protein synthetic requirements for the outgrowth of trophoblast cells from mouse blastocysts

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 224:401-408 (1982)

Protein Synthetic Requirements for the Outgrowth of Trophoblast Cells From Mouse Blastocysts

EMILY J. BLAKE, JOKL SCHINDLER, AND MICHAEL I. SHERMAN Roehe Institute of Molecular Biology, Nutley, New Jersey 07110 (J.S., M.I.S.) and Deprtment of Human. Genetics and Dmelopment, Colum.bia University College of Physicians and Surgeons, New York, New York 10052 (E,.J.B,, M . I.S.)

ABSTRACT Mouse blastocysts cultured under optimal conditions attach to the surface of the culture dish and subsequently give rise to outgrowths of trophoblast cells. The migration of trophoblast cells appears to be analogous to their behavior during the invasive phase of implantation in utero. In these studies, we have attempted to determine the time of synthesis and nature of the products required for trophoblast outgrowth. Fourth-day blastocysts were cultured in two different media: cNCTC, a nutrient medium supplemented with fetal calf serum, and PCMF, a simple preimplantation medium lacking amino acids and serum. Trophoblast outgrowth occurs in the former, but not in the latter, medium. Most blastocysts will give rise to outgrowths following as little as 12 hr of exposure to cNCTC at the appropriate time, even if they are subsequently placed in PCMF. Outgrowth fails to occur if blastocysts are treated with cycloheximide during the interval of exposure to cNCTC, whereas treatment with the antimetabolite after the period of culture in cNCTC does not block outgrowth. These observations are consistent with the view that protein(s) essential for trophoblast outgrowth are synthesized many hours prior to the actual event.

Successful implantation of the mammalian embryo requires that both the mother and the conceptus undergo a series of specific, ordered changes in a coordinated fashion. Disruption of the endocrinological balance in the uterus can prevent implantation and interrupt the developmental program of the blastocyst. In mice, implantation delay occurs naturally when a pregnant female suckles a previous litter or it can be induced by ovariectomy; in both instances, implantation can be achieved, and the developmental program of the conceptus restored, when the hormonal insufficiency is rectified (Whitten, '55; Yoshinaga and Ad- ams, '66). During implantation delay, the developmental program of the blastocyst appears to be disrupted by virtue of a generalized suppression of metabolic rates (Menke and McLaren, '70; Weitlauf, '74) and biosynthetic activities (McLaren, '68; Weitlauf and Green- wald, '68; Sherman and Barlow, '72; Weitlauf, '73; Chavez and Van Blerkom, '79; Van Bler- kom et al., '79). Although some early experiments suggested that hormone administration reverses implantation delay through a direct effect upon the blastocyst (Psychoyos, '67;

Prasad et al., '68; Smith, '68; Smith and Smith, '71), it now appears more likely that hormones act indirectly by altering the uterine milieu, thereby permitting restoration of normal metabolic processes in the conceptus (see Sher- man and Wudl, '76; Van Blerkom et al., '79).

During implantation in the mouse, trophoblast cells attach to, and subsequently invade,. the uterine wall. Under appropriate culture conditions, trophoblast cells will behave in an apparently analogous way (see Sherman and Wudl, '76): They initially become adherent to the substratum and then migrate out along it. These events can be blocked by placing blastocysts in suboptimal culture medium, e.g., in the absence of serum factors (Gwatkin, '66; Rizzino and Sherman, '79; Sellens and Sher- man, '80), amino acids (Gwatkin, '66; Spindle and Pedersen, '73; Naeslund, '79; Sellens and Sherman, '80; Sherman and Matthaei, '80), or

Joel Schindler is now at the Department of Anatomy. University

Address reprint requests t o Michael I. Sherman, Roche Institute of Cincinnati Medical Center, Cincinnati. OH 45267.

of Molecular Biology, Nutley, NJ 07110.

