Pituitary Adrenal Axis Activity in the Male Libyan Jird, Meriones Libycus: Seasonal Effects and Androgen Mediated Regulation

ISSN 0015-5497, e-ISSN 1734-9168 Folia Biologica (Kraków), vol. 65 (2017), No 2 https://doi.org/10.3409/fb65_2.95

Pituitary Adrenal Axis Activity in the Male Libyan Jird, Meriones libycus: Seasonal Effects and Androgen Mediated Regulation

Naouel AKNOUN-SAIL, Yamina ZATRA, Arezki KHEDDACHE, Elara MOUDILOU, Farida KHAMMAR, Jean-Marie EXBRAYAT, and Zaina AMIRAT

Accepted April 28, 2017 Published online September 06, 2017 Published September 20, 2017

AKNOUN-SAIL N., ZATRA Y., KHEDDACHE A., MOUDILOU E., KHAMMAR F., EXBRAYAT J.M., AMIRAT Z. 2017. Pituitary adrenal axis activity in the male Libyan jird, Meriones libycus: Seasonal effects and androgen mediated regulation. Folia Biologica (Kraków) 65: 95-105.

Many wild species exhibit seasonal cycles of testicular and adrenal functions suggesting important interrelationships between the two. However, data on desert rodents are very scarce. We investigated the pituitary adrenal (PA) axis activity of the Libyan jird from the Sahara desert, during the breeding and the non-breeding seasons. We explored, during the breeding season, the effects induced by orchidectomy and testosterone replacement (75 µg/40 µl sesame oil/100g BW). We found that the PA axis is more active during the non-breeding season and after orchidectomy with high ACTH and cortisol plasma concentrations (P<0.05). In the quiescent period, adrenal cells were hypertrophied in all cortical zonae (P<0.05), while orchidectomized jirds had thin zona fasciculata and thick zona reticularis with hypertrophied cells. Testosterone reduces the activity of the PA axis, probably by acting in the brain and/or pituitary. Here, we found that testosterone treatment led to a significant decrease in adrenocorticotropin levels together with structural remodeling of the adrenal cortex; analysis of androgen receptor (AR) densities in the adrenal cortex suggests that the AR mediates the direct effect of testosterone on adrenocortical morphology. This action may allow for homeostatic adjustments to occur, increasing individual fitness under extreme conditions in which this Saharan rodent species lives.

Key words: ACTH, cortisol, season, orchidectomy, testosterone replacement, Libyan jird.

Naouel AKNOUN-SAIL, Yamina ZATRA, Arezki KHEDDACHE, Farida KHAMMAR, Zaina AMIRAT, Arid Zones Research Laboratory (LRZA), Faculty of Biology, the University of Sciences and Technology Houari Boumediene (USTHB), BP 32, 16111, Algiers, Algeria. E-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected] Elara MOUDILOU, Jean-Marie EXBRAYAT, UMRS 449, Laboratory of General Biology, The Catholic University of Lyon (UCLy), Reproduction and Comparative Development, Ecole Pratique des Hautes Etudes (EPHE), 10 place des archives, 69288 Lyon Cedex 02, France E-mail: [email protected]; [email protected]

Environmental endocrinology has developed in energy balance but also increase in response to order to better understand how hormones modu- stress and could directly have an impact upon re- late and control physiological processes in animals productive function by inhibiting GnRH release exposed to extreme environmental conditions. (BUBENIK et al. 1999). Elsewhere, recent studies The adrenocortical function plays a crucial role in have shown that the adrenocortical response can response to physiological demand leading to the be controlled by various endocrine and paracrine peripheral release of corticosteroids. Mineralocor- factors which participate in remodeling and ticoids are involved in mineral homeostasis regu- changing functional zonation, such as the transfor- lation while glucocorticoids, mainly controlled by mation of glomerular cells into fascicular ones by adrenocorticotropic (ACTH) hormone, maintain subcapsular progenitor cells (PIHLAJOKI et al.

Ó Institute of Systematics and Evolution of Animals, PAS, Kraków, 2017 Open Access article distributed under the terms of the Creative Commons Attribution License (CC-BY) OPEN Ð ACCESS http://creativecommons.org/licences/by/4.0 96 N. AKNOUN-SAIL et al.

