The expression of β-glucuronidase during preimplantation development of mouse embryos

DEVELOPMENTAL

BIOLOGY

48,

The Expression

104-109 (1976)

of /3-Glucuronidase during Preimplantation Development of Mouse Embryo+ LINDA

Roche

Institute

WUDL

of Molecular Biology Nutley, Roswell Park Memorial Accepted

AND VERNE New Jersey 07110 Institute Buffalo, August

CHAPMAN and Department of Molecular New York 14263

Biology,

11,1975

P-Glucuronidase activity was measured in mouse embryos during the preimplantation period of development by using a microfluorometric assay. A IOO-fold increase in activity was observed between 57 @-cell stage) and 84 hr (morulael of development. Activity changes between 30 and 60 hr were also significant. Genetic variants of p-glucuronidase occur between the strains of mice C57BL/6J and C3H/HeJ which differ in levels of activity and heat denaturation kinetics. Activity changes and heat denaturation kinetics of p-glucuronidase in C57BL/6, C3H/HeJ and F, hybrid embryos were compared, and it was demonstrated that paternal genes were expressed during the loo-fold increase in activity and that embryonic genes may be functioning between 30 and 60 hr of development. INTRODUCTION

The timing of paternal gene expression provides an experimental means of estimating when the embryonic genome becomes functional and potentially capable of directing development. In Amphibia, paternal gene products are not observed until advanced stages of embryogenesis (Wright and Subtelny, 1971, Johnson and Chapman, 1971a,b). There is considerable evidence that amphibian and echinoderm eggs contain stored messenger RNA that directs protein synthesis during early embryogenesis (Gross et al., 1973; Skoultchi and Gross, 1973). Early mammalian embryonic development appears to differ from the amphibian model in a number of respects. For example, RNA synthesis is initiated in mouse embryos at earlier stages. Incorporation of isotope-labeled precursors into RNA occurs by the 2-cell stage (Knowland and Graham, 1972), and by the 4-cell stage all classes of RNA synthesis can be determined (Woodland and Graham, 1969). Unlike amphibian embryos that are resistant to inhibitors of RNA synthesis before gas1 This work was supported by Grants from the National Institutes of Health, No. GM-19521 and HD 08768-01. 104 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

trulation, such inhibitors can block mammalian cell division as early as the first cleavage, indicating that RNA synthesis is required for early development (Golbus et al., 1973; Ellem and Gwatkin, 1968). By using electrophoretic variants of the enzyme glucosephosphate isomerase (Gpi-1) it has been possible to demonstrate expression of a paternal gene product before implantation and possibly as early as the 8cell stage (Chapman, et al., 1971 and Brinster, 1973). In this report we describe changes in /3glucuronidase activity during early cleavage stages of mouse embryo development. Using a microfluorometric assay procedure that is sensitive enough to measure enzyme activity of single embryos (Wudl and Paigen, 1974), we observed that glucuronidase activity increases loo-fold between the 8-cell and morulae stages. By using genetic variants of the glucuronidase structural gene we were able to determine that the paternal allele was expressed during this early increase in activity. These results indicate that embryonic gene expression accounts for the increase in glucuronidase activity as opposed to the activation of a stored message synthesized prefertilization.

