AU636318B2 - Inhibin isolated from ovarian follicular fluid - Google Patents

Description

P/00/01 1 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATIO STANDARD PATENT 63 3 A 0 Invention Title: Inhibin Isolated from Ovarian Follicular Fluid The following statement is a full description of this invention, including the best method of performing it known to me/us: GH&CO REF: P21001-AX ADK INHIBIN ISOLATED FROM OVARIAN FOLLICULAR FLUID This invention relates to the isolation of a biologically active factor from mammalian ovarian follicular fluid, and valuable uses of that factor.

On the basis of circumstantial evidence and limited experimentation it was suggested as early as 1932 that the gonads produced a non-steroidal factor, termed inhibin, which was capable of selectively suppressing the pituitary gland secretion of follicle stimulating hormone (FSH) [McCullagh, Science 76, (1932) 19]. Since that time the development of radioimmunoassays to measure FSH has led to the accumulation of a substantial body of evidence to suggest that inhibin exists, but it was not until the early 1970s that any attempt to isolate and identify this substance was made. Since that time a number of groups of investigators have attempted to purify inhibin from several gonadal sources with conflicting results [de Jong, Mol.

Cell, Endocrinol. 13, (1979) Some investigators have claimed to have isolated and sequenced inhibin from human seminal plasma, with the molecular weights of their species of inhibin being 5,000 and 14,000 daltons [Seidah et al, FEBS Letters 167, (1984) 98; Sheth et al, FEBS Letters, 165, (1984) 11]. Furthermore, the gonadal origin of these 25 materials has been seriously questioned [Beksac et al, Int.

S. J. Andrology L, (1984) 389; Lilja and Jeppsson, FEBS Letters 182, (1985) 181]. Other groups of investigators have utilized fluid collected from the seminiferous tubules of o* the testis (rete testis fluid) and also ovarian follicular fluid to attempt to isolate gonadal inhibin. As yet these attempts, despite being carried out over a period of 12 years, have been unsuccessful in obtaining a purified S material. This background indicates that there is no general agreement as to the nature, chemical features or 35 site of production of the substance defined as inhibin.

The properties of bovine follicular fluid extracts have led to the postulate that there is a substance or substances, "inhibin", with specific functions. We have now 1 7523S isolated a material from a gonadal source which satisfies all the biological criteria which are characteristic of inhibin.

The present invention relates to the purification and characterization of inhibin and to the use of the purified material to raise antibodies, the use of inhibin and anti-inhibin antiserum in a quantitative radioimmunoassay, and applications in vitro and in vivo of inhibin and antibodies raised against inhibin.

0 According to one broad form of the invention there is provided a proteinaceous material, inhibin, consisting essentially of two subunits, characterized in that one subunit has an apparent molecular weight of about 14,000 2,000, wherein the purified proteinaceous material 15 is of a purity sufficient to yield a single major band on non-reducing SDS-PAGE, and can suppress production of follicle stimulating hormone without suppressing production of luteinising hormone.

Preferably, the proteinaceous material is glycosylated.

Preferably, the subunits are joined by at least one disulfide bridge.

Preferably, the proteinaceous material is at least 0 pure.

In one preferred form of the invention the 25 proteinaceous material has an apparent molecular weight of about 56,000 1,000.

Preferably the proteinaceous material is obtained from follicular fluid, more preferably from mammalian follicular S" fluid, most preferably from ovarian follicular fluid.

Preferred proteinaceous material of the invention is obtained from a bovine, an ovine or a human.

2 In a preferr:ed form of the invention the proteinaceous material comprises a purified inhibin of a purity sufficient to yield a single major band on non-reducing SDS-PAGE and having a molecular weight of between about 54,000 and 57,000, the purified inhibin consisting essentially of two subunits, and being capable of suppressing production of follicle stimulating hormone by rat anterior pituitary cells in culture without inhibiting the production of luteinising hormone by the cells.

LO Preferably the proteinaceous material comprises a first subunit having an apparent molecular weight of about 44,000 3,000.

More preferably the first subunit has an isoelectric point of between about pH 6.0 and Most preferably the second through sixteenth N-terminal amino acids of the subunit are: i e.

9 99 9 *9 9 991@

*SCR

'S

'04 Amino Acid Position 2 3 4 6 7 8 9 11 12 13 14 16 Amino Acid Ala Val Gly Gly Phe Met Arg Arg Gly Ser Glu Pro Glu Asp Gin 3 8031S A I Preferably the proteinaceous material comprises a second subunit having an apparent molecular weight of about 14,000 2,000.

More preferably the second, third, fifth, sixth, seventh, eighth, ninth, tenth, thirteenth and fourteenth N-terminal amino acids of the subunit are: Amino Acid Position Amino Acid 2 Leu 3 Glu Asp 6 Gly 7 Lys 8 Val 9 Asp or Asn Ile 13 Lys 14 Lys In accordance with a more preferred form of the invention there is provided a purified protein, inhibin, characterized in that: the apparent molecular weight as determined by S SDS-PAGE is 56,000 1,000; the isoelectric point is in the range 6.9-7.3; S. it can bind specifically to Concanavalin A-Sepharose; S. it consists of two sub-units separable on reduction as eo,. determined by SDS-PAGE, characterised in that: their apparent molecular weights are 44,000 3,000 and 14,000 2,000 respectively, (ii) the isoelectric point of the 44,000 molecular weight subunit is in the range 6.0-7.0, (iii) the N-terminal amino acid sequences of the two subunits are as described hereinbelow.

.35 the proteinaceous material can suppress follicle 0 stimulating hormone but not luteinising hormone, 4 7523S r thyroid stimulating hormone or prolactin in an in vitro bioassay system; and it can be labelled with radioactive iodine.

The invention also provides fragments, subunits, homologues and derivatives of inhibin.

According to another aspect of the invention, there is provided a method for isolating and purifying inhibin from mammalian ovarian follicular fluid, characterized by the following steps: a) one or more gel permeation chromatography steps; b) one or more reverse-phase high performance liquid chromatography steps; c) one or more preparative polyacrylamide gel electrophoresis steps; d) electrophoretic elution of the purified inhibin.

Preferably the method comprises: a) subjecting the starting material to a first gel permeation chromatography to yield a first eluent; b) subjecting the first eluent to a second gel permeation chromatography to yield a second eluent; c) subjecting the second eluent to reverse phase high performance liquid chromatography to yield a third eluent; d) subjecting the third eluent to preparative o* *25 polyacrylamide gel electrophoresis to obtain a single major band; and e) assaying the single major band for ability to suppress production of follicle stimulating hormone by rat 69 *anterior pituitary cells in culture without inhibiting the production of luteinising hormone by the cells.

The invention also provides a purified material comprising purified inhibin obtained by one of the above methods.

Preferably the gel permeation chromatography step is .35 carried out using a gel permeation support with high pore volume, for example Sephacryl S200 (a gel permeation matrix comprising covalently cross-linked allyl dextran and 5 7523S 1 ji .I 1 N,N'-methylene bis acrylamide) or Sephadex G100 (a bead formed gel of cross-linked dextran and epichlorohydrin).

