J. Embryol. exp. Morph. Vol. 16,1, pp. 211-29, August 1966
Printed in Great Britain
211
Modification of embryonic development of reproductive and lymphoid organs in the chick
By ALAN E. ERICKSON & GREGORY PINCUS
Worcester Foundation for Experimental Biology, Shrewsbury
Following the publication of Lillie's (1917) observations and theories on the
endocrine basis of the development of the bovine freemartin, experiments were
initiated to determine if heterosexual gonad grafts to the chorio-alantoic membrane of chicks could stimulate formation of intersexes (Willier, 1927). Hormone
extracts of testes and urine were used by Dantchakoff (1935) and Kozelka &
Gallagher (1934), and synthetic hormones were tested in the chick by Wolff &
Ginglinger (1935). Estrogens feminized the left gonad of male embryos and
resulted in retention of Mullerian ducts in males and occasional cystic development of Mullerian ducts in females. Less extensive feminization of the left
testis, dilation of Wolffian ducts, and partial deletion of Mullerian ducts
occurred in androsterone-treated embryos (Wolff, 1935). Sensitivity of the
genital papilla to exogenous steroids was described by Reinbold (1951).
Riddle & Dunham (1942) demonstrated that a single injection of estradiol
benzoate into laying birds resulted in feminization of genetic males which
developed from ova released shortly thereafter. Lutz & Lutz-Ostertag (1959)
described spontaneous chick freemartins developing in double-yolked eggs.
Studies of individual male embryos which had access to the resources of two
yolks in double-yolked eggs (Ruch, 1961) revealed the occurrence of gonadal
modifications similar to those resulting from administration of estrogens or
from implantation of female gonads adjacent to the genital ridge of young male
embryos (Wolff, 1946-47). When heterosexual implants of gonads of sufficient
size were placed on the chorioallantoic membrane, male left testes developed
into ovotestes and Mullerian ducts were reduced in the female (GroenedijkHuijbers, 1960). The immunological consequences of vascular anastomoses between bovine twins discussed by Owen (1945), and more recently by Fecheimer,
Herschler & Gilmore (1963) and Goodfellow, Strong & Stewart (1965) afford
an additional point of departure in studying the freemartin.
For several years we have been studying the effects of steroid compounds on
the development of the reproductive system of the chick embryo (Pincus &
Hopkins, 1958; Pincus & Erickson, 1962). The sensitivity of the testis and
Mullerian duct of the avian embryo to estrogens, the contrast of 'feminization'
1
Authors' address: Worcester Foundation for Experimental Biology, Shrewsbury, Mass.,
U.S.A.
212
A. E. ERICKSON & G. PINCUS
of embryonic chick testes by ovarian tissue to the 'defeminization' of fetal
ovary by mammalian testes, and the asymmetric structure of the reproductive
system of the avian female have all been related theoretically to hormonal
regulation of sex differentiation. Demonstration that normal regulation is attributable to molecules of a steroid nature, however, has not yet been made. The
detection (Price, Ortiz & Deane, 1964) of 3/?-hydroxysteroid dehydrogenase in
early embryonic adrenal and gonadal tissue (31 days) and the in vitro evidence
of androgenic activity of fetal guinea-pig testis as early as initial formation of
primary sex cords (25 days) (Price, Ortiz & Zaaijer, 1964) add impetus to the
search for and evaluation of natural and synthetic steroids which might afford
effective and lasting control of fetal sex differentiation without compromising
fertility of the treated individual.
In this report, the results of exposing 4-day chick embryos to a number of
steroid and non-steroid compounds are described.
MATERIALS AND METHODS
All eggs were obtained from a cross between Rhode Island Red males and
Barred Plymouth Rock females to give a strain known as Merryknoll (Merryknoll Farms, East Freetown, Mass., U.S.A.). This strain is characterized by a
sex-linked white feather spot on the head of the male chick.
Eggs were placed in a Robbins Hatchomatic Incubator which was equipped
for automatic turning of eggs every hour and for controlled temperature and
humidity of 99-100 and 83-86 °F (wet bulb) respectively. After 96 h of incubation the eggs were injected and returned to the incubator until the nineteenth
day when they were removed for candling. Eggs containing viable embryos were
transferred to the hatcher, the temperature and humidity of which were maintained at 98-99 and 85-88 °F wet bulb. Records were made of hatchability of
fertile eggs, sex of chicks, and approximate age of embryos which failed to
survive. The mean numbers of fertile eggs in test and control groups were 66
and 60 respectively.
Compounds tested were dissolved in sesame oil. The solutions were sterilized
by exposure to 100 °C for 1 h on each of three successive days prior to use. The
broad end of the eggs was washed with 95 % ethanol and punctured with an egg
punch. The compounds were introduced into the eggs in 0-1 c.c. of sesame oil
with a 23-gauge, \ in. needle. After injection the opening was washed and sealed
with a small drop of collodion. Solvent controls were treated similarly with
0-1 c.c. of sesame oil.
The extent of development of the genital papilla was determined in all chicks.
The technique used, known as vent-sexing, involved applying sufficient pressure
around the cloaca to cause eversion of the ventral fold from which the papilla
arises. Note was made of those instances in which vent-sexing was difficult or
impossible because of an abnormally small cloacal opening.
Chick reproductive and lymphoid organs
213
Most chicks from each treatment group were sacrificed by chloroform and
dissected within 24 h of hatching. Modifications of the reproductive organs and
gonads were noted whenever such occurred. Alterations in morphology of the
left testis apparent at dissection were confirmed by histological study. A continuous cortex on the male left gonad warranted tabulation of the specimen as
an ovotestis. Only those testes with more limited cortical development were
entered under the heading of 'Cortex, Partial' in the tables.
