Morphodynamics of the contract plate in the course of oocyte maturation in the scyphozoan Aurelia aurita (Cnidaria: Semaeostomae)

The structure forming in the area of contact between the oocyte and the germinal epithelium in the course of oocyte maturation of the scyphozoan Aurelia aurita is termed the contact plate. This study traces the successive stages of contact plate formation in the course of oocyte maturation at the light microscopic and ultrastructural levels. At early stages ofoocyte development, the appearance of granules is observed in the peripheral cytoplasm of the oocyte; these granules accumulate at the pole, which retains its connection with the germinal epithelium of the gonads. Two types of these granules are recognized: (1) granules with homogeneous content and (2) granules containing loose shapeless material in the form of thick cords. The transformation of type two granules into larger structures, as well as the consolidation of type one and type two granules at later stages of oocyte development, are probably the processes that lead to the formation of the characteristic structure and contact plate, visible in paraffin and semithin sections. It remains unclear where exactly the contact plate is localized at the moment of fertilization: inside or outside the oocyte. The content of granules and components of the plate specifically bind the antibodies (RA47) against mesoglein, the ZP domain-containing protein of the mesoglea of A. aurita. The contact plate, covering only the anomalous pole of the oocyte but detected by the presence of ZP domain-containing proteins, may prove to be the simplest egg membrane of the zona pellucida type.     

Plate in the zone of oocyte and germinal epithelium contact in Scyphomedusa Aurelia aurita binds antibodies to ZP-domain protein mesoglein

The morphological features of oocyte and germinal epithelium (epithelial wall of germinal sinus) contact area in Scyphomedusa Aurelia aurita are described. Growing oocytes were divided into seven stages based on oocyte size. The structure revealed the area of contact between the oocyte and the germinal epithelium called the contact plate. During oocyte growth, single granules are fused into the homogeneous mass area of oocyte contact with epithelium. Plate components bound antibodies to mesoglein. It was assumed that the plate material contained ZP-domain proteins. Electrophoresis and immunoblotting results show that proteins immunologically similar to mesoglein have higher molecular masses, probably due to post-translation modifications, which are common for extracellular proteins. On the other hand, however, gonad proteins may be other representatives of jellyfish ZP-domain proteins. Further experiments should be conducted to clarify which alternative is true.     

Oogenesis: From Oogonia to Ovulation in the Flagfish,Jordanella floridae Goode and Bean, 1879 (Teleostei: Cyprinodontidae)

We provide histological details of the development of oocytes in the cyprinodontid flagfish, Jordanella floridae. There are six stages of oogenesis: Oogonial proliferation, chromatin nucleolus, primary growth (previtellogenesis [PG]), secondary growth (vitellogenesis), oocyte maturation and ovulation. The ovarian lamellae are lined by a germinal epithelium composed of epithelial cells and scattered oogonia. During primary growth, the development of cortical alveoli and oil droplets, are initiated simultaneously. During secondary growth, yolk globules coalesce into a fluid mass. The full‐grown oocyte contains a large globule of fluid yolk. The germinal vesicle is at the animal pole, and the cortical alveoli and oil droplets are located at the periphery. The disposition of oil droplets at the vegetal pole of the germinal vesicle during late secondary growth stage is a unique characteristic. The follicular cell layer is composed initially of a single layer of squamous cells during early PG which become columnar during early vitellogenesis. During primary and secondary growth stages, filaments develop among the follicular cells and also around the micropyle. The filaments are seen extending from the zona pellucida after ovulation. During ovulation, a space is evident between the oocyte and the zona pellucida. Asynchronous spawning activity is confirmed by the observation that, after ovulation, the ovarian lamellae contain follicles in both primary and secondary growth stages; in contrast, when the seasonal activity of oogenesis and spawning ends, after ovulation, the ovarian lamellae contain only follicles in the primary growth stage. J. Morphol. 277:1339–1354, 2016. © 2016 Wiley Periodicals, Inc.     