WUDL MATERIALS

AND

AND CHAPMAN

p-Glucuronidase

METHODS

Inbred mouse strains C3H/HeJ (C3H) and C57BLKJ (B6) from the Jackson Laboratory were used for these studies. The C3H strain carries the Gush structural allele for p-glucuronidase which is more heat labile than the Gush structural gene product found in B6 mice. Ovulation was induced in 8-16-week-old females by injecting 4 IU pregnant mare’s serum (PMS; Pregnyl, Ayerst) followed 48 hr later with 4 IU human chorionic gonadotropin, (HCG; Sigma). The females were paired with stud males late in the afternoon following gonadotropin injection and checked the following morning for vaginal plugs. The midpoint of the dark cycle is taken as the approximate time of mating and is used as the t = 0 of development. Embryos were flushed from the fallopian tubes or uterine horns with phosphate-buffered saline (GIBCO), pH 7.2, containing 0.3% bovine serum albumin. Embryos were collected at 36 hr (2-4-cell), three times on the third day at approximately 57 hr, 60 hr and 65 hr (8-16-cells) and at 84 hr (morulae or early blastocyst). Embryos were separated from the cellular debris with micropipets and washed twice in fresh phosphate-buffered saline with bovine serum albumin. p-Glucuronidase activity was assayed in single embryos by a single cell microfluorometric assay technique (Wudl and Paigen, 1974). For the enzyme assay, intact embryos in buffered saline-albumin solution were picked up with micropipets in a volume of less than 2 ~1 and transferred to 0.5 ml of enzyme assay mixture that contained 0.1 M acetate, pH 6.0, 0.9% NaCl, 0.1% bovine serum albumin and 3 x lo-” M 4-methylumbelliferyl-b-n-glucuronic acid. The embryos were picked up with finely drawn polyethylene tubing (0.3-0.4mm inside diameter), and deposited singly in microdroplets of l-10 nl under oil on a glass microscope slide. The embryos were disrupted by alternating freezing and

in Mouse

Embryos

105

thawing the slides in an atmosphere of N,. The liberated enzyme was allowed to react with substrate in the droplet by incubating the slides in a moist chamber at 37°C for 1 or 2 hr. The slide was then placed in a closed chamber with a reservoir of triethylamine, which diffuses through the oil and raises the pH of the droplet to approximately 10. This stops the enzyme reaction and optimizes the fluorescence of the reaction product, 4-methylumbelliferone (4-MU). The fluorescence of droplets with and without embryos was measured by using a Nikon fluorescence microscope equipped with an Aminco photomultiplier microphotometer. Interference filters (Baird Atomic) were positioned to obtain monochromatic light for excitation (365 nm) and emission (455 nm). The fluorescence of a droplet is directly proportional to the number of moles of product in the droplet. Heat denaturation kinetics of p-glucuronidase were examined in day-4 (84-hrj embryos. For these assays a modification of the microassay technique was used. Mice 21-25 days of age were superovulated and embryos collected at the 2-cell stage (36 hr) and grown in in vitro culture (Whitten and Biggers, 1968) to the late morulaeearly blastocyst stage (early day 4). Embryos were then collected, counted, and washed in phosphate buffered saline. Approximately 100 embryos of each genotype (C3H homozygous, B6 homozygous, and C3B6F, heterozygous) were placed in 60 ~1 of buffer (0.1 M acetate, pH 4.6) in small (0.9 ml) test tubes. The embryos were frozen and thawed five times by alternating placing the test tubes in Dry Ice-acetone and warm water. The test tubes were then placed in a controlled-temperature circulating water bath preheated to 70°C. At t = 0 and t = 30 min, lo-p1 samples were removed and added to a second micro test, tube containing 10 ~1 of assay buffer plus substrate (0.1 M acetate, pH 6.0, 6 x lo-+ M 4-MU-glucuronic acid). These tubes

106

DEVELOPMENTAL

BIOLOGY

VOLUME

were covered with Parafilm and placed in a closed chamber with a reservoir of buffer and incubated at 37°C for 24 hr. p-Glucuronidase is extremely stable at 37°C and activity is linear for more than 100 hr. The concentration of product in the micro test tube assay was determined by the following procedure. The assay mixture was deposited in nanoliter droplets under oil on microscope slides and made basic by incubating the slides in the presence of triethylamine. The fluorescence and diameter of several droplets from each assay mixture were measured. Fluorescence is directly proportional to the number of moles of product present in a droplet, and volume can be derived from the droplet diameter. From these data the concentration of 4-MU in the droplets is calculated.

