Natural Model for the Study of Germ Cell Sexual Differentiation

Proc. NatL Acad. Sci. USA Vol. 79, pp. 6584-6588, November 1982 Developmental.Biology

Ectopic germ cells: Natural model for the study of germ cell sexual differentiation (meiosis/oocyte development/adrenal gland) SHAKTI UPADHYAY AND LUcIANO ZAMBONI Department of Pathology, Harbor-UCLA Medical Center, Torrance, California 90509; and University ofCalifornia School of Medicine, Los Angeles, California 90024 Communicated by Susumo Ohno, August 2, 1982

ABSTRACT In the course of a study on the morphogenesis of Table 1. Distribution of ectopic germinal cells in adrenal glands the adrenal gland in random-bred Swiss albino mice, we-noted the of fetal and postnatal mice presence ofectopic germ cells in the adrenal cortexes and medullas in animals of both sexes, from day 121/2 of fetal development to Sex postnatal day 12. Up to day 15 of fetal development, the cells ex- Day of Not hibited characteristics of primordial germ cells. At day 17, and development differen. Female Male irrespective ofthe sex ofthe fetus, they all entered.meiosis in syn- Fetal mice chrony with those in the ovary. Postnatally, in females as well as 121/2 1 males, all ectopic germ cells displayed morphologic characteristics 13 1 identical to those ofyoung oocytes in unilaminar ovarian follicles. 14 1 No germinal elements were seen in the adrenal glands past day 15 3 12 of life. Our study shows that mammalian germ cells are capa- 17 1 ble ofundergoing.sustained differentiation outside the gonads and 18 2 that, in ectopic sites, they all differentiate into oocytes as they 19 1 normally would in the ovary, even in males. Postnatal mice The initiation ofmeiosis has received considerable attention in 2 1 3 1 recent years. It is known that in the female the germinal cells 4 2 enter meiosis during fetal or, at the latest, very early postnatal 6 1 life whereas in the male this process is delayed until puberty. 9 1 Several authors consider this sexual dimorphism in germinal 11 1 cell behavior to be the result of the relative predominance of 12 1 1 one of two substances that are predicated to be present in the gonads of both sexes-a meiosis-inducing substance and a meiosis-preventing substance-which would stimulate the ger- minal cells to enter meiosis or would prevent them from doing mesonephric origin and identical differentiation patterns makes so (1-3). On the basis ofexperimental studies, Byskov and Saxen it much less easy to accept the hypothesis that they may be the (4), Byskov (5), Fajer et al. (6), Grinsted et al. (7), and Byskov source of substances having antagonistic influences on the ger- and Grinsted (8) suggested that the inducer is produced by minal cells insofar as meiotic maturation is concerned. mesonephros (the fetal excretory organ)-derived elements Recently, we had the opportunity to monitor the differen- which, according to these authors, are the somatic cells of the tiation of ectopic germ cells in the adrenal gland and to make ovigerous cords in the fetal ovary or, in the male, the cells of observations that are directly pertinent to this subject. They the rete testis and the epididymis; the preventer would be pro- allow us to view the sexual differentiation ofthe germ cells from duced predominantly by the somatic cells of the seminiferous a perspective broader than that from which this very complex cords of the fetal testis. issue has been approached previously. The exposure ofgerminal cells, such as those in the ovigerous cords of the fetal ovary, to abundant inducer would stimulate MATERIALS AND METHODS early meiosis; meiosis initiation would be delayed in the testis, due to predominance of preventer activity in the developing The observations reported here were made incidentally in the seminiferous cords (3, 5, 7). At puberty, preventer activity in course of a study on the fetal and postnatal development of the the testis would decrease, thus releasing the germinal cells to mouse adrenal gland from day 121/2 of intrauterine develop- the meiotic process (3, 5). We recently found, however, that ment to sexual maturity. Commercially obtained, random-bred the somatic cells ofthe seminiferous cords ofthe testis are meso- Swiss albino mice were used throughout. The adrenal glands nephric in origin (9-11) and, in fact, derive from the same cel- were fixed by vascular perfusion or immersion in 2.5% glutar- lular elements that in the female differentiate into the precur- aldehyde/0. 1 M cacodylate buffer, postfixed in 1% buffered sors of the cells The OS04, and embedded in Epon 812. Each gland was sectioned granulosa (12, 13). demonstration that the forlight and electron microscopy. The light microscopic sections somatic cells of the male and female sex cords have the same were cut approximately 1 Aim thick, stained with 1% aqueous The publication costs ofthis article were defrayed in part by page charge toluidine blue, and studied and photographed with a Zeiss Ul- payment. This article must therefore be hereby marked "advertise- traphot; the ultrathin sections were stained with lead hydroxide ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. and studied with a Hitachi HUllE electron microscope. 6584 Downloaded by guest on September 29, 2021 Developmental Biology: Upadhyay and Zamboni Proc. Natl. Acad. Sci. USA 79 (1982) 6585 OBSERVATIONS the genital ridge, entered the path followed by the mesonephric cells colonizing the area offuture adrenal cortical development Ectopic germinal cells were found in the cortexes and medullas (14) and consequently became trapped in the Anlage ofthe ad- of the adrenal glands of 10 fetuses and 9 postnatal mice (Table renal gland. The observation that in early fetal stages they 1). Ofthe fetuses, one was studied prior to sexual differentiation showed ameboidal features such as irregular profiles and the of the gonad, five were male, and four were female; the post- presence ofcytoplasmic pseudopods confirms this hypothesis. natal animals included seven females and two males. In spite of their extragonadal location in the adrenal cortex Through day 15 of intrauterine development, three or four or in the medulla, not only did the germ cells survive but they germ cells were seen in each adrenal gland (Fig. 1 a and b). They also underwent sustained differentiation for nearly 3 weeks, displayed normal features and had morphologic characteristics becoming progressively fewer in number and eventually dis- of