the populatio onf germ cell isn the developing female chick · fig. j. oocyte ats leptoten e (18...

J. Embryol. exp. Morph., Vol. 11, Part 3, pp. 513-536, September 1963Printed in Great Britain

The Population of Germ Cells in the Developing

Female Chick

by GILLIAN C. HUGHES1

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 divisionsof oogonia) and the subsequent cytological changes in oocytes associated withthe prophase of meiosis, have been described in detail for many mammalianspecies (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 criticalstudies of these processes in avian ovaries have been undertaken sinceD'Hollander's (1904) classic description. D'Hollander's careful study of the left(functional) ovary of the chick supported Waldeyer's (1870) thesis that theprocess of oogenesis is completed by the time of hatching, and that the ovaries ofbirds resemble those of eutherian mammals in that no new germ cells are generatedafter 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 ovariancortex. Morphological changes heralding the onset of meiotic prophase werefirst seen at 14 days' incubation, while many oocytes appeared to be passingthrough the leptotene, zygotene and pachytene stages between the sixteenth andtwentieth days. D'Hollander also noted that in the chick, as in other animalsbearing yolky eggs, the diplotene phase is a very lengthy one and is characterizedby 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 pachytene1 Author's address: Department of Anatomy, The Medical School, Birmingham, 15, U.K.

514 GILLIAN C. HUGHES

stage (as seen in squash preparations) at 19 days' incubation; on the twentiethday, the ovary contained oocytes at both pachytene and diplotene stages. Ohnoconsiders, however, that at the time of hatching, most oocytes are in an interphase-like 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. Forinstance, Firket (1914) observed that many germ cells degenerate between thethirteenth and fifteenth days of incubation, and postulated that most if not allthe oocytes originally present degenerate, to be replaced later by' definitive' germcells proliferated from the germinal epithelium. Corresponding claims have alsobeen 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 andexperimental studies have provided incontrovertible evidence that the definitivegerm 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 chickembryo between 14 and 18 days' incubation represent synizesis leading to theleptotene phase. He also believed that a long 'resting' phase occurs between thetime of hatching and the age of 65-69 days, when germ cells pass into the pachy-tene phase; and that during the final rapid growth phase immediately beforeovulation, a second 'resting' period ensues without an intervening diplotenephase. Goldsmith's controversial views have not since been substantiated, norhas anyone confirmed Sluiter's (1940) claim that the entire series of stages ofmeiotic 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 ofgerm cells during ovarian development. Beaumont & Mandl (1962) and Baker(1963) have shown that in rat and human foetuses alike, (i) a high mitotic activityof 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 largenumbers of oocytes escape atresia and constitute the finite 'stock' of oocyteswhich gradually dwindles with age.

The only relevant quantitative study of avian oocytes that has been undertakenhitherto is that of Faure-Fremiet & Kaufman (1928). They estimated that at2 days after hatching, the left ovary of the White Leghorn chick contains about3 • 6 x 106, and that of the Rhode Island Red some 12 • 5 x 106 oocytes. At the ageof 15 days, the numbers are said to have decreased to approximately 5,300 and5,800 respectively. Since these workers did not use precisely the same techniquefor counting germ cells in the 2-day-old and 15-day-old specimens, and since theyprovide 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 over-estimate.

The present study was therefore undertaken in order (1) to re-examine, bymeans of histological and squash preparations, the sequence of morphologicalchanges occurring in the left ovary of the chick during oogenesis and during theprophase of meiosis; (2) to enumerate germ cells in the left ovarian cortex, atvarious stages of development, between the ninth day of incubation and the firstday after hatching; and (3) to correlate the quantitative and qualitative observa-tions.

MATERIALS AND METHODS

Animals

Fertile eggs (F1 generation White Leghorn x Rhode Island Red) were incubatedat 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 respec-tively. The room containing the brooders was constantly illuminated, but thechicks were given free access to an area covered by felt which transmitted littleor no light.

