The influence of the anti-androgen flutamide on early sexual differentiation of the marsupial male. J. C. Lucas, M. B. Renfree, G. Shaw and C. M. Butler.
The influence of the anti-androgen flutamide on early sexual differentiation of the marsupial male J. C. Lucas, M. B. Renfree, G. Shaw and C. M. Butler Department of Zoology, The University of Melbourne, Parkville, Victoria, Australia,
3052
The development of the prostate and the normal descent of the testes in the tammar wallaby (Macropus eugenii) were influenced by treatment with the non-steroidal anti-androgen flutamide. Male pouch young were treated daily from day 9 to day 45, or from day 20 to day 45, of pouch life. Prostate development was inhibited in both treatment groups to a similar extent. Since prostatic buds do not form until day 25 of pouch life, these results suggest that there is a window of androgen sensitivity operating between day 20 and day 25 of pouch life. The number of prostatic buds was significantly lower, but despite the duration of treatment there was never complete abolition of prostate development. Although testes had descended to the same position in treated and control pouch young, inguinal hernias developed in three of four animals treated with flutamide from day 9. These data demonstrate that virilization of the male reproductive tract in this marsupial is dependent on a relatively brief exposure to androgens. Blocking androgen receptor action interferes with normal development of the inguinal canal, which suggests that it is this aspect of inguinoscrotal testicular descent that is androgen dependent. Introduction In
mammals, androgens play a central role in sexual differen¬ of the male, regulating differentiation of the internal and
tiation
external
genitalia
and testicular descent. Testosterone and its
metabolite, 5a-dihydrotestosterone (DHT), act via the same androgen receptor, but DHT has a higher binding affinity for the androgen receptor and, consequently, a higher androgenic activity (George and Wilson, 1994). Testosterone is thought to
act directly on the Wolffian duct where it stimulates differen¬ tiation of the epididymides and vasa deferentia. Other male sex accessory tissues develop from the urogenital sinus where the presence of the enzyme 5a-reductase means that it is DHT that
masculinization (Cunha et al, are also bound by the androgen receptor in female urogenital tissues and treatment with either androgen can induce prostate formation. In males of both eutherian and marsupial mammals, andro¬ gens stimulate the prostate to develop as multiple solid cords of cells budding from the epithelium of the urogenital sinus. Distinct groups of these cords (buds), corresponding to future lobes of the prostate, branch extensively and invade the surrounding mesenchyme, which will develop both connective tissue and smooth muscle fibres. Differentiation and growth of the prostate continue slowly until an increase in androgen production at puberty induces growth and maturation of the prostate (Cunha and Lung, 1979). Treatment with an antiandrogen, such as flutamide, or castration inhibits this growth, without affecting androgen receptor expression (Husmann is the active
hormone
inducing
1987). Both testosterone and DHT
al, 1991). "Correspondence.
et
Received 14 June 1996.
In both male and female
mammals, the gonads develop high
in the abdomen and move caudally, but in most males, the testes migrate much further down the abdomen to the internal ring and then through the inguinal canal into the scrotum. This second, inguinal phase of descent appears to be androgendependent in eutherian mammals (Hutson et al, 1994; Heyns
and Hutson, 1995). In eutherians with androgen insensitivity syndrome (AIS), or in those treated with anti-androgens, the testes are typically retained at the internal inguinal ring (Muntzing et al, 1974; Peets et al, 1974; Wensing et al, 1975; Symes et al, 1978; Viguier-Martinez et al, 1983; Fentener van Vlissingen et al, 1988; Hutson et al, 1990; van der Schoot, 1992; van der Schoot and Elger, 1992; Martel et al, 1993). In humans with AIS, androgen receptors are defective and there is abnormal development of androgen-dependent tissues (Griffin and Wilson, 1980; Hutson, 1986). The tammar wallaby, Macropus eugenii, is the best under¬ stood marsupial with respect to its sexual differentiation. The testes develop distinct seminiferous cords by 2 days after birth (Rentree and Short, 1988; Rentree et al, 1996). Androgen production by testes is low at birth, and increases rapidly to a plateau between day 5 and day 10 after birth, remaining high until about day 45, after which it falls. The main androgen produced by the developing testis is testosterone, with little or no DHT production. Testosterone is undetectable in ovaries over this period (Rentree el al, 1992). The prostate is present as small buds in male urogenital sinuses by day 25 after birth and, after daily treatment with testosterone from birth to day 25, similar structures develop in females (Shaw et al, 1988), demonstrating that androgens can induce virilization of the urogenital sinus. The similarity of prostate development in marsupial and eutherian mammals suggests that there may be
common
et
factors
controlling
al, 1988; Rentree
et
virilization in both groups (Short
Mortality (days after birth) of Macropus eugenii pouch
Table 1.
