Advances in Bioscience and Biotechnology, 2013, 4, 21-35 ABB
http://dx.doi.org/10.4236/abb.2013.410A3004 Published Online October 2013 (http://www.scirp.org/journal/abb/)
RNA species whose transcription is totally silent in
pre-MBT stage are not mRNA but rRNA and possible
involvement of weak bases (ammonium salts and/or amines)
in the transcriptional silence of rRNA genes during the
pre-MBT stage in Xenopus early embryos
Koichiro Shiokawa1,2
1Department of Judo Therapy, Faculty of Medical Technology, Teikyo University, Utsunomiya, Japan
2Department of Biosciences, School of Science and Engineering, Teikyo University, Utsunomiya, Japan
Received 23 July 2013; revised 24 August 2013; accepted 15 September 2013
Copyright © 2013 Koichiro Shiokawa. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
In Xenopus laevis embryogenesis, fertilized eggs un-
dergo 12 cycles of synchronous divisions and reach
the stage called midblastula transition (MBT). It has
long been believed that during the first 12 cycles of
cleavage (pre-MBT stage), transcriptional activity of
the zygotic nuclei is totally absent. However, hetero-
geneous mRNA-like RNA is synthesized in pre-MBT
stage embryos, and exogenously-injected bacterial
CAT genes with SV40 promoter are expressed from the
cleavage stage. Nevertheless, the synthesis of rRNA as
detected by rRNA-specific 2’-O-methylation does not
take place in pre-MBT embryos and starts only from
the latter half of the MBT stage, corroborating the
fact that formation of definitive nucleoli as well as the
transcription of microinjected rRNA genes starts only
at and after MBT stage. Thus, while mRNA-like RNA
synthesis occurs from pre-MBT stage, synthesis of
rRNA is controlled in the way that transcription of
rRNA genes is totally silent during pre-MBT stage
and is initiated only at the latter half of MBT stage.
Once initiated, the rate of the synthesis of rRNA is
constant throughout later stages on a per-cell basis.
We searched substances which are responsible for the
transcriptional silence of rRNA genes during the pre-
MBT stage. Weak bases such as ammonium ion and
amines selectively inhibited rRNA synthesis at the
transcriptional level in post-MBT stage embryo cells.
Since we found that the level of ammonia extracted
from embryos is much higher in pre-MBT embryos
than in post-MBT embryos, we suggest that weak
bases like ammonium ion could be responsible for the
transcriptional silence of rRNA genes by slightly in-
creasing intracellular pH during the pre-MBT.
Keywords: Pre-MBT Transcription; Absence of rRNA
Synthesis; Initiation of rRNA Synthesis; Nucleolus
Formation; Weak Bases; Amines; Ammonium Ion;
Xenopus Embryogenesis
1. INTRODUCTION
MBT is the important time point of transition from the
phase of cleavage division to the phase of morphogenetic
cell interactions. After 12 rounds of cleavage cell cycles,
Xenopus fertilized eggs reach midblastula stage (4096
cells/embryo), or the stage of so-called midblastula tran-
sition (MBT), when G1 and G2 phases reappear in the
cell cycle [1,2]. During the cleavage stage, translation of
maternal mRNA takes place actively [3,4]. At MBT, cell
division shifts from synchronous to asynchronous one [5,
6], cell cycles shift from a checkpoint-unregulated to
checkpoint-regulated state [7,8], and cells, especially
those at the dorsal marginal zone, acquire motility [9].
Also, strongly activated, at least on a per-embryo basis
but not necessarily on a per-cell basis, is the transcriptio-
nal activity from zygotic nuclear genes [10-17]. Also, the
per-embryo activity of the transcription of exogenously-
introduced bacterial CAT (chloramphenicole acetyltrans-
ferase) genes with pan-expression promoter of SV40 vi-
rus starts to increase from cleavage stage on [18,19].
Since the reports of Newport and Kirschner [5,6], it
has long been believed that there is no transcription at all
before MBT stage. However, recent results about this

K. Shiokawa / Advances in Bioscience and Biotechnology 4 (2013) 21-35 22
issue led to acceptance of the view that some transcrip-
tion takes place during the pre-MBT stage. In our old
studies, for instance, we noticed that heterogeneous non-
mitochondrial RNA is pulse-labeled during the pre-MBT
stage [12], and recently Peter Klein and his group [17]
reported that there is a transcription of TGF-β family mem-
ber mRNAs, which is important for the post-MBT mor-
phogenesis. If these are all correct, it follows that tran-
scription of RNA polymerase II-dependent RNAs occurs
even before the MBT. In the original paper by Newport
and Kirschner [5,6], it is described that cleavage em-
bryos make a leaky transcription, since overexposure of
autoradiogram to X-ray film made faint signals in the
high-molecular-weight RNA region visible. In the reports
of Newport and Kirschner [5,6], however, this was not
interpreted as indicating the transcription of small amount
of heterogeneous mRNA-like RNA in pre-MBT stage.
The idea that transcription takes place even during the
pre-MBT stage is similar to that in sea urchin embryo-
genesis in which new transcription starts right after the
initiation of development or even before the fertilization.
If we think this way, we encounter the new idea that
MBT in Xenopus embryogenesis is not a remarkable
stage of the development at least concerning the tran-
scriptional activation of zygotic genome on a per-cell ba-
sis.
In our studies, however, based on 3H-uridine-labeled
RNA labeling profiles we previously proposed our work-
ing hypothesis that Xenopus early development consists
of three characteristic phases of RNA synthesis: The first
one is pre-MBT stage which is characterized by the syn-
thesis of heterogeneous mRNA-like RNA and low level
of small-molecular-weight RNA probably due to the ac-
tivity of DNA-dependent RNA polymerase II, and the
second phase is MBT stage which is characterized by
additional activation of tRNA synthesis due to the activ-
ity of DNA-dependent RNA polymerase III, and the third
phase is post-MBT stage which is characterized by addi-
tional active synthesis of rRNA due to the activity of
DNA-dependent RNA polymerase I (Figure 1). In 4 - 5
hrs after MBT, embryos reach early gastrula stage and
enter the phase of extensive morphogenesis including the
neural inductions due to invaginating Spemann organizer,
which results in the establishment of mesodermal and neu-
ral structures [20,21]. These induction processes are re-
sults of various cellular cross-talk involving interactions
of various growth factors and their receptors [2,22,23].
