In nearly all somatic cells, the ribosome biosynthesis is a key activity. The same is true also for mammalian oocytes and early embryos. This activity is intimately linked to the most prominent nuclear organelles — the nucleoli. Interestingly, during a short period around fertilization, the nucleoli in oocytes and embryos transform into ribosome-biosynthesis-inactive structures termed nucleolus-like or nucleolus precursor bodies (NPBs). For decades, researchers considered these structures to be passive repositories of nucleolar proteins used by the developing embryo to rebuild fully functional, ribosome-synthesis competent nucleoli when required. Recent evidence, however, indicates that while these structures are unquestionably essential for development, the material is largely dispensable for the formation of active embryonic nucleoli. In this mini-review, we will describe some unique features of oocytes and embryos with respect to ribosome biogenesis and the changes in the structure of oocyte and embryonic nucleoli that reflect this. We will also describe some of the different approaches that can be used to study nucleoli and NPBs in embryos and discuss the different results that might be expected. Finally, we ask whether the main function of nucleolar precursor bodies might lie in the genome organization and remodelling and what the involved components might be.

Introduction

The nucleolus is the most prominent nuclear organelle in almost all cells, including mammalian oocytes and embryos. It is primarily responsible for the ribosome production. At the same time, additional non-canonical functions have been described and the list of these functions is slowly but constantly growing. However, because nucleoli are primarily associated with the ribosome biogenesis, elucidating the mechanism of the non-ribosome-biogenesis-related functions is often tricky and challenging. Indeed, ribosome biogenesis is a vital cellular function and under some conditions, the synthesis of ribosomal proteins accounts for up to nearly 10% of total protein synthesis [1–4]. The difficulty in dissecting the canonical and non-canonical functions inevitably calls for new approaches and alternative cellular models. Based on the methodological approach and the system's attributes, however, these can give seemingly different results. In this mini-review, we will focus on mammalian oocytes and embryos as these offer a unique opportunity to study the ribosome-biogenesis-independent processes due to the fact that during a very specific period in development, ribosome production is spontaneously shutdown.

General features of nucleoli

Although the nucleolus is intimately associated with the action of RNA polymerase I which transcribes ribosomal genes (rDNA), it is apparent that the activity of all three polymerases is required for the formation and/or maintenance of this organelle and its composition. While RNA polymerase I produces most of the ribosomal RNAs (rRNAs) from the rDNA genes, the messenger RNAs encoding RNA polymerase I subunits and other nucleolar proteins are produced by RNA polymerase II, and the remaining rRNA is produced by RNA polymerase III. In turn, the rRNAs and additional proteins in the form of a ribosome produce RNA polymerase subunits. Therefore, nucleolar structure, function and activity integrates and reflects all the various aspects of the cell's (metabolic) state [5–9].

Because the nucleolus lacks a distinct membrane separating it from the rest of the nucleoplasm and because it is naturally protein dense, one of the original notions was that it can be used by cells to sequester or concentrate various proteins as part of their regulation. Indeed, the first detailed nucleolar proteome identified a large fraction of proteins that are not strictly associated with ribosome biogenesis [10,11]. Since then, nucleoli have been implicated in various cellular processes [12,13].

Nucleoli in mammalian oocytes and embryos

Mammalian oocytes and embryos are highly specialized cells with a unique biology. Initially, oocytes are not much larger than other cells in the body. However, at a specific time oocytes are recruited and enter a growth phase. During this phase, they accumulate an enormous amount of different materials such as mRNAs and proteins. During the growth phase, nucleolar morphology does not differ significantly from that which can be found in somatic cells; all the nucleolar sub-components, i.e. fibrillar centers, granular and dense-fibrillar components, can be identified [14,15]. As expected, and in agreement with results obtained in somatic cells, the major RNA species found in oocytes is rRNA [16]. However, as oocytes reach their full size, e.g. ∼70–80 µm in mice or 100–120 µm in humans [17–19], both RNA polymerase II and I transcription ceases [20]. This coincided with a change in the oocyte chromatin morphology. The process is generally known as the non-surrounded nucleolus (NSN)- to- surrounded nucleolus (SN) transition, during which the oocytes gain their full developmental competence [21,22]. This is accompanied by extensive chromatin condensation [15] and the transformation of the nucleolus into a so-called nucleolus-like body (NLB; Figure 1). NLBs have no inner structure and, under the electron microscope, they appear as spherical dense-fibrillar masses inside the oocyte nucleus, called germinal vesicles [14,15]. Although the details of this process remain elusive, empirically, if the oocytes fail to reach the SN stage, they will have a reduced maturation capacity and the embryos arising from such oocytes rarely progress beyond the two-cell embryonic stage [23–25]. Whether NLBs play an active role in this process is unknown, but their function as a DNA structural support, at least, is obvious. When oocytes start to mature and resume meiosis, concomitantly with the disassembly of the germinal vesicle, the NLB material becomes dispersed in the cytoplasm. Unlike in somatic cells, oocyte meiotic divisions are intrinsically asymmetrical. Therefore, the vast majority of NLB material is retained by the large egg cell and only a fraction is lost in the form of the polar body.

The dynamic nuclear and nucleolar morphology during early embryogenesis.

Figure 1.
The dynamic nuclear and nucleolar morphology during early embryogenesis.

Summarizes changes in the nucleus/nucleolus morphology during the final phases of oogenesis and during early embryonic development. In growing oocytes, the chromatin is dispersed throughout the nucleus, DAPI-dense chromocenters (top) can be observed and the rRNA synthetic activity is high. As the oocytes reach their full size, their chromatin condenses and closely associates with transcriptionally inactive ‘nucleolus-like bodies’ (NLBs). The majority of nucleolar material is retained by the huge egg cell around the time of fertilization. The NLB material reappears again in both parental pronuclei in the form of ‘nucleolus precursor bodies’ (NPBs). At this phase, these spheres serve as the major structural support for centric and pericentric chromatin, no chromocenters can be detected. Chromocenters form at later embryonic stages coinciding with the time of embryonic genome activation (EGA). Finally, around the blastocyst stage, the individual nuclei contain fully differentiated nucleoli and multiple chromocenters and the overall morphology is reminiscent of that of somatic cells.

Figure 1.
The dynamic nuclear and nucleolar morphology during early embryogenesis.

Summarizes changes in the nucleus/nucleolus morphology during the final phases of oogenesis and during early embryonic development. In growing oocytes, the chromatin is dispersed throughout the nucleus, DAPI-dense chromocenters (top) can be observed and the rRNA synthetic activity is high. As the oocytes reach their full size, their chromatin condenses and closely associates with transcriptionally inactive ‘nucleolus-like bodies’ (NLBs). The majority of nucleolar material is retained by the huge egg cell around the time of fertilization. The NLB material reappears again in both parental pronuclei in the form of ‘nucleolus precursor bodies’ (NPBs). At this phase, these spheres serve as the major structural support for centric and pericentric chromatin, no chromocenters can be detected. Chromocenters form at later embryonic stages coinciding with the time of embryonic genome activation (EGA). Finally, around the blastocyst stage, the individual nuclei contain fully differentiated nucleoli and multiple chromocenters and the overall morphology is reminiscent of that of somatic cells.

In embryos, the NLB material reappears in nuclei after fertilization (Figure 1). With regard to what is known about the re-assembly of somatic nucleoli after mitosis, the emergence of structures similar to NLBs in parental pronuclei is interesting. Just like in full-grown oocytes and eggs, it is generally accepted that RNA polymerase I is not active immediately after fertilization. Moreover, oocyte NLBs have been shown to be depleted for rRNA [26], the RNA specie which could at least partly aid the re-assembly process [27,28]. At this stage, the structures are known as nucleolus precursor bodies (NPBs). The name stems from the observation that when ribosome production reinitiates with the onset of major embryonic genome activation (EGA) at later embryonic stages, active embryonic nucleoli are always found topologically associated with the NPB mass, either inside the mass or on its surface, depending on the species [29–32]. For these reasons, developmental biologists long considered NLBs and NPBs to be passive repositories where nucleolar components are stored. In this model, oocytes would simply store the nucleolar material in the form of NLBs and after fertilization, when the embryo needs to synthesize new ribosomes to fuel its development, the nucleolar components would be readily available. However, the repository role of NLBs/NPBs has never been really experimentally substantiated and in light of the recent results NLBs/NPBs seem to have additional and perhaps even more important function(s), which manifest well before EGA and the embryonic nucleologenesis.

