Cellular associations in the rat oocyte-cumulus cell complex: Morphology and ovulatory changes

Cumulus-oocyte complexes were isolated from the follicles of proestrous rats or from the oviducal ampullae of estrous rats. The zona pellucida of some complexes was dissolved before fixation. The follicular cumulus cells were seen to be held together mainly by long processes, which often extended over a distance of several cells. Large numbers of straight processes from the corona radiata cells, passing to the oocyte, surface, were seen in the space formerly occupied by the zona pellucida. Oocyte microvilli were uniformly short; none traversed the zona. The postovulatory complexes were covered by amorphous extracellular material which also filled the spaces between the cells. By lysis of this material with hyaluronidase the cumulus cells were detached. The surfaces of these cells were covered with blebs. By testing the ability of hyaluronidase to remove the corona cells from the zona pellucida of complexes isolated around the time of ovulation, it was found that the completion of retraction of the corona cells processes occurred in the oviduct, immediately after ovulation. It is suggested that the oviducal environment may influence the final step of the withdrawal of the corona cells' projections from the zona pellucida.     

Late preovulatory synthesis of proteoglycans by the human oocyte and cumulus cells and their secretion into the oocyte-cumulus-complex extracellular matrices

Light- and electron-microscope autoradiography using 3H-glucosamine and 3H-fucose as precursors was employed to investigate proteoglycan synthesis and secretion by late preovulatory human oocytes and cumulus cells. Both the oocyte and cumulus cells were found to be important cellular sources supplying proteoglycans to the oocyte-cumulus-complex extracellular matrices, i.e., the zona pellucida and the cumulus intercellular matrix. Both the oocyte and cumulus cells were shown to secrete labelled proteoglycans into the zona pellucida. Labelled proteoglycans were also detected in the cumulus intercellular matrix. Chase experiments revealed the labelled molecules to be relatively closely associated with both the zona pellucida and the cumulus intercellular matrix. Staining with chromic acid and phosphotungstic acid showed proteoglycan material to penetrate from the cumulus intercellular matrix into pores of the zona pellucida. This material is thought to be a structural equivalent of the newly synthesized proteoglycans secreted by cumulus cells and migrating into the zona pellucida (as detected by autoradiography). It is concluded that newly synthesized proteoglycans secreted by the oocyte and cumulus cells in the late preovulatory period are a component of the microenvironment in which fertilization takes place.     

Late preovulatory synthesis of proteoglycans by the human oocyte and cumulus cells and their secretion into the oocyte-cumulus-complex extracellular matrices

Summary Light- and electron-microscope autoradiography using ³H-glucosamine and ³H-fucose as precursors was employed to investigate proteoglycan synthesis and secretion by late preovulatory human oocytes and cumulus cells. Both the oocyte and cumulus cells were found to be important cellular sources supplying proteoglycans to the oocytecumulus-complex extracellular matrices, i.e., the zona pellucida and the cumulus intercellular matrix. Both the oocyte and cumulus cells were shown to secrete labelled proteoglycans into the zona pellucida. Labelled proteoglycans were also detected in the cumulus intercellular matrix. Chase experiments revealed the labelled molecules to be relatively closely associated with both the zona pellucida and the cumulus interecellular matrix. Staining with chromic acid and phosphotungstic acid showed proteoglycan material to penctrate from the cumulus intercellular matrix into pores of the zona pellucida. This material is thought to be a structural equivalent of the newly synthesized proteoglycans secreted by cumulus cells and migrating into the zona pellucida (as detected by autoradiography). It is concluded that newly synthesized proteoglycans secreted by the oocyte and cumulus cells in the late preovulatory period are a component of the microenvironment in which fertilization takes place.     

