BIOLOGY

OF REPRODUCTION

Rabbit

18, 371 -378

Endometrial

RNA-

(1978)

and DNA-Dependent S. CHILTON’

BEVERLY

J.

and

Department

of

University

DNA

Polymerase

C. DANIEL,

Activity

JR.2

Zoology,

of Tennessee,

Knoxville,

Tennessee

37916

ABSTRACT Changes

endometrial RNA- and DNA-dependent DNA polymerase activity have been studied during the first 9 days of pregnancy. Both enzyme activities are low at estrus and 1 day postcoitum, but measurable increases in activity occur by 48 h after coitus. The RNA-dependent DNA polymerase (RDDP) activity reaches a maximum on Days 3-4 postcoitum, a level it maintains except for slight drops on Days 5 and 8-9. The DNA-dependent DNA polymerase (DDDP) activity initiates a rise on Day 1 postcoitum, to reach a peak on Day 6 and remains at this level through Day 8, but drops significantly on Day 9. When polymerase levels are obtained for pseudopregnant animals, the RDDP activity parallels that of pregnant animals up to Day 5 and the DDDP activity attains levels comparable to Day 6 pregnancy levels. In pseudopregnancy, RDDP activity drops precipitously on Day 6 resuming its usual high level again on Day 8, while DDDP activity remains high through Day 6, dropping on Day 8 of pregnancy. Low levels of both RNA-dependent and DNA-dependent DNA polymerase activity can be induced

in rabbit

by

either

estrogen

high levels of enzyme through the synergistic

or progesterone

activity

administration

normally of estrogen

effects

INTRODUCTION Much

evidence

to

found in Days and progesterone.

3-4

ovariectomized

association

supports

the

concept

that

(1975),

in

with

gene

gested reverse

differentiative

amplification/modification

phases

Hammond,

of

1928,

early

Kirby,

1970;

Adams,

1970,

1973;

Daniel,

1971).

Whether

this

direction

specific proteins, supplied by the urn, it is assumed at the genetic understanding recent

level. Many gaps of gene regulation. has

chromosomal

Howk

and

focused

role of association

reproductive tract that I3-polymerase tone

is

tiation.

1962;

tissue

Hafez,

related

Wang,

on

proteins

Accepted Received

September 20, 1977. June 23, 1977.

‘Present

Address:

Division

of

ideal

system with

events interaction

concomitantly. Yang et al. sence in extracts

the

potential

from

(Bellair,

thereby

an

in our However,

inferring

rabbits

DNA-dependent tivity profiles

beand

and

Gallo

cell

uterine

differen-

endometrial

where

the

role

hormonally

within can

that be

of

domi-

tissue and investigated

(1976) demonstrated the preof endometrial homogenates of

a

and

DNA changed

(6-8s

and

polymerases predictably

3-4s)

whose during

acthe

first 7 days of pregnancy. Also a 5-6s RNAdirected DNA polymerase, (RDDP) which showed a preference for synthetic viral specific template-primers (Baltimore and Smoler, 1971), was isolated from high salt (0.5 M KCI)

1968; an

treated activity Reproductive

Wu

during the

associated

nated cellular materno-fetal

tissues. It has been suggested is associated with the nonhis1969)

offers

exist

DNA polyrnerases with embryonic

the

be induced

activation.

Accordingly,

polymerases

to

RNAs or other moieties cells of the uterine endometrithat the ultimate regulation is

attention

developmental cause of their

embryogenesis

1952;

However,

can only

in a recent review article, have sugthat what they have termed “intrinsic” transcriptase may be employed in gene

mammals, the uterus provides essential environmental direction integral to the growth and (e.g.

does.

of pregnancy

extracts and showed precise from Days 0-7 postcoitum.

changes Bedigian

in et

al. (1976) also found RDDP in uterine tissues of pregnant rabbits, which was “. . . associated with particles that band at a density characteristic of type C RNA viruses.” These data

Biolo-

gy, Department of Obstetrics and Gynecology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104. 2Please address reprint requests to Dr. J. C. Daniel,

coupled in

Jr. 371

the

with

the

endometriurn

evidence of

of virus-like pregnant

rabbits

particles (Dan-

CHILTON

372

iel,

unpublished

et al.,

observation)