2015). The formation of ectopic gonadal-like tis- Psammomys obesus (AMIRAT et al. 1980). We sue in the adrenal gland, due to the action of LH found (unpublished personal data) 2-fold higher elevated in response to gonadectomy, can be concentrations for cortisol (50 ng/mg) vs corticos- viewed as an extreme example of adrenocortical terone (25 ng/mg) in adrenal tissue homogenate remodeling leading to an increased androstenedi- and 3-fold higher (28 ng/ml vs 8.7 ng/ml) at the cir- one level (R_HRIG et al. 2015). culating blood level. The aim of this study was to Small mammals living in arid lands require per- understand how testicular androgens can modulate manent physiological adjustments ensuring both the pituitary adrenal axis in response to the physio- survival and successful reproduction. In arid habi- logical needs of free-living Meriones libycus.We tats, rodents show seasonal reproductive activity compared PA axis activity during the reproductive only during the most favorable conditions cycle and explored the influence of orchidectomy (BELHOCINE et al. 2007; BOUFERMES et al. 2014; during the breeding season followed by testoster- FUENTES et al. 1991; GERNIGON et al. 1994; one replacement, upon the adrenal structure and KENAGY et al. 1999; KHAMMAR &BRUDIEUX morphometric changes occurring in the adrenal 1984, 1987). Seasonal reproduction is generally cortex, the immunolocalization of androgen re- correlated with adrenocortical activity as reported ceptors (ARs) in the adrenal cortical zonae, and the in the free-living degus Octodon degus (KENAGY patterns of plasma ACTH and cortisol concentra- et al. 1999), sand rat Psammomys obesus (AMIRAT tions. et al. 1980; KHAMMAR &BRUDIEUX 1984), viscacha Lagostomus maximus maximus (RIBES et al. 1999) and South African striped mice Rhabdomys Material and Methods pumilio (SCHRADIN 2008). The variation of glucocorticoid levels during the breeding season (re- Animals viewed in ROMERO 2002) might influence gonadal androgens. Interestingly, injection of tes- Experiments were carried out according to the tosterone was found to reduce corticosterone lev- guidelines of the Federation of European Labora- els in the free-living striped mice, Rhabdomys tory Animal Science Associations (FELASA), fol- pumilio, a south African desert species (RAYNAUD lowing approval by the Institutional Animal Care et al. 2012) and to increase cortisol levels, while Committee of the Algerian Higher Education and ACTH decreases in the Saharan sand rat Scientific Research. The permits and ethical rules (BENMOULOUD et al. 2014). These observations were in accordance to the Executive Decree n° 10-90 suggest functional relationships between the testes completing the Executive Decree n°04-82 of the and the adrenal cortex. In fact, many previous Algerian Government, establishing the terms and studies have explored gonad-adrenal relationships approval modalities of animal welfare in animal in order to explain the X-zone disappearance in facilities. Furthermore, it was approved by the local males after puberty (CRAMER &HORNING 1937) university ethical committee “Association Algéri- and sexual dimorphism affecting adrenal structure enne des Sciences en Experimentation Animale” AASEA and function as reported in many small mammals (Agreement Number 45/DGLPAG/DVA.SDA.14). (MALENDOWICZ &JACHIMOWICZ 1982; NIKICICZ Meriones libycus, commonly named the Libyan et al. 1984). Moreover, previous studies have re- jird, is a nocturnal, herbivorous and granivorous vealed a modulatory effect of sex steroids on pitui- Saharan gerbil. It is of great interest because of its tary adrenocortical response to ACTH under stress tolerance to water deprivation, thus providing an both for males and females in which androgens in- excellent model of adaptation to water restriction. hibit and estrogens stimulate this axis (HANDA et al. Jirds live in superficial burrows arranged under 1994a; VIAU &MEANEY 1996). Furthermore, large bushes of halophilic desert plants. They were AJDZANOVIC et al. (2015) reported that testoster- trapped using Sherman traps in the Beni-Abbes one decreases the capacity for ACTH and corticos- area (30° 07¢N;2°10¢W, 497 m elevation) in the terone secretion in a rat model of andropause. Algerian Sahara desert during the breeding season In the Libyan jird Meriones libycus, living in the (February-March) and the middle of the non- Sahara desert, reproduction is characterized by breeding season (September-October). After trap- a short breeding period, from the end of winter to ping, animals were systematically identified ac- the beginning of summer, followed by a long non- cording to the visual zoological criteria of PETTER breeding season from June to the middle of winter (1961) and LE BERRE (1990); this desert rodent is (BELHOCINE et al. 2007; BOUFERMES et al. 2014). characterized by a soft coat of light tawny color on Cortisol was the predominantly produced gluco- the back, white on the belly. The tail is as long as corticoid from the adrenals of this species, similar the body, and ends with a bundle of dark brown to reports in other desert rodents including Merio- hair. The plantar soles are covered with white nes unguiculatus (OLIVER &PERON 1964) and hairs, and the nails are clear. The tympanic bullae Pituitary Adrenal Axis of Libyan Jird 97 are hypertrophied. The captured females and juve- Morphometric analysis nile males were systematically released near their burrows. Only adult male jirds were used in our Adrenal glands were dehydrated in increasing study. They were weighed and housed in individual concentrations of ethanol, cleared in butanol and cages in a temperature-controlled room (20-22°C) embedded in paraffin. Sections of 5 µm thickness with a light/dark regime representing the natural were stained by routine Masson’s trichrome col- circadian L/D cycle (11/13 in February March; oration. The specificity of the coloration was con- 13/11 in September October). They were fed with firmed, and the sections were observed and barley, bread, dates, some carrots, and vegetables. photographed under a light microscope (Nikon This diet furnishes all body requirements without Eclipse E 400 connected to a Nikon DXM 1200 the necessity of providing a source of drinking wa- digital camera). Morphometric analysis was per- ter in the laboratory. Daily control of their health formed on six animals/experimental group using status and hygienic maintenance was applied dur- Axio vision software Rel. 4.6.3. Tissue thickness ing the duration of the experiment. was measured under a 10x objective from twenty serial sections randomly chosen from the middle adrenal gland including medullary tissue. Eight Experimental design measurements were taken around each adrenal section firstly for whole cortical thickness and then for All experiments began after one week of adapta- each zonae thickness. Measurements were aver- tion to captive conditions. Seven adult males were aged for each animal. Cell and nuclear areas were caught during the non-breeding season (nBC) and measured in 100 randomly chosen cells for each kept under conditions similar to that of breeding zonae /adrenal section under a 100x objective. control. During the breeding season, 21 M. libycus individuals were divided into three groups: breed- Androgen receptor (AR) immunohistochemistry ing control (BC, n=7), orchidectomized (ORX, n=7) and orchidectomized followed by testoster- Immunohistochemistry was performed by using one replacement (ORX+T, n=7). Fourteen animals the avidin-biotin complex method. Adrenal paraf- were orchidectomized bilaterally under anesthesia fin sections of 5 µm thickness were mounted on induced by intraperitoneal injections of ketamine sterile water coated superfrost glass slides hydrochloride (50 mg/kg, Ketalar, Pfizer, NY, (Thermo Scientific, Menzel-Gläser, Braussch- USA). Spermatic arteries were ligated and the tes- weig, Germany). Slide sections were deparaf- tes were rapidly removed. Muscular and cutaneous finized in cyclohexane, rehydrated sequentially in incisions were closed separately by silk sutures. 95% ethanol, 70% ethanol, distilled water and Surgery wounds were disinfected and sprinkled rinsed in 0.01 M phosphate buffered saline (PBS, with an antibiotic powder. The animals were then pH 7.4). After antigen retrieval in citrate buffer held under supervision until they awoke and then (pH 6.0) using a pressure cooker as described in put in individual cages (50 cm in length, 35 cm in the kit prospectus (Vector Antigen Unmasking So- width and 30 cm in height), for 50 days, under lutions, Vector Laboratories, CA, H 3300), the en- daily control. After this period, seven of them un- dogenous peroxidase activity was blocked with derwent hormonal replacement treatment by sub- 3% hydrogen peroxide for 10 min. In order to cutaneous injections of testosterone enanthate block non-specific site binding, the sections were (75 ìg/40 ìl sesame oil/100g BW, Androtardyl, washed in PBS pH 7.4 and then incubated with Bayer HealthCare) for seven days. normal horse serum for 20 min at room temperature. The sections were incubated overnight at 4°C At the end of the experiments, all animals were with primary antibody in a wet chamber. All anti- euthanized under anesthesia between 9:00 and bodies were provided by Santa Cruz Biotechnol- 11:00 a.m. The testes of control groups (nBC and ogy (Santa Cruz, CA, USA). A rabbit polyclonal BC) and the seminal vesicles of all animals were antibody (N20) raised against a peptide within the removed and weighed. Adrenal glands were N-terminal domain of the human AR (sc-816), quickly removed, cleaned from their surrounding diluted at 1:300 in PBS, was used as the primary fat and weighed. Right adrenal glands were fixed antibody. The slides were then washed in PBS and in 10% formalin solution for immunohistochemi- incubated with corresponding biotinylated secon- cal studies, while left ones were stored at -20 ºC in dary antibodies (Anti-Mouse IgG/Rabbit IgG; 1 ml of phosphate buffered saline (pH 7.4). Blood BA-1400, Vectastain Universal) for1hinawet was collected on ice, either in EDTA tubes for chamber. Visualization was performed by apply- plasma ACTH assay or in lithium heparinized ing the ready to use Vectastain® Elite ABC tubes for plasma cortisol assay. Biological sam- Reagent at room temperature for 30 min. The ples were stored at -20 ºC until assays performed in binding sites of the primary antibody were visu- the same series for each hormone. alized by using the chromogenic substrate 98 N. AKNOUN-SAIL et al.

