Genital abnormalities in castrated fetal rats and their prevention by means of testosterone propionate.

GENITAL ABNORAIALITIES I N CASTRATED F E T A L
IL4TS AND T H E I R PREVENTION BY M E A N S
O F TESTOSTERONE PROPIOXATE 1,
L. J. WELLS, MARGARET m7. CAVANAUGlI
AKD EDWARD L. MAXWELL
Depaitmeiit of A x a t o m y , Uni c e r s i t y of M z n w s o l n , M 11111f a p o l i s
T W E N T Y FIGURES
In a study of castrated fetal rats, it has been virtually
proven that the fetal testis produces a hormone (androgen)
which stimulates the prenatal growth of such accessory reproductive organs a s the seminal vesicles and bulbourethral
glands (Wells, ’5Ob; Wells and F’ralick, ’51). The fetuses
were castrated during developmental stages in which the
primordia of the accessory reproductive organs had already
appeared and in which the Miillerian ducts had disappeared.
It could not be determined whether the testicular androgen
induces the formation of these primordia and causes the
retrogression of the Rliillerian ducts.
Accordingly, new work was undertaken with a view to
solving the problem by studying fetuses in which the operation was performed before the appearance of these primordia
and before the disappearance of the i\liillerian ducts. I n our
attempt to extend the work to the earliest possible stage of
prenatal development, the available methods were “pushed
to the limit. ” Technical difficulties prevented us from solving
the problem, but we were successful in obtaining fetuses
‘Aided by grants from the United States Public Health S e n i c e (RG-2801) and
from the Graduate Nedical Research Fund of the Vniversity.
The authors are indcbted to Ciba Pharmacrutieal Products, Inc. for supplying
the testosterone propionate (Perandren) and t o Mrs. J u n i t a Mortensen n h o assisted as laboratory technologist.
109
110
L. .J. WELLS, 31. CAVAPTh17(:II A S U F;.
L. MAXWELL
which lived until near term, after liaving been subjected to
castration prior to the appearance of prostatic buds and
coagulating glands. h preliminary report of the observations
was presented at the Providence meeting of the American
Association of Aiiatomists ( T e l l s , Cavanaugh and Rlnxwell,
’52).
JIATERIA1,S AND METHODS
In rats of the Sprague-Dawley strain, 113 preg*nancieswere
timed by witnessing the mating. Forty-five yielded a closely
graded series of control fetuses. Sixty-eight were used in
the experinients on castration; a nunibcr of them also served
as sources of control fetuses which were takrii at the time
of the operation.
At operation a gravid female was anesthetized with ether,
arid the uterus exposed by optliiing the peritoneal cavity at
the liiiea alba. Each fetus to be castrated was brought into
view by incising the uterus and fetal rnenilii*aiies, and was
then delivered into the peritoneal cavity of the mother in
such mariner that the placental circulation remained intact
(“estrauterinized fetus”). On each side of the fetal body
an oblique inguinal incision was made, the testis removed
arid the wound closed by mc’aiis of interrupted sutures of
silk; for. additioiial details of the method, two earlier papers
may be examined ( V7ells, 50a ; X-ells arid Fralicalr, ’51, Method
2 ) . In each of 4 cases a pellet of testosterone propionate
(Perandren), weighing about 1.0 nig, was implanted under
the fetal skin. I n all cases the treated fetus, still attached to
the uterus by the placenta and urnhilical cord, was allowed
to remain in the maternal peritoneal cavity. I11 each of three
cases the fetus was covered, however, by nieaiis of a tent
created by stitching a thin sheet of cellulose acetate to the
uterine m ~ l l(artificial uterus). Similarly, in each of -2 cases,
the fetus was covered by a tent which was fashioned out of
a thin sheet of rubber and stitched to the uterine wall. When
all steps of the fetal surgery w ~ completed,
i ~
the maternal
wound was closed by suturing and the etherization stopped.
111
C H A N G E S IN O R C I I I E C T O M I Z E D FETlTSES
The experiments were terminated by killing the mothers.
This was done near term arid usually when the fetuses had
attained the age of 21 days and 15 hours. It was found that
14 of the castrated fetuses were alive and quite active and
that the “artificial uteri’ ’ had not increased the percentage
of survival. The survival was a s follows. Of two fetuses
castrated at the age of about 19: days, both were alive (litter16 h,
mates). Of 61 castrated a t ages of 18 d 2 h u p to 18 d
1 2 were living. Of 15 castrated a t 1 7 d
21 h to 18 d
1h,
none was alive.
I n coiiducting the autopsy of a n individual litter, a living
castrated fetus and a n unoperated male were taken by severance of the umbilical cords. They were promptly weighed
and then killed. The reproductive tracts were fixed i7z situ
in Bouiri’s solution, and sectioned transversely i n perfect
series at 10 p. The sections were mounted on microscope
slides and stained with hernatoxyliri and eosin.
With the aid of microscopes, data were obtained from the
stained sections. The length of the RIiillerian ducts and/or
prostatic utricle was determined by simple calculation (numher of transverse sections x thickness of section), on the
assumption that they were straight vertical structures, which,
of course, they were not; nevertheless the data would seem
to have comparative value. Such data were not obtained in
the case of the fetuses of group C (table 2) since the reproductive tracts had been sectioned in the frontal, rather than
the transverse, plane. In 20 fetuses the counting of the prostatic buds was facilitated by making transparent models of
the prostatic urethra and outgrowths. The models were prepared by projecting the sections with a n Edinger projector,
by stippling the desired outlines on thin sheets of cellulose
acetate with inks of various colors and by stacking these
sheets. The models were abnormally short in vertical dimension because the acetate sheets were thin and because suitable
spacers of cardhoard were delihera tely omitted. The models
were: however, accurate in two dimensions and in the number
of prostatic buds shown.
