The development of teratomas from parthenogenetically activated ovarian mouse eggs

DEVELOPMENTAL

BIOLOGY

37, 369-380 (1974)

The Development

of Teratomas

Activated

Ovarian

from Parthenogenetically Mouse

Eggs”

2

LEROY C. STEVENS AND DON S. VARNUM The Jackson Laboratory. Accepted

Bar Harbor,

December

Maine 04609

3, 1973

Ovarian teratomas developed spontaneously in about half of the females of the inbred strain LT. Some of them began to develop at about 30 days of age, and the incidence rose to about 50% in animals 90 days old. They originated from ovarian eggs that began to develop parthenogenetically. They resembled normal embryos until the blastocyst stage. after which most became disorganized. The most advanced ovarian embryo observed had a primitive streak and resembled a normal embryo of 7.5 days’ gestation. Most of the teratomas were benign and composed of many types of well differentiated tissues of embryonic and extraembryonic origin. but some of them contained proliferating undifferentiated cells. Parts of many of them were grafted subcutaneously, but only one gave rise to a transplantable teratoma. It produced several tissue types and undifferentiated stem cells. Parthenogenesis also occurs spontaneously in a small percentage of ovulated LT eggs. The! undergo cleavage and implant in the uterus. Most of them die at 5 to 7 days of gestation. INTRODUCTION

Only a few ovarian teratomas in the mouse have been described previously. Slye et al. (1920) found two solid ovarian teratomas among 25,000 autopsied mice. Jackson and Brues (1941) grafted a spontaneous teratoma from the left ovary of a C3H mouse and found that during the course of serial transplanation the composition of the tumor did not change. All grafts developed into tumors composed of undifferentiated embryonic cells and diverse types of immature and mature tissues. When well differentiated areas were selected for grafts, they either did not grow progressively or grew extremely slowly. They concluded that the most immature cells were responsible for growth, since they contained virtually all of the mitotic activity. ‘Supported in part by research grant CA 02662 from the National Cancer Institute and in part by grants from the Cancer Society of Greensville County, South Carolina, the Ladies Auxiliary of the Veterans of Foreign Wars, the Al Rose Cancer Aid, and the Endowment Fund of the Jackson Laboratory. ‘The Jackson Laboratory is fully accredited by The American Association for Accreditation of Laboratory Animal Care.

Fawcett (1950) described a case of bilateral teratomas of the ovary in a mouse of the “Swiss” albino strain. The tumors were composed of several recognizable embryonic tissues in varying degrees of maturation including large areas of nervous tissue, glandlike structures, cysts filled with mucoid secretion, gastric mucosa, squamous cells forming epithelial pearls, and small nodules of cartilage. He noted that germinal epithelium was continuous with mucus-secreting epithelium with tubular structures resembling typical gastric glands. This arrangement suggested that the cells of the germinal epithelium may have been capable of differentiating directly into the various embryonic tissues found in ovarian teratomas. This is not the case in this study. Fekete and Ferrigno (1952) described an ovarian teratoma in a C3HeB strain mouse. It was composed of diploid undifferentiated embryonic cells and many types of differentiated tissues. They selected small pieces of pigmented parts, soft white parts, or hard bony parts to graft in an unsuccessful attempt to separate the different components. They concluded that the stem cells were undifferentiated pluri--

370

DEVELOPMENTAL.

BIOLOGY

potent cells. Direct evidence to support this theory for testicular teratomas was obtained by Kleinsmith and Pierce (1964) and others (see Evans, 1972). When this teratocarcinoma was converted to an ascitic form, it lost its pluripotency and formed only parietal endoderm with Reichert’s membrane (Pierce et al., 1960). Thiery (1963) found two ovarian teratomas in CSH/N mice, one of which was pregnant. Meier et al. (1970) found two unique teratomas in the ovaries of CBA/J and DBA/W females. They contained parieta1 endoderm carcinoma with Reichert’s membrane. Pierce and Dixon (1959b) first identified this type of tumor in a growth derived from a testicular teratoma of a strain 129/Sv mouse. Our colleagues at the Jackson Laboratory have been aware of our interest in teratomas, and on about six occasions during the last 15 years they have drawn our attention to chance findings of ovarian teratomas. In March, 1971, Dr. S. E. Bernstein found a teratoma in the ovary of an LT/ChReSv (hereafter referred to as LT) mouse. He presented us the mouse and several of her close relatives. It soon became apparent that ovarian teratocarcinogenesis occurred in about half of LT females, and that for the first time we had an animal model available for developmental and experimental studies of this disease. This article will deal with the incidence, composition, and development of these tumors. It will show that teratomas originate from eggs that develop parthenogenetically within the ovary. It will also briefly describe the development of embryos that undergo parthenogenesis in the oviduct and implant in the uterus, but this will be the subject of another article. MATERIALS

AND

METHODS

Origin of inbred LT mice. MacDowell (1950) described a mutation in strain C58 called light (Bit), which affected coat color.

