Oogenesis in hydra. II. Cytophotometric determination of DNA concentration in the nuclei of somatic and sex cells

During sexual reproduction in Hydra, interstitial cells in the female sex zone of the body (i-cells) undergo mitotic division and form a thickening in the epiderm. The proliferation of i-cells is accompanied by the increase of cytoplasm volume and by the appearance in the cytoplasm of a great number of membranous structures (rough endoplasmic reticulum, Golgi apparatus and mitochondria), enzymatic granules, lipid inclusions and glycogen. All cells of the epidermal thickening soon (in approximately twenty four hours) acquire the characteristics of typical phagocytes. However it is the cell situated inside the group of syncytially connected ones and adjacent to mesogloea that begins to grow rapidly and phagocytize surrounding cells. The cells of the epidermal thickening, though they are often given the name of oogonia, were found to have a tetraploid DNA content in their nuclei. The presence of four unseparated centrioles of equal size suggests that all preparatory processes for division were completed. A conclusion was drawn that cells of the epidermal thickening undergo premeiotic DNA synthesis prior to their phagocytizing by the growing oocyte and, thus, are oocytes themselves. The oogonial stage in Hydra coincides with the early period of mitotic reproduction of i-cells. The data obtained are discussed from the viewpoint of the formation of the accessory gonad apparatus.     

Oogenesis of Tilapia mossambica. III. The cytoplasm of previtellogenic oocytes]

Using methods of light and electron microscopy and of autoradiography, the morphology of cytoplasm in previtellogenic oocytes of tilapia mossambique was studied. Similar to other bony fishes, mitochondria at the early previtellogenic oocytes are mostly located in the perinuclear cytoplasm to be later distributed over the whole volume of growing oocytes. The Golgi complex is poorly developed. In the peripheral regions of the late previtellogenic oocytes, stickform mitochondria, pinocytotic vesicles and microvilli are observed, along with the perioocyte space formation. In the cytoplasm of previtellogenic oocytes polyribosomes appear. No differences in 3H-leucine incorporation intensity was noticed in oocytes of different previtellogenic stages. The characteristic feature of tilapia mossambique previtellogenic oocytes, in comparison with other bony fishes, is the presence of fat droplets in their cytoplasm.     

Oogenesis in the leech Glossiphonia heteroclita (Annelida, Hirudinea, Glossiphoniidae) II. Vitellogenesis, follicle cell structure and egg shell formation

By the end of previtellogenesis, the oocytes of Glossiphonia heteroclita gradually protrude into the ovary cavity. As a result they lose contact with the ovary cord (which begins to degenerate) and float freely within the hemocoelomic fluid. The oocyte's ooplasm is rich in numerous well-developed Golgi complexes showing high secretory activity, normal and transforming mitochondria, cisternae of rER and vast amounts of ribosomes. The transforming mitochondria become small lipid droplets as vitellogenesis progresses. The oolemma forms microvilli, numerous coated pits and vesicles occur at the base of the microvilli, and the first yolk spheres appear in the peripheral ooplasm. A mixed mechanism of vitellogenesis is suggested. The eggs are covered by a thin vitelline envelope with microvilli projecting through it. The envelope is formed by the oocyte. The vitelline envelope is produced by exocytosis of vesicles containing two kinds of material, one of which is electron-dense and seems not to participate in envelope formation. The cortical ooplasm of fully grown oocytes contains many cytoskeletal elements (F-actin) and numerous membrane-bound vesicles filled with stratified content. Those vesicles probably are cortical granules. The follicle cells surrounding growing oocytes have the following features: (1) they do not lie on a basal lamina; (2) their plasma membrane folds deeply, forming invaginations which eventually seem to form channels throughout their cytoplasm; (3) the plasma membrane facing the ovary lumen is lined with a layer of dense material; and (4) the plasma membrane facing the oocyte forms thin projections which intermingle with the oocyte microvilli. In late oogenesis, the follicle cells detach from the oocytes and degenerate in the ovary lumen.     

