Cytology of Oocyte and Somatic Cell

Development in Mammalian Ovarian Follicles

Summary
Cellular interactions in the mammalian ovarian follicle
between its germ-line and somatic cell components are
crucial for its development and function. These
interactions are mediated by both membrane gap
junctions and paracrine factors. Somatic cell-to-oocyte
communication is essential for oocyte growth and the
regulation of meiotic maturation. In particular, granu-
losa cells provide nutrients and molecular signals that
regulate oocyte development. Oocytes, on the other
hand, promote the organization of the follicle, the
proliferation of granulosa cells, and the differentiation
and function of cumulus cells, a subset of granulosa cells.
Determining the nature of the oocyte-to-granulosa cell
signals remains a key challenge for future work.
Oogonial mitosis ceases at some time during fetal
development, the germ cells then entering meiosis and
becoming oocytes. 'The progression of meiosis in
oocytes is arrested at prophase and remains stopped at
this stage until preovulatory or atretic changes occur in
the large follicle. Oocytes, therefore, can remain in
prophase for years, depending upon the species. The
initial, or primordial, ovarian follicles consist of an
oocyte, 15 to 20pm in diameter, surrounded by a few
flattened primitive granulosa cells (Fig. 1) and consti-
tute a non-renewable source of germ cells. These
primordial follicles can persist during the functional lifc
of the ovary, but cohorts can begin development at any
time during this life in response to a signal or signals
whose origin or nature are unknown. Mammalian
oocytes grow to 70 to 100 pm in diameter, depending on
the species. and this growth is accompanied by first, a
thickening of the flattened primitive granulosa cell to a

Introduction
Ovarian follicles, the fundamental functional units of
ovaries, do not form in the absence of oocytes. When
female germ cells are destroyed or fail to develop
appropriately, somatic support cells do not assemble
into follicles. In sharp contrast: seminiferous tubulcs,
the male counterparts of ovarian follicles, do form in
the absence of germ cells, although the germ cell-
deficient tubules probabIy do not function normally(').
Clearly somatic and germ-line cells interact from the
earliest stages of ovarian development. Subsequently,
in normal development, the oocyte and somatic cells in
thc follicle develop coordinately and interdependently
in situ, although some aspects of the development of
each can be separated experimentally('). Significantly.
the interactions of follicular germ and somatic elements
are regulated by extrafollicular signals, most notably
the gonadotropic hormones, FSH' and LH secreted by
the anterior pituitary.
This review will focus on the nature of the cellular
interactions in the development of the ovarian follicle.
Current concepts of the regulation of mammalian
oocyte development by follicle cells and the new
findings on the role of the oocyte in the development
and function of follicular somatic cells will be discussed. Fig. I . Stages of mammalian ovarian follicular devclopmcnt.
A. Primordial follicles (arrow heads) and preantral follicle (arrow);
Abbreviations: CAMP, cyclic adenosine monophosphate; FSH, follicle- B. Preovulatory antral follicle; thc arrowhead indicates the cumulus
stimulating hormone: GVB, gcrniiiial vesicle breakdown; LH. luteiiiizing cell-enclosed oocyte and the arrows delineate the mural granulosa
hormone. cells. Scale bars equal 100prn.
cuboidal shape, and then proliferation of the granulosa
cells (Fig. 1).The oocytes remain arrested in prophase
of meiosis throughout their growth. Because these early
stages of oocyte and follicular development can occur in
the absence of gonadotropins, they are generally
considered gonadotropin-independent . However, these
follicles can rcspond to gonadotropins and generate
CAMP and steroid hormones in response to them in
vitrd3,"). When the oocyte nears the end of its growth
phase, a cavity, or antrum, forms among the granulosa
cells in most mammalian species (Fig. 1). Subsequent
growth of antral follicles is gonadotropin-dependent.
