Granulosa Cell Deficient Follicles

The diameters of oocytes in follicles having a single layer of granulosa cells were measured hi four week old mice of various strains. There is a unique population of these follicles hi strains LT/Sv and C58/J in which the oocytes are significantly larger than the oocytes in single granulosa cell layered follicles of other common strains (C57BL/6J, BALB/cJ, and DBA/2J). These unique follicles are referred to as granulosa cell deficient (GCD) follicles since oocytes of these sizes are usually found in follicles with more than a single layer of granulosa cells. The parthenogenetic embryos that give rise to ovarian teratomas in strain LT/Sv are usually found in GCD-follicles. Some of the ova of strains LT/Sv and LTXBP, but not the ova of the other strains, are capable of spontaneous parthenogenetic activation after meiotic maturation. Although the ovulated ova of strain LTXBP are capable of spontaneous parthenogenetic development, the frequency of GCD-follicles and teratocarcinogenesis is low. Therefore, the frequency of ovarian teratocarcinogenesis is correlated with the simultaneous occurrence of two atypical conditions: first, the capability of the matured ova to undergo spontaneous parthenogenetic activation and, second, the high frequency of GCD-follicles.
GCD-follicles containing oocytes with a diameter greater than 65 μm were studied by electron microscopy. The follicles are usually enclosed within a layer of flattened theca-like cells. A basal lamina separates these cells from a single layer of cuboidal granulosa cells. Granulosa cell processes traverse the zona pellucida to contact the oocyte which shows ultrastructural characteristics typical of oocytes in the final growth stages. It is proposed that the GCD-follicles are competent to participate in the normal functions of follicular cells relating to oocyte growth and meiotic maturation.     

Light and electron microscope studies on ovarian follicles in the lizard Chalcides ocellatus

The development of ovarian follicles in a skink has been studied with light and electron microscopy. In early stages the previtellogenic oocyte has a follicular covering (granulosa) comprising only two cell types, small cells and pyriform cells. A complex microvillous interdigitation between follicle cells and oocyte is present from very early stages but regresses as a mature size is reached. The outer thecal layer differentiates into distinct interna and externa as growth proceeds. Occasional biovular follicles are formed. Pyriform cells establish direct continuity with the oocyte via cytoplasmic bridges which traverse the layer of microvilli interdigitating in the zona pellucida. Such bridges appear most frequently just before the onset of yolk deposition; the organelles and cytoplasmic constituents presumed to be transferred across them may stimulate this activity. As the follicles grow, the pyriform cells shrink and disappear to leave just the small cells forming the single layered granulosa. There is asynchrony in recruitment and/or early growth rates of follicle crops and uniformity of oocyte size appears only as vitellogenesis nears completion (with up to five oocytes, about 1 cm in diameter, on each side). Yolk deposition may involve transformation of golgi vesicles or pinocytotic vesicles but there is no evidence to show mitochondria as foci for deposition.     

Ultrastructural observations of previtellogenic ovarian follicles of dove

Dove ovarian follicle is a complex structure composed of oocyte surrounded by a somatic compartment consisting of theca externa, theca interna and granulosa. The structure of ovarian follicle (1 and 2 mm) of dove was studied by electron microscopy. The granulosa was pseudostratified in the 1-mm-diameter follicles and stratified with two or three irregular rows of cells in the 2-mm-diameter follicles. In the larger follicle indentations between oocyte and granulosa cells become more numerous and the microvilli of granulosa cell elongated to form a zona radiata with similarly elongated oocyte microvilli. Lining bodies were present at the tips of granulosa microvilli and in the cortical region of the oocyte. In the oocyte cortex were observed coated pits, coated vesicles, dense tubules, multivesicular bodies and primordial yolk spheres. Primordial yolk spheres may contain lining bodies and were observed fused with dense tubules and multivesicular bodies or associated with smooth cisternae.     

