/. Embryo/, exp. Morph. Vol. 36, l,pp. 87-99, 1976
Printed in Great Britain
87
Effects of testosterone implants in pregnant
ewes on their female offspring
By I. J. CLARKE, 1 R. J. SCARAMUZZI 2 AND R. V. SHORT 1
From the M.R.C. Unit of Reproductive Biology, Edinburgh
SUMMARY
Pregnant ewes were implanted with 1 g testosterone between days 30-80, 50-100, 70-120
or 90-140 of gestation. Ewes treated between days 30-80 and 50-100 showed increased
aggressive behaviour and clitoral enlargement, whereas this was not seen in the day 70-120
or 90-140 groups. Although the implants released similar amounts of testosterone at all
stages of gestation, plasma testosterone concentrations were lower in the day 70-120 and
90-140 groups. The increased testosterone clearance in late gestation could be a result of
increased placental aromatization. Masculinization of the genital tubercle was complete in
female lambs in the day 30-80 group, partial in the day 50-100 and 70-120 groups, and
absent in the day 90-140 group. These results therefore suggest that the 'critical period' for
masculinization of the external genitalia is between days 40 and 50 of foetal life. In contrast
to this anatomical masculinization, one aspect of behavioural masculinization had a later
critical period, since female lambs of 30-80, 50-100 and 70-120 day groups adopted male-like
urination postures, whereas the day 90-140 offspring showed the normal female pattern.
INTRODUCTION
The first suggestion that female foetuses might be masculinized by testicular
hormones was provided by Lillie's classical study of the bovine freemartin
(Lillie, 1916,1917). Pfieflfer (1936) demonstrated a similar effect by transplanting
testicular tissue to neonatal female rats. Later studies revealed that a single
neonatal injection of testosterone propionate would masculinize female rats.
Since this time the androgen-sterilized rat has been the subject of many detailed
investigations (Harris, 1964; Barraclough, 1968; Harris, 1970; Beach, 1974;
Brown-Grant, 1974; Davidson, 1974). Androgenization of the female foetus or
neonate has also been studied in the guinea-pig (Barraclough & Gorski, 1961;
Goy, Bridson & Young, 1964), hamster (Paup, Coniglio & Clemens, 1972;
Swanson & Brayshaw, 1973), mouse (Manning & McGill, 1974), dog (Beach &
Kuehn, 1970; Beach, 1975), sheep (Short, 1974), cow (Jost, Chodkiewicz &
Mauleon, 1963), monkey (Goy, 1970) and man (Money & Erhardt, 1972).
Androgenized sheep have a number of advantages for experimental studies.
1
Authors' address: M.R.C. Unit of Reproductive Biology, 2 Forrest Road, Edinburgh
EH1 2QW, U.K.
2
Author's address: C.S.I.R.O. Division of Animal Research Laboratory, Prospect,
P.O. Box 239, Blacktown, New South Wales, Australia.
88
I. J. CLARKE, R. J. SCARAMUZZI AND R. V. SHORT
They are large enough to permit serial blood sampling over long periods of time;
this is essential if one is to investigate pituitary gonadotrophin secretion and
gonadal feedback mechanisms. They are also ideal for observing the behavioural
consequences of androgenization, since the social and sexual behaviour of
sheep reveals a clear-cut dimorphism between the two sexes. The purpose of the
present investigation has been to document as fully as possible the anatomical
and behavioural changes in newborn female sheep exposed to testosterone at
different stages of intrauterine life, as a prelude to subsequent studies on their
endocrinology and reproductive behaviour in adulthood.
MATERIALS AND METHODS
During the 1973 mating season the oestrous cycles of twenty-four Finnish
Landrace x Dorset Horn ewes were synchronized with progestagen-impregnated
intravaginal sponges (Synchromate; G. D. Searle & Co., High Wycombe,
Bucks, England) containing 30 mg fluorogestone acetate (SC 9880; 17a-acetoxy9a-fluoro-l l/?-hydroxypregn-4-ene-3:20 dione). Mating to Finn-Dorset rams
took place at the second oestrus following sponge withdrawal. The ewes were
then randomly divided into three groups of eight. Implants of 1 g (1-010 ±S.E.
