Molecular Human Reproduction, Vol.16, No.2 pp. 63–67, 2010
Advanced Access publication on October 9, 2009 doi:10.1093/molehr/gap088
NEW RESEARCH HORIZON Review
Nucleolar transplantation in oocytes
and zygotes: challenges for further
research
Helena Fulka and Josef Fulka Jr 1
Institute of Animal Science, Pratelstvi 815, CS-104 00, Prague 10, Czech Republic
1
Correspondence address. Tel: þ420-267-009-606; Fax: þ420-267-710-779; E-mail: [email protected]
abstract: In germinal vesicles of immature mammalian oocytes, including humans, as well as in pronuclei in 1-cell stage embryos, prominent nuclear organelles, nucleoli, can be easily detected even under a relatively low magnification. In humans, it has been clearly documented that their number, position and distribution in pronuclei can be used as an indicator of embryonic developmental potential. In the light of
some recent experiments showing the feasibility of nucleolar manipulation we discuss here if these new approaches can be used to rescue
those embryos with abnormal pronuclear nucleolar patterns.
Key words: oocytes / zygotes / pronuclei / nucleolus
Introduction
Nucleoli in mammalian oocytes and embryos
Fully grown mammalian oocytes in large antral follicles are arrested at
germinal vesicle (GV) stage where they are awaiting the gonadotrophin
signal or release from the follicle in the case of in vitro culture. In both
these cases, oocyte begins to mature and can reach the metaphase II
stage (Fulka, Jr et al., 1998). It has been convincingly demonstrated
that oocytes where the chromatin closely surrounds the nucleolus
have much higher developmental potential after fertilization when
compared with oocytes where the chromatin is loosely distributed
in the germinal vesicle karyoplasms (De La Fuente, 2006). It is,
however, not fully understood why such a close association—
nucleolus:chromatin—is so important and which role the nucleolus
plays in the establishment of developmental potency (Miyara et al.,
2003). That this role indeed exists has been demonstrated in human
assisted reproduction, where the implantation rate of 1-cell stage
human embryos can be predicted, beside some other markers,
according to the number of nucleoli, their position in pronuclei and
their distribution between both pronuclei—maternal/paternal
(Tesarik et al., 1999; Gianaroli et al., 2003).
When compared structurally and functionally to fully differentiated
somatic cell nucleoli, nucleoli in oocytes are very different. In
somatic cells, the nucleolus contains three distinct compartments—
fibrillar centers that contain ribosomal RNA (rRNA) genes and
enzymes important for transcription, granular component containing
ribosomal subunits and dense fibrillar material containing the
nascent rRNA and enzymes necessary for its processing. On the
other hand, nucleoli in fully grown oocytes and early cleavage stage
embryos are composed only of the fibrillar material (HernandezVerdun, 2006; Oestrup et al., 2009). For this reason, these unique
structures have been named the ‘nucleolar precursor bodies
(NPBs)’ (Tesarik et al., 1987; Kopecny, 1989). Their transformation
into fully differentiated typical nucleoli occurs gradually and reflects
the transition from the maternal to embryonic genome control. This
transition occurs at different embryonic developmental stages,
depending on a given species (mouse, 2-cell stage; bovine, 8-cell
stage; pig; human, 4-cell stage, etc.). Whilst typical differentiated
nucleoli are involved in the regulation of many cellular processes, i.e.
cell cycle progression, pluripotency, gene activation and so on, the
function of NPBs is unknown (Boisvert et al., 2007).
It has been shown recently that nucleoli in fully grown oocytes can
be easily isolated from germinal vesicles. This gives us an unrivalled
opportunity to explore the function of these specific structures and
opens new avenues for the treatment of oocytes and zygotes with
aberrant nucleoli.
New developments
Manipulating the nucleolus
The feasibility of nucleolar manipulation has been investigated by
Fulka, Jr. et al. (2003) who found that these organelles can be microsurgically isolated from germinal vesicles of fully grown porcine
oocytes. The enucleolation pipette is inserted into the oocyte
& The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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64
cytoplasm in a close vicinity to the nucleolus, which is then slowly aspirated
from GV. During this aspiration the nucleolus penetrates the GV envelope
(membrane), and as sand in an hourglass, it is gradually translocated from
GV into the oocyte cytoplasm from which it can be removed in the form of
a so-called ‘nucleoloplast’. This means that the nucleolus is still enclosed
with the oocyte vitelline membrane and therefore the nucleoloplast
also contains a minimum volume of the oocyte cytoplasm (Figs 1–3).
