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Morphogenesis of the ovarian interstitial tissue in the neonatal mouse.

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Morphogenesis of the Ovarian Interstitial Tissue
in the Neonatal Mouse '
Departments of Anatomy and Obstetrics and Gynecology, T h e Milton S.
Hershey Medical Center, T h e Pennsylvania State University,
Hershey, Pennsylvania 17033
The development of the mouse ovarian interstitial tissue was
examined by light and electron microscopy at postnatal days 9, 10, 11, 12, and
18. Prior to day 1 2 the ovarian stroma is composed of fibroblast-like cells. These
cells contain rough endoplasmic reticulum, elongate mitochondria with lamellar
cristae, and lipid bodies which appear uniformly electron opaque. On postnatal
day 12 islands of lipid containing cells are seen deep in the interfollicular stroma.
These cells referred to as interstitial cells have rounded mitochondria with tubular cristae, smooth endoplasmic reticulum, lipid bodies with extracted centers,
well-developed Golgi complexes, and solitary cilia lacking the central tubular
elements. By postnatal day 18 similar cells are seen in the thecal regions of
normal appearing follicles. Also seen in the theca are cells whose cytoplasm
contains characteristics of both fibroblasts and interstitial cell.
It is proposed that the interstitial cells develop from the fibroblast-like stromal
cells, that this development proceeds in a wave from deep in the interfollicular
tissue toward the follicle, and that the interstitial cells are the source of ovarian
steroid production during this period of development.
Fetal development of the mammalian
ovary involves the organization of the
stromal cells into follicular epithelia and
interfollicular stroma (Odor and Blandau,
'66). During postnatal life these units are
subject to drastic structural and functional
changes which may result in the formation of a steroidogenic tissue. Investigations of such changes have primarily been
concerned with the postpubertal period.
Little emphasis has been given to the development and function of the prepubertal
ovary and its role in the establishment of
the postpubertal cellular components. The
present study pertains to structural
changes in the ovarian stroma of the
mouse which by the end of the second
week of postnatal life facilitate the recognition of the ovarian interstitial tissue. Cellular and subcellular organization has
been examined by light and electron microscopy with particular emphasis on the
pattern of tissue differentiation and the
appearance of subcellular structures generally associated with steroidogenesis
(Fawcett et al., '69). The correlation of
ANAT. REC., 177: 569-584.
structure and the proposed function of the
interstitial cells of the immature mouse
will be discussed.
The mice used for this study were of
the C57 BL/6J strain. They were maintained on Purina Lab Chow ad libitum in
a 1 2 : 1 2 hour light-dark cycle. Animals
were killed by cervical dislocation between
9 and 10 A.M. on postnatal days 9, 10, 11,
12, and 18 (day of birth = day 0). Sampling
of a litter for ages 9-12 was done by one
of two methods: all females were killed on
a given day or one half the females were
removed on two successive days. Litters
used were of six to eight pups. Ovaries
were removed and fixed by immersion in
a mixture of 3% glutaraldehyde and 1%
paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for one hour at
room temperature. Tissues were subsequently washed, fixed with 1% OsOd in
0.1 M Sodium cacodylate for one hour deReceived May 21, '73. Accepted July l l , '73.
1 Aided in part by NICHD grant 00282 Post Doctoral
Training Program in the Physiology of Reproduction.
hydrated in alcohol and embedded in
Araldite (Durcupan-ACM, Fluka). Thin
sections were stained with uranyl acetate
and lead hydroxide and viewed with a
Philips 300 electron microscope. Sections
in thickness were examined by
light microscopy after staining with toluidine blue (Trump et al., '61). Frozen sections of unfixed tissues were stained with
oil red-0. The identification of intracellular granules as lipid was based on the following criteria : positive staining with oil
red-0 on frozen sections, metachromatic
staining with toluidine blue and increasing staining following treatment with
periodic acid using 0.5-1 sections of plastic embedded tissue, increasing homogeneity of granules with prolonged exposure to osmium during fixation.
Although cell types and tissue organization change during ovarian development,
much of the terminology used to describe
the ovary of the immature animal is identical to that used for the adult. Because
such a nomenclature can lead to confusion,
the following terminology has been
adopted, The term interfollicular region
refers to that area between follicles and
delineated from the follicles by the basal
lamina. The term undifferentiated stroma
will be used to describe the accumulations
of cells in the interfollicular region which
have not exhibited any sign of specialization or tissue organization. As the animal
matures, cells of the interfollicular region
differentiate and become organized into a
variety of tissues. The lamina of cells
adjacent to the basal lamina of the follicle
becomes the theca.
