Differential transcriptional activity associated with chromatin configuration in fully grown mouse germinal vesicle oocytes.

It was previously shown that fully grown ovarian germinal vesicle (GV) oocytes of adult mice exhibit several nuclear configurations that differ essentially by the presence or absence of a ring of condensed chromatin around the nucleolus. These configurations have been termed, respectively, SN (surrounded nucleolus) and NSN (nonsurrounded nucleolus). Work from our and other laboratories has revealed ultrastructural and functional differences between these two configurations. The aims of the present study were 1) to analyze the equilibrium between the SN and the NSN population as a function of the age of the mice and the time after hCG-induced ovulation and 2) to study the polymerase I (pol I)- and polymerase II (pol II)-dependent transcription in both types of oocytes through the detection of bromouridine incorporated into nascent RNA. We show 1) that ovarian GV oocytes exhibiting the SN-type configuration can be found as soon as 17 days after birth in the C57/CBA mouse strain and 2) that the SN:NSN ratio of ovarian GV oocytes is very low just after hCG-induced ovulation and then increases progressively with the time after ovulation. Furthermore, we demonstrate that the SN configuration correlates strictly with the arrest of both pol I- and pol II-dependent transcription in mice at any age. Finally, we show that ribosomal genes are located at the outer periphery of the nucleolus in the NSN configuration and that pol I-dependent perinucleolar transcription sites correspond to specific ultrastructural features of the nucleolus. Altogether, these results provide clear-cut criteria delineating transcriptionally active GV oocytes from those that are inactive, and confirm that the SN-type configuration is mostly present in preovulatory oocytes.

Nuclear envelope removal/maintenance determines the structural and functional remodelling of embryonic red blood cell nuclei in activated mouse oocytes.

Nuclei of embryonic red blood cells (e-RBC) from 12-day mouse fetuses are arrested in G0 phase of the cell cycle and have low transcriptional activity. These nuclei were transferred with help of polyethylene glycol (PEG)-mediated fusion to parthenogenetically activated mouse oocytes and heterokaryons were analysed for nuclear structure and transcriptional activity. If fusion proceeded 25-45 min after oocyte activation, e-RBC nuclei were induced to nuclear envelope breakdown and partial chromatin condensation, followed by formation of nuclei structurally identical with pronuclei. These 'pronuclei', similar to egg (female) pronuclei, remained transcriptionally silent over several hours of in vitro culture. If fusion was performed 1 h or later (up to 7 h) after activation, the nuclear envelope of e-RBC nuclei remained intact and nuclear remodelling was less spectacular (slight chromatin decondensation, formation of nucleolus precursor bodies). These nuclei, however, reinforced polymerase-II-dependent transcription within a few hours of in vitro culture. Our present experiments, together with our previous work, demonstrate that nuclear envelope breakdown/maintenance are critical events for nuclear remodelling in activated mouse oocytes and that somatic dormant nuclei can be stimulated to renew transcription at a time when the female pronucleus remains transcriptionally silent.

In vivo aging of oocytes influences the behavior of nuclei transferred to enucleated rabbit oocytes.

The present study examined nuclear remodeling in rabbit nuclear transfer (NT) embryos formed from metaphase II (MII) oocytes aged in vivo until 19 hr postcoitum (hpc), enucleated, and fused at 22-26 hpc with 32-cell morula blastomeres by means of electric fields, which also induced recipient oocyte activation. Post-activation events observed during the first hour following the fusion/activation pulse were studied in terms of chromatin, lamins, and microtubules, and revealed that transferred nuclei underwent premature chromosomes condensation (PCC) in only one-third of NT embryos and remained in interphase in others. Recipient oocytes were mostly not activated by manipulations performed before the fusion/activation pulse. The persistence of transferred nuclei in interphase resulted from the rapid progression of recipient oocytes to interphase after activation, suggesting that the cytoplasmic state of MII oocytes aged in vivo was poised for the approach to interphase. Studying microtubular organization in MII oocytes before nuclear transfer manipulations, we found that 19 hpc MII oocytes aged in vivo differed from 14 hpc MII oocytes (freshly ovulated) and from 19-hpc MII oocytes aged in vitro (collected at 14 hpc and cultured for 5 hr), notably by the presence of microtubule asters and tubulin foci or only tubulin foci dispersed throughout the cytoplasm. When PCC was avoided, remodeling of the transferred nucleus was well advanced 1 hr after nuclear transfer, and NT embryos developed better to the blastocyst stage.

