- Email: [email protected]
Follicular growth in fetal and prepubertal ovaries of humans and other primates
1 Follicular Growth in Fetal and Prepubertal Ovaries of Humans and Other Primates H. P E T E R S A. G. B Y S K O V J. G R I N S T E D
FORMATION OF THE OVARY
The ovary differentiates during fetal life and establishes its two functions: to produce gametes and to synthesize hormones. It consists of germ cells and somatic cells, some of which become the hormone producing components. The germ cells migrate from the yolk sac to the gonadal ridge (Witschi, 1948; Zamboni and Merchant, 1973). The somatic components presumably arise from three different types of tissues: mesenchyme, coelomic epithelium and cells of mesonephric origin. It is probable that the cells which come from the mesonephros trigger two major events inthe developing ovary: the onset of meiosis and the formation of follicles (K611iker, 1898; Byskov, 1975). The ovarian anlage is established during the first trimester of pregnancy in primates, and many germ cells start meiotic prophase during the third month of pregnancy (Baker, 1963, 1966). Within the second trimester of fetal life most of the germ cells enter meiosis, and the first small follicles form and start to grow (Sauramo, 1954; van Wagenen and Simpson, 1965). During the third trimester of pregnancy the pool of oocytes is being established. Many antral follicles appear and differentiation of three types of hormone producing cells takes place: the granulosa cells, the theca cells and the interstitial cells. CLASSIFICATION OF FOLLICLES
Follicular growth and atresia begins during embryonic life in the primate ovary and continues throughout childhood (Potter, 1963; van Wagenen and Simpson, 1965; Valdes-Dapena, 1967). Only during certain diseases and Clinics in E n d o c r i n o l o g y a n d M e t a b o l i s m - -
Vol. 7, No. 3, November1978.
469
470
H. PETERS, A. G. BYSKOV AND J. GRINSTED
medication is follicular growth held in abeyance (Peters, Himelstein-Braw and Faber, 1976; Himelstein-Braw, Peters and Faber, 1978). The ovary contains follicles in several stages of development. Three main groups can be distinguished: (1) the small, non-growing follicle, in which the oocyte and its envelop are at rest; (2) the pre-antral follicle, in which the oocyte and its envelop grow and differentiate; and (3) the antral follicle, in which the oocyte has terminated its growth, while the envelop continues to differentiate.
The small follicle. The small follicle (types 1 and 2, Figure 1) (also described as primordial and primary follicles respectively) consists of a small oocyte, a few or a whole ring of flat granulosa cells and a basement membrane (Figure 2). A theca layer is usually not defined at this stage. The small follicles lie in the outer cortex of the ovary during childhood and represent the pool of resting follicles from which all growing follicles emerge. Although they are often overlooked on casual examination they are by far the largest group representing 97 per cent of the total follicular complement in children and young women (Block, 1952).
i
.-is<.,,
i"
+~
~i'
i
<.,,
Figure 1. Classification of follicles in the human ovary.
The pre-antralfollicle. The pre-antral follicle (type 3 to 5, Figure 1) (also called secondary, medium or growing follicle) is one that has entered the growth phase (Figure 3). Its oocyte has begun to enlarge and the granulosa cells have started to multiply, forming one or more layers around the oocyte. The zona pellucida forms, and a theca layer differentiates as cells from the stroma become concentrically arranged outside the basement membrane.
F O L L I C U L A R G R O W T H IN FETAL AND P R E P U B E R T A L OVARIES
Figure 2. Small follicles in the ovary of a 22-week-old fetus. 1/am plastic section. X 380.
Figure 3. A pre-antral and a small follicle in the ovary of a seven-year-old child. × 280.
47t
472
H. PETERS, A. G. BYSKOVAND J. GRINSTED
Fluid begins to collect between the granulosa cells. Multiple areas of distended intercellular spaces eventually coalesce and form the fluidcontaining antrum.
The antralfollicle. The antral follicle (type 6 to 8, Figure 1) (also called tertiary, vesicular or Graafian follicle) contains a full grown oocyte, many granulosa cells, a cavity filled with fluid and outside the basement membrane a well developed theca layer (Figure 4).
Figure 4. A large antral folliclein the ovaryof a four-year-oldchild. X 40.
FOLLICULAR GROWTH IN FETAL LIFE The first small follicles appear during the fourth month of gestation at the inner part of the cortex and start to grow almost immediately. Around the sixth month of pregnancy many pre-antral follicles have developed at the border between cortex and medulla. Antral follicles often appear during the last two months of fetal life and the ovary of a newborn child may be crowded with large antral or cyst-like follicles (Potter, 1963). The number of germ cells increases until about the fifth month of gestation when a maximum of about six million is reached (Baker, 1963). Thereafter their number is progressively reduced. Many oocytes die in the early stages of meiosis. When they enter the diplotene stage they must be furnished with granulosa cells in order to survive (Ohno and Smith, 1964). Naked oocytes in diplotene are frequently seen in the fetal ovary but they are always in process
FOLLICULARGROWTHIN FETALAND PREPUBERTALOVARIES
473
of degeneration (Figure 5). Also small follicles often become atretic in the fetus while this is rarely seen in a child's ovary (Himelstein-Braw et al, 1976). In late fetal life many of the pre-antral and antral follicles show signs of atresia as pyknotic granulosa cells or shrunken oocytes. The pre-antral and antral follicles of the fetus are often morphologically different from follicles seen in childhood and in the adult. The shape of the follicle can be irregular with 'tail-formations' of the granulosa layer (Figure 6). The formation of the basal lamina which encloses the granulosa layer is
Figure 5. Naked oocytesin degeneration and two small folliclesin the ovary of a 21-week-old fetus. I gm plastic section. × 400.
apparently poorly controlled. This may explain the occurrence of bi- or polyovular follicles, which in homo is frequent in the fetus but is an unusual phenomenon after birth. In macaqua, however, polyovular follicles persist (van Wagenen and Simpson, 1973). Follicles may in addition to a large diplotene oocyte also contain smaller ones in earlier stages of meiosis. This suggests that the mechanisms which control the formation of the follicular envelop are not yet fully differentiated (Figure 7). Other growing follicles in the fetal human ovary possess a regular granulosa layer but an abnormal theca layer, which is either extremely hypertrophied with large rounded cells (Figure 8a) (Watzka, 1957) or thin without differentiated cells (Figure 8b). These two types of follicles may lie close to each other. It is possible that factors within the oocyte or the granulosa cells locally influence the differentiation of their immediate surroundings and induce the formation of the theca.
474
H. PETERS, A. G. BYSKOV AND J. GRINSTED
Figure 6. Pre-antral follicle with 'tail formation' of the granulosa layer. Ovary of a 31-week-old fetus. × 280.
Figure 7. A pre-antral follicle containing a large oocyte in its centre and several small ones in the granulosa layer. Ovary of a 31-week-old fetus. × 220.
FOLLICULARGROWTHIN FETALAND PREPUBERTALOVARIES
(a)
475
(b)
Figure 8. Small antral folliclesin the fetal ovary. (a) With a hypertrophiedtheca, 31-week-old fetus. × 80. (b) The folliclehas a thin undifferentiatedtheca, 39-week-oldfetus. × 80. FOLLICULAR G R O W T H IN C H I L D H O O D
It has long been the impression of endocrinologists, paediatricians and gynaecologists that the ovary during childhood is a quiet organ, in which no follicular growth occurs. Stieve (1949) states that up to the twelfth year of life the human ovary is at rest. Van Wagenen and Simpson (1973) report that 'the first indication that small follicles had begun to grow and form small antral follicles was at 10 years of age', while they found follicle growth in the monkey (Macaca mulatta) ovary throughout childhood. They speculate that the absence of pre-antral and antral follicles in human children of their series might be 'connected with the morbidity characteristics of the donors of much of the human material in contrast with the good health of the monkey donors'. The opportunity to evaluate the normal development of the ovary is given in three reported series of children who had died in accidents or after a brief acute disease (Block, 1952; Sauramo, 1954; Peters, Himelstein-Braw and Faber, 1976). From these studies it can be deduced that also in human childhood the ovary is a very active organ in which follicular growth and follicular atresia is taking place at all ages. The size which antral follicles reach varies, but large pre-ovulatory follicles do not occur. Quiescent ovaries without follicular growth are not seen in normal children. The ovary increases in size during childhood (Figure 9a, b and c). This results from an increase in the number and size of fluid-filled follicles and an augmentation of the stroma which partly consists of rests and scars of antral follicles. At ten months the ovary might show three large antral follicles (Figure 9b); at seven years this number has doubled, and at nine years often quadrupled (Figure %).
476
H. PETERS, A. G. BYSKOVAND J. GRINSTED
All follicles that enter the growth phase during childhood and before ovulation has set in are destined to degenerate. However, they do not grow up to a certain size and then degenerate, but they can become atretic at any stage of their development in the h u m a n as ,well as in Macaca mulatta (Stevens, 1903; Allen et al, 1930; Watzka, 1957; Potter, 1963; ValdesDapena, 1967; Koering, 1969). Tt/e incidence of atresia becomes larger as
(a)
(b)
(c)
Figure 9. Ovaries of children at different ages. × 4.5. (a) Newborn. (b) Age 10 months. (c) Age 9 years.
the size of the follicles increases (Vermande-van Eck, 1956; Himelstein-Braw et al, 1976). Only two per cent of the follicles in the earliest growth stages are atretic, while all large antral follicles show signs of atresia. We conclude that during childhood there is concomitantly follicular growth and atresia. Follicles start to grow at all ages, develop and can become atretic at any stage of growth.