Precocious loss of cortical granules during mouse oocyte meiotic maturation and correlation with an egg-induced modification of the zona pellucida

Fertilization results in cortical granule exocytosis, which is thought to be involved in modifications of the zona pellucida that constitute the zona pellucida block to polyspermy. A previous report demonstrated that a decrease in the number of Lens culinaris agglutinin-staining granules, which are likely to be cortical granules, occurred during in vivo mouse oocyte maturation with arrest at metaphase II, as well as the formation of a cortical granule-free domain in the area of the metaphase II spindle (T. Ducibella, E. Anderson, D.F. Albertini, J. Aalberg, and S. Rangarajan, 1988, Dev. Biol. 130, 184-197). We extend these observations by reporting here that germinal vesicle-intact oocytes matured in vitro to metaphase II in either the absence or the presence of serum develop a cortical granule-free domain and have reduced numbers of cortical granules when compared to germinal vesicle-intact oocytes; these changes are similar to those of oocytes matured in vivo. The reduction in the number of cortical granules requires germinal vesicle breakdown, since it is prevented by dibutyryl cAMP, which inhibits germinal vesicle breakdown in vitro. The ability of oocytes to respond to the calcium ionophore A23187 with a reduction in the number of cortical granules is also associated with meiotic maturation and develops between 7 and 12 hr after initiation of maturation. The maturation-associated reduction in the number of cortical granules is likely to represent cortical granule exocytosis, since this reduction is accompanied by the formation of a cortical granule-free domain and a conversion of ZP2 to ZP2f when the oocytes are matured in vitro in serum-free medium; this zona pellucida modification occurs following fertilization and is thought to be due to cortical granule exocytosis. In contrast, the loss of cortical granules and development of the cortical granule-free domain of oocytes matured in vitro in the presence of serum is not accompanied by the modification of ZP2. The inhibitory effect of serum on the ZP2 modification may afford in vivo a physiological mechanism to prevent a precocious modification of the zona pellucida that could result in a premature block to polyspermy and hence inhibit fertilization.     

Dynamics of Mitotic Apparatus Formation and Tubulin Content during Oocyte Maturation in Starfish

To follow the topo-temporal behavior of structures containing tubulin and the change in tubulin content during oocyte maturation, starfish oocytes were extracted with a medium containing detergent so that morphological observation and biochemical analysis could be conducted on the same residual oocyte preparation simultaneously. Before 1-methyladenine (1-MeAde) stimulation, "pre-meiotic asters" were observed on the germinal vesicle at the animal pole. 1-MeAde caused the appearance of distinct asters at the position of the aster precursor. When germinal vesicle breakdown (GVBD) took place, chromosomes were condensed. Chromosome gathering was concurrent with a reduction in the size of nuclear matrix. The mitotic apparatus was first constructed parallel to the cortex and then changed its axis perpendicularly. Fluorescence of tubulin due to indirect immunofluorescence in the cytoplasm other than the mitotic apparatus decreased rapidly along the course of maturation at least up to the first metaphase. Despite these dynamic morphological change, the tubulin content in the whole oocyte and the residual structures, measured by SDS-PAGE and immunostaining, did not show remarkable (statistically significant) changes through the course of maturation, although the content tended to decrease a little before the second polar body formation and to increase thereafter in the latter.     

Centriole behavior during meiosis in oocytes of the sea urchin Hemicentrotus pulcherrimus

