EP0272309A4

EP0272309A4 - Ovine inhibin.

Info

Publication number: EP0272309A4
Application number: EP19870904454
Authority: EP
Inventors: Joachim Spiess; Jean Edouard Frederic Rivier; Wayne C Bardin; Wylie Walker Vale Jr
Original assignee: Salk Institute for Biological Studies
Current assignee: Salk Institute for Biological Studies
Priority date: 1986-06-24
Filing date: 1987-06-23
Publication date: 1989-12-19
Also published as: EP0272309A1; AU605162B2; JPH01500121A; WO1988000208A1; AU7640887A; CA1305829C

Abstract

A 34,500-dalton protein with inhibin activity is isolated from ram rete testis fluid using reverse-phase high-performance liquid chromatography and gel filtration. The isolated molecule is composed of two chains having molecular weights of about 18,000 and about 16,500 Daltons, which are bound together by disulfide bonding and the longer of which is likely glycosylated. Microsequencing revealed the NH2-terminal portion of the 18kD chain to be Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gl n-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His-Ala-Asp-Cys and of the 16.5kD chain to be Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe. This 34.5kD protein specifically inhibits basal secretion of FSH, but not of LH, in a rat anterior pituitary monolayer culture system, exhibiting a half-maximal effective dose of 0.3 ng/ml. Furthermore, antibodies raised against a synthetic replicate of the N-terminal six residues of the 18kD chain are effective to reduce the activity of highly purified 34.5kD ovine inhibin and might be used to exert a profertility effect in rams, ewes and other mammals.

Description

OVINE INHIBIN The present invention relates to a protein having inhibin activity isolated to substantial homogeneity from material obtained from ovine animals. BACKGROUND OF THE INVENTION The existence of inhibin as a water-soluble substance of gonadal origin which acts specifically at the pituitary level to suppress the secretion of follicle-stimulating hormone (FSH) was postulated by McCullagh more than 50 years ago. Science, 76, 19-20 (1932) . There has been great interest in it, and many laboratories have attempted to isolate and characterize this substance. In Nature, 318, 659-663, (1985) Mason et al. published sequences for the subunits of porcine inhibin derived from studies of cDNA. In B.B.R.C. , 135, 3, 957-964, March 28, 1986, Mason et al. published sequences for the subunits of human inhibin similarly derived from cDNA. Inhibin may be used to regulate fertility, gonadotropin secretion or sex hormone production in mammalians, both females and particularly males.

SUMMARY OF THE INVENTION In accordance with the present invention, a protein having a molecular weight of about 34,500 daltons (34.5kD) and having inhibin activity has been successfully isolated from ram rete testis flui (RTF). The protein has been partially characterized using microsequencing methods.

The protein was isolated to substantial homogeneity from material obtained from RTF and is hereinafter referred to as ovine inhibin. The protein has a molecular weight of about 34.5kD and is composed of two polypeptide chains having molecular weights of about 18,000 and about 16,500 Daltons, respectively, the chains being linked together in the biologically active protein by disulfide bonding. The amino-terminal residue sequence of the larger 18kD chain of the protein is believed to be Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp- Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu- Pro-Ala-Ala-His-Ala-Asp-Cys. The amino-terminal of the 16.5kD chain begins Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn- Ile-Cys-Cys-Lys-Lys-Gln-Phe-Tyr-Val-Ser-Phe-Lys-Asp-Σle- Gly. The 34.5kD protein exhibits inhibin activity in that it specifically inhibits the basal secretion of FSH but does not inhibit secretion of luteinizing hormone (LH). Purification of ovine inhibin to substantial homogeneity, i.e., about 90% by weight of total protein in the fraction, was achieved through a combination of protein separation procedures including gel filtration and reverse-phase, high-performance liquid chromatography (RP-HPLC) .

