The Culture in vitro of Urogenital Organs of Pleurodeles waltlii

Transcription

1 The Culture in vitro of Urogenital Organs of Pleurodeles waltlii by CHARLES L. FOOTE and FLORENCE M. FOOTE 1 From the Laboratoire d'embryologie experimentale du College de France et du C.N.R.S., Paris WITH ONE PLATE INTRODUCTION EARLIER reports (Foote & Foote, 1958a, b, 1959) describe growth and maintenance in vitro of larval organs, particularly gonads, of Rana catesbeiana and Xenopus laevis. Immature germ cells of both testes and ovaries are well maintained in vitro, especially if the culture medium is supplemented with watersoluble sex-hormonal substances, although germ cells in process of maturation become necrotic. Recently some urogenital organs from the salamander, Pleurodeles waltlii, have been grown in vitro. Tissues and organs from this amphibian might prove to be more suitable for tissue and organ culture investigations than those of Anurans. MATERIALS AND METHODS Animals at three different ages were used in this study: recently hatched larvae, metamorphosing animals, and adults. Recently hatched larvae (10-12-mm. length) To determine whether sex differentiation would occur in vitro, trunk portions of young larvae of Pleurodeles waltlii of developmental stages (Gallien & Durocher, 1957) were placed in organ cultures. The head, tail, skin, and most of the yolk endoderm were removed from recently hatched larvae. These larvae had been made bacteriologically sterile by placing them in water containing 20 /xg./ml. of streptomycin and 1-0 per cent, (w/v) sulphadiazine for 24 hours prior to cultivation of organs. Trunk portions of larvae composed of neural tube, notochord, myotomes, nephrogenic areas, and a few yolk-filled cells were placed in cultures. Four trunks of larvae were enclosed in a small square of vitelline membrane taken from an unincubated hen's egg according to the recent procedure of Wolff (1960). Fourteen packets, each containing 4 trunk portions of larvae surrounded by vitelline membranes from chick eggs, were placed on 1 Authors' address: Department of Zoology, Southern Illinois University, Carbondale, Illinois, U.S.A. [J. Embryol. exp. Morph. Vol. 10, Part 4, pp , December 1962] H h

2 466 C. L. FOOTE AND F. M. FOOTE CULTURE OF the culture medium. The medium consisted of 3 parts embryo extract from 9-day chick embryos diluted to EE-50 in Tyrode's solution; 3 parts Tyrode's solution diluted 2:1 with double distilled water, 4 parts double distilled water, 5 parts 1 per cent, agar in Gey's solution, penicillin, and mycostatin. Culture dishes used were embryological watch-glasses sealed with paraffin wax according to the method of Wolff & Haffen (1951). Thirty trunks of larvae were placed directly on the medium, in groups of 4 or as single isolated explants. Cultures were maintained for 57 days with transfers to fresh medium at 2-week intervals. Metamorphosing animals (80-mm. length) To investigate the fate of tissues of older larvae 22 pairs of gonads with attached mesonephroi and associated ducts of Pleurodeles at the stage of metamorphosis (approximately 4-5 months of age) were placed in organ cultures with each of two pairs in contact with each other. Sex differentiation occurs during this developmental stage (Gallien, 1954). Twenty-two portions of the same kidneys and gonads were left as single isolated explants in the same culture dishes as the pairs. Cultures were maintained for 21 days without transfer. Adult animals Thirty-two explants of small pieces of testis from adult Pleurodeles were maintained on the standard medium for a period of 21 days. RESULTS Recently hatched larvae Following the 57 days in culture in the vitelline membranes, the trunks of the 4 larvae in each packet had grown together to form a single mass of tissues and organs (Plate 1, fig. A). The neural tubes and mesonephroi showed progressive differentiation and many mitotic figures were observed in sectioned material. The 4 neural tubes and notochords retained their positions in relation to each other, but the original form of the trunks was not apparent. The greater part of the mass of cells and tissues was made up of mesonephric tubules, but Wolffian ducts could not be identified positively. Many pigment cells and granules were distributed throughout the mass. No definitely developed gonads could be recognized, but in several areas groups of large cells, 7-10 in number, had the appearance of primordial germ cells (Plate 1, fig. B). Striated muscle fibres had not formed except in some larvae grown directly on the culture medium. In many of these latter larvae little differentiation had taken place, and in some the general body form was lost because of the dispersion of cells over the surface of the medium. While urogenital organs did not show typical form in larvae enclosed in vitelline membranes, there had been growth and differentiation, not only of these structures, but of nervous tissues also. They showed a greater degree of