Two morphologically distinct subpopulations of
granulosa cells exist upon formation of the antrum;
mural granulosa cells that line the follicle wall and
cumulus granulosa cells that encompass the oocyte
(Fig. 1). Many of the mural granulosa cells appear to be
organized as a pseudostratified epithelium contacting a
basal lamina, while many of the cumulus granulosa cells
are similarly organized contacting the oocyte. The
mural granulosa cells are a heterogeneous population in
which some cells express some molecules more than
other cells. For example, the mural granulosa cells
closest to the basal lamina develop more LH-receptors
than do the more centrally located cells that line the
antrum(').
Throughout follicular development the granulosa
cells can communicate with each other and the oocyte
via gap junctionsf6-'). These channels through the
plasma membranes allow the exchange of low molecu-
lar weight molecules including those that are known to
partici ate in cellular signaling mechanisms, such as
cAMPr9). This network of gap junctions unites the
mural and cumulus granulosa cells with the oocyte as a
structural and functional syncytium. Communication
via gap junctions is. therefore, a cornerstone for cellular
interactions governing the functions of follicular
components, particularly those regulated by endocrine
signaling. Paracrine mechanisms, however, also play a
part in intrafollicular differentiation and function.
Cellular Interactions in the Regulation of Oocyte
Growth
Oocytes isolated at mid-growth grow normally in vitro
only when the usual association of the oocytcs with their
companion granulosa cells is maintained. Even when
granulosa cell-denuded oocytes are co-cultured with
granulosa cells, there is little, if any, oocyte growth. I n
contrast, culture of oocyte-granulosa cell complexes
under conditions that maintain essentially normal cell-
to-cell associations promotes oocyte growth(lO.").
Furthermore, the rate of oocyte growth in vilro is
directly correlated with the number of granulosa cclls
coupled to it(12).
Oocytes are large cells having a low plasma
membrane area to ooplasmic volume ratio. The plasma
membrane of oocytes, therefore. is probably unable to
maintain an adequate inflow of nutrients and outflow of
metabolic waste to support optimal oocyte growth.
Granulosa cells enhance the uptake of some amino
acids into the oocyte via gap junctions(13."). The plasma
membrane of the granulosa cells. therefore, acts as an
extension of the oocyte's plasma membrane providing
for the uptake of nutrients, which diffuse into the
oocyte through the heterologous gap junctions that
couple the oocyte to the granulosa cells.
Gap junctions also mediate metabolic cooperativity
between oocytes and their companion somatic cells.
Oocytes denuded of their companion somatic cells can
utilize pyruvate and other intermediaries of the
tricarboxylic acid pathway for energy producticm. but
not g1ucose(l5). In contrast. the granulosa cells can
utilize glucose and apparently provide glucose metab-
olites to the oocyte for energy production. Somatic cells
might secrete glucose metabolites such as pyruvate and
oxaloacetate for uptake by the oocyte or the metab-
olites could be transferred through the gap junctions. In
addition, adenosine triphosphate, which is not taken up
efficiently through the oocyte plasma membrane, is
probably delivered to the oocyte using the gap
junctional pathway. The distribution pattern of mono-
to tri-phosphorylated derivatives of uridine and guano-
sine in granulosa cell-enclosed oocytes is different from
that of denuded oocytes, but similar to that in the
granulosa cells themselves("). Maintenance of the
normal gap junctional association between the granu-
losa cells and the oocyte is essential for granulosa cell-
like distribution in the oocyte. The granulosa cells,
therefore, cooperate in oocyte metabolic processes and
this cooperation is probably essential for normal oocyte
growth and development.
Oocyte development is also affected by regulatory
signals from the granulosa cells. Protein phosphoryl-
ation and dephosphorylation are common mechanisms
for the regulation of cell function. The pattern of
protein phosphorylation in oocytes is regulated by
granulosa cells and this is dependent upon the
maintenance of gap junctional coupling(17).In addition,
signals from the granulosa cells regulate the progression
of meiofis in oocytes.
