J. Embryol. exp. Morph., Vol. 11, Part 3, pp. 513-536, September 1963
Printed in Great Britain
The Population of Germ Cells in the Developing
Female Chick
by GILLIAN C. HUGHES 1
From the Department of Anatomy, University of Birmingham
WITH THREE PLATES
INTRODUCTION
T H E process of oogenesis (i.e. the formation of new germ cells by mitotic divisions
of oogonia) and the subsequent cytological changes in oocytes associated with
the prophase of meiosis, have been described in detail for many mammalian
species (e.g. Winiwarter, 1901; Winiwarter & Sainmont, 1909<z, b; Kingery, 1917;
Pratt & Long, 1917; Cowperthwaite, 1925; Brambell, 1927; Swezy & Evans,
1930; Martinovitch, 1938; Slizynski, 1957, 1961; Ohno, Kaplan & Kinosita,
1960,1961; Beaumont &Mandl, 1962; Baker, 1963). In contrast, very few critical
studies of these processes in avian ovaries have been undertaken since
D'Hollander's (1904) classic description. D'Hollander's careful study of the left
(functional) ovary of the chick supported Waldeyer's (1870) thesis that the
process of oogenesis is completed by the time of hatching, and that the ovaries of
birds resemble those of eutherian mammals in that no new germ cells are generated
after sexual maturity (see Franchi, Mandl & Zuckerman, 1962).
D'Hollander observed that from the fourteenth day of incubation onwards,
differentiation of germ cells is most advanced in the central portion of the ovarian
cortex. Morphological changes heralding the onset of meiotic prophase were
first seen at 14 days' incubation, while many oocytes appeared to be passing
through the leptotene, zygotene and pachytene stages between the sixteenth and
twentieth days. D'Hollander also noted that in the chick, as in other animals
bearing yolky eggs, the diplotene phase is a very lengthy one and is characterized
by the presence of lampbrush chromosomes (see also Loyez, 1906; King, 1908;
Van Durme, 1914; Koltzoff, 1938; Dodson, 1948, Duryee, 1950; Gall, 1952,
1954; Callan & Lloyd, 1960).
More recently, Ohno (1961) has described numerous cells at the pachytene
1
Author's address: Department of Anatomy, The Medical School, Birmingham, 15, U.K.
514
G I L L I A N C. H U G H E S
stage (as seen in squash preparations) at 19 days' incubation; on the twentieth
day, the ovary contained oocytes at both pachytene and diplotene stages. Ohno
considers, however, that at the time of hatching, most oocytes are in an interphaselike or 'dictyotene' stage, usually considered to be typical of mammalian oocytes
(see Franchi et al, 1962; Mandl, 1963).
D'Hollander's findings have been the subject of much controversy. For
instance, Firket (1914) observed that many germ cells degenerate between the
thirteenth and fifteenth days of incubation, and postulated that most if not all
the oocytes originally present degenerate, to be replaced later by' definitive' germ
cells proliferated from the germinal epithelium. Corresponding claims have also
been made for mammalian species (e.g. rat: Butcher, 1927; Swezy, 1929; Hargitt,
1930a, b; mouse: Kingery, 1917; Allen, 1923; guinea-pig: Papanicolaou, 1924;
Bookhout, 1945; Aron, Marescaux & Petrovic, 1952, 1954a, b; cat: Sneider,
1940; man: Swezy & Evans, 1930). More recently, however, quantitative and
experimental studies have provided incontrovertible evidence that the definitive
germ cells in mammals arise solely from primordial germ cells (see Franchi et al.,
1962; Beaumont & Mandl, 1962; Baker, 1963).
D'Hollander's cytological observations were disputed by Goldsmith (1928),
who claimed that the nuclear changes occurring in the oocytes of the chick
embryo between 14 and 18 days' incubation represent synizesis leading to the
leptotene phase. He also believed that a long 'resting' phase occurs between the
time of hatching and the age of 65-69 days, when germ cells pass into the pachytene phase; and that during the final rapid growth phase immediately before
ovulation, a second 'resting' period ensues without an intervening diplotene
phase. Goldsmith's controversial views have not since been substantiated, nor
has anyone confirmed Sluiter's (1940) claim that the entire series of stages of
meiotic prophase can be seen in any single specimen at 14 to 15 days' incubation.
Until recently, little was known about the fluctuations in the population of
germ cells during ovarian development. Beaumont & Mandl (1962) and Baker
(1963) have shown that in rat and human foetuses alike, (i) a high mitotic activity
of oogonia is responsible for a sharp increase in the total population of oocytes;
(ii) as oogenesis ceases, and many oocytes undergo spontaneous degeneration,
the total number of germ cells decreases rapidly; and (iii) sufficiently large
numbers of oocytes escape atresia and constitute the finite 'stock' of oocytes
which gradually dwindles with age.