Autopsy

Embryos were removed from eggs between 4 and 20 days' incubation. Noattempt was made to dissect the ovaries of embryos aged less than 8 days' in-cubation; the caudal half of the embryo was fixed and the left ovaries identifiedin the serial histological sections. The left ovaries of embryos incubated for 8days or more were dissected out, and fixed together with some of the underlyingkidney tissue. Ovaries of older chicks (up to 17 days after hatching) were alsoprepared for histological studies.

Ovaries of specimens aged 9 days' incubation to 2 days after hatching weredissected free from all adjoining tissue before being processed for squash pre-parations.

Histological procedures

The specimens were first fixed in Allen's B15 solution for ^-1 hr., followed byBouin's aqueous solution for |—1 hr. Thereafter, the tissues were transferred to70 per cent alcohol which was changed a number of times until the yellow colourstopped seeping out of the specimens. The tissues were then dehydrated andembedded in the usual way in wax. Serial sections 10/u thick were stained withWeigert's iron haematoxylin and 'chromotrop 2R\

Squash preparations

The tissues were subjected to a modification of the methods described byFord & Hamerton (1956) and Beaumont & Mandl (1962). The material was firstplaced in a hypotonic solution of 1 -2 w/v sodium citrate at room temperature for


30 min. before being transferred for 15 min. to a 10 per cent, solution of trypsinmaintained at 37°C. The tissues were then fixed for 1 hr. in a chilled 3:1 mixtureof absolute ethyl alcohol and glacial acetic acid. After hydration in 30 per centalcohol and distilled water (3-5 min. for each fluid), they were hydrolysed inN-HC1 at 60°C. for 8 min. The final procedures of staining and of making thesquash 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 embryosremoved between the ninth and twentieth days of incubation. In general, twospecimens were examined for each day of incubation (three for the thirteenth andseventeenth days). The left ovaries of two chicks which hatched on the twentiethday were removed the following day and the sections used for counting.

Serial sections were examined under a magnification of x 1250, and germ cellssituated in the ovarian cortex counted in every fortieth section. In order toestimate the total number of germ cells, the counts were simply multiplied by 40(see Mandl & Zuckerman, 1951; Green & Zuckerman, 1954). The sections weresystematically moved on the mechanical stage so that the whole of the corticalarea was scanned once; an eye-piece graticule was used as a guide. Care was takento focus at all levels through the thickness of the section, and to record only thosecells whose nucleus was largely included in the section.

The germ cells were classified as follows: oogonia at interphase; oogonia atprophase, metaphase or anaphase of mitosis; oocytes at the pre-leptotene,leptotene, zygotene, pachytene and diplotene phases of meiosis; and germ cellsundergoing degeneration.

PLATE 1

FIGS. 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).

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)

J. Embryol. exp. Morph. Vol. 11, Part 3

PLATE 2

J. Einbryol. exp. Morph. Vol. 11, Part 3

PLATE 3


RESULTS

Qualitative observations4-8 days'1 incubation

On the fourth day of incubation, the gonad primordia are clearly recognizableon the median surfaces of the Wolffian bodies (Plate 1, Fig. A). Germ cells arepresent 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 fromthe smaller somatic cells; moreover, the nuclei of the germ cells are large, eitherround or ovoid, arid contain proportionately less chromatin than do those ofsomatic cells.

Medullary and cortical proliferations appear on the fifth and sixth to seventhdays respectively. In the left ovary, germ cells are more numerous in the cortexthan in the medulla. A few germ cells divide mitotically between the fourth andeighth days of incubation. Oogonia are smaller than the primordial germ cells,and when at interphase they usually contain two large masses of chromatin withintheir nuclei (Plate 1, Fig. B). Oogonial mitoses are distinguishable from those ofsomatic cells by virtue of their larger size (Plate 1, Figs. C-D). The appearance ofnuclei of resting and dividing oogonia, as seen in squash preparations, is shownin Plate 1, Figs. E-G.