VOLUME

37,1974

He outcrossed an affected mouse to strain BALB, and after 7 generations of brothersister matings, he sent a litter to Dr. Herman Chase. After 28 generations of brother-sister matings, Chase sent mice in 1957 to Dr. E. S. Russell of the Jackson Laboratory, who shared her colony with Dr. S. E. Bernstein. We received LT mice from Bernstein in 1971 at the 75th generation of brother-sister mating. The present LT colony is derived from these animals. They are good breeders with an average litter size of 5.3. Identification of teratomas. Teratomas were identified by two methods. One was by examination of ovaries with a dissecting microscope. The other was by examination of serial histological sections. Some tumors could be detected only by this method. Most of the teratomas were composed of several tissue types and were easy to recognize. In a few cases, however, the designation as teratoma was arbitrary. They originated from ovarian eggs which began to develop parthenogenetically, and the first recognizable stage was composed of two blastomeres. All adult LT ovaries contained cleaving eggs, but it was clear that most of them did not continue to develop into tumors composed of several types of tissues. We did not classify cleaving ovarian eggs as teratomas since most of them apparently died before primary germ layer formation. However, if the early growths had advanced to form endoderm and ectoderm, as in normal 6-day embryos, we classified them as teratomas because we believed they would have continued to develop and produce several tissue types. Some growths were composed only of trophoblastic giant cells. We arbitrarily did not classify these as teratomas since they were composed of differentiated cells that had stopped proliferating and had lost the ability to produce other cell types. Females were superovulated using 5 IU pregnant mares serum followed 55 hours later by 5 IU human chorionic gonadotropin.

Parthenogenetic

STEVENS AND VARNUM

of Teratomas

in LT Mice

Ovarian teratomas of strain LT mice ranged in size from microscopic to about 3 cm in diameter. Grossly identifiable tissues and cell types included epithelial cysts with clear or brown fluid; and solid parts with pigment cells, hair, cartilage, bone, and adipose tissue. Hemorrhagic areas in some small teratomas indicated the presence of trophoblastic giant cells. Serial sections of 146 LT ovarian teratomas were examined histologically. Table 1 summarizes the composition of the teratomas according to the age of the host. Animals from 5 to 14 days old were classified as being 10 days old. Animals 15 to 24 days old were classified as 20 days old, etc. Many types of cells and tissues were represented in the teratomas (Figs. 1-9). The most commonly observed was immature and adult neural tissue. Retina with pigmented and nuclear layers and choroid plexus were present in some tumors. Various types of epithelia; cartilage; bone with marrow; skeletal, smooth, and cardiac TABLE

371

Development

muscle; pigment cells; and trophoblastic giant cells were present in many teratomas. Undifferentiated embryonic cells were observed in some teratomas, but they were not common in tumors of older mice. Early teratomas were observed in histological sections of ovaries of five 30-day-old mice. They were composed of groups ot undifferentiated embryonic cells that resembled cells in 6- or 7-day embryos. The cells were arranged as primative epithelium (Fig. 9) spreading through normal ovarian tissue. Teratomas were not observed in serial sections of ovaries from 42 mice younger than 30 days. Nine teratomas from 40-day-old mice were examined histologically. Several types of cells and tissues were observed including undifferentiated embryonic cells. trophoblastic giant cells, distal endoderm with Reichert’s membrane, neural tissue. skeletal and cardiac muscle, and immature epithelium. In one of the tumors. all the cells were differentiated, and it appeared to have stopped growing. A teratoma in an ovary of a X)-day-old mouse consisted only of embryonic ec-

OBSERVATIONS

Descriptions

Teratoma

1

COMPOSI~ON OF LT OVARIAN TERATOMAS ACCORDING TO AGE Age (days ) No. of teratomas Embryoid bodies Undifferentiated cells Trophoblastic giant cells Reichert’s membrane Immature neuroepithelium Mature neural tissue Proximal endoderm Organized epithelium Cardiac muscle Skeletal muscle All mature tissues Choroid plexus Glandular epithelium Skin Bone Cartilage Pigment Retina Bone marrow Uterine decidua

