The measurement of the activity of the X-linked enzyme HPRT has been widely used as an indicator of X-chromosome activity during preimplantation development in the mouse. More recently, the concomitant measure-ment of the activity of the autosomally-encoded enzyme APRT has been used in an attempt to decrease the variability inherent in the measurement of enzyme activity from minute samples such as preimplantation embryos.

In this study the use of the HPRT-deficient mouse mutant, Hprtb-m3, allowed the unequivocal identification of the parental origin of HPRT activity measured in embryos derived from crosses between wild-type mice, and mice which were homozygous or hemizygous for the Hprtb-m3 allele. Results were similar to those of a previous study, where oocyte-encoded HPRT activity accounted for about 10% of total HPRT activity at 76 hours post human chorionic gonadotrophin injection and the paternally-derived Hprt allele was shown to be transcriptionally active by the late 2-cell stage.

In contrast to other studies, differential expression of the two Hprt alleles was detected during the preim-plantation period, in embryos derived from crosses between wild-type and HPRT-deficient mice. Evidence was also found for the existence of an X-linked locus which influences the amount of APRT activity in the unfertilized oocyte. We propose that the expression pat-tern of this locus may be influenced by its parental origin.

Hypoxanthine phosphoribosyltransferase (HPRT: E.C. 2.4.2.8) and adenine phosphoribosyltransferase (APRT: E.C. 2.4.2.7) activities have been studied extensively in the preimplantation mouse embryo, both in relation to the early genetic events postfertilization such as embryonic genome activation, and to the timing of X-chromosome inactivation (Epstein, 1970, 1972; Epstein et al., 1978; Harper and Monk, 1983; Kratzer and Gartler, 1978; Monk and Kathuria, 1977; Monk and Harper, 1978, 1979).

These studies have shown that the transition from oocyte-encoded to embryo-encoded HPRT activity occurs during the early preimplantation period, between the late 2-cell and morula stages of development. Although these observations are consistent with the finding that degradation of oocyte-encoded Hprt mRNA occurs during the 2-cell stage (Payn-ton et al., 1988), the question of the precise timing of the initiation of transcription from the paternal Hprt allele has not been resolved, because Kratzer (1983) detected embryonic HPRT activity at the 2-cell stage, whereas Harper and Monk (1983) did not detect embryonic HPRT activity until the 4-to 8-cell stage. In the mouse embryo, the paternal allele of the X-linked Pgk-1 gene is first expressed on day 6 of development, two days later than the maternal allele (Papaioannou et al., 1981; Krietsch et al., 1982; Krietsch et al., 1986). Pravtcheva et al. (1991) have proposed that this is a secondary consequence of the close linkage of the Pgk-1 locus to the X-chromosome controlling element (Xce), and is not a property intrinsic to the Pgk-1 gene itself. This view is consistent with the detection of expression of the paternal alleles of the X-linked Hprt and α-galactosi-dase genes at the 8-cell stage (Chapman, 1986; Epstein et al., 1978; Kratzer and Gartler, 1978; Monk and Harper, 1978), since these loci are not closely linked to Xce. How-ever, the possibility remains that the delayed activation of the paternal Pgk-1 allele represents an extreme case of a more general suppression of the paternal X-chromosome during preimplantation development, because previous studies of Hprt and α-galactosidase gene dosage may not have been of sufficient sensitivity to detect subtle differences in the expression of the parental alleles.

The simultaneous measurement of both HPRT and APRT activity in individual samples (see Monk, 1987, for a review) purports to decrease the variability between samples due to variable recovery of enzyme activity from embryo lysates, by using APRT activity as a baseline against which HPRT activity is normalized. However, this treatment of the data may be invalid in certain experimental situations because HPRT and APRT may interact through their common substrate, phosphoribosylpyrophos-phate (PRPP), (Greene et al., 1970). Thus, Monk and Harper (1978) measured the expected two-fold difference in HPRT activity between oocytes from XX and XO female mice, but the APRT level was higher in XO oocytes, resulting in a three-fold rather than the predicted two-fold difference in the HPRT/APRT ratio between XX and XO oocytes. In this study we sought to examine some of the questions discussed above, using the Hprtb-m3 mutant as a baseline against which HPRT expression derived from different parental sources can be directly assayed in early mouse embryos. Specifically, we sought to determine the precise timing of activation of the paternal Hprt allele, and to determine whether its expression was similar to the maternal allele during the preimplantation period. We also sought to determine whether the level of APRT activity in unfertilized oocytes was influenced by the level of oocyte HPRT activity.

