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Control of chromosome behavior in amphibian oocytes
DEVELOPMENTAL
BIOLOGY
Control I. The Activity
35, 283-292 (1973)
of Chromosome of Maturing
Behavior
Oocytes
in Amphibian
Inducing
in Transplanted
Chromosome
Oocytes Condensation
Brain Nuclei’
DAVID ZIEGLER AND YOSHIO MASUI Department
of Zoology,
University
of Toronto,
Toronto,
Ontario,
Canada
Accepted June 30, 1973
The capability of oocyte cytoplasm to induce chromosome condensation was studied by transplantation of isolated brain nuclei into Rana pipiens oocytes induced to undergo maturation in vitro by progesterone treatment. It was found that the chromosome condensation activity (CCA) first appeared in the cytoplasm of maturing oocytes shortly after germinal vesicle breakdown (GVBD), persisted in fully mature oocytes, but rapidly disappeared when the oocytes were artificially activated. A comparison of the time course of the oocyte chromosome condensation cycle and of brain chromosome condensation in maturing and activated oocytes revealed a close temporal correlation between the two, suggesting that both are under the control of the same cytoplasmic factor(s). Oocytes enucleated before GVBD always failed to develop CCA. The CCA could be restored in enucleated oocytes by injecting nucleoplasm obtained from oocytes that had not yet undergone GVBD although this same nucleoplasm was incapable of producing CCA when mixed with the cytoplasm of oocytes that had not reached the stage of GVBD. It was therefore suggested that the CCA had a dual origin involving both cytoplasmic maturation and GV materials. INTRODUCTION
The existence of cytoplasmic control over nuclear behavior during the mitotic cell cycle has been well documented (Graham, 1966; Graham et al., 1966; Gurdon and Woodland, 1968; deTerra, 1969; Johnson and Rao, 1971). This regulatory effect of the cytoplasm is realized through nucleocytoplasmic interaction where substances preformed in the cytoplasm are thought to interact with nuclei at specific times in the cell cycle to affect the subsequent behavior of the nuclei. Compared to the abundant information on cytoplasmic regulation of nuclear activity in mitotic cells, the evidence has been relatively scant with regard to meiotic cells. However, Masui and Markert (1971) and Masui (1972), by transferring cytoplasm from maturing Ram pipiens oocytes into immature oocytes and blastomeres of ‘This research was supported by a grant award to Y. M. from the National Cancer Institute of Canada.
cleaving eggs, have shown that the initiation of germinal vesicle breakdown (GVBD), the arrest of the chromosomes at second meiotic metaphase and their release from the arrest at the time of egg activation are all controlled by cytoplasmic factors that develop during meiotic maturation. Some observations have been made indicating that the chromosome condensation occurring during oocyte maturation is also regulated by cytoplasmic factors. Brachet (1922) observed that when sperm prematurely entered eggs of the sea urchin Paracentrotus lividus which were still undergoing the maturation divisions, the sperm chromatin rapidly condensed into chromosomes similar to those of the oocyte. The same phenomenon was reported by Bataillon (1928) and Bataillon and Tchou-Su (1934) in precociously inseminated Hyla and Triton eggs. Since the nuclei of sperm that enter fully mature eggs at the normal time of fertilization always transform into
283 Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
284
DEVELOPMENTALBIOLOGY
swelling pronuclei, it is assumed that a unique cytoplasmic environment prevailing in premature oocytes is responsible for condensation of sperm chromosomes in premature fertilization. Gurdon (1967, 1968) demonstrated the existence of such a unique cytoplasmic condition in maturing Xenopus laevk oocytes by microinjection of isolated brain nuclei. He found that nuclei injected into oocytes after GVBD were rapidly transformed into condensed chromosomes. In the present experiments, the cytoplasmic activity that induces chromosome condensation in transplanted brain nuclei, briefly called the chromosome condensation activity (CCA), was studied in maturing Rana pipiens oocytes. Details of its time course, some aspects of its origin and its temporal coordination with oocyte chromosome condensation were examined cytologically. MATERIALS
AND
METHODS
Hibernating, sexually mature Rana pipiens, obtained from dealers in Minnesota, Wisconsin and Vermont in the fall and winter and then maintained in the cold, were used as oocyte donors. A modified frog Ringer’s solution containing antibiotics (Masui, 1967), called the standard solution, was used for all operations. Ovarian oocytes were defolliculated, induced to mature with progesterone and incubated according to the methods described by Masui (1967). In order to obtain the synchronously maturing oocytes that were used in all of the experiments, the synchrony of maturation was tested beforehand using a group of ovarian oocytes excised from each prospective donor frog. The progress of maturation and its synchrony in the test oocytes was monitored with respect to the timing of GVBD at 18°C checked by fixing the oocytes in hot 70% ethanol and breaking them open with watchmaker’s forceps. When the test oocytes maturing fastest underwent GVBD by 12-14 hours post
VOLUME 35, 1973
hormone treatment (PHT) and all the test oocytes had completed GVBD by 16-18 hours PHT, the remaining oocytes of the frog were used for experimentation. Adult Rana pipiens brain nuclei were isolated according to the procedure of Graham et al. (1966), but with an improved isolation medium. Our medium consisted of 0.20 M sucrose, 0.0024 M MgC&, 0.01 M Tris, and 0.005 M maleic acid (final pH 6.7). The nuclear suspension was injected while the recipient oocytes were submerged in the standard solution using a micropipette attached to a micromanipulator (Prior). The oocytes were injected with a volume of nuclear suspension that was sufficient to introduce 50-300 nuclei per oocyte without causing perceptible swelling of the oocytes. Following injection, the oocytes were incubated at 18°C. When fully mature oocytes were injected with nuclei, they were immersed in phosphate buffer (0.025 M Na2HP0,-NaH2. PO1, pH 6.2) from 30 min prior to injection to 15 min after injection for the purpose of preventing activation which would otherwise result from the injury produced by the injecting micropipette. This method proved to be effective in reversibly suppressing activation in 80-92s of the cases (Masui, unpublished). To study the effect of the host nucleus on the behavior of the injected nuclei, oocytes were manually enucleated prior to the induction of maturation according to the method described by Dettlaff et al. (1964). All the operated oocytes were incubated at 18°C. For cytological examination, oocytes were fixed in an aqueous formalih fixative composed of 7.5% formalin, 4.5% sucrose, and 0.5% cetylpyridinium chloride. They were fixed in this solution for 3 hr, washed in distilled water overnight, dehydrated by the alcohol-amyl acetate procedure (Drury, 1941) and embedded in 52°C paraffin. Sections were prepared at a thickness of 10 pm, mounted on glass slides, stained according to the Feulgen procedure and
ZIEGLER AND MASUI
counterstained 1940).
