Paper - Studies on guinea pig oocytes 1
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Adams EC. and Hertig AT. Studies On Guinea Pig Oocytes. I. Electron Microscopic Observations On The Development Of Cytoplasmic Organelles In Oocytes Of Primordial And Primary Follicles. (1964) J Cell Biol. 21:397-427. PMID 14189912
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Studies on Guinea Pig Oocytes I. Electron Microscopic Observations on the Development of Cytoplasmic Organelles in Oocytes of Primordial and Primary Follicles
Eleanor C. Adams And Arthur T. Hertig, MD.
From the Department of Pathology, Harvard Medical School, Boston
Oocytes in primordial and primary follicles of young adult guinea pig ovaries ﬁxed in osmium tetroxide and embedded in Epon 812, have been observed by electron microscopy. The gradual differentiation of a series of cytoplasmic organelles has been correlated with the growth in size of the oocyte and the development of the follicular wall. The most immature primordial oocyte is morphologically a simple cell consisting of a large slightly eccentric nucleus, a few large spherical mitochondria, a profusion of granular cytoplasmic vesicles, and free RNP particles. At the primary follicle stage, abundant endoplasmic reticulum, clusters of mitochondria proliferating around a rosette formation, multiple Golgi complexes, vesicular aggregates forming cortical granules, and a profusion of micro— villi have been differentiated. Concentrations of cytoplasmic organelles at the periphery of the oocyte in the primary follicle suggest that it is equipped for the absorption, utilization, and intracellular transport of material delivered to its surface membrane. The juxtaposition of components of the ultrastructure during the development and growth of this large cell appears to follow a precise pattern and provides an unusual opportunity to study the
interrelationships of differentiating organelles.
The development of oocytes in a variety of species has long been examined by workers successively employing newly developed techniques in the ﬁelds of morphology, physiology, or biochemistry. Electron microscopic studies of the development and interrelationships of ultrastructure in invertebrate oocytes have been extensively reported by Rebhun (1961), Afzelius (1957), and others. Yamada et al. (1957) and Sotelo and Porter (1959) were the ﬁrst to report on mammalian oocytes (mouse and rat, respectively), followed by Odor (I960), Chiquoine (1960), Anderson and Beams (1960), and others working on a
variety of species. Their reports were concerned primarily with oocytes developing in secondary follicles or with tubal ova. The observations presented here are on guinea pig oocytes in earlier stages when a variety of cytoplasmic organelles are beginning to be differentiated.
Following the cessation of mitotic division of oogonia in fetal gonads, the primordial mammalian oocyte enters a series of phases of meiotic prophase followed soon after birth by a prolonged resting stage of prophase known as the dictyate stage (Franchi, Mandl, and Zuckerman, 1962). In the adult guinea pig ovary, primordial oocytes in this
397 stage lie singly or in clusters just beneath the surface epithelium of the ovary and may remain quiescent for very extended periods of time surrounded by a thin layer of attenuated follicular cells. The development of a mature follicle with its oocyte takes place in two phases. The ﬁrst is primarily one of growth of the oocyte itself which occurs during the transformation of the singlelayered primordial follicle into a primary follicle with multiple layers of widely separated granulosal cells. During the second phase it is the follicle that grows most rapidly due primarily to an expanding antrum while the contained oocyte shows relatively less enlargement. Although it is generally believed that the ﬁrst period of growth is independent of the hypophysis, pituitary-ovarian control of the second phase of growth is well docu~ mented (Everett, 1961). Reported here is the initial phase of growth and differentiation of oocytes from the primordial follicle stage, sur~ rounded by a single layer of epithelial cells, to the primary follicle stage with solid multiple layers of granulosal cells.
Our study of the variety of forms of primordial oocytes found at all stages of the estrous cycle of the guinea pig reveals the gradual differentiation of a series of cytoplasmic organelles that is corre~ lated with the growth in size of the oocyte and the development of the granulosal cells of the follicle wall. The most immature primordial oocyte is morphologically a very simple cell, probably repre~ senting a state of “maintenance metabolism.” In contrast, the primary oocyte in the primary follicle has tripled its diameter, has elaborated complex surface specializations and many cyto~ plasmic organelles, and thus potentially is competent to complete the stages of development leading to ovulation and subsequent fertilization.
MATERIAL AND METHODS
The 30 primordial and primary follicles examined for this study were found in ovaries from animals 3 to 9 months of age, sacriﬁced by a blow on the head at known stages of their estrous cycle. A variety of early stages of follicular development is found at all times in guinea pig ovaries without regard to the stage of the estrous cycle. In contrast, the growth of those secondary follicles destined to ovulate their contained oocyte appears to be initiated soon after estrus and to
be completed within the 15-16 day interval of one estrous cycle (Myers, Young, and Dempsey, 1936). The oocytes in primordial and primary follicles reported here were found in ovaries from animals
sacriﬁced at a variety of known stages of their estrous cycle so that the oocytes in the developing secondary follicles could also be studied.
One ovary from each animal was ﬁxed in Bouin’s ﬂuid for hematoxylin and eosin staining and subsequent histologic examination. The other ovary was placed in a small glass evaporating dish containing Caulﬁeld’s (l957) modiﬁcation of Palade’s ﬁxative buffered at pH 7.5-7.6 and kept chilled in an ice bath. Here it was cut into small pieces with iris scissors or a ﬁne scalpel. After ﬁxation for _1/§ to 1 hour, the tissue was rapidly dehydrated in ice-cold graded (50 to 100 per cent) alcohols and was inﬁltrated with a 5:5 mixture of Epon 812 according to the method of Luft (1961). The embedded tissues were hardened 24 hours at 60°C. Using a Porter-Blum microtome and a DuPont diamond knife, the blocks were ﬁrst “thick” sectioned to search for primordial and primary follicles. These M to l—micron sections were mounted on glass slides, dried for a few minutes, stained brieﬂy with dilute alkaline toluidine blue (Trump at al., l96l), rinsed with tap water, mounted in 50 per cent aqueous glycerin, and examined immediately with a phase microscope to select suitable follicles. The thick sections of those follicles that were subsequently examined in the electron microscope were photographed to record their location and histology. Step—serial thin (light to dark gold) sections were then cut through the oocytes, mounted on 100-mesh grids, and stained with lead according to the method of Karnovsky (1961). The grids were examined in an RCA EMU-3F using initial magniﬁcations of 1250 to 9,000. The thin sections were alternated with thicker ones mounted on glass slides for phase photography.