Mice

The following strains of mice were used in this study: random-bred MF1/Lac, which carry the wild-type Hprtballele, and HPRT-deficient mice carrying the Hprtb-m3 mutant allele (Hooper et al., 1987). The Hprtb-m3 allele on the 129/Ola background was back-crossed to the MF1/Lac genetic background for four generations and was subsequently maintained as a random-bred HPRT-deficient colony. The mice were maintained under constant conditions of lighting [14 hours of daylight (05.00 to 19.00) and 10 hours of darkness (19.00 to 05.00)] and temperature (18-22°C).

Superovulation of females and collection of embryos

Superovulation of females and collection of embryos was carried out as described in Pratt (1987). Briefly, immature (21-24 day-old) or mature (>6 weeks old) females were superovulated using an intraperitoneal injection of 5 i.u. of pregnant mares’ serum gonadotrophin (PMSG) (Folligon, Intervet, UK), followed 48 hours later by 5 i.u. of human chorionic gonadotrophin (hCG) (Chorulon, Intervet, UK). The age of fertilized embryos was expressed as the number of hours after the injection of hCG (hours post hCG) or as the number of days after the detection of the vaginal plug (day of detection = day 1 of pregnancy). Hormones were injected at either 12.00 or 17.00 hours depending on the requirements of individual experiments.

Unfertilized oocytes and fertilized 1-cell embryos were collected from superovulated females at about 22 hours post hCG. Cleavage stage embryos were flushed on day 2, 3, 4 and 5 of pregnancy, from either the oviduct or uterine horns, depending on the stage of development.

Assay of HPRT and APRT activity in embryo lysates

Embryos which were required for HPRT or APRT assay were flushed and washed as described above, and pipetted into 10 μl Drummond microcapillary tubes (Microcaps, Marathon supplies, UK) which were partially filled (3-5 μl) with M2 + BSA. Embryos were stored at –70°C until use, and assayed in batches of ten, unless otherwise stated.

The conditions under which HPRT and APRT activity was measured were similar to those described by Monk (1987). The reaction mixture for the simultaneous measurement of HPRT and APRT activity consisted of sodium phosphate buffer (38.75 mM, pH 7.4), magnesium chloride (5 mM), phosphoribosyl pyrophos-phate (PRPP, Na salt, 1 mM), [3H]hypoxanthine (10 μM) and [14C]adenine (10 μM). The reaction mixture for measuring HPRT activity alone was similar, with sodium phosphate buffer replacing [ 14C]adenine. Non-radioactive chemicals were obtained from Sigma Chemical Co., UK, and radiolabelled hypoxanthine and adenine from Amersham International, UK. The specific activity of the radiolabelled chemicals was the highest available and was usually in the range 3-8 Ci/mmol.

Samples (3-5 μl) were added to 50 μl of reaction mixture in 1.5 ml microcentrifuge tubes and incubated in a waterbath at 37°C for 3-5 hours (2-cell to blastocyst stage), or overnight (16-18 hours; unfertilized oocytes). The activity of both enzymes remained linear over the incubation periods used (data not shown). Negative controls consisted of sample tubes containing M2 + BSA. The reaction was stopped and the product(s) precipitated by the addition of 1 ml of an ice-cold solution containing 0.1 M lan-thanum chloride, 1 mM unlabelled hypoxanthine and 1 mM unla-belled adenine. The tubes were placed on ice for at least 30 minutes.

The reaction product(s) were collected on Whatman GF/C microfibre filters, and the radioactivity was counted in an LKB 1211 Minibeta liquid scintillation counter, using Optiphase scin-tillation fluid (Pharmacia, UK).