BRIEF DESCRIPTION OF DRAWING FIGURE 1 is a chromatogram of the final RP-HPLC purification of an inhibin protein active fractions which was applied directly onto a 0.46 x 25 cm Vydac Cg column with a 5 μ particle size and a 300A pore size, and eluted at 40°C with a gradient of TFA/CH₃CN buffers from 25% Buffer B to 95% Buffer B in 45 minutes, at a flow rate of 0.7 ml/min. with a back pressure of about 890 psi. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Using a multi-step procedure, the 34.5kD peptide was isolated to substantial homogeneity from ram rete testis f uid (RTF) . The protein is composed of two chains of 18 D and 16.5kD, and the chains of the intact molecule are held together by disulfide bonding, the linkage between the chains being necessary for biological activity. A ino acid analysis of the total protein has been performed, and a partial amino acid residue sequence of each chain has also been determined, beginning at the amino-terminus. The chains are rich in Cys residues, and it is believed that internal disulfide bonding is also present. The amino-terminal sequence of the 18kD chain is Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp- Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu- Pro-Ala-Ala-His-Ala-Asp-Cys. The 18kD chain is estimated to be about 135 residues in length, is likely glycosolated and is linked by one or more disulfide bridges to the 16.5kD chain. The 16.5kD chain has between about 115 and about 130 residues and begins at the N-terminus with the following sequence: Gly-Leu-Glu- Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-(Tyr or Phe) . The next residues following these are believed to be: Val-Ser-Phe-Lys-Asp-Ile-Gly. The C-terminus of either chain may be amidated or free acid.

The sharp elution peak of the protein which was obtained in the final chromatographic purification step is evidence that the protein has been purified to at least about 90% by weight of total protein. The 34.5kD protein is water-soluble, and one of the subunits of the native protein is likely glycosylated. A second isolated molecule appears to have an N-shortened version of the 18kD chain, that is shorter by 15 residues, but is linked to an identical 16.5kD chain.

The 34.5kD protein exhibits inhibin activity in that it specifically inhibits basal secretion of FSH but not LH in a rat anterior pituitary monolayer culture system and exhibits a half-maximal effective dose (EC_5Q) of about 0.3 ng/ml (10 pM.), based upon the assay described in detail in Endocrinology, 113, 1121-31 (1983). The isolated 34.5kD protein, as well as partially purified inhibin preparations, blocks the secretion of both LH and FSH _in vitro when cells are stimulated by gonadotropin releasing hormone. Iri vivo, partially purified inhibin preparations are highly selective to decrease plasma FSH and not LH levels. The effects of inhibin on basal gonadotropin secretion ju vitro appears to best reflect the ii vivo situation.

The 34.5kD protein is useful for regulating gonadotropin secretion and thus fertility and/or sex hormone production of both male and female mammalians. The possibility that inhibin might have direct gonadal actions on gametogenesis or steroidogenesis is also likely, and some brain actions of inhibin are suggested. A purification procedure was used to isolate ovine inhibin from crude RTF which utilized successive purification steps that include Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) with different stationary phases and/or mobile phases and also include gel filtration or permeation Fast Protein Liquid Chromatography(FPLC) .

The starting material for the procedure was about 3950 ml. of RTF that was first dialyzed against Milli Q H₂0 with 0.02% dimethylsulfide at 4^ΘC using dialysis bags with molecular weight cut off (MWCO) ca. 1000. The retentate was divided into two equal batches and lyophilized. Each half of the lyophilized pool was resuspended in column eluant and subjected to large scale gel permeation using a 5.0 by 150 cm glass column packed with 140 cm Sepharose CL-6B, V_fc=2750 mis.

Column eluant was 6 M Guanidine*HCl, 0.1 M ammonium acetate, 0.05% dimethylsulfide in Milli Q H₂0, ph 4.75. Eluant was 0.22 Jim filtered and degassed before use. Flow rate was 50 mis/hour. Active fractions from the two batches were pooled and further processed.

