Placenta (1989), IO, 37-43

Stimulatory Effect of Prolactin on Human Placental Progesterone Secretion at Term In Vitro: Possible Inhibitory Effect on Oestradiol Secretion

EYTAN R. BARNEAaJ, FUAD FAREPb & KOL SHAHAR”

u Rappaport Family Institute fbr Research in the Medical Sciences, Technion-Israel Institute qf Technology, P.O.B. 9697, Hatfa 31096, Israel

h Department of Pharmacology, Faculty of Medicine, Technion- Israel Institute of Technology, P.O.B. 9649, Hatfa 31096, Israel

( To whom correspondence should be addressed

Paper accepted 20.6. I 988

INTRODUCTION

Prolactin (PRL) is a polypeptide (mol. wt 22 500) which belongs to a group of lactogenic hor- mones that includes growth hormone and human placental lactogen (Catt and Pierce, 1987). In addition to the maternal pituitary, other sources of PRL production during pregnancy have been identified. These include the fetal pituitary, the decidua and possibly the placenta (Albrecht, 1980). Receptors for PRL have been found in several endocrine organs, including ovary, milk ducts, uterus, hypophysis, kidney, adrenal gland, and placenta (Herington, Graham and Healy, 1980; Kelly et al, 1984).

Multiple roles have been attributed to PRL; few, however, are firmly established. Among others, PRL’s involvement in lactation, stress and osmotic processes has been shown (Yen, 1987). In addition, its involvement in steroid hormone secretion has been extensively investi- gated in the non-pregnant state. In different animal species and hormonal states, PRL’s effect on androgen, progesterone (P,) and oestrogen secretion differs. In the rat postovulatory ovary, PRL is luteotrophic, promoting P, secretion, while its effect on oestradiol (E,) secretion is mainly inhibitory in the follicular stage (Wang and Chan, tg8z; Kalison, Warshaw and Gibori, 1985; I,ee et al, 1986). In the adrenal gland it promotes dehydroepiandrosterone sulphate secretion.

During pregnancy PRL is present in high concentrations in the amniotic fluid, having a role in osmotic processes (Josimovich, Merisko and Bocella, tg77), and may promote local prosta- glandin secretion, aiding in initiation of labour (McCoshen and Bare, 1985). PRL’s role in lac- togenesis is well established (Lyons, 1958). In the fetus, PRL may promote lung maturation (Hamosh and Hamosh, 1977).

Although very high levels of PRL are present in the circulation and amniotic fluid, its effect on placental function has been little investigated. At term, PRL incubated with placental

0143~4004/89/010037 fo, $05.00/o 61 1989 Baillitre Tindall Ltd

38 Plurnta (tgtly), Vol. ~0

explants suppresses human chorionic gonadotrophin (hCG) secretion (Pride et al, 1986). Its effect upon placental steroidogenesis is not yet known.

In the present study, the effect of PRL upon placental steroidogenesis was examined by measuring E, and P, production and secretion from placental explants at term. Results show that PRL has a significant stimulatory effect upon P, secretion.

MATERIALS AND METHODS

Tissues Placental tissues were obtained from seven healthy subjects following elective caesarean section at term. Small placental fragments were dissected out in a sterile fashion and placed in a large excess of 0.9 per cent (v/v) NaCl solution to remove all blood. Subsequently, tissues were rinsed again in Dulbecco’s modified Eagle’s medium containing I per cent antibiotic solution (5000 U penicillin, 50 ,ng streptomycin and IO ooo U Fungizone). Incubation was carried out with 30 mg placental tissue in I ml medium containing either 0.5 per cent bovine serum albu- min (BSA) or IO per cent fetal calf serum (FCS), with or without PRL (ovine PRL, Lot 123F0320; Sigma Chemical Co., St. Louis, MO, USA) (2 to 20 ooo ng), for 2 to 24 h at 37°C in a gas mixture of 95 per cent air and 5 per cent CO,. Following incubation, dishes were placed on ice, and the media and tissues were collected and stored at - 2o°C until assayed.

Placental explant viability was previously reported (Barnea et al, 1986). There was a pro- gressive decrease in glucose concentration during incubation, but the pH of the culture medium unchanged. As shown by haematoxylin and eosin staining, placental explants were confirmed to be viable.