Composition of NLBs

Over the decades, researchers have tried to elucidate the composition of NLBs in a simple presumption that this information will provide the answer as to what the function of this huge protein structure might be. Because of the morphological link to active oocyte nucleoli, mainly nucleolar proteins and rRNA were in focus. Interestingly, several of the canonical nucleolar components, such as 18S and 28S rRNA, ITS2 (internal transcribed spacer 2; part of the pre-rRNA), UBTF or RPL26, become undetectable in NLBs of developmentally competent SN oocytes, which coincides with the RNA polymerase I transcription shutdown [26]. Since somatic nucleoli contain a large fraction of proteins not directly involved in ribosome biogenesis, the same is likely true for NLBs and NPBs as indicated by the first proteomic analysis of NLBs [33]. Unfortunately, due to the difficulty of obtaining a sufficient amounts of the NLB material with a sufficient purity, the NLB/NPB proteome is still far from perfect. The identified components are summarized in Table 1.

Table 1.
Summary of identified NLB and NPB components
Protein associated with NLBs/NPBsSpeciesReference
Fibrillarin, NPM1 (B23), nucleolin (C23), RNA polymerase I, UBTF (UBF), coilin Bovine Fair et al. [103
nucleolin, PGRMC1 Bovine Terzaghi et al. [104
SRSF2 (SC35), Sm antigen Porcine Kopecny et al. [105
NPM1 Porcine Hyttel et al. [106
p130, PAF53, UBF Porcine Bjerregaard and Maddox-Hyttel, [107
Fibrillarin Porcine Laurincik et al. [108
Nucleolin Porcine Ogushi et al. [49
UBF, RPA116, fibrillarin Mouse Zatsepina et al. [63
Fibrillarin, coilin Mouse Zatsepina et al. [64
UBF, fibrillarin, nucleolin Mouse Shishova et al. [26
Oct4, SRSF2, RNA polymerase II Mouse Parfenov et al. [109
Nucleoplasmin2 (NPM2) Mouse Inoue and Aoki [45
NPM1 Mouse Ogushi and Saitou [59
NPM1, nucleolin, fibrillarin, UBF Mouse Fulka and Langerova [57
Histone H3.3, NPM1 Mouse Lin et al. [110
Multiple (most proteins identified not confirmed by other methods) Mouse Ogushi et al. [33
RNA found in NLBs/NPBs 
RNA (species undetermined) Mouse Kopecny et al. [111
RNA (species undetermined, but not 18S/28S rRNA and not ITS1/ITS2) Mouse Shishova et al. [26]; Shishova et al. [52
snRNA (small nuclear RNAs) Rat Kopecny et al. [112
 Porcine Kopecny et al. [105
RNA (species undetermined) Rabbit Sutovsky et al. [113
Protein associated with NLBs/NPBsSpeciesReference
Fibrillarin, NPM1 (B23), nucleolin (C23), RNA polymerase I, UBTF (UBF), coilin Bovine Fair et al. [103
nucleolin, PGRMC1 Bovine Terzaghi et al. [104
SRSF2 (SC35), Sm antigen Porcine Kopecny et al. [105
NPM1 Porcine Hyttel et al. [106
p130, PAF53, UBF Porcine Bjerregaard and Maddox-Hyttel, [107
Fibrillarin Porcine Laurincik et al. [108
Nucleolin Porcine Ogushi et al. [49
UBF, RPA116, fibrillarin Mouse Zatsepina et al. [63
Fibrillarin, coilin Mouse Zatsepina et al. [64
UBF, fibrillarin, nucleolin Mouse Shishova et al. [26
Oct4, SRSF2, RNA polymerase II Mouse Parfenov et al. [109
Nucleoplasmin2 (NPM2) Mouse Inoue and Aoki [45
NPM1 Mouse Ogushi and Saitou [59
NPM1, nucleolin, fibrillarin, UBF Mouse Fulka and Langerova [57
Histone H3.3, NPM1 Mouse Lin et al. [110
Multiple (most proteins identified not confirmed by other methods) Mouse Ogushi et al. [33
RNA found in NLBs/NPBs 
RNA (species undetermined) Mouse Kopecny et al. [111
RNA (species undetermined, but not 18S/28S rRNA and not ITS1/ITS2) Mouse Shishova et al. [26]; Shishova et al. [52
snRNA (small nuclear RNAs) Rat Kopecny et al. [112
 Porcine Kopecny et al. [105
RNA (species undetermined) Rabbit Sutovsky et al. [113

The table does not include proteins localized to NLBs/NPBs by introducing exogenous mRNA encoding their fluorescent-tagged versions or proteins localized to embryonic/growing oocyte nucleoli. While some of the proteins were repeatedly found in NLBs/NPBs by independent groups, the localization of others was not reported beyond a single study. Also note that while the first NLB proteome reported by Ogushi and colleagues in 2017 [33] identified a substantial number of proteins not directly involved in ribosome biogenesis, with the exception of NPM2 and nucleolin, it failed to detect the already known and corroborated NLB/NPB components.

The different approaches to study the nucleolar components in embryogenesis

When assessing the function of an organelle, analyzing knockout animal models for a gene(s) of interest is usually the method of choice. However, when working with early embryos, this approach has serious limitations. The main reason is the very basic biology of eggs and early embryos. As mentioned, oocytes accumulate a substantial amount of protein and RNA. Thus, prior to EGA, embryos rely on the maternal stores of these molecules. To assess the role of a gene soon after fertilization, one has to target the female (mother) and ablate the gene product in eggs. However, if the gene of interest is an essential one, as is often the case for genes involved in ribosome biogenesis, generating full knockout females is impossible since the homozygous deletion will be lethal. The lethality has to be thus addressed by maintaining the deletion in a heterozygous state but this naturally preserves the protein and mRNA pool present in the oocytes and eggs. To date, several mouse lines heterozygous for a deletion of genes involved in ribosome biogenesis, such as fibrillarin or RNA polymerase I subunits, have been reported [34–37]. Other lines targeting ribosomal proteins, such as Rpl24, Rps6 or Rpl22, are also available [38–42]. Additional information can also be obtained from the International Mouse Phenotyping Consortium (https://www.mousephenotype.org). In summary, with a few exceptions, the homozygote deletions are indeed incompatible with life and the null embryos generated by heterozygote mating often arrest in development around the blastocyst stage or only slightly later. The limitation of this approach is that null embryos can thus tell us if the selected gene is essential and when the maternal pool is depleted, not if the gene product is required by embryos prior to EGA or whether embryos tend to rely on the maternal mRNA or rather the protein.

A more refined approach is to use conditional knockouts targeting the female germline (mother). In this scheme, the gene of interest, or its part, is usually flanked by loxP sites. Then, the animal is mated to a so-called driver mouse. When focusing on oocytes and embryos, ZP3-Cre or Gdf9-Cre driver lines are typically used [43]. However, even this approach has its limitations and again will not be useful if the gene of interest is essential to basic cellular function since the females will not produce any eggs from which embryos could be generated. Nevertheless, this approach was successfully used to characterize Npm2 (nucleoplasmin 2), the oocyte-specific nucleoplasmin paralog [44], which constitutes the majority of the NLB/NPB mass [33,45].

Additional approaches frequently used in oocytes, eggs and early embryos, such as siRNA injection or the recently developed Trim-Away method [46], do not offer the solution either as the NLB/NPB proteins are not readily accessible due to the dense structure of NLBs/NPBs. Moreover, the confirmed NLB components are also rather abundant and likely relatively stable [47]. All these characteristics make the use of the above-mentioned approaches problematic [47]. It should also be mentioned that irrespectively of the method used, organelles are complex multi-protein structures and eliminating a single component might not give the answer as to what the overall function of the organelle might be. Thus, to answer whether NLBs and NPBs are essential at the final stages of oogenesis and for the development in pre-EGA embryos, an alternative approach has to be used.

It has been shown that the NLB mass can be microsurgically removed from the germinal vesicles and pronuclei in zygotes without causing major damage to either the nucleus or the chromatin inside [48]. During this procedure, the NLB/NPB is slowly aspirated from the germinal vesicle or pronucleus using a fine glass pipette. This process has been termed ‘enucleolation’ and was first applied to porcine and later to mouse and human oocytes [49,50]. It is conceivable that the success of this procedure is correlated with RNA polymerase I activity: In oocytes, when transcription is spontaneously shut down, the rDNA is reorganized and extruded from NLBs [51,52]. This probably allows NLBs to be removed without causing DNA damage. Indeed, even growing, actively transcribing oocytes can be enucleolated [53,54] when treated with actinomycin D, a well-known DNA-intercalating agent that is highly effective against RNA polymerase I [55,56].

Experiments with porcine and mouse oocytes showed that removing NLBs has no effect on the rate of oocyte maturation: comparable numbers of control, non-manipulated and enucleolated oocytes were able to complete meiosis and reach the metaphase II stage [48,57]. Furthermore, when analyzed in more detail, it was shown that enucleolated oocytes complete meiosis with the correct number of chromosomes [57]. Thus, NLBs are dispensable for meiotic division and its regulation.

