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Control of sperm nuclear behavior in physiologically polyspermic newt eggs: Possible involvement of MPF
DEVELOPMENTALBIOLOGY142, 301-312 (1990)
Control of Sperm Nuclear Behavior in Physiologically Polyspermic Newt Eggs: Possible Involvement of MPF YASUHIRO IWAO1 AND RICHARD P. ELINSON*
Biological Institute, Faculty of Science, Yamaguchi University, Yamaguchi 753,Japan; and *Department of Zoology, University of Toronto, Toronto, Ontario M5S 1A1, Canada Accepted August 2, 1990 We have studied the mechanism controlling the behavior of accessory sperm nuclei in physiologically polyspermic eggs of the newt, Cynops pyrrhogaster. Our approach was to identify cytoplasmic components which would prevent the usual degeneration of accessory sperm nuclei. Injection of cytoplasm from unfertilized eggs, but not fertilized ones, induced multipolar cleavage in polyspermically fertilized eggs as well as centrosome separation and formation of extra bipolar spindles in accessory sperm nuclei. Cytosols extracted from unfertilized Cynops or Xenopus eggs also were active in inducing multipolar cleavage, as were germinal vesicle materials from oocytes of the frogs Xenopus or Rana or of Cynops. In all of these cases, the nuclear cycle as well as the onset of first cleavage was delayed relative to those in control eggs. In contrast, injection of an extract with maturation-promoting factor (MPF) activity, prepared from unfertilized Xenopus eggs, induced precocious and multipolar cleavage when injected into fertilized Cynops eggs. Injection of the MPF-containing extract caused acceleration of the nuclear cycle as well as formation of extra bipolar spindles by the accessory sperm nuclei. These results suggest that a local deficiency of MPF may lead to the degeneration of accessory sperm nuclei in physiologically polyspermic eggs. ©1990AcademicPress,Inc. INTRODUCTION Most animals exhibit m o n o s p e r m y . In n o r m a l fertilization, only one s p e r m is i n c o r p o r a t e d into the egg and m o n o s p e r m y is e n s u r e d by several blocks to p o l y s p e r m y o p e r a t i n g before s p e r m - e g g fusion (for a review, Jaffe and Gould, 1985). However, some a n i m a l s exhibit physiological polyspermy. Since several s p e r m e n t e r an egg in n o r m a l fertilization, a p o l y s p e r m y block in the egg cytop l a s m is i m p o r t a n t for e n s u r i n g n o r m a l d e v e l o p m e n t in those species. Physiologically p o l y s p e r m i c eggs provide a s y s t e m to analyze the m e c h a n i s m s which control cent r o s o m e s e p a r a t i o n and bipolar spindle f o r m a t i o n . A unique m e c h a n i s m to p r e v e n t p o l y s p e r m y has been r e p o r t e d in u r o d e l e a m p h i b i a n s ( F a n k h a u s e r , 1932; F a n k h a u s e r and Moore, 1941a; W a k i m o t o , 1979; Street, 1940; Iwao et al., 1985; Y a m a s a k i and Iwao, 1987). Two to t w e n t y s p e r m nuclei e n t e r t h e egg c y t o p l a s m , f o r m s p e r m pronuclei, u n d e r g o D N A synthesis (Wakimoto, 1979; Iwao et al., 1985), and develop asters. However, a f t e r f o r m a t i o n of a zygote nucleus between one s p e r m pronucleus and the egg pronucleus, all of the o t h e r accessory s p e r m nuclei become pycnotic and d e g e n e r a t e before first cleavage. Thus, in the s a m e egg cytoplasm, the zygote nucleus behaves quite differently f r o m the accessory s p e r m nuclei.
1To whom correspondence should be addressed.
Two h y p o t h e s e s were proposed to explain these phen o m e n a ( B a t a i l l o n a n d T c h o u Su, 1930; F a n k h a u s e r , 1948; reviewed by Elinson, 1986). F a n k h a u s e r proposed t h a t an i n h i b i t o r y activity e m a n a t e s f r o m the zygote nucleus. This activity s u p p r e s s e s accessory s p e r m nuclei and p r e v e n t s t h e m f r o m p a r t i c i p a t i n g in cleavage a n d development. In c o n t r a s t , Bataillon and Tchou Su suggested t h a t active c y t o p l a s m n e c e s s a r y for mitosis is c o n c e n t r a t e d a r o u n d the zygote nucleus, so t h a t the zygote nucleus can e n t e r mitosis, but accessory s p e r m nuclei c a n n o t . T h e a c c e s s o r y s p e r m nuclei a r e o u t of s y n c h r o n y with metabolic c h a n g e s linked to the zygotic cell cycle, and this a s y n c h r o n y leads to nuclear degeneration of the accessory s p e r m nuclei. Few e x p e r i m e n t s have been m a d e to d i s t i n g u i s h bet w e e n t h e s e h y p o t h e s e s . We h a v e c o n f i r m e d s o m e of F a n k h a u s e r ' s results, including the survival of accessory s p e r m nuclei in a n d r o m e r o g o n i c egg f r a g m e n t s or in p a r t i a l l y c o n s t r i c t e d egg halves (Iwao et al., 1985). While these results s u p p o r t F a n k h a u s e r ' s h y p o t h e s i s , t h e y do not prove it (Elinson, 1986). In addition, we have r e c e n t l y f o u n d t h a t nuclear d e g e n e r a t i o n in Cynops eggs is specific for h o m o l o g o u s s p e r m nuclei. N e i t h e r homologous s o m a t i c nuclei, s p e r m nuclei of the m o n o s p e r m i c s a l a m a n d e r Hynobius, nor s p e r m nuclei of the toad B u f o d e g e n e r a t e in Cynops egg c y t o p l a s m ( Y a m a s a k i a n d Iwao, 1987). I t is not known, however, w h a t kinds of c y t o p l a s m i c activities u n d e r l i e the c o n t r o l of n u c l e a r d e g e n e r a t i o n in fertilized n e w t eggs. 301
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Several cytoplasmic factors are known which regulate nuclear events occurring during oocyte maturation and early cleavage in amphibians (for a review, Masui and Shibuya, 1987). These include m a t u r a t i o n - p r o m o t i n g factor (MPF) which induces nuclear membrane breakdown, chromosome condensation and spindle assembly, and cytostatic factor (CSF) which arrests the cell cycle at metaphase. Recent work on the control of cell division has demonstrated t h a t MPF is a dominant and universal regulatory factor for entering M-phase (for reviews, Dunphy and Newport, 1988; Lohka, 1989; Murray and Kirschner, 1989a), and MPF activity is regulated by cyclin in the early embryonic cell cycle (Murray and Kirschner, 1989b; Murray et al., 1989). In this study, we report that several different cytoplasmic fractions can rescue accessory sperm nuclei, allowing them to form bipolar spindles and induce extra cleavage furrows. Use of fractions derived from Xenopus eggs suggests t h a t MPF may be involved in the survival of these nuclei. MATERIALS AND METHODS Sexually m a t u r e newts, Cynops pyrrhogaster, were collected near Yamaguchi in Japan during hibernation season. Leopard frogs, Rana pipiens, were purchased from dealers in Vermont at the start of hibernation and stored at 4°C until use. Xenopus laevis were purchased from dealers and maintained in our laboratories. To obtain unfertilized Cynops eggs, ovulation was induced by two injections each of 100 IU of human chorionic gonadotropin (HCG, Teikoku Zoki; Tokyo or Sigma) at intervals of 48 hr at 23°C. The mature eggs were obtained from the lowest portion of the oviducts by squeezing the female or by dissection. Ovulation in Xenopus was induced by injection of HCG (500 IU/female) at 18°C. In vitro maturation of defolliculated Rana oocytes was induced by exposure to 10 #g/ml progesterone in De Boer's solution (in mM; 110.3 NaC1, 1.3 KC1, 1.3 CaC12, 5.7 Tris-HC1, pH 7.4) at 18°C. To perform artificial insemination, Cynops sperm suspension was prepared by macerating the vas deferens in 20% Steinberg's solution (in mM; 11.6 NaC1, 0.13 KC1, 0.068 Ca(NOB)2, 0.17 MgSO4, and 0.92 Tris-HCI, pH 7.4). The unfertilized eggs were placed in a dry petri dish (5 cm in diameter), to which about 1 mt of the sperm suspension (1-5 × 105 cells/ml) was added. Fifteen minutes later, 20% Steinberg's solution was added to the dish. After removing their jelly coats with 2.5% Na-thioglycolate, pH 9.0, 30 min after fertilization, the fertilized eggs were allowed to develop in 20% Steinberg's solution at 23 _+ 0.5°C. To transfer egg cytoplasm, unfertilized or fertilized Cynops eggs were centrifuged on 10% Percoll in 20%
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Steinberg's solution at 950g for 10 min at 4°C. The unbroken eggs were stratified into a lipid layer, clear cytoplasm, pigment, and yolk granules. Fifty nanoliters of clear cytoplasm was injected into each recipient egg with a glass micropipet (30 ~m in tip diameter), and the injected eggs were incubated in Ca2+-rich 100% Steinberg's solution (3.4 mM Ca(NOB)2) for 30 min to enhance wound healing, followed by culturing in 20% Steinberg's solution. All injections were made into the animal hemisphere of the eggs which has a diameter of about 2.2 mm. With sham injections of 50 nl of 1% Nile blue sulfate, the dye spread through § of the animal hemisphere within 1 hr and over the whole animal hemisphere by 1.5 hr. To inject germinal vesicles (GVs), full-grown oocytes obtained from ovaries of mature Rana, Xenopus, or Cynops females were pricked at the animal pole with a fine glass pipet, and their GVs were squeezed out into extraction buffer by gently pressing the equatorial region of the oocyte with forceps (in mM; 250 sucrose, 100 KC1, 30 MgSO4, 25 Tris-HC1, pH 7.4). In some cases, aliquots of a concentrated GV extract were made by first drawing up a small volume of paraffin oil, then about 50 GVs, followed by another small volume of paraffin oil into a capillary tube. The orifice of the capillary tube was smaller than a GV, and this caused disruption of the GV membrane and release of its contents. The end of the tube was heat sealed and the tube was centrifuged at 600g for 10 min. Each fertilized egg was injected with 50 nl of GV extract, approximately equivalent to one GV/egg. ~ To extract cytoplasm from unfertilized eggs, Cynops eggs were manually dejellied in DeBoer's solution, or Xenopus eggs were de jellied with 45 mM2-mercaptoethanol in Ca-free Barth's solution (in mM," 88 NaC1, 1.0 KC1, 2.4 NaHCOa, 1.5 EDTA, and 7.5 Tris-HC1, pH 8.5), followed by thorough washing with Ca-free Barth's solution. The eggs were washed three times with the GVextraction buffer and packed in an Eppendorf tube. After removing excess buffer by light centrifugation (4.5g, 10 min), the eggs were centrifuged at 15,000g for 20 min at 4°C. Fifty nanoliters of slightly red cytosol was injected into each fertilized Cynops egg. A crude preparation of maturation-promoting factor was a generous gift from Dr. M. J. Lohka (University of Colorado, School of Medicine) and was prepared from unfertilized Xenopus eggs according to the methods of Lohka et al. (1988). The 250,000g supernatant was precipitated with 0-34% or 35-80% saturated ammonium sulfate, resuspended in approximately one-third or onefourth the original volume, respectively, and then dialyzed against dialysis buffer (in mM; 100 Na fl-glycerophosphate, 15 MgC12, 1.0 dithiothreitoI, 0.1 phenylmethyl sulfonylfluoride, and 20 Hepes-NaOH,
Iwno ANDELINSON SpermNuclear Behavior pH 7.5). Samples were stored at -70°C. Fifty nanoliters of the MPF fractions was injected into each fertilized Cynops egg after dilution with the GV-extraction buffer just before injection. MPF activity was determined either by injection of 50 nl of extract into defolliculated Rana oocytes to observe germinal vesicle breakdown (GVBD) 10 hr after injection at 18°C, or by adding 50 pl of the extract to the cell-free assay system to observe nuclear envelope breakdown (NEBD) and chromosome condensation of decondensed sperm nuclei (cf., Lohka and Maller, 1985). For cytological observations, eggs were fixed in Smith's solution for 24 hr and embedded in paraffin. The 12-tLm-thick serial sections were stained with Feulgen's reagent and fast green, and were observed at 400× magnification. RESULTS Since normal fertilization of Cynops is physiologically polyspermic, 2-10 sperm were incorporated into each egg fertilized by artificial insemination. Sperm entered the eggs randomly and uniformly as judged by the distribution of sperm entrance spots. Each sperm nucleus moved into the egg cytoplasm about 200 #m deep from the surface, but only one sperm pronucleus (the principal sperm nucleus) reached the center of the egg to contact the egg pronucleus and make a zygote nucleus. Progress of nuclear events in fertilized Cynops eggs is summarized in Table 1. All sperm nuclei underwent chromatin decondensation to form sperm pronuclei with well-developed asters and DNA synthesis (Iwao et al., 1985) with the same chronological schedule. However, when the zygote nucleus underwent chromosome condensation, the accessory sperm nuclei became pycnotic and degenerated before first cleavage. In some accessory sperm nuclei, chromosomes were associated with small monopolar spindles, but they never formed bipolar spindles. The degenerating sperm nuclei moved to the equatorial region or the vegetal hemisphere around the time of cleavage.