+
+
+
+
B
A
16d
C
18d.Zh
17d .I0 h
I
I
I
I
I
I
I
I
I
I
E
D
C
IOd,lZh
crest.
utricle
21 d , 15h
F
lad, Zh
18d,Zh
H
Zld,l5h
I
21d115h
Fig. 1 Conventionalized diagraiiis of the genital ducts and derivatives ill control
fctuscs ( A t o 0) and experimental fetuses ( H and I). A t the age of 18 d
2 11, thc
prevailing pattern ~ v a slike that illustrated in drawing C (12 of the 14 fetuses). Only
m e of the castrated fetuses showed a “ V ” a t the upper elid of the prostatic ntricle
(drawing H)
+
.
C H A N G E S I N O R C H I E C T O M I Z E D FETUSES
11.3
TERMINOLOGY
The coagulating glands, most easily defined ‘ny reference
to the adult rat, are those paired parts of the prost a t‘IC complex which are topographically associated with the seminal
vesiclcs and which release into the urethra a substance that
coagulates ejaculatcd semen to produce the “vaginal plug’’
Moore, ’39, fig. 26). The prostatic utricle (uterus masculinus),
honiologue of the vagina in the female and derived from the
lfiillerian ducts, is present in the newborn male. The M U Z Zeriun tubercle in a fetus is that hillock in the urogenital sinus
which receives the Miillerian ducts and Wolffian ducts (fig.
1 A ) and which persists after birth as a mound (colliculus
seminalis) on the urethral crest. The balcmo-preputial lamella
is a solid plate of epithelium from which the epithelium of
the glans penis and that of the inner surface of the prepuce
develop (insert in fig. 17).
OBSERVATIONG
I n preview it should be noted that in 6 of the 14 castrated
fetuses to be considered the operation was performed at
developmental stages younger than 18d
11h (table 2). I n
11h, the epialmost all of the control males under 1 8 d
thelial buds of the prostate and the epithelium of the coagulating glands had not yet appeared (table 1);the oblique
and horizontal portions of the Miillerian ducts were still
present (fig. 1C, see text).
+
+
Cont r o 1 m t 1es
+
Prostate. I n controls younger than 18 d
11h, there was
only one instance in which a prostatic bud was found. This
structure, noted in a fetus of 1 8 d
4 h , appeared in only
three of the l o p sections. I t was situated in a region comparable to the one indicated by the arrow in figure 13. Up
14 h, the largest number
to and including the stage of 18 d
of buds in an individual male was 6. Such buds were smaller
and shorter than those illustrated in figure 16.
+
+
18
18
18
18
18
18
18
18
18
18
18
19
19
21
17
16
16
16
1i
17
17
17
17
17
+ 11
+ 12
+ 13
+ 14
+ 12
+ 14
+ 15
+5
+7
+1
+2
+3
+4
+ 21
+ 23
+ 10
+ 12
+ 14
+ 18
+ 20
+ 12
+ 16
DAYSA~~o;OCRS
1.25i (1)
1.2i4 (2)
1.650
1.434
1.590
1.631
2.453
2.487
5.003
.700
.880
.716
1.035
1.087
.985
BODY W T .
415
40 (12)
458 (1)
458
429 (1)
PREPUTIAL
FRENULUM,
WIDTH
102 X
107 X
102 X
111 X
127 x
116 X
104 X
126 X
112 x
139 X
153 X
83 X
82
89
84
82
73
78
81
70
77
109
95
7i
_
(2)
(2)
V
V
Vc f
v
V
V
v
V
V
V
(13)
;
;
V
V
V
V
V
;
D
A
D
SEJLINAL
VESICLES,
EPITHELIUM
_
E
;::;:“,
_
p
0
0
0
0
0
~
+++
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
._
~
~
<
<
0
0
0
0
0
0
0
0
1 4
0
0
4
34
44
6
20
20
61 (6)
0
0
0
0
0
0
PROSTATIC
RUDS,
~E P I~T H E~L I U~M
___
A , absent (Wolffian ducts not dilated a t sites of future vesicles) ; D, dilatation of Wolffian ducts a t sites of future vesicles;
V, vesicular outgrowths; Vcf, vesicles each with a “cranial flexure” (flexure illustrated in fig. 1).
Actual nuniber in one case (not an average number) ; no buds in the other cases.
* RL, right-left diameter; DV, dorsal-ventral diameter.
980
1050
940
880
890
720
860
1040
660
930
420
480
258 (12)
P
P
*
PROSTATIC
UTRICLE,
R L X DV
+
~
M ~ L L DUCTS
.
PROST. U T R I C L E ,
LENGTH
Kumbers in parentheses indicate the number of cases.
3
1
2
2
2
1
1
13
-___
5
I
1
1
1
3
1
14
13
1
2
8
2
3
1
1
FETUSES
TlnLE 1
Observations in 8.3 control fetirses
~
r
A
f
115
C H A K G E S I N O R C H I E C T O M I Z E D FETC'SES
I n fetuses so young that the prostatic buds had not yet
appeared, it was not possible to identify any well defined
211
boundaries of the future prostate. Controls of 1 8 d
and older showed, however, in the region surrounding the
epithelium of the future prostatic urethra, a relatively thick
area of densely packed cells which must have originated from
mesenchyme (fig. 13). That most of these cells a r e eventually
used in forming the non-parenchymal component of the prostate (stroma, etc.), was suggested by the minute anatomy
of the developing prostate i n older fetuses (figs. 16 and 14).
The prostatic buds in fetuses of 2 1 d
15 h were numerous. Distally, some of them had distinct branches (fig. 2).