48, 1976

strains C3H and B6 and the F, hybrids from C3H females mated with B6 males. Strain C,H possesses the Gush structural alleles for glucuronidase that codes for a more thermolabile form of the enzyme than the Gush allele present in B6. If embryonic and, particularly, paternally derived structural genes are being transcribed and translated, the enzyme from hybrid embryos should have heat denaturation characteristics different from C3H embryos. However, if the increased activity in hybrid embryos from C3H mothers during early embryogenesis is the result of translation of stored message, activation of stored enzyme or the product of differentially activated maternal genes then the enzyme in C3H and F, embryos would have the same activity and heat lability. The mean glucuronidase activities of emRESULTS bryos between 36 and 84 hr from B6, C3H p-Glucuronidase activities of single em- and F, hybrids from C3H mothers is shown bryos were measured between 36 and 84 hr in Table 1. These data show that C3H and of preimplantation development of hybrid embryos from C3H mothers have C57BL/6 mice. The distribution of embrybetween one-fifth and one-tenth the activonic enzyme activity at various stages of ity of B6 at the 2-cell stage, which is simidevelopment is shown in Fig. 1. The most lar to activity differences observed in adult striking feature of these data is the lOO- tissues between C3H and B6. A slight but fold increase in activity that occurs be- significant increase in activity occurred between 57 and 84 hr. Even though the em- tween 36 and 57 hr for all embryos. The bryos are cleaving during this time, the average increases in activity during this increase in cell number is only three- to period were 0.3, 0.8 and 1.0 fmoles per hr fourfold, thus the increased activity repre- per embryo for C3H, F, hybrid and B6 sents an increase in activity per cell. embryos, respectively. These data suggest To determine whether message for the that the paternal B6 gene may be exincrease in glucuronidase was transcribed pressed by this stage of development. before or after fertilization we examined The greater increase in glucuronidase of glucuronidase activities in embryos from F, embryos compared with C3H could also represent a generalized faster rate of development due to hybrid vigor. A qualitative determination of B6 gene expression in F, embryos was made from the heat denaturation kinetics of glucuronidase in 84-hr embryos. It became apparent that the F, embryos contain p-glucuronidase which is intermediate in heat sensitivity to that of single FIG. 1. p-Glucuronidase activity of the two parental strains and similar to C57BLKJ embryos during preimplantation developthat of adult hybrid animals (Table 2, Expt ment. Midnight of the day of mating is arbitrarily used as time

__ . of fertilization.

1).

WUDL AND CHAPMAN

~-Glucuronidase

in Mouse

107

Embryos

Since the embryos used in Expt 1 were The B6 homozygotes were omitted in this because it was difficult to obobtained at 36 hr from superovulated 21- experiment tain a sufficient number of 84-hr embryos 25-day-old females and grown in vitro, it from these females. Again, the F, hybrids was possible that we were not looking at contained a heat-stable form of p-glucuronthe normal developmental pattern. Thereidase indicating that the paternal gene for fore, we repeated the heat inactivation exp-glucuronidase is expressed with a 100” periment using 6-&week-old females induced to ovulate and mated according to fold increase which begins at 57 hr of develthe standard procedure. Embryos devel- opment. oped in viuo and were collected from the DISCUSSION uterus after 84 hr. The procedure for testThe loo-fold increase in activity in ing heat inactivation of /?-glucuronidase mouse embryos between 57 and 84 hr is the described for Expt 1 was followed. The re- most striking feature of p-glucuronidase sults are presented in Table 2 as Expt 2. expression in preimplantation development. To determine whether this expresTABLE 1 sion is due to stored message in the egg at P-GLUCURONIDASE ACTIVITY (lo-l5 MOLES HR-* fertilization or to the transcription and EMBRYO-') translation of embryonic genes we examStrain Hours postfertilizationa ined the properties of enzyme in embryos carrying different alleles of the glucuroni-, 36 57 60 65 84 dase gene. On the basis of the heat dena-, C57BLKJ turation kinetics of hybrid embryos from. Mean 0.8 1.8 5.5 20.2 150.2 C3H females which carry the heat-labile SE 0.05 0.1 2.7 3.5 31.2 Gus” alleles, we determined that the emF,: 32 49 5 31 20 bryonic genes including the paternally deMean 0.2 1.0 1.8 3.1 98.5 rived allele are expressed during the lOOSE 0.03 0.2 0.3 0.4 9.5 fold increase in activity. C3kHeJ 16 19 18 42 34 The increase in activity which occurs after 57 hr is greater on both a per cell and Mean 0.1 0.4 0.6 0.8 65.9 SE 0.01 0.02 0.07 0.09 6.4 a per embryo basis than the increase n 16 10 19 30 29 which occurs before this time. These findings could be the result of either (i) a devela Time of fertilization is arbitrarily set at midopmental induction of glucuronidase activnight after mating. ity which occurs at 57 hr or (ii) an initiab F, embryos from CeH/HeJ females x C57BLKJ males. tion of embryonic transcription and transTABLE