Cytoplasm of Unfertilized Cynops Eggs Induced Multipolar Cleavage and Rescue of Accessory Sperm Nuclei Initially, we asked whether Cynops eggs had any activities in their cytoplasm which would promote or prevent nuclear degeneration of accessory sperm nuclei. This was tested by various types of cytoplasmic transfer between unfertilized and fertilized eggs. Cytoplasm taken directly from eggs had little effect on cleavage, so each donor egg was stratified by centrifugation before sucking out their cytoplasm. When clear cytoplasm obtained from the unfertilized eggs was injected into eggs 1-2 hr
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after fertilization (G1), most eggs cleaved normally (Table 2). In contrast, when this cytoplasm was injected into eggs just after contact of sperm and egg nuclei (4-5 hr, G2), multipolar division occurred in 56% of the eggs at first cleavage within 9 hr after fertilization. (For ease of communication, we refer to the period 4-5 hr after fertilization as G2, even though the end of S-phase has not been strictly defined. See Table 1.) The onset of multipolar cleavage was always delayed about 1-2 hr in comparison with t h a t of bipolar cleavage. Cytoplasm obtained from fertilized eggs in G 1 or M had little activity to induce multipolar cleavage, so t h a t most recipient eggs cleaved normally and on schedule. These results demonstrate t h a t cytoplasm of unfertilized eggs has a strong activity to induce multipolar cleavage, but t h a t activity disappears after fertilization. To determine whether the induced multipolar cleavage was caused by the survival of the accessory sperm nuclei, nuclear behavior in eggs t h a t had been injected at G2 with unfertilized egg cytoplasm was examined with cytological sections. Surviving accessory sperm nuclei, including swollen pronuclei or accessory bipolar spindles with a haploid set of metaphase chromosomes, were found in 7 out of 10 eggs examined at 7-8 hr after fertilization (Fig. 1). In one egg, for example, there was a metaphase plate of the zygote nucleus with a bipolar spindle in the center of the egg (Fig. 1A), four swollen accessory sperm pronuclei (Fig. 1B), three accessory smaller bipolar spindles with haploid sets of chromosomes (Fig. 1C), and a pycnotic accessory sperm nucleus (Fig. 1D). These accessory sperm nuclei and spindles were found in the equatorial region or vegetal hemisphere of the egg. The different stages of sperm nuclei in the injected eggs might be due to the distribution of injected materials, caused by diffusion after injection. Some delay (1-2 hr) was observed in the progress of nuclear events in both zygote and accessory nuclei. The number of eggs with undegenerated accessory sperm nuclei and the delay of nuclear events corresponded with the delayed multipolar cleavages observed in living eggs. We did not detect undegenerating accessory sperm nuclei in the eggs t h a t had been injected with egg cytoplasm, but which cleaved in a normal, bipolar way. We conclude t h a t accessory sperm nuclei were rescued by the cytoplasmic injection and induced extra furrow formation.
Germinal Vesicle Materials of Cynops or Xenopus Oocytes Induced Multipolar Cleavage Since contents of the germinal vesicie are spread out in the cytoplasm during oocyte maturation, the effects of GV contents on the degeneration of accessory sperm nuclei were examined. When a whole Cynops GV was
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TABLE 1 TIMECOURSEOFNUCLEAREVENTSIN FERTILIZEDEGGSORIN EGGSINJECTEDWITHMPF FRACTION MPF fraction-injected eggs Time (hours after fertilization) 1
Cell cycle phase Telo II, GI
2
Normalized time 0.15 0.31
2-3
S
4
"G2''a
(4-4.25) 4.5
0.31-0.46 0.62
0.77
5.5
0.85
6.5 (6-7.5)
M
0.85-0.92
1.0 (0.92-1.15)
Zygote nucleus
Accessory sperm nuclei
Second polar body emission Pronuclei with decondensed chromatin. No differences between sperm nuclei with respect to decondensation and aster formation DNA synthesis starts in all nuclei (Iwao et al., 1985) Contact of sperm and egg pronuclei to produce zygote nucleus
(0.62-0.66) 0.69
5
5.5-6
Normal development
Separation of centrosomes associated with zygote nucleus
(MPF fraction injected) Centrosome separation Pronuclei with monoaster or one centrosome Bipolar metaphase Some centrosome spindle separation Cleavage beginning decondensing cleavage nuclei
Zygote nucleus: Chromosome condensation and formation of a bipolar spindle Accessory sperm nuclei: Lack bipolar spindle and pycnotic. Occasional small monopolar spindle 50% of eggs cleaved (range of cleavage time)
Cleavage nuclei
Some bipolar spindles; other nuclei pycnotic with one centrosome Mixture of cleavage nuclei, bipolar spindles, pycnotic nuclei
a The exact time for the start of G2 is not known, since the end of S phase has not been determined.
injected into a Cynops fertilized egg at G 2, 70% of the eggs u n d e r w e n t m u l t i p o l a r division at first cleavage (Table 3). Whole Xenopus GV or m a t e r i a l s e x t r a c t e d f r o m Rana GVs also induced m u l t i p o l a r cleavage (Fig. 2A and Table 3). The onset of cleavage was delayed 1-2 hr, so t h a t cleavage b e g a n 7.5 h r a f t e r fertilization and 55% of the eggs (24/44) u n d e r w e n t first cleavage by 8.5 h r a f t e r fertilization. I n c o n t r a s t , n e i t h e r cytosol e x t r a c t e d f r o m enucleated Xenopus oocytes nor c y t o p l a s m of enucleated Rana oocytes affected cleavage. The n u c l e a r cycle w a s also d e l a y e d 1-2 hr, so t h a t m o s t of the nuclei in the injected eggs r e m a i n e d in G2 with decondensed c h r o m a t i n enclosed by n u c l e a r enve-
lopes (Fig. 2B). F o r m a t i o n of b i p o l a r s p i n d l e s a n d f u r r o w i n g f r o m accessory s p e r m nuclei were confirmed with cytological sections. In 10 eggs which h a d been injected w i t h Rana GV materials, zygote nuclei f o r m e d a p a i r of c l e a v a g e nuclei (80%) or b i p o l a r m e t a p h a s e plates (20%), while the accessory s p e r m nuclei exhibited a v a r i e t y of behaviors at 8 h r a f t e r fertilization. The 37 accessory s p e r m nuclei f o r m e d a bipolar spindle (14%), s e p a r a t e d c e n t r o s o m e s (5%) (Fig. 2C), a pair of small cleavage nuclei associated with (14%) (Fig. 2D) or without (38%) f u r r o w f o r m a t i o n , pronuclei with one centrosome (14%), or pycnotic c h r o m a t i n (15%). These results d e m o n s t r a t e t h a t injected GV m a t e r i a l s delay the nu-
IWAOANDELINSON TABLE 2 TRANSFER OF CYTOPLASM AMONG UNFERTILIZED AND FERTILIZED CYNOPS EGGS No. of b
Donor egg~ Unfertilized (Meta II) Unfertilized (Meta II) G1 G2 M
Recipientegg
eggs
% multipolar cleavagec
--) G1
34
3
--~ G2 --~ Gz --~ G2 --~ G1
68 20 20 17
56 10 5 12
a Clear cytoplasm 50 nl from a stratified egg was injected into each recipient egg. Times after fertilization used were: G1 (1-2 hr), G2 (4-5 hr), M (5-6 hr). bBased on experiments using 10 different females. Cleavage delay of 1-2 hr.
clear cycle by delaying e n t r y into M-phase and lead to bipolar spindle f o r m a t i o n in accessory s p e r m nuclei.
Cytosol Extracted from Unfertilized Cynops or Xenopus Eggs Induced Multipolar Cleavage To c h a r a c t e r i z e the p r o p e r t i e s of the m u l t i p o l a r cleavage-inducing activity, we tried to isolate active cytosol
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f r o m unfertilized eggs. W h e n cytosol (15,000g s u p e r n a t a n t ) o b t a i n e d f r o m unfertilized Cynops eggs was injected into eggs at G2, 76% of the eggs u n d e r w e n t multipolar division at first cleavage (Table 3). N e i t h e r ext r a c t i o n buffer n o r a s i m i l a r c y t o s o l o b t a i n e d f r o m fertilized Cynops eggs affected cleavage, a n d h e a t i n g (60°C, 15 min) the cytosol i n a c t i v a t e d it c o m p l e t e l y (Table 3). As with injected clear c y t o p l a s m or GV m a t e r i a l s , the onset of m u l t i p o l a r cleavage was delayed 1-2 h r in all of t h e i n j e c t e d eggs, a n d e x t r a b i p o l a r s p i n d l e s f o r m e d f r o m accessory s p e r m nuclei. The presence of m u l t i p o l a r cleavage a c t i v i t y in cytosol f r o m u n f e r t i l ized, but not fertilized eggs (Table 3) c o r r e l a t e d with the presence of activity in clear c y t o p l a s m f r o m unfertilized, b u t not fertilized eggs (Table 2). Cytosol o b t a i n e d f r o m unfertilized Xenopus eggs also h a d a s t r o n g activity to induce m u l t i p o l a r cleavage (Table 3). Since Xenopus is an a n u r a n , its fertilization is m o n o s p e r m i c , and the egg n o r m a l l y does not have to deal with accessory s p e r m nuclei. This suggests t h a t the activity is a g e n e r a l cellular one, not specifically r e l a t e d to physiological polyspermy. To d e t e r m i n e the ionic r e q u i r e m e n t s for isolation of cytosol c o n t a i n i n g the m u l t i p o l a r cleavage-inducing activity, we e x a m i n e d effects of divalent cations in the e x t r a c t i o n buffer (Table 4). As the c o n c e n t r a t i o n of Ca 2+
FIG. 1. Nuclei in a Cynops egg that had been injected at G2 with cytoplasm of an unfertilized Cynopsegg. (A) A bipolar spindle of a zygote nucleus with metaphase chromosomes. (B) A swelling accessory sperm nucleus. (C) An extra bipolar spindle of an accessory sperm nucleus with a haploid set of chromosomes. (D) A pycnotic accessory sperm nucleus at 7 hr after fertilization. Scale line: 10 ttm.
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DEVELOPMENTALBIOLOGY TABLE 3 INDUCTION OF MULTIPOLARCLEAVAGEBY INJECTION
Injected materials a A. Germinal vesicles (GV) Cynops GV Xenopus GV Rana GV extract (600g, supernatant) Cytoplasm (15,000g, supernatant) from enucleated Xenopus oocytes Cytoplasm from enucleated Rana oocytes (uncentrifuged) Extraction buffer No injection B. Cytosols Unfertilized Cynops eggs Unfertilized Cynops eggs (60°C, 15 min) Fertilized Cynops eggs Unfertilized Xenopus eggs Extraction buffer No injection
No. of eggsb
% multipolar cleavage c
75 17
71 65
20
95
19
0
20 20 20
0 0 0
51
76
17 20 17 26 20
0 0 76 0 0
a Fifty nanoliters or 1 GV injected at Gz (4-5 hr after fertilization). b Based on experiments using 21 different females. c Cleavage delay of 1-2 hr.
in the buffer was increased, recovery of the activity in the cytosol was reduced, so t h a t no activity was obtained with buffer containing 20 mM Ca2+. In contrast, Mg z+ was important for retaining the activity in the cytosol, because no activity was detected with the MgZ+-free buffer in which MgS04 was replaced by NaC1. These results suggest t h a t this activity requires Mg 2+ but is sensitive to Caz+ ions.