Figs. 1 to 5 are phase photomicrographs, taken at a uniform magniﬁcation (X 560), of ﬁve of the 30 oocytes that were examined by electron microscopy. These photographs illustrate the growth of the oocyte during the transition from a primordial to a primary follicle. Occasional reference to these ﬁgures is made in the text so that size relationships can be kept in mind as cytoplasmic changes take place in the oocyte.
The simplest oocyte, even smaller than the stage shown in Fig. l, is an elongate cell measuring about 20 X 14 p. and surrounded by a single layer of flat follicle cells (Figs. 6 to 7). It often occurs in clusters, within a narrow band of connective tissue—ethe ovarian cortex—~lying just beneath the surface epithelium. The large, some
398 THE JOURNAL or CELL BIOLOGY - VOLUME 21, 1964 what eccentric nucleus is packed with granular material of varying density and size and has a wavy envelope which, on tangential section, shows prominent pores frequently containing a central granule. None of our electron micrographs of oocytes of this stage, however, show a nucleolus. The outer nuclear membrane has many adherent ribonucleoprotein granules and often appears to be budding off the numerous granular vesicles seen in the cytoplasm (Fig. 9). Many RNP granules are distributed uniformly in the cytoplasmic ground substance. There are also aggregates of dense granular material (DA) scattered in the cytoplasm. The large and often dense mitochondria, more numerous at one pole, are usually spherical with only one or two short cristae, but there are occasional elongated oval forms with multiple parallel cristae. There are no intramito— chondrial granules. One variety of large mitochondria has an internal vesicular structure as though the cristae had been detached from the peripheral membrane and subsequently inﬂated (F ig_ 6). The cytoplasmic vesicles are at times in close contact with mitochondria. Larger smooth—mem— braned vacuoles containing a dense material possibly phospholipid are seen occasionally. The surface of the primordial oocyte is closely enveloped by the thin primordial follicle cells, and at intervals the apposing membranes of oocyte and follicle cell show desmosome—like thickenings similar to that seen in Fig. 10. Occasional small outpocketings of the oocyte indent the follicle cells (Fig. 7). In the region of the junction of two follicle cells, a concentration of vesicles is often seen beneath the ovular surface (Fig. 7). The follicle cells are covered exteriorly by an amorphous basement mem~ brane that blends into the intercellular substance of the ovarian connective tissue (Fig. 7).
Formation of M itochorzdrial Rosettes and PC!/?i?‘6d ll/Iembranes
The ﬁrst indications of developmental processes (Fig. 8) are seen in primordial oocytes of about the same size as the simplest stage previously described, but whose follicle wall is somewhat thicker. This thickening, in the absence of observed follicular cell mitosis at this stage, is apparently due to the addition of cells differentiating from the surrounding stroma. Foot processes of stromal cells appear to join the periphery of the follicle (Fig. 8). Two cytoplasmic conﬁgurations have been observed in all oocytes examined in
E. C. ADAMS AND A. T. HERTIG Guinea Pig Oocytes. I
this stage: (1) Mitochondria often clustered in rosettes around a very dense core of slightly granular material (Figs. 9, 10, and 13), perhaps the dense aggregates previously described and illustrated (Fig. 6, DA); and (2) a few short paired rough membranes seen in close association with mitochondria (Figs. 10 and 11). These membranes of endoplasmic reticulum are not associated with the mitochondria in rosettes. In the central cytoplasm of some oocytes there is a slight concentration of granular vesicles, and multi—vesicular bodies may also be present (Fig. 1'2). The ovular nucleus contains a dense reticu— lated nucleolus (Fig. 8). Vesicles, which are prominent and numerous in the cytoplasm of the follicle cells and at the periphery of the oocyte, are fre~ quently seen in rows along the apposing cellular membranes (Fig. ll). The interdigitating mem~ branes between adjoining follicular cells often show localized small tight junctions (Fig. 1 l) Which may restrict the direct passage to the oolemma of substances that have crossed the perifollicular basement membrane. As the single layer of cuboidal follicle cells becomes more crowded, the individual cells begin to send out projections be~ tween adjacent follicle cells and the ovular sur~ face. A single rudimentary cilium was seen at the base of one such projection (Figs. 8 and 9')» The actual relationship, however, between the cilium and this cytoplasmic projection is not clear from a single section.
Formation of Golgi Complex
The next constantly recognizable change in the primordial oocyte is the development of a large concentration of smooth—membraned vesicles and short broad channels localized in a juxtanuclear position (Figs. 14 and 15). This area is interpreted as an early stage of the Golgi complex. Its possible relationship to the “yolk nucleus” seen by previous workers in various species of oocytes was discussed by Anderson and Beams (1960). This simple Golgi body appears in oocytes as they begin to increase in size and to have a more constantly spherical shape (Fig. 1). We have not, as yet, been fortunate enough to observe the earliest formation of this Golgi area other than a slight condensation of the granular vesicles in the central cytoplasm (Fig. 12). Many of the Golgi vesicles contain material much less dense than do the granular forms (Fig. 15). As the oocyte begins
to enlarge (25 p.) the Golgi area becomes lobular
399 and apparently splits into two or more groups of vesicles (Fig. 16) that subsequently assume a more peripheral location (Fig. 17).
Further remodeling at the ovular surface is apparent at this stage. The follicle cells increasingly project long watery appearing extensions between the oocyte and contiguous follicle cells, creating broadening irregular periovular spaces (Figs. 18 to 20). The coalescence of elements of the rough endoplasmic reticulum in the follicular cytoplasm near the origin of these projections suggests the synthesis of protein and, moreover, that this may be a mechanism for the production of
the material of the zona pellucida (Fig. 19). Other areas of the follicle cells are still in close Contact with the oocyte and the indentations of one cell type into the other appear to be areas of height ened pinocytie (or reverse pinocytic) activity in the oocyte (Fig. 20). The follicular Golgi area often is oriented toward the oocyte (Fig. 20). Desmosome-like thickenings of apposed ovular and follicular membranes and of adjacent follicular membranes are frequently seen (Figs. 18 to 19).