Ultrastructural changes in the maturing oocyte of the sea urchin Hemicentrotus pulcherrimus were observed, with special reference to the behavior of centrioles and chromosomes, using oocytes that had spontaneously started the maturation division process in vitro after dissection from ovaries. The proportion of oocytes entering the maturation process differed from batch to batch. In those eggs that accomplished the maturation division, it took ~4.5-5 h from the beginning of germinal vesicle breakdown to the formation of a second polar body. Serial sections revealed that a young oocyte before germinal vesicle breakdown had a pair of centrioles with procentrioles, located between the presumed animal pole and the germinal vesicle and accompanied by amorphous aggregates of moderately dense material and dense granules (granular aggregate). Just before germinal vesicle breakdown, a pair of fully grown centrioles located in the granular aggregate, which is present until this stage and then disappears, had already separated from another pair of centrioles. In meiosis I, each division pole had two centrioles, whereas in meiosis II each had only one. The two centrioles in the secondary oocyte separated into single units and formed the mitotic figure of meiosis II. The first polar body had two centrioles and the second had only one. The two centrioles in the first polar body did not form the mitotic figure nor did they separate at the time of meiosis II. These results indicate that, in sea urchins, duplication of the centrioles does not occur during the two successive meiotic divisions and the egg inherits only one centriole from the primary oocyte, confirming the results previously reported for starfish oocytes.     

FORMATION AND BEHAVIOR OF PINOSOMES IN THE SEA URCHIN OOCYTE DURING OOGENESIS

Formation and behavior of the pinosomes at the surface of the oocyte during oogenesis in the 4 species of sea urchins, Anthocidaris crassispina, Temnopleurus toreumaticus, Mespilia globulus and Pseudocentrotus depressus, were studied. The plasma membrane of the oocyte is almost smooth at the early stage of oogenesis, although a small number of cytoplasmic processes appear on it, facing the germinal epithelium. At the beginning of vitellogenetic stage many processes appear on the whole surface of the oocyte. Near the base of the fully grown process, the pinosome designated as the α-pinosome is formed. The α-pinosome may play a part in maturation of the yolk granule. The processes shorten as a whole at the time of the breakdown of the germinal vesicle. Formation of the pinosome designated as the β-pinosome begins just before vitellogenetic stage and continues during this stage. The β-pinosome may be directly concerned with the formation of cortical granules.     

Fetuin inhibits zona pellucida hardening and conversion of ZP2 to ZP2f during spontaneous mouse oocyte maturation in vitro in the absence of serum.

The zona pellucida of mouse oocytes becomes resistant to chymotrypsin digestion, or "hardened", when spontaneous maturation occurs in serum-free medium (De Felici and Siracusa, Gam Res 1982; 6:107). The hardened zona pellucida is refractory to sperm penetration, thus preventing fertilization. Conversion of the zona pellucida glycoprotein ZP2 to ZP2f by a protease from precociously released oocyte cortical granules appears to be a major contributory factor of zona pellucida hardening (Ducibella et al., Dev Biol 1990; 137:46). Fetal bovine serum (FBS) prevents zona hardening and the ZP2 to ZP2f conversion during oocyte maturation in vitro (Downs et al., Gam Res 1986; 15:115; Ducibella et al., Dev Biol 1990; 137:46). This study was conducted to determine whether fetuin, a major glycoprotein constituent of FBS and a protease inhibitor, could prevent zona pellucida hardening during murine oocyte maturation in serum-free medium. Commercially available preparations of fetuin purified by three different methods were all active in inhibiting zona pellucida hardening in a concentration-dependent manner. Further chromatographic purification of one of these preparations indicated that the activity preventing zona pellucida hardening was associated specifically with fetuin. Fetuin also inhibited the conversion of ZP2 to ZP2f in a concentration-dependent manner during oocyte maturation in serum-free medium. Moreover, oocytes that matured in serum-free medium containing fetuin could be fertilized and could undergo preimplantation development to the blastocyst stage. These results indicate that fetuin, a component of FBS, inhibits zona pellucida hardening during oocyte maturation, and suggest that fetuin acts by preventing the proteolytic conversion of ZP2 to ZP2f by precociously released cortical granules.     

Modifications of the mouse zona pellucida during oocyte maturation and egg activation: effects of newborn calf serum and fetuin.

