The proportions of glucose phosphate isomerase (GPI-1) allozymes produced by early (Gpi-1sa/Gpi-1sb ♀ × Gpi-1sc/Gpi-1sc ♂)F1 mouse embryos were analysed by quantitative cellulose acetate electrophoresis. Technical controls showed that this system is extremely sensitive, quantitatively reproducible and quite accurate. Genetic controls established that the Gpi-1sa/Gpi-1sb mothers were homozygous for the Gpi-1tb temporal allele, that produces relatively high GPI-1 activity in the oocyte. The oocyte-coded enzyme lasted until about days post coitum (p.c.) or shortly thereafter. The maternally derived, embryonic Gpi-1s allele was expressed no earlier than the paternally derived allele. This was first expressed between and days p.c. In this cross, most of the transition from oocyte-coded to embryo-coded GPI-1 occurred between and days p.c.

Chapman, Whitten & Ruddle (1971) showed that the paternally derived allele of Gpi-1s, which codes for the dimeric enzyme glucose phosphate isomerase, GPI-1 (E.C. 5.3.1.9), was expressed in mouse embryos by the late blastocyst stage. By using groups of 500 embryos, Brinster (1973) was able to show that the paternally derived allele was expressed as early as the 8-cell stage at days post coitum (p.c.). These important observations provided some of the first evidence for expression of the embryonic genome in preimplantation mouse embryos.

In both experiments embryos were produced by crossing homozygous Gpi-1sa/Gpi-1sa females with homozygous Gpi-1sb/Gpi-1sb males. The onset of paternal gene expression was demonstrated by the observation of both the GPI-1B homodimer and the GPI-1AB heterodimer. This indicates synthesis of both paternally coded GPI-1B and maternally coded GPI-1A monomers at this time (assuming that GPI-1 A monomers are not recycled from GPI-1 A homodimers). Prior to this, the embryo produced only the GPI-1 A allozyme. It is likely that most, if not all, of this is synthesized in the diploid oocyte but some may be produced in the early embryo, either from oocyte messenger RNA or by the early transcription of the maternally derived, embryonic Gpi-1sa allele.

The discovery of a third allele of Gpi-1s by Padua, Bulfield & Peters (1978) provides an opportunity to examine the contributions of the oocyte-coded enzyme and both the maternally and paternally inherited forms of the embryo-coded enzyme. In this paper we report the results of an electrophoretic analysis of GPI-1 in embryos produced by crossing heterozygous Gpi-1sa/Gpi-1sbfemales to homozygous Gpi-1sc/Gpi-1sc males. Qualitative observations on the time of disappearance of the GPI-1 AB allozyme and the time of appearance of the GPI-1C allozyme respectively allow us to monitor the disappearance of the oocyte-coded enzyme and the onset of embryonic, paternally derived gene expression. In addition, the quantitative increase in the ratio of GPI-1 A: GPI-1AB allozymes indicates the onset of embryonic, maternally derived gene expression in Gpi-1sa/Gpi-1sc embryos and allows us to evaluate whether the maternal allele is expressed before the paternal allele.

(i) Mice

Two new partially congenic strains were produced by backcrossing mice, carrying different Gpi-1s alleles to the C57BL/O1a inbred strain for eight generations. (C57BL/Ola mice are homozygous for Gpi-1sb.) Mice carrying the Gpi-1sc allele were kindly supplied by Dr G. Bulfield and used to produce the C57BL/O1a-Gpi-1sc/Ws stock (abbreviated to B-Gpi-1sc). The C57BL/ Ola.l29-Gpi-1sa/Ws strain (abbreviated to B-Gpi-1sa) was derived from a 129 stock (129/Sv-CP/Pas-FukiVe) kindly provided by Dr V. E. Papaioannou. These two stocks and the inbred strains C57BL/O1a, 129/Sv-S1J-CP (abbreviated to B and 129 respectively) and DB A/2O1a were maintained at the Sir William Dunn School of Pathology. (The C57BL/O1a stock is also known as C57BL/6O1a. We use the former name because it now seems likely that the stock is derived from the British C57BL line rather than the American C57BL/6 line.)