0022-104W82/2243-0401$02 50 'c, 1982 ALAN R. LISS. INC

402 E.J. BLAKE, J. SCHINDLER, AND M. 1. SHERMAN

glucose (Wordinger and Brinster, '76; Naes- lund, '79; Van Blerkom et al., '79). Sellens and Sherman ('80) provided evidence that blastocysts cultured in these suboptimal media re- semble those blocked from implanting in utero in several respects. Preliminary studies suggested that trophoblast outgrowth was blocked in suboptimal culture medium because blastocysts could not enter a critical biosynthetic phase (Sherman and Matthaei, '80). The purpose of the present study was to define this biosynthetic period further and to learn the nature of the biosynthetic products that are critical for trophoblast outgrowth.

MATERIALS AND METHODS Materials

NCTC-109 medium and fetal calf serum (heat-inactivated at 56°C for 20 min) were purchased from Microbiological Associates (Be- thesda, MD). Antibiotics were purchased from Gibco (Grand Island, NY). Bovine serum al- bumin (BSA; PentexlMiles, Elkhart, IN) and fetuin (Calbiochem, La Jolla, CA) were solu- bilized and dialyzed extensively as described by Sellens and Sherman ('80). Cycloheximide (Calbiochem) was dissolved in distilled water at 1 mglml and filter sterilized. Fetuin and cycloheximide stock solutions were stored at -2O"C, thawed once and maintained at 4°C during use for a maximum of 1 (cycloheximide) or 2 (fetuin) weeks. Plastic tissue culture dishes (35 mm) were purchased from Falcon Plastics (Oxnard, CA). [3"S]Methionine ( - 1000 Cilmmol) and unlabled methionine were purchased from Amersham (Arlington Heights, IL) and Calbiochem, respectively. Eppendorf centrifuge tubes were obtained from Fisher. Scientific (Pittsburgh, PA). Hydrofluor (Na- tional Diagnostics, Somerville, NJ), NCS (Amersham), and LSC (Yorktown Research, Hackensack, NJ) were used for scintillation spectrometry, as indicated.

Culture media cNCTC is NCTC-109 medium supplemented

with penicillin (100 ulml), streptomycin (100 pglml), kanamycin (100 ulml), and 10% heat- inactivated fetal calf serum. PCM is preimplantation culture medium formulated as described by Goldstein et al. ('75) except that the BSA was dialyzed prior to use (Sellens and Sherman, '80). PCM supplemented with 0.05% dialyzed fetuin is referred to as PCMF.

Embryos SWRlJ female mice (Jackson Laboratory,

Bar Harbor, ME) were superovulated (Run-

ner and Palm, '53) and mated with SJLlJ males (Jackson Laboratory). The day of observation of the sperm plug was considered the first day of pregnancy. Blastocysts were collected on the afternoon of the fourth day of pregnancy. All embryos were obtained by flushing uterine horns with phosphate-buffered saline [solution A of Dulbecco and Vogt ('54)]. Embryos were washed in PCM and then transferred for culture into either PCMF or cNCTC.

Cultur-e conditions Embryos were cultured in groups of 20 to

35 in 3 ml of PCMF or cNCTC medium, as indicated, in 35-mm tissue culture-grade plastic dishes and maintained at 37OC in a humid- ified atmosphere of 5% CO, in air. Embryos were scored as positive for outgrowth when at least one trophoblast cell and its nucleus could be seen flattened out on the culture dish surface.

Radioactive labehzg of embryos Labeling of embryos with [35S]methionine

was carried out in 1.5-ml Eppendorf centrifuge tubes in 30-50 p1 of radioactive medium. The [35S]methionine concentration ranged from 0.25 to 0.35 pCilpl in PCMF and from 1.0 to 1.5 pCi/pl in cNCTC for incubations of 16- 24 hr. Different concentration ranges were used in the two media because of differences in protein synthetic rates and because of the presence of unlabeled methionine in cNCTC but not PCMF. Under these conditions, blastocysts cultured in PCMF or cNCTC incorpo- rated approximately 24,000 and 13,000 c p d embryo, respectively, into trichloroacetic acid- insoluble material (see below). Following the labeling periods, the embryos were washed twice with 1 ml PCM lacking sodium pyru- vate, sodium lactate and BSA but supplemented with 0.02 mglml cold L-methionine, and stored frozen at -70°C.

