Nucleolar Development Research Articles

Transcriptome Dynamics of Epidermal Reprogramming during Direct Shoot Regeneration in Torenia fournieri.

abstractShoot regeneration involves reprogramming of somatic cells and de novo organization of shoot apical meristems (SAMs). In the best-studied model system of shoot regeneration using Arabidopsis, regeneration is mediated by the auxin-responsive pluripotent callus formation from pericycle or pericycle-like tissues according to the lateral root development pathway. In contrast, shoot regeneration can be induced directly from fully differentiated epidermal cells of stem explants of Torenia fournieri (Torenia), without intervening the callus mass formation in culture with cytokinin; yet, its molecular mechanisms remain unaddressed. Here, we characterized this direct shoot regeneration by cytological observation and transcriptome analyses. The results showed that the gene expression profile rapidly changes upon culture to acquire a mixed signature of multiple organs/tissues, possibly associated with epidermal reprogramming. Comparison of transcriptomes between three different callus-inducing cultures (callus induction by auxin, callus induction by wounding and protoplast culture) of Arabidopsis and the Torenia stem culture identified genes upregulated in all the four culture systems as candidates of common factors of cell reprogramming. These initial changes proceeded independently of cytokinin, followed by cytokinin-dependent, transcriptional activations of nucleolar development and cell cycle. Later, SAM regulatory genes became highly expressed, leading to SAM organization in the foci of proliferating cells in the epidermal layer. Our findings revealed three distinct phases with different transcriptomic and regulatory features during direct shoot regeneration from the epidermis in Torenia, which provides a basis for further investigation of shoot regeneration in this unique culture system.

Morphological assessment of preimplantation embryo quality in cattle

The extensive use of embryo technologies has emphasized the need for assessing embryo quality by morphological techniques, such as transmission electron microscopy, immunocytochemistry for confocal laser scanning microscopy and fluorescence in situ hybridization. By a combination of these techniques, it has been possible to demonstrate: (i) that rRNA gene activation, as monitored by embryonic nucleolar development, is comparable in bovine embryos developed in vivo and produced in vitro, whereas reconstructed nuclear transfer embryos may be deviant, (ii) that generating embryos by both in vitro production and reconstruction by nuclear transfer is associated with increased occurrence of apoptosis, in particular in the inner cell mass of blastocysts, and (iii) that these two embryo production techniques are associated with increased occurrence of mixoploidy that is, embryos presenting a large population of normal diploid cells and a small population of abnormal haploid or polyploid cells. It is clear that blastocysts that appear healthy at stereomicroscopy may have subcellular defects. Therefore, the possibility of long-term evaluation in vitro of embryos after hatching has been examined. However, whereas embryos developing in vivo after hatching present a number of well defined developmental milestones, such as elongation of the trophoblast, formation of hypoblast and epiblast followed by differentiation of endoderm, mesoderm and ectoderm, in vitro culture systems for development beyond the blastocyst stage currently allow the embryo to complete only a single milestone, namely hypoblast formation.

