Calmodulin synthesis and accumulation during oogenesis and maturation of Xenopus laevis oocytes

The calmodulin levels in stage 6 Xenopus oocytes averaged 89 +/- 24 (SD) ng/oocyte and had largely accumulated by stage 3 of oogenesis. From stage 3 to early stage 6, calmodulin levels did not increase further. However, in large stage 6 oocytes (greater than 1.25 mm diam) calmodulin levels again rose to a level as high as 121 ng/oocyte. Calmodulin levels did not change during the maturation of stage 6 oocytes and the results of measurements on animal and vegetal oocyte halves from control and mature oocytes showed no evidence of a redistribution of calmodulin during maturation. Measurements of calmodulin synthesis in stages 1 and 2 oocytes, stage 4 oocytes, and stage 6 oocytes indicated that calmodulin was being synthesized continuously during oogenesis and that the rate of synthesis increased during oogenesis. In stage 1 and 2 oocytes (combined), the synthesis rate was 3.5 pg/hr/oocyte; in stage 4 oocytes it was 48 pg/hr/oocyte, and in large stage 6 oocytes the rate had increased to 160 pg/hr/oocyte. These changes in the rates of synthesis were discussed as they relate to the pattern of calmodulin accumulation during oogenesis.     

Oocyte-granulosa cell heterologous gap junctions are required for the coordination of nuclear and cytoplasmic meiotic competence

Homologous gap junctions are generally recognized as a means of coordinating cellular behavior under developmental and homeostatic conditions. In the mammalian ovary, heterologous gap junctions between the oocyte and the granulosa cells have been widely implicated in the regulation of meiotic maturation late in oogenesis. However, the role of oocyte-granulosa cell gap junctions at earlier stages of oogenesis is poorly understood. Stage-specific defects in both oocyte and follicle development have been identified in juvenile mice deficient in heterologous oocyte-granulosa cell gap junctions due to targeted deletion of Gja4, the gene encoding connexin-37. Follicle development arrests at the type 4 preantral stage and although oocytes commence growth, oocyte growth ceases at a diameter of 52 microm (74.3% of control size). Analysis of cell cycle and cytoskeletal markers indicates that oocytes arrest in a G(2) state based on uniform decondensed GV chromatin, interphase microtubule arrays, and nonphosphorylated cytoplasmic centrosomes. Functional assays of meiotic competence confirm that oocytes from connexin-37-deficient mice are unable to enter M phase (initiate meiotic maturation) unless treated with the phosphatase inhibitor okadaic acid (OA). Unlike growing oocytes from heterozygous control animals, OA-treated oocytes from connexin-37-deficient mice respond acutely and progress rapidly to the circular bivalent stage of meiosis I and upon removal from OA rapidly revert to an interphase state. In contrast, OA-treated control incompetent oocytes are slow to respond, exhibit a lower proportion of chromosomal bivalent stage oocytes, but remain in and progress into meiotic M phase upon removal from OA. This study demonstrates that heterologous gap-junctional communication is required for the completion of oocyte growth and the acquisition of cytoplasmic meiotic competence.     

On the synthesis and destruction of A- and B-type cyclins during oogenesis and meiotic maturation in Xenopus laevis

We have measured the levels of cyclin mRNAs and polypeptides during oogenesis, progesterone-induced oocyte maturation, and immediately after egg activation in the frog, Xenopus laevis. The mRNA for each cyclin is present at a constant level of approximately 5 x 10(7) molecules per oocyte from the earliest stages of oogenesis until after fertilization. The levels of polypeptides show more complex patterns of accumulation. The B-type cyclins are first detectable in stage IV and V oocytes. Cyclin B2 polypeptide is present at approximately 2 x 10(9) molecules (150 pg) per oocyte by stage VI. The amount increases after progesterone treatment, but returns to its previous level after GVBD and undergoes no further change until it is destroyed at fertilization. Cyclin B1 is present at 4 x 10(8) molecules per oocyte in stage VI oocytes, and rises steadily during maturation, ultimately reaching similar levels to cyclin B2 in unfertilized eggs. Unlike the B-type cyclins, cyclin A is barely detectable in stage VI oocytes, and only starts to be made in significant amounts after oocytes are exposed to progesterone. A portion of all the cyclins are destroyed after germinal vesicle breakdown (GVBD), and cyclins B1 and B2 also experience posttranslational modifications during oocyte maturation. Progesterone strongly stimulates both cyclin and p34cdc2 synthesis in these oocytes, but whereas cyclin synthesis continues in eggs and after fertilization, synthesis of p34cdc2 declines strongly after GVBD. The significance of these results is discussed in terms of the activation and inactivation of maturation-promoting factor.     

