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LOCALIZATION OF ATRIAL NATRIURETIC FACTOR (ANF) IN RATTESTIS AFTER LEYDIG CELL DESTRUCTION: EVIDENCE FORA POTENTIAL ROLE IN REGULATING GONADAL FUNCTION

M. BAKALSKA, M. MOURDJEVA1, A. RUSSINOVA, S. KYURKCHIEV, I. KEHAYOV1

Institute of Experimental Morphology and Anthropology and 1Institute of Biology and Immunology of Reproduction,Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

Objective. Since the atrial natriuretic factor (ANF) is synthesized in various peripheral tissues,where it acts in an autocrine or paracrine fashion, the aim was to gain new insight into ANF expres-sion and function in rat testis after Leydig cell destruction (LCD).

Methods. Leydig cell destruction was performed by the treatment with ethane dimethane sul-phonate (EDS).

Results. ANF was expressed after Leydig cell destruction and total elimination (24 h and 7 daysafter EDS treatment) and also after Leydig cell regeneration (21 and 45 days after EDS treatment).ANF staining in the interstitial compartment was observed in apoptotic Leydig cells 24 h after treat-ment. In seminiferous epithelium ANF labeling was detected in Sertoli and germ cell cytoplasm witha more prominent labeling in spermatids. The degenerating germ cells were totally labeled.

Conclusions. The demonstration of ANF staining in seminiferous epithelium after Leydig cellselimination and androgen deprivation suggests that Leydig cells are not the sole source of ANF inrat testis and that the seminiferous epithelium may be a new site in which ANF may be synthe-sized. The result indicates that ANF plays a role in regulating gonadal function.

Key words: ANF � Testis � Immunocytochemistry � Ethane dimethane sulfonate � Rat

ENDOCRINE REGULATIONS, Vol. 33, 183�191, 1999

In the rat atrial natriuretic factor (ANF), a 28-amino acid peptide or its related species, is producedand secreted by cardiac atriocytes (ATLAS et al. 1984)and exerts natriuretic, diuretic and vasorelaxant ac-tivity (GARCIA et al. 1982; GRAMMER et al. 1983).

The physiological responses of ANF are associatedwith an increase in intracellular cGMP and a decreasein cAMP (INAGAMI 1989). Several studies showed themodulatory role of ANF in steroidogenesis in a varietyof tissues as bovine adrenal glands (DE LEAN et al.1984), mouse Leydig cells (PANDEY et al. 1986) andhuman granulosa cells (PANDEY et al. 1987). Theseactions, although shown to be mediated by guanylatecyclase coupled ANF receptors, have been observedat ANF concentrations far above those present in thecirculation. These findings suggest that locally syn-thesized ANF acts in an autocrine or paracrine man-ner to produce a physiological response. Increasing

evidence suggests that ANF influences the testoster-one production in Leydig cells (MUKHOPADHYAY et al.1986; PANDEY et al. 1986; SHUMACHER et al. 1992;DAVIDOFF et al. 1993). In this context KHURANA et al.(1993) demonstrated that ANF stimulates Leydig cellsteroidogenesis beginning from the cholesterol side-chain cleavage enzyme. This effect was found to beelicited by ANF coupled to a guanylate cyclase activ-ity containing plasma membrane receptor, termed GC-A (CHINKERS et al. 1989), and a functional relation-ship seems to exist between testicular GC-A expres-sion and testosterone production (KAPASI et al. 1996).The presence of GC-A in Leydig cells could be estab-lished by a variety of methods (PANDEY et al. 1986;PANDEY and SINGH 1990; KAPASI et al. 1996). Howev-er, MULLER and MIDDENDORFF (1997) demonstrated thatseminiferous tubules rather than Leydig cells repre-sent the predominant sites of functionally active ANF

184 ANF IN RAT TESTES

receptors in rat testis. Moreover, GC-A concentrationswere found to be five-fold higher in tubular than inLeydig cell membranes. These results raise the ques-tion whether circulating Leydig cell produced ANFor seminiferous tubule produced ANF predominatesin the interaction with tubular ANF receptors. On theother hand, ANF immunoreactivity in rat testis andspecific localization in spermatids and acrosomal caps,but not in Leydig cells was observed by PANDEY et al.(1991) and in rat Leydig cells by MULLER and MID-DENDORFF (1997). Despite of this discrepancy, the abovefindings suggest a novel role for ANF related to germcell development in addition to steroidogenesis.Recent studies in our laboratory have shown that ANFwas constantly expressed, with some differences instaining intensity, in Leydig cell cytoplasm and in dif-ferent tubular cell types from birth to adult stage(MOURDJEVA et al., submitted for publication).

