Anat Embryo1 (1990) 182:503-507

Anatomy and Embryology 9 Springer-Verlag1990

In vitro survival of cells derived from isodiploid uniparental half embryos of the mouse after aggregation with normal embryos T. K o c h - A l b r e c h t and U . P e t z o l d t Fachbereich Biologic der Philipps-Universitfit Marburg, Postfach 1929, D-3550 Marburg/Lahn, Federal Republic of Germany Accepted July 31, 1990

Summary. Fertilized mouse eggs, heterozygous for two

allozymes of glucose phosphate isomerase (GPI) were bisected, and the resulting half eggs were diploidized with cytochalasin B. After separate aggregation with normal embryos carrying a third allozyme of GPI, the resulting chimaeras were kept in culture up to 10 days. The majority grew out on the culture dish during this period. By GPI analysis, 7.7% of the embryos were found to be chimaeric. Both types of uniparental cells, from gynogenetic and from androgenetic half eggs, were capable of surviving in chimaeras in vitro. These results are comparable with published data obtained by using uniparental embryos generated by micromanipulation. Key words: Mouse eggs - Bisection - Uniparental em-

bryos - Chimaeras - In vitro development

Introduction

The developmental capacity of uniparental mouse eggs has been analysed extensively in recent years (for review see Solter 1988; Surani et al. 1987). These embryos were mainly generated by microsurgical removal of one pronucleus from a fertilized or parthenogenetically activated egg and by either diploidizing the remaining pronucleus by cytochalasin treatment, or by reconstituting the egg with another male or female pronucleus obtained from a second egg. Such uniparental embryos were not capable of developing to term. At midgestation, gynogenetic embryos showed poor development of trophectodermderived tissues, whereas under androgenetic conditions mainly the growth of the embryo was affected. For the completion of normal development, both maternal and paternal genomic contributions appeared to be necessary (Solter 1988; Surani et al. 1987). By formation of chimaeras with normal embryos it was possible, however, to rescue cells of gynogenetic emOffprint requests to: U. Petzoldt

bryos up to term (Anderegg and Markert 1986; Otani et al. 1987), as it has also been shown for parthenogenetic embryos (Stevens 1978; Surani et al. 1977). Androgenetic cells became restricted to trophectoderm-derived tissues also in chimaeras, and did not show up in newborn mice (Surani et al. 1987, 1988). Another technique to generate uniparental eggs is the bisection of fertilized eggs. Andro- and gynogenetic haploid or isodiploid embryos were capable of cleaving, and some of reaching the blastocyst stage (Barton and Surani 1983; Tarkowski 1977). In the present study we examined the development of bisected, uniparental, half eggs over a longer period. Two days after diploidization, the half embryos were separately aggregated with normal embryos, and the development of the resulting chimaeras observed in vitro up to day 10. The survival of the uniparental part of the chimaeras was verified by using different allozymes of glucose phosphate isomerase (GPI) as markers. For this purpose, the egg for bisection was heterozygous for two GPI allozymes, and the normal embryo for chimaera formation carried a third allozyme.

Materials and methods Mice. The following mouse strains with different alleles for the enzyme GPI were used for the experiments: BALB/cJHan (Gpilsa/a), 129Sv (Gpi-lsa/"), NMRI (selected for Gpi-ls"/"), C57BL/ 6JHan (Gpi-lsb/b) and C57BL10 (Gpi-lsC/~).Females were normally

mated without hormone treatment, and checked for the presence of a vaginal plug the following morning (day 1 of pregnancy). Collection and culture of eggs and embryos. Fertilized eggs for bisection were collected on the morning of day 1, released from the cumulus masses by treatment with hyaluronidase (Sigma, St. Louis, MO, USA; 150 IU ml 1) and washed several times in Hepes-buffeted mouse embryo culture medium M2 (Fulton and Whittingham 1978). Then the zona pellucida was removed with pronase (Calbiochem, La Jolla, CA, USA; 3,6 mg ml-1) in M2 with polyvinylpyrrolidone but without bovine serum albumin (BSA). The eggs were washed in M2 with BSA, and transferred into the embryo culture medium M16 (Whittingham 1971). Two-cell embryos were flushed

504 from the oviduct at day 2, and the zona pellucida was removed as described above. Eggs and embryos were cultured in plastic dishes (Greiner, Niirtingen, FRG) in droplets of M16 under paraffin oil (Fisher Scientific, Fair Lawn, N.J., USA) at 37~ in an atmosphere of 5% CO2, 5% 02 and 90% N2. For in vitro attachment and outgrowth, the embryos were transferred to droplets of Eagle's minimum essential medium (MEM, Flwo Laboratories, UK) with 20% foetal calf serum (Monk and Anselt 1976) in Falcon plastic dishes (Becton Dickinson, Oxnard, CA, USA) and cultured up to day 10.

