Proc. Natl. Acad. Sci. USAVol. 83, pp. 5464-5468, August 1986Biochemistry

Cloning and expression of cDNA for anti-Mullerian hormone(Xgtll expression screening/Miillerian-inhibiting substance/fetal testis/adult ovary)

JEAN-YVES PICARD*, RICHARD BENAROUSt, DANIEL GUERRIER*, NATHALIE JOSSO*, AND AXEL KAHNt*Unite de Recherches sur l'Endocrinologie du Ddveloppement, Institut National de la Sante et de la Recherche Medicale, H6pital des Enfants-Malades, 75743Paris Cedex 15, France; and tUnit6 de Recherches de Pathologie Moldculaire, and tUnitt de Recherches de Gdndtique et Pathologie Moldculaires, InstitutNational de la Sante et de la Recherche Mddicale, CHU Cochin-Port-Royal, 24 rue du Fg St Jacques, 75014 Paris, France

Communicated by Alfred Jost, April 17, 1986

ABSTRACT Messenger RNA, prepared from fetal bovinetesticular tissue, was used to construct a cDNA library in Kgtllphage. The library was screened with an antibody probedirected against bovine anti-Mullerian hormone and threepositive clones were isolated. Cross-hybridizing cDNA insertscarried by clones 4 and 5 (1.2 and 0.08 kilobases long,respectively) code for a fragment of authentic anti-MUllerianhormone, as shown by the ability of the anti-epitope antibodieseluted from fusion protein 4 to bind strongly to anti-Muillerianhormone on immunoblots and by the capacity of anti-epitopeantibodies 4 and 5 to precipitate radioiodinated bovine anti-Mullerian hormone. A probe prepared from insert 4 hybridizeswith an mRNA present only in tissues that are known produc-ers of anti-Miillerian hormone, such as the fetal testis and adultovarian follicles. The amount of specific mRNA in tissues ofmales and females is related to the rate of their anti-Muillerianhormone production. The 2.1-kilobase size of this mRNAspecies is large enough to code for theMr 62,000 anti-Mullerianhormone polypeptide chain. Insert 4 also hybridizes with anmRNA of similar size in human and rat fetal testicular tissue.The third isolated clone, clone 8, which does not cross-hybridize with the others, carries a cDNA insert coding for aubiquitous protein, smaller than anti-Mullerian hormone, withwhich it apparently shares an epitope.

Male sex differentiation is not limited to the development ofmale reproductive organs. It also involves the regression ofprimordia, which would otherwise give rise to the femalereproductive tract. The fetal testis accomplishes both tasksby secreting two different hormones (1): testosterone, aproduct of fetal Leydig cells, is responsible for the develop-ment of male-specific organs, while anti-Mullerian hormone(AMH) suppresses the Mullerian ducts, the primordia foruterus and tubes. As recently reviewed (2), the hormone, aMr 145,000 glycoprotein homodimer containing 13.5% car-bohydrate, has been purified from bovine fetal testes, and itsontogeny has been studied by radioimmunoassay. AMH isproduced by Sertoli cells, not only during the period whenMullerian ducts regress in the male fetus, but also in latepregnancy, after birth, and even, albeit at a much reducedrate, in adulthood. Low amounts of AMH are also releasedinto follicular fluid by mature granulosa cells. Cloning of thegene responsible for the synthesis of this intriguing differen-tiation factor is of obvious physiological interest. We haveisolated two cross-hybridizing cDNA clones for bovineAMHby antibody screening ofa Xgtll expression library and testedtheir specificity by study of the capacity of anti-epitopeantibodies, eluted from the fusion proteins produced by theseclones, to recognize purified AMH. One of these clones hasbeen tested by RNA blot hybridization to RNA extractedfrom various organs and shown to bind to a discrete 2.1-

kilobase (kb) mRNA species found primarily in testiculartissue and also, at a lower concentration, in bovine ovarianfollicles. This mRNA species is tissue specific but not speciesspecific, and it is sufficiently large to contain the 1700 basesrequired to encode the Mr 62,000 polypeptide chain of theAMH monomer.

