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J. Embryo/, exp. Morph. Vol. 23, 2, pp. 419-26, 1970
Printed in Great Britain
419
Granulosa cell-germ cell relationship in
the developing rabbit ovary
By B E R N A R D G O N D O S 1
From the Department of Pathology, Harbor General Hospital,
and the University of California, Los Angeles
Granulosa cells play an important part in the nutrition of the oocyte during
oogenesis (Raven, 1961). Anderson & Beams (1960) suggested that, in the
primary follicle, nutritive material synthesized by the granulosa cells reaches the
oocyte by diffusion across the zona pellucida. In the rabbit, follicle formation
and elaboration of the zona pellucida begin at the end of the second week after
birth (Gondos & Zamboni, 1967). Prior to this time germ cells and granulosa
cells are clustered together within the ovarian cortex. Although participation of
the granulosa cells in zona pellucida formation has been described in detail
(Merker, 1961), the function of granulosa cells in the earlier stages of oogenesis
is unclear. The following is an electron microscope study of the relationship
between granulosa cells and germ cells in the prefollicular rabbit ovary.
MATERIAL AND METHODS
Ovaries were removed from New Zealand white rabbits, 1-15 days of age,
and processed for electron microscope examination. Fixation was accomplished
by one of the following techniques: (1) mincing of tissue fragments and immersion in 1% Os0 4 with salts added (Zetterqvist, 1956); (2) vascular perfusion
with 2-5% glutaraldehyde in cacodylate buffer (Sabatini, Bensch & Barnett,
1963) and postfixation in osmic acid. All specimens were dehydrated in graded
alcohols, embedded in Epon 812 and sectioned with an LKB ultramicrotome.
For orientation purposes, sections 0-5-1 JLI in thickness were stained with
toluidine blue and examined by light microscopy. Ultrathin sections of suitable
areas were stained with lead citrate and studied with a Hitachi HU-11 C electron
microscope. Of the two fixation methods described, direct osmium immersion
produced more satisfactory sections for light microscopy, while initial vascular
perfusion resulted in superior preservation of cellular ultrastructure. Accordingly, the electron micrographs reproduced here are all prepared from sections
of perfused ovaries.
1
Author's address: Department of Pathology, Harbor General Hospital, Torrance, California, U.S.A.
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RESULTS
At the junctions between granulosa cells and germ cells, cell membranes are
closely apposed (Fig. 1 A, B). The width of the intercellular space measures
250-300 Â. Adjacent cell membranes follow the contour of one another in such
a manner as to maintain an almost constant intercellular distance. This relationship is slightly altered by a widening of the intercellular space in areas where
three cells come together. Granulosa cells and oogonia in division are closely
related by tight application of adjacent cell membranes (Fig. 1C).
Surface contact between germ cells and granulosa cells is enhanced by the
presence of interdigitating cytoplasmic projections arising from neighboring
cells. These projections are shown at low magnification in Fig. 1 A and at higher
magnification in Fig. 2 A and B. Extensive intermingling of projections often
makes it difficult to determine which is the cell of origin. Fig. 2 A shows projections arising from a granulosa cell, while Fig. 2B shows origin from a germ
cell. The projections are long and slender, resembling microvilli in their shape,
but with a less regular arrangement. They are folded and flattened to adapt to
the narrow intercellular space. The long axis of a projection generally lies parallel
to adjacent cell membranes. The projections are filled with ribonucleoprotein
particles (ribosomes), the cytoplasm of each projection blending in with that of
its cell body. Desmosome-like attachments between oocytes and granulosa cells
are rarely seen in the prefollicular stage of development. However, condensations of adjacent cell membranes resembling desmosomes occasionally appear
between interdigitating cytoplasmic projections.
Extending around the germ cells are prominent elongations of the granulosa
cells. These elongations intervene between adjacent oocytes and between oocytes
and basement membranes of the cortical clusters (Fig. 1B). Tips of granulosa
cells extending in this manner make contact with one another to envelop
individual oocytes in the process of follicle formation. The close relationship
between granulosa cell extensions and adjacent oocytes is indicated by the direct
apposition of cell membranes and the presence of numerous cytoplasmic projections (Fig. 1B).