FOLLICULARGROWTHIN FETALANDPREPUBERTALOVARIES
477
ROLE OF HORMONES IN FOLLICULOGENESIS AND FOLLICULAR GROWTH Fetal life Only a little is known about the role that extra- or intra-ovarian hormones play in the organization of follicles. Gonadotrophins are detectable in the human fetus after the third month of gestation (see Chapter 2). In the female FSH and LH rise to a maximal level between the fifth and seventh month of pregnancy (Grumbach and Kaplan, 1974; Hagen and McNeilly, 1975; Clements et al, 1976; Takagi et al, 1977). This rise in gonadotrophins coincides in time with the onset of follicular growth. There is a parallel but slight increase in the fetal serum oestrogens (Weniger, Chouragui and Zeis, 1972). It is possible that the fetal oestrogens are not secreted by the ovary but are of placental origin (Shutt, Smith and Shearman, 1974). This is supported by the finding that the content of oestradiol-17/3 in fetal human ovaries is low or nil as shown by radioimmunoassays (Reyes, Winter and Faiman, 1973). However, histochemical tests seem to provide evidence of ovarian steroidogenic activity; the enzyme 3/3-hydroxysteroid dehydrogenase appears in granulosa cells at the beginning of the fourth month of gestation and is reported to increase thereafter (Goldman, Yakovac and Bongiovanni, 1965). The role of fetal gonadotrophins in normal follicular development was examined in the rhesus monkey (Macaca mulatta) by hypophysectomy during gestation (Gulyas et al, 1977). Fetal hypophysectomy done at a time when folliculogenesis was already in progress caused marked changes in the ovary. Ovaries of normal monkeys close to term contained many small, preantral as well as antral follicles. Forty-five days after fetal hyophysectomy the ovaries at term contained only one-third of the normal number of oocytes (Gulyas et al, 1977). Small follicles had formed, but their number was also reduced. No antral follicles were present. It is uncertain whether the absence of pituitary hormones directly prevented follicle formation, it is also possible that the reduction of the number of oocytes played a contributory role (Peters, 1978). The absence of pre-antral follicles after hypophysectomy suggests that gonadotrophins from the fetal pituitary are necessary for granulosa cell multiplication and fluid formation in the fetal ovary. Anencephalic fetuses or children provide a model to study the effect of low pituitary function on the ovarian development in the embryo (Ch'in, 1938; Ross, 1974). The anterior pituitary of anencephalics is small and contains only two per cent of the normal amount of gonadotrophic hormones (Grumbach and Kaplan, 1973). The ovarian development is impaired and the organs are small. Follicle development does not go beyond the early preantral stages and antra do not form (Ch'in, 1938; Himelstein-Braw, 1977, personal communication). The anencephalic model adds to the evidence that gonadotrophins are essential for follicular growth. Childhood Although follicles develop and become atretic at all ages during childhood, little is known about bow active the ovary is in terms of hormone production. Hormone levels in follicle fluid during childhood cannot be determined, but
478
H. PETERS,A. G. BYSKOVANDJ. GRINSTED
there is circumstantial evidence of the endocrine activity of the prepubertal ovary. In the adult, gonadotrophins and steroid hormones have been measured in the fluid of follicles of different sizes; they are present not only in the largest follicles, but also in those measuring 4 to 8 mm in diameter (Sanyal et al, 1974; McNatty et al, 1975). Similar sized follicles are common in the ovary during childhood and they increase in number after the age of six years. In the adult, FSH enters the antral follicle and stimulates production of oestrogen and the growth of granulosa cells (for discussion see Chapter 6). That this might also happen during childhood is suggested by the fact that gonadotrophin and oestrogen levels rise steadily after the age of six or eight years respectively (Bidlingsmaier and Knorr, 1973; Faiman and Winter, 1974). This is also the age when the number of antral follicles increases in the ovary. One can therefore speculate that with rising circulating gonadotrophin levels, FSH in some of the antral follicles increases, which will promote their growth. This in turn will cause an increase in oestrogen production of the follicles which is mirrored in the rising circulating oestrogen and urinary excretion levels (Presl, 1974). Thus already in childhood a close correlation between follicle growth, hormone response and hormone production seems to exist. In Chapter 3, Winter and his colleagues have reviewed other evidence for early ovarian function.
OVARIAN DEVELOPMENT AND FOLLICULAR GROWTH IN DISEASE
Little is as yet known about the influence of illness on ovarian development. Some diseases and their treatment have been identified as causing inhibition of follicular growth and even permanent damage to the ovary. Childhood leukaemia and abdominal tumours (nephroblastoma, i.e. Wilms' tumour and neuroblastoma) deserve special mention. Considerable progress in the treatment of these diseases has been made in recent years and more children are successfully treated and reach adulthood. The cure of these diseases makes the use of drastic, lifesaving therapeutic measures imperative which might, however, have effects on the ovary that will in later years present problems. Leukaemia The treatment of acute leukaemia involves corticosteroids and cytotoxie agents. Several of these drugs have been reported to cause ovarian atrophy and amenorrhoea in women (Sieber and Adamson, 1975). Ovaries of children who died of leukaemia after courses of treatment of varying length were recently studied to determine whether the duration of treatment used in leukaemia influenced the ovarian development (Himelstein-Braw, Peters and Faber, 1978). The girls who had received treatment only for a short period of time had normal ovaries with ample follicular growth and many small, nongrowing follicles. This suggested that the disease itself probably does not disturb ovarian development and follicle growth. Treatment for more than
FOLLICULARGROWTHIN FETALAND PREPUBERTALOVARIES
479
two months caused some or complete inhibition of follicle growth and at times reduction in the number of small follicles. Cytotoxic drugs inhibit cell proliferation in actively growing cells through a direct action on different phases of the cell cycle (Spiers, 1974). It is likely that the cytotoxic drugs act directly on the granulosa cells and prevent their proliferation, thereby inhibiting follicular growth. How long such inhibition is maintained after the cessation of treatment is not known, but it is likely that follicles will again develop some time after the treatment is discontinued. Siris, Leventhal and Vaitukaitis (1976) reported several conceptions after the cessation of chemotherapy for leukaemia: one patient had been off treatment for three to five years when she conceived and subsequently delivered a normal baby. A second patient conceived twice while on maintenance chemotherapy but chose to end the pregnancies by induced abortion. This suggests that growth and maturation can take place after treatment is discontinued and moreover that the oocytes in such follicles are undamaged. However, a reduction in the number of small follicles might have serious sequelae (see below). Abdominal turnouts
The treatment of solid abdominal tumours in children necessitates irradiation as well as surgery and chemotherapy (Harrison et al, 1974; d'Angio et al, 1976). During radiotherapy the ovaries are in (or close to) the field of exposure and the possibility of permanent damage to the ovaries must be considered. Pearson, Duncan and Pointon (1964) observed that three out of five women who had received radiation therapy in childhood for Wilms' tumour later presented primary amenorrhoea. Two women began cyclic bleeding after menarche, became pregnant, but were unable to carry the conceptus to term and aborted (D. Pearson, personal communication). There was evidence of ovarian failure (Shalet et al, 1976) in 18 patients who had received radiotherapy during childhood for abdominal tumours. Serum FSH and LH levels were elevated and oestradiol levels were low. It is likely that ovarian failure induced by abdominal irradiation in childhood is irreversible (Shalet et al, 1976). We must raise the question whether the ovary itself shows evidence of damage which could account for the ovarian failure. An investigation of ovaries from children who had been treated for abdominal tumours showed that in all those cases in which abdominal irradiation was used, either alone or in conjunction with chemotherapy, the ovaries were severely damaged (Himelstein-Braw, Peters and Faber, 1977). Follicle growth was inhibited in all cases and the number of small nongrowing follicles was markedly reduced (Figure 10a and b). A reduction in the number or disappearance of small follicles represents permanent ovarian damage as new formation of germ cells does not take place after birth in humans (Franchi, Mandl and Zuckerman, 1962; van Wagenen and Simpson, 1973; Peters, 1976; Zuckerman and Baker, 1977). Oocytes and small follicles are therefore irreplaceable and their destruction early in life causes severe complications in adulthood. We must conclude that abdominal radiation leads to the destruction of small follicles and inhibition of follicle
480
H. PETERS, A. G. BYSKOVAND J. GRINSTED
growth. This in turn causes low oestrogen secretion and high FSH and LH serum levels giving the clinical picture of ovarian failure. Other diseases in childhood are known to cause ovarian abnormalities. Ataxia telangiectasia is associated with immunological, endocrine and ovarian abnormalities (Dunn et al, 1964). The ovaries of girls afflicted with this disease are characterized by the paucity of small as well as antral follicles (Bowden, Danis and Sommers, 1963). Miller and Chatten (1967) emphasize the frequent association of this disease with ovarian follicular agenesis.
(a)
(b) Figure 10. Ovarian cortex of one and a half year-old children. X 40. (a) The ovary of a normal child killed in an accident has many small folliclesin the cortex. (b) The ovary of a child treated for Wilms' tumour with radiation has only a few small folliclesleft.