HPRT and APRT activities are expressed in picomoles per hour per embryo (pmoles–1 h–1embryo–1). All measurements were made after incubation of the samples in the reaction mixture for a specified period at 37°C.

HPRT activity in the Hprtb-m3 mutant

To determine whether preimplantation embryos homozygous or hemizygous for the Hprtb-m3 allele are HPRT-deficient, HPRT activity was assayed in unfertilized oocytes collected at 22 hours post hCG from Hprtb-m3/Hprtb-m3 and Hprtb/Hprtb females, and in embryos homozygous and hemizygous for the Hprtb-m3 or Hprtb allele, collected at 24, 48, 72 and 96 hours post hCG. The results (Table 1) confirmed the HPRT-deficient status of unfertilized oocytes and preimplantation embryos obtained from Hprtb-m3/ Hprtb-m3 females. The HPRT activity detected in embryos homozygous or hemizygous for the Hprtb allele was similar to that reported by Harper and Monk (1983).

Table 1.

HPRT activity in unfertilized oocytes and preimplantation embryos homozygous for the mutant (Hprtb-m3) or normal gene (Hprtb)

HPRT activity in unfertilized oocytes and preimplantation embryos homozygous for the mutant (Hprtb-m3) or normal gene (Hprtb)
HPRT activity in unfertilized oocytes and preimplantation embryos homozygous for the mutant (Hprtb-m3) or normal gene (Hprtb)

Hprt and Aprt gene dosage in unfertilized oocytes

In the first experiment HPRT and APRT activities were measured in unfertilized oocytes from the reciprocal heterozygous females, Hprtb/Hprtb-m3 and Hprtb-m3/Hprtb. Oocytes were assayed in batches of ten using incubation periods of ∼18 hours. In a second experiment, HPRT and APRT activities were measured in unfertilized oocytes from Hprtb/Hprtb, Hprtb-m3/Hprtb-m3, Hprtb/Hprtb-m3 and Hprtb-m3/Hprtb females. The data and statistical analyses are summarized in Tables 2 and 3, respectively. Intercross differences in enzyme activity were analysed using Anova, with replicate number and either HPRT or APRT as covariates. HPRT activity in oocytes from Hprtb/Hprtb females was significantly higher than in oocytes from both heterozygous groups, which had similar levels of HPRT activity (Table 3).

Table 2.

HPRT and APRT activity in unfertilized oocytes from Hprtb/Hprtb, Hprtb-m3/Hprtb-m3, Hprtb/Hprtb-m3 and Hprtb-m3/Hprtb females

HPRT and APRT activity in unfertilized oocytes from Hprtb/Hprtb, Hprtb-m3/Hprtb-m3, Hprtb/Hprtb-m3 and Hprtb-m3/Hprtb females
HPRT and APRT activity in unfertilized oocytes from Hprtb/Hprtb, Hprtb-m3/Hprtb-m3, Hprtb/Hprtb-m3 and Hprtb-m3/Hprtb females
Table 3.

Summary of Anova analysis of data from Table 2

Summary of Anova analysis of data from Table 2
Summary of Anova analysis of data from Table 2

Significant intercross differences were detected in oocyte APRT activity, which was highest in oocytes from Hprtb-m3/Hprt b-m3 females and lowest in oocytes from Hprtb/Hprtb females. Oocytes from the heterozygous females had intermediate levels of APRT which were significantly different from each other (F[1]=12.732, P<0.001). APRT activity in oocytes from Hprtb-m3/Hprtb-m3 females was significantly different from oocytes derived from Hprtb/Hprtb and Hprtb-m3/Hprtb, but not Hprtb/Hprtb-m3 females (Table 3).

Transition from oocyte-encoded to embryonic HPRT activity

The measurement of HPRT activity in single embryos from the cross: Hprtb/Hprtb-m3 × Hprtb-m3/Y, allows an estimation of the stage of preimplantation development at which oocyte-encoded HPRT activity disappears, because 50% of the embryos will not inherit a Hprtb allele and will not pro-duce embryo-encoded HPRT activity. These can be identi-fied by HPRT assay once oocyte-encoded activity has dis-appeared. In a preliminary experiment, a bimodal distribution of HPRT activity was detected in embryos derived from this cross at 76 hours and 96 hours post hCG, but not at 54 hours post hCG (data not shown).