The longest buds were those of the ventral series.
CoaguEating glands. The coagulating glands mere observed
in 15 of the controls (19 d
12 h t o 21 d
15 h ) . They
consisted of two componeiits, iiiner and outer. The inner
component, a solid core of epithelium, had originated from
the dorsal aspect of the embryonic prostatic urethra (figs. 6
and 2 ) . The outer component was a thick iiivestment of
densely packed cells of mesenchymal origin.
The question arose a s to whether the primordia of the
coagulatiiig glands might exist, in advance of epithelial outgrowths, in the form of clusters of densely packed cells (e.g.,
primordia of the outer componeiit of the glands). A positive
answer was suggested by the fact that two cellular condensations were observed in each of the two fetuses of 18 d
14 h
and that they were situated in the region of the future coagulating glands, 01' near it. Also, they had undoubtedly originated from mesenchyme. Incidentally, they were smaller a i d
less distinct than those marked by the arrows in figure 7.
In fetuses younger than 1 8 d
l l h , there did not seem
to be any paired cellular coiidensatioiis in what was regarded
as the region of the future coagulating glands (fig. 5).
SciniqzaZ v r s i c Z ~ s . Vesicular outgrowths from the Wolffian
ducts were regularly present in fetuses killed at ail age of
18 d
2 h, hence the developing vesicles need only be given
brief consideration in the present paper. At 2 1 d
15 h, the
+
+
+
+
+
+
+
+
116
L. J. \YELLS, &I. CAVANAUGH
A N D E. L. MAXWELL
original cranial end of each vesicle had a flexure which passed
niedially arid inferiorly (“cranial flexure, ” fig. 1G).
Bulbouret7irul gl(112tls. The primordia of the bulbourcthral
16 11.
glands were not yet present in controls of 16 d to 16 d
Either the primordia or the developing glands themselves
were observed in all inales of 17 d
10 h to 21 d
15 h.
il1iillcriun ducts u n d prostcitic utricle. The normal retrogression of the Miillerian ducts in the male was already in
2 h to 1s d
14 h. The pattern
progress in controls of 18 d
usually observed in such fetuses is illustratedl i n figure 1C.
The stages shown in figures I D and 1 E were rarely seen.
These observations suggested that in the castrated fetuses
to be considered all, or almost all, of the vertical portions of
the Rliilleriari ducts had already dropped out before the time
of the operation.
In the prevailing pattern depicted in figure 1 C, the prostatic utricle had a lumen. A t the niliillerian tubercle, the
utricle ended by opening bilaterally into the urogenital sinus
(embryonic prostatic urethra, fig. 13).
15 h (figs. 1(3,
The prostatic utricle was present at 21 d
1C and 14). Inferiorly, below the level of section shown in
figure 10, the utricle usually had a small lumen which forlied
and then opened bilaterally into the urethra.
Pmile urethra and prryiitial frenulzcnz. I n the fetuses of
18 d
2 h to 19 d
14 h the distal end of the urethra was
a solid plate of epithelium situated in the developing glans
penis (urethral plate, see insert in fig. 17). J u s t proximal to
the urethral plate, the floor (ventral wall) of the urethra
was still missing (open urethral groove, not illustrated in
the photographs). The developing preputial frenulum was
a ventral structure in the region where the right and left
portions of the balano-prcputial lamella were separated from
each other by developing connective tissue. ‘This frenuluni
was broadest proxirnally, at the site of the open urethral
groove, and it was here that the data on the width of the
frenulum were obtained.
+
+
+
+
+
+
+
+
2E
2c
18+
19+
2E
1c
18
7 [ 2-13]
8
111-151
6 [ 3-9 ]
+ 13
+
5E
5C
18
3.178
3.211
4.509
5.010
4.408
5.442
406
41
517 (4)
37
7 8 [74-851
+
4.114
5.289
41
46
Castrate4 fetuses gi\en aiidiogeii
75 [T4-77]
82 [79-841
61
P
P
500
340
340
232
596
253
94
81
113 X 109
80 X 66
135 X 79
82 X 78
223 X
81 X
P
(4)
60 [39-62]
66 (1)
(a)
55
(2)
(1)
9-20]
13
48 (1)
26 [23-27]
(5)
(2)
10
3-16]
63 ( 3 )
(1)
(3)
(5)
~ _ ~ _ _ _ ~ ~
______
Age of the fetuses a t autopsy was about 21 d
13 h. Xumbers in parentheses indic;ite tlic iiuml~trr of c a w s . Smnbers in
b1acltrts indicate the range. E, experiniental; C, control (litter-mate).
RL, right left diameter; DV, dorsal-.rentral diameter.
A
5E
5c
glVL
Castrated fetuses n o t gireii androgen
__hours
m
m
r:U;
e
M
a
w
M
~
s4F;
H
Q
k
Q
W
3
z
m
m
_
_
_
118
I,. J . WELLS, M. CAVANAUGH AKD E. L. MAXWELL
At 21 d
+ 13 h
the urethra was a continuous tube. The
urethral plate had acquired a lumen, a i d the earlier urethral
groove had been closed by the union of the right and left
urethral folds.
(
L(
s t TC!t (3cl
fr t u s c s 9%o t g iz;e+%ugt d r 0.9 em
( g r o u p s d t o C, tccblr 2 )
Prostutic buds. Epithelial buds of the prostate wei*e observed in each of the 12 fetuses of groups A to C. All buds
except one to 4 of the ventral series were relatively short
(fig. 3 ) . The buds were usually unbranched. In each of three
exceptional fetuses, one of the ventral buds ~ v w sbegimiiiiq to
branch (see fig. 15).
