Qualitative patterns of protein synthesis in the preimplantation mouse embryo

DEVELOPMENTAL

BIOLOGY

Qualitative

46, 446-451 (1975,

Patterns

of Protein

Synthesis

Mouse II. During

Release from

in the Preimplantation

Embryo

Facultative

Delayed

Implantation

JONATHAN VAN BLERKOM' Department

of Pediatrics.

B. F. Stolinsk,v

Laboratories. Colorado

C’niuersit.v 80220

of Colorado

Medical

Center.

DentIer.

AND GARS 0. BROCKWAI Department

of Molecular,

Cellular

and

Deuelopmental

Biology.

Chicersity

of Colorado.

Boulder,

Colorado

80302 Accepted

May

27, 1975

Qualitative patterns of newly synthesized protein were examined in mouse emhryos released from facultative delayed implantation either by removal from the uterus with subsequent culture of progesterone and estradiol 17(3 (in cico) to the ovariectomized in aitro, or by the administration mother. It was found that the qualitative pattern of protein synthesis during the first 25 hr following release from delay is the same as the pattern ohtained from normal hlastocysts immediately prior to implantation. These results are discussed in relation to possible mechanisms of activation of delayed hbstocysts. INTRODUCTION

thesis (Weitlauf, 1969) and carbon dioxide The ability of the mouse embryo to enter production (Menke and McLaren, 1970). A blastocyst in delay may be released from a state of facultati\re delayed implantation makes this species particularly useful for this condition and resume metabolic activof estrostudies of embryonic protein synthesis in ity either by the administration gen to the mother (McLaren, 1973), in relation to preimplantation development. follows within The delayed state may occur naturally as which case implantation 23 hr, or by removal of the during lactational delay (Enders, 1963), or approximately may be induced experimentally if a mouse embryos from the uterus followed by culis ovariectomized during the first few days ture in uitro (Gulyas and Daniel, 1969: Weitlauf, 1973). of the preimplantation period (McLaren, Three questions are central td an under1971). During this period of delayed implantation, the unattached blastocyst. free- standing of this regulated period of develliving in the uterus has been shown to enter opmental and metabolic arrest and its a state of arrested development or dor- control. 1) How similar are the qualitative mancy characterized by a cessation of mi- patterns of protein synthesis of an embryo tosis and DNA synthesis (McLaren, 1968) as it “comes out” of delay to the patterns of (cell division is arrested in Gl or early S; protein synthesis in embryos just prior to 2) Are there qualitative difSherman and Barlow, 1972) and by a dra- implantation? matic depression in the levels of RNA syn- ferences in the pattern of protein synthesis thesis (Prasad et al., 1968), protein syn- in embryos released from delay in oico and in uitro? 3) After the termination of the delay, do embryos synthesize proteins ‘Send reprint requests to ,Jonathan \‘an Blerkom. characteristic of earlier preimplantation Department of Pediatrics. B. F. Stolinskl. Laboratostages? We have attempted to examine ries. L’niversit?. of Colorado hledical Center, Denver. these questions by making a detailed comColorado 80220. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

446

447

BRIEF NOTES

parison of the qualitative patterns of protein synthesis in embryos released from the delay of implantation either by hormonal intervention or by removal from the uterus and culture in vitro. MATERIALS