Determination of trichloroacetic acid-soluble and -insoluble radioactivity

To determine the total uptake of [Y3]methionine by embryos, 0.5 ml of a 1.0 mglml BSA solution was added to the embryo samples. Following three rounds of freezing and thaw- ing, ice-cold trichloroacetic acid (TCA) was added to yield a final concentration of 10%. The samples were placed on ice for 30 min and centrifuged, and then supernates were re- moved and saved. Pellets were resuspended in hot TCA, placed in a boiling water bath for 15 min, centrifuged, and the supernates pooled with those from the 10% cold TCA extraction.

CONTROL OF TROPHOBLAST OUTGROWTH 403

Hydrofluor was added to the pooled supernates, and samples were counted to determine TCA-soluble counts. The precipitated material was digested for 1 hr at 37OC with NCS, transferred to scintillation vials, and counted with LSC to determine the extent of incorporation into TCA-insoluble material.

RESULTS Conditions that allow blastocysts to form

trophoblast outgrowths Blastocysts cultured in cNCTC hatch from

their zona pellucida, attach to the substratum, and form outgrowths of trophoblast cells that migrate away from the embryonic mass (Sher- man, '75). Blastocysts cultured in PCMF hatch and attach to the substratum, but fail to produce any trophoblastic outgrowths (Sellens and Sherman, '80; Sherman and Matthaei, '80). In order to demonstrate that the blastocysts cultured in PCMF remained viable and maintained their potential for trophoblast outgrowth, they were subsequently transferred to cNCTC. As seen in Figure 1, blastocysts cultured for up to 140 hr in PCMF fail to produce trophoblastic outgrowths, whereas blastocysts kept in PCMF delay for as long as 96 hr yield outgrowths when transferred to cNCTC. In general, blastocysts from the delayed cultures outgrow slightly faster and more synchronously than embryos placed di- rectly into cNCTC following isolation.

Since embryos transferred from PCMF to cNCTC give a high frequency of trophoblast

outgrowths, attempts were made to determine if pulse exposures to cNCTC are equally effective. Blastocysts were incubated in cNCTC for up to 24 hr either immediately after collection or following various periods of time in PCMF. The embiyos were subsequently returned to PCMF and scored for outgrowth, Experiments were continued until all blastocysts showed evidence of trophoblast outgrowth or until no further increase in the number of outgrowths was observed for 36 hr. The results are summarized in Table 1. The percentage of outgrowths from a series of experiments in which various incubation periods in cNCTC followed a minimum of 16 hr of incubation in PCMF is shown in Figure 2. Taken together, these data indicate that prior culture of blastocysts in PCMF ultimately in- creases the effectiveness of pulse exposure to cNCTC in promoting trophoblast outgrowth. Additionally, whereas a 4-hr exposure to cNCTC fails to promote trophoblast migration, incubation in cNCTC for 12 hr or more allows the majority of blastocysts to produce outgrowths. Almost all blastocysts give rise to trophoblast outgrowths if incubated in cNCTC for 16 hr or longer following an initial 16-hr incubation period in PCMF (Table 1 and Fig. 2). I t should be noted that, as reported previously in preliminary pulse experiments (Sherman and Matthaei, '80), the vast majority of blastocysts initiate trophoblast outgrowth after they have been returned to PCMF (e.g., Fig. 3). Finally, although there

TOTAL HOURS IN CULTURE

Fig. 1. Trophoblast outgrowth in cNCTC medium following initial culture in PCMF. Blastocysts were collected on the fourth day of pregnancy and cultured as described under Materials and Methods. After the indicated periods

of culture in PCMF, blastocysts were transferred to cNCTC. A control group of blastocysts remained in PCMF throughout.