0-003) testosterone (Organon Laboratories Ltd., Newhouse, Lanarkshire,
Scotland) were placed subcutaneously in the neck of two groups of ewes, using
aseptic procedures under local Xylocaine anaesthesia. One group was implanted
on day 30 (range 29-30) of gestation and the implants were removed on day 80
(range 80-81); another group received implants on day 50 (range 48-51) which
were removed on day 100 (range 98-101). The third group of ewes served as
controls and were not implanted. One ewe in the control group died, and one
animal in the D3O-8O group was not pregnant. During April 1974 the remaining
22 ewes produced 54 lambs (24 male, 30 female) over a ten-day period. Large
litters were reduced to two lambs and the surplus animals were reared artificially.
All the females were retained as experimental subjects. Six males were kept as
controls, and were bilaterally vasectomized at 8 months of age.
During the 1974 mating seasons 1 g testosterone was implanted into two
further groups of six pregnant ewes on days 70 or 90 of gestation and the implants were removed on days 120 or 140 respectively. Oestrus had not been
synchronized in these ewes but they all lambed in February 1975 over a fourweek period, giving 21 lambs (9 male, 12 female). All the females were kept and
the males were discarded. Table 1 summarizes these results.
Blood samples were taken from the jugular veins of all the pregnant sheep at
the time of implant removal, and plasma testosterone levels were measured by
the technique of Corker & Davidson (1976). Single samples were taken from the
ewes lambing in 1974 and three half-hourly samples were taken from those
lambing in 1975.
The size of the clitoris in the implanted ewes was subjectively assessed on a
Testosterone
implants in pregnant
89
ewes
Table 1. Effects of subcutaneous testosterone implants in pregnant
ewes on length of gestation and number of lambs born
Experimental
group
Control
D3O-8O
D50-100
D70-120
D90-140
Year
of
mating
Number
of ewes
lambing
Length of
pregnancy
(days)
M ± S.E.M.tt
Male
Female
Number
of cases of
intrauterine
death* %
1973
1973
1973
1974
1974
Total
7
7
8
6
6
32
142-8 ±0-85
143-3 ±0-47
143-5 ±0-42
143-2 ±0-58
141-6±0-51
143-0 + 0-27
5
9
11
6
3
34
10
9
10
6
6
41
0
1
1
2
4
8
Number of
lambs born
K
* Group difference, x* = 9-319; 005 < P < 010.
f Non-significant group difference.
% Non-significant difference between years.
0-5 scale at the time of implant removal and again just prior to parturition.
After removal, the testosterone implants were dried and weighed to determine
the amount of testosterone released over the implantation period, and hence it
was possible to estimate the daily release rate.
The appearance of the external genitalia of all the lambs was recorded at
birth, and the size of the clitoris assessed at 10 months of age. The penile
diameters of the female offspring showing complete masculinization were compared with those of normal male controls at 10 months of age, using callipers to
obtain three measurements immediately anterior to the scrotum.
The D30-80, D50-100, D70-120 and D90-140 and control lambs were run
together after weaning (12 weeks of age) and their urination behaviour was
observed under field conditions.
The internal genitalia of the D3O-8O, D50-100 and control lambs were
examined at 12-18 months of age by laparotomy under nembutal anaesthesia.
Two sheep from each of the D30-80 and D50-100 groups were killed during the
course of study. Post-mortems were carried out immediately following death,
and ovaries, accessory glands, uterus and vagina were fixed in 10 % formal
saline, sectioned at 6 /im and stained with haematoxylin and eosin.
RESULTS
The effects of testosterone on the implanted pregnant ewes
Following implantation the D3O-8O and the D50-100 ewes showed increased
aggressive behaviour. This was not shown by the control ewes or those implanted
later in gestation. No objective methods were used to quantitate this behaviour.