3,3’-diaminobenzidine-tetra-hydrochloride (SK-4100, assay coefficients of variation for the ACTH as- DAB substrate kit for peroxidase) (DAB) and H2O2 say were respectively 3.6 and 7.2 %, and the assay solution for 1 min. Sections were counterstained sensitivity was less than 1.0 pg/ml. The intra- and with hematoxylin (Hematoxylin QS, H-3404; Vector inter-assay coefficients of variation for the corti- Laboratories, Inc. Burlingame, CA 94010, USA) sol assay were, respectively, 2-3 % and 1.4-1.6 %. for 30 s before dehydration in a series of graded The lower limit of cortisol detection was 0.018 ethanol solutions, and cleared in cyclohexane. Im- µg/dl. For the antibody derivate used in the kit of ages were captured by using a light microscope assay, the cross reactivities were 5.8% for corti- (Nikon Eclipse E 400 connected to a Nikon DXM costerone, 4.1% for 11-deoxycortisol, 1.50% for 1200 digital camera). Sections incubated with nor- 17á-hydroxyprogesterone and less than 1% for mal horse serum instead of primary antibody were all other tested compounds. used as negative controls. AR positive sections were defined as nuclear staining while cytoplasmic staining was non-specific. The number of AR- Statistical analysis positive cells was counted and averaged by using All numerical data are expressed as means ± Cell Target software, a bioinformatics application standard error of the mean (S.E.M.). Data were for morphometric analysis of cells (University of normally distributed and obtained values were Alcala, Spain). Positively stained cells vs. a total analyzed by one-way analysis of variance number of 100 cells in each of the adrenal zonae (ANOVA). The significance of differences be- were counted within a defined region of 45442.78 tween groups were verified using Tukey’s test µm2, under a x40 objective, from six animals/ex- which was considered significant when P<0.05. perimental group. The staining reaction was subdi- Statistical analyses were performed by using vided according to the percentage of stained nuclei GraphPad Prism v.5 software (GraphPad, San Di- as follows: Negative (-) = no nuclear immunostain- ego, CA). ing, weak staining (+) = less than 25% of positive nuclear staining, moderate staining (++) = 25-50% positive nuclear staining and strong staining (+++) Results = more than 50% of positive nuclear staining.

