J Comp Physiol B (2014) 184:10551063

DOI 10.1007/s00360-014-0859-3

ORIGINAL PAPER

Androgen receptormediated regulation ofadrenocortical activity

inthe sand rat, Psammomys obesus
AbdelouafiBenmouloud ZainaAmirat
FaridaKhammar AlexandreV.Patchev
JeanM.Exbrayat OsborneF.X.Almeida

Received: 6 May 2014 / Revised: 13 August 2014 / Accepted: 19 August 2014 / Published online: 2 September 2014
Springer-Verlag Berlin Heidelberg 2014

Abstract The wild sand rat, Psammomys obesus, displays seasonal variations in adrenocortical activity that
parallel those of testicular activity, indicating functional
cross-talk between the hypothalamo-pituitary-adrenal and
hypothalamo-pituitarygonadal axes. In the present study,
we examined androgen receptor (AR)-mediated actions of
testicular steroids in the regulation of adrenocortical function in the sand rat. Specifically, we examined the expression of AR in the adrenal cortex, as well as adrenal apoptosis in male sand rats that had been surgically castrated or
castrated and supplemented with testosterone; biochemical indices of adrenocortical function and hormone profiles were also measured. Orchiectomy was followed by
an increase in adrenocorticotropic hormone secretion from
the anterior pituitary and subsequently, increased adrenocortical activity; the latter was evidenced by orchiectomyinduced increases in the adrenal content of cholesterol and
lipids as well as adrenal hypertrophy (seen as an elevation
of the RNA/DNA ratio). Further, androgen deprivation
Communicated by G. Heldmaier.
A.Benmouloud Z.Amirat F.Khammar
Laboratoire de recherche sur les zones arides, Facult des
Sciences Biologiques, Universit des Sciences et de la
Technologie Houari Boumediene (USTHB), BP 32,
ElAlia, 16111Algiers, Algeria
A.V.Patchev O.F.X.Almeida(*)
Max Planck Institute ofPsychiatry, Kraepelinstrasse 210,
80804Munich, Germany
e-mail: osa@mpipsykl.mpg.de
J.M.Exbrayat
Universit de Lyon, UMRS 449, Laboratoire de Biologie
Gnrale, Universit Catholique de Lyon, Reproduction et
Dvelopment Compar, EPHE, 25 rue du Plat,
69288Lyon Cedex 02, France

respectively up- and downregulated the incidence of apoptosis within the glucocorticoid-producing zona fasciculata
and sex steroid-producing zona reticularis. Interestingly,
orchiectomy resulted in increased expression of AR in the
zona fasciculata. All of the orchiectomy-induced cellular
and biochemical responses were reversible after testosterone substitution therapy. Together, these data suggest that
adrenocortical activity in the sand rat is seasonally modulated by testicular androgens that act through AR located in
the adrenal cortex itself.
Keywords Androgen receptor Adrenal cortex
Testosterone Apoptosis Sand rat

Introduction
Seasonal reproduction during favorable periods of the year
ensures survival of offspring and maximizes reproductive
success. The wild sand rat, Psammomys obesus, found in
the Sahara desert, displays seasonal variations in testicular activity; plasma testosterone levels start to rise in early
summer, peak during the autumnwinter transition, and
gradually decrease to a minimum in the spring (Khammar
and Brudieux 1984). These seasonal changes parallel those
seen in the steroidogenic activity of the adrenal cortex
(Amirat etal. 1980). Thus, glucocorticoid secretion fluctuates as a function of the availability of food to meet the
energy demands of breeding (Schradin 2008).
Glucocorticoids and mineralocorticoids play a key role
in integrating metabolic activity and energy balance, saltion metabolism, and reproductive function (Miller 1988;
Dallman etal. 1999). Glucocorticoids, such as cortisol
and corticosterone, are critical for the organism to cope
with the energetic demands of stressful stimuli. Increased

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adrenocortical secretion is accompanied by dynamic structural changes, namely cell proliferation and apoptosis, that
are regulated by central, pituitary, and other physiological
signals (Wolkersdrfer and Bornstein 1998).
Several field studies reported increased glucocorticoid
levels during the breeding season (Romero 2002; Schradin
2008) and implicated gonadal androgens in the regulation
of seasonal changes in glucocorticoid secretion. Interestingly, testosterone was found to suppress corticosterone
levels in free-living striped mice (Rhabdomys pumilio), a
desert species (Raynaud etal. 2012). However, the mechanisms underlying the postulated interactions between
gonadal and adrenocortical steroids in desert rodents
remain unknown. Here, it is pertinent to mention that the
adrenals and gonads have a common embryologic (mesodermal) origin (Keegan and Hammer 2002), and that functional cross-talk between the secretory products of these
endocrine glands have been previously shown to influence
the magnitude and duration of the physiological (glucocorticoid secretion) response to stress (Patchev and Almeida
1996; Handa and Weiser 2014).
Apart from their better-known actions in the regulation
of male sexual development, bone and muscle growth, testicular androgens also influence adrenal gland size, steroidogenesis, and secretory activity in a variety of species
(Kitay 1975). For example, circulating androgens suppress
the activity of adrenal 3-hydroxysteroid dehydrogenaseisomerase (3-HSD) (Stalvey 2002), a key enzyme in the
biosynthetic pathways that produce estradiol, testosterone,
cortisol, corticosterone, and aldosterone (Simard etal.
2005; Thomas etal. 2011). Androgen receptors (AR) mediate the actions of androgens such as testosterone (Lubahn
etal. 1988). These receptors are ligand-activated transcription factors that regulate gene expression in immune, cardiovascular, metabolic, skin, and neural tissues (Chang etal.
2013). Interestingly, AR have been localized in the adrenal
gland of several species, including humans (Rossi etal.
1998), rhesus monkeys (Hirst etal. 1992) and laboratory
rats (Rattus norvegicus) (Bentvelsen etal. 1996).
We previously demonstrated that androgen deprivation (by orchiectomy) in the sand rat induces significant
changes in the adrenal cortex, characterized by hypertrophy
and enhancement of adrenocorticosteroid content (Benmouloud etal. 2006). There are reports that activity of the
hypothalamo-pituitary-adrenal (HPA) axis in this species
correlates with sexual activity (Gernigon etal. 1992), but
the role of androgens in this respect are not known.
The present study was designed to examine whether the
adrenal cortex of the sand rat expresses androgen receptors that could provide an explanation for the postulated
role of gonadal androgens in the seasonal regulation of
adrenocortical function. Our studies involved bilateral
orchiectomy, testosterone replacement, and biochemical

