Expression of human pregnancy specific β1 glycoprotein (PSG) genes during placental development

Vol. 166, No. 3, 1990 February 14, 1990

Expression

BIOCHEMICAL

of inmsn

Received

RESEARCH COMMUNICATIONS Pages 1265-1273

pregnancy specific p glycoprotein placental deve Ifopmentl

Catherine Streydio Institut

AND BIOPHYSICAL

and Gilbert

(PSG)

genes

during

Vassar-t*

de Recherche Interdisciplinaire and *Service de Genetique Medicale, Universite Libre de Bruxelles, 808 route de Lennik, B-1070 Brussels, Belgium December

14,

1989

Using gene-specific oligonucleotide probes, the expression of four pregnancy specific B glycoprotein (PSG) genes termed A, B, C-D and E (Streydio et al., Ed8 and in press) and of some of their splice variants Ci, C and D were analysed during human placental development. Except for a stronger hybridization signal obtained at 9 weeks of gestation, which might be correlated to the development of the placenta, the relative amounts of the different PSGmRNAsshowed little variation throughout pregnancy as revealed by Northern blots performed at 6, 13, 18 and 40 weeks of gestation. The expression of the different PSGgenes does not seem to be developmentally regulated, in contrast to placental lactogen, used as a control, the expression of which is clearly correlated with the age of gestation. PSG D, A, E transcripts seem equally abundant, while PSGB expression was much lower. Moreover, the proportion of the PSGC-D variants resulting from alternative splicing remained constant during gestation. 01990 Academic Press, Inc.

The humanpregnancy specific major placental

reaching HO-300 pg/ml at term (1).

heterogenous set of proteins,

still

presenting

points and carbohydrate

unknown, it

induce abortion

(PSG) constitutes

product found in the maternal circulation

with concentrations

isoelectric

Bl glycoprotein

different

content

is thought to be essential

the

during pregnancy, It consists of a

molecular weights,

(2). While the function since antisera

of PSG is

against PSG

in pregnant monkeys (3).

Previous molecular cloning studies (4-12) have revealed the existence

of a large multigene family

including

at least 11 genes, which

1 From the new nomenclature for the carcinoembryonic antigen gene family (I.S.O.B.M. XVII meeting), PSG A, C, D and E correspond to PSG 3, PSG lc, PSGla and PSG2, respectively. Abbreviations: PSG, pregnancy specific Bl glycoprotein; CEA, carcinoembryonic antigen; hPL, human placental lactogen; hGH, human growth hormone. 0006-291X/90 1265

$1.50

Copyright 0 I990 by Academic Press. Inc. All rights of reproduction in an!, form reserved.

Vol. 166, No. 3, 1990

constitutes itself

BIOCHEMICAL

AND BIOPHYSICAL

a subgroup of the carcinoembryonic

RESEARCH COMMUNICATIONS

antigen (CEA) gene family,

a memberof the Immunoglobulin supergene family In view of the close structural

similarities

(13). between CEA.sand PSGs,

claims of the presence of PSGs in neoplasms of various origins reevaluated.

However, a PSG transcript

promyelocytic as in testis

leukemia cell

has recently

been found in a human

line (14) and in humanfoetal

(15). Fibroblasts

(2) must be

(16) and normal granulocytes

liver

(10) as well

(17) were also

shown to express PSG genes. The particularity heterogeneity Using specific we report

of the PSGs as compared to the CRA family and multiplicity

of their

short carboxyl

probes corresponding to the different

is the

terminal

ends (12).

car-boxy1 domains of PSG,

here analysis of the expression of four PSGgenes (A, B, C-D and E)

and three splice variants

of the PSGC-D gene, during normal placental

development.

