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Immunohiostochemical localization of two types of fatty acid-binding proteins in rat ovaries during postnatal development and in immature rat ovaries treated with gonadotropins.

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THE ANATOMICAL RECORD 241235-243 (1995)
lmmunohistochemical Localization of Two Types of Fatty
Acid-Binding Proteins in Rat Ovaries During Postnatal Development
and in Immature Rat Ovaries Treated With Gonadotropins
Department of Anatomy, School of Medicine, Kanazawa University, Kanazawa (S.I., O.A.),
and Department of Biochemistry, Niigata University School of Medicine, Niigata (H.F.,
T.K., T.O.), Japan
Background: The ovary of adult rats expresses two types of
cytoplasmic fatty acid binding proteins (FABP),i.e., heart FABP (H-FABP)
and intestinal 15 kDa protein (I-15P). We studied immunohistochemically
the cellular localizations of these FABPs in the ovaries of rats at various
postnatal ages and in the ovaries of immature (3-week-dd) rats treated with
pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG).
Methods: The cryosections of ovaries were incubated with polyclonal antibodies against H-FABP and I-l5P, and the immunoreactions were visualized at both light and electron microscopic levels.
Results: The immunoreactivity for H-FABP occurred temporarily in the
follicular epithelial (granulosa) cells from 3 days to 2 weeks post partum,
and then was localized exclusively to the thecahnterstitial gland cells from
2 weeks to adulthood. In contrast, the immunoreactivity for I-15P appeared
temporarily in a small subset of theca/interstitial gland cells from 2 to 3
weeks, disappeared at 4 weeks, and was localized exclusively to the corpus
luteum cells after the onset of ovulation in the animal around 5 weeks. In
the immature rat ovaries induced to ovulate by treatment with gonadotropins, I-l5P-immunoreactive cells were first recognized in the luteinized
granulosa layer of large preovulatory follicles, and increased in number
progressively in the developing corpora lutea after the ovulation.
Conclusions: Two types of FABPs are expressed in distinct steroid-producing cell types of rat ovary, and their expressions seem to be regulated in
coincidence with the expressions of respective steroid hormones. These
results suggest that FABPs play specific roles in the ovarian hormone
synthesis. o 1995 Wiley-Liss, Inc.
Key words: Fatty acid-binding proteins, Immunocytochemistry, Ovary,
Postnatal development, Gonadotropins, Rat
Fatty acid-binding protein (FABP) represents a family of low molecular weight (14-15 kDa) cytosolic proteins with high binding affinity for hydrophobic
ligands (Ockner et al., 1972). FABP is produced abundantly in a wide variety of mammalian tissues and
implicated in the uptake, intracellular transport, and
metabolism of long-chain fatty acids (Sweetser et al.,
1987; Veerkamp et al., 1991). The members of FABP
family, with varying degree of sequence similarity, are
expressed specifically in different organs and tissues.
Some organs express more than one type of FABP, either in the same or different cell populations. For example, the absorptive epithelial cell of r a t intestine
produces at least three types of FABP, i.e., liver FABP
(L-FABP), intestinal FABP (I-FABP),and intestinal 15
kDa protein (I-15P) (Shields et al., 1986; Iseki et al.,
1989; Amano et al., 1992). In the adult cycling rat
ovary, both heart FABP (H-FABP) and I-15P are
known to occur. They have been localized immunohistochemically to the thecahnterstitial gland and corpora
lutea, respectively, suggesting their specific roles in
distinct steroid hormone-producing cell types (Watanabe et al., 1991; Amano e t al., 1992). The present study
has been undertaken to clarify further the relationship
between the type of steroid-producing cell and the type
of FABP being expressed, by applying immunohisto-
Received July 5, 1994; accepted September 7, 1994.
Address reprint requests to Dr. Shoichi Iseki, Department of Anatomy, School of Medicine, Kanazawa University, Kanazawa, Ishikawa
920, Japan.