A precocious but limited loss of cortical granules (CG) occurs during mouse oocyte maturation both in vivo and in vitro. Although CG loss during maturation in vivo is not associated with changes in the zona pellucida (ZP), a maturation-associated conversion of ZP2 to ZP2f occurs during oocyte maturation in vitro in serum-free medium. We now demonstrate that a maturation-associated change of ZP3 to ZP3f, as assessed by a reduction in sperm binding, also occurs during maturation in vitro in serum-free medium, and that both newborn calf serum (NCS) and fetuin, each of which inhibits the ZP2 conversion, also inhibit the ZP3 conversion. The concentration-dependence of the NCS- and fetuin-mediated inhibition of the ZP2 conversion, coupled with the concentration of fetuin present in NCS, is consistent with fetuin being the component present in NCS that is primarily responsible for this inhibition. Although NCS can inhibit the ZP modifications that occur during oocyte maturation in vitro, ionophore treatment of eggs, which results in an extensive release of CGs over a short period of time, overcomes the inhibitory effect of NCS on the ZP2 conversion. Results of these studies suggest a potential regulatory function of serum-derived components in the formation of a fertilizable egg.     

Subcortical microtubule network separates the periplasm from the endoplasm and is responsible for maintaining the position of accessory nuclei in hymenopteran oocytes

Oocytes of hymenopterans are equipped with peculiar organelles termed accessory nuclei. These organelles originate from the germinal vesicle (oocyte nucleus) and gather preferentially at the anterior pole. To gain insight into the mechanism of uneven (asymmetrical) distribution of accessory nuclei, the organization of the microtubule cytoskeleton in the oocytes of two hymenopterans Chrysis ignita and Cosmoconus meridionator has been studied. It is shown that during late previtellogenesis two networks of microtubules are present along the contact zone between the oocyte and enveloping follicular epithelium. The external one is associated with belt desmosomes connecting neighbouring follicular cells. The internal network is composed of randomly orientated microtubules and separates transparent, organelle-free periplasm from the endoplasm. All cellular organelles and the germinal vesicle are localized in the endoplasm. Accessory nuclei are accumulated in the anterior endoplasm; they always lie in direct contact with the subcortical network. Treatment with colchicine results in the disappearance of the periplasm as well as in the redistribution of cellular organelles including accessory nuclei. Presented findings suggest that subcortical microtubules play an important role in the positioning of accessory nuclei throughout the ooplasm.     

Early oogenesis in the ascidian Ciona savignyi

Summary

Morphology of the germinal epithelium and the early follicular oocyte in the ascidian Ciona savignyi as examined by electron microscopy. The oogenetic part of the germinal epithelium contains oocytes at two different stages and the dark and clear cells. The smaller oocyte contains synaptonemal complexes. The larger oocyte in the initial phase of growth has a conspicuous nucleolus, electron-dense materials and some mitochondria close to the nuclear envelope. The nucleus of the larger oocyte is round and has the smooth contour. The dark cell contains a relatively large nucleus and is sometimes connected to each other by an intercellular bridge. Therefore, the dark cell, which has been suggested to be the progenitor cell of two kinds of accessory cells, may be also the oogonium. The early follicular oocyte just after migration from the germinal epithelium retains most of cytological features similar to those of the larger oocyte. However, the nuclear contour of the early follicular oocyte is uneven. This difference in the nuclear contour probably indicates that such a follicular oocyte is in the second phase of growth.     

Mitochondrial trafficking through Rhot1 is involved in the aggregation of germinal granule components during primordial germ cell formation in Xenopus embryos

In many animals, the germ plasm is sufficient and necessary for primordial germ cell (PGC) formation. It contains germinal granules and abundant mitochondria (germline‐Mt). However, the role of germline‐Mt in germ cell formation remains poorly understood. In Xenopus, the germ plasm is distributed as many small islands at the vegetal pole, which gradually aggregates to form a single large mass in each of the four vegetal pole cells at the early blastula stage. Polymerized microtubules and the adapter protein kinesin are required for the aggregation of germ plasm. However, it remains unknown whether germline‐Mt trafficking is important for the cytoplasmic transport of germinal granules during germ plasm aggregation. In this study, we focused on the mitochondrial small GTPase protein Rhot1 to inhibit mitochondrial trafficking during the germ plasm aggregation. Expression of Rhot1ΔC, which lacks the C‐terminal mitochondrial transmembrane domain, inhibited the aggregation of germline‐Mt during early development. In Rhot1‐inhibited embryos, germinal granule components did not aggregate during cleavage stages, which reduced the number of PGCs on the genital ridge at tail‐bud stage. These results suggest that mitochondrial trafficking is involved in the aggregation of germinal granule components, which are essential for the formation of PGCs.     

