Cytoplasmic and biochemical differentiation of the human villous
cytotrophoblast in the absence of syncytium formation
Harvey J. Kliman+, Jerome F. Strauss III*+, Lee-Chuan Kao*, Stephen Caltabiano+, and Samuel Wu
+Departments of Pathology and Laboratory Medicine, and Obstetrics
and Gynecology*, University of Pennsylvania, Philadelphia, PA 19104
Running head: Trophoblast differentiation
Key words: placenta, extracellular matrix, hCG
Supported by USPHS Grants HD-00715 (HJK), HD-06274 (JFS) and DK-07314
(LCK).
Address all correspondence to :
Harvey J. Kliman, M.D.-Ph.D.
Dept of Pathology and Lab. Medicine
6 Founders Pavilion
Hospital of the University of Pennsylvania
3400 Spruce Street
Philadelphia, PA 19104-4823
(215) 662-6518
Human villous cytotrophoblasts form syncytial trophoblasts through
a process of cell fusion (Boyd and Hamilton, 1970 and Kliman et al., 1986). There has been speculation that biochemical differentiation,
such as the ability to secrete chorionic gonadotropin (hCG) (Pierce
and Midgley, 1963, and Morrish et al., 1987) and placental lactogen (Hoshina et al., 1983, and Hoshina et al., 1985), is tied to syncytia formation. We have demonstrated that
cultured human cytotrophoblasts differentiate biochemically and
morphologically in a time-dependent fashion (Kliman et al., 1986, Feinman et al., 1986, and Kliman et al., 1987). In the presence of fetal calf serum (FCS), these villous
cytotrophoblasts flattened out onto the culture surface, aggregated
and formed syncytia over a 24 to 96 h period. Concurrently with
these morphological changes, the trophoblasts differentiated biochemically,
evidenced by the secretion of increasing amounts of hCG and progesterone
into the culture media (Kliman et al., 1986). This in vitro system, however, did not allow us to dissociate the morphologic
and biochemical changes. Here we demonstrate that by culturing
cytotrophoblasts under serum-free conditions and manipulating
the culture surface by precoating with a variety of extracellular
matrix proteins, morphologic and biochemical differentiation can
be uncoupled.
Human cytotrophoblasts were purified from normal term placentae
obtained following spontaneous vaginal delivery or uncomplicated
Cesarean section using the method of Kliman et al. (1986). The cytotrophoblasts were diluted to a final concentration
of 106 cells/ml with Dulbecco's Modified Eagles' Medium containing 25
mM HEPES and 25 mM glucose (DMEM-H-G) with glutamine (4 mM) and
gentamicin (50 µg/ml) added, and, in serum-supplemented media,
1 to 20% (vol/vol) heat-inactivated fetal calf serum. 8Bromo-cAMP
(1.5 mM, Sigma Chemical Co., St. Louis, MO) was added to some
media. One ml of the cell suspension was plated into each well
of a 16 mm Nunclon multidish (Nunc, Roskilde, Denmark) for biochemical
studies, while 0.2 to 1.0 ml was plated onto 22 mm square coverslips
(#1 or 11/2) placed in each well of a 35 mm Nunclon multidish for histologic
studies. For electron microscopy, 10 ml of the cell suspension
was cultured in 80 cm2 Nunclon flasks without precoating in serum-free media, with or
without added 8-bromo-cAMP. The cells were incubated at 37_C in
humidified 5% CO2/95% air with media changes every 24 h.
Extracellular matrix proteins
Fibronectin (Collaborative Research, Bedford, MA) was dissolved
in DMEM-H-G to a final concentration of 50 µg/ml. Laminin (Collaborative
Research) was dissolved in DMEM-H-G to a final concentration of
20 µg/ml. Collagens (Sigma Chemical Co.) were dissolved in 0.15%
NaCl (w/vol) to a final concentration of 80 or 100 µg/ml. Bovine
serum albumin, fraction 5 (Miles Scientific, Naperville, IL) was
dissolved in deionized H2O to a final concentration of 80 µg/ml.
All solutions contained 50 µg/ml gentamicin and were filtered
with a 0.2 µm syringe filter, applied to either cover glasses
or plastic culture surfaces until covered, allowed to stand overnight
in a humidified incubator at 37_C, aspirated and dried in a culture
hood for 1-2 h prior to use.