Centrosomes with striated rootlets in rabbit zygotes.

An electron microscopic study of the rabbit zygote has shown the presence of numerous paracrystalline structures (PSs) around the pronuclei. The majority of these structures are situated in the narrow space between pronuclei. The PSs during interphase are associated with small dense knobs, and filamentous material; some of them, namely those situated in the internuclear space, are also associated with striated rootlets. The PS and its appendages form a complex which nucleates microtubules during interphase and phase M. The structure of these complexes changes with the cell cycle. Striated rootlets disappear at G2/M. Dense knobs and filamentous material separate from the PS, become loose and associate with numerous microtubules at the poles of the first mitotic spindle. PSs and their associated structures are considered to be a newly discovered morphological form of the centrosome.

Conditions favoring RNA polymerase I transcription in permeabilized cells.

RNA synthesis can be detected in nuclei using modified RNA precursors (Br-UTP) introduced in permeabilized cells. Surprisingly, RNA pol I transcripts are detected only after inhibition of RNA pol II or salt enhancement of RNA pol I activity. By modifying a previously reported protocol, we found that RNA pol I transcripts can be detected selectively or simultaneously with RNA pol II transcripts without any drug treatment. Removing glycerol from the permeabilization and transcription buffers and improving the permeabilization using Triton X-100 revealed RNA pol I transcription in two cell lines (mammalian and Xenopus) and in isolated mouse oocytes. The transcripts were most probably rRNA because they were detected in the nucleoli, digested by RNase, sensitive to actinomycin D, and resistant to alpha-amanitin. We found by microinjection of the Br-UTP precursors in living cells that low ionic strength allows the detection of RNA pol I transcription. Electron microscopy of mouse oocytes showed that the "looseness" of the nucleolar organization is associated with the detection of the RNA pol I transcription; this detection does not necessarily need nucleolar disorganization. The data obtained with both permeabilized cells and microinjections of RNA precursors in the absence of glycerol support the hypothesis that the degree of hydration of the cell plays a role in RNA pol I transcription.

Fusion with activated mouse oocytes modulates the transcriptional activity of introduced somatic cell nuclei.

We have analyzed the transcriptional activity of somatic cell nuclei fused with artificially activated mouse oocytes. Two types of somatic cells have been used: transcriptionally silent thymocytes, obtained from the thymus of newborn mice, and transcriptionally active murine erythroleukemia cells (MEL) from in vitro culture. Cells were fused with activated oocytes, either less than 1 h, or 3 h post-ethanol-treatment. When the fusion occurred early after activation (1 h or less), the transferred somatic cell nuclei reacted by nuclear envelope breakdown (NEBD), which exposed their chromatin to the cytoplasmic environment of the oocytes, and the reconstituted nuclei underwent significant ultrastructural remodeling. No transcriptional activity was ever detected in these reconstituted nuclei during the subsequent 3-4 h of culture of the resulting hybrid cells. In the case of MEL, this means that transcriptional activity ceased as soon as they entered the cytoplasm. In contrast, somatic nuclei which entered the cytoplasm of activated oocytes 3 h postactivation did not undergo NEBD and their remodeling was less pronounced. In contrast to the first group, these nuclei were transcriptionally active during the following 3-4 h of culture. In both cases, the female pronucleus remained transcriptionally silent.

Development-dependent localization of nuclear antigens in growing mouse oocytes.