1977)

The

prompted

following

acterize

the

merase

an

to

of

elucidated

induction time

for

of

The

hormones

castrate

DNA

poly-

was

after

for

effects

on

does of

poly-

enzymes

pseudopregnancy

sequence.

steroid

char-

DNA

of

the the

polymerase

also

in

same

hormone

in

Tissue

third

MATERIALS

AND

reagents

METHODS

were

obtained

from

the

following sources: RNAase free sucrose from Sigma (St. Louis, MO); (rA)n, (dT)n, (dT)9 and nonlabeled deoxyribonucleotide triphosphate (dTTP) from P-L Biochemicals (Milwaukee, WI): (3 HI -methyl dTTP from New England Nuclear (Boston, MA); nonionic detergent NP-40 from Particle Data Laboratories, Ltd. (Elmhurst, IL); Whatman 3mm filter paper discs from Reeve Angel and Co. (Clifton, NJ); crystalline bovine serum albumin from Calbiochem (San Diego, CA). All

other

chemicals

purchased

were

of standard

reagent

Treatment

polymerase

activity

to one of the following

were

pretreated

was

procedures.

TABLE

1. Hormone

by

of 15 IU of human cervical stimulation.

injection

6 and

8,

along the mesometrial

border

and the endometri-

motor-driven

teflon

pestle.

The homogenate

chorionic Two-

centrifuged

schedulea.

Days of treatment

No. of animals

1

2 2 2 2

5Does progesterone

injection followed

5,

again for 10 mm at 2000 X g. The nuclear pellet was resuspended in 1 ml of homogenization buffer. The supernatant fractions were pooled and spun at 10,000 X g for 15 mm. The mitochondrial pellet from this centrifugation was resuspended in 0.5 ml of homogenization buffer. The supernatant fraction was divided into 2 aliquots, 1 of which was salt treated to a final concentration of 0.25 M KCI. These supernatant fractions were then spun at 109,000 X g for 90 mm in a Spinco Type 50 or Titanium 50 rotor.

Pregnancy was accomplished by placing each virgin doe with 1-3 males successively and observing a minimum of 2 matings. Each doe was then left with I male for 60 mm. Two-4 animals were used for each day of pregnancy. B. Pseudopregnancy was induced by giving each vein

4,

administration.

Elvejhem

according

A.

doe an ear gonadotropin

2,

um was scraped free using a scalpel blade and then weighed. For estrus and 1 day pregnant animals, tissues from both uterine horns were pooled, but the horns were treated separately on Days 2-9 of pregnancy and pseudopregnancy. Endometrial scrapings were homogenized (11 strokes) in 3 volumes (wlv) of homogenization buffer containing 0.01 M Tris, pH 8.0; 0.1 M KCI; 3 mM MgCl2; 0.25 M sucrose and 5 mM dithiothreicol (DTT) using a Potter-Elvejhem motor driven teflon pestle at 0-4”C. All subsequent procedures were done at this temperature. The tissue homogenate was centrifuged for 10 mm at 2000 X g. The supernatant fraction was reserved and the nuclear pellet resuspended and re-homogenized in the original volume of buffer using 5 strokes of the Potter-

Young, mature, virgin New Zealand does were maintained in an animal holding facility for 3-4 weeks postshipment to insure against disease and pseudopregnancy at the time of experimentation. Groups of animals that were ultimately assayed for uterine

Days

Extraction

open

grade.

Animal

on

Does were killed by embolism and immediately opened midventrally. The uteri were removed, flushed with 0.9% saline solution and placed on ice. Pregnancy was verified by the presence of embryos in uterine or oviductal flushings. Embryos found in these flushings were saved and also assayed for polymerase activity as described for endometrium. Each uterine horn was cut

experiments.

Biochemical

sacrificed

Dukelow (1972) when measuring endometrial proliferation (mitotic index) and DNA synthesis (incorporation of I HI methylthymidine) in response to steroid

ovarian

a

were

a midventral incision and maintained for 3 prior to experimentation. After this time, animals were given suprascapular, s.c. injections of either corn oil or the appropriate steroid dissolved in corn oil according to the schedule in Table 1. This is the same injection schedule employed by Lee and

the

activity

examined

does

through weeks

extracts during the Activity profiles were

both

JR.

respectively, of pseudopregnancy. C. When testing for hormone induction of polymerase activity, does were bilaterally ovariectomized

and the RNA or (rA)n(dT)9polymerase in subcellular frac-

of rabbit endometrial 9 days of pregnancy.

