Proc. Nati. Acad. Sci. USA Vol. 86, pp. 3654-3658, May 1989 Cell Biology Mechanism of programmed cell death in the blastocyst (blastocyst regulation of embryonal carcinoma/preimplantation development/apoptosis) G. BARRY PIERCE*, ANDREA L. LEWELLYN, AND RALPH E. PARCHMENT Department of Pathology, University of Colorado School of Medicine, 4200 East Ninth Avenue, Denver, CO 80262 Communicated by David M. Prescott, February 13, 1989 (receivedfor review October 17, 1988) ABSTRACT The malignant growth potential ofembryonal carcinoma cells may be controlled by environmental factors. zP For example, embryonal carcinoma cells placed into normal blastocysts may not exhibit the continued growth expected of T malignant cells but rather may lose all aspects ofthe malignant phenotype and become apparently normal embryonic cells. E Loss of the malignant phenotype of embryonal carcinoma cells occurs early in these I jected blastocysts and has been used as the basis of assays to study the mechanisms of regulation of embryonal carcinoma by the blastocyst. In this regard, P19, an _G I~CM a embryonal carcinoma that makes midgestation chimeras, was b regulated by blastocele fluid plus contact with trophectoderm but not by blastocele fluid plus contact with inner cell mass FIG. 1. (a) Diagram of a mouse blastocyst 3.5 days after fertili- (ICM). In contrast, ECa 247, which makes trophectoderm, was zation. The zona pellucida (ZP) is a soft egg shell lined by 52 regulated by exposure to blastocele fluid plus contact with trophectodermal cells (T), which will form the placenta. The 12 1CM cells will form the embryo, but at this stage they have the potential trophectoderm or ICM. During the course of these experi- to form trophectoderm as well. (b) Diagram of a late stage blastocyst. ments, dead embryonal carcinoma and ICM cells were ob- Programmed cell death has occurred and one of the ICM cells has served, and blastocele fluid was then shown to kill ECa 247 and become apoptotic~~~~~~AC(AC). After apoptosis, the 1CM no longer has the normal ICM cells of early blastocysts with trophectodermal potential to differentiate trophectoderm; instead it differentiates a potential. P19 cells and ICM cells with potential to make the layer of endoderm (E). The blastocele cavity contains 1 nl of fluid. embryo were not killed by blastocele fluid. Programmed cell death occurs in the ICM of the blastocyst during the transition In the course of the current investigation, it was observed from early (when ICM has the potential to make trophecto- that P19 cells, unlike ECa 247 cells, were not regulated by derm) to late (when the ICM lacks the potential to make blastocele fluid plus contact with the 1CM. Furthermore, trophectoderm). It is postulated that this programmed cell some ECa 247 cells, but not P19 cells, were killed during death is designed to eliminate redundant 1CM cells with incubation in blastocele fluid. Blastocele fluid also selectively trophectodermal potential, and its mechanism of action is killed some ICM cells, after which the potential of the ICM mediated by epigenetic factors in blastocele fluid. to make trophectoderm was lost. Our ultimate goal is to ascertain the mechanism by which the blastocyst regulates embryonal carcinoma cells, which have MATERIALS AND METHODS been injected into the blastocele, to the point that they Eight-cell eggs at 2 days of pregnancy and blastocysts at 3.5 respond to embryonic signals as ifthey were normal inner cell days of pregnancy were obtained from 8-week-old CD-1 mass (ICM) cells as exemplified by the production of chi- (Charles River Breeding Laboratories) mice by flushing the meric mice (1-7). The blastocyst is a cystic stage of embry- oviducts or uteri, respectively, with a few drops of Eagle's onic development occurring in the mouse 3.5 days after minimal essential medium containing penicillin G (100 units/ fertilization (Fig. la). ml), streptomycin (100 Ag/ml), 0.1 mM nonessential amino We have previously shown that blastocele fluid plus con- acids, and 1 mM pyruvate (MEM). The eggs and blastocysts tact with trophectoderm are required for regulation of em- were held in MEM, with 10% heat-inactivated fetal bovine bryonal carcinoma (loss of malignant attributes such as the serum (MEM + 10) under washed mineral oil. ability to form colonies of malignant cells in vitro) (8). The methods for injecting blastocysts and producing tro- However, ECa 247, the embryonal carcinoma cells used in phectodermal vesicles have been described (8, 11). Briefly, that study, failed to produce chimeras when injected into cancer cells in a drop ofMEM + 10 under washed mineral oil blastocysts because they preferentially localized in and dif- were injected into blastocysts by