Endocrinol Japon 1991,38(6),589-595

Gestational Profiles of Rat Placental Lactogen-Il (rPL-ll) andGrowth Hormone (GH) in Maternal and Fetal Serum,Amniotic fluid, and Placental Tissue

KURAJIRO KISHI, MASAHIRO HIRASHIBA ANDYASUHIKO HASEGAWA

Kanzakigawa Laboratory, Shionogi Research Laboratories,Shionogi & Co., Ltd., Osaka 561, Japon

Abstract. Rat placental lactogen-II (rPL-II) and growth hormone (rGH) in maternal and fetal serum,

amniotic fluid, and placental tissue were measured by a homologous radioimmunoassay during the last

half of pregnancy. rPL-II appeared first in maternal circulation and the placental tissue on day 11 of

pregnancy. The maternal serum rPL-II concentration increased progressively and reached the peak

value (684•}76 ng/ml) on day 19, and declined thereafter up to term. rPL-II content in the tissue had a

similar pattern to the maternal serum profile of rPL-II, while its concentration in the tissue increased

dramatically on day 12 and remained high until day 19. Fetal serum rPL-II was detected on days 17 and

18, though its concentration was much lower (ranged between 3-10ng/ml) than that of maternal serum.

rPL-II in amniotic fluid was also detectable only on days 12-14 of pregnancy, and the peak value on day

13 was 22% of the maternal serum rPL-II concentration. The rGH concentration increased gradually as

pregnancy advanced with. a decline on the day before parturition. Although rGH in fetal serum

increased on day 20 with a decline on the following day, it was slightly detectable in amniotic fluid on the

last two days of pregnancy. The molecular profile of rPL-II in amniotic fluid and maternal serum of day

13 pregnant rats were examined by Western blotting. Anti-rPL-II serum detected two proteins with

molecular weights (mol wt) of 19.5K and 20.5K in amniotic fluid and one protein with a mol wt of 20.5K

in maternal serum under nonreducing conditions. Their migrations coincided with those of purified

rPL-II, which migrated as three components of proteins with mol wts of 19.5K, 20.0K, and 20.5K under

nonreducing conditions. These results indicate that gestational profiles of rPL-II in maternal serum and

the placental tissue were different in their overall pattern and the absolute concentration of rPL-II from

those in fetal serum and amniotic fluid, in which rPL-II consisted of monomeric forms of variants.

Key words: rPL-II, Gestational profile, Maternal and fetal circulation, Amniotic fluid, rGH.(Endocrinol Japon 38: 589-595, 1991)

THE PLACENTA secretes a variety of

polypeptide hormones [ 1, 2]. Placental lactogens(PLs) were identified as prolactin (PRL)-like and

growth hormone (GH)-like hormones from the

placenta and are thought to be involved in theregulation of maternal intermediary metabolism

and fetal growth in addition to other biological

functions [3]. Since PLs are structurally very

similar to PRL and GH, they are postulated tohave PRL-like and GH-like activities in the tissue.

The biological activities of PLs, however, differbetween species; some PLs being functionallymore PRL- than GH-like and others are more GH-

than PRL-like [3].In rodents, PLs were estimated to have PRL-like

activity in the beginning [4, 5]. Recently two formsof PLs, placental lactogen-I (PL-I) and placentallactogen-II (PL-II), have been purified in the rat

[6],the mouse[7,8], and the hamster[9]. Rat(r)

Received: June 25, 1991Accepted: October 18, 1991Correspondence to: Dr. Kurajiro KISHI, Kanzakigawa Labora-tory, Shionogi Research Laboratories, Shionogi & Co., Ltd.,3-1-1 Futaba-cho, Toyonaka, Osaka 561, Japan.

