Regulation of Mouse Preimplantation Development: Differential Effects ...

BIOLOGY

OF REPRODUCTION

41, 3 17-322

(1989)

Regulation of Mouse Preimplantation Development: Differential Effects of CZB Medium and Whitten’s Medium on Rates and Patterns of Protein Synthesis in 2-Cell Embryosl WILLIAM

T. POUEYMIROU,

JOANNE

C. CONOVER,

Department

and RICHARD

M. SCHULTZ2

of Biology

University of Pennsylvania Philadelphia, Pennsylvania 19104-6018 ABSTRACT In contrast

to Whitten’s medium, CZB medium, which lacks glucose but is supplemented with glutainine, oil-cell embryos beyond the 2-cell stage for embryos obtained from mice that exhibit the 2-cell block. The molecular basis for this effect, however, is not known. We report that correlated with the ability of CZB medium to support development beyond the 2-cell stage is an enhanced rate of total protein synthesis and higher level of synthesis of proteins that reflect activation of transcription of the embryonic genome when compared to embryos cultured in Whitten’s medium ± ethylenediaminetetraacetate (EDTA). The overall patterns of protein synthesis of embryos cultured in CZB or Whitten’s medium ± EDTA are similar, although the synthesis of the transcripti on-d ependent proteins is markedly delayed in embryos cultured in Whitten’s-EDTA. Last, there are no signcaiu differences in the adenosine 5’-triphosphase (ATP) levels in 2-cell embryos cultured from the I-cell stage in each of the media. supports

development

mine tent,

(EDTA) can foster, to a modest exdevelopment of 1-cell embryos to the blastocyst stage (Abramczuk et al., 1977). We have found, however, a large degree of variability in the extent of development when EDTA is included (Poueymirou and Schultz, unpublished observations). Recently, a medium (CZB) has been developed that supports a high level of development of 1-cell embryos obtained from outbred strains of mice to the blastocyst stage (Chatot et al., 1989). Virtually all of the embryos develop beyond the 2-cell stage and at least 50% of the embryos reach the blastocyst stage after 96 h of culture in vitro (Chatot et a!., 1989). Compared to a medium such as Whiuen’s medium, the apparent key modifications of CZB are a high lactate:pyruvate ratio, the omission of glucose, and the addition of 1 mM glutamine. The biochemical basis for why this medium supports extensive development beyond the 2-cell stage is not known. It should also be noted that omission of both glucose and phosphate from a chemically defined medium overcomes to a large extent the 2-cell block exhibited by hamster embryos; omission of glucose alone has a lesser effect (Schini and Bavister, 1988). We report here results of experiments that demonstrate that CZB supports a higher level of total protein synthesis in 2-cell embryos when compared to Whitten’s medium ± EDTA. The time of onset of transcriptional activation of the embryonic genome, which is

INTRODUCTION When cultured in vitro in a defmed medium, such as Whiuen’s medium (Whitten, 1971), 1-cell embryos obtained from most outbred strains of mice cleave to the 2-cell stage but do not develop to a high extent beyond this point. This failure to cleave beyond the 2-cell stage is termed the “2-cell block.” Reciprocal crosses with strains of mice that do not possess a 2-cell block with strains that do indicate that the block is due to a maternal effect (Goddard and Pratt, 1983). In addition, injection of cytoplasm obtained from blastomeres of 2-cell embryos that do not possess a 2-cell block into blastomeres of a 2-cell embryo that do possess the block rescues the injected embryo, which then can develop to the blastocyst stage (Muggleton-Harris et al.,

1982). The 2-cell block, however, is due in part to the composition of the culture medium, For example, although Whitten’s medium poorly supports cleavage beyond the 2-cell stage of embryos from outhred strains of mice, inclusion of iM concentrations of ethylenedia-

Accepted April 3, 1989. Received Febniaiy 3, 1989. tThis research was supported 21355 to R.M.S.). 2Reprnt requests.

by grants

from

the NIH

(HD22681

and

317

tetraacetate in vitro

POUEYMIROU

318

characterized by the synthesis of a set of protein whose synthesis is inhibited by a-amanitin, is similar for 2-cell embryos cultured in either CTh or Whinen’s + EDTA; there is, however, a significant delay in the onset of synthesis of these proteins in embryos incubated in Whitten’s EDTA. Increased synthesis of these proteins occurs in embryos incubated in CTh compared to the other two media, since the level of total protein synthesis is greater in CTh. These increases in the level of total protein synthesis and the level of proteins that are products of zygotic transcription are associated with the ability of embryos cultured in the media to cleave beyond the 2-cell stage. Last, there are no significant differences in ATP levels in 2-cell embryos cultured from the 1-cell stage in the different media, MATERIALS Embryo

