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Summary. Anti-Mullerian hormone (AMH) producedby the immature Sertoli cells negatively regulates thepostnatal Leydig cell (i.e. adult Leydig cells/ALC)differentiation, however, the mechanism is sparselyunderstood. AMH negatively regulates the steroidogenicfunction of fetal Leydig cells (FLC) and ALC. However,when this function is established in the ALC lineage andwhether AMH has a function in FLC in the postnataltestis are not known. Therefore, the objectives of thisstudy were to examine the presence of AMH receptortype II (AMHR-II) in FLC and cells in the ALC lineagein the postnatal mammalian testis using the rat modelMale Sprague Dawley rats of days 1, 5, 7-21, 28, 40, 60and 90 were used. AMHR-II in testicular interstitial cellswas detected in testis tissue using immunocytochemistry.Findings showed that the mesenchymal and theprogenitor cells of the ALC lineage, were negative forAMHR-II. The newly formed ALC were the first celltype of the ALC lineage to show positive labeling forAMHR-II, and the first detection was on postnatal day13, although they were present in the testis from day 10.From days 13-28, labeling intensity for AMHR-II in theALC was much weaker than those at days 40-90. FLCwere also positive. The time lag between the firstdetection of the newly formed ALC in the testis and thefirst detection of AMHR-II in them suggests that theestablishment of the negative regulatory role of AMH onALC steroidogenesis does not take place immediatelyupon their differentiation; no change in cell size occursduring this period. The absence of AMHR-II inmesenchymal cells suggests that it is unlikely that thenegative regulatory effect of AMH on ALCdifferentiation in the postnatal testis is achieved via adirect action of AMH on mesenchymal cells. Thepresence of AMHR-II in postnatal FLC suggests a

possible role by AMH on FLC, which warrants futureinvestigations.Key words: AMHR-II, Leydig cells, Mesenchymalcells, Progenitor cells, Sertoli cells, Postnatal testis,Light microscopy, Immunohistochemistry

Introduction

Anti-Mullerian hormone (AMH), a glycoprotein anda member of the transforming growth factor-beta (TGF-) family, is an important factor in male sexualdifferentiation. In the male, AMH is produced by theSertoli cells from the time of fetal sex differentiation topuberty (Josso et al., 2001). As other members of TGF-family, AMH signals through two related but distinctreceptors, both serine/threonine kinases with a singletransmembrane domain, called type I and type II. Type IIreceptor is solely expressed in AMH target organs (Jossoet al., 2001) such as the testis.

The prenatal effect of AMH in males is welldocumented and includes the regression of Mullerianducts, the anlagen of the female internal reproductivetract in male fetuses (Jost, 1953). Rouiller-Fabre et al.,(1998) have also showed that AMH represses aromataseactivity of fetal Sertoli cells and inhibits testosteronesynthesis by fetal Leydig cells (FLC). The role of AMHin the postnatal testis is not clearly identified at present.Male transgenic mice expressing very high levels ofhuman AMH (hAMH) under the control of the mousemetallothionein-1 promoter (MT-hAMH), areincompletely masculinized externally and rapidlybecome infertile (Behringer et al., 1990). It is also seenthat male MT-hAMH mice expressing lower levels ofAMH show a reduced number of postnatallydifferentiated Leydig cells or adult Leydig cells (ALC,Racine et al., 1998). In contrast, AMH deficient miceexhibit marked Leydig cell hyperplasia (Racine et al.,1998). Taken together, it is suggested that AMH is a

Detection of anti-mullerian hormone receptor II protein in the postnatal rat testis from birth to sexual maturityS.M.L.C. Mendis-Handagama1, N. Di Clementi2, H.B.S. Ariyaratne1,3 and L. Mrkonjich11Department of Comparative Medicine, College of Veterinary Medicine, The University of Tennessee, Knoxville, USA and2Department de Biologie, Ecole Normale Superieure, INSERM U 493, 912120, Montrouge, France3Present address: Department of Basic Sciences, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, SriLanka

Histol Histopathol (2006) 21: 125-130

Offprint requests to: Chamindrani Mendis-Handagama, Department ofComparative Medicine, College of Veterinary Medicine, The Universityof Tennessee, 2407 River Drive, Knoxville, TN 37996, USA. e-mail:[email protected]

DOI: 10.14670/HH-21.125

http://www.hh.um.es

Histology andHistopathologyCellular and Molecular Biology

negative regulator of ALC differentiation in the postnataltestis (Racine et al., 1998).