MATERIALS ANDM3THODS Tissue sampling. Humanplacental tissues were obtained from induced abortions carried out at 6. 9 and 13 weeks of gestation and from term deliveries (40 weeks). The placenta of 18 weeks was obtained from a patient who had cardiac problems but presented a normal pregnancy. Placenta tissue of 23 and 31 weeks was collected from a miscarriage and from a premature birthoccurringfrom an alcoholic mother, respectively. All samples were carefully dissected to avoid contamination by non syncytiotrophoblast tissue. Northern blot analysis. Poly(A) mRNAwas extracted by the thiocyanate guanidinium method (18) and purified on oligo(dT) cellulose chromatography (Boebringer). After denaturation using glyoxal (19), 100 ng of poly(A) mRNA or 10 ug of total RNA, with 5 c(g of RNA ladder (BRL) used as molecular weight markers, were separated by electrephoresis on a 1% agarose gel and transferred to a nylon membrane(Pall Biodyne A). The filters were prehybrized during 4 hours at 50"~ in NETx6 (NETxl: NaCl O.l5M, Tris-HCL 15mM ph 7.5, EDTA 1mMph 7.5), SDS 0.5X, Na pyrophosphate 0.05X, Denhardt x 5 bovine serum albumin 100 rig/ml, and (0.1% Ficoll, 0.1% polyvinylpyrr&idone), P labelled 40-mer oligonucleotides for 60 hybridized with the appropriate hours at 50'~. The washes were performed once in NETx6 at room temperature and three times for 30 min. at 55-c, 6O'c, and 65"~ in NETxl. Between each washing step, filters were exposed to X-ray film at -70-c, using an intensifying screen. The fact that the oligonucleotide probes could distinguish the different PSG-specific mHNAswas checked by hybridisation to dot blots of the corresponding cDNAclones. Because of the high sequence homology between human growth hormone (hGH) and humanplacental lactogen WIJ), a 800 bp fragment of the hGH cDNA (20) was used to 3!iobe the hPL The hGH probe labelled with [a P] dATP expression used as control. (Amersham, 800 Ci/mmole) by the random priming procedure (21) was hybridised to the Northern blots at 30'~ during 60 hours in formamide 50X, Denhardt x 5, SSC x 5, phosphate buffer 5omMph 6.5, SDS O.l%, salmon sperm DNA lOOug/ml. 1266

BIOCHEMICAL

Vol. 166, No. 3, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

The washes were performed at 55'~ in SSCx 2, SDS 0.1% and the filters exposed to X-ray film.

were

Oligonucleotide labeling. The various 40-m oligonucleotide probes (Applied Biosystem 381 A) were end-labelled with r/ SEP] dATP (Amersham, 3000 Ci/mmole) by T po ynucleotide kinase (22) to achieve a similar specific activity of O.B-4x10' dpm/ug. Measurement of PSGand hPL. Except for the placenta of 9 weeks for which the corresponding mother serum sample was taken at 6 weeks, the PSGand hPL concentrations were determinated in serum samples collected the day before medical intervention. Determination of PSG and hPL concentrations was performed with commercial radioimmunoassays kits: RIA-GNOSTSPl (Berhing) and AMKRLEXRPL IRMAKIT (Amersham) respectively, according to the manufacturer's specification. RESULTS In order to discriminate

between the different

a high degree of sequence similarity have defined specific

PSGmRNAspresenting

(>90% at the nucleic

acid level),

we

probes corresponding to segments of the carboxyl

terminal

domains (Table 1). These have been demonstrated to display the

greatest

variability

between the individual

stepwise washes to a final

PSGmolecular species (12). After

temperature of 65'~ in NKTxl (see Material

Methods), the probes revealed one, two or three transcripts legend) with the following and

size:

(fig.

and

1 and

A: 2.05 Kb; B: 2.3 and 2.0 Kb; Ci: 2.5, 2.0

Kb; C: 2.5, 2.0 and (2.2) Kb; D: 2.3 and 2.0 Kb; E: 1.8 Kb.

