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Tissue and Cell 44 (2012) 308– 315

Contents lists available at SciVerse ScienceDirect

Tissue and Cell

j our na l ho me p age: www.elsev ier .com/ locate / t i ce

hrelin regulates Bax and PCNA but not Bcl-2 expressions following scrotalyperthermia in the rat

rash Kheradmanda,∗, Omid Dezfoulianb, Masoud Alirezaei c

Department of Clinical Sciences, School of Veterinary Medicine, Lorestan University, P.O. Box: 465, Khorram Abad, IranDepartment of Pathobiology, School of Veterinary Medicine, Lorestan University, P.O. Box: 465, Khorram Abad, IranDivision of Biochemistry, School of Veterinary Medicine, Lorestan University, P.O. Box: 465, Khorram Abad, Iran

r t i c l e i n f o

rticle history:eceived 21 January 2012eceived in revised form 23 April 2012ccepted 29 April 2012vailable online 31 May 2012

eywords:hrelinaxcl-2CNAestis

a b s t r a c t

More recently, we have reported the beneficial effects of ghrelin in improvement of histopathological fea-tures of the rat testis following local heat exposure. However, the exact mechanism and the precise roleof apoptosis- and proliferation-specific proteins in this regeneration process remained to be explored.Thus, thirty adult male Wistar rats were allotted for the experiment and subdivided equally into threegroups: control-saline (CS), heat-saline (HS) and heat-ghrelin (HG). The scrota of HS and HG groups wereimmersed once in water bath at 43 ◦C for 15 min. HG animals received 2 nmol of ghrelin subcutaneouslyimmediately after heating every other day until day 60 and the other groups were given physiologicalsaline using the same method. The testes of all groups were taken after rat killing on days 30 and 60 afterheat treatment for immunocytochemical detection of pro-apoptotic factor Bax, anti-apoptotic proteinBcl-2 and proliferation-associated peptide PCNA in the germ cells. Ghrelin could significantly suppressthe Bax expression in spermatocytes compared to the HS group at day 30 (P < 0.05). Likewise, the meanpercentages of spermatogonia containing Bax substance were lower in ghrelin-exposed animals, how-ever the differences were not statistically significant. There were immunoreactive cells against Bcl-2 in

each germ cell neither in the control nor in the heated animals of experimental groups. In contrast, thenumber of PCNA immunolabeling cells were higher in HG group in compared to HS or CS animals onboth experimental days (P < 0.001). Down-regulation of Bax expression concurrent with overexpressionof PCNA in HG group indicates the ability of ghrelin in acceleration of testicular germ cells regenerationfollowing heat stress. These findings indicate that ghrelin may be used as a novel and efficient antioxidant

on of

agent to induce resumpti

. Introduction

Germ cell death has been recognized as a constant and signifi-ant feature of mammalian spermatogenesis (Russell et al., 1990).n adult normal rats this loss is occurred mostly during spermato-onial development (up to 75%) and to a lesser extent duringaturation divisions of spermatocytes and spermatid development

Huckins, 1978). It has been claimed that both spontaneous (duringormal spermatogenesis) and increased germ cell death triggeredy various stimuli such as exposure to local heat in rats occur viapoptosis (Sinha Hikim et al., 2003a,b; Vera et al., 2004). More-ver, it has been well shown that short term exposure (43 ◦C for5 min) of the rat testis to mild heat results within 6 h in stage- and

ell-specific activation of germ cell apoptosis (Sinha Hikim et al.,003b).

∗ Corresponding author. Tel.: +98 661 6200109; fax: +98 661 6200109.E-mail address: arashkheradmand@yahoo.com (A. Kheradmand).

040-8166/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.tice.2012.04.009

spermatogenesis upon environmental heat exposure.© 2012 Elsevier Ltd. All rights reserved.

Members of the Bcl-2 family of proteins play a major role ingoverning the mitochondrial-dependent apoptotic pathway, withproteins such as Bax functioning as an inducer and proteins suchas Bcl-2 as a suppressor of cell death (Adams and Cory, 1998;Hengartner, 2000; Reed, 2000). It has been clearly demonstratedthat initiation of apoptosis is preceded by a redistribution of Baxfrom a cytoplasmic to paranuclear localization in heat-susceptiblegerm cells and early in apoptosis, a significant amount of Bax accu-mulates in endoplasmic reticulum (Sinha Hikim et al., 2003b).

