Dose-dependent relationship between oocyte cytoplasmic volume and transformation of sperm nuclei to metaphase chromosomes

We have studied the chromosome condensation activity of mouse oocytes that have been inseminated during meiotic maturation. These oocytes remain unactivated, and in those penetrated by up to three or four sperm, each sperm nucleus is transformed, without prior development of a pronucleus, into metaphase chromosomes. However, those penetrated by more than four sperm never transform any of the nuclei into metaphase chromosomes (Clarke, H. J., and Y. Masui, 1986, J. Cell Biol. 102:1039-1046). We report here that, when the cytoplasmic volume of oocytes was doubled or tripled by cell fusion, up to five or eight sperm nuclei, respectively, could be transformed into metaphase chromosomes. Conversely, when the cytoplasmic volume was reduced by bisection of oocytes after the germinal vesicle (GV) had broken down, no more than two sperm could be transformed into metaphase chromosomes. Thus, the capacity of the oocyte cytoplasm to transform sperm nuclei to metaphase chromosomes was proportional to its volume. The contribution of the nucleoplasm of the GV and the cytoplasm outside the GV to the chromosome condensation activity was investigated by bisecting oocytes that contained a GV and then inseminating the nucleate and anucleate fragments. The anucleate fragments never induced sperm chromosome formation, indicating that GV nucleoplasm is required for this activity. In the nucleate fragments, the capacity to induce sperm chromosome formation was reduced as compared with whole oocytes, in spite of the fact that the fragments contained the entire GV nucleoplasm. This implies that non-GV cytoplasmic material also was required for chromosome condensation activity. When inseminated oocytes were incubated in the presence of puromycin, the sperm nuclei were transformed into interphase-like nuclei, but no metaphase chromosomes developed. However, when protein synthesis resumed, the interphase nuclei were transformed to metaphase chromosomes. These results suggest that the transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of mouse oocytes requires both the nucleoplasm of the GV and non-GV cytoplasmic substances, including proteins synthesized during maturation.     

Lectin-mediated uptake of lysosomal hydrolases by genetically deficient human fibroblasts.

A protein factor named S-II that stimulates RNA polymerase II was previously purified from Ehrlich ascites tumor cells [1]. In this work using an antibody prepared against purified S-II, the localization of S-II in the cell was investigated by an indirect immunofluorescence technique. In 3T3 cells, specific immunofluorescence was detected only in the nucleoplasm where RNA polymerase II is located, and not in the nucleoli where RNA polymerase I is present. In Ehrlich ascites tumor cells fluorescence was detected mainly in the nucleoplasm, although some fluorescence was also detectable in the cytoplasm, possibly due to leak of S-II from the nuclei during preparation of the immunofluorescent samples. In metaphase cells fluorescent was not found on chromosomes but throughout the cytoplasm. These findings suggest that S-II is a nuclear protein and that it spreads into the cytoplasm without being attached to chromosomes in metaphase, but is reassembled into the nucleoplasm in the interphase. Specific immunofluorescence was also detected in the nuclei of HeLa cells and salivary glands cells of flesh-fly larvae, suggesting that the nucleoplasm of these heterologous cells contains proteins immunologically cross-reactive with the antibody against S-II.     

Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.     

Control of chromosome behavior in amphibian oocytes. II. The effect of inhibitors of RNA and protein synthesis on the induction of chromosome condensation in transplanted brain nuclei by oocyte cytoplasm