Forrnat-ion. of Locailsized E=rga.stoplas-rm and Zena Pellucida
When the oocyte has grown to a 35-1.; sphere (Fig. 2) and the follicular cells are beginning to be crowded, a specialization of rough endoplasmic reticulum appears in the oocyte. Its elongating proﬁles, still in close association with one or more mitochondria, are also associated with oval smooth-membraned vesicles ﬁlled with a moderately dense homogeneous substance. These proﬁles
CG, Cortical granules
DA, Dense aggregate
ER, Endoplasmic reticulum
E V, Expanded Vesicle of endoplasmic
ret.iculun1 F, Follicle cell FN, Follicle cell nucleus 0, Golgi area
I M , Intermediate mitochondrion
M, Mitochondrion MM, Modiﬁed mitochondrion
ma, Ovular microvilli
mvb, Multivesicular bodies NP, Nuclea.r pores
ON, Ooeyte nucleus
P, Follicle cell projection R, Mitochondrial rosette RNP, Ribonucleoprotein particle TJ, Tight junction
VA, Vesicular aggregate ZP, Zona pellucida
FIGURES 1 to 5 Phase micrographs of }§ to 1 p. T oluidin blue—sta.ined Epon sections of developing oocytes in primordial and primary follicles. X 560.
FIGURE 1 Cluster of 5 small primordial oocytes probably in stage of early primitive Golgi development, similar to those illustrated in Figs. 14 to 9.0, but more mature than those illustrated in Figs. 6 to 13. Guinea pig 38.
FIGURE ‘:2 The developing primordial follicle a11d oocyte whose ultrastructure is illustrated
i11 Figs. ‘:21 and ‘:22. The oocyte is enlarging and the follicular wall is formed by cuboidal cells. Note also two smaller oocytes. Guinea. pig 38-11.
FIGURE 3 The cuboidal cells i11 the wall of this oval follicle are beginning to be doublelaycred at the poles. This same follicle and oocyte are illustrated in Fig. 96 a.nd are similar to the others illustrated in Figs. 25 to 332. Guinea pig 45-4.
FIGURE 4 A primary follicle with a solid mult.ilayered granulosa wall. This follicle and oocyte are illustrated in Figs. 33 to 36. A mitotic ﬁgure in a granulosa cell was observed in a tangential section through this follicle. Guinea pig 45-3.
FIGURE 5 A larger primary follicle just before antrum formation. The int.ercellular spaces between the granulosa cells are expanding. The ultrastructure of this oocyte is illustrated in Figs. 40 to 432. Guinea pig 38-5.
THE JOURNAL or CELL BIOLOGY - VOLUME 91, 1964: E. C. ADAMS AND A. T. HERTIG Guinea Pig 00:.-gtes. I 401 are frequently seen as looped formations scattered in the cytoplasm (Fig. 21). In localized areas in the Central cytoplasm they may form either parallel arrays or concentrically arranged layers which en— close the smooth-membraned vesicles between the narrow cisternae of the ergastoplasm, i.e. on the ribosomal side of the membrane (Fig. 22). The oocyte illustrated in Figs. 23 and 24 has the most extreme concentration observed in any of our specimens.
Aggregates of small smooth—membraned vesicles containing an internal structure or granule begin to be apparent in the central cytoplasm (Fig. 22, VA). Mitochondrial rosettes are numerous throughout the cytoplasm and seem to be associated with a variety of mitochondrial forms. The dense granular material in the center of the
rosettes is most closely associated with mitochondria whose peripheral membranes and cristae appear incomplete (Figs. 13 and 39). RNP granules are diffusely distributed in the cytoplasm. Occasional dense aggregates of variable size are scattered in the perinuclear ground substance and may represent various formative stages of the rosette. Multiple Golgi complexes, now larger, are seen at the periphery of the oocyte (Fig. 23). The granular cytoplasmic vesicles previously seen in profusion are decreasing in number, but chains or clusters of short, rather broad forms are increasing.
The amorphous material of the zona pellucida is seen in the enlarging space between the oocyte and the follicular cells. Broad conical projections, containing clusters of small vesicles, and stubby microvilli begin to extend into the zona from the oocyte (Fig. 22).
Development of Specialized Structures in the Zona, Pellucida, Cortical Granules, and “Modiﬁed Mitochondria”
This phase, illustrated in Figs. 3 and 25 to 32, is found in oocytes within an oval follicle, 0.08 mm in length, at whose poles there is the earliest indication of a double—layered wall. The follicle cells at the poles also are beginning to separate, forming wide irregular intercellular spaces traversed by broad cytoplasmic projections that attach these primitive granulosa cells to each other (Fig. 25).
As the increasing breadth of the zona pellucida progressively separates the oocyte from the internal layer of follicle cells, intimate contact between these two cell types is still maintained by the irregular projections of the follicle cells. These projections (Fig. 32) traverse the zona, often in a radial fashion, and end in a broad desmosome—like thickening at the cell membrane of the oocyte. Ovular microvilli, now better formed, also extend for a short distance into the zona pellucida, making this amorphous membrane a bed of intercellular projections from both types of cells (Fig. 32). Loop formations of long proﬁles of rough endoplasmic reticulum are dispersed throughout the cytoplasm (Fig. 25). In addition, concentrations of these loops with associated oval dense vesicles are seen in the central cytoplasm where they often encircle an expanded vesicle of rough endoplasmic
reticulum (Fig. 30). A prominent feature of this stage is a perinuclear area devoid of larger organelles but containing free RNP particles and granular vesicles (Fig. 25). Frequently, aggregates of dense material are seen in this rather structureless area (Figs. 25 and 28, DA). Mitochondria
FIGURE 6 Low—power micrograph of the simplest form of oocyte observed in this series,
even less mature than those in Fig. 1. The primitive follicle cells are thin and closely applied
to the surface membrane of the oocyte. The large nucleus has a wavy envelope. Note
spherical mitochondria of varied size and internal structure. Note also a few dense aggre gates i11 cytoplasm. The occasiona.l foci of a slightly increased density at apposing
membranes of follicle cells and oocytes are desmosome—like areas. Vacuoles containing a
dense material possibly phospholipid are seen above the nucleus and at the center below the nucleus. Guinea pig 45-5. X 5000.
