BIOLOGY

OF

43,

REPRODUCTION

Expression

210-218

(1990)

of Trophoblastic

Genes

in Sheep

1. R HANSEN,4 J. J. MCDONNELL,5 P. W. FARIN,5 and R M. ROBERTS2’4

C. E. FARIN,4

Department

Interferon

K. IMAKAWA,3’4

of Animal Sciences4 Department Universijy of M&ouri-Columbia,

and Cattle1

C. N. MURPHY,5

of Veterinary Medicine Columbia, Missouri 65211

& Surgery5

ABSTRACT The

trophoblastic

as mediators duration

of gene

135SJ-labeled translated

1 mRNA

at Day

13.

to Day of this

mRNA

a coding from

Days

low

on Day

together,

pregnancy

in this

was Days

Day

may

initiation

noted 15,

19,

that

21,

Maternal been

conceptus luteolysis

and cattle which the

must be resident within the and establish pregnancy (Short,

to prevent The major

secretory

maternal

products

recognition

13 and

21 of gestation 15 and

and have responses extension al., 1984b; changes

et al.,

initiation and

(Godkin

(Bartol

are

protein

synthesis

Salamonsen

1982)

and

March

Received

January

‘Research doctoral

of maternal that include (Godkin et et a!., 1989),

(Godkin

et al.,

1988;

protein-encoding

some

requests:

of Missouri, 3Present

of Medicine,

address: Wichita,

Dr.

NIH

Grant This

RM.

Columbia, The

with

blotting

of RNA

on

all

days

12 of pregnancy,

Day

25.

maternal

The

increase

recognition

of

paper

#HD21896. is #11,114

C.E,F.

supported

from

the

by

Missouri

NIH

Roberts, MO

Women’s

158

Animal

Science

Research

infusion Thatcher

et

Post-

Uni-

65211. Research

Institute,

The

University

of Kansas

regions

and

bTP-1 with

exhibit bovine

approximately

physiological

studies 166-amino administration actions

of

of the

interestrous

in nonpregnant et al., 1989)

gel

by

(Plante (Stewart

ovine

or

bTP-1

in-

specifIc ovine endoet a!., 1988) and

interval

cows and ewes

of oTP-i

oTP-1

acid IFNa1 of IFNa1 can

after et

conceptuses

electrophoresis,

intrauterine

al., 1988, et al., 1989).

1989;

as detected

begins

at approx-

imately Day 13 of pregnancy (Godkin et al., 1982). By using a more sensitive radioimmunoassay, however, Ashworth and Bazer (1989) reported that oTP-1 is produced as early as Days 8 and 10 of gestation. Similarly, although ma-

Agricultural Center,

IFNa1 subclass. oTP-i and identity in cDNA sequence

introduction of synthesis of proteins in culture (Salarnonsen

prolongation

et a!., Sharif

IFNct11 in their

cluding metrial

1990. by

and

acid 85%

by two-dimensional

#HD07217.

detected

detected

as Day

at least

through

blastocyst

prior induction

in all conceptuses

by Northern as early

continuing

mRNA,

was

probe

conceptuses

present

mRNA

the 172-amino approximately

mimic

Station.

2Reprint versity

12,

confirmed

longer

13, massive

bTP-i

ilar to those of the extensively subclass proteins. Furthermore,

1985)

1990.

supported

Fellowship

Experiment

16,

to Day

markedly

the

ovine

seven

and 3’-unexamined,

increased

13 when

and

bTP-1

Production Accepted

of the prior

of the

and

70% sequence identity in their 3 ‘-untranslated regions (Imakawa et al., 1987, 1989; Stewart et a!., 1987). These trophoblast proteins also possess potent antiviral and antiproliferative activities (Roberts et a!., 1989) which are sim-

and

et al.,

of the corpus luteum et a!., 1986a; Thatcher

1987;

of

cattle

et al.,

in cattle

in triggering a series of the conceptus

of the lifespan Knickerbocker

Vallet

for

in sheep

in sheep

in endometrial

1984a;

expression to Day

in conceptuses

elongation

and

al., 1989), and inhibition of prostaglandin F2,. production by the uterus (Fincher et a!., 1986; Knickerbocker et al., 1986b; Salamonsen et a!., 1988). In addition, oTP-1 and bTP1 have been identified as interferons (IFNs) belonging to

protein-i (oTP-1) and bovine respectively. These proteins of conceptuses between Days