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J Comp Physiol B (2014) 184:10551063

and immunohistochemical approaches to strengthen the

evidence for androgen receptor-mediated regulation of the
HPA axis in the sand rat.

Materials andmethods
Animals
Adult male fat sand rats (Psammomys obesus; Cretzschmar 1828), were collected during breeding season in the
Beni-Abbes arid area (Wilaya of Bechar, Algeria; 307N,
210W) of the Sahara desert. This diurnal species belongs
to the Muridae family, has a life expectancy of 3years, and
feeds exclusively halophil plants of the Chenopodiaceae
family (especially, Suaeda mollis, Traganum nudatum,
and Salsola foetida; Daly and Daly 1973). Animals weighing 126.53.4g were individually housed in a temperature-controlled environment (22C) in a ventilated room
under a 12:12 lightdark cycle (lights on at 07:00). All
experiments were performed according to the guidelines
of the Federation of European Laboratory Animal Science
Associations.
Orchiectomy andhormone treatment
Three groups of sand rats (n =12/group) were set up:
Group I (control) was sham-castrated and received no further treatment; Group II (ORX) was surgically castrated;
group III (ORX+T) was castrated and testosterone-supplemented. Bilateral surgical orchiectomy was performed
under anesthesia (mixture of ketamine hydrochloride,
Ketalar, Pfizer, NY, USA; 50mg/kg and xylazine hydrochloride, Rompun, Bayer, Toronto, Canada, 10mg/kg;
mixture administered i.p.), using a ventral approach. The
third group (ORX+T) was injected with testosterone
oenanthate (Testo Enant, Geymonat SpA, Anagni, Italy,
150g/100g BW., diluted in 80l olive oil; i.m.) daily for
7 consecutive days, starting 3weeks after orchiectomy.
Blood sampling andpostmortem tissue collection
At the end of the experiment (4weeks after orchiectomy), animals were euthanized (09:0011:00h) by
decapitation after inducing deep anesthesia with intravenous sodium pentobarbital (Abbot, Saint-Rmy-sur-Avre,
France; 100mg/kg BW.). Trunk blood was collected and
the plasma fractions stored at 20C until analysis. The
adrenal glands were quickly removed and weighed. The
right adrenal of each animal was immersion fixed in 4%
paraformaldehyde (Sigma, St Louis, USA) until immunohistochemical analysis and assessment of apoptosis.
The left adrenal cortex (excluding medulla) was weighed

J Comp Physiol B (2014) 184:10551063

and placed in cold saline until the biochemical assays (see

below) were performed. Seminal vesicles and pituitary
glands were removed and weighed.
Biochemical assays onadrenal cortex
Lipids were extracted from pre-weighed adrenocortical
tissue that was homogenized in a mixture of chloroform/
methanol (Bligh and Dyer 1959). The cholesterol content
of adrenocortical tissues was determined using the LibermannBurchard method (Burke etal. 1974). As a measure
of adrenocortical growth, we measured total adrenal protein and RNA and DNA content (Winick and Noble 1965)
as well as the RNA/DNA ratio as an index of cell size
(Schmidt and Schibler 1995). Protein contents were quantified using the Bradford method (Bradford 1976), with
bovine serum albumin as standard. Adrenocortical RNA
and DNA were extracted using a previously described procedure (Shibko etal. 1967); briefly the left adrenal cortex
of each animal was homogenized in 4 volumes of 0.5M
perchloric acid (Panreac Quimica S.A, Barcelona, Spain)/g
fresh tissue, and the purity of extracted RNA and DNA was
estimated by photometry (260/280nm).
Blood plasma hormone assays
Plasma testosterone and cortisol levels were analyzed by
electro-chemiluminescence immunoassay (Roche Diagnostics, Meylan, France), using an automated hormone
analyzer Elecsys 1010 (Roche Diagnostics). Intra- and
inter-assay coefficients of variation were 13%/11.4%
and 23%/1.41.6% for testosterone and cortisol, respectively. The lower limits of detection were 0.06ngml
and 0.018g/dl for the androgen and glucocorticoids,
respectively. Plasma levels of luteinizing hormone (LH)
were measured by microparticle enzyme immunoassay
(AxSYMLH; Abbott Laboratories, USA) using an Axsym
automated analyzer system, with average intra-assay
and inter-assay coefficients of variation of 8.2 and 13%,
respectively, and a sensitivity of approximately 0.6ng/ml.
Plasma levels of ACTH were measured using the Roche
ACTH sandwich electro-chemiluminescence immunoassay
ECLIA (Roche Diagnostics) on a Roche Cobas e 411 analyzer (intra- and inter-assay coefficients of variation of 3.6
and 7.2%, respectively; assay sensitivity <1.0pg/ml).
In situ endlabeling ofDNA todetect apoptosis
The highly sensitive TUNEL method (Gavrieli etal. 1992)
was used to detect apoptosis at the single cell level. Adrenal tissue Sections (4m thick) were cut from paraffin
blocks, deparaffinized and rehydrated in a graded series of
ethanol, followed by washing in phosphate-buffered saline