To gain new insight into ANF expression and func-tion in rat testis we investigated ANF localization af-ter Leydig cell destruction by treatment with ethanedimethane sulphonate (EDS). A single intraperitonealinjection of EDS results in destruction of all Leydigcells within 36 hours which regenerate about 2 to6 weeks later (SHARPE et al. 1990). This model hasmany advantages over the hypophysectomized rat inthat the destruction of the Leydig cells is highly se-lective and has no direct effect on proliferating germcells, as well as on hemopoiesis (JACKSON et al. 1984).

Materials and Methods

Monoclonal antibody preparation. The produc-tion and characterization of the specificity of Mab 6C3used in this study have been described previously (KE-HAYOV et al. 1998). Briefly, BALB/c mice were immu-nized by s.c. injections of ANF conjugate to bovineserum albumin (BSA) emulsified in Freund�s adju-vant. Immune spleen lymphocytes showing the high-est titer of anti-ANF antibodies were fused with P3U1mouse myeloma line. Supernatants from wells withgrowing hybridomas were screened for the presenceof anti-ANF antibodies by ELISA. As a result Mab6C3, which was of IgG type, was selected.

EDS model. Mature Wistar rats were housed un-der the conditions of 12-hour light and dark cy-cles. Food and water were provided ad libitum. Theanimals received single i.p. injection of EDS (75

mg/kg body weight) in 1:3 ratio of dimethyl sul-phoxide (DMSO) and water or of vehicle alone.EDS is not commercially available and was kind-ly synthesized from methane sulphonyl chloride,ethylene glycol and pyridine and supplied by Prof.M.Davidoff from the Institute of Anatomy, Uni-versity of Hamburg, Germany. The animals werekilled 24 hours, 7, 21 and 45 days after treatment.Tissue blocks were fixed by immersion in Bouin�sfluid for 24 h at 20 oC. Subsequently, the blockswere embedded in paraffin, and 5 µm sectionswere mounted on chrome-gelatin precoated slides.The paraffin sections were stained with acridinorange and the morphology of apoptotic cells wasanalyzed by a Zeiss epifluorescence microsrope.Zeiss MC-100 camera system and Kodak T-MAX400 film were used for the microphotographs.

Immunocytochemistry. Paraffin sections fixed inthe Bouin�s solution were processed for immunocy-tochemical analysis using avidin-biotin-peroxidasetechnique described by HSU et al., (1981). In this pro-cedure methanol hydrogen peroxide solution wasused to block endogenous peroxidase activity, andnormal rabbit serum was used to block nonspecificbinding of the secondary antibody (biotinylated rab-bit anti-mouse IgG). Sections were incubated withMab 6C3 (hybridoma supernatant) for 18 h at 4 oCthen rinsed with phosphate buffered saline (PBS) andincubated for 60 min with biotinylated anti-mouseIgG (Vector, Burlingam) diluted 1:250 in PBS. Afterrinsing in PBS avidin-biotin-peroxidase conjugate(Vector, Burlingam) diluted 1:250 in PBS was ap-plied for 60 min. The binding sites were visualizedwith 3,3'-diaminobenzidine tetrahydrochloride(DAB) in 0.5 M Tris-HCl buffered saline (pH 7.6)0.01% H

2O

2, dehydrated and coverslipped. Control

sections were incubated either with normal mouseserum or with control antibodies � Mab 5G5 (IgG)which bound 59 kDa antigen localized on Leydigcell surface (RUSSINOVA et al. 1995) and Mab 4E6(IgG) which bound 40 kDa antigen localized in ova-rian steroid producing cells (RUSSINOVA et al. 1999)instead of the primary antibody.