An autoradiographic study of gonadotrophin regulation of labelled glycoconjugates within preovulatory mouse follicles during the final stages of oocyte maturation, using [3H]glucosamine as the radioactive precursor

Immature mice were treated with PMSG and hCG to induce follicular development and ovulation. [3H]Glucosamine was injected at the same time as the PMSG or 2 h before autopsy. The synthesis and distribution of labelled glycoconjugates within the preovulatory follicles was hormonally dependent. PMSG stimulated a rapid uptake of [3H]glucosamine into the zona pellucida and follicular fluid of the largest antral follicles although labelled macromolecules could not be demonstrated in any of the cellular components of these follicles. The injection of hCG stimulated a rapid incorporation of labelled macromolecules into the cellular components of the preovulatory follicle, namely into thecal, granulosa and especially the cumulus cells surrounding the oocyte. The density of labelled macromolecules within the follicular fluid also increased, while the specific and uniform labelling of the zona pellucida which was so characteristic of the period of PMSG stimulation changed. Between 4 and 8 h after the injection of hCG, labelled glycoconjugates containing [3H]glucosamine, became increasingly associated with the outer surface of the zona pellucida and with the region of the egg plasma membrane, even in Graafian follicles not destined to ovulate. The change in distribution of labelled macromolecules on the zona surface may be a prerequisite for successful sperm-zona binding and the specific association of labelled glycoconjugates in the region of the egg plasma membrane may be involved in the preparation of the egg surface for sperm-egg interactions involving cortical granule exocytosis and the block to polyspermy.     

Visualization of the three-dimensional distribution of collagen fibrils over preovulatory follicles in the hamster

Before an oocyte can escape from a preovulatory follicle, the apical wall must thin to the point of rupture. Although numerous layers of cells are present, it is the collagen fibrils in the theca externa that provide most of the strength to the developing follicle. The three-dimensional distribution and integrity of these fibrils over a follicle cannot be appreciated with standard used methods such as examination of thin sections by transmission electron microscopy In this paper we describe a technique that removes cells superficial to the collagen fibrils so that their distribution may be examined by scanning electron microscopy. On the third day of the hamster's 4-day estrous cycle, bundles of fibrils pass from intrafollicular areas and ascend follicles. Approximately halfway up the follicle wall, the bundles fan out and form a meshwork of fibrils which covers the apex. As the time of ovulation approaches, the number of layers of fibrils decreases over the apex until a tear forms in the weakened matrix. Experimental results demonstrating that the meshwork is composed of collagen fibrils are presented. The usefulness of this technique in visualizing the collagen content in preovulatory follicles is discussed as well as factors that may aid in weakening this layer so that follicle rupture may occur.     

A histochemical study of the changes occurring in the protein-carbohydrate composition of the cumulus-oocyte complex and zona pellucida in immature mice in response to gonadotrophin stimulation

Summary A histochemical account is presented of the changes that occur in the protein—carbohydrate composition of the cumulus—oocyte complex in immature mice after gonadotrophin treatment. The distribution and nature of the glycosaminoglycans (GAG) present was established by enzymic digestion of tissue sections with testicular orStreptomyces hyaluronidase prior to staining with periodic—acid Schiff (PAS) or Alcian Blue. Treatment with exogenous gonadotrophins [pregnant mare's serum and human chorionic gonadotrophin (hCG)] induced gross changes in the appearance of the zona pellucida (and in the histochemical staining of the cumulus—oocyte complex). A reduction was observed in the amount of PAS-positive material present within the zona pellucida of oocytes located in large Graafian follicles examined 40 h after stimulation with pregnant mare's serum. After the injection of hCG, the zona pellucida was further depleted of PAS-positive naterial. Most of the PAS-positive material became confined to the plasma membrane of the oocyte, while the oocyte itself also became increasingly PAS-positive. All the GAGs disappeared from zona pellucida within 4 h of hCG stimulation. The changes observed in the protein—carbohydrate composition of the zona pellucida in preovulatory oocytes immediately prior to ovulation may be a prerequisite for successful sperm-egg interactions.     