Fine Structures of Oocytes and Accessory Cells of the Ovary in the Starfish Patiria (Asterina) pectinifera at Different Stages of Oogenesis and After 1-Methyladenine-Induced Maturation

Morphological changes in the growing and maturing oocytes of Patiria ( Asterina ) pectinifero were studied by electron microscopy. Oogenesis is of the solitary type. An extensive system of rough endoplasmic reticulum (ER) and Golgi complex (GC) develops in the ooplasm forming the cortical, yolk and secretory granules in its peripheral regions. The contents of the latter granules are released from the oocyte and form the vitelline membrane. At early stages of oogenesis, extensive multiplication of mitochondria results in formation of a large aggregate of these organelles in the perinuclear cytoplasm ("yolk nucleus"). After maturation of full grown oocytes has been induced by 1-methyladenine, the membranous cell structures are rapidly rearranged: vast aggregates of ER cisternae in the surface cytoplasm layer and single ER cisternae among yolk granules are disintegrated to small vesicles; the GC is reduced. These processes are suggested to be somehow related to changes in hydration of the cytoplasm and in rigidity of its surface layer. In maturing oocytes, the yolk granules form characteristic linear rows, trabeculae, traversing the cytoplasm and their boundary membranes fuse in zones of contact. Some granules are converted to multivesicular bodies, thus suggesting the activation of hydrolytic enzymes that form part of the yolk in echinoderms.     

Breakdown of starfish ovarian follicle induced by maturation-promoting factor

Immature starfish oocytes are surrounded by envelopes consisting of follicular cells. These cells adhere to each other and to the oocyte, immobilizing the latter within the ovary. When isolated oocytes in their follicles are treated with 1-methyladenine (1-MeAde), germinal vesicle breakdown (GVBD) and follicular envelope breakdown (FEBD) occur simultaneously. The 1-MeAde acts on the oocyte surface to produce a maturation-promoting factor (MPF) in the cytoplasm, which brings about GVBD. In the present study, MPF was found to induce FEBD as well as GVBD when injected into immature oocytes with their follicles in Asterina pectinifera. Although GVBD was induced by MPF in the presence of cytochalasin D, this drug prevented MPF-induced FEBD, and each follicular cell remained in situ on the surface of the oocyte. However, desmosomes connecting the processes of the follicle cell with the oocyte surface were disrupted following MPF injection even in the presence of cytochalasin D, and the processes became detached from the oocyte. FEBD occurred in these oocytes when cytochalasin D was removed, resulting in the formation of a small follicular clump by microfilament-mediated contraction of the follicle cells. These results show that FEBD is not brought about by the direct action of 1-MeAde but by the action of MPF. Therefore, in starfish, spawning as well as oocyte maturation is directly triggered by MPF produced under the influence of 1-MeAde.     

Yolk formation in Isohypsibius (Eutardigrada)

Summary In Isohypsibius granulifer, yolk is autosynthesized. The Golgi apparatus is mainly responsible for the formation of yolk, which consists of irregular platelets with heterogeneous contents and a diameter of about 1 m. Dense globules, 300 nm in diameter, are visible among yolk platelets. These develop in the vesicles of the rough endoplasmic reticulum. The genesis of these vesicles is associated with the outer membrane of the nuclear envelope, which forms blebs intensively during previtellogenesis and early vitellogenesis. The developing oocytes are assisted by nurse cells, to which they are jointed by cytoplasmic bridges. For every oocyte, there are a number nurse cells, which are sister cells of the oocyte. In addition to rRNA, nurse cells transfer to the oocyte lipids, platelets of yolk formed in their cytoplasm, mitochondria and cortical granules.     

