BIOLOGY OF REPRODUCTION 58, 664-669 (1998)
Postnatal Development of Leydig Cells in the Opossum (Monodelphis domestica):
An Immunocytochemical and Endocrinological Study'
4
3
Q. Xie, 3 S. Mackay, 2 3, S.L. Ullmann, 3 D.P. Gilmore,3 A.P. Payne, and C. Gray
Institute of Biomedical & Life Sciences, 3 University of Glasgow, Glasgow G12 8QQ, United Kingdom
Department of Pathological Biochemistry, 4 Glasgow Royal Infirmary, Glasgow G4 OSF United Kingdom
ABSTRACT
This study involved characterization of Leydig cells of the
opossum Monodelphis domestica, functionally by immunocytochemical identification of the enzyme 3f1-hydroxysteroid dehydrogenase (39-HSD) and by measurement of testosterone levels
using RIA. Immunostaining for 3p-HSD was first detected in a
few Leydig cells on Day 16, was increased by Day 24, reached
a peak at 4 mo, and was present even in senescent (3 yr) animals. Plasma testosterone was first measurable (0.35 nM) at prepuberty (3.5 mo). Prior to that, plasma testosterone concentrations were uniformly below the level of detection (< 0.3 nM) in
both sexes from Day 5 to 2.5 mo. By 4 mo (puberty), plasma
testosterone levels in males had risen significantly to 1.53 +
0.35 nM, continuing to increase to 1.79 + 0.4 nM at 6 mo and
peaking at 2.71 + 0.29 nM in the adult (1-2 yr). Ovarian testosterone concentrations were consistently lower than those in
the testis, as were those of adrenals of both sexes. Thus the testis
would appear to be the major source of androgen production
throughout life in this species. Our immunocytochemical study
suggests that in Monodelphis, puberty is reached at 4 mo, and
this was further supported by a rise in circulating testosterone
levels at this time.
INTRODUCTION
The course of sexual differentiation in male eutherians
involves testis formation followed by masculinization of the
reproductive tract, external genitalia, and brain; much of
this development occurs prenatally [1-3]. The major androgen responsible for the masculinization of the reproductive
tract is testosterone, secreted by Leydig cells of the fetal
testis [4, 5]. At least two populations of Leydig cells have
been proposed for eutherians: a transient fetal one responsible for primary somatic masculinization and a postnatal
population that initiates puberty and remains active during
adult life [6].
Marsupials are potentially useful experimental animals,
as their offspring are born at a stage comparable in many
respects to embryonic stages of eutherian species. It is
therefore important to ascertain whether their developmental processes are the same as or different from those of
eutherian mammals. To date, research has shown that sexual differentiation in marsupials appears to deviate from the
eutherian pattern in that some sexually dimorphic structures
(scrotum, mammary primordia, gubernaculum, and processus vaginalis) develop prior to gonadal differentiation [711] and therefore, apparently, independently of hormone
secretion. Moreover, because marsupials are born relatively
undeveloped, gonadal sex differentiation occurs perinatally
(tammar wallaby) [12] or postnatally (bandicoots) [13] rather than during gestation.
The American gray short-tailed opossum, Monodelphis
domestica, is increasingly being used as a laboratory marsupial because of its small size, easy maintenance, and high
rate of reproduction in comparison to many other species
(14-day gestation period and litters of 3-14 pups), and it is
important to establish baseline information on its reproductive biology. Previous investigations of reproductive function in Monodelphis have focused on sexual behavior [1417] and morphological aspects of gonadal development
[18-21]. The mechanism and time course of sexual differentiation in this species are largely unknown. Studies have
produced conflicting data, with testicular differentiation being reported as occurring on the day of birth (about 50%
of males show gonadal sex differentiation at birth) [21] or
postnatally (Days 3-4) [19, 221. Testosterone is the principal gonadal androgen produced during marsupial sexual
differentiation [12, 23]. George et al. [24] have reported the
synthesis of testosterone by the Virginia opossum testis
from Day 10 postnatally. In the tammar wallaby, gonadal
testosterone levels are low in both sexes at birth, but in
males they rise between Days 2 to 10 with the formation
of seminiferous tubules [12]. Testosterone levels remain
high in the testis until after Day 40, by which time sexual
differentiation of the internal genitalia is essentially complete [10, 12].