24 of gestation

been implicated to the presence

uterus 1969).

responsible

of pregnancy

known as ovine trophoblast trophoblast protein-I (bTP-i), are major secretory products Days

studies,

of pregnancy. were

is present

with

mRNA.

trophectoderm

results

onset

hybridization

both coding At all stages

representing

one

of gestation.

implicated

the

species.

recognition of pregnancy in sheep defined as the critical period during

conceptus

25

of pregnancy

coincident

INTRODUCTION has

and

been

to describe

by in situ

0TP-l

conceptuses

to the

These

23, mRNA

bTP-l

15/16

occurs

15/16.

=

prior

recognition

confined

have

was

10, 11, 12, 13, and 15 of gestation (n 2, 4, 5), were hybridized to specific

earlier

in only

in ovine

of maternal was

at Day

17,

on Day

15/16

be present

on Days 19 (n

conceptuses

detected

study

conceptuses

bovine

with

respectively)

of this

442-9 18 and used to detect

ovine

was

mRNA

236-913),

occuring

on

mRNA

the

demonstrate

data

in expression

increase

of bTP-1

1 mRNA

(bases

on

these

Consistent

of seven

oTP-1

with

probe

conceptuses

conceptuses. in seven

and

collected 15/16, and bases

bTP-1,

and

objective

ovine

were

levels

of oTP-

The

one encompassing (bases 650-912)

however,

expression

Maximal

Taken

a marked

amounts

region

bovine

in expression

probe,

(oTP-1

cattle.

ovine conceptuses, on Days 12/13,

of ovine

at low

and

preimplantation

1 in

probes, probe

13 coincident

3’-noncoding

12/13-19.

examined.

detected

protein-i

in sheep

bTP-

different 3’-speciflc

3’-speciflc

although

from

1 and

to trophectoderm was

the

occurs

plus

extracted with

bovine

With

trophoblast

bovine

from paraffin-embedded conceptuses, collected

confined

13. Thus,

0TP-

probes. Two and a second

oTP- 1 mRNA

employed.

was

and

was

and

of pregnancy

for

Sections

cDNA regions,

ovine

recognition

expression

Northern analysis. = 1, 3, 3, 2, 2),

oTP-

interferons

of maternal

jor

School

production

gestation

KS 67214-4716.

210

(Barto!

of

bTP-1

et a!.,

appears 1985),

to begin bTP-1

has

at Days been

15-17

reported

of to

EXPRESSION

be produced as ear!y activity in media from (Betteridge Analysis ing the

12 on the of cultured

et a!., 1987). of changes in oTP-1

period

blot and increase

of maternal

Northern in oTP-1

pregnancy

et al., method

changes

basis of antivira! bovine embryos

messenger

RNA

recognition

by a decline

between

mRNA

16 and

single ization

concentrations,

good

agree-

changes

in bTP-1

The objectives lyze the onset more precise!y

mRNA

MATERIALS

AND

of

ceptuses

conceptuses

the uterus described

were

on

Days

2), and

=

15 (n

Day 0). Within 15 mm immobilized in blocks of (Fisher Scientific, St. Louis, phate-buffered

saline

localization the period

collected

by

whiteface Murphy

ewes (1987).

10 (n

and of

ice

1004760, cDNA

from

were agarose M phosin

2 h. Conceptuses

4% were

Messenger RNA (Farm et al., Prime Labeling Indianapolis,

either

3),

=

(estrus

immersion-fixed for

Boehringer-Mannheim, probes

using Con-

1), ii (n

=

washed in PBS and embedded in paraffin. for oTP-1 was detected by in situ hybridization 1989) by using random-primed (Random [35S]-labeled

surgical

2) of pregnancy

=

and

on

dark-field

a coding

IN),

plus

3’-un-

illumination

resent each bridization value

conceptus in the signal represents

(optical

density)

included

within

of tissue

(i.e.