Numerous studies have recently documented the direct actionof ghrelin in modulation of apoptosis in different cell types. Forexample, it has been reported that ghrelin inhibits apoptosis inseveral cells such as cardiomyocytes (Huang et al., 2009; Kui et al.,2009), pancreatic � cells (Granata et al., 2007; Wang et al., 2010),pituitary lactotrophs (Granado et al., 2009) and adipocyte cells(Kim et al., 2004), but promotes it in chick ovarian granulosa

cells (Sirotkin and Grossmann, 2008), endothelial cells (Nagayaand Kanagawa, 2003), aldostroma and adenocarcinoma derivedcells (Belloni et al., 2004). Ghrelin, the endogenous ligand for thegrowth hormone (GH) secretagogue receptor (GHS-R), is expressed

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rimarily in various tissues (van der Lely et al., 2004). Within theestis, expression of ghrelin has been demonstrated in mature Ley-ig cells of rat and human. In addition, expression of the functionalhrelin receptor, the GHS-R type 1a, has been shown in Sertolind Leydig cells (Barreiro and Tena-Sempere, 2004; Tena-Sempere,005). Furthermore, expression of GHS-R 1a in the seminiferousubules strongly suggests that the seminiferous epithelium mighte a target for ghrelin action and directly regulates seminiferousubules function (Barreiro and Tena-Sempere, 2004). Thus, it isxpectable that ghrelin may control the main gonadal functionsuch as apoptosis and proliferation in the rat testis.

On the other hand, the beneficial action of ghrelin in attenuationf degenerative effects and restoration of histopathological featuresf testicular cells following heat stress was recently demonstratedn our laboratory (Kheradmand et al., 2011). However, in thattudy the exact role of apoptosis-associated factors or proliferation-ssociated peptide PCNA in the modulation of adverse effects ofeat was not fully explored and remained for further studies. There-

ore, the present study was designed to clarify the ghrelin abilityn regulation of Bax, Bcl-2 and PCNA expressions in the rat testisollowing local heat exposure.

. Materials and methods

.1. Experimental animals

All investigations were conducted in accordance with the Guid-ng Principles for the Care and Use of Research Animals and werepproved by the Animal Care Committee at the Lorestan Univer-ity of Medical Sciences (Khorram Abad, Iran). Thirty 60-day olddult male Wistar rats bred in the vivarium of the Pasture Instituten Tehran, Iran were used for this study. The animals were main-ained under constant conditions of light (12 h light:12 h darkness)n an animal room in groups of five rats per cage and controlledemperature (21–24◦ C) with free access to pelleted food and tapater ad libitum. All animals were treated humanely and in com-liance with the recommendations of Animal Care Committee forhe Lorestan University of Medical Sciences.

.2. Drugs and reagents

Rat lyophilized acylated ghrelin (n-octanoylated research grade)as purchased from Tocris Cookson Ltd. (Bristol, UK). Ghrelin wasissolved in sterile physiologic saline solution before injection.ax, Bcl-2 and PCNA expressions were determined using availabletandard immunohistochemical kits provided from Dako CompanyGlostrup, Denmark).

.3. Heat treatment

Heat stress of testes was induced as described previouslyLue et al., 1999). In short, after rat anesthesia, their testes weremmersed once in a thermo-digitally controlled water bath at 43 ◦Cor 15 min.The animals were randomly divided into three equalroups (each containing five rats) as the following order: group

was assigned as control-saline, group 2 was defined as heat-aline and group 3 was designed as heat-ghrelin regimens. Groups

and 3 were anesthetized intraperitonealy using a combinationf ketamin (Alfasan, Woerden, Holland, 70 mg kg−1) and diazepamCaspian Tamin Pharmaceutical Co., Rasht, Iran, 2.5 mg kg−1) andnderwent to scrotal heating process as described. Immediatelyfter heat exposure, HG rats were given ghrelin subcutaneously

2 nmol/100 �l saline) and this treatment was continued everyther day till day 60. This period of treatment time was chosenccording to the duration or rat spermatogenesis cycle, which isanged between 48 and 52 days (Kerr et al., 1992). Likewise, the

d Cell 44 (2012) 308– 315 309

dose of ghrelin used in our in vivo experiments was comparablewith amounts of ghrelin secreted into the blood during starvation:exogenous administration of 1 nmol of ghrelin is able to inducea significant elevation (2.4–2.6-fold increase) in serum levels oftotal ghrelin 1 h after injection (Fernandez-Fernandez et al., 2005),whose magnitude is in the range of that induced by fasting (Wrenet al., 2001). The animals in HS and CS groups were treated by ster-ile saline instead of ghrelin as the same method. Five rats from eachgroup were killed upon diethyl ether anesthesia (May & Baker Ltd.,Dagenham, England) by decapitation on days 30 and 60 after scro-tal heat treatment and their testes were taken for detection of Baxand Bcl-2 levels as well as PCNA immunolabeling.