AND

METHODS

Embryos used in this study were obtained from sexually mature, randomly bred HS mice (Heterogeneous Stock described by McLearn et al., 1970). Females were placed with males at 6 PM and checked for vaginal plugs at 8 AM the following morning. The day of detection of the vaginal plug was considered day 1 of pregnancy with implantation normally beginning on mid day 5. Bilateral ovariectomy was performed during the evening of day 3 of pregnancy and ovariectomized animals were not maintained on any hormonal regimen. Blastocysts in delay were collected on day 13 (8 days in delay), day 18 ( 13 days in delay I and day 22 ( 17 days in delay) of pregnancy. Animals on day 13 were divided into two groups; one group received a subcutaneous injection of estradiol 170 (1 pug) and progesterone (2 mg) in either propylene glycol or sesame oil, while animals in the second group were uninjected. Embryos recovered from the uteri of uninjected animals on the days of pregnancy indicated above were immediatel! placed into unlabeled culture medium, or medium containing YS-L-methionine and exposed to isotope for 1 hr. Embryos from uninjected animals were initially cultured in unlabeled medium for between 1 and 15 hr. and then transferred to labeled medium and exposed to ‘“S-t.-methionine for 1 hr. Animals that had been injected on day 13 of pregnancy (8 days in delay) were sacrificed at the following times post injection: 10 min. 30 min. 1, 5, 10. 13, 15. 20 and 25 hr. Blastocysts collected from these animals were immediately incubated for 1 hour in culture medium containing label. The culture medium used in this study was that described by Whitten (1971). Labeled

medium containing 200 &i/ml of YS-Lmethionine (specific activity 150 Ci/ mmole, New England Nuclear) was prepared as previously described (Van Blerkom and Brockway, 1975). Following exposure to radiolabeled methionine, embryos were prepared for elect rophoresis (Van Blerkom and Brockway, 1975). Electrophoresis was performed on either 7 to 14, or 8 to 15%’exponential gradient, SDS, polyacrylamide slab gels (described by Van Blerkom and Manes, 1974) and protein bands were visualized by autoradiography on Kodak x-ray film (type NSBT) (Van Blerkom and Manes. 1974). Each well of the slab gel contained the total amount of labeled protein obtained from four blastocysts exposed to label for 1 hr (between 36,000 and 40.000 cpm/lOO rig/protein). The gels were calibrated for molecular weight distribution as was previously described (V’an Blerkom and Manes, 1974). Each series was repeated at least four times, with the exception of embryos recovered on day 22. or 17 days in delay, in which only one experiment was performed. All embryos examined in this study were well-expanded, hatched, of normal appearance, and of approximately the same size. To ensure that the administration of estradiol 170 and progesterone to ovariectomized animals resulted in the implantation of delayed blastocysts, some animals that had been given an initial injection on day 13 of pregnancy were given daily injections of progesterone to maintain pregnancy. At various times later (days 15. 18 and ‘LO). these animals were sacrificed and the uteri examined for implantation sites. RESULTS

The qualitative patterns of protein synthesis from embryos which had been in delay for up to 17 days and recovered from the uteri of uninjected animals are presented in Fig. 1. Figure 2 shows the protein synthetic patterns of embryos obtained from animals treated with hormone to terminate the delay, prior to the collection

448

DEVELOPMENTAL BIOLOG\

-100

-70

-45

8

13

17

N.B

DAYS IN DELAY FIG. 1. This autoradiograph demonstrates the qualitative pattern of protein synthesis in embryos during release from facultative delayed implantation in uitro (after removal from the uterus of an uninjetted animal followed by culture in vitro for 1 hr in labeled medium). Embryos were in the delay of implantation for 8, 13 and 17 days (days 13, 18 and 22 of pregnancy). The pattern of protein synthesis obtained from embryos initially cultured in unlabeled medium for between 1 and 15 hr following removal from the uterus, and then exposed to labeled medium for 1 hr, is identical to the patterns presented in this figure. The pattern of protein synthesis of a normal -lay 5 blastocyst (just prior to implantation) is shown