young treated with flutamide
al, 1995).
Androgens play role in testicular descent in eutherians, but it is not known whether this is the case in tammars. In tammars, transabdominal testicular descent is essentially complete and the testes enter the internal inguinal ring by day 20 after birth (Shaw et al, 1988; Rentree et al, 1996). The testes pass through the inguinal canal between day 25 and day 35 after birth, and testicular descent is complete by day 60 (Shaw et al, 1988; Rentree et al, 1996). In marsupials, the scrotum forms anterior to the penis, rather than forming posterior to the penis from the labioscrotal folds as in eutherians (Rentree, 1992), so that marsupial testes enter the scrotum via a more direct route (Griffiths et al, 1993). The relatively long period after birth before testicular descent is complete in marsupials allows analysis of the control of these important developmental a
Total number treated
Treatment
group
Days after birth 45
_
10-25
26-35
36-44
(autopsy)
Control
Days Days
20-45 9-45
5 2
4
6 6
5 4 1 1
1
Flutamide
Days Days Days Days
20-45 9-45 12-45 17-45
Total
2 1 22
lo
events.
This
study aimed
to
clarify
the role of
androgens
in
early
marsupial male development using the non-steroidal antiandrogen, flutamide, during the time when the first signs of androgen action are seen in the reproductive tract.
Treatments Male
pouch
young (n 22) of 20 days after birth (late treatment) and 9 days after birth (early treatment), were treated daily with flutamide (n 6 at day 9; 6 at day 20) or vehicle 5 at day 20) until 45 days after birth. (n 2 at day 9; Additional animals were treated from day 12 (n 2) or day 17 (n 1). Of the 19 animals treated, five were not treated for the full 45 days. Two control, one late and two early treated animals either died or were autopsied early due to illness of either the pouch young or the mother (Table 1). The late treatment group (days 20—45) was treated from just before the testes entered the inguinal canal and 5 days before the first signs of prostate development (Shaw el al, 1988). The early treatment group (days 9-45) was treated from the time of the first indication of transabdominal testicular descent as deter¬ mined by the characteristic changed shape of the Wolffian duct (Rentree et al, 1996). No significant difference was detected between the scores of the controls for long or short treatment groups. Since there were too few controls of the short treatment group, their scores were combined with treated animals for statistical comparison. =
=
Materials and Methods
=
=
=
=
Animals
=
Tammar wallabies of Kangaroo Island, South Australia origin held in open grassy yards. Their diet was supplemented
were
with lucerne cubes, oats and fresh vegetables. Fresh water was supplied ad libitum. Pouch young were removed from female adults in Iactational quiescence. Removal of pouch young results in reactivation of blastocysts in Iactational diapause and births occur 26-27 days later (Tyndale-Biscoe and Rentree, 1987). Females were checked daily for births (day 0) or were checked intermittently and any pouch young were aged using the growth curves of Poole et al. (1991). The sex of pouch young was determined using an otoscope to detect the presence of scrotal bulges, which are confined to males (O et al, 1988). Care and treatment of the animals conformed to the National Health and Medical Research Council of Australia/ Commonwealth Scientific and Industrial Research Organisation/Australian Research Council 1990 guidelines Australian Code of Practice for the Care and use of Animals for Scientific Purposes and all experiments were approved by institutional animal experimentation ethics committees.