Both Sephacryl and Sephadex are trade marks of Pharmacia.

Preferred elution methods utilize volatile solvents containing, for example, ammonium acetate, acetic acid, or similar compounds, to allow direct recovery of biological activity by lyophilization or vacuum drying.

Preferably the reverse-phase high-performance liquid chromatography is carried out usinc chemically-bonded N-alkylsilica column packing of narrow particle size distribution, most suitably 5-10l. The eluents used may be volatile or non-volatile, and contain ionic modifiers such as trifluoroacetic acid (TFA), ammonium bicarbonate, ammonium acetate, or sodium phosphate, in a gradient of water with a miscible organic solvent such as methanol, acetonitrile, or isopropanol. A preferred procedure utilizes a gradient of 0-50% acetonitrile in 0.1% TFA.

Various preparative polyacrylamide gel electrophoresis (PAGE) methods can be employed in the presence of sodium dodecyl sulphate (SDS) using PAGE gels of various porosites and cross-linking content. A preferred buffer system for electrophoresis is based on the method of Laemmli, as described in Nature 227, (1970) 680.

There is further provided a method for raising specific antibodies to inhibin or a subunit of inhibin, said antibodies having the ability to neturalize the activity of inhibin in the in vitro bioassay, and to cause an increase in gonadal weight in vivo.

There is still further provided a method for the radioimmunoassay of inhibin in biological samples such as plasma, serum or urine.

The invention also provides an antiserum containing antibodies specific to inhibin or a subunit of inhibin.

The invention further provides a method for 35 producing monoclonal antibodies specific to inhibin, comprising using a proteinaceous material of the invention.

6 7523S The invention still further provides monoclonal antibodies which preferentially bind to an immunological determinant of inhibin, of the present invention.

According to the invention there is provided a method for assaying for inhibin in a sample, comprising a radioimmunoassay procedure using an effective amount of a proteinaceous material of the invention.

The invention also provides a method for detecting and diagnosing infertility in a non human mammal using the above described radioimmunoassay procedure.

The invention further provides a non human mammal having a titre of anti-inhibin antibodies that is greater than the naturally-ocurring titre of the anti-inhibin antibodies in the mammal, wherein the anti-inhibin antibodies recognise inhibin as herein defined.

According to the invention, there is also provided a method for increasing the fertility of a mammal comprising passively immunizing the mammal with an effective amount of an antiserum of the invention or actively immunizing the mammal with a proteinaceous material of the invention in combination with a suitable adjuvant whereby antibodies to the proteinaceous material are produced.

There is further provided a method for advancing sexual maturity in a sexually immature mammal comprising 25 actively immunizing the mammal with an effective amount of a preparation of a proteinaceous material or the invention or a subunit of inhibin, in combination with an adjuvant, or S. passively immunizing the mammal with an antiserum of the invention.

The invention provides: a method for inhibiting gonadal function in a mammal; 1 postponing the onset of puberty in a mammal; suppressing ovulation in a female mammal; or 0 suppressing spermatogenesis in a male mammal, 35 which method comprises administering an effective amount of a proteinaceous material of the inventionto the mammal.

0 0 0*6* 000 7 The invention also provides: a method for increasing the rate of ovulation in a female mammal which method comprises actively immunizing che female inammal with an effective amount of a proteinaceous material of the invention or passively inmunizing the female mammal with an antiserum of the invention.

The invention further provides A composition effective as a contraceptive in a mammal, comprising an effective amount of a proteinaceous material of the invention together with a rharmaceutically acceptable carrier or diluent.

The invention provides a method for effecting contraception in a mammal comprising administering to the mammal a composition of the invention.

One embodiment of the present invention will nuw be described in detail by way of example only with reference to the following non-limiting examples, and the accompanying drawings in which: Figure 1 shows the elution profile of inhibin activity in bovine ovarian follicular fluid (bFF) fractionated on Sephacryl S-200.

Figure 2 shows the elution profile of inhibin activity in bFF fractionated on Sephadex G100.

MSSO

*Figure 3 shows the elution profile of inhibin 25 activity in bFF fractionated by reverse-phase high performance liquid chromatography (RP-HPLC) after prior gel So,. chromatography.

Figure 4 shows reduced and non-reduced SDS-polyacrylamide gel electrophoretic patterns of four sequential fractions obtained by preparative PAGE.

aQ 4 C *C as o 8 7523S Abbreviations used herein are as follows: bFF bovine ovarian follicular fluid RP-HPLC reverse-phase high performance liquid chromatography PAGE polyacrylamide gel electrophoresis FSH follicle stimulating hormone LH luteinising hormone U units GF gel filtration kD kilo Daltons TFA trifluoroacetic acid SDS sodium dodecyl sulphate EXAMPLE 1 Purification of inhibin from bovine ovarian follicular fluid The purification procedure is based on the sequential application of one or more gel permeation steps, one or more reverse phase high resolution chromatography steps and one or more PAGE steps.

Collection of bovine follicular fluid (bFF) Bovine ovaries were obtained from local abattoirs and bFF aspirated into a chilled vessel containing the .25 protease inhibitors Trasylol (10 U/ml) and phenylmethylsulphonyl fluoride (24gg/ml). The bFF was stored frozen at -20 0

C.

.2 The procedure for purification of inhibin consisted 900 of 4 stages or steps. In the sections below an outline of the procedure is described and details of the purification procedure are presented.

The steps in the purification procedure are as S" follows: gel permeation chromatography on Sephacryl 35 S-200.

gel permeation chromatography on Sephadex G-100.

*o* 9 7523S reversed phase high performance liquid chromatography.

preparative polyacrylamide gel electrophoresis.

electrophoretic elution of samples.

The purification of inhibin was monitored by the bioassay method of Scott et aL, [Endocrinology 107, (1980) 1536], used with minor modifications [Au et al, Endocrinology 112, (1983) 239]. The method is based on the ability of inhibin to cause dose-dependent reduction of FSH but not LH cellular content of rat anterior pituitary cells in culture.

Step A: Gel Permeation Chromatography on Sephacryl S200: Elution Buffer 0.05M Ammonium Acetate pH bFF (50-100ml) was diluted with 0.05M ammonium acetate pH 7.0 (25-50 ml) and centrifuged (12000 g x 30 min at 4 0 C. The supernatant (75-150 ml) was fractionated on a Sephacryl S200 gel filtration column (9 x 90 cm) at a flow rate of 70-100 ml per hour. As seen in Fig. 1, inhibin activity was located in a void volume region (MW>90,000) of this column. 90% recoveries of inhibin activity were obtained with a 3-4 fold increase in specific activity.

Step B: S Gel Permeation Chromatography on Sephadex G100: S. Elution Buffer 4M Acetic Acid Void volume fractions B, C and D, Fig. 1) were combined and 25-30% of the pooled fractions were acidified with glacial acetic acid (chromogen-free) to a final concentration of 4M acetic acid and kept at 4 0 C for one hour. The remaining 50-75% was stored frozen at -20 0 C prior to subsequent fractionation. The acidified pool (approximately 120ml) was applied to a Sephadex G100 gel filtration column (9 x 90 cm) with 4M acetic acid as eluting buffer at a flow rate of 70-100 ml per hour. All operations with both gel filtration columns were performed at 4 0

C.