Leydig cells can first be identified ultrastructurally in
Monodelphis on postnatal Day 3 [21]. Surprisingly, Fadem
and Harder [23] reported that there were high levels of
testosterone (comparable to adult male levels) in the peripheral plasma of newborn Monodelphis. However, there
is no reported evidence of testicular hormone production at
birth in either Australian (e.g., tammar wallaby [7]) or
American marsupials (e.g., Virginia opossum [25]); moreover, Leydig cells cannot be distinguished prior to Day 3
in Monodelphis, so the source of the testosterone measured
by Fadem and Harder [23] remains unclear.
The aim of the present investigation was to shed further
light on early sexual differentiation in Monodelphis by 1)
immunocytochemical identification of the enzyme 3[3-hydroxysteroid dehydrogenase (3-HSD) involved in steroid
hormone synthesis and 2) measurement of androgen levels
in the gonads, adrenal glands, and peripheral plasma of both
sexes.
MATERIALS AND METHODS
Animals
Adult opossums weigh between approximately 75 and
140 g, and generally males are heavier than females; the
size of the opossum is intermediate between that of a mouse
and a rat. The young are born in a very immature state of
development, each one measuring approximately 100-150
mm crown-rump in length and weighing about 100 mg.
Accepted October 22, 1997.
Received February 17, 1997.
'This work was generously supported by a grant from the Wellcome
Trust, 039933/Z.
2Correspondence: Sarah Mackay, Laboratory of Human Anatomy,
IBLS, University of Glasgow, Glasgow G12 8QQ, Scotland. FAX: 0141330-4299; e-mail: [email protected]
664
665
LEYDIG CELLS IN POSTNATAL OPOSSUM
Table 1. Details of animals used for immunocytochemistry (ICC) and
testosterone measurements.*
Age
Number of
animals for ICC t
Day 3-5
Day 8-10
Day 16
Day 24
4Wk
7-10 Wk
3-3.5 Mo
4 Mo
6 Mo
1-3Yr
11 M
4M
5M
4M
3M
9M
4M
5 M
8M
Number of plasma
samples for RIA t
2 M
2 M
4M
1M
3M
7M
5M
10M
3 M
20M+
2
2
3
1
F
F
F
F
10F
* For testosterone measurement, blood samples were collected from a
total of 81 animals from Day 5 to 4 wk of age; plasma from animals up
to 4 wk was pooled.
t M, male; F,female.
They remain firmly attached to the nipple until about 16
days of age, at which time they measure on average 300350 mm in length and weigh about 800 mg. The juveniles
can be weaned by 7-8 wk. The prepubertal stage (3-3.5
mo) is defined as that period when testicular cords become
patent and form the seminiferous tubules, though sperm are
still absent. Puberty occurs at 4 mo, when sperm can be
seen for the first time and body weight reaches 55-90 g.
The animals used in the present study were bred at Glasgow University. For immunocytochemistry, male opossums
of the following ages were used (birth = Day 0): Days 35, 8-10, 16, 24; 4 wk, 7-10 wk; 3-3.5 mo (prepubertal); 4
mo (pubertal); and 1-3 yr (adult). At least 3 animals were
used at each age point (see Table 1). Animals up to 24 days
after birth were killed by inhalation of CO 2 or halothane,
and gonads were fixed by immersion. Older animals were
killed by an i.p. injection of 4% sodium pentobarbitone and
fixed by perfusion. For testosterone measurements, animals
from Day 5 to 4 wk were decapitated, and older animals
from 7 wk onward (as listed above and also including 6
mo) were terminally anesthetized.
Tissue Preparation
Testes were immersion- or perfusion-fixed with 4%
formaldehyde in 0.1 M phosphate buffer (pH 7.2-7.4) before being stored in fixative for over 24 h at room temperature (20°C). Specimens were then rinsed in buffer for 1030 min, dehydrated through an ascending ethanol series,
and embedded in paraffin wax at 57C.