1057

(PBS; 10mM, pH 7.4). Endogenous peroxidase was inactivated by covering the specimens with 3% H2O2 for 5min
at room temperature. An in situ cell death detection kit
(Roche Molecular Biochemicals Co., East Sussex, UK) was
used following the manufacturers protocol and then incubated for 90min in a humid atmosphere at 37C. Fluorescein-labeled dUTP was visualized using an alkaline phosphatase-conjugated anti-fluorescein antibody (from sheep;
Roche) for 30min. After rinsing in Tris-buffered saline
(TBS; 50mM, pH 7.4), the slides were developed with the
chromogens nitro-blue tetrazolium (NBT) or 5-bromo-4chloro-3-indolyphosphate (BCIP) in TBS to produce black
or purple stained products; NBT and BCIP were obtained
from Sigma. Sections were finally counterstained with
hematoxylin (Vector Laboratories, Burlingame, CA, USA),
dehydrated, cleared in xylene and mounted using Crystal
mount (Biomeda, Foster City, CA). Images were captured
using a light microscope (Nikon Eclipse E 400, connected
to a Nikon DXM 1200 digital camera).
To generate positive controls for the TUNEL assay, sections were pretreated with DNase I (3,000IU/ml; Sigma).
Sections that were not treated with TdT (Roche cell detection kit) were used as negative controls. Three different
fields containing 100 cells per field were examined at 400X
magnification. The number of positively stained cells was
counted in a total area of 22,429m2. By evaluating the
ratio of positively stained cells vs. total cell number, sections were categorized as negative () if no cells were
stained, weakly positive (+) if <25% of cells were stained,
moderately positive (++) if >25% of cells were stained,
or strongly positive (+++) if >50% of cells were stained.
Androgen receptor immunohistochemistry
Paraffin Sections (4m) were deparaffinized, dehydrated through a series of graded ethanol and washed in
TBS. Immunohistochemistry was performed after antigen
retrieval in sodium citrate buffer (10mM, pH 6.0; boiled
for 15min in a 750W microwave oven). Endogenous
peroxidase activity was blocked with 0.3% H2O2 in TBS
(20min, room temperature) before incubation (60min)
with 10% non-immune goat serum to block non-specific
binding sites. Sections were then incubated overnight at
4C with a mouse monoclonal antibody directed against
the androgen receptor (AR) (antibody F39.4.1 from AbCys
S.A., Paris, France, diluted 1:500 in TBS). Immunostaining was visualized by incubating sections with biotinylated
secondary antibody (Vectastain Elite ABC kit, Vector Laboratories, CA, USA) for 60min, followed by the chromogen
3,3-diaminobenzidine-tetra-hydrochloride (DAB; 0.05%
in TBS containing 0.01% H2O2,Vector Laboratories, CA,
USA). Sections were counterstained with hematoxylin
(Vector) for 30s before dehydration in a series of graded

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4.60.2
155.911.3
12.81.1#

*, # P<0.05; **P<0.01; ***P<0.001

*ORX vs. control, #ORX +T vs. control

Data are reported as meanSEM for 12 sand rats in each group

37.42.2
40.22.1
129.94.3
ORX+T (n=12)

77.64.3

22.22.5

0.330.08

41.84.6***

158.416.5

18.82.9***

6.90.5
0.260.08

0.430.72***
26.83.0*

18.91.8
72.62.4

86.32.7*
115.66.5**
ORX (n=12)

35.51.2
37.11.2

44.51.3** 41.91.5*

133.95.9
Control (n=12)

Cortex
Total
Right

Adrenal weights were significantly increased (15.9%;

