DEVELOPMENTALBIOLOGY

148,620-624(1991)

BRIEF NOTES Effects of Exogenous Putrescine Preimplantation Development JANET A. SAWICKI, The Lankenau

Medical

Research

Center,

ANITA~MPELLIZERI, 100

Lalacaster Avenue Accepted

After first demonstrating V&J,

fertilized

eggs were

August

on Murine in Vitro

ANDTHOMASG.O’BRIEN West of City 16,

Line,

Wynnewood,

Pennsyluania

19096

1991

that murine embryos take up putrescine from the medium in which they are cultured in

placed

in culture

and maintained

for 4 days in medium

supplemented

with

varying

amounts

of

putrescine. Their development was monitored each day. While embryos that were cultured in putrescine-supplemented medium developed at the same rate as control embryos, a significantly higher percentage of the putrescine-treated embryos attained the blastocyst stage as compared to the control group. o 1992 Academic PWS, h. INTRODUCTION

inhibitor of ornithine decarboxylase (ODC), a rate-limiting enzyme in the polyamine biosynthetic pathway, was included in the drinking water of females in the early stages of pregnancy (Fozard et ah, 1980). The effects of elevated levels of polyamines in cells, tissues, and developing embryos are unknown. Although some interconversion of polyamines has been noted (Pegg et ah, 1982), polyamines are not typically converted to other metabolites in the cell. Thus one consequence of unregulated polyamine synthesis or uptake of exogenous polyamines would presumably be a rise in intracellular polyamine concentrations. Given the tight controls usually exerted on ODC and polyamine synthesis, and the precise interactions required of embryonic cells during the processes of differentiation and organogenesis, one might expect to find profound developmental defects as a result of abnormally high polyamine levels. With these thoughts in mind, we studied whether culturing preimplantation stage mouse embryos in the presence of exogenous putrescine would have any effects on very early development. A significantly higher percentage of embryos that were cultured in the presence of exogenous putrescine attained the blastocyst stage as compared to control embryos cultured in unsupplemented medium.

The diamine putrescine (1,4-diaminobutane) and the polyamines spermidine [H,(CH,),NH(CH,)4NH.J are present in all eukaryotic cells. In sum, they represent the major di- and polyvalent cations present in mammalian cells. Presumably because of their charge separation via methylene carbons, polyamines bind very tightly to nucleic acids, much more so than inorganic divalent cations such as M$+. While very little is known with certainty about the regulatory functions of polyamines in eukaryotic cells, it seemslikely that their interaction with macromolecules, especially nucleic acids and structures containing nucleic acids, is critical to their ability to modulate cell function. Despite our lack of knowledge of the precise mechanisms that are involved, it is well documented that polyamines are essential for normal growth processes. When the levels of polyamines in cultured mammalian cells are experimentally reduced or depleted, either by the use of specific inhibitors of polyamine synthesis (Coward and Pegg, 1987; Metcalf et ah, 1978) or by mutation (Snow, 19’78), cell proliferation is inhibited and changes in differentiation and development are observed. Several studies have addressed the effects of limiting polyamine levels during embryonic development (reviewed by Heby, 1987). Not surprisingly, a reduction in polyamine levels usually adversely affects development. In vitro culture of cleavage stage embryos in the MATERIALS AND METHODS presence of either of two inhibit,ors of polyamine synt,heStandard procedures were used to induce superovulasis affected the mitotic rate of cleaving cells and the processes of cavitation and hatching (Alexandre, 1978, tion in CD-l female mice (Charles River, Kingston Facil1979). In a study of postimplantation development, con- ity). Females were then mated with CD-l males. Eggs tragestational effects were observed in mice when an were harvested from the oviducts 12 hr postcoitum. 0012.1606/92 Copyright All rights

$3.00

0 1992 by Academic Press, Inc. of reproduction in any form reserved.