Antibodies
The polyclonal rabbit anti-human placental 33-HSD antibody was a gift from Prof. J.I. Mason (Department of
Biochemistry, University of Edinburgh). Previous work has
demonstrated cross-reactivity with Monodelphis domestica
tissues [26].
Immunocytochemistry
Immunostaining was carried out by the avidin-biotin
technique on 5-7-pgm paraffin sections. Briefly, sections
were deparaffinized and incubated in 0.1% hydrogen peroxide (H 20 2 ) for 15-30 min to eliminate endogenous peroxidase activity. After rinsing in 0.01 M PBS, sections were
treated with 10-20% normal goat serum (NGS) in PBS for
30-60 min to clear background staining; they were then
incubated in a humidity chamber overnight at 40C with the
Table 2. Concentrations of gonadal and adrenal testosterone (T) in the
developing opossum.*
Aget
(day)
No. of
gonads
Gonadal T in
supernatant
(ng/mg protein)
Number
of
adrenals
Adrenal T in
supernatant
(ng/mg protein)
5M
5F
8 M
8 F
16 M
16 F
28 M
28 F
10 testes
4 ovaries
6 testes
6 ovaries
6 testes
2 ovaries
5 testes
4 ovaries
0.1
<0.01
0.3
<0.01
0.45
<0.01
0.7
<0.01
8
4
6
6
14
2
7
4
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.11
0.11
* For the testosterone measurements, samples from animals of the same
sex and age were pooled.
t M, male; F,female.
primary antibody (polyclonal rabbit anti-human placental
3-HSD antibody). The primary antibody was diluted 1:
1500 in PBS (containing 1% NGS + 0.3% Triton X-100).
Sections were then washed in PBS and incubated at room
temperature for 1-2 h with secondary antibody (peroxidaselabeled goat anti-rabbit) at a dilution of 1:200 in PBS (containing 1% NGS and 0.3% Triton X-100). After PBS rinses,
sections were left in ABC for 1 h. Peroxidase activity was
revealed following a 5- to 10-min incubation at room temperature in a medium containing 0.05% 3,3'-diaminobenzidine, 0.01% H 20 2 , and 0.02% NiC1 3 in 0.01 M phosphate
buffer. The slides were counterstained with 0.5% methyl
green, dehydrated, and mounted in Histomount (Hughes &
Hughes, Ltd., Willington, Somerset, UK). Control experiments were performed on young and adult testis sections
by substituting preimmunized 10-20% NGS in PBS for the
primary antibody. All photographs were taken on a Leitz
Vario-Orthomat photomicroscope (Leica, Milton Keynes,
Buckinghamshire, UK).
Blood, Gonadal, and Adrenal Samples
Blood samples were obtained from the younger animals
following decapitation and from animals 7 wk of age onward by cardiac puncture (see Table 1). From Day 5 to 4
wk, the blood was collected in heparinized capillary tubes
and pooled because of the very small volumes available
(10-50 l plasma/pool). From 7 wk onward, blood was
obtained from terminally anesthetized individual animals
by cardiac puncture with a heparinized syringe. Plasma was
separated from chilled blood by centrifugation and stored
in heparinized tubes at -200 C.
At birth, gonads in both sexes are elongate in shape.
From Day 3 onward, the testis becomes more rounded and
grows faster than the ovary. By Day 5, testis length is 0.4
± 0.03 mm, increasing to 0.99 + 0.02 mm by Day 16 [21].
In animals up to 28 days of age, the gonads were dissected
and pooled, as were the adrenals (see Table 2). Gonads
from older animals were assayed individually. Upon removal, the organs were placed in 0.1 ml of 0.9% NaCI and
homogenized. After centrifugation, the supernatant was removed and stored at -200 C before being assayed.