P<0.05) 4weeks after orchiectomy (ORX) (Table1). The
relative weight of the left adrenal gland in control animals
was greater than that of the right one (P<0.05) and ORX
potentiated this dimorphism further (left adrenal: 16.5%
increase, P<0.01 vs. right adrenal: 15.2% increase,
P<0.05). The weight of the seminal vesicles and body
weight were significantly reduced in ORX animals (seminal vesicles: 73.6%, P<0.001; body weight:13.6%,
P<0.01), and ORX also resulted in a significant increase
in the pituitary weight of ORX animals (12.7%, P<0.05);
these findings indicate the efficacy of ORX.
Castration also stimulated metabolism in the adrenal
cortex (Table1). Biochemical analysis revealed that the
observed increase in adrenocortical weight was paralleled by increases in lipid content (62.7%, P<0.001) and
cholesterol synthesis (39.4%, P<0.001), indicative of
increased adrenosteroid biosynthesis. In addition, increases
in total protein content (69.4%, P<0.001) and DNA and
RNA contents (56.8%, P<0.01 and 69.4%, P<0.001,
respectively) of the adrenal cortex were observed after
ORX (Fig.1a), and ORX was accompanied by an increase
in the RNA/DNA ratio in the adrenal cortex (33.9%,
P<0.05; Fig.1b).
Notably, with the exception of the adrenal lipid contents, all of the above ORX-induced changes were restored

Left

Orchiectomy (ORX) alters various indices ofadrenal

function ina testosteronereversible manner

Body weight (g) Adrenal weight (mg/100g BW)

Results

Group

All numerical data are expressed as meanstandard error

of the mean (S.E.M.). Data were normally distributed and
values obtained were analyzed by one-way analysis of variance (ANOVA). Repeated measure tests were performed
using the Bonferroni test for multiple comparisons. Differences were considered significant when P<0.05. Statistical analyses were performed using GraphPad Prism v.5
software (GraphPad, San Diego, CA).

Table1Orchiectomy (ORX) increases adrenal and pituitary weights, adrenal lipid content and reduces seminal vesicles weight and body weight

Statistical analysis

Adrenal cholesterol Adrenal lipid (mg/100g

(mg/100g BW)
BW)

Seminal vesicles
(mg/100g BW)

Pituitary gland
(mg/100g
BW)

ethanol solutions, clearing in xylene, and mounting in DPX

mounting (Fluka, St Louis, USA). Images were obtained
with an Olympus BX60 microscope (Olympus, Hamburg,
Germany). Negative control sections were generated by
omitting the anti-AR (replaced with normal goat serum).
The number of AR-positive cells was counted and averaged within a defined region of 10,322m2. The degree of
staining was categorized according to the criteria described
above for evaluation of TUNEL-stained cells.

4.70.2

J Comp Physiol B (2014) 184:10551063

5.30.2*

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Control

ORX

(***)

ORX + T

RNA/DNA ratio

Biochemical parameters
(mg/adrenal cortex/100g BW)

1059

(***)
(#)

(**)

3
2
1
0

Total Protein

DNA

RNA

Fig.1Biochemical changes in the adrenal cortex of the sand

rat following orchiectomy (ORX) and testosterone replacement
(ORX+T). a Total protein, DNA and RNA contents, normalized to
adrenal cortex weight/100g BW. b Increased RNA/DNA ratio after

(*)

Control

ORX

ORX + T

ORX indicates hyperplasia and hypertrophy of the adrenal cortex.

Data are reported as meanSEM for 12 sand rats in each group.
*ORX vs. control; #ORX +T vs. control; *, #P<0.05; **P<0.01;
***P<0.001

Table2Orchiectomy-induced alterations in plasma levels of testosterone, cortisol, LH, and ACTH in the sand rat
Treatment group

Plasma concentration (ng/ml)

Testosterone

Cortisol

LH

ACTH

Control
ORX

462.646.7
115.619.9***

35.73.7
24.02.9*

5.70.7
34.23.3***

0.880.19
3.030.36***

ORX+T

499.844.5

37.22.5

6.30.9

1.480.18

Data shown are meanSEM for 12 sand rats in each group

*P<0.05; ***P<0.001 compared to control

to control levels after supplementation with testosterone

(ORX+T), as shown in Table1; Fig.1a , b.
Testosteronedependent modulation ofadrenocortical
hormone secretion

the zona reticularis where sex steroids are synthesized

(62.0%; P<0.001 vs. control; Fig.2e). All of the ORXinduced effects were reversed after supplementation of ORX
animals with testosterone (ORX+T; Fig.2c, f).
Adrenocortical tissue expresses androgen receptors (AR)

As shown in Table2, androgen deprivation by orchiectomy

(ORX) for 4weeks resulted in a significant decrease in the
plasma levels of cortisol (32.7%, P<0.05) and testosterone (75.0%, P<0.001). These changes were accompanied by significant increases in the circulating levels of LH
(83.2%, P<0.001) and ACTH (71.2%, P<0.001). Control levels of all these hormones were restored after supplementation of ORX animals with testosterone (ORX+T)
(Table2).
Altered adrenal cortex proliferation andapoptosis
Apoptosis in the adrenal cortex, detected by TUNEL histochemistry, was greatest (501.8% more than in controls)
in the innermost zone (zona reticularis) that synthesizes
androgens (Fig.2d). Orchiectomy (ORX) led to a significant
increase in the apoptotic index in the zona fasciculata which
synthesizes glucocorticoids (76.3% vs. control; P<0.001;
cf. Fig2a, b) and a concomitant decrease in apoptosis in

