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Immunoelectron microscope observations on secretion of human placental lactogen (hPL) in the human chorionic villi.

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THE ANATOMICAL RECORD 216:68-72 (1986)
Immunoelectron Microscope Observations on
Secretion of Human Placental Lactogen (hPL) in
the Human Chorionic Villi
Department of Anatomy (S.F., H. U., H.K.) and Department of Obstetrics and Gynecology
(K.H.), University of Occupational and Environmental Health, School of Medicine,
Kitakyushu 807, Japan
Immunoelectron microscope localization of human placental lactogen (hPL) was investigated in the chorionic villi from week 7 to full-term gestation
with the protein A-gold technique. With specific antiserum against hPL, immunoreactive gold particles were found to be preferentially located in Golgi-derived,
electron-dense small granules of 80-180 nm in the syncytiotrophoblast. Our electron
micrographs indicate that these small granules increase in number in the course of
gestation and are released by exocytosis from the apical cell surface. The present
study reveals that hPL is segregated from the Golgi apparatus, stored in the syncytiotrophoblast as secretory granules, and released into the maternal blood.
Since Josimovich and MacLaren (1962) identified human chorionic somatomammotropin, i.e., human placental lactogen (hPL), many attempts to reveal the
localization of this hormone in human placentas have
been performed. By using specific antisera against hPL,
both immunofluorescence (Swanson-Beck et al., 1969;
Kim et al., 1971; Gaspard et al., 1980) and immunoperoxidase (De Ikonicoff et al., 1971; De Ikonicoff and Cedard, 1973; Watkins, 1978) approaches have demonstrated that hPL is localized in the syncytiotrophoblast
of human placental chorionic villi. Autoradiographic
studies with labeled cDNA probes derived from hPL
mRNA also identified the syncytiotrophoblast as the site
of synthesis of hPL (Boime et al., 1976; Hoshina et al.,
1982). In spite of these light microscope investigations,
there has been only one brief ultrastructural report concerned with localization of hPL in the syncytiotrophoblast by the enzyme-labeled antibody technique
(Hayashi, 1980).
We demonstrate in this paper the ultrastructural localization of hPL in the chorionic villi from week 7 to
full-term placentas by the application of protein A-gold
technique (Roth et al., 1978) with special reference to
the synthesis and release of hPL in the syncytiotrophoblast.
Tissue Preparation
Human placentas of 7, 9, and 12 weeks of gestation
obtained from natural and therapeutic abortion, and
placentas from full-term normal pregnancies, were used
for the present study. For light microscope observations,
specimens were fixed in Bouin's solution for 24 h r a t
room temperature and routinely embedded in paraffin.
Specimens for electron microscopy were fixed in Zamboni solution (Zamboni and De Martino, 1967) or PLP
(periodate-lysine-paraformaldehyde)solution (McLean
and Nakane, 1974) for 18 h r at 4°C. After washing in
0 1986 ALAN R. LISS, INC.
0.1 M phosphate buffer (pH 7.4) with 10% sucrose a t
least 2 hr at 4"C, some of the specimens were postfixed
with 1% osmium tetroxide in 0.1 M phosphate buffer for
1 hr at 4°C. All the specimens were dehydrated in graded
concentrations of acetone and embedded in epoxy resin.
The unlabeled antibody peroxidase-antiperoxidase
(PAP) technique according to Ueda et al. (1983)was used
for light microscope immunocytochemistry. The working dilutions of the antiserum against hPL (DAKO Immunoglobulins, Denmark) ranged from 1500 to 1:2,000.