Extracts with M P F Activity Induced Precocious and Multipolar Cleavage The absence of multipolar cleavage-inducing activity in fertilized eggs and its ionic dependence suggested a relationship with m a t u r a t i o n - p r o m o t i n g factor, a known cytoplasmic regulator of nuclear behavior whose levels are high in unfertilized amphibian eggs, but decrease at fertilization. As a first step, we asked whether a fraction containing MPF activity extracted from unfertilized Xenopus eggs could induce multipolar cleavage. The first fraction tested was a 0-34% saturated ammonium sulfate precipitate of a 250,000g supernatant. When the undiluted MPF fraction was injected into Cynops eggs at G2, none of the 35 injected eggs cleaved in the 20 hr after injection. Abortive cortical wrinkles were observed in some eggs (7/35) at 6-8 hr after fertilization, but true cleavage furrows were never
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formed. Condensed chromosomes were scattered in the cytoplasm t h a t contained a disordered fibrous network resembling astral or spindle fibers. In contrast, diluted MPF fractions induced precocious and multipolar cleavage (Figs. 3A and 4A). When a 1:20 dilution of the MPF fraction was injected at G2 (4-4.5 hr after fertilization), 90% of the eggs cleaved 30-60 min earlier than eggs injected with a 1:20 dilution of the dialysis buffer (Fig. 5A). Three quarters of the eggs underwent multipolar division at first cleavage (Figs. 3A and 4A). In most cases, a main furrow appeared first in the center of the animal hemisphere, and then, 30-60 min later, additional furrows formed eccentrically on the animal hemisphere. The eggs developed to abnormal blastulae, but arrested before gastrulation. An injection of a 1:10 dilution of the MPF fraction at Gz also induced precocious and multipolar cleavage on all injected eggs (Fig. 4A). The activity to induce either precocious or multipolar cleavage decreased as the MPF fraction was diluted further (Fig. 4A), so t h a t these activities could not be detected after dilution to 1/160. The MPF activity of our fraction, measured by GVBD in Rana full-grown oocytes, corresponded well with the activities inducing both precocious and multipolar cleavage (Fig. 4A). Furthermore, the MPF activity measured by NEBD in the cell-free system was detected at least after dilution to 1/16. When a fraction precipitated by 35-80% s a t u r a t e d ammonium sulfate containing only a small amount of MPF activity (Lohka et al., 1988) was injected at Gz, the undiluted fraction caused precocious and multipolar cleavage in the Cynops eggs, but these activities could not be detected after dilution to 1/20 (Fig. 4B). Loss of the activities corresponded well with t h a t of MPF activity measured by GVBD in Rana oocytes (Fig. 4B). MPF activity measured by NEBD in the cell-free system was detectable only in the undiluted fraction. These relationships between the activities suggest t h a t MPF is a candidate for inducing precocious and multipolar cleavage in injected Cynops eggs. The acceleration of the onset of cleavage by the MPF fraction is shown in detail in Fig. 5. When the 1:20 dilution of the MPF fraction was injected 3 or 4 hr after fertilization, the onset of cleavage was accelerated 1.5-2 hr or 0.5-1 hr, respectively (Fig. 5A). The injection at 5 hr did not accelerate the onset of cleavage, but induced multipolar cleavage in 95% (19/20) of the eggs. It should be noted t h a t the furrows in the eggs t h a t had been injected with the MPF fraction 3 hr after fertilization formed more eccentrically and simultaneously, so t h a t a precocious main furrow was not formed. This may be due to a failure of the sperm and egg nuclei to join together in the zygote. When the 1:20 dilution of the MPF fraction was injected into the eggs 2 hr after fertiliza-
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FIG. 2. Effects of injection of Rana GV materials into fertilized (G2) Cynops eggs. (A) Multipolar cleavage 9 hr after fertilization. (B) Decondensed egg and principal sperm nuclei forming a zygote nucleus 7 hr after fertilization. (C) An accessory sperm nucleus (N) undergoing centrosome separation (c) 8 hr after fertilization. (D) A pair of small cleavage nuclei (CN) derived from an undegenerated accessory sperm nucleus just under an extra cleavage furrow (F) 9 hr after fertilization (tangential section). Scale line: (A) 1 mm; (B-D) 10 ttm.
tion, increased acceleration did not occur (Fig. 5B). Twelve out of 20 (60%) injected eggs underwent multipolar cleavage before 6 hr after fertilization, but the rest of the eggs (40%) cleaved bipolarly on schedule between 6.5-7.5 hr after fertilization. Furthermore, injection of the 1:10 dilution of the MPF fraction 2 hr after fertilization did not cause any cleavage before 4 hr after fertilization (Fig. 5B). In those eggs, all of the sperm nuclei remained as pronuclei until 3 hr after fertilization, but their centrosomes were unclear. Some accessory sperm nuclei underwent centrosome separation around 3.5 hr after fertilization, and then most sperm
TABLE 4 EFFECT OF DIVALENT CATIONSIN EXTRACTIONBUFFER ON INDUCING CLEAVAGE No. ofb
% multipolar
MgS04
Ions in extraction buffera CaC12
NaC1 (mM)
eggs
cleavage
30 30 30 0
0 10 20 0
0 0 0 30
51 68 25 29
76 26 0 0
Extraction medium also contained 250 m M Sucrose, 100 mM KC1, and 25 m M Tris-HC1 (pH 7.4). Based on experiments using 12 different females.
nuclei formed bipolar spindles 4-4.5 hr after fertilization. It should be mentioned that centrosome separation normally does not occur before 3.5 hr, the time for completion of DNA synthesis (Iwao et al., 1985).
Extracts with M P F Activity Induced Precocious Formation of Bipolar Spindles and Chromosomes in Accessory Sperm Nuclei The MPF fraction t h a t resulted in multipolar cleavage also caused accelerated bipolar spindle formation in the accessory sperm nuclei. Five Cynops eggs, injected with the 1:10 dilution of the MPF fraction 4-4.25 hr after fertilization (G2), were fixed at 0.5, 1.0, 1.5, or 2.0 hr after injection to observe nuclear events (Table 1). The zygote nuclei underwent centrosome separation 0.5 hr after injection, while all accessory sperm nuclei stayed as pronuclei with a monoaster or one centrosome. The zygote nuclei formed metaphase chromosomes incorporated on bipolar spindles in the center of the injected eggs 1 hr after injection (Fig. 3B), about I hr earlier than in noninjected eggs. At the same time, some accessory sperm nuclei in the equatorial region or vegetal hemisphere underwent centrosome separation (Fig. 3C). At 1.5 hr after injection, cleavage furrowing began, and each anaphase zygote nucleus was separated into two swelling cleavage nuclei. Some of the accessory sperm nuclei
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DEVELOPMENTALBIOLOGY
VOLUME142, 1990
FIG. 3. Effects of an injected MPF fraction (1:10 dilution of 0-34% saturated ammonium sulfate fraction, 4-4.25 hr after fertilization) on cleavage and nuclear behavior in fertilized Cynops eggs. (A) Multipolar cleavage 7 hr after fertilization. (B) A bipolar spindle of a zygote nucleus. (C) An accessory sperm nucleus undergoing centrosome separation (c) 1 hr after injection. (D) An extra spindle of an accessory sperm nucleus 1.5 hr after injection. Scale line: (A) 1 mm; (B-D) 10 ~m.
w e r e p y c n o t i c , b u t m a n y o f t h e o t h e r s f o r m e d e x t r a bip o l a r s p i n d l e s ( F i g . 3D). A t 2.0 h r a f t e r i n j e c t i o n , all t h e zygote nuclei f o r m e d swelling cleavage nuclei, while the accessory s p e r m nuclei e x h i b i t e d a v a r i e t y of behaviors.
T h e 38 n u c l e i f o r m e d a p a i r of c l e a v a g e n u c l e i ( 2 9 % ) , a m e t a p h a s e p l a t e on a b i p o l a r s p i n d l e ( 1 0 % ) , a p r o n u cleus with two centrosomes (3%) or with one centros o m e ( 8 % ) , o r p y c n o t i c n u c l e i (50%). T h e s e o b s e r v a t i o n s
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FIG. 4. MPF fractions induce precocious and multipolar furrowing in fertilized Cynops eggs. Twenty eggs at G2 (4-4.5 hr after fertilization) were injected with 50 nl of each dilution of the MPF fraction, and scored for precocious (-- • --) and multipolar (-- • --) cleavage. Cleavage was considered precocious if it occurred before 6 hr, when non-injected eggs never cleaved. The dilutions of the MPF fraction were also assayed for their activity to induce germinal vesicle breakdown (GVBD) in full-grown Rana oocytes (-- © --). In (A), the MPF fraction was a 0-34% saturated ammonium sulfate fraction, while in (B), a 35-80% cut was used. The former has MPF activity, as assayed in the cell-free system, at a dilution of 1/16, while the latter only had activity when undiluted.
IWAO AND ELINSON
309
Sperm Nuclear Behavior
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FIG. 5. Acceleration of the onset of cleavage by the injection of an MPF fraction precipitated by 0-34% saturated ammonium sulfate. (A) Fifty nanoliters of a 1:20 dilution of the MPF fraction was injected into each of 20 eggs 3 hr ( - - • --), 4 hr ( - - O --), and 5 hr ( - - • - - ) after fertilization. The injections at 4 hr are equivalent to those in Fig. 4. Precocious cleavage of eggs injected with the MPF fraction is shown as a function of time in comparison with cleavage in the eggs injected with a 1:20 dilution of buffer ( - - - O - - - ) or non-injected eggs ( - - - • - - - ) . (B) Fifty nanoliters of a 1:10 dilution of the MPF fraction was injected into each of 20 eggs 2 hr ( - - O - - ) or 4 hr ( - - • --) after fertilization. Fifty nanoliters of a 1:20 dilution of the MPF fraction was injected 2 hr after fertilization ( - - • --). Precocious cleavage of eggs injected with the MPF fraction is shown as a function of time in comparison with cleavage in the eggs injected with a 1:10 dilution of buffer ( - - - O - - - ) or non-injected eggs ( - - - • - - - ) .
demonstrate that 42% of the accessory sperm nuclei underwent centrosome separation and mitosis in response to the injection of the MPF fraction. Although the nuclear events in the MPF-injected eggs were greatly accelerated, the progress of the zygote nucleus was more advanced than t h a t of the accessory sperm nuclei. The difference of acceleration in the nuclear cycle corresponded well with the timing of multipolar furrowing, mentioned previously in which the formation of a main furrow in the center was followed by extra furrows at the periphery.
DISCUSSION
Rescue of Accessory Sperm Nuclei with M P F Containing Fractions We have demonstrated t h a t various cytoplasmic extracts can rescue accessory sperm nuclei from degeneration in polyspermic Cynops eggs. These extracts include clear cytoplasm or cytosol from unfertilized eggs, GV contents, and fractions with MPF activity from Xenopus eggs. While rescue includes centrosome separation, formation of bipolar spindles with metaphase chromosomes, and induction of furrowing, the different extracts may not be acting in the same way. Clear cytoplasm, cytosol, and GV contents all delayed the onset of cleavage, while MPF fractions accelerated it. As discussed later, the delay itself may permit the accessory sperm nuclei to proceed through the cell cycle.