Immunoreactive androgen receptor (AR) was detectable

in the adrenal cortex (Fig.3); semi-quantitative evaluation of the immunohistochemical staining is summarized
in Table3. All experimental groups displayed strong
AR immunoreactivity in both, the zona glomerulosa and
zona fasciculata, and all groups showed only weak AR
staining in the zona reticularis. Immunoreactivity was
clearly restricted to cell nuclei within the zona glomerulosa (Fig.3a, d, g) and zona fasciculata (Fig.3b, e, h).
In contrast, the zona reticularis displayed only weak and
diffuse AR immunostaining (Fig.3c, f, i). After orchiectomy (ORX), immunoreactive AR signal was increased
in the zona glomerulosa and zona fasciculata (13%,
P<0.05 and 25.6%, P<0.01, respectively) (Fig.3d, e).
Testosterone replacement in ORX animals (ORX+T)
partly reversed the effects of ORX in the zona fasciculata (Fig.3h) and resulted in a slight upregulation of AR
expression in the zona reticularis (Fig.3i).

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J Comp Physiol B (2014) 184:10551063

Control

ORX

ORX + T

ZG

C
ZG

ZG

ZF

ZF

ZF

ZF

E
ZF

ZR

ZR

ZR
M

M
Fig.2Distribution of apoptotic cells in the adrenal cortex of the
testis-intact (control), orchiectomized (ORX) and testosteronesupplemented orchiectomized (ORX+T) sand rat. Apoptotic cells
labeled using TUNEL histochemistry; sections counterstained with
hematoxylin-eosin. Control (a, d), ORX (b, e), and ORX+T (c, f).
Black arrows indicate apoptotic nuclei; white arrows indicate normal nuclei. The highest apoptotic index was detected in the inner-

most zona reticularis of control animals (d). Androgen deprivation

by ORX induced an increase in apoptosis in the zona fasciculata (b),
an effect which gradually decreased toward the zona reticularis (e).
Inserts in (a) and (b) show TUNEL-negative and -positive controls,
respectively. ZG Zona Glomerulosa, ZF Zona Fasciculata, ZR Zona
Reticularis, M Medulla. Scale bar=20m

Discussion

and RNA/DNA ratio in the adrenal cortex. All of these

alterations may be attributed to ORX-induced stimulation
of pituitary adrenocorticotropic hormone (ACTH) secretion; ACTH is known to increase the weight and RNA, protein and DNA contents of the adrenal cortex (Alario etal.
1987), and to induce adrenal hypertrophy and hyperplasia
(Kobayashi etal. 2006; Hoeflich and Bielohuby 2009). Previous work demonstrated that seasonal changes in adrenocortical mass are strongly correlated with corticosteroid
secretion (Amirat etal. 1980). On the other hand, since
ORX increases the secretion of pituitary luteinizing hormone (LH) in the sand rat (see Table2) and other species,
the contribution of this gonadotropin to ORX-associated
adrenocortical hyperactivity cannot be discounted. This
view is supported by the fact that LH, acting through adrenocortical LH receptors (Carlson 2007), stimulates proliferation and activity of the adrenal cortex in humans (Rao etal.
2004).
Our results show that ORX induces structurefunction
remodeling of the adrenal cortex (cf. Freedman etal.
2013) involving both, cell proliferation and apoptosis.
Interestingly, ORX regulated apoptosis in a layer-specific fashion. Whereas ORX did not influence the rate of
apoptosis in the outermost layer of the adrenal cortex, the
zona glomerulosa which is the primary site of aldosterone
(mineralocorticoid) synthesis, the manipulation reduced

The primary aim of this study was to confirm our initial

observations that adrenocortical activity in the male sand
rat, Psammomys obesus (Benmouloud etal. 2006), like
in other rodents (Patchev and Almeida 1998; Viau 2002;
Handa and Weiser 2014), is sensitive to gonadal androgens and participates in the physiological regulation of the
reproductive system. In addition, we explored a potential
mechanism that could account for such effects.
Using a combination of surgical castration (ORX) and
testosterone supplementation therapy (ORX+T), we
show here that testicular steroids exert a strong influence
over adrenocortical activity in the sand rat. Specifically, we
found that castration induces an increase in adrenal weight
similar to that previously observed in laboratory mice
(Johnsen etal. 2006), rats (Duda etal. 1985) and rabbits
(Kandsi-Bouhadad and Hadj-Bekkouche 2010). This finding most likely reflects a proportional increase in the size of
the adrenal cortex (Duda etal. 1985). Indeed, ORX caused
a significant increase in the cholesterol and lipid content of
the adrenal cortex, suggesting stimulation of adrenocortical
steroidogenesis in the absence of testicular hormones.
The changes observed in the adrenal size after ORX may
be ascribed to hyperplasia and hypertrophy since they were
accompanied by increases in the DNA and RNA content

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J Comp Physiol B (2014) 184:10551063

1061

Control

ZG

ZF

ZR

ZF

ORX

ZG
ZF

ZF

ZR

ORX + T

ZG
ZF

ZR

ZF

Fig.3Immunoreactive androgen receptor (AR) in the adrenal cortex

of the intact, castrated (ORX) and testosterone-supplemented castrated (ORX+T) sand rats. Sections counterstained with hematoxylin-eosin. Black arrows show examples of AR-positive cell nuclei;
AR-negative cell nuclei are marked with white arrows. Control (a, b,
c), ORX (d, e, f), and ORX+T (g, h, i). Zona glomerulosa and outer
zona fasciculata are shown in a, d, g; zona fasciculata is shown in

b, e, h; zona reticularis is shown in c, f, i. Large numbers of ARpositive cells were observed in the zona glomerulosa and outer layers
of the zona fasciculata in testis-intact (control) animals (a, b). Orchiectomy (ORX) upregulated AR staining in the zona fasciculata (d, e).
ZG Zona Glomerulosa, ZF Zona Fasciculata, ZR Zona Reticularis, M
Medulla. The insert shows the negative control. Scale bar=10m