using holding and injecting ferentiated into trophectoderm (9). It was decided to compare pipettes attached to micromanipulators. Only blastocysts the mechanism of regulation of an embryonal carcinoma that with a clearly visible cancer cell were used in the assay for makes chimeras to that of ECa 247. In contrast to ECa 247, cancer cell regulation as measured by abrogation of colony- ==60% of blastocysts injected with P19 cells develop into forming ability (8, 12). As for controls, the cancer cells were midgestation chimeras as evidenced by glucose phosphate cloned in 96-well Linbro plates, where they grew equally well isomerase assays, but most of them were abnormal and few as in the perivitelline space. For production of trophectoder- came to term (10). Nevertheless, the malignant phenotype mal vesicles containing an embryonal carcinoma cell, sepa- was at least partially suppressed during the early develop- rate drops of medium containing either blastocysts or cancer ment of these chimeras. cells were used to obviate transfer of contaminating embry- onal carcinoma cells. Blastocysts were injected with a cancer The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: ICM, inner cell mass. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 3654 Downloaded by guest on September 29, 2021 Cell Biology: Pierce et al. Proc. Natl. Acad. Sci. USA 86 (1989) 3655 cell, which was placed on the mural trophectoderm opposite Table 2. Effect of trophectodermal vesicles on colony formation the 1CM; the 1CM was then amputated with a 26-gauge of single P19 cells needle. If the tip of the needle was pressed into the bottom No. of colonies/no. of attempts of the plastic Petri dish, the edges of the trophectoderm were sealed to form a vesicle. When the vesicles reexpanded, P19 cells in those with a visible cancer cell were assayed for regulation of trophectodermal P19 cells colony formation (8). Exp. vesicles cultured alone To prepare giant blastocysts (13), the zonae pellucidae of 1 3/12 17/40 eight-cell eggs were removed by incubation in 0.25% Pronase 2 0/4 20/40 at 370C, after which the eggs were washed three times in 3 4/14 17/40 MEM + 10. Eight eight-cell eggs were fused in a small drop 4 1/6 27/40 of 0.1% phytohemagglutinin (Sigma) in MEM in 35-mm 5 3/10 25/40 bacteriologic Petri dishes and incubated for 1-2 min. The Total 11/46 (24%) 106/200 (53%) dishes were flooded with MEM + 10 to dilute the phytohe- P= 0.001. magglutin and were incubated at 370C in 100% humidity with 5% C02/95% air on a rocking platform (22 cycles per min). from the first one to avoid mixing of the cell types. A zona The resultant giant blastocysts were used after either 48 or 72 pellucida was oriented on a holding pipette so that the hole hr of incubation. through which the blastocyst had been evacuated faced the Zonae pellucidae, to be used as carriers of the cellular injecting pipette. Then, depending on the experiment, em- preparations to be studied in the giant blastocysts, were bryonal carcinoma cells alone or 2 embryonal carcinoma cells prepared from blastocysts, morulae, and abnormal eggs plus 12 ICM cells were injected through the hole into the unsuitable for other experiments. The blastocysts, attached cavity of the zona pellucida. One-half of the specimens were by gentle suction to holding pipettes, were penetrated by a incubated in MEM + 10 for 7 days in 5% C02/95% air with pipette through which the fluid and cells of the embryo were 100% humidity (controls), and half were injected into giant evacuated (see Fig. 2). The zonae pellucidae were flushed blastocysts. To this end, they were placed in a large drop of with MEM + 10 by inserting a pipette through the hole used MEM + 10 with the giant blastocysts. A giant blastocyst was to evacuate their contents and then stored at 4°C in MEM + attached to a holding pipette by gentle suction, and a trian- 10. In contrast to the method of Rossant (14), the method gular-shaped hole was made in its wall using three fine glass described here has the advantage of usually producing only needles. A zona pellucida carrying the cells to be tested was a single hole in the zona pellucida. attached by suction to a pipette and pushed through the Two lines of embryonal carcinoma were used. ECa 247, The which was derived from OTT6050 (15), is aneuploid (a triangular hole into the blastocele ofthe giant blastocyst. bimodal chromosome number of 57 and 74 chromosomes), preparations were placed in the incubator until the wound in grows well in vitro, and its neoplastic attributes including the giant blastocysts had healed and the blastocele had tumor and colony formation are regulated by the blastocyst re-formed. This usually took 1-3 hr. The expanded blasto- (11, 12).