with
Light
Chromosome
Green (Moore,
RESULTS
Condensation of the Oocyte Chromosomes during Meiotic Maturation Prior to and immediately after treatment of the oocytes with progesterone, the oocyte chromosomes appeared long and slender, exhibited many chiasmata and were always found to be confined within a small, distinct area of nucleoplasm. This structure, composed of chromosomes and surrounding nucleoplasm, has been designated by Duryee (1950) as the chromosome frame (Fig. la). During the early stages of maturation, the chromosome frame was always located in the portion of the GV facing the vegetal hemisphere (basal portion of the GV). At approximately 12 hr PHT, the chromosome frame had disappeared and the chromosomes were found scattered from the middle region of the GV to the region close to the animal pole (apical portion of GV). The chromosomes at this stage still appeared long and slender. By 16 hr PHT, the 13 paired homologs were obviously contracting, and many exhibited a morphology typical of diakinesis (Fig. 1B). At this time, breakdown of the nuclear membrane, which had started in the basal region of the GV at 12 hr PHT, had progressed toward the apex of the GV and involved at least half of the GV membrane in most of the oocytes. As GVBD proceeded, the process of chromosome contraction continued and it reached its maximum by 20 hr PHT culminating in chromosomes possessing a distinct globular form. At the initial stage of maximum chromosome contraction, the globular chromosomes were still well dispersed and embedded in a long, thin patch of uniformly Light-Green stained nucleoplasm located just below the animal pole cortex (Fig. 1C). However, between 20 and 22 hr PHT, the globular chromosome pairs started to aggregate, forming a diminutive
Condensation
Activity
285
cluster of Feulgen-positive bodies just beneath the animal cortex (Fig. 1D). The chromosomes were found within the cortex by 24 hr PHT. This cluster of chromosomes represented the first meiotic metaphase. The first polar body was formed approximately 1 hr later and, without undergoing interphase decondensation, the chromosomes were found arrested at 2nd metaphase by 36 hr PHT. A coordination between GVBD and oocyte chromosome condensation was observed. When individual cases were closely examined, it was found that the chromosomes of oocytes with intact GVs had never begun further condensation, always remaining as diplotene type chromosomes within intact chromosome frames. On the other hand, oocytes in which GVBD had already progressed had no chromosome frames and, depending upon the extent of GVBD, exhibited varying degrees of chromosome condensation. Even in a few exceptional cases where GVBD was considerably delayed, this temporal relation was invariably maintained. Formation of Chromosomes from Brain Nuclei in Maturing Oocyte Cytoplasm In order to determine when the cytoplasm of a maturing oocyte first acquired the capability of inducing chromosome condensation, isolated brain nuclei were injected into the cytoplasm of ovarian oocytes, and these were then induced to mature in vitro. The injected oocytes were examined cytologically at various stages of maturation with respect to the behavior of the injected brain nuclei. The results of this experiment are summarized in Fig. 2. Some of the oocytes injected with nuclei were incubated for 24, 48, or 72 hr without treatment with progesterone. The nuclei injected into these fully grown but immature oocytes failed to form chromosomes, and an increased proportion became pycnotic as the incubation period was extended. The same type of behavior was displayed by the nuclei injected into the
286
DEVELOPMENTALBIOLOGY
VOLUME 35, 1973
FIG. 1. The oocyte chromosome cycle. (A) The oocyte chromosomes and chromosome frame immediately after progesterone treatment. x 1400. (B) Obviously contracting oocyte chromosomes displaying typical morphology of diakinesis 16 hr after progesterone treatment. x 2500. (C) Maximally contracted, globular, oocyte chromosomes dispersed in a thin area of nucleoplasm just under the animal cortex. The specimen was fixed 20 hr after progesterone treatment. x 1500. (D) The maximally contracted oocyte chromosomes after aggregation at 22 hr posthormone treatment. x 1400. All specimens stained with the Feulgen Light-Green technique. (-), oocyte’s chromosomes; M, limiting membrane of the chromosome frame; GV, germinal vesicle; AP, animal pole.
ZIEGLER AND MASIJI o-o 100 -
GVED
Chromosome
Condensation
Activity
287
always found accumulated at the periphery of the clear cytoplasm.
SO-
Chromosome Condensation (CCA) of Oocyte Cytoplasm Stages of Maturation
Activity at Various
In order to determine the time of disappearance of the CCA, brain nuclei were , I I 12 16 20 24 28 32 36 40 44 40 4 8 injected into maturing oocytes at various HO”rS times after progesterone treatment. The Fbc. 2. The initial appearance of chromosome conresults of this experiment are presented in densation activity in the cytoplasm of maturing Fig. 5. oocytes. Oocytes obtained from 5 different females When oocytes were injected with brain were injected with brain nuclei and then induced to nuclei prior to 19 hr PHT, neither a 2- nor mature in vitro by progesterone treatment. Ordinate: the proportion of oocytes exhibiting complete GVBD 4-hr incubation after nuclear injection was or induced chromosomes. Abscissa: hours after horsufficient to induce chromosome condensamone treatment when the oocytes were fixed for tion in the injected nuclei. However, when cytological examination. Each point on the graph injected at the indicated times between 19 represents observations on a minimum of 25 oocytes. and 30 hr, the brain chromosomes were GVBD, proportion of the oocytes with complete GVBD; CCA, proportion of the oocytes with coninduced to condense during both 2- and densed brain chromosomes. 4-hr periods. When brain nuclei were injected into progesterone-treated oocytes which were mature oocytes 36 and 48 hr PHT, the fixed 18 hr or less PHT. However, when oocytes were treated with phosphate buffer oocytes were fixed between 19 and 48 hr (pH 6.2) to prevent them from activating. after progesterone treatment, chromoOocytes which failed to show the external somes derived from the injected brain nu- signs of activation after nuclear injection clei (induced chromosomes) were found in still possessed second metaphase chromovarious regions of the cytoplasm. somes. It was these oocytes that contained The induced chromosomes and the oo- condensed brain chromosomes (Fig. 5). cyte chromosomes were so different in Apparently, once the oocyte cytoplasm appearance and location that the two could acquires the capability of inducing chrobe easily distinguished. From 20 hr on- mosome condensation, it retains the capaward, the oocyte chromosomes were globu- bility even after the fully mature state is lar, whereas the majority of the induced attained. chromosomes resembled the prometaphase On the other hand, when recipient ooto metaphase chromosomes of mitotic cells cytes did activate, rarely were the brain (Fig. 3). Occasionally, some of the induced chromosomes induced to condense. The chromosomes were anaphaselike (Fig. 4). main features of brain nuclear behavior in The induced chromosomes occurred in this environment were swelling and disperclusters that may have been derived from 1 sal of chromatin (Fig. 6). These results, or more nuclei, and lo-15 clusters were summarized in Table 1, clearly indicate usually found in an oocyte. The chromo- that the cytoplasmic CCA rapidly disapsome clusters were usually located well pears when an oocyte is activated. below the cortex in the animal region. They Parameters Affecting CCA were always found within clear, particlefree, cytoplasmic areas that sometimes The efficiency with which chromosome possessed a fibrous structure resembling a condensation was induced in the brain spindle (Fig. 4). Pigment granules were nuclei was influenced by parameters other 20-
288
DEVELOPMENTALBIOLOGY
VOLUME 35, 1973
FIG. 3. Prometaphase to metaphase type of induced chromosomes. (A) Chromosomes derived from brain nuclei that were exposed to maturing oocyte cytoplasm for 19 hr. x 3150. (B) Chromosomes derived from brain nuclei that were injected into an oocyte treated with progesterone 24 hr earlier. The specimen was fixed 2 hr after nuclear injection. x 2500. Feulgen Light-Green staining. (-), induced chromosomes; CA, clear area. FIG. 4, Anaphase type of induced chromosomes. The induced chromosomes were derived from brain nuclei injected into an oocyte which had been maturing for 24 hr when injected. The specimen was fixed 2 hr after nuclear injection. x 3000. Feulgen, Light-Green staining. C, induced chromosomes; CA, area of clear cytoplasm (note spindlelike fibrous structure within the clear area).