Reconstitution of the Golgi apparatus after microinjection of rat liver Golgi fragments into Xenopus oocytes

We have studied the reconstitution of the Golgi apparatus in vivo using an heterologous membrane transplant system. Endogenous glycopeptides of rat hepatic Golgi fragments were radiolabeled in vitro with [3H]sialic acid using detergent-free conditions. The Golgi fragments consisting of dispersed vesicles and tubules with intraluminal lipoprotein-like particles were then microinjected into Xenopus oocytes and their fate studied by light (LM) and electron microscope (EM) radioautography. 3 h after microinjection, radiolabel was observed by LM radioautography over yolk platelet-free cytoplasmic regions near the injection site. EM radioautography revealed label over Golgi stacked saccules containing the hepatic marker of intraluminal lipoprotein-like particles. At 14 h after injection, LM radioautographs revealed label in the superficial cortex of the oocytes between the yolk platelets and at the oocyte surface. EM radioautography identified the labeled structures as the stacked saccules of the Golgi apparatus, the oocyte cortical granules, and the plasmalemma, indicating that a proportion of microinjected material was transferred to the surface via the secretion pathway of the oocyte. The efficiency of transport was low, however, as biochemical studies failed to show extensive secretion of radiolabel into the extracellular medium by 14 h with approximately half the microinjected radiolabeled constituents degraded. Vinblastine (50 microM) administered to oocytes led to the formation of tubulin paracrystals. Although microinjected Golgi fragments were able to effect the formation of stacked saccules in vinblastine-treated oocytes, negligible transfer of heterologous material to the oocyte surface could be detected by radioautography. The data demonstrate that dispersed fragments of the rat liver Golgi complex (i.e., unstacked vesicles and tubules) reconstitute into stacked saccules when microinjected into Xenopus cytoplasm. After the formation of stacked saccules, reconstituted Golgi fragments transport constituents into a portion of the exocytic pathway of the host cell by a microtubule-regulated process.     

Activity of maturation promoting factor in mammalian oocytes after its dilution by single and multiple fusions

Mouse and porcine fully grown oocytes at metaphase I(MI) were fused to one or more fully grown oocytes of the same species that contained an intact germinal vesicle (GV). In fused cells containing one GV, premature chromosome condensation (PCC) was observed. In fused cells containing more than one GV, germinal vesicle breakdown (GVBD) and PCC were delayed. Fusion of an MI fully grown oocyte with a growing oocyte resulted in rapid PCC, whereas, fusion of an MI fully grown oocyte with more than one growing oocyte resulted in neither PCC nor GVBD. Moreover, MI chromosomes formed a clump of chromatin. Results of these experiments suggest that the delay in GVBD in fusions of MI oocytes with multiple GV-intact oocytes was due to dilution of maturation promoting factor (MPF) by the cytoplasm of the GV-intact oocytes and that the cytoplasm of growing oocytes can inhibit MPF present in MI oocytes.     

Cytochemistry of the Golgi apparatus in developing ovarian germ cells of the Syrian hamster

Summary A cytochemical study of the Golgi apparatus in the developing oocyte of the golden hamster was carried out using the TPPase, AcPase and zinc iodide-osmium tetroxide (ZnOs) techniques. Tissue from both immature and sexually mature animals was investigated.Peak TPPase activity was found in pre-growth oocytes in ovaries from sexually mature adults. Some activity was also present in SER in the peripheral cytoplasm of growing oocytes. AcPase activity was found only after the onset of oocyte growth. It was present in Golgi cisternae and associated vesicles and in some profiles of peripheral SER. No structures corresponding to GERL were identified. Strong staining with ZnOs was seen, at all stages studied, in certain Golgi vesicles and short tubules but not in the cisternae unless the oocyte was atretic. Weaker ZnOs staining was characteristic of ER throughout the oocyte.With all techniques there was a falling off of reactivity as oocyte size increased. Within a single oocyte some Golgi bodies were negative while others were positive, with both TPPase and AcPase techniques. This suggests that two or more functional types of this organelle are present within the developing oocytes.We would like to thank Dr. K.N. Christie for his interest and helpful suggestions regarding the enzyme techniques     