Despite the fact that in a normal fetus taken froin H 11011experimental litter at I 8 d
4 h a single bud was already
pixsent (table l ) , the data secnied to indicate that in each
of the castrated fetuses of group A all of the prostatic buds
had appeared subsequent to the orchiectomy. To illustrate
what was found in individual litters, three fetuses of litter
941 may be used. il normal control, killed and preserved a t
the beginning of the experiment, 18 d
7 h, had no buds.
A second fetus, castrated a t 18 d
7 h, aiid killed at 2 1 d
15 h, had 7 huds. The third, a n uiioperatcld control killed
a t 21 d
15 h, had many buds (the buds could scarcely be
counted accurately since a model was not made).
Similarly, the data indicated that in each of the castrated
fetuses of group B most of the buds had appeared after the
time of the operation. The findings in one litter, no. 932, may
bc presented. A normal control, killed a t the beginning of the
experiment, 18 d
12 11, showed three buds. h second fetus,
castrated at 18 d
12 h and killed at 21 d
15 h, showed
16 buds. The third, an unoperated control killed a t 21 (1
15 h, showed 48 buds.
The huds in the castrated fetuses of group C were longer
and more numerous than those of a normal fetus from which
figure 16 was obtained. This fact together with the tabulated
data seemed to shorn that any h d s existing a t the time of
+
+
+
+
+
+
+
+
+
119
C H A N G E S I N OR C HIE C T OMIZEU FETUSES
the operation had continued to grow during the experimental
period and also that some new buds had originated during
this period.
(‘ongulating glancls. In regions comparable to those where
the coagulating glands of controls were observed, only paired
clusters of cells of riieseiichymal origin were found in 4 of
the 5 castrated fetuses of group A (fig. 7). It was problematical whether these clusters constituted primordia of the outer,
non-epithelial coniponent of the glands.
The coagulating glands were present in 6 of the castrated
fetuses of groups A and B. The tabulated data were regarded
as ample evidence that the epithelial coniponent of the glands
had developed after the orchiectoniy.
The glands were likewise present in the two castrated
fetuses of group C. It was likely that these glands had already appeared at the time the operation was performed.
Jfiillfirian ducts ~ i v dprostatic utricle. The Miillerian ducts,
a s such, had largely disappeared, and were mainly represented
by a large derivative, the prostatic utricle (fig. 1 H ) . That
retrogression of the Niillerian ducts which had occurred during the experimental period was approximately the same iu
each castrated fetus of groups 9and B ; it usually proceeded
from a stage shown in figure 1C up to that shown in figwe
1 H . In the castrated fctusm of group C such retrogression
had already occurred, however, at the time of the operation,
or shortly thereafter (fig. 1F).
The prostatic utricles of the castrated fetuses mere larger
than those of the litter-mate controls of the same age (fig. 1H,
fig. 1G and table 2 ) . Utricles of the castrated fetuses of
group A were the largest of all (fig. 7 ) , even larger than those
of two normal males which were Idled at 19 d
1 2 11 and
19 d
14 h, respectively. The utricles of the castrated fetuses
of giwups B and C were only somewhat larger than those of
15 h.
the litter-mate controls obtained at 31 d
The distal end of the utricle had the shape of an inverted
Y in each castrated fetus, and, in this respect, was normal.
+
+
+
120
L. * J . WELLS, M. CAVANAUGH A X D E. L. MAXWELL
It was abnormal in each of the castrated fetuses of groups h
and €3 i n that the luiiieii was missing.
Xiilleriun tirO(~rc~lo
nird c~icrcztlirfory ducts. The Mullerian
tubercle was usually abnornial in shape, being so flat that
i n section it scarcely yesunbled a hillock. The cjaculatory
ducts also were usually ahnormal. I n each of the castrated
fetuses of groups X and €3, the distal end of each duct lacked
a lumen. In 7 of these 10 fetuses the ducts were dilated,
either hilaterally (5 fetuses) o r unilaterally ( 2 fetuses) ;
sometimes the corresponding seminal vesicles were also dilated. I n each of two fetuses of group A, though, a t a region
m l l above the lllulleriaii tubercle, the ejaculatory ducts and
the seminal vesicles were more or less solid structures (luinina
missing or almost missing, figs. 11 and 15).
Penile urethra and p r e p u f i u l frrnulum. The penile urethra
was normal in one respect but abnormal in a second. It was
iiormal i n that the urethral plate had undergone cavitation
to produce the fossa navicularis. It was abnormal i n that the
urethral groove was still open (hypospadias). The existence
of a condition of hypospadias was actually observed in only
8 of the 1 2 castrated fetuses (three in group A, three in B
and two in C), and was, f o r one reason or another, doubtful
in the other 4. This anomaly was regarded as an abnormal
persistence of an earlier normal embryonic condition. The
proximal p a r t of the developing prcputial frenulum was
usually very wide, thus reflecting the fact that the floor of
the urethra was missing in this region.
Castrated fetuses given. androgen
(group D, table 2 )
I n each of the two experimental fetuses of group D, a
pellet of testosterone propionate mas implanted under the
skin immediately after the testes had been removed. When
the stained sections were studied microscopically, it mas found
that i n each case all those effects of castration noted in the
experimental fetuses of groups A to C had been prevented,
C H A K G E S I S 0I:CHIECTOMIZEl)
FETVSES
121
01' largely
prevented. Thus, the prostatic buds were iiuiiierous
(fig.1 aiid table 2 ) . Tlic coagulutiiig glands were present
(figs. 1and 8). The ejaculatory ducts had continuous lumens,
a i d were not almornially dilated (fig. 12). The floor of the
peiiilc~ urethra \vas pi-esciit (abseiice of hypospadias ) . The
priinitive preputial frcnuluin was iiarrow (fig. 20).