Growing Oocytes Need Zinc: Zinc Deficiency in the Preantral Ovarian Follicle

ObjectiveTo evaluate the effect of zinc deficiency on oocyte development in the preantral ovarian follicle – a novel developmental stage for micronutrient investigation.MethodsOocyte‐granulosa cell complexes were collected from preantral ovarian follicles of 14 day old CD‐1 mice and cultured on collagen membranes under control, zinc‐deficient, or rescue conditions. Oocyte diameter was measured from brightfield images taken with an Olympus DP20 microscope digital camera. Chromatin arrangement and nucleolar development was observed after DAPI staining. Gene expression was measured with qPCR. Meiotic competence was measured by exposing cultured oocyte‐granulosa cell complexes to Epidermal Growth Factor (EGF) to mimic the in‐vivo ovulatory signal (maturation media). The ability of the oocytes to undergo meiotic division was assessed 24 hours after EGF treatment.ResultsZinc deficiency disrupted oocyte growth in vitro. Zinc deficient oocytes were smaller than control or rescue on day 4 (p<0.001) and 6 (p<0.05), but not day 10 of cell culture. Zinc deficiency impacted nuclear development; zinc deficient oocytes were more likely to have multiple nucleoli on day 6 (p<0.001), but not day 10 of cell culture. Differentiation of somatic cells was also impaired; zinc‐deficient granulosa cell gene expression was consistent with a preantral, undifferentiated phenotype, even after 10 days of culture. Preantral somatic cell markers lhcgr, ar, pcsk6, and fosb were elevated compared with control (p<0.05 for all). Preantral zinc deficiency reduced the meiotic potential of cultured oocytes. After EGF treatment, oocytes matured in zinc deficient media were less likely to resume meiosis and reach the meiotic stage required for fertilization (chi square p<0.001). The meiotic defect was seen in zinc‐deficient oocytes even when transferred to maturation media containing zinc.ConclusionsPrior to entering the pool of antral ovulatory follicles, oocytes and the surrounding somatic cells undergo a period of growth and differentiation. This phase is known as preantral oocyte development and may last 3–5 months in humans. This study shows that zinc deficiency impairs multiple preantral developmental milestones including oocyte growth, oocyte nuclear maturation, and somatic cell differentiation. Most notably, the acquisition of meiotic competence – a benchmark of oocyte fertility and viability – was severely reduced by zinc deficiency. The meiotic defect was noted in zinc‐deficient cultured oocytes even when they were transferred to maturation media containing zinc, indicating a zinc requirement during development, rather than during meiotic resumption. These results demonstrate that zinc is required in oocyte development earlier than previously reported, and that even short periods of zinc deficiency may impact fertility months later. These results may impact our understanding of prenatal micronutrient supplementation, as well as optimal methods for in‐vitro oocyte maturation.Support or Funding InformationNIH ‐HD074831‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nucleolar Dominance and Repression of 45S Ribosomal RNA Genes in Hybrids between Xenopus borealis and X. muelleri (2n = 36)

Nucleolar dominance is a dramatic disruption in the formation of nucleoli and the expression of ribosomal RNA (rRNA) genes, characteristic of some plant and animal hybrids. Here, we report that F₁ hybrids produced from reciprocal crosses between 2 sister species of Xenopus clawed frogs, X. muelleri and X. borealis, undergo nucleolar dominance somewhat distinct from a pattern previously reported in hybrids between phylogenetically more distant Xenopus species. Patterns of nucleolar development, 45S rRNA expression, and gene copy inheritance were investigated using a combination of immunostaining, pyrosequencing, droplet digital PCR, flow cytometry, and epigenetic inhibition. In X. muelleri × X. borealis hybrids, typically only 1 nucleolus is formed, and 45S rRNA genes are predominantly expressed from 1 progenitor's alleles, X. muelleri, regardless of the cross-direction. These changes are accompanied by an extensive (∼80%) loss of rRNA gene copies in the hybrids relative to their parents, with the transcriptionally underdominant variant (X. borealis) being preferentially lost. Chemical treatment of hybrid larvae with a histone deacetylase inhibitor resulted in a partial derepression of the underdominant variant. Together, these observations shed light on the genetic and epigenetic basis of nucleolar dominance as an underappreciated manifestation of genetic conflicts within a hybrid genome.

Early Aberrations in Chromatin Dynamics in Embryos Produced UnderIn VitroConditions

In vitro production of porcine embryos by means of in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) is limited by great inefficienciy. The present study investigated chromatin and nucleolar dynamics in porcine embryos developed in vivo (IV) and compared this physiological standard to that of embryos produced by IVF, parthenogenetic activation (PA), or SCNT. In contrast to IV embryos, chromatin spatial and temporal dynamics in PA, IVF, and SCNT embryos were altered; starting with aberrant chromatin-nuclear envelope interactions at the two-cell stage, delayed chromatin decondensation and nucleolar development at the four-cell stage, and ultimately culminating in failure of proper first lineage segregation at the blastocyst stage, demonstrated by poorly defined inner cell mass. Interestingly, in vitro produced (IVP) embryos also lacked a heterochromatin halo around nucleolar precursors, indicating imperfections in global chromatin remodeling after fertilization/activation. Porcine IV-produced zygotes and embryos display a well-synchronized pattern of chromatin dynamics compatible with genome activation and regular nucleolar formation at the four-cell stage. Production of porcine embryos under in vitro conditions by IVF, PA, or SCNT is associated with altered chromatin remodeling, delayed nucleolar formation, and poorly defined lineage segregation at the blastocyst stage, which in turn may impair their developmental capacity.