The predominant use of mouse oocytes proved essential for further functional study of NLBs. The relative ease with which the biological material can be obtained, together with the availability of well-advanced reproductive technologies and detailed genetic information in the mouse, has been essential for advancing our understanding of the pre-EGA NLB function. This finally led to the rejection of the long-accepted dogma in developmental biology that NLBs serve exclusively as passive repositories of nucleolar protein, which is later utilized by the developing embryo [57,58]. The experiments that led to the shift in our understanding of NLB function will be described in the next sections.

It has been shown that when NLBs are microsurgically removed from oocytes, these are then matured to metaphase II and then fertilized, the resulting embryos fail to replace the NLB material, i.e. no structure equivalent to NPBs can be observed in the pronuclei, even at the ultrastructural level, and embryos arrest at the two-cell stage [49,59] (Figure 2). Although a fraction of the original NLB material remains associated with the maternal chromosomes, this is not sufficient to support the development [57]. The NLB material can also be introduced back: when reinjected into metaphase II eggs originating from enucleolated oocytes, the embryos are able to develop further [49]. A simple explanation for these results is that the main function of NLBs, and then NPBs, is to indeed stockpile the nucleolar material, and this material is then required to build functional embryonic nucleoli. If this material is absent, embryos are unable to produce new ribosomes and arrest in development. While this explanation seems logical, an important question is whether mammalian eggs actually need to stockpile the ribosome biogenesis-related proteins for their later use in embryogenesis and, in turn, whether embryos do indeed need the full range of proteins and their exact quantity, which is found in eggs. This might not be the case because in contrast with species such as Xenopus, Danio or Drosophila, which undergo many cell divisions depending on the maternal stores, EGA occurs quite early in mammals [60–62].

NLB and NPB removal and transplantation schemes and the effect on early development.

Figure 2.
NLB and NPB removal and transplantation schemes and the effect on early development.

Depicts the major NLB/NPB removal and transplantation experiments and their developmental outcome in the mouse model. (A) Under normal conditions, the NLB material found in germinal vesicle stage oocytes reappears after fertilization and persists until around the eight-cell stage [57]. (B) When NLBs are removed, this material is not replaced by embryos and these arrest typically around two-cell stage [49,59]. No live offspring was ever reported [49,58,59]. (C) When the NLB material was removed from oocytes, but introduced back prior to fertilization, live offspring was obtained in 13% of cases. In controls, where a small amount of the nucleoplasm was removed, 8% of the embryos was able to give rise to live pups (‘sham-operated’). This control simulated the nuclear envelope rupture, which occurs during enucleolation [49]. This result indicated that the NLB material is critical for embryogenesis but not oocyte maturation. (D) When NPBs were removed from zygotes ∼10 h post fertilization, 32% of the manipulated embryos were able to develop to term. Again, a small amount of nucleoplasm was removed from controls. In this case, 49% of live offspring was obtained (‘sham-operated’) [58]. These experiments were crucial in determining the timing when NPBs are essential for development, i.e. during the first 10 h but not later. (E) Finally, when NLBs were removed from oocytes, these were them matured in vitro, injected with a high dose of Npm2 mRNA and fertilized after ∼4 h post injection, live animals were obtained in 21% of cases. When a control eGfp mRNA of the same high concentration or low concentration of Npm2 mRNA was injected to previously enucleolated eggs, no offspring was obtained [33]. For comparison, when a standard intracytoplasmic sperm injection (ICSI) is performed, ∼50–60% of live animals can be expected [114,115]. After in vitro maturation and in vitro fertilization, between 21% and 34% of live offspring was reported using F1 hybrid mice [116].

Figure 2.
NLB and NPB removal and transplantation schemes and the effect on early development.

Depicts the major NLB/NPB removal and transplantation experiments and their developmental outcome in the mouse model. (A) Under normal conditions, the NLB material found in germinal vesicle stage oocytes reappears after fertilization and persists until around the eight-cell stage [57]. (B) When NLBs are removed, this material is not replaced by embryos and these arrest typically around two-cell stage [49,59]. No live offspring was ever reported [49,58,59]. (C) When the NLB material was removed from oocytes, but introduced back prior to fertilization, live offspring was obtained in 13% of cases. In controls, where a small amount of the nucleoplasm was removed, 8% of the embryos was able to give rise to live pups (‘sham-operated’). This control simulated the nuclear envelope rupture, which occurs during enucleolation [49]. This result indicated that the NLB material is critical for embryogenesis but not oocyte maturation. (D) When NPBs were removed from zygotes ∼10 h post fertilization, 32% of the manipulated embryos were able to develop to term. Again, a small amount of nucleoplasm was removed from controls. In this case, 49% of live offspring was obtained (‘sham-operated’) [58]. These experiments were crucial in determining the timing when NPBs are essential for development, i.e. during the first 10 h but not later. (E) Finally, when NLBs were removed from oocytes, these were them matured in vitro, injected with a high dose of Npm2 mRNA and fertilized after ∼4 h post injection, live animals were obtained in 21% of cases. When a control eGfp mRNA of the same high concentration or low concentration of Npm2 mRNA was injected to previously enucleolated eggs, no offspring was obtained [33]. For comparison, when a standard intracytoplasmic sperm injection (ICSI) is performed, ∼50–60% of live animals can be expected [114,115]. After in vitro maturation and in vitro fertilization, between 21% and 34% of live offspring was reported using F1 hybrid mice [116].

It has been shown that several nucleolar proteins are, at least partially, degraded during oocyte maturation and their levels often decline further following fertilization [47,57,63,64]. This rather speaks against the need to accumulate the RNA polymerase I machinery by eggs to fuel the embryonic ribosome biogenesis. If this was so, one would expect the level of the proteins to be maintained during the RNA polymerase I inactivity but this is not the case [47]. Where analyzed, the respective mRNAs behave similarly [3,57]. Accordingly, the total number of ribosomes decreases during the period prior to EGA [3,65].

Furthermore, a set of elegant experiments by Kyogoku et al. [58] showed that when NPBs are removed ∼10 h post fertilization, the manipulated mouse embryos are able to develop to term despite the absence of the NPB material (Figure 2). This time point is well before EGA and de novo rRNA synthesis. These experiments indicate that, while being essential for development, the reestablishment of embryonic nucleoli and ribosome production is functionally largely independent of NLBs and NPBs. Concomitantly with the maternal materials and ribosome clearance, the embryo likely synthetizes new nucleolar proteins based on maternal mRNAs and these might be sufficient for the initial embryonic ribosome production [3,65,66]. The details on how the first wave of embryonic ribosomes is produced in the absence of the NLB material are, however, unclear. Nevertheless, these experiments also show that the NLB material is critical in the early post-fertilization phase, well before EGA. Regarding the NPB's putative repository role, the factors ensuring the developmental competence would have to be released exclusively during the first few hours following fertilization. Additional experiments, however, show that the critical NLB/NPB factors are likely neither components of the RNA polymerase I machinery nor pre-ribosomes or ribosomes.

That NLBs do not serve as RNA polymerase I transcriptional machinery repository is indicated by inter-species NLB transfer experiments. It has been shown that rDNA transcription is highly species-specific, both in vitro and in vivo, i.e. mouse rDNA and human RNA polymerase I machinery are not compatible and vice versa [67–69]. Specifically, it is the SL1 factor, an essential component of the RNA polymerase I machinery, which is composed of TBP (TATA-binding protein) and several TAFs (TBP-associated factors), that provides the species-selectivity [70]. The other components, e.g. core RNA polymerase I or upstream binding factor UBF (nucleolar transcription factor 1, UBTF), can substitute for each other across species [70]. In light of these results, one would hardly expect the porcine rDNA promoter and the mouse NLB components to show compatibility. In agreement, experience with inter-species nucleus transfer experiments between mouse and pig also supports this presumption; inter-species embryos, in general, do not contain well-formed active nucleoli [71,72]. Still, mouse NLBs can rescue the development of porcine embryos derived from previously enucleolated oocytes [73]. Naturally, the full-term development was not followed due to the technical challenges of using porcine embryos as a model. Nevertheless, these embryos progress well beyond enucleolation-associated developmental block. This result is striking and in light of these results, one would expect the opposite combination, i.e. transfer of porcine NLBs to mouse eggs originating from enucleolated oocytes, to also be developmentally permissive. Indeed, this combination is also developmentally permissive, however, only under the condition that the NLB material amount is adjusted based on the species origin of the oocyte [74,75]: Only when two porcine NLBs are transferred per one enucleolated mouse egg, the embryos are able to pass the developmental block and progress to the blastocyst stage at rates comparable to intraspecies-NLB transfer embryos, i.e. manipulated embryos containing one mouse NLB per one mouse enucleolated egg (M. Benc, submitted). Although it seems highly unlikely for the porcine rDNA promoter and mouse RNA polymerase I machinery, and vice versa, to be compatible, this has been neither directly investigated nor demonstrated. Nevertheless, it seems reasonable to assume that only a limited number of NLBs factors, or even a single protein, is needed by the embryos to pass the developmental block. This factor or factors should be evolutionary well conserved.