I -15P
Fig. 1. Immunoblot analysis of the specificity of anti-H-FABP and anti-I-15P antibodies. Purified rat
H-FABP (lane 1,O.Z pg), I-15P (lane 2,O.Z fig), and homogenate of adult rat ovary (lane 3,lO pg protein)
were subjected to a SDSipolyacrylamide-gel electrophoresis, blotted onto a nitrocellulose membrane, and
immunostained with the respective antibodies.
chemistry to rat ovaries a t postnatal development and
to immature rat ovaries induced to ovulate by gonadotropins.
H-FABP was purified from the heart cytosol of
Wistar rats according to Offner et al. (1986). The detail
of this procedure was described previously (Kanda et
al., 1989). Polyclonal antisera against H-FABP were
raised in rabbits by subcutaneous injection of the protein emulsified with Freund’s complete adjuvant, and
the IgG fraction of antisera was obtained through a
protein A column (Watanabe et al.,, 1991). I-15P was
purified from the rat small intestinal epithelium by gel
filtration, ion-exchange chromatography, and hydroxyapatite chromatography as described in detail previously (Kanda e t al., 1990, 1991). Antisera against
I-15P were obtrained in the same way as for anti-HFABP (Amano e t al., 1992).
Female Wistar rats were used in the present study at
the postnatal day 0, 1, 2, 3, and 4, and the postnatal
week 1,2 , 3 , 4 , 5 , 6 , and 10 (adult). Adult animals were
either used without consideration of ovarian cycle (for
immunoblotting) or monitored by daily vaginal smears
and used after two consecutive 4-day cycles (for immunohistochemistry). Groups of three 3-week-old animals
were treated with gonadotropins according to the procedure described by Ishimura et al. (1990). Briefly, the
animals reveived a single subcutaneous injection of 10
IU pregnant mare serum gonadotropin (PMSG; UCB
Bioproducts, Braine-I’Alleud, Belgium) and three
groups were sacrificed successively at 12, 24, and 48 h
after the PMSG treatment. The rest of PMSG-treated
groups were then injected intraperitoneally with 10 IU
human chorionic gonadotropin (HCG; UCB Bioproducts) and sacrificed at 6,12,24, and 48 h after the HCG
treatment. All animals, three for each age or experimental group, were sacrificed under a nembutal anesthesia by a transcardial perfusion with cold physiological saline.
Fig. 2. Light micrograph of the ovary from a rat at 3 days post
partum immunostained for H-FABP (a)and I-15P (b).The epithelial
cells of primary follicles (F) are weakly immunopositive for H-FABP
but negative for I-15P. x 660.
Fig. 3. Immuno-electron micrograph showing an H-FABP-positive
epithelial cell of primary follicle in the 7-day old rat ovary. Note the
absence of features of steroid-endocrine cell. A weak immunoreactivity is discerned in the cytoplasmic matrix. M, mitochondrion.
x 21,000. Bar, 500 nm.
Fig. 4. Light micrograph of the ovary from a rat a t 2 weeks post
partum immunostained for H-FABP (a)and I-15P (b).H-FABP reactivity is weakly positive in the granulosa cells of secondary follicles
(F) and intensely positive in the thecaiinterstitial gland cells (I),
whereas I-15P reactivity is localized in a subset of thecaiinterstitial
gland cells (arrow). x 660.
Flg. 5. Electron micrograph showing thecaiinterstitial gland cells in
the 2-week-old rat ovary immunostained for I-15P. An immunopositive cell (asterisk),as well as the adjacent cells, have numerous lipid
droplets and oval mitochondria with tubular cristae. x 5800. Bar, 1
Localization of FA5Ps in the Developing Ovaries
The ovaries removed from a group of untreated adult
rats were combined and homogenized in ice-cold 10 mM
potassium phosphate buffer (pH 7.4) supplemented
with 150 mM KC1. The homogenate samples containing 10 pg protein, together with samples of purified
H-FABP and 1-15P, were separated by electrophoresis
in a 15%acrylamide slab gel a t pH 8.8 in the presence
of 0.1% SDS according to Laemmli (1970). They were
then transferred electrophoretically to a nitrocellulose
membrane (Schleicher and Schuell, Dassel, FRG) according to Towbin et al. (1979). After blotting, the samples were incubated overnight at 4°C with anti-HFABP or anti-I-15P antibody at appropriate dilution.