The implanted ewes all lambed after a normal gestation of approximately
143 days and no dystokia was encountered. There was some intrauterine death,
90
I. J. CLARKE, R. J. SCARAMUZZI AND R. V. SHORT
Table 2. Plasma testosterone levels and daily rate of release in ewes
implanted at different stages of gestation
Experimental
group
Year of
mating
Control
D3O-8O
D50-100
D70-120
D90-140
1973
1973
1973
1974
1974
Plasma testosterone
concentration (ng/ml)
M + S.E.M.*J
Rate of release of
testosterone (mg/day)
M±S.E.M.f§
< 01
ll-4±2-59
7-8 ±1-84
4-2 ±0-49
3-8 ±0-52
6-9 ±0-45
71 ±0-48
7-6 ±0-69
70 ±0-44
* Significant group difference, P < 005.
t Non-significant group difference.
% Significant year difference, P < 005.
§ Non-significant year difference.
Table 3. Clitoral hypertrophy in pregnant ewes 50 days after
subcutaneous implantation of testosterone
Size of clitoris (0-5 range)*
Period of
implantation
(days of
gestation)
0 (control)
D3O-8O
D50-100
D70-120
D90-140
At implant removal
At lambing
A
Mean
Range
Mean
Range
0
3-4
3-5
0-4
0
0
d-5)
(1-5)
(0-1)
0
0
0
0
0
0
0
0
0
0
0
* 0 = normal; 5 = marked hypertrophy.
and although this was higher in the groups implanted later in gestation (Table 1),
the group differences did not achieve significance (0-05 < P < 010; Table 1).
There was no difference in the number of dead lambs born during 1974 or 1975.
All the ewes lactated, although two D90-140 mothers did not produce enough
milk to feed even a single lamb, so supplementary feeding was necessary.
Testosterone concentrations in the jugular venous plasma of the ewes at the
time of implant removal are given in Table 2, together with the estimated daily
release rate. The variability was greater in the single samples collected in 1974,
and the three half-hourly samples taken in 1975 reduced the standard errors
considerably (Table 2). The D3O-8O ewes had significantly (P < 0-05) higher
testosterone levels than the D70-120 and the D90-140 animals. Correlation of
plasma testosterone levels with time of implant removal was significant
(r = - 0-526; P < 0-05). The daily release rate of testosterone was similar in all
Testosterone implants in pregnant ewes
91
Table 4. The effects of subcutaneous implantation of testosterone in pregnant
ewes on the external genitalia of their genotypically female lambs
Period of exposure
to testosterone
(days of pregnancy)
Size of clitoris
(0-5 range)*
Control
D3O-8O
D50-100
D70-120
D90-140
0,0,0,0,0,0,0,0
Penis
5,5,5,5,5,5,5,5,5
2,3,1,1,0,0
0,0,0,0,0
Vaginal opening
Normal
Absent
Very constricted in all cases
Constricted in all cases
Normal
* 0 = normal; 5 = marked hypertrophy.
groups. The plasma testosterone levels in the control ewes were below the limit
of sensitivity of the assay (< 0-1 ng/ml).
Subjective assessment of clitoral size in pregnant ewes at implant removal are
given in Table 3. Enlargement was greater in the D3O-8O and D50-100 groups
than in the D70-120 and D90-140 groups. The hypertrophy diminished rapidly
following withdrawal of the implants and the clitoral size had returned to normal
by the time of parturition.
External genitalia of androgenized female lambs
The genetically female lambs could be identified at birth by the fact that
regardless of the extent of masculinization of the external genitalia, the ovaries
remained in their normal intra-abdominal position; thus the scrotum of the
masculinized animals was always empty, whereas the normal males had palpable
testes present in the scrotum from birth onwards.
Females exposed to testosterone between days 30-80 of gestation showed
complete masculinization of the external genitalia; they were born with a penis
and an empty scrotal sac (Fig. 1C). The processus urethrae appeared similar to
that of the normal male, but the prepuce could not be drawn back from the
galea capitis, and the preputial opening appeared abnormal (Fig. 1C and D).