9-15 days' incubation

The germ cells in the left ovarian cortex have become arranged in small groupsor 'nests', each of which is surrounded by a layer of connective tissue, theprimitive tunica albuginea. These 'nests' show a tendency towards merging onabout the fifteenth day.

From the ninth to the thirteenth day, many dividing germ cells appear through-out the left ovarian cortex. Nuclear changes indicating the onset of meioticprophase 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 tothe 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).


As seen in histological preparations, oocytes at the pre-leptotene stage containchromatin granules which are closely packed together at the centre of the nucleus.A fine reticulum which bears a number of small chromatin granules radiatesbetween the central mass of chromatin and the nuclear membrane (Plate 1,Fig. H). Germinal nuclei at more advanced stages of development appear onthe fifteenth day. The central mass of chromatin appears to have spread outalong the reticulum (Plate 1, Fig. I), resulting in a number of large chromatingranules becoming scattered throughout the nucleus. Nuclei at the pre-leptotenestage, as seen in squash preparations (Plate 3, Figs. P, Q), show the beginningsof 'thread' formation.

Age(days'

incubation)

9

10

11

12

13

14

15

16

17

18

19

20

1 day afterhatching

Interphase

/ 22,920\ 27,280

/ 59,920\42,680

/ 71,840\ 65,720

/144,920\ 156,200

f511,640< 222,520L 224,760

/197,800\ 250,520

/ 250,800\ 256,880

/ 263,440\ 158,200

f301,640-I 395,320L 139,480

/118,600\ 475,600

/ 255,040\ 73,320

/ 2,440\33,68O

{ =

Numbers of germ

Prophase

1,0801,000

1,720320

2,5601,560

6,5603,320

5,9603,0404,640

2,9605,720

3,9605,920

4,1606,360

7,2808,4804,040

3,6805,640

1,8001,120

1,360520

—

Metaphase

720800

1,0801,280

1,9601,240

3,0003,240

6,0403,9202,360

2,7203,600

2,0404,520

2,1603,880

3,3203,1204,280

2,2804,480

2,640920

640320

—

TABLE

cells at various stages of development

Numbers of germ cells in the following

Anaphase

40400

360400

840400

760840

1,5601,240

960

1,3201,120

8001,400

7201,280

1,040600840

240960

440200

200120

—

EarlyPre-leptotene

—

—

—

—

17,7201,4004,160

3,1609,920

83,64037,640

27,32079,320

269,72072,76078,920

47,480102,600

148,80038,360

14,28023,240

6,6001,240

LatePre-leptotene

—

—

—

—

—

6,84040

28,92017,760

191,20062,72048,160

24,84078,320

79,48051,240

35,12021,400

17,1606,920


16-18 days' incubation

Oocytes at the leptotene stage appear on the sixteenth day. Each nucleuscontains a tangled mass of long fine chromosomes (Plate 2, Fig. J), and a hetero-pyknotic body; the latter is seen particularly clearly in squash preparations(Plate 3, Fig. R). Since the female chick is heterogametic, the deeply staining bodyprobably represents the Z-chromosomes.

Some nuclei at the zygotene and pachytene stages appear on the seventeenthday of incubation. The chromosome threads become thicker (Plate 3, Fig. S),presumably due to synapsis. Polarization of chromosomes ('bouquet' forma-tion) 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

1,4801,600

250,28038,1203,200

44,04058,000

53,680108,120

33,92053,200

20,4806,280

40

27,88020,320

120

28,88011,200

8,96056,480

229,520130,240

131,16044,200

—

1,2401,360

40,24049,720

440183,280

245,440128,480

264,240274,080

—

—

—

9,960

4,16011,400

7,60049,360

Nos. of at re ticgerm cells

600480

640520

1,0802,120

1,0001,360

5,720520

1,600

12,3209,880

9608,600

16,88024,520

48,80023,20033,040

13,96039,160

60,32016,880

23,80033,360

61,52067,400

Total numbersof germ cells

25,36029,960

63,72045,200

78,28071,040

156,240164,960

548,640232,640238,480

220,280280,760

349,040315,000

345,120292,920

1,102,400626,000312,080

324,240825,680

611,600539,880

590,880435,960

508,760449,480


TABLE

Mean numbers of germ cells at various stages ofMean numbers of germ cells in the following