In the present article, we describe that rRNA is proba-
bly the only RNA species which is totally not transcribed
before MBT (or during pre-MBT stage) but starts to be
synthesized just after the MBT stage. Therefore, rRNA
fits the RNA whose gene was first described to be totally
silent during pre-MBT stage but is activated only after
MBT stage. In this sense, the general idea of the tran-
Figure 1. Characteristic patterns of RNA synthesis in early
Xenopus development. It is shown that there are three ma-
jor developmental stages, with respect to changes in RNA
synthetic pattern before and after MBT. From Shiokawa et
al. [13].
scriptional silence of zygotic nuclear DNA in Xenopus
development before MBT is still valid as far as the tran-
scriptional control of rRNA genes is concerned. It seems
to be valid that MBT is the stage when G1 phase appears
in the cell cycle and is the stage when the maternal pro-
gram of apoptosis is first executed as a fail-safe mecha-
nism of development but not before. However, we pro-
pose here additionally that MBT is the stage when rRNA
genes are first transcribed but not before.
2. TECHNICAL DIFFICULTIES OF
STUDYING RNA SYNTHESIS IN
XENOPUS EMBRYOS DURING
PRE-MBT STAGE OF DEVELOPMENT
Embryos of Xenopus laevis were first utilized as materials

K. Shiokawa / Advances in Bioscience and Biotechnology 4 (2013) 21-35 23
for molecular biological studies of developmental chang-
es in nucleic acid metabolism, especially in the field of
the control of RNA synthesis by Brown and Littna [24,
25]. These authors first isolated 32P-labeled RNA by phe-
nol treatment. Gurdon and Brown [26] proved that em-
bryos, which do not contain rRNA genes and therefore
do not form nucleoli, do not synthesize rRNA, and thus
showed that nucleoli formation is the cytological mani-
festation of the functioning of ribosomal RNA genes.
Shiokawa and Yamana [27,28] and Shiokawa et al. [29]
introduced dissociated embryonic cell system as a new
experimental system for studies of Xenopus embryonic
RNA synthesis, and after comparing the pattern of RNA
synthesis between 14CO2-labeled whole embryos and (3H)
uridine-labeled dissociated embryonic cells, Shiokawa and
Yamana [28] showed that developmental activation of
rRNA synthesis is not affected by the artificial cell dis-
sociation and proceeds normally in the dissociated cell
system just as in the whole embryos.
The utilization of the dissociated cell system is to
overcome the presence of impermeable surface coat which
prevents uptake of various radioactive RNA precursors
into the whole embryo. The inability of the whole em-
bryo to uptake radioactive precursors made detection of
newly synthesized RNA very difficult [28,29]. Further-
more, the previous idea of the total transcriptional silence
of pre-MBT stage embryos also partly came from the
situation in which cleavage stage embryos contain only a
very small number of nuclei. This latter situation of the
occurrence of only a small number of nuclei at the stages
before MBT (pre-MBT stage) tends to lead to estimation
of apparently undetectable or extremely low transcrip-
tional activity per embryo even though transcription per
nucleus may not be so small as compared with that in
cells in post-blastula stage embryos. Also, it is a very
characteristic feature of Xenopus fertilized eggs that they
contain several 1000-folds amount of ribosomes per cell
pre-formed by active transcription of extra-chromoso-
mally amplified ribosomal DNA [30], and this tends to
make it difficult to detect new formation of ribosomes in
early stages.
3. DEVELOPMENTAL CONTROL OF
RRNA GENE EXPRESSION AS
STUDIED USING DISSOCIATED
XENOPUS EMBRYONIC CELLS
Ribosomes are supramolecular structures which are large
cellular machinery to produce proteins. Cellular activity
to produce ribosomes sensitively reflects the cellular
physiological conditions for cell growth. In eukaryotic
cells ribosomes consist of 60S and 40S particles, and
RNA moiety of 60S particles consists of 28S rRNA and
two smaller RNAs, 5.8S RNA and 5S RNA, and that of
40S particles consists only of 18S rRNA. In Xenopus
oocytes rRNA producing genes, rDNA, are specifically
amplified at stage III of oogenesis [30] and mature oo-
cytes accumulate ca. 1012 ribosome particles which are
103 times larger than that of the adult-type cell and is
enough for supporting protein synthesis for development
up to the stage 42 feeding tadpoles [26].
Gurdon and Brown [26] proved that anucleolate X-
enopus mutant embryos which do not have rDNA [31,32]
neither synthesize rRNA nor form nucleoli, and thus
showed that nucleoli formation is the cytological mani-
festation of the functioning (transcription) of rDNAs.
Shiokawa and Yamana [27,28] and Shiokawa et al. [29]
used dissociated embryonic cells, instead of whole em-
bryos, as an experimental system for studies of Xenopus
embryonic RNA synthesis. Embryos were dejellied by
treatment with sodium thioglycolate (pH 8.3), and then
dissociated in the Ca-free medium containing 0.02 M
EDTA [28]. During culture isolated cells formed aggre-
gates of various sizes depending on the cell density, but
the relative rate of rRNA synthesis was the same, irre-
spective of the size of the aggregates finally formed [28].
Dissociated embryonic cells were labeled very extensive-
ly by simply adding radioactive precursor of RNA into
their culture medium, although to label whole embryos
14CO2 has to be used [24,28]. Shiokawa and Yamana [28]
compared the overall RNA synthetic pattern in 3H-
uridine-labled dissociated cells and 14CO2-labeled whole
embryos, and concluded that as long as the overall pro-
file of RNA-labeling pattern, which mainly reflects rRNA
synthetic activity, is concerned, developmental change in
the pattern of RNA synthesis is the same between the
dissociated cell system and the whole embryos. Shioka-
wa and Yamana [28] found that per embryo activity of
rRNA synthesis in dissociated blastula increases in the
dissociated cell system just like that in the developing
whole embryos, as embryos develop from the blastula
stage to the neurula stage.
