On the infiltration of Golgi bodies from the follicular epithelium to the egg

Summary 1. In a well advanced oocyte of the tortoise the egg membranes besides the theca and the single-layered epithelium consist of a zona pellucida often differentiated into zona striata and a homogeneous layer; underlying these two layers is a layer of cortical fibrillae or fibrillar layer, Next to this layer, is the limiting membrane of the egg which is not present in all stages and generally disappears in a well developed oocyte. In certain animals either the homogeneous layer or fibrillar layer is absent. 2. In certain animals,Golgi bodies seem to be extruded into the follicle cells from the theca cells. 3. At a particular stage of development the follicle cells become very active and produce a large number of smallGolgi bodies. TheseGolgi granules filter through canalicular passages of the zona radiata into either the homogeneous layer and from thence into the fibrillar layer or where a homogeneous layer is not present directly to the fibrillar layer. Where a fibrillar layer is not present they are transferred directly to the limiting membrane and from thence to the egg. 4. In certain cases e. g. in Fowl, Calotes and Uromastix, fairly large lumps ofGolgi bodies are extruded from the follicle cells through the zona pellucida into the egg. Here the fine canilicular passages do not seem to form a vehicle for the passage of these comparatively larger bodies. 5. The fine canalicular passages in the zona radiata ofTestudo graeca andKachuga smithii and the fibrillar prolongation of the cytoplasm which we have called the fibrillar layer are marked features of the egg membranes at certain stages of development of the egg. During the period when infiltration ofGolgi bodies through these passages takes place slides prepared by silver nitrate and osmic methods show black beaded chains ofGolgi granules in various stages of descent. 6. It is claimed that the extrusion and infiltration ofGolgi bodies from the follicular epithelium to the egg are established phenomena at least in the Vertebrates.     

An electron microscope study of the formation of the zona pellucida in oocytes from Triturus viridescens

Summary The formation and morphology of the zona pellucida around the amphibian oocyte was studied by means of electron microscopy. The onset of formation is indicated by undulation of the basal membrane of the follicle cells and the plasmalemma. Gradually a homogeneous ground substance is deposited between the aforementioned layers. The plasmalemma becomes so undulated as to form microvilli which project up into the continually accumulating ground substance. As maturation proceeds, sites on the basal membrane remain in contact with the plasmalemma and eventually extend down as macrovilli. At the time the zona reaches its maximal development and complexity, it consists of two layers, a more peripheral homogeneous layer and an adjacent thicker and more dense zona radiata. The microvilli, which are 0.1 in diameter, project up deeply into the zona radiata. The macrovilli, which are 0.2 in diameter, by this time extend down through the entire width of the zona pellucida and into (but not in direct contact with) peripheral cytoplasm. As the oocyte reaches maturity, the microvilli will gradually recede and the ground substance of the zona pellucida will serve to form the fertilization membrane. The problem of the site of synthesis and function of the zona is discussed.This investigation was supported by a Public Health Service research grant (05803-04) from the National Institute of General Medical Sciences and a research career program award (5-K3-HD-5356-02) from the National Institute of Child Health and Human Development.     

牦牛卵泡细胞及其卵母细胞不同发育时期的结构变化

采集成年母牦牛卵巢,通过光镜和电镜对牦牛卵泡及其卵母细胞不同发育时期的结构变化进行了观察。结果发现当卵母细胞被单层立方卵泡细胞包围时,微绒毛开始出现,而皮质颗粒、透明带则在包被2-4层卵泡细胞时开始出现。随着卵母细胞的继续发育,透明带增厚,微绒毛由粗短变为细长,密度增加;皮质颗粒、线粒体、滑面内质网等细胞器的数目不断增加,并逐渐移行到质膜下;在移行的过程中,皮质颗粒成团存在。在囊状卵泡中,卵母细胞皮质颗粒呈线形分布于质膜下,线粒体、滑面内质网又移向胞质中央。卵母细胞借助微绒毛穿过透明带与卵泡细胞胞质突起相联系。结果证明牦牛卵泡和卵母细胞不同发育时期的结构变化与其它哺乳动物的基本相似。     

The ultrastructure of the layers enveloping yolk-forming oocytes from Triturus viridescens