DEVELOPMENT OF THE NUCLEAR STRUCTURE AND METABOLISM DURING OOGENESIS OF PERINEREIS CULTRIFERA (ANNELIDA,POLYCHAETA)

An ultrastructural study of the nucleus was carried out, during oogenesis of Perinereis cultrifera, accompanied by an autoradiographic and biochemical study of the syntheses of RNA. The nucleus encloses formations deriving from the dispersal of meiotic chromosomes and a voluminous nucleolus. The latter undergoes morphological development of which each stage is characteristic of a stage of oogenesis. The autoradiographic study shows that the synthesis of RNA of extra-nucleolar origin is highly intense in young oocytes (during the stages of previtellogenesis and vitellogenesis) and that it decreases in older oocytes. The synthesis of RNA of nucleolar origin is very weak during previtellogenesis, increases during vitellogenesis, which is the stage at which it reaches its peak, and then decreases during the stages of the development of cortical alveoli and of maturity. These autoradiographic results are confirmed by a biochemical study which shows that once an oocyte diameter of 80 μm is reached (mid-vitellogenesis), the specific radioactivity of 18 and 28 S rRNA and of 4 and 5 S RNA decreases progressively up to the end of oogenesis.     

Heat production and respiration of the oocyte of Xenopus laevis L. in various stages of oogenesis]

The data are given on the respiration and heat production intensity in the X. laevis oocytes by which the value and patterns of change of psiu-function (function of bound dissipation) during oogenesis are calculated. All 3 values were shown to decrease rapidly from the stage of previtellogenesis till the stage of mature oocyte what agrees with the thermodynamic theory of development.     

Differentiation and distribution of annulate lamellae in growing oocytes in the newt, Cynops pyrrhogaster

Stages of oocyte development in Cynops pyrrogaster are defined, and changes of annulate lamellae in their fine structure, number, sizes and locations during oogenesis are described. The results show that two different types of annulate lamellae occur during oogenesis. One type differentiates in or at the periphery of vesicle-rich cytoplasm at the early stages of vitellogenesis and increases in number and size. The maximum number of about 40 stacks per median section of oocyte is reached at the stage of complete differentiation of the animal and the vegetal hemispheres. In these growing oocytes, all the stacks show elongate appearances and tetragonal arrangements of annuli as common characteristics. A second type of stacks of annulate lamellae is added anew in full-grown oocytes, increasing the number of stacks per median section of the oocyte to about 90. The new stacks occur in close contact with electron-dense bodies in the cytoplasm and have a massive appearance and hexagonal array of annuli. It is suggested that they appear coincidentally with the onset of oocyte maturation. The possible significance of the observed results is discussed.     

Oogenesis and the ovarian cycle in Salamandra salamandra infraimmaculata Mertens (Amphibia; Urodela; Salamandridae) in fringe areas of the taxon's distribution

Reproductive cycle and oogenesis were studied in specimens of Salamandra salamandra infraimmaculata Mertens that inhabit fringe areas of the taxon's distribution in the Mediterranean region. Both ovarian mass and length are correlated significantly with body mass and length. Ovarian length is also correlated with the number of oocytes. During the oogenetic cycle six stages in oocyte development were recognized. Three occur during previtellogenesis: stage 1, in which oogonia divide and form cell nests; stage 2 in which oogonia differentiate into oocytes; and stage 3, in which the oocyte cytoplasm increases in volume. In the vitellogenic phase two additional stages, 4 and 5, were recognized: stage 4, in which lipid accumulates in vacuoles in the periphery followed by the appearance of yolk platelets near the cytoplasmic margin; and stage 5, in which oocyte volume increases rapidly due to increased number of yolk platelets until it reaches its maximal size. During postvitellogenesis one stage was recognized: stage 6, in which the beginning of maturation is characterized by movement of the nucleus toward the animal pole. Oogenesis continues year-round. The first four stages were seen in all ovaries examined. The ovarian cycle is independent of season and reproductive stage apart from the number of mature, postvitellogenic oocytes that increases following gestation toward the beginning of spring (March-April). J. Morphol 231:149–160, 1997. © 1997 Wiley-Liss, Inc.     