Age(days'

incubation)

9

10

11

12

13

14

15

16

17

18

19

20

1 day afterhatching

Interphase

25,100

51,300

68,780

150,560

319,640

224,160

253,840

210,820

278,813

297,100

164,180

18,060

Prophase

1,040

1,020

2,060

4,940

4,547

4,340

4,940

5,260

6,600

4,600

1,460

940

Metaphase

760

1,180

1,600

3,120

4,107

3,160

3,280

3,020

3,573

3,380

1,780

480

Anaphase

220

380

620

800

1,253

1,220

1,100

1,000

827

600

320

160

EarlyPre-le'ptotene

—

—

—

—

7,760

6,540

60,640

53,320

140,467

75,040

93,580

18,760

3,920

LatePre-leptotene

—

—

—

—

—

—

3,440

23,340

100,693

51,580

65,360

28,260

12,040

individual chromosomal threads are short and thick, and the bivalents againspread to occupy the whole of the nucleus (Plate 2, Fig. L). 'Banding' of thechromosomes is apparent in squash preparations (Plate 3, Fig. T), each bivalentappearing 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 ofincubation. Separation of homologous chromosomes and the resulting chiasmatamay be seen in histological preparations (Plate 2, Fig. M), though the individualthreads are very fine. The nucleoli are prominent (Plate 2, Fig. N). It is notpossible to recognize individual chromosomes and chiasmata in squash pre-parations (Plate 3, Fig. U), probably because treatment with trypsin removessome 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 dictyatestage as they do in many mammals. Follicular development sets in on about thefourth day after hatching. Since the germ cells grow rapidly from the time ofhatching 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;


development in the left ovarian cortex of the chickstages of development:

Leptotene Zygotene Pachytene Diplotene

1,540

97,200

51,020

80,900

43,560

20

16,107

20,040

32,720

179,880

—

867

44,980

91,860

186,960

—

—

—

4,980

7,780

13,380 87,680 269,160 28,460

Mean numberatretic

germ cells

540

580

1,600

1,180

2,613

11,100

4,780

20,700

35,013

26,560

38,600

28,580

64,460

Means oftotal numberof germ cells

27,660

54,460

74,660

160,600

339,920

250,520

332,020

319,020

680,160

574,960

575,740

513,420

479,120

the nuclear configuration clearly indicates that, as judged by the presence ofchiasmata in the lampbrush chromosomes, the oocyte is undoubtedly at thediplotene stage.

Degeneration of germ cellsMany germ cells degenerate in the course of development. Such cells are

characterized by clumping of the chromatin within the nucleus, and by thewrinkling of the nuclear membrane (Plate 3, Fig. V). Some atretic germ cells arederived from resting oogonia; some from mitotically active oogonia, and somefrom oocytes at various stages of meiotic prophase ('Z' cells, as defined byBeaumont & 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 moreadvanced in development than those at the extremities of the cortex. Thus, forexample, the left ovary on the twentieth day of incubation contains restingoogonia at the extremities of the cortex which co-exist with oocytes at thediplotene stage in the centre of the cortex (see Tables 1 and 2). As a result, thereis a great overlap between the various stages of meiosis, each stage appearingduring several days of incubation in different regions of the ovary (see below).


600,000

t

30,000 -

15Age in days

D

TEXT-FIG. 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.

