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Acta Histochemica 116 (2014) 559–569

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Acta Histochemica

jo ur nal homepage: www.elsev ier .de /ac th is

mmunolocalization of estrogen and androgen receptors in the caputpididymidis of the fat sand rat (Psammomys obesus): Effects ofeasonal variations, castration and efferent duct ligation

afik Menada,∗, Souaâd Smaïa, Elara Moudilouc, Farida Khammarb,ean-Marie Exbrayatc,∗∗, Thérèse Gernigon-Spychalowicza

Small Vertebrates’ Reproduction, Laboratory of Research on Arid Areas, Faculty of Biological Sciences, Houari Boumediene University of Sciences andechnology, El Alia, Algiers, AlgeriaMammal Ecophysiology, Laboratory of Research on Arid Areas, Faculty of Biological Sciences, Houari Boumediene University of Sciences and Technology,l Alia, Algiers, AlgeriaUniversity of Lyon, UMRS 449, Laboratory of General Biology, Catholic University of Lyon, Reproduction and Comparative Development/EPHE, Lyon Cedex2, France

r t i c l e i n f o

rticle history:eceived 12 August 2013eceived in revised form0 November 2013ccepted 13 November 2013

eywords:aput epididymidisstrogen receptorsndrogen receptors

mmunohistochemistry

a b s t r a c t

The fat sand rat (Psammomys obesus) is a model to study seasonal reproductive cycle changes and severalmetabolic disorders. In order to show a possible involvement of estrogens in the male reproductive func-tions, the expression of estrogen receptors (ESR1 and ESR2) and androgen receptor (AR) were investigatedin the caput epididymidis of fat sand rats during the breeding season, resting season, after castration, aftercastration followed by testosterone treatment, and after ligation of efferent ducts. In the breeding season,principal cells presented a strong immunostaining of AR in both nuclei and cytoplasm, a strong stainingof ESR1, mainly in the apical zone, and a strong immunoexpression of ESR2, mainly in nuclei. In the rest-ing season, a moderate immunostaining of AR in both cytoplasm and nuclei was observed. ESR1 stainingshowed a strong immunoreactivity in the nuclei. In contrast, the nuclei were negative for ESR2. After cas-tration, a low and selective signal distribution was observed: the nuclei were moderately positive for AR

at sand rat and ESR2, and negative for ESR1. After castration and testosterone treatment, an androgen-dependencefor AR and the restoration of ESR1 but not ESR2 immunoexpression were observed. After ligation of theefferent ducts, a considerable reduction of AR immunoreactivity was observed in contrast to ESR1 andESR2, which gave a strong immunostaining signal. These results illustrate the complexity of the regula-tion of the androgen and estrogen receptor expression in the epididymis and argue for the coexistenceof both androgenic and estrogenic pathways.

ntroduction

Epididymal development and physiology are regulated by aomplex interplay of hormones and testicular factors, among whichestosterone is crucial for epididymal functions (Ezer and Robaire,

002). However, some studies have provided compelling evidenceor the role of estrogens, in regulating the functions of the efferentucts and the epididymis (Hess, 2003; Shayu et al., 2005). Estrogen

∗ Corresponding author at: Reproduction des Petits Vertébrés, Laboratoire deecherche sur les Zones Arides, Faculté des Sciences Biologiques, Université desciences et de la Technologie Houari Boumédiene, DZ-16111 EL Alia, Algeria.∗∗ Corresponding author at: Université de Lyon, UMRS 449, Laboratoire de Biologieénérale, Université Catholique de Lyon, Laboratoire de Reproduction et Développe-ent Comparé, E.P.H.E. 69288 Lyon Cedex 02, France.

E-mail addresses: menadrafik@gmail.com (R. Menad),mexbrayat@univ-catholyon.fr (J.-M. Exbrayat).

065-1281/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.acthis.2013.11.004

© 2013 Elsevier GmbH. All rights reserved.

biosynthesis is catalyzed by a microsomal P450 enzyme complex,called aromatase, which is responsible for the irreversible trans-formation of androgens into estrogens. In the male reproductivetract of immature animals, estrogens are produced in Sertoli cells(Van der Molen et al., 1981), whereas in mature animals, they arepresent in germinal cells, in spermatozoa and in the Leydig cells(Payne et al., 1976; Levallet et al., 1998; Carreau et al., 2006, 2007).The epididymis and efferent ducts express moderate to high levelsof estrogen receptors (Hess et al., 1997; Mowa and Iwanaga, 2001)and estradiol (E2) is one of the key hormones regulating their func-tion (Hess et al., 2001a,b; Lee et al., 2001). The estrogens are ofmajor importance for male fertility. The inactivation of estrogenreceptor 1 (ESR1) in mice results in an abnormal epididymal phe-

notype (Eddy et al., 1996) and infertility (Hess et al., 1997) followingdefective fluid absorption in the efferent ducts. The estrogen recep-tor 2 (ESR2) knockout mice are fertile and have a normal epididymalphenotype (Krege et al., 1998). In several species, ESR2 is expressed

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60 R. Menad et al. / Acta Hist

n all epididymal regions (Yamashita, 2004), whereas the expres-ion of ESR1 is localized in efferent ducts and in different segmentsf the epididymis, depending on the species (Hess, 2003). ESR2 washown to be expressed in principal cells in mice (Zhou et al., 2002),tallions (Parlevliet et al., 2006) and boars (Pearl et al., 2007).