Cellular Interactions in the Regulation of Meiosis
Throughout thc growth pha5e of thc oocyte, thc
progression of meiosis is arrested at the diplotene. or
germinal vesicle-stage, of prophase I. Nevertheless, the
oocyte becomes competent to resume meiosis at
approximately the same time as the formation of the
follicular antrum("). Competent oocytes undergo
GVB, the fir\t inorphological manifestation of the
resumption of meiosis, spontaneously upon removal
from follicles and cultured in supportive niedia(lg*").
Following GVB, the chromosomes condense and
meiosis progresses to metaphase IT and the cytoplasm
undergoes changes that prepare for fertilization. These
processes are generally referred to as 'oocyte matu-
ration'. The spontaneous maturation of oocytes upon
removal from the follicle and culture led to the
speculation, in 1939, that somatic cells in the follicle
function to maintain the oocyte in meiotic arrest('").
Since then, numerous studies have been conducted to
determine the mechanism for the maintenance of
meiotic arrest and identify substances that participatc in
the process. There is very strong evidence that cAMP
(see ref. 21 for review and other purines, particularly
guanyl play critical roles in the
maintenance of meiotic arrest. It is now generally
believed that meiosis-arresting substances arc relayed
through gap junctions from mural granulosa cells to the
oocyte via the cumulus cells. Cumulus cell-enclosed
oocytes undergo spontaneous maturation unless they
are attached to mural granulosa Thus the
integrity of the gap junction-mediated syncytium of the
mural and cumulus granulosa cells with the oocyte is
vital to the maintenance of meiotic arrest.
Oocyte maturation in vivo is induced by the
preovulatory surge of gonadotropins, primarily LH.
Since, oocytes do not have receptors for LH, the
maturation-inducing action of L H must be mediated by
granulosa cells. Two hypotheses for the mechanism of
LH-induced maturation have been proposed.
The first postulates that LH induces the degradation
of gap junctions that couple mural granulosa cells to
cumulus cells. This would result in a reduction in the
flow of meiosis-arresting substances from the mural
granulosa cells to the oocyte by way of the cumulus
cells, and resumption of meiosis would then commence.
There is clear evidence that the disassembly of gap
junctions correlates temporally with the initiation of
GVB(25).This hypothesis is supported by the obser-
vation that maturation actually does commence when
the oocyte-cumulus cell complex is experimentally
separated from the follicle all(^^,^^). Nevertheless, it
remains to be demonstrated that L H induces sufficient
degradation of the gap junctional system to reduce the
delivery of meiosis-arresting substances and cause
GVB. In fact, oocytes become committed to undergo
GVB significantly before it occurs. Moreover, a
reduction in the amount of putative meiosis-arresting
substances in oocyte-cumulus cell complexes prior to
GVB has not been demonstrated.
The second hypothesis is that LH induces the
generation of a positive maturation-inducing signal in
the granulosa cells that is introduccd into oocytes via
the gap junctional pathway. Although some evidence
has been presented that such a mechanism exists('6): the
identity of such a signal remains elusive. Mobilization
of calcium is thought to play an important role in many
cellular process including cell division("-29). A candi-
date, therefore, for the maturation-inducing signal
produced in the granulosa cells after LH-stimulation is
calcium, or a calcium mobilizing substance, which could
be introduced into the oocytes through the gap
junctions. Two observations are consistent with this
idea. First, gonadotropins induce an increase in free
calcium and calcium mobilizing substances in granulosa
~ e l l s ( ~ ' - ~Second,
~). increasing free calcium in oocytes
results in GVB when spontaneous maturation is
blocked by analo 5 of CAMPor CAMP-phosphodiester-
4.
ase inhibitor^(^^,-^) and inhibitors of calmodulin block
spontaneous maturation(35). In addition, treatment of
cultured intact follicles with calcium ionophore induces
oocyte maturation(").
Role of Oocytes in the Regulation of Follicular
Development and Function
A question that has intrigued reproductive biologist for
many years has been whether the oocyte participates in
the development and functioning of the granulosa cells.