Estimates of the total population of germ cells

The results indicate that the total population of germ cells in the left ovariancortex increases about twenty-five-fold between the ninth and seventeenth daysof incubation (Tables 1 and 2; Text-fig. 1); subsequently, up to 1 day afterhatching, the number decreases by some 30 per cent.


The increase in the numbers of germ cells is associated with oogonial mitoticactivity (Tables 1 and 2; Text-figs. 1 and 2). Assuming that all the dividing germcells counted are destined to give rise to two daughter-cells, i.e. they will not

10,000

5,000

1,000

15 io~ i r

Age in days15 ni ' ' ' 20 ID

TEXT-FIG. 2. Mean numbers of oogonial mitoses at different stages ofdevelopment.

degenerate in the course of division, it may be calculated that the duration ofgerminal 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 thecounts, this estimate covers only the interval between late mitotic prophase andthe end of anaphase.

34


The number of degenerating germ cells rises considerably as the time ofhatching approaches (Tables 1 and 2; Text-fig. 1). The elimination of atreticgerm cells, together with the marked decrease in mitotic activity, accounts for the

600,000

450,000

xE| 300,000

150,000

30,000

•

\

9 10 20 ID15Age 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.

fall in the total population of germ cells which occurs from the seventeenth dayonwards (see Text-fig. 1). The fact that the total population of germ cells in-creases between days 13 and 17, at a time when the absolute numbers of germinal


mitoses remain relatively constant, whereas those of degenerating cells rise,suggests that the time taken for an atretic germ cell to 'disappear' from the ovaryis greater than that occupied by oogonial mitosis. Assuming that the duration

600,000 -

30,000 -

\

15Age in days

20 ID

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 seven-teenth day of incubation and the first day after hatching, degenerating germ cellsare eliminated from the ovary within some 10 hr. of becoming histologicallyrecognizable as atretic.


The numbers of germ cells in various stages of the prophase of meiosis areshown in Tables 1 and 2, and Text-figs. 3 and 4. The degree of overlap of thevarious stages of prophase (see above) is clearly illustrated. The percentages of

100

50

10

9 10 15Age in days

20 ID

TEXT-FIG. 5. Mean percentages of germ cells at different stages ofdevelopment. , at interphase; , undergoing mitotic

division; , atretic.

50

10

9 10 . 15

Age in days20 ID

TEXT-FIG. 6. Mean percentages of germ cells at different stages ofdevelopment. , at early pre-leptotene; , at late pre-

leptotene; , at leptotene.

oocytes at different stages of meiotic prophase were calculated in order to dis-cover the ages at which their incidence is maximal (Table 3; Text-figs. 5-7). Theresults show that until about the eighteenth day of incubation, most of the germcells present are oogonia atinterphase (Table 3), this stage remaining predominant

TA

BL

E

3

Est

imat

ed p

ropo

rtio

ns o

f ge

rm c

ells

at

vari

ous

stag

es o

f de

velo

pmen

t in

the

lef

t ov

aria

n co

rtex

of

the

chic

k

Per

cent

ages

of g

erm

cel

ls a

t the

foll

owin

g st

ages

ofd

evel

opm

ent:

Age

(day

s'in

cuba

tion

)

9 10 11 12 13 14 15 16 17 18 19 20

1 da

y af

ter

hatc

hing

Inte

r pha

se

90-7

94-2

92 1

93-7

94

0

89-5

76-5

66

1

41 0

51-7

28-5

3-5

1 Lar

ge'