(ii) Sample preparation

Unfertilized eggs and some preimplantation embryos were produced by induced ovulation following injections of 5i.u. pregnant mares’ serum gonadotrophin, PMS (Folligon, Intervet), followed 48 h later by 5 i.u. human chorionic gonadotrophin, HCG (Chorulon, Intervet). These injections were given at approximately 11a.m. The time of mating is taken to be 11 p.m., when ovulation is expected to begin. Most embryos were produced by natural matings that were timed from 1a.m., which is the middle of the dark period (7 p.m. to 7 a.m.) preceding the detection of the vaginal plug.

Unfertilized eggs and embryos were collected at approximately 10a,m. Cumulus cells were removed from the eggs in a solution containing 100 units of hyaluronidase (Sigma) per ml of phosphate-buffered saline, (PBS). Preimplantation embryos were flushed from the reproductive tract and postimplantation embryos ( days p.c.) were removed from decidual swellings under a dissecting microscope. Eggs and embryos were washed twice in PBS or 0·9 % saline and stored at −20 °C in less than 1 /A of PBS or saline, held between two small volumes of paraffin oil, in finely drawn-out Pasteur pipettes as recommended by Dr M. Buehr (personal communication). All samples were frozen and thawed three times in liquid nitrogen vapour before analysis.

Adult livers were homogenized in distilled water (3 mis per gram of tissue) using a hand-held glass homogenizer. After low-speed centrifugation the supernatant was stored at −20 °C and used undiluted for starch gel electrophoresis or diluted 1/80 with distilled water for cellulose acetate electrophoresis. Supernatants from mice homozygous for different Gpi-1s alleles were mixed in various proportions and the mixtures coded and stored at −20 °C prior to electrophoresis. These mixtures were used for various technical controls.

(iii) Electrophoresis and densitometry

Starch gels were prepared in rectangular (100×80×2mm) moulds from 12 % electrostarch and run with a pH 6·4 Tris-citrate buffer, essentially as described by Chapman, Whitten & Ruddle (1971). Bands of GPI were detected by staining an overlying nitrocellulose membrane, applied to the blotted surface of the inverted gel, as described by Peterson, Friar & Wong (1978). A 90 ×30 × 5 mm staining well was formed by sealing a rectangular perspex frame to the 100 × 40 mm overlay (cut from a 200 × 160 mm sheet of Sartorius SM11306) with petroleum jelly (Vaseline). Approximately 6ml of aqueous stain, prepared as described by Peterson et al. 1978 but with a different grade of glucose-6-phosphate dehydrogenase (Sigma Type XI) were used per well. Normally, twelve samples were stained per well.

Cellulose acetate electrophoresis was done on 76 × 60 mm Helena, Titan III electrophoresis plates, using the ‘pH 8·5’ Tris-glycine buffer (3·0 g Tris base plus 14·4 g glycine per litre) described by Eicher & Washburn (1978). (In our hands, this recipe produced a pH 8·1 buffer which we used without any pH adjustment.) After electrophoresis (1 h at 200 V) the plates were stained for 3 to 30 min in 62×78 mm staining dishes, using the same aqueous staining mixture that was used for starch gels. To avoid problems of overstaining, samples with widely differing GPI-1 activities were not run on the same plate. Both the cellulose acetate plates and nitrocellulose membranes were washed, fixed and dried after staining.

The proportions of the different GPI-1 allozymes were quantified by scanning densitometry using an absorbance maximum of 550nm. A Vitatron MPS, fitted with a densitometer unit, analogue integrator and chart recorder was used for the earlier experiments (control mixtures of liver homogenates run on starch gels) and a Gelman DCD-16 computing densitometer with automatic integration was used for the later experiments (all of the cellulose acetate plates). For densitometry with the Vitatron MPS system the nitrocellulose overlays were soaked in glacial acetic acid, methanol and water (1:1:3 by volume) and sandwiched between two glass plates. For densitometry with the Gelman DCD-16 both the nitrocellulose membranes and cellulose acetate plates were scanned dry and uncleared.