The only relevant quantitative study of avian oocytes that has been undertaken
hitherto is that of Faure-Fremiet & Kaufman (1928). They estimated that at
2 days after hatching, the left ovary of the White Leghorn chick contains about
3 • 6 x 106, and that of the Rhode Island Red some 12 • 5 x 106 oocytes. At the age
of 15 days, the numbers are said to have decreased to approximately 5,300 and
5,800 respectively. Since these workers did not use precisely the same technique
for counting germ cells in the 2-day-old and 15-day-old specimens, and since they
provide no evidence that the two methods are strictly equivalent, the reported
GERM CELLS IN THE FEMALE CHICK
515
reduction in the population of oocytes with age may be a gross under- or overestimate.
The present study was therefore undertaken in order (1) to re-examine, by
means of histological and squash preparations, the sequence of morphological
changes occurring in the left ovary of the chick during oogenesis and during the
prophase of meiosis; (2) to enumerate germ cells in the left ovarian cortex, at
various stages of development, between the ninth day of incubation and the first
day after hatching; and (3) to correlate the quantitative and qualitative observations.
MATERIALS AND METHODS
Animals
Fertile eggs (F1 generation White Leghorn x Rhode Island Red) were incubated
at 39°C. and 60 per cent humidity. Chicks were housed in brooders at 35°,
29-33° and 23-27°C. for the first, second and third weeks after hatching respectively. The room containing the brooders was constantly illuminated, but the
chicks were given free access to an area covered by felt which transmitted little
or no light.
Autopsy
Embryos were removed from eggs between 4 and 20 days' incubation. No
attempt was made to dissect the ovaries of embryos aged less than 8 days' incubation; the caudal half of the embryo was fixed and the left ovaries identified
in the serial histological sections. The left ovaries of embryos incubated for 8
days or more were dissected out, and fixed together with some of the underlying
kidney tissue. Ovaries of older chicks (up to 17 days after hatching) were also
prepared for histological studies.
Ovaries of specimens aged 9 days' incubation to 2 days after hatching were
dissected free from all adjoining tissue before being processed for squash preparations.
Histological procedures
The specimens were first fixed in Allen's B 15 solution for ^-1 hr., followed by
Bouin's aqueous solution for |—1 hr. Thereafter, the tissues were transferred to
70 per cent alcohol which was changed a number of times until the yellow colour
stopped seeping out of the specimens. The tissues were then dehydrated and
embedded in the usual way in wax. Serial sections 10/u thick were stained with
Weigert's iron haematoxylin and 'chromotrop 2 R \
Squash preparations
The tissues were subjected to a modification of the methods described by
Ford & Hamerton (1956) and Beaumont & Mandl (1962). The material was first
placed in a hypotonic solution of 1 -2 w/v sodium citrate at room temperature for
516
GILLIAN C. HUGHES
30 min. before being transferred for 15 min. to a 10 per cent, solution of trypsin
maintained at 37°C. The tissues were then fixed for 1 hr. in a chilled 3:1 mixture
of absolute ethyl alcohol and glacial acetic acid. After hydration in 30 per cent
alcohol and distilled water (3-5 min. for each fluid), they were hydrolysed in
N-HC1 at 60°C. for 8 min. The final procedures of staining and of making the
squash preparations were the same as those described by Beaumont & Mandl
(1962).
Quantitative histologicalprocedures
The numbers of germ cells were estimated in the left ovaries of embryos
removed between the ninth and twentieth days of incubation. In general, two
specimens were examined for each day of incubation (three for the thirteenth and
seventeenth days). The left ovaries of two chicks which hatched on the twentieth
day were removed the following day and the sections used for counting.
Serial sections were examined under a magnification of x 1250, and germ cells
situated in the ovarian cortex counted in every fortieth section. In order to
estimate the total number of germ cells, the counts were simply multiplied by 40
(see Mandl & Zuckerman, 1951; Green & Zuckerman, 1954). The sections were
systematically moved on the mechanical stage so that the whole of the cortical
area was scanned once; an eye-piece graticule was used as a guide. Care was taken
to focus at all levels through the thickness of the section, and to record only those
cells whose nucleus was largely included in the section.
The germ cells were classified as follows: oogonia at interphase; oogonia at
prophase, metaphase or anaphase of mitosis; oocytes at the pre-leptotene,
leptotene, zygotene, pachytene and diplotene phases of meiosis; and germ cells
undergoing degeneration.
PLATE 1
A-D, H, I. Histological preparations (10/A). Figs. E-G. Squash preparations.
FIG. A. Dorsal gut mesentery and gonad primordia of a 4-day-old embryo.
FIG. B. Oogonium at interphase (18 days' incubation).
FIG. C. Prophase of germinal mitosis (18 days' incubation).
FIG. D. Metaphase of germinal mitosis (18 days' incubation).
FIG. E. Oogonium at interphase (19 days' incubation).
FIG. F. Germinal prophase (13 days' incubation).
FIG. G. Germinal metaphase (11 days' incubation).
FIG. H. Oocytes at early pre-leptotene (18 days' incubation).
FIG. I. Oocytes at late pre-leptotene (18 days' incubation).
FIGS.
PLATE 2
Histological preparations (10//.).
FIG. J. Oocytes at leptotene (18 days' incubation).
FIG. K. Oocytes at zygotene showing 'bouquet' formation (18 days' incubation).
FIG. L. Oocyte at pachytene (18 days' incubation).
FIGS. M, N. Oocytes at diplotene (2 days after hatching).