During the first 11 days of postnatal life,
the mouse ovary is composed of numerous
small follicles and an apparently undifferentiated stroma. The follicles generally
contain one or two layers of follicle cells
and have no antra. They are delineated by
a basal lamina (fig. 1). Cords of follicle
cells, also delineated by the basal lamina
of the follicle (figs. 1, 2 ) are frequently
seen projecting into the interfollicular
regions. The cytoplasm of the cord cells,
as well as that of the cells of the follicle
proper, frequently exhibit lipid granules
which are uniformly electron dense. These
cells also contain elongate mitochondria
with lamellar cristae and rough endoplasmic reticulum. In the interfollicular region
of the ovary at this time numerous fibroblast-like cells are seen (figs. 1, 2, 4). They
are elongate cells which exhibit a basophilic cytoplasm after staining with toluidine blue. Some intracellular lipid is found
in these cells by postnatal day 11 but not
as much is found in the follicular tissue.
When present this lipid is uniformly electron opaque. While the cytoplasm of these
interfollicular cells is not as abundant as
that of the follicle cells, the mitochondria
and endoplasmic reticulum are similar in
appearance to those described above.
As the animal matures, the amount of
interfollicular tissue increases. Mitotic
figures are frequently seen (fig. 1). By
postnatal day 12, some of the cells of this
tissue are arranged in a thecal layer adjacent to the basal lamina of the follicle (fig.
3 ) . Most of the cells comprising this layer
are similar to those described above as
fibroblast-like cells, and collagen fibers are
seen frequently in the intercellular spaces.
Some of the thecal cells, however, have a
more rounded appearance and contain
larger numbers of lipid bodies. Unlike the
fibroblast-like cells, the endoplasmic reticulum of these cells (fig. 5) is predominantly
of the smooth variety; the mitochondria
are more rounded with some tubular as
well as lamellar cristae.
In the deeper regions of the interfollicular tissue at post-natal day 12, individual
and clusters of cells are seen which are
readily distinguished from the surrounding tissue (fig. 3 ) . These cells shall be
referred to as interstitial cells. They are
more rounded than the cells of the theca
and contain larger numbers of lipid bodies.
This lipid is localized in regions deep in
the cytoplasm of the cell rather than being
evenly distributed. Since it is refractile to
staining with toluidine blue, it imparts a
frothy appearance to the cytoplasm. Electron microscopic examination of this lipid
reveals an electron-lucent core and opaque
periphery (figs. 6-8). The Golgi complex in these cells (fig. 8) contains greater
numbers of lamellae stacks and vesicular
elements than have been seen either in
follicle cells or interfollicular cells of
younger animals. A single cilium lacking
the central tubular elements is frequently
seen in an indentation of the interstitial
cell. Similar cilia have been observed in
follicle cells and fibroblast-like cells at all
ages studied. The basal structures of such
cilia are found in the region of the Golgi
The endoplasmic reticulum is more
highly developed in the interstitial cells
than in other cells of the ovary. It is tubular and predominantly of the smooth surfaced variety although some ribosome
studded segments are seen. It is evenly dispersed throughout the cytoplasm and is
often found closely applied to the lipid
granules and mitochondria (figs. 7, 8).
The mitochondria which also appear to be
mwe numerous are spherical with tubular
The cords of follicle cells seen in
younger animals are still present at postnatal day 12, and are still delineated by a
basal lamina (fig. 3). The cord cells contain lipid granules, but they are not as
numerous as those of the interstitial cells,
are more evenly dispersed, and are uniformly electron dense. Mitochondria and
endoplasmic reticulum are similar to those
seen in similar cells in younger animals.
By postnatal day 18, the clusters of interstitial cells are more prominent due to
an increase in their number and the number of cells comprising them (figs. 9, 10).
The mitochondria of these cells are generally larger and contain a greater density
of tubular cristae than was seen in younger
In the thecal region at 18 days of age
a third cell type is seen, in addition to
those which were seen in postnatal day 12
(fig. 10). The cytoplasmic components of
these cells are similar in structure to those
of the interstitial cells found clustered in
the deeper regions of the interfollicular
region. These cells shall be referred to as
thecal interstitial cells.
The thecal interstitial cells have not
been found juxtaposed to the basement
lamina of the follicle. There are generally
two or more layers of cells between them
and the follicle (fig. 10). Frequently,
capillary endothelial cells are found in
this area. Adjacent to the follicle fibroblasts are generally found. In addition to
the fibroblasts and the thecal interstitial
cells, cells which appear to be transitional
between these two cell types are seen in
the theca. These transitional cells contain
some lipid which is generally uniformly
electron opaque. Their endoplasmic reticulum is predominantly of the rough variety
although smooth elements are evident.