Giemsa C-banding and silver-staining for cytological studies in Pisum

Technical hints are given for Giemsa C-banding of the Pisum chromosomes and for silver-staining of NORs and nucleoli. C-band positive heterochromatin appears as pairs of centromeric dots on each of the seven chromosomes and as heavily stained C-bands at the secondary constrictions in the long arms of chromosomes 4 and 7. In the latter arm, there is also an intercalary proximally situated C-band. The centromeric dots are attached to chromosomal spindle fibres, which supports the supposition that they are kinetochores. The C-blocks of the SAT regions are also detectable at metaphase I of meiosis and hence facilitate the interpretation of configurations in structural hybrids involving the SAT chromosomes. In mitotic interphase nuclei, the SAT blocks appear as four chromocentres, and in the giant nuclei of the suspensor, as four endochromocentres. Ag-NORs coincide with the C-bands at the SAT regions. Moreover, the centromeric dots are stained by silver nitrate. In Pisum, nucleoli may be formed from the centromeric regions of the chromosomes. At late mitotic prophase (diminishing stage of nucleolar development), the nucleoli sometimes appear as paired heavily silver-stained bodies budding out from the chromosomes. At meiotic prophase, darkly stained intranucleolar bodies occur in slides prepared according to a suitable silver-staining technique.

The role of RNA polymerase I transcription and embryonic genome activation in nucleolar development in bovine preimplantation embryos

The aim of the present study was to investigate the role of RNA polymerase I (RPI) transcription in nucleolar development during major transcriptional activation (MTA) in cattle. Late eight-cell embryos were cultured in the absence (control group) or presence of actinomycin D (AD) (RPI inhibition, AD 0.2 microg/ml; total transcriptional inhibition, AD 2.0 microg/ml). Late four-cell embryos were cultured to late eight-cell stage in 0.2 microg/ml AD (MTA prevention, ADLT (long-term total transcriptional inhibition group). Embryos were processed for autoradiography, transmission electron microscopy, fluorescent in situ hybridization (ribosomal RNA, rRNA), silver staining (nucleolar proteins), and immunofluorescence (RPI). Control embryos displayed extranucleolar and nucleolar transcription, functional nucleoli, and distinct RPI localization. Nuclei (97%) showed large rRNA clusters, in 94.1% co-localized with nucleolar proteins deposits. In AD 0.2 group, only extranucleolar transcription was detected. Segregated dense-fibrillar and granular components, but no fibrillar centers, were observed. RPI was dispersed. Nuclei (55%) presented rRNA clusters, in 38.8% co-localized with silver-stained deposits. AD 2.0 and ADLT groups displayed no transcription and disintegrating nucleolar precursors. AD 2.0 (34%) and 14% (ADLT) of nuclei presented clusters of maternally inherited rRNA. In AD 2.0 group, RPI was dispersed, but 17.2% of nuclei showed colocalization of rRNA with nucleolar proteins. In ADLT group, RPI was lacking and clustering of nucleolar proteins was hampered. In conclusion, rDNA transcription is not required for targeting of rRNA processing proteins, rRNA is maternally inherited and target to rDNA independent of transcription, and de novo transcription is required for proper nucleologenesis in cattle.

Nucleolar proteins and ultrastructure in bovine in vivo developed, in vitro produced, and parthenogenetic cleavage‐stage embryos