FIG. O. Oocyte at diplotene in primordial follicle (14 days after hatching).
J. Embryol. exp. Morph.
Vol. 11, Part 3
PLATE 1
GILLTAN C. HUGHES
{Facing page 516)
Vol. 11, Part 3
J. Embryol. exp. Morph.
PLATE 2
J. Einbryol. exp. Morph.
Vol. 11, Part 3
PLATE 3
GERM CELLS IN THE FEMALE CHICK
517
RESULTS
Qualitative observations
1
4-8 days' incubation
On the fourth day of incubation, the gonad primordia are clearly recognizable
on the median surfaces of the Wolffian bodies (Plate 1, Fig. A). Germ cells are
present in the dorsal mesentery, at the coelomic angles and in the genital ridges
(Plate 1, Fig. A). The large size of the germ cells clearly distinguishes them from
the smaller somatic cells; moreover, the nuclei of the germ cells are large, either
round or ovoid, arid contain proportionately less chromatin than do those of
somatic cells.
Medullary and cortical proliferations appear on the fifth and sixth to seventh
days respectively. In the left ovary, germ cells are more numerous in the cortex
than in the medulla. A few germ cells divide mitotically between the fourth and
eighth days of incubation. Oogonia are smaller than the primordial germ cells,
and when at interphase they usually contain two large masses of chromatin within
their nuclei (Plate 1, Fig. B). Oogonial mitoses are distinguishable from those of
somatic cells by virtue of their larger size (Plate 1, Figs. C-D). The appearance of
nuclei of resting and dividing oogonia, as seen in squash preparations, is shown
in Plate 1, Figs. E-G.
9-15 days' incubation
The germ cells in the left ovarian cortex have become arranged in small groups
or 'nests', each of which is surrounded by a layer of connective tissue, the
primitive tunica albuginea. These 'nests' show a tendency towards merging on
about the fifteenth day.
From the ninth to the thirteenth day, many dividing germ cells appear throughout the left ovarian cortex. Nuclear changes indicating the onset of meiotic
prophase first appear in oocytes at the centre of the cortex at 13 to 14 days'
incubation, at a time when actively dividing oogonia become more restricted to
the extremities of the cortex.
PLATE 3
FIGS. P-U, X. Squash preparations. Figs. V, W. Histological preparations (10//,).
FIGS. P, Q. Oocytes at pre-leptotene (17 days' incubation).
FIG. R. Oocyte at leptotene (18 days' incubation).
FIG. S. Oocyte at zygotene (19 days' incubation).
FIG. T. Oocyte at pachytene (19 days' incubation^.
FIG. U. Oocyte at diplotene (2 days after hatching).
FIG. V. Germ cell at an advanced stage of degeneration (18 days' incubation).
FIG. W. 'Z' cells (cells in meiotic prophase undergoing degeneration; 18 days' incubation).
FIG. X. 'Z' cell (19 days' incubation).
GILLIAN C. HUGHES
518
As seen in histological preparations, oocytes at the pre-leptotene stage contain
chromatin granules which are closely packed together at the centre of the nucleus.
A fine reticulum which bears a number of small chromatin granules radiates
between the central mass of chromatin and the nuclear membrane (Plate 1,
Fig. H). Germinal nuclei at more advanced stages of development appear on
the fifteenth day. The central mass of chromatin appears to have spread out
along the reticulum (Plate 1, Fig. I), resulting in a number of large chromatin
granules becoming scattered throughout the nucleus. Nuclei at the pre-leptotene
stage, as seen in squash preparations (Plate 3, Figs. P, Q), show the beginnings
of 'thread' formation.
TABLE
Numbers of germ cells at various stages of development
Numbers of germ cells in the following
Age
(days'
incubation)
Interphase
Prophase
Metaphase
Anaphase
Early
Pre-leptotene
Late
Pre-leptotene
—
9
/ 22,920
\ 27,280
1,080
1,000
720
800
40
400
—
10
/ 59,920
\42,680
1,720
320
1,080
1,280
360
400
—
11
/ 71,840
\ 65,720
2,560
1,560
1,960
1,240
840
400
—
12
/144,920
\ 156,200
6,560
3,320
3,000
3,240
760
840
—
13
f511,640
< 222,520
L 224,760
5,960
3,040
4,640
6,040
3,920
2,360
1,560
1,240
960
17,720
1,400
4,160
—
14
/197,800
\ 250,520
2,960
5,720
2,720
3,600
1,320
1,120
3,160
9,920
—
15
/ 250,800
\ 256,880
3,960
5,920
2,040
4,520
800
1,400
83,640
37,640
6,840
40
16
/ 263,440
\ 158,200
4,160
6,360
2,160
3,880
720
1,280
27,320
79,320
28,920
17,760
17
f301,640
-I 395,320
L 139,480
7,280
8,480
4,040
3,320
3,120
4,280
1,040
600
840
269,720
72,760
78,920
191,200
62,720
48,160
18
/118,600
\ 475,600
3,680
5,640
2,280
4,480
240
960
47,480
102,600
24,840
78,320
19
/ 255,040
\ 73,320
1,800
1,120
2,640
920
440
200
148,800
38,360
79,480
51,240
20
/ 2,440
\33,68O
1,360
520
640
320
200
120
14,280
23,240
35,120
21,400
—
—
6,600
1,240
17,160
6,920
1 day after
hatching
{=
—
—
—
GERM CELLS IN THE FEMALE CHICK
519
16-18 days' incubation
Oocytes at the leptotene stage appear on the sixteenth day. Each nucleus
contains a tangled mass of long fine chromosomes (Plate 2, Fig. J), and a heteropyknotic body; the latter is seen particularly clearly in squash preparations
(Plate 3, Fig. R). Since the female chick is heterogametic, the deeply staining body
probably represents the Z-chromosomes.