It has been demonstrated that estrogens play an important rolen the absorption of luminal fluid and in pubertal developmentParlevliet et al., 2006). They also have other functions, includingegulating the expression of lactoferrin, cystatin 12, and oxytocineceptor in rabbits (Yu and Chen, 1993; Filippi et al., 2002; Li et al.,005) or of androgen receptors and ESR1 in rats (Oliveira et al.,004).

Androgens control epididymal activity. Castration and efferentuct ligation result in the reduction of epididymal weight, cellulartrophy, tissue remodeling and changes in gene expression. A mas-ive apoptosis in the epididymis following such manipulations waseported in Sprague-Dawley rats beginning 18 h after surgery in thenitial segment and it ending on the seventh day in the distal epi-idymis (Fan and Robaire, 1998). Androgen receptors are present

n principal cells of all epididymal regions in mice (Zhou et al.,002), goats (Goyal et al., 1997), rats (Zhu et al., 2000), and stal-

ions (Parlevliet et al., 2006). They are expressed in principal cells,asal cells and the smooth muscle cells in boars (Pearl et al., 2007).ndrogens are essential for the maintenance of the principal cellorphology and prevention of apoptosis (Fan and Robaire, 1998;

zer and Robaire, 2002). They regulate the expression of proteinsnvolved in motility, storage and membrane maturation of sperma-ozoa (Briz et al., 1995; Syntin et al., 1999; Castellon and Huidobro,999). Androgens also control the expression of androgen receptorsGoyal et al., 1998; Zhu et al., 2000; Oliveira et al., 2004).

The fat sand rat (Psammomys obesus) is a diurnal rodent whichives in the North-West of the Algerian Sahara, near Wadis. Itas a seasonal reproduction cycle with a breeding period fromutumn through early spring and a resting phase from late springhrough summer. This cycle has been described in both hormonalKhammar, 1987) and cytophysiological terms (Gernigon et al.,991; Gernigon, 1992; Menad, 2008). The aim of this study waso investigate the immunoexpression of estrogen and androgeneceptors, as well as their seasonal and castration-induced varia-ions in fat sand rats (P. obesus) as a model to study seasonal changesn the reproductive cycle and several metabolic disorders.

aterial and methods

nimals and samples

Adult male fat sand rats (P. obesus) of average weight 145 g wererapped in the wild region of Béni Abbès (30◦07′ N 2◦10′ W) dur-ng the breeding (n = 30) or resting season (n = 8). Eight out of 30nimals caught during the breeding season and all the eight ani-als caught during the resting season (groups 1 and 2 respectively)ere euthanized 48 h after capture, their caput epididymides were

uickly excised, cleaned from the surrounding fat and weighed.ut of the remaining 22 animals caught during the breeding season,ight were castrated, eight castrated and treated with testosterone,nd six subject to efferent duct ligation before euthanasia (groups 3,, and 5, respectively). All experiments were carried out in compli-nce with the guidelines of the Federation of European Laboratorynimal Science Associations (FELASA) following approval by the

ocal Ethical Committee of the Houari Boumediene University ofciences and Technology, Algeria.

astration and hormonal replacement

Eight adult male fat sand rats (P. obesus) were bilaterally cas-rated (group 3). Surgical procedures were performed under sterile

ica 116 (2014) 559–569

conditions. Animals were anesthetized by an intraperitoneal injec-tion of ketamine hydrochloride (50 mg/kg, Ketalar, Pfizer, NY, USA)and xylazine hydrochloride (10 mg/kg, Rompun, Bayer, Toronto,Canada). The efferent ducts and the testicular vessels were ligatedclose to the testes, which were then removed. The epididymideswere returned to the scrotum and the incisions closed with silksutures. The animals were left to recover for 30 days, kept on a nat-ural diet consisting of Chenopodiacae (low-calorie salt bush rich insalt and water) as described elsewhere (Daly and Daly, 1973), inconditions corresponding to their natural environment in termsof temperature and the photoperiod. After 30 days, they wereeuthanized. The capita epididymidis were subsequently removed,weighed and prepared for histological and immunohistochemicalanalyses.

A further group of eight animals (group 4) were castrated in thesame manner. However, after 30 days and prior to euthanasia, theywere submitted to daily intramuscular injections of testosteroneoenantate (75 Ug per day dissolved in 40 �l olive oil) for 15 days ashormonal replacement.

Ligation of efferent ducts

Bilateral ligation of the efferent ducts for one month was car-ried out following castration on a group of six animals (group 5).After anesthesia, the testes and epididymides of rats were exposedthrough an incision of the anterior abdominal wall. Using a magni-fying glass, a ligature was carefully placed around both right and leftefferent ducts. The ligation was made simultaneously with castra-tion and animals were maintained in the same conditions as thoseof groups 3 and 4 (castrated only or castrated and treated).