2.4. Determination of Bax, Bcl-2 and PCNA expressions

To evaluate the possible role of ghrelin on germ cells apopto-sis and proliferation, cell counting on Bax, Bcl-2 and PCNA positivecells was conducted. Paraffin embedded tissues of all groups weresectioned into 3-�m thickness and after deparaffinization, theslides were immersed in target retrieval solution (pH 9.0) andheated in water bath for 20 min at 98 ◦C to unmasked antigens. Thesections were then dipped in 3% H2O2 for 10 min to block endoge-nous peroxidase and nonspecific background staining was blockedby incubating the sections for 3 min in 1% normal rabbit serum.The slides were incubated with the following antibodies: poly-clonal rabbit antibody against Bax protein (dilution 1:40; DAKOCorporation Carpinteria, CA, USA), mouse monoclonal anti-PCNA(Clone PC 10; dilution 1:100; DAKO, Glostrup, Denmark) and mon-oclonal antibody against Bcl-2 (Clone 124; dilution 1:50; Dako,Glostrup, Denmark) followed by rinsing in TBS. Thereafter, all sec-tions incubated with biotinylated secondary antibody with usingan Envision method (Dako, Glostrup, Denmark) at room temper-ature for 30 min. The preparations were incubated with liquiddiaminobenzidine tetrahydrochloride substrate (DAB, ready to use)for 10 min and were counterstained with Mayer’s hematoxylin,dehydrated, and mounted in Diatex at the end of processing. Neg-ative control slides in the absence of primary antibody were alsoincluded (Linderoth et al., 2003; Yoshino et al., 2006). The popu-lations of all positive cells were calculated randomly in 7 fields bylow power field (LPF) for PCNA and high power field (HPF) for Bax.

2.5. Statistical analysis

Results were analyzed using the SPSS/PC computer (version 16)program. All data were tested for normality followed by homo-geneity of variances by Levene static test. When the variances werehomogenous, the percentage of cells immunostained against Bax,Bcl-2 and PCNA peptides among CS, HS and HG groups were com-pared using one-way ANOVA and Tukey’s test as post hoc, in orderto determination of difference among groups (Petrie and Watson,1999). Data are presented as the mean ± SEM. Values were consid-ered to be statistically significant at P < 0.05.

3. Results

The immunoreactivity of seminiferous germ cells includingspermatogonia and spermatocytes against Bax, Bcl-2 and PCNAproteins are depicted in Figs. 1–3. Both the quality and quantityof immunostaining were conducted in these cells. Under normalconditions in CS rats, individual spermatogonia and spermatocytesunderwent apoptosis, as indicated by brown intensive cytoplasmic

stained cells for Bax protein. Similarly, the positive cells againstBax were also seen in HS group and with a minimum level in HGanimals (Fig. 1). Immunolabeling of cytoplasmic protein Bax waspresented prominently in the spermatocytes and to a lesser extent

310 A. Kheradmand et al. / Tissue and Cell 44 (2012) 308– 315

Fig. 1. Bouin-fixed, paraffin-embedded testis sections were subjected to imunocytochemical staining using polyclonal rabbit antibody against Bax. (A) Dark brown stainedcytoplasmic protein Bax were mainly localized in the spermatocytes (arrows) in the control group on day 30 (400×). (B) Extensive and strong Bax expression in the cytoplasmicregion of majority of spermatocytes following scrotal heating in HS rats on day 60 (400×). (C) Lower immunoreactive spermatocytes labeled by anti-Bax antibody in ghrelin-exposed animals on day 30 (400×). (D) Reduced number of positive-cells to Bax in HG group on day 60 after heat removal (400×). All of spermatogonia cells were stainedn tocytt