In addition to the qualitative evaluation of structures in all birds, a quantitative study of organ weights of comb, thymus, spleen, bursa of Fabricius and
gonads (left in the female, left and right in the male) was made of 10 males and
10 females from each test and control group. Organs were excised, trimmed,
and weighed on a Roller-Smith torsion balance. Means and standard errors
were computed, and the Tukey test (Snedecor, 1956) was used for comparison
among means to determine significance of differences of weights.
Whenever possible, a few males and females were retained for sacrifice at
approximately 210 days of age for study of the long-term effects of the treatment
to which they had been subjected as embryos.
RESULTS
The dose-levels of each compound tested and percentage hatchability of
fertile eggs in test and control groups have been listed in Table 1. In subsequent
tables compounds are all grouped according to the type of activity of each
steroid as determined by traditional mammalian assay. Table 2 contains a listing
of all active compounds with an index of the effects of treatment as noted in
day-old chicks.
Hatchability
Mean hatchability for control groups was 75+1-5 (standard error). Seven
per cent of control embryos died prior to 10 days of incubation and 19 % after
that age. A similar excess of deaths among older embryos was noted in all test
groups except the following: XXXVI, XLI, XLII, XLIII, and XLIV. All
embryos treated with 0-8 mg of pregn-4-ene-3,ll,20-trione-6a-methyl were dead
before the tenth day of incubation. Of the steroid compounds, the nortestosterones (XIII, XVII), progestins (XXI, XXVIII, XXXVI, XXXIX, XLIII and
XLVI) and estrone (I) were the most toxic materials administered.
Effects on the reproductive systemGenital papilla. All estrogens listed in Table 3 A affected gonads, Mullerian
ducts, and the genital papilla. Female-type vents were seen in approximately
5 % of male controls. Female controls rarely possessed a male-type papilla, but
in most instances a miniature papilla or small eminence could be detected at
sexing. Reduction of the genital papilla of the male was the most frequently
encountered modification in estrogen-treated chicks. Enlargement of the genital
14-2
214
A. E. ERICKSON & G. PINCUS
Table 1. List of compounds tested and effect on hatchability
Hatchability (%)
Identifying
code
I
Compounds
Estrone
VIII 2-Oxa-5a-androstane-3-one-17/?-hydroxy-17amethyl
X Estra-l,3,5-(io)-triene-16,17-diol-3-methoxy16a-methyl
XIII 17a-Vinyl-19-nortestosterone
XIV
2a-Ethinyl-2/?, 17/#-dihydroxy- A-norandrostane
2a-Ethinyl-2y#, 17/tf-dihydroxy-2/?, 17/?-diacetateA-norandrostane
XVIII Testololactone-1 -dehydro
XV
XXI
A-norprogesterone
XXIII 6a-Methyl testosterone
XXIV Testosterone-ll/?-hydroxy-17a-methyl
XXV Testololactone
5
XXVI 17a-Ethinyl-A -androsten-3/?, 17>5-diol
Dose
(mg)
01
0-8
01
0-4
01
0-8
01
0-8
01
0-8
01
0-8
01
0-8
01
0-8
01
0-8
01
0-4
01
0-4
01
0-4
01
0-8
K
t
,
Test
Control
68
2
83
74
60
60
67
3
70
83
83
81
68
81
49
56
40
73
63
76
4
82
77
72
74
77
80
81
73
81
79
73
84
79
XXVIII Pregn-5-en-20-one-3& 17-diol
01
0-4
68
67
13
0
44
31
XXIX Pregnenolone-3-methyl-ether
01
0-8
01
0-8
74
76
77
65
69
01
0-8
70
67
68
69
74
78
0-8
62
47
0
004
010
01
0-8
01
0-8
27
0
68
71
59
1
XXVII 17-Ethyl-19-nortestosterone
XXX
Androstan-3-one-17/?-hydroxy acetate
XXXI 4-Chloro-17a-methyl testosterone
XXXII A5-Androsten-3/?,17a-diol
XXXIII N;NM>is(dicMoroacetyl)-N,NMiethyl-l,4xylylene-diamine
XXIV N.N^bisCdichloroacetyl)-!, 8-octa-methylene
diamine
XXXV 3/?-Acetoxy-17a hydroxy-pregn-5-en-20-one
XXXVI Pregn-4-ene-l ,3,20-trione
01
0-4
01
79
68
81
78
65
65
Chick reproductive and lymphoid organs
215
Table 1 (cont.)
Hatchability (%)
Identifying
code
Compounds
XXXVII 2/3,17/?-Dihydroxy-2a, 17a-diethinyl-A-nor5a-androstane
XXXVIII 1,1 -Dichloro-2(o-chlorophenyl)-2-(/?-chlorophenyl)-ethane
XXXIX 1,2a-Methylene-6-chloro-l 7a-acetoxy-4,
6-diene-3,20-dione
XL SJ^'^'-Tetrahydroxy flavone
2H-l-Benzopyran-7-ol-3-p-(methoxy-phenyl-)
2-2-4-trimethyl acetate
XLII Erucic acid
XLI
XLIII 6a-Methyl-pregn-4-en-3,l 1,20-trione
XLIV Diethylsti 1 bestrol
XLV
Testosterone
XLVI Progesterone
Dose
(mg)
004
0-20
01
0-8
001
008
01
0-8
0-19
0-08
01
0-8
01
0-8
01
0-8
01
0-8
0002
002
010
0-8
(
Test
Control
60
39
74
84
40
0
68
48
74
86
74
69
46
0
82
51
69
62
61
47
35
0
70
84
73
73
64
82
84
81
72
60
papilla or of the supporting cloacal fold was common in andro gen-treated
chicks (Table 3B). The hypertrophied structure was characterized by a broadened head with a medial trough on the free dorsal margin. A frequent accompaniment of papillary enlargement was reduction in size of the vent opening,
making it difficult to adequately evert the genital papilla for sexing.