Oogenesis of Tilapia mossambica. I. Oogonia and meiotic prophase oocytes

Using light and electron microscopy and autoradiography, the morphology and synthesis of DNA, RNA and proteins in oogonia and early meiotic prophase oocytes in Tilaria mossabique were studied. According to dimensions and morphological features observed it is possible to distinguish between two groups of oogonia: large oogonia corresponding to type A spermatogonia of mammals, and small actively dividing oogonia, located in groups and identical to type B spermatogonia. The morphology of oogonia and of the early meiotic prophase oocytes well compares with the pattern described for other species of bony fishes. In the cytoplasm of these cells dense bodies, nuage-material, free ribosomes, large mitochondria with lamellar cristae and Golgi cisterns are available. In the oocyte nuclei at zygotene and pahytene stages 3H-thymidine incorporation was seen mainly into the nucleolus-associated chromatin. Besides, the formation of a heterochromatin cape and the synaptonemal complex was observed. Incorporation of 3H-uridine and 3H-leucine in the nuclei of these cells was very poor.     

The cytoskeleton organizes germ nuclei with divergent fates and asynchronous cycles in a common cytoplasm during oogenesis in the chordate Oikopleura

Germline cysts are conserved structures in which cells initiating meiosis are interconnected by ring canals. In many species, the cyst phase is of limited duration, but the chordate, Oikopleura, maintains it throughout prophase I as a unique cell, the coenocyst. We show that despite sharing one common cytoplasm with meiotic and nurse nuclei evenly distributed in a 1:1 ratio, both entry into meiosis and subsequent endocycles of nurse nuclei were asynchronous. Coenocyst cytoskeletal elements played central roles as oogenesis progressed from a syncytial state of indistinguishable germ nuclei, to a final arrangement where the common cytoplasm had been equally partitioned into resolved, mature oocytes. During chromosomal bouquet formation in zygotene, nuclear pore complexes clustered and anchored meiotic nuclei to the coenocyst F-actin network opposite ring canals, polarizing oocytes early in prophase I. F-actin synthesis was required for oocyte growth but movement of cytoplasmic organelles into oocytes did not require cargo transport along colchicine-sensitive microtubules. Instead, microtubules maintained nurse nuclei on the F-actin scaffold and prevented their entry into growing oocytes. Finally, it was possible to both decouple meiotic progression from cellular mechanisms governing oocyte growth, and to advance the timing of oocyte growth in response to external cues.     

Formation of lining bodies and oocyte bodies during avian oogenesis

Oocytes, 80 μm-30 mm in diameter, from ovaries of White Leghorn hens have been examined. Structures are described that appear to represent intermediate stages in lining body formation. The formation (or assembly) of lining bodies occurs in the follicle cell cytoplasm, a few microns distant from the follicle cell plasma membrane. In the very late stages of their formation or after their formation the lining bodies become associated with follicle cell plasma membrane. A parallel process takes place in the oocyte cytoplasm. Structures we have termed oocyte bodies are being formed or assembled. Oocyte bodies are seen forming near Golgi complexes and many oocyte bodies ultimately become associated with Golgi cisternae. The evidence presented in this paper demonstrates that the two structures, lining bodies and oocyte bodies, are distinct and originate in the follicle cell and oocyte cytoplasm, respectively.     

Cytochemical studies of developmental stages during oogenesis in Culex pipiens molestus (Forsk?l). I. Lipids and carbohydrates