In the present study, ribosomal RNA (rRNA) gene activation, monitored through nucleolus development, was studied by autoradiography following (3)H-uridine incubation, transmission electron microscopy, and immunofluorescence confocal laser scanning microscopy of key nucleolar proteins involved in rRNA transcription (topoisomerase I, upstream binding factor, and RNA polymerase I) and processing (fibrillarin, nucleolin, and nucleophosmin) in in vivo developed, in vitro produced, and parthenogenetic bovine embryos. In general, in vivo developed embryos displayed formation of fibrillo-granular nucleoli during the 4th post-fertilization cell cycle. During the previous stages of development, nucleolus precursor bodies (NPBs) were observed. However, on some occasions the initial steps of nucleolus formation were observed already at the 2- and 4-cell stage in cases where such embryos were collected from superovulated animals together with later embryonic stages presenting nucleolar development and autoradiographic labeling. The in vitro produced embryos displayed very synchronous formation of fibrillo-granular nucleoli and autoradiographic labeling during the 4th cell cycle. In vivo developed and in vitro produced embryos displayed allocation of nucleolar proteins to fibrillar and granular compartments of the developing nucleoli during the 4th cell cycle. The parthenogenetic embryos typically displayed formation of fibrillo- granular nucleoli during the 5th cell cycle and autoradiographic labeling was not observed until the morula stage. Moreover, the 1-, 2-, and 4-cell parthenogenetic embryos practically lacked NPBs. On the other hand, parthenogenetic embryos displayed allocation of nucleoar proteins to nuclear entities during the 4th cell cycle. In conclusion, both in vivo developed and in vitro produced bovine embryos displayed activation of transcription and nucleolar development during the 4th cell cycle. However, in vivo developed embryos flushed together with later developmental stages displayed premature activation of these processes. Parthenogenetic bovine embryos, on the other hand, displayed a delayed activation.

On the formation of amplified nucleoli during early Xenopus oogenesis

In Xenopus the genes for ribosomal RNA are selectively amplified during the early stages of oogenesis and give rise to over 1000 extrachromosomal nucleoli. These oocyte nucleoli are unique in that they contain very high copy numbers of rRNA genes, are not attached to chromosomes, and lack nonribosomal DNA. How the amplified rRNA genes induce the formation of multiple nucleoli is as yet poorly understood. To gain some more insight into this assembly process we have studied nucleolar development in early previtellogenic Xenopus oocytes. By using light and electron microscopic immunocytochemistry, in situ hybridization, and the TdT-method for detection of DNA, we have identified three separate structural entities which are related to the three components of the functionally active nucleoli present in midsized oocytes. Besides fibrillar and granular bodies we describe a novel rod-like structure which contains the pol I-specific transcription factor UBF. Notably, rDNA emerges from these rods forming a filamentous layer. These results reinforce UBF’s role as an architectural element involved in the spatial organization of the rRNA genes. We consider the rod-like rDNA/UBF complexes as extrachromosomal nucleolar organizers which are first juxtaposed to preformed fibrillar bodies before both elements gradually fuse into a coherent nucleolar structure.

Nucleolar proteins and nuclear ultrastructure in preimplantation bovine embryos produced in vitro.

The aim of the present investigation was to describe the basic cell biology of the postfertilization activation of rRNA genes using in vitro-produced bovine embryos as a model. We used immunofluorescence confocal laser scanning microscopy and transmission electron microscopy to study nucleolar development in the nuclei of embryos up to the fifth postfertilization cell cycle. During the first cell cycle (1-cell stage), fibrillarin, upstream binding factor (UBF), nucleolin (C23), and RNA polymerase I were localized to distinct foci in the pronuclei, and, ultrastructurally, compact spherical fibrillar masses were the most prominent pronuclear finding. During the second cell cycle (2-cell stage), the findings were similar except for a lack of nucleolin and RNA polymerase I labeling. During the third cell cycle (4-cell stage), fibrillarin, UBF, nucleophosmin, and nucleolin were localized to distinct foci. Ultrastructurally, spherical fibrillar masses that developed a central vacuole over the course of the cell cycle were observed. Early in the fourth cell cycle (8-cell stage), fibrillarin, nucleophosmin, and nucleolin were localized to small bodies that with time developed a central vacuole. UBF and topoisomerase I were localized to clusters of small foci. Ultrastructurally, spherical fibrillar masses with a large eccentric vacuole and later small peripheral vacuoles were seen. Late in the fourth cell cycle, nucleophosmin and nucleolin were localized to large shell-like bodies; and fibrillarin, UBF, topoisomerase I, and RNA polymerase I were localized to clusters of small foci. Ultrastructurally, a presumptive dense fibrillar component (DFC) and fibrillar centers (FCs) were observed peripherally in the vacuolated spherical fibrillar masses. Subsequently, the presumptive granular component (GC) gradually became embedded in the substance of this entity, resulting in the formation of a fibrillo-granular nucleolus. During the fifth cell cycle (16-cell stage), a spherical fibrillo-granular nucleolus developed from the start of the cell cycle. In conclusion, the nucleolar protein compartment in in vitro-produced preimplantation bovine embryos is assembled over several cell cycles. In particular, RNA polymerase I and topoisomerase I are detected for the first time late during the fourth embryonic cell cycle, which coincides with the first recognition of the DFC, FCs, and GC at the ultrastructural level.