the

toderm was hybridization tion

were signals

Experiment

650-912 of the oTP-1 cDNA; sp. act.: 1.2 X io Farm et al., 1989). For each conceptus, adjacent were hybridized (50% formamide, 0.6 M sodium 42#{176}C) with pjg; positive (sp.

act.:

dpm/p.g; sections chloride,

either [35S]-y-actin cDNA (sp. act.: 9.7 X 108 control) or [35S1-pBS M13 plasmid (vector) 1.1

X i09

dpm/tg;

negative

control)

pair mRNA bases

probes.

of all pixels area

of each

yolk

sac,

type

embry-

with

conceptuses oTP- 1 and

by subtraction with

the specific actin cDNA

of the

the negative

control

hybridiza-

(vector

DNA)

II

Holstein

heifers

were

synchronized

Kalamazoo, Eleven

Day

=

0). The

flush

by

MI) bovine flush 19 (n

medium

injection

and bred conceptuses on =

of Lu-

at estrus were

by ob-

Day 12/13 (n 5) of pregnancy

used

was

=

Modified

2), (esDul-

becco’s PBS supplemented with pennicilin and streptomycm (Seidel et a!., 1980). Conceptuses were recovered within 3 h of flushing and immersion-fixed in ice-cold 4% paraformaldehyde ceptuses for bTP-1 amide, beled lated

in PBS were was

0.6

for

M sodium (bases

chloride, 236-913)

PstI/SspI region

2 h. After

washing

in PBS,

con-

then embedded in paraffin. Messenger detected by in situ hybridization (50%

bTP-1

cDNA

RNA form-

42#{176}C)by using a [35S]-lacoding plus 3’-untrans-

probe

(sp.

act.:

1.2

X

iO

dpm/

rig; Day 13-19 conceptuses) or the [35S1-0TP-266 cDNA probe described in Experiment I (Day 12 conceptus). The oTP266 cDNA probe was derived from the unique 3’-untranslated region of the oTP-1 mRNA that shows >92% identity with awa

the

corresponding

et al., 1989)

control

region

and

tion

a 266-base of oTP-1 including

for the hygray-scale

probe.

from the 3’-untranslated a BglII/SspI restriction

or

image

Nashville, used to rep-

on measurement endoderm,

determined associated

sp.

region fragment

based on measured

value pixel

cross-sectional

4.21%. For individual signals associated

1 cDNA;

dpm/g)

based entire

after hy-

onic disc) present in each randomly selected section. Measurements on all conceptus sections were done at the same magnification (12.5 X). The coefficient of variation for repeated measures (n = 4) of a standard section of trophec-

fragment (oTP-266,

X i09

of

video

analysis. The the average

trophectoderm,

published results). (actmn cDNA) and

1.2

by computerized

analysis (Bioquant System IV, R&M Biometrics, TN). A single, randomly selected section was

translated region fragment of oW-i mRNA (oTP-560, a PstI/ RstI restriction fragment including bases 442-918 of the 0TPact.:

by autoradiography relative intensity

as optical density silver grains, was

tained by nonsurgical uterine Day 15/16 (n = 4), and Day

of recovery, conceptuses 2% (w/v) low-melting MO) dissolved in 0.01

(PBS)

paraforma!dehyde

Kit, No.

under

probes

METHODS

of crossbed by Smith and

collected

3), 13 (n

signals were detected of exposure at 4#{176}C. The

talyse (Upjohn Co., artificial insemination.

were

=

(w/v)

the during

211

GENES

bridization signals, quantified the reflectance of hybridized

trus

ovine

flushing techniques =

in conceptuses.

studies were, first, to anaproduction, characterizing of trophoblastic IFN gene

I

Eleven

12 (n

by in situ hybridbeen reported re-

levels

of the present of oTP-i mRNA the initiation

expression, and, second, to describe pattern of bTP-i mRNA production maternal recognition of pregnancy.