' h e effects of castration upon the prostatic utricle were
largely prevented. The utricles of the experimental fetuses
i n group D were smaller than those of fetuscs which had been
castrated for a similar period of time and which had not
heeii given ancirogeii (figs. 11 and 1 2 ) . The utriclrs were,
however, somewhat larger than those of the unoperated littermates of the same age (table 2 ) . At the Jliillerian tuhercle
there ~ v a sa lumen i n tlie utricle of each fetus in group I>;
the tubcrcle and associated ducts were
ciitially lilrc those
illustrated in f i g u i ~14.
1)ISC'IJRSTOS
The genital ahriormalities in the castrated fetuses jncludpd
a reduction of the number of prostatic buds, absence of tlie
coagulating glands, iiicoiiiplete formation of the floor of the
penile urethra (hypospadias) and eiilargenient of the pi*ostatic utricle. The first threc of these abnormalities could lie
prevented by means of testosterone propionate, and the 4th
largely prevented.
Should these observations be regarded as evidence that in
normal development the testicular androgen is a causative
factor in the formation of the primordia of the male accessory reproductive organs? We would answer in the negative
despite the absence of the coagulating glands in 4 of the
castrated fetuses and despite the prevention of this anomaly
l ~ piniplanted testosterone propionate ; such observations on
the coagulating glands, though intriiisically sound, must he
halanced against the current lack of demonstration that the
fetal testis produces testosterone propionate. Also, a negRtive aiiswer is suggested by the existence of prostatic buds
122
1,. J. WELLS, ill. C A V A N A U G E AND E. L. MAXWELL
in castrated fetuses in which tlie operation had lxen performed
prior to the time of expectation of buds.
A second question is mliethei~the observations should bc
regarded as evidence that in normal development the testicular androgen is an importaiit factor in tlie retrogression of
the ~ l u l l c r i a nducts in the male. IVc have arrived at a negative answer. All castrated fctuses of group A showed a lack
of persistence of those olilique and horizontal segments of
the right and left ducts which must have existed at the time
these fetuses were subjected to castration (jig. 1C ) . Also,
the enlargement of the prostatic utricle aiid the prevention
of this effect by testosterone propionate must be considered
in the light of what was found iii the case of the ejaculatory
ducts. I n most of tlir cxperimental fetuses of‘ groups A and
R, the ejaculatory ducts were dilated. Such dilatation could
be prevented hy testosterone propionate.
Although a satisfactory cxplanation of the enlargement of
the piaostatic utricle and of the s i n d t a n e o u s dilatation of
the ejaculatory ducts is iiot a p p a r m t , clues are afforded in
the fact that distally these structures lacked lumciis and that
in two illstarices the seminal vcsicles \vei*e somewhat dilated.
It occurs to us that the utricles aiid ejaculatory ducts, as a
consquence of lack of pa teiicy distally, might have been expandcd a t some time during the experiinental period by an
abnorrnal accumulation of fluid in their cavities. If so, it is
reasonaldr to suppose that the testosterone propionate prevented such expansion hy keeping open, in some indirect
manner, the 0rifict.s of the utricles and ejaculatory ducts.
The observed hypospadias, explained ernlryologicall~a s
a n abiiorrnal persistence of the enihryonic urethral groove, is
undoubtedly a consequence of the orchiectomy. On the other
hand, it may not he due solely to the experimental deprivation of testicular androgeii. Its prevention by testosterone
pi-opionate does riot entirely iwle out the possibility that some
lion-spcific “physiological insult ” during the experimental
surg:.ci.j~might hare acted as a coiitrihtiiiy factor. Other
workcrs have found that hypospadias and other genital anomn-
C H A N G E S I N OBCHIECTOMIZED FETGSES
123
lies may be produced experimentally in the male ofYspring
of r a t s by feeding the gravid female a diet deficient in vitamin
A (Wilson and Warkany, '48).
Our data support the view that in rats testicular androgen
is produced before birth and that it stimulates the prenatal
growth of the male accessory reproductive organs (Wells,
'50b; Wells and Fralick, '51; see Maxwell and TTells, '51).
This view, incidentally, is also supported by the fact that
when the fetal testis is grafted into the seminal vesicle of an
adult castrated host it is capable of producing androgen at
once (Jost, '48 ; Moore, '53).
The reduced number of prostatic buds in our series of
castrated fetal rats is at variance with certain ohservatioris
in yourig opossums (Xoore, '50a, '50h). After the removal
of the testes of an opossum on the 20th day of life the existing
prostatic buds (about a half-dozen) continue t o grow, new
buds are rapidly added and the buds undergo extensive
1)ranching; u p to the 100th day the prostate of a castrated
opossum is similar to that of a normal control. The present
authors do not know why there should have been such a
discrepancy in the results obtained in these two species of
mamnials, r a t and opossum (placental mammal vs. marsupial).
On the other hand, the lack of persistence of the Miillerian
ducts in our castrated fetuses and also the actual formation
of prostatic buds during the experimental period a r e in
keeping with similar observations in the castrated opossunis
(11oore, '50b).
Absence of the coagulating glands and the presence of hypospadias in our r a t s a r e anomalies like those found in fetal
mice in which the testes were destroyed by irradiation (Raynaud and Frilley, '48; Raynaud, 'SO). This castration by
irradiation was performed on the 13th day after conception,
and the fetal mice were killcd o n the 18th day. The ahnormalities, more extensive than those in our specimens, included
absence of the seniinal vesicles and prostate and, incidentally,
the presence of a n cpithcllial cord derived from the Miillerian
ducts ("cordon vaginal"). Rayiiaud ('50) states that the
genital tubercle of a castrated mouse was structurally similar
to that of a normal female control. The authors did iiot present a record of having prevented these effects by means of
introduced atidrogeii.