Rescuing activity in a Xenopus egg extract was associated with MPF activity, assayed both by oocyte maturation in vivo and by nuclear envelope breakdown and chromosome condensation in vitro. Since the MPF fraction injected was crude, the factor responsible for centrosome separation and formation of bipolar spindles may not be MPF itself, but some other activity which copurifies with MPF. However, several recent reports suggest t h a t MPF can act on the centrosome. The p34 cdc2 protein kinase, which is homologous to a component of MPF (Gautier et al., 1988; Dunphy et al., 1988), is accumulated in centrosomes of mammalian cultured cells during M-phase (Raibowl et al., 1989). MPF activity in Xenopus egg cytoplasm appears at 0.65 normalized time (NT) and reaches a maximum at 0.80 NT (Gerhart et al., 1984) encompassing the time of centrosome separation (0.77 NT). In addition, some proteins of the centrosome are highly phosphorylated during M-phase in sea urchin eggs (Kuriyama, 1989) and in mammalian cultured cells (Vandre et al., 1984). Hyperphosphorylation occurs in fertilized Xenopus eggs during M-phase (Karsenti et al., 1987) due to M-phase-specific protein kinase(s) which probably include MPF protein kinase activity (Lohka et al., 1988; Labbe et al., 1988). These observations suggest t h a t MPF plays a role in centrosome separation, directly or indirectly, by participating in protein kinase cascades in the egg cytoplasm during M-phase. To account for a role of MPF in the control of nuclear behavior of physiologically polyspermic newt eggs, we propose t h a t a higher concentration of active MPF in
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DEVELOPMENTALBIOLOGY
the center of the fertilized egg leads to the separation of the centrosome in the zygote nucleus, but its lower concentration in the periphery is not enough to induce normal mitosis in the accessory sperm nuclei.
Hypotheses on Control of Nuclear Behavior in Polyspermic Newt Eggs Two hypotheses were proposed in the past to explain the mechanism of suppression of accessory sperm nuclei. Fankhauser (1948) proposed an inhibitory factor emanating from the zygote nucleus which caused the degeneration of the accessory sperm nuclei, and this hypothesis was supported by extensive work on the development of egg fragments. Against Fankhauser's idea, Bataillon and Tchou Su (1930) proposed t h a t there is a limited amount of active cytoplasm in the animal hemisphere of the egg. The large aster of the principal sperm nucleus occupies the cytoplasmic-rich center of the animal hemisphere and may collect active cytoplasm. This active cytoplasm promotes entry into mitosis of the zygote nucleus earlier t h a n the accessory sperm nuclei which are in a poorer cytoplasmic area at the periphery. Some aspect of egg metabolism, such as O2 consumption or C02 production, is linked to the zygote nucleus, and it changes at mitosis. The accessory sperm nuclei are out of synchrony with the new metabolism, so they degenerate. Our results fit well with Bataillon and Tchou Su's hypothesis, since we could rescue accessory sperm nuclei by supplying extra cytoplasmic components by injection. We would revise Bataillon and Tchou Su's hypothesis by substituting "the ability to activate MPF" for "active cytoplasm." Instead of egg metabolism (02, C02), we hypothesize t h a t the MPF cycle is linked to the zygotic nucleus. Although our experiments did not detect the inactivator, MPF inactivation has been demonstrated in Xenopus eggs (Gerhart et al., 1984). The accessory sperm nuclei would be out of synchrony with the wave of MPF inactivation, and this a s y n c h r o n y may cause their degeneration. Our results and hypothesis raise a number of questions to be tested in the future. For instance, there should be an unequal distribution of MPF activity in the newt egg cytoplasm, with a higher concentration in the center. At least in frog oocytes, such an unequal distribution can exist (Masui, 1972). A second question is w h e t h e r mistimed exposure to MPF inactivation can cause nuclear degeneration or whether additional factors are involved. The possibility of more complex regulatory interactions is suggested by our present observations on the heterogeneity of nuclear response to injected factors, and by the fact t h a t degeneration
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depends on the type of nucleus (Yamasaki and Iwao, 1987).
Acceleration of Cleavage with MPF-Containing Fractions An interesting result obtained by injection of the MPF fraction was the acceleration of nuclear cycles and precocious cleavage in fertilized newt eggs. In the normal course of Cynops development, the egg cortex is determined to make furrows about 40 min before first cleavage in response to a signal transmitted from subcortical cytoplasm (Sawai, 1972). Our results show t h a t the cortex can acquire competence to make furrows at least 3.5 hr before the first cleavage. Since all of the nuclear events after centrosome separation occurred early, the MPF fraction seems to trigger a cascade of reactions t h a t had been ready by early G2. Injection of the MPF fraction either 2 or 3 hr after fertilization accelerated cleavage to the same extent, and centrosome separation was not detected before 3.5 hr after fertilization. Since nuclei undergo DNA synthesis 2-3 hr after fertilization (Iwao et al., 1985), the results imply t h a t the MPF fraction can accelerate events of the cell cycle, which occur after S-phase but not before.
Delay of Cleavage and Rescue of Accessory Sperm Nuclei with Other Cytoplasmic Fractions Except for the injection of the MPF fraction, all rescues of accessory sperm nuclei both in our experiments and in previous ones (Elinson, 1986) were correlated with a delay in the cell cycle. The early injection (2-3 hr) of MPF also elicited multipolar cleavage, while the injection of cytoplasm obtained from unfertilized eggs into the G1 eggs had little effect. Perhaps a greater stability or a stronger activity of the MPF fraction could account for this difference. The detailed action of the MPF fraction as well as t h a t of extracted cytosol and GV materials should be further examined during the early embryonic cell cycle. Our injections of GV materials and of cytoplasm or cytosol from unfertilized eggs caused delayed, multipolar cleavage. Similar results were obtained with andromerogonic egg f r a g m e n t s (Fankhauser, 1934a,b; F a n k h a u s e r and Moore, 1941b; Iwao et al., 1985), eggs with the female nucleus removed by suction (Kaylor, 1937; Iwao et al., 1985), and cycloheximide-treated eggs (Iwao et al., 1985). These observations suggest t h a t prolonging the cell cycle allows centrosome separation or bipolar spindle formation in the accessory sperm nuclei. To fit this in with our MPF hypothesis, we would say t h a t in the absence of mitosis of a dominant zygote nucleus, MPF inactivation is not triggered. This either permits a rise in MPF activity at the periphery or allows
IWAO AND ELINSON
more time for the accessory sperm nuclei to interact with a low level of MPF. In either case, the accessory sperm nuclei would undergo centrosome separation and enter mitosis on a delayed schedule. We do not know whether rescue by injected GV materials or unfertilized egg cytoplasm is effective simply because they delay the cell cycle or because they contain a cell cycle regulator, normally involved in the control of sperm nuclear behavior. There are numerous reports showing that the GV contains factors involved in the cell cycle (Smith and Ecker, 1969; Katagiri and Moriya, 1976; Masui et al., 1979; Lohka and Masui, 1983, Skoblina et al., 1984; Ohsumi et al., 1986; Gautier, 1987), and Imoh and Miyazaki (1984) noted the restricted distribution of GV materials in the center of the animal hemisphere in Cynops eggs. Similarly, unfertilized, but not fertilized, egg cytoplasm had MPF and CSF, which are involved in cell cycle regulation (Masui and Markert, 1971; Sawai and Higuchi, 1989). Further analysis of the active components of the GV and unfertilized egg cytoplasm will be necessary to determine how they rescue accessory sperm nuclei and induce multipolar furrowing. Through this study, we have tried to elucidate how the nuclear behavior is regulated in physiologically polyspermic newt eggs based on current views of cell cycle regulation. Although the precise mechanisms have not been clarified, the physiologically polyspermic newt egg provides one of the best systems to analyze molecular mechanisms of centrosome replication and separation during mitosis. We thank Dr. Manfred J. Lohka, University of Colorado School of Medicine, for his courteous gift of the MPF fractions and for performing the in vitro assay on them, Dr. Christian Aimar for participating in initial attempts at these experiments, and Dr. Yoshio Masui for stimulating discussions. This work was supported by a JSPS-NSERC Bilateral Exchange Grant to Y.I., a NSERC Operating Grant to R.P.E., and a grant-in-aid for Scientific Research from the Japanese Ministry of Education, Science and Culture to Y.I. REFERENCES BATAILLON, E., and TCHOU Su (1930). Etudes analytiques et experimentales sur les rytbmes cinetiques dane l'oeuf. Arch. BioL 40, 439540. DUNPHY, W. G., and NEWPORT,J. (1988). Unraveling of mitotic control mechanisms. Cell 55, 925-928. DUNPHY, W. G., BRIZUELA,L., BEACH, D , and NEWPORT,J. (1988). The Xenopus homologue of cdc2 is a component of MPF, a cytoplasmic regulator of mitosis. Cell 54, 423-431. ELINSON, R. P. (1986). Fertilization in amphibians: The ancestry of the block to polyspermy. Int. Rev. Cytol. 101, 59-100. FANKHAUSER, G. (1932). Cytological studies on egg fragments of the salamander Triton. II. The history of supernumerary sperm nuclei in normal fertilization and cleavage of fragments containing the egg nucleus. J. Exp. Zoo£ 62, 185-235. FANKHAUSER,G. (1934a). Cytological studies on egg fragments of the salamander Triton. IV. The cleavage of egg fragments without the egg nucleus. J. Exp. ZooL 67, 159-215.
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FANKHAUSER, G. (1934b). Cytological studies on egg fragments of the salamander Triton. V. Chromosome number and chromosome individuality in the cleavage mitoses of merogonic fragments. J. Exp. Zool. 67, 349-393. FANKHAUSER, G. (1948). The organization of the amphibian egg during fertilization and cleavage. Ann. N. Y. Acad. ScL 49, 684-708. FANKHAUSER,G., and MOORE, C. (1941a). Cytological and experimental studies of polyspermy in the newt, Tritrus viridescens. I. Normal fertilization. J. MorphoL 68, 347-385. FANKHAUSER,G., and MOORE, C. (1941b). Cytological and experimental studies of polyspermy in the newt, Tritrus viridescens. II. The behavior of the sperm nuclei in androgenetic eggs. J. MorphoL 68, 387-423. GAUTIER,J. (1987). The role of the germinal vesicle for the appearance of maturation-promoting factor activity in the axolotl oocytes. Dev. BioL 123, 483-486. GAUTIER, J., NORBURY, C., LOHKA, M., NURSE, P., and MALLER, J. (1988). Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2 +. Cell 54, 433-439. GERHART, J., WU, M., and KIRSCHNER, M. (1984). Cell cycle dynamics of an M-phase-specific cytoplasmic factor in Xenopus laevis oocytes and eggs. J. Cell BioL 98, 1247-1255. IMOH, H., and MIYAZAKI,Y. (1984). Distribution of the germinal vesicle material during progesterone-induced oocyte maturation in Xenopus and Cynops. Dev. Growth Differ. 26, 157-165. IWAO, Y., YAMASAKI,H., and KATAGIRI, C. (1985). Experiments pertaining to the suppression of accessory sperm in fertilized newt eggs. Dev. Growth Differ. 27, 323-331. JAFFE, L. A., and GOULD, M. (1985). Polyspermy-preventing mechanisms. In "Biology of Fertilization" (Metz, C. B. and Monroy, A., Eds), Vol. 3, pp. 223-250, Academic Press, San Diego. KARSENTI, E., BRAVO,E., and KIRSCHNER,M. (1987). Phosphorylation changes associated with the early cell cycle in Xenopus eggs. Dev. Biol. 119, 442-453. KATAGIRI, C., AND MORIYA,M. (1976). Spermatozoan responses to the toad egg matured after removal of germinal vesicle. Dev. BioL 50, 235-241. KAYLOR, C. T. (1937). Experiments on androgenesis in the newt, Tritutus virideseens. J. Exp. Zool. 76, 375-394. KURIYAMA,R. (1989). 225-Kilodalton phosphoprotein associated with mitotic centrosomes in sea urchin eggs. Cell Motil. Cytoskeleton 12, 90-103. LABBE, J. C., PICARD, A., KARSENTI, E., and DOREE, M. (1988). An M-phase-specific protein kinase ofXenopus oocytes: Partial purification and possible mechanism of its periodic activation. Dev. BioL 127, 157-169. LOHKA, M. J. (1989). Mitotic control by metaphase-promoting factor and cdc proteins. J. Cell Sci. 92, 131-135. LOHKA, M. J., and MAILER, J. L. (1985). Induction of nuclear membrane breakdown, chromosome condensation and spindle formation in cell-free extract. J. Cell Biol. 101,518-523. LOHKA, M. J., AND MASUI, Y., (1983). The germinal vesicle material required for sperm pronuclear formation is localized in the soluble fraction of egg cytoplasm. Exp. Cell Res. 148, 481-491. LOHKA, M. J., HAYES, M. K., and MALLER, J. L. (1988). Purification of maturation-promoting factor, an intracellular regulator of early mitotic events. Proc. NatL Acad. Sci. USA 85, 3009-3013. MASUI, Y. (1972), Distribution of the cytoplasmic activity inducing germinal vesicle breakdown in frog oocytes. J. Exp. ZooL 179, 365378. MASUI, Y., and MARKERT, C. L. (1971). Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. ZooL 177, 129-146. MASUI, Y., and SHIBUYA, E. K. (1987). Development of cytoplasmic
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activities that control chromosome cycles during maturation of amphibian. In "Molecular regulation of nuclear events in mitosis and meiosis" (Schlegel, R. A., Halleck, M. S., and Rao, P. N., Eds.), pp. 1-35. Academic Press, San Diego. MASUI, Y., MEYERHOF, P. G., and ZIEGLER, D. H. (1979). Control of chromosome behavior during progesterone induced maturation of amphibian oocytes. J. Steroid Biochem. 11, 715-722. MURRAY,A. W., and KIRSCHNER,M. W. (1989a). Dominoes and clocks: The union of two views of the cell cycle. Science 246, 614-621. MURRAY, A. W., and KIRSCHNER, M. W. (1989b). Cyclin synthesis drives the early embryonic cell cycle. Nature (London) 339, 275-280. MURRAY,A. W., SOLOMON,M. J., KIRSCHNER,M. W. (1989). The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature (London) 339, 280-286. OHSUMI, K., SHINAGAWA, A., AND KATAGIRI, C. (1986). Periodic changes in the rigidity of activated eggs depend on germinal vesicle materials. Dev. Biol. 118, 467-473. RAIBOWL, K., DRAETTA,G., BRIZUELA,L., VANDRE, D., and BEACH, D. (1989). The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells. Cell 57, 393-401.
