DEFINED MEDIA AND DEVELOPMENT OF MAMMALIAN EGGS IN VITRO '

R. B. L. Gwatkin King Ruirch Laboratory of Reprodirctive Physiology, Department of Animal Biology,

School of Veterinary Medicine, University o/ Pennsylvania, Philadelphia, Pa.

Compared with tissue cells in culture, the nutritional requirements of mam- malian ova have scarcely been investigated. Yet the potential significance of doing so is enormous, for defined media permitting the routine development of ova through the free-living stage before implantation would allow many experi- ments now difficult, or impossible, in vivo.

Omitting the special problem of in vitro fertilization, this paper will outline recent progress made in developing defined media for fertile mammalian eggs. Use of these media to study the effect of viruses on early development will then be considered briefly. Finally, some previously unpublished experiments on the culture of mouse eggs beyond the blastocyst stage will be presented.

The Culture of Mammalian Ova

Although the eggs of many species have been seen to cleave once or twice in vitro, only those of the rabbit and the mouse undergo appreciable develop- ment. Rabbit eggs will cleave readily from one-cell to morula in a medium containing large proportions of serum. However, blastocyst expansion fails to take place (Austin, 1961). Mouse ova, on the other hand, can be cultured to fully expanded blastocysts, but one must start from the two-cell stage. The first division is refractory. Cleavage from the one-cell stage through to blasto- cyst will take place within organ cultures of the oviduct (Biggers, Gwatkin & Brinster, 1962). Under these conditions much of the cellular integrity of the duct is preserved (Gwatkin & Biggers, 1963) and, presumably, one or more essential factors are made available by the oviduct for this first cleavage division.

In 1949, Hammond cultured eight-cell mouse ova to blastocysts in balanced salt solution with glucose and egg white. Whitten in 1956 made the medium more defined by using crystalline bovine serum albumin instead of egg white, in Krebs-Ringer bicarbonate with glucose (TABLE 1 ) . At least some of the blasto- cysts produced in this medium were normal, since they developed into mice on transfer to the uteri of foster mothers. In these experiments, carried out by McLaren and Biggers in 1958, a genetic marker was used to distinguish between the embryos originating from the transplanted and native blastocysts.

A basic clue to the successful culture of mouse eggs from the two-cell stage was discovered by Whitten in 1957, when he showed that the addition of one mg. of lactic acid per ml. of his medium (Krebs-Ringer bicarbonate with glucose and albumin) allowed the development of some eggs from the two-cell stage. Whitten speculated that lactic acid might be made available to the egg in vivo by sperm glycolysis, but it is more likely that lactate is produced by the oviduct itself. Data are not available for the mouse, but the secretions of rabbit (Bishop, 1961) and human oviducts (Mastroianni, Winternitz & Lowi, 1958) both con- tain large supplies of lactate. Of special significance is the recent finding of Mastroianni and Wallach (1961) that the lactate concentration in the oviduct fluid of the rabbit increases during the first three days of pregnancy.

Brinster (1963, 1965a, b, c ) has much extended Whitten's original observa- tion that the early cleavage stages of the mouse egg require a 3-carbon compound

I9

80 Annals New York Academy of Sciences TABLE 1

PARTIALLY DEFINED MEDIA FOR THE CULTURE OF MOUSE O V A TO THE ELASTOCYST STAGE

Component From 2-cell stage From

S-cell stage whitten* Whittent Brinsterz Brinsters mM mM mM mM

NaCl 119.32 119.32 109.23 119.32 KCl 4.78 4.78 4.78 4.78 CaCl, 2.54 - 1.71 1.71 KH,PO, 1.19 1.19 1.19 1.19 MgSO, * 7H20 1.19 1.19 1.19 1.19 NaHCOS 25.07 25.07 25.07 25.07 Glucose 5.5 5.5 Calcium lactate - 2.54 Sodium lactate - - 10.15 - Sodium pyruvate - - - 0.316 Crystalline bovine serum albumin 1 gm/L 1 gm/L 1 gm/L l g m / L Penicillin - - 100 U/ml 100 U/ml Streptomycin - - 50 pg/ml 50 pg/ml

* Whitten, W. K. 1956. Nature 177: 96. t Whitten, W. K. 1957. Nature 179: 1081. $ Brinster, R. L. 1963. Exp. Cell Res. 32: 205. § Brinster, R. L. 1965. J. Exp. Zool. 158: 59.

- - - -

for development. He has established that this requirement may be met by pyru- vate, oxaloacetate, and phosphoenolpyruvate, but not by a number of other compounds of the Krebs cycle, Embden-Meyerhof, or pentosephosphate path- ways (Brinster, 1965b). The reason for this specificity was not established. It may be that the enzymes dealing with some of these inactive compounds are not yet developed at the two-cell stage. However, other possibilities may also be involved. The egg membrane may be impermeable to some of the compounds. A third possibility, which does not appear to have been mentioned previously, is that such compounds as pyruvate may be readily lost from the intracellular pool to the surrounding medium, a situation that has been shown to limit the growth of isolated tissue cells in cloning experiments (Harris, 1964).