Purification by semi-preparative RP-HPLC was carried out first at 50°C and thereafter at room temperature (RT) using 133 ml-equivalents per run. The inhibin protein fractions from the various columns used for each step are pooled for each following step. Each run was applied directly onto a 1 x 30 cm Vydac 5-μm particle-size C₄ column (The Separations Group, Hesperia, CA.) and eluted using a gradient of TEAP buffer. In this TEAP system. Buffer A consists of 0.1 N. triethylammonium phosphate(TEAP) pH 5, and Buffer B is 60% CH₃C in Buffer A. After all the filtrate had been loaded, the column was washed with the aqueous Buffer A until the UV absorption reached baseline. Flow through the column is maintained at 2.5 ml per minute. The column is loaded at 30% Buffer B, and a gradient for the mobile phase was then begun gradually changing to 95% over 30 minutes. The fractions are separated by an Altex 420 gradient liquid chromatography system equipped with a Spectroflow 773 UV detector (Kratos Analytical Instruments, Ramsey, N. J.) and a Servocoder SR 6253 strip chart recorder and are collected and tested for substantial inhibin activity.

Inhibin protein fractions from the various individual columns were pooled and further purified by a 1 x 30 cm Vydac 5-um-particle-size C. column and a heptafluorobutyric acid (HFBA) buffer system at RT. In the HFBA system. Buffer A contains 1 ml of HFBA in 999 ml water, and Buffer B is 400 ml of water, 1 ml of HFBA and 599 ml of acetonitrile. Columns were loaded at 30% B followed by a gradient to 58% B in 25 minutes.

Active zones from reversed phase HPLC were lyophilized and resuspended in column eluant for processing on Pharmacia FPLC system by applying to two 1 x 30 cm Superose(FPLC) 12-B columns, 10 pm (Pharmacia Fine Chemicals, Piscataway, N. J.) linked in series.

Each column was eluted with 6M guanidineΗCl, 0.1M ammonium acetate, pH 4.75, and 0.5% DMS in Milli Q H₂0 at a flow rate of 0.4 ml per minute for about 50 min. The column fractions were monitored by UV absorption and by bioassay. Active fractions eluted between

K_AV=0.31-0.36.

Inhibin protein fractions from the various individual columns were pooled and further purified by RP-HPLC using 5-μm-particle-size C. column, 1 x 30 cm, and 0.5% TFA/CH₃CN buffer system.

After such HPLC desalting, the active fraction was lyophilized and subjected to FPLC cation exchange by being brought up in Buffer A which was 50 mM sodium acetate, 4 M urea, 1 mM CHAPS (3-[ (Cholamidopropyl) dimethylammonio]-1-propanesulfonate) in Milli Q H₂0, pH 5.3. Buffer B was 1 M NaCl in Buffer A. A Pharmacia FPLC system equipped with a Mono S HR 5/5 column, V. = 1 ml, was used at a flow rate of 1 ml per min. Column was loaded at 0% B followed by a gradient to 30% B in 25 minutes and then to 100% B in 5 minutes.

Next, the active fraction was applied to a 0.46 x 25 cm Vydac C_g Column of reversed phase material with a 5 um particle size and a 300A pore size. Buffer A is 0.5% (v/v) TFA in water and Buffer B is 1 ml TFA, 200 ml of water and 799 ml of acetonitrile. Flow rate was 0.7 ml/min at 40°C with a back pressure of 900 psi. Buffer B was used at 25 volume % for the initial loading, followed by a gradient to 50% in 25 minutes. Two zones of active inhibin protein eluted, and both were separately processed thereafter. Each active fraction was applied to a 0.46 x 25 cm Vydac C_g Column of reversed phase material with a 5 um particle size and a 300A pore size_* Buffer A is 0.1% (v/v) TFA in water, and Buffer B is 1 ml TFA, 200 ml of water and 799 ml of acetonitrile. Buffer B was used at 25 volume % for the initial loading at 40°C and a flow rate of 1.2 ml/min. Then a gradient to 95% is run in 45 minutes at a flow rate of 0.7 ml/min at 40°C with a back pressure of 890 psi, and a detector setting of 215 n , 2.9 AUFS, which was slightly changed to 214 nm. before elution of the peak. The purified inhibin protein eluted generally between about 27.0 minutes and about 28.3 minutes after start of the gradient, which is equal to between about 18.9 ml and about 19.8 ml of elutant after start of the gradient. A chromatogram of the final step, for the later eluting active fraction from the previous step, is depicted in FIGURE 1 and was generated using an Altex 420 System, two Beckman Model 100A pumps, a Datamark, Servocoder SR 6253 strip chart recorder, a Kratos, Spectroflow 773 variable wavelength, UV/visible detector and a Rheodyne 7125 injector with a 2.0 ml loop. The inhibin protein fractions accumulated from all batches, respectively for the earlier and later eluting fractions, were pooled, resulting in a total of approximately 195 ug of inhibin protein, including about 120 ug of the earlier eluting active inhibin fraction and about 75 ug of the later eluting fraction. The 195 pg was estimated to have been obtained from about 2700 ml of RTF, with the remainder having been used in the biological testing that was carried out to identify the active fractions during purification.

