/ . Embryol. exp. Morph. Vol. 20, 3, pp. 329-41, November 1968 3 2 9With 4 plates

Printed in Great Britain

The development of teratomas from intratesticulargrafts of tubal mouse eggs

By LEROY C. STEVENS1

The Jackson Laboratory, Bar Harbor, Maine

Grafts of cleaving tubal ova from non-inbred mice to ectopic sites usuallyresult in growths composed of extra-embryonic but not embryonic tissues(Fawcett, Wisloki & Waldo, 1947; Fawcett, 1950; Jones, 1951; Whitten 1958;Kirby, 1960, 1962a; Billington, 1965; and others). Runner (1947) grafted tubalmouse ova to the anterior chamber of the eye and one developed the threeprimary germ layers and then regressed, probably because the host and donorwere histo-incompatible. This is the only report of an ectopically graftedpre-uterine egg that developed intra-embryonic derivatives. Kirby (1962&, 1965)grafted oviducal segmenting mouse eggs to the kidney and obtained only tropho-blast and extra-embryonic membranes. He concluded that a 'uterine factor'is necessary for the development of intra-embryonic structures from mouse eggs.Kirby (1965) and Billington (1965) grafted morulae and blastocysts to the testis,and the morulae never gave rise to embryonic shield derivatives.

Kirby (1963) found that the proportion of successful grafts of blastocystsis higher in the testis than for any other extra-uterine site that he tried. He men-tioned the ease and certainty with which the blastocyst can be introduced intothe testis, and that the vascularity of the testis is such that it affords a particu-larly good bed for blastocyst development. I have also found (Stevens, 1964)that embryonic tissues develop well as intratesticular grafts, and that the testicu-lar environment exerts a strong teratocarcinogenetic influence on 12-day genitalridges of strain 129/Sv mice. This influence results in the initiation of develop-ment of male primordial germ cells. They proliferate and give rise to undif-ferentiated embryonic cells which in turn give rise to the primary germ layers.The primary germ layers differentiate into disorganized mixtures of many kindsof tissues characteristic of teratomas.

Testicular teratomas develop spontaneously from primordial germ cells inabout 10 % of the males of strain 129 mice (Stevens, 1967 a) during the 13th dayof gestation. They never develop in females of this strain. It is possible thatteratomas do not develop from female primordial germ cells because they mayhave already entered into meiosis. Fetal female germ cells remain in meiotic

1 Author's address: The Jackson Laboratory, Bar Harbor, Maine 04609, U.S.A.

330 L. C. STEVENS

prophase, and do not undergo mitotic division until fertilization (Borum, 1961).The prenatal cessation of mitosis in female germ cells may prevent the develop-ment of ovarian teratomas.

The initiation of development of strain 129 male primordial germ cells by thetesticular environment suggested the possibility that the development of femalegerm cells might be similarly initiated after meiosis is complete, i.e. afterfertilization. To explore this possibility, fertilized strain 129 single- and two-cellova were grafted into the testes of adult males. The influence of the sex hor-mones on teratocarcinogenesis is not yet understood, but the results presentedhere demonstrate that strain 129 two-cell eggs can develop in a disorganizedmanner in the testis. Cells in these growths may remain as proliferating un-differentiated embryonic cells for remarkably prolonged periods of time. In onecase the growth from a two-cell egg was serially transplanted for five generations,and it retained its embryonic nature, like a malignant teratoma, for 5£ months.The disorganized nature of the cells and tissues within the grafts may preventsome cells from differentiating. Alternatively, the undifferentiated cells mayhave arisen from cells with characteristics of primordial germ cells.

MATERIALS AND METHODS

Most of the animals used in this investigation were from a stock of micecongenic with inbred strain 129/Sv. This stock, 129/Sv-SFCP, was developedby introducing the genes, C, P, and SlJ into the strain 129 genome, and ischaracterized by its relatively high incidence (10 %) of spontaneous testicularteratomas. Hereafter these mice will be referred to as strain 129. Other inbredstrains used were obtained from colonies maintained at The Jackson Laboratoryby other investigators. They include strains A/HeJ, C57BL/10Sn, C57BL/6J,and C57BL/6K.S. An Fx hybrid, 129 x A/He, was also used.

