Development 112, 471-476 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

471

Developmental profile of glucose phosphate isomerase allozymes in

parthenogenetic and tetraploid mouse embryos

ULRICH PETZOLDT

Department of Biology, Philipps-University of Marburg, FOB 1229, D-3550 Marburg, Germany

Summary

Glucose phosphate isomerase (GPI) allozymes werecompared in eggs and embryos of the mouse strainsC57BL/6-JHan (GPI-1BB) and 129/Sv (GPI-1AA)under different experimental conditions. The quantitat-ive differences in eggs of the two strains disappeared bythe blastocyst stage at day 4 to 5, both in fertilized anddiploid parthenogenetic embryos. The degree of degra-dation of oocyte-coded enzyme molecules and theactivation of the embryonic genome for GPI appeared tobe equivalent in parthenogenetic embryos from hetero-

zygous females when only one or other maternal alleletype remained in the egg after meiosis. Also in tetraploidembryos, generated by electrofusion of homozygousfertilized eggs from the two strains, both genomesseemed to be activated at the same time at day 4; here,however, the GPI-1BB allozyme remained predominantup to day 6.

Key words: mouse embryos, parthenogenesis,electrofusion, tetraploidy, glucose phosphate isomerase.

Introduction

Early mammalian development is directed by pre-formed maternal molecules synthesized duringoogenesis and by gene products appearing afteractivation of the embryonic genome (for review seeJohnson et al. 1984). Particular attention has been givento the developmental profile of the enzyme glucosephosphate isomerase (GPI) in the mouse. Functionalenzyme molecules are synthesized during oogenesis andare present after ovulation (Brinster, 1973; Buehr andMcLaren, 1985). This material is used and degradedduring preimplantation development (Brinster, 1973),but can still be detected in late blastocysts and earlyimplanting embryos (West and Green, 1983; Dubouleand Burki, 1985; Gilbert and Solter, 1985; West et al.1986; West and Flockhart, 1989a; Petzoldt, 1990). It ishowever, not clear whether, in addition, mRNA forGPI from oogenesis is present and translated afterfertilization (Gilbert and Solter, 1985).

Embryo-coded GPI was first detected between the8-cell stage (Brinster, 1973) and the morula stage (Westand Green, 1983; Duboule and Burki, 1985) whencrossing mouse strains homozygous for different GPIallozymes (Chapman et al. 1971). The total amount ofactive GPI molecules, however, decreases up to the lateblastocyst stage (Brinster, 1973; West et al. 1986).

The activity of genes for GPI during mouse oogenesisis controlled by a cw-acting temporal gene (Petersonand Wong, 1978; McLaren and Buehr, 1981). Thisregulation varies considerably in different mouse strainsresulting in varying amounts of active GPI molecules in

ovulated eggs obtained from these strains (Peterson etal. 1985). When the embryonic genome is activated,however, these quantitative GPI differences largelydisappear by the early egg cylinder stage (West andFlockhart, 1989a) and are not found in somatic cells(Peterson and Wong, 1978).

It was the aim of the present study to compare theloss of oocyte-coded GPI and the increase of embryo-coded GPI in embryos with different levels of oocyte-coded enzyme. For this purpose, we used normal andparthenogenetic embryos from the two mouse strainsC57BL/6-JHan (Gpi-lsb/b) and 129/Sv {Gpi-ls"1")which synthesize defined but different quantities of theenzyme during oogenesis (Peterson et al. 1985). Theactivity of the enzyme was either quantitativelydetermined during preimplantation development or therelative amount of the allozymes was compared by co-electrophoresing eggs and embryos of the same agefrom both strains in the same sample. To compare theloss of maternal molecules and the activation of thedifferent embryonic alleles under similar cellularconditions, either eggs from heterozygous females(129/Sv xC57BL/6-JHan) F! were used after partheno-genetic activation or homozygous fertilized eggs fromboth strains were combined by electrofusion tech-niques.

Materials and methods

Mouse strains, collection and culture of eggsMice of the strains 129/Sv {Gpi-ls"1") and C57BL/6-JHan

472 U. Petzoldt

(Gpi-lsb/b) and heterozygous females (129/SvxC57BL/6-JHan)F! (Gpi-ls"^) were used for the experiments. To obtainfertilized eggs, females were mated in the afternoon andexamined for vaginal plugs the following morning (=day 1 ofgestation).