The first positive suggestion of this was the observation
in the early 1970's that removal of oocytes from sheep
follicles in situ resulted in the precocious differentiation
of granulosa cells to corpus luteal ~ e l l s ( ~ ~The
, ~ ' corpus
).
luteum is a structure formed by remnants of the ovarian
follicle after ovulation and whose function is essential
for the maintenance of pregnancy. It was concluded
that the oocyte produces factors that prevent the
luteinization of thc granulosa cells. These findings were
later questioned and discounted(333J0).However, recent
studies have demonstrated the essential participation of
the oocyte in thc development and function of
granulosa cells.
The cumulus cells undergo a dramatic confor-
mational change prior to ovulation in which they
secrete hyaluronic acid, a nun-sulfated glycosaminogly-
can, which expands in the spaces between the cumulus
cclls and embeds the cells in a mucinous matrix (Fig. 2).
This process is often called cumulus expansion or
mucification. Although cumulus expansion in vivo is
probably stimulated by L H vin an indirect mechanism,
it i s stimulated directly in v i m by FSH. FSH, however,
fails to stimulate the production of hyaluronic acid by
mouse cumulus cells when the cumulus cells are
mechanically stripped from the oocytes and cultured as
single cells or small clumps of cells("). Likewise,
cumulus expansion fails to occur in response to FSH
when the oocyte is removed from inside the zona
pellucida, the acellular coat that surrounds the oocytc,
by a microsurgical procedure (oocytectomy)('2). These
studies demonstrated, therefore, that the oocyte
participates in the process of cumulus expansion in
mice. Culture medium conditioned by cumulus cell-
denuded oocytes enabled the cumulus cells to produce
hyaluronic acid and promoted the expansion of
oocytectomized cumulus complexes in response to
FSH("?"). It is clear, therefore, that the oocyte secretes
a soluble factor(s) that enables the cumulus cells to
respond to gonadotropic stimulation and secrete
hyaluronic acid. 'The cumulus expansion-stimulating
activity of FSH is thought to be mediated by CAMP.
The action of the cumulus expansion enabling factor is
probably downstream from the generation of CAMP
since stimulation of cumulus expansion by cAMP
analogs does not occur in the absence of oocytes or the
Fig. 2. A . An oocyte-cumulus cell complex isolated from a
preovulatory anlral follicle. Note that the cumulus cells are tightly
packcd around the oocyte. B. An ovulated secondary oocyte (egg)
recovered from the oviduct. The oocyte has undcrgonc maturation
(arrowhead indicates the polar body) and the cumulus oophorus is
dispersed (expanded) and embedded in a mucinous matrix containing
hyaluronic acid. Scale bar equal 100pm.
enabling The cumulus expansion enabling
factor is produced specifically by oocytes; media
condition by other cell types, including spermatocytes.
did not exhibit expansion-enabling activity("542).
Oocytes incapable of spontaneous GVB are also
incompetent to produce cumulus expansion enabling
factor. The factor is produced by GVB-competent
oocytes at the GV-stage, while undergoing GVB, and at
metaphase 11, but production of detectable enabling is
lost after fertilization("). The oocyte's production of
enabling factor is, therefore, developmentally regu-
lated.
The enabling factor i s secreted by denuded GV-stage
oocytes maintained in meiotic arrest with a CAMP-
phosphodiesterase inhibitor, but it is not clear whether
the GV-stage oocyte in the follicle constitutively
produces enabling factor. Intact oocyte-cumulus cell
complexes are extremely sensitive to FSH stimulation,
and FSH is present in the follicle before the
preovulatory surge of LH, yet the cumulus oophorus
does not expand until after the LH-surge(33).Perhaps
granulosa cells normally suppress the secretion of
enabling factor until oocyte maturation is induced.