Eggs were removed from the oviduct 0-2 days after the mothers were foundwith mating plugs and were stored in Hank's solution. As pointed out by Kirby(1963), eggs and embryos can be held in saline solution for several hourswithout deleterious effects. Follicular cells adhering to the zygotes were alsografted and the zona pellucida was left intact. A single egg was grafted to eachtestis.

Hosts ranged in age from about 1 month to fully mature males. They wereanesthetized with Avertin, and testes were exposed through a median ventralincision in the skin and body wall. Eggs were drawn into a micropipette attachedto rubber tubing with a mouthpiece and were expelled through a tear in thetunica albuginica into the testis. All grafts were isogenic and were recoveredfrom the testis 7 to 60 days after transplantation, fixed in Vandegrift's solution,serially sectioned at 7 fi and stained with hematoxylin and eosin. Parts of somegrafts were retransplanted subcutaneously to male hosts.

Egg graft teratomas 331

RESULTS

The results of grafting eggs to adult testes are summarized in Table 1. Onlyfour of 250 grafts of single cell eggs were recovered. Some of the two-cell strain129 eggs underwent development, but none from strains other than 129 exceptfor one from a 129 x A/He hybrid.

Table 1. Results of grafting early mouse embryos to adult testes

Stage

1-cell

2-cell

4- to 8-cell

Strain

129

A/Hex 129

129

A/HeJ

129 x A/He

C57BL/10Sn

C57BL/6JC57BL/KS

129

A/HeJ

Age ofgraft(days)

—

—

714172130405060

3060

3060

3060

20

20

203060

30

Numbergrafted

230

20

144

1211490

14151

243

13096

7827

459

14

10

3315057

27

Extraembryonic

deriva-tivesonly

3

1

2022148

105

00

00

00

0

0

6200

2

Em-bryonicderiva-tives

0

0

13178

1458

00

10

00

0

0

1171

0

Withundifferen-

tiatedembryonic

cells

0

0

23165522

00

00

00

0

0

1101

0

The histologic composition of the grafts varied according to the length oftime the graft was left in the testis. As would be expected, grafts of embryosrecovered a short period of time after transplantation contained embryonic andimmature cells. Most grafts which contained embryonic derivatives and wererecovered after long residence in the testis were composed of adult-type tissues.Unexpectedly, grafts of embryos left for as long as 30-60 days containedundifferentiated embryonic cells and immature tissues. The undifferentiatedembryonic cells in these long-term grafts were still proliferating and giving rise

332 L. C. STEVENS

to undifferentiated as well as to differentiated cells. They retained the capacityto grow progressively for long periods like the stem cells of teratomas.

The embryonic cells that developed from the graft were not confined byReichert's membrane, but were free to migrate through the interstitial areasaway from the original graft site (Plate 1, fig. E). This migratory activity dis-rupted normal intercellular relationships.

The behaviour of the grafts of eggs to the testis is described below accordingto their age. Since all were isografts, they did not succumb to the homograftreaction, but were able to survive indefinitely.

Single-cell eggs (day 0)

Two hundred and fifty zygotes were removed from the oviducts of mice onthe day a copulation plug was found and they were transplanted into adulttestes (Table 1). Only four of these were recovered 14-20 days later, and theywere all composed solely of trophoblastic giant cells (Plate 1, fig. A).

Two-cell eggs (day 1)

Of 669 grafts of strain 129 two-cell eggs, 99 were recovered 7-60 days aftertransplantation. Fifty-two of these were composed of extraembryonic deriva-tives only, but 47 had embryonic shield derivatives as well. Twenty-six of thosewith embryonic derivatives contained undifferentiated embryonic cells, evenafter 21-60 days, when all cells might be expected to be differentiated. Nineteenlooked grossly like teratomas that can be serially transplanted indefinitely, andthey were regrafted into other hosts. One (LS5364) behaved as a transplantableteratoma and retained undifferentiated proliferating cells for at least 165 days.