FIGURE 1
Scale line represents 0-5 /t throughout
(A) Closely apposed germ cells (o) and granulosa cell (g) in ovarian cortex.
Granulosa cell is distinguished by its dense cytoplasm and irregular nucleus. Cytoplasmic projections line granulosa cell-germ cell junction. Boundary between germ
cells is regular except for intercellular bridge, at arrow.
(B) Granulosa cell elongation extends over surface of germ cell. Close intercellular
relationship is maintained.
(C) Section shows dividing oogonium, with chromosomes (ch) oriented on mitotic
spindle, and granulosa cell (nucleus at right). Cell membranes are directly applied
to one another.
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B. GONDOS
Cell relationship
423
Invaginations of oocyte cell membranes by cytoplasmic projections of
granulosa cells are frequently seen. One such invagination producing contact
between granulosa cell membrane and oocyte nucleus is shown in Fig. 2C.
Contact is facilitated by the peripheral location of the oocyte nucleus. The
relationship thus established provides an extremely close association between
cytoplasm of the granulosa cell and nuclear contents of the oocyte. The presence
of numerous vesicles in the oocyte cytoplasm suggests the possibility of pinocytotic exchange between the adjacent cells. The granulosa cell also shows
abundant vesicle formation and a prominent Golgi complex next to the nucleus.
In Fig. 2D, a deep, narrow indentation of the oocyte membrane is produced by
a granulosa cell projection directed toward the oocyte nucleus. In this section
contact is not demonstrated, but the distal portion of the projection approaches
the border of the nucleus. A similar indentation is shown in Fig. 2E. In this case
the cytoplasmic projection is sectioned obliquely producing the appearance of
an inclusion in the oocyte cytoplasm.
Direct cytoplasmic continuity between germ cells and granulosa cells was not
found. Intercellular bridges could frequently be seen between germ cells (see
Fig. 1 A), but extensive search for bridges communicating with granulosa cells
yielded negative results.
DISCUSSION
The fine structural relationship between germ cells and granulosa cells in the
developing rabbit ovary is similar to that described in the rat (Franchi, 1960),
guinea-pig (Anderson & Beams, 1960), mouse (Odor & Blandau, 1969), hamster
(Weakley, 1966) and human (Stegner & Wartenberg, 1963; Lanzavecchia &
Mangioni, 1964). The close apposition of cell membranes of adjacent germ cells
and granulosa cells and the presence of interdigitating cytoplasmic projections
seem to be consistent features of mammalian oogenesis. The granulosa cell
projections are similar to those seen in mature follicles, but in the latter the
projections are more elongated, extending across the zona pellucida to make
FIGURE 2
(A) Cell junction showing cytoplasmic projections of granulosa cell (g) extending
along surface of germ cell (<?).
(B) Projections arising from cytoplasm of germ cell (o) make contact with cell
membrane of granulosa cell (g).
(C) Section shows deep invagination of germ cell membrane by cytoplasm of
granulosa cell (nucleus at right). Arrow indicates point of contact between outer
nuclear membrane of germ cell and cell membrane. Numerous large vesicles are
seen in germ cell cytoplasm. Smaller vesicles are present in granulosa cell cytoplasm.
(D) Arrow indicates long, narrow granulosa cell projection directed toward germ cell
nucleus, part of which is shown in lower right corner.
(E) Obliquely sectioned granulosa cell projection (arrow) simulating intracytoplasmic inclusion.
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B. GONDOS
contact with microvilli on the oocyte surface (Baca & Zamboni, 1967). The
function of cytoplasmic projections in the adult follicle has been considered to
be the exchange of material between granulosa cell and oocyte (Hertig & Adams,
1967). It seems likely that a similar function applies in the developing ovary.
Granulosa cell projections extending deep into the cytoplasm of oocytes have
been described in hamster (Weakley, 1966) and human (Baca & Zamboni,
1967) follicles, but were not seen in fetal and early postnatal mouse ovaries
(Odor & Blandau, 1969). Extension of such projections to the point of nuclear
contact was observed in the present study. An extremely close relationship was
thus demonstrated between granulosa cells and germ cells at a time when germ
cells are known to undergo active DNA synthesis (Peters, Levy & Crone, 1965).