Morphology day
45 the pouch young were removed from the teat, their head length was recorded. The position of and weighed the testes through the skin was recorded, and the pelvic region
At
photographed.
Solutions
The young were killed by decapitation, and the abdomen opened and examined before the lower portion of diges¬ tive tract was removed. The abdominal cavity was photo¬ graphed and internal position of the testes noted. The pelvic region containing the testes was fixed in either Bouin's fixative or neutral buffered formalin (10% v/v). Samples were decalci¬ fied in Gooding's décalcification solution, processed and embedded in paraffin wax. Each animal was serially sectioned and sections mounted on gelatine coated slides before staining with Ehrlich's haematoxylin and eosin. The morphology of the Wolffian ducts and the urogenital sinus in the region of the developing prostate was observed was
Flutamide (2-rnethyI-N-(4-nitro-3-[trifluoromethyl]phenyl) propanamide) (Schering-Plough, Baulkham Hills, NSW) was
dissolved in alcohol (40 mg ml" ) before mixing 1:3 with methyl cellulose (0.4%, v/v). The flutamide solution was vortexed before drawing 5 µ into a fine polythene capillary (inner diameter, 0.4 mm; outer diameter, 0.8 mm). A daily dose of 10 mg kg was given orally to the pouch young via the the capillary using technique described by Shaw et al. (1988). Control animals were given vehicle only. ~
1. Prostatic bud formation. In control animals (a) prostatic bud formation (solid arrow heads) is well advanced with deep invasion of the surrounding mesenchyme. This section was scored 5. In flutamide-treated animals (b) prostatic bud formation is much reduced. This section was scored 1. ugs: urogenital sinus, m: mesenchyme. Scale bars represent 480 µ .
Fig.
and recorded. Prostatic development was assessed by blind subjective scoring. All slides with prostate sections were selected from the region of the urogenital sinus, and 10 evenly spaced sections marked. Slides from all animals were mixed together and were assessed blind by a second observer. The scale was as follows: 0 no bud formation; 1 one bud present with low invasion of the mesenchyme; 2 = one or more bud present showing low to moderate invasion of the mesen¬ chyme; 3 numerous buds showing moderate invasion of the mesenchyme; 4 numerous buds with deep invasion of the mesenchyme; 5 prolific bud formation with the presence of at least one bud section showing in the mesenchyme separated from the rest of the epithelium, indicating invasion of the bud to at least the known thickness of one section (see Fig. 1). Scores were averaged for each animal. =
=
=
=
=
Morphometry The cross-sectional areas of sections of testis were calculated the point counting method and from this the volume of each testis was calculated using Cavalieri's principle (Gunderson et al, 1988). Length of the testis was taken as the distance (calculated from known thickness of each section) between the first and last section with seminiferous tubules.