590 KISHI et al.

PL-II is structurally similar to rPRL rather than

rGH [ 10] and is synthesized by the trophoblast

giant cells in the basal zone [ 11, 12, 13] in contactwith the maternal circulation. The gestational

profile of rPL-II in maternal circulation detectedwith a homologous radioimmunoassay (RIA) ischaracterized by a sustained increase as pregnancy

advances [14]. The binding sites for ovine (o) PRLand human (h) PL have been identified in thematernal rat liver [ 15, 16]. Therefore, rPL-II

seems likely to act primarily on maternal sites inthe rat. However, these binding sites for oPRL and

hPL have also been identified in the fetal rat liver

[15,16].Furthermore, oPL stimulates ornithinedecarboxylase activity in rat fetal liver [17]and

amino acid intake in rat fetal diaphragm[18]. It

would be interesting to examine whether rPL-II

plays a role directly in fetal sites.The biological function of rGH in rat fetal

development is poorly understood. In contrast tothe function in tissues of postnatal rats [ 19, 20],

GH is reported to lack biological activity in thefetal rat tissue [ 17, 18], though binding sites forhGH in fetal rat liver membranes have been

identified [15]. Furthermore, hGH, bovine (b) GHand oPL are reported to stimulate production ofIGF-I in adult rat fibroblast [21] and postnatal

hypophysectomized rats in a dose-dependentmanner [20]. Although insulin-like growth factor-

I (IGF-I) stimulates the growth of the fetus [22]and postnatal hypophysectomized rat [23], it is not

clear whether rGH and rPL-II are involved inregulating IGF-I synthesis and release in thematernal and fetal compartment during pregnan-

cy in the rat. It also still remains unknown whetherrGH plays a role in regulating rPL-II secretion

during the last half of pregnancy in the rat,although mouse (m) GH is considered to be a

potent regulator of mPL-II secretion in the mouse

[24].The present study was undertaken to investigate

the distributions of rPL-II and rGH in fetal serum

and amniotic fluid as well as maternal serum,

which would permit further examination of their

physiological role and regulation during the lasthalf of pregnancy in the rat.

Materials and Methods

Animals

Mature virgin (8-week-old) Sprague-Dawley

(Jcl: SD) rats were purchased from CLEA Japan

Inc. (Ishibe, Shiga). The animals were maintained

under controlled conditions of temperature

(23•}1•Ž) and humidity on a 12-h light, 12-h dark

schedule and provided with ordinary laboratory

chow and water ad libitum. Timed pregnancies

were determined by daily vaginal examination of a

female housed with a male. The females with

sperm in the vaginal smear were considered

pregnant and this day was designated day 0 of

pregnancy. The number of pregnant animals

examined was 3 to 14 on days 10-21 of pregnancy.

Sample collectionPregnant rats were bled on days 10-21 of

pregnancy under ether anesthesia. After amnioticfluid was collected, the placentae and fetuses were

removed from the uterus and fetal blood was

collected from the umbilical cord on days 17-21.

The serum was separated and stored at -20•Ž

until assayed. Placentae and fetuses were blotted to

remove excess blood and fluid, and weighed. The

number of placentae and fetuses amounted to

15.9•}0.3/animal. The placentae were stored at

-20℃Cuntil assayed F The placentaefrom days

10-21 pregnant rats were homogenized in 100

mM NH4HCO3, 10 mM EDTA, 10 mM EGTA, 1

mM phenylmethylsulfonyl fluoride (PMSF), pH

9.3 for 30 seconds with the ULTRA-TURRAX

homogenizer and then for 30 seconds with a glass

homogenizer. Extraction was carried out for 2 h at

4•Ž with slow stirring. The homogenate was

centrifuged at 100,000•~g for 60min and the

supernatant was assayed.

Hormones and RIA

rPL-II was purified in this laboratory [25], and

rGH (NIADDK-rGH-RP-2) RIA kit was a gener-

ous gift from the NIADDK. Antibody to rPL-II

was raised in rabbits and a homologous RIA for

rPL-II was developed as described previously [25].

No displacement was observed with rPRL, rGH,

oPRL, and other pituitary hormones up to 1,000

ng/ml. The displacement curves for sera and

placental extracts from day 13 and 18 pregnant

rats were parallel to the rPL-II standard curve.