FIGURE 7 Higher power of oocyte—follicle junction to show the profusion of granular
vesicles most prominent in cytoplasm near surface of oocyte. Note RNP granules on ex ternal nuclear membrane and pore—formation of this membrane on tangential section.
Many RNP granules are free in cytoplasmic ground substance. Guinea pig 45-5. X 14,500.
THE JOURNAL or CELL BIOLOGY ~ VOLUME 921, 1964 E. C. ADAMS AND A. T. HERTIG Guinea Pig Oocytes. I 403 are present in large clusters within which are multiple rosette formations.
Two new cytoplasmic structures characteristic of all later stages of guinea pig oocytes are first seen in this stage of development. One consists of moderately dense membrane—enclosed spheres of uniform size found exclusively at the extreme periphery of the oocyte (CG, Fig. 26). The content of these spheres is homogeneous or only slightly granular. They are morphologically similar to structures observed by electron microscopy in tubal ova of golden hamsters (Austin, 1961) and of rabbits (Hadek, 1963) and identiﬁed as cortical granules by these authors. The relationship of these granules to the isolated vesicular aggregates (VA) described previously (Fig. 22) becomes apparent by the frequent observation of similar membrane—bounded spheres of varying size contained within these aggregates (Fig. 27). Vesicles comprising the aggregates are variable in size and contain one or more internal dense areas or tiny vesicles. The coalescence of these vesicles, followed by the concentration of the internal material, appears to form the dense single membrane—cov— cred granules. Although these granules are often seen near, or in the Golgi complex of our guinea pig material, they are not conﬁned to this location.
The other newly formed structure is a large elongated pleomorphic organelle (Fig. 31, MM) whose peripheral double membrane and cristae resemble those of mitochondria, but which additionally contains multiple central ﬁbrillar structures running longitudinally. Anderson and Beams (1960) also observed these structures in their guinea pig oocytes, calling them modiﬁed mitochondria. In this stage of development, we believe intermediate forms between these large structures and mitochondria conﬁrm their origin from the
latter. These intermediate forms (Fig. 30) appear
among clusters of mitochondria. Their cristae are indistinct, often detached from the peripheral membrane, and could be forming the longitudinal central ﬁbrillar structure.
Two specimens in this stage of development each showed a rudimentary cilium within a follicle cell, one emerging from the cell at the base of a projection (Fig. 29).
Interconnection of Cytoplasmic Areas by M embrane Systems during Primary F ollicle Development
The transformation from a primordial follicle to one designated as a primary follicle is marked by the gradual development of a multi—layered follicular wall composed of cells which, having differentiated into a granulosa type, then begin to proliferate by mitosis and to become separated by widening irregular intercellular spaces (Figs. 4 and 5). In the course of this growth, the follicle has sunk slightly deeper into the ovary and usually rests near a mass of interstitial tissue. Within the oocyte, a diverse system of membranes appears to be developing (Fig. 38). Although the nucleus frequently shows blebbing of the outer membrane (Fig. 26) and is still surrounded by a ground substance containing RNP particles and small vesicles, its envelope does not appear to be directly connected with the more peripheral membranous structures (Fig. 33). Varied forms of mitochondria continue to be present in increasing numbers. There are localized concentrations of rough-membraned endoplasmic reticulum closely associated both with smooth—membraned vesicles containing a homogeneous dense substance and with mito— chondria (Fig. 39). These concentrations seen in the central cytoplasm of oocytes in the smaller primary follicles (Figs. 4 and 36) appear to disperse gradually (Fig. 37) and subsequently to
FIGURE 8 The follicle cells are somewhat cuboidal. The oocyte cytoplasm co11tains several mitochondrial rosettes, and paired membranes of rough endopla.smic reticulum are beginning to form. Note foot processes of ovarian stromal cells at top and lower left that ap~ parently attach t.o the periphery of the follicle and become incorporated into it.s wall.
Guinea pig 38-7. X 5000.
FIGURE 9 A higher power of Fig. 8, showing cytoplasmic details including a strand of endoplasmic reticulum developing in association with mitochondria, and mitochondrial rosettes formed by mitochondria of various internal structures surrounding a dense somewhat granular core. The outer nuclear membrane appears to be budding off granular vesicles. Note rudimentary cilium projecting from a centriole near base of follicle cell at right. Guinea pig 38-7. X 14,500.
404 THE JOURNAL or CELL BIOLOGY VOLUME Q1, 1964 E. C. ADAMS AND A. T. HERTIG Guinea Pig Oocytes. I 4-05 406 THE JOURNAL OF CELL BIOLOGY - VOLUME 921, 1964 FIGURE 14 A low—power micrograph of an oocyte containing a single juxtanuclear aggregate of vesicles
interpreted as an early Golgi complex. Guinea pig 51-1. X 5,000.
FIGURE 10 Note close association of paired membra.nes of endoplasmic reticulum with membranes of mitochondria, profusion of granular Vesicles, and the variety of int.erna1 structures and sizes of spherical mitochondria. Guinea pig 51-11. X 923,000.
FIGURE 11 Note concentration of Vesicles beneath oocyte membrane near junction of
two follicle cells. Also note close association of a mitochondrion and endoplasmic reticulum. Guinea pig 51-11. X 23,000.
FIGURE 12 Occasionally multivesicular bodies are seen either singly or in groups. This is the greatest conce1'1t.ra.t.ion seen in any oocyte of these stages. Guinea pig 38—1‘2. X 23,000.
FIGURE 13 A mitochondrial rosette, illustrating the slightly granular dense core material and the close association of this material with the two mitochondria at right whose internal or external membranes appear incomplete. Guinea pig 51. X 33,000.
E. C. ADAMS AND A. T. HERTIG Guinea Pig Oocytes. I
I become transformed into connecting links between areas of the cytoplasm in oocytes in large primary follicles (Figs. 5 and 40). The paired rough membranes also occasionally form expanded vesicles which contain accumulations of moderately dense material and possibly represent isolation areas for storage and /or synthesis (Figs. 35 and 40).
