Proteolytically Activated, Recombinant Anti-Mullerian Hormone Inhibits Androgen Secretion,Proliferation, and Differentiation of Spermatogoniain Adult Zebrafish Testis Organ Cultures

K. S. Skaar,* R. H. Nobrega,* A. Magaraki, L. C. Olsen, R. W. Schulz,and R. Male

Department of Molecular Biology (K.S.S., L.C.O., R.M.), University of Bergen, N5020 Bergen, Norway;Department of Biology (R.H.N., A.M., R.W.S.), University of Utrecht, NL-3584 CH Utrecht, The Netherlands;and Sars International Centre for Marine Molecular Biology (L.C.O.), N5008 Bergen, Norway

Anti-Mullerian hormone (Amh) is in mammals known as a TGF� type of glycoprotein processed toyield a bioactive C-terminal homodimer that directs regression of Mullerian ducts in the male fetusand regulates steroidogenesis and early stages of folliculogenesis. Here, we report on the zebrafishAmh homologue. Zebrafish, as all teleost fish, do not have Mullerian ducts. Antibodies raisedagainst the N- and C-terminal part of Amh were used to study the processing of endogenous andrecombinant Amh. The N-terminally directed antibody detected a 27-kDa protein, whereas theC-terminally directed one recognized a 32-kDa protein in testes extracts, both apparently notglycosylated. The C-terminal fragment was present as a monomeric protein, because reducingconditions did not change its apparent molecular mass. Recombinant zebrafish Amh was cleavedwith plasmin to N- and C-terminal fragments that after deglycosylation were similar in size toendogenous Amh fragments. Mass spectrometry and N-terminal sequencing revealed a 21-residueN-terminal leader sequence and a plasmin cleavage site after Lys or Arg within Lys-Arg-His atposition 263–265, which produce theoretical fragments in accordance with the experimental re-sults. Experiments using adult zebrafish testes tissue cultures showed that plasmin-cleaved, but notuncleaved, Amh inhibited gonadotropin-stimulated androgen production. However, androgensdid not modulate amh expression that was, on the other hand, down-regulated by Fsh. Moreover,plasmin-cleaved Amh inhibited androgen-stimulated proliferation as well as differentiation oftype A spermatogonia. In conclusion, zebrafish Amh is processed to become bioactive and hasindependent functions in inhibiting both steroidogenesis and spermatogenesis. (Endocrinology152: 3527–3540, 2011)

Anti-Mullerian hormone (Amh) is a member of theTGF� superfamily engaged in the regulation of cell

proliferation, differentiation, growth, and apoptosis (1,2). The namesake function of Amh is to induce regressionof the Mullerian ducts during male sex differentiation intetrapod vertebrates (3, 4).

Amh was first isolated as a 123-kDa dimeric glycopro-tein secreted from bovine testes and from bovine testes

incubation medium (3, 5–7). Recombinant human Amhproduced in Chinese hamster ovary cells is processed bycleavage of the 24-amino acid (aa) leader sequence, fol-lowed by a second cleavage after the RAQR motif at po-sition 424–427 (8, 9). Functional experiments have re-vealed that Amh is strongly activated by cleaveage (10,11). Amh is glycosylated in the N-terminal part of thehuman and avian protein (12, 13), and the N-terminal

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in U.S.A.Copyright © 2011 by The Endocrine Societydoi: 10.1210/en.2010-1469 Received December 23, 2010. Accepted June 23, 2011.First Published Online July 26, 2011

* K.S.S. and R.H.N. contributed equally to this work.Abbreviations: aa, Amino acid; Adiff, A differentiated; ALK, activin-receptor kinase; Amh,anti-Mullerian hormone; AmhRII, Amh type II transmembrane receptor; ar, androgen re-ceptor; Aund, A undifferentiated; BrdU, bromodeoxyuridine; cyp17a1, cytochrome P450,family 17, subfamily A, polypeptide 1; ER, endoplasmatic reticulum; HEK293, humanembryonic kidney 293; insl3, insulin-like 3; 11-KT, 11-ketotestosterone; PKA, protein ki-nase A; star, steroidogenic acute regulatory protein; wpf, week postfertilization.

R E P R O D U C T I O N - D E V E L O P M E N T

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region (present as a homodimer after cleavage) enhancesthe activity of the C-terminal fragment (14). The C-ter-minal fragment contains the conserved TGF� domain (2)with highly conserved cysteines involved in forming acystine-knot structure of the active part (12, 15). A re-cent report has verified that cleavage is necessary forefficient receptor binding and that the N-terminal pro-region is present at receptor interaction but dissociatesfrom the C-terminal homodimer as a consequence ofreceptor binding (16).

Amh signals through binding to a Amh type II trans-membrane receptor (AmhRII) with serine/threonine ki-nase activity (17). This complex recruits and phosphory-lates one of the activin-receptor kinases (ALK) mediatingstimulatory (ALK2 and ALK3) or inhibitory (ALK6) ef-fects (17, 18) that involve Smad proteins in the down-stream signaling cascade (19). In the testis, AmhRII is ex-pressed in Sertoli (20) and Leydig (21) cells. In fish, anAmhRII has been identified in medaka (Oryzias latipes)(22) and reported from black porgy, Acanthopagrusschlegeli (23).

In mammalian testes, Amh is highly expressed by im-mature Sertoli cells until prepuberty, when at the start ofmeiosis, increasing testosterone levels down-regulateAmh, mRNA and protein levels, that remain low duringlate puberty and adulthood (24). Amh expression in ju-venile males is stimulated by Fsh when the androgen re-ceptor is not activated in Sertoli cells (25). Moreover, Amhis a negative regulator of postnatal Leydig cell differenti-ation and steroidogenesis. Mice transgenic for humanAmh show a decreased number of adult-type, differenti-ated Leydig cells, low plasma levels of testosterone, andreduced mRNA levels of steroidogenic enzymes, includingCyp17 (21). Also, Amh reduced LH-induced testosteroneproduction by fetal and adult Leydig cells in rodents (26,27). Amh-deficient mice, on the other hand, showed aremarkable hyperplasia of Leydig cells (28, 29). Theseobservations suggest that in mammals, Amh has a negativeregulatory role in the postnatal differentiation and func-tion of Leydig cells.