GESTATIONAL PROEILES OE rPL-II AND rGH 591

Electrophoresis and immunoblottingAmniotic fluid from day 13 pregnant rats was

concentrated 10-fold by ultrafiltration throughADVANTEC UK-10 membrane and by ULTRA-CENT-10 (TOSOH, Tokyo) before loading. Thesamples of concentrated amniotic fluid and mater-nal serum of day 13 pregnant rats were elec-trophoresed on discontinuous SDS-poly-acrylamide gel as described previously [26]. As acontrol for non-specific binding, pooled serumfrom normal cyclic rats was loaded. Molecularweight standards (SDS-polyacrylamide gel elec-trophoresis low mol wt standard) and prestainedmarkers (low ranges) were obtained from Pharma-cia (Uppsala, Sweden) and Bio-Rad (Richmond,U.S.A.), respectively. The proteins elec-trophoresed on the gel were transferred to PVDFmembrane (Clear blot membrane-p, ATTO,Tokyo) for 1 h at 0.27 A. The membrane wasincubated with rabbit anti-rPL-II serum and goatanti-rabbit immunoglobulin-alkaline phosphataseconjugate (Bio-Rad), and then developed with theBio-Rad AP color development reagents described

previously [25]. Identical samples were also elec-trophoresed separately on another gel and stainedwith silver nitrate [27] for to estimate molecularsize.

Results

Gestational profilesThe gestational profile of the rPL-II concentra-

tion of maternal and fetal serum and amnioticfluid is shown in Fig.1. rPL-II appeared in

maternal circulation on day 11 of pregnancy. Its

concentration increased progressively and reachedthe peak value on day 19. After day 19 of

pregnancy, the rPL-II concentration of the mater-nal serum decreased by term, with values rangingbetween days 16 and 17. Fetal serum rPL-II wasdetectable only on the first day of sampling (day

17) and the following day (day 18), and itsconcentration ranged between 3 and 10ng/ml.rPL-II in amniotic fluid was first detectable on day

12 and increased dramatically the following day

(40 ng/ml, corresponding to 22% of the maternalserum rPL-II concentration). After day 13 of

pregnancy, the rPL-II concentration of the amnio-tic fluid declined rapidly, and it was undetectablefrom day 15 through term.

Maternal Serum

Fetal Serum

Amniotic Fluid

Fig. 1. Gestational profiles of rPL-II and rGH in maternal

and fetal serum and amniotic fluid. Values are

means•}SEM for 4-15 rats.

Fig. 2. The content (yg/placenta) and concentration(ƒÊ/g

tissue) of rPL-II in the placental tissue during the

last half of pregnancy. Each point represents

rPL-II content and concentration of the samples

pooled each day of pregnancy as described in Fig.

1.

592 KISHI et al.

In the placental tissue, rPL-I I was first detect-able on day 11 of pregnancy. Its content increased

progressively until day 19 of pregnancy anddeclined rapidly thereafter up to term (Fig. 2).The gestational profile of rPL-II content in the

placental tissue is similar to that of maternalcirculation. The rPL-II concentration in the

placental tissue, however, increased dramaticallyon day 12 and remained high until day 19 of

pregnancy.The rGH concentration of maternal serum

remained low from day 10 to day 15 and ranged

between 10 and 21 nglrnl (Fig. 1). It increased

gradually until day 20 of pregnancy and fellrapidly on the day before parturition. Fetal serum

rGH was first detectable on day 18 and its

1 2 3 4

Fig. 3. Immunoblotting of rPL-1I from amniotic fluid

and maternal serum of day 13 pregnant rats.

The Sarnples Were diluted in the buffer With-

out mercaptoethanol, electrophoresed, trans-

ferred to PVDF membrane, and detected with

anti-rPL-Il serum. Lane 1, rPL-II (50ng); lane

2, concentrated amniotic fluid (5 Al corres-

ponding to 50ƒÊl of the initial fluid); lane 3,

maternal serum (0.5 p.l); lane 4, serum pool

from cyclic rats as a control for non-specific

binding (0.5ƒÊl).

concentration increased dramatically on day 20 of

pregnancy (28•}2ng/ml, corresponding to 18% of

the maternal rGH concentration). rGH in amniotic

fluid was slightly detectable for the last two days

(<5ng/ml).

