Steroids involved with final oocyte maturation in the winter flounder.

A number of androgens and progestogens including 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20-P) were examined in female winter flounder as possible maturation inducing steroids (MIS). During final oocyte maturation serum levels of testosterone (T) and 17 beta-hydroxy-5 beta-androsten-3-one (5 beta-T) peaking at over 200 ng/ml and pregnenolone (PE) at 40 ng/ml were the predominant steroids found from each major group. High levels of T and 5 beta-T were correlated with oocyte stages characterized by germinal vesicle migration. Of the PEs measured, maximum serum levels of PE, 3 beta,17 alpha-hydroxy-5-pregnen-20-one (17-PE) and 3 beta,17 alpha, 20 beta-dihydroxy-5-pregnene (17,20-PE) were found during later oocytes stages associated with germinal vesicle breakdown. Levels of 17,20-P, an established MIS in most fish, were almost non-detectable (less than 0.1 ng/ml serum) in females throughout all stages of final oocyte maturation. Incubations of ovarian follicles in vitro with physiological concentrations of T and 5 beta-T indicated that these steroids could induce all stages of final oocyte maturation. Similar in vitro incubations showed that 17-PE and 17,20-PE were only effective on germinal vesicle breakdown. The principal conclusions are that T, 5 beta-T and the PEs can be considered as MISs in winter flounder and the PE pathway predominates during the final stages of oocyte maturation in winter flounder in contrast to progesterones which predominate in other fish species, mostly salmonids, studies to date.     

泥螺卵子发生的超微结构研究

利用透射电镜观察了泥螺卵子发生过程。结果表明 ,泥螺的卵子发生可划分为卵原细胞、卵黄发生早期、卵黄发生中期及卵黄发生后期卵母细胞 4个时期。卵原细胞核大而圆 ,胞质内分布有少量的线粒体和高尔基囊泡 ,细胞表面具微绒毛。卵黄发生早期的卵母细胞 ,胞质中各类细胞器发达 ,并出现数量较多的类朦胧子。卵黄发生中期的卵母细胞胞体迅速增大 ,核伸出伪足状突起 ,卵质中各种细胞器活动活跃 ,并参与形成卵黄粒和脂滴。此期还可观察到卵母细胞与滤泡细胞间的物质交换现象。卵黄发生后期的卵母细胞体积增至最大 ,细胞器数量减少。本文就卵黄发生前后卵母细胞内部构造的变化、意义及滤泡细胞与卵母细胞蛋白来源间的关系作了探讨     

Morphology of immature bovine oocytes

Bovine cumulus oocyte complexes (COCs) as used for in vitro maturation and fertilization can be classified into different categories by light microscopical inspection. We have distinguished four categories based on compactness and transparency of the cumulus investment and homogeneity and transparency of the ooplasm. The four categories were studied for their morphological characteristics at the ultrastructural level and for their developing capacity in an in vitro maturation system. In categories 1 and 2 oocytes, organelles were evenly distributed. In categories 3 and 4, oocytes organelles were clustered and the distribution of the organelles mimicked the characteristics of oocytes during final maturation. Cumulus cell process endings penetrated the cortex of the oocyte or were located superficial to the cortex of the oocyte. In category 1 oocytes, most of the process endings penetrated the cortex. In category 4 oocytes, most of the process endings did not penetrate. In categories 2 and 3 oocytes, both forms of process endings did occur. After in vitro maturation, only category 4 oocytes showed a decreased developing capacity. Categories 1–3 oocytes showed equal developing capacity in an in vitro maturation system.     

Differential amplification of antifreeze protein genes in the pleuronectinae

Summary The organization of antifreeze protein (AFP) genes in the yellowtail flounder was investigated by Southern blotting and the characterization of clones from a genomic library. This flounder, like the closely related winter flounder, has a set of 10–12 linked but irregularly spaced AFP genes. However, it lacks the tandemly amplified set of 20 such genes that are present in the winter flounder. DNA sequence analysis of a tandemly repeated gene from winter flounder showed that it can code for one of the two most abundant AFP components in the serum. Consistent with this higher AFP gene dosage, the peak serum AFP level in midwinter was 9 mg/ml in the winter flounder and only 4 mg/ml in the yellowtail flounder. A recent amplification of the AFP gene in the winter flounder lineage might be responsible for the higher serum AFP levels in this fish. This increase in gene dosage might have helped the winter flounder colonize the ice-laden, shallow-water niche that it currently occupies along the east coast of North America. Genomic Southern blotting of two other righteye flounders, the smooth flounder and the American plaice, illustrates another example of a differential amplification of AFP genes that correlates with a species' exposure to ice.     