Nucleologenesis in the cleaving bovine embryo: immunocytochemical aspects.

In vivo nucleologenesis was studied in bovine embryos by electron microscopic immunogold labelling of DNA, RNA, protein C23 and protein B23. We have used the classification of Kopecný et al. (1989b) and Kopecný (1990) dividing nucleologenesis in four steps: compact nucleolar precursor body (NPB), monovacuolated NPB, NPB containing secondary vacuoles and fully reticulated nucleolus. These different features of early bovine embryo nucleologenesis were mainly observed during the eight-cell stage. In the first step of nucleolar development, the association of compact NPB with DNA structures was observed. DNA was also labelled in some small secondary vacuoles appearing during the third developmental step. From the second step onward, the labelling of protein C23 was observed in the compact fibrillar network of the NPB. Protein B23 started to be labelled in the compact fibrillar mass at the third step. RNA labelling was also observed for the first time in NPB containing secondary vacuoles. Labelled RNA was located in the peripheral region of compact fibrillar mass as well as along the border of the vacuoles. In the reticulated nucleolus, the dense fibrillar component was found to contain both proteins and RNA.

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 microCi/ml) for 10 hr, while eight-cell embryos (n = 2), morulae (n = 2), and blastocysts (n = 3) were incubated with 280 microCi/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 counterstained 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.

Intranuclear inclusions in paramyxovirus-induced encephalitis: evidence for altered nuclear body differentiation.

Intranuclear inclusion bodies (INB) are frequently encountered in viral infections, where they are thought to be accumulations of viral particles. However, for RNA viruses replicating in the cytoplasm, this compartmentalization represents a paradox not consistent with the viral replication cycle. To define the basis for intranuclear paramyxoviral inclusion bodies in astrocytes, natural cases of canine distemper virus subacute encephalitis were examined by light and transmission electron microscopy, and by quantitative confocal immunofluorescence microscopy. Although INB were viral antigen positive, they were not composed of structurally recognizable paramyxoviral nucleocapsids. The structural basis for the INB was instead viral antigen-associated forms of nucleolar development known as nuclear bodies. Three variants of the light microscopic Cowdry type A INB were complex nuclear bodies, giant beaded nuclear bodies (sphaeridia), and nuclear body-associated granulofilamentous matrices. In the latter, the granulofilamentous matrix frequently filled the nucleus, resulting in a fourth morphological INB variant, and was associated with morphological evidence of nuclear degeneration. These findings suggest a novel mechanism of virus-induced cytopathology whereby intranuclear viral protein exerts deleterious effects upon nucleolar differentiation in infected cells and hence altered host cell RNA metabolism.

U3 snRNPs and nucleolar development during oocyte maturation, fertilization and early embryogenesis in the mouse: U3 snRNA and snRNPs are not regulated coordinate with other snRNAs and snRNPs

U3 small nuclear ribonucleic acids (snRNA) and U3 small nuclear ribonucleoprotein (snRNP), which are thought to be responsible for ribosomal RNA processing, are quantitated and localized during oocyte maturation, fertilization, and early embryogenesis in the mouse. On the basis of Northern blot and nuclease protection experiments, it is estimated that there are about 5 × 10 4 U3 snRNA molecules in an ovulated oocyte and in a two-cell embryo. This number then increases roughly 50-fold to 2.7 × 10 6 molecules per embryo by the blastocyst stage. At all stages of development U3 snRNP antigens colocalize with nucleoli, as defined by differential interference contrast microscopy and an antibody to a nucleolar epitope. The synthesis and distribution of U3 snRNA and U3 snRNP follow a pattern independent from other major U snRNPs and snRNAs.