Changes in subcellular localization of mtlrRNA outside mitochondria in oogenesis and early embryogenesis of Drosophila melanogaster

Mitochondrial large ribosomal RNA (mtlrRNA) has been identified as a cytoplasmic factor inducing pole cells in ultraviolet (UV)-sterilized Drosophila embryos. In situ hybridization studies have revealed that mtlrRNA is present outside mitochondria localized on the surface of polar granules during the cleavage stage. In the present study, we describe the developmental changes in extramitochondrial mtlrRNA distribution through early embryogenesis using in situ hybridization at the light and electron microscopic level. No mtlrRNA signal was discernible on polar granules in the mature oocyte, unless the oocyte was activated for development. mtlrRNA was localized on the surface of polar granules during a limited period of stages from oocyte activation to pole bud formation and disappeared as soon as being detached from polar granules without entering pole cells. These changes in the temporal and spatial distribution of mtlrRNA outside mitochondria are compatible with the idea that mtlrRNA is required for pole cell formation but not for the differentiation of pole cells as functional germ cells.     

Development of the germinal epithelium and early folliculogenesis during ovarian morphogenesis in the cichlid fish Cichlasoma dimerus (Teleostei,Perciformes)

Formation of the germinal epithelium and folliculogenesis during ovarian development in Cichlasoma dimerus were described at the light‐ and electron‐microscopic levels. Prior to gonadal differentiation, germ cells and enveloping support cells reside within an inpocketing of the coelomic epithelium. Separation of the germinal and interstitial compartments of the gonad by a basement membrane is apparent from early gonadal development. Upon ovarian differentiation, oogonia undergo cyst‐forming divisions leading to the formation of clusters of interconnected cystocytes that synchronously enter meiosis, becoming oocytes. At the pachytene step, each oocyte becomes individualized by cytoplasmic extensions of prefollicle cells, thereby developing as an ovarian follicle. Subsequent somatic reorganization leads to the formation of the ovarian lumen in a cephalo‐caudal gradient. As a result, the germinal epithelium becomes internalized and lines the ovarian lumen. As defined by its origin from the germinal epithelium, the ovarian follicle is composed of an oocyte and the surrounding follicle cells. Thecal cells derived from the stroma encompass the basement membrane outside the follicle, thus forming a follicle complex. A common basement membrane is shared by the germinal epithelium and the follicle complex along a small portion of its surface. This point of attachment represents the site at which the oocyte would be released to the ovarian lumen during ovulation.     

Maturation conditions and boar affect timing of cortical reaction in porcine oocytes

The cortical reaction induces changes at the egg's Zona pellucida (ZP), perivitelline space and/or oolemma level, blocking polyspermic fertilization. We studied the timing of sperm penetration and cortical reaction in pig oocytes matured under different conditions and inseminated with different boars. Immature (germinal vesicle stage) and in vitro matured (IVM) (metaphase II stage) oocytes were inseminated and results assessed at different hours post insemination. Penetrability and polyspermy rates increased with gamete coincubation time and were higher in IVM oocytes. A strong boar effect was observed in IVF results. Cortical reaction (assessed as area occupied by cortical granules) and galactose-β(1-3)-Nacetylgalactosamine residues on ZP (area labeled by peanut agglutinin lectin, PNA) were assessed in IVM and in vivo matured (IVV) oocytes at different hours post insemination. After maturation, IVM and IVV oocytes displayed similar area occupied by cortical granules and it decreased in fertilized oocytes compared to unfertilized ones. Cortical reaction was influenced by boar and was faster in polyspermic than in monospermic oocytes, and in IVM than in IVV oocytes. The outer ZP of inseminated oocytes appeared stained by PNA and the labeled area increased along with gamete coculture time. This labeling was also observed after insemination of isolated ZP, indicating that this modification in ZP carbohydrates is not induced by cortical reaction. The steady and maintained cortical reaction observed at 4 to 5 h post insemination in IVV monospermic oocytes might reflect the physiological time course of this important event in pigs. Both maturation conditions and boar affect cortical granules release.     