ZIEGLERANDMASUI
ChromosomeCondensationActivity
289
o--oGVBD F.ll.1 j-J2 hrs than a simple time factor. It was found O-O GVBD F 25.1 .-. GVED F.17.1 that the longer the nuclei were exposed to the oocyte cytoplasm, the more heteropycnotic they became. This adverse cytoplasmic effect may explain why chromosome condensation was induced with a higher frequency when the exposure period was only for 2 or 4 hr (Fig. 5) than when the 2 4 6 8 10 nuclei were injected prior to hormone treatment (Fig. 2). FIG. 5. Duration of chromosome condensation acIn addition, it was observed that there tivity in maturing and fully mature oocytes. Oocytes was a difference in the CCA of the cyto- obtained from 3 different females were injected with plasm in the animal and vegetal hemis- brain nuclei at various times after hormone treatment pheres. When oocytes were injected with and fixed for cytological examination 2 or 4 hr after brain nuclei at various times after GVBD injection of the nuclei. The rates of maturation of the oocyte donors are plotted as the frequency of GVBD (24,30,36, and 48 hr PHT) and fixed 2 or 4 on the graph. The cytoplasmic chromosome condenhr later, those containing nuclei in the sation activity of the oocytes appears on the histoanimal hemisphere were always found to gram. Ordinate: the proportion of oocytes with compossess induced chromosomes. On the plete GVBD and the proportion possessing induced chromosomes. Abscissa: hours after hormone treatother hand, oocytes of the same maturation ment when the nuclei were injected. Maturing oocytes age, but with the nuclei located deep were also injected at 12, 14, and 16 hr after hormone within the vegetal hemisphere never in- treatment, but neither a 2- nor 4-hr exposure resulted duced chromosome condensation. How- in chromosome formation. The numbers above the ever, it was found that when the nuclei bars represent the number of oocytes examined in were exposed to the vegetal hemisphere each case. The notations in the upper left-hand corner of the graph are the authors’ identifications of the cytoplasm for 24 hr, chromosome conden- donors. sation did occur. These results suggest that the vegetal region does possess CCA but that the activity is much lower there than recipients for nuclear injection in the present experiment. As controls, normal and in the animal hemisphere. sham-operated oocytes in which a compaContribution of the GV to the Developrable amount of cytoplasm was squeezed ment of CCA out instead of the GV were also prepared. In the foregoing experiments, it was Brain nuclei were injected into the animal found that GVBD always preceded the region of all these recipient oocytes at 24 hr appearance of CCA (Figs. 2 and 5). In order PHT. When the oocytes were fixed 3 hr to examine the possibility of a causal after nuclear injection, 19 out of 20 (95%) of relation between the GV and development the normal and 15 out of 20 (75%) of the of the CCA, oocytes enucleated prior to sham-operated oocytes were found to conhormone treatment were tested for the tain induced chromosomes, and these perability to develop CCA. Enucleated oo- centages were increased to 100% (20 of 20) cytes that survived the operation devel- and 85% (17 of 20), respectively, when the oped a shiny surface by 24 hr PHT and oocytes were fixed 24 hr after nuclear they later became activatable. Thus, as injection. In contrast to these results, not previously shown by Skoblina (1969) and one of the 40 enucleated oocytes was found Smith and Ecker (1969), enucleated oo- to be able to induce chromosome condensacytes that develop a surface gloss after tion during the same periods of incubation. hormone treatment are in the process of In a complementary experiment, it was maturation. Such oocytes were selected as demonstrated that the capability of induc-
290
DEVELOPMENTALBIOLOGY
ing chromosome condensation was recovered in enucleated oocytes by reintroduction of the GV contents. Here, enucleated oocytes that had been maturing for 22 hr were injected with the GV contents from lo-hr maturing oocytes, and then, 2 hr later, i.e., 24 hr after hormone treatment
VOLUME 35, 1373
of the recipient, brain nuclei were injected. The oocytes thus operated were fixed 3 hr after nuclear injection. In this case, the condensed brain chromosomes were found in 22 of 34 (65%) of the oocytes. These results clearly indicate that the GV substance is necessary for the development of the CCA. On the other hand, however, the following experiment demonstrated that maturation of the cytoplasm was also important for the development of the CCA. In this experiment, enucleated oocytes (40) that had been maturing for 10 hr were injected with GV contents obtained from oocytes of the same age. Brain nuclei were injected 2 hr later. The recipient oocytes were examined 3 and 5 hr after nuclear injection and in neither case were condensed brain chromosomes found. DISCUSSION
FIG. 6. (A) Brain nuclei in situ. x1006. (B) An isolated brain nuoleus injected into a maturing oocyte that was fixed immediately after nuclear injection. x 1666. (C) Swollen nuclei in activated egg cytoplasm. The brain nuclei were injected into a fully mature oocyte 48 hr after hormone treatment. The oocyte activated, underwent pseudocleavage and was fixed 2 hr after nuclear injection. x1660. Feulgen, LightGreen staining.
The results of this study confirm Gurdon’s finding (1967, 1968) that chromosome condensation in brain nuclei can be induced by maturing oocyte cytoplasm. The mitotic index in the brain of a hibernating frog was approximately 0.001, and none of the brain nuclei incubated in the isolation medium at 18°C for up to 24 hr formed chromosomes. It is therefore unlikely that the chromosomes of the brain nuclei were induced to condense as a result of the isolation procedure itself nor as a consequence of their own intrinsic mitotic rhythm. In the present study, it was found that while a 2-hour exposure of brain nuclei to the animal hemisphere cytoplasm of an oocyte that been treated with progesterone 24 hr earlier always results in chromosome condensation, brain nuclei exposed to immature or activated egg cytoplasm for extended periods of time never formed condensed chromosomes. Therefore, it is concluded that the cytoplasm of a maturing oocyte develops a unique condition which provides mitotically quiescent nuclei with an environment capable of inducing chromosome condensation.