Mitochondria are more variable in size,
shape and cristae configuration in these
cells than in other thecal cell types. They
are generally small, elongate to oval, with
lamellar and/or tubular cristae.
The appearance of subcellular structures characteristic of steroidogenic cells
has been taken as the basis for the identification of the ovarian interstitial cells of
the immature mouse. These structures include the tubular smooth endoplasmic
reticulum, rounded mitochondria with tubular cristae, and lipid granules. The selection of such a criterion for interstitial cell
identification is not based on direct proof
of its steroidogenic function in the immature mouse, but rather its generally
accepted role in the adult.
Bouin ('02) was among the first to recognize the interstitial tissue on the basis
of lipid inclusions. Subsequent identification has also involved histochemical demonstration of steroid dehydrogenase activity (Hart et al., '66). There are, however,
problems in utilizing these techniques for
the identification of the interstitial tissue
in the immature mouse. Both lipid and
steroid dehydrogenase activity are found
in follicle cells as well as interfollicular
regions of the immature ovary (Mendelson and Leathem, '70). Thus as demonstrated by the present techniques of light
microscopy, the presence of lipid and
steroid dehydrogenase are insufficient to
distinguish the interstitial cells.
Ultrastructural analysis of the lipid indicates differences between the cells of
the follicular and interfollicular regions of
the ovary. The lipid granules of the interstitial cells exhibit an electron lucent
center. Lipid granules in nearby follicle
cells or fibroblasts are uniformly electron
dense, as are granules in presumptive interstitial cells which are seen prior to postnatal day 12. This pattern is apparently
due to extraction during the dehydration
and embedding procedures. Care was taken
that block size and tissue preparation
schedules were uniform. When tissues
were osmicated for a greater period of
time, interstitial cells did not exhibit the
extraction pattern, It is assumed that the
alteration in lipid granule structure is a
reflection of qualitative changes in the
lipid and a result of metabolic changes in
the differentiating interstitial cells.
The observation of single cilia in the
interstitial cells of the immature mouse
ovary is not unique among steroidogenic
cells. Wheatley (’67) reported similar
structures in the adrenal cortical cells and
Crisp and Browning (‘68) observed them in
the cells of the corpus luteum. Such cilia
have also been seen in the follicle and
stromal cells as well as oogonia (Anderson, ’70; Motta et al., ’71). In all such
cases the cilia are found in the Golgi
region, lack the central tubular elements,
and are assumed to be non-motile. Speculations have been made concerning the
function of these ubiquitous structures
(Archer and Wheatley, ’71; Rash et al.,
’69; Crisp and Browning, ’68). At this
time, however, the function of the cilia
found in the interstitial tissue is unknown.
The development of an identifiable interstitial cell is dependent on the hypertrophy
and hyperplasia of certain membranous
components within the presumptive interstitial cell. Membranes of the endoplasmic
reticulum mitochondria1 cristae and Golgi
complex are greatly increased in interstitial cells of 12 day old animals. The close
spatial association between the smooth endoplasmic reticulum and the mitochondria
and lipid implies a functional correlation.
This is supported by the close timing of
their appearance. We are unable, however,
on the basis of this data to identify the
functions of these cell components.
The presence of these organelles leads
to the assumption that the interstitial cells
are involved in steroidogenesis. Histochemical studies support this contention
(Hart et al., ’66). Studies with immature
rats indicate the presence of an extractable, biological active estrogen within the
ovary (Cieciorowska and Russfield, ’68).
Incubations of ovaries from immature rats
indicate an ability to convert progesterone
to estrone and estradiol (Quattropani and
Weisz, ’73). The amount of progesterone
converted to estradiol at the various ages
examined is correlated directly with the
increase in recognizable interstitial cells
in this species. Preliminary work with
incubations of mouse ovaries indicate a
similar ability to produce estrogens on
postnatal day 16 (Quattropani, unpublished observations).
The obvious structural changes have
been considered as an end point in the differentiation of the interstitial tissue. The
factors leading to these changes are not
so obvious. From the data presented herein,
it would appear that the interstitial tissue
develops from a wave of differentiation
which is initially apparent in the region
of the interfollicular stroma most distant
from the follicles and subsequently progresses toward them.
While we have referred to the cells of
the interfollicular stroma as the source of
interstitial cells in the immature mouse,
other sites of origin have been proposed.
These thories have involved the classification of the interstitial cells into “primary”
and “secondary” (Dawson and McCabe,
’51; Rennels, ’51). The primary, being the
first to appear, is generally thought to arise
from stromal cells (Stegner, ’70), although
cords of cells projecting from the follicle
have also been proposed as their site of
origin (Rennels, ’51). The present investigation indicates that during the time that
the interstitial cells are developing, the
cells of these cords exhibit an ultrastructure similar to that of the follicle cells and
that the cords are delineated by a basal
lamina. This dissimilarity with the interstitial cells had previously been noted
(Brandau, ’70; Merchant and Zomboni,
’72). The secondary interstitial tissue has
been described as arising from the theca
of atretic follicles (Rennels, ’51). It is
this class which supposedly makes up the
majority of the adult tissue.