(iv) Calculations

Statistical tests were done on a Hewlett Packard 97 programmable calculator, programmed, by Mr D. G. Papworth of the M.R.C. Radiobiology Unit, for Student’s t-test and Smith & Satterthwaite’s variation of Welch & Aspin’s t-test for samples with different variances (Satterthwaite, 1946).

(i) Technical controls

The sensitivity of the two electrophoresis systems was tested by comparing the time taken before GPI-1 staining was visible for samples of eggs. In all such tests the enzyme staining developed much more quickly and fewer samples failed to stain on the cellulose acetate plates than on the starch gels with nitrocellulose overlays.

Both electrophoresis systems produced good estimates of the proportions of GPI-1 allozymes in coded mixtures of liver homogenates (Fig. 1) although there is a tendency for the minor component to be overestimated particularly on the cellulose acetate plates. Little variation occurred when the same samples were run repeatedly on the same or different gels or plates and even less variation occurred when the same staining pattern was repeatedly scanned. There was also excellent agreement between results obtained using the two densitometers.

Fig. 1.

Comparison of two electrophoresis systems for the relationship between the expected percentage of GPI-1A in a mixture of liver homogenates and the percentage estimated by quantitative electrophoresis. Each point represents the mean of three separate, coded mixtures. The line shows the expected relationship if GPI-1A and GPI-1B have equal specific activities.

Fig. 1.

Comparison of two electrophoresis systems for the relationship between the expected percentage of GPI-1A in a mixture of liver homogenates and the percentage estimated by quantitative electrophoresis. Each point represents the mean of three separate, coded mixtures. The line shows the expected relationship if GPI-1A and GPI-1B have equal specific activities.

For both electrophoresis systems, however, deliberate overstaining produced an overestimation of the minor component in the mixtures where the two allozymes were present in unequal proportions. This so-called stain saturation effect occurs in other systems at high enzyme activities or at lower activities when staining is prolonged (Markert & Masui, 1969) and probably contributes to the sigmoidal shape of the curves shown in Fig. 1.

These technical controls indicate that the starch-gel method offers slightly more accurate quantitation, at least for liver homogenates. This advantage is outweighed, however, by the greater sensitivity of the cellulose acetate plates. For this reason the cellulose acetate plates were used for the analysis of GPI-1 allozymes in early embryos.

(ii) Genetic controls

The activity of GPI-1 in the oocyte is regulated by a cis-acting temporal gene, Gpi-1t which is closely linked to the structural gene (Peterson & Wong, 1978; McLaren & Buehr, 1981). This gene will, therefore, affect the relative contributions of oocyte- and embryo-coded enzyme. Oocytes from females that are heterozygous for both the structural Gpi-1s and temporal Gpi-1t loci produce a skewed distribution of GPI-1 allozymes. The proportions of the allozymes produced by unfertilized eggs from females of various genotypes were compared with those from eggs from (DBA×B)F1 females, which are known to be heterozygous, Gpi-1saGpi-1ta/Gpi-1sbGpi-1tb, in order to determine the Gpi-1t genotype of the stocks of mice used in our experiments.

The results, shown in groups 1 and 2 in Table 1, indicate that eggs from B-Gpi-1sa/Gpi-1sb heterozygotes produce allozymes in an approximately 1:2:1 ratio as expected if the B-Gpi-1b strain carries the Gpi-1tb allele which is characteristic of the B strain. Certainly B-Gpi-1sa/Gpi-1sb eggs do not show the dramatic skewing of the (DBA × B)F1 eggs that is produced by unequal proportions of GPI-1 A and GPI-1B monomers (Peterson & Wong, 1978; group 2 Table 1).

Table 1.