Teleost fish lack Mullerian ducts, but Amh homologueshave been identified in several species, such as Japanese eel(30), zebrafish (31, 32), tilapia (33), medaka (22), sea bass(34), Iberian chub (35), pejerrey (36), and rainbow trout(37, 38). In primary cultures of Japanese eel testis frag-ments, recombinant eel Amh (originally named spermato-genesis-preventing substance) inhibited 11-ketotestoster-one (11-KT), the main androgen in zebrafish (39) andother fish species (40), and induced spermatogenesis byblocking the proliferation of type B spermatogonia (30). Inmedaka, there is an additional aspect of Amh bioactivityduring early gonad differentiation in both sexes: gene

knockdown experiments targeting Amh or AmhRII re-duced germ-cell proliferation, and recombinant eel Amhcounteracted this effect when added to medaka gonad tis-sue fragments from amh knockdown animals (41). In con-trast, the medaka amhrII/hotei loss-of-function mutantdisplayed an increased number of germ cells in both sexes(42). Moreover, male mutants showed premature initia-tion of meiosis, and 50% underwent sex reversal.

Taken together, there is evidence for effects of Amh ongerm-cell proliferation in fish, whereas the quality of theeffect differs, being stimulatory during early stages of de-velopment (41) but inhibitory at the onset of puberty (30).However, it has remained unexplored whether Amh bio-activity described in mammals, such as the inhibitory ef-fect on Leydig cell function (43), can be observed in fishand may thus be an evolutionary conserved feature. More-over, it is not known whether Amh has an effect on sper-matogenesis in adult animals. In this work, we first haveinvestigated how zebrafish Amh is processed to becomebioactive, before testing plasmin-cleaved, recombinantAmh in zebrafish testis cultures. To this end, we have in-vestigated whether Amh modulates steroidogenesis orspermatogenesis in adult zebrafish testis. We also inves-tigated the ontogenesis of amh expression and aspects ofthe endocrine regulation of amh expression, and we pro-pose a model on the role of Amh on adult zebrafish testisfunctions.

Materials and Methods

Detailed material and methods are provided in the Supplementaldata, published on The Endocrine Society’s Journals Online website at http://endo.endojournals.org. In brief, antisera directed toN-terminal and C-terminal regions of Amh were raised againstpeptides and provided as purified rabbit antibodies by BioGenes(Berlin, Germany). The C-terminal-directed antibody was usedfor immunocytochemical detection of endogenous Amh in adultzebrafish testis. Recombinant zebrafish Amh was produced instably transfected human embryonic kidney 293 (HEK293) cellsand purified from culture medium via a 6xHis tag introducedafter Pro33 (AY721604). The presumed proteolytic cleavagesite, based on sequence comparison, was optimized from RAQRmotif (position 439–442) to RARR using QuickChange II Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA), and theproduct was named His-Amh-Q441R. Plasmin treatments wereconducted as indicated in figure legends. The reaction wasstopped with aprotinin (A3428; Sigma, St. Louis, MO) and theextract frozen at �80 C until further use.

Amh in distilled water was deglycosylated using the Enzy-matic Carbo Release kit from QA-Bio (Palm Desert, CA)(KE-DG01). N-terminal sequencing using Edman degrada-tion was performed (Proteomics Facility, University of Leeds,Leeds, UK) on SDS-PAGE separated Amh followed by elec-troblotting onto a sequencing-grade polyvinylidene fluoridemembrane. Matrix-assisted laser desorption/ionization-time-

3528 Skaar et al. Amh Function in Zebrafish Testis Endocrinology, September 2011, 152(9):3527–3540

of-flight analyses (PROBE Proteomics Facility, University ofBergen) were performed on acetone precipitated purified un-treated and plasmin-cleaved peptide-in-gel samples. Untreatedand plasmin-cleaved, recombinant zebrafish Amh (10 �g/ml or140 nM, 70 nM if dimer) were added to primary zebrafish testisorgan cultures (as described in Ref. 44) in the presence of re-combinant zebrafish Fsh (100–500 ng/ml). Cultures were ana-lyzed for 11-KT in media (44), and testis tissue for expression ofselected genes [cytochrome P450, family 17, subfamily A, poly-peptide 1 (cyp17a1), steroidogenic acute regulatory protein(star), insulin-like 3 (insl3), and androgen receptor (ar)] by real-time quantitative PCR (44). Relative mRNA levels of target genes(cyp17a1, star, insl3, and ar) were normalized to 18S rRNA (ref-erence endogenous control gene) and expressed as fold inductioncompared with the control groups.

Gonadal amh mRNA was quantified as previously (44) insamples collected 4 wk postfertilization (wpf) (after completionof sex differentiation), 8 wpf (pubertal gonad growth ongoing),and 12 wpf (young adults), detailed in Supplemental Methods.To address the endocrine regulation of amh expression, adulttestis tissue was incubated with androgens or with recombinantzebrafish Fsh in the absence or presence of a protein kinase Ainhibitor H89 under different conditions (see SupplementalMethods), before amh mRNA levels were quantified as previ-ously (44).

Effects of recombinant zebrafish Amh on spermatogenesis[bromodeoxyuridine (BrdU) incorporation into type A undiffer-entiated (Aund) spermatogonia and frequency of germ-cell types]were analyzed using a previously described tissue culture system(45). Testes were collected from untreated, adult males, and tis-sue was incubated in the presence of 200 nM 11-KT to test theeffect of recombinant zebrafish Amh (10 �g/ml), or testes werecollected from adult males exposed for 3 wk to 10 nM estradiol-17� to induce androgen insufficiency and inhibit spermatogen-esis (46) before the tissue was incubated in the absence or pres-ence of Amh (10 �g/ml). Incubation conditions, fixation, andmorphological and morphometrical analysis of the samples aredetailed in the Supplemental Methods.

Significant differences between two groups were identifiedusing Student’s t test (paired and unpaired) (P � 0.05). Com-parisons of more than two groups were performed with one-wayANOVA followed by Student-Newman-Keuls test (P � 0.05).GraphPad Prism 4.0 (GraphPad Software, Inc., San Diego, CA)was used for all statistical analysis. Sequence analyses were per-formed as described in Supplemental Methods.