The coiiiplcs of abnormalities iioted in our material is less
extensi\Te than that found iii fetal rabbits (Jrost, '30). In
rabbit fetuses castrated 0x1 the 19th to 21st day of life and
killed oil tlie 28th day, ,Tost reported absence of the prostate
arid the difierentiation of thc Jliillcrian ducts into a vagina
(prostatic utricle ?) and into certain segments of uterine horns
and tubes. Such effects i n rabbit fetuses were piwwited by
implanted testosterone propionate.
Tn our ~ v o r k the
, forniation of prostatic buds in tlie absence
of testes is in harmony n.ith the esisteiice of "feniale prostates" in adult feniale rats. Prostates occur spontaneously
i n a high percentage of the females in certain selected stiaains
of inl)red r a t s (Price, '44).
It is clear that the enlargement of the prost,atic utricle in
the castrated fetal r a t is attributalile to the castration. It is
probleniatical, however, i ~ ~ h e t l i ethis
r
change is due to the
experimental deprivation of testicular androgen, a s was implied earlier i n the discussioii (paragraphs 3 aiid 4). This
over-all interpretation is supported by the fact that in the
rhesus monkey, for example, the introduction of androgen
into the circulation of a gravid female fails t o cause i n a
female fetus any retrogression of the lliillerian ducts and
derivatives but does produce a high degree of intersexuality
(Wells aiid van Wagenen, i n press). Our over-all interpretation was formulated in spite of certain observations in chick
embryos and in a single rabbit fetus. In a female chick ernbryo in which a developing testicle has l)een grafted into the
peritoneal cavity, the graft may hring about a complete retrogression of the Miillerian ducts of the host (Wolff, '50).
Similarly, when a n ernbryonic testis is grafted onto the soniatopleure (a membrane wliich later participates in the formation
of the chorioallaiitoic niem1)rane) of a female chick embryo,
the graft may cause a complete disappearance of the 1zliil-
lerian ducts (Huijbers, ’51). Also, in a single case of N fem;tlc
rabbit fetus in which a testicle had been grafted onto the
developing iiiesosalpirix, it was fouiid that a sclgmeiit of the
Miillerian duct was iiiissing on that side of the body (,Tost,
,r,O).
In closing the discussion, our data 1)riiig into shar1) focus
tlic question of whether in nornial niale fetuses thc testicular
aridrogcii is produced early enough to, and at such early
stages i n suficiciit quantity to, induce the formation of the
primordia of “niale orgaIls” and t o cause the nornial retrogression of the bliilleriaii ducts. We think this is an open
quest ion.
SI’MMARY ANT) (“ONCIiT7SIOhTS
l’etal r a t s so young that they still lacked 1)i.ostatic buds
and coagulating glands and so young that they still had Iong
segnimts of the 3Iiillerian ducts, were subjected to castration
and then permitted to live until near temi.
It w a s found that pi-ostatic buds had appwwed during the
experimental period hut that the numhei- of l ~ u d swas significantly smaller than the number in controls of the same age
and litter. Thc coagulating glaiids were soinetinies ahsent.
The fjoor of the penile urethra was usually niissing (hypospadias). A11 portions of the Niillerian ducts except the
prostatic utriclc had disappeared. The utricle was relatively
large.
These effects of castration could be pi-evented, or largely
prevented, by means of iinplarited testostei-one propioilate.
Our ohservatioiis support the view that in r a t s testicular
androgen is produced before birth and that it stimulates the
prenatal g ~ o w t hof tlic male accessory 1-eproductive organs.
These observations a r e inadequate to determine, however,
I t would be beyond the scope of the present paper t o undeitalte n review of
t h e vast literature 011 the role of liornioiies in sex differentiation in mammals.
Tlrr wliolc subjeet w a s consideircl a t a recent Colloquiuiii ; the I’roreedinys of
this syinposium appeared serially in 1950 (Arch. (1’Aiiat. iiiier. Xorph. esp., 39),
and the papers were assembled under o i i ~cover in 1931 (La Differenciation S e v u e l l ~
cliez les VBrtCbres, Centre National de la. Rrcherclie Scientifiqur, P‘niis i ).
126
L. J. WELLS, XI. CAVAXAUGH AXTI E. L. i\lAS\\’ELL
whether androgen from the fetal testis acts a s N causativc
factor in the formation of the priinoi.dia of these organs and
in the iiornial retrogression of the lZliillerian ducts.
LITERATTJRE CTTEI)
HLJIJBERS,
M. M.
1951 I)e inilord van de gonade np de ontwikkcliiig x a i i llet
geslachtsapparaat b i j het kippenembr) o. Thebis, Amsterdam. A h stracted in English in 1952: Exccrpta l\ledica, G (sect. 1) : 31-33.
J o w , A. 1948 Activitk a n d l o g h e dn testicule foetal de rat gieffk SUT l’adultr
castri.. Compt. rend. Soe. de Riol., 143: 196-198.
19.50 Sur le eontr6lr hormonal de la, diffkrenciation sexuclle d u 1:ii)in.
drrli. d’Aiiat. inicr. et de Morph. eup., 39: 577-607.
~ T A X T \ E I , I , E. L., AND L. J. TVTLLS 1951 Effects o f graftct3 testes n1mn the
iepiodnctive organs of castrated fetal iats. Anat. Rec., 1 0 9 : 378 ( a b st1act).
PRICE,
I)O:IOTIIY
194.2 The ieartivity of the prostat? gland of the feiiialr rat in
relation t o age. Am. J. Anat., 7 5 : 207-931.