6‘20

621

BRIEF NOTES TABLE

1

UPTAKEOFEXOGENOUS [~H]PIJTRE~~INE BYPREIMPLANTATIONEMBRYOS A. cpm in polyamines

after

Stage

# embryos

Two-cell

in [aH]putrescine cpm [3H]putrescine taken up

28 45 60 13 21 40 20 21 30 10 13 17

Four-cell

Morula

Blastocyst

B. [3H]Putrescine

incubation

uptake

per embryo

Stage

cpm [aH]putrescine taken up/embryo

Two-cell Four-cell

1049 +- 290 2320 +- 792

20,800 73,280 46,500 21,600 81,840 56,000 97,000 90,600 67,400 42,900 26,000 60,900

[‘H]Spermidine

[aH]Putrescine 1,600 5,360 2,500 7,000 3,120 16,000 1,840 2,160 15,200 6,600 8,000 9,800

0 0 0 800 0 180 0 0 0 0 0 0

[aH]Spermine 0 0 0 0 0 440 0 0 0 0 0 0

and per cell cpm [aH]putrescine taken up/cell 525 580

Stage

cpm [3H]putrescine taken up/embryo

Morula” Blastocyst*

3804 -+ 794 3291 1 1172

cpm [3H]putrescine taken up/cell 127-190 51-110

n Morula have 20-30 cells. b Blastocysts have 30-64 cells.

Only fertilized eggs, identified by the presence of two pronuclei, were used in these experiments. Eggs and embryos were cultured in drops of medium under oil in humidified 5% CO,, balance air at 37°C (20-30 eggs or embryos/30 ~1 medium). Initial culture was in CZB medium (Chatot et ak, 1989). Once embryos reached the four-cell stage they were transferred to modified Whitten’s medium (Epstein et ah, 1969; Golbus and Epstein, 1974). Groups of test embryos were cultured in the presence of exogenous putrescine (Sigma Co., St. Louis, MO) at varying concentrations (5 PM, 25 wM, and 100 PM), while control embryos were cultured in unsupplemented medium. Each day the embryos were scored for the stages of preimplantation development that had been attained (two cell, four cell, morula, blastocyst). A statistical analysis for ranked categorical data using a one degree of freedom x2 was applied to the data. The analysis was carried out on SAS (Statistical Analysis System) using the CATMOD procedure. In radiolabeling experiments, four-cell CD-l embryos were cultured in vitro for 4 hr in modified Whitten’s medium containing 50 &i [3H]putrescine (Amersham, Arlington Heights, IL) and then washed six times in phosphate-buffered saline. Polyamines were extracted with 0.2 N perchloric acid, dansylated, and separated by thin layer chromatography according to previously described procedures (O’Brien et ak, 1980) to determine

rH] cpm incorporated spermine.

into putrescine,

spermidine,

and

RESULTS

To determine whether preimplantation embryos take up polyamines from the culture medium, two-cell, fourcell, morula, and blastocyst stage embryos were cultured in vitro for 4 hr in the presence of [3H]putrescine. After washing the embryos extensively, polyamines were extracted, dansylated, and the [3H] cpm in benzene extracts of the dansylated samples were determined. Benzene extracts were spotted on a thin layer chromatography plate to separate dansylated polyamines. Spots corresponding to putrescine, spermidine, and spermine, identified using uv light, were scraped into tubes, eluted, and the [3H] cpm were determined. The results of these analyses, summarized in Table lA, indicate that [‘Hlputrescine is taken up by cultured embryos at all stages. Furthermore, most of the rH] cpm in the polyamines extracted from embryos remains as rH]putrescine; there is very little conversion of putrescine to spermidine or spermine under these conditions. On a per embryo basis, two-cell embryos take up less PH]putreseine than do later stages. Two- and four-cell embryos, however, take up significantly more putrescine per cell than do morula and blastocysts (Table 1B).