For fusion experiments, eggs were collected at day 1,released from the cumulus masses by treatment withhyaluronidase (Sigma, St. Louis, MO, USA; 150i.u. mP1)and washed 3 times with Hepes-buffered mouse embryoculture medium M2 (Fulton and Whittingham, 1978).Cleaving embryos, morulae and blastocysts were flushed fromthe oviduct or uterus. To remove the zona pellucida, eggs andembryos were treated with pronase (Calbiochem, La Jolla,CA, USA; S^mgml"1) in M2 with polyvinylpyrrolidone(PVP) but without bovine serum albumin (BSA). Afterseveral washings in M2, the eggs were directly treated foranalysis or transferred into mouse embryo culture mediumM16 (Whittingham, 1971) in droplets under paraffin oil(Fisher Sci., Fair Lawn, NJ, USA or British Drug Houses,Poole, UK) and cultured at 37°C in an atmosphere of 5%CO2 in air or 5% CO2, 5% O2 and 90% N2. For in vitroattachment, morulae and blastocysts were transferred todroplets of Eagle's minimum essential medium (MEM, FlowLaboratories, UK) with 20% fetal calf serum (Monk andAnsell, 1976).

Parthenogenetic activationFemales were superovulated by an intraperitoneai injection of5 or 7.5i.u. pregnant mare serum gonadotropin (PMSG,Intergonan, Vemie, Kempen, Germany) and 5 i.u. of humanchorionic gonadotropin (hCG, Ekluton, Vemie) administeredabout 48 h later. Unfertilized eggs were recovered 16 to 17 hpost hCG and parthenogenetically activated according toCuthbertson (1983) using an incubation in M2 with 7%absolute ethanol (Roth, Karlsruhe; Germany) for a period of7min. Then eggs were cultured in M16 containing 5figml"'cytochalasin B (Aldrich, Milwaukee, Ml; USA or Sigma).After removal of the cumulus cells, they were examined forthe presence of pronuclei. Only eggs with two pronuclei wereselected and further cultured in M16 up to the blastocyststage.

ElectrofusionAfter removal of the zona pellucida, single eggs of the strains129/Sv and C57BL/6-JHan were transferred as pairs todroplets of M16 with phythaemagglutinin (PHA, Sigma;lO^gml"1) and stored at 37°C for several minutes (Mintz etal. 1973). When they were well agglutinated they were washedthree times in M2 and put into a simple fusion chamber(produced in the institute's workshop) between two platinumwires (200 fan diameter) with a fixed distance apart of 160 fan.Fusion was performed in M2 without BSA with square pulsesgenerated from a dual impedance research stimulator(Harvard; Edenbridge, Kent, UK) connected to an oscillo-scope (Rhode and Schwarz, Munich, Germany). Two pulsesof lkV cm"1 intensity, a duration of 0.3 ms each and with aninterval of about 1 s, were used (Kubiak and Tarkowski, 1985;Kurischko and Berg, 1986; Ozil and Modlinski, 1986). Thefused eggs were either directly frozen for analysis or culturedup to day 6.

Enzyme analysisFor GPI analysis, eggs and embryos were washed in M2without BSA and stored frozen in micropipettes at -20 to— 70°C. For photometric determinations only eggs andembryos without zona pellucida were used and stored notlonger than 4 days at -70°C (Peterson et al. 1985). After a

triple cycle of freezing and thawing, GPI activity was assayedaccording to Peterson et al. (1985) but using reagents fromBoehringer, Mannheim, Germany; Merck, Darmstadt, Ger-many and Sigma. The reaction was performed in a Kontron,Uvicon 860 (Kontron Instruments, Zurich, Switzerland) at35 °C and monitored at 340 nm over a period of 30min. Tocalculate the background, each cuvette was pre-run with thereaction mixture but without the sample for 30min underidentical conditions. These values were calculated against acontrol cuvette containing only the reaction mixture in bothdeterminations.

For electrophoresis, the samples were disrupted by freezingand thawing and directly applied to cellulose acetate plates(Titan III, Helena, Beaumont, TX, USA). After allozymeseparation and staining according to Eppig et al. (1977), gelswere scanned on a Laser Densitometer Ultrascan XL (LKB,Bromma, Sweden). Peaks were cut from the paper, andquantitative estimates were obtained by weight determi-nations.

Results

For quantitative GPI analysis, eggs and embryos of thestrains C57BL/6-JHan (GPI-1BB) and 129/Sv (GPI-1AA) were obtained from the maternal genital tract atdenned times. At day 1, both fertilized and/orunfertilized eggs were used in all experiments. Bothstrains showed developmental variations at day 3 and 4,but differences between the strains were not consideredto be significant. Peterson et al. (1985) found that GPIactivity was higher in C57BL/6 eggs than in 129/Sveggs. Fig. 1 shows that this difference remained duringcleavage up to the afternoon of day 3 but disappeared atday 4. During this period the total activity of both typesof embryo decreased significantly (day 1, 9.00 to day 4,9.00: GPI-1AA: P=0.01, GPI-1BB: P=

GPI allozymes in early mouse embryos 473

Aext.