The cortical granules, increasing in number, are present in a variety of forms. Those within a vesicular aggregate frequently contain remnants of vesicles, suggesting that this is a formative stage of the granules (Fig. 34). Those free—1ying in the extreme peripheral cytoplasm occasionally show ruptured membranes, suggestive of breakdown or utilization of the granules (Fig. 35).
At the ovular surface a profusion of slender microvilli extend into the zona (Figs. 35, 38, and 41). The projections of the granulosal cells that traverse the zona terminate in desmosome—like thickenings at the oolemma (Figs. 38 and 40) or indent the cytoplasm of the oocyte (Figs. 41 and 42). The double-membraned appearance of these attachments or indentations is evidence that there is no syncytial relationship between the oocyte and the granulosa cells. This is in agreement with previous observations by Anderson and Beams (1960) on later stages of guinea pig oocyte development.
The largest primary follicles observed to date are about 0.15 mm in diameter and have multiple layers of granulosa cells often widely separated by localized lakes of intercellular ﬂuid (Fig. 5). A theca interna layer is differentiating. The oocyte measures 50 to 55 p. in diameter, which corresponds to an approximate 30~fold increase in volume over that of the resting primordial stage.
The growth and development of primordial and primary guinea pig follicles is accompanied by a continuous process of differentiation of organelles
within the oocyte. The changing juxtaposition of various classes of these organelles and the re modeling of membranous forms within the cytoplasm and at the cell surface are morphologic of the differentiating (maturation) processes. In the following discussion, some speculations are presented to interpret these morphologic changes in terms of their possible physiologic signiﬁcance.
1. Mz'toch0ndriaZ rosettes appear to be Concerned with the proliferation of mitochondria in guinea pig oocytes developing in primordial and primary follicles. This conclusion is based not only on the observation of the initial formation of rosettes at the time when mitochondria are beginning to in evidences
crease in number, but also on the presence of rosettes within the many clusters of mitochondria at later stages. In addition, the close association of the dense core of the rosettes with primitiveappearing mitochondria whose peripheral membranes or cristae look incomplete suggests the participation of this material in the elaboration of mitochondria. The variation in size and internal structure of mitochondria illustrated has previously been observed in various mammalian oocytes by Anderson and Beams (1960), Odor (1960), and Blanchette (1961).
Although the origin of the core substance is conjectural on the basis of our present micrographs, we suggest that it may appear ﬁrst as the isolated dense aggregates seen in the cytoplasm of even the simplest oocyte. The somewhat granular appearance of both these aggregates and the core substance might indicate an origin from a concentration of RNP particles, but a lipoprotein complex is also possible. The possibility of an RNP component is of particular interest in view of reports of configurations in other species which may be analogous to mitochondrial rosettes. Miller (1962) and Lanzavecchia (1962) both describe in Rana oocytes material passing through nuclear pores to become condensed into perinuclear masses which have associated mitochondria. Such observations are reminiscent of light microscopic observations i11 tadpole oocytes reported by Ornstein (1956) who suggested that the nucleus and mitochondria
FIGURE 15 Higher power of oocyte shown in Fig. 14, illustrating the rough form of vesicles surrounding and blending with the smooth forms of the Golgi area. Note endoplasmic reticulum associated with mitochondria. Note also mitochondrial rosettes. Guinea pig
51-1. X 14,500.
FIGURE 16 Illustrating the Golgi area of oocyte slightly more advanced than that in Fig. 14. Note that this oocyte appears to have two Golgi complexes, one of which is lobu—
lated. Guinea pig 38-8. X 5000.
THE JOURNAL oF CELL BIOLOGY - VOLUME 21, 1964 E. C. ADAMS AND A. T. HERTIG Guinea Pig Oocytes. I 409 410 THE JOURNAL o1«‘ CELL BIOLOGY - VOLUME 21, 1964 FIGURE 19 A higher power of Fig. 17, illustrating a. projection of a follicle cell between an adjacent follicle cell (left) and the oocyte (right). Note concentration of concentric lamellae of ergastoplasm at the base of this watery appearing projection. Note also desmosome-like attachment of oocyte to this projection and the concentration of ovular vesicles at right just beneath this thickening. Guinea pig 45-]. X 33,000.
FIGURE 90 Indentation of oocyte (left) by follicle cell (right) and increasing vesicle formation in oocyte cytoplasm at this interdigitation. Note Golgi complex of follicle cell oriented toward oocyte. Guinea pig 45-1. X 33,000.
FIGURE 17 An oocyte containing multiple dispersed Golgi areas, two of which are shown here. Note increase in size of oocyte. A proﬁle of endoplasmic reticulum is seen near nuclear membrane at top. Guinea pig 45-1. X 5000.
FIGURE 18 A higher power of Fig. 17 to show various arrangements of cristae in mitochondria and the close approximation of some mitochondria to endoplasmic reticulum. Note that there is an increasingly prominent oocyte-follicle cell intercellular space at the junction of two follicle cells (right) and that follicular projections often show tight junctions (left). Guinea pig 451. X 14,500.
E. C. ADAMS AND A. T. HERTIG Guinea Pig Oocytes. I
411 cooperate to produce a dense material seen streaming from the nuclear membrane. In contrast to these reports, however, RNP particles in our material are not seen associated with nuclear pores in such concentrated masses, but appear diffusely and evenly distributed in the cytoplasm. It is possible that the dense aggregates are formed at least in part by the agglomeration of these particles. Also possibly analogous to guinea pig oocyte rosettes are compact groups of mitochondria reported by Andre (1962) in developing rat spermatocytes. He observed that endoplasmic vesicles and RNP granules join in the formation of new mitochondria and emphasized the necessity for preexisting mitochondria in the process of proliferation.
2. Endoplasmic reticulum appears as a variety of interrelated forms during the developmental stages examined. In the simplest oocyte, the pervading cytoplasmic element is in the form of slightly granular vesicles. Since these vesicles occasionally appear contiguous with the nuclear, cellular, and mitochondrial membranes, one is tempted to imagine them as circulating between these membranes. They may represent the major mechanism for intake of materials required for a “maintenance metabolism.”
The earliest formation of paired membranes is seen in close morphological relationship to one or more mitochondria. Granular vesicles occasionally seen in chains near this developing ergastoplasm may possibly contribute to this endoplasmic reticulum. In a few micrographs these paired proﬁles are seen near the nuclear membrane, but most frequently they are found more peripherally. No annulate lamellae were observed.