Quantitative analysis of GPI-1 allozymes in eggs from heterozygous females

Quantitative analysis of GPI-1 allozymes in eggs from heterozygous females
Quantitative analysis of GPI-1 allozymes in eggs from heterozygous females

The Gpi-1sc allele also affects GPI-1 activity because the GPI-1C allozyme is thermolabile. Padua et al. (1978) showed that the specific activity of GPI was lower in a variety of tissues from Gpi-1sc/Gpi-1sc mice than in those from other genotypes. This is illustrated in Fig. 2 which shows the results of quantitative electrophoresis of mixtures of GPI-1A or GPI-1B and GPI-1C in crude liver homogenates. The results indicate that under our experimental conditions the specific activity of GPI-1 in Gpi-1s′c/Gpi-1sc is about 50 % of that in Gpi-1sa/Gpi-1sa livers and less than 50 % of that in Gpi-1sb/Gpi-1sb livers.

Fig. 2.

Relationship between the expected percentage of GPI-1C in mixtures of liver homogenates and the percentage estimated by quantitative cellulose acetate electrophoresis. Each point represents the mean of three separate coded mixtures. The straight line shows the expected relationship if the specific activity of GPI-1C is equal to the other allozyme in the mixture. .The curved line shows the expected relationship if the specific activity of GPI-1C is only 50 % of the other allozyme in the mixture.

Fig. 2.

Relationship between the expected percentage of GPI-1C in mixtures of liver homogenates and the percentage estimated by quantitative cellulose acetate electrophoresis. Each point represents the mean of three separate coded mixtures. The straight line shows the expected relationship if the specific activity of GPI-1C is equal to the other allozyme in the mixture. .The curved line shows the expected relationship if the specific activity of GPI-1C is only 50 % of the other allozyme in the mixture.

Results of quantitative electrophoresis of brain and liver homogenates from Gpi-1sb/Gpi-1sc males are shown in Table 2. The GPI-1C allozyme is significantly reduced, as expected, but the other two allozymes survive in a 2:1 ratio which suggests that they are equally active and stable. Essentially similar results were obtained for unfertilized eggs (Table 1, groups 3 to 5). In this case the GPI-1C allozyme was more completely reduced and the heteropolymer was present in slight excess of the expected 2:1 ratio in G pi-1 sa/Gpi-1sc eggs. (It is unclear whether this small excess is trivial or, for example, represents a novel Gpi-1t allele associated with Gpi-1sc.)

Table 2.

Quantitative analysis of GPI-1 allozymes in brains and livers from heterozygous male mice

Quantitative analysis of GPI-1 allozymes in brains and livers from heterozygous male mice
Quantitative analysis of GPI-1 allozymes in brains and livers from heterozygous male mice

These controls are relevant, in two respects, to the analysis of (Gpi-1sa/Gpi-1sb ♀ ×Gpi-1sc/Gpi-1sc ♂)F1 embryos, discussed in the next section. First, they show that the G pi-Ia/Gpi-1sb mothers are Gpi-1sa, Gpi-1tb/Gpi-1sb, Gpi-1tb and so will produce oocytes with relatively high GPI-1 activities. Second, the controls indicate that the GPI-1 AC and GPI-1BC allozymes are stable and so can be used in the quantitative analysis. The GPI-1C allozyme, however, is unstable and cannot easily be used in the quantitative analysis because the amount of activity lost in embryos cannot be predicted from experiments on other tissues.

(iii) GPI-1 allozymes in (Gpi-1sa/Gpi-1sb ♀ ×Gpi-1sc/Gpi-1sc ♂)F1 embryos

a) Qualitative analysis

The results from the qualitative analysis of GPI-1 allozymes in (B-Gpi-la/ Gpi-1sb ♀ × B-Gpi-1sc/Gpi-1sc ♂)F1 embryos is shown in Table 3. Both Gpi-1s*/Gpi-1sc and Gpi-1sb/Gpi-1sc embryos are produced so samples containing embryos of both genotypes may produce GPI-1AC, GPI-1BC and GPI-1C allozymes as well as the three oocyte allozymes (GPI-1 A, GPI-1 AB and GPI-1B). In practice the GPI-1AC allozyme co-migrates with GPI-1B so only five bands are distinguishable.

Table 3.