10 7523S |1 Under these conditions the bulk of the inhibin activity eluted in a lower molecular weight region (elution volume 1760-1880ml, MW range 20,000-60,000, Fig. 2) with a 10-20 fold increase in specific activity and 45% recovery of inhibin activity in this region. Using analytical columns 2.5 x 100 cm) similar profiles of activity with higher specific activities (10-50 fold) have been observed.

Step C Reverse Phase High Performance Liquid Chromatography

(RP-HPLC)

The column fractions (elution volume approximately 1760-1910 ml) from Step B were pooled prior to loading onto the RP-HPLC column. The column employed was an Ultrapore RPSC (Beckman, Berkeley, California). The mobile phase used was a linear gradient between 0.1% TFA in water and acetonitrile in 0.1% TFA; the flow rate was 1 ml/per minute and 0.5 ml fractions were collected. Three loading procedures were employed: the sample was lyophilised and 1 mg dissolved in 4M acetic acid to a concentration of 8-10 mg dry weight/ml, centrifuged in approximately 100~ 4M acetic acid and applied to the HPLC column via the injector; the lyophilised material (5-10 mg) was dissolved in 20 ml of 4M acetic acid, centrifuged and loaded onto the column via a solvent port on the HPLC; the unlyophilised material (approximately 100ml) was filtered through a 0.5 m filter (FH: Millipore Corp) prior to loading via a solvent port on to the column at a flow rate S..n of 2 ml/minute.

The inhibin regions from the bulk runs and (c) were rechromatographed. Each fraction was combined with the contents of the corresponding tubes for the repeat runs (if required) and aliquots were taken from bioassay, amino acid analysis and SDS-PAGE. The acetonitrile from each fraction was then removed by evaporation under N 2 and the sample lyophilised. As seen in Fig. 3, inhibin activity was found in one region of the chromatogram, corresponding to 1 7523S approximately 30% acetonitrile. The sample load in this experiment was 1 mg.

Recoveries of inhibin of 40% were obtained with the various loading procedures, although the HPLC column performance was markedly influenced by the latter two procedures. A 10-fold increase in specific activity was attained with this HPLC step, with an overall 160-fold increase in specific activity.

Step D: Preparative Polyacrylamide Gel Electrophoresis The inhibin-containing fractions obtained by RP-HPLC were dissolved in non-reducing sample buffer (0.06 M Tris-HC1 pH 6.8, 12.5% glycerol, 1.25% w/v SDS and 0.006% bromophenol blue) and fractionated on a vertical polyacrylamide gel electrophoresis apparatus [Reid and Bieleski, Analytical Biochemistry, 22, (1968) 374] with modifications. The SDS polyacrylamide gel electrophoresis solutions [Laemmli, Nature 227 (1970) 680] consisted of a stacking gel (0.125M Tris-HC1 pH 6.8, 0.1% w/v SDS, acrylamide, 01.3% Bis acrylamide, 0.1% ammonium persulphate) and a separating gel (0.38M Tris-HC1 pH 8.8, 0.1% w/v SDS, acrylamide, 0.2% Bis acrylamide, 0.03% ammonium persulphate). The electrophoretic buffer was .25 Tris-glycine buffer containing 0.05% SDS. The protein load (500-700 Vg) was divided between the eight sample slots.

**The gels were electrophoresed initially at until the sample had migrated into the separating gel then the current was increased to 30mA for the duration of the run (4h) until the bromophenol blue marker reached the bottom of the gel. The gel was stained with 0.5% Coomassie blue in acetic acid: isopropyl alcohol:water 1:3:6 v/v (15 min) and destained with acetic acid: methanol:water, 50:165:785 v/v, and the inhibin region (molecular weight approximately 56,000) which was visualised by this procedure was sectioned into 2 mm slices using a 12 7523S scalpel and ruler. Gel slices above and below the inhibin region were also taken. The gel slices were stored in sealed tubes at -15 to -20 0 C prior to electrophoretic elution.

Figure 4 shows reduced and non-reduced SDS-gel electrophoretic patterns of 4 sequential fractions B, C and D) outained by preparative PAGE purification. Inhibin activity was located primarily in fraction B (apparent molecular weight 56,000 1,000 mean SD; 5 purified inhibin preparations). Under reducing conditions, fraction B reduced into two major bands with apparent molecular weights of 44,000 3,000 and 14,000 2,000 (n 5) (Lane The Laemmli (1970) SDS-PAGE system was employed.

Proteins were localized by silver staining. Protein standards used were: bovine serum albumin (molecular weight 67,000); ovalbumin (43,000); carbonic anhydrase (29,000); goose lysozyme (21,000); hen egg lysozyme (14,500). The reductant was 0.1% 2-mercaptoethanol.

Step E: Electropheretic Elution at Room Temperature The method used was modified from that of Hunkapiller et al. [Methods in Enzymology, 91 (1983) 227].

Gel slices were diced in distilled water with a razor blade, washed in elution buffer SDS in 0.05M NH 4 HCO3) for 5 min and placed in an electrophoretic elution cell fitted with dialysis membrane discs (6,000-8,000 molecular weight 0. cut off). The gel slices were covered with soaking buffer SDS in 0.4M NH 4

HCO

3 and overlayed with elution buffer SDS in 0.05M NH 4

HCO

3 Solid sodium thioglycollate, to a final concentration of 0.5mM, was added to the elution buffer. Gel pieces were allowed to soak for 3-5 hours prior to the initiation of the electrophoretic elution process at 50V (direct current). After 12-16 hours the elution buffer was replaced with dialysis buffer (0.02% SDS in 0.01M NH 4

HCO

3 followed by further electrophoretic elution at 80V (direct current) for 20-24 13 7523S hours until the Coomassie blue stain and protein had migrated into the sample collection well. The eluted sample was removed from the collection well by means of a bent-tipped 50l Hamilton syringe, aliquoted and either frozen or lyophilised. Samples of the purified fractions were set aside for the in vitro bioassay, amino acid analysis and molecular weight determination using SDS-PAGE as employed with the silver staining technique.

Using purification procedures based on the above methods, i.e. a combination of gel permeation chromatography,RP-HPLC and preparative PAGE, inhibin activity was recovered as a single protein band on SDS-PAGE (Fig. 4, with an apparent molecular weight of 56,000 1,000 preparations).

The purified preparations of inhibin suppress FSH but not luteinising hormone, thyroid stimulating hormone or prolactin in the in vitro bioassay, indicating that the purified product is specific in suppressing FSH. The suppression is not due to non-specific toxic effects.