DANIEL,

4

(Fowler

attempt

activity

13, (rA)0-(dT)-dependent

also

group

mice

investigations.

describes biological

merase (DDDP) dependent DNA tions first

and

further

AND

3

4

5

6

7

8

9

Co

CO

CO

CO

CO

CO

...

CO

Co

e

e

e

e

e

e

...

e

e

e

e

e

e

e

e

...

...

...

...

...

...

...

...

p p

p p

were killed (

2

24 h after

4-pregnene-3,20-dione).

the last injection.

co

=

1 ml

corn

oil;

e

=

100

pg/kg

1 713-estradiol;

p

=

3 mg/kg

RABBIT

ENDOMETRIAL

POLYMERASE

The resulting microsomal pellets were resuspended in 0.5 ml of homogenization buffer and homogenized using 3 strokes of a conical, ground glass homogenizer. As controls, lung, liver and spleen from the same animals on Days 0-9 of pregnancy were homogenized and fractionated according to the procedure described above and then assayed for both RNA-dependent and DNA-dependent DNA polymerases.

Polymerase

subcellular

activity

subcellular

fractionation

by differential

50 Ml aliquots of each fraction were added to 0.2 ml lysis buffer consisting of 0.05 M Tris, adjusted to pH 8.0 with 0.1 N HCI and 0.1 N NaOH; 0.5 M KU; 5 mM MgCI2; 50 mM DTT; 0.2% NP-40 and 15% glycerol. Samples were allowed to lyse for 5 mm in an ice bath. The remaining portion of each fraction was stored at -20#{176}C. Samples were later thawed and aliquots were used to determine protein content (Lowry et al., 1951). To assay for polymerase activity, 10 M’ samples were removed from the lysis buffer, added to 30 M1 of a reaction mixture containing 0.17 mg/mI bovine serum albumin (BSA); 25 Mg/mi of the appropriate synthetic template-primer, (rA)n’(dT)s for RDDP and (rA)n.(dT)n for DDDP; 2 mM MgCI2; 5 mM DTT and water and allowed to sit on ice for 5 mm. After this time, 10 MI [3HI-dTTP (37.5 iCi/ml) was added for a total reaction volume of 50 MI and incubated at 35#{176}Cfor 30 mm. Following incubation, 40 MI of the reaction mixture was spotted onto Whatman 3 mm filter paper disks. Paper disks were subsequently washed (4#{176}C)for 10 mm each in 4

represents

(Table M

into filter

I 2-( 5-tert-butyl

DNA (polymerized) paper disks, while

benzoxazoyl)l

in toluene for liquid scintillation LS-230. The external standard for quench and a background subtracted from each sample.

DNA:

5:1

(dT)0 small

template-primers ribohomopolymer

(rA).(dT),

thawing

of stock

counting in a was used to Control was

contained the followto homo-oligomer

for

RDDP

and 11 (rA). were stored

for DDDP. All template-primers aliquots at -20#{176} C to eliminate

repeated

activity

nuclear

and

oJ’ Poly;n to

is

observed

mal

KC1

erase

Yang

concentrated

chondrial

fraction in

presence

those

of

this

has

microsomal

particulate

also mainly

a!.

Analysis

expression

of

of Polymerase

During

reported with the postTherefore 3 fractions

fractions

and

microsomal KCI treated supernatant. we summarized the activity in these as a collective

salt

pellets

a

(1976) be associated

to

in high

microsomal

enzyme et

(pM

3 fractions

RDDP

of

point

activity

DDDP

activity.

Activity

Pregnancy

The profile of RDDP activity, expressed as the sum of the specific activities (pM [3H] -dTTP/mg protein) of the mitochondrial, microsomal and KCI-treated postmicrosomal supernatant

from

represented

in

is low

polynierase on

Day

sum

Days

1 of

of

1. at

day

postcoitum

(73.7

embryos

± 9.7-67.6

5 to

Day (71.1

enter

the

to

blastulate.