Conclusion The peripheral membranous and extracellular layers of oocytes at the onset of yolk formation were studied by electron microscopy. It was shown that three cellular layers are present at this time. The outer or surface epithelium contains typical squamous cells. The middle or theca is the connective tissue layer which contains fibroblasts, blood vessels, and collagen fibers. The inner or follicular epithelium proper consists of compactly arrayed follicle cells that have distinct cell boundaries. Two extracellular layers were observed, a coarse granular homogeneous layer and a dense zona radiata. Macrovilli (0.2 in diameter), extensions from the follicle cells, project through the extracellular layers into the peripheral cytoplasm while more numerous microvilli (0.1 in diameter) project up to the dense matrix of the zona radiata. The plasmolemma separating the peripheral cytoplasm from the follicle cells is completely irregular; it forms microvilli. The relations of the enveloping layers as seen with both light and electron microscopes are discussed.This investigation was supported by a Public Health Service research grant (5803-C3) and research career program award (K-3-5356) from the Division of General Medical Sciences.     

Elektronenmikroskopische und histotopochemische Untersuchungen über Struktur und Bildung der Zona pellucida menschlicher Eizellen

Summary Examined with the electron microscope the zona pellucida of human oocytes represents an extracellular, amorphous substance with slight differences in density. There is a greater consolidation in the inner parts and a drecrease of density towards the periphery. The plasmalemma of the oocyte forms a large number of slender projections (microvilli) penetrating the homogeneous groundsubstance of the zona pellucida. Plasmatic elongations of the follicle cells extending towards the oocyte traverse the zona in oblique or tangential directions and end at the oocytes surface forming a contact relationship (partially characteristic desmosomes). No syncytial communication between ooplasma and follicle cell cytoplasm can be demonstrated. The follicle cell processes contain finely granular material. The cytoplasm of numerous follicle cells facing the oocyte containes branched deposits of compact and dense substances with a granular ground-structure. Histochemically these substances react like anionic polysaccharides. Alternating zones of the follicle cell membranes show vaguely outlined lesser density. Intra- and extraplasmatic granular concentrations in these areas probably represent secretion processes. Acid mucopolysaccharides are the primary substrat of the zona pellucida whose deposition begins almost in the state of a bilaminar secondary follicle. Possibly this material is incorporated in the definite complex of glycoproteins by means of loss or binding of the acid groups (uronic acids, ester sulphates). PAS-positive inclusions about 1,0 to 1,5 in diameter lying in the cytoplasm of the follicle cells immediately adjacent to the plasmalemma or in open communication with the perivitelline space, represent probably paraplasmatic components for the building of the zona. Studied with the electron microscope these inclusions resemble strikingly the amorphous and cloudy ground substance of the zona pellucida. Stratified structures which would embody an appositional growth of the zona have not been demonstrated.     

Muco-follicle cells of the jelly coat in the oocyte envelope of the sheatfish (Silurus glanis L.)

During early vitellogenesis of the oocytes of Silurus glanis, the follicular cells proliferate, their epithelial organization becomes disrupted, and they transform into an irregularly structured large mass of cells engaged in intensive secretory activity. They contain nuclei, rough endoplasmic reticulum, Golgi bodies, and secretory inclusions termed “acorn bodies,” which are synthesized in the cytoplasm. The acorn bodies have two components: an electron-dense cap and a moderately electron-dense body. As development proceeds, the acorn bodies become modified into spherules of mucous material, the mucosomes. The electron-dense part persists as a small calotte or crescent often irregularly structured at the periphery of the mucosome, and fragments of it are dispersed into the interior of the mucosomal body. The mucosomes are membrane-bound and contain small granules, 55 nm in diameter. At the end of vitellogenesis, the follicle cells are filled with mucosomes, and cytoplasmic residua can only sparingly be observed among them. Oocytic microvilli extend through the zona radiata and intermingle with follicular cell processes in the cleft between the zona radiata and the belt of mucosomes during growth of the oocyte. Capillaries develop in connective tissue of the theca layer as vitellogenesis proceeds. © 1993 Wiley-Liss, Inc.     