Cytoplasmic reorganization during the resumption of meiosis in cultured preovulatory rat oocytes

Changes in organelle topography and microtubule configuration have been studied during the resumption and progression of meiosis in cultured preovulatory rat oocytes. Germinal vesicle breakdown (GVBD) was reversibly inhibited by dibutyryl cAMP (DcAMP) or nocodazole, a microtubule-disrupting agent. The microtubule stabilizing agent taxol did not inhibit GVBD, but did impair further maturation. The migration of acidic organelles and chromatin in living oocytes was analyzed using the vital stains acridine orange and Hoechst 33258, respectively. Germinal vesicle stage oocytes undergo perinuclear aggregation of acidic organelles during GVBD and these organelles subsequently disperse into the cell cortex as the first meiotic spindle migrates to the oocyte periphery. DcAMP and nocodazole block the perinuclear aggregation of acidic organelles, whereas, in taxol-treated oocytes, organelle aggregation and GVBD occur but the dispersion of acidic organelles was arrested. Dose-response studies on the effects of nocodazole showed that GVBD was generally retarded and that a 50% inhibition of GVBD was achieved at concentrations in excess of 1.0 microM. Concentrations of taxol at 10 microM or above effectively inhibited both chromatin condensation and meiotic spindle formation. Indirect immunofluorescence microscopy with anti-tubulin antibodies revealed dissolution of microtubules with 1.0 microM nocodazole. Taxol had little effect on microtubule organization in germinal vesicle or chromatin condensation stage oocytes; however, when oocytes that had formed first meiotic spindles were treated with taxol, numerous microtubule asters appeared which were preferentially associated with the oocyte cortex. The changes in organelle topography, microtubule configuration, and drug sensitivity are discussed with respect to the regulation of cytoplasmic reorganization during the meiotic maturation of rat preovulatory oocytes.     

Oocyte development in summer flounder: seasonal changes and steroid correlates

Seven ovarian stages are described in summer flounder Paralichthys dentatus. In the prespawning season plasma oestradiol levels increased in maturing fish with lipidogenic oocytes and gonadosomatic index increased in fish undergoing vitellogenesis. Atretic oocytes present in the postspawning season indicated which individuals may have spawned. The pattern of oocyte development is similar to that of other flatfishes and some teleosts. The summer flounder was unusual in having a long lipid uptake phase (oocyte diameter up to 301 μm) prior to any indication of vitellogenin (yolk protein) uptake. This information will be useful in the construction of an updated maturity schedule for the wild population.     

Morphological markers of anteroposterior and dorsoventral polarity in developing oocytes of the hymenopteranCosmoconus meridionator (Ichneumonidae)

Summary The progressive establishment of anteroposterior and dorsoventral polarity in developing oocytes ofCosmoconus meridionator is described. In fully grown oocytes, the asymmetrical (polar) organization is apparent in the localization of the oocyte nucleus (germinal vesicle) and oosome, and in the uneven (graded) distribution of lipid droplets, yolk spheres and specific organelles termed accessory nuclei (AN). The latter structures occur preferentially within the anteroventral periplasm. The developmental significance of AN is discussed.     

Winter Hibernation and UCHL1-p34cdc2 Association in Toad Oocyte Maturation Competence

Currently, it is believed that toad oocyte maturation is dependent on the physiological conditions of winter hibernation. Previous antibody-blocking experiments have demonstrated that toad ubiquitin carboxyl-terminal hydrolase L1 (tUCHL1) is necessary for germinal vesicle breakdown during toad oocyte maturation. In this paper, we first supply evidence that tUCHL1 is highly evolutionarily conserved. Then, we exclude protein availability and ubiquitin carboxyl-terminal hydrolase enzyme activity as factors in the response of oocytes to winter hibernation. In the context of MPF (maturation promoting factor) controlling oocyte maturation and to further understand the role of UCHL1 in oocyte maturation, we performed adsorption and co-immunoprecipitation experiments using toad oocyte protein extracts and determined that tUCHL1 is associated with MPF in toad oocytes. Recombinant tUCHL1 absorbed p34^cdc2, a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13^suc1 was isolated from tUCHL1 with a dependence on the ATP regeneration system, suggesting that still other functions may be involved in their association that require phosphorylation. In oocytes from hibernation-interrupted toads, the p34^cdc2 protein level was significantly lower than in oocytes from toads in artificial hibernation, providing an explanation for the different quantities isolated by recombinant tUCHL1 pull-down and, more importantly, identifying a mechanism involved in the toad oocyte’s dependence on a low environmental temperature during winter hibernation. Therefore, in toads, tUCHL1 binds p34^cdc2 and plays a role in oocyte maturation. However, neither tUCHL1 nor cyclin B1 respond to low temperatures to facilitate oocyte maturation competence during winter hibernation.     