Histology and immunohistochemistry
Cultured cells grown on coverslips and tissue were fixed with
Bouin's solution as previously described (Kliman et al., 1986). The coverslips and tissue sections were then stained
and photographed (Kliman et al., 1986). Dilutions and sources of antibodies against ß-hCG, SP1,
hPL, low molecular weight cytokeratin, alpha1-antichymotrypsin
(ACT), vimentin (Kliman et al., 1986); 34 kd protein (Roy-Choudhury et al., 1988), and P-450scc (Nulsen et al., 1989) have been described previously. The percentage of nuclei
in syncytia was determined at the light microscopic level by counting
500 nuclei in sequential high power fields and determining whether
they were in single isolated cells, single cells of an aggregate
(2 or more cells attached to each other or single cells attached
to syncytia), or syncytia (defined as a cell with two or more
nuclei within one obvious cytoplasmic border). The total number
of nuclei in syncytia was divided by 500.
Following 24 h of culture, the trophoblasts were washed once with
warm DMEM-H-G, and then fixed with half-strength Karnovsky's fixative
(Karnovsky, 1965) for 15 min at room temperature, followed by
incubation overnight at 4_C . The fixed cells were removed from
the flasks by adding approximately 100 pre-cleaned 3 mm glass
beads to the tissue culture flasks, agitating gently for 1 minute,
then aspirating off the cell suspension. The cells were then pelleted,
transferred to 0.1 M cacodylate buffer, pH 7.4, postfixed with
1% osmium for 1 h, dehydrated in graded ethanols and propylene
oxide, and embedded in Epon. Silver sections were stained with
uranyl acetate and lead citrate and examined at 75 kV with a Hitachi
600 electron microscope (Hitachi Corp., Tokyo, Japan).
Analytical and statistical methods
Media were assayed for progesterone by RIA as previously described
(DeVilla et al., 1972). hCG was quantitated using reagents obtained from Serono
(hCG MAIA clone; Braintree, MA). The assay was calibrated to the
First International Reference Preparation. DNA content was quantified
using the dye Höescht 33258 and a DNA mini-flourometer (Hoefer
Scientific Instruments, San Francisco, CA) using sperm whale DNA
as a standard as described by Labarca and Purgen (1980). Sample
means within coating regimens were compared using the Student's
t-Test. Sample means between ECM treatment groups were compared
using the nonparametric Mann-Whitney U test.
Characterization of purified cytotrophoblasts
Since this work attempts to show that mononuclear cytotrophoblasts
purified from term placentae are capable of differentiating into
functional syncytiotrophoblasts in the absence of syncytia formation,
it is critical that the starting material is proven to be unequivocally
cytotrophoblasts and not, for example fragments of syncytiotrophoblasts.
Figure 1 is a compilation of immunocytochemical studies performed
on matched sets of starting placentae (left figure of each pair)
and the resultant cytotrophoblast preparations (right figure of
each pair). In the left column, four syncytiotrophoblast markers
are used: ß-hCG, hPL, SP1, and low molecular weight cytokeratin
(1 a-d). In each case, the villous syncytiotrophoblasts stain
(arrow heads), while the cytotrophoblasts in situ (arrows) and the purified cytotrophoblasts do not stain, ruling
out the possibility that the purified cytotrophoblasts are fragments
of syncytiotrophoblasts. Figures 1 e and 1 f illustrate the results
of two mesenchymal markers: ACT (1 e) and vimentin (1 f). ACT
stains the tissue macrophages of the villous core (Hofbauer cells),
but not the villous cytotrophoblasts (arrows), syncytiotrophoblasts
(arrow heads), or the purified cytotrophoblasts. In a similar
pattern, vimentin (1 f) stains endothelial cells (small arrows)
and fibroblasts (arrow head) of the villous core, but not the
villous cytotrophoblasts (large arrow), syncytiotrophoblasts,
or purified cytotrophoblasts. These last two sets of figures rule
out contamination of the cytotrophoblast preparation by macrophages,
endothelial cells or fibroblasts. Finally, two positive markers
for villous cytotrophoblasts are illustrated: P-450scc (1 g) and 34 kD protein (1 h). Anti-P-450scc stains villous cytotrophoblasts (arrows), syncytiotrophoblasts
(arrow heads), and the isolated cytotrophoblasts. Since this antibody
stains an enzyme present in the mitochondria of these cells (Nulsen
et al., 1989), the staining pattern is granular. Anti-34 kD stains only
the cytoplasm of villous cytotrophoblasts (1 h, arrows). The cytoplasm
of villous syncytiotrophoblasts are negative (arrow heads). Consistent
with this, the purified cytotrophoblasts exhibit strong cytoplasmic
staining for 34 kD (1 h, right figure). These results clearly
show that the purified trophoblast preparation described here
represents a pure population of villous cytotrophoblasts without
contamination by mesenchymal cells. In addition, these markers
indicate that the preparation is not the result of fragmentation
of syncytiotrophoblasts, and these cells do not represent differentiated
trophoblasts since they exhibit the same staining pattern as villous
cytotrophoblasts.