Table3Semi-quantitative assessment of apoptosis and androgen receptor (AR) staining in the adrenal cortex of the sand rat after orchiectomy
(ORX) and testosterone replacement (ORX+T)
Group

Control
ORX
ORX+T

Apoptotic index

Androgen receptor labeling index

Zona glomerulosa

Zona fasciculata

Zona reticularis

Zona glomerulosa

Zona fasciculata

Zona reticularis

+
+

+
++

+++
+

+++
+++

++
+++

++

+++

++

Crosses refer to positively stained cells: () no staining; (+) weakly positive staining of <25% cells; (++) moderately positive staining of
>25% cells; (+++) strongly positive staining of >50% cells

the occurrence of apoptosis in the innermost adrenocortical LH-responsive and sex steroid-synthesizing layer
(zona reticularis). In marked contrast, apoptosis was
upregulated by ORX in the zona fasciculata where cortisol (and corticosterone) is biosynthesized. This observation may account for the significantly reduced plasma
levels of cortisol in castrated sand rats (this study) and

laboratory rats (Malendowicz and Mlynarczyk 1982).

The fact that these changes occur in the face of elevated
plasma ACTH levels (Amirat and Brudieux 1993) and
adrenocortical hyperactivity suggests complex negative
feedback regulatory mechanisms at the level of the hypothalamo-pituitary unit; investigation of these mechanisms
deserves attention in future studies.

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The above-described ORX-induced cellular and biochemical adjustments in the sand rats adrenal cortex were
found to be reversible after testosterone substitution therapy (ORX+T). These data suggest that testosterone, the
main steroid secreted by the sand rat testes, exerts a significant inhibitory action on adrenocortical structure and
function. Indeed, previous studies in the laboratory rat have
described reciprocal cross-regulatory interactions between
gonadal steroids and glucocorticoids (Handa etal. 1994;
Almeida etal. 1997), the molecular basis of which is still a
subject of intense research (Bagamasbad and Denver 2011).
Although not determined here, we estimate that the adrenocortical adjustments to ORX+T required 34weeks to
become fully manifest; this estimate is based on similar
studies in laboratory rats (Bingaman etal. 1994) and mice
(Dalterio etal. 1983).
Notwithstanding that gonadal steroids (e.g., testosterone) can act at the hypothalamic and pituitary levels to
modulate adrenocortical activity (Patchev and Almeida
1998; Handa and Weiser 2014), an important question is
whether testicular steroids can act directly on adrenocortical cells. Testosterone (T) can either be aromatized to
estradiol or transformed into the potent androgen dihydrotestosterone (DHT) through the actions of 5- reductase. The cellular actions of both T and DHT are mediated
by androgen receptors (AR), but T is less potent than DHT
(Askew etal. 2007). The novel finding in this study was
that AR is strongly expressed in the zona glomerulosa of
testis-intact sand rats. Following ORX, AR expression
increases in the glucocorticoid-producing zona fasciculata
(no changes in the zona glomerulosa and zona reticularis)
in a T-reversible fashion, an observation that opens new
lines of research. Interestingly, previous authors working on the laboratory mouse suggested that DHT may
inhibit adrenocortical activity, albeit at the hypothalamicpituitary level, by activating estrogen receptors after its
3-hydroxysteroid dehyhrogenase-mediated conversion to
estrogenic 3-diol (Lund etal. 2004). Thus, an interesting
question to be answered in future is whether the adrenal
cortex of the sand rat has the enzymatic machinery to convert T into either DHT or 3-diol and expresses estrogen
receptors. It will also be of interest to examine whether
the role of adrenocortical AR extends beyond their herein
reported inhibitory effects on glucocorticoid synthesis and
secretion.
In summary, the results of this study provide the first
evidence that gonadal androgens modulate adrenocortical
activity in the sand rat by acting on AR located on cells
within the glucocorticoid-producing zona fasciculata. This
modulation is assumed to serve as a mechanism to maximize reproductive fitness and survival in the harsh desert
environment inhabited by this species.

13

J Comp Physiol B (2014) 184:10551063

Acknowledgments The authors thank all the personnel of BeniAbbes Station in the Algerian Sahara for trapping the sand rats used
in this study. They also thank M.T. Laurent, N. Mouterfi, and R. Stoffel for technical assistance. The support of the Algerian Ministry of
Higher Education and Scientific Research, the Max Planck Institute
of Psychiatry (Munich, Germany), the Life and Health Sciences
Research Institute (ICVS) of the University of Minho (Braga, Portugal) and the Universit Catholique de Lyon (France) is acknowledged.
Support from the AlgerianFrench collaborative Project 09 MDU 756
is also acknowledged.

