Re-localization of nuclear DNA helicase II during the growth period of bovine oocytes

Nuclear DNA helicase II (NDH II) is the bovine homolog of human RNA helicase A. The aim of this study was to compare NDH II localization between somatic cells (bovine embryonal fibroblasts) and female germ cells (oocytes), with the main focus on the dynamic changes in the redistribution of NDH II during the growth phase of the bovine oocytes. The fine granular staining of NDH II was spread in the whole nucleoplasm of fibroblasts, excluding the reticulated nucleoli. In contrast, the large reticulated nucleoli of the growing oocytes isolated from early antral follicles exhibited strong positivity for NDH II together with the immunostaining signals of upstream binding factor (UBF) and RNA polymerase I subunit (PAF53), documenting the high synthetic activity of these nucleoli. At the time of termination of oocyte growth, NDH II was preferentially located at the nucleolar periphery together with proteins of fibrillar centres. In fully grown oocytes, NDH II was still present in the thin periphery shell around the compact nucleolar core. The semiquantitative RT-PCR revealed that the average signal of NDH II mRNA in fully grown oocytes was only at 40% level in comparison with growing oocytes. Western blot analysis further confirmed that a 140 kD NDH II protein was abundant in growing oocytes, while the signal was substantially weaker in fully grown oocytes. The significant decrease in NDH II gene expression and in NDH II mRNA translation correlates with a termination of the oocyte growth. Altogether, the results demonstrate that NDH II expression parallels the activity of ribosomal RNA biosynthesis in the bovine growing oocytes.     

An autoradiographic and cytophotometric study of oogenesis in a pulmonate snail,Planorbarius corneus

Summary The spatial and temporal patterns of macromolecular syntheses in oocytes and somatic auxiliary cells of the snail Planorbarius corneus have been investigated by autoradiography and cytophotometry. Oogenesis has been divided into three stages, comprising early meiosis up to diplotene (stage I), previtellogenetic growth phase (stage II), and vitellogenesis (stage III). No DNA synthesis was found in any oocyte stage. In stage-I oocytes, only nucleoli were found labelled with ³H-uridine. Oocyte nuclei of stage II and III actively synthesize RNA in nucleoli and chromosomes. The most intense incorporation of uridine in chromatin probably occurs during the previtellogenesis — vitellogenesis transition period during which cytological findings suggest well developed lampbrush chromosomes. RNA synthesis in amphinucleoli of stage-III oocytes is restricted to basophilic nucleolar parts, whereas acidophilic parts (protein bodies) neither synthesize nor store RNA. During vitellogenesis oocytes incorporate amino acids into yolk platelet proteins. Radioactive proteins are found in yolk platelet precursors 5 h after injection of the tracer and in yolk platelets 3 h thereafter. The labelling pattern suggests that oocytes synthesize certain hitherto unidentified yolk components. No evidence for the participation of follicle cells in synthesis and transport of vitellogenic proteins has been obtained from autoradiography. Cytological findings suggest an important role for these cells in oogenesis. They are highly active in RNA and protein synthesis. Cellular differentiation is accompanied by polyploidization of the nuclei which attain a highest DNA content of 256 c. Polyploidization probably occurs in incremental steps as indicated by complete endomitotic chromosomal cycles. Autoradiographs show that, during vitellogenesis, oocytes do not incorporate significant amounts of glucose, and only certain follicle cells were labelled with glucose, probably indicating the synthesis of glycogen.     

RNA SYNTHESIS IN THE MOUSE OOCYTE

RNA synthesis in the oocyte and granulosa cell nuclei of growing follicles has been studied in the mouse ovary. The RNA precursor [³H]uridine was administered intraperitoneally to adult mice and the amount of label incorporated into ovarian RNA was quantitated autoradiographically using grain-counting procedures. Uridine incorporation into the nucleus is low in oocytes of small, resting follicles but increases during follicle growth and reaches a peak prior to the beginning of antrum formation. Thereafter uptake rapidly declines and is very low in the oocytes of maturing follicles. Uridine incorporation into granulosa cell nuclei, in contrast to that found in the oocyte, increases gradually during most of the period of follicle growth. Qualitative studies of the activity of endogenous, DNA-dependent RNA polymerases have also been made in fixed oocytes isolated from follicles at different stages of growth. Polymerase activity is demonstrable in the nucleolus and nucleoplasm of oocytes from growing follicles, but is absent from maturing oocytes of large follicles.     

