Testosterone-Estradiol Filled Polydimethylsiloxane Subdermal Implants: Effect on Fertility and Masculine Sexual and Aggressive Behavior of Male Rats

Abstract

Simultaneous administration of specific doses of testosterone and estradiol via subdermal Silastic implants created a reversible azoospermia in male rats without increasing the seminal vesicle and ventral prostate weights or serum concentrations of testosterone and estradiol (Ewing et al., 1977). Herein we report that a single implantation of 2.5 cm testosterone and 0.1 cm estradiol subdermal polydimethylsiloxane (PDS, Silastic) capsules rendered male rats reversibly sterile for 8 months. The onset or cessation of sterility occurred abruptly 6 weeks following either the implantation or removal of steroid filled capsules, respectively. This reversible sterility was attained without any significant change in either masculine sex behavior or shock induced aggressive behavior.

Full text

BIOLOGY OF REPRODUCTION 21, 765-772 (1979)
765
Testosterone-Estradiol Filled Polyd imethylsi loxane Subdermal Implants:
Effect on Fertility and Masculine Sexual and
Aggressive Behavior of Male Rats
LARRY L. EWING,’ ROGER A. GORSKI,2 ROBERT J. SBORDONE,3
JULIA V. TYLER,4 CLAUDE DESJARDINS5 and BERNARD ROBAIRE6
Division of Reproductive Biology, Department of Population Dynamics,’
The Johns Hopkins School of Hygiene and Public Health,
Baltimore, Maryland 21205
Department of Anatomy,2 UCLA School of Medicine,
Los Angeles, California 90024
Department Psychiatry/Biobehavioral Sciences,3
Neuro psychiatric Institute, UCLA School of Medicine,
Los Angeles, California 90024
Department of Psychology,4 University of California,
Los Angeles, California 90024
Department of Zoology,5 University of Texas, Austin
Austin, Texas 78712
Department of Pharmacology and Therapeutics and Department of Obstetrics and Gynecology,6
McGill University, Montreal, Canada
ABSTRACT
Simultaneous administration of specific doses of testosterone and estradiol via subdermal
Silastic implants created a reversible azoospermia in male rats without increasing the seminal
vesicle and ventral prostate weights or serum concentrations of testosterone and estradiol (Ewing
et al., 1977), Herein we report that a single implantation of 2.5 cm testosterone and 0.1 cm estra-
diol subdermal polydimethylsioxane (PDS, Silastic) capsules rendered male rats reversibly sterile
for 8 months. The onset or cessation of sterility occurred abruptly 6 weeks following either the
implantation or removal of steroid filled capsules, respectively. This reversible sterility was attained
without any significant change in either masculine sex behavior or shock induced aressive be-
havior.
INTRODUCTION
Ludwig (1950) showed that low doses of
testosterone propionate decreased spermato-
genesis in rats. When testosterone was adminis-
tered to rats via sustained-release capsules
(polydimethylsiloxane, PDS; Silastic) oligo- but
not azoospermia was achieved (Ewing et al.,
1977). By contrast, testosterone and estradiol
interacted synergistically to inhibit completely
spermatogenesis in the rat when administered
Accepted July 5, 1979.
Received February 9, 1979.
by subdermal PDS implants (Ewing et al.,
1977). Some doses of testosterone and estradiol
rendered male rats azoospermic without in-
creasing either the seminal vesicle and ventral
prostate weights or the serum concentrations of
testosterone and estradiol (Ewing et al., 1977).
It occurred to us that subdermal Silastic
implants containing testosterone and estradiol
might prove an effective male contraceptive in
the rat if the observed azoospermia resulted in a
reversible sterility that left masculine sexual
behavior intact (Ewing and Robaire, 1978).
Herein, we show that specific testosterone-
estradiol doses, administered via subdermal
Silastic implants, caused sterility in male rats