Ultrastructural and histochemical study of previtellogenic oogenesis in the desert lizard Scincus mitranus (Squamata,Sauropsida)

The structure of the granulosa in reptilian sauropsids varies between groups. We investigated the follicle development in the desert lizard Scincus mitranus. In the germinal bed, oogonia, and primary oocytes were identified and found to be interspersed between the epithelial cells. Previtellogenesis was divided into three stages: early, transitional, and late previtellogenic stages. During the early previtellogenic stage (diplotene), the oocyte is invested by small epithelia cells that formed a complete single layer, which may be considered as a young follicle. The transitional previtellogenic stage was marked by proliferation and differentiation of the granulosa layer from a homogenous layer consisting of only small cells to a heterogeneous layer containing three cell types: small, intermediate, and large cells. The late previtellogenic stage was marked by high-synthetic activity of large cells and the initiation of cytoplasmic bridges between large granulosa cells and the oocyte. Small cells were the only type of granulosa cells that underwent division. Thus, these cells may be stem cells for the granulosa cell population and may develop into intermediate and subsequently large cells. The intermediate cells may be precursors of large cells, as suggested by their ultrastructure. The ultrastructure of the large granulosa was indicative of their high synthetic activity. Histochemical analysis indicated the presence of cholesterol and phospholipids in the cytoplasm of large cells, the zona pellucida, among the microvilli, in the bridges region, and in the cortical region of the oocyte cytoplasm. These materials may be transferred from large cells into the oocyte through cytoplasmic bridges and provide nutritive function to large cells rather than functioning in steroidogenesis or vitellogenesis.     

Lectin binding sites of the mouse ovary, intraovarian and ovulated ova

Fluorescein isothiocyanate (FITC) labeled lectins were used to study the distribution of specific binding sites in histologic sections of mouse ovaries as well as ovulated ova. Four distinct patterns of reactivity of the components of the follicle (exclusive of the ovum) and the surrounding ovarian stroma were recognized: uniform staining of granulosa cells, theca cells and surrounding stroma; weak to moderate staining of the granulosa cells and strong staining of the theca cells and stromal cells; no reactivity of the granulosa cells coupled with strong reactivity of the theca and stromal cells; no reactivity with any component of the cumulus oophorus. Three lectins (from Triticum vulgare, Arachis hypogaea and Maclura pomifera) distinctly accentuated the basal lamina of the cumulus oophorus. The reaction of lectins with oocytes and zona pellucida revealed six distinct patterns: no reactivity with either structure; weak reactivity with the cytoplasm of the oocyte and no reactivity with the zona pellucida; very strong reactivity with the cytoplasm of the oocyte and no reactivity with the zona pellucida; moderate reactivity with both the oocyte and the zona pellucida; moderate reactivity with the oocyte and very strong reactivity with the zona pellucida; no reactivity with the oocyte and moderate reactivity with the zona pellucida. The same pattern of reactivity was seen in the ovulated ova in the oviduct except for the lectin from Arachis hypogaea, the reactivity of which changed upon ovulation and/or fertilization of the ovum. These data provide a list of lectin markers for distinct components of the mouse ovary.     

Ultrastructural and quantitative dynamics of the granulosa of ovarian follicles of the lizard Gerrhonotus coeruleus (family Anguidae)