Mit

oses

7-3

4-7

5-7

5-5

2-9

3-5

2-8

2-9

1-6

1-5

0-6

0-3

Ear

lyP

re-l

epto

tene

— 2-3

2-6

18-3

16-7

20-7

13-1

16-3

3-7

0-8

Lat

eP

re-l

epto

tene

— — — 10

7-3

14-8

90

11 -

4

5-5

2-5

Lep

tote

ne

— — — 0-5

14-3

8-9

14-1

8-5

2-8

Zyg

oten

e

— — — — 2-4

3-5

5-7

35

0

18-3

Pac

hyte

ne

— — — — 01

7-8

16

0

36-4

56-2

Dip

lote

ne

— — — — — 0-9

1-5

5-9

Atr

etic

germ

cel

ls

20

11

21

0-7

0-8

4-4

1-4

6-5

51

4-6

6-7

5-6

13-5

GERM CELLS % H X tfl >n w > r m o HICK


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 decreasesfrom the ninth day of incubation onwards (Table 3; Text-fig. 5). The proportionof cells undergoing degeneration increases from the thirteenth day of incubationonwards (Table 3; Text-fig. 5).

Maximal proportions of oocytes in early pre-leptotene occur between days 15and 19 (Text-fig. 6). Presumably this is a transitory stage since the proportionsof 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 incidenceof oocytes at the latter stage occurs between the seventeenth and nineteenth days

50

10

9 10 15Age in days

1 120 ID

TEXT-FIG. 7. Mean percentages of germ cells at different stages of development., at zygotene; , at pachytene; , at diplotene.

of incubation (Text-fig. 6), an observation which suggests that their duration isbrief.

The incidence of cells at the zygotene stage is maximal on the twentieth day ofincubation (Text-figs. 4 and 7). A large proportion are in the pachytene stagebetween the nineteenth day of incubation and 1 day after hatching (Table 3;Text-fig. 7). This suggests that pachytene is a relatively lengthy stage. Qualitativeobservations indicate that the numbers of oocytes at diplotene continue to in-crease beyond the first day after hatching.

Sources of error in quantitative studies

The present estimates of the population of germ cells in the developing ovaryare subject to several sources of error.

(1) Owing to the time-consuming nature of the method of counting, it has notbeen possible to analyse more than two or three ovaries for each age group. Nohighly inbred strains of chicks were readily available. On the other hand, vari-ability due to genetic factors is likely to have been reduced somewhat by the use


of Fx hybrids between the two 'strains'. Nonetheless, in the case of two age-groups (13 and 17 days' incubation), the estimate for one individual was twice ashigh as that for the second. For this reason, a third embryo of each age groupwas added, and the overall mean for the three used.

(2) The possibility exists that at early developmental stages the proportion ofgerm cells migrating to the right and left ovaries varies between individuals. Thisfactor alone may be responsible for some of the variability in the population ofoocytes in the left ovaries.

(3) Since oocytes frequently extend over more than one section, care was takento record only those cells whose nucleus was largely contained within the selectedsection. Even so, the decision whether more or less than half of any given cellwas included is entirely subjective. The classification of germ cells into variousstages of meiotic prophase is also a somewhat subjective process.

(4) It is difficult to make accurate counts of cells packed together in largenumbers at all levels throughout the thickness of the section. This appliesparticularly to the extremities of the cortex which contain very large numbers ofclosely crowded oogonia. In spite of the precautions taken to minimize thissource of error, it is likely that an unknown (but probably small) proportionof germ cells was missed, while some may have been counted twice.

DISCUSSION

The qualitative histological and cytological observations made in the courseof 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 Fx crosses of theWhite Leghorn x Rhode Island Red breeds, the number of germ cells in the leftovarian cortex increases from about 28,000 at 9 days' to approximately 680,000at 17 days' incubation. Thereafter, the population declines sharply until about480,000 remain 1 day after hatching. The increase in the number of germ cells isassociated with a high incidence of oogonial mitoses, and the subsequent declinewith a high incidence of degeneration of oocytes together with a sharp decrease inthe number of germinal mitoses.

Faure-Fremiet & Kaufman's (1928) estimates of the population of germ cellsin the ovaries of 2-day-old chicks differ from those in 1-day-old chicks in thepresent study by a factor of about x 16. This discrepancy can only be accountedfor by the differences between the two methods of estimating the populations ofoocytes. Faure-Fremiet & Kaufman estimated the number of oocytes per unitarea of cortex and from this calculated the numbers of oocytes in the entireovaries. Their method of estimating the number of oocytes, however, rests onthe assumptions that the cortex is uniformly filled with germ cells, and that germcells of differing sizes are randomly distributed throughout the cortical area.