Bucky ball functions in Balbiani body assembly and animal-vegetal polarity in the oocyte and follicle cell layer in zebrafish

The Balbiani body is an evolutionarily conserved asymmetric aggregate of organelles that is present in early oocytes of all animals examined, including humans. Although first identified more than 150 years ago, genes acting in the assembly of the Balbiani body have not been identified in a vertebrate. Here we show that the bucky ball gene in the zebrafish is required to assemble this universal aggregate of organelles. In the absence of bucky ball the Balbiani body fails to form, and vegetal mRNAs are not localized in oocytes. In contrast, animal pole localized oocyte markers are expanded into vegetal regions in bucky ball mutants, but patterning within the expanded animal pole remains intact. Interestingly, in bucky ball mutants an excessive number of cells within the somatic follicle cell layer surrounding the oocyte develop as micropylar cells, an animal pole specific cell fate. The single micropyle permits sperm to fertilize the egg in zebrafish. In bucky ball mutants, excess micropyles cause polyspermy. Thus bucky ball provides the first genetic access to Balbiani body formation in a vertebrate. We demonstrate that bucky ball functions during early oogenesis to regulate polarity of the oocyte, future egg and embryo. Finally, the expansion of animal identity in oocytes and somatic follicle cells suggests that somatic cell fate and oocyte polarity are interdependent.     

Identification of two forms of vitellogenin-derived phosvitin and elucidation of their fate and roles during oocyte maturation in the barfin flounder, Verasper moseri

A new method for visualizing small and multiple phosvitins (Pvs) in oocytes from a marine teleost was developed by a combination of gel filtration, alkaline phosphatase treatment, and SDS-PAGE followed by silver staining. Three distinct Pv polypeptides having molecular masses of 15 kDa, 8 kDa, and 7 kDa were visualized in vitellogenic follicle extract of barfin flounder, Verasper moseri. N-terminal amino acid sequencing identified two different N-termini that fell into the PvA (7 kDa) and PvB (15 kDa and 8 kDa) groups, which were derived from two forms of vitellogenin (Vg), VgA and VgB, respectively. Analysis of time-course change in phosphorus-rich peaks of gel chromatography fractions of follicle extracts from different maturational stages demonstrated a rapid degradation of Pvs during mid-phase of oocyte maturation. Quantitative analysis of free amino acids in maturing follicles revealed an increment of serine content but not of phosphoserine, indicating the occurrence of dephosphorylation concomitant with Pv degradation. Measurement of phosphatase activity in follicles and eggs at different maturational stages demonstrated a significant activation of phosphatase especially under acidic conditions. This suggested that Pv degradation and dephosphorylation are regulated by changes in ooplasm pH during oocyte maturation. Our results also suggested that the Pvs in barfin flounder vitellogenic oocytes bind to much lower amounts of calcium and magnesium than those of masu salmon, Oncorhynchus masou. This indicates that the Pvs in the barfin flounder, a marine teleost spawning its eggs in seawater, do not play a role in the transport and deposition of calcium and magnesium into oocytes.     

Developmental and ultrastructual characteristics of mouse oocytes grown <Emphasis Type="Italic">in Vitro</Emphasis> from primordial germ cells

The aim of this study was to clarify the developmental and ultrastructual characteristics of oocytes grown in vitro from primordial germ cells. The female genital ridges at 12.5 days post coitus were cultured for 18 days on an insert membrane in Waymouth's MB752/1 medium, supplemented with 15% fetal bovine serum and 1 mM sodium pyruvate; subsequently, the follicles isolated from the tissue were cultured for eight days in Waymouth's medium supplemented with 5 microg/ml insulin, 5 microg/ml transferrin, 5 ng/ml selenium, 10 mIU/ml follicle stimulating hormone, and 100 ng/ml stem cell factor. The primordial germ cells developed in vitro into oocytes of more than 60 microm in diameter. The transmission electron microscopic analysis indicated that the oocytes, which developed in vitro, showed no obvious abnormality in their ultrastructure and had organelles appropriate for the oocyte size. However, a delay in the progressive changes of morphology in some of the organelles during oocyte growth was often found when comparing them to oocytes grown in vivo.     