Four hundred and nine two-cell eggs from mice other than strain 129 were

PLATE 1

Fig. A. Trophoblastic giant cells derived from a single-cell egg grafted to the testis for 20 days.xlOO.Fig. B. Growth from a two-cell embryo grafted for 7 days. The proamniotic cavity (P) issurrounded by ectoderm (EC) which is enveloped by proximal endoderm (PE). Reichert'smembrane (R) has been secreted by distal endoderm (DE). The embryo is surrounded bytrophoblastic cells and it resembles a normal 6-day mouse embryo, x 430.Fig. C. Growth from a two-cell embryo grafted for 14 days. An ectodermal vesicle (EC)surrounds a proamniotic cavity (P) filled with cellular debris. The ectoderm (EC) andendoderm (PE) are separated by mesodermal cells (M). The distal yolk sac has secreted athick Reichert's membrane (R). x 430.Fig. D. Growth from a 2-cell embryo grafted for 14 days. It is composed of ectoderm (EC),mesoderm (M), proximal endoderm (PE), distal endoderm, Reichert's membrane (R), andtrophoblastic cells; in approximately normal relationships to one another. x200.Fig. E. Growth from a two-cell embryo grafted for 14 days. It is similar to the growth repre-sented in Fig. D. The darkly stained cells at the right are undifferentiated cells that havemigrated away from the original graft site, x 100.

/ . Embryol. exp. Morph., Vol. 20, Part 3 PLATE 1

L. C. STEVENS facing p. 332

/ . Embryol. exp. Morph., Vol. 20, Part 3 PLATE 2

L. C. STEVENS

Egg graft teratomas 333grafted into the adult testis, and only one graft was recovered (Table 1). Thiswas a two-cell ¥1 hybrid, 129 x A/He, recovered 30 days after transplantation,and it consisted of adult nerve.

The histological composition and the transplantation behavior of the graftsof strain 129 eggs are described according to the length of time they were allowedto remain in the host.

(1) 7-day-old grafts of two-cell eggs. Three grafts of two-cell eggs were re-covered after 7 days of development in the testis. Two were composed of a fewtrophoblastic giant cells like the graft of a single-cell egg represented in Plate 1,fig. A. The third was much larger than the others and contained embryonic andextra-embryonic cells arranged very much as in normally developing embryos(Plate 1, fig. B). A network of trophoblastic cells bathed in a pool of blood wasattached to Reichert's membrane which was secreted by a single layer of distalyolk sac cells. A cavity separated the distal from the proximal endoderm. Theproximal endoderm enveloped the ectodermal epithelium which surrounded theproamniotic cavity. There were a few cells of the ectoplacental cone. The embryowas well organized, and the relationship of the ectoderm and endoderm wassimilar to that in 6-day embryos and in embryoid bodies of teratomatous origin(Stevens, 1967 a).

(2) 14- to 17-day-old grafts of two-cell eggs. Six grafts of two-cell eggs wererecovered 14-17 days after grafting. One consisted of trophoblastic giant cellsin a pool of blood. Another consisted of trophoblastic giant cells and a fewdistal yolk sac cells embedded in a blob of Reichert's membrane. Four otherscontained both extra-embryonic and embryonic immature tissues in variousdegrees of organization. In some areas of the grafts, ectodermal, mesodermal,and endodermal components were arranged in the normal relationship to oneanother. In other areas, on the other hand, the cells were arranged as dis-organized chaotic mixtures. One contained an ectodermal vesicle surrounding aproamniotic cavity filled with cellular debris (Plate 1, fig. C). The ectoderm was

PLATE 2

Fig. A. Growth from a two-cell egg grafted for 21 days. Embryonic epithelia, mesenchymalcells, and immature neuro-epithelium are represented, x 200.Fig. B. Growth from a two-cell egg grafted for 21 days. Embryonic ectodermal vesicles.Testicular tubule of the host at lower left, x 430.Fig. C. Growth from a two-cell egg grafted 30 days. Lower left, bone; top, adipose tissue andpart of a hair follicle; upper right, neural tissue; lower right, striated muscle, x 200.Fig. D. Growth from a two-cell egg grafted 30 days. Alimentary epithelium and fetal testiculartubules. x200.Fig. E. Growth from a two-cell egg grafted for 50 days. Proliferating undifferentiated cells.x430.Fig. F. Growth from a two-cell egg grafted for 60 days. Undifferentiated and immature cells.x200.