Gonads. Cortical development of the left testis was stimulated by six estrogens
and one compound from the androgenic series, 17a-ethinyl-A5-androsten-3/?,17/?diol. The extent of cortical growth varied from small patches to continuous
bands of cortex occupied by typical-appearing oogonia. With the aid of lowpower magnification, it was possible to identify the translucent rim of cortical
material characteristic of ovary and ovotestis at the time of dissection. Variations of weight of left gonads of males and females (Table 4) did not follow any
discernible pattern. The elevated weight of the left testis in males treated with a
low dose of 2a-ethinyl-2/?,17/?-dihydroxy-A-norandrostane (XIV) coincided
with a histological demonstration of a substantial cortex and medulla with an
abundance of intervening lacunae which were not seen in the more compact left
testis from the high-dose group. Disruption of the normal medullary structure
14-3
216
A. E. ERICKSON & G. PINCUS
of the testis was far more apparent than the rather limited cortical organization
in chicks treated with non-steroid compound XLI. Reduction in weight of the
right testis was apparent in all groups with ovotestes even though the difference
was not always significant.
Table 2. Summary of developmental modifications seen in 1-day-old chicks
Reproductive system
Mullerian duct
Male Male
right
JV1 I
gonad gonad
with recortex duced
A
f
9
I
1
1
I
1
I
XIV
I
I
li
I
I
XLII
XLIV
XLV
Deletion
^
3
I
XLI
Genital papilla
9
Hypertrophy
tion*
A
/
— —\
3
9
3
I
[
I
I
i
I
I
A
t
3
\
••
Thymus
r
•*
J
9
x
9
i
i
i
I
it
1
it
i
[
OlililULdllUU
Bursa
,
'" ^
c?
9
I
I
I
I
I
I
•
*»
Spleen
A
i
3
-\
9
I
I
t
Lymphoid organs
A
Cf l m i l l citizen
OlIUIUlclllUJl
I
VIII
X
XIII
XV
XVIII
XXIII
XXIV
XXVI
XXVIII
XXIX
XXX
XXXI
XXXIII
XXXIV
XXXVII
XXXIX
Wolffian duct
I—
I
I
i
i
I
[
I
I
I
I
I
1
1
I
I
I
l
I
I
I
1
It
it
i
I
I
i
i
;1
I
I
I
I
I
I
I
I
i
L
I
i
i
I
i
I
I
i
I
I
I
1
l
I
I
I
I
I
I
i
It
[
I
i
i
* Feminization of 3 vent recorded only if incidence was 10 ( {, or higher.
t Stimulated , not inhibited.
Mullerian ducts. Retention of some Mullerian elements in the male and cystic
development of both these remnants and Mullerian ducts in the females were
common in the groups treated with compounds which stimulated ovotestis
development. Deletion of portions of the female Mullerian duct followed treatment with a few androgens and two of the A-norandrostanes. An anterior
extension of the Mullerian duct, running parallel to the ovary along the Mullerian ligament was observed in many females in which the posterior portions of
the duct were absent.
Wolffian ducts. Dilation of the Wolffian ducts was seen in some of the androgen-treated groups. The contents of the ducts varied from a transparent amber
color to an opaque, white coagulum.
Long-term results. Table 5 has been included to summarize the long-term
effects observed in birds hatched from treated eggs. The birds reported on are
those which survived for 210 days. Among the females the most frequent
I
Chick reproductive and lymphoid organs
217
abnormality was a defective oviduct. The findings included cystic ducts and
ducts in which there was apparent blockage preventing normal oviposition.
The extreme anterior Mullerian elements described above persisted in both
males and females. Adult males from the ovotestis groups usually had asymmetric left testes. Mean weights of left and right testes of adult controls were
20-05 ± 2-25 g and 14-95 ± 1-63 g. This weight differential was absent in many of
the estrogen-treated birds.
The left vasa deferentia were not always comparable in development to the
right vasa, even in cases where the left testis contained fully mature sperm in an
essentially normal organ.
Table 3. Effects of compounds on morphology of the reproductive system of
the chick as seen at hatching frequency of modifications ( %)
Left testis
Right
x
Mullerian ducts
Genital papilla
\\7/"i1 flRon Hiir*tc
VVUllllall UULlc
A'
ICSHS
A
c?Some $ Cysts
Hypertrophy Reduction Hyperl-lupiiy
PP.
rptpn
A
UULCU
1CICI1- or elon- $ JVC
8
$
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<j
tion gations due:tions
Part A. Estrogenic compounds
25
0
89
43
100
100
0
100
+
re-
Code
I
X
XIV
XV
XXXVII
XLIV
Dose
Partial
testis
01
01
0-8
01
0-8
01
23
0
0
33
0
4
27
30
10
4
0
38
100
94
63
90
0
0-8
004
0-20
01
0-8
VIII
XII [
XX11I
XXVI
01
0-8
01
01
0-8
01
0-4
46
62
55
70
40
96
100
11
23
+
+
+
+
+
+
94
73
50
0
45
100
91
100
0
0
9
100
0
27
100
90
91
0
0
0
64
0
94
86
95
15
100
100
*
96
100
100
100
0
+
Part B. Androgenic compounds
4
+
54
XXXI
XLV
100
74
0*8
01
0-8
83
100
0
0
64
9
95
0
0
4
50
0
70
0
0
0
0
54
0
0
0
3
0
35
0
0
0
0
65
0
0
0
4
0
0
0
0
0
0
0
46
100
0
0
0
96
93
20
21
100
0
0
25
0
35
PartC . Progestins
XLIII
XLI
+
01
0-8 (toxic)
01
0-8
0
80
0
0
t
§
Part D. Non-steroids
0
0
20
31
* Could not be sexed.