Three—dimensional reconstruction of the developing granular endoplasmic reticulum would probably reveal a wavy plate—like structure. Our micrographs do not indicate whether or not this develops as one continuous structure in the cytoplasm of the oocyte.
The juxtaposition of developing endoplasmic reticulum to mitochondria was also noted by Bernhard and Rouiller (1956) who reported the close topographical relationship between mitochondria and ergastoplasm in livers of animals refed after starvation. These authors concluded that this juxtaposition indicates a physiologic relationship between these organelles and that the mitochondria provide energy for regrowth of ergastoplasm.
Localized concentrations of narrow cisternae of ergastoplasm with associated small vesicles and mitochondria often form parallel arrays or elaborate concentric loops in the central cytoplasm. Fawcett and Ito (1958), using phase and electron microscopy, observed that membranes of endoplasmic reticulum develop into parallel arrays and ultimately into elaborate concentric systems in preparations of guinea pig spermatocytes allowed to stand at room temperature for 4 hours. Waddington and Okada (1960) reported that the formation of double membranes into parallel or concentric membranes was related to degenera~ tive phenomena in Drosophifa ovaries. These two reports have made us question whether or not the localized accumulations of ergastoplasm in our oocyte micrographs might be caused by either atretic processes or technical procedures. Neither of these causes can be ruled out. Nevertheless, we feel that the presence of localized arrays of closely packed membranes in one form or another, in all of the nine oocytes observed in certain stages of development, may well be a normal manifestation of development in the oocytes. The formation of the zona and the peripheral location of the Golgi complex may bring about not only increased hydration of the cytoplasm reflected in increased ovular size but also other physiologic adaptations reflected in changing morphologic patterns of endoplasmic reticulum.
The localized ergastoplasm with associated mitochondria and small vesicles containing a moderately dense homogeneous material may be
The endopla.smic reticulum in this oocyte (see also Fig. Q) ha.s begun to form
loops dispersed in the cytoplasm as well as parallel arrays and con.centric loops in a. localized area of the cytoplasm. A vesicular aggregate is also present. The zona pellucida is beginning to appear in the widening space between the oocyte and the follicle cells. Guinea
pig 38-11. X 5,000.
FIGURE 91% Note that the location of the srnooth—membraned vesicles containing a dense amorphous material is between. and not within the cisternae of the paired rough membranes of endoplasmic reticulum. A vesicular aggregate is seen at the lower center. Note projections of oocyte and follicle cell into zona pellucida. Guinea pig 38-11. X 14,500.
THE JOURNAL or CELL BIOLOGY - VOLUME 21, 1964 E. C. ADAMS AND A. T. HERTIG Guinea Pig Oocytes. I 4-13 analogous to basophilic elements of the yolk nucleus described in molluscan oocytes by Rebhun (1961) and to the cortex of the vitelline body of some spider oocytes described by Sotelo and Trujillo—Cen6z (1957). The observation by Rebhun that “yolk particles contained in the yolk nucleus are never found within the cisternae, but are bounded away from the interior of the cisternae by a particle-studded membrane” is similar to our observations of vesicles containing an amorphous dense material located between, i.e. not within, the cisternae of the multilaminar ergastoplasmic arrays. Both Rebhun and Sotelo and Trujillo-Cenéz point out that there is no evidence that the yolk nucleus is concerned with the production of yolk. Nevertheless, it is possible that in the guinea pig oocytes these ergastoplasmic membranes are involved in the elaboration of the associated oval vesicles. It is also possible that the contents of the vesicles may be a lipid substance related to the large fat vacuoles seen in guinea pig oocytes developing in These fat vacuoles have been considered by previous workers as questionably analogous to yolk.
The reports of Sotelo and Porter (1959) and Odor (1960) on rat oocytes, Yamada et al. (1957) on mouse oocytes, Blanchette (1961) on rabbit oocytes, and Wartenburg and Stegner (1960) on human oocytes have indicated that endoplasmic reticulum is present only in meager amounts. In contrast, we ﬁnd in guinea pig oocytes the development, by the primary follicle stage, of a complex ergastoplasm. A species difference in the development of organelles in mammalian oocytes would, in itself, be of interest since it would suggest species differences in physiologic mechanisms. Although much of the older literature on developing mammalian oocytes emphasized the morphologic similarities, recent reports of histochemical studies indicate species differences. deGeeter (1954) commented that the apparent reserve of fat in guinea pig oocytes implies a physiology different from that in rat and rabbit oocytes in which poly secondary follicles.
saccharide is probably the principal reserve material. Jacoby (1962) in a review of ovarian histo— chemistry reports that “there appears to be a lack of uniformity amongst different species as regards the RNA—‘linked’ mechanism of protein increase in the growing oocyte.” The development of Epon 812 as an embedding agent is, at least, partially responsible for the preservation of the endoplasmic reticulum. Our comparable guinea pig oocytes embedded in rnethacrylate, but otherwise handled in identical fashion, showed only a scant amount similar to that reported by Anderson and Beams (1960) on guinea pig oocytes embedded in methacrylate.
The variety of forms of endoplasmic reticulum visualized during these differentiating stages of the guinea pig oocyte is an illustration of the dynamic state of these membranes discussed by Ito (1962) and is consistent with the functions, discussed by Porter (1961), currently assigned to elements of the endoplasmic reticulum, namely protein synthesis and intracellular transport. This concept would be particularly well adapted to explain the metabolism of a cell as large as an oocyte in which a system for intracellular transport of synthesized or absorbed material might be expected.
3. Corticaﬁ granules located just beneath the oolemma are ﬁrst seen in guinea pig oocytes as the follicle wall is just beginning to be double~layered. Morphologically similar granules observed in golden hamster tubal ova by Austin (1961) and in rabbit tubal ova by Hadek (1963) were thought to be involved in the zona reaction of fertilization since they were present in unpenetrated and absent in penetrated ova. Similar granules just beneath the oolemma of mature human oocytes were identiﬁed as yolk platelets by Tardini at al. (1961) who discussed their possible relationship to yolk globules and multivesicular bodies. Although Austin (1956) reported phase—microscopic observation of similar granules in golden hamster oocytes collected from follicles without antra, we know of no previous electron microscopic obser
FIGURE 23 An oocyte showing pronounced localizations of parallel arrays or concentric loops of ergastoplasm closely associated with dense vesicles. One group encloses an expanded vesicle of rough endoplasmic reticulum and mitochondria. A large Golgi complex is seen in the peripheral cytoplasm. This is the most extreme concentration of endoplasmic reticulum observed in any of our oocytes. Guinea. pig 38-3. )< 5000.