22 ! E K M 2Q

334 L. C. STEVENS

separated from proximal endoderm by a sheet of mesodermal cells. There werenucleated red blood cells in primitive blood vessels. The distal endoderm hadsecreted a thick Reichert's membrane, and trophoblastic giant cells were attachedto it. Other undifferentiated cells were located distal to the embryo. There wereseveral areas of necrotic material. Plate 1, figs, D and E illustrate growths com-posed of ectoderm, proximal endoderm, distal endoderm, Reichert's membraneand trophoblastic cells, all in approximately normal relationships to one another.Another 14-day graft of a two-cell egg developed proximal and distal endoderm,Reichert's membrane, disorganized masses of immature epithelial and mesen-chymal cells, and a large area of fetal heart muscle. The remaining two graftswere disorganized growths with extra-embryonic and embryonic derivatives.

(3) 21-day-old grafts of two-cell eggs. Twenty-eight growths from two-cell eggswere recovered 21 days after grafting. All contained extra-embryonic derivatives.Twenty-one were composed solely of extra-embryonic cells. Seven grafts con-tained embryonic derivatives, and six of these had undifferentiated and imma-ture cells. In addition to extra-embryonic derivatives and undifferentiated cells,the following were represented: embryonic ectoderm and endoderm, mesenchy-mal cells, immature neuro-epithelium (Plate 2, figs. A and B), and fetal heartmuscle. One graft contained a small nodule of cartilage.

(4) 30-day-old grafts of two-cell eggs. Twelve growths were recovered 30 daysafter grafting. Four were composed solely of extra-embryonic derivatives, andeight had embryonic derivatives. The only embryonic shield derivative in threegrafts was immature and adult neural tissue. Five other grafts were pleomorphicand contained undifferentiated embryonic cells and immature and adult neuraltissue, various types of epithelium, striated muscle, cartilage, heart muscle,glandular tissues, bone, and adipose tissue (Plate 2, figs. C and D). One graftwas very pleomorphic and the tissues were highly differentiated. It contained animmature testis (Plate 2, fig. D). This is the only growth that contained gonadaltissue.

(5) 40- to 50-day-old grafts of two-cell eggs. Thirty-seven grafts were recovered40-50 days after transplantation. Nineteen of them contained embryonic shield

PLATE 3

Fig. A. Growth from a two-cell egg grafted for 60 days. Immature neuro-epithelium. x 200.Fig. B. Growth from a two-cell egg grafted for 60 days. Immature muscle fibers, x 200.Fig. C. Growth from a two-cell egg grafted for 60 days. Notochord (center) and epithelialcysts. x200.Figs. D-F. First subcutaneous transplant generation from a growth derived from a two-cellegg grafted to the testis for 60 days.Fig. D. Mature neural tissue, x 430.Fig. E. Adipose tissue, hair follicles, and epithelium, x 100.Fig. F. Immature muscle fibers and epithelial cyst, x 200.

/. Embryo!, exp. Morph., Vol. 20, Part 3

L. C. STEVENS facing p. 334

/. Embryol. exp. Morph., Vol. 20, Part 3 PLATE 4

wmsm^

L. C. STEVENS

Egg graft teratomas 335derivatives and seven had undifferentiated embryonic cells (Plate 2, fig. E).Among the tissue types represented were immature and adult nerve, notochord,thyroid, muscle, and various types of epithelium. One of these grafts gave riseto a metastatic growth of adult neural tissue in the left renal lymph node.

