2
1
LH
h¥
初 切 ︒
¥
︒
︒
2 Iー
1 3
LS¥
旬
¥凶ω
柄
︒
同X
Ea u
48 72
development 2 4
post‑diapause Hours
。
• n
Fig. 14. lncorporation of radioactive amino acid into acid‑insoluble fraction o[ post‑diapause eggs. Eggs were incubated for 48 hr at 25
・
c after ovipos1 t
ion，
chilled for 180 days 8nd then made to initiate the post‑diapause develop闘ent. Solid circles represent the incorporation va!ues for the post‑diapause eggs incubated at 25・
C. Open circles show the values of the simliar eggs ‑but activated by the hot‑HCl treatment after chilling，
followed by 25・
C incubation. The procedures of experiments were sim孟larto those described in Fig. 1272
DISCUSSION
The changes of incorporation of a radioactive precursor into egg proteins were measured. The value was low io unfertょlized eggs. It rose during the pre‑diapause develop‑
ment in the deposited
，
fertilized eggs until 20 hr of age，
when a part of the eggs were treated w
ェ
th hot acid. In thus‑treated eggs the incorporation augmented rapidly making a peak on 36 hr but low at 48 hr. Since the eggs at 36 hr of artificial non‑diapause development are at the m且ddle gastrulation stage and those at 48 hr at the onset of orga nogenesis，
the present data indicated that the rate of incorporation was most active at the time preceding the extensive morphogenetic alterations Somewhat similar results have previously reported (Kawaguchi and Fujii，
1984)
，
where the incorporation of tritiated leucine into the acid‑insoluble fraction (labeling time was 6 hr) became minimum at the age of 48 hr after oviposition (hot‑acid treated at 20 hr)，
although the temporal decrease of incor poration was not 50 5harp and i乞5 time was shorter compared to the present data. In contrast，
the result5 of similar experiments reported from another laboratory (Sonobe and Odake，
1986) gave the incorporation rate of [35S1methionine(for 3 hr) which rose linearly after the hot‑HCl treatment These discrepancies among the different investigators may be due lo the d且fferentstrains used and/or experimental conditions
，
in part斗cular the specific activity of labeled73
precursors and the duration of labeling time. In the previ‑
ous experiments cited above
，
the labeling time was much longer than that (1 hr) applied in this experiment. Under the presenL conditions，
the incorporation rose linearly until 30 min，
leveled off until 1 hr and fell thereafter (data not shown). In future，
the amino acid incorporation into proteins should be normalized by measuring the pool size of direct precursor (amino‑acyl tRNA) of protein syn thes孟s，
to obtain the absolute rate of protein synthesis，
although measuring the pool size requires tedious work so that the method has previously been applied for only limited species
，
e .g. the sea urchin embryos (Fry and Gross，
1970; Regier and Kafatos
，
1977).Nevertheless
，
the present investigation，
together with those reported previously，
confirmed that the rate of incor‑poration increased rapidly after the activation of eggs by hot acid
On the other hand
，
the eggs which were allowed to enter diapause without hot‑acid treatment displayed a decreasing pattern，
leaving a temporal maximum at 20 to 30 hr of age. This result was in substantial agreement with those reported previously (Kawaguchi and Fuj i i，
1984;Sonobe and Odake
，
1986). After the long‑term chilling of hibernating eggs，
the apparent ability of amino acid incorporation had already recovered as seen in the O‑hr plot of Fig. 14. The time and mode when and how this recovery occurs during the period of cold treatment remain to be74
investigated
The incorporation was measured a1so for the post‑
diapause development. In comparison between the eggs with and without the hot‑IIC1 treatment
，
no difference was observed until 48 hr after the onset of the post‑diapause development. The chilled eggs before the start of the development already had the activity of amino acid incorporation as discussed above. In the chilled eggs，
the incubation for labeling was brought about at 25・
C，
and thevalue could be understood as an intrinsic potential of protein synthesis
，
since under the natural conditions these eggs would undergo limited protein synthesis at the low temperature The value rose rapidly without a lag period in agreement with the previous report for the acid‑treated post‑diapause eggs (Saito et 81.
，
1982). The incor poration made a peak aL 12 hr，
fell thereafter and rose again from 24 hr，
when the extensive organogenesis com mences The value attained another peak at 72 to 96 hr(patterns omitted). The marked fluctuat孟on curve observed here is in contrast to the pattern seen in a prevl.ous investigation
，
in which the increase continued 1inearly until 96 hr ¥athollt tempornl minimum (Saito et 81.，
1982).The discrepancy again may be ascribed to the different strnins and/or experimental conditions as discllssed above.