Light microscopic observations of alterations in staining of the zona pellucida of porcine follicular oocytes: Possible early indication of atresia

Serial sections of porcine ovaries were examined in an attempt to detect early signs of oocyte degeneration/atresia using special staining. Porcine ovaries were fixed in Bouin's fixative and embedded in paraffin using routine techniques. Serial sections (8 μm) were mounted on glass slides and stained with Shorr's S3 and hematoxylin stain. Several criteria were used for examining general histology of the antral follicles: condition of the granulosa layer, antral cavity, the oocyte and its surrounding zona pellucida, and the cumulus layers. A change in the staining characteristic of the zona pellucida was the single most striking observation in all ovaries examined. In presumably healthy follicles, the zona pellucida was uniformly stained green, the granulosa layer was intact with fewer than three pyknotic cells per section, and a uniform basement membrane (stained green) separated the intact theca layers from the remainder of the follicle. In those follicles showing some degree of degenerative changes in the follicular wall, the zona pellucida was stained a bright orange. In the last stages of degeneration, follicles exhibited many pyknotic nuclei throughout the granulosa layers, the layer of granulosa cells was in many cases separated from the basement membrane, and the antrum was infiltrated with lymphocytes. In these follicles, the zona pellucida was always stained orange. Frequently, the zona pellucida was partially stained orange before any detectable changes could be seen in other elements of the follicular wall. Additionally, many non-antral (primary) follicles exhibited oocytes with orange-stained zonae pellucidae. In terminal stages of follicular degeneration, collapsed follicles were infiltrated by connective tissue elements stained bright orange and green. These structures very often contained dying oocytes always with bright orange-stained zonae pellucidae. Scattered throughout the ovarian stroma were many orange-stained remnants of zonae pellucidae. It is thought that perhaps the characteristic staining of the zona pellucida with Shorr's S3 stain may give an early, previously undetectable indication of follicular atresia.     

The oocyte of a new world marsupial, Monodelphis domestica: structure, formation, and function of the enveloping mucoid layer

Ovulated oocytes of the gray short-tailed opossum Monodelphis domestica are surrounded by a thin zona pellucida and are devoid of a cumulus oophorus. In the ampulla of the oviduct, oocytes acquire a thick mucoid layer composed of concentrically arranged fibrillar material. Exocytosis by the secretory cells of the oviductal epithelium occurs in the region of the oviduct adjacent to the egg. This suggests that the oocyte-zona-mucus layer complex may influence the oviductal epithelium to secrete. During secretion, fibrillar contents of the secretion granules appear to be transformed into membranous material which presumably becomes fibrillar again as it is incorporated into the forming mucoid layer. Spermatozoa (which are known to pair in the cauda epididymis) are found in pairs and with intact acrosomes in the mucoid layer of fertilized eggs. This suggests that spermatozoa of Mondelphis remain paired until they reach the zona pellucida and that the acrosome functions in zona binding and/or penetration.     

Quantitative and qualitative cytochemical analysis of changes in polysaccharides-proteins in rat ovulable and atretic ovarian follicles during the estrous cycle