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egatively against antibody to Bax in these figures. Sg: Spermatogonia; Sp: Spermahe reader is referred to the web version of the article.)

n spermatogonia, exclusively in HS group with no reaction in Ser-oli cells. Surprisingly, ghrelin treatment could significantly reducehe number of cells exhibiting Bax expression when compared to HSnimals. So that, cell counting revealed that 5.55 ± 1.49% of sperma-ocytes in HG rats expressed Bax substance versus 14.68 ± 2.05% ofhese cells in HS group on day 30 (P < 0.05). Although the mean per-entage of spermatocytes containing Bax was still lower on day 60n HG rats, however the difference was not statistically significantFig. 4). Similar results were also observed in the spermatogonia,n which despite of lower Bax-positive cells in HG group, however,hrelin did not significantly (P = 0.07) down-regulate the expres-ion of Bax in these cells. Overall, the number of cells with apparentax accumulation was much higher in spermatocytes than the sper-atogonia and also in HS animals than the HG rats. In other words,

he expression of apoptosis-protein Bax was negligible in the sper-atogonia cells of all experimental groups (Fig. 4).The proliferation-associated peptide PCNA were mainly local-

zed in spermatogonia and primary spermatocytes, whereas, lowerontents of PCNA were detected in the second spermatocytes orpermatids. Attempts for in situ detection of PCNA expressionhowed an overexpression of this peptide in HG group either onay 30 or at day 60 after heat exposure (Fig. 2). Indeed, ghrelinould strongly up-regulate the PCNA expression both in spermato-onia as well as spermatocytes in compared to the other groups.or instance, the mean percentage of immunoreacted spermatogo-ia against PCNA was 16.87 ± 2.53, 13.58 ± 1.67 and 42.32 ± 6.53 inS, HS and HG groups on day 30, respectively (P < 0.001, Fig. 5).

Expression of Bcl-2 identified neither in spermatogonia nor in

permatocytes in each experimental groups. In fact, Bcl-2 proteinas absent in the various germ cells of seminiferous tubules and

hrelin could not affect the cytoplasmic levels of Bcl-2 amountFig. 3).

e; L: Leydig cell. (For interpretation of the references to color in this figure legend,

4. Discussion

The present study examined for the first time the pro-tective ability of ghrelin against apoptotic effects of elevatedtesticular temperature parallel to compensatory function of thishormone on germ cells proliferation in the rat testis. The resultsclearly indicated that these actions of ghrelin are mainly medi-ated through down-regulation of Bax expression in the germcells particularly in spermatocytes and up-regulation of nuclearassociated-proliferation peptide PCNA in spermatogonia and sper-matocytes without any change in Bcl-2 expression. The impact ofthese ghrelin modulatory effects resulted in quick regenerationof seminiferous epithelial germ cells to resume spermatogenesisearlier than the HS group.

Spermatogenesis is a temperature-dependent process andraised scrotal temperature in the rat (Lue et al., 2000) and mouse(Jannes et al., 1998) causes decrease of testes weight and reduc-tion of viability, morphology and sperm motility. Most studies havebeen shown that single short exposure of the testis principallyaffects primary spermatocytes and early spermatids, as the mostsusceptible germ cells to heat (Yamamoto et al., 2000). Histopatho-logically, we have similarly observed that pachytene spermatocytesand spermatids are the most susceptible cells to heat when mea-sured by miotic index (Kheradmand et al., 2011). Wang et al. (1998)demonstrated that during experimentally induced cryptorchidism,as an elevated testicular temperature disorders, the most affectedgerm cells were spermatocytes. Similar observation was also foundby Tomomasa et al. (2002), in which they reported apoptosis is

more prominent in spermatocytes of undescended rat testis.

It has widely accepted that the mechanism of cell deathfollowing increased testicular temperature is through apopto-sis not necrosis and may involve reactive oxygen species (ROS),

A. Kheradmand et al. / Tissue and Cell 44 (2012) 308– 315 311

Fig. 2. Nuclear immunostaining using specific mouse anti-PCNA monoclonal antibody in the testicular germ cells. (A) In situ determination of brown stained positive cells inthe spermatogonia and spermatocytes (arrows) in the normal control testes on day 30 (100×). (B) Heat-treated testes on day 30 represent lower levels of PCNA with severedestruction of seminiferous epithelial germ cells (100×). (C) Strong immunoreaction of spermatogonia and spermatocytes in response to ghrelin on day 30 (400×). (D) Asignificant number of spermatogonia and spermatocytes show PCNA immunolabling in HG group on day 60. Spermatids were also generated and stained less intensivelyt atoc( and wS ces to