Some nuclei at the zygotene and pachytene stages appear on the seventeenth
day of incubation. The chromosome threads become thicker (Plate 3, Fig. S),
presumably due to synapsis. Polarization of chromosomes ('bouquet' formation) is seen in histological preparations (Plate 2, Fig. K). During pachytene,
1
in the left ovarian cortex of the chick
stages of development:
Leptotene
Zygotene
Pachvtene
Diplotene
Nos. of at re tic
germ cells
Total numbers
of germ cells
600
480
25,360
29,960
640
520
63,720
45,200
1,080
2,120
78,280
71,040
1,000
1,360
156,240
164,960
5,720
520
1,600
548,640
232,640
238,480
12,320
220,280
9,880
280,760
960
8,600
349,040
315,000
16,880
24,520
345,120
292,920
1,480
1,600
40
250,280
38,120
3,200
27,880
20,320
120
1,240
1,360
—
48,800
23,200
33,040
1,102,400
626,000
312,080
44,040
58,000
28,880
11,200
40,240
49,720
—
13,960
39,160
324,240
825,680
53,680
108,120
8,960
56,480
440
183,280
9,960
60,320
16,880
611,600
539,880
33,920
53,200
229,520
130,240
245,440
128,480
4,160
11,400
23,800
33,360
590,880
435,960
20,480
6,280
131,160
44,200
264,240
274,080
7,600
49,360
61,520
67,400
508,760
449,480
—
—
GILLIAN C. HUGHES
520
TABLE
Mean numbers of germ cells at various stages of
Mean numbers of germ cells in the following
Age
(days'
incubation)
Interphase
Prophase
9
25,100
1,040
10
51,300
11
Anaphase
Early
Pre-le'ptotene
Late
Pre-leptotene
760
220
—
—
1,020
1,180
380
—
—
68,780
2,060
1,600
620
—
—
12
150,560
4,940
3,120
800
—
—
13
319,640
4,547
4,107
1,253
7,760
—
14
224,160
4,340
3,160
1,220
6,540
—
15
253,840
4,940
3,280
1,100
60,640
3,440
16
210,820
5,260
3,020
1,000
53,320
23,340
17
278,813
6,600
3,573
827
140,467
100,693
18
297,100
4,600
3,380
600
75,040
51,580
19
164,180
1,460
1,780
320
93,580
65,360
20
18,060
940
480
160
18,760
28,260
3,920
12,040
1 day after
hatching
Metaphase
individual chromosomal threads are short and thick, and the bivalents again
spread to occupy the whole of the nucleus (Plate 2, Fig. L). 'Banding' of the
chromosomes is apparent in squash preparations (Plate 3, Fig. T), each bivalent
appearing to consist of hetero- and euchromatin in alternating bands.
19 days' incubation until hatching
Oocytes at the diplotene stage appear on the nineteenth and twentieth days of
incubation. Separation of homologous chromosomes and the resulting chiasmata
may be seen in histological preparations (Plate 2, Fig. M), though the individual
threads are very fine. The nucleoli are prominent (Plate 2, Fig. N). It is not
possible to recognize individual chromosomes and chiasmata in squash preparations (Plate 3, Fig. U), probably because treatment with trypsin removes
some chromosomal elements (see Callan & Macgregor, 1958).
0-17 days after hatching
The diplotene stage is very lengthy, and the oocytes do not pass into a dictyate
stage as they do in many mammals. Follicular development sets in on about the
fourth day after hatching. Since the germ cells grow rapidly from the time of
hatching onwards, the cortical area of the ovary enlarges considerably. Plate 2,
Fig. O shows an enlarged oocyte in a primordial follicle, 14 days after hatching;
GERM CELLS IN THE FEMALE CHICK
521
development in the left ovarian cortex of the chick
stages of development:
Leptotene
Zygotene
Pachytene
—
Diplotene
Mean number
atretic
germ cells
Means of
total number
of germ cells
540
27,660
580
54,460
1,600
74,660
1,180
160,600
2,613
339,920
11,100
250,520
4,780
332,020
—
20,700
319,020
1,540
20
97,200
16,107
867
—
35,013
680,160
51,020
20,040
44,980
—
26,560
574,960
80,900
32,720
91,860
4,980
38,600
575,740
43,560
179,880
186,960
7,780
28,580
513,420
13,380
87,680
269,160
28,460
64,460
479,120
the nuclear configuration clearly indicates that, as judged by the presence of
chiasmata in the lampbrush chromosomes, the oocyte is undoubtedly at the
diplotene stage.