48.4

±

through

14.3) the

trophoblast When

The

time

of

the

change

(Days in

the 3-4

respectivewhen

the

morulae

activity rises

blastocyst

on

Days

cleaving

drops

again

remains

invasion

and The

rises

time

as

SSA,

and

this

11.2)

on

± 15.3 the

uterus

± 12.3

±

a peak

with

is

of

h postcoitum).

(SSA)

reaches

coincidentally

pregnancy,

activity (29.2

(24

activities

and

of

The

estrus

pregnancy

specific

second

0-9

Fig.

on

on

6

Day

relatively

high

attachment

and

7-9

of

polymerase

pregnancy). activity

was

TABLE 2. Distribution of RNAand DNA-dependent DNA polymerase activities (pM(3 HI -dTTP/mg protein) in 0.5 M KCI extracted fractions of one Day 4 endometrial homogenate.

Fraction

RDDP activity

DDDP activity

8.0 32.7

720.Oa 494.0

solutions.

According activity

from

extracts

data

specific

in

freeze

RESULTS

Localization

the

polymerase

each

thiophene

(BBOT) Beckman correct

of

of

Yang

DDDP

beginning

was the nonpolymerized material remained soluble and was washed away (Waters and Yang, 1974). The paper disks were dehydrated in 2 10 mm changes of a 1:1 alcohol ether solution and 1 change of ether (10 mm). They were then dried under a heat lamp for 15 mm and subsequently placed in vials with 5 ml of 0.4%

Synthetic ratios

The

the

but

protein)

that

had been precipitated

incorporated onto the

sum

KCI)

implies

ly)

ing

the

2).

(0.5

of

determined,

[31-I1-dTFP/mg

changes of an acid wash solution consisting of 5% acetic acid, 0.7% hydrochloric acid and 1% sodium pyrophosphate. In the acid wash the (3Hl-dTTP that

2, 5-bis

distribution

was

association.

Assay

Following centrifugation,

the

373

ACTIVITY

the

treated

but microsomal

fractions.

.4 ctivity et

al.

(1976),

primarily

in

significant

the

mito-

levels

and

Thus,

RDDP

are

Nuclear Mitochondrial MiCrosomal Normal 0.25 M KU treated Post-microsomal supernatant Normal 0.25 M KCI treated Sum of specific activities from 3 fractions noted

7.8a 1.8 0.6 1.4 41.9

203.2a 83.2 10.8 18.2a 941.4

postmicroso-

for

each

assay,

aSum

of specific

activities

from

3 fractions

noted.

CHILTON

374

AND

DANIEL,

JR.

Nen

‘ONSO enr.4tn N

+444+1

+4+4+4

50

0’

‘30

60

+1+1+1

40

+1+1+4

en 00

Co

0

#{149}f5 ‘0

0

en

20

+1

-,

-

-

‘ow”,’ N +4

+4

+1

+1

+1

“*0’

N

‘r-r

I

0

2

3

4

3

0.,,

34

6

7

0

‘0’

0

9

P?.gI.tc,

.0

FIG. 1. Distribution of RDDP activity beginning at estrus and continuing through Day 9 of pregnancy. Activity is expressed as pM[3H1-dTTP/mg protein on a logarithmic scale and plotted showing standard error

(S.E.). Included is the activity profile for lung samples assayed as controls and the curve for cellular protein

-4,-’,’

E V

en

a

+4+1-H

V

0 0.

+4+4+4

000en

‘0

-

Co

011

E

CONN

0.

II-9

content.

-40-

en

+1+1+1

+1+1+1

-

1

00 N N

compared

with

endometrial days

the

total

protein

homogenates

of

the cellular

pregnancy,

as determined (1951)

by

and

the

relatively

firming

that

as

of

mg

constant

first

9

et a!.

protein/g thus

1);

0

0.

NN*

U,

0

content,

Lowry

(Fig.

N

for

the

protein

methods

expressed

remains

profile

during

N

‘ONen

+1-44+1

.0

E

+4+4+4

0’X 004’3*0

*

V 0

tissue, con-

a

U

C

‘5

the

specific

RDDP

activity

has

a

‘0411:

t=enN

a

pattern

of

during

change

early

relative

to

the

total

protein

0 0. be

pregnancy.