(6) 60-day-old grafts of two-cell eggs. Thirteen grafts were recovered. Fivewere composed of extra-embryonic derivatives only. Six developed into tera-tomas composed of mature tissues. Two others were pleomorphic, but hadundifferentiated embryonic cells and immature tissues (Plate 2, fig. F, andPlate 3, figs. A-C). They were large growths, approximately 25 mm indiameter, and superficially they resembled transplantable testicular teratomas.Portions of them were minced and grafted subcutaneously to 12 male hosts. Mostof these grafts survived until the hosts were killed 5-7 months later, and theywere composed of adult type epithelium, cartilage, muscle, and hair follicles.One, however, contained adult type tissues (Plate 3, figs. D-F, and Plate 4,fig. A) and undifferentiated proliferating cells, and it grew to a retransplantablesize 1 month after grafting. It was regrafted subcutaneously to six males, andgrew to a large size in three of them. These were pleomorphic, but, like trans-plantable teratomas, they contained undifferentiated embryonic cells in additionto immature and mature tissues. They were maintained as transplantableteratomas (Plate 4, figs. B-E) and contained undifferentiated and immatureelements for five subcutaneous transplant generations (165 days). After thefifth generation, all of the cells differentiated and the tumors failed to growprogressively. The two-cell egg that gave rise to this teratoma was grafted intothe testis August, 1966, and the last growth derived from it was retransplantedin July 1967. Seventy mice were recipients of grafts derived from that egg.

Four- to eight-cell eggs grafted to the testis

Strains 129 and A/HeJ eggs were removed from the oviduct 2 days afterfinding copulation plugs and were grafted to the testes of adults (Table 1).Forty-seven growths were recovered 20, 30, and 60 days after transplantationand prepared for histological examination. Results were similar to those observed

PLATE 4

Figs. A-E. Subcutaneous transplant generations from a growth derived from a two-cell egggrafted to the testis for 60 days.Fig. A. First generation. Nodule of cartilage, primitive epithelium, and undifferentiated cells.x200.Fig. B. Third generation. Immature epithelium, mesenchyme, and undifferentiated cells.x200.Fig. C. Fourth generation. Immature neuro-epithelium and undifferentiated cells, x 200.Figs. D-E. Fifth generation. Mesenchyme, primitive epithelium, immature neuro-epithelium,and undifferentiated cells, x 200.

336 L. C. STEVENS

after grafting two-cell eggs. Some growths were pleomorphic and containedundifferentiated embryonic cells and immature tissues as well as well-differen-tiated tissues. The grafts of A/HeJ eggs yielded only two growths of extra-embryonic derivatives. The descriptions below pertain only to strain 129eggs.

(1) 20 days after grafting. Seven grafts were recovered and six of them con-tained extra-embryonic derivatives only. One, in addition to trophoblast anddistal endoderm with Reichert's membrane, had immature neuroepithelium andundifferentiated cells.

(2) 30 days after grafting. Thirty-seven grafts were recovered. Twenty hadonly extra-embryonic derivatives. Seventeen had many types of adult tissues,and ten of these had undifferentiated cells and immature tissues.

(3) 60 days after grafting. A single large growth resulting from a 2-day egggraft was recovered. It was highly pleomorphic, containing immature and adultnerve, many kinds of epithelia, striated muscle, cartilage, bone with marrow,adipose tissue, pigment cells, and undifferentiated cells. There was a metastaticgrowth composed of adult nerve in the left renal lymph node.

DISCUSSION

Two main findings are reported here: (1) Tubal two-cell strain 129 mouseeggs are able to develop into disorganized growths in an extra-uterine site; and(2) Differentiation of some cells in the growths may be delayed for remarkablylong periods of time.

Single-cell mouse eggs did not develop embryonic derivatives when graftedto adult testes. Growths from only four of 250 zygotes grafted to the testis wererecovered, and they were composed solely of trophoblastic cells. Similarly,intratesticular grafts of two-cell (1-day) and four- to eight-cell (2-day) eggs fromstrains A/HeJ, C57BL/Ks, and C57BL/10Sn failed to develop embryonicderivatives. In contrast, several growths containing embryonic derivativesdeveloped from grafts of two-cell and four- to eight-cell eggs of strain 129 origin.Apparently the 'uterine factor' postulated by Kirby (19626, 1965) is not alwaysnecessary for the development of grafted strain 129 eggs.

The donors and hosts were genetically identical, except for Y-linked genes, sothat the growths from grafts of two-cell eggs were not rejected, and they couldbe observed over extended periods of time. Seven days after transplantation, agraft was composed of trophoblastic giant cells, Reichert's membrane, distaland proximal endoderm, and a layer of ectoderm surrounding a proamnioticcavity. The extra- and intra-embryonic derivatives were organized as in normal6-day mouse embryos. After 7 days, the grafts became disorganized, and theintra- and extra-embryonic components were arranged as a chaotic mixture.