The immunoreactions were visualized by incubating
the blots successively with biotinylated anti-rabbit IgG
(Vector Laboratories, Inc., Burlingame, CA) for 1 h,
streptavidin-conjugated horseradish peroxidase (Dakopatts, Glostrup, Denmark) for 1 h, and finally with
3'3'-diaminobenzidin tetrahydrochloride in the presence of 0.01% hydrogen peroxide for a few minutes.
The ovary of newborn rat contained numerous primary follicles scattered in the interstitial tissue (ovarian stroma). No immunoreactivity for H-FABP or
I-15P was recognized in any cell population of newborn
rat ovary, including the oocyte, follicular epithelial cell
(granulosa cell), and stromal cell. At 3-4 days post
partum, most of the granulosa cells began to exhibit a
weak immunoreactivity for H-FABP (Fig. 2a), which
increased in intensity a t 1 week and continued to be
present, although with decreasing intensity, until 2
weeks. In contrast, no reactivity for I-15P occurred in
any cell population before 2 weeks (Fig. 2b). On immuno-electron microscopy, the K-FABP-positive granulosa cells at 1 week had abundant free ribosomes,
small elements of rough-surfaced endoplasmic reticulum, poorly developed Golgi apparatus, and small mitochondria with lamellar cristae (Fig. 3).
At 2 weeks, many secondary follicles with multilayer
of granulosa cells were seen. The theca interna layer
around the follicular epithelium, as well a s the interstitial glands in the matrix, began to develop. In addition to the weak H-FABP immunoreactivity remaining
in the granulosa cells, almost all the thecahnterstitial
gland cells exhibited intense reactivity against anti-HFABP antibody (Fig. 4a). On the other hand, a small
subpopulation of thecahnterstitial gland cells was immunostained with anti-I-15P antibody (Fig. 4b). It was
not clear whether this subpopulation was reactive only
for I-15P or simultaneously for both H-FABP and
I-15P. Immuno-electron microscopy further demonstrated that the I-15P-positive cells had the appearance
of steroid-secreting cell, with numerous lipid droplets,
oval mitochondria with tubular cristae, and many profiles of smooth-surfaced endoplasmic reticulum (Fig. 5).
There was no apparent difference in ultrastructure between the I-15P-positive and -negative thecahnterstitial cells.
At 3-4 weeks, some antral follicles were seen in addition to the primary and secondary follicles, and the
interstitial glands were further developed. The immunoreactivity for H-FABP disappeared from the granulosa cells by 3 weeks, whereas the thecahnterstitial
gland cells continued to be immunostained intensely
with anti-H-FABP antibody (Figs. 6a, 7a). On the other
hand, the I-15P-immunoreactivity in the small subset
of thecahnterstitial gland cells was still present a t 3
weeks but diappeared by 4 weeks (Figs. 6b, 7b).
Around 5 weeks, the ovarian cycles started and
many corpora lutea began to be seen in the ovary. The
luteal cells were immunostained intensely with anti-115P antibody, while being entirely immunonegative
for H-FABP (Figs. 8a,b). In the adult cycling rats, the
immunoreactivities for H-FABP and I-15P continued
to be present in the thecahnterstitial gland cells and
corpus luteum cells, respectivitly, throughout the
stages of cycle. In contrast, the granulosa cells were
devoid of immunostaining with either antibody, even
in the large antral follicles of the proestrous phase.
For light microscopic immunohistochemistry, the animals were fixed by a transcardial perfusion with 4%
paraformaldehyde in 0.1 M phosphate buffer, pH 7.2.
The ovaries were excised out and further immersed in
the same fixative for 4 h at 4°C. The tissue blocks, after
rinsing overnight with phosphate buffer containing
30% sucrose, were frozen, cut into 15 pm sections with
a cryostat, and mounted on gelatin-coated glass slides.
