Endocrine Journal 1995, 42 (4), 537-543

Expression of Prolactin Gene During Pregnancy Studied by Histocemistry

in In

Human Decidua Situ Hybridization

NOZOMU TADOKORO, NORIYUKI KOIBUCHI*, HIDEKI OHTAKE*,

TAKESHI KAWATSU, YuMo KATO* *, SADAO YAMAOKA*, AND

TAKAHIRO KUMASAKA

Departments of Obstetrics and Gynecology, and *physiology, Dokkyo University School of Medicine, Tochigi 321-02, and **Institute of Molecular and Cellular Regulation, Gunma University, Maebashi 371, Japan

Abstract. The prolactin (PRL) gene is known to be expressed not only in the anterior pituitary but also in the decidualized human endometrium. This study was designed to detect the site of synthesis of PRL during pregnancy by in situ hybridization histochemistry. Decidual and trophoblast tissues from early

pregnancy were obtained from patients undergoing therapeutic abortion at 8-10 weeks of gestation. Term placentae were obtained from patients with uncomplicated deliveries at 38-40 weeks. Sections of these tissues were hybridized with 355-labeled RNA probe complementary to human PRL mRNA. Specific hybridization signals were distributed over the decidual cells in early and term pregnancy. In the decidua capsularis of early pregnancy, labeled cells were concentrated close to the amniotic cavity, although decidual cells were distributed evenly. In the decidua parietalis, almost all decidual cells were labeled, but no specific labeling was seen in the endometrial glands or capillary endothelium. In the decidua basalis, greater signals were always detected over the decidual cells in early pregnancy than in term pregnancy, when sections, which were hybridized with the same probe and exposed simultaneously, were compared. No specific hybridization was detected in the trophoblast cells. These results not only confirm that PRL is specifically synthesized in the decidual cells but also indicate that

there are regional and periodical differences in PRL gene expression in the decidual cells during

pregnancy.

Key words: Prolactin, Decidua, Placenta, In situ hybridization histochemistry (Endocrine Journal 42: 537-543,1995)

PROLACTIN (PRL) is known as a peptide

hormone which is secreted from the anterior

pituitary [1]. Pituitary PRL (pPRL) is involved in many physiological functions, including reproduction, growth, lactation, immune function,

and metabolism [2]. The PRL gene is also expressed in the endometrium in several different

Received: January 19, 1995 Accepted: April 13, 1995 Correspondence to: Dr. Nozomu TADOKORO, Department of Obstetrics and Gynecology, Dokkyo University School of Medicine, Mibu, Tochigi 321-02,

Japan

species [3]. The human endometrial PRL (hePRL)

gene has already been cloned [4, 5]. The amino acid sequence of hePRL is identical to that of pPRL, whereas hePRL mRNA is 150 nucleotides larger

because of the difference in the 5' untranslated region of each mRNA [6]. hePRL is synthesized in

the decidualized endometrium and secreted into the amniotic fluid during pregnancy [7, 8]. The

physiological role of hePRL has not been fully clarified yet. A previous study has indicated that hePRL has functions analogous to those in the

pituitary, but may be specifically related to fetal

growth and maternal adaptations to pregnancy [9].

538 TADOKORO et al.

In particular, since PRL influences water and ion

transport in lower vertebrates and binds specifically

with high affinity to amniotic membrane, amniotic fluid PRL may play a role in regulating amniotic

fluid volume and ion content [9].

It is generally accepted that hePRL is a product of the decidual cells of human endometrium [9].

Studies have shown PRL immunoreactivity in the decidual cells [10-16], and in situ hybridization

histochemistry (ISH) showed the localization of PRL mRNA in the decidual cells [16]. However,

regional differences in PRL gene expression, such as in the decidua basalis, decidua parietalis and

decidua capsularis, have not been clarified yet. In the present study, we have collected decidual

tissues of all regions in early pregnancy and applied ISH. In addition, we have compared the

localization of the PRL gene expressing cells located in the decidua basalis in early and term

pregnancies.