The progression of ovarian follicular development in the Northern Alligator Lizard has been documented ultrastructurally and by enumeration of cells, with a focus on changes in the granulosa component of the follicle. The pattern of cellular differentiation of the granulosa entails, as in other lizards, the transformation of a simple, cuboidal epithelium in small follicles into a complex layer consisting of three types of cells. Marked differences in size and ultrastructure of the cell types indicate different functional states: the smallest cells are little differentiated and serve primarily as stem cells to other granulosa cells throughout follicular growth, whereas the larger "intermediate" and "pyriform" cells do not divide and show ultrastructural features indicative of synthetic activity. Contrary to some views that this latter cell type is the final step in cellular differentiation and provides organelles and cytoplasm to the oocyte through an intercellular bridge, the results of this study suggest that only relatively small molecules such as ribosomal RNA might pass between cells. Further, these observations support the interpretation that a heterogeneous granulosa results from the fusion in early follicular stages of some cells that are in surface contact with the oocyte. Several of the cytological features of the larger granulosa cell types are seen in the oocyte and in germ-line cells generally, such as highly dispersed chromatin, large nucleoli, abundant nuclear pores, mitochondrial "rosettes," annulate lamellae, "ribosome bodies," and surface microvilli. This strongly suggests that the cytology of large granulosa cells is induced by the oocyte. The heterogeneous granulosa persists only through previtellogenesis and at the onset of exogenous yolk uptake by the oocyte it becomes a secondarily homogeneous layer. The appearance of the granulosa at this stage is similar to that of reptiles whose granulosa remains a single-cell layer throughout folliculogenesis (e.g., turtles and crocodilians). Thus, although follicular development has been scrutinized in only a few representative genera of reptiles to date, the course of follicular development among lizards is similar in detail and involves the transitory development of a heterogeneous population of cells. This feature appears to be exclusive to the squamate reptiles.     

Ultrastructure of follicular atresia in the rat

Observations were made on the sequence of morphologic changes in atresia of medium-sized preantral follicles in the rat. Ultrastructural studies indicated that in both control and hormonally treated animals granulosa cell changes, including nuclear condensation and alterations in cytoplasmic organelles, occurred prior to effects on the oocyte. In more advanced stages of atresia, extensive disruption of granulosa cell cytoplasm was associated with loss of microvilli and cytoplasmic vacuolization in oocytes. The findings are consistent with the view that follicular atresia begins with alterations in granulosa cells, effects on the oocyte occurring later in the atretic process.     

Study of yolk precursor transport in the avian ovary with the use of horseradish peroxidase.

Ovaries of adult Japanese quails were exposed in vivo to the exogenous protein horseradish peroxidase (HRP) for varying lengths of time to investigate ultrastructurally the permeability of the wall of these follicles, the protein uptake capacity of granulosa and oocyte and the kinetics of protein uptake in different stages. There is a sudden increase in permeability of the follicle wall from previtellogenesis to vitellogenesis. This is not due to a loss of sealing (tight) junctions in the granulosa cell layer, but is probably related to a permeability change in the basement membrane. The transition from the slow growth phase to the rapid growth during vitellogenesis is accompanied by a limited widening of the intercellular channels and the concomitant development of a complex endocytotic apparatus in the ooplasm. The slowing down of yolk deposition during the last day before ovulation is accompanied by a narrowing of the intercellular channel width. The granulosa cells show a high intracellular HRP uptake during intermediary yolk formation. Transcytosis through the granulosa cannot be excluded but is probably a minor pathway at certain stages. The light microscopically detectable uptake of HRP by the oocyte coincides with the start of exogenous vitellogenesis. After 90 sec of exposure to HRP (intravenous injection) the tracer can be found in the intercellular channels of the granulosa and in superficially located yolk spheres. On the other hand it takes 10 min for the tracer to traverse the cortex of the oocyte.     