We studied the effects of actinomycin D, alpha-amanitin, puromycin, and cycloheximide on the cytoplasmic activity of maturing Rana pipiens oocytes that induces chromosome condensation in transplanted brain nuclei. Treatment of oocytes with each inhibitor suppressed the chromosome condensation induced by metaphase oocytes to varying degrees depending upon the dose of inhibitor, despite the fact that untreated metaphase I oocytes already possessed chromosome condensation activity (CCA). Treatment of brain nuclei before injection completely suppressed condensation at all doses used. Chromosome condensation induced by metaphase II oocyte cytoplasm, however, was insensitive to all the inhibitors, even when the brain nuclei were pretreated. Oocytes treated with alpha-amanitin throughout maturation induced chromosome condensation when tested at metaphase II. Removal of the oocyte chromosomes after the germinal vesicle (GV) broke down did not prevent the development of CCA, whereas removal of the entire GV before initiation of maturation deprived oocytes of CCA. The results suggest that metaphase I oocyte cytoplasm stimulates synthesis of brain nuclear RNAs that are translated into proteins necessary for chromosome condensation, whereas metaphase II oocytes possess all the factors for chromosome condensation. In both cases, GV nucleoplasm appears indispensable for the development of CCA, whereas immediate activity of the oocyte genome is not required.     

Changes in nuclear localization of An3, a RNA helicase,during oogenesis and embryogenesis in Xenopus laevis

The immunolocalization of An3 protein, an ATP-dependent RNA helicase and a member of the DEAD box family, was compared with the localization of fibrillarin, a protein essential for rRNA processing, and snRNPs, which are involved in mRNA splicing reactions, during oogenesis and embryogenesis in Xenopus laevis. Although An3 protein was detected in the cytoplasm of all stages of oocytes, in most stages An3 protein was also present in the nucleus. Prior to stage I An3 protein was uniformly dispersed throughout the entire germinal vesicle; from stages I to V it was in nucleoli. By stage VI nucleolar labeling with anti An3 disappeared and the protein was no longer present within nuclei. An3 reactivity was also present throughout the nuclei of follicle cells surrounding prestage I to stage VI oocytes. Both cytoplasmic and nuclear An3 staining were present in cells of stages 8 to 35 embryos; however, nuclear staining was punctate and uniformly distributed throughout the nucleoplasm. Fibrillarin was diffusely distributed throughout the entire germinal vesicle prior to stage I, localized exclusively to nucleoli of oocytes between stages I and VI and in nucleoli of stages 12 and 35 embryonic cells. Reactivity for snRNPs (anti-Sm) in germinal vesicles of prestage I oocytes was diffuse, and similar to the distribution of An3 and fibrillarin; in later stage oocytes anti-Sm staining was restricted to a population of granules, much fewer in number and more heterogeneous in size than nucleoli. Anti-Sm activity was apparent in nuclei of embryonic cells of stages 8 to 35 embryos. Although colocalization of the Sm epitope and An3 was not observed in developing oocytes and in embryonic cells, Sm reactive material was frequently found in close association with An3-positive nucleoli (oocytes) and nuclear deposits (embryonic cells). In stage IV and V oocytes treated with actinomycin D (4 μg/ml) to inhibit rRNA synthesis, nucleoli, which continued to possess fibrillarin, lacked An3; staining of follicle cell nuclei for An3 was unchanged. Treatment with 200 μg/ml actinomycin D to block mRNA synthesis, inhibited An3 but not fibrillarin staining in nuclei of prestage I oocytes and follicle cells. The changing patterns of An3 reactivity and the differential effects of actinomycin D on such localizations observed here are consistent with a role for An3 in the processing/production of RNA. © 1996 Wiley-Liss, Inc.     

肌动蛋白存在于车蝗精母细胞核和染色体中

以兔抗肌动蛋白抗体为一抗,FITC偶联的羊抗兔IgG抗体为二抗进行间接免疫荧光实验,观察到车蝗（Oedaleus asiaticus）精母细胞核及减数分裂Ⅰ细线期、终变期、减数分裂Ⅱ中期染色体上均发出明亮的黄经发色荧光,说明其中含有肌动蛋白。本文结果证明肌动蛋白是车蝗减数分裂细胞核和染色体的组成成分。     

Effects of cyclic AMP on the spontaneous meiotic maturation of cumulus-free bovine oocytes cultured in chemically defined medium