primordial germ cells-i.e., a relatively large, vesicular nu- appearing after postnatal day 12 as result of degeneration. In cleus with one or two prominent nucleoli and an expanded cy- males as well as in females, the ectopic cells differentiated and toplasm with abundance offree ribosomes and sparsity ofother matured as germinal cells normally do in the fetal ovary. In syn- organelles. The germ cells frequently had irregular profiles and chronized fashion, they entered meiosis at day 17 of develop- cytoplasmic pseudopodia. ment and laterdifferentiated into elements that, exceptfor their From day 17 through day 19 of development, the number localization and for being surrounded by adrenal cells, were of the germinal cells in each adrenal gland appeared to be es- undistinguishable from quiescent oocytes in unilaminar ovarian sentially unchanged (Fig. ic). All cells were in meiosis as evi- follicles. The capability of the germinal cells to undergo sus- denced by the presence ofsynaptinemal complexes and typical tained differentiation in extragonadal locations is in itself chromosomal patterns (Figs. 1 c-fand 2a). This surprising ob- unique; even more remarkable, however, is the fact that, al- servation was made not only in the three female fetuses studied though in the female the ectopic germinal cells differentiated at days 18 and 19 (Figs. 1 c and d and 2a) but also in the one into oocytes just as those in the ovary, in the male the gonadal male at day 17 of development (Fig. 1 e andf). The cells dis- and extragonadal germinal cells differentiated along different played also an increased cytoplasmic organization. The mito- lines-the first as male germinal elements and the second as chondria were larger and more numerous and showed a distri- bution of the cristae reminiscent of that seen in immature oocytes. oocytes (Fig. 1 d andf); the ergastoplasmic reticulum was more These observations are important especially because they developed and, in one cell, a prominent Golgi complex was were made in a natural model and were not the result ofgenetic present in juxtanuclear position (Fig. ld). A few germinal ele- manipulations or any other experimental situation. They ments were in advanced stages of degeneration and showed strongly suggest that the germinal cells differentiate along a karyolysis and cytoplasmic swelling and vacuolization (Fig. 2a). male line only in the testis and that it is only in this organ that In the postnatal animals, the occurrence ofectopic germinal they are prevented from entering meiosis during fetal life and cells was noted less frequently than during the fetal period; differentiating into oocytes. From this perspective, the timing when present, no more than one germinal element was seen of the initiation of meiosis in the testicular germinal elements in each adrenal gland. Irrespective of the sex ofthe animal, all should be considered simply as one ofthe many manifestations germinal cells studied at these stages had morphologic char- of the highly complex process of differentiation undergone in acteristics ofyoung oocytes in unilaminar ovarian follicles (Fig. the testis by a cell that otherwise and elsewhere would have 2 b-e), these features being more pronounced in the older an- become an oocyte, the other manifestations being mitotic di- imals than in the younger. They were round (50-85 ,um in di- visions occurring throughout the reproductive period, a second ameter) and had regular outlines. The nucleus was spheroidal meiotic division following the first in rapid succession, and and located centrally or slightly eccentrically; the chromatin elaborate structural changes whereby the cell becomes pro- showed typical dyctiate patterns, and one or two nucleoli were gressively more differentiated and is eventually transformed evident (Fig. 2 b-d). Each cell had a prominent Golgi complex into a spermatozoon. in juxtanuclear position (Fig. 2c), numerous mitochondria with Thus, it is difficult to justify study approaches that narrow transversely oriented or arching cristae, and short ergasto- a process ofcell differentiation down to only one ofits multiple plasmic cisternae (Fig. 2 c and d); the plasma membrane had manifestations, ignore the others, and attempt to explain the sparse and short microvilli (Fig. 2e). The "oocytes" were closely one under consideration simply on the basis ofsecretory mech- surrounded by the adrenal cortical cells or by the chromaffin anisms. Incidentally, the postulated occurrence of substances cells of the medulla (Fig. 2 c and d) from which they were sep- capable of favoring or delaying early initiation of meiosis, es- arated by narrow, slit-like spaces (Fig. 2 c-e). Desmosomal pecially the first, becomes even more questionable now that we junctions were frequently seen in the areas where germinal and have shown that meiosis does occur in fetal life also when the adrenal cell plasma membranes were in contact (Fig. 2e). The germinal cells are associated with neuroectodermal elements adrenal cells that surrounded the oocytes occasionally displayed such as the chromaffin cells of the adrenal medulla. a crescentic shape similar to that of the granulosa cells lining Reporting on the occurrence ofXY oocytes in chimeric XX/ the walls of unilaminar ovarian follicles (Fig. 2 b and c). XY female mice, Evans et al. (15) stated that "the sex of a germ No ectopic germinal elements were seen in the adrenal cell is not an autonomous property but is determined by the glands of a 13-day-old female and two 14-day-old male mice. nature of the gonad in which it finds itself." This concept may Sections from the adrenals of male and female mice 3, 4, 5, 6, now be expanded as follows: the differentiation potential ofthe 7, and 8 weeks old similarly failed to reveal presence ofectopic mammalian germ cell is predominantly female and is an auton- germinal elements. omous property that the cell appears to lose only in the testis. The mechanism(s) responsible for the male sexual differentia- tion of the germ cells in the testis remain(s) to be identified. DISCUSSION This study was supported by a General Research Support Grant (MG- It is likely that the ectopic germinal cells noted in this study 3751) from Harbor-UCLA Medical Center Research and Education In- consisted of elements that, in the course of their migration to stitute to S.U. Downloaded by guest on September 29, 2021 6586 Developmental Biology: Upadhyay and Zamboni Proc. Natl. Acad. Sci. USA 79 (1982)