4. MBT IS ALSO CHARACTERIZED BY
THE FIRST STAGE WHEN
MATERNALLY PRESET PROGRAM
OF APOPTOSIS IS EXECUTED AS A
FAIL-SAFE MECHANISM OF
DEVELOPMENT
MBT is characterized not only as the stage when G1
phase first appear in the cell cycle, but also the stage
when maternally-preset program of apoptosis is executed.
The execution of apoptosis in Xenopus embryos in the
very early development could be summarized as in Fig -
ure 2. As shown in Figure 2, it appears that embryonic

K. Shiokawa / Advances in Bioscience and Biotechnology 4 (2013) 21-35 24
Newmeyer et al. [43] showed that nuclear events typical
of apoptosis can be reproduced in the cell-free extract of
Xenopus eggs, the apoptosis mechanism seems to be a
maternally-preset device in Xenopus unfertilized eggs.
cells check themselves to see if they are capable of con-
tinuing further development at MBT, and if some cells
find themselves physiologically aberrant, they are re-
moved into the blastocoel and disappear from the em-
bryo by executing the apoptotic program [33]. In the cel-
lular activities to be checked here are included the level
of the methyl donor S-adenosylmethionine (SAM), DNA
structure, DNA replication, DNA methylation, RNA tran-
scription, and translation, as shown in the experiments
which utilized SAMDC mRNA [33-35], γ-ray [36-38],
cycloheximide [36-39], and 5-azadeoxycytidine (5-Aza-
CdR) [40] as stimuli for execution of the apoptosis. From
the experiments to trace cell lineage, we reached the con-
clusion that this apoptosis is to check and eliminate da-
maged cells shortly after MBT, and constitutes a surveil-
lance or a “fail-safe” mechanism to save the rest of the
embryo for normal development [33,35,41,42]. Since
5. EXTENSIVE MRNA CAP
METHYLATION TAKES PLACE, BUT
SYNTHESIS OF RRNA IS TOTALLY
ABSENT IN THE PRE-MBT STAGE AS
REVEALED BY THE ANALYSIS OF
RRNA-SPECIFIC 2’-O-METHYLATION
IN XENOPUS EARLY
EMBRYOS
Ribosomal RNA synthesis was first considered to be ini-
tiated at the gastrula stage during Xenopus embryogene-
sis [26]. However, evidence that rRNA synthesis does
ot occur in pre-gastrular stages was not completely n
Figure 2. Changes which take place during Xenopus early embryonic development. This model shows how early development
proceeds by indicating occurrence of apoptotic checkpoint at the midblastula stage or the stage of MBT. Fertilized eggs cleave
rapidly until the early blastula stage, and when they reached MBT stage, the “first developmental checkpoint” comes when G1
phase first appears. We assume that this check mechanism determines cell-autonomously if each embryonic cell may continue or
may not continue development. If there is a cell which is not good for continued development, the cell is eliminated by execution
of the maternal program of apoptosis. If the number of apoptotic cells was large, the whole embryo stops development and dies.
However, if the number of apoptotic cells was small, such cells are confined within the blastocoel and disappear due to apoptosis,
permitting the rest of the embryo continues on development. From Shiokawa [42].

K. Shiokawa / Advances in Bioscience and Biotechnology 4 (2013) 21-35 25
conclusive, because RNA fractionation methods routine-
ly used in previous experiments were sucrose density
gradient centrifugation and agarose or polyacrylamide
gel electrophoresis, and these methods did not complete-
ly separate rRNA from heterogeneous mRNA-like RNA,
which is synthesized very actively especially in embryos
during blastula stages [10,11].
18S and 28S rRNA molecules contain a relatively
large number of 2’-O-methyl groups (90 per 40S pre-
rRNA), and almost all of these 2’-O-methyl groups are
conserved during pre-rRNA processing and in the ma-
tured rRNAs. Most eukaryotic mRNAs, on the other
hand, contain no 2’-O-methyl group but the one in the
cap terminus position. Therefore, rRNA and mRNA can
be separately quantitated if nucleotides having 2’-O-me-
thylation were determined in nuclease digests of these
RNAs. Since rRNA precursor is methylated immediately
after its transcription, the detection of rRNA-specific
2’-O-methylation is a sensitive method to detect rRNA
gene products shortly after its transcription. Thus, the
methods to detect rRNA-specific 2’-O-methylation is the
methods to detect rRNA synthesis without being affected
by the presence of inevitably-contaminating mRNA in
the rRNA samples.
We labeled Xenopus embryonic cells with (methyl-3H)
methionine at the morula, blastula, gastrula and neurula
stages, and purified 3H-labeled high-molecular-weight
RNA from each embryos on sucrose density gradient
centrifugation. We digested the RNAs pooled from the
high-molecular-weight RNA regions with RNases A and
T2, and analyzed the nucleotide digests on DEAE-Se-
phadex columns. Figure 3 shows a set of results of such
experiments. Judging from the sucrose gradient profiles
(inset in each figure) rRNA synthesis indicated by the
presence of distinct 18S and 28S rRNA radioactivity
peaks occurred only at post-gastrula stages. In the pro-
files of morula (definitely, this stage is the pre-MBT
stage) and blastula RNAs, however, radioactivity was
widely distributed in the high-molecular-weight RNA
region, and one can not discriminate if this is the labeling
of rRNA or mRNA. We found here that about 80% of the
radioactivity was resistant to alkali hydrolysis, which
implies that most of the label in the high-molecular-
weight RNA region at these early developmental stages
(morula and blastula stages) was due to methylation of
DNA which was fractionated also in this region. Incor-
poration of the label into 4S RNA, which is another RNA
with ample 2’-O-methylation, occurred at all the stages
examined (from morula to neurula stages). The DEAE-
Sephadex column chromatographic profiles of morula
cell RNA labeled for 3 hr showed methylation peaks at
regions of charge value-2 (mononucleotide due to base
methylation) and charge value-5 (mRNA cap), but there
was no appreciable amount of 2’-O-methylation in the
region of charge value-3 (rRNA-specific dinucleotides;
NpmNp) or charge value-4 (rRNA-specific trinucleotides;
NmpNmpNp) nucleotides. This shows that morula cells
do not synthesize rRNA at all, although they synthesize a
considerably large amount of capped mRNA. In RNA
from early blastula cells which were labeled for 4 hrs
until the end of late blastula stage, the largest component
obtained in the DEAE column chromatography was the
charge value-3 compound (rRNA-specific dinucleotides).
