Differentiation of mouse trophoblast does not require cell-cell interaction

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Experimental

DIFFERENTIATION REQUIRE MICHAEL

Cell Research 123 (1979) 73-77

OF MOUSE

TROPHOBLAST

CELL-CELL

INTERACTION

DOES NOT

I. SHERMAN and SUI BI ATIENZA-SAMOLS

Roche Institute

of Moleculur

Biology,

Nufley,

NJ 07110, USA

SUMMARY Almost one-fifth of cells disaggregated from 16-cell mouse embryos neither divide nor die. On the basis of a number of criteria, it is concluded that these cells differentiate to tronhoblast. Thus, neither cell-cell interaction nor cell division is required for execution of the-programme for trophoblast differentiation from the 16-cell stage.

Trophoblast cells derive from the outer following the fate of trophectodermal vestrophectoderm layer of the blastocyst [ 1,2]. icles, structures which never had any ICM In the rodent, these cells, which comprise cells [19], Sherman [20] and Wudl & Shermuch of the fetal component of the pla- man [21] demonstrated that the trophectocenta, are distinguishable from other cells derm-to-trophoblast conversion occurs in derived from the zygote by their giant size the absence of contact with any other cell [3], polyploid nuclei [4, 51, invasive nature type. The experiments described below [6,7] and expression of specialized enzyme were undertaken to determine whether any activities, including A5,3P-hydroxysteroid cell contact is necessary for the differentiadehydrogenase (3P-HSD) [g-l l] and plas- tion of trophectoderm cells to trophoblast. minogen activator [ 121.Neither uterine factors nor contact with maternal cells is reMATERIALS AND METHODS quired for the expression by trophoblast cells of these and other properties; in fact, Embryo disaggregation and culture Two-cell embryos were removed from oviducts of trophoblast cells can differentiate from sunerovulated I221 SWRlJ females mated with SJL/J trophectoderm progenitors even when the males on the second day of pregnancy, i.e., the day observation of a sperm plug. Embryos were embryos fail to hatch from the zona pellu- following cultured for 24 h at 37°C in 5% CO&r in the precida [5, 13, 141.When cells of the inner cell implantation culture medium described by Goldstein al. [16]. The resultant 4- to &cell embryos were mass (ICM) of the mouse blastocyst are et transferred to the same medium with the exception selectively killed such that only trophecto- that Mg2+ and Ca*+ had been omitted and appropriate of NaCl had been added to maintain the ionic derm cells and their progeny survive [15- amounts strength. This medium prevents compaction of the em181, the cells resulting from these treat- bryos [23], but allows the blastomeres to undergo at one further division during an incubation period ments resemble trophoblast by several cri- least of 24 h. Embryos were then pipetted up and down in teria [ U-181, suggesting that continued con- drawn out capillary tubes, first to break the zona pellucida and then to separate the blastomeres. Only tact with ICM derivatives is not required those embryos containing 15-18 cells were used for for trophoblast differentiation. Finally, by analysis. In some cases, incompletely disaggregated Exp Cell Rcs 123 (1979)

74

Sherman and Atienza-Samols

Table 1. Fate of blastomeres isolated from sixteen-cell embryosa Population Total blastomeres Blastomeres undivided after 24 h

No. scored

Time following disaggregation (hours)

% Divided

% Viable but undivided

240

24

536

43

5

102

48

13

39

48

% Dead

’ Blastomeres, disaggregated as described in Materials and Methods, were cultured in the preimplantation medium of Goldstein et al. [16] for 24 h following disaggregation. Survivors were then transferred to supplemented NCTC-109 medium. b Of these blastomeres that did divide. one of the resultant daughter cells died within the subsequent 24 h period at a frequency of 56 %. groups of blastomeres were saved as controls. Follow&g disaggregation, blastomeres were cultured for 24 h in the medium of Goldstein et al. [16]. Thereafter, medium was changed to NCTC-109 [24] supplemented with antibiotics and 10% heat-inactivated fetal calf serum (FCS) [12].

A5,3@Hydroxysteroid dehydrogenase determinations Blastomeres were cultured individually, or in some cases (controls) in groups, in wells of Terasaki tissue culture plates (Falcon Plastics, Oxnard, Calif.). Cell numbers were determined daily. At the indicated times, recrystallized pregnenolone (1 pg/ml final concentration) was added to the medium. After 24-40 h, medium was collected and progesterone levels were determined by radioimmunoassay [lo].

Plasminogen activator assays Individual (experimental), or groups (control) of, blastomeres were seeded in 3.5 mm tissue culture dishes (Falcon Plastics) and assayed for plasminogen activator production either after 24 h (controls) or 96 120 h (experimentals) of incubation. Assays were for 24 h periods by the fibrin-agar overlay procedure described previously [12, 251. Cells and overlay were then stained with Coomassie brilliant blue [25].