10 0

20 0

30 5 5 5

40 9 5 6 3 17 2 4 7 1 1 1 1 0 0 0 0 0 0 0 0 0

50 17 2 5 14 3 9 14 3 6 1 2 2 4 4 1 4 7 5 1 0 0

60 17 3 3 0 0 5 17 1 14 1 5 8 5 4 0 4 2 9 4 3 0

70 12 0 4 1 1 3 9 1 5 2 5 5 1 2 0 4 5 7 1 2 0

80 5 0 1 1 0 1 4 1 3 0 1 3 1 0 0 2 1 2 0 0 0

go 4 1 2

loo87 12 18

1

‘1 13

1 1 3 1 3 0 0 I 1 1 0 0

24 68 3 61 7 24 37 9 46 6 20

1

23

2 0 0 0

27 10 7 4

FIGS. 1-6. Well differentiated tissues observed in LT ovarian teratomas. neuroepithelium and undifferentiated embryonic cells. FIG. 2. Cyst lined with cuboidai and stratified squamous epithelium. FIG. 3. Retinal tissue. FIG. 4. High power photomicrograph of retina showing pigmented and nuclear layers. FIG. 5. Choroid plexus. FIG. 6. Thyroid.

FIG. 1. Immature

312

FIG. 7. Undifferentiated embryonic cells in teratoma of a 3-month-old mouse. FIG. 8. Skin with hair follicles and sebaceous glands (top) and bone (b) with marrow. FIG. 9. Early teratoma in a l-month-old mouse. Note similarity to Fig. 7. Remnant of egg, left center. FIGS. 10-13. Cleaving eggs in ovarian follicles. Note polar body (?) in Fig. 10. 373

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VOLUME 37, 1974

toderm and endoderm arranged as in a normal 6day embryo. Other tumors of this age group had several tissue types of various degrees of maturation. A few of the teratomas contained uterine decidual tissue and lens. We had not observed these in testicular teratomas.

The early stages in the development of ovarian teratomas were strikingly similar to early stages of normal mouse embryos (Figs. 10-21). Healthy appearing 2-cell eggs, some apparently with polar bodies, were observed in all ovaries of mice older than 18 days, Later cleavage stages and morulae were also observed. They were Incidence and later&y of spontaneous composed of cells of equal size, and mitotic ovarian teratomas in LT mice figures were common. Blastocysts were less The incidence of teratomas in mice from common than earlier stages, but not rare. 10 days to 4 months old is presented in This indicated that some embryos in early stages of development did not survive. Table 2. No tumors were observed histologically in mice 10 to 20 days of age. No Early blastocysts with only a few inner cell tumors were detected with a dissecting mass cells were seen, as well as later stages microscope in ovaries of l-month-old mice. with a layer of recognizable proximal enEleven percent of mice 2 months old, 42% dodermal cells. Distal endodermal cells of mice 3 months old, and 40% of mice 4 TABLE 2 months old had teratomas detectable with INCIDENCE OF SPONTANEOUS OVARIAN TERATOMAS ~-4 a dissecting microscope. LT MICE The ovaries of 63 of the 3-month-old Number of mice (%) Number mice were examined with a dissecting miAge with teratomas observed of mice croscope and also histologically. ThirtyHistoWith Disseven percent of the mice had teratomas logically setting detectable with a dissecting microscope, mic.roscope but 52% of them had tumors observable 0 42 0 lo-20 Days histologically. 52 0 Month The incidence of teratomas reached a 21 Months 17 (11%) 148 peak of 52% at 3 months of age, and about 3 months 95 40 (42%) 15% were microscopic and could not be 33 (52%) 63” 23 (37%) 4 months 100 40(40%) recognized with a dissecting microscope. Thirty-one of the teratomas were in the DOvaries of 63 of the 95 three-month-old mice were left ovary, 53 in the right, and 13 were examined with a dissecting microscope and histologibilateral. Apparently the right ovary is cally. more likely to develop tumors than the left. TABLE 3

Origin of Teratomas

in LT mice

Teratomas in LT mice originated from ovarian oocytes that began to develop parthenogenetically in single- and many-layered follicles. Condensed chromosomes, multinucleate eggs, and early cleavage stages (Figs. 10-13) were observed in all of 7 serially sectioned ovaries 20-24 days old, in about half of those 18 days of age, in only 5 of 26 ovaries 17 days old, but not in any of 54 ovaries 16 days of age and younger (Table 3). There were no corpora lutea in any of these younger ovaries.