The sections were incubated with anti-H-FABP or antiI-15P rabbit antibody a t the concentrations of 2-10 kg
IgG/ml overnight a t room temperature. The sites of
immunoreactions were visualized by incubating the
sections successively with porcine anti-rabbit IgG antibody (Dakopatts) for 1 h and peroxydase-antiperoxydase complex (PAP, Dakopatts) for 1h, according to the
method of Sternberger (1974). For negative control, the
primary antibody was replaced by non-immune rabbit
serum. For electron microscopic immunocytochemistry, the sections immunostained with PAP procedure
were postfixed with 0.5% OsOI for 20 min a t room temperature. After en block staining with uranyl acetate,
the sections were dehydrated in graded ethanol series
and embedded in epoxy resin. Ultrathin sections were
made for observation with Hitachi H-700 electron microscope. Details of the immunohistochemical procedures were described previously (Iseki et al., 1989).
Specificity of the Antibody
On the Western blotting analysis, anti-H-FABP and
anti-I-15P antibodies reacted not only with the respective purified FABPs but also with the homogenate of
adult rat ovary, forming single protein bands with molecular weights of 14-15 kDa (Fig. 1).There was no
cross reaction between the two antibodies. This result
indicated that the prcscnt antibodies specifically detect
H-FABP and I-15P that are produced in the rat ovary.
In the subsequent immunohistochemical study, the
control slides incubated with non-immune rabbit serum as primary antibody showed no staining in any
cell or extracellular element.
Localization of FABP in Immature ovary Induced to Ovulate
It is well established that treatment of the non-cylcing animals such as immature rats or hypophysectomized rats with gonadotropins gives rise to a synchro-
Figs. 6-8.Light micrographs of the ovaries from rats a t 3 weeks (6),
4 weeks (7), and 5 weeks (8)postpartum immunostained for H-FABP
(a) and I-15P (b). F, follicle; I, interstitial gland; L, corpus luteum.
x 330. ( 6 )H-FABP immunoreactivity is positive in all thecaiinterstitial gland cells, whereas I-15P reactivity is positive in a subset of
nized process of follicle development, ovulation, and
corpus luteum formation in the ovary (Zarrow et al.,
1958). In the present system, the follicles of immature
rat ovaries are induced to develop into large preovulatory (Graafian) follicles by 48 h after administration of
PMSG, and subsequent treatment with HCG brings
about a synchronized ovulation of many follicles that
peaks around 12 h after administration of HCG (Yoshinaga-Hirabayashi e t al., 1990).
thecaiinterstitial gland cells. (7) All thecaiinterstitial gland cells are
positive for H-FABP but negative for I-15P. ( 8 ) H-FABP reactivity is
localized in the thecaiinterstitial gland cells, whereas I-15P reactivity
is localized in the corpus luteum cells.
The ovary of unstimulated 3 week-old-rats contained
many secondary follicles and some small antral follicles surrounded by the theca interna layer, a s well as
moderately developed interstitial glands. All cells of
thecaiinterstitial glands were intensely immunostained with anti-H-FABP antibody (Fig. 6a), whereas
a weak I-15P-immunoreactivity was recognized in a
small subpopulation of thecaiinterstitial gland cells
(Fig. 6b). In contrast, the granulosa cells of small and
FIgS. 9-12.
TABLE 1. Immunohistochemical localization of two types of FABP in various cell types of rat ovarv
Age (days) and hormone treatment'
-' +
'The hormones are administered a t 21 days post partum.
2The immunoreaction is evaluated a s negative (-), some cells positive (?), and most cells positive ( + )
3Theca interna cells and interstitial gland cells.
large follicles were completely devoid of either immunoreactivity. By 24 h after PMSG administration,
many antral follicles were formed, but none of them
showed either immunoreactivity in the granulosa cells.
Forty-eight hours after PMSG treatment, maturation of follicles had further progressed and many
Graafian follicles with abundant follicular fluid were
seen. These follicles had not ovulated, as proved by the
presence of oocyte in serial sections. The immunoreactivity for I-15P was recognized in some of the granulosa
cells of Graafian follicles (Fig. 9b). On immunoelectron
microscopy, the granulosa cells of this stage contained
numerous smooth-surfaced endoplasmic reticulum,
lipid droplets, and round mitochondria with tubulovesicular cristae, which are characeristics of the luteinized cells (Fig. 10). All the immunopositive granulosa
cells showed luteinization, whereas the immunonegative population contained both luteinized and less
luteinized cells.