Nucleologenesis and the onset of transcription in the eight-cell bovine embryo: fine-structural autoradiographic study.

Eight-cell cow embryos were isolated and cultured in vitro in a medium enriched with 200 microCi of [5-3H]uridine for 20 min. Epon ultrathin sections of the embryos were investigated for the nucleolar morphology and for the appearance and localization of the sites of [5-3H]uridine incorporation by means of electron microscopic autoradiography. In addition to this, a general pattern of replicated embryonal DNA distribution was revealed by [methyl-3H]thymidine incorporation and light microscopic autoradiography. The essential phases of the transformation of the small nucleolus precursor body (NPB) into a vast, functionally fully active nucleolus, characterized by typical nucleolar substructural components, are taking place within the eight-cell stage. This process differed in its morphology from the nucleologenetic process in early embryogenesis of other mammals, especially of that in the mouse. The first sign of NPB transformation was the appearance of a large central vacuole followed later on by perinucleolar chromatin penetration into NPB, documented by both morphology and [3H]thymidine autoradiography. In some cases, concentration of dense fibrillar material forming clumps or stalks was seen in the central vacuole. The following rapid nucleolar development was characterized by the formation of secondary vacuoles concomitant with the onset of [5-3H]uridine incorporation into the dense fibrillar component and with the appearance of the first granules in the otherwise fibrillar structure of the nucleolus. During the late eight-cell stage, the still-rounded nucleolus developed features of a reticulated nucleolus known from somatic cells intensively synthesizing rRNA: a dense fibrillar component with associated labeling encircling fibrillar centers and a well-developed granular component. The labeled dense fibrillar component was observed mostly in the central area of the nucleolus; early embryonic NPB dense fibrous material not involved in transcription was disappearing rapidly. At the transition to the 16-cell stage the nucleoli lost their rounded shape because of the accumulation of a large amount of granular component, and they occupied a considerable part of the nucleus. In conclusion, the appearance of the nucleolar vacuole in eight-cell cow embryo is the starting point for following morphogenetic events linked with the onset of transcription.

Quantitative analysis of Sertoli cells in neonatally oestrogen-treated rats.

On Day 1 of age rats were treated with 500 micrograms oestradiol benzoate. Oestrogen-treated rats had increased numbers of Sertoli cells per reference area or volume, whereas the total number of cells per testis was unchanged. The mean nuclear size was significantly smaller in oestrogen-treated rats than in control rats, at 22 and 45 days of age. The volume density of the heterochromatin clumps decreased from 22 to 45 days of age in control rats (68% fall), the decrease being slower in oestrogenized animals (30% fall) during the same period. The differences were significant at 45 days of age only. The relative volume occupied by the nuclear membrane infoldings was significantly less in oestrogenized rats than in control ones at the two ages considered. Nucleolar development was delayed in oestrogen-treated rats, which had lower numbers of nuclear sections showing nucleoli, as well as a decrease in the nucleolar diameter. We suggest that these Sertoli cell alterations are due to the altered gonadotrophin and testosterone concentrations induced by the steroid treatment rather than to a direct effect of oestrogen.

Nucleologenesis in the human embryo developing in vitro: Ultrastructural and autoradiographic analysis

Nucleolar development during cleavage of human embryos in vitro was analyzed using combined autoradiographic and ultrastructural approaches. Human 2- to 4-cell embryos lack functionally active nucleoli. They possess nucleolus-like bodies formed by a homogeneous mass composed of densely packed fine fibrils. The nucleolus-like bodies contain no recently replicated embryonic DNA and do not show any detectable RNA synthesis. Their transformation into nucleoli is a relatively rapid process characterized by progressive infiltration of these bodies by adjacent chromatin, followed by the beginning of RNA synthesis and processing reflected by the appearance of the first nucleolar granules. These changes take place in 6- to 8-cell embryos, probably starting after the third cleavage division. Finally, most frequently in 10- to 12-cell embryos, typical nucleolar structure is established as a result of intranucleolar differentiation giving rise to distinct fibrillar and granular components as well as to nucleolar interstices. It is suggested that this pattern of nucleologenesis, different from that pertinent to mouse embryos (M. Geuskens and H. Alexandre (1984). Cell Differ. 14, 125–134) , might be related to the relatively late activation of embryonic rRNA genes transcription and to the expressed rapidity of this process.