Results

In testicular sections 24 h after EDS treat-ment a variety of changes in Leydig cell morphology

185ANF IN RAT TESTES

Fig.1. Acridine orange staining 24 h after EDS treatment and after Leydig cell degeneration. Arrow in A and B points to Leydigcells with intense, well demarcated areas of nuclear fluorescence suggesting that condensation of chromatin has occurred � a

hallmark of apoptosis (24 h after EDS treatment). Some Leydig cells exhibited fragmented nuclei which is an indicator ofapoptosis (C). Arrow in E points to an apoptotic cell located in the seminiferous lumen, exhibiting also condensed chromatin (24

h after EDS treatment). Using acridin orange staining the chromatin of new Leydig cell population showed normalmorphological appearance (D � 45 days after EDS treatment). Magnification: x 200.

was detectd by acridine orange staining. The majorityof Leydig cells were much larger with intense, wellremarkable areas of nuclear fluorescence suggesting

that the condensation of chromatin has occurred, whichis an indicator of apoptosis (Fig. 1a,b). Some Leydigcells exhibited fragmented nuclei � a hallmark of ap-

186 ANF IN RAT TESTES

optosis (Fig. 1C). In selected tubules apoptotic germcells were found located in the lumen which were de-fined as exhibiting condensed chromatin, while nonapoptotic cells showed a fine network of chromatinin the entire nuclear area (Fig.1 E)

Using ABC method and anti-ANF antibody themost intensive staining was observed 24 h after ESDtreatment. In interstitial compartment ANF labelingwas detected in apoptotic Leydig cells which seemedto be totally labeled (Fig. 2A). By light microscopethe structural damage of Leydig cells was often somarked that it became difficult to identify individualcells, since their cytoplasm was fragmented and dis-persed throughout large areas of the interstitial spacesa feature not seen in normal interstitial tissues. ANFstaining was observed in Sertoli cell cytoplasm, sper-matids and in totally labeled degenerating germ cells,specifically in tubules at stage VII of the seminifer-ous cycle (Fig. 2B, C).

In testicular sections 7 days after EDS treatmentthree novel morphological characteristics were detect-ed: 1. no Leydig cells were observed in the interstitialcompartment; 2. appearance of a ring of small baselylocated vacuoles in the seminiferous tubules, specifi-cally in the tubules at stage VII -VIII of seminiferouscycle; 3. a comparable decrease was inferred fromreduction in the diameter of seminiferous tubule lu-men at stage VII. This morphological picture was notseen in vehicle treated control rats (not shown). ANFimmunoreactivity was detected in Sertoli and germcell cytoplasm with a more prominent staining in sper-matids. Strong labeling was observed in the region oflamina propria. The region of basely located vacuoleswhich coincides with the location of Sertoli cell bar-rier was also stained. Labeled degenerative cells wereoften observed (Fig. 3A, B).

At the 21st day after EDS treatment small labeledLeydig cells were observed in the interstitial compart-ment. ANF staining in seminiferous tubules was de-tected in Sertoli and germ cells cytoplasm. However,the labeling was more significant in the region of lam-ina propria and in the seminiferous epithelium regionsituated above labeled spermatogonia (Fig. 3C).

At the 45th day after EDS treatment the vacuoliza-tion of the Sertoli cell barrier area was not detected.The seminiferous tubule diameter increased. TheLeydig cells were more abundant and arranged inclusters. Using acridin orange staining the chroma-

tin of new Leydig cell population showed normalmorphological appearance (Fig. 1D).The strongestANF staining was detected in Leydig cell repopula-tion. In seminiferous tubules ANF labeling was ob-served in Sertoli and germ cell cytoplasm and in sper-matids embedded within the apical invaginations ofthe Sertoli cells. The reaction intensity in the regionof lamina propria decreased. The labeled degenerat-ing germ cells were not observed. No reaction wasobserved in control sections (Fig. 2D).