Bisection

~, [ A)

Day

of fertilized

pronuclearstage egg

Dipl0idization

Bisection of eggs. Bisection of fertilized eggs was performed around noon or in the afternoon of day 1, combining the methods of Tarkowski (1977) and Barton and Surani (1983) with slight modifications (Petzoldt and Muggleton-Harris 1987). The plane of bisection was chosen in such a way that one pronucleus was in each egg half (Fig. 1), generating a haploid androgenetic egg (containing the male pronucleus) and a haploid gynogenetic egg (containing the female pronucleus). For diploidization, the half eggs were treated overnight with cytochalasin B (5 gl/ml; Aldrich, Milwaukee, MI/USA). Then the eggs were cultured in normal medium (Fig. 1).

Gynogenetic embryo

Androgenetic

Normal

embryo

embryo

Day

Chimaera formation. To form aggregation chimaeras, the two selected embryos were placed in a single droplet of M16 under oil. They were gently pushed toegether (Hillman et al. 1972; Mintz 1971) and kept at 37~ C. They were observed twice more on the same day for successful aggregation, and thereafter once daily.

formation

Enzyme analysis. For analysis, single embryos were washed in M2 without BSA and stored frozen in micropipettes. After homogenization by freezing and thawing, they were applied directly to cellulose acetate plates (Titan III, Helena, Beaumont, TX, USA), and GPI allozymes were separated and stained for enzyme activity as described by Eppig et al. (1977). Day

6

Experimental design. Fig. I gives the general outline of the experiments. Fertilized eggs, heterozygous for two GPI allozymes were bisected at day 1, thus generating half-eggs with either the paternal pronucleus carrying one GPI alMe, or with the maternal pronucleus carrying the other GPI allele. After treatment with cytochalasin B, the embryos were isodiploid gynogenetic and androgenetic respectively. They cleaved in normal medium to the 3- to 4-cell stage at day 4. Embryos with lower and higher cell numbers were also used in these experiments. At day 4, the uniparental half embryos were aggregated with normal embryos homozygous for a third GPI allele. These embryos were one day younger but had reached the 2- to 8-cell stage after the same period. After chimaera formation, the embryos were transferred to MEM at day 6. They were collected at day 10 and analysed for their GPI allozyme patterns. If possible, egg cylinder and trophoblast were separated before analysis. It was thus possible to determine by GPI analysis whether the uniparental part of the chimaera was of gyno- or androgenetic origin.

Transfer to MEN

1 Day

1

10

GPI analysis

Fig. 1. Experimental scheme to form aggregation chimaeras between uniparental embryos from bisected half eggs and normal embryos. Fertilized eggs for bisection were heterozygous for 2 GPI allozymes, the normal embryos were homozygous for a third GPI allozyme, o, GPI allozyme in normal embryos; *, maternal type of GPI allozyme in heterozygous eggs, remaining in isodiploid form in gynogenetic embryos; ~, paternal type of GPI allozyme in heterozygous eggs, remaining in isodiploid form in androgenetic embryos

Results

U n i p a r e n t a l m o u s e e m b r y o s were g e n e r a t e d b y bisection o f fertilized eggs a n d s u b s e q u e n t t r e a t m e n t w i t h c y t o c h a lasin B. A f t e r this p r o c e d u r e , the eggs were i s o d i p l o i d a n d h a d the d o u b l e n u c l e o c y t o p l a s m i c r a t i o o f n o r m a l one-cell eggs. T h e i n h e r e n t i n a c c u r a c y o f m a n u a l bisect i o n resulted in half-eggs o f v a r y i n g size ( n o t determined). N e v e r t h e l e s s t h e y were c a p a b l e o f cleaving a n d r e a c h i n g the 3- to 4-cell stage at d a y 4 a n d s o m e o f t h e m h a d s t a r t e d to c o m p a c t . T h e r e was, h o w e v e r , considerable e m b r y o n i c loss f r o m b i s e c t i o n to cleavage (see also T a r k o w s k i 1977). In o r d e r to increase the n u m b e r