Histology

In all groups of animals, the epididymides were removed aftereuthanasia (in the morning), fixed in Bouin’s solution, dehydratedin increasing concentrations of ethanol (70%, 95%, and 100%),cleared in toluene, and finally embedded in paraffin wax. Sections(5 �m thick) were cut with a Leitz microtome and mounted onhistological slides or Superfrost® glass slides (Thermo Scientific,Menzel-Gläser, Brausschweig, Germany) for immunohistochem-istry. All the organs were cut according to the sagittal plan to retainregionalization as previously described by Menad (2008). Afterhydration, the sections were stained with Masson’s trichrome asdescribed elsewhere (Martoja and Martoja, 1967; Gabe, 1968).

Morphometry and statistical analysis

A morphometric study of histological slides was carried outusing a calibrated Zeiss microscope. The principal cell height wasmeasured on 150 cells per batch using a 40× objective. Resultswere expressed as means ± standard measurement error (SEM).Comparisons were performed using a one-way variance analysis(Anova) followed by the Scheffe post hoc test. P values lower than0.05 (P < 0.05) were considered as indicative of a significant dif-ference. All calculations were performed using the Origin pro 7.5software program (OriginLab Corp., Northampton, MA, USA).

Immunohistochemistry

The expression of estrogen receptors (ESR1 and ESR2) andandrogen receptors (AR) was analyzed by immunohistochemistry.Sections were deparaffinized with cyclohexane and rehydrated

with decreasing concentrations of ethanol. The slides were thenwashed in tap water for 10 min. For antigen retrieval, the slideswere incubated at 95 ◦C in a 10 mM sodium citrate solution (H-3300, pH 6.0) for 45 min (for ESR1 and ESR2 analysis) or 30 min

ochemica 116 (2014) 559–569 561

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Fig. 1. Effects of seasonal variations, castration, castration then testosterone treat-ments and efferent duct ligation on weight of the caput epididymidis. (BS) Breedingseason, (RS) resting season, (C) castrated, (Ct) castrated then treated animals, ani-

thick, the intertubular space contained tight and dense connectivefibers grouped into bundles, and many fibroblasts and blood vessels(Fig. 3f). The epithelial tube was mainly composed of principal cellswith a highly significant reduction in height (12.03 ± 0.275 �m,

Fig. 2. Effects of seasonal variations, castration, castration then testosterone treat-

R. Menad et al. / Acta Hist

for AR). After heating, the slides were left to cool down for 20 minnd then washed in distilled water. Endogenous peroxidase activityas quenched with a 3% peroxidase solution for 20 min followed

y two baths in distilled water. Sections were encircled using aakoPen (Dako, Glostrup, Denmark) and incubated with a 10% nor-al goat serum (S-1000) for 1 h at room temperature to block

on-specific binding sites. Afterwards, the slides were incubatedith primary polyclonal antibodies against human AR (ab74272,bcam plc, Cambridge, UK), ESR1 (H-184: sc-7207, Santa Cruziotechnology, Santa Cruz, CA, USA), or ESR2 (H-150: sc-8974,anta Cruz Biotechnology, USA). All antibodies were used as 1:50ilutions in PBS. Next, tissue sections were incubated overnightt 4 ◦C in a wet chamber. Then, the slides were washed in PBSolution and incubated with corresponding secondary biotinylatedntibodies (Anti-Mouse IgG/Rabbit IgG; BA-1400, Vectastain Uni-ersal, Vector Laboratories, Burlingame, CA, USA) for 1 h in a wethamber. After rinsing three times in PBS for 5 min, slides werencubated with a streptavidin-biotin-peroxidase complex for 1 h.ach tissue section was washed in PBS and stained by the DABhromogen (3,3′-diaminobenzidine, kit for peroxidase; Vector Lab-ratories) for 1 min. The reaction was stopped by rinsing in PBSolution. Tissue sections were then counterstained with hema-oxylin (Hematoxylin QS, H-3404; Vector Laboratories) for 1 min,ehydrated and preserved using the Permount mounting mediumThermo Fisher Scientific, Waltham, MA, USA). The immunostain-ng was observed under a Nikon Eclipse E 400 light microscope withhe Nikon DXM 1200 digital camera. Samples incubated with nor-

al goat serum instead of the primary antibody served as negativeontrols. The intensity of the immunostaining was scored as null−), weakly positive (+), moderately positive (++) or strongly pos-tive (+++) by two independent observers blinded to the receptorype under analysis.

esults

The effects of the seasonal reproductive cycle as well as of castra-ion, castration followed by testosterone treatment and of efferentuct ligation on tissue remodeling and the immunoexpression ofSR1, ESR2 and AR were studied in fat sand rats.

aput epididymidis weight

The weight of the caput epididymidis varied depending on thehysiological state of the animals (Fig. 1). In spite of these varia-ions, the variance analysis by the Scheffe post hoc test did not showignificant differences in mean weight between particular groups:reeding season (BS), resting season (RS), castrated (C), castratednd treated animals (Ct) and animals having undergone efferentuct ligation (EDL).