Discussion

Although originally isolated from mammalianatria, ANF is not exclusively an atrial hormone. Itspresence and local synthesis have been establishedin several tissues and organs (GUTKOWSKA et al. 1989,1993). The Leydig cells could be target cells for sucha local production since ANF has been shown tostimulate testosterone synthesis and release (PANDEY

et al. 1996; KHURANA et al. 1993). Moreover, pres-ence of ANF receptors in Leydig cells has been dem-onstrated (PANDEY and SINGH 1990; KAPASI et al.1996). Recently, MULLER and MIDDENDORFF (1997)established that the seminiferous tubules representthe predominant sites of ANF receptors in rat testis.

In the present study the use of immunocytochem-ical analysis of ANF expression following EDS treat-ment has provided a step forward in elucidating ANFsource in rat testis. We use the EDS model for fourreasons. First, the Leydig cells are the main candi-date for ANF production in the testis. Second, afterEDS treatment Leydig cells have undergone ad-vanced disintegration which leads to their completeelimination from the testis (KERR et al. 1985). Ley-dig cells are absent from the testis for at last 7 days.Regeneration of these cells occurs within 2-6 weeksresulting in a substantial restoration of their totalvolume normally seen in adult rat testis (SHARPE etal. 1990). Third, it is interesting to follow up ANFexpression in the seminiferous epithelium to a periodof androgen deprivation due to temporally depletionof the Leydig cell population. Fourth, there is no in-formation about data on ANF staining pattern inLeydig cell repopulation.

In our earlier studies it has been suggested that inimmature and adult rat testis ANF was localized inLeydig, germ and Sertoli cell cytoplasm, as well as

187ANF IN RAT TESTES

Fig.2. ABC staining with anti-ANF antibody 24 h after EDS treatmentThe reaction was observed in apoptotic Leydig cells (A) and in Sertoli cell cytoplasm, spermatids and degenerating germ cells

(arrows) (B and C). No reaction was observed in control section after replacement of the anti-ANF antibody with normal mouseserum (D). Magnification: A,B and C x 200; D x140

in degenerating germ cells (MOURDJEVA et al., sub-mitted for publication). The latter were observed atthe 23 day of age. It is known that in rat at 16 -24

days of age spermatocytes in some selected tubulesshowed increased DNA fragmentation � a hallmarkof apoptosis (BILLIG et al. 1995), and germ cell de-

188 ANF IN RAT TESTES

Fig.3. ABC staining. Seven days after EDS treatment. No Leydig cells were observed in interstitial compartment. The ANFstaining was observed in Sertoli and germ cell cytoplasm with more prominent staining in spermatids. The degeneratinggerm cells were also labeled. Strong ANF labeling was observed in the region of basely situated vacuoles which coinsides

with the location of Sertoli cell barrier (A and B). Small labeled Leydig cells were observed in interstitial compartment 21days after EDS treatment. The ANF labeling in seminiferous tubules was more significant in the region of lamina propria

(C). The strong labeled new Leydig cell population was detected 45 days after EDS treatment (D).Magnification: A,B and C x140; D x200.    

189ANF IN RAT TESTES

generation is an integral and important part of nor-mal spermatogenesis. Compared to the above results,the present study revealed similar cell type ANFstaining pattern. ANF staining was detected in apop-totic cells, germ and Sertoli cell cytoplasm. The func-tional significance of ANF staining observed in de-generating cells during normal testicular develop-ment and after EDS treatment remains to be eluci-dated. The demonstration of ANF staining inseminiferous epithelium after Leydig cell elimina-tion suggests that Leydig cells are not the sole sourceof ANF in rat testis and the seminiferous epitheliummay be a new site which ANF may be synthesized.The results also suggest a broad spectrum of ANFactivity in rat testis.

EDS has been recognized to exert an unusual phar-macological action. A single dose killed Leydig cellswhich showed morphological features of the pro-grammed cell death or apoptosis (GAYTAN et al. 1995a), to inhibit Leydig cell testosterone production, andto suppress serum androgen levels 24 h after admin-istration (KERR et al. 1986).