404 E.J. BLAKE, J. SCHINDLER, AND M.I. SHERMAN

v) I

[L

3 0 (3

loo-

z 0 75- 3 0

2 n

5 0 - > V

E 3 25- m

6." b

TABLE 1. Percentage of blastocysts producing outgrowths following iiarious culture regimens'

Hours of culture in cNCTC 0 16-18 20-24 25-32 48 72 96

24 53 98 f 1.8 100 - 97 * 2.2 - 100 - -

16 46 f 5.9 - 94 f 1.9 95 f 1.5 100 97 94 12 - - 87 i 9.0 81 k 1.4

- - 22 i 16.8 14 +_ 7.1 - - - 8 4 - - 4 * 3.4 0 2

Hours of culture in PCMF prior to transfer to cNCTC'

- - - -

18-20 66

- - -

- - - - - - - - - 0

'Values given in the table represent the average percentage of blastocysts giving rise to trophohlast outgrowths ( 5 SE). Data for each time point are taken from 1 to 14 experiments. 'Blastocysts were cultured i n PCMF for the length of time indicated. transferred tr, cNCTC for the nirmber of hnurs shown in the first column, and then returned to PCMF.

I I I I

--PtMFfcNCTC I-f'CMF

I 0 25 50 75 to

1 1 1 1 1 1 100

L 24

HOURS OF EXPOSURE TO cNCTC

Fig. 2. Relationship between length of exposure to cNCTC medium and trophoblast outgrowth. Embryos were collected and incubated in PCMF for a t least 16 hr. The embryos were then transferred to cNCTC for the indicated periods of time and returned to PCMF for further incubation. The points represent pooled data from up to 14 separate experiments, each with 25-35 embryosiculture.

are usually many trophoblast cells in these blastocyst outgrowths, the migration and increase in size of the cells do not progress in PCMF to the extent observed when blastocysts are cultured continuously in cNCTC medium (Sherman and Matthaei, '80).

Effects of cycloheximide on trophoblast outgrowth

It seemed reasonable to assume that blastocysts fail to outgrow in PCMF, an amino acid- free medium, because depletion of amino acid pools leads to a reduction in protein synthesis

(Sellens et al., '81). The observation that blastocysts outgrow in PCMF after prior exposure to cNCTC suggests either that proteins critical for trophoblast migration were synthesized prior to outgrowth, when the blastocysts were in cNCTC, or that exposure to exogenous amino acids allows the embryos to build up amino acid pools in order to synthesize necessary proteins at the time of outgrowth. To distinguish between these alternatives, incubations were carried out in the presence of the protein synthesis inhibitor cycloheximide. Cycloheximide treatment during culture in cNCTC interferes with trophoblast outgrowth (Fig. 4A) and protein synthesis, as measured by [:%fmethionine incorporation into


100-

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HIS-, v) c v) > 0 75- 0

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z

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B 25

100 I I I I

A

-

i5 50 75 100 125 0 I 0.2 0.3 0.4

25 t O' 215 ;o 715 Id0

% INHIBITION OF OUTGROWTH

Fig. 4. Effects of cycloheximide on cultured blastocysts. (A) Rlastocysts were collected and cultured for 24 hr in PCMF, transferred for a 16-hr incubation in cNCTC containing various concentrations of cycloheximide, washed, returned to fresh PCMF and scored for outgrowth. Each point represents the average of three separate experiments. (B) To determine the inhibitory effect of cycloheximide upon protein synthesis, blastocysts were cultured in the presence of [ "Slmethionine during exposure to cNCTC

cycloheximide as in (A) and incorporation into TCA- insoluble material was measured (see Materials and Meth- ods). Results are averaged from two independent experiments. (C) Inhibition of trophoblast outgrowth (from A) is plotted vs inhibition of [''Slrnethionine incorporation (from B).