CHROMOSOMAL ABNORMALITIES AND OVARIAN DEVELOPMENT Turner's syndrome, 45,X Abnormal gonadal development in children with chromosomal aberrations is not uncommon. The syndrome of gonadal dysgenesis in girls of 45,X karyotype is usually characterized by lack of sexual development and streak gonads with few if any oocytes or follicles. Serum FSH is usually elevated early in infancy, tends to fall in mid childhood and rises to castrate levels around the age of 10 years ( G r u m b a c h and van Wyk, 1974). Not all girls show complete absence of sexual development. A few cases in which cyclic bleeding occurred have been reported (Court Brown et al, 1964; Weiss, 1971). Birth of a child to a woman of 45,X karyotype has been reported (Balmer et al, 1960). The evolution of gonadal dysgenesis in Turner's syndrome and the variety seen in its clinical development have been widely discussed. It was originally
FOLLICULARGROWTHIN FETALAND PREPUBERTALOVARIES
481
thought that the ovary did not develop because of a failure of primordial germ cells to migrate into the primitive gonad (Jones, Ferguson-Smith and Heller, 1963). However, Singh and Carr (1966) showed that early in fetal life the gonads of 45,X individuals are indistinguishable from those of normal fetuses. Up to the third month of embryonic life, germ cells populate the gonads, but they disappear prematurely after accelerated atresia. The ovarian picture at term is highly variable (Carr, Haggar and Hart, 1968). The ovary of a new born baby might already be entirely devoid of or still contain some follicles. In most cases all germ cells disappear before the age of maturity. The loss of germ cells in 45,X individuals is attributed to a direct effect of X-dosage deficiency in the oocyte. Two X chromosomes are active in normal 46,XX oocytes during the growth phase (Gartler, Andina and Gant, 1975) and the presence of only a single X is thought to be responsible for a rapid oocyte degeneration (Burgoyne and Biggers, 1976). However, Kohn et al (1977) recently reported a 23-week-old fetus with 45,X/47,XXX chromosomal mosaicism whose ovaries contained very few oocytes. Oocytes in various stages of degeneration were characteristic. Kohn et al (1977) argue that the presence of a triple X cell line, even in a high percentage of ovarian cells, does not necessarily protect the ovary from abnormal early oocyte degeneration and depletion. Why the rate of oocyte degeneration varies in different 45,X individuals causing some to be without germ cells at birth and others at varying times later is not known.
Trisomy 18 Other chromosomal abnormalities are frequently associated with ovarian dysgenesis. Trisomy 18 causes a syndrome which involves skeletal and facial characteristics and severe cardiovascular anomalies (Edwards et al, 1960; Smith et al, 1960). Ovarian dysgenesis in trisomy 18 was thought to be a rare manifestation (Alexiou et al, 1971), but has recently been described as rather common (Russel and Altschuler, 1975). The ovarian changes might show only a reduction in the number of oocytes and small follicles and loci of germ cell necrosis. In other cases total absence of germ cells, invagination of the surface epithelium as well as cords and ducts lying in the cortex characterize the gonad already early in life (Figure 11).
Down's syndrome, Trisomy 21 Children with Down's syndrome often show late sexual development and poorly developed sexual characteristics (Smith and Berg, 1976). Menarch is usually delayed and fertility is uncertain (Benda, 1960). A study of the development of ovaries in children with Down's syndrome at different ages has recently been undertaken (Hc~jager et al, 1978). In all eases the number of small follicles was reduced with an abrupt fall after the age of three years. However, ovaries completely depleted of germ cells did not occur. Follicle growth was partially or totally inhibited. Ovarian function and the gonadal-pituitary feedback mechanisms are so far ill defined as serum hormone levels in children with Down's syndrome have not yet been determined. However, a disturbance in the gonadotrophin metabolism is
482
H. PETERS, A. G. BYSKOVAND J. GRINSTED
suggested by abnormalities in the basophil cells of the anterior lobe of the pituitary (Benda, 1960; Purees, 1966), and oestrogen deficiencies are likely as the number and sizes of antral follicles are reduced in the ovaries.
Figure 11. Ovaryof an eight-week-oldgirl with Trisomy18. The cortexcontains only a few small folliclesand cell cords. × 120.
SUMMARY Follicular growth begins in the fetal ovary as soon as the first follicles are formed. Although orderly follicular growth is found in the fetal ovary, many of the early growing follicles show abnormalities. Follicles with irregular granulosa layers, with hypertrophied or with underdeveloped theca layers, are characteristic. Such follicles are rarely seen after birth. The ovary during childhood is an active organ in which follicular growth and follicular atresia normally take place. Follicles begin to grow at all ages, differentiate to preantral and antral follicles, but degenerate at various stages of their development before they reach pre-ovulatory sizes. Follicular growth in the fetus and children is dependent on hormones. Fetal gonadotrophins are necessary to ensure normal and sequential follicular growth before birth. During childhood a close correlation between follicle growth, hormone response and hormone production seems to exist. Certain diseases and treatment with cytotoxic agents or radiation to the abdomen influence ovarian development and follicular growth. Chromosome abnormalities, especially Turner's syndrome, trisomy 18 or 21, alter normal ovarian development by reducing the pool of available follicles and inhibiting follicular growth. Treatment with cytotoxic drugs inhibits follicular growth, while abdominal irradiation in childhood unless the ovaries are adequately shielded causes permanent damage by destroying the small follicles.
FOLLICULAR GROWTH IN FETAL AND PREPUBERTAL OVARIES
483
ACKNOWLEDGEMENT Some of the work reported here was supported by Euratom contract 120-73-1 BIO DK.
REFERENCES Alexiou, D., Chrysostomidou, O., Vlachos, I. & Deligeorgis, D. (1971) Trisomy 18 with ovarian dysgenesis. Acta Paediatrica Scandinavica, 60, 93-97. Allen, E., Pratt, J. P., Newell, Q. V. & Bland, L. J. (1930) Human ova from large follicles; including a search for maturation divisions and observations on atresia. American Journal of Anatomy. 46, 1-53. Baker, T. G. (1963) A quantitative and cytological study of germ cells in human ovaries. Proceedings of the Royal Society B, 158,417-433. Baker, T. G. (1966) A quantitative and cytological study of oogenesis in the rhesus monkey. Journal of Anatomy, 100, 761-776. Balmer, F., Schwarz, G., Hienz, H. A. & Walter, K. (1960) Turner syndrome with fully developed secondary sex characteristics and fertility. Acta Endocrinologica, 35, 397-404. Benda, C. E. (1960) The Child with Mongolism. New York: Grune and Stratton. Bidlingsmaier, F. & Knorr, D. (1973) Plasma estrogens in childhood and adolescence. Acta Paediatrica Scandinavica, 62, 86. Block, E. (1952) Quantitative morphological investigations of the follicular system in women. Acta Anatomica, 16, 108-123. Bowden, D. H., Danis, P. G. & Sommers, S. C. (1963) Ataxia telangiectasia: case with lesions of ovaries and adenohypophysis. Journal of Neuropathology and Experimental Neurology,
22,549-554. Burgoyne, P. S. & Biggers, J. D. (1976) The consequences of X-dosage deficiency in the germ line: impaired development in vitro of preimplantation embryos from XO mice. DevelopmentalBiology, 51, 109-117. Byskov, A. G. (1975) The role of the fete ovarii in meiosis and follicle formation in different mammalian species. Journal of Reproduction and Fertility, 45, 201-209. Carr, D. H., Haggar, R. A. & Hart, A. G. (1968) Germ cells in the ovaries of XO female infants. American Journal of Clinical Pathology, 49, 521-526. Ch'in, K. Y. (1938) The endocrine glands of anencephalic foetuses. A quantitative and morphological study of 15 cases. ChineseMedical Journal 2 (Supplement), 63-90. Clements, J. A., Reyes, F. I., Winter, J. S. D. & Faiman, C. (1976) Studies on human sexual development. III. Fetal pituitary and serum, and amniotic fluid concentrations of LH, CG and FSH. Journal of Clinical Endocrinology and Metabolism, 42, 9-19. Court Brown, W. M., Harndon, D. G., Jacobs, P. A., Maclean, N. & Mantel, D. J. (1964) Abnormalities of the sex chromosome complement in man. Medical Research Council SpecialReport, Series 305. London: Her Majesty's Stationery Office. d'Angio, G. J., Evans, A. E., Breslow, N., Beckwitt, B., Bishop, H., Beigl, P., Goodwin, W., Leape, L. L., Sinks, L. F., Sutow, W., Tefft, M. & Wolff, J. (1976) The treatment of Wilms tumor. Cancer, 38, 633-646. Dunn, H. G., Menwissen, H., Livingstone, C. S. & Pump, K. K. (1964) Ataxia-telangiectasia. Canadian Medical Association JournaL 91, 1106-1118. Edwards, J. H., Harndon, D. G., Cameron, A. H., Crosse, V. M. & Wolff, O. H. (1960) A new trisomic syndrome. Lancet, i, 787-790. Faiman, C. & Winter, J. S. D. (1974) Gonadotropins and sex hormone pattern in puberty clinical data. In The Control of the Onset of Puberty (Ed.) Grumbach, M. M., Grave, G. D. & Mayer, F. E. pp. 32-61. London: John Wiley. Franchi, L. L., Mandl, A. M. & Zuckerman, S. (1962) The development of the ovary and the process of oogenesis. In The Ovary, Vol. 1 (Ed.) Zuckerman, S. pp. 1-88. New York; London: Academic Press. Gartler, S. M., Andina, R. & Gant, N. (1975) Ontogeny of X-chromosome inactivation in the female germ line. Experimental Cell Research, 91,454-457. Goldman, A. S., Yakovae, W. C. & Bongiovanni, A. M. (1965) Development of activity of 3/3-hydroxysteroid dehydrogenase in human fetal tissues and in two anencephalic newborns. Journal of Clinical Endocrinology, 26, 14-22.