Here, the charge value-4 compound (specific to 2’-O-
methylation in 28S rRNA) was also detected though in a
much smaller amount. These results show that rRNA
synthesis occurs already in embryos at the blastula stage.
The relative amount of the charge value-5 (mRNA cap)
component showing cap methylation in the blastula stage
was smaller than in morula stage. At the gastrula stage,
the relative amounts of charge value-3 and charge value-
4 substances, both indicating rRNA synthesis, increased
greatly and the relative amount of the cap methylation
became much smaller. The methylation profile of neurula
cells was essentially the same as that of gastrula cells.
6. RRNA SYNTHESIS IS ACTIVATED IN
THE LATTER HALF PERIOD OF
BLASTULA STAGE AS
DEMONSTRATED BY ANALYSIS OF
2’-O-METHYLATION
Since we found that embryos starts to synthesize rRNA
during the 4 hr from early blastula to late blastula stage,
we divided this period into two 2 hr-periods, and studied
rRNA synthesis by detecting 2’-O-methylation in each
period. The results obtained showed that embryos con-
tained only a trace amount of rRNA-specific dinucleo-
tides in the former 2 hrs of the blastula stage but con-
tained a very large amount of rRNA-specific dinucleo-
tides in the latter 2 hrs, indicating that rRNA synthesis
starts in the latter half of the MBT stage (Figure 4).
These results are consistent with the timing of the ex-
pression of exogenously-injected ribosomal RNA genes
[44]. By contrast, the extent of cap formation and base-
methylation were not greatly different in the former and
the latter half periods of blastula stage, indicating that
only rRNA synthesis is activated during the latter 2 hr
period of the blastula stage. From these results we con-
cluded that rRNA synthesis starts during the midblastula
to late blastula stage [10,11].
7. ONCE STARTED IN THE LATTER
HALF PERIOD OF THE MBT STAGE,
THE RATE OF RRNA
SYNTHESIS PER CELL IS CONSTANT
THROUGHOUT LATER STAGES
From the amounts of incorporation of the (methyl-3H)-
roup into rRNA-specific dinucleotides (NmpNp) and g

K. Shiokawa / Advances in Bioscience and Biotechnology 4 (2013) 21-35 26
Figure 3. Analysis of 2’-O-methylation in Xenopus early embryonic cells.DEAE-Sephadex A25 column chroma-
tographic profiles were obtained for nuclease digests of (methyl-3H)methionine-labeled high-molecular weight
RNAs, which had been purified by sucrose density gradient centrifugation (insets). Embryos were dissociated and
their cells were labeled with (methyl-3H)methionine. Numbers indicated are positions of marker oligonucleotides of
the indicated charge values. The number of embryos used for each labeling experiment was 3000 morulae (a); 300
early blastulae (b); 50 gastrulae (c) or 20 neurulae (d). The use of the changing number of embryos at different stage
was in order to obtain RNA from 106 cells at all the stages. In the case of the experiment in (a), one labeling experi-
ment was carried out using cells from 300 morulae and RNA extracted was subjected to sucrose density gradient cen-
trifugation, and high-molecular-weight RNA was pooled. This was repeated 10 times, and 10 high-molecular-weight
RNA preparations were pooled, and subjected to digestion with RNase A and T2, and analyzed on DEAE Sephadex
column at a time. Yellow peak is charge value-3 rRNA-specific dinucleotides (Nm
pNp). The −4 charge value compo-
nent is also for rRNA-specific methylated trinucleotides (Nm
pNm pNp). Green peak (−5 component) is for methylated
type I cap structure (m7GpppNm
pNp). Abscissa, Fraction number. From Shiokawa [42].
mRNA-specific cap, and the specific radioactivity of S-
adenosylmnethionine which was determined by sepa-
rate column chromatographic analyses, the rates of syn-
theses of 18S rRNA and 28S rRNA and capped mRNA
were estimated, assuming that the specific radioactivity
of the methyl-3H in S-adenosylmethionine is the same as
the specific activity of methyl-3H in the methyl group
contained in 2’-O-methylated nucleotides. The rate of
rRNA synthesis per embryo was about 1 ng/embryo/hr at
the blastula stage when rRNA synthesis starts in the latter
2 hr of the MBT stage, and this continued to increase
greatly from the late blastula stage to the neurula stage.
When the rate per cell of rRNA synthesis was calculated,
it is undetectable (less than 0.02 pg/cell/hr) during the
morula stage, 0.02 pg/cell/hr in the former 2 hr-period of
the MBT stage, and about 0.12 pg/cell/hr in the latter 2
hr-period of the MBT stage, and was constantly about
0.2 pg/cell /hr in the following 15 hrs of development
(from gastrula stage to early tailbud stage). Since the
appearance of nucleoli can be correlated with the start of
rRNA synthesis, we corrected the rate of rRNA synthesis
for the number of cells with definitive nucleoli. The ap-
proximate percentage of nucleolated cells increased
sharply during the blastulas stage: it is about 50% in the
late blastula stage and in later stages most cells have nu-
cleoli. Thus, the rate of rRNA synthesis was about 0.2
pg/nucleolated cell/hr, and this did not change greatly in
the later stages. It appears, therefore, that once rRNA
synthesis starts, its rate per cell does not change greatly
throughout development (Figure 5).