The rate of spontaneous meiotic maturation and the period of commitment to this process were determined in bovine oocytes devoid of surrounding cumulus cells, cultured in chemically defined medium with bovine serum albumin in the absence of serum. The effects of compounds that are known to elevate levels of intracellular cyclic adenosine monophosphate (cAMP) on the resumption and progression of meiosis were investigated. Bovine oocytes were mass-harvested, denuded of cumulus cells, and cultured in 2A-BMOC medium supplemented with 0.5% bovine serum albumin. Intracellular cAMP levels were indirectly modified using 8-bromo-cAMP, dibutyryl cAMP (dbcAMP), forskolin, or 3-isobutyl-1-methyl xanthine (IBMX). Meiotic maturation was scored cytogenetically. Ninety percent of denuded bovine oocytes mature after 24 h, with 65% progressing beyond anaphase I. These oocytes remain at the germinal vesicle (GV) stage for up to 8 h in culture. GV breakdown (GVBD) occurs in 40.5% of oocytes at 9 h. The peak times for the different meiotic stages were 12 h for diakinesis, 15 h for late diakinesis to metaphase I, 20 h for metaphase I, and 24 h for telophase I. By 48 h, most had reached metaphase II. There is a 2-h lag period between the time at which they become irreversibly committed to mature (at 7 h) and when they demonstrate GVBD (at 9 h). Incubation for 12 h with high concentrations of 8-bromo-cAMP and forskolin significantly inhibited GVBD, while the effect of dbcAMP was similar but less pronounced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytoskeletal organization of rat oocytes during metaphase II arrest and following abortive activation: A study by confocal laser scanning microscopy

In metaphase II arrested rat oocytes (M il), microtubles were found in the taper-shaped meiotic spindle and in the cytoplasm as asters and free microtubules. Whereas spindle microtubules were acetylated, those located in the cytoplasm were not. Cytoplasmic microtubules were also labile as assessed by mild cooling. In contast to mouse oocytes, rat microtubule organizing centers (MTOCs) did not react with MPM-2 antibody by immunofluorescence despite the fact that this antibody reacts with several proteins as shown by immunoblot. However, cytoplasmic MTOCs in M II-arrested rat oocytes could be detected by their nucleating capacity in the presence of taxol, a drug that induced the formation of numerous cytoplasmic asters. In addition, taxol caused a change in the spindle shape and the formation of astral microtubules at the spindle poles. Meiotic spindles (as well as chromosomes devoid of microtubules after nocodazoletreatment) were overlaid by an actin-rich domain. Spontaneous abortive activation led to the extrusion of the second polar body followed by another metaphase arrest— metaphase III; however, normal spindles did not form and dispersed chromosomes surrounded by microtubles were observed. Electron microscopic studies confirmed these observations and revealed that the kinetochores are located deep within the chromosomes in contrast to mouse kinetochores, and this might be responsible for the absence of a metaphase III spindle in the rat oocyte. Induced activation caused transition to interphase with the formation of a characteristic microtubule network. This study shows that there are several significant differences in the cytoskeletal organization of rat and mouse oocytes. © 1993 Wiley-Liss, Inc.     

Structural changes in oocyte nucleoli ofXenopus laevis during oogenesis and meiotic maturation

Immunoelectron microscopy with anti-nucleolin defined substructures within the multiple nucleoli of biosynthetically active stage II–III oocytes and within the nucleoli of relatively quiescent stage VI oocytes of Xenopus laevis. Dense fibrillar components (DFCs) of nucleoli from stage II–III oocytes consisted of nucleolonemas that radiated from a continuous DFC sheath surrounding fibrillar centers (FCs). Discernible granular regions (GRs) were absent in these same nucleoli. Conversely, stage VI oocyte nucleoli displayed compacted DFCs and prominent GRs. Immunofluorescence microscopy then tracked fibrillarin, nucleolin, and condensed DNA through oogenesis and into progesterone-induced meiotic maturation and nuclear breakdown. In stage II–III oocyte nucleoli, fibrillarin was enriched near the FC-DFC boundaries, while nucleolin was distributed throughout these same DFCs. Both proteins were enriched within the compacted DFCs of stage VI oocyte nucleoli. Staining with (DAPI) 4′,6-diamidino-2-phenylindole showed condensed DNA within nucleolar FCs of both stage II–III and stage VI oocyte. Upon nuclear breakdown, we found fibrillarin and nucleolin in small particles and in the surrounding cytoplasm. Although we saw no trace of fibrillarin or nucleolin in nuclear remnants prepared just minutes later, DAPI-stained particles remained within these preparations, thus suggesting that FCs were at least slow to disassemble. Received: 18 March 1996 / Accepted: 16 April 1996     