Example 2: Alternative Purification Procedures Inhibin was isolated as in Example 1, except that inhibin was precipitated by adjusting the pH of the void *25 volume fraction from Step A to pH 4.75 with 4M acetic acid and centrifuged at 12,000 x g for 30 min at 4 0 C. The resulting pellet was dissolved in 0.05M ammonium acetate pH 7.0. The solubilization of the pellet was aided by homogenization and sonication in buffer at room temperature. The sample was adjusted to 4M acetic acid with glacial acetic acid and centrifuged prior to application to the column for the second gel permeation step (Step The overall recoveries of inhibin activity including pH precipitation and acidification of the dissolved pellet was 34%. The modification has the advantage that the column sample volume is reduced by 75% allowing a greater throughput of material. Inhibin activity was recovered in e-14 14 7523S similar column fractions (elution volumes 1700-2100 ml) to that in the procedure of Example 1 following fractionation by gel filtration with 4M acetic acid as elution buffer.

The subsequent behaviour of inhibin on the RP-HPLC and the PREP-PAGE procedure was not influenced by the various modificatons examined in this example.

The following Purification Summary demonstrates the results achieved by the above-described purification at each step thereof.

go..

OSS

C

eo O* e 0* 0 to O 4 15 7523S Table 1 Specific Total Recoveries at each Activity Protein stage Preparation U/mg (mg) Purification Factor 1. bFF 63 a (4) 3350 100 2. Gel Filtration a) Sephacryl S200 (0.05 M M ammnonium acetate 21b(7) 800 3.7 to ael filtration Extraction Drior to gel filtration b) Sephadex G100 (4M acetic acid) 25 3. RP-HPLC o Solubilization 0 prior to all, .a PREP-PAGE 6 66 4. PREP-PAGE, 9 4 4 c (12) 5-15 1 0 2 6 0 c (12) 0.4-0.7 40 163 212 000 c 0.01 33655 *~i S S4 *0 S S

S.

*0 0 0 0*

S.

S

000000

S

0000 0 @000 a) U/mg protein determined by Lowry et. al, biol. Chemn.

193, (1951) 265).

b) U/mg dry wt.

c) U/mg protein based on amino acid content--- 16 7523S Example 3: Further Chemical Characterization of Inhibin Analytical SDS-PAGE of the final product under non-reducing conditions gave a single band with an apparent molecular weight of 56,000 1,000 (mean SD, preparations) while under reducing conditions two major bands with apparent molecular weights of 44,000 3,000 and 14,000 2,000 (5 preparations) were observed. Evidence of heterogeneity was observed as assessed from electrophoresis of the 56kD band under non-reducing conditions and the 44kD band under reducing conditions. The apparent molecular weight range for the 56kD material was between 54,000 and 57,000 while the 44kD material ranged from 42,000 to 46,000. A single band was observed with the 14kD band.

These findings are consistent with the glycoprotein nature of this molecule.

The pi values of intact inhibin and the larger subunit were determined using the 2 dimensional PAGE system of O'Farrell, J. Biol. Chem, 250 (1975), 4007-4021.

Intact inhibin was detected by silver stain and showed a single band with an apparent molecular weight of 56,000 but with several closely associated spots with pi values in the pH range 6.9-7.3. These data are suggestive of a glycoprotein preparation. The 44,000 molecular weight subunit showed a single band with an apparent molecular weight of 46,000 with several closely associated spots with pi values in the pH range 6.0-7.0, suggesting that this subunit is a glycoprotein.

Further evidence that inhibin is a g.ycoprotein was established by: The ability of radiolabelled irhibin to bind to the lectin Concanavalin A imrr oilized on Sepharose (Trade Mark of Pharmacia, Uppsala, Sweden). From several experiments 15-17% of the 35 tracer bound to the lectin and was released following elution with the sugar 17 7523S methyl-a-D-glucopyrancside (Calbioch-m, San Diego, USA).

Binding of horseradish peroxidase-labelled wheat germ lectin to inhibin, following fractionation of inhibin following fractionation of inhibin on SDS-PAGE and electrotransfer of the protein on to nitrocellulose. The binding of the lectin was monitored by the intensity of the peroxidase colour reaction. Lectin binding was associated with the 56kD intact protein and with the 44kD subunit.

Example 4: The N-Terminal Amino Acid Sequence of the Two Subunits of Inhibin A purified preparation of inhibin was reduced and carboxymethylated and the two subunits, of apparent molecular weight 44,000 and 14,000 respectively, were separated by PAGE and recovered from the gel by an electroelution process as described in Step E, Example 1 above. The SDS was removed by methanol precipitation of inhibin, and the N-terminal amino acid sequence determined.

0000 The sequence of the two sub-units are:

S

*0 00 0000 00: 0 0 :00 0 0 000 18 7523S Residue 1 2 3 4 6 7 8 9 11 12 13 14 16 44 kD Subunit xxx Ala Val Gly Gly Phe Met Arg Arg Gly Ser Glu Pro Glu Asp Gln 14 kD Subunit Leu Glu yyy Asp Gly Lys Val Asx Ile yyy Lys Lys xxx ambiguous yyy unable to be determined these experiments Asx Asn or Asp insufficient material in SS S 0S S S

S

S

*SSS

25 Example Raising of Antibodies to Purified Inhibin 0**S

S

55 S. S

S*

SS

S

19Lg of inhibin purified as described above was dissolved in 6001 of Dulbecco's Phosphate Buffer pH 7.4 and emulsified with an equal volume of an oil-based adjuvant (for example, Marcol 52: Montanide 888 in the ratio 9:1.

Marcol 52 is a Trade Mark of Esso, and Montanide 888 is a Trade Mark of SEPPIC, Paris). Two hundred 1 was injected into each of four intramuscular sites and 200 ±l injected subcutaneously into a rabbit. The animal was boosted six weeks later with 18Ag of purified inhibin, using the same injection procedure as above. The titre of antibody in the rabbit serum was assessed by its ability to bind to 19 7523S iodinated inhibin (for details see below), or by its ability to neutralize inhibin activity in vitro. The highest titre was observed two weeks post booster (week 8 sample), returning to preimmunization levels by 17-18 weeks.

During immunization, the rabbit increased its testicular volume from 3.0 to 3.5 ml, indicating that immunization against inhibin can increase gonadal weight, presumably by neturalization of endogenous inhibin, thus allowing FSH levels to rise.

Example 6: Antiserum Characterise,,'p The week 8 antiserum from the rabbit, prepared as described above, war Investigated for its ability to neutralize inhibin activity in vitro. A charcoal-treated bovine follicular fluid preparation was used as inhibin standard in an inhibin in vitro bioassay [Scott et al, Endorcrinology, 107, (1980), 1536]. It was found that 2[pl of antiserum was sufficient to neutralize a dose of inhibin (2 units) known to give a maximal response in the assay.

This neutralizing activity was not present in preimmunization serum. One other rabbit was immunized initially with a less pure inhibin preparation (340Rg obtained after the RP-HPLC purification step) and boosted 25 with 22 Rg of pure inhibin. The.initial immunizing injection was in complete Freund's adjuvant, using the immunizing method of Vaitukaitas et al (Journal of Clinical Endocrinology and Metabolism, 33, 1971, 988), while the booster injection procedure was the same as cited above.

Antiserum (week 9) from this animal also showed neturalizing capabilities in vitro.