Amino acid analyses of the substantially homogeneous, later eluting inhibin protein was performed after acid hydrolysis in 4M methane sulfonic acid and

0.2% tryptamine at 110°C. for 24 hours. Norleucine was used as an internal standard. The amino acid analyzer was a Beckman Model 121M with ninhydrin post-column derivatization. The results are shown in the Table below.

TABLE AMINO ACID COMPOSITION OF PURIFIED INHIBIN

PROTEIN FROM RAM RETE TESTIS FLUID

Amino Acid Inhibin Protein

As 22

Thr 11

Ser 17

Glx 22

Gly 16

Ala 16

Val 11

Met 5

He 8

Leu 22

Tyr 8

Phe 10

His 8

Trp 4 Inhibin Protein

10 10 12 23

Although the results of amino acid analysis of a large protein are by no means definitive of the precise composition because amino acid analysis can only provide a rough estimate, the results give a fairly accurate portrayal of relative residue compositions and together with other SDS and sequence data accurately define a pure compound.

A portion of the purified, later eluting fraction highest peak was exposed to 2% sodium dodecyl sulfate (SDS) with and without 5% β-mercaptoethanol at neutral pH in a boiling water bath for 2-3 minutes? both aliquots were subsequently applied to a slab gel and subjected to SDS polyacrylamide gel electrophoresis (PAGE) as described by Laemmli, U.K., Nature, 227,

680-685(1970). Protein bands were discovered by silver staining.

On SDS-PAGE under non-reducing condition, the inhibin protein showed a single band migrating at about 34.5kD. Under reducing condition, the inhibin protein separated into two bands, one migrating at 18kD and the other at 16.5kD. Electrophoresis showed the protein was more than 90% pure.

NH_-terminal sequence analyses of the 18kD and 16.5kD chains of the 34.5kD inhibin protein were accomplished by first separating the two chains by SDS-PAGE under reducing conditions. Microsequencing, as described in Spiess, J. et al. Biochemistry, 20, 1982-1988 (1981), of the intact inhibin protein beginning at the NH₂-terminus consistently revealed two residues of approximately equal concentration at every cycle, indicating that the protein is composed of two chains. Based upon the results from multiple sequencing analyses of both the intact and reduced inhibin protein, the sequence of the NH₂~terminal residues of the 18kD chain of the inhibin protein is Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala- Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala- His-Ala-Asp-Cys. The first NH₂-terminal residues of the 16.5kD chain of the inhibin protein are Gly-Leu- Glu-Cys, and it is believed that the next residues are Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-(Tyr or Phe) . The next residues following these are believed to be: Val-Ser-Phe-Lys-Asp-Ile-Gly. The C-terminal portion of the 16.5KD chain is believed to be: Trp-Asn-Asp-Trp- Ile-Ile-Ala-Pro-Ser-Gly-Tyr-His-Ala-Asn-Tyr-Cys-Glu-Gly- Glu-Cys-Pro-Ser-His-Ile-Ala-Gly-Thr-Ser-Gly-Ser-Ser-Leu- Ser-Phe-His-Ser-Thr-Val-Ile-Asn-His-Tyr-Arg-Met-Arg-Gly- His-Ser-Pro-Phe-Ala-Asn-Leu-Lys-Ser-Cys-Cys-Val-Pro-Thr- Lys-Leu-Arg-Pro-Met-Ser-Met-Leu-Tyr-Tyr-Asp-Asp-Gly-Gln- Asn-Ile-Ile-Lys-Lys-Asp-Ile-Gln-Asn-Met-Ile-Val-Glu-Glu- Cys-Gly-Cys-Ser-OH.