Ultrastructural localization of rRNA in HeLa cells, rat liver cells and Xenopus laevis oocytes by means of the monoclonal antibody--protein a--gold technique

Summary The postembedding localization of rRNA was investigated in ultrathin sections of HeLa cells, rat liver andXenopus laevis oocytes by means of the monoclonal antibody to rRNA and protein A-gold technique. The incidence of gold particles was highest in nucleoli and cytoplasmic areas containing ribosomes. The chromosomes were labelled less than the surrounding cytoplasm in mitotic HeLa cells. In nucleoli of HeLa cells and rat hepatocytes, the labelling of areas containing ribonucleoprotein components was greater than the labelling of fibrillar centres. In segregated nucleoli ofX. laevis oocytes, the labelling of the granular region substantially exceeded that of the fibrillar regions. The incidence of nucleoplasmic gold particles in interphasic HeLa cells was found to be slightly increased in the vicinity of nucleoli. The labelling of clusters of interchromatin granules in rat hepatocytes was not significantly different from that of the rest of the nucleophasmic interchromatin spaces.A part of this study was presented as the poster and abstract at the 8th European Congress on Electron Microscopy 1984 in Budapest.     

Chromosome configuration during in vitro maturation of goat, sheep and buffalo oocytes

The competence of meiotic chromosome configuration at the time of co-culture of oocytes with spermatozoa is an essential prerequisite for successful in vitro fertilization (IVF). Although this technology has been used in several livestock species, various intrinsic and extrinsic factors affecting the high repeatablity of IVF have yet to be understood. The present study was conducted to determine the appropriate time for coculture of oocytes and spermatozoa in order to optimize the fertilization rate in sheep, goats and buffalo. Oocytes were collected from the ovaries of slaughtered animals. The oocytes were divided into 10 groups and cultured for maturation in TCM-199 supplemented with estrous cow serum for different durations at 38.5 x 0.5/C in a CO(2) incubator. Sheep and goat oocytes were removed from culture medium after 0,6,12,22,24,26,28,30,32 and 36 and buffalo oocytes after 0,6,12,16,20,22,24,26,28, and 36 h. The oocytes were treated with hypotonic solution (0.75 M KCl) and fixed in Carony's fixative on glass slides. The fixed oocytes were stained with Giemsa solution, and the meiotic chromosomes were evaluated under a compound microscope at x 1000 magnification. Observations were recorded on a total of 1328 oocytes (sheep, 409; goat, 727 and buffalo, 192). The sequential configurations of diffused chromatin, pachytene, diplotene (along with nucleoli), diakinesis and metaphase II (MII) were analyzed at different durations of culture. Control oocytes (fixed at 0 h without incubation) were mostly at the pachytene stage, and as the duration of culture increased the instances of diplotene, diakinesis and finally MII increased. Oocytes at the MII stage of meiosis are known to be at the optimal stage of development for co-culture with spermatozoa and successful in vitro fertilization. On the basis of sequential configuration of chromosomes, it was found that the optimal duration of in vitro maturation of oocytes is 32, 30 and 24 h for sheep, goats and buffalo, respectively.     