TCA-insoluble material (Fig. 4B), but appears not to inhibit methionine uptake by cells of the blastocyst (measured by cell-associated, TCA- soluble [:%]methionine): In the presence of 0.4 pg/ml cycloheximide, there were 44,570 ,35S

I I I I I I

HOURS IN CULTURE

Fig. 5. Effects of cycloheximide during initial culture of blastocysts. Blastocysts were collected and cultured for 24 hr in PCMF containing 0 (O), 0.05 (0). 0.1 (O), 0.2 (A), or 0.4 ( V ) pg/ml cycloheximide. They were then cultured according to the following regimens: (A) cNCTC, 24-40 hr; PCMF, 40 hr and beyond; (B) PCMF, 24-48 hr; cNCTC, 48-64 hr; PCMF, 64 hr and beyond. Embryos were washed thoroughly between treatments.

cpmlembryo compared to 42,887 d5S cpdem- bryo in control samples (averages of duplicate measurements in two independent experiments). As Figure 4C indicates, protein synthesis must be inhibited substantially before any effect is observed on trophoblast outgrowth; nevertheless, the presence of 0.4 p g l d cycloheximide during the cNCTC pulse almost completely inhibits both outgrowth and protein synthesis.

The effects of cycloheximide upon trophoblast outgrowth when the antimetabolite is added before (Fig. 5) or after (Fig. 6) exposure to cNCTC medium are much less severe. Ad- dition of cycloheximide to PCMF medium during the 24-hr incubation period prior to exposure to cNCTC ultimately delays trophoblast outgrowth and reduces the percentage of blastocysts able to give rise to them. How- ever, even at 0.4 pglml cycloheximide, almost half of the blastocysts eventually give rise to outgrowing trophoblast cells (Fig. 5A). If a 24- hr recovery period in PCMF is inserted be-

406 E.J. BLAKE, J. SCHINDL ,ER, AND M.I. SHERMAN

cyst behavior in the two media: cNCTC con- tains serum, amino acids, vitamins, and other components that are lacking in PCMF. Even the formulation of salts is different. However, we have demonstrated previously that trophoblast outgrowth can occur in the absence of vitamins and that the serum requirement can be satisfied by the fetuin fraction that is in- cluded in PCMF (Rizzino and Sherman, ’79; Sellens and Sherman, ’80). Indeed, PCMF supplemented with amino acids can support outgrowth, but we have chosen to utilize cNCTC as a pulse medium in these studies, because initiation of trophoblast migration takes substantially longer in PCMF plus amino acids than it does in cNCTC (Sellens and Sher- man, ’80). Although other factors in cNCTC might render this medium optimal for outgrowth, we conclude that an exogenous source of amino acids is the single component essential for initiation of this phenomenon. Studies with cycloheximide described above allow us to state further that a period of culture during which protein synthesis can occur at a reasonable rate must precede trophoblast outgrowth (on a molar basis, net incorporation of methionine during a 16-hr incubation is five times greater in cNCTC than in PCMF).

Trophoblast outgrowth can be blocked almost completely if blastocysts are exposed to cycloheximide during culture in cNCTC. Ad- dition of cycloheximide before or after exposure to cNCTC is much less effective: treatment with the antimetabolite during the initial period of culture in PCMF inhibits only a fraction of blastocysts from producing outgrowths, even though their protein synthetic rate has been reduced to approximately 10% that of blastocysts in cNCTC. Cycloheximide treatment following exposure to cNCTC is even less disruptive: outgrowth consistently takes place at high frequency, but the average time required for initiation of outgrowth is prolonged.

In order to explain the inhibitory effect on outgrowth of cycloheximide added prior to exposure to cNCTC, two other observations must be considered. The first is that pulse exposure to cNCTC immediately upon place- ment of blastocysts into culture is a relatively ineffective regimen for promoting trophoblast outgrowth: only half of the blastocysts will do so upon subsequent transfer to PCMF (Table 1); the second is that a 24-hr “recovery period” between cycloheximide treatment (in PCMF) and subsequent culture in cNCTC ne- gates the adverse effect of the antimetabolite (Fig. 5B). There are two possible interpreta-

5 HOURS IN CULTURE

Fig. 6. Effects of cycloheximide during or after incubation of blastocysts in cNCTC. Bbastocysts were collected and cultured in PCMF from 0 to 24 hr, in cNCTC from 24 to 40 hr, and Once again in PCMF after 40 hr of culture. Embryos were exposed to 0.4 pgiml cycloheximide as fol- lows: (O), no elposure; (0). 24-40 hr; (A) , 40-56 hr; (A), 56-70 hr. Emhryos were washed in PCM before each transfer. Results are typical of four independent experiments carried out according to the same protocols.

tween the initial exposure to cycloheximide and subsequent culture in cNCTC (free of antimetabolite), there is virtually no interfer- ence by cycloheximide with trophoblast outgrowth (Fig. 5B).