ACTIVITY (lo-i5 Strain

F,

C3H/HeJ

n Embryos h Embryos

from from

superovulated mature female

HR-* PL-') % Survival

to

t30

1” 1 26

3.1 43.4 27.5

1.7 16.9 11.6

54.8 38.9 42.2

1 2

16.9 50.0

2.4 5.0

14.2 10.0

ExpeZ?=t

C57BLi6.J (C3H x C57BL/6)

2

MOLES

mature induced

females grown to blastocyst stage to ovulate and grown to blastocyst

Survival (%I Adult liver enzyme (Paigen anfgt;nschow

in uitro. stage in uivo.

60 40 10

108

DEVELOPMENTAL

BIOLOGY

lation soon after fertilization which appears as a burst of activity at 57 hr due to the accumulation of glucuronidase and glucuronidase message. While our present data do not rule out either of these possibilities, there is evidence for the latter in that gene function may begin very early, possibly between 30 and 57 hr. Between 30 and 57 hr there is a greater increase in enzyme activity in B6 and F, embryos than in C3H. This difference is consistent with the relative differences observed between these genotypes in their adult tissue levels of glucuronidase (Ganschow and Paigen, 1968). Ganschow (1975) has further established that the difference in adult enzyme levels between C3H and B6 is due to a difference in the number of enzyme molecules present and that this difference results from a higher rate of synthesis in B6 compared with C3H. Three other enzymes that have been studied during preimplantation development have a similar timing of increase in activity: uridine kinase (Daentl and Epstein, 19711, hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (Epstein, 1970) and adenine phosphoribosyltransferase (Epstein, 1970). These enzymes increase about six- to tenfold during the period that glucuronidse increase lOO-fold. However, since no genetic variants of these enzymes are available it has not been possible to determine whether the messages for these enzymes were synthesized before or after fertilization. One possible exception is the expression of HGPRT in embryos from X0 and XX mothers (Epstein et al., 1972). In this case there is some inference that the message for HGPRT is produced soon after fertilization. Electrophoretic variants of structural genes have been used to study the timing of paternal gene expression for several loci including glucosephosphate isomerase, Gpi-1 (Chapman, et al., 1971; Brinster, 1973) isocitrate dehydrogenase Id-l (Donahue and Stern, 1971; Epstein et al; 1972), supernatant malic enzyme, Mod-l (Wolf

VOLUME

48, 1976

and Engle, 1972) and cytoplasmic glutamate oxaloacetate transaminase Got-l (Chapman and Graham, unpublished). Of these only the Gpi-1 paternal gene product has been detected in preimplantation embryos. Paternal gene expression for Id-l, Mod-l and Got-l has been observed after implantation but no data are available which indicate whether these enzymes are active preimplantation and if any activity changes occur before paternal gene products can be detected. Thus, it is not possible to determine whether these loci are not active until later in development or whether the assay techniques used were not sensitive enough to measure activity levels in earlier stages of development. The microassay system now available for p-glucuronidase can measure activity levels in single embryos and, potentially, in single blastomeres. This sensitivity coupled with the marked activation of Gus gene function we have now demonstrated and the availability of genetic variation for this enzyme suggest that glucuronidase will prove to be a very useful system for studying the control of expression of a specific structural gene during early embryogenesis. The authors thank Dr. Kenneth Paigen for his helpful suggestions in these experiments and for criticism of the manuscript. The technical assistance of Christine Heller is gratefully acknowledged. Valuable assistance was also rendered by Dr. Margaret Raney Holmes. REFERENCES BRINSTER, R. L. (1973). Paternal glucose phosphate isomerase activity in three-day mouse embryos. Biochem. Genet. 9, 187-191. CHAPMAN, V. M., WHITTEN, W. K., and RUDDLE, F. H. (19711. Expression of paternal glucose phosphate isomerase-1 (GPI-1) in preimplantation stages of mouse embryos. Develop. Bid. 26, 153158. DAENTL, D., and EPSTEIN, C. J. (1971). Developmental interrelationships of uridine uptake, nucleotide formation and incorporation into RNA by early mammalian embryos. Develop. Bid. 24, 428-442. DONAHUE, R. P., and STERN, S. (1970). Isocitrate dehydrogenase in mouse embryos: Activity and