484
H. PETERS, A. G. BYSKOV AND J. GRINSTED
Grumbach, M. M. & Kaplan, S. L. (1973) Ontogenesis of growth hormone, insulin, prolactin and gonadotropin secretion in the human foetus. In Fetal and Neonatal Physiology. pp. 462-487. Cambridge: Cambridge University Press. Grumbach, M. M. & Kaplan, S. L. (1974) Fetal pituitary hormones and the maturation of central nervous system regulation of anterior pituitary function. In Modern Perinatal Medicine (Ed.) Gluck,L. pp. 247-256. Chicago: Yearbook Medical Publishers. Grumbach, M. M. & van Wyk, J. J. (1974) Disorders of sex differentiation. In Textbook of Endocrinology (Ed.) Williams, R. H. pp. 423-501. Philadelphia, London, Toronto: W. B. Saunders. Gulyas, B. J., Hodgen, G. D., Tullner, W. W. & Ross, G. T. (1977) Effects of fetal or maternal hypophysectomy on endocrine organs and body weight in infant Rhesus monkeys (Macaca mulatta): with particular emphasis on oogenesis. Biology of Reproduction, 16, 216-227. Hagen, C. & McNeilly, A. S. (1975) Identification of human luteinizing hormone, folliclestimulating hormone, luteinizing hormone fl-subunit and gonadotrophin a-subunit in foetal and adult pituitary glands. Journal of Endocrinology, 67, 49-57. Harrison, J., Myers, M., Rowen, M. & Vermund, H. (1974) Results of combination chemotherapy, surgery and radiotherapy in children with neuroblastoma. Cancer, 34, 485-490. Himelstein-Braw, R., Peters, H. & Faber, M. (1977) Influence of irradiation and chemotherapy on the ovaries of children with abdominal turnouts. British Journal of Cancer, 36, 269-275. Himelstein-Braw, R., Peters, H. & Faber, M. (1978) Morphological appearance of the ovaries of leukemic children. British Journal of Cancer, in press. Himelstein-Braw, R., Byskov, A. G., Peters, H. & Faber, M. (1976) Follicular atresia in the infant human ovary. Journal of Reproduction and Fertility, 46, 55-59. H~jager, B., Peters, H., Byskov, A. G. & Faber, M. (1978) Follicular development in ovaries of children with Down's syndrome. Acta Paediatrica Scandinavica, in press, Jones, H. W., Ferguson-Smith, M. A. & Heller, R. H. (1963) The pathology and cytogenetics of gonadal agenesis. American Journal of Obstetrics and Gynecology, 87, 578-600. Koering, M. J. (1969) Cyclic changes in ovarian morphology during the menstrual cycle in Macaea mulatta. American Journal of Anatomy, 126, 73-101. Kohn, G., Cohen, M. M., Beyth, Y. & Ornoy, A. (1977) Prenatal diagnosis and gonadal findings in X/XXX mosaicism. Journal of Medical Genetics, 14, 120-123. KSlliker, A. yon (1898) Uber die Markkangle und Markstrange in den Eierst~cken junger hundinnen. Verhandlungen der anatomischen Gesellschaft(Kiel), 14, 149-156. McNatty, K. P., Hunter, W. M., McNeilly, A. S. & Sawers, R. S. (1975) Changes in the concentration of pituitary and steroid hormones in the follicular fluid of human Graafian follicles throughout the menstrual cycle. Journal of Endocrinology, 64, 555-571. Miller, M. E. & Chatten, J. (1967) Ovarian changes in ataxia telangiectasia. Acta Paediatrica Scandinavica, 56, 559-561. Ohno, S. & Smith, J. B. (1964) Role of fetal follicular cells in meiosis of mammalian oocytes. Cytogenesis, 3, 324-333. Pearson, D., Duncan, W. B. & Pointon, R. C. S. (1964) Wilms tumour - - a review of 96 consecutive cases. BritishJournal of Radiology, 37, 154-160. Peters, H. (1976) Intrauterine gonadal development. Fertility and Sterility, 27, 493-500. Peters, H. (1978) Folliculogenesis. In The Vertebrate Ovary (Ed.) Jones, R. E. London: Plenum. In press. Peters, H., Himelstein-Braw, R. & Faber, M. (1976) The normal development of the ovary in childhood. Acta Endocrinologica, 82, 617-630. Potter, G. (1963) The ovary in infancy and childhood. In The Ovary (Ed.) Grady, H. G. & Smith, D, E. pp. 11-23. Baltimore: Williams and Wilkins. Presl, J. (1974) Development of the Ostrogen Feedback. Prag: Avicenum Zdravotnick6 Nakladatelstvi. Purves, H. D. (1966) Cytology of the adenohypophysis. In The Pituitary Gland Vol. 1 (Ed.) Harris, G. W. & Donovan, B. T. pp. 147-232. London: Butterworth. Reyes, F. I., Winter, J. S. D. & Faiman, C. (1973) Studies on human sexual development. I. Fetal gonadal and adrenal sex steroids. Journal of Clinical Endocrinology and Metabolism, 37, 74-78. Ross, G. T. (1974) Gonadotropins and preantral follicular maturation in women. Fertility and Sterility, 25, 522-543.
FOLLICULAR GROWTH IN FETAL AND PREPUBERTAL OVARIES
485
Russell, P. & Altschuler, G. (1975) The ovarian dysgenesis of Trisomy 18. Pathology, 7, 149-155. Sanyal, M. K., Berger, M. J., Thompson, I. E., Taymor, M. L. &Horne, H. W. (1974) Development of Graafian follicles in adult human ovary. I. Correlation of estrogen and progesterone concentration in antral fluid with growth of follicles. Journal of Clinical Endocrinology and Metabolism. 38,828-835. Sauramo, H. (1954) Histology and function of the ovary from the embryonic period to the fertile age. Acta Obstetrica Gynecologia Scandinavica, 33 (Supplement 2), 1-25. Shalet, S. M., Beartwell, C. G., Morris Jones, P. H., Pearson, D. & Orrell, D. H. (1976) Ovarian failure following abdominal irradiation in childhood. British Journal of Cancer. 33, 655-658. Shutt, D. A., Smith, I. D. & Shearman, R. P. (1974) Oestrone, oestradiol-17p and oestriol levels in human foetal plasma during gestation and term. Journal of Endocrinology, 60, 333-341. Sieber, S. M. & Adamson, R. H. (1975) Toxicity of antineoplastic agents in man: chromosomal aberrations, antifertility effects, congenital malformations and carcinogenic potential. In Advances in Cancer Research, 22, 57-155. Singh, R. P. & Carr, D. H. (1966) The anatomy and histology of XO human embryos and fetuses. AnatomicalRecords, 155,369-384. Siris, E. S., Leventhal, B. G. & Vaitukaitis, J. L. (1976) Effects of childhood leukemia and chemotherapy on puberty and reproductive function in girls. New England Journal of Medicine. 294, 1143-1146. Smith, D. W., Patau, K., Therman, E. & Inhorn, S. L. (1960) A new autosomal trisomy syndrome: multiple congenital anomalies caused by an extra chromosome. Journal of Pediatrics, 57,338-345. Smith, G. F. & Berg, J. M. (1976) Down's Anomaly. Edinburgh; London; New York: Churchill Livingstone. Spiers, A. S. D. (1974) Mode of action and clinical uses of therapeutic agents in leukemia. In Leukemia (Ed.) Gunz, F. & Baike, A. G. pp. 561-654. New York; San Francisco; London: Grune and Stratton. Stevens, T. G. (1903) The fate of the ovum and Graaflan follicle in premenstrual life. Obstetrical Transactions. 45, 465-482. Stieve, H. (1949) Anatomisch nachweisbare Vorg~inge im Eierstock der Menschen und ihre Umweltbedingte Steuerung. Geburtshilfe undFrauenheilkunde, 9, 639-644. Takagi, S., Yoshida, T., Tsubata, K., Ozaki, H., Fujii, T. K., Nomura, Y. & Sawada, M. (1977) Sex differences in fetal gonadotropins and androgens. Journal of Steroid Biochemistry, 8, 609-620, Valdes-Dapena, M. (1967) The normal ovary of childhood. Annals of the New York Academy of Sciences, 142, 597-613. van Wagenen, G. & Simpson, M. E. (1965) Embryology of the Ovary and Testis in Homo sapiens and Macaca mulatta. New Haven; London: Yale University Press. van Wagenen, G. & Simpson, M. E. (1973) Postnatal Development of the Ovary in Homo sapiens and Macaca mulatta. New Haven; London: Yale University Press. Vermande-van Eck, G. J. (1956) Neo-ovogenesis in the adult monkey. Consequences of atresia of ovocytes. AnatomicalRecords, 125,207-224. Watzka, M. (1957) Weibliche Genitalorgane. Das Ovarium. Handbuch der mikroskopische Anatomie der Menschen, Vol. 7/1. Berlin: Springer. Weiss, L. (1971) Additional evidence of gradual loss of germ cells in the pathogenesis of streak ovaries in Turner's syndrome. Journal of Medical Genetics. 8, 540-544. Weniger, J. P., Chouragui, J. & Zeis, A. (1972) Sur l'activit6 s6cr&oire de l'ovaire embryonnaire de mammif6re. Annales d'Endocrinologie, 33, 243-250. Witschi, E. (1948) Migration of the germ cells of human embryos from the yolk sac to the primitive gonadal folds. Carnegie Institute Contributions to Embryology, 32, 67-80. Zamboni, L. & Merchant, H. (1973) The fine morphology of mouse primordial germ cells in extragonadal locations. American Journal of Anatomy, 137, 299-336. Zuckerman, S. & Baker, T. G. (1977) The development of the ovary and the process of oogenesis. In The Ovary, Vol. 1 (Ed.) Zuckerman, S. & Weir, B. J. pp. 41-76. New York; San Francisco; London: Academic Press.