Figure 6 illustrates that contrary to the re- sult when cycloheximide is present during exposure to cNCTC, the antimetabolite is ineffective in blocking trophoblast outgrowth if added after the blastocysts had been cultured in cNCTC. There is, however, a delay in the time of initiation of outgrowth. I t should be noted that [ “Slmethionine incorporation is not inhibited as markedly when cycloheximide is added to PCMF rather than cNCTC: levels of inhibition are only 40-50%. However, since the extent of [ Y3lmethionine incorporation in PCMF is only about 20% that in cNCTC (data not shown), the residual amount of protein synthesized by blastocysts during exposure to PCMF plus cycloheximide is extremely low.

DISCUSSION

Trophoblast outgrowth from blastocysts can occur in a normally nonpermissive medium following exposure for as little as 8 hr to a nutritive, serum-containing medium. There are many differences between PCMF and cNCTC that might explain differential blasto-


tions of these results: the first, which we be- lieve most reasonable, is that many fourth-day blastocysts at the time of collection are not capable of producing adequate amounts of outgrowth-related proteins, even in cNCTC, and that prerequisite developmental processes re- quiring protein synthesis must occur. A second explanation, which we cannot yet eliminate, is that fourth-day blastocysts are normally capable of producing outgrowth-related proteins in vivo, but that the trauma of being placed in culture precludes their doing so; cycloheximide treatment during the initial culture period exacerbates the effect, whereas recovery incubations in PCMF prior to exposure to cNCTC overcome it.

The observation that cycloheximide has a minimal effect upon trophoblast outgrowth when added after exposure to cNCTC suggests that the critical time for synthesis of outgrowth-related proteins is several hours prior to the event per se (for example, Fig. 3 illustrates that about 30 hr elapsed between the midpoint of the pulse exposure to cNCTC and the time at which half the blastocysts initiated outgrowth). It would appear that there is also a substantial hiatus between the time at which outgrowth-related proteins are translated and the time at which their mRNA had been transcribed, since exposure to a-amanitin at any time after collection of blastocysts on the fourth day fails to influence either the frequency or time of initiation of outgrowth (Schindler and Sherman, '81; Sher- man e t al., '81). Although the critical period for production of outgrowth-related proteins appears to be during the cNCTC pulse, protein synthesis (even if a t reduced rates) following transfer to PCMF appears to be beneficial for outgrowth, since cycloheximide treatment during this period delays initiation of outgrowth. Furthermore, the extent of trophoblast outgrowth in PCMF following exposure to cNCTC is rarely equal to that observed when blastocysts are maintained in cNCTC. Whether this means that outgrowth-related proteins must be continually replenished or that other types of outgrowth-promoting proteins are made after return of blastocysts to PCMF remains to be determined.

Since levels of protein synthesis in PCMF are much lower than those in cNCTC, failure to outgrow in the former medium could be due to either qualitative or quantitative differences between the proteins synthesized by blastocysts in the two media. In preliminary studies we have attempted to distinguish between these two possibilities by incubating

cells with [."S]methionine in cNCTC or PCMF during the synthetic period critical for outgrowth and analyzing the products by two- dimensional gel electrophoresis (O'Farrell, '75). Analyses of these gels did not reveal ob- vious qualitative differences (unpublished observations). It is possible, therefore, that the synthesis of outgrowth-related proteins can take place in PCMF at the appropriate developmental time, but that outgrowth is pre- cluded because insufficient quantities of these proteins are produced.

ACKNOWLEDGMENTS

We are grateful to Ms. Perla Pine for assist- ance. We also thank Drs. J. Monahan and C. Coleclough for comments on the manuscript.

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protein synthetic requirements for the outgrowth of trophoblast cells from mouse blastocysts

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