Homogenates of lung, liver and spleen were taken from animals on Days 0-9 of pregnancy and assayed as control tissues. Little RDDP activity was found in the lung (Fig. 1) and liver and spleen had similar low levels of activity (Table 3). It may be further noted that there was no detectable activity recovered from either oviducal ings on Days 1-9 postcoitum. The

change

in

the

in

activity

coitum,

going

maximum remaining of

of at this

from

DDDP

within

180.5

2074.6

±

high

level

were

±

lung

yielded

the

U

+4

+1

,-

-

+1

a 0

NI4’5N 43

0000

E

en

a

I.I-’

U ‘4-

N0Co

0

e

N

‘00

a 0 0

0.

43

00.-’

en

.0

N +4+4+4

+4+4+4

0

04-40’

0

activity

24

16.1

282.8

through

and dropping to 832.5 9. Homogenates of lung, liver tested on all days of pregnancy

these,

+1

V

3-4s

initiated

+4

en

the embryos or uterine wash-

pregnancy

Day

Of

is

+4

0.

greatest

h

SSA on

the

Day

8th

0

a

during the same time course of pregnancy is depicted in Fig. 2. Upon comparison of polymerase activity with the cellular protein levels, a dramatic increase in the amount of polymerase

0

N*N

V

postto

a 7,

day

0

(.3

en +4+4+4

-

+4+4(3

P-N U (5

oar-en

.0

43 4-

V

+4

0.

>4

V

0

0 04

amount

I-’

of

V

1-en

>4

on and spleen (Table 3).

± 139.6

U,

0040

a

(3

en

0 (3

444

0

u (3

.(3

V 00 C

> C 43

;1’.,;v.;

:;cl;

RABBIT

ENDOMETRIAL

POLYMERASE

ACTIVITY

375

‘.0 #{149}0 p 7004

5so 4,

4#{176}

S

a

9

#{149}0

S

#{163}2 3 0.7.

FIG. polymerase the curve of

C

4

3

2

4

4

04

6

6

3. Overlaying

which

the

value

of

lowest

tissue.

When

were

assayed,

able

on

Days

it

is

of pregnancy. for solid

the

injection

± 101.6

and

of

ranged

normal

RDDP Fig. 1) is 5,6 and 8

(see 2,4,

total line.

induction 5A

as compared

pregnant

to

Day

of Polymerase

represents

2001.1

8 animals.

Activity

RDDP

activity

in ovari-

from 4000

for

uterine

was

detect-

embryonic

tissues

activity

pregnancy

hCG

normally

below

± 16.1

enzyme

0-9

for

during pregnancy activity on Days

of

for

Hormone

considerably

180.5

embryos

no

I

9

on Days 0-9 endometrium is shown by scale.

but

SSA,

I

7

,4 04 444C5 04 00 7

activity,

18.9-89.2

6

4P07I7CI,Ofl

the profile

activity representing

Figure polymerase

I

pseudopregnancy.

8 post FIG. 2. DDDP analysis The mg soluble protein/g endometrial homogenates The Y-axis is a logarithmic

#{149}

4

#{163}a4i0’

fj

Pl.g..oo

P90u00p,.904n07

-

:‘

I

60

____‘_

NO,,,,.’

‘0

,00

,_.

..__

.*

3).

(Table

(000

Polymerase

ProJIles

The

results

amine

the

are

polymerase

found nancy.

during

representing

in

animals

2,

SSA

on

71.7

±

activity

on

Day

33.0

±

but

has

value

of

of

The

5 day

the

on comparable days However, by comparison 14.3

on normal

activity

4 and

is essentially

the

ex-

3. Superimposed

8 of pseudopregnancy.

activity

pregnant

to

pseudopregnant

activity

curve

2, 4, 5, 6 and

in

Fig.

enzyme

is the

pregnancy

designed

activity in

for

Pseudopregnancy

experiments

represented

profile

Days

of RDDP

5-6s

does

During

pseudo-

same

00

-

as that

normal pregwith a value of

6 day pregnant animals, 6 of pseudopregnancy is

for

Normal

P’.qn400,

,00, .--.