Ultrastructural and autoradiographic studies of nucleolar development and rDNA transcription in preimplantation mouse embryos

The development of the nucleoli and the sites of rDNA transcription have been studied by high-resolution autoradiography during the cleavage stages of mouse embryos. The appearance of fibrillar centres at the periphery of the fibrillar primary nucleoli has been observed at the 4-cell stage. Several fibrillar centres, interconnected by electron-dense fibrillar strands, form a reticulated region around the fibrillar mass at the 6- to 8-cell stage. After a 10 min pulse with [ 3H]uridine, only this peripheral network is labelled. At the late morula and at the blastocyst stage, the fibrillar component (nucleolonema) of the reticulated nucleoli is labelled after 10 min [ 3H]uridine incorporation. When the embryos are reincubated for 2 h in cold medium, the label is localized mainly in the granular component. Fibrillar centres are not labelled. Autoradiograms of in vitro developed embryos pulsed for 2 h with [ 3H]uridine confirm that the central fibrillar core of the nucleoli of 6-to 8-cell embryos is never labelled. Thus, the fibrillar constituent of this core is not homologous to the fibrillar component of the nucleoli of later stage embryos, which is the site of active rDNA transcription. An interpretation of nucleologenesis during early mouse embryogenesis is proposed.

Reproduction and sexual development in a freshwater gastrotrich

Post-embryonic development of parthenogenic eggs of Lepidodermella squammata was studied by light and electron microscopy in animals of known age and reproductive history. Each bilateral gonad initially contains eight cells. No mitotic proliferation occurs during parthenogenic egg development. Germ cells are tightly clustered, have smooth plasma membranes with no interconnections, and are uninucleate. There is no surrounding ovary or oviduct. At hatching, two cells in each gonad are identifiable as parthenogenic eggs. The enlarged nucleolus of the most mature egg has already attained the morphology that persists throughout vitellogenesis, with intertwined granular and fibrillar threads. Less mature eggs have earlier stages of nucleolar development, and lack indications of meiotic events. Parthenogenic eggs enter vitellogenesis singly, with formation of RER and active Golgi complexes, and the accumulation of lipid, yolk, and various granules. The shell is formed in situ, whereas the spines elongate after egg deposition. Most animals produce four parthenogenic eggs, which undergo immediate development (tachyblastic eggs). Resting (opsiblastic) eggs are rare in isolation culture. Both types of eggs are produced only prior to the formation of sperm and primary oocytes. The absence of synaptonemal complexes, which would indicate synapsis of homologous chromosomes in prophase of meiosis I, implies that parthenogenesis is by apomixis in L. squammata.

Effects of polyamine limitation on nucleolar development and morphology in early chick embryos

Inhibition of polyamine synthesis in early chick embryos by in ovo treatment with dl-α-difluoromethylomithine (DFMO), injected beneath the blastoderm after 5 h of incubation, permanently blocks post-gastrular development. After the first day of polyamine limitation, the embryos possess a thickened primitive streak. Further morphogenesis is blocked and the ectoderm and mesoderm are condensed around the streak. There is obvious suppression of nucleolar formation in 24 h DFMO-treated embryos. In the mesoderm obliquely in front of Hensen's node the frequency of nucleolus-possessing cells is only a few percent lower in DFMO-treated than in control embryos. However, in the same area the frequency of mesoderm cells possessing multiple nucleoli is about 50% lower in the polyamine-depleted embryos. At the ultrastructural level, mesoderm cells from 24 h DFMO-treated embryos show a reduction of the fibrillar component of the nucleolus with a resulting segregation of the nucleolar material. Our data indicate that stimulation of polyamine synthesis is an obligatory step in the differentiation of epiblast cells into mesoderm cells.