In the present study the classifications of
primary and secondary have not been
adopted. The ovary of the 12 day old
mouse does exhibit a theca. This layer is
distinguished by the high proportion of
fibroblasts arranged about the follicle.
These cells are similar in subcellular structure to the stromal cells seen earlier. However, by postnatal day 18 interstitial cells
which are indistinguishable ultrastructurally from those found deep in the interfollicular region are also found in the
theca. The follicles around which these
cells are found do not appear to be atretic.
The data presented herein indicate postnatal day 12 as the period during which
interstitial cells are first recognized. It
should be pointed out, however, that this
schedule of differentiation is quite likely
peculiar to the conditions of this study. Indeed, in a previous study with C57BL/6Ra
mice, the first appearance of interstitial
cells was noted on postnatal day 10 (Quattropani, unpublished observations). Stenger ('70:i using Swiss Albino mice found
similar cells on postnatal day 7.
While we have no data to explain the
induction of the changes which result in
the development of the interstitial cells,
there are two factors which should be considered: the contribution of the oocyte, or
follicular unit, and the effect of circulating gonadotrophins. In light of the pattern
of the appearance of the interstitial cells
(distal and then proximal to the follicle)
the inhibiting effect of the oocyte or follicle might be considered. Evidence has
been presented by Nekola and Nalbandov
('71) that the oocyte has an inhibitory
effect on the luteinization of the granulosa cells in vitro. In the rat correlations
of interstitial cell development and circulating gonadotrophin levels neither support nor refute a causal relationship
(Quattropani and Weisz, '73). While peak
levels occur after the development of the
tissue, less than peak may be sufficient.
Unfortunately, gonadotrophin levels in
C57BL/6J mice are not yet available.
While the suddenness of the structural
changes suggests a role for alterations in
hormonal milieu, the pattern or distribution of these changes also suggests an
oocyte effect. It would, therefore, appear
that the differentiation of the interstitial
tissue of the young mouse is the result of
the interaction of several factors in a labile
The author wishes to express his appreciation to Dr Everett Anderson under
whose guidance this study was initiated.
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phosphatase and the uptake of horseradish
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interne de l'ovaire; l a glande interstitielle et le
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Light micrograph of an ovary from a n 11 day old mouse. Cords of
follicle cells (FC) and mitotic figures i n the interfollicular tissue
(arrows) are present X 480.
Electron micrograph of the periphery of a follicle from and 11 day
old animal showing zona pellucida (ZP), follicle cells ( F ) and
follicle cord cell (FC) with basal lamina (EL) and fibroblast-like
cells ( S ) of the stroma. X 12,000.
Section from 12 day old animal showing islands of interstitial cells
(arrows) and follicle cell cords (FC). X 480.
Steven L. Quattropani
Stroma of 11 day old animal. Endothelial cell of capillary ( C ) and
fibroblast-like cells ( S ) with lipid ( L ) rough endoplasmic reticulum
and elongate mitochondria with lamellar cristae. x 19,500.
L. Quattropani
5 Lipid (L) containing thecal cell from 12 day old animal. Elements
of smooth (arrow) and rough endoplasmic reticulum and mitochondria with tubular cristae are seen. x 18,500.
Interstitial cell of 12 day old animal found in a n island of lipid containing stromal cells. Lipid ( L ) is extracted centrally, mitochondria
are rounded with numerous tubular cristae, the endoplasmic reticulum is predominantly of the smooth variety. x14,500.
Steven L. Quattropani
Interstitial cells of a 12 day old animal exhibiting close apposition
of smooth endoplasmic reticulum to lipid and mitochondria
(arrows). x 18,500.
The region of the Golgi apparatus ( G ) is seen in this interstitial cell
of a 12 day old animal. A cross section of a cilium ( C ) is found
i n a depression in the cell. X 18,500.
Steven L. Quattropani
Light micrograph of ovary froin a n 18 day old animal showing
islands of interstitial cells ( I ) and thecal interstitial cells (arrows).
x 570.
The theca adjacent to a follicle (F) from a n 18 day old animal.
Fibroblast-like cells ( S ), interstitial cell ( I ) , intermediate cell types
( P ) , and endothelial cells ( C ) are seen. X 13,000.
Steven L. Quattropani
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ovarian, interstitial, mouse, tissue, neonatal, morphogenesis
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