The protein content of the tissue pellet remaining after
centrifugation was determined from its absorbance at 595
nm after reaction with Coomassie blue G250 (Pierce; Life
Science Laboratories Ltd., Luton, UK). The weight of protein was calculated from a standard curve prepared using
BSA [27]. The sample pellets were dispersed in 210 ,il 0.1
M NaOH and incubated overnight at 4°C. On the following
666
XIE ET AL.
day, duplicate 100-ll samples were transferred to two separate tubes, and each was treated with 0.9 ml of distilled
water and 1 ml of Coomassie blue. The protein concentration in each tube was calculated by substitution of the absorbance into the regression equation of the standard curve.
The protein content of the duplicate tubes was summed to
give the total protein content of the original sample. The
testosterone concentrations were expressed as ng/mg protein.
Testosterone Measurements
Testosterone was measured in diethyl ether extracts of
plasma and in the supernatant from the homogenized gonadal and adrenal tissue. Twenty-five-microliter volumes of
plasma were taken from the pooled samples; 50-pl plasma
volumes were taken from the individual older opossums
and from the supernatant, and correction was made for the
difference in volumes. The assay was a double-antibody
RIA originally developed by Cook and Beastall [28] for
human studies and more recently utilized by Gilmore et al.
[29] for studies in the sloth and by Kassim et al. [30] for
work on the rat. The antiserum was raised against testosterone-3-(o-carboxymethyl)oxime-BSA conjugate, and 25Ihistamine-3-testosterone was used as tracer. The assay
cross-reacts with So-dihydrotestosterone by 16%, 5a-androstane-3a,173-diol by 5.8%, 5-androstane-3[i,1713-diol
by 3.7%, androstenedione by 2.1%, dehydroepiandrosterone by 0.04%, and cortisol by < 0.01%. The intra- and
interassay precision was calculated as coefficients of variation of 8% and 12%, respectively.
Testosterone was spiked into adult male opossum plasma
samples at levels of 2 and 5 nM; 90% recovery of added
testosterone was achieved. Furthermore, adult male opossum plasma samples were analyzed at doubling dilutions
and diluted parallel to the standard curve used for the assay.
RESULTS
33-HSD Immunocytochemistry
At birth, testicular cords in Monodelphis are composed
of germ cells and Sertoli cells delimited by a basement
membrane and surrounded by peritubular cells. Opossum
Leydig cells are first distinguishable morphologically in the
interstitial spaces by Day 3 [21]. Our present study of the
immunolocalization of 33-HSD during postnatal testis development showed that from Days 3 to 8 there was no
specific immunostaining for the enzyme in the interstitial
tissue but that it was present in the testicular cords (Fig. 1).
By Day 16, immunopositive staining was apparent in both
the cytoplasm of the Leydig cells and in the testicular cords
(Fig. 2). On Day 24, immunostaining of the Leydig cells
was more intense (Fig. 3). Staining intensity continued to
increase between 7 and 12 wk (Fig. 4); by contrast, the
immunopositive staining within testicular cords declined
during this period. By 4-5 mo (puberty), immunostained
Leydig cells reached peak numbers (Fig. 5), and some residual staining was still found peripherally in the testicular
cords. In the adult opossum (1-3 yr), positively reacting
cells were abundant in the interstitial tissue only (Fig. 6).
In control incubations (with the primary antibody omitted),
no positive staining was found.
Testosterone Levels
Plasma testosterone concentrations during early postnatal development of the opossum (Day 5 to 2.5 mo) were
uniformly below the level of detection, i.e., < 0.3 nM. Testosterone levels in the male from Week 4 onward are illustrated in Figure 7. By 4 mo, testosterone levels had risen
to 1.53 + 0.35 nM. After 4 mo, levels continued to increase
steadily, to 1.79 + 0.4 nM at 6 mo, and reached a peak of
2.71 + 0.29 nM in the adult (1-2 yr).
Gonadal and adrenal testosterone concentrations are
shown in Table 2. Ovarian testosterone concentrations were
uniformly low (< 0.01 ng/mg protein). Testicular testosterone levels at Day 5, 8, 16, and 28 were, respectively, 0.1,
0.3, 0.45, and 0.7 ng/mg protein. The adrenal testosterone
concentrations were uniformly low in both sexes until Day
28.