3
15
76
0
10
18
0
80
0
0
7
45
6
44
218
A. E. ERICKSON & G. PINCUS
Table 4. Mean weights (mg) and standard errors of selected organs of
1-day-old chicks*
Gonads
Dose
Compound (mg)
I
Control
01
0-8
X
Control
01
0-8
Control
01
0-8
Control
01
0-8
Control
01
0-8
Sex
Left
Right
Thymus
Bursa
Spleen
A. Effects of estrogen-type compounds on selected organs
<J
3.50 ±0-256
<J
4-57±0-361f
(Toxic)
XIV
XV
Control
01
0-8
Control
01
2-79±0-147
4-32±0-325
6-74±0-446tt 2-34 ±0-248
3.67 ±0-254
0-66±0-054*f
6-29 ±0-390
6.82±0-540
4.73±0-476{
2-79±0-147
2-37 ±0-190
l-40±0-206tt
VIII
XIII
74-30 ±7-24
5408 ±4-58
56-34±5-52
71-92 ±4-94
72-22 ±5-26
74-91 ±8-49
74-30 ±7-24
85-36±4-93
61.36±3-11J
71-92 ±4-94
77.36±6-91
58.04 ±6-49
4416±4-51
33-3O±4-26
28-70 ±2-42f
37-54±l-85
33-3O±l-8O
25-70 ±2-24ft
44-16±4-51
43-39±3-89
24-64 ±2-56ft
37-54±l-85
30-75±2-14f
12-96±l-75tJ
44-16±4-51
37-22 ±2-76
28-46±l-56f
37-54±l-85
38-52±2-21
20-72 ±2-75ft
39-22 + 3-52
12-12+1-10
10-99 ±0-911
12-12 ±0-708
12-28 ±0-977
11-04 ±0-578
13-74±1-14
12-12±M0
11-43 ±0-668
9-65 ±0-402
12-28 ±0-977
11 -73 ±0-726
10-57±l-15
Control
2-57 + 0-142
0-04
0-20
0-48 ±0-052
59-6O±5-37
0-56±0-091
50-86 ±4-08
68-10+6-00
67-38 ±5-55
5203 ±5-76
17-37±1-82|
32-60±2-93
22-74 ±2-49t
16-84±2-09t
13-20±0-631
11 -45 ±0-527
12-28 ±0-977
12-36±0-522
11 -70 ±0-656
10-67 ±0-770
10-98 ±0-697
8-72±0-533
10-94 ±0-496
10-70 ±0-726
9-66±0-96l
2-79±0-147 74-30 ±7-24
0-76 ± 0-0731 77-80±3-67
0-470 ± 0-0641 69-15±3-88
71-92 ±4-94
62-34±6-31
7202 ±5-49
44-16±4-51
38-94 ±2-66
32-66 ±2-89
37-54±l-85
29-10±3-27
22-02 ±2-14f
ll-45±0-377
11-48 ±0-663
12-28 ±0-977
10-90±0-793
11 -48 ±0-663
6-29 ±0-390
5-95 ±0-328
4-77±0-369t
Control
004
0-20
XLIV
2-79 ±0-147
0-89 ±0073*
0-78 ±0162*
74-30 ±7-24
64-98±516
66-34±5-75
71-92 ±4-94
77-44 ±8-04
65-24 ±7-42
57-62 + 3-70
0-8
XXXVII
4-32±0-325
2-97 ±0-257*
3-57 ±0-350
Control
01
08
Control
01
0-8
4-32±0-325
4-08 ±0-239
2-96±0-262ft
26-81 ±2-70t
B. Effects of androgen-type compounds on selected organs.
Control
5.53±0-512
3-73 ±0-394
c?
01
3-91±O-383t 2-35±0-317f
0-8
2-86 + 0-174
4-34±0-252
Control
76-90±5-78
40-70±4-06
01
6810±4-60
8-5O±l-83f
(?
0-8
(Toxic)
Control
62-88±4-54
30-71 ±2-45
9
01
9
80-71 ±5-54
12-41 ±2-97f
0-8
(Toxic)
12-12+110
12-12 + 1-30
10-58±0-516
ll-16±0-65l
9-86±0-547
10-96±0-718
Chick reproductive and lymphoid organs
219
Table 4 (cont.)
Gonads
Compound
XVIII
Control
01
0-8
Control
01
0-8
Control
01
0-8
Control
01
0-8
XXX
XXXI
XLV
9
2
3-45 ±0-349
5-3O±O-3O3f
5-39±0-660f
2-20±0152
2-88 + 0-218ft
l-86±0-190
Thymus
76-25±4-71
74-88 + 6-25
6408±3-71
81-43 + 7-16
67-36 + 5-38
62-64 ±3-61
79-18 + 9-51
78-70 + 7-67
65-26±5-33
71-34 + 5-17
76-78 + 3-80
63-49 ±4-37
6710 + 404
62-06 + 3-64
55-38 ±3-20
51-60 + 5-49
54-35 + 4-76
5O-8O±5-38
6*
<?