Lipids and carbohydrates were studied in the polytrophic ovaries of Culex pipiens molestus during oogenesis. The cytoplasm of both the oocyte and the nurse cells contains lipid structures at all stages of development--granules in the early stages and spheres in the later stages. Intranuclear lipid bodies can be demonstrated in the oocyte and in the nurse cells. After leaving the nucleus, lipids are deposited in the peripheral cytoplasm. From the third to the seventh adult phase, lipid granules are concentrated in the area of the nurse cell and oocyte junction, indicating that lipids originate in the nurse cells and are transported from these to the oocyte. The follicular epithelial cells provide the oocyte with lipid material for fatty yolk synthesis and formation of the egg envelopes. Lipids are distributed similarly to the Golgi apparatus, indicating that there is a relationship between this organelle and fat formation. In the early stages, the cytoplasm of the oocyte, the nurse cells and the follicular epithelium contains glycogen granules. In the later stages these cells also contain mucopolysaccharides. The mucopolysaccharide yolk spheres are enclosed in vacuoles, while the chorion is composed of acid mucopolysaccharides. The follicular epithelium and vitelline membrane are of a mucopolysaccharide nature. A topographical relationship exists between the Golgi apparatus and the glycogen granules, indicating that this organelle also plays a role in glycogen synthesis.     

Ultrastructure of growing oocytes and accessory cells in Austrognathia (Gnathostomulida, bursovaginoidea)

The ovary of Austrognathia cf. riedli consists of 4-6 oocytes which are located in the mid-body region between the epidermis and the gut epithelium. The ovary is not enveloped by a tunica and each growing oocyte is surrounded by one or more accessory cells, the function of which is hypothesized in this study. Oogenesis is not synchronous and can be subdivided into a previtellogenic phase and a vitellogenic phase. Previtellogenic oocytes undergo a number of cell differentiations which consist mainly of an increase in size of the nucleus and nucleolus and the appearance in the cytoplasm of chromatoid bodies, annulate lamellac and short cisternae of rough endoplasmic reticulum (RER). Vitellogenic oocytes are characterized by the increase of RER, the appearance of numerous Golgi complexes and the accumulation of electron-dense globules, glycogen and lipid droplets. The electron-dense globules have been interpreted as yolk on the basis of both their localization and composition. Yolk synthesis occurs mainly by an endogenous mechanism and, to a lesser extent, by micropinocytosis. No shell-granules have been identified in the oocytes. The present ultrastructural findings are discussed in comparison with those from other lower metazoans.     

Cytochemical studies of oocyte development in Sarcophaga lineatocollis Macq. (Muscidae: Diptera)

The ovary of Sarcophaga lineatocollis is a typical polytrophic ovary. Each of its 25-30 ovarioles is composed of a small terminal filament, a small germarium and a vitellarium consisting of the egg follicle. The tunica propria is a noncellular, PAS-positive membrane. The ovarian follicle contains fifteen trophocytes and one oocyte. RNA is synthesized with the aid of the nuclei in the trophocyte cytoplasm, which are RNA- and PAS-positive. Protein is deposited intensively in the early stages of the trophocytes. The trophocytes of Sarcophaga lineatocollis synthesize RNA and protein more actively than the oocyte. In this fly, protein yolk precursor (PYP) bodies are supplied by the trophocyte cytoplasm to the ooplasm at an advanced stage of development. Nucleolar budding and vacuolation are observed in the trophocytes. RNA, DNA, protein and PYP bodies appear to be transported to the ooplasm from the trophocytes. Pyknotic trophocyte nuclei can be seen entering the ooplasm. The perinuclear Golgi bodies of the trophocytes help in the production and maturation of PYP bodies in the trophocytes before they are organized and passed on to the oocytes. Some RNA is contributed to the oocyte by the follicular epithelium. All these processes leading to maturation and development of the oocyte are discussed and interpreted.     

A network system for vitellogenin synthesis in the mussel Mytilus galloprovincialis (L.)