Degeneration of germ cells
Many germ cells degenerate in the course of development. Such cells are
characterized by clumping of the chromatin within the nucleus, and by the
wrinkling of the nuclear membrane (Plate 3, Fig. V). Some atretic germ cells are
derived from resting oogonia; some from mitotically active oogonia, and some
from oocytes at various stages of meiotic prophase ('Z' cells, as defined by
Beaumont & Mandl, 1962; see Plate 3, Figs. W, X).
Distribution of germ cells
The germ cells in the central parts of the ovarian cortex are consistently more
advanced in development than those at the extremities of the cortex. Thus, for
example, the left ovary on the twentieth day of incubation contains resting
oogonia at the extremities of the cortex which co-exist with oocytes at the
diplotene stage in the centre of the cortex (see Tables 1 and 2). As a result, there
is a great overlap between the various stages of meiosis, each stage appearing
during several days of incubation in different regions of the ovary (see below).
522
GILLIAN C. HUGHES
600,000
t
30,000 -
15
Age in days
D
1. Mean numbers of germ cells at different stages of development.
, total;
, at interphase;
, undergoing mitotic division;
, atretic. In this, and in Text-figs. 2-7, 9-20 = days of incubation,
ID = 1 day after hatching.
TEXT-FIG.
Estimates of the total population of germ cells
The results indicate that the total population of germ cells in the left ovarian
cortex increases about twenty-five-fold between the ninth and seventeenth days
of incubation (Tables 1 and 2; Text-fig. 1); subsequently, up to 1 day after
hatching, the number decreases by some 30 per cent.
523
GERM CELLS IN THE FEMALE CHICK
The increase in the numbers of germ cells is associated with oogonial mitotic
activity (Tables 1 and 2; Text-figs. 1 and 2). Assuming that all the dividing germ
cells counted are destined to give rise to two daughter-cells, i.e. they will not
10,000
5,000
1,000
15 io~
TEXT-FIG.
i
r
15
ni
'
'
'
20
ID
Age in days
2. Mean numbers of oogonial mitoses at different stages of
development.
degenerate in the course of division, it may be calculated that the duration of
germinal mitosis is of the order of some 2 - 2 | hr. It should be noted, however,
that since oogonia in early prophase and telophase were not included in the
counts, this estimate covers only the interval between late mitotic prophase and
the end of anaphase.
34
GILLIAN C. HUGHES
524
The number of degenerating germ cells rises considerably as the time of
hatching approaches (Tables 1 and 2; Text-fig. 1). The elimination of atretic
germ cells, together with the marked decrease in mitotic activity, accounts for the
600,000
•
\
450,000
x
E
| 300,000
150,000
30,000
15
20 ID
Age in days
TEXT-FIG. 3. Mean numbers of germ cells at different stages of development.
, total;
, at early pre-leptotene;
, at late pre-leptotene;
, at leptotene.
9 10
fall in the total population of germ cells which occurs from the seventeenth day
onwards (see Text-fig. 1). The fact that the total population of germ cells increases between days 13 and 17, at a time when the absolute numbers of germinal
525
GERM CELLS IN THE FEMALE CHICK
mitoses remain relatively constant, whereas those of degenerating cells rise,
suggests that the time taken for an atretic germ cell to 'disappear' from the ovary
is greater than that occupied by oogonial mitosis. Assuming that the duration
600,000 -
\
30,000 -
15
20 ID
Age in days
TEXT-FIG. 4. Mean numbers of germ cells at different stages of development.
, total;
, at zygotene;
, at pachytene;
, at
diplotene.
of mitosis is of the order of 2 • 5 hr., it may be calculated that between the seventeenth day of incubation and the first day after hatching, degenerating germ cells
are eliminated from the ovary within some 10 hr. of becoming histologically
recognizable as atretic.
526
GILLIAN C. HUGHES
The numbers of germ cells in various stages of the prophase of meiosis are
shown in Tables 1 and 2, and Text-figs. 3 and 4. The degree of overlap of the
various stages of prophase (see above) is clearly illustrated. The percentages of
100
50
10
9
10
15
Age in days
20
ID
5. Mean percentages of germ cells at different stages of
development.
, at interphase;
, undergoing mitotic
division;
, atretic.
TEXT-FIG.
50
10
9
10
. 15
Age in days
20
ID
6. Mean percentages of germ cells at different stages of
development.
, at early pre-leptotene;
, at late preleptotene;
, at leptotene.