MATERIALS AND METHODSPreparation of RNA. Total RNA was prepared by the

guanidine chloride method (3), as modified by Kahn et al. (4),from testicular tissue and isolated seminiferous tubules offetal or 2-week-old calves, from bovine ovarian follicles, andfrom testicular tissue of human fetuses, products of electiveabortions. RNA was also prepared from various other bovinetissues: intestine, brain, liver, heart, skeletal muscle, ovary,uterus, adrenal, lung, thymus, and thyroid. Poly(A)+ RNAswere isolated by oligo(dT)-cellulose (Bethesda ResearchLaboratories) chromatography (5).

Construction ofa Xgtll cDNA Expression Library. A cDNAclone library was obtained in the phage expression vectorXgtll according to Young and Davis (6, 7). Double-strandedDNA complementary to total poly(A)+ RNA extracted from2-week-old calf testes was prepared according to the RNaseH method of Darlix et al. (8), methylated at EcoRI sites,ligated to EcoRI linkers according to Huynh et al. (9),digested by EcoRI restriction enzymes (Boehringer Mann-heim), and sized by HPLC on an Ultropak TSK-G4000 SWcolumn (LKB). A 100-ng aliquot of fractions >0.35 kb wasligated to 2 ug of EcoRI-cut dephosphorylated Xgtll DNA(Protoclone kit, Promega Biotec, Madison, WI). LigatedDNA was packaged using Gigapack (Vector Cloning Sys-tems, San Diego, CA). A library of 1,500,000 primary cloneswas obtained, with 94% recombinants.

Screening of the Xgtll Library. Clones (500,000) wereplated at a density of 50,000 plaque-forming units per 15-cmdiameter Petri dish and were screened according to Youngand Davis (6). Two nitrocellulose filters, soaked in 10 mMisopropyl P-D-thiogalactopyranoside, were successivelyoverlaid on each Petri dish (10), the first one for 2 hr at 37°C,and the second one overnight at 4°C. The filters, saturatedwith 0.05M Tris HCl, pH 8.1/0.15M NaCl, containing 0.05%sodium azide, 2% glycine, and 3% non-fat dry milk (TBSbuffer), were soaked for 2 hr at room temperature in ananti-bovine AMH rabbit antiserum (11) diluted 1:250 in TBSbuffer. A 1:2000 dilution of the antiserum detects 1 ng ofAMH spotted on a nitrocellulose membrane. After thoroughrinsing, the filters were revealed by autoradiography, usingradioiodinated protein A (specific activity, 10 ,uCi/,g;1 Ci37 GBq), at a concentration of 5 x 105 cpm/ml. Those phageplaques producing a signal on duplicate nitrocellulose filterswere replated at a lower density up to homogeneity.

Abbreviations: AMH, anti-Mullerian hormone; kb, kilobase(s).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Selection of Anti-Epitope Antibodies. Nitrocellulose filtersexposed to homogeneous positive phages plated at highdensity were used for the selection from rabbit antiserum ofantibodies reacting specifically with the AMH epitopespresent in the different fusion proteins (anti-epitope antibod-ies). After incubation of the filters with anti-AMH antiserum,bound antibodies were eluted according to Weinberger et al.(12) and concentrated by exposure and subsequent elutionfrom protein A-Sepharose beads (Pharmacia). The specificityof the anti-epitope antibodies for AMH was tested by twoindependent techniques. The anti-epitope antibodies wereused as immunological reagents to reveal bovine AMHelectrophoresed and blotted on nitrocellulose as described(13). They were also used as the first antibodies to precipitateradioiodinated AMH (specific activity, 15 uCi/,ug), in theconditions used for liquid phase RIA for bovine AMH (11).

Characterization of cDNA Clones. Preparation of phageDNA was carried out according to Maniatis et al. (14). Afterdigestion by EcoRI, cDNA inserts were sized by electropho-resis on 1% agarose gels in the presence of ethidium bromide(0.5 pug/ml) and purified by electroelution of the resultingbands. Insert 5, not visualized under these conditions, wassized by electrophoresis on a 6% polyacrylamide gel. Label-ing of purified cDNA inserts was performed by nick-trans-lation (15) for inserts 4 and 8, and by fill-in of the EcoRI sitesfor insert 5. The inserts were tested for cross-hybridizationby blot-hybridization according to de Keyzer et al. (16).cDNA insert 4 was subcloned in M13 mplO vector (17), andcomplementary single-strand DNA probes were obtained byprimer extension (16).