622

DEVELOPMENTAL BIOLOGY 2-cell . . . . .. . . . *

100 1

F z7

60-‘h

-

4.ce,,

-____ r*--.

morula

-_--

blastocyst

l m--

VOLUME 148.1991

response then plateaus at some higher concentration. As shown in Fig. 2, the rate at which putrescine-treated embryos attained different developmental stages did not differ from the control embryos. In all embryo groups including the control group, most embryos are two-cell embryos on Day 1 in culture, four-cell embryos on Day 2, morula on Day 3, and blastocysts on Day 4. DISCUSSION

The results presented in this work add to a body of indirect evidence suggesting that polyamines play an important role in early development and differentiation ,,--------------in mammals. The mechanisms by which these molecules --------------------e Yi ,*' i; effect changes on cellular metabolism and/or gene ex/' pression are not known. z ,/ 20 j While the number of preimplantation mouse embryos required for direct determination of the levels of putreseine or polyamines in early developmental stages is proof . , . , . , . , . , . , hibitive, it is likely that putrescine levels are elevated in 0 20 40 60 60 100 120 cleavage stage embryos cultured in the presence of exogpM putrescine enous putrescine. The radiolabeling experiments with FIG. 1. Attainment of different stages of preimplantation develop[3H]putrescine reported here indicate that substantial ment by embryos cultured in medium containing putrescine. Fertiamounts of putrescine are taken up from the medium lized eggs were placed in culture and incubated for 4 days. Culture and that there is little conversion of putrescine to spermedia contained varying amounts of putrescine (0, 5, 25, or 100 PM). midine or spermine over a 4-hr period, suggesting that The vertical bars represent the standard deviations from the means. The experiment was repeated three times. (Sample sizes: no putrethe observed increase in viability of embryos cultured in seine, 122; 5 pM putrescine, 128; 25 pM putrescine, 122; 100 pM putreexogenous putrescine may be solely the result of eleseine, 112). vated levels of putrescine in the cells. A study by Zwierzchowski et al. (1986) demonstrated that an inhibitor of putrescine biosynthesis, DL-cY-difluoromethylorTo assess whether elevated polyamine levels affect nithine (a-DFMO), had no effect on mouse embryos cultured for 1 or 2 days, but on the third day, the DNA preimplantation development, fertilized eggs were harvested and cultured in the presence of varying concen- synthesis rate was markedly lower in ol-DFMO-treated embryos than in control embryos. Perhaps, as these autrations of putrescine. Each day, embryos were observed and scored for the developmental stage that had been thors suggest, the endogenous pool of putrescine is relaattained, two-cell, four-cell, morula, or blastocyst. The tively high at early cleavage stages. Then, at later stages, this pool may become depleted and render the percentage of cultured embryos that attained a particular developmental stage after 4 days in culture was de- embryos sensitive to the inhibition of putrescine biosyntermined. The results of this study are shown in Fig. 1. thesis. Repletion of this pool by the uptake of exogenous putrescine, as is likely the case in this report, may enEmbryos cultured in the presence of exogenous putreseine had a growth advantage over control embryos in able some embryos that could not thrive in culture to continue to develop. The fact that not all embryos are that significantly more of the treated embryos attained rescued by culture in putrescine-supplemented medium the four-cell, morula, and blastocyst stages. Statistical suggests that there are perhaps other metabolic pools analysis of the data using a x2 test for ranked categorical data and one degree of freedom indicates that, for all that are depleted to varying amounts in cultured embryos. concentrations of putrescine, the differences in developAnother possible explanation for the increased rigor ment between the control embryos and the treated emcultured in putrescinebryos are highly significant (5 PM putrescine, P < 0.015; associated with embryos medium is that these embryos simply 25 pM putrescine, P < 0.00005; 100 pM putrescine, P -c supplemented 0.0002). Further application of both the x2 test and a have more cells than nontreated embryos. This appears modification of the Wilcoxon Rank Sum procedure to not to be the case, however. When nuclei of embryo compare the test groups with each other supports a spreads are stained with acetoorcein or Hoechst stain dose-response relationship up to 25 PM putrescine; the there does not appear to be a significant increase in the

:0’ : I1’

623

BRIEF NOTES

4-cell

1.0

1.0

morula

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

1

blastocyst

0.0 1

2

3

4

FIG. 2. Rate of development of preimplantation embryos cultured in media containing varying amounts of putrescine. n , no putrescine control; 5 PM putrescine; 0, 25 pM putrescine; B, 100 KM putrescine. In each panel, the X axis is the number of days in culture and the Y axis is percentage of cultured embryos at particular stage x lo-‘. The vertical bars represent the standard deviations from the means. This data is taken from the same experiments as described in the legend to Fig. 1.