6
Right
74-00 + 3-52
81-34 + 6-77
80-91 ±705
74-59±719
67-99 ±5-20
74-67 ±9-28
<5
Of Of Of
Control
01
0-4
Control
01
0-4
a
Of Of Of
XXVIII
6
Of Of Of
Control
01
0-4
Control
0-1
0-4
Control
01
0-8
Control
01
0-8
Control
01
0-8
Control
01
0-8
Control
01
0-8
Control
01
0-8
3-56±0172
4-32 + 0-364
3-26±O-311t
Of Of Of
XXVI
<J
<J
Of Of Of
Control
01
0-4
Control
01
0-4
Left
OI- Of Of
XXIV
Sex
Of Of Of
XXIII
Dose
(mg)
7-10±0-471
5.68±0-277t
5-70±0-312t
74-30 + 7-24
8808 + 4-81
73-48 ±5-84
71-92 ±4-94
68-84 + 5-50
65-32±5-14
76-90±5-78
57-84 ±6-21
58-52±4-12
62-88 ±4-54
63-55 + 4-49
68-77±4-73
79-74 + 3-28
80-18 + 4-04
70-85 ±4-74
83-56±6-01
77-80 + 5-14
64-76 + 4-93
C. Effects of progestins on selected organs
76-90 + 5-78
63-94 + 2-37
71-93±4-82
62-88 + 4-54
61-36 + 5-09
66-14 + 8-26
Bursa
3918±l-62
42-71 + 3-63
29-14±2-10ft
29-63 ±2-45
34-24 ±1-89
29-39 ±2-87
45-41+4-73
34-20+ 3-84f
o-oon
35-74±2-85
29-70+1-71
O-OOfJ
Spleen
12-12 + 0-709
11-28 + 0-848
10-70 ±0-800
11-45 + 0-822
11-89+1-02
11-40 ±0-880
12-50±l-06
10-50 + 0-615
12-34 + 0-647
11-66 + 0-684
9-79±0-767f
46-58 + 3-69
26-48+ 2-62f
10-31 ± 1 0 6 t t
41-30±5-31
20-94 + 2-61f
8-28±0-54f
36-46 ±2-34
32-37 + 3-18
39-37±l-34
28-93 + 2-56
27-85 + 1-74
20-32 ±2-44f
44-16 + 4-51
39-87 + 3-19
30-46±3-12f
37-54+1-85
34-36 + 2-45
22-62 ±2-98|J
40-70 ±406
9-64+l-95f
1306+101
12-43 + 0-744
12-42±M6
12-58 + 0-786
12-46 ±1-29
10-78±l-19
40-70 ±406
36-25 + 2-42
43-36±3-55
30-71+2-45
30-68 ±2-56
24-74 ±3-25
10-58 ±0-516
12-60 + 0-983
15-70±l-28tt
10-10 + 0-607
12-25 ±1-22
9-86 ±0-547
10-84 ±0-698
10-90 + 0-752
9-35±0-658
9-38 ±0-740
9-56 ±0-774
8-98 ±0-720
12-12 + 1-10
12-26 + 0-525
12-88 ±0-654
12-28 ±0-977
11-94+1-14
ll-80±0-679
10-58±0-516
10-23 ±1-14
9-68 ±0-598
o-oott
30-71+2-45
9-86 + 0-547
11-07 + 0-955
10-16+l-89f
10-71 ±8-05
o-oott
44-29 ±3-83
13-24±l-29
37-10±4-12
11-98 ±0-828
12-02 ±l-97fj 11-34 ±0-820
41-15±2-58
13-79±1-19
33-69 + 5-07
12-66 ±0-676
ll-89±3-08fJ 10-76±0-888
220
A. E. ERICKSON & G. PINCUS
Table 4 (cont.)
Gonads
XXIX
XXXIX
Control
S
$
Control
o
01
0-8
01
0-8
Left
Sex
-to
Compound
Dose
(mg)
Right
9
Of
<y
Control
001
S
008 (Toxic)
Control
9
001
008 (Toxic)
XLII1
6
$
08
(Toxic)
<y
Control
Control
01
XLVI
-to
0002
o
002
ci
01
(J
0-8
(Toxic)
o
Control
0002
002
9
01
9
XXXIII
XXXIV
XLII
Bursa
Spleen
76-90 ±5-78
84-36 ±4-50
77-30±7-67
62-88 ±4-54
75-31±5-31
85-40 ±5-43f
8315±6-32
54-50±4-53t
40-70 ±4-06
42-40 ±2-66
40-12±3-18
30-71 ±2-45
35-8O±2-53
34-16 + 2-14
10-58±0-516
12-15±0-759
15-70±l-28tt
9-86±0-547
ll-28±0-957
1215±0-757
46-27 ±3-10
31-16±2-62t
13-18±0-550
10-34±0-677f
9514±8-46
59-64±6-58f
42-48 ±2-62
24-20 ±l-97f
13-00±0-787
10-47 ±0-656t
70-48 ±5-07
77-38 ±5-22
66-96±7-71
6M4±5-56
40-87±4-31
42-48 ±402
35-74±3-91
31-84±2-50
12-42 ±0-780
12-20 ±0-929
1206 ±1-28
10-90 ±0-710
68-46 ±205
78-66±5-82
62-24 ±4-18
57-07 + 4-70J
27-88 ±1-41
3601 ±1-91
30-17 ±2-40
27-30±3-53J
13-06±0-880
11-20 ±0-777
12-53±0-864
9-46 ±1-22
Thymus
3-41 ±0-246
2-36±0151f
D. Effects of non-steroidal compounds on selected organs
67-81 ±5-36
Control
38-90±3-77
01
62-56 ±403
35-40±2-34
0-8
(Toxic)
82-24 ±5-45
Control
35-60±2-10
9
o
36-06 ±4-44
01
54-50±5-01t
0-8
(Toxic)
Control
004
010 (Toxic)
Control
9
o
004
010 (Toxic)
Control
010
008
Control
010
008
<?
o
9
9
12-61 ±1-22
10-64 ±00789
12-52 ±0-607
9-80± 0-755|
67-81 ±5-36
45-73 ±3-8Of
38-90±3-77
30-26 ±3-34
12-16±l-22
9-82 ±0-905
82-24 ±5-45
41-51 ±3-60f
35-60±210
24-04±3-17|
12-52 ±0-607
9-08±l-22f
88-35 ±6-43
79-25 ±8-84
67-51 ±10-4
76-97±5-15
78-42±5-53
6405 ±3-39
49-64 ±2-63
44-89 ±4-63
36-54±3-51
38-78 ±3-32
38-41 ±3-53
34.43+ 2-41
13-57±0-785
12-32±0-814
11-32 ±0-773
11-85 ±0-871
13-70±0-919
10-49±0-713J
* = lOd1 and 10$ at each dose level.
f = Significant difference from control value (P < 95).
