kinases at various stages the seminiferous epithelium. The different scripts detected with the four PKA-R probes PKA-R tionated the
mRNAs previously rat germ cells
in situ
hybridization
oligonucleotides labeling when to BDNF The
mRNA
when
with
the
four
that
seen we
is not
with
added
expressed
the
labeled
blots and of mRNAs of cAMP-
of the cycle of sizes of tranagree with the
PKA-R
mRNA-specific
(1)
There was complementary
in the PKA-R
a 30- or 100-fold
rat
probes
molar
testis. was
excess
no (2)
abolof the
same, but unlabeled, probe. (3) Under the same hybridization conditions, adjacent sections-hybridized to a 48-mer oligonucleotide specific for the 75K low-affinity nerve growth factor (NGF) receptor mRNA-showed, in agreement with our earlier studies [34], no labeling over germ cells, while labeled cells corresponding to Sertoli cells were found close to the basement membrane. Combined, vide strong evidence that the labeling the various PKA-R mRNA-specific probes is further
supported
expression hybridization,
by
the
fact
that
of the four PKA-R mRNAs, agrees with the results
analyses. The results of the expression of mRNAs
Northern for the
these results patterns seen are specific. the
prowith This
stage-dependent
as revealed by in situ of the Northern blot
blots revealed four isoforms
TESTIS
than differentiating germ cells. In agreement with results from Northern blot analysis of fractionated cells
and
a differential of the regula-
dependent tional both
role Riot
the
different
at different and RI3 were
times found
matogenesis zvme could
in specific
ers of germ cells of these stages. Labeling over could not be assessed with certainty because culties
of delineating
the
border
of individual
Sertoli of the Sertoli
the
ontogeny
laycells difficells
in the fixed sections after in situ hybridization. Though Sertoli cells are known to express PKA-R subunit mRNAs [40], the lack of intensely labeled Sertoli cells suggested that cells of this type constitutively express lower levels of PKA-R
the
of
the
PKA-R
were found and sperin the stage-
four
subunits
(Fig.
subunits
play
a func-
of spermatogenesis. to be expressed
Although in pachytene
R113 gene.
This
suggestion
as well
presR1j3 peak 1(113sper-
a functional activation
enof
as all functional
inter-
pretations from the present data require, however, that the PKA-R mRNA expression documented here lead to the Synthesis able
of functional on the
cells are however,
tein are present erous epithelium, mRNA in germ for
proteins.
possibility
that
immediately it has been
Limited newly
information
synthesized
translated shown that
is avail-
mRNAs
in germ
into protein. For NGF, both NGF mRNA and pro-
in germ cells at all stages of the indicating an efficient translation cells [41].
seminifof this
In contrast to the two wpe I subunits, expression of mRNAs both isoforms of the pe II enzyme appeared to he
strictly
regulated.
stage
Thus
IV with
peak
the
levels
level
of RiI3
at stages
mRNA
VI-VHc,d.
increased RIIt
at mRNA
expression contrast
showed a marked increase at stage VIIc,d. In to R1113, Riot mRNA was also present at high levels
at later pressed
times in spermatogenesis in elongating spermatids.
pare
the
distributions
pachvtene XIV)
are
of PKA-R
when it was It is interesting isoenzvmes
activities during meiosis are active during haploid
with
highly exto comthe
known
and spermiogenegene expression
1-8 of spermiogenesis [42, 43]. RIot seems to cover of the meiotic transcriptionallv active phase from mid(stage
VII
of the
cycle)
to meiotic
as well as step 1-8 spermatids, limited to late phases (steps
Of particular
interest
is the
expression
of Ri(3 mRNA
have a very specific role in the hut not in meiotic division.
potheses
could
specific niferous
antisera tubules
lowed by It is also mRNA is [44]. The
he experimentally
divisions
(stage
whereas 1(113 and 7-8) of spermiogenesis.
late pachytene stages of meiosis followed appearance before diakinesis and meiotic therefore prophase,
FIG. 3. Cells expressing RIlt mRNA in the adult rat testis. IA) Dark-field photomicrographs showing R113 mRNA-expressing cells of the indicated stages of the rat seminiferous epithelium. Note the unlabeled tubules (circle) characteristic of R1l3 mRNA expression during stages 1-VI. (B) Higher magnification bright-field photomicrographs diplaying Rl)3 mRNA expression over the layers of late pachytene spermatocytes (Ips) and round spermatids (rs) at stages VIl-VIlI. Note the absence of labeling at stages 1-VI. Scale bars in (A) and IB) and other symbols are as in Figure 2.