Lectin binding sites of the mouse ovary,intraovarian and ovulated ova

Summary Fluorescein isothiocyanate (FITC) labeled lectins were used to study the distribution of specific binding sites in histologic sections of mouse ovaries as well as ovulated ova. Four distinct patterns of reactivity of the components of the follicle (exclusive of the ovum) and the surrounding ovarian stroma were recognized: 1. uniform staining of granulosa cells, theca cells and surrounding stroma; 2. weak to moderate staining of the granulosa cells and strong staining of the theca cells and stromal cells; 3. no reactivity of the granulosa cells coupled with strong reactivity of the theca and stromal cells; 4. no reactivity with any component of thecumulus oophorus. Three lectins (from Triticum vulgare, Arachis hypogaea and Maclura pomifera) distinctly accentuated the basal lamina of thecumulus oophorus. The reaction of lectins with oocytes and zona pellucida revealed six distinct patterns: 1. no reactivity with either structure; 2. weak reactivity with the cytoplasm of the oocyte and no reactivity with the zona pellucida; 3. very strong reactivity with the cytoplasm of the oocyte and no reactivity with the zona pellucida; 4. moderate reactivity with both the oocyte and the zona pellucida; 5. moderate reactivity with the oocyte and very strong reactivity with the zona pellucida; 6. no reactivity with the oocyte and moderate reactivity with the zona pellucida. The same pattern of reactivity was seen in the ovulated ova in the oviduct except for the lectin from Arachis hypogaea, the reactivity of which changed upon ovulation and/or fertilization of the ovum. These data provide a list of lectin markers for distinct components of the mouse ovary.     

Ultrastructure of cortical granule release and zona interaction in monospermic and polyspermic human ova fertilized in vitro

Cortical granule release and interaction with the zona pellucida are reported in monospermic and polyspermic fertilized ova and early human embryos cultured in vitro. Twenty-seven preovulatory oocytes from women with tubal or idiopathic infertility were recovered by laparoscopy, after induction of follicular maturation with clomid and human chorionic gonadotropin. These were then inseminated with husband's or donor sperm, cultured for 3–72 hr, routinely fixed in glutaraldehyde/osmium and examined ultrastructurally. Evidence of cortical granule release was observed in all ova and embryos investigated and their contents were identified either at the egg surface or in the perivitelline space or interacting with the inner zona, apparently reinforcing its structure. The latter is very likely the morphological expression of the zona reaction. Delayed release was seen in certain regions of normally fertilized ova and particularly in polyspermic ova, where massive “explosions” of granules occurred. This was attributed to delayed cortical maturation. The mechanics of release were similar in both monospermic and polyspermic ova. Spontaneous dehiscence was also described in one injured unfertilized oocyte. The significance of the cortical and zona reactions as an effective block to polyspermy at the level of the inner zona, which becomes more impenetrable to supplementary sperm, is discussed.     

Comparison of the penetrability of the egg vestments in follicular oocytes, unfertilized and fertilized ova of the rabbit

Mixed populations of rabbit ovulated eggs and follicular oocytes, one labelled with a fluorescent marker, were transferred to the same tubal ampulla of an inseminated recipient female and were then recovered 3 hr later. There was no significant difference in the number spermatozoa penetrating to the perivitelline space or within the substance of the zona pellucida of follicular oocytes (immature or atretic) and mature ovulated ova. In contrast to mature ovulated ova, however, none of the spermatozoa reaching the perivitelline space of vesicular (dictyate) oocytes had attached to or penetrated the vitelline surface to enter the ooplasm.The same approach involving transfer of nonpenetrated eggs together with eggs penetrated previously in a donor female, demonstrated that prior entry of spermatozoa does not reduce the penetrability or receptivity of the rabbit zona pellucida to subsequent spermatozoa.These experiments indicate: (a) that the penetrability of the granulosa cell investment and/or zona pellucida of the rabbit follicular oocyte does not change from the time of antrum formation until the point at which follicular atresia ensures; (b) that between the time of initial LH stimulation and ovulation important changes mediating the onset of the fertizability of the dictyate oocyte of the rabbit probably occur at the vitelline surface; and (c) that in neither a qualitative nor quantitative sense has the demonstrably greater resistance of the rabbit zona pellucida to proteolysis following fertilization any physiological significance for sperm penetration.     