Results

Amh is processed by proteolytic cleavageThe zebrafish Amh cDNA sequence (AY721604) pre-

dicts a 549-aa protein with a molecular mass of 61.1 kDa,including a 21-residue leader sequence, that after cleavagegives a 58.7-kDa protein (Fig. 1A and Supplemental Fig.1). Two N-glycosylation sites were predicted at N334(NSST) and N510 (NRSL), where the first site is conservedamong fish species and is close to the human Amh glyco-sylation site (NLSD position 329–332, BC049194) (Sup-plemental Fig. 1). The predicted C-terminal TGF� domain

(aa 457–549) has seven conserved cysteine residues whereC514 is expected to be involved in dimer formation. Hu-man AMH is known to be cleaved after RAQR at position448–451, which align to a putative cleavage site in ze-brafish Amh at position 439–445 (Supplemental Fig. 1B).

Immunoblots of testes protein extracts under reducingand nonreducing conditions revealed two fragments proba-bly generated by proteolytic cleavage of the full-length pro-tein: N-27 and C-32 (Fig. 1, B and C). An alternative lysisbuffer produced two candidate full-length Amh proteinsof 66 and 71 kDa detected with the anti-C antibody (Fig.1C). A 140-kDa protein detected under nonreducingconditions may represent a dimerized form. We ob-served no molecular mass shift between reducing andnonreducing conditions of the presumptive processedC-32 form of the protein. This finding implies that the32-kDa C-fragment is present as a monomer in ze-brafish testes and not as the expected cysteine-bridgeddimer typically seen in the TGF� class of protein(IPR021203; http://www.ebi.ac.uk/interpro).

Recombinant zebrafish AmhRecombinant Amh (wild-type sequence) produced in

HEK293 cells was recognized as a 70-kDa precursor incells lysates and in concentrated medium by the two spe-cific antibodies anti-N and anti-C (see Fig. 1, E and F).Treatment with plasmin, a serine protease that cleavesafter arginine or lysine (9), gave three cleavage productsfrom the N terminus (N-24.5-26.5-28.5), whereas theC-terminal antibody detected three matching products (C-34-36-39) (Fig. 1, E and F). Increased plasmin concentra-tions produced several less abundant C-fragments (C-16-19-21.5), all larger than the predicted C-product of 11.7kDa assuming TGF� class of maturation (Fig. 1A andSupplemental Fig. 1C).

Amh in testis extracts from zebrafish was apparentlycleaved by an endogenious enzyme to give the C-terminal32-kDa protein. This fragment was susceptible to plasmintreatment, because it was degraded even at low proteaseconcentration and no degradation products were ob-served (see Fig. 1D).

Recombinant zebrafish Amh optimized fordownstream processing

To produce a biologically active recombinant zebrafishAmh designed for simple purification and use in functionalexperiments, a strategy used with human recombinantAMH (47) was adapted. The putative protease cleavagemotif RAQR at aa position 439–442 was changed to aRARR motif, and a histidine tag was inserted just beforeproline at position 33 (similar to 47). Medium from stablytransfected HEK293 cells producing His-Amh-Q441R re-

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vealed both the apparent 70-kDa precursor form and dif-ferent processed forms (Fig. 2A). Nonreducing conditionsgave a 140-kDa protein, suggesting a dimerized form ofthe 70-kDa protein. A 50-kDa protein (N-50) was de-tected with anti-N corresponding to the predicted 48-kDaproduct after cleavage at the RARR site (Fig. 2A). Also,additional fragments were detected, suggesting at least oneadditional cleavage site. Three low molecular mass frag-ments were detected with the C-terminal antibody at re-ducing conditions, which were similar to fragments seenafter plasmin cleavage (compare Figs. 1F and 2A).

The inserted histidine tag was recog-nized by an antihistidine antibody (Fig.2B) and revealed a pattern similar to theanti-N antibody. The histidine tag wasused for purification of the recombinantprotein and revealed a 140-kDa possibledimer at nonreducing conditions and amajor70-kDaandastrong50-kDaprod-uct also seen in a silver stained gel (Fig.2C). Plasmin treatment produced threefragments similar to unmodified recom-binant Amh (Fig. 1, E and F), N-24.5-26.5-28.5 detected with anti-N, whereasanti-C detected C-34-36-39 plus a weakC-16. Interestingly, the C-terminal frag-ments shifted to a higher molecular massatnonreducingconditions,andC-16wasonlydetectedat reducingconditions (Fig.2C). The above results show that some ofthe 70-kDa full-length and the C-termi-nal C-34-36-39 plasmin products pro-duceputativehomodimers linkedbyadi-sulfidebridge intheC-terminalhalfof theprotein (Fig. 2C).

Identification of signal peptideand possible site of proteolyticcleavage

The predicted 21-aa leader sequence(Fig. 1 and Supplemental Fig. 1) was ver-ified by N-terminal sequencing (Edmandegradation) of purified 70-kDa ze-brafishHis-Amh. Identificationof the sixfirst aa showed that the signal peptidehadbeencleavedoffbetweenaaC21andA22 (see Supplemental Figs. 1 and 2A).

Two products from plasmin-treatedrecombinant Amh, N-28.5 and C-36,were analyzed by mass spectrometry toreveal the cleavage site (SupplementalFig. 2B). Aligning the fragments fromthe mass spectrometry analysis indicated

cleavage after K269 or R270 in the sequence of the modifiedprotein (K263 or R264 in the native Amh). Theoretically,thiscleavageshouldgiveanN-fragmentof27.8kDa(27kDawithout histidine tag) and a C-fragment of 31.8 kDa (Fig. 1and Supplemental Fig 1).

Zebrafish Amh is glycosylated in human culturedcells but not in zebrafish testes

Mammalian Amh is glycosylated, and two N-glycosy-lation sites were predicted in the zebrafish protein (Sup-

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FIG. 1. Proteolytic cleavage of Amh. A, Schematic representation of zebrafish Amh withpredicted and experimental cleavage products. Positions of N-terminal leader sequence,peptides used to generate antisera, possible glycosylation sites, and the conserved structuralTGF� domain are indicated. Testis extracts (B–D) and recombinant Amh (E and F) weresubjected to immunoblotting with antibodies anti-N (B, left panel, and E) and anti-C (B, rightpanel, C, D, and F). Extracts of testes from adult zebrafish males were homogenized in Ringersolution (extracts 1 and 2; B) or prepared in lysis buffer (C) under reducing (B and C, lane R)or nonreducing conditions (C, lane NR). Testis extracts (D) and concentrated (22�) mediumfrom HEK293 cells secreting recombinant zebrafish Amh (E and F) were cleaved withincreasing concentrations of plasmin indicated in milligrams per milliliter. Controls withoutplasmin are indicated as immediately frozen (0/Fr) or room temperature incubated (0/RT). Thec lane (E) contains concentrated (20.5�) medium from nontransfected cells. Testes proteinextracts 1 and 2 were made from 5.5- and 12-month-old fish in B and 9-month-old fish in Cand D. Arrows indicate detected precursor protein and processed forms in kilodaltons.Migration of molecular mass standards are indicated to the left in kilodaltons.