These findings suggest that the granulosa cells in the prefollicular rabbit ovary
may have a role in the regulation of oocyte maturation. Flickinger & Fawcett
(1967) proposed that Sertoli cells in the mouse testis have a combined function
of germ-cell nutrition and co-ordination of cytological events during spermatogenesis. The close relationship between granulosa cells and germ cells during
oogenesis has similarities to the relationship between Sertoli cells and testicular
germ cells during spermatogenesis (Nicander, 1967). Evidence to indicate that
Sertoli cells in the fetal guinea-pig may be capable of steroid biosynthesis was
presented by Black & Christensen (1969). Since Sertoli cells are considered to be
the male homologues of the granulosa cells (Witschi, 1951), it is of interest that
granulosa cells in the neonatal rabbit exhibit a complex cytoplasmic organization
suggesting capacity for synthetic and secretory activity (Gondos, 1969). During
the period of follicular growth, granulosa cells have been observed to undergo
rapid cell multiplication and actively synthesize protein (Björkman, 1962).
Oocytes and nurse cells in the developing ovaries of Drosophila and other
invertebrates are connected by intercellular bridges (King, 1964). These bridges
allow flow of nutrients from one cell to another during oocyte maturation (King
& Mills, 1962). Although Kemp (1958) stated that he found intercellular connexions between germ cells and granulosa cells in developing mammalian
ovaries, Franchi (1960) was unable to confirm thesefindingsin the rat. TrujilloCenóz & Sotelo (1959) found no evidence of cytoplasmic continuity between
projections of granulosa cells and oocytes in mature follicles of the rabbit, nor
was any such continuity found in the prefollicular rabbit ovaries of the present
study. The discrepancy between findings in invertebrates and vertebrates is not
surprising. In Drosophila, 15 nurse cells and a single oocyte arise from a stemline oogonium by four consecutive incomplete divisions, leaving the 16 cells
interconnected (Brown & King, 1964). In all vertebrates that have been investigated, the supporting granulosa cells arise within the gonad, while primordial germ cells originate extragonadally (Franchi, Mandl & Zuckerman,
1962). Incomplete divisions of oogonia can give rise to interconnected germ
cells in the ovaries of mammals (Gondos & Zamboni, 1969), but there is no
evidence to suggest that granulosa cells can be formed in this manner.
Cell relationship
425
SUMMARY
1. Ovaries of New Zealand white rabbits, 1-15 days of age, were studied with
the electron microscope.
2. The relationship between granulosa cells and germ cells was characterized
by close apposition of adjacent cell membranes, the presence of numerous interdigitating cytoplasmic projections and extension of granulosa cell projections
deep into the cytoplasm of germ cells.
3. These observations indicate that, in addition to their role in germ cell
nutrition and zona pellucida formation, granulosa cells of the developing ovary
may have a direct influence on the maturation of germ cells.
4. Intercellular bridges, such as those connecting developing germ cells, were
not found between germ cells and granulosa cells.
RÉSUMÉ
Les rapports entre les cellules germinales et la granulosa pendant
le développement de l'ovaire de lapin
1. Des ovaires de lapins blancs de Nouvelle Zélande, âgés de 1-15 jours, ont
été étudiés au microscope électronique.
2. Les rapports entre les cellules de la granulosa et les cellules germinales
sont caractérisés par une intime juxtaposition des membranes cellulaires
adjacentes, par la présence de nombreux prolongements cytoplasmiques digités
et la pénétration profonde des prolongements cellulaires de la granulosa dans
le cytoplasme des cellules germinales.
3. Ces observations indiquent que, en plus de leur rôle dans la nutrition des
cellules germinales et dans la formation de la zone pellucide, les cellules de la
granulosa de l'ovaire en développement peuvent avoir une influence directe sur
la maturation des cellules germinales.
4. Des ponts intercellulaires analogues à ceux qui établissent des connexions
entre les cellules germinales, n'ont pas été observés entre les cellules germinales
et les cellules de la granulosa.
I wish to thank Mrs Lucy A. Conner for technical assistance. The work was supported by
a Ford Foundation grant in Reproductive Biology.
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{Manuscript received 11 June 1969)