The penile diameters of the D3O-8O females (0-77 ±0-10 cm; M ± S . E . M . ) were
significantly smaller (P < 0-01) than the male controls (1-20±0-10cm; M ±
S.E.M.) when measured at 10 months of age.
The external genitalia of the androgenized females are compared with normal
female controls at 10 months of age in Table 4. In the normal ewe the clitoris
is short and lies in the fossa clitoridis, surrounded by the labia (Fig. 1 A). The
D50-100 females showed marked elongation and fusion of the labia within
which the enlarged clitoris could be palpated, although it could not be extruded.
In addition to the small vaginal opening, these ewes displayed a second opening
at the tip of the fused labia (Fig. 1 B). The D70-120 animals showed less marked
92
I. J. CLARKE, R. J. SCARAMUZZI AND R. V. SHORT
Testosterone implants in pregnant ewes
93
Table 5. Urination patterns of androgenized female lambs at
12 weeks of age
Experimental
group
n
Voiding
patterns
Female controls
Male controls
D3O-8O
D50-100
D70-120
D90-140
8
6
8
9
6
5
Continuous
Pulsatile
Pulsatile
Pulsatile
Pulsatile
Continuous
Type of
urination
posture
Female
Male
Male
Male
Male
Female
external masculinization, and the external genitalia of the D90-140 group did not
differ from the normal females (Table 4).
Internal genitalia of androgenized female lambs
Laparotomy revealed that all the androgenized ewes possessed ovaries. The
uteri and vaginae of the D3O-8O ewes were markedly distended with fluid but
this was not so in the other experimental groups. One D50-100 ewe had a congenitally absent left uterine horn, with a fallopian tube that was occluded and
distended with fluid.
Two D30-80 animals were killed at 10 months of age, during their first mating
season. A single corpus luteum was present in the ovary of one animal, and it
appeared functional when examined histologically; the uterine and vaginal
histology was also characteristic of the luteal phase of the cycle (McKenzie &
Terrill, 1937; Robinson, 1959). The other ewe showed a heavily luteinized
follicle in one ovary and no corpora lutea. The uterus was grossly distended; the
endometrium was attenuated, with sparse glandular tissue. Normal follicles and
oocytes were identified in the ovaries of both animals. In both sheep the caudal
part of the vagina merged into the base of the penis, and at this point histologically recognizable bulbo-urethral glands were present. Small seminal
vesicles were also located on the lateral vaginal walls of both sheep.
FIGURE 1
The external genitalia of androgenized female sheep compared to those of the
normal ewe and ram at 6 months of age.
(A) The perineal area of the normal female showing the anus (a), vaginal opening
(b), vulva (c) and ventral commissure (d).
(B) The perineal area of a D50-100 female showing a constricted vaginal opening (e)
and fused labia (/).
(C) Ventral view of a D3O-8O female showing an empty scrotum (;) and abnormal
preputial opening (fi).
(D) Ventral view of a normal ram showing a well developed scrotum containing
testes (/) and a normal preputial opening (g).
94
I. J. CLARKE, R. J. SCARAMUZZI AND R. V. SHORT
Two D50-100 ewes were killed during the summer anoestrous period of 1975.
The ovaries of both animals were small and inactive, although follicles and
oocytes were present. The uteri were histologically typical of anoestrus (McKenzie & Terrill, 1937); neither bulbo-urethral glands nor seminal vesicles were
evident.
Urination behaviour of androgenized female lambs
Sheep show a distinct sexual dimorphism in their pattern of urination (Short,
1974). Whereas the female normally arches her back and crouches with her hind
legs whilst voiding urine in a continuous stream, the male stands normally
whilst voiding urine in pulsatile jets. Table 5 shows the urination behaviour of
the androgenized female sheep. All those animals with either partial or complete
masculinization of their external genitalia (D3O-8O, D50-100, D70-120) showed
a male urination pattern, whereas the D90-140 females showed a female
pattern.