Twelve hours after the HCG treatment following 48
h PMSG treatment, the ovary contained both the preovulatory Graafian follicles and newly formed corpora
lutea. I-15P-immunoreactivity was present in the majority of the granulosa cells of Graafian follicles, a s well
as in cells of corpora lutea (Fig. l l b ) . Twenty-four
hours after HCG treatment, the ovary was filled with
corpora lutea in their developing stages. Almost all the
Fig. 9. Light micrograph of the ovary from a 21-day-old rat treated
with PMSG for 48 h and immunostained for H-FABP (a)and I-15P
(b).Some of the granulosa cells of large preovulatory follicle are positive for I-15P (arrow). F, follicle; I, interstitial gland. x 330.
Fig. 10. Immuno-electron micrograph showing an I-15P-positive
granulosa cell of large preovulatory follicle in the ovary from a 21day-old rat treated with PMSG for 48 h. Note the lipid droplets (L),
abundant smooth endoplasmic reticulum (sER), and large mitochondria with tubulovesicular cristae (M). A strong immunoreactivity is
distributed in the cytoplasmic matrix. x 21,000. Bar, 500 nm.
Figs. 11, 12. Light micrographs of the ovaries from 21-day-old rats
12 h (11)and 24 h (12) after HCG injection immunostained for
H-FABP (a)and I-15P (b).F, follicle; I, interstitial gland; L, corpus
luteum; 0, oocyte. x 165. (11)H-FABP reactivity is localized in the
thecaiinterstitial gland cells, whereas I-15P reactivity is localized in
the granulosa cells of large preovulatory follicle and in the corpus
luteum cells. (12) H-FABP reactivity is localized in the thecaiinterstitial gland cells, whereas I-15P reactivity is localized in the corpus
luteum cells.
luteal cells were strongly immunostained with anti-115P antibody (Fig. 12b).
Throughout the course of follicle maturation, ovulation, and luteinization induced by the hormone treatment, H-FABP-immunoreactivity was localized exclusively in the thecahnterstitial gland cells (Figs. 9a,
l l a , 12a). Also, a small number of I-15P-positive cells
continued to be present in the thecahnterstitial compartment of hormone-stimulated ovaries.
Table 1 summarized the results of immunohistochemistry for the two FABPs in rat ovary.
The steroid-secreting endocrine cells in the rat ovary
are composed of four populations, i.e., theca interna
cells, interstitial gland cells, corpus luteum cells, and
granulosa cells of antral follicles (Guraya, 1972). The
interstitial gland cells, which are thought to be derived
from the theca interna of atretic follicles, share many
common morphological and functional features with
the theca interna cells (Erikson e t al., 1985). The thecalinterstitial gland cells are considered the main
source of ovarian androgens, because they express the
enzymes, 17a-hydroxylase/C17-C20 lyase which synthesizes androstenedione from progesterone (Hedin et
al., 1987; Ishimura et al., 1990) and 17P-hydroxysteroid dehydrogenase which converts androstenedione to
testosterone (Yoshinaga-Hirabayashi et al., 1987),
while lacking aromatase which converts testosterone
into estradiol (Ishimura et al., 1989). In the present
study, immunoreactivity for H-FABP has been localized primarily to the thecahnterstitial gland cells, irrespective of the rat age and hormonal condition. This
result, together with an earlier observation of the occurrence of H-FABP in the Leydig cells of rat testis
(Watanabe et al., 1991), suggest a role of H-FABP in
the metabolism of androgens. On the other hand, the
temporary occurrence of weak H-FABP-reactivity in
the granulosa cells of 0-2-week-old rat ovaries cannot
be interpreted along this line. The granulosa cells of
this stage have no characteristics of steroid-endocrine
cells, a s shown by their morphological features (this
report) and lack of cholesterol side-chain cleavage enzyme (Farkash et al., 1986). Therefore, the role of
H-FABP in the granulosa cells of infantile rat ovary
may be rather related to the transportation and metabolism of long-chain fatty acids a s energy sources, as is
postulated for other cell populations like heart and
skeletal muscle cells (Veerkamp et al., 1991).