Mechanical continuity and reversible chromosome disassembly within intact genomes removed from living cells

Chromatin is thought to be structurally discontinuous because it is packaged into morphologically distinct chromosomes that appear physically isolated from one another in metaphase preparations used for cytogenetic studies. However, analysis of chromosome positioning and movement suggest that different chromosomes often behave as if they were physically connected in interphase as well as mitosis. To address this paradox directly, we used a microsurgical technique to physically remove nucleoplasm or chromosomes from living cells under isotonic conditions. Using this approach, we found that pulling a single nucleolus or chromosome out from interphase or mitotic cells resulted in sequential removal of the remaining nucleoli and chromosomes, interconnected by a continuous elastic thread. Enzymatic treatments of interphase nucleoplasm and chromosome chains held under tension revealed that mechanical continuity within the chromatin was mediated by elements sensitive to DNase or micrococcal nuclease, but not RNases, formamide at high temperature, or proteases. In contrast, mechanical coupling between mitotic chromosomes and the surrounding cytoplasm appeared to be mediated by gelsolin-sensitive microfilaments. Furthermore, when ion concentations were raised and lowered, both the chromosomes and the interconnecting strands underwent multiple rounds of decondensation and recondensation. As a result of these dynamic structural alterations, the mitotic chains also became sensitive to disruption by restriction enzymes. Ion-induced chromosome decondensation could be blocked by treatment with DNA binding dyes, agents that reduce protein disulfide linkages within nuclear matrix, or an antibody directed against histones. Fully decondensed chromatin strands also could be induced to recondense into chromosomes with pre-existing size, shape, number, and position by adding anti-histone antibodies. Conversely, removal of histones by proteolysis or heparin treatment produced chromosome decondensation which could be reversed by addition of histone H1, but not histones H2b or H3. These data suggest that DNA, its associated protein scaffolds, and surrounding cytoskeletal networks function as a structurally-unified system. Mechanical coupling within the nucleoplasm may coordinate dynamic alterations in chromatin structure, guide chromosome movement, and ensure fidelity of mitosis. J. Cell. Biochem. 65:114–130. © 1997 Wiley-Liss, Inc.     

Sperm chromatin acquires an activity that induces microtubule assembly during residence in the cytoplasm of metaphase oocytes of the mouse

Evidence from several cell types indicates that chromatin can induce microtubule assembly in its vicinity. To determine whether this activity is present in sperm chromatin, whose biochemical composition differs from somatic chromatin, mouse oocytes that were undergoing meiotic maturation were inseminated. Maturing oocytes are not activated by sperm penetration but remain arrested at metaphase. The sperm chromatin within the oocyte cytoplasm initially became dispersed and later, under the influence of oocyte cytoplasmic factors, recondensed into a small mass of individual chromosomes. When inseminated oocytes were processed for immunofluorescence using an anti--tubulin antibody, microtubules were never associated with dispersed sperm chromatin, although the chromosomes of the oocyte were arranged on a spindle. In contrast, microtubules were associated with the majority of sperm nuclei that had become recondensed, and were frequently arranged into a spindle-like structure. When oocytes had been penetrated by more than three sperm, most sperm nuclei remained at the dispersed chromatin stage and these were never associated with microtubules. Exposure of polyspermic oocytes to taxol, which promotes microtubule assembly, failed to induce microtubule assembly around dispersed sperm chromatin. Exposure of monospermic oocytes to nocodazole, which inhibits tubulin polymerization, prevented resolution of the recondensed sperm chromatin into individual chromosomes. These results suggest that sperm chromatin lacks an activity that can induce local microtubule assembly, and that it acquires this activity once modified by oocyte cytoplasmic factors.     