Radioimmunoassays Measurements of the steroid E, and P, in the media and the tissues were carried out using pre- viously described standard radioimmunoassay techniques (Barnea and Fakih, 1985; Barnea et al, 1986). Steroid measurements in the tissues were carried out by first homogenizing the tissue in I ml of buffer used for radioimmunoassay. Subsequently the homogenate was sedimented by centrifugation at 2400 rpm for IO min. The supernatant was removed and served as a source of steroid measurements. Steroid recovery from the tissue was determined using an internal reference standard, which was 75 per cent for E, and 50 per cent for P,; results were corrected accordingly. For a given placenta, all samples were run in a single assay. Interassay variability for E, and P, was IO per cent and I I per cent, respectively. Data were expressed as pg/mg pro- tein for E, and as ng/mg protein for P,.

Statistical analyses The data were analysed by means of a one-way analysis of variance (ANOVA) and Student’s t test. P value of < 0.05 was considered statistically significant.

RESULTS

In preliminary experiments, the basal secretion of P, in the medium at 2 to 24 h was compared

50 1 0 0.5% BSA - 10% FCS I

39

2 4 24 hrs * ~(0.05 vs. 2 hours # ~(0.05 vs. 0.5% BSA

Ftgurr I. E&t of different culture medium compositions upon placental explant I’, secretion. There was a significant increase in the steroid secretion in both media used. In the presence of IO per cent FCS, the secretion of P, was signi- ficantly higher than in the prcsencc of 0.; per cent RSA.

in the presence of media with either 0.5 per cent BSA or IO per cent FCS. Although P, pro- duction in the IO per cent FCS-supplemented medium was higher than that in 0.5 per cent BSA in both cases, a significant linear increase in the steroid production was found (Figure I). However, since FCS is not a defined medium, we preferred to use 0.5 per cent BSA in sub- sequent experiments.

Subsequently, the effect of a fixed dose of PRL (200 ng/ml) for various incubation periods was examined, and a highly significant (P < 0.005) time-dependent increase in P, secretion was found follow-ing exposure to PRL (Figure 2).

Exposure of placental explants for 24 h to various concentrations of PRL (z to 20 ooo ng/ml) resulted in a significant dose-dependent increase in P, secretion (Figure 3) (P < 0.05). The maximal effect (two-fold) was noted at physiologic concentrations of 200 ng/ml. In order to de- termine PRL’s effect on P, accumulation in the tissue, the effect of PRL (200 and IOOO ng, final concentration) was examined. Exposure of the placental explants to 200 ng/ml PRL caused a significant increase in P_ accumulation compared with controls (88 f 28 versus 384 f 14 ng/mg protein, mean f SEM) (P < 0.001). However, exposure to IOOO ng/ml PRL caused a significant drop in P, accumulation (88 f 28 versus 35 f 14ng/mg protein) (P < 0.05).

The effect of PRL on E, secretion in a total of seven placentae was examined. In four cases a significant inhibitory effect was noted (Figure 4) (P < 0.05); in two cases no significant change was present, and in only one case was a stimulatory effect apparent.

DISCUSSION

The present data show that PRL has a significant stimulatory effect on placental P, synthesis and secretion into the medium. This is evidenced by the increase in explants/P, content and secretion into the media after only 4 h. The response was bell-shaped, with a maximal response

Plmenta (1989), Vol. 10

# * T

I Ocontrol -200 ng prolactin

# *

I

I

2 4 * ~(0.05 vs. 2 hours

24 hours

# ~(0.05 vs. control Figure z. Effect of zoo ng/ml PRL upon placental explant P, secretion for the first 24 h. There was a significant time- dependent increase in P, secretion at 4 and 24 h compared with controls.

120-

T$lOO- 2 k ao-

*

01 I /a / I I

control 2 20 200 / / 20 000 ng prolactin

* ~(0.05 vs. control

Figure3. Effect of various concentrations of PRL upon placental explant P, secretion. At 200 ng/ml, there was a signi- ficant increase in the secretion of the steroid compared with controis (one-way ANOVA; P < 0.05). Results represent four separate experiments.

noted at 200 ng/ml (physiologic concentrations). The effect on E, secretion was mainly inhibit- ory.

Circulating levels of PRL increase between the first trimester and term, reaching levels of 200 to 300 ng/ml in both mother and fetus. The levels in the amniotic fluid peak at 20 weeks, reaching IOOO to 2000 ng/ml, then decrease towards term (Albrecht, 1980). We examined PRL’s effect on P, and E, secretion using these concentrations, and the highest response was noted at 200 ng/ml (Figure I), while at pharmacologic doses (20 ooo ng/ml) no significant eff& was noted (Figures 2 and 4).