Body mass and organs weight Plasma ACTH and cortisol assay Data for weight parameters are summarized in ACTH and cortisol assays were performed ac- Table 1. Body mass, relative testes and vesicular cording to the electro-chemiluminescence immu- weights were significantly lower during the non- noassay ECLIA (Roche Diagnostics, Meylan, breeding season vs breeding season (-19% and France) protocol by using a Roche cobas e 411 ana- -46% respectively for BW and testes weight, P<0.05; lyzer for ACTH and an automated Elecsys 1010® -86%, P<0.001 for seminal vesicles weight). hormone analyzer for cortisol. The intra-and inter- However, the relative adrenal weight didn’t change

Table 1 Characteristics of some weight parameters in Libyan jird Meriones libycus during the reproductive cycle. Effect of orchidectomy and testosterone replacement. The body weight, testes and seminal vesicles were lower duringnon-breeding season while adrenal weight was higher. Or- chidectomy caused no significant effect on adrenal weight; however, testosterone replacement increased it

Body weight (BW) mg/100g BW Period of the year Animal groups (g) Seminal Vesicles Adrenals weight weight Testes weight Non-breeding season nBC 88±2* 27.8±1.3 54±4*** 833±28* BC 108±8 25.7±1.9 394±68 1529±215 Breeding season ORX 106±4 25.4±1.9 92±14** – ORX+T 93±3 30.6±1.6 378±25## –

nBC: non-breeding control, BC: breeding control, ORX: orchidectomized, T: testosterone replacement. Data is reported as mean±SEM, n=7 animals/group *nBC vs BC, *ORX vs BC. * P<0.05, **P<0.01, ***P<0.001 #ORX+T vs ORX. ## P<0.01. Pituitary Adrenal Axis of Libyan Jird 99 significantly between the non-breeding and the tex by a band of connective tissue which sends breeding periods (+8% in nBC vs BC; P>0.05). strands into the medulla and which is developed The weight of the seminal vesicles was used as a during the breeding season (Fig 1B). The zona reference in order to confirm androgen reduction glomerulosa occupied approximately 7-8 layers of together in the control non-breeding jirds and in vacuolated cells during the non-breeding season the orchidectomized animals. Vesicle weight was (Fig. 2A) and 4-5 layers during the breeding season drastically reduced 50 days after orchidectomy (Fig. 2C). The cell cords of the zona fasciculata (-76% of relative vesicular weight in ORX vs BC, were closely packed. In the outer half of this zona, P<0.01) and it was fully restored after 7 days of tes- the cells were highly vacuolated mainly during the tosterone replacement therapy (P<0.01) (Table 1). non-breeding season but larger than those of the The orchidectomy didn’t significantly influence zona glomerulosa (Fig. 2A). The vacuolization body mass or adrenal weight although testosterone markedly decreased in the inner half of the zona replacement caused a non-significant increase in fasciculata.Thezona reticularis, formed of deeply adrenal weight (+20% vs ORX). stained cells arranged in irregular short rows, is narrow during the breeding season (Fig. 2D) and larger during the non-breeding season (Fig. 2B). Histology of jird adrenal gland Orchidectomy induced cell disorganization in In the intact jird, histological study showed that the outer zonae with disappearance of cell cords in the adrenal cortex has a thick connective tissue the zona fasciculata and extreme vacuolization of capsule (Fig. 1A, 1B) and was divided into three glomerular and fasciculata cells (Fig. 2E). The zones as commonly described for all other mam- zona reticularis showed numerous small-sized mals. It’s arranged into three concentric zonae, the cells which were infiltrated with connective tissue outermost zona glomerulosa, the middle zona fas- cells with elongated nuclei (Fig. 2F). Testosterone ciculata and the inner zona reticularis (Fig. 1B). replacement restores the changes affecting adrenal The medulla was sharply demarcated from the cor- structure (Fig 2G, 2H).