TABLE THE
BEHAVIOR
treatment
in
vitro (nuclear
injection
time)
36
46
Activated Total no.~
I’b’b$ come.+
2 4 Total:
1
BRAIN NUCLEI IN ACTNATEDAND UNACTIVATEDEGG CYTOPLASM Hours after hormone
Nuclear exposure time” (hr)
0
OF
-
10 15 25
---
1 1 2
10 7 8
-
Total no.
i’I’Ik~ some*
15
0
20 20 40
19 20 39
Unactivated
Activated
Unactivated % “car;? 1 em km)
291
Chromosome Condensation Activity
ZIEGLER AND MASUI
% Average diameter (A
o95 190 98
-
Total no.
10 15 25
No. with chmmosomes
---5 1 3 4
% A;my 1 et.3 hm)
10 11.5 20 14.5 16 -
Total no.
15 10 20 30
bJiowi”,l
W A;g;te
8ollx8
eter km)
0 10 20 30
05 196 4.5 199 4.5 199 -
n Hours after nuclear injection when fixed. b Number of oocytes examined. Only oocytes with brain nuclei in the animal hemisphere were considered. c Number of oocytes with chromosome clusters derived from injected nuclei.
Since the appearance and disappearance of the CCA closely coincided with the beginning and end of the oocyte chromosome condensation cycle, it appears that the condensation of the oocyte chromosomes as well as brain chromosomes was regulated by the same cytoplasmic condition. However, it must be noted that the oocyte and brain chromosomes did not begin condensation in exact synchrony. The oocyte chromosomes always entered diakinesis 3-4 hr prior to the earliest time at which condensation of the brain chromosomes was observed. This time lag may be ascribed to the fact that, prior to the period of maturation, the oocyte chromosomes are at the diplotene stage of meiotic prophase and are therefore already partially condensed, while the brain chromosomes, being in interphase, are completely dispersed . The results demonstrate unequivocally that the development of the CCA depends upon the release of some component from the GV. However, the development of CCA cannot be solely attributed to the GV. The GV contents that restored CCA in enucleated oocytes that had been maturing for 22 hr failed to restore CCA within the same period when injected into enucleated oocytes which had been maturing for only 10 hr. This indicates that the cytoplasm also contributes to the development of the
CCA. Furthermore, the fact that no progress of oocyte chromosome condensation was observed until after the GV membrane had begun to break down at the basal region also suggests that both cytoplasm and GV contents are involved in the development of the CCA. This coordination between GVBD and oocyte chromosome condensation could be explained as a consequence of the breakdown of the GV membrane which would remove the barrier to the interaction of GV material and cytoplasm at the proper time in maturation. Nuclear behavior during oocyte maturation and activation seems to be controlled by various cytoplasmic factors. Initiation of GVBD and arrest of the chromosomes at Metaphase II are caused by cytoplasmic factors which appear in the course of maturation independently of the GV (Masui and Markert, 1971). Also, the factor that stimulates DNA synthesis appears during this period and persists after activation (Gurdon, 1968). The factor responsible for chromosome condensation studied here seems to be distinct from the factors previously described because its production is GVdependent and it disappears at activation. ACKNOWLEDGMENTS The authors are indebted to Mr. William J. Wasserman for stimulating discussions and to Janice Ziegler for help with preparing the manuscript.
292
DEVELOPMENTALBIOLOC,Y REFERENCES
BATAILLON, E. (1928). Etudes analytiques
sur la maturation des oeufs de batraciens. C. R. Acad. Sci. 187, 520-523. BATAILL~N, E., and TCHOIJ-SU(1934). L’analyse experimentale de la fecondation et sa definition par les processus cinetiques. Ann. Sci. Nat. 2001.17,9-36. BRACHET,A. (1922). Recherches sur la fecondation prematuree de l’oeuf d’oursin (Pamcentrotus lividw). Arch. Biol. 32, 205-244. DETERRA, N. (1969). Cytoplasmic control over the nuclear events of cell reproduction. Znt. Rev. Cytol. 25, l-29. DE’ITLAFF,T. A., NIKITINA, L. A., and STROEVA,0. G. (1964). The role of the germinal vesicle in oocyte maturation in anurans as revealed by the removal and transplantation of nuclei. J. Embryol. Exp. Morphol. 12, 851-872. DRURY,H. F. (1941). Amylacetate as a clearing agent for embryonic material. Stain Technol. 16, 21-22. DURYEE,W. R. (1950). Chromosomal physiology in relation to nuclear structure. Ann. N. Y. Acad. Sci. 59,920-953. GRAHAM, C. F. (1966). The regulation of DNA synthesis and mitosis in multinucleate frog eggs. J. Cell Sci. 1, 363-374. GRAHAM,C. F., ARMS, K., and GURDON,J. B. (1966). The induction of DNA synthesis by frog egg cytoplasm. Develop. Biol. 14, 349-381. GURDON,J. B. (1967). On the origin and persistence of a cytoplasmic state inducing DNA synthesis in frogs’ eggs. Proc. Nut. Acad. Sci. U.S. 58,545-552.
VOLUME35, I973
GURDON,J. B. (1966). Changes in somatic cell nuclei inserted into growing and maturing amphibian oocytes. J. Embryol. Exp. Morphol. 20, 401-414. GURDON,J. B., and WOODLAND,H. R. (1968). The cytoplasmic control of nuclear activity in animal development. Biol. Rev. Cambridge Phil. Sot. 43, 233-267. JOHNSON,R. T., and ho, P. N. (1971). Nucleo-cytoplasmic interactions in the achievement of nuclear synchrony in DNA synthesis and mitosis in multinucleate cells. Biol. Rev. Cambridge Phil. Sot. 46, 97-155. M&uI, Y. (1967). Relative roles of the pituitary, follicle cells and progesterone in the induction of oocyte maturation in Rana pipiens. J. Exp. 2001. 166, 365-376.
MASUI, Y. (1972). Distribution of the cytoplasmic activity inducing germinal vesicle breakdown in frog oocytes. J. Exp. 2001. 179, 365-378. MASUI, Y., and MARKERT,C. L. (1971). Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. 2001. 177, 129-146. MOORE, B. (1940). Chromosomes of frog eggs and embryos stained by the Feulgen method to avoid excessive staining of yolk granules. Anut. Rec. 78, Suppl. 122. SKOBLINA,M. N. (1969). Independence of the cortex maturation from germinal vesicle material during the maturation of amphibian and sturgeon oocytes. Exp. Cell Res. 55, 142-144. SMITH, L. D., and ECKER,R. E. (1969). Cytoplasmic regulation in early events of amphibian development. Proc. Con. Cancer Res. Conf., 8, 103-129.