Collection

and

AND

METHODS

Culture

Female mice (CF-i; 6-8 wk old; Harlan-Sprague Dawley, Indianapolis, IN) were injected with 5 IU pregnant mare’s serum gonadotropin (PMSG; CalBiochem-Behring, La Jolla, CA) and then with 5 IU human chorionic gonadotropin (hCG; Sigma Chemical Co., St. Louis, MO) 48 h later. A single female was placed with a single male (B6D2FJ/J; Jackson Lab, Bar Harbor, ME), and 1-cell embryos were recovered the next morning from the oviducts of females possessing a vaginal plug as previously described (Poueymirou and Schultz, 1987); the collection medium was bicarbonate-free minimal essential medium (Earle’s salts) supplemented with pyruvate (100 j.Lg/ml), gentamicin (10 jig/mi), polyvinylpyrrolidone (3 mg/mI), and 25 mM 2-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES), pH 7.2 (MEM/PVP). The embryos were cultured in Whitten’s medium (Whitten, 1971), Whitten’s medium containing 100 jiM EDTA (Abramczuk et a!., 1977), or CZB medium at 37C in a humidified atmosphere of 5% CO2 in air. To obtain a fairly synchronous population of 2-cell embryos, the “pick-off’ method was employed (Bolton et a!., 1984). Briefly, recently cleaved 2-cell embryos were culled every 1-2 h. The half-point of the time of pick off was arbitrarily set as 0 h post pick-off (hppo). (35SjMethionine Incorporation

Two-cell times

after

Radiolabeling and by Mouse Embryos

embryos, pick-off,

which were collected at defined were incubated in the appropriate

ET AL. medium act.>800

containing 1 mCi/ml of Trans-35S-label (sp. Ci/mmol; ICN, Irvine, CA) for 2 h. Trans-35 S-label is a mixture of about 85% methionine and 15% cysteine. After the period of radiolabeling the embryos were washed through six 200-p.! drops of bicarbonate-free MEM/PVP. Acid-soluble and acid-insoluble radioactivity were determined as previously described (Poueymirou and Schultz, 1987). One-

and

Two-Dimensional

Gel

Electrophoresis

One-dimensional gel electrophoresis was performed with a 4% stacking gel and a 10% separating gel in the presence of sodium dodecyl sulfate according to the method of Laemmli (Laemmli, 1970). Two-dimensional nonequilibrium pH gradient electrophoresis on mini-gels was performed with 11% separating gels as previously described (Bornslaeger et al., 1986). Radiolabeled [35S]proteins were detected by fluorography (Bonner and Laskey, 1974) at -70C using Kodak X AR5 X-ray film. Typically, 10-22 and 15-50 embryos were analyzed for one- and two-dimensional gel electrophoresis, respectively. Equal amounts of radioactivity were loaded onto the gels. Exposure times were usually about 18 h and 2-3 days for the one- and two-dimensional gels, respectively.

ATP

Determination

ATP was measured as previously described (Spielmann et al., 1984; Manejwala et a!., 1989). Briefly, groups of 2-cell embryos (5-6/sample), cultured in the appropriate medium, were transferred at various times to tubes containing 30 p.1 of tris(hydroxymethyl)aminomethane (Tns)-EDTA buffer (100 mM Tris-HCI, pH 7.75, containing 0.2 mM EDTA and 9 mM MgSO4). The samples were then frozen rapidly in dry ice-ethanol. Prior to the ATP assay, the samples were thawed rapidly, the volume was adjusted to 100 p.! with Tris-EDTA buffer, and the samples were boiled for 5 miii to inactivate phosphatases. Ten microliters of luciferin-luciferase assay mix (Sigma) was added and the luminescence was measured in a Turner photometer. RESULTS Effect of Different Culture Media on Cleavage of 1-Cell Embryos