A comparative histochemical study of fish (Channa maruleus) and amphibian (Bufo stomaticus) oogenesis

Summary Comparative histochemical studies on the fish (Channa maruleus) and amphibian (Bufo stomaticus) oogenesis demonstrate a great similarity in the growth and differentiation of their egg follicle. The ooplasm, germinal vesicle and egg-membranes show distinct morphological and cytochemical changes during previtellogenesis and vitellogenesis.During previtellogenesis the various components of the follicle are engaged in the synthesis of protoplasm as shown by the proliferation of yolk nucleus substance, mitochondria and some lipid bodies in the ooplasm and of nucleoli in the germinal vesicle. The substance of the yolk nucleus consisting of proteins, lipoproteins and RNA first appears adjacent to the nuclear membrane. Numerous mitochondria of lipoprotein composition, and some lipid bodies consisting of unsaturated phospholipids lie in association with the yolk nucleus which forms substratum for the former. The lipid bodies, present inside the germinal vesicle, follicular epithelium, and adjacent to the plasma membrane in association with some pinocytotic vacuoles, have been considered to play a significant role in the active transport of some substances from the environment into the ooplasm and from the latter into the germinal vesicle. The follicular epithelium itself is very poorly developed, negating its appreciable role in the contribution of specific substances into the oocyte, which seem to be contributed by the germinal vesicle showing a considerable development of nuclear sap, basophilic granules and nucleoli consisting of RNA and proteins; many large nucleoli bodily pass into the cytoplasm during the previtellogenesis of Channa, where their substance is gradually dissolved. The intense, diffuse, basophilic substance of the cytoplasm is believed due to free ribosomes described in many previous ultrastructural studies.During vitellogenesis, the various deutoplasmic inclusions, namely carbohydrate yolk, proteid yolk and fatty yolk, are deposited in the ooplasm. The carbohydrate yolk bodies rich in carbohydrates originate in association with the plasma membrane and correspond to vesicles and cortical granules of previous studies. The proteid yolk consisting of proteins and some lipoproteins, and fatty yolk containing first phospholipids and some triglycerides and then triglycerides only are deposited under the influence of yolk nucleus substance, mitochondria and cytoplasm. The mitochondria and yolk nucleus substance foreshadow in some way the pattern of these two deutoplasmic inclusions and persist at the animal pole of mature egg while the other inclusions of previtellogenesis disappear from view. The pigment granules, which also show a gradient from the animal to vegetal pole in Bufo, are also formed in association with yolk nucleus substance and mitochondria. Some glycogen also appears in both the species. The nuclear membrane becomes irregular due to the formation of lobes. The lipid bodies of the germinal vesicle come to lie outside the nuclear membrane, suggesting active transport of some substances into the ooplasm; many nucleoli bodily pass into the ooplasm of Bufo, where they are gradually absorbed. The amount of basophilic granules is considerably increased in the germinal vesicle during vitellogenesis. Various egg-membranes such as outer epithelium, thin theca, single-layered follicular epithelium, zona pellucida or vitelline membrane surround the vitellogenic oocytes. The zona pellucida formed between the oocyte and follicle cells consists of a carbohydrate-protein complex. The follicle cells show lipid droplets, mitochondria and basophilic substance in their cytoplasm. The various changes that occur in the components of the follicle during vitellogenesis seem to be initiated by gonadrotrophins formed under the influence of specific environmental conditions.The author wishes to express sincere appreciation and gratitude to Dr. Gilbert S. Greenwald, who has made the completion of this investigation possible.Ph. D. Population Council Post-doctoral Fellow.     