All of the cells in the growths were derived from the grafted eggs. The growthswere readily distinguished from host cells, and the two-cell eggs were free of

Egg graft teratomas 337follicle cells. Approximately 10 % of strain 129 males have spontaneous testicularteratomas. These tumors are all detectable grossly in adults, and mice withteratomas were not used as hosts.

The term ^ undifferentiated embryonic cells' is used here to refer to cells whichcannot be identified by morphological criteria as being derived from any of thethree primary germ layers. They are indistinguishable from the stem cells ofteratocarcinomas which Pierce (1967) and Stevens (1967 a), have shown to bepluripotent. They give rise to differentiated tissues and to more undifferentiatedproliferating cells like themselves.

After 20 to 60 days some of the growths derived from two-cell egg graftsconsisted of mixtures of well-differentiated adult-type tissues. Others containedadult tissues and in addition, immature and undifferentiated cells, even after60 days. It was unexpected that proliferating undifferentiated and immaturecells would be found in such old grafts. In one case, a graft of a two-cell egggave rise to a growth that grew subcutaneously for five transplant generations,and retained undifferentiated elements for at least 165 days. Many of the 20- to60-day grafts looked histologically like, and one behaved like the transplantableteratomas derived from the testes of strain 129 mice (Stevens, 1958).

We are currently maintaining transplantable teratomas derived from 3- to6-day embryos grafted to the testes of adults. One is still growing progressivelyafter 2 years of serial transplantion, and several others continue to grow pro-gressively after 1-1/2 years. The transplantation behavior of these tumors willbe the subject of a later article.

Kirby (1963) found that when mouse blastocysts were grafted to the adulttest is, development usually ceased at the morphological stage at which thedefinitive placenta should develop. He thought that failure of the placenta todevelop is presumably a contributory cause of death of the graft. Our resultsshow that the grafts of two-cell mouse embryos will develop and survive in-definitely. Apparently an adequate blood supply develops along with the growthof the graft as it does for transplantable tumors.

The embryonic cells that developed from the grafts were not confined byReichert's membrane. They were free to migrate through the interstitial areasaway from the original graft site. This migratory activity disrupted the normalintercellular relationships, and may have resulted in the delay in the onset ofdifferentiation. Possibly some of the cells which remained morphologicallyundifferentiated for prolonged periods of time failed to receive stimuli fromother cells they would normally be in contact with. An alteration of histogeneticinteractions may underlie the delay in differentiation.

Abbot & Holtzer (1966) found that if differentiated chondrocytes are estab-lished as monodispersed cultures, they cease to synthesize chondroitin sulfateand collagen, and begin to synthesize DNA and to proliferate. This change inbehavior is reversible. They proposed that a chondrocyte whose cell membraneis engaged in amoeboid movement cannot make chondroitin sulfate, but that

338 L. C. STEVENS

DNA synthesis and proliferation is promoted. They advanced a theory thatadherent chondrocytes reciprocally stabilize their cell membranes which allowsthem to make chondroitin sulfate. They point out that this theory and theconcept of contact inhibition proposed by Abercrombie & Heaysman (1954)are obviously analogous. This kind of alteration of intercellular relationshipmay be involved in the delay of differentiation reported here for cells in growthsfrom two-cell eggs.

Bernfield & Fell (1967) found a delay in development to the fully differen-tiated state in pancreatic re-aggregates and the significance of this was unclearto them. They suggested the possibility that the small size of the explant may beinvolved, but that it seemed more likely that interactions between differentiatingand proliferating cells may be a factor in the control of the expression of genomicfunction during development.

The disorganization of the embryo after the 6-day stage disrupted normalcellular relationships and may have delayed the determination of the cells sothat they remained in an undifferentiated state for prolonged periods of time.This interpretation could be used to support the theory, sometimes referred toas the 'misplaced blastomere theory,' proposed by Askanazy (1907), and upheldby Needham (1950), Nicholson (1950), Willis (1962), Collins & Pugh (1964),and Pugh & Smith (1964) that teratomas originate from embryonic totipotentcells that have escaped the influence of embryonic organizers. It is the onlyexperimental evidence to support this view, but I do not believe that it isproof.