Hybridization of cDNA to RNA Blots. RNA blots (18) wereprepared on GeneScreen Plus from various preparations ofRNA with 10 mM hydroxymethylmercury as denaturingagent (19). The blots were incubated with double- or single-stranded cDNA probes at 1 to 3 x 106 cpm/ml and wereexposed to XAR-5 Kodak film at -80°C. The amount ofspecific mRNA was estimated by densitometric scanning ofautoradiograms on a Shimadzu Dual Wavelength TLC scan-ner CS-930.

RESULTSThree clones, reacting at various intensities with the anti-AMH antiserum and positive after three rounds of replating,were identified by screening of 500,000 clones (Fig. 1).

Binding of Anti-Epitope Antibodies to AMH. Anti-epitopeantibodies eluted from the fusion protein produced by clones4 and 5 (anti-epitope antibodies 4 and 5) bind to AMH onimmunoblots. AMH is characterized by its reduction-sensi-

tive behavior, which is due to its disulfide-bonded structure.In the presence of 2-mercaptoethanol, AMH polymers arereplaced by a Mr 72,000 monomer (reviewed in ref. 2).Positive results are obtained with anti-epitope antibodies 4and 5 after 4 and 24 hr of exposure, respectively. In contrast,visualization ofAMH using antibodies eluted from the fusionprotein of clone 8 (anti-epitope antibody 8) requires 19 daysof exposure (Fig. 2). Antibodies 4 and 5 precipitate radio-iodinated bovine AMH in a dose-dependent fashion, whereasantibody 8 has little or no effect (Fig. 3). Precipitation ofradioiodinated AMH by anti-epitope antibodies 4 and 5 isabolished by an excess of unlabeled AMH (results notshown).

Characterization of cDNAs and RNA Blot Analysis. Asshown by agarose gel electrophoresis (Fig. 4), inserts 4 and8 are 1.2 and 0.5 kb long, respectively. Insert 5, not visibleunder these conditions, is 0.08 kb long, as demonstrated byPAGE (results not shown). Insert 4 cross-hybridizes withinsert 5, but not with insert 8 (results not shown). For thisreason, the short insert 5 was not studied further. Afternick-translation, insert 4 recognizes a 2.1-kb mRNA speciespresent in bovine fetal testicular tissue but not in the 12 othertissues tested. In contrast, insert 8 binds to a 1-kb mRNAspecies present in all tissues investigated (Fig. 5). OtherRNAblots were prepared by using insert 4 subcloned in M13 mplO.High-sensitivity single-stranded probes prepared from thecoding strand allowed us to detect a 2.1-kb mRNA species inbovine, rat, and human fetal testes; in bovine seminiferoustubules; and in bovine ovarian follicles (Fig. 6). The propor-tion ofAMH-specific versus total mRNAs extracted from thefetal calf testis was evaluated by comparison with a standardmRNA of known concentration coding for L-type pyruvatekinase, and hybridized under the same conditions (data notshown). According to this estimation, AMH mRNA couldrepresent 0.01-0.05% of total testicular mRNAs. SpecificmRNA present in various tissues was related to the amountof immunoreactive AMH produced, estimated from pub-lished data (21-23) (Table 1).

DISCUSSIONAMH represents an extremely low proportion of the proteinssynthesized by the fetal testis. To clone the gene responsiblefor its synthesis, a powerful technique, adapted to thedetection of rare mRNAs, is required. Use of antibodyprobes for this purpose has benefited from the developmentof Xgtll as an efficient and convenient expression cloningvector. Since its description by Young and Davis (6), theXgtll phage has been the key to the success of major cloning

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FIG. 1. Immunological screening of nitrocellulose filters applied on plates of purified Xgtll clones 4, 5, and 8. Filters were incubated in rabbitanti-AMH antiserum diluted 1:250, followed by incubation in radioiodinated protein A (5 x 101 cpm/ml). The strongest autoradiographic signalis given by clone 5, perhaps partly due to the fact that the insert is very small and does not affect the speed of phage replication.

Biochemistry: Picard et al.