5 3 0 GILLIAN C. HUGHES

TABLE 4

A comparison of the present observations with those of D'Hollander (1904)

Observations of D'Hollander Corresponding observations made in the present study

Cell-types

' a '' a "' b '*V'd '' e 'T'g ''h '

' i 'T

' j ' in primordialfollicles

Days of incubationwhen cell-types

were first observed

101014151516

16-1717-18

1921

Days after hatching034

Germ cells at various stagesof de ve lop men t

primordial germ cellsinterphase oogoniaoocytes in early pre-leptotene

„ „ late pre-leptotene

,, „ leptotene„ „ zygotene,, ,, pachytene

,, ,, diplotene

,, ,, diplotene

,, „ diplotene inprimordial follicles

Days of incubationwhen developmental

stages were firstobserved

99

13-1415

1616-1717-19

19-20

Days after hatching0-3

4

* Cells described, but not designated by a letter.

Both these assumptions are unlikely to be valid. First, the nuclear diameter ofgerm cells tends to increase as development proceeds; and second, there is adifferential spatial distribution of germ cells within the cortex, the larger germcells occurring mostly near the centre and the smaller ones at the extremities ofthe cortex.

Faure-Fremiet & Kaufman also paid no attention to the possibility that thesame oocyte may be seen in more than one section. It has since been shown thata correction factor (Abercrombie, 1946) needs to be applied in order to accountfor the relative thickness of the sections with respect to the size of the nuclei (seeMandl & Zuckerman, 1951). Sources of error in the present quantitative studywere kept to a minimum.

The observation that the population of germ cells begins to decrease steeply atan early age confirms that of Faure-Fremiet & Kaufman (1928) on the ovaries ofhatched chicks. These authors, as well as Brambell (1925), provide ampleevidence 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 therepeated 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 oocyteswhich had already become invested in follicles.

The present observations thus indicate that while a high proportion of germcells degenerate during the latter part of embryonic life, sufficiently large numbersescape atresia and constitute the 'stock' of oocytes which will subsequentlybecome depleted with age. Firket's (1914) thesis that all or nearly all the germcells present in the left ovary of the embryonic chick degenerate, and are replacedlater by proliferations from the germinal epithelium, is thus finally invalidated.

The pattern of ovarian development observed in the chick is essentially thesame as that in the rat (Beaumont & Mandl, 1962) and in man (Baker, 1963). Ifthe total numbers of germ cells present in the ovaries of these species are plottedgraphically against time ('birth' and 'hatching' being superimposed), the shapesof the graphs are strikingly similar (Text-fig. 8).

Germ cells degenerate throughout development in all three species. The closesynchronization of developmental stages in the rat has made it possible to detectfour' waves' of degeneration: the first involves oogonia at interphase; the second,actively dividing oogonia ('atretic divisions'); the third, oocytes at the pachytenestage ('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 thediplotene phase is seen in full-term and neonatal specimens (Baker, 1963).

No attempt was made in the present study to classify germ cells undergoingdegeneration. Possibly because degenerating germ cells in the chick are elimin-ated from the ovary relatively quickly (see above), early stages of atresia seemedto be less common than those at advanced stages, by which time classification wasnot feasible. Qualitative observations, however, indicated that the increase in thenumber of atretic oocytes 1 day after hatching was due to a high incidence of cellsdegenerating at diplotene (cf. human ovary at birth; rat, 2 days after birth).Asynchronization in the development of germ cells may cause an overlap betweenany 'waves' of degeneration and thus render the detection of individual peaksdifficult. The results indeed show that while the numbers of atretic germ cells inthe 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 earlystages of development and (with a few possible exceptions) does not occur afterthe onset of sexual maturity. In contrast, in all amphibians, many fishes (par-ticularly teleosts) and probably in many reptiles, oogenesis occurs continuouslyduring adult reproductive life (see Franchi et ah, 1962). It is of interest to notethat in Sphenodon, a primitive diapsid reptile, primordial germ cells enter theprophase of meiosis shortly after their migration into the genital ridges (Tribe &Brambell, 1932). It is not known whether all the oogonia become transformedinto 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