766 EWING ET AL.
Upon completion of the first phase of the fertility
that was reversible on removal of the implant.
Steroid treatments, which caused this reversible
sterility, did not alter either masculine sexual or
aggressive behavior of rats.
General
MATERIALS AND METHODS
Ninety Sprague-Dawley male rats (250 g; range
240-260 g) were purchased on June 1, 1976 from
Charles River Co. Upon receipt, the rats were ear-
marked, placed in individual cages, provided access to
food and water ad libitum and maintained on a
13L:1ID schedule (lights on at 0700 h and off at
2000 h). Sixty-two males were selected for fertility by
placing each for 24 h with a proestrous female, which
littered 21-23 days following this single exposure to
the male.
Sixty of these males were allotted randomly (10
males/treatment) to 1 of 6 treatment groups includ-
ing: 1) control (4.0 cm cholesterol implant); 2) 2.5 cm
testosterone (T) implant; 3) 4.0 cm T implant; 4) 1.0
cm T-0.1 cm estradiol (E) implants; 5) 2.5 cm T-0.1
cm E implants and 6) 2.5 cm T-0.3 cm E implants.
The 2 remaining males were used in a second experi-
ment designed to test the time of onset and cessation
of sterility following either the implantation or
removal of steroid filled capsules. One of these animals
(control) received a 4.0 cm cholesterol implant and
the other (treated) 2.5 cm T-0.1 cm E implants. The
implants were prepared from Dow Corning PDS tubing
#602-305 as described by Stratton et al. (1973). The
release rates for this batch of tubing were determined
by incubating representative T and E capsules in Krebs
Ringer bicarbonate bovine serum albumin solution in
vitro. The release rates for testosterone and estradiol
were 30 pg cm /day and 2.4 pg cm /day,
respectively. The capsules were implanted on Sep-
tember 1, 1976. The 60 rats in the first experiment
were maintained in individual cages for 3 months.
Measurement of fertility in the 2 rats in the second
experiment was begun immediately.
Fertility
Three months after capsule implantation, each of
the 60 males in the first experiment was placed with 5
females for 20 days in a large cage. Twenty days
later the females were sacrificed and 1) the number of
corpora lutea present in both ovaries, 2) the number
of implantation sites and 3) the number of viable
embryos determined. A similar fertility trial was
carried out on control rats and rats from among
selected treatment groups 4 months after removal of
the PDS implants.
The 2 males in the second experiment were placed
with 5 females each for 20 days immediately following
capsule implantation. Five different females were
provided to each of these males every 3 weeks during
the following 48 weeks. The same 3 criteria described
above were measured.
Behavior
study, the 60 males were shipped to the Department
of Anatomy, University of California School of
Medicine at Los Angeles by air freight. They were
housed individually and given access to food and water
ad libitum. The animals were kept under reversed
lighting conditions with lights on at 2200 h and off at
1100 h. Each individual cage was coded so that the
experimenters who did the behavioral analyses were
unaware of the treatment groups.
Masculine Sexual Behavior
After a 2 week adaptation period, the animals were
tested for masculine sexual behavior. They were
placed in the testing arena (43.2 X 58.4 cm plexiglas
arena) and allowed 15 mm to adapt to the new sur-
roundings. All animals were tested between 1 300-
1600 h (during the animal’s early evening) under the
dim illumination of the testing room provided by a 25
watt red bulb. After the period of adaptation, a
steroid primed receptive stimulus female was intro-
duced into the testing arena. The stimulus females
were ovariectomized and had been given ‘\10 pg
estradiol benzoate and 0.5 mg progesterone to induce
receptivity. Each stimulus female was placed with a
stud male to verify her receptivity before being used
experimentally. Stimulus females that failed to show
strong lordosis responses to the stud male were not
used. The experimental males were tested for a period
of 20 mm and the stimulus females were changed after
10 min in an attempt to minimize the influence of the
female on male behavior. During the sex behavior
tests, scores were recorded for the time to first mount,
to first intromission and to first ejaculation and for
the postejaculatory interval; the total number of
mounts. intromissions and ejaculations were also
recorded. The animals were allowed 1 week to recover
and were then retested. Prior to the initial test, we
decided to terminate behavioral testing at the point
when more than half of the control males ejaculated
during a particular test. This criterion was met on the
second test, in which 80% of the controls ejaculated at
least once.
Aggressive Behavior
On February 3, 1977, the body weights were
recorded for all the experimental animals. Following
this, they were moved in their same cages into an
animal room under normal lighting conditions (lights
on at 0500 h and off 1900 h). The animals were
handled for 2 mm/day for two 5 day periods, with
Saturday and Sunday being omitted, to ensure that
the animals would not be stressed by the handling
incurred upon their transfer to the shock-elicited
aggression testing situation.
The apparatus used for the shock-induced aggres-
sion tests consisted of a cylindrical test chamber with
inside dimensions of 30 cm diameter X 30 cm height.
The entire chamber was constructed of plexiglas which
allowed an unobstructed view of its interior. The grid
floor of this chamber consisted of 0.634 cm stainless
steel rods spaced 1.27 cm apart (center to center).
Electric shock was delivered through the floor grids
operating through a Davis model 255 grid scrambler.
The duration of shock and the interval between shocks
was controlled by two Davis Model D 501 time