Summary The glycosaminoglycan (periodic acid — Schiff, PAS) and hyaluronic acid (alcian blue) content of the membrana granulosa, zona pellucida and antrum of rat ovarian follicles was analyzed qualitatively and quantitatively during the estrous cycle in three types of follicles: ovulable, early atretic and late atretic. The qualitative analysis consisted of the conjunctive localization of PAS-reactive, fluorescent granules within the membrana granulosa. The quantitative analysis consisted of microdensitometric measurements of PAS and alcian blue staining within the zona pellucida and antrum of the ovulable and atretic follicles. For the localization of PAS granules within the granulosa cells, ovaries were removed on the day of proestrus, fixed in 6% paraformaldehyde, embedded in methacrylate and sectioned. Following the examination of the cells for fluorescence, the same section was stained with PAS and lead-hematoxylin. In ovulable follicles there was no fluorescence in the membrana granulosa while PAS granules occurred exclusively within the cells of the cumulus and corona radiata. In late atretic follicles, fluorescent-PAS reactive granules were located in the granulosa cells at the periphery of the follicle. During early atresia no fluorescence and very few PAS granules were observed in the granulosa cells. Since fluorescence is a marker for some lysosomes, these observations suggest that the PAS granules in the ovulable follicles may not be a type of lysosome. The amount of stain in the zona pellucida and antrum of the three follicular types was quantified using a scanning and integrating microdensitometer. On all days of the estrous cycle, PAS intensity was higher in the zona pellucida than in the antrum of the three follicular types. PAS staining in the respective antra was the same on all days of the estrous cycle. Intrafollicular PAS staining in the zonae pellucidae differed during the cycle. With respect to the zonae pellucidae, staining intensity in the three follicles was identical on estrus. On diestrus-1, staining intensity was the same in the ovulable and early atretic follicles and less in the late atretic follicle. By diestrus-2 and on proestrus, PAS intensity was highest in the zona pellucida of the ovulable follicle and less in the zona pellucida of both types of atretic follicle. In contrast to this pattern of staining, alcian blue staining intensity was identical in the zona pellucida of all follicles throughout the cycle. There was no difference in intra-antral alcian blue staining intensity on estrus and diestrus-2. On diestrus-1 and proestrus, staining intensity was greater in the antrum of the late atretic follicle than in the antra of the other follicular types. These studies indicate that glycosaminoglycan content is greater in the zona pellucida of the ovulable follicle of the rat on the last two days preceding ovulation than in the zona pellucida of either the early or late atretic follicles. In contrast, hyaluronic acid content remains constant in the zona pellucida of the three follicular types throughout the estrous cycle. These studies also give the first indication that, in the rat, the localization of PAS granules exclusively in the cumulus oophorus and corona radiata may be used to identify ovulable follicles.This work was supported by a research grant from the National Institute of Child Health and Human Development, HD-12684     

The oocyte-cumulus complex: Ultrastructure of the extracellular components in hamsters and mice

To enhance preservation of the extracellular materials, we have fixed hamster and mouse oocyte cumulus complexes (OCC) for transmission electron microscopy in the presence of ruthenium red. Ruthenium red had four effects on the extracellular components of the freshly ovulated hamster OCC. It interacted with the surface of cumulus and corona radiata cells; it stabilized the extracellular matrix (ECM) that was comprised of granules and filaments; it produced moderate electron density and good structural definition in the zona pellucida, and it revealed occasional smalls granular depsits on the oolemma. The ECM observed between cells of the cumulus and corona radiata layers extended into the outer one third of the zona pellucida. The granule and filament matrix was removed from the cumulus layer, corona radiata, and pores of the zona pellucida by brief treatment with hyaluronidase. The extracellular components of oviducal OCC from hamsters and mice appeared similar to OCC removed from follicles of the hamster shortly before ovulation. However, oviducal OCC did show increased aggregation of granules in the ECM. In most cases where females had been mated and oocytes were fertilized, the extracellular components appeared similar to those seen in fresh OCC. Exceptions were noted in some oocytes that lacked cumulus and corona radiata cells. In these instances, the zona pellucida generally lacked the granule/filament matrix. After fertilization numerous small electrondense granules were noted in the perivitelline space. These were presumed to originate in the cortical granules and formed a new investing layer around the zygote. Our data suggest that the OCC becomes more difficult for a sperm to penetrate as it approaches the oocyte. The significance of these results is discussed with respect to sperm traffic in the OCC and the cortical reaction.     

Topographical localisation of glucidic residues and their variations in the canine zona pellucida during folliculogenesis

SummaryIn the present ultrastructural study, horseradish peroxidase-labelled lectins, in conjunction with antiperoxidase antibody and protein A-gold, were used to characterise and localise the oligosaccharide sequences of zona pellucida glycoproteins at different stages of follicular development in the canine ovary. Deacetylation and sialidase digestion were also performed before lectin cytochemistry. The zona pellucida of oocytes present in unilaminar primary follicles reacts with WGA- and RCA-I-lectins. The zona pellucida of oocytes present in bilaminar and trilaminar secondary follicles displays positivity to WGA, RCA-I, Con-A, UEA-I, and sialidase/SBA. This labelling pattern persists in the zona pellucida of oocytes present in antral tertiary follicles with the exception of WGA and RCA-I reactive sites which are differently distributed throughout the zona pellucida. The topographical distribution of these carbohydrates is not uniform throughout the zona pellucida, indicating the regionalization of oligosaccharide chains within three concentric bands of the zona matrix: an inner surface close to the oocyte plasmamembrane, an intermediate portion and an outer layer in contact with the follicular cells. Our results demonstrated variations in the presence and distribution of the carbohydrate residues in the canine zona pellucida during different stages of follicular growth. We also observed the presence of vesicles in both the ooplasm and granulosa cells, showing a similar lectin binding pattern to that of the zona pellucida.     