DISCUSSION
Although Leydig cells can be identified on the basis of
ultrastructural features (numerous lipid droplets, abundant
smooth endoplasmic reticulum) on Day 3 [21], 3-HSD
immunoreactivity is not detectable in the opossum testis
before Day 8. By Day 16, positive staining is present in a
few Leydig cells. This 3-HSD reaction increases by Day
24, reaches a peak at 4 mo, and remains maximal throughout adulthood. These data would therefore suggest that puberty is reached around 4 mo, confirming our previous
demonstration that sperm also first appear at this time [21].
The achievement of puberty at this age is further confirmed
by the rise in circulating testosterone levels to adult values.
In mammals, the main source of testicular androgen is
the Leydig cells of the interstitial tissue [5, 31]. However,
some steroidogenic enzymes are also found within the seminiferous tubules [5, 32]. Although both the seminiferous
tubules and the interstitial cells in rat testes are thus capable
of converting progesterone to testosterone and androstenedione, the interstitial cells are considerably more efficient
[31, 32]. Whole testis and interstitial cells are capable of
converting cholesterol to androgens, whereas seminiferous
tubules cannot synthesize androgens de novo from cholesterol; however, they are capable of converting more immediate precursors such as progesterone to testosterone
[32]. In the Hokkaido brown bear, Tsubota et al. [33] reported that the cytochrome P450 enzymes cholesterol sidechain cleavage, 17a-hydroxylase/C 1 7_
2 0 lyase, and aromatase were localized in Leydig cells but that spermatids also
stained very intensely in the testis. They suggested that
steroidogenesis may occur not only in Leydig cells but also
in spermatids prior to the mating season, and that Leydig
PLATE I. Immunostaining in testes sections.
FIG. 1. Section through the testis (Day 8), immunostained for 313-HSD.
Note the positive reaction (arrows) within the testicular cords (Tc) and the
absence of 3P-HSD in the interstitial tissue (It). x200.
FIG. 2. Section through the testis (Day 16). Immunopositive staining
has appeared in the interstitial tissue (arrowheads) and is still present in
the testis cords (arrows). x240.
FIG. 3. Section through the testis (Day 24). Positive staining has increased in the interstitial tissue (arrows) and decreased in the testis cords
(Tc). X230.
FIG. 4. Section through the prepubertal testis (12 wk) showing immunostaining of Leydig cells (Lc) and at the periphery of the seminiferous
tubules (arrows). x200.
FIG. 5. Section through the pubertal testis (4 mo). Note immunostaining in the numerous closely packed Leydig cells and residual staining in
the seminiferous tubules (arrows). X200.
FIG. 6. Section through adult testis (1.5 yr). Note intense immunostaining in the Leydig cells and its absence in the seminiferous tubules.
x100.
LEYDIG CELLS IN POSTNATAL OPOSSUM
667
668
XIE ET AL.
n +SEM)
le
-J
0A
E
O
2
a=
U
0
(5)
(4)
NO
1
1.8
2to 2.5
3 to 3.5
4.0
6.0
12 to
Age in months
SEM testosterone concentl rations in plasma of develFIG. 7. Mean
oping male opossums. Bars represent mean v.alues of testosterone for animals aged from 1 to 24 mo after birth. Nunnber of pools or animals is
shown in parentheses. *ND, nondetectable (<
< 0.3 nM).
cells and spermatids are the predomi nant sites of androgen
and estrogen synthesis, respectively.
Surprisingly, during the first weelk after birth in Monodelphis, positive immunostaining for 3P3-HSD is present in
the testicular cords, whereas the intererstitial tissue remains
negative for this enzyme. This contr asts with observations
in other species, such as the rat, in w1hich 3[-HSD staining
is restricted to Leydig cells [34]. Durring subsequent development, positive immunostaining in the opossum testicular
cords declines gradually, being abser it in the adult seminiferous tubules. This finding suggest ts that, in marsupials,
there may be transient enzyme act tivity in the testicular
cords that disappears during developCement.