The post‑diapause eggs treated by hot‑HCl develop normally as do those without the treatment. Therefore
，
little difference in the developmental changes in amino ncid
75
incorporation rate between the eggs with and without hot acid treatment
，
as seen until 48 hr of the development，
is not a matter of surprise. From this point of view，
the dispariLy between these kinds of eggs as observed after 72 hr (the gap continued until 120 hr，
patterns not shown) offers a peculiar question: Irrespcctive of whatever the present result mirrors some difference in absolute rate of protein synthesis or that in the pool size of methionine directly affecting the apparent rate of its in corporation into proteins (serg and Mertes，
1970)，
some delayed effects of hot‑acid treatment are suggested. More detailed investigation will be needed to solve the problem.The overall results of the experiments described in this chapter confirm that marked fluctuation in rate of amino acid incorporation into whole proteins (thus probably in rate of protein synthesis) occurs at the stages investi‑ gated in the previous chapters.
GENERAL DISCUSSION
The extent of protein synthesis in the diapause eggs of B. mori has previously been assessed by the measurements of both the incorporation of radioactive amino acids into the acょd‑insoluble fraction (Kawaguchi and Fujii
，
1984; Sonohe，
1986; Saito et 81.
，
1985a) and the proportion of polysomal ribosomes per total population of ribosomes (Saito et 81.，
1982). It was concluded that the rate of protein synthesis in diapause eggs is suppressed，
although these have translatable mRNAs (Saito et 81. I 1984)，
suggesting the regulat斗on or protein synthesis at the translation level is involved at the developmental switches. $oon after the onset of post‑diapause development I these mRNAs w
エ
11 be utilized for translation in the cells，
although contribution by newly transcribed mRNAs must gradually become importanl. On the other hand，
the start of diapause and its intensification may accompany the dissociation of mRNAs from polysomes (Saito
，
unpublished results). Therefore
，
the author is interested in the problem whether mRNAs change qualitatively at the different stages of dェ
apause and development in the eggs of B. mori.The present study was mainly aimed at the analyses of the species of RNAs which could be translated in an in vitro prote且n synthesizing system. The patterns of in vitro translational products were compared between the stages of
77
pre‑diapause
，
artificial non‑diapause and post‑diapause development The post‑diapause eggs were divided in lwo groups: those activated by chi11ing a10ne and those treated by hot HC1 after chi11ing. RN^s from a1l states of eggs gave more than 130 countable spots on the 2D fluorograms Ionly a part of which underwent the changes in intensity.
No large d孟fference of the pattern was observed among pre diapause eggs
，
chilled eggs and post‑diapause eggs. Thesefacts indicate that the extensive interchange of the pre‑
existing set of mRNAs does not take place in the eggs
，
in which physiological and morphological alteration are actえvely progressing. Moreover，
the rate of in vivo in‑corporation of [~~S]methionine 35 into the acid‑insoluble
fraction altered extensively
，
suggesting that protein syn‑thesis is changed in terms of quantity. This situation was typical at the first 24 hr after the onset of post‑
diapause development; the initial activation of protein synthesis at this stage does not require the appearance of new set of mRNA species.
The present results
，
together with the previous knowl‑edge of the presence of active mRNA cited above
，
a1so indi‑cate that eggs during diapause and chilling has 1imited activity of transcription (and of mRNA metabolism); thus the mRNAs 且n the eggs after terminotion of diapause may contain the population present in the eggs at the beginning of diapause
1n addition to the species of trans1atable mRNAs
，
those78
of the proteins synthesized in vivo were analyzed. 1n order to compare the diapausing stage (the eggs chilled for 11 days were prepared as diapause eggs) and the artificial non‑diapause eggs were used for the experiments. Conse‑
quently
，
most of the spots were not different throughout the stages tested. These results seem to imply that a little changes in quality of proteins are required to the devel‑opment. This situation is different from that in D me1anogaster (Summers et 81.
，
1986) and in the sea urchin (Bedard and Brandhorst，
1983) I Xenoplls 1aevis (Bal‑lantine et ld.
，
1979); in these organisms，
the increase of incorporation rate of radioactive amino acid is augmented in paral1el with the occurrence of new protein species until the stage of gastrulation. As to the quantitative aspect，
the B. mori eggs also exhibited an decrease in in‑corporation of amino acid into proteins at an early stage of gastrulation (48 hr after oviposょtion) and this mode of changes resembled those of the above organisms
1n the f]uorograph孟c study
，
chilled eggs (stil1 had no developmental ability when transferred to 25・
C) possessed the potency of incorporation to give an identical pattern to that of the developing eggs. This incorporation in the dormant eggs would be c8used by the breakage of chorion.It has been argued that the diapause phase is maintained by the limited supply of oxygen (Okada
，
1971; Sonohe et 81. I 1979). lndeed，
when diapause eggs are incuhated in nitrogen gas and polyols (the laLter are specifically ac‑79
cumulated in relation to diapause)
，
apparent dormancy lasts (Okada，
1971; Kageyama and Ohnishi，
1973) The present results suggest that the diapausing eggs，
in particular those during chilling，
are equipped with the ability to recover protein synthesis，
and the regulation mechanism could be immediately turned on，
although the actual rate ofprotein synthesis 且n intact eggs would be low enough t
。
avoid consuming the energy sources (Chino
，
1957，
1978).The presence of mRNA is cons且dered to be a strategy for rapid re‑activation when the environmental conditions become adequate to ini t五ate development. The low rate of protein synthesis in a dormant phase is explained
，
at least partly，
by the limited access of mRNAs to the translat
エ
on system. In this connection is of importance the finding that the cap site of mRNA molecules from dormant eggs are lacking in methylation (Saito et 81.，
1985b)，
which is necessary fornormal translation of mRNAs
In the meantime
，
the post一
diapauseeggs activated by the hot‑HCl treatment gave temporally appearing spots as revealed by the in vitro translation experlments. Such spots (bl，
b2，
b3 and b4) disappeared in the pattern at the subsequent stages tested. The spots b1 to b4 did not appear at the comparable stages after the onset of the post‑diapause development that was evoked by chilling alone. On the basis of these modes of appearance
，
the components bl tob4 are assumed to be stress‑shock protelns like hsp70 of D.