Insemination,intrafollicular fertilization and development of the fertilization plug during gestation in Heterandria formosa (Poeciliidae)

The viviparous teleost Heterandria formosa is a remarkable species for its reproductive characters including: (a) the smallest oocyte in viviparous fish species; (b) a high level of matrotrophy with a complex placenta; and (c) the highest level of superfetation. Superfetation involves (d) the continuous development of oocytes and fertilization at the same time with embryos in gestation. The sequential fertilization of oocytes requires (e) storage of spermatozoa in the ovary. Among these characteristics, fertilization is of fundamental interest, specifically the intrafollicular fertilization of poeciliids, species that do not present micropyle, and the consequent formation of the fertilization plug, a structure developed at the periphery of the follicle where the entrance of spermatozoa occurs. Both processes intrafollicular fertilization and formation of the fertilization plug have been rarely described. There is only one study illustrating, the fertilization plug of H. formosa with a drawing. In the context of reproductive aspects of H. formosa, the goal of this study is to describe the morphology of the ovary during insemination, intrafollicular fertilization and development of the fertilization plug. After insemination, spermatozoa enter the ovary and occupy folds of the lamella near follicles of all stages of oogenesis, the delle, where the germinal epithelium establishes contact with the follicular epithelium. The results of the present study provide evidence that both epithelia open at the distal end of the delle, this morphological change allow that the spermatozoa to make contact with the zona pellucida of the oocyte. After fertilization, the delle becomes blocked by proliferation of cells of the germinal epithelium, to form the fertilization plug that persists throughout gestation. Abundant reticular fibers and blood vessels are seen around the fertilization plug. Persistence of the fertilization plug suggests that it could be the site where the juvenile will gain entrance to the ovarian lumen during birth.     

Nuclear requirement of post-maturational cortical differentiation of amphibian oocytes: effects of cycloheximide.

Cycloheximide induced a complex series of alterations in the cortical cytoplasm of amphibian (Rana pipiens) oocytes undergoing steroid induced nuclear and cytoplasmic maturation in vitro. The morphological changes were described and the role of nuclear-cytoplasmic interactions in the induction of these changes was investigated in intact, enucleated and enucleated-reinjected oocytes. Three stages of cortical changes were ascertained on the basis of: localized alterations at the animal pole, redistribution of pigment and localized contractility (furrow formation) primarily along the animal:vegetal pole axis. The extent and type of cortical alterations varied depending upon the time at which oocytes were examined following hormonal stimulation and cycloheximide treatment. Cycloheximide did not produce cortical alterations in non-hormone treated oocytes nor in steroid treated oocytes until after germinal vesicle breakdown. Nuclear and cytoplasmic maturation and the appearance of cortical alterations were all inhibited when cycloheximide was added to oocytes at the time of steroid treatment. Cycloheximide induction of cortical alterations occurred only after the inhibitor was no longer effective in preventing germinal vesicle breakdown. Enucleated oocytes underwent cytoplasmic maturation in response to the steroid but exhibited no cortical alterations following the delayed addition of cycloheximide. Simultaneous administration of cycloheximide and steroid to enucleated oocytes inhibited cytoplasmic maturation and all observable cortical alterations. Reinjection of nuclear material into enucleated oocytes restored the ability of cycloheximide to induce cortical alterations following steroid induction of cytoplasmic maturation. Without steroid treatment, such reinjected oocytes did not exhibit cortical changes in response to cycloheximide. The data demonstrate that the nucleus is required for and contains a factor(s) which controls the cycloheximide response and post-maturation differentiation of the oocyte. The maturational changes in the cortical cytoplasm appear to be dependent on the intermixing of the germinal vesicle nucleoplasm materials with mature cytoplasm following germinal vesicle breakdown. The results further suggest that the cortical effects of cycloheximide are dependent upon the initiation of protein synthesis during this period of oocyte development. The significance of these observations and experimental studies are discussed in relation to current understanding of the molecular mechanisms controlling meiosis induction and the composition of the germinal vesicle.