What is important is that the oocyte is not damaged during this manipulation and can thus be used for some other experiments (enucleolated
oocyte). The possibility of using the enucleolation approach in some
other species has been demonstrated later in the mouse (Fulka et al.,
2004; Mohammed et al., 2008; Ogushi et al., 2008). Thus we can
assume that this approach can be used in oocytes of those mammals
where nucleoli are clearly visible in GVs.
As mentioned above, nucleoli in fully grown mammalian oocytes are
enclosed with chromatin (Bouniol-Baly et al., 1999). Thus, it cannot be
excluded that some parts of chromatin are removed during the
enucleolation procedure. Labeling (anti-trimethylated H4/K20,
marker of pericentric heterochromatin) or staining (Hoechst) of isolated nucleoli in nucleoloplasts, however, showed that this happens
only exceptionally (Ogushi et al., 2008) since the positive signals
were usually only detected inside the enucleolated GVs. Moreover,
Ogushi et al. (2008) have demonstrated that healthy mice can be
obtained from enucleolated oocytes that were later transplanted
with nucleoli. This is clear evidence against the possibility of chromatin
removal during the enucleolation procedure. On the other hand, we
cannot, however, exclude the possibility that some traces of nucleolar
material remained inside the GV.
Fulka and Fulka
Figure 1 The oocyte before the enucleolation. The oocyte is
stabilized with the holding pipette (H), the enucleolation pipette (E)
is on the right. Intact germinal vesicle (GV) with a prominent nucleolus (arrow) is clearly visible. 500.
Analyzing the function of oocyte nucleoli
in the process of oocyte maturation
In yeasts and somatic cells, the nucleolus (nucleolar material) is
involved in the cell cycle regulation, namely it is essential for the transition from metaphase to anaphase (Ma and Pederson, 2008). In yeast,
inactive Cdc14 (protein phosphatase) is localized in the nucleolus
during interphase but in anaphase Cdc14 is activated, released from
the nucleolus and controls the dissolution of cohesins. In somatic
cells, NuSAP (nucleolar-spindle associated protein) is localized in the
nucleoli in the interphase but in the M-phase it is detected in
central spindle microtubules. Depletion of NuSAP results in aberrant
mitotic spindles and abnormal chromosome segregation and cytokinesis (Raemaekers et al., 2003; Ribbeck et al., 2007). Thus, it is reasonable to suppose that enucleolated oocytes, because they do not
contain nucleoli at all, will exhibit some defect when exiting either
from metaphase I to anaphase I or possibly later on, during the exit
from metaphase II to anaphase II. Our results however show that
the nucleolus is not essential for these transitions in oocytes. No morphological differences between metaphases I and II were found
between control (intact) and enucleolated oocytes. In both cases,
perfect metaphase groups with normal arrangements of chromosomes
and well visible spindles were detected. Karyotyping also showed no
differences between control and experimental groups. Similarly, the
frequency of oocytes reaching metaphase II was essentially the same
in both groups. Thus, we can conclude that nucleoli in fully grown
mammalian oocytes are not essential for final phases of oocyte maturation (Ogushi et al., 2008).
Figure 2 The enucleolation pipette first penetrates through zona
pellucida and when its tip is in the vicinity of the nucleolus very
slow suction is applied. This preferentially aspirates the nucleolus
into the pipette (arrow). Note that the nucleolus penetrates the
GV envelope (membrane) and is slowly translocated into the
cytoplasm. 500.
No nucleoli are detected in enucleolated
oocytes in which the formation of (pro)nuclei
was induced
If the decondensation of chromatin and formation of nuclei (pronuclei)
is induced either by incubating metaphase I oocytes with protein synthesis inhibitors (Clarke and Masui, 1983; Clarke and Masui, 1985) or
after parthenogenetic activation or fertilization of metaphase II
oocytes, clearly visible nucleoli can be detected in newly formed
nuclei. This is, however, not the case when this decondensation is
induced in previously enucleolated oocytes. In all those cases
65
Nucleolar transplantation
Figure 3 After the entire nucleolus escapes from GV it can be
removed from the oocyte by increasing the distance between both pipettes. The products of enucleolation are then the enucleolated oocyte
with an intact GV (arrowhead) and the nucleoloplast containing the
nucleolus (arrow). Note that the nucleoloplast is enclosed with the vitelline membrane and contains a minimum volume of the cytoplasm. 500.