Morphometric and ultrastructural characterization of Bos indicus preantral follicles

The aim of the present study was to characterize the ultrastructure of zebu cow preantral follicles (PAFs). Ovarian cortex samples were processed for light and transmission electron microscopy. Primordial follicles consisted of an oocyte surrounded by one layer of flattened or flattened-cuboidal granulosa cells. The oocyte contained a large and usually eccentric nucleus. Most organelles were located at the perinuclear ooplasm. Round shaped mitochondria, which contained electron-dense granules, smooth and rough endoplasma reticulum and a Golgi apparatus were also observed. Vesicles and coated pits were often observed in the cortical ooplasm. In primary follicles, the oocyte was surrounded by one layer of cuboidal granulosa cells. Short microvilli were observed on the oolema. Secondary follicles consisted of an oocyte surrounded by a variable number of layers of cuboidal granulosa cells. Small secondary follicles had an ultrastructure very similar to that observed in primary follicles. At this follicular stage, the zona pellucida was beginning to form around the oocyte. In large secondary follicles, the zona pellucida was totally developed around the oocyte. Several granulosa cell projections could be detected that were encroaching into the zona pellucida and protruding towards the oocyte, where gap junctions were observed between oocyte and granulosa cell membranes. Organelles within the oocyte were located at the periphery of the ooplasm, and clusters of cortical granules were observed. Round mitochondria were abundant in all developmental stages. In conclusion, this study described the ultrastructure of zebu cow PAFs, and some unique characteristics could be observed as compared with what has been reported for follicles of Bos taurus cattle.     

A correlative microscopical analysis of differentiating ovarian follicles of mammals

The mammalian ovary has been studied by optical microscopy and by scanning and transmission electron microscopy with the purpose of presenting an integrated view of the differentiating mammalian follicle. During follicular development, changes in the granulosa cells are particularly noteworthy and include dramatic modifications in cell shape coincident with antrum formation. The cytoplasmic processes of those granulosa cells immediately surrounding the oocyte, as well as the more peripheral granulosa cells comprising a second and third layer, traverse the zona pellucida, infrequently interdigitate with the microvilli of the egg, and make both desmosomal and gap junction contacts with the oocyte. The zona pellucida is thus distinguished by numerous fenestrations of varying diameters. The membrana limitans (basal lamina) is a bipartite structure composed of (a) a homogeneous stratum upon which the peripheral layer of granulosa cells rests, and (b) an outer region of collagen-like fibers. The specific advantages and limitations of the different methodologies utilized to study folliculo-genesis are discussed.     

Ultrastructural characteristics of the follicle cell-oocyte interface in the oogenesis of Ceratophrys cranwelli

In this work we carried out an ultrastructural analysis of the cell interface between oocyte and follicle cells during the oogenesis of the amphibian Ceratophrys cranwelli, which revealed a complex cell-cell interaction. In the early previtellogenic follicles, the plasma membrane of the follicle cells lies in close contact with the plasma membrane of the oocyte, with no interface between them. In the mid-previtellogenic follicles the follicle cells became more active and their cytoplasm has vesicles containing granular material. Their apical surface projects cytoplasmic processes (macrovilli) that contact the oocyte, forming gap junctions. The oocyte surface begins to develop microvilli. At the interface both processes delimit lacunae containing granular material. The oocyte surface has endocytic vesicles that incorporate this material, forming cortical vesicles that are peripherally arranged. In the late previtellogenic follicle the interface contains fibrillar material from which the vitelline envelope will originate. During the vitellogenic period, there is an increase in the number and length of the micro- and macrovilli, which become regularly arranged inside fibrillar tunnels. At this time the oocyte surface exhibits deep crypts where the macrovilli enter, thus increasing the follicle cell-oocyte junctions. In addition, the oocyte displays coated pits and vesicles evidencing an intense endocytic activity. At the interface of the fully grown oocyte the fibrillar network of the vitelline envelope can be seen. The compact zone contains a fibrillar electron-dense material that fills the spaces previously occupied by the now-retracted microvilli. The macrovilli are still in contact with the surface of the oocyte, forming gap junctions.     

Mouse oocytes promote proliferation of granulosa cells from preantral and antral follicles in vitro.