8. WEAK BASES SUCH AS AMMONIUM
SALTS AND AMINES SELE CTIVELY
INHIBIT RRNA SYNTHESIS IN
XENOPUS LAEVIS NEURULA CELLS
As for the mechanism of the developmental control of

K. Shiokawa / Advances in Bioscience and Biotechnology 4 (2013) 21-35 27
(a)
(b)
Figure 4. Comparison of RNA methylation pattern between the
former half and the latter half of the blastula stage. (a) Blastula
cells were labeled for 2 hrs with (methyl-3H)methionine imme-
diately after cell dissociation; (b) Blastula cells were first cul-
tured for 2 hrs, then labeled as in (A) for 2 hrs. RNA was ex-
tracted and DEAE-Sephadex column chromatography was per-
formed as in Figure 4 to find out 2’-O-methylatiom. Charge
value-3 component which is specific for rRNA synthesis occurs
only in the latter half of the blastula stage, whereas the labeling
of charge value-5 substance, which is mRNA cap-specific me-
thylation, occurred equally in both (a) and (b). From Shiokawa
et al. [10].
rRNA gene expression, the classical nuclear transplanta-
tion experiment by Gurdon and Brown [26] suggested
that a cytoplasmic factor is involved, because a nucleus
from later stage embryos transplanted into the unfertil-
ized egg ceased rRNA synthesis during cleavage and re-
stored it at and after the gastrula stage. Shiokawa and
Yamana [45] prepared dissociated blastula and neurula
cells and cultured these cells together or separately and
obtained results which suggested that Xenopus blastula
cells may release some factor that inhibits rRNA synthe-
sis in neurula cells [45]. Since the boiled culture medium
(conditioned medium) from the blastula cell culture was
effective as the unheated culture medium, we thought
Figure 5. Rates of syntheses of three major RNA species dur-
ing Xenopus development. Early stages were divided into three
periods with respect to RNA synthetic activity. Phases I-III are
characterized by active synthesis of mRNA, tRNA and then
rRNA by DNA-dependent RNA polymerases II, III, and I, re-
spectively. From Shiokawa et al. [13].
that the active substance that inhibits rRNA synthesis in
neurula cells is a low-molecular-weight substance. The
experiments utilizing the conditioned medium, however,
were not of high reproducibility [45,46]. Therefore, we
homogenized early embryos with 0.5 N perchloric acid
(PCA) and after neutralizing it with KOH, applied it onto
a small charcoal column, and eluted acid-soluble materi-
als from the charcoal column with ammonia-alcohol (0.2
N ammonia-50% ethanol). The white powder obtained
by lyophilization of the ammonia-alcohol eluate selec-
tively inhibited rRNA synthesis in neurula cells [45,46].
After a long lasting efforts for purification of the active
substance, we finally reached the conclusion that the ac-
tive substance in the charcoal eluate was ammonium per-
chlorate that was formed artifactually during the charcoal
column chromatography [47]. In these results, however,
the inhibition of rRNA synthesis induced by the ammo-
nium perchlorate was quite strong and in spite of the
inhibition of rRNA synthesis 4S RNA synthesis was not
affected appreciably. Therefore, we continued to study
how ammonium salts affect rRNA synthesis in Xenopus
neurula cells.
The concentration of ammonium perchlorate which

K. Shiokawa / Advances in Bioscience and Biotechnology 4 (2013) 21-35 28
selectively inhibits rRNA synthesis turned out to be ca.
2.5 mM. We compared the effects of the same concen-
tration (2.5 mM) of ammonium perchlorate and sodium
perchlorate in Xenopus neurula cells, with the finding that
only the former but not the latter inhibited rRNA synthe-
sis selectively (Figure 6). This implied that the effective
part of the ammonium perchlorate is not the perchlorate
moiety, but the ammonium moiety. We cultured Xenopus
neurula cells in the medium which contained 2.5 mM of
ammonium chloride and ammonium sulfate, and found
that both of these inhibit rRNA synthesis, with little in-
hibition on 4S RNA synthesis [47-49]. We tested ammo-
nium dihydrogenphosphate, ammonium monohydrogen-
phosphate, and ammonium phosphate, using as referenc-
es, potassium dihydrogenphosphate, potassium monohy-
drogenphosphate and potassium phosphate all at 2.5 mM.
We found that only ammonium ion-releasing salts were
effective in inhibiting rRNA synthesis, and the effective-
ness was roughly proportional to the number of ammo-
nium ions to be released. We also found that while am-
monium acetate and ammonium aspartate selectively
inhibited rRNA synthesis at 2.5 mM, again, their potas-
sium salt were ineffective. We also found that ammo-
nium salts of all the four major ribonuleotide monopho-
sphates (5’-AMP, 5’-GMP, 5’UMP, and 5’-CMP) at 5
mM were effective in selectively inhibiting rRNA syn-
thesis in neurula cells. We also tested the effects of amin-
es such as monomethylaminehydroperchloride, dime-
thylaminehydroperchloride, and trimethylaminehydrop-
erchloride, and found that all of these are active as selec-
tive inhibitors of rRNA synthesis at 2.5 mM. Similar po-
sitive results were obtained when three kinds of ethyla-
mines were tested. From these results, we concluded
Figure 6. Effects of ammonium perchlorate and sodiumn per-
chlorate on RNA synthetic pattern of neurula cells.Neurula
cells were labeled for 3 hrs with 20 μCi of 3H-uridine for 3 hrs
in the presence of 2.5 mM of ammonium perchlorate (B) or
sodium perchlorate (C); RNAs were extracted and electropho-
resed on 0.5% agarose-2.4% polyacrylamide gels; (A) Control
neurula cells. Dotted peaks are for 28S and 18S rRNAs, and
black peaks are for 4S RNA (tRNA). From Shiokawa et al.