The question is whether we can rescue those oocytes where the
nucleolar material is absent. Our experiments showed that the
re-injection of nucleoli into previously enucleolated maturing (metaphase I) oocytes can indeed save them. The injected nucleoli are
quickly disassembled in the oocyte cytoplasm but when these
oocytes are then fertilized, the newly formed pronuclei contain
visible nucleoli and embryos develop normally, giving rise to healthy
offspring (Ogushi et al., 2008).
We have also tested if somatic or embryonic stem cell nucleoli can
substitute for the original oocyte nucleolar material. The GV stage
oocytes were enucleolated and matured up to metaphase II. Thereafter, the metaphase II chromosome groups were removed and into
the resulting cytoplasts, nuclei from somatic or ES (embryonic stem)
cells were injected. In controls (non-enucleolated) the newly formed
pseudo-pronuclei contained clearly visible nucleoli, but no nucleoli
were detectable in the experimental groups. This clearly confirms
the maternal (oocyte) origin of nucleoli in somatic cell nuclear transfer
embryos, but we cannot exclude the minimum participation of nucleolar material from somatic or embryonic stem cells (Ogushi et al.,
2008). Essentially, the same results were obtained by Mohammed
et al. (2008).
Implications
mentioned above, well-formed (pro)nuclei were formed but they
never contained nucleoli. Thus, for example, both the maternal as
well as paternal pronuclei are formed after ICSI of enucleolated
mature oocytes but neither of them contained nucleoli. This clearly
indicates the limited supply of nucleolar material in the oocyte and
once this material is removed it is not synthesized again (Fulka et al.,
2004; Maddox-Hyttel et al., 2007; Mohammed et al., 2008).
Development of enucleolated oocytes after
parthenogenetic activation or fertilization
In the mouse as well as in the pig, the enucleolated and subsequently
matured oocytes can be fertilized or activated parthenogenetically.
The resulting embryos however undergo one or two cleavages and
then their development ceases. As expected, no nucleoli are visible in
these cleaving embryos. The in vitro transcription assay with BrUTP
(or direct injection) showed essentially the same intensity of labeling
at 2-cell stage mouse embryos. The BrUTP incorporation can be
mostly assigned to the RNA polymerase II as it is not confined to nucleoli
and is consistent with the major genome activation. However, there was
no detectable labeling at 4-cell stage ‘enucleolated’ embryos, when
compared with controls where clearly visible active nucleoli were
detected. Additional approaches confirmed these observations (RNA
fluorescent in situ hybridization with Cot1 DNA as a probe). As mentioned above, the nucleolus is the site of ribosomal RNA synthesis
and maturation of RNA polymerase III transcripts. These processes
cannot logically occur in embryos with no nucleoli and the embryonic
genome cannot be thus activated (Lefevre, 2008).
Oocytes without nucleoli can be rescued
by transplanting a nucleolus
The results mentioned above indicate that the oocyte nucleolar
material is necessary for normal embryonic development.
Can assisted reproductive techniques rescue
human oocytes or zygotes with abnormal
nucleoli?
In human assisted reproduction the mature oocytes are typically collected, i.e. after reaching the metaphase II stage. Thus, nobody can say
how nucleoli looked like before the oocyte begins to mature. The
evaluation can be done only after IVF or ICSI when well-developed
pronuclei are formed. It is, however, unclear why in some zygotes
the number of nucleoli is essentially equal when both pronuclei are
compared and why they show the same pattern of distribution and
why in some zygotes the distribution of nucleoli is unequal. It may
well be that the aberrant pattern reflects the asynchronous decondensation of chromatin and formation of pronuclei. Do these zygotic
nucleoli have some role in further embryonic development?
We have enucleolated mouse 1-cell stage embryos where each pronucleus contained just a single nucleolus. Nucleoli were either
removed from the maternal or paternal pronucleus or from both pronuclei. When we removed nucleoli from both pronuclei, the manipulated embryos never reached the blastocyst stage and were arrested at
4–8 cells. When only a single nucleolus from one pronucleus was
removed and the other pronucleus was left intact, embryos developed
slightly better, but blastocysts obtained were aberrant, i.e. much
smaller when compared with controls, with no prominent inner cell
mass and some cells outside the embryo. There was no difference
in development when nucleolus was removed either from the
maternal or paternal pronucleus. The re-injection of isolated nucleoli
restored the developmental potential of manipulated zygotes, but
this does not mean that these embryos will develop to offspring
(Fulka and Fulka, Jr, in preparation). This type of manipulation of
zygotes is, however, only possible with mononucleolar zygotes.
Most typically, pronuclei contain several nucleoli and in this case
their manipulation is rather complicated. These observations clearly
66
suggest the importance of pronuclear nucleoli for further embryonic
development.