melanogaster (Buzln and Petersen
，
1982; Ashburner and Bonー80
ner
，
1979) These componenLs were characterized by having pI of 5 to 6 and size of about 70 kOa on the 20 gel，
the values resembling those of hsp70 (loc. cit.)The RNA(s) which was hybridizable with a probe contain‑
ing the D. melanogaster hsp70 gene were 81so detected by the Northern blotting in the post‑diapause eggs at 6 hr after the hot‑HCl treatment. Such a signal was not found in the eggs at the same age without the hot‑acid Lreatment. This situation is in paral1el with that of the appearance of the bl
，
b2，
b3 and b4 spot 】n the above described in vitro translation study. Although the band w孟th the hybridization signal was single，
and whether it constituted of plural components is unknown，
it is tempting to suppose that the band contained the mRNAs for any or al1 of the b1，
b2，
b3 and b4 proteins1n addition
，
a trace signal of hybridization was de‑tected ln all stages examined
，
suggesting that the relevant RNA has a basa! expression. Ilowever，
in the in vitro trans‑lation experiments
，
the corresponding appearance was not detected. 1t should be noted here，
however，
that the bl，
b2 8nd b3 spots were detected in pre‑diapause stage，
indi caLing the presence of mRNAs at this period.The author suggests that the tentative stress‑shock proteins found in the present study may be related to the recovery from the stress that 1s forced to occur by the hot‑IICI treatment. Also hsp70 has been discussed in rela‑
Lion to the survival at an e!evated temperature (Mitchell et
81
81. I 1979). In genera1 the stress‑shock proteins in deve1‑
oping embryos can be produced when the transcriptional ability of the embryonic genome is estab1ished; in the 2‑
cell slagc in case of the mouse embryo (Petzoldt et 81. I
1981). Also io the post‑d込apause eggs of B. mori
，
the appearance of bl etc. may offer an indication of trans‑criptional ability necessary for the recovery from the stress. In the light of this inference
，
the fact that any of the bl，
b2，
b3 and b4 was not detected in the artificial non‑diapause eggs after the hot‑HCl treatment is rather incomprehensible. In European races，
a spot induced by hot‑IICl treaしment has been reported (Dorel and Cou1on，
1988) as discussed in Chapter II. The difference would be due to some evo1utional background of Lhe races used.The in vitro trans1alion products named b5
，
b6，
b8，
b15
，
b19，
b20，
b22，
b23，
b27 and b30 appeared or became intense in early periods (6 or 24 hr) in the post‑diapause eggs when treated with hoL HC1. These components were a1so seen or became strong in the post‑diapause eggs without hot‑I
IC1 treatment
，
but only at 72 hr. Thus the time when these spots become marked was shifted by the hoL‑HCl treatment，
suggesting that the hoL‑acid treatment might play a role to acce1erate the expression of some mRNAs
On the whole
，
the experiments described in the present article indicated very limiLed changes in quality of both mRNAs and proteins synthesized 斗n different stages of B.mori eggs. On1y a small fraction of the mRNAs and proteins
82
exhibited fluctuation. These include the molecules of which appearance strictly dependent upon the hot‑HCl treatment
，
which may be a severe stress to the eggs but generally conducted to break successfully the diapause in B. mori These proteins are suggested to have a role in the estab‑
lishment of the normal post‑diapause development after the recovery from the stress‑shock.
ACKNOWLEDGMENTS
The author expresses his sincerest appreciation to Professor K. Koga for h且sencouragement
，
excellent direction and helpful discussion throughout the investigation，
and for his guidance for，
and critical reading of，
the manuscript. Specia1 thanks shou1d be due to Professor H. Doira and Dr.Y. Kawaguchi for their continuous encouragement and advice
，
and for their critica1 reading of the manuscript. The author a1so wish to thank Dr. 11. Fujii and Dr. Y. Banno for their kind encouragement and technica1 he1p. The author a1so thanks to Mr. T. Kawabata for generous supply of the silkworms used且n the present study
，
to Dr. A. Kuroiwa for the gift of the plasmid containing the hsp7D gene，
and to Dr. Y. Suglmoto for his technical advice and generous offers to use his equipmenLs. The author is gratefu1 t。
a number of former and present colleagues of the Laboratory of Sericul tural Science，
Kyushu Universi ty，
whose advice has been of invaluable help in these experiments，
84
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