q,

number of cells in morula or inner cell masses of blastocysts cultures in putrescine-supplemented medium as compared to control embryos. For all the stages that were tested, the percentage of total counts taken up that was actually found to be associated with extracted polyamines was quite low (532%). Furthermore, the only labeled polyamine that was consistently detected was putrescine. Recently, Gramzinski et al. (1990) demonstrated that hydrogen peroxide is generated during oxidation of the polyamines spermine and spermidine by amine oxidases in the blastocoel-like fluid of embryoid bodies derived from mouse embryonal carcinoma cells. We are currently investigating the nature of the nonpolyamine associated radiolabel in blastocysts and earlier stage embryos to determine whether it is associated with oxidized polyamines.

We thank Spencer M. Free, Jr., for his assistance cal analysis. This work was supported by NIH (J.A.S.) and ES01664 (T.G.O.).

with the statistiGrants CA40402

REFERENCES ALEXANDRE, H. (1978). Effets de deux inhibiteurs de la biosynthese des polyamines sur la differentiation primaire de I’oeuf de souris. C R. Acad. Sci. Ser. D 286. 1215-1217. A LEXANDRE, H. (1979). The utilization of an inhibitor of spermidine and spermine synthesis as a tool for the study of the determination of cavitation in the preimplantation embryo. J. Embryol. Erp. Mmphol. 53, 145-162. CHATOT, C. L., et al. (1989). An improved culture medium supports development of random-bred I-cell mouse embryos in vitro. J. Rrprod. Ferfil. 86, 679-688. COWARD, J. K., and PEGG, A. E. (1987). Specific multisubstrate adduct inhibitors of aminopropyltransferases and their effect on poly-

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DEVELOPMENTAL BIOLOGY

amine biosynthesis in cultured cells. Adv. Enzyme Repel. 26, 107 113. EPSTEIN, C. J., et al. (1969). Biochemical development of preimplantation mouse embryos: In vivo activities of fructose 1,6-diphosphate aldolase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, and lactate dehydrogenase. Biochem. Genet. 3,271-281. FOZARD, J. R., et al. (1980). L-Ornithine decarboxylase: An essential role in early mammalian embryogenesis. Science 208,505-508. GOLBUS, M. S., and EPSTEIN, C. J. (1974). Effect of 5-bromodeoxyuridine on preimplantation mouse embryos development. D$erentiation 2,143-149. GRAMZINSKI, R. A., et al. (1990). Evidence linking programmed cell death in the blastocyst to polyamine oxidation. D&erentiation. 43, 59-65. HEBY, O., et a/. (1987). Polyamine synthesis inhibitors act as both inducers and suppressors of cell differentiation. In “Inhibition of

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Polyamine Metabolism” (P. P. McCann, A. E. Pegg, and A. Sjoerdsma, Eds.), pp. 165-186. Academic Press, New York. METCALF, B. W., et al. (1978). Catalytic irreversible inhibition of mammalian ornithine decarboxylase (EC 4.1.1.17.) by substrate and product analogues. J. Am. Chem. Sot. 100,2551-2553. O’BRIEN, T. G., et al. (1980). Regulation of polyamine biosynthesis in normal and transformed hamster cells in culture. Biochem. Biophys.

Acta 632,270-283.

PEGG, A. E., et al. (1982). Polyamine biosynthesis and interconversion in rodent tissues. Fed. Proc. 41,3065-3072. SNOW, M. H. L. (1978). Proliferative centres in embryonic development. In “Development in Mammals” (M. H. Johnson, Ed.), Vol. 3, pp. 337-362. Elsevier/North-Holland, Amsterdam. ZWIERZCHOWSKI, L., et ccl. (1986). Effect of polyamine limitation on DNA synthesis and development of mouse preimplantation embryos in z&o. J. Reprod. Fetiil. 76,115-121.