Given the morphological link between NLBs/NPBs and active oocyte nucleoli, it is possible that ribosomes or pre-ribosomal particles can also be found inside of these structures. However, this does not make the NLB material essential for embryogenesis as demonstrated recently. It has been shown that the enucleolation phenotype can be reversed by replacing the NLB material with artificially produced NPM2-only spheres. In 2017, Ogushi et al. [33] injected a high dose of Npm2 mRNA into previously enucleolated and then in vitro matured metaphase II eggs. When fertilized, the embryos formed NPB-like structures in their pronuclei and 20% of them was able to give rise to live animals. Given the technical difficulty of this experiment, the developmental rates are stunning (Figure 2). Thus, a simple presence of NPB-like structures was sufficient to restore the developmental competence. It seems logical that the NPM2-only spheres contain neither the full extent of factors nor their quantity normally found in NLBs or NPBs since this is removed well before the Npm2 mRNA injection. NPM2 itself has no known direct role in ribosome biogenesis. Instead, it is generally considered to be a histone chaperone [76–78]. Therefore, when investigating the pre-EGA NPB's essential function in embryogenesis one should look beyond the ribosome biogenesis.

Embryonic nucleoli and their effect on the genome

With the advent of more sophisticated microscopy techniques, it became apparent that the genome is not organized randomly in the nucleus, for review see [79,80]. As the largest sub-nuclear organelles, nucleoli seem to play an important role in the spatial organization of nuclei. One might intuitively envisage that such large structures could restrict the mobility of the DNA within the nucleus, thus possibly preventing long-distance DNA interactions or functioning as an epigenetic hub organizing epigenetically similar sequences [81,82]. Indeed, a recent study focusing on long-range DNA interactions showed that this is at least one of the effects of having nucleoli in the nucleus [81]. In somatic cells, the nucleolus is at the core of a unique nuclear environment where, as well as in the vicinity of the nuclear envelope, the majority of heterochromatin is located [82–84]. Heterochromatin is mostly defined as a subset of DNA regions, together with the associated proteins, that are mostly transcriptionally inactive and usually replicate during mid-to-late S-phase of the cell cycle [85].

As in somatic cells, also in early embryos, a subset of sequences is known to associate with NPBs. The interesting question is, how individual sequences associate with nucleoli or are retained in their vicinity? As for rDNA gene clusters, their association with these organelles is logical because the nucleolus is in principle the result of their activity. The localization of rDNA clusters has also been studied after fertilization. Even in mouse zygotes, where it is generally accepted, there is no RNA polymerase I activity, rDNA clusters associate with NPBs [86]. This association is not surprising for the female rDNA genes because they relatively recently participated in ribosome production and could be thus binding various nucleolar proteins, which can aid the association with NPBs. However, the situation is more complicated in the case of the male pronucleus: It is generally accepted that apart from the paternal genome, mature sperm contribute very little to the newly formed embryo. Moreover, the sperm DNA is compacted by protamines to almost a crystalline state leaving practically no room for other DNA-binding factors [87,88]. Whether fertilization-competent sperm contain rDNA-bound nucleolar factors, which could be responsible for the association of paternal rDNA clusters with NPBs, is arguable. Several nucleolar proteins were identified is some proteomic studies of fertilization-competent sperm, but not in others [89,90]. Irrespectively of this, even the paternal rDNA clusters were found in the vicinity of NPBs [86]. Because not all NPBs are associated with rDNA cluster under normal conditions, however, an interesting idea was put forward by Romanova and colleagues: These authors propose that NPBs are heterogeneous in their ability to recruit rDNA clusters [86]. While this idea definitely has merit, perhaps a more simple explanation lies in a certain degree of randomness of the association. Assuming that the surface of the NPB can topologically accommodate multiple rDNA clusters, why should one expect all NPBs to associate with rDNA clusters in the first place? Thus, whether NPB are indeed non-equivalent in their rDNA-recruiting capability or whether the association is caused by a random effect remains an open question.

However, the localization of rDNA gene clusters in pronuclei can be simply the result of a general tendency of centric and pericentric chromatin to associate with NPBs and thus be rather passive. In mice, rDNA clusters are distributed within the centromeric regions of several chromosomes [91–93]. These sequences progressively associate with NPBs in both pronuclei as they form and pass through individual sub-stages [94]. What exactly drives the association of centromeres with NPBs is unknown, but it seems to be important as it has been shown that when these regions are relocated from NPBs and artificially tethered to the nuclear periphery, the embryos fail to develop [95]. In Drosophila somatic cells the centromere clustering and their tethering to the nucleolus was shown to be important for genome stability. In this species, the centromere clustering was shown to depend on the nucleoplasmin-like protein (NLP) and the insulator CCCTC-binding factor (CTCF) [83]. Whether the same proteins are involved in the centromere positioning in mouse zygotes is unknown but it is clear that the association cannot, in principle, depend on rDNA clusters as these are located on only a subset of chromosomes [91,93], while the vast majority of chromosomes associate with NPBs via their centric and pericentric regions [94,96,97].

When NPBs are missing, the centric and pericentric chromatin collapses and forms clusters morphologically similar to chromocenters [33,59]. Just like somatic nucleoli, NPBs might thus serve as an epigenetic hub as their major constituent, NPM2, was shown to be important for the epigenetic remodelling of centric/pericentric chromatin after fertilization. This possibility first emerged in the Npm2 conditional knockout mice [44]. Here, structures equivalent to NPBs are missing in early embryonic nuclei and the knockout females were reported to be subfertile or infertile [44]. NPM2 is a member of the nucleoplasmin family and an oocyte-specific paralogue [78]. It was first identified in Xenopus where it was shown to have a histone-chaperone activity and to be essential for sperm head decondensation [76,77,98]. Although the phenotype of the mouse Npm2-null embryos is by no means as dramatic as in Xenopus (in mice, the male pronucleus forms), defects in chromatin remodelling were also detected [33,44]. Burns and colleagues observed the absence of a certain histone H3 epigenetic mark in peri-NPB chromatin in the Npm2-null mouse embryos [44]. Unfortunately, this result was obtained using a polyclonal antibody and the exact H3 residues affected were unspecified [44]. To date, which residues are affected is still unknown despite a significant profiling effort (Fulka, unpublished; and [59]). Irrespectively of the exact residue, further analysis uncovered prominent chromatin bridges in the centric region of chromosomes, delayed first embryonic mitosis and chromosome segregation problems in the Npm2-null embryos [33]. The same phenotype is also typical for embryos derived from enucleolated oocytes [33,57]. Thus, the chromatin remodelling activity linked to NLBs/NPBs seems critical. However, whether the observed effects can be attributed exclusively to the NPM2 chaperone activity is unclear as another histone chaperone, DAXX (death domain-associated protein 6), was found to be absent from the pronuclei when NPBs are missing [57]. DAXX was reported to be centromere- and telomere-specific H3.3 histone chaperone [99–101]. Unlike NPM2, DAXX is expressed in various tissues. In oocytes, neither DAXX nor its main interaction partner ATRX (alpha thalassemia/mental retardation syndrome X-linked) was found in NLBs/NPBs [57]. Thus, the absence is likely secondary.

Embryonic nucleoli — where do we go from here?

For several years, the prevailing notion in the field was that a detailed understanding of NLB/NPB composition would provide the answers as to what their essential function in development might be. While NLBs/NPBs obviously contain a full range of nucleolar factors, the rescue experiment performed by Ogushi and co-workers demonstrates that neither the RNA nor the full range of proteins present in NLBs and NPBs [26,33,102] are essential for successful embryonic development. Concomitantly, the question is whether NPM2 is the only factor responsible for the resulting developmental arrest in embryos lacking NPBs or whether additional proteins, perhaps acting downstream of NPM2, are involved. It is, therefore, important to dissect the roles of these two chaperones, i.e. NPM2 and DAXX, and elucidate, which of the two is directly responsible for the abnormal remodelling of the centromeres. This may give an important clue as to whether a similar process occurs in somatic cells or whether the effect is strictly embryo-specific. Elucidating the mechanism of the developmental arrest thus remains to be the major challenge; indeed, in light of the results obtained so far, the question arises whether we should even consider NLBs and NPBs to be ‘nucleoli’. Perhaps embryos have re-purposed these structures entirely. And if not, is the NLB's as yet undefined function somehow mirrored in somatic cells? In any case, the intriguing story of these embryonic structures shows that our understanding of the biology of nucleoli in the context of complex cellular behaviors is far from complete.