Comparative cytochemistry of the developing ovarian follicles of the dog,rabbit, and mouse: Origin of the zona pellucida

The reactivity of the glycoproteins of ovarian follicles of the dog, rabbit, and mouse were compared, using the Alcian blue (pH 0.5) and Alcian yellow (pH 2.5) technique at the light-microscope level and the periodic acid-chromic acid-silver methenamine technique at the electron-microscope level. In paraffin sections, the rabbit and mouse show the appearance of both zona pellucida and follicular fluid in the earliest growing follicles. In the dog, there is a sequence of development with the follicular fluid appearing late, after much of the zona has been secreted. The zona and follicular fluid are highly sulfated in all animals. Zonae pellucidae of atresia appear to lose all traces of sulfation and become highly acidic. At the electronmicroscope level, oocytes contain little if any reactive glycoprotein material which can be related to zona pellucida formation. The initial appearance of the zona material occurs between follicle cell membranes extending outwards and away from the oocyte. Follicle cells of all species consistently contain a variety of reactive Golgi bodies and granules, with exocytotic vesicles, suggesting active synthesis and secretion of zona material. Our observations suggest that in the early stages of oogenesis, the follicular epithelium is responsible for at least part of the synthesis of the zona pellucida. It is possible therefore that both the oocyte and its follicle cells participate, probably on the sequential basis, in the synthesis of the mammalian zona pellucida.     

A glycoprotein from the liver constitutes the inner layer of the egg envelope (zona pellucida interna) of the fish, Oryzias latipes

A glycoprotein from the liver, which shares epitopes with chorion (egg envelope or zona pellucida) glycoproteins, is present only in the spawning female fish, Oryzias latipes, under natural conditions. This spawning female-specific (SF) substance is distinct from vitellogenin but closely resembles a major glycoprotein component, ZI-3, of the inner layer (zona radiata interna) of the ovarian egg envelope with respect to some biochemical and immunochemical characteristics. Here we report that the [125I]SF substance, injected into the abdominal cavity of the spawning female fish, was rapidly transported by the blood circulation into the ovary and incorporated into the inner layer of egg envelope of the growing oocytes. The result strongly suggests that the SF substance from the liver is a precursor substance of the major component, ZI-3, of the inner layer of egg envelope in the fish.     

Electron microscopic study of the formation of the egg membranes of the oocytes of the last year of development in the peliad]

After ovulation the egg membrane of the pelled consists of 3 layers. The homogeneous electron-dense layer comprising its major mass zona radiata, is laid as early as the stage of protoplasmatic growth of the oocyte as a result of accumulation of an electron-dense substance of a high electron density in the basement of the oocyte microvilli. The next, pectinate layer is formed from the highly electron-dense substance formed in the vacuoli of the follicular cell Golgi complex released into the subfollicular space. The most external layer - a fibrous layer - is formed due to release of large vacuoles, formed in the apical area of the follicular cells not long before ovulation. The obtained data suggest a great role of the follicular epithelium in the formation of egg membranes of the pelled.     