Because a substantial portion of the sequence of both chains of an inhibin protein is accurately known, the mRNA encoding the chains can be isolated, and the cDNA's can be synthesized by recombinant DNA techniques. Messenger RNA (mRNA) is obtained from ovarian follicules or from ram testes which produce inhibin, and then cDNA is synthesized from the mRNA by reverse transcription. The cDNA is inserted into a cloning vector which is used to transform a suitable host to create a cDNA library.

Based upon the known partial amino acid residue sequence of the inhibin chains, labelled oligonucleotides are synthesized for detecting cDNA corresponding to each chain. Because of the degeneracy of the genetic code, mixed hybridization probes are prepared and used as probes. These probes are then used to select, from the library, cDNA clones that contain gene sequences encoding the chains. cDNA libraries may also be screened by im unological expression assay with an antibody raised against inhibin or one of the two inhibin chains. Immunological expression assay may also be used to confirm screening with hybridization probes. From selected clones, cDNA is excised and inserted into appropriate vectors under the control of suitable promotor sequences, and the vectors are transformed into cell lines for expression of the recombinant inhibin chains. Although vectors containing the genes for both chains could conceivably be transformed into the same cell line, for simplicity, vectors for expression of each chain are preferably transformed separately into cell lines. The two inhibin chains can then be isolated from the cellular material and/or the cell culture medium. The two chains are then subjected to oxidizing conditions which promote disulfide bonding between the chains. The foregoing molecular biology techniques may also be used to read the gene sequences encoding the separate inhibin chains, and thereby completely characterize the protein chains. Substantially pure 34.5kD inhibin or the nontoxic salts thereof, combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition, may be administered to mammals, including humans, either intravenously, subcutaneously, percutaneously, intramuscularly or orally for control of fertility, gonadotropin secretion or sex hormone production.

Furthermore antibodies raised against synthetic fragments of inhibin, e.g. the six N-terminal residues of the 18KD chain, namely Ser-Thr-Pro-Pro-Leu-Pro, have been shown to neutralize the activity of purified inhibin. Thus passive (administration of antibodies) or active (administration of immunogenic inhibin as antigen) immunization methods could be employed to block endogenous inhibin and thereby elevate endogenous gonadotropin secretion and exert a profertility effect in sheep (both rams and ewes) , in human beings and in other vertibrate animal species having inhibin of a similar polypeptide structure. Administration of inhibin induces decreased fertility in female mammals and decreases spermatogenesis in male mammals, and administration of a sufficient amount of inhibin could be employed to induce infertility in sheep, including rams and ewes, and in other mammals. Inhibin is also useful for tests to diagnose infertility.

Such peptides are often administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g., with zinc, iron or the like (which are considered as salts for purposes of this application) . Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If administration in liquid form is desired, sweetening and/or flavoring may be used, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.

Inhibin should be administered under the guidance of a veterinarian or a physician, and pharmaceutical compositions will usually contain an effective amount of the peptide in conjunction with a conventional, pharmaceutically-acceptable carrier. The dosage will vary depending upon the specific purpose for which the protein is being administered, and dosage levels in the range of about 0.1 to about 1 milligrams per Kg. of body weight may be used when the protein is administered on a regular basis as a male contraceptive. Although the method of purification of inhibin has been described primarily in terms of isolation from RTF, inhibin can be similarly purified from other crude extracts, for example follicular fluid. Although the invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto.