TEXT-FIG.
oocytes at different stages of meiotic prophase were calculated in order to discover the ages at which their incidence is maximal (Table 3; Text-figs. 5-7). The
results show that until about the eighteenth day of incubation, most of the germ
cells present are oogonia atinterphase (Table 3), this stage remaining predominant
16-7
3-5
2-8
2-9
1-6
1-5
93-7
940
89-5
76-5
661
41 0
51-7
28-5
3-5
12
13
14
15
16
17
18
19
20
1 day after
hatching
18-3
2-9
92 1
11
2-5
5-5
3-7
0-3
0-8
14-1
11 -4
16-3
0-6
2-8
8-5
3-5
8-9
90
13-1
18-3
350
5-7
2-4
14-3
—
—
14-8
—
—
—
20-7
10
—
—
—
0-5
2-6
2-3
—
—
Zygotene
7-3
5-5
5-7
4-7
—
94-2
10
7-3
90-7
Early
Late
Pre-leptotene Pre-leptotene Leptotene
9
Large'
Mitoses
Inter phase
1
Age
(days'
incubation)
5-9
1-5
36-4
56-2
0-9
—
—
—
—
—
Diplotene
160
7-8
01
—
—
—
—
Pachytene
Percentages of germ cells at the following stages ofdevelopment:
Estimated proportions of germ cells at various stages of development in the left ovarian cortex of the chick
TABLE 3
13-5
5-6
6-7
4-6
51
6-5
1-4
4-4
0-8
0-7
21
11
20
Atretic
germ cells
>
r
m
o
>n
w
H
X
tfl
%
GERM CELLS
HICK
GILLIAN C. HUGHES
528
until the nineteenth day. The actual peak percentage of oogonia at interphase
(cf. total population) occurs between the ninth and fourteenth day of incubation
(Text-fig. 5). In contrast, the percentage of mitotically active oogonia decreases
from the ninth day of incubation onwards (Table 3; Text-fig. 5). The proportion
of cells undergoing degeneration increases from the thirteenth day of incubation
onwards (Table 3; Text-fig. 5).
Maximal proportions of oocytes in early pre-leptotene occur between days 15
and 19 (Text-fig. 6). Presumably this is a transitory stage since the proportions
of cells entering this phase, from the fifteenth to the nineteenth day of incubation,
are roughly equal to those passing on into late pre-leptotene. The peak incidence
of oocytes at the latter stage occurs between the seventeenth and nineteenth days
50
10
TEXT-FIG.
1
1
15
20 ID
Age in days
7. Mean percentages of germ cells at different stages of development.
, at zygotene;
, at pachytene;
, at diplotene.
9
10
of incubation (Text-fig. 6), an observation which suggests that their duration is
brief.
The incidence of cells at the zygotene stage is maximal on the twentieth day of
incubation (Text-figs. 4 and 7). A large proportion are in the pachytene stage
between the nineteenth day of incubation and 1 day after hatching (Table 3;
Text-fig. 7). This suggests that pachytene is a relatively lengthy stage. Qualitative
observations indicate that the numbers of oocytes at diplotene continue to increase beyond the first day after hatching.
Sources of error in quantitative studies
The present estimates of the population of germ cells in the developing ovary
are subject to several sources of error.
(1) Owing to the time-consuming nature of the method of counting, it has not
been possible to analyse more than two or three ovaries for each age group. No
highly inbred strains of chicks were readily available. On the other hand, variability due to genetic factors is likely to have been reduced somewhat by the use
GERM CELLS IN THE FEMALE CHICK
529
of F x hybrids between the two 'strains'. Nonetheless, in the case of two agegroups (13 and 17 days' incubation), the estimate for one individual was twice as
high as that for the second. For this reason, a third embryo of each age group
was added, and the overall mean for the three used.
(2) The possibility exists that at early developmental stages the proportion of
germ cells migrating to the right and left ovaries varies between individuals. This
factor alone may be responsible for some of the variability in the population of
oocytes in the left ovaries.
(3) Since oocytes frequently extend over more than one section, care was taken
to record only those cells whose nucleus was largely contained within the selected
section. Even so, the decision whether more or less than half of any given cell
was included is entirely subjective. The classification of germ cells into various
stages of meiotic prophase is also a somewhat subjective process.
(4) It is difficult to make accurate counts of cells packed together in large
numbers at all levels throughout the thickness of the section. This applies
particularly to the extremities of the cortex which contain very large numbers of
closely crowded oogonia. In spite of the precautions taken to minimize this
source of error, it is likely that an unknown (but probably small) proportion
of germ cells was missed, while some may have been counted twice.
DISCUSSION
The qualitative histological and cytological observations made in the course
of the present study fully confirm those of D'Hollander (1904; see Table 4 below).
They are at variance, however, with Goldsmith's (1928) and Sluiter's (1940)
claims mentioned on page 514.
The results of the present quantitative study show that in F x crosses of the
White Leghorn x Rhode Island Red breeds, the number of germ cells in the left
ovarian cortex increases from about 28,000 at 9 days' to approximately 680,000
at 17 days' incubation. Thereafter, the population declines sharply until about
480,000 remain 1 day after hatching. The increase in the number of germ cells is
associated with a high incidence of oogonial mitoses, and the subsequent decline
with a high incidence of degeneration of oocytes together with a sharp decrease in
the number of germinal mitoses.
Faure-Fremiet & Kaufman's (1928) estimates of the population of germ cells
in the ovaries of 2-day-old chicks differ from those in 1-day-old chicks in the
present study by a factor of about x 16. This discrepancy can only be accounted
for by the differences between the two methods of estimating the populations of
oocytes. Faure-Fremiet & Kaufman estimated the number of oocytes per unit
area of cortex and from this calculated the numbers of oocytes in the entire
ovaries. Their method of estimating the number of oocytes, however, rests on
the assumptions that the cortex is uniformly filled with germ cells, and that germ
cells of differing sizes are randomly distributed throughout the cortical area.