Immunoblotting

The charactcristics of molecular forms of rPL-II

in amniotic fluid and maternal serum of day 13

pregnant rats were examined by silver staining

(data not shown) and immunological detection

(Fig. 3). Under nonreducing conditions, purified

rPL-II had one major band and two minor bands

with mol wts of 19.5K, 20.0K, and 20.5K, respec-

tively, on the dye-stained gel. These three bands

were also detected by immunological staining.

Although a 24.5K protein was slightly visible in the

same lane, it may be a reduced form of rPL-II

because the 24.5K migrated at the same position as

a major form of purified rPL-II with 2-

mercaptoethanol treatment (data not shown).

In the nonreduced sample of amniotic fluid,

major and minor bands were detected at the same

positions as those of purified rPL-II with mol wts

of 20.5K and 19.5K, respectively (Fig. 3, lane 2).

The largest mol wt component of rPL-II was the

most abundant in amniotic fluid, whereas the

smallest one was the most abundant in the purified

preparations. In the nonreduced serum sample

from day 13 pregnant rats, only the largest

component of protein (20.5K) was slightly de-

tected when 0.5ƒÊl (Fig. 3, lane 3) and 2.0ƒÊl (data

not shown) of serum were loaded on the SDS-gel.

Theothcrbands(ln(,rcthan43K,aroulld30Kand

14K) with slight intensity compared to a majorform of rPL-II were visible in the samples ofamniotic fluid and maternal serum of day 13pregnant rats. These bands, however, were alsoobserved when a 4-fold amount of serum sampleof cyclic rats was loaded (data not shown). There-fore, these proteins are not considered to repre-sent the specific ones in the samples of pregnantanimals.

Discussion

The present study examines the gestational

profiles of rPL-II detected with a homologous RLSin the placental tissue and biological fluids during

the last half of pregnancy in the rat. The overall

GESTATIONAL PROFILES OF rPL-II AND rGH 593

pattern of rPL-II in the maternal circulation ischaracterized by a progressive increase with asmall decline just before parturition (Fig. I). This

profile is similar to that of mPL-II in the mouse[28]with the exception of a small decline justbefore parturition and to the previous report of

Robertson and Friesen in the rat [14] with theexception of a small peak during mid gestation.rPL-II mRNA has been reported to be first

detectable on day 10 [13] or day 11 [10] of

pregnancy. rPL-II appeared first in maternalcirculation and the placental tissue on day 11 of

pregnancy. Therefore, the day when rPL-II pro-tein starts being synthesized in the tissue coincides

with the day when rPL-II mRNA is first detectable.The concentration of rPL-II in the placental

tissue remained relatively constant during days12-19 of pregnancy while total production ofrPL-II per placenta increased gradually until day

19 as pregnancy advanced (Fig. 2), which suggeststhat the gestational increase in total rPL-II pro-

duction in the placenta results primarily from anincrease in the mass of the placenta. The gestation-

al profile of total production of rPL-II per

placenta parallels that of the rPL-II concentrationin the maternal circulation. The newly synthesized

rPL-II may be secreted rapidly to the maternalcirculation without a stable storage pool as in

another rodent, the mouse [29]. The induction oftotal rPL-II mRNA from the placenta increases as

pregnancy advances, particularly dramatically onday 13, and remains relatively constant thereafter

[10].Therefbre, the gestational pattern of total

production of rPL-II per placenta is not complete-ly parallel to that of the amount of rPL-II mRNAinduction during the first half of the profiles,suggesting that post translational efficiency maychange between mid and late pregnancy.