Fig. 1. Structure of the adrenal gland in the Libyan jird Meriones libycus during breeding and non-breeding seasons. Effect of orchidectomy and testosterone replacement. Orchidectomy weakly increased the adrenal cortex (C) with a hypertrophy of the zona reticularis and regression of zona fasciculata. Testosterone replacement causes a further increase in adrenal cortex height with restoration of zona fasciculata status. A: nBC; B: BC, C: ORX, D: ORX+T. ZG: zona glomerulosa, ZF: zona fasciculata, ZR: zona reticularis. Scale bar: 100 = µm. 100 N. AKNOUN-SAIL et al.

nBC BC

ORX ORX+T

Fig. 2. Immunolocalization of androgen receptors (AR) in the adrenal cortex of Meriones libycus during breeding and non-breeding seasons. Non-breeding control nBC (A, B), BC (C, D), ORX (E, F), ORX+T (G, H). Zonae glomerulosa and fasciculata were shown in figure 2 (A, C, E, G) and zona reticularis in figure 2 (B, D, F, H). Red circles indicate immunostained nuclei, black circles indicate negatively stained (hematoxylin) nuclei. Large numbers of AR positive cells were observed in all zonae in nuclei; orchidectomy intensified the immunopositive AR in nuclei of zona reticularis and reduced it in the zona glomerulosa and fasciculata. Testosterone caused important labeling in all cortical zonae. Scale bar = 20 µm.

Morphometric analysis of the adrenal cortex by an enlarged cortex (+9% vs BC, P<0.01) (Table 2) compared to the breeding season (Fig. 1B). The Data for morphometric measurements are sum- zona glomerulosa was significantly more devel- marized in Table 2. During the non-breeding sea- oped (+26% vs breeding season, P<0.05) and the son, the adrenal gland (Fig. 1A) was characterized cell and nuclear areas were significantly higher in Pituitary Adrenal Axis of Libyan Jird 101 all cortical zonae (respectively +14% and +12% in Testosterone replacement induced an enlargement zona glomerulosa, P<0.01; +22%, P<0.001 and +6%, (Fig. 1D) of the adrenal cortex (+10%, P<0.01). It P<0.05 in zona fasciculata and +41%, P<0.001 clearly restored the zona fasciculata morphometry and +17%, P<0.01 in zona reticularis) (Table 2). (+66% in thickness, P<0.001 and +51% and +22% respectively in cell and nuclear areas, P<0.01) Orchidectomy induced a slight increase in the while no changes occurred in zonae glomerulosa adrenal cortex thickness (Fig. 1C) (+6%, P<0.05) and reticularis which showed a significant de- due to a significant hypertrophy of the zona reticu- crease in cell area (-13%, P<0.05) (Table 2). laris (+125%, P<0.001) associated with the regression of zona fasciculata (-31%, P<0.001) and Immunolocalization of ARs in the adrenal cor- a weak development of zona glomerulosa (+23%, tex of the Libyan jird P>0.05). Indeed, cell and nuclear areas were reduced after orchidectomy, respectively in the zona Semi-quantitative evaluation of the adrenal AR glomerulosa (-6%, -8% P<0.05) and especially in immunostaining is summarized in Table 3. Immu- the zona fasciculata (-21%, -24%, P<0.001), but noreactive ARs were detected in all adrenal zonae only cell area increased significantly in the zona although they were less abundant during the non- reticularis (+30%, P<0.001) (Table 2). breeding season as well in the zona glomerulosa