In a further experiment, HPRT and APRT activities were measured simultaneously in single embryos from the same cross at 76 hours post hCG. 50% of embryos with the lowest HPRT activity were assigned to the “low” activity group, and the remainder were assigned to the “high” activity group (Table 4). The validity of presenting the data in this manner was based upon the demonstration that 50% of embryos do not inherit a Hprtb allele and are HPRT-deficient on day 5 of pregnancy (data not shown). Inspection of the data in Table 4 suggests that in embryos derived from this cross the contribution of oocyte-encoded HPRT activity is not greater than about 10% of total measurable HPRT activity at 76 hours post hCG.

Table 4.

HPRT and APRT activity measured in single embryos from the cross: Hprtb/Hprtb-m3 × Hprtb-m3/Y at 76 hours post hCG

HPRT and APRT activity measured in single embryos from the cross: Hprtb/Hprtb-m3 × Hprtb-m3/Y at 76 hours post hCG
HPRT and APRT activity measured in single embryos from the cross: Hprtb/Hprtb-m3 × Hprtb-m3/Y at 76 hours post hCG

An accurate estimation of the earliest appearance of paternally-encoded HPRT activity in the embryo was made by measuring HPRT activity during preimplantation development in embryos derived from mating Hprtb-m3/Hprtb-m3 females to Hprtb/Y males (Table 5). The appearance of HPRT activity will depend on the transcriptional activation of the paternally-derived Hprtb allele in female embryos. Embryos were flushed at 46, 52 and 57 hours post hCG. Embryos from Hprtb-m3/Hprtb-m3 females mated to Hprtb-m3/Y males, were used as the negative control to allow as accurate a comparison as possible between back-ground counts min–1 and the appearance of paternal HPRT activity. The data were similar between the three replicates (F[2]=0.174, P>0.841) and were combined and analysed by one-way analysis of variance. A significant level of HPRT activity above background was detected in embryos (including putative Hprtb-m3/Hprtb female embryos) collected at 52 and 57 (P<0.05), but not at 46 hours post hCG. The increase in activity between 52 and 57 hours post hCG was also significant (P<0.05). The results indicate that paternally-encoded HPRT activity is first detectable between 46 and 52 hours post hCG, which corresponds to the late 2-cell stage in the embryos used in this study.

Table 5.

Appearance of paternally-encoded HPRT activity on day 2 of pregnancy

Appearance of paternally-encoded HPRT activity on day 2 of pregnancy
Appearance of paternally-encoded HPRT activity on day 2 of pregnancy

Expression of maternal and paternal Hprt alleles during preimplantation development

Table 6 shows the level of HPRT activity measured in embryos from the crosses: (1) Hprtb/Hprtb × Hprtb/Y, (2) Hprtb/Hprtb × Hprtb-m3/Y and (3) Hprtb-m3/Hprtb-m3 × Hprtb/Y, at 50, 74 and 98 hours post hCG. The data were log10 transformed and analysed using Anova. The data suggest that differential expression of maternal and paternal Hprt alleles may occur during preimplantation development, because the absence of the paternal Hprtb allele in group 2 embryos did not significantly reduce total activity until day 4 of pregnancy. In the absence of significant amounts of oocyte-encoded HPRT, the ratio of the HPRT activity in the three groups on day 3 of development is predicted to be 3:2:1 due to Hprtb gene dosage. The observed ratio (calculated by dividing the activity in group 3 into groups 1 and 2), is 7.7:4.3:1. Assuming some residual maternally-encoded activity in groups 1 and 2, this ratio approximates reasonably closely to the ratio 6:4:1 predicted if the maternal Hprtb allele is twice as active as the paternal Hprtb allele during the early cleavage stages. Since the interpretation of these data may be complicated by the presence of oocyte-encoded HPRT in group 1 and group 2 embryos prior to day 3 of pregnancy, and by the preferential inactivation of the paternal Hprtb allele in the trophec-toderm around day 4 of pregnancy, HPRT activity was measured in group 2 and group 3 embryos collected at 64, 68, 72 and 76 hours post hCG, on day 3 of pregnancy. The ratio of HPRT activity between the two crosses was calculated from the means at each timepoint (Table 7). The mean of the combined ratios at all timepoints and from all replicates was 3.84±1.28:1, which is in reasonably close agreement with the ratio of 4.3:1 described above.