BIOLOGY
Control I. The Activity
35, 283-292 (1973)
of Chromosome of Maturing
Behavior
Oocytes
in Amphibian
Inducing
in Transplanted
Chromosome
Oocytes Condensation
Brain Nuclei’
DAVID ZIEGLER AND YOSHIO MASUI Department
of Zoology,
University
of Toronto,
Toronto,
Ontario,
Canada
Accepted June 30, 1973
The capability of oocyte cytoplasm to induce chromosome condensation was studied by transplantation of isolated brain nuclei into Rana pipiens oocytes induced to undergo maturation in vitro by progesterone treatment. It was found that the chromosome condensation activity (CCA) first appeared in the cytoplasm of maturing oocytes shortly after germinal vesicle breakdown (GVBD), persisted in fully mature oocytes, but rapidly disappeared when the oocytes were artificially activated. A comparison of the time course of the oocyte chromosome condensation cycle and of brain chromosome condensation in maturing and activated oocytes revealed a close temporal correlation between the two, suggesting that both are under the control of the same cytoplasmic factor(s). Oocytes enucleated before GVBD always failed to develop CCA. The CCA could be restored in enucleated oocytes by injecting nucleoplasm obtained from oocytes that had not yet undergone GVBD although this same nucleoplasm was incapable of producing CCA when mixed with the cytoplasm of oocytes that had not reached the stage of GVBD. It was therefore suggested that the CCA had a dual origin involving both cytoplasmic maturation and GV materials. INTRODUCTION
The existence of cytoplasmic control over nuclear behavior during the mitotic cell cycle has been well documented (Graham, 1966; Graham et al., 1966; Gurdon and Woodland, 1968; deTerra, 1969; Johnson and Rao, 1971). This regulatory effect of the cytoplasm is realized through nucleocytoplasmic interaction where substances preformed in the cytoplasm are thought to interact with nuclei at specific times in the cell cycle to affect the subsequent behavior of the nuclei. Compared to the abundant information on cytoplasmic regulation of nuclear activity in mitotic cells, the evidence has been relatively scant with regard to meiotic cells. However, Masui and Markert (1971) and Masui (1972), by transferring cytoplasm from maturing Ram pipiens oocytes into immature oocytes and blastomeres of ‘This research was supported by a grant award to Y. M. from the National Cancer Institute of Canada.
cleaving eggs, have shown that the initiation of germinal vesicle breakdown (GVBD), the arrest of the chromosomes at second meiotic metaphase and their release from the arrest at the time of egg activation are all controlled by cytoplasmic factors that develop during meiotic maturation. Some observations have been made indicating that the chromosome condensation occurring during oocyte maturation is also regulated by cytoplasmic factors. Brachet (1922) observed that when sperm prematurely entered eggs of the sea urchin Paracentrotus lividus which were still undergoing the maturation divisions, the sperm chromatin rapidly condensed into chromosomes similar to those of the oocyte. The same phenomenon was reported by Bataillon (1928) and Bataillon and Tchou-Su (1934) in precociously inseminated Hyla and Triton eggs. Since the nuclei of sperm that enter fully mature eggs at the normal time of fertilization always transform into
283 Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
284
DEVELOPMENTALBIOLOGY
swelling pronuclei, it is assumed that a unique cytoplasmic environment prevailing in premature oocytes is responsible for condensation of sperm chromosomes in premature fertilization. Gurdon (1967, 1968) demonstrated the existence of such a unique cytoplasmic condition in maturing Xenopus laevk oocytes by microinjection of isolated brain nuclei. He found that nuclei injected into oocytes after GVBD were rapidly transformed into condensed chromosomes. In the present experiments, the cytoplasmic activity that induces chromosome condensation in transplanted brain nuclei, briefly called the chromosome condensation activity (CCA), was studied in maturing Rana pipiens oocytes. Details of its time course, some aspects of its origin and its temporal coordination with oocyte chromosome condensation were examined cytologically. MATERIALS
AND
METHODS
Hibernating, sexually mature Rana pipiens, obtained from dealers in Minnesota, Wisconsin and Vermont in the fall and winter and then maintained in the cold, were used as oocyte donors. A modified frog Ringer’s solution containing antibiotics (Masui, 1967), called the standard solution, was used for all operations. Ovarian oocytes were defolliculated, induced to mature with progesterone and incubated according to the methods described by Masui (1967). In order to obtain the synchronously maturing oocytes that were used in all of the experiments, the synchrony of maturation was tested beforehand using a group of ovarian oocytes excised from each prospective donor frog. The progress of maturation and its synchrony in the test oocytes was monitored with respect to the timing of GVBD at 18°C checked by fixing the oocytes in hot 70% ethanol and breaking them open with watchmaker’s forceps. When the test oocytes maturing fastest underwent GVBD by 12-14 hours post
VOLUME 35, 1973
hormone treatment (PHT) and all the test oocytes had completed GVBD by 16-18 hours PHT, the remaining oocytes of the frog were used for experimentation. Adult Rana pipiens brain nuclei were isolated according to the procedure of Graham et al. (1966), but with an improved isolation medium. Our medium consisted of 0.20 M sucrose, 0.0024 M MgC&, 0.01 M Tris, and 0.005 M maleic acid (final pH 6.7). The nuclear suspension was injected while the recipient oocytes were submerged in the standard solution using a micropipette attached to a micromanipulator (Prior). The oocytes were injected with a volume of nuclear suspension that was sufficient to introduce 50-300 nuclei per oocyte without causing perceptible swelling of the oocytes. Following injection, the oocytes were incubated at 18°C. When fully mature oocytes were injected with nuclei, they were immersed in phosphate buffer (0.025 M Na2HP0,-NaH2. PO1, pH 6.2) from 30 min prior to injection to 15 min after injection for the purpose of preventing activation which would otherwise result from the injury produced by the injecting micropipette. This method proved to be effective in reversibly suppressing activation in 80-92s of the cases (Masui, unpublished). To study the effect of the host nucleus on the behavior of the injected nuclei, oocytes were manually enucleated prior to the induction of maturation according to the method described by Dettlaff et al. (1964). All the operated oocytes were incubated at 18°C. For cytological examination, oocytes were fixed in an aqueous formalih fixative composed of 7.5% formalin, 4.5% sucrose, and 0.5% cetylpyridinium chloride. They were fixed in this solution for 3 hr, washed in distilled water overnight, dehydrated by the alcohol-amyl acetate procedure (Drury, 1941) and embedded in 52°C paraffin. Sections were prepared at a thickness of 10 pm, mounted on glass slides, stained according to the Feulgen procedure and
ZIEGLER AND MASUI
counterstained 1940).