We have analyzed the distribution of nuclear and nucleolar proteins during the period of oocyte's growth. Oocytes were isolated mechanically or enzymatically from ovaries of juvenile mice of various ages (from 1 to 28 days after birth). Small nuclear ribonucleoproteins (snRNPs), the splicing factor SC-35, and a protein linked to cell proliferation (p-120) were detected by indirect immunofluorescence. snRNP distribution is consistent with the prophase state of oocyte's nuclei, while SC-35 (and p-120) exhibit a "speckled" distribution throughout the entire period of growth. The number of speckles (or foci) appears maximal around 10 days after birth, i.e., in the period of maximal transcriptional activity, and is sensitive to alpha-amanitin treatment. On the other hand, the immunofluorescent distribution of of nucleolin and p-103 (a nucleolar marker of the granular component) is compared to the ultrastructural distribution of the granular component analyzed by electron microscopy on oocytes of the same age.

Relationship between sperm nucleus remodelling and cell cycle progression of fragments of mouse parthenogenotes.

Nucleate and anucleate fragments of parthenogenetically activated mouse oocytes, as well as cybrids obtained by fusion of anucleate fragments (cytoplasts) of maturing and activated matured oocytes were fertilized at different time after activation. Remodelling of the sperm nucleus was studied by electron microscopy at 1.5 and 3 h after fertilization and, in addition, at 14 h in cybrids. Results show that 1) the nuclear envelope of the sperm nucleus can break down when the insemination takes place after the end of M-phase, but the capacity of the parthenote cytoplasm to remodel the sperm nucleus is restricted in time. 2) Male chromatin can decondense within the old, unbroken nuclear envelope, but in such cases formation of a male pronucleus, one of the two nuclei of zygote possessing inactive nucleoli, is never observed.

Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics.

After labelling DNA with the specific vital fluorophore Hoechst 33342, oocytes, isolated by puncture from antral follicles in adult mice, have two essentially different configurations of their nuclear fluorescence images. These have been called SN (where the nucleolus is surrounded by chromatin) and NSN (where the nucleolus is not surrounded by chromatin). Intermediate configurations are also found, although with a lower frequency. The proportion of each class is on the average equal and depends neither on the presence of cumulus cells nor on the age of the mouse. Electron microscopy confirms several ultrastructural differences between these two nuclear configurations, namely, the structure of the nucleolus, which is vacuolated in NSN-type and compact in SN-type oocytes. Using video-enhanced fluorescence microscopy at low level of excitation light, we could follow directly in vitro the meiotic maturation of both classes, without impairing their viability. We show that in germinal vessicle (GV) state, the chromatin does not change from one configuration into the other and that both classes are able to mature to metaphase II, although the maturation has slightly different characteristics.

Inhibition of protein kinases by 6-dimethylaminopurine accelerates the transition to interphase in activated mouse oocytes.

Mouse oocyte activation is followed by a peculiar period during which the interphase network of microtubules does not form and the chromosomes remain condensed despite the inactivation of MPF. To evaluate the role of protein phosphorylation during this period, we studied the effects of the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP) on fertilization and/or parthenogenetic activation of metaphase II-arrested mouse oocytes. 6-DMAP by itself does not induce the inactivation of histone H1 kinase in metaphase II-arrested oocytes, and does not influence the dynamics of histone H1 kinase inactivation during oocyte activation. However, 6-DMAP inhibits protein phosphorylation after oocyte activation. In addition, the phosphorylated form of some proteins disappear earlier in oocytes activated in the presence of 6-DMAP than in the activated control oocytes. This is correlated with the acceleration of some post-fertilization morphological events, such as sperm chromatin decondensation and its transient recondensation, formation of the interphase network of microtubules and pronuclear formation. In addition, numerous abnormalities could be observed: (1) the spindle rotation and polar body extrusion are inhibited; (2) the exchange of protamines into histones seems to be impaired, as judged by the morphology of DNA fibrils by electron microscopy; (3) the formation of a new nuclear envelope around the sperm chromatin proceeds prematurely, while recondensation is not yet completed. These observations suggest that the 6-DMAP-sensitive kinase(s) is (are) involved in the control of post-fertilization events such as the formation of the interphase network of microtubules, the remodelling of sperm chromatin and pronucleus formation.

Changes in embryonic 8-cell nuclei transferred by means of cell fusion to mouse eggs.