Nuclear structures in the telotrophic ovarioles of nocturnal ground beetles (Tenebrionidae, Polyphaga). II. The oocyte nucleus of Blaps lethifera and Gnaptor spinimanus. Light optical data]

Changes in the nuclear structures and their participation in RNA synthesis in the growing oocytes were followed in two species of beetles Blaps lethifera and Gnaptor spinimanus. In the oocytes of both the species, the chromosomes join into the karyosphere following the short-term lampbrush stage. A large capsule appears around the karayosphere which consists of the fibrous substance, granules and karyosphere nucleoli. The latter form in the karyosphere and contain RNP but they are not true nucleoli since they do not include 3H-uridine. RNA synthesis on the chromosomes, active at the lampbrush stage, falls markedly following their joining into the karyosphere. The oocyte nuclei of these beetles are, thus, characterized by the absence of RNA synthesizing nucleolar system and, as compared with the trophocytes, by the low level of RNA synthesis on the chromosomes.     

Zellwachstum und RNS-Synthese im Ei-Nährzellverband von Ophryotrocha puerilis

Summary During its growth phase, the oocyte is associated with a single highly polyploid nurse cell. Although the nurse cell contains high amounts of cytoplasmic ribonucleic acid, no RNA seems to be transferred to the oocyte. Autoradiographs prepared after pulse labelling with ³H-uridine indicate that the nuclei of both cell types are actively engaged in RNA synthesis during the whole period of oogenesis. Chromosomal RNA synthesis is most intense in oogonia before onset of the growth period. It still proceeds, although at a lower rate, after termination of oocyte growth when all RNA is lost from the nucleoli. Nucleolar RNA synthesis, on the other hand, is strictly correlated with the growth phase proper. Grain counts on chromatin and nucleoli of both egg cells and nurse cells at all stages of oogenesis indicate that nucleolar and chromosomal RNA synthesis are independent of each other to a large extent. It is thought that the type of RNA synthesized within the nucleolus is essentially ribosomal while the chromosomes are primarily engaged in the formation of messenger RNA.     

The mechanism of regulation of fibroblastic cell replication. II. Participation of the nucleoli

Cultures of human diploid fibroblasts (HDFs) exhibiting density dependent inhibition of replication (DDIR) resumed their progression through the cell cycle following medium replacement and, after a lag period of two hours, showed a dramatic increase in the incidence of isonucleolinar ⁴ cells and in the levels of uptake of ³H-uridine into the nucleoli. Between five and ten hours after refeeding these nucleolar changes were maximal, leveling off at the highest values, in periods corresponding to late G1 and early S. Concomitantly, a parallel increase in the number of nucleolini per cell occurred. As cells progressed through S and G2 phases the nucleolini decreased in number and reverted to the aniso-nucleolinar type. The intensity of nucleolar labeling by ³H-uridine and its correlate, the frequency of cells with labeled nucleoli, also decreased during these cell cycle stages. Both pre- and postreplicative periods of mitotic quiescence were characterized by high levels of anisonucleolinosis (60–80% of the cells) and by very low levels of nucleolar ³H-uridine incorporation. The magnitude of these nucleolar changes occurring during G1 stage was found to be strongly dependent on: (1) the length of time of contact between the cells and the fresh medium, at least eight hours of contact being necessary for a maximal response; (2) the amount of serum in the medium, the optimal serum concentration being between 10 and 50%, and (3) the pH of the medium. The nucleolar response was completely abolished at pH values below 7.0. These nucleolar changes were very sensitive to the presence of cycloheximide (10 μg/ml) and actinomycin D (0.003 μg/ml). The behavior of the nucleoli in response to these parameters was similar to the activation response of the cells to initiate DNA synthesis. During the time period of maximal nucleolar (activation) the onset of DNA synthesis as well as the morphological and autoradiographic manifestations of the nucleolar activation were completely inhibited by very low levels of actinomycin D (Ellem and Mironescu, '72), a selective inhibitor of nucleolar RNA synthesis (Perry, '65). This suggested a possible role of nucleolar metabolism, in normal diploid cells, in the initiation of DNA synthesis. Our results, however, seem to indicate that the nucleolar changes are necessary but not sufficient for the subsequent initiation of DNA synthesis, since with graded serum concentrations or medium volumes, smaller levels of a stimulus were needed to produce maximal isonucleolinosis than to effect a maximum replicative response in the cells.     