Experiment

on Day 23 (Farm

however, oTP-1 mRNA was not deon Day 12 of gestation by dotet al., 1988) but was detected in a

conceptus on Day 11 of gestation (Farm et a!., 1989). No data have

garding

22

When the more was used to ana-

ment was found in that mRNA increased markedly 13 of gestation and dec!ined gradual!y through Day et a!., 1989). In contrast, tected in six conceptuses blot analysis (Hansen

durby dot-

Days

INTERFERON

bridization 10 days

demonstrated a marked on Day 13 or Day 14 of

1988; Stewart et a!., 1989). of in situ hybridization in oTP-i

levels

of pregnancy

techniques have mRNA beginning

followed

(Hansen sensitive lyze

as Day groups

TROPHOBLSTIC

OF

sections

of the

hybridizes

bTP-1

to bTP-1

mRNA

mRNA

In addition, for each conceptus, negative (vector DNA) in situ were

included.

Specificity

(Imak-

(Farm,

un-

positive hybridizaof the

bTP-

1 cDNA, and vector DNA probes was confirmed by Northern hybridization analysis with total RNA from Day 19 bovine conceptuses (Fig. 1) according to procedures described by Farm et al. (1989). were detected and quantified individual

conceptuses

dpm/ DNA

bridization

by

Hy-

bridization

of the

In situ hybridization signals as in Experiment I. Data from

were

subtraction negative

corrected of

signals

control

for

background

associated (vector

DNA)

with probe.

hyhy-

212

FARIN

ET AL.

at 20#{176}C. RNA was collected 20 mm, 4#{176}C) and resuspended

by centrifugation in 1-2 ml GTC

-

2

3

ter addition were extracted saturated)

of sodium sequentially

and

(24:1).

acetate with

0.2 volumes

Samples

were

(pH 4) 1 volume (10000

sedimented,

vacuum-dried,

Five

micrograms for

each

Bio-Rad

1590.

RNA

day

pregnancy

of

830-

Richmond,

Blots restriction

(w/v)

pooled for

(Imakawa

et al.,

lated,

poly(A)

and

1 and umn

actin and

1989),

which

regions

presence FIG. 1. Northern blot analysis of Day 19 bovine conceptus total RNA (2 g/lane) demonstrating specificity of hybridization for the bTP-1 cDNA probe (Lane 1; bTP-1 mRNA approximately 1 kilobase (lmakawa at al., 19891). -y-actin cDNA probe (Lane 2; actin mRNA approximately 2 kilobase (Gunning et al., 19831), and lack of hybridization with the vector (negative) control DNA probe (Lane 3). Estimated base pair lengths, based on EcoRl-Hindllldigested lambda DNA, are indicated on the left margin. All cDNA probes used in this analysis were (32P)-labeled by random prime procedures (Farm et al., 1989).

cDNA were

from

beef

detection 3), 19 (n reproductive

=

cows

were

artificially

of estrus

(Day

0).

3), 21 (n = tracts were

On

inseminated Days

15 (n

12 h =

1),

17

3), and 25 (n = 3) of gestation, removed at slaughter, placed

standards were 1982) and used

of total RNA to electro(Zetaprobe,

purified

end-labeled to determine

3’-untrans-

cDNA.

In addition,

on

(Maniatis

protocols

outlined

were used The conditions

triphosphate 108

by the

colin the

(dCTP) and 1.0 lambda

with [32P]-ATP (Maniatis the size of message.

to

x

108

DNA et al., In ad-

(Maniatis (Stratagene synthesized

manufacturer.

as a positive control for hybridization

et al., gene’ bTP-

a Nensorb

et al., 1982) x

PstI/ cDNA

coding,

‘y-actin cDNA (Gunning to be a ‘housekeeping to assess mRNA integrity.

were

blot

hybridization

dition, the bTP-i cDNA insert was subcloned al., 1982) into a pBS M13 transcription vector Inc., San Diego, CA) and cRNA transcripts were

et

cRNA

in the Northern and autoradiog-

raphy were as described previously (Hansen et al., 1988). Following autogradiography of the bTP-1 hybridization, nylon membranes were washed in 0.15 M sodium chloride! 0.015 M sodium citrate, 0.5% (v/v) SDS at 95#{176}C for 20 mm to remove bound bTP-i cDNA probe. These membranes

on ice and returned to the laboratory where conceptuses were recovered by uterine flushing (Bartol et al., 1985; Hel-

were

mer et a!., 1987). Total RNA was isolated from these conceptuses by using a modification of the methods described by Chirgwmn et al. (1979) and Chomczynski and Sacchi (1987).

ovine

Briefly, individual conceptuses were homogenized in 4 M guanidine thlocyanate, 25 mM potassium citrate (pH 7), 0.5% (v/v) sarcosyl, and 0.1 M 2-mercaptoethanol (GTC) and precipitated in the presence of 25 mM acetic acid and ethanol

bTP-i

activities of approximately 0.5 respectively. HindIII/EcoRI-digested

transcripts analysis.