For immunoelectron microscopy, the protein A-gold
technique moth et al., 1978) was carried out with some
slight modifications (Ueda and Fujimoto, 1984; Ueda et
al., 1985). Colloidal gold was prepared by the method of
Frens (1973). First, 200 ml of 0.01 % chloroauric acid
(Wako Pure Chemicals, Osaka, Japan) was boiled in a
clean siliconized flask, and 2 ml of 1% sodium citrate
was added. After cooling, the gold solution was adjusted
to pH 6.9 with 0.2 M potassium carbonate. Then 1mg of
protein A (Pharmacia Fine Chemicals, Uppsala, Sweden) was dissolved in 10 ml of the colloidal gold solution
in a siliconized centrifuge bottle. After 3 min, 1 ml of
polyethylene glycol was added and the mixture was
centrifuged at 20,OOOg for 15 min at 4°C. The protein
A-gold complex was resuspended in 10 ml of 0.1M phosphate-buffered saline (PBS).
Ultrathin sections mounted on nickel grids were labeled as follows. The grids were placed on 1%egg albumin in PBS for 10 min to reduce nonspecific adsorption
of protein on the sections. In osmium-postfixed specimens, pretreatment with saturated sodium metaperiodate for 1 hr a t room temperature to restore the
hormonal antigenicity was performed just prior to the
Received September 12, 1986; accepted February 25, 1986.
Fig. 1. Serial 4 - ~ msections of the chorionic villi at 12 weeks of gestation stained with Masson’s
trichrome (A) and by PAP method with anti-hPL serum at a concentration of 1:1,000(B). Immunoreactions
of hPL are localized only in the syncytiotrophoblast (ST). CT, Cytotrophoblast; IT, Interstitial tissue. x
incubation with egg albumin (Bendyan and Zolliger,
1983).This pretreatment was a fairly efficient way both
to restore high resolution of labeling with immunoreactive gold particles and to preserve the cytoplasmic fine
structure. The grids were then incubated for 2 h r at
room temperature with the antiserum against hPL
(DAKO Immunoglobulins, Denmark). From the intensity of the preliminary PAP labeling, it was decided to
use a 1:1,000 dilution of the antiserum. After washing
in distilled water, the grids were stained with uranyl
acetate and lead citrate and examined with a JEM 100
CX electron microscope. The specificity of the present
immunolabeling was confirmed by the following controls: 1)The antiserum was absorbed with hPL; 2) normal rabbit serum, antisera against human prolactin
(PRL), and human growth hormone (GH) (DAKO Immunoglobulins, Denmark) were substituted for the antihPL; 3) the ultrathin sections were incubated only with
the protein A-gold complex solution for 1 hr.
Figure 1A shows human placental chorionic villi at 12
weeks of gestation stained with Masson’s trichrome. In
the adjacent 4-pm section incubated with anti-hPL a t a
concentration of 1:1,000 and stained by the PAP technique, positive reactions of hPL were found exclusively
in the syncytiotrophoblast (Fig. 1B). Neither cytotrophoblasts nor interstitial tissue showed any positive re-
actions. No reactions were observed if normal rabbit
serum or other antisera (anti-PRL and anti-GH) replaced the specific antiserum. The reactions became
heavier in the course of gestation and were maximal in
the full-term specimens.
At 12 weeks of gestation, the syncytiotrophoblast contains abundant membrane-bounded granules of various
sizes and electron densities in the cytoplasm as follows:
electron-dense small granules (SG) of 80-180 nm in diameter, less-electron-dense middle-sized granules (MG)
of 200-300 nm and large bodies (LB) of 500-1,000 nm,
as shown in Figure 2. Among these three kinds of granular inclusions, the immunoreactive gold particles were
preferentially located on the small granules (Fig. 2). The
heavy reaction often appeared on this type of granule
aggregated near the apical cell surface (Fig. 3A). These
granules appear to be released from the apical cell surface by exocytosis (Fig. 3B). In the Golgi regions, the
gold particles were often observed on the small granules
segregating from the Golgi lamellae (Fig. 3C). In the
specimens at 7 and 9 weeks of gestation, the gold particles were also localized in the small granules. However,
there were fewer granules present than a t 12 weeks.