Qualitative analysis of GPI-1 allozymes produced by (B-Gpi-1sa/ Gpi-1sb ♀ xB-Gpi-1sc/Gpi-1sc ♂) F1 embryos

Qualitative analysis of GPI-1 allozymes produced by (B-Gpi-1sa/ Gpi-1sb ♀ xB-Gpi-1sc/Gpi-1sc ♂) F1 embryos
Qualitative analysis of GPI-1 allozymes produced by (B-Gpi-1sa/ Gpi-1sb ♀ xB-Gpi-1sc/Gpi-1sc ♂) F1 embryos

At 2·4 and 2·5 days p.c. (8-cell stage) only three allozyme bands were detected (Groups 1-4 in Table 3). Assuming that the bands of GPI-1B activity produced by groups 1-4 do not contain any GPI-1AC allozyme this implies that, at this stage, all of the detectable GPI-1 activity is oocyte coded.

Some samples of older embryos produced five bands of GPI-1 as shown in Fig. 3 and groups 5, 7 and 9 in Table 3. This indicates that both oocyte-coded and embryo-coded enzymes are present in these samples. Other samples, containing small numbers of embryos, produced no GPI-1BC heteropolymer presumably because all of the embryos in the sample were Gpi-1sa/Gpi-1sc (groups 6, 8 and 10 in Table 3). The GPI-1 AB allozyme is produced entirely by the diploid oocyte genome and was absent from four of the ten 5·4-day embryos. It, therefore, seems likely that no oocyte-coded enzyme remained in these samples.

Fig. 3.

The five bands of GPI-1 activity produced by three (Gpi-1sa/Gpi-1sb ♀ × Gpi-1sc/Gpi-1sc ♂)F1 4·4-day mouse embryos (below) and the densitometer tracing (above). Migration is towards the cathode (left) and in this sample the proportions (from right to left) were estimated as 9·9 % GPI-1A, 11·2 % GPMAB, 34·7 % GPI-1B plus GPMAC, 32·7 % GPI-1BC, 11·5 % GPI-IC.

Fig. 3.

The five bands of GPI-1 activity produced by three (Gpi-1sa/Gpi-1sb ♀ × Gpi-1sc/Gpi-1sc ♂)F1 4·4-day mouse embryos (below) and the densitometer tracing (above). Migration is towards the cathode (left) and in this sample the proportions (from right to left) were estimated as 9·9 % GPI-1A, 11·2 % GPMAB, 34·7 % GPI-1B plus GPMAC, 32·7 % GPI-1BC, 11·5 % GPI-IC.

The qualitative results indicate that the oocyte-coded enzyme is exhausted by about days p.c. and the embryonic genome is expressed by 3·4 days p.c. The presence of the GPI-1C allozyme in 3·4-day embryos indicates expression of the paternally derived allele. The formation of the GPI-1BC allozyme suggests that the maternally derived, embryo-coded Gpi-1sb allele is also expressed in 3·4-day Gpi-1sb/ Gpi-1sc embryos although it is possible that GPI-1B monomer is produced from oocyte-coded mRNA.

The observation that one group of three 4·4-day embryos has no GPI-1BC allozyme suggests that no significant oocyte GPI-1 mRNA remained after the paternally derived Gpi-1sc allele was expressed.

b) Quantitative analysis

The quantitative analysis of the allozyme patterns is shown in Table 4 and a scan from a group of three 4·4-day embryos is illustrated in Fig. 3. The quantitative results provide estimates of the proportion of embryo-coded enzyme at different stages and gives us some indication of when the maternally derived, embryonic Gpi-1s allele is first expressed.

Table 4.