istology

During the breeding season, the caput epididymidis of fat sandats was highly convoluted. It consisted of several sections con-aining spermatozoa surrounded by intertubular connective tissueomposed of scattered connective fibers, fibroblasts and blood ves-els (Fig. 3a). The tube was surrounded by a thin layer of smoothuscle tissue with adherent pseudostratified epithelium com-

osed mainly of principal cells (19.69 ± 0.534 �m in height; Fig. 2)ith microvilli. This epithelium also included basal cells, apical

ells, clear cells and halo cells (Fig. 3b). However, the principalells were the most numerous and occupied almost the entire

pithelium. The basal cells, characterized by a dense ovoid nucleus,ere discontinuously and irregularly distributed along the basal

amina. During the resting season, the caput epididymidis had amall diameter and the lumen was devoid of sperm (Fig. 3c). The

mals having undergone efferent duct ligation (EDL). BS = reference group. Probabilityfor all pairs (BS-RS, BS-C, BS-CT, BS-EDL) > 0.05. Samples sizes (n): BS = 8, RS = 8, C = 8,CT = 8, EDL = 6.

epithelial tube was mainly composed of principal cells with a sig-nificantly reduced height (11.21 ± 0.21 �m, p < 0.001; Fig. 2). Theconnective tissue and smooth muscle layer surrounding the tubewere very developed and the outer serous membrane was verythick. The intertubular space contained tight and dense connec-tive fibers grouped into bundles surrounded by many fibroblastsand blood vessels (Fig. 3d). In the animals caught and castratedduring the breeding season, the caput epididymidis showed epithe-lial alterations, a connective intertubular tissue increase and adecrease in tube diameter (Fig. 3e). Like during the resting sea-son, the serous membrane outside the caput epididymidis was very

ments and efferent duct ligation on height of principal cells. (BS) Breeding season,(RS) resting season, (C) castrated, (Ct) castrated then treated animals, animalshaving undergone efferent duct ligation (EDL). BS = reference group. Probability;BS-RS < 0.001, BS-C < 0.001, 0.01 > BS-CT > 0.001, BS-EDL < 0.001, Sample size (n) ofeach batch = 150.

562 R. Menad et al. / Acta Histochemica 116 (2014) 559–569

Fig. 3. Histology of caput epididymidis of sand rat Psammomys obesus. (a and b) Breeding season: (a) general appearance showing a whole of large epithelial tubes (ept)maintained among themselves it by a connective tissue (ct) (green color) and connective capsule (cc). (b) Pseudostratified epithelium presents a various epithelial cells; pc:principal cells witch are most numerous, bc: basal cells, hc: halo cells, ac: apical cells. In each epithelial section, sperms are presents (spz). The connective tissue surroundingthe epithelial tube is reduced. (c and d) Resting season: (c) general topography showing two portion; caput epididymidis (pe) and efferent ducts (efd). (d) Epididymis showsa developed connective tissue (green color) and smooth muscle cells (smc). Epithelial tube is narrow and formed with reduced epithelium height surrounding lumen (lu)deprived of sperm. (e and f) Castrated animals: (e) general appearance present a predominance of connective tissue, (f) compartments of epididymis showing very denseconnective tissue surrounding a hollow epithelial tube with narrow and empty lumen covered by a disorganized epithelium (dep). (g and h) Castrated then treated animals:(g) general topography showing a clear reduction of connective tissue. (h) Epithelium presents numerous alterations (epa) and apocrine secretion (as). (i and j) Animalh minac

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aving undergone ligation of efferent ducts: (i) general appearance showing a predoontaining a stained substance and extruded cells (exc).

< 0.001; Fig. 2). It was characterized by a small diameter and thebsence of sperm. The smooth muscle layer surrounding the tubeas developed and the basal lamina separating the epithelium wasrinkled. The nuclei located at the basal pole were superimposed,

iving the appearance of a stratified epithelium. In some sections,he principal cells preserved their secretory character. In animalshich were castrated and then treated with testosterone, the struc-

ure of the caput epididymidis was partially restored (Fig. 3g and), principal cells showed a very significant increase in height21.53 ± 0.198 �m, p < 0.01; Fig. 2) and their nuclei were locatedentrally. The apocrine secretion was also restored. The inter-

ubular connective tissue was less developed than in non-treatedastrated animals. Different cell types and a few extruded cells werebserved. Some alterations persisted in several sections and theonnective tissue was scarce (Fig. 3h). In animals having undergone

nce of connective tissue (green color), (j) Atrophied epithelium surrounding lumen

a ligation of efferent ducts during the breeding season, various his-tological effects were observed in the caput epididymidis after onemonth. In the ligature zone, two regions were visible: the first pre-sented a large lumen with reduced epithelium, and the second onea narrow lumen and principal cells of normal height (Fig. 3i and j).In the first region, the sperm were absent and a highly significantdecrease of principal cell height was observed (10.88 ± 0.141 �m;p < 0.001; Fig. 2). It contained many extruded cells and developedconnective tissue (Fig. 3j). No effects were observed near the regionof the ligature zone.

Immunohistochemistry

All results of immunohistochemistry for estrogen and androgenreceptors are summarized in Table 1.

R. Menad et al. / Acta Histochemica 116 (2014) 559–569 563

Table 1Immunolocalization of estrogen receptors (ESR1, ESR2) and androgen receptors (AR) in caput epididymidis.