0.03-

0.02-

0.01-

day 19.00

day 2 day 39.00 17.00

day 49.00 17.00

Fig. 1. Quantitativedeterminations of GPI activityin eggs and embryos from themouse strains C57BL/6-JHan(solid line) and 129/Sv (brokenline). Activity is given in Aextinction by one egg orembryo per hour. Embryonicstages: day l = l-cell eggs(fertilized or unfertilized); day2=2-cell embryos; day 3,9.00=4- to 8-cell embryos andbeginning compaction; day 3,17.00=4-cell stage tocompaction; day 4,9.00=morulae and blastocysts;day 4, 17.00= morulae andblastocysts. Bars give thestandard deviation; numbers inbrackets=number of samplesper stage.

embryos between day 3 and 4 (Fig. 4). A considerablebackground of oocyte-coded GPI molecules is stillvisible at day 5 (Fig. 3 and 4).

When one-cell eggs of both strains were combined byelectrofusion to form one tetraploid egg, both diploidhomozygous genomes were united under equivalentcellular conditions. These eggs were capable of cleav-age and reached the early blastocyst stage by day 4 andstarted to attach to the culture dish at day 6. Using

70 '

60-

50-

40-

30

(9)

day 1 day 2 day 3 day 4 day 5

Fig. 2. Co-electrophoresis of single eggs and diploidparthenogenetic embryos from the strains C57BL/6-JHan(GPI-1BB, solid line) and 129/Sv (GPI-1AA, broken line).GPI activity of the allozymes is given in % of the totalactivity after densitometric evaluation of the gels.Embryonic stages: day l=l-cell unfertilized eggs; day 2=2-cell embryos; day 3=4- to 8-cell embryos; day4=compaction to morula; day 5=(expanded) blastocysts.Bars give the standard deviation; numbers inbrackets=number of samples per stage.

t •

#Fig. 3. GPI gels and densitometer curves from diploidparthenogenetic embryos after activation of eggs fromheterozygous females (129/SvxC57BL/6-JHan)F1. a andsolid line=2-cell embryo, day 2 showing oocyte-coded GPI;b and broken line=blastocyst, day 5, expressing theGpi-lal" genes; c and dotted line=blastocyst, day 5,expressing the Gpi-lblb genes.

474 U. Petzoldt

80 H

70 -

GO

50 •

30 -

20 -

10

*

day 2 day 3 day U day 5 day 2 day 3 day£ day 5 day 2 day 3 day£ day 5

GPI-1AA GPI-1AB GPI-1BB

Fig. 4. Electrophoresis of single parthenogenetic embryos after activation of eggs from heterozygous females (129/SvXCSTBL/o-JHan)?!. GPI activity for the 3 different allozymes is given in % of total activity after densitometric evaluationof the gels. Number of samples: day 2=9; day 3=18; day 4=29; day 5=10.

Table 1. GPI activity in tetraploid eggs and embryos generated by electrofusion of homozygous fertilizedC57BL/6-JHan eggs with homozygous fertilized 129/Sv eggs

Age

Day 1Day 2Day 3Day 4

Day 5

Day 6

* GPI-1AB peak!

Stage

1-cell2-cell

4- to 5-cellMorula toblastocyst

(Expanded)blastocystExpandedblastocyst,beginning

attachment

No. ofembryos(samples)

6(6)4(4)6(6)

17(5)

3(3)

5(5)

% GPI-1AA % GPI-1AB

44.6±6.144.2±8.444.0±7.241.3±7.0 7.0±l.l*

30.9±7.2 22.2±5.6

20.1±6.1 42.6±4.4

> were barely visible, electropherograms were approximately evaluated.t Calculated as GPI-lAA+GPI-lAB/2 to GPI-lBB+GPI-lAB/2 in embryos of days 4 to 6.