[47].
that weak bases such as ammonium ion and amines are
similarly effective as selective inhibitors of rRNA syn-
thesis in Xenopus neurula cells.
9. WEAK BASES ALSO SUPPRESS
DEVELOPMENT AL ACTIVA TION OF
RRNA SYNTHESIS IN XENOPUS
BLASTULA CELLS
Weak bases inhibit rRNA gene expression in Xenopus
neurula cells in which rRNA synthesis is fully activated.
We cultured blastula cells for 5, 10, 15 hrs in the medium
which contained 2.5 mM of ammonium salts. When con-
trol blastula cells were labeled with 3H-guanosine for 5
hrs a small amount of incorporation was detected in 28S
and 18S rRNA with a large amount of incorporation into
4S RNA and DNA (Figures 7(A)-(C)). Ammonium
chloride, but not potassium chloride, inhibited rRNA
synthesis but not 4S RNA and DNA syntheses. When we
first cultured blastula cells in the ammonium chloride-
containing medium for 5 hrs, and then labeled them for 5
hrs in the continued presence of the ammonium chloride,
much clearer inhibition of rRNA synthesis was obtained.
This is an indication of the inhibition of post-MBT acti-
vation of rRNA synthesis (Figures 7(D)-(F)). Essentially
the same results were obtained when we cultured blastula
cells for 10 hrs in the presence of ammonium chloride
and then labeled them for 5 hrs (Figures 7(G)-(I)). Im-
portantly, syntheses of 4S RNA (tRNA) and heteroge-
neous nuclear RNA, and in addition, DNA were not in-
terfered greatly under these conditions. Using 10% poly-
acrylamide gel, we separated small-molecular-weight
RNAs in the RNA preparations obtained and confirmed
that ammonium chloride did not inhibit the labeling of
4S RNA, 5S RNA, and U1, U2, and U5 small nuclear
RNAs.
When we cultured blastula cells for 5 hrs in the am-
monium ion-containing medium and after this transferred
them into the normal medium which contained 3H-gua-
nosine. We found here that rRNA synthesis can be re-
stored to a large extent, indicating that the effects of
ammninium salts are reversible. By HPLC analysis of the
acid-soluble faction of the embryonic cells, we confirm-
ed here that ammonia was incorporated into neurula cells
within 1 hr. We also confirmed that ammonium salts at
ca. 3 mM did not inhibit protein synthesis, cell division,
and cellular reaggregating activity at least for the first 10
hrs of incubation, although after treatment for 10 hrs, cell
reaggregation was found to be significantly inhibited.
These data indicates that ammonium salt actually selec-
tively inhibits not only the initiation of rRNA synthesis
at MBT but also subsequent activation of rRNA synthe-
sis in post-MBT stages.

K. Shiokawa / Advances in Bioscience and Biotechnology 4 (2013) 21-35 29
Figure 7. Inhibition of the initiation and activation of rRNA
gene expression by ammonium chloride but not by potassium
chloride in Xenopus blastula cells. Blastula cells were treated
with 2.5 mM of either ammonium chloride ((B), (E), (H)) or
potassium chloride ((C), (F), (I)) and labeled with 3H-guano-
sine. ((A)-(C)) Blastula cells were labeled for 5 hrs. ((D)-(F))
Cells were cultured for 5 hrs in the presence of ammonium
chloride (E) or potassium chloride (F), and then labeled for 5
hrs with 3H-guanosine in the continued presence of the salts.
((G)-(I)) Cells were cultured for 10 hrs in the presence of the
salts and then labeled for 5 hrs in the continued presence of the
salts. RNAs were extracted and electrophoresed on 0.5% aga-
rose-2.4% polyacrylamide gels. In this profile the radioactivity
appeared also in DNA. ((A), (D), (G)) Control cells. Black
peaks are for 28S and 18S rRNA, and white peaks are for 4S
RNA, and shaded peaks are for DNA. From Shiokawa et al.
[48].
10. WEAK BASES INHIBIT RRNA
TRANSCRIPTION, BUT NOT
POST-TRANSCRIPTIONAL
PROCESSING
Xenopus neurula cells were first inhibited for 2.5 hrs by 5
mM of ammonium chloride or monomethylamine hydro-
perchloride and then pulse-labeled for 2.5 hrs in the con-
tinued presence of the weak bases [47]. We obtained
active labeling of heterogeneous nuclear RNA (hnRNA),
and 40 S rRNA primary transcript, in addition to the la-
beling of 28S and 18S mature rRNAs and 4S RNA. Un-
der these conditions, both of the weak bases inhibited la-
beling of 40S pre-rRNA, 28S rRNA, and 18S rRNA al-
most completely (Figure 8), suggesting that the inhibi-
tion of rRNA synthesis by these weak bases is at the
transcription level.
We then selected conditions of partial inhibition of
rRNA synthesis by these weak bases. When we labeled
the control neurula cells for 1 hr (Figure 9(A)), we ob-
tained 40S pre-rRNA (black peak), 30 S rRNA interme-
diate (shaded peak) and 18S rRNA (dotted peak), in ad-
dition to a large amount of heterogeneous mRNA-like
RNA [50]. The appearance of 18S mature rRNA before
28S mature rRNA is due to the fact that the processing of
the former is finished before that of the latter [51]. The
identification of 30S component is based on slight re-
Figure 8. Effects of ammonium salts and amine on pulse-la-
beled RNA synthesis. Xenopus neurula cells were treated with
5 mM each of potassium chloride (B); ammonium chloride (C);
or monomethylaminehydroperchloride (D) for 2.5 hrs and then
pulse-labeled with 3H-uridine for 2.5 hrs in the continued pre-
sence of the potassium and ammonium salts. RNA was extract-
ed and gel electrophoresed, and radioactivity determined using
the sliced gels. Densitometric scanning of gels before slicing
are omitted from the radioactivity profiles. Peaks of 40S rRNA
precursor are marked by black colour. A, Control untreated
cells. From Shiokawa et al. [47].