Localizations Breeding S Resting S Castrated Castrated +T Ligatured

ESR1PC N ++/− +++/− +/− ++/− +++/−

C +++ AC ++/AC + ++ +++/−BC N ++ ++ − ++ +++SMC N − + − + −F N − + − ++ −Lu Spz +/− / / / /

EC − + / / N+++/−, C+

ESR2PC N +++/− − +/− − +++/−

C − ++ − ++ +++BC N +++ − + +/− +++SMC N + ++ + − /F N +++ +/− + − /Lu Spz + / / / /

EC / / / / /

ARPC N +++ ++ +/− ++/− ++/+/−

C +++ G ++ +/− +++ G +BC N ++/− ++ + ++/− ++SMC N ++ +++ + + +F N ++ − + + −Lu Spz + / / / /

EC / / +/− +/− /

S MC: sC

E

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: season, T: testosterone treatment, PC: principal cells, BC: basal cells, F: fibroblast, S: cytoplasm, N: nucleus, G: granular.

strogen receptor 1 (ESR1)

During the breeding period, ESR1 showed a pronouncedmmunoreactivity in the apical cytoplasm of principal cells inhe caput epididymidis. The nuclei of these cells showed mod-rate (++) or null (−) staining (Fig. 4a). The nuclei of basal cellsere immunoreactive for ESR1. In the lumen, sperms were weakly

tained. During the resting period, the immunohistochemical sig-al was located in the nuclei and cytoplasm of principal cells with

higher intensity in the apical area. The staining intensity was theame in principal and basal cells. Smooth muscle cells and fibro-last nuclei presented a weak (+) immunostaining (Fig. 4b). In theastrated group, reduced immunoexpression was shown in princi-al cells, basal cells, smooth muscle cells, fibroblasts and extrudedells. Persisting immunostaining was shown in the cytoplasm ofrincipal cells (Fig. 4c). In animals which were castrated and thenreated with testosterone, the apical cytoplasm and some nucleif principal cells, the nuclei of basal cells, fibroblasts and smoothuscle cells were immunoreactive (Fig. 4d). In the group of animals

aving undergone a ligation of efferent ducts, a strong immunos-aining was observed in the cytoplasm and some nuclei of principalells as well as in the nuclei of basal and extruded cells (Fig. 4e).

strogen receptor 2 (ESR2)

Contrary to the results obtained for ESR1, we observed no stain-ng in the apical cytoplasm for ESR2 in all the groups of fat sand rats.uring the breeding season, the immunohistochemistry for ESR2ave a strong signal in the nuclei of principal cells, basal cells andbroblasts (Fig. 5a). A weak staining was observed in the cytoplasmf principal cells and sperm. In the epithelium, some principal cellsere not stained. During the resting season, a moderate stainingas localized in the cytoplasm of principal cells and in smoothuscle cells. The nuclei of principal cells and basal cells were neg-

tive. The fibroblasts were weakly stained (Fig. 5b). In castratednimals, a weak immunoreactivity was detected in principal cells,asal cells, smooth muscle cells and the nuclei of fibroblasts. Cyto-lasm of principal cells and some nuclei were not stained (Fig. 5c).

mooth muscle cells, SPZ: sperm, Lu: lumen, EC: extruded cells, AC: apical cytoplasm,

In castrated and then treated animals, both cytoplasm of principalcells and nuclei of basal cells showed a weak immunoreactivity.There was no staining in the nuclei of principal cells, smooth musclecells and fibroblasts (Fig. 5d). In animals having undergone a liga-tion of efferent ducts, strong immunostaining was detected in thenuclei of basal cells and in both cytoplasm and nuclei of principalcells. The nuclei of some principal cells were not stained (Fig. 5e).

Androgen receptors (AR)

During the breeding season, androgen receptors (AR) werehighly immunoreactive in both cytoplasm and nuclei of principalcells. In the cytoplasm, the immunostaining was clearly granular.The basal cells showed moderate staining or no staining. A positiveimmunohistochemical signal was localized in smooth muscle cellsand fibroblasts. The sperm showed a weak staining (Fig. 6a). Duringthe resting period, the immunostaining persisted in both princi-pal and basal cells, however with a lower intensity of the signal.Smooth muscle cells showed a higher immunoreactivity (Fig. 6b).In castrated animals, no or weak staining was observed in prin-cipal cells. Basal cells and smooth muscle cells showed reducedimmunoreactivity (Fig. 6c). In animals which were castrated andthen treated with testosterone, a moderate immunohistochemicalsignal in the nuclei of principal cells was accompanied by a gran-ular immunostaining in cytoplasm. Some nuclei of principal cellswere negative. The nuclei of basal cells were moderately stainedor negative. Smooth muscle cells and fibroblasts were not stained(Fig. 6d). In animals having undergone ligation of efferent ducts, amoderate immunohistochemical staining was observed in nucleiof principal and basal cells. A weak immunoreaction was localizedin smooth muscle cells as well as in the cytoplasm and some nucleiof principal cells. Fibroblasts were not stained (Fig. 6e).