530
GILLIAN C. HUGHES
TABLE 4
A comparison of the present observations with those of D'Hollander (1904)
Observations of D'Hollander
Cell-types
'a'
'a"
'b'
*
V
'd'
'e'
T
'g'
'h'
Days of incubation
when cell-types
were first observed
10
10
14
15
Corresponding observations made in the present study
Germ cells at various stages
of de ve lop men t
primordial germ cells
interphase oogonia
oocytes in early pre-leptotene
„
„ late pre-leptotene
T
' j ' in primordial
follicles
9
9
13-14
15
15
16-17
17-18
,,
„
,,
„ leptotene
„ zygotene
,, pachytene
16
16-17
17-19
19
21
,,
,, diplotene
19-20
,,
,, diplotene
Days after hatching
0-3
,,
„ diplotene in
primordial follicles
16
Days after hatching
'i'
Days of incubation
when developmental
stages were first
observed
0
3
4
4
* Cells described, but not designated by a letter.
Both these assumptions are unlikely to be valid. First, the nuclear diameter of
germ cells tends to increase as development proceeds; and second, there is a
differential spatial distribution of germ cells within the cortex, the larger germ
cells occurring mostly near the centre and the smaller ones at the extremities of
the cortex.
Faure-Fremiet & Kaufman also paid no attention to the possibility that the
same oocyte may be seen in more than one section. It has since been shown that
a correction factor (Abercrombie, 1946) needs to be applied in order to account
for the relative thickness of the sections with respect to the size of the nuclei (see
Mandl & Zuckerman, 1951). Sources of error in the present quantitative study
were kept to a minimum.
The observation that the population of germ cells begins to decrease steeply at
an early age confirms that of Faure-Fremiet & Kaufman (1928) on the ovaries of
hatched chicks. These authors, as well as Brambell (1925), provide ample
evidence that atresia affects large oocytes in the young chick. Evidence that the
'stock' of oocytes in adult hens is further depleted with age is provided by the
repeated observation that the numbers of eggs laid progressively decline with age
(Brody, Henderson & Kempster, 1923; Faure-Fremiet & Kaufman, 1928;
Greenwood, 1937; Clark, 1940; Insko, Steele & Wightman, 1947). Pearl &
Schoppe's (1921) claim that the numbers of oocytes in the chick increase with age
GERM CELLS IN THE FEMALE CHJCK
531
can be accounted for by the fact that their counts were limited to large oocytes
which had already become invested in follicles.
The present observations thus indicate that while a high proportion of germ
cells degenerate during the latter part of embryonic life, sufficiently large numbers
escape atresia and constitute the 'stock' of oocytes which will subsequently
become depleted with age. Firket's (1914) thesis that all or nearly all the germ
cells present in the left ovary of the embryonic chick degenerate, and are replaced
later by proliferations from the germinal epithelium, is thus finally invalidated.
The pattern of ovarian development observed in the chick is essentially the
same as that in the rat (Beaumont & Mandl, 1962) and in man (Baker, 1963). If
the total numbers of germ cells present in the ovaries of these species are plotted
graphically against time ('birth' and 'hatching' being superimposed), the shapes
of the graphs are strikingly similar (Text-fig. 8).
Germ cells degenerate throughout development in all three species. The close
synchronization of developmental stages in the rat has made it possible to detect
four' waves' of degeneration: the first involves oogonia at interphase; the second,
actively dividing oogonia ('atretic divisions'); the third, oocytes at the pachytene
stage ('Z' cells); and the fourth, oocytes at the diplotene stage (Beaumont &
Mandl, 1962). In the human ovary, peak numbers of 'atretic divisions' and ' Z '
cells overlap, and a further 'wave' of degeneration affecting oocytes at the
diplotene phase is seen in full-term and neonatal specimens (Baker, 1963).
No attempt was made in the present study to classify germ cells undergoing
degeneration. Possibly because degenerating germ cells in the chick are eliminated from the ovary relatively quickly (see above), early stages of atresia seemed
to be less common than those at advanced stages, by which time classification was
not feasible. Qualitative observations, however, indicated that the increase in the
number of atretic oocytes 1 day after hatching was due to a high incidence of cells
degenerating at diplotene (cf. human ovary at birth; rat, 2 days after birth).
Asynchronization in the development of germ cells may cause an overlap between
any 'waves' of degeneration and thus render the detection of individual peaks
difficult. The results indeed show that while the numbers of atretic germ cells in
the left ovaries increase more or less linearly between 13 and 19 days' incubation,
there are no signs of significant 'peaks' in the incidence of atresia (see Text-fig. 1).