0 Example 7: Radioifmunoassay of bFF Inhibin 35 Purified preparations of inhibin were iodinated either Sby a mild chloramine-T procedure or by using the Bolton-Hunter reagent [Bolton and Hunter, Biochem J. 133 20 7523S (1973) 529] to a specific activity of 0.5 RCi/pg as determined by a self displacement procedure in the radioimmunoassay method described below. The iodinated material showed the same apparent molecular weight as the non-iodinated molecule as assessed by SDS-PAGE under reducing and non-reducing conditions. Using the iodinated tracer, a radioimmunoassay procedure was derived using a polyethylene glycol-facilitated second antibody precipitation step to separate antibody-bound and -unbound iodinated hormone [Peterson M.A. and Swerdloff, Clin.

Chem. 25 (1979) 1239-1241]. Characteristic displacement curves were obtained for purified and for unfractionated bFF preparations. The dose response curve for the purified material showed a sensitivity (ED 10 of 2 ng/tube with ED50 of 25 ng/tube. The dissociation constant of the -10 inhibin-antibody interaction was 4.5 x 10 0M at 20 0

C.

Example 8: Inhibition of ovulation in human chorionic gonadotrophin-stimulated 5-day pregnant mice Crude extracts of bovine follicular fluid have previously been shown to inhibit ovulation. The inhibition can be competitively reversed with FSH Cummin3, Ph.D.Thesis, 1983, University of New England (Armidale)].

5-7 day pregnant mice were given 1.5 Rg inhibin g, subcutaneously at 9 followed by subcutaneous injection of 10 IU of HCG at 6.00 p.m. The following morning thb number of ova in the ampulla of the Fallopian tube was counted. Inhibin administration significantly inhibited ovulation, as shown in Table 2.

S

S C 21 7523S *e a

S

0! 1e 0 00 d 44* I 0040

OS

0 9 *o Sample Number of Number of Ova Animals in Ampulla Control (Solvent Alone) 11 4.91 2.66 Purified Inhibin* 7 2.43 2.22 bFF** (50 8 3.38 1.80 bFF (100 4 2.0 0.0 Inhibin preparation is 75% pure based on intensity of silver stain on SDS-PAGE. The contaminants consist of higher molecule weight material (M.wt 65-70 kD) which is biologically inactive in the inhibin in vitro bioassay. Dose approximately 1.5 [g protein/animal.

Containing 20pg/ml inhibin based on inhibin in vitro bioassay.

Purified inhibin and 100pl bFF both resulted in significant inhibition of ovulation compared to the control 25 (p 0.01 and p 0.05 respectively by Wilcoxon's test).

Example 9: Effect of Immunization on Plasma FSH Levels In Example 5 above, one rabbit was immunized, and 30 boosted on two further occasions with purified inhibin. The antiserum so obtained neutralized inhibin activity in the in ,itr bioassay. It would be expected that in vivo, the a.tiserum would neutralize circulating inhibin, leading to an elevation of circulating FSH. The rabbit's plasma FSH 35 showed a rise and fall in concert with the titre of the inhibin antibody in the rabbit serum. The results are shown in Table 3.

22 7523S 00 9 so* 0 0 0* 0 .0 0**S* of 9.

Weeks Post Number of Antibody Plasma FSH Booster Iniection Serum Samples Titre* ng/ml 3-6, 17, 21 6 0.25 4.87 0 76 a 11-14 3 0.67 0.19 5.44 0.24 b 7-10 3 2.13 0.45 6.15 0.44 c Reciprocal of antiserum volume (pl) required to neutralise 1.5 U inhibin in the inhibin in vitro bioassay a vs c; a vs b and c; p 0.05 Example Suppression of circulating FSH following acute administration of inhibin to castrated male rats It is expected that purified inhibin, as seen in the experiment below with bovine follicular fluid, should suppress circulating FSH within 4-8 hours of administration. Inhibin (bovine follicular fluid) was administered via the jugular vein into the circulation of 34-day old male rats which had been 25 castrated 3 days earlier, and the levels of plasma FSH 5 hours later were determined by FSH radioimmunoassay.

There was a significant dose-dependent decrease in FSH associated with increasing doses of bovine follicular fluid.

Results are shown in Table 4.

23 7523S Table 4 0

OSS*

*0*S 0r S 0* S. 0 *0

S

S0 S

S

S

S. *5

S

*505

LO

Sample Number of Plasma FSH Animals Expressed as of Pre-injection levels Control (Solvent Alone) 5 99.3 12.8a bFF* (62.5Ll) 5 80.8 5 bFF (125 il) 5 66.6 10.3 c bFF (250 Rl) 5 51.9 7 .0d containing 20Rg/ml inhibin based on inhibin in vitro bioassay a vs b; b vs c; c vs d p 0.05 Example 11: Purification of Inhibin From Ovine Follicular Fluid We have found that inhibin activity from ovine follicular fluid is purified in a similar manner to bFF 25 inhibin using purifications steps A, B and C above. Its characteristics following steps D and E are similar to those of bFF inhibin. By extension it is expected that purification steps A to E would be applicable to other mammalian inhibins including that from human.

30 The Webster's Third New International Dictionary (1981 unabridged ed.) defines a homologue as "one that exhibits homology", and defines homology as "a similarity often attributable to common origin..., a likeness short of identity in structure or function between parts of different 35 organisms due to evolutionary differentiation from the same or a corresponding part of a remote ancestor", thus it can be seen that ovine and human inhibins are homologues of the 24 7523S bovine inhibin purified according to Example 1.

Since FSH is important in the stimulation of ovarian and testicular function, the main potential applications of purified inhibin lie in its ability to specifically inhibit FSH secretion, or in its use as an antigen such that immunization against inhibin will elicit antibodies capable of neutralizing endogenously occurring inhibin, thereby raising FSH levels. Many studies have been performed in vivo using crude or partially purified extracts of gonadal tissues or fluids in attempts to study the action and physiology of inhibin. In these experiments, effects attributed to but not proven to be due to inhibin or antibodies against inhibin include: 1. Inhibin of gonadal function [Moudgal at al, 1985 in Gonadal Proteins and Peptides and their Biological Significance (ed. Sairam), World Scientific Publishing, Singapore (in press].

2. An increase in ovulation rate [Henderson et al, J.

Endocrinol. 102, (1984) 305; Cummins et al, Proc.

Aust. Soc. Reprod. Biol 15 (1983) 81].

3. An advancement of the onset of puberty [Al Obaidi et al, Proc. Aust. Soc. Reprod. Biol, 15 (1983) The known properties of inhibin and of FSH suggest a number of possible applications for the purified inhibin and 25 antibody to inhibin according to the present invention: Increase of ovulation rate: It is recognized that FSH stimulates the development of ova in mammalian ovaries [Ross e. a 1, (1981) in Textbook of Endorcrinology, ed.

Williams, p. 3551 and that excessive stimulation of the ovaries with FSH will lead to multiple ovulations [Gemzell, Induction of ovulation with human gonadotrophins, Recent Prog. Hormone Res. 21 (1965) 179]. We have demonstrated that inhibin will suppress FSH both in vitro and in vivo and that inhibin can be used as an immunogen to raise neutralizing antibodies against inhibin. The immunization of mammals, e.g.

cattle and sheep, with the purified preparation of 25 7523S inhibin and a suitable adjuvant leads to the development of antibodies in immunized animals. These antibodies neutralize the animal's own inhibin production, thereby removing the suppresive effect on FSH secretion. The resultant elevation in FSH leads to increased stimulation of follicular development in the ovary with an increase in ovulation rate.