FORMATION OF THE OVARY
The ovary differentiates during fetal life and establishes its two functions: to produce gametes and to synthesize hormones. It consists of germ cells and somatic cells, some of which become the hormone producing components. The germ cells migrate from the yolk sac to the gonadal ridge (Witschi, 1948; Zamboni and Merchant, 1973). The somatic components presumably arise from three different types of tissues: mesenchyme, coelomic epithelium and cells of mesonephric origin. It is probable that the cells which come from the mesonephros trigger two major events inthe developing ovary: the onset of meiosis and the formation of follicles (K611iker, 1898; Byskov, 1975). The ovarian anlage is established during the first trimester of pregnancy in primates, and many germ cells start meiotic prophase during the third month of pregnancy (Baker, 1963, 1966). Within the second trimester of fetal life most of the germ cells enter meiosis, and the first small follicles form and start to grow (Sauramo, 1954; van Wagenen and Simpson, 1965). During the third trimester of pregnancy the pool of oocytes is being established. Many antral follicles appear and differentiation of three types of hormone producing cells takes place: the granulosa cells, the theca cells and the interstitial cells. CLASSIFICATION OF FOLLICLES
Follicular growth and atresia begins during embryonic life in the primate ovary and continues throughout childhood (Potter, 1963; van Wagenen and Simpson, 1965; Valdes-Dapena, 1967). Only during certain diseases and Clinics in E n d o c r i n o l o g y a n d M e t a b o l i s m - -
Vol. 7, No. 3, November1978.
469
470
H. PETERS, A. G. BYSKOV AND J. GRINSTED
medication is follicular growth held in abeyance (Peters, Himelstein-Braw and Faber, 1976; Himelstein-Braw, Peters and Faber, 1978). The ovary contains follicles in several stages of development. Three main groups can be distinguished: (1) the small, non-growing follicle, in which the oocyte and its envelop are at rest; (2) the pre-antral follicle, in which the oocyte and its envelop grow and differentiate; and (3) the antral follicle, in which the oocyte has terminated its growth, while the envelop continues to differentiate.
The small follicle. The small follicle (types 1 and 2, Figure 1) (also described as primordial and primary follicles respectively) consists of a small oocyte, a few or a whole ring of flat granulosa cells and a basement membrane (Figure 2). A theca layer is usually not defined at this stage. The small follicles lie in the outer cortex of the ovary during childhood and represent the pool of resting follicles from which all growing follicles emerge. Although they are often overlooked on casual examination they are by far the largest group representing 97 per cent of the total follicular complement in children and young women (Block, 1952).
i
.-is<.,,
i"
+~
~i'
i
<.,,
Figure 1. Classification of follicles in the human ovary.
The pre-antralfollicle. The pre-antral follicle (type 3 to 5, Figure 1) (also called secondary, medium or growing follicle) is one that has entered the growth phase (Figure 3). Its oocyte has begun to enlarge and the granulosa cells have started to multiply, forming one or more layers around the oocyte. The zona pellucida forms, and a theca layer differentiates as cells from the stroma become concentrically arranged outside the basement membrane.
F O L L I C U L A R G R O W T H IN FETAL AND P R E P U B E R T A L OVARIES
Figure 2. Small follicles in the ovary of a 22-week-old fetus. 1/am plastic section. X 380.
Figure 3. A pre-antral and a small follicle in the ovary of a seven-year-old child. × 280.
47t
472
H. PETERS, A. G. BYSKOVAND J. GRINSTED
Fluid begins to collect between the granulosa cells. Multiple areas of distended intercellular spaces eventually coalesce and form the fluidcontaining antrum.
The antralfollicle. The antral follicle (type 6 to 8, Figure 1) (also called tertiary, vesicular or Graafian follicle) contains a full grown oocyte, many granulosa cells, a cavity filled with fluid and outside the basement membrane a well developed theca layer (Figure 4).
Figure 4. A large antral folliclein the ovaryof a four-year-oldchild. X 40.
FOLLICULAR GROWTH IN FETAL LIFE The first small follicles appear during the fourth month of gestation at the inner part of the cortex and start to grow almost immediately. Around the sixth month of pregnancy many pre-antral follicles have developed at the border between cortex and medulla. Antral follicles often appear during the last two months of fetal life and the ovary of a newborn child may be crowded with large antral or cyst-like follicles (Potter, 1963). The number of germ cells increases until about the fifth month of gestation when a maximum of about six million is reached (Baker, 1963). Thereafter their number is progressively reduced. Many oocytes die in the early stages of meiosis. When they enter the diplotene stage they must be furnished with granulosa cells in order to survive (Ohno and Smith, 1964). Naked oocytes in diplotene are frequently seen in the fetal ovary but they are always in process
FOLLICULARGROWTHIN FETALAND PREPUBERTALOVARIES
473
of degeneration (Figure 5). Also small follicles often become atretic in the fetus while this is rarely seen in a child's ovary (Himelstein-Braw et al, 1976). In late fetal life many of the pre-antral and antral follicles show signs of atresia as pyknotic granulosa cells or shrunken oocytes. The pre-antral and antral follicles of the fetus are often morphologically different from follicles seen in childhood and in the adult. The shape of the follicle can be irregular with 'tail-formations' of the granulosa layer (Figure 6). The formation of the basal lamina which encloses the granulosa layer is
Figure 5. Naked oocytesin degeneration and two small folliclesin the ovary of a 21-week-old fetus. I gm plastic section. × 400.
apparently poorly controlled. This may explain the occurrence of bi- or polyovular follicles, which in homo is frequent in the fetus but is an unusual phenomenon after birth. In macaqua, however, polyovular follicles persist (van Wagenen and Simpson, 1973). Follicles may in addition to a large diplotene oocyte also contain smaller ones in earlier stages of meiosis. This suggests that the mechanisms which control the formation of the follicular envelop are not yet fully differentiated (Figure 7). Other growing follicles in the fetal human ovary possess a regular granulosa layer but an abnormal theca layer, which is either extremely hypertrophied with large rounded cells (Figure 8a) (Watzka, 1957) or thin without differentiated cells (Figure 8b). These two types of follicles may lie close to each other. It is possible that factors within the oocyte or the granulosa cells locally influence the differentiation of their immediate surroundings and induce the formation of the theca.
474
H. PETERS, A. G. BYSKOV AND J. GRINSTED
Figure 6. Pre-antral follicle with 'tail formation' of the granulosa layer. Ovary of a 31-week-old fetus. × 280.
Figure 7. A pre-antral follicle containing a large oocyte in its centre and several small ones in the granulosa layer. Ovary of a 31-week-old fetus. × 220.
FOLLICULARGROWTHIN FETALAND PREPUBERTALOVARIES
(a)
475
(b)
Figure 8. Small antral folliclesin the fetal ovary. (a) With a hypertrophiedtheca, 31-week-old fetus. × 80. (b) The folliclehas a thin undifferentiatedtheca, 39-week-oldfetus. × 80. FOLLICULAR G R O W T H IN C H I L D H O O D
It has long been the impression of endocrinologists, paediatricians and gynaecologists that the ovary during childhood is a quiet organ, in which no follicular growth occurs. Stieve (1949) states that up to the twelfth year of life the human ovary is at rest. Van Wagenen and Simpson (1973) report that 'the first indication that small follicles had begun to grow and form small antral follicles was at 10 years of age', while they found follicle growth in the monkey (Macaca mulatta) ovary throughout childhood. They speculate that the absence of pre-antral and antral follicles in human children of their series might be 'connected with the morbidity characteristics of the donors of much of the human material in contrast with the good health of the monkey donors'. The opportunity to evaluate the normal development of the ovary is given in three reported series of children who had died in accidents or after a brief acute disease (Block, 1952; Sauramo, 1954; Peters, Himelstein-Braw and Faber, 1976). From these studies it can be deduced that also in human childhood the ovary is a very active organ in which follicular growth and follicular atresia is taking place at all ages. The size which antral follicles reach varies, but large pre-ovulatory follicles do not occur. Quiescent ovaries without follicular growth are not seen in normal children. The ovary increases in size during childhood (Figure 9a, b and c). This results from an increase in the number and size of fluid-filled follicles and an augmentation of the stroma which partly consists of rests and scars of antral follicles. At ten months the ovary might show three large antral follicles (Figure 9b); at seven years this number has doubled, and at nine years often quadrupled (Figure %).
476
H. PETERS, A. G. BYSKOVAND J. GRINSTED
All follicles that enter the growth phase during childhood and before ovulation has set in are destined to degenerate. However, they do not grow up to a certain size and then degenerate, but they can become atretic at any stage of their development in the h u m a n as ,well as in Macaca mulatta (Stevens, 1903; Allen et al, 1930; Watzka, 1957; Potter, 1963; ValdesDapena, 1967; Koering, 1969). Tt/e incidence of atresia becomes larger as
(a)
(b)
(c)
Figure 9. Ovaries of children at different ages. × 4.5. (a) Newborn. (b) Age 10 months. (c) Age 9 years.
the size of the follicles increases (Vermande-van Eck, 1956; Himelstein-Braw et al, 1976). Only two per cent of the follicles in the earliest growth stages are atretic, while all large antral follicles show signs of atresia. We conclude that during childhood there is concomitantly follicular growth and atresia. Follicles start to grow at all ages, develop and can become atretic at any stage of growth.