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FIG. 2. Capacity of anti-epitope antibodies eluted from nitrocel-lulose filters exposed to clones 4, 5, and 8 to recognize AMH onimmunoblots. AMH is characterized by its reduction-sensitive be-havior, which is due to its disulfide-bonded structure. In the presenceof 2-mercaptoethanol, AMH polymers are replaced by a M, 72,000monomer (reviewed in ref. 2). Markers are from the high molecularweight protein kit (Pharmacia). The concentration of the anti-epitopeantibodies was too low for accurate determination, but it did notdiffer significantly, asjudged by the intensity of the autoradiographicsignal given by radioiodinated protein A on the nitrocellulose filtersprior to elution. Pure AMH (1 ug) was denatured either with or

without 5% 2-mercaptoethanol (ME + or -), electrophoresedovernight on 4-30%o polyacrylamide gradient gels (Pharmacia) in thepresence of NaDodSO4, and blotted on a nitrocellulose sheet. Thenitrocellulose sheet was incubated first in a 1:200 dilution ofantibodies eluted from fusion proteins of clones 4, 5, and 8, and thenin a solution of radioiodinated protein A (5 x i0s cpm/ml). Anautoradiographic pattern typical of the reduction-sensitive behaviorof AMH is revealed after various periods of exposure: 4 hr foranti-epitope antibodies 4, 24 hr for anti-epitope antibodies 5, and 19days for anti-epitope antibodies 8. This indicates that the affinity ofanti-epitope antibodies 8 for AMH is much lower than that of clones4 and 5.

FIG. 4. Sizing of cDNA inserts prepared by electroelution:electrophoresis on 1% agarose gels in the presence of ethidiumbromide (0.5 ,ug/ml). Boehringer Mannheim DNA XIII markers wereused for calibration. kbp, Kilobase pairs.

by 1 g of bovine testicular tissue during a 4-hr incubationperiod (27). Given the fact that AMH is a Sertoli cell product(28-30), Sertoli cells or isolated seminiferous tubules mightconceivably be preferable to total testicular tissue as startingmaterial. However. testicular microdissection has the disad-

1 2 3 4 5 6 78 9 10 11 12 13

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ventures (24-26). We therefore elected to screen a cDNAlibrary in this vector with a previously characterized high-affinity anti-bovine AMH antiserum (11).An important point to consider in planning cloning strategy

is the choice of starting material for preparation of RNA.Even at the period of its maximal output, AMH represents anextremely low proportion of testicular protein production.One RIA unit ofAMH, corresponding to 3 ,ug (21), is released

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FIG. 3. Precipitation of radioiodinated AMH (125I-AMH) (spe-cific activity, 15 pCi/,ug) by anti-epitope antibodies eluted fromnitrocellulose filters exposed to clones 4, 5, and 8. The concentra-tions of the anti-epitope antibodies were too low for accuratedetermination, but they did not differ significantly, as judged by theintensity of the autoradiographic signal given by radioiodinatedprotein A on the nitrocellulose filters prior to elution. Effect ofanti-AMH rabbit antiserum (*) is included for comparison. Anti-epitope antibodies 4 (A) and 5 (o) precipitate AMH in a dose-dependent manner, while the effect of anti-epitope antibody 8 (o) isnot distinguishable from nonspecific binding (NSB).

A

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FIG. 5. RNA blot-hybridization analysis of RNA prepared fromvarious tissues of 140- to 210-day-old fetal calves. Fetal age was

assessed from crown-rump length according to Prepin (20). Seven to20 ,g of poly(A)+ RNA was electrophoresed on 1% agarose gels, in50 mM borate buffer (pH 8.2) containing 10 mM hydroxymethyl-mercury, and was blotted onto GeneScreen Plus. Lanes: 1, intestine;2, brain; 3, liver; 4, heart; 5, skeletal muscle; 6, testis; 7, ovary; 8,uterus; 9, cerebellum; 10, adrenal gland; 11, lung; 12, thymus; 13,thyroid gland. Ribosomal prokaryotic and eukaryotic RNAs (2 j."geach) were run as size markers in an adjacent lane; their positions areindicated on the left. (A) The blot was hybridized with a 32P-labeledprobe nick-translated from cDNA insert 4 (1 x 106 cpm/ml) (specificactivity, 2.1 x 108 cpm/,lg). (B) After dehybridization, the same blotwas hybridized with a 32P-labeled probe nick-translated from cDNAinsert 8 (0.4 x 106 cpm/ml) (specific activity, 0.85 x 108 cpm/hg).Probe 8 hybridizes to a ubiquitous 1-kb mRNA species, too small toencode bovine AMH. In contrast, probe 4 hybridizes to an mRNAspecies present only in fetal testicular tissue and not in the othertissues investigated.