% = Significant difference from other dose (P < 95).
Chick reproductive and lymphoid organs
221
Table 5. Long-term effects observed in birds (7 months old)
hatched from treated eggs
Compound
type*
o
o
o
A
S
P
O
s
s
Number
of
Code
number animals
Number
of
Female birds
VIII
Mature. Both with
oviduct abnormalities
X
One immature. Both
with oviduct abnormalities
Mature. Both with incomplete oviducts.
One with extreme left
anterior cyst
Typical mature
animals
Male birds
Testes undersize. Two mature
birds; one with atypical left
vas deferens
Three mature. Three with
equal size left and right testes.
Two with retarded vasa def.
Two with extreme anterior cysts
Mature. One with right testis
XV
heavier than left. One with
with atypical left vas deferens.
Two with cystic remnants of
Mullerian duct
Mature. Left testes grossly
Mature. Three with
XXVI
irregular form; microscopic
cystic oviducts
variations in three
Three
mature; one maturing.
XXVIII
0
All with equal-sized left and
right testes
Mature.
One with left and right
XXIX
testes equivalent size. One with
hard mass in left vas deferens
Typical
mature
XXX
0
Mature.
Two with testes
3 Typical mature
XXXIII
equal-sized left and right
Mature but with small equal1 Typical mature
XXXIV
sized testes
XXXVI
0
•
5 Typical mature
5 Mature. Two with cysts 4 Mature. Four with left and
XXXVII
three with right testes
and one with shortened
grossly and histologically
left oviduct
atypical. Sperm present in all
testes. Wolffian duct
abnormalities in all four
XL
6 Typical mature
5 Typical mature
Mature. One with grossly and
2
XLI
4
Mature. One with
microscopically irregular left
cystic right oviduct
testis
Mature. Four with small left
XLIV
2
Mature. Both with
testes. One with cystic, sterile,
abnormal oviducts
and fertile tubules adjacent
to moderate-sized follicles.
Some thickening of interstitial connective tissue
*O = Estrogen; A = androgen; P = progestin; S = non-steroid.
XIV
222
A. E. ERICKSON & G. PINCUS
Effects on lymphoid organs
All estrogens active in reducing the weight of the right testis of male chicks
were also active in inhibiting development of the bursa of Fabricius. Other
lymphoid organs did not differ significantly from control values.
Androgens were more active as inhibitors of bursa development than as
modifiers of the reproductive tract. The effect on the lymphoid bursa was not
consistently reflected by reduction of the other lymphoid organs of the body.
The activity of progestins on lymphoid development was almost negligible.
Progesterone was inactive. l,2a-Methylene-6-chloro-17a-acetoxy-4,6-diene3,20-dione (XXXIX) was the only progestin to produce a consistent drop in
weight of all three lymphoid organs of both sexes at the 0-08 mg doselevel. The non-steroid compound XXIV almost paralleled the activity of compound XXXIX in its effects on the lymphoid tissue. A significant difference
between weights of test thymus, bursa and spleen and control organs was seen
only in female chicks, however.
Reduction in size of cloaca! vent
Vent-sexing was difficult in many of the birds which had been treated with
androgens. The cloacal aperture was small in many of these birds and could not
be opened sufficiently to permit eversion of the genital papilla for study. This
defect was generally associated with bursa reduction. An unfortunate consequence of treatment of chick embryos with compounds affecting these changes
was the rather unthrifty condition of the birds at hatching and the high frequency
of blockage of the vent with dried droppings. Special attention had to be given
the chicks during their first few weeks of life to keep the vents unplugged and
even then the mortality rate was higher than that encountered among chicks
which hatched following treatment with compounds which did not affect the
bursa.
DISCUSSION
The wide variety of steroid compounds and the several non-steroid compounds
tested in this series were selected because of (a) their interesting structural
characteristics and (b) reports, published and unpublished, of activities disclosed through assays performed for a variety of purposes. Our goal has been
to determine if the structural uniqueness of some of these compounds would
modify the well-documented responses of the chick embryo to the natural
androgens and estrogens which have been studied. Several compounds were first
dissolved in isopropyl alcohol and administered to the embryos through the
dipping technique described by us in earlier papers (Pincus & Hopkins, 1958;
Pincus & Erickson, 1962). In all instances the qualitative information obtained
by this method of testing has been duplicated when the compounds were administered by injection. Standardization of the injection procedure has per-
Chick reproductive and lymphoid organs
223
mitted evaluation of relative activity of the compounds on the reproductive
system and developing lymphoid organs. It is important to emphasize that while
these compounds have all been tested on the 96 h chick embryo, development
of the genital papilla, extent of growth of the cortex of ovotestes, and modification of the Mullerian ducts, can be affected by treatment at other stages in
embryonic development (Pincus & Erickson, 1962). While we have not had
occasion to observe the sensitivity of the bursa of Fabricius to steroid treatment at other than 96 h of incubation, others (Rao, Aspinall & Meyer, 1962)
have reported that this structure will be inhibited by appropriate treatment
during the period from the fifth to the thirteenth day of incubation.
Effects on the reproductive system
If stimulation of cortical development is selected as the criterion of most
drastic feminization, compounds X, XIV and XLIV (Table 3) were outstanding
in this regard. Five of the eight steroids with benzenoid ring A, in addition to the
three A-nor compounds, caused some degree of cortical stimulation of the male
left testis. Cortical tissue in ovotestes ranged in extent from small patches to
broad, continuous bands of oogonial cells with an underlying intermediate zone
of loose connective tissue and lacunae derived from former medullary tubules.