FOLLICULARGROWTHIN FETALANDPREPUBERTALOVARIES
477
ROLE OF HORMONES IN FOLLICULOGENESIS AND FOLLICULAR GROWTH Fetal life Only a little is known about the role that extra- or intra-ovarian hormones play in the organization of follicles. Gonadotrophins are detectable in the human fetus after the third month of gestation (see Chapter 2). In the female FSH and LH rise to a maximal level between the fifth and seventh month of pregnancy (Grumbach and Kaplan, 1974; Hagen and McNeilly, 1975; Clements et al, 1976; Takagi et al, 1977). This rise in gonadotrophins coincides in time with the onset of follicular growth. There is a parallel but slight increase in the fetal serum oestrogens (Weniger, Chouragui and Zeis, 1972). It is possible that the fetal oestrogens are not secreted by the ovary but are of placental origin (Shutt, Smith and Shearman, 1974). This is supported by the finding that the content of oestradiol-17/3 in fetal human ovaries is low or nil as shown by radioimmunoassays (Reyes, Winter and Faiman, 1973). However, histochemical tests seem to provide evidence of ovarian steroidogenic activity; the enzyme 3/3-hydroxysteroid dehydrogenase appears in granulosa cells at the beginning of the fourth month of gestation and is reported to increase thereafter (Goldman, Yakovac and Bongiovanni, 1965). The role of fetal gonadotrophins in normal follicular development was examined in the rhesus monkey (Macaca mulatta) by hypophysectomy during gestation (Gulyas et al, 1977). Fetal hypophysectomy done at a time when folliculogenesis was already in progress caused marked changes in the ovary. Ovaries of normal monkeys close to term contained many small, preantral as well as antral follicles. Forty-five days after fetal hyophysectomy the ovaries at term contained only one-third of the normal number of oocytes (Gulyas et al, 1977). Small follicles had formed, but their number was also reduced. No antral follicles were present. It is uncertain whether the absence of pituitary hormones directly prevented follicle formation, it is also possible that the reduction of the number of oocytes played a contributory role (Peters, 1978). The absence of pre-antral follicles after hypophysectomy suggests that gonadotrophins from the fetal pituitary are necessary for granulosa cell multiplication and fluid formation in the fetal ovary. Anencephalic fetuses or children provide a model to study the effect of low pituitary function on the ovarian development in the embryo (Ch'in, 1938; Ross, 1974). The anterior pituitary of anencephalics is small and contains only two per cent of the normal amount of gonadotrophic hormones (Grumbach and Kaplan, 1973). The ovarian development is impaired and the organs are small. Follicle development does not go beyond the early preantral stages and antra do not form (Ch'in, 1938; Himelstein-Braw, 1977, personal communication). The anencephalic model adds to the evidence that gonadotrophins are essential for follicular growth. Childhood Although follicles develop and become atretic at all ages during childhood, little is known about bow active the ovary is in terms of hormone production. Hormone levels in follicle fluid during childhood cannot be determined, but
478
H. PETERS,A. G. BYSKOVANDJ. GRINSTED
there is circumstantial evidence of the endocrine activity of the prepubertal ovary. In the adult, gonadotrophins and steroid hormones have been measured in the fluid of follicles of different sizes; they are present not only in the largest follicles, but also in those measuring 4 to 8 mm in diameter (Sanyal et al, 1974; McNatty et al, 1975). Similar sized follicles are common in the ovary during childhood and they increase in number after the age of six years. In the adult, FSH enters the antral follicle and stimulates production of oestrogen and the growth of granulosa cells (for discussion see Chapter 6). That this might also happen during childhood is suggested by the fact that gonadotrophin and oestrogen levels rise steadily after the age of six or eight years respectively (Bidlingsmaier and Knorr, 1973; Faiman and Winter, 1974). This is also the age when the number of antral follicles increases in the ovary. One can therefore speculate that with rising circulating gonadotrophin levels, FSH in some of the antral follicles increases, which will promote their growth. This in turn will cause an increase in oestrogen production of the follicles which is mirrored in the rising circulating oestrogen and urinary excretion levels (Presl, 1974). Thus already in childhood a close correlation between follicle growth, hormone response and hormone production seems to exist. In Chapter 3, Winter and his colleagues have reviewed other evidence for early ovarian function.
OVARIAN DEVELOPMENT AND FOLLICULAR GROWTH IN DISEASE
Little is as yet known about the influence of illness on ovarian development. Some diseases and their treatment have been identified as causing inhibition of follicular growth and even permanent damage to the ovary. Childhood leukaemia and abdominal tumours (nephroblastoma, i.e. Wilms' tumour and neuroblastoma) deserve special mention. Considerable progress in the treatment of these diseases has been made in recent years and more children are successfully treated and reach adulthood. The cure of these diseases makes the use of drastic, lifesaving therapeutic measures imperative which might, however, have effects on the ovary that will in later years present problems. Leukaemia The treatment of acute leukaemia involves corticosteroids and cytotoxie agents. Several of these drugs have been reported to cause ovarian atrophy and amenorrhoea in women (Sieber and Adamson, 1975). Ovaries of children who died of leukaemia after courses of treatment of varying length were recently studied to determine whether the duration of treatment used in leukaemia influenced the ovarian development (Himelstein-Braw, Peters and Faber, 1978). The girls who had received treatment only for a short period of time had normal ovaries with ample follicular growth and many small, nongrowing follicles. This suggested that the disease itself probably does not disturb ovarian development and follicle growth. Treatment for more than
FOLLICULARGROWTHIN FETALAND PREPUBERTALOVARIES
479
two months caused some or complete inhibition of follicle growth and at times reduction in the number of small follicles. Cytotoxic drugs inhibit cell proliferation in actively growing cells through a direct action on different phases of the cell cycle (Spiers, 1974). It is likely that the cytotoxic drugs act directly on the granulosa cells and prevent their proliferation, thereby inhibiting follicular growth. How long such inhibition is maintained after the cessation of treatment is not known, but it is likely that follicles will again develop some time after the treatment is discontinued. Siris, Leventhal and Vaitukaitis (1976) reported several conceptions after the cessation of chemotherapy for leukaemia: one patient had been off treatment for three to five years when she conceived and subsequently delivered a normal baby. A second patient conceived twice while on maintenance chemotherapy but chose to end the pregnancies by induced abortion. This suggests that growth and maturation can take place after treatment is discontinued and moreover that the oocytes in such follicles are undamaged. However, a reduction in the number of small follicles might have serious sequelae (see below). Abdominal turnouts
The treatment of solid abdominal tumours in children necessitates irradiation as well as surgery and chemotherapy (Harrison et al, 1974; d'Angio et al, 1976). During radiotherapy the ovaries are in (or close to) the field of exposure and the possibility of permanent damage to the ovaries must be considered. Pearson, Duncan and Pointon (1964) observed that three out of five women who had received radiation therapy in childhood for Wilms' tumour later presented primary amenorrhoea. Two women began cyclic bleeding after menarche, became pregnant, but were unable to carry the conceptus to term and aborted (D. Pearson, personal communication). There was evidence of ovarian failure (Shalet et al, 1976) in 18 patients who had received radiotherapy during childhood for abdominal tumours. Serum FSH and LH levels were elevated and oestradiol levels were low. It is likely that ovarian failure induced by abdominal irradiation in childhood is irreversible (Shalet et al, 1976). We must raise the question whether the ovary itself shows evidence of damage which could account for the ovarian failure. An investigation of ovaries from children who had been treated for abdominal tumours showed that in all those cases in which abdominal irradiation was used, either alone or in conjunction with chemotherapy, the ovaries were severely damaged (Himelstein-Braw, Peters and Faber, 1977). Follicle growth was inhibited in all cases and the number of small nongrowing follicles was markedly reduced (Figure 10a and b). A reduction in the number or disappearance of small follicles represents permanent ovarian damage as new formation of germ cells does not take place after birth in humans (Franchi, Mandl and Zuckerman, 1962; van Wagenen and Simpson, 1973; Peters, 1976; Zuckerman and Baker, 1977). Oocytes and small follicles are therefore irreplaceable and their destruction early in life causes severe complications in adulthood. We must conclude that abdominal radiation leads to the destruction of small follicles and inhibition of follicle
480
H. PETERS, A. G. BYSKOVAND J. GRINSTED
growth. This in turn causes low oestrogen secretion and high FSH and LH serum levels giving the clinical picture of ovarian failure. Other diseases in childhood are known to cause ovarian abnormalities. Ataxia telangiectasia is associated with immunological, endocrine and ovarian abnormalities (Dunn et al, 1964). The ovaries of girls afflicted with this disease are characterized by the paucity of small as well as antral follicles (Bowden, Danis and Sommers, 1963). Miller and Chatten (1967) emphasize the frequent association of this disease with ovarian follicular agenesis.
(a)
(b) Figure 10. Ovarian cortex of one and a half year-old children. X 40. (a) The ovary of a normal child killed in an accident has many small folliclesin the cortex. (b) The ovary of a child treated for Wilms' tumour with radiation has only a few small folliclesleft.