The spontaneous and experimentally induced testicular teratomas of strain129 mice are derived from primordial germ cells during the 13th day of fetal lifeand not later (Stevens, 1966, 19676). All of the spontaneous teratomas arisewithin the seminiferous tubules (Stevens, 1962). When male genital ridges withprimordial germ cells from 12-day (for strain 129) or 13-day (for strain A/HeJ)fetuses are grafted into the testes of adults, they develop into testes with tera-tomatous foci, and, as in the early spontaneous tumors, these foci also arisewithin the seminiferous tubules (Stevens, 1964).

Homozygous steel (57/57) mice have very few if any primordial germ cells.When genital ridges from SI I SI mice are grafted into adult testes, they developedinto testes without teratomas (Stevens, 19676). Their fertile littermates ( + / +and 5//+) have primordial germ cells and approximately 75 % of intratesticulargenital ridge grafts from them developed into testes with teratomas. These de-velopmental and genetic studies demonstrate that testicular teratomas arederived from primordial germ cells.

The development of teratomas from two-cell eggs may be explained in twodifferent ways. They may develop directly from undetermined disorganizedproducts of the two-cell egg. Alternatively, the grafted two-cell egg may giverise to cells which become determined and develop characteristics of primordialgerm cells, and it is these that give rise to the teratomas. When genital ridges

Egg graft teratomas 339

with primordial germ cells are grafted to the adult testis, as were the two-celleggs, they do develop teratomas. We are attempting to obtain evidence that willhelp decide between these alternative explanations.

SUMMARY

1. A small proportion of two-cell (but not one-cell) strain 129 mouse eggsdevelop intra- and extra-embryonic derivatives when grafted to adult testes. Thedevelopment of grafted one-cell zygotes was extremely infrequent, and em-bryonic derivatives were never observed.

2. Two-cell eggs develop in the testis quite normally for about a week, butlater they become disorganized mixtures of many kinds of embryonic and adulttissues.

3. Some growths derived from grafts of two-cell eggs have undifferentiatedcells and immature tissues for at least 60 days, and in one case, 165 days.

4. The marked delay in the onset of differentiation of some cells derived fromtwo-cell egg grafts may be attributed to the disruption of normal intercellularrelationships. Alternatively, the undifferentiated and immature cells may havebeen derived from cells with characteristics of primordial germ cells.

RESUME

Formation de teratome a partir de greffe d'ceufs de Souris,dans le testicule de Souris adultes

1. Une petite quantite d'ceufs de Souris de la souche 129, au stade deux cellules(mais jamais au stade une cellule) greffes dans le testicule de Souris adultesse developpent des tissus embryonnaires et des tissus extra embryonnaires.Les zygotes greffes ne se developpent que tres rarement, et des tissus embryon-naires n'y sont jamais observes.

2. Les oeufs du stade deux cellules se developpent normalement dans letesticule pendant une semaine, mais au-dela, ils forment des amas inorganisesdans lesquels on retrouve divers tissus embryonnaires et adultes.

3. Certaines tumeurs issues de la greffe d'ceufs du stade deux cellules contien-nent des cellules non differenciees, et des tissus immatures, jusqu'a 60 jours, etmeme dans une cas, jusqu'a 165 jours.

4. Le retard de certaines cellules a se differencier peut etre du a la rupture desrelations normales intercellulaires. Les cellules non differenciees peuventegalement deriver de cellules semblables aux cellules germinales primordiales.

This investigation was supported in part by Public Health Service Research GrantCA-02662, from the National Cancer Institute, and a grant from The William H. DonnerFoundation, Tnc.

The principles of laboratory animal care as promulgated by the National Society forMedical Research, are observed in this Laboratory.

I gratefully acknowledge the expert assistance of Don S. Varnum.

340 L. C. STEVENS

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JONES, I. C. (1951). Endocrine effects in the virgin female host of fertilized mouse ova de-veloping in extra-uterine sites. / . Endocrinol. 7, lxiii.

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{Manuscript received 25 March 1968)