Sperm Nuclear Transformation and Aster Formation Related to Metabolic Behaviour in Amphibian Eggs

Bufo arenarum oocyte maturation is related to important modifications in the metabolism of carbohydrates. Such changes involve a different relative participation of the main oxidative routes and are under the influence of seasonal variations of the pituitary activity.
Ovulated coelomic oocytes obtained during the winter period, were unable to initiate cleavage after injection of a sperm suspension. The extent of sperm head transformation and aster formation in the cytoplasm of oocytes with a different metabolic behaviour (obtained during the winter and summer periods) were studied.
Our results show that sperm nuclear transformation and DNA synthesis were quite similar in both types of oocytes. In contrast to summer oocytes, in which the pronucleus was sourranded by an aster, no aster structure was formed in winter ooctyes notwithstanding pronucleus formation occurred.
These results suggest that the failure to develop aster may explain the lack of cleaving in winter oocytes. It appears that the metabolic changes, aster formation and the capability to cleave are closely related and could be dissociated from oocyte nuclear maturation in this species.     

The occurrence of intercellular bridges during oogenesis in the mouse

A fine structural analysis of fetal mouse ovaries reveals the presence of intercellular bridges between developing oocytes. These bridges, which connect two or more oocytes, are most frequently seen prior to the dictyate stage of meiotic prophase. The intercellular connections are limited by a tri-laminar membrane which is continuous with the oocyte plasmalemma. A characteristic feature of all bridges is the presence of an electron-dense material on the cytoplasmic side of the limiting membrane. Since this dense material is a constant and conspicuous component of the entire bridge, identification of these connections is possible in all planes of section. In cross section, the bridges are usually cylindrical, while in longitudinal section, a variety of configurations are observed. Oocytes connected by intercellular bridges exhibit a highly developed Golgi complex which is frequently localized in the region of the cytoplasmic continuities. Vesicular elements, apparently derived from the Golgi, are routinely observed within the boundaries of the bridges. Other cytoplasmic organelles, including rough and smooth endoplasmic reticulum, free ribosomes and mitochondria, are also seen in these bridges. The presence of these vesicles and organelles within intercellular bridges suggests that these connections may provide a means for transfer of organelles and other substances from one oocyte to another. It may be, therefore, that intercellular bridges are important for the nourishment and maturation of certain selected oocytes as well as for the synchronization of meiotic events.     

A-kinase anchor proteins as potential regulators of protein kinase A function in oocytes

In the mammalian oocyte, the cAMP-dependent protein kinase (PKA) has critical functions in the maintenance of meiotic arrest and oocyte maturation. Because PKA is spatially regulated, its localization was examined in developing oocytes. Both regulatory subunits (RI and RII) and the catalytic subunit (C) of PKA were found in oocytes and metaphase II-arrested eggs. In the oocyte, RI and C were predominantly localized in the cortical region, while RII showed a punctate distribution within the cytoplasm. After maturation to metaphase II, RI remained in the cortex and was also localized to the meiotic spindle, while RII was found adjacent to the spindle. C was diffuse within the cytoplasm of the egg but was enriched in the cytoplasm surrounding the metaphase spindle, much like RII. The polarized localization and redistribution of RI, RII, and C suggested that PKA might be tethered by A-kinase anchor proteins (AKAPs), proteins that tether PKA close to its physiological substrates. An AKAP, AKAP140, was identified that was developmentally regulated and phosphorylated in oocytes and eggs. AKAP140 was shown to be a dual-specific AKAP, having the ability to bind both RI and RII. By compartmentalizing PKA, AKAP140 and/or other AKAPs could spatially regulate PKA activity during oocyte development.     

Cathepsin B-mediated yolk protein degradation during killifish oocyte maturation is blocked by an H+-ATPase inhibitor: effects on the hydration mechanism