3530 Skaar et al. Amh Function in Zebrafish Testis Endocrinology, September 2011, 152(9):3527–3540

plemental Fig. 1). In HEK293 cells, recombinant Amh co-localized with disulfide isomerase to the endoplasmicreticulum, which is necessary for the secretory pathwayand glycosylation (Supplemental Fig. 3). To examine gly-cosylation, purified recombinant and endogenous ze-brafish Amh were subjected to enzymatic deglycosylation(Fig. 3). Size analysis showed that recombinant Amh wasreduced by approximately 5 kDa after treatment with theN-deglycosylation enzyme (anti-N; Fig. 3A). Plasmin frag-mentation and immunoblotting revealed both N- and pos-sibly also O-glycosylation but only in the C-terminal halfof the protein. The results are in agreement with glycosyl-ation taking place at the conserved NSST site at position340–343 and possibly at the NRSL site at position 516–519 and that Amh is proteolytically cleaved at KRH263–265 as suggested (Fig. 3). Similar enzyme treatment had noeffect on the molecular masses of the endogenous Amh,suggesting that it is not glycosylated (Fig. 3B).

Expression and localization of endogenous Amhand biological activity of recombinant zebrafishAmh

In males, amh mRNA levels increased approximately80-fold from 4 to 8 wpf and another approximately 8-fold

from 8 to 12 wpf (see figure 5A). Infemales, expression levels increased ap-proximately 10-fold from 4 to 8 wpfbut then remained constant (see figure5A). In adult zebrafish testes, Amh wasdetected in Sertoli cells surroundingearly germ-cell generations, such astype Aund (Fig. 4, A–E) and type A dif-ferentiated (Adiff) spermatogonia (Fig.4, F and G), the former often locatednear to the interstitial compartment(Fig. 4, B and C). The Amh-specificstaining was much weaker or absentfrom Sertoli cells surrounding laterstages of germ-cell development, suchas type B spermatogonia, spermato-cytes, and spermatids (Fig. 4, A, D, andF). Preabsorption of the antibody withthe peptide fragment used to generatethe antiserum abolished the staining(Supplemental Fig. 4), demonstratingthe specificity of the immunocytochem-ical reaction.

Testes from adult zebrafish werestudied in a primary, short-term tissueculture system to investigate the effectof Amh on gonadotropin-stimulatedandrogen release. In zebrafish, the pi-tuitary gonadotropin Fsh has a strong

steroidogenic potency that exceeds the one of LH, andLeydig cells express the receptor for both gonadotropins,Lh and Fsh (44). Thus, the release of 11-KT, the majorandrogen in fish, was stimulated using recombinant ze-brafish Fsh. Preincubation of zebrafish testes with purifiedplasmin-treated recombinant zebrafish Amh for 6 or 24 hsignificantly reduced, or abolished, respectively, Fsh-stim-ulated androgen release (Fig. 5B). When testing uncleavedrecombinant Amh, Fsh-stimulated 11-KT production wasnot compromised (Fig. 5B, to the right of dashed line).Expression analysis revealed that the Fsh-induced up-reg-ulation of cyp17a1, star, and insl3 transcript levels wassignificantly reduced after 24 h of preincubation withAmh (Fig. 5C, Supplemental Fig. 5); expression of ar re-mained unaltered under all conditions.

Amh mRNA in testis tissue cultures was down-regu-lated by Fsh treatment, independent of the steroidogenicactivity of Fsh (Fig. 5, D and E). Increasing doses of 11-KTdid not change amh mRNA levels significantly. Moreover,the down-regulatory effect of Fsh is possibly also inde-pendent of the cAMP/PKA pathway (Fig. 5, D and E).

To examine whether zebrafish Amh prevents andro-gen-stimulated adult spermatogenesis, akin to the inhibi-

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FIG. 2. Processing of purified recombinant zebrafish Amh. Recombinant zebrafish Amh inup-concentrated cell culture medium (A and B) or as purified protein (C) were Westernblotted and detected using anti-N, anti-C, or anti-His antibodies under reducing (R) ornonreducing (NR) conditions. Recombinant His-Amh-Q441R (R/H) was analyzed except in B,where unmodified recombinant Amh (Unm) or recombinant Amh with optimized cleavagesite RARR but no histidine tag (�His) was included in addition to negative control (c) culturemedium from nontransfected HEK293 control cells. Purified Amh was treated in vitro withplasmin (C), untreated refer to no plasmin digestion and L15 represents no protein, i.e.purified fractions using culture medium as input during purification. Silver-stained gel (C,right) shows purified His-Amh-Q441R, untreated, and plasmin-treated compared with culturemedium L15. Medium in A had been concentrated 32.5 times, whereas in B, the �His, R/H,Unm, and c-medium had been concentrated 20, 37.5, 21.4, and 27.3 times the initialvolume, respectively.

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tion of the onset of spermatogenesis described in juvenileJapanese eel (30), we incubated adult zebrafish testis tissue(45) for 7 d with 11-KT only (control), or with 11-KT andAmh. All stages of spermatogenesis were present (i.e. un-differentiated and differentiated type A spermatogonia,type B spermatogonia, spermatocytes, spermatids, andspermatozoa) (see Fig. 6, A, C, and E) in testes incubatedwith 11-KT only. Qualitatively, all these stages were alsopresent after incubation with 11-KT and Amh. However,morphometric analysis revealed significant differences.The number of cysts with type Aund spermatogonia washigher in testis tissue exposed to Amh, whereas the numberof cysts containing type B spermatogonia, spermatocytes,

and spermatids was significantly reduced (Fig. 6, A, D, andF). The increased number of type Aund spermatogonia canreflect a block of their differentiation, leading to their accu-mulation, or an increase in their proliferation. Therefore, westudied BrdU incorporation, which revealed a reduction ofthe BrdU-labeling index of Aund spermatogonia in the pres-ence of Amh (Fig. 6B), excluding the possibility that Amhstimulated the proliferation of Aund spermatogonia. The in-hibitory effect of Amh on 11-KT-induced proliferation offurther advanced germ cells was reflected in the reducednumberofBrdU-positivecysts in testisexposedto11-KTandAmh (Fig. 6, D and F).

