the development in vitro of the mammalian gonad. ovary and ovogenesis

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  • The Development in vitro of the Mammalian Gonad. Ovary and OvogenesisAuthor(s): P. N. MartinovitchSource: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 125, No.839 (Apr. 27, 1938), pp. 232-249Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/82192 .Accessed: 07/05/2014 16:42

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  • 232 E. C. Dodds and W. Lawson

    Mills, W. H. and Nixon, I. G. 1930 J. Chem. Soc. p. 2520. Norris, J. F., Thomas, R. and Brown, B. M. 1910 Ber. dtsch. chem. Ges. 43, 2958. Robson, J. M. and Schonberg, A. 1937 Nature, Lond., 140, 196. Russanow, A. 1889 Ber. dtsch. chem. Ges. 22, 1944. Schiff, H. 1874 Liebigs Ann. 172, 357. Schlenk, W. 1909 Liebigs Ann. 368, 303. Schmidlin, J. and Garcia-Banus, A. 191a Ber. dtsch. chem. Ges. 45, 3199. Schmidlin, J. and Massini, P. 1909 Ber. dtsch. chem. Ges. 42, 2381. Schneider, F. 1899 Ber. dtsch. chem. Ges. 32, 689. Schopf, M. 1894 Ber. dtsch. chem. Ges. 27, 2324. Serini, A. and Steinruck, K. 1937 Naturwissenschaften, 25, 682. Smith, A. 1893 Ber. dtsch. chem. Ges. 26, 68. Stoermer, R. and Kippe, B. 1903 Ber. dtsch. chem. Ges. 36, 4009. Thiele, J. and Schleussner, K. 1899 Liebigs Ann. 306, 198. Ullmann, F. and Mourawiew-Vinigradoff, A. 1905 Ber. dtsch. chem. Ges. 38, 2218. Ullmann, F. and Stein, A. 1906 Ber. dtsch. chem. Ges. 39, 624. Ullmann, F. and Wurstemberger, R. 1905 Ber. dtsch. chem. Ges. 38, 4105. Werner, G. 1895 Ber. dtsch. chem. Ges. 28, 1999. Wislicenus, W. and Endres, A. 1903 Ber. dtsch. chem. Ges. 36, 1194. Zincke, T. and Munch, S. 1904 Liebigs Ann. 335, 184. Zincke, T. and Muhlhausen, G. 1903 Ber. dtsch. chem. Ges. 36, 131.

    611.013.i6:6ii.013.9

    The development in vitro of the mammalian gonad.

    Ovary and ovogenesis*

    By P. N. Martinovitch

    Strangeways Research Laboratory, Cambridge

    {Communicated by F. H. A. Marshall, F.E.S.?Received 15 December 1937)

    [Plates 9-11]

    Introduction

    Several investigators have tried to cultivate in vitro the mammalian and

    avian gonad, usually with the object of obtaining the growth and differenti?

    ation of the germ cells under in vitro conditions. The results so far

    obtained, however, have been disappointing. Champy (1920) is the only * This work has been made possible by a fellowship granted by the Rockefeller

    Foundation.

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  • The development in vitro of the mammalian gonad 233

    author, so far as we know, who has definitely claimed to observe some

    differentiation of the germ cells in vitro. He states that in testicular ex-

    plants from the mature rabbit he observed the transformation of spermato-

    gonia into spermatocytes, but the process did not advance beyond the

    leptotene phase of meiosis. Champy's findings, however, have not been

    confirmed by other investigators, probably owing to the difficulty of

    cultivating mature testicular tissue.