We next investigated whether purified extracellular matrix proteins could support syncytia formation in serum-free medium. Precoating glass coverslips with fibronectin or a variety of collagens permitted cytotrophoblasts to form syncytia in serum-free media (Fig. 2). Although culture for 96 h in the presence of FCS produced the greatest degree of syncytial formation (83% ), approximately 50% of the trophoblast nuclei were found in syncytia when the culture surface was precoated with fibronectin; placental collagens types I, IV and V; calf skin collagen type I; and rat tail collagen. Approximately 33% of the trophoblast nuclei were found in syncytia when the culture surface was precoated with placental collagens types III and V. In contrast, without serum or precoating, only 2.5% of the trophoblast nuclei were found in syncytia at the end of the 96 h of culture period. Precoating with bovine serum albumin yielded results similar to no precoating. These results suggests that fibronectin and collagens do not support syncytia formation by, for example, neutralization of negative charges on the coverslip, but mediate flattening, aggregation and syncytia formation by a more specific mechanism.
Stimulation of hCG and progesterone secretion in the absence of
syncytia formation
We had shown previously that 8-bromo-cAMP stimulates hCG and progesterone
secretion by cultured human trophoblasts in the presence of serum
(Feinman et al., 1986). We wondered if trophoblasts could be stimulated to secrete
hCG and progesterone under serum-free conditions where 1) they
could not form syncytia or 2) they could syncytialize because
they were plated on fibronectin, laminin or type IV collagen-treated
surfaces. Figures 3 and 4 illustrate the results of several such
experiments. Trophoblasts were cultured over a 48 h period under
serum-free conditions without pretreatment, or in serum-free medium
on surfaces coated with fibronectin, type IV collagen, or laminin.
In addition, half of the cultures were treated with 1.5 mM 8-bromo-cAMP.
As we have shown previously, 8-bromo-cAMP induces increased secretion
of hCG and progesterone in cultured trophoblasts (Feinman et al., 1986), but now we demonstrated the same effect under serum-free
conditions. We wondered if this effect of 8-bromo-cAMP would be
the same if the cells were plated on different surfaces. We therefore
compared the effects of 8-bromo-cAMP on cells plated on uncoated
coverslips and coverslips coated with either fibronectin, laminin
or type IV collagen. Statistical analysis comparing the secretion
of hCG and progesterone at 24 and 48 h in the absence or presence
of 8-bromo-cAMP revealed no significant (p<0.05) differences between
the uncoated coverslips and those with the different coverslip
treatments. These data establish that trophoblasts secrete hCG
and progesterone in a similar fashion under serum-free conditions
whether cultured in the absence or presence of extracellular matrix
proteins. Furthermore, these results indicate that trophoblasts
which do not flatten down on the culture surface and do not subsequently
form syncytia because they are grown under serum-free conditions
still express endocrine activities associated with the syncytial
trophoblast.
One goal of this work was to develop a serum-free culture system
which could be used to investigate the factors which regulate
the endocrine functions of the human trophoblast. In the process
of achieving that goal, we discovered that in the absence of serum,
purified human cytotrophoblasts do not flatten out, aggregate
or form syncytia-as is seen in the presence of serum. We thought
it likely that the fibronectin in the serum played a role in the
adhesion of these cells to the culture surface. We verified that
fibronectin-as well as a variety of collagens-could, to a variable
extent, replace the adhesion factors in serum (Fig. 2).
Our experiments revealed that we could modulate morphologic differentiation-that is, progression of mononuclear cytotrophoblasts to multinuclear syncytial trophoblasts-by modifying the surface on which the cells were cultured. We wondered whether cytotrophoblasts cultured in the absence of serum without the precoating of the surface (thus constraining them to remain as single cells) could still differentiate biochemically (i.e., could still be stimulated to secrete hCG and progesterone) in spite of their inability to form syncytial structures. The results presented in figures 3 and 4 clearly demonstrate that trophoblasts grown in the absence of serum can secrete hCG and progesterone and that 8-bromo-cAMP augments this secretory activity whether they are placed on uncoated surfaces or surfaces coated with fibronectin, type IV collagen, or laminin. Thus, trophoblasts which remain as single mononuclear cells can express endocrine functions of the syncytial trophoblast.