with
Light
Chromosome
Green (Moore,
RESULTS
Condensation of the Oocyte Chromosomes during Meiotic Maturation Prior to and immediately after treatment of the oocytes with progesterone, the oocyte chromosomes appeared long and slender, exhibited many chiasmata and were always found to be confined within a small, distinct area of nucleoplasm. This structure, composed of chromosomes and surrounding nucleoplasm, has been designated by Duryee (1950) as the chromosome frame (Fig. la). During the early stages of maturation, the chromosome frame was always located in the portion of the GV facing the vegetal hemisphere (basal portion of the GV). At approximately 12 hr PHT, the chromosome frame had disappeared and the chromosomes were found scattered from the middle region of the GV to the region close to the animal pole (apical portion of GV). The chromosomes at this stage still appeared long and slender. By 16 hr PHT, the 13 paired homologs were obviously contracting, and many exhibited a morphology typical of diakinesis (Fig. 1B). At this time, breakdown of the nuclear membrane, which had started in the basal region of the GV at 12 hr PHT, had progressed toward the apex of the GV and involved at least half of the GV membrane in most of the oocytes. As GVBD proceeded, the process of chromosome contraction continued and it reached its maximum by 20 hr PHT culminating in chromosomes possessing a distinct globular form. At the initial stage of maximum chromosome contraction, the globular chromosomes were still well dispersed and embedded in a long, thin patch of uniformly Light-Green stained nucleoplasm located just below the animal pole cortex (Fig. 1C). However, between 20 and 22 hr PHT, the globular chromosome pairs started to aggregate, forming a diminutive
Condensation
Activity
285
cluster of Feulgen-positive bodies just beneath the animal cortex (Fig. 1D). The chromosomes were found within the cortex by 24 hr PHT. This cluster of chromosomes represented the first meiotic metaphase. The first polar body was formed approximately 1 hr later and, without undergoing interphase decondensation, the chromosomes were found arrested at 2nd metaphase by 36 hr PHT. A coordination between GVBD and oocyte chromosome condensation was observed. When individual cases were closely examined, it was found that the chromosomes of oocytes with intact GVs had never begun further condensation, always remaining as diplotene type chromosomes within intact chromosome frames. On the other hand, oocytes in which GVBD had already progressed had no chromosome frames and, depending upon the extent of GVBD, exhibited varying degrees of chromosome condensation. Even in a few exceptional cases where GVBD was considerably delayed, this temporal relation was invariably maintained. Formation of Chromosomes from Brain Nuclei in Maturing Oocyte Cytoplasm In order to determine when the cytoplasm of a maturing oocyte first acquired the capability of inducing chromosome condensation, isolated brain nuclei were injected into the cytoplasm of ovarian oocytes, and these were then induced to mature in vitro. The injected oocytes were examined cytologically at various stages of maturation with respect to the behavior of the injected brain nuclei. The results of this experiment are summarized in Fig. 2. Some of the oocytes injected with nuclei were incubated for 24, 48, or 72 hr without treatment with progesterone. The nuclei injected into these fully grown but immature oocytes failed to form chromosomes, and an increased proportion became pycnotic as the incubation period was extended. The same type of behavior was displayed by the nuclei injected into the
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DEVELOPMENTALBIOLOGY
VOLUME 35, 1973
FIG. 1. The oocyte chromosome cycle. (A) The oocyte chromosomes and chromosome frame immediately after progesterone treatment. x 1400. (B) Obviously contracting oocyte chromosomes displaying typical morphology of diakinesis 16 hr after progesterone treatment. x 2500. (C) Maximally contracted, globular, oocyte chromosomes dispersed in a thin area of nucleoplasm just under the animal cortex. The specimen was fixed 20 hr after progesterone treatment. x 1500. (D) The maximally contracted oocyte chromosomes after aggregation at 22 hr posthormone treatment. x 1400. All specimens stained with the Feulgen Light-Green technique. (-), oocyte’s chromosomes; M, limiting membrane of the chromosome frame; GV, germinal vesicle; AP, animal pole.
ZIEGLER AND MASIJI o-o 100 -
GVED
Chromosome
Condensation
Activity
287
always found accumulated at the periphery of the clear cytoplasm.
SO-
Chromosome Condensation (CCA) of Oocyte Cytoplasm Stages of Maturation
Activity at Various
In order to determine the time of disappearance of the CCA, brain nuclei were , I I 12 16 20 24 28 32 36 40 44 40 4 8 injected into maturing oocytes at various HO”rS times after progesterone treatment. The Fbc. 2. The initial appearance of chromosome conresults of this experiment are presented in densation activity in the cytoplasm of maturing Fig. 5. oocytes. Oocytes obtained from 5 different females When oocytes were injected with brain were injected with brain nuclei and then induced to nuclei prior to 19 hr PHT, neither a 2- nor mature in vitro by progesterone treatment. Ordinate: the proportion of oocytes exhibiting complete GVBD 4-hr incubation after nuclear injection was or induced chromosomes. Abscissa: hours after horsufficient to induce chromosome condensamone treatment when the oocytes were fixed for tion in the injected nuclei. However, when cytological examination. Each point on the graph injected at the indicated times between 19 represents observations on a minimum of 25 oocytes. and 30 hr, the brain chromosomes were GVBD, proportion of the oocytes with complete GVBD; CCA, proportion of the oocytes with coninduced to condense during both 2- and densed brain chromosomes. 4-hr periods. When brain nuclei were injected into progesterone-treated oocytes which were mature oocytes 36 and 48 hr PHT, the fixed 18 hr or less PHT. However, when oocytes were treated with phosphate buffer oocytes were fixed between 19 and 48 hr (pH 6.2) to prevent them from activating. after progesterone treatment, chromoOocytes which failed to show the external somes derived from the injected brain nu- signs of activation after nuclear injection clei (induced chromosomes) were found in still possessed second metaphase chromovarious regions of the cytoplasm. somes. It was these oocytes that contained The induced chromosomes and the oo- condensed brain chromosomes (Fig. 5). cyte chromosomes were so different in Apparently, once the oocyte cytoplasm appearance and location that the two could acquires the capability of inducing chrobe easily distinguished. From 20 hr on- mosome condensation, it retains the capaward, the oocyte chromosomes were globu- bility even after the fully mature state is lar, whereas the majority of the induced attained. chromosomes resembled the prometaphase On the other hand, when recipient ooto metaphase chromosomes of mitotic cells cytes did activate, rarely were the brain (Fig. 3). Occasionally, some of the induced chromosomes induced to condense. The chromosomes were anaphaselike (Fig. 4). main features of brain nuclear behavior in The induced chromosomes occurred in this environment were swelling and disperclusters that may have been derived from 1 sal of chromatin (Fig. 6). These results, or more nuclei, and lo-15 clusters were summarized in Table 1, clearly indicate usually found in an oocyte. The chromo- that the cytoplasmic CCA rapidly disapsome clusters were usually located well pears when an oocyte is activated. below the cortex in the animal region. They Parameters Affecting CCA were always found within clear, particlefree, cytoplasmic areas that sometimes The efficiency with which chromosome possessed a fibrous structure resembling a condensation was induced in the brain spindle (Fig. 4). Pigment granules were nuclei was influenced by parameters other 20-
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FIG. 3. Prometaphase to metaphase type of induced chromosomes. (A) Chromosomes derived from brain nuclei that were exposed to maturing oocyte cytoplasm for 19 hr. x 3150. (B) Chromosomes derived from brain nuclei that were injected into an oocyte treated with progesterone 24 hr earlier. The specimen was fixed 2 hr after nuclear injection. x 2500. Feulgen Light-Green staining. (-), induced chromosomes; CA, clear area. FIG. 4, Anaphase type of induced chromosomes. The induced chromosomes were derived from brain nuclei injected into an oocyte which had been maturing for 24 hr when injected. The specimen was fixed 2 hr after nuclear injection. x 3000. Feulgen, Light-Green staining. C, induced chromosomes; CA, area of clear cytoplasm (note spindlelike fibrous structure within the clear area).