REVERSIBLE STERILITY IN MALE RATS 767
The effect of the steroid treatments on total
interval generators. The intensity of the shock current
was continuously monitored throughout each experi-
mental session. Each aggressive episode and the
delivery of shock was recorded concurrently on Davis
digital counters, a standard electric cumulative timer
and an Esterline-Angus 20 channel event recorder.
The experimental animals were paired on the basis
of similar body weight within 1 treatment group. The
experimenters knew the pairings for the animals
by body weight within treatment groups, but were
unaware of the experimental code. For aggression
testing, a pair of rats was placed on the grid floor of
the experimental chamber and received 100 shocks of
4.0 mA for a total of 1.5 seconds duration every 5
seconds. Two observers recorded each experimental
session. The first pressed 1 of 6 microswitches to
indicate which of 6 types of aggressive behavior
occurred and released the microswitch when the
behavior terminated. The criteria for aggressive be-
havior were as follows: 1) shoving-lunging (rat forcibly
pushes or shoves opponent with one or both forepaws
or lunges toward opponent, striking him with fore-
paws; 2) boxing (rat strikes opponent by means of
rapid boxing-like movements with its forepaws); 3)
lunge and bite attempt (rat lunges toward opponent
and attempts to bite it); 4) mild biting (biting occurs
but bite does not penetrate opponent’s skin); 5)
moderate biting (biting penetrates opponent’s skin but
does not draw blood or produce visible physical
damage); 6) severe biting (biting penetrates opponent’s
skin drawing blood and resulting in visible physical
damage). An interobserver reliability coefficient of
0.96 obtained prior to the start of the experiment
indicated that each of these categories was easily
observable and distinguishable from the other. The
second observer recorded the presence or absence of
genital sniffing or licking and sexual mounting during
the experimental session. A third observer initiated the
experimental session and recorded any idiosyncratic
behavior or events which occurred during an experi-
mental session. Each pair of rats was tested once, and
each testing session lasted 11 minutes.
To compare the treatment groups in terms of the
severity of aggressive behavior, an index of “patho-
logical” aggression was constructed for each pair of
rats. This index was computed by assigning numerical
weights to each type of aggressive behavior as a
function of their respective position on an ordinal
scale of severity. For example, the categories shoving-
lunging, boxing, attempt to bite, mild biting, moderate
biting and severe biting were assigned numerical
weights of 1-6, respectively. The sum of all cateogires
was then divided by the total number of observations
to provide a measure of the severity of aggressive
behavior that characterized each pair during aggression
testing.
After completion of the behavioral tests, the
animals were shipped back to Baltimore where the
PDS implants were removed. Four months later, the
males were retested for fertility.
Fertility
RESULTS
embryo number, implantation sites, embryos!
female and the ratio of implantation sites to the
number of embryos are shown in Table 1. This
table also presents the number of corpora lutea
observed in the ovaries of the females. The
lack of a significant difference in the number of
corpora lutea suggests that differences in
fertility were not due to spurious differences in
ovarian function. In contrast, important differ-
ences existed in total number of implantation
sites, in the number of embryos and embryos!
female. All steroid treatments significantly
reduced these measures of fertility. Two
treatments, namely, 2.5 cm T-0.3 cm E and 2.5
cm T-0.1 cm E, created sterile male rats. The
ratio of the number of implantation sites to the
number of embryos was one for all treatment
groups in which fertilization occurred, suggest-
ing that there was no defect in fertilizability of
spermatozoa remaining in the ejaculate of
animals rendered oligo- rather than azoospermic.
Fertility of animals rendered sterile by the
2.5 cm T-0.1 cm E was tested again 4 months
after removal of implants. Fifty females mated
with 10 control males produced 12.4 ± 0.6
embryos/female and 50 females mated with 10
previously sterile but now reversed males
produced 12.1 ± 0.3 embryos/female.
The duration of interfertility and the time
course of sterility onset and fertility recovery
are shown in Fig. 1. Cohabitation of a single
control male with 5different females resulted
in 50-60 embryos for each 3 week period
tested. In contrast, the male receiving the 2.5
cm T-0.1 cm E treatment was sterile for 33
weeks. The fertility of this male was similar to
that of the control at the end of the third and
sixth weeks after capsule implantation, dropped
to zero embryos between the sixth and ninth
week after capsule implantation and returned
to the control level six weeks after removal of
the T and E filled Silastic implants.
Masculine Sex Behavior
The results in Table 2 show the effects of all
steroid treatments on masculine sex behavior
including: the time to first mount, time to first
intromission, time to first ejaculation and the
total number of mounts, intromissions and
ejaculations. All animals exhibited masculine
sex behavior. There was no significant effect of
any steroid treatment on the behavioral para-
meters measured. Obvious “improvements” in
masculine sex behavior occurred when the