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SAWAI, T. (1972). Role of cortical and subcortical components in cleavage furrow formation in amphibia. J. Cell Sc4 11,543-556. SAWAI, T., and HIGUCHI, K. (1989). Cytostatic effect of the cytoplasm of mature oocytes in the newt, Cynops pyrrhogaster. ZooL Sc4 6, 709-714. SKOBLINA, M. N., PIVNITSKY, K. K., and KONDRATIEVA,O. T. (1984). The role of germinal vesicle in maturation of Pleurodeles waltUi oocytes induced by steroids. Cell Differ. 14, 153-157. SMITH, L. D., AND ECKER, R. E. (1969). Role of the oocyte nucleus in physiological maturation in Rana pipiens. Dev. BioL 19, 281-309. STREET, J. C. (1940). Experiments on the organization of the unsegmented eggs of Triturus pyrrhogaster. J. Exp. ZooL 85, 383-408. VANDRE, D. D., DAVIS, F. M., RAO, P. N., and BORISY, G. G. (1984). Phosphoproteins are components of mitotic microtubule organizing centers. Proc. NatL Acad~ Sci. USA 81, 4439-4443. WAKIMOTO, B. T. (1979). DNA synthesis after polyspermic fertilization in the axolotl. J. Embryol. Exp. MorphoL 52, 39-48. YAMASAKI, H., and IWAO, Y. (1987). Behavior of "accessory" somatic and sperm nuclei introduced into physiologically polyspermic newt eggs. J. Exp. ZooL 234, 331-338.
Control of Sperm Nuclear Behavior in Physiologically Polyspermic Newt Eggs: Possible Involvement of MPF YASUHIRO IWAO1 AND RICHARD P. ELINSON*
Biological Institute, Faculty of Science, Yamaguchi University, Yamaguchi 753,Japan; and *Department of Zoology, University of Toronto, Toronto, Ontario M5S 1A1, Canada Accepted August 2, 1990 We have studied the mechanism controlling the behavior of accessory sperm nuclei in physiologically polyspermic eggs of the newt, Cynops pyrrhogaster. Our approach was to identify cytoplasmic components which would prevent the usual degeneration of accessory sperm nuclei. Injection of cytoplasm from unfertilized eggs, but not fertilized ones, induced multipolar cleavage in polyspermically fertilized eggs as well as centrosome separation and formation of extra bipolar spindles in accessory sperm nuclei. Cytosols extracted from unfertilized Cynops or Xenopus eggs also were active in inducing multipolar cleavage, as were germinal vesicle materials from oocytes of the frogs Xenopus or Rana or of Cynops. In all of these cases, the nuclear cycle as well as the onset of first cleavage was delayed relative to those in control eggs. In contrast, injection of an extract with maturation-promoting factor (MPF) activity, prepared from unfertilized Xenopus eggs, induced precocious and multipolar cleavage when injected into fertilized Cynops eggs. Injection of the MPF-containing extract caused acceleration of the nuclear cycle as well as formation of extra bipolar spindles by the accessory sperm nuclei. These results suggest that a local deficiency of MPF may lead to the degeneration of accessory sperm nuclei in physiologically polyspermic eggs. ©1990AcademicPress,Inc. INTRODUCTION Most animals exhibit m o n o s p e r m y . In n o r m a l fertilization, only one s p e r m is i n c o r p o r a t e d into the egg and m o n o s p e r m y is e n s u r e d by several blocks to p o l y s p e r m y o p e r a t i n g before s p e r m - e g g fusion (for a review, Jaffe and Gould, 1985). However, some a n i m a l s exhibit physiological polyspermy. Since several s p e r m e n t e r an egg in n o r m a l fertilization, a p o l y s p e r m y block in the egg cytop l a s m is i m p o r t a n t for e n s u r i n g n o r m a l d e v e l o p m e n t in those species. Physiologically p o l y s p e r m i c eggs provide a s y s t e m to analyze the m e c h a n i s m s which control cent r o s o m e s e p a r a t i o n and bipolar spindle f o r m a t i o n . A unique m e c h a n i s m to p r e v e n t p o l y s p e r m y has been r e p o r t e d in u r o d e l e a m p h i b i a n s ( F a n k h a u s e r , 1932; F a n k h a u s e r and Moore, 1941a; W a k i m o t o , 1979; Street, 1940; Iwao et al., 1985; Y a m a s a k i and Iwao, 1987). Two to t w e n t y s p e r m nuclei e n t e r t h e egg c y t o p l a s m , f o r m s p e r m pronuclei, u n d e r g o D N A synthesis (Wakimoto, 1979; Iwao et al., 1985), and develop asters. However, a f t e r f o r m a t i o n of a zygote nucleus between one s p e r m pronucleus and the egg pronucleus, all of the o t h e r accessory s p e r m nuclei become pycnotic and d e g e n e r a t e before first cleavage. Thus, in the s a m e egg cytoplasm, the zygote nucleus behaves quite differently f r o m the accessory s p e r m nuclei.
1To whom correspondence should be addressed.
Two h y p o t h e s e s were proposed to explain these phen o m e n a ( B a t a i l l o n a n d T c h o u Su, 1930; F a n k h a u s e r , 1948; reviewed by Elinson, 1986). F a n k h a u s e r proposed t h a t an i n h i b i t o r y activity e m a n a t e s f r o m the zygote nucleus. This activity s u p p r e s s e s accessory s p e r m nuclei and p r e v e n t s t h e m f r o m p a r t i c i p a t i n g in cleavage a n d development. In c o n t r a s t , Bataillon and Tchou Su suggested t h a t active c y t o p l a s m n e c e s s a r y for mitosis is c o n c e n t r a t e d a r o u n d the zygote nucleus, so t h a t the zygote nucleus can e n t e r mitosis, but accessory s p e r m nuclei c a n n o t . T h e a c c e s s o r y s p e r m nuclei a r e o u t of s y n c h r o n y with metabolic c h a n g e s linked to the zygotic cell cycle, and this a s y n c h r o n y leads to nuclear degeneration of the accessory s p e r m nuclei. Few e x p e r i m e n t s have been m a d e to d i s t i n g u i s h bet w e e n t h e s e h y p o t h e s e s . We h a v e c o n f i r m e d s o m e of F a n k h a u s e r ' s results, including the survival of accessory s p e r m nuclei in a n d r o m e r o g o n i c egg f r a g m e n t s or in p a r t i a l l y c o n s t r i c t e d egg halves (Iwao et al., 1985). While these results s u p p o r t F a n k h a u s e r ' s h y p o t h e s i s , t h e y do not prove it (Elinson, 1986). In addition, we have r e c e n t l y f o u n d t h a t nuclear d e g e n e r a t i o n in Cynops eggs is specific for h o m o l o g o u s s p e r m nuclei. N e i t h e r homologous s o m a t i c nuclei, s p e r m nuclei of the m o n o s p e r m i c s a l a m a n d e r Hynobius, nor s p e r m nuclei of the toad B u f o d e g e n e r a t e in Cynops egg c y t o p l a s m ( Y a m a s a k i a n d Iwao, 1987). I t is not known, however, w h a t kinds of c y t o p l a s m i c activities u n d e r l i e the c o n t r o l of n u c l e a r d e g e n e r a t i o n in fertilized n e w t eggs. 301
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Several cytoplasmic factors are known which regulate nuclear events occurring during oocyte maturation and early cleavage in amphibians (for a review, Masui and Shibuya, 1987). These include m a t u r a t i o n - p r o m o t i n g factor (MPF) which induces nuclear membrane breakdown, chromosome condensation and spindle assembly, and cytostatic factor (CSF) which arrests the cell cycle at metaphase. Recent work on the control of cell division has demonstrated t h a t MPF is a dominant and universal regulatory factor for entering M-phase (for reviews, Dunphy and Newport, 1988; Lohka, 1989; Murray and Kirschner, 1989a), and MPF activity is regulated by cyclin in the early embryonic cell cycle (Murray and Kirschner, 1989b; Murray et al., 1989). In this study, we report that several different cytoplasmic fractions can rescue accessory sperm nuclei, allowing them to form bipolar spindles and induce extra cleavage furrows. Use of fractions derived from Xenopus eggs suggests t h a t MPF may be involved in the survival of these nuclei. MATERIALS AND METHODS Sexually m a t u r e newts, Cynops pyrrhogaster, were collected near Yamaguchi in Japan during hibernation season. Leopard frogs, Rana pipiens, were purchased from dealers in Vermont at the start of hibernation and stored at 4°C until use. Xenopus laevis were purchased from dealers and maintained in our laboratories. To obtain unfertilized Cynops eggs, ovulation was induced by two injections each of 100 IU of human chorionic gonadotropin (HCG, Teikoku Zoki; Tokyo or Sigma) at intervals of 48 hr at 23°C. The mature eggs were obtained from the lowest portion of the oviducts by squeezing the female or by dissection. Ovulation in Xenopus was induced by injection of HCG (500 IU/female) at 18°C. In vitro maturation of defolliculated Rana oocytes was induced by exposure to 10 #g/ml progesterone in De Boer's solution (in mM; 110.3 NaC1, 1.3 KC1, 1.3 CaC12, 5.7 Tris-HC1, pH 7.4) at 18°C. To perform artificial insemination, Cynops sperm suspension was prepared by macerating the vas deferens in 20% Steinberg's solution (in mM; 11.6 NaC1, 0.13 KC1, 0.068 Ca(NOB)2, 0.17 MgSO4, and 0.92 Tris-HCI, pH 7.4). The unfertilized eggs were placed in a dry petri dish (5 cm in diameter), to which about 1 mt of the sperm suspension (1-5 × 105 cells/ml) was added. Fifteen minutes later, 20% Steinberg's solution was added to the dish. After removing their jelly coats with 2.5% Na-thioglycolate, pH 9.0, 30 min after fertilization, the fertilized eggs were allowed to develop in 20% Steinberg's solution at 23 _+ 0.5°C. To transfer egg cytoplasm, unfertilized or fertilized Cynops eggs were centrifuged on 10% Percoll in 20%
VOLUME142, 1990