3 UROGENITAL ORGANS 467 differentiation and were better maintained than trunk portions of larvae in groups or as individual explants placed directly on the culture medium. However, no larvae in culture were as completely differentiated as intact larvae of comparable ages. Pleurodeles embryos and larvae, grown in chick vitelline membranes in vitro, appear to be the most promising for studies of cellular differentiation in tissue and organ cultures of the various amphibian species used for this type of experiment to date. Metamorphosing animals From the older group of larvae 22 isolated explants (16 males and 6 females) and 11 pairs of gonads and mesonephroi were recovered following the 21-day period in culture. Of the 11 pairs of gonads 7 were male-male, 2 female-female, and 2 male-female combinations. There was much distention of the ovarial sacs in ovaries paired with testes, as well as those of isolated explants. Some auxocytes were present, but many germ cells were in premiotic stages with distinct chromosomes (Plate 1, fig. C). Mesonephroi were less well maintained than gonads, perhaps because of their size, and there was necrosis of some cells in the kidney tubules (Plate 1, fig. D). In general, the Mullerian and Wolffian ducts were in good condition, but the lumina of some were obliterated by cell fragments (Plate 1, fig. D). In the two heterosexual pairs there had been no apparent modification of either gonad, since they appeared similar to those pairs of like sex. This suggests that at this developmental stage these gonads grown together in organ cultures for 21 days were not modified, and no evidence of sex reversal was apparent. The gonads and kidneys of the isolated explants were identical to those grown in contact. It is possible that the period of time the organs were in culture was too short for one gonad to exert a modifying influence on another. One of the principal points of interest is that these gonads were better maintained and in better condition at the end of the culture period than urogenital organs from other species of amphibians cultivated in vitro (Foote & Foote, 1958o, 1959). Adults Since it is apparent that the gonads of Pleurodeles, at the time of sex differentiation, are very well maintained in organ cultures for up to 3 weeks, explants of adult testes were placed on similar media for the same period of time, in order to compare the maintenance in vitro of larval and adult organs. Those explants of adult testis containing primordial germ cells and spermatogonia, together with their associated somatic cells, appeared to be in as good condition as those of larval gonads (Plate 1, figs. E, F). However, areas containing mature spermatozoa were disorganized and the sperm fragmented, due, at least in part, to necrosis of the Sertoli cells. Other somatic cells were well maintained. Those germ cells undergoing cell-division at the time they were placed in culture were

4 468 C. L. FOOTE AND F. M. FOOTE CULTURE OF not visible at the end of the culture period, and only somatic cells were present in the explants. No attempt was made to culture ovaries of adult Pleurodeles since previous experiments have shown that ovaries containing auxocytes are not well supported in vitro (Foote & Foote, 1957). The results given here for testes of adult Pleurodeles are similar to those reported for gonads of adult Triturus cristatus carnifex (Foote & Foote, 1957). DISCUSSION While the embryonic gonads of birds and mammals have been grown successfully in vitro on both natural and synthetic media (Wolff & Haffen, 1951; Wolff, 1952; Stenger-Haffen, 1957), the cultivation of ovaries and testes of amphibians in vitro often results in the degeneration of the older germ cells and the transformation of younger germ cells into fibroblast-like cells (Foote & Foote, 1958«). The cultivation of embryonic gonads of birds and mammals has perhaps been more successful than that of amphibians because the length of time required for sex differentiation to occur is shorter in the former groups, whether in vivo or in vitro. When amphibian gonads are maintained on media with components from natural sources, growth and maintenance are better than on any of the synthetic media used to date (Foote & Foote, 1958a, b, 1961). However, when hormonal substances are added to the media, natural or synthetic, both germ cells and somatic cells retain their characteristic form for as long as 2 months, but with little progressive differentiation (Foote & Foote, 1959). Wolf, Quimby, Pyle, & Dexter (1960) obtained poor results in attempts to prepare monolayer cell cultures from gonads of adult fish and amphibians, but found that immature gonadal cells readily attached and divided. The results from the present experiments indicate that for studies of cellular growth and differentiation of amphibian tissues in vitro, P. waltlii may be a favourable species. Gallien (1959) has shown that this urodele is excellent for in vivo experimental studies. The technique of enclosing explants in chick vitelline membranes (Wolff, 1960) gives promise of growing amphibian cells, tissues, and organs in good condition for long periods of time. The appearance of primordial germ cells in these cultured larval tissues suggests a method for investigation into the origin of these cells, as well as their differentiation and ultimate fate. SUMMARY The trunk portions of Pleurodeles waltlii, stages 37-38, were grown in groups of 4 in chick vitelline membranes in organ cultures for 57 days. Growth and differentiation of urogenital structures and nervous tissues occurred, including some cells thought to be primordial germ cells. Trunks of larvae placed directly on culture medium were less well differentiated and less well maintained.