References
Alario P, Gamallo A, Beato MJ, Trancho G (1987) Body weight gain,
food intake and adrenal development in chronic noise stressed
rats. Physiol Behav 40:2932
Almeida OFX, Canoine V, Ali S, Holsboer F, Patchev VK (1997)
Activational effects of gonadal steroids on hypothalamo-pituitary-adrenal regulation in the rat disclosed by response to dexamethasone suppression. J Neuroendocrinol l9:129134
Amirat Z, Brudieux R (1993) Seasonal changes in in vivo cortisol
response to ACTH and in plasma and pituitary concentrations
of ACTH in a desert rodent, the sand rat (Psammomys obesus).
Comp Biochem Physiol 104A:2934
Amirat Z, Khammar F, Brudieux R (1980) Seasonal changes in
plasma and adrenal concentrations of cortisol, corticosterone,
aldosterone and electrolytes in the adult male sand rat (Psammomys obesus). Gen Comp Endocrinol 40:3643
Askew EB, Gampe RT, Stanley TB, Faggart JL, Wilson EM (2007)
Modulation of androgen receptor activation function 2 by testosterone and dihydrotestosterone. J Biol Chem 282:2580125816
Bagamasbad P, Denver RJ (2011) Mechanisms and significance of
nuclear receptor auto- and cross-regulation. Gen Comp Endocrinol 170:317
Benmouloud A, Zahaf S, Khammar F, Amirat Z (2006) Influence de
la castration sur la surrnale du rat des sables Psammomys obesus
mle adulte. Bull Soc Hist Nat Afr Nord 73:197205
Bentvelsen FM, McPhaul MJ, Wilson CM, Wilson JD, George FW
(1996) Regulation of immunoreactive androgen receptor in the
adrenal gland of the adult rat. Endocrinology 137:26592663
Bingaman EW, Magnuson DJ, Gray TS, Handa RJ (1994) Androgen
inhibits the increases in hypothalamic corticotropin-releasing
hormone (CRH) and CRH-immunoreactivity following gonadectomy. Neuroendocrinology 59:228234
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction
and purification. Can J Biochem Physiol 37:911917
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 72:248254
Burke RW, Diamondstone BI, Velapoldi RA, Menis O (1974) Mechanisms of the Liebermann-Burchard and zak color reactions for
cholesterol. Clin Chem 20:794801
Carlson HE (2007) Human adrenal cortex hyperfunction due to
LH/hCG. Mol Cell Endocrinol 269:4650
Chang C, Yeh S, Lee SO, Chang T (2013) Androgen receptor (AR)
pathophysiological roles in androgen related diseases in skin,
bone/muscle, metabolic syndrome and neuron/immune systems:
lessons learned from mice lacking AR in specific cells. Nucl
Recep Signaling 11:126
Cretzschmar J (1828) In: Atlas zu der Reise im Nrdlichen Afrika von
Eduard Rppel, Sagethier, Frankfurt am Main. pp 5859
Dallman MF, Akana SF, Bhatnagar S, Bell ME, Choi S, Chu A, Horsley C, Levin N, Meijer O, Soriano LR, Strack AM, Viau V (1999)

J Comp Physiol B (2014) 184:10551063

Starvation: early signals, sensors and sequelae. Endocrinology
140:40154023
Dalterio SL, Mayfield DL, Michael SD, Macmillan BT, Bartke A
(1983) Effects of delta 9-THC and castration on behavior and
plasma hormone levels in male mice. Pharmacol Biochem Behav
18:8186
Daly M, Daly S (1973) On the feeding ecology of Psammomys obesus (Rodentia, Gerbillidae) in the Wadi Saoura, Algeria. Mammalia 37:545561
Duda T, Waliszewska A, Trzeciak WH, Malendowicz LK (1985) Sex
differences in adrenocortical structure and function. XX. The
effects of gonadectomy and testosterone or estradiol replacement
on cholesterol content and distribution in the gland. J Steroid
Biochem 23:577581
Freedman BD, Kempna PB, Carlone DL, Shah MS, Guagliardo NA,
Barrett PQ, Gomez-Sanchez CE, Majzoub JA, Breault DT (2013)
Adrenocortical zonation results from lineage conversion of differentiated zona glomerulosa cells. Dev Cell 26:666673
Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ: a specific labelling of nuclear DNA
fragmentation. J Cell Biol 119:493501
Gernigon T, Ablaoui R, Boudoucha D, Kandsi F (1992) Cytological
and biochemical effects of castration of a Saharan rodent with a
seasonal cycle Psammomys obesus. Bull Soc Zoo Fr 117(3):343
Handa RJ, Weiser MJ (2014) Gonadal steroid hormones and the
hypothalamo-pituitary-adrenal axis. Front Neuroendocrinol
35:197220
Handa RJ, Burgess LH, Kerr JE, OKeefe JA (1994) Gonadal steroid
hormone receptors and sex differences in the hypothalamo-pituitary-adrenal axis. Horm Behav 28:464476
Hirst JJ, West NB, Brenner RM, Novey MJ (1992) Steroid hormone
receptors in the adrenal glands of fetal and adult rhesus monkeys.
J Clin Endocrinol Metab 75:308314
Hoeflich A, Bielohuby M (2009) Mechanisms of adrenal gland
growth: signal integration by extracellular signal regulated
kinases1/2. J Mol Endocrinol 42:191203
Johnsen IK, Slawik M, Shapiro I, Hartmann MF, Wudy SA, Looyenga BD, Hammer GD, Reincke M, Beuschlein F (2006) Gonadectomy in mice of the inbred strain CE/J induces proliferation
of sub-capsular adrenal cells expressing gonadal marker genes. J
Endocrinol 190:4757
Kandsi-Bouhadad F, Hadj-Bekkouche F (2010) valuation du contenu surrnalien en androstnedione et effets de la castration
chez le lapin domestique (Oryctolagus cuniculus). C R Biologies
333:591596
Keegan CE, Hammer GD (2002) Recent insights into organogenesis
of the adrenal cortex. Trends Endocrinol Metab 13:200208
Khammar F, Brudieux R (1984) Seasonal changes in testicular contents of testosterone and androstenedione and in metabolic clearance rate of testosterone in the sand rat (Psammomys obesus). J
Reprod Fert 71:235241
Kitay JI (1975) Effects of various hormones on the pituitary-adrenal
axis. Adv Exp Med Bio 54:155167
Kobayashi H, Kambe F, Imai T, Hibi Y, Kikumori T, Ohmori S, Nakao
A, Seo H (2006) Differential expression of cyclin-dependent
kinase inhibitors, p27Kip1 and p57Kip2, by corticotropin in rat
adrenal cortex. J Endocrinol 189:671679
Lubahn DB, Josef DR, Sullivan PM, Willard HF, French FS, Wilson EM (1988) Cloning of human androgen receptor complementary DNA and localization to the X chromosome. Science
240:327330
Lund TD, Munson DJ, Haldy ME, Handa RJ (2004) Dihydrotestosterone may inhibit hypothalamo-pituitary-adrenal activity by