Discussion

The caput epididymidis of fat sand rats in our study showeda decrease in weight during the resting season, in castrated ani-mals and in animals having undergone efferent duct ligation. A

564 R. Menad et al. / Acta Histochemica 116 (2014) 559–569

Fig. 4. Estrogen receptor 1 (ESR1) in caput epididymidis. (a) During the breeding season, principal cells nuclei show a moderate or null staining. Apical cytoplasm gives avery strong signal. Basal cells show a moderate staining. (b) In resting season, the immunoreactivity is localized in apical cytoplasm and nuclei of principal cells. A weakimmunoreaction is localized in smooth muscle cells, fibroblasts and extruded cells. (c) In principal cells of castrated group the immunoexpression is weaker compared withbreeding season and resting season. The basal cells, smooth muscle cells and fibroblasts are not stained. (d) In castrated then treated group, a moderate immunolabeling inprincipal cells, basal cells and fibroblasts is localized. Principal cells show an individualized positive signal in apical cytoplasm and for the majority of principal cells nuclei.Other principal cells show a negative signal in nuclei. (e) In animals having undergone efferent ligation, the majority of principal cells, extruded cells and basal cells areintensely immunostained with the opposite of certain principal cells, smooth muscle cells and fibroblasts which are negative. No immunostaining was observed in negativec musca

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ontrols (insert). Legends: pc: principal cell, bc: basal cell, f: fibroblast, smc: smoothrrow head: absence of staining.

omparable variation had been previously reported for mice (Abou-aila, 1987) and jirds (Belhocine, 1998). In Sprague-Dawley rats,poptosis was shown to be the main cause of the epididymal weighteduction (Holland et al., 1992; Fan and Robaire, 1998). In con-rast, castration followed by a testosterone treatment resulted inhe caput epididymidis weight increase. In rats, testosterone treat-

ent had been previously shown to partially restore the epididymiseight and prevent apoptotic cell death in all epididymal regions

xcept the initial segment (Robaire et al., 1977). It is probablehat the absence of sperm, the apoptosis of principal cells, theack of androgen-dependent protein expression and of the apoc-ine secretion were responsible for the decrease in weight of the

aput epididymidis.

The height of principal cells in our study was similar to thateported for the hamster (Beu et al., 2003) and for other sand ratsSprando et al., 1999). Our results indicate that principal cells in

le cell, spz: sperm, ec: extruded cells, full arrow head: presence of staining, hollow

the fat sand rat are very affected by testosterone deprivation. Thesame decrease was observed in the caput epididymidis of jirds inthe resting season or after orchidectomy (Belhocine, 1998) and ofchimpanzees after treatment with a GnRH antagonist (Smithwickand Young, 2001). We have previously shown that testosteronealone was insufficient for maintaining the structure and function ofprincipal cells (Gernigon, 1992; Menad, 2008) since the testicularfactors were also essential.

Numerous extruded cells were observed in animals havingundergone efferent duct ligation in our study. This phenomenonhas been previously described in the mouse epididymis (Agnes andAkbarsha, 2001; Jara et al., 2002). According to Grossmann et al.

(1998) and Jara et al. (2002), extrusion is the mechanism of apop-totic cell elimination from epithelium. Our results do not supportthis hypothesis because extruded cells were immunoreactive forestrogen ESR1 and androgen receptors.

R. Menad et al. / Acta Histochemica 116 (2014) 559–569 565

Fig. 5. Estrogen receptor 2 (ESR2) in caput epididymidis. (a) In breeding season, an important staining pattern is located in nuclei of principal cells. In contrast, nuclei of otherprincipal cells show a negative immunoreaction. The same intensity of immunostaining was observed in fibroblasts and basal cells. Sperm show a weak immunoreactivity.(b) In resting season, a no staining was observed in principal and basal cells nuclei. A moderate immunoreaction was observed in principal cells cytoplasm and smooth musclenuclei. (c) In castrated group, a moderate immunoreaction was observed in all cells of caput epididymidis excepted certain principal cells that were negative. (d) In castratedthen treated animals, moderate immunostaining is present in principal cells cytoplasm and certain nuclei of basal cells. The nuclei of principal cells, smooth muscle cellsa ry strP ined

p rrow

iess2ro1lOp

noc(m

nd fibroblasts do not present any labeling. (e) In efferent duct ligated animals, a verincipal cells which do not show any signal in nuclei are inserted between the starincipal cell, bc: basal cell, f: fibroblast, smc: smooth muscle cell, spz: sperm, full a

The epithelium disorganization in castrated animals could resultn modification of protein junctions. In Sprague-Dawely rats,xperiments with castration and castration followed by treatmenthowed that Claudin 1 is an androgen-dependent protein but exclu-ively in the apical pole and not in the lateral pole (Gregory et al.,001). Several protein junctions of lateral plasma membrane wereeported to be dependent on androgens, e.g. Occludins, Zonulaccludens 1, and E-cadherin (Cyr et al., 1993; Levy and Robaire,999). The occludin and the connexin levels decreased after the

igation of efferent ducts (Pointis et al., 2005; Turna et al., 2006).ur results show that ligation of efferent ducts induces atrophy ofrincipal cells, but not the epithelium disorganization.