Cytochemical localization of GalNAc and GalNAcβ1,4Galβ1,4 disaccharide in mouse zona pellucida

Carbohydrate residues contained in the zona pellucida play a key role in the process of sperm-egg interaction. In vitro fertilization experiments have shown that a specific monoclonal antibody against GalNAcş,4Galş,4 disaccharide inhibits fertilization in mice. In the present study, the ultrastructural cytochemical localization of GalNAc residues and the GalNAcş,4Galş,4 disaccharide was carried out in ovarian and postovulatory oocytes by using lectin-gold cytochemistry and immunocytochemistry. Plant lectins SBA and DBA showed an affinity for the entire zona pellucida matrix of ovarian oocytes throughout the follicular maturation; however, immunoreactivity for GalNAcş,4Galş,4 disaccharide was not detected in ovarian oocytes at the earliest stages of follicular development but was found to be associated with the inner region of the zona matrix at the trilaminar primary follicle stage. The Golgi apparatus, vesicular aggregates, and cortical granules of the oocyte were intensely labeled by SBA and DBA throughout follicular development. Immunoreactivity to GalNAcş,4Galş,4 disaccharide was first observed in the Golgi apparatus and vesicular aggregates in trilaminar primary follicles. No immunoreactivity was observed in the cortical granules. In postovulatory oocytes, results were similar to those observed in ovarian oocytes. Our results thus suggest that (1) GalNAcş,4Galş,4 disaccharide residues are present only in the inner region of the zona pellucida and, therefore, might be involved in sperm penetration through the zona pellucida, (2) the inner and outer regions of the zona pellucida contain different oligosaccharide chains, (3) the vesicular aggregates detected in the oocyte could represent an intermediate step in the secretory pathway of zona pellucida glycoproteins and might be involved in the formation of cortical granules.     

A novel set of structures within the elasmobranch,ovarian follicle

Elasmobranch fishes produce some of the largest oocytes known, exceeding 10 cm in diameter. Using various microscopy techniques we investigated the structural adaptations which facilitate the production of these large egg cells in three species of shark: the Atlantic sharpnose shark, Rhizoprionodon terraenovae, dusky smoothound, Mustelus canis and the little gulper shark, Centrophorus uyato. The ovarian follicle of elasmobranchs follows the typical vertebrate pattern, with one notable exception; the zona pellucida reaches extreme widths, over 70 μm, during early oogenesis. Contact between the follicle cells and the oocyte across the zona pellucida is necessary for oogenesis. We describe here a novel set of large, tube‐like structures, which we named follicle cell processes that bridge this gap. The follicle cell processes are more robust than the microvilli associated with the follicle cells and the oocyte plasma membrane and much longer. During early oogenesis the follicle increases in size relatively quickly resulting in a wide zona pellucida. At this stage the follicle cell processes appear taut, uniform and radially oriented. As oogenesis continues the zona pellucida narrows and the follicle cell processes change their orientation, appearing to wrap around the oocyte. The presence of the contractile protein actin within the follicle cell processes and their change in orientation may well be an adaptation for maintaining the integrity of these large oocytes. The follicle cell processes also contain electron dense material, identical to material found within the follicle cells, suggesting a role in the transport of metabolites to the developing oocyte. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Elektronenmikroskopische Untersuchungen an den Hüllen der Oozyten und Eier des Flussbarsches Perca fluviatilis

Zusammenfassung Die Hüllen von Oozyten und reifen Eiern des Fußbarsches wurden licht- und elektronenmikroskopisch untersucht. In Oozyten vor der Bildung der Zona radiata sind die meisten der keulenförmigen Mikrovilli gerade auf die Follikelzellen ausgerichtet. Gelegentlich verlaufen Mikrovilli auch parallel zur Oozytenoberfläche. Die Zona radiata reiferer Eizellen besteht aus zwei Schichten, der elektronendichten Zona radiata externa und der weniger dichten Zona radiata interna. Während die erstere aus einem hochorganisierten Material besteht, ist die letztere aus einer Substanz von geringerer Dichte zusammengesetzt. In der Matrix der Zona radiata interna liegen unregelmäßig angeordnete flaschenbürstenähnliche Einschlüsse. Nach Kontrastierung mit l%iger Phosphorwolframsäure und 0,5% igem Uranylazetat sind feine Fibrillen in der Matrix nachweisbar. Die Zona radiata externa ist außen von einer Gallerthülle umgeben. Sie ist etwa fünfmal so stark wie die gesamte Zona, radiata. Diese Gallerthülle besteht aus homogenem Material von geringerer Dichte. Nach Anfärbung mit 0,2%-igem Rutheniumrot färbt sich diese Schicht intensiv rot. Daraus läßt sich auf das Vorhandensein von sauren Mukopolysacchariden schließen. In der Gallerthülle liegen elektronendichte Körnchen, in denen sich bei hoher Auflösung eine hochorganisierte, kristalloide Struktur nachweisen läßt. Die Gallerthülle wird von den zytoplasmatischen Fortsätzen der kegelförmigen Follikelzellen durchdrungen. Diese Fortsätze gehen in Ausläufer über, die in die Porenkanäle der Zona radiata eindringen. In einigen Fällen umhüllen die Ausläufer der Follikelzellen die Mikrovilli der Oozyte vollständig. Normalerweise liegen die Mikrovilli und die Ausläufer aber deutlich voneinander getrennt. Die Mikrovilli enden entweder direkt an der Zellmembran der Follikelzellen oder sie dringen tief in Membraneinstülpungen ein. Mikrovilli und die Ausläufer der Follikelzellen befinden sich in den Porenkanälen fast reifer Eier. Kurz vor der Ovulation werden sie wahrscheinlich zurückgezogen. Die Porenkanäle reifer Barscheier sind im äußeren Drittel der Zona radiata interna verschlossen.