The effect of hormones and growth factor on the course of meiosis in murine oocytes in culture]

The role of some intraovarian regulators of the final stages of gametogenesis is analysed. It is shown that the epidermal growth factor (EGF) in concentration of 1 and 10 ng/ml is able to induce reinitiation of meiosis from dictyotene stage during cultivation of the ovarian follicles of prepuberal mice in the serum-free medium after gonadotrophic stimulation. The pattern of maturation was analogous to that of maturation after HCG (LH) administration. Also, the EGF is able to stimulate meiosis reinitiation in the culture of cumulus-free oocytes blocked with cAMP at the stage of dictyotene. At the same time fibroblast growth factors and insulin do not demonstrate such an activity. Taking into consideration a high sensibility of oocytes to the EGF action, and also the fact that the character of changes of steroid hormones secreted by the ovary in culture under the action of EGF is the same as that under the influence of LH it is suggested that, the EGF and EGF-like proteins secreted by somatic follicle cells are the paracrinic regulators of the mammalian oocyte maturation which modulate neuroendocrine factors of the oogenesis control.     

Comparative study of reproductive biology in single- and multiple-spawner cyprinid fish. I. Morphological and histological features

To clarify the dynamics and regulation of oogenesis in single- and multiple-spawning cyprinid fish with group-synchronous oocyte development, a multidisciplinary approach to their reproduction was undertaken using three species from the River Meuse (Belgium): the roach Rutilus rutilus as a single spawner, and the bleak Alburnus alburnus and the white bream Blicca bjoerkna as multiple spawners. The gonadosomatic index (GSI) and histomorphometric changes (distribution of oocyte size, relative proportion of the various oocyte stages) in the ovary are compared. Different patterns of GSI and oocyte growth were observed both between the single- and multiple-spawner fish and between the two multiple spawners. Maximum GSIs were higher in roach (21%) than in bleak and white bream (17.7 and 14.5%, respectively), and compared to the rapid decline of GSI in the roach population, the GSI of multiple spawners decreased progressively during the spawning season. In roach, a short gonadal quiescent period and an early onset of vitellogenesis was recorded from late summer onwards whereas, in bleak and white bream, exogenous vitellogenesis was not systematically observed before winter. A protracted spawning season and/or a low water temperature in autumn are hypothesized to explain this long period of gonadal quiescence. In bleak, during the spawning season, the oocytes recruited arose from the stock of endogenous vitellogenesis and attained the final maturation stage very rapidly. This recruitment occurred during the whole spawning season. In white bream, the differentiation of vitellogenic oocytes from smaller oocytes was completed before the onset of the spawning season. During the spawning period, the proportion of vitellogenic oocytes decreased progressively whereas the percentage of oocytes in the final maturation stage remained approximately constant.     

Oogenesis of microlecithal oocytes in the viviparous teleost Heterandria formosa

Viviparous teleosts exhibit two patterns of embryonic nutrition: lecithotrophy (when nutrients are derived from yolk that is deposited in the oocyte during oogenesis) and matrotrophy (when nutrients are derived from the maternal blood stream during gestation). Nutrients contained in oocytes of matrotrophic species are not sufficient to support embryonic development until term. The smallest oocytes formed among the viviparous poeciliid fish occur in the least killifish, Heterandria formosa, these having diameters of only 400 μm. Accordingly, H. formosa presents the highest level of matrotrophy among poeciliids. This study provides histological details occurring during development of its microlecithal oocytes. Five stages occur during oogenesis: oogonial proliferation, chromatin nucleolus, primary growth (previtellogenesis), secondary growth (vitellogenesis), and oocyte maturation. H. formosa, as in all viviparous poeciliids, has intrafollicular fertilization and gestation. Therefore, there is no ovulation stage. The full‐grown oocyte of H. formosa contains a large oil globule, which occupies most of the cell volume. The oocyte periphery contains the germinal vesicle, and ooplasm that includes cortical alveoli, small oil droplets and only a few yolk globules. The follicular cell layer is initially composed of a single layer of squamous cells during early previtellogenesis, but these become columnar during early vitellogenesis. They are pseudostratified during late vitellogenesis and reduce their height becoming almost squamous in full‐grown oocytes. The microlecithal oocytes of H. formosa represent an extreme in fish oogenesis typified by scarce yolk deposition, a characteristic directly related to matrotrophy. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

Metabolism of inositol pentakisphosphate to inositol hexakisphosphate in Xenopus laevis oocytes