DNA determinations Blastomeres were cultured either in Terasaki tissue culture plates or in Lab-Tek tissue culture eight-chamber slides (Miles Laboratories, Nanerville, Ill.). Seven days after the blastomeres had been disag&egated, the plates and slides were washed, cells were fixed and stained with SchilTs reagent, and nuclear DNA contents in single cells were measured by microfluorometrv, all as described ureviouslv f5. 211. Fluorescence units were converted to ploidy values by comparison with liver nuclei, which have dinloid, tetraplaid or octaploid DNA contents (see ref.-[5]). ‘Due to normal scatter in the values obtained for tetraploid liver nuclei, trophoblast nuclei or cells were considered to be unequivocally in the polyploid range only if they contained more than six times the haploid amount of DNA. Exp Cell Res 123 (IY7Y)

RESULTS AND DISCUSSION Under appropriate conditions of disaggregation and culture (see Materials and Methods), substantial numbers of individual blastomeres from 16-cell mouse embryos survive for several days, and some of these cells do so without further division. For example, table 1 illustrates that in a typical series of experiments involving isolation and 24 h culture of a total of 240 blastomeres from 15 to l&cell embryos, 102 blastomeres failed to divide but remained viable. After transfer to serum-supplemented NCTC-109 medium and a subsequent 24 h incubation, almost half of the blastomeres that had originally failed to divide died and some of the other blastomeres showed one or more delayed cell division(s). However, 39% of the blastomeres, (that is, 17% of the original population of the blastomeres under study) remained as single cells. These cells generally attached to the culture dish within 72 h of disaggregation and flattened down on the substratum about 24 h thereafter. We observed that about half of the blastomeres that divided in the first 24 h following disaggregation subsequently lost one of the progeny, resulting in a single blastothereafter from mere indistinguishable those that had not divided during the period of observation. Although these blastomeres

Differentiation of mouse trophoblast cells

75

B ri)

Q

Q C

d

1 0

I

@m 1

50

100

I 150

fg

I

I

200

250

0 I 300

pg progesterone produced124 h; ordifrequency. A5,3P-Hydroxysteroid dehydrogenase activity in blastomeres from sixteen-cell embrvos. (A) Control blastomeres (individuals and groups), assayed from 2U8 h after disaggregation; (B) individual blastomeres, assayed from 96-120 h after disaggregation; (C) individual blastomeres, assayed from 120-160 h after disaggregation. Note that regardless of the number of cells present (numbers inside symbols) in (A), the culture media did not contain above-background (25 pg) amounts of progesterone (the values observed are due to low levels of cross-reactivity of the antibody with the substrate, pregnenolone [lo]). The squares and circles in (B) and (C) refer to blastomeres which had never divided or divided once followed by the death of a daughter cell, respectively. Nuclear numbers are indicated inside symbols in (B) and (C).

Fig. I. Abscissa: nate:

had become single cells once again prior to the time at which differentiated markers were detectable (see below), they had not, strictly speaking, developed in the absence of cell contact from the 16-cell stage. Consequently, in some experiments, cells were inspected daily to determine which had never divided and which had divided once and then lost a daughter cell. We also noted that the majority of single blastomeres which developed successfully were multinucleate: of 55 blastomeres monitored in two typical experiments, only 11 (20%) were mononucleate, whereas 39 (71%) were binucleate, and 5 (9%) had more than two nuclei. Therefore, we have compared the developmental capacity of mononucleate and multinucleate cells.

Fig. 2. Production of plasminogen activator by an iso-

lated cell developing from a 16-cell blastomere. The assay procedure is as described in Materials and Methods. The cell, in this base binucleate, has secreted plasminogen activator, leading to activation of plasmin and formation of a clear zone of lysis of the fibrin originally surrounding the cell.

We reported previously that prior to the time of their attachment to, and outgrowth along, the substratum, trophoblast cells do not possess 3/3-HSD activity [I 11. Consistent with this observation, fig. 1A illustrates that within 48 h of disaggregation, that is, prior to the time equivalent to that required for trophoblast outgrowth, neither single cells nor groups of up to four cells developing from disaggregated blastomeres were capable of converting significant amounts of pregnenolone to progesterone. However, 34 days later, 68% of individually cultured blastomeres produced detectable amounts of progesterone from pregnenolone (fig. lB, C), illustrating that they had acquired 3/3-HSD activity. Activity was detectable both in cells developing from blastomeres which had never divided and those which had divided once followed by death of a daughter cell. No correlation was Exp Cell Rrs 123 (1979)