Mintz (1964a) has shown that mouse ova, as early as the two-cell stage, incorporate H3-leucine, and one might therefore expect that there is a require- ment for amino acids which is being met by the albumin. Brinster ( 1 9 6 5 ~ ) has in fact shown that the albumin in his media can be replaced by polyvinyl- pyrrolidone (PVP), a synthetic polymer providing some of the physical proper- ties of protein, and a mixture of the 21 individual amino acids of albumin. Omitting either the polymer or all of the amino acids blocked development. However, no one amino acid was proved to be essential, since when the amino acids were omitted singly, development still occurred.

Osmolarity and hydrogen ion concentration are not particularly critical for the development of two-cell ova to blastocysts. In Krebs-Ringer bicarbonate with lactate and albumin, Brinster (1965a) found the optimum osmolarity to be 0.2760 osmols with a range of 0.2718 to 0.2801 osmols. The optimum pH was about 6.82 with a range of 5.87 to 7.78

Effect of Viruses on Eggs In Vitro Brinster’s media, chemically defined except for their albumin component,

have been applied in studies to determine the effect of viruses on early develop-

Gwatkin : Development of Mammalian Eggs 81

ment. The mammalian egg, perhaps due partially to its regulative capacity, is relatively resistant to radiations and chemical carcinogens (Wilson, 1964). However, no one knows whether the egg can become infected with viruses, which may remain latent to cause damage in later development, or even in adult life. Recently, I have shown that infection of the mouse ovum is possible, at least with one virulent virus (Gwatkin, 1964; Gwatkin and Auerbach, 1966).

Two-cell mouse ova were cultured in drops of Brinster’s lactate medium under mineral oil and in the presence of Mengo virus. This RNA-virus penetrated the zona pellucida, the thick acellular membrane which surrounds the egg until the time of implantation, and blocked further development of the egg in vitro (Gwatkin, 1963). In subsequent experiments the egg was found to support the synthesis of new Mengo virus particles even before fertilization (Gwatkin, 1964). When a one-step growth curve of this virus in the egg was determined, a relatively long latent period and low virus yield per unit volume were encountered. These characteristics set the egg apart, as a host, from other mouse cells which have been studied. However, the significance of this phenomenon is not understood. There is some evidence that the ribosomes are forming during cleavage in the rat (Szollosi, 1965) and only reach a high concentration in the trophoblast cells. Since virus-protein synthesis presumably occurs on the ribosomes, their relatively low numbers early in cleavage could account for the peculiar behavior of the egg as a host. I hope soon to test this hypothesis by infecting late blastocysts.

In contrast to the cytopathology exhibited by Mengo virus, there was no obvious reaction to high concentrations of Polyoma or Rubella viruses (Gwatkin, unpublished). Whether latent infection may have occurred which will affect later development is being studied by transfer of virus-treated eggs to foster mothers. Since this technique is tedious, and since usually only a fraction of the eggs develop, I have recently attempted to continue in vitro culture beyond the blastocyst stage. The remainder of this paper will report some of these experiments.

Cellular Outgrowth f r o m Mouse Blastocysts In Vitro

Cole, Edwards, and Paul (1965) have succeeded in growing cell strains from rabbit blastocysts using reconstituted collagen and Waymouth’s medium 7521 1

FIGURE 1. Free-floating blastocysts which have shed their zonae pellucidae. Shown after culture for seven days in pyruvate-lactate medium (Brinster, 1965d) with 0.1 per cent glucose. x 60.

82 Annals New York Academy of Sciences

with calf and human serum. Mintz (1964b) recently noted that mouse blasto- cysts grow out into cell cultures in a medium consisting of equal parts of fetal calf serum and Earle’s balanced salt solution with lmg. lactic acid per ml. This process was not described in detail and no attempt was made to determine the conditions necessary for it. In Brinster’s media attachment and outgrowth does not take place. In his most recent formulation, combining the optimal interact- ing concentrations of both lactate and pyruvate (Brinster, 1965d), and which I supplement with 0.1 per cent glucose, the two-cell eggs grow into blastocysts in three days. The zona pellucida is shed by 3?h days, and the blastocysts then remain free-floating (FIGURE 1) for at least ten days before they show signs of collapse and become disorganized. The free-floating arrested condition of these blastocysts may be analogous to delayed implantation (Enders, 1963).