Perspectives

  • Nucleoli are major nuclear organelles. Apart from the canonical function, i.e. ribosome production, nucleoli also participate in additional vital cellular processes. These non-canonical functions might be masked by an ongoing RNA polymerase I activity, making them difficult to study. Mammalian oocytes and embryos offer a unique opportunity to explore these functions because RNA polymerase I activity naturally shuts down around the time of fertilization.

  • Instead of serving as a depot for nucleolar proteins, embryonic nucleoli are essential just after fertilization, when they participate in parental genome epigenetic remodelling. In light of the current results, the long-standing dogma of developmental biology should be reconsidered.

  • Future directions should include elucidating the essential function of oocyte/embryonic nucleoli and determining whether this function is paralleled in somatic cells.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Acknowledgements

H.F. is supported by the GACR 20-04465S, J.R. is supported by the Ministry of Agriculture of the Czech Republic: QK1910156, and P.L. is supported by the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreements GA 734434 (DRYNET), and project ‘DEMETRA’ (MIUR) 2018–2022.

Abbreviations

     
  • EGA

    embryonic genome activation

  •  
  • NLB

    nucleolus-like body

  •  
  • NPBs

    nucleolus precursor bodies

References

References
1
LaMarca
,
M.J.
and
Wassarman
,
P.M.
(
1979
)
Program of early development in the mammal: changes in absolute rates of synthesis of ribosomal proteins during oogenesis and early embryogenesis in the mouse
.
Dev. Biol.
73
,
103
119
2
Santon
,
J.B.
and
Pellegrini
,
M.
(
1981
)
Rates of ribosomal protein and total protein synthesis during Drosophila early embryogenesis
.
Dev. Biol.
85
,
252
257
3
Taylor
,
K.D.
and
Pikó
,
L.
(
1992
)
Expression of ribosomal protein genes in mouse oocytes and early embryos
.
Mol. Reprod. Dev.
31
,
182
188
4
Kressler
,
D.
,
Hurt
,
E.
and
Bassler
,
J.
(
2010
)
Driving ribosome assembly
.
Biochim. Biophys. Acta
1803
,
673
683
5
Tsang
,
C.K.
,
Bertram
,
P.G.
,
Ai
,
W.
,
Drenan
,
R.
and
Zheng
,
X.F.S.
(
2003
)
Chromatin-mediated regulation of nucleolar structure and RNA Pol I localization by TOR
.
EMBO J.
22
,
6045
6056
6
Tiku
,
V.
and
Antebi
,
A.
(
2018
)
Nucleolar function in lifespan regulation
.
Trends Cell Biol.
28
,
662
672
7
Gertz
,
H.J.
,
Siegers
,
A.
and
Kuchinke
,
J.
(
1994
)
Stability of cell size and nucleolar size in Lewy body containing neurons of substantia nigra in Parkinson's disease
.
Brain Res.
637
,
339
341
8
Tiku
,
V.
,
Kew
,
C.
,
Mehrotra
,
P.
,
Ganesan
,
R.
,
Robinson
,
N.
and
Antebi
,
A.
(
2018
)
Nucleolar fibrillarin is an evolutionarily conserved regulator of bacterial pathogen resistance
.
Nat. Commun.
9
,
3607
9
Derenzini
,
M.
,
Trerè
,
D.
,
Pession
,
A.
,
Montanaro
,
L.
,
Sirri
,
V.
and
Ochs
,
R.L.
(
1998
)
Nucleolar function and size in cancer cells
.
Am. J. Pathol.
152
,
1291
1297
PMID:
[PubMed]
10
Andersen
,
J.S.
,
Lam
,
Y.W.
,
Leung
,
A.K.L.
,
Ong
,
S.-E.
,
Lyon
,
C.E.
,
Lamond
,
A.I.
et al (
2005
)
Nucleolar proteome dynamics
.
Nature
433
,
77
83
11
Andersen
,
J.S.
,
Lyon
,
C.E.
,
Fox
,
A.H.
,
Leung
,
A.K.L.
,
Lam
,
Y.W.
,
Steen
,
H.
et al (
2002
)
Directed proteomic analysis of the human nucleolus
.
Curr. Biol.
12
,
1
11
12
Boisvert
,
F.M.
,
van Koningsbruggen
,
S.
,
Navascues
,
J.
and
Lamond
,
A.I.
(
2007
)
The multifunctional nucleolus
.
Nat. Rev. Mol. Cell Biol.
8
,
574
585
13
Dundr
,
M.
(
2012
)
Nuclear bodies: multifunctional companions of the genome
.
Curr. Opin. Cell Biol.
24
,
415
422
14
Chouinard
,
L.A.
(
1971
)
A light- and electron-microscope study of the nucleolus during growth of the oocyte in the prepubertal mouse
.
J. Cell. Sci.
9
,
637
663
PMID:
[PubMed]
15
Chouinard
,
L.A.
(
1975
)
A light- and electron-microscope study of the oocyte nucleus during development of the antral follicle in the prepubertal mouse
.
J. Cell. Sci.
17
,
589
615
PMID:
[PubMed]
16
Bachvarova
,
R.
(
1974
)
Incorporation of tritiated adenosine into mouse ovum RNA
.
Dev. Biol.
40
,
52
58
17
Hirao
,
Y.
and
Miyano
,
T.
(
2008
)
In vitro growth of mouse oocytes: oocyte size at the beginning of culture influences the appropriate length of culture period
.
J. Mamm. Ova. Res.
25
,
56
62
18
Wassarman
,
P.M.
and
Josefowicz
,
W.J.
(
1978
)
Oocyte development in the mouse: an ultrastructural comparison of oocytes isolated at various stages of growth and meiotic competence
.
J. Morphol.
156
,
209
235
19
Levi
,
M.
,
Ghetler
,
Y.
,
Shulman
,
A.
and
Shalgi
,
R.
(
2013
)
Morphological and molecular markers are correlated with maturation-competence of human oocytes
.
Hum. Reprod.
28
,
2482
2489
20
Bouniol-Baly
,
C.
,
Hamraoui
,
L.
,
Guibert
,
J.
,
Beaujean
,
N.
,
Szöllösi
,
M.S.
and
Debey
,
P.
(
1999
)
Differential transcriptional activity associated with chromatin configuration in fully grown mouse germinal vesicle oocytes
.
Biol. Reprod.
60
,
580
587
21
Debey
,
P.
,
Szöllösi
,
M.S.
,
Szöllösi
,
D.
,
Vautier
,
D.
,
Girousse
,
A.
and
Besombes
,
D.
(
1993
)
Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics
.
Mol. Reprod. Dev.
36
,
59
74
22
Monti
,
M.
,
Zanoni
,
M.
,
Calligaro
,
A.
,
Ko
,
M.S.H.
,
Mauri
,
P.
and
Redi
,
C.A.
(
2013
)
Developmental arrest and mouse antral not-surrounded nucleolus oocytes
.
Biol. Reprod.
88
,
2
23
Inoue
,
A.
,
Nakajima
,
R.
,
Nagata
,
M.
and
Aoki
,
F.
(
2008
)
Contribution of the oocyte nucleus and cytoplasm to the determination of meiotic and developmental competence in mice
.
Hum. Reprod.
23
,
1377
1384
24
Zuccotti
,
M.
,
Giorgi Rossi
,
P.
,
Martinez
,
A.
,
Garagna
,
S.
,
Forabosco
,
A.
and
Redi
,
C.A.
(
1998
)
Meiotic and developmental competence of mouse antral oocytes
.
Biol. Reprod.
58
,
700
704
25
Zuccotti
,
M.
,
Garagna
,
S.
,
Merico
,
V.
,
Monti
,
M.
and
Alberto Redi
,
C.
(
2005
)
Chromatin organisation and nuclear architecture in growing mouse oocytes
.
Mol. Cell. Endocrinol.
234
,
11
17
26
Shishova
,
K.V.
,
Lavrentyeva
,
E.A.
,
Dobrucki
,
J.W.
and