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Summary The envelope of oocytes and mature eggs of the perch was examined by light and electron microscope. In oocytes before the formation of the zona radiata most of the clubshaped microvilli project straightly towards the follicular cells. Some, however, run parallel to the oocyte surface. The zona radiata of more mature oocytes consists of the electron dense zona radiata externa and the less electron dense zona radiata interna. While the former consists of a highly organized material, the latter is composed of a substance of low density, with the exception of a few brush-shaped inclusions which are scattered irregularely within the matrix of the zona radiata interna. After treatment with 1% phosphotungstic acid and 0.5% uranyl acetate tiny fibres become visible. External to the zona radiata externa the oocytes are enveloped by a jelly layer, about five times as thick as the zona radiata. The jelly layer appears to consist of a rather homogeneous material of low density. After treatment with a solution of 0,2% ruthenium red the jelly layer is stained intensely red, suggesting the presence of acidic mucopolysaccharides. Within the matrix of the jelly layer are embedded electron dense granules of unknown function. At high resolution they reveal a well organized, crystalloid structure. The jelly layer is penetrated by cytoplasmic processes of the cone-shaped follicular cells. These processes develop extensions which enter the pore canals of the zona radiata. In some cases these extensions envelope the microvilli of the oocytes completely. But normally the microvilli and the extensions are clearly separated. The microvilli make either direct contact with the membrane of the follicular processes or penetrate deeply into membrane interdigitations. Microvilli together with follicular extensions are present in the pore canals of almost mature eggs. They seem to be withdrawn just before ovulation. The pore canals of the zona radiata are partly closed in mature eggs.

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Ich verdanke die Anregung zu dieser Arbeit einer Diskussion, die ich mit Herrn Prof. Dr. E. A. Arndt, Rostock, anläßlich seines Vortrages am 21. Februar 1966 in Kiel führte.

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Untersuchungen mit Unterstützung durch die Deutsche Forschungsgemeinschaft. Herrn Prof. Dr. O. Moritz, Institut für Pharmakognosie, danke ich für die Möglichkeit, das Elektronenmikroskop seines Institutes zu benutzen. Frau R. Bardenhewer, geb. Schmidtke, danke ich für die Hilfe bei der Präparation und für die Anfertigung der photographischen Vergrößerungen.     