Quantitatively clearer results as regards spermatogoniatype Aund were obtained when using testis tissue for pri-mary cultures from males pretreated with estrogen in vivo.Previous studies showed that estrogen treatment blockeddifferentiation and reduced proliferation of Aund but alsoof Adiff spermatogonia (46). Spermatogonial proliferationand differentiation recovered from the estrogen-inducedinhibition when testis tissue was cultured ex vivo underbasal conditions, as indicated by the presence of clones oftype B spermatogonia (Fig. 7B), a BrdU-labeling index ofspermatogonia type Aund of 40% (Fig. 7C), and a highnumber of BrdU-positive germ cells (Fig. 7E), both singlecells and differentiating clones of spermatogonia. Thisspontaneous recovery was suppressed in the presence ofAmh: differentiating germ cells were rare and spermato-gonia type Aund were frequently present (Fig. 7A),whereas their BrdU-labeling index was reduced morethan 3-fold (Fig. 7B). In general, the number of BrdU-positive cells was lower (Fig. 7D). We conclude thatAmh reduced proliferation and prevented differentia-tion of spermatogonia type Aund.

Discussion

This communication focuses on how Amh is processed toa biologically active protein and elucidating its functionsin fish. Using zebrafish as model organism, Amh was an-alyzed in testes extracts and as recombinant protein. Wefound that zebrafish Amh, proteolytically cleaved to be-come fully active, near abolished Fsh-stimulated androgenproduction, inhibited spermatogenesis, and was down-regulated by Fsh but not by androgens.

Endogenous AmhThe endogenous zebrafish protein was detected as a

N-27 and a C-32 fragment in contrast to processed en-dogenous rat Amh, reported to be 48 and 12 kDa, respec-tively, at reducing conditions (11). The eel homologue wasdetected as a 30-kDa protein in immature testes using an

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FIG. 3. Glycosylation analyses of Amh. Purified His-Amh-Q441R (A) andendogenous Amh in testes extracts (B) treated with N-deglycosidase (�N)or with N and O-deglycosidases in combination (�N/O) analyzed withimmunoblotting using anti-N (A) and anti-C (A and B). Molecular massshifts are indicated with asterisks and fragment sizes in kilodaltons.Recombinant Amh had either been proteolytically cleaved with plasmin(0.01 or 0.2 mg/ml) or were untreated. l.b., Lysis buffer; R/H, His-Amh-Q441R; e, endogenous.

3532 Skaar et al. Amh Function in Zebrafish Testis Endocrinology, September 2011, 152(9):3527–3540

antiserum against the N-terminal 243 aa of the protein(30). This suggests that Amh is proteolytically processedboth in mammals and fish but with different cleavage sites.In human Amh, the C-terminal fragment includes 109 aaand forms a homodimer (2), whereas the zebrafish proteinC terminus extends over 280 residues and is a monomer.This is unusual for members of the TGF� family, becausedimerization of a C-terminal fragment is considered a gen-eral characteristic of this class of proteins (48). However,the recombinant variant of zebrafish Amh appeared as adisulfide linked dimer in the range of 65–76 kDa andmonomer size of 34, 36, and 39 kDa detected with C-ter-minal-directed antibodies.

Two endogenous precursor candidate proteins of 66and 71 kDa were recognized with the anti-C antibody

(Figs. 1C and 3B) and a possible dimerof 140 kDa at nonreducing conditions.The eel Amh is 60 kDa at reducing con-ditions and 120 kDa at nonreducingconditions due to disulfide bonding(37). Full-length zebrafish Amh con-sists of 549 aa, whereas full-length eelprotein consists of 614 aa, coincidingwith theoretical molecular masses of58.7 and 62.6 kDa without signal pep-tides, respectively.

Proteolytic activation of zebrafishAmh differs from human Amh

Signal peptides of secreted proteinsare cleaved off by signal peptidase in theendoplasmatic reticulum (ER) beforesecretion (49). N-terminal sequencingof the purified recombinant Amh veri-fied cleavage of a 21-residue long signalpeptide. The recombinant Amh wasfurther located to ER in the HEK293cells and colocalized with the ER-pro-tein disulfide isomerase (SupplementalFig. 3).

Zebrafish His-Amh-Q441R was de-signed to be in vivo processed in theHEK293 cells similar to modifiedmammalian Amh (47). Although mam-malian Amh was cleaved at the opti-mized site only (47), we never detectedthe predicted C-terminal 11.7-kDa ze-brafish fragment (Supplemental Fig. 1).The N-terminal-directed antibody de-tected a major 50-kDa protein and sev-eral weaker fragments (N-32-34-36).In addition, C-fragments (with C-19 asmajor product) were found. The pat-

tern of fragments agrees with the occurrence of at least oneextra cleavage of zebrafish Amh in HEK293 cells com-pared with modified human protein. Proteolytic activa-tion of human AMH takes place by cleavage after residue451 at RAQR/S, although a potential alternative cleavagemay occur after R254 at PR/S (50). Human AMH with theoptimized RARR451/S cleavage site is spontaneouslycleaved in HEK293 cells to give a biologically active pro-tein (51). The sequence variant RAQR451/R needed plas-min cleavage to be activated. Treating zebrafish Amh withplasmin resulted in a different cleavage pattern comparedwith human AMH, with preference for larger C-fragmentsthan the reported bioactive 25-kDa human homodimerprotein. The predicted plasmin cleavage site (KR264/H) in

FIG. 4. Immunocytochemical localization of Amh on zebrafish testis sections. A, D, and F,Low magnifications show Amh in Sertoli cells surrounding early stages of germ-celldevelopment (arrows), such as type Aund spermatogonia and type Adiff spermatogonia. Notethat Amh staining was much weaker or absent from Sertoli cells surrounding furtheradvanced germ cells, such as type B spermatogonia (B) and spermatocytes (SC). Interstitialcompartment (IC) and sperm (SZ) are also shown. B, C, E, and G, High magnifications ofAmh-positive Sertoli cells surrounding one (B and C) or two Aund spermatogonia (E), as well asa pair of Adiff spermatogonia (G). B and C, Nucleus (blue) and cytoplasm (red) of type Aund

spermatogonia are colored in C to illustrate that Amh is restricted to the Sertoli cellcytoplasm. The interstitial compartment (IC) is colored yellow.