Steinberg's solution at 950g for 10 min at 4°C. The unbroken eggs were stratified into a lipid layer, clear cytoplasm, pigment, and yolk granules. Fifty nanoliters of clear cytoplasm was injected into each recipient egg with a glass micropipet (30 ~m in tip diameter), and the injected eggs were incubated in Ca2+-rich 100% Steinberg's solution (3.4 mM Ca(NOB)2) for 30 min to enhance wound healing, followed by culturing in 20% Steinberg's solution. All injections were made into the animal hemisphere of the eggs which has a diameter of about 2.2 mm. With sham injections of 50 nl of 1% Nile blue sulfate, the dye spread through § of the animal hemisphere within 1 hr and over the whole animal hemisphere by 1.5 hr. To inject germinal vesicles (GVs), full-grown oocytes obtained from ovaries of mature Rana, Xenopus, or Cynops females were pricked at the animal pole with a fine glass pipet, and their GVs were squeezed out into extraction buffer by gently pressing the equatorial region of the oocyte with forceps (in mM; 250 sucrose, 100 KC1, 30 MgSO4, 25 Tris-HC1, pH 7.4). In some cases, aliquots of a concentrated GV extract were made by first drawing up a small volume of paraffin oil, then about 50 GVs, followed by another small volume of paraffin oil into a capillary tube. The orifice of the capillary tube was smaller than a GV, and this caused disruption of the GV membrane and release of its contents. The end of the tube was heat sealed and the tube was centrifuged at 600g for 10 min. Each fertilized egg was injected with 50 nl of GV extract, approximately equivalent to one GV/egg. ~ To extract cytoplasm from unfertilized eggs, Cynops eggs were manually dejellied in DeBoer's solution, or Xenopus eggs were de jellied with 45 mM2-mercaptoethanol in Ca-free Barth's solution (in mM," 88 NaC1, 1.0 KC1, 2.4 NaHCOa, 1.5 EDTA, and 7.5 Tris-HC1, pH 8.5), followed by thorough washing with Ca-free Barth's solution. The eggs were washed three times with the GVextraction buffer and packed in an Eppendorf tube. After removing excess buffer by light centrifugation (4.5g, 10 min), the eggs were centrifuged at 15,000g for 20 min at 4°C. Fifty nanoliters of slightly red cytosol was injected into each fertilized Cynops egg. A crude preparation of maturation-promoting factor was a generous gift from Dr. M. J. Lohka (University of Colorado, School of Medicine) and was prepared from unfertilized Xenopus eggs according to the methods of Lohka et al. (1988). The 250,000g supernatant was precipitated with 0-34% or 35-80% saturated ammonium sulfate, resuspended in approximately one-third or onefourth the original volume, respectively, and then dialyzed against dialysis buffer (in mM; 100 Na fl-glycerophosphate, 15 MgC12, 1.0 dithiothreitoI, 0.1 phenylmethyl sulfonylfluoride, and 20 Hepes-NaOH,
Iwno ANDELINSON SpermNuclear Behavior pH 7.5). Samples were stored at -70°C. Fifty nanoliters of the MPF fractions was injected into each fertilized Cynops egg after dilution with the GV-extraction buffer just before injection. MPF activity was determined either by injection of 50 nl of extract into defolliculated Rana oocytes to observe germinal vesicle breakdown (GVBD) 10 hr after injection at 18°C, or by adding 50 pl of the extract to the cell-free assay system to observe nuclear envelope breakdown (NEBD) and chromosome condensation of decondensed sperm nuclei (cf., Lohka and Maller, 1985). For cytological observations, eggs were fixed in Smith's solution for 24 hr and embedded in paraffin. The 12-tLm-thick serial sections were stained with Feulgen's reagent and fast green, and were observed at 400× magnification. RESULTS Since normal fertilization of Cynops is physiologically polyspermic, 2-10 sperm were incorporated into each egg fertilized by artificial insemination. Sperm entered the eggs randomly and uniformly as judged by the distribution of sperm entrance spots. Each sperm nucleus moved into the egg cytoplasm about 200 #m deep from the surface, but only one sperm pronucleus (the principal sperm nucleus) reached the center of the egg to contact the egg pronucleus and make a zygote nucleus. Progress of nuclear events in fertilized Cynops eggs is summarized in Table 1. All sperm nuclei underwent chromatin decondensation to form sperm pronuclei with well-developed asters and DNA synthesis (Iwao et al., 1985) with the same chronological schedule. However, when the zygote nucleus underwent chromosome condensation, the accessory sperm nuclei became pycnotic and degenerated before first cleavage. In some accessory sperm nuclei, chromosomes were associated with small monopolar spindles, but they never formed bipolar spindles. The degenerating sperm nuclei moved to the equatorial region or the vegetal hemisphere around the time of cleavage.
Cytoplasm of Unfertilized Cynops Eggs Induced Multipolar Cleavage and Rescue of Accessory Sperm Nuclei Initially, we asked whether Cynops eggs had any activities in their cytoplasm which would promote or prevent nuclear degeneration of accessory sperm nuclei. This was tested by various types of cytoplasmic transfer between unfertilized and fertilized eggs. Cytoplasm taken directly from eggs had little effect on cleavage, so each donor egg was stratified by centrifugation before sucking out their cytoplasm. When clear cytoplasm obtained from the unfertilized eggs was injected into eggs 1-2 hr
303
after fertilization (G1), most eggs cleaved normally (Table 2). In contrast, when this cytoplasm was injected into eggs just after contact of sperm and egg nuclei (4-5 hr, G2), multipolar division occurred in 56% of the eggs at first cleavage within 9 hr after fertilization. (For ease of communication, we refer to the period 4-5 hr after fertilization as G2, even though the end of S-phase has not been strictly defined. See Table 1.) The onset of multipolar cleavage was always delayed about 1-2 hr in comparison with t h a t of bipolar cleavage. Cytoplasm obtained from fertilized eggs in G 1 or M had little activity to induce multipolar cleavage, so t h a t most recipient eggs cleaved normally and on schedule. These results demonstrate t h a t cytoplasm of unfertilized eggs has a strong activity to induce multipolar cleavage, but t h a t activity disappears after fertilization. To determine whether the induced multipolar cleavage was caused by the survival of the accessory sperm nuclei, nuclear behavior in eggs t h a t had been injected at G2 with unfertilized egg cytoplasm was examined with cytological sections. Surviving accessory sperm nuclei, including swollen pronuclei or accessory bipolar spindles with a haploid set of metaphase chromosomes, were found in 7 out of 10 eggs examined at 7-8 hr after fertilization (Fig. 1). In one egg, for example, there was a metaphase plate of the zygote nucleus with a bipolar spindle in the center of the egg (Fig. 1A), four swollen accessory sperm pronuclei (Fig. 1B), three accessory smaller bipolar spindles with haploid sets of chromosomes (Fig. 1C), and a pycnotic accessory sperm nucleus (Fig. 1D). These accessory sperm nuclei and spindles were found in the equatorial region or vegetal hemisphere of the egg. The different stages of sperm nuclei in the injected eggs might be due to the distribution of injected materials, caused by diffusion after injection. Some delay (1-2 hr) was observed in the progress of nuclear events in both zygote and accessory nuclei. The number of eggs with undegenerated accessory sperm nuclei and the delay of nuclear events corresponded with the delayed multipolar cleavages observed in living eggs. We did not detect undegenerating accessory sperm nuclei in the eggs t h a t had been injected with egg cytoplasm, but which cleaved in a normal, bipolar way. We conclude t h a t accessory sperm nuclei were rescued by the cytoplasmic injection and induced extra furrow formation.
Germinal Vesicle Materials of Cynops or Xenopus Oocytes Induced Multipolar Cleavage Since contents of the germinal vesicie are spread out in the cytoplasm during oocyte maturation, the effects of GV contents on the degeneration of accessory sperm nuclei were examined. When a whole Cynops GV was
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TABLE 1 TIMECOURSEOFNUCLEAREVENTSIN FERTILIZEDEGGSORIN EGGSINJECTEDWITHMPF FRACTION MPF fraction-injected eggs Time (hours after fertilization) 1
Cell cycle phase Telo II, GI
2
Normalized time 0.15 0.31
2-3
S
4
"G2''a
(4-4.25) 4.5
0.31-0.46 0.62
0.77
5.5
0.85
6.5 (6-7.5)
M
0.85-0.92
1.0 (0.92-1.15)
Zygote nucleus
Accessory sperm nuclei
Second polar body emission Pronuclei with decondensed chromatin. No differences between sperm nuclei with respect to decondensation and aster formation DNA synthesis starts in all nuclei (Iwao et al., 1985) Contact of sperm and egg pronuclei to produce zygote nucleus
(0.62-0.66) 0.69
5
5.5-6
Normal development
Separation of centrosomes associated with zygote nucleus
(MPF fraction injected) Centrosome separation Pronuclei with monoaster or one centrosome Bipolar metaphase Some centrosome spindle separation Cleavage beginning decondensing cleavage nuclei
Zygote nucleus: Chromosome condensation and formation of a bipolar spindle Accessory sperm nuclei: Lack bipolar spindle and pycnotic. Occasional small monopolar spindle 50% of eggs cleaved (range of cleavage time)
Cleavage nuclei
Some bipolar spindles; other nuclei pycnotic with one centrosome Mixture of cleavage nuclei, bipolar spindles, pycnotic nuclei
a The exact time for the start of G2 is not known, since the end of S phase has not been determined.
injected into a Cynops fertilized egg at G 2, 70% of the eggs u n d e r w e n t m u l t i p o l a r division at first cleavage (Table 3). Whole Xenopus GV or m a t e r i a l s e x t r a c t e d f r o m Rana GVs also induced m u l t i p o l a r cleavage (Fig. 2A and Table 3). The onset of cleavage was delayed 1-2 hr, so t h a t cleavage b e g a n 7.5 h r a f t e r fertilization and 55% of the eggs (24/44) u n d e r w e n t first cleavage by 8.5 h r a f t e r fertilization. I n c o n t r a s t , n e i t h e r cytosol e x t r a c t e d f r o m enucleated Xenopus oocytes nor c y t o p l a s m of enucleated Rana oocytes affected cleavage. The n u c l e a r cycle w a s also d e l a y e d 1-2 hr, so t h a t m o s t of the nuclei in the injected eggs r e m a i n e d in G2 with decondensed c h r o m a t i n enclosed by n u c l e a r enve-
lopes (Fig. 2B). F o r m a t i o n of b i p o l a r s p i n d l e s a n d f u r r o w i n g f r o m accessory s p e r m nuclei were confirmed with cytological sections. In 10 eggs which h a d been injected w i t h Rana GV materials, zygote nuclei f o r m e d a p a i r of c l e a v a g e nuclei (80%) or b i p o l a r m e t a p h a s e plates (20%), while the accessory s p e r m nuclei exhibited a v a r i e t y of behaviors at 8 h r a f t e r fertilization. The 37 accessory s p e r m nuclei f o r m e d a bipolar spindle (14%), s e p a r a t e d c e n t r o s o m e s (5%) (Fig. 2C), a pair of small cleavage nuclei associated with (14%) (Fig. 2D) or without (38%) f u r r o w f o r m a t i o n , pronuclei with one centrosome (14%), or pycnotic c h r o m a t i n (15%). These results d e m o n s t r a t e t h a t injected GV m a t e r i a l s delay the nu-
IWAOANDELINSON TABLE 2 TRANSFER OF CYTOPLASM AMONG UNFERTILIZED AND FERTILIZED CYNOPS EGGS No. of b
Donor egg~ Unfertilized (Meta II) Unfertilized (Meta II) G1 G2 M
Recipientegg
eggs
% multipolar cleavagec
--) G1
34
3
--~ G2 --~ Gz --~ G2 --~ G1
68 20 20 17
56 10 5 12
a Clear cytoplasm 50 nl from a stratified egg was injected into each recipient egg. Times after fertilization used were: G1 (1-2 hr), G2 (4-5 hr), M (5-6 hr). bBased on experiments using 10 different females. Cleavage delay of 1-2 hr.
clear cycle by delaying e n t r y into M-phase and lead to bipolar spindle f o r m a t i o n in accessory s p e r m nuclei.