Barnea et al: Effbct qf‘prolactzn on placental sterordogenem

CJ 10 W I \

\i/

I * i/

??

0’

04 6 I

control 2 20 200 200-g prolactin

* ~(0.05 vs. control Figm 4. Effect of various concentrations of PRL upon placental E, secretion. The inhibitory effect of PRL was par- ticularlq observed at mo ng/ml (one-way ANOVA; P < 0.05). Results represent four separate experiments.

This is in accord with data from Pride et al (1986), who found a similar type of effect of PRL upon explants/hCG secretion, although it was inhibitory at term. In their experiments, doses of IOO to zoo ng/ml had the maximal effect. This can be explained, as shown by Kelly et al (1984), by the fact that PRL up-regulates its receptor at low doses, but has the opposite effect at high concentrations. Incubation of PRL with mammary glands causes a decreased receptor concentration at IOOO ng (Kelly et al, 1984).

The relationship between PRL and steroidogenesis in the ovary has been widely investi- gated, yielding contradictory data. However, most authors report that PRL stimulates P, secretion in both rat and human corpus luteum-acting there alone or, in concert with lutei- nizing hormone, having a luteotrophic role (Rotchild, 1981).

The effect of PRL on steroidogenesis is believed to be exerted via binding to specific recep- tors, which have previously been detected in the placenta (Herington, Graham and Healy, 1980). The mediators of PRL’s action at the cellular level have not yet been conclusively iden- tified.

Some reports suggest that in the ovary, besides promoting P, synthesis, PRL prevents con- version of P, into zocl-hydroxyprogesterone (Eckstein and Nimrod, 1979). The first event is accomplished by favouring cholesterol ester uptake and processing by the side-chain-cleavage enzyme system, leading to pregnenolone formation, which rapidly converts to P,. Such a path- way has been described in the placenta, where it is believed to be the major avenue of P, syn- thesis (Davies and Ryan, 1972). The conversion to r7-hydroxyprogesterone has been shown in the placenta, especially in sheep, where this process is promoted by cortisol (Anderson, Flint and Turnbull, 1975). In the human, such a pathway has been suggested only recently by Scho- field and Auletta (1984), who showed enzymes capable of carrying out the process in term pla- cental microsomes. Based on our data, we are unable to establish whether in placental explants the dominant process is de novo P, synthesis, prevention of degradation of P,, or both.

The role of PRL in parturition has recently been investigated. PRL of decidual origin that reaches the amniotic fluid has been shown to have a promoting effect upon local prostaglandin synthesis (McCoshen and Bare, 1985). In our view, PRL would have a regulatory effect by promoting local P, production, needed for maintaining uterine quiescence and counteracting E,-induced effects. Furthermore, PRL decreases E, secretion (Figure 4), thus increasing the

42 Placenta (19K9). Vol. 10

P,/E, ratio. On the other hand, in early labour PRL levels in circulation drop precipitously (Rigg, Lein and Yen, 1977). This major drop could lead to a drop in placental P, secretion, causing a decreased P,/E, ratio, thereby promoting effective uterine contractions and delivery of the infant.

In conclusion, PRL acts upon placental steroidogenesis, increasing P, secretion while in certain cases inhibiting E, secretion. The mechanism of this interaction is under investigation.

SUMMARY

Maternal and fetal circulating prolactin (PRL) increases ro-fold compared with the non-preg- nant state. We examined the effect of PRL upon placental steroidogenesis. It had a significant (P < 0.05) time-dependent stimulatory effect upon placental explants/P, accumulation and secretion into the medium. The maximal stimulatory effect (two-fold) in dose-dependent ex- periments was found to be 200 ng/ml. The effect of PRL upon oestradiol secretion was mainly inhibitory. This inhibition was most pronounced at 200 ng/ml. In conclusion, placental steroid secretion is modulated by PRL. This effect occurs mainly at concentrations seen in the pla- centa at term, suggestive of its physiologic role.

ACKNOWLEDGEMENT

This work was supported by a grant to E. R. Barnea from the Lady Davis Foundation.

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Bnrnea et al: Effect ofprolactin on placental sternidogenesis 43

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