ZIEGLERANDMASUI
ChromosomeCondensationActivity
289
o--oGVBD F.ll.1 j-J2 hrs than a simple time factor. It was found O-O GVBD F 25.1 .-. GVED F.17.1 that the longer the nuclei were exposed to the oocyte cytoplasm, the more heteropycnotic they became. This adverse cytoplasmic effect may explain why chromosome condensation was induced with a higher frequency when the exposure period was only for 2 or 4 hr (Fig. 5) than when the 2 4 6 8 10 nuclei were injected prior to hormone treatment (Fig. 2). FIG. 5. Duration of chromosome condensation acIn addition, it was observed that there tivity in maturing and fully mature oocytes. Oocytes was a difference in the CCA of the cyto- obtained from 3 different females were injected with plasm in the animal and vegetal hemis- brain nuclei at various times after hormone treatment pheres. When oocytes were injected with and fixed for cytological examination 2 or 4 hr after brain nuclei at various times after GVBD injection of the nuclei. The rates of maturation of the oocyte donors are plotted as the frequency of GVBD (24,30,36, and 48 hr PHT) and fixed 2 or 4 on the graph. The cytoplasmic chromosome condenhr later, those containing nuclei in the sation activity of the oocytes appears on the histoanimal hemisphere were always found to gram. Ordinate: the proportion of oocytes with compossess induced chromosomes. On the plete GVBD and the proportion possessing induced chromosomes. Abscissa: hours after hormone treatother hand, oocytes of the same maturation ment when the nuclei were injected. Maturing oocytes age, but with the nuclei located deep were also injected at 12, 14, and 16 hr after hormone within the vegetal hemisphere never in- treatment, but neither a 2- nor 4-hr exposure resulted duced chromosome condensation. How- in chromosome formation. The numbers above the ever, it was found that when the nuclei bars represent the number of oocytes examined in were exposed to the vegetal hemisphere each case. The notations in the upper left-hand corner of the graph are the authors’ identifications of the cytoplasm for 24 hr, chromosome conden- donors. sation did occur. These results suggest that the vegetal region does possess CCA but that the activity is much lower there than recipients for nuclear injection in the present experiment. As controls, normal and in the animal hemisphere. sham-operated oocytes in which a compaContribution of the GV to the Developrable amount of cytoplasm was squeezed ment of CCA out instead of the GV were also prepared. In the foregoing experiments, it was Brain nuclei were injected into the animal found that GVBD always preceded the region of all these recipient oocytes at 24 hr appearance of CCA (Figs. 2 and 5). In order PHT. When the oocytes were fixed 3 hr to examine the possibility of a causal after nuclear injection, 19 out of 20 (95%) of relation between the GV and development the normal and 15 out of 20 (75%) of the of the CCA, oocytes enucleated prior to sham-operated oocytes were found to conhormone treatment were tested for the tain induced chromosomes, and these perability to develop CCA. Enucleated oo- centages were increased to 100% (20 of 20) cytes that survived the operation devel- and 85% (17 of 20), respectively, when the oped a shiny surface by 24 hr PHT and oocytes were fixed 24 hr after nuclear they later became activatable. Thus, as injection. In contrast to these results, not previously shown by Skoblina (1969) and one of the 40 enucleated oocytes was found Smith and Ecker (1969), enucleated oo- to be able to induce chromosome condensacytes that develop a surface gloss after tion during the same periods of incubation. hormone treatment are in the process of In a complementary experiment, it was maturation. Such oocytes were selected as demonstrated that the capability of induc-
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DEVELOPMENTALBIOLOGY
ing chromosome condensation was recovered in enucleated oocytes by reintroduction of the GV contents. Here, enucleated oocytes that had been maturing for 22 hr were injected with the GV contents from lo-hr maturing oocytes, and then, 2 hr later, i.e., 24 hr after hormone treatment
VOLUME 35, 1373
of the recipient, brain nuclei were injected. The oocytes thus operated were fixed 3 hr after nuclear injection. In this case, the condensed brain chromosomes were found in 22 of 34 (65%) of the oocytes. These results clearly indicate that the GV substance is necessary for the development of the CCA. On the other hand, however, the following experiment demonstrated that maturation of the cytoplasm was also important for the development of the CCA. In this experiment, enucleated oocytes (40) that had been maturing for 10 hr were injected with GV contents obtained from oocytes of the same age. Brain nuclei were injected 2 hr later. The recipient oocytes were examined 3 and 5 hr after nuclear injection and in neither case were condensed brain chromosomes found. DISCUSSION
FIG. 6. (A) Brain nuclei in situ. x1006. (B) An isolated brain nuoleus injected into a maturing oocyte that was fixed immediately after nuclear injection. x 1666. (C) Swollen nuclei in activated egg cytoplasm. The brain nuclei were injected into a fully mature oocyte 48 hr after hormone treatment. The oocyte activated, underwent pseudocleavage and was fixed 2 hr after nuclear injection. x1660. Feulgen, LightGreen staining.
The results of this study confirm Gurdon’s finding (1967, 1968) that chromosome condensation in brain nuclei can be induced by maturing oocyte cytoplasm. The mitotic index in the brain of a hibernating frog was approximately 0.001, and none of the brain nuclei incubated in the isolation medium at 18°C for up to 24 hr formed chromosomes. It is therefore unlikely that the chromosomes of the brain nuclei were induced to condense as a result of the isolation procedure itself nor as a consequence of their own intrinsic mitotic rhythm. In the present study, it was found that while a 2-hour exposure of brain nuclei to the animal hemisphere cytoplasm of an oocyte that been treated with progesterone 24 hr earlier always results in chromosome condensation, brain nuclei exposed to immature or activated egg cytoplasm for extended periods of time never formed condensed chromosomes. Therefore, it is concluded that the cytoplasm of a maturing oocyte develops a unique condition which provides mitotically quiescent nuclei with an environment capable of inducing chromosome condensation.