In the Zamboni osmium-fixed full-term human placentas, the small granules increase remarkably in number
and most of them show heavy reactions although the
other types of granules have almost completely disappeared (Fig. 4).Very few or no gold particles were ob-
Fig. 2. Immunoelectron microscope localization of hPL in the syncytiotrophoblast (ST) at 12 weeks of
gestation. The gold particles are concentrated on the small granules (SG)near the apical region of the
cell. GA, Golgi apparatus; LJ3, large body; MG, middle-sized granule. x 19,000.
Fig. 4. Immunoelectron microscope localization of hPL in the syncytiotrophoblast (ST)of a full-term
specimen. The small granules appear to be more abundant at term than a t 12 weeks of gestation. LD,
Lipid droplets. x 19,000,
served on the cytoplasmic organelles or matrix in the
It is well known from biochemical data derived by
radioimmunoassay that serum and urine concentrations
of hPL gradually increase during the course of gestation
(Biswas et al., 1972; Gaspard et al., 1973). There were
The present immunoelectron microscopy indicates that relatively few small granules in our specimens at 7
the syncytiotrophoblast is the only hPL antigenic site. weeks, but they had remarkably increased in number a t
Hayashi (1980) briefly described the ultrastructural lo- full term. This may argue for the conclusion based on
calization of hPL on the nuclear membrane, endo- the present immunological study that the only the small
plasmic reticulum, and Golgi complex in the syn- granules are the WL-containing granules in the
cytiotrophoblast. However, our data demonstrated that syncytiotrophoblast.
Although the immunological properties of GH have
the specific immunoreaction of the gold particles occurred mainly on the small granules derived from the similarities to those of hPL (Sciarra et al., 1963; Currie
Golgi lamellae and often aggregated near the apical et al., 1966; Hou and Swanson-Beck, 1967), there were
region of the syncytiotrophoblast. These findings indi- no cross-reactions between GH and hPL in the present
cate that the small granules are segregated from the study. Anti-GH and anti-PRL did not show any specific
Golgi apparatus as hPL-containing granules and re- reactions in the present specimens by either light or
leased by exocytosis from the apical cell surface into the electron microscope immunocytochemistry.
As described above, the syncytiotrophoblast contains
maternal blood.
a t least the other two types of membrane-bounded granular inclusions in our specimens. Besides hPL, human
placentas also generate human chorionic gonadotropin
(hCG) (Boime et al., 1978; Ashitaka et al., 1980). In our
Fig. 3. Immunoelectron microscope localization of hPL in the syncy- laboratory, the ultrastructural localization of hCG has
tiotrophoblast at 12 weeks of gestation. The gold particles occur on the been observed in the syncytiotrophoblast at 7 weeks of
small granules. (A). These granules appear to be released by exocytosis gestation by methods similar to those in the present
(B). The gold particles are also observed on the terminal expansions of
study but with anti-hCG (DAKO immunoglobulins,
the Gulgi cisternae (GA) (C). SG, Small granules. A, X 38,000; B, X
Denmark; UCB Bioproduct, Belgium); the “less elec63,000;C, x 46,000.
tron-dense middle-sized granules” (MG)described in this
paper were the most likely hCG storage site (unpublished data). Considering the differences in chemical
nature of hCG and hPL (a glycoprotein and a simple
protein, respectively), and in the blood concentration
(hCG levels are high in the earlier stages of gestation),
it is unlikely that these two hormones were included in
the same granules. Large bodies do not seem to be secretory granules, since no evidence of their release from
the cell could be obtained. Hamasaki et al. (1985) demonstrated the presence of iron in this inclusion by energy-dispersive X-ray microprobe analysis. At any rate,
more detailed studies by histochemical and immunoelectron microscope techniques are necessary to elucidate
the precise function of these two types of granules.
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chorionic, immunoelectron, observations, microscopy, ville, lactogen, secretion, placental, hpl, human
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