Beneath the intermediate zone varying numbers of typical medullary tubules
were found in which there was no apparent histological variation from normal.
Although cortical elements of ovotestes did not generally persist in male birds
held to maturity, the irregular form of the testes at 7 months of age was evidence
of the earlier exposure to test materials. Ovocytes in follicles were seen only in a
small number of genetic males which had been exposed to diethylstilbestrol.
Normal spermatogenesis was observed in all mature testes. The marked reduction in size of the right testis noted in some day-old chicks did not appear to
prevent essentially normal development and growth to almost full size of that
organ by maturity, This potential for marked subsequent development of an
initially inhibited right gonad may be similar to that observed in the rudimentary
right ovary when the latter is released from the 'inhibition' of the left ovary
early in life of the chick (Domm, 1929).
Remnants of Mullerian ducts observed in estrogen-treated males and androgen-treated females persist through the life of the birds. Cysts containing up to
500 c.c. of clear fluid have been observed in some experimental birds of 210
days of age. Compounds XIV and XXXVII followed the pattern of stimulation
of male Mullerian ducts with simultaneous inhibition of female ducts described by Hamilton (1951, 1963) as typical for androstanes when administered
in large doses.
Two of the A-nor compounds (XIV and XV), which paralleled in part the
action of the most potent estrogens, were first investigated by Jacques & Pincus
(1962) who referred, to them as 'inverted steroids'. Compound XIV was described as having a feeble uterotropic activity in the immature mouse assay but
224
A. E. ERICKSON & G. PINCUS
caused a slight inhibition of uterotropic activity of estrone in combined assay.
Compound XV was described as antiprogestational. Pincus, Banik & Jacques
(1964) indicate that the third A-nor compound (XXXVII) is anti-progestational
and estrogenic.
At the time of examination of the 1-day chicks, hypertrophy of the Wolffian
duct was noted only after treatment with certain androgens and one progestin.
Since the mesonephros is considered to be non-functional at hatching, it is
suggested that the contents causing the dilation might be due to a back-up of
material from the ureters. The inadequacy of the cloaca to permit normal flow
of wastes in some androgen-treated birds has already been mentioned.
Examination of 7-month-old males from groups VIII, XIV, XV and XXXVII
disclosed deficiencies of vasa deferentia which had not been observed in controls
nor been anticipated from changes noted at the time of post-hatch examination.
It is possible that incomplete development of the vasa in chicks exposed to the
two A-nor compounds might be a reflexion of reduced production of or response to intrinsic androgens during some phase of maturation.
Effect on lymphoid organs
Sixteen of the steroid compounds demonstrated biological activity on the
morphological level by inhibiting normal development of the bursa of Fabricius.
Meyer, Rao & Aspinall (1959), Aspinall, Meyer & Rao (1961) and Glick (1961)
had described this effect earlier. Meyer et al. (1959) reported that the structural
defects of the cloacal area included stunted growth of the ventral anal lip, increased musculature of the dorsal lip, and an attenuated proctodeal canal with
few anal mucoid glands. The sequence of normal and abnormal embryological
development of the bursa-vent complex is well described by Meyer et al.
Ackerman (1962) and Ackerman & KnoufT (1959) have reported on light and
electron microscopic studies of bursa development, describing the effect of
steroid administration on the appearance of alkaline phosphatase.
A comparison of the six steroid compounds (VIII, XIII, XXIII, XXIV,
XXXI and XXXIX) causing significant differences between control and both
male and female bursa weights at a dose of 0-1 mg or less reveals the following
common structural characteristics listed in decreasing order of frequency;
(a) keto group at C-3 (six out of six compounds), (b) hydroxyl or ester at C-17
(6/6), (c) C-19 carbon (5/6), and (d) unsaturation in ring A (5/6). Aspinall et al.
(1961) reported that under their experimental conditions the most active bursainhibiting compounds on a strain of White Leghorns were those with a 3-keto
group, fully saturated steroid nucleus, and 17-hydroxyl (with one exception in
which a keto group was located at C-17). Our experience has been comparable
except with regard to reduction of ring A.
While testosterone possesses all the characteristics described as common
among our most active androgenic compounds, it lacks the alkyl groups found
associated with ring B, A, and/or D in the most active materials. Compounds X
Chick reproductive and lymphoid organs
225
and XLIV, both with benzenoid ring A, were active bursa inhibitors. Compound
XXXI was a potent inhibitor of the bursa and of normal Mullerian development. Dorfman & Kind (1963) reported that the relative potency of this compound compared with testosterone was of a lower order in the castrate rat
assay. One of the most active comopunds was XIII, reported by Edgren,
Calhoun, Elton & Colton (1959) as an estrogen antagonist in the immature
female mouse assay. Saunders & Drill (1956) found that the androgenic and
myotropic potency of compound XIII was considerably lower than that of
testosterone propionate in the castrate rat assay.
The capacity of anti-spermatogenic substance XXXIV (Beyler, Potts, Coulston & Surrey, 1961) and the progestin XXXIX to inhibit development of all
three lymphoid organs in one or both sexes is of interest, particularly in view of
the rather scattered effects of other steroids on lymph organs other than the
bursa. From the teratologic point of view there is no reason to expect an inhibition of thymus, bursa and spleen through a single treatment with a single
compound administered at 96 h of incubation.
The evaluation of compounds described in this report presents an aspect not
currently available in routine assays for determination of androgenicity or
estrogenicity of steroids. The morphological response of the left testis of the
chick embryo to estrogens is not duplicated by testes of other experimental
animals used in current assays. One is led to consider the possible long-term
effects of steroids on fertility of offspring born to mothers treated with such
compounds during pregnancy. One wonders if attrition of gonia might be
accelerated, fertility of mature sex cells would be compromised, or competence
of sex ducts affected by such treatment, x2 analysis of the relative toxicity of each
compound for male as against female embryos as indicated by hatchability
demonstrated that only in group XXXIX was hatchability of females significantly less than that of males.