In attempting to obtain further development of mouse blastocysts, Ham’s F10 tissue culture medium (Ham, 1963) supplemented with fetal calf serum was investigated. Two-cell eggs did not develop well in this medium, so they were first cultured in pyruvate-lactate-glucose medium to the blastocyst stage. When these blastocysts were transferred to F10 with ten per cent fetal calf serum, they attached to the bottom of the dish, and 70 to 100 per cent of them produced an extensive outgrowth over the next three to four days. Suitable substrates were glass, even when siliconized, and wettable polystyrene plastic. To facilitate histological examination, groups of ten blastocysts were cultured in a 0.1 ml. standing drop of medium on a circular coverglass (diameter 22mm.). Evapora- tion was prevented by supporting the coverglass in a plastic organ culture dish (Falcon Plastics, Div. of B-D Labs. Inc.), which contained 2 ml. of medium in the outer well. Dishes were incubated at 37” C in water-saturated 5 per cent C 0 2 in air.

Eventually Eagle’s medium of 30 components was found to replace Ham’s F10 medium of 36 components, thereby simplifying the task of determining the factors which were essential for outgrowth (FIGURE 2a). These two-dimen- sional “implantations,” if I may use this term, stained with May-Grunwald- Giemsa, were found to consist of cells with nuclei of widely varying size, some in division. Nucleoli were prominent, multiple, and often irregular in shape; some of them protruded from their nuclei (FIGURE 2b). The significance of this protrusion is unknown. It has been observed by many others, particularly in oocytes and developing eggs (Izquierdo & Vial, 1962; Szollosi, 1965).

In the center of each outgrowth was extracellular material, which stained a deep blue in the May-Grunwald-Giemsa procedure. The nature of this sub- stance has not been established. However, it does not seem to be predom- inantly a ribonucleoprotein, since the deep blue staining was not affected by an RNAse treatment that removed cytoplasmic staining (FIGURE 3a,b). The presence of polysaccharide was suggested by deep staining in the Periodic acid- Schiff technique (FIGURE 3c). Staining for lipid with Sudan black B revealed lipid granules in the cytoplasm of the cells (FIGURE 3d) but none in the extra- cellular material. Most outgrowths also exhibited a central group of relatively small cells, the nature of which will be considered in another publication.

Macromolecztlar Requirements. Both Ham’s F10 medium and Eagle’s medium required a macromolecular component (TABLE 2). This was met by fetal calf serum even after exhaustive dialysis, by new born calf serum; by fetuin (an a- globulin which makes up a large part of the protein of fetal calf serum; see FIGURE 4); and by Cohn fractions 111-0, IV-1, and IV-4 of bovine serum. The optimal concentration of the sera was about 1 per cent. Fetuin and the serum

Gwatkin: Development of Mammalian Eggs 83

FIGURE 2. a. Group of outgrowths three days after blastocysts were added to a drop of Eagle’s basal medium with 10 per cent fetal calf serum. May-Griinwald-Giemsa. X 100. b. Higher magnification of area in a. Note partially extruded nucleoli (marked by arrows). x 1000.

84 Annals New York Academy of Sciences

FIGURE 3. a. Outgrowth (three days in Eagle’s basal medium with 5 per cent dialyzed calf serum). Note deeply stained extracellular material at center. May-Grunwald-Giemsa. x 162.5. b. Similar outgrowth to a, but treated with crystalline ribonuclease (Worthington) in buffered saline (pH 6.1) prior to staining. Note that extracellular material at center of outgrowth is unaffected, but cytoplasmic staining of outgrowing cells is markedly reduced. x 162.5. c. Simi- lar outgrowth stained by Periodic acid-Schif? procedure. Note that only the central extracellular material is stained. X 162.5. d. Lipid granules within cytoplasm of outgrowing cells. Culture conditions same as in a, b, c. Sudan black B after fixation in calcium chloride formalin. x 650.

Gwatkin: Development of Mammalian Eggs 85

fractions exerted their maximum effect at about 0.1 per cent, except for fraction IV-I which was optimal at 0.01 per cent. It is of interest that fraction IV-1 contains mainly the serum lipoproteins. No outgrowth whatsoever occurred with calf serum fraction 11, crystalline bovine serum albumin, gelatin, or polyvinyl- pyrrolidone (PVP) tested repeatedly at concentrations of 1 .O, 0.1 and 0.01 per cent.

Micromolecular Requirements. Since after exhaustive dialysis against physio- logical saline fetal calf serum still met the macromolecular requirements for out- growth, it was possible to determine the micromolecular requirements. With a supplement of one per cent dialyzed fetal calf serum, removal of all 12 amino acids (the L-stereoisomers of arginine, cystine, tyrosine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine) from Eagle’s medium (Eagle, 1955) completely prevented attachment and out- growth of the blastocysts in repeated experiments. Determination of the indi- vidual amino acids essential for implantation in vitro is still in progress. However, clearly not all 12 that Eagle has established as essential for tissue cultures of both freshly isolated and established cells are needed for blastocyst attachment and outgrowth. Blastocysts arrested in a free-floating condition for five days by removal of one group of amino acids (arginine, lysine and histidine) implanted when transferred to complete medium (FIGURE 5 ) . These results suggest that implantation might be controlled in vivo by the specific free amino acid content of the uterus. This hypothesis will be elaborated elsewhere.