FIGURE 24 A higher power of Fig. Q3 to illustrate that the dense vesicles are between the cisternae of the parallel and concentric loops of the ergastoplasm. An expanded vesicle of rough endoplasmic reticulum is present. Guinea pig 3843. X 14,500.
THE JOURNAL OF CELL BIOLOGY - VOLUME 21, 1964 E. C. ADAMS AND A. T. HERTIG Guinea Pig Oocytes. I 415 vations of such granules so early in the development of any mammalian oocyte.
In the guinea pig oocyte, they form at a time when its increased surface activity is indicated by the elevation of microvilli and the profusion of vesicles in the peripheral cytoplasm, when the Golgi areas are enlarging in dispersed peripheral groups, and when the developing zona pellucida and follicle projections are providing a probable mechanism for the transport of materials to the oocyte membrane. The activities of all these devices for the regulation of exchange between the oocyte and its immediate environment might require the availability of membrane~forming material. We suggest that the cortical granules may represent reservoirs of such material and that they may contain a lipoprotein substance formed in peripheral aggregates of vesicles containing an internal structure. The coalescence of groups of vesicles followed by the condensation of their internal material appears to form the membrane-bounded dense spheres.
Somewhat similar aggregates have been described in the mouse oocyte by Yamada at al. (1957) and Rhodin (1963) and in rat oocytes at much later developmental stages by both Odor (1960), who called them a “vesicular complex,” and Sotelo and Porter (1959) who noted their resemblance to similar concentrations of vesicles in the centrosphere region of somatic cells. Sotelo and Porter postulated that these clumps of vesicles in mature or fertilized rat ova are derived from earlier accumulations of vesicles liberated from multivesicular bodies. In guinea pig oocytes of these early stages, multivesicular bodies are infrequently seen, but the proximity of even an occasional one to the aggregates of vesicles suggests that multivesicular bodies may, likewise, be a stage in the formation of the guinea pig vesicular aggregates. The apparent end products of these vesicular
aggregates in the guinea pig-“the cortical granules-—are not seen in their rat oocyte material.
Since these aggregates of vesicles apparently elaborate a granule, they might functionally be considered part of a Golgi complex, according to the suggestion of Dalton and Felix (1956) that this complex is concerned with the segregation and removal of water from maturing secretory products in certain cell types or from absorbed substances in others. The lack of any tubular elements, however, and the presence elsewhere in the oocyte of large aggregates of both vesicles and stacked tubules have led us to distinguish them, at least on a morphological basis, as separate entities. Their secretory products, the cortical granule, are frequently seen within a Golgi area and may serve to provide membrane material for the enlarging Golgi areas.
Our suggestion that the cortical granules in primary guinea pig oocytes represent a reservoir of membrane—forming material does not preclude the possibily related role proposed for the tubal ovum stage in golden hamsters (Austin, 1961) and in rabbits (Hadek, 1963). In these species as well as in sea urchin oocytes there is evidence that the contents of the cortical granules, released at the time of fertilization, may unite with or alter the vitelline membrane to form a barrier to further sperm penetration.
4. The Golgi compiex first appears as a single aggregate of smooth—membraned vesicles and short broad channels. Although the juxtanuclear location of this simple Golgi structure might be evidence for its derivation from the nuclear mem~ brane, our micrographs indicate a more probable derivation from a remodeling of granular vesicles since these surround and blend with the closely packed smooth—membraned forms. We have no evidence that the occasional large vacuoles which appear to contain phospholipid are involved in
FIGURE ‘25 An oocyte (comparable to the one illustrated in Fig. 3) containing dispersed loops of endoplasmic reticulum. Note that mitochondria appear to be in clusters containing many rosette forms and that the Golgi complexes lie in the peripheral cytoplasm. Note perinuclear zone that is free of larger organelles. Guinea pig 38-4. X 3,400.
FIGURE 26 A higher power of an oocyte (Fig. 3) in a stage of development similar to that
in Fig. 25. Note Inicrovilli extending from oocyte into zona pellucida, the profusion of vesicles just underneath the oolemma, the cortical granule, the peripheral Golgi complex, the clusters of mitochondria containing rosette forms, and the perinuclear area relatively free of larger organelles but containing RNP particles. Not.e also blebbing of external nuclear membrane. Guinea pig 45-4. X 9,700.
THE JOURNAL OF CELL BIoLoGY - VOLUME 21, 1964 417
E. C. ADAMS AND A. T. HERTIG Guinea Pig Oocytes. I the elaboration of this primitive form of the Golgi complex as proposed by Mercer (1962). The
elaborate multiple large Golgi complexes seen in"
the periphery of oocytes in primary follicles develop curved stacks of tubules and, in addition, often enclose a cortical granule which we have suggested may represent storage material.
Since the Golgi complex develops at the time when the primordial oocyte begins its growth phase and when the intercellular space between the oocyte and follicle is enlarging, it would seem reasonable to assign to the Golgi areas the adjustment of ﬂuid reserves of the oocyte.
5. The development of the interrelationship between follicle cells‘ and the oocyte follows the pattern previously illustrated and described by Anderson and Beams (1960) for guinea pig oocytes, by Odor (1960) and Franchi (1960) for rats, by Merker (1961) for rabbits, and by Chiquoine (1960) for a variety of mammals. As the cytoplasmic organelles in the oocyte develop, the widening zona pellucida may well begin to act as a ﬁlter between the oocyte and the substances collecting in the expanding intercellular spaces of the follicle wall. A profusion of slender microvilli extends from the ovular membrane into the zona and presumably increases the absorptive capacity at the surface of the oocyte. In addition, nutritive material also may be transported directly to the surface of the oocyte by the
projections of the granulosal cells. Although there is no evidence of a syncytial relationship between the oocyte and the granulosal cells, the presence of a network of delicate vesicles and channels at the extreme periphery of the oocyte could be interpreted as evidence that a surface mechanism has been established for the transport or absorption of material across the surface membrane of the oocyte.