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zebrafish Amh (Supplemental Fig. 1) seems to be in a po-sition similar to the secondary mammalian cleavage site(R254) (50).

Modification by glycosylationEndogenous and recombinant Amh appeared as 70-

kDa proteins, whereas sequence predictions suggestedproteins of 58.7 and 59.6 kDa (excluding leader se-quences), respectively. Glycosylation of the recombinantprotein was evident in HEK293 cells and could explain

much of the observed differences (Fig. 3A). The discrep-ancy between observed and theoretical molecular mass ofendogenous Amh remains unclear, because glycosylationwas not detected (Fig. 3C). The in vivo function of glyco-sylation remain obscure, but studies of gonadotropinspoint at a general impact on improved secretion and sta-bility (52, 53).

Deglycosylation of recombinant Amh C-terminalcleavage product C-34-36-39 yielded a 32-kDa productsimilar to the theoretical size and endogenous nonglyco-

FIG. 5. Amh ontogeny, Amh role on androgen synthesis, and Amh endocrine regulation on zebrafish testes. A, Relative expression of amh during 4, 8,and 12 wpf of vasa::egfp zebrafish males and females. The individuals were selected according to their enhanced green fluorescent protein expressionpattern and shape of the gonad. mRNA levels were determined by quantitative PCR, normalized to the levels of 18S rRNA, and expressed as relative levelsto the amh mRNA levels of 4-wpf males. Bars indicate mean � SEM (4 wpf, n � 4�/3�; 8 wpf, n � 7�/6�; 12 wpf, n � 5�/6�). Small and capitalletters indicate significant differences (P � 0.05) during the evaluated period for males and females, respectively. Asterisks indicate differences betweenmales and females (***, P � 0.001). B, Bars (mean � SEM) represent the amounts of Fsh-induced 11-KT (ng/mg testis) release in the presence or absenceof cleaved (cl) (left) or uncleaved (uncl) (right) His-Amh-Q441R (10 �g/ml) (rzfAmh) for 6 or 24 h of incubation. The biological activity of uncleaved Amh(to the right of the vertical dotted line) was tested using different concentrations (100, 250, and 500 ng/ml) of a different batches of recombinantzebrafish Fsh (rzfFsh). Different letters denote significant differences (P � 0.05) between the different experimental groups. C, Relative mRNA expressionof cyp17a1, star, insl3, and ar in testis tissue incubated in the absence of Amh and Fsh (Basal), in the presence of cleaved Amh (Amh 42 h), in thepresence of Fsh (basal 24 h � Fsh 18 h), or preincubated with cleaved Amh before addition of Fsh (Amh 24 h � Fsh 18 h). Gene expression levels(mean � SEM) were normalized to 18S rRNA levels (Supplemental Fig. 5). Different letters denote significant differences (P � 0.05) between the differentexperimental groups. D, Fsh (500 ng/ml) effects on steroid release (11-KT, left ordinate), and mRNA levels of cyp17a1, and amh (right ordinate) in thepresence of absence of 100 �M PKA inhibitor H89 for short-term incubation (24 h). Bars indicate mean � SEM (n � 8 for 11-KT; n � 6 for geneexpression) nanograms of 11-KT released per milligram of tissue, or fold induction of the control (without Fsh for Fsh only, or without H89 for Fsh �H89). The dotted line crossing the ordinate at 1 (right axis) indicates no stimulation. Asterisks denote significant differences (P � 0.05) between controland treated groups using a paired t test, whereas a hash symbol (#) indicates differences (P � 0.05) between Fsh only and Fsh � H89. The down-regulated levels of amh by Fsh did not change in the presence of H89 (P � 0.05). For each gene, mRNA levels were normalized with the geometric meanof the housekeeping genes (18S, ef1, and �-actin), calibrated with the PCR threshold values as � CT mean value obtained from all samples, and expressedas fold induction of control. E, The effect of different concentrations of androgens (50, 100, 200, and 400 nM 11-KT) on zebrafish amh mRNA levels after7 d of in vitro testis tissue culture. Bars (n � 21 for basal; n � 4 for 50 nM; n � 6 for 100 nM; n � 4 for 200 nM; n � 7 for 400 nM) indicate the relativeexpression of amh transcript levels (mean � SEM), which were normalized to the 18S rRNA levels. No significant difference (P � 0.05) of amh expressionwas found among the different concentrations of androgens.

3534 Skaar et al. Amh Function in Zebrafish Testis Endocrinology, September 2011, 152(9):3527–3540

sylated Amh. This suggests that the verified cleavage siteKR/H could be a natural cleavage site of zebrafish Amhyielding a 27-kDa N-fragment and a 32-kDa C-fragment.

Endogenous protease(s), which have Amh as natural sub-strate, is/are not known. However, proprotein convertase5 has been suggested as the natural protease in rat (11).

FIG. 6. Biological activity of recombinant zebrafish Amh (rzfAmh) on zebrafish spermatogenesis in testis tissue culture from untreated adultmales. A, Ex vivo spermatogenesis supported by 11-KT (200 nM; white bars) was inhibited by Amh (10 �g/ml; black bars). Bars indicate thenumber (mean � SEM) of cysts/mm2 of testis containing type Aund spermatogonia, type Adiff spermatogonia, type B spermatogonia,spermatocytes (SC), and spermatids (ST). B, BrdU-labeling index of type Aund spermatogonia from zebrafish testes cultured for 7 d with11-KT or 11-KT�Amh. *, Values are significantly different (P � 0.05) between 11-KT and 11-KT � Amh in A and B. C and D, Lowmagnifications of zebrafish testis sections immunostained for BrdU (brown) and counterstained with hematoxylin. Amh decreased thenumber of BrdU-positive spermatogenic cysts containing germ cells advanced beyond the stage of type A spermatogonia (arrows) and led toan accumulation of type Aund spermatogonia. E and F, High magnifications illustrate that type Aund spermatogonia are often BrdU-positive(black arrowheads) in tissue incubated with 11-KT, whereas type Aund spermatogonia accumulated in the presence of Amh and were thenoften BrdU-negative (white arrowheads).