by
Testicular position A fixed reference
point and
a
unit of measurement
to the caudal end of the testis was calculated. This distance was converted into a standardized measure as follows. The distance between the sacral vertebra located above and the most caudal vertebra in which the section contained testicular tissue was found. By counting the number of vertebrae separating these two points, the distance between individual vertebrae was calculated and defined as a vertebral unit. The testicular position was converted to vertebral units to allow comparisons between animals that varied in size. The blocks were cut transversely. However, because not all specimens were cut at an angle exactly 90° to the cranialcaudal axis, it was necessary to apply a correction factor to the raw data calculated from the angle of the cut (Fig. 2). The position of the widest point of the dorsal ganglia (left and right) posterior to the sacral vertebrae was found. It was assumed that the dorsal ganglia were level and equidistant to the midline. Taking a line through the spinal cord and dorsal aorta as the midline, the distance to the far side of the dorsal ganglion was found (x). The distance between the section on which the right ganglion was widest and the section on which the left ganglion was widest was calculated using the number of sections separating the two points and defined as y. The midline was extended ventrally to the level of the posterior end of each testis, and the distance from the midline to the middle of the testis found (d). A number of right angled triangles were now drawn as shown in Fig. 2. Using Pythagorus' theorem, a relationship was derived as below:
point
z2 + d2 x1
were
required to determine accurately and subsequently to compare the position of each testis histologically. The widest point of each of the dorsal root ganglia caudal to the sacral vertebrae that was fused to the pelvis was found. The distance from this
(1)
=
and,
since sin
=
alx=>à
yllz —
=
xyl2z
(2)
4 -]
Midline
3 -
Level c
0)
E
2
-
CL O >
D
of method used to correct for angle of cut in testes may appear to have different positions Level morphometry. when the animal is cut obliquely. With the dorsal ganglia as fixed reference points, assumed to be level, the angle of the cut (a) was calculated. The midline was extrapolated to the testes and congruent triangles were used to correct for the distance in error (z). DG: dorsal ganglia, d: distance between midline and ganglia, y. distance between sections on which the widest points of the left and right ganglia
Fig.
2.
appear.
Substituting
for d in (1)
zV + xV/4
=
0
(3)
-
Solving
for
and
=
zlx
to find a, the cutting angle, which may be used with congruent triangles to calculate the distance along the midline that was in error.
it is then
possible
Statistical
analysis
A Mann-Whitney U test was applied to compare prostate growth across all groups, and also testicular length and volume.
Kruskal-Wallis tests were used to compare testicular position between groups. Wilcoxon tests were used to compare the left and right testicular positions in each animal.
Results Prostate and
-1-1-1-
Days 9-45
Days 20-45 Treatment group
Control
3. Developmental scores of prostatic bud development in pouch young. Values shown are the means for each animal derived from assessment of ten sections, ranked 0 (no development) to 5 (advanced
Fig.
development). Control animals had significantly higher scores, that is, greater development, than either of the groups of flutamide-treated animals (Mann-Whitney U test, < 0.001). There was no significant difference between the short (days 20-45) and long (days 9-45) treat¬ ment groups. ·, Animals given a complete treatment, and which were used in the statistical analysis. A, Values for two additional animals that died on day 39 (early treatment) or day 38 (control treatment).
using the relationship cos
Wolffian duct
days, epithelial buds had invaded mesenchyme surrounding the urogenital sinus (Fig. la).
In control animals at 45
the
I
Diagram
lower in both groups of animals treated with flutamide than in controls. (Average scored: controls, 3.42 ± 0.17; short treatment group, 1.40 ± 0.28; long treatment group, 1.13 ± 0.30; Mann-Whitney U test, < 0.001, Fig. 3). No significant difference was detected between the two treatment groups. No histological differences in Wolffian duct development were detected between control and experimental animals of either treatment group. Prostatic bud formation
Testicular position In all animals, the
testes were visible through the skin and situated at or near the neck of the scrotum, having entered the inguinal canal (Fig. 4). In the early treatment group, three out of the four animals that completed treatment developed inguinal hernias on their right-hand side (Fig. 4). When the abdominal cavity was opened and the gut removed the testes were no longer in the abdomen but had passed into the inguinal canal in all animals autopsied at day 45. In animals with inguinal hernias, the intestines had passed through the patent internal inguinal ring and into the scrotum. The herniated intestine was removed before the animals were fixed for sectioning. Testicular position relative to the vertebrae was not signifi¬ cantly different between controls and either group of treated animals or between treatment groups (Fig. 5). However, there was a significant difference between left and right testes in both groups of treated animals, with right testes descended further than left (one sided Wilcoxon tests: control animals =0.14, pooled expérimentais =0.006, days 9-45 =0.050, days 20-45
=
0.053).
scores were
Testicular volume and
length
was observed in the volume and length of the of control or treated animals (Fig. 6). There was considerable variation in the length and volume of the testes of control animals, while the two treatment groups showed less interanimal variation.