Cytosol Extracted from Unfertilized Cynops or Xenopus Eggs Induced Multipolar Cleavage To c h a r a c t e r i z e the p r o p e r t i e s of the m u l t i p o l a r cleavage-inducing activity, we tried to isolate active cytosol
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305
f r o m unfertilized eggs. W h e n cytosol (15,000g s u p e r n a t a n t ) o b t a i n e d f r o m unfertilized Cynops eggs was injected into eggs at G2, 76% of the eggs u n d e r w e n t multipolar division at first cleavage (Table 3). N e i t h e r ext r a c t i o n buffer n o r a s i m i l a r c y t o s o l o b t a i n e d f r o m fertilized Cynops eggs affected cleavage, a n d h e a t i n g (60°C, 15 min) the cytosol i n a c t i v a t e d it c o m p l e t e l y (Table 3). As with injected clear c y t o p l a s m or GV m a t e r i a l s , the onset of m u l t i p o l a r cleavage was delayed 1-2 h r in all of t h e i n j e c t e d eggs, a n d e x t r a b i p o l a r s p i n d l e s f o r m e d f r o m accessory s p e r m nuclei. The presence of m u l t i p o l a r cleavage a c t i v i t y in cytosol f r o m u n f e r t i l ized, but not fertilized eggs (Table 3) c o r r e l a t e d with the presence of activity in clear c y t o p l a s m f r o m unfertilized, b u t not fertilized eggs (Table 2). Cytosol o b t a i n e d f r o m unfertilized Xenopus eggs also h a d a s t r o n g activity to induce m u l t i p o l a r cleavage (Table 3). Since Xenopus is an a n u r a n , its fertilization is m o n o s p e r m i c , and the egg n o r m a l l y does not have to deal with accessory s p e r m nuclei. This suggests t h a t the activity is a g e n e r a l cellular one, not specifically r e l a t e d to physiological polyspermy. To d e t e r m i n e the ionic r e q u i r e m e n t s for isolation of cytosol c o n t a i n i n g the m u l t i p o l a r cleavage-inducing activity, we e x a m i n e d effects of divalent cations in the e x t r a c t i o n buffer (Table 4). As the c o n c e n t r a t i o n of Ca 2+
FIG. 1. Nuclei in a Cynops egg that had been injected at G2 with cytoplasm of an unfertilized Cynopsegg. (A) A bipolar spindle of a zygote nucleus with metaphase chromosomes. (B) A swelling accessory sperm nucleus. (C) An extra bipolar spindle of an accessory sperm nucleus with a haploid set of chromosomes. (D) A pycnotic accessory sperm nucleus at 7 hr after fertilization. Scale line: 10 ttm.
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DEVELOPMENTALBIOLOGY TABLE 3 INDUCTION OF MULTIPOLARCLEAVAGEBY INJECTION
Injected materials a A. Germinal vesicles (GV) Cynops GV Xenopus GV Rana GV extract (600g, supernatant) Cytoplasm (15,000g, supernatant) from enucleated Xenopus oocytes Cytoplasm from enucleated Rana oocytes (uncentrifuged) Extraction buffer No injection B. Cytosols Unfertilized Cynops eggs Unfertilized Cynops eggs (60°C, 15 min) Fertilized Cynops eggs Unfertilized Xenopus eggs Extraction buffer No injection
No. of eggsb
% multipolar cleavage c
75 17
71 65
20
95
19
0
20 20 20
0 0 0
51
76
17 20 17 26 20
0 0 76 0 0
a Fifty nanoliters or 1 GV injected at Gz (4-5 hr after fertilization). b Based on experiments using 21 different females. c Cleavage delay of 1-2 hr.
in the buffer was increased, recovery of the activity in the cytosol was reduced, so t h a t no activity was obtained with buffer containing 20 mM Ca2+. In contrast, Mg z+ was important for retaining the activity in the cytosol, because no activity was detected with the MgZ+-free buffer in which MgS04 was replaced by NaC1. These results suggest t h a t this activity requires Mg 2+ but is sensitive to Caz+ ions.
Extracts with M P F Activity Induced Precocious and Multipolar Cleavage The absence of multipolar cleavage-inducing activity in fertilized eggs and its ionic dependence suggested a relationship with m a t u r a t i o n - p r o m o t i n g factor, a known cytoplasmic regulator of nuclear behavior whose levels are high in unfertilized amphibian eggs, but decrease at fertilization. As a first step, we asked whether a fraction containing MPF activity extracted from unfertilized Xenopus eggs could induce multipolar cleavage. The first fraction tested was a 0-34% saturated ammonium sulfate precipitate of a 250,000g supernatant. When the undiluted MPF fraction was injected into Cynops eggs at G2, none of the 35 injected eggs cleaved in the 20 hr after injection. Abortive cortical wrinkles were observed in some eggs (7/35) at 6-8 hr after fertilization, but true cleavage furrows were never
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formed. Condensed chromosomes were scattered in the cytoplasm t h a t contained a disordered fibrous network resembling astral or spindle fibers. In contrast, diluted MPF fractions induced precocious and multipolar cleavage (Figs. 3A and 4A). When a 1:20 dilution of the MPF fraction was injected at G2 (4-4.5 hr after fertilization), 90% of the eggs cleaved 30-60 min earlier than eggs injected with a 1:20 dilution of the dialysis buffer (Fig. 5A). Three quarters of the eggs underwent multipolar division at first cleavage (Figs. 3A and 4A). In most cases, a main furrow appeared first in the center of the animal hemisphere, and then, 30-60 min later, additional furrows formed eccentrically on the animal hemisphere. The eggs developed to abnormal blastulae, but arrested before gastrulation. An injection of a 1:10 dilution of the MPF fraction at Gz also induced precocious and multipolar cleavage on all injected eggs (Fig. 4A). The activity to induce either precocious or multipolar cleavage decreased as the MPF fraction was diluted further (Fig. 4A), so t h a t these activities could not be detected after dilution to 1/160. The MPF activity of our fraction, measured by GVBD in Rana full-grown oocytes, corresponded well with the activities inducing both precocious and multipolar cleavage (Fig. 4A). Furthermore, the MPF activity measured by NEBD in the cell-free system was detected at least after dilution to 1/16. When a fraction precipitated by 35-80% s a t u r a t e d ammonium sulfate containing only a small amount of MPF activity (Lohka et al., 1988) was injected at Gz, the undiluted fraction caused precocious and multipolar cleavage in the Cynops eggs, but these activities could not be detected after dilution to 1/20 (Fig. 4B). Loss of the activities corresponded well with t h a t of MPF activity measured by GVBD in Rana oocytes (Fig. 4B). MPF activity measured by NEBD in the cell-free system was detectable only in the undiluted fraction. These relationships between the activities suggest t h a t MPF is a candidate for inducing precocious and multipolar cleavage in injected Cynops eggs. The acceleration of the onset of cleavage by the MPF fraction is shown in detail in Fig. 5. When the 1:20 dilution of the MPF fraction was injected 3 or 4 hr after fertilization, the onset of cleavage was accelerated 1.5-2 hr or 0.5-1 hr, respectively (Fig. 5A). The injection at 5 hr did not accelerate the onset of cleavage, but induced multipolar cleavage in 95% (19/20) of the eggs. It should be noted t h a t the furrows in the eggs t h a t had been injected with the MPF fraction 3 hr after fertilization formed more eccentrically and simultaneously, so t h a t a precocious main furrow was not formed. This may be due to a failure of the sperm and egg nuclei to join together in the zygote. When the 1:20 dilution of the MPF fraction was injected into the eggs 2 hr after fertiliza-
IWAO AND ELINSON
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307
FIG. 2. Effects of injection of Rana GV materials into fertilized (G2) Cynops eggs. (A) Multipolar cleavage 9 hr after fertilization. (B) Decondensed egg and principal sperm nuclei forming a zygote nucleus 7 hr after fertilization. (C) An accessory sperm nucleus (N) undergoing centrosome separation (c) 8 hr after fertilization. (D) A pair of small cleavage nuclei (CN) derived from an undegenerated accessory sperm nucleus just under an extra cleavage furrow (F) 9 hr after fertilization (tangential section). Scale line: (A) 1 mm; (B-D) 10 ttm.
tion, increased acceleration did not occur (Fig. 5B). Twelve out of 20 (60%) injected eggs underwent multipolar cleavage before 6 hr after fertilization, but the rest of the eggs (40%) cleaved bipolarly on schedule between 6.5-7.5 hr after fertilization. Furthermore, injection of the 1:10 dilution of the MPF fraction 2 hr after fertilization did not cause any cleavage before 4 hr after fertilization (Fig. 5B). In those eggs, all of the sperm nuclei remained as pronuclei until 3 hr after fertilization, but their centrosomes were unclear. Some accessory sperm nuclei underwent centrosome separation around 3.5 hr after fertilization, and then most sperm
TABLE 4 EFFECT OF DIVALENT CATIONSIN EXTRACTIONBUFFER ON INDUCING CLEAVAGE No. ofb
% multipolar
MgS04
Ions in extraction buffera CaC12
NaC1 (mM)
eggs
cleavage
30 30 30 0
0 10 20 0
0 0 0 30
51 68 25 29
76 26 0 0
Extraction medium also contained 250 m M Sucrose, 100 mM KC1, and 25 m M Tris-HC1 (pH 7.4). Based on experiments using 12 different females.
nuclei formed bipolar spindles 4-4.5 hr after fertilization. It should be mentioned that centrosome separation normally does not occur before 3.5 hr, the time for completion of DNA synthesis (Iwao et al., 1985).
Extracts with M P F Activity Induced Precocious Formation of Bipolar Spindles and Chromosomes in Accessory Sperm Nuclei The MPF fraction t h a t resulted in multipolar cleavage also caused accelerated bipolar spindle formation in the accessory sperm nuclei. Five Cynops eggs, injected with the 1:10 dilution of the MPF fraction 4-4.25 hr after fertilization (G2), were fixed at 0.5, 1.0, 1.5, or 2.0 hr after injection to observe nuclear events (Table 1). The zygote nuclei underwent centrosome separation 0.5 hr after injection, while all accessory sperm nuclei stayed as pronuclei with a monoaster or one centrosome. The zygote nuclei formed metaphase chromosomes incorporated on bipolar spindles in the center of the injected eggs 1 hr after injection (Fig. 3B), about I hr earlier than in noninjected eggs. At the same time, some accessory sperm nuclei in the equatorial region or vegetal hemisphere underwent centrosome separation (Fig. 3C). At 1.5 hr after injection, cleavage furrowing began, and each anaphase zygote nucleus was separated into two swelling cleavage nuclei. Some of the accessory sperm nuclei
308
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FIG. 3. Effects of an injected MPF fraction (1:10 dilution of 0-34% saturated ammonium sulfate fraction, 4-4.25 hr after fertilization) on cleavage and nuclear behavior in fertilized Cynops eggs. (A) Multipolar cleavage 7 hr after fertilization. (B) A bipolar spindle of a zygote nucleus. (C) An accessory sperm nucleus undergoing centrosome separation (c) 1 hr after injection. (D) An extra spindle of an accessory sperm nucleus 1.5 hr after injection. Scale line: (A) 1 mm; (B-D) 10 ~m.
w e r e p y c n o t i c , b u t m a n y o f t h e o t h e r s f o r m e d e x t r a bip o l a r s p i n d l e s ( F i g . 3D). A t 2.0 h r a f t e r i n j e c t i o n , all t h e zygote nuclei f o r m e d swelling cleavage nuclei, while the accessory s p e r m nuclei e x h i b i t e d a v a r i e t y of behaviors.
T h e 38 n u c l e i f o r m e d a p a i r of c l e a v a g e n u c l e i ( 2 9 % ) , a m e t a p h a s e p l a t e on a b i p o l a r s p i n d l e ( 1 0 % ) , a p r o n u cleus with two centrosomes (3%) or with one centros o m e ( 8 % ) , o r p y c n o t i c n u c l e i (50%). T h e s e o b s e r v a t i o n s
A. 0-34% Fraction
100
B. 35-80% Fraction
100
TT __
&&
50
_.
_
~
50
_
= -~ JL "_:=~
0
0 1/10
1/20
1/40 1/80 Di l u t i o n
1/180
1/1
1/10 1/20 Di [ u t i o n
1/40
" 1/80
FIG. 4. MPF fractions induce precocious and multipolar furrowing in fertilized Cynops eggs. Twenty eggs at G2 (4-4.5 hr after fertilization) were injected with 50 nl of each dilution of the MPF fraction, and scored for precocious (-- • --) and multipolar (-- • --) cleavage. Cleavage was considered precocious if it occurred before 6 hr, when non-injected eggs never cleaved. The dilutions of the MPF fraction were also assayed for their activity to induce germinal vesicle breakdown (GVBD) in full-grown Rana oocytes (-- © --). In (A), the MPF fraction was a 0-34% saturated ammonium sulfate fraction, while in (B), a 35-80% cut was used. The former has MPF activity, as assayed in the cell-free system, at a dilution of 1/16, while the latter only had activity when undiluted.