The beneficial ment of embryos

effect of CTh medium on developfrom the 1-cell stage was confirmed

MOUSE

PREIMPLANTATION

(Chatot et a!., 1989). The kinetics and extent of cleavage of 1-cell embryos to the 2-cell stage was fairly similar for embryos incubated in either CZB, Whitten’s + EDTA, or Whitten’s EDTA (data not shown). In contrast to Whitten’s ± EDTA, CZB supported development beyond the 2-cell stage. For example, 68% of the embryos cultured in CZB (n = 279) reached the 4-cell stage by 37 hppo, whereas only 27% and 1% of the embryos cultured in Whitten’s + EDTA (n = 330) or Whitten’s EDTA (n = 150), respectively, reached the 4-cell stage. These experiments were conducted for technical reasons in 5% CO2 in air, and not 5% 02,5% CO2. 90% N2, which supports better development (Chatot et a!., 1989; Conover, unpublished observations). In addition, CZB medium supported extensive development of these 4-cell embryos to the blastocyst stage (about 60%), following transfer to Whitten’s + EDTA at the morula stage, whereas only about 0-8% of the 4-cell embryos continuously cultured in Whitten’s + EDTA developed to the blastocyst stage (data not shown).

DEVELOPMENT

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[35S]Methionine

Culture Uptake

Media and

Incorporation

Protein synthesis is required for progression through the cell cycle. The effect of the different culture media on [35S]methiornne uptake and incorporation into protein was therefore examined to ascertain if differences in the extent of total protein synthesis supported by the different media correlated with the ability of a given medium to foster cleavage of 2-cell embryos. Although uptake of [35S]methionine into the acid-soluble pool was similar for 2-cell embryos incubated in each of the media, CZB supported the higher level of incorporation of [355]methionine into acid-insoluble material (Fig. 1A,B). Moreover, the extent of this incorporation was associated with the ability of the different media to support cleavage of 2-cell embryos. The decreased incorporation of [35S]methionine into acid-insoluble material with increasing time after cleavage to the 2-cell stage probably reflected the destruction of maternal RNA that occurs during the 2-cell stage (Bachvarova and DeLeon, 1980; Piko and Clegg, 1982; Giebelhaus et a!., 1983). Expressing the data as the ratio of the number of cpm in the acid-insoluble material to that in the acid-soluble material, which corrects for differences in the specific activity of the [35S]methionine in the intracellular methionine pool, reinforced the conclusion that CZB supported a higher

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Time (hppo) FIG. I. Effect of different culture media on uptake and incorporation of [35Slmethionine into acid-soluble and acid-insoluble radioactive material. At the indicated time after cleavage to the 2-cell stage, embryos were cultured for 2 h in the appropriate medium and radioactive material was determined in the acid-soluble and acid-insoluble fractions as described under MaurlaLs and Methodj. A) Acid-soluble fraction. B) Acid-insoluble fraction. C) The ratio of the number of cpm in the acid-insoluble fraction (P) to the number of cpm in the acid-soluble fraction (S). The experiment was performed 3 times, and similar qualitative results were obtained in each case; shown is a representative experiment. S. CZB; 0 Whitten’s EDTA A, Whiuen’s EDTA; #qpo, half-point of the time of pick-off.

+

POUEYMIROU

320

rate of protein synthesis throughout the second cell cycle when compared to embryos incubated in Whitten’s ± EDTA (Fig. 1C). Effect

of the

Different

on Activation of the Pattern

Culture

Media

of Transcription

Embryonic of Protein

Genome

and

Overall

Synthesis

In the 2-cell mouse embryo changes occur in the pattern of protein synthesis that are due in part to activation of the embryonic genome (Flach et a!., 1982; Bensaude et a!., 1983; Bolton et a!., 1984). The synthesis of these protein, which is inhibited by a-amanitin (Flach et a!., 1982; Bensaude et al., 1983; Bolton et a!., 1984; Poueymirou and Schultz, 1987) provides a marker for transcriptional activation. The transcription-requiring proteins (TRPs) in the 70,000 dalton region are likely to be members of the heat-shock family (HSP-70) (Bensaude et a!., 1983; Bolton et a!., 1984) (Fig. 2). In addition, there is another protein of apparent molecular weight (Mr) of 74,000 whose synthesis is also inhibited by a-amanitin (Poueymirou and Schultz, 1987). The time for the onset of TRP synthesis was similar for embryos cultured in either CTh or Whitten’s + EDTA, but was markedly delayed in embryos cultured in Whitten’s EDTA (Fig. 2). The absolute amount of synthesis of the TRPs, however, was greater in embryos cultured in CTh as compared to Whiuen’s + EDTA, since the rate of total protein synthesis was greater in CZB than in Whitten’s + EDTA and equal amounts of radioactivity were applied to the gels. The overall temporal patterns of protein synthesis during the 2-cell stage were similar in both CZB and Whitten’s + EDTA (Fig. 2). In addition, the temporal pattern of protein synthesis of embryos incubated in Whiuen’s EDTA seemed retarded relative to that exhibited by embryos cultured in either CZB medium or Whitten’s + EDTA (Fig. 2; for example, see regions of the fluorograms in the Mr range of 116,000 and 25,000). These conclusions were also borne out by a two-dimensional nonequilibrium pH electrophoresis (Bornslaeger et a!., 1986) analysis performed at each of the time points indicated in Figure 2 (data not shown). Effect ATP