for PKA-R
and that the appearance of be regulated by a transcriptional
(steps most
expression
on
spermatocytes and round spermatids, RIot mRNA was ent at similar levels at all stages of the cycle whereas mRNA expression increased markedly at stage VI with a at stages \THIXI. This suggests that the activity of the containing holoenzvme is important at this time of
to the
mRNA
studies
of mRNAs
that
transcriptional sis. All isoforms
of PKA-R
from
expression
7), indicating
ton’ subunits of cAMP-dependent protein kmnases at various stages of the cycle of the seminiferous epithelium. The in situ hybridization analysis showed that this specificity is due induction
1063
expression in the testis [20], all four subunits to be predominantly expressed in spermatocytes matids. However, clear differences were seen
rat testis or in fracof the specificity of
revealed the following: we used an oligonucleotide
labeling
ished
seen in fetal [39]. Assessment
mRNAs previous germ
DISCUSSION In the present study we have used Northern in situ hybridization to analyze the expression for the four isoforms of the regulatory subunits
IN THE
tested
1(11(3
during
by an abrupt disdivisions. 1(1(3 may regulation of meiotic The functional hyby the
addition
of
against the four PKA-R subunits to semifrom different stages cultured in vitro fol-
an analysis of the progression of meiosis in vitro. intriguing to note that the distribution of Rut and virtually identical to that of protamine 1 mRNA synthesis of protamine 1 mRNA starts at early stage
FIG. 4. RHo mRNA-expressing cells in the adult rat testis. IA) Dark-field photomicrographs of cells expressing RIle mRNA at the indicated stages of the seminiferous epithelium. Note the presence of unlabeled tubules (circle) at stages 1-VI. (B) Bright-field photomicrographs showing RIle mRNA expression over round spermatids (rs) at stages VII-VIll and elongated spermatids (esl at stages IX-XIV. Note the absence of labeling at stages 1-VI. Symbols and scale bars as in Figure 2.
A
B
FIG. 5. Cells expressing RII)3 mRNA in the adult rat testis. (A) Dark-field photomicrographs showing of the seminiferous tubules of stages 1-VI and VIl-VIll. Note the reduced intensity of labeling seen over tomicrographs showing RII)3 mRNA expression at the level of round spermatids of stages 1-VIlI. Although tubules,
as shown
in the
figure.
contained
intense
labeling
over
round
spermatids.
Symbols,
abbreviations,
RII)3 mRNA-expressing cells in the middle the tubule of stages IX-XIV. IBI Bright-field most tubules at stages 1-VI were unlabeled, and
scale
bars
as
in Figure
2.
parts phosome
1066
LONNERBERG
ET AL.
VII of the during comitant terest gene
cycle;
to know expression
Expression found ferent tion
in the brain of mRNA
both Pilot and than Riot and the testis the tially expressed functions may PKA in both expression of ferent subunits cific stimulus
FIG. 6. Control for the specificity of the in situ hybridization. Brightfield photomicrograph showing absence of labeling over any cells in the rat seminiferous epithelium after hybridization to a 48-mer oligonucleotide specific for rat BDNF mRNA. The BDNF oligonucleotide probe has the same length and GC content as the four PKA-R probes and hybridization was performed in parallel with those shown in Figures 2-5. The tubule shown in the figure is from stage VIl-VIll, but identical results were obtained with tubules in the other stages. Bar = 20 pm.
the
transcript
is stored
stages IX-XIV and translated decrease in the mRNA
in the
whether RIIot has any effect or its mRNA stabilization. of all four brain, regions for the
PKA-R
subunit
predominantly [22]. However, four
subunits
spermatids
in stages I-lV with conlevels. It would be of inon protamine
genes
has
also
1 been
in neurons of many difthe regional distribudiffers
within
the
brain,
1(11(3 showing
a more restricted expression Ri(3 mRNAs. Thus both in the brain and in various PKA-R genes appear to be differenin different cells, indicating that discrete be associated with a particular subtype of locations. It has been suggested that the PKA-R in the brain reflects a role of the difin modulating neuronal responses to a spe[22]. It is worth noting that the 1(1(3 and 1(11(3
genes are predominantly expressed in the brain and in reproductive tissues, notably the testis [9, 20], suggesting that these subunits play a key role in second messenger systems used in common between these two systems. In this context, it is interesting to note that genes encoding two of the most abundant and widespread neurotransmitter-synthesizing enzymes-the ‘y-aminobutyric-acid-synthesizing enzyme glutamic acid decarboxylase [45] and the acetylcholine-synthesizing
enzyme
choline
acetyl
transferase
[46]
(as
FIG. 7. Relative level of expression of mRNAs for various PIKA-R subunits in different cell types of the seminiferous epitelium in the adult rat testis. The intensity of shading reflects the relative level of expression of the indicated mRNA in the particular cell type as assessed by Northern blot and in situ hybridization. (See text for comments on the individual PKA-R subunits.) The 14 cell associations or stages of the cycle of the seminiferous epithelium are defined according to the morphological development of the acrosome and of the shape of the spematid nuclei )49). IFrom Dym and Clermont 1501. Reproduced by permission from Alan R. Liss, Inc.)
EXPRESSION well
as the
neuroactive
all expressed ential gions
expression together
sion
peptide
cholecystokinin
in differentiating
male
cells.
The
genes
in the
testis
opens
up
F, Beavo J, Bechtel
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Cl-I, Cadd
form
of the
crease same
Sertoli
cells
30- to 40-fold in response treatment elicits a 2- to
Rut
mRNA expression of PKA-R mRNA
lation may
in cultured
be responsible,
been
for the
and the RIot and
marked
shown
to dramatically
down-regulate
of the 75-kDa low-affinity NGF result is a marked stage-specificity ceptor, Whether
which is a similar
expression and RII(3 The
restricted to stages androgen-mediated
is responsible mRNA at stages
stage-dependent
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