K. Shiokawa / Advances in Bioscience and Biotechnology 4 (2013) 21-35 30
Figure 9. Effects of ammonium chloride on the labeling of 40S
pre-rRNA, 30 S pre-rRNAs and 18S and 28S mature rRNAs.
Neurula cells were labeled for 1 hr ((A), (B)) or for 3 hrs ((C),
(D)) with 3H-uridine in the presence of 2.6 mM ammonium
chloride. RNAs were gel electrophoresed for 2 hrs ((A), (B)) or
1 hr ((C), (D)). Longer electrophoresis was for obtaining better
resolution in the high-molecular-weight RNA region. Densi-
tometric scanning of gels before slicing are omitted from the
radioactivity profiles. Black peaks are for 40S pre-rRNA, shad-
ed peaks are for 30S pre-rRNA, and dotted peaks are for 28S
and 18S mature rRNAs. From Shiokawa et al. [50].
tardation of this peak from 28S optical density peak on
the agarose-polyacrylamide gel. When neurula cells pre-
treated for 2.5 hrs with 2.6 mM ammonium chloride
(which was the conditions for partial inhibition of rRNA
synthesis) were labeled with 3H-uridine for 1 hr, the ex-
tent of the inhibition of labeling was 60%, 59%, and 70%
for 40s pre-rRNA, 30s rRNA intermediates and 18S rRNA,
respectively (Figures 9(A) and (B)). When the same cul-
tures were labeled for 3 hrs, labeling of 18 S and 28 S
mature rRNAs but not 40S and 30S pre-rRNA became
predominant, and inhibition here was 72%, 69%, and 7%
for 28S and 18S rRNAs and 4S RNA, respectively (Fig-
ures 9(C) and (D)). When we performed a parallel expe-
riment using 1 mM trimethylammonium perchloride, the
inhibition was 67%, 70%, 75%, 73% and 8%, for 40S
pre-rRNA, 30S rRNA intermediate, 28S rRNA, 18S
rRNA and 4S RNA, respectively. Thus, the extents of the
inhibition of the labeling of 40S pre-rRNA, 30S interme-
diate rRNA, 18S and 28S mature rRNA were much the
same (67% - 75%), and in spite of the large inhibition of
the labelinbg of rRNA species, inhibition of the labeling
of 4S RNA (tRNA) was only 8%. Therefore, we con-
cluded that inhibition was at the level of the formation of
40S primary transcript and [50] probably not at the proc-
essing of 40S pre-rRNA.
Approximately 30 - 60 min is needed for the pulse-
labeled 40S pre-rRNA to be processed completely into
18S and 28S rRNAs [50,51]. Therefore, we first pulse-
labeled neurula cells for 35 min and chased the label in
rRNA species for 2 hrs. Labeled RNAs in neurula cells
under these conditions were 40S pre-rRNA and hetero-
geneous mRNA-like RNA (Figure 10(A)). These neu-
rula cells were administered with actinomycin D (10
μg/ml) as a transcription inhibitor, and treated either with
ammonium chloride (5 mM) or with trimethylammonium
perchloride (5 mM) for 2 hrs in the continued presence
of actinomycin D. During the chase period of 2 hrs, the
label in the 40S pre-rRNA in the control culture com-
pletely disappeared and 18S and 28S mature rRNAs ap-
peared (Figure 10(B)). Such changes in the labeling pro-
file were observed also in the cultures treated with 10
mM ammonium chloride (Figure 10(C)) and trimethyl-
ammonium perchloride (Figure 10(D)). These results
show that weak bases used here inhibited rRNA tran-
scription but did not inhibit the processing of 40S pre-
rRNA into two mature rRNAs [50]. Also, these results
exclude the possibility of aberrant processing or rapid
wastage of the mature rRNAs.
Figure 10. Effects of ammonium chloride on the processing of
40S pre-rRNA which was labeled before administration of am-
monium chloride. Neurula cells were pulse-labeled with 3H-
uridine for 35 min. One culture was then immediately frozen as
a zero-time control (A). Three cultures were then administrered
with 10 μg/ml of actinomycin D and further cultured for 2 hrs
in the presence of either 10 mM ammonium chloride (C) or 10
mM of trimethylammonium perchloride (D). Control cells were
incubated for 2 hrs without being exposed to these weak bases
(B). RNA were extracted and electrophoresed on agarose-poly-
acrylamide gels. Densitometric scanning of gels before slicing
are omitted from the radioactivity profiles. A black peak is 40S
pre-rRNA and dotted peaks are 28S and 18S rRNAs. From Shi-
okawa et al. [50].

K. Shiokawa / Advances in Bioscience and Biotechnology 4 (2013) 21-35 31
11. POSSIBLE INVOLVEMENT OF PH
CHANGE IN THE WEAK
BASE-TREATED NEURULA CELLS
From these results, the mechanism by which weak bases
inhibit rRNA synthesis in Xenopus embryonic cells can
be summarized as in Figure 12.
In Xenop us embryos, a slight increase of intracellular pH
has various important effects [52]. Such pH changes
have been implicated as a necessary step in oocyte ma-
turation [53], fertilization [54], and commencement of
cleavages [55]. We suspected that a slight elevation of
intracellular pH might be involved in the inhibition of
rRNA synthesis. Since it is known that pH-mediated
changes are abolished when Na+ was eliminated from the
surrounding medium [53,56], we tested the effect of 3
mM ammonium chloride and 1.5 mM trimethylammo-
nium perchloride on neurula cells in the medium in
which all the Na+ was replaced by choline ions. Results
of the labeling experiment showed that approximately
80% of the inhibitory activity of both ammonium chlo-
ride (Figure 11(B)) and trimethylammonium perchloride
(Figure 11(C)) completely disappeared when their ef-
fects were tested in the medium whose Na+ had been re-
placed by choline ions (respectively, Figures 11(E) and
(F)). Therefore, these weak bases seem to exert their
rRNA synthesis-inhibiting effects via slight pH elevation
within the neurula cells.