Metaphase II and activated mouse oocytes were fused with 8-cell blastomeres, and morphological changes in the transferred nuclei were followed using light and electron microscopy. In metaphase II oocytes, blastomere nuclei underwent premature chromosome condensation (PCC) typical for S-phase nuclei: chromatin pulverization. Then an abortive spindle was formed without evident microtubule organizing centers. Blastomere chromosomes condensed to a lesser degree than meiotic chromosomes and lacked mature functional, trilaminar kinetochores. After parthenogenetic activation of these oocytes, blastomere chromosomes followed, in synchrony with oocyte chromatin, a similar route of changes (anaphase, telophase) and then reformed interphase nuclei of the pronuclear type. Remodeling of 8-cell nucleus thus occurred, but the integrity of the chromatin set was frequently disturbed by formation of micronuclei. If blastomere fusion with oocytes was done close to activation (either before or after parthenogenetic stimulation), the chances of remodeling of the nuclei decreased, because PCC was not regularly induced in all oocytes. In hybrids produced 60 min or later after oocyte activation, blastomere nuclei were maintained in interphase without any structural modifications. Multiple experiments in the mouse have shown that the nuclei from 8-cell stage transferred to enucleated oocytes and egg cells are not capable of substituting for pronuclear functions. Possible reasons for impaired functional reprogramming of 8-cell nucleus in the mouse are discussed in light of our present findings on the morphology of nuclei transferred before and after oocyte activation.

Chromatin behaviour under influence of puromycin and 6-DMAP at different stages of mouse oocyte maturation.

Preovulatory mouse oocytes were cultured in vitro up to each subsequent stages of maturation: germinal vesicle (GV), germinal vesicle breakdown (GVBD), groups of not yet individualized bivalents, circular bivalents, late prometaphase I, metaphase I, anaphase I and telophase I. The stages were identified in living oocytes by fluorescence microscopy using Hoechst 33342 as a specific vital dye. Oocytes from each stage of development developed in vitro and ovulated metaphase II oocytes were subsequently cultured in the presence of puromycin or 6-dimethylaminopurine (6-DMAP), an inhibitor of protein phosphorylation. The effects on chromatin of these drugs were studied during and at the end of culture by fluorescence and electron microscopy. We found that puromycin and 6-DMAP stop meiosis when applied at all stages of oocyte maturation, except for metaphase II. Oocytes at this stage are activated by puromycin. Reaction of the oocytes to the two drugs is different at GV and at metaphase II. All of the other stages react to the drugs by chromatin compaction, which can be followed by chromatin decondensation to form a nucleus. Our results suggest that late prophase chromatin condensation, bivalent individualization and retention of their individuality, as well as individualization of monovalents from telophase and retention of their individuality at metaphase II, are dependent on protein phosphorylation. The events occurring between metaphase I and telophase I are independent of protein synthesis and phosphorylation. The events occurring between metaphase II and formation of the nucleus are independent of protein synthesis.

Dynamics of paternal chromatin changes in live one-cell mouse embryo after natural fertilization.

Using video-enhanced fluorescence microscopy, we describe in live mouse zygotes the paternal chromatin changes undergone after fertilization. We focus on the sperm recondensation process and the formation of the paternal pronucleus, in relationship with the progression of maternal chromatin. Chromatin is labeled with the vital fluorophore Hoechst 33342. Our conditions of dye concentration and irradiation allow a continuous following of the dynamics of changes without major perturbation. We combine these observations with ultrastructural analysis performed by electron microscopy of the same eggs fixed at chosen stages. We show that the highly recondensed state corresponds to the appearance of the nuclear envelope and therefore the beginning of the pronuclear stage.

Sperm penetration into immature mouse oocytes and nuclear changes during maturation: an EM study.