INCIDENCE OF CLEAVING EGGS IN OVARIES OF YOUNG LT MICE EXAMINED HISTOLOGICALLY

in days

Age

Number of ovaries

Number of ovaries with cleaving eggs

12 13 14 15 16 17 18 20 24

10 2 12 18 12 26 32 2 5

0 0 0 0 0 5 15 2 5

FIGS. 14-18. Blastocyst formation in ovaries of LT mice. Note crystal in dead blastomere in Fig. 14 (lower left). In Fig. 18 proximal endoderm is beginning to form on ventral surface of inner cell mass. FIG. 19. Ovarian embryo equivalent to about 6 days of gestation. 355

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VOLUME 31,1974

FIGS. 20 and 21. An advanced organized ovarian embryo with ectoderm, mesoderm forming from the primitive streak (top), proximal and distal endoderm, Reichert’s membrane, and trophoblastic giant cells. FIGS. 22 and 23. Clusters of disorganized embryonic cells.

STEVENSANDVARNUM

Parthenogenetic Teratoma Development

were observed migrating from the inner cell mass along the inner surface of the trophectoderm. The inner cell mass grew down into the yolk cavity. A proamniotic cavity appeared (Fig. 19) and the embryos were composed of an inner layer of ectoderm surrounded by a layer of proximal endoderm in turn surrounded by distal endoderm, Reichert’s membrane, and trophoblastic giant cells. The most advanced organized ovarian embryo observed also had mesoderm forming from a primitive streak (Figs. 20 and 21). When the growths reached the stage comparable to normal 6- to 7-day embryos, they became disorganized (Figs. 22-24). Undifferentiated cells migrated away from the site of origin and continued to proliferate or to differentiate. The structure of some of the parthenogenetic ovarian growths indicated that some developed in a more normal manner than others. For example, some were composed only of trophoblastic giant cells (Fig. 25), others of trophoblastic giant cells and neural tissue of various degrees of maturity, and one was composed solely of trophoblastic giant cells and yolk sac with immature red blood cells (Fig. 26). Some early tumors lacked trophoblastic cells. Frequently the distal endodermal cells were not arranged as a sheet, and Reichert’s membrane was secreted in solid masses. Fifteen of 126 ovaries serially sectioned and examined histologically had foci of trophoblastic giant cells and distal endoderm with Reichert’s membrane without other extraovarian cell types and were not classified as teratomas. No teratomas were seen in LT mice other than in the ovary Transplantable

LT Teratoma

Most LT ovarian teratomas were benign because their embryonic stem cells differentiated and ceased to proliferate. However, 1% teratomas appeared grossly as it they might grow progressively, and pieces

377

of them were grafted subcutaneously into 38 adult LT hosts. Only one of these has grown for more than a few transplant generations. After 13 generations. this t.umor is currently composed of’ several tissue types including neural tissue. epithelia of various types, muscle, pigment cells, retina, lens. mesenchymal, and undifferentiated cells. We are maintaining about 20 sublines derived from this tumor, and no conspicuous histological differences have evolved among them. Transplant able teratomas of testicular and embryonic origin changed in composition with increased numbers of transplant generations (Stevens 1958, and personal observations since then), and the transplantable LT ovarian teratoma may also change. In Utero Development of Parthenotes During postmortem examination of virgin female LT mice a small percentage were found to be pregnant. The first parthenote was detected by a localized swelling in the uterus. It was dissected from the decidua and a yolk sac enclosing an embryolike structure was recognized. Histologically the swelling was composed of uterine decidua, trophoblastic giant cells, Reichert’s membrane, distal endoderm. proximal endoderm, disorganized ecmesenchymal cells, and cells toderm, which looked like early fetal heart muscle. This first parthenote was the most advanced we have seen. Most of’ them, such as the one represented in Fig. 27. resembled 5- or B-day embryos. Some parthenotes were composed only 01’ trophoblastic cells, Reichert’s membrane. and cellular debris. Apparently other embryonic and extraembryonic elements had already died. LT females were superovulated. and one-cell eggs were recovered f’rom the oviduct. In most cases 2-3 days later some of the eggs had undergone cleavage and two-cell eggs. eight-cell eggs, moruiae. or blastocysts were present. Chromosomes (Jf’ some of’ the hlastocysts were obser\:ed. and cells were haploid, diploid. or polyploid.

