Subunits of Cyclic Adenosine 3', 5'-Monophosphate-Dependent ...

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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

the

kinase

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

mouse.

Ribonucleic

rat testis.

Biochem

genes Cellular

in the mammalian testis. In: Endocrinology of the Testis. B.V. (Biomedical Division), pp. 199-

epididymal

sperm.

J Biol

Chem

259:832-

Kleene

Schiltz

E, Browner cDNA

the

Proc Levy

DF,

Krebs

J, Schulz

U, Dunica

regulatory

rat

1986.

subunit

granulosa

of 3’:5’-cyclic

Molecular

(Rll,)

of type

J Biol

cells.

P, Einig

(Rh)

other

F, Hanks

Chem

1983.

deadenylation

Dev

Isolation

Biol

of

105:71-79

of a cDNA

cAMP-dependent

clone

protein

kinase,

AM, Gilbert

sequence

I, Walter proteins.

A cDNA

subunit

U, 1984.

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and

pre-

regulatory

protein

Proc

kinase

J, 1982.

NatI Acad

from

I),

of the testic-specific

USA

c-abl

(A Laboratory

Manual).

DNA.

Proc

Klein

A, Mackenbach

1, de

G, Grosveld mRNA

pro-

Laboratory

Agthoven

Valle

81:6723-27

CAMP-dependent

Cloning

for sequencing

M, van Della

reg-

Res 16:8189-90

Acids Harbor

method

A, von Lindern

Sci

of the

to microtubule-asso-

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Molecular

Spring

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catalytic

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L)e Camilli subunit

T, Fritsch

McKnight

acid

Endocninol

kinase

regulation in the

M, Task#{233}nK, Eskild

Mol

and

Translational

EG,

amino

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ciated

SM, Walter

spermiogenesis

complementary

human

Maldonado

flagellum.

Sci USA 80:3605-12

subunit

SM,

subunit

sperm

SL, RichardsJS,

of the from

subunit

sandberg

cloning,

ulatory

regulation

1984.

I regulatory

full-length

Lohmann

regulatory

mammalian

D, Ratoosh

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Acad

Molecular

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T, 1987. and age-dependent changes in mRNA for cAMP-dependent protein kinases in rat testis. Biol Reprod 37:947-56 0, Mvklebust F, Scott JD, Hansson V. Jahnsen T, 1989. Human testis cDNA for the regulatory subunit Rn,, of cAMP-dependent protein kinase encodes an alFr#{248}ysaA, Sandberg

Cellular

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#{248}yen0,

sandberg

T, 1988a. pendent

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Lohmann

WG,

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RJ, Hecht

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Nati

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M, Goldman

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KC, Distel

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GA,

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VJ, Beattie

CAMP-dependent

a protamine Lee

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263:2098-104 L, Kidd

261:12352-6

testis-repre-

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nu-

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FFG, Van der spermatocytes

haploid-specific

ti CAMP-dependent

T, Hedin

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M, Eskild

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FEBS

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Len

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V, Jahnsen

D, Hansson

FO, Knutsen

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Oyen nibonucleoproteins

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Ronanden.

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for

RNA synthesis

RNA during

AH, Itomments

protein

HonowitzJA.

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MA, Kistler W5, 1987. Transcriptional and translational control of the mesfor transition protein 1, a major chromosomal protein of mammalian spermatids. J Biol Chem 262:13309-15 Horowitz JA, beg H, Orn GA, 1984. Characterization and localization of cAMP-de-

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