Distribution of c-myc, c-myb, and Ki-67 antigens in interphase and mitotic human cells evidenced by immunofluorescence staining technique

Using specific antibodies and the immunofluorescence staining technique we found a similar subcellular distribution pattern of the cellular proto-oncogene proteins c-myc and c-myb in interphase and mitotic HL60 and Molt4 cells. Antibodies against c-myc as well as those against c-myb protein gave rise to a nuclear staining excluding the nucleoli. In mitotic cells both proteins are apparently not associated with the chromatin of the condensed chromosomes, but appear diffusely distributed throughout the cytoplasm. In contrast, immunostaining using the proliferation marker antibody Ki-67 yielded in both cell lines several prominent specks in the nucleus and a weak finely dispersed staining throughout the nucleoplasm. No fluorescence was detectable in the cytoplasm. In dividing cells Ki-67 immunofluorescence was found to be associated with the surface of the chromosomes. The functional significance of the different localizations of the proteins is discussed in light of what is currently known about nuclear antigens.     

Ultrastructure of bovine oocytes exposed to Taxol prior to OPS vitrification

Our objective was to document potential subcellular consequences of treatment with the microtubule stabilizer Taxol with or without subsequent vitrification of cow and calf oocytes by the open pulled straw (OPS) method. Oocytes were divided into four experimental groups for cows and four groups for calves: (1) a control group fixed immediately after maturation; (2) an OPS group cryopreserved by conventional OPS; (3) a Taxol/CPA group exposed to 1 microM Taxol and cryoprotective agents (CPAs); and (4) a Taxol/OPS group vitrified by OPS including 1 microM Taxol to the vitrification solution. All oocytes were processed for light and transmission electron microscopy. The main injuries were observed on the metaphase plate and the spindle. In control oocytes, the metaphase appeared as condensed chromosomes arranged in a well-organized metaphase plate and the spindle showed well organized microtubules in both cow and calf oocytes. However, in cow OPS oocytes, the metaphase plate was disorganized into scattered chromosomes or the chromosomes were condensed into a single block of chromatin. In addition, microtubules were not organized as typical spindles. In contrast, cow Taxol/OPS oocytes as well as both cow and calf Taxol/CPAs oocytes showed well-organized metaphase plates and normal spindle morphology. All calf OPS and calf Taxol/OPS oocytes displayed a single block of chromatin and no microtubules could be observed around the chromosomes. In conclusion, treatment with 1 microM Taxol before and during vitrification did not induce adverse changes in the oocyte cytoplasm or metaphase spindles in adult bovine oocytes, but stabilized the metaphase and spindle morphology.     

Ovarian nests in cultured Russian sturgeon Acipenser gueldenstaedtii and North American paddlefish Polyodon spathula comprised of previtellogenic oocytes

Ovarian nests in the ovaries of sexually maturing Russian sturgeon Acipenser gueldenstaedtii and North American paddlefish Polyodon spathula were investigated. They comprised early previtellogenic, diplotene stage oocytes and somatic cells. In the nucleoplasm, these oocytes contained chromatin in the form of grains, threads and lampbrush chromosomes, primary nucleoli and multiple nucleoli. Two stages of oocytes in nests were distinguished by differences in the distribution of mitochondria with distorted cristae and lipid droplets in the ooplasm. These stages were as follows: pre‐early stage 1 (PE 1) and early stage 1 (EP 1) previtellogenic oocytes. In PE 1 oocytes few mitochondria with distorted cristae and lipid droplets were distributed randomly. The ooplasm of PE 1 oocytes was not differentiated into homogeneous and granular compartments. In EP 1 oocytes, mitochondria with distorted cristae were more numerous and grouped in the vicinity of the nucleus, lipid droplets accumulated near these mitochondria. In the nucleoplasm of EP 1 oocytes several low electron‐dense spherical bodies, possibly Cajal bodies, were present.     