FIG. 1. (a and b) Ectopic germ cells (arrows in a; GC in b) in the Anlage of the adrenal cortex of a 12'/2-day-old mouse embryo. (a, x 700; b, x2,500.) (c-f) Germinal cells undergoing meiotic prophase in the adrenal glands of two female fetuses at day 18 (c) and day 19 (d) of development and one male fetus at day 17 (e and f). In c and e, the germinal elements are indicated by arrowheads. Arrows in d and fpoint to synaptinemal complexes. (c, x400; d, x6,200; e, x380; f, x6,200.) Downloaded by guest on September 29, 2021 Developmental Biology: Upadhyay and Zamboni Proc. Natl. Acad. Sci. USA 79 (1982) 6587

FIG. 2. (a) Degenerating germinal elements in the adrenal cortex of an 18-day-old female fetus. Arrow, synaptinemal complex. (x2,600.) (b- d) "Oocytes" in the adrenal medullas of 11-day-old (d) and 12-day-old (b) male mice and 4-day-old female mouse (c). Note the crescentic shape of the adrenal medullary cells surrounding the "oocytes" in b and c and the numerous chromaffin granules in their cytoplasm (c, d). (b, X 700; c, x7,500; d, x3,500.) (e) This micrograph illustrates the relationship between an "oocyte" (0) and surrounding adrenal cortical cells in a 12-day-old female mouse. Microvilli protruding from the oocyte surface are shown at arrows and a desmosomal junction is indicated by the arrowhead. (x 13,000.) Downloaded by guest on September 29, 2021 6588 Developmental Biology: Upadhyay and Zamboni Proc. Natl. Acad. Sci. USA 79 (1982)

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