III

inserts

each

Northern

for

included of the

nick-translated

and

of total RNA from the sin15, only 4 tg total RNA was

of [a-32P]-deoxycytididmne

specific cpm/ig,

by standard

Crossbred

CA)

SDS

determined

were hybridized with a nick-translated, fragment (bases 241-1035) of bTP-i

blots were hybridized with 1983). Actin was considered whose expression was used

=

were

4#{176}C)

with 1 x g, 10 in 0.3

1 volume of 80% ethanol,

were

of total

Laboratories,

analyzed.

(n the

in 0.5%

concentrations

analysis. Because of low recovery gle conceptus collected on Day

EcoRI

after

with with

analysis. Thereafter, 7-p.g samples of each pool from Days 17, 19, 21, 23, and 25 were subjected phoresis and transferred to nylon membranes

1980..

Experiment

again washed

dissolved

stored at -80#{176}C. RNA spectrophotometrically. conceptus

alcohol

X g, 20 mm,

from the aqueous phase After centrifugation (10000 RNA pellets were dissolved

volumes of GTC and precipitated isopropanol. The RNA pellets were

g, Af-

X

to 0.2 M, proteins of phenol (water-

of chloroform-isoamyl

centrifuged

and RNA was precipitated volume of isopropanol. mm, 4#{176}C) the resulting

(5000 solution.

subsequently

FIG.

2.

rehybridized

Experiment

with

the -y-actin

cDNA

probe.

I. In situ localization of 0TP-1 and actin mRNA5 in Day 10 (A-El and Day 13 (F-J) of pregnancy. (A and F) Bright-field micrograph of Day 10 and Day 13 ovine conceptuses, respectively; trophectoderm (closed arrow), and endoderm (open arrow). (B and G) Hybridization with IS]--y-actmn cDNA (dark-field optics). (C and H) Hybridizatitin with (Sj-oTP-560 cDNA. (D and I) Hybridization with 135S10TP-266 cDNA. (E and J) Hybridization with (Sl-vector DNA. conceptuses

from

EXPRESSION

OF

TROPHOBIASTIC

INTERFERON

GENES

213

214

FARIN

Statistical

Analyses

For

ET AL. significant

Experiments

I and

II,

after

correction

for

back-

actin

change

mRNA

ground hybridization levels, changes in relative hybridization signals on different days of pregnancy were analyzed

Experiment

by one-way analysis of variance within each probe a significant F-statistic was encountered, means arated by Least Significant Difference (Snedecor

tuses examined RNA for bTP-1

ran,

type. When were sepand Coch-

1967).

RESULTS Experiment

I

Consistent

for trophoblastic trophectoderm

mRNA

in the was

previous

not

present

in either

endoderm

(Fig. probe

tion

one

in only

localized exclusively conceptuses and (Fig.

2) or embryonic

2c, Fig. 3). In contrast, use of resulted in a positive hybridiza-

of three

Day

and in none of the conceptuses 2d, Fig. 3). Thus, prior to Day contained

et al., 1989),

Trophoblastic interferon mRNA was in all seven conceptuses between Days when the oTP-560 cDNA probe was

used for hybridization the oTP-266 cDNA

ceptuses

(Farin

interferon was of the developing

disc (data not shown). detected at low levels 10 and 12 of gestation

signal

observations

11 ovine

from Days 13, only one

detectable

amounts

conceptuses

of oTP-1

mRNA

as

assessed by use of a specific cDNA probe that recognizes the unique 3’-noncoding region of the oTP-1 mRNA. An in-

(p

crease both Day

0.01)




2.8,

re-

did

not

AC11N

III

Experiment The

bTP-1

and

‘y-actin

analysis hybridized approximately 1.1 bases

for

actin

transcripts than the 10

11

12

13

sult since subcloned

15

EMBRYO AGE (days)