To confirm this finding, we performed transmission electron microscopy on cytotrophoblasts grown without serum or precoating, with or without added 8-bromo-cAMP, to evaluate whether the cells when stimulated acquire the necessary machinery for synthesis and secretion of hormones. Cytotrophoblasts grown in serum-free media exhibited relatively simple fine structural features after 24 h of culture. Cells cultured in the presence of 8-bromo-cAMP remained spherical and mononuclear, but they acquired the features typical of the syncytiotrophoblast. Therefore, we have demonstrated that these cells differentiated both biochemically and structurally in culture in the absence of syncytium formation. To rule out the possibility that the purified cytotrophoblasts used in these experiments represented fragments of more mature syncytiotrophoblasts, we performed extensive immunocytochemical characterization of our starting preparation (Figure 1). This figure makes it clear that the initial preparation represents a highly pure population of cells which do not show the features of villous syncytiotrophoblasts, but stain as villous cytotrophoblasts.
It is generally accepted that the cytotrophoblast is the undifferentiated
stem cell of the villous trophoblast and that the syncytial trophoblast
is the fully differentiated, end-stage trophoblast (Pierce and
Midgley, 1963 and Boyd and Hamilton, 1970). Some have suggested
that biochemical differentiation can occur only after syncytial
formation (Morrish et al., 1987). Our work with human trophoblasts and Sherman and Atienza-Samols'
(1979) work with mouse trophectoderm contradicts this notion.
We believe that the trophoblasts of the human chorionic villi
form a continuum of differentiated cells, some of which are mononuclear
and others multinuclear. In addition, our results support the
hypothesis that syncytia formation is not a prerequisite for biochemical
differentiation. Therefore, the formation of the syncytiotrophoblast
is not the trigger for biochemical differentiation, but is only
one of the consequences of the differentiation program. This scheme
parallels very closely the biology of myoblast differentiation
(Holtzer et al., 1980). The serum-free system described here, and its ability
to disassociate morphologic from biochemical differentiation in
the human trophoblast, should permit us to elucidate the mechanisms
of trophoblast regulation and maturation.
Human syncytiotrophoblasts are derived from villous cytotrophoblasts
by cell fusion. Simultaneous with this morphologic transformation,
trophoblasts acquire specific endocrine functions, including elaboration
of chorionic gonadotropin (hCG) and progesterone. We wondered
if syncytia formation was a prerequisite for biochemical differentiation,
or simply was one part of the differentiation program. By growing
purified human cytotrophoblasts under serum-free conditions and
manipulating the culture surface, we were able to disassociate
morphologic from biochemical differentiation. We have shown previously
(Endocrinology 118:1567, 1986) that human cytotrophoblasts grown in the presence
of fetal calf serum flatten out, aggregate and form functional
syncytiotrophoblasts in vitro over 24-96 h. Here we demonstrate that when grown in the absence
of serum, the cells do not undergo these morphologic changes,
but remain as individual spherical cells. If the culture surface
was precoated with fibronectin or a variety of collagens, but
not albumin, the cells regained their ability to flatten, aggregate
and form syncytia. Although syncytia formation did not occur when
cytotrophoblasts were cultured under serum-free conditions in
the absence of extracellular matrix (ECM) proteins, biochemical
differentiation was not affected. These cells secreted hCG and
progesterone at the same rate under serum-free conditions whether
they were plated on plastic only-which prevented syncytia formation-or
fibronectin, laminin or type IV collagen-which allowed syncytia
formation to occur. Furthermore, cytoplasmic differentiation in
the absence of syncytia formation was confirmed by performing
transmission electron microscopy on cytotrophoblasts grown under
serum-free conditions in the presence of 8-bromo-cAMP. We conclude
that syncytia formation is not a prerequisite for biochemical
differentiation, but simply part of the trophoblast differentiation
program.
We gratefully acknowledge Justina Minda for assistance in the
electron microscopy and Julia Haimowitz for the preparation of
the manuscript.
Boyd, J. D., and Hamilton W. J. (1970) In The human placenta, pp. 167-174. London: MacMillan.