Sexual differentiation in eutherian .s is believed to be initially controlled genetically when th[e indifferent gonad is
transformed into a testis or an ovary. Further differentiation
of the wolffian and miillerian duct sy stems occurs under the
influence of testicular hormones [35 5-37]. The differentiation of wolffian duct derivatives is c aused by testosterone,
secreted by the fetal Leydig cells [38 ]. In Monodelphis, this
functional aspect of Leydig cells has been demonstrated by
the activation of the 3-HSD enzyime, which is first detected by Day 16. Testicular androgeen production in Monodelphis presumably starts around D ay 16 when regression
of the female wolffian duct is app; rent [39]. This result
indicates the correlation between differentiation of the
wolffian duct and 3P3-HSD enzyme s:vnthesis by developing
Leydig cells.
Our results indicate that in Mornodelphis, testosterone
synthesis at an early stage does not cdiffer appreciably from
that reported in other marsupials, iincluding the Virginia
opossum [24] and the tammar wallaaby [12]. Although the
testes are undoubtedly the major sourrce of testosterone production throughout life, the adrenal glands have also been
implicated as a source of this steroid I in the eutherian fetus
[40]. Fadem and Harder [23] have suggested that the adrenals may also be a major source olf androgen synthesis in
the newborn opossum. However, this explanation is unlikely to hold, since, as the present stuidy shows, there is no
evidence to indicate substantial tes tosterone secretion by
the developing adrenal glands in eitl her sex.
We were unable to detect the presence of testosterone in
peripheral plasma from Monodelphis until 3.5 mo (prepubertal). However, by 4 mo, testosterone levels had risen
significantly, to reach a peak at adulthood. Fadem and
Harder [23] reported that androgen levels in Monodelphis
are measurable in the circulation from the day of birth;
curiously, they found levels to be significantly higher in
mixed-sex plasma pools from animals on Day 4 than in
adult males. In our study, animals were readily sexed by
external morphological features after Day 3; prior to this,
karyotyping is a useful confirmatory adjunct [21]. Blood
samples were obtained by pooling plasma from male and
female animals separately, and the testosterone levels were
also measured separately in both sexes.
Our results differ from those of Fadem and Harder [23]
in several respects. Firstly, we found no evidence to indicate plasma testosterone levels at or above those of adult
males in developing opossums of either sex. Secondly, Fadem and Harder [23] found levels of testosterone from
males on Day 16 to be higher than those of adult males; in
contrast, at this stage we were unable to detect testosterone
in the peripheral plasma-and even in the testes, levels
were relatively low (0.45 ng/mg protein). However, testosterone levels from adults of both sexes were broadly comparable in the two studies. If confirmed, Fadem and Harder's results are surprising: extremely high testosterone levels in the female at the time of sexual differentiation would
result in masculinization unless the testosterone-sensitive
tissues were in some way protected. Our findings are consistent with those of previous studies on testosterone synthesis by gonadal tissue in marsupials [12, 24]. Although
the testosterone assay employed by Fadem and Harder exhibited more cross-reactivity with other steroids than did
ours, this would be unlikely to fully explain the major discrepancy between the two studies. Moreover, the authors
themselves were unable to account for the very high levels
of testosterone they measured in mixed-sex plasma pools
from animals 4 and 8 days of age.
Previous researchers [21] described morphological differences between Leydig cells in early postnatal development and adulthood, the change occurring at about 3.5 mo.
However, it is unclear whether these differences betokened
1) different populations of cells or 2) different stages of
development of a single population. The present work
shows positive 3[P-HSD staining from Day 16 but does not
resolve the continuing problem of Leydig cell differentiation. Although testosterone synthesis is underway in the
opossum testis from early postnatal life, levels in the plasma remain low until prepuberty (3.5 mo). Future studies on
the role of steroid hormones in sexual differentiation in this
species could perhaps involve the use of substances blocking the effects of androgens and estrogens.
ACKNOWLEDGMENT
We are grateful to Prof. J.I. Mason (Department of Biochemistry, University of Edinburgh) for the 3P-HSD polyclonal antibody.
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