Quantitative analysis of GPI-1 allozymes produced by (B-Gpi-1sa/Gpi-1sb♀ × B-Gpi-1sc/Gpi-1sc ♂) F1 embryos

Quantitative analysis of GPI-1 allozymes produced by (B-Gpi-1sa/Gpi-1sb♀ × B-Gpi-1sc/Gpi-1sc ♂) F1 embryos
Quantitative analysis of GPI-1 allozymes produced by (B-Gpi-1sa/Gpi-1sb♀ × B-Gpi-1sc/Gpi-1sc ♂) F1 embryos

In unfertilized eggs (Group 1, Table 4) approximately 50 % of the GPI-activity is in the GPI-1 AB allozyme, so in embryos the proportion of oocyte-coded enzyme is approximately twice the proportion of enzyme represented by the GPI-1AB allozyme. This is likely to be overestimated, however, because the GPI-1C allozyme is thermolabile. The genetic controls discussed in the previous section do not allow us to predict the extent of GPI-1 C loss so in Table 5 two estimates of the proportion of embryo-coded enzyme are given. One assumes that no GPI-1 C is preferentially lost and the other assumes that 67 % of this allozyme is lost.

Table 5.

Estimated proportion of embryo-coded GPI-1 produced by (B-Gpi-1sa/Gpi-1sb× B-Gpi-1sc/Gpi-1sc) F1 embryos

Estimated proportion of embryo-coded GPI-1 produced by (B-Gpi-1sa/Gpi-1sb× B-Gpi-1sc/Gpi-1sc) F1 embryos
Estimated proportion of embryo-coded GPI-1 produced by (B-Gpi-1sa/Gpi-1sb× B-Gpi-1sc/Gpi-1sc) F1 embryos

These calculations indicate that at 3·4 days p.c. 22–25 % of the GPI-1 activity is embryo coded. Presumably the paternally derived Gpi-1sc allele is active earlier than this time and may be expressed as early as days p.c. This is when Brinster (1973) detected paternally derived GPI-1A allozyme in pooled samples of 500 Gpi-1sa/Gpi-1sb embryos. The absence of GPI-1C in our -day embryo samples may reflect the small sample size even with our more sensitive technique.

The results shown in Table 5 also indicate that 88–94 % of the GPI-1 is embryo coded in the six 5·4-day embryos that showed some residual oocyte-coded GPI-1AB heteropolymer (groups 8 and 9 in Table 3 and groups 7 and 9 in Table 4). Since the other four 5·4-day embryos had no oocyte GPI-1 AB heteropolymer, oocyte GPI-1 is normally exhausted at or shortly after days p.c. in the embryos studied. This conclusion is not necessarily valid for all embryos since the oocyte genome will have a profound effect on both the initial activity and stability of the oocyte-coded enzyme. For example, Gpi-la/Gpi-lafemales will produce oocytes with much less GPI-1 enzyme which is likely to be exhausted before days p.c. unless oocyte mRNA is translated in early embryos.

The expression of the embryonic, maternally derived Gpi-1sa allele will result in an elevated ratio of GPI-1A: GPI-1AB allozymes in those samples containing Gpi-1s′d/Gpi-1sc embryos. The results, shown in Table 6, indicate that for 4·4-day embryos the ratio is significantly higher than for unfertilized eggs from Gpi-1sa/ Gpi-1sb females so the embryonic Gpi-1scallele is expressed. At 3-4 days, when the expression of the paternally derived Gpi-1sc allele is first detected, the ratio is higher than for eggs but not significantly so. At 2·4 and 2·5 days the ratio is no higher than for eggs.

Table 6.

Expression of maternally and paternally derived Gpi-1s alleles in (B-Gpi-1sa/Gpi-1sb♀ × B-Gpi-1sc/Gpi-1sc ♂)F1 embryos

Expression of maternally and paternally derived Gpi-1s alleles in (B-Gpi-1sa/Gpi-1sb♀ × B-Gpi-1sc/Gpi-1sc ♂)F1 embryos
Expression of maternally and paternally derived Gpi-1s alleles in (B-Gpi-1sa/Gpi-1sb♀ × B-Gpi-1sc/Gpi-1sc ♂)F1 embryos

Although earlier expression of the embryonic Gpi-1sa allele may go undetected if the samples contained mainly Gpi-1sb/Gpi-1sc embryos, these results suggest that the maternally derived, embryonic Gpi-1sa allele is expressed no earlier than the paternal Gpi-1sc allele.