DeVilla, G. O., Roberts K., Wiest W. G., Mikhail G. and Flickinger
G. (1972) A specific radioimmunoassay of plasma progesterone.
J. Clin. Endocrinol. Metab., 35, 458-60.
Feinman MA, Kliman HJ, Caltabiano S and Strauss JF III. (1986)
8-Bromo-3'5'-adenosine monophosphate stimulates the endocrine
activity of human cytotrophoblasts in culture. J. Clin. Endocrinol. Metab., 63, 1211-1217.
Hoshina, M., Hussa, R., Pattillo, R., and Boime, I. (1983) Cytologic
distribution of chorionic gonadotropin subunit and placental lactogen
messenger RNA in neoplasms derived from human placenta. J. Cell Biol., 97, 1200-1206.
Holtzer, B., Croop, J., Toyama, Y., Bennett, G. H., Fellini, S.,
and West, C. (1980) Differences in differentiation programs between
presumptive myoblasts and their daughters, the definitive myoblast
and myotubes. In Plasticity of Muscle (ed.) Pete, D., p 133. New York: de Gruyter.
Hoshina, M., Boothby, M., Hussa, R., Pattillo, R., Camel, H. M.,
and Boime, I. (1985) Linkage of human chorionic gonadotrophin
and placental lactogen biosynthesis to trophoblast differentiation
and tumorigenesis. Placenta, 6, 163-172.
Kao, L-C, S. Caltabiano, S. Wu, J. F. Strauss III and H. J. Kliman
(1988) The human villous cytotrophoblast: interactions with extracellular
matrix proteins, endocrine function and cytoplasmic differentiation
in the absence of syncytium formation. Dev. Biol., in press.
Karnovsky, M. (1965) A formaldehyde-glutaraldehyde fixative of
high osmolarity for use in electron microscopy. J. Cell Biol., 27, 127A.
Kliman, H. J., Nestler, J. E., Sermasi, E., Sanger, J. M., and
Strauss, J. F. III. (1986) Purification, characterization and
in vitro differentiation of cytotrophoblasts from human term placentae.
Endocrinology, 118, 1567-1582.
Kliman, H. J., Feinman, M. A., and Strauss, J. F. III. (1987)
Differentiation of human cytotrophoblasts into syncytiotrophoblasts
in culture. In Trophoblast Research (ed.) Miller, R. K. and Thiede, H., Vol. 2, pp. 407-421. New
York: Plenum Medical.
Labarca, C., and Paigen, K. (1980) A simple, rapid, and sensitive
DNA assay procedure. Anal. Biochem., 102, 344-52.
Morrish, D. W., Bhardwaj, D., Dabbagh, L. K., Marusyk, H., and
Siy, O. (1987) Epidermal growth factor induces differentiation
and secretion of human chorionic gonadotropin and placental lactogen
in normal human placenta. J. Clin. Endocrinol. Metab., 65, 1282-1290.
Nulsen, JC, Silavin S. L, Kao L-C, Ringler GE, Kliman, HJ, and
Strauss JF III (1989) Control of the steroidogenic machinery of
the human trophoblasts by cyclic AMP. J. Reprod Fert, Suppl.,
37: 147-153.
Pierce, G. B., Jr., and Midgley A. R., Jr. (1963) The origin and
function of human syncytiotrophoblastic giant cells. Amer. J. Path., 43, 153-173.
Roy-Choudhury, S., Sen-Majumdar A., Murthy U, Mishra VS, Kliman
HJ, Nestler JE, Strauss JF III, and Das M. (1988). Biosynthesis
and turnover of a 34,000 molecular weight protein growth factor
in human cytotrophoblasts. Eur J. Biochem. 172: 777-783.
Sherman, M. I., and Atienza-Samols S. B. (1979) Differentiation of mouse trophoblast does not require cell-cell interaction. Exp. Cell Res., 123, 73-77.