Table 2 Morphometric study of the adrenal cortex in the Libyan jird Meriones libycus during the reproductive cycle. Effect of orchidectomy and testosterone replacement. Adrenal cortex was higher in non-breeding season with hypertrophied cells of all cortical layers. Or- chidectomy weakly increased the adrenal cortex with significant hypertrophy of zona reticularis and regression of zona fasciculata, testosterone restores this effect. Cell and nuclear areas: orchidectomy reduced the cell status in zona glomerulosa and zona fasciculata when reticularis increased. Testosterone restores the effect Tissue thickness (µm) Cell and Nuclear areas (µm2) Groups ZG ZF ZR Total ZG ZF ZR Cortex CA NA CA NA CA NA nBC 1194±25** 110±3* 826±19 242±11 277±4** 94±1,3** 540±6*** 125±1.2* 404±4*** 117±1.3** BC 1092±16 87±12 824±10 206±9 243±5 84± 1.7 444±8 118±2.4 286±4 100±1.4 ORX 1156±34* 107±4 567±20*** 463±22*** 227±3* 77±1.3** 351±6*** 90±1.4** 366±6*** 91±1.6** ORX+T 1272±40## 99±2 944±27### 427±13 232±3 73±1.2 532±6## 110±1.6## 320±3# 94±1.2 BC: breeding control, nBC: non-breeding control, CA: cell area, NA: Nuclear area, ORX: orchidectomized, T: testosterone replacement. ZG: zona glomerulosa, ZF: zona fasciculata, ZR: zona reticularis. * nBC vs BC, * ORX vs BC. * P<0.05, **P<0.01, ***P<0.001 # ORX+T vs ORX. #P<0.05, ## P<0.01, ### P<0.001. Data used 6 animals/group with 20 adrenal serial sections for tissue thickness and 100 cells/zonae for cell and nuclear areas

Table 3 Semi-quantitative assessment of AR immunostaining in the adrenal cortex of the Libyan jird Meriones libycus during the breeding and non-breeding seasons. The immunostaining was less abundant in non-breeding (nBC) than breeding season (BC). Orchidectomy (ORX) reduces it in ZG and ZF and increases it in ZR. Testosterone replacement (ORX+T) intensified immunostaining strongly in all cortical layers Groups Zona glomerulosa Zona fasciculata Zona reticularis nBC Non-breeding season ++++ BC ++ +++ + ORX Breeding season + ++ +++ ORX+T +++ +++ +++