II-1

1

5

i

1i i

i i

i

I15-

i i

i

5

i5

i i

i i

i

i

i

i

I20-

1

1

5

i

, 9

t

i

1i

1

PP

PP

1

2pp

2pp

9 10 15 20 ID

,Man age (months)

Rat age (days)

Chick age (days)

Hatching

TEXT-FIG. 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.


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 arestrictly synchronized within the ovary. Differentiation is always more advancedat the centre of the cortex than at the cortical extremities. The same is true ofamphibians, where oocytes in the more central areas of the cortex co-exist withoogonia at the periphery (Witschi, 1914). A similar tendency has been reportedfor some mammals (e.g. rabbit: Winiwarter, 1901; mouse: Borum, 1961; man:Baker, 1963), although the differences in the stages of development attained byperipherally, as compared with centrally placed germ cells may only be slight(see also Cowperthwaite, 1925; Brambell, 1927; Mintz, 1959). The close syn-chronization 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 andsquash preparations. Counts were made of the numbers of oogonia and oocytesat various stages of meiotic prophase in specimens aged 9 days' incubation to1 day after hatching.

2. Neither the mitotic activity of oogonia nor the onset of the successive stagesof meiotic prophase are synchronized throughout the ovary. The developmentof the germ cells is more advanced at the centre than at the extremities of theovarian cortex.

3. The process of oogenesis ceases at about the time of hatching. Appreciablenumbers of oocytes enter the pre-leptotene phase on the thirteenth day, zygoteneand pachytene on the seventeenth, and diplotene on the nineteenth day ofincubation.

4. Degeneration of germ cells occurs throughout the developmental periodstudied.

5. Oogonial mitoses are associated with an increase in the total population ofgerm cells from about 28,000 at 9 days' incubation to some 680,000 on theseventeenth day. Subsequently, a high incidence of degenerating germ cells,together with a decline in the number of oogonial mitoses, account for a decreasein the total population between the seventeenth day of incubation and 1 day afterhatching, when only about 480,000 oocytes persist.

RESUME

La population de gonocytes chez la Poule au cours de son developpementembryorinaire

1. On a etudie les modifications morphologiques et cytologiques surven-ant 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 lesoogonies et les oocytes a divers stades de la prophase meiotique, du 9e jour del'incubation au ler jour apres Peclosion.

2. Ni l'activite mitotique des oogonies, ni le debut des stades successifs de laprophase meiotique ne sont synchronises dans l'ovaire entier. Le developpementdes 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 nombresappreciates d'oocytes entrent en phase de pre-leptotene le 136me jour, de zygoteneet 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 dedeveloppement etudiee.

5. Les mitoses oogoniales sont associees a un accroissement de la populationtotale de gonocytes, d'environ 28.000 le 9e jour de l'incubation, a environ 680.000le 1T jour. Ensuite, une importante degenerescence de gonocytes, conjointementa une baisse du nombre des mitoses oogoniales, est la cause d'une diminution deleur population totale entre le 17e jour de l'incubation et le ler jour apresl'eclosion, ou persistent seulement environ 480.000 oocytes.

ACKNOWLEDGEMENTS

The expenses incurred in this study were defrayed from grants, made to Professor Sir SollyZuckerman, 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 ofGenetics, University of Birmingham, for his advice on cytological techniques.

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{Manuscript received 18th January 1963)

the populatio onf germ cell isn the developing female chick · fig. j. oocyte ats leptoten e (18...

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