Zatsepina
,
O.V.
(
2015
)
Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA
.
Dev. Biol.
397
,
267
281
27
Hernandez-Verdun
,
D.
(
2011
)
Assembly and disassembly of the nucleolus during the cell cycle
.
Nucleus
2
,
189
194
28
Falahati
,
H.
,
Pelham-Webb
,
B.
,
Blythe
,
S.
and
Wieschaus
,
E.
(
2016
)
Nucleation by rRNA dictates the precision of nucleolus assembly
.
Curr. Biol.
26
,
277
285
29
Geuskens
,
M.
and
Alexandre
,
H.
(
1984
)
Ultrastructural and autoradiographic studies of nucleolar development and rDNA transcription in preimplantation mouse embryos
.
Cell Differ.
14
,
125
134
30
Tesarík
,
J.
,
Kopecný
,
V.
,
Plachot
,
M.
,
Mandelbaum
,
J.
,
Da Lage
,
C.
and
Fléchon
,
J.E.
(
1986
)
Nucleologenesis in the human embryo developing in vitro: ultrastructural and autoradiographic analysis
.
Dev. Biol.
115
,
193
203
31
Kopecný
,
V.
,
Fléchon
,
J.E.
,
Camous
,
S.
and
Fulka
,
J.
(
1989
)
Nucleologenesis and the onset of transcription in the eight-cell bovine embryo: fine-structural autoradiographic study
.
Mol. Reprod. Dev.
1
,
79
90
32
Tománek
,
M.
,
Kopecný
,
V.
and
Kanka
,
J.
(
1989
)
Genome reactivation in developing early pig embryos: an ultrastructural and autoradiographic analysis
.
Anat. Embryol.
180
,
309
316
33
Ogushi
,
S.
,
Yamagata
,
K.
,
Obuse
,
C.
,
Furuta
,
K.
,
Wakayama
,
T.
,
Matzuk
,
M.M.
et al (
2017
)
Reconstitution of the oocyte nucleolus in mice through a single nucleolar protein, NPM2
.
J. Cell. Sci.
130
,
2416
2429
34
Newton
,
K.
,
Petfalski
,
E.
,
Tollervey
,
D.
and
Cáceres
,
J.F.
(
2003
)
Fibrillarin is essential for early development and required for accumulation of an intron-encoded small nucleolar RNA in the mouse
.
Mol. Cell. Biol.
23
,
8519
8527
35
Chen
,
H.
,
Li
,
Z.
,
Haruna
,
K.
,
Li
,
Z.
,
Li
,
Z.
,
Semba
,
K.
et al (
2008
)
Early pre-implantation lethality in mice carrying truncated mutation in the RNA polymerase 1-2 gene
.
Biochem. Biophys. Res. Commun.
365
,
636
642
36
Hamdane
,
N.
,
Tremblay
,
M.G.
,
Dillinger
,
S.
,
Stefanovsky
,
V.Y.
,
Németh
,
A.
and
Moss
,
T.
(
2017
)
Disruption of the UBF gene induces aberrant somatic nucleolar bodies and disrupts embryo nucleolar precursor bodies
.
Gene
612
,
5
11
37
Herdman
,
C.
,
Mars
,
J.-C.
,
Stefanovsky
,
V.Y.
,
Tremblay
,
M.G.
,
Sabourin-Felix
,
M.
,
Lindsay
,
H.
et al (
2017
)
A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription
.
PLoS Genet.
13
,
e1006899
38
Oliver
,
E.R.
,
Saunders
,
T.L.
,
Tarlé
,
S.A.
and
Glaser
,
T.
(
2004
)
Ribosomal protein L24 defect in belly spot and tail (Bst), a mouse minute
.
Development
131
,
3907
3920
39
Matsson
,
H.
,
Davey
,
E.J.
,
Draptchinskaia
,
N.
,
Hamaguchi
,
I.
,
Ooka
,
A.
,
Levéen
,
P.
et al (
2004
)
Targeted disruption of the ribosomal protein S19 gene is lethal prior to implantation
.
Mol. Cell. Biol.
24
,
4032
4037
40
Panić
,
L.
,
Tamarut
,
S.
,
Sticker-Jantscheff
,
M.
,
Barkić
,
M.
,
Solter
,
D.
,
Uzelac
,
M.
et al (
2006
)
Ribosomal protein S6 gene haploinsufficiency is associated with activation of a p53-dependent checkpoint during gastrulation
.
Mol. Cell. Biol.
26
,
8880
8891
41
Anderson
,
S.J.
,
Lauritsen
,
J.P.H.
,
Hartman
,
M.G.
,
Foushee
,
A.M.D.
,
Lefebvre
,
J.M.
,
Shinton
,
S.A.
et al (
2007
)
Ablation of ribosomal protein L22 selectively impairs alphabeta T cell development by activation of a p53-dependent checkpoint
.
Immunity
26
,
759
772
42
Kirn-Safran
,
C.B.
,
Oristian
,
D.S.
,
Focht
,
R.J.
,
Parker
,
S.G.
,
Vivian
,
J.L.
and
Carson
,
D.D.
(
2007
)
Global growth deficiencies in mice lacking the ribosomal protein HIP/RPL29
.
Dev. Dyn.
236
,
447
460
43
Lan
,
Z.-J.
,
Xu
,
X.
and
Cooney
,
A.J.
(
2004
)
Differential oocyte-specific expression of Cre recombinase activity in GDF-9-iCre, Zp3cre, and Msx2Cre transgenic mice
.
Biol. Reprod.
71
,
1469
1474
44
Burns
,
K.H.
,
Viveiros
,
M.M.
,
Ren
,
Y.
,
Wang
,
P.
,
DeMayo
,
F.J.
,
Frail
,
D.E.
et al (
2003
)
Roles of NPM2 in chromatin and nucleolar organization in oocytes and embryos
.
Science
300
,
633
636
45
Inoue
,
A.
and
Aoki
,
F.
(
2010
)
Role of the nucleoplasmin 2 C-terminal domain in the formation of nucleolus-like bodies in mouse oocytes
.
FASEB J.
24
,
485
494
46
Clift
,
D.
,
McEwan
,
W.A.
,
Labzin
,
L.I.
,
Konieczny
,
V.
,
Mogessie
,
B.
,
James
,
L.C.
et al (
2017
)
A method for the acute and rapid degradation of endogenous proteins
.
Cell
171
,
1692
1706.e18
47
Israel
,
S.
,
Casser
,
E.
,
Drexler
,
H.C.A.
,
Fuellen
,
G.
and
Boiani
,
M.
(
2019
)
A framework for TRIM21-mediated protein depletion in early mouse embryos: recapitulation of Tead4 null phenotype over three days
.
BMC Genomics
20
,
755
48
Fulka
,
J.
,
Moor
,
R.M.
,
Loi
,
P.
and
Fulka
,
J.
(
2003
)
Enucleolation of porcine oocytes
.
Theriogenology
59
,
1879
1885
49
Ogushi
,
S.
,
Palmieri
,
C.
,
Fulka
,
H.
,
Saitou
,
M.
,
Miyano
,
T.
and
Fulka
,
J.
(
2008
)
The maternal nucleolus is essential for early embryonic development in mammals
.
Science
319
,
613
616
50
Fulka
,
J.J.
,
Benc
,
M.
,
Loi
,
P.
,
Langerova
,
A.
and
Fulka
,
H.
(
2019
)
Function of atypical mammalian oocyte/zygote nucleoli and its implications for reproductive biology and medicine
.
Int. J. Dev. Biol.
63
,
105
112
51
Bonnet-Garnier
,
A.
,
Feuerstein
,
P.
,
Chebrout
,
M.
,
Fleurot
,
R.
,
Jan
,
H.-U.
,
Debey
,
P.
et al (
2012
)
Genome organization and epigenetic marks in mouse germinal vesicle oocytes
.
Int. J. Dev. Biol.
56
,
877
887
52
Shishova
,
K.V.
,
Khodarovich
,
Y.M.
,
Lavrentyeva
,
E.A.
and
Zatsepina
,
O.V.
(
2015
)
High-resolution microscopy of active ribosomal genes and key members of the rRNA processing machinery inside nucleolus-like bodies of fully-grown mouse oocytes
.
Exp. Cell Res.
337
,
208
218
53
Kyogoku
,
H.
,
Ogushi
,
S.
and
Miyano
,
T.
(
2010
)
Nucleoli from growing oocytes support the development of enucleolated full-grown oocytes in the pig
.
Mol. Reprod. Dev.
77
,
167
173
54
Kyogoku
,
H.
,
Ogushi
,
S.
,
Miyano
,
T.
and
Fulka
,
J.
(
2011
)
Nucleoli from growing oocytes inhibit the maturation of enucleolated, full-grown oocytes in the pig
.
Mol. Reprod. Dev.
78
,
426
435
55
Sobell
,
H.M.
(
1985
)