1063
acting through estrogen receptor in the male mouse. Neurosci
Lett 365:4347
Malendowicz LK, Mynarczyk W (1982) Sex differences in adrenocortical structure and function. X. Lipid and corticosterone in the
rat adrenal as affected by gonadectomy and testosterone or estradiol replacement. Endokrinologie 79:292300
Miller WL (1988) Molecular biology of steroid hormone synthesis.
Endocr Rev 9:295318
Patchev VK, Almeida OFX (1996) Gonadal steroids exert faciliting
and buffering effects on glucocorticoid-mediated transcriptional regulation of corticotropin-releasing hormone and corticosteroid receptor genes in rat brain. J Neurosci 16:70777084
Patchev VK, Almeida OFX (1998) Gender specificity in the neural
regulation of the response to stress: new leads from classical paradigms. Mol Neurobiol 16:6377
Rao CV, Zhou XL, Lei ZM (2004) Functional luteinizing hormone/
chorionic gonadotropin receptors in human adrenal cortical
H295R cells. Biol Reprod 71:579587
Raynaud J, Muller K, Schradin C (2012) Experimental increase of
testosterone levels in free-ranging juvenile male African striped
mice (Rhabdomys pumilio) induces physiological, morphological, and behavioral changes. Gen Comp Endocrinol 178:108115
Romero LM (2002) Seasonal changes in plasma glucocorticoid concentrations in free-living vertebrates. Gen Comp Endocrinol
128:124
Rossi R, Zatelli MC, Valentini A, Cavazzini P, Fallo F, del Senno L,
degli Uberti EC (1998) Evidence for androgen receptor gene
expression and growth inhibitory effect of dihydrotestosterone on
human adrenocortical cells. Endocrinology 159:373380
Schmidt EE, Schibler U (1995) Cell size regulation, a mechanism that
controls cellular RNA accumulation: consequence on regulation
of the ubiquitous transcription factor Oct1 and NF-Y and liver
enriched transcription factor DBP. J Cell Biol 28:467483
Schradin C (2008) Seasonal changes in testosterone and corticosterone levels in four social classes of a desert dwelling sociable
rodent. Horm Behav 53:573579
Shibko S, Koivistoven P, Tratnyek CA, Newhall AR, Friedman L
(1967) Method for sequential quantitative separation and determination of protein, RNA, DNA, lipid and glycogen from a single liver homogenate or from subcellular fraction. Anal Biochem
19:415528
Simard J, Ricketts ML, Gingras S, Soucy P, Feltus FA, Melner MH
(2005) Molecular biology of the 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene family. Endocr Rev
26:525582
Stalvey JRD (2002) Inhibition of 3-hydroxysteroid dehydrogenaseisomerase in mouse adrenal cells: a direct effect of testosterone.
Steroids 67:721731
Thomas JL, Bucholtz KM, Kacsoh B (2011) Selective inhibition of
human 3-hydroxysteroid dehydrogenase type 1 as a potential treatment for breast cancer. J Steroid Biochem Mol Biol
125:5765
Viau V (2002) Functional cross-talk between the hypothalamic-pituitary-gonadal and -adrenal axes. J Neuroendocrinol 14:506513
Winick M, Noble A (1965) Quantitative change in DNA, RNA and
protein during prenatal and postnatal growth in the rat. Dev Biol
12:451466
Wolkersdrfer GW, Bornstein SR (1998) Tissue remodelling in the
adrenal gland. Biochem Pharmacol 56:163171

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