76

Sherman and Atienza-Samols

octaploid,

or greater, amounts of DNA

We conclude that at least some cells isolated at the 16-cell stage have the capacity to differentiate to trophoblast in the absence of cell division or cell-cell interaction. This reinforces our earlier proposal [ 18,201 that the program for trophoblast dif2 4 8 16 32 64 128 ferentiation is an intrinsic property of proFig. 3. Abscissa: DNA content (log, scale); ordinate: genitor cells, perhaps from as early as the freauencv. DNA contents of individual cells developing from two-cell stage. The data indicate further isolated 16cell blastomeres after 7 davs of culture. that if a ‘quanta1 cell division [27] is re(A) Ploidy values of individual nuclei. Numbers withquired for the trophectoderm-to-trophoin the circles indicate whether the nuclei were from mono-, bi- or tri-nucleate cells. (B) Total DNA con- blast conversion, it occurs prior to the 16tent of each cell. Numbers in the circles refer to the number of nuclei that each cell contained. The dotted cell stage, and many days earlier than line separates values considered to be in the diploid the time at which differentiation-related and polyploid ranges. markers are detectable. (Of course, since polyploidization takes place following disaggregation, a quanta1 endoreduplicative evident between nuclear numbers and lev- cycle might be involved instead.) Finally, els of 3P-HSD activity. since all cells in trophectodermal vesicles We have also demonstrated that produc- appear to differentiate to trophoblast [20], tion of plasminogen activator by mouse presumably because they all occupy an embryos begins during in vitro stages equi- ‘outside’ position [see ref. 21, the same valent to implantation [ 12, 261. When 2 1 should apply to the individually cultured blastomeres or groups of 2-4 blastomeres blastomeres in this experiment. Whether were assayed for plasminogen activator ac- the small proportion of cells which did not tivity by the fibrin-agar overlay assay [25, express characteristic trophoblast markers 261 between 24 and 48 h following disag- failed to do so because they were in poor gregation, all were negative. On the other health or because they had already been hand, of 44 individual cells assayed for programmed to produce ICM cells remains plasminogen activator 3 or 4 days later, to be determined. after the cells had flattened on the culture wish to thank Dr Marilyn Monk, MRC Mamdish, 22 cells gave clear plaques (e.g. fig. 2) We malian Development Unit, London, for suggesting the and 5 showed traces of activity. use of calcium-free medium for facilitating embryo disaggregation and Drs A. Weissbach and J. Monahan We determined, by microfluorometry fol- for comments on the manuscript. lowing Feulgen staining [21], nuclear ploidy in individual cells following their attachment to the culture dish. More than 70% REFERENCES of nuclei. whether from mononucleate or 1. Gardner, R L, Adv biosci 6 (1971) 279. multinucleate cells, were in the polyploid 2. Hillman, N, Sherman, M I & Graham, C F, J embryo1 exp morph01 28 ( 1972)263. range (fig. 3A). When nuclear DNA con3. Duval, M J, Anat physio127 (1891) 279. tents from multinucleate cells were totalled, 4. Zybina, E V, Dokl acad nauk SSSR 153 (1963) 85 % of the cells were found to contain 1428. ExpCdlRes

/23(1979)

Differentiation of mouse trophoblast cells

77

5. Barlow, P W & Sherman, M I, J embryo1 exu 18. Rizzino, A & Sherman, M I, Exp cell res 121 (1979) 221. morph01 27 (1972) 447. 6. Kirby, D R S, The early conceptus normal and 19. Tarkowski, A K & Wroblewska, J, J embryo1 exp abnormal (ed W W Park & S Livingstone) p. 68. morphol 18 (1967) 155. Universitv of St Andrews Press, Edinburgh 20. Sherman, M I, The early development of mam(1965). _ mals (ed M Balls & A E Wild) p. 145. Cambridge 7. Salomon, D S & Sherman, M I, Exp cell res 90 University Press, London (1975). (1975) 261. 21. Wudl, L R & Sherman, M I, J embryo1 exp morph01 48 (1978) 127. 8. Deane, H W, Rubin, B L, Driks, E C, Lobel, B L 22. Runner, M N & Palm, J, J exp zoo1 124(1953) 303. & Leipsner, G, Endocrinology 70 (1962) 407. 9. Chew, N J & Sherman, M I, Biol reprod 12 (1975) 23. Ducibella, T & Anderson, E, Dev biol 47 (1975) 351. 45. 10. Marcal, J M, Chew, N J, Salomon, D S & 24. Evans, V J, Bryant, J C, Kerr, H A & Schilling, Sherman, M I, Endocrinology 96 (1975) 1302. E L, Exp cell res 36 (1964) 439. 11. Sherman, M I & Atienza, S B, Biol reprod 16 25. Beers, W H, Strickland, S & Reich, E, Cell 6 (1977) 190. (1975) 387. 12. Strickland, S, Reich, E & Sherman, M I, Cell 9 26. Sherman, M I, Proteins and steroids in early (1976) 231. mammalian development (ed H M Beier & P Karlson). In press. 13. Sherman, M I, Dev bio127 (1972) 337. 14. - Exp cell res 75 (1972) 449. 27. Bischoff, R & Holtzer, H, J cell bio141 (1969) 188. 15. Sherman, M I & Atienza, S B, J embryo1 exp morph01 34 (1975) 467. 16. Goldstein, L S, Spindle, A I & Pedersen, R A, Received January 31, 1979 Radiat res 62 (1975) 276. 17. Rowinski, J, Solter, D & Koprowski, H, J exp zoo1 Revised version received May 3, 1979 192(1975) 133. Accepted May 8, 1979

Exp Cd Res 123 (1979)