Actinomycin and DNA transcription
.
Proc. Natl. Acad. Sci. U.S.A.
82
,
5328
5331
56
Perry
,
R.P.
and
Kelley
,
D.E.
(
1970
)
Inhibition of RNA synthesis by actinomycin D: characteristic dose-response of different RNA species
.
J. Cell. Physiol.
76
,
127
139
57
Fulka
,
H.
and
Langerova
,
A.
(
2014
)
The maternal nucleolus plays a key role in centromere satellite maintenance during the oocyte to embryo transition
.
Development
141
,
1694
1704
58
Kyogoku
,
H.
,
Fulka
,
J.
,
Wakayama
,
T.
and
Miyano
,
T.
(
2014
)
De novo formation of nucleoli in developing mouse embryos originating from enucleolated zygotes
.
Development
141
,
2255
2259
59
Ogushi
,
S.
and
Saitou
,
M.
(
2010
)
The nucleolus in the mouse oocyte is required for the early step of both female and male pronucleus organization
.
J. Reprod. Dev.
56
,
495
501
60
Tadros
,
W.
and
Lipshitz
,
H.D.
(
2009
)
The maternal-to-zygotic transition: a play in two acts
.
Development
136
,
3033
3042
61
Lee
,
M.T.
,
Bonneau
,
A.R.
and
Giraldez
,
A.J.
(
2014
)
Zygotic genome activation during the maternal-to-zygotic transition
.
Annu. Rev. Cell Dev. Biol.
30
,
581
613
62
Langley
,
A.R.
,
Smith
,
J.C.
,
Stemple
,
D.L.
and
Harvey
,
S.A.
(
2014
)
New insights into the maternal to zygotic transition
.
Development
141
,
3834
3841
63
Zatsepina
,
O.V.
,
Bouniol-Baly
,
C.
,
Amirand
,
C.
and
Debey
,
P.
(
2000
)
Functional and molecular reorganization of the nucleolar apparatus in maturing mouse oocytes
.
Dev. Biol.
223
,
354
370
64
Zatsepina
,
O.
,
Baly
,
C.
,
Chebrout
,
M.
and
Debey
,
P.
(
2003
)
The step-wise assembly of a functional nucleolus in preimplantation mouse embryos involves the cajal (coiled) body
.
Dev. Biol.
253
,
66
83
65
Pikó
,
L.
and
Clegg
,
K.B.
(
1982
)
Quantitative changes in total RNA, total poly(A), and ribosomes in early mouse embryos
.
Dev. Biol.
89
,
362
378
66
Knowland
,
J.
and
Graham
,
C.
(
1972
)
RNA synthesis at the two-cell stage of mouse development
.
J. Embryol. Exp. Morphol.
27
,
167
176
PMID:
[PubMed]
67
Sullivan
,
G.J.
,
Bridger
,
J.M.
,
Cuthbert
,
A.P.
,
Newbold
,
R.F.
,
Bickmore
,
W.A.
and
McStay
,
B.
(
2001
)
Human acrocentric chromosomes with transcriptionally silent nucleolar organizer regions associate with nucleoli
.
EMBO J.
20
,
2867
2874
68
Miller
,
O.J.
,
Miller
,
D.A.
,
Dev
,
V.G.
,
Tantravahi
,
R.
and
Croce
,
C.M.
(
1976
)
Expression of human and suppression of mouse nucleolus organizer activity in mouse-human somatic cell hybrids
.
Proc. Natl. Acad. Sci. U.S.A.
73
,
4531
4535
69
Mishima
,
Y.
,
Financsek
,
I.
,
Kominami
,
R.
and
Muramatsu
,
M.
(
1982
)
Fractionation and reconstitution of factors required for accurate transcription of mammalian ribosomal RNA genes: identification of a species-dependent initiation factor
.
Nucleic Acids Res.
10
,
6659
6670
70
Murano
,
K.
,
Okuwaki
,
M.
,
Momose
,
F.
,
Kumakura
,
M.
,
Ueshima
,
S.
,
Newbold
,
R.F.
et al (
2014
)
Reconstitution of human rRNA gene transcription in mouse cells by a complete SL1 complex
.
J. Cell. Sci.
127
,
3309
3319
71
Lagutina
,
I.
,
Fulka
,
H.
,
Lazzari
,
G.
and
Galli
,
C.
(
2013
)
Interspecies somatic cell nuclear transfer: advancements and problems
.
Cell Reprogram.
15
,
374
384
72
Jiang
,
Y.
,
Kelly
,
R.
,
Peters
,
A.
,
Fulka
,
H.
,
Dickinson
,
A.
,
Mitchell
,
D.A.
et al (
2011
)
Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors
.
PLoS One
6
,
e14805
73
Morovic
,
M.
,
Strejcek
,
F.
,
Nakagawa
,
S.
,
Deshmukh
,
R.S.
,
Murin
,
M.
,
Benc
,
M.
et al (
2017
)
Mouse oocytes nucleoli rescue embryonic development of porcine enucleolated oocytes
.
Zygote
25
,
675
685
74
Fulka
,
H.
,
Martinkova
,
S.
,
Kyogoku
,
H.
,
Langerova
,
A.
and
Fulka
,
J.
(
2012
)
Production of giant mouse oocyte nucleoli and assessment of their protein content
.
J. Reprod. Dev.
58
,
371
376
75
Murin
,
M.
,
Strejcek
,
F.
,
Bartkova
,
A.
,
Morovic
,
M.
,
Benc
,
M.
,
Prochazka
,
R.
et al (
2019
)
Intranuclear characteristics of pig oocytes stained with brilliant cresyl blue and nucleologenesis of resulting embryos
.
Zygote
27
,
232
240
76
Dingwall
,
C.
and
Laskey
,
R.A.
(
1990
)
Nucleoplasmin: the archetypal molecular chaperone
.
Semin. Cell Biol.
1
,
11
17
PMID:
[PubMed]
77
Philpott
,
A.
,
Leno
,
G.H.
and
Laskey
,
R.A.
(
1991
)
Sperm decondensation in xenopus egg cytoplasm is mediated by nucleoplasmin
.
Cell
65
,
569
578
78
Frehlick
,
L.J.
,
Eirín-López
,
J.M.
and
Ausió
,
J.
(
2007
)
New insights into the nucleophosmin/nucleoplasmin family of nuclear chaperones
.
Bioessays
29
,
49
59
79
Cremer
,
T.
and
Cremer
,
C.
(
2006
)
Rise, fall and resurrection of chromosome territories: a historical perspective. Part I. The rise of chromosome territories
.
Eur. J. Histochem.
50
,
161
176
PMID:
[PubMed]
80
Serizay
,
J.
and
Ahringer
,
J.
(
2018
)
Genome organization at different scales: nature, formation and function
.
Curr. Opin. Cell Biol.
52
,
145
153
81
Quinodoz
,
S.A.
,
Ollikainen
,
N.
,
Tabak
,
B.
,
Palla
,
A.
,
Schmidt
,
J.M.
,
Detmar
,
E.
et al (
2018
)
Higher-order inter-chromosomal hubs shape 3D genome organization in the nucleus
.
Cell
174
,
744
757.e24
82
Vertii
,
A.
,
Ou
,
J.
,
Yu
,
J.
,
Yan
,
A.
,
Pagès
,
H.
,
Liu
,
H.
et al (
2019
)
Two contrasting classes of nucleolus-associated domains in mouse fibroblast heterochromatin
.
Genome Res.
29
,
1235
1249
83
Padeken
,
J.
and
Heun
,
P.
(
2014
)
Nucleolus and nuclear periphery: velcro for heterochromatin
.
Curr. Opin. Cell Biol.
28
,
54
60
84
Guetg
,
C.
and
Santoro
,
R.
(
2012
)
Formation of nuclear heterochromatin: the nucleolar point of view
.
Epigenetics
7
,
811
814
85
Rhind
,
N.
and
Gilbert
,
D.M.
(
2013
)
DNA replication timing
.
Cold Spring Harb. Perspect. Biol.
5
,
a010132
86
Romanova
,
L.
,
Korobova
,
F.
,
Noniashvilli
,
E.
,
Dyban
,
A.
and
Zatsepina
,
O.
(
2006
)
High resolution mapping of ribosomal DNA in early mouse embryos by fluorescence in situ hybridization
.
Biol. Reprod.
74
,
807
815
87
Fuentes-Mascorro
,
G.
,
Serrano
,
H.
and
Rosado
,
A.
(
2000
)
Sperm chromatin
.
Arch. Androl.
45
,
215
225
88
Ward
,
W.S.
(
2010
)
Function of sperm chromatin structural elements in fertilization and development