Initiation of transcription and nucleologenesis in equine embryos

The time of activation of the embryonic genome (maternal-embryonic transition) in equine embryos was investigated by assessing incorporation of ³H-uridine and nucleolar development. In Experiment 1, embryos were recovered from the oviduct (n = 15) and the uterus (n = 3). Recovered embryos were assessed for morphologic development and quality score. Recovered embryos with less than 8 cells (two cells, n = 4; four cells, n = 5; five cells, n = 2) were incubated with ³H-uridine (560 μCi/ml) for 10 hr, while eight-cell embryos (n = 2), morulae (n = 2), and blastocysts (n = 3) were incubated with 280 μCi/ml for 0.5–1 hr. At the end of incubation, embryos were washed twice in PBS with 10% FBS and incubated for 30 min with 2.5 mg/ml of unlabelled uridine. Embryos were spread onto glass slides, dipped into emulsion, and exposed for 8 d, then developed and counter-stained with Giemsa and propidium iodide. Embryos at the blastocyst, morula, eight-cell, and five-cell stages incorporated ³H-uridine into their cell nuclei as detected by autoradiography. In a second experiment, nucleologenesis in equine embryos was examined by transmission electron microscopy. Nucleoli or nucleolar precursors were found in 12 of 23 embryos examined. Most embryos in the four- to six-cell stage (n = 7) had nucleolar precursor bodies (npb) consisting of homogeneous fibrillar structures. Two five- to six-cell embryos also possessed reticulated nucleoli with both fibrillar and granular components as did all eight-cell embryos (n = 3). Nucleoli in one morula and one blastocyst were reticulated with prominent granular components, fibrillar components, and apparent fibrillar centers. These results indicate that incorporation of ³H-uridine and the formation of functional nucleoli with typical fibrillar and granular components occurs between the four- to eight-cell stage in equine embryos. © 1995 wiley-Liss, Inc.     

Ultrastructural cytochemistry of the nucleus and nucleolus in growing rabbit oocytes

Summary— Ultrastructural changes of the germinal vesicle during the growth of rabbit oocytes were studied by means of light and electron microscopy, ³H-uridine autoradiography, Ag-NOR staining and E-PTA staining. Particular interest was paid to the nucleologenesis and condensation of chromatin. In contrast to other mammalian species, chromosome condensation in rabbit oocytes occurred concomitantly with rRNA synthesis-dependent nucleolar compaction and preceded nuclear envelope breakdown and resumption of meiosis.     

Oogenesis of Mozambique tilapia. II. Synthesis of RNA and development of the nucleoli

Using methods of light and electron microscopy and authoradiography, morphology and biosynthetic activity of nucleoli and RNA synthesis in developing oocytes of Tilapia mossambique were studied. In previtellogenic oocytes nucleoli are mostly composed of fibrils, while the granular component is poorly developed. 3H-uridine is poorly incorporated in their peripheral parts. With the start of vitellogenesis, fibrils and granules are seen randomly located throughout the whole volume of the nucleoli, with primary granule concentrations in their peripheral region. 3H-uridine is intensely incorporated in the whole volume of nucleoli, and the labeled RNA migrates to the cytoplasm. In early previtellogenic oocytes chromosomes are strongly labeled with 3H-uridine, and RNA quickly migrates to the cytoplasm.     

On the nucleolar size and density in human early granulocytic progenitors, myeloblasts

Human myeloblasts were studied in bone marrow of patients suffering from chronic phase of chronic myeloid leukaemia to provide more information on the nucleolar diameter in these early granulocytic progenitors. These cells are a convenient model for such study since the number of myeloblasts in diagnostic bone marrow smears of investigated patients is larger than in not-leukemic persons because of the increased granulopoiesis. The nucleolar diameter was measured in myeloblasts after various cytochemical procedures such as methods for visualisation of RNA, DNA and proteins of AgNORs using digitized images and image processing. The results clearly demonstrated that values of the nucleolar diameter depended on the procedures used for visualising nucleoli. It seems to be also clear that a close relationship exists between the diameter of nucleoli and their number since the larger the number of nucleoli per cell the smaller their mean size. However, one of multiple nucleoli present in the nucleus is usually significantly larger. Moreover, the possibility exists that the variability of nucleolar diameter of leukemic myeloblasts and thus the heterogeneity of these cells might depend on various stages of the cell cycle as supported by nucleolar measurements on aging leukemic myeloblasts (K 562 cells) in vitro. Since the staining density of small and large nucleoli did not differ substantially after staining for RNA, it seems to be likely that the nucleolar size is directly related to the total RNA content in myeloblasts. In addition, karyometry combined with RNA cytochemistry still appears to be an useful tool to study nucleoli at the single cell level.     