The spectacular change of intertubular connective tissue, con-ective capsule and smooth muscle cells suggests a broad spectrum

f androgen intervention in extracellular matrix remodeling. Aomparable response was observed in the epididymis of the jirdBelhocine, 1998). In this species, collagen I and collagen III accu-

ulate in seminal vesicles (Belhocine et al., 2007). In chimpanzees,

ong immunohistochemical reaction was observed in principal cells and basal cells.cells. No immunostaining was observed in negative controls (insert). Legends: pc:head: presence of staining, hollow arrow head: absence of staining.

an inflammatory reaction in the connective tissue was reportedafter castration (Smithwick and Young, 2001). The matrix metal-loproteinases (MMP), enzymes implicated in extracellular matrixremodeling, were previously detected in the male reproductivetract (Belhocine et al., 2007, 2010). Modulation of MMPs is a veryplausible theory. In a recent study, Belhocine et al. (2010) reportedthe immunoexpression of MMP2 and MM9 in Libyan jirds duringthe resting season or following castration, and showed a pro-nounced immunoresponsiveness of both of these gelatinases.

The distribution of the AR, ESR1, and ESR2 expression in thefat sand rat is undoubtedly an indicator of testosterone and17�-estradiol implication in the epididymal function. These twohormones were previously reported to be present in the rat epi-didymis at inversely proportional concentrations between initial

segment, caput, corpus and cauda epididymidis (Yasuhara et al.,2008).

During the breeding season, we observed a strong AR expres-sion in all cells of the epithelium. Principal cells showed an

566 R. Menad et al. / Acta Histochemica 116 (2014) 559–569

Fig. 6. Androgen receptors (AR) in caput epididymidis. (a) A pronounced immunohistochemical signal is localized in all the compartments of epididymis in breeding seasonwith more intensity and granular aspect in principal cells. Basal cells show a moderate or negative immunoreaction. (b) Immunoreactivity in resting season is moderate inprincipal cells and basal cells. The smooth muscle cells show a high immunoreaction. (c) Two opposite immunoreaction is observed in principal cells of castrated group;negative and weak response. (d) In castrated then treated animals, principal cells nuclei present a moderate or a lack of labeling. In cytoplasm of principal cells a stronggranular staining is observed. Basal cells show a same response with animal in breeding season. A weak immunostaining is localized in smooth muscle cells, fibroblasts andextruded cells. (e) In efferent duct ligated group, a moderate immunoreactivity is observed in principal cells, basal cells and smooth muscle cells. Certain nuclei of principalc trols

c of sta

ieoeaeaelSieoeTob

ells and fibroblasts are negative. No immunostaining was observed in negative conell, spz: sperm, full arrow head: presence of staining, hollow arrow head: absence

mmunostaining in the nuclei and cytoplasm. In other species, ARxpression was shown to be localized nearly ubiquitously through-ut the epididymis, in epithelium and stromal cells (Ungefrorent al., 1997; Goyal et al., 1998; Zhou et al., 2002). This reflects

global androgen dependency of the caput epididymidis. How-ver, estrogens are present in the epididymis, luminal fluid, spermnd epithelium (Carpino et al., 2004; Shayu and Rao, 2006; Josepht al., 2011). This enzyme was also immunolocalized in epithe-ial cells and interstitium of several species (Carpino et al., 2001;hayu et al., 2007; Joseph et al., 2011), including by our own stud-es on sand rats (unpublished data). In the present study, the ESR1xpression was concentrated in both nuclei and apical cytoplasmf principal cells and in basal cells, whereas the ESR2 was mainly

xpressed in the nuclei of principal cells, basal cells and fibroblasts.he ESR1 expression was previously described in the epididymisf the mouse (Zhou et al., 2002), hamster (Joseph et al., 2011) andoar (Pearl et al., 2007). The distribution of the ESR1 expression in

(insert). Legends: pc: principal cell, bc: basal cell, f: fibroblast, smc: smooth muscleining.

nuclear and apical cytoplasm was described in caput and corpusepididymidis of rats (Hess et al., 2011). Hess reported that ESR2was expressed in all regions of epididymis with almost the sameintensity between initial segment, caput, corpus and cauda epi-didymidis, contrarily to ESR1, the expression of which showed aregional distribution with the highest mRNA expression in the cor-pus (Hess et al., 2011). Our results illustrate a selective distributionof the ESR1 and ESR2 expression in principal cells in contrast to ARwhich has a ubiquitous distribution. In some previous studies, sim-ilar patterns were reported for ESR2, which was shown to be widelyexpressed throughout the male reproductive tract and was foundin nearly every cell type of the testis, efferent ducts, and epididymis(Nie et al., 2002; Zhou et al., 2002).

Compared to the breeding season, during the resting season,the AR and ESR1 expression was not much affected. In contrast,the expression of ESR2 was clearly decreased and delocalized. Itseems that the resting season induces the ESR2 translocation from

ochem

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speaclaa(to

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R. Menad et al. / Acta Hist

he nucleus toward the cytoplasm of principal cells and does notuch affect the AR and ESR1 immunoexpression. There are very

ew studies addressing the impact of seasonal variations on thexpression of ESRs. A clear seasonal dependency of the ESR1 expres-ion was observed in roe deers, whereas the ESR2 expression wasot season-dependent (Schön and Blottner, 2008).