o f e x p e r i m e n t a l e m b r y o s , 1- to 2-cell u n i p a r e n t a l emb r y o s were also u s e d for c h i m a e r a f o r m a t i o n ( 3 0 - 3 5 % of aggregations). F o r c a l c u l a t i o n o f the survival rate o f the u n i p a r e n t a l e m b r y o s , the n u m b e r o f successful a g g r e g a t i o n s at d a y 4 was t a k e n as 100% (Table 1). O f these e m b r y o s , 97.1% survived u p to d a y 6, a n d u s u a l l y r e a c h e d the b l a s t o c y s t stage b y t h a t time. F r e q u e n t l y , after a g g r e g a t i o n a n d d u r i n g b l a s t o c y s t f o r m a t i o n , b l a s t o m e r e s were e x c l u d e d o r f r a g m e n t e d . A f t e r t r a n s f e r to M E M , 95.2% survived u p to d a y 9 or 10 (Table I) a n d grew o u t on the c u l t u r e

505

Fig. 2A-D. Chimaeras between uniparental and normal embryos, outgrowing in vitro (day 10). A Embryo with well developed egg cylinder, composed of cells from the normal embryo only (GPI electrophoresis in Fig. 3 B and C). B Embryo with discernible egg cylinder structures, consisting of gynogenetic and normal compo-

nents (GPI in Fig. 3F). C Embryo with some egg cylinder structures, consisting of gynogenetic and normal components (GPI in Fig. 3E). D Embryo with mainly trophoblast cells, consisting of androgenetic and normal components (GPI in Fig. 3 H). Arrows, egg cylinder (structures). Bar about 50 gm

Table 1. Chimaera formation between uniparental half embryos from bisected eggs and normal embryos Combination of GPI allozymes

Chimaera formation at day 4

Transfer to MEM at day 6 a

Outgrowth at day 10 b

Chimaeras at day 10 c

Bisected egg

Normal embryo

Total

Both half embryos

Total

Both half embryos

Total

Both half embryos

Total

BC or CB

AA AA CC

29

16

27

16

25

14

75

42

74

42

74

42

1 (gynogen.) 7 (4 x androgen., 3 • gynogen.)

58 (55.8%)

99 (95.2%)

56 (53.4%)

8 (7.7%)

AB

Total

104 (100%)

58 (55.8%) 101 (97.1%

Both half embryos

m

" In one series, transfer already at day 5 b 2 embryos were harvested at day 9 c 1 chimaera was harvested at day 9 dish. T h e e m b r y o s s h o w e d a c o n s i d e r a b l e v a r i a t i o n m the s t r u c t u r e o f egg cylinders (Fig. 2). A t o t a l o f eight ( 7 . 7 % ) o f the a g g r e g a t e d e m b r y o s was f o u n d to be c h i m a e r i c after o u t g r o w t h (Table 1), f o u r c o n t a i n i n g g y n o g e n e t i c a n d f o u r a n d r o g e n e t i c cells,

as revealed b y G P I e l e c t r o p h o r e s i s (Fig. 3). O n l y emb r y o s expressing two clearly d e f i n e d G P I b a n d s were c o n s i d e r e d as chimaeric. In six cases (e.g. Fig. 3 D , E, F, G), the n o r m a l p a r t was s t r o n g e r t h a n the u n i p a r e n t a l c o n t r i b u t i o n ; in two cases (e.g. Fig. 3 H) the t w o c o m p o -

506

Fig. 3. Electrophoreticseparation of GPI allozymesfrom chimaeras between uniparental and normal mouse embryos,grown in vitro (day 10). A, I, Control kidney homogenates; B, egg cylinderfrom an embryo composed only of cells from the normal partner (GPI1CC, see Fig. 2A); C, trophoblast from the embryo of slot B, D, chimaera between gynogenetic(GPI-IBB) and normal (GPIIAA) embryo, collected at day 9 (see Table 1); E, F, chimaeras between gynogenetic(GPI-1AA) and normal (GPI-1CC) embryos (see Fig. 2 B and C); G, chimaerabetween androgenetic(GPI-1BB) and normal (GPI-ICC) embryo; H, chimaerabetweenandrogenetic (GPI-1BB) and normal (GPI-1CC) embryo (see Fig. 2D). Composite picture from differentgels