DISCUSSION
There were strong indications that the effects of testosterone on the pregnant
ewes were less noticeable when implants were inserted during the later stages of
gestation. For example, ewes implanted on day 30 or 50 developed marked
aggressive behaviour, whereas those implanted on day 70 or 90 showed none;
maternal clitoral hypertrophy was also most marked in the day 30 and 50 animals,
less marked in the day 70 ones, and totally absent in the day 90 group. There
was also a significant decline in the maternal plasma testosterone levels at later
gestational ages, although the estimated daily release rate of hormone from the
implant remained constant. This strongly suggests that the clearance rate of
testosterone from the maternal circulation increases with advancing gestational
age. It is known that the ovine foeto-placental unit is capable of metabolizing
testosterone to oestrogen (Findlay & Seamark, 1971), and urinary oestrogen
excretion increases dramatically in the ewe after the 70th day of gestation
(Fevre, Piton & Rombauts, 1965), suggesting an increase in placental aromatizing activity. Another factor may be the protective effect of progesterone.
Diamond & Young (1963) found that progesterone protected guinea-pigs
against the masculinizing action of testosterone propionate, and it is well known
that there is a marked rise in the peripheral plasma progesterone levels in ewes
after the 80th day of gestation (Bassett, Oxborrow, Smith & Thorburn, 1969).
These facts need to be taken into account when seeking to define apparent
'critical periods' in foetal development during which maternally administered
testosterone can exert a masculinizing effect.
Another limitation of the present study is that the experiments were carried
out over two different years, and so the results could be confounded by unknown seasonal effects. For example, it could be argued that the lower blood
testosterone levels in the day 70-120 and day 90-140 ewes was a year difference
and not due to the duration of gestation. Fortunately, the anatomical and
95
Testosterone implants in pregnant ewes
\ \ \ \ \ \ \ \ \ \ \ \
K \
\
0
4
Conception
20
60
\ \ \ \ \ \ \ \ V \ X\
\ \ \ \
X X X X X X
40
\j
x x x \ x \ x \ \ \ V \ \ \ \ \ l
X X X X X X X \ \
\ \ x
X X \
80
\
\ \ \ \ \ \ \ \ \ \ X J
100
120
140
Birth
Davs of ucstalion
Fig. 2. Periods during which pregnant ewes were implanted with testosterone.
§3, Short (1974); • , present study. Exposure of the female foetus to testosterone
from period 1 or 2 onwards caused complete external masculinization. Exposure
from period 3 onwards caused partial masculinization. Exposure during period 4
caused no masculinization. Period 2 appears to be the critical time for complete
masculinization of the external genitalia, and is the time when the external genitalia normally begin to develop in male lambs.
behavioural results obtained in both years of the present study are entirely
concordant with those obtained in a previous year by Short (1974), when
ewes were implanted on days 20, 40, 60 or 80 of gestation and the implants left
in until term (see Fig. 2). Therefore we feel that seasonal effects can be
discounted.
Short (1974) showed that ewes implanted with testosterone on day 20 or 40
of gestation gave birth to female lambs with completely masculinized external
genitalia, whereas ewes implanted on day 60 or 80 produced female lambs with
essentially female-type external genitalia. In the present investigation the implants were only left in the ewe for 50 days, and these two approaches complement one another (see Fig. 2); the day 30-80 lambs in the present study were
completely masculinized, whereas the day 50-100, 70-120 and 90-140 animals
has essentially female type genitalia. We can now begin to suggest a period of
development during which the genital tubercle of the female sheep foetus is most
sensitive to testosterone. If exposure begins in either periods 1 or 2 and continues
96
I. J. CLARKE, R. J. SCARAMUZZI AND R. V. SHORT
into period 3 then complete masculinization takes place. Exposure during
period 4 causes no masculinization. Thus the 'critical period' for masculinization of the genital tubercle appears to be between days 40-50 (period 2; Fig. 2).
However, androgen may continue to cause partial masculinization until day
80 of gestation (period 3; Fig. 2). Failure to induce masculinization after day 80
could be due either to the refractoriness of the target tissue, or to the failure of
testosterone to reach the foetus in sufficient quantities as a result of the increased
maternal clearance rate.