FIG. 24. Ectodermal (upper) and endodermal (lower) vesicles in an LT teratoma. FIG. 25. Tumor composed solely of trophoblastic giant cells. FIG. 26. Simple teratoma containing only trophoblastic giant cells and yolk sac with immature blood cells. FIG. 27. Parthenote implanted in uterine descidua composed of ectoderm, proximal and distal endoderm, Reichert’s membrane (R), and trophoblastic giant cells. 378

STEVENS ANDVARNUM

Parthenogenetic Teratoma Development

DISCUSSION

There are four sources of teratomatous tissues in mice. (I) Testicular teratomas occur spontaneously in some sublines of strain 129, and a small proportion of them grow progressively when serially transplanted. About one-third of the males of a new inbred subline of mice, 129/terSv, develop spontaneous congenital testicular teratomas (Stevens, 1973), and we are attempting to establish transplantable tumors from them. (2) Teratomas can be experimentally induced in grafted male genital ridges of several strains of mice (Stevens, 1970; Stevens and Varnum, unpublished). (3) Male and female teratomas of any inbred strain or hybrid may be obtained by grafting B-day embryos to various sites in histocompatible adults (Stevens, 1968, 1970; Damjanov et al., 1971a,b). We are attempting to obtain transplantable teratomas by grafting parthenote embryos to adult sites. (4) Ovarian teratomas are rare in mice, but they occur spontaneously in about half of inbred strain LT females 3 months and older. Most were composed of several types of well differentiated tissues and are similar histologically and in growth pattern to the spontaneous testicular teratomas of strain 129 mice (Stevens and Hummel, 1957; Stevens, 1973). There are a few conspicuous histological differences. Most spontaneous testicular teratomas have notochord which has not been observed in ovarian teratomas. Well organized retina was observed in several ovarian teratomas, but was rare in testicular teratomas. Parieta1 endoderm with Reichert’s membrane, trophoblastic giant cells, and uterine decidua were observed in ovarian but not in primary testicular tumors. Teratomas were first observable in males at 15 days of gestation. Oocyte cleavage stages were not observed in ovaries of LT mice 17 days and younger, but they were common in oocytes in immature follicles of older animals. Cleaved eggs were interpreted as representing early stages of tera-

379

toma development. The youngest females with teratomas were about 30 days. Tumors were more frequent in 2-month-old mice, and the incidence rose to about 50°C in S-month-old mice. Probably cleaving eggs in ovaries of young mice did not survive to form teratomas as frequently as in older mice. Teratocarcinogenesis in the testis is under strong genetic control (Stevens and Mackensen, 1961; Stevens, 1967a). At least 3 genetic loci influence susceptibility to teratocarcinogenesis in the testis (Stevens, 1973). We are trying to determine whether specific genes influence teratocarcinogenesis in the ovaries of LT mice. Preliminary evidence indicates that parthenogenetic development in LT mice is not due to the action of the gene B”. Sixty-two F, hybrids between strains LT and C57BLI6 were examined and five of them had ovarian teratomas. Probably a small number of genes determine susceptibility to ovarian teratocarcinogenesis in LT mice. Early in the course of this investigation it was apparent that teratomas did not appear until females were old enough to have corpora lutea. A hormonal component was considered as a possible etiological agent, but we have not yet identified any environmental factor involved in teratocarcinogenesis of the ovary. Until recently, implantation of parthenogenetic mouse embryos had not been described. Tarkowski et al. (1970) activated eggs in situ by electric shock, and obtained implantations of embryos which developed for about 7 days. Graham ( 1970) obtained parthenogenesis in vitro by removing the cumulus cells with hyaluronidase and culturing eggs. Both observed haploid and diploid blastocyst cells. The parthenotes of LT mice were also composed of haploid and diploid cells, and they died at the same developmental stage as those induced by Tarkowski (1971) and Graham (1970). The reasons for this limited life-span are not yet known; however, lethality must be at the organismic rather