CHROMOSOMAL ABNORMALITIES AND OVARIAN DEVELOPMENT Turner's syndrome, 45,X Abnormal gonadal development in children with chromosomal aberrations is not uncommon. The syndrome of gonadal dysgenesis in girls of 45,X karyotype is usually characterized by lack of sexual development and streak gonads with few if any oocytes or follicles. Serum FSH is usually elevated early in infancy, tends to fall in mid childhood and rises to castrate levels around the age of 10 years ( G r u m b a c h and van Wyk, 1974). Not all girls show complete absence of sexual development. A few cases in which cyclic bleeding occurred have been reported (Court Brown et al, 1964; Weiss, 1971). Birth of a child to a woman of 45,X karyotype has been reported (Balmer et al, 1960). The evolution of gonadal dysgenesis in Turner's syndrome and the variety seen in its clinical development have been widely discussed. It was originally
FOLLICULARGROWTHIN FETALAND PREPUBERTALOVARIES
481
thought that the ovary did not develop because of a failure of primordial germ cells to migrate into the primitive gonad (Jones, Ferguson-Smith and Heller, 1963). However, Singh and Carr (1966) showed that early in fetal life the gonads of 45,X individuals are indistinguishable from those of normal fetuses. Up to the third month of embryonic life, germ cells populate the gonads, but they disappear prematurely after accelerated atresia. The ovarian picture at term is highly variable (Carr, Haggar and Hart, 1968). The ovary of a new born baby might already be entirely devoid of or still contain some follicles. In most cases all germ cells disappear before the age of maturity. The loss of germ cells in 45,X individuals is attributed to a direct effect of X-dosage deficiency in the oocyte. Two X chromosomes are active in normal 46,XX oocytes during the growth phase (Gartler, Andina and Gant, 1975) and the presence of only a single X is thought to be responsible for a rapid oocyte degeneration (Burgoyne and Biggers, 1976). However, Kohn et al (1977) recently reported a 23-week-old fetus with 45,X/47,XXX chromosomal mosaicism whose ovaries contained very few oocytes. Oocytes in various stages of degeneration were characteristic. Kohn et al (1977) argue that the presence of a triple X cell line, even in a high percentage of ovarian cells, does not necessarily protect the ovary from abnormal early oocyte degeneration and depletion. Why the rate of oocyte degeneration varies in different 45,X individuals causing some to be without germ cells at birth and others at varying times later is not known.
Trisomy 18 Other chromosomal abnormalities are frequently associated with ovarian dysgenesis. Trisomy 18 causes a syndrome which involves skeletal and facial characteristics and severe cardiovascular anomalies (Edwards et al, 1960; Smith et al, 1960). Ovarian dysgenesis in trisomy 18 was thought to be a rare manifestation (Alexiou et al, 1971), but has recently been described as rather common (Russel and Altschuler, 1975). The ovarian changes might show only a reduction in the number of oocytes and small follicles and loci of germ cell necrosis. In other cases total absence of germ cells, invagination of the surface epithelium as well as cords and ducts lying in the cortex characterize the gonad already early in life (Figure 11).
Down's syndrome, Trisomy 21 Children with Down's syndrome often show late sexual development and poorly developed sexual characteristics (Smith and Berg, 1976). Menarch is usually delayed and fertility is uncertain (Benda, 1960). A study of the development of ovaries in children with Down's syndrome at different ages has recently been undertaken (Hc~jager et al, 1978). In all eases the number of small follicles was reduced with an abrupt fall after the age of three years. However, ovaries completely depleted of germ cells did not occur. Follicle growth was partially or totally inhibited. Ovarian function and the gonadal-pituitary feedback mechanisms are so far ill defined as serum hormone levels in children with Down's syndrome have not yet been determined. However, a disturbance in the gonadotrophin metabolism is
482
H. PETERS, A. G. BYSKOVAND J. GRINSTED
suggested by abnormalities in the basophil cells of the anterior lobe of the pituitary (Benda, 1960; Purees, 1966), and oestrogen deficiencies are likely as the number and sizes of antral follicles are reduced in the ovaries.
Figure 11. Ovaryof an eight-week-oldgirl with Trisomy18. The cortexcontains only a few small folliclesand cell cords. × 120.
SUMMARY Follicular growth begins in the fetal ovary as soon as the first follicles are formed. Although orderly follicular growth is found in the fetal ovary, many of the early growing follicles show abnormalities. Follicles with irregular granulosa layers, with hypertrophied or with underdeveloped theca layers, are characteristic. Such follicles are rarely seen after birth. The ovary during childhood is an active organ in which follicular growth and follicular atresia normally take place. Follicles begin to grow at all ages, differentiate to preantral and antral follicles, but degenerate at various stages of their development before they reach pre-ovulatory sizes. Follicular growth in the fetus and children is dependent on hormones. Fetal gonadotrophins are necessary to ensure normal and sequential follicular growth before birth. During childhood a close correlation between follicle growth, hormone response and hormone production seems to exist. Certain diseases and treatment with cytotoxic agents or radiation to the abdomen influence ovarian development and follicular growth. Chromosome abnormalities, especially Turner's syndrome, trisomy 18 or 21, alter normal ovarian development by reducing the pool of available follicles and inhibiting follicular growth. Treatment with cytotoxic drugs inhibits follicular growth, while abdominal irradiation in childhood unless the ovaries are adequately shielded causes permanent damage by destroying the small follicles.
FOLLICULAR GROWTH IN FETAL AND PREPUBERTAL OVARIES
483
ACKNOWLEDGEMENT Some of the work reported here was supported by Euratom contract 120-73-1 BIO DK.
REFERENCES Alexiou, D., Chrysostomidou, O., Vlachos, I. & Deligeorgis, D. (1971) Trisomy 18 with ovarian dysgenesis. Acta Paediatrica Scandinavica, 60, 93-97. Allen, E., Pratt, J. P., Newell, Q. V. & Bland, L. J. (1930) Human ova from large follicles; including a search for maturation divisions and observations on atresia. American Journal of Anatomy. 46, 1-53. Baker, T. G. (1963) A quantitative and cytological study of germ cells in human ovaries. Proceedings of the Royal Society B, 158,417-433. Baker, T. G. (1966) A quantitative and cytological study of oogenesis in the rhesus monkey. Journal of Anatomy, 100, 761-776. Balmer, F., Schwarz, G., Hienz, H. A. & Walter, K. (1960) Turner syndrome with fully developed secondary sex characteristics and fertility. Acta Endocrinologica, 35, 397-404. Benda, C. E. (1960) The Child with Mongolism. New York: Grune and Stratton. Bidlingsmaier, F. & Knorr, D. (1973) Plasma estrogens in childhood and adolescence. Acta Paediatrica Scandinavica, 62, 86. Block, E. (1952) Quantitative morphological investigations of the follicular system in women. Acta Anatomica, 16, 108-123. Bowden, D. H., Danis, P. G. & Sommers, S. C. (1963) Ataxia telangiectasia: case with lesions of ovaries and adenohypophysis. Journal of Neuropathology and Experimental Neurology,
22,549-554. Burgoyne, P. S. & Biggers, J. D. (1976) The consequences of X-dosage deficiency in the germ line: impaired development in vitro of preimplantation embryos from XO mice. DevelopmentalBiology, 51, 109-117. Byskov, A. G. (1975) The role of the fete ovarii in meiosis and follicle formation in different mammalian species. Journal of Reproduction and Fertility, 45, 201-209. Carr, D. H., Haggar, R. A. & Hart, A. G. (1968) Germ cells in the ovaries of XO female infants. American Journal of Clinical Pathology, 49, 521-526. Ch'in, K. Y. (1938) The endocrine glands of anencephalic foetuses. A quantitative and morphological study of 15 cases. ChineseMedical Journal 2 (Supplement), 63-90. Clements, J. A., Reyes, F. I., Winter, J. S. D. & Faiman, C. (1976) Studies on human sexual development. III. Fetal pituitary and serum, and amniotic fluid concentrations of LH, CG and FSH. Journal of Clinical Endocrinology and Metabolism, 42, 9-19. Court Brown, W. M., Harndon, D. G., Jacobs, P. A., Maclean, N. & Mantel, D. J. (1964) Abnormalities of the sex chromosome complement in man. Medical Research Council SpecialReport, Series 305. London: Her Majesty's Stationery Office. d'Angio, G. J., Evans, A. E., Breslow, N., Beckwitt, B., Bishop, H., Beigl, P., Goodwin, W., Leape, L. L., Sinks, L. F., Sutow, W., Tefft, M. & Wolff, J. (1976) The treatment of Wilms tumor. Cancer, 38, 633-646. Dunn, H. G., Menwissen, H., Livingstone, C. S. & Pump, K. K. (1964) Ataxia-telangiectasia. Canadian Medical Association JournaL 91, 1106-1118. Edwards, J. H., Harndon, D. G., Cameron, A. H., Crosse, V. M. & Wolff, O. H. (1960) A new trisomic syndrome. Lancet, i, 787-790. Faiman, C. & Winter, J. S. D. (1974) Gonadotropins and sex hormone pattern in puberty clinical data. In The Control of the Onset of Puberty (Ed.) Grumbach, M. M., Grave, G. D. & Mayer, F. E. pp. 32-61. London: John Wiley. Franchi, L. L., Mandl, A. M. & Zuckerman, S. (1962) The development of the ovary and the process of oogenesis. In The Ovary, Vol. 1 (Ed.) Zuckerman, S. pp. 1-88. New York; London: Academic Press. Gartler, S. M., Andina, R. & Gant, N. (1975) Ontogeny of X-chromosome inactivation in the female germ line. Experimental Cell Research, 91,454-457. Goldman, A. S., Yakovae, W. C. & Bongiovanni, A. M. (1965) Development of activity of 3/3-hydroxysteroid dehydrogenase in human fetal tissues and in two anencephalic newborns. Journal of Clinical Endocrinology, 26, 14-22.