nents were more or less equivalent. In one case of a chimaera containing androgenetic cells, the aggregated embryos started to grow out separately on the culture dish, but formed a kind of common structure by the end. In one experiment only the uniparental part survived (not shown). Even though in more than 50% of the cases the two aggregates derived from both halves of a bisected egg survived the full 10 days (Table 1), there were never two positive chimaeras derived from the same egg. In five cases of true chimaeras, the partner aggregates survived too but showed no uniparental cells. Isolation of egg cylinders and trophoblasts of 10-dayold embryos was possible in 20 cases (Fig. 3 B and C), but was never achieved from proven chimaeras. In one case of a chimaera, only the trophoblast was analysed and found to contain gynogenetic cells (not shown).

Discussion The method of bisection allowed the production of andro- and gynogenetic embryos from the same fertilized egg. This operation halved the total content of each resulting egg. If the half eggs remained haploid, the normal nucleocytoplasmic ratio was maintained, but if they were diploidized the ratio was twice that of a normal egg. Haploid uniparental half-eggs are known to cleave and develop up to the blastocyst stage, though with decreasing viability (Tarkowski and Rossant 1976; Tarkowski 1977). Isodiploid half eggs cleaved well, especially when they received more than half of the cytoplasm keeping them closer to the normal nucleocytoplasmic ratio (Barton and Surani 1983). In general, decreasing the nucleocytoplasmic ratio affects cleavage and development (McGrath and Solter 1986), whereas an approximate doubling after bisection of eggs did not change the onset of embryonic gene activ-

ity (Petzoldt and Muggleton-Harris 1987). Barton and Surani (1983) showed that diploid eggs with less than 50% of the cytoplasm, i.e. the nucleocytoplasmic ratio had at least doubled, cleaved well to the morula stage but blastocyst formation was grossly affected. Since manual halvings of eggs are rarely equal, this might explain why we never obtained positive chimaeras from both isodiploid halves of a bisected egg at day 10. Correspondingly, Tarkowski (1977) described survival of two haploid blastocysts from the same egg only in one case. It seems there is always a certain advantage for one of the two parts. Additionally, there is a different developmental potential of andro- and gynogenetic eggs. Only 50% of haploid or isodiploid androgenetic eggs are reported to survive early cleavage (Tarkowsky 1977), since the vital X-chromosome is lacking. In isodiploid androgenetic half-eggs the survival rate appears to be even lower. Barton and Surani (1983) did not get any blastocyst development, whereas 18% of their gynogenetic half-eggs reached this stage. In our experiments, chimaera formation worked well in rescuing cells from isodiploid androgenetic eggs up to day 10. From our studies we obtained no information as to whether in out-growing chimaeras the gynogenetic and the androgenetic cells were concentrated in the egg cylinder and in the trophoblast respectively. Recent experiments with transgenic markers have shown that in chimaeras between normal and uniparental embryos, the partheno- and androgenetic cells were equally well distributed in trophectoderm and inner cell mass of a blastocyst. Of the experimental blastocysts, 69% contained parthenogenetic and 33% androgenetic contributions (Thomson and Solter 1989). After implantation at day 8 to 9, gynogenetic cells were confined to inner cell massderived structures and androgenetic cells to trophectoderm-derived tissues (Thomson and Solter 1988). This also holds true for chimaeras containing parthenogenetic cells (Clarke et al. 1988; Thomson and Solter 1988). The percentage of positive chimaeras up to day 9 in vivo (Thomson and Solter 1988) is about the same we found in vitro. It is possible that in vitro development favours the survival of androgenetic cells. McGrath and Solter (1986) showed, that pure reconstructed androgenetic embryos grew out slower than gynogenetic embryos, but survived longer, probably due to their potential to support trophoblast development. In conclusion, cells derived from isodiploid androgenetic and gynogenetic embryos which were generated by the relatively simple technique of bisection were capable of suriviving in vitro up to 10 days, when they formed chimaeras with normal embryos. These results are comparable with published data obtained by using uniparental embryos formed by micromanipulations. It should be clarified, however, whether or not cells from bisected half eggs can also survive to later stages or even to term, when such chimaeras are transferred to foster mothers and develop under in vivo conditions. Acknowledgements. We wish to thank Ms Heike Luft for the drawings and typingthe manuscript. The work was supported by grants from the Deutsche Forschungsgemeinschaftto U.P.

507

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