5 UROGENITAL ORGANS 469 Larval gonads containing young germ cells or gonads with immature germ cells from adult P. waltlii were well maintained in organ culture for 21 days. Spermatozoa and meiotic stages in explants of mature testes became disorganized and necrotic. Immature gonads of P. waltlii are better maintained in organ culture than gonads of the other species of amphibians studied to date. RESUME Culture in vitro d'organes urogenitaux de Pleurodeles waltlii Des fragments de tronc de Pleurodeles waltlii, aux stades 37-38, ont ete cultives par groupes de 4 dans des membranes vitellines de poulet pendant une periode de 57 jours en culture d'organes. On observe la croissance et la differentiation de structures urogenitales et de tissus nerveux, parmi lesquels certaines cellules paraissent representer les cellules germinales primordiales. Des troncs de larves places directement sur le milieu se differencient et se maintiennent moins bien. Des gonades larvaires contenant de jeunes cellules germinales ou des gonades de l'adulte contenant des cellules germinales pas mures se maintiennent bien en culture d'organes pendant 21 jours. Les spermatozoides et les stades meiotiques des testicules murs se desorganisent et se necrosent. Des gonades de P. waltlii avant la maturite sexuelle se maintiennent mieux en culture d'organes que les gonades d'autres especes d'amphibiens etudiees jusqu'a ce jour. ACKNOWLEDGEMENT The authors wish to express their appreciation to Professor Etienne Wolff, Director, Laboratoire d'embryologie experimentale du College de France et du C.N.R.S., Paris, for the hospitality of his laboratory during the time the senior author was Special Research Fellow, U.S. Public Health Service, REFERENCES FOOTE, C. L., & FOOTE, F. M. (1957). In vitro cultivation of gonads of adult amphibia. Anat. Rec. 127,415. (1958a). In vitro cultivation of gonads of larval anurans. Anat. Rec. 130, (19586). Maintenance of gonads of frog larvae on synthetic media. Proc. Soc. exp. Biol. Med. N.Y. 98, (1959). Effects in vivo and in vitro of a water soluble testosterone on gonads of two species of anurans. Arch. Anat. micr. Morph. exp. 48, (1961). Culture d'ovaires d'axolotl sur differents milieux. C.R. Acad. Sci. Paris, 253, GALLIEN, L. (1954). Inversion experimentale du sexe, sous l'action des hormones sexuelles, chez le triton, Pleurodeles waltlii, Michah. Bull. Biol. 88, (1959). Analyse des effets des hormones steroides dans la differentiation sexuelle des amphibiens. Arch. Anat. micr. Morph. exp. 48, & DUROCHER, M. (1957). Table chronologique du developpement chez Pleurodeles waltlii. Bull. Biol. 91, STENGER-HAFFEN, K. (1957). Etude des besoins nutritifs des gonades embryonnaires d'oiseau cultivees en milieux synthetiques. Arch. Anat. micr. Morph. exp. 46, WOLF, K., QUIMBY, M. C, PYLE, E. A., & DEXTER, R. P. (1960). Preparation of monolayer cell cultures from tissues of some lower vertebrates. Science, 132,

6 470 C. L. FOOTE AND F. M. FOOTE WOLFF, T. (1952). Sur la differenciation sexuelle des gonades de Souris explantees. C.R. Acad. Sci. Paris, 234, (1960). Sur une nouvelle modalite de la culture organotypique. C.R. Acad. Sci. Paris, 250, & HAFFEN, K. (1951). Sur la culture in vitro des glandes genitales des embryons d'oiseau: obtention de la differenciation sexuelle normale et de l'intersexualite experimentale des gonades explantees. C.R. Acad. Sci. Paris, 233, EXPLANATION OF PLATE FIG. A. Section through mass formed by 4 P. waltlii larvae grown together in culture in vitelline membrane for 57 days. Note neural tubes (N), notochords (c), mesonephric tubules (M), primordial germ cells (p). xll5. FIG. B. Section through mesonephric tubules and primordial germ cells observed in another group of 4 larvae cultivated under the same conditions as those shown in fig. A. x 320. FIG. C. Section of ovary of larval P. waltlii maintained 21 days in culture, paired with testis shown in fig. D. Note distention in 2 lobes of the ovary, x 80. FIG. D. Section of testis and mesonephros of larval P. waltlii maintained 21 days in culture paired with ovary shown in fig. C. Note Mullerian and Wolffian ducts at lower left, x 70. FIG. E. Section through anterior end of testis of adult P. waltlii showing immature germ cells, x 130. FIG. F. Section of explant taken from same area of testis as fig. E after 21 days in culture, x 130. (Manuscript received 9:i: 62)

7 J. Embryol. exp. Morph. Vol. 10, Part 4 C. L. FOOTE and F. M. FOOTE