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results from test 2 were compared with those of
test 1. However, no significant effects of steroid
treatment on this “improvement” in sex
behavior were discovered when the results were
subjected to analysis of variance. No treatment
effects were observed in the postejaculatory
interval.
Aggressive Behavior
The effect of the steroid treatments on the
shock-elicited aggressive behavior of rats is
shown in Table 3. A one-way analysis of
variance revealed a significant difference
between the groups in terms of the numbers of
fights which occurred during the experimental
session [F (5,22) =3.27; P<0.05] -Subsequent
Newman-Keuls tests revealed that rats given 2.5
cm T engaged in significantly more fights than
did any of the other treatment groups or
controls (P<0.05). None of the remaining
treatment groups, however, was found to be
significantly different from controls (P>0.25).
Parametric and nonparametric one-way analysis
of variance tests indicated that there was no
difference between the treatment groups
in either the time spent fighting [F (5,22) =
1.64; P>0.15] or the index of “pathological”
aggression (H =3.60, df =5, P>0.50).
DISCUSSION
Appropriate doses of androgens have been
shown to be antispermatogenic in numerous
species including man (Ewing and Robaire,
1978). In 1973, it was discovered that a specific
dose of testosterone administered via PDS
subdermal implants rendered male rabbits
reversibly azoospermic (Ewing et al., 1973) and
consequently sterile (Desjardins et al., 1973).
The sterile rabbit’s body weight, accessory sex
organ weight, accessory sex organ secretion,
seminal volume and at least one component of
masculine sexual behavior (latency to mount)
were indistinguishable from these factors in
control rabbits (Ewing et al., 1973; Desjardins
et al., 1973).
When male rats and rhesus monkeys were
treated similarly, an inconsistent azoospermia
resulted. This observation was similar to results
reported by many investigators who injected
human males with esterified derivatives of
testosterone (Reddy and Rao, 1972; Mauss et
al., 1973; Swerdloff et al., 1977; Cunningham
et al., 1977; Paulsen et al., 1977; Steinberger
and Smith, 1977). Taken together, these results
768 EWING ET AL.