WUDL

AND CHAPMAN

B-Glucuronidase

electrophoretic variation. J. Reprod. Fert. 22, 575-577. ELLEM, R. A. O., and GWATKIN, R. B. L. (1960). Patterns of nucleic acid synthesis in the early mouse embryo. Deuelop. Biol. 18, 311-330. EPSTEIN, C. J. (1970). Phosphoribosyltransferase activity during early mammalian development. J.

Biol. Chem. 245, 3289-3294. EPSTEIN, C. J., WESTON, J. A., WHITTEN, W. K., and RUSSELL, E. S. (1972). The expression of the isocitrate dehydrogenase. Develop. Biol. 27,430-433. GANSCHOW, R. (1975). Simultaneous genetic control of the structure and rate of synthesis of murine pglucuronidase. In Proceedings, III International Conference on Isozymes, (Markert, C. L. ed.). Academic Press, New York. GANSCHOW, R., and PAIGEN, K. (1968). Glucuronidase phenotypes of inbred mouse strains. Genetics 59,335-349. GOLBUS, M. S., CALARCO, P. G., and EPSTEIN, C. J. (1973). The effects of inhibitors of RNA synthesis (a-amanitan and actinomycin) on preimplantation mouse embryogenesis J. Exp. 2001. 186: 207216. GROSS, K. W., JACOBS-LORENA, M., BAGLIONI, C., and GROSS, P. R. (1973). Cell free translation of maternal messenger RNA from sea urchin eggs. Proc. Nat. Acad. Sci. USA 70, 2614-2618. EPSTEIN, D. J. (1972). Expression of the mammalian X chromosome before and after fertilization. Sci-

ence

175,

1467-1468.

JOHNSON, K. E., and CHAPMAN, V. M. (1971a). Expression of paternal genes during embryogenesis in the viable interspecific hybrid amphibian embryo Rana pipiens x Rana palustris. Electronhoretic analvsis of five enzvme svstems. J. -Exe. ” *1~

2001.

in Mouse Embryos 178,

109

313-318.

E., and CHAPMAN, V. M. (1971b). Expression of the paternal genes for glutamate oxaloacetate transaminases (GOT) during embryogenesis inxenopus laevis. J. Exp. Zool. 178,319324. KNOWLAND, J., and GRAHAM, C. F. (1972). RNA synthesis at the two-cell stage of mouse development. J. Embryol. Exp. Morphol. 27, 167-176. PAIGEN, K., and GANSCHOW, R. (1965). Genetic factors in enzyme realization. Brookhaven Symp. Biol. 18, 99-115. PAIGEN, K., SWANK, R. T., TOMINO, S., and GANSCHOW, R. E. (1975). The molecular genetics of mammalian glucuronidase, J. Cell Physiol. X5, JOHNSON,

K.

379-392. SKOULTCHI, A., and GROSS, P. R. (19731. Maternal histone messenger RNA detection by molecular hybridization. Proc. Nat. Acad. Sci. USA 70, 2840-2844. WHITTEN, W. K., and BIGGERS, J. B. (19681. Complete development in vitro of the preimplantation stages of the mouse in a simple chemically defined medium. J. Reprod Fert. 17, 399-401. WOLF, U., and ENGEL, W. (1972). Gene activation during early development of mammals. Human

Genet. 15, WOODLAND, synthesis

99-118.

H. R., and GRAHAM, C. F. (1969). RNA during early development of the mouse.

Nature (London)

221,

327-332.

WRIGHT, D. A., and SUBTELNY, S. (19711. Nuclear and cytoplasmic contributions to dehydrogenase phenotypes in hybrid frog embryos. Develop. Biol. 24.119-140. WUDL, L., and PAIGEN, K. (1974). Enzyme measurements on single cells. Science 184, 992-994.