Table 6.

Changes in HPRT activity in embryos on day 2, 3, and 4 of pregnancy

Changes in HPRT activity in embryos on day 2, 3, and 4 of pregnancy
Changes in HPRT activity in embryos on day 2, 3, and 4 of pregnancy
Table 7.

HPRT activity in embryos from reciprocal crosses between mice homozygous for the mutant (Hprtb-m3) or normal (Hprtb) gene on day 3 of pregnancy

HPRT activity in embryos from reciprocal crosses between mice homozygous for the mutant (Hprtb-m3) or normal (Hprtb) gene on day 3 of pregnancy
HPRT activity in embryos from reciprocal crosses between mice homozygous for the mutant (Hprtb-m3) or normal (Hprtb) gene on day 3 of pregnancy

To determine whether the ratio at each timepoint had departed significantly from the 2:1 ratio predicted from Hprt gene dosage, the data from each timepoint were transformed to log10 and analysed using the independent Stu-dent’s t-test to determine whether log2 (0.3010) fell within the 95% confidence interval for the difference of the means. Only three out of twelve timepoints had data consistent with a 2:1 ratio, suggesting that the HPRT activity difference in the two groups had departed significantly from the expected ratio of 2:1.

The HPRT/APRT double assay has been widely used in the study of gene expression in the early mouse embryo (Monk, 1987) and as a model for preimplantation diagnosis in the human (Monk et al., 1988, 1990). However, the accuracy of this method of identifying Hprt gene dosage effects depends on the validity of using APRT activity as a stan-dard against which HPRT activity is normalized. As dis-cussed above, the results of Monk and Harper (1978) and Green et al. (1970) suggest that this manipulation of the data might not be valid in certain experimental situations. Although there was some indication of increased APRT activity in embryos with low HPRT activity in this study, the results were variable and do not provide conclusive evi-dence for the occurrence of the process of substrate stabi-lization in the mouse embryo.

Imprinting of APRT in oocytes

HPRT activity in oocytes from reciprocal heterozygous Hprtb/Hprtb-m3 and Hprtb-m3/Hprtb females was identical and was significantly lower than in oocytes from homozy-gous Hprtb/Hprtb females. However, the ratio of the activity in oocytes from Hprtb/Hprtb females relative to those from Hprtb/Hprtb-m3 and Hprtb-m3/Hprtb females was 3:1 rather than the 2:1 ratio predicted by a simple gene dosage effect. The detection of different APRT, but not HPRT, levels in oocytes from Hprtb/Hprtb-m3 and Hprtb-m3/Hprtb females was unexpected. Since both groups had identical HPRT activity, the process of substrate stabilization cannot account for this anomaly. These results suggest that the APRT activity of the egg cytoplasm may be influenced by a locus displaying parent-of- origin specific effects similar to the reported imprinting of hyaluronidase sensitivity of the cumulus mass in reciprocal (C57BL/By × BALB/cBy)F1 hybrids (Bander et al., 1988). However, this explanation requires the existence of different Aprt alleles segregating in MF1 and HPRT-deficient mice. The back-crossing of the Hprtb-m3 allele to the MF1 strain, in addition to the fact that only one electrophoretic variant (from wild mice) of the APRT enzyme is known (Green, 1989) renders this explanation unlikely.