ZG: zona glomerulosa, ZF: zona fasciculata, ZR: zona reticularis. The intensity of ARs positive stained cells is expressed as a percentage: (-) no staining; (+) weakly positive staining of <25% cells; (++) moderately positive staining of >25 % cells; (+++) strongly positive staining of >50 % cells; Data used 6 animals/ experimental group. 102 N. AKNOUN-SAIL et al. and fasciculata (Fig. 2A) than in the zona reticu- Discussion laris (Fig. 2B), compared to the breeding control (Fig. 2C, 2D) for which positive AR immunoreac- Previous papers have reported that the Libyan tivity was found in all adrenocortical zonae. How- jird Meriones libycus is a seasonal breeder ever, the labeling was expressed moderately in the (BELHOCINE et al. 2007; BOUFERMES et al. 2014) zona glomerulosa and strongly in the zona fascicu- with a short breeding period during spring. In this lata (Fig. 2C), while the zona reticularis (Fig. 2D) paper, we reported for the first time that during the was weakly immunostained. non-breeding period (in autumn), the pituitary ad- Orchidectomy induced more immunostaining in renal axis of the male of this desert rodent was the zona reticularis (Fig. 2F) and reduced it in the more active than during the breeding season. In- outer zonae (Fig. 2E) while testosterone replace- deed, circulating ACTH as well as cortisol plasma ment caused important recurrence of labeling (Ta- concentrations were elevated when testicular ac- ble 3) in all cortical zonae (Fig. 2G, 2H). tivity was reduced. These results suggest negative relationships between male sexual hormones and pituitary adrenal axis. Therefore, we focused on ACTH and cortisol concentrations of the Libyan jird how adrenal androgens could modulate the activ- In the Libyan jird, plasma ACTH and cortisol ity of the PA axis involved in the response to the concentrations were about 5 folds higher (P<0.05) physiological homeostatic needs of the male Lib- during the non-breeding than during the breeding yan jird Meriones libycus, living in its natural Sa- season (Fig. 3) revealing important seasonal varia- haran environment. tions of the PA axis activity of this desert gerbil. Several papers have reported relationships be- Orchidectomy performed on sexually active jirds tween pituitary-adrenal axis activity and gonads also induced an increase in plasma concentrations (SEALE et al. 2004a). On the other hand, sexual of ACTH (148%, P<0.001) and cortisol (121%, differences in HPA axis activity are well estab- P<0.05) while testosterone replacement drasti- lished in several mammal species, such as sheep cally reduced (Fig. 3) plasma ACTH levels (-94%, (CANNY et al. 1999) and rodents (HANDA et al. P<0.001), whereas cortisol concentrations showed 1994a). These differences are partly due to circu- only a slight reduction (-26%, P>0.05) (Fig. 3). lating sex hormones (BURGESS &HANDA 1992; HANDA et al. 1994a). Our study shows an important role for the androgen testosterone in the sup- pression of adrenocortical activity in male jirds. Future studies should examine the role of female sex steroids on this parameter. Studies in laboratory rats reported that estrogens stimulate adrenocortical function and those females generally display higher PA axis activity. Unfortunately, in addition to the lack of information on female desert rodents, there is also very little data that refers to how rodents living in arid environments respond to environmental stress. Our results are at odds with previous studies in species showing seasonal glucocorticoid variation similar to that of androgens, in order to accommo- date and maintain energy for reproduction as reported in the sand rat Psammomys obesus (AMIRAT et al. 1980; KHAMMAR &BRUDIEUX 1984), in the male red deer Cervus elaphus (HUBER et al. 2003) and the viscacha Lagostomus maximus maximus (FILIPPA &MOHAMED 2006; RIBES et al. 1999). Nonetheless, our results agree Fig. 3. Plasma concentrations of cortisol (ng/ml) and ACTH with those found in the European beaver Castor fi- (pg/ml) during the reproductive cycle of Meriones libycus. ber L (CZERWINSKA et al. 2015), free living degus Plasma ACTH and cortisol were higher during the Octodon degus (KENAGY et al. 1999), the noctur- non-breeding season. Orchidectomy raised plasma concentrations of both ACTH and cortisol. Testosterone nal garden dormouse (BOULOUARD 1971) and the replacement restores this effect. male South African striped mouse Rhabdomys nBC: non-breeding control, BC: breeding control, ORX: orchidectomy, T: testosterone replacement. pumilio (SCHRADIN 2008). *: nBC vs BC; ORX vs BC, * P<0.05, **P<0.01 #ORX+T vs ORX, ### P<0.001. During the non-breeding period, the increase in Data used 7 animals /group adrenocortical thickness was due, in part, to cell Pituitary Adrenal Axis of Libyan Jird 103 hypertrophy in the three zonae, while plasma pituitary cells, especially in corticotroph cells. In- ACTH and cortisol concentrations were enhanced. deed, some previous studies have shown that few This observation is probably related to the need of if any ARs were present in corticotrophs glucocorticoid and mineralocorticoid hormones in (SHERIDAN &HERBERT 1980). O’HARA et al. order to maintain adequate homeostasis under un- (2015) also reported an increase in the percentage favorable environmental conditions (outside of the volume of the corticotroph pituitary cell type in breeding season). AR knock-out mice. However, others studies have On the other hand, during the breeding season, reported the colocalization of ARs with cortico- orchidectomy induced regression of zona fascicu- troph cells, suggesting the direct effect of andro- lata thickness due to cell area decrease, probably gens on corticotroph function although the associated with apoptotic cells, as reported in molecular mechanism remains unclear (MAEJIMA Psammomys obesus living in the same desert area et al. 2009). (BENMOULOUD et al. 2014) while the zona reticu- In our rodent model, further investigations are laris became hypertrophied. In future studies, necessary to emphasize the localization of the an- TUNEL assays and BrDU labeling could confirm drogen receptors at the corticotroph level. We also if the morphometric changes occurring in the adre- believe that the presence of androgen receptors in nal cortex zonae result from a decrease in apopto- the three adrenocortical layers is largely involved sis and/or the cell proliferation increase, as