    In birds, progressive differentiation of the germ cells in vitro has not

    been recorded. Cultures of ovarian and testicular tissue of the embryonic fowl have been studied by Fano and Garofolini (1927 and 1928). The

    explants formed an epithelial outgrowth which was soon replaced by what

    appeared to be fibroblasts. Dantchakoff (1932) cultivated the undif-

    ferentiated gonad of 3|-day fowl embryos and described three types of

    cells in her cultures: (a) large amoeboid cells?Waldeyer's "Ureier" or "

    Urkeimzellen"?which in vitro developed pseudopodia and showed

    active amoeboid movement, (b) connective tissue strands, (c) epithelium? like tissue. Wermel (1933) made cultures of the undifferentiated gonad and of embryonic and adult testes and observed growth of both the somatic

    and germinal tissue. Muratori (1935 and 1937) cultivated gonad tissue

    from embryonic and newly hatched fowls, but although he describes the

    nuclear pattern of the germ cells emigrating from the explants, he makes

    no mention of any differentiation occurring in the explanted gonocytes. Several workers have grown mammalian gonad tissue in vitro.

    Maccabruni (1913) studied cultures of the human foetal ovary and

    observed proliferation from the explant of connective tissue only. Wolff

    and Zondek (1925) obtained proliferation of both connective tissue and

    epithelium from explants of human foetal ovaries and from the ovary of a

    45-year old woman. Olivo (1934) obtained successful cultures of the

    cumulus oophorus tissue containing the egg from a human embryo, but

    failed to cultivate the granulosa tissue without the egg, from the same

    ovary. He also noted that the egg enlarged somewhat during cultivation

    and two large nuclei appeared in it.

    Mjassojedoff (1925) found that the follicular cells of the rabbit ovary when growing in vitro, quickly lose their epithelial character and in

    6-8 days become very like fibroblasts. Champy (1920, 1926, 1927) and

    Champy and Morita (1928) explanted ovarian and testicular tissue from

    mammals (rabbit, rat) and from some of the lower vertebrates, and

    observed that the germinal tissue proliferated in vitro in an undifferentiated

    form. Champy regarded this observation as evidence in support of his

    dedifferentiation theory. As stated above, Champy also described one

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  • 234 P. N. Martinovitch

    stage of spermatogenesis in vitro which took place in a 9-day culture of

    adult rabbit testis after all the spermatocytes present at the time of

    explantation had degenerated. Esaki (1928) studied the fate of interstitial

    and Sertoli cells in explants of rabbit and guinea-pig testis. He stated that

    the interstitial cells are derived from the mesenchyme and that in vitro

    they become transformed into spindle-shaped elements indistinguishable from ordinary fibroblasts; the Sertoli cells became rounded and phago-

    cytic in culture. Mihailoff (1937) recorded the survival in vitro, for about

    70 days, of the seminiferous tubules of the immature rabbit. A few

    primitive germ cells (gonocytes) persisted in a more or less normal state

    during this period, but failed to differentiate. Both mesenchyme derivative

    and Sertoli cells grew and migrated actively. The object of the present investigation was to cultivate the entire ovary

    of embryonic and new-born rats and mice and to study its behaviour

    in vitro with special reference to the growth and differentiation of the germ cells. It was clear that if the germinal tissue could be made to differentiate

    in vitro, it would provide a new method for analysing the complex factors

    responsible for the growth and development of the ovary (Martinovitch

    1937)- Of recent years something has been learned of the influence of the

    gonadotropic hormones on the normal functions of the ovary. Little is

    known, however, of the possible influence of the organ as a whole on the

    germinal tissue which it contains or of the inherent capacity of the germ cells for independent growth and differentiation. The results of the present

    experiments shed some light on both these problems and also on the vexed

    question of the length of survival of the mammalian ovum.

    Material and technique

    Forty ovaries from rats and mice ranging in age from the 16th day of

    embryonic life to the 4th day after birth were cultivated in vitro by the

    watch-glass method (Fell and Robison 1929). The culture medium was

    composed of equal parts of chicken embryo extract prepared in Pannett

    and Compton's solution and chicken plasma. In some experiments 1 %

    glucose was added to the medium. One ovary from each animal was

    explanted in vitro and the corresponding ovary was fixed as a control. In

    order to compare the rate of growth of the ovum in vitro and in vivo, the

    normal ovaries of 8-, 5- and 2-day-old rats were also fixed and sectioned.

    The explants and some of the controls were fixed in Allen's modification

    of Bouin's fluid and serially sectioned. The remaining controls were fixed

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  • The development in vitro of the mam