VOLUME 46, 1975

of embryos. From an examination of these figures, it is apparent that there are no major qualitative differences in the pattern of protein synthesis in embryos released from delay either by culture in vitro after removal from the uterus (Fig. 1) or by hormonal intervention (in uivo) (Fig. 2). The pattern of protein synthesis obtained from embryos which were recovered from uninjected animals and initially cultured in unlabeled medium (for between 1 and 15 hr) was identical to the pattern obtained from embryos exposed to label directly after removal from the uterus (Fig. 1). A comparison of protein synthetic patterns of normal day 5 blastocysts with patterns obtained from delayed embryos shows no obvious qualitative differences (Fig. 1). From an examination of the protein patterns presented in Fig. 2, it is evident that the exposure of blastocysts to a uterine environment for up to 25 hr after stimulation by estradiol 170 and progesterone has no qualitative effect upon the pattern or protein synthesis at the level of resolution available in this electrophoretic system (Van Blerkom and Manes, 1974). The autoradiographic patterns also indicate that during the first 25 hr. following release from the delay in viva (or 15 hr in embryos cultured in vitro), blastocysts do not synthesize proteins which were characteristic of earlier preimplantation stages (Van Blerkom and Brockway, 1975). In this regard, not only is the qualitative pattern of protein synthesis during the immediate pre- and postdelay period unchanged, but also, this pattern appears both very rapidly, and in its entirety, following the termination of the delay; there is no time lag before certain bands appear. It is also of interest to note that the relative intensities of the autoradiographic bands during the immediate pre- and postdelay period are essentially the same. in the right-hand column (N.B.). Electrophoresis was performed on 7-14!%, exponential gradient slab gels. Approximate molecular weights (X 10m3) are given on the far right.

449

BRIEF NOTES

100 70

45

No 0.5 Hormone

5

10

15

20

25

HOURS POST INJECTION FIG. 2. This autoradiograph demonstrates the qualitative pattern of protein synthesis during the first 25 hr following release from delayed implantation in uiuo (following the injection of estradiol 176 and progesteronel. Embryos were in the delay of implantation for 8 days (day 13 of pregnancy) and the protein samples were electrophoresed on 8-150; exponential gradient slab gels. The pattern of protein synthesis in blastocysts released from delay in oitro shown in the left-hand column for comparison. This sample was from the same preparation of labeled embryonic proteins presented in the first column in Fig. 1. Protein patterns from blastocysts recovered at 10 min postinjection were identical to the patterns shown in this series. A slight difference in the resolution and position of some bands between Figs. 1 and 2 resulted from a slight difference in the gradient (for a detailed discussion of how minor differences in the gradient can effect the resolution of certain bands, see Van Blerkom and Manes. 1974). In Fig. 2, proteins denoted by arrow b have separated into two bands (only one band in Fig. 1) and the band at arrow a appears somewhat sharper in Fig. 2 than in Fig. 1. Approximate molecular weights ( x lo- 7 are given on the far right.

450

DEVELOPMENTAL Bro~ocl-

An examination of the uteri of animals injected with estradiol l’ip and progesterone on day 13 of pregnancy, as well as those of animals which were given subsequent injections, demonstrated that implantation had been accomplished by the administration of hormone. In addition, many blastocysts recovered at 25 hr following the injection of hormone to animals on day 13 of pregnancy appeared to be undergoing giant cell transformation. It is not known whether embryos released from delay in vitro retain the capacity to implant upon transfer to a suitable host. DISCUSSION

VOLUME 46. 1975

lyas and Daniel. 1969). Alternatively, the mRNAs which code for the proteins resolved in this electrophoretic system could be unusually stable, and the release from the delay may be associated simply with the termination of some other limiting condition(sj such as 1) the resumption or acceleration of rRNA and/or tRNA synthesis or, 2) from a marked change in the rate of uptake of nucleic acid and/or protein precursors (i.e. change in pool size). Until more is known about the fate of mRNA during delay, these interpretations, alremain speculative. though suggestive, However, it seems likely that at the current attainable resolution, the reactivation of delayed mouse blastocysts is accompanied by a quantitative rather than qualitative change in protein synt.hesis.