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.     

Multiple nucleoli and enhanced nucleolar activity in the nurse cells of the insect ovary

Origin and function of the nucleolar apparatus in nurse cell nuclei of Calliphora erythrocephala have been investigated by cytological and autoradiographic methods in some inbred lines of laboratory blowflies with well paired polytene chromosomes in the nurse cell nuclei. Besides the nucleolus at chromosome VI large numbers of multiple free nucleoli develop in the highly polyploidized nurse cells during oocyte growth. The nucleoli incorporate H³-uridine in a considerable amount producing a homogeneous and RNase-sensitive label even after short time incubation. Their capacity of RNA synthesis is independent of their spatial relationships to other nuclear components. DNA particles in the nucleoli could be identified by the Feulgen reaction and by fluorescence staining with N,N'-diethylpseudoisocya-ninchloride, which also demonstrates the existence of own templates for autonomous RNA synthesis. There are indications that the nucleolus' own DNA is produced by gene amplification beyond the level of endomitotic polyploidization in the nurse cell nuclei. A quantitative estimation of grain density in the autoradiograms shows a rigorous shift of rRNA synthesis: at least 72% of all newly synthesized macromolecular RNA in nurse cell nuclei as contrasted to 13 % of nucleolar RNA synthesis in bristle forming cells with a similar degree of polyploidy. It seems that the nurse cell nuclei of Calliphora in addition to polyploidization increase their template capacity for synthesizing rRNA in a similar way as has repeatedly been demonstrated for Amphibia. Cytological and physiological peculiarities of the nurse cells have been discussed from the viewpoint of their functional similarity to the oocyte nucleus.     

In vitro culture of secondary follicles and prematuration of cumulus–oocyte complexes from antral follicles increase the levels of maturation‐related transcripts in bovine oocytes

This study evaluates the levels of messenger RNA (mRNA) for eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1 in oocytes from secondary and antral follicles at different stages of development. The effects of in vitro culture, in vitro prematuration, and in vitro maturation on the expression of these genes on oocytes were also analyzed. The results showed that mRNA levels for H1FOO, GDF9, and PARN were higher in oocytes from small, medium, and large antral follicles, respectively, than those seen in secondary follicles. Oocytes from small, medium, and large antral follicles had higher levels of CCNB1 than oocytes from secondary follicles. Oocytes from cultured secondary follicles had higher levels of GDF9, CMOS, PARN, eIF4E, CCNB1, and H1FOO than before culture. Prematured oocytes from small antral follicles had higher levels of mRNA for GDF9, PARN, and eIF4E than before culture. In addition, higher levels of cMOS and H1FOO were identified in prematured oocytes from medium antral follicles. In conclusion, follicular growth is associated with an increase in the expression of H1FOO, GDF9, CCNB1, and PARN. The culture of secondary follicles, prematuration, and maturation of oocytes from antral follicles increase the expression of eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1.     

Fine structural and cytochemical analysis of the processes of cell death of oocytes in atretic follicles in new born and prepubertal rats

The process of cell death of oocytes was studied in atretic ovarian follicles of rats aged from 1 to 28 days using light and electron microscope and cytochemical methods. These methods were TUNEL procedure for DNA breaks, active caspase-3 and lysosome-associated membrane protein 1 (LAMP-1) immunolocalizations. The structural features of the process of oocyte death are mainly characterized by the presence of abundant clear vacuoles and autophagosomes, as well as by the absence of large clumps of compact chromatin associated to the nuclear envelope and apoptotic bodies. These features are common to oocytes in all types of follicles studied. Cytochemical features consisting in positive reactions to TUNEL method, active caspase-3 and LAMP-1 immunolocalizations, are common to the cell death of oocytes in all types of follicles. Particular features of the process of cell death of oocytes are found in different types of follicles. Two morphological patterns of cell death occur in pre-follicular oocytes of the new born and in primordial follicles in 1 to 5 days old rats. One is distinguished by clear nucleoli and moderate compaction of chromatin in clumps frequently resembling meiotic bivalents. The second pattern is characterized by nucleolar condensation and by the absence of compact chromatin. The process of cell death of oocytes in antral follicles is characterized by ribonucleoprotein ribbon-like cytoplasmic structures, pseudo-segmentation, and loss of contact with granulosa cells.