In the castrated group, we observed a low and selective expres-ion: some nuclei were moderately positive for AR, stronglyositive for ESR2 and negative for ESR1. A decline in the AR lev-ls had been previously reported in the rat epididymis one weekfter castration, with a striking expression decrease in stromal cellsompared to epithelial cells (Zhu et al., 2000). In other studies, uni-ateral or bilateral castration did not affect the expression of ESR1nd ESR2 in rats and goats. Moreover, bilateral castration caused

sharp decrease in both AR and ESR immunostaining intensityGoyal et al., 1998; Oliveira et al., 2004). Our observations supporthe hypothesis that AR and ESR immunoexpression is dependentn both androgens and testicular factors.

In animals which were castrated and then treated by testos-erone, we observed androgen dependence for AR and a recovery ofSR1 immunoexpression but not of the ESR2 expression which wasxclusively cytoplasmic. The androgen-dependent AR expressionas decreased in castrated animals and restored by testosterone

reatment. In a study by Zhu et al. (2000), after androgen replace-ent, nuclear AR levels recovered to more than 90% of those of

ontrols in both epididymal cell types. We show that, althoughhe epididymis is an androgen-dependent tissue (Ezer and Robaire,002; Robaire et al., 2007), testosterone alone is not sufficient forhe ESR1 and ESR2 immunoexpression in the fat sand rat and thatesticular factors are also important. It has been shown in rats thatndrogen treatment does not induce a significant change in theSR1 expression. On the other hand, testosterone alone or com-ined with estradiol did not significantly affect the ESR2 expressionOliveira et al., 2004). However, testosterone replacement restoredhe expression of both AR and ESRs to normal levels in orchidec-omized goats (Goyal et al., 1998). We found a pronounced granularmmunoreactivity of AR in the cytoplasm of principal cells dur-ng the breeding season and in animals which were castrated andhen treated by testosterone. A comparable immunoreaction wasbtained in the nuclei of principal cells of the rat by others (Zayat al., 2012). These authors suggested that hormone binding andeceptor dimerization were responsible for a punctuate staining.

e think that this immunoreaction results from a down-regulationf AR by degradation in endosomal/lysosomal structures, followingigh rates of testosterone in both cases.

The ligation of efferent ducts reduced the intensity of the ARmmunohistochemical signal in all epithelial cells, but did noteduce the immunoreactivity of ESR1 and ESR2. On the contrary,hey were increased. This strong immunoreaction could be dueo low concentrations of estradiol resulting from lack of luminaluid following ligation. The autocrine action of estradiol on the ESR

mmunoexpression is very probable. Oliveira et al. (2004) showedhat estradiol treatment results in a negative feedback in the ESRxpression. However, the source of estrogens in the epididymalumen is primarily the germ cells (Janulis et al., 1996; Carreaut al., 2003, 2006; Carreau and Hess, 2010). The immunoexpres-ion of ESR1 during the resting season, as well as in animals whichere castrated or castrated and treated with testosterone, suggests

hat testicular factors play an important role in the ESR1 regula-ion. However, the fact that the efferent duct ligation did not resultn a decreased immunostaining, but rather in its increase, arguesgainst the hypothesis of the lumicrine regulation. We have shown

very strong immunoreaction of ESR1 in the apical cytoplasm.urthermore, it is probable that apical ESR1 staining results fromhe existence of coated pits and vesicles implicated in internaliza-ion and/or externalization phenomena, which are abundant in this

ica 116 (2014) 559–569 567

region of principal cells and which define receptor sites. This distri-bution may reflect the existence of another, non-nuclear, signalingpathway. These results are in agreement with those obtained byothers (Lucas et al., 2008, 2010). ESR1 and ESR2 in the male repro-ductive tract may play a role in the rapid action of estrogen (Levin,2009). According to Lucas et al. (2008), estradiol at physiologicalconcentrations activates SRC-mediated translocation of ESRs to theplasma membrane. Contrary to the results obtained for ESR1, weobserved no staining in the apical cytoplasm for ESR2 in the allgroups of fat sand rats.

Our results argue for the coexistence of androgenic and estro-genic pathways in the caput epididymidis of the fat sand rat. Theyillustrate the complexity of the regulation of androgen and estrogenreceptor immunoexpression, and support concurrent involvementof estrogens and androgens in the epididymal function control. Thesporadic distribution of the ESR1 and ESR2 expression in the nucleiof principal cells suggests the existence of an alternative responsepathway in the epididymis of the fat sand rat. In addition, theimmunolocalization of ESR1 in the apical cytoplasm and probablyin the plasma membrane strengthens the theory of the involvementof estradiol in the functioning of the fat sand rat epididymis by thefast pathway. Further immunocytochemical studies are needed todetermine the apical and/or membrane localization of ESR1 and totest for possible ESR colocalizations.

Acknowledgements

The authors thank all the personnel of Beni-Abbes station inthe Algerian Sahara, especially M. Yaïche and H. Salmi for trap-ping the animals. Thanks to F. Meknaci, A. Boufersaoui, M. Raquetand N. Edaïkra for their contribution. Thanks to M.T. Laurent, C.Bouchot and H. Serclerat for their assistance. The histological partwas performed on Laboratory of Research on Arid Areas, Faculty ofBiological Sciences, University of Sciences and Technology HouariBoumediene (USTHB) Algiers. Immunohistochemical studies wereperformed in the laboratory of general biology in the Catholic Uni-versity of Lyon/EPHE.

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