Studies by Weitlauf (19’i4) demonstrated that actinomycin D prevented delayed embryos from becoming fully activated and thereby attaining normal levels This work was supported hg a grant from the of protein synthesis in vitro. These experiNational Institutes of Health, U.S. Public Health ments were interpreted as indicating that Service (Grant No. HD-01274~. LVe would like to the activation of dormant embryos may thank Drs. Cole Manes and Martin H. Johnson for require the synthesis of new embryonic their helpful suggestions. RNA. Weitlaufs’ results, together with the REFERENCE finding that the qualitative pattern of ENDERS. A. C. (ed.) (19631. “Delayed Implantation” protein synthesis is the same during the Univ. of Chicago Press. Chicago, Illinois. immediate pre- and postdelay period sug- GULYAS. B. J.. and DANIEL, J. C., Jr. (1969). Incorporation of lahelled nucleic acid and protein precurgest that if new mRNA synthesis is resors by diapausing and nondiapausing hlastocysts. quired for full developmental activation, Bid. Reprod. 1, 11-20. then such new templates apparently code MCLEARN. G. E.. WILSON. J. R., and MEREDITH. W. for the same major species of proteins (1970). The use of isogenic and heterogenic mouse synthesized before the delay was initiated. stocks in behavioural research. In “Contrihutions to Behavior--Genetic Analysis-The Mouse as a ProThe rapidity with which the full pattern of totype”. (G. Lindzey and D. D. Thiessen. eds.1. pp. protein synthesis appears following the 3-22. Appleton-Century-Crofts. termination of delay could indicate that MCLAREN, A. (1968). A study of blastocgsts during the release from delay may not require new delay and subsequent implantation in Lactating mRNA synthesis, but possibly the activamice. J. Endocr. 42. 453-464. MCLAREN, A. (1971) Blastocysts in the mouse uterus: tion of preformed templates. This possibilthe effect of ovariectomy; progesterone and oestroity is especially interesting because it imgen. d. Endocrinol. 50, 515-526. plies that the onset of the delay is associMCLAREN, A. (1973) Blastocyst activation In “The ated with a storage or “masking” of Regulation of Mammalian Reproduction” (S. J. mRNA. Although at present there is no Sepal. R. Crozier. P. A. Corfman. and P. G. Condliffe, eds.). pp. 321-328. Charles C. Thomas evidence to support the existence of such a Pubs.. Springfield. Illinois. population of mRNAs in mammalian emMENKE, T. M., and MCLAREN. A. (19’iO1 Carbon bryos, such a storage process could be an dioxide production by mouse hlastocysts during option available to a class of mammalian lactational delay of implantation or after ovariecembryos when conditions for continued tomy. J. Endocrinol. 47, 287-294. PRASAD, M. N. R., DASS, C. M. S.. and MOHLA, S. development become unfavourable (Gu-

BRIEF NOTE> I 196XI. Action of oestrogen on the blastocvsts and uterus in delayed implantation: An autoradiographic study. J. Reprod. Fert. 16, 97-104. SHERMAN. M. I., and BARLOW. P. W. (19721. Deoxyribonucleic acid content in delayed mouse blastocysts. J. Reprod. Fert. 29. l’L?~L%. VAN BLERKOM. ,I., and MANES. C. t197-l). Development ot preimplantation rabbit embryos in ciL>o and in ~‘itro. II. .A comparison ot’qualitative aspect> ot’ protein synthehis. DeL’elop. Biol. 40, -1Wi1. VAN HLERKOM. J., and BHOCKM..A\. G. 0. I 1!);51. Qualitative patterns or protein synthesis in the preimplantation mouse embryo. I. Normal pregnancy. DeLlelop. Biol. 41, 148~157.

451

H. M. I 1969). Temporal changes in protein synthesis hy mouse hlastocysts transterred to ovariectomized recipients. J. Exp. Zool. 171, 481486. WEITLACIF. H. M. (197:31. In vitro uptake and incorporation of amino acids by hlastocysts from intact and ovariectomized mice. J. Exp. ZOOS.183, W-308. LVEITLAI'F. H. M. (19711. Effect ot’ actinomycin D on protein synthesis by delayed implanting mouse embryos in vitro. J. hp. &ml. 189, 19;-‘Lo’?. N'HITTEN, ItI’. K. t 197 1 I Nutrient requirements ot the culture of preimplantation embryos in Llitr0. In “Advances in the Bioscience” Yol. 6 \G. Raape. ed.1. pp. 129~111. Pergamon Prexh. England. WEITLACF.