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han the other germ cells (400×). (E) Low percentages of spermatogonia and spermF) Predominant accumulation of PCNA within spermatogonia and spermatocytes

permatogonia; Sp: Spermatocyte; Sd: Spermatid. (For interpretation of the referen

ranslocation of proapoptotic factor Bax from the cytoplasm to per-nuclear position and release of cytochrome c from mitochondriaSinha Hikim et al., 2003b; Setchell, 2006). Mouse and rat spermato-ytes are sensitive to the heat, independent of or dependent on thepermatogenic stage of the seminiferous tubules (Lue et al., 1999;zu et al., 2004). Neither Sertoli nor Leydig cell numbers appear toe reduced after testicular heating (Morgentaler et al., 1999). Nev-rtheless, there are some evidences for effects on spermatogonia,ertoli and Leydig cells (Setchell, 1998; Aktas and Kanter, 2009).

It seems that apoptotic peptide Bax plays a major function ineat-induced testicular damage in the germ cells. Accordingly, weypothesized that ghrelin may have a possible role in the modu-

ation of Bax/Bcl-2 ratio, which is the critical determinant of cellate. Bcl-2 family members are the key regulators of apoptosishat may either promote cell survival (Bcl-2, Bcl-w) or stimu-

ate (Bax, Bak, Bad) cell death (Zamzami et al., 1998; Adams andory, 1998). It appears that members of Bcl-2 family interactmong each other to form a dynamic equilibrium between homo-nd heterodimers (Yan et al., 2000). In the present study, ghrelin

ytes represent proliferation-associated peptide PCNA in HS rats on day 60 (100×).ith a lesser extent in spermatids in ghrelin-treated animals on day 60 (100×). Sg:

color in this figure legend, the reader is referred to the web version of the article.)

treatment could significantly reduce the expression of Bax proteinin the spermatocytes on day 30 after heat removal. Furthermore,non significant decrease was also observed in the spermatogoniacells, by which ghrelin down-regulated the cytoplasmic substanceBax in contrast to the heated group. Spermatogonia are generallybelieved to be unaffected during hyperthermia (Setchell, 2006).Thus, the little expression and non pronounced alterations in theBax levels in the spermatogonia against the spermatocytes could bejustified in the present work. This result is in consistent with ourprevious histopathological evaluation of the rat testis, where sper-matogonia cells were more resistant to heat than the other germcells and remained unchanged during the experimental period(Kheradmand et al., 2011). Likely, longer or repeated exposureto heat is required for higher Bax accumulation in the cytoplasmof spermatogonia. It should be considered that the lower level of

Bax amount in spermatogonia than the spermatocytes, even in HSgroup, provides the possibility of regeneration for seminiferoustubules to return to their normal and full spermatogenesis capac-ity. The obtained results may suggest that ghrelin can suppress the

312 A. Kheradmand et al. / Tissue and Cell 44 (2012) 308– 315

Fig. 3. Cytoplasmic determination of Bcl-2 expression using monoclonal antibodyaha

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Fig. 4. The mean percentages of spermatogonia (A) and spermatocytes (B)immunosatined against Bax substance in CS, HS and HG groups on days 30 and 60

sibly exerts its protective functions mainly through downstream of

gainst Bcl-2 in experimental groups. Germ cells neither in the control (A) nor ineated animals (B) nor in ghrelin-exposed rats (C) immunolabeled against Bcl-2ntigen (200×).

eat-induced apoptotic events in the spermatocytes, the most sus-eptible cells to heat, and also explain that this hormone may be

elpful in the attenuation of harmful effects upon testicular heatxposure. To our knowledge, there is no available data concerninghe ghrelin influence on the heat-induced apoptotic effects in the

after exposure to heat. Bars represent mean ± SEM. Comparison among groups wasmade using one-way ANOVA followed Tukey’s post hoc test. The means marked withasterisk indicate significant difference from each other (P < 0.05). NS: not significant.

testis as yet to compare with our study, and the present investiga-tion is probably the first among current reports.