The

FIG. 3. Changes in relative hybridization Signals (silver grain reflectance; Mean ± SE) for 0TP-1 and actin mRNAs in ovine conceptuses from Day 10 to Day 15 of pregnancy. Changes in 0TP-1 mRNA was assessed by using both the 0TP-560 and 0TP-266 cDNA probes (see text for details). All data

corrected

(negative

for

control)

background

DNA.

hybridization

signals

associated

with

vector

loaded 25, gave 1 mRNA

mRNA

(positive full-length only into

Day (4

(Fig.

probes

6a,b).

The

bases 241-1035 the transcription than

a relatively was present

the

used

for

Northern

bands of the expected for bTP-1 mRNA and

control, Fig. bTP-1 mRNA.

15 conceptus p.g)

cDNA

to mRNA kilobases

sample, conceptus

low hybridization at all stages

majority

size of 2 kilo-

of bTP-1

cRNA

6a, Lane C) were shorter This was an expected reof the vector.

bTP-1

which

had

samples

cDNA

less from

were

total

RNA

Days

17-

signal (Fig. 6a). bTPof pregnancy examined

from Day 15 to Day 25 (Fig. 6a). Although cedures failed to remove all of the bTP-1

the washing cDNA probe

profrom

EXPRESSION

FIG. 4. Experiment Bright-field micrograph trophectoderm (closed ization with IS1-oTP-266

the

nylon

it

to detect

gestation

studied

siderably

larger

TROPHOBLASTIC

an

(Fig.

was

possible

actin

signal

6b),

than

the

because bTP-1

in a subsequent at each the

actin

hy-

of the

days

of

mRNA

is con-

mRNA.

From

the

11, and ons, least

15, oW-i

results

presented

herein, during

expression embryonic

12 of pregnancy,

although 8-fold

present, lower than mRNA

215

GENES

was

mRNA

correlated

closely

earlier

(Farm

et

al.,

1989)

of oW-i mRNA occurs with development. On Days 10, for

trophoblastic

expressed,

interfer-

at concentrations 13. From Days although

at 13-

concen-

nancy

also

spherical Although hybridized

Similarly, at the

the

morphological

coincided

et al., 1989). on Day 13

transition

of the

sphere on Days found on Day 13

increased production of bTP-1 mRNA time of maternal recognition of pregwith

to filamentous

a morphological

change

conceptuses conditions

59.07#{176}C), it was

from

form.

both the oTP-560 and oTP-266 to oW-i mRNA, only the former

tent signal with the hybridization oTP-560:

with

(Farm expression

conceptus from a 1-2-mm-diameter to a 3-5-mm tubular form usually

of pregnancy. by conceptuses

was expressed found on Day strongly

trations of mRNA fall shortly thereafter The dramatic increase in oW-i gene ovine 10-12

DISCUSSION

and extended rapid onset

INTERFERON

II. In situ localization of bTP-1 and actin mRNA5 in bovine conceptuses on Day 12 (A-D) and Day 19 (E-L) of pregnancy. (A, E, I) of (A) Day 12 conceptus, (E) Day 19 conceptus trophectoderm (closed arrow) with endoderm (open arrow), and (I) Day 19 conceptus arrow) with yolk sac tissue (open arrow), respectively. (B, F, J) Hybridization with 135S1-y-actin cDNA (dark-field optics). (C) HybridcDNA (92% identity with bTP-1 mRNA), (G, K) Hybridization with lS)-bTP cDNA. (0, H, L) Hybridization with (SJ-vector DNA.

membrane,

bridization

OF

cDNA probes gave a consis-

younger than Day used in the present possible

that

the

13. Under study (Tm

oTP-560

cDNA

a

w 216

FARIN

-

U

ET AL.

could

bTP

be

tion

ACTIN

The

possible cDNA

whereas

theoretically,

between

sequences

probe

(Tm

identity with the et a!., 1989). Under

in this

experiment,

(I)