The ultrastructure of oocyte and sperm nuclei was studied in mouse ovarian oocytes inseminated in vitro and cultured for 1 1/2 and 3 h in a medium containing dbcAMP or lacking the maturation inhibitor. In oocytes blocked at the germinal vesicle (GV) stage, certain maturation-linked changes were noted. Sperm apposition and sperm-oocyte fusion were similar to that during fertilization of ovulated oocytes. The sperm nucleus and its nuclear envelope remained intact after penetrating into the ovarian oocyte. One and a half h after removal of the drug (time 0 of maturation) the germinal vesicle (GV) and sperm nucleus remained intact. In oocytes maturing for 3 h, the nuclear envelopes of the GV and sperm nucleus had fragmented. The NE of the oocyte formed quadruple membranes while the NE of the sperm remained as flat vesicles. Oocyte chromatin condensed to form chromosomes, whereas at the same time the sperm chromatin was in the process of decondensation and was surrounded by fragments of the sperm NE. The sperm chromatin, composed of DNA complexed with protamines, consisted of thin fibrils; the individual fibrils measured 3.8 nm in diameter. Near the penetrated spermatozoa only occasional Mts were detected which were not related to the proximal centriole which was recognizable in the neck-piece of the flagellum. Thus in mouse oocytes the introduced sperm centriole is not capable of behaving as a centrosome and organizing microtubules in the form of an aster.

Remodeling of mouse thymocyte nuclei depends on the time of their transfer into activated, homologous oocytes.

The potential of parthenogenetically activated mouse oocytes to remodel somatic cell nuclei was studied by ultrastructural means using oocyte-thymocyte hybrids. Complete nuclear remodeling, initiated by nuclear envelope breakdown and chromosome condensation (which is followed by formation of pronucleus-like nucleus) is possible only during a short time gap between metaphase II and telophase of meiotic division. Maturation-promoting factor activity is high during this period. The thymocyte nucleus can follow the sequence of morphological changes only in concert with the development of the native nucleus and only after exposure of the chromatin to the ooplasm. If hybridization is effected with pronucleate oocytes, the thymocyte nucleus retains its interphase character but shows particular modifications in nucleolar morphology (identical to changes observed during reactivation of the nucleolus in stimulated lymphocyte) and in the activity of the nuclear envelope (blebbing). Thus the nucleus not exposed to maturation-promoting factor activity may be influenced by a 'programme' specific for oocyte (blebbing) and by a programme inherent in the introduced somatic cell nucleus.

'Blebbing' of the nuclear envelope of mouse zygotes, early embryos and hybrid cells.

In the mouse zygote and in two-cell stage embryos the inner leaflet of the nuclear envelope of pronuclei and that of blastomere and polar body II nuclei evaginate, forming multiple blebs within the perinuclear space, which contains a granular material. Blebbing exists only in oocytes activated by sperm in vivo or in vitro, or parthenogenetically by treatment with ethanol or puromycin. The germinal vesicle and an interphase nucleus formed after treatment of the oocyte at metaphase I by puromycin do not form blebs. Formation of blebs is specifically located in the cell cycle. The burst of the blebbing activity occurs during the first half of the cell cycle in one-cell embryos and in the earliest interphase period in the second cell cycle. Blebbing ceases from the beginning of the third cell cycle. The occurrence in the cytoplasm of 'double-layered' vesicles containing granular material resembling bleb contents and the disappearance of blebs from the nuclear envelope by the end of the cell cycle provide evidence that blebs represent a step in the transport of some material from the nucleus to the cytoplasm. Ethanolic phosphotungstic acid does not stain blebs, suggesting the absence of basic protein in their contents. Blebbing can be induced in somatic (thymocyte) and embryonic (blastomere of 8-cell stage embryo) nuclei following cell hybridization with activated oocytes. Their response to the oocyte cytoplasm by initiating blebbing depends on: (1) the position of the host cell in its cell cycle at the moment of hybridization, and (2) the time spent by the foreign nuclei in the host cytoplasm following cell fusion. If donor nuclei are introduced close to the time of activation, they start to produce blebs at the time corresponding to the initiation of blebbing by the female pronucleus in the first cell cycle. If foreign nuclei are introduced a few hours after activation they must be incubated in the host cytoplasm for some time before initiation of bleb formation, provided that the host pronucleus has initiated blebbing by that time. The existence of blebbing in nuclei formed only after oocyte activation, and the timing and the general occurrence of this event during the earliest cleavage stages of almost every mammalian embryo, suggest that this special nucleocytoplasmic transport plays a specific role at the beginning of development.