,

-.-.-..,_..

a

activity

0.6, 57.3

± 3.8.

profile

for

risen Figure the

3-4s

again

by

Day

4 illustrates

8 to that

polymerase

the

during S

Days parallels

2, 4, 5 and the

change

of pregnancy. curve established DDDP

drops

6 of

pseudopregnancy

in activity

on

the

to a level

of 896.3

± 136.9

‘

closely same

However, inconsistent with for the RDDP enzyme, by

50

a

days

the the Day

2 3 4 06vs1.

FIG.

4.

The

pseudopregnancy profile for pregnancy.

the

QaSco’

profile

of

is superimposed same

enzyme

5

6

7

S 6

pO,,-,471.oI!Oo

DDDP

activity

during

on the characteristic activity

during

normal

0

CHILTON

376

AND

DANIEL,

JR.

RNA-

and

activity

DNA-dependent

on

which

Days

has

teristics

some

on

et

Days

morulae

as

1967)

measured

(Hilliard previous through

the

DNA

time

increases the

again

on

primed DNA the ovariectobar graphs. A.

does

treated

progesterone,

or

according

rabbits,

animals

small

but

activity

injected

tion

of

the

same effect

high

polymerase

measured

in

profile

polymerase

activity

directed

DNA to

animals

the

levels

equivalent

mals.

Estrogen

of

sucrose

gradient

they

polymerase

13 activity

postcoitum,

not

(Table

1)

while

normal

treatment

activity.

oil

those

have

seen

in

comparable

with

estrogen in

for

RNA-

When

com-

polymerase

corn

resulted

DNA

described

progesterone in

same

DNA-dependent

slightly

curve,

ani-

these

quantitative

results changes

it

rabbit

the

utilized However,

DNA

synthesis

from

(rA)n

then

be

by activity

that endometrial

there

at

.

(dT)9

have differ

reflect

to

the

in

on

the

Days

ported

by

Bedigian

and

placenta

pregnancy,

but

Since

a!.

was

these

of

RDDP (1976)

prominent

essentially

the as

pregnancy

interaction

was

it could

measured of

The et

the

specificity,

6-9

sequential

of

requirements

activity

enzymes.

uterus

DDDP.

template

al.,

converts which

cofactor

RDDP

polymerase

(dT)n,

same

et initiated

terminus rapidly

but (rA)n

has

.

The

(dT)n

(Yang

RDDP

(rA) by

the only

of

a

template

RDDP

then

the

exists.

3’-OH

it

beof

(rA)n

the

the

recognized

pM[3H]-dTTP

followed

the once

uter-

pregnancy

specific

by

ap-

the

interaction

template

viral

resucrose

nearly in

molecules the

synthetic

1976).

and

the

ho-

endometrial

more

early

for

recognizes

enzyme

higher

during

(dT)9

of

is apparent in

not

on

activity

polymerase

DDDP

levels

DISCUSSION From

different

administration levels,

polymerase

potential

enzymes

levels.

are

the

tissue activity

from may

endometrium

ribohomopolymer,

polymerase

estrous

the

noted

whole

obtained

in

activity

fractionated

data

3

increase

enzyme

fractionation

proximate ine

that

the

The

in

Day

polymerase

to

polymerases

gradients.

cause

yields

illustrates

3-4s

with

results

progesterone

5B

as that

to

an

syner-

to

for

to

further

of

opposed

increase

discrepancies

fractions

as

slight

by

in

of

fractions

estrus

the

subcellular

corded

a

followed

a

5-7

centrifugation.

from

differences

mogenates

report

Days

postnuclear

report

Presumably, reflect

dropping

on

extracts

Additionally,

pregnancy,

(1976)

activity

to steadily

of

al.

in

subcellular

postcoitum.

Fig.

for

or

the

progesterone comparable

polymerase

injected

reveal

produces

However,

3-4

graph

alone

and

activity

induction

pared

activity.

estrogen

on Days bar

oil

polymerase

progesterone

of

of

corn

6

to

from

in Table 1. in pregnant

endometrial

or

level

gistic

progesterone

RDDP

7-9).

1976),

constant,

pregnancy

here

to that detected in pregnant animal. Administra-

estrogen

The

with

measurable

equivalent or 1 day

estrous

and

estrogen,

outlined levels

the

to

either

estrogen

schedule daily activity

to

Compared

with

(Days

1-Day

et

high

corresponds

et a!.,

Yang

its

remains

which

subjected

activity. ectomized

9.

of

and

become

Day

decrease

FIG. 5. Characteristics of hormonally polymerase activity in the uterus of mized rabbit are represented by the RDDP activity. B. DDDP activity.