It is interesting that the rPL-II concentration inamniotic fluid increased dramatically and tempo-rarily during days 12-14, though rPL-II wasundetectable during the rest of the days of

pregnancy (Fig. 1). The amniotic fluid proteinsdetected with anti-rPL-II serum consisted ofmonomeric forms, which had mol wts of 19.5Kand 20.5K under nonreducing conditions (Fig. 3).The 20.5K protein was predominant in amnioticHuid in contrast to the puri丘ed rPL-II. A protein

with the same mol wt as the 20.5K was alsodetected in maternal serum of day 13 pregnantrats, indicating that the largest form (20.5K) of

monomeric rPL-II is predominant in amniotic

Huid as well as in maternal circulation during mid

pregnancy. These data also indicate that most ofthe rPL-II exists as a monomeric form in maternalserum and amniotic fluid during mid pregnancy incontrast to the observation that the dimer oraggregated forms of rPL-II are predominant inmaternal serum on day 18 of pregnancy [25]. The

physiological roles and biological activities of rPL-II and its variants in amniotic fluid and maternalcirculation remain to be determined.

It is not clear what profile rPL-II takes in fetalcirculation during mid pregnancy because of theunavailability of adequate amounts of fetal serumfor rPL-II RIA. During late pregnancy, fetalserum rPL-II was slightly detectable only on days17 and 18, and was less than 1% of the maternalcirculation, and undetectable thereafter. rPL-II issynthesized by the trophoblast giant cells in thebasal zone [ 11, 12, 13] in contact with the maternalcirculation. Therefore, the majority of rPL-IIsecreted by the cells seems to have access to thematernal blood supply and may act primarily onmaternal sites. However, it does not rule out the

possibility that rPL-II may play a direct role infetal development during pregnancy, becausebinding sites for oPRL and hPL have been iden-tified in the rat fetal liver [ 15, 16] and in the fetalmouse liver for oPRL and mPL-II [30, 31].Absolute concentrations of PLs and PL-IIs in fetalcirculation are species dependent. The PLs con-centrations of fetal serum are higher than those ofmaternal circulation for the first half of pregnancyin sheep [32] and throughout pregnancy in cattle

[33,34],while hPL and mPLII concentrations infetal serum are less than 1 and 10% of thematernal circulation [2, 35], respectively. Amongthe former group of species, particularly in sheep,oPL has been reported to stimulate glycogensynthesis by the ovine fetal liver while oGH hadlittle or no activity in ovine hepatocytes [36],suggesting that oPL is involved in regulating themetabolism and growth of the ovine fetus. Howev-er, it is not clear whether rPL-II plays a direct rolein regulating rat fetal development. According tothe experiment with heterologous species hor-mones, oPL but not hGH stimulates IGF-II syn-thesis in rat fetal fibroblasts [21], in whose fetalserum the concentrations of IGF-II have beenreported to be 20- to 100-fold higher than those inmaternal serum [37] and increase gradually dur-

594 KISHI et al.

ing pregnancy [38].The gestational profile of rGH was characte-

rized by a progressive increase in maternal circula-tion with a decline on the day before parturitionand by its appearance and dramatic increase in the

fetal serum during only the last three days of

pregnancy (Fig. 1). These patterns observed hereare similar to those observed in previous reports

[39, 40]. Although fetal pituitary rGH is detectedby RIA [39] and rGH mRNA-positive cells appear

in the fetal pituitary [41] during the last three daysof pregnancy, the source of fetal serum rGH is not

clear. Furthermore, no information is availableabout the physiological role of rGH in the fetalcirculation. During post natal life, the actions ofrGH are mediated by IGF-I and synthesis and

release of IGF-I is under the control of rGH [42,43]. However, it is also not clear whether rGH also

plays a role in regulating IGF-I secretion in thefetal and maternal compartment during pregnan-cy in the rat. The fact that the gestational profilesof rGH and rPL-II are similar to those of mGH

and mPL-II [28, 44] and that mGH is consideredto be a potent regulator of mPL-II secretion in the

mouse [24] does not rule out the possibility thatrGH in maternal circulation may be involved inregulating rPL-II secretion during the last half of

pregnancy in the rat, though it still remains to bedetermined.

Acknowledgments

We thank the NIADDK for providing the rGH

RIA kit and Ms. Kae Ikeuchi for excellent techni-

cal assistance.

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