Note: Descriptions are shown in the official language in which they were submitted.
1305829
OVINE INHIBIN
The present invention relates to a protein
having inhibin activity isolated to substantial
homogeneity from material obtained from ovine animals.
BACKGROUND O _THE INVENTION
The existence of nhibin 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 yreat 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 fluid(RTF).
The protein has been partially characterized using
microsequencing methods.
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In a first embodiment, the invention
provides a substantially pure ovine inhibin protein
consisting essentially of greater than 90~ by weight of total
protein,
said inhibin protein consisting of a dimer of a
first polypeptide chain having an apparent molecular weight of
about 18,000 Daltons, as measured by gel electrophoresis, and
a second polypeptide chain having a molecular weight of about
16,500 Daltons, as measured by gel electrophoresis, said first
chain being linked to said second chain through disulfide
bonding,
said dimer, when subjected to reverse phase HPLC
elutes from a C8 column of 5 micron particle size and 300
angstrom pore size at between about 27 and about 28 minutes
following the instigation of a straight line gradient flow of
Buffers A and B, which gradient varies from 25% Buffer B to
90~ Buffer B over a period of 45 minutes, at a flow rate of
0.7 ml per minute and a back pressure of about ~90 psi, with
Buffer A being 0.1~ trifluoroacetic acid (TFA) and Buffer B
being 80% acetonitrile in 0.1% TFA;
said first chain 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-Ala-Asp-Cys-His-Arg-Ala and
said second chain having about the formula:
H-Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-
Phe-Tyr-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-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, said protein
specifically inhibiting basal secretion of follicle-
stimulating hormone while not inhibiting basal secretion of
luteinizing hormone.
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2a
In a second embodiment, the invention
provides a protein subunit of ovine inhibin having a
purity of greater than 90% by weight based upon total protein
selected from the group consisting of (a) a first polypeptide
having an apparent molecular weight of about 18kD, as measured
by gel electrophoresis, 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-Ala-Asp-Cys-
His-Arg-Ala, and (b) a second polypeptide having an apparent
molecular weight of about 16.5kD, as measured by gel
electrophoresis, and having the sequence Gly-Leu-Glu-Cys-Asp-
Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-Tyr-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-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, wherein the first polypeptide, when
combined through disulfide bonding with the second
polypeptide, creates a dimer, which dimer elutes from a Ca
reverse phase ~PLC column of 5 micron particle size and 300
angstrom pore size, at between about 27 and about 28 minutes
following the instigation of a straight line gradient flow of
Buffers A and B, which gradient varies from 25% Buffer B to
90% Buffer B over a period of 45 minutes, at a flow rate of
0.7 ml per minute and a back pressure of about 890 psi, with
Buffer A being 0.1% trifluoroacetic acid (TFA) and with Buffer
B being 80% acetonitrile in 0.1% TFA.
JJ:
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130~9
2~
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* C8
column with a 5 ~m 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.
DET~ILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Using a multi-step procedure, the 34.5kD peptide
was isolated to substantial homogeneity from ram rete
testis fluid (RTF). The protein is composed of two
chains of 18kD 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. Amino 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
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.5~6~
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 ~he 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
(EC50) 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. In vivo,
partially purified inhibin preparations are highly
selective to decrease plasma FSH and not LH levels. The
effects of inhibin on basal gonadotropin secretion in
vitro appears to best reflect the in vivo situation.
The 34.5kD protein is useful for regulating gonadotropin
secretion and thus fertility and/or sex hormone
13~)5~
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 cruds 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 4C using
dialysis bags with molecular weight cut off (MWC0) 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, Vt=2750 mls.
Column eluant was 6 M ~uanidine HCl, 0.1 M ammonium
acetate, 0.05% dimethylsulfide in Milli Q H20, ph
4.75. Æluant was 0.22 ~m filtered and degassed before
use. Flow rate was 50 mls/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-~m
particle-si2e 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% CH3CN in Buffer A. After all the filtrate had been
loaded, the column was washed with the aqueous
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1~05~
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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 W 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-~m-particle-size C4 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 ~m (Pharmacia
Fine Chemicals, Piscataway, N. J.) linked in series.
Each column was eluted with 6M guanidine HCl, O.lM
ammonium acetate, pH 4.75, and 0.5~ DMS in Milli ~ 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
KAV=0.31-0.36.
Inhibin protein fractions from the various
individual columns were pooled and further purified by
RP-HPLC using 5-~m-particle-size C4 column, 1 x 30 cm,
and 0.5% TFA/CH3CN buffer system.
After such ~PLC 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, ~ mM CHAPS (3-[(Cholamidopropyl)
dimethylammonio]-l-propanesulfonate) in Milli Q H20,
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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,
Vt = 1 ml, was used at a flow rate of l ml per min.
Column was loaded at 0% B followed by a gradient to 30% B
in 25 minutes and then to lOQ% B in 5 minutes.
Next, the active fraction was applied to a
0.46 x 25 cm Vydac* C8 Column of reversed phase
material with a 5 ~m particle SiZ8 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 40C. 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* C8 Column of reversed phase
material with a 5 ~m 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 40C 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 40C with a back pressure of 890 psi, and a detector
setting of 215 nm, 2.9 AUFS, which was slightly ch~nged
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 lOOA pumps, a Datamark* Servocoder SR
6253 strip chart recorder, a Kratos Spectroflow* 773
variable wavelength, W/visible detector and a Rheodyne
7125 injector with a 2.0 ml loop.
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~.,
82~
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 pg of inhibin protein, including about
120 ~g of the earlier eluting active inhibin fraction
and about 75 ~g of the later eluting fraction. The 195
~ug 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 110C. 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 AcidInhibin Protein
Asx 22
Thr 11
Ser 17
Glx 22
Gly 16
Ala 16
~al 11
Met 5
Ile 8
Leu 22
Tyr 8
Phe 10
His 8
Trp 4
~ ;~O~S8~:~9
--8--
Amino Acid Inhibin Protein
Lys 10
Arg 10
Cys 12
Pro 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 accura~ely
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% ~-mercaptoethanol at
neutral p~ 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.I 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 migratin~ 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.
NH2-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
58~3
g
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
:1 ~05~
--10--
immunological 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 ~ith 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, na~e~y Se~-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
i :~C~5&2~
other vertibrate animal species having inhibin of a
similar polypeptide structure. Administration of
inhibin ind~ces 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
~0~ 2~
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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.