TABLE THE
BEHAVIOR
treatment
in
vitro (nuclear
injection
time)
36
46
Activated Total no.~
I’b’b$ come.+
2 4 Total:
1
BRAIN NUCLEI IN ACTNATEDAND UNACTIVATEDEGG CYTOPLASM Hours after hormone
Nuclear exposure time” (hr)
0
OF
-
10 15 25
---
1 1 2
10 7 8
-
Total no.
i’I’Ik~ some*
15
0
20 20 40
19 20 39
Unactivated
Activated
Unactivated % “car;? 1 em km)
291
Chromosome Condensation Activity
ZIEGLER AND MASUI
% Average diameter (A
o95 190 98
-
Total no.
10 15 25
No. with chmmosomes
---5 1 3 4
% A;my 1 et.3 hm)
10 11.5 20 14.5 16 -
Total no.
15 10 20 30
bJiowi”,l
W A;g;te
8ollx8
eter km)
0 10 20 30
05 196 4.5 199 4.5 199 -
n Hours after nuclear injection when fixed. b Number of oocytes examined. Only oocytes with brain nuclei in the animal hemisphere were considered. c Number of oocytes with chromosome clusters derived from injected nuclei.
Since the appearance and disappearance of the CCA closely coincided with the beginning and end of the oocyte chromosome condensation cycle, it appears that the condensation of the oocyte chromosomes as well as brain chromosomes was regulated by the same cytoplasmic condition. However, it must be noted that the oocyte and brain chromosomes did not begin condensation in exact synchrony. The oocyte chromosomes always entered diakinesis 3-4 hr prior to the earliest time at which condensation of the brain chromosomes was observed. This time lag may be ascribed to the fact that, prior to the period of maturation, the oocyte chromosomes are at the diplotene stage of meiotic prophase and are therefore already partially condensed, while the brain chromosomes, being in interphase, are completely dispersed . The results demonstrate unequivocally that the development of the CCA depends upon the release of some component from the GV. However, the development of CCA cannot be solely attributed to the GV. The GV contents that restored CCA in enucleated oocytes that had been maturing for 22 hr failed to restore CCA within the same period when injected into enucleated oocytes which had been maturing for only 10 hr. This indicates that the cytoplasm also contributes to the development of the
CCA. Furthermore, the fact that no progress of oocyte chromosome condensation was observed until after the GV membrane had begun to break down at the basal region also suggests that both cytoplasm and GV contents are involved in the development of the CCA. This coordination between GVBD and oocyte chromosome condensation could be explained as a consequence of the breakdown of the GV membrane which would remove the barrier to the interaction of GV material and cytoplasm at the proper time in maturation. Nuclear behavior during oocyte maturation and activation seems to be controlled by various cytoplasmic factors. Initiation of GVBD and arrest of the chromosomes at Metaphase II are caused by cytoplasmic factors which appear in the course of maturation independently of the GV (Masui and Markert, 1971). Also, the factor that stimulates DNA synthesis appears during this period and persists after activation (Gurdon, 1968). The factor responsible for chromosome condensation studied here seems to be distinct from the factors previously described because its production is GVdependent and it disappears at activation. ACKNOWLEDGMENTS The authors are indebted to Mr. William J. Wasserman for stimulating discussions and to Janice Ziegler for help with preparing the manuscript.
292
DEVELOPMENTALBIOLOC,Y REFERENCES
BATAILLON, E. (1928). Etudes analytiques
sur la maturation des oeufs de batraciens. C. R. Acad. Sci. 187, 520-523. BATAILL~N, E., and TCHOIJ-SU(1934). L’analyse experimentale de la fecondation et sa definition par les processus cinetiques. Ann. Sci. Nat. 2001.17,9-36. BRACHET,A. (1922). Recherches sur la fecondation prematuree de l’oeuf d’oursin (Pamcentrotus lividw). Arch. Biol. 32, 205-244. DETERRA, N. (1969). Cytoplasmic control over the nuclear events of cell reproduction. Znt. Rev. Cytol. 25, l-29. DE’ITLAFF,T. A., NIKITINA, L. A., and STROEVA,0. G. (1964). The role of the germinal vesicle in oocyte maturation in anurans as revealed by the removal and transplantation of nuclei. J. Embryol. Exp. Morphol. 12, 851-872. DRURY,H. F. (1941). Amylacetate as a clearing agent for embryonic material. Stain Technol. 16, 21-22. DURYEE,W. R. (1950). Chromosomal physiology in relation to nuclear structure. Ann. N. Y. Acad. Sci. 59,920-953. GRAHAM, C. F. (1966). The regulation of DNA synthesis and mitosis in multinucleate frog eggs. J. Cell Sci. 1, 363-374. GRAHAM,C. F., ARMS, K., and GURDON,J. B. (1966). The induction of DNA synthesis by frog egg cytoplasm. Develop. Biol. 14, 349-381. GURDON,J. B. (1967). On the origin and persistence of a cytoplasmic state inducing DNA synthesis in frogs’ eggs. Proc. Nut. Acad. Sci. U.S. 58,545-552.
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GURDON,J. B. (1966). Changes in somatic cell nuclei inserted into growing and maturing amphibian oocytes. J. Embryol. Exp. Morphol. 20, 401-414. GURDON,J. B., and WOODLAND,H. R. (1968). The cytoplasmic control of nuclear activity in animal development. Biol. Rev. Cambridge Phil. Sot. 43, 233-267. JOHNSON,R. T., and ho, P. N. (1971). Nucleo-cytoplasmic interactions in the achievement of nuclear synchrony in DNA synthesis and mitosis in multinucleate cells. Biol. Rev. Cambridge Phil. Sot. 46, 97-155. M&uI, Y. (1967). Relative roles of the pituitary, follicle cells and progesterone in the induction of oocyte maturation in Rana pipiens. J. Exp. 2001. 166, 365-376.
MASUI, Y. (1972). Distribution of the cytoplasmic activity inducing germinal vesicle breakdown in frog oocytes. J. Exp. 2001. 179, 365-378. MASUI, Y., and MARKERT,C. L. (1971). Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. 2001. 177, 129-146. MOORE, B. (1940). Chromosomes of frog eggs and embryos stained by the Feulgen method to avoid excessive staining of yolk granules. Anut. Rec. 78, Suppl. 122. SKOBLINA,M. N. (1969). Independence of the cortex maturation from germinal vesicle material during the maturation of amphibian and sturgeon oocytes. Exp. Cell Res. 55, 142-144. SMITH, L. D., and ECKER,R. E. (1969). Cytoplasmic regulation in early events of amphibian development. Proc. Con. Cancer Res. Conf., 8, 103-129.