Actin synthesis is not regulated by granulosa cells in mouse growing and preovulatory oocytes

The synthesis and intracellular distribution of actin were studied in isolated dictyate and metaphase II mouse oocytes by (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of newly synthetized oocyte protein and (2) cytochemical F-actin labeling by fluorescent phalloidin. Unpermeabilized, fully grown oocytes bound phalloidin intensely at the level of the zona pellucida (ZP), such ZP-associated actin representing a significant portion of total actin found in these cells. In contrast, phalloidin binding to ZP was very low in growing oocytes and was undetectable in ovulated, metaphase II eggs. When ZP-associated actin of fully grown oocytes was removed by prolongedly exposing oocytes to α-chymotrypsin, the amount of newly synthesized actin displayed by cumulus-enclosed oocytes was reduced to a level comparable to that shown by oocytes isolated from granulosa cells. We demonstrate that ZP-associated actin belongs to granulosa cell processes that remain within the ZP as a consequence of oocyte isolation procedures. We conclude that actin synthesis of mouse oocytes is not regulated by granulosa cells.     

Die Entstehung multipler Oocytennukleolen aus akzessorischen DNS-Körpern bei Gryllus domesticus

The early stages of female and male germ cells have been investigated in Feulgen squash preparations, in unfixed state with phase contrast optics and in the electron microscope. The DNA axes of the ring-shaped multiple nucleoli in the growing oocytes of Gryllus arise from compact DNA bodies which are found in oogonia of young larvae and in oocytes prior to the growth period. The nuclei of the early oogonia contain several little DNA bodies whereas young oocytes at leptotene, zygotene and pachytene have only one body which is bigger than at earlier stages (Pig. 3). At metaphase and anaphase during oogonial mitosis the DNA body has a filamentous shape distinguishable from the compact chromosomes (Fig. 5). In oogonia as well as at leptotene and zygotene stages, nucleoli are produced in the peripheral, uncoiled parts of each DNA body whereas the compact interior is completely free of nucleolar material (Figs. 4, 12). At pachytene, the whole DNA body begins to despiralize, and single DNA strands are released into the nucleoplasm. These strands form hundreds of multiple nucleoli which finally are dispersed in the germinal vesicle (Fig. 11). — Incorporation studies with radio-active thymidine have shown that DNA synthesis in the DNA body is not synchronous with the S-phase of the chromosomes (Fig. 7). — The DNA body is an own formation distinct from the sex chromosomes (in contrast to the opinion of Sotelo and Wettstein, 1964). Although the positive heteropycnotic X-chromosome in the germ cells of the male cricket is very similar to the DNA body of the female (Fig. 8), there is no regular contact between sex chromosome and nucleolus neither in spermatogonia nor in spermatocytes (Figs. 9, 14). In all probability, the site of the nucleolar organizer is autosomal. — It is suggested that the amplification of the nucleolar genes in Gryllus oocytes results in an accumulation of ribosomal RNA for use during the early cleavage stages of the embryo     

Time-lapse and retrospective analysis of DNA methylation in mouse preimplantation embryos by live cell imaging

Genome-wide change of DNA methylation in preimplantation embryos is known to be important for the nuclear reprogramming process. A synthetic RNA encoding enhanced green fluorescence protein fused to the methyl-CpG-binding domain and nuclear localization signal of human MBD1 was microinjected into metaphase II-arrested or fertilized oocytes, and the localization of methylated DNA was monitored by live cell imaging. Both the central part of decondensing sperm nucleus and the rim region of the nucleolus in the male pronucleus were highly DNA-methylated during pronuclear formation. The methylated paternal genome undergoing active DNA demethylation in the enlarging pronucleus was dispersed, assembled, and then migrated to the nucleolar rim. The female pronucleus contained methylated DNA predominantly in the nucleoplasm. When the localization of methylated DNA in preimplantation embryos was examined, a configurational change of methylated chromatin dramatically occurred during the transition of 2-cell to 4-cell embryos. Moreover, retrospective analysis demonstrated that a noticeable number of the oocytes reconstructed by round spermatid injection (ROSI) possess small, bright dots of methylated chromatin in the nucleoplasm of male pronucleus. These ROSI oocytes showed a significantly low rate of 2-cell formation, thus suggesting that the poor embryonic development of the ROSI oocytes may result from the abnormal localization of methylated chromatin.