Evidence is now emerging that the oocyte plays a role in the development and function of granulosa cells. This study focuses on the role of the oocyte in the proliferation of (1) undifferentiated granulosa cells from preantral follicles and (2) more differentiated mural granulosa cells and cumulus granulosa cells from antral follicles. Preantral follicles were isolated from 12-day-old mice, and mural granulosa cells and oocyte-cumulus complexes were obtained from gonadotropin-primed 22-day-old mice. Cell proliferation was quantified by autoradiographic determination of the 3H-thymidine labeling index. To determine the role of the oocyte in granulosa cell proliferation, oocyte-cumulus cell complexes and preantral follicles were oocytectomized (OOX), oocytectomy being a microsurgical procedure that removes the oocyte while retaining the three-dimensional structure of the complex or follicle. Mural granulosa cells as well as intact and OOX complexes and follicles were cultured with or without FSH in unconditioned medium or oocyte-conditioned medium (1 oocyte/microliter of medium). Preantral follicles were cultured for 4 days, after which 3H-thymidine was added to each group for a further 24 h. Mural granulosa cells were cultured as monolayers for an equilibration period of 24 h and then treated for a 48-h period, with 3H-thymidine added for the last 24 h. Oocyte-cumulus cell complexes were incubated for 4 h and then 3H-thymidine was added to each group for an additional 3-h period. FSH and/or oocyte-conditioned medium caused an increase in the labeling index of mural granulosa cells in monolayer culture; however, no differences were found among treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early programming of maturation competence in mouse oogenesis

Growing mouse oocytes incompetent to mature were freed of attached granulosa cells at different stages of growth, and cultured in vitro in the presence of fibroblast monolayers and/or their products. In these culture conditions, although growth was arrested, isolated oocytes survived in vitro for several days, and finally resumed meiosis spontaneously, progressing up to metaphase I. The culture time length needed for in vitro acquisition of the capacity to mature was inversely related to the initial oocyte size at the time of isolation from granulosa cells, and closely corresponded to developmental timing of acquisition of such ability in vivo. We conclude that the acquisition of mouse oocyte competence to mature follows a definite time program, which is independent of the presence of granulosa cells and of heterologous cell contacts, at least within the developmental stages studied.     

Ultrastructural evaluation of oocytes during atresia in rat ovarian follicles

Mammalian females are born with a finite number of ovarian oocytes, the vast majority of which ultimately undergo degeneration by atresia. The overall process of ovarian follicular atresia has been morphologically well described only in large antral follicles. Additionally, little attention has been focused on ultrastructural changes in the oocyte. Furthermore, most such morphological studies were performed prior to identification of apoptosis as a mechanism of physiological cell death. Therefore, the purpose of this study was to use electron microscopy to compare the process of atretic oocyte degradation in ovarian follicles of female Fischer 344 rats (38 days old) with ultrastructural characteristics of apoptosis. Examination of ovarian follicles revealed that nucleolar segregation, cytoplasmic or nuclear condensation, apoptotic body formation, and chromatin margination along the nuclear membrane are never observed in atretic oocytes during the degenerative process. Instead, early morphological changes in atretic oocytes include retraction of granulosa cell- and oocyte-derived microvilli and condensation of mitochondria and loss of cristae. These occurrences coincide with initiation of granulosa cell apoptosis. After most granulosa cells are lost, more severe changes occur, including segmentation of the oocyte and cytoplasmic vacuolization as atresia progresses. Thus, these results suggest that, during atresia, oocytes are removed by physiological oocyte cell death, a method that does not involve classically described apoptosis.     

Populations of granulosa cells in small follicles of the sheep ovary.