Materials and Methods

PRL cDNA fragment (bases 1-611), was obtained by digesting the plasmid with Pst I restriction endonuclease. This fragment was subcloned into Pst I site of a pBluescript SK II (+) transcription vector (Stratagene, La Jolla, CA) and amplified in E. coli (XLI-Blue, Stratagene). For Northern blot hybridization, this Pst I fragment was labeled with Klenow fragment (Oligolabeling KitTM, Pharmacia LKB Biotechnology) and 32P-deoxy CTP, according to the protocol prepared by the manufacturer. To transcribe the RNA probe (riboprobe) for ISH, the vector was linearized with either Cla I or Barn HI restriction endonuclease. Then sense and antisense riboprobes were transcribed with T3 and T7 RNA

polymerases (Stratagene), respectively. 355-labeled UTP was incorporated. Specific activities of the

probes were approximately 250-375 Ci/mmol. The sense probe was used as a control since it is chem-ically similar, has the same percentage G/C, and can be manufactured with the same specific activi-ty as the antisense probe, but does not hybridize with any known species. The riboprobes were then hydrolyzed to approximately 250 nucleotides according to the procedure by Cox et al. [18].

Sample collection

Decidual and trophoblast tissues from early

pregnancy were obtained by curettage from

patients undergoing therapeutic abortion at 8-10 weeks of gestation (n=6). Term placentae were

obtained from patients with uncomplicated deliveries at 38-40 weeks (n=4). Informed consent

for the use of utero-placental tissues was obtained from all the patients before the treatment. Tissues were trimmed to approximately 10 mm x 10 mm x

10 mm cubes and immediately frozen with dry

ice. Tissues containing maternal surface were cut from the term placenta, because decidual cells are

located close to the maternal surface (termed as the decidua basalis). These tissues were kept in liquid nitrogen until use.

Probe preparation

The full length cDNA for human pPRL mRNA in a pOkayama-Berg vector was kindly donated from Dr. K. Nakajima, Mie University, Tsu, Japan. Its characterization has been described elsewhere [17]. The cDNA fragment (627 base pair (bp) in size), which contains 16 by nucleotides from the

plasmid in the 5'-flanking region and 612 by of the

Extraction of total RNA and Northern blot hybridization

Total RNAs of the decidua, chorionic villi and

placenta were extracted by the acid guanidinium thiocyanate-phenol-chloroform method [19]. Total RNA was also extracted from the anterior pituitary

of adult male Wistar rats (8 weeks of age, n=3), which were sacrificed under pentobarbital

anesthesia (50 mg/kg body weight). The homology of nucleotide sequences of the human and rat PRL within the portion used in the present study was

approximately 70% [4, 20]. Twenty-five jig of total

RNA from each human tissue and five ug from the rat anterior pituitary was electrophoresed in 1.5%

agarose gel containing 2% formaldehyde. The RNAs were then transferred to a nylon membrane

(Gene Screen PIusTM, E. I. du Pont de Nemours & Co., Boston, MA). The membrane was hybridized

with the radiolabeled probe (prepared as above) for 24 h at 42 °C with 10 ml hybridization buffer

(50% formamide, 1% SDS, 1 M sodium chloride, 10% dextran sulfate and 100 jig/ml calf thymus DNA), rinsed twice with 2 x standard saline citrate

buffer (SSC,1 x SSC contains 0.15 M NaCI, 0.015 M sodium citrate) for 5 min at room temperature,

PROLACTIN GENE EXPRESSION IN HUMAN DECIDUA 539

twice with 2 x SSC containing 1% SDS for 30 min at 60 °C and twice with 0.1 x SSC for 30 min at room temperature and exposed on Fuji X-ray film for 24 h at -85 °C. The film was then developed with Kodak D-19 developer and fixed with Kodak Fix.