FIGURE LEGENDS
Figure 1. Immunocytochemical staining of intact placentae (left figure of each pair) and resultant purified cytotrophoblast preparation (right figure of each pair). a) ß-hCG. Syncytiotrophoblasts stain (arrow heads), while cytotrophoblasts are negative (arrows). Purified cells are negative. b) hPL. Syncytiotrophoblasts stain (arrow heads), while cytotrophoblasts are negative (arrows). Purified cells are negative. c) SP1. Syncytiotrophoblasts stain (arrow heads), while cytotrophoblasts are negative (arrows). Note cytotrophoblast in metaphase. Purified cells are negative. d) Low molecular weight cytokeratin. Syncytiotrophoblasts stain (arrow heads), while cytotrophoblasts are negative (arrows). Purified cells are negative. e) ACT. Hofbauer cells are positive, while villous cytotrophoblasts (arrows) and syncytiotrophoblasts (arrow heads) are negative. Purified cytotrophoblasts are negative. f) Vimentin. Villous core endothelial cells (small arrows) and fibroblasts (arrow head) stain, while cytotrophoblasts (large arrows) and syncytiotrophoblasts are negative. The purified cytotrophoblasts are also negative. g) P-450scc. The cytoplasm of villous cytotrophoblasts (arrows) and syncytiotrophoblasts (arrow heads) stain positively. In a similar fashion, the cytoplasm of the purified cytotrophoblasts also stains. The granular staining pattern is due the fact that P-450scc is present only in the mitochondria of these cells. h) 34 kD protein. Villous cytotrophoblast cytoplasm is stained (arrows), while syncytiotrophoblast cytoplasm is negative (arrow heads). The cytoplasm of the purified cytotrophoblasts are strongly positive. Bars represent 10 µm.
Figure 2. Effects of coverslip pretreatment on trophoblast syncytium formation. Cytotrophoblasts were cultured under serum-free conditions for 96 h as described in the Material and Methods. Coverslips were pretreated with the following: albumin (ALB); fibronectin (FIB); Types I, III, IV and V collagen from placenta [Type I (P), Type III (P), Type IV (P), Type V (P)]; Type I collagen from calf skin [Type I (CS)]; and rat tail collagen (RT) In addition, the results of control cultures grown without serum (SF) or with fetal calf serum (FCS) with no coverslip pretreatment are shown in the first two bars. Data presented for the serum-free, fetal calf serum and albumin treatments are means±SEM from 3-5 separate experiments. Data for the fibronectin and collagen treatment groups are from a single experiment in which these treatments were examined simultaneously with the same cytotrophoblast preparation. Similar results were obtained with fibronectin and placental types I and IV collagens from at least five other experiments. (From Kao et al., 1988)
Figure 3. hCG secretion as a function of culture surface. Cytotrophoblasts were cultured in plastic wells without precoating (A), or within wells precoated with fibronectin (B), laminin (C) or type IV collagen (D) in media without (q) or with (Æ) 1.5 mM 8-bromo-cAMP for 48 h as described in the Materials and Methods. Values presented are the means ±S.D. of (n=4) separate experiments. All hCG differences between cultures treated or not treated with 8-bromo-cAMP were statistically significant to at least the 95th percentile (p<0.05), except for the day 1 values in panel D (p=0.067).
Figure 4. Progesterone secretion as a function of culture surface. Cytotrophoblasts were cultured in plastic wells without precoating (A), or within wells precoated with fibronectin (B), laminin (C) or type IV collagen (D) in media without (q) or with (Æ) 1.5 mM 8-bromo-cAMP for 48 h as described in the Materials and Methods. Values presented are the means ±S.D. of (n=4) separate experiments. All progesterone differences between cultures treated or not treated with 8-bromo-cAMP were statistically significant to at least the 95th percentile (p<0.05), except for the day 2 values in panel A (p=0.21), the day 1 values in panel B (p=0.11), and the day 2 values in panel D (p=0.07).
Figure 5. Transmission electron microscopy of cytotrophoblasts cultured in serum-free media. Cytotrophoblasts were cultured in serum-free media for 24 h without (A) or with (B-D) 1.5 mM 8-bromo-cAMP and processed as described in the Materials and Methods. A) Cross-section of spherical control cytotrophoblast exhibiting microvilli, coated pits (arrow heads), golgi (G), mitochondria (M), numerous free polyribosomes, and short stacks of irregular RER (arrows). B) Cross-section of treated cytotrophoblast exhibiting many RER stacks (small arrows), active golgi apparatus with numerous small vesicles (large arrow), bundles of peri-nuclear intermediate filaments (small arrow heads), and many small mitochondria (large arrow heads). C) Cross-section of large treated cytotrophoblast showing many dilated, almost tubular, RER profiles, golgi apparatus (G), peri-nuclear intermediate filaments and lipid droplets (L). D) Enlargement from C showing RER (arrows), intermediate filament bundles (arrow heads) and lipid droplets (L). Bars represent 1 µm. (Adapted from Kao, et al., 1988).