The aim of this study in sheep ovaries was to determine the total number of granulosa cells in primordial follicles and at subsequent stages of growth to early antrum formation. The second aim was to examine the interrelationships among the total number of granulosa cells in the follicles, the number of granulosa cells in the section through the oocyte nucleolus, and the diameter of the oocyte. A third aim was to examine whether proliferating cell nuclear antigen labelling occurred in flattened granulosa cells in primordial follicles or was confined to follicles containing cuboidal granulosa cells. The follicles were classified using the section through the oocyte nucleolus by the configuration of granulosa cells around the oocyte as type 1 (primordial), type 1a (transitory), type 2 (primary), type 3 (small preantral), type 4 (large preantral), and type 5 (small antral). In type 1 follicles, the number of granulosa cells and oocyte diameter were highly variable in both fetal and adult ovaries. Each type of follicle was significantly different from the others (all P < 0.05) with respect to oocyte diameter, number of granulosa cells in the section through the oocyte nucleolus and total number of granulosa cells. Follicles classified as type 2, 3, 4 or 5 each corresponded to two doublings of the total granulosa cell population. The relationships between oocyte diameter and the number of granulosa cells (that is, in the section through the oocyte nucleous or total population per follicle) could all be described by the regression equation loge chi = a + b loge gamma with the correlation coefficients R always > 0.93. For each pair of variables the slopes (b) for each type of follicle were not different from the overall slope for all types of follicle pooled. Immunostaining for proliferating cell nuclear antigen was observed in granulosa cells in type 1 follicles, as well as in the other types of follicle. These findings indicate that 'flattened' granulosa cells in type 1 follicles express an essential nuclear protein involved in cell proliferation before assuming the cuboidal shape. Thus, when considering factors that regulate specific phases of early follicular growth, it is important to consider: (i) the follicle classification system used; (ii) the animal model studied; (iii) whether type 1 follicles are all quiescent; and (iv) the likelihood that each follicle type represents more than one doubling of the population of granulosa cells.     

The cellular envelope of oocytes in teleosts

Summary Structural and functional relationships between oocytes and their envelopes were studied by means of electron microscopy in several teleost species after injection of live fish with horseradish peroxidase. The marker first appeared in the capillaries and the pericapillary spaces of the ovarian stroma. It then entered the collagen-filled spaces between the granulosa and theca cells; these spaces are in direct connection with the pericapillary spaces. The marker penetrated between the follicle cells and into the channels of the zona radiata surrounding the microvilli which traverse these channels. The marker was never found inside the microvilli or in the follicle cells; finally, it reached the surface of the oocytes and was internalized via micropinocytosis. Six stages in the course of folliculogenesis were observed, determined by (1) the formation of follicular and thecal cellular layers and a collagen-filled space between them, (2) the development of microvilli of oocytal and follicular origin, (3) the differentiation of the vitelline envelope and the pore channels, (4) pinocytotic activity of the oocytes, and (5) rapid growth of the oocyte and its envelopes during vitellogenesis.This research was supported by a grant from the National Council for Research and Development, Israel, and the GKSS GeesthachtTesperhude, Federal Republic of Germany     

Ovarian follicle ultrastructure in the teleost Synbranchus marmoratus (Bloch, 1795), with special reference to the vitelline envelope development

Synbranchus marmoratus is a protogynous diandric teleost fish widely distributed throughout South America. The aim of this work was to study the ultrastructure of the vitelline envelope and the relationship among oocyte and their follicular cells during oogenesis. During perinucleolar stage, the oocyte and the follicular cells form microvillar processes that project into the perivitelline space. The oocyte secretes a dense and amorphous material, which appears as the first evidence of the vitelline envelope (VE) development. The VE passes from a double to a multilayered structure during oocyte growth. In mature oocytes, the VE reach a mean thickness of 11 microm, having up to 30 layers. Oocyte microvilli are thinner than the follicular ones and were seen in contact with the follicular plasmalema, however we could not find any contact between the follicular microvilli and the oolemma. Before ovulation, microvillar processes retract and the pore canals seem to collapse. An outer electron dense layer occludes the superficial pore and forms a continuous layer. No jelly or adhesive coatings were seen at least in ovulated eggs sampled from ovarian lumen. Follicular cell and oocyte cytological characteristics do not differ from those described in other teleosts species.