R A Y X ; \ C ~ D , A. 1950 Rec~heichcs exp6rimentsles sin l e d6\ eloppenirnt de l’ap
pareil g6iiital et lr fonctionriement des glandes endocrines des foetus
de souris et de mulot. h ~ h d. ’ 8 n a t . iriici. et d e Morlih. exp., 3 9 :
318-5 76.
R I Y N ~ ~A.,
D , A N D Jf. FRILLEY
1948 Effrt, sur le d6ieloppcment deq divcrscs
glande? iinnexes du tractus g6nit:il rles emhryons tle Souris, tle la dr-truetion, au nioycii des rayons X, d ~ glandes
s
gknitnles des embr:, ons.
Coinpt. rend. 8oc. d e Biol., Zg?: 424-486.
hfOoRE, c‘. I;. 1939 Biologv of the Testes. Sex and Internal lieeretion?. Ed. by
E. Allen, C. H. Danforth and E. A. Doisp. \Villiains and Wilkiiis Co.,
Kaltinioie. Chap. VIT, 353-431.
195Oa Sturlics on sex hormones aiid sexual diffcientiation in inamnials. Arch. d’Anat. niirr. Xorph. rxp., 39: 484-4!38.
- l930b
The iole of the fetal indocrine glands in dexelolment. J.
Clin. Endocrinol., 10 : 942-985.
__-__
1953 Adrenal cortical secretions in relation to t h r reprotlurtive
sFstcrn of rats. J. C’liii. Endocrinol. :ind Metnbol., 13’: 330-368.
WELLS,L. J . 1950a Subjection of fetal rats to surgery and repeated subcutaneous injertions: mctliod and survival. Anat. Rec., 208: 309-331.
_
_ l930h
~ Hormones and srxual clevelopment in placental mainiiials.
Aich. d’Anat. d, niicr. Morph. exp., 3<9: 499-517.
WELLS,L. J., 31. CAVANAUGH
A ~ D
B. L. ~ ~ Z B X W T P L L 1959 1)~relopnientof the
reproductive organs in castrated fctal rats: prostate, coagulating glands,
genital papilla anti prostatic utricle. Anat. Rec., 113: 467 (abstract).
WELLS, L. J., A N D R. L. FRALICK
1932 Prodnction of androgen h: t h e testes
of fetal rats. Am. J. Anat., 8.9: 63-107.
WEI,I,S.L. J., ~ N D
G. TAN WVGENFSA4ndrogen-i~iducec~
female pseudohrrmaphroditisin in the monkey ( M a m r a mulattff) : anatomy o f the ieproiluctive
organs. Contiih. t o Enibiyol., Carnegie Tnit., Wash., 9 . 7 : ( i n press).
OIIAN(;ES 1K OItCtilECTOMIZED FETUSES
127
WIr.sox, J. G., KND J. WARKANY1940 hlulformations in the genito-urinary
tract induced by matcrnal ritainin A dcficieucy in the rut. Am. J.
Anat., 83: 357-395.
WOLFF, E. 1950 Le r6le des hormones emhryonaires dans la diffbrcneiation
sexut!lle tlcs oisc:iiix. Arch. d'Annt. micr. Morph. exp., 89: 427-450.
+
x
+
75.
+ 2 11 and killed a t 21 tl + 15 11.
4 Fetus castrated a t 18 d
2 11, given n pellet of androgen at 18 d
62 buds (66 buds in an unoperated litter-mate of the same age).
+
Litter 967. Thcrc were
Fetus castrated a t the age of 18 (1 3 11 :cntl killed :it the age of 2 1 d
15 11. Litter 966. T l ~ e r cwere 9 prostt:itic buds,
all shown in the drawing. The coagiilating glands were absent, a s illustrated. X i 5 .
3
Litter 966. There weie 61 prostatic buds, some of which are hidden in the drawing.
Unoperated control of 21 d
x 75.
2
+ 15 h.
The drawings depict the uretlir;r (t')and prostatic buds as they appeared, when vievet1 f l om above, in transparent models
which were prepared by stippling onto sheets of cellulose acetate the configuration of the epithelium (see text). The orientation is the same as that shown in figure 14, i.e., dorsal aspect down and right aspect on the observer's right. A t the
Mtillerian tubercle ( M ) , the prostatic utricle and tlie ejaculatory ducts were not modeled. C, coagulating gland.
ESPL.lNATION OF FIGUXES
PLATE 1
I.. if.
WILGB, X. CAVAYAUBII A X 0 E. Is YAXWEIaL
CHANGES IN ORCHIECTOXIZEI) FETUSES
PLATE 1
11.
+2
+ 13 h.
Littei 1346. X 60.
+
+2
11,
gireii andlogen a t 18 d
+ 2 11 and killed
at 21 d
+ 15 11.
Litter 967. ('ongulating glands
+
4 11 and killed at 2 1 d
+
+ 13h.
Litter 967. Photo suppleniciits
15 h. Litter 953. Left ejaculatory duct solid, right duct almost
2 11, gircn andlogen a t 18 d X 2 h and killctl a t 2 1 d
12 Fetus eastiated a t 18 d
figure 8 bj shoi\ing addition.il 1)rostatic buds (see fig. 4). X 60.
11 Fctnq c : i c t i a t c d a t 18 (1
solid. X 60.
+
Litter 953. White arrow, epithcliuin of the prostatic utiicle. El:ick airow, outer
ixirgin of utiicle. C a ~ i t jof utricle not shown i n the section presented. X 60.
Xoiniai ni:ilc of 18 ci
+ 2 11. i i
of 21 d + 15 11.
Fetns castiated a t 18 d
piesent. X 60.
Fetus castiated a t 18 d
3 11 and killed at 2 1 d
1 3 h. Littei 966. Prostatic utriele large. Ejaculator: ducts dilated.