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5466 Biochemistry: Picard et A

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1 2 3 4 5 6

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FIG. 6. RNA blot-hybridization analysis ofmRNA prepared fromgonadal tissue of various species, performed as described in thelegend to Fig. 5. Lanes: 1, poly(A)+ RNA from testicular tissue of200-day-old fetal calf; 2, poly(A)+ RNA from testicular tissue of15-day-old calf; 3, poly(A)+ RNA from seminiferous tubules isolatedfrom testicular tissue of 20-day-old calf; 4, poly(A)+ RNA frombovine ovarian follicles; 5, total RNA from testicular tissue of20-day-old fetal rats; 6, total RNA from human fetal testicular tissue.Samples were 5 ,ug for poly(A)+ RNAs and 10 ,ug for total RNAs.Lanes 1-3 were exposed 18 hr, and all others were exposed 6 days.The 2.1-kb mRNA hybridizing with insert 4 is tissue specific but notspecies specific. Comparable amounts are found in testicular tissuefrom fetal and neonatal calves, and much lower amounts are foundin bovine ovarian follicles. The same mRNA is also present in humanand rat testicular tissue. The RNA blot was hybridized with asingle-stranded M13 mplO probe from subclone 4-3 at 1.5 x 106cpm/ml (specific activity, 1.1 x 109 cpm/ltg).

vantages oflow quantitative recovery and potential alterationof RNA by tissue RNases. We therefore chose to preparemRNA from total testicular tissue. Because we have shownthat production ofAMH by the calf testis is relatively stablefrom mid-fetal life to -1 month after birth (22), RNA forcDNA construction was prepared from 2-week-old calf testesfor reasons ofconvenience. Justification for this decision wasprovided later by the fact that the amount of RNA blot-detectable mRNA coding for AMH is similar in fetal or15-day-old bovine testicular tissue (Fig. 6; Table 1) andrepresents 0.01-0.05% of total mRNA. This figure is con-sistent with the 0.01% proportion of [3H]leucine incorporatedin AMH versus total proteins released into incubation me-dium by fetal bovine testicular tissue (unpublished data).To establish the specificity ofthe cDNA inserts, we studied

the ability of antibodies eluted from immobilized fusionproteins produced by individual clones (anti-epitope antibod-ies) to bind to purified AMH. Anti-epitope antibodies havebeen used successfully to identify cDNA clones for humanglucocorticoid receptor (12, 25). Another independent argu-ment for establishing specificity of the cDNA clones is basedon size, amount, and tissue localization ofmRNA hybridizingwith the cDNA probes.Three clones reacting with anti-AMH antiserum have been

Table 1. Relationship between amount of AMH and specificmRNA produced by various bovine tissues

Specific mRNA,tAMH released,* % of fetal

Tissue ,ug per g per 4 hr testicular mRNAFetal testis 3 10015-day-old testis 3 77Ovarian follicles 0.0012 1.6Liver 0 0

*The amount of immunoreactive AMH released into incubation

isolated. However, specificity studies indicate that only twocross-reacting inserts, 4 and 5, 1.2 and 0.08 kb, respectively,code for a part of the AMH polypeptide chain. The third one,insert 8 (0.5 kb), does not cross-hybridize with the twoothers, and it encodes a smaller ubiquitous protein, whichshares an epitope with AMH, allowing it to be recognized byan anti-AMH antiserum and allowing anti-epitope antibodies8 to bind weakly to AMH on immunoblots. Using the lesssensitive technique of immunoprecipitation ofradioiodinatedAMH, we found anti-epitope antibodies 8 to be ineffective.Definitive proof that clone 8 does not code for the AMHpolypeptide chain is provided by the results of RNA blotanalysis (Fig. 5). Not only is the 1-kb mRNA specieshybridizing with cDNA insert 8 too small to encode the Mr72,000AMH monomer, but its absence of tissue specificity isan additional argument for discarding it.