In contrast to these amino acid requirements, removal of L-glutamine or all of the vitamins from Eagle’s medium did not affect blastocyst attachment and

TABLE 2 NUTRIENT FACTORS AFFECTING CELLULAR OUTGROWTH FROM MOUSE BLASTOCYSTS

Essential for Outgrowth Macromolecular Component

Met by Not met by* Calf serum (1%)t Fetal calf serum ( 1 % ) Dialyzed fetal calf serum (1 % ) Fetuin (0.1%) Gelatin

Calf serum Fr. I1 Crystalline bovine plasma

albumin

Calf serum F;. 111-0 (0.1%) Polyvinylpyrrolidone (PVP) IV-1 (0.01%) IV-4 (0.1%)

Micromolecular Component Met by some of Eagle’s 12 “essential” amino acids.$

Not Essential for Outgrowth L-glutamine Vitamins (Biotin, folk acid, choline C1,

nicotinamide, Ca-D-pantothothenic acid, pyridoxal-HC1, thiamine-HCl, riboflavin, i-inositol.)

Some of Eagle’s 12 “essential” amino acids.$

* Tested at concentrations of 1.0, 0.1 and 0.01 per cent. t Optimal concentrations in parentheses. $ Experiments still in progress.

86 Annals New York Academy of Sciences

FIGURE 4. Blastocyst outgrowth after four days in Eagle’s basal medium with 1 per cent fetuin ( a-globulin) . May-Griinwald-Giemsa. X 250.

outgrowth. The fact that glutamine is not required is of particular interest since it has been reported as essential for many tissue cells in culture, including the L-strain which is of mouse origin (Harris, 1964). Failure to detect a vitamin requirement is not surprising, since vitamins are needed in relatively small amounts which could probably be supplied from cellular pools in these short term experiments.

Prolonged Culture of Outgrowths. Even with regular replenishment of the medium, cellular proliferation declined after the fourth day in culture. After 1 1 days some cells assumed a fibroblast-like condition, or a bizarre shape (FIGURE 6d). Whether this was due simply to degeneration of the cells or represented cellular differentiation cannot be decided at the present time. Also, to what extent these outgrowths resemble implantation of the blastocysts in the endo- metrium is an open question. Development never proceeded to germ layer formation, as sometimes occurred in the experiments of Bryson (1964), who observed the outgrowth and differentiation of mouse blastocysts in intraperi-

Gwatkin: Development of Mammalian Eggs 87

toneal diffusion chambers. In my experiments the outgrowth may be largely due to trophoblast cells, but there are no adequate criteria for saying that embryonic cells may not also be present. Perhaps further differentiation can be obtained by implanting the blastocysts on cell cultures of endometrium. This is being explored currently.

In conclusion, I should add that these blastocyst outgrowths are ideal for examining early embryonic chromosomes (Gwatkin and Meckley, to be pub- lished). Since the cells are firmly attached to the coverslips, histological prepara- tion is routine, and the flat condition of the cells permits the chromosomes to be spread by simple air drying. There is no need for squashing, as with whole blastocysts.

FIGURE 5 . Blastocysts were maintained in a free-floating condition in Eagle’s basal medium (lacking arginine, lysine and histidine) with 1 per cent dialyzed fetal calf serum. They were then transferred to complete Eagle’s basal medium with 5 per cent fetal calf serum. The blastocysts then “implanted” and one such “implantation” is shown six days later. Note presence of multinuclear cells and absence of extracellular material. May-Griinwald-Giemsa. x 250.

88 Annals New York Academy of Sciences

FIGURE 6. Appearance of blastocyst-outgrowths after varying periods of time in Eagle’s basal medium with 1 per cent fetal calf serum. All stained with May-Grunwald-Giemsa. x 162.5. a. Three days. b. Four days. c. Six days. d. Eleven days.

Gwatkin: Development of Mammalian Eggs 89 Acknowledgments

The author wishes to thank Mr. Dorsey Williams and Mr. Martin Ratowski for their technical aid. Mr. Paul Meckley assisted in these studies during the summer of 1965 as part of the Summer Research Program for students in the School of Veterinary Medicine. This research was supported by Grant GB-617 from the National Science Foundation. The author is a research career develop- ment awardee of the National Institute of Child Health and Human Develop- ment (l-K3-HD-21,269.-01Al).

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