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REVERSIBLE STERILITY IN MALE RATS 769
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0 3 6 9 2 15 IS 21 24 21 30 33 36 39 42 45 48
WEEKS OF TREATMENT
FIG. 1. The temporal effect of 2.5 cm testosterone-
0.1 cm estradiol implants on fertility of male rats.
X-X equals 1 control male rat. 0 0 equals 1
treated rat. Each male cohabited with 5 females for
20 days. Five different females were provided for
each male every 3 weeks. By courtesy of U.S. Depart-
ment of Health, Education and Welfare.
suggested that the effective contraceptive dose
range of testosterone or its esterified derivatives
was so narrow as to preclude its use as the sole
constituent of a male contraceptive in several
species including man. This suggested the need
for a second component with antigonadotropic
properties which would broaden the effective
contraceptive dose range. Others have used
progestins (Terner and MacLaughlin, 1973;
Coutinho and Melo, 1973;Frick, 1973;Johans-
son and Nygren, 1973) or Danazol (Paulsen and
Leonard, 1976; Skoglund and Paulsen, 1973;
Ulstein et al., 1975) as the second component.
We chose estradiol because it is a naturally
occurring and potent antigonadotropic steroid
which is synthesized and secreted by testes of
all species examined thus far (Ewing and
Brown, 1977).
Subsequently, it was shown (Ewing et al.,
1977; Ewing, 1977) that testosterone-estradiol
PDS subdermal implants of appropriate dimen-
sions created azoospermia consistently in all
rats tested. Moreover, this azoospermia was
achieved without any chronic elevation in
either serum testosterone and estradiol or in
accessory sex organ size.
The next step in evaluating the contraceptive
efficacy of testosterone-estradiol PDS sub-
dermal implants was to test whether azoo-
spermic animals were sterile and to test the
duration of sterility achieved after a single
implantation of steroid-filled capsules, the
reversibility of the sterile state and, finally, the
effect of the steroid treatment on aggressive
and masculine sexual behavior of male rats.
Fertility
Male rats of proven fertility were rendered
sterile within 6 weeks after the subdermal
implantation of a 2.5 cm testosterone and a 0.1
cm estradiol Silastic capsule (Fig. 1). The males
remained sterile for over 8 months as the result
of a single placement of subdermal T and E
capsules. Fertility returned to control levels
within 6 weeks after removal of the implants.
There was no sign of defects in sperm fertilizing
ability in oligospermic males receiving either
the 2.5 cm T or the 1.0 cm T plus 0.1 cm E
implants (Table 1). Although the number of
embryos/female was reduced to 1.2 and 1.7,
respectively, the ratio of implantation sites!
embryo remained constant. This observation
suggests that accidental pregnancies resulting
from the occasional spermatozoan present in an
ejaculate from an oligospermic male rat were
not resorbed and would go to term.
Behavior
Numerous antispermatogenic agents cause a
reversible sterility in males of several species
(Ewing and Robaire, 1978). Unfortunately,
several of these promising antifertility agents
also inhibit sexual behavior in many species
including man (Heller et at., 1959; Morse et al.,
1973; Michael et al., 1973). Consequently, it
was important to show that the testosterone-
estradiol PDS implants which rendered males
reversibly sterile left masculine sex behavior
intact. Testosterone-estradiol subdermal PDS
implants of various dimensions, which partially
or totally inhibited spermatogenesis and fertil-
ity, did not interfer with masculine sex be-
havior of male rats.
The data in the present study indicate that
rats which received 2.5 cm testosterone im-
plants engaged in more fights than did any
other treatment group or controls. While the
number of fights has been the most frequently
used measure of aggression in rodents exposed
to foot shock, recently it has been shown to be
an inadequate measure of aggressive behavior
(Carder and Sbordone, 1975; Ghiselli and Thor,
1974; Krsiak and Steinberg, 1969; Sbordone,
1976; Sbordone and Carder, 1974; Sbordone
and Garcia, 1977) and may instead by a be-
havioral artifact due to the constraints of the
testing situation (Sbordone and Garcia, 1978).

770 EWING ET AL.
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REVERSIBLE STERILITY IN MALE RATS 771
TABLE 3. The effect of testosterone (T)-estradiol (E) Silastic subdermal implants on foot shock-induced aggres-
sion in male rats.
Treatment Fights (n)
Duration of
fighting (seconds)
Index of pathological
aggressiona
Cholesterol
control 23,6±9,7b 9.0±47 1.8±0.2
4.0 cmT 20.5 ±9.8 8.4 ±4.0 1.7 ±0.4
2.5 cm T 54.5 ±8.6 24.1 ±7.7 1.7 ±0.2
2.5 cm T
+0,3cmE 10.8±3.0 5.9±2.4 1.8±0.5
2.5 cm T
+0.1 cm E 19.0 ±9,3 9,3 ±6.4 1.5 ±0.4
1.0cm T
+0,lcmE 9.8±2.5 2.4±0.4 1.9±0.3
alndex of pathological aggression equals a weighted index of aggressive behaviors divided by the total num-
bers of observations. See Materials and Methods.
bEh value represents the mean ±SEM of 4-5 pairs of males from each treatment.
Since no difference was found between any of
the treatment groups on other measures of
aggression that have been shown to be more
sensitive to drug treatment effects (Sbordone
and Garcia, 1977; Sbordone et at., 1978), it
seems unlikely that any of the treatments used
in this study significantly increases aggressive
behavior in male rats.
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