Also the data presented by Tesarik et al. (1999 and Gianaroli et al.
(2003) and some others suggest that the role of nucleoli in pronuclei is
important. But it is still unclear why. Before the onset of the first
mitotic division, the extrachromosomal content of both pronuclei dissolves in the cytoplasm, so we may suppose its equal distribution in
both blastomere nuclei and the rescue of those embryos exhibiting
the aberrant nucleolar distribution pattern. But this apparently does
not occur and it is evident that nucleoli must already be present correctly in pronuclei.
When we compare the association of chromatin with nucleoli in
fully grown GV oocytes with the association in pronuclei, certain
differences are evident. In GV-staged fully competent oocytes essentially all chromatin surrounds the nucleolus. On the other hand, in pronuclei it is just the pericentric heterochromatin. The oocytes where
the chromatin is rather diffuse in germinal vesicles (non-surrounded
nucleoli) are developmentally much less competent than those
oocytes with chromatin closely surrounding the nucleolus. This may
indicate certain role of this association for further development after
fertilization (Martin et al., 2006a, 2006b).
The question is, if this close association, nucleolus:chromatin, is
‘absolutely’ essential for embryonic development. Burns et al. (2003)
demonstrated that in NPM22/2 (nucleoplasmin 2) mice no typical
nucleoli are visible in GVs and pronuclei. The NPM22/2 embryos
developed very poorly when compared with controls but because a
few NPM22/2 embryos developed to birth, the authors suggested
the presence of some compensatory mechanisms.
So, can we transfer some nucleolar material into pronuclei? As
mentioned above, the injection of nucleoli into cytoplasm, which
does not contain the disassembled nucleolar material, rescues these
oocytes. In our experiments, we have moved nucleoli from GVs
into the cytoplasm. To our surprise, these nucleoli were very
rapidly targeted back into GVs but in the GV karyoplasms several
tiny nucleoli were then detected instead of a single nucleolus as it is
typical for non-manipulated oocytes. Similarly, the fusion of nucleoloplasts to enucleolated GV-staged oocytes resulted in the same rapid
movement of nucleoli back into GVs. Interestingly, when we transplanted extra nucleoli into non-enucleolated GVs, these extranucleoli
were also translocated into the nucleus. This means that GVs can
accept some extra nucleolar materials. Similarly, nucleoli are targeted
back into pronuclei from which they were removed. Thus, in theory,
nucleoli (isolated for example from spare GV-staged oocytes as they
are morphologically similar to pronuclear nucleoli) can be injected into
the cytoplasm of pronuclear stage embryos and we can expect that
they will move into pronuclei. The mechanism of transporting back
the transplanted (translocated) nucleoli back into GVs is currently
unknown, but it is well known that nuclear proteins contain the
‘so-called’ nuclear localization signals that are responsible for transport of nuclear proteins through nuclear pore complex and direct
these proteins to the nucleus (Alberts et al., 2008).
Can we then expect that the injected nucleolar material will be
equally distributed between both pronuclei? Evidently, this must be
tested first in laboratory animals. Moreover, it will be very useful to
know if these injected nucleoli will rescue the pronuclear stage
embryos that originate from GV-stage enucleolated oocytes.
Fulka and Fulka
Conclusion
From the technical point of view, the manipulation of nucleoli is simple
in GV-staged oocytes and rather difficult in 1-cell stage embryos where
pronuclei contain multiple nucleoli. The injection of isolated nucleolar
material into the cytoplasm of 1-cell stage embryos is technically feasible and we can expect that the injected nucleoli will be translocated
into pronuclei. It is, however, unclear if these injected nucleoli will be
able to establish the functional communication—chromatin:nucleolus.
This association, specifically between the nucleolus and pericentric
heterochromatin domains even at 1-cell stage embryos, seems to be
important for further embryo development and it is often aberrant
in cloned embryos (Martin et al., 2006a, b; Probst et al., 2007).
Even if this communication is not absolutely essential at this stage, it
must be tested whether the injected embryos will be rescued or
will develop better. Another important aspect is the safety of
suggested approach from the epigenetic point of view and consequent
normality of offspring born when this method is applied. It is still an
open issue, whether or not the assisted reproductive techniques can
lead to some abnormalities in children born (Gosden et al., 2003).
Clearly, some additional experiments in laboratory animals, primates
and ungulates are thus evidently necessary.
Funding
J.F. Jr’s laboratory is supported by GACR 523/09/1878 and MZE
0002701404.
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Submitted on August 20, 2009; resubmitted on September 29, 2009; accepted on
October 7, 2009