The avian ovary thus resembles that of eutherian mammals in that oogenesis,
i.e. the formation of new germ cells from pre-existing oogonia, ceases at early
stages of development and (with a few possible exceptions) does not occur after
the onset of sexual maturity. In contrast, in all amphibians, many fishes (particularly teleosts) and probably in many reptiles, oogenesis occurs continuously
during adult reproductive life (see Franchi et ah, 1962). It is of interest to note
that in Sphenodon, a primitive diapsid reptile, primordial germ cells enter the
prophase of meiosis shortly after their migration into the genital ridges (Tribe &
Brambell, 1932). It is not known whether all the oogonia become transformed
into oocytes at this stage, or whether some persist and divide later. It is possible,
532
GILLTAN C. HUGHES
750
600
^ 450
E
JO
E
I 300
(2
150
30
Birth
i
i
i
i
1
i
i
I
i
i
i
10
i
i
i
I
,
9
i
20- 5
i
15
1
i
1PP 2pp
15-5
1
i
5
II- 5
9
i
i
1
1
i
i
i
1
1PP 2pp
t
20
,Man age (months)
Rat age (days)
Chick age (days)
ID
Hatching
8. Means for the total populations of germ cells at different stages of development.
, chick;
, rat (Beaumont & Mandl, 1962);
, man (Baker, 1963).
* chick x 103; ratx 102; manx 104. ID = 1 day after hatching, pp = post par turn.
TEXT-FIG.
GERM CELLS IN THE FEMALE CHICK
533
however, that Sphenodon hatches with a finite' stock' of oocytes, as does the chick.
Although all oogonia in the chick are transformed into oocytes by the time of
hatching (i.e. all the germ cells present have entered the prophase of meiosis)
neither the oogonial divisions, nor the successive stages of meiotic prophase are
strictly synchronized within the ovary. Differentiation is always more advanced
at the centre of the cortex than at the cortical extremities. The same is true of
amphibians, where oocytes in the more central areas of the cortex co-exist with
oogonia at the periphery (Witschi, 1914). A similar tendency has been reported
for some mammals (e.g. rabbit: Winiwarter, 1901; mouse: Borum, 1961; man:
Baker, 1963), although the differences in the stages of development attained by
peripherally, as compared with centrally placed germ cells may only be slight
(see also Cowperthwaite, 1925; Brambell, 1927; Mintz, 1959). The close synchronization of development observed in the rat (Beaumont & Mandl, 1962)
would thus appear to be an exception rather than the rule.
SUMMARY
1. Morphological and cytological changes occurring in the left (functional)
ovary of the developing chick were studied by means of both histological and
squash preparations. Counts were made of the numbers of oogonia and oocytes
at various stages of meiotic prophase in specimens aged 9 days' incubation to
1 day after hatching.
2. Neither the mitotic activity of oogonia nor the onset of the successive stages
of meiotic prophase are synchronized throughout the ovary. The development
of the germ cells is more advanced at the centre than at the extremities of the
ovarian cortex.
3. The process of oogenesis ceases at about the time of hatching. Appreciable
numbers of oocytes enter the pre-leptotene phase on the thirteenth day, zygotene
and pachytene on the seventeenth, and diplotene on the nineteenth day of
incubation.
4. Degeneration of germ cells occurs throughout the developmental period
studied.
5. Oogonial mitoses are associated with an increase in the total population of
germ cells from about 28,000 at 9 days' incubation to some 680,000 on the
seventeenth day. Subsequently, a high incidence of degenerating germ cells,
together with a decline in the number of oogonial mitoses, account for a decrease
in the total population between the seventeenth day of incubation and 1 day after
hatching, when only about 480,000 oocytes persist.
RESUME
La population de gonocytes chez la Poule au cours de son developpement
embryorinaire
1. On a etudie les modifications morphologiques et cytologiques survenant dans l'ovaire gauche (fonctionnel) de l'embryon de poulet, au moyen
534
GTLLIAN C. HUGHES
preparations a la fois histologiques et par ecrasement. On a denombre les
oogonies et les oocytes a divers stades de la prophase meiotique, du 9e jour de
l'incubation au ler jour apres Peclosion.
2. Ni l'activite mitotique des oogonies, ni le debut des stades successifs de la
prophase meiotique ne sont synchronises dans l'ovaire entier. Le developpement
des gonocytes est plus avance au centre qu'aux extremites du cortex ovarien.
3. Le processus de l'oogenese cesse vers le moment de l'eclosion. Des nombres
appreciates d'oocytes entrent en phase de pre-leptotene le 136me jour, de zygotene
et de pachytene le 17e jour, et de diplotene le 19e jour de l'incubation.
4. La degenerescence de gonocytes survient tout au long de la periode de
developpement etudiee.
5. Les mitoses oogoniales sont associees a un accroissement de la population
totale de gonocytes, d'environ 28.000 le 9e jour de l'incubation, a environ 680.000
le 1T jour. Ensuite, une importante degenerescence de gonocytes, conjointement
a une baisse du nombre des mitoses oogoniales, est la cause d'une diminution de
leur population totale entre le 17e jour de l'incubation et le ler jour apres
l'eclosion, ou persistent seulement environ 480.000 oocytes.
ACKNOWLEDGEMENTS
The expenses incurred in this study were defrayed from grants, made to Professor Sir Solly
Zuckerman, F.R.S., by the Population Council, Inc. and by the Medical Research Council.
The Author is grateful to Professor Sir Solly Zuckerman, F.R.S., for his valuable criticisms,
to Dr A. M. Mandl for her help and encouragement, and to Dr B. John, Department of
Genetics, University of Birmingham, for his advice on cytological techniques.
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