Tissue sectioning and in situ hybridization

Ten ,um thick frozen sections were cut on a cryostat and mounted onto organosilane-coated

glass slides, thawed, and dried at 48 °C for 10-60 min. The tissues were then fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) containing 0.02% diethylpyrocarbon-ate (DEP) for 5 min, rinsed with PBS (3 min), and dehydrated through a graded series of ethanol (50%, 75%, 95%,1 min each). The slides were then immersed in a solution of 0.25% acetic anhydride containing 0.1 M triethanolamine (pH 8.0, 10 min) to decrease nonspecific binding of the riboprobes by neutralizing the positive charge on the tissues and slides [21]. The sections were then rinsed with 0.2 x SSC for 10 min, dehydrated through a series of ethanol, and dried in a desiccator. They were then kept at - 85 °C until use. Several sections of each tissue block were stained with hematoxylin-eosin (H-E) solution to identify which part of the decidual tissue is contained, such as the decidua capsularis, decidua parietalis or decidua basalis. The protocol for ISH was essentially the same as described elsewhere [22]. The sections were

prehybridized for 1.5-2 h at room temperature with 40 µl of a prehybridization buffer [22]. The

prehybridization solution was then removed and 40 p1 of heat-denatured hybridization buffer [22] containing sense or antisense riboprobe (7 x 105 cpm/section) was pipetted onto each section. Hybridization continued for 24 h at 50 °C in humidified boxes. Following hybridization, the sections were rinsed

with 2 x,1 x and 0.5 x SSC (10 min each), treated with ribonuclease A (RNase A, Sigma type X-A, 20 jug/ml in 10 mM Tris pH 8.0, 0.5 M NaCl,1 mM EDTA) for 30 min at 45 °C, rinsed with RNase-free buffer for 30 min at 45 °C, rinsed with 2 x,1 x, and 0.5 x SSC (10 min each), and rinsed with 0.1 x SSC containing 10 mM dithiothreitol at 45 °C overnight. The next day, the sections were rinsed for 1 min each in 300 mM ammonium acetate:ethanol 1:1, 3:7,1:9, and finally in absolute ethanol. The sec-

tions were then dried in a desiccator, dipped in

Kodak NTB3 photographic emulsion (45 °C), and

stored dry in light-tight boxes at 4 °C for 10-60 days. Slides were then developed with Kodak D-

19 developer diluted 1:1 with water (10 min at 15 °C) , fixed with Kodak Fix (6 min), stained with H-E solution, dehydrated through a graded series of

ethanol and coverslipped.

Results

Northern blot hybridization

In order to confirm whether the probe used in

the present study specifically hybridizes with PRL mRNA, Northern blot hybridization was

performed. As shown in Fig. 1, an approximately 1.3 kilobase (kb) size band was detected in the decidua of early pregnancy (lanes c and d), and

the placenta of term pregnancy (lane f). No hybridization signal was detected in the chorionic

villi in early pregnancy (lane e). A hybridization signal (approximately 1.2 kb in size) was also detected in the rat anterior pituitary (lanes a and

b). These results are consistent with those of a

previous study [6].

Fig. 1. Northern blot analysis of prolactin mRNA. Five

(lanes a and b) or 25 µg (lanes c-f) total RNA obtained from the rat anterior pituitary (lanes a and

b), decidua of 8 weeks of gestation (lanes c and d),

chorionic villi of 8 weeks (lane e) and term placenta

of 38 weeks (lane f) were electrophoresed. The solid arrowhead shows the position of the 185 ribosomal

RNA. The open arrowheads show the

hybridization signals for PRL mRNA. Note that the

sizes of the pituitary PRL mRNA and endometrial PRL mRNA are different.