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The proprotein convertases 1 (enzyme commission num-ber 3.4.21.93) and 2 (enzyme commission number3.4.21.94) cleave proteins at KR-X sites. Further investi-gations will have to reveal if these proteases are present inzebrafish testes and if they can cleave zebrafish Amh.

Zebrafish Amh, monomer, or dimer?Based on the gel patterns of recombinant zebrafish

Amh under reducing and nonreducing conditions (Fig.2C), it seems that a cysteine close to the C terminus isinvolved in dimerization of the protein. The N-terminal

fragments failed to form homodimers,in contrast to human AMH (Fig. 4C)(9). Homodimerization of the recombi-nant Amh C-peptide correlates well todata on mammalian AMH (9) but dis-agrees with our observation that the en-dogenous C-32 is present as a monomerin zebrafish testes. Japanese eel Amhwas detected as a 120-kDa cysteine-bridged dimer in its full-length form intestes extracts but the cysteine-bridgewhere absent in a 30-kDa N-terminalpart in the recombinant eel Amh vari-ant, suggesting a similar organizationas for the recombinant zebrafish Amh(30). The human Amh required cleav-age for biological activity to generatethe active C-terminal 25-kDa ho-modimer, the N-terminal part remainsin the complex and enhances receptorbinding but is lost during receptor ac-tivation (16).

Cleaved Amh inhibits Fsh-stimulated androgen productionand inhibits spermatogenesis

Teleost fish lack Mullerian ducts butnevertheless express Amh, suggestingthat other than the namesake functionscan be investigated in fish. In mammals,AMH inhibited Leydig cell differentia-tion and LH-stimulated androgen re-lease (27, 43, 54). Our studies revealedfor the first time in a lower vertebrate asuppressive role of Amh on gonadotro-pin-stimulated Leydig cell gene expres-sion (star, insl3, and cyp17a1) and an-drogen release (Fig. 5). In vivo (55) andin vitro (27) experiments demonstratedthat Amh inhibition of testosteroneproduction also in rodents involved re-duced Cyp17 expression, and low lev-

els of testosterone in Amh-overexpressing mice coincidedwith decreased expression of steroidogenic genes (21). Weconclude that Amh-mediated down-regulation of star andcyp17a1 in Leydig cells is dominant over stimulatory effectsof steroidogenic gonadotropin on the expression of thesegenes and is an evolutionary conserved function of Amh.

Amh reduced Fsh-induced increases in insl3 mRNA lev-els. In mammals, INSL3 is required for the testicular de-scent in embryonic life and acts as a male germ-cell sur-vival factor in adults (56, 57). Transcription factors such

FIG. 7. Biological activity of recombinant zebrafish Amh (rzfAmh) on zebrafishspermatogenesis in testis tissue culture from estrogen pretreated adult males. A and B,Zebrafishtestis sections stained with toluidine blue from adult males, which were first exposedto estrogen in vivo to reduce spermatogonial proliferation and differentiation, before ex vivoincubation for 7 d with basal medium (A) or Amh (B). In the absence of estrogen,spermatogonial proliferation and differentiation recovered ex vivo (arrows) in basal medium(A) but not in the presence of Amh (B), where mainly of type Aund spermatogonia were found(inset in B), whereas several cysts of type B spermatogonia (B) were found in the testesincubated with basal medium (inset in A). C, Amh inhibited the proliferation of type Aund

spermatogonia. Bars represent the % of BrdU-positive Aund expressed as mean � SEM; **,Values between Amh and basal are significantly different. D and E, Zebrafish testis sectionsimmunostained for BrdU showed a decrease in the number of BrdU-positive cysts containingtype Aund spermatogonia (arrowheads) and more advanced germ cells (asterisks) in Amh-treated tissue (E) compared with the basal condition (D).

3536 Skaar et al. Amh Function in Zebrafish Testis Endocrinology, September 2011, 152(9):3527–3540

as steroidogenic factor 1 and NUR77 (a nuclear receptorsubfamily 4 group A member 1) regulate certain steroid-ogenic enzyme-encoding genes and regulate the INSL3promoter in mouse, rat, and humans (58). Hence, al-though there is no mechanistic information regardinginsl3 regulation in zebrafish, we speculate that Amh mod-ulates insl3 expression via reduced androgen production.

Amh transcripts are present in Sertoli cells in fish (31,58). Our work verified this for the first time at the proteinlevel (Fig. 4). Amh was prominently present in Sertoli cellsaround early spermatogonia, close to the interstitial area,which fits well to other observations we have made on thelocation of stem cell candidates in the testis (59). BecauseSertoli cells contacting later germ-cell generations showedlittle/no Amh protein, Amh function may be related tospermatogonial stages of germ-cell development, espe-cially type A spermatogonia.

In prepubertal Japanese eel, Amh inhibited the 11-KT-induced proliferation of type B spermatogonia, therebyinhibiting the onset of spermatogenesis (30). This is in linewith AmhrII loss-of-function experiments in medaka thatresulted in a germ-cell overproliferation phenotype (42).Our observations verify this in a third and unrelated order

of teleost fish, suggesting that Amh-me-diated inhibition of spermatogenesismay be a typical aspect of Amh action infish. In addition, we report for the firsttime on effects of Amh on adult sper-matogenesis. BrdU incorporation anal-ysis showed that although Amh re-duced the mitotic index of type Aund

spermatogonia, that are the germ-cellpopulation containing the spermatogo-nial stem cells (59), still the number oftype Aund spermatogonia increased.This apparent contradiction can be ex-plained by the Amh-mediated block ofthe further differentiation of the slowly(viz. low mitotic index) accumulatingtype Aund spermatogonia. Inhibitingdifferentiation of type Aund spermato-gonia would also explain the decreasednumber of more differentiated germcells observed after Amh exposure. Thepresent data, however, do not allow ex-cluding an independent effect of Amhon later germ-cell stages. Taken to-gether, our studies provide direct evi-dence for an inhibitory effect of Amh onthe proliferation of type Aund spermato-gonia and moreover for a block of theirdifferentiation. Hence, type Aund sper-

matogonia accumulate while rapidly proliferating type Bspermatogonia (and later stages of spermatogenesis) becomedepleted. Inrat testis, thehighest levelsofexpressionofAMHand its receptor are in Sertoli cells in epithelial stage VII (60),postulating a relation between AMH signaling and the lowmitotic activity of spermatogonia in stage VII. It might there-fore be interesting to examine experimentally the possibilitythat AMH modulates proliferation/differentiation of sper-matogonia also in adult mammals.