Figure 11. Disappearance of the inhibitory effect on rRNA
synthesis of ammonium chloride and trimethylammonium-
perchloride in the medium in which Na+ was replaced by
choline ion. Neurula cells were treated for 2.5 hrs either in the
normal medium (0.1 × Steinberg’s solution) ((A)-(C)) or in
the choline+-containing Na+-free medium ((D)-(F)) with 3
mM of ammonium chloride ((B), (E)) or 1.5 mM of trime-
thylammonium perchloride ((C), (F)). ((A), (D)) are untreated
control neurula cells. All the cultures were administered with
3H-udirine and cells were labeled for 3 hrs. Dotted peaks are
18S and 28S rRNAs. From Shiokawa et al. [50].
We prepared acid-soluble fractions of these weak
base-treated neurula cells, and found that the level of
ATP as well as other ribonucleotidetriphosphates re-
mained unchanged. Therefore, neither ammonium chlo-
ride nor trimethylammonium perchloride disturbed the
energy generating system in Xenopus neurula cells [50].
Figure 12. A working hypothesis that weak bases, most probably ammonium ion within the embryonic cells, may be involved in the
regulation of rRNA transcription in Xenopus early embryos. Left: Weak bases do not inhibit TCA cycle and do not induce wastage of
40S pre-rRNA and 18S and 28S mature rRNAs. Also, processing of pre-rRNAs is not inhibited. Instead, weak bases inhibit transcrip-
tion of rRNA genes (rDNA). This inhibition is probably mediated by intracellular slight pH elevation. From Shiokawa et al. [50].
Right: Developmental changes in the amount of ammonium ion within the embryo determined by amino acid analyzer using the
PCA-soluble fraction of the embryo homogenates. The changing amount of the ammonium ion is expressed as ammonia (ng/egg or
embryo). From Shiokawa et al. [48].

K. Shiokawa / Advances in Bioscience and Biotechnology 4 (2013) 21-35 32
12. UPTAKE OF AMMONIUM ION AND
AMINES IN XENOPUS NEURULA
CELLS
Using amino acid analyzer, we analyzed ammonia and
amines in acid-soluble fractions of Xenopus neurula cells
which had been treated with 2.5 mM of either ammo-
nium chloride, ammonium phosphates, or methylamines.
In the amino acid profiles, it has been shown that the
peak representing ammonia became quite large in the
cells treated with ammonium salts. In cells treated with
monomethylamine hydroperchloride, a large amount of
monomethylamine was detected, with no increase in the
amount of ammonia. However, amounts of other com-
ponents such as ornithine and other amino acids in the
neurula cells did not change appreciably in spite of the
treatments with ammonium salts and amines. These re-
sults indicate that both ammonium ion and amines selec-
tively inhibit rRNA synthesis after being uptaken into
Xenopus neurula cells.
13. AMMONIA IS A CANDIDATE OF THE
REGULATOR OF RRNA SYNTHESIS
WHICH CONTROLS THE
INITIATION AND ACTIVA TION O F
RRNA SYNTHESIS AT AND AFTER
MBT
When we examined ninhydrin-positive materials in the
acid-soluble fraction of Xenopus early embryos, there
was a sizable amount of ammonia but not amines within
the embryo. When we performed similar analyses at va-
rious stages of development, we found that the level of
ammonia in an egg was ca. 55 ng/egg before fertilization
and this value did not change greatly during cleavage,
but decreased promptly to the level as low as ca. 20 ng/
embryo. The occurrence of ca. 55 ng/egg of ammonia
corresponds ca. 3 mM of ammonia as an intra-egg con-
centration, and 20 ng/embryo of ammonia corresponds
ca. 1 mM as an intra-embryo concentration, since the vo-
lume of an egg is ca. 1 μl. The lowered level of ammonia
was maintained throughout later stages until the muscu-
lar response stage (Figure 12).
We attempted to isolate ammonia (and amines if any)
from cleavage stage embryos. Starting from 25,000 clea-
vage embryos, we isolated about 10 mg of residual mate-
rials after evaporating the hydrochloric acid solution that
was used to capture the cellular volatile ammonia com-
ponents. Mass spectrometric analysis revealed the pres-
ence of ammonium chloride but not of amines. Ten mil-
ligrams of ammonium chloride obtained here corre-
sponds to ca. 4 mg of ammonia. We tested effects of the
ammonium chloride isolated from cleavage stage em-
bryos for its activity to inhibit rRNA synthesis in Xeno-
pus neurula cells at 1.0 and 5.0 mM. The results showed
that the synthesis of rRNA was inhibited by 60% (at 1
mM) and 90% (at 5 mM) with only a slight inhibition
(less than 10%) in 4S RNA (tRNA) synthesis. These ob-
servations also suggest that ammonium ion is a candidate
for the factors that regulate initiation and activation of
rRNA synthesis in Xenopus embryogenesis.
14. CONCLUSION
It has been long believed that RNA synthesis never takes
place from zygotic nuclei during pre-MBT stage or clea-
vage stage in early Xenopus embryogenesis. However,
Shiokawa’s old data [12] and Klein’s new data [17] show
that there is RNA synthesis in pre-MBT embryos. How-
ever, not all the RNA is synthesized during the pre-MBT
stage. For example, rRNA is the RNA whose genes are
totally silent during the pre-MBT stage, and transcription
of rRNA genes is initiated from the latter half of the
MBT stage. This developmental change in the initiation
of rRNA genes coincides with the developmentally con-
trolled appearance of nucleoli not before but after MBT.
Weak bases were found to selectively inhibit rRNA syn-
thesis in neurula cells and also inhibit the developmental
activation of rRNA synthesis which takes place after MBT.
Based on some evidence, weak bases such as ammonia
could be a factor that constitutes the control mechanism
of the developmental regulation of rRNA genes at the
transcriptional level, but not at the step of the post-tran-
scriptional processing level. Our data suggest that ammo-
nia and amines exert their effect via slight elevation of
intracellular pH in the early Xenopus embryos.
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