Although our tests did not show any consistent effect on the thymus (or
spleen) we cannot dismiss the possibility that by careful selection of treatment
time and of compound it might be possible to develop a thymus-inhibiting
procedure which could be of potential value to the further investigation of
activity of this organ (and of the bursa of Fabricius). Angervall & Lundin (1964)
have recently reported on studies of this type using pregnant rats as recipients
of corticoids.
The compounds used in this series were no more successful in maintaining
basic gonadal reverses than those described by previous workers. 'Gonadal
feminization' in the male chick as a response to treatment with a variety of
compounds lacks stability under present experimental conditions. Contrary to
the findings of others who have reported studies of the effects of steroids on the
reproductive system of the chick embryo, we have seen no evidence of masculinization of ovaries in the experimental groups reported upon in this paper.
Modifications of the sex ducts appear to be permanent. Analytical approaches
226
A. E. ERICKSON & G. PINCUS
to mechanisms of duct differentiation, such as described by Hamilton (1961),
will doubtless shed light on some aspects of our experimental results.
SUMMARY
1. A total of 25 steroids and six non-steroids were administered to chick
embryos after 96 h of incubation.
2. The most potent estrogenic compounds tended to cause : (a) stimulation
of cortical development on the male left gonad; (b) decrease in size of the male
right testis; (c) retention and cyst formation of the Mullerian ducts of both sexes;
and (d) reduction of the genital papilla of the male.
3. The androgens caused no observable modification of gonad morphology.
Hypertrophy of the genital papilla and reduction of the vent opening were
frequently encountered in androgen-treated chicks. Dilation of the Wolffian duct
and deletions of the Mullerian ducts of the female were observed in some chicks.
4. All estrogens which were active in significantly reducing the weight of the
right testis of the male, and most of the active androgens, also caused significant
reduction of the bursa of Fabricius.
5. The combination of vent reduction, hypertrophy of the genital papilla, and
reduction of the bursa of Fabricius were seen most commonly in the androgentreated birds.
6. Modification of the bursa was not consistently accompanied by reduction
of other lymphoid elements of the body. Mortality was higher in chicks suffering
from bursa reduction than in the other groups.
7. Progestins were generally toxic and did not have any characteristic effect
on the reproductive system.
8. Long-term effects seen in 210-day birds hatched from treated eggs included abnormalities of the oviducts, absence of a normal differential in left
and right testis weights, disruption of the normal histological architecture of the
left testis, and defects of the left vas deferens. Persistent follicles in testes which
had been stimulated by estrogens were seen only in a limited number of males
exposed to diethylstilbestrol.
RESUME
Modification du developpement embryonnaire des organs
reproducteurs et lympho'ides chez le poulet
1. On a administre un total de 25 steroides et 6 non-steroides a des embryons
de poulet apres 96 h d'inclusion.
2. Les composes oestrogenes les plus puissants ont tendu a provoquer:
(a) une stimulation du developpement cortical de la gonade male gauche;
(b) une diminution de la taille du testicule droit; (c) la retention et la formation
de cystes dans les canaux de Miiller des deux sexes; et (d) la reduction de la
papille genitale du male.
Chick reproductive and lymphoid organs
227
3. Les androgenes n'ont pas provoque de modification observable de la
morphologie des gonades. L'hypertrophie de la papille genitale et la reduction
de l'orifice cloacal ont ete frequemment rencontrees chez les poulets traites aux
androgenes. On a observe chez quelques poulets une dilatation du canal de
Wolff et des destructions du canal de Miiller chez les femelles.
4. Tous les oestrogenes qui etaient actifs en reduisant de maniere significative
le testicule droit du male, et la plupart des androgenes actifs ont egalement
provoque une reduction significative de la bourse de Fabricius.
5. La combinaison de la reduction de l'orifice cloacal, de l'hypertrophie de la
papille genitale et de la reduction de la bourse de Fabricius a ete observee tres
communement chez les oiseaux traites aux androgenes.
6. La modification de la bourse n'etait pas forcement accompagnee d'une
reduction d'autres elements lymphoi'des du corps. En outre, la mortalite etait
plus forte chez les poulets souffrant d'une reduction de la bourse que chez les
autres groupes.
7. Les progestatifs ont ete en general toxiques et n'ont pas eu d'effets caracteristiques sur l'appareil reproducteur.
8. Des effets a long terme observes sur des oiseaux de 210 jours, nes d'oeufs
traites, comprenaient des anomalies des oviductes, l'absence d'une difference
de poids normale entre les testicules gauche et droit, une-rupture de l'architecture histologique normale du testicule gauche et des defectuosites du canal
deferent gauche. On a observe des follicules persistants dans des testicules qui
avaient ete stimules par les oestrogenes mais seulement dans un nombre limite
de males exposes au diethylstilboestrol.
This work was supported by a grant from the National Foundation.
The authors wish to thank the following persons and organizations for supplying the
compounds indicated:
G. D. Searle Co.: VIII, XIII.
Dr A. L. Beyler, Sterling Winthrop Co.: XXXIII, XXXIV.
Merck and Co., Inc.: XXXVII.
Schering Laboratories: XXXIX.
Dr sa G. Baldratti, Farmitalia, Milano, Italy: XXXI.
Dr Jean Jacques, Laboratoire de Chimie Organique des Hormones, College de France:
XIV, XV.
Cancer Chemotherapy National Service Center, National Institutes of Health: XXXIV,
XXXVIII, XLI, XLIII.
We wish to acknowledge the technical assistance of Mrs Virginia Kingsbury.
228
A. E. ERICKSON & G. PINCUS
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{Manuscript received 4 November 1965, revised 23 January 1966)