540 TADOKORO et al.

In situ hybridization detection of human PRL mRNA

Examples of in situ hybridization detection for PRL mRNA are shown in Figs. 2-4. In the sections which were hybridized with the antisense probe

(Figs. 2A, 3 and 4), dense concentrations of silver

grains were detected over the specific cells, whereas no such specific localizations of grains were seen

in the sections which were hybridized with the sense (control) probe (Fig. 2B). These results

suggest that a specific hybridization signal was detected.

In early pregnancy (Figs. 2, 3 and 4A), decidual cells exclusively showed hybridization signals. In

the decidua capsularis (Fig. 2A), although decidual cells were distributed throughout the decidua, the

labeled cells were exclusively located around the region close to the chorion laeve (Ch). On the

other hand, no specific hybridization signal was

seen over the Ch or amnion (Am). In the decidua

parietalis (Fig. 3), most of the decidual cells were strongly labeled. No specific hybridization signal was detected in the endometrial gland (G, Fig. 3A)

or the capillary endothelium (arrowhead) (Fig. 3B). In the decidua basalis, infiltrated by the trophoblast

Fig. 2. Examples of in situ hybridization detection for human prolactin mRNA in the decidua capsularis in

early pregnancy. Sections were hybridized with

either 355-labeled antisense (A) or sense (B) probe. These probes were synthesized simultaneously with

the same radioisotope. Hybridizations were

performed simultaneously for 24 h at 50 °C. Autoradiography was continued simultaneously for

60 days at 4 °C. The hybridization signal consists of

dark grains over the individual cells (A). Scale bar length=100 µm. Am, amnion; Ch, chorion laeve;

UC, uterine cavity.

Fig. 3. Examples of in situ hybridization detection for human PRL mRNA in the decidua parietalis in early

pregnancy. These two sections were hybridized with the same probe and exposed simultaneously for 45 days. Note that no hybridization signal was

observed in the epithelial cells in the uterine gland

(G) (3A) or the capillary endothelial cells

(arrowhead) (3B). Scale bar length=100 ,um.

PROLACTIN GENE EXPRESSION IN HUMAN DECIDUA 541

cells, the decidual cells showed a strong

hybridization signal as in the decidua parietalis, but no hybridization signal was detected in the

trophoblast cells (Fig. 4A). In the decidua basalis of term placenta, specific

hybridization signals were detected over the

decidual cells as in early pregnancy (Fig. 4B). When the intensity of the hybridization signals of the

decidua basalis in early and term pregnancy were compared in sections which were hybridized and

exposed simultaneously, the signals in term

pregnancy were much weaker than those in early

pregnancy (Fig. 4A). As in early pregnancy, no specific hybridization signal was detected in the

trophoblast cells.

Discussion

The results of the present study show that

hybridization signals are located over the decidual cells in early and term pregnancy in the human

decidua. There was no labeling in the other subsets of cells. Under our experimental conditions, the

melting temperature (Tm) of the riboprobe used in

the present study and PRL mRNA hybrids is estimated to be 78 °C [23]. Because the optimum rate of reassociation of RNA-RNA hybrids for

ISH is approximately 25 °C below the Tm [18], hybridization was performed at 50 °C. Although

PRL, growth hormone (GH) and human placental lactogen (hPL) have similar amino acid sequences

and, therefore, their immunological and biological

properties overlap [4], the homology of the nucleotide sequence between the PRL cDNA

fragment used in the present study and the other two genes was less than 40% [4]. Because a 1%

base mismatch reduces the Tm of the RNA-RNA hybrids by 1.4 °C [23], the estimated Tm of riboprobe for the PRL used in the present study

and GH or hPL mRNA hybrids is below 0 °C. The

possibility that this probe cross-hybridizes with hPL or GH can therefore be excluded when sections are hybridized at 50 °C. Moreover, no significant hybridization signals were seen over the sections

which were hybridized with the sense (control)

probe. This indicate that the specific hybridization signal for PRL was detected over the decidual cells.