Immunohistochemically, Bax and Bak were found to be presentin the cytoplasm of Sertoli cells, spermatogonia and spermatocytesbut not in spermatids, where it was more significant in the twolast cell types. The high levels of Bax expression in spermatogoniaand spermatocytes showed that these cells are more sensitive toheat and subsequent apoptosis compared to the Sertoli cells (Yanet al., 2000). Dundar et al. (2005) indicated that during experi-mental cryptorchidism in the rats, Bax expression was increasedin spermatogonia and spermatocytes in the first month and mainlyseen in spermatocytes in the second and third months. Ito et al.(1997) have also been confirmed that apoptosis is more prominentin spermatocytes of cryptorchid mice. In contrast, Rockett et al.(2001) determined a reduction in Bax expression following heatingin mice testicular cells.

Bcl-2 is an important member of Bcl-2 family, which acts as anti-apoptotic factor. However, Bcl-2 did not change in response to anyof treatments (heat or ghrelin) in the current work and we coulddetect Bcl-2 expression in various germ cell types neither in heatnor in ghrelin-exposed animals. It has been well documented thatBcl-2 is not expressed in the normal rat testis (Rodriguez et al.,1997; Yan et al., 2000). Thus, it could be concluded that the anti-apoptotic effects of ghrelin in the rat testis is not probably mediatedby Bcl-2 peptide regulation during heat-induced apoptosis. Thisresult may indicate that the ratio of Bax/Bcl-2 is only balancedtoward suppression of Bax expression. In other words, ghrelin pos-

Bax level without modifying Bcl-2 levels in heated testes.The amount of proliferation-associated peptide PCNA (marker

of G1-phase and S-phases of cell cycle) was another main factor

A. Kheradmand et al. / Tissue an

Fig. 5. Percentages of spermatogonia (A) and spermatocytes (B) containing PCNAin all experimental groups calculated at 30 and 60 days after heat treatment. Barsrepresent mean ± SEM. Comparison among groups was made using one-way ANOVAfs

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ollowed Tukey’s post hoc test. All means marked with ** (P < 0.01) and *** (P < 0.001)ignificantly differ from each other.

etermined in the present study. Short heat exposure couldignificantly reduce the mean percentage of immunostained sper-atocytes against PCNA. The concurrent relative up-regulation of

ax in these cells resulted in severe depletion of spermatocytes inhe HS group. Similar observations were also found in an elevatedesticular temperature model in human, in which the expressionf PCNA were significantly decreased in varicocele (up to 70%)ompared to the control (Shiraishi et al., 2010). Of particular note,hrelin strongly up-regulated the PCNA levels both in spermato-onia and spermatocytes either on day 30 or on day 60. Reductionn Bax substance level consistent with increased PCNA expressionn the HG animals on day 30 are closely associated to cause quickegeneration of seminiferous tubules germ cells compared to theS rats, and therefore stimulate testicular recovery and resumptionf spermatogenesis.

Two distinct forms of PCNA are recognized; organic solvent-ensitive and insensitive, which could be detected by IHC ormmunoblotting. These methods might be specific to detect PCNAn all germinal cells from mitotically dividing spermatogoniao spermatids, however, its concentration is gradually reducedoward the center of seminiferous tubules (Bravo and McDonald-ravo, 1987). On the other hand, Chapman and Wolgemuth (1994)eported that localization of PCNA was not found in the pre-eptotene spermatocytes (after the last mitotic division) while it

as detected again in leptotene, zygotene, and pachytene sper-atocytes (the stages of meiotic prophase). They concluded thatNA synthesis in pre-meiotic cells in contrast to pre-mitotic, is not

elated to PCNA and replication in this stage is engaged with otherrotein complexes that are able to veil PCNA. Nevertheless, PCNA isetectable in meiotic prophase cells after pre-leptotene spermato-ytes probably either continuing or newly synthesis of PCNA which

d Cell 44 (2012) 308– 315 313

is required for DNA replication or repair in testicular germ cells. Inthis regard we realized that localization of PCNA was not restrictedto the mitotic cells, and it could be found in lower levels in themeiotic end product cells, like round spermatids.