The formation and metabolism of inositol pentakis-and hexakisphosphates (InsP5 and InsP6) were investigated in Xenopus laevis oocytes. After [3H]inositol injection, [3H]InsP5 and subsequently [3H]Insp6 increased progressively over 72 h. In intact oocytes, [3H]InsP5 was progressively converted to [3H]InsP6 from 6 to 72 h of incubation and was not metabolized to lower inositol phosphates. In contrast, [3H]InsP6 remained unmetabolized for up to 72 h. These data are consistent with the kinetics of the increases in [3H]InsP5 and [3H]InsP6 in [3H]inositol-labeled oocytes. The highly phosphorylated inositols showed significant changes during oogenesis and maturation. In oocytes incubated for 48 h after [3H]inositol injection, the radioactive incorporation into polyphosphoinositols increased progressively from stage 3 to stage 6, with 5- and 6-fold rises (cpm/mg protein) for [3H]InsP5 and [3H]InsP6, respectively. These developmental changes were associated with 5-fold increases in [3H]inositol tetrakisphosphate between stages 3 and 6 of oogenesis. Induction of oocyte maturation by progesterone (1 microM) during the last 12 of a 36-h incubation with [3H]inositol doubled the levels of [3H]InsP6 relative to [3H]InsP5, suggesting that the activity of inositol pentakisphosphate kinase increases during maturation. These results provide direct evidence for metabolic conversion of InsP5 to InsP6 in animal cells and show that the higher inositol polyphosphates, unlike the lower phosphoinositols, are extraordinarily stable. These species increase markedly during ovum development and may play a regulatory role in oogenesis and maturation.     

南美白对虾卵子发生的组织学

采用组织学方法研究了南美白对虾的卵子发生过程,根据卵细胞大小、核仁形态、卵黄粒的有无、皮质棒的出现以及卵母细胞与滤泡细胞的关系,将南美白对虾的卵子发生划分为卵原细胞、卵黄发生前的卵母细胞和卵黄发生的卵母细胞三个时期,并描述了各期卵细胞的形态特征。     

Energy Metabolism in Fish Development

SYNOPSIS. During oogenesis the fish oocytes accumulate severalsubstances of which lipids and glycogen are the major energysubstrates. Oocyte maturation is accompanied by an increasein all the enzymes of carbohydrate metabolism. After fertilization,respiration and glycogenolysis are increased and the energycharge is decreased. During early embryogenesis glycogen appearsto be the only substrate of glycolysis. Glycolysis and the citricacid cycle are the main sources of energy for the biosyntheticactivities and for the maintenance of embryo morphology. Thereare two patterns of ontogeny of glycolytic enzymes in troutembryos. One group of enzymes does not undergo appreciable changeswhereas enzymes within the second group exhibit variable activities.Marked changes in enzyme activity occur during fertilizationand gastrulation. Lactate dehydrogenase (LDH) is of particularinterest. Its activity increases during gastrulation. This increasein LDH activity is followed by a change in the isozyme patternand in the adenylate charge. 1mmunochemical and histochemicallocalization of LDH revealed that its cellular distributiondepends on the position of the cells in the embryo. Moving cellshad higher levels of LDH activity. The lactate dehydrogenaseisozymes appear to play an important role in the regulationof energy metabolism during fish embryogenesis. These gene productsare useful biochemical markers of cellular differentiation andorganogenesis.     

Nuclear pore flow rate of ribosomal RNA and chain growth rate of its precursor during oogenesis of Xenopus laevis

The number of ribosomal RNA molecules which are transferred through an average nuclear pore complex per minute into the cytoplasm (nuclear pore flow rate, NPFR) during oocyte growth of Xenopus laevis is estimated. The NPFR calculations are based on determinations of the increase of cytoplasmic rRNA content during defined time intervals and of the total number of pore complexes in the respective oogenesis stages. In the mid-lampbrush stage (500–700 μm oocyte diameter) the NPFR is maximal with 2.62 rRNA molecules/pore/minute. Then it decreases to zero at the end of oogenesis. The nucleocytoplasmic RNA flow rates determined are compared with corresponding values of other cell types. The molecular weight of the rRNA precursor transcribed in the extrachromosomal nucleoli of Xenopus lampbrush stage oocytes is determined by acrylamide gel electrophoresis to be 2.5 × 10⁶ daltons. From the temporal increase of cytoplasmic rRNA (3.8 μg per oocyte in 38 days) and the known number of simultaneously growing precursor molecules in the nucleus the chain growth rate of the 40 S precursor RNA is estimated to be 34 nucleotides per second.     