Claims

WHAT IS CLAIMED:

1. A substantially 34,500-Dalton ovine inhibin protein composed of a first polypeptide chain having a molecular weight of about 18,000 Daltons and a second polypeptide chain having a molecular weight of about 16,500 Daltons, said first and second chains being linked to each other through disulfide bonding, said first chain having an amino-terminal sequence beginning with Ser-Thr-Pro-Pro and said second chain having an amino-terminal sequence beginning with Gly-Leu-Glu- Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe, said protein specifically inhibiting basal secretion of follicle-stimulating hormone while not inhibiting basal secretion of luteinizing hormone.

2. A protein according to Claim 1 wherein said second chain has an amino terminal sequence beginning with Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys- Lys-Lys-Gln-Phe-Tyr-Val-Ser-Phe-Lys-Asp-Ile-Gly.

3. A protein according to Claim 1 wherein said second chain has an amino terminal sequence beginning with Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys- Lys-Lys-Gln-Phe-Phe-Val-Ser-Phe-Lys-Asp-Ile-Gly.

4. A protein according to any one of Claims 1-3 wherein said first chain has an amino terminal sequence beginning with Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp- Ser-Pro.

5. A protein according to any one of Claims 1-3 having a relative amino acid composition in the approximate amounts as follows: Asx 22, Thr 11, Ser 17, Glx 22, Gly 16, Ala 16, Val 11, Met 5, He 8, Leu 22, Tyr 8, Phe 10, His 8, Trp 4, Lys 10, Arg 10, Cys 12, and Pro 23.

6. A protein according to any one of Claims 1-3 eluting from a C_g, RP-HPLC column at the approximate region indicated in the accompanying FIGURE i. 7. A protein according to Claim 1 wherein said first chain has an amino terminal sequence beginning with Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala- Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala. 8. A protein according to Claim 1 wherein said first chain has an amino terminal sequence beginning with Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala- Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala- His-Asp-Cys. 9. A protein according to any one of Claims 1,

7 and 8 wherein said second chain has the formula: H-Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys- Lys-Gln-Phe-Phe-Val-Ser-Phe-Lys-Asp-Ile-Gly-Trp-Asn-Asp- Trp-Ile-Ile-Ala-Pro-Ser-Gly-Tyr-His-Ala-Asn-Tyr-Cys-Glu- Gly-Glu-Cys-Pro-Ser-His-Ile-Ala-Gly-Thr-Ser-Gly-Ser-Sβr- Leu-Ser-Phe-His-Ser-Thr-Val-Ile-Asn-His-Tyr-Arg-Met-Arg- Gly-His-Ser-Pro-Phe-Ala-Asn-Leu-Lys-Ser-Cys-Cys-Val-Pro- Thr-Lys-Leu-Arg-Pro-Met-Ser-Met-Leu-Tyr-Tyr-Asp-Asp-Gly- Gln-Asn-Ile-Ile-Lys-Lys-Asp-Ile-Gln-Asn-Met-Ile-Val-Glu- Glu-Cys-Gly-Cys-Ser-OH.

10-. A protein according to any one of Claims 1, 7 and 8 wherein said first chain is glycosylated.

11. A protein subunit of ovine inhibin selected from the group consisting of (a) a polypeptide having a molecular weight not greater than about 18kD, having about 135 amino acid residues, and having an amino terminal sequence beginning with Ser-Thr-Pro-Pro- Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln- Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His-Asp-Cys and (b) a polypeptide having a molecular weight not greater than about 16.5kD, having between about 115 and about 130 amino acid residues, and having an amino terminal sequence beginning with Gly-Leu-Glu-Cys-Asp-Gly-Lys- Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe. 12. Antibodies raised against Ser-Thr-Pro-Pro-

Leu-Pro which neutralize the biological activity of ovine inhibin.