of Different Levels

in 2-Cell

The main difference CZB is the omission

Culture

Media

on

Embryos

between of glucose

Whitten’s medium and and addition of gluta-

ET AL. mine. Although 2-cell embryos take up and phosphorylate glucose, the metabolism beyond fructose-6-phosphate is apparently inhibited, due to an inhibition in phosphofructokinase activity (for review, see Kaye, 1986). Glutamine, which is a primary N donor, could also be an energy source due to its metabolism to glutamate, which can enter the TCA cycle as a-ketoglutarate. ATP levels were accordingly measured in embryos cultured in the different media, since ATP levels are a general indicator of metabolic health. Determination of ATP levels in 2-cell embryos, which were cultured from the 1-cell stage in the different media, at three different periods during the second cell cycle revealed no striking differences (Fig. 3), i.e., the levels were relatively constant during the 2-cell stage and were similar for embryos cultured in the different media. The ATP levels obtained in this study were lower than those previously reported by (Spielman et al. (1984). In that study, values of ATP of about 0.63 and 0.67 pmol/embryo were obtained for 2-cell embryos cultured in Whitten’s medium (no EDTA) for NMRI and C57BL strains of mice. We do not have an explanation for this difference, although strain differences may account for it. DISCUSSION Results of experiments reported here confirm the observation that CTh medium supports cleavage beyond the 2-cell stage of embryos obtained from mice that exhibit a 2-cell block. More importantly, our results extend those obtained in the previous study (Chatot et a!., 1989) by demonstrating that a positive correlation exists between the ability of CZB, Whitten’s + EDTA, and Whitten’s EDTA to support further development of 2-cell embryos and the rate of total protein synthesis, including the synthesis of proteins indicative of activation of the embryonic genome. In contrast, ATP levels were apparently unaffected by the different culture media. The ability of CZB to support cleavage of 2-cell embryos obtained from outbred strains of mice that possess a 2-cell block may be due in part to CTh’s ability to support a higher rate of protein synthesis; the molecular basis for CTh’s ability to foster increased levels of protein synthesis is not known, Inherent in this increase is an increase in the absolute amount of synthesis of proteins that reflect activation of transcription of the embryonic genome. Transcription, and thus synthesis of these proteins, is requisite for cleavage of 2-cell embryos (Bolton et a!., 1984). Thus, the greater levels of synthesis of these proteins may be related to

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PREIMPLANTATION

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FIG. 2. Fluorograms of 2-cell embryos cultured and radiolabeled in different media. At the indicated time after cleavage to the 2-cell atage, embryos were radiolabeled for 2 bin the appropriate media, and prepared for and subjected to one-dimensional gel eleclrophoresis as described under Materials ard Methods. The experiment was performed 3 times and similar results were obtained in each case; shown is a representative example. In one experiment, the increased synthesis of the zranscnplion-requiring protein (TRPs) at the later time periods was net observed for embryos cultured in Whinen’sEDTA. The arrowhead points to the region of synthesis of the TRPs. C, B medium; W, Whittcn’s medium EDTA, W-, Whitten’s medium - EDTA; hppo, half-point of the time of pick-off.

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POUEYMIROU

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total nucleotide reveal differences

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ET AL. pool, and hence energy charge, may not observed in the present study.

ACKNOWLEDGMENT

0.40

J.C.C. and R.M.S. would like to thank Clam Otatot and Carol Ziomek for informing us of their results prior to publication and encouragmg us to perform these studies, and W.T.P., i.C.C., and RJvtS. would like to thank Fazal Manejwala for performing the AlP assays.