The present study confirmed previously describedmorphological alterations in rat testis after EDS treat-ment as Leydig cell apoptosis, appearance of degen-erating germ cells at stage VII � VIII, reduction oftubule lumen diameter at stage VII and the presenceof vacuoles in the region of Sertoli cell barrier atstage VII (GHOSH et al. 1992; KERR et al. 1993; SHARPE

1994). These results suggest that testosterone actsspecifically at around stage VII of the spermatoge-nic cycle and that the degenerating germ cells at thisstage are the most sensitive index of testosterone in-sufficiency (SHARPE 1994). However, using anti-ANFantibody we established strong staining intensity inrat testis after testosterone withdrawal, which sug-gests that ANF expression does not depend on test-osterone production.

It is known that the destruction of Leydig cellselicits an immune response involving macrophage-lymphocyte interaction (KERR et al. 1985), anda significant increase in the number and total mac-rophage volume during Leydig cell apoptosis wasdescribed (GAYTAN et al. 1995 a). It was also foundthat the resident macrophages prevent the inflam-matory reaction elicited Leydig cell death (GAYTAN

et al. 1995 b). In this regard, KERR et al. (1985) spec-ulated that EDS may be an allergen specific to the

macrophages which precipitates their phagocyticactivities and interaction with lymphocytes. The ob-served strong ANF immunoreactivity after EDS treat-ment was an unexpected result. However, VOLLMAR

et al. (1993) demonstrated intensive ANF stainingafter X-ray irradiation, which induces apoptosis ofthymocytes followed by extensive phagocytosis andelevation of ANF expression. Evidence was present-ed that spleen macrophages produce ANF (THROSBY

et al.1991), and these results support the hypothesisthat ANF may play a functional role in the immuneresponse. The authors speculated that ANF may playthe role of a signaling molecule for interaction be-tween macrophages and lymphocytes. It is likely thatthe strongest ANF staining observed 24 h after EDStreatment was at least in part a result of high phago-cytic activity.

The regeneration of Leydig cells 2-6 weeks afterEDS treatment following their total depletion raisesthe question of their origin. There is increasing evi-dence that newly differentiated Leydig cells may arisefrom peritubular cells, subsequently leaving laminapropria to gain access to the surrounding interstitialtissues (KERR et al. 1985). In this context, we ob-served increased ANF staining in the region of peri-tubular lamina propria 21 days after EDS treatmentat which time Leydig cells regeneration occurs (KERR

et al. 1985). The observed ANF labeling in the re-gion of peritubular lamina propria indicates that ANFmay play a role in Leydig cell regeneration. It isknown that ANF, when chronically administered,cause a notable hypertrophy of rat Leydig cell dueto increases in the volume of all organelles involvedin cholesterol and testosterone synthesis, and thesefindings suggest that ANF stimulates growth and ste-roidogenic capacity (MAZZOCCHI et al. 1990). Evi-dence that seminiferous tubules are thought to be thesite of production of factors modifying both the sizeand the secretory properties of Leydig cells was es-tablished by SHARPE and COOPER (1984). Our resultstaken together with those mentioned above giveground to assume that the seminiferous tubule ANFin some way exerts a local stimulatory effect uponthe development and function of new Leydig cellpopulation.

In conclusion, the immunological detection ofANF in seminiferous epithelium after Leydig cellelimination and androgen deprivation raises the pos-

190 ANF IN RAT TESTES

sibility of a local production of ANF which seemsto play a role in regulating gonadal function. 

Acknowledgments

We thank Professor Davidoff from the Institute ofAnatomy, University of Hamburg, Germany, for hissupport in immunocytochemical investigations. Thisresearch was supported by grants B 815/98 andK 803/98.. from the National Foundation for Scien-tific Research.

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Corresponding Author: Mariana Bakalska, M.D.Institute of Experimental Morphology andAnthropologyBulgarian Academy of SciencesAcad. G.Bonchev str., bl.251113 SofiaPhone: 00359-2-979 23 29Fax: 00359-2-719 007