Day

levels

plasma

regains

implantation

DDDP

13 (Yang

from

when

!

of

the

polymerase

venous

6

the

(Krishnan

Activity

Day

of

estradiol-173

ovarian 1971).

on

levels

increased

rabbit

on

Concurrently,

environslightly

blastokinin

Eaton,

level

cleaving

uterine

maximum

and

in and

a maxi-

when

drops

protein

Daniel,

charac-

reaches

activity with

uterine

and

1976),

the

enzyme

RDDP,

cellular

postcoitum

5, correlating

rabbit

1-1

al.,

The

some

encounter

The

Day

and

3-4

first

ment.

polymerase

postcoitum.

viral

(Yang

mum

DNA

1-9

may the

2

activity in

the on

absent

rerabbit Day by

10 Day

25. The

rationale

behind

using

homogenates

of

RABBIT

lung,

liver

the

and

the

viral

source

of

merase.

the

Also,

consistent, to

of

verify

specific

phenomena

Embryos

were

merase

enzyme

particles

have

early

Calarco

activity

from

Days

were

flushed

uterus.

and Chase

be

of

Furthermore,

Days (1973)

the

of

difference

age

Sherman

time for

the

the

of

protein

content of

herein

reported

(based

are

expressed

embryos) enzyme

tissue

activity

tissues

no

the

profile

of

pseudopregnancy for

comparison

of

presence

and

As

in

activities

of

protein/em-

also

resulted

with

uterine

and

levels

embryonic 4,

curve in

tissues

the

factor

the

Day

5 levels

production. protein

reported

to of

normal days

Yang

uterine treatment.

(100

pg/kg)

This

ovariectomized

3 mg/kg

of

body

protein

did

profiles

not

Embryos

for

intro-

the

et

in

high

utilize

the

to

antigen

p30

the

some in-

particulate (0.5

M

detergent from

KCI)

(NP-40)

other

assayed

spleen).

This

distinguishing

this

y which

last RDDP

is ubiqui-

et

al.,

1972).

demonstrated

that

polymerase

include

70s

achieve

endometrial

RNA

and

requirement

salt

and

this

tumor

is

template the

RNA

of

viral cells

rhabdomyosarcoma of

viral

(Fridlender

of

DDDP, polymerase

subcellular

(1976)

of

absence

stages

characteristics

polymerase

failure

the

1976),

liver

properties to

early

(rC)(dG)6

of

occurrence al.

is

associa-

reproductive

some

viral

absence

cellular its

the

DNA

nonionic

lung,

cocultivation

suspect merase,

the

has

use

virtual

embryonic

enhance

true

inability

viruses,

weight

Pretreatment

was especially

blastokinin.

that

with

studies,

al.,

and

the

cellular (1972)

et

is important

in

func-

eukaryotes

maternal

from

(i.e.

The

in for

and

(Yang

its

relate

reproductive

reason

the

Its

solubilization

tous

as they

of

RDDP

(rA)n(dT)9

from

polymerase

data

nuclear

properties.

and

these

the

normal

concentrations

the

influence.

both

pregnancy

Daniel

estradiol-17j3

uterine

of and

administered

4

protein

the permits

polymerase

a concentration in

following

on

during

DNA

proliferation.

unresolved.

the

by

function of

cell

2 polymerases

specificity

the

hormon-

with

entities

remains

the

to

aspect

RDDP

or

13. However,

for

pseudopregnancy.

progesterone at

activity

pregnancy

Figs.