380

DEVELOPMENTAL BIOLOGY

than cellular level. The cells of parthenotes which develop in the ovary survive in teratomas indefinitely, and parthenogenetic blastocysts can be rescued from the oviduct by grafting them to adult testes where some cells have survived (Stevens and Varnum, unpublished). Other attempts to rescue parthenogenetic cells are being made by fusing parthenotes with normal embryos to produce chimeras. We will also attempt to graft parthenote embryonic cells into the blastocoels of normal blastocysts. Mosinger (1961) discussed the origin of ovarian teratomas other than in the mouse, and concluded that they (embryomes) originated parthenogenetically in the guinea pig, because all stages from two blastomeres to morulae were observed near complex teratomas. The teratomas of LT ovaries are a particularly good illustration of this, because all stages from dividing eggs to blastocysts to germ layer formation to complex large teratomas are observed and furthermore, LT eggs undergo spontaneous parthenogenetic cleavage in the oviducts and implant in the uteri. The genetic basis, chromosomal constitution, developmental capacities, and other characteristics of these parthenotes are under investigation. REFERENCES DAMJANOV,I., SOLTER,D., BELICZA,M., and SKREB,N. (1971a). Teratomas observed through extrauterine growth of seven-day mouse embryos. J. Nut. Cancer Inst. 46, 471-480. DAMJANOV,I., SOLTER,D., and SKREB, N. (1971b). Teratocarcinogenesis as related to the age of embryos grafted under the kidney capsule. Wilhelm Roux’ Arch. Entwicklungsmech. Organismen 167, 288-290. EVANS,M. J. (1972). The isolation and properties of a clonal tissue culture strain of pluripotent mouse teratoma cells. J. Embryol. Exp. Morphol. 28, 163-176. FAWCETT,D. W. (1950). Bilateral ovarian teratomas in a mouse. Cancer Res. 10, 705-707. FEKETE,E., and FERRIGNO,M. A. (1952). Studies on a transplantable teratoma of the mouse. Cancer Res. 12, 438-440.

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GRAHAM,C. F. (1970). Parthenogenetic mouse blastocysts. Nature (London) 226, 165-167. JACKSON,E. B., and BRUES,A. M. (1941). Studies on a transplantable embryoma of the mouse. Cancer Res. 1, 494-498.

KLEINSMITH,L. J., and PIERCE,G. B. (1964). Multipotentiality of embryonal carcinoma cells. Cancer Res. 24, 1544-1551. MACDOWELL,E. C. (1950). “Light’‘-A new mouse color. J. Hered. 41, 35-36. MEIER, H., MYERS, D. D., Fox, R. R., and LAIRD, C. W. (1970). Occurrence, pathological features, and propagation of gonadal teratomas in inbred mice and the rabbit. Cancer Res. 30, 30-34. MOSINGER,M. (1961). Sur la carcinoresistance du cobaye. I. Les tumeurs spontanes du cobaye. Bull. Cancer 48, 217-235.

PIERCE,G. B., and DIXON, F. J. (195913).Testicular teratomas. II. Teratoma as an ascitic tumor. Cancer 12, 584-589. PIERCE,G. B., DIXON, F. J., and VERNEY,E. L. (1960). An ovarian teratocarcinoma as an ascitic tumor. Cancer Res. 20, 106-111. SLYE, M., HOLMES,H. F., and WELLS,H. G. (1920). Primary spontaneous tumors of the ovary in mice. Studies on the incidence and inheritability of spontaneous tumors in mice. 14th Communication. J. Cancer Res. 5, 205-226.

STEVENS,L. C., and HUMMEL,K. P. (1957). A description of spontaneous congenital testicular teratomas in strain 129 mice. J. Nut. Cancer Inst. 18,719-747. STEVENS,L. C. (1958). Studies on transplantable testicular teratomas of strain 129 mice. J. Nut. Cancer Inst. 20, 1257-1276. STEVENS,L. C., and MACKENSEN,J. A. (1961). Genetic and environmental influences on teratocarcinogenesis in mice. J. Nat. Cancer Inst. 27, 443-453. STEVENS,L. C. (1967a). The biology of teratomas. Aduan. Morphog. 6, 1-31, (1967). STEVENS,L. C. (1968). The development of teratomas from intratesticular grafts of tubal mouse eggs. J. Embryol. Exp. Morphol. 20, 329-341. STEVENS,L. C. (1970). Experimental production of testicular teratomas in mice of strains 129, A/He, and their F, hybrids. J. Nut. Cancer Inst. 44, 923-929.

STEVENS,L. C. (1973). A new inbred subline of mice (129/terSv) with a high incidence of spontaneous congenital testicular teratomas. J. Nut. Cancer Inst. 50, 235-242.

TARKOWSKI,A. K. (1971). Recent studies on parthenogenesis in the mouse. J. Reprod. Fert., Suppl. 14, 31-39. TARKOWSKI,A. K., WITKOWSKA,A., and NOWICKA,J. (1970). Experimental parthenogenesis in the mouse. Nature (London) 226, 162-165. THIERY, M. (1963). Ovarian teratomas in the mouse. Brit. J. Cancer 17, 231-234.