In contrast, anti-epitope antibodies 4 produce a strongautoradiographic signal with AMH on immunoblots, visiblewithin 4 hr of exposure, compared to 19 days for anti-epitopeantibodies 8 (Fig. 2). They, and anti-epitope antibodies tocross-hybridizing clone 5, also immunoprecipitate radioiodi-nated AMH (Fig. 3). The specificity of cDNA insert 4 isconfirmed by RNA blot of mRNA of various tissues. Thelocalization and abundance of the 2.1-kb mRNA hybridizingto insert 4 is well-correlated with the site, ontogeny, and levelof production of AMH by gonadal tissue in both males andfemales. This mRNA is present only in tissues that are knownproducers of AMH, such as immature testicular tissue,seminiferous tubules, and adult ovarian follicles (see ref. 2 forreview). The amount of mRNA coding for AMH in varioustissues is related to their rate ofAMH production (Table 1).More specific mRNA was isolated from ovarian follicles thanwould be expected from the AMH production of bovinegranulosa cells (23), but the latter were isolated from follic-ular fluid and not from the follicles themselves.The size of the testicular and ovarian mRNA cross-

hybridizing with insert 4 is large enough to encode the AMHpolypeptide chain. The Mr 72,000 AMH monomer contains13.5% carbohydrate (21). Therefore, the expected size of thepolypeptide chain is Mr 62,000, corresponding to 560 aminoacids and requiring a 1.7-kb length of coding mRNA. The2.1-kb mRNA hybridizing with cDNA insert 4 is coherentwith this requirement, taking into account the putativenoncoding regions of the messenger, the poly(A) tail, and themRNA nucleotides coding for a signal peptide, probablycleaved from the AMH polypeptide before its secretion.Single-stranded insert 4-3 is complementary to slightly morethan one-half of the mRNA coding for AMH. It also hybrid-izes with an mRNA of similar size, present in human and ratfetal testicular tissue, indicating that it encodes a fragment ofthe AMH polypeptide chain conserved during evolution. Thisis interesting in view of the fact that bovine AMH carriesstrongly immunogenic zoospecific antibodies, and only asmall proportion of monoclonal antibodies raised against itrecognize AMH of other nonrelated mammalian species (31),which therefore are difficult to study. Tools for the study ofthese non-bovine AMHs will soon be provided by molecularbiology.

Note Added in Proof. Isolation of the bovine and human genes foranti-Mullerian hormone has been reported recently by Cate et al (32).The size of the mRNA they show is in agreement with our own

findings. The cDNA 4 we describe in the present paper has now beensequenced. It corresponds to the two COOH-terminal one-thirds ofthe bovine cDNA isolated by Cate et al. from base 609 to the poly(A)tail.

We are grateful to Dr. Yves de Keyzer for helpful advice, to Dr.Bernard Vigier for carrying out immunoprecipitation of radioiodi-nated AMH with anti-epitope antibodies, and to Mr. Chesnais

medium was estimated from published data (21-23).tAn autoradiogram of an RNA blot obtained from 10 ,ug of poly(A)IRNA was scanned on a densitometer. Integrated optical density isexpressed as percentage of that read for fetal testicular mRNA.

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(Institut National de la Recherche Agronomique, Jouy-en-Josas) forallowing us to collect ovarian follicles from superovulated cows.

1. Jost, A. (1953) Recent Prog. Horm. Res. 8, 379-418.2. Josso, N. & Picard, J. Y. (1986) Physiol. Rev., in press.3. Cox, R. A. (1968) Methods Enzymol. 12, 120-129.4. Kahn, A., Cottreau, D., Daegelen, D. & Dreyfus, J. C. (1981)

Eur. J. Biochem. 116, 7-12.5. Aviv, H. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69,

1408-1412.6. Young, R. A. & Davis, R. W. (1983) Proc. Natl. Acad. Sci.

USA 80, 1194-1198.7. Young, R. A. & Davis, R. W. (1983) Science 222, 778-782.8. Darlix, J. L., Bromley, P. A. & Spahr, P. F. (1977) J. Virol.