No difference
testes
Fig. 4. Position of the testis (t), kidney (k), bladder (b) inguinal canal (i), scrotum (s), and phallus (p) in 45-day-old male pouch young, (a) External appearance of control; (b) the abdominal cavity of a control animal; testes have entered inguinal canal and are not visible; (c) external appearance of a pouch young treated with flutamide between day 9 and day 45 with inguinal hernia (h), the coils of intestine are visible through the skin; (d) the inguinal canal with the intestines removed and replaced by a probe to show the position of the hernia, (a, c and d): scale bar represents 2.5 mm; (b): scale bar represents 5 mm.
Discussion needed for prostate development in the tammar Androgens wallaby since prostate development is substantially inhibited by anti-androgens in this marsupial. Androgens may also play a role in some aspects of testicular descent. Although testicular are
delayed, animals treated from before testicular initiated until after the normal time of testicular migration descent developed inguinal hernias. Prostatic buds were well developed by day 45 and had deeply invaded the surrounding mesenchyme in the control descent
was
not
was
(a)
1.5 2.5
e _
> m
2O
c =>
* ce = -O
2
"-
1.0
1.5 E E
-c CD
>
*-'
E
c
CO
_5
0.5
o
>
-
0.5
Control
Days 20-45
Days 9^t5
Treatment group in tammar pouch young. This was descent of testicular 5. Fig. Degree measured as the distance of the testis in vertebral units (mean ± sem) from the sacral vertebra. There were no significant differences observed in the position of testes between any group. In both treatment groups there was a significant difference between the position of the left testis and that of the right testis. , left testes; right testes.
0.0
Control
1.5
E S>
In normal males, the prostatic buds first become at obvious about day 25 after birth (Shaw et al, 1988; Rentree et al, 1996). Prostate development therefore appears to be initiated in a narrow window between day 20 (the latest start
prostate.
development. Similarly, prostate development in rats is severely curtailed but not entirely eliminated by neonatal
Days
20-45
(b)
2.0
pouch young. Prostatic bud development was greatly reduced in treated pouch young. The two treatment groups were not significantly different from one another, suggesting that extended administration of the inhibitor does not significantly enhance the inhibitory effects of flutamide on the developing
of treatment) and day 25 after birth (the earliest day of observed prostatic buds). Since flutamide induces its antiandrogenic effects by blocking the androgen receptor, it is possible that the receptor may be absent or inactive in the urogenital sinus before day 20 after birth. Flutamide never completely prevented prostatic bud forma¬ tion. Flutamide can never totally abolish androgen binding, and like many other competitive steroid inhibitors, may also act as a weak androgen or oestrogen (Di Monaco et al, 1995) which would explain the stimulation of the observed slight prostatic
Days
9-45
1.0
C
0.5
1
0.0
Control
Days 9-45
Days
20-45 testes of ± sem). There were Volume and 6. (mean (a) (b) length Fig. no significant differences observed among control, long treatment (days 9-45) and short treatment (days 20-45) groups in either length or volume. Growth of testes has not been affected by treatment with
flutamide.