% GPI-1BB

55.4±6.155.8±8.456.0+7.251.7±6.1

46.9+4.3

37.3±9.2

Ratio of GPImonomers

A:Bt44.6:55.444.2:55.844.0:56.044.8:55.2

42.0:58.0

41.4:58.6

electrophoresis, the first signs of the heteromere GPI- decreased in tetraploid fusion embryos from the one-1AB band were visible at day 4, although the results are cell stage to blastocyst at day 5 (data not shown).at the limit of densitometrical evaluation (Table 1).They became stronger at day 5 and 6. Up to day 4 thequantitative correlation between GPI-1AA and GPI- DiscussionIBB remained unchanged, but at day 5 and 6 thisrelation was slightly shifted in favour of GPI-1BB The results of our study largely confirm data obtained(Table 1). As in diploid controls, total GPI activity by other groups but using different experimental

procedures. When taking the quantitative GPI determi-nations from Peterson et al. (1985), the correlationbetween the activity of 1.92 in oocytes from the strainC57BL/6-J (GPI-IBB) to that of 1.54 in oocytes fromthe strain 129/Sv (GPI-1AA) is 55.5 % to 44.5 %. Thisis very close to our correlations determined afterelectrophoresis (57.8% to 42.2% in Fig. 2 and 55.4%to 44.6 % in Table 1) as well as to the correlation of Aextinction per egg in Fig. 1 (0.0305 to 0.0243=55.7 % to44.3%). This agreement additionally supports theprocedure of West et al. (1989) who normalized datafrom electrophoresis by using the standard values fromPeterson et al. (1985).

The GPI activity during subsequent in vivo develop-ment remained at approximately the level of therespective egg up to compaction at day 3 in both strainsand declined at the morula to blastocyst stage at day 4.This was also shown by Brinster (1973) and West et al.(1986). By that time, however, the total GPI activitywas almost equivalent in both strains, earlier thanfound by West and Flockhart (1989a). This similar levelof activity at the blastocyst stage was also apparent afterco-electrophoresis of diploid parthenogenetic eggs andembryos from the two strains, though with some delayafter in vitro culture. Obviously, degradation of oocyte-coded GPI and activation of the embryonic genome isregulated in parthenogenetic embryos without a pa-ternal genome in a similar way to that in fertilized eggs.

West and Flockhart (1989a) compared the GPIallozyme relations in heterozygous embryos withdifferent allele combinations and different amounts ofoocyte-coded enzyme present. In all cases, the totalGPI activity was quite similar at day 5. This wasachieved by a relatively slower degradation of oocyte-coded material and a higher activity of the embryonicgenome in embryos with a low level of oocyte GPI. Theembryonic gene activity, nevertheless, was initiatedabout the same time. These results might be addition-ally influenced by different GPI dimer stabilities of theallozyme combinations used (West and Flockhart,19896).

Our data from parthenogenetic embryos using eggsfrom heterozygous females with one or the othermaternal allele remaining in diploid form confirm thissimilar timing of initiation of embryonic gene activity.This is more clearly shown after fusion of homozygousfertilized eggs from the two strains. The GPI-1AB bandappeared at day 4, when the ratio of both oocyte-codedallozymes is still the same as after ovulation. The ratioremained, however, in favour of GPI-IBB until the lateblastocyst stage with attachment beginning at day 6.This is in contrast to our own results comparingallozyme ratios between homozygous diploid embryosfrom the two strains (Fig. 1 and 2), but agrees withobservations of West et al. (1986), who crossedequivalent mouse strains and analysed the allozymeratio in heterozygous diploid embryos. They found amonomer ratio between GPI-1A and GPI-1B of46.5%:53.5% in day 7 egg cylinders grown in vivo, astage almost all GPI was embryo-coded. Therefore, itmight be possible, that in heterozygous embryos from

GPI allozymes in early mouse embryos 475

these two strains the Gpi-lsb allele is slightly strongerexpressed than the Gpi-1 f allele. This might be thecase also in our heterozygous tetraploid embryos.

In conclusion, our experiments show that in homo-zygous diploid normal and parthenogenetic embryosthe differences in total GPI activity between the strains129/Sv (GPI-1AA) and C57BL/6-JHan (GPI-IBB)disappeared by the blastocyst stage at day 4 to 5. Theembryonic genome was activated in diploid partheno-genetic as well as in tetraploid heterozygous embryos atday 4. In the tetraploid embryos generated by fusion ofeggs from the two strains, GPI-IBB activity remainedpredominant up to day 6. This might be caused by ahigher expression of the Gpi-lsb gene in heterozygousembryos, although we can not exclude differentdegradation rates of the oocyte-coded molecules.

I wish to thank Ms H. Luft for excellent technical assistanceand Ms D. Georg for help with GPI electrophoresis. Theresearch was supported by the Deutsche Forschungsgemein-schaft. I am grateful to one of the referees, Dr J. D. West, forconstructive criticism of the manuscript.

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{Accepted 13 March 1991)