Voagu1:iting glands niiwng (cl~itlieliunimissing). Arroirs indicate t i 7 o ni:isses of cells in a region n h e i e the coagulating
gl'lllds llllgllt be expected ( s e e text). x GO.
+
Unopeiated eontiol of 2 1 tl
Litter 9GG (cf. fig. 2). Piostatic utricle not slionn since it mas below the level of tlie
transvcise section pwsented ( w e figs. 10 n i i d 1 G I . Coagulating g1:inds piesent (black o b ~ e c t sin top third of photo).
X GO.
10 Unopeiated coutiol
:I
8
7
G
5 h'oinial malt fetus of 18 d
The prost:itic utiicle appears in e:icli illustration except figure 6 (near center of photo). Right and left ejaculatory ducts
in 5 of tlie photos (figs. 7 arid 9-12'); right duct in three (figs. 2, 6 and 8 ) . Left d u c ~ u sdeferens and tlie duct of the
left seminal iesicle iii figures G aiid 8.
Each section is oriented in such manner that the dorsal aspect is down (note rectum) and the right aspect on the observer's right. U i e t n s and wall of bladder in figures 5 and 9. Urethia in 6 of the illustrations (up-figs.
6-8 and 10-12).
Eight tr:insversc sections of the pel1 ic region of several fetuses piesented f o r the purpose of illustrating the prostatic
utricle, ejaculatorj d u r t s , coagulating glands aiid prostatic buds.
ESPLANATIOK O F FIGURES
PLATE 2
L.
J. W E L L S , 11 C A T A B A U G H A N D E L. MAXWELL
C’HANGES I N OKCHIECT0311ZLI) FETUSES
PLATE 2
+
:ciitl
+
light :tspect
011
+
+
Uiioperatetl control obtained a t eiid of experilneiit, 21 d
15 11. Litter 941. Prost:ctic urethra, ~ i ~ ~ r i i c ~
1)rostatic
nus
buds,
R.lulleriaii tuhcrcle, bilateral teriiiinatioii of prostatic. utriclc (nimketl by two black ink dots) :riicl terniiiiatiou of tlic
ejaculatory ducts (sites about ti 111111 helow ink dots). X 50.
+
Norlnal fetus of 18 (1
2 h, obtained a t I)cgi~iiiii~g
of exl~c~rinient.Litter 041. Urethra i n ob!iquc scc.tioii. Alwmce of
prostatic buds. Arrow illarks approxii1i;ltc site where a single but1 \zws fouiid in aiiotlier fetus (iiornial fetus of 18 (1 4 11,
table 1). White blister on the right aspect of the Miillerinii tuhwcle is the prcstatic utricle (cf. fig. 1, drawing c). X 50.
0iiriit:itioii : t1ors:il aspect do~vii (notr rcctulii)
Noriiial fetus of 19 cl + 1 2 h. l’rost:itic urctlirn
the Miillerian tulicrrlc. x 60.
aiid
biids ( I 9 I)uds counted in the scri:rl sections), :1ud
ii
portioii of
+
+
20
+
Fetus castrated :it 18 d
2 11, given aiidrogcii a t 18 d
frenulurii (obserier’s right). X 30.
+ 2h
and killed a t 2 1 d
+ 13 11.
Litter 967. Narrow I)reliutial
311 a n d killed at % I d
35h. Titter 966. Glniis penis, ~ i r e p ~ cniid
c ~ penile uretllrLi (latter
Fetus castr:itctl at 1 8 d
o n observer’s iiglit) . The section presented does not prss tlirougli tlie euistiiig ( a l ~ n o r i i i n l ~
l ~) e ~ s i s t i i i gurctlir:ll
)
gloove.
X 30.
+
Unoperatrd vniitrol of 21 d
15 11. Litter 96ti. Prepiitial g1:iiids (oliservcr’s l e f t ) , glans peuis :nit1 :rtt:iclied prepuce,
pcwile ni.ethra ;mtl a nxrroiv prrpritial frei~uliim (latter on observer’s right). x 30.
+
(Iiisrrteil srllaii piloto.) ?;ornial fetus of 18 tl
2 11. Litter 133.5. t i l a m l i e n i s :iiitl the prcpuw ai’e :itt:tclid to c a d i
other h y the haiailo-prel~utial1aniell:i. Note urethral I’latc (~iiedimisolid epithelium). x 20.
+
(Large photo.) Normal fetus of 18 d
2 11. Litter 1355. Astcxrisk indic*ates left ]ircpntial gl:iiitl. I’lioto :~Isoslioivs
tlie cavernous hntlirs of the ~)ciiisaiitl tlic peuile iiretlir:~ (1:itter srctioiic~ddorsal t o tlic region of tlic uret1ir:il groove
- groove not illustrated). X 3 0 .
19
18
1i
17
Fiw sections of the de\clopiug penis (pliotos oht:iiiicd froiii tralimcwe set-tioiis of 5 fetuses). 0rieiit:itiou : clorsuiri of
penis oil obser\er ’s left; left aspect do\\n.
lti
15 Fetus castrated a t 18 d
7 11 aiid killed :it 2 1 (1
15 11. Litter 941. Prostatic uretlirn, :I sii~alliiunihrr of prostatic
buds (total of 7 hurls in iuodcl) , 1i:’ostatic. utricle (iiiarkcd by tlie two white ink (lots) : ~ i i i le j a c i h t o r y ducts (absence
of lumen in right duct). x 50.
14
13
Four trausvcrse wctioiis of the p e l 1 ic region.
obserler ’s right.
EXPLANATION OF FIGURES
PLATE 3
L. J . WELLS. M CAT.4NAUUH AND E. L. MAXWELL
CTI-IaNOES I N ORCHIECTOMIZED E’ETUYES