13

15/18

EMBRYO

the oW-266 probe guishing oW-i and However, it should

19

AGE

(days)

oTP-560

FIG. 5. Changes in the relative hybridization signals for bTP-1 mRNA in trophectoderm of bovine conceptuses between Days 12/13 and 19 of pregnancy (Mean ± SE). All data corrected for background hybridization signals associated with vector (negative control) DNA.

and

only

sequences

could

was

able

IFNa11 class and an 85% identity of the

embryonic

IFNa11s mRNA

(Imakawa sequences

to recognize

other

not just oW-i, of sequence

since there is approximately between the coding regions

interferons

interferons

(oW-i

and

et al., 1989). Under with 75% identity

within

bW-1)

the conditions to the oW-560

the

and

the

used, probe

A

that

pregnancy

B Day $

C 151719212325

1980

#{149}

137

$

Day

of Pregnancy C

of our

1517

than

80%

represented bTP-i) mRNA, it

are

media

comparable

oW-560 oTP-266

probe probe

for each

mRNA

signal

very

consistent as early

an oW-i-like

of

the of the

a positive hybridization with the oW-560 probe, with

study

culture

were

as much

Therefore, visualized

conceptuses

produce

of conceptus

probes

dpm/g), the length

conceptuses

ovine

least 70%

region conditions

of greater

io

up to twice

in

oW-i mRNA. The results

RIA

56.6#{176}C)reprewhich was only about

Thus, because of 0TP-1 (and

cDNA x twice

hybridized. more easily

particularly

vation

(-1

provide

molecule might be

distincalike.

was most likely more specific, distinbW-1 mRNAs from other IFNa mRNAs. be recognized that although both the

oW-266

specific activities was approximately and

oTP-266:

IFNa11 3’-untranslated the hybridization

identity would be hybridized. the highly conserved 3’ end

-J

no 85%

that segment of the oTP-1 mRNA to other IFNa11 mRNAs showing

sequence (Imakawa used

probe

be

oW-266

sented identical

U

distinguished,

should

low with as Days

protein,

amounts the 8 and

as determined

(Ashworth

and

Bazer,

of obser10 of by 1989).

of Pregnancy S

19 212325

-1980 #{149} I.

94

0

560

#{149} -560 p

FIG. 6. Northern blot analyses of bTP-1 mRNA (A) and actin mRNA (B) in bovine conceptuses between Days 15 and 25 of pregnancy. Lane S represents molecular sizing standards; Lane C is bTP-1 cRNA transcripts used as a positive hybridization control (2 ng). In B, the same nylon filter was used as in A, but was probed with y-actin cDNA. Lane C (Panel B) represents hybridization to synthetic y-actin cRNA transcript (2 ng), which was used as a positive control.

OF TROPHOBLASTIC

EXPRESSION

Production

of this

ceptuses

at this

tein produced to 1000-fold

per less

conceptuses,

protein

stage

appears

respectively.

results

since

conceptus per than that produced The

serum used by Ashworth with ovine IFNct11s other The

extremely

of pregnancy

hour

to

of Experiments

REFERENCES

of pro-

which

the

anti-

III demonstrated

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the bovine conceptus, like the ovine conceptus, produced low amounts of hIP-i mRNA at least three days prior to the time of maternal recognition of pregnancy, which is considered

to be Day

15/16

of gestation

(Betteridge

et al., 1978,

1980; Northey and French, 1980). These results are consistent with detection of antiviral activity as early as Day 12 of pregnancy by cultured bovine trophoblast tissues (Betteridge et al., 1988). bovine conceptus

Unlike the produced

ovine bW-i

interval, with message detected nancy in this study. Furthermore, proteins secreted by cultured strated that (i.e. bTP-1) nancy

proteins cross-reactive are secreted at least

(Godkin

this

et al.,

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toderm

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The

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associated

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localized

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25 of preganalysis of demon-

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the

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19 bovine

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trophoblast,

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bW-i

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Gunning

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(Lif-

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Series

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The authors would like to acknowledge Dr. L Kedes (Department of Medicine, Stanford University, Palo Alto, CA) for generously contributing the -y-actin cDNA used in this study. In addition, the authors express their gratitude to the Upjohn Company (Kalamazoo, MI) and CEVA I.aboratories, Inc. (Overland Park, KS) for donating materials

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