Green & Winters (1945) considered that the gonad of the sheep foetus first
began to differentiate into a testis or an ovary between days 25 and 29 of pregnancy, whereas Mauleon (1961) found that the gonads could first be sexed
histologically on day 35. Attal (1969) reported that testosterone could be
detected in the testes of male sheep foetuses from day 30 of pregnancy and that
the external genitalia begin to differentiate on day 45. These results are concordant with our findings that the external genitalia of the female sheep foetus
can be masculinized between days 40 to 50 of pregnancy.
Short (1974) showed that ewes implanted from days 20, 40, 60 or 80 of
gestation until term had difficulties at lambing and many of them failed to
lactate. Such problems were not encountered in the present experiment where
the implants were removed before parturition. Beach & Kuehn (1970) injected
bitches for 10 days during mid-pregnancy with large doses of testosterone propionate and also encountered foetal death and lactational failure. The estimated
daily dose of testosterone received by all the ewes in the present study was
approximately 0-1 mg/kg/day, which is at least an order of magnitude less than
that used to masculinize the females of other species (see Beach & Kuehn, 1970).
Inspection of the D30-80 offspring at 10 months of age revealed that the
prepuce was adhering to the galea capitis, and the penile diameters were
significantly smaller than in entire rams of a similar age. This is presumably due
to a difference in the degree of androgenization between normal males and
androgenized females. Ashdown (1957a, b) has shown that the growth of
the bull's penis and separation of the prepuce is dependent on post-natal
testosterone secretion.
The pattern of urination behaviour in sheep is sexually dimorphic, and can
be influenced by testosterone between days 20-80 of gestation (Short, 1974);
in the present study this behavioural pattern was not masculinized after day 90
(Table 5). Only ewes showing complete or partial masculinization of their
external genitalia voided urine in a pulsatile manner, and this could be a consequence of androgen-induced development of urethral musculature. However,
the different urination postures presumably reflect a more fundamental
influence on behaviour.
It appears that the nervous reflexes involved in urination are directly influenced
by testosterone, and that this behavioural system differs from those involved in
mating behaviour, since Short (1974) showed that an androgenized ewe with a
Testosterone implants in pregnant ewes
97
male urination pattern was nevertheless capable of showing normal female
oestrous behaviour. The urination posture is also sexually dimorphic in dogs,
and the bitch will show a male pattern if treated with testosterone during foetal
or early post-natal life (Berg, 1944; Martins & Valle, 1948; Beach, 1975).
Beach suggests that the sexual dimorphism of urination postures is predominantly
determined post-natally in the dog, but we have shown that the urination posture
of the sheep can be masculinized during early (day 20-80) foetal life.
In conclusion, we have shown that maternally administered testosterone is
capable of affecting various aspects of development of the female foetus in
different ways up until day 90. The spectrum of effects could be due to the
existence of 'critical periods' of development of the foetus, during which different systems pass through a stage of androgen sensitivity, but the results are
confused by apparent changes in the maternal rate of testosterone metabolism
with advancing gestation, and changing progesterone levels. It will be necessary
to measure the amount of testosterone actually reaching the foetus before the
existence of 'critical periods' can be unequivocally proven.
The authors gratefully acknowledge the help of the Animal Breeding Research Organisation's farm staff for the care and management of the sheep. We also thank Mr W. G. Davidson,
Mrs R. Cunningham, Mr L. McKenzie and Mr D. W. Davidson for technical assistance.
One of us (I. J.C.) is in receipt of a William Georgetti Scholarship.
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{Received 3 December, 1976; revised 18 March 1976)
Testosterone implants in pregnant ewes
99
Note added in proof. The concentrations of testosterone and dihydrotestosterone have recently been measured in ovine foetal gonads and plasma by
Pomerantz & Nalbandov (Proc. Soc. exp. Biol. N.Y. 149, 413-419, 1975).
The foetal testis contains more androgen than the foetal ovary during the first
half of gestation and plasma levels in male foetuses are significantly higher than
in females at this time. Testicular and plasma concentrations fall during the
second half of gestation and this is compatible with our observations that the
critical period during which testosterone causes masculinization is during
the first half of gestation.
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