23, 659-668.9. Huynh, T. V., Young, R. A. & Davis, R. W. (1985) in DNA

Cloning: A Practical Approach, ed. Glover, D. M. (IRL,Oxford), Vol. 1, pp. 49-78.

10. Walter, P., Green, S., Greene, G., Krust, A., Bornert, J. M.,Jeltsch, J. M., Staub, A., Jensen, E., Scrace, G., Waterfield,M. & Chambon, P. (1985) Proc. Natl. Acad. Sci. USA 82,7889-7893.

11. Vigier, B., Picard, J. Y., Campargue, J., Forest, M. G., Hey-man, Y. & Josso, N. (1985) Mol. Cell. Endocrinol. 43, 141-150.

12. Weinberger, C., Hollenberg, S., Ong, E. S., Harmon, J. M.,Brower, S. T., Cidlowski, J., Thompson, E. B., Rosenfeld,M. G. & Evans, R. M. (1985) Science 228, 740-742.

13. Picard, J. Y. & Josso, N. (1984) Mol. Cell. Endocrinol. 34,23-29.

14. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) MolecularCloning: A Laboratory Manual (Cold Spring Harbor Labora-tory, Cold Spring Harbor, NY), pp. 75-97.

15. Rigby, B. W. J., Dieckmann, M., Rhodes, C. & Berg, P.(1977) J. Mol. Biol. 113, 237-251.

16. de Keyzer, Y., Bertagna, X., Lenne, F., Girard, F., Luton,

J. P. & Kahn, A. (1985) J. Clin. Invest. 76, 1892-1898.17. Messing, J. (1983) Methods Enzymol. 101, 20-78.18. Thomas, P. (1980) Proc. Natl. Acad. Sci. USA 77, 5201-5205.19. Bailey, J. M. & Davidson, N. (1976) Anal. Biochem. 70,

75-85.20. Prdpin, J. (1983) Dissertation (Universitd de Paris).21. Picard, J. Y., Goulut, C., Bourrillon, R. & Josso, N. (1986)

FEBS Lett. 195, 73-76.22. Vigier, B., Tran, D., Du Mesnil du Buisson, F., Heyman, Y. &

Josso, N. (1983) J. Reprod. Fertil. 69, 207-214.23. Vigier, B., Picard, J. Y., Tran, D., Legeai, L. & Josso, N.

(1984) Endocrinology 114, 1315-1320.24. Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L.,

Clauser, E., Ou, J. H., Masiarz, F., Kan, Y. W., Goldfine,I. D., Roth, R. A. & Rutter, W. J. (1985) Cell 40, 747-758.

25. Govindan, M. V., Devic, M., Green, S., Gronemeyer, H. &Chambon, P. (1985) Nucleic Acids Res. 13, 8293-8304.

26. Sadler, J. E., Shelton-Inloes, B. B., Sorace, J. M., Harlan,J. M., Titani, K. & Davie, E. W. (1985) Proc. Natl. Acad. Sci.USA 82, 6394-6398.

27. Vigier, B., Legeai, L., Picard, J. Y. & Josso, N. (1982)Endocrinology 111, 1409-1411.

28. Blanchard, M. G. & Josso, N. (1974) Pediatr. Res. 8, 968-971.29. Tran, D. & Josso, N. (1982) Endocrinology 111, 1562-1567.30. Hayashi, H., Shima, H., Hayashi, K., Trelstad, R. L. &

Donahoe, P. K. (1984) J. Histochem. Cytochem. 32, 649-654.31. Legeai, L., Vigier, B., Tran, D., Picard, J. Y. & Josso, N.

(1986) Biol. Reprod., in press.32. Cate, R. L., Mattalino, R. J., Herssion, C., Tizard, R.,

Farber, N. M., Cheung, A., Ninfa, E. G., Frey, A. Z., Gash,D. J., Chow, E. P., Fisher, R. A., Bertonis, J. M., Torris, G.,Wallner, B. P., Ramachandran, K. L., Ragin, R. C.,Manganaro, T. F., MacLaughlin, D. T. & Donahoe, P. K.(1986) Cell 45, 685-698.

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