Immunocytochemical studies have shown that

decidual cells are strongly immunoreactive against PRL antisera [10-16]. In addition, some studies

have shown PRL immunoreactivity in the non-decidual cells such as in the trophoblast [10, 14,

16] and amnion [15], whereas other studies have shown that only decidual cells are immunoreactive

[11, 13]. PRL mRNA was detected only in decidual tissues by Northern blot hybridization [24] and ISH

[16]. In the present study, we also showed by ISH that only decidual cells have a hybridization signal for PRL mRNA. It is therefore most likely that

only decidual cells express the PRL gene during

pregnancy, but in the present study it seems that not all decidual cells expressed the PRL gene. In

the decidua capsularis (Fig. 2A), labeled cells were exclusively concentrated around the area close to

Fig. 4. Examples of in situ hybridization detection of the PRL mRNA in the decidua basalis (DB) in early (A)

and term pregnancy (B). These sections and the

sections shown in Fig. 3 were hybridized

simultaneously with the same probe and exposed

together for 45 days. Note that a greater number of

grains were concentrated in the sections in (A). Also note that no hybridization signals were

detected in the chorionic villi (CV). Scale bar length=100 µm.

542 TADOKORO et al.

the amniotic cavity, although decidual cells are

distributed throughout the stromal tissue. A

previous study did not report any such difference [16]. Probably this is because they did not collect all regions of decidua in early pregnancy.

As in a previous study [16], the decidual cells

specifically showed a hybridization signal not only in early pregnancy but also in term pregnancy. In

addition, we have found a difference between decidual cells in early and term pregnancy in the

relative levels of the cellular hybridization signals. In the decidua basalis (Fig. 4), a greater

hybridization signal was detected in early

pregnancy. It has been reported that the PRL concentration in amniotic fluid and decidual PRL content are decreased in parallel from 26 weeks of

gestation until delivery [25]. Maternal and fetal

pituitary PRL make only a minor contribution to the total amount of PRL in the amniotic fluid [26]. The results of the present study are consistent with the hypothesis that the decrease in the PRL

concentration in amniotic fluid and decidual PRL content in term pregnancy is induced through the

decrease in the transcription of PRL mRNA. However, the results of the present study cannot fully prove this hypothesis. Because we collected

only placental tissues in term pregnancy, the

possibility that decidual cells located in other regions, such as the fetal membrane and decidua

parietalis, express the same level of the PRL gene as in early pregnancy, cannot be excluded. This

might be possible because the influence of amniotic fluid in decidual cells located in the decidua basalis

and fetal membrane in term pregnancy is different. With the advance of pregnancy, the chorionic villi

keep on growing and the distance between the decidual cells in the decidua basalis and amnion is

extended. Under such conditions, if PRL synthesis

is influenced by some factors from amniotic fluid as well as autocrine and/or paracrine factors

produced in the utero-placental unit [9], PRL synthesis and secretion of decidual cells in the decidua basalis may be different from those located

close to the amniotic cavity. Trials to detect the difference between the decidua basalis and fetal

membrane in term pregnancy in PRL gene expression are currently under way.

The functional significance of PRL in the amniotic fluid has not been fully understood. A previous

study has shown that PRL plays a role in water transport by fetal membrane [27]. On the other

hand, a ligand binding study showed that PRL receptor was located in the chorion laeve [28] and

PRL receptor mRNA, which was cloned from hepatoma and breast cancer cDNA libraries, was

detected in the chorion laeve by Northern blot hybridization [29]. Whether other endometrial structures express the PRL receptor gene has not

yet been determined. Attempts to detect the localization of PRL receptor gene expressing cells

in human utero-placental tissues are currently underway.

Acknowledgement

We wish to thank Dr. Kunio Nakajima, Depart-ment of Biochemistry, Mie University School of Medicine, Tsu, Japan, for generously providing cDNA for PRL mRNA. We are also grateful to Ms. M. Sakai and Ms. N. Ohmori, Department of Physiology, Dokkyo University School of Medicine, for their technical assistance.

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