东北梅花鹿初级卵母细胞的超微结构

研究东北梅花鹿初级卵母细胞发育的超微结构变化,目的是为探索东北梅花鹿初级卵母细胞的发育规律提供组织学和形态学依据。本研究于2003年和2004年的6月初到8月末取3只、9月中旬到10月初取4只,共计7只健康经产2~3胎的成年东北梅花鹿卵巢;卵巢经2·5%戊二醛固定液固定后,切取约1mm3的卵巢皮质和直径0·5~1·5mm及1·5~3mm的卵泡作为电镜观察用材料;该材料经0·1MpH7·2的PBS漂洗、1%锇酸固定、不同浓度乙醇脱水后,再经Epon812和丙酮等量混合液浸透,最后用Epon812包埋制块,并用半薄切片机切成0·5~2μm半薄切片;再经亚甲基兰-天青Ⅱ染色后,在光镜下进行卵泡分类和卵母细胞定位;将经定位的材料用超薄切片机切成厚度为700~800的切片,经醋酸铀和柠檬酸铅双重染色后,用透射电镜观察、记录并照相。观察时将卵泡依其直径大小、透明带的形成、卵泡腔的出现等分为原始卵泡、初级卵泡、次级卵泡和三级卵泡4类。研究结果表明,在原始卵泡阶段,卵母细胞为较规则的圆形,质膜与卵泡细胞膜紧密相贴,有时形成桥粒,细胞器多分布于近核区,高尔基体不典型,线粒体多为圆形,嵴较少;在次级卵泡阶段,2~4层的卵泡细胞局部开始形成透明带,4层以上时形成薄的透明带,微绒毛斜伸入透明带内,方向不规律;在直径为0·5~1·5mm的三级卵泡阶段,卵母细胞的透明带增厚,各种细胞器在皮质区内数量较多,皮质区内高尔基体的数目增多,粗面内质网明显减少;在直径为1·5~3mm的三级卵泡阶段,卵母细胞的透明带继续加厚,微绒毛缩短变弯,开始从透明带退出,许多皮质颗粒开始排列在卵母细胞膜下。     

The Differentiation of the Zona Pellucida (Vitelline Envelope) in the Lizard Tarentola mauritanica

The organization of the zona pellucida in the lizard Tarentola mauritanica was studied at the transmission electron microscope. Evidence is provided in support of the hypothesis that follicle cells and the oocyte work together to synthesize and release components that give rise to the zona .
The components of the zona consist of fibrils and amorphous electron-dense material, which are first observed in young previtellogenic oocytes. These components seem to be released by coated vesicles that are formed by the Golgi complex in both the oocyte and the follicle cells. The material relased by the coated vesicles forms patches around the microvilli that project from the oocyte and the folds of follicle cells. During the following previtellogenic stages, the patches merge together to form a continuous coat around the oocyte. The coat persists until the end of vitellogenesis.     

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.     

The cellular origin of the zona pellucida antigen in the human and hamster

The origin of the zona pellucida in follicles at different stages of maturation was studied in the hamster and human by indirect immunofluorescence microscopy using a specific anti-hamster zona pellucida serum. The earliest detection of the zona pellucida material occurred in the ooplasm of oocytes in primordial follicles. In primary follicles, the fluorescence was localized at the periphery of the oocyte. In secondary and in mature follicles a deposition of fluorescent material was visualized on the inner and outer regions of the zona pellucida. Follicular cells, other than the oocyte, failed to exhibit a fluorescent reaction with the antizona pellucida serum. It is concluded that the oocyte is an important source of the antigenic material of the zona pellucida of hamster and human ova.     

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.     

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.     

Cx37 and Cx43 Localize to Zona Pellucida in Mouse Ovarian Follicles

In the ovarian follicle, granulosa cells adjacent to the oocyte extend processes through the zona pellucida matrix, and these projections establish gap junctions both with the oocyte and with neighboring transzonal projections. The identity of connexins contributing to gap junctions between transzonal projections has not been extensively studied. Here, we examined the expression pattern of Cx37 and Cx43 in mouse zona pellucida using multiple connexin-specific antibodies. Immunofluorescence staining revealed abundant Cx37 and Cx43 puncta within the zona pellucida of both preantral and antral follicles. Cx37 persisted in the zona pellucida of mature follicles up to 5 h after an ovulatory stimulus whereas Cx43 was reduced in the zona pellucida by 3 h after an ovulatory stimulus. We suggest that in addition to its role in oocyte-granulosa cell communication, Cx37 could enable a distinct communication pathway between those granulosa cells that are in direct contact with the oocyte.     