Three rudimentary cilia have been seen in longitudinal section in cells of the wall of developing primordial follicles. Two of these are developing within a centriole and the third has emerged from the cell. They are similar to stages of cilia developing in ﬁbroblasts and smooth muscle cells discussed by Sorokin (1962). All three of the cilia in our material appear to be related to projections of the follicle cells. Their function as well as their prevalence remains unknown on the basis of our material. They may serve to circulate the inter cellular ﬂuid in periovular spaces although motility in rudimentary cilia has not been established. Centrioles with associated rudimentary cilia have also been observed in the parietal cells of the granulosal wall of mature rat follicles (Bjorkman, 1962).
In summary, by the beginning of secondary follicle formation in the guinea pig, the complex development and the peripheral location of the cyto— plasmic organelles of the primary oocyte suggest
FIGURE 27 A vesicular aggregate containing two gra.nules. Most of the vesicles show either an internal vesicle or a dense spot. Cortical granules are present beneath the oolemma. Guinea. pig 51-8. >(Q3,000.
FIGURE ‘28 Dense aggregate in the perinuclear cytoplasm. Not.e RNP particles on external nuclear membrane and free in cytoplasmic ground substance and the variety of granules in the nucleus. Guinea pig 51-8. X 23,000.
FIGURE 29 Guinea pig 38-3. X 14,500.
A rudimentary cilium emerging from a follicle cell at the base of projection.
FIGURE 30 A group of mitochondria showing intermediate forms converting to the structure seen in Fig. 31. Also present are loops of endoplasmic reticulum that are associated with small smooth~membraned vesicles and an expanded rough—membraned vesicle.
Guinea pig 51-8. X 23,000.
Two structures interpreted as modiﬁed mitochondria showing double external
membranes, peripheral cristae, and longitudinal ﬁbrils internally. They are joined by short strands of endoplasmic reticulum. Guinea pig 51-8. >< Q3,000.
FIGURE 32 Note the broad foot—like process of a follicle cell attached to oocyte membrane by a desmosome—like thickening of both membranes and that the microvilli are projecting into the zona pellucida. Guinea pig 51-8. X 14,500.
THE JOURNAL or‘ CELL BIOLOGY - VOLUME Q1, 1964 E. C. ADAMS AND A. T. HERTIG Guinea. Pig Oocytes. I 419 that it is equipped for the absorption, utilization, and intracellular transport of materials delivered to its surface membrane.
The authors gratefully acknowledge the participation in this project by Miss Susanne Foster and Mr. Arlo Collins who faithfully recorded their observations on the estrous cycles of these guinea pigs. Special thanks are due to Mrs. Audrey Hadﬁeld for the preparation of the micrographs and to Miss Edith Lowry for secretarial assistance. The authors
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also wish to acknowledge the generosity of Drs. Morris Karnovsky and Guido Majno who made many helpful suggestions during the preparation of the manuscript.
This research was supported entirely by a grant from National Institutes of Health, National Cancer
Institute, C—2451, now transferred to National Insti— tute of Child Health and Human Development,
HD00l37. Received for publication, August 2, I963.
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FIGURE 33 A 1ow—power view of the oocyte in a primary follicle illustrated in Fig. 4, showing a zona pellucida containing projections from follicle cells a.nd microvilli from oocyte, vesicular aggregates containing granules, clusters of mitochondria containing rosette formations, a.nd a tangential section through the nucleus. Guinea pig 4-5~3.
FIGURE 34 A vesicular aggregate containing granules in a variety of stages of formation. The vesicles each with an internal vesicular structure appear to coalesce, with subsequent condensations of a dense homogeneous substance and the elaboration of a granule with a single external membrane. A multivesicular body is seen at the periphery of the aggregate.
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The periphery of the oocyte showing cortical granules, one of which appears
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THE JOURNAL or CELL BIOLOGY - VOLUME 21, 1964 421
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FIGURE 36 An oocyte in a primary follicle. Localized concentrations as well as dispersed peripheral proﬁles of endoplasmic reticulum are present. Guinea pig 45-3. X 3400.
FIGURE 37 An oocyte in a somewhat larger primary follicle to show more dispersed loops of endoplasmic reticulum associated with clusters of mitochondria. Note tangential section through the nucleus. Guinea pig 38-6. >( 3400.
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FIGURE 38 Note variation in density and form of the vesicles and tubules in this Golgi area. Note also the diversity of membranous structures around the periphery of this complex. An intact cortical granule is seen near the surface of the oocyte. A projection from a gratlulosal cell is attached t_o the surface of the oocyte by a desmosome-like thickening. Guinea pig 45-3. X 14,500.
FIGURE 39 Small oval Vesicles containing dense Inaterial appear to form in relation to the ribosomal side of these tangentially sectioned, paired rough membranes. Note also free RNP gra.nules in the ground substance. The dense cores in the two mitochondrial rosettes at top left are closely associated with mitochondria that appear to be forming. Guinea pig 3843. X 22,000.
FIGURE 40 A low—power View of the oocyte in a mature primary follicle illustrated in Fig. 5. Zona pellucida is well formed and is traversed by the projections of follicular cells. These interdigitate with the ovular microvilli. The peripheral cytoplasm of the oocyte contains a concentration of Golgi complexes interspersed with vesicular aggregates and clusters of mitochondria all of which appear to be connected by proﬁles of rough-memhraned endoplasmic reticulum. Guinea. pig 38-5. x 5,000.
FIGURE 41 A higher power of the surface of the oocyte illustrat.ed in Fig. 40 showing the ovular microvilli, the tip of a follicular projection that is indenting the oolemma, a. cortical granule, a Vesicular aggregate, and proﬁles of endoplasmic reticulum. Guinea pig 38-5. x 14,500.
FIGURE 42 A follicular projection is indenting the oocytc, and delicate channels project. into the cytoplasm from the oolemma covering this projection. Proﬁles of endoplasmic reticulum show delicate branches that invade a cluster of mitochondria. Guinea pig 38-5. x ~23,000.