5 of

Arthur

does

normal

absence 3 approximate

seen Day

polymerase superimposed

established

the

tract

fractions The

and

ani-

that

limited.

of

virus-like

clude

bryo.

on

of

an

obviously

embryogenesis

cellular

2-11

amount

mg

are

significance

polymerase

pregnant

understanding

understood

of

time,

biological

to

of

total

does

suggested

their

implications

our

and

adminis-

is subject

concurrent

apparently

results

of

the to

to

be

that

Lee

in decreased

normally

limited

and

by

resulted

enzymes

be

the

proliferation

this

in

could

and

not

Of

to the

assays

as

normalized

direction

tion

Because

initially, on

these

not

from

according

used

of

correction

present.

varies

amount

it

tional

and subsequently exactivity as cpm/unit embryos

Thus

phenomena

embryos.

subjects,

with

mals. activity

high

day

ovariectomized

1972) During

remain

3-4

continucd to

cas-

with

reported

progesterone Dukelow,

of

progesterone

normal

cell

be early

appears

correlated

as

proliferation.

may

produces

However,

levels

The

developmental

pooled

of

It

and

activity

index

of

treatment

endometrial

replication

in

1)

(1972).

and

cell

DNA-de-

activities

mouse

protein

numbers

Kang

estrogen

mitotic

tration

al

on

and

and

(1973)

pM/mm/mg

the DNA

embryonic

concepnises the measurable or

from

and

day

their

Kang

they

in embryos

enzyme

species

of

and

20-100 pressed

or

RNA-

11.5

obvious

no

with

the

can

events does.

sequential

in

(Lee

embryos

whether

Sherman

both

cells

Besides

the

detected

polymerase

proliferating

it poly-

However,

with

postcoitum.

DNA

1974),

DNA-dependent

was

reported

pendent

Dukelow

oviduct

no

activity 1-9

the

1973;

detected.

the

increase

1965;

DNA

ovariectomized

polymerase

Schlafke, Piko,

in

on

verifies

treatment

uterine

5 that Table

DNA

This

hormone the

normal

implantation

treatment).

which

does

hormonally

underwent

and

induce

Fig.

and

pregnancy,

from

to to

(schedule,

with

Manes,

associated

1-9

poly-

association

1973;

degree

synchronized, does

8 of

(Day

from

virus-like

of

differentiation 7

377

uteri

growth,

trated

both

RNA-directed

might

polymerase

for

the

ovariectomized

pregnancy

uterine uterine-

pregnancy.

in

1973;

was

in

to

used

tissues

Because

found

the

other

ACTIVITY

primed,

Day

poly-

of

assayed

that

activity

such

early

Szollosi,

anticipated

merase

during

duced

be a

determine

changes

(Enders al.,

and

was

the

reflective

been

et

might

in

activities.

embryos

Biczysko

to

were also

reduce

DNA

RDDP

that

activity

POLYMERASE

a general-

which

necessary

levels

to

had

infection

was

it

polymerase

was

animals

RNA-dependent

low

order

as controls

that

systemic

ized

in

spleen

possibility

ENDOMETRIAL

RNA

its tumor

expression with

cells virus

the

group

by human

and

the

specific

in Day 4 endometrial samples. We that the RNA-dependent DNA polybecause of its viral properties, is associ-

CHILTON

378

ated

with

this

remains

the to

uterine be

virus-like

particles,

AND

ACKNOWLEDGMENTS with of Ridge Grant

gratitude, Drs. Wen National #5

ROl

JR.

Hellman,

but

demonstrated.

The authors wish to acknowledge the help, criticism and encouragement Yang and Richard Tyndall of Oak Laboratory. This work was supported by NIH HD 06226.

DANIEL,

A.

(1977).

RNA

tumor

virus

expression

in mouse uterine tissue during pregnancy. Biol. Reprod. 16, 344-348. Fridlender, B., Fry, M., Bolder, A. and Weissbach, A. (1972). A new synthetic RNA-dependent DNA polymerase from human tissue culture cells. Proc. Nat. Acad. Sci. 69, 452-455. Hafez, E. S. E. (1971). Some maternal factors affecting physiochemical properties of blastocysts. In.: The Biology of the Blastocyst. (R. J. Blandau, ed.), The Univ. of Chicago Press, Chicago, pp. 139-191.

Hammond,

J. (1928). Die Kontrolle der Fruchtbarkeit Zuechtungskunde 3, 523. Hammond, J. (1952). Marshall’s Physiology of Reproduction Vol. II, 648, Longnaur, London. Hilliard, J. and Eaton, L. W., Jr. (1971). Estradiol-173, progesterone and 20o-hydroxypregn-4-en-3-one in rabbit ovarian venous plasma. II. From mating bei Tieren.

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