Cx37 and Cx43 localize to zona pellucida in mouse ovarian follicles

In the ovarian follicle, granulosa cells adjacent to the oocyte extend processes through the zona pellucida matrix, and these projections establish gap junctions both with the oocyte and with neighboring transzonal projections. The identity of connexins contributing to gap junctions between transzonal projections has not been extensively studied. Here, we examined the expression pattern of Cx37 and Cx43 in mouse zona pellucida using multiple connexin-specific antibodies. Immunofluorescence staining revealed abundant Cx37 and Cx43 puncta within the zona pellucida of both preantral and antral follicles. Cx37 persisted in the zona pellucida of mature follicles up to 5 h after an ovulatory stimulus whereas Cx43 was reduced in the zona pellucida by 3 h after an ovulatory stimulus. We suggest that in addition to its role in oocyte-granulosa cell communication, Cx37 could enable a distinct communication pathway between those granulosa cells that are in direct contact with the oocyte.     

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。     

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.     

Formation of the rupture site in preovulatory hamster and mouse follicles: Loss of the surface epithelium

Changes in the morphology of epithelial cells covering the sides and apex of ovarian follicles were examined in mice and hamsters during the final 13 hr before rupture using light and electron microscopy. At the time of the surge of luteinizing hormone, approximately 13 hr before follicle rupture, epithelial cells along the follicle sides are spherical, covered with microvilli, and remain so throughout the entire cycle. As ovulation approaches, cells at the apex become progressively flatter, increase in diameter, and undergo a reduction in the number and length of microvilli. By 2 hr before ovulation, the microvilli are present only along the boundary between adjacent cells and the cells are in different stages of degeneration. In some cells, the cytoplasm is electron dense and the nuclei are pyknotic. Other cells become electron lucent and cytoplasmic elements are leached from the cell. The apical plasma membrane is lost first over the center of the cell and later over the periphery. Epithelial cells detach from the apex individually until a large patch devoid of cells is formed. This includes the site of eventual rupture. The loss of epithelial cells from the apex of ovarian follicles of other species is compared with our results, and the processes involved in stretching, degeneration, and sloughing of the epithelial cells are discussed.     

Ultrastructural and histochemical changes in the murine zona pellucida during the final stages of oocyte maturation prior to ovulation

The ultrastructural morphology of the mouse zona pellucida was studied in preovulatory follicles from the ovaries of immature mice treated with exogenous gonadotrophins. The ovaries were fixed in the presence of cetylpyridinium chloride, which precipitates carbohydrates, so that their loss during fixation and processing is substantially reduced. The semi-thin araldite sections obtained from osmicated material were viewed by conventional light microscopy, while the ultra-thin sections were examined by transmission electron microscopy. A parallel series of semi-thin sections of non-osmicated ovaries was deresined and subsequently stained with periodic acid Schiff (PAS). The morphological appearance of the zona pellucida in preovulatory oocytes changed during the final stages of oocyte maturation. A close correlation was observed between the ultrastructural appearance of the zona pellucida and that observed following PAS staining during the period studied. Real differences were observed in the location, density, and distribution of glycoconjugates within the zona pellucida during the final stages of oocyte maturation prior to and immediately following germinal vesicle breakdown. Similar changes in the zona were observed in adult females autopsied during proestrus and oestrus. The changes in the density and distribution of glycoconjugates are likely to have important consequences for fertilization by affecting sperm-zona binding and sperm-egg interactions.     

A quantitative and qualitative cytochemical analysis of glycosaminoglycan content in the zona pellucida of hamster ovarian follicles

In this study the amount of neutral and acidic glycosaminoglycans in the zona pellucida and antrum of primary, preovulatory, and atretic follicles was analyzed. Serial sections of preovulatory hamster follicles were stained with PAS or alcian blue to estimate the content of neutral and acidic glycosaminoglycans, respectively. The amount of hyaluronic acid, chondroitin sulfate and sialic acid was determined using enzyme digestion procedures followed by alcian blue staining. Microdensitometric analyses showed that the strongest PAS staining was in the zona pellucida of atretic follicles, less staining in preovulatory follicles but more than in the antrum of preovulatory follicles. No hyaluronic acid was found in the zona pellucida of any follicular type, but there was a measurable amount in the antrum of preovulatory follicles. Chondroitin sulfate was present in the zona pellucida of primary and atretic follicles, as well as in the antrum of preovulatory follicles. Sialic acid was present in the antrum and zona pellucida of all follicular types. Sialic acid plays a role in receptor recognition and its presence may reflect the role of the zona pellucida in sperm recognition and fertilization.