A further possibility is the existence of an X-linked locus which influences the level of APRT in the ooplasm. Such a locus might display allelic differences between the 129/Ola inbred strain from which the Hprtb-m3 mutant is derived, and the MF1 strain. Close linkage of such a locus to Hprt would result in MF1 and HPRT-deficient mice maintaining different alleles at this locus during the crossing of the Hprtb-m3 allele to the MF1 genetic background. The existence of such a locus is supported by the fact that Monk and Harper (1978) detected differences in Aprt gene dosage between oocytes from XX and XO mothers that shared an identical genetic background.

Transition from oocyte-encoded to embryonic HPRT activity

The rate of decline of oocyte-encoded HPRT activity measured in this study indicates that at 76 hours post hCG about 10% of total activity is derived from oocyte-encoded Hprt mRNA. This result is similar to that reported previ-ously (Kratzer, 1983; Monk and Harper, 1978).

The earliest stage at which paternally-encoded HPRT activity was detected was at 52 hours post hCG. This implies that the paternal Hprt allele is transcriptionally active by the late 2-cell stage. This result is in agreement with that of Kratzer (1983) who also detected embryo-encoded HPRT activity at the 2-cell stage. The detection of paternally-encoded HPRT activity as early as 52 hours post hCG suggests that the maternal and paternal alleles are activated simultaneously in the mouse embryo.

Expression of maternal and paternal alleles during preimplantation development

The evidence for the differential expression of the parental Hprt alleles on day 3 and 4 of pregnancy may reflect differences in transcriptional activity of the two alleles, or in the stability or efficiency of translation of their respective mRNAs. The variation in the extent to which the observed ratio departed from the expected ratio of 2:1 might be explained by the inherent developmental variability in batches of embryos, or might reflect a “catch-up” phase, whereby expression of the parental alleles becomes identical at the later time-points examined. The latter suggestion is supported by the observation that the deviation from the expected 2:1 ratio occurred less frequently at the later time-points (Table 7). Differential expression of the parental Hprt alleles was not detected in other studies (Chapman, 1986; Kratzer, 1983; Monk and Harper, 1978). However, the use of the HPRT-deficient mutant Hprtb-m3, in this study, may have resulted in increased sensitivity to the detection of relatively subtle differences in the expression of the parental Hprt alleles. These results could also be explained by different rates of development of embryos from Hprtb/Hprtb × Hprtb-m3/Y and Hprtb-m3/Hprtb-m3 X Hprtb/Y crosses, which might indirectly cause the observed differences in HPRT activity between the two crosses. However, such a difference was not detected in this study.

Pravtcheva et al. (1991) have suggested that the delayed activation of the paternal Pgk-1 allele might be due to its coincidental regulation with the Xce locus. They postulate a localized spread of inactivation around Xce which encompasses the Pgk-1 locus, but not the more distant Hprt or α-galactosidase loci. Although the finding of differential expression of the parental Hprt alleles suggests that the postulated inactivation of Xp-linked alleles may be more wide-spread than proposed by Pravtcheva et al. (1991), it does not invalidate the essential features of their model. The existence of an imprint on Xp which may be responsible for its preferential inactivation (and the coincidental suppression of paternal Pgk-1 expression) prior to day 6 of pregnancy is contrary to the proposal of Lyon and Rastan (1984) that Xm carries an imprint which protects it from inactivation, since the suppression of Xp-linked alleles prior to the occurrence of preferential Xp-inactivation is not predicted by their model. However, an earlier proposal, namely, that Xp is imprinted in the oocyte cytoplasm shortly after fertilization (Chandra and Brown, 1975), is consistent with the suppression or delayed activation of Xp-linked alleles, and with the adaptive function of X-inactivation proposed by Moore and Haig (1991).

In conclusion, we have shown significant differences in Hprt and Aprt gene dosage in unfertilized oocytes and preimplantation embryos from mice carrying the normal and mutant forms of the Hprt gene in homozygous and het-erozygous combinations. Further work is required to deter-mine whether all of these results reflect processes which occur in normal wild-type embryos or whether they are found only in embryos carrying the Hprtb-m3 allele.

The authors thank Dr. Nigel Brown for help with statistical analysis. T.F. Moore acknowledges receipt of a Training Fellow-ship from Action Research.

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