Characterization of the O-GlcNAc protein modification in Xenopus laevis oocyte during oogenesis and progesterone-stimulated maturation

Little information exists about single N-acetylglucosamine modifications on proteins in growth and developmental model systems. To explore these phenomena, Xenopus laevis oocytes from stages I-VI of oogenesis were isolated and proteins analyzed on SDS-PAGE. The proteins were probed with antibodies specific for O-GlcNAc. Levels of the O-GlcNAc protein modification were highest in stages I and II, while decreasing in stages III-VI. The reduction in amount of O-GlcNAc-modified proteins was correlated to increases in apparent O-GlcNAcase (streptozotocin-inhibitable neutral hexosaminidase), activity involved in removing protein monoglycosylations. The O-GlcNAc modification was also characterized during progesterone-stimulated oocyte maturation. Although O-GlcNAcase activity appeared relatively constant between quiescent and matured stage VI oocytes, a small decrease in the levels of both total and specific O-GlcNAc-modified proteins was observed. Investigating the function of O-GlcNAc during maturation, oocytes were incubated with compounds known to modulate the levels of the O-GlcNAc protein modification and then stimulated to mature. Oocytes treated with compounds known to increase O-glycosylation consistently matured slower than non-treated controls, while oocytes treated with compounds that decrease O-glycosylation matured slightly faster than controls. The O-GlcNAc modification may play important roles in both the developmental and cell division processes of X. laevis oocytes.     

Dynamics of the oocyte cortical cytoskeleton during oogenesis in Rhodnius prolixus

The oocyte cortex undergoes dramatic changes during oogenesis in Rhodnius prolixus. Despite numerous studies examining oogenesis in the telotrophic ovariole, none has investigated the ultrastructural details of the oocyte cortex, in particular, the lateral cortical cytoskeleton. Indirect immunofluorescent staining of sections, rhodamine phalloidin staining of whole mounts and scanning and transmission EM of permeabilized and unpermeabilized preparations revealed the dynamic changes of the oocyte cortex from early previtellogenesis through to late vitellogenesis. During early previtellogenesis, oocytes 50-150 mum in length have a smooth oolemma, with no discernible cortical cytoskeleton. During mid to late previtellogenesis (oocytes 150-350 mum in length) a tightly woven network of microfilaments and microtubules forms, excluding mitochondria and Golgi complexes from the lateral cortex. At the onset of vitellogenesis, the follicuiar epithelium becomes patent, and there is an increase in microvilli covering the lateral oocyte surface. The microfilament cores form a discrete pattern that corresponds to the imprint of the follicle cells on the oocyte surface. While the lateral microfilament cytoskeleton becomes more elaborate, the lateral microtubule cytoskeleton diminishes, remaining sparse throughout vitellogenesis. The oocyte cortical cytoskeleton undergoes dramatic changes during oogenesis. These cortical dynamics are intricately related to the cellular and molecular processes that occur during oogenesis.     

Changes in zinc,copper and metallothionein contents during oocyte growth and early development of the teleost Danio rerio (zebrafish)

In the present report, we investigated zinc, copper and metallothionein (MT) contents in zebrafish oocytes and embryos. Our results demonstrate that the metal content increases during oocytes maturation. Zinc increases from 30 ng/oocyte (stage-1 oocytes) to 100 ng/oocyte (stage-3 oocytes); copper varied from 1 ng/oocyte (stage-1 oocytes) to 3.5 ng/oocyte (stage-3 oocytes). During embryogenesis, zinc and copper contents dramatically increase after fertilisation around the 512-cells stage, then slowly decrease until the mid-gastrula stage. During oocyte growth, the changes in the MT level are proportional to metal content, whereas during embryogenesis the pattern of MT accumulation does not parallel that of the two metals. Indeed, the maternal pool of MT decreases steadily during the early stages of the development until the gastrula stage. We have examined the effect of cadmium on the expression of MT during zebrafish development. After cadmium exposure, MT content increases in embryos at the blastula stage, whereas no induction occurs in embryos at the gastrula stage. However, pre-treatment of embryos at the gastrula stage with 5-aza-2'-deoxycytidine induces MT synthesis following exposure to cadmium. These observations show that changes in metal levels are not correlated to MT content in the embryo, whereas DNA methylation is one of the factors regulating MT expression.     

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.