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REFERENCES

0.10 0.00 CZB

W+EDTA

W-EDTA

Medium FIG. 3. Effect of the different culture media on adenosine 5’-monophosphate (ATP) levels in 2-cell embryos. ATP levels were determined as described under Materials and Methods. 1 experiment was performed 2 times, and uiplicatc measurements made for each experiment. Similar results were obtained and the data were pooled. Black bars, early 2-cell embryo; open bars, mid 2-cell embryo; suppled bars, late 2-cell embryo. The data are expressed as the mean ± SEM none of the differences between and among groups are significant by the Kruskal-Wallis teat.

the cleavage of these 2-cell embryos. In addition, the overall greater rate of total protein synthesis supported by CZB may also be the basis for the increased extent of cleavage, since protein synthesis is also required for cleavage. The synthesis of the TRPs in embryos cultured in Whitten’s EDTA is markedly delayed relative to that in embryos cultured in either CZB or Whiuen’s + EDTA. This delayed expression, the reduced level of total protein synthesis, and/or the reduced level of synthesis of the TRPs may account for the inability of these embryos to cleave extensively beyond the 2-cell stage. An obvious explanation for the better development supported by CZB medium would be that the overall metabolic health of the embryos is enhanced and that this might be reflected in differences in the ATP levels. No dramatic differences in the ATP levels are found, however, in 2-cell embryos cultured in the different media. A more thorough analysis that would entail determining the effect of the different media on the

Abramczuk J, Softer D, Koprowski H, 1977. The beneficial effect of EDTA on development of mouse one-cell embryos in chemically defined medium. DevBiol6l:378-83 BachvarovaT, DeLeon V. 1980. Polyadenylated RNA of mouse ova and loss of maternal RNA in early development. Dev Biol 74:1-8 Bensaude 0, Babinet C, Morange M Jacob F, 1983. Heat shock proteins, first major products of zygotic gene activity. Nature (Loud) 305:331-33 Bolton VN, Osdes PJ, Johnson MH, 1984. The relationship between cleavage, DNA replication, and gene expression in the mouse 2-cell embryo. J Embiyol Exp Morphol 79:139-63 Sooner WM, Laskey RA, 1974. A film detection method for tritium-labeled protein and nucleic acids In polyacrylamide gels. EuriBiochem46:83-88 Sonislaeger EA. Mauei PM. Schultz RM. 1986. Involvement of cAMP-dependent protein kinase A and protein phosphorylation in regulation of mouse oocyte maturation. Dcv Biol 114:453-62 Cbatoi CL, Ziomek CA, Bavister BD, Lewis JL, Torres I, 1989. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J Reprod Fertil 86:679-88 Flach G. Johnson MR. Braude PR, Taylor RAS, Bolton VN, 1982. The transition from maternal to embryonic control in the 2-cell mouse embryo. EMBOJ 1:681-86 Gielbelhaus DH, Heikkila ii, Schultz GA, 1983. Changes in the quantity of histone and actin messenger RNA during development of preimplantation mouse embryos. Dev Biol 98:148-54 Goddard Mi, Pratt RPM. 1983. Control of events during early cleavage of the mouse embryo at analysis of the “2-cell block”. I Einbsyol Exp Morphol 73:111-33 Kayc PL, 1986. Metabolic aspects of the physiology of preimplantation development. In: Rossant I, Pedersen RA (eds.), Experimental Approaches to Mammalian Embryonic Development. New York: Cambridge University Press, pp.267-92 Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond)227:680-85 Manejwala FM, Cragoc EJ Jr. Schultz P.M. 1989. Blastocoel expansion in the preimplantalion mouse embryo: role of cxtracellular sodium and chloride and possible apical mutes of their entry. Dcv Biol 133:210-20 Muggleton-Harris A, Whiuingham DG, Wilson L, 1982. Cyloplasmic control of preimplantation development in vitro in the mouse. Nature (Loud) 299:460-62 P11w L, Clegg KB, 1982. Quantitative changes in total RNA, total poly(A), and ribosomes in early mouse embryos. Dcv Biol 89:362-78 Poueymirou WT, Schultz RM, 1987. Differential effects of activators of cAMP-dependent protein kinasc and protein kinase C on cleavage of one-cell mouse embryos and protein synthesis and pbosphorylaticm in one- and two-cell embryos. Dcv Biol 121:489-98 Schini SA, Bavister BD, 1988. Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose. Biol Reprod 39:1183-92 Speilman H, Jacob-Mueller U, Schulz P, Schimmcl A, 1984. Changes of the adenine ribonucleotide content during preimplantation development of mouse embryos in vivo and in vitro. J Reprod Fertil 71:467-73 Whiuen WK, 1971. Nutrient requirements for the culture of preimplantation mouse embryos in vitro. Adv Biosci 6:129-39