One unexplained peculiarity of the results shown in Table 4 is that the proportion of GPI-1 AC or GPI-1BC heteropolymer is more than twice the proportion of GPI-1 A or GPI-1B in 5·4-day Gpi-1sb/Gpi-1sc and Gpi-1sb/Gpi-1sc embryos respectively (groups 7–10 in Table 4). This is clearest in groups 8 and 10 which have no residual oocyte-coded GPI-1 activity. This effect was not seen in adult brain and liver from Gpi-1sb/Gpi-1sc mice (see Table 2) and is more extreme than the slightly elevated heteropolymer levels seen in oocytes (groups 3–5 in Table 1). It, therefore, seems unlikely to be caused by a difference in allozyme stability but could reflect a greater production of GPI-1 C monomers at this stage of development.

The expression of a number of paternally derived genes, in addition to Gpi-1s, has been demonstrated in the mouse and this subject has been extensively reviewed (Epstein, 1975; McLaren, 1976; Chapman, West & Adler, 1977; Sherman, 1979; Johnson, 1981; Kidder, 1981; Magnuson & Epstein, 1981). As far as we know the X-chromosome-linked Pgk-1 gene is the only other system in the mouse where consideration has been given to the relative timing of the onset of expression of the maternally and paternally derived genes. Krietsch et al. (1982) interpret their results to suggest that the maternally derived allele is expressed before the paternal allele and this issue has also been raised by Papaioannou, West, Bucher & Linke (1981).

Considerable attention has been paid to the transitions from oocyte-coded to embryo-coded mRNA and protein (see reviews by Epstein, 1975; Sherman, 1979; Johnson, 1981; Magnuson & Epstein, 1981). Although most oocyte mRNA is degraded by the 4-cell stage (see for example Johnson, 1981) some mRNA persists until the early blastocyst stage (Bachvorova & De Leon, 1980).

As yet little is known about specific mRNA or protein gene products. Harper & Monk (1983) have suggested that oocyte mRNA for hypoxanthine phosphoribosyl transferase (HPRT) is present until the late 2-cell and possibly until the 4- to 8-cell stage. Although our experiments with GPI-1 allozymes indicate that oocyte-coded GPI-1 mRNA is absent by the time the paternally derived Gpi-1sc allele is expressed they shed no light on whether oocyte GPI-1 mRNA is present in younger embryos.

The recent experiments of Harper & Monk (1983) are also relevant to the duration of oocyte-coded HPRT enzyme which, they suggest, is depleted by the early morula stage ( days p.c.). This is based on extrapolation of a biphasic curve, for increasing HPRT activity per embryo with time, to derive hypothetical curves for oocyte-coded and embryo-coded contributions. Our more direct approach conclusively shows that oocyte-coded GPI-1 enzyme persists somewhat longer.

Oocyte-coded and embryo-coded enzyme both contribute to the glucose phosphate isomerase activity in preimplantation embryos. In our experiments oocyte-coded enzyme was present throughout preimplantation development but had disappeared by days p.c. in some embryos and was almost exhausted in other embryos at this stage. We confirmed that embryo-coded GPI-1 is produced before days p.c. and found no evidence that the maternally derived Gpi-1s’ allele is expressed before the paternally derived Gpi-1s allele in our crosses. During the three-day period between and days p.c., there is a transition from almost entirely oocyte-coded enzyme to 90-100 % embryo-coded enzyme. It seems likely that there is no oocyte-coded GPI-1 mRNA present during this transition period.

We thank Mrs L. Ofer and Mrs M. Carey for performing many of the starch gel technical controls, Drs M. Buehr, R. Holmes, A. C. Peterson and P. Thorogood for advice and technical demonstrations and Drs G. Bulfield and V. E. Papaioannou for supplying mice. We are also grateful to Professor R. L. Gardner, Dr M. F. Lyon and Mr G. Fisher for reading the manuscript. This work was supported by an M.R.C. programme grant awarded to Professor R. L. Gardner.

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