The aim of this study is to assess, by RT‐PCR, in situ hybridization, electron microscopy, and immunohistochemistry, the site/s of vitellogenin (VTG) synthesis in the mussel Mytilus galloprovincialis. Our investigations demonstrate that, among the analyzed tissues, the synthesis of VTG occurs only in the female gonad, that is, within the oocyte and follicle and connective cells. Such a synthesis is just evident in early vitellogenic oocytes, whose cytoplasm is characterized by numerous RER cisternae and an extended Golgi complex surrounded by nascent yolk platelets. The synthesis of VTG goes on in vitellogenic oocytes assuming a pear form, and progressively reduces once the oocyte shows the pear or polygonal form, typical of those oocytes that have concluded the growth. The expression of VTG occurs also within follicle (auxiliary) and connective cells. In particular, it is noteworthy that follicle cells are characterized by numerous RER cisternae and an active Golgi complex surrounded by numerous vesicles and vacuoles containing electron dense material. The same material is also present along their plasma membrane, within the intercellular space between oocyte and follicle cells, and finally within invaginations of the oocyte surface, thus suggesting a VTG transfer to the oocyte via endocytosis. Differently, no VTG synthesis was observed within digestive gland. All together the findings here reported strongly suggest that in M. galloprovincialis, inside the gonad, the VTG synthesis occurs in the oocyte (autosynthesis) and in the follicle and adipogranular cells (heterosynthesis). J. Cell. Physiol. 228: 547–555, 2013. © 2012 Wiley Periodicals, Inc.     

Myelin-like structures as a possible source of the smooth endoplasmic reticulum in early amphibian oocytes

A comparative study of amphibian oocyte ultrastructural organization has shown a significant accumulation of elements of the smooth endoplasmic reticulum in the oocyte cytoplasm at the third stage of development. The analysis of oocytes of two frog species, Xenopus laevis and Rana temporaria, at the first and second stages of their development enabled us to recognize in the cytoplasm of the oocyte some myelin-like structures (MLs) made of 30-40 densely packaged membranous layers and shaped as dense bodies. MLs are also present in the adjacent follicular cells and in the intercellular space. In the oocyte cytoplasm these structures are located near the nuclear envelope and other intracellular organelles. At the third stage of oogenesis, which is characterized by a high functional activity of the cells, MLs are seen to unwrap sequentially into double-layer membranes similar to the smooth endoplasmic reticulum cisternae. Intermediate steps of this process being also observed. It is supposed that MLs may play the role of membrane stocks to be used eventually for the formation of nascent endoplasmic membranes in the amphibian oocytes.     

Cytoarchitecture of the ovarioles in scale insects (Hemiptera,Coccinea)

Telotrophic ovarioles of scale insects are subdivided into tropharia (=trophic chambers) and vitellaria that contain single developing oocytes. Tropharium encloses trophocytes (=nurse cells) and arrested oocytes. The central area of the tropharium, termed the trophic core, is devoid of cells. Both trophocytes and oocytes are connected to the trophic core: trophocytes by cytoplasmic processes, oocytes by means of nutritive cords. The trophic core, processes and nutritive cords are filled with bundles of microtubules. The trophocytes contain large lobated nuclei with giant nucleoli. Fluorescent labelling with DAPI has shown that trophocyte nuclei are characterized by high contents of DNA. In the cortical cytoplasm of trophocytes, numerous microfilaments are present. The developing oocyte is surrounded by a simple follicular epithelium. The cortical cytoplasm of follicular cells contains numerous microtubules and microfilaments.     

Activation of mammalian oocytes by a factor obtained from rabbit sperm

In this study a fraction was prepared from rabbit sperm that activated rabbit and mouse oocytes following injection into the cytoplasm. The sperm factor activated oocytes exhibited cortical granule exocytosis, pronuclear formation, and cleavage. The sperm factor was soluble in aqueous solution and was not active extracellularly. Unlike most artificial activation methods that are only effective with aged oocytes, the sperm factor activated recently ovulated oocytes. The factor appears to be a protein or associated with a protein but not an acrosomal protein. Fractions from both mouse and bull sperm did not activate rabbit or mouse oocytes. Their inactivity may be owing to the techniques used to recover the fractions or differences between species in sperm morphology and fertilization processes. These observations support the hypothesis that oocyte activation is induced by a factor within sperm that is released into the cytoplasm of the oocyte at the time of sperm-oocyte fusion.