and Cunha, 1981), treatment of
castration (Price, 1936; Lung neonates with the anti-androgen
cyproterone
acetate
(Chung
Ferland-Raymond, 1975) or treatment with oestradiol (Lung and Cunha, 1981). These authors suggest that other factors may be involved in initial development of the prostate,
and
unable to eliminate the possibility of a 'priming' or conditioning effect of androgens on the urogenital sinus before prostatic bud development. Other hormones, such as GH or prolactin, may be involved in prostate development. GH is known to affect prostate function in adult rats in an androgen independent manner (Chung and Ferland-Raymond, 1975; Reiter et al, 1995). However, tammar females treated with androgens form prostate buds at the same time as males (Shaw et al, 1988). If non-androgenic influences were the cause of the limited prostatic bud formation in flutamide treated males, then a similar degree of development would be observed in females. Since females lack any prostate bud formation, it seems more
but
were
that the flutamide did not totally block androgen action the treated males. The influence of androgens on either transabdominal (days 9—25) or inguinoscrotal (days 25—60) testicular descent in tammars is not as marked as it is in those eutherians so far studied. In marsupials, the scrotum is anterior to the penis and, consequently, the route of descent of the testes is much more direct than in eutherians (Griffiths et al, 1993). In eutherians, testicular descent is thought to be controlled by the gubernaculum, a cord of mesenchyme attaching the caudal pole of the testis to the scrotum, which appears to be directed or guided by calcitonin gene-related peptide released from the genitofemoral nerve (Heyns and Hutson, 1995; Terada et al, 1995). The scrotum is post penile and during development the gubernaculum must actively migrate to the scrotum. By
likely in
contrast, in marsupials, it is already firmly attached to the scrotal wall during late fetal life before descent begins (Griffiths et al, 1993). A hormone controlled directional component of descent may therefore be unnecessary in marsupials. Although blocking androgens did not prevent inguino—scrotal descent of the testis, it did induce inguinal hernia, as is commonly seen in eutherian mammals with AIS or after treatment with antiandrogen. Thus, anti-androgen treatment may affect the size, flexibility or closure of the inguinal canal. Testicular size was not affected by flutamide treatment in tammars (this study) or in rats (Spencer et al, 1993). In the early treated tammars, the two testes descended to slightly different positions. The difference in the position of right and left testes caused by the flutamide treatment may be explained by an amplification of a normal handedness which is usually so slight as to pass unnoticed. Turnbull et al. (1981) reported the descent of the left testis before the right in brushtail possums (Trichosurus vulpécula). In tammars, it is the right testis that is lower. The presence of unilateral inguinal hernias in three out of four wallabies treated from day 9 may be a consequence of the differences in position between right and left testes. The hernias are all on the right hand side and it is the right testis that is lower in eight out of nine treated animals. This asymmetrical testicular descent may predispose the animal to hernia. Inguinal hernias are associated with AIS in pigs, mice and humans, in which maldescent of the testes is also common (Wensing and Colenbrander, 1973; Hutson, 1986) and is thought to be related to failure of the gubernaculum to regress fully (Wensing, 1973, 1988; Wensing et al, 1975; McMahon et al, 1995). The results of our study add another explanation, that the hernias may also be due to some effect on inguinal canal development. As in humans (Hutson et al, 1996), the inguinal canal in male tammar pouch young becomes obliter¬ ated after testicular descent (C. M. Butler, G. Shaw and M. B. Rentree, unpublished). Flutamide may be acting to delay or prevent this obliteration. Thus, the flutamide-treated tammar pouch young may provide a good model for the human inguinal hernia. The influence of flutamide on prostatic development clearly demonstrates its effectiveness as an androgen antagonist in tammars. Sensitivity to androgens appears to begin in the male urogenital sinus between day 20 and day 25 after birth. Androgen independent factors may also be involved in differ¬ entiation of sex accessory tissues in tammars. Since inguinal hernias develop in both AIS eutherians and flutamide-treated tammars, androgens are implicated in the normal development of the inguinal canal. However, testes of AIS eutherians fail to descend, while in tammars, testicular decent is not prevented, possibly reflecting the relatively short distance and direct path the testis must take to reach the scrotum in this marsupial. We are grateful to Schering-Plough for the kind gift of Flutamide. We thank A. Duns, R. Moyle, L. J. Parry and D. Whitworth for assistance with animal handling; C. Kenyon, F-X. Jiang and R. Birks for advice with histology, morphometry and statistics; and J. M. Hutson and R. Short for helpful discussions. This work was supported by grants to M. B. Rentree, R. V. Short and G. Shaw from the National Health and Medical Research Council. Animals were held under permit number RP-92-099 from the Department of Conservation and Natural Resources, Victoria, Australia.
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