Collection of serum from animals immunized against inhibin also provides an antiserum which can be used for passive immunization of other animals. By this method, the injection of the inhibin antiserum neutralizes the animal's own inhibin and hence leads to an elevation of FSH and the subsequent events in stimulating ovulation. Both the passive and the active methods of immunization may be used to increase ovulation rate.

The potential to use inhibin for active immunization to achieve gonadal growth is illustrated by the increase in testicular size of the rabbit tested during immunization against inhibin (Example (ii) Inhibition of ovarian and testicular function: The recognized importance of FSH in the stimulation of follicular development in the ovary and sperm production in the testis [Ross et al, (1981), in 25 Testbook of Endocrinology, ed. Williams, p. 355; 5 Bardin and Paulsen, (1981), The Testes, in Textbook of Endocrinology, ed. Williams, p. 293] supports a potential role for inhibin in the suppression of gonadal function. It is expected that the administration of inhibin will lead to a suppression of ovarian and testicular function and a disruption of fertility. This action of inhibin can be used in males and females of the human, ovine and bovine species and is likely to be applicable to other .:35 species.

(iii) Advancement of the onset of fertility: It is recognized that one of the earliest events in the 0:00 26 7523S onset of puberty is the rise in FSH levels which leads to ovarian and testicular stimulation [Ross et al, (1981) in Textbook of Endocrinology, ed. Williams, p.

355; Bardin and Paulsen, (1981), The Testes, in Textbook of Endocrinology, ed. Williams, p. 293]. The potential exists that immunization of sexually immature mammals against inhibin, by active or passive techniques, will lead to a premature onset of puberty with attendant stimulation of ovarian and testicular function.

The lifetime reproductive performance of domestic animals such as cows, sheep and pigs depends upon the age of onset of puberty, and intervals between each conception, and the potential for subsequent ovarian failure with advancing age. Immunization of young animals before puberty with inhibin, either by active or passive immunization, neutralises the animal's own inhibin production and leads to an elevation of FSH.

This elevation in FSH levels induces pubertal development at an earlier age than normal by stimulation of the gonad. This method can be used to induce precocious puberty in male or female mammals.

(iv) Suppression of puberty: Since FSH is recognized as a crucial factor in the onset of puberty, administration e 25 of inhibin may be used as a means of suppressing S*puberty in unwanted situations, e.g. precocious pubertal development in humans, or in delaying the onset of puberty.

Inhibin can be used as an immunogen to raise antisera or monoclonal antibodies which can be used to develop radioimmunoassays or enzyme-linked immunoassays to measure inhibin, and to develop immunoadsorbent columns to aid in the purification of inhibin.

(vi) Using the above-described antisera a radioimmunoassay .35 system to measure inhibin has been devised which enables the measurement of inhibin in biological samples plasma, serum or urine), which is not 27 7523S possible using the previously known in vitro bioassay system of Scott et al (1980). Inhibin levels in plasma or serum will provide an index of Sertoli cell and granulosa cell function for use in the diagnosis of fertile status.

(vii) It is possible that administration of high doses of inhibin may inhibit the secretion of LH. This would further support the ability of inhibin to suppress ovulation.

It will be clearly understood that the invention in its general aspects is not limited to the specific details referred to hereinabove.

0e 6 0O e 0 28 e 0

SS

h 28 7523S

Claims (90)

1. A proteinaceous material consisting essentially of purified inhibin of a purity sufficient to yield a single major band on non-reducing SDS-PAGE, wherein the purified inhibin consists of two subunits, one subunit has an apparent molecular weight of 14,000 2,000, and the inhibin is capable of suppressing production of follicle stimulating hormone without suppressing production of luteinizing hormone.

2. A proteinaceous material according to claim 1 which is glycosylated.

3. A proteinaceous material as defined by claim 1 or claim 2, wherein said first subunit and said second subunit are joined by at least one disulfide bridge.

4. A proteinaceous material according to any one of claims 1 to 3, wherein the proteinaceous material is at least 75% pure. A proteinaceous material according to any one of claims 1 to 3, wherein the proteinaceous material is pure.

6. A proteinaceous material as defined by any one of claims 1 to 3, wherein the proteinaceous material has an *apparent molecular weight of 56,000 1,000.

7. A proteinaceous material as defined by claim 6, having an apparent molecular weight of 56,000. S. 8. A proteinaceous material as defined by any one of claims 1 to 3, wherein the proteinaceous material does not suppress thyroid stimulating hormone or prolactin in an in vitro bioassay system. 30 9. A proteinaceous material as defined by claim 8, wherein the proteinaceous material does not produce a non-specific, toxic suppression of hormone production.

10. A proteinaceou material, inhibin, of a purity sufficient to yield a single major band on non-reducing SDS-PAGE, characterized in that: the apparent molecular weight as determined by SDS-PAGE is 56,000 1,000; the isoelectric point is in the range 6.9-7.3; 30 the proteinaceous material can bind specifically to Concanavalin A-Sepharose; the proteinaceous material consists of two sub-units separable on reduction as determined by SDS-PAGE, characterised in that: their: apparent molecular weights are 44,000 3,000 and 14,000 2,000 respectively, (ii) the isoelectric point of the 44,000 molecular weight subunit is in the range 6.0-7.0, (iii) the N-terminal amino acid sequences of the two subunits are: Amino Acid Position 44 kD Subunit 14 kD Subunit 2 Ala Leu 3 Val Glu 4 Gly Gly Asp 6 Phe Gly 7 Met Lys 8 Arg Val 9 Arg Asp or Asn Gly Ile 11 Ser 12 Glu 13 Pro Lys 14 Glu Lys Asp 16 Gin the proteinaceous material can suppress follicle stimulating hormone but not luteinising hormone, thyroid stimulating hormone or prolactin in an in vitro bioassay system; and the proteinaceous material can be labelled with radioactive iodine. *5 S*

11. A proteinaceous material as defined by any one of claims 1 to 3, wherein the band is visualised with silver staining. ''^tx 21001AX I 31

12. A proteinaceous material as defined by any one of claims 1 to 3, wherein the band is visualised with Coomassie blue staining.

13. A proteinaceous material as defined by any one of claims 1 to 3, wherein injection of a preparation of the material in a pharmaceutically acceptable carrier into a mammal causes the mammal to produce antibodies to the mammal's native inhibin.

14. A proteinaceous material as defined by any one of claims 1 to 3, having a pi of between about 6.9 and 7.3. A proteinaceous material as defined by any one of claims 1 to 3, which can bind specifically to Concanavalin A-conjugated Sepharose.

16. A proteinaceous material as defined by any one of claims 1 to 3, which can bind with horseradish peroxidase-labelled wheat germ lectin following fractionation of the proteinaceous material into fractions on SDS-PAGE and electrotransfer of the fractions to nitrocellulose.