Recent immunohistochemical studies have shown
that aromatase is absent in the granulosa cells of small
follicles but is localized exclusively in those of larger
antral follicles, which occur in the adult cycling rat
ovary and in the immature rat ovary stimulated with
PMSG for 24 h (Ishimura et al., 1989; Yoshinaga-Hirabayashi et al., 1990). Since the granulosa cells are
lacking in cholesterol side chain-cleavage enzyme, they
are thought to synthesize estrogen from androgen that
is provided by the surrounding thecalinterstitial gland
cells (Richards, 1980). The present study has shown
no immunoreactivity for H-FABP or I-15P in the granulosa cells of antral follicles, either in the cycling
rats or in the immature rats stimulated with PMSG
for 24 h. This finding suggests that the expression of
either of two FABPs does not correlate with the synthesis of estrogen. However, 48 h after stimulation
with PMSG, I-15P-immunoreactivity is found in some
of the granulosa cells of large preovulatory follicles.
Immunoelectron microscopy further indicates that the
immunopositive cells have the morphological features
of steroid-producing cells and are thus on the way to
luteinization. This is followed by further increase in
the number of immunopositive granulosa cells in the
preovulatory follicles 12 h after HCG treatment. This
process of I-15P occurrence is coincident with the conversion of estrogen-producing granulosa cells into progesterone-producing luteal cells, which is represented
by the occurrence of cholesterol side-chain cleavage enzyme in the granulosa cells 48 h after PMSG treatment
(Zlotkin et al., 1986), and the loss of aromatase in the
corpus luteum cells 15 to 18 h after HCG treatment
(Yoshinaga-Hirabayashi et al., 1990). The present findings, therefore, implicate that I-15P is a histochemical
marker of progesterone-endocrine cells. If this is the
case, the small subpopulation of I-15P-positive theca/
interstitial cells observed in the 2-3 week ovaries
might represent a progesterone-producing subpopulation. The physiological significance of the temporary
occurrence of such a minor cell population is unknown.
The present study has revealed that two types of
FABPs are expressed in distinct steroid-endocrine cell
types of rat ovary, and that their expression seems to
be regulated cooperatively with the changes in steroidhormone metabolism during postnatal development
and periovulatory period. In this conjunction, there is a
cytoplasmic protein named sterol carrier protein 2
(SCP2, also called nonspecific lipid-binding protein)
which has a similar molecular weight but has no sequence homology with FABP family (Vahouny et al.,
1987). SCP2 binds cholesterol and is implicated in the
transfer of cholesterol among membranes as well a s the
metabolism of cholesterol. SCP2 is present not only in
the liver and intestine but also in major steroid-endocrine organs, including the ovary (Tanaka et al., 1984).
Interestingly, SCP2 is expressed in both the corpus luteum and thecalinterstitial cells of rat ovary, in coincidence with the expression of cholesterol side-chain
cleavage enzyme (Rennert et al., 1991). This is consistent with the notion that SCP2 facilitates the initial
step of steroid biosynthesis, i.e., conversion of cholesterol to pregnenolone (Vahouny et al., 1987). On the
other hand, the present study suggests that H-FABP
and I-15P are involved defferencially in the syntheses
of androgen and progesterone, respectively. The most
likely explanation for this result is that the two types
of FABPs bind different metabolic intermediates of steroid biosynthesis. So far, however, little evidence is
available for the affinity of FABP family with cholesterol and its derivatives. Of the members of FABP family, only L-FABP has been demonstrated to bind cholesterol (Nemecz and Schroeder, 1991). In contrast,
H-FABP is known to bind only long-chain fatty acids
(Veerkamp et al., 1991). As for I-l5P, some researchers
have documented its binding affinity with cholesterolderived bile components such a s chenodeoxycholate
(Sacchettini et al., 1990; Lin et al., 1991), but others
have failed to find this (Kanda et al., 1991). In order to
clarify the roles played by the two FABPs in rat ovary,
i t is of primary importance to investigate further their
binding affinities with various metabolic intermediates of steroid biosynthesis.
This study was supported by Grant-in-Aid 04670009
from the Ministry of Education, Science and Culture of
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development, acid, two, typed, rat, fatty, ovaries, immunohiostochemical, protein, localization, immature, postnatal, binding, treated, gonadotropin
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