There are some underlying mechanisms to justify the directapoptotic-modulatory effects of ghrelin in the heated testis. Theinvolvement of oxidative stress and subsequent radical oxygenspecies (ROS) generation in the induction of apoptosis followingheat stress (Zhang et al., 2007; Paul et al., 2009; Shiraishi et al.,2010) and the protective effects of various antioxidant to repairdisrupted spermatogenesis (Ikeda et al., 1999; Setchell, 2006) havebeen fully described. It has also been demonstrated that heat stressinduces an elevation in intracellular peroxide levels as early as5 min after exposure to 43 ◦C and showed the possibility that ele-vation of intracellular peroxide levels may be a signal triggeringapoptosis (Ikeda et al., 1999). The level of MDA, a stable product oflipid peroxidation, in mice testicular germ cells (Zhang et al., 2007)or in cultured mouse spermatozoa (Alvarez and Storey, 1985) hasbeen suggested to increase with temperature elevation. In addi-tion, activities of several scavenging enzymes in testes of rats withexperimentally induced cryptorchidism were impaired, accom-panied by increased peroxidation of cellular lipids (Ahutopa andHuhtaniemi, 1992). Antioxidant treatment, which enhances theendogenous antioxidant defense systems within cells, can inhibit avariety of the apoptotic pathways (Verhaegen et al., 1995; Gormanet al., 1997). Among some antioxidants investigated by Ikeda et al.(1999), catalase (CAT) and catalase pluse superoxide dismutase(SOD) significantly reduced intracellular peroxide levels in testicu-lar cells following heat stress and therefore, provided a significantprotective effect against heat or oxidative stress-induced apopto-sis. It is also relevant that mice with knocked out SOD were moresensitive to effects of experimental cryptorchidism (Ishii et al.,2005). These evidences highlight the potential role of antioxidantenzymes in neutralization of ROS produced during heat stress. Onthe other hand, the presence of different antioxidant enzymes hasbeen investigated in the rat testis. As compared to the liver, therat testis expressed equivalent levels of SOD activity but only 5% ofthe GPx activity and 2% of CAT activity (Peltola et al., 1992) and ascompared to somatic cells, such as Sertoli or peritubular cells, thetesticular germ cells presented low glutathione-dependent enzymeactivity (Bauche et al., 1993). These data indicate a low capacityfor scavenging ROS in the testicular germ cells. Despite of testicu-lar response to the thermal stress through increased expression ofantioxidants (Paul et al., 2009) or even unchanged in total antiox-idative capacity (Zhang et al., 2007), however, germ cell death stilloccurs. One explanation is that these promotions are insufficient tocompensate the elevated levels of ROS produced within the testis,the factor that is responsible for reduced fertility.

Antioxidant properties of ghrelin have been recently demon-strated in our laboratory in the rat testis (Kheradmand et al., 2009a)and ovary (Kheradmand et al., 2010) and also upon local testicu-lar hyperthermia (Kheradmand et al., 2011). In the testis, we haveindicated that ten days administration of ghrelin increases antiox-idant enzyme activities and reduce MDA content. Likewise, wehave proved that prolonged exposure to ghrelin increases func-tional membrane integrity of rat spermatozoa (Kheradmand et al.,2009b) and also that ghrelin enhances viability of rat spermatozoaduring incubation at 37 ◦C up to 5 h (Kheradmand et al., 2009c). Itseems that many of these physiological effects are due to its potentantioxidant actions. These findings confirm and further support theantioxidant characteristics of ghrelin in the rat testes and also showthat protective effects of the hormone against heat possibly may be

mediated through its antioxidative properties.

Ghrelin also directly stimulates the secretion of bothgonadotropins at the pituitary and differentially affects theresponse to LHRH; the LH response is inhibited, while the FSH

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esponse is enhanced (Barreiro and Tena-Sempere, 2004). In addi-ion, hormonal regulation of testicular GHS-R mRNA expression

ay involve interactions between different modulatory signalssuch as ghrelin and FSH). Evidence for the stage-specific expres-ion of the mRNA encoding the functional ghrelin receptor inat seminiferous tubules, under the regulation of pituitary FSH,trongly suggests that ghrelin signaling might be involved in theunctional regulation of the tubular compartment of the testisBarreiro et al., 2003).

In conclusion, ghrelin treatment was shown to suppress redis-ribution of apoptotic mediated factor Bax and stimulate testicularerm cell regeneration through upstream of PCNA levels follow-ng local heat exposure. Our study further evidenced that harmfulffects of short thermal stress in the rat testis could be attenuatedy ghrelin antioxidant therapy.

cknowledgments

This study was financially supported by research project (No.050245) of Research Vice-Chancellery of Lorestan University, Iran.

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