in the structural remodeling of the gland rather observed in the Androgen Receptor Knock out than the regulation of steroidogenesis which may (ARKo) mice (MIYAMOTO et al. 2007) or in the or- be regulated by pituitary ACTH. This fact does not chidectomized sand rat (BENMOULOUD et al. 2014). exclude the role of testicular androgens on steroi- The hypertrophy of zona reticularis could be due dogenesis by interfering with other factors which to the need for androgens in order to cope with lack deserve to be studied. of gonadal steroids occurring after gonadectomy Some papers have shown that testicular andro- (KANDSI-BOUHADAD &HADJ-BEKKOUCHE 2010). gens can act either on adrenocortical enzymes, Our results suggest an inhibitory effect of testicular suchas21and11â-hydroxylases (PROVENCHER androgens on glucocorticoid activity as reported et al. 1992), 3âHSD (STALVEY 2002), or on by VIAU and MEANEY (1996) in laboratory rats. growth factors. Recently JOPEK et al. (2017) Exogenous testosterone replacement reduced showed that testosterone replacement stimulates the pituitary adrenal axis activity. ACTH fell dras- expression of numerous genes in the rat, mainly tically, whereas the structure of the zona fascicu- those associated with lipids and cholesterol me- lata was restored. Slight zona reticularis tabolism. Testosterone could also reduce adrenal hyperplasia occurred since thick zona with small- sensitivity to ACTH (NOWAK et al. 1995) via ARs sized cells was observed. The influence of testos- present in the adrenocortical cells (BENMOULOUD terone on cortical cell remodeling may depend on et al. 2014; ROSSI et al. 1998; TREJTER et al. the ability of androgen receptors localized in the 2015). In the desert rodent, Psammomys obesus, adrenocortical zona to interact with the circulating AMIRAT and BRUDIEUX (1993) reported a reduc- testosterone hormone. tion in adrenal sensitivity to ACTH during spring Several studies have shown that testosterone de- when testosterone production was higher in this creases the corticosterone response to stress via species (KHAMMAR &BRUDIEUX 1984). Such a AR-mediated central mechanisms involving both phenomenon could also occur in the Libyan jird as CRH and AVP (VIAU &MEANEY 1996; LUND et suggested by immunostaining after testosterone al. 2004). In fact, the CRH contents and expres- treatment in all the adrenal zonae. sion, as well as the number of immunoreactive Recent studies have indicated that adrenal con- neurons, increased in gonadectomized male trol is under the influence of various endo- (SEALE et al. 2004b). At the pituitary level, the crine/paracrine factors, allowing the remodeling overproduction of ACTH observed in the and zonation of the adrenal cortex. However, the gonadectomized jird was probably driven by im- molecular basis of this process remains unknown paired negative feed-back of testosterone through (PIHLAJOKI et al. 2015). Factors involved in this the HPA axis. differentiation are expressed by stem/progenitor Numerous data have shown the inhibitory effect cells, but they can also be generated from other of androgens on adrenocorticotropic hormone molecules. The stem/progenitor cells reside either (AJDZANOVIC et al. 2015; HANDA et al. 1994a) in the adrenal capsule or in the sub-capsular region and the level of expression of the POMC transcript and can alter their shape, structure and hormone (HANDA et al. 1994b; SHARMA &CHATURVEDI secretions in response to physiological needs and 2011). However, the mechanism by which testos- experimental manipulations. terone acts remains unclear. Few studies have re- In conclusion, this study provides further evi- ported the expression of AR immunoreactivity in dence that in the male Libyan jird inhabiting Sa- 104 N. AKNOUN-SAIL et al. hara desert areas, adrenocortical activity is BENMOULOUD A., AMIRAT Z., KHAMMAR F., PATCHEV negatively correlated with testicular activity dur- A.V., EXBRAYAT J.M., ALMEIDA O.F. 2014. Androgen receptor-mediated regulation of adrenocortical activity in ing the annual reproductive cycle. The orchidec- the sand rat, Psammomys obesus. J. Comp. Physiol. B. 184: tomy followed by testosterone replacement 1055-1063. performed during the sexual season suggests a BOUFERMES R., RICHARD N., LE MOGUEN K., AMIRAT Z., strong inhibitory action of the testicular androgens KHAMMAR F., KOTTLER M.L. 2014. Seasonal expression of which act firstly at the pituitary level and also di- KiSS-1 and the pituitary gonadotropins LHâ and FSHâ in adult male Libyan jird (Meriones libycus). Anim. Reprod. rectly via their own receptors present in all adreno- Sci. 147: 56-63. cortical zonae. These actions lead especially to the BOULOUARD R. 1971. Study of Adrenocortical Function Dur- remodeling of the adrenal cortex activity, probably ing the Annual Cycle in a Hibernant: The Garden Dormouse by triggering paracrine factors that drive the re- (Eliomys Quercinus L.). II. Activity During Autumn: Period lease of steroids required to maintain adequate ad- of Hibernation Preparation. J. Physiol-Paris 63: 77-86. aptation in response to extreme physiological BUBENIK G.A., BROWN R.D., SCHAMS D., BARTOS L., 1999. The effect of ACTH on the GnRH-induced release of LH and demands under arid environment. testosterone in male white-tailed deer. Comp. Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol. 122: 173-179. BURGESS L.H., HANDA R.J. 1992. Chronic estrogen-induced alterations in adrenocorticotropin and corticosterone secre- Acknowledgements tion, and glucocorticoid receptor-mediated functions in female rats. Endocrinology 131: 1261-1269. The Authors declare no conflicts of interest. We CANNY B.J., O’FARRELL K.A., CLARKE I.J., TILBROOK A.J. 1999. The influence of sex and gonadectomy on the hypotha- are grateful to the staff of Research station of Bé- lamo-pituitary-adrenal axis of the sheep. J. Endocrinol. 162: ni Abbès, in the Algerian Sahara desert, for their 215-225. help in trapping jirds. We want also to thank CRAMER W., HORNING E. 1937. On the relationship between C. BOUCHOT in Lyon for her technical assistance, the male gonads and the adrenal gland. The Lancet. 5: O. SAIL for his help and T. KHALDOUN for the 1330-1331. graphs. Comments by two anonymous reviewers CZERWINSKA J., CHOJNOWSKA K., KAMINSKI T., BOGACKA I., PANASIEWICZ G., SMOLINSKA N., KAMINSKA B. 2015. significantly improved our manuscript. 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