In teleost oocytes, yolk proteins (YPs) derived from the yolk precursors vitellogenins are partially cleaved into free amino acids and small peptides during meiotic maturation before ovulation. This process increases the osmotic pressure of the oocyte that drives its hydration, which is essential for the production of buoyant eggs by marine teleosts (pelagophil species). However, this mechanism also occurs in marine species that produce benthic eggs (benthophil), such as the killifish (Fundulus heteroclitus), in which oocyte hydration is driven by K+. Both in pelagophil and benthophil teleosts, the enzymatic machinery underlying the maturation-associated proteolysis of YPs is poorly understood. In this study, lysosomal cysteine proteinases potentially involved in YP processing, cathepsins L, B, and F (CatL, CatB, and CatF, respectively), were immunolocalized in acidic yolk globules of vitellogenic oocytes from the killifish. During oocyte maturation in vitro induced with the maturation-inducing steroid (MIS), CatF disappeared from yolk organelles and CatL became inactivated, whereas CatB proenzyme was processed into active enzyme. Consequently, CatB enzyme activity and hydrolysis of major YPs were enhanced. Follicle-enclosed oocytes incubated with the MIS in the presence of bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, underwent maturation in vitro, but acidification of yolk globules, activation of CatB, and proteolysis of YPs were prevented. In addition, MIS plus bafilomycin A1-treated oocytes accumulated less K+ than those stimulated with MIS alone; hence, oocyte hydration was reduced. These results suggest that CatB is the major protease involved in yolk processing during the maturation of killifish oocytes, whose activation requires acidic conditions maintained by a vacuolar-type H+-ATPase. Also, the data indicate a link between ion translocation and YP proteolysis, suggesting that both events may be equally important physiological mechanisms for oocyte hydration in benthophil teleosts.     

Preliminary ultrastructural and autoradiographic evidence that the trophonema of the sea anemone Actinia fragacea has a nutritive function

The developing oocyte of the sea anemone Actinia fragacea is associated with a distinct group of gonad epithelial cells which constitute the trophonema. Electron microscopy has shown that the cells of the trophonema extend through a pore in the mesoglea which surrounds the oocyte, and make intimate contact with the oocyte surface. The ooplasm beneath this region of contact differs from the rest of the oocyte in containing numerous small vesicles, but few yolk granules or other organelles. Light microscope autoradiography has shown that oocytes within gonads can take up and incorporate tritiated glucose and leucine from solution. The cells of the trophonema appear more active in precursor incorporation than other gonad epithelial cells. The evidence therefore suggests that these cells have a nutritive function during oogenesis.     

Mouse early oocytes are transiently polar: three-dimensional and ultrastructural analysis

The oocytes of many invertebrate and non-mammalian vertebrate species are not only asymmetrical but also polar in the distribution of organelles, localized RNAs and proteins, and the oocyte polarity dictates the patterning of the future embryo. Polarily located within the oocytes of many species is the Balbiani body (Bb), which in Xenopus is known to be associated with the germinal granules responsible for the determination of germ cell fate. In contrast, in mammals, it is widely believed that the patterning of the embryo does not occur before implantation, and that oocytes are non-polar and symmetrical. Although the oocytes of many mammals, including mice and humans, contain Bbs, it remains unknown how and if the presence of Bbs relates to mouse oocyte and egg polarity. Using three-dimensional reconstruction of mouse neonatal oocytes, we showed that mouse early oocytes are both asymmetrical and transiently polar. In addition, the specifics of polarity in mouse oocytes are highly reminiscent of those in Xenopus early oocytes. Based on these findings, we conclude that the polarity of early oocytes imposed by the position of the centrioles at the cytoplasmic bridges is a fundamental and ancestral feature across the animal kingdom.     

东方扁虾卵子发生的超微结构

根据卵细胞的形态、内部结构特征及卵母细胞与滤泡细胞之间的关系,东方扁虾的卵子发生可划分为卵原细胞、卵黄发生前卵母细胞、卵黄发生卵母细胞和成熟卵母细胞等四个时期。卵原细胞胞质稀少,胞器以滑面内质网为主。卵黄发生前卵母细胞核明显膨大,特称为生发泡;在靠近核外膜的胞质中可观察到核仁外排物。卵黄发生卵母细胞逐渐为滤泡细胞所包围;卵黄合成旺盛,胞质中因而形成并积累了越来越多的卵黄粒。东方扁虾卵母细胞的卵黄发生是二源的。游离型核糖体率先参与内源性卵黄合成形成无膜卵黄粒。粗面内质网是内源性卵黄形成的主要胞器。滑面内质网、线粒体和溶酶体以多种方式活跃地参与卵黄粒形成。卵周隙内的外源性物质有两个来源:滤泡细胞的合成产物和血淋巴携带、转运的卵黄蛋白前体物。这些外源性物质主要通过质膜的微吞饮作用和微绒毛的吸收作用这两种方式进入卵母细胞,进而形成外源性卵黄。内源性和外源性的卵黄物质共同参与成熟卵母细胞中富含髓样小体的卵黄粒的形成。卵壳的形成和微绒毛的回缩被认为是东方扁虾卵母细胞成熟的形态学标志。