17. A proteinaceous material as defined by any one of claims 1 to 3, or 10 obtained from follicular fluid.

18. A proteinaceous material as defined by claim 17, obtained from mammalian follicular fluid.

19. A proteinaceous material as defined by claim 18, 25 obtained from ovarian follicular fluid.

20. A proteinaceous material as defined by claim 19, obtained from a bovine.

21. A proteinaceous material as defined by claim 19, obtained from an ovine. 30 22. A proteinaceous material as defined by claim 19, obtained from a human.

23. A proteinaceous material comprising a purified inhibin of a purity sufficient to yield a single major band on non-reducing SDS-PAGE and having a molecular weight of between about 54,000 and 57,000, the purified inhibin comprising two subunits and being capable of suppressing production of follicle stimulating hormone by rat anterior pituitary cells in culture without S21001AX 32 inhibiting the production of luteinising hormone by the cells.

24. A proteinaceous material as defined in claim 23, wherein the apparent molecular weight is about 56,000.

25. A subunit, of a purity sufficient to yield a single major band on non-reducing SDS-PAGE, of a proteinaceous material, inhibin, consisting of two subunits, one of. which has an apparent molecular weight of 14,000 2,000, which inhibin is capable of suppressing production of follicle stimulating hormone without suppressing production of luteinizing hormone.

26. A subunit as defined by claim 25, which is glycosylated.

27. A subunit as defined by claim 25, wherein the subunit has an apparent molecular weight of 44,000 1 3,000.

28. A subunit as defined by claim 27, wherein the subunit has an apparent molecular weight of 44,000.

29. A subunit as defined by claim 28, wherein the subunit has an isoelectric point of between about pH and

30. A subunit as defined by claim 29, wherein the second through sixteenth N-terminal amino acids of the subunit are: Amino Acid Position Amino Acid 2 Ala 3 Val 4 Gly 5 Gly 6 Phe 7 Met 8 Arg 9 Arg 10 Gly 11 Ser 12 Glu 13 Pro 14 Glu 21001AX 33 Asp 16 Gin

31. A subunit as defined by claim 25, wherein the subunit has an apparent molecular weight of 14,000 2,000.

32. A subunit as defined by claim 31, wherein the subunit has an apparent molecular weight of 14,000.

33. A subunit as defined by claim 31, wherein the second, third, fifth, sixth, seventh, eighth, ninth, ±0 tenth, thirteenth and fourteenth N-terminal amino acids of the subunit are: Amino Acid Position Amino Acid 2 Leu 3 Glu 5 Asp 6 Gly 7 Lys 8 Val 9 Asp or Asn 10 Ile 13 Lys 14 Lys

34. A fragment, of a purity sufficient to yield a single major band on non-reducing SDS-PAGE, of a proteinaceous material, inhibin, consisting of two subunits, one of which has an apparent molecular weight of 14,000 2,000, which inhibin is capable of suppressing production of follicle stimulating hormone without suppressing production of luteinizing hormone.

35. A fragment according to claim 34 wherein said fragment can suppress production of follicle stimulating hormone without suppressing production of luteinizing hormone.

36. A fragment as defined by claim 34 or 35, which is glycosylated.

37. A derivative, of a purity sufficient to yield a single major band on non-reducing SDS-PAGE, of a proteinaceous material, inhibin, consisting of two 0 Q0 0* 0 0 1 r 21001AX 34 subunits, one of which has an apparent molecular weight of 14,000 4 2,000, which inhibin is capable of suppressing production of follicle stimulating hormone without suppressing production of luteinizing hormone.

38. A homologue, of a proteinaceous material, inhibin, as defined by claim 10, of a purity sufficient to yield a single major band on non-reducing SDS-PAGE, which homologue is capable of suppressing production of follicle stimulating hormone without suppressing production of luteinizing hormone.

39. A homologue as defined in claim 38, which is glycosylated. A proteinaceous material as defined by any one of claims 1 to 3, wherein the proteinaceous material is labelled.

41. A proteinaceous material as defined by claim labelled with radioactive iodine.

42. A meLhod for purifying a proteinaceous material, inhibin, as defined in claim 1, from a starting material containing the inhibin comprising: a) subjecting the starting material to a first gel permeation chromatography to yield a first eluate; too b) subjecting the first eluate to a second gel permeation chromatography to yield a second eluate; 25 c) subjecting the second eluate to reverse phase high performance liquid chromatography to yield a third eluate; d) subjecting the third eluate to preparative polyacrylamide gel electrophoresis to obtain a single 30 major band; and e) assaying the material of the single major band for ability to suppress production of follicle stimulating se hormone by rat anterior pituitary cells in culture without inhibiting the production of luteinising hormone by the cells.

43. A proteinaceous material comprising purified inhibin obtained according to the method defined by claim 42. T 44. A proteinaceous material as defined by claim 43, 21001AX 35 wherein the purified inhibin has a specific activity of at least about 3 times greater than the specific activity of the starting material. A proteinaceous material as defined by claim 44, wherein the purified inhibin has a specific activity of at least about 10 times greater than the specific activity of the starting material.

46. A proteinaceous material as defined by claim wherein the purified inhibin has a specific activity of at least about 20 times greater than the specific activity of the starting material.

47. A proteinaceous material as defined by claim 46, wherein the purified inhibin has a specific activity of at least about 50 times greater than the specific activity of the starting material.

48. A proteinaceous material as defined by claim 47, wherein the purified inhibin has a specific activity of at least about 160 times greater than the specific activity of the starting material.

49. The method defined by claim 42, wherein the starting material is follicular fluid. The method defined by claim 49, wherein the starting material is mammalian follicular fluid.

51. The method defined by claim 50, wherein the starting 25 material is bovine follicular fluid.

52. The method defined by claim 50, wherein the starting material is ovine follicular fluid.

53. The method defined by claim 50, wherein the starting material is human follicular fluid.

54. The method defined by any one of claims 42 to 53 wherein a gel permeation matrix comprising Sephacryl S-200 is used to perform the first gel permeation chromatography. The method defined by any one of claims 42 to 54 wherein the first eluate comprises a void volume fraction from the gel permeation chromatography.

56. The method defined by any one of claims 42 to wherein Sephadex G-100 is used to perform said second gel 0* 9 0* K21001AX 36 permeation chromatography, in the presence of acid.

57. The method defined by claim 56 wherein .he acid is 4M acetic acid.

58. The methcd defined by any one of claims 42 to 57, wherein the second gel permeation chromatography is performed on an analytical column.

59. The method defined by any one of claims 42 to 58, further comprising performing the reverse phase high performance liquid chromatography using a chemically-bonded N-alkyl silica column materia The method defined by any one of claims 42 to 59, further comprising isolating a gel portion from the preparative polyacrylamide gel containing inhibin and subjecting the inhibin contained in the gel portion to electrophoretic elution.

61. The method defined by claim 60, wherein the electrophoretic elution comprises: washing the gel portion in an elution buffer; soaking the gel portion in a soaking buffer overlaid with an elution buffer; initiating electrophoretic elution of the gel portion; replacing the elution buffer with a dialysis buffer; continuing electrophoretic elution of the gel 25 portion until the inhibin has migrated out of the gel portion; and collecting the inhibin. i: 62. A method for producing antibodies specific to a proteinaceous material as defined in any one of claims 1 to 3, comprising immunizing a mammal with an effective amount of a preparation of a proteinaceous material as S" defined by any one of claims 1 to 3, in combination with an adjuvant.