Sertoli cell AMH expression is high before puberty inmammals (2), is up-regulated by FSH (25), and greatlyreduced at the pubertal increase in androgen levels andSertoli cell androgen receptor expression (2). In zebrafish,however, amh expression progressively increased with pu-berty and adulthood. Two related observations seem im-portant in this regard. First the Amh protein level is highin Sertoli cells contacting type A spermatogonia. And sec-ond, these Sertoli cells are not terminally differentiated infish. Therefore, increasing amh expression during onto-genesis can be explained with the increase in the numberof spermatogenic cysts containing type A spermatogoniathat accompanies pubertal testis growth and the furtherdevelopment toward adulthood (61).

FIG. 8. Amh effects on zebrafish testis functions. Fsh stimulates Leydig cell androgen (11-KT)release but down-regulates Amh expression in Sertoli cells, whereas Amh suppresses Leydigcell androgen (11-KT) production by decreasing the expression of key genes involved inandrogen biosynthesis, such as star and cyp17a1, also in the presence of Fsh. In the absenceof Amh, androgens stimulate germ-cell differentiation from type Aund spermatogonia intotype Adiff spermatogonia, and type B spermatogonia (B). The suppressive role of Amh onsteroidogenesis might have secondary effects on the expression of other Leydig cell genes,such as insl3 (partially down-regulated). Similar to the mammalian testis, Amh receptors areexpressed by Sertoli cells but not by germ cells in fish (22), so that an autocrine Amh loopmight trigger a downstream signaling mechanism in Sertoli cells, which would prevent earlyspermatogonial differentiation. On the other hand, when Sertoli cells expressing high levels ofAmh respond to Fsh, Amh-mediated inhibition of germ-cell development and androgenproduction would wane, whereas Fsh-stimulated androgen and possibly growth factor releasewould stimulate the progression of germ cells toward meiosis.

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Amh expression patterns during ontogenesis changedifferently in mammals vs. zebrafish and may reflect dif-ferences in the regulation of expression. In mammals, Fsh-stimulated androgen hormone excretion and inhibits Amhexpression, whereas in zebrafish Fsh, down-regulatedamh expression and androgens had no significant effect.For androgens, we speculate that the cystic mode of sper-matogenesis in fish, characterized by undifferentiated Ser-toli cells in contact with type A spermatogonia (i.e. show-ing high levels of Amh) also in the adult testis, is a situationincompatible with a mechanism where the pubertal in-crease in androgen production would induce a testis-widedown-regulation of amh expression. The down-regula-tion triggered by zebrafish Fsh contrasts with the up-reg-ulation observed in mammals (25). We have not examinedif cAMP is required in zebrafish in this context, but PKAsignaling appears to be of minor relevance. Fsh-mediateddown-regulation of amh expression fits well into the spec-trum of biological activities of Amh. Two aspects are rel-evant in this context, namely that Fsh is a potent steroid-ogenic hormone in fish (58), including zebrafish (44), andthat unpublished work in our laboratory shows that theproliferation/differentiation of spermatogonia is stimu-lated by Fsh in testis tissue culture in an androgen-indepen-dent manner (Nobrega, R. H., and R. W. Schulz, unpub-lished data). Both these effects of Fsh would be counteractedby Amh, so that Fsh-mediated down-regulation of amhexpression seems an integral component of Fsh-mediatedstimulation of zebrafish testis functions.

Despite the different regulatory mechanisms, reducedAmh signaling may permit germ-cell development both infish (continuously occurring after puberty on the level ofindividual spermatogenic cysts) and mammals (occurringas a testis-wide event during puberty) and could be thesecond aspect of evolutionary conserved Amh bioactivityacross vertebrates.

The biological activities of Amh in zebrafish are sum-marized graphically in Fig. 8. Amh effects in zebrafish areconsistent with keeping germ cells in an immature stateof development. In view of the Fsh-mediated suppression ofAmh, we predict that Sertoli cells expressing high levels ofAmh may have a limited responsiveness to Fsh. In Leydigcells, Amh inhibits androgen production, so that in thevicinity of Sertoli cells expressing high levels of Amh (i.e.contacting type A spermatogonia), androgen levels may belocally lower, resulting in an area were germ-cell differ-entiation is less likely to occur. In Sertoli cells, yet elusivesignaling mechanisms would be activated in response toAmh to prevent differentiation of early spermatogonialgenerations (Fig. 8). When Sertoli cells expressing highlevels of Amh become responsive to Fsh, Amh would bedown-regulated, thereby permitting Fsh to stimulate

germ-cell proliferation/differentiation and androgen pro-duction. For future work, several aspects are of interest,such as the biological activity of fish Amh as monomericprotein; information on the signaling systems used byAmh to modulate steroidogenesis via Leydig cells, andspermatogenesis via Sertoli cells; or finally, the integrationof the mainly inhibitory Amh signaling with presumablyexisting, stimulatory signaling to achieve a coordinatedregulation of testis functions.

Acknowledgments

We thank M. Niere for the help in establishing permanentlytransfected; W. Telle, E. Langelandsvik, and D. Jensen for ex-cellent technical assistance; Wytske van Dijk and Caaj DouweGreebe for technical assistance during the in vitro Amh biolog-ical studies and radio immunoassays; Fernanda Loureiro deAlmeida for performing the negative controls of the immunocy-tochemistry reactions; and Henk Schriek and Co Rootselaar forassistance during the in vivo experiments and maintaining thezebrafish stocks.

Address all correspondence and requests for reprints to: RuneMale, Department of Molecular Biology, University of Bergen,P.O. Box 7800, N-5020 Bergen, Norway. E-mail: addressrune.male@mbi.uib.no; or Rudiger W. Schulz, Department ofBiology, Utrecht University, Padualaan 8, 3584 CH, Utrecht,The Netherlands. E-mail: r.w.schulz@uu.nl.

This work was supported by the Norwegian Research Coun-cil Project no. 159045, the European Union LIFECYCLE projectno. FP7-222719, and the National Council for Scientific andTechnological Development (R.H.N.).

Disclosure Summary: The authors have nothing to disclose.

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3540 Skaar et al. Amh Function in Zebrafish Testis Endocrinology, September 2011, 152(9):3527–3540