Isolating the oocytes from the follicle might, therefore,
trigger the precocious release of enabling factor from
GV-stage oocytes. In this speculative model, cumulus
expansion requires the presence of an enabling factor
released by the oocyte, but the granulosa cells suppress
release of the enabling factor until maturation is
Fig. 3. Oocytc-granulosa cell complexes from isolated preantral
follicles and cultured using two culture systems that allow
maintenance o f cell-to-cell communication and interaction with
extracellular matrix. A. Complexes cultured in a gel of rat tail
collagen as described by Torrence B. Complexes cultured on
a collagen imprcgnatcd membrane as described by Eppig and
Schroeded"). When the complexes are grown o n the membranes the
oocyte becomcs bccomcs extended on a stalk and the cells that
surround the oocyte differentiate as functional cumulus cells while the
cells that are attached to the membrane behave more like mural
granulosa cells(43). Thanks to Evelyn Telfer for providing Fig. 3A.
Scale bar equal 1OOum.
stimulated by LH. This would prevent precocious
cumulus expansion in response to the FSH present in
the follicle before the LH surge.
The granulosa cells adjacent to the oocyte acquire
competence to expand in response to the cocktail of
FSH plus the enabling factor at the same time that the
oocyte acquires competence to undergo GVB("j. Does
the oocyte influence the differentiation of the subset of
granulosa cells that become functional cumulus cells?
To test this idea, GVB-incompetent oocytes were
microsurgically removed from the isolated oocyte-
granulosa cell complexes from preantral follicles, hence
before any granulosa cells become competent for
cumulus expansion. Intact and oocytcctomiLed com-
plexes were then cultured for ten days. The developing
oocytes in the intact complexes acquired competence to
 0 n
Fig. 4. Diagrammatic representation of the processes in oocytes that are dependent upon factors from the graiiulosa cells and the functions of
granulosa cells that are affected by factors from the oocyte.
undergo GVB during this culture period. When FSH
was added to the intact complexc? after the 10 day
culture period, the granulosa cells adjacent to the
oocyte underwent expansion, indicating that they had
differentiated into functional cumulus cells. In contrast,
when FSH and enabling factor were added to the
cultures of the oocytectomized complexes after the 10
day culture period, no expansion was olxerved(43).The
presence of an oocyte, therefore. is required for the
differentiation as well as the function of cumulus cells.
The oocytectomizcd complcxcs were also cultured 10
days in medium conditioned by either growing or fully
grown oocytes to determine whether the oocytes
produce a soluble factor that would promote the
differentiation of functional cumulus cells, but the
results were negative("). Evidently, the close associ-
ation of an oocyte with the granulosa cells is essential
for the differentiation of the cumulus cell subset.
However, while oocyte conditioned medium did not
induce differentiation of the granulosa cells, it did
stimulatc the proliferation of the granulosa cells and
promoted the maintenance of a three-dimensional
organization of the granulosa cell complex. This was
observed whether the medium was conditioned by
growing GVB-incompetent oocytes, or fully grown
GVB-competent. oocytes("). Since GVB-incompetent
oocytes do not secretc cumulus expansion enabling
factor, the factor(s) that stimulate granulosa cell
proliferation is different from the enabling factor. At
prcsent, however, the chemical nature of these factors
has not been resolved.
Conclusion
Methods are now available(45346)for studying oocyte
and somatic cell development and function in ways that
allow intercommunication between cellular compart-
ments and maintain intcractions with components of
extracellular matrix (Fig. 3). It is clear that the
development and function of both the somatic and germ
cell compartments of the mammalian ovarian follicle
require bidirectional communication (Fig. 4) and it is
essential to understand how they interact. Although the
requirement of the somatic cells for oocyte develop-
ment has long been appreciated, the potential influ-
ences of the oocyte on thc function of the somatic cells,
and their responses to stimulation by endocrine,
paracrine, or autocrine factors, adds a new layer of
complexity to an already complex cellular SJ stem. This
new perspective of bidirectional interactions will
certainly promote novel approaches to resolving the
fundamental mechanisms of ovarian development and
function, and contribute to analyzing fertility problems
in clinical, agricultural and zoological fields.
Acknowledgements
My thanks to Evelyn Telfer and Janan Eppig for
critically reading this essay and the NIH for $upporting
research in my laboratory through grants HD20575,
HD23839 and HD21970.
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