Theoretically, the inhibitory action of AMH on thedifferentiation of Leydig stem cells to progenitor cells tobegin the process of ALC differentiation could beexerted either directly or indirectly. Mesenchymal cells,especially the ones in the peritubular region of thepostnatal testis interstitium are shown as the primaryprecursors for the ALC in the adult testis (Ariyaratne et.al., 2000). Therefore, if AMH action on thesemesenchymal cells is direct, they should possess AMHreceptors (AMHR), specifically, AMHR-II (Josso et al.,2001). However, there is no information available on thepresence of AMHR type II in mesenchymal cells in thepostnatal testis to determine whether AMH acts directlyor indirectly to inhibit the ALC differentiation. Bycontrast, it is known that the mesenchymal cells aroundthe Mullerian ducts in the fetal testis contain AMHR-II(Josso and di Clementi, 2003).

AMH has also been suggested as a negativeregulator of ALC steroidogenesis. Addition of AMH topurified adult mouse Leydig cells (Racine et al., 1998)and R2C and MA-10 adult Leydig cell lines (Teixeira etal., 1999; Fynn-Thompson et al., 2003) decreases steroidhormone production (Racine et al., 1998; Rouiller-Fabreet al., 1998) by down regulating steady-state mRNAlevels of enzymes in the steroidogenic pathway (Racineet al., 1998) and interfering with cAMP signaling (Fynn-Thompson et al., 2003). However, it is also reported thathigher levels of intra-testicular testosterone is present inethane dimethane sulphonate-treated adult rats after 35days who were treated with AMH/MIS from day 11-14after EDS treatment (Salva et al., 2004).

AMHR-II mRNAs, but not the protein AMHR-II,have been detected in developing and adult rat testis(Barrends et al., 1995) adult Leydig cells (Lee et al.,1999). Yet, no precise information is available on whenand/or which cell type of the ALC lineage first gainAMHR-II. In addition to the cells of ALC lineage, thepostnatal testis interstitium contains FLC (Mendis-Handagama et al., 1987, 1998; Kerr and Knell, 1988;Ariyaratne and Mendis-Handagama, 2000; Baker et al.,2003) which also have the steroidogenic potential.However, to our knowledge, there is no documentationon the presence of AMHR-II in FLC. In this study, wehave used the first polyclonal antibody against AMHR-IIto address this important issue which is crucial tounderstand the establishment of the regulatory role ofAMH in ALC and FLC in the postnatal testisinterstitium, and on ALC differentiation. Materials and methods

Animals

Pregnant female Sprague Dawley rats werepurchased from Harlan Industries (Madison, WI). Theywere housed one rat per cage, under conditions of

controlled temperature (25C) and lighting (14L:10D) inthe animal facility of The University of TennesseeCollege of Veterinary Medicine. Rats were providedfood (Agway Prolab rat formula, Syracuse, NY) andwater ad libitum. These rats were examined for litterstwice daily (morning and evening) and the day of birthof pups was considered as Day 1 of postnatal life.AMHR-II Antibody

A complete description of the antibody used toimmunolocalize AMHR-II is published elsewhere (18).Briefly, a bacterially produced protein constituted by theextracellular domain of the human AMHR-II fused to aHis6 tag was used to immunize a rabbit, and theresulting serum after 3 months was purified by affinitywith the fusion protein as indicated in the OQuick PureSystem kit (Sterogene, Isnes, Belgium). As previouslydescribed (Gouedard et al., 2000; di Clemente et al.,2003), This antibody recognizes both the immature andmature forms of AMHR-II in CHO cells permanentlytransfected with AMHR-II and not in parental CHOcells, by western-blotting. Testicular tissue preparation

Twenty one groups of male rats of the followingages were used (n=5 per group); 1, 5, 7-21, 28, 40, 60and 90 days of age. They were killed by CO2 inhalationand both testicles of each rat were harvested and fixed inBouins fluid by immersion fixation for 5-6 hours,processed and prepared for immunocytochemistry(Mendis-Handagama et al., 1998; Ariyaratne andMendis-Handagama, 2000). Sections of 5 m inthickness were cut from these testis blocks and adheredon ProbeOn Plus glass slides (Fisher Scientific,Pittsburgh, PA). The animal protocol used(protocol#731) was approved by the Institutional AnimalCare and Use Committee. Immunocytochemistry

To immunolocalize AMHR-II, testis tissue sectionswere deparaffinized, incubated in 3% hydrogen peroxidefor 20 minutes to quench endogenous peroxidaseactivity, and then incubated in goat serum (Biogenex,San Ramon, CA) overnight at 4C to eliminate non-specific binding. AMHR-II antibody was used at anoptimum dilution of 1:100, and the incubations werecarried out at 4C overnight. In control incubations,tissue sections were incubated with preimmune serum.The antigen-antibody complex was visualized using asuper sensitive biotin-streptavidin-peroxidase method(Biogenex, San Ramon, CA). Finally, the sections werecounter-stained with Mayers hematoxylin (Sigma, St.Louis, MO), dehydrated in a series of increasingconcentrations of ethanol and cover-slipped underPermount (Fisher Scientific, Fair Lawn, NJ).

126AMHR-II in testicular intestitial cells

Results

Immunocytochemistry

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