484
H. PETERS, A. G. BYSKOV AND J. GRINSTED
Grumbach, M. M. & Kaplan, S. L. (1973) Ontogenesis of growth hormone, insulin, prolactin and gonadotropin secretion in the human foetus. In Fetal and Neonatal Physiology. pp. 462-487. Cambridge: Cambridge University Press. Grumbach, M. M. & Kaplan, S. L. (1974) Fetal pituitary hormones and the maturation of central nervous system regulation of anterior pituitary function. In Modern Perinatal Medicine (Ed.) Gluck,L. pp. 247-256. Chicago: Yearbook Medical Publishers. Grumbach, M. M. & van Wyk, J. J. (1974) Disorders of sex differentiation. In Textbook of Endocrinology (Ed.) Williams, R. H. pp. 423-501. Philadelphia, London, Toronto: W. B. Saunders. Gulyas, B. J., Hodgen, G. D., Tullner, W. W. & Ross, G. T. (1977) Effects of fetal or maternal hypophysectomy on endocrine organs and body weight in infant Rhesus monkeys (Macaca mulatta): with particular emphasis on oogenesis. Biology of Reproduction, 16, 216-227. Hagen, C. & McNeilly, A. S. (1975) Identification of human luteinizing hormone, folliclestimulating hormone, luteinizing hormone fl-subunit and gonadotrophin a-subunit in foetal and adult pituitary glands. Journal of Endocrinology, 67, 49-57. Harrison, J., Myers, M., Rowen, M. & Vermund, H. (1974) Results of combination chemotherapy, surgery and radiotherapy in children with neuroblastoma. Cancer, 34, 485-490. Himelstein-Braw, R., Peters, H. & Faber, M. (1977) Influence of irradiation and chemotherapy on the ovaries of children with abdominal turnouts. British Journal of Cancer, 36, 269-275. Himelstein-Braw, R., Peters, H. & Faber, M. (1978) Morphological appearance of the ovaries of leukemic children. British Journal of Cancer, in press. Himelstein-Braw, R., Byskov, A. G., Peters, H. & Faber, M. (1976) Follicular atresia in the infant human ovary. Journal of Reproduction and Fertility, 46, 55-59. H~jager, B., Peters, H., Byskov, A. G. & Faber, M. (1978) Follicular development in ovaries of children with Down's syndrome. Acta Paediatrica Scandinavica, in press, Jones, H. W., Ferguson-Smith, M. A. & Heller, R. H. (1963) The pathology and cytogenetics of gonadal agenesis. American Journal of Obstetrics and Gynecology, 87, 578-600. Koering, M. J. (1969) Cyclic changes in ovarian morphology during the menstrual cycle in Macaea mulatta. American Journal of Anatomy, 126, 73-101. Kohn, G., Cohen, M. M., Beyth, Y. & Ornoy, A. (1977) Prenatal diagnosis and gonadal findings in X/XXX mosaicism. Journal of Medical Genetics, 14, 120-123. KSlliker, A. yon (1898) Uber die Markkangle und Markstrange in den Eierst~cken junger hundinnen. Verhandlungen der anatomischen Gesellschaft(Kiel), 14, 149-156. McNatty, K. P., Hunter, W. M., McNeilly, A. S. & Sawers, R. S. (1975) Changes in the concentration of pituitary and steroid hormones in the follicular fluid of human Graafian follicles throughout the menstrual cycle. Journal of Endocrinology, 64, 555-571. Miller, M. E. & Chatten, J. (1967) Ovarian changes in ataxia telangiectasia. Acta Paediatrica Scandinavica, 56, 559-561. Ohno, S. & Smith, J. B. (1964) Role of fetal follicular cells in meiosis of mammalian oocytes. Cytogenesis, 3, 324-333. Pearson, D., Duncan, W. B. & Pointon, R. C. S. (1964) Wilms tumour - - a review of 96 consecutive cases. BritishJournal of Radiology, 37, 154-160. Peters, H. (1976) Intrauterine gonadal development. Fertility and Sterility, 27, 493-500. Peters, H. (1978) Folliculogenesis. In The Vertebrate Ovary (Ed.) Jones, R. E. London: Plenum. In press. Peters, H., Himelstein-Braw, R. & Faber, M. (1976) The normal development of the ovary in childhood. Acta Endocrinologica, 82, 617-630. Potter, G. (1963) The ovary in infancy and childhood. In The Ovary (Ed.) Grady, H. G. & Smith, D, E. pp. 11-23. Baltimore: Williams and Wilkins. Presl, J. (1974) Development of the Ostrogen Feedback. Prag: Avicenum Zdravotnick6 Nakladatelstvi. Purves, H. D. (1966) Cytology of the adenohypophysis. In The Pituitary Gland Vol. 1 (Ed.) Harris, G. W. & Donovan, B. T. pp. 147-232. London: Butterworth. Reyes, F. I., Winter, J. S. D. & Faiman, C. (1973) Studies on human sexual development. I. Fetal gonadal and adrenal sex steroids. Journal of Clinical Endocrinology and Metabolism, 37, 74-78. Ross, G. T. (1974) Gonadotropins and preantral follicular maturation in women. Fertility and Sterility, 25, 522-543.
FOLLICULAR GROWTH IN FETAL AND PREPUBERTAL OVARIES
485
Russell, P. & Altschuler, G. (1975) The ovarian dysgenesis of Trisomy 18. Pathology, 7, 149-155. Sanyal, M. K., Berger, M. J., Thompson, I. E., Taymor, M. L. &Horne, H. W. (1974) Development of Graafian follicles in adult human ovary. I. Correlation of estrogen and progesterone concentration in antral fluid with growth of follicles. Journal of Clinical Endocrinology and Metabolism. 38,828-835. Sauramo, H. (1954) Histology and function of the ovary from the embryonic period to the fertile age. Acta Obstetrica Gynecologia Scandinavica, 33 (Supplement 2), 1-25. Shalet, S. M., Beartwell, C. G., Morris Jones, P. H., Pearson, D. & Orrell, D. H. (1976) Ovarian failure following abdominal irradiation in childhood. British Journal of Cancer. 33, 655-658. Shutt, D. A., Smith, I. D. & Shearman, R. P. (1974) Oestrone, oestradiol-17p and oestriol levels in human foetal plasma during gestation and term. Journal of Endocrinology, 60, 333-341. Sieber, S. M. & Adamson, R. H. (1975) Toxicity of antineoplastic agents in man: chromosomal aberrations, antifertility effects, congenital malformations and carcinogenic potential. In Advances in Cancer Research, 22, 57-155. Singh, R. P. & Carr, D. H. (1966) The anatomy and histology of XO human embryos and fetuses. AnatomicalRecords, 155,369-384. Siris, E. S., Leventhal, B. G. & Vaitukaitis, J. L. (1976) Effects of childhood leukemia and chemotherapy on puberty and reproductive function in girls. New England Journal of Medicine. 294, 1143-1146. Smith, D. W., Patau, K., Therman, E. & Inhorn, S. L. (1960) A new autosomal trisomy syndrome: multiple congenital anomalies caused by an extra chromosome. Journal of Pediatrics, 57,338-345. Smith, G. F. & Berg, J. M. (1976) Down's Anomaly. Edinburgh; London; New York: Churchill Livingstone. Spiers, A. S. D. (1974) Mode of action and clinical uses of therapeutic agents in leukemia. In Leukemia (Ed.) Gunz, F. & Baike, A. G. pp. 561-654. New York; San Francisco; London: Grune and Stratton. Stevens, T. G. (1903) The fate of the ovum and Graaflan follicle in premenstrual life. Obstetrical Transactions. 45, 465-482. Stieve, H. (1949) Anatomisch nachweisbare Vorg~inge im Eierstock der Menschen und ihre Umweltbedingte Steuerung. Geburtshilfe undFrauenheilkunde, 9, 639-644. Takagi, S., Yoshida, T., Tsubata, K., Ozaki, H., Fujii, T. K., Nomura, Y. & Sawada, M. (1977) Sex differences in fetal gonadotropins and androgens. Journal of Steroid Biochemistry, 8, 609-620, Valdes-Dapena, M. (1967) The normal ovary of childhood. Annals of the New York Academy of Sciences, 142, 597-613. van Wagenen, G. & Simpson, M. E. (1965) Embryology of the Ovary and Testis in Homo sapiens and Macaca mulatta. New Haven; London: Yale University Press. van Wagenen, G. & Simpson, M. E. (1973) Postnatal Development of the Ovary in Homo sapiens and Macaca mulatta. New Haven; London: Yale University Press. Vermande-van Eck, G. J. (1956) Neo-ovogenesis in the adult monkey. Consequences of atresia of ovocytes. AnatomicalRecords, 125,207-224. Watzka, M. (1957) Weibliche Genitalorgane. Das Ovarium. Handbuch der mikroskopische Anatomie der Menschen, Vol. 7/1. Berlin: Springer. Weiss, L. (1971) Additional evidence of gradual loss of germ cells in the pathogenesis of streak ovaries in Turner's syndrome. Journal of Medical Genetics. 8, 540-544. Weniger, J. P., Chouragui, J. & Zeis, A. (1972) Sur l'activit6 s6cr&oire de l'ovaire embryonnaire de mammif6re. Annales d'Endocrinologie, 33, 243-250. Witschi, E. (1948) Migration of the germ cells of human embryos from the yolk sac to the primitive gonadal folds. Carnegie Institute Contributions to Embryology, 32, 67-80. Zamboni, L. & Merchant, H. (1973) The fine morphology of mouse primordial germ cells in extragonadal locations. American Journal of Anatomy, 137, 299-336. Zuckerman, S. & Baker, T. G. (1977) The development of the ovary and the process of oogenesis. In The Ovary, Vol. 1 (Ed.) Zuckerman, S. & Weir, B. J. pp. 41-76. New York; San Francisco; London: Academic Press.