IWAO AND ELINSON
309
Sperm Nuclear Behavior
100
100
/ --o--,,20 ,r.ct,oo ,, -......
J
TI2o fr,°t~oo 15 h,~ ~r 1/20 b o . o , I
--o--
f /7/ I F? I I// a=l
(2 hr)
1/10 fracfion
- - o - - 1/20 fraction
(2 hr)
--I--
1/10 f r a c f i o n
(4 hrJ
--o--
]/10
--a--
None
buffer
50
! iI
D
0e4Z
t !
, ,
I
I
0 3 hr
t. after
5 6 7 fertilization
8
3
hr
I
T
I
4 after
T
I
T
/® T
I
I
5 6 7 fertilization
I
8
FIG. 5. Acceleration of the onset of cleavage by the injection of an MPF fraction precipitated by 0-34% saturated ammonium sulfate. (A) Fifty nanoliters of a 1:20 dilution of the MPF fraction was injected into each of 20 eggs 3 hr ( - - • --), 4 hr ( - - O --), and 5 hr ( - - • - - ) after fertilization. The injections at 4 hr are equivalent to those in Fig. 4. Precocious cleavage of eggs injected with the MPF fraction is shown as a function of time in comparison with cleavage in the eggs injected with a 1:20 dilution of buffer ( - - - O - - - ) or non-injected eggs ( - - - • - - - ) . (B) Fifty nanoliters of a 1:10 dilution of the MPF fraction was injected into each of 20 eggs 2 hr ( - - O - - ) or 4 hr ( - - • --) after fertilization. Fifty nanoliters of a 1:20 dilution of the MPF fraction was injected 2 hr after fertilization ( - - • --). Precocious cleavage of eggs injected with the MPF fraction is shown as a function of time in comparison with cleavage in the eggs injected with a 1:10 dilution of buffer ( - - - O - - - ) or non-injected eggs ( - - - • - - - ) .
demonstrate that 42% of the accessory sperm nuclei underwent centrosome separation and mitosis in response to the injection of the MPF fraction. Although the nuclear events in the MPF-injected eggs were greatly accelerated, the progress of the zygote nucleus was more advanced than t h a t of the accessory sperm nuclei. The difference of acceleration in the nuclear cycle corresponded well with the timing of multipolar furrowing, mentioned previously in which the formation of a main furrow in the center was followed by extra furrows at the periphery.
DISCUSSION
Rescue of Accessory Sperm Nuclei with M P F Containing Fractions We have demonstrated t h a t various cytoplasmic extracts can rescue accessory sperm nuclei from degeneration in polyspermic Cynops eggs. These extracts include clear cytoplasm or cytosol from unfertilized eggs, GV contents, and fractions with MPF activity from Xenopus eggs. While rescue includes centrosome separation, formation of bipolar spindles with metaphase chromosomes, and induction of furrowing, the different extracts may not be acting in the same way. Clear cytoplasm, cytosol, and GV contents all delayed the onset of cleavage, while MPF fractions accelerated it. As discussed later, the delay itself may permit the accessory sperm nuclei to proceed through the cell cycle.
Rescuing activity in a Xenopus egg extract was associated with MPF activity, assayed both by oocyte maturation in vivo and by nuclear envelope breakdown and chromosome condensation in vitro. Since the MPF fraction injected was crude, the factor responsible for centrosome separation and formation of bipolar spindles may not be MPF itself, but some other activity which copurifies with MPF. However, several recent reports suggest t h a t MPF can act on the centrosome. The p34 cdc2 protein kinase, which is homologous to a component of MPF (Gautier et al., 1988; Dunphy et al., 1988), is accumulated in centrosomes of mammalian cultured cells during M-phase (Raibowl et al., 1989). MPF activity in Xenopus egg cytoplasm appears at 0.65 normalized time (NT) and reaches a maximum at 0.80 NT (Gerhart et al., 1984) encompassing the time of centrosome separation (0.77 NT). In addition, some proteins of the centrosome are highly phosphorylated during M-phase in sea urchin eggs (Kuriyama, 1989) and in mammalian cultured cells (Vandre et al., 1984). Hyperphosphorylation occurs in fertilized Xenopus eggs during M-phase (Karsenti et al., 1987) due to M-phase-specific protein kinase(s) which probably include MPF protein kinase activity (Lohka et al., 1988; Labbe et al., 1988). These observations suggest t h a t MPF plays a role in centrosome separation, directly or indirectly, by participating in protein kinase cascades in the egg cytoplasm during M-phase. To account for a role of MPF in the control of nuclear behavior of physiologically polyspermic newt eggs, we propose t h a t a higher concentration of active MPF in
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the center of the fertilized egg leads to the separation of the centrosome in the zygote nucleus, but its lower concentration in the periphery is not enough to induce normal mitosis in the accessory sperm nuclei.
Hypotheses on Control of Nuclear Behavior in Polyspermic Newt Eggs Two hypotheses were proposed in the past to explain the mechanism of suppression of accessory sperm nuclei. Fankhauser (1948) proposed an inhibitory factor emanating from the zygote nucleus which caused the degeneration of the accessory sperm nuclei, and this hypothesis was supported by extensive work on the development of egg fragments. Against Fankhauser's idea, Bataillon and Tchou Su (1930) proposed t h a t there is a limited amount of active cytoplasm in the animal hemisphere of the egg. The large aster of the principal sperm nucleus occupies the cytoplasmic-rich center of the animal hemisphere and may collect active cytoplasm. This active cytoplasm promotes entry into mitosis of the zygote nucleus earlier t h a n the accessory sperm nuclei which are in a poorer cytoplasmic area at the periphery. Some aspect of egg metabolism, such as O2 consumption or C02 production, is linked to the zygote nucleus, and it changes at mitosis. The accessory sperm nuclei are out of synchrony with the new metabolism, so they degenerate. Our results fit well with Bataillon and Tchou Su's hypothesis, since we could rescue accessory sperm nuclei by supplying extra cytoplasmic components by injection. We would revise Bataillon and Tchou Su's hypothesis by substituting "the ability to activate MPF" for "active cytoplasm." Instead of egg metabolism (02, C02), we hypothesize t h a t the MPF cycle is linked to the zygotic nucleus. Although our experiments did not detect the inactivator, MPF inactivation has been demonstrated in Xenopus eggs (Gerhart et al., 1984). The accessory sperm nuclei would be out of synchrony with the wave of MPF inactivation, and this a s y n c h r o n y may cause their degeneration. Our results and hypothesis raise a number of questions to be tested in the future. For instance, there should be an unequal distribution of MPF activity in the newt egg cytoplasm, with a higher concentration in the center. At least in frog oocytes, such an unequal distribution can exist (Masui, 1972). A second question is w h e t h e r mistimed exposure to MPF inactivation can cause nuclear degeneration or whether additional factors are involved. The possibility of more complex regulatory interactions is suggested by our present observations on the heterogeneity of nuclear response to injected factors, and by the fact t h a t degeneration
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depends on the type of nucleus (Yamasaki and Iwao, 1987).
Acceleration of Cleavage with MPF-Containing Fractions An interesting result obtained by injection of the MPF fraction was the acceleration of nuclear cycles and precocious cleavage in fertilized newt eggs. In the normal course of Cynops development, the egg cortex is determined to make furrows about 40 min before first cleavage in response to a signal transmitted from subcortical cytoplasm (Sawai, 1972). Our results show t h a t the cortex can acquire competence to make furrows at least 3.5 hr before the first cleavage. Since all of the nuclear events after centrosome separation occurred early, the MPF fraction seems to trigger a cascade of reactions t h a t had been ready by early G2. Injection of the MPF fraction either 2 or 3 hr after fertilization accelerated cleavage to the same extent, and centrosome separation was not detected before 3.5 hr after fertilization. Since nuclei undergo DNA synthesis 2-3 hr after fertilization (Iwao et al., 1985), the results imply t h a t the MPF fraction can accelerate events of the cell cycle, which occur after S-phase but not before.
Delay of Cleavage and Rescue of Accessory Sperm Nuclei with Other Cytoplasmic Fractions Except for the injection of the MPF fraction, all rescues of accessory sperm nuclei both in our experiments and in previous ones (Elinson, 1986) were correlated with a delay in the cell cycle. The early injection (2-3 hr) of MPF also elicited multipolar cleavage, while the injection of cytoplasm obtained from unfertilized eggs into the G1 eggs had little effect. Perhaps a greater stability or a stronger activity of the MPF fraction could account for this difference. The detailed action of the MPF fraction as well as t h a t of extracted cytosol and GV materials should be further examined during the early embryonic cell cycle. Our injections of GV materials and of cytoplasm or cytosol from unfertilized eggs caused delayed, multipolar cleavage. Similar results were obtained with andromerogonic egg f r a g m e n t s (Fankhauser, 1934a,b; F a n k h a u s e r and Moore, 1941b; Iwao et al., 1985), eggs with the female nucleus removed by suction (Kaylor, 1937; Iwao et al., 1985), and cycloheximide-treated eggs (Iwao et al., 1985). These observations suggest t h a t prolonging the cell cycle allows centrosome separation or bipolar spindle formation in the accessory sperm nuclei. To fit this in with our MPF hypothesis, we would say t h a t in the absence of mitosis of a dominant zygote nucleus, MPF inactivation is not triggered. This either permits a rise in MPF activity at the periphery or allows
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more time for the accessory sperm nuclei to interact with a low level of MPF. In either case, the accessory sperm nuclei would undergo centrosome separation and enter mitosis on a delayed schedule. We do not know whether rescue by injected GV materials or unfertilized egg cytoplasm is effective simply because they delay the cell cycle or because they contain a cell cycle regulator, normally involved in the control of sperm nuclear behavior. There are numerous reports showing that the GV contains factors involved in the cell cycle (Smith and Ecker, 1969; Katagiri and Moriya, 1976; Masui et al., 1979; Lohka and Masui, 1983, Skoblina et al., 1984; Ohsumi et al., 1986; Gautier, 1987), and Imoh and Miyazaki (1984) noted the restricted distribution of GV materials in the center of the animal hemisphere in Cynops eggs. Similarly, unfertilized, but not fertilized, egg cytoplasm had MPF and CSF, which are involved in cell cycle regulation (Masui and Markert, 1971; Sawai and Higuchi, 1989). Further analysis of the active components of the GV and unfertilized egg cytoplasm will be necessary to determine how they rescue accessory sperm nuclei and induce multipolar furrowing. Through this study, we have tried to elucidate how the nuclear behavior is regulated in physiologically polyspermic newt eggs based on current views of cell cycle regulation. Although the precise mechanisms have not been clarified, the physiologically polyspermic newt egg provides one of the best systems to analyze molecular mechanisms of centrosome replication and separation during mitosis. We thank Dr. Manfred J. Lohka, University of Colorado School of Medicine, for his courteous gift of the MPF fractions and for performing the in vitro assay on them, Dr. Christian Aimar for participating in initial attempts at these experiments, and Dr. Yoshio Masui for stimulating discussions. This work was supported by a JSPS-NSERC Bilateral Exchange Grant to Y.I., a NSERC Operating Grant to R.P.E., and a grant-in-aid for Scientific Research from the Japanese Ministry of Education, Science and Culture to Y.I. REFERENCES BATAILLON, E., and TCHOU Su (1930). Etudes analytiques et experimentales sur les rytbmes cinetiques dane l'oeuf. Arch. BioL 40, 439540. DUNPHY, W. G., and NEWPORT,J. (1988). Unraveling of mitotic control mechanisms. Cell 55, 925-928. DUNPHY, W. G., BRIZUELA,L., BEACH, D , and NEWPORT,J. (1988). The Xenopus homologue of cdc2 is a component of MPF, a cytoplasmic regulator of mitosis. Cell 54, 423-431. ELINSON, R. P. (1986). Fertilization in amphibians: The ancestry of the block to polyspermy. Int. Rev. Cytol. 101, 59-100. FANKHAUSER, G. (1932). Cytological studies on egg fragments of the salamander Triton. II. The history of supernumerary sperm nuclei in normal fertilization and cleavage of fragments containing the egg nucleus. J. Exp. Zoo£ 62, 185-235. FANKHAUSER,G. (1934a). Cytological studies on egg fragments of the salamander Triton. IV. The cleavage of egg fragments without the egg nucleus. J. Exp. ZooL 67, 159-215.
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