.
Mol. Hum. Reprod.
16
,
30
36
89
Chauvin
,
T.
,
Xie
,
F.
,
Liu
,
T.
,
Nicora
,
C.D.
,
Yang
,
F.
,
Camp
,
D.G.
et al (
2012
)
A systematic analysis of a deep mouse epididymal sperm proteome
.
Biol. Reprod.
87
,
141
90
Skerget
,
S.
,
Rosenow
,
M.A.
,
Petritis
,
K.
and
Karr
,
T.L.
(
2015
)
Sperm proteome maturation in the mouse epididymis
.
PLoS One
10
,
e0140650
91
Suzuki
,
H.
,
Sakurai
,
S.
,
Nishimura
,
M.
,
Kominami
,
R.
and
Moriwaki
,
K.
(
1992
)
Compensatory changes in silver-stainability of nucleolar organizer regions in mice
.
Jpn. J. Genet.
67
,
217
232
92
Kurihara
,
Y.
,
Suh
,
D.S.
,
Suzuki
,
H.
and
Moriwaki
,
K.
(
1994
)
Chromosomal locations of Ag-NORs and clusters of ribosomal DNA in laboratory strains of mice
.
Mamm. Genome
5
,
225
228
93
Cazaux
,
B.
,
Catalan
,
J.
,
Veyrunes
,
F.
,
Douzery
,
E.J.
and
Britton-Davidian
,
J.
(
2011
)
Are ribosomal DNA clusters rearrangement hotspots?: a case study in the genus Mus (Rodentia. Muridae)
.
BMC Evol. Biol.
11
,
124
94
Aguirre-Lavin
,
T.
,
Adenot
,
P.
,
Bonnet-Garnier
,
A.
,
Lehmann
,
G.
,
Fleurot
,
R.
,
Boulesteix
,
C.
et al (
2012
)
3D-FISH analysis of embryonic nuclei in mouse highlights several abrupt changes of nuclear organization during preimplantation development
.
BMC Dev. Biol.
12
,
30
95
Jachowicz
,
J.W.
,
Santenard
,
A.
,
Bender
,
A.
,
Muller
,
J.
and
Torres-Padilla
,
M.-E.
(
2013
)
Heterochromatin establishment at pericentromeres depends on nuclear position
.
Genes Dev.
27
,
2427
2432
96
Martin
,
C.
,
Beaujean
,
N.
,
Brochard
,
V.
,
Audouard
,
C.
,
Zink
,
D.
and
Debey
,
P.
(
2006
)
Genome restructuring in mouse embryos during reprogramming and early development
.
Dev. Biol.
292
,
317
332
97
Ahmed
,
K.
,
Dehghani
,
H.
,
Rugg-Gunn
,
P.
,
Fussner
,
E.
,
Rossant
,
J.
and
Bazett-Jones
,
D.P.
(
2010
)
Global chromatin architecture reflects pluripotency and lineage commitment in the early mouse embryo
.
PLoS One
5
,
e10531
98
Laskey
,
R.A.
,
Honda
,
B.M.
,
Mills
,
A.D.
and
Finch
,
J.T.
(
1978
)
Nucleosomes are assembled by an acidic protein which binds histones and transfers them to DNA
.
Nature
275
,
416
420
99
Drané
,
P.
,
Ouararhni
,
K.
,
Depaux
,
A.
,
Shuaib
,
M.
and
Hamiche
,
A.
(
2010
)
The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3
.
Genes Dev.
24
,
1253
1265
100
Lewis
,
P.W.
,
Elsaesser
,
S.J.
,
Noh
,
K.M.
,
Stadler
,
S.C.
and
Allis
,
C.D.
(
2010
)
Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres
.
Proc. Natl. Acad. Sci. U.S.A.
107
,
14075
14080
101
Voon
,
H.P.J.
and
Wong
,
L.H.
(
2016
)
New players in heterochromatin silencing: histone variant H3.3 and the ATRX/DAXX chaperone
.
Nucleic Acids Res.
44
,
1496
1501
102
Fulka
,
H.
and
Aoki
,
F.
(
2016
)
Nucleolus precursor bodies and ribosome biogenesis in early mammalian embryos: old theories and new discoveries
.
Biol. Reprod.
94
,
143
103
Fair
,
T.
,
Hyttel
,
P.
,
Lonergan
,
P.
and
Boland
,
M.P.
(
2001
)
Immunolocalization of nucleolar proteins during bovine oocyte growth, meiotic maturation, and fertilization
.
Biol Reprod.
64
,
1516
25
104
Terzaghi
,
L.
,
Luciano
,
A.M.
,
Dall’Acqua
,
P.C.
,
Modina
,
S.C.
,
Peluso
,
J.J.
and
Lodde
,
V.
(
2018
)
PGRMC1 localization and putative function in the nucleolus of bovine granulosa cells and oocytes
.
Reproduction.
155
,
273
282
105
Kopecny
,
V.
,
Biggiogera
,
M.
,
Laurincik
,
J.
,
Pivko
,
J.
,
Grafenau
,
P.
,
Martin
,
T.E.
et al (
1996
)
Fine structural cytochemical and immunocytochemical analysis of nucleic acids and ribonucleoprotein distribution in nuclei of pig oocytes and early preimplantation embryos
.
Chromosoma.
104
,
561
574
106
Hyttel
,
P.
,
Laurincik
,
J.
,
Rosenkranz
,
C.
,
Rath
,
D.
,
Niemann
,
H.
,
Ochs
,
R.L.
et al (
2000
)
Nucleolar proteins and ultrastructure in preimplantation porcine embryos developed in vivo
.
Biol Reprod.
63
,
1848
1856
107
Bjerregaard
,
B.
and
Maddox-Hyttel
,
P.
(
2004
)
Regulation of ribosomal RNA gene expression in porcine oocytes
.
Anim Reprod Sci.
82–83
,
605
16
108
Laurincik
,
J.
,
Bjerregaard
,
B.
,
Strejcek
,
F.
,
Rath
,
D.
,
Niemann
,
H.
,
Rosenkranz
,
C.
et al (
2004
)
Nucleolar ultrastructure and protein allocation in in vitro produced porcine embryos
.
Molecular Reproduction and Development.
68
,
327
334
109
Parfenov
,
V.N.
,
Pochukalina
,
G.N.
,
Davis
,
D.S.
,
Reinbold
,
R.
,
Schöler
,
H.R.
and
Murti
,
K.G.
(
2003
)
Nuclear distribution of Oct-4 transcription factor in transcriptionally active and inactive mouse oocytes and its relation to RNA polymerase II and splicing factors
.
J Cell Biochem.
89
,
720
732
110
Lin
,
C.J.
,
Koh
,
F.M.
,
Wong
,
P.
,
Conti
,
M.
and Ramalho-
Santos
,
M.
(
2014
)
Hira-mediated H3.3 incorporation is required for DNA replication and ribosomal RNA transcription in the mouse zygote
.
Dev Cell.
30
,
268
279
111
Kopecný
,
V.
,
Landa
,
V.
and
Pavlok
,
A.
(
1995
)
Localization of nucleic acids in the nucleoli of oocytes and early embryos of mouse and hamster: an autoradiographic study
.
Mol Reprod Dev.
41
,
449
458
112
Kopecný
,
V.
,
Landa
,
V.
,
Malatesta
,
M.
,
Martin
,
T.E.
and
Fakan
,
S.
(
1996
)
Immunoelectron microscope analyses of rat germinal vesicle-stage oocyte nucleolus-like bodies
.
Reprod. Nutr. Dev.
36
,
667
679
.
113
Sutovský
,
P.
,
Jelínková
,
L.
,
Antalíková
,
L.
and
Motlík
,
J.
(
1993
)
Ultrastructural cytochemistry of the nucleus and nucleolus in growing rabbit oocytes
.
Biol. Cell.
77
,
173
180
114
Yamagata
,
K.
,
Suetsugu
,
R.
and
Wakayama
,
T.
(
2009
)
Assessment of chromosomal integrity using a novel live-cell imaging technique in mouse embryos produced by intracytoplasmic sperm injection
.
Hum. Reprod.
24
,
2490
2499
115
Ogonuki
,
N.
,
Mori
,
M.
,
Shinmen
,
A.
,
Inoue
,
K.
,
Mochida
,
K.
,
Ohta
,
A.
et al (
2010
)
The effect on intracytoplasmic sperm injection outcome of genotype, male germ cell stage and freeze-thawing in mice
.
PLoS One
5
,
e11062
116
Eppig
,
J.J.
,
O'Brien
,
M.J.
,
Wigglesworth
,
K.
,
Nicholson
,
A.
,
Zhang
,
W.
and
King
,
B.A.
(
2009
)
Effect of in vitro maturation of mouse oocytes on the health and lifespan of adult offspring
.
Hum. Reprod.
24
,
922
928