Architecture of the hamster oocyte-cumulus complex

The structure of the hamster oocyte-cumulus complex (OCC) has been analyzed to help understand how mammalian sperm penetrate the investing coats of the oocyte. At ovulation, oocytes of most mammalian species are surrounded by a zona pellucida, corona radiata, and cumulus layer. Cells of the cumulus and corona radiata are separated by an extracellular matrix (ECM) that contains hyaluronic acid. In hamsters, the diameter of mature follicular OCCs is 0.61 ± 0.12 mm, whereas in freshly ovulated OCCs in culture medium it is 0.78 ± 0.15 mm. This indicates the OCC expands at or after ovulation. The corona radiata is 1–4 cell layers deep, and corona radiata cells are closely packed (center-to-center distances between adjacent cells in follicular OCCs averaged 14 ± 3 μm). The cumulus layer is 5–8 cell layers deep, and intercellular spaces are much larger (center-to-center distances between adjacent cumulus cells in follicular OCCs averaged 50 ± 20 μm). An ovulated OCC has an approximate volume of 248 × 10⁶ μm³ and was estimated to contain approximately 5,700 cells. Studies with stretched OCCs show that ink particles can readily penetrate the extracellular spaces of the cumulus and corona radiata layers and interact with the ECM, staining it black. At the periphery of the OCC, the ECM appeared discontinuous and formed “cords” containing cumulus cells, whereas closer to the corona radiata the matrix completely filled intercellular spaces. Ink penetrated the corona radiata, but the ECM was difficult to visualize because of the close packing of cells in this layer. Our observations on unfixed OCCs show that cell packing becomes more dense proceeding from the periphery of the OCC to the zona pellucida and that the ECM at the periphery differs from the matrix deeper in the OCC. These data suggest that an incoming sperm would find penetration of the investing coats to increase in difficulty (that is physical resistance would increase) as it got closer to the oocyte. We have also examined the effects of processing for microscopy on the structure of the OCC. Both standard fixation procedures and fixation in the presence of ruthenium red caused condensation of the ECM, especially in the cumulus layer, and thus produced a significant decrease in the diameter of the OCC.     

Differential expression of ZPC in the bovine ovary,oocyte, and embryo

Using nonradioactive in situ hybridization (ISH), the mRNA encoding the zona glycoprotein bZPC was localized in bovine ovaries, oocytes, and embryos. In the ovary, the distribution of the mRNA was correlated with the developmental stage of the follicle. Whereas in primordial and primary follicles the mRNA was predominantly seen in the oocyte, it was found in both the oocyte and the follicle cells of secondary and tertiary follicles. In 2-day-old embryos produced by in vitro fertilization (IVF), no mRNA encoding ZPC could be demonstrated. Immunoblotting using monospecific polyclonal antibodies against porcine ZPC revealed a distinct band at a molecular weight of 47 kD in the ovarian cortex of cows, calves, and fetuses as well as in bovine follicle cells. Immunohistochemistry using the ZPC antibody displayed a strong signal in the zona pellucida of bovine oocytes and 2- to 6-day-old embryos as well as in the follicle cells. Our results show that during follicular development bovine ZPC is synthesized by the oocyte of the primary follicle and by both the oocyte and the follicle cells of the secondary and tertiary follicle. After fertilization, the synthesis of the zona protein is finished. Mol. Reprod. Dev. 49:435–443, 1998. © 1998 Wiley-Liss, Inc.     

Influence of follicle size on the penetrability of immature pig oocytes for homologous in vitro penetration assay

In order to improve the performance of homologous in vitro penetration (hIVP) assays using immature oocytes to assess the penetrating ability of boar sperm, the present study was designed to evaluate the influence of oocyte and follicle size on the penetrability of immature pig oocytes obtained from slaughterhouse ovaries. Nonatretic antral follicles were isolated, measured with a computerized image analysis system and grouped according to their diameter: Group 1 (0.40-0.99 mm), Group 2 (1.00-2.19 mm), Group 3 (2.20-2.79 mm), and Group 4 (2.80-6.50 mm). After sperm coincubation and before penetrability evaluation, the immature oocytes were classified into four size categories according to their diameter excluding zona pellucida: <105, 105-109, 110-114, and > or =115 microm. As regards follicle size, the highest viability and penetrability were obtained with oocytes from follicles >2.20 mm (P>0.05). Regarding oocyte size, significant differences (P<0.05) were observed for all parameters evaluated between oocytes with a diameter above or below 110 microm. However, our results revealed that such differences were due to follicle size rather than oocyte diameter, since oocytes with the same diameter but from different follicle size groups showed different penetration rates. With increasing follicle size, the percentage of penetrated oocytes increased (P<0.05). Finally, our results showed that the greater penetrability of immature oocytes from larger follicles is not due to variations in the thickness of the zona pellucida. There were no significant differences in zona pellucida thickness between oocytes from the four follicular size groups. In summary, these results indicate that follicle size directly affects the penetrability of immature pig oocytes used in hIVP.