63. The method defined by claim 62, further comprising the steps of: dissolving the purified inhibin in buffer; emulsifying the purified inhibin dissolved in the buffer with adjuvant tu yield an emulsion; and 21001AX 37 injecting the emulsion into the mammal.

64. The method defined by claim 62 or 63, further comprising isolating antiserum containing said antibodies from the mammal.

65. An antiserum comprising antibodies specific to a proteinaceous material as defined in any one of claims 1 to 3, the antiserum being produced according to the method of any one of claims 62 to 64.

66. The antiserum defined by claim 65, wherein the antiserum neutralizes inhibin activity in vitro.

67. A method for producing monoclonal antibodies specific to inhibin, comprising producing the monoclonal antibodies using the proteinaceous material defined by any one of claims 1 to 3.

68. Antibodies obtained according to the method as defined by claim 67.

69. Monoclonal antibodies which bind to an immunological determinant of the proteinaceous material defined in any one of claims 1 to 3.

70. A method for assaying for inhibin in a sample, comprising a ra~ioinmmnoassay procedure using an effective amounL of a proteinaceous material as defined by any one of claims 1 to 3.

71. A method for detecting and diagnosing infertility in a mammal, comprising using the radioimmunoassay procedure as defined by claim 70 to assay for inhibin in a sample from the mammal.

72. A non-human mammal having a raised titre of anti-inhibin antibodies induced by immunizing the mammal with a proteinaceous material as defined in any one of claims 1 to

73. A proteinaceous material as defined by claim 13, further wherein the mammal experiences an increase in serum levels of follicle stimulating hormone caused by the antibodies.

74. A method for increasing the fertility of a mammal, comprising immunizing the mammal with an effective amount of an antiserum as defined by claim t. 21001AX 38 A method for increasing the fertility of a mammal, comprising immunizing the mammal with a proteinaceous material as defined by any one of claims 1 to 3, in combination with a suitable adjuvant, whereby antibodies to the proteinaceous material are produced.

76. A method for advancing sexual maturity in a sexually immature mammal, comprising immunizing the mammal according to the method as defined by any one of claims 62 to 64.

77. A method for advancing sexual maturity in a sexually immature mammal, comprising injecting the mammal with an antiserum as defined by claim

78. A method for suppressing or delaying the onset of sexual maturity in a mammal, comprising administering to the mammal an effective amount of a proteinaceous material as defined by any one of claims 1 to 3.

79. A method for inhibiting gonadal function in a mammal, comprising injecting the mammal with an effective amount of a preparation comprising a proteinaceous material as defined by any one of claims 1 to 3. A method for postponing the onset of puberty in a mammal, comprising administering to the mammal an effective amount o2 a proteinaceous material as defined by any one of claims 1 to 3. 25 81. A method for suppressing ovulation in a female mammal, comprising administering to the female mammal an effective amount of a proteinaceous material as defined by any one of claims 1 to 3.

82. A method for increasing the rate of ovulation in a mammal, comprising immunizing the mammal with an effective amount of a proteinaceous material as defined by any one of claims 1 to 3.

83. A method for increasing the rate of ovulation in a mammal, comprising administering to the mammal an antiserum as defined by claim

84. A method for suppressing spermatogenesis in a male mammal, comprising administering to the male mammal an effective amount of a proteinaceous material as defined 21 001oo AX 39 by any one of claims 1 to 3. A composition effective as a contraceptive in a mammal, eromprising an effective amount of a proteinaceous material defined by any one of claims 1 to 3, together with a pharmaceutically acceptable carrier or diluent.

86. A method for effecting contraception in a mammal, comprising administering to the mammal a composition as defined by claim

87. A proteinaceous material or antiserum, substantially as herein described with reference to any one of the Examples.

88. A subunit of inhibin, substantially as herein described with reference to any one of the Examples.

89. A fragment of inhibin, substantially as herein described with reference to any one of the Examples. A derivative of inhibin, substantially as hereinbefore described with reference to Example 7.

91. A homologue, substantially as hereinbefore described with reference to Example 11.

92. A method of purifying inhibin, raising antibodies or assaying for inhibin, substantially as hereinbfore described with reference to any one of the Examples.

93. A method of assay for inhibin characterized by the use of an antibody against inhibin made by the method of 25 Claim 62.

94. A method for constructing oligonucleotide probes for subsequent cloning and expression of inhibin genes using recombinant DNA technology, characterized by the use of the amino acid sequences of the protein subunits according to any one of claims 25 to 33.

95. A method for suppressing FSH levels in a mammal, characterized by the administration of a proteinaceous material according to any one of claims 1 to 3 to the mammal.

96. A method of raising FSH levels in a mammal, characterized by administering to that mammal an antiserum according to claim S 97. A therapeutic composition comprising a proteinaceous 21001AX 40 material according to any one of claims 1 to 3, together with a pharmaceutically-acceptable diluent.

98. A therapeutic composition comprising a proteinaceous material according to any one of claims 1 to 3, together with a pharmaceutically acceptable delayed release composition.

99. A therapeutic or diagnostic composition comprising an antiserum according to claim 65, together with a pharmaceutically-acceptable diluent.

100. A therapeutic composition comprising an antiserum according to claim 65, together with a pharmaceutically acceptable delayed release composition.

101. A method for the isolation of clones expressing all or part of inhibin genes produced using recombinant DNA technology, characterized by the use of an antiserum according to claim

102. A method for detection of inhibin mRNA in biological samples, characterized by the use of the amino acid sequences of a subunit according to any one of claims to 33. 103 Antibody to inhibin according to claims 65 or 68 labelled according to any previously known method.

104. A diagnostic method characterized by the use of a S* labelled antibody according to claim 103. 25 105. A radioimmunoassay for inhibin characterized in that a radiolabelled inhibin according to claim 41 is reacted with an antibody to inhibin.

106. A radioimmunoassay for inhibin characterized in that inhibin as herein defined is reacted with a radiolabelled antibody according to claim 103.

107. A radioimmunoassay according to claim 105 or claim 106, characterized in that a polyethylene glycol-facilitated second antibody precipitation step is used.

108. An enzyme-linked immunoassay characterized by the use of a proteinaceous material according to any one of claims 1 to 3 coupled to an enzyme by any previously kn known method. 21001 AX 41

109. An enzyme-linked immunoassay characterized by the use of an antibody according to claim 68 or 69 coupled to an enzyme by any previously known method.

110. A method according to claim 42 which method comprises subjecting the first eluate to a pH adjustment to pH 4.75 prior to the second gel permeation chromatography. DATED this 7th day of September 1992 MONASH UNIVERSITY, MONASH MEDICAL CENTRE, ST VINCENT'S INSTITUTE OF MEDICAL RESEARCH and BIOTECHNOLOGY AUSTRALIA PTY LTD, By their Patent Attorneys GRIFFITH HACK CO 1.) 2101A