(2.35)

For the same amount of mR.NAloaded per lane (lOOng), the different hybridization

signals showed little

variation

PSG

when samples from 6, 13, 18 and

Table 1. Regionsof mRNAs corresponding to the different gene and splice variant specific oligonucleotide probes Genes

Splice variants

sequence5'- 3'

position

ref.

PSG A

TTCCTCAAAGTATTTACCATCAGCTACAGTCCAMATTGC

1344-1383

12

PSG B

GGTCCCTGCCATGGAGACCTACAGAGTClCAG'lCATGAC

1243-1282

12

Ci

GGTAAGTGGATCCCAGCATCGTTGGCAATAGGGTTTTAGG

1243-1282

12

C

1250-1289 CCAGTTCAATAAACTATACT!lWWGAACCGTAAl'lGAAA

5

D

ACTGGACAGTTCCCTGMTTCTACTAGTACCTCCAATTCC

1244-1283

5

-

TTCTACAAGAATAGGACTTCTTCCCTCTCCTTAAWCAACA

9661005

5

PSG C-D

PSG B

1267

Vol.

166, No. 3, 1990

BIOCHEMICAL

6

9

13

18 23’31’

AND BIOPHYSICAL

40

6

9

13

la

RESEARCH COMMUNICATIONS

23’31.

40 weeks

2.4 PSG A

PSG a 1.4 hPL

hPL

6

9

13

18

23.31’

40

6

9

13

18 23’31”

40

PSG 0

PSG C

hPL

hPL

Fig. 1. PSG mBNA (upper part of each panel) and hPL d?NA (lower part of each panel) levels during normal gestational development at 6, 9, 13, 18 and 46 weeks of human placenta. Two ssmples from pathological conditions are indicated by * (see Material and Methods). The letters correspond to gene-specific probes: PSG A, B, B and to the splice variants of the PSG C-D gene: Ci, C, D. The Northern blots, shown here, correspond to washes performed at 55-c in Ssme results were obtained at 65-c, except NBTxl, except for PSG A (60'~). that hybridization with the 2.35 Eb mBNA, revealed by the Ci probe was unstable at this temperature. The autoradiographs were exposed 4 h. (PSG A, B and II), 16 h. (PSG B) and 46 h. (PSG C and Ci), respectively. The sample from the 6 week placenta hybridized to probe PSG C (omitted from the Northern blot shown on pannel PSG C) displayed a pattern identical to those observed at results were obtained when other times of gestation (not shown). Identical the same blots were rehybridized with different oligonucleotide probes.

40

weeks

of gestation

systematically the

were

observed

hybridization

gestation

(Fig.

corresponding (except

for

in the

signal

of human placental

(Table

2).

However,

of 9 weeks

stronger

they

of 9 weeks) were

in the

found

normal

1268

shown range

For the

with

in sera medical

on Table for

1).

signal

In contrast,

steadily

the day before are

a stronger

(fig

at term.

mENA increased

and collected

placenta

at 6 weeks,

(hPL)

1).

PSG and hPL concentrations

mothers

sample

(fig.

placenta

was also

lactogen 1).

the

compared

the

PSG E,

the the from

was

amount age of

the

intervention 2. Except

for

the

age of pregnancy

Vol.

166, No. 3, 1990

BIOCHEMICAL Table

PSG

PlO - P90

Serum sampling

hPL

interval

PlO - F90 interval

w/ml

6

6

0.3

1.2 -

4.0

nd.

9

6

1.4

1.2 -

4.0

nd.

13

13

13

10.8 -

35.7

<0.6

0.5 -

1.1

18

18

44

17.7 -

58.3

2.0

1.1 -

2.3

23

23

6

39.9 - 132

3.2

2.0 -

4.2

31

31

175

84.1 - 278

3.3

4.2 -

8.6

40

40

130

- 442

7.0

4.9 - 10

PSG and hPL concentrations PI0 - P90 intervals.

pathological genes:

case

the expression transcript

of all

level

expression collected

seemed

hybridization

an alcoholic

signal

which

other

samples.

transcripts

data

was obtained in

hybridization

signals to X-ray

2).

the

the

smoothly

found

of the using

film

obtained at 7O'c,

for

In the

in agreement 2).

In contrast,

placenta

which

of 31 weeks,

was normal presented

or

a weaker

pattern

observed

with serum amounts similar

the

levels (Table

of the specific

and Methods):

PSG D, A and E probes an intensifying

1269

with

increasing

(Material

using

PSG E the

hybridization

with

the

(23 weeks),

weaker

relative

the same conditions

cervix

a slightly

patient's

probes

the PSG and hPL

the

PSG expression

correlated

in the

is

from

of

for

(Table

PSG E species

are well

estimation

radiolabelled

exposure

with

proteins, A gross

1, Table

gave

collected

of the

This

serum

the

were

except

mother's

The hPL probe,

constrasts These

for

opening

slightly.

pregnancy,

except

signal.

with

(Fig

the corresponding

expression

was diminished,

in the

with

which

differential

to increase

of hPL was normal

increased,

corresponding

PSG genes

of PSG found

from

slightly

revealed

of the miscarriage

of which

low

sera are indicated

of 23 and 31 weeks

pregnancies,

in the

134

in patient

The two placentae

the

RESEARCH COMMUNICATIONS

2. PSG and hPL concentrations

Age of gestation Tissue sampling

AND BIOPHYSICAL

screen

for

the

of the 2). different

PSG

activity, figure

1 shows

after

4 hours

(Fig.

1,

left);

of

Vol.

166, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

exposure of 16 and 46 hours in the same conditions the PSGB and the PSGCi or C hybrids,

RESEARCH COMMUNICATIONS

were necessary to observe

respectively

(Fig 1).

DISCUSSION The PSGfamily,

a subgroup of the CEA family,

11 genes coding for highly organisation

similar

composed of one variable

immunoglobulin-like the diversity

domains (4-12).

of the small carboxyl

the remarkable conservation advantage of the variability specific

proteins

consists of at least

presenting a general structural

(v) and two or three constant (c) A

peculiar

feature

of the PSGsubgroup is

domains of the proteins,

in contrast

to

of the rest of the sequences (12). Taking in the carboxyl

regions of PSGto define gene-

probes (Table 1), we have shown in this study that the PSG termed A,

B, C-D and E (5,12) were expressed constantly 18 and 40 weeks of gestation, same amount of hybridization

mRNA

throughout pregnancy: at 6, 13,

the hybridization

loaded on the gels (Fig.

signals were similar

for the

1). However, stronger

signals were obtained with DNA from the placenta of 9 weeks:

each qRNA studied was found to be more abundant at this stage as compared to the other times of gestation

(6, 13, 18, 40 weeks)(Fig.

were obtained with 10 pg of total seems that this result

1). Similar

DNA on a separate blot

(data not shown). It

could be due to a sampling problem related

development of the placenta.

The stronger hybridization

explained by a higher proportion

of syncytiotrophoblast

results

to the

signals could be cells

at this stage.

Our data demonstrating constant expression of the PSG genes during placental

development disagree with the conclusion of J.L.

S.C. Niemann (24) who observed increasing

levels

gestation.

in the intensity

While there is some variations

hybridization is likely

signals observed between

that these are related

variability

(l),

The pattern

of hybridization

control,

Bocco (23) and

of PSG expression during

the different

of the

placentae (Fig.

l),

to the well known inter-individual

rather than to a developmental regulation of the human placental

of expression.

lactogen qRNA,

used as a

demonstrated the expected increase with the age of gestation

Thus, in contrast

it

to the developmentally 1270

(25).

regulated expression of hPL, PSG

vol.

No. 3, 1990

166,

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

premessenger PS

NH2

messengers

Ci C

I

0’

rlFig. 2. Schematic representation of the structure of the 5 splice variants of the PSG C-D gene. The hypothetical structure of the premessenger is based on the structure of the genamic clone CM35 of Oikawa et al., (8): exon (D), intron (X:), polyadenylation site (*), specific probes (-). The FL-RCA-l has been described by Khan et al., (10). expression,

the

during

pregnancy.

of the

two genes

where

opposite

(Fig.

1).

stringent the

function

of which

The existence can also changes

in the

In spite

of the

washing

conditions,

been latter

correspond

demonstrated transcript

size

two

identical

difference

polyadenylation while

the

(10).

Both

2.0

of the

two pathological

of the different mRNA species

AI domain

On the

them could the

result

2.5

(279

nucl.)

other

from

pregnancies,

probes have

the

and 2.0 Kb, correspond

FL-NCA-1 the

apply

revealed

with

of 2.3

and 2.0

(Fig.

2).

It

out

from

the

to the

In this

to a differential correspond

of

of

revealed case,

use of

to the

cDNA reported

has

detection

PSG Ci and C probes

respectively.

transcription

1271

spliced

observed

and the

been

variants

is

could

hand,

Kb mRNA could

be the

expression

of PSG and hPL mRRAs have been

PSG II and D' cDNA, respectively

Kb mRNA could

mRNAs would

the

The two PSG D splice

mRNAs of 2.5

between sites:

levels

seems to be constant

regulation

The same explanation

two PSG B RNA messengers. mainly

from

several

the

(12).

unknown,

of a different

specificity

to the that

still

be inferred

PSG B, C-i, C and D probes.

Kb could

is

PSG Ci cDNA

by Khan et al., the same gene

but

the

Vol.

166, No. 3, 1990

would use different

BIOCHEMICAL

polyadenylation

PSG C splice variant,

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

sites 505 bp from each other (Fig 2). The

having spliced-out

the Ci intron

(355 nucl.)

is very

low in abundance, as has been observed by Zimmermannet a1.,(26). perhaps correspond to the discrete probe reveals to a lesser extent

It could

band at 2.2Kb (Fig 1). The corresponding the major 2.0 Kb mRNAin contrast

to the Ci

probe (Fig 1). This is perhaps because its sequence overlaps the first polyadenylation

site of the PSG C-D premessenger (Fig 2). However, we cannot

exclude that the various mRNAsrevealed by a given probe in this study could correspond to closely

related,

still

Using probes presenting

unknown genes.

similar

specific

activities,

the PSGD, A and B mRNA seem to be the major transcripts

we found that

as compared to the

PSGB mRNA. Our study demonstrates also that throughout placenta,

the third

preferentially This result a Agtll

polyadenylation

site

of the PSGC-D premRNAis

used leading to the excision agrees with the representation

library

of term placenta

the development of the

of "intron"

Ci and exon C.

of cDNAclones as observed in

(5) where 13 D, one Ci and one C clones had

been obtained using a 5' PSG D probe. Although the alternative is not developmentally

regulated,

have been detected in foetal In conclusion, the placental preferential

liver

it could be tissue specific

splicing

since PSGmRNA

(data not shown and (10)) and testis

from the onset of pregnancy,

tissue accumulates PSG transcripts

(15).

(6 weeks in this study),

in a constant way with

expression of PSGD, A and E genes. Future studies will

needed to confirm whether PSGE could be regulated suggested by its behaviour in two pathological

step

in a specific

be

way as

conditions.

We are grateful to Dr. 5.13. Dumont for continual support and interest. We greatly thank Dr. F. Rodesch for providing the placental tissues, Dr. C. Gervy for her help with the PSG and BPL radioinmrunoassays and Ch. Christophe for the synthesis of oligonucleotides. This work was supported by the Oeuvre Belge du Cancer, the Minister